Visit our Expo - Redox and Inflammation signaling 2012

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Luxembourg,
January 25th to 28th, 2006
European Conference Center
Kirchberg - Luxembourg
Cell Signaling World 2006
Signal Transduction Pathways as therapeutic targets
Proceedings
and
Program
Editor
Marc Diederich
Organized by
Recherches Scientifiques Luxembourg
Printing of the Proceedings sponsored by the
Fondation de Recherche Cancer et Sang (Luxembourg)

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Table of content
PREFACE ......................................................................................................................................................................5
ACKNOWLEDGMENTS ...........................................................................................................................................7
GENERAL INFORMATION .....................................................................................................................................8
EXHIBITION ..............................................................................................................................................................11
SCIENTIFIC PROGRAM ........................................................................................................................................14
WEDNESDAY JANUARY 25TH, 2006.......................................................................................................................14
THURSDAY JANUARY 26TH, 2006 (EARLY MORNING SESSION) .........................................................................15
THURSDAY JANUARY 26TH, 2006 (MORNING SESSION)......................................................................................15
THURSDAY JANUARY 26TH, 2006 (EVENING SESSION) .......................................................................................17
FRIDAY JANUARY 27TH, 2006 (EARLY MORNING SESSION) ...............................................................................18
FRIDAY JANUARY 27TH, 2006 (MORNING SESSION) ...........................................................................................18
FRIDAY JANUARY 27TH, 2006 (EVENING SESSION).............................................................................................20
SATURDAY JANUARY 28TH, 2006 (EARLY MORNING SESSION)..........................................................................21
SATURDAY JANUARY 28TH, 2006 (MORNING SESSION)......................................................................................21
ORAL PRESENTATIONS........................................................................................................................................23
WORKSHOP PRESENTATIONS.........................................................................................................................107
POSTER PRESENTATIONS .................................................................................................................................117
SESSION I. PROTEIN DOMAINS ............................................................................................................................118
SESSION II. RECEPTOR SIGNALING AND G PROTEINS.......................................................................................127
SESSION III. PROTEIN KINASE CASCADES AS THERAPEUTIC TARGETS ...........................................................183
SESSION IV. PROTEIN KINASE INHIBITORS: INSIGHTS INTO DRUG DESIGN FROM STRUCTURE....................257
SESSION V. PHOSPHATASES AS KEY CELL SIGNALING INTERMEDIATES.........................................................266
SESSION VI. PROTEASOME DEGRADATION PATHWAYS ....................................................................................276
SESSION VII. STEM CELL SPECIFIC CELL SIGNALING MECHANISMS...............................................................281
SESSION VIII. INFLAMMATION SPECIFIC SIGNALING .......................................................................................284
SESSION IX. NOVEL COMPOUNDS TARGETING INFLAMMATORY SIGNALING PATHWAYS.............................324
SESSION X : CELL DEATH IN CANCER .................................................................................................................333
SESSION XI : CELL DEATH AND CARDIOVASCULAR DISEASES.........................................................................445
SESSION XII : CELL DEATH AND NEURODEGENERATIVE DISEASES ................................................................458
SESSION XIII : CELL SIGNALING PATHWAYS LEADING TO REGULATED CHROMATIN MODIFICATIONS .....488
SESSION XIV : TRANSCRIPTIONAL AND TRANSLATIONAL CONTROL..............................................................503
SESSION XV : REACTIVE OXYGEN SPECIES AND CELL SIGNALING..................................................................585
SESSION XVI : CHEMOPREVENTIVE AGENTS ....................................................................................................592
SESSION XVII : CELL SIGNALING IN HEALTH AND DISEASE ............................................................................607
LIST OF PARTICIPANTS .....................................................................................................................................672
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Preface
In 1998, we organized the first specialized meeting in the field of signal tranduction and gene
expression in Luxembourg. This type of meeting was originally thought to teach doctoral
students involved in the cellular and molecular biology teaching program (DEA de
Pharmacologie moléculaire) of the University of Nancy I (France).
Since then, 3500 fundamental and clinical researchers were gathering in Luxembourg for
different meeting to discuss therapeutic applications in the field of signal transduction,
transcription and translation. These meeting allowed new insights into a rapidly moving field.
Novel antibodies against receptors, protein kinase inhibitors, antisense oligonucleotides
targeting both signal transduction and gene expression will certainly enhance therapeutic
approaches for the next century.
For our 2006 edition of meeting, with more than 950 participants on site, I am convinced that
this meeting will be a great success.
Welcome to Luxembourg !
Marc Diederich
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Acknowledgments
This meeting has been realized under the aegis of :
Recherches Scientifiques asbl
The Fondation de "Recherche Cancer et Sang"
Assosciation An Haerz fir kriibskrank Kanner
We have the pleasure to acknowledge support from :
Kuwait Petroleum SA - Luxembourg
The City of Luxembourg
The Fondation de Recherche "Cancer et Sang"
Alexis Biochemicals
Novartis Luxembourg
Your onsite organization team :
Franck Morceau, Romain Blasius, Estelle Henry, Marie-Hélène Teiten, Serge Eifes, Simone
Reuter, Silvia Cristofanon, Isabelle Buck, Florence Folmer, Anne-Laure Joly, Delphine
Magard, Emilie Courtine, Audrey Richard, Athanase Visvikis and Marc Diederich
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Meeting Venue
General Information
All meeting sessions are held at the European Congress Center.
European Congress Center - Quartier Européen Sud,
1, rue du Fort Thüngen
L-1499 Luxembourg-Kirchberg
Registration will take place in the area outside of the "Hémicycle" (Level 0) at the registration desk
open daily (9h00-19H30).
Coffee breaks will be served in the exhibit area daily (See general program for details).
Lunch
For the participants that pre-paid lunch, lunch is served from 12h00 - 14h00 at two locations :
Salon Bleu (Level -1) and Bar de l'Hémicycle (Level -1)
There is no difference between the two lunch sites, both take about 350 participants. Additional tickets
are NOT available at the registration desk.
Exhibits are open daily form January 25 th to 28 th , 2006.
The Exhibit opens on Wednesday January 25 th (Welcome reception) and ends on Friday January 27 st ,
2006 after the afternoon coffee break. Exhibitors are nevertheless welcome to stay on Saturday
January 28th, 2006.
Lecture rooms (see expo map for locations) :
- Main lecture room : Hemicycle : Level 0
- Lecture room Havanne : Level 0
Posters (levels -1 and 0)
There will be four poster viewings :
Thursday January 26th
Group A (13h00 - 15h00)
Session I. Protein Domains
Session II. Receptor signaling and G proteins
Session III. Protein kinase cascades as therapeutic targets
Session IV. Protein kinase inhibitors: insights into drug design from structure
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Group B (15h00 - 17h00)
Session V. Phosphatases a key cell signaling intermediates
Session VI. Proteasome degradation pathways
Session VII. Stem cell specific cell signaling mechanisms
Session VIII. Inflammation specific signaling
Session IX. Novel compounds targeting inflammatory signaling pathways
Session XV. Reactive oxygen species and cell signaling
Session XVII : Cell signaling in health and disease
On Thursday evening, after Group B, the poster boards need to be cleared in order to allow re-
numbering of the boards for the next day. The organizers will otherwise need to take the posters off
and will not be responsible for lost or damaged posters.
Friday January 27th
Group C (13h00 - 15h00)
Session X. Cell death in cancer
Session XI. Cell death and cardiovascular diseases
Session XII. Cell death and neurodegenerative diseases
Group D (15h00 - 17h00)
Session XIII. Cell signaling pathways leading to regulated chromatin modifications
Session XIV. Transcriptional and translational control
Session XVI. Chemopreventive agents
Welcome reception (expo area)
On Wednesday January 25, 200 from 19H30-20H30 at the expo surface.
Gala Diner
The Gala Diner will take place at the Hotel Le Royal (12, Boulevard Royal, L-2449 Luxembourg) on
Thursday, January 26th starting at 21H00. Additional tickets are NOT available at the registration
desk.
Transportation
Our bus shuttles will bring the participants from the hotels to the meeting center in the morning and
back to the hotels or city center in the evening. Please note that the busses leave between 6h30 and
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8h00 from your hotel depending on the distance to the meeting center. The busses are red, orange and
yellow and are from the bus company "Demy Cars".
Taxis can be called from the registration desk.
How to reach the congress center ?
- by taxi :
from the airport (Luxembourg - Findel) in about 15 minutes.
from the railroad station (about 15 minutes)
- by bus : take bus line number 16 (every 20 minutes from the city center)
- by car :
from France : Highway A4 from Metz, take Highway A1 (E44) direction Trier Plateau de
Kirchberg Aéroport, choose exit number "8", orient towards "Quartier Europeen Sud Luxembourg-
Centre"
from Belgium : Highway A411 from Brussels, take Highway A6 (E44) direction Trier Plateau
de Kirchberg Aéroport, choose exit number "8", orient towards "Quartier Europeen Sud Luxembourg
Centre"
from Germany : Highway A6 (E44) from Trier, direction Luxembourg, choose exit number
"8", orient towards "Quartier Europeen Sud Luxembourg Centre".
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Exhibition

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Visit our Expo
(in alphabetical order)
Abgent Europe 11
Active Motif 7
ALEXIS Biochemicals 2
amaxa GmbH 37
AMBION 6
Ariadne Genomics 44
AXXORA Platform 3
BD Biosciences 14 and 15
Berthold Detection Systems GmbH 29
Biobase GmbH 10
Biochemical Journal/Portland Press 23
Bio-Connect BV 27
Biognost bvba 9
Bio-Rad Laboratories NV/SA 18
Biosource 20
CCSBE NV Sales 31
Cell Signaling Technology 40
Diagenode 30
Eurogentec 24
GENTAUR Molecular Products 32
Greiner Bio One 21
Invitrogen 19
InvivoGen 5
Lecuit Opto-Technical S.A. 16 and 17
Merck Biosciences GmbH 39
Miltenyi Biotec 4
MP Biomedicals 41
nanoTools Antikoerpertechnik 13
New York Academy of Sciences 25
Olympus Belgium NV 43
Perbio Science 35
PerkinElmer NV/SA 42
Polyplus-transfection 22
Prophac 1
R&D Systems Europe Ltd 26
Science/AAAS 36
SEROTEC LTD 28
Sigma-Aldrich 38
tebu-bio 33 and 34
VWR International 8
Westburg 12
Internet café and wireless internet access generously
offered by Cadolto, prefabricated buildings
http://www.cadolto.com
http://www.prefabriques.lu
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Scientific Program
Wednesday January 25th, 2006
9h00 – 14h00 : Registration of the participants
12h00 – 14h30 : Lunch is served
14h00 - 19h00: Keynote session
14h00 – 15h00 : Gregg L. Semenza (Institute for Cell Engineering, The Johns Hopkins
University School of Medicine, Baltimore, USA):
Keynote: Hypoxia Signal Transduction in Health and Disease
15h00 – 15h30 : Michael B. Yaffe (Center for Cancer Research, Department of Biology
Massachusetts Institute of Technology, Cambridge, MA, USA):
Phosphoserine/threonine-binding domains: Integrators of protein kinase signaling pathways in
cell cycle control and cancer
15h30 – 16h00 : Mathieu Bollen (Afdeling Biochemie, Faculteit Geneeskunde, Katholieke
Universiteit Leuven, Leuven, Belgium):
Targeting of Polycomb repressive complexes by the moonlighting protein NIPP1
16h00 – 16h30 : Coffee break and visit of the expo
16h30 – 17h00 : Jurg Tschopp (Department of Biochemistry, University of Lausanne, BIL
Biomedical Research Center, Epalinges, Switzerland):
Conserved signaling mechanisms in plant and vertebrate innate immunity
17h00 – 17h30 : Roger J. Davis (Howard Hughes Medical Institute, UMASS Medical
School, Worcester, USA):
Signal transduction by stress-activated MAP kinases
17h30 – 18h00 : Laurent Meijer (CNRS, Roscoff, France):
Multiple intracellular effects of pharmacological inhibitors of cyclin-dependent kinases
18h00 – 18h30 : François Fuks (Free University of Brussels, Faculty of Medicine,
Laboratory of Molecular Virology, Brussels, Belgium) :
Mechanisms of DNA methylation in mammals
19h00 : Official inauguration and welcome reception
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Thursday January 26th, 2006 (Early morning session)
7h30 – 8h30 : Receptor Signaling : Short communications
7h30 – 7h40 : Jan. S. Jepsen (Danish Cancer Society, Copenhagen, Denmark ) :
Protein Kinase C alpha as a potential target in the treatment of acquired antiestrogen
resistance of human breast cancer.
7h40 – 7h50 : Raymond R. Mattingly (Department of Pharmacology and Karmanos Cancer
Institute, Wayne State University, Detroit, Michigan, U.S.A.)
Increased EGF Receptor expression and activity is induced by deregulated N-Ras and may
provide a therapeutic target in NF1 neurofibrosarcoma
7h50 – 8h00 : Shyamala Maheswaran (Department of Surgical Oncology, Massashusetts
General Hospital, Boston, MA , USA) :
Loss of the Antiproliferative Gene BTG2 in Estrogen Receptor Positive Breast Carcinoma is
Associated with Tumor Grade and Overexpression of Cyclin D1 Protein
8h00 – 8h10 : Xiao C. Li (Division of Hypertension and Vascular Research, Henry Ford
Hospital, Detroit, MI 48202, USA ) :
Cross-talk between angiotensin II and glucagon receptor signaling mediates activation of
mitogen-activated protein kinases ERK 1/2 in rat glomerular mesangial cells.
8h10 – 8h20 : Metoda Lipnik-Stangelj (Department of Pharmacology and Experimental
Toxicology, Faculty of Medicine, University of Ljubljana, Slovenia) :
Multiple role of histamine H1 receptor-PKC-MAPK signalling pathway in histaminestimulated
nerve growth factor secretion from glial cells
8h20 – 8h30 : Ying E. Zhang (Laboratory of Cellular and Molecular Biology, Center for
Cancer Research, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland, USA) :
Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting
MEKK2-JNK pathway
Thursday January 26th, 2006 (Morning session)
8h30 - 10h30: Receptor signaling
8h30 – 9h00 : Walter Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin,
Germany):
The role of Met-Gab1 Signalling in vivo.
9h00 – 9h30 : Ulf R. Rapp (Institut fuer Medizinische Strahlenkunde und Zellforschung
(MSZ), Wuerzburg, Germany):
Role of Prohibtin and E-cadherin in Raf mediated oncogenesis
9h30 – 10h00 : Johannes L. Bos (Dept of Physiological Chemistry, Utrecht, The
Netherlands):
Epac and Rap1 in the control of cell adhesion
10h00 – 10h10 : Jean-Paul Borg (Molecular Pharmacology, Marseille Cancer Institute,
UMR599 Inserm-Institut Paoli-Calmettes, Marseille, France) :
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The mammalian tumor suppressor Scribble has a key role in cell migration
10h10 – 10h20 : Tarik Issad (CNRS UMR8104, INSERM U567, Université René Descartes
Paris 5, Institut Cochin, Paris, France) :
The use of BRET (Bioluminescence Resonance Energy Transfer) for the study of tyrosinekinase
receptors
10h20 – 10h30 : Atsufumi Kawabata (Division of Physiology and Pathophysiology, School
of Pharmaceutical Sciences, Kinki University, Japan) :
Cell signaling by proteinase-activated receptor-2 for interleukin-8 formation in human lung
epithelial cells
10h30 – 11h00 : Coffee break and visit of the expo
11h00 - 13h00: Protein kinase cascades as therapeutic targets
11h00 – 11h30 : Dario Alessi (MRC Protein Phosphorylation Unit, School of Life Sciences,
MSI/WTB Complex, University of Dundee, Scotland):
Regulation and function of the WNK1 and WNK4 protein kinases that are mutated in
Gordon’s hypertension syndrome.
11h30 – 12h00 : Jacques Pouysségur (Institute of Signaling, Developmental Biology and
Cancer Research CNRS UMR 6543, Centre A. Lacassagne, Nice, France):
Nutrient stress signaling and tumor progression
12h00 – 12h30 : Brian Hemmings (Friedrich Miescher Institute for Biomedical Research,
Basel, Switzerland):
The NDR kinase family: Regulators of cell proliferation and morphogenesis
12h30 – 13h00 : Ying Xia (Center for Environmental Genetics and Department of
Environmental Health, University of Cincinnati Medical Center, Cincinnati, USA):
The MEKK1-JNK Pathway Regulates Mouse Eyelid Morphogenesis
12h00 – 14h30 : Lunch is served
13h00 – 17h30 : Workshops
Thursday January 26th, HEMICYCLE room
13h00-14h00 : Becton-Dickinson
14h00-15h00 : Ambion 1
15h00-16h30 : Biobase 1
16h30-17h30 : Amaxa
Thursday January 26th, HAVANNE room
13h00-14h00 : CST 1
14h00-15h00 : CST 2
15h00-16h00 : Miltenyi Biotech 1
13h00 – 15h00 : Poster session I
15h00 – 17h00 : Poster session II
17h00 – 17h30 : Coffee break and visit of the expo
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Thursday January 26th, 2006 (Evening session)
17h30 - 20h00: Cell death in cancer
17h30 – 18h00 : Caroline Dive (Cellular and Molecular Pharmacology Group, Paterson
Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom):
title to be announced
18h00 – 18h30 : Peter H. Krammer (Tumorimmunology Program, German Cancer
Research Center, Heidelberg, Germany):
Signaling of life and death in T cells
18h30 – 19h00 : Guido Kroemer (CNRS, UMR8125, Institut Gustave Roussy, Villejuif,
France):
Mechanisms of caspase-independent cell death
19h00 – 19h30 : Michael J. Pinkoski (MRC Toxicology Unit, Leicester, United Kingdom):
Live and let die by 73 - mechanisms of degradation and chemoresistance
19h30 - 19h45 : Sidhartha Ray (Arnold & Marie Schwartz College of Pharmacy & Health
Sciences Div. of Pharmaceutical Scs/Long Island University, Brooklyn, NY, USA):
How Antitoxic and Anticancer signals Maneuver Programmed and Unprogrammed Cell
Death In Vivo?
19h45 – 20h00 : Lina Ghibelli (University Tor Vergata, Rome, Italy):
Oxidative Bax activation as the trigger of the stress-induced intrinsic apoptotic pathway
21-00 – 24h00 : Gala diner at Hotel Le Royal
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Friday January 27th, 2006 (Early morning session)
7h30 – 8h30 : Cell Signaling in health and disease : short communications I
7h30 – 7h40 : Bernd Schmeck (Department of Internal Medicine/Infectious Diseases,
Charité, Universitätsmedizin Berlin, Germany ) :
Listeria monocytogenes induced Rac1-dependent endothelial histone modification and
cytokine release
7h40 – 7h50 : Mayra Solis (Terry Fox Molecular Oncology Group, Lady Davis Institute
McGill University, Montreal Canada) :
Distinct roles of IRF-3 and IRF-7 in the activation of anti-tumor properties of human
macrophages
7h50 – 8h00 : Maria C. Albertini (Istituto di Chimica Biologica A. Fornaini, Universita' di
Urbino, Urbino and Universita' di Roma Tor Vergata, Rome, Italy) :
Mechanisms involved in the anti-apoptotic effect of magnetic fields
8h00 – 8h10 : Michal Hayun (Safdié Institute for AIDS and Immunology Research, Faculty
of Life Sciences, Israel) :
The immunomodulator AS101 induces growth arrest and apoptosis in Multiple Myeloma cells
via the PI3-K/Akt pathway.
8h10 – 8h20 : Livio Mallucci (Cell Signaling Laboratory, Pharmaceutical Sciences Research
Division, King's College London, Franklin Wilkins Building, London, UK) :
PI3K Targeting by the Beta-GBP Cytokine. Mitogenic Input and Therapeutic Response
8h20 – 8h30 : Chantal Schwartz (Laboratoire d’Hémato-Cancérologie Expérimentale, CRP-
Santé, Luxembourg) :
Survey on in vitro cell death induction of multiple myeloma cells upon valproic acid
treatment.
Friday January 27th, 2006 (Morning session)
8h30 - 10h30: Inflammation specific signaling
8h30 – 9h00 : Bharat B. Aggarwal (Cytokine Research Section, The University of Texas M.
D. Anderson Cancer Center, Houston, USA):
Targeting Inflammatory Cell signaling pathway for Prevention and Treatment of cancer
9h00 – 9h30 : Mark Ginsberg (Department of Medicine, University of California San
Diego, La Jolla, CA, USA):
Integrin Signaling in Cancer and Inflammation
9h30 – 10h00 : Young-Joon Surh (National Research Laboratory of Molecular
Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University,
South Korea):
Redox-Sensitive Transcription Factors as Prime Targets for Chemoprevention and
Cyoprotection
10h00 – 10h15 : Irfan Rahman (Department of Environmental Medicine, Lung Biology and
Disease Program, University of Rochester Medical Center, Rochester, NY, USA):
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Regulation of inflammation and glucocorticoid signaling by curcumin and resveratrol
10h15 – 10h30 : Moncef Zouali (INSERM U606, Centre Viggo Petersen, Hôpital
Lariboisière, Paris, France):
Signaling pathways controlling self-tolerance: On the road to systemic autoimmune diseases
10h30 – 11h00 : Coffee break and visit of the expo
11h00 - 13h00: Cell signaling in health and disease
11h00 – 11h30 : Bernd Groner (Georg-Speyer-Haus, Institute for Biomedical Research,
Frankfurt am Main, Germany):
Signaling events in mammary epithelial cell differentiation and breast cancer
11h30 – 12h00 : John Hiscott (Molecular Oncology Group Lady Davis Institute for Medical
Research, Jewish General Hospital, Montreal, Canada):
TLR-dependent and -independent pathways leading to the interferon antiviral response
12h00 – 12h20 : Anna Krichevsky (Center for Neurologic Diseases, Brigham and Women's
Hospital
Harvard Medical School, Boston, USA):
A role of microRNAs in brain tumors (preliminary title)
12h20 – 12h40 : Dominique Stehelin (CNRS UMR 8526, Institut de Biologie de Lille,
France):
New steps toward the use of parvovirus H1 as a therapeutic anti-cancer drug.
12h40 – 13h00 : Jacques Emile Dumont (Institut de Recherche Interdisciplinaire (IRIBHM),
Universite Libre de Bruxelles (ULB), Belgique):
Molecular Biology of Thyroid Cancer
12h00 – 14h30 : Lunch is served
13h00 – 17h30 : Workshops
Friday January 27th, HEMICYCLE
13h00-14h00 : GenOway
14h00-15h00 : Ambion 2
15h00-16h30 : Biobase 2
16h30-17h30 : Polyplus Transfection
Friday January 27th, HAVANNE
13h00-15h00 : Ariadne Genomics 1
13h00-15h00 : Ariadne Genomics 2
15h00-16h00 : IBA 1
16h00-17h00 : IBA 2
13h00 – 15h00 : Poster session III
15h00 – 17h00 : Poster session IV
17h00 – 17h30 : Coffee break and visit of the expo
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Friday January 27th, 2006 (Evening session)
17h30 – 19h00 : Cell signaling pathways leading to regulated chromatin modifications
17h30 – 17h50 : Richard Pestell (Lombardi Comprehensive Cancer Center, Georgetown
University, Washington, USA):
Cyclins and HDAC/HATs
17h50 – 18h10 : Luciano Di Croce (ICREA and Centre de Regulacio Genomica (CRG),
Barcelona, Spain):
Targeting chromatin machines to promoters in leukemias
18h10 – 18h30 : Willem Voncken (Research Institute Growth and Development, University
Maastricht, The Netherlands):
Talking to chromatin: Silencers Silenced.
18h30 – 18h50 : Guy Haegeman (Laboratory for Eukaryotic Gene Expression and Signal
Transduction, Department of Molecular Biology, Ghent University, Belgium):
Chromatin impact on NFkB-driven gene expression
23h00 – 3h00 : Science Party at Melusina Night Club
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Saturday January 28th, 2006 (Early morning session)
7h30 – 8h30 : Cell signaling un health and disease : short communications II
7h30 – 7h40 : Réjane Paumelle (Département d’Athérosclerose, U545 Inserm, Institut
Pasteur de Lille and Faculté de Pharmacie, Université de Lille II, Lille, France) :
Acute anti-inflammatory properties of statins involve Peroxisome Proliferator-Activated
Receptor-alpha via inhibition of the PKC signalling pathway
7h40 – 7h50 : Alexander L. Pukhalsky (Research Centre for Medical Genetics, Moscow ) :
Alkylating drugs applied in non-cytotoxic doses as a novel compounds targeting
inflammatory signal pathways
7h50 – 8h00 : Eric Adriaenssens (Institut Pasteur, Lille, France.) : A new antisense
transcript is involved in the genomic imprinting control at the H19/IGF2 locus.
8h00 – 8h10 : Caroline Rouaux (Laboratoire de Signalisation Moléculaire et
Neurodégénérescence – INSERM U692 - Faculté de Médecine, Strasbourg, France) :
Inhibition of histone deacetylase activity as a therapeutic tool in amyotrophic lateral sclerosis.
8h10 - 8h20 : Peter J. van den Elsen (Leiden University Medical Center, Leiden, The
Netherlands ) :
Epigenic control of MHC2TA transcription: varying contributions of DNA and histone
modifications in cell-activation and interferon-gamma-mediated induction of gene expression
in cancer cells
8h20 – 8h30 : Joydeb Kumar Kundu (National Research Laboratory of Molecular
Carcinogenesis and Chemoprevention, College of Pharmacy, Seoul National University,
South Korea) :
Resveratrol inhibits phorbol ester-induced expression of COX-2 through inactivation of NF-
!B and AP-1 in mouse skin: role of I!B kinase and MAP kinases
Saturday January 28th, 2006 (Morning session)
8h30 - 10h30: Transcriptional control
8h30 – 9h00 : Yinon Ben-Neriah (The Lautenberg Center for Immunology, Hebrew-
University-Hadassah Medical School, Jerusalem, Israel):
Tissue specific control of NF-kappaB activation
9h00 – 9h30 : Sankar Ghosh (Yale University School of Medicine, New Haven, CT , USA):
Regulation of NF-kB-dependent transcription
9h30 – 10h00 : Moshe Oren (Department of Molecular Cell Biology, The Weizmann
Institute, Rehovot, Israel):
Oncogenic signaling by mutant p53
10h00 – 10h10 : Hyeyoung Kim (Department of Pharmacology and Brain Korea 21 Project
for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea) :
Crosstalk between NF-kB and AP-1 in Helicobacter pylori-mediated inflammation in human
gastric epithelial cells
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10h10 – 10h20 : Gerhard Müller-Newen (Institut für Biochemie, Universitätsklinikum der
RWTH Aachen, Aachen, Germany) :
Real-time analysis of Jak/STAT signal transduction in single cells.
10h20 – 10h30 : Claude P. Muller (Institute of Immunology, Laboratoire National de Santé,
Luxembourg) :
Transcriptional control of differential glucocorticoid receptor expression: determining tissue
specific expression, and protein isoforms.
10h30 – 11h00 : Coffee break and visit of the expo
11h00 - 13h00: Transcriptional control
11h00 – 11h30 : Iris Behrman (University of Luxembourg):
Jaks and cytokine receptors - an intimate relationship.
11h30 – 12h00 : Jacques Piette (Laboratory of Virology & Immunology, Institute of
Pathology B23, University of Liege, Liege, Belgium):
The phosphatase SHIP-1 is an important mediator in the oxidative stress-induced NF-kB
activation.
12h00 – 12h30 : Peter Angel (Deutsches Krebsforschungszentrum, DKFZ, Division of
Signal Transduction and Growth Control, Heidelberg, Germany):
Regulation of gene expression in skin remodeling and tumourigenesis
12h30 – 12h40 : Michèle J. Hoffmann (Urologische Klinik und Institut für Pathologie*,
Heinrich-Heine-Universität Düsseldorf, Germany) :
Epigenetic Regulation of Stem Cell Maintenance Genes in Prostate Carcinoma
12h40 – 12h50 : Jose Aramburu (Departament de Ciències Experimentals i de la Salut,
Universitat Pompeu Fabra, Barcelona, Spain) :
Regulation of the cell cycle by the transcription factor NFAT5 in response to osmotic and
genotoxic stress.
12h50 – 13h00 : Wim Vanden Berghe (Laboratory for Eukaryotic Gene Expression and
Signal Transduction (LEGEST), Department of Molecular Biology, Ghent University,
Belgium ) :
Attenuation of MSK1-driven NF-kB gene expression by soy isoflavones does not require
estrogenic activity
13h00: End of the meeting
12h00 – 14h30 : Lunch is served
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Oral presentations
(by alphabetical order)
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A new antisense transcript is involved in the genomic imprinting control at the
H19/IGF2 locus
Nathalie Berteaux 1 , Nicole Aptel 2 , Thierry Dugimont 1 , Guy Cathala 2 , Céline Janton 1 ,
Jean Coll 3 , Hubert Hondermarck 1 , Jean-Jacques Curgy 1 , Thierry Forné 2 and Eric
Adriaenssens 1 .
1 ERI-8 INSERM, UPRES-EA 1033, IFR 118, USTL, 59655 Villeneuve d’Ascq cedex,
France ; 2 Institut de Génétique Moléculaire, UMR 5535 CNRS, Université Montpellier
II, IFR 122, Montpellier, France ; 3 UMR 8527 CNRS, IBL, Institut Pasteur, Lille,
France.
E-mail : eric.adriaenssens@univ-lille1.fr
Natural antisense transcripts have been implicated in many aspects of eucaroytic gene
expression including genomic imprinting and RNA interference. Our group has identified a
new antisense transcript at the human and mouse H19/IGF2 locus, that we called 91H. This
locus belongs to an imprinting domain located in human chromosome 11p15.5 (homologue to
the mouse distal chromosome 7). H19 is expressed from the maternal allele and Igf-2 is
paternally expressed. Both genes share common regulatory sequences including the H19 5’
differentially methylated domain ICR (Imprinting Control Region) and the 3’ downstream
enhancer sequences.
In human, 91H is an 120 kb-long transcript, antisense to H19, spanning the entire H19
transcribed region and extending 69Kb and 39 kb downstream and upstream, respectively.
This includes the ICR and the 3’ enhancer regions but not the IGF2 transcription unit. The
promoter region is contained in the first intron of the MrpL23 gene (the H19 3’neighbouring
gene). In addition, we show that in mouse 91H is a biallelic transcript.
We demonstrate that this antisense RNA is a nuclear and short-lived transcipt in human and
mouse normal cells, but we observe its stabilization and accumulation in human breast
epithelial cells. Consistently, an in vivo study confirms these data revealing a 91H
overexpression in breast cancer biopsies. Furthermore, the developmental expression of 91H
is very similar to those of the H19 RNA.
Knock-down of 91H by RNA interference leads to loss of silencing of the Igf-2 gene on the
paternal chromosome and a down-regulation of Igf-2 expression. We investigate the effect of
91H on DNA methylation in ICR and differentially methylated region of this locus but also in
heterochromatin formation leading to gene silencing.
Our results indicate that 91H plays a key role in genomic imprinting at the H19/IGF2 locus
and we discuss the role of long intergenic antisense transcript in chromatin remodelling and
imprinting maintenance.
Publications:
1: Berteaux N, Lottin S, Monte D, Pinte S, Quatannens B, Coll J, Hondermarck H, Curgy JJ, Dugimont T,
Adriaenssens E. H19 mRNA-like noncoding RNA promotes breast cancer cell proliferation through positive
control by E2F1. J Biol Chem. 2005, 280:29625-36.
2: Lottin S, Adriaenssens E, Berteaux N, Lepretre A, Vilain MO, Denhez E, Coll J, Dugimont T, Curgy JJ. The
human H19 gene is frequently overexpressed in myometrium and stroma during pathological endometrial
proliferative events. Eur J Cancer. 2005, 41:168-77.
3: Berteaux N, Lottin S, Adriaenssens E, Van Coppenolle F, Leroy X, Coll J, Dugimont T, Curgy JJ. Hormonal
regulation of H19 gene expression in prostate epithelial cells. J Endocrinol. 2004, 183:69-78.
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4: : Lottin S, Vercoutter-Edouart AS, Adriaenssens E, Czeszak X, Lemoine J, Roudbaraki M, Coll J,
Hondermarck H, Dugimont T, Curgy JJ. Thioredoxin post-transcriptional regulation by H19 provides a new
function to mRNA-like non-coding RNA. Oncogene. 2002, 21:1625-31.
5: Yazidi-Belkoura IE, Adriaenssens E, Vercoutter-Edouart AS, Lemoine J, Nurcombe V, Hondermarck H.
Proteomics of breast cancer: outcomes and prospects.Technol Cancer Res Treat. 2002, 1:287-96.
6: Lottin S, Adriaenssens E, Dupressoir T, Berteaux N, Montpellier C, Coll J, Dugimont T, Curgy JJ.
Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells.
Carcinogenesis. 2002, 23:1885-95.
7 Hondermarck H, Dolle L, El Yazidi-Belkoura I, Vercoutter-Edouart AS, Adriaenssens E, Lemoine J.
Functional proteomics of breast cancer for signal pathway profiling and target discovery. J Mammary Gland Biol
Neoplasia. 2002, 7:395-405.
8: Adriaenssens E, Lottin S, Berteaux N, Hornez L, Fauquette W, Fafeur V, Peyrat JP, Le Bourhis X,
Hondermarck H, Coll J, Dugimont T, Curgy JJ. Cross-talk between mesenchyme and epithelium increases H19
gene expression during scattering and morphogenesis of epithelial cells. Exp Cell Res. 2002, 275:215-29.
9: Adriaenssens E, Lemoine J, El Yazidi-Belkoura I, Hondermarck H. Growth signaling in breast cancer cells:
outcomes and promises of proteomics. Biochem Pharmacol. 2002, 64:797-803.
10: Adriaenssens E, Lottin S, Dugimont T, Fauquette W, Coll J, Dupouy JP, Boilly B, Curgy JJ. Steroid
hormones modulate H19 gene expression in both mammary gland and uterus. Oncogene. 1999, 18:4460-73.
11: Adriaenssens E, Dumont L, Lottin S, Bolle D, Lepretre A, Delobelle A, Bouali F, Dugimont T, Coll J,
Curgy JJ. H19 overexpression in breast adenocarcinoma stromal cells is associated with tumor values and steroid
receptor status but independent of p53 and Ki-67
expression. Am J Pathol. 1998, 153:1597-607.
12: Dugimont T, Montpellier C, Adriaenssens E, Lottin S, Dumont L, Iotsova V, Lagrou C, Stehelin D, Coll J,
Curgy JJ. The H19 TATA-less promoter is efficiently repressed by wild-type tumor suppressor gene product
p53. Oncogene. 1998, 16:2395-401.
- 25 -

Targeting Inflammatory Transcription Factor by Dietary Agents for
Prevention and Treatment of Cancer
Bharat B. Aggarwal, Ph.D.
Cytokine Research Laboratory, Department of Experimental Therapeutics, The
University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, U.S.A.
NF-kB, a transcription factor, is present normally in the cytoplasm as an inactive
heterotrimer consisting of p50, p65 and IkBa subunits. When activated, NF-kB translocates
to the as a p50-p65 heterodimer. This factor regulates the expression of various genes that
control apoptosis, viral replication, tumorigenesis, various autoimmune diseases, and
inflammation. NF-kB has been linked to the development of carcinogenesis for several
reasons. First, various carcinogens and tumor promoters have been shown to activate NF-kB.
Second, activation of NF-kB has been shown to block apoptosis and promote proliferation.
Third, the tumor microenvironment can induce NF-kB activation. Fourth, constitutive
expression of NF-kB is frequently found in tumor cells. Fifth, NF-kB activation induces
resistance to chemotherapeutic agents. Fifth, several genes involved in tumor initiation,
promotion, and metastasis are regulated by NF-kB. Sixth, various chemopreventive agents
have been found to downregulate the NF-kB activation. All these observation suggest that
NF-kB could mediate tumorigenesis and thus can be used as a target for chemoprevention and
for the treatment of cancer. Besides NF-kB, we have also targeted AP-1 and STAT3, other
transcription factors that mediate tumorigenesis. We will present the data which shows that
phytochemicals derived from fruits, vegetables and spices are important inhibitors of NF-kB
activation, and can suppress the expression of genes involved in carcinogenesis and
tumorigenesis in vivo.
Recent references:
1 Shishodia, S., and Aggarwal B.B. Diosgenin Inhibits Osteoclastogenesis, Invasion,
and Proliferation Through the Downregulation of Akt, IkB Kinase Activation and NF-kB-
Regulated Gene Expression, Oncogene (in press)
2 Aggarwal BB, Shishodia S., Takada Y., Banerjee S., Newman RA, Bueso-Ramos CE,
and Price JE Curcumin Suppresses the Paclitaxel-induced NF-!B Pathway in Breast Cancer
Cells and Inhibits Lung Metastasis of Human Breast Cancer in Nude Mice, Clinical Cancer
Research (in press).
3 Aggarwal BB, Ichikawa H. Molecular Targets and Anticancer Potential of Indole-3-
Carbinol and Its Derivatives. Cell Cycle. 2005 Sep 6;4(9) 1201-1215
4 Takada Y, Kobayashi Y, and Aggarwal BB. Evodiamine abolishes constitutive and
inducible NF-kappa B activation by inhibiting Ikappa Balpha kinase activation, thereby
suppressing NF-kappa B-regulated antiapoptotic and metastatic gene expression, upregulating
apoptosis, and inhibiting invasion.J. Biol Chem. 2005 Feb 14; 2005;280(17):17203-12
5 Takada Y, Andreeff M, Aggarwal BB. Indole-3-carbinol suppresses NF-{kappa}B
and I{kappa}B{alpha} kinase activation causing inhibition of expression of NF-{kappa}Bregulated
antiapoptotic and metastatic gene products and enhancement of apoptosis in
myeloid and leukemia cells. Blood. 2005 Apr 5; 2005 Jul 15;106(2):641-9.
6 Siwak DR, Shishodia S, Aggarwal BB, Kurzrock R. Related Articles, Links Abstract
Curcumin-induced antiproliferative and proapoptotic effects in melanoma cells are associated
with suppression of IkappaB kinase and nuclear factor kappaB activity and are independent of
- 26 -

the B-Raf/mitogen-activated/extracellular signal-regulated protein kinase pathway and the
Akt pathway. Cancer. 2005 Aug 15;104(4):879-90.
7 Takada Y., and Aggarwal B. B., Zerumbone Abolishes NF-kappaB and IkappaB
Kinase Activation Leading to Suppression of Antiapoptotic and Metastatic Gene Expression,
Upregulation of Apoptosis, and Downregulation of Invasion, ONCOGENE (in press)
8 Haruyo Ichikawa, Yasunari Takada, Akira Murakami and Bharat B. Aggarwal
Identification of a Novel Blocker of I!B" Kinase That Enhances Cellular Apoptosis and
Inhibits Cellular Invasion Through Suppression of NF-kB Regulated Gene Products, Journal
of Immunology (2005 Jun 1;174(11):7383-92.
9 Shishir Shishodia, Hesham M. Amin, Raymond Lai, and Bharat B. Aggarwal,
Curcumin (Diferuloylmethane) Inhibits Constitutive NF-!B Activation, Induces G1/S Arrest,
Suppresses Proliferation, and Induces Apoptosis in Mantle Cell Lymphoma, Biochemical
Pharmacology (2005 Sep 1;70(5):700-13.
10 Yan, C., Jamaluddin Md S., Aggarwal, B.B., Myers JN, Boyd D.D., Gene Expression
Profiling Identifies ATF3 as a Novel Mediators of the Anti-Tumor Effect of Curcumin,
Molecular Cancer Therapeutics 2005 Feb;4(2):233-41.
11 Aggarwal B. B., Nuclear Factor NF-!B: Enemy Within, Cancer Cell 2004
Sep;6(3):203-8.
12 Takada Y., Bhardwaj A., Potdar P., and Aggarwal B. B., Nonsteroidal
Antiinflammatory Agents Differ in Their Ability To Suppress NF-!B Activation, Inhibition
of Expression of Cyclooxygenase-2 and Cyclin D1, and Abrogation of Tumor Cell
Proliferation, ONCOGENE (2004) 23, 9247-9258)
13 Takada Y, Aggarwal B. B. Evidence that Genetic Deletion of the TNF Receptor p60
or p80 in Macrophages Modulates RANKL-Induced Signaling. BLOOD. 2004 Dec
15;104(13):4113-21
14 Aggarwal B. B., Takada Y.and Ommen O. V. From Chemopreventive to
chemotherapy: common targets and common goals. Expert Opinion in Investigational
Drugs 13, (10), 2004, 1-12
15 Shishodia S. and Aggarwal B. B., Guggulsterone Inhibits NF-!B and I!B" Kinase
Activation, Suppresses Expression of Antiapoptotic Gene Products and Enhances Apoptosis;
Journal of Biological Chemistry. 2004 Nov 5;279(45):47148-58.
16 Shishodia S., and Aggarwal B.B., Nuclear Factor-!B: A Friend or a Foe in Cancer?
Biochemical Pharmacology 2004 Sep 15;68(6):1071-80.
17 Aggarwal, B.B., A. Kumar, and A. Bharti. Therapeutic potential of curcumin derived
from turmeric (Curcuma longa), Herbal and Traditional Medicine: Molecular Basis of
Health (ed by Lester Packer, Choon Nam Ong and Barry Halliwell)
18 Aggarwal B. B., A. Kumer, M.S. Aggarwal, S. Shishodia, Curcumin derived from
turmeric (Curcuma longa): A spice for all seasons, in Phytochemicals in Cancer
Chemoprevention (ed by Debasis Bagchi, Ph.D., and Harry G. Preuss, M.D.) CRC Press.
19 Dorai T., and Aggarwal B. B., Role of Chemopreventive Agents in Cancer Therapy.
Cancer Letters 2004 Nov 25;215(2):129-40.
20 Li, L., B.B. Aggarwal, S. Shishodia, J. Abbruzzese and R. Kurzrock. Nuclear factor-
!B and I!B kinase are constitutively active in human pancreatic cells and their downregulation
by curcumin (diferuloyl methane) is associated with suppression of proliferation
and induction of apoptosis Cancer 2004 Nov 15;101(10):2351-62
21 Aggarwal B.B., A. Bhardwaj, R.S. Aggarwal, N.P. Seeram, S. Shishodia, and Y.
Takada, Role of resveratrol in prevention and therapy of cancer: preclinical and clinical
studies Invited review. Anticancer Research 2004 Sep-Oct;24(5A):2783-840
- 27 -

Mechanisms involved in the anti-apoptotic effect of magnetic fields
Maria C. Albertini, Claudia Cerella *, Sonia Cordisco*, Augusto Accorsi, Antonio
Bergamaschi#, Maria D'Alessio*, Milena De Nicola*, Silvia Nuccitelli*, Lina Ghibelli*
Istituto di Chimica Biologica A. Fornaini, Universita' di Urbino, Urbino, Italy
*Dipartimento di Biologia, #Cattedra di Medicina del Lavoro, Universita' di Roma Tor
Vergata, Rome, Italy 00133; *. E-mail: cemetbio@uniurb.it
Concern for possible health hazard due to increasing exposure to magnetic fields (MFs), came
from epidemiological reports indicating a correlation between exposure to MFs and
development of some types of tumors. This led to great interest in the interaction of MFs with
living matter, especially focusing on possible interference on cell metabolism that may
account for a pro-tumoral role of MFs. Research on this issue is made quite difficult since
different types of MFs (i.e., static vs oscillating; different oscillation frequencies; different
intensities; different vector directions; etc.) may differently interact with cells, thus giving rise
to controversial results. We showed that 0.3-60 mTesla (mT) static (i.e., magnete-induced)
MFs may well act as tumor promoting agents, tumor promotion being the epigenetic effect
that allows tumor growth by favoring the growth/survival of transformed cells. Indeed, MFs
strongly reduce the extent of damage-induced apoptosis on a set of cell lines, thus increasing
survival of possibly transformed cells (Fanelli et al., FASEB J 1999, 133:95-102). We
describe here the events triggered by exposure to MFs on a reporter cell line (U937), which
lead to impairment of damage-induced apoptosis, compared to an insensitve one (Jurkat). We
show that MFs anti-apoptotic effect requires a correct redox equilibrium, a working
phospholipase C (PLC, a key actor of receptor-mediated signal transduction), and nitric oxide
syntase (NOS, a key actor of intracellular inflammatory signal transduction), the antiapoptotic
effect being abolished in the presence of reducing (DTT) or oxidizing (BSO)
agents; of PLC inhibitors (neomycin and U73122); and NOS inhibitors (L-NAME),
respectively. Accordingly, the sensitive cells show increased Ca2+ influx, increased IP3
production, plasma membrane hyperpolarization and alterations of free radicals and
glutathione levels. Jurkat cells may be rendered sensitive upon activation with
phytohemagglutinin; the normal/activated Jurkat provide an ideal system where to look for
the primary determinants for the sensitivity to MFs anti-apoptotic effect. Intriguingly,
extremely low frequency (50Hz) electroMFs (ELF) exert similar effects, reducing apoptosis
in a Ca2+ dependent fashion.
LIST OF RECENT PAPERS
Piacentini MP, Piatti E, Fraternale D, Ricci D, Albertini MC, Accorsi A.
Phospholipase C-dependent phosphoinositide breakdown induced by ELF-EMF in Peganum
harmala calli.
Biochimie. 2004 Apr-May;86(4-5):343-9.
Ghibelli L, Teodori L, Cerella C, De Nicola M, D'Alessio M, Clavarino G, Cordisco S,
Albertini MC, Accorsi A, Magrini A, Bergamaschi A.
Epigenetic role of magnetic field exposure in tumor progression: fine-tuning experimental
models]
G Ital Med Lav Ergon. 2003 Jul-Sep;25 Suppl(3):277-8.
- 28 -

Albertini MC, Accorsi A, Citterio B, Burattini S, Piacentini MP, Uguccioni F, Piatti E.
Morphological and biochemical modifications induced by a static magnetic field on Fusarium
culmorum.
Biochimie. 2003 Oct;85(10):963-70.
Albertini MC, Teodori L, Piatti E, Piacentini MP, Accorsi A, Rocchi MB.
Automated analysis of morphometric parameters for accurate definition of erythrocyte cell
shape.
Cytometry A. 2003 Mar;52(1):12-8.
Piatti E, Albertini MC, Baffone W, Fraternale D, Citterio B, Piacentini MP, Dacha M,
Vetrano F, Accorsi A.
Antibacterial effect of a magnetic field on Serratia marcescens and related virulence to
Hordeum vulgare and Rubus fruticosus callus cells.
Comp Biochem Physiol B Biochem Mol Biol. 2002 Jun;132(2):359-65.
Colussi C, Albertini MC, Coppola S, Rovidati S, Galli F, Ghibelli L.
H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells
from apoptosis.
FASEB J. 2000 Nov;14(14):2266-76.
- 29 -

Regulation and function of the WNK1 and WNK4 protein kinases that are mutated in
Gordon’s hypertension syndrome.
Dario R Alessi
MRC Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, UK
The WNK family of protein kinases comprise 4 members (WNK1, WNK2, WNK3 and
WNK4) and were originally identified as serine-threonine protein kinases that lack a
conserved Lys residue normally found in subdomain II of the catalytic domain. Subsequent
studies, identified mutations in the genes encoding WNK1 and WNK4, in families with an
inherited hypertension and hyperkalemia (elevated plasma K + ) disorder, called
pseudohypoaldosteronism type II (PHAII, also known as Gordon’s syndrome). WNK
isoforms are large protein kinases (WNK1-2382 residues, WNK4-1243 residues), in which
the catalytic domain is located at the N-terminus (residues 221 to 479 for WNK1 and 174 to
432 for WNK4). Apart from two putative coiled-coil domains, the remainder of the WNK
polypeptides possess no obvious structural features. Mutations in the WNK1 gene found in
PHAII subjects, are deletions in intron-1, which reportedly elevate the expression of the
WNK1 protein, indicating that hypertension could result from increased expression of
WNK1. Consistent with this notion, mice lacking one allele of WNK1, had lower blood
pressure. The WNK1 knockout is an embryonic lethal, indicating that WNK1 is also required
for normal development. Thus far, the mutations in the WNK4 gene found in PHAII subjects,
lie distal to both of the putative coiled-coil domains. Little is known about the molecular
mechanism by which WNK isoforms regulate cellular processes. In my talk I will present our
recent results that indicate that the WNK protein kinases are activated by osmotic stress and
phosphorylate and activate protein kinases of the STE20 family, termed STE20/SPS1-related
Proline-Alanine-rich Kinase (SPAK) and the Oxidative Stress Response kinase-1 (OSR1).
SPAK and OSR1 were previously reported to be activated in cells in response to osmotic
stress such as high salt and phosphorylate and regulate the activity of ion transporters such as
NKCC1. It had also been previously shown that treatment of cells with high salt concentration
activated WNK1. I will discuss the possibility that osmotic stress might control the activity of
ion transporters through the WNK-SPAK/OSR1 pathway. It is possible that mutations in
WNK1 and WNK4 genes in humans disrupt this pathway, thereby affecting ion transporter
activity in organs such as the kidney, which could account for the development of
hypertension in subjects with these mutations.
- 30 -

AP-1-dependent gene expression in skin remodelling and tumourigenesis
Peter Angel
Deutsches Krebsforschungszentrum, Division Signal Transduction and Growth Control
(A100), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
The transcription factor AP-1, which is composed of members of the Fos, Jun and ATF
protein families participates in physiological and pathophysiological processes due to its
central role as a cellular switch of genetic programs in response to extracellular signals. The
distinct expression pattern of AP-1 subunits in the skin, the loss of chemically induced
carcinogenesis in AP-1 compromised mice, and the large number of putative AP-1 target
genes in epidermal cells, indicate that AP-1 members play a pivotal role in epidermal
organisation, skin homeostasis and tumourigenesis (1, 2). We have employed a heterologous
organotypic coculture system containing fibroblasts from c-Jun or JunB-deficient mice to
identify trans-regulatory activities of c-Jun and JunB in the paracrine interaction between
keratinocytes and dermal fibroblasts (3). Large-scale expression profiling of these cultures
revealed novel AP-1 target genes in fibroblasts including cytokines, growth factors and
chemoattractants (4). Mice lacking the AP-1 member JunB in skin exhibit defects in wound
healing, which is associated with aberrant expression of these newly identified target genes
(5). The mouse skin was also the model of choice to study the regulation and function of AP-1
subunits in tumourigenesis in vivo (1). Upon combination of the well-established chemically
induced mouse model of epithelial skin tumours and expression profiling we have identified a
novel signalling pathway activating AP-1, which is initiated by S100A8 and S100 A9, two
members of the S100 family of Ca2+ binding proteins (6), which are also upregulated in a c-
Jun/JunB-dependent mouse model of human psoriasis (7). Both genes are also overexpressed
in human epithelial tumours of the skin and other organs, suggesting an important role of this
pathway in tumour progression and metastasis.
1. Angel, P., Szabowski, A., and Schorpp-Kistner M. (2001). Function and regulation of AP-1 subunits in skin
physiology and pathology. Oncogene 20, 2413-23
2. Hess, J., Angel, P. and Schorpp-Kistner, M. (2004) Functions of AP-1 subunits: Quarrel and Harmony among
Siblings. J. Cell Sci 117:5965-73
3. Szabowski, A., Maas-Szabowski, N., Andrecht, S., Kolbus, A., Schorpp-Kistner, M., Fusenig, N.E. and
Angel, P. (2000) c-Jun and JunB antagonistically control cytokine regulated mesenchymal-epidermal interaction
in skin.” Cell 103, 745-55
4. Florin, L., Hummerich, L., Dittrich, B., Kokocinski, F., Wrobel, G., Gack, S., Schorpp-Kistner, M., Werner,
S., Hahn, M., Lichter, P., Szabowski, A., Angel, P. (2004) Identification of novel AP-1 target genes in
fibroblasts regulated during cutaneous wound healing. Oncogene 23:7005-17
5. Florin, L., Knebel, J., Zigrino, P., Vonderstrass, B., Mauch, C., Schorpp-Kistner, M., Szabowski, A. and
Angel P. (2005) Delayed wound healing and epidermal hyperproliferation in mice lacking JunB in the skin. J Inv
Dermatol, in press
6. Gebhardt, C., Breitenbach, U., Tuckermann, J. P., Dittrich, B. T., Richter, K. H., and Angel, P. (2002).
Calgranulins S100A8 and S100A9 are negatively regulated by glucocorticoids in a c-Fos-dependent manner and
overexpressed throughout skin carcinogenesis. Oncogene 21, 4266-4276.
7. Zenz R., Eferl R., Kenner L., Florin L., Hummerich, Mehic D., Scheuch H., Angel P., Tschachler E. and
Wagner, E.F. (2005) Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun
proteins. Nature (2005) 437, 369-75
- 31 -

Regulation of the cell cycle by the transcription factor NFAT5 in response to osmotic
and genotoxic stress.
Jose Aramburu, Katherine Drews, Beatriz Morancho and Cristina López-Rodriguez.
Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, 08003
Barcelona, Spain. E-mail: jaramburu@imim.es; katherine.drews@upf.edu;
beatriz.morancho@upf.edu; cristina.lopez-rodriguez@upf.edu.
The transcription factor NFAT5 belongs to the Rel family, that comprises NF-kappaB and the
calcineurin-activated NFATc proteins (1). NFAT5 is activated by hypertonicity and regulates
the expression of osmoprotective genes, but also responds to mitogenic signals and is
expressed in proliferating cells (2, 3). As osmotic stress can cause DNA breaks, and activation
of NFAT5 by hypertonicity is regulated by PI3-kinase related kinases (PIKK) (4), which
control cellular responses to DNA damage, we hypothesized that NFAT5 might play a role in
the cellular response to genotoxic stress. Cell cycle arrest is a hallmark of genotoxic stress and
is often exacerbated by the disruption of proteins involved in DNA damage sensing and
repair. We have studied whether the inhibition of NFAT5 had an effect in the cell cycle of
cells exposed to osmotic and genotoxic stress. Our results showed that proliferating NFAT5-/-
T cells did not display substantial cell cycle defects under non-stress conditions, but arrested
at G1 and G2/M when subjected to hypertonicity and ionizing radiation doses that did not
arrest wild-type T lymphocytes. Likewise, suppression of NFAT5 with shRNA in HEK293
cells caused them to accumulate in G2/M when exposed to hypertonic stress or ionizing
radiation. We found that ionizing radiation and the genotoxic drugs etoposide and
hidroxyurea caused NFAT5 to accumulate in the nucleus in HEK293 cells and that its
overexpression rendered cells more resistant to cell cycle arrest when exposed to
hypertonicity or ionizing radiation. Our results indicate that NFAT5 can regulate the cell
cycle in response to genotoxic stress, and we are currently exploring the mechanisms
underlying this function.
References.
1) López-Rodríguez C, Aramburu J, Rakeman AS, Copeland NG, Gilbert DJ, Thomas S,
Disteche C, Jenkins NA, and Rao A. 1999. NF-AT5: the NF-AT family of transcription
factors expands in a new direction. Cold Spring Harb Symp Quant Biol. 1999; 64:517-526.
2) López-Rodríguez C, Aramburu J, Jin L, Rakeman AS, Michino M, Rao A. 2001. Bridging
the NFAT and NF-kappaB families: NFAT5 dimerization regulates cytokine gene
transcription in response to osmotic stress. Immunity. 15:47-58.
3) López-Rodríguez C, Antos C. L, Shelton J, Richardson J. A, Fangming L, Novobrantseva
T. I, Bronson R.T, Igarashi P, Rao A, and Olson E. N. 2004. Loss of NFAT5 results in renal
atrophy and lack of tonicity-responsive gene expression. Proc. Natl Acad of Sci USA. 101;
2392-2397.
4) Irarrazabal C.E, Liu J.C, Burg M.B, and Ferraris J.D. 2004. ATM, a DNA damageinducible
kinase, contributes to activation by high NaCl of the transcription factor
TonEBP/OREBP. Proc. Natl. Acad. Sci. U. S. A 101, 8809-8814.
- 32 -

Jaks and cytokine receptors – an intimate relationship
Claude Haan 1 , Simone Radtke 2 , Bernd Giese 2 , Christiane Margue-Wurth 1 , Andreas
Herrmann 2 , Gerhard Müller-Newen 2 , Serge Haan 2 , Peter C. Heinrich 2 , Iris Behrmann 1
1 Laboratoire de Biologie et Physiologie Intégrée, University of Luxembourg, 162a, av. de
la Faïencerie, 1511 Luxembourg, Grand-Duchy of Luxemburg,
2 Institute of Biochemistry, RWTH-Aachen Medical School, 52074 Aachen, Germany
E-mail: iris.behrmann@uni.lu
Many cytokines signal via Janus kinases (Jaks). These tyrosine kinases are associated with
cytokine receptor subunits, they become activated upon receptor triggering and subsequently
activate downstream signalling events, e. g. the phosphorylation of STAT transcription
factors.
Using gp130 (the common receptor subunit for IL-6-type cytokines) and Jak1 as an example,
we analysed the structural requirements for association between Jaks and cytokine receptors,
as well as the dynamics of this interaction. Subdomains F1 and F2 of the N-terminal FERM
domain of Jak1 are crucially involved in binding to gp130 as well as to other cytokine
receptors. On the receptor side, the conserved box1 and box2 regions as well as the
intervening sequences are crucial for Jak association. The receptor plays an active role in
activation of the bound Jaks, as shown by the mutant gp130-W652A that does not elicit
signalling in spite of associating Jaks (1,2).
Jaks are found exclusively in membrane fractions. The plasma membrane localization of Jaks
is dependent on their ability to associate with cytokine receptors since the non-receptor
binding Jak1 mutant L80A/Y81A is only found within the cytoplasm. Using FRAP analysis
with fluorescent gp130 and Jak1 proteins we show that Jak1 has the same diffusion behaviour
as the transmembrane protein gp130, and that there is no rapid exchange of Jaks between
different receptors. Thus, this cytokine receptor/Jak complex can be regarded to be equivalent
to a receptor tyrosine kinase (3,4).
To assess the role of the predicted SH2 domain in Jak1 we exchanged the conserved arginine
residue by lysine, a mutation known to abrogate the phosphotyrosine binding. This R466K
mutation did not affect the signal transducing capacities of the kinase, nor its localization,
indicating that the Jak1-SH2 domain does not fulfil the “classical” SH2 domain function.
However, it may play a structural role for association with the oncostatin M receptor
(OSMR)(5).
Jaks are not only crucial for signal transduction of cytokines, but they are also involved in the
regulation of the surface expression of at least some cytokine receptors, including the OSMR.
We identified three dileucine motifs within the interbox1/2 region that prevent efficient
OSMR surface expression in the absence of associated Jaks, possibly by lysosomal targeting
and destabilization of the receptor. This may provide a quality control ensuring that
signalling-competent receptors (i. e. those with an associated Jak) would be enriched at the
cell surface (6).
Refs.:
(1) Haan et al., 2001, JBC 276: 37451
(2) Haan et al., 2002, Biochem. J. 361: 105
(3) Behrmann et al., 2004, JBC 279: 35486
(4) Giese et al., 2003, JBC 278: 39205
(5) Radtke et al., 2005, JBC 280: 25760
(6) Radtke et al., 2005, JBC online.
- 33 -

Tissue specific control of NF-kB activation
Yinon Ben-Neriah1, Jongdae Lee2, Elad Horwitz1, Gady Cojocaru1, Naama Kanarik1,
Matti Davis1, Irit Alkalay1, Kyoko Katakura2, Lars Eckmann2, Eli Pikarsky3 and Eyal
Raz2
1The Lautenberg Center for Immunology and 3Dept of Pathology, Hebrew-University-
Hadassah Medical School Jerusalem 91120, Israel; 2Department of Medicine University
of California San Diego, La Jolla, CA 92093-0663
Canonical NF-kB activation targets IkB and p105/NF-kB1 to degradation via the ubiquitin
proteasome system. We and others have previously shown that #-TrCP is a key regulator in
this system; its two isoforms #-TrCP1 and 2 are both necessary and sufficient to control IkB
stability. Ongoing experiments in our lab attempt to unveil the unique functions of each #-
TrCP isoform in different tissues and determine the consequence of specific isoform
inhibition. So far, #-TrCP-controlled degradation has been mainly studied in standard tissue
culture conditions, yet many tissues and particularly epithelial cells are configured in vivo in a
specific orientation. We will describe a cellular mechanism whereby the polarity of gut
epithelium dictates the outcome of NF-kB signaling, thereby playing a major role in colonic
homeostasis. TLR9 activation via apical and basolateral surface domains have distinct
transcriptional responses. Whereas basolateral TLR9 signals NF-kB activation, apical TLR9
invokes intracellular tolerance against subsequent basolateral TLR9 and other TLRs'
challenges. TLR9-deficient mice lacking the tolerizing receptor are triggered much more
rapidly for NF-kB activation and are highly susceptible to experimental colitis. Our data
provide a case for an organ-specific innate immunity where TLR regulation evolved to
maintain colonic homeostasis. This is achieved by a novel cellular mechanism, where the
different surface domains of a polarized cell dictate an opposing signaling outcome to a
similar stimulus.
- 34 -

The Role of Met-Gab1 Signalling in Vivo
Walter BIRCHMEIER, Jolanta CHMIELOWIEC, and Ute SCHAEPER
Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin,
Germany
We examined wound healing in conditional mutant mice, in which the Met tyrosine kinase,
the receptor of HGF/SF, was ablated in the epidermis. In the Met-deficient epidermis,
keratinocytes were completely unable to contribute to re-epithelialization of the wounds.
However, wound closure could occur by fast proliferation and migration of the few (5%)
remaining wild-type keratinocytes. These results show that the HGF/SF and Met signalling
system, which is not important for normal skin formation and maintenance, is crucial for skin
regeneration.
Gab1 is a signalling protein that belongs to the class of docking proteins, which function
downstream of tyrosine kinases. Gab1 was discovered as a direct target of the tyrosine kinase
c-Met, the receptor for SF/HGF. It was not known, which downstream signalling pathways
are important for Gab1 function in vivo, and what are the in vivo contributions of the c-Met
and Grb2 binding sites for coupling Gab1 to specific tyrosine kinases. To address these
questions, we generated Gab1 knock-in mice that express Gab1 cDNAs, which carry
mutations in the c-Met, Grb2, PI3K or Shp-2 binding sites. Mutant mice display distinct, but
also overlapping phenotypes, which demonstrate that Gab1 acts in several tyrosine kinase
signalling pathways. Of particular importance in vivo is the binding site of Gab1 to the Shp-2
downstream substrate, which is essential for the migration of muscle precursor cells into the
limbs.
- 35 -

Targeting of Polycomb repressive complexes by the moonlighting protein NIPP1
Mathieu Bollen
Division of Biochemistry, Faculty of Medicine, Catholic University of Leuven, B-3000
Leuven, Belgium. E-mail: Mathieu.Bollen@med.kuleuven.be
NIPP1 is a nuclear protein that is ubiquitously expressed in metazoans and plants. The
knockout of NIPP1 in mice is embryonic lethal before gastrulation and NIPP1-/- cell lines are
not viable. NIPP1 interacts in vivo with a protein kinase (MELK), a protein phosphatase
(PP1), two pre-mRNA splicing factors (CDC5L, SAP155) and two transcriptional repressors
(EED, EZH2). We previously reported that NIPP1 is associated with storage sites for splicing
factors and is required for a late step of spliceosome assembly. In addition, NIPP1 functions
as a transcriptional repressor by a mechanism that does not require functional interaction sites
for splicing factors, MELK nor PP1, but depends on the presence of EED and EZH2, two core
components of the Polycomb Repressive Complexes 2-4 (PRC2-4). The latter complexes
initiate the heritable repression of genes that are important for development and cell
proliferation, at least in part by the EZH2-mediated trimethylation of histone H3 on Lys27
(H3K27). Consistent with a role for NIPP1 in Polycomb signalling, we found that NIPP1 is
associated with H3K27-trimethylated chromatin and that NIPP1-/- mouse blastocyst
outgrowths and cultured cells with a decreased concentration of NIPP1 are severely deficient
in H3K27 trimethylation, but are normally trimethylated on H3K9. We also show that NIPP1
is a nucleic-acid binding protein and interacts strongly with intron-1 of the MSMB gene,
which encodes the prostatic tumor growth suppressor PSP94. We identified the MSMB gene
as a novel Polycomb target gene and show that the siRNA-mediated knockdown of NIPP1 is
associated with a decreased recruitment of EZH2 to this gene. These findings are consistent
with the notion that NIPP1 is involved in the recruitment of PRC2 to its target genes. Our data
suggest that NIPP1 functions as a moonlighting protein and has independent functions in premRNA
splicing and transcription.
- 36 -

The mammalian tumor suppressor Scribble has a key role in cell migration
Sébastien Nola, Marie-Josée Santoni, Claire Nourry, Christelle Navarro and Jean-Paul
Borg.
Molecular Pharmacology, Marseille Cancer Institute, UMR599 Inserm-Institut Paoli-
Calmettes, Marseille, France 13009. E-mail : borg@marseille.inserm.fr
Genetic studies in Drosophila and mice have shown that the PDZ protein Scribble has a
crucial role in many aspects of cell polarity. Loss of function of Scribble in Drosophila
provokes profound defects in epithelial apico-basal polarity, as well as the formation of
neoplastic tumors. Mice deficient for Scribble show abnormal planar cell polarity, and die
within a few days after birth from craniorachischisis, a dramatic neural tube defect. We have
shown that mammalian Scribble is a cytoplasmic protein tighly associated to the plasma
membrane, and that it is part of a protein complex containing betaPIX, an exchange factor for
Rac and Cdc42, and GIT1, a GTPase Activating Protein (GAP) for Arf6. The PDZ domains
of Scribble associate directly to the carboxy-terminus of betaPIX, and beta-PIX is bound to
GIT1. Here, we demonstrate that PAK, a serine-threonine kinase, is also associated to
Scribble in epithelial cells. The Scribble-betaPIX-GIT1-PAK protein complex is located at the
leading edge of migratory T47D epithelial cells, and, using siRNA and dominant-negative
constructs, we demonstrate that localization of betaPIX is dependant on the correct
positioning of Scribble at the plasma membrane. Using a modified Boyden chamber assay, we
show that Scribble, as well as the members of the associated protein complex, are required for
the effective migration of T47D cells towards a chemoattractant stimulus. In conclusion,
Scribble and the associated betaPIX-GIT1-PAK protein complex is required for cell migration
of epithelial cells. The unproper positioning of the betaPIX-GIT1-PAK signalling machinery
in Scribble deficient cells probably accounts for the cell migration defect in T47D cells and
may explain the neural tube defect observed in Scribble deficient mice.
Nourry C, Grant SG, Borg JP (2003). PDZ domain proteins: plug and play! Science’s STKE
179: RE7.
Legouis R, Jaulin-Bastard F, Schott S, Navarro C, Borg J-P, and Labouesse M (2003).
Basolateral targeting by Leucine Rich Repeat domains in epithelial cells. EMBO Reports 4:
1096–1100.
Audebert S., Navarro C., Nourry C., Chasserot-Golaz S., Lécine P., Bellaiche Y., Dupont J.-
L., Premont R.T., Sempéré C., Strub J.-M., Van Dorsselaer A., Vitale N. and Borg J.-P.
Mammalian Scribble forms a tight complex with the bPIX exchange factor (2004). Current
Biology 14: 987-95.
Navarro C., Nola S., Audebert S., Santoni M.-J., Arsanto J.-P., Ginestier C., Marchetto S.,
Jacquemier J., Isnardon D., Le Bivic A., Birnbaum D., and Borg J.-P. Junctional recruitment
of mammalian Scribble relies on E-cadherin engagement. (2005) Oncogene, 24: 4330-4339.
O.Lahuna*, Quellari M., Achard C., Nola S., Méduri G., Navarro C., Vitale N., Borg J.-P.*
and Misrahi M*. Thyrotropin receptor trafficking relies on the hScrib-bPIX-GIT1-ARF6
signaling pathway. (2005) The EMBO Journal, 24: 1364-1374. (*) co-corresponding authors.
Margolis B. and Borg J.-P. Apico-basal polarity complexes. J Cell Science, 118: 5157-5159.
- 37 -

Epac and Rap1 in the control of cell adhesion
Johannes L. Bos
Department of Physiological Chemistry and Centre for Biomedical Genetics, University
Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
Rap1 is a Ras-like small GTPase originally identified as a revertant of Ras-induced cell
transformation, but current studies in both lower eakaryotes and mammalian cells have
revealed that the main function of Rap in the control of cell adhesion. Rap1 is activated by a
variety of different stimuli, some of which are mediated by cAMP. We have identified
previously two guanine nucleotide exchange factors (GEFs) for Rap1, Epac1 and Epac2.
These two GEFs have a cAMP-binding domain and respond to cAMP. Structural analyses
revealed that the cAMP binding domain functions as an auto-inhibitory domain, preventing
binding of Rap by steric hindrance. By structure-based drug design we developed a cAMP
analog 8-pCPT-2'OMe-cAMP (007) that efficiently activates Epac but not protein kinase A
(PKA). Indeed in various cell lines 007 was able to induce Rap1 activation, but not PKA. 007
did not induce the activation of ERK, challenging the model that cAMP-induced activation of
ERK is mediated by Rap1. Using this analog we could show that both Epac and Rap1
regulates integrin-mediated cell adhesion and E-cadherin and VE-cadherin-mediated cell
junction formation. Our results suggest that Rap1 may have a more general function in cell
adhesion, perhaps in the recruitment of cell adhesion molecules.
- 38 -

SIGNAL TRANSDUCTION BY STRESS-ACTIVATED MAP KINASES
ROGER J. DAVIS
Howard Hughes Medical Institute & University of Massachusetts Medical School,
Worcester, MA 01605 USA, E-mail : Roger.Davis@Umassmed.Edu
The JNK group of stress-activated MAP kinases consists of ten protein kinases that
phosphorylate the NH2-terminal activation domain of c-Jun on Ser-63 and Ser-73 causing
increased transcriptional activity. JNK protein kinase activity is increased in response to
treatment of cells with pro-inflammatory cytokines or exposure to environmental stress.
Activated JNK is phosphorylated on Thr and Tyr within the tripeptide motif Thr-Pro-Tyr
located in kinase sub-domain VIII. Mutational analysis demonstrates that JNK activation
requires the phosphorylation of both Thr and Tyr within this motif. This phosphorylation is
mediated by dual specificity protein kinases, including MKK4 and MKK7.
The function of the JNK signaling pathway has been studied using a combination of
biochemical and genetic approaches. Genetic analysis of JNK signaling in Drosophila
demonstrates that JNK is required for early embryonic morphogenesis. Similarly, disruption
of the JNK signaling pathway in mice using homologous recombination demonstrates that
JNK is required for embryonic viability. In contrast, mice with genetically engineered
selective defects in JNK signaling are viable, but exhibit changes in stress-induced gene
expression and apoptosis. These studies provide insight into the role of the JNK stressactivated
MAP kinase pathway in the cellular response to environmental stress, including
apoptosis and cell survival.
- 39 -

Targeting chromatin machines to promoters in leukemias
R. Villa, L. Morey, M. Buschbeck, A. Gutierrez, and L. Di Croce
ICREA and Center for Genomic Regulation (CRG), Barcelona, Spain
Many human cancers are characterized by alterations in the balance of DNA methylation. In
cancer cells, a large part of the genome undergoes dramatic hypomethylation, which is often
linked to genome instability, concomitantly with regional gain of methylated sequences at
sites usually unmethylated (CpG islands). The major outcome of promoter hypermethylation
appears to be long-term silencing of the associated gene. Silencing of tumor suppressors due
to such a mechanism can provide a growth advantage to cancer cells.
The ability of the PML-RARa fusion protein to block hematopoietic differentiation and to
induce acute promyelocytic leukemia is based on aberrant gene repression. Mechanistically,
PML-RARa inactivates its target genes by recruiting histone deacetylase (HDAC) and DNA
methyltransferase activities to the promoters.
We will show that MBD1, a member of a conserved family of proteins able to bind
methylated DNA, cooperates with PML-RARa in transcriptional repression and cellular
transformation. PML-RARa recruits MBD1 to its target promoter through an HDAC3mediated
mechanism. Binding of HDAC3 and MBD1 is not confined to the promoter region
but instead is spread over the locus. Retroviral expression of dominant negative mutants of
MBD1 in hematopoietic precursors compromised the ability of PML-RARa to block their
differentiation and thus restored cell differentiation. Our results demonstrate that PML-RARa
functions by recruiting an HDAC3-MBD1 complex that contributes to the establishment and
maintenance of the silenced chromatin state. Our results identify MBD1 as a critical mediator
of PML-RARa-induced gene silencing subsequent to promoter hypermethylation.
Further characterization of the PML-RARa!co-repressor complex, which establishes and
allows spreading of the silenced state via Polycomb complex, will be additionally discussed,
together with the implication of crosstalk among the different epigenetic layers to the
molecular pathology of leukemia.
- 40 -

Title to be announced
Caroline Dive
Cellular and Molecular Pharmacology Group, Paterson Institute for Cancer Research,
Christie Hospital NHS Trust, Manchester, United Kingdom
(no abstract provided)
- 41 -

MOLECULAR BIOLOGY OF THYROID CANCER
Detours, V., Delys, L., Hebrant, A., Weiss, D., Maenhaut, C., Dumont, J.E.
IRIBHM, University of Brussels, School of Medicine, Campus Erasme, B - 1070
Brussels.
Thyroid tumors are interesting for several reasons :
1) there are several well defined tumors from benign to very malignant, some former
evolving in the latter
2) the primary cause of the majority of these tumors is known
3) the signal transduction pathways involved have been well studied
4) there are good in vivo and in vitro models.
The types, pathogeneses of the various tumors are described.
Gene expression has been studied in human tumors, in mice transgenic models and in
vitro models (primary cultures of human and dog thyroid cells, human cancer cell lines). The
comparison of the results on the various models allows to draw several general conclusions on
the evolution in vitro of all lines, the role of negative feedback in tumorigenesis and the
importance of taking into account cell populations in the interpretation of results of
microarray studies on solid tumors. A pathogenic scheme of the evolution of papillary
carcinomas into anaplastic carcinomas is presented.
- 42 -

Mechanisms of DNA methylation in mammals
FRANÇOIS FUKS
Free University of Brussels, Faculty of Medicine, 808 route de Lennik, 1070 Brussels,
Belgium.
In mammals, DNA methylation plays an important role in development and is associated with
transcriptional silencing. The goal of our current work is to elucidate the mechanisms by
which the DNA methylation machinery - the DNA methyltransferases (DNMTs) and the
MBDs- functions. In particular, we wish to address two questions: 1. How do the DNMTs and
the MBDs lead to gene silencing? 2. How is the DNMT enzymatic activity regulated?
1. One mechanism by which the DNA methylation machinery brings about transcriptional
repression is through recruitment of HDAC and histone H3 Lys9 methyltransferase activities
(1, 2). Our recent work indicates that DNMTs are mechanistically linked to the Polycomb
Group (PcG) proteins (3). We find that the PcG protein EZH2 interacts with DNA
methyltransferases and associates with DNA methyltransferase activity in vivo. ChIPs
indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of
EZH2. Furthermore, we show that EZH2 is required for DNA methylation of EZH2-target
promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA
methyltransferases. They highlight a previously unrecognized direct connection between two
fundamental epigenetic repression systems.
2. A key question still poorly understood is how are the DNMTs, and in particular their
enzymatic activity, regulated. Data will be presented that suggest a new mechanism for the
regulation of DNA methylation by post-translational modification.
References
1. Fuks F. Curr Opin Genet Dev. 2005 Oct;15(5):490-5.
2. Brenner C, Deplus R, Didelot C, Loriot A, Vire E, De Smet C, Gutierrez A, Danovi D,
Bernard D, Boon T, Pelicci PG, Amati B, Kouzarides T, de Launoit Y, Di Croce L, Fuks F.
(2005). EMBO J. 24:336-46.
3. Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A,
Bernard D, Vanderwinden JM, Bollen M, Esteller M, Di Croce L, de Launoit Y, Fuks F.
Nature. 2005 Dec 14 [Epub ahead of print].
- 43 -

Oxidative Bax activation as the trigger of the stress-induced intrinsic apoptotic pathway
Lina Ghibelli
Dipartimento di Biologia, Universita' di Roma Tor Vergata
Bax translocation is the most upstream event of the intrinsic apoptotic pathway so far
described. Despite the importance of the issue, the mechanisms that push Bax to mitochondria
are still unknown. The intrinsic pathway of apoptosis is mostly triggered by cell damage,
involving gross environmental alterations such as uncontrolled Ca2+ or ROS overload. This
suggests that its activation may be due not to sophisticated interactions (such as those
involved in the receptor-mediated extrinsic apoptotic pathway), but rather to sudden
environmental changes, thus rendering quite difficult to catch the mechanisms involved. The
idea was to check whether Bax may be a protein "sensing" intracellular chemical/physical
alterations, rather that being activated by specific molecular interactions. Bax translocation to
mitochondria is induced by conformational changes that may be caused by Bax
homodimerisation. We show by computer simulation that the 2 cysteine residues of Bax may
form disulfide bridges, producing conformational changes that favour Bax translocation.
Oxidative, non apoptogenic treatments produce an upshift of Bax migration compatible with
homodimerisation that is reverted by reducing agents; this is accompanied by translocation to
mitochondria. Dimers also appear in pure cytosolic fractions of cell lysates treated with
H2O2, showing that Bax dimerisation may take place in the cytosol. Bax dimers-enriched
lysates support Bax translocation to isolated mitochondria much more efficiently than
untreated lysates, indicating that dimerisation may promote Bax translocation. The absence of
apoptosis in our system allows to demonstrate that Bax moves because of oxidations even in
the absence of apoptosis. This provides the first evidence that Bax dimerisation and
translocation respond to oxidative stimuli, suggesting a novel role for Bax as a sensor of
redox imbalance.
- 44 -

Blocking the "4 Integrin-Paxillin Interaction Selectively Impairs Mononuclear
Leukocyte Recruitment to an Inflammatory Site
Chloé C. Féral*, David M. Rose*, Jaewon Han, Norma Fox, Gregg J. Silverman,
Kenneth Kaushansky, and Mark H. Ginsberg.
The University of California San Diego, La Jolla, CA 92093.
"4 integrin antagonists show promise for several autoimmune and inflammatory diseases but
may exhibit mechanism-based toxicities. We tested the capacity of blockade of "4 integrin
signaling to perturb functions involved in inflammation, while limiting potential adverse
effects. We generated and characterized mice bearing an "4(Y991A) mutation that blocks
paxillin binding and inhibits "4 signals that support leukocyte migration. In contrast to the
embryonic-lethal phenotype of "4 null mice, mice bearing the "4(Y991A) mutation were
viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes
into thioglycollate-induced peritonitis. "4 integrins are essential for definitive hematopoiesis;
however the "4(Y991A) mice had intact lympho-hematopoiesis and, with the exception of
reduced Peyer’s patches, normal architecture and cellularity of secondary lymphoid tissues.
We conclude that interference with "4 integrin signaling can selectively impair mononuclear
leukocyte recruitment to sites of inflammation while sparing vital functions of "4 integrins in
development and hematopoiesis.
- 45 -

Regulation of NF-kB-dependent transcription
Sankar Ghosh
Yale University School of Medicine, New Haven, CT , USA
(no abstract provided)
- 46 -

Regulation of NF-kB-dependent transcription
Sankar Ghosh
Yale University School of Medicine, New Haven, CT , USA
(no abstract provided)
- 47 -

Chromatin impact on NF-kB-driven gene expression
Guy HAEGEMAN, Wim VANDEN BERGHE, Ruben HOYA ARIAS, ‘Matladi
NDLOVU & Linda VERMEULEN
Laboratory for Eukaryotic Gene Expression and Signal Transduction (LEGEST),
Department of Molecular Biology, Ghent University, Ledeganckstraat 35, 9000 GENT
(Belgium)
E-mail: guy.haegeman@ugent.be
Interleukin-6 (IL6) is a pluri-potent cytokine, of which the expression needs to be tightly
regulated in order to keep the organism in a balanced and homeostatic condition. Indeed,
dysregulation of IL6 leads to a variety of affections, like chronic inflammation, autoimmune
and cardiovascular diseases, cancer progression, osteoporosis, etc.
The IL6 gene promoter contains a plethora of transcription factor-binding sites, among which
NF-kB is the most important factor for induction of the gene by infammatory stimuli, like e.g.
TNF. NF-kB is in majority a cytoplasmic protein complex (composed of a p50 and a p65
subunit), which upon inflammatory stimulation migrates to the nucleus and occupies its
position on a variety of gene promoters. In previous work, we have shown that, in addition to
this cytoplasmic activation step, the transcriptional potency of NF-kB is codetermined by the
activated MAP kinase pathway, more particularly by the nuclear kinase MSK1, that
phosphorylates the p65 subunit at Ser 276 (to generate a fully transcription-competent
enhanceosome), as well as the Histon-3 tails at Ser 10 (which is the onset of chromatin
relaxation).
As it was recently published that IKK-a is the Histon-3 phosphorylating kinase after TNF
induction, we investigated the role and impact of these two different kinases for IL6 gene
expression. We found that MSK1 is the primary initiating kinase for temporary chromatin
opening and relaxation (even in the absence of activated NF-kB), whereas IKK-a (only
released after TNF induction) takes over and continues the status of chromatin relaxation,
when activated MSK1 levels off. Sustained chromatin opening and arrival of NF-kB at the
gene promoter upon TNF induction thus lead to abundant IL6 gene expression, whereas
enhanced IL6 gene expression and/or temporary chromatin relaxation by MSK1 extinguishes
in the absence of TNF signalization.
Not only the local (i.e. the gene-specific) chromatin relaxation, but also the entire
nucleosomal arrangement along the chromatin fiber is determinative for the (cell-specific)
levels of gene expression. Indeed, upon comparison of the IL6 gene promoter status in two
different breast cancer cell lines, i.e. the benign, low IL6-expressing cell type MCF-7 and the
aggressive tumor cell line MDA-MB-231 that shows abundant IL6 expression, we found that,
upon DNaseI digestion and restriction enzyme accessibility assays, high IL6 expression
correlates with an increased number of hypersensitive sites, constitutive NF-kB/DNA binding,
and elevated MSK1 activity. Moreover, chromatin of the low-expressing cell line can be
converted to a more open configuration upon treatment of the cells with inhibitors of DNA
methylation, thus leading to augmented levels of IL6 gene expression in the restrictive cell
type MCF-7.
- 48 -

The immunomodulator AS101 induces growth arrest and apoptosis in Multiple
Myeloma cells via the PI3-K/Akt pathway.
Michal Hayun1, Merav Weil1, Yaniv Naor1, Yona Kalechman1, Michael Albeck2,
Nechama Haran-Ghera3 and Benjamin Sredni1
1Safdié Institute for AIDS and Immunology Research, Faculty of Life Sciences,
2Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel; 3Department of
Immunology, The Weizman Institute of Science, Rehovot, Israel
Multiple Myeloma (MM) is a clonal B-cell malignancy affecting both the immune and the
skeletal systems, and accounts for 10% of all hematological cancers. The immunomodulator
ammonium trichloro (dioxoethylene-o,o') tellurate (AS101) is a non toxic compound which
has been shown to have direct anti-tumoral properties in several tumor models. The present
study examined the anti-tumoral activity of AS101 compound by targeting certain signaling
pathway (PI3-K/Akt) crucial for survival of MM cells. We showed that AS101 induced a
significant inhibition of cell proliferation in MM cells which was time-and dose-dependent.
AS101 induced G2/M arrest, an effect associated with an increase of the p53 tumor
suppressor gene, cyclin-dependent kinase inhibitor p21WAF1, and CDK1 phosphorylation.
Longer incubation of MM cells with AS101 resulted in an increase in the early apoptotic cell
population and caspase 9 activity. PI3-K/Akt pathway is of particular interest because of its
role in inhibiting apoptosis and promoting cell proliferation. Downregulation of Akt, decrease
in Survivin expression and suppression of the antiapoptotic effect of exogenously addition of
IGF-1 were observed following AS101 treatment. AS101 was also found to sinergize with
Rapamycin in inducing G2/M arrest in MM cells. Rapamycin is a selective inhibitor of the
phosphoprotein mammalian target of rapamycin (mTOR), a downstream target of pAkt. Our
results indicate that the immunomodulator AS101, currently being used in clinical studies
alone or combined with Rapamycin may be effective in the treatment of MM patients.
- 49 -

THE NDR KINASE FAMILY: REGULATORS OF CELL PROLIFERATION AND
MORPHOGENESIS
Hemmings, B.A., Hergovich, A., Schmitz, D., Kohler, R., Vichalkovski, A., Cornils, H.,
Stegert, M.R., Tamaskovic, R., and Bichsel, S.J.
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
The NDR (nuclear-Dbf2-related) family of proteins is a group of serine/threonine kinases
conserved from yeast to mammals which are important regulators of cell morphogenesis and
proliferation. They are members of AGC kinases (protein kinases A, G, and C) and include
the following protein kinases NDR, LATS, Cbk1, Orb6, Cot-1 and Dbf2. Although, the
precise function(s) of the mammalian NDR kinases still needs to be defined, evidence
suggests that NDR might be involved in tumor progression. NDR1 is hyper-activated in
several S100B-positive melanoma cell lines, and thus given that S100B is over expressed in
more than 80% of metastatic melanomas, NDR1 could potentially influence tumor metastasis
potential. Moreover, disruption of the murine NDR2 gene by insertional mutagenesis was
found to result in B-cell lymphomas, suggesting that both mammalian forms of NDR kinase
might be involved in tumor initiation or progression.
Interestingly, in Saccharomyces cerevisiae, the NDR relative Dbf2 constitutes an integral part
of mitotic exit network (MEN), whereas the other NDR homologue in budding yeast, Cbk1, is
required for regulating morphological changes (RAM network). Recent genetic studies of
yeast have unraveled many key players in processes regulating these NDR-related proteins.
Importantly, these studies demonstrated that the budding yeast relatives of human NDR,
interact with Mob1 (Mps one binder 1) and Mob2, respectively. A crucial interaction required
for the activity and biological functions of these yeast kinases.
Recent work from our laboratory has shown human NDR1 and NDR2 to be regulated in a
similar fashion. Both proteins are efficiently activated upon treatment of cells with the protein
phosphatase 2A inhibitor, okadaic acid (OA). Upon activation, phosphorylation occurs on the
activation segment site, Ser281 (Ser282 for NDR2), and the hydrophobic motif site, Thr444
(Thr442 for NDR2). Phosphorylation of Ser281 occurs by autophosphorylation, whereas
Thr444 is targeted by an upstream kinase, recently identified as the Ste20-like kinase MST3.
Significantly, phosphorylation of both sites is crucial for NDR activity in vitro and in vivo.
Further, we found evidence that human MOB1 (hMOB1), the closest relative of yeast Mob1
and Mob2, stimulates NDR kinase activity and interacts with NDR both in vivo and in vitro.
hMOB1 activates NDR1 at the membrane and membrane association of NDR1 was dependent
on the domain required for hMOB1 binding.
Recent developments regarding the regulation and function of this emerging signaling
pathway will be presented.
- 50 -

Signaling pathways leading to the activation of the interferon antiviral response
John Hiscott, Molecular Oncology Group
Lady Davis Institute –McGill University, Montreal Canada H3T 1E2
IRF-3 and IRF-7 transcription factors are essential for the induction of type I interferon (IFN)
and development of the innate antiviral response. Retinoic acid inducible gene I (RIG-I)
contributes to virus-induced IFN production independent of the Toll-like receptor pathways in
response to RNA viruses and dsRNA. We recently demonstrated that the NF-kB inducible,
anti-apoptotic protein A20 functioned as a negative regulator of RIG-I and blocked RIG-Imediated
NF-kB and IRF gene activation, but only weakly interfered with TLR-3 mediated
IFN activation. Expression of A20 completely blocked CARD domain containing DRIG-Iinduced
IRF-3 Ser396 phosphorylation, homodimerization and DNA binding. The level of
A20 inhibition was upstream of the TBK1/IKKe kinases that phosphorylate IRF3 and IRF7
but downstream of RIG-I itself, since RIG-I was not a target for the ubiquitin-editing
functions of A20.
To identify the A20 target, a BLAST search was performed that identified an uncharacterized
family of proteins – including the prototypical KIAA1271 – that contains a single CARD-like
domain at the N-terminus. Coexpression of KIAA1271 strongly activated IRF and NF-kB
dependent promoters and appeared to be the adapter that links RIG-I sensing to downstream
signaling through the TBK1/IKKe kinases. Four independent groups demonstrated that
MAVS/VISA/IPS-1/CARDIF acts downstream of RIG-I and stimulates the expression of
IFNb through the activation of NF-kB and IRF-3. MAVS localizes to the mitochondria via a
C-terminal transmembrane domain that is required for its function. Within the context of
hepatitis C virus infection, KIAA1271 appears to be the direct target of the HCV NS3/4A
protease; NS3/4A strongly inhibited KIAA1271-mediated activation of IFNB promoter and a
mutated form of KIAA1271, KIAA1271(C508A) which altered the site of NS3/4A proteolytic
cleavage was completely resistant to cleavage. Cleavage of KIAA1271 by NS3/4A disrupts
the C-terminal sequence involved in mitochondrial localization of the adapter. The
characterization of MAVS/KIAA1271 provides the first link between mitochondria and the
innate immune response.
How cross-talk between the mitochondrial apoptotic pathways and the innate response is
achieved remains unknown. Using vesicular stomatitis virus as a model, we demonstrated that
the intrinsic apoptotic cascade, specifically the BAX-BCL-2-Caspase-9 cascade, was the
primary pathway of VSV-induced apoptosis. Cell death was significantly reduced in BaxBak -/-
murine embryonic fibroblasts (MEFs). Conversely, virus replication was enhanced in the
BaxBak -/- MEFs compared to wild-type cells. Accompanying increased viral replication,
expression of antiviral and cytokine genes was also attenuated in infected BaxBak -/- MEFs.
BAX but not BAK was required for IRF-3 phosphorylation and DNA binding, thus
substantiating a role for apoptotic signaling in the activation of innate immune response.
Therefore, virus-induced apoptosis through a BAX-dependent mitochondrial pathway
represents an early signal for the initiation of IFN antiviral response.
- 51 -

The use of BRET (Bioluminescence Resonance Energy Transfer) for the study of
tyrosine-kinase receptors
Tarik ISSAD
CNRS UMR8104, INSERM U567, Université René Descartes Paris 5, Institut Cochin,
Department of Cell Biology, 22 rue Méchain, 75014 Paris
e.mail: issad@cochin.inserm.fr
During these last years, we have developed an expertise in the use of BRET technology for
the study of protein-protein interaction, particularly in the field of tyrosine kinase receptors
and their interaction with regulatory partners. We first demonstrated that the BRET
methodology can be used to monitor ligand-induced conformational changes within the
insulin receptor (Boute et al, Mol.Pharm., 2001; Tips, 2002 ; Biochem.Pharm., 2002). We
then used BRET to study, in real time, in living cells, the interaction of the insulin receptor
(IR) with the protein tyrosine phosphatase PTP1B, which is anchored on the cytosolic face of
the endoplasmic reticulum (Boute et al., EMBO Report, 2003). We showed that insulin
rapidly and dose-dependently stimulated the interaction of the IR with PTP1B and we
demonstrated that internalisation of the IR is necessary for this interaction. In addition, we
demonstrated that PTP1B plays an important role in the regulation of the insulin receptor
immature precursor during its biosynthesis in the endoplasmic reticulum (Boute et al., EMBO
Report, 2003 ; Issad et al., Biochimie 2005). We more recently demonstrated that the BRET
methodology can also be used to monitor ligand-induced conformational changes within the
IGF1 Receptor, as well as the interaction of this receptor with PTP1B (Blanquart et al., Mol.
Pharm. 2005).
The receptor-like plasma membrane protein-tyrosine phosphatases PTPalpha and PTPespilon
may also play a role in the regulation of the IR. We have shown that in the basal state, the IR
and these PTPases are pre-associated at the plasma membrane. Using BRET saturation
experiments, we have shown that insulin does not stimulate the recruitment of these PTPases
to the receptor but rather induced conformational changes within these pre-associated
complexes (Lacasa et al., Mol. Pharm. 2005).
Finally, we also provide evidence that the BRET methodology can be use to monitor the
interaction of the insulin receptor with intracellular adaptors positively or negatively involved
in insulin signaling.
Publications
Boute, N., Pernet, K., and Issad, T. (2001) The activation state of the insulin receptor
monitored using the Bioluminescence Resonance Energy Transfer methodology Molecular
Pharmacology, 60: 640-645
Tavaré, J.M. and Issad, T. (2001) Two-dimensional phosphopeptide mapping of receptor
tyrosine kinases Methods in Molecular Biology 124: 67-85 (Humana Press)
Zilberfarb, V., Siquier, K., Strosberg, A.D., and Issad, T. (2001) Effect of dexamethasone on
adipocyte differentiation markers and tumour necrosis factor alpha expression in human
PAZ6 cells Diabetologia, 44 : 377-386
Grosfeld, A.,. Zilberfarb, V., Turban, S., André, J., Guerre-Millo, M., and Issad, T. (2002)
Hypoxia increases leptin expression in human PAZ6 adipose cells Diabetologia 45: 527-530
Lahlou,N., Issad,T., Carel, J-C., Camoin, L., Lebouc,Y., Roger, M., and Girard, J. (2002)
Mutations in the human leptin and leptin receptor genes as models of serum leptin receptor
regulation Diabetes 51: 1980-1985
- 52 -

Boute, N., Jockers, R. and Issad, T. (2002) The use of Resonance Energy Transfer in highthroughput
screening: BRET versus FRET. Trends in Pharmacological Sciences 23: 351-
354
Issad, T., Boute, N. and Pernet, K (2002) A homogenous assay to monitor the activity of the
insulin receptor using Bioluminescence Resonance Energy Transfer. Biochemical
Pharmacology 64: 813-817
Guerre-Millo, M., Grosfeld, A., and Issad, T. (2002) Stimulatory effect of hypoxia on leptin
release in cultured adipose cells. Obesity Research 10: 856
Issad T., Boute N, Pernet K. (2002) The activity of the insulin receptor assessed by
bioluminescence resonance energy transfer. Ann. N. Y. Acad. Sci. 973: 120-123
Boute N., Boubekeur S., Lacasa D. and Issad T. (2003) Dynamics of Interaction between
Insulin Receptor and Protein Tyrosine Phosphatase-1B in Living Cells. EMBO reports 4 :
313-319.
Issad T., Boute N., Boubekeur B., Lacasa D. and Pernet K. Looking for an insulin pill? Use
the BRET methodology! (2003) Diabetes and Metabolism 29: 111-117.
Boute N, Zilberfarb V, Camoin L, Bonnafous S, Le Marchand-Brustel Y, Issad T. The
formation of an intrachain disulfide bond in the leptin protein is necessary for efficient leptin
secretion. Biochimie. 2004;86:351-56.44.
Lacasa D, Boute N and Issad T. (2005-a) Interaction of the insulin receptor with the
receptor-like protein tyrosine phosphatases PTPalpha and PTPepsilon in living cells. Mol.
Pharmacol. 4:1206-1213.
Issad T, Boute N, Boubekeur S, Lacasa D. (2005) Interaction of PTPB with the insulin
receptor precursor during its biosynthesis in the endoplasmic reticulum. Biochimie. 87:111-
116.
Blanquart C, Boute N, Lacasa D and Issad T. (2005-b) Monitoring the activation state of the
Insulin-like Growth Factor-1 Receptor and its interaction with PTP1B by using the BRET
methodology. Mol. Pharmacol. Jun 23; [Epub ahead of print]
Issad T. and Jockers R. Bioluminescence Resonance Energy Transfer (BRET) to monitor
protein-protein interactions (In Press) Methods in Molecular Biology: Transmembrane
signaling protocols (Humana Press)
- 53 -

Protein Kinase C alpha as a potential target in the treatment of acquired antiestrogen
resistance of human breast cancer.
Jan. S. Jepsen 1 , Lisa Frankel 1 , Bo Hansen 2 , Anne E. Lykkesfeldt 1
1 Danish Cancer Society, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
2 Santaris Pharma A/S, Bøge Allé 3, DK-2970, Hørsholm, Denmark.
E-mail: jsj@cancer.dk, lfr@cancer.dk, bh@santaris.com, al@cancer.dk
Initially, antiestrogen therapy is an efficient treatment of hormone-dependent breast cancer.
However, many patients eventually acquire resistance. This presents a serious therapeutic
problem and current research aims at elucidating the underlying molecular mechanisms in
order to identify new targets in treatment of patients with resistant tumors.
The Protein Kinase C alpha (PKC alpha) level is increased in several cancer types. However,
limited information is available on the involvement of PKC alpha in antiestrogen resistance.
We screened nine different antiestrogen resistant breast cancer cell lines and found that
elevated PKC alpha expression was a general phenomenon. Two of the resistant cell lines
were chosen for further studies: MCF-7/TAM R -1 and MCF-7/182 R -6, resistant to tamoxifen
and ICI 182,780 (faslodex), respectively. These two cell lines posses highly elevated PKC
alpha protein expression and kinase activity levels in comparison to parental MCF-7 cells.
A potent and relatively specific PKC alpha inhibitor, Ro-32-0432, resulted in a significant
growth inhibition of these antiestrogen resistant cell lines relative to MCF-7. Moreover,
specific down-regulation of PKC alpha by transient transfection of antisense oligonucleotides
resulted in a 30-40% growth inhibition of TAM R -1 and 182 R -6, while MCF-7 remained
unaffected. Additionally, using small hairpin RNA, we generated stable PKC alpha knockdown
cells with almost no PKC alpha protein expression and found that PKC alpha knock
down restored the sensitivity of TAM R -1 to tamoxifen. Finally, preliminary results indicate
that MCF-7 clones which overexpress PKC alpha are more resistant to antiestrogen mediated
growth inhibition than parental MCF-7 cells. These results suggest that PKC alpha may be
causally involved in the growth of antiestrogen resistant cells and that PKC is a potential
therapeutic target in the treatment of antiestrogen resistant breast cancer.
Recent papers (papers from 2004, 2005)
Frogne T, Jepsen JS, Larsen SS, Fog CK, Brockdorff BL and Lykkesfeldt AE. Antiestrogenresistant
human breast cancer cells require activated protein kinase B/Akt for growth. Endocr
Relat Cancer. 2005 12(3): 599-614.
Christensen GL, Jepsen JS, Fog CK and Lykkesfeldt AE. Sequential versus combined
treatment of human breast cancer cells with antiestrogens and the vitamin D analogue
EB1089 and evaluation of predictive markers for vitamin D treatment. Breast Cancer
Research and Treatment. 2004 85(1):53-63.
Juncker-Jensen A, Lykkesfeldt AE, Worm J, Ralfkiær U, Espelund U and Jepsen JS. Insulinlike
growth factor binding protein 2 is a marker for antiestrogen resistant human breast cancer
cell lines but is not a major growth regulator. Submitted to Growth Hormone & IGF Reseach
Jepsen JS, Pfundheller HM and Lykkesfeldt AE: Specific down regulation of p21 (WAF1/CIP1)
and the estrogen receptor " in MCF-7 cells by antisense oligonucleotides containing locked
nucleic acid (LNA). Oligonucleotides. 2004 14(2): 147-156.
- 54 -

Jespsen JS, Sorensen MD and Wengel J. Locked Nucleic Acid: a potent nucleic acid
analogue in therapeutics and biotechnology. Oligonucleotides. 2004 14(2): 130-146.
Vollmer J, Jepsen JS, Uhlmann E, Schetter C, Jurk M, Wader T, Wullner M and Krieg AM.
Modulation of antisense or CpG oligodeoxynucleotide-mediated immune stimulation by
locked nucleic acid (LNA). Oligonucleotides. 2004 14(1):23-32.
Jepsen JS and Wengel J. LNA-Antisense rivals siRNA for gene silencing.
Current Opinion in Drug Discovery and Development. 2004 7(2):188-194.
Jepsen JS. Locked nucleic acid (lna) as a therapeutic tool to knock down gene expression.
Encyclopedia of medical genomics and proteomics. Accepted.
Jepsen JS and Kauppinen S. Locked nucleic acid (lna) as a tool in biotechnology research
and diagnostics. Encyclopedia of medical genomics and proteomics. Accepted.
Stein CA, Dias N, Benimetskaya L, Jepsen JS, Lai J and Raffo A. LY900003 (Isis 3521) and
G3139 (Genasense; Oblimersen)- Phosphorothioate Antisense Oligonucleotides With
Pleiotropic Mechanisms of Action. Nucleic Acid Therapeutics in Cancer (2004), pp. 177-198.
Edited by Gerwirtz, A., Humana Press (ISBN: 1-58829-258-4).
Hrdlicka, P.J., Jepsen, J.S., Nielsen, C and Wengel, J.: Synthesis and biological evaluation of
conformationally restricted and nucleobase-modified analogs of the anticancer compound 3'-
C-ethynylcytidine (ECyd). Nucleosides Nucleotides Nucleic Acids. 2005 24(5-7): 397-400.
Hrdlicka PJ, Jepsen JS, Nielsen C and Wengel J. Synthesis and biological evaluation of
nucleobase-modified analogs of the anticancer compounds 3’-C-ethynyluridine (EUrd) and
3’-C-ethynylcytidine (ECyd).
Bioorganic & Medicinal Chemistry. 2005 13(4): 1249-1260
Hrdlicka PJ, Andersen NK, Jepsen JS, Hansen FG, Haselmann KF, Nielsen C and Wengel J.
Synthesis and biological evaluation of branched and conformationally restricted analogs of
the anticancer compounds 3’-C-ethynyluridine (EUrd) and 3’-C-ethynylcytidine (ECyd).
Bioorganic & Medicinal Chemistry. 2005 13(7): 2597-2621
Kragelund BB, Poulsen K, Andersen KV, Baldursson T, Kroll JB, Neergard TB, Jepsen J,
Roepstorff P, Kristiansen K, Poulsen FM and Knudsen J. Conserved residues and their role in
the structure, function, and stability of acyl-coenzyme A binding protein. Biochemistry. 1999
Feb 23; 38(8):2386-94.
Jepsen JS Locked nucleic acid (LNA) as cancer-therapeutic agent. Dan Med Bull 2004
51:139.
- 55 -

Cell signaling by proteinase-activated receptor-2 for interleukin-8 formation in human
lung epithelial cells
Atsufumi Kawabata, Mami Nagataki, Kazumi Moriyuki, Fumiko Sekiguchi, Hiroyuki
Nishikawa*.
Division of Physiology and Pathophysiology, School of Pharmaceutical Sciences, Kinki
University, Higashi-Osaka 577-8502, Japan, and *Fuso Pharmaceutical Industries Ltd.,
Osaka 536-8523, Japan. E-mail: kawabata@phar.kindai.ac.jp
Proteinase-activated receptor-2 (PAR2) plays a dual role in the respiratory system, being pro-
and anti-inflammatory. Stimulation of PAR2 in the airway epithelial cells causes
formation/release of prostaglandins and cytokines, leading to bronchodilation and/or
inflammation. The present study investigated cell signaling pathways responsible for
formation of interleukin-8 (IL-8) following PAR2 stimulation in human lung epithelial cells
(A549), as compared with those for formation of prostaglandin E 2 (PGE 2). SLIGRL-NH 2, a
PAR2-activating peptide, but not LSIGRL-NH 2, a scrambled peptide, elicited release of IL-8
as well as PGE 2, which reached a plateau at 18 h and 3 h, respectively. PAR2 stimulation also
increased the levels of mRNA for IL-8. Inhibition experiments indicated possible involvement
of MEK-ERK, JNK, Src, protein kinase C and EGF receptor tyrosine kinase, but not p38
MAP kinase and COX, in PAR2-triggered IL-8 formation, being not necessarily the same as
those for PGE 2 formation. Phosphorylation of JNK, ERK and EGF receptors was confirmed
following PAR2 stimulation. Upon activation, PAR2 thus triggers activation of multiple
signaling pathways including EGF receptors, JNK and MEK-ERK in A549 cells, resulting in
delayed up-regulation and release of IL-8 that is pro-inflammatory in the respiratory system.
Recent publications :
1. Kawao, N., Nagataki, M., Nagasawa, K., Kubo, S., Cushing, K., Wada, T., Sekiguchi, F.,
Ichida, S., Hollenberg, M.D., MacNaughton, W.K., Nishikawa, H. and Kawabata, A.* (2005)
Signal transduction for proteinase-activated receptor-2-triggered prostaglandin E 2 formation
in human lung epithelial cells. J. Pharmacol. Exp. Ther., in press.
2. Nishikawa, H. and Kawabata, A.* (2005) PAR-2 as a target for gastric mucosal
protection. Drugs of Future, in press. (Review)
3. Sekiguchi, F., Hasegawa, N., Inoshita, K., Yonezawa, D., Inoi, N., Kanke, T., Saito, N.
and Kawabata, A.* (2005) Mechanisms for modulation of mouse gastrointestinal motility by
proteinase-activated receptor (PAR)-1 and -2 in vitro. Life Sci., in press.
4. Kanke, T.*, Ishiwata, H., Kabeya, M., Saka, M., Doi, T., Hattori, Y., Kawabata, A. and
Plevin, R. (2005) Binding of a highly potent protease-activated receptor-2 (PAR2) activating
peptide, [ 3 H]-2-furoyl-LIGRL-NH 2, to human PAR2. Br. J. Pharmacol., 145, 255-263.
5. Kawabata, A.*, Oono, Y., Yonezawa, D., Hiramatsu, K., Inoi, N., Sekiguchi, F., Honjo,
M., Hirofuchi, M., Kanke, T. and Ishiwata, H. (2005) 2-furoyl-LIGRL-NH 2, a potent agonist
for proteinase-activated receptor-2, as a gastric mucosal cytoprotective agent. Br. J.
Pharmacol., 144, 212-219.
6. Kawabata, A.* and Kawao, N. (2005) Physiology and pathophysiology of proteinaseactivated
receptors (PARs): PARs in the respiratory system: cellular signaling and
physiological/pathological roles. J. Pharmacol. Sci., 97, 20-24. (Review)
7. Kanke, T.*, Takizawa, T., Kabeya, M. and Kawabata, A. (2005) Physiology and
pathophysiology of proteinase-activated receptors (PARs): Proteinase-activated receptor-2
(PAR-2) as a potential therapeutic target. J. Pharmacol. Sci., 97, 38-42. (Review)
- 56 -

8. Nishikawa, H., Kawai, K., Tanaka, M., Ohtani, H., Tanaka, S., Kitagawa, C., Nishida,
M., Abe, T., Araki, H. and Kawabata, A.* (2005). Protease-activated receptor-2 (PAR-2)related
peptides induce tear secretion in rats: involvement of PAR-2 and non-PAR-2
mechanisms. J. Pharmacol. Exp. Ther., 312, 324-331.
9. Kimura, T., Arai, M., Masuda, H. and Kawabata, A.* (2004). Impact of a pharmacistimplemented
anemia management in outpatients with end-stage renal disease in Japan. Biol.
Pharm. Bull., 27, 1831-1833
10. Kawabata, A.*, Nakaya, Y., Ishiki, T., Kubo, S., Kuroda, R., Sekiguchi, F., Kawao, N.,
Nishikawa, H. (2004) Receptor-activating peptides for PAR-1 and PAR-2 relax rat gastric
artery via multiple mechanisms. Life Sci., 75, 2689-2702.
11. Sekiguchi, F. and Kawabata, A.* (2004) Protease-activated receptors (PARs) as
therapeutic targets: development of agonists/antagonists and modulation of gastrointestinal
functions. Drug Design Reviews, 1, 287-296. (Review)
12. Kawabata, A.*, Kubo, S., Ishiki, T., Kawao, N., Sekiguchi, F., Kuroda, R., Hollenberg,
M.D., Kanke, T. and Saito N. (2004) Proteinase-activated receptor-2-mediated relaxation in
mouse tracheal and bronchial smooth muscle: Signal transduction mechanisms and distinct
agonist sensitivity. J. Pharmacol. Exp. Ther., 311, 402-410.
13. Kawabata, A.*, Itoh, H., Kawao, N., Kuroda, R., Sekiguchi, F., Masuko, T., Iwata, K.,
Ogawa, A. (2004). Activation of trigeminal nociceptive neurons by parotid PAR-2 activation
in rats. NeuroReport, 15, 1617-1621.
14. Sekiguchi, F., Mita, Y., Kamanaka, Y., Kawao, N., Matsuya, H., Taga, C. and
Kawabata, A.* (2004). The potent iNOS inhibitor ONO-1714 inhibits nNOS and exerts
antinociception in rats. Neurosci. Lett., 365, 111-115.
15. Kawabata, A*, Kanke, T., Yonezawa, D., Ishiki, T., Saka, M., Kabeya, M., Sekiguchi,
F., Kubo, S., Kuroda, R., Iwaki, M., Katsura, K. and Plevin, R (2004) Potent and
metabolically stable agonists for protease-activated receptor-2: Evaluation of activity in
multiple assay systems in vitro and in vivo. J. Pharmacol. Exp. Ther. 309, 1098-1107.
16. Kawao, N., Ikeda, H., Kitano, T., Kuroda, R., Sekiguchi, F., Kataoka, K., Kamanaka, Y.
and Kawabata, A.* (2004) Modulation of capsaicin-evoked visceral pain and referred
hyperalgesia by protease-activated receptors 1 and 2. J. Pharmacol. Sci. 61, 683-692.
17. Kawabata, A.*, Kubo, S., Nakaya, Y., Ishiki, T., Kuroda, R., Sekiguchi, F., Kawao, N.
and Nishikawa, H. (2004). Distinct roles for protease-activated receptors 1 and 2 in vasomotor
modulation in rat superior mesenteric artery. Cardiovasc. Res. 61, 683-692.
18. Kawabata, A.*, Nishikawa, H., Saitoh, H., Nakaya, Y., Hiramatsu, K., Kubo, S.,
Nishida, M., Kawao, N., Kuroda, R., Sekiguchi, F., Kinoshita, M., Kakehi, K., Arizono, N.,
Yamagishi, H. and Kawai, K. (2004) A protective role of protease-activated receptor-1 in rat
gastric mucosa. Gastroenterology 126, 208-219.
19. Kawao, N., Hiramatsu, K., Inoi, N., Kuroda, R., Nishikawa, H., Sekiguchi, F. and
Kawabata, A.* (2003) The PAR-1-activating peptide facilitates pepsinogen secretion in rats.
Peptides 24, 1449-1451.
20. Kamanaka, Y., Kawabata, A.*, Matsuya, H., Taga, C., Sekiguchi, F. and Kawao, N.
(2003) Effect of a potent iNOS inhibitor (ONO-1714) on acetaminophen-induced
hepatotoxicity in the rat. Life Sci. 74, 793-802.
21. Kawabata, A.*, Nakaya, Y., Kuroda, R., Wakisaka, M., Masuko, T., Nishikawa, H. and
Kawai, K. (2003) Involvement of EDHF in the hypotension and increased gastric mucosal
blood flow caused by PAR-2 activation in rats. Br. J. Pharmacol. 140, 247-254.
22. Kuroda, R.*, Kawabata, A. (2003) Pain information pathways from the periphery to the
cerebral cortex. Yakugaku Zasshi 123, 533-546 (Review) (in Japanese)
- 57 -

Crosstalk between NF-kB and AP-1 in Helicobacter pylori-mediated inflammation in
human gastric epithelial cells
Hyeyoung Kim
Department of Pharmacology and Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea. Email:
kim626@yumc.yonsei.ac.kr
Previously we have shown that inflammatory enzymes such as cyclooxygenase-2 (COX-2)
and inducible nitric oxide syntahse (iNOS) were expressed in H. pylori-infected gastric
epithelial cells. The expression of COX-2 and iNOS may be regulated by NF-kB and AP-1.
Present study aims to investigate whether H. pylori-induced expressions of COX-2 and iNOS
are regulated by NF-kB and AP-1 in gastric epithelial AGS cells, and whether the
transcriptional regulation of COX-2 or iNOS is inhibited by transfection of antisense
oligodeoxynucleotide (AS ODN) for NF-kB subunit p50 or c-jun dominant negative mutant
gene (TAM-67). As a result, H. pylori induced the expression of COX-2 and iNOS via
activation of NF-kB and AP-1, which was inhibited in the cells transfected with either p50 AS
ODN or TAM-67. The activated NF-kB complex was p65/p50 hetero- and p50/p50 homo-
dimmers while the activated AP-1 complex was a c-jun/c-fos heterodimer in H. pyloriinfected
AGS cells. In conclusion, NF-kB and AP-1 share H. pylori-mediated inflammation
signaling by inducing COX-2 and iNOS expression in gastric epithelial cells. Either
suppression of NF-kB or AP-1 may inhibit H. pylori-induced inflammation in gastric
epithelial cells.
Publication list (2002-2005)
1) NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar cells. JH Yu, JW
Lim, KH Kim, T Morio, H Kim. Free Rad Biol Med 39: 590-602, 2005
2) NADPH oxidase mediates interleukin-6 expression in cerulein-stimulated pancreatic acinar
cells. JH Yu, JW Lim, H Kim, KH Kim, Int J Biochem Cell Biol 37:1458-1469, 2005
3) Cellular stress - related protein expression in Helicobacter pylori - infected gastric
epithelial AGS cells. JW Lim, H Kim, JM Kim, JS Kim, HC Jung, KH Kim, Int J Biochem
Cell Biol 36:1624-1634, 2004.
4) Oxidative stress - related proteome changes in Helicobacter pylori - infected human gastric
mucosa. HY Baek, JW Lim, H Kim, JM Kim, JS Kim, HC Jung, KH Kim, Biochem J
379:291-299, 2004
5) Helicobacter pylori in a Korean isolate activates mitogen-activated protein kinases, AP-1,
and NF-!B and induces chemokine expression in gastric epithelial AGS cells. JH Seo, JW
Lim, H Kim, KH Kim, Lab Inv 84:49-62, 2004
6) The Ku antigen-recombination signal-binding protein J! complex binds to the nuclear
factor-!B p50 promoter and acts as a positive regulator of p50 expression in human gastric
cancer cells. JW Lim, H Kim, KH Kim, J Biol Chem 279(1):231-237, 2004
7) Role of oxygen free radicals in patients with acute pancreatitis. BK Park, JB Chung, JH
Lee, JH Suh, SW Park, SY Song, H Kim, KH Kim, JK Kang, World J Gastroenterology
9(10):2266-2269, 2003
8) Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis
in pancreatic acinar AR42J cells. JY Song, JW Lim, H Kim, T Morio, KH Kim.. J Biol Chem
278 (38):36676-36687, 2003 Oct.
- 58 -

9) Proteome analysis of rat pancreatic acinar cells: Implication for cerulein - induced acute
pancreatitis, JH Yu, SY Yun, H Kim, KH Kim, Proteomics 3(12):2446-2453, 2003
10) Mass spectrometry and tandem mass spectrometry analysis of rat pancreatic
mitochondrial ATP synthase: upregulation in pancreatic acinar cells treated with cerulein, JH
Yu, SY Yun, H Kim, KH Kim, Proteomics 3(12):2437-2445, 2003
11) The effect of p38 mitogen-activated protein kinase on mucin gene expression annd
apoptosis in Helicobacter pylori-infected gastric epithelial cells, H Kim, JH Seo, KH Kim,
Ann NY Acad Sci 1010:90-94, 2003
12) Role of NF-!B and DNA repair protein Ku on apoptosis in pancreatic acinar cells, JY
Song, JW Lim, H Kim, KH Kim, Ann NY Acad Sci 1010:259-262, 2003
13) Calcium-dependent apoptotic gene expression in cerulein-treated AR42J cells, JH Yu, H
Kim, KH Kim, Ann NY Acad Sci 1010:66-70, 2003
14) Signal transduction of cerulein-induced cytokine expression and apoptosis in pancreatic
acinar cells, J Lee, J Seo, H Kim, JB Chung, KH Kim, Ann NY Acad Sci 1010:104-108,
2003
15) NF-kB and Bcl-2 in Helicobacter pylori-induced apoptosis in gastric epithelial cells, SH
Chu, JW Lim, KH Kim, H Kim, Ann NY Acad Sci 1010:568-575, 2003
16) Diagnostic significance of antibodies to heat shock proteins, H Kim, Clinica Chemica
Acta, 337:1-10, 2003
17) Cell adhesion - related gene expression by Helicobacter pylori in gastric epithelial AGS
cells, JW Lim, H Kim, KH Kim, Int J Biochem Cell Biol, 35:1284-1296, 2003
18) Role of NF-kB and AP-1 on Helicobacter pylori-induced IL-8 expression in AGS cells.
SH Chu, H Kim, JY Seo, JW Lim, N Mukaida, KH Kim, Dig Dis Sci 48(2): 257-265, 2003
19) Expression of Ku70 and Ku80 mediated by NF-kB and cyclooxygenase-2 are related to
cell proliferation in human gastric cancer cells. JW Lim, H Kim, KH Kim, J Biol Chem
277(48):46093-46100, 2002
20) Transcriptional regulation by thiol compounds in Helicobacter pylori induced IL-8
production in human gastric epithelial cells. JY Seo, H Kim, KH Kim. Ann New York Acad
Sci 973:541-545, 2002
21) Cyclooxygenase-2 expression by transcription factors in Helicobacter pylori-infected
gastric epithelial cells: comparison between HP99 and NCTC 11637. JH Seo, H Kim, KH
Kim. Ann New York Acad Sci 973:477-480, 2002
22) Suppression of cerulein-induced cytokine expression by antioxidants in pancreatic acinar
cells. JH Yu, JW Lim, W Namkung, H Kim, KH Kim. Lab Inv 82(10):1359-1368, 2002
23) Effect of mannitol on Helicobacter pylori-induced cyclooxygenase-2 expression in gastric
epithelial AGS cells. H Kim, JY Seo, KH Kim Pharmacology 66(4):182-189, 2002 Dec.
24) Oxidative stress-induced cytokine production in isolated rat pancreatic acinar cells;
Effects of small molecule antioxidants. JY Seo, H Kim, JT Seo, KH Kim, Pharmacology
64(2):63-70, 2002 Feb.
25) Oxidant-sensitive transcription factor and cyclooxygenase-2 by Helicobacter pylori
stimulation in human gastric cancer cell lines. H Kim, JW Lim, JY Seo, KH Kim, J
Environmental Pathology, Toxicology and Oncology 21:121-129, 2002
- 59 -

Signaling of life and death in T cells
Peter H. Krammer
Tumorimmunology Program, German Cancer Research Center, Heidelberg, Germany):
(no abstract provided)
- 60 -

MicroRNA is an anti-apoptotic factor in human brain tumors
Anna M. Krichevsky1, Jennifer A. Chan2, & Kenneth S. Kosik3
1 Department of Neurology, Brigham and Women's Hospital, Harvard Medical School;
2 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical
School, Boston, MA 02115 and 3 Neuroscience Research Institute, University of
California Santa Barbara, Santa Barbara, CA 93106, USA
MicroRNAs (miRNAs) are a recently discovered class of small 18-23 nucleotide non-coding
RNA molecules that have been shown to regulate target gene expression in various
organisms. By targeting the mRNA of protein-coding genes for either cleavage or repression
of translation, miRNAs are thought to play critical roles in development, control of growth,
proliferation, and cell lineage determination. Expression data and location in the human
genome suggested the potential for this new class of regulatory RNAs to influence the
processes of proliferation, differentiation and cell death in a human malignancy.
Using our homemade oligonucleotide array designed to detect the majority of mammalian
miRNAs identified thus far, we measured the expression levels of miRNAs during normal
brain development, in the process of neurogenesis in vitro and in high-grade brain tumors,
glioblastomas. Our study identified a specific miRNA, miR-21, that is markedly elevated in
glioblastomas. We demonstrate that glioblastomas strongly overexpress this specific miRNA
compared to low-grade brain tumors, non-neoplastic fetal and adult brain, neuronal and glial
cultured cells, and stem cell-derived neural precursors, clearly linking this miRNA to
malignancy. Sequence-specific knock-down of miR-21 with modified antisense
oligonucleotides triggers activation of caspases and leads to increased apoptotic cell death of
glioblastoma cells in culture, implying a role for this miRNA in gliomagenesis. Wholegenome
expression profiling revealed that multiple proapoptotic genes as well as genes
negatively regulating cell proliferation have been specifically induced after the knock-down
of this miRNA. These findings suggest that the over-expressed miRNA may function as a
micro-oncogene, presumably by blocking expression of key apoptosis-enabling genes.
Moreover, our studies not only validate a functional role of miRNA in brain tumors, but also
raise the possibility that miRNAs may serve as novel attractive targets in oncology.
- 61 -

Mechanisms of caspase-independent apoptosis
Guido Kroemer
CNRS-UMR8125, Institut Gustave Roussy, 94805 Villejuif, France
Chemotherapy resistance has often been related to disabled apoptosis with deficient caspase
activation. However, tumor cells tend to die in response to most conventional
chemotherapeutic agents even when caspase activation is inhibited. This caspase-independent
cell death is mediated by a series of metabolic changes, as well as by the mitochondrial
release of caspase-independent death effectors such as AIF. We have recently found that,
even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit
an effective anti-tumor immune response that precludes the growth of inoculated tumors or
leads to the regression of established neoplasia. Caspase inhibition by Z-VAD-fmk or
transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin-induced cell death
(as measured as clonogenic survival), yet suppressed the immunogenicity of dying tumor cells
in a variety of different rodent models of neoplasia. Depletion of DC in vivo curtailed the
immune response against doxorubicin-treated apoptotic tumor cells. Caspase inhibition
suppressed the capacity of doxorubicin-killed cells to be phagocytosed by dendritic cells
(DC), yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors
became immunogenic upon doxorubicin treatment in vitro, and intratumoral inoculation of
doxorubicin could trigger the regression of established tumors in immunocompetent mice.
These results delineate a procedure for the generation of cancer vaccines and the stimulation
of anti-neoplastic immune responses in vivo. Moreover, they suggest that inhibition of
caspase activation (which leads to a shift from apoptotic to non-apoptotic death modalities)
can abrogate the immunogenic nature of cell death, thus favoring the escape of tumors from
immune surveillance.
- 62 -

Resveratrol inhibits phorbol ester-induced expression of COX-2 through inactivation of
NF-!B and AP-1 in mouse skin: role of I!B kinase and MAP kinases
Joydeb Kumar Kundu, Young Kee Shin and Young-Joon Surh
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
Resveratrol, an antioxidant and anti-inflammatory phytoalexin present in red wine and grapes,
was reported to inhibit chemically-induced carcinogenesis in mouse skin. The present study
was aimed to elucidate molecular mechanisms of inhibitory effects of resveratrol on 12-Otetradecanoylphorbol-13-acetate
(TPA)-promoted mouse skin carcinogenesis. Topical
application of resveratrol significantly inhibited TPA-induced COX-2 expression. Resveratrol
diminished the DNA binding of NF-!B by suppressing phosphorylation and subsequent
degradation of I!B", and resultant nuclear translocation of p65. Resveratrol also blunted
TPA-induced phosphorylation of p65 and subsequent interaction of p65 with cyclic AMP
response element binding protein-binding protein. The TPA-induced DNA binding of AP-1
and the expression of c-Jun and c-Fos were also diminished by resveratrol. Moreover,
pretreatment with resveratrol suppressed the activation of ERK, p38 MAP kinase and JNK.
Topical application of TPA caused rapid induction of IKK activity, which was suppressed by
pretreatment with resveratrol or cotreatment with Bay 11-7082, a pharmacological inhibitor of
IKK. Topically applied Bay 11-7082 also abrogated TPA-induced activation of NF-!B and
AP-1 as well as expression of COX-2, suggesting a regulatory role of IKK in TPA-induced
COX-2 expression in mouse skin in vivo. The TPA-induced phosphorylation of ERK, JNK
and p38 MAP kinases was also abrogated by Bay 11-7082 treatment.
List of Publications :
1. Kundu JK, Choi K-Y., and Surh Y-J. #-catenin-mediated signaling: a novel
molecular target for chemoprevention with anti-inflammatory substances. Biochemica et
Biophysica Acta - Rev. Cancer. 1765 (1): 14-24, 2006.
2. Kundu JK, and Surh Y-J. Molecular mechanisms underlying chemoprevention with
resveratrol, Cancer Prev. Res.. 10 (2): 89-98; 2005.
3. Surh Y-J., Kundu JK, Na H-K., and Lee J-S. Redox-sensitive transcription factors as
prime targets for chemoprevention with anti-inflammatory and antioxidative phytochemicals,
J. Nutr. 135 (12): 2993S-3001S; 2005.
4. Prawan A, Kundu JK and Surh Y-J. Molecular basis of heme oxygenase-1 induction:
implications for chemoprevention and chemoprotection, Antiox. Redox Signal. (in press),
2005.
5. Kim SO, Kundu JK, Shin YK, Park J-H., Cho M-H, Kim T-Y. and Surh Y-J. [6]-
Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-
!B in phorbol ester-stimulated mouse skin. Oncogene 24(15): 2558-67, 2005.
6. Kundu JK and Surh Y-J. Breaking the relay in deregulated cellular signal
transduction as a rationale for chemoprevention with anti-inflammatory phytochemicals,
Mutat. Res. 591 (1-2): 123-146; 2005
7. Chowdhury KK, Saha A, Bachar SC and Kundu JK. Analgesic and antiinflammatory
activity of Desmodium triflorum DC. J. Biol. Sci., 5 (5) : 581-583; 2005
8. Surh Y-J. and Kundu JK. Signal transduction network leading to COX-2 induction: a
road map in search of cancer chemopreventives. Arch. Pharm. Res. 28: 1-15; 2005.
- 63 -

9. Kundu JK, Mossanda KA, Na H-K and Surh Y-J. Inhibitory effects of extracts of
Sutherlandia frutescens and Harpagophytum procumbens on phorbol ester-induced COX-2
expression in mouse skin : AP-1 and CREB as potential targets. Cancer Lett. 218: 21-31;
2005
10. Kundu JK and Surh Y-J. Molecular basis of chemoprevention by resveratrol : NF-!B
and AP-1 as potential targets, Mutat. Res. 255: 65-80; 2004
11. Kundu JK, Chun K-S, Kim SO and Surh Y-J. Resveratrol inhibits phorbol esterinduced
cyclooxygenase-2 expression in mouse skin: MAPKs and AP-1 as potential
molecular targets. Biofactors 21: 33-39; 2004
12. Kim SO, Chun K-S, Kundu JK and Surh Y-J. Inhibitory effects of [6]-gingerol on
PMA-induced COX-2 expression and activation of NF-!B and p38 MAPK in mouse skin.
Biofactors 21: 27-31; 2004
13. Datta BK, Dutta SK., Khan TH, Kundu JK, Rashid MA, Nahar L and Sarker SD.,
Anti-cholinergic, cytotoxic and anti-HIV activities of a sesquiterpene and a flavone glycoside
from the aerial parts of P. viscosum, Pharmaceutical Biol., 42: 18-23; 2004.
14. Datta BK, Datta SK, Chowdhury MM, Khan TH, Kundu JK, Rashid MA, Nahar L,
Sarker SD. Analgesic, anti-inflammatory and CNS depressant activities of sesquiterpenes and
a flavonoid glycoside from Polygonum viscosum Die Pharmazie, 59: 222-225; 2004
15. Kundu JK, Na H-K, Chun K-S, Kim Y-K, Lee S-J, Lee S-S, and Surh Y-J . Inhibition
of phorbol ester-induced COX-2 expression by epigallocatechin gallate in mouse skin and
cultured human mammary epithelial cells. J Nutr. 133: 3805S-3810S; 2003
16. Das AK, Ahmed F, Bachar SC, Kundu JK and Dev S.; Anti-inflammatory effect of
Albizzia lebbeck (Benth.) Bark J. Biol. Sci. 3: 685-687, 2003
17. Shaphiullah M, Bachar SC, Kundu JK, Begum F, Uddin A, Roy SC, Khan MTH.
Antidiarrheal activity of the methanol extract of Ludwigia hyssopifolia Linn Pak. J. Pharm.
Sci 16:7-12; 2003.
18. Datta BK, Datta SK, Rashid M.A, Kundu JK, Hasan CM and Sarker SD. Further
Sesquiterpenes from Polygonum viscosum (Polygonaceae), Nat. Prod. Lett. 16: 143-148,
2002
19. Datta BK, Rashid MA, Kundu JK, Rouf ASS, Sarker SD and Datta SK. Isolation and
structure elucidation of viscoazucine, a novel sesquiterpene from Polygonum viscosum.
Pharmazie, 56: 578-9, 2001
20. Kundu JK, Rouf ASS., Hossain MN, Hasan CM and Rashid MA. Antitumor activity
of epifriedelanol from Vitis trifolia. Fitoterapia 71: 574-576; 2000
21. Ahmed M, Datta BK, Sadhu SK, Kundu JK and Bachar SC. Preliminary biological
studies on stagninol a sesquiterpene isolated from Persicaria stagnina Linn., Pharmazie, 52:
472-75, 1997.
- 64 -

Cross-talk between angiotensin II and glucagon receptor signaling mediates activation
of mitogen-activated protein kinases ERK 1/2 in rat glomerular mesangial cells.
Xiao C. Li, Oscar A. Carretero, and Jia L. Zhuo.
Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, MI
48202, USA. E-mail: xcli1@hfhs.org
Agonist-induced phosphorylation of MAP kinases extracellular signal-regulated protein
kinases 1 and 2 (p-ERK 1/2) plays an important role in the regulation of cell differentiation,
transformation and proliferation. We hypothesized that angiotensin II (Ang II), glucagon and
high glucose (HG) induce ERK 1/2 phosphorylation in cultured rat glomerular mesangial cells
via cross-talk between their signaling pathways. Ang II (1 nM), glucagon (1 nM) and high
glucose (HG, 25 mM) induced ERK 1/2 phosphorylation (Ang II: 174 ± 16%; glucagon: 214
± 14%; and HG: 190 ± 15%; p < 0.01 vs. control), and all responses were inhibited by the
AT 1 receptor blocker losartan (Ang II + losartan: 62 ± 21%; glucagon + losartan: 77 ± 14%;
and HG ± losartan: 98 ± 10%; p < 0.01 vs. Ang II, glucagon or HG). Pretreatment of
mesangial cells with a phospholipase C (PLC) inhibitor U73122 (10 -6 M), a cAMP-dependent
protein kinase A (PKA) inhibitor H89 (10 -6 M), or a phosphatidylinositol 3-kinase (PI 3kinase)
inhibitor wortmannin (10 -6 M), all markedly attenuated phosphorylation of ERK 1/2
induced by Ang II (Ang II + U73122: 111 ± 13%; Ang II + H89: 86 ± 10%; and Ang II +
wortmannin: 114 ± 8%; p < 0.01 vs. Ang II), glucagon (glucagons + U73122: 109 ± 15%;
glucagon + H89: 113 ± 16%; and glucagons + wortmannin: 115 ± 9%; p < 0.01 vs. glucagon)
and HG (HG + U73122: 90 ±18%; HG + H89: 117 ± 7%; and HG + wortmannin: 139 ± 11%;
p < 0.01 vs. HG). These results suggest that AT 1 receptor-activated PLC, cAMP-dependent
PKA and PI 3-kinase signaling pathways mediate Ang II-, glucagon- and hyperglycemiainduced
MAP kinases ERK 1/2 phosphorylation in glomerular mesangial cells. Parts of this
work were supported by a grant from National Institutes of Health (RO1DK067299).
Addendum: A list of recent publications
Widdop, R.E., X.C. Li, and B. Jarrott. Regional haemodynamic effects of AT1 angiotensin II
receptor antagonist, CV-11974, in conscious rats. Blood Press. 3 (Suppl 5): 15-20, 1994.
Li, X.C., and R.E. Widdop. Regional hemodynamic effects of angiotensin II AT1 receptor
antagonist, CV-11974, in conscious renal hypertensive rats. Hypertension 26: 989-997, 1995.
Li, X.C., and R.E. Widdop. Angiotensin type 1 receptor antagonists CV-11974 and EXP 3174
cause selective renal vasodilatation in conscious spontaneously hypertensive rats. Clin. Sci.
91: 147-154, 1996.
Widdop, R.E., and X.C. Li. A simple versatile method for measuring tail cuff systolic blood
pressure in conscious rats. Clin. Sci. 93: 191-194, 1997.
Li, X.C., P.M. Beart, A.M. James, N.M. Nicole, and R.E. Widdop. Type I and II metabotropic
receptor agonists and antagonists evoke cardiovascular effects after intrathecal administration
in conscious rats. Br. J. Pharmacol. 128: 823-829, 1999.
Paull, J.R.A., X.C. Li, D.B. Sampey, and R.E. Widdop. Pharmacodynamic contribution to the
vasodilator effect of chronic AT1 receptor blockade in SHR. Hypertension 37: 91-98, 2001.
Rey, F.E., X.C. Li, O.A. Carretero, J.L. Garvin, and P.J. Pagano. Perivascular superoxide
anion contributes to impairment of endothelium-dependent relaxation. Role of gp91phox.
Circulation 106: 2497-2502, 2002.
Li, X.C., and R.E. Widdop. AT2 receptor-mediated vasodilatation is unmasked by AT1
receptor blockade in conscious SHR. Br. J. Pharmacol. 142: 821-830, 2004.
- 65 -

Li, X.C., M.S. Karadsheh, P.M. Jenkins, and J.A. Stitzel. Genetic correlation between the
free-choice oral consumption of nicotine and alcohol in C57BL/6J x C3H/HeJ F2 intercross
mice. Behav. Brain Res. 157: 79-90, 2005.
Li, X.C., M.S. Karadsheh, P.M. Jenkins, and J.A. Stitzel. Chromosomal loci that influence
free-choice nicotine consumption in C57BL/6J x C3H/HeJ F2 intercross mice. Brain Res.
(Invited for revision)
Li, X.C., Shao, Y., Ohishi, M., Campbell, D.J., and Zhuo, J.L. AT1 receptor-activated
signaling mediates angiotensin IV-induced responses in renal microvascular and glomerular
mesangial cells. Am. J. Physiol. Renal Physiol. 2005 (in press)
Zhuo, J.L., Li, X.C., Garvin, J.L., Navar, L.G., and Carretero, O.A. Intracellular angiotensin II
induces cytosolic Ca2+ mobilization by stimulating intracellular AT1 receptors in proximal
tubule cells. Am. J. Physiol. Renal Physiol. 2005 (in press).
Zhuo, J.L., Carretero, O.A., Peng, H., Li, X.C., Regoli, D., Neugebauer, W., and Rhaleb, N-R.
Characterization of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) receptor binding sites
using 125I-Hpp-Aca-SDKP in rat cardiac fibroblasts. Hypertension 2005 (invited for
revision)
Li, X.C., Carretero, O.A., Navar, L.G., and Jia L. Zhuo. AT1A receptor-mediated
accumulation of extracellular angiotensin II in proximal tubule cells: role of cytoskeleton
microtubules and tyrosine phosphatase. Am. J. Physiol. Renal Physiol. 2005 (Invited for
revision, F-00405, 2005)
Li, X.C., Carretero, O.A., Shao, Y. and Zhuo, J.L. Glucagon receptor-mediated MAP kinase
ERK 1/2 phosphorylation in mesangial cells: role of protein kinase A and phospholipase C
signaling. Hypertension (2005) (In Press)
- 66 -

Multiple role of histamine H 1 receptor-PKC-MAPK signalling pathway in histaminestimulated
nerve growth factor secretion from glial cells
Metoda Lipnik-Stangelj
Department of Pharmacology and Experimental Toxicology, Faculty of Medicine,
University of Ljubljana, Slovenia. E-mail: metoda.lipnik-stangelj@mf.uni-lj.si
Histamine is a potent stimulator of nerve growth factor (NGF) synthesis and secretion which
is essential for differentiation, regeneration and survival of neurons. The main signalling
pathway, involved in this process, includes histamine-H 1 receptor (H1R)-activated stimulation
of Ca 2+ -dependent protein-kinase C (PKC) and mitogen-activated protein-kinase (MAPK).
Several other mediators contribute to the regulation of NGF secretion, and there is intense
crosstalk between them. IL-1beta and IL-6 also showed strong influence on NGF secretion.
Although interactions between histamine, IL-1beta and IL-6 are common in different
physiological and pathological responses, they remained unclear in the regulation of NGF
release from glial cells, so far. In the present study, we investigated interactions between
histamine, IL-1beta and IL-6 in the regulation of NGF secretion from astroglial cells in
primary culture, and the role of H1R-PKC-MAPK signalling pathway in this process.
Histamine, IL-1beta or IL-6, all showed dose- and time-dependent stimulation of NGF
secretion from cultured astrocytes. Concomitant treatment of the cells with both, histamine
and IL-1beta, significantly enhanced NGF secretion after 24 hours of incubation, in
comparison to the treatment with either histamine or IL-1beta alone. The effect was dosedependent
and additive, and was effectively blocked by H1R antagonists/inverse agonists
(mepyramine and triprolidine), PKC inhibitors (GF 109203X and Go 6976 which is Ca 2+
dependent, PKC-alpha isoform selective inhibitor) and MAPK kinase inhibitor (PD 98059),
but they have no effect on NGF secretion, stimulated by IL-1beta alone. Simultaneous
treatment of the cells with histamine and IL-6 did not show any interaction in the stimulation
of NGF secretion after 24 hours of incubation. On the contrary, significant enhancement of
NGF secretion, in comparison to the treatment with either histamine or IL-6 alone, occurred
after 48 hours of incubation, which indicates long-lasting interactions between histamine and
IL-6. Using H1R antagonists, PKC inhibitors and MAPK kinase inhibitor they were also able
to effectively block the enhancement of NGF secretion after 48 hours of incubation. Our
study suggests that activation of histamine H1R-Ca 2+ -dependent PKC-MAPK signalling
pathway plays a crucial role not only in direct stimulation of NGF secretion by histamine, but
also in indirect stimulation, via different types of interactions between histamine, IL-1beta
and IL-6. Thus, H1R could be important therapeutic target in the treatment of certain
neurodegenerative diseases.
1. Lipnik-!tangelj M, "arman-Kr#an M. Histamine and IL-6 interaction in the stimulation of
nerve growth factor secretion from cultured astrocytes. Inflamm Res 2005; 54(Suppl 1):S36-7.
2. Lipnik-!tangelj M, "arman-Kr#an M. Histamine-stimulated nerve growth factor secretion
from cultured astrocytes is blocked by protein kinase C inhibitors. Inflamm Res 2004; 53:
S57-8.
3. Lipnik-!tangelj M, "arman-Kr#an M. Activation of histamine H1-receptor enhances
neurotrophic factor secretion from cultured astrocytes. Inflamm Res 2004; 53: 245-52.
4. Lipnik-!tangelj M, "arman-Kr#an M. Pharmacological modulation of NGF secretion from rat
cortical astrocytes in primary culture by histamine H1-H2-and-H3-receptor ligands.
Pharmacologist 2002; 44(2 Suppl 1):A110.
5. Lipnik-!tangelj M, Juri$ DM, "arman-Kr#an M. Histamine-induced synthesis and secretion
of nerve growth factor from astrocytes. Inflamm Res 1998; 47(Suppl 1):S34-5.
- 67 -

Loss of the Antiproliferative Gene BTG2 in Estrogen Receptor Positive Breast
Carcinoma is Associated with Tumor Grade and Overexpression of Cyclin D1 Protein
Hirofumi Kawakubo, Elena Brachtel, Paul Walden and Shyamala Maheswaran
Department of Surgical Oncology, Massashusetts General Hospital, Boston, MA 02114
The B-cell Translocation Gene-2 (BTG2) is present in the epithelium of many tissues
including the mammary gland where its expression is regulated during glandular proliferation
and differentiation in pregnancy. Estrogen and progestin suppress BTG2 expression
suggesting that these steroids, which stimulate proliferation and lobuloalveolar development
of mammary epithelial cells, may down-regulate BTG2 in the mammary gland. Expression
analysis of BTG2 protein in breast cancer revealed the loss of nuclear expression in 46% of
tumors whereas it was readily detectable in the nuclei of adjacent normal glands. Loss of
BTG2 in estrogen receptor positive breast tumors correlated significantly with increased
histological grade and tumor size. Consistent with its ability to inhibit cyclin D1 transcription,
suppression of BTG2 mRNA in the mammary gland during gestation, and by estrogen and
progestin correlated with stimulation of cyclin D1. In estrogen receptor positive breast
carcinomas, loss of nuclear BTG2 expression demonstrated a significant correlation with
cyclin D1 overexpression suggesting that BTG2 may be a factor involved deregulating cyclin
D1 expression in breast cancer. Moreover, cyclin D1 reversed BTG2-mediated inhibition of
breast cancer cell growth indicating a functional antagonism between the two proteins. Thus
coordinated inhibition of BTG2 and enhancement of cyclin D1 would constitute a mechanism
to increase estrogen-responsiveness and proliferation in breast cancer cells.
1. Kawakubo, H., Carey, J. L., Brachtel, E., Gupta, V., Green, J. E., Walden, P. D., and Maheswaran, S. (2004).
Expression of the NF-kappaB-responsive gene BTG2 is aberrantly regulated in breast cancer. Oncogene 23,
8310-8319.
2. Kawakubo, H., Brachtel, E., Kish, J., Muzikansky, A., Gupta, V., Walden, P. D., and Maheswaran, S. (2005).
Loss of BTG2 in estrogen receptor positive breast carcinoma is associated with tumor grade and overexpression
of cyclin D1 protein. Oncogene.
3. Hoshiya, Y., Gupta, V., Kawakubo, H., Brachtel, E., Carey, J. L., Sasur, L., Scott, A., Donahoe, P. K., and
Maheswaran, S. (2003a). Mullerian inhibiting substance promotes interferon gamma-induced gene expression
and apoptosis in breast cancer cells. J Biol Chem 278, 51703-51712.
4. Gupta, V., Carey, J. L., Kawakubo, H., Muzikansky, A., Green, J. E., Donahoe, P. K., MacLaughlin, D. T.,
and Maheswaran, S. (2005). Mullerian inhibiting substance suppresses tumor growth in the C3(1)T antigen
transgenic mouse mammary carcinoma model. Proc Natl Acad Sci U S A 102, 3219-3224.
5. Segev, D. L., Ha, T. U., Tran, T. T., Kenneally, M., Harkin, P., Jung, M., MacLaughlin, D. T.,
Donahoe, P. K., and Maheswaran, S. (2000). Mullerian inhibiting substance inhibits breast cancer cell growth
through an NFkappa B-mediated pathway. J Biol Chem 275, 28371-28379.
6. Segev, D. L., Hoshiya, Y., Hoshiya, M., Tran, T. T., Carey, J. L., Stephen, A. E., MacLaughlin, D. T.,
Donahoe, P. K., and Maheswaran, S. (2002). Mullerian-inhibiting substance regulates NF-kappa B signaling in
the prostate in vitro and in vivo. Proc Natl Acad Sci U S A 99, 239-244.
7. Segev, D. L., Hoshiya, Y., Stephen, A. E., Hoshiya, M., Tran, T. T., MacLaughlin, D. T., Donahoe, P. K., and
Maheswaran, S. (2001). Mullerian inhibiting substance regulates NFkappaB signaling and growth of mammary
epithelial cells in vivo. J Biol Chem 276, 26799-26806
- 68 -

PI3K Targeting by the Beta-GBP Cytokine. Mitogenic Input and Therapeutic Response
Livio Mallucci, and Valerie Wells
Cell Signaling Laboratory, Pharmaceutical Sciences Research Division, King's College
London, Franklin Wilkins Building, 150 Stamford Street, London SE1 9NH, UK. Email:
livio.mallucci@kcl.ac.uk
Beta-GBP (beta-galactoside binding protein), a newly identified antiproliferative cytokine
produced by activated CD4 + and CD8 + T cells, can enforce programmed cell death in cancer
cells without harming normal cells. We show that the prime target of beta-GBP activated
signaling is the p110 catalytic subunit of PI3 kinase. We find that PI3 kinase has a permissive
role in controlling cell cycle and survival signaling and that high mitogenic input, whether
endogenous or enforced, facilitates beta-GBP induced apoptosis via inhibition of PI3 kinase
activity and consequent inhibition of Ras-ERK and Akt/protein kinase B signaling.
References
Mallucci L and Wells V (2005) beta-GBP: potential role in cancer therapy. Curr. Opin.
Investig. Drugs 6; 1228-1233.
Ravatn R et al. (2005) Circumventing multidrug resistance in cancer by beta-galactoside
binding protein, an antiproliferative cytokine. Cancer Res. 65; 1631-1634.
Mallucci L et al. (2003) Turning cell cycle controller genes into cancer drugs. A role for an
antiproliferative cytokine (beta-GBP). Biochem. Pharmacol. 66; 1563-1569.
- 69 -

Increased EGF Receptor expression and activity is induced by deregulated N-Ras and
may provide a therapeutic target in NF1 neurofibrosarcoma
Joshua T. Dilworth, Janice M. Kraniak, Michael A. Tainsky, John J. Reiners, Jr., and
Raymond R. Mattingly
Department of Pharmacology and Karmanos Cancer Institute, Wayne State University,
Detroit, Michigan 48201, U.S.A. Email: r.mattingly@wayne.edu
Neurofibromatosis Type 1 (NF1) is the most common inherited cancer predisposition
syndrome. NF1 is caused by functional loss of neurofibromin (Nf1), which compromises Ras
inactivation and drives deregulated Ras-dependent signaling pathways. We have investigated
a panel of neurofibrosarcoma-derived cell lines and have previously demonstrated that two
Nf1-deficient lines have increased basal N-Ras and ERK MAPK activation, relative to a
control line that is wild-type for Nf1. We now show that Nf1-deficient cells have an increased
amount and activity of the epidermal growth factor receptor (EGFR) and demonstrate a
hypersensitive and prolonged response to EGF stimulation. Our studies indicate that EGFR
expression is modulated as a result downstream of Ras signaling. Maintenance of elevated
EGFR expression depends on serum stimulation and the MAPK pathway. Furthermore, using
tetracycline-repressible expression vectors, we show that expression of constitutively active
N- or K-Ras but not H-Ras is sufficient to increase EGFR expression. Inhibition of the EGFR
causes a selective reduction in the proliferation of Nf1-deficient cells. Our research proposes
a mechanism to explain the clinical observation that malignant Schwann cells from NF1
patients overexpress the EGFR. In addition, we provide evidence that supports the EGFR as a
potential pharmacological target in the treatment of this disease.
DAMD170310182
Recent papers ;
S. Beqaj, S. Jakkaraju, R.R. Mattingly, D. Pan & L. Schuger. High RhoA activity maintains
the undifferentiated mesenchymal phenotype, whereas RhoA down-regulation by laminin-2
induces smooth muscle myogenesis. J. Cell Biol. 156: 893-903 (2002)
V.G. Keshamouni, R.R. Mattingly & K.B. Reddy. Mechanism of 17-#-estradiol-induced
ERK1/2 activation in breast cancer cells: A role for HER2 and PKC- . J. Biol. Chem. 277:
22558-22565 (2002)
R.R. Mattingly, R.A. Gibbs, R.E. Menard & J.J. Reiners Jr.. Potent suppression of the
proliferation of A10 vascular smooth muscle cells by combined treatment with lovastatin and
3-allylfarnesol, an inhibitor of protein farnesyltransferase. J. Pharmacol. Exp. Ther. 303: 74 -
81 (2002)
H. Yang, D. Cooley, Q. Ge, R. Andrade & R.R. Mattingly. Phosphorylation of the Ras-
GRF1 exchange factor at Serine-916/898 reveals activation of Ras signaling in the cerebral
cortex. J. Biol. Chem. 278: 13278-13285 (2003)
R.E. Menard & R.R. Mattingly. Cell surface receptors activate p21-activated protein kinase
I (PAKI) via multiple Ras and PI3-kinase-dependent pathways. Cell. Signal. 15: 1099-1109
(2003)
R.E. Menard & R.R. Mattingly. Gbetagamma subunits stimulate p21-activated kinase1
(PAK1) through activation of PI3-kinase and Akt but act independently of Rac1/Cdc42.
FEBS Lett. 556: 187-192 (2004)
- 70 -

D.P. Chopra, R.E. Menard, J. Januszewski & R.R. Mattingly. TNF-alpha mediated
apoptosis in normal human prostate epithelial cells and tumor cell lines. Cancer Lett. 203:
145-154 (2004)
D.R. Yingst, K.J. Massey, N.F. Rossi, M.J. Mohanty & R.R. Mattingly. Angiotensin II
directly stimulates activity and alters phosphorylation of Na,K-ATPase in rat proximal tubule
with a rapid time course. Am. J. Physiol. 287: F713-F721 (2004)
J.H. Norum, T. Methi, R.R. Mattingly & F.O. Levy. Endogenous expression and PKAdependent
phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in HEK293
cells. FEBS J. (formerly Eur. J. Biochem.) 272: 2304-2316 (2005)
R.E. Menard & R.R. Mattingly. Active p21-activated protein kinase I (PAKI) rescues
MCF10A breast epithelial cells from undergoing anoikis. Neoplasia 7: 638-645 (2005)
R.R. Mattingly, J.A. Kraniak, J.T. Dilworth, P. Mathieu, B. Bealmear, J.E. Nowak, J.A.
Benjamins, M.A. Tainsky & J.J. Reiners, Jr. The MEK inhibitor PD184352/CI-1040
selectively induces apoptosis in malignant schwannoma cell lines. J. Pharmacol. Exp. Ther. In
press (2005)
S.E. Ziemba, R.R. Mattingly, M.J. McCabe Jr & A.J. Rosenpire. Inorganic mercury inhibits
the activation of LAT in T cell receptor-mediated signal transduction. Toxicol. Sci. In press
(2005)
H. Yang & R.R. Mattingly. The Ras-GRF1 exchange factor coordinates activation of H–Ras
and Rac1 to control neuronal morphology. In revision for Mol. Biol. Cell (2005)
- 71 -

Pharmacological inhibitors of DISEASE-RELEVANT CYCLIN-dEPENDENT
PROTEIN KINASES (CDks) and GLYCOGEN SYNTHASE KINASE -3 (GSK-3)
Laurent MEIJER
Cell Cycle Laboratory, Station Biologique de Roscoff, C.N.R.S., BP 74, 29682
ROSCOFF cedex, Bretagne, France. E-mail: meijer@sb-roscoff.fr
(http://www.sb-roscoff.fr/CyCell/)
Phosphorylation represents one the most common post-translational mechanism used by cells
to regulate their enzymatic and structural proteins. Frequent alteration of protein
phosphorylation in human disease is the reason for an exponentially growing investment in
the optimization and therapeutic evaluation of small molecular weight, pharmacological
inhibitors of protein kinases, the enzymes responsible for protein phosphorylation. It is
estimated that nowadays 30-35 % of drug discovery programs in the pharmaceutical industry
target a protein kinase! Currently 55 kinase inhibitors are undergoing clinical evaluation
against diseases such as cancers, inflammation, diabetes, neurodegeneration.
Among the 518 human protein kinases we have selected a few disease-relevant kinases to
screen for small molecular weight inhibitors. Among these, cyclin-dependent kinases (CDKs)
regulate the cell division cycle, apoptosis, transcription, differentiation, nervous system
functions, and GSK-3, an essential element of the Wnt signaling pathway, is involved in
multiple physiological processes including cell cycle regulation by controlling the levels of
cyclin D1 and -catenin, insulin action on glycogen synthesis, axonal outgrowth, neuronal
cell death. Both families of enzymes regulate -amyloid formation and tau
hyperphosphorylation, two hallmarks of Alzheimer’s disease. Inhibitors of these kinases have
a strong potential to treat cancers, neurodegenerative diseases (Alzheimer’s, Parkinson’s,
Nieman-Pick type III, stroke, etc…), unicellular parasites. Some of our early CDK/GSK-3
inhibitors have reached the pre-clinical and clinical stages of pharmaceutical evaluation. For
instance, roscovitine (CYC202, Seliciclib), is currently undergoing phase 2 clinical trials
against leukaemia, lung and breast cancers, and phase 1 trials against various kidney diseases.
It is undergoing pre-clinical animal evaluation against Alzheimer’s disease stroke and
Niemann-Pick’s disease type III. Several other families of kinase inhibitors are currently
being developed in the laboratory, a special focus on the bis-indole indirubins will be
provided.
Over a hundred CDK inhibitors [1] and about thirty GSK-3 inhibitors [2] have been
identified, among which more than forty have been co-crystallized with CDK2 or CDK5 and
four with GSK-3. These co-crystal structures are extremely helpful to design further
derivatives with increased potency and selectivity. These kinase inhibitors all target the ATPbinding
pocket of the catalytic site of their targets. The actual selectivity of most compounds,
and thus the underlying mechanism of their cellular effects, is poorly known. We have
developed affinity chromatography using immobilized inhibitors as a straightforward
approach to identify the actual targets of kinase inhibitors [3,4]. Results show that although
some compounds are quite selective, single target products are very unlikely to be discovered.
This may in fact turn out to be an advantage as cells are unlikely to develop resistance to
multiple target drugs.
[1] Knockaert, M., Greengard, P. and Meijer, L., 2002. Pharmacological inhibitors of cyclindependent
kinases. Trends Pharmacol. Sci. 23, 417-425.
- 72 -

[2] Meijer, L., Flajolet, M. and Greengard, P., 2004. Pharmacological inhibitors of glycogen
synthase kinase-3. Trends Pharmacol. Sci. 25, 471-480.
[3] Knockaert, M. and Meijer, L., 2002. Identifying in vivo targets of cyclin-dependent kinase
inhibitors by affinity chromatography. Biochem. Pharmacol. 64, 819-825.
[4] Bach, S., Knockaert, M., Lozach, O., Reinhardt, J., Baratte, B., Schmitt, S., Coburn, S.P.,
Tang, L., Jiang. T., Liang, D.C., Galons, H., Dierick, J.F., Totzke, F., Schächtele, C., Lerman,
A.S., Carnero, A., Wan, Y., Gray, N. and Meijer, L., 2005. Roscovitine targets: protein
kinases and pyridoxal kinase. J. Biol. Chem. 280, 31208-31219.
- 73 -

Real-time analysis of Jak/STAT signal transduction in single cells.
Andreas Herrmann 1 , Michael Vogt 1 , Martin Mönnigmann 2 , Bernd Giese 1 , Michael
Sommerauer 1 , Peter C. Heinrich 1 , Gerhard Müller-Newen 1
1 Institut für Biochemie, Universitätsklinikum der RWTH Aachen, Pauwelsstraße 30,
52057 Aachen, Germany. e-mail: mueller-newen@rwth-aachen.de
2 Lehrstuhl für Prozesstechnik, RWTH Aachen, 52056 Aachen, Germany
The Jak/STAT signal transduction pathway plays a central role in acute and chronic
inflammation. The pro-inflammatory cytokine interleukin-6 (IL-6) signals through the
cytokine receptor gp130. Upon activation, the receptor-associated tyrosine kinases of the
Janus kinase (Jak) family phosphorylate STAT transcription factors. STAT3 (signal
transducer and activator of transcription 3) is preferentially activated in response to IL-6.
Activated STAT3 translocates into the nucleus where it induces target genes that exert
multiple functions in inflammation and carcinogenesis.
We have tagged the cytokine IL-6, its receptor gp130 and the transcription factor STAT3 with
different fluorescent proteins (yellow fluorescent protein (YFP) or cyan fluorescent protein
(CFP)). These fusion proteins enabled us to study ligand-binding and receptor dimerization
(Giese et al. (2005) J. Cell. Sci. 118, 5129-40) as well as nuclear translocation of STAT3
(Pranada et al. (2004) J. Biol. Chem. 279, 15114-23) in single cells by the use of confocal
laser-scanning microscopy and advanced photo-bleaching techniques. We demonstrated that
non-phosphorylated STAT3 constitutively shuttles between the cytoplasm and the nucleus.
Persistent activation of STAT3 is observed in chronic inflammation and cancer. To analyze
persistent activated STAT3 we cotransfected double labelled STAT3-CFP-YFP with v-Src
resulting in constitutive tyrosine phosphorylation and nuclear accumulation of the fluorescent
transcription factor. By bleaching selectively the YFP moiety of STAT3-CFP-YFP in one
cellular compartment and by monitoring the distribution of the CFP and YFP fluorescence
over time with high spatial resolution, we show that persistently activated STAT3 shuttles
between cytoplasm and nucleus. Computational evaluation of the data by model-based
parameter estimations revealed that activated STAT3 shuttles more rapidly than non-activated
STAT3. We propose that inhibition of nucleocytoplasmic shuttling of persistently activated
STAT proteins is a new target for the treatment of chronic inflammation and cancer.
List of recent papers
original papers 2003 -2005 (a selection)
[1] Ancey, C., Küster, A., Haan, S., Herrmann, A., Heinrich, P. C., and Müller-Newen, G. (2003)
A fusion protein of the gp130 and interleukin-6Ra ligand-binding domains acts as a potent interleukin-6
inhibitor.
J. Biol. Chem. 278, 16968-16972
[2] Giese, B., Au-Yeung, C.-K., Herrmann, A., Diefenbach, S., Haan, C., Küster, A., Wortmann, S. B.,
Roderburg, C., Heinrich, P. C., Behrmann, I., and Müller-Newen, G. (2003)
Long-term association of the cytokine receptor gp130 and the Janus kinase Jak1 revealed by FRAP analysis.
J. Biol. Chem. 278, 39205-39213
[3] Haug, K., Warnstedt, M., Alekov, A. K., Sander, T., Ramirez, A., Poser, B., Maljevic, S., Hebeisen, S.,
Kubisch, C., Rebstock, J., Horvath, S., Hallmann, K., Dullinger, J. S., Rau, B., Haverkamp, F., Beyenburg, S.,
Schulz, H., Janz, D., Giese, B., Müller-Newen, G., Propping, P., Elger, C. E., Fahlke, C., Lerche, H., and Heils,
A. (2003)
Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized
epilepsies.
Nat. Genet. 33, 527-532
- 74 -

[4] Niemand, C., Nimmesgern, A., Haan, S., Fischer, P., Schaper, F., Rossaint, R., Heinrich, P. C., and
Müller-Newen, G. (2003)
Activation of STAT3 by IL-6 and IL-10 in primary human macrophages is differentially modulated by SOCS3.
J. Immunol. 170, 3263-3272
[5] de Graaf, K., Hekermann, P., Spelten, O., Herrmann, A., Packman, L. C., Büssow, K., Müller-Newen,
G., and Becker, W. (2004)
Characterization of cyclin L2, a novel cyclin with an arginine/serine-rich (R/S) domain: Phosphorylation by
Dyrk1A and colocalization with splicing factors.
J. Biol. Chem. 279, 4612-4624
[6] Krüger, T., Benke, D., Eitner, F., Lang, A., Wirtz, M., Hamilton-Williams, E. E., Engel, D., Giese, B.,
Müller-Newen, G., Floege, J., and Kurts, C (2004)
Identification and functional characterization of dendrititc cells in the healthy murine kidney and in experimental
glomerulonephritis.
J. Am. Soc. Nephrol. 15, 613-621
[7] Herrmann, A., Sommer, U., Pranada, A. L., Giese, B., Küster, A., Haan, S., Becker, W., Heinrich, P. C.,
and Müller-Newen, G. (2004)
STAT3 is enriched in nuclear bodies.
J. Cell Sci. 117, 339-349
[8] Hebeisen, S., Biela, A., Giese, B., Müller-Newen, G., Hidalgo, P., and Fahlke, C. (2004)
The role of the C-terminus in ClC chloride channel function.
J. Biol. Chem. 279, 13140-13147
[9] Behrmann, I., Smyczek, T., Heinrich, P. C., Schmitz-Van De Leur, H., Komyod, W., Giese, B., Müller-
Newen, G., Haan, S., Haan, C. (2004)
Jak subcellular localisation revisited: The exclusive membrane-localisation of endogenous Janus Kinase 1 by
cytokine receptor interaction uncovers the Jak/receptor complex to be equivalent to a receptor tyrosine kinase.
J. Biol. Chem. 279, 34586-35493
[10] Selander, K. S., Li, L., L., W., Merrell, M., Dahmen, H., Heinrich, P. C., Müller-Newen, G., and Harris,
K. W. (2004)
Inhibition of gp130 signaling in breast cancer blocks constitutive activation of STAT3 and inhibits in vivo
malignancy.
Cancer Res. 64, 6924-6933
[11] Pranada, A. L., Metz, S., Herrmann, A., Heinrich, P. C., and Müller-Newen, G. (2004)
Real-time analysis of STAT3 nucleocytoplasmic shuttling.
J. Biol. Chem. 279, 15114-15123
[12] Giese, B., Roderburg, C., Sommerauer, M., Wortmann, S. B., Heinrich, P. C. and Müller-Newen, G.
(2005)
Dimerization of the cytokine receptors gp130 and LIFR analysed in single cells.
J. Cell. Sci. 118, 5129-5140
recent reviews (a selection)
[R1] Heinrich, P. C., Behrmann, I., Haan, S., Hermanns, H. M., Müller-Newen, G., and Schaper, F. (2003)
Principles of interleukin (IL)-6-type cytokine signalling and its regulation.
Biochem. J. 374, 1-20
[R2] Müller-Newen, G. (2003)
The cytokine receptor gp130: faithfully promiscuous.
Science STKE 2003, pe40
[R3] Hermanns, H. M., Müller-Newen, G., Heinrich, P. C. and Haan, S. (2005)
Bow to you partner for signaling
Nature Struct. Mol. Biol. 12, 476-478
- 75 -

Transcriptional control of differential glucocorticoid receptor expression: determining
tissue specific expression, and protein isoforms.
Jonathan D. Turner 1 and Claude P. Muller 1,2
1
Institute of Immunology, Laboratoire National de Santé, 20A rue Auguste Lumière, L-
1011, Luxembourg
2
Department of Immunology, Graduate School of Psychobiology, University of Trier, D-
54290, Germany
Email: jonathan.turner@LNS.ETAT.LU and claude.muller@LNS.ETAT.LU
The 5’ UTR of the glucocorticoid receptor plays a key role in determining tissue specific
expression, and protein isoforms. Analysis of the 5’ UTR of the hGR has revealed 11 splice
variants of the hGR exon 1, based on 7 exon 1s, 4 of which (1-D to 1-F and 1-H) were
previously unknown. All of the exon 1 variants have unique splice donor sites and share a
common exon 2 splice acceptor site. Due to an upstream in-frame TGA stop codon the
predicted translation from all splice variants is identical. The four new exon 1s show
remarkable similarity with their rat homologues. Exon 1-D starts and finishes 17- and 36bp
upstream of the corresponding ends of the rat exon 1 4. Exon 1-E is only 6bp longer than its
homologue 1 5. Exon 1-F contains two short inserts of 11- and 6bp when compared to the rat
1 7. 1-H is 18bp longer than the corresponding rat 1 11. In addition to these new exons, we
found that the human exon 1-C occurs as 3 distinct splice variants, covering the region
homologous to the rat exons 1 9 and 1 10. All of the alternative hGR exons 1s presented here
were found to be transcribed in human tissue. The human hippocampus expresses mRNA of
all the exon 1 variants, whilst the expression of the other exon 1s seems to be tissue-specific.
While exon 1-D is only in the hippocampus, exons 1-E and 1-F are also detected in the
immune system, and exon 1-H additionally in the liver, lung and smooth muscle. The 5’
region of the hGR is more complex than previously thought, and we suggest that each of these
untranslated first exons have a distinct proximal promoter region, providing additional depth
to the mechanisms available for tissue specific expression of the hGR isoforms.
- 76 -

Oncogenic signaling by mutant p53
Moshe Oren
Department of Molecular Cell Biology, The Weizmann Institute, Rehovot, Israel
(no abstract provided)
- 77 -

Acute anti-inflammatory properties of statins involve Peroxisome Proliferator-Activated
Receptor-alpha via inhibition of the PKC signalling pathway
Réjane Paumelle 1 , Christophe Blanquart 1 , Olivier Briand 1 , Olivier Barbier 1 , Gaëtane
Woerly 2 Christian Duhem 1 , Frédéric Percevault 1 , Jean-Charles Fruchart 1 , David
Dombrowicz 2 , Corine Glineur 1 and Bart Staels 1,3 .
1 Département d’Athérosclerose, U545 Inserm, Institut Pasteur de Lille and Faculté de
Pharmacie, Université de Lille II, Lille, France. 2 Inserm U547, Institut Pasteur de Lille,
Lille, France. E-mail: Bart.Staels@pasteur-lille.fr
Statins are inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase used in the
prevention of cardiovascular diseases (CVD). In addition to their cholesterol-lowering
activities, statins exert pleiotropic anti-inflammatory effects, which might contribute to their
beneficial effects not only on CVD, but also on lipid-unrelated immune and inflammatory
diseases, such as rheumatoid arthritis, asthma, stroke and transplant rejection. However, the
molecular mechanisms involved in these anti-inflammatory properties of statins are
unresolved. The nuclear receptor Peroxisome Proliferator-Activated Receptor (PPAR)-alpha
that is expressed in inflammation regulatory cell types, such as macrophages, exerts potent
anti-inflammatory activities. In this study we present genetic evidence for a role for this
nuclear receptor in mediating the lipid-independent anti-inflammatory activities of statins in
two in vivo models of acute inflammation. Furthermore, the inhibitory effects of statins on
lipopolysaccharide (LPS)-induced inflammatory response genes were abolished in PPARalpha-deficient
macrophages and neutrophils, two cell types involved in acute inflammatory
responses. Moreover, we provide a molecular mechanism explaining these observations by
showing that simvastatin inhibited PPAR-alpha phosphorylation by LPS-activated PKCalpha.
A constitutive active form of PKC-alpha inhibited NF-kappaB transrepression by
PPAR-alpha whereas simvastatin enhanced transrepression activity of wild-type PPAR-alpha,
but not of PPAR-alpha mutated in its PKC phosphorylation sites. These data indicate that the
acute anti-inflammatory effect of simvastatin occurs via PPAR-alpha by a mechanism
involving inhibition of PKC-alpha inactivation of PPAR-alpha transrepression activity.
PUBLICATIONS
- Tulasne, D., Paumelle, R., Weidner, M., Vandenbunder, B. and Fafeur, V. The
multisubstrate docking site of the MET receptor is dispensable for MET-mediated RAS
signaling and cell scattering. 1999. Mol. Biol. Cell. 10, 551-565.
- Paumelle, R., Tulasne, D., Leroy, C., Coll, J., Vandenbunder, B. and Fafeur, V. Sequential
activation of ERK and repression of JNK by SF/HGF in MDCK epithelial cells. 2000. Mol.
Biol. Cell. 11, 3751-3763.
- Paumelle, R., Tulasne, D., Kherrouche, Z., Plaza, S., Leroy, C., Reveneau, S.,
Vandenbunder, B. And Fafeur, V. Hepatocyte growth factor/scatter factor activates the ETS1
transcription factor by a RAS-RAF-MEK-ERK signaling pathway. 2002. Oncogene. 21:
2309-2319.
- Blanquart, C., Mansouri, R., Paumelle, R., Fruchart, J. C., Staels, B. The protein kinase C
signalling pathway regulates a molecular switch between transactivation and transrepression
- 78 -

activity of the Peroxisome Proliferator-Activated Receptor alpha (PPAR{alpha}). 2004. Mol
Endocrinol.18(8):1906-18.
-Teissier, E., Nohara, A., Chinetti, G., Paumelle, R., Cariou, B., Fruchart, J.C., Brandes, R.,
Shah, A., Staels, B. Peroxisome proliferator-activated receptor induces NADPH oxydase
activity in macrophages leading to the generation of LDL with PPAR activation properties.
2004. Circ. Res. 95(12):1174-82.
- Paumelle, R., Blanquart, C., Briand, O., Barbier, O, Woerly, G, Duhem, C., Percevault, F.,
Fruchart, J. C., Dombrowicz, D., Glineur, C., and Staels, B. Acute anti-inflammatory
properties of statins involve Peroxisome Proliferator-Activated Receptor-alpha via
inhibition of the PKC signalling pathway. In revision in Circ. Res.
- 79 -

Cyclins and HDAC/HATs
Richard Pestell
Lombardi Comprehensive Cancer Center, Georgetown University, Washington, USA
(no abstract provided)
- 80 -

Restauration of SHIP-1 activity in human leukemic cells modifies NF-!B activation
pathway and cellular survival upon oxidative stress treatment
Geoffrey Gloire*, Edith Charlier*, Souad Rhamouni*, Cédric Volanti*, Alain Chariot*,
Christophe Erneux $ and Jacques Piette*
*University of Liège, B-4000 Liège, $ University of Brussels, B-1070 Brussels
Since the discovery that NF-!B 1 could be activated by H 2O 2, several laboratories put a lot of
efforts into dissecting the molecular mechanisms underlying this activation, particularly in
Jurkat leukemic cells. These works led to the observation that NF-!B activation by H 2O 2
involved tyrosine phosphorylation of the inhibitor I!B" through an IKK-independent
mechanism. In the present work, we investigated with more details NF-!B redox regulation
in human leukemic cells. By using different cell lines (CEM, Jurkat and Jurkat JR), we clearly
showed that NF-!B activation by H 2O 2 is cell-type dependent: it activates NF-!B through
tyrosine phosphorylation of I!B" in Jurkat cells, but induces an IKK-mediated IkB"
phosphorylation on serines 32 and 36 in CEM and Jurkat JR cells. We showed that this H 2O 2induced
IKK activation in CEM and Jurkat JR cells is mediated by SHIP-1, a lipid
phosphatase which is absent in Jurkat cells. Indeed, the genetic complementation of SHIP-1 in
Jurkat cells made them shifting to an IKK-dependent mechanism upon oxidative stress
stimulation. The phosphatase domain of SHIP-1 is crucial in that process, since the
complementation of Jurkat cells with a mutant of SHIP-1 lacking catalytic activity failed to
mediate IKK activation upon H 2O 2 treatment. We also showed that Jurkat expressing SHIP-1
are much more resistant to H 2O 2-induced apoptosis than the parental cells, suggesting that
SHIP-1 have an important role in protecting leukemic cells from ROS-induced apoptosis.
Overall, our study highlight a novel role for SHIP-1 in leukemic cells ROS responses in terms
of signal transduction pathways and apoptosis resistance, which can be of interest in
improving ROS-mediated chemotherapies.
- 81 -

Live and let die by 73 - mechanisms of degradation and chemoresistance
Michael J. Pinkoski
MRC Toxicology Unit
Leicester, United Kingdom
p73 belongs to a family of transcription factors, including p53 and p63, that mediates
responses to DNA damage and cellular stress by inducing DNA repair, cell cycle arrest and
apoptosis. p73 is expressed as two isoforms, TA and DN, that have opposing biological
effects with DNp73 has a dominant negative/repressive effect on TAp73. Therefore, TA/DN
ratios dictate p73-dependent cellular responses to such insults as genotoxic chemotherapeutic
agents. In fact, DNp73 is associated with reduced survival in hepatocellular carcinoma (HCC)
patients and is therefore an indicator of a negative prognosis. Mechanistically, p73 is capable
of engaging both intrinsic and extrinsic apoptosis pathways. TAp73 induces transcription of
CD95 and increased sensitivity to CD95-induced apoptosis. TAp73 also induces expression of
PUMA, Noxa and Scotin to engage death pathways via the mitochondria and apoptosome.
Unlike p53, p73 mutations are rarely observed in human cancers, however, p73 activity is
influenced by protein stability. p73 protein degradation is regulated by the E3 ubiquitin ligase,
Itch, whereas stabilization of p73 protein is positively regulated by the nuclear body protein,
PML. Both TA and DNp73 proteins are degraded in an Itch dependent manner, but
interestingly, both Itch and DNp73 are downregulated following genotoxic stress.
Degradation of DNp73 under these conditions is not mediated by Itch. TAp73, is not
decreased following DNA damage, lending further support to the hypothesis TAp73 acts as a
tumour suppressor while DNp73, which can inhibit both TAp73 and p53 would represent an
oncogenic activity. Therefore, in addition to DNp73 as a prognostic indicator, these data
suggest that DNp73 and Itch are potential targets for cancer therapy.
- 82 -

Hypoxia Signaling. Impact on Tumor metabolism, Cell survival and Cell death.
N. M. Mazure. F. Dayan, D. Roux, E. Berra, C. Brahimi-Horn, and J. Pouysségur
Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR 6543,
Centre Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice, France
The function of the Hypoxia-Inducible Factor-1 (HIF-1), the key transcription factor involved
in cellular adaptation to hypoxia is restricted to low oxygen tension (pO 2). As such, this
transcription factor is central in modulating the tumor microenvironment, sensing nutrient
availability and controlling anaerobic glycolysis, intracellular pH, and cell survival.
Degradation and inhibition of the limiting HIF-1" subunit are intimately connected in
normoxia. Hydroxylation of two proline residues by Prolyl Hydroxylase Domain protein 2
(PHD2) earmarks the protein for degradation while hydroxylation of an asparagine residue by
Factor Inhibiting HIF-1 (FIH-1) reduces its transcriptional activity. Indeed, silencing in
normoxia, of either PHD2 or FIH-1, partially induced hypoxic genes, whereas combined
PHD2/FIH-1 silencing generated a full hypoxic gene response. Given the fact that HIF-1"
possesses two transactivation domains (N- and C-TAD), we hypothesized on a possible bifunctional
activity of HIF-1a that could be discriminated by FIH-1, a modifier of the C-TAD.
In human cell lines, engineered to overexpress or silence FIH-1, in response to tetracyclin, we
demonstrate by quantitative RT-PCR that a set of hypoxic genes (CAIX, PHD3, PGKI and
BNIP3) respond differently towards FIH-1 expression. This finding, extended to 30 hypoxiainduced
genes, indicates differential gene expression by the N- and C-TAD in response to the
hypoxic gradient.
We propose that the oxygen-sensitive attenuator FIH-1, together with 2 distinct TADs are
central in setting the gene expression repertoire dictated by the cell pO 2.
In the tumor microenvironment context, we are intrigued by the duality of HIF-1 that can
induce either cell survival or cell death. We will discuss our unpublished work on the
physiological roles played by the HIF-induced pro-apoptotic gene product BNIP3, autophagy
and necrotic cell death.
Ways to exploit this basic knowledge to magnify tumor regression will be presented ?
- 83 -

Alkylating drugs applied in non-cytotoxic doses as a novel compounds targeting
inflammatory signal pathways
Alexander L. Pukhalsky, Galina V. Shmarina, Vladimir A. Alioshkin and Alex
Sabelnikov
Research Centre for Medical Genetics, Moscow 115478, Gabrichevsky Institute of
Epidemilogy and Microbilogy, Moscow 125212, Russia, East Carolina University,
Greenvill, NC 27858, U.S.A. E-mail: osugariver@medgen.ru
Alkylating drugs (ADs) belonging to the nitrogen mustard family are commonly used as
cytostatic and immunosuppressive agents. Our recent in vitro studies demonstrated that in the
case of gradual dose decrease, the number of targets for alkylation in the cell is also reduced
and the drug switches from crucial cytostatic to cell growth modifier. At doses of 0.3 mkg/ml
and lower, the effects of ADs are no longer associated with DNA damage or altered cytokine
production. Instead, the disruption of signal transduction by the IL-2beta and/or TNF-alpha
cell surface receptors is observed. As a result ADs in the doses 100-fold lower than cytostatic
ones are capable to modify lymphocyte activity including the activity of regulatory T cells.
We hypothesized that ADs may have a beneficial effect in the treatment of inflammatory
diseases. Indeed, the application of non-cytotoxic doses of an AD melphalan (MP) reduces
the severity of murine experimental colitis induced by the replacement of drinking water with
5% solution of dextran sulphate sodium (DSS). Daily administration of MP (0.025 mg/kg
body weight) markedly reduced the severity of DSS-colitis as determined by clinical and
quantitative histological criteria. Moreover, the beneficial effect of MP was also shown in
asthmatic patients. Forty-two patients with moderate and severe asthma were enrolled in
randomized placebo-controlled trial: 21 patients were treated with MP (5 daily inhalation of
the drug in the dose of 0.1 mg) and 21 subjects were inhaled with placebo. Improvement of
exercise tolerance as well as a marked reduction of exacerbation severity and requirement for
inhaled beta2-agonists has been found in MP-treated patients. In 60% of these patients
histological and ultrastructural signs of bronchial epithelium regeneration were also revealed.
Thus, ADs at non-cytotoxic concentrations exert beneficial effect both in acute and chronic
inflammatory diseases. Such anti-inflammatory activity is thought to be due to blocking of
signal transduction through various cytokine surface receptors. Consequently, different steps
of inflammatory cascade turn up inhibited.
List of publications
1. Toptygina AP, Pulhalsky AL, Alioshkin VA. Immunoglobulin G subclass profile of antmeasles
response in vaccinated children and in adults with measles history. Clin Diagn Lab
Immunol, 2005; 12: 845-847.
2. Pukhalsky AL, Shmarina GV, Kapranov NI, Kokarovtseva SN, Pukhalskaya DA,
Kashirskaja NJ Anti-inflammatory and immunomodulating effects of clarithromycin in
patients with cystic fibrosis lung disease. Med Inflam 2004; 13:111-117.
3. Pukhalsky AL, Shmarina GV, Bliakher MS, Fedorova IN, Toptygina AP, Fisenko JJ,
Alioshkin VA Cytokine profile after rubella vaccine inoculation: evidence of
immunosuppressive effect of vaccination. Med Inflam 2003, 12(4): 203-207.
4. Sokolov EI, Pukhalsky AL, Tsyplenkova VG, Shevelev VI. Inhalation of ultra-low
doses of alkylating drugs in the treatment of asthma. Pulmonology 2002; 12(3):82-88
(Russian).
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5. Sokolov EI, Zykov KA, Pukhalsky AL, Zykov KA, Shmarina GV, Matko LY, Demidov
YI, Kokarovtseva SN, Shevelev VI. Immunomodulating effects of inhaled alkylating drug
melphalam in asthmatic patients. Pulmonology 2002; 12(5):81-86 (Russian).
6. Pukhalsky AL, Shmarina GV. Stimulatory and protective effects of alkylating agents
applied in ultra-low concentrations. Pharmacology 2001; 62: 129-132.
7. Shmarina GV, Pukhalsky AL, Kokarovtseva SN, Pukhalskaya DP, Kalashnikova EA,
Kapranov NI, Kashirskaja SJ. Improvement of nutrient absorption may enhance systemic
oxidative stress in cystic fibrosis patients. Med Inflam 2001; 10: 61-67.
8. Shmarina GV, Pukhalsky AL, Kokarovtseva SN, Pukhalskaya DP, Shabalova LA,
Kapranov NI, Kashirskaja SJ. Tumor necrosis factor-"/interleukin-10 balance in normal and
cystic fibrosis children. Med Inflam 2001; 10: 191-197.
9. Pukhalsky AL, Kapranov NI, Kalashnikova EA, Shmarina GV, Shabalova LA,
Kokarovtseva SN, Pukhalskaya DA, Kasirskaja NJ, Simonova OI. Inflammatory markers in
cystic fibrosis patients with lung Pseudomonas aeruginosa infection. Med Inflam 1999; 8:
159-167.
10. Pukhalsky AL, Shyian SD, Kalashnikova EA, Shmarina GV, Pukhalskaya DA, Bovin
NV Immunomodulating activities of a natural " 1-acid glycoprotein and its carbohydrate
chains attached to the protein-free polymer. Med Inflam 1998, 7:115-118.
11. Pukhalsky AL, Vikulova VK, Toptygina AP, Lyashko VN. Genetic heterogeneity of
heat shock protein synthesis and the spleen cell sensitivity to antiproliferative effect of
alkylating agents in mice. Bull Exp Biol 1997; 124:561-565.
12. Pukhalsky A, Toptygina A, Khaidukov S. Interleukin-2 receptor # chain as a possible target for low doses
of mafosfamide. Mediators Inflamm 1995; 4:175-180.
13. Pukhalsky AL, Toptygina AP. The comparative study of mechanisms of
antiproliferative effect of mafosfamide and cyclosporin A applied in low doses. Bull Exper
Biol 1993; 115 (5): 514-515.
14. Pukhalsky AL, Toptygina AP, Viktorov VV. Immunosuppressive action of cyclophosphamide in mice:
contribution of some factors to determination of strain differences. Int J Immunopharmac 1993; 15: 509-514.
15. Pukhalsky AL, Toptygina AP, Viktorov VV. Pharmacokinetics of alkylating metabolites
of cyclophosphamide in different strains of mice. Int J Immunopharmac 1990; 12:217-223.
16. Yushkov SF, Pukhalsky AL, Rozenberg IB. The effect of sarcolysin and endoxan on the
resorption of the tadpoles’ tail muscles in metamorphosis. Pharmacol Toxicol 1970; 6:723-
726 (Russian).
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REGULATION OF INFLAMMATION AND GLUCOCORTICOID SIGNALING BY
CURCUMIN AND RESVERATROL
Dr. Irfan Rahman
Department of Environmental Medicine, University of Rochester Medical Center,
Rochester, NY. Email: irfan_rahman@urmc.rochester.edu
Reactive oxygen species (ROS) play a key role in enhancing the inflammation through the
activation NF-kappaB and AP-1 transcription factors, and nuclear histone acetylation and
deacetylation (chromatin remodeling) in various inflammatory diseases. We have recently
shown that oxidative stress enhances lung inflammation via expression of pro-inflammatory
mediators through inhibition of histone deacetylase activity and NF-kappaB transactivation in
monocytes/macrophages and epithelial cells. We also show the antioxidant and/or antiinflammatory
effects of dietary polyphenols (curcumin-diferuloylmethane, a principal
component of turmeric) and resveratrol (a flavanoid found in red wine), in the control of NFkB
activation, upregulation of glutathione biosynthesis gene via Nrf2 activation, scavenging
effect of ROS by inducing glutathione peroxidase activity, chromatin remodeling and
subsequently inflammatory gene regulation in macrophages and lung epithelial cells. We
further demonstrate that these dietary polyphenols restore glucocorticoid functions (antiinflammatory
property) in response to oxidative stress by upregulation of histone deacetylase
activity. The anti-inflammatory property of curcumin and resveratrol may be due to its ability
to induce histone deacetylase activity by reversing the post-translationally modified proteins.
These data provide new information on regulation of inflammatory response by dietary
polyphenols at the molecular level and possibly a way forward to inhibit chronic
inflammatory response in various diseases.
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HOW ANTITOXIC AND ANTICANCER AGENTS MANEUVER PROGRAMMED
AND UNPROGRAMMED CELL DEATH IN VIVO?
Sidhartha D. Ray
Molecular Toxicology Laboratories, Division of Pharmaceutical Scs,
Arnold & Marie Schwartz College of Pharmacy & Health Sciences, Long Island
University, Brooklyn, NY 11201
Most pro-toxic and pro-carcinogenic signals fundamentally share a common platform and use
genome-dependent and genome-independent events to deregulate the intracellular
microenvironment to ultimately articulate macroscopic changes in vivo. Pro-death signals
tend to perturb the level of oxidative stress (deplete glutathione & induce lipid peroxidation),
stimulate mitochondrial pore formation (release Cyt c), tilt antioxidant balance, jeopardize
energy metabolism (drop ATP production) and mishmash ion homeostasis to achieve their
goals. At the molecular level, various cytokines (TNF- ) and cell-death controlling elements
(caspases, bcl-XL, p53) dominate in masterminding programmed or unprogrammed cell
death. Using in vivo models, our decades of research have shown that select single entity
phytochemicals or a combination of phytochemicals can rescue cells from such chaotic
aftermath and propagate antitoxic and anticarcinogenic signals in favor cell survival. Using a
carcinogen and a non-carcinogen, our laboratory has investigated the patterns of programmed
and unprogrammed cell death and their counteraction by a series of phytochemicals
(curcumin, silymarin, hesperidin, rutin) in the liver in vivo. Regulated pre-exposure (days to
weeks) or the continuous presence (months) of these phytochemicals in vivo carefully
propagate target-specific anti-toxic and anti-carcinogenic signals creating a potential sink for
the processes that orchestrate apoptotic/necrotic/apocrotic cell deaths. Although activation or
inhibition of Caspases, select expression of pro and anti-apoptotic genes, modulation of DNA
damage/DNA repair processes and maintenance of pro- and antioxidant balance are the
ultimate key to survival signals, modulation of fundamental biochemical events serve as the
basis in shaping/micromanaging anti-toxic and/or anti-carcinogenic outcomes. This talk will
emphasize local and global changes (of the parameters described above) at the cellular,
organellar, and molecular levels to understand how cytoprotective agents selectively
disseminate anti-toxic and/or anti-carcinogenic signals to trigger specific molecular switches
in vivo prior to hepatotoxic or hepatocarcionogenic reactions and obliterate lethal
consequences.
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Inhibition of histone deacetylase activity as a therapeutic tool in amyotrophic lateral
sclerosis.
Caroline ROUAUX, Irina PANTELEEVA, Frédérique RENE, Jose-Luis GONZALEZ
de AGUILAR, Anne-Laurence BOUTILLIER & Jean-Philippe LOEFFLER
Laboratoire de Signalisation Moléculaire et Neurodégénérescence – INSERM U692 -
Faculté de Médecine - 11, rue Humann - 67085 Strasbourg Cedex FRANCE. Tel.: (+33)
390 24 30 88 Fax: (+33) 390 24 30 65.
Motoneuronal death in the spinal cord is a characteristic of Amyotrophic Lateral Sclerosis
(ALS). Our recent data showed a decrease in histone acetylation levels, as well as in protein
levels of the CREB-Binding Protein (CBP), a specific Histone Acetyl Transferase (HAT), in
motoneurons from the lumbar spinal cord of an in vivo mouse model of ALS (SOD1 G86R
mice) when compared to those of wild type animals. The aim of this study was to investigate
whether preventing the loss of acetylation by blocking histone deacetylases with specific
inhibitors (HDACi) could have an impact on motoneuronal death. Therefore, SOD1 G86R
mice were treated with the HDACi Sodium Valproate (VPA) at 250mg/kg/j, IP. VPA
treatment induced a delay in the onset of the symptoms, an increase in motor performances
and allowed to significantly reduce motoneuronal death. To study the mechanism of action of
this HDACi, we used a cellular model of motoneurons (the NSC34 neuroblastoma X Spinal
cord cell line) in which oxidative-stress induced-death was mimicked with a pulse of
hydrogen peroxide (H 2O 2). H 2O 2-induced motorneuronal death was accompanied by histone
deacetylation and CBP loss. Moreover, this loss resulted from a direct cbp gene repression.
Several HDACi (Trichostatin A: TSA, Sodium Valproate: VPA and Sodium Butyrate: NaBu)
were tested. We found that a long-term treatment with each compound had a significant
protective effect on H 2O 2-induced motoneuronal death, reversing histone acetylation decrease
and promoting cbp gene expression. Consequently, despite the effects of HDACi on the
neuromuscular junction remain to be elucidated, we believe that their beneficial effects in the
ALS model may result from a protection against motoneurons degeneration.
Rouaux et al., 2003, EMBO 22(24):6537-49
Rouaux et al., 2004, Biochem Pharmacol. 2004, 68(6):1157-64.
Supported by AFM and ARS.
- 88 -

Listeria monocytogenes induced Rac1-dependent endothelial histone modification and
cytokine release
Bernd Schmeck*, Wiebke Beermann*, Vincent van Laak*, Bastian Opitz*, Andreas
Hocke*, Julia Eitel*, Trinad Chakraborty+, Gudula Schmidt#, Holger Barth#, Norbert
Suttorp*, and Stefan Hippenstiel*
*) Department of Internal Medicine/Infectious Diseases, Charité – Universitätsmedizin
Berlin, 13353 Berlin, Germany (E-mail: Bernd.Schmeck@charite.de); +) Institute for
Medical Microbiology, Justus-Liebig University of Giessen, 35392 Giessen, Germany; #)
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-
Ludwig-Universitat Freiburg, 79104 Freiburg, Germany.
Infection of endothelial cells by Listeria monocytogenes is an essential step in the
pathogenesis of listeriosis. Small GTPases of the Rho family act as molecular switches in
signal transduction. We tested the hypothesis that Rho GTPases contribute to the regulation of
cytokine expression in Listeria monocytogenes infection.
L. monocytogenes induced release of distinct CC and CXC, as well as Th1 and Th2 cytokines
and growth factors by endothelial cells whereas non-invasive L. innocua did not induce
cytokine release and activated Rac1 as shown by pulldown assay. Cytokine expression was
reduced byinhibition of RhoA, Rac and Cdc42 (Clostridium difficile toxin B (TcdB)), and
Rac1-inhibitor Nsc23766. Activating Rho proteins by Escherichia coli CNF1 increased
Listeria-dependent cytokine expression. We analyzed regulation of IL-8 expression in more
detail: Listeria-induced IL-8 release was reduced by inhibition of RhoA, Rac1 and Cdc42
(TcdB) or Rac1while blocking of RhoA/B/C by Clostridium limosum C3 fusion toxin or Rho
kinase by Y27632 had no effect. Activation of RhoA, Rac and Cdc42 (CNF1), but not of
RhoA alone (CNF Y), enhanced Listeria-dependent IL-8 release. Next, we analyzed histone
modifications at the gene-promoter of IL-8. Inhibition of RhoA, Rac1 and Cdc42 (TcdB) and
Rac1 (Nsc23766) reduced Listeria-related recruitment of NF-kappaB/p65 and RNA
Polymerase II to the IL-8 promoter, as well as Lys-8-acetylation of histone H4 and Ser-
10/Lys-14-phosphorylation/acetylation of histone H3 at the IL-8 gene promoter in HUVEC.
In conclusion, Rac1 contributed to epigenetic regulation of L. moncytogenes-induced
cytokine expression by human endothelial cells.
Recent papers:
1: Opitz B, Puschel A, Beermann W, Hocke AC, Forster S, Schmeck B, van Laak V,
Chakraborty T, Suttorp N, Hippenstiel S. Listeria monocytogenes Activated p38 MAPK and
Induced IL-8 Secretion in a Nucleotide-Binding Oligomerization Domain 1-Dependent
Manner in Endothelial Cells. J Immunol. 2006 Jan 1;176(1):484-90.
2: Brell B, Hippenstiel S, David I, Pries AR, Habazettl H, Schmeck B, Suttorp N,
Temmesfeld-Wollbruck B.
Adrenomedullin treatment abolishes ileal mucosal hypoperfusion induced by Staphylococcus
aureus alpha-toxin--an intravital microscopic study on an isolated rat ileum. Crit Care Med.
2005 Dec;33(12):2810-016.
3: Krull M, Bockstaller P, Wuppermann FN, Klucken AC, Muhling J, Schmeck B, Seybold J,
Walter C, Maass M, Rosseau S, Hegemann JH, Suttorp N, Hippenstiel S. Mechanisms of
Chlamydophila pneumoniae Mediated GM-CSF Release in Human Bronchial Epithelial Cells.
Am J Respir Cell Mol Biol. 2005 Dec 9; [Epub ahead of print]
- 89 -

4: Schmeck B, Huber S, Moog K, Zahlten J, Hocke AC, Opitz B, Hammerschmidt S,
Mitchell TJ, Kracht M, Rosseau S, Suttorp N, Hippenstiel S. Pneumococci induced TLR- and
Rac1-dependent NF-{kappa}B-recruitment to the IL-8 promoter in lung epithelial cells. Am J
Physiol Lung Cell Mol Physiol. 2005 Nov 18; [Epub ahead of print]
5: Schmeck B, Beermann W, van Laak V, Zahlten J, Opitz B, Witzenrath M, Hocke AC,
Chakraborty T, Kracht M, Rosseau S, Suttorp N, Hippenstiel S. Intracellular bacteria
differentially regulated endothelial cytokine release by MAPK-dependent histone
modification. J Immunol. 2005 Sep 1;175(5):2843-50.
6: N'Guessan PD, Schmeck B, Ayim A, Hocke AC, Brell B, Hammerschmidt S, Rosseau S,
Suttorp N, Hippenstiel S. Streptococcus pneumoniae R6x induced p38 MAPK and JNKmediated
caspase-dependent apoptosis in human endothelial cells. Thromb Haemost. 2005
Aug;94(2):295-303. contributed equally
7: Slevogt H, Tiwari KN, Schmeck B, Hocke A, Opitz B, Suttorp N, Seybold J. Adhesion of
Moraxella catarrhalis to human bronchial epithelium characterized by a novel fluorescencebased
assay. Med Microbiol Immunol (Berl). 2005 Jul 30;:1-11 [Epub ahead of print]
8: Brell B, Temmesfeld-Wollbruck B, Altzschner I, Frisch E, Schmeck B, Hocke AC, Suttorp
N, Hippenstiel S.
Adrenomedullin reduces Staphylococcus aureus alpha-toxin-induced rat ileum
microcirculatory damage. Crit Care Med. 2005 Apr;33(4):819-26.
9: Opitz B, Forster S, Hocke AC, Maass M, Schmeck B, Hippenstiel S, Suttorp N, Krull M.
Nod1-mediated endothelial cell activation by Chlamydophila pneumoniae. Circ Res. 2005
Feb 18;96(3):319-26. Epub 2005 Jan 13.
10: Krull M, Kramp J, Petrov T, Klucken AC, Hocke AC, Walter C, Schmeck B, Seybold J,
Maass M, Ludwig S, Kuipers JG, Suttorp N, Hippenstiel S. Differences in cell activation by
Chlamydophila pneumoniae and Chlamydia trachomatis infection in human endothelial cells.
Infect Immun. 2004 Nov;72(11):6615-21.
11: Schmeck B, Zahlten J, Moog K, van Laak V, Huber S, Hocke AC, Opitz B, Hoffmann E,
Kracht M, Zerrahn J, Hammerschmidt S, Rosseau S, Suttorp N, Hippenstiel S. Streptococcus
pneumoniae-induced p38 MAPK-dependent phosphorylation of RelA at the interleukin-8
promotor. J Biol Chem. 2004 Dec 17;279(51):53241-7. Epub 2004 Oct 13.
12: Walter C, Zahlten J, Schmeck B, Schaudinn C, Hippenstiel S, Frisch E, Hocke AC,
Pischon N, Kuramitsu HK, Bernimoulin JP, Suttorp N, Krull M. Porphyromonas gingivalis
strain-dependent activation of human endothelial cells. Infect Immun. 2004 Oct;72(10):5910-
8.
13: Schmeck B, Gross R, N'Guessan PD, Hocke AC, Hammerschmidt S, Mitchell TJ,
Rosseau S, Suttorp N, Hippenstiel S. Streptococcus pneumoniae-induced caspase 6dependent
apoptosis in lung epithelium. Infect Immun. 2004 Sep;72(9):4940-7.
- 90 -

Epigenetic Regulation of Stem Cell Maintenance Genes in Prostate Carcinoma
Michèle J. Hoffmann, Mirko Müller, Rainer Engers*, Wolfgang A. Schulz
Urologische Klinik und Institut für Pathologie*, Heinrich-Heine-Universität Düsseldorf,
Germany
E-mail: michele.hoffmann@uni-duesseldorf.de; muellemi@uni-duesseldorf.de;
engers@med.uni-duesseldorf.de ; wolfgang.schulz@uni-duesseldorf.de;
We have previously reported that hypomethylation of LINE-1 retrotransposons is associated
with the progression of prostate carcinoma. As in other cancers, causes and consequences of
this alteration are not understood. Most likely, retrotransposon hypomethylation in cancer
cells is related to a global change in chromatin structure that interacts with alterations in DNA
methylation in a mutual fashion, i.e. altered expression of chromatin regulator proteins could
alter DNA methylation and/or vice versa. In particular, some genes contributing to the stem
cell character of germ cells and early embryonic cells appear to be controlled by DNA
methylation, but also influence chromatin structure and DNA methylation in turn, e.g. the
BMI1 polycomb gene and CTCFL/BORIS. Altered expression of these genes has been
postulated to occur in many human cancers. We have therefore investigated their expression
and relationship to DNA methylation in prostate cancer.
In primary prostate cancer tissues, expression of polycomb EZH2 and BMI1 mRNAs was on
average increased and decreased, respectively. Expression of CTCFL was increased in a small
number of the cases and OCT4 was slightly diminished. No significant association with
overall DNA methylation changes in prostate cancers was observed. However, CTCFL as
well as OCT4 were less methylated in testes than in somatic cells and accordingly expressed
much more strongly. Additional experiments using DNA methylation inhibitors showed DNA
methylation to represent the essential mechanism for control of CTCFL expression, whereas
OCT4 down-regulation was more dependent on other factors. No significant changes in DNA
methylation of these two genes were found in prostate cancers, but OCT4 was
hypomethylated in testicular cancers.
Taken together, our results argue against a general reactivation of the investigated chromatin
regulators in prostate cancer, with the known exception of EZH2. Since we have investigated
bulk primary tumors, it remains of course possible that such a reactivation occurs in a small
subset of the tumor cells or in metastases.
Funded by the Deutsche Krebshilfe.
Recent Publications
Schulz WA: Molecular Biology of Human Cancers. An Advanced Student‘s Textbook. Springer, 2005
Maas S, Warskulat U, Steinhoff C, Mueller W, Grimm MO, Schulz WA, Seifert HH (2004) Decreased FAS
expression in advanced stage bladder cancer is not related to p53 status. Urology 63, 392-397
Cronauer MV, Schulz WA, Burchardt T, Ackermann R, Burchardt M (2004) Inhibition of p53 function
diminishes androgen receptor-mediated signaling in prostate cancer cell lines. Oncogene 23, 3541-3549.
Betz B, Florl AR, Seifert HH, Dall P, Schulz WA, Niederacher D (2004) DHPLC as a reliable high-throughput
pre-screening method for aberrant promoter methylation in cancer. Hum. Mutat. 23, 612-620
Zhang J, Zotz RB, Li Y, Wang R, Kiel S, Schulz WA, Wen D, Chen Z, Zhang L, Wang S, Gabbert HE, Sarbia
M (2004) Methylenetetrahydrofolate reductase C677T polymorphism and predisposition towards esophageal
squamous cell carcinoma in a German Caucasian and a northern Chinese population. J. Cancer Res. Clin.
Oncol. 130, 574-580
Florl AR, Steinhoff C, Müller M, Seifert HH, Hader C, Engers R, Ackermann R, Schulz WA (2004) Coordinate
hypermethylation at specific sites in prostate carcinoma precedes LINE-1 hypomethylation. Brit. J. Cancer 91,
985-994
- 91 -

Santourlidis S, Kimura F, Fischer J, Schulz WA (2004) Suppression of clonogenicity by the PCNA-binding
domain of mammalian Dnmt1. Biochem. Cell Biol. 82, 589-596
Raschke S, Balz V, Efferth T, Schulz WA, Florl AR (2005) Homozygous deletions of CDKN2A caused by
alternative mechanisms in different human cancer cell lines. Genes Chromosomes Cancer, 42, 58-67
Thievessen I, Wolter M, Prior A, Seifert HH, Schulz WA (2005) Hedgehog signaling in normal urothelial cells
and in urothelial carcinoma cell lines. J. Cell. Physiol. 203, 372-377
Hoffmann MJ, Florl AR, Seifert HH, Schulz WA (2005) Multiple mechanisms inactivating CDKN1C in bladder
cancer. Int. J. Cancer 114, 406-413
Kindich R, Florl AR, Jung V, Engers R, Müller M, Schulz WA, Wullich B (2005) Application of a modified
real-time PCR technique for relative gene copy number quantification to the determination of the relationship
between NKX3.1 loss and MYC gain in prostate cancer. Clin. Chem. 51, 649-652
Cronauer MV, Schulz WA, Ackermann R, Burchardt M (2005) Effects of WNT/#-catenin pathway activation on
signaling through T-cell factor (TCF) and androgen receptor in prostate cancer cell lines. Int. J. Oncol. 26,
1003-1040
Rahnenführer J, Beerenwinkel N, Schulz WA, Hartmann C, von Deimling A, Wullich B, Lengauer T (2005)
Estimating cancer survival and clincial outcome based on genetic tumor progression scores. Bioinformatics 21,
2438-2446
Wu Q, Hoffmann MJ, Hartmann FH, Schulz WA (2005) Amplification and overexpression of the ID4 gene at
6p22.3 in bladder cancer. BMC Mol. Cancer 4, 16 (online)
Sarbia M, Geddert H, Kiel S, Kandemin Y, Schulz WA, Vossen S, Zotz RD, Willers R, Baldus SE, Schneider
PM, Gabbert HE (2005) Methylenetetrahydrofolate reductase C677T polymorphism and risk of adenocarcinoma
of the upper gastrointestinal tract. Scand. J. Gastroenterol. 40, 109-111
Laner T, Schulz WA, Engers R, Müller M, Florl AR (2005) Hypomethylation of the XIST promoter in prostate
cancer. Oncol. Res. 15, 257-264
Kindich R, Florl AR, Kamradt J, Lehmann J, Müller M, Wullich B, Schulz WA (2005) Relationship of NKX3.1
and MYC gene copy number ratio and DNA hypomethylation to prostate carcinoma stage. Eur. Urol., in press
Wethkamp N, Ramp U, Geddert H, Schulz WA, Florl AR, Suschek CV, Hassan M, Gabbert HE, Mahotka C
(2006) Expression of Death-Associated Protein Kinase during tumor progression of human renal cell
carcinomas: clues for hypermethylation-independent mechanisms of inactivation. Eur. J. Cancer, in press
Schulz WA, Seifert HH (2004) DNA methylation in urological cancers. In: “DNA methylation and cancer
therapy” (Szyf M, Hg.), pp.42-58, Landes Biosciences
Cronauer MV, Schulz WA, Burchardt T, Anastasiadis AG, De La Taille A, Ackermann R, Burchardt M (2003)
The androgen receptor in hormone-refractory prostate cancer: Relevance of different mechanisms of androgen
receptor signaling. Int. J. Oncol. 23, 1095-1102
Grimm MO, Hartmann FH, Schulz WA (2004) Microarrays: Technik und Potenzial beim Prostatakarzinom.
Urologe 43, 653-658
Schulz WA, Müller M (2004) Molekulare Karzinogenese des Prostatakarzinoms. in: Borchers H, Jakse G (eds)
“Die Therapie des Prostatakarzinoms: Die Zukunft hat begonnen”. Akt. Onkol. 122, 39-53.
Hoffmann MJ, Schulz WA (2005) Causes and consequences of DNA hypomethylation in human cancer.
Biochem. Cell Biol. 83, 296-321
Schulz WA (2005) Qualified promise: DNA methylation assays for cancer detection and classification
(editorial). J. Biomed. Biotechnol. 3, 227-229
Schulz WA, Steinhoff C, Florl AR (2005) Methylation of endogenous human retroelements in health and
disease. Curr. Topics Microbiol. Immunol., in press
Schulz WA (2006) L1 retrotransposon in human cancers. J. Biomed. Biotechnol., in press
- 92 -

Survey on in vitro cell death induction of multiple myeloma cells upon valproic acid
treatment.
Chantal Schwartz, Valérie Palissot, René N.H.C. Brons and Guy Berchem.
Laboratoire d’Hémato-Cancérologie Expérimentale, CRP-Santé, 84, Val Fleury, L-1526
Luxembourg. E-mail :berchem.guy@chl.lu
Multiple myeloma (MM) is, despite the emergence of new treatments in recent years, still a
lethal disease in 2005. Over the last several years, significant insights into the dysregulation
of various signal transduction pathways of MM have emerged and have evolved the
development of new agents. Histone deacetylase (HDAC) inhibitors as valproic acid (VPA)
are compounds that inhibit HDAC enzymes that, in conjunction with histone acetylases,
regulate the acetylation state of histones and, by extension, the conformational status of
chromatin. VPA is clinically known to be used in treatment of different types of epileptic
diseases. The inhibitory functions of VPA on class I HDAC in combination with its moderate
secondary effects in therapy was recently exploited (Gottlincher M, 2004). VPA has been
described as inducer of apoptosis and differenciation in various cancer types and leukaemia.
In our study, effects of cell death have been studied in cells harvested and purified from
routine bone marrow aspirates of several patients with multiple myeloma, as well on the
myeloma cell lines OPM2, RPMI and U266 treated with a physiological range of VPA (1-2
mM). Using an XTT based cytotoxicity assay, we observe significant in vitro toxicity already
starting at doses of 1 mM for 24h in the cell lines OPM2 (60% viability) and RPMI (80%
viability), as well as on CD138+ selected MM cells from 2 patients. The cell line U266 was
more resistant (90% viability). Incubating in presence of ZVAD, a pan-caspase inhibitor, had
a partial protective effect, but did completely inhibit the cytotoxicity of VPA on OPM2 and
RPMI, which indicates that cell death through VPA is not only triggerd by the apoptotic
pathway. This was confirmed by flow cytometric analysis with PI/Annexin V staining, where
the cell lines U266 and OPM2 did not show apoptosis features, nor in DNA fragmentation
assays. However Western blot analysis of OPM2 and RPMI with antibodies against caspase 9
and 3 showed an accumulation of their cleaved forms within a time course of 72h of VPA
treatment, indicating that cell death induced by VPA in MM is not completely independent of
caspase activity.
In summary, these data suggest that VPA has a cytotoxic effect on MM cells, and partially but
not primarily induces apoptosis on these cells. Further experiments exploring other possible
mechanisms of cell death will be conducted.
- 93 -

Hypoxia Signal Transduction in Health and Disease
Gregg L. Semenza
Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205 USA. Email:
gsemenza@jhmi.edu
In mammals, delivery of O 2 to cells is regulated by multiple homeostatic mechanisms, which
maintain a balance between O 2 supply and demand. O 2 concentrations must be tightly
regulated because of the risk of energy depletion or oxidative damage associated with
inadequate and excess O 2, respectively. Hypoxia-inducible factor 1 (HIF-1) is a master
transcriptional regulator of O 2 homeostasis. The half-life and transcriptional activity of the
HIF-1 subunit are controlled by O 2-dependent hydroxylation, which provides a
molecular mechanism for transducing changes in oxygenation into changes in gene
expression. HIF-1 controls the expression of genes whose protein products control O 2
delivery through the control of erythropoiesis (erythropoietin) and vascularization (vascular
endothelial growth factor, placental growth factor, stromal derived factor 1, platelet-derived
growth factor B, and angiopoietin-1and -2) growth. In addition to controlling the production
of cytokines that activate vascular endothelial cells, HIF-1 also controls cell-autonomous
responses of endothelial cells and bone marrow-derived progenitor cells. In preclinical
studies, administration of an adenovirus encoding a constitutively active form of HIF-
1 promotes both angiogenesis and arteriogenesis. A genetic polymorphism in the
human HIF1A gene is associated with absence of coronary collaterals in patients with
ischemic heart disease. HIF-1 overexpression is associated with increased risk of
mortality in many different human cancers and HIF-1 contributes to immortalization, genetic
instability, angiogenesis, glycolysis, autocrine growth factor signaling, invasion/metastasis,
and treatment failure. In the clear cell type of renal cell carcinoma, loss of function of the von
Hippel-Lindau tumor suppressor protein results in dysregulated HIF-1 activity that is
associated with the repression of E-cadherin gene expression, leading to the loss of cell-cell
adhesion that is required for invasion/metastasis. Several novel anti-cancer agents have antiangiogenic
effects that are related to their inhibition of HIF-1 activity. Thus, strategies
designed to inhibit or activate HIF-1 may represent a novel therapies in cancer and ischemic
cardiovascular disease, respectively, which are the major causes of mortality in industrialized
societies.
- 94 -

Distinct roles of IRF-3 and IRF-7 in the activation of anti-tumor properties of human
macrophages
Mayra Solis 1,* Raphaëlle Romieu-Mourez 1,* , Alessandra Nardin 2,* , Véronique Baron-
Bodo 2 , Delphine Goubau 1 , Bernard Massie 3 , Margarita Salcedo 2 and John Hiscott 1 .
* R.R-M, M.S. and A.N. contributed equally to this work 1 Terry Fox Molecular Oncology
Group, Lady Davis Institute McGill University, Montreal Canada H3T 1E2 2 IDM, Paris
France 3 Biotechnology Research Institute, Montreal Canada H4P 2R2
Type I interferons (IFN-alpha, –beta) exert strong antiviral, antiproliferative and
antiangiogenic effects and are widely used in clinical settings as adjuvants for therapy against
cancer. IFN-beta induction requires the activation of latent transcription factors, including the
interferon regulating factor (IRF)-3, NF-!B and ATF-2/c-Jun. Secretion of the newly
produced IFN-beta and binding to cognate adjacent type I IFN receptors induces transcription
of the irf7 gene. IRF-7 induces the expression of delayed IFN-alpha genes and elicits the full
induction of type I IFN production. When properly activated, macrophages can be
tumoricidal, thus making attractive additions to standard cancer therapies. Tolerance and
activity of human autologous IFN-gamma-activated macrophages, (MAK ® cells), have been
assessed in pilot trials in cancer patients. Here, we tested the hypothesis that activation of
IRF-3 and IRF-7, with subsequent type I IFN production, may be involved in the acquisition
of new antitumor functions by macrophages. We generated adenoviral vectors for the delivery
of constitutive active mutants of IRF-3 (Ad-F3) or IRF-7 (Ad-F7) into primary human
macrophages. Rapid cell death was observed in Ad-F3- transduced macrophages, whereas
Ad-F7-transduction produced type I IFNs and displayed increased expression of genes
encoding TRAIL, IL-12, IL-15 and CD80, persisting for at least 72 hours. Expression of
iNOS, TNF-alpha, FasL, IL-1 and IL-6 genes was unaltered by Ad-F7 transduction. In
addition, Ad-F3 and Ad-F7 transduction appeared to negatively regulate the transcription of
the pro-tumorigenic genes encoding IL-8, VEGF or MMP-2. Ad-F7- expressing macrophages
exerted a cytostatic activity on SK-BR3, MCF-7 and COLO-205 cancer cell lines. The latter
cells were previously shown to be insensitive to the action of non-activated macrophages or
MAK cells. In conclusion, transduction of active forms of IRF-3 or IRF-7 appears to
differentially regulate the apoptosis and the antitumor properties of primary macrophages,
with IRF-7 active mutant leading to the acquisition of novel anti-tumor effector functions.
- 95 -

New steps toward the use of parvovirus H1 as a therapeutic anti-cancer drug
D.Stéhelin, N.Martin, G.Muharram, A.Bègue, C.Lagrou, A-C.Flourens A.Roussel,
I.Loison.
CNRS-UMR 8526. Institute of Biology of Lille, 1 rue Calmette-BP 447, 59021 Lille
Cedex. dominique.stehelin@ibl.fr
Recent emphasis (Int J Oncol. 4, 2005, 901; Nature Medicine 8, 2000, 862 ; Nature
Biotechnology 18, 2000, 723) has brought into focus the intriguing property of some viruses
that are able to quite selectively destroy many types of tumour cells. Our laboratory has been
studying such a virus, namely parvovirus H1, which opportunistically infects the human
population, but apparently without any known pathology in adults.
Parvovirus H1 exhibits quite remarkable features in cell cultures: 1)It infects preferentially
many types of human tumour cells. 2)The cells replicating the virus are rapidly killed (ex vivo
in c.a. 3-5 days), releasing viral progeny. 3)The new virus readily infects the neighbouring
tumour cells in a recurrent fashion
In vivo experiments showed that SCID mice injected with human tumour cell-lines (i.e.
HeLa) develop tumours and rapidly die, unless they are injected with Parvovirus H1, in which
case the tumours regress, then disappear definitely, preventing the animal from dying.
A small Phase1 clinical trial carried on with T.Tursz at the IGR Hospital in Villejuif, in
collaboration with J.Rommelaere (Heidelberg), gave encouraging results: 1)High doses of
parvovirus could be injected, apparently without hampering essential life parameters.
2)Repeated injections could be applied, without observing deleterious high antibody levels.
3)A systemic spread of the virus was observed, where the virus could reach distal metastases
which sustained its replication. 4)The virus disappeared following the last injection.
These promising results encouraged us to further investigate the mechanism by which the
virus is able to selectively destroy tumour cells, and to decipher why so many different types
of tumour cells are efficient targets for parvoviral mediated oncolysis. We will also present
recent achievements concerning the preparation of purified parvovirus grown on cells without
any animal fraction, and our preliminary predictive test to determine if fresh human tumours
are responsive to parvoviral kelling.
We recently obtained an important research grant that should allow us to progress toward a
potential use of this virus in anticancer therapy.
References:
FAISST, S., D.GUITTARD, A.BENNER, Y.Y.CESBRON, J.R.SCHLEHOFER,
J.ROMMELAERE and
T.DUPRESSOIR
Dose-dependent regression of HeLa cell-derived tumours in scid mice after parvovirus
H-1 infection
International Journal of Cancer (1998) 75 : 584-589
DUTHEIL, N., F.SHI, T.DUPRESSOIR and R.M.LINDEN
Adeno-associated virus site-specifically integrates into a muscle-specific DNA region
Proc. Natl. Acad. Sci. USA (2000) 97 : 4862-4866
- 96 -

Redox-Sensitive Transcription Factors as Prime Molecular Targets for
Chemoprevention and Cytoprotection
Young-Joon Surh (surh@plaza.snu.ac.kr)
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention
College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
There are multiple lines of compelling evidence supporting the association between
inflammatory tissue damage and cancer. A new horizon in chemoprevention research is the
recent discovery of molecular links between inflammation and cancer. Components of the
cell signaling network, especially those converge on redox-sensitive transcription factors
including nuclear factor-kappaB (NF-!B) and AP-1 involved in mediating inflammatory
response, have been implicated in carcinogenesis. A wide variety of chemopreventive and
chemoprotective agents can alter or correct undesired cellular functions caused by abnormal
pro-inflammatory signal transmission mediated by NF-!B and AP-1. Modulation of cellular
signaling involved in chronic inflammatory response by anti-inflammatory agents hence
provides a rational and pragmatic strategy in molecular target-based chemoprevention.
Induction of phase-2 detoxifying or antioxidant enzymes represents an important cellular
defence response to oxidative and electrophilic insults. Nrf2 plays a crucial role in regulating
phase-2 detoxifying/antioxidant gene induction. Many chemopreventive and chemoprotective
agents have been found to activate this particular redox-sensitive transcription factor, thereby
potentiating cellular antioxidant capacity.
References
1. Surh, Y.-J. (2003) Cancer chemoprevention with dietary phytochemicals. Nature
Reviews Cancer, 3: 768-780.
2. Lim, S.-Y., Jang, J.-H., Na, H.-K., Lu, S., Rahman, I., and Surh, Y.-J. (2004) 15-deoxy-
12,14 -prostaglandin J2 protects against nitrosative PC12 cell death through up-regulation of
intracellular glutathione synthesis. J. Biol. Chem., 279: 46263-46270.
3. Kim, S.-O., Kundu, J.K., Shin, Y.K., Park, J.-H., Cho, M.-H., Kim, T.-Y., and Surh, Y.-J.
(2005) [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP
kinase. Oncogene, 24, 2558-2567.
- 97 -

Gout-associated uric acid crystals activate the NALP3 inflammasome
Jurg Tschopp
Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland
Development of the acute and chronic inflammatory responses known as gout and pseudogout
are associated with the deposition of monosodium urate (MSU) or calcium pyrophosphate
dihydrate (CPPD) crystals, respectively, in joints and periarticular tissues. Although MSU
crystals were first identified as the etiologic agent of gout in the 18th century1 and more
recently as a “danger signal” released from dying cells2, little is known on the molecular
mechanisms underlying MSU- or CPPD-induced inflammation. We will show that MSU and
CPPD engage the caspase-1 activating NALP3 inflammasome, resulting in the production of
active IL-1b and IL-18. Macrophages from mice deficient in various components of the
inflammasome such as Caspase-1, ASC and NALP3 are defective in crystal induced IL-1b
activation. Moreover, an impaired neutrophil influx is found in an in vivo model of crystalinduced
peritonitis in inflammasome-deficient mice or mice deficient in the IL-1b receptor
(IL-1R). These findings provide insight into the molecular processes underlying the
inflammatory conditions of gout and pseudogout and further support a pivotal role of the
inflammasome in several autoinflammatory diseases.
- 98 -

Epigenic control of MHC2TA transcription: varying contributions of DNA and histone
modifications in cell-activation and interferon-$-mediated induction of gene expression
in cancer cells
Peter J. van den Elsen 1,3 , Marja C.J.A. van Eggermond 1 , Martine J. Jager 2 and Tjadine
M. Holling 1
1 Division of Molecular Biology, Department of Immunohematology and Blood Transfusion, and
2 Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
3 Department of Pathology, VU University Medical Center, Amsterdam, The
Netherlands.
The products of major histocompatibility complex class II (MHC-II) genes encode cell
surface glycoproteins, which play an essential role in the initiation of antigen-specific
immune responses by virtue of their ability to present antigenic peptides to CD4 + T
lymphocytes. Lack of MHC-II molecule expression results in a severe immunodeficiency as
is observed in pediatric patients suffering from the bare lymphocyte syndrome.
Essential for the transcriptional activation of MHC-II genes is the MHC2TA encoded class II
transactivator (CIITA), a co-activator, which interacts with the MHC-enhanceosome bound to
the SXY-promoter module of all MHC-II and accessory genes. Lack of MHC-II gene
transcription, therefore, is due to lack of CIITA expression. Constitutive and induced
expression of MHC2TA is governed through the employment of several independent
promoters, which are activated following cell type-specific signaling pathways (e.g. human T
cells) or in the response to inflammatory cytokines of which interferon-$ (IFN-$) is the most
potent (e.g. fibroblasts and epithelial cells).
In human T cell leukemia, the lack of CIITA expression was found to result from epigenetic
modifications at the MHC2TA locus. Besides DNA methylation, we also observed
trimethylation of lysine residue 27 of histone H3 (K27-H3) of the T cell employed MHC2TA
promoter to correlate with lack of MHC2TA transcription.
Interestingly, in uveal melanoma cells different epigenetic phenotypes were observed
involving MHC2TA. In several uveal melanoma cell lines (Mel285 and OCM-1) despite
hypermethylation of the uveal melanoma employed MHC2TA promoters, gene transcription
was readily noted following IFN-$ treatment. Interestingly, in these uveal melanoma cells
IFN-$ induction of MHC2TA transcription was associated with a strong increase in
acetylated-histone H3 and H4, and induction in dimethyl-K4-H3 levels in MHC2TA
chromatin as determined by chromatin immunoprecipitation analyses. In contrast, OMM1.3
cells lacked MHC2TA transcription after IFN-$ stimulation. Surprisingly, the genomic
MHC2TA promoter DNA was unmethylated, which is in contrast to other non IFN-$inducible
cell types. However the lack of MHC2TA transcription correlated with relative high
levels of trimethyl-K27-H3 in MHC2TA promoter chromatin.
Together our results indicate varying levels of epigenetic control of MHC2TA transcription in
cancer cells, which may contribute to evasion of host-mediated immunosurveillance.
- 99 -

Recent papers:
1. Holling TM, Van der Stoep N, Quinten E and Van den Elsen PJ.
Activated T cells accomplish MHC class II expression through T cell specific occupation of
CIITA promoter III. J. Immunol. 2002; 168: 763-770.
2. Van der Stoep N, Quinten E and Van den Elsen PJ.
Transcriptional regulation of the MHC class II transactivator (CIITA) promoter III:
Identification of a novel regulatory region in the 5’-UTR and an important role for cAMP
reponsive element binding protein 1 and activating transcription factor-1 CIITA promoter III
transcriptional activation in B-lymphocytes. J. Immunol. 2002; 169: 5061-5071.
3. Holling TM, Schooten E, Langerak AW and Van den Elsen PJ.
Regulation of MHC class II expression in human T cell malignancies. Blood 2004; 103:
1438-1444.
4. Van der Stoep N, Quinten E, Marcondes Rezende M and Van den Elsen PJ.
E47, IRF-4 and PU.1 synergize to induce B cell-specific activation of the Class II
Transactivator promoter III (CIITA-PIII). Blood 2004; 104: 2849-2857.
5. Van den Elsen PJ, Holling TM, Kuipers HF and Van der Stoep N.
Transcriptional regulation of antigen presentation. Curr. Opin. Immunol. 2004; 16: 67-75.
6. Holling TM, Schooten E and Van den Elsen PJ.
Function and regulation of MHC class II molecules in T-lymphocytes: of mouse and man.
2004; 65: 282-290.
7. Holling TM, Van der Stoep N and Van den Elsen PJ.
Epigenetic control of CIITA expression in leukemic T cells. Biochem. Pharmacol. 2004; 68:
1209-1213.
8. Kuipers HF, Biesta PJ, Mommaas AM, Groothuis T, Neefjes J, and Van den Elsen PJ.
Statins affect cell surface expression of MHC-II molecules by disrupting cholesterolcontaining
microdomains. Hum. Immunol. 2005; 66: 653-665.
9. Kuipers HF and Van den Elsen PJ
Statins and control of MHC2TA transcription. Nature Medicine 2005; 11: 365-366.
10. Schooten E, Klous P, Van den Elsen PJ and Holling TM.
Lack of MHC class II expression in activated mouse T cells correlates with hypermethylation
at the CIITA-PIII region. Immunogenetics 2005; in press.
- 100 -

Attenuation of MSK1-driven NF-kB gene expression by soy isoflavones does not require
estrogenic activity
Wim Vanden Berghe, Nathalie Dijsselbloem, Linda Vermeulen, 'Matladi N. Ndlovu,
Elke Boone, Guy Haegeman
Laboratory for Eukaryotic Gene Expression and Signal Transduction (LEGEST),
Department of Molecular Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000
Gent, Belgium. Email: w.vandenberghe@ugent.be
We have analysed in molecular detail how soy isoflavones (genistein, daidzein, biochanin A)
suppress NF-kB-driven interleukin-(IL)6 expression. In addition to its physiological immune
function as an acute stress cytokine, sustained elevated expression levels of IL6 promote
chronic inflammatory disorders, aging frailty and tumorigenesis. Our results in estrogenunresponsive
fibroblasts, mitogen- and stress-activated protein kinase (MSK) knock-out cells
and estrogen receptor (ER)-deficient breast tumor cells, demonstrate that phytoestrogenic
isoflavones can selectively block nuclear NF-kB transactivation of specific target genes (in
particular IL6), independently of their estrogenic activity. This occurs via attenuation of
mitogen-activated protein/ERK kinase (MEK) and extracellular signal-regulated kinase
(ERK) activity, which further downregulates MSK-dependent NF-kB p65 and histone H3
phosphorylation. As constitutive NF-kB and MSK activity are hallmarks of aggressive
metastatic ER-deficient breast cancer, the MSK signaling pathway may become an attractive
target for chemotherapy.
Recent references
Vermeulen, L., De Wilde, G., Van Damme, P., Vanden Berghe, W. & Haegeman, G.
Transcriptional activation of the NF-!B p65 subunit by mitogen- and stress-activated protein
kinase-1 (MSK1). EMBO Journal, 22(6), 1313-1324, 2003.
De Bosscher, K., Vanden Berghe, W. & Haegeman, G. The interplay between nuclear receptors
and NF-!B or AP1: molecular mechanisms for gene repression.
Endocr. Rev., 24(4), 488-522, 2003.
Dijsselbloem, N., Vanden Berghe, W., De Naeyer, A. en Haegeman, G. Phyto-estrogens:
multi-purpose compounds at the crossroad of hormone replacement, anti-cancer and antiinflammatory
therapy? Biochem. Pharm., 68, 1171-1185, 2004.
De Bosscher, K., Vanden Berghe, W., Beck, I., Lauw, A., Hapgood, J. & Haegeman, G. Antiinflammatory
capacities of a plant-derived, non-steroidal contraceptive compound. Proc Natl
Acad Sci U S A. [Epub ahead of print 21 october] , 2005
De Naeyer, A., Vanden Berghe, W., Pocock, V., Milligan, S., Haegeman, G., De
Keukeleire, D. : Estrogenic and anticarcinogenic properties of kurarinone, a
lavandulyl flavanone from the roots of Sophora flavescens. Journal of Natural Products, 67
(11): 1829-1832, 2004.
De Naeyer, A., Vanden Berghe, W., De Keukeleire, D. & Haegeman, G. Phytoestrogens in
cancer chemoprevention; characterization and beneficial effects of a new flavanone,
- 101 -

kurarinone, a major phytoestrogen constituent of Sophora Flavescens ait. in
“Phytopharmaceuticals in Cancer Chemoprevention” (Modern Nutrition Science) edited by
Debasis Bagchi, USA, CRC Press, 428-448, 2004.
Vanden Berghe, W., Dijsselbloem, N., De Naeyer, A., Vermeulen, L. en Haegeman, G.
Regulation of NF-kB-driven inflammatory genes by phytoestrogens. In “Oxidative stress,
Inflammation and Health”, edited by Y-J. Surh,, Marcel Dekker Inc, 61-107, 2005
Vanden Berghe, W.*, Dijsselbloem, N.*, Vermeulen, L., Ndlovu, M., Boone, E. &
Haegeman, G. Phytoestrogenic soy isoflavones attenuate MSK1-driven NF-kB gene
expression in estrogen receptor deficient breast tumor cells. Cancer Research, in revision
(2005) (*) equally contributed
- 102 -

Talking to chromatin: Silencers Silenced.
Jan Willem Voncken
Div. Molecular Genetics, University of Maastricht, Universiteitssingel 50, 6229 ER,
Maastricht, The Netherlands. Email: w.voncken@molgen.unimaas.nl
Polycomb Group (PcG) proteins modify chromatin structure. Best known for their ability to
maintain transcriptional silencing, PcG proteins are critically involved in many other
important biological processes, ranging from X-chromosome inactivation to stem cell
regeneration. The molecular mechanisms involved in PcG-directed chromatin modification
repression are starting to unravel. Functional regulation of PcG-proteins is poorly understood.
We previously found hat PcG-complexes are phosphorylated and dissociate from chromatin
in vivo in a cell cycle-dependent manner. Recent evidence from our lab suggests that PcG
proteins are phosphorylated in response to cell signalling independent of cell cycleprogression.
This link suggests a novel mechanism by which PcG-mediated gene silencing
can be dynamically modulated. The implications of our findings will be discussed.
- 103 -

A Systems Approach to Protein Kinase Signaling Networks
Michael B. Yaffe M.D., Ph.D.
Howard S. and Linda E. Stern Associate Professor
Center for Cancer Research
Depts of Biology and Biological Engineering
Associate Member, Broad Institute
Massachusetts Institute of Technology
Cambridge, Massachusetts, USA
Many protein kinases and phosphoserine/threonine-binding domains such as 14-3-3
proteins, WW domains, FHA domains, Polo-box domains, and BRCT domains function
together within signaling networks to control cell cycle progression, the response to DNA
damage, and the onset of apoptosis. How signals emerging from these pathways are integrated
and processed as a network is unclear. To address this, we have been developing systems
models of signaling where kinase activities, protein phosphorylation, binding of substrates to
phosphoserine/threonine binding domains, and cellular responses such as cell cycle arrest and
apoptosis are quantitatively measured at densely sampled points in time, and related
mathematically using partial least squares regression and principal components analysis.
Using cytokine-induced apoptosis in HT-29 cells as a starting point, we used this approach to
construct a systems model of 7980 intracellular signaling events that directly links
measurements to 1440 response outputs associated with apoptosis. The model accurately
predicted multiple time-dependent apoptotic responses induced by a combination of the
death-inducing cytokine tumor necrosis factor (TNF) with the pro-survival factors epidermal
growth factor (EGF) and insulin. The model revealed new molecular mechanisms connecting
signaling to apoptosis including the role of unsuspected autocrine circuits activated by TGF-"
and IL-1", All of the molecular signals could be divided along two primary signaling axes
that constitute fundamental dimensions (molecular “basis axes”) within the apoptotic
signaling network. Projections of different stimuli along these axes capture the entire
observed apoptotic response, suggesting that cell survival is determined by signaling through
this canonical basis set. We are currently applying this methodology to study signaling events
that control cell cycle arrest and apoptosis after DNA damage.
- 104 -

Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting
MEKK2-JNK pathway
Ying E. Zhang 1 , Motozo Yamashita 1 , Sai-Xia Ying 1 , Cuiling Li 2 , Chuxia Deng 2 , and
Steven Y. Cheng 1
1 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National
Cancer Institute, National Institutes of Health, Bethesda, MD 20892
2 Genetics of Development and Disease Branch, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Smad ubiquitin regulatory factor (Smurf), a HECT domain ubiquitin ligase, was previously
identified as E3 ligase targeting TGF-beta/BMP pathway through in vitro biochemical
experiments. To investigate the physiological function of Smurf1, we disrupted the mouse
Smurf1 allele through homologous recombination. Smurf1-deficient mice are born normal,
fertile and have normal life span. Given the fact that members of the TGF-#eta/BMP
superfamily are crucial inductive signals during embryonic development, the seemingly
normal development of Smurf1 -/- mice and the concurrent increase of the Smurf2 transcript
in Smurf1 -/- suggest that the loss of Smurf1 function might have been compensated for, at
least in part, by the elevated Smurf2. Despite of no phenotypic developmental abnormalities
or health problems, Smurf1 -/- mice displayed age-dependent increase of bone mass. The
cause of this increase can be traced to enhanced activities of osteoblasts, which become
sensitized to bone morphogenesis protein (BMP) in the absence of Smurf1. Surprisingly, this
skeletal abnormality is not due to alteration in the Smad-mediated canonical TGF-beta or
BMP signaling. Instead, loss of Smurf1 leads to accumulation of phosphorylated MEKK2
and activation of the downstream JNK signaling cascade. We found that Smurf1 physically
interacts with MEKK2 and promotes the ubiquitination and turn-over of phosphorylated
MEKK2, which resulted in downregulation of osteoblast activity and response to BMP.
These results reveal a novel function of Smurf1 in the regulation of bone homeostasis through
directly targeting MAPK signaling pathway, thereby regulating the biological response of the
TGF-beta superfamily signaling.
Selected Recent Publications from My Group:
Yu, L., Hebert, M. and Zhang, Y. E.: TGF-beta receptor-activated p38 MAP kinase mediates
Smad-independent TGF-beta responses. EMBO J. 21: 3749-3759, 2002.
Ying, S.-X., Zareena J. H., and Zhang, Y. E.: Smurf1 facilitates myogenic
differentiation and antagonizes the Bone Morphogenetic Protein-2-induced osteoblast
conversion by targeting Smad5 for degradation. J. Biol. Chem. 278: 39029-39036, 2003.
Derynck, R. and Zhang, Y. E.: TGF-beta family signaling: Smad-dependent and
Smad independent pathways. Nature 425: 577-584, 2003.
Yamashita, M., Ying, S.-X., Zhang, G.-M., Li, C., Cheng, S. Y., Deng, C.-X., and
Zhang, Y. E.: Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by
targeting MEKK2 for degradation. Cell 121: 101-113, 2005
- 105 -

Signaling pathways controlling self-tolerance: On the road to systemic autoimmune
diseases
Moncef ZOUALI
INSERM U606, Centre Viggo Petersen, Hôpital Lariboisière,
2, rue Ambroise Paré, 75475 Paris Cedex 10, France,
The immune receptors of lymphocytes are able to sense the nature of bound ligands. Through
coupled signaling pathways the generated signals are appropriately delivered to the
intracellular machinery, allowing specific functional responses. A central issue in
contemporary immunology is how the fate of B lymphocytes is determined at the successive
developmental stages and how the B cell receptor distinguishes between signals that induce
immune response or tolerance. Experiments with mice expressing transgenes or lacking
signal transduction molecules are providing important clues to the mechanisms that regulate
signaling thresholds at different developmental stages. Their relevance for the pathogenesis
of autoimmune diseases in clinical settings is currently the focus of interest, particularly since
in some instances, similar defects have been identified in human autoimmune diseases. The
fact that altered signaling has been described in both T cells and B cells of autoimmune
patients strengthens the view that the abnormalities observed are relevant to the pathogenesis.
The studies are also revealing novel potential mechanisms of induction of autoimmunity,
which may have a bearing on the understanding of human diseases.
- 106 -

Workshop presentations
(by alphabetical order)
- 107 -

Efficient DNA and siRNA delivery into primary cells and cell lines
Amaxa
Gene transfer into primary cells and difficult-to-transfect cell lines has up to now been
hampered by inefficient transfection methods. The Nucleofector technology presents a
powerfull tool, which has been successfully used worldwide to transfer DNA plasmids or
siRNA duplexes into primary cells such as neurons, T-cells and many cancer cells. Here, we
discuss the latest results and applications, that have been obtained by using this method.
Furthermore, we will introduce amaxa’s new Nucleofector 96-well shuttle, which implies all
advantages of the Nucleofector technology in a high throughput manner.
- 108 -

Ambion
MicroRNAs (miRNAs) are small, RNA molecules encoded in the genomes of plants and
animals. In mammalians, these highly conserved, ~21-mer RNAs regulate the expression of
genes by binding to the 3'-untranslated regions (3'-UTR) of specific mRNAs resulting in
translation inhibition.
In this tsudy, we describe methods to study their expression and to investigate their functional
implications.
Expression profile can be easily performed at array scale allowing miRNA global gene
expression studies of different biological processes like cancer, differenciation, viral
infection, etc.
Functional analysis was performed using miRNA Precursor (Pre-miR) molecules that mimic
and activate miRNA inhibitory action. Additionally, miRNA Inhibitor (Anti-miR) molecules
that inhibit miRNA action on target mRNAs were used to further confirm the functionality of
certain miRNAs.
Using these tools, we were able to demonstrate the implication of let-7 in the regulation of
Ras protein expression in lung cancer patients.
- 109 -

Molecular Networks in Mammals: Extraction from Literature and Pathway Analysis
Ariadne Genomics, Inc.
Using the proprietary high-content linguistics tool MedScan we compiled a comprehensive
database of molecular networks by extracting the information from scientific literature.
MedScan is capable of extracting functional associations between proteins, small molecules,
and pathways, recognizes types of regulatory mechanisms involved, effects of regulation and
experimental conditions.
The resulting database stores 700,000 relationships between mammalian proteins and
chemicals including facts about protein interactions, promoter binding, molecular
biosynthesis and trafficking, and cell process regulation. Different approaches towards
reconstructing individual pathways or cascades from this database and assigning functional
categories to proteins will be described.
Our visualization software is capable of systematically mining this database for small network
motifs that are robust in regard to the effects induced at the gene expression levels. We have
also developed a Bayesian framework for integration of microarray data and binary gene-togene
regulatory relationships. The approach allows the reduction of expression pattern
complexity and finds the minimal set of regulatory proteins that are responsible for
differential expression of other genes.
Finding mesoscopic communities in sparse networks
I. Ispolatov, I. Mazo, and A. Yuryev
q-bio.MN/0512038, submitted to Phys. Rev. E
Identifying Local Gene Expression Patterns in Biomolecular Networks
A. Y. Sivachenko, A. Yuryev, N. Daraselia, I. Mazo
2005 IEEE Computational Systems Bioinformatics Conference, Aug. 8-11, 2005
Cliques and duplication-divergence network growth
Ispolatov, I ; Krapivsky, P L ; Mazo, I ; Yuryev, A
New J.Phys. 7(2005) 145
Binding properties and evolution of homodimers in protein-protein interaction networks
Ispolatov, Iaroslav; Yuryev, Anton; Mazo, Ilya; Maslov, Sergei
Nucleic Acids Research 2005 33(11):3629-3635
Extracting Protein Function Information from MEDLINE Using a Full-Sentence Parser
Nikolai Daraselia, Sergei Egorov, Andrey Yazhuk, Svetlana Novichkova, Anton Yuryev, Ilya Mazo
published in Proceeding of the Second European Workshop on Data Mining and Text Mining for
Bioinformatics, pp11-18, 2004
A Simple and Practical Dictionary-Based Approach for Identification of Proteins in MEDLINE Abstracts
Sergei Egorov PhD, Anton Yuryev PhD, and Nikolai Daraselia PhD
Journal of the American Medical Informatics Association 2004
Pathway studio - the analysis and navigation of molecular networks
Alexander Nikitin, Sergei Egorov, Nikolai Daraselia and Ilya Mazo
BIOINFORMATICS APPLICATIONS NOTE Vol. 19 no. 0 2003, pages 1-3
Extracting human protein interactions from MEDLINE using a full-sentence parser
Nikolai Daraselia, Anton Yuryev, Sergei Egorov, Svetalana Novichkova, Alexander Nikitin and Ilya Mazo
BIOINFORMATICS Vol. 19 no. 0 2003, pages 1–8
- 110 -

Organizer: BIOBASE GmbH
Date: January, 26 2006
Time: 3 pm – 4:30 pm
Duration: ca 1 hour 30 minutes
Attendees: 110 - 140
Topic: Cutting-edge Biology for Explaining Expression Data
Chair: Alena Kemper, BIOBASE, Wolfenbüttel
Speakers:
Prof. Dr. Edgar Wingender, BIOBASE, Wolfenbüttel
„Making Sense of Transcriptomics and Proteomics - New Frontiers of Integrative Biology”
Dr. Hauke Stephan Busch, German Cancer Research Center – DKFZ – TP3, Heidelberg
“CD95-induced Appoptosis: An Example for Large-Scale Modeling of Signal Transduction
Networks”
Dr. Alexander Kel/ Holger Karas, BIOBASE, Wolfenbüttel
„Presentation of the ExPlain System”
Organizer: BIOBASE GmbH
Date: January, 27 2006
Time: 3 pm – 4:30 pm
Duration: ca 1 hour 30 minutes
Attendees: 110 - 140
Topic: Cutting-edge Biology for Explaining Expression Data
Chair: Alena Kemper, BIOBASE, Wolfenbüttel
Speakers:
Prof. Dr. Edgar Wingender, BIOBASE, Wolfenbüttel
„Making Sense of Transcriptomics and Proteomics - New Frontiers of Integrative Biology”
Prof. Dr. Jürgen Borlak, Fraunhofer Institute of Toxicology and Experimental Medicine,
Center for Drug Research and Medical Biotechnology, Hannover
„Regulatory Networks of Liver-enriched Transcription Factors in Liver Biology and Disease”
Dr. Alexander Kel/ Holger Karas , BIOBASE, Wolfenbüttel
„Presentation of the ExPlain System”
- 111 -

A Novel Fluorescence-based Kinetic Kinase Assay for Use with Recombinant Protein
and Cell Lysates
M. Li, J.A. Vrana, M. Surby, X-D Qian, C.J. Clark, M. Schults, B. Imperiali, E.M.
Schaefer
Signal Transduction Division, Invitrogen Corporation, Hopkinton, Massachusetts and
Department of Chemistry, Massachusetts Institute of Technology
Protein phosphorylation plays a critical role in signal transduction in normal and disease
states. Recent advances in the development of specific protein kinase inhibitors illustrates the
potential for a new generation of drugs to treat human cancers. We have adapted a novel
fluorescent peptide substrate-based assay for the rapid, homogeneous and sensitive detection
of the activity of a broad range of protein kinases. This assay platform uses the chelationenhanced
fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred
to as Sox), which is incorporated into the substrate peptide. A series of specific Ser/Thr and
Tyr substrate peptides are utilized to measure a range of kinases covering all families of the
kinome. Several key features of this assay include measurement of kinase activity under
optimal kinetics, physiological (mM) ATP, and in real time without the use of radioactive
tracer, beads, antibodies or specialized equipment. Importantly, activity increases result in a
corresponding increase in fluorescence. Because the assay can be performed with a wide
range of ATP concentrations it can be used to select for both ATP-competitive and ATP-noncompetitive
kinase inhibitors. We have applied this system for the analysis of multiple sample
types including the determination of kinase activity of recombinant proteins and using crude
cell lysates in a 96 or 384 well format. This technology, used in conjunction with phosphospecific
antibody based profiling, provides a valuable set of tools for rapidly increasing our
understanding of the kinome and the nature of signaling networks.
- 112 -

Reliable and efficient gene Knock-down and inducible overexpression in vivo.
Ben Davies#, Kader Thiam*
# Senior Scientific consultant, *Director of Transgenic Technologies
genOway, 181 avenue Jean Jaures, 69362 Lyon cedex 07, France. info@genoway.com
Over the past decades, gene Knock-out and overexpression animal models have become
standard laboratory tools for in vivo functional gene analysis. More recently, attempts have
been made to extend the repertoire of transgenic techniques using RNA interference for gene
Knock-down and inducible promoter systems for temporally regulated gene expression,
allowing the compensation and developmental problems associated with classical approaches
to be overcome. Despite initial encouraging reports, the application of both these technologies
in vivo has been unreliable. Here we describe two new approaches which allow both
technologies to be robustly and consistently applied in a transgenic context.
A major requirement for successful and reliable shRNA mediated gene Knock-down in vivo
is a low copy number of integrating constructs. We have established and validated a
technology which allows the generation of a high number of independent founder lines with a
pre-screened copy number. Furthermore a second Knock-in based technology has been
designed which takes advantage of ready-to-use validated targeting vectors to introduce a
single copy of the shRNA cassette within a specific permissive genomic site. With both these
technologies, gene knock-down of up to 80% has been reliably demonstrated.
The tetracycline system has been used successfully in cell culture models to achieve
regulated, inducible gene expression. Unfortunately, the application of this technology in vivo
has been plagued by a lack of predictability. The resulting leakiness and inconsistency of
expression in the resulting transgenic lines necessitates the generation and analysis of many
independent founder lines before a convenient line with the appropriate induction kinetics is
found. We report the development, of a new system which uses a preferential genomic
context to achieve reliable and tight regulation of transgene expression. This reliable and
reproducible inducible systems is reversible and is not restricted to preferential tissues.
Through the use of these technologies, the transgenic tool box is now complete to address
gene function in vivo whilst minimizing complicating compensatory and developmental
effects. With these methods in place, gene activity can now be reliably and exquisitely finetuned,
allowing the most appropriate model for a specific scientific question to be designed.
- 113 -

New tags for transfection and protein:protein interaction analysis
T. Schmidt
IBA GmbH, Rudolf-Wissell-Str. 28, D-37079 Göttingen
Magnet Assisted Transfection (MATra) is a new, easy-to-handle, very fast and highly
efficient technology to transfect cells in culture. In a first step, nucleic acids are associated
with specific magnetic nanoparticles (MagTag). Exploiting magnetic force the full nucleic
acid dose is then drawn towards and delivered into the target cells leading to efficient
transfection without disturbing the membrane architecture, without causing chromosomal
damage or leaving a hole in the cell membrane like other transfection technologies. All types
of nucleic acids from plasmid DNA or siRNA to oligonucleotides can be used with the
MATra approach. Data from a variety of species using cell lines or primary tissue culture
have accumulated including human, monkey, mouse, rat, xenopus, pig, cat or fish.
One-STrEP-tag is an extremely fast and efficient method to isolate functional protein
complexes which have formed in the cellular environment after expression of a tagged bait
protein. After disruption of the cells the intact protein complex is separated from the crude
lysate on Strep-Tactin affinity columns. While other systems require tedious optimization
because of high background or two successive purification steps, the One-STrEP system
requires one STEP only. The fast purification under physiological conditions makes even
weakly associated protein complexes amenable to functional studies or mass spectroscopic
identification. To even improve this unsurpassed system, new monoclonal antibodies against
the One-STrEP-tag have been developed. One antibody called Strep-MAB binds nearly
irreversibly to the One-STrEP-tag while the other exhibits easy controllable reversible
binding properties. These complementary and Strep-Tactin independent One-STrEP-tag
binding tools open new assay opportunities in protein:protein interaction studies which will be
presented.
- 114 -

Exploring ubiquitin signaling with dedicated PIQOR Ubiquitin-PS microarrays
Hartmut Scheel, Corinna B. Scholz, Kay Hofmann
Bioinformatics Group, Miltenyi Biotec GmbH, MACSmolecular Business Unit,
Stöckheimer Weg 1, D-50829 Köln, Germany. E-mail: kay.hofmann@miltenyibiotec.de
The MACSmolecluar business unit of Miltenyi Biotec offers smart products and services for
molecular biology research and gene expression analysis via the proprietary PIQOR
Microarray platform for topic-defined or custom expression profiling solutions.
Here, we introduce the PIQOR Ubiquitin-PS microarray with a focus on the ubiquitinproteasome
system (UPS). By developing this microarray, we are addressing the growing
need for a deeper understanding of the UPS and a comprehensive monitoring of its
components. It has become clear in the recent years that ubiquitylation plays a key role in
processes like cell cycle progression, protein quality control, DNA repair, stress response,
apoptosis and regulation of signaling.
The importance of the UPS for signal transduction is not restricted to its well-known role in
the selective degradation of regulatory proteins. Equally important is the role of ubiquitin
modification as a signal by itself. The high complexity of the UPS is underscored by the large
number of components involved in ubiquitin attachment, removal and recognition, all similar
to the numbers known for phosphorylation signaling. A deregulation of the UPS is associated
with a multitude of diseases, including cancer, catabolic diseases, inflammation and
neurodegeneration. Therefore, the UPS becomes more and more a rich source for discoveries
with therapeutic impact and ubiquitin research increases steadily.
A hallmark of the UPS is that most proteins involved belong to a limited number of protein
families characterized by common homology domains. The bioinformatics group of Miltenyi
MACSmolecular possesses appropriate tools like the profile technique to mine sequence
databases for proteins with a given homology domain. In a systematic UPS analysis
programme applying this technique, a list of ~1200 genes was generated covering all aspects
of the UPS and including many unpublished candidate genes, e.g. approximately 600 E3
ligases were identified. With this catalogue of genes at hand, a new microarray was designed,
which allows the systematic detection of mRNA expression changes in the entire UPS. This
new microarray fits nicely into the portfolio of other topic-defined microarrays offered by
Miltenyi Biotec, which already includes microarrays designed for oncology and immunology.
- 115 -

Antibody and protein delivery in living cells: presentation of a new potent reagent and
potential applications.
Claire Weill
Polyplus-transfection – Bioparc – BP 90018 Illkirch – FRANCE
In cells, main molecular functions are carried out by proteins. Studying proteins, in terms of
localizations, functions or regulations implies the possibility of transfecting such
biomolecules into cells. Usually, cDNA corresponding to the coding sequence of the protein
is transfected in cells by various methods. However in some cases, the expected protein is
finally not expressed for diverse reasons. The function of a protein may also be elucidated by
an indirect approach: inhibiting RNA coding for the protein, using gene silencing technology
(siRNA). However, the more direct approach consists in introducing the protein of interest
into the cells.
Therefore, we have recently developed a potent reagent, named PULSin, for
cytoplasmic protein delivery. This new protein delivery reagent forms non covalent
complexes with proteins or antibodies. PULSin/protein complexes are internalized via
anionic cell-adhesion receptors present on virtually all cells and are released into the
cytoplasm where they disassemble. The protein ends up free, able to diffuse in the cytoplasm
and to proceed to its function or target organelle. We will demonstrate that PULSin delivers
efficiently anionic proteins in a large variety of living eukaryotic cells, including primary cells
and suspension cells, without any side effect. Moreover, PULSin is also able to transfect
antibodies, allowing users to study proteins or to block their activity in living cells.
Using PULSin opens large areas of interest in various cell biology research domains
like studying lethal proteins or controlling protein level and time course in living cells.
- 116 -

Poster presentations
Poster are classifiied by session
I. Protein Domains
II. Receptor signaling and G proteins
III. Protein kinase cascades as therapeutic targets
IV. Protein kinase inhibitors: insights into drug design from
structure
V. Phosphatases a key cell signaling intermediates
VI. Proteasome degradation pathways
VII. Stem cell specific cell signaling mechanisms
VIII. Inflammation specific signaling
IX. Novel compounds targeting inflammatory signaling pathways
X. Cell death in cancer
XI. Cell death and cardiovascular diseases
XII. Cell death and neurodegenerative diseases
XIII. Cell signaling pathways leading to regulated chromatin
modifications
XIV. Transcriptional and translational control
XV. Reactive oxygen species and cell signaling
XVI. Chemopreventive agents
XVII : Cell signaling in health and disease
and then by alphabetical order (first author)
LATE abstracts are at the end of each session
- 117 -

Session I. Protein Domains
- 118 -

Session I : protein domains Poster I, 1
Synthetic corticotropin-like peptide GKVLKKRR, corresponding to the fragment 81-88
of human pro-interleukin-1alpha, is an antagonist of corticotropin receptor
Elena V. Navolotskaya, Vera I. Vanina, Alexander A. Kolobov*, Elena A. Kampe-
Nemm*, Valery M. Lipkin
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the
Russian Academy of Sciences, Science Avenue, 6, 142290 Pushchino, Moscow Region,
Russia, navolots@fibkh.serpukhov.su
*State Research Center for Institute of Highly Pure Bioprepararions, 197110 St.
Petersburg, Russia, kolobov@vs3283.spb.edu
The corticotropin-like octapeptide GKVLKKRR (termed as leucorticotropin), which
corresponds to the amino acid sequence 81-88 in the precursor of human interleukin-1alpha,
was synthesized on automatic peptide synthesizer. Peptide was purified to homogeneity by
reverse-phase HPLC (chromatograph Gilson, France) on Delta Pack C18 column. The
peptide purity was > 99%. Leucorticotropin was labeled with tritium by the high-temperature
solid-state catalytic isotope exchange reaction to specific activity of 20 Ci/mmol. Receptor
binding studies revealed that [3H]leucorticotropin bound with high affinity and specificity to
corticotropin receptor on rat adrenal cortex membranes (Kd = 2.2 nM). Leucorticotropin at
concentrations of 0.1 – 1000 n! was found to have no influence on the adenylate cyclase
activity in adrenal cortex membranes, while intranasal injection of leucorticotropin to rats at
doses of 10 - 50 µg/kg was found to inhibit the secretion of 11-oxycorticosteroids from the
adrenals to the bloodstream. Thus, leucorticotropin is an antagonist of corticotrophin
receptor.
- 119 -

Session I : protein domains Poster I, 2
Preparation and characterization of recombinant chicken growth hormone (chGH) and
its putative antagonist chGH G119R mutein
Helena E. Paczoska-Eliasiewicz1, Gili Salomon2, Shay Reicher2, Eugene E.
Gussakowsky3, Anna Hrabia1 and Arieh Gertler2
1Department of Animal Physiology, University of Agriculture, Kraków, Poland,
2Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew
University of Jerusalem, Israel and 3Department of Botany, University of Manitoba,
Winnipeg, MB R3T 2N2, Canada.
Synthetic cDNA of chicken GH (chGH) and its G119R mutein was synthesized according to
published sequence but optimized for expression in E. coli. The respective cDNAs were
inserted into pMON expression vector and transformed into Mon 105 E. coli strain. The
proteins expressed upon induction with nalidixic acid were found almost entirely in the
insoluble inclusion bodies (IBs). The IBs were isolated, the proteins solubilized in 4.5 M urea,
at pH 11.3 in presence of cysteine, refolded and purified to homogeneity by anion-exchange
chromatography on Q-Sepharose. The overall yields were 400 to 500 mg from 5 liters of
fermentation. Both proteins were > 98% pure as evidenced by SDS-PAGE and contained at
least 95% monomers as documented by gel-filtration chromatography on a SuperdexTM75
column under not denaturing conditions. Circular dichroism analysis revealed that both
proteins have identical secondary structure characteristic of cytokines, namely > 50% of alpha
helix content. Chicken GH was capable of forming a 1:2 complex with recombinant oGHR-
ECD (oGH receptor extracellular domain) though its affinity to ECD as determined by RRA
was 11-fold lower than that of ovine GH (oGH). Correspondingly, its bioactivity, assessed
using PDF-P1 3B9 cells stably transfected with rabbit GHR was 20-25 fold lower, whereas
chGH G119R mutant did not bind to oGHR-ECD and was devoid any biological activity in
PDF-P1 3B9 cells. In contrast, in binding experiments carried-out using chicken liver
membranes both oGH and chGH showed similar EC50 values in competition with 125I-oGH.
These EC50 and were 5-9 fold lower than that of G119R mutein. These results emphasize the
importance of species specificity and indicate the possibility of antagonistic activity of chGH
G119R.
- 120 -

Session I : protein domains Poster I, 3
Preparation of leptin antagonists by site-directed mutagenesis of human, ovine, rat and
mouse leptin's site III: implications on blocking undesired leptin action in vivo.
Gili Salomon1, Leonora Niv-Spector1, Dana Berger1, Isabelle Callebaut2, Jean Djiane3
and Arieh Gertler1*
1Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew
University of Jerusalem, 2LMCP, CNRS UMR7590, Universites Paris 6 & Paris 7,
France, 3Institut National de la Recherche Agronomique, NOPA, France.
As no structural information of the 3D structure of leptin receptor (LEPR) is available the
model of interleukin 6 (IL6) was applied. In that model, a hexameric complex is formed
gradually, first by IL6 molecule which interacts with the IL6R-alpha, then with gp130
forming an inactive trimer which subsequently dimerizes forming an active hexamer, whose
formation is achieved due to interaction of IL6 bound in one trimer (through its site III) with
the immunoglobulin domain (IGD) of gp130 of the other trimer. We have identified the
putative leptin's binding site III by modeling LEPR, on the basis of its alignment with gp130,
and fitting leptin on IL6 in the IL6/gp130 complex as leptin's amino acids 39-42 (LDFI),
which are preserved in all leptin species. To verify this hypothesis and to test its generality we
have prepared and purified to homogeneity human, ovine, rat and mouse triple
(L39A/D40A/F41A) and quadruple (L39A/D40A/F41A/I42A, human and ovine only) leptin
muteins. All six muteins had typical cytokine secondary structure, acted as true antagonists,
namely they interacted with LEPR with affinity similar to the wild type hormone (as
evidenced by SRP and RRA), were devoid of biological activity in several leptin-responsive
bioassays and specifically inhibited leptin action. Those muteins can be prepared in gram
amounts and thus serve as a novel tool of studying leptin function in vitro and in vivo. To
prolong their life in circulation some muteins were pegylated using 40 kDa polyethylene
glycol. Although pegylation decreases the affinity, increasing circulation half-life can
recompensate this deficit so pegylated antagonists are expected to be more potent in vivo.
Antagonizing leptin has been suggested as a possible therapy in auto-immune diseases and
heart failure. Thus leptin antagonists not only offer a novel tool to elucidate the role of leptin
in mammalian physiology and pathology but have a potential of becoming a drug.
- 121 -

Session I : protein domains Poster I, 4
A novel Mitogen-Activated Protein Kinase docking site in the N terminus of MEK5alpha
organizes the components of the Extracellular Signal-Regulated Kinase 5 signaling
pathway
Jan Seyfried, Xin Wang, Giorgi Kharebava, and Cathy Tournier
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford
Road, Manchester M13 9PT, UK
The alternative splicing of the mek5 gene gives rise to two isoforms. MEK5beta lacks an
extended N terminus present in MEK5alpha. Comparison of their activities led us to identify a
novel mitogen-activated protein kinase (MAPK) docking site in the N terminus of MEK5 that
is distinct form the consensus motif identified in the other MAPK kinases. It consists of a
cluster of acidic residues at position 61 and positions 63-66. The formation of the
MEK5/extracellular signal-regulated kinase 5 (ERK5) complex is critical for MEK5 to
activate ERK5, to increase transcription via MEF2, and to enhance cellular survival in
response to osmotic stress. Certain mutations in the ERK5 docking site that prevent
MEK5/ERK5 interaction also abrogate the ability of MEKK2 to bind and activate MEK5.
However, the identification of MEK5 alpha mutants with selective binding defect
demonstrates that the MEK5/ERK5 interaction does not rely on the binding of MEK5a to
MEKK2 via their respective PB1 domains. Altogether these results establish that the N
terminus of MEK5a is critical for the specific organization of the components of the ERK5
signaling pathway.
- 122 -

Session I : protein domains Poster I, 5
Analysis of transiently overexpressed Src Y527F on phosphoproteome signalling
Jörg von Hagen, Uwe Michelsen, Sven Andrecht, Anja Seiler and Rob Hendriks
Merck KGaA, Darmstadt, Germany
Protein phosphorylation and dephosphorylation is a major signaling method for activating and
inactivating cell proteins. Here we describe the overexpression of constitutively activated Src
Y527F and a novel method to identify target proteins down-stream of Src. Src plays important
roles in cell differentiation, proliferation, and survival. For example, stimulation of the
epidermal growth factor receptor, which leads to cell proliferation, results in Src activation.
Src is activated during the G2/M transition. Src plays a role in cell adhesion, cell morphology
and motility, and bone resorption.
- 123 -

Notes
- 124 -

Notes
Notes
- 125 -

- 126 -

Session II. Receptor signaling and G proteins
- 127 -

Session II : Receptor signaling and G proteins Poster II, 1
Developmental Responses of Chicken Ductus Arteriosus to Oxygen and Vasoactive
Mediators
Pia Ågren, Bea Zoer, Lilian Kessels, Jo GR De Mey, Carlos E Blanco and Eduardo
Villamor.
Departments of Pediatrics and Pharmacology, GROW Research Institute, University of
Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. eiv@paed.azm.nl
Background: At birth, the mammalian ductus arteriosus (DA) undergoes alterations in its
pharmacological responsiveness. This phenomena has not been characterized in the chicken
embryo.
Objective: To characterize DA reactivity in the chicken embryo from the last stage of prenatal
development and throughout the perinatal period.
Design/Methods: Isolated DA from 15-d, 19-d non-internally-pipped embryos (total
incubation: 21-d) and from 21-d externally-pipped embryos were mounted in a myograph and
bubbled with 5% O2 /5% CO2/90% N2. The contractile responses induced by O2 (0 to 21%),
KCl, the adrenergic agonist norepinephrine (NE), the "1-adrenoceptor agonist phenylephrine
(Phe), the inhibitor of Kv channels 4-aminopyridine (4-AP), the inhibitor of KATP channels
glibenclamide, the inhibitor of KCa channels tetraethylammonium (TEA), the non-selective
cyclooxygenase (COX) inhibitor indomethacin, the COX-1 inhibitor valeryl salicylate, the
COX-2 inhibitor nimesulide and the relaxations (during 62.5 mM KCl-induced contraction)
induced by acetylcholine (ACh), the NO donor sodium nitroprusside (SNP), the adenylate
cyclase activator forskolin, and the #-adrenergic receptor agonist isoproterenol were tested.
Results: Contractions induced by O2, KCl, and NE increased significantly between 15- and
21-d. but 4-AP-induced contractions did not change. Glibenclamide and TEA did not contract
DA. Indomethacin, valeryl salicylate and nimesulide induced a weak contraction that was not
significantly different to the one induced by the vehicle (ethanol). Relaxations induced by
ACh, SNP and isoproterenol were markedly reduced in the 21-d embryo. In contrast,
forskolin-induced relaxation was not affected by age. When the DA was divided in two
segments, it was observed that the response to O2 and NE was significantly higher in the
pulmonary end and the response to KCl and SNP was higher in the aortic end.
Conclusions: Transition to ex ovo life is accompanied by dramatic changes in chicken DA
reactivity. These changes are characterized by an increase in the contractile responses and a
decrease in relaxation. The reactivity of chicken DA is not homogeneous and depends on the
segment studied. Response to oxygen appears to be located in the pulmonary end of the DA.
- 128 -

Session II : Receptor signaling and G proteins Poster II, 2
Galpha12 interaction with alphaSNAP induces VE-cadherin localization at endothelial
junctions and regulates barrier function
Alexandra V. Andreeva, Mikhail A. Kutuzov, Rita Vaiskunaite, Jasmina Profirovic,
Sanda Predescu, Asrar B. Malik and Tatyana A. Voyno-Yasenetskaya
Department of Pharmacology, College of Medicine, University of Illinois at Chicago,
Chicago, Illinois 60607, U.S.A. Email: aandreev@uic.edu
In vascular endothelium, the endothelial-specific cadherin (VE-cadherin) is essential for the
control of permeability and angiogenesis. Newly synthesized VE-cadherin is transported to
the plasma membrane and forms a homotypic interaction with VE-cadherin from other
endothelial cells, resulting in the assembly of adherens junctions. The involvement of
heterotrimeric G proteins in the regulation of adherens junction function is unclear. Galpha12
has recently been reported to bind directly to the C-terminal region of E-cadherin. In this
study, we identified alphaSNAP, an essential component of the membrane fusion machinery,
as an interactive partner of Galpha12 using yeast two hybrid screening. GST pull-down assays
showed the selective interaction of alphaSNAP with Galpha12 in COS-7 as well as human
umbilical vein endothelial cells (HUVECs). Using domain swapping experiments, we
demonstrated that the N-terminal region of Galpha12 (1-37 aa) was necessary and sufficient
for its interaction with alphaSNAP. Galpha13 with its N-terminal extension replaced by that
of Galpha13 acquired the ability to bind to alphaSNAP, whereas Galpha12 with its Nterminus
replaced by that of Galpha13 lost this ability. Using four point mutations of
alphaSNAP, which alter its ability to bind to the SNARE complex, we determined that the
convex rather than concave surface of alphaSNAP was involved in its interaction with
Galpha12. Co-transfection of HUVECs with Galpha12 and alphaSNAP stabilized VEcadherin
at the plasma membrane, whereas downregulation of alphaSNAP using siRNA
resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with
Galpha12 overexpression, decreased endothelial barrier function. Our results demonstrate a
direct link between the alpha-subunit of G12 and alphaSNAP, and suggest a role for this
interaction in regulating the membrane localization of VE-cadherin and hence formation of
adherens junctions, control of cell confluence and endothelial barrier function.
- 129 -

Session II : Receptor signaling and G proteins Poster II, 3
Rap1: a turnabout for the crosstalk between cadherin and integrin signaling
Fiorella Balzac*, Maria Avolio*, Simona Degani*, Floriana Francalanci*, Irina
Kaverina†, Guido Tarone*, J. Victor Small† and S. Francesco Retta*
*Department of Genetics, Biology and Biochemistry, University of Torino, 10126
Torino, Italy;
†IMBA - Institute of Molecular Biotechnology, Vienna, Austria.
e-mail: francesco.retta@unito.it (S.F. Retta)
The coordinate modulation of cadherin and integrin functions plays an essential role in
fundamental physiological and pathological processes, including morphogenesis and cancer.
However, the molecular mechanisms underlying the functional crosstalk between cadherins
and integrins are still elusive.
We show that the small GTPase Rap1, a crucial regulator of the inside-out activation of
integrins, is a target for E-cadherin-mediated outside-in signaling. In particular, a strong
activation of Rap1 occurs upon adherens junction disassembly and is triggered by E-cadherin
internalization and trafficking along the endocytic pathway. In contrast, Rap1 activity is not
influenced by integrin outside-in signaling. Moreover, the activation of Rap1 is associated
with and controlled by an increased Src kinase activity, and is paralleled by the colocalization
of Rap1 and E-cadherin at the perinuclear recycling endosome compartment, and the
association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain
p120ctn. Finally, the E-cadherin endocytosis-dependent activation of Rap1 is associated with
and is required for the formation of integrin-based focal adhesions.
Our findings provide the first evidence of an E-cadherin-modulated endosomal signaling
pathway involving Rap1, and suggest that cadherins may have a novel modulatory role in
integrin adhesive functions by fine-tuning Rap1 activation.
- 130 -

Session II : Receptor signaling and G proteins Poster II, 4
CXC receptors and chemokines expression in human meningioma: SDF1/CXCR4
signaling activates ERK1/2 and stimulates meningioma cell proliferation.
Federica Barbieri1, Adriana Bajetto1, Carola Porcile1, Alessandra Pattarozzi1,
Alessandro Massa1, Gianluigi Lunardi1, Gianluigi Zona2, Alessandra Dorcaratto3,
Jean Louis Ravetti3, Renato Spaziante2, Gennaro Schettini1 and Tullio Florio1.
1Pharmacology - Dept. of Biology, Oncology and Genetics and 2Neurosurgery - Dept.
Neuroscience, Ophthalmology and Genetics, University of Genova, 3Pathology, San
Martino Hospital – 16132 Genova, Italy. E-mail: federica.barbieri@unige.it
Recent evidence indicates that cancer cells express chemokine (CK) receptors and that their
signaling is crucial for tumor growth regulation, migration and angiogenesis. Here we
analyzed the profiles of expression of CXC CK receptors and related CKs in surgical samples
of human meningiomas. RT-PCR was used to identify the presence of mRNA for CXCR1-5
and their main ligands (growth-related oncogene, GRO1-2-3/CXCL1-2-3; interleukin 8, IL-
8/CXCL8; monokine-induced g-interferon MIG/CXCL9; $-interferon-inducible-protein-10,
IP-10/CXCL10; stromal cell-derived factor-1, SDF1/CXCL12; B-cell activating chemokine-
1, BCA-1/CXCL13). All the five receptors displayed high percentages of positive cases.
CXCR1 was expressed in 34/37, CXCR2 in 31/35, CXCR3 in 30/36, CXCR4 in 43/55 and
CXCR5 in 34/36 cases. Conversely, their ligands showed a lower pattern of expression: IL-8
and GRO1-3 were detected in 14/35 and 15/36 cases, respectively; IP-10 in 15/36, MIG in
10/36, SDF1 in 29/55 and BCA-1 in 1/35. The exclusive binding between SDF1 and CXCR4
suggests that the SDF1/CXCR4 interaction might play a uniquely biological role. To
investigate the signaling mechanisms activated by SDF1 binding to CXCR4 in meningioma,
four short term cultures were obtained from tumor tissues. [3H]-thymidine uptake and
Western blot were carried out to evaluate the proliferative activity of SDF1 and the
involvement of extracellular signal-regulated kinase (ERK1/2) activation. CXCR4 was
functionally coupled as demonstrated by the significant increase of DNA synthesis in primary
cultures which occurred in response to 12.5 nM SDF1. The SDF1-dependent proliferation was
associated with a marked phosphorylation of ERK1/2 and was significantly reduced by
PD98059 (a MEK inhibitor), thus linking the SDF1/CXCR4 pathway to meningioma cell
proliferation via ERK1/2 signal transduction. The simultaneous expression of CXCR1-5 and
their CKs in meningiomas suggests that these receptor-ligand pairs could sustain the tumor
proliferation and, in particular, the SDF1/CXCR4 axis may be important for this process.
- 131 -

Session II : Receptor signaling and G proteins Poster II, 5
Dysregulation of GABAergic Neurotransmission in Mood Disorders
Hendrik Bielau1, Tomasz Gos1, Christian Mawrin2, Kurt Trübner3, Ralf Brisch1,
Gabriela Meyer-Lotz1, Henrik Dobrowolny1, Bruno Baumann1, Hans-Gert Bernstein1,
Bernhard Bogerts1
1Department of Psychiatry, Psychotherapy und Psychosomatic Medicine and 2Institute
of Neuropathology; Otto-von-Guericke-University, Leipziger Str. 44, 39120 Magdeburg,
Germany, E-mail: Hendrik.Bielau@Medizin.Uni-Magdeburg.de
3Institute of Legal Medicine, University of Essen, Hufelandstr. 55, 45122 Essen,
Germany
Alterations of GABAergic neurotransmission are assumed to play a crucial roll in the
pathophysiology of mood disorders. GABA acts via binding to A and B receptors, whereas
the B receptor is G-protein-coupled. Glutamatdecarboxylase (GAD) is the key enzyme of
GABA synthesis. The goal of our postmortem investigation was the bihemispherical
immunohistochemical staining of GAD neurons in brain structures which are important for
mood regulation. In 20 brains of patients with mood disorders and 19 controls (C) an
immunohistochemical staining of GAD-65/67 neurons was performed in dorsolateral
prefrontal cortex, orbitofrontal cortex, anterior cingulate cortex, superior temporal gyrus,
hippocampus and medial and lateral thalamus with consecutive determination of neuronal
density. In the diagnosis group were 11 patients with bipolar disorder (BD) and 9 patients
with major depressive disorder (MDD). The data were tested statistically using ANOVA und
post-hoc Tukey tests. The evaluation revealed significant differences between the groups (C,
BD, MDD) in dorsolateral prefrontal cortex, orbitofrontal cortex, superior temporal gyrus and
hippocampus. The post-hoc tests demonstrated higher neuronal densities in unipolar patients
compared to bipolar patients and controls in dorsolateral prefrontal cortex, superior temporal
gyrus and hippocampus. In the orbitofrontal cortex a higher neuronal density was found in
bipolar and unipolar patients compared to controls. In the bipolar group dose equivalents of
antidepressants given prior to death correlated positively with the neuronal density in the
hippocampus. In conclusion, the current data on GAD-65/67 point on a dysregulation of the
GABAergic system in mood disorders. Possibly, existing deficits of GABAergic
neurotransmission will be compensated or overcompensated by antidepressants. Moreover,
preliminary data point toward a diminished density of GAD terminals.
- 132 -

Session II : Receptor signaling and G proteins Poster II, 6
GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier
dysfunction.
Anna A. Birukova, Alexander D. Verin, Konstantin G. Birukov
Department of Medicine, University of Chicago, Chicago, Illinois 60637, U.S.A. E-mails:
abirukov@medicine.bsd.uchicago.edu, averin@medicine.bsd.uchicago.edu,
kbirukov@medicine.bsd.uchicago.edu,
Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements (actin
filaments, microtubules, intermediate filaments) and cell contact protein complexes (focal
adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic
agonist thrombin caused partial microtubule (MT) disassembly, which was linked to
activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction and
dysfunction of lung EC barrier. GEF-H1 is a MT-associated Rho-specific guanosine
nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity.
In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho
activation, myosin light chain phosphorylation, actin remodeling and EC barrier dysfunction
associated with partial MT disassembly. Our results show that depletion of GEF-H1 or
expression of dominant negative GEF-H1 mutant significantly attenuated permeability
increase, actin stress fiber formation and increased MLC and MYPT1 phosphorylation
induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild
type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on
stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in
the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule
involved in crosstalk between MT and actin cytoskeleton in agonist-induced Rho-dependent
EC barrier regulation.
- 133 -

Session II : Receptor signaling and G proteins Poster II, 7
Role of PAK1 and PAK2 in HGF stimulation of cancer cell motility
Michael D. Bright, Anne J. Ridley
Ludwig Institute for Cancer Research (UCL Branch), 91 Riding House Street, London,
W1W 7BS, England. E-mail : michael@ludwig.ucl.ac.uk
p21-activated kinases (PAKs) are a family of six serine/threonine protein kinases which signal
downstream of Rac and Cdc42. PAKs are divided into two groups with PAK1-3 in group 1
and PAK4-6 in Group 2. Each PAK isoform has a C-terminal kinase domain and a Cdc42/Rac
interacting binding (CRIB) domain with an N-terminal regulatory region. Group 1 PAKs also
have an autoinhibitory domain (AID) which overlaps the CRIB domain and is essential to
their regulation by Rac and Cdc42. PAKs are also regulated by phosphorylation at multiple
sites and by localization via adapter proteins. PAKs are important in the regulation of
cytoskeletal dynamics and are particulary interesting because they are overexpressed in some
cancers and have been shown to regulate cancer cell migration and invasion.
HGF stimulation induces scattering, enhanced motility and morphological changes in several
epithelial cell types. We have studied a variety of human cancer cell lines that respond to
HGF by scattering, and found that HGF stimulation of these cells rapidly stimulates
phosphorylation of PAK1 and PAK2. In subsequent studies we have focussed on DU145
prostate cancer cells, where HGF induces redistribution of #-catenin from cell:cell junctions
to membrane ruffles as cells detach from each other and scatter. Interestingly, the scattering
response of DU145 cells to HGF is dependent on substrate; they scatter when plated on
plastic but not on glass. The effect of siRNA knockdown of PAK1 and PAK2 on HGFinduced
scattering and motility is being investigated.
- 134 -

Session II : Receptor signaling and G proteins Poster II, 8
Expression of chemokine receptor CXCR4 (CD184) in B chronic lymphocytic leukaemia
cells.
Nicolaas H. C. Brons1, Valérie Palissot1, Chantal Schwartz1, Manon Bosseler1,
Bernadette Leners1, Guy J Berchem1,2.
1Laboratoire d'Hémato-Cancérologie Expérimentale, CRP-Santé, 84 Val Fleury, L1526
Luxembourg. E-mail : berchem.guy@chl.lu
2Hémato-Cancérologie, Centre Hospitalier de Luxembourg, 4 rue barblé, L1210
Luxembourg.
Chronic lymphatic leukaemia (CLL) accounts for about 30% of adult leukemias in Western
countries. This lymphoma is very indolent but relentless, with median survivals of almost a
decade. The disease is characterized by the accumulation of a monoclonal population of CD5
positive neoplastic B cells in secondary lymphoid organs, marrow, and blood. Although the
slowly proliferating cells are sensitive to chemotherapeutic agents, chemotherapy is almost
never curative and relapse inevitably follows. In 1975 Kanti Rai has described a clinical
staging system which until now serves as basis for treatment decisions.
The study was aimed to explore the CD184 also known as the CXCR4 chemokine receptor on
B-lineage chronic lymphocyte leukaemia (B-CLL) cells of various disease stages and its
relationship with others receptor expressions. CD 184 is expressed on CLL cells and normal
B cells, it is one of the main receptors that mediate B cell entry in a secondary lymphoid
tissues and their homing to T cell and B cell zones. Marrow stromal cells or nurselike cells
constituvely secrete CXCR12, the ligand for CXCR4, thereby attracting and rescuing CLL B
cells from apoptosis in a contact dependent fashion. Therefore, CD184 has been mentioned as
a possible prognostic marker in CLL. Flow cytometry was used to detect the CXCR4
expression in patients of different Rai stages The samples were measured on a FACSCanto
(BD) and up to 6 markers per tube were analysed by the DivaSoftware (BD). Calibration
curves for the MFE were obtained with Fluorospheres (DakoCytomation, K0110). More than
thirty CD markers have been used to characterized B leukemic cells and correlation was
researched with CD184. Of the 44 persons , 11 were healthy donors, 5 had Rai stage 0
disease, 12 had Rai stage I disease, 3 had Rai stage II disease, 4 had Rai stage III disease, 9
had Rai stage IV disease. CXCR4 was consistently expressed on circulating B-CLL with a
mean fluorescence intensity higher than in cells from healthy volunteers. There was a
correlation between CXCR4 expression and late stage of the disease. Interestingly,
chemotherapeutic treatments seem to decrease level of this marker down to level of earliest
stages. However, there was no correlation of CD184 level and quantitative expressions of
known CD markers tested. Different correlations with previous treatments and known
prognostic factors will be described and clinical implications will be discussed.
- 135 -

Session II : Receptor signaling and G proteins Poster II, 9
Genetically-encoded, FRET-based sensors for monitoring protein kinase signaling
Justin J. Brumbaugh, Christiane Jost, Carsten Schultz
Gene Expression Program, European Molecular Biology Laboratory (EMBL),
Heidelberg 69117, Germany. Email: brumbaug@embl.de, jost@embl.de,
Schultz@embl.de
Protein phosphorylation and dephosphorylation are important forms of posttranslational
regulation and a fundamental means for controlling cellular fate and function. Deviant kinase
and phosphatase activities are responsible for numerous diseases, and in particular, cancer.
Due to the far reaching implications of phosphorylation and dephosphorylation, it is
imperative to develop tools to closely monitor these dynamic processes in real time.
Previously, we reported the construction of a genetically encoded FRET probe for monitoring
protein kinase C (PKC) activity in living cells (Schleifenbaum et al., J. Am. Chem. Soc.,
2004). To help unravel complex and often interdependent, intracellular signaling we are
working to adapt this probe to other kinase specificities. To this end we have developed two
novel protein kinase A (PKA) sensors and, to the best of our knowledge, the first genetically
encoded dual parameter FRET probe that responds independently to PKA and PKC
stimulation with differential output. Using an array-based approach and automated
microscopy, we have also developed a method using in vivo imaging to screen potential
probes under more comparable conditions and with higher throughput. This technology may
lead to more versatile, cell-based assays for kinase activators and inhibitors.
- 136 -

Session II : Receptor signaling and G proteins Poster II, 10
Stimulation of signal transduction by mono(ADP-ribosyl)ation inhibitors: ecto-ADPRT
as modulators of receptor stimulation?
Claudia Cerella, Maria C. Albertini*, Augusto Accorsi*, Antonio Bergamaschi#, Maria
D'Alessio, Milena De Nicola, Silvia Nuccitelli, Lina Ghibelli
Dipartimento di Biologia, #Cattedra di Medicina del Lavoro, Universita' di Roma Tor
Vergata, Via Ricerca Scientifica 1, 00133, Rome, Italy; *Istituto di Chimica Biologica A.
Fornaini, Universita' di Urbino, Urbino, Italy. E-mail: cerella@uniroma2.it
A set of mono(ADP-ribosyl)ation inhibitors (m-ADPRI) such as 3-aminobenzamide (3-
ABA); nicotinamide (NA); vitamin K1 (K1); vitamin K2 (K2); novobiocin (NVB); miodobenzylguanidine
(mIBG), but not PARP inhibitors (DPQ; low 3-ABA or NA doses)
rapidly and specifically mobilise Ca2+ from a thapsigargin-sensitive store in U937 monocytic
and Jurkat T lymphocitic cells, as well as their normal counterpart (peripheral blood
monocytes and lymphocytes). A capacitative Ca2+ influx ensues. m-ADPRI-induced Ca2+
mobilisation is insensitive to dantrolene, inhibitor of cyclic(ADP-ribose)-sensitive ryanodine
channels. Instead, it is sensitive to U7322 and neomycin (neo), inhibitors of phospholipase C
(PLC), the enzyme responsible for production of the endoplasmic reticulum (ER) Ca2+
channels agonist IP3. Accordingly, m-ADPRI stimulate IP3 production. NA-, K2-, NVB- and
mIBG-induced Ca2+ mobilisation is also sensitive to pertussis toxin (PTX), indicating that
these compounds activate PLC by stimulating G proteins (subtypes Gi). 3-ABA and K1 do
not, thus possibly activating PLC by other means (tyrosin kinases?). The direct target(s) of m-
ADPRI, may possibly be ecto-ADPRTs, a set of enzymes with poorly known functions
localised on the external face of plasma membrane. Indeed, competition experiments between
ADPRT substrate (NAD) and inhibitor (3-ABA) showed that extracellular NAD dosedependently
inhibited 3-ABA-induced Ca2+ mobilisation. Our current model is that m-
ADPRI may stimulate signal transduction by freeing receptors from a negative modulator
(ADP-ribose) provided by ecto-ADPRTs.
- 137 -

Session II : Receptor signaling and G proteins Poster II, 11
Phospholipase C-g1 attenuates growth hormone signaling by forming a ternary complex
with Jak2 and protein tyrosine phosphatase-1B
Jang Hyun Choi1, Hyeon Soo Kim1, Sun-Hee Kim1, Yun Soo Bae2, H. Moo Kwon3,
Sung Ho Ryu1, and Pann-Ghill Suh1*
1 Department of Life Science, Pohang University of Science and Technology, Pohang,
Kyungbuk, 790-784, Republic of Korea, E-mail : pgs@postech.ac.kr
2 Divisions of Molecular Life Sciences, Center for Cell Signaling Research, Ewha
Womans University, Seoul 120-750, Republic of Korea
3 Divisions of Nephrology, University of Maryland, Baltimore, Maryland 21201, USA
Growth hormone (GH) binds to its membrane receptor (GHR) and regulates many cellular
functions such as proliferation, differentiation and chemotaxis. While the activation of GHmediated
signaling is well understood, the precise mechanism responsible for its regulation
has not been well assessed. In this study, we demonstrate that PLC-$1 modulates the action of
GH-mediated signaling by interacting with tyrosine kinase Jak2 in a GH-dependent manner.
In the absence of PLC-$1 (PLC-$1-/- MEFs), GH-induced JAK2 and STAT5
phosphorylations significantly increased and the re-expression of PLC-$1 reduced GHinduced
Jak2 activation. Furthermore, GH-induced JAK2 phosphorylation was enhanced by
the specific knock-down of PLC-$1 using siRNA. Interestingly, PLC-$1 physically linked
Jak2 with protein tyrosine phosphatase-1B (PTP-1B), which was implicated in the modulation
of cytokine signaling via Jak2. In PLC-$1-/- MEFs, GH-dependent activation of both STAT-5
and c-Fos were up-regulated, and GH-induced proliferation was potentiated. These results
suggest that PLC-$1 plays a key role in regulation of GH-mediated signaling by attenuating
the activation of JAK2.
- 138 -

Session II : Receptor signaling and G proteins Poster II, 12
ATP-dependent mechanotransduction by chondrocytes seeded in agarose constructs and
subjected to dynamic compression
T T Chowdhury and M M Knight
Cell and Tissue Engineering, Department of Engineering, Queen Mary, University of
London, Mile End Road, London. E1 4NS. UK E-mail : t.t.chowdhury@qmul.ac.uk
Introduction: Mechanical stimulation is essential in maintaining the biochemical properties of
articular cartilage. Both .NO and calcium are well known intracellular signaling molecules
which play an important role in chondrocyte mechanotransduction. It has been shown that
chondrocytes respond to mechanical stimuli by releasing ATP. Extracellular ATP binds to the
P2Y2 purinoreceptors, which may activate intracellular calcium levels and release of .NO,
therefore influencing chondrocyte metabolism. The current study examines whether inhibition
of mechanosensitive ATP by receptor antagonists will influence .NO levels, cell proliferation
and proteoglycan synthesis by chondrocytes cultured in agarose constructs and subjected to
dynamic compression.
Methods: Bovine chondrocytes were seeded in agarose gel and equilibrated in culture for 24
hrs. Using a full characterized cell-straining system, constructs were subjected to 15%
dynamic compression at a frequency of 1Hz for 48 hours in 1 ml of unsupplemented culture
medium or in medium supplemented with 20 µM gadolinium chloride (putative
mechanosensitive ion channel blocker), 100 µM suramin (P2 receptor antagonist) and 10
units.ml-1 apyrase (catalyses extracellular ATP). Medium was additionally incubated with 1
µCi.ml-1 [3H]-thymidine and 10 µM 35SO4, for the assessment of cell proliferation and
proteoglycan synthesis, respectively. Nitrite, a stable end product of .NO was measured using
the Griess assay.
Results: The application of dynamic compression resulted in the strain-induced stimulation of
[3H]-thymidine incorporation in unsupplemented constructs (p

Session II : Receptor signaling and G proteins Poster II, 13
The tyrosine 974 within the LIF-R-chain of the gp130/LIF-R heteromeric receptor
mediates negative regulation of LIF signalling
Thomas Clahsen1, Ute Lehmann1, Claudia Stross1, Heike M. Hermanns1, Rudolf
Volkmer-Engert2, Jens Schneider-Mergener2, Peter C. Heinrich1, Fred Schaper1
1 Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52074 Aachen,
Germany, E-mail: Thomas.Clahsen@post.rwth-aachen.de
2 Department of Medical Immunology, Universitätsklinikum Charité, Molecular
Libraries and Recognition, Ziegelerstrasse 5-9, Berlin D-10117, Germany
Signalling of interleukin-6 and interleukin-11 through gp130 homodimeric receptor
complexes has been analysed with respect to initiation and termination of signalling in great
detail. Gp130 contains a crucial motif around tyrosine Y759 which mediates negative
regulation through the feedback inhibitor SOCS3 and the protein tyrosine phosphatase SHP2.
Signalling of LIF, CNTF, CT-1, CLC or OSM through gp130/LIF-R is believed to be similar
due to the presence of the common signal transducer gp130 within the receptor complexes
utilized, but the difference in the composition of gp130/gp130-homodimers and gp130/LIF-Rheterodimers
is likely to be reflected in different signalling. Here, we analysed the
contribution of the LIF-R within the gp130/LIF-R complex to negative regulation mediated
by SHP2 and SOCS3. We show that SHP2 contributes to the negative regulation of signalling
through gp130/LIF-R complexes. The inhibitory tyrosine motifs within the cytoplasmic parts
of gp130 and the LIF-R act independently. Whereas SHP2 and SOCS3 bind directly to the
inhibitory motif of gp130, only SHP2 was found to bind to the corresponding inhibitory
sequence of the LIF-R. This observation was further corroborated by experiments indicating
that mainly gp130 contributes to the inhibition of signalling by SOCS3.
- 140 -

Session II : Receptor signaling and G proteins Poster II, 14
Serotonin-induced inhibition of voltage-gated K+ currents and contraction in
pulmonary arteries. Signalling via 5-HT(2A) receptors.
Laura Cobeño, Angel Cogolludo, Laura Moreno, Giovanna Frazziano, Tizziana Russo,
Juan Tamargo and Francisco Perez-Vizcaino.
Department Pharmacology. School Medicine. Universidad Complutense de Madrid.
28040 Madrid. SPAIN. E-mail: acogolludo@ift.csic.es
Multiple lines of evidence indicate that serotonin (5-HT) and voltage-gated potassium (Kv)
channels independently play a central role in the pathogenesis of pulmonary hypertension
(PH). We hypothesized that 5-HT might module the activity of Kv channels, therefore,
establishing a link between these pathogenetic factors in PH. We studied the effects of 5-HT
on Kv currents in rat pulmonary artery smooth muscle cells (PASMC) and on contractile
force in isolated pulmonary arteries (PA). 5-HT reduced native Kv currents, depolarized
PASMC and caused a contraction of PA. The 5-HT(2A) receptor antagonist ketanserin and
the 5-HT(1B) receptor antagonist SB224289 inhibited 5-HT-induced contraction by 72 ± 4%
and 22 ± 2%, respectively. The contraction induced by 5-HT was also attenuated by the 5-HT
transporter (5-HTT) inhibitor fluoxetine, which also antagonizes 5-HT(2A) receptors, but not
by other 5-HTT inhibitors such as fluvoxamine or citalopram. The inhibitory effects of 5-HT
on Kv currents were prevented by ketanserin and fluoxetine, but not by any of the other drugs.
Furthermore, ketanserin inhibited the depolarizing effect induced by 5-HT. 5-HT induced
inhibition of Kv current was insensitive to the PKCzeta pseudosubstrate inhibitor. In contrast,
inhibition of phospholipase C (PLC, U-73122), classic PKCs (Go-6976) or tyrosine kinases
(genistein and tyrphostin 23) prevented the effects of 5-HT on Kv currents and contractions.
Altogether these results indicate that 5-HT inhibits native Kv currents following the activation
of 5-HT(2A) receptors via PLC, classic PKCs and tyrosine kinase. The inhibition of Kv
channels contributes to 5-HT-induced pulmonary vasoconstriction and might play a role in
PH.
- 141 -

Session II : Receptor signaling and G proteins Poster II, 15
Thromboxane A2-induced inhibition of Kv currents via PKCzeta: Role of reactive
oxygen species and Kv1.5 channels
Angel Cogolludo, Laura Moreno, Giovanna Frazziano, Federica Lodi, Laura Cobeño,
Valeria Queirolo, Eduardo Villamor, Juan Tamargo and Francisco Perez-Vizcaino.
Department Pharmacology. School Medicine. Universidad Complutense de Madrid.
28040 Madrid. SPAIN. E-mail: acogolludo@ift.csic.es.
Voltage-gated potassium channels (Kv) and thromboxane A2 (TXA2) have been involved in
several forms of human and experimental pulmonary hypertension. We have reported that the
TXA2 analog U46619, via activation of TP receptors, inhibited Kv currents in rat pulmonary
artery smooth muscle cells (PASMC), increased cytosolic calcium and induced a contractile
response in isolated rat and piglet pulmonary arteries (PA). These effects were inhibited by
non selective PKC inhibitors and by the selective PKCzeta pseudosubstrate inhibitor. Herein,
we have analyzed the role of reactive oxygen species and Kv1.5 channels, a major channel
protein contributing to Kv currents in PASMC, in the signalling pathway. U46619 inhibited
Kv currents in native PASMC and these effects were strongly inhibited by addition of catalase
to the internal solution in the patch pipette. The membrane permeable hydroperoxide t-butyl
hydroperoxide also inhibited Kv currents. The contractile responses to U46619 in isolated
pulmonary arteries were inhibited by polyethylenglycol-catalase (PEG-catalase, a membrane
permeable form of catalase) and the NADPH oxidase inhibitors DPI and apocynin. U46619
increased dichlorofluorescein fluorescence, an indicator of intracellular hydrogen peroxide, in
pulmonary arteries and this effect was prevented PEG-catalase. U46619 also inhibited Kv
currents in Ltk(-) cells stably expressing hKv1.5 channels. These cells strongly expressed
PKCzeta as measured by Western blot. In conclusion, activation of NADPH oxidase and the
subsequent production of hydrogen peroxide are involved in the Kv channel inhibition and the
contractile response induced by the TXA2 analog U46619 in rat PA. Kv1.5 channels are
possible targets for the inhibitory effect of TXA2 in native pulmonary arteries.
- 142 -

Session II : Receptor signaling and G proteins Poster II, 16
Purification of G protein-coupled receptors and associated protein complexes under
native condition
Avais M. Daulat*, Jean-Luc Guillaume*, Pascal Maurice*, Philippe Delagrange$, Ralf
Jockers*#.
* Institut Cochin, Département Biologie Cellulaire. Inserm, U567. CNRS, UMR 8104.
Université Paris Descartes, Faculté de Médecine René Descartes, UMR-S 8104, Paris, F-
75014 France. # Email jockers@cochin.inserm.fr
$ Institut de Recherche SERVIER, Suresnes, France
G Protein-Coupled Receptors (GPCRs) constitute the largest family of membrane receptors
and are targeted by about half of the drugs prescribed for human diseases. In contrast to other
protein families the “interactom”, the repertoire of proteins that interact with a given protein,
is only poorly known for GPCRs. Faster progress in the field has been mainly hampered by
the fact that “classical” protein-protein interaction approaches such as yeast two-hybrid and
affinity purification techniques, are not well-adapted for these multi-transmembrane-spanning
proteins.
Recently, the tandem affinity purification (TAP) method has been developed to successfully
purify soluble protein complexes. Here, we adapted this method to the purification of GPCRassociated
protein complexes. This technique relies on a two-step purification protocol of
proteins that associate with the TAP-tagged bait protein in living cells. We established two
HEK 293 clones each stably expressing a distinct C-terminally TAP-tagged GPCR. Among
different detergents tested, digitonin and Brij 96 were selected for their ability to solubilize
high amounts of functional receptors. Typical purification yields varied between 20 and 30%.
The successful co-purification of receptor-associated proteins was shown by the presence of
heterotrimeric G proteins as determined by western blot and mass spectrometric analysis.
Large-scale purifications yielded in coomassie blue stainable protein amounts that were
identified by mass spectrometry.
In conclusion, we established a purification procedure for the isolation of protein complexes
that associated with GPCRs in living cells under basal and activated conditions.
- 143 -

Session II : Receptor signaling and G proteins Poster II, 17
Functional overlapping between caspase cleavage site and CBL binding sequence in the
juxtamembrane domain of the MET receptor.
Julien DEHEUNINCK, Catherine LEROY, Zongling JI, Bénédicte FOVEAU, Vincent
VILLERET, David TULASNE and Véronique FAFEUR
CNRS UMR 8117, Institut de Biologie de Lille, Institut Pasteur de Lille, B.P.447, 59021
Lille, FRANCE. E-mail : veronique.fafeur@ibl.fr
Hepatocyte growth factor/ scatter factor (HGF/SF) binds and activates the MET tyrosine
kinase receptor to transduce cell survival. Nonetheless, we previously demonstrated that stress
stimuli cause a caspase-dependent cleavage in the MET juxtamembrane domain, which
converts MET into a pro-apoptotic p40 MET fragment. The caspase 3 cleavage occurs at
aspartic acid residue D1000. This residue is juxtaposed to tyrosine Y1001, which is rather
involved in binding CBL upon MET activation by its ligand, allowing MET trafficking and
recycling. We wished to examine the functional relationships between caspase-dependent
cleavage and CBL binding at the ESVDY sequence of the juxtamembrane domain of MET.
We first established that the caspase cleavage- and CBL binding- sites overlapped, since a
MET D1000N mutant lost the ability both to generate p40 MET and to recruit CBL. We then
individually mutated all amino acids of the ESVDY sequence of MET constituting the
caspase and CBL sites. While a MET V999A mutant was selectively defective in generating
p40 MET, a MET Y1001F mutant was selectively defective in recruiting CBL, demonstrating
that caspase-dependent cleavage and CBL recruitment are dissociable mechanisms. We
further examined the consequence of MET phosphorylation at CBL binding site on the
caspase-dependent cleavage. Using in vitro MET phosphorylation and dephosphorylation, we
demonstrated that generation of p40 MET is unfavored when MET is phosphorylated, and
vice versa. These data were strengthened by modeling studies using the MET peptide
sequence ESVDYR and the known structure of caspase 3 or CBL in complex with specific
peptide sequences. These studies show that a phosphorylated Y1001 is not compatible with
specific association of caspase-3 to MET and also exclude simultaneous fixation of both
caspase 3 and CBL to MET.
In conclusion, we demonstrated that the caspase-dependent cleavage of MET and binding of
CBL to the activated MET receptor are dissociable and incompatible mechanisms. As a
consequence, we propose that HGF/SF can protect MET against caspase-dependent cleavage
by activating autophosphorylation of the receptor.
- 144 -

Session II : Receptor signaling and G proteins Poster II, 18
Identification of PAR1-G protein signalling pathways involved in thrombin-induced
CCL2 release
Xiaoling Deng, Paul F Mercer, Geoffrey J Laurent and Rachel C Chambers.
Centre for Respiratory Research, University College London, 5 University Street,
WC1E 6JJ, London, England. E-mail: x.deng@ucl.ac.uk
Introduction: In experimental models of lung injury, activation of proteinase-activated
receptor-1 (PAR1) by coagulation proteinases is critical in driving both the inflammatory and
fibrotic response. PAR1 exerts its pluripotent cellular effects by concomitant activation of
G"i/o, G"q and G"12/13. We have previously shown that protection from lung inflammation
and fibrosis in PAR1 deficient (PAR1 KO) mice is accompanied by reduced lung levels of the
potent thrombin-inducible protein CCL2 (MCP-1/JE). The aim of this study was to examine
the signalling pathways by which PAR1 activation induces the release of this chemokine by
cultured mouse lung fibroblasts. Methods and Results: Wild type mouse lung fibroblasts
(MLFs) were stimulated with thrombin, TFLLR-NH2 and RLLFT-NH2 (PAR1 peptide
agonist and control peptide respectively), and CCL2 protein release into cell culture
supernatants was assessed by ELISA. Both thrombin and TFLLR induced CCL2 release by
MLFs in a time- and dose-dependent manner. No response was obtained with the control
peptide RLLFT-NH2 or in PAR1 KO fibroblasts. The PAR1 specific antagonist RWJ-58259
(1µM) completely blocked thrombin induced-CCL2 release. In order to investigate potential
signalling pathways involved the effects of pertussis toxin (PTX, G"i/o inhibitor), Ro-318425
(Protein Kinase C (PKC) inhibitor and U0126 (MEK1/2 inhibitor) on PAR1 activation
induced-CCL2 release was also assessed. PTX inhibition of G"i/o had no effect on CCL2
release induced by thrombin, whereas Ro-318425 inhibition of PKC reduced CCL2 release by
49±6%, and U0126 inhibition of MEK1/2 reduced CCL2 release by 53±8% (all p

Session II : Receptor signaling and G proteins Poster II, 19
Human Recombinant Vasostatin 1 May Interfere with Cell-Extracellular Matrix
Interactions
Valentina Di Felice1, Francesco Cappello1, Antonella Montalbano1, Nella Ardizzone1,
Claudia Campanella1, Angela De Luca1, Daniela Amelio 2, Bruno Tota2, Angelo Corti3
and Giovanni Zummo1
1Human Anatomy Section, Di.Me.S., University of Palermo, Via del Vespro 129, 90127
Palermo, Italia, e-mail: valentina.difelice@unipa.it; 2 Sezione di Fisiologia Organismale,
Dipartimento di Biologia Cellulare, Università della Calabria CAP 87036, Arcavacata di
Rende, Cosenza, Italia, e-mail: tota@unical.it; 3 DIBIT –Department of Oncology, San
Raffaele H Scientific Institute Via Olgettina 58, 20132, Milano, Italia, E.mail
corti.angelo@hsr.it.
Vasostatin 1 (VS-1), the N-terminal fragment derived from the cleavage of Chromogranin A
(CgA), has been shown to exert several biological activities on several tissues and organs [1].
Recently, it has been reported that human recombinant VS-1 (STA-CgA 1-78) may alter
myocardial contractility in eel, frog and rat hearts [2]. In this study we have explored if STA-
CgA 1-78 can induce intracellular cascades interacting both with adhesion molecules and/or
extracellular matrix components (ECM), i.e. involvement of the heat shock protein 90
(HSP90) and the endothelial NOS (eNOS), known to be implicated in signal transduction
mechanisms affecting myocardial contractility. We used 3D-cultured adult rat cardiomyocytes
cultivated over fibronectin or fibroblasts or embedded in Matrigel or Collagen type I. Aurion
conjugated VS-1 (Au-STA-CgA1-78) has been used to identify possible sites of interaction of
this molecule with the cell membrane. We found that in our 3D-colture, cell-ECM
interactions played a crucial role in the cellular localization of HSP90 as well as in the
expression of eNOS. VS-1 appeared to modulate cell-ECM interactions, thereby remarkably
leading to a different cellular localization of HSP90. Moreover, Au-STA-CgA1-78 was never
detected inside the cell nor overlapping the plasma membrane, but nearby the outer side of the
cardiomyocyte plasmalemma, at a particular distance, typical of integrins. On the whole, these
data suggest that VS-1 does not have a classic receptor on the membrane but that integrins
may represent a non-conventional VS-1 receptor modulating eNOS signalling pathway.
[1] Helle KB (2004) Biol. Res 79 :769-794; [2] Cerra MC, De Iuri L, Angelone T, Corti A,
Tota B (2005) Bas Res Cardiol 100:1-10.
- 146 -

Session II : Receptor signaling and G proteins Poster II, 20
Angiotensin receptor signal transduction involving src and MAPK in rat nucleus tractus
solitarius
Takayuki Endoh and Takashi Suzuki
Department of Physiology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba
261-8502, Japan. E-mail : tendoh@tdc.ac.jp
It is recognized that brain contains all the components of the renin-angiotensin systems
(RAS). The nucleus tractus solitarius (NTS) is known to plays a major role in the regulation
of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltagedependent
calcium channels (VDCCs) serve as crucial mediators of membrane excitability
and Ca2+-dependent functions. The purpose of this study was to investigate the effects of
Angiotensin (Ang) on VDCCs currents (ICa) in the NTS using patch-clamp recording
methods. An application of Ang caused facilitation of L-type ICa. AT1 receptors antagonist,
Losartan antagonized the Ang-induced facilitation of ICa. Intracellular dialysis of the Giprotein
antibody attenuated the Ang-induced facilitation of ICa. Both Src tyrosine kinase
inhibitor and mitogen activated protein kinase (MAPK) inhibitor attenuated the Ang-induced
facilitation of ICa. p38 MAPK inhibitor also attenuated the Ang-induced facilitation of ICa.
These results indicate that Ang facilitates L-type VDCCs via Gi-proteins involving Src
tyrosine kinase and p38 MAPK kinase mediated by AT1 receptors in NTS.
- 147 -

Session II : Receptor signaling and G proteins Poster II, 21
Alzheimer’s Amyloid Precursor Protein in Human Sperm
Margarida Fardilha, Sandra I. Vieira, Sara C. T. S. Domingues, Odete A. B. da Cruz e
Silva and Edgar F. da Cruz e Silva
Center for Cell Biology, University of Aveiro, 3810-193 Aveiro, Portugal (E-mail:
dacruze@bio.ua.pt)
The Alzheimer’s Amyloid Precursor Protein (APP) is a type I transmembrane glycoprotein
with receptor-like characteristics that originates the Abeta peptide by proteolytic processing.
Abeta is potentially cytotoxic and the major component of the cerebral amyloid plaques in
Alzheimer’s Disease (AD) patients. Nonetheless, APP is ubiquitously expressed in
mammalian cells with a broad tissue distribution, and Abeta deposition has been reported to
occur in many cells outside the nervous system. Many putative functions have been suggested
for APP, although its precise physiological role remains to be elucidated. As several results
point to a role of chronic inflammation in AD pathogenesis and suggest that AD might be a
systemic disorder, the importance of APP function in non-neuronal cells/tissues has gained
further relevance. Previous studies have shown that APLP2 is found in testis and sperm. Our
results are consistent with those observations but also point to the presence of APP itself in
human sperm. The present immunocytochemical studies using a variety of antibodies that
either recognize APP-specific epitotes or epitopes shared with other APP family members
reveal quite distinct distributions in human sperm, thus suggesting a putative role for this
important protein in sperm function. We will present the results obtained and discuss their
significance for sperm signal transduction mechanisms.
Supported by FCT and CBC.
- 148 -

Session II : Receptor signaling and G proteins Poster II, 22
Sequential caspase cleavages generate pro-apoptotic factor from MET tyrosine kinase
receptor
Bénédicte FOVEAU*, Catherine LEROY*, Julien DEHEUNINCK, Zongling JI,
Véronique FAFEUR and David TULASNE.
*These authors contributed equally to the study
CNRS UMR 8117, Institut de Biologie de Lille, Institut Pasteur de Lille, B.P.447, 59021
Lille, FRANCE.
The receptor of hepatocyte growth factor/scatter factor (HGF/SF), the MET tyrosine kinase
receptor, is known to be essential for normal development and cell survival. We previously
showed that stress stimuli cause a cleavage in the MET juxtamembrane domain that converts
the receptor into a pro-apoptotic 40 kDa fragment (p40 MET). We report here an additional
caspase cleavage of MET occurring in its extreme C-terminal region. The cleavage occurs at
aspartic residue 1374 within the DNID sequence of mouse MET. Although this cleavage
removes only the five last amino acids, it has important functional consequences. First, the Cterminal
cleavage favors the juxtamembrane one, leading to efficient generation of the proapoptotic
p40 MET fragment. Second, the p40 MET generated by the two steps cleavage has
enhanced pro-apoptotic activity. These results revealed a tight regulation of MET caspase
cleavages resulting in the reshaping of a survival receptor to a pro-apoptotic factor.
- 149 -

Session II : Receptor signaling and G proteins Poster II, 23
Involvement of hydrogen peroxide in the activation of BKCa currents and vasodilator
effects of quercetin in rat coronary arteries
Giovanna Frazziano, Angel Cogolludo, Laura Moreno, Ana Briones, Federica Lodi,
Laura Cobeño, Mercedes Salaices, Juan Tamargo, and Francisco Perez-Vizcaino.
Department Pharmacology. School Medicine. Universidad Complutense de Madrid.
28040 Madrid. SPAIN. E-mail: frazzygio@jumpy.it
Calcium-activated potassium channels (BKCa) regulate coronary artery tone in vivo, play a
key role in blood pressure regulation and have been suggested as novel potential drug targets
in hypertension. The dietary flavonoid quercetin exerts systemic and coronary vasodilator
effects in vitro, reduces blood pressure in several rat models of hypertension and its
consumption is associated with a lower mortality rate from coronary heart disease in
epidemiological studies. We hypothesized that quercetin might activate BKCa channel in
isolated myocytes from rat coronary arteries and that this mechanism might be involved in its
coronary artery relaxant effects. Membrane currents were measured using the whole-cell
configuration of the patch-clamp technique. Quercetin (1-30 microM) increased the outward
currents in the whole range of test potentials and increased the frequency of spontaneous
transient outward currents (STOCs) carried by BKCa channels. These effects were abolished
by the selective BKCa blocker iberiotoxin and by the hydrogen peroxide scavenger catalase in
the internal pipette solution. The membrane permeable hydroperoxide t-butyl hydroperoxide
also increased outward currents. Quercetin increased dichlorofluorescein fluorescence, a
widely used indicator of intracellular hydrogen peroxide, in coronary arteries with a similar
magnitude and time-course than t-butyl hydroperoxide. Quercetin-induced increase was
prevented by polyethylenglycol-catalase, indicating the role of cytosolic hydrogen peroxide.
The vasodilator effect of low concentrations of quercetin in isolated rat coronary arteries was
inhibited by iberiotoxin. In conclusion, quercetin increased BKCa currents via production of
intracellular hydrogen peroxide. This effect is involved, at least partly, in the coronary
vasodilator effects of quercetin.
- 150 -

Session II : Receptor signaling and G proteins Poster II, 24
Role of rafts on µ and %-opioid receptor pharmacology in plasma membrane of
mammalian cells.
G.Gaibelet, A.Andre, C.Lebrun, B.Lagane, C.Millot, S.Poupard, A.Saulière, A.Lopez
and L.Cezanne
IPBS/CNRS, 205 route de Narbonne, 31062 TOULOUSE cedex, France.
A large body of evidence indicates lateral heterogeneity of lipid and protein distribution in the
biological membranes. In this way, lipid assemblies as rafts composed mainly of
sphingomyelin and cholesterol have been proposed as lipid-platforms which segregate
membrane components within the cell membrane. In this context these confinements are
expected to be relevant by concentrating interacting molecules in particular regions.
Assuming that lipid rafts contain specific sets of protein including for example Galpha
subunits of heterotrimeric G-protein, we explored the role of rafts in regulating GPCR
functionnality. The RCPGs chosen for our studies are the human µ (hMOR) and % opioid
receptors (hDOR) expressed in mammalian plasma membrane.
Effect of cholesterol has been evaluated in term of ligand binding on both receptors.
Cholesterol depletion affects agonist binding. Surprisingly, for hMOR, this effect is observed
only in presence of Gpp(NH)p. Cholesterol re-complementation restored binding properties in
the two cases, indicating a cholesterol specific effect. Conversely, antagonist binding did not
decrease after cholesterol depletion. These results suggest that a population of high-affinity
receptor state require a rich-cholesterol environment. However, in the case of hMOR,
cholesterol is not sufficient and require the presence of G-proteins. In addition, GTP$S
binding experiments, carried out on plasma membrane from cells expressing hMOR, showed
that the coupling efficiency is affected after depletion, suggesting the existence of two distinct
pharmacological behaviour. The distribution of receptors in rich and poor cholesterol domains
have been investigated. We propose a correlation between the pharmacological properties and
the distribution of the components of cells membrane expressing hMOR or hDOR.
- 151 -

Session II : Receptor signaling and G proteins Poster II, 25
THE SIGNALING GATEWAY MOLECULE PAGES DATABASE
Clare Garvey1, Brenda Riley1, Monica Hoyos-Flight1, Bernd Pulverer1, Barbara
Marte1, Nicole Tindill2, Brian Saunders3, Warren Hedley3, Shankar Subramaniam3,
Timo Hannay1.
1. Nature Publishing Group, 4 Crinan Street, London, N1 9XW. United Kingdom. 2.
Nature Publishing Group, 75 Varick Street, 9th Floor, New York, NY 10013, USA. 3.
San Diego Supercomputer Center, University of California, San Diego, 9500 Gilman
Drive, Mail Code 0505, La Jolla, CA 92093-0505
C.Garvey@nature.com
Signaling@nature.com
Signal transduction is at the core of most biological processes and represents a vibrant area of
biological investigation. The Signaling Gateway Molecule Page database is an open access
relational database that aims to provide all relevant published information pertaining to almost
4000 cell signaling molecules. The Molecule Pages are authored by experts, anonymously
peer reviewed, and published online by Nature Publishing Group. Access to all information
in the Signaling Gateway Molecule Pages is available completely free, making this a resource
driven by the signaling community for the signaling community.
Individual Molecule Pages contain both automated data and peer reviewed author-entered
data. The automated data component integrates information about the molecule, such as
DNA and protein sequence information, structural information, sequence comparisons and
related sequences, and basic biophysical and biochemical properties, which are obtained from
external database records. The author-entered component summarizes all significant
published information on a given molecule and includes interlinked formalized descriptions
of all its biochemical states.
Over 150 full Molecule Pages have been published so far, 150 are undergoing peer review
and 400 are currently being authored. The Signaling Gateway Molecule Page database
integrates and summarizes the vast amount of published information on key signal
transduction molecules, linking up not only signaling molecules but also signaling
communities to further accelerate the pace of discovery in cellular signaling.
- 152 -

Session II : Receptor signaling and G proteins Poster II, 26
Molecular mechanism of CRF induced calcium mobilization in CRF1 and CRF2
expressing cells: involvement of different signalling pathways
Eric Gutknecht1-2, Ilse Van der Linden1, Georges Vauquelin2 and Frank M.
Dautzenberg1
1CNS Discovery, Johnson & Johnson Research & Development, Turnhoutsweg 30, 2340
Beerse and 2MBFA, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium
Corticotropin releasing factor (CRF), the most important mediator of the body’s stress
response activates two receptors (CRF1 and CRF2) both belonging to the B-class family of
the GPCRs. CRF1 and CRF2 mainly couple to the stimulatory G-protein Galpha s which
activates adenylate cyclase and protein kinase A. However, we reported recently that in stably
transfected HEK293 cells, both receptors induce transient calcium (Ca2+) mobilization in the
fluorimetric imaging plate reader (FLIPR) format. Ca2+ mobilization was completely
abolished after depletion of intracellular Ca2+ stores or inhibition of IP3 receptors. In
contrast, a specific inhibitor of the PKA pathway had no effect, whereas blockade of
phospholipase C abolished the Ca2+ mobilization. To better understand the involvement of
the various G-proteins, we incubated cells with pertussis toxin and cholera toxin, two well
known inhibitors of Gi and Gs proteins. While pertussis toxin partially blocked Ca2+ (40%),
cholera toxin almost completely (80%) inhibited the transient Ca2+ response. To further
assess the contribution of Galpha i in this process and the role of beta-gamma subunits, we
transiently transfected the C-terminal part of the G-protein receptor kinase type 2 GRK2 (as a
beta-gamma scavenger) which resulted in a potent reduction of the Ca2+ response. To
elucidate the mechanism of the phospholipase C activation, we transfected different alphasubunits
of the Gq family in CRF1 and CRF2(a) expressing cells. While a potentiation of the
Ca2+ signal was observed with Galpha-16, only a weak or no increase in the signal could be
monitored with Galpha o, Galpha olf, Galpha q, or Galpha 11. Thus, in CRF1 and CRF2
expressing HEK293 cells, both receptors seems to act through different G-proteins indicating
that several signalling pathways, including Galpha s, are involved in the activation of
phospholipase C.
- 153 -

Session II : Receptor signaling and G proteins Poster II, 27
South of Ras: Identification and Characterization of Genes Deregulated by Oncogenic
Ras Signaling
Minh-Cam Ha-Thi, Oleg Tchernitsa, Anja Schramme, Balázs Györffy, Anastassia
Malek, Christine Sers and Reinhold Schäfer
Laboratory of Molecular Tumor Pathology, Charité Universitätsmedizin Berlin,
Schumannstr. 20/21, D-10117 Berlin, Germany. E-mail: reinhold.schaefer@charite.de
The small GTPase Ras is a molecular signaling switch known to play a critical role in tumor
growth and development. The oncogenic activity of Ras signaling pathways is tightly coupled
with the deregulation of the genetic program. Therefore, it is essential to identify deregulated
target genes and to study their contribution to transformed phenotypes. We obtained a
consensus list of genes responding to oncogenic Ras signaling in pairs of normal versus Rastransformed
cells of rat, mouse and human origin by analyzing differential gene expression
using suppression subtractive hybridization (SSH) and by interrogating high-density
microarrays. To systematically study the deregulation of Ras-responsive genes in Rastransformed
cells under various experimental conditions, we established species-specific
customized microarrays representing approx. 300 Ras targets. We used Ras target microarrays
for studying the transcriptional response to conditional Ras activation in a time-resolved
manner and for identifying groups of target genes (signal-regulated transcriptional modules)
controlled by specific downstream pathways such as the mitogen-activated kinase (MAPK)
pathway or the phosphatidylinositol-3 kinase (PI3K) pathway in rodent cells. We also
identified 45 genes showing increased transcriptional expression and 56 down-regulated
genes in normal (BJELB) and H-Ras transformed human embryo fibroblasts (BJELR). To
narrow-down the number of critical target genes, we targeted signaling effectors and
individual Ras-responsive genes by RNA interference. Silencing by siRNA of the
transcriptional regulators Fra-1 and HMGA2, frequently up-regulated in Ras-transformed
cells, impairs the proliferative capacity suggesting that these targets are necessary for cellular
transformation. Likewise, knocking-down transformation-sensitive genes in non-transformed
precursor cells can result in neoplastic transformation. Using this approach, a still growing set
of genes capable of negatively affecting cellular transformation and also Ras signal
transduction is being established.
- 154 -

Session II : Receptor signaling and G proteins Poster II, 28
S100C/A11, an essential element of the signal transduction of TGFb-induced growth
suppression and a potential target for therapy against hyper-proliferative human
diseases.
Nam-ho Huh, Eiichi Makino, and Masakiyo Sakaguchi.
Department of Cell Biology, Okayama University Graduate School of Medicine,
Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan E-mail :
namu@md.okayama-u.ac.jp
TGFb is known to be a potent growth suppressor to epithelial cells, including epidermal
keratinocytes. We have demonstrated that S100C/A11, an EF-hand-type Ca++-binding
protein, plays a critical role for the signal transduction. On exposure of normal human
keratinocytes to TGFb, S100C/A11 was specifically phosphorylated at 10Thr by PKCa. The
phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear
translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The
resulting free Sp1/3 transcriptionally activated p21(WAF1), a representative negative
regulator of cell growth. The new pathway involving S100C/A11 was shown to be
indispensable for the growth suppression by TGFb together with the well-known Smadmediated
pathway.
Since the phosphorylated N-terminal domain of S100C/A11 was demonstrated to be
critical for exhibiting the function described above, we introduced into cells a peptide
composed of N-terminal 19 amino acids flanked by HIV-TAT protein transduction domain.
The peptide induced apoptotic cell death in a number of human normal and tumor cells. The
induction of apoptotic cell death was apparently independent of p53, p21(WAF1), and
caspase activity, but treatment with the peptide resulted in partial translocation of apoptosisinducing
factor (AIF) from the cytoplasm to nuclei. These results indicate that the peptide
may lead to the establishment of a new molecular target for the treatment of human cancer
and other hyper-proliferative diseases.
References: J Cell Biol 24;163(4):825-35, 2003; J Cell Biol 164(7):979-84, 2004; J Mol
Med 82(9):612-20, 2004; Proc Natl Acad Sci USA. 102(39):13921-6, 2005.
- 155 -

Session II : Receptor signaling and G proteins Poster II, 29
Epac activation converts cAMP from a proliferative into a differentiation signal in PC12
cells
Simone Kiermayer, Ricardo M. Biondi, Guido Plotz, Jörg Haupenthal, Stefan Zeuzem,
and Albrecht Piiper.
Universitätsklinikum des Saarlandes, Department of Internal Medicine II, Kirrberger
Str., Building 41, 66421 Homburg/Saar, Germany. E-mail: inskie@uniklinikumsaarland.de
cAMP plays, amongst others, an important role in the regulation of cell proliferation and
differentiation. How an elevated intracellular cAMP concentration [cAMP]i induces distinct
biological responses is still unknown. Thus, we investigated the qualitative effects of an
elevated [cAMP]i using cAMP-analoga, which specifically activate the main cAMP effectors,
protein kinase A (PKA) or Epac1 and Epac2, exchange factors activating the small GTPases
Rap1 and Rap2. As a model we used neuroendocrine PC12 cells, in which elevation of
[cAMP]i activates extracellular signal-regulated kinase (ERK) 1/2 and induces low-degree
neurite outgrowth. Our studies revealed that the specific stimulation of PKA triggered
ERK1/2 activation. It was considerably more transient than that, observed upon simultaneous
activation of both PKA and Epac. Unexpectedly, the PKA-specific cAMP analog induced cell
proliferation rather than neurite outgrowth. This proliferative signaling pathway involved
activation of the epidermal growth factor receptor and ERK1/2. Activation of Epac and its
down-stream effector Rap1 appeared to extend the duration of PKA-dependent ERK1/2
activation and converted cAMP from a proliferative into an anti-proliferative, neurite
outgrowth-promoting signal. Thus, we found strong evidence that the outcome of cAMP
signaling can depend on the set of cAMP effectors activated.
- 156 -

Session II : Receptor signaling and G proteins Poster II, 30
Expression of the insulin-like growth factor-I receptor in human colorectal cancer and
associations with Cx26 and antiapoptotic marker Bcl-xL.
Mariusz Koda, Stanislaw Sulkowski, Luiza Kanczuga-Koda, Andrzej Wincewicz,
Mariola Sulkowska, Waldemar Famulski, Wojciech Kisielewski.
Department of Pathology, Medical University of Bialystok, Waszyngtona 13, 15-269
Bialystok, Poland. E-mail: sulek@zeus.amb.edu.pl
Insulin-like growth factor (IGF) and its receptor (IGF-IR) play an important role in
mitogenesis, apoptosis, growth and proliferation of several types of cancers. Overexpression
of IGF-IR in colorectal cancer is associated with increase of cancer cells proliferation and
migration as well as inhibition of apoptosis. In our previous reports we demonstrated
correlations between IGF-IR and apoptosis. Moreover we observed relationships between
connexin26 (Cx26) expression and apoptotic markers in human colorectal cancer. Recently, it
has been shown that expression of connexins and gap junction functions are also regulated by
growth factors including IGF-I. Therefore, in this study we have focused on the relationships
between IGF-IR and Cx26 as well as Bcl-xL expression.
A total number of 115 cases of colorectal cancer were examined by immunohistochemistry,
using the avidin-biotin-peroxidase method. Associations among above proteins were assessed
in the entire group of colorectal cancer patients and its subgroups depending on lymph node
involvement (N0 and N1), histological grade (G2 and G3), extent of tumor growth (pT1+pT2
and pT3+pT4), histopathologic type (adenocarcinoma and mucinous carcinoma), sex, age
(%60 and >60)and tumor site (colon and rectum).
The expression of IGF-IR, Cx26 and Bcl-xL was noted in 47%, 56.5% and 75.6% of the
tumors, respectively. In the entire group of patients we found the positive correlation between
IGF-IR and Cx26 (p

Session II : Receptor signaling and G proteins Poster II, 31
Expression of insulin signaling transmitters and glucose transporters at the protein level
in the rat testis.
Kersti Kokk1, 2, Esko Veräjänkorva2, Xiao-Ke Wu2, 4, Helle Tapfer1, Elle Põldoja1,
Helle-Evi Simovart1 and Pasi Pöllänen2, 3
1Department of Anatomy, University of Tartu, Tartu, Estonia, E-mail:
Kersti.Kokk@ut.ee
2Department of Anatomy, University of Turku, Turku, Finland
3Center for Development/Administration, Kymi Dale (Kymenlaakso) Health Care
District,
FIN-48210 Kotka, Finland
4Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang
Traditional Medicine University, Harbin 150040, China.
Insulin Receptor Substrate (IRS) proteins are key mediators in insulin signaling from the
insulin receptor and play a central role in maintaining basic cellular functions such as growth,
survival and metabolism. It takes place through receptor-mediated tyrosine phosphorylation
of the IRS proteins. IRS-1 and IRS-2 genes have been considered plausible candidates
involved in the pathogenesis of Type 2 diabetes. A family of glucose transporters (GLUT)
mediates the cellular uptake of glucose. The aim of present study is to demonstrate the
distribution of Insulin Receptor Substrates 1-3 (IRS 1-3), glucose transporters 1-4 (GLUT 1-
4), SIRP1alpha, PKB kinase and PI3 kinase in the rat testis to see if signal transduction
mediated by these proteins is active in testicular cells. Wistar rats were used as donors of
testis tissue. Expression of these genes was studied at the protein level using
immunohistochemistry and Western blotting. IRS-1, IRS-2, GLUT 1, GLUT 2, GLUT 3 and
SIRP1alpha were strongly expressed in the different types of cells in all the testes investigated
by immunochemistry. Positive immunoreactions for IRS-3, GLUT 4, PKB kinase and PI3
kinase were not found in the rat testis. Immunoblotting experiments demonstrated the
presence of about 26-67 kD reactive with anti- IRS-1, IRS-2, GLUT 1, GLUT 2, GLUT 3,
PKB kinase and SIRP 1alpha, but no proteins reactive with IRS-3, GLUT 4 and PI3 kinase
antibodies were detected in the rat testis. The present result suggest that proteins like insulin
and certain cytokines using IRS-1, IRS-2, GLUT 1, GLUT 2, GLUT 3, PKB kinase and
SIRP1alpha in their signal transduction can have effects on the epithelial cells of the male
reproductive tract in the rat.
- 158 -

Session II : Receptor signaling and G proteins Poster II, 32
Distinct signaling and actions caused by intracellular ligands (pepducins) for proteinaseactivated
receptor-1 in multiple cells/tissues
Satoko Kubo, Tsuyoshi Ishiki, Ichiko Doe, Fumiko Sekiguchi, Hiroyuki Nishikawa*,
Kenzo Kawai*, Atsufumi Kawabata.
Division of Physiology and Pathophysiology, School of Pharmaceutical Sciences, Kinki
University, Higashi-Osaka 577-8502, Japan and *Fuso Pharmaceutical Industries Ltd.,
Osaka 536-8523, Japan. E-mail: 0544410001t@kindai.ac.jp
Proteinase-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, can be
activated by an N-palmitoylated peptide based on the intracellular loop 3 of human PAR-1,
Pal-RCLSSSAVANRSKKSRALF-amide (P1pal-19), in a C-terminal-dependent manner in
human platelets or PAR1-transfected cells. In contrast, the shorter peptide, Pal-
RCLSSSAVANRS-amide (P1pal-12), could be a PAR1 antagonist. We thus examined the
actions of those peptides in multiple cells/tissues known to express PAR1. P1pal-19, like the
extracellular PAR1 agonist TFLLR-amide (TF), caused Ca2+ signal in human lung epithelial
cells and rat gastric mucosal epithelial cells, and also delayed prostaglandin E2 formation in
the latter cells. P1pal-19 and TF produced endothelial NO-dependent vasodilation and
epithelial prostanoid-dependent bronchodilation. However, P1pal-19 failed to mimic the
contractile activity of TF in the endothelium-denuded blood vessels and gastric smooth
muscle. In contrast, P1pal-12 partially inhibited vasorelaxation by TF and P1pal-19. In sum,
P1pal-19 mimics the actions of TF in the endothelial and epithelial, but not smooth muscle,
cells/tissues, and P1pal-12 may act as a PAR-1 antagonist in the vascular endothelium.
- 159 -

Session II : Receptor signaling and G proteins Poster II, 33
Small GTPase Ras and Rho expression in rat osteoblasts during spaceflight
Yasuhiro Kumei 1 , Sadao Morita 1 , Hitoyata Shimokawa 1 , Kei-ichi Ohya 1 ,
Hisako Katano 2 , Hideo Akiyama 3 , Masahiko Hirano 3
1 Tokyo Medical and Dental University, Tokyo, Japan
2 University of Tokyo Medical Science Institute, Tokyo, Japan
3 Toray Research Center, Kamakura, Japan
Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized after a 24
hr-treatment with 1",25 dihydroxyvitamin D3. The mRNA levels of Ras and Rho were
analyzed by quantitative RT-PCR normalized to glyceraldehyde 3-phosphate dehydrogenase.
The small GTPase Ras and Rho are activated by GEF (guanine nucleotide exchange factor)
and deactivated by GAP (GTPase-activating factor). Recently we reported that expression of
Ras GEF, Ras-GRF (guanine nucleotide releasing factor) increased to 2- to 5-fold of the
ground controls, during spaceflight. Ras-GRF acts downstream G-protein coupled receptors,
whereas another Ras GEF, SOS (son of sevenless) activates Ras after binding to adaptor
protein Grb2 of receptor tyrosine kinase. The SOS mRNA levels also increased to 3- to 5-fold
during spaceflight, although Ras GAP expression was not altered. Ras activation was
followed by MAP kinase cascades including ERK1/2 expression, and FAK (focal adhesion
kinase) increased during spaceflight. Cdc42 is a Rho family protein that responds to
environmental stress and induces JNK (Jun-N-terminal kinase). Both Cdc42 GAP and GEF
expression was increased 2-fold in the flight cultures, as compared to the ground controls.
Accelerated Cdc42 turnover might result in 2-fold increase of JNK expression. Rho plays
important roles in actin rearrangement. Microgravity may alter physiological change of rat
osteoblasts at least partly via small GTPase.
- 160 -

Session II : Receptor signaling and G proteins Poster II, 34
SDF1 controls pituitary cell proliferation through the activation of ERK1/2 and the
Ca2+-dependent cytosolic tyrosine kinase, Pyk2
°Alessandro Massa, *Silvia Casagrande, °Adriana Bajetto, °Carola Porcile, °Federica
Barbieri, °Stefano Thellung, °Sara Arena, °Alessandra Pattarozzi, °Monica Gatti,
°Alessandro Corsaro, *Mauro Robello, °Gennaro Schettini, °Tullio Florio.
°Section of Pharmacology, Dept. Oncology Biology and Genetics, University of Genova,
Italy. *Unit INFM, Dept. of Physics, University of Genova, Italy .
Stromal cell-Derived Factor 1 (SDF1) is a chemokine of the CXC subfamily involved in the
recruitment of immune cells to sites of inflammation. SDF1 exerts its effects interacting with
CXCR4, a G-protein coupled receptor. CXCR4 is often expressed by tumor cells and its
activation causes tumor cell proliferation. To date, no evidence have been provided on the
possible role of SDF1 in pituitary adenoma funcion, although it was reported the expression
of CXCR4 in rat pituitary. In this work, we used GH4C1 cells as a model to study the effects
of SDF1 in pituitary functions. In these cells, SDF1 induced dose-dependent proliferation.
Thus we evaluated the intracellular signaling involved in this effect. SDF1 increased cytosolic
[Ca2+] and activated Pyk2, ERK1/2 and BKCa channels. To correlate these intracellular
effectors with the proliferative activity we inhibited their activity using BAPTA-AM (Ca2+
chelator), PD98059 (MEK inhibitor), salicylate (Pyk2 inhibitor) and TEA (K+ channel
blocker). All these compounds reverted SDF1-induced proliferation, suggesting the
involvement of multiple intracellular pathways. To identify a possible cross-talk and a
molecular ordering among this pathways, we tested these antagonists on SDF1 dependent
activation of ERK1/2, Pyk2 and BKCa channels. We report that: the inhibition of [Ca2+]i
increase or BKCa channels activity did not affect ERK1/2 activation by SDF1; Pyk2
activation was purely Ca2+ dependent, not involving ERK1/2 or BKCa channels; BKCa
channels activity was antagonized by Pyk2 but not by ERK1/2 inhibitors. These data suggest
that SDF1- dependent increase of [Ca2+]i activates Pyk2, that, in turn, regulates BKCa
channel activity. Conversely, ERK1/2 activation is an independent phenomenon. In
conclusion we demonstrate that SDF1 causes proliferation of GH4C1 cells, suggesting that
the activation of CXCR4 may represent a novel regulatory mechanism for pituitary cell
proliferation, that may contribute to pituitary adenoma development.
- 161 -

Session II : Receptor signaling and G proteins Poster II, 35
Involvement of Kv1.5 channel endocytosis in serotonin-induced inhibition of voltagegated
K+ currents and contraction in pulmonary arteries.
Laura Moreno, Angel Cogolludo, Laura Cobeño, Federica Lodi, Giovanna Frazziano,
Juan Tamargo, and Francisco Perez-Vizcaino.
Department Pharmacology. School Medicine. Universidad Complutense de Madrid.
28040 Madrid. SPAIN. E-mail: lmoreno@ift.csic.es
Serotonin (5-HT) and voltage-gated K+ (Kv) channels play a central role in the pathogenesis
of pulmonary hypertension (PH). We showed (abstract, this meeting) that 5-HT inhibits Kv
channels in rat pulmonary artery smooth muscle cells (PASMC) establishing a link between
these pathogenetic factors in PH. These effects were mediated by activation of 5-HT(2A)
receptors via PLC, classic PKC and tyrosine kinase. Herein we analyzed whether 5-HT
inhibited human Kv1.5 channels stably expressed in Ltk- and the possible role of Kv1.5
endocytosis in native PASMC. 5-HT reduced Kv1.5 currents and this effect was prevented by
the 5-HT(2A) receptor antagonist ketanserin and by the tyrosine kinase inhibitor tyrphostin
23. However, we did not find changes in the Kv1.5 protein phosphorylation in tyrosine
residues using an anti-phosphotyrosine antibody in Kv1.5 immunoprecipitates after treatment
with 5-HT. Inhibition of caveolar disruption (#-cyclodextrin) or inhibition of endocytotic
processes (concanavalin A), prevented the inhibitory effects of 5-HT on Kv currents in native
PASMC. Concanavalin A also inhibited the vasoconstrictor effect of 5-HT in isolated rat
pulmonary arteries. In homogenates from pulmonary arteries, 5-HT(2A) receptors and
caveolin-1 co-immunoprecipitated with Kv1.5 channels and this was increased upon
stimulation with 5-HT. Moreover, confocal microscopy revealed that Kv1.5 channels were
internalized when PASMC were stimulated with 5-HT. In conclusion, activation of 5-HT(2A)
receptors inhibits rat native Kv currents as well as cloned human Kv1.5 currents. 5-HTinduced
inhibition of Kv currents and the subsequent vasoconstriction of PA result from the
interaction of 5-HT(2A) receptors with Kv1.5 channels within caveolar membrane
microdomains and involve tyrosine phosphorylation and endocytotic processes.
- 162 -

Session II : Receptor signaling and G proteins Poster II, 36
CLA/PUFA and Deoxycholic Acid (DCA)-Induced Signaling in Colorectal Cancer via
the Insulin-Like Growth Factor 1 Receptor (IGF-1R).
Sherif S Morgan, Shawna Comer, and Emmanuelle J Meuillet.
The Cancer Biology Graduate Interdisciplinary Program, Nutritional Sciences
Department, and Molecular and Cellular Biology Department. University of Arizona,
Tucson, Arizona 85721
Colorectal cancer is the second leading cause of cancer-related deaths in the US. Clinical
studies have shown that colorectal cancer patients have higher levels of bile acids (BA) in
colonic lumen, and animal studies substantiate the role of BA as tumor promoters. The
molecular mechanism of deoxycholic acid (DCA), which is a hydrophobic cholesterol
derivative remains unclear. Recently, DCA has been shown to perturb the plasma membrane,
which leads to EGFR activation. We sought to investigate a link between the presence of
DCA and IGF-IR activity in colorectal cancer cells. Conjugated linoleic acid (CLA) and n-3
polyunsaturated fatty acids (PUFA) have been shown to have anti-tumorigenic effects. We
hypothesize that DCA can perturb the plasma membrane, leading to the activation of the IGF-
IR and its downstream signaling pathways, promoting survival and proliferation of colorectal
cancer cells. Conversely, nutritional modulation by increasing intake of PUFA/CLA may
counteract these effects by restoring normal IGF-IR signaling. We show that treatment with
DCA leads to activation of IGF-IR and ERK-1/2 through the MEK-1 pathway. Counterintuitively,
DCA does not activate AKT; in fact, it counteracts the IGF-induced AKT
activation. DCA appears to preferentially activate ERK, while inhibiting AKT. Treatment
with CLA, on the other hand, activates both ERK and AKT. Further studies are required to
identify the functional role of activating these pathways, because they may lead to different
cellular responses. These results support the role of DCA as a tumor promoter and sheds light
on the potential mechanisms of CLA chemoprevention. These data have important
implications since they support the significant role of nutrition in the prevention and treatment
of colorectal cancer.
- 163 -

Session II : Receptor signaling and G proteins Poster II, 37
Cellular prion protein signaling in serotonergic neuronal cells
Sophie Mouillet-Richard1, Benoît Schneider1, Elodie Pradines1, Mathéa Pietri1,
Myriam Ermonval1, Jacques Grassi2, Vincent Mutel3, Jean-Marie Launay4 & Odile
Kellermann1.
1 Différenciation cellulaire et prions, CNRS UPR1983 94801 Villejuif, France & Institut
Pasteur, Département de biologie cellulaire et Infections, 75724 Paris Cedex 15, France.
mouillet@vjf.cnrs.fr 2 Commissariat à l’Energie Atomique Saclay, DSV-SPI, 91 191 Gif
sur Yvette Cedex, France 3 F. Hoffmann-LaRoche, Basel, Switzerland. 4 Hôpital
Lariboisière 75009 Paris, France.
The cellular prion protein PrPC is the normal counterpart of the scrapie prion protein (PrPSc), itself
the major if not the only component of the infectious agent of transmissible spongiform
encephalopathies (TSE). PrPC is a ubiquitous glycoprotein abundantly expressed in neuronal cells,
which are the targets of TSE pathogenesis. It is attached to the outer membrane leaflet through a GPI
anchor, and primarily resides in lipid rafts, which represent subcellular microdomains highly enriched
in cell signalling molecules.
To gain insight into the physiological role of PrPC in relation with the onset, maintenance and
regulation of neuronal functions, we exploit the properties of the 1C11 neuroectodermal cell line,
endowed with the capacity to convert into fully-functional serotonergic neuronal cells upon
appropriate induction. Within 4 days of differentiation, almost 100% 1C11 cells implement 5-HT
synthesis activity, storage, catabolism, uptake as well as three serotonergic receptors of the 5-HT2B,
5-HT1B/D and 5-HT2A subtypes. These G-protein coupled receptors sustain an autoregulation of the
overall serotonergic functions by external 5-HT.
Antibody-mediated ligation of PrPC at the cell surface of 1C11 cells provides a means to mimic the
interaction with a yet-to-be-identified ligand and to monitor downstream signal transduction events.
While PrPC-associated signalling cascades occur in 1C11 precursor cells as well as other nonneuronal
cell systems, PrPC fulfils some neuronal-specific function in mature 1C115-HT serotonergic
cells. By interacting with the membrane protein caveolin and the Fyn tyrosine kinase within a platform
located on neuritic extensions, PrPC mobilizes a PI3kinase-PKC - NADPHoxidase module and other
downstream effectors targeting the MAPK kinases ERK1/2.
In addition to recruiting signalling pathways, antibody-mediated ligation of PrPC has impact on the
couplings of the serotonergic receptors present on 1C115-HT serotonergic cells. Concomitant
exposure of 1C115-HT serotonergic cells to 5-HT receptor agonists and anti-PrP antibodies
significantly alters the G-protein-mediated couplings associated to all three receptors. PrPC ligation
has diverse outcome depending on the receptor and the pathway considered. First, PrP antibodies
selectively augment the PLA2 response imparted by 5-HT2B receptors. Second, PrP ligation fully
abrogates the PLC coupling to 5-HT2A receptors. Third, PrP antibodies reduce the 5-HT1B/D
negative coupling to adenylate cyclase. Because the 5-HT1B/D receptor function is modulated through
antagonising crosstalk that arise from 5-HT2B and 5-HT2A receptors, PrPC ligation also impacts on
the functional interactions occurring between the three receptor subtypes.
Hence, PrPC not only mobilizes transduction cascades controlling the cellular redox state and ERK1/2
kinases but also interferes with the signalling activity of serotonergic receptors that regulate 5-HT
synthesis, storage and uptake. Overall, our findings support the notion that PrPC contributes to the
homeostasis of serotonergic neurons. Eventually, they provide a foundation for uncovering the impact
of prion infection on serotonergic neurons.
- 164 -

Session II : Receptor signaling and G proteins Poster II, 38
Transformation by oncogenic Ras exhibits JNK-isoform specificity
Christina Nielsen, Jacob Thastrup, Marja Jäättelä and Tuula Kallunki
Apoptosis Department, Danish Cancer Society, Strandboulevarden 49, 2100
Copenhagen, Denmark. E-mail: tk@cancer.dk
JNK family consists of at least 10 members: JNK1, JNK2 and JNK3 isoforms and their splice
variants. Active Ras oncogene is expressed in 30-40% of human cancers. Ras, among others,
can activate JNK pathway.
We have recently found out that active Ras cannot transform cells that carry inactivated JNK2
isoform of JNK. Using knockout iMEFs we have observed that JNK2 is essential for Ras
transformation whereas JNK1 is not. Various individual Jnk2-/- iMEFs are severely deficient
in multilayer growth as measured by focus formation and soft agar assays. The mouse
tumorigenesis assay further proved that Ras transduced Jnk2-/- iMEFs are clearly less
tumorigenic than the Ras transformed wt and Jnk1-/- iMEFs in vivo. We have further verified
our findings by down regulating the endogenous JNK2 in wt iMEFs utilizing the short hairpin
(sh) RNA technique. For this we have prepared two JNK2 specific retroviral sh RNA
constructs which both are efficient and specific in JNK2 down regulation. Introduction of
either of the JNK2 specific sh RNAs into the wt iMEFs reduced their tumorigenicity clearly.
Novel data strongly suggesting that JNK2 is necessary for Ras induced transformation and
tumorigenesis will be presented.
- 165 -

Session II : Receptor signaling and G proteins Poster II, 39
Identification of suramin as a direct inhibitor of adenylyl cyclase and a competitive
inhibitor of [3H]-forskolin binding
Jiri Novotny, Jiri Stöhr, Lenka Bourova and Petr Svoboda
Department of Biochemistry of Membrane Receptors, Institute of Physiology, Academy
of Sciences, Videnska 1083, Prague 4 and Department of Physiology, Faculty of Natural
Sciences, Charles University, Vinicna 7, 120 00 Prague 2, Czech Republic
E-mail: novjiri@biomed.cas.cz
Activity of adenylyl cyclase (AC) is markedly higher in brain cortex from young than from
adult rats. Here we compared the modulatory effects of many different compounds on AC
activity in cerebrocortical preparations from young (12-day-old) and adult (90-day-old) rats.
We did not found any substantial difference between the effects of all tested agents and AC
activity measured in various conditions was always about two-fold higher in samples from
young animals. Our experiments analysing the presumed modulatory role of suramin revealed
that this pharmacologically important drug may act as a direct inhibitor of AC. The enzyme
activity was diminished to the same extent by suramin in membranes from both tested age
groups. Moreover, suramin was able to reduce AC activity stimulated by Mn2+ or forskolin
in membranes derived from S49 cyc- cells lacking Gs-alpha protein. We also determined
characteristics of [3H]-forskolin binding in cerebrocortical membranes and these
measurements allowed us to detect high-affinity, as well as super-high-affinity, [3H]forskolin
binding sites. The binding parameters of these sites differed quite significantly in
samples from both age groups tested. We also assessed the potential effect of suramin and
results of these analyses suggested that this compound may act as a potent competitive
inhibitor of [3H]-forskolin binding. In summary, our present studies extend the existing array
of suramin cellular tagets by identifiying this drug as a potent direct inhibitor of AC and [3H]forskolin
binding.
This investigation was supported by Centrum of Neuroscience (LC 554).
- 166 -

Session II : Receptor signaling and G proteins Poster II, 40
Oxytocin- and vasopressin-induced mitogenic signalling in human small-cell lung
carcinoma
Christel Péqueux1, Marie-Thèrèse Hagelstein2, Jean-Claude Hendrick2 and Jean-
Jacques Legros2.
1Tumour and Development Biology Laboratory, CRCE, Pathology Institute, CHU-B23,
University of Liège, B-4000 Liège, Belgium, e-mail: C.Pequeux@ulg.ac.be;
2Neuroendocrine unit, CIL, Pathology Institute, CHU-B23, University of Liège, B-4000
Liège, Belgium, e-mail: Jean-Jacques.Legros@ulg.ac.be
Neuroendocrine tumour cells usually develop very potent autocrine/paracrine signalling
pathways that play a crucial part in the dysregulation of cellular growth. In the study
presented here, we demonstrated, on small cell lung carcinoma (SCLC) cell lines, that the
oxytocin (OT) as well as the vasopressin (VP) genes, normally transcriptionally restricted in
their expression, are activated in SCLC, leading to synthesis and secretion of OT and VP.
Concomitantly, these tumour cells express the various neuropeptide receptors: OTR, V1aR,
V1bR/V3R and V2R. We observed that OT, as well as VP, induces a dose dependent and
time persistent increase of tumour cell proliferation. This mitogenic effect of OT was
completely abolished by the OTR antagonist OVTA. We demonstrated that OT- and VPinduced
mitogenic effects on SCLC are largely mediated by the OTR and the V1aR
respectively and that this mitogenic signalling passes through the phosphorylation of ERK1/2
and p90RSK in a PLC-, Ca++-, PKC- and MEK1/2-dependent pathway. Moreover, we
evidenced that growth of OT- and VP-stimulated SCLC could be down regulated by
signalling inhibitors and was not a consequence of toxicity. Additionally, we reported that
86% of primary SCLC biopsies analyzed expressed at least the OTR and that 71% expressed
the OTR, the V1aR and the V2R altogether. Interestingly, in normal human lung, OTR
colocalized with vascular endothelial cells of the lung. Among SCLC plasma samples, the
distribution of elevated OT is 40%, of elevated VP is 43.3% and of both elevated hormones is
16.7%. Altogether, these results clearly demonstrate that, in addition to the vasopressinergic
system, OT and particularly OTR contribute to give the SCLC tumour a survival advantage.
Our data identify pathways by which OT and VP induced their mitogenic action on SCLC and
they highlight that blocking of their receptor signalling represents viable pharmacological
targets.
- 167 -

Session II : Receptor signaling and G proteins Poster II, 41
Endoglin modulates Transforming Growth Factor-beta signaling pathways in L6E9
myoblasts
Alicia Rodríguez-Barbero, Soraya Velasco, José M. López-Novoa
Instituto Reina Sofía de Investigación Nefrológica. Dpto de Fisiología y Farmacología.
Universidad de Salamanca. Campus Unamuno, Edificio Departamental. Avda. Campo
Charro s/n. 37007. Salamanca. Spain. E-mail: barberoa@usal.es
Endoglin is a transmembrane accessory receptor for transforming growth factor-# (TGF-#).
TGF-# elicits cellular responses via specific type I and II serine/threonine kinase receptors
and their downstream nuclear effectors, termed Smads. Type I receptors act downstream of
type II receptors and determine the signalling specificity within the receptor complex. In most
cells, TGF-# signals via TGF-# type II receptor and activin receptor-like kinase (ALK) 5 to
induce Smad2/3 phosphorylation. However, it has recently been shown that in endothelial
cells TGF-# activates two distinct types I receptor pathways, that is, ALK5-inducing Smad2/3
phosphorylation and ALK1-promoting Smad1/5 phosphorylation. Endoglin, as well as other
TGF-# signaling components, is involved in several pathophysiological processes such as
angiogenesis and fibrosis. Mutations in endoglin and ALK1 have been linked to the vascular
disorder, hereditary hemorrhagic telangiectasia type 1 (HHT1). However, the function of
endoglin in TGF-#/ALK signalling has remained unclear. To assess the function of endoglin
in TGF-#/ALK signalling, we select a non-endoglin expressed cell type such as L6E9 rat
myoblasts, and transfected them with L-endoglin. We found that both, the classic TGF-
#/ALK5 and the endothelial specific TGF-#/ALK1 pathways are present and functional in
L6E9 myoblasts. TGF-#1 activated both Smad1 y Smad2/3 and also induced Smad2/3 and
Smad4 nuclear translocation without differences between mock and endoglin transfected
myoblasts. Nevertheless, endoglin promote Id1 expression and reduce PAI-1 activity. Our
results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling. The
progression in our understanding on the functional role of endoglin might have important
relevance in the regulation of pathophysiological processes in which endoglin is involved.
- 168 -

Session II : Receptor signaling and G proteins Poster II, 42
Galphaz subunits are essential partners for permanent association of receptor-activated
Galphai subunits to RGS9-2 proteins.
María Rodríguez-Muñoz, David Bermúdez, Elena de la Torre-Madrid, Pilar Sánchez-
Blázquez and Javier Garzón.
Laboratorio de Neurofarmacología. Instituto Cajal. Avenida Doctor Arce 37, 28002
Madrid. Spain. Email: mrodriguez@cajal.csic.es
The in vivo administration of an ED80 of morphine induces the transfer of mu-opioid
receptors (MOR) that are associated with Galpha subunits to RGS9-2 proteins (1). The
phosphorylation of specific residues in or near the RGS box and the subsequent binding to 14-
3-3 impedes RGS9-2 GAP activity and stabilizes this interaction. Thus, by retaining MORactivated
Galphai2 subunits, RGS9-2 proteins play a key role in the onset of opioid tolerance.
Besides the role of RGS9-2 in these processes, morphine also promotes the association of
Galpha subunits with RGS-Z proteins. Since recent studies have shown that Galphaz subunits
are stably associated with RGSZ2, we have examined the contribution of Galphaz to the
sequestering of Galphai subunits by RGS9-2 proteins. Proteins from mouse PAG
synaptosomal membranes were immunoprecipitated with anti-RGS9-2 IgGs and analysed by
Western blotting. An anti Phospho-Serine antiserum recognised a 76Kda protein that
corresponded to RGS9-2. Impairing the expression of Galphaz through the subchronic
administration of selective ODNs provoked a 45% reduction in the Ser phosphorylation of
RGS9-2 and reduced tolerance to repeated administration of morphine. Conversely, in
Galphai2 knockdown animals and mice receiving exogenous Galphaz subunits, the
phosphorylation of RGS9-2 augmented by 36 and 42%, respectively. Our data show that
activated GalphazGTP subunits are required for agonist-dependent phosphorylation of RGS9-
2 proteins, which leads to the stabilization and sequestering of MOR-activated G-alpha
subunits by RGS9-2 proteins.
(Supported by MEC SAF2003-01121 and BMC2002-03228;Instituto de Salud Carlos III
G03/005)
1. Garzón J, Rodríguez-Muñoz M, López-Fando A, Sánchez-Blázquez P (2005) J. Biol.
Chem. 280: 8951-8960
- 169 -

Session II : Receptor signaling and G proteins Poster II, 43
Conditionnal expression of wild type or mutated Gsalphalpha induced MAPKinases
ERK-1/2, prolactin and growth hormone promoters activation in a somato-lactotroph
cell line.
David Romano, Morgane Pertuit, Karine Magalon, Anne Barlier, Ramahefarizo
Rasolonjanahary, Alain Enjalbert et Corinne Gérard.
Interactions Cellulaires Neuroendocriniennes, IFR Jean Roche, Faculté de Médecine
Nord, 13916 Marseille cedex 20, France. E-mail : romano.d@nord.univ-mrs.fr
Mutations of the Gsalphalpha (Gsa) protein, named gsp oncogene, are present in 40% of
human somatotroph adenomas. These mutations induced a constitutive activation of the Gsa
protein and an increase of adenylyl cyclase (AC) activity. However its role in tumorigenesis is
not clear because of an overlap of clinical phenotype (hormonal hypersecretion, proliferation)
between tumors bearing or not the gsp oncogene. This overlap might be due to an
overexpression of the wild type (wt) Gsa protein, mimicking the activating mutation, or to the
compensatory mechanisms like the increase of phosphodiesterase activity. To understand the
molecular mechanisms induced by the gsp oncogene, as well as the putative role of
overexpressed wt Gsa, a somato-lactotroph cell line (GH4C1) was stably transfected with wt
Gsalpha or constitutively activated Gsalpha by the R201C mutation, under the control of the
tetracycline operon (GH4C1 Tet-off).
After induction, we observe a similar increase of both wild type (wt) and mutated transgene
mRNA levels. Moreover, the time course of both transgene proteins expression follows their
mRNA synthesis with a maximum after 7 days of induction. However, the wt transgene
protein is more expressed than the mutated one, suggesting a negative post-transcriptionnal
regulation of the gsp oncogene. At a functional level, cAMP level and AC activity are
strongly increased after induction of the mutated protein, with a time course pattern similar to
the expression of the gsp oncogene. However, (i) basal MAPKinases ERK-1/2
phosphorylation, (ii) human prolactin (PRL) and growth hormone (GH) promoters activity are
progressively and similarly increased by induction of both wt and mutated Gsa proteins.
Moreover, using the MAPKinase specific inhibitor U0126, we show that this increase in both
PRL and GH promoters activity is MAPKinase-dependent.
So far, these cell lines seem to be interesting models to study the molecular mechanisms
involved in the tumorigenesis of human somatotroph adenomas bearing or not the gsp
oncogene.
- 170 -

Session II : Receptor signaling and G proteins Poster II, 44
UVB-induced EGFR-Signaling is mediated via the AhR
Claudia Schäfer1, Ulrike Hübenthal1, Jason Cline1, Melanie Wurm2, Christian Calles1,
Peter Schroeder1, Lars-Oliver Klotz3, Helmut Hanenberg2, Jean Krutmann1, Josef
Abel1 and Ellen Fritsche1
Institut für umweltmedizinische Forschung, Auf’m Hennekamp 50; 2Department of
Paediatric Oncology, 3Institute of Biochem. and Mol. Biol., University of Düsseldorf,
Universitätsstrasse 1, 40225 Düsseldorf, Germany; e-mail: claudia.schaefer@uniduesseldorf.de
The aryl hydrocarbon receptor (AhR) is a ligand-dependend transcription factor that rests in
its inactive form as a multiprotein complex in the cytoplasm of most cells. AhR ligands
include polycyclic aromatic hydrocarbons (PAH) and halogenated PAH. Ligand binding leads
to dissociation of the interacting proteins, AhR nuclear translocation, heterodimerization with
the AhR nuclear translocator (ARNT) and activation of AhR-dependent genes like
cytochrome P450 (CYP) 1A1. CYP1A1 induction by UVB irradiation was recently described
and the formation of an endogenous AhR ligand by UVB was proposed. Epidermal growth
factor receptor (EGFR)-signaling is also initiated by UVB irradiation. Considering that the
AhR ligand TCDD causes EGFR-activation in human mammary carcinoma cells, we
investigated whether UVB-initiated EGFR-signaling is mediated by activation of the AhR
pathway.
Signaling was studied in the immortalized keratinocyte cell line HaCaT. Irradiation of cells
was carried out with 100 J/m2 UVB. Irradiation led to an AhR-dependent increase in
CYP1A1 and COX-2 mRNA as determined by quantitative real-time PCR. Because COX-2
expression can be modulated by EGFR activation, we performed immunocytochemical
analyses of the EGFR. These experiments revealed that clustering and internalization of the
EGFR after UVB irradiation are also –at least partially- AhR dependent. Further EGFR
downstream targets which are activated after UVB irradiation are Erk1/2 phosphorylation and
COX-2 protein expression. We determined by Western blotting that Erk1/2 phosphorylation
and COX-2 expression are partially dependent on AhR signaling. Involvement of the AhR in
UVB-induced signaling was verified by utilization of the AhR antagonist MNF and by AhR
gene knockdown. In summary we found that the AhR is involved in UVB-induced activation
of the EGFR signaling pathway. Therefore we conclude that the AhR serves as a photosensor
in the cytoplasm of keratinocytes mediating UVB-induced signal transduction.
- 171 -

Session II : Receptor signaling and G proteins Poster II, 45
Dual activity of phosphorothioate CpG-oligodeoxynucleotides on HIV: Reactivation of
latent provirus and inhibition of productive infection in human T cells
Carsten Scheller*1, Stefan Lamla1, Ulf Dittmer2, Axel Rethwilm1, Eleni Koutsilieri1
Institute of Virology and Immunobiology, University of Wuerzburg, Versbacher Strasse
7, 97078 Wuerzburg, Germany
Institute of Virology, UK Essen, Hufelandstrasse 55, 45122 Essen, Germany
* correspondence to: Carsten Scheller, email: scheller@vim.uni-wuerzburg.de
CpG oligodeoxynucleotides (CpG ODNs) bind to toll-like receptor 9 (TLR-9) and trigger
cellular activation in immune cells with antigen-presenting activity, including B-cells and
dendritic cells. Here we show that treatment of the latently HIV-infected T cell line ACH-2
with the CpG ODNs 2006 or 2040 trigger activation of viral gene expression, demonstrating
that CpG-signaling activity can also be found in T cells. In contrast to the stimulating effects
on viral gene transcription in latently-infected cells, CpG ODNs potently suppressed HIV
replication in productively-infected T cells. Inhibition of virus replication was not related to
CpG motifs but was likely caused by the phosphorothioate backbone of the CpG ODNs,
probably by interfering with the viral enzyme reverse transcriptase. These results point to a
novel role of CpG-sequences in the development of immune activation therapies to
accomplish eradication of HIV from its latent T cell reservoir in vivo.
- 172 -

Session II : Receptor signaling and G proteins Poster II, 46
5-HT2B and alpha1D receptors control redox equilibrium and TNF-alpha shedding in
bioaminergic neuronal cells.
Benoit Schneider1, Mathéa Pietri1, Sophie Mouillet-Richard1, Myriam Ermonval1,
Vincent Mutel2, Jean-Marie Launay3 and Odile Kellermann1
1Institut André Lwoff-Institut Pasteur, CNRS UPR 1983, Laboratoire Différenciation
Cellulaire et Prions, Villejuif Cedex, France. 2Pharma Research Department, F.
Hoffman-La-Roche Ltd., Base, Switzerland. 3Service de Biochimie, EA3621 Hôpital
Lariboisière, Faculté de Pharmacie, Université apris V, Paris, France. E-mail:
bschneid@vjf.cnrs.fr.
Loss of neuronal homeostasis in neurodegenerative disorders, such as Alzheimer’s,
Parkinson or Prion diseases, may arise from dysregulations of physiological signaling
pathways due to the accumulation of aggregation-prone proteins. For instance, alterations
with serotonin (5-hydroxytryptamine, 5-HT)- or norepinephrine (NE)- dependent signaling
pathways have been reported in these pathologies. In addition, oxidative stress is also widely
suspected to be a major cause of neuronal cell dysfunction or death, since post-mortem brain
studies from affected patients exhibit enhanced indices of reactive oxygen species (ROS). A
possible origin of oxidative stress could hence relate to deviation of ROS-coupled signaling
pathways that normally take part in the control of neuronal functions.
We took advantage of a neuroectodermal progenitor, 1C11, endowed with the capacity
to differentiate into serotonergic or noradrenergic neuronal cells to probe the possible
involvement of 5-HT or NE in regulating the cellular redox state in neurons. Upon induction
by Bt2cAMP, almost 100% of 1C11 cells acquire, within 4 days, a complete serotonergic
phenotype (1C115-HT) including 5-HT synthesis, storage and uptake as well as three Gprotein
coupled receptors of the 5-HT1B/1D, 5-HT2B and 5-HT2A subtypes. Under
combined addition of Bt2cAMP and DMSO, 100% of 1C11 cells convert within 12 days into
fully functional noradrenergic neurons (1C11NE) able to synthesize, store and take up NE.
1C11NE cells transduce NE signals through a single alphalpha1D adrenoceptor. Along either
differentiation program, the bioaminergic receptors act as autoreceptors and mediate the effect
of 5-HT or NE in the coordination and/or onset of all neurotransmitter-associated functions.
By using a panel of specific agonists towards each receptor subclass, we establish that
5-HT2B receptors and alphalpha1D adrenoceptors are functionally coupled to ROS synthesis
through NADPH oxidase activation in 1C115-HT and 1C11NE cells. The ROS response
imparted by each receptor is restricted to 1C11-derived progenies that have implemented a
complete serotonergic or noradrenergic phenotype. These observations indicate that 5-HT2B
and alphalpha1D autoreceptors take part in the control of the cellular redox equilibrium in
neuronal cells. In addition, our data identify TACE (TNF-alphalpha Converting Enzyme), a
member of a disintegrin and metalloproteinase (ADAM) family, as a downstream target of the
5-HT2B and alphalpha1D receptor-NADPH oxidase signaling pathway. Upon 5-HT2B or
alphalpha1D receptor stimulation, ROS act as second message signals that fully govern TNFalphalpha
shedding in the surrounding milieu of 1C115-HT and 1C11NE cells. TNFalphalpha
may in turn contribute to neuronal homeostasis. Eventually, our study may have
implications regarding the origin of oxidative stress as well as up-regulated expression of
proinflammatory cytokines in neurodegenerative disorders, which may relate to the deviation
of normal signaling pathways coupled to neurotransmitters.
- 173 -

Session II : Receptor signaling and G proteins Poster II, 47
Signaling pathway utilized by HSV-1 to induce NF-kB activation: possible role of
herpesvirus entry receptor A
M. Teresa Sciortino,1 M. Antonietta Medici,1 Francesca Marino-Merlo,1 Daniela
Zaccaria,1 Maria Gioffrè,1 Assunta Venuti,1 A. Pia Camuti,1 Sandro Grelli,2 Antonio
Mastino1
1Dept. of Microbiol., Genet. and Mol. Sci., Univ. of Messina, Salita Sperone 31, 98166
Messina, Italy. E-mail: antonio.mastino@unime.it. 2Dept. of Exp. Med. and Bioch. Sci.,
Univ. of Rome “Tor Vergata”, Rome, Italy.
We have previously demonstrated that wild type herpes simplex 1 (HSV-1), as well as nonreplicating
UV-inactivated HSV-1, promptly activate the nuclear factor kappa B (NF-kB) in
U937 monocytoid cells and that glycoprotein D (gD) of HSV-1 is sufficient by itself to exert a
similar effect. We have then investigated the signaling pathway utilized by HSV-1 to initiate
NF-kB activation and, particularly, whether our observation could be related to the capability
of HSV-1-gD to directly stimulate NF-kB through its interaction with the herpesvirus entry
receptor A (HveA). Here we report that: a) exposure to UV-inactivated HSV-1 induced a
MOI-dependent activation of NF-kB in HveA expressing THP-1 monocytoid cells, b)
exposure to soluble gD induced a dose-dependent activation of NF-kB in THP-1 cells that
was inhibited by an antibody able to interfere with gD/HveA interaction, c) cocultivation of
THP-1 cells with an adherent cell line forced to express wild type gD on its surface by stable
transfection led to NF-kB activation, d) cell-to-cell contacts of THP-1 cells with the same
adherent cell line forced to express on its surface, by stable transfection, a mutated form of gD
lacking the capability to interact with HveA, did not activate NF-kB. These results suggest
that HSV-1-gD/HveA interaction is sufficient for triggering a signal transduction pathway
leading to NF-kB activation.
- 174 -

Session II : Receptor signaling and G proteins Poster II, 48
Expression of leptin, leptin receptor and hypoxia-inducible factor 1 in human
endometrial cancer.
Stanislaw Sulkowski, Mariusz Koda, Andrzej Wincewicz, Luiza Kanczuga-Koda,
Mariola Sulkowska, Boguslaw Musiatowicz.
Department of Pathology, Medical University of Bialystok, Waszyngtona 13, 15-269
Bialystok, Poland. E-mail: sulek@zeus.amb.edu.pl
Recent studies suggested that leptin (Ob) and its receptor (ObR) could be involved in the
pathogenesis of various human malignancies, among others in endometrial cancer. Moreover,
hypoxia, which is associated with solid tumors, might stimulate, through hypoxia-inducible
factor 1alpha (HIF-1alpha), expression of Ob and ObR. In the present study, we analyzed by
immunohistochemistry the expression of Ob, ObR and HIF-1alpha in 60 cases of human
endometrial cancer tissues. Additionally, we assessed correlations among studied proteins as
well as relationships with selected clinicopathological features. Immunoreactivity for Ob,
ObR and HIF-1alpha protein was observed in 56.7%, 30.0% and 78.3% of endometrial
cancers, respectively. The expression of HIF-1alpha showed a significant positive correlation
with Ob (p

Session II : Receptor signaling and G proteins Poster II, 49
Activated melanocortin 3 receptor is endocytosed and leads to modification of
transcription factor forkhead (Drosophila) homolog (rhabdomyosarcoma) like 1
James M. Wachira, Bindhu Guruswamy, Lawrence C. Uradu and Cleo A. Hughes-
Darden
Department of Biology, Morgan State University, 1700 E. Cold Spring Lane, Baltimore
MD 21251, U.S.A. E.mail: jwachir@morgan.edu, binduguru25@yahoo.com,
lawrenceuradu@yahoo.com, chughes@moac.morgan.edu
Melanocortins play a central role in autonomic modulation of metabolism by acting through a
family of highly homologous G-protein coupled receptors. Studies with gene knockout mice
have implicated melanocortin receptors, MC3R and MC4R, in the etiology of obesity, insulin
resistance and salt-sensitive hypertension. In an attempt to better understand the mechanisms
of function of these receptors, we expressed MC3R and MC4R in neuronal cells and
demonstrated their colocalization to several membrane regions. We now show that in
cultured neuronal cells, MC3R localizes to lipid rafts and undergoes endocytic internalization
upon activation by gamma-MSH through a protein kinase sensitive pathway. The appearance
of the internalized receptor in lysosomes suggests that it is subsequently degraded. The
expression of protein kinase A regulatory subunits and of c-Jun and c-Fos was analyzed by
either immunoblotting or real-time PCR. No discernable changes were observed in the
expression levels of these protein kinase A and protein kinase C responsive genes. However,
an observed modification of the protein kinase B responsive FKHRL1 in MC3R expressing
cells suggests an important role for the PI3K/AKT pathway in melanocortin mediated effects.
Immunohistochemical studies showed a robust expression of MC3R protein in brain nuclei
with relevance to cardiovascular function and fluid homeostasis thereby suggesting that the
physiological effects of melanocortins on the cardiovascular system arise from effects on the
central nervous system.
Supported by MBRS-Score grant #2SO6-GM051971-08 and RCMI grant # G12RR017581.
Lawrence Uradu is supported by NIH/MARC grant #5T34GM007977-21
- 176 -

Session II : Receptor signaling and G proteins Poster II, 50
Aberrant activation of Wnt/#-catenin pathway in esophageal squamous cell carcinoma
(ESCC): evidence of FRAT1 overexpression
Yihua Wang1, Shuang Liu1, Wei Zhang1, Hongxia Zhu1, Xiaobo Zhou1, Cuiqi Zhou1,
Guo Zhang1, Lanping Quan1, Jinfeng Bai1, Liyan Xue2, Ning Lu2 and Ningzhi Xu1
1Laboratory of Cell and Molecular Biology; 2Department of Pathology; Cancer
Institute & Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100021, China. E-mail: xningzhi@public.bta.net.cn
Esophageal squamous cell carcinoma (ESCC) ranks among one of the most frequent cause of
cancer death in the world. Despite recent advances in therapy and management, esophageal
cancer remains a killer. Therefore, new therapies based on a better understanding of the
biology of esophageal cancer are most required. Recently, accumulation of nuclear and
cytoplasmic #-catenin has been observed in ESCC. However, mechanisms underlying this
phenomenon remain unknown. Frequently rearranged in advanced T-cell lymphomas-1
(FRAT1), strikingly overexpressed in some human esophageal cancer cell lines, is a positive
regulator of the Wnt/#-catenin pathway. Whereas FRAT1 is a regulator of cytoplamsic #catenin,
little is known with regard to the molecular relationship between FRAT1 and #catenin
in human cancers, including ESCC.
In this study, FRAT1 cDNA was cloned and transfected into human ESCC cells. The effects
of FRAT1 overexpression on cellular growth and transcriptional activity of #-catenin/T-cell
factor (TCF) were analyzed. RNA interference (RNAi) was also used to determine the
functions of FRAT1. In situ hybridization and immunohistochemical analysis were performed
to identify the level of FRAT1 mRNA expression and localization of #-catenin in surgical
specimens of ESCC respectively.
We observe that FRAT1 is overexpressed in ESCC and speculate that aberrant activation of
Wnt/#-catenin pathway in esophageal cancer appears to be due to upstream events such as
FRAT1 overexpression. Analysis of freshly resected ESCC specimens showed that FRAT1
was overexpressed in approximately 74% of tumor samples compared to matched normal
ones. We found that overexpression of FRAT1 in ESCC cell line KYSE150 significantly
promoted cell growth, whereas suppression of FRAT1 level by RNAi markedly inhibited
growth of esophageal tumor cells. In addition, FRAT1 overexpression induced the nuclear
accumulation of #-catenin and promoted the transcriptional activity of #-catenin/TCF. These
effects were reversed by coexpression of GSK3# or &NTCF4. Furthermore, aberrant
expression of #-catenin was correlated with FRAT1 overexpression in primary human ESCC.
Taken together, our data support the novel hypothesis that FRAT1 overexpression is critical
to Wnt/#-catenin pathway activation in ESCC. These findings identify components of Wnt/#catenin
pathway, such as FRAT1 overexpression, as possible therapeutic targets for the
development of novel molecular treatments for human esophageal cancer.
- 177 -

Session II : Receptor signaling and G proteins Poster II, 51
PDGFRbetaY857F mutant mediates PDGF-BB-induced signalling and chemotaxis,
despite severely reduced kinase activity.
Piotr Wardega, Carl-Henrik Heldin, Johan Lennartsson.
Ludwig Institute for Cancer Research, Uppsala University, Box 595, SE-751 24 Uppsala,
Sweden
Piotr.Wardega(AT)LICR.uu.se,C-H.Heldin(AT)LICR.uu.se,
Johan.Lennartsson(AT)LICR.uu.se
Platelet-derived growth factor receptor (PDGFR) becomes dimerized and activated upon
ligand binding, which leads to receptor transphosphorylation and increases its enzymatic
activity. In the present study, we studied the effects of mutation of tyrosine residue 857 in the
activation loop of PDGFRbeta, to phenylalanine. In agreement with published work, our
studies show, that PDGFRbetaY857F has much lower kinase activity in vitro in comparison
to PDGFRbetaWT , both as measured as receptor autophosphorylation and as phosphorylation
of an exogenous substrate. Interestingly, PDGF-induced tyrosine phosphorylation of
PDGFRbeta, in a cellular context, is not affected by the Tyr857 mutation. There are, however
significant changes in ability of PDGFRbetaY857F to induce signal transduction compared to
the wild-type receptor. For example, we could show that Stat5 signalling differs dramatically
between PDGFRbetaWT and PDGFRbetaY857F. We observed almost complete loss of
PDGF-induced Stat5 phosphorylation by the mutant receptor. Surprisingly Stat3, which
belongs to the same group of transcription factors as Stat5, was phosphorylated to the same
extent by mutant and wild-type receptor. In addition, we have also found that the Akt and
JNK signalling pathways are not affected by the PDGFRbetaY857F mutation. Moreover, the
Erk pathway was activated by slower kinetics in the mutant receptor. Interestingly, in spite of
the defect in the PDGFRbetaY857F receptors kinase activity and the changes observed in
signal transudction it could still efficiently induce cell chemotaxis. It is known that many
cytoplasmic tyrosine kinases are capable of phosphorylating the PDGFR and it is possible that
these may compensate for the lowered enzymatic activity of PDGFRbetaY857F . In summary,
we have demonstrated that the PDGFRbetaY857F has defect kinase activity, but is
nevertheless phosphorylated in the cell, presumably by cytoplasmic kinases and can induce
signals sufficient for normal chemotaxis toward PDGF-BB.
- 178 -

Session II : Receptor signaling and G proteins Poster II, 52
AF6 negatively regulates Rap1-induced cell adhesion
Zhongchun Zhang, Holger Rehmann, Leo S. Price, Jurgen Riedl, and Johannes L. Bos
From the Department of Physiological Chemistry and Centre of Biomedical Genetics,
University Medical Centre Utrecht, The Netherlands.
Address correspondence to Johannes L. Bos, Department of Physiological Chemistry
and Centre of Biomedical Genetics, University Medical Centre Utrecht, Universiteitsweg
100, 3584 CG Utrecht, The Netherlands. Tel. +31302538988; Fax. +31302539035 E-mail:
J.L.Bos@med.uu.nl
Small GTPases of the Ras family are molecular switches, whose functions are guiding
extracellular signals towards cellular responses. Rap, one of the most extensively-studied
members of the family, regulates cell proliferation, cell differentiation, cell death, and most
notable cell-matrix and cell-cell adhesion.
AF6 is involved in the connection of membrane-associated proteins to the actin cytoskeleton.
It binds to Ras-like small GTPases and is suggested to be an effector of both Ras and Rap.
In the absence of an immune challenge, T cells circulate the body in a non-adhesive state.
The maintenance of this non-adhesive condition prevents inappropriate immune responses
from occurring. An increase in Rap activation is sufficient to rapidly upregulate T cell
adhesiveness, demonstrating that Rap signaling must be tightly controlled in unstimulated T
cells.
In this study, we show that knockdown of AF6 in T lymphocyte by RNAi enhances Rap1induced
integrin-mediated cell adhesion, whereas overexpression of AF6 has the opposite
effect. We conclude that AF6 is a negative regulator of Rap-induced cell adhesion. In
addition, our results suggest that endogenous AF6 may function to buffer GTP-Rap in resting
cells, maintaining it in a non-productive complex. Therefore, loss of AF6 function may result
in immunological disorders.
- 179 -

Notes
- 180 -

Notes
Notes
- 181 -

- 182 -

Session III. Protein kinase cascades as therapeutic targets
- 183 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 1
Cyclic nucleotide-dependent inhibition of ecto-nucleotide
pyrophosphatase/phosphodiesterase 1 (E-NPP1) expression.
I. Aerts1, P.P. De Deyn2and H. Slegers1
Department of Biomedical Sciences, Cellular Biochemistry1, Neurochemistry and
behaviour2, University of Antwerp, Belgium
Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (E-NPP1) is a member of a multigen
family which contains NPP1 (PC-1), NPP2 (autotaxine) and NPP3 (B10; gp130RB13-6).
These are type II transmembrane metalloenzymes that hydrolyse ATP into AMP and PPi or
lysophosphatidylcholine into lysophosphatidic acid. NPP1 is expressed in different tissues
and abnormal expression is observed in several pathologies (1).
Glioblastoma multiforme (GBM) is the most aggressive glioma with an expected lifespan of
less than one year. Immunohistochemistry demonstrated that NPP1 expression is not detected
in normal tissue while its expression increased with the gradation of glioma. NPP1 is highly
expressed in reactive astrocytes of GBM.
The rat C6 glioma cell line, an experimental ‘in vitro’ and ‘in vivo’ model system for GBM
(2), is used for the study of the expression of NPP1. Cyclic AMP-dependent induction of
differentiation resulted in inhibition of NPP1 expression. The use of specific PKA inhibitors
indicated that this decrease in expression is PKA-dependent. Nitroprusside, a nitric oxide
(NO) donor, inhibited the expression of NPP1 suggesting that NO may be involved in the
latter mechanism. BAY 41-2272, a NO-independent activator of soluble guanylate cyclase
(sGC), attenuated NPP1 expression. The presented data indicated that inhibition of NPP1
expression may be due to NO-induced activation of sGC. In addition dibutyryl cGMP, also
attenuated the expression of NPP1, characterizing that NO/sGC/cGMP as elements of the
signal transduction pathway that modulates the expression of NPP1. The signal transduction
components of the NO-dependent pathway that effects NPP1 expression remain to be
determined.
The elucidation of the mechanism of NPP1 expression may lead to new medications or
therapies for the treatment of patients with GBM.
- 184 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 2
Multiple roles of PI-3’ kinase / Akt signaling in human hepatoma cell proliferation and
drug-induced apoptosis
Catherine alexia 3, Marlène Bras 2, Guillaume Fallot 3, Nathalie Vadrot 3, Fanny
Daniel 3, Malika Lasfer 3, Houda Tamouza 3 & André Groyer 1
1 Inserm U.683 and 3 Inserm U.481, Faculté de Médecine Xavier Bichat, 75870 Paris
Cédex 18, France ; 2 Institut Pasteur, Département d'Immunologie, 75724 Paris Cedex
15, France. e-mail: groyer@bichat.inserm.fr
IGF-II and type I-IGF receptor (IGF-IR) gene expression is increased in primary liver
tumours and transgenic mice over-expressing IGF-II in the liver develop hepatocarcinoma
(HCC) spontaneously, suggesting that alterations of IGF-IR signaling in vivo may play a role
in the auto/paracrine control of hepatocarcinogenesis. We have addressed the contribution of
PI-3’K/Akt signaling on the proliferation of HepG2 human hepatoma cells and on their
protection against doxorubicin-induced apoptosis. Both basal HepG2 cell DNA replication
and that stimulated by IGF-IR signaling were inhibited by the specific PI-3’K inhibitor
Ly294002 (Ly). In the former case, PI-3’K signaling overcame cell cycle arrest in G1 via
increased cyclin D1 protein and decreased p130Rb and p27kip1 gene expression. Doxorubicin
treatment induced apoptosis in HepG2 cells and was concomitant with the proteolytic
cleavage of Akt-1 and -2. Drug-induced apoptosis was reversed by IGF-I and was (i)
dependent on Akt-1 and -2 phosphorylation and (ii) accompanied by the inhibition of initiator
caspase-9 activity, suggesting that IGF-IR signaling interferes with mitochondria-dependent
apoptosis. Accordingly, Ly enhanced doxorubicin-induced apoptosis and suppressed its
reversal by IGF-I. Altogether, the data emphasize the crucial role of PI-3’K/Akt signaling (i)
in basal as well as IGF-IR-stimulated HepG2 cell proliferation and (ii) in controlling both
doxorubicin-induced apoptosis (e.g., drug-induced cleavage of Akt) and its reversal by IGF-I
(protection against apoptosis parallels the extent of Akt phosphorylation). They suggest that
targeting Akt activity or downstream Akt effectors (e.g. GSK3-beta, FOXO transcription
factors) may help define novel therapeutic strategies of increased efficacy in the treatment of
HCC-bearing patients.
- 185 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 3
Grb2 associated binder 1 (Gab1) adaptor/scaffolding protein regulates Erk signal in
human B cells
Adrienn Angyal1, David Medgyesi2, Gabriella Sarmay1
1Department of Immunology, Lorand Eotvos University, Budapest 1117, Hungary, email:
a1117@freemail.hu, 2Research Group of the Hungarian Academy of Sciences at
the Department of Immunology, Lorand Eotvos University, Budapest 1117, Hungary
Grb2-associated binder 1 (Gab1) is a member of the Gab/dos family of adapter molecules
involved in the signal transduction pathways of a variety of growth factors, cytokines, and
antigen receptors. In contrast to its positive role upon cytokine and growth factor receptor
signaling, Gab1 was shown to be a negative regulator of T independent 2 response of
marginal zone B cells in the spleen and to participate in a negative feedback loop of T cell
activation. Gab adaptor proteins have several tyrosine residues that are phosphorylated upon
activation of tyrosine kinases and consequently bind signaling molecules with SH2 domains,
such as SHP-2 tyrosine phosphatase and phosphatidyl inositol 3-kinase (PI3-K); and also
have pleckstrin homologue (PH) domain, that binds to PIP3 in the cell membrane. Gab1 was
shown to participate in activating ras/MAPK pathway upon EGFR signaling that was
mediated by the SHP-2 tyrosine phosphatase and the ras regulating protein, rasGAP.
Furthermore, via positive feed back regulation, Gab1 enhances PI3-K activity, resulting in a
high level of phosphatidyl inositol 3,4,5 trisphosphate (PIP3) in the plasma membrane.
The function of Gab1 in B cell signaling has not been clarified yet. We explored this question
by using siRNA to temporarily knock down Gab1 in BL41 Burkitt lymphoma cell line. The
transfer of siRNA to the cells was carried out by nucleofector technology (Amaxa). 24 h after
the transfection with Gab1 siRNA the efficiency of silencing and its functional consequences
were tested by Western blot analysis. Activation of Erk and Akt was followed by using
antibodies specific for the phosphorylated forms of the kinases. Erk activation was almost
completely blocked while Akt phosphorylation was partially reduced in the Gab1 knocked
down samples. These results suggest that in human B cells Gab1 is a major regulator of Erk,
and a less potent regulator of Akt/PKB.
- 186 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 4
Mallory Body Keratin Aggresomes Form in Response to Integrin Initiated Cell
Signaling MAP Kinase pathway
Fawzia Bardag-Gorce, Yong Wu, Latoiya Wilson, Jun Li, Li Nan, Barbara A. French
and Samuel W. French.
LABioMed at Harbor UCLA Medical Center. Torrance, CA, 90502. USA.
Mallory body formation is observed in the liver cells of alcoholic patients, as well as in other
non-alcoholic diseases. Several factors, including proteasome inhibition, are hypothesized to
be the cause of this phenomenon of MB formation. Feeding mice the drug diethyl-1, 4dihydro-2,
4, 6-trimethyl-3, 5-pyridinedicarboxylate (DDC) for 10 weeks, is a good model to
study Mallory body formation. Recent study showed that the primary culture of hepatocytes
isolated from the liver of these drug primed mice is also a good in vitro model to study this
phenomenon.
The MB formation was in several case associated with tumor formation. In these drugprimed
mice tumor growth was noticed in the liver of mice where the tumor cells showed also
formation of MB. The mitogen–activated protein kinase pathway, a central regulator of
tumor growth and cell differentiation, was studied to further examine the array of factors
leading to Mallory body formation. MEK is a central focus of the pathway and it is
associated with the up regulation by phosphorylating of the map kinase P44/42 (ERK &).
Western blot analysis exhibited increased phosphorylation of p44/42, which was associated
with an increase of Mallory body formation in liver cells of mice fed DDC. In the DDCprimed
primary culture of hepatocytes there was a marked MBs formation and this formation
was significantly reduced when U0126, an inhibitor of MEK1 protein, which reduced
significantly the phosphorylated forms of ERK1/2. Other tumor markers such as AFP, was
examined and showed an increase in the protein level when the mice were fed DDC and
where a significant amount of MBs were formed. This data are consistent with the conclusion
that Mallory body formation is associated with preneoplastic state of the cell.
- 187 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 5
Sanguinarine inhibits VEGF-induced Akt phosphorilation
Giuseppina Basini, Sujen E. Santini, Simona Bussolati, Francesca Grasselli
Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualità e Sicurezza
degli Alimenti, Sezione di Fisiologia Veterinaria, Università di Parma, 43100 Parma,
Italy. E-mail: basini@unipr.it
Angiogenesis, the process by which new blood vessels develop from pre-existing ones, plays
an essential role during embryogenesis, adult vascular remodelling and in several pathological
disorders including cancers, ischemic cardiovascular disease, diabetic retinopathy, wound
healing and inflammation. Unraveling the pro- and anti-angiogenetic mechanisms would offer
therapeutic options to ameliorate or perhaps even cure disorders that are now leading causes
of mortality. Research conducted over the past 10 years has demontrated that VEGF is a
major regulator of angiogenesis by interacting with cellular receptors and communicating
with the nucleus through a network of intracellular signaling pathways. Sanguinarine (SA), an
alkaloid isolated from Sanguinaria Canadensis, is known for its antiangiogenetic effects by
suppressing basal and VEGF-induced new vessel growth. The present study was aimed to
evaluate possible effect of SA on Akt phosphorylation, a critical event in VEGF-mediated
angiogenesis. Briefly, 2'105 immortalized porcine aortic endothelial cell (AOC) generously
provided by José Yelamos (Hospital Universitario Virgen de la Arrixaca, El Palmar, 30120
Murcia, Spain) were seeded in 1 ml M199 and treated with: 1) VEGF (100 ng/ml); 2) SA (300
nM); 3) VEGF+SA at room temperature. After a 10 min incubation, Akt activity was
evaluated by Akt/PKB Kinase Activity Assay Kit (Stressgene, Victoria, Canada). VEGF
treatment significantly (p

Session III : Protein kinase cascades as therapeutic targets Poster III, 6
Constitutively active cytoplasmic c-Jun N-terminal kinase 1 is a dominant regulator of
dendritic architecture: role of microtubule-associated protein 2 as an effector.
Benny Bjorkblom, Nina Ostman, Vesa Hongisto, Vladislav Komarovski, Jan-Jonas
Filen, Tuula Nyman, Tuula Kallunki, Michael J. Courtney, Eleanor T. Coffey.
Turku Centre for Biotechnology, Åbo Akademi and Turku University, Turku FIN-
20521, Finland. Email:ecoffey@btk.fi
Normal functioning of the nervous system requires precise regulation of dendritic shape and
synaptic connectivity. Here, we report a severe impairment of dendritic structures in the
cerebellum and motor cortex of c-Jun N-terminal kinase 1 (JNK1)-deficient mice. Using an
unbiased screen for candidate mediators, we identify the dendrite-specific high-molecularweight
microtubule-associated protein 2 (MAP2) as a JNK substrate in the brain. We
subsequently show that MAP2 is phosphorylated by JNK in intact cells and that MAP2
proline-rich domain phosphorylation is decreased in JNK1-/- brain. We developed
compartment-targeted JNK inhibitors to define whether a functional relationship exists
between the physiologically active, cytosolic pool of JNK and dendritic architecture. Using
these, we demonstrate that cytosolic, but not nuclear, JNK determines dendritic length and
arbor complexity in cultured neurons. Moreover, we confirm that MAP2-dependent process
elongation is enhanced after activation of JNK. Using JNK1-/- neurons, we reveal a dominant
role for JNK1 over ERK in regulating dendritic arborization, whereas ERK only regulates
dendrite shape under conditions in which JNK activity is low (JNK1-/- neurons). These
results reveal a novel antagonism between JNK and ERK, potentially providing a mechanism
for fine-tuning the dendritic arbor. Together, these data suggest that JNK phosphorylation of
MAP2 plays an important role in defining dendritic architecture in the brain.
- 189 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 7
Proliferation of human melanoma cells related to high expression of PKA regulatory
subunit 1A protein
Sara Bondioni, Erika Peverelli , Monica Rodolfo1 ,Stefano Ferrero 2, Silvano Bosari 2,
Paolo Beck-Peccoz Anna Spada, Andrea Lania and Giovanna Mantovani.
Institute of Endocrine Sciences, University of Milan, Ospedale Maggiore IRCCS,
1Istituto Nazionale dei Tumori and 2Pathology Unit, Department of Medicine, Surgery
and Dentistry, A.O. San Paolo and Ospedale Maggiore IRCCS; Milan, Italy.
The two regulatory subunits (R1 and R2) of PKA are differentially expressed in several
cancer cell lines and studies indicate distinct roles for these subunits in growth control. The
aim of this study was to evaluate the expression of the different PKA regulatory subunits
(R1A, R2A, R2B) in human melanomas, as well as the effects of selective subunit activation
on cell proliferation. Immunohystochemistry demonstrated high expression levels of the three
PKA regulatory subunits studied in all melanoma samples. On the contrary, in normal
melanocytes R1A was absent or expressed at very low levels, suggesting a higher R1/R2 ratio
in tumoral tissue compared to normal one. The effect of R1/R2 ratio on proliferation was
assessed in 6 human melanoma cell lines that show the same PKA regulatory subunits
expression pattern of melanoma samples. The R2-selective cAMP analogue 8-Cl-cAMP dosedependently
inhibited cell proliferation (60% inhibition at 5 M 8-Cl-cAMP) through the
induction of apoptosis. Inhibition of cell proliferation was also obtained by R1A silencing
through siRNA technique. Our data indicate that a high R1/R2 ratio promotes proliferation in
human melanoma cells.
- 190 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 8
Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on ERK1/2
(p44/p42 Mitogen-Activated Protein Kinases) cascade in pancreatic beta-cells
Christophe Broca, Safia Costes, Nathalie Linck, Joel Bockaert, Stephane Dalle and
Gyslaine Bertrand.
Equipe Avenir INSERM; IGF; INSERM U661; CNRS UMR5203; 141 rue de la
Cardonille, 34094 Montpellier cedex 5, France. E-mail : christophe.broca@igf.cnrs.fr
PACAP, neuropeptide belonging to the vasoactive intestinal peptide (VIP)/glucagon/secretin
family and interacting with both specific (PAC1) and VIP-shared (VPAC) receptors, plays an
important role in pancreatic beta-cell function. The ERK1/2 cascade, which modulates cell
proliferation and gene transcription but also cytoplasmic processes such as insulin granules
exocytosis, could thus mediate part of PACAP pancreatic effects. So, the aim of our study
was to investigate in pancreatic beta-cells (INS-1E and rat islets) the relationships between
PACAP and ERK1/2 activation (measured by western blotting of doubly phosphorylated
activated forms of these kinases) as well as the signaling cascade hereby activated.
First, in the absence of glucose, PACAP (100 nM) transiently (15 min) stimulated ERK1/2
phosphorylation whereas in the presence of a stimulating glucose concentration (8.3 mM),
PACAP induced a sustained and long lasting (( 3 h) stimulation of ERK1/2 phosphorylation
following a biphasic pattern. The activation of ERK1/2 cascade by PACAP was clearly
glucose-dependent (0-20 mM), and was totally abolished by the MEK (ERK1/2 kinases)
inhibitor PD98059 (20 µM). Moreover, PACAP-induced ERK1/2 phosphorylation was
clearly sensitive to the inhibition of the protein kinase A (PKA) by 10 µM H89 (-40%), the
inhibition of L-type calcium channel by 2 µM nifedipin (-65%), but also sensitive to the
inhibition of the phosphatidylinositol 3-kinase (PI3K) by 100 nM wortmannin or 10 µM
LY294002 (-40% and -50% respectively). Finally, compared to PACAP and forskolin, we
demonstrated that VIP exerts only minor effects on ERK1/2 cascade either in the absence or
the presence of glucose.
We thus concluded that PACAP exerts a sustained and prolonged effect on the pancreatic
beta-cell ERK1/2 cascade, mainly through the PAC1 receptor, and also through the
stimulation of both calcium-, PKA- and PI3K-cascade. The coupling of PAC1 receptors to
ERK1/2 could play an important role in the insulinotropic function as well as in the survival
of pancreatic beta-cells, and thus be relevant in diabetes physiopathology.
- 191 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 9
Reduced Bcr-Abl function leads to erythroid differentiation of K562 cells via downregulation
of ERK
A. Brózik*, Cs. Heged!s**, N.P. Casey***, A. Bors*, M. Geiszt****, K. Német* and M.
Magócsi*
* National Medical Centre, Dept. of Molecular and Cell Biology, Budapest, Hungary
** Eötvös Loránd Univ. Fact. of Science
*** Univ. of Tasmania, Division of Medicine, Australia
**** Semmelweis. Univ. Dept. of Physiology, Budapest, Hungary
E-mail: magocsi@biomembrane.hu
The chimeric bcr-abl gene encodes a constitutively active tyrosine kinase that leads to
abnormal transduction of growth and survival signals. Selective inhibition of this tyrosine
kinase with Gleevec reduces the autophosphorylation of Bcr-Abl protein and subsequently the
activity of ERK1/2 MAP kinases in K562 cells. According to our earlier observation that
down-regulation of ERK1/2 activities is able to induce differentiation of various erythroid cell
lines, we show here that inhibition of the MEK/ERK signalling pathway by the specific MEK
inhibitor UO126 is sufficient to induce hemoglobin expression in both mRNA and protein
levels - similarly to the effect of Gleevec treatment. Gleevec strongly reduces Bcr-Abl,
STAT-5, and ERK phosphorylation, decreases Bcl-xl level and induces caspase-3 dependent
apoptosis. In contrary, UO126 reduces only ERK activity and does not induce cell death.
For studying the effect of reduced Bcr-Abl function on erythroid differentiation at the level of
bcr-abl transcript we applied siRNA approach. Based on our FISH studies, K562 cells carry
more than 10 copies of the fusion bcr-abl gene. We used retroviral vector with GFP reporter
and H1 promoter driven DNA templates for siRNA mediated degradation of bcr-abl mRNA.
Despite the high (> 90%) transduction efficiency we could detect only a transient decrease in
Bcr-Abl protein and in phosphorylated ERK1/2 levels. This transient change in Bcr-Abl
signalling was sufficient to induce hemoglobin mRNA expression at 96-144 hours after
transduction without significant cell death. These results suggest that reduced Bcr-Abl
function can influence the differentiation blockade in chronic myeloid leukemia cells via the
ERK pathway.
- 192 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 10
Protein Kinase B (c-akt): a molecular switch in regulation of lineage choice decisions
during myelopoiesis
Miranda Buitenhuis, Liesbeth Verhagen, Christian Geest, Hanneke van Deutekom and
Paul J. Coffer.
Molecular Immunology Lab, Dept. of Immunology, University Medical Center Utrecht,
Lundlaan 6, 3584 EA Utrecht, The Netherlands.
Phosphatidylinositol 3-kinase (PI3K) has been reported to play a critical role in
proliferation and survival of a variety of cell types; however, a role in regulating blood cell
production is largely unknown. We have utilized an ex-vivo differentiation system, using
human umbilical cord blood derived CD34+ hematopoietic progenitor cells, to investigate the
role of PI3K and its downstream effectors in myeloid differentiation.
Inhibition of PI3K, with the pharmacological inhibitor LY294002, completely blocked
proliferation and differentiation during granulopoiesis. To pin-point the molecular
mechanisms responsible for this effect, we utilized specific pharmacological inhibitors of
PKB/c-akt or mTOR, both down-stream effectors of PI3K. Inhibition of PKB blocked
progenitor proliferation without affecting cell survival. Interestingly, inhibition of PKB,
abrogated neutrophil maturation, but conversely dramatically enhanced terminal
differentiation of eosinophils. Retroviral transductions were performed to ectopically express
a constitutively active PKB (myrPKB) in CD34+ cells. Transduction of cells with myrPKB
resulted in enhanced neutrophil differentiation and monocyte development compared to cells
transduced with empty vector alone. Conversely, ectopic expression of myrPKB resulted in an
almost complete block in eosinophil differentiation, surprisingly demonstrating neutrophil
differentiation even in the presence of IL-5. In order to investigate the role of PKB in
regulation of hematopoiesis in vivo, #2-microglobulin-/-NOD/SCID mice were transplanted
with CD34+ cells ectopically expressing active myrPKB or dominant-negative PKBcaax.
Transplantation of mice with CD34+ cells ectopically expressing myrPKB resulted in
enhanced neutrophil and monocyte development, whereas ectopic expression of PKBcaax
induced eosinophil development in vivo. To investigate which downstream signal
transduction pathways could be involved in PKB mediated regulation of myelopoiesis,
hematopoietic progenitors were either retrovirally transduced with myrPKB or treated with
pharmacological inhibitors, and western blot analysis was performed. Preliminary data
indicate that phosphorylation of C/EBPalpha, a transcription factor known to play an
important role in regulation of myelopoiesis, is inhibited upon PKB activation in
hematopoietic progenitors.
Taken together, these results demonstrate that PKB activity plays a critical role in the
regulation of cell fate choices during myeloid lineage commitment, most likely via regulation
of phosphorylation of C/EBPalpha. High PKB activity promotes neutrophil differentiation
and monocyte development, while reduction of PKB activity is required to induce eosinophil
differentiation.
- 193 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 11
Anaphylactic shock critically depends on PI3K and eNOS-derived NO
Anje Cauwels1, Ben Janssen2 and Peter Brouckaert1
1Department for Molecular Biomedical Research, Ghent University and VIB,
Technologiepark 927, B-9052 Ghent, Belgium; 2Department of Pharmacology and
Toxicology, University of Maastricht, Maastricht 6200 MD, The Netherlands
Anaphylaxis is an acute, severe and life-threatening systemic allergic reaction that occurs
after the reexposure to a specific antigen. Allergens include insect stings, medications,
antibiotics, anesthetics, contrast materials, certain foods, latex exposure or even exercise. A
serious anaphylactic reaction is associated with severe hypotension and requires immediate
parenteral treatment with epinephrine and aggressive volume resuscitation. However,
anaphylactic cardiovascular collapse can be resistant to epinephrine and intravenous fluids.
Platelet activating factor (PAF) is implicated in the cardiovascular dysfunctions occurring in
various shock syndromes, including anaphylaxis. It is generally accepted that the excessive
production of the vasodilator nitric oxide (NO) causes inflammatory hypotension and shock.
NO may be produced by the constitutive NO synthases eNOS (NOS3) or nNOS (NOS1), or
by the transcriptionally regulated inducible iNOS (NOS2). At this time, iNOS is regarded to
be the major producer of shock-inducing NO. Nevertheless, the contribution of NO to PAFinduced
or anaphylactic shock is still ambiguous. We studied PAF and anaphylactic shock in
conscious mice. Surprisingly, hyperacute PAF or anaphylactic shock and mortality entirely
depended on NO, produced not by the inducible iNOS but by the constitutive eNOS, rapidly
activated via the (alternative) phosphatidylinositol-3-kinase (PI3K) pathway. In contrast to the
unsubstantiated axioma that only excessive iNOS-derived NO underlies cardiovascular
collapse in shock, our data support the surprising concept that eNOS-derived NO is the
principal vasodilator in PAF-induced or anaphylactic shock and define eNOS and/or PI3K as
new possible targets for treating anaphylaxis.
- 194 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 12
Small GTPases Ras modulate transforming growth factor-beta 1-induced extracellular
matrix síntesis.
Begoña Cenador, Isabel Fuentes-Calvo, Eugenio Santos1, José M. López-Novoa and
Carlos Martínez-Salgado2.
Departamento de Fisiología y Farmacología, Universidad de Salamanca, Avda. Campo
Charro s/n, 37007 Salamanca, 1Centro de Investigación del Cáncer, Campus Miguel de
Unamuno s/n, 37007 Salamanca, and 2Unidad de Investigación, Hospital Universitario
de Salamanca, Paseo San Vicente 58-182, 37007 Salamanca.
Ras proteins are small GTPases with prooncogenic effect that act as transducers of
extracellular signals that regulate cell survival, growth and differentiation. Transforming
growth factor beta 1 (TGF-beta 1) has a relevant role in the origin and maintenance of
glomerulosclerosis (GSC) and tubule-interstitial fibrosis (TIF). TGF-beta and Ras signalling
pathways are close related: TGF-beta 1 overcomes Ras mitogenic effects and Ras counteracts
TGF-beta signalling. TIF is associated to increases in Ras, and with the activation of its
signalling pathways Erk/MAPK (mitogen activated protein kinases) and phosphatidylinositol-
3-kinase (PI3K)/Akt in a renal fibrosis model. We have studied the role of Ras, Erk/MAPK
and Akt in TGF-beta 1 mediated extracellular matrix (ECM) synthesis, using embrionary
fibroblasts from knockout (KO) mice for Sos 1 (sos 1-/-), a guanidine nucleotide exchange
factor that is the main Ras activating protein. Fibronectin and collagen type I expression, as
well as total collagen synthesis, is much bigger in sos 1-/- fibroblasts than in control sos 1+/+
fibroblasts, both in basal conditions and after TGF-beta 1 treatment. Ras activation is higher
in sos 1-/- fibroblasts than in controls. Phosphorylated Akt expression is also bigger in sos 1-/-
fibroblasts than in controls, but there are no differences in phosphorylated Erk expression.
These studies show that Ras may down-regulate TGF-beta 1-induced ECM synthesis, and the
Akt signalling pathway participates in the increase in ECM synthesis in sos 1-/- fibroblasts.
- 195 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 13
Yeast mitochondrial biogenesis is regulated via the cAMP protein kinase tpk3p
Cyrille Chevztoff, Michel Rigoulet and Anne Devin
IBGC du CNRS, 1 rue Camille Saint Saëns, 33077 BORDEAUX cedex, France. e-mail :
anne.devin@ibgc.u-bordeaux2.fr
In most organisms, the cellular mitochondrial content is modulated by environmental stimuli
and/or in response to energy demand changes. Several cAMP targets and transcription factors
have been shown to be involved in the up-modulation of mitochondrial biogenesis when
energy demand increases. In the yeast Saccharomyces cerevisiae, the Ras/cAMP/protein
kinase pathway is involved in many physiological adaptations of cells upon environmental
changes. Among the adaptation processes occurring during the transition between exponential
growth to stationary phase, the down-modulation of the cytochrome content and of the
respiratory activity of yeast cells plays a role in the maintenance of a high growth yield. The
question is therefore raised as to the role of the Ras/cAMP-protein kinase A pathway in this
adaptative process. We have previously shown that mutant strains exhibiting an increased or
decreased activity of this pathway have a coordinated change in mitochondrial enzyme
content. The question thus arose as to whether a specific kinase might be involved in this
process. We show that out of the three cAMP protein kinases in yeast, tpk3p is the one
involved in mitochondrial biogenesis. Indeed, when grown on non-fermentescible carbon
source TPK3- cells have a significantly decreased amount of mitochondria. Respiratory rates,
enzymatic equipment as well as mitochondrial DNA amount are decreased in this strain.
Mitochondria isolated from the TPK3- strain display distinct energetic features when
compared to the wild type, pointing out a role of Tpk3p in the biogenesis of the mitochondrial
respiratory chain. Moreover, this signaling pathway clearly involves reactive oxygen species,
as shown by the full reversal of the decreased amount of mitochondria in the TPK3- strain by
anti-oxidant. The activity of transcription factors involved in mitochondrial biogenesis is
decreased in the TPK3- strain and restored by anti-oxidant addition, thus indicating that
mitochondria to nucleus signaling might go through reactive oxygen species. This study
points out a crucial role of reactive oxygen species generated in a tpk3p deficient strain for the
adjustment of mitochondrial biogenesis to cellular energy demand.
- 196 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 14
MAPK pathways influence the release of .NO and PGE2 in IL-1# stimulated
chondrocyte/agarose constructs subjected to dynamic compression
T T Chowdhury, K Elliot*, D M Salter*, D L Bader and D A Lee
Queen Mary, University of London, Mile End Road, London. E1 4NS. UK
*Edinburgh University Medical School, Edinburgh. EH16 4TJ
E-mail : t.t.chowdhury@qmul.ac.uk
Introduction: Nitric oxide (.NO) and prostaglandin E2 (PGE2) are catabolic mediators
induced by IL-1# and are therefore potentially important pharmacological targets in
osteoarthritis (OA). In chondrocytes, the application of mechanical load has been shown to
counteract the IL-1# induced .NO and PGE2 release. Furthermore, the cytokine can activate
all three members of the MAPK family. However, the role of the MAPK subtypes in the
release of .NO and PGE2 in chondrocytes is complex and may primarily involve
phosphorylation of p38 or JNK and activation of NF!B and AP-1. The current study
examines whether inhibition of these pathways will abolish the IL-1# induced .NO and PGE2
release in mechanically stimulated chondrocyte/agarose constructs.
Methods: Bovine chondrocytes seeded in agarose gel were cultured under free-swelling
conditions, for 48 hrs in medium containing 0 or 10 ng.ml-1 IL-1#, supplemented with 0 to
1000 ng.ml-1 SB203580 (inhibitor of p38 MAPK) or 0 to 50 nM JNK II (inhibitor of JNK-1,
2, 3), or 0 to 1000 ng.ml-1 PDTC (inhibits NFkB activation) or 0 to 1000 ng.ml-1 curcumin
(inhibitor of AP-1). For the mechanical loading experiments, all constructs were subjected to
15 % dynamic compression (1 Hz) for 48 hrs under the following conditions: 0 or 10 ng.ml-1
IL-1#, +/- 10 ng.ml-1 SB203580 or 5 nM JNK II or 10 ng.ml-1 PDTC or 10 ng.ml-1
curcumin.
Results: For constructs cultured under free-swelling conditions, SB203580 and JNK II
resulted in a dose-dependent inhibition of nitrite and PGE2 release in IL-1# stimulated
constructs. PDTC and curcumin reversed the IL-1# induced nitrite release whereas PGE2
levels were only partially inhibited. For the mechanical loading experiments, dynamic
compression could counteract the IL-1# induced nitrite release. Compression-induced
inhibition of nitrite release was abolished by SB203580, PDTC and curcumin and partially
reduced by JNK II. The application of dynamic compression resulted in the strain-induced
inhibition of PGE2 release in IL-1# stimulated constructs. This effect was abolished by
SB203580 and JNK II and partially reduced by PDTC and curcumin.
Discussion: These results suggest the involvement of MAPKs and the transcription factors,
AP-1 and NFkB in the IL-1# induced release of .NO and PGE2. Further work will determine
the sequential activation of the signalling cascades associated with mechanical load and IL-
1#. Moreover, the temporal organization of the multiple pathways will provide important
information for the pharmacological and biophysical treatment of degenerative joint disease
as observed in OA.
Acknowledgements:
This work was supported by a project grant from the Wellcome Trust, project number,
073972
- 197 -

Session III : Protein kinase cascades as therapeutic targets Poster III,
The carcinogen Lindane disrupts autophagy at the maturation step by triggering the
MAPK ERK pathway
Elisabeth A. Corcelle1, Marielle Nebout1, Soumeya Bekri2, Nils Gauthier3, Paul
Hofman4, Philippe Poujeol5, Patrick Fénichel1 and Baharia Mograbi1
1INSERM, U670, IFR 50, Faculté de Médecine, Avenue de Valombrose, F-06107 Nice
Cedex 02, France, 2Groupe Appareil Digestif et Environnement (EA3234), Faculté de
Médecine, Bd Gambetta F-76183 Rouen cedex, 3INSERM, U627, IFR50, F-06107 Nice,
4INSERM, E0215/Laboratoire de Pathologie Clinique et Expérimentale, Hôpital
Pasteur, Nice and 5CNRS UMR 6548, Faculté des Sciences, Nice. E-mail :
mograbi@unice.fr
Macroautophagy (hereafter referred to as autophagy) has emerged as a key tumor suppressor
pathway. During this process, the cytosolic constituents are sequestered into autophagosomes,
which subsequently fuse with lysosomes to become autolysosomes where their contents are
finally degraded. Although a reduced autophagy has been demonstrated in human tumors or
in response to oncogenes and carcinogens, the underlying mechanism(s) remain(s) unknown.
Here, we show that widely used carcinogen Lindane promotes vacuolation of Sertoli cells. By
electron and immunofluorescent microscopy analyses, we demonstrated that these structures
are acid autolysosomes, containing cellular debris, and labeled by LC3, Rab7 and LAMP1,
markers of autophagosomes, late endosomes and lysosomes respectively. Such Lindaneinduced
vacuolation results from significant delay in autophagy degradation, in relation with a
decline of the lysosomal activity of Aryl Sulfatase A. At molecular level, we show that this
defect in autolysosomal maturation is independent of mTOR and p38 inhibitions. Rather, the
activation of the mitogen-activated protein kinase (MAPK)/ERK pathway is required for
Lindane to disrupt the autophagic pathway. Most importantly, we provide the first evidence
that sustained activation of ERK pathway is sufficient to commit cell to autophagic
vacuolation. Taken together, these findings strongly support that the aberrant sustained
activation of ERK by the carcinogen Lindane disrupts the maturation of autophagosomes into
functional autolysosomes. Our findings therefore suggest the possibility that high constitutive
ERK activity found in all cancers may provide a malignant advantage by impeding the tumor
suppressive function of autophagy.
- 198 -

Session III : Protein kinase cascades as therapeutic targets Poster III,
DJ-1, a negative regulator of PTEN, is widely expressed in ovarian carcinoma
Ben Davidson1, Vivi Ann Flørenes1, Martina Skrede1, Reuven Reich2
Department of Pathology1, the National Hospital-Norwegian Radium Hospital,
University of Oslo, Montebello N-0310 Oslo, Norway
2 Department of Pharmacology and Experimental Therapeutics, School of Pharmacy,
Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel
E-mail: ben.davidson@radiumhospitalet.no; vivi.ann.florenes@radiumhospitalet.no;
martina@skrede.name; reich@cc.huji.ac.il
DJ-1 has recently been shown to be a negative regulator of PTEN in Drosophila and in human
lung and breast carcinoma. The objective of this study was to analyze DJ-1 mRNA expression
in primary and metastatic ovarian carcinoma. Seventy-two peritoneal and pleural effusions,
43 primary tumors and 14 solid metastases were analyzed for DJ-1 mRNA expression using
RT-PCR. p-AKT and PTEN protein expression was analyzed in 70 effusions using Western
blotting. DJ-1 mRNA was frequently expressed at all anatomic sites (65/72 effusions, 35/43
primary carcinomas and 11/14 metastases), with similar expression at these sites (p>0.05,
Kruskal-Wallis test). In effusions, expression was higher in peritoneal compared to pleural
specimens (p=0.047, Mann-Whitney test) and in post-chemotherapy compared to primary
diagnosis specimens (p=0.012, Mann-Whitney test). Western blotting showed p-AKT
expression in 80% of effusions, with loss of PTEN in 90% of specimens. Patients with postchemotherapy
effusions expressing higher DJ-1 levels had shorter progression-free survival in
univariate analysis (p=0.027). Our data show that DJ-1 expression is frequent in ovarian
carcinoma, that its expression is associated with aggressive clinical course, and that it is
associated with AKT phosphorylation and silencing of PTEN in metastatic cells in effusions.
DJ-1 expression may therefore mediate enhanced AKT signaling in this cancer type, in
addition to AKT2 amplification.
- 199 -

Session III : Protein kinase cascades as therapeutic targets Poster III,
The mitogen-activated protein kinases (MAPK) p38 and JNK are markers of tumor
progression in breast carcinoma
Ben Davidson1, Sophya Konstantinovsky2, Lilach Kleinberg1, Mai TP. Nguyen1, Assia
Bassarova1, Gunnar Kvalheim3, Jahn M. Nesland1, Reuven Reich2
Departments of Pathology1 and Oncology3, the National Hospital-Norwegian Radium
Hospital, University of Oslo, Montebello N-0310 Oslo, Norway
2 Department of Pharmacology and Experimental Therapeutics, School of Pharmacy,
Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel
E-mail: ben.davidson@radiumhospitalet.no; sophya@md.huji.ac.il;
lilachkl@odont.uio.no; assia.bassarova@radiumhospitalet.no;
gunnar.kvalheim@klinmed.uio.no; j.m.nesland@medisin.uio.no; reich@cc.huji.ac.il
The objective of this study was to investigate the activation of mitogen-activated protein
kinases (MAPK) in breast carcinoma effusions, and to analyze its relationship to anatomic site
and clinical parameters. Activated MAPK (p-ERK, p-JNK and p-p38) expression was studied
in 42 effusions and 51 corresponding solid tumors (23 primary, 28 metastases) using
immunohistochemistry. Hormone receptor and HER2 status, proliferation (Ki-67) and
apoptosis (p85-PARP fragment) were assessed. MAPK levels, activity and activation ratio
(phospho/pan-MAPK ratio) were analyzed in 19 effusions using Western blotting (WB).
Nuclear expression of p-p38 and p-JNK was significantly higher in effusions compared with
both primary tumors (p

Session III : Protein kinase cascades as therapeutic targets Poster III, 15
A cross-talk between the Wnt and the adhesion-dependent signaling pathways governs
the chemosensitivity of acute myeloid leukemia
Fabienne De Toni , Loïc Ysebaert, Stéphane Manenti and Claire Racaud Sultan.
Centre de Physiopathologie de Toulouse Purpan, IFR 30, INSERM U563, Département
“Oncogenèse et signalisation dans les cellules hématopoïétiques”, CHU PURPAN, 3
place du Dr Baylac 31059 TOULOUSE Cedex 03, FRANCE. E-mail :
fadetoni@toulouse.inserm.fr
Relapses following chemotherapy are a major hindrance to patients' survival in acute myeloid
leukemia (AML). To investigate the role of the hematopoietic niche in the chemoresistance of
leukemic cells, we examined two pathways: one mediated by adhesion molecules/integrins,
and the other by soluble factors of the morphogen Wnt pathway. In our study, both the
adhesion of leukemic blasts to fibronectin and the addition of Wnt antagonists induced,
independently, resistance of AML cells to daunorubicin in a cell survival assay. Using
pharmacological inhibitors and siRNA, we showed that both resistance pathways required the
activity of the Glycogene Synthase Kinase 3b (GSK3b). Moreover, the AML cell protection
downstream of GSK3b was mediated by NF-kB. A link between the adhesion and the Wnt
pathway was found, as adhesion of U937 on human osteoblasts, a component of the
hematopoietic niche, triggered the secretion of the Wnt antagonist sFRP-1 and supported
resistance to daunorubicin. The osteoblast-conditioned medium could also confer
chemoresistance to U937 cells cultured in suspension, and this cell protective effect was
abrogated after depletion of sFRP-1. In the context of this potential double in vivo resistance,
modulators of the common signal GSK3b and of its target NF-kB could represent important
novel therapeutic tools.
F. De Toni et al, Oncogene 2006 in press
- 201 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 16
Repeated exposures of human skin fibroblasts to UVB at subcytotoxic level trigger
premature senescence through the TGF-ß1 signaling pathway.
Florence Debacq-Chainiauxa, Céline Borlona, Thierry Pascala, François Eliaersa,
Noëlle Ninannea, Françoise de Longuevilleb, José Remaclea and Olivier Toussainta
a Laboratory of Biochemistry and Cellular Biology, Department of Biology, University
of Namur (FUNDP), Rue de Bruxelles 61, 5000 Namur, Belgium
b Eppendorf Array Technologies, Rue du Séminaire 12, 5000 Namur, Belgium
e-mail: florence.chainiaux@fundp.ac.be
Premature senescence of human diploid fibroblasts (HDFs) can be induced by exposures to a
variety of oxidative stress and DNA damaging agents. In this study we developed a model of
UVB-induced premature senescence of skin HDFs. After a series of 10 subcytotoxic (nonproapoptotic)
exposures to UVB at 250 mJ/cm2, the so-called biomarkers of senescence were
markedly expressed : growth arrest, senescence associated ß-galactosidase activity,
senescence-associated gene overexpression, deletion in mitochondrial DNA. Using a low
density DNA microarray to study gene expression profiles of 240 senescence- and stressrelated
genes, a set of 44 genes were found as differentially expressed in this model among
which Transforming Growth Factor-ß1 (TGF-ß1). Neutralizing antibodies against TGF-ß1 or
its receptor II (TßRII) sharply attenuated the senescence-associated features, suggesting a role
for TGF-ß1 in UVB-induced premature senescence. Both the latent and active forms of TGFß1
were increased with time after the last UVB-stress. This model represents a proof-ofconcept
as alternative in vitro model in photoaging research for screening potential antiphotoaging
compounds.
- 202 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 17
Deguelin controls angiogenic and invasive properties of endothelial cells by modulating
the Akt/IkappaB kinase pathway cells.
Raffaella Dell’Eva1, Claudia Ambrosini 1, Douglas M. Noonan2, Adriana Albini,1 and
Nicoletta Ferrari1
1Experimental Oncology A Laboratory, National Cancer Research Institute (IST), L.go
R. Benzi 10, 16132 Genova, Italy. 2Università degli Studi dell'Insubria, Varese, Italy. Email:
nicoletta.ferrari@istge.it, raffaella.delleva@istge.it
Tumor growth relies on angiogenesis a process controlled by multiple growth factors
generating a multitude of signals that require careful coordination to guarantee a proper
vascular network. Here we evaluate the potential of deguelin, a natural plant productisolated
from Mondulea seriacea (Leguminosae) effective on both premalignant and malignant HBE
cells, and investigate its mechanisms of action. Deguelin causes extensive biological
modifications of endothelial cells by decreasing their growth rate while protecting them from
apoptosis, repressing their ability to migrate and invade but not affecting their capacity to
organize into capillary like structures. Inhibition of invasion by deguelin was associated with
decreased metalloprotease MMP- 2 activity. In vivo, deguelin blocks angiogenesis in the
Matrigel plug assay. Basal levels of phosphorylated Akt (pAkt) were reduced after brief
exposure to deguelin (1h) and inhibition of phosphorylation of IkB by TNFalpha in the
presence of deguelin correlates well with the ability of deguelin to decrease activation of NFkB
in the nucleus. Our data suggest that inhibition of NF-kB by deguelin may be an effective
strategy to control angiogenesis. Our results justify further studies to evaluate the utility of
deguelin as a chemopreventive agent.
- 203 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 18
Different ability to up-regulate FasL expression may confer differential response of
glioma cells to CB2-selective and non-selective cannabinoids
Aleksandra Ellert-Miklaszewska 1, Bozena Kaminska 2, Liliana Konarska 1:
1Dept. of Biochemistry and Clinical Chemistry, Medical University, Banacha 1 str., 02-
097 Warsaw, Poland, Email: aellert@nencki.gov.pl, 2Lab. of Transcription Regulation,
Nencki Institute, Pasteura 3 str., 02-093 Warsaw, Poland
Cannabinoids induce apoptosis of glioma cells in vitro and tumor regression in vivo. Despite
a growing interest, the molecular mechanisms of their pro-apoptotic action, induced by
stimulation of either of receptor type, CB1 and/or CB2, still remain elusive. We have
observed that synthetic cannabinoids: WIN-55,212-2 (non-selective CB1/CB2 agonist) and
JWH133 (CB2-selective), affect growth and viability of glioma cells. However, the
morphological changes and the time course of apoptotic cell death were different, depending
on the drug added. Thus, the aim of the present study was to identify pathways that
distinguish cannabinoid actions triggered by CB1/CB2 and selective CB2 stimulation in C6
glioma cells. We found that both compounds induce similar pattern of changes in MAPK
activation (p42/44 Erk, p38, JNK). However, the levels of phosphorylated (active) protein
kinase B (PKB/Akt) decreased significantly during incubation with WIN55,212-2, while it
was less pronounced after exposure to JWH133. The increase of the active form of Forkhead
transcription factor (FKHR), one of Akt downstream targets, and its translocation to the
nucleus occur after WIN55,212-2 but not JWH133 addition. Since FKHR has been implicated
in inducing the expression of genes critical for cell death, such as FasL gene, we studied the
role of Akt/FKHR pathway in regulation of FasL expression during WIN55,212-2- and
JWH133-induced apoptosis. RT-PCR analysis revealed the up-regulation of fasL mRNA level
only after WIN55,212-2 treatment, whereas it did not occur after JWH133, unless the cells
were pre-treated with LY294002, phosphatidylinositol-3 kinase (PI3K) inhibitor. The extent
of FasL expression correlated with the degree of PI3K/Akt inhibition, Forkhead activation
and apoptosis induction, suggesting that the observed difference in Akt inhibition might be
responsible for the diversified effects of the two cannabinoids. Our findings help to
understand differences in anti-tumor efficiency of cannabinoid receptor agonists, which in
turn might be of clinical relevance in glioma treatment. Supported by the grant No. 06/2002
from the Polish Pharmacy and Medicine Development Foundation by Polpharma S.A.
- 204 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 19
The ERK5 pathway promotes cell survival by down-regulating FasL expression
following osmotic stress stimulation
Katherine G Finegan1, Xin Wang1, Andrew C. Robinson1, Lynette Knowles1, Roya
Khosravi-Far2, Katherine A. Hinchliffe1, Raymond P. Boot-Handford3 and Cathy
Tournier1
Faculty of Life Sciences1 and Wellcome Trust Center for Cell Matrix3, University of
Manchester, Michael Smith Building, Oxford Road Manchester M130PT, UK;
Deparment of Pathology2, Harvard Medical School, Boston MA 02215, USA
Extracellular signal-regulated kinase 5 (ERK5) is a mitogen-activated protein kinase (MAPK)
that is activated by dual phosphorylation via a unique MAPK/ERK kinase, MEK5. The
analysis of the targeted deletion of the erk5 and mek5 genes has demonstrated a physiological
significance in their role in promoting cell survival. We have identified how the ERK5
signalling pathway regulates this process using as our models fibroblasts deficient in either
ERK5 or MEK5 expression. We found that ERK5 was required for mediating the survival of
fibroblasts under basal conditions and in response to sorbitol treatment. Increased FasL
expression acted in a positive feedback loop to enhance apoptosis of ERK5- or MEK5deficient
cells under conditions of osmotic stress. Compared to wild type cells, erk5-/- and
mek5-/- fibroblasts treated with sorbitol displayed a reduced Protein Kinase B (PKB) activity
associated with increased Foxo3a activity. Based on these results we conclude that the ERK5
signalling pathway promotes cell survival by down-regulating FasL expression via a
mechanism that implicates PKB-dependent inhibition of Foxo3a downstream of
Phosphatidylinositol 3-Kinase.
- 205 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 20
Constitutive active EGFR and Erk1/2 is promising targets for treatment of antiestrogen
resistant breast cancer cell lines
Thomas Frogne, Jan S. Jepsen and Anne E. Lykkesfeldt
Department of Tumor Endocrinology, Institute of Cancer Biology, Danish Cancer
Society Strandboulevarden 49, DK-2100 Copenhagen, Denmark E-mail: tfr@cancer.dk
Breast cancer patients with advanced disease often benefit from endocrine therapy. However,
after an initial response, most patients will develop resistance towards treatment. In our
department, we have established a large panel of antiestrogen resistant cell lines, hereby
mimicking antiestrogen resistant tumor growth. We have previously shown that these cell
lines are dependent on activated Protein Kinase B (PKB/Akt) to maintain growth in the
presence of antiestrogen and we are currently investigating the importance of the ErbB
receptor family. When we compare the parental and resistant cell lines under basal growth
conditions we see an upregulation of the Epidermal Growth Factor Receptor (EGFR) protein
and an increase in activity of the prominent downstream targets Extracellular Regulated
Kinase 1 & 2 (Erk1/2), in our antiestrogen resistant cells. Furthermore, the resistant cell lines
possessed increased activation of ErbB2 and had lost protein expression of ErbB4. To asses
the importance of EGFR and Erk1/2 in growth of the resistant cell lines we used the small
molecule inhibitors ZD1839 and U0126, which inhibits activation of EGFR and Erk1/2,
respectively. These experiments showed a significantly stronger growth inhibition of the
antiestrogen resistant cell lines when compared to parental cells. Further studies with these
inhibitors showed that activation of EGFR and Erk1/2 is not only required for growth of the
antiestogen resistant cell lines but also for the development of antiestrogen resistance.
Based on these observations, we conclude that constitutive activation of EGFR and Erk1/2 is
required for both development and growth of antiestrogen resistant cell lines.
We are currently evaluating expression of activated Akt and Erk1/2 in clinical material in
order to evaluate whether antiestrogen resistant tumors overexpress active Akt and/or Erk1/2
and whether these two kinases may be a target for treatment of antiestrogen resistant breast
cancer.
- 206 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 21
H- and N-Ras isoforms modulate transforming growth factor-beta 1-induced
proliferation and extracellular matrix síntesis.
Isabel Fuentes-Calvo, Begoña Cenador, Alejandro Esteller, Eugenio Santos1, José M.
López-Novoa and Carlos Martínez-Salgado2.
Departamento de Fisiología y Farmacología, Universidad de Salamanca, Avda. Campo
Charro s/n, 37007 Salamanca, 1Centro de Investigación del Cáncer, Campus Miguel de
Unamuno s/n, 37007 Salamanca, and 2Unidad de Investigación, Hospital Universitario
de Salamanca, Paseo San Vicente 58-182, 37007 Salamanca.
Transforming growth factor beta 1 (TGF-beta 1) has a relevant role in the origin and
maintenance of glomerulosclerosis (GSC) and tubule-interstitial fibrosis (TIF). Ras proteins
are small GTPases with prooncogenic effect that act as transducers of extracellular signals
that regulate cell survival, growth and differentiation. TGF-beta and Ras signalling pathways
are close related: TGF-ß1 overcomes Ras mitogenic effects and Ras counteracts TGF-beta
signalling. TIF is associated to increases in Ras, Erk and Akt activation in a renal fibrosis
model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors
Erk and Akt, in TGF-ß1-mediated ECM synthesis and proliferation, using embrionary
fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras-/-/N-ras-/-) isoforms
and from heterozygote mice (H-ras+/-/N-ras+/-). ECM synthesis is increased in basal
conditions in H-ras-/-/N-ras-/- fibroblasts, this increase being bigger after stimulation with
TGF-beta 1. TGF-beta 1-induced fibroblast proliferation is smaller in H-ras-/-/N-ras-/- than in
H-ras+/-/N-ras+/- fibroblasts. Erk activation is decreased in H-ras-/-/N-ras-/- fibroblasts;
inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double
KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM
synthesis. We can suggest that H- and N-ras isoforms down-regulate ECM synthesis, and
mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may
be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.
- 207 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 22
The possible role of PI3-kinase in melanoma cell migration.
Dorota Gil1, Joanna Duli"ska-Litewka1, Anna Pituch Noworolska2 and Piotr Laidler1
1Departament of Medical Biochemistry, Kopernika 7, 31-034 Kraków 2Department of
Clinical Immunology, Polish-American Children's Hospital, Jagielonian University
Medical College, e-mail: dgil@poczta.fm
Integrins are heterodimeric ("/# subunits) cell surface glycoproteins that bind to ECM
proteins and activate members of intracellular signalizing pathways cascades that can
stimulate or regulate motility and invasivnes, cell growth, and survival. Cancer cells very
often switch the types of extracellular matrix receptors (integrins) they express, favouring
ones that transmit progrowth signals. The expression of "3#1 integrin has been indicated as
possibly associated with invasive phenotype not only trough formation of a new cell-matrix
contact sites but also by regulating the expression of matrix-degrading enzymes, MMP-2 or
MMP-9 metalloproteinases. "3#1 integrin is a laminin (LN) receptor and recent studies
showed that laminin–5 and "5–containing laminins, such as laminin–10, and –11, are
preferred ligands for "3#1 integrin. Interaction of "3#1 integrin with LN–5 may provide
evidences to how it transduces signals that affect cell behavior.
METHODS: Studies were carried out on human melanoma metastatic cell lines: A 375 (from
solid tumor) and 1205Lu (from mouse lung). Cells were culture on plastic dishes alone or
covered with LN-5. Expression of integrins was studied by flow cytometry, secretion of
MMP-2 and MMP-9 by zymography, and the invasive potential by using conventional
Boyden chambers. Expression of cell signaling proteins was analyzed using Western Blot.
RESULTS: LN–5 stimulated up regulation of MMPs activity in both cell lines. Signaling
pathway necessary for migration of melanoma cells likely involved PI3-kinase activity as the
addition of the PI3-K specific inhibitor, LY294002, decreased the level of active MMP-2 in
A 375 and LU1205 cells. Western blot analyses of whole cell lysates confirmed the inhibition
of PI3-kinase activity after addition of LY294002, as determined with anti-phospho–AKT
(Ser 473) antibodies and observed increase in the level of phospho–AKT in the cells cultured
on LN–5.
CONCLUSION: Activation of PI3-kinase in response to ECM signals carried through
specific integrin receptor is likely an important event involved in metastasis of melanoma.
Understanding the intracellular signaling pathways in response to ECM proteins may be
useful in the development of tools for selective blocking of some signaling pathways.
This work is suported by the KBN: Jagiellonian University Medical College (W)/256/P/L)
and FP5 EU project “European Searchable Tumor Cell Lines Data Bank” – ESTDAB – NAS:
QLRI – CT-2001-01325.
- 208 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 23
CK2 modulates AKT kinase activity: implication in cell survival and death
Barbara Guerra
Dep. of Biochemistry & Molecular Biology, University of Southern Denmark, DK-5230
Odense, Denmark. E-mail: bag@bmb.sdu.dk
Protein kinase CK2 is a Ser/Thr kinase ubiquitously distributed and highly conserved in all
eukaryotic organisms so far investigated. CK2 is implicated in many cellular functions e.g.
cell growth, cell cycle progression, transformation and cell survival. Mounting evidence has
indicated that CK2 is also implicated in cancer. For instance, a potent biological synergy in
the development of primitive multiclonal lymphoid leukemias has been observed in bitransgenic
mice overexpressing CK2alpha-catalytic subunit and c-Myc. Accelerated
lymphomagenesis has been reported in mice overexpressing CK2 and deficient in the
expression of p53. These and other data have indicated that the inappropriate expression of
CK2, in cooperation with other proteins, might augment the oncogenic potential of cells. In
line with these observations, the phosphatidylinositol 3-kinase (PI3K)/AKT signal
transduction pathway has also been shown to be central in many intracellular processes such
as proliferation, angiogenesis, motility and cell survival. The fact that a search for substrates
of AKT has led to the identification of several components of the apoptotic machinery, and
that the PI3K pathway is negatively regulated by PTEN, a dual-specificity lipid and protein
phosphatase considered to be a tumour suppressor gene product, gave, indeed, rise to the idea
that AKT is a key element in the regulation of cell survival. Recently, we were able to show
for the first time that the specific inhibition of the PI3K-AKT signaling pathway in
combination with the depletion of either CK2 subunits by antisense oligodeoxynucleotides
leads to an enhanced drug-induced apoptotic response. In vitro as well as in vivo studies have
revealed that the individual CK2 subunits interact with AKT enhancing AKT kinase activity
and that the complex formation is not modulated by the phosphorylation status of AKT. These
data identifies a novel molecular mechanism that leads to modulation of AKT activation
raising the possibility that CK2 and AKT might be implicated in common pathways that
control cell proliferation and survival. Moreover, they underline the importance of developing
efficient strategies to directly inhibit AKT and CK2 as valuable therapeutic approach in
cancer therapy.
- 209 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 24
Animal models and drug design for therapy of Met-triggered cancers
Hessameh Hassani1, Nicolas Pietrancosta1, Bernard Maigret2, Jean-Louis Kraus1,
Flavio Maina1 & Rosanna Dono1
1IBDML, Campus de Luminy-Case 907, 13288 Marseille Cedex 09 France - 2UMR
CNRS/UHP 7565, Université Henri Poincare, Nancy 1, BP 239, 54506 Vandoeuvre-les-
Nancy Cedex, France. E-mails: dono@ibdm.univ-mrs.fr
Hyperactivity of the receptor tyrosine kinase (RTK) Met is crucial for development of
invasive/metastatic phenotype of neoplastic cells. Met-expressing cells acquire invasive
phenotype during progression of breast cancer, and autocrine activation by its ligand HGF is
responsible for spontaneous metastasis to the lung. Mutations or amplifications of Met human
gene have been found in different types of cancer cells and Met is a prognostic marker for
different carcinomas. To date, small-molecule drugs can selectively and efficiently inhibit
RTK activity both in vitro and in vivo and act as anticancer drugs. So far, there are no
pharmaceutical compounds able to interfere with Met activity in human cancers. We have
undertaken a multi-disciplinary approach, which combines cell biology, mouse genetics,
chemistry and non-invasive monitoring of primary tumours and metastasis, to develop
chemical compounds that specifically inhibit Met-triggered oncogenic events both in vitro
and in vivo. By performing biological and biochemical studies, we identify two compounds
that block Met-induced cell scattering, receptor trans-phosphorylation and intracellular
signalling in culture cells. We are currently performing chemical modifications in order to
optimize their efficacy and specificity. Computer-assisted molecular modelling has allowed
us to understand the mechanism of action of the novel Met inhibitors and maximize the
chance of predicting the most efficient chemical changes. This approach has led to the
generation of a Met inhibitor one log more active than the previous ones. The characterization
of this novel Met inhibitor is ongoing. We are also generating mouse models of Met-mediated
tumor and metastasis suitable to study activity of Met inhibitor in vivo. By taking advantage
of Cre-mediated recombination, transgenic mice will over-express Met in a temporal and
spatial regulated manner together with the Luciferase reporter to allow non-invasive
monitoring of tumours and metastasis.
- 210 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 25
Novel Involvement of the Immunomodulator AS101 in IL-10 Signal Transduction, Via
the Tyrosine Kinase Fer
Rami Hayun, Sali Shpungin, Hana Malovani, Michael Albeck, Uri Nir and Benjamin
Sredni
Safdié Institute for AIDS and Immunology Research, Faculty of Life Sciences, Bar-Ilan
University, Ramat-Gan, Israel. E-mail: srednib@mail.biu.ac.il
Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel.
Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel. E-mail:
nir@mail.biu.ac.il
Interleukin-10 (IL-10) plays a major role in proliferation of many tumor cells and activates
the transcription factor Stat3 by tyrosine phosphorylation. The immunomodulator ammonium
trichloro(dioxoethylene-o,o') tellurate (AS101) sensitizes several tumors to chemotherapy, by
inhibiting the tumor IL-10 autocrine loop. The tyrosine kinase Fer, is essential for the
proliferation of some malignant cell-lines and its connection to Stat3 activation
was demonstrated in a few cases. This study examined the role of AS101 in IL-10 signal
transduction and the correlation between Fer and Stat3, in human peripheral blood
mononuclear cells (PBMC). We showed that Fer was associated with Stat3 in PBMC and in
the RAW 264.7 cell line. Recombinant IL-10 increased the tyrosine phosphorylation of Stat3,
up regulated the levels of Fer, and increased the association of Fer with phosphorylated Stat3
(pYStat3). The immunomodulator AS101 as well as IL-10 receptor antagonist, Inhibited IL-
10 secretion, reduced the phosphorylation of Stat3, decreased the levels of Fer protein and
decreased the association between Fer and pYStat3. All AS101 activities were totally
abrogated by exogenous addition of recombinant IL-10. These results indicate that AS101
plays as a negative regulator in IL-10 signal transduction, and able to dephosphorylate the
proto-oncogene Stat3, and modulates the ability of Fer to associate with pYStat3. Therefore,
anti-IL-10 treatment using AS101 may be effective in certain malignancies and other
pathologies where IL-10 secretion is elevated and Stat3 is continuously phosphorylated.
- 211 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 26
Different signaling pathways mediates the strain-induced CREB activation in cardiac
fibroblasts
Britta E. Husse, Gerrit Isenberg
Julius Bernstein Institute of Physiology, Martin Luther University Halle-Wittenberg, D-
06097 Halle/S., Germany. E-mail: britta.husse@medizin.uni-halle.de
The transcription factor CREB (cAMP response element binding protein) mediates the
mechanical strain-induced gene expression in the heart. We investigated which signaling
pathways are involved in the CREB activation by strain using cultured ventricular fibroblasts
from adult rat hearts. The analysis of phospho-CREB by immunocytochemistry showed a
nearly complete CREB activation (93.2±6.1%) by cyclical mechanical strain (1 Hz, 5%
elongation, 15 min) which did not distinguish from the whole number of CREB-positive
fibroblasts (87.8±9.6%). The strain-induced CREB activation was partially reduced by PKA
inhibition (19.9±11.2%), by PKC inhibition (31.2±11.2%) but not by CaMK II inhibition. The
inhibition of several members of the MAPK cascade revealed a reduction of strain-induced
CREB phosphorylation by 24.4±5.8% (Src-inhibition), by 27.7±5.8% (Raf1-inhibition) and
by 23.8±8.6% (MEK-inhibition). The PI3-K inhibition reduced strain-caused phospho-CREB
by 19.3±5.4%. The inhibition of p38, the stress-sensitive pathway, decreased the straininduced
CREB phosphorylation by 21.5±12.8%. The time-dependent CREB activation by
cAMP stimulation with 10µM forskolin was transient with a peak after 5 min; from
18.5±6.3% to 42.9±15.9% phospho-CREB positive cells. The PKC-induced CREB activation
by 500 nM PMA was sustained beginning after 10 min; from 12.1±5.0% to 54.3±10.4%
phospho-CREB positive cells. Our results suggest that the complete strain-induced CREB
phosphorylation includes several signaling pathways. We discuss this wide-ranging
possibilities of CREB activation as safety for CREB-mediated gene expression in the heart.
- 212 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 27
Signal transduction pathway of CNP-dependent inhibition of upregulation of PAI-1
expression by TNF-alpha in endothelial cells
Hanna Jerczynska, Czeslaw Cierniewski, Zofia Pawlowska
Department of Molecular & Medical Biophysics, Medical University in Lodz, Poland. Email:
pawlow@zdn.am.lodz.pl
Plasminogen activator inhibitor type 1 (PAI-1) plays a fundamental role in regulation of the
fibrinolytic system. PAI-1 synthesis is upregulated by multiple factors, but only few are
known to be able to decrease PAI-1 expression. In our previous study we found that C-type
natriuretic peptide (CNP), which modulates salt and water balance as well as vascular tone,
was an effective inhibitor of PAI-1 synthesis and release from endothelial cells, it also reveals
stronger inhibitory activity for TNF-alpha - stimulated cells. The signaling pathways required
for this effect have not been elucidated. It was reported that NF-kB is involved in TNFregulated
PAI-1 production. We have evidenced that the activation of MAPK kinase cascade
is a necessary step in induction of PAI-1 expression by TNF in endothelial cells. In this study,
the effects of signal transduction inhibitors on TNF-induced PAI-1 expression in the presence
of CNP were examined. We used PD 98059, an ERK1/2 inhibitor, LY 294002, a PI3K/Akt
inhibitor, and 8-bromo-cGMP, a soluble analog of cGMP to characterize the signal
transduction pathway involved in CNP action in human endothelial cells. Involvement of NFkB
activation in this process was also investigated.
Our results imply that CNP inhibition of TNF-activated PAI-1 gene expression takes place via
regulation of signal transduction pathways involving PI3K/Akt and cGMP-dependent protein
kinase, possibly by inhibiting NF-kB-dependent pathways. The activation of ERK seems not
to be implicated in this process. Thus, our results explain at least in part the mechanism
involved in PAI-1 expression inhibition by CNP in TNF-stimulated endothelial cells.
Supported by grants: QLK3-CT-2002-30326 (5th EU FP) and Medical University of Lodz,
Poland.
- 213 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 28
PI3K$ - A Novel Target for Rheumatoid Arthritis. Closing the loop - From genetic
validation and rationale drug design to therapeutic proof of concept.
Ji H. 1, Rückle T. 1, Camps M. 1, Rintelen F. 1, Ardissone A. 2, Gillieron C. 1, Bertschy-
Meier D. 1, Cirillo R. 2, Hirsch E. 3, Wymann MP. 4, Vanhaesebroeck B. 5, Schwarz
M.K. 1 and Rommel C. 1*
1Serono Pharmaceutical Research Institute, Serono International S.A., 14, Chemin des
Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland 2LCG-RBM, Serono
International S.A., Via Ribes 1, 10010 Colleretto Giacosa, Italy. 3Department of
Genetics, Biology and Biochemistry, University of Torino, Via Santena 5bis, 10126
Torino, Italy. 4Department Clinical & Biological Sciences, Institute of Biochemistry &
Genetics, Centre of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel,
Switzerland. 5 Ludwig Institute for Cancer Research, 91 Riding House Street, London,
W1W 7BS, UK.
*christian.rommel@serono.com
Small molecule inhibitors targeting class I PI3K isoforms have vast potential use for the
treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular
diseases. However, the lack of specificity, isoform selectivity and poor biopharmaceutical
profile of PI3K inhibitors has so far hampered rigorous disease-oriented and
pharmacologically relevant target validation. PI3K$ plays a crucial role in mediating
leukocyte chemotaxis as well as mast cell degranulation, mainly in response to G-protein
coupled receptor activation and hence represents a high value target for autoimmunity and
inflammation.
In our laboratories we have studied PI3K$ deficient mice in various murine models of
rheumatoid arthritis (RA). We show that these mice are largely protected in these models,
which feature the effector phase of RA, resulting from impaired neutrophil migration. These
findings further validate PI3K$ as a therapeutic target.
Exploiting the interface of Molecular Medicine, Medicinal Chemistry and Experimental
Pharmacology, we have developed potent and selective small molecule PI3K$ inhibitors, that
upon oral treatment suppress the progression of joint inflammation and cartilage damage in
murine models of RA, reproducing the protective effects exhibited by PI3K$ deficient mice.
SAR of two generations of inhibitors, supported by X-ray crystallography and structure based
design, has led to the identification of the lead compound AS-605240 (Ki = 7.8 nM), active in
animal models of cell recruitment, mast cell activation and inflammation.
Our results identify selective PI3K$ inhibitors as potential therapeutics for the treatment of
RA.
- 214 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 29
Multiple roles for beta-arrestins in FSH signalling : a 5 S/T cluster in the C-terminus of
the receptor is important for beta-arrestin recruitment, desensitization and
internalization but not for beta-arrestin-mediated Erk activation.
E. Kara, P. Crepieux, C. Gauthier, N. Martinat, V. Piketty, F. Guillou, E. Reiter
UMR INRA-CNRS-Univ. Tours-Haras Nationaux, PRC, Equipe " Mécanismes d’action
des Gonadotropines ", 37380 Nouzilly France
The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning
receptor that couples to G"s upon agonist stimulation. #-arrestins (#-arr) are rapidly recruited
to the FSH-activated receptor and play key roles in the desensitization and internalization
processes. In addition, #-arr are able to activate the ERK pathway through a G proteinindependent
mechanism.
We have identified a cluster of five Ser/Thr residues in the c-terminus of the FSH-R (T638,
T640, S641, S642 and T644) which is important for #-arr recruitment. When these residues
were mutated into Alanines (5A), the FSH-induced recruitment of #-arr 1 and #-arr 2 was
reduced by 90% as assessed by co-immunoprecipitation with the FLAG-receptor. As
expected, cAMP accumulation by the 5A mutant was enhanced when compared to the wildtype
receptor. Consistantly, internalization of the 5A mutant was reduced when compared to
the wild-type control. In striking contrast however, the ability of the 5A mutant to activate
ERK via the #-arr pathway was actually increased when compared to the wild-type receptor.
Together, these results suggest that at least two functionally distinct pools of #-arr are
recruited to the activated FSH-R : 1) the predominant part interacts with the Ser638-Thr644
cluster, involved in both desensitization and internalization and exerts a negative role on the
ERK activation by both Gs and #-arr-dependent mechanisms, 2) a smaller proportion of the
#-arr does not interact with the Ser/Thr cluster and is required for ERK activation through a
#-arr-dependent pathway.
- 215 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 30
Design and functional activity of phosphopeptides with potential immunomodulating
capacity, based on the sequence of Grb2 associated binder 1 (Gab1)
Akos Kertesz1, Balazs Takacs1, Gyorgyi Varadi2, Gabor K. Toth2, Gabriella Sarmay1
1Department of Immunology, Lorand Eotvos University, Budapest 1117, Hungary, email:
sarmayg@cerberus.elte.hu and 2Department of Medical Chemistry, University of
Szeged, Szeged 6720, Hungary, e-mail: tgabor@mdche.szote.u-szeged.hu
Grb2-associated binder 1 (Gab1) is a scaffolding/adaptor protein involved in the signal
transduction pathways of growth factors, cytokines, and antigen receptors. Gab adaptor
proteins have several tyrosine residues which are phosphorylated upon ligand mediated
tyrosine kinase activation and bind signaling molecules with SH2 domains, such as SHP-2
tyrosine phosphatase, and phosphatidyl inositol 3-kinase (PI 3-K). We hypothesized that
phosphopeptides corresponding to motifs of Gab1 may interfere with the corresponding
signaling molecules in B cells, thus modulating the cell activation. Our main goal is to design
cell membrane permeable phosphopeptides based on the sequence of Gab1 that may regulate
the B-cell response.
Phosphopeptides representing Gab1’s tyrosine phosphorylated motifs were synthesized and
tested. Using peptide pull down assay, we identified SHP-2, Lyn and PLCg binding to the
same motif, 621GD-LD633. Furthermore, the 621GD-LD633 phosphopeptide bound and
powerfully activated SHP-2, while the PI3-K binding 442EL-PN453 and 467IQ-GP478
peptides less efficiently activated the phosphatase. In order to test the role of the 621-633
motif on B-cell signaling, the phosphopeptide was coupled to a membrane permeable
transporting peptide, octanoyl-R8 (OR8). Confocal laser scanning microscopy analysis of
cells exposed to the BODIPY dye labeled peptide showed that the peptide is taken up by B
cells and it is localized mostly in lysosome. Next, B cells were treated with membrane
permeable phosphopeptide, OR8(GD-LD) then the alteration of the intracellular free [Ca2+]
and tyrosine phosphorylation of intracellular proteins were tested. OR8(GD-LD) transiently
raised the level of [Ca 2+] in B-cells, and selectively modified the phosphorylation of certain
intracellular proteins.
Conclusion: cell membrane permeable phosphopeptides designed based on the sequence of
Gab adaptor protein may selectively modulate intracellular signaling pathways thus may be a
source of further drug development.
- 216 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 31
LRRK1 protein kinase activity is stimulated upon binding of GTP to its Roc domain
Daniel Korr, Luisella Toschi, Peter Donner, Hans-Dieter Pohlenz, Bertram Weiss, and
Bertolt Kreft
Research Laboratories, Schering AG, Muellerstr.178, 13342 Berlin, Germany,
E-mail: bertolt.kreft@schering.de
Human leucine-rich repeat kinase 1 (LRRK1) is a multi-domain protein of unknown function
belonging to the ROCO family of complex proteins. Here, we report the molecular
characterization of human LRRK1 and show, for the first time, that LRRK1 is both a
functional protein kinase and a GDP/GTP-binding protein. Binding of GTP to LRRK1 is
specific, requires the GTPase-like Roc domain, and leads to a stimulation of LRRK1 kinase
activity. LRRK1 is the first example of a GTP-regulated protein kinase harboring both the
kinase effector domain and the GTP-binding regulatory domain. Hence, we propose a model
in which LRRK1 cycles between a GTP-bound active and a GDP-bound inactive state.
Moreover, we mutated LRRK1 to mimic mutations previously identified in LRRK2/dardarin,
the only human paralogue of LRRK1, that have been linked to autosomal-dominant
parkinsonism. We demonstrate that three of four mutations analyzed significantly
downregulate LRRK1 kinase activity. Ultimately, the results presented for LRRK1 may
contribute to the elucidation of LRRK2’s role in the pathogenesis of Parkinson’s disease.
- 217 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 32
Activation of p38 MAP kinase in diosgenin-induced apoptosis in human rheumatoid
synovial cells with increased COX-2 expression
Bertrand Liagre1, David Y. Léger1, Pascale Vergne-Salle2, Philippe Bertin2, Richard
Trèves2 and Jean-L. Beneytout1.
1Laboratoire de Biochimie, UPRES EA 1085, Faculté de Pharmacie, Limoges, France.
E-mail: bertrand.liagre@unilim.fr; 2Service de Rhumatologie et Thérapeutique, CHRU
Dupuytren, Limoges, France.
Alterations in the apoptosis of synovial cells have been described in resident synoviocytes and
associated with the pathogenesis of RA. The role of cyclooxygenase-2 (COX-2) and
prostaglandins in synoviocyte death is still unknown. Recently, we showed that diosgenin, a
plant steroid, induced apoptosis in human RA FLS with COX-2 overexpression (Liagre B, et
al. (2004) Arthritis Res. Ther. 6:R373-R383). In a new work, we studied the kinase (Akt and
MAPK) signalling pathway after diosgenin-induced apoptosis in the human RA FLS.
Particular attention was paid to the modulation of COX-2 expression and activity in RA
synoviocyte viability. Our results showed that 40 µM diosgenin caused an inhibition of Akt
phosphorylation. Moreover, after diosgenin treatment, p38 MAP kinase activation was
increased in contrast with JNK or ERK activation. Phosphorylation of p38 was correlated
with an increase of COX-2 expression and activity. However, diosgenin inhibited nuclear
factor-kB (NF-kB) in apoptotic conditions in human RA FLS. In order to clarify if activation
of p38 was directly associated with diosgenin-induced RA FLS apoptosis, we used a selective
inhibitor of p38 (SB 203580) to verify DNA fragmentation after diosgenin treatment. We also
studied in the same conditions the level of COX-2 expression and prostaglandin E2 (PGE2)
production. Pre-treatment with SB 203580 before diosgenin treatment reduced the production
of mononucleosomes and oligonucleosomes in comparison with diosgenin alone.
Furthermore, this inhibition of diosgenin-induced apoptosis was correlated with an inhibition
of COX-2 expression and a decrease of PGE2 synthesis.
In conclusion, we showed for the first time that diosgenin-induced apoptosis in human RA
FLS is associated with an increase of p38 activity and a decrease of Akt phosphorylation and
NF-kB activation. Overexpression of COX-2 is consecutive at the activation of p38 after
diosgenin treatment. Future studies should evaluate whether diosgenin could be used as a
therapeutic agent for RA in association with a COX-2 inhibitor.
This work was supported by La Société Française de Rhumatologie.
- 218 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 33
Protein kinase C-eta as a possible therapeutic target in breast cancer
Etta Livneh, Galia Oberkobitz, Noa Rotem, Galia Karp, Adva Maissel and Galit Shahaf.
Department of Microbiology and Immunology, Ben Gurion University, Beer Sheva
84105, Israel, e-mail:etta@bgumail.bgu.ac.il
Protein kinase C (PKC) is involved in several major signal transduction pathways that control
gene expression, cell growth and differentiation. However, these kinases were not thoroughly
explored for their chemotherapeutic values. Here we have explored PKCeta as a possible
target for chemotherapy in breast cancer. PKCeta was recently suggested as a therapeutic
target in glioblastoma multiforme. PKCeta, expressed primarily in epithelial cells, appears as
a candidate regulator of mammary gland proliferation or differentiation, as its expression is
specifically up-regulated in the mammary gland during the transit from resting to pregnant
state. This prompt us to examine the hormonal regulation of its expression by estrogen and
progesterone as their levels are also up-regulated during pregnancy. We show that estradiol
gradually increased the expression of PKCeta in the estrogen-responsive lines MCF-7 and
T47D, but not in the estrogen non-responsive line MDA-MB 231. In contrast to the observed
up-regulation of PKCeta, the PKCdelta isoform was down-regulated while PKCalpha
expression was unaltered, demonstrating different response to estradiol stimulation.
Moreover, the presence of progesterone, involved in the differentiation of the mammary
gland, reduced the estrogen- induced PLCeta expression in a time-dependent manner.
Interestingly, our recent studies suggest also a role in apoptosis for PKCeta. Here we have
investigated its role in cell death induced by the DNA damaging agents UV irradiation and
the anti-cancer drug– Camptothecin (CPT). Our studies showed that the inducible expression
of PKCeta in MCF-7 cells provided partial resistance against cell death, which was
accompanied by increased cell survival and PARP cleavage. Furthermore, we show that JNK
activity required for apoptosis in MCF-7 cells was inhibited by the inducible expression of
PKCeta. Thus, our studies suggest that PKCeta has an important role in the hormonal
regulation, cell cycle control and resistance to DNA damage in breast cancer cells. These
properties could make PKCeta a target for chemotherapeutic intervention in breast cancer.
- 219 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 34
Spatial regulation of PKCeta: C1b domain and the pseudosubstrate containing fragment
target PKCeta to the Golgi and the Nuclear Envelope
Adva Maissel, Mairav Marom, Marat Shtutman, Galit Shahaf and Etta Livneh
Department of Microbiology and Immunology, Faculty of Health Sciences and the
Cancer Research Center, Ben Gurion University, Beer Sheva 84105, Israel
Protein kinase C (PKC) represents a family of serin/threonine kinases, playing a central role
in the regulation of cell growth, differentiation and transformation. These enzymes differ in
their primary structure, biochemical properties, tissue distribution and sub-cellular
localization. The specific cellular functions of PKC isoforms is largely controlled by their
localization. PKCeta, a member of the novel subfamily, is expressed predominantly in
epithelial tissues. However, not much is known with respect to its mechanism of activation
and regulation. Our recent studies suggest its role in cell cycle control. In this work we show
that PKCeta is localized at the Golgi apparatus, ER and the nuclear envelope. Furthermore,
using GFP-fusion proteins of the different functional domains of PKCeta we deciphered the
specific structural domains of the protein responsible for its apparent localization. We show
that the cysteine-rich repeat C1b is responsible for its Golgi localization, while for its
presence at the ER/nuclear envelope the pseudosubstrate containing fragment coupled to the
C1 domain is required. In response to short-term activation by PMA we show translocation of
PKCeta to the plasma membrane and the nuclear envelope. Moreover, we demonstrate that
the C1b is sufficient for its translocation to the plasma membrane. Using an antibody specific
for phosphorylated S675 (the hydrophobic site described as one of the priming sites of PKC
enzymes), we show that, PKCeta is hyperphosphorylated on the hydrophobic site under these
conditions of short-term PMA treatment. Interestingly, accumulation of PKCeta at the nuclear
envelope as well as hyperphosphorylation on the hydrophobic site also occurred in response
to serum-starvation. It should be noted that interaction of PKCeta with the cyclin E/Cdk2
complex at the perinuclear region was recently reported by us in response to serum-starvation.
Thus, our studies demonstrate translocation of PKCeta to the nuclear envelope, and suggest
that the spatial regulation of PKCeta could be important for its cellular functions including
effects on cell cycle control and involvement in tumor promotion.
- 220 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 35
Dlk/ZIP Kinase is involved in Mitosis and Cytokinesis
Steve Manderscheid, Gerd Landsberg, Dominik Brinckmann and Karl Heinz
Scheidtmann
Institute of Genetics, University of Bonn, Roemerstr. 164 D-53117 Bonn, Germany. Email:
stevemander@web.de
Dlk/ZIP Kinase is a Serin /Threonin specific Kinase of the proapoptotic DAP Kinase family.
Dlk/ZIP Kinase does not induce apoptosis per se, rather it seems to be involved in regulation
of transcription, contractile processes and mitosis, particularly cytokinesis. During mitosis
Dlk/ZIP Kinase associates with centrosomes, centromeres and the contractile ring. In order to
elucidate the biological role of these interactions and to map the interaction domains of
Dlk/ZIP Kinase with these structures we established cell lines stably expressing N- and Cterminal
truncation mutants at a low level. Mutant &N1 consisting of the extracatalytic
domain colocalized with centrosomes and the contractile ring but not with
centromere/kinetochore structures. Constitutive expression of this mutant did not reveal any
phenotype indicating that it did not act in a dominant negative manner, at least not at the low
expression level.
On the other hand, &C2NLS lacking the C-terminal 110 amino acid residues including the
leucine zipper, but containing a heterologous NLS did not associate with centrosomes or the
contractile ring, though it was found associated with chromatin. In contrast to &N1-expressing
cells, the cell line expressing &C2NLS exhibited a multinucleation phenotype. Interestingly,
the &C2NLS cell line appeared heterogeneous. The majority of cells expressed the GFP
fusion construct at a low level whereas about a quarter of the cells exhibited strong
overexpression, mainly in the nucleus. Overexpression strongly correlated with generation of
large round or deformed nuclei. Multinucleated cells were observed too.
Cells were additionally analysed for phosphorylation of histone H3, expression and
localization of chromosomal passenger proteins (aurora B, INCENP, and survivin). H3
phosphorylation of Serin 10 and Threonin 11 was normal, the chromosomal passenger
showed the known distribution pattern. Our data show for the first time that Dlk might be
involved in mitotic processes, prior to cytokinesis.
Acknowledgement : This work is supported by DFGGrant Sche 246/16 and “bourse de
formation-recherche” BFR 03/021
- 221 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 36
The JNK/NF-kB balance controls the life span of retinoic acid-treated APL cells.
Julie Mathieu1, Stéphane Giraudier2, Michel Lanotte1 and Françoise Besançon1
1INSERM U685, Centre Hayem, Hôpital St Louis, 1 avenue Claude Vellefaux, 75475
Paris, France; 2 INSERM U362, Institut Gustave Roussy, 39 rue Camille-Desmoulins,
94805 Villejuif, France.
E-mail : Julie.mathieu@stlouis.inserm.fr;Francoise.besancon@stlouis.inserm.fr
All-trans retinoic acid (ATRA) significantly improves the survival of patients with acute
promyelocytic leukemia (APL) by inducing granulocytic differentiation of leukemia cells.
Since an activation of the transcription factor NF-kappa B occurs during ATRA-induced
maturation of APL cells, a mechanistic link between these two processes was investigated.
Using an in vitro model for APL, we report that ectopic overexpression of a repressor of NFkappa
B activation did not affect granulocytic differentiation. Importantly, NF-kappa B
inhibition markedly resulted in a decreased viability of the differentiated cells which
correlated with increased apoptosis. Apoptosis was accompanied by a sustained activation of
the c-Jun N-terminal kinase (JNK). Inhibition of JNK by the specific inhibitor SP600125 or
by transfection of a dominant-negative mutant of JNK1 reduced the percentage of apoptotic
cells, thus showing that JNK activation constitutes a death signal. Furthermore, impairment of
NF-kappa B activation resulted in increased levels of reactive oxygen species (ROS) upon
ATRA treatment. ROS accumulation was suppressed by the antioxidant butylated
hydroxyanisol, which also abolished ATRA-induced JNK activation and apoptosis.
Altogether, our results demonstrate an anti-apoptotic effect of NF-kappa B activation during
ATRA-induced differentiation of NB4 cells and identify repression of ROS-mediated JNK
activation as a mechanism for this effect. Our observations also suggest that NF-kappa B
signaling may contribute to an accumulation of mature APL cells and participate in the
development of ATRA syndrome.
- 222 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 37
Determination of human epidermal growth factor receptors downstream signaling
phosphoproteins in breast cancer using suspension beads protein array.
Jean-Louis Merlin, Elisabeth Longavenne, Fadila Chergui, Carole Ramacci, Marie
Rouyer, Sanae Bouali, Pascal Genin, Agnès Leroux.
Centre Alexis Vautrin, EA3452 Université Henri Poincaré, Nancy, France
jl.merlin@nancy.fnclcc.fr
Human epidermal growth factor receptors (HER) have major therapeutic implications in
breast cancer. Receptor-directed monoclonal antibodies as well as tyrosine kinase inhibitors
are designed to block HER downstream signaling and achieve proliferation control and
apoptosis induction. In this study, we validate the use of multiplex phosphoproteins singlestep
analysis using suspension beads protein array to explore the functionality of HER
downstream signaling in two human breast cancer cell lines, MDA-MB231 and MDA-
MB468 : bearing different status of PTEN expression : MDA-MB468 are PTEN-null cell
lines.
The kinetics of expression of phosphorylated key-proteins of EGFR downstream signaling
(EGFR, AKT, p70S6 kinase, ERK1/2, GSK3 and p38MAPK) was measured by multiplex
analysis using suspension beads protein array in EGF treated-cells. EGF exposure led to
elevated expression of phospho-EGFR in both cell lines. Variations of expression of HER
downstream signaling phosphoproteins were detected among the cell lines. EGF exposure led
to increased phospho-AKT expression in PTEN expressing cells within the 5 minutes
following exposure to EGF. No variation in phospho-AKT expression was observed in the
PTEN-null cell line.
In breast cancer specimens, great variations of phosphoproteins expression were observed
showing that before treatment initiation HER downstream signaling functionality can differ
from patient to patient. Such variations could probably be implicated and explain differences
in clinical response to anti-HER drugs.
All suspension beads protein array results, achieved in cell lines or breast cancer specimens,
were compared and found fully consistent with western blot.
This study demonstrates the great interest of suspension beads protein array for single-step
phosphoproteins multiplex analysis. Suspension beads protein array can be used with sizelimited
tumor specimens and yield accurate determination of phosphorylation rates. These
results validate the use of multiplexed phosphoproteins analysis using suspension beads
protein array to assess functionality of HER dowstream signaling as predictive and/or
surrogate marker for clinical response to anti-HER drugs.
- 223 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 38
Phosphorylation by p42/44 MAPK regulates the activity and localization of human
hypoxia inducible factor HIF-1alpha.
Ilias Mylonis1, Georgia Chachami1,2, Martina Samiotaki3, George Panayotou3,
Efrosyni Paraskeva2, Eleni Georgatsou1, Sophia Bonanou1 and George Simos1
1Laboratory of Biochemistry and 2Laboratory of Physiology, Department of Medicine,
University of Thessaly, Larissa, Greece.
3Protein Chemistry Laboratory, B.S.R.C. «Alexander Fleming», Vari, Greece
E-mail: simos@med.uth.gr
Hypoxia inducible factor 1 (HIF-1) is a heterodimeric transcriptional activator that controls
the expression of most of the genes induced by hypoxic conditions. The molecular
mechanisms that regulate the expression and activity of its inducible subunit HIF-1alpha,
involve several post-translational modifications including hydroxylation, acetylation and
phosphorylation. In order to study the role of HIF-1alpha phosphorylation we have used
human recombinant HIF-1alpha as a substrate in kinase assays with cell extracts. Our data
show that at least two different nuclear protein kinases can interact with and modify HIF-
1alpha and one of them was identified as the p42/p44 MAPK. Analysis of phosphorylated
HIF-1alpha by mass spectroscopy revealed two serine residues as possible MAPK
phosphorylation sites in its carboxy-terminal part. Site-directed mutagenesis of these two
residues significantly reduced the phosphorylation of HIF-1alpha by either cell extracts or
purified MAPK, showing that they represent major in vitro modification sites. When the
mutant forms of HIF-1alpha were introduced into HeLa cells, they exhibited much lower
transcriptional activity than the wild-type. Furthermore, the GFP-tagged HIF-1alpha mutants
were excluded from the nucleus in contrast to the predominant nuclear localization of the
wild-type form. These data suggest that phosphorylation of the identified sites by MAPK is
required for nuclear accumulation of HIF-1alpha and subsequent activation of the hypoxia
target genes.
- 224 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 39
Doxorubicin-induced MAP kinase activation in hepatocyte cultures is independent of
oxidant damage
Rosaura Navarro, Rosa Martínez, Idoia Busnadiego, M. Begoña Ruiz-Larrea and José
Ignacio Ruiz-Sanz
Department of Physiology, Medicine and Dentistry School, University of the Basque
Country, 48080-Bilbao, Spain. E-mail: mbego.ruizlarrea@ehu.es
Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited due to its
toxicity. DOX cytotoxic effects have been associated with reactive oxygen species (ROS)
generated during the drug metabolism. ROS induce signaling cascades leading to changes in
the phosphorylation status of target proteins, which are keys for cell survival or apoptosis.
The mitogen-activated protein kinase (MAPK) cascades are routes activated in response to
oxidative stress. In this work the effects of DOX on cytotoxicity, indicators of oxidative stress
(malondialdehyde –MDA- and GSH), and the phosphorylation status of extracellular signalregulated
kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 kinases were analyzed in
primary cultures of rat hepatocytes. DOX (1- 50 µM) did not modify LDH release into the
medium during the incubation time up to 6 h. The levels of MDA, determined by HPLC, and
the intracellular GSH were constant during the treatment with the drug. GSH levels from
mitochondria extracted by Percoll gradient from cultured hepatocytes were not modified by
DOX, thus excluding its depletion or any impaired mitochondrial uptake. Characterization of
proteins by Western blot analysis revealed that DOX increased phosphorylation of p38
kinases, JNK1/2 and ERK1/2. In conclusion, DOX triggers activation of ERK, JNK and p38
kinases in primary hepatocyte cultures independently of oxidant damage.
This work was supported by the Basque Government (Research Project and Predoctoral
Training Grant to R.N.) and the University of the Basque Country (UPV00081.327-E-
15294/2003).
- 225 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 40
Superoxide anions are involved in doxorubicin-induced ERK activation in hepatocyte
cultures
Rosaura Navarro, Idoia Busnadiego, M. Begoña Ruiz-Larrea and José Ignacio Ruiz-
Sanz
Department of Physiology, Medicine and Dentistry School, University of the Basque
Country, 48080-Bilbao, Spain. E-mail: joseignacio.ruizs@ehu.es
Doxorubicin (DOX), an antineoplastic agent widely used for the treatment of cancer, belongs
to the anthracycline family of antitumor antibiotics. DOX may undergo one-electron
reduction to the corresponding semiquinone free radical by flavin-containing reductases.
Under aerobic conditions, the semiquinone radical reacts rapidly with oxygen to generate
superoxide anion, undergoing redox-cycling. At moderate concentrations, ROS play an
important role as regulatory mediators in signaling processes. We have shown that DOX
increased phosphorylation of enzymes comprising MAP kinase cascades in primary
hepatocyte cultures, and that this action was independent of oxidant damage. In particular,
extracellular signal-regulated kinase (ERK) was phosphorylated by the drug treatment. In this
work, we have determined the possible involvement of particular free radicals in DOXinduced
ERK phosphorylation in hepatocyte cultures by using specific free radical
scavangers. The levels of ERK phosphorylation were measured by Western blot analysis with
an anti-Thr202/Tyr204-phosphorylated 44/42 MAPK antibody. Deferoxamine (iron chelator),
catalase (hydrogen peroxide removing enzyme) or alpha-tocopherol (peroxyl-radical
scavenger) did not affect DOX-increased ERK phosphorylation levels. However, the cellpermeable
superoxide dismutase mimetic MnTBAP, and the flavin-containing enzyme
inhibitor diphenyleneiodonium, reverted DOX-induced effects. These results suggest that
superoxide anions, probably generated by DOX metabolism, are involved in the effects of the
anthracycline on the MAP kinase cascade activation.
This work was supported by the Basque Government (Research Project and Predoctoral
Training Grant to R.N.) and the University of the Basque Country (UPV00081.327-E-
15294/2003).
- 226 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 41
Doxorubicin transitorily triggers Akt/PKB activation in primary cultures of rat
hepatocytes
Rosaura Navarro, Idoia Busnadiego, M. Luisa Hernández, M. Begoña Ruiz-Larrea and
José Ignacio Ruiz-Sanz
Department of Physiology, Medicine and Dentistry School, University of the Basque
Country, 48080-Bilbao, Spain. E-mail: joseignacio.ruizs@ehu.es
The serine-threonine protein kinase Akt/PKB (protein kinase B) has received much interest in
recent years because it is a key mediator of cell proliferation and seems to play an important
role in tumorigenesis and resistance to chemotherapeutic drugs. The kinase suppresses
chemotherapy-triggered apoptosis through interaction with critical molecules that regulate or
execute apoptosis. Although most reports have shown that chemotherapy decreases Akt
activity, the potent anticancer drug doxorubicin can increase Akt activity, depending on the
cell type. Thus, in several cancer cell lines, the drug triggers a transient phosphorylation and
activation of Akt at early time points, prior to the detection of apoptosis. In this work, we
have determined in primary cultures of rat hepatocytes the effects of doxorubicin on the
phosphorylation status of Akt. The levels of Akt phosphorylation were measured by Western
blot analysis with an anti-Ser473-phosphorylated Akt antibody. Doxorubicin (10 µM) had a
biphasic behaviour, increasing (60%) Akt phosphorylation at 30 min and decreasing it (40%)
at 1h. At later times, the Akt phosphorylation status remained similar to control cells (without
doxorubicin). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented Akt
activation. The profile of the phsophorylation levels of Akt with doxorubicin was completely
different from that found for the pro-apoptotic JNK, which increased in a dose- and timedependent
manner.
This work was supported by the Basque Government (Research Project and Predoctoral
Training Grant to R.N.) and the University of the Basque Country (UPV00081.327-E-
15294/2003).
- 227 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 42
Phosphoinositide 3-kinase/Akt pathway as a potential therapeutic target in patients with
high risk myelodysplastic syndromes
Maria Nyåkern1, Carlo Finelli2, Pier Luigi Tazzari3, Costanza Bosi2, Matilde Yung
Follo1, Lucio Cocco1, Alberto M. Martelli1
1Department of Anatomy, Cell Signalling Laboratory, Università di Bologna, 40126
Bologna, Italy;2Institute of Hematology and 3Blood Bank, Policlinico S.Orsola-
Malpighi, Università di Bologna, 40138 Bologna, Italy. E-Mail:
alberto.martelli@unibo.it
The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase
(PI3K), is known to play an important role in anti-apoptotic signaling and has been implicated
in the aggressiveness of a number of different human cancers including acute myeloid
leukemia (AML). Myelodysplastic syndromes are hematopoietic stem cell disorders
characterized by ineffective hematopoiesis, leading to blood cytopenias, and by a high risk of
progression to overt AML. The progression of MDS to AML is thought to be associated with
abrogation of apoptotic control mechanisms. However, little is known about the signal
transduction pathways which may be involved in enhanced survival of MDS cells. In this
report, we have performed immunocytochemical and flow cytometric analysis, to evaluate the
levels of activated Akt in bone marrow or peripheral blood mononuclear cells from patients
diagnosed with MDS. We observed high levels of Ser473 phosphorylated Akt (p-Akt)
staining in 90 % of the cases (n=22) diagnosed as high risk MDS, whereas mononuclear cells
from normal bone marrow or low risk MDS patients showed low or absent Ser473 p-Akt
staining. Furthermore, all high risk MDS patients also demonstrated high expression of the
Class I PI3K p110& catalytic subunit and a decreased expression of PTEN. Pharmacological
inhibitors of the PI3K/Akt pathway (SH-5, perifosine) caused apoptotic cell death of bone
marrow mononuclear cells isolated from high risk MDS patients. Taken together, our results
suggest that Akt activation might be one of the factors contributing to the decreased apoptosis
rate observed in patients with high risk MDS. PI3K/Akt inhibition may constitute a novel
therapeutic strategy if apoptosis induction of MDS mononuclear cells is the goal.
- 228 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 43
Expression of HPV E5 protein induces expression of EP4 prostanoid receptor through
PKA- and CREB-dependent pathway in C33A cervical cancer cells.
Jung Min OH and Yong-Sung JUHNN
Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul
National University College of Medicine, Seoul 110-799, Korea
HPV (Human Papilloma Virus) infection is the major cause of cervical cancer development.
The role of HPV E6 and E7 protein in the carcinogenesis has been extensively studied,
however, the role of E5 protein in cervical carcinogenesis is not understood clearly. The
prostaglandin signaling pathway mediated by cyclooxygenase-2(COX-2) is activated in
cervical cancer cells and many other types of cancer cells, therefore, this study aimed to
analyze the effect of E5 protein on prostaglandin signaling pathway.
Stable expression of E5 protein caused an increase in COX-2 protein and EP4 prostanoid
receptor in C33A cervical cancer cells. Stable expressing COX-2 protein also increased
mRNA expression of EP4 and prostaglandin E synthase and secretion of prostaglandin E2
(PGE2), major product of COX-2 in inflammatory process, in C33A cervical cancer cells.
Treatment of E5 expressing cells with NS-398, a COX-2 inhibitor, decreased the expression
of EP4, and furthermore, treatment of HeLa cells that expresses high level of COX-2 with
NS-398 also resulted in decrease in EP4 mRNA expression and PGE2 secretion. Treatment
with H89, a PKA inhibitor or decoy CRE reduced expression of EP4 mRNA in COX-2
expressing cells. However, expression of COX-2 in HaCaT cells, immortalized human
keratinocytes did not induce expression of EP4.
Because the EP4 protein level did not increase in parallel with EP4 mRNA following E5
overexpression, the degradation rate of EP4 protein was analyzed. The EP4 protein in COX-2
expressing cells was found to degrade faster in the presence of cycloheximide than that of
vector-transfected control cells. The degradation of EP4 protein was blocked by treatment
with MG-132, a proteasome inhibitor.
From this result, we conclude that HPV E5 protein induces EP4 mRNA through COX-2-,
PKA-, and CREB-dependent pathway in cervical cancer cells, not in HaCaT cells.
- 229 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 44
Differential modulation of AMPK signaling pathways by low or high levels of exogenous
reactive oxygen species in colon cancer cells
In-Ja Park, Jin-Taek Hwang 1 , Young Min Kim 2 , Joohun Ha 1 and Ock Jin Park
Department of Food and Nutrition, Hannam University, 133 Ojeong-dong Daedeok-gu,
Daejeon 306-791, Korea, E-mail: ojpark@hannam.ac.kr
1 Department of Biochemistry and Molecular Biology, Medical Research Center for
Bioreaction to Reactive Oxygen Species, Kyung Hee University College of Medicine,
Seoul 130-791, Korea
2 Department of Biological Sciences, Hannam University, Daejeon 306-791, Korea
ROS have been regarded as undesirable metabolic byproducts often linked to macromolecule
damage, aging or degenerative diseases. However, ROS emerge as important intracellular
signaling molecules, which act as mediators or second messengers at nontoxic concentrations
through receptor-transducing pathways. In cancer cell system, ROS exert a paradoxical effect.
ROS can promote tumor growth by transforming normal cells through activation of
transcription factors or inhibition of tumor suppressor genes, whereas the elevated levels
would inhibit tumor cells through the stimulation of proapoptotic-signals. The excess ROS
generate cell cycle arrest and apoptotic cell death or even necrosis in severe cases. Therefore,
the maintenance of ROS homeostasis is extremely important to cell signaling and the
regulation of cell death.
The present study was undertaken to examine the effect of low and high concentrations of
H 2O 2 on cancer cell proliferation and apoptosis, and AMPK signaling pathways in HT-29
human colon cancer cells. Non-toxic dose of H 2O 2 (10 uM) induced cancer cell proliferation,
whereas the toxic level of 1000 uM H 2O 2 induced apoptosis. The stimulation of cell
proliferation was accompanied with an increase in cyclooxygenase-2 (COX-2), and apoptosis
induced by high-dose H 2O 2 was correlated with the activation of AMPK and negatively
correlated with COX-2 expression. These results suggest that ROS at non-toxic levels can
stimulate cancer cell growth by regulating AMP-activated protein kinase (AMPK) and/or
COX-2, and the abundant exogenous ROS linked to the growth inhibition through modulating
AMPK signaling pathways.
- 230 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 45
Identification of signaling pathway leading to increase of mitochondrial cytochrome-c
oxidase (COX) and mitofusin-2 (Mfn-2) proteins during insulin-mediated myogenesis in
vitro
Patrycja Pawlikowska1, Barbara Gajkowska2, Micha# M. Godlewski1, Arkadiusz
Orzechowski1*
1*Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw
Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland, e-mail:
orzechowski@alpha.sggw.waw.pl, 2Department of Cell Ultrastructure MRC, Polish
Academy of Sciences, Warsaw, Poland, *corresponding author.
Mitochondrial fusion mediated by Mfn-2 has been recently shown to play a crucial role in the
embryonic development, antiapoptosis through DNA protection against free radicals-induced
lesions and maintaining unchanged mtDNA pool. The purpose of this study was to examine
the role of mitochondria in insulin-dependent myogenesis in vitro. Two differently encoded
(nuclear and mitochondrial) subunits of cytochrome-c oxidase (COX), myogenin and Mfn-2
expressions were determined by Western blots. Our studies with LY294002 confirmed
assumption that insulin stimulates myogenesis though PI3-K-dependent signaling pathway.
Similarly, if MAPKK (MEK) was inhibited by PD98059 insulin-mediated myogenesis from
C2C12 muscle cells was accelerated. Additionally, immunoblots revealed that insulin raised
the expression of subunit I and IV of COX in L6 muscle cells. Apparently, insulin-dependent
induction of COX I was mediated by PI3-K, since blockade of insulin action with LY294002
resulted in decreased level of mitochondrial but not nuclear encoded subunit of COX.
Moreover, electron and confocal microscopy showed that insulin activates formation of a
network of elongated, tubular mitochondria. Increased mitochondrial length and
interconnectivity was not observed upon PI3-K inhibition with LY294002. Subsequently, we
decided to study transmembrane GTPase – Mfn-2 which seems to be essential for
mitochondrial fusion. We observed that insulin induces Mfn-2 protein in L6 myoblasts.
Moreover, we found that inhibition of MAPKK-dependent signaling pathway additionally
elevated both Mfn-2 and myogenin protein levels. Although correlation does not prove
causation, we speculate that Mfn-2 participates in insulin-mediated muscle differentiation. We
conclude that insulin-dependent myogenesis is associated with increased level of COX I and
Mfn-2 as well as augments fusion of mitochondria. The above-mentioned effects are inhibited
by LY294002, whereas inhibition of MAPKK with PD98059 led to additional amplification
of this phenomenon.
- 231 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 46
Tyrosine nitration of MEK and ERK induces their autophosphorylation and activation
in vivo and in vitro
Elena Pinzar*, Maria Garrido°, Daniela Urrecheaga°*, Guillaume Bonnot*, Patrick
Levy* and Serge P. Bottari *#
*Laboratoire HP2, UFR de Médecine, Grenoble Universités, Domaine de la Merci,
38700 La Tronche, France, °Facultad de Farmacia, Universidad Central de Venezuela,
Caracas, Venezuela, #Département de Biologie Intégrée, CHU de Grenoble, 38043
Grenoble, France. E-mail: Serge.Bottari@ujf-grenoble.fr
Angiotensin II (Ang II) has been shown to activate MAPK pathways and to induce production
of reactive oxygen and nitrogen species. Nitric oxide (NO) and superoxide (O2-.) as well as
peroxinitrite (ONOO-) have been previously reported to stimulate MAPK in a few cell types.
We therefore investigated whether ERK could be nitrated in rat vascular smooth muscle cells
(VSMC) upon exposure to Ang II. Stimulation of cells with 10 nM Ang II induced a
prominant and sustained nitration of ERK1/2 as revealed by immunoprecipitation and
immunoblotting. ERK nitration was accompanied by activation of the enzyme and showed a
kinetic profile different than that of phosphorylation. Both Ang II-induced ERK nitration and
phosphorylation were mediated by the AT1 receptor. The ONOO- donor 3morpholinosydnonimine
hydrocloride (SIN-1) also stimulated nitration and activation of
recombinant unactive ERK and MEK. Nitration and phosphorylation of ERK1/2 by Ang II
was significantly inhibited by the NAD(P)Hoxidase inhibitors 4-(2-aminoethyl)
benzenesulfonyl fluoride (AEBSF) and diphenylene iodonium (DPI). Moreover, the selective
inducible nitric-oxide synthase (iNOS) inhibitor 1400W but not the endothelial NOS inhibitor
L-NAME, completely inhibited nitration and activation of ERK. The ONOO- and O2-.
scavengers, respectively ebselen and myricetin, drastically blunted Ang II-stimulated nitration
and activation of ERK. Conversely, the MEK inhibitor U0126, did not affect ERK nitration,
but completely blocked ERK phosphorylation and activation. Taken together these data
indicate that Ang II can activate ERK1/2 both through nitration via reactive nitrogen speciessensitive
pathways and by phosphorylation through the Ras-Raf-MEK pathway.
Interestingly, ERK nitration appears to be a prerequisite for its activating phosphorylation by
MEK in response to Ang II in VSMC.
- 232 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 47
HSP27 SCAFFOLDS MK2 TO THE AKT SIGNAL COMPLEX
Madhavi J. Rane
Department of Medicine, Molecular Signaling Group, University of Louisville,
Louisville, KY 40202, U.S.A. E-mail:mrane@louisville.edu
The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB) signaling pathway
plays a critical role in cell growth, proliferation, and cell survival. Up regulation of this
pathway has been demonstrated in various carcinomas. Thus, inhibiting Akt activation and
promoting cell death provides a strategy for targeted cancer therapy. We have shown
previously that Akt/Hsp27 interaction regulates neutrophil apoptosis and that MK2 acts as
PDK2 for Akt. The current study tested the hypothesis that Hsp27 regulates Akt activation
and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that
loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment in neutrophils or by
transfection of Hsp27 siRNA in HK-11 cells resulted in induction of cellular apoptosis.
Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt Ser473
phosphorylation, activation, and Hsp27 Ser82 phosphorylation. Co-transfection of AktCA
and Hsp27siRNA in HK-11 cells specifically silenced Hsp27 expression, without altering
expression of Akt. Silencing Hsp27 expression resulted in the loss of Akt/MK2 interaction,
loss of Akt Ser473 phosphorylation, activation, Hsp27 Ser82 phosphorylation, and induced
HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (117-128) on
Akt as an Hsp27 binding region. Deletion of amino acids 117-128 on Akt resulted in loss of
its interaction with Hsp27 and MK2, but not Hsp90 as demonstrated by immunoprecipitation
and GST pull-down studies. Co-transfection studies in HEK-293 cells demonstrated that
constitutively active MK2 (MK2EE) phosphorylated Aktwt on Ser473 but failed to
phosphorylate Akt&117-128 mutant. In conclusion, Hsp27 scaffolds MK2 to Akt signal
complex and promotes Akt activation and cell survival. Veterans Administration Grant and
AHA 0335278N
- 233 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 48
Transforming Growth Factor-#1 Induces Cyclooxygenase-2 Gene Expression In Human
Mesangial Cells: Role of MAPK And PI3K pathways
Alicia Rodríguez-Barbero, Fernando Dorado, Atanasio Pandiella*, José M. López-
Novoa.
Instituto Reina Sofía de Investigación Nefrológica. Dpto de Fisiología y Farmacología.
*Centro de Investigaciones Biológicas. (CISC). Universidad de Salamanca. Campus
Unamuno, Edificio Departamental. Avda. Campo Charro s/n. 37007. Spain. E-mail:
barberoa@usal.es
Transforming growth factor-# (TGF-#) plays a fundamental role in the progression of renal
diseases. Mesangial cells play a major role in physiological and pathophysiological renal
processes. Accumulating evidence has suggested that eicosanoids derived from
cyclooxygenase 2 (COX-2) participate in a number of pathological processes in immunemediated
renal diseases. Mesangial cells express receptors for TGF-# and COX-2 expression
can be induced in mesangial cells. However, there are no studies on the possible role of TGF-
# on COX-2 expression in human mesangial cells (HMC). The aims of this study were to
assess whether TGF-#1 stimulates COX-2 expression in primary HMC, and to determine the
routes involved in TGF-#1-induced COX-2 expression and PGE2 synthesis. TGF-#1 induced
COX-2 promoter activity, COX-2 mRNA and protein expression in HMC. COX-2 induction
was accompanied by increased PGE2 synthesis. To establish the role that ERK1/2 and p38
MAPK as well as PI3K/Akt play in TGF-#1-dependent COX-2 expression, HMC were pretreated
with inhibitors of these pathways. Inhibition of ERK1/2 by the MEK inhibitors,
PD98059 or U0126 and inhibition of p38 MAPK activity by SB203580 notably decreased the
TGF-#1-dependent COX-2 protein induction and PGE2 synthesis in HMC. In addition, pretreatment
of HMC with the PI3K inhibitor LY294002 attenuated TGF-#1-induced COX-2
expression and PGE2 synthesis. These results demonstrate that TGF-#1 regulates COX-2
expression in HMC through the activation of ERK1/2, p38 MAPK, and PI3K. These data can
help to elucidate the molecular mechanisms underlying the regulation of COX-2 and open up
specific strategies for the treatment of glomerular disease.
- 234 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 49
Yeast as a Tool for the Study of the Mammalian PI3K-PTEN-Akt Pathway
Isabel Rodríguez-Escudero,1 Françoise M. Roelants,2 César Nombela,1 Jeremy
Thorner,2 María Molina1 and Víctor J. Cid1
1Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense
de Madrid, Pza. de Ramón y Cajal s/n, 28040 Madrid, Spain. E-mail:
vicjcid@farm.ucm.es
2Department of Molecular and Cell Biology, Division of Biochemistry and Molecular
Biology, University of California, Berkeley, California 94720 USA.
Generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) by class I
phosphatidylinositol 3-kinase (PI3K) is at the head of crucial signaling pathways in metazoa,
like those involved in the control of cell proliferation and apoptosis. Therefore, this lipid
kinase, its antagonist the phosphatidylinositol 3-phosphatase (PTEN), and its main
downstream effector protein kinase B (PKB/c-Akt) are key therapeutic targets. With the aim
of facilitating genetic, molecular and pharmacological analyses on these proteins, we have
expressed mammalian PI3K (p110alpha), PTEN and c-Akt in the model organism
Saccharomyces cerevisiae. Expression of mammalian PI3K dramatically inhibited yeast cell
growth, an effect that depended on conversion of intracellular phosphatidylinositol (4,5)bisphosphate
(PIP2) to PIP3, a lipid that does not naturally exist in S. cerevisiae. All cellular
effects were fully reverted by co-expression of catalytically-active human PTEN, and
ameliorated by a PI3K inhibitor (LY294002). This system allows an easy platform for in vivo
pharmacological studies and screens on these proteins. We have studied the effects of PIP2
depletion on the yeast cytoskeleton and signaling. Interestingly, conversion of PIP2 to PIP3
causes activation of a yeast MAPK pathway, suggesting a role of phosphoinositides upstream
MAPK signaling in yeast. We were able to reproduce PIP3-dependent re-localization to the
plasma membrane and phosphorylation of heterologous c-Akt in yeast. Endogenous yeast
protein kinases are responsible for phosphorylation of heterologous c-Akt, suggesting that
Akt-activating kinases are conserved through phylogeny. Thus, yeast might also provide a
new tool for the discovery of novel Akt activators.
- 235 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 50
Competition effects shape the response sensitivity and kinetics of phosphorylation cycles
in cell signaling
Carlos Salazar and Thomas Höfer
Theoretical Biophysics, Institute of Biology, Humboldt University Berlin, Invalidenstr.
42, 10115 Berlin, Germany. E-mail: carlos.salazar@rz.hu-berlin.de
Activation-inactivation cycles of signaling proteins and transcription factors catalyzed by
kinases and phosphatases are a core component of cellular signal transduction. We present a
concise kinetic description of phosphorylation cycles that starts by considering the elementary
steps of enzyme-target binding and catalysis. A rapid-equilibrium approximation for protein
interactions is used to reduce the set of parameters. Generally no explicit rate laws exist for
the kinase and phosphatase; linear or Michaelis-Menten rate equations can be obtained in
special cases. Key parameters that determine system behavior are the concentrations of kinase
and phosphatase relative to the target protein and the affinities of the two enzymes for the
different phosphorylation states of the target. By scanning the space of these parameters, we
obtain a phase diagram that shows the existence of graded, ultrasensitive and bistable
behaviors as well as a previously undescribed biphasic response. Two kinds of competition
effect turn out to shape the behavior: (1) the degree of product inhibition of each enzyme, and
(2) the competition between kinase and phosphatase to bind the target protein, as determined
by their relative target affinities and concentrations. Like the steady-state response curve, the
response time was found to be strongly dependent on the kinetic design of the
phosphorylation cycles, and the order in which (de)phosphorylation of multiple residues
proceed. Sequential mechanisms of phosphate processing are characterized by a higher degree
of competition between phosphorylation and dephosphorylation, which results in sharper
response curves and slower response times than in random mechanisms. Taken together, our
kinetic analysis allows us to identify key parameters that should be given priority in
experimental measurements and to arrive at experimentally testable predictions concerning
both the steady-state response and the kinetics of phosphorylation cycles and cascades.
- 236 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 51
JNK1 isoform is required for M-CSF and LPS induced MKP1 (DUSP1) expression
Ester Sánchez-Tilló, Mónica Comalada, Consol Farrera, Jordi Xaus, Annabel F.
Valledor, Carme Caelles, Jorge Lloberas and Antonio Celada
Macrophage Biology Group, Institute of Biomedical Research, Barcelona Science Park,
University of Barcelona, Spain. Phone 34934037165; Fax 34934034747; acelada@ub.edu
Macrophages proliferate in the presence of M-CSF in a process that depends on early (5 min)
and short ERK activation. The addition of LPS arrests proliferation and prolongs ERK
activation, as well as p38 and JNK, which are required for an inflammatory response. The
decision to proliferate or to become activated is correlated with the extent and duration of
MAPK regulation, which is controlled by mitogen kinase phosphatases (MKPs). In our
model, the phosphatase responsible for ERK deactivation is MKP1 (DUSP1), which is
induced by M-CSF or LPS with different time-course and independent of ERK activation. We
observed that MKP1 induction by both M-CSF and LPS is transcriptionally dependent on
JNK activity while other MAPKs are not involved. The inhibition of MKP1 by JNK caused a
slight elongation of ERK-1/2 and p38 activation, as well as inhibition of M-CSF-dependent
proliferation. Using macrophages from PKC' knock-out mice, we observed that although M-
CSF and LPS activate the same pathway to induce MKP1, only crosstalk between PKC' and
JNK is present in LPS-stimulated macrophages. The two JNK genes, jnk1 and jnk2 are
constitutively expressed in macrophages, but only the JNK1 isoform is involved in MKP1
induction by M-CSF or LPS, as determined using single knock-out mice. Moreover, JNK1 is
required for pro-inflammatory cytokine biosynthesis (TNF-", IL-1# and IL-6) and NO
production triggered by activation of LPS and TNF-". The induction of these cytokines is
independent of MKP1 expression, as determined in MKP1 knock-out mice. Our results show
that stimuli that induce MAPK activation also lead to the induction of attenuators, which
provides a negative feedback mechanism through which to control the activation of other
MAPKs. JNK could be a novel therapeutic target to regulate MKP1 expression in tumours
and in chronic inflammatory responses.
EN.REFLIST
- 237 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 52
MAPKinase gene expression – as determined by microarray analysis - distinguishes
uncomplicated from complicated reconstitution after major surgical trauma
E. Marion Schneider1, Manfred Weiss2, Weidong Du1, Fabian Kriebel1, Gerhard
Leder3, Klaus Buttenschön3, Ulrich C. Liener3, Uwe Bernd Brückner3
fabiankriebel@web.de
1Sektion Experimentelle Anaesthesiologie; 2Abteilung Klinische Anaesthesiologie,
3Abteilung Chirurgie, Universitaetsklinikum Ulm, Steinhoevelstrasse 9; 89075 Ulm,
Germany
Objective: A prospective pilot study in patients with major surgical trauma at an university
hospital was set up to identify signaling pathways by microarray expression analysis, which
may be distinct in individual patients and may be indicative for complicated versus uneventful
reconstitution post trauma.
Methods: RNA was prepared from peripheral blood drawn into PAXgene tubes from three
patients before and exactly 24h after trauma. These RNA were then transcribed into cDNA,
Cy3- or Cy5-labeled, and hybridized onto a cDNA microchip, red vs. green fluorescence was
quantified using a microarray scanner. Patient (patients before and post trauma) versus control
(healthy donors) blood cell gene expressions were compared using fluorescence intensities
and appropriate normalizations.
Results: In addition to a number of rather non-specific stress response genes, activated in all
patients, we found a remarkable number of differences in gene expression patterns of
individual patients. Some of the differing genes were associated with uncomplicated
convalescence such as up-regulation of both the ERK5 pathway (MAPK7, mitogen-activated
protein kinase-7) and transcription factors which stimulate hematopoiesis and tissue
remodeling (MEF2, myocyte enhancer factor-2, BMP-2, bone morphogenic protein-2,
TNFRSF11a (TNF-R superfamily, syn. RANK), and RUNX-1, runt related transcription
factor-1). Chemokine genes active in stem cell recruitment from the bone marrow as well as
dendritic cell and NK maturation (SCYA14 (HCC-1, hemofiltrate cc chemokine )), were
increased as well as activators of the lymphoid compartment (TNFRSF7 (CD27), CD3zeta
and perforin (PRF1)). In contrast, all these transcripts were down-regulated in complicated
reconstitution and later development of septic shock. Moreover, p38 kinase (MAPK14), S100
molecules and members of the lipoxygenase pathway were associated with a more eventful
outcome. In none of the patients, we found c-Jun-stress activated factor (JNK) pathway to be
markedly activated, also p105 (Rel) or the “NFkB inducible kinase” (NIK, MAP3K14) were
not found to be up-regulated post surgical trauma.
Conclusions: Microarray expression studies are a promising tool for screening and then
selecting differentially regulated genes in favorable as compared to complicated reconstitution
post trauma. Gene up-regulation patterns may emphasize previously unrecognized molecules
and signaling pathways encouraging the appropriate protein and functional assays in an
individual patient but also in larger patient cohorts.
- 238 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 53
Modulation of MPF and MAPK pathways activities by physiological pHi changes.
Chantal Sellier1, Anne F Antoine1, Stéphane Flament2, Arlette Lescuyer1, Jean-Pierre
Vilain1 and Jean-François Bodart1.
1 - Laboratoire de Régulation des signaux de division, EA 1033, IFR 118, UTSL, SN3, F-
59655 Villeneuve d’Ascq cedex, France. E-mail : chantal_sellier@yahoo.fr ; Anne-
Frederique.Antoine@univ-lille1.fr ; Arlette.Lescuyer@univ-lille1.fr ; Jean-
Pierre.Vilain@univ-lille1.fr ; Jean-François.Bodart@univ-lille1.fr
2 - Faculté des Sciences, EA 3442, UHP, Bvd des Aiguillettes, BP 239, F-54506
Vandoeuvre Lès Nancy cedex, France. E-mail : stephane.flament@scbiol.uhp-nancy.fr
Intracellular pH (pHi) plays a key role in mitogenic signal transduction, development and
maintenance of transformed phenotypes. Nevertheless, cell cycle mechanisms sensitive to pHi
remain to be determined. Analogous to G2/M transition, Xenopus oocyte maturation is
characterized by germinal vesicle breakdown (GVBD), MPF (Cyclin B/Cdk1) and Mos-
MAPK pathways activation. These events are accompanied by a transient alkalization. We
determined that an acidifying compound, NH4Cl, delayed progesterone-induced GVBD in a
dose-dependent manner. Cyclin B phosphorylation, Cdk1 Y-15 dephosphorylation as well as
p39Mos accumulation, MAPK and p90Rsk phosphorylations induced by progesterone were
delayed by acidification. The delay induced by NH4Cl was prevented by injection of MOPS
buffer pH 7.7. In contrast, alkalyzing treatment such as Tris buffer pH 9 injections,
accelerated GVBD, MPF and MAPK activation. Strickingly, we observed that NH4Cl
strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation
while it had no or little effect on the MPF autoamplification loop and GVBD triggered by egg
cytoplasm injection. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or
active MAPK-induced GVBD. Thus, dynamic of early events driving MAPK and MPF
activation induced by progesterone may be negatively or positively regulated by pHi changes
whereas only MAPK-induced GVBD pathway was inhibited by acidification. Finally, using
electrophysiological approch, we observed that NH4Cl induced an activation of Ca2+activated
Cl current suggesting an elevation of intracellular Ca2+ concentration. Therefore,
effects of pHi on early meiosis dynamic might be related to subsequent change in Ca2+
homeostasis.
- 239 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 54
Protective effect of a peptide derived from the endogenous PKC inhibitor PKI55 on the
neurosecretory function in ischemic brain slices
Rita Selvatici1, Sofia Falzarano1, Lara Franceschetti1, Remo Guerrini2, Silvia Marino3
and Anna Siniscalchi3
1Department of Experimental and Diagnostic Medicine, Section of Medical Genetics;
2Department of Pharmaceutical Sciences, 3Department of Clinical and Experimental
Medicine, Section of Pharmacology, University of Ferrara, Italy. E-mail: svr@unife.it
We have recently identified the PKI55 protein, coding for 55 amino acids, that is normally
poorly translated in vivo and acts as a specific modulator of either cPKC-" and nPKC-&
isozymes. PKI55 remains relatively inactive until PKC attains an active conformation and
reaches a critical concentration in the cell; it is not modified by the enzyme but irreversibly
associates with its target, thus behaving as a suicidal inhibitor. The inhibition and degradation
of over-activated PKC isozymes may prevent unfavourable changes in cellular phenotypes,
resulting from PKC overexpression (Selvatici, J Mol Evol 2003 57:131). In the present work
we compared the in vitro biochemical activity of the PKI55 protein, of its 39-amino acids Nterminal
fragment (G39) and of its 16 amino acids C-terminal fragment (G16) on specific
PKC recombinant isozymes, by measuring the initial rate of phosphate incorporation from
32P-ATP into saturating amounts of histone IIIS. PKI55 concentration-dependently inhibited
PKC activity, provided that calcium ions were present in the assay medium (IC50=6µM). The
inhibitory activity was retained by G16 (IC50=50µM), but not by G39. The active fragment
G16 was tested in an in vitro model of brain ischemia: superfused guinea pig cerebral cortex
slices, continuously electrically (10 Hz) stimulated, were exposed to 20 min of oxygenglucose
deprivation (OGD, Badini, Neurochem Int 1997 31:817), and then reperfused for 1
hour (REP). PKC activity was increased to 244±36% at the end of OGD, while acetylcholine
(ACh) release, taken as an index of the neurosecretory function, was reduced to 35±2% of the
controls. Following REP, a reduction in PKC activity to 54±7% was displayed, indicating a
down regulation of previously activated PKC (Selvatici, J Neurosci Res 2003 71:64); ACh
release only partially recovered (REP=69±6% of the controls), suggesting persistence of
neuronal suffering. PKC activation during OGD was attenuated by 50µM G16 and the
consequent down regulation following REP was prevented. Moreover, ACh release fully
recovered to normal values (REP+G16=91±4% of the controls). These data suggest a
neuroprotective action for G16, the active fragment of the endogenous PKC inhibitor PKI55.
- 240 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 55
Effects of chemical ischemia in cerebral cortex slices. Focus on mitogen activated
protein kinase cascade.
Anna Siniscalchi1, Sabrina Cavallini1, Silvia Marino1 Sofia Falzarano2, Lara
Franceschetti2,
and Rita Selvatici2
1Department of Clinical and Experimental Medicine, Section of Pharmacology,
2Department of Experimental and Diagnostic Medicine, Section of Medical Genetics;
University of Ferrara, Italy. E-mail: snn@unife.it
Mitochondrial toxins could model, in vitro, the energy failure that occurs during transient
brain ischemia in vivo, in alternative to either oxygen and glucose deprivation, or to high
glutamate treatment. In our laboratory, sodium azide (NaN3), combined with the glycolysis
blocker, 2-deoxyglucose (2-DOG), has been used to induce chemical ischemia (CI) in rat
cerebral cortex slices (Cavallini, Neurochem Int 2005, 47:482), and the involvement of
glutamate overflow, calcium overload and nitric oxide efflux in its mechanism of action has
been shown. In the present work, the downstream events following CI have been addressed:
superfused rat cerebral cortex slices, continuously electrically (5 Hz) stimulated, were treated
with 10 mM NaN3 plus 2 mM 2-DOG for 5 min, then reperfused with normal medium for
one hour (REP). For Ras/MAP kinases analysis by Western blotting, membranes were
incubated with rabbit polyclonal antibodies against p21Ras, ERK1/2 (p44/42), phospho-
ERK1/2, p38, phospho-p38, SAPK/JNK and phospho-SAPK/JNK. Densitometric analysis of
autoradiographic bands was performed with a Bio-Rad densitometer. The level of p21 Ras
was increased by 40 % immediately after CI, and did not recover to control values following
REP. The total levels of both ERK1 and ERK2 were reduced by CI and partially recovered to
control values in REP; their phosphorylation degree (phosphorylated to total protein level
ratio, about 50 % in the controls) did not significantly change either under CI or under REP
conditions. The phosphorylation degree of MAPK p38, very low in control slices (17%), was
not modified by either CI or REP. The activation of SAPK/JNK was full in the controls and
was reduced to 70% after CI and to 64% following REP. All these effects were prevented by
the NMDA receptor antagonist MK801, 10 µM, suggesting the involvement of glutamate.
The present findings show that CI reduces the MAPK levels, possibly because of acute energy
depletion, although the p21Ras protein is increased. The decrease in SAPK/JNK, observed
under CI/REP conditions, may lead to necrotic neuronal death, since its activation has been
related to both apoptosis and neuroprotection mechanisms. These results could be of interest
in view of preventive treatments of ischemia/reperfusion-induced brain damages.
- 241 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 56
Hantavirus infection of lung microvascular endothelial cells activates the extracellular
regulated kinase 1 /2 (ERK1/2) and phosphoinositol3-kinase (PI3K) pathways leading to
proinflammatory cytokine/chemokine production.
Christina F. Spiropoulou, Karen L. Hutchinson, Cesar Albarino, Thomas G. Ksiazek
and Pierre E. Rollin
Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for
Disease Control and Prevention, Atlanta, GA 30333, USA. E-mail:ccs8@cdc.gov
Hantavirus pulmonary syndrome (HPS), is a severe respiratory illness with approximately
40% mortality which is caused by New World hantaviruses. In humans, the primary target
organ for virus infection is the lung, and severe disease symptoms have been attributed to
massive lung endothelial cell capillary leakage. Immunopathology studies have shown the
presence of monocytic and lymphocytic cell infiltrates in the microvasculature of patients but
no evidence of endothelial cell disruption. Hantavirus infection of primary microvascular
endothelial cells in vitro also does not cause cell disruption but induces the upregulation of a
number of proinflammatory chemokines. These observations have led to the hypothesis that
host immune responses play a crucial role in the pathogenesis of hantavirus diseases. This
study was designed to examine pathways participating in the induction of proinflammatory
chemokines by hantavirus infection. Hantavirus entry and replication activates the
extracellular regulated kinase 1 /2 (ERK1/2) and phosphoinositol3-kinase (PI3K) pathways.
By using specific pharmacological inhibitors we have shown that both pathways contribute to
the induction of proinflammatory cytokines/chemokines by hantaviruses. However, we
observed a partial inhibition using inhibitors specific for the ERK1/2 pathway whereas total
inhibition of virus induced cytokines and chemokines was seen with LY294002, an inhibitor
of PI3K. Studies leading to discovery of possible pharmacotherapeutic interventions for HPS
are important given the lack of effective hantavirus vaccines.
- 242 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 57
Extracellular pH changes activate the p38-MAPK signalling pathway in the vertebrate
heart
Konstantina Stathopoulou, Catherine Gaitanaki and Isidoros Beis
Department of Animal and Human Physiology, School of Biology, Faculty of Sciences,
University of Athens, Panepistimioupolis, Athens, 157 84, GREECE. E-mail:
kstath@biol.uoa.gr, cgaitan@biol.uoa.gr, ibeis@biol.uoa.gr
The acid-base balance is one of the most important physiological parameters that affect the
proper cellular function. In the case of heart, ischemia is closely related to acidosis, whereas
growth factors and peptide hormones induce alkalinization. In the present study we
investigated the effect of extracellular pH changes on the activation of the p38-MAPK
signalling pathway in the isolated perfused amphibian heart. Alkalosis (pH 8.5 or 9.5)
maximally activated p38-MAPK within 2 min (4.17- and 3.20-fold, compared to control
values, respectively) and this effect was reversible since the kinase phosphorylation levels
decreased upon heart reperfusion with normal Tris-Tyrode’s buffer. On the contrary, acidosis
activated p38-MAPK moderately, but persistently (1.65-fold, at 1 min and 1.91-fold, at 60
min). Na+/H+ exchanger inhibitors amiloride and HOE642 (a kind gift from Aventis Pharma
Deutschland GmbH) abolished p38-MAPK phosphorylation induced by alkalosis, whereas the
Na+/K+-ATPase inhibitor ouabain partially attenuated this activation, results indicating that
this signalling pathway depends on these cation exchangers. What is more, alkalosis (pH 8.5)
induced a significant MAPKAPK2 (2.59-fold, 2 min) and HSP27 phosphorylation (5.33-fold,
2 min) in a p38-MAPK-dependent manner, as demonstrated with experiments using 1 µM
SB203580. Furthermore, the phosphorylated forms of p38-MAPK and HSP27 were
immunohistochemically detected in the perinuclear region and dispersedly in the cytoplasm of
ventricular cells during alkalosis. All the above data suggest that the p38-MAPK signalling
pathway is activated by extracellular pH changes and in the case of alkalosis this activation
seems to function protectively in the amphibian heart.
Acknowledgements: This study was funded by the Special Research Account of the
University of Athens and the Pythagoras I grant (70/3/7399). Ms Stathopoulou is a recipient
of a State Scholarships Foundation fellowship.
- 243 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 58
The MEKK1-JNK Pathway Regulates Mouse Eyelid Morphogenesis
Atsushi Takatori and Ying Xia
Department of Environmental Health, University of Cincinnati Medical Center,
Cincinnati, Ohio 45267 U.S.A
MEK kinase 1 (MEKK1) is a mitogen-activated protein kinase kinase kinase, known as an
upstream regulator for the c-Jun NH2-terminal kinases (JNKs). Knocking out Mekk1 in mice
results in eye-open at birth (EOB) phenotype that leads to severe eye pathologies. There are
two Jnk isoforms, Jnk1 and Jnk2, that are ubiquitously expressed in various tissues. This
study is aimed at understanding the distinct role for JNK1 and JNK2 in transmitting the
MEKK1 signals during eyelid morphogenesis. Mekk1-/-, Jnk1-/- and Jnk2-/- mice were
backcrossed to C57BL/6 background and intercrosses were carried out to generate Mekk1+/-
Jnk1-/-and Mekk1+/-Jnk2-/- mice. We find that the Mekk1+/-Jnk1-/-, but not Mekk1+/-
Jnk2-/-, mice displayed EOB similar to the Mekk1-/- mice. Unlike the Mekk1+/-Jnk2-/-
fetuses, which showed a thin layer of eyelid epithelium covering the ocular surface at
embryonic day 16.5, the Mekk1+/-Jnk1-/- fetuses lacked eyelid closure and had fully exposed
cornea. Immunohistochemistry studies showed similar levels of JNK phosphorylation, but a
much reduced c-Jun phosphorylation and plasminogen activator inhibitor-1 (PAI-1)
expression in the developing eyelid epithelium of the Mekk1+/-Jnk1-/-, compared to that of
the wild type and the Mekk1+/-Jnk2-/- fetuses. Considering the crucial role of PAI-1 in
epithelial cell migration, we studied the keratinocyte migration using an in vitro wound
healing assay. Our results indicated that the Mekk1+/-Jnk1-/- keratinocytes had slower
wound closure than the wild type and Mekk1+/-Jnk2-/- keratinocytes. Taken together, our
results show that in the absence of JNK1, one functional Mekk1 allele is insufficient to cause
c-Jun phosphorylation and epithelial cell movement, responsible for defective eyelid
morphogenesis of the Mekk1+/-Jnk1-/- fetuses. JNK1 appears to be more critical than JNK2
in transmitting the MEKK1 signals to c-Jun phosphorylation and target gene expression
during mouse embryonic eyelid development.
Support: NIH EY 15227
- 244 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 59
Erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) acts through redistribution
of protein kinases within lipid rafts and signal transduction changes in leukemic cells
Iana Tsoneva1, Spiro M. Konstantinov2, Hansjörg Eibl3, Martin R. Berger4
1,4Inst. of Biophysics, BAS, Acad. G. Bonchev St., bl 21, 1113 Sofia, Bulgaria e-mail:
Itsoneva@obzor.bio21.bas.bg
2,4Lab for Experimental Chemotherapy, Dept. of Pharmacology, Medical University of
Sofia, 2 Dunav St., 1000 Sofia, Bulgaria; e-mail: Pgen@TU-Sofia.bg
4 !Unit of Toxicology and Chemotherapy, German Cancer Research Center, INF 280,
69120 Heidelberg, Germany; e-mail: M.Berger@DKFZ.de
3Max Planck Inst. of Biophysical Chemistry, Am Faßberg, 37077, Göttingen, Germany
ErPC3 belongs to the group of alkylphosphocholines which are membrane targeting
antineoplastic agents and act as signal transduction modulators. Our hypothesis was that
ErPC3 can modulate the protein content of lipid rafts. The non receptor tyrosine kinase c-Abl
is widely expressed in malignant hematopoietic cells and its oncogenic variant Bcr-Abl has
enough leukemogenic potential for the development of acute lymphoblastic and chronic
myeloid leukemia. Therefore, the panel of cell lines included two groups: cells with bcr-abl
rearrangement (K-562 and LAMA-84) and cells with c-Abl only (HL-60 and SKW-3). Whole
cell lysates were analyzed by Western blotting. Lipid rafts were isolated from membranes by
sucrose gradient centrifugation and analyzed for cAbl (p160) and BCR-ABL (p210). Whole
cells mounted by cytospin were stained for the ganglioside GM1 content by cholera toxin B
subunit conjugated to FITC. ErPC3 elicited changes in Rb protein status that included
increased expression and fragmentation. Furthermore, the alkylphosphocholine caused time
dependently increased association of cAbl and BCR-ABL proteins to lipid rafts. This process
was accompanied by reorganisation of lipid rafts containing ganglioside GM1. When
comparing ErPC3 with cytosine arabinoside, the antimetabolite did not induce significant
changes in the cAbl content of the lipid rafts. The mechanism of action of ErPC3 is related to
its effects on lipid rafts as well as on Rb activation and induction. A deeper understanding of
molecular mechanisms especially those related to modulation of signal molecule content in
membrane lipid rafts will contribute to better evaluation and selection of anticancer lysolipids
such as ErPC3.
- 245 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 60
Molecular mechanisms of the deleterious action of ceramide on insulin signalling:
involvement of caveolae
Sophie Turban1, Xavier Lelièpvre2, Soazig Le Lay3, Olivier Bourron2, Harinder S.
Hundal1 & Eric Hajduch2.
1Department of Molecular Physiology, Dundee, UK, e-mail: s.turban@dundee.ac.uk,
2Inserm U671, Paris, France, 3Max Planck Institute, Dresden, Germany
Several studies have revealed that in peripheral tissues, insulin resistance might be associated
with intracellular formation of ceramide, the main secondary messenger of the sphingomyelin
transmembrane signalling pathway. We have recently shown that ceramide inhibited insulininduced
activation of protein kinase B (PKB), a key protein in insulin signalling, by
promoting its association and phosphorylation by protein kinase Cz (PKCz).
Caveolae are small invaginations of the plasma membrane that are enriched in cholesterol and
sphingolipids like ceramide. Interestingly, many signalling proteins such as PKCz have been
found in those microdomains. The purpose of the study was to investigate the mechanisms
involved in the negative effect of ceramide on the insulin pathway and to determinate whether
caveolae would be the place where the lipid inhibits PKB via the activation of PKCz. We
show that ceramide induces the recruitment of PKCz in the caveolin rich domain. Moreover
the concomitant recruitment of PKB is observed in those compartments. We report that
cholesterol depletion and destruction of caveolae structures in 3T3-L1 adipocytes, L6 muscle
cells and isolated human adipocytes with methyl-beta-cyclodextrin prevents the deleterious
effect of ceramide on the insulin-induced translocation and activation of PKB in the plasma
membrane. In addition we demonstrate that in isolated adipocytes from caveolin-1 knock-out
mice, ceramide did not alter the insulin-induced phosphorylation of PKB. All together these
findings indicate that in insulin sensitive tissues, ceramide inhibit PKB via PKCz within
caveolae.
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Session III : Protein kinase cascades as therapeutic targets Poster III, 61
Involvement of NF!B activating pathways in DLBCL pathogenesis
Joost C. van Galen1, Bauke Ylstra1, Chris J.L.M. Meijer1, Jettie J.F. Muris1, Cindy
P.E, Giroth1, Gert J. Ossenkoppele2, Joost J. Oudejans1.
Departments of Pathology1 and Hematology2, VU university medical center, De
Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. E-mail: j.vangalen@vumc.nl.
Intrinsic resistance to apoptosis of lymphoma cells is a probable mechanism causing
chemotherapy resistance and eventual fatal outcome in patients with diffuse large B cell
lymphomas (DLBCL). Microarray studies have demonstrated that DLBCL can be classified
in activated B-cell (ABC) like or germinal center B-cell (GCB) like DLBCL. Clinical
outcome in ABC like DLBCL is worse than for GCB like DLBCL.
Poor outcome in ABC like DLBCL is explained by constitutive activation of the NF!B
pathway resulting in high expression levels of many apoptosis inhibitory genes. The
mechanism behind this NF!B activation however is unknown. In this study we determined
expression profiles of possible pathways leading to NF!B activation in these DLBCL.
We investigated whether an ABC like phenotype correlates with expression of genes involved
in NFkB activation.
Using dual channel oligo arrays we performed genome-wide microarray analysis in a group of
46 primary nodal DLBCL to determine GCB versus ABC like phenotype. Next we performed
hierarchical cluster analysis of only genes coding for intracellular signal transduction factors
involved in NF!B activation. Both profiles were compared to each other.
We found that in ABC like DLBCL expression of NFkB target genes correlated with the
expression of genes upstream from NFkB activation that are implicated in B-cell receptor
(BCR) signaling. No correlation was observed with activation of pathways downstream of the
TNF receptor family.
We conclude that NF!B activation in ABC DLBCL is correlated with constitutive BCR
receptor signaling. Thus interfering with the function or expression of BCR target genes
might provide new possibilities to restore apoptosis sensitivity in ABC like DLBCL cells and
thus improve clinical outcome in these patients.
- 247 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 62
Reciprocal Relationship Between RhoC GTPase and Caveolin-1 Leads to Altered
MAPK Signaling During Pancreatic Cancer Cell Migration and Invasion.
Cynthia M. van Golen and Kenneth L. van Golen.
Department of Internal Medicine, Division of Hematology/Oncology, The University of
Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109 email:
kgolen@umich.edu
BACKGROUND: In the current study we investigated the role of caveolin-1 (cav-1) in
pancreatic adenocarcinoma (PC) cell migration and invasion; initial steps in metastasis. Cav-
1 is the major structural protein in caveolae; small *-shaped invaginations within the plasma
membrane. Caveolae are involved in signal transduction, wherein cav-1 acts as a scaffolding
protein to organize multiple molecular complexes regulating a variety of cellular events.
Recent evidence suggests a role for cav-1 in promoting cancer cell migration, invasion and
metastasis; however, the molecular mechanisms have not been described. The small
monomeric GTPases are among several molecules which associate with cav-1. Classically,
the Rho GTPases control actin cytoskeletal reorganization during cell migration and invasion.
RhoC GTPase is overexpressed in aggressive cancers that metastasize and is the predominant
GTPase in PC. Like several GTPases, RhoC contains a putative cav-1 binding motif.
RESULTS: Analysis of 10 PC cell lines revealed high levels of cav-1 expression in lines
derived from primary tumors and low expression in those derived from metastases.
Comparison of the BxPC-3 (derived from a primary tumor) and HPAF-II (derived from a
metastasis) demonstrates a reciprocal relationship between cav-1 expression and p42/p44 Erk
activation with PC cell migration, invasion, RhoC GTPase and p38 MAPK activation.
Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration
of cav-1 expression. CONCLUSIONS: Cav-1 expression inhibits RhoC GTPase activation
and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting
migration and invasion. In contrast, loss of cav-1 expression leads to RhoC-mediated
migration and invasion in metastatic PC cells.
- 248 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 63
Functional Genomics of Coronavirus-Host Interactions using a High-Throughput
siRNA Strategy
Hélène Verheije1, Matthijs Raaben1, Lucas Pelkmans2, Eberhard Krausz3,4, Eugenio
Fava3,4, Peter J.M. Rottier1, Cornelis A.M. de Haan1
1Virology Division, Department of Infectious Diseases & Immunology, Faculty of
Veterinary Medicine, Utrecht University, The Netherlands; 2EHT Zurich, Institute for
Molecular Systems Biology, Zürich, Switzerland; 3Max Planck Institute for Molecular
Cell Biology and Genetics, Dresden, Germany; 4MPI-CBG High-Throughput
Technology Development Studio (HT-TDS), Dresden, Germany. E-mail:
h.verheije@vet.uu.nl
Viruses replicate within cells by using the cellular machinery to their own advantage. In
general much is known about the pathogens, however, only little is known about the
contributions of the host. Our research aims to analyse coronavirus-host interactions by using
a functional genomics screen, based on a recently developed high throughput genome-wide
RNAi strategy (Pelkmans et al. 2005). The screen is designed to identify genes whose direct
loss-of-function phenotypes result in altered viral entry and replication. As a proof of
principle 54 individual kinases, known to be involved in endocytosis, were analyzed for their
role in coronavirus entry. The extensively characterized mouse hepatitis virus (MHV), a close
relative of the severe acute respiratory syndrome (SARS) virus, was used for the initial
screen. HeLa cells were transfected with the different siRNAs and 72h after transfection the
cells were infected with MHV at a low multiplicity of infection. MHV infection was
monitored by analyzing reporter gene expression at an early time point post infection.
Downregulation of several kinases resulted in inhibition of infection, suggesting a role for
these kinases in MHV internalization. Also an increase in infection efficiency was found for
some kinases. In a follow-up study, a genome-wide kinase and phosphatase siRNA library,
targeting 750 kinases and 180 phosphatases, will be used to identify more host genes whose
loss-of-function results in impaired entry and replication. This might provide interesting leads
for the development of anti-coronaviral agents.
- 249 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 64
PDGFRbetaY857F mutant mediates PDGF-BB-induced signalling and chemotaxis,
despite severely reduced kinase activity.
Piotr Wardega, Carl-Henrik Heldin, Johan Lennartsson.
Ludwig Institute for Cancer Research, Uppsala University, Box 595, SE-751 24 Uppsala,
Sweden
Piotr.Wardega@LICR.uu.se , C-H.Heldin@LICR.uu.se,
Johan.Lennartsson@LICR.uu.se
Platelet-derived growth factor receptor (PDGFR) becomes dimerized and activated upon
ligand binding, which leads to receptor transphosphorylation and increases its enzymatic
activity. In the present study, we studied the effects of mutation of tyrosine residue 857 in the
activation loop of PDGFRbeta, to phenylalanine. In agreement with published work, our
studies show, that PDGFRbetaY857F has much lower kinase activity in vitro in comparison
to PDGFRbetaWT , both as measured as receptor autophosphorylation and as phosphorylation
of an exogenous substrate. Interestingly, PDGF-induced tyrosine phosphorylation of
PDGFRbeta, in a cellular context, is not affected by the Tyr857 mutation. There are, however
significant changes in ability of PDGFRbetaY857F to induce signal transduction compared to
the wild-type receptor. For example, we could show that Stat5 signalling differs dramatically
between PDGFRbetaWT and PDGFRbetaY857F. We observed almost complete loss of
PDGF-induced Stat5 phosphorylation by the mutant receptor. Surprisingly Stat3, which
belongs to the same group of transcription factors as Stat5, was phosphorylated to the same
extent by mutant and wild-type receptor. In addition, we have also found that the Akt and
JNK signalling pathways are not affected by the PDGFRbetaY857F mutation. Moreover, the
Erk pathway was activated by slower kinetics in the mutant receptor. Interestingly, in spite of
the defect in the PDGFRbetaY857F receptors kinase activity and the changes observed in
signal transudction it could still efficiently induce cell chemotaxis. It is known that many
cytoplasmic tyrosine kinases are capable of phosphorylating the PDGFR and it is possible that
these may compensate for the lowered enzymatic activity of PDGFRbetaY857F . In summary,
we have demonstrated that the PDGFRbetaY857F has defect kinase activity, but is
nevertheless phosphorylated in the cell, presumably by cytoplasmic kinases and can induce
signals sufficient for normal chemotaxis toward PDGF-BB.
- 250 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 65
Leukocyte mitogen activated protein kinase cascade activity associated with the LRRK2
G2019S mutation and Parkinson's disease
Linda R. White1, Sylvia N. Kvam2, Mathias Toft1,3, Matthew Farrer3, Jan O. Aasly2
1Department of Neuroscience, Norwegian University of Science and Technology,
2Department of Neurology, University Hospital of Trondheim, N-7006 Trondheim,
Norway, and 3Department of Neuroscience, Mayo Clinic College of Medicine,
Jacksonville, Florida, USA. E-mail: linda.white@ntnu.no
Several pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2, Park8) gene have
now been identified in connection with familial and sporadic parkinsonism. Of these, the
substitution 6055G>A is the hitherto most common cause of genetically-related Parkinson's
disease (PD) to be found, accounting for over 2 % of PD patients in Norway (Aasly et al.,
Ann Neurol 2005;57:762-5). The mutation is autosomal dominant, the clinical features
including asymmetric resting tremor, bradykinesia and rigidity, with a good response to
levodopa treatment. It is thus indistinguishable from idiopathic PD. LRRK2 is a large,
multidomain protein, and the 6055G>A mutation results in a single amino acid substitution
(G2019S) in the mitogen-activated protein kinase kinase kinase (MAPKKK) domain, at the
start of the kinase activation site (a highly-conserved region). This is therefore expected to
affect enzyme activity. Since PD develops in the later decades of life, it is likely that changes
in MAPK cascades attributable to the mutation might be identifiable prior to disease
development, and in tissues other than the brain's dopaminergic system. We have therefore
isolated leukocytes from the blood of Norwegian patients bearing the Park8 G2019S mutation
and diagnosed as suffering from PD, as well as from patients identified as mutation carriers,
but who have not yet developed any movement disorder, and from relatives identified as not
carrying the mutation. These groups have been compared with a positive control group
(patients with typical PD of no known cause, genetic or otherwise (idiopathic PD)), and age
and sex-matched healthy controls. The phosphorylation of proteins central to MAPKmodulated
signal transduction has been assayed in these samples by protein microarray and
ELISA, and highly significant differences between the groups identified.
- 251 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 66
Inhibition of gap junction intercellular communication by capsaicin in rat liver
epithelial cells are mediated by phosphorylation of Cx43 and activation of ERK1/2
through activation of NADPH oxidase
Ji Hyuck Yim, Ki Won Lee, Jung Yeon Kwon, and Hyong Joo Lee*
Department of Food Biotechnology, School of Agricultural Biotechnology, and
Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742,
Republic of Korea
Many studies have focused on the antimutagenic or anticarcinogenic activities of capsaicin
(trans-8-methyl-N-vanillyl-6-nonenamide) which is a major pungent ingredient in red pepper.
Recently, several studies have shown that anticarcinogenic effects of capsaicin may be partly
involved in the induction of apoptosis in several cancer cell lines. A recent study suggests that
the mechanism that makes antiproliferative activity of capsaicin in a human hepatocarcinoma
cells is due to their ability to produce hydrogen peroxide (H2O2) via activation of NADPH
oxidase. However, it can be argued that such inappropriate H2O2 generation may also have a
deleterious effect on nonmalignant cells as well. The present study investigated whether
capsaicin generates H2O2 at the intracellular levels and, if so, whether the formation of H2O2
by capsaicin induces carcinogenicity in normal rat liver epithelial cells. Capsaicin at the
concentration of 50µM completely inhibited gap junction intercellular communication (GJIC;
a carcinogenic phenomenon) in WB-F344 normal rat liver epithelial cells (WB cells) in a
dose-dependant manner. The inhibition of GJIC by capsaicin was mediated by
hyperphosphorylation of connexin 43 (Cx43) and activation of extracellular-regulated kinase
1/2 (ERK1/2) as demonstrated using pharmacological inhibitors of ERK1/2. Pretreatment of
WB cells with catalase and inhibitors of NADPH oxidase significantly reversed capsaicininduced
inhibition of GJIC and hyperphosphorylation of Cx43, suggesting that ROS may
mediate the inhibition of GJIC induced by capsaicin. These results indicate that the
antiproliferative effects of capsaicin on cancer cells are partially due to their prooxidant
actions, thereby indicating their potential toxicity and carcinogenicity.
- 252 -

Session III : Protein kinase cascades as therapeutic targets Poster III, 67
Renal Akt kinase signaling in Zucker diabetic fatty rats (ZDF).
Jana Zdychova1, Jessie N. Lindsley2, Sharon Anderson2, Radko Komers1,2.
1Diabetes Center, Institute for Clinical and Experimental Medicine, 140 21 Prague 4,
Czech Republic; 2Division of Nephrology and Hypertension, Oregon Health&Science
University, 97239 Portland, OR, USA. E-mail: rako@medicon.cz
Impaired insulin signaling via PI-3-kinase (PI3K) and Akt in skeletal muscle, liver and fat has
been implicated in the pathophysiology of insulin resistance (IR) including Type 2 diabetes
(DM2). Kidney is also a target of insulin actions. However, unlike the skeletal muscle, renal
Akt signaling in DM2 has been so far less studied. To address this issue, renal cortical activity
and protein expression of Akt, its down-stream effector mTOR, and Akt antagonist PTEN
were studied in 4 and 12-week old ZDF (ZDF4 and ZDF12), a model of DM2, and in agematched
controls (Zucker lean, ZL4 and ZL12). Akt and mTOR activities were also studied in
separate groups of 12 week old animals after a short-term treatment with PI3K inhibitor
wortmannin (W, 100 µg/kg bwt) or vehicle (DMSO). Akt activity (in vitro kinase assay) and
expression of active Akt (P-Akt) were increased in ZDF12 as compared to ZL12 (p

Notes
- 254 -

Notes
Notes
- 255 -

- 256 -

Session IV. Protein kinase inhibitors: insights into drug
design from structure
- 257 -

IV. Protein kinase inhibitors: insights into drug design from structure Poster IV, 1
The mechanism of anti-proliferation by epidermal growth factor receptor tyrosine
kinase inhibitor ZD1839 in human oral squamous cell carcinoma.
Nam K. Jeon, Jin Kim and Eun J. Lee
Department of Oral Pathology, Oral Cancer Research Institute, BK21 project for
Medical Science, Yonsei University College of Dentistry, Seoul, 120752, KOREA Email:e16lee@yumc.yonsei.ac.kr
Activation of the epidermal growth factor receptor (EGFR) and downstream signaling
pathways have been implicated in causing resistance to EGFR-targeted therapy in solid
tumors, including the head and neck tumors. To investigate the mechanism of
antiproliferation to EGFR inhibition in oral cancer, we compared EGFR tyrosine kinase
inhibitor (Gefitinib, Iressa, ZD1839) with respect to its inhibitory effects on three kinases
situated downstream of EGFR: MAPK, Akt, and glycogen synthase kinase-3ß (GSK-3ß). We
have demonstrated that ZD1839 induces growth arrest in oral cancer cell lines by independent
of EGFR-mediated signaling. Cell cycle kinetic analysis demonstrated that ZD1839 induces a
delay in cell cycle progression and a G1 arrest; this was associated with increased expression
of both p27KIP1 and p21CIP/WAF1 proteins. We found that the resistance to the
antiproliferative effects of gefitinib, in vitro was associated with uncoupling between EGFR
and MAPK inhibition, and that GSK-3ß activation and degradation of its target cyclin D1
were indicators of a high cell sensitivity to gefitinib. In conclusion, our data show that the
uncoupling of EGFR with mitogenic pathways can cause resistance to EGFR inhibition in
oral cancer. (This study was supported by the BK21 project for Medical Science, and by a
grant of the national Cancer control R&D Program 2003(No. 0320230), Ministry of Health &
Welfare, Republic of Korea.)
- 258 -

IV. Protein kinase inhibitors: insights into drug design from structure Poster IV, 2
Three dimensional structures of Pim-1 inhibitor complexes give new insights for the
development of target specific kinase inhibitors
Stefan Knapp, A.N. Bullock, J. É. Debreczeni, O. Y. Fedorov, B. D. Marsden. E.
Meggers
University of Oxford, Structural Genomics Consortium (SGC), Botnar Research Center,
Oxford OX3 7LD, UK. E-mail: stefan.knapp@sgc.ox.ac.uk
The Structural Genomics Consortium (SGC) is a not-for-profit organization that aims to
determine the three dimensional structures of proteins of medical relevance, and place them in
the public domain without restriction. Due to the important role in current drug development
we solve 12 human kinases so far and selected human Pim-1 kinase for further follow up
studies.
The kinase Pim-1 plays a pivotal role in cytokine signaling and is implicated in the
development of a number of tumors. The three dimensional structure of Pim-1 is
characterized by unique structural features within the ATP binding pocket which makes it
particularly important to determine how inhibitors bind to this kinase. We determined 6 high
resolution crystal structures of Pim-1 in complex with a potent inhibitors of the
bisindolylmaleimide class (BIM-1), a imidazo [1,2-b]pyridazine, three novel ruthenium half
sandwich organometallic complexes as well as a complex with a high affinity substrate
peptide.
The determined structural data revealed a number of rather unexpected features of Pim-1
inhibitor interaction. For example the imidazo[1,2-b]pyridazine inhibitor formed no hydrogen
bond with the hinge region of Pim-1 as is usually observed for this inhibitor class and other
kinase inhibitors. Instead the imidazo[1,2-b]pyridazine forms a bifurcated hydrogen bond
with the active site lysine K67 and a water molecule that is coordinated by E89 and D186.
Structures of ruthenium half sandwich organometallic complexes which inhibit Pim-1 with
low pico molar affinity revealed that all three inhibitors fit tightly into the binding pocket with
a shape complementarity that would not be possible to achieve with a conventional organic
compound. The presented data suggest a number of strategies for the further development of
potent and selective Pim-1 inhibitors which may find applications for the treatment of Pim-1
dependent cancer types.
- 259 -

IV. Protein kinase inhibitors: insights into drug design from structure Poster IV, 3
The Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor ZD1839 (‘Iressa)
Suppresses Proliferation and Invasion of Human Oral Squamous Carcinoma Cells via
MMP, uPAR Dependent Mechanism
Eun J. Lee, Jin H. Whang, Nam K. Jeon, Young E. Kwak and Jin Kim
Department of Oral Pathology, Oral Cancer Research Institute, BK21 project for
Medical Science, Yonsei University College of Dentistry, Seoul, 120-752, KOREA
E-mail:e16lee@yumc.yonsei.ac.kr
Oral squamous cell carcinomas (OSCCs) are characterized by a marked propensity for local
invasion and dissemination to cervical lymph nodes. Overexpression of the epidermal growth
factor receptor (EGFR) and high levels of certain matrix metalloproteinases (MMP) have
been implicated in the development of squamous-cell carcinoma of oral cancer. This study
attempted to determine the mechanisms underlying the effects of ZD1839 on the cellular
level, and to characterize the effects of ZD1839 with regard to human OSCC cell growth and
invasion/migration. Cell cycle kinetic analysis demonstrated that ZD1839 induces a delay in
cell cycle progression and a G1 arrest, which was associated with the up-regulation of cyclindependent
kinase inhibitors (CDKI) p27KIP1 and p21CIP1/WAF1. The up-regulation of
CDKI may be mediated by a p53-independent and hnRNPC1/C2-dependent pathway. In
addition, 100nM ZD1839 demonstrated that both MMP-2 and MMP-9 enzyme activity were
decreased by ~25-30%. The current in vitro study indicates that the inhibition of proliferation
and invasion/migration in OSCC cell lines by ZD1839 results in an anticancer effect via
multiple cellular and molecular mechanisms, and suggests that ZD1839 may be useful in
inhibiting and /or preventing metastasis. (This study was supported by the BK21 project for
Medical Science, and by a grant of the national Cancer control R&D Program (02-PJ1-PG3-
20801-0015) and 2003(No. 0320230), Ministry of Health & Welfare, Republic of Korea.)
- 260 -

IV. Protein kinase inhibitors: insights into drug design from structure Poster IV, 4
Aberrant methylation of p15INK4B and p14ARF and deletion of p16INK4A are
frequent events in liver fluke related cholangiocarcinoma
Temduang Limpaiboon1,4, Preeda Prakrankamanant1, Inthira Tussakhon1, Patcharee
Chinnasri1, Patcharee Jearanaikoon1,4, Chawalit Pairojkul2,4, Banchob Sripa2,4,
Vajarabhongsa Bhudhisawasdi3,4
1Department of Clinical Chemistry, Centre for Research and Development of Medical
Diagnostic Laboratories, Faculty of Associated Medical Sciences, 2Department of
Pathology, 3Department of Surgery, 4Liver Fluke and Cholangiocarcinoma Research
Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
E-mail:temduang@kku.acth
Cholangiocarcinoma (CCA) is the highest incidence cancer in Northeast Thailand. CCA is
caused by liver fluke (Opisthorchis viverrrini) infection resulting in both genetic and
epigenetic alterations. The INK4 locus located on chromosome 9p21 consisting of three
genes, p14ARF, p16INK4A and p15INK4B, which are involved in cell cycle regulation and
reported to be inactivated in many human cancers through genetic and epigenetic events. To
understand mechanisms of inactivation of these three genes in CCA, we analyzed loss of
heterozygosity (LOH) at chromosomal region 9p21 using three polymorphic microsatellite
markers; D9S1752, D9S171 and D9S161 and determined promoter hypermethylation of
p14ARF, p16INK4A and p15INK4B using methylation-specific PCR in 79 CCA. LOH was
found at least one or more loci in 32 of 74 informative cases (43.2%) in which high frequency
was found in D9S1752 (34.9%) and less frequency was observed in D9S171 (27.6%).
Aberrant methylation was present predominantly in p15INK4B (50.6%) and p14ARF (41.2%)
and less frequently in p16INK4A (25.3%). Our results suggest that promoter
hypermethylation is a predominant inactivation mechanism of p15INK4B and p14ARF,
whereas, deletion is a major event for inactivation of p16INK4A during CCA development.
This finding may lead to a new approach of CCA treatment.
- 261 -

IV. Protein kinase inhibitors: insights into drug design from structure Poster IV, 5
Exploring the chemotherapeutic benefit of combining Gemcitabine (Gemzar) and
Rapamycin
Ofer Merimsky1,2, Yaara Gorzalczany2 and Ronit Sagi-Eisenberg2
1Department of Oncology, The Tel-Aviv Sourasky Medical Center, Sackler School of
Medicine, Tel Aviv University, Tel Aviv 69978 Israel and 2Dept. of Cell and
Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978
Israel
Two major obstacles encountered while treating cancer patients are achieving the right
balance between the effectiveness of the drug and its cytotoxic side effects as well as
overcoming mechanisms of existing or acquired resistance. The emerging solution appears to
constitute a combination of a “traditional” cytotoxic drug with a “signaling” drug.
Gemcitabine (Gemzar), is an antimetabolite drug that inhibits DNA synthesis and displays
some activity as a single agent in treating pancreatic and sarcoma cancer. However,
gemcitabine treatment is often associated with acquired resistance. Rapamycin is a selective
inhibitor of mTOR, a phosphatidylinositol-3-kinase (PI3K)-related kinase that controls cell
proliferation, cell survival and adhesion-independent survival and migration. Combining
Gemzar and rapamycin might thus provide a therapeutic advantage. Here we report a patient
who underwent resection of abdominal leiomyosarcoma at the time of renal transplantation.
While on rapamycin for rejection prevention, no sarcoma re-growth was evident. On
interruption of rapamycin, after spontaneous kidney rejection, the sarcoma relapsed, and
metastasized. Re-introduction of rapamycin and co-administration of gemcitabine yielded a
prolonged remission and symptoms alleviation. To define the molecular impact of this drug
combination, we have used SK-LMS cells, a human leiomyosarcoma cell line as a model.
Indeed, we show that both gemcitabine and rapamycin inhibit cell proliferation, only
rapamycin alone or when combined with gemcitabine reduce significantly the amount of
phosphorylated ERK as well as a the amount of bcl-2. In view of the fact that gemcitabineinduced
cell death is determined by bcl-2 content, our results provide a strong basis for a
novel regimen based on Gemzar/rapamycin combination in cancer treatment.
- 262 -

Notes
- 263 -

Notes
Notes
- 264 -

- 265 -

Session V. Phosphatases as key cell signaling intermediates
- 266 -

V. Phosphatases as key cell signaling intermediates Poster V, 1
SHP-1 tyrosine phosphatase in human erythrocytes.
Bragadin M.1, Clari G.2, Ion Popa F.2, Bordin L.2
1 Dept. of Environmental Sciences, Ca’ Foscari University of Venice, Calle Larga
S.Marta Dorsoduro 2137, Venice, Italy; e-mail: bragadin@unive.it
2 Dept. of Biological Chemistry, University of Padua, Viale G. Colombo 3, 35121 Padua,
Italy; e-mail: giulio.clari@unipd.it ; luciana.bordin@unipd.it
SHP-1 is a SH2-domain containing protein Tyr-phosphatase expressed in hematopoietic cell
lines, which is hypothesized to play a largely negative role in signal transduction. In human
erytrocytes, the phospho-Tyr level of proteins, mainly transmembrane band 3 protein, is
closely controlled by the antithetic activity of Tyr-protein kinases and phosphatases, resulting
in a dephosphorylated state. Only after particular stimuli, as with oxidizing agents, diamide or
pervanadate, or thiol alkylating compound, N-ethyl maleimide (NEM), Tyr-phosphorylation
of band 3 can be triggered, inhibiting Tyr-phosphatase action and inducing erytrocyte
membrane reorganization.
We demonstrate that, in human red blood cells, SHP-1 is present in membranes from resting
cells, but in 5 percent of the protein amount. Interstingly, this amount increases up to
threefold following NEM treatement of intact cells, whereas diamide and pervanadate do not
alter the normal protein location. In addition, SHP-1 translocation from cytosol to membrane
is not affected by band 3 P-Tyr level, since it is not mediated by the SH2-P-tyr recruiment
mechanism, and localizes into the cytoskeletal compartment. Band 3 is the target of SHP-1,
which dephosphorylates tyrosines located in acidic sequences such as Tyr 8,21 and 904.
All together, these findings support the idea that, in human erytrocytes, the normal level of
Tyr-phosphorylation of membrane protein, mainly band 3, must be down regulated. We
hypothesize that the simultaneous presence of both SHP-2 and SHP-1 ensures band 3
dephosphorylation in different conditions: SHP-2, through interaction of its SH2 domain/s to
P-Tyr protein, is regulated by the band 3 Tyr-phosphorylation level; SHP-1 may be involved
by simple membrane rearrangement.
- 267 -

V. Phosphatases as key cell signaling intermediates Poster V, 2
Novel Cdc25 inhibitors: activity in MCF-7 cells and potentiation of cytotoxic drugs
Souhayla El Maâdidi, Laurent Brault and Denyse Bagrel
Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, UFR SciFA,
Université Paul Verlaine-Metz, Campus Bridoux, rue du Général Delestraint, 57070
Metz, France. E-mail: bagrel@univ-metz.fr
The Cdc25s dual-specificity phosphatases are key regulators of cell cycle progression which
dephosphorylate and activate cycline dependent kinases. Three homologs of Cdc25
phosphatases exist in humans: Cdc25A, Cdc25B, and Cdc25C. Cdc25B and Cdc25C regulate
the G2/M cell cycle transition while Cdc25A is responsible for regulating the G1/S transition.
The overexpression of Cdc25A and B is frequently associated with a wide variety of cancers.
Taken into account there regulatory role, the aim of this study is a development of new Cdc25
inhibitors. In our laboratory, novel maleic anhydride derivatives have been synthetised
(named 6a to 6j) and tested in vitro using pNPP as substrate on the three recombinant GST-
Cdc25A, GST-Cdc25B and GST-Cdc25C . These inhibitors differ in the size of their fatty
acid chain, the most active of them having the longest chain. The in vitro inhibitory activity
was confirmed by testing those compounds on two breast cancer cell lines: MCF-7 and its
counterpart resistant to vincristine Vcr-R. Four compounds were chosen: 6a presenting the
shortest fatty acid chain (6 carbons) and inactive in vitro, 6e having a mid-long chain (11
carbons) showing an intermediate activity, and the 2 most potent inhibitors in vitro (6i and 6j)
with the longest side chain (17 carbons). These two compounds induced a cell cycle blockage
in G0/G1 phase and a decrease of cell proportions in S and G2/M phases after 24 and 48h
treatments. Moreover, apoptosis was triggered within 48h-treatment, without oxidative burst.
We also showed that 6i and 6j are able to potentiate the effects of cisplatin and adriamycin, at
least by an additional effect. Thus, we can conclude that maleic anhydride-derivatives may
mediate apoptosis through a cell cycle blockage via inhibition of the phosphatases Cdc25 and
may present a potential interest in therapeutic strategies against cancer.
- 268 -

V. Phosphatases as key cell signaling intermediates Poster V, 3
Cell adhesion to the extracellular matrix increases the proliferation of acute myeloid
leukemia (AML) cells and up-regulates the CDC25A phosphatase.
Anne Fernandez-Vidal, Loïc Ysebaert, Christine Didier, Stéphane Manenti.
Centre de physiopathologie Toulouse-Purpan, INSERM U563-IFR30, Département
« Oncogénèse et signalisation dans les cellules hématopoïétiques », CHU Purpan, 3 place
du Dr Baylac 31024 Toulouse Cedex 3, France. E-mail : fervidal@toulouse.inserm.fr.
Microenvironmental stimuli are key factors in normal and leukemic hematopoietic
differenciation. In this contexte, we investigated the influence of the major extracellular
matrix component, fibronectin, on the proliferative state of acute myeloid leukemia and
normal hematopoietic cells. Although fibronectin inhibited the proliferation rate of immature
normal CD34+ cells, it stimulated the proliferation of immature leukemic cells at similar
differentiation state (KG1a cell line). These data suggest that hematopoietic cells shift from a
negative to a positive proliferative response to cell adhesion during the leukemogenic
transformation process. As a general feature, we found that cell adhesion to fibronectin matrix
increases the proliferation of various cell lines. Importantly, we identified the dual specificity
phosphatase Cdc25A as a key effector of this effect. Indeed, adhesion to fibronectin of
leukemic cells dramatically up-regulates the cellular level of this protein, in good correlation
with the proliferative response. By contrast, Cdc25A protein down-regulation was observed in
normal CD34+ cells, nicely fitting with the inhibition of the proliferation observed in these
cells. Furthermore, siRNA- and pharmacological inhibitor-based experiments strongly suggest
that Cdc25A regulation in this context is indeed involved in the adhesion –dependent
proliferation process. Altogether, these data demonstrate for the first time an integrindependent
Cdc25A protein regulation, making of this cell cycle effector an important link
between extracellular stimuli and the cell cycle machinery. This also make of this phosphatase
and of its molecular regulators, a potential therapeutic target in the acute myeloid leukemia
pathologies.
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V. Phosphatases as key cell signaling intermediates Poster V, 4
The Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase is upregulated
in cervix cancer cells lines.
Rachel Henkens, Philippe Delvenne, Jacques Boniver, Tomas Mustelin, Michel
Moutschen and Souad Rahmouni.
Department of Pathology, Liège University, Liège, Belgium.
Protein tyrosine phosphatases (PTPases) regulate a variety of important processes in cells
including cell cycle progression and several are involved in cancer. The 21-kDa Vaccinia
virus VH1-related (VHR) dual-specific protein phosphatase plays a critical role in cell cycle
progression and is itself regulated during its progression. Using RNA interference, it has been
demonstrate that cells lacking VHR arrest in G1 and G2 phases of the cell cycle and show
signs of beginning of cell senescence. We report here for the first time that VHR PTPase is
upregulated in several cervix cancer cell lines. Using immunohistochemistry and
immunoblotting techniques, we show that this dual-specific phosphatase is upregulated in
HPV positive cells lines such as Hela S3, CaSki, SiHa as well as in HPV negative cervix
cancer cell lines such as C33 and HT3 compared to normal keratinocytes (NK). In addition to
the upregulation of this protein, we report that VHR has a cytoplasmic localization in NK
while its localized in the cytoplasm and in the nucleus of the cancer cell lines investigated.
The semi-quantitative RT-PCR analysis shows no difference between the mRNA levels in the
different cell lines compared to NK. Finally, we report that the VHR upregulation observed is
at least partially due to the half life differences between the normal keratynocytes (+/- 7h) and
the different cervix cancer cell lines (>12h).
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V. Phosphatases as key cell signaling intermediates Poster V, 5
Regulation of apoptosis signal-regulating kinase 1 (ASK1) by polyamine levels via
protein phosphatase 5
Mikhail A. Kutuzov, Alexandra V. Andreeva and Tatyana A. Voyno-Yasenetskaya.
Department of Pharmacology, College of Medicine, University of Illinois at Chicago,
Chicago, Illinois 60607, U.S.A. Email: kutuzov@uic.edu
Protein phosphatase 5 (PP5) is a member of the PPP family of protein Ser/Thr phosphatases,
conserved from protists to plants and animals. Recent years have seen increasing evidence for
PP5 involvement in a variety of signaling pathways, suggesting that PP5 is multifunctional
phosphatase, similarly to more extensively characterized PPP phosphatases PP1 and PP2A. A
number of proteins have been identified that interact with PP5 and either regulate its activity,
or are PP5 substrates, or play a role of scaffold. PP5 may also be regulated, at least in vitro,
by low molecular weight compounds, such as polyunsaturated fatty acids and fatty acyl-CoA
esters. We found that PP5 is strongly inhibited by micromolar concentrations of a natural
polyamine spermine. This inhibition was observed both with p-nitrophenyl phosphate and
protein substrates phosphocasein and apoptosis signal-regulating kinase 1 (ASK1, thought to
be a physiological substrate of PP5). Inhibition of polyamine biosynthesis in COS-7 cells by
alpha-difluoromethylornithine (DFMO) led to accelerated dephosphorylation of oxidative
stress-activated ASK1. This effect of DFMO was suppressed by okadaic acid and by siRNAmediated
PP5 depletion, indicating that the effect of polyamine levels on ASK1
dephosphorylation was mediated by PP5. Polyamine depletion in COS-7 cells abrogated
oxidative stress-induced activation of caspase-3 (which executes ASK1-induced apoptosis), as
well as caspase-3 activation induced by ASK1 overexpression, but had no effect on basal
caspase-3 activity. These results implicate polyamines, emerging intracellular signaling
molecules, as potential physiological regulators of PP5. Our findings also suggest a novel
mechanism of the antiapoptotic action of a decrease in polyamine levels via de-inhibition of
PP5 and accelerated dephosphorylation and deactivation of ASK1.
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V. Phosphatases as key cell signaling intermediates Poster V, 6
Regulation of SHP-2 protein tyrosine phosphatase by Angiotensin II-derived reactive
oxygen species in hypertensive and normotensive rats- Evidence of oxidation of protein
tyrosine phosphatases in hypertension
Fatiha Tabet1, Ernesto L. Schiffrin2, Rhian M. Touyz1.
1Ottawa Health Research Institute, University of Ottawa, Health Sciences Building,
304/451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada. E-mail: ftabet@uottawa.ca,
E-mail: rtouyz@uottawa.ca
2Experimental Hypertension Laboratory, Clinical Research Institute of Montreal, 110
Pine Av. West, Montreal, Quebec H2W 1R7, Canada. E-mail:
ernesto.schiffrin@ircm.qc.ca
Reactive Oxygen Species (ROS) act as signaling molecules that regulate activation of MAP
kinases. We recently demonstrated that angiotensin II (Ang II)-stimulated tyrosine kinases
play a role in redox-sensitive activation of MAP kinases and that in vascular smooth muscle
cells (VSMCs) from hypertensive rats (SHR) responses are enhanced. In the present study we
tested the hypothesis that Ang II-induced ROS production augments tyrosine
phosphorylation-dependent signaling in SHR as compared to normotensive rats WKY through
differential activation of intracellular SHP-2 tyrosine phosphatase expression and activation,
and through reversible inactivation of cellular PTPs. Mesenteric artery VSMCs of 16-week
WKY and SHR rats were studied. Cells were stimulated with Ang II (10-7mol/l). The
expression and activation (0-30 min) of SHP-2 was evaluated using immunoblotting analysis.
The role of AT-1 and AT-2 receptors in Ang II-induced activation de SHP-2 was evaluated
using AT-1 and AT-2 receptor inhibitors Valsartan and PD123319, respectively. The “In-Gel”
PTP Assay was used to determine whether PTPs in response to Ang II (10-7mol/l, 0-60 min)
or H2O2 (0.1-200µM, 10 min), are susceptible to oxidation. GST-FER fusion protein was
used as a protein tyrosine kinase source for phosphorylation of the [$-32P]-labeled poly (4:1)
Glu-Tyr substrate. In the basal state, SHP-2 expression (p

Notes
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Notes
Notes
- 274 -

- 275 -

Session VI. Proteasome degradation pathways
- 276 -

VI. Proteasome degradation pathways Poster VI, 1
Dactylin, a F-box/WD ubiquitin ligase regulates osteoblast differentiation and limb
development
Steven Y Cheng1, 2 and Ying E. Zhang2
1 Cellogenetics. Inc., Gaithersburg, MD 20898
2 Laboratory of Cellular & Molecular Biology, National Cancer Institute, NIH,
Bethesda, MD 20892
Dactylin is a member of the F-box/WD-40 gene family, which recruit specific target proteins
through their WD-40 protein-protein binding domains for ubiquitin mediated degradation.
Disruption of the mouse dactylin gene results in the absence of central digits, underdeveloped
or absent metacarpal/metatarsal bones and syndactyly. This phenotype is remarkably similar
to split hand-split foot malformation in humans, a clinically heterogeneous condition with a
variety of modes of transmission. We found BMP signaling downregulates dactylin
expression through Smad pathways. Moreover, we showed that downregulation of Dactylin
is required for BMP-2-mediated C2C12 myoblast differentiation into osteoblast since
overexpression of Dactylin inhibited osteoblast differentiation. Thus, through its F-Box and
WD protein interaction domains, Dactylin regulates osteoblast differentiation by modulating
BMP signaling. These results offer new insights into how BMP elicits biological effects, in
particular into the mechanism of inhibition of myoblast differentiation and stimulation of
osteoblast differentiation, and important role of Dactylin in controlling mesenchymal cell fate.
- 277 -

VI. Proteasome degradation pathways Poster VI, 2
p14ARF triggers G2 arrest through ERK-mediated Cdc25C phosphorylation,
ubiquitination and proteasomal degradation.
Beatrice Eymin, Christian Brambilla, Elisabeth Brambilla and Sylvie Gazzeri.
INSERM U578, Institut Albert Bonniot, 38706 La Tronche Cedex, France. Université
Joseph Fourier, Faculté de Medecine, Groupe de Recherche sur le Cancer du Poumon,
La Tronche Cedex, France. E-mail: Beatrice.Eymin@ujf-grenoble.fr
The Cdc25C phosphatase is a key regulator of mitotic entry which activity is highly regulated
by phosphorylation. In response to DNA damage, phosphorylation of Cdc25C at serine 216
induces cytosolic retention through 14-3-3 binding. We previously reported the ability of the
p14ARF tumor suppressor to induce accumulation of inactive phospho-Cdc25C(Ser216)
protein as well as decrease of Cdc25C steady state level and correlated these events with a
p53-independent G2 arrest. The aim of this study was to investigate the cellular signaling
pathways involved in this process. By using specific pharmacological inhibitors, we
demonstrate that activation of the MAP kinases ERK1/2 pathway is involved in the p53independent
G2 checkpoint induced by p14ARF. Moreover, we identify Cdc25C as a new
ERK1/2 target by providing evidence that activated P-ERK1/2 bind and phosphorylate
Cdc25C on its ser216 residue following p14ARF expression. Importantly, we further show
that phosphorylation at Ser216 by phospho-ERK1/2 promotes Cdc25C ubiquitination and
proteasomal degradation, suggesting that Cdc25C proteolysis is required for a sustained G2
arrest in response to p14ARF. Taken together, these results demonstrate that the MAPK ERK
signaling pathway plays a role in the p53-independent antiproliferative functions of p14ARF.
Furthermore, they identify a new mechanism by which phosphorylation at serine 216
contributes to Cdc25C inactivation.
- 278 -

VI. Proteasome degradation pathways Poster VI, 3
Ubiquitination as a priming process of PKC alpha and epsilon degradation in the
alphalphaT3-1 gonadotrope cell line.
Benoit Poulin, Hélène Maccario, Brice Junoy, Bénédicte Boyer, Alain Enjalbert and
Sophia V. Drouva.
CNRS UMR 6544, Université de la Méditerranée, Faculté de Médecine, IFR Jean Roche
Bd Pierre Dramard 13916 Marseille, France. E-mail: drouva.sv@jean-roche.univ-mrs.fr
Protein kinase C (PKC) is a family of isoenzymes playing a key role in the regulation of
gonadotrope cell functions. We have previously demonstrated that gonadotrope cell lines
(alphaT3-1, LbT2) possess several PKC isoenzymes which are selectively sensitive to
gonadotropin-releasing hormone (GnRH) and phorbol ester TPA. Indeed, specific PKC
isoforms are differentially phosphorylated, activated and down regulated by GnRH (delta,
epsilon, theta) or TPA (alpha, betaII, delta, epsilon, theta). Furthermore, we have shown that
under desensitization conditions the degradation of PKC alpha, epsilon and delta isoenzymes
may involve proteolytic processing implicating ubiquitin/proteasome system. Specific
proteasome inhibitors prevented GnRH- and TPA-elicited kinase depletion and induced
membrane accumulation of the respective isoenzymes in a phosphorylated form. In the
present study we investigated in alphaT3-1 gonadotrope cells, the mechanisms underlying
proteasome-dependent TPA-sensitive down-regulation of the PKC alpha and epsilon
isoenzymes, and of the GnRH-activated PKC epsilon isoform. By a pull-down assay using
beads conjugated with a GST-fusion protein containing ubiquitin-associated domains (Rad23)
that bind polyubiquitinated proteins, we show that TPA induced rapid (15min) and sustained
(up to 4h) PKC alpha and PKC epsilon polyubiquitination. Moreover, GnRH provoked a
selective polyubiquitination of PKC epsilon, in a receptor-dependent manner, but was unable
as expected to induce any polyubiquitination of PKC alpha The GnRH-evoked PKC epsilon
polyubiquitination was important and rapid (as early as 10min) and progressively decreased
over time (but remained detectable after a 4h treatment). The empty beads, used as control,
did not retain any polyubiquitinated protein. Thus in alphaT3-1 gonadotrope cells, the event
priming TPA-induced PKC alpha and PKC epsilon down-regulations as well as GnRHevoked
PKC epsilon desensitization, is their respective polyubiquitination, it preceeds the
isoenzymes degradation by the proteasome complex. Surprisingly proteasome inhibition
counteracted the TPA- as well as GnRH- induced polyubiquitination of their target PKC
isoenzymes ; this may reflect either high activity of deubiquitinating enzymes or direct
interaction with ubiquitin conjugation at the proteasome level.
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Notes
- 280 -

Session VII. Stem cell specific cell signaling mechanisms
- 281 -

VII. Stem cell specific cell signaling mechanisms Poster VII, 1
Murine marrow-derived mesenchymal stem cells:Isolation,Characterisation
*Raza Haji Hosseini ,**Samad Nadri
*Biology Dept. Faculty of science, Payame Noor University, Tehran, Iran 19569
*E.mail:hosseini@pnu.ac.ir,
**Biology Dept. Faculty of science, Payame Noor University, Tehran, Iran 19569
**E.mail:nadrisamad@yahoo.co.uk
Introduction: Many researchers have employed different methods to isolate and purify
mesenchymal stem cells in mouse bone marrow but there is a lack of consensus on
mesenchymal stem cells express surface markers. There is evidence for the affect cell density
on surface markers in human mesenchymal stem cells. IN present study, Expression of
CD34,CD44,Sca-1,c-Kit,Vcam-1 on MSCs purified by Low density and High density from
NMRI and BALB/c was studied.
Method and Material:6-8 week-old NMRI andBALB/c were killed by cervical dislocation and
bone marrow were flushed and cultured using low glucose DMEM medium by Low density
and High density methods then MSCs were purified by continue passages.To examine the
mesenchymal nature of isolated cells,The cells were differentiated into bone and
adipocyte.the cells were abeled by CD34,CD44,Sca-1,c-Kit,Vcam-1 antibody conjugated to
PE or FITC and analyzed by flow cytometry method using FACS-analysis Machine.
Result:Spindle –shaped purified cells were observed in Low density and High density cultures
at the end of the fourth and seven passage,respectively.The cells from both NMRI
andBALB/c were easily differentiated into osteogenic and adipogenic Lineages.Flow
cytometry analysis indicated that The MSCs from either NMRI and BALB/c mice are CD44+
,Sca-1+ CD34- and c-Kit- in Low density and High density culture.The mMSCs from tow
strains differed in their expression of the cell-surface epitope Vcam-1.
Conclusion:In present investigation,The presence of five surface markers was investigated on
MSCs purified by Low density and High density culture from NMRI and BALB/c.The results
presented here,indicated up to 90% cells is CD44+ , It seems That It could be useful markers
in isolation of the MSCs.Comparisons of MSCs isolated from 2 strains demonstrated That
some features of The cells were strain specific also They isolation more rapidly if plated at
Low density culture. In this studies, In spite of previously research,Cell density is not affected
on expression surface markers.
Key Word:marker,mesenchymal stem cell,bone marrow
- 282 -

Notes
- 283 -

Session VIII. Inflammation specific signaling
- 284 -

VIII. Inflammation specific signaling Poster VIII, 1
Analysis of tissue distribution of TNF-alpha, TNF-alpha-Receptors and the activating
TNF-alpha-Converting Enzyme (TACE) suggest activation of the TNF-alpha system in
the ageing intervertebral disc
B.Bachmeier (1), A. Nerlich (2), Ch. Weiler (3), N. Boos (4)
Dept. of Clinical Chemistry Clin. Biochemistry, Univ. Munich, Germany,
bachmeier@med.uni-muenchen.de
Dept. of Pathology, Academic Hospital Munich-Bogenhausen, Germany
Institute of Pathology, Ludwig-Maximilians-University Munich, Germany
Spinal Surgery Unit, Orthopaedic Clinic, Univ. Zürich, Switzerland
The expression and up-regulation of the pro-inflammatory cytokine TNF-alpha recently has
been suggested to play a major role in the degeneration of the intervertebral disc. In order to
further elucidate the role of TNF-alpha in intervertebral discs and to find out, if the TNFalpha
activation by the corresponding converting enzyme TACE and the two TNF-alphareceptors
I and II are indeed potentially involved in the cytokine action, we
immunohistochemically analyzed the expression and localization of all four factors in human
discs. In parallel, the quantitative levels of TNF-alpha-mRNA (by quantitative RT-PCR) were
determined.
Therefore, a series of 43 human lumbar intervertebral disc samples with complete crosssection
of the motion segment were immunohistochemically stained for TNF-alpha, TNFR-I
and –II and TACE (all antibodies: Santa Cruz Biotech., CA, USA) and morphometrically
evaluated for the proportion of positively labelled cells in five anatomic sub-settings: nucleus
pulposus, inner and outer anterior and posterior annulus fibrosus. In a further series of
surgical material from 20 symptomatic patients the level of TNF-alpha-mRNA was
determined by quantitative RT-PCR (Light Cycler, Roche, Penzberg) and correlated to the
corresponding immunohistochemical staining pattern.
Thereby, we detected a significant expression of TNF-alpha beginning in advanced
adolescence (ca. 17 years) and being most extensive in the nucleus pulposus. Likewise, the
TNF-alpha expression in adult nucleus pulposus ranges between 27 – 80% of all cells until
senile age. In the inner and outer annulus fibrosus significantly less cells are labelled (0 –
50%). In fetal and juvenile discs no TNF-alpha expression is detectable. The parallel analyses
both TNF-alpha-receptors revealed a similar distribution pattern with respect to age and
localization. Additionally, the TNF-alpha converting enzyme TACE, which is essential for the
shedding and activation of the cytokine, are up-regulated similarly. The number of TNFalpha-transcripts
was elevated in most cases with immunohistochemical TNF-alpha
expression confirming the synthesis of TNF-alpha protein in the discs.
Our present study clearly supports the recent notion that TNF-alpha is expressed in discs of
increasing age. Furthermore, we also can confirm the previous observation that TNF-alpha
correlates with histomorphological signs of disc degeneration. Additionally, the present study
provides unambiguous evidence that TNF-alpha is activated (by the converting enzyme
TACE). Finally, the presence of both biologically active TNF-alpha receptors (TNFR-I and –
II) in similar proportion to each other supports the idea that TNF-alpha can also exhibit its
action within the disc.
- 285 -

VIII. Inflammation specific signaling Poster VIII, 2
A role for TNF-alpha in VLDL apoB but not lipid secretion modulation in primary rat
hepatocytes under non-promoting triacylglycerol production conditions
Nerea Bartolomé, Lorena Rodríguez, Cristina López, Virginia Martínez, Begoña Ochoa,
María J. Martínez and Yolanda Chico
Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque
Country, P. Box 669, Bilbao, Bizkaia, Spain. ofbbaesn@lg.ehu.es
The cytokine TNF-alpha has been reported to play a pivotal role in the septic inflammatory
response. It is rapidly liberated to bloodstream triggering the production of other cytokines
and initiating the liver acute phase reaction. Hipertriglyceridemia is an early hallmark of
sepsis associated to high levels of VLDL particles, due to both enhanced secretion by the liver
and decreased peripheral clearance, but the time response of hepatocytes and the underlying
mechanisms are not completely understood. The biogenesis of VLDL is a complex and timerequiring
process that depends on lipid availability and the activity of microsomal triglyceride
transfer protein (MTP) for correct apoB lipidation. The aim of this work was to asses in vitro
whether TNF-alpha has a sustained role in VLDL production regulation and the putative
implication of apoB and MTP transcription. Rat primary hepatocytes were cultured in
conditions mimicking the fed situation, with 5, 20 or 100 ng/ml TNF-alpha for 8 or 16 h.
VLDL were isolated from the medium and apoB-48, apoB-100 and lipids were quantified.
Total RNA was extracted from cells and apoB and MTP mRNA levels measured by Northern
blotting. At 8 h, there was an increase of 17%, 22% and 24% in the secretion of VLDL
particles by 5, 20 or 100 ng/ml TNF-alpha-treated hepatocytes, respectively. However, basal
secretion was recorded after 16 h. Cell apoB mRNA content increased by 1.88-, 1.54- and
1.44-fold in cells at 8 h of TNF-alpha treatment with 5, 20 or 100 ng/ml, and 1.43 and 1.38
fold after 16 h of treatment with 20 or 100 ng/ml. The total amount of lipids secreted into
VLDL, as well as the lipid composition of each particle and the MTP transcript levels did not
change significantly with any of the TNF-alpha doses and periods of treatment tested. In
conclusion, this is another finding supporting for a correlation between the hepatocyte level of
apoB mRNA and the secretion of its protein without enhanced lipid secretion vinculated to a
sepsis-promoted cytokine.
Supported by grants G03/015, PE02UN04 and a personal grant to N.B.
- 286 -

VIII. Inflammation specific signaling Poster VIII, 3
Immune-complexes induce monocyte survival through defined intracellular pathways
Giordano Bianchi, Fabrizio Montecucco, Maria Bertolotto, Luciano Ottonello and
Franco Dallegri.
Clinic of Internal Medicine I, Department of Internal Medicine, University of Genoa
Medical School, Genoa, Italy
Regulation of inflammation is a crucial event since its alteration determines severe tissue
damage. The fine balance among several microenvironment components establishes the
resolution or perpetuation of inflammatory reactions. In a variety of diseases, immunecomplex
(IC) deposition induces an inflammatory response with tissue injury which is
mediated by inflammatory cell infiltration, proliferation, survival or apoptosis. In particular,
the interaction between ICs and the Fc gamma receptor triggers the activation of monocytes
and macrophages, favoring their tissue accumulation by inhibiting the apoptosis. To elucidate
the intracellular pathways governing this process, on the basis of our previous findings
regarding the dose-dependent inhibition of apoptosis in IC-activated monocytes, we have
investigated the role of phosphatidylinositol 3-kinase (PI3K)/Akt pathway showing its
involvement in survival of activated monocytes. As confirmation, molecular inhibitors of
PI3K and Akt suppressed the anti-apoptotic effect of ICs. In parallel we have demonstrated
the activation of the NF-kB system in triggered monocytes, directly by EMSA assay and
indirectly by showing the increased expression of X-IAP. Evenly NF-kB and IKK-b inhibitors
induced the apoptosis of activated monocytes at a rate comparable to that of resting
monocytes. In agreement with previous data showing the Fc triggered activation of MAPK
pathway, we have found that ICs induce the activation of erk1/2 and p38 conferring apoptosis
resistance to activated monocytes. Finally the activity of caspase 3, 8 and 9 resulted inhibited
in IC-activated monocytes. Even though further experiments are needed to identify a possible
link among these pathways, these results disclose a signaling route triggered by ICs which can
be involved in the pato-physiology of inflammatory diseases.
- 287 -

VIII. Inflammation specific signaling Poster VIII, 4
Modulation of Host Responses to Bacterial Endotoxin
Herbert Bosshart and Michael Heinzelmann
Innate Immunity Research Laboratory, Department of Surgery, Zurich University
Hospital, CH-8091 Zurich, Switzerland, e-mail: h_bosshart@bluewin.ch
The term sepsis describes a potentially lethal clinical condition that develops as a result of a
dysregulated host response to bacterial infection. The commonest bacterial component
implicated in initiating the septic syndrome is a cell wall molecule derived from Gramnegative
bacteria, known as lipopolysaccharide (LPS) or endotoxin. Like all mammals,
humans are equipped with an LPS-sensing machinery consisting, primarily, of LPS-binding
protein (LBP), CD14, a glycosylphosphatidylinositol-anchored monocyte differentiation
antigen, and Toll-like receptor 4 (TLR4), a signal transducing integral membrane protein.
Modest stimulation of TLR4 facilitates the elimination of invading microorganisms. Potent
TLR4 stimulation, however, produces severe reactions in the host, often leading to multiple
organ failure and death. The search for pharmaceuticals that reduce mortality in septic
patients has been a painstaking process. Thus far, only a few compounds have been found to
significantly reduce mortality rates. Perhaps one of the more promising therapeutic strategies
currently pursued is based on the identification of synthetic or naturally-occurring substances
that neutralize LPS or inhibit LPS-mediated activation of host immune cells such as
monocytes and macrophages. Here, we describe a number of divers molecular structures with
a capacity to either enhance or blunt LPS-induced monocyte activation. The underlying
molecular mechanisms are discussed.
- 288 -

VIII. Inflammation specific signaling Poster VIII, 5
CD3 activation alters T cell responses to LFA-1 engagement: regulation of T cell
polarization and migration
Eva M. Cernuda-Morollón1, Federica Marelli-Berg2 and Anne J. Ridley1.
(1) Ludwig Institute for Cancer Research-UCL Branch, 91, Riding House Street, W1W
7BS London, UK. eva@ludwig.ucl.ac.uk; anne@ludwig.ucl.ac.uk (2) Department of
Immunology, Division of Medicine, Imperial College London, Du Canne Road, London
W12 ONN, UK. f.marelli@imperial.ac.uk.
Recognition of cognate antigen by the T cell receptor induces T cell proliferation. The
recruitment of these activated T cells to and retention at sites of inflammation are crucial for
the inflammatory response and therefore play an important role in pathological conditions
such as cardiovascular disorders and chronic inflammation. T cell recruitment requires them
to cross the endothelium, a process that involves multiple receptors including the integrin
LFA-1 on T cells, which binds to ICAM-1 on endothelial cells. We have observed that T cell
receptor activation, mimicked by cross-linking with anti-CD3 antibody, inhibits T cell
transendothelial migration. CD3 activation also reduces T cell migration speed on ICAM-1.
This effect is not due to changes in T cell adhesive properties and correlates with an
impairment in T cell polarization, as revealed by staining for different markers, including
CD44, ICAM-3 and lipid raft markers. Rho GTPases regulate actin and microtubule dynamics
and therefore cell polarization and migration. We are investigating the activation status of the
Rho GTPases RhoA, Rac1 and Cdc42. Pre-treatment with CD3 antibodies increases Rac1
activity and modulates T cell responses to LFA-1 engagement. Furthermore, CD3 activation
promotes stathmin/Op18 phosphorylation and increases the levels of acetylated alpha-tubulin,
both of which indicate an increase in microtubule stability.
We propose that CD3 activation affects actin and microtubule dynamics, thereby impairing
normal T cell polarization and migration.
- 289 -

VIII. Inflammation specific signaling Poster VIII, 6
The stress-activated protein kinase pathway in dendritic cell activation.
Benjamin M. Chain, Matt Handley, Jane Rasaiyaah, Gabriel Pollara and David R Katz
Dept. Immunology and Molecular Pathology, UCL, London, UK E.mail :
b.chain@ucl.ac.uk
The activation and subsequent maturation of dendritic cells via Toll-like receptors (TLRs) is a
key step in the initiation of adaptive immunity. The canonical pathway requires activation and
nuclear translocation of NFkB transcription factor. However there is accumulating data that
activation of members of the stress-activated kinase p38 family is also necessary. In contrast,
the role of another family of stress activated kinases, the c-Jun N-terminal kinases (JNKs) , in
TLR signalling remains obscure.
Our laboratory has re-examined the function of the stress-activated protein kinase pathway in
human in vitro cultured monocyte-derived dendritic cells. Activation of dendritic cells with
high concentrations of lipopolysacharide (LPS, a TLR4 agonist) or poly I:C (TLR3 agonist)
induced both p38 and JNK phosphorylation. Low concentrations of these agonists activated
p38 but not JNK. In contrast zymosan (TLR2/dectin agonist) induced selective p38 but no
JNK activation at any concentration. The activation of p38 by LPS correlated with the degree
of dendritic cell maturation, while activation of JNK was associated with some cell death.
Both kinases were strongly induced by reactive oxygen (H2O2) species, but this was not
associated with any dendritic cell maturation, but rather with extensive dendritic cell
apoptosis. A selective competitive inhibitor of p38 blocked dendritic cell maturation induced
via TLR2, 3 or 4. In contrast a selective competitive inhibitor of JNK had no effect on
dendritic cell maturation, but partially protected dendritic cells from reactive oxygen induced
apoptosis.
Unexpectedly, an inhibitor of mixed linage kinases (MLKs) , previously considered to be
predominantly upstream activators of JNK activation, strongly inhibited dendritic cell
maturation in response to LPS. Dendritic cells were found to express several members of the
MLK family, and furthermore, both LPS and poly I:C induced MLK3 phosphorylation. In
contrast, zymosan induced no MLK3 phosphorylation. Inhibition of MLK activity resulted in
a block in maturation (as measured by cell surface phenotype) and cytokine secretion in
response to LPS and poly I:C, but not in response to zymosan.
In conclusion, we have documented selective activation of individual modules of the stress
activated kinase family in dendritic cells depending both on TLR ligand and signal strength.
This plasticity in molecular response may be crucial in allowing these key sentinels of the
immune system to optimise the quantitative and qualitative parameters of the ensuing immune
response.
Handley et al. Free Radic Biol Med. 2005 Jun 15;38(12):1637-52. Epub 2005 Mar 30. JNK
activation limits dendritic cell maturation in response to reactive oxygen species by the
induction of apoptosis.
- 290 -

VIII. Inflammation specific signaling Poster VIII, 7
Elastin peptides pro-inflammatory role in melanoma progression.
Romain Debret#§, Richard Le Naour§, Jean Michel Sallenave‡, William Hornebeck#,
Moncef Guenounou§, Philippe Bernard# and frank Antonicelli#
# Department of Dermatology, CNRS UMR 6198, § Department of Immunology, EA
3796, IFR 53 Biomolécules, University of Reims, Champagne-Ardenne, France. ‡ Rayne
Laboratories, Medical School, The University of Edinburgh, Edinburgh, UK.
E-mail: frank.antonicelli@univ-reims.fr
Background: Melanoma is a serious skin cancer. If not treated, it can be fatal. Melanoma is
not the most common cancer, but its incidence is increasing faster than any other. Although
surgery can be curative for early-stage melanoma, complete surgical excision of advanced
disease is often followed by recurrence. This is because surgery is a local treatment; it reduces
tumour burden (and tumour-associated immune suppression), but it does not eliminate tumour
cells that have entered the circulation and spread to distant sites. Prior to metastasize to the
regional nodes which are the single most important prognostic factor in solid neoplasms,
melanoma cells ought to degrade the surrounding extra-cellular matrix releasing therefore
protein fragments so-called matrikines because of their biological activities. Initial studies of
the “architecture” of the tumour front indicated certain structural changes such as elastin
fibres degradation in this front. We, then, focussed our work on the effects of these elastin
fragments on the complex interaction between immune system (lymphocytes) and melanoma
progression in the purpose to help to explain how certain tumours are more aggressive than
others.
Results: We investigated the contribution of EFs on IL-1 beta expression, a cytokine known
for playing a key role in melanoma cells activation. Our results first evidenced that high
tumorigenic melanoma cells (M3Da cells) treated with EFs led to IL-1 beta mRNA and
protein up-regulation. The effects of EFs on M3Da cells were found to be mediated by
receptor (S-gal) occupancy, as being suppressed by lactose and reproduced by cell stimulation
with the VGVAPG peptide. Binding of EFs to their receptor induced a rapid activation of Erk
& and p38 MAP Kinase pathways. However, activation of these pathways was not associated
with IL-1 beta mRNA up-regulation by EFs. Concomitantly, we demonstrated that EFs
stimulation induced NF-kB nuclear translocation (Western blot) and DNA binding on IL-1
beta promoter region (EMSA and ChIP techniques) whereas inhibition of NF-kB with the
specific chemical inhibitor SN-50 or by over-expression of IkB, the endogenous inhibitor of
NF-kB pathway, totally abolished EFs-mediated IL-1 beta mRNA over-expression. In setting
with IL-1 beta results, EFs also enhanced IL-8 and GRO-alfa expressions in melanoma cells.
However, increased concentration of IL-1 beta in culture media inhibited this EFs-induced IL-
8 expression. Interestingly, EFs also enhanced PHA-stimulated lymphocyte IL-1 beta
expression, but addition of this media on melanoma cells failed to activate IL-8 expression
compared to media from PHA-treated lymphocytes.
Conclusion: These data further illustrate that elastic fibres are important sources of matrikines
in pathologic conditions such as melanoma invasion by interfering in the relation
cancer/inflammation. Depending on their concomitant localisation with large amount of IL-1
beta, EFs could intervene in different stages of melanoma progression according to the
presence or not of infiltrated inflammatory cells in melanoma microenvironment.
- 291 -

VIII. Inflammation specific signaling Poster VIII, 8
Specific contribution of the signalling pathways NF-kappaB and PI3K / mTOR on the
gene expression profile of LPS stimulated murine macrophages
Sofia Dos Santos1, Anne-Isabelle Delattre, Françoise de Longueville2 and Martine
Raes1
1Laboratory of Biochemistry and Cellular Biology, University of Namur (F.U.N.D.P.),
Rue de Bruxelles, 61, 5000 Namur, Belgium. Email : martine.raes@fundp.ac.be,
2Eppendorf Array Technologies, Rue du Séminaire, 20a, 5000 Namur, Belgium.
Inflammation is the first response of the immune system to a variety of external or internal
insults, such as infectious agents, physical injury, hypoxia, or pathological processes.
In this study, we have simulated an inflammatory response by incubating RAW264.7 murine
macrophages with LPS (lipopolysaccharide), a major component of the outer membrane of
Gram-negative bacteria. The DNA microarray "DualChipTM Mouse Inflammation"
(Eppendorf) was used to characterize the gene expression profiles of LPS-stimulated
macrophages in the presence or not of BAY 11-7082 (a specific inhibitor of NF-kappaB) or
LY294002 (a specific inhibitor of the PI3K / mTOR pathway).
NF-kappaB is known to be a key transcription factor in the response to inflammation, but
little is known about the specific contribution of the PI3K / mTOR pathway in LPS signalling.
Results suggest a particularly important role for the transcription factor NF-kappaB, as
expected, but to a lesser extent the PI3K pathway is also involved. Differences between BAY
11-7082 and LY294002 were underlined. For example, genes encoding the PAF receptor,
PAI-1, PLA2 (group V), IL13 receptor (alpha 2) and GTP cyclohydrolase 1, which were upregulated
after LPS treatment, were mainly repressed by LY294002.
NF-kappaB plays an important role in the macrophage response to LPS, but we also have
shown that the PI3K pathway partially contributes to it. Further experiments with the specific
inhibitor of mTOR (rapamycin) will provide more information on the specific contribution of
the PI3K / mTOR pathway in the inflammatory response in LPS stimulated macrophages.
S. Dos Santos is a Research Fellow of FNRS (Fonds National de la Recherche Scientifique,
Brussels, Belgium).
- 292 -

VIII. Inflammation specific signaling Poster VIII, 9
Interleukin-31: Characterisation of signalling capacities and relevance for inflammatory
skin diseases
Alexandra Dreuw1, Barbara Lippok1, Mark M. Neis2, Bettina Peters3, Hans F. Merk2,
Andreas Bosio3, Jens M. Baron2, Peter C. Heinrich1 and Heike M. Hermanns1
1Institut für Biochemie and , Uniklinik der RWTH Aachen, Aachen, Germany;
2Hautklinik, Uniklinik der RWTH Aachen, Aachen, Germany
3Miltenyi Biotec GmbH, MACSmolecular Business Unit, Köln, Germany
Interleukin-31 is a recently identified cytokine which is produced by activated T lymphocytes,
preferentially by Th2 cells and to a minor extent by Th1 cells. It signals via a receptor
complex containing the specific IL-31 receptor and as the second signal transducing receptor
the oncostatin M-receptor #. In the present study we characterised in depth the molecular
mechanisms underlying IL-31R-mediated signal transduction. IL-31R is a strong activator of
STAT3 and STAT5, while STAT1 is only marginally tyrosine phosphorylated. We identify
tyrosine residues 652 and 721 in the cytoplasmic region of the longest isoform of IL-31R as
the major STAT5 and STAT3 activating sites, respectively. Additionally, we demonstrate
Jak1 binding to IL-31R and its activation in heteromeric complexes with the OSMR#, but
also in a homomeric receptor complex. Interestingly, unlike OSMR# and gp130, IL-31R is
insufficient to mediate ERK1/2 phosphorylation. We propose that this is due to a lack of
recruitment of the tyrosine phosphatase SHP-2 or the adapter protein Shc to the cytoplasmic
domain of IL-31R. Since transgenic mice overexpressing IL-31 develop a phenotype closely
resembling atopic dermatitis in humans we evaluated the potential importance of this novel
cytokine in the pathogenesis of T cell-mediated skin diseases. Confirming the results so far
obtained only in transgenic mice we find statistically elevated mRNA levels of IL-31 in
biopsies taken from patients with atopic dermatitis. However, no increased transcription of
IL-31 can be detected in biopsies taken from psoriatic plaques, which strengthens the
hypothesis, that IL-31 might be an important Th2-cytokine.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 542; TP B6 and
C11)
- 293 -

VIII. Inflammation specific signaling Poster VIII, 10
Mitochondrial uncoupling protein UCP2 regulates the LPS-induced ROS signaling in
innate imunity.
Yalin Emre, Corinne Hurtaud, Tobias Nübel, François Criscuolo, Daniel Ricquier, and
Anne-Marie Cassard-Doulcier
CNRS-UPR 9078, Faculté de Médecine Paris 5 Necker-Enfants Malades, 156 rue de
Vaugirard, 75730 Paris Cedex 15, France. E-mail : emre@necker.fr,
hurtaud@necker.fr, nuebel@necker.fr, criscuolo@wanadoo.fr, ricquier@necker.fr and
cassard-doulcier@necker.fr
Macrophages constitute the first line of defense against pathogens in innate immunity. The
recognition by Toll-like receptor-4 (TLR4) of lipopolysaccharides (LPS), a chief pathogenassociated
molecular pattern of Gram negative bacteria, triggers innate activation of
macrophages. Efficient activation is largely dependent upon the concomitant reactive oxygen
species (ROS) signaling. Since mitochondria is the main site of ROS production in resting
cells and the inner mitochondrial membrane protein UCP2 (uncoupling protein 2) is a
negative regulator of mitochondrial ROS production, we investigated a possible involvement
of UCP2 in macrophage innate activation and more precisely in the underlying ROS
signaling. We showed that UCP2 was quickly downregulated in macrophages in response to
LPS in order to increase the mitochondrial ROS production thus promoting MAPK
phosphorylation and activation. Consistent with this, UCP2-deficient macrophages have
increased nitric oxide production, cytokine release and their ablity to migrate. Based on our
data we conclude that the mitochondrial UCP2 plays an important role in pathogen-mediated
innate activation of macrophages by negatively regulating the LPS-induced ROS signaling.
- 294 -

VIII. Inflammation specific signaling Poster VIII, 11
Specific integrin alpha and beta chain phosphorylations regulate LFA-1 activation
through affinity-dependent and –independent mechanisms
Susanna C. Fagerholm, Susanna M. Nurmi, Tiina J. Hilden, and Carl G. Gahmberg§
Division of Biochemistry, Faculty of Biosciences, PB56 (Viikinkaari 5), 00014 University
of Helsinki, Finland
Integrins are heterodimeric adhesion receptors that are crucial for the functions of
multicellular organisms. The activation of integrin-mediated adhesion is a complex process
that involves both affinity regulation and cytoskeletal coupling, but the molecular
mechanisms have remained incompletely understood. The four members of the beta2integrins
are expressed exclusively on leukocytes and mediate adhesion and signaling events
that are essential for leukocyte function. In T cells, the main beta2-integrin is LFA-1
(alphaLbeta2-integrin), which can become activated after cell stimulation through the T cell
receptor, chemokine receptors or other signals. LFA-1 mediates crucial T cell functions like
the contact between the T cell and the antigen presenting cell and T cell extravasation from
the blood stream. Here, we report that phosphorylation of each cytoplasmic domain of the
leukocyte integrin LFA-1 mediates different modes of integrin activation. Alpha-chain
phosphorylation on Ser1140 is needed for conformational changes in the integrin after
chemokine- or integrin ligand-induced activation, or activation induced by active Rap1
(Rap1V12). In contrast, the beta-chain Thr758 phosphorylation mediates selective binding to
14-3-3 proteins in response to inside-out activation through the T cell receptor, resulting in
cytoskeletal rearrangements. Thus, site-specific phosphorylation of the integrin cytoplasmic
domains is important for the dynamic regulation of these complex receptors in cells.
- 295 -

VIII. Inflammation specific signaling Poster VIII, 12
Molecular characterization of synovial fibroblasts from rheumatoid arthritis and their
reponse to diverse anti-inflammatory drugs
Valerie Gossye1, Karolien De Bosscher1, Dirk Elewaut2 and Guy Haegeman1
1Laboratory for Eukaryotic Gene Expression and Signal Transduction (LEGEST),
Department of Molecular Biology, Ghent University, Gent, Belgium. E-mail:
valerie.gossye@ugent.be, karolien.debosscher@ugent.be, guy.haegeman@ugent.be;
2Laboratory for Molecular Immunology and Inflammation, Department of
Rheumatology, Ghent University Hospital, Gent, Belgium. E-mail:
dirk.elewaut@ugent.be
Objective. Inflammation is a central feature of many chronically developing inflammatory and
immune diseases, including rheumatoid arthritis (RA). Although the cause of RA is at present not
completely understood, previous studies have shown that fibroblast-like synoviocytes (FLS) as part of
a complex cellular network, are crucial for disease progression as well as for joint destruction.
To date, the goal of treatment is to reduce joint inflammation and pain, maximize joint function, and
prevent joint destruction and deformity. However, current treatment regimens suffer major drawbacks.
For instance, the usage of glucocorticoids (GCs) is overshadowed by severe side effects, such as
diabetes, glaucoma and osteoporosis. Furthermore, occurrences of resistance to the therapeutic effects
of corticosteroids, as well as disease reactivation after cessation of GC therapy, have been reported.
Therefore, there is a particular need for the development of a new generation of drugs to treat these
diseases.
Hence, we undertook a detailed study of the molecular characteristics of primary FLS isolated
from the inflamed synovium of RA patients and investigated whether anti-inflammatory drugs, such as
GCs and Compound A (CpdA), a fully dissociated compound of plant origin for inflammatory gene
repression (see De Bosscher et al. PNAS, 102, 15827-15832, 2005), exert their anti-inflammatory
effects on these effector cells and examined their putative mode of action.
Results and Conclusions
1. Immunofluorescence studies demonstrate that, upon dexamethasone (DEX) or CpdA stimulation for
1 hour, the glucocorticoid receptor (GR) shifts from the cytoplasm to the nucleus, indicating that GR
translocation proceeds normally.
2. RT-PCR results showed that in FLS93 and FLS118 TNF-induced Interleukin-6 mRNA expression
levels were repressed by DEX more adequately than by CpdA, but vice versa in FLS120. Along the
same line, CpdA was found to be more effective in downregulating RANTES in FLS93 and FLS120,
whilst in FLS118 DEX appeared to be more effective. These data suggest that different patients show
a different response/sensitivity towards different therapeutics. Furthermore, we can also conclude that
in the same FLS different cytokines are differently modulated by either therapeutic agent.
3. Finally, we observed in all investigated FLS a fast ‘homologous’ down-regulation of GR, already
apparent after 7 hours of DEX induction. In addition, Western blot analysis results showed that
induction of FLS with TNF, in combination with DEX, results in downregulation of IkappaB-alpha
protein expression, as opposed to the TNF-only condition. We thus confirmed previous results of our
group, demonstrating that in L929sA fibroblasts DEX stimulation does not lead to upregulation of
IkappaB-alpha protein expression (De Bosscher et al., PNAS, 94, 13504-13509, 1997).
Altogether, these findings highlight the complexity of molecular signaling cascades suitable for
therapeutic intervention that underly the chronic inflammatory and destructive processes in RA.
- 296 -

VIII. Inflammation specific signaling Poster VIII, 13
The JAK1 SH2 domain plays a structural role in the upregulation of OncostatinM
receptor surface expression
Serge Haan1*, Simone Radtke1*, Angela Jörissen1*, Heike M. Hermanns1, Sandra
Dieffenbach1, Tanja Smyczek1, Peter C. Heinrich1, Iris Behrmann2* and Claude
Haan1,2*
1 Institut für Biochemie, Uniklinik Aachen, Pauwelsstraße 30, 52074 Aachen, Germany
2 Laboratoire de Biologie et Physiologie Intégrée, Fac. Sciences Techno. & Com.,
Université du Luxembourg, 162A avenue de la Faïencerie, L-1511 Luxembourg
*contributed equally
The Janus kinase family of protein tyrosine kinases comprises four mammalian members.
Three, Jak1, Jak2 and Tyk2, are expressed in a wide variety of tissues, whereas Jak3
expression is restricted to cells of the haematopoietic system. Jak1 is involved in signal
transduction of the interferons IFN" and IFN$ and many other cytokines such as IL-6-type
cytokines (IL-6, OSM, LIF, IL-11, CNTF, CT-1, CLC) signal via the receptor chains gp130,
LIFR and OSMR. All these signal transducing receptors have been described to bind Jak1,
Jak2 and Tyk2. Among these, Jak1 is essential for signal transduction. Interestingly the
surface expression of the OncostatinM receptor (OSMR) has been described to be dependent
on Jak binding. Recent data also suggest that Jak1 and gp130 form an indissociable complex
which might best be compared to a receptor tyrosine kinase. It is still under debate which
functional domains exist in the Jaks and the interplay of these domains in kinase activation is
not well understood. Although the presence of an SH2 domain is discussed since the first
decription of Jaks elaborate functional studies have not yet been published.
In the present study we investigated the functionality of the Jak1 SH2 domain using an
inactivating mutant of the Jak1 SH2 domain, Jak1-R466K. No effect on cytokine signal
transduction could be observed. The R466K mutant also had no effect on receptor binding, on
subcellular distribution as well as on Jak1-mediated upregulation of the OSMR. However, the
SH2 domain seems to be structurally important to ensure cytokine receptor binding and
surface expression.
(This work has been supported by the Deutsche Forschungsgemeinschaft (SFB542 TP B1 and
HA3433/1) and the Fonds der Chemischen Industrie (Frankfurt am Main, Germany))
- 297 -

VIII. Inflammation specific signaling Poster VIII, 14
Real-time analysis of Jak/STAT signal transduction in single cells
Andreas Herrmann 1 , Michael Vogt 1 , Martin Mönnigmann 2 , Bernd Giese 1 , Michael
Sommerauer 1 , Peter C. Heinrich 1 , Gerhard Müller-Newen 1
1 Institut für Biochemie, Universitätsklinikum der RWTH Aachen, Pauwelsstraße 30,
52057 Aachen, Germany. e-mail: mueller-newen@rwth-aachen.de
2 Lehrstuhl für Prozesstechnik, RWTH Aachen, 52056 Aachen, Germany
The Jak/STAT signal transduction pathway plays a central role in acute and chronic
inflammation. The pro-inflammatory cytokine interleukin-6 (IL-6) signals through the
cytokine receptor gp130. Upon activation, the receptor-associated tyrosine kinases of the
Janus kinase (Jak) family phosphorylate STAT transcription factors. STAT3 (signal
transducer and activator of transcription 3) is preferentially activated in response to IL-6.
Activated STAT3 translocates into the nucleus where it induces target genes that exert
multiple functions in inflammation and carcinogenesis.
We have tagged the cytokine IL-6, its receptor gp130 and the transcription factor STAT3 with
different fluorescent proteins (yellow fluorescent protein (YFP) or cyan fluorescent protein
(CFP)). These fusion proteins enabled us to study ligand-binding and receptor dimerization
(Giese et al. (2005) J. Cell. Sci. 118, 5129-40) as well as nuclear translocation of STAT3
(Pranada et al. (2004) J. Biol. Chem. 279, 15114-23) in single cells by the use of confocal
laser-scanning microscopy and advanced photo-bleaching techniques. We demonstrated that
non-phosphorylated STAT3 constitutively shuttles between the cytoplasm and the nucleus.
Persistent activation of STAT3 is observed in chronic inflammation and cancer. To analyze
persistent activated STAT3 we cotransfected double labelled STAT3-CFP-YFP with v-Src
resulting in constitutive tyrosine phosphorylation and nuclear accumulation of the fluorescent
transcription factor. By bleaching selectively the YFP moiety of STAT3-CFP-YFP in one
cellular compartment and by monitoring the distribution of the CFP and YFP fluorescence
over time with high spatial resolution, we show that persistently activated STAT3 shuttles
between cytoplasm and nucleus. Computational evaluation of the data by model-based
parameter estimations revealed that activated STAT3 shuttles more rapidly than non-activated
STAT3. We propose that inhibition of nucleocytoplasmic shuttling of persistently activated
STAT proteins is a new target for the treatment of chronic inflammation and cancer.
- 298 -

VIII. Inflammation specific signaling Poster VIII, 15
Sulforaphane inhibits phorbol ester-induced COX-2 expression in human breast
epithelial cells: I!B kinase # as a potential target
Ha-Na Kim, Eun-Hee Kim, Hye-Kyung Na, Young-Joon Surh
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
Sulforaphane (SFN; 1-isothiocyanato-4-(methylsulfinyl)-butane), occurring naturally as a
precursor glucosinolate in cruciferous vegetables, is a member of the isothiocyanate family of
chemopreventive agents. Recent studies suggest that inflammation is causally linked to
carcinogenesis. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of
prostaglandins, is inappropriately expressed in various cancers. In the present study, we
investigated the effect of SFN on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced
COX-2 expression and underlying molecular mechanisms in human breast epithelial (MCF-
10A) cells. Treatment of MCF-10A cells with SFN (12.5 and 25 µM) significantly reduced
TPA-induced COX-2 expression. The DNA binding of NF-!B and degradation of IkappaBalpha
(I!B-") were suppressed by SFN in TPA-stimulated MCF-10A cells. SFN dosedependently
inhibited TPA-induced I!B kinas # (IKK#) activity. In addition, SFN inhibited
the phosphorylation of extracellular signal-regulated kinase (ERK) and the activity of ERK
and p38 mitogen-activated protein kinase (MAPK) in TPA-treated MCF-10A cells. U0126
and SB203580 that are specific inhibitors of ERK and p38 MAPK, respectively blunted TPAinduced
COX-2 expression. In conclusion, SFN inhibited TPA-induced COX-2 expression
and NF-!B activation in MCF-10A cells by targeting IKK# and MAPKs.
- 299 -

VIII. Inflammation specific signaling Poster VIII, 16
Redirecting adenovirus encoding dominant-negative inhibitor kappaB to inflamed
endothelial cells inhibits endothelial gene expression
J.M. Ku#do1, S.A. Asgeirsdottir1, A.R. Bellu2, M.G. Rots2, K.I. Ogawara3, T. Peneder1,
C. Trautwein4, H. Haisma2, and G. Molema1
University Medical Center Groningen, University of Groningen, The Netherlands;
1Department of Pathology and Laboratory Medicine, Medical Biology Section,
Hanzeplein 1, 9713 GZ Groningen, j.m.kuldo@med.umcg.nl; 2Department of
Therapeutic Gene Modulation; 3Department of Pharmaceutics, Faculty of
Pharmaceutical Sciences, Okayama University, Japan; 4Medical School of Hanover,
Germany
In glomerulonephritis endothelium expresses inflammatory molecules responsible for harmful
leukocyte infiltration. We propose that inhibition of expression of a broad spectrum of genes
by endothelial cells will inhibit disease progression by strong suppression of leukocyte
recruitment into the kidney. To accomplish this multiple gene inhibition, we used an
adenovirus encoding a therapeutic transgene that interferes with nuclear factor kappaB
signaling, a major pathway involved in inflammatory gene expression control. For retargeting
to the activated endothelial cells, we introduced endothelial cell-specific anti-E-selectin or
anti-VCAM-1 antibody-polyethylene glycol (PEG) modifications and studied effects of
constructs in endothelial and messangial cells in vitro, as well as in vivo in mouse model of
glomerulonephritis. In vitro retargeted adenoviruses selectively infected cytokine-activated
endothelial cell and no transgene was detected in (activated or non-activated) messangial
cells. E-selectin directed adenovirus was able to deliver more transgene to activated
endothelium than VCAM-1 directed virus. In vivo, PEGylated, anti-E-selectin-retargeted
adenovirus selectively homed to glomeruli in glomerulonephritis, resulting in downregulation
of inflammatory genes at two days after start of disease. These studies demonstrate the
potential of tropism-modified adenovirus to deliver therapeutic genes to endothelial cells in
inflammation.
- 300 -

VIII. Inflammation specific signaling Poster VIII, 17
Novel Serine Protease-Induced Signaling in Human Peripheral Monocytes
Yves Laumonnier, Tatiana Syrovets, Thomas Simmet
Department of Pharmacology of Natural Products & Clinical Pharmacology, University
of Ulm, D-89081 Ulm, Germany. E-mail: yves.laumonnier@uni-ulm.de
Besides its important role in fibrinolysis, plasmin is a potent proinflammatory serine protease
activating human peripheral monocytes. Plasmin induces lipid mediator release, expression of
proinflammatory genes and chemotaxis. However, the exact nature of its receptor in
monocytes remains elusive. In this context we have now investigated the role of annexin A2
described as a surface protein involved into the binding of the inactive zymogen plasminogen.
In contrast to neutrophils, primary monocytes respond to plasmin and express the annexin A2
heterotetramer consisting of annexin A2 and S100A10 as shown by Western immunoblot,
flow cytometry, immunofluorescence microscopy and co-immunoprecipitation. Using a
biotin-labeled tri-functional plasmin cross-linker, we were able to biotinylate both annexin A2
and S100A10 suggesting that plasmin is indeed binding to the subunits of the annexin A2
heterotetramer. To assess the role of the annexin A2 heterotetramer as a receptor for plasmin
signaling, plasmin-induced chemotaxis was analyzed after treatment with neutralizing
antibodies. Antibodies directed against either annexin A2 or S100A10 impaired the plasmininduced
signaling but not that induced by FMLP. Similarly, antisense oligodeoxynucleotides
(ODN) directed against annexin A2 or S100A10, but not the respective sense controls,
reduced the plasmin-induced chemotaxis, but not that to FMLP. Consistently, monocytes
pretreated with antisense ODN showed a decreased release of the proinflammatory cytokine
tumor necrosis factor (TNF)-alpha confirming that annexin A2 and S100A10 are essential for
the plasmin-induced signaling. At the molecular level plasmin induced cleavage of the
annexin A2 heterotetramer at position 27 as demonstrated by point mutation. Finally, we
found that the plasmin-mediated cleavage triggers dissociation of the annexin A2
heterotetramer.
These data identify the annexin A2 heterotetramer as a signaling receptor for plasmin in
human peripheral monocytes. The dissociation of the heterotetramer after the plasmininduced
cleavage of annexin A2 is apparently the molecular initiator for the plasmin-induced
proinflammatory signaling in human peripheral monocytes.
- 301 -

VIII. Inflammation specific signaling Poster VIII, 18
Molecular mechanisms underlying inflammatory responses to Helicobacter pylori
infection
Jeong-Sang Lee1, Ki-Baik Hahm2, Jeffrey A. Johnson3, and Young-Joon Surh1
1National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University. Seoul 151-742, 2Genomic Research
Center for Gastroenterology, School of Medicine, Ajou University, Suwon 442-749,
South Korea, and 3School of Pharmacy, University of Wisconsin, Madison, WI, USA
The Helicobacter pylori (H. pylori) were identified by Marshall and Warren in 1983. The H.
pylori survive in the forbidding harsh acid environment of the stomach and duodenum by
hiding in the mucus layer and neutralizing stomach acid in its local surrounding environment.
Multiple lines of evidences suggest that H. pylori infection is one of the primary causes of
gastritis and peptic ulcer, which are provoked by oxidative stress and inflammation. More
than 50% of the world's population is infected by this bacterium. The H. pylori–induced
inflammation has been implicated in the pathogenesis and progression of gastric cancer. In the
present study, we investigated molecular mechanisms responsible for H. pylori-induced
inflammation in human gastric cancer (AGS) cells. H. pylori induced inflammatory response
accompanied up-regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase
(iNOS), and both the secretion and protein expression of interleukin-8 (IL-8) in AGS cells.
The nuclear translocation and subsequent DNA binding of nuclear factor-kappaB (NF-!B), a
eukaryotic transcription factor known to regulate aforementioned pro-inflammatory enzymes
and mediators, were increased after H. pylori treatment. Similarly, the DNA binding of
eukaryotic transcription factor activator protein-1 (AP-1) was induced by H. pylori treatment.
In addition, c-Jun that is the major component of AP-1 was detected by super shift assay, and
the level of phospho-c-Jun was found to be increased. H. pylori infection accelerated the
DNA binding of another transcription factor CREB, whose interacting partners were detected
as CREB and CBP as determined by the super shift assay. In another experiment, H. pylori –
infected AGS cells exhibited increased phosphorylation of upstream kinases, ERK and AKT.
However, long term administration of H. pylori resulted in the activation of proliferating cell
nuclear antigens (PCNA) and Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End
Labeling (TUNEL) in C57BL6 mice, suggesting that H. pylori infection may create a balance
between proliferation and apoptotic process in the stomach. Taken together, H. pylori
treatment can cause the inflammatory response through up-regulation of proteins involved in
pro-inflammatory signal transduction.
- 302 -

VIII. Inflammation specific signaling Poster VIII, 19
Lack of association of the cyclooxygenase-2 and inducible nitric oxide synthase gene
polymorphism with risk of cervical cancer in Korean population.
Taek Sang Lee a,b, Jae Weon Kima,b, Noh Hyun Parka,b, Soon Beom Kanga,b, Hyo
Pyo Leea,b, Yong Sang Songa,b*
aDepartment of Obstetrics and Gynecology, bCancer Research Institute, College of
Medicine, Seoul National University, Seoul, 110-744, Korea
E-mail: yssong@snu.ac.kr
In recent years, plenty of studies have shown that nitric oxide (NO) and prostaglandin (PG) as
the main inflammatory mediators are involved in various pathophysiological processes
including inflammation and carcinogenesis. In this study, we have explored the impact of
polymorphisms of two representative inflammatory mediators on the risk of cervical cancer.
This study includes 176 cases of histologically confirmed invasive cervical cancer and 172
healthy controls. Allele frequency of twelve SNPs of COX-2 and 5 of iNOS gene in 43
normal populations were analyzed. 14 of 17 shows monomorphic or minimal minor allele
frequency, and therefore we did not perform further analysis for them. Three SNPs ( 2 for
COX-2, 1 for iNOS) were thus chosen for the study. Genotyping of three SNPs (SNP-rs5275
in the untranslated region of exon 10 and rs5277 in the coding region of exon 3 of COX-2,
and iNOS Ser608Leu allele C/T polymorphism within exon 16 of the iNOS reductase
domain) was performed. No significant increase was found in any genotype of the COX-2 and
iNOS in cancer group. We analyzed the correlation between the genotypes and the
clinicopathologic parameters of cervical cancer. No significant association of genotype was
found with respect to each phenotype including clinical stage, lymph node (LN) status,
histologic type, or parametrial invasion. In conclusion, our data shows the lack of significant
evidence of association of the COX-2 and iNOS polymorphisms with cervical cancer in
Korean population.
- 303 -

VIII. Inflammation specific signaling Poster VIII, 20
Helicobacter pylori induced IFN gamma production in CD8- NK cells
Åsa Lindgren, Åsa Sjöling & Samuel Lundin
Department of Medical Microbiology and Immunology, Göteborg University,
Medicinaregatan 7A, 413 90 Göteborg, Sweden. E-mail: asa.lindgren@microbio.gu.se
Helicobacter pylori is a gram-negative bacterium which causes a chronic infection of the
gastric and duodenal mucosa and is associated with both development of ulcers and an
increased risk of gastric adenocarcinomas.
Natural killer (NK) cells are lymphocytes that play a critical role for the innate immunity
against viral and bacterial infections as well as against tumour cells. NK cells are present in
the mucosa of both healthy and H. pylori infected individuals and because of their ability to
secrete IFN gamma (IFNg), the presence of NK cells in the gastric mucosa may indicate that
this cell type is involved in the immune response to H. pylori infection.
It has been shown that there are two different subpopulations of NK cells; CD8+ and CD8-
and we found that CD8- dominates in the stomach mucosa. Our attention is thus directed
towards elucidating how CD8- and CD8+ NK cells respond to H. pylori and if there are any
differences between the two populations.
Stimulations of NK cells with H. pylori have revealed that only the CD8- subpopulation
seems to respond with IFNg production. Inhibition of MAPK p38 and of PI3K revealed
opposite effects in CD8- NK cells, where inhibition of p38 gave rise to decreased and
inhibition of PI3K gave rise to increased IFNg secretion. We have also detected a degradation
of the NFkB inhibitor IkBa upon stimulation of NK cells with H. pylori, which strengthens
the hypothesis that the above mentioned signalling cascades are associated with the immune
response to
H. pylori. Upstream recognition of H. pylori and initiation of the response are also studied.
We are currently studying Toll-like receptors (TLRs) that are bacterial recognition receptors
that might be involved in the induction of IFNg production.
In conclusion, we have shown that CD8- NK cells produce IFNg in response to H. pylori
stimulation, in a p38-dependent manner. We believe that elucidation of the signalling
pathways in NK cells activated by H. pylori will be important for the further study of the
development of H. pylori-induced diseases.
- 304 -

VIII. Inflammation specific signaling Poster VIII, 21
Insulin primes human neutrophils for CCL3-induced migration: crucial role for JNK.
Fabrizio Montecucco, Giordano Bianchi, Luciano Ottonello, Maria Bertolotto, Giorgio
L. Viviani and Franco Dallegri.
Clinic of Internal Medicine I, Department of Internal Medicine, University of Genoa
Medical School, I-16132 Genoa, Italy. E-mail: fabriziomontecucco@tiscali.it
Recent studies showing that neutrophil infiltration in unstable atherosclerotic lesions is
associated with plaque rupture have suggested a pathogenetic role for neutrophils in
atherosclerosis. Nevertheless, the mechanisms regulating the recruitment of neutrophils at
sites of atherosclerotic lesions are still unknown. In this regard, it has been recently shown
that insulin is involved in actin reorganization of neutrophil locomotory response. Thus, we
studied the effects of > 50 microU/ml insulin, i.e. concentrations occurring in
hyperinsulinemic conditions characterized by an increased atherosclerotic risk, on neutrophil
migration to CCL3, a chemokine produced in atherosclerotic plaques and considered to be
incapable of attracting human neutrophils. Neutrophils from healthy volunteers became
capable of migrating to CCL3 after a short (15 min) exposure to 300 microU/ml insulin.
Accordingly, although no [Ca2+]i mobilization was observed in response to CCL3 or insulin,
the combination of insulin and CCL3 stimulation results in a well detectable [Ca2+]i spike.
Furthermore, the exposure of neutrophils to serum from hyperinsulinemic and euglycaemic
adult patients with metabolic syndrome (as defined by ATP III and WHO criteria), induced
neutrophil responsiveness to CCL3. A strict correlation (p:

VIII. Inflammation specific signaling Poster VIII, 22
Streptococcus pneumoniae-induced p38 MAPK and NF-kappaB dependent COX-2
expression in human lung epithelium
P. Dje N´Guessan, S. Hippenstiel, M. O. Etouem, J. Zahlten, W. Beermann, D. Lindner,
B. Opitz, M. Witzenrath, S. Rosseau, N. Suttorp, B. Schmeck
Department of Internal Medicine/Infectious Diseases, Charité – Universitätsmedizin
Berlin, 13353 Berlin, E-mail: Stefan.hippenstiel@charite.de
S. pneumoniae is a major cause of community-acquired pneumonia and death due to
infectious diseases in industrialized countries. Cyclooxygenase (COX) derived prostaglandins
like prostaglandin E2 (PGE2) are considered as important regulators of lung function. Herein
we tested the hypothesis that pneumococci induced COX-2 dependent PGE2 production in
pulmonary epithelial cells. Pneumococci infected human pulmonary epithelial BEAS-2B cells
released PGE2. Expression of COX-2 but not COX-1 was dose- and time-dependently
increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with
pneumococcal pneumonia. S. pneumoniae induced degradation of IkappaBalpha and DNAbinding
of NF-kappaB. Specific inhibition of the IKK kinase complex blocked pneumococciinduced
PGE2 release and COX-2 expression. p38 MAP kinase and cJun-NH2-terminal
kinase (JNK) were activated in pneumococci infected cells. PGE2 release and COX-2
expression was reduced by p38 MAP kinase-inhibion but not by JNK-inhibition. Dominant
negative mutants of p38 MAP kinase isoforms alpha, beta2, gamma, and delta blocked S.
pneumoniae-induced NF-kaapB activation. In addition, recruitment of NF-kappaB subunit
p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAP kinase but not
on JNK activity. In summary, p38 MAP kinase and NF-kappaB controlled COX-2 expression
and subsequent PGE2 release by lung epithelial cells may contribute significantly to the host
response in pneumococcal pneumonia.
Supported by German Federal Ministry of Education and Research, Competence Network
CAPNETZ.
- 306 -

VIII. Inflammation specific signaling Poster VIII, 23
Non-receptor tyrosine kinases play a role in zymosan (yeast) induced macrophage
activation
Sandra Olsson and Roger Sundler
Macrophage Signaling, Exp. Med. Science, Lund University, BMC B12, 221 84 Lund,
Sweden. E-mail: Sandra.Olsson@med.lu.se, Roger.Sundler@med.lu.se
Macrophages play an essential role in defending us against microorganisms, by phagocytosis
and by production of proinflammatory mediators, such as TNF-" and eicosanoids. The
proinflammatory mediators also play a role in chronic inflammation. It is therefore of
importance to study the signaling to regulation of their production.
Fungi are typical microorganisms causing opportunistic infections and zymosan beads,
carbohydrate-rich cell wall preparations from Saccharomyces cerevisea, are often used to
study the interaction of yeast with the innate immune system. Members of the Src family
kinases are known to play a critical role in many signaling pathways, with a putative role in
inflammation. We therefore studied their role in zymosan-induced activation of macrophages
using different Src family kinase inhibitors. Our results demonstrate that Src kinases play
differential roles in both arachidonate release for eicosanoid formation and TNF-"
production. Zymosan may signal through several receptors and the postulated receptors
include mannose receptor, Toll-like receptor 2 (TLR2), #-glucan receptors (Dectins) and
SIGNR1. We show that the Dectin-1 receptor is involved in zymosan signaling to arachidonic
acid release, while the receptor used for TNF-" formation may be different. Tec kinase, a
downstream kinase of Src, also seems to be involved in zymosan-induced activation of
macrophages, since it is phosphorylated upon zymosan stimulation and arachidonate release is
reduced after inhibition of Tec. MAP kinases are able to contribute to the production of
eicosanoids and TNF-" and we show here that inhibition of Src kinases, but not Btk,
decreased the zymosan-induced phosphorylation of MAP kinases. These data indicate that
non-receptor tyrosine kinases such as the Src family kinases and Btk play a role in zymosaninduced
formation of inflammatory mediators in macrophages.
- 307 -

VIII. Inflammation specific signaling Poster VIII, 24
Microsomal prostaglandin E synthase-1 expression induced by TNFalpha does not
involve PKC or p38 MAP kinase in gingival fibroblasts
Tove Olsson, Tomomi Kawakami and Tülay Yucel-Lindberg
Department of Pediatric Dentistry, Karolinska Institutet, Box 4064, SE-141 04
Huddinge, Sweden. E-mail: tove.olsson@ki.se; tomomi.kawakami@ki.se;
tulay.lindberg@ki.se
Prostaglandin E2 (PGE2) plays a pivotal role in several inflammatory conditions including
rheumatoid arthritis and periodontitis. The inducible microsomal prostaglandin E synthase-1
(mPGES-1) is the recently identified terminal enzyme regulating the synthesis of PGE2. We
have previously reported that the cytokine tumor necrosis factor alpha (TNFalpha) induces the
expression of mPGES-1 in human gingival fibroblasts. In this study we further investigated
the regulation of mPGES-1 with special regards to protein kinase C (PKC) and p38 MAP
kinase (p38 MAPK) in relation to PGE2. The results showed that TNFalpha induced the
expression of mPGES-1 in gingival fibroblasts accompanied by increased PGE2 production.
Treatment of the cells with the PKC activator PMA (phorbol-12-myristate-13-acetate)
increased the production of PGE2 as well as upregulated the stimulatory effect of TNFalpha,
without increasing mPGES-1 expression. In addition, the PKC inhibitor Bisindolylmaleimide
markedly decreased the TNFalpha-induced PGE2 production but not mPGES-1 expression.
Furthermore, the p38 MAPK inhibitor SB203580 caused an abrogation of the TNFalphainduced
PGE2 production whereas mPGES-1 expression remained unaffected. These findings
indicate a lack of co-regulation of mPGES-1 and its product PGE2 regarding PKC and p38
MAPK signal pathways in human gingival fibroblasts.
- 308 -

VIII. Inflammation specific signaling Poster VIII, 25
The effect of Epidermal Growth Factor (EGF) implantation in incisional wounds of
rabbits on leucocyte chemotaxis
Nesrin Özsoy 1, $ule Co%kun 2, Nursel Gül 1
1 Department of Biology, Faculty of Science, Ankara University, 06100, Tando&an,
Ankara, Turkey. E-mail: ozsoy@science.ankara.edu.tr
2 Department of Biology, Faculty of Science and Art, Gazi University, 06500 Be%evler,
Ankara, Turkey.
Epidermal growth factor (EGF) is mitogenic for many cells of mesodermal and ectodermal
origin. It is a polypeptide that increases ion change, glycolysis, RNA and DNA production
and wound healing in effected cells. It especially promotes cell migration into the wound area.
EGF plays a fundamental role in the process of wound repair by stimulating chemotaxis. In
this study, the impact of EGF to the leucocyte chemotaxis at the wounded tissues has been
studied. Mucous membranes of rabbits were wounded by incision. EGF formulation was
submucosally localized and afterwards, the wound was closed with double suture. The
significance of this study is that it is the first wound model applied to the mucosa of rabbits.
Oral implantation of EGF is original in this wound model. In this research, New-Zealand male
rabbits were used weighting to 2.5 ± 0.4 kg. After submucosal incisions were made, the
rabbits were divided into three groups. 1- Only incision group 2- polyethylene glycol (PEG)
implanted group 3-EGF into PEG implanted wounds. On the 5th day of operation, the rabbits
were killed by excess of sodium pentobarbitale anesthesia and the wounded tissue was
immediately removed for Transmission Electron Microscope (TEM) preparation. The tissues
were embedded into araldite and blocked. Semi-thin and thin sections were prepared from the
blocks, and leucocyte chemotaxis was investigated. It was observed that, in the connective
tissues of the “incision only” group, leucocyte cells were in minority. Leucocytes
accumulated in the connective tissues of the group that received both incision and EGF
application. That all leucocytes were observable attracted attention. It was found that EGF
stimulated leucocyte chemotaxis during wound healing and attracted all leucocytes to the
healing area.
- 309 -

VIII. Inflammation specific signaling Poster VIII, 26
Regulations of Notch2, 3, 4 and Dll1 expression by TNF" in Endothelial Cells are
specifically dependant on PI3K, NFkB and JNK MAPK pathways.
Thibaut Quillard, Stéphanie Coupel, Juliette Fitau and Béatrice Charreau
INSERM, U643, Nantes, France ; ITERT, Institut de Transplantation Et de Recherche
en Transplantation, CHU, Nantes, France ; Université de Nantes, UFR de médecine,
Nantes, France. E-mail : thibaut.quillard@univ-nantes.fr
Notch signaling pathway is a key player in cell communication through molecular cell/cell
interactions. Vertebrates express multiple Notch receptors (Notch1 - 4) and ligands including
Delta-like (Dll) (1-4) and Jagged 1 & 2. Notch plays a role in adult in several contexts
involving cell plasticity including vascular morphogenesis and remodeling. However,
regulation of the Notch receptors and ligands in vascular endothelial cells (EC) upon
inflammation remains unknown. AIM : In the present study, we investigated the expression
and regulation of Notch receptors and ligands in EC upon activation with TNF" in vitro and
in vivo. METHODS & RESULTS : Expression of Notch receptors and ligands was
investigated in primary cultures of human EC, treated with TNF", by quantitative RT-PCR
and Western Blotting. We show that TNF" down-regulated Notch1,3,4 and up-regulated
Notch2 and the ligand Dll1. A concomitant decrease in mRNA level for effector genes Herp1
and Hes1 was observed reflecting a reduced activity of Notch signaling. Similar regulations
were obtained in HUVEC and vascular SMC. Moreover, comparison with IL1", IFN$ and
VEGF indicate a regulation pattern specific of inflammatory stimuli. In EC, TNF" activates
several signaling pathways including the PI3Kinase (PI3K), NF!B and JNK MAPK
pathways. The respective involvement of these signaling pathways in Notch regulations
mediated by TNF" in EC was examined. Our findings suggest that TNF"-mediated
regulation of Notch2 transcription was dependant of PI3K as changes for Notch3, 4 and Dll1
mRNA levels involved NF!B and JNK MAPK. Notch regulations were further confirmed in
vivo, in rats treated with TNF"..
CONCLUSION : Our results show for the first time that inflammation triggers a dysregulated
expression of Notch molecules in endothelium that could contribute to EC/immune cells
interactions and promote vascular remodeling as observed in chronic inflammatory disorders.
- 310 -

VIII. Inflammation specific signaling Poster VIII, 27
Melphalan reduces the severity of experimental colitis in mice by blocking tumor
necrosis factor-alpha signaling pathway
Galina V. Shmarina, Alexander L. Pukhalsky, Vladimir A. Alioshkin and Alex
Sabelnikov
Research Centre for Medical Genetics, Moscow 115478, Gabrichevsky Institute of
Epidemilogy and Microbilogy, Moscow 125212, Russia, East Carolina University,
Greenvill, NC 27858, U.S.A. E-mail: osugariver@medgen.ru
In acute DSS-induced colitis nuclear factor (NF)-kappaB-dependent inflammatory cytokines
including tumor necrosis factor (TNF)-alpha are known to be up-regulated. We examined the
effects of alkylating drug melphalan on the sensitivity of mouse fibroblasts to TNF-alphainduced
cytitoxicity in vitro and on intestinal inflammation and NF-kappa B activation in
vivo. In vitro, low concentrations of melphalan protected fibroblastoid cells (L929) against
TNF-alpha-mediated apoptosis. The cells were pre-incubated with 0.0001 mg/ml of
melphalan for 1 h and then treated with recombinant TNF-alpha. A significant decrease in the
number of dead cells in comparison to the control (TNF-alpha treated cells without preincubation
with melphalan) was observed. In vivo, daily administration of melphalan
markedly reduced the severity of murine experimental colitis as determined by clinical and
quantitative histological criteria. Male BALB/c mice were exposed to 5 % (w/v) dextran
sulfate sodium (DSS) dissolved in drinking water for 5 days with or without daily melphalan
injections (0.025 mg/kg body weight, i.p.). Time-matched controls consisted of mice received
melphalan only, and those injected with phosphate buffered saline. At day 6 the mice were
sacrificed and the general signs of colitis had been evaluated. DSS-mice exhibited a
significant loss of the intestine length, pronounced elevation in blood white cells and marked
increase in relative spleen weight. Such changes were not so evident in DSS-mice treated with
melphalan. Histological analysis of colon biopsies showed that melphalan protected against
both the neutrophil infiltration and mucosal destruction. Furthermore, colonic NF-kappaB
DNA-binding activity, up-regulated in DSS-induced colitis, was suppressed by melphalan
treatment. Taken together, these results suggest that alkylating drug melphalan applied in noncytotoxic
concentrations may reduce the severity of experimental colitis in mice by blocking
TNF-alpha signaling pathway.
- 311 -

VIII. Inflammation specific signaling Poster VIII, 28
Experimental sepsis: characteristics of activated macrophages and apoptotic cells in the
rats spleen
Helle E. Simovart1, Andres Arend1, Kersti Kokk1, Helle Tapfer1, Marina Aunapuu1,
Elle Poldoja1, Gunnar Selstam2, Aade Liigant1
1Department of Anatomy, University of Tartu, Biomedicum , 19, Ravila St. Tartu
50411, Estonia. E-mail : helle-evi.simovart@ut.ee
2Department of Molecular Biology, University of Umea, Sweden
Sepsis, being characterized by massive translocation of bacteria into tissues, induces the
suppression of the function of both leukocytes and macrophages.
The aim of the study was to count activated macrophages (AM) and apoptotic (Ao) cells in
the rat spleen during the period of experimental sepsis and to clarify the associations of these
parameters with the leukocyte migration and the bacterial translocation into different organs.
The Wistar rats were intraperitoneally inoculated with E. coli and were sacrificed after 2, 6,
24, 48 and 120 hours. Bacteria and leukocytes in tissues were specifically stained. AM were
identified by immunohistological staining and Ao cells were detected with the «In Situ Cell
Death Detection Kit».
The high counts of E. coli were strongly associated with a low level of the total counts of
leukocytes at 6h, accompanied by the high translocation of microbes and migration of
leukocytes into tissues. In the spleen, the count of AM was highest at 24h after the inoculation
with E. coli, at the same time the Ao cell count began to rise and achieved the highest level 24
h later when the AM amount has diminished.
In conclusion, our investigation indicates that the molecular peculiarities of macrophages and
their response to the inflammation process are tissue-specific. In the spleen the activation
process was remarkable at the late stage of sepsis accompanied by a high count of apoptotic
cells, the process which involved the haematopoietic cells, localized in the spleen as
macrophages themselves.
- 312 -

VIII. Inflammation specific signaling Poster VIII, 29
Angiotensin II Type 1 Receptor as a target for the resolution of disease induced
wounding
Gary R. Smith
Chemistry Department, The Open University, Walton Hall, Milton Keynes MK7 6AA,
UK.
E-mail : gary.smith@persescomms.com
The critical role of inappropriate inflammation is becoming accepted in many diseases,
including cardiovascular diseases, inflammatory and autoimmune disorders,
neurodegenerative conditions, infection and cancer.
A review of laboratory and clinical evidence demonstrates that cancer up-regulates the
angiotensin II type 1 (AT1) receptor through oxidative, hypoxic and steer stress mechanisms,
thereby triggering a wound response that remodels surrounding tissue and subdues the
immune system.
With regard to cancer being a systemic disease, an examination of supporting evidence for a
systemic role of AT1 in relationship to the wound response is presented as a logical
progression. The evidence suggests that regulation of the mutually antagonistic angiotensin II
receptors (AT1 and AT2) is an essential process in the management of ‘chronic’ inflammation
and wound recovery, and that an imbalance in the expression of these receptors will lead to
progress in disease conditions.
Manipulation of the angiotensin system with existing anti-hypertensive drugs has been found
to have therapeutic effect in clinical studies in type 2 diabetes, arteriosclerosis, renal disease,
sarcoidosis and hormone refractory prostate cancer. It is anticipated that in the foreseeable
future Angiotensin Receptor Blockade will provide a new approach to the treatment of many
of the diseases that afflict mankind.
- 313 -

VIII. Inflammation specific signaling Poster VIII, 30
A role for Src and receptor tyrosine kinases but not for Janus kinases in SOCS3 tyrosine
phosphorylation
Ulrike Sommer1, Christine Schmid1, Radoslaw M. Sobota1, Ute Lehmann1, James A.
Johnston2, Fred Schaper1, Peter C. Heinrich1 and Serge Haan1
1 Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen,
Pauwelsstrasse 30, D-52074 Aachen, Germany
2 Dept. Immunology, Queen's University Belfast, 97 Lisburn Rd., Belfast BT9 7BL,
Northern Ireland
The suppressors of cytokine signalling (SOCS) are negative feedback inhibitors of cytokine
signal transduction. SOCS3 is a key negative regulator of IL-6 signal transduction.
Furthermore, SOCS3 was shown to be phosphorylated upon treatment of cells with IL-2 and
this has been reported to regulate its function and half-life. We set out to investigate whether
SOCS3 phosphorylation may play a role in IL-6 signalling. Tyrosine phosphorylated SOCS3
was detected upon treatment of mouse embryonic fibroblasts (MEF) with IL 6. Interestingly,
the observed SOCS3 phosphorylation does not require SOCS3 recruitment to phosphotyrosine
pY759 of gp130 and the kinetics of SOCS3 phosphorylation do not match the activation
kinetics of the Janus kinases. This suggests that other kinases may be involved in SOCS3
phosphorylation. Using Src and Janus kinase inhibitors as well as Src kinase deficient MEF
cells, we provide evidence that Src kinases, which we found to be constitutively active in
these cells, are involved in the phosphorylation of IL-6 induced SOCS3. In addition, we found
that receptor tyrosine kinases (RTKs) such as PDGFR or EGFR can very potently
phosphorylate IL-6 induced SOCS3. Taken together, these results suggest that SOCS3
phosphorylation is not a JAK-mediated phenomenom but is dependent on the activity of other
kinases such as Src kinases or RTKs which can either be constitutively active or activated by
an additional stimulus.
This work has been supported by the Deutsche Forschungsgemeinschaft (HA3433/3) and the
Fonds der Chemischen Industrie (Frankfurt am Main, Germany).
- 314 -

VIII. Inflammation specific signaling Poster VIII, 31
Increased Cyclooxygenase-2 Expression Associated with Inflammatory Cellular
Infiltration in Elderly Patients with Vulvar Cancer
Taek Sang Lee a,b, Jae Weon Kima,b, Jae Kyung Wonc, Noh Hyun Parka,b,
In Ae Parkc, Soon Beom Kanga,b, Hyo Pyo Leea,b, Yong Sang Songa,b*
aDepartment of Obstetrics and Gynecology, bCancer Research Institute, cDepartment
of pathology, College of Medicine, Seoul National University, Seoul, 110-744, Korea
E-mail: yssong@snu.ac.kr
As the relationship between inflammation and carcinogenesis grows stronger, the role of
cyclooxygenase-2 (COX-2), and epidermal growth factor (EGFR) has been highlighted in the
pathogenesis and progression of human cancer. In view of the fact that vulvar cancer is
characterized by pre-cancerous inflammatory changes in elderly patients, the expressions of
COX-2 and EGFR are expected to show different pattern of distribution according to age and
other prognostic factors. To verify whether there was a relationship between their expressions
and clinico-pathologic parameters in vulvar cancer, we investigated the inflammatory cellular
infiltration and the expression of COX-2 and EGFR and by immunohistochemical analysis.
Eleven of 19 samples (57.8%) were stained positive for COX-2, and 17(89.4%) for EGFR.
The portion of inflammatory cellular infiltration in adjacent normal tissue was also higher in
old age group and showed strong correlation with COX2 positivity (p=0.002). Furthermore,
COX-2 expression was significantly more frequent in over 60 year old group than in under 50
(p=0.009). COX-2 revealed high expression in the moderately and well differentiated cases,
whereas, poorly differentiated carcinoma were negative (p=0.023). On the other hand, EGFR
expression was not differently distributed according to stage, age, tumor grading, or presence
of lymph node metastasis. Our study suggests that vulvar cancer in elderly patients may be
associated with inflammation, and thus with increased COX-2 expression. In light of these
data, a clinical trial designed to assess the addition of COX-2 targeted therapy to conventional
treatment in vulvar cancer seemed worthwhile, avoiding serious surgical morbidity in the
elderly patients.
- 315 -

VIII. Inflammation specific signaling Poster VIII, 32
Role of the host body cytokine TNF-alpha in the mechanism of bacterial growth
activation.
Alexandra A. Tomova, Yulia M. Romanova, Alexandr L. Gintsburg
Laboratory of Genetic Engineering of Pathogenic Microorganisms, The Gamaleya
Research Institute of Epidemiology and Mycrobiology, Russian Academy of Medical
sciences, 18 Gamaley street, Moscow 123098, Russia; e-mail: altomova@abv.bg
Resting or nonculturable microbes are thought to represent about 60% of the biomass on
Earth. How to stimulate such microbes to exit from dormant state may aid the culturing of
such organisms. Why and how nonculturable forms of bacteria resuscitate and propagate in
the host body with a lot of defense factors of immunity? The aim of this work was to identify
the role of the host body cytokine TNF-" in the mechanism of bacterial growth activation in
vitro and in vivo. During evolutional coexistence of micro- and macroorganisms pathogenic
bacteria developed the adaptive mechanisms that allowed them to utilize even immune
defense factors (cytokines) as growth activation factors. We studied the dynamics of the
reproduction of Salmonella vegetative forms and the resuscitation of Salmonella
nonculturable forms in infected animals. We demonstrated that the increase in the level of
TNF-" in host body leads to the activation of the Salmonella cell’s growth. We have
discovered that the incubation of Salmonella cells in vitro in a media complemented with
TNF-" leads to a higher growth rate of bacterial cells. To realize the mechanism of
pathogenic bacteria activation in host body we investigated the growth rates of vegetative and
resuscitation of Salmonella nonculturable forms after action of TNF-". Direct interaction of
the bacteria with cytokines in vitro during incubation the cells with TNF-" must be occurring
through the bacterial receptor and the increase in the bacterial growth rate in the presence of
cytokines must be a result of cytokine induced bacterial cell gene and protein expression. By
the method of molecular display we identified several genes that were significantly up
regulated in the presence of TNF-". By the proteome analysis of S. typhimurium cultures
incubated with/without addition of TNF-" we have shown the increase expression of some
proteins. We identified the protein that might participate in the binding of Salmonella to
cytokine in vitro. The investigation of mechanisms of reproduction and growth activation of
culturable and nonculturable forms of pathogenic bacteria in host body is very important and
actual problem.
- 316 -

VIII. Inflammation specific signaling Poster VIII, 33
Receptor interacting protein RIP1, a crucial initiator of inflammation and necrotic cell
death
Authors: Tom Vanden Berghe, Miki Kalai, Nele Festjens, Jill Demeulemeester, Kathleen
D’Hondt and Peter Vandenabeele
Molecular Signaling and Cell Death Unit, Department of Molecular Biomedical
Research, VIB, Ghent University, Technologiepark 927, 9052 Zwijnaarde, Belgium
Receptor interacting protein (RIP1) is recruited to tumor necrosis factor-" receptor 1
(TNFR1) complex upon stimulation and known to play a crucial role in the receptor-induced
NF-!B activation. Interaction of RIP1 with the scaffold protein Hsp90 protects the adaptor
kinase from proteasome-dependent degradation. We showed that pretreatment of L929
fibrosarcoma cells with the Hsp90 inhibitor geldanamycin, induces a shift of TNF-induced
necrosis to apoptosis, suggesting a crucial role of RIP1 also in necrotic signaling. Using an
RNA interference approach, we demonstrate that RIP1 is crucial for the initiation of necrosis.
Moreover, retroviral transduction of kinase-death mutants shows that RIP1-induced necrosis
is dependent on its kinase activity. The importance of RIP1 has also been shown in FasL- and
dsRNA-induced caspase-independent cell death. Analogous to the formation of the
apoptosome, one can propose the formation of a “necrosome-complex” in which the
activation of RIP1 is a crucial event. We are currently trying to unravel the activation
mechanism of RIP1 and how RIP1 recruitment finally triggers downstream execution
mechamisms.
- 317 -

VIII. Inflammation specific signaling Poster VIII, 34
ABIN-3 is a novel LPS/TNF-inducible inhibitor of NF-kB activation
Lynn Verstrepen, Andy Wullaert, Sofie Van Huffel&, Mira Haegman, Matthew
Sanders, Karen Heyninck and Rudi Beyaert
Unit of Molecular Signal Transduction in Inflammation, Department for Molecular
Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent
University, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium, E-mail:
lynnv@dmbr.ugent.be
& Current address: Membrane Biology Lab (HWJ), Institute of Molecular and Cell
Biology, 61 Biopolis Drive, Proteos, Singapore 138673
The transcription factor NF-kB is responsible for upregulation of different genes involved in
inflammation, immune responses, differentiation, proliferation and apoptosis. Dysregulation
of NF-kB activation plays an important role in a number of diseases like asthma, Crohn’s
disease, and rheumatoid arthritis. Therefore, a tight regulation of the NF-kB signaling
pathway is necessary. Via in silico cloning, we identified a novel protein (termed ABIN-3)
that shows partial sequence homology to ABIN-1 and ABIN-2, which were previously
identified in our group as NF-kB modulatory proteins. Similar to the other ABIN’s, ABIN-3
bound the ubiquitin-editing protein A20 and inhibited NF-kB activation induced by TNF, IL-
1 and TPA upon overexpression. ABIN-3 was also found to inhibit the LPS/TLR4-initiated
pathway to NF-kB. Unlike the other ABIN’s, ABIN-3 expression could only be detected
upon stimulation with LPS and to a lesser extend with TNF. Furthermore, we found a NF-kB
site in the promoter of ABIN-3. Luciferase-reporter and gelretardation assays showed that the
ABIN-3 promoter was activated upon stimulation with TNF or LPS in a NF-kB dependent
way. Taken togheter, these results implicate ABIN-3 as a novel negative feedback regulator
of LPS-/TNF- induced NF-kB activation.
- 318 -

VIII. Inflammation specific signaling Poster VIII, 35
Stimulus Specificity of Gene Expression Programs Determined by Temporal Control of
IKK activity
Shannon L. Werner, Derren Barken, and Alexander Hoffmann
Signaling Systems Laboratory, Department of Chemistry and Biochemistry, University
of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0375, USA
Email: slwerner@ucsd.edu; dbarken@ucsd.edu; ahoffmann@ucsd.edu
The transcription factor nuclear factor-kappa B (NF-kB) regulates genes having critical roles
in cellular stress responses, adaptive and innate immunity, lymphoid organ development, cell
growth, survival, and apoptosis. Misregulation of NF-kB plays a role in chronic
inflammatory diseases and cancers. Many different stimuli induce the NF-kB, including
inflammatory cytokines (TNF, IL-1), bacterial endotoxin (LPS), oxidative stress, UVirradiation,
and chemotherapeutic drugs. This begs the question: how do different stimuli
elicit unique gene expression programs by activating the same signaling pathway? One
possibility is that the same signaling pathway can transmit temporally distinct signals that
have different physiological effects.
NF-kB-inducing stimuli utilize specific signaling pathways that converge at IKK (inhibitor of
kappaB kinase). Do different stimuli result in unique IKK activity profiles? Do these, in
turn, generate temporally distinct, stimulus-specific nuclear NF-kB activity and gene
expression profiles? This study provides evidence that the temporal control of IKK is
modulated by stimulus-specific signal processing mechanisms that play a role in determining
downstream specificity in gene expression. While TNF-induced IKK activity is rapidly
attenuated by negative feedback by the A20 protein, LPS-signaling and LPS-specific gene
expression programs are dependent on a cytokine-mediated positive feedforward mechanism.
- 319 -

VIII. Inflammation specific signaling Poster VIII, 36
T cell aggregation induced through CD43: Intracellular signals and inhibition by the
immunomodulatory drug leflunomide.
Esther Layseca-Espinosa1,2, Gustavo Pedraza-Alva1, José Luis Montiel1, Roxana del
Río1, Nora A. Fierro1, Roberto González-Amaro2, and Yvonne Rosenstein1.
1Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca,
Mor., and 2Department of Immunology, Facultad de Medicina, Universidad Autónoma
de San Luis Potosí, San Luis Potosí, S.L.P., México
The CD43 co-receptor molecule has been shown to participate in lymphocyte adhesion and
activation. Leukocyte homotypic aggregation results from a cascade of intracellular signals
delivered to the cells following engagement of different cell surface molecules with their
natural ligands. This phenomenon requires an active metabolism, reorganization of the
cytoskeleton and relocalization of cell surface molecules. The aim of this study was to
identify some of the key members of the signaling cascade leading to T lymphocyte
homotypic aggregation following CD43 engagement. CD43-mediated homotypic aggregation
of T lymphocytes required the participation of Src kinases, PLC-$2, PKC, PI3-K as well as
ERK1/2 and p38. Data shown here suggest that these signaling molecules play a central role
in regulating actin cytoskeleton remodeling after CD43 ligation. We also evaluated the ability
of immunomodulatory drugs such as leflunomide to block the CD43-mediated homotypic
aggregation. Leflunomide blocked the recruitment of targets of the Src family kinases as well
as actin polymerization, diminishing the ability of T lymphocytes to aggregate in response to
CD43 specific signals, suggesting that this drug might control the migration and recruitment
of lymphoid cells to inflamed tissues.
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Notes
- 321 -

Notes
Notes
- 322 -

- 323 -

Session IX. Novel compounds targeting inflammatory
signaling pathways
- 324 -

Session IX. Novel compounds targeting inflammatory signaling pathways Poster IX, 1
Oxidized phospholipids reduce the vascular leak and inflammation in the rat model of
LPS-induced acute lung injury
1Anna A. Birukova, 2Stephanie Nonas, 1Joe G.N. Garcia and 1Konstantin G. Birukov
1Department of Medicine, University of Chicago, Chicago, Illinois 60637, U.S.A. Emails:
abirukov@medicine.bsd.uchicago.edu, jgarcia@medicine.bsd.uchicago.edu,
kbirukov@medicine.bsd.uchicago.edu, 2Department of Medicine, Johns Hopkins
University, Baltimore, Maryland 21224, U.S.A. E-Mail: snonas1@jhmi.edu
Acute inflammation and vascular leak are cardinal features of acute lung injury (ALI) and the
acute respiratory distress syndrome (ARDS). Non-specific tissue inflammation and injury in
response to a variety of infectious and non-infectious insults leads to oxidative stress and the
generation lipid oxidation products. We showed that, besides antagonistic effects on toll-like
receptor 4 (TLR4) which triggers LPS-induced inflammatory cascade, oxidized 1-palmitoyl-
2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) enhances lung endothelial cell
(EC) barrier via activation of small GTPases Rac and Cdc42. Using EC cultures and rat
models of lipopolyscharide (LPS)-induced lung injury, we tested the hypothesis that OxPAPC
may affect acute lung inflammatory response to LPS and recover lung vascular barrier
properties. LPS induced 200-fold increase in broncho-alveolar lavage (BAL) cell count
(p

Session IX. Novel compounds targeting inflammatory signaling pathways Poster IX, 2
NAC prevents cardiac maladaptative responses to hypertension and promotes TNFR-2
signaling in cardiomyocytes
Nicole Defer1, Gabriele Candiani1, Jin-Bo Su2, Marie Bourraindeloup2, Luc Hittinger2,
Françoise Pecker1 and Catherine Pavoine1
1INSERM U581; Université Paris 12, Faculté de Médecine; Créteil, F-94000 France;
2INSERM U660; Université Paris 12, Faculté de Médecine; Créteil, F-94000 France;
The aim: Our previous in vivo studies showed that N-acetylcysteine (NAC) was a potential
therapeutical tool for the management of hypertension–induced heart failure. However, early
mechanisms of NAC action remained to be defined.
Results and Methods: Rats were given a four week treatment with the nitric oxide synthase
inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg/day in drinking water) and
high salt diet (HS, 8% NaCl). HS/L-NAME rats displayed hypertension-induced adaptative
responses, characterized by thickening of ventricular walls and eccentric hypertrophy of
cardiomyocytes, without left ventricular dysfunction. This was associated with changes in two
early biochemical markers of adaptative cardiac responses, namely decrease in beta-1adrenergic
(AR) stimulation of adenylyl cyclase (AC) and increase in the number of the type
1 TNF receptors (TNFR-1) over TNFR-2. NAC treatment (125 mg/kg/day in drinking water)
given during weeks 3 and 4 of HS/L-NAME administration, had no effect on hypertension,
but triggered a proportional hypertrophy of cardiomyocytes and prevented alterations of both
beta-1-AR/AC signaling and TNFR-1/TNFR-2 ratio. Imaging studies performed on
electrically stimulated cardiomyocytes isolated from control rats treated for 2 weeks with
NAC, showed that NAC blunted TNF- induced production of ROS and its associated negative
effect on Ca transient amplitude. Neutralizing anti-TNFR-1 antibodies mimicked these effects
of NAC in cardiomyocytes isolated from control untreated rats. Furthermore, NAC
uncovered a strong enhancing effect of TNF on Ca transients, that was associated with
phosphorylation of Erk, Akt and PKC zeta and activation of the cPLA2. These TNF effects
were impaired by anti TNFR-2 antibodies.
Conclusion: Our results highlighted NAC as a tool favouring TNFR-2 while blunting TNFR-1
signaling pathways, and counteracting early maladaptative responses to hypertension.
- 326 -

Session IX. Novel compounds targeting inflammatory signaling pathways Poster IX, 3
Wogonin prevents immunosuppressive action but not anti-inflammatory effect induced
by glucocorticoid
Riyo Enomoto1, Chie Suzuki1, Chika Koshiba1, Takayuki Nishino1, Mikiko
Nakayama1, Hiroyuki Hirano2, Toshio Yokoi2 and Eibai Lee1.
1Department of Pharmacology and 2Department of Pharmaceutical Chemistry, Faculty
of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe
651-2180 Japan. E-mail : enomoto@pharm.kobegakuin.ac.jp
Glucocorticoid such as dexamethasone has anti-inflammatory and immunosuppressive action
as major pharmacological effects. The latter action caused by lymphocyte apoptosis is not
only therapeutic effect but also adverse reaction. Wogonin, a plant flavone, inhibited
dexamethasone-induced apoptotic changes such as DNA fragmentation, nuclear condensation,
phosphatidylserine exposure and caspase activation in rat thymocytes. Since wogonin
inhibited the dexamethasone-induced DNA fragmentation in the noncompetitive manner, a
target of this flavone is unlikely to the glucocorticoid receptor. This flavone also protected
apoptosis induced by other glucocorticoids. Since wogonin reduced one of major
pharmacological effects of dexamethasone, we examined whether this flavone diminishes the
anti-inflammatory action, another pharmacological effect. The anti-inflammatory action was
measured by the carrageenan-induced edema model. Although dexamethasone significantly
suppressed paw edema induced by carrageenan, wogonin had no effect on the antiinflammatory
action of dexamethasone. These results suggest that wogonin may be an useful
compound to reduce the immunosuppressive side effect of glucocorticoids.
- 327 -

Session IX. Novel compounds targeting inflammatory signaling pathways Poster IX, 4
The Cyclooxygenase-2 Selective Inhibitor Celecoxib Suppresses matrix
Metalloproteinase and Inhibits in Vitro Invasion of the Human Oral Squamous
Carcinoma cells
Young E. Kwak, Nam K. Jeon, Jin Kim and Eun J. Lee
Department of Oral Pathology, Oral Cancer Research Institute, BK21 project for
Medical Science, Yonsei University College of Dentistry, Seoul, KOREA, 120-752
E-mail: e16lee@yumc.yonsei.ac.kr
Cyclooxygenease-2(COX-2) expression is a critical factor in inflammation, and plays an
important role in defense against exogenous stimuli, while overexpression of COX-2 causes
cells to exhibit changes in tumor phenotype. Recently, celecoxib was shown to reduce the risk
of development of cancer and mortality from it. Tumor metastasis is the most important cause
of cancer death. The present study attempted to determine the effects of celecoxib with regard
to human oral squamous cell carcinoma(OSCC) cell growth and invasion/migration. The YD-
10B cells represented a highly-invasive human oral squamous cell carcinoma(OSCC) cell line
which was found to express the COX-2 protein. Celecoxib inhibited cell invasion/migration
through the type 1 collagen matrix by ~ 60% within 24 hours. Gelatin-based zymography
assay reveal that, in the presence of 10uM celecoxib, both MMP-2 and MMP-9 enzyme
activity decreased by ~25-30%. The current in vitro study indicated that the inhibition of
invasion/migration in OSCC cell lines by the cyclooxgenase-2 specific inhibitor, celecoxib,
results in anticancerous effects via a MMP-2 and MMP-9 suppression. In addition, YD-10B
cells could be useful to study the pathological mechanism of OSCC. (This study was
supported by the BK21 project for Medical Science, and by a grant of the national Cancer
control R&D Program 2003(No. 0320230), Ministry of Health & Welfare, Republic of
Korea.)
- 328 -

Session IX. Novel compounds targeting inflammatory signaling pathways Poster IX, 5
Inhibition of ICAM-1-mediated metastasis by thalidomide in human lung cancer
Yi-Chu Lin and Ching-Chow Chen
Department of Pharmacology, College of Medicine, National Taiwan University,
Taipei 10018, Taiwan. E-Mail: f92443016@ntu.edu.tw
Our previous study demonstrated that TNF-alpha-induced expression of Intercellular
Adhesion Molecule-1 (ICAM-1) not only increased monocyte adherence to the lung epithelial
cells, but also elicited tumor cell invasion. In this report, we examined the role of ICAM-1 in
carcinogenesis and the effect of thalidomide. The in vitro cell invasion and the in vivo tumor
metastasis induced by ICAM-1-overexpressing A549 cells were both inhibited by
thalidomide. The tumors grown in nude mice induced by s.c. human lung cancer xenografts
were detected to express ICAM-1 and attenuated by oral administration of thalidomide (200
microg/kg/day). Moreover, highly expressed ICAM-1 was detected in the human specimens
of lung cancer patients. The inhibitory effect of thalidomide on the TNF-alpha-induced
protein and mRNA expressions of ICAM-1 was seen, and this inhibition attributed to the
reduced activity and binding of NF-kappaB to the ICAM-1 promoter. However, IkappaBalpha
degradation and translocation of NF-kappaB to the nucleus were not affected by
thalidomide. These studies provide a framework for targeting ICAM-1 gene by thalidomide as
a biologically based therapy for lung cancer.
- 329 -

Session IX. Novel compounds targeting inflammatory signaling pathways Poster IX, 6
Statins selectively upregulate TNFR2 expression in primary human endothelial cells
Tobias Nübel1*, Steffen Schmitt2 and Gerhard Fritz1
1 fritz@mail.uni-mainz.de, Department of Toxicology, University of Mainz, Obere
Zahlbacher Str. 67, D-55131 Mainz, Germany and 2 facslab@uni-mainz.de, University
of Mainz, Naturwissenschaftlich-Medizinisches Forschungszentrum, Obere Zahlbacher
Str. 67, D-55131 Mainz, Germany
* Current address: nuebel@necker.fr, CNRS-UPR 9078, Faculté de Médecine Necker-
Enfants malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are
clincally widely used as lipid-lowering drugs. Apart from their cholesterol lowering capacity,
statins exert pleiotropic effects which are clearly independent from lipid-lowering. Besides
influencing angiogenesis, thrombosis, apoptosis and metastasis-related mechanisms, an antiinflammatory
effect is also ascribed to statins. The cytokine tumor necrosis factor alpha
(TNFalpha) plays a key role in inflammatory processes. Upon binding of TNFalpha to its
receptors TNFR1 and TNFR2 the activity of the transcription factor NF-kB gets stimulated
leading to transcriptional activation of genes involved in the regulation of inflammatory
processes.
Here we addressed the question of whether modulation of TNFR expression might be a
molecular mechanism of statins contributing to their anti-inflammatory potency. To this end,
we investigated the effect of the HMG-CoA reductase inhibitor lovastatin on the cell surface
expression of TNFRs in primary human endothelial cells (HUVEC). We showed by ELISA,
FACS and immunocytochemical analyses that lovastatin selectively upregulates TNFR2
expression in HUVEC without affecting the cell surface expression of TNFR1. This effect of
lovastatin appears to be independent of statin mediated inhibition of cell cycle progression
from G1 to S-phase since cells both in G1 and G2 phase showed elevated levels of TNFR2
expression after lovastatin treatment. Analyzing TNFalpha provoked induction of the cell
adhesion molecule E-selectin as an important endothelial inflammatory stress response, we
observed a dose dependent promoting effect of lovastatin on the TNFalpha stimulated
increase in the frequency of E-selectin positive endothelial cells.
This finding indicates that lovastatin sensitizes HUVEC towards TNFalpha induced signaling
mechanisms by upregulation of TNFR2 expression. Based on our data we therefore suggest
that statins have impact on endothelial responses to inflammatory stress by modulating the
expression of cytokine receptors.
- 330 -

Session IX. Novel compounds targeting inflammatory signaling pathways Poster IX, 7
DiC14-amidine cationic lipid induces the production of IL-12 by murine bone-marrow
derived dendritic cells: importance of TLR-4 and JNK
Tetsuya Tanaka1), Michel Vandenbranden2), Jean-Marie Ruysschaert2) and Alain
Jacquet1).
1)Service de Génétique Appliquée, Institut de Biologie et de Médecine Moléculaires,
Université Libre de Bruxelles, B-6041 Gosselies, Belgium and 2)Laboratoire "Structure
et Fonction des Membranes Biologiques," Université Libre de Bruxelles, Campus
Plaine, B-1050 Brussels, Belgium E-mail : tetsuyat2003@hotmail.com
DiC14-amidine cationic lipid has been shown to act as a Th1-adjuvant. A recombinant form
of the major mite allergen Der p 1 (ProDer p 1) complexed with diC 14-amidine prevented the
development of typical Th2-biased allergic response in mice. The present study investigates
the effect of diC14-amidine on murine bone-marrow-derived dendritic cells (BMDC) and
particularly at the level of the pro-Th1 cytokine IL-12 production.
BMDC exhibited IL-12 but not IL-10 secretion when stimulated with diC14-amidine,
complexed or not with ProDer p 1. The recombinant allergen did not induce cytokine
production. Using BMDCs from TLR4- and TLR2-deficient mice, we clearly demonstrated
that diC14-amidine stimulation of dendritic cells involved TLR4 but not TLR2. Although all
three MAP kinases, ERK1/2, p38 and JNK were activated, diC14-amidine induced IL-12
release exclusively via the JNK signaling pathway. Finally, IL-12 release induced by the
cationic lipid correlated with degradation of IkB, a critical step in NF-kB activation. In
conclusion, the activation of dendritic cells by the complex ProDer p 1-amidine leading to IL-
12 production could explain the development of Th1-biased immune response by this vaccine.
- 331 -

Notes
- 332 -

Session X : Cell death in cancer
- 333 -

Session X : Cell death in cancer Poster X, 1
Rosiglitazone antagonizes IGF-I effects and induces growth arrest and apoptosis in
anaplastic cancer cells
Aurora Aiello, Giuseppe Pandini*, Francesco Frasca*, Marco Genua, Antonella
Murabito, Riccardo Vigneri*, Antonino Belfiore
Dip. di Medicina Clinica e Sperimentale, University of Catanzaro, Policlinico Mater
Domini, via T. Campanella 115. 88100 Catanzaro, Italy and *Dip. di Medicina Interna e
Medicine Specialistiche, University of Catania, Catania, Italy. E-mail: belfiore@unicz.it
Anaplastic thyroid cancer (ATC) is an extremely aggressive tumor characterized by resistance
to chemotherapy and a very poor prognosis. PPAR-$ agonists have recently emerged as
potential antineoplastic drugs. In order to establish whether ATC could be a target of PPARgamma
agonists we examined PPAR-gamma protein expression in a panel of six ATC cell
lines and then studied the biologic effects of a PPAR-gamma agonist, rosiglitazone, currently
used as antidiabetic drug. PPAR-gamma protein was present and functional in all ATC cell
lines. Rosiglitazone induced complex biological effects in ATC cells, including inhibition of
anchorage dependent and independent growth and migration, and increased apoptosis rate.
Moreover, rosiglitazone antagonized the mitogenic and anti-apoptotic effect of IGF-I. These
effects of rosiglitazone were associated with up-regulation of the lipid phosphatase PTEN and
inhibition of the PI3K/AKT signaling pathway. Moreover, rosiglitazone induced changes in
cell cycle regulators, such as an increase of cdk inhibitors p21cip1 and p27kip1, a decrease of
cyclin D1 and inactivation of Rb protein and changes in apoptosis regulators, such as a
decrease of Bcl-XL expression and caspase 3/7 activation.
In conclusions, these data suggest that rosiglitazone induces multiple beneficial effects in
ATC cells, among which up-regulation of PTEN and inhibition of IGF-I effects, and may
have, therefore, a role in the multimodal therapy currently used to slow down ATC growth
and dissemination.
This work was partially supported by grants from AIRC and MIUR (Cofin 2003) to A.B.
- 334 -

Session X : Cell death in cancer Poster X, 2
Induction of apoptosis and activation of p53-dependent and independent pathways by a
new mithramycin analogue
Veronica Albertini1, Sara Vignati1, Sara Napoli1, Jurgen Rohr2, Giuseppina M.
Carbone1 and Carlo V. Catapano1
1Laboratory of Experimental Oncology, IOSI, Bellinzona, CH and 2Department of
Pharmaceutical Sciences, University of Kentucky, Lexington, KY
Aureolic acid antibiotics, like chromomycin and mithramycin (MTM), bind selectively to
GC-rich DNA and block preferentially activity of GC-rich DNA binding transcription factors,
like Sp1. Combinatorial biosynthesis approaches applied to the MTM producer S. Argillaceus
have recently yielded new analogues that exhibit greater transcriptional inhibitory activity and
might have an improved therapeutic index compared to MTM. A new compound, MTM SDK,
down-regulated transcription of multiple genes implicated in cancer pathogenesis, inhibited
growth of a variety of human cancer cell lines and was a potent inducer of apoptosis. SDK
was equally active against cancer cell lines with wild type or mutated p53. Interestingly, the
pro-apoptotic activity of SDK in cancer cells was not associated with significant toxicity in
normal fibroblasts. Although the activity of SDK was apparently p53-independent, we
investigated whether the p53 pathway could modulate cell responses to SDK and MTM in
p53wt cells. Both compounds increased p53 protein level by a post-transcriptional
mechanism. Activation of p53 by MTM resulted in up-regulation of p21, while the level of
p21 did not increase in SDK-treated cells due to inhibition of p21 transcription. Increased
levels of p21 attenuated the apoptotic response to MTM, while its absence in SDK-treated
cells was associated with increased apoptosis. The protective role of p21 could also explain
the limited cytotoxicity of SDK in normal fibroblasts, in which SDK did not block p21
induction. Thus, SDK is a potent antiproliferative and pro-apoptotic agent due to its ability to
interfere with critical growth and survival genes. SDK activity is also modulated by p53dependent
pathways in p53wt cells. The ability of SDK to simultaneously activate p53 and
block p53-dependent activation of p21 favors apoptosis in cancer cells and contributes to its
greater activity compared to MTM. On the other hand, in normal fibroblasts p53-dependent
up-regulation of p21 can have a protective role and contribute to the reduce toxicity of SDK.
- 335 -

Session X : Cell death in cancer Poster X, 3
Comaprative Proteomics of Ovarian Epithelial Tumors
!
Hee Jung An, Nam Hoon Cho, Park, Choi YP, Kang S, Ding O, Kim DS
Dept of Pathology, Bundang CHA Medical Hosiptal
Dept of Pathology, Yonsei Univresity College of Medicine
Brain Korea 21 Project for Medical Science, Yonsei University!!
!
We analyzed twelve ovarian epithelial tumors using 2D PAGE-based comparative proteomics
to construct intra- and inter-tumoral distance map trees, and to discover surrogate biomarkers
indicative of an ovarian tumor. The analysis was performed after laser microdissection of 12
fresh-frozen tissue samples, including 4 serous, 5 mucinous and 3 endometrioid tumors, with
correlation with their histopathological characteristics. Ovarian epithelial tumors and normal
tissues showed an apparent separation on the distance map tree. Mucinous carcinomas were
closest to the normal group, whereas serous carcinomas were located furthest from the normal
group. All mucinous tumors with aggressive histology were separated from the LMP group.
The benign-looking cysts adjacent to the IEC showed an expression pattern identical to that of
the IEC area. The extent of change on the lineages leading to the mucinous and serous
carcinoma was 1.98-fold different. The overall gene expression profiles of serous or
endometrioid carcinomas appeared to be less affected by grade or stage than by histologic
type. The surrogate biomarkers screened in ovarian tumors and found to be significantly upregulated
in comparison to normal tissues were as follows: NM23, annexin-1, protein
phosphatase-1, ferritin light chain, proteasome alpha-6, and NAGK (N-acetyl glucosamine
kinase). In conclusion, ovarian mucinous tumors are distinct from other ovarian epithelial
tumors. LMP mucinous tumors showing histologically aggressive features belong to
mucinous carcinoma on the proteomic basis.
- 336 -

Session X : Cell death in cancer Poster X, 4
Endosulfan inhibits cell growth and apoptosis through activation of ERK1/2 and AKT
signaling pathways
S. Antherieu 1, N. Ledirac 1, P. Lenormand 2 and R. Rahmani 1
1 INRA UMR-1112, Equipe de Toxicologie Cellulaire et Moléculaire, et Génomique,
Sophia-Antipolis, France, e-mail: antherieu@antibes.inra.fr
2 CNRS UMR-6543, Centre Antoine Lacassagne, Nice, France.
Endosulfan is an organochlorine insecticide reported to be a potential carcinogen in humans.
This insecticide was previously described to alterate MAP kinases signaling pathways and
suspected to affect cell growth and differentiation in keratinocytes. In the present study, we
assessed the mitogenic and apoptogenic potentials of endosulfan in HaCaT keratinocytes.
We first confirmed its mutagenicity by using the AMES assay, which demonstrated that
unchanged endosulfan rather than metabolites, was at the origin of mutagenic effects
(negative results in the presence of S9 hepatic fractions).
Moreover, we showed that endosulfan led to a persistent ERK1/2 phosphorylation with an
accumulation of the phosphorylated form both in cytosol and nucleus. This sustained
activation is responsible for a decreased cell proliferation, since untreated cells reached
confluency after 3 days, whereas confluency is reached after 4 days in endosulfan-treated
cells. These data were also confirmed by BrdU incorporation which was strongly decreased
after insecticide treatment.
In addition, reactive oxygen species (ROS) has been shown to be involved in endosulfaninduced
ERK1/2 activation; we demonstrated that blockade of this oxidative stress by NAC
(N-acetylcystein) strongly prevented inhibition of proliferation by endosulfan. These results
clearly showed that endosulfan-induced ROS were involved in cell growth decrease.
Finally, endosulfan apoptogenic potential was investigated and a reduction of caspases 3/7
activation and PARP cleavage was evidenced. This inhibition of apoptosis is probably
correlated to endosulfan-induced AKT pathway, since a 3-fold induction of phospho-AKT
was observed after 1 h of treatment.
On the whole, this study demonstrates that endosulfan is a mutagenic agent, responsible of a
ROS-induced proliferation decrease and apoptosis inhibition. These processes in turn prevent
elimination of altered cells and therefore may contribute to carcinogenesis.
- 337 -

Session X : Cell death in cancer Poster X, 5
Apoptosis associated with reduced Bcl-2 expression in colorectal cancer tumors
Márcia M. Aranha1, Pedro M. Borralho1, Paula Ravasco2, Isabel B. Moreira da Silva1,
Luís Correia3, Afonso Fernandes4, Maria E. Camilo2, Cecília M.P. Rodrigues1
1Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, 1600-083
Lisbon, Portugal; 2Unidade de Metabolimo e Nutrição, IMM; Centro de
3Gastrenterologia e de 4Anatomia Patológica, Santa Maria Hospital, Lisbon, Portugal;
E-mail: maranha@ff.ul.pt
Compelling evidence implicates loss of cell-cycle control and changes in apoptotic pathways
in the development and progression of most human cancers. In this study, several apoptotic
factors were examined in normal and tumor tissues from patients diagnosed for colorectal
cancer. Apoptosis as assessed by the transferase mediated dUTP-digoxigenin nick-end
labeling (TUNEL) assay was significantly higher in tumor tissue compared with normal
mucosa (p < 0.01). TUNEL-positive cells were increased from adenoma to poorly
differentiated adenocarcinoma, but decreased in association with higher degree of
adenocarcinoma differentiation. Similar results were observed for NF-!B/RelA expression
detected by immunohistochemistry (p < 0.01). Further, Bcl-2 expression was consistently
higher in normal mucosa than in tumor tissue (p < 0.01). In both adenoma and
adenocarcinoma tissues, Bcl-2 immunoreactivity was almost undetectable, which inversely
correlated with the percentage of apoptotic cells (r = - 0.54; p < 0.01). In addition, expression
analysis of phosphorylated p53 and Bax by western blot revealed almost no variation of
protein expression in tumor tissue compared to normal mucosa. These results demonstrate
increased apoptosis in pre-malignant and poorly differentiated malignant tissues. Apoptosis
may proceed through a p53- and Bax-independent fashion but is correlated with suppression
of Bcl-2 expression. The histological progression of colorectal cancer from adenoma to
adenocarcinoma involves also NF-!B activation. Whether these changes are associated with
a better prognosis deserves further investigation. (Supported by Sociedade Portuguesa de
Gastrenterologia, Portugal)
- 338 -

Session X : Cell death in cancer Poster X, 6
Expression of Bcl-xL, Bax and P53 in primary tumors and lymph node metastases in
oral squamous cell carcinoma.
Marek Baltaziak1, Ewa Duraj1, Mariusz Koda1, Mariola Sulkowska1, Marcin
Musiatowicz2, Luiza Kanczuga-Koda1, Andrzej Wincewicz1, Boguslaw Musiatowicz1,
Stanislaw Sulkowski1.
Departments of Pathology1, Pediatric Otorhinolaryngology2; Medical University of
Bialystok, Waszyngtona 13, 15-269 Bialystok, Poland. E-mail: sulek@zeus.amb.edu.pl
Disturbances in expression of apoptosis associated proteins, take part in development and
progression of many human malignancies. The aim of the present study was assessment of
correlations among proteins involved in apoptosis: Bcl-xL, Bax and P53 as well as
relationships of these proteins with selected clinicopathological features in oral squamous cell
carcinoma. Consequently, we examined by immunohistochemistry, using the avidin-biotinperoxidase
method, Bcl-xL, Bax and P53 expression in 56 samples of primary oral squamous
cell carcinoma and in 22 matched pairs of primary and metastatic tumors. The evaluation of
immunostaining of Bcl-xL, Bax and P53 was analyzed in 10 different tumor fields and the
mean percentage of tumor cells with positive staining was evaluated. The significance of the
associations were determined using Spearman correlation analysis and the Chi-square test.
We found positive, Bcl-xL, Bax and P53 immunostaining in 44.6%, 28.6% and 58.9% of
studied primary tumors and in 63.6%, 45.5% and 72.7% of lymph node metastases,
respectively. Analysis of associations among studied proteins revealed positive correlation
between Bcl-xL and Bax in primary tumors (p

Session X : Cell death in cancer Poster X, 7
Salicylic acid and oxidative stress susceptibility within colon epithelial cell cultures
Charles S. Bestwicka, Lesley Milnea, Mary P. Moyerc, Garry Duthiea and Susan J.
Duthieb
Molecular Nutrition Groupa and Nutrition and Epigenetics Groupb, The Rowett
Research Institute, Aberdeen AB21 9SB, Scotland, UK. INCELL Corporationc, LLC
12000 Network Blvd., Ste B-200 San Antonio, TX 78249 210-877-0100.
Email:C.Bestwick@rowett.ac.uk
Salicylic acid (SA) is a widely occurring dietary phyto-phenol that may contribute to the
cancer preventative effects of a fruit and vegetable rich diet. In planta, SA is a critical
component of pathogen resistance responses, affecting the generation of reactive oxygen
species (ROS) and programmed cell death development. The colonic epithelium may be
unusually resistant to stress-induced apoptosis, thereby potentially increasing the propensity
for tumour development and we hypothesise that SA exposure may enhance the response to
oxidative DNA damage, ultimately facilitating more effective entry into apoptosis. Here, we
describe preliminary observations on the effect of non-cytotoxic SA exposure on cellular
ROS, antioxidant activity and resistance to oxidative insult within both non-transformed
(NCM460) and malignantly transformed (HT-29) colon epithelial cells. Exposure to SA for
24h decreased intracellular ROS levels within both cell types. By 72h, no significant
difference was observed between control and SA treated NCM460 cells but ROS levels in
HT-29 were slightly elevated in response to >100micromolar SA. No change to catalase
activity was detected but glutathione peroxidase activity had declined by 72h in both SA
treated cell types. DNA strand breaks were significantly increased in NCM460 cells but not
within HT-29 cells variously exposed to SA. Following a 72h SA exposure, cultures were
challenged with varying levels of hydrogen peroxide. SA significantly enhanced DNA
damage within NCM460 but not HT-29cells subject to mild oxidative stress. In conclusion,
SA modifies antioxidant and ROS levels within both transformed and non-transformed colon
epithelial cells, though there is no intuitive correlation between these changes and effects on
DNA stability. Nevertheless, SA does marginally enhance DNA susceptibility to oxidative
damage within the non-transformed colon cell line.
- 340 -

Session X : Cell death in cancer Poster X, 8
Bisnaphthalimidopropylspermidine induces DNA instability and apoptosis within colon
carcinoma derived cell lines
Charles S. Bestwick a, Lynda Ralton,c Lesley Milne,a Susan J. Duthie,b and Paul Kong
Thoo Linc
Molecular Nutrition Groupa and Nutrition and Epigenetics Groupb, The Rowett
Research Institute, Greenburn Road, Aberdeen AB29 9SB, Scotland, UK. The Robert
Gordon Universityc, School of Life Sciences, St. Andrew Street, Aberdeen AB25 1HG,
Scotland, UK. Email: C.Bestwick@rowett.ac.uk
DNA intercalators are potentially potent apoptosis inducers. Bisnaphthalimido compounds
bisintercalate to DNA via the major groove. However, solubility is poor and we have
modified the linker chain connecting the two naphthalimido ring moieties to increase
solubility. One such modification has been the incorporation of polyamines within the linker.
Polyamines are ubiquitous polycations critical for cell growth and also exhibit apoptotic
regulatory activity. Thus, in addition to facilitating cellular uptake, incorporating polyamines
within the linker may lead to an altered or extended spectrum of activity. Here, we describe
the effect of a spermidine analogue, bisnaphthalimidopropylspermidine (BNIPSpd), on DNA
stability and survival of Caco-2 and HT-29 colon carcinoma derived cells. After 48h
exposure, an IC50 of 0.15 and 1.64 micromolar was determined for Caco-2 and HT-29
respectively. Analogue uptake was rapid and essentially homogenous within the cell
populations by 4h. Uptake was associated with a dose dependent increase in DNA strand
breaks (0.1 –100micromolar) and decreased intracellular polyamines (0.01-100micromolar).
Following treatment with 0.5micromolar BNIPSpd or greater, cells expressed enhanced active
caspase-3, increased nuclear instability, internucleosomal DNA fragmentation and apoptoticmorphological
features. Pre-treatment with non-overtly cytotoxic or direct DNA damaging
concentrations of BNIPSpd retarded DNA repair capacity, as well as inducing a depletion of
cellular polyamines. The potential to exploit such analogues as chemotherapeutic agents and
as probes to assess the role of polyamines in cell survival regulation is discussed
- 341 -

Session X : Cell death in cancer Poster X, 9
Par-4-mediated Recruitment of Amida to the Actin Cytoskeleton Leads to the Induction
of Apoptosis
Meike Boosen1, Susanne Vetterkind1, Ansgar Koplin1, Susanne Illenberger2 and Ute
Preuss1*
1Institute of Genetics, University of Bonn, Roemerstr. 164 D-53117 Bonn, Germany,
2Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092
Braunschweig, Germany
email:meike.boosen@uni-bonn.de
Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact
mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we
identified Amida as a novel interaction partner, a ubiquitously expressed protein which has
been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida
occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the
leucine zipper of Par-4. Amida resides mainly in the nucleus, but displays nucleo-cytoplasmic
shuttling in heterokaryons. Upon co-expression with Par-4 in REF52.2 cells, Amida
translocates to the cytoplasm and is recruited to actin filaments by Par-4, resulting in
enhanced induction of apoptosis. The synergistic effect of Amida/Par-4 complexes on the
induction of apoptosis is abrogated when either Amida/Par-4 complex formation or
association of these complexes with the actin cytoskeleton are impaired, indicating that the
Par-4-mediated relocation of Amida to the actin cytoskeleton is crucial for the pro-apoptotic
function of Par-4/Amida complexes in REF52.2 cells. The latter results in enhanced
phosphorylation of the regulatory light chain of myosin II (MLC) as has previously been
shown for Par-4-mediated recruitment of DAP-like kinase (Dlk), suggesting that the
recruitment of nuclear proteins involved in the regulation of apoptotic processes to the actin
filament system by Par-4 represents a potent mechanism how Par-4 can trigger apoptosis.
- 342 -

Session X : Cell death in cancer Poster X, 10
2-Methoxyestradiol induces autophagy in Ewing sarcoma-derived cell lines:
an anti-apoptotic mechanism
Amélie Borges1, Chantal Bauvy1, Sylvie Souquére2, Gérard Pierron2, Patrice
Codogno1, Mojgan Djavaheri-Mergny1
1INSERM U504, Institut André Lwoff, Villejuif
mergny@vjf.inserm.fr
2 UPR-1983, Institut André Lwoff, Villejuif,
2-Methoxyestradiol (2-Me) is a naturel estrogen metabobolite which acts as antiproliferative
agent in various tumor cell lines. We have previously shown that 2-Me induced apoptosis in
Ewing sarcoma cells through mitochondrial death pathway. In the present study, we report
that 2-Me-induced macroautophagy in Ewing sarcoma-derived cells independently of their
p53 status. Macroautophagy has been characterized by ultrastructural analysis, accumulation
of LC3-II protein and proteolysis assay. In addition, we found that the inhibition of autophagy
by either using 3-methyladenine or by silencing of autophagy genes enhances 2-Me-induced
apoptosis. These results suggest that 2-Me-induced autophagy is an anti-apoptotic
mechanism.
- 343 -

Session X : Cell death in cancer Poster X, 11
5-Fluorouracil activates death receptor and mitochondrial pathways of apoptosis in
colon cancer cells in a p53 proficiency-dependent manner
Pedro M. Borralho,1 Isabel B. Moreira da Silva,1 Márcia M. Aranha,1 Cristina
Albuquerque,2 Carlos Nobre Leitão,2 Cecília M.P. Rodrigues1
1Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, 1600-083
Lisbon, Portugal; 2Centro de Patobiologia Molecular, IPO, Lisbon, Portugal; E-mail:
borralho@ff.ul.pt
5-Fluorouracil (5-FU), the most important drug used for colorectal cancer (CRC)
chemotherapy, is a potent antitumor agent that affects pyrimidine synthesis by inhibiting
thymidylate synthase. Treatment of cancer cells with 5-FU is thought to induce apoptosis.
However, resistance to 5-FU is still a major limitation to its clinical use. In these studies, we
used HCT116 and SW480 human colorectal cancer cell lines, with wild-type and mutant-type
p53 expression, respectively. Supplementation with 8 µM 5-FU was more cytotoxic in
HCT116 than in SW480 cells (p < 0.001). Nuclear fragmentation and caspase-3, -8 and -9
activities were markedly increased in HCT116 cells after 5-FU exposure (p < 0.01). In
addition, wild-type p53 expression in 5-FU-treated HCT116 cells was almost 25-fold
increased compared with control cells (p < 0.001), whereas mutant p53 expression was not
altered in SW480 cells. Pro-apoptotic Bax and anti-apoptotic Bcl-2 remained unchanged
during 5-FU treatment. Interestingly, 5-FU resulted in a 4-fold increased expression of the
death receptor Fas in HCT116 cells (p < 0.05). siRNA-mediated Fas gene expression
knockdown significantly reduced nuclear fragmentation as well as caspase-3, -8 and -9
activities. Finally, western blot analysis of mitochondrial extracts showed a 6-fold increase in
Bax, together with a 3-fold decrease in cytochrome c in HCT116 cells exposed to 5-FU (p <
0.05). In conclusion, these results suggest that 5-FU exerts its cytotoxic effects through a
p53/Fas-dependent apoptotic pathway, which is independent from modulation of Bax and
Bcl-2 protein expression. In addition, p53 might also play a transcriptional independent role,
tipping the Bax/Bcl-2 balance, allowing Bax to translocate to the mitochondria and
consequently activating the mitochondrial pathway of apoptosis. Thus, 5-FU activates and
modulates classical pathways of apoptosis in a p53 proficiency-dependent manner.
(Supported by Fundação Calouste Gulbenkian, Portugal)
- 344 -

Session X : Cell death in cancer Poster X, 12
Antiproliferative effects of retinoic acid and histone deacetylase inhibitor Bml-210 on
human cervical cancer HeLa cells
Veronika V. Borutinskaite1, 2, Ruta Navakauskiene 1 and Karl-Eric Magnusson2
1Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius,
Lithuania. E-mail: verbo@imk.liu.se ruta.navakauskiene@bchi.lt
2Division of Medical Microbiology, Department of Molecular and Clinical Medicine,
Linköping University, SE-581 85 Linköping, Sweden. E-mail: karma@imk.liu.se
Human papillomaviruses (HPVs) have been associated with a number of neoplastic lesions,
most notably cervical cancer which is one of the major forms of cancer world wide. Human
cervical carcinoma HeLa cells contain integrated human papillomavirus type 18 (HVP18).
Retinoic acid is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro
of HeLa cervical carcinoma cells. Cellular responses to RA are mediated by nuclear retinoic
acid receptors (RARs) and retinoid X receptors (RXRs). On the other hand, histone
deacetylase inhibitors have been shown to be chemopreventive agents for the treatment of
cancer cells.
In this study, we have studied the antiproliferative effect of retinoic acid (RA) and histone
deacetylase inhibitor Bml-210 on HeLa cells, and particularly the effects on the protein
expression that may be involved in the cell cycle control and apoptosis. Our data suggest that
a combination of RA and Bml-210 leads to cell growth inhibition with following apoptosis in
a treatment time- dependent manner. We confirm that Bml-210 alone or in combination with
RA cause a marked increase in the level of p21, which coincides with the increased level of
transcription factor Sp1. The changes in p53 level are only under RA influence. We also
discovered that histone deacetylase inhibitor Bml-210 causes increased levels of antiapoptotic
protein Bcl-2 and phosphorylated p38 MAPK; these link in cell cycle arrest with
response to extracellular stimuli. Our results suggest that RA and Bml-210 are involved in
different signalling pathways that regulate cell cycle arrest and lead to apoptosis of HeLa
cells.
- 345 -

Session X : Cell death in cancer Poster X, 13
Targeting of the IFNgammaR2 dileucine-based (LI276-277) internalization motif
reinstates T cell sensitivity to the IFNgamma/STAT1-dependent apoptosis in vitro and
in vivo
Daniela Boselli1,2, Josiane Ragimbeau4, Paola Bernabei1, Roberto Chiarle1,3, Sandra
Pellegrini4, Mirella Giovarelli1,2, and Francesco Novelli1,2
1Center for Experimental Research and Medical Studies (CeRMS), San Giovanni
Battista Hospital, Via Santena 5, 10126 Turin, Italy; 2Department of Medicine and
Experimental Oncology, 3Department of Pathology, University of Turin, 10126 Turin,
Italy; 4Unit of Cytokine Signaling, Institut Pasteur, 75724 Paris, France
E-mail: daniela.boselli@unito.it
Activation of the IFNgamma (IFNg)/STAT1 pathway switches on many pro-apoptotic and
anti-proliferative genes. The downregulation of the IFNg receptor 2 chain (IFNgR2) has been
described in T lymphocytes and is thought to prevent activation of the apoptotic pathway. We
have previously shown that in human T lymphocytes IFNgR2 is internalized in a clathrindependent
manner and that this internalization is ligand-independent. Here we show that, in
Jurkat T cells, an IFNgR2 mutant in which the LI267-277 motif had been substituted with two
alanines (LI276-277AA) accumulated at the cell surface. The defective internalization of the
LI267-277AA mutant determined a strong and sustained IFNg-dependent response in terms of
STAT1 activation, IRF-1 and MHC class I induction, Fas and FasL cell surface up-regulation
and apoptosis. All these functions were weakly or not induced by IFNg in Jurkat cells
expressing wild type IFNgR2. The growth into SCID mice of ST4 malignant T cells
expressing the LI276-277AA mutant receptor, but not that of cells expressing the wild type
receptor, was inhibited by IFNg. These data indicate that the LI276-277 motif critically
controls ligand-independent internalization of IFNgR2 in T cells. This internalization motif
may provide a valuable therapeutic target for the reinstatement of IFNg/STAT1 apoptotic
signaling in autoreactive or neoplastic T cells.
- 346 -

Session X : Cell death in cancer Poster X, 14
Endogenous FGF1 inhibits p53-dependent apoptosis
Sylvina Bouleau, Vincent Rincheval, Bernard Mignotte, Jean-Luc Vayssière, Flore
Renaud.
Laboratoire de Génétique et Biologie Cellulaire, CNRS FRE2445, Ecole Pratique des
Hautes Etudes, Université de Versailles, 45 Av des Etats Unis, 78035 Versailles, France.
E-mail : bouleau@genetique.uvsq.fr
The oncosuppressor p53 is an important regulator of apoptosis and is associated with few
diseases (like cancers, neurodegenerative diseases…). In contrast to p53, survival factors
inhibit the cell death process. Their action is usually mediated via paracrine and/or autocrine
pathways. However, some survival factors, as FGF1 and FGF2, do not contain secretion
peptide signal but a nuclear localization signal (NLS) and may act via an intracrine pathway.
We have initiated the study of the role of FGF1 in p53-dependent apoptosis in a rat embryo
fibroblast cellular model. We have shown that : (1) p53 represses fgf1 gene expression; (2)
FGF1 overexpression inhibits pro-apoptotic and anti-proliferative p53 activities via an
intracrine pathway; (3) FGF1 increases the level of MDM2, which induced p53 degradation;
(4) FGF1 inhibits the p53-dependent trans-activation of the bax gene, which code a major
pro-apoptotic protein (Bouleau S. et al. , 2005, Oncogene, 24(53) : 7839-7849).
Then, we persue this study in a neuronal cellular model: the PC12 cells (issued from a rat
pheocromocytoma). These cells can differentiate in sympathetic like neurons in presence of
neurotrophic factors like NGF or FGF1. First, we have characterized the apoptosis process
induced by etoposide, to ascertain the implication of p53 in this process. Second, we have
shown that the addition of recombinant FGF1 in the culture medium protects the cells from
p53-dependent cell death, suggesting a protective activity of exogenous FGF1. Actually, we
initiate the study of the activity of endogenous FGF1. In particular, we currently analyze the
activity of intracellular and nuclear FGF1 on p53-dependent apoptosis.
Bouleau S, Grimal H, Rincheval V, Godefroy N, Mignotte B, Vayssière JL, Renaud F. “FGF1
inhibits p53-dependent apoptosis and cell cycle arrest via an intracrine pathway”. 2005,
Oncogene, 24(53) : 7839-7849.
- 347 -

Session X : Cell death in cancer Poster X, 15
hGTSE-1 protein can modulate the cellular response to stress by regulating the stability
of the cyclin-dependent kinase inhibitor p21CIP1/WAF1
Bublik DR, Scolz M, Monte M and Schneider C.
Human GTSE-1 (G2 and S phase-expressed-1) is a cell cycle-regulated protein with increased
expression during S and G2 phases of the cell cycle. We have previously demonstrated that
hGTSE-1 can negatively regulate p53 transactivation function, protein levels and p53dependent
apoptosis during the S/G2 cell cycle window. More recently we have observed
that, although hGTSE-1 represses p53 activity, it’s able to stabilize the cyclin-dependent
kinase inhibitor p21CIP1/WAF1. p21CIP1/WAF1 protein was shown to be strongly
regulated by hGTSE-1: p21CIP1/WAF1 levels were increased in cells with Ponasterone Ainducible
expression of hGTSE-1, and concomitantly downregulated in hGTSE-1-knocked
down cells, in a proteasome-dependent manner. As a functional consequence,
p21CIP1/WAF1 stabilization observed in hGTSE-1-induced cells, conferred resistance to
taxol-induced apoptosis; conversely, loss of p21CIP1/WAF1 after hGTSE-1 knock-down by
small interference RNA sensitized cells to taxol-induced apoptosis.
Besides, we have also found out that hGTSE-1-dependent stabilization of p21CIP1/WAF1
requires a functional Heat Shock Protein 90 (Hsp90) given that the specific Hsp90 inhibitor
Geldanamycin completely prevented hGTSE-1’s effects on p21CIP1/WAF1. We propose the
recently discovered Hsp90-binding and p21CIP1/WAF1-interactor TPR protein WISp39, as a
mediator that acts downstream of hGTSE-1 in regulating p21CIP1/WAF1 stability; indeed in
the absence of WISp39 hGTSE-1 failed to induce p21CIP1/WAF1 accumulation. Moreover,
a direct interaction between hGTSE-1, p21CIP1/WAF1 and WISp39 was confirmed in vitro
and in vivo suggesting that hGTSE-1 might be associated to the multi-protein Hsp90
chaperone complex.
These findings may support an important role of hGTSE-1 in maintaining and tightly
regulating p21CIP1/WAF1 levels thereby modulating its functions, and indicate that hGTSE-
1 could fine tune the cellular response to stress through regulation of p21CIP1/WAF1
turnover.
- 348 -

Session X : Cell death in cancer Poster X, 16
The effects of selenium diet on expression of selenoproteins in the respiratory tract of
the rat
Katarzyna Bukalis, , Dorothea Alber, Dietrich Behne, Antonios Kyriakopoulos
Hahn-Meitner-Institut, Department “Molecular Trace Element Research in the Life
Sciences”, Glienicker Str. 100, D-14109 Berlin, Germany, e-mail: k.bukalis@hmi.de)
The organs of the respiratory tract lung and trachea, are constantly exposed to many gases and
particular matter present in the atmosphere or toxins such as asbestos or tobacco smoke.
Many of these pollutants are powerful oxidants. Several lung and trachea diseases have been
associated with oxidative stress and linked to oxidant insults. In addition, long-term exposure
to environmental toxicants can induce mutation, or changes in the DNA of the exposed cells,
resulting in altered gene functions and in the further step lead to altered patterns of protein
production that might allow for growth of precancerous cells. The tissues of the respiratory
tract therefore require a specific defence system against oxidants and free radicals.
The trace element selenium plays an important role in this defence system. In the form of
selenocysteine it is an essential component of glutathione peroxidase, an enzyme involved in
the detoxification of peroxides. Selenium has been also reported to have diverse anticancer
actions and to inhibit cancer growth in animals. In the patients with many cancers including
those of respiratory tract the low blood levels of selenium have been observed. This finding
indicate that low selenium levels can associated with increased cancer incidence in humans.
There is evident relationship between selenium deficiency, oxidative stress and lung and
trachea diseases. Therefore it is necessary to investigate the impact of the selenium status in
the diet on the incidence of the lung and trachea diseases. It is of grate interest to analyse the
selenium containing proteins to understand the role of the selenium and its compounds in the
protective mechanisms. In addition, there may be further selenoproteins which may likewise
be of significance in the antioxidant defence system of the respiratory tract.
- 349 -

Session X : Cell death in cancer Poster X, 17
Pirfenidone suppresses TGF-beta and is cytotoxic in malignant glioma cells
Isabel Burghardt, Steffen A. Aulwurm, Brigitte Frank, Michael Weller and Wolfgang
Wick
Laboratory of Molecular Neuro-Oncology, Department of General Neurology, Hertie
Institute for Clinical Brain Research, University of Tuebingen, Medical School, Otfried-
Mueller-Str. 27, 72076 Tuebingen, Germany
Presenting author: Tel. +49 7071 2981969, FAX: +49 7071 295260,
E-mail: isabel.burghardt@gmx.net
Among the essential characteristics of human malignant gliomas are infiltrative growth,
angiogenesis and suppression of antitumor immune surveillance. Transforming growth factorbeta
(TGF-beta) is intimately involved in the regulation of these processes.
The novel antifibrotic drug 5-methyl-1-phenyl-2-(1H)-pyridone (Pirfenidone) significantly
reduces TGF-beta2 mRNA and protein levels in four human glioma cell lines (LN18, LNT-
229, LN-308 and T98G) in vitro. In good accordance, Pirfenidone between 1 and 5 mM also
shows a dose dependent decrease in the growth inhibition of TGF-beta-sensitive CCL-64 cells
mediated by TGF-beta-containing glioma cell supernatant. TGF-beta2, the major isoform in
malignant gliomas, is synthesized as a pre-pro-TGF-beta polypeptide at 55 kDa that is
processed to a 12,5 kDa polypeptide by cytoplasmic and secreted furin-like proteases. 5 mM
Pirfenidone decreases activity of recombinant furin by 40% in vitro.
In addition, Pirfenidone at 1, 2.5 and 5 mM leads to reduced cell density in LN18, LNT-229,
LN-308 and T98G by 10, 20 and 40% after 48 hours of treatment.
The interaction with cell survival pathways is further demonstrated by reduction of the levels
of the endogenous phosphorylation of protein kinase B(PKB)/Akt and ribosomal protein S6
(RPS6) at a concentration of 5 mM, thus possibly influencing the phosphatidyl-inositol-3
kinase (PI3K) cascade downstream of the epidermal growth factor receptor (EGFR).
Investigation of effects on the invasive phenotype of glioma reveals that Pirfenidone has no
effect on protein levels of “pro-migratory” matrix metalloproteinases matrix
metalloproteinase (MMP)-2 and MMP-9, as well as on tissue inhibitor of metalloproteinase
(TIMP)-2.
- 350 -

Session X : Cell death in cancer Poster X, 18
Dosage Effects of Ginkgolide B on Ethanol-Induced Cell Death in Human Hepatoma G2
Cells
Wen-Hsiung Chana and Yan-Der Hsuuwb
a. Department of Bioscience Technology, Chung Yuan Christian University, Chung Li,
Taiwan 32023. E-mail: whchan@cycu.edu.tw
b. Department of Animal Science, National Pingtung University of Science and
Technology, Neipu, Pingtung, Taiwan 91201. E-mail: hsuuw@yahoo.com.tw
Ginkgolide B is a major active component of Ginkgo biloba extracts, which have been shown
to confer anti-cancer effects by inducing apoptosis or inhibiting oxidative stress generation.
Ethanol induces a wide range of cellular toxicities, many of which have been linked to free
radical generation. To further elucidate the cellular effects of ginkgolide B, we examined the
dose-response effect of ginkgolide B on ethanol-induced toxicity in human Hep G2 cells.
TUNEL and MTT assays revealed that ethanol (50-400 mM) induced apoptotic cell death in
human Hep G2 cells, and that this effect was inhibited by low (5-25 µM) doses of ginkgolide
B, but enhanced by high (50-100 µM) doses of ginkgolide B. Additional experiments revealed
that ethanol treatment directly increased intracellular oxidative stress; this effect was
enhanced by high doses of ginkgolide B but remained unchanged following treatment with
low concentrations of ginkgolide B. The dose-response effects of ginkgolide B on reactive
oxygen species (ROS) generation was directly correlated with cell apoptotic biochemical
changes including c-Jun N-terminal kinase (JNK) activation, caspase-3 activation, and DNA
fragmentation. These results indicate that treatment dosage may determine the effect of
ginkgolide B on ethanol-induced ROS generation and cell apoptosis, and support the notion
that an appropriate dosage of ginkgolide B may aid in decreasing the toxic effects of ethanol.
- 351 -

Session X : Cell death in cancer Poster X, 19
Downregulation of Lifeguard leads to sensitization against TRAIL induced apoptosis in
tumor cell lines
Claudia YU Choi, Kerstin Reimers, Susanne Kall, Peter M. Vogt
Department of Plastic, Hand and Reconstructive Surgery, Medical School Hannover,
Podbielskistr. 380, 30659 Hannover, Germany, E-mail: Choi.Claudia@MH-
Hannover.de
A dysbalance between apoptosis and proliferation plays an essential role in tumor
development. The expression of anti-apoptotic proteins promotes tumorigenesis and
resistance to chemotherapy. TRAIL is the most promising candidate for tumor selective death
receptor-activation. It activates the death receptors TRAIL-1 and TRAIL 2 inducing apoptosis
preferentially in tumor cells but not in normal tissue. Specific means to sensitize tumor cells
for TRAIL are desirable. Lifeguard (LFG) is a protein with inhibitory function on Fasmediated
apoptosis. We hypothesized that LFG inhibits TRAIL-induced apoptosis. We
determined high LFG-expression-levels in A549 cells and comparing tumor cell lines of
different origins we found that TRAIL-sensitivity correlated with high LFG-expressionlevels.
To selectively interfere with expression of LFG, an antisense construct with effective
down-regulation was introduced to A549 cells.After treatment, the sensitivity to TRAIL was
enhanced with up-regulation of the apoptotic activity. In conclusion, LFG is expressed in
many TRAIL-resistant tumor cell lines and the inhibition of LFG-expression was sufficient to
sensitize A549 cells from TRAIL-mediated apoptosis. Our findings indicate that LFG is a
protein with pathogenic function in tumorigenesis with new therapeutic options in cancer
therapy targeting LFG.
- 352 -

Session X : Cell death in cancer Poster X, 20
Relationship between apoptotic and autophagic programmed cell death: role of Bcl-2
and Beclin 1 proteins
Iwona A. Ciechomska, Christoph Goemans, Aviva Tolkovsky
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge,
CB2 1QW, UK
Email: iac23@mole.bio.cam.ac.uk; cgg20@mole.bio.cam.ac.uk;
amt@mole.bio.cam.ac.uk
Programmed cell death (PCD) mechanisms can be divided into several categories including
type I (apoptosis) and type II (autophagic cell death). The mutual relationship between
apoptotic and autophagic death is currently debated because Beclin 1, an important regulator
of autophagy, is also a Bcl-2 interacting protein. Recent studies revealed that the interaction
between Bcl-2 and Beclin 1 regulates starvation-induced autophagy.
In this study we investigated how the interactions between Bcl-2 and Beclin-1 influence the
apoptotic machinery. In order to investigate this effect first we have studied how each of these
proteins individually, Bcl-2 or Beclin 1, influence the level of apoptosis in HeLa cells induced
by a variety of apoptotic stimuli. The Bcl-2 protein is localized in the different cellular
membranes, therefore wild-type and Bcl-2 variants that are exclusively targeted to the
endoplasmic reticulum (ER) membrane or the mitochondrial outer membrane (MOM) were
used. We have confirmed that Bcl-2 decreases the level of apoptosis induced by staurosporine
independently on the localization, although we have found some toxic effect of wild-type Bcl-
2 after transient overexpression in untreated cells. Overexpression of either wild-type or
Beclin 1 mutants that cannot bind to Bcl-2 resulted in increased apoptosis. Furthermore, Bcl-2
protein binds to Beclin 1 and the intracellular localization of Beclin 1 was changed according
to Bcl-2 localisation. Next we examined how the interaction between Beclin 1 and Bcl-2
influence the level of apoptosis. Beclin 1 mutants that are defective in Bcl-2 binding induced
less apoptosis than wild-type Beclin 1 after overexpression of ER- or MOM-targeted Bcl-2.
Thus, our data suggest that Beclin 1 not only functions as a proautophagy protein, but also as
a proapoptotic protein via disruption of the anti-apoptotic function of Bcl-2.
- 353 -

Session X : Cell death in cancer Poster X, 21
Human soluble TRAIL/Apo2L induces apoptosis in chemotherapy refractory nodal
diffuse large B-cell lymphomas.
Saskia AGM Cillessen1, Chris JLM Meijer1, Gert J Ossenkoppele2, Kitty CM
Castricum1, August H Westra2, Petra Niesten1, Jettie JF Muris1, Hoite F Nijdam3,
Klaas G van der Hem4, Marcel Flens5, Erik Hooijberg1, Joost J Oudejans1
Department of Clinical Pathology1 and Hematology2, VU University Medical Center,
Amsterdam. Department of Otolaryngology, Westfries Gasthuis, Hoorn3, Department
of Internal Medicine4 and Clinical Pathology5, Zaans Medical Center de Heel,
Zaandam. The Netherlands. Email: SAGM.Cillessen@vumc.nl
Part of Diffuse Large B-Cell Lymphomas (DLBCL) is resistant to chemotherapy. Human
soluble (hs) TRAIL/Apo2L might be an alternative form of therapy for these lymphomas. In
isolated lymphoma cells of DLBCL and B-cell lines we investigated whether
hsTRAIL/Apo2L induces apoptosis in lymphomas that are resistant to chemotherapy induced
cell death.
Twelve out of twenty-two DLBCL samples including seven chemotherapy refractory
lymphomas were sensitive for hsTRAIL/Apo2L. hsTRAIL/Apo2L induced apoptosis was
preferentially observed in DLBCL samples and B-cell lines that were relatively or completely
resistant to Etoposide induced apoptosis. Furthermore, hsTRAIL/Apo2L induced apoptosis in
DLBCL and B-cell lines showing high expression levels of inhibitors of the caspase 9
mediated pathway Bcl-2 and/or XIAP. In hsTRAIL/Apo2L sensitive cells expression of the
TRAIL receptor R1 and/or R2 and absence of R3 and R4 was observed.
We conclude that hsTRAIL/Apo2L induces apoptosis in part of chemotherapy refractory
nodal DLBCL and that disruption of the caspase 9 mediated pathway and expression of Bcl-2
and XIAP does not confer resistance to hsTRAIL/Apo2L induced apoptosis in DLBCL and
B-cell lines. Thus, based on our results hsTRAIL/Apo2L appears to be a valuable alternative
treatment for patients with chemotherapy refractory DLBCL.
- 354 -

Session X : Cell death in cancer Poster X, 22
In the absence of Insulin-like Growth Factor (IGF)-1 signaling, Interferon-gamma (IFNg)
inhibits the growth of human malignant T cells
Laura Conti1,2, Angela Longo1, Gabriella Regis1,2, Mirella Giovarelli1,2, Roberto
Chiarle1 and Francesco Novelli1,2.
1Center for Experimental Research and Medical Studies (CERMS), San Giovanni
Battista Hospital-Molinette, via Santena 5, 10126 Turin, Italy, and 2Department of
Medicine and Experimental Oncology, Section of Pathology, University of Turin, C.so
Raffaello 30, 10125 Turin, Italy. E-mail: laura.conti@unito.it.
IGF-1 is a growth factor that promotes the survival and proliferation of many tumors and cell
types, and several approaches to target IGF-1 signaling have resulted in the induction of
apoptosis in a broad range of tumor cells. However, we have found that human malignant T
cells, which are insensitive to the antiproliferative and apoptotic effects of IFN-g, still grow in
the absence of IGF-1 signaling. T cell refractoriness to the IFN-g/Signal Transducer and
Activator of Transcription (STAT) 1 pathway mainly rests on internalization of the IFN-g
Receptor 2 (IFN-gR2) signaling chain, which is critically induced by IGF-1. Here we show
that retrovirus-mediated gene transfer of a dominant negative IGF-1 Receptor (IGF-1R DN)
inhibits IFN-gR2 internalization and induces its cell surface accumulation. This results in a
strong and sustained IFN-g-induced STAT1 activation and consequently in the high
expression of pro-apoptotic molecules that render malignant T cells susceptible to the
antiproliferative and apoptotic effects of IFN-g, both in vitro and in vivo. These data indicate
that inhibition of IGF-1 signaling associated with IFN-g administration could be a useful
approach to inhibit the growth of neoplastic T cells resistant to each treatment on its own.
- 355 -

Session X : Cell death in cancer Poster X, 23
Mechanisms of Bcl-2 up-regulation in normal and tumor monocytes.
Silvia Cristofanon#*, Silvia Nuccitelli*, Maria D’Alessio*, Marc Diederich# and Lina
Ghibelli*.
#Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC), Fondation
Recherche sur le Cancer et les Maladies du Sang, Hôpital Kirchberg, 9, rue Edward
Steichen, L-2540 Luxembourg
*Dipartimento di biologia, Università di Roma “Tor Vergata”, Via della Ricerca
Scientifica, 00133 Rome, Italy
Glutathione (GSH) depletion (i.e., with BSO) stimulates Bcl-2 up-regulation in a set of tumor
cells (D’Alessio et al, 2004). These cells are able to survive to BSO and to resist to BSOinduced
chemo-sensitisation: indeed, a) inhibition of Bcl-2 trans-activation sensitises U937
cells to apoptosis; and b), GSH depletion up-regulates Bcl-2 in BSO resistant but not in BSOsensitive
cells. Here we show that freshly isolated peripheral blood monocytes from of
healthy donors up-regulate Bcl-2 as a response to the strong ROS production due to the
separation protocol. BSO treatment further up-regulate Bcl-2, with a mechanism involving $glutamyl-transpeptidase
(GGT) and the transcription factor NF!B. On the one side, acivicin, a
specific inhibitor of GGT, though speeding up BSO-induced GSH depletion, completely
abrogates Bcl-2 up-regulation. On the other side, the involvement of NF!B in the BSOdependent
Bcl-2 up-regulation was previously suggested in our system by the ability of its
inhibitor CAPE of impairing Bcl-2 over-expression and sensitizing U937 to BSO. The
mechanism involved seems to imply a non-canonical activation of a p50-p50 NF!B
homodimer (as opposed to the canonical p50-p65 heterodimer), recently demonstrated to be
responsible of the NF!B mediated BCL-2 up-regulation (Kurland et al, 2001).
- 356 -

Session X : Cell death in cancer Poster X, 24
Apoptotic GSH extrusion as a switch between extrinsic and intrinsic apoptotic pathway:
possible involvement of caspase 2
Milena De Nicola*, Claudia Cerella*, Maria D’Alessio*, Antonio Bergamaschiª, Andrea
Magriniª, Lina Ghibelli*
* Dipartimento di Biologia; ª Cattedra Medicina del Lavoro, Universita' di Roma Tor
Vergata, Via Ricerca Scientifica 1, 00133 Roma, Italia. E.mail:
milena.de.nicola@uniroma2.it
A current paradigm of apoptotic signaling is that different types of apoptogenic stimuli,
physiologic vs damaging, trigger different apoptotic pathways, extrinsic or intrinsic,
respectively. Many recent reports describe cross-talks between these pathways, depending on
cells and/or inducers. Here we describe that damaging agents such as puromycin or etoposide
are able to elicit both apoptotic pathways in a mutually exclusive fashion on human tumor
monocytic U937 cells, and that the switch between the two pathways depends on the
extrusion vs non-extrusion of glutathione. In the first instance, the redox disequilibrium
following GSH loss activates Bax which in turn causes cytochrome c release and caspase 9
activation, i.e., the intrinsic pathway. In the second instance, apoptosis occurs without redox
imbalance and requires caspase 8, in a fashion that is undistinguishable from a canonical
extrinsic pathway triggered by Fas stimulation. Thus, glutathione extrusion is a trigger of the
intrinsic pathway; as such, it is upstream to caspase 9 or 3 activation, but requires caspase 2.
We propose that caspase 2 activates the intrinsic pathway by promoting GSH extrusion; it
may additionally promote caspase 8 activation, possibly explaining why puromycin or
etoposide are able to elicit also the extrinsic pathway.
- 357 -

Session X : Cell death in cancer Poster X, 25
The secret life of caspase-14
G. Denecker, E. Hoste, T. Hochepied, B. Gilbert, P. Ovaere, K. D’Herde1, C.
VandenBroucke, S. Lippens, L. Schoonjans2, C. Libert, P. Vandenabeele and W.
Declercq
Molecular Signalling and Cell Death Unit, Department for Molecular Biomedical
Research, Technologiepark 927, B-9052 Ghent-Zwijnaarde (http://www.dmbr.ugent.be),
VIB, Ghent University. E-mail: wim.declercq@dmbr.Ugent.be
1 Department of Anatomy, Embryology, Histology, Medical Physics, Ugent. 2
Department of Transgene Technology, KUL, Leuven, Belgium.
Caspase-14 is expressed in the differentiating keratinocytes of the epidermis and the
hairfollicles. In addition, caspase-14 could also be detected in the Hassall's body's of the
thymus. In the skin caspase-14 activation occurs in the uppermost layers of the epidermis and
correlates with epidermal cornification. To unravel the possible role of caspase-14 during skin
differentiation, we have generated caspase-14-deficient mice. Caspase-14 knock-out mice
were born normally with the expected Mendelian ratio. The absence of caspase-14 protein
was confirmed both by western blot and immunohistochemistry. Histological sections of the
back skin and thymus of newborn wild-type and caspase-14-deficient mice revealed no
macroscopical differences. Immunohistochemical analysis of other early and late
differentiation markers demonstrated a normal expression pattern of K1, K10, K14, loricrin,
involucrin and filaggrin. However, the skin of caspase-14-deficient mice had a more shiny
appearance and showed a lichiniform phenotype. Electron microscopic analysis indicated the
presence of high numbers composite keratohyalin granules in the caspase-14-deficient
epidermis. These granules are normally used as profilaggrin stores. In accordance, caspase-14
mice have an altered profilaggrin-processing pattern. These data argue for a role of caspase-
14 in the final steps of keratinocyte differentiation. Further analysis of different physiological
parameters is ongoing in order to unravel the secret life of caspase-14.
- 358 -

Session X : Cell death in cancer Poster X, 26
Involvement of PI-3 Kinase signaling in polyunsaturated fatty acid-induced promotion
of apoptosis in CaCo-2 cells.
Joe-Lin du Toit, Louise Louw and Anna-Mart Engelbrecht
Department of Physiological Sciences, University of Stellenbosch, Stellenbosch, 7600,
South Africa. E-mail: jl@sun.ac.za
The phosphoinositide 3-kinase (PI3-kinase) signaling pathway plays a pivotal role in the
regulation of cell growth. A downstream component of the PI3-kinase pathway, PKB/Akt,
phosphorylates and regulates the function of various substrates involved in metabolism,
proliferation and apoptosis. Evidence has shown that PKB/Akt signaling also contributes to
tumourigenesis and cancer progression and that PI3-kinase signaling is up regulated in colon
cancer cells. This study aims to elucidate the mechanisms by which various fatty acids
influence the PI3-kinase pathway in order to promote apoptosis of colon adenocarcinoma
cells and slow down cancer progression.
NCM 460 and CaCo-2 cells were cultured and treated with arachidonic acid (AA), oleic acid
(OA), palmitic acid (PMA) and docosahexanoic acid (DHA) respectively, and incubated for
48 hours. Samples were analysed by Western blotting with antibodies directed against total
PBK/Akt, phospho-PKB/Akt (Ser473 and Thr308), PI3-kinase and various substrates of
PKB/Akt as well as their phosphorylated counterparts. Wortmannin (100 nM) was used to
inhibit PI3-kinase. Cell viability was assessed with MTT assays.
DHA was most effective to significantly increase NCM 460 cell viability whilst decreasing
CaCo2 cell viability, followed by AA. With fatty acid treatment, PI3-kinase signaling was up
regulated in NCM460 cells and down regulated in CaCo-2 cells, and for both cell lines this
was most profound in cells treated with DHA. Phosphorylation of the substrates of PKB/Akt
involved in apoptosis (Bad, FKHR) as well as other substrates such as GSK-3# and mTOR
were altered in CaCo-2 cells treated with DHA. We propose a potential role for DHA as a
therapeutic agent in the treatment of colon cancer.
- 359 -

Session X : Cell death in cancer Poster X, 27
Cationic surfactants induced apoptosis in normal and cancer cells.
Riyo Enomoto 1 , Chie Suzuki 1 , Masataka Ohno 1 , Toshinori Ohasi 1 , Ryoko Futagami 1 ,
Keiko Ishikawa 1 , Mika Komae 1 , Takayuki Nishino 1 , Yasuo Konishi 2 and Eibai Lee 1 .
1 Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin
University, Ikawadani-cho, Nishi-ku, Kobe 651-2180 Japan, 2 Biotechnology Research
Institution, National Research Council Canada, 6100 Royalmount Avenue, Montreal,
Quebec, H4P 2R2, Canada. E-mail : suzuki@pharm.kobegakuin.ac.jp
Cationic surfactants such as benzethonium chloride which possess quaternary ammonium salt
are used as a preservative and an antimicrobial agent. Recently, these compounds in eye drops
have been reported to induce apoptosis in conjunctive cells. Here, we examined whether
various types of surfactants induces apoptosis or not in normal and cancer cells.
Benzethonium chloride induced apoptosis at the concentration lower than its critical micelle
concentration (CMC) in rat thymocytes. Other quaternary ammonium surfactants such as
cetyltrimethylammonium bromide similarly increased biochemical and morphological
features of apoptosis whereas both anionic and amphoteric surfactants had no significant
effect on these apoptotic changes. These results suggest that the cationic charge of quaternary
ammonium surfactants rather than the surface action of these surfactants is involved with
onset of the apoptotic process. Benzethonium chloride increased the release of cytochrome c
and followed the activation of caspase-9 and -3. The treatment of benzethonium chloride also
led to apoptotic cell death in Jurkat cells. These results indicate that quaternary ammonium
surfactants induce apoptosis in normal and cancer cells.
- 360 -

Session X : Cell death in cancer Poster X, 28
Butylated hydroxyanisole is more than a reactive oxygen species scavenger
Nele Festjens1, Michael Kalai1,2, Joél Smet3, Ann Meeus1, Rudy Van Coster3, Xavier
Saelens1,4 and Peter Vandenabeele1
1Molecular Signalling and Cell Death Unit, Department for Molecular Biomedical
Research, Ghent University and Flemish Interuniversity Institute for Biotechnology
(VIB), Technologiepark 927, 9052 Ghent, Belgium; E-mail:
Nele.Festjens@dmbr.Ugent.be
2Laboratory of Cellular Microbiology, Unit of Molecular Microbiology, Institute
Pasteur of Brussels, Engelandstraat 642, 1180 Brussels, Belgium
3Department of Pediatrics, Division of Pediatric Neurology and Metabolism, Ghent
University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
4Molecular Virology Unit, Department for Molecular Biomedical Research, Ghent
University and VIB, Technologiepark 927, 9052 Ghent, Belgium
L929 fibrosarcoma cells treated with TNF or dsRNA die by necrosis, a process in which
mitochondria-produced reactive oxygen species (ROS) are crucial. We studied the effects of
two related chemical antioxidants, butylated hydroxyanisole (BHA) and butylated
hydroxytoluene (BHT). Although they reduced ROS levels similarly, BHA was clearly more
anti-necrotic than BHT, showing that inhibition of necrosis does not exclusively depend on
scavenging ROS. While BHA delays TNF-induced necrosis, it apparently does not block
dsRNA-induced cytotoxicity. However, cell death was shifted from necrosis to apoptosis.
BHT did not modulate cell death type indicating that inhibition of necrotic cell death does not
exclusively depend on scavenging ROS and implying that BHA has additional features
lacking in BHT. We then compared the effects of both antioxidants on the activities of
complexes of the mitochondrial electron transport chain (ETC) with those of specific ETC
inhibitors. BHA is more effective than BHT, but less than rotenone, in the inhibition of
NADH-dehydrogenase activity. However, rotenone is less efficient as anti-necrotic agent,
suggesting that other features of BHA contribute to its protective effect. We finally
demonstrate that phospholipase A2 and lipoxygenases play a role in signalling to or execution
of necrotic cell death. We conclude that the strong anti-necrotic effect of BHA reflects not
only its ROS scavenging property, but also its ability to inhibit complex I and lipoxygenases.
- 361 -

Session X : Cell death in cancer Poster X, 29
Combination of doxorubicin and sulforaphane for reversing doxorubicin-resistant
phenotype in mice fibroblasts with p53Ser220 mutation
Carmela Fimognari, Giorgio Cantelli-Forti, Patrizia Hrelia.
Department of Pharmacology, University of Bologna, Bologna, Italy. E-mail:
carmela.fimognari@unibo.it
Traditional cytotoxic chemotherapeutic approaches cannot cure most advanced solid
malignancies. The major factor that limits the effectiveness of chemotherapy in patients with
advanced cancer is the acquisition of resistance. Chemoresistance is a multifactorial process,
which includes alterations in drug accumulation, increased activity of gluthatione Stransferases,
loss of function and mutations of p53, etc. One strategy for reversing drug
resistance is the concomitant use of chemopreventive agents (i.e. non-cytotoxic agents able to
block the progression to invasive cancer) that are by themselves non-toxic but that cause a
better response rates than either reagent alone. Sulforaphane is one of the most promising
chemopreventive agent. Sulforaphane inhibits cell-cycle progression and induces apoptosis in
different tumor cell lines. The pro-apoptotic potential of sulforaphane could be effective
either alone or in combination with other therapeutic strategies in reversing chemoresistance.
We firstly investigated the effects of sulforaphane on mouse fibroblasts bearing a different
p53 status (wild-type, knock-out, mutated) for understanding whether its activity is prevented
by a mutated p53 status. p53-knock-out fibroblasts from newborn mice transfected with the
p53Ser220 mutation, observed in different human cancers, were used as a model of mutated
p53 status. Moreover, since p53Ser220 mutation fibroblasts showed a doxorubicin-resistant
phenotype, we secondly treated the cells with a combination of doxorubicin plus
sulforaphane. To clarify the optimal schedule of combination, we studied the effects of
simultaneous and sequential exposure to doxorubicin and sulforaphane. Taken together, our
results suggest that a mutated p53 status did not prevent the induction of apoptosis by
sulforaphane and that sulforaphane was able to reverse the resistance to doxorubicin when
administered simultaneously or after doxorubicin. The association sulforaphane-doxorubicin
may therefore allow doxorubicin to be administered at lower doses, thereby reducing its
potential toxicity.
- 362 -

Session X : Cell death in cancer Poster X, 30
Alterations in the mRNA expression of the novel, apoptosis-related gene, BCL2L12,
after treatment of human leukemic cell line HL60 with the antineoplasitic agent
etoposide.
Kostas V. Floros1, Hellinida Thomadaki1, Dimitra Florou1, Maroulio Talieri2 , and
Andreas Scorilas1
1 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of
Athens, GR-15701 Athens, Greece. E-mail: ascorilas@biol.uoa.gr
2‘G. Papanicolaou’ Research Center for Oncology, Saint Savas Hospital, GR-11522
Athens, Greece.
Apoptotic cell death is a high-regulated process, which plays a crucial role in many biological
events. Etoposide is an antineoplastic drug which targets the DNA unwinding enzyme,
topoisomerase II. It is observed that in specific concentrations it rapidly causes death of
myeloid leukemia cells by apoptosis. HL-60, a human promyelocytic cell line, is very
sensitive to a number of apoptosis-inducing agents. We investigated the possible alterations in
the expression of the novel apoptosis-related gene, BCL2L12, which was cloned by our
group, after treatment of the HL-60 cell line with etoposide. Apoptosis was assessed by DNA
laddering and cell toxicity was investigated by the MTT method. Total RNA was extracted
and cDNA was prepared by reverse-transcription. BCL2L12 and some other apoptosis-related
genes were amplified by PCR, using specific primers. #-Actin was used as a control gene.
Analysis of RT-PCR product demonstrated a gradual downregulation of BCL2L12 and more
than 5 fold decrease 4h after drug treatment. Our results suggest that mRNA expression
analysis of BCL2L12 in human promyelocytic leukemia cells HL-60 after treatment with
etoposide, may serve, after further studies, as new molecular factor predicting chemotherapy
outcome in human leukemia in the future.
- 363 -

Session X : Cell death in cancer Poster X, 31
Apoptotic cell death after Foscan(-induced photosensitization in respect of Foscan
subcellular localization
Aurélie François, Sophie Marchal, François Guillemin, Lina Bolotine
Centre Alexis Vautrin, CRAN UMR 7039 CNRS, INPL UHP Nancy I, Vandoeuvre-Lès-
Nancy
Subcellular localisation of photosensitizers is a main factor involved in cell death in response
to the photodynamic therapy (PDT), since the primary damage sites are closely related to the
distribution of sensitizers. In this study, we have studied the influence of the time of
incubation on the subcellular localisation of Foscan( and on the apoptotic cell death after
PDT.
At short incubation times (0.5 h to 3 h), Foscan( displays diffuse pattern with preferential
localisation in the endoplasmic reticulum (ER) and the Golgi apparatus. From 12h incubation
Foscan( is progressively excluded from the Golgi, and is tightly confined to ER by 24 hours.
Considering ER localization we further evaluated the response to ER stress induced by
Foscan(-PDT through the expression of the ER lumen protein GRP 78 (Bip). For the same
levels of photoinduced cell death the GRP 78 expression, assessed by Western Blot, was
much more pronounced for 24h Foscan( incubation. Apoptotic cell death after Foscan(-
PDT was assessed by the activation of caspases 12, 6 and 7 along with the PARP cleavage.
Similar to GRP 78 expression, a greater activation of caspase-dependent apoptotic pathway
was observed for 24h incubation compared to 3h.
In conclusion, for the given level of photocytotoxicity with Foscan( after 3h or 24h
incubation, the better ER stress was observed at prolonged incubation time, consistent with
ER localization of Foscan(. An enhanced apoptosis through caspase-12 activation with the
subsequent involvement of post-mitochondria caspases at 24h of incubation indicates that
apoptotic cell death is favoured from ER rather than Golgi localization.
- 364 -

Session X : Cell death in cancer Poster X, 32
Alternative splicing of TRAIL and DR5: first clues for their role in human renal cell
carcinoma
N Göbel, A Krieg, U Ramp, N Wethkamp, T Kempf, HE Gabbert, C Mahotka
Institute of Pathology, Heinrich Heine-University, Moorenstr.5, 40225 Düsseldorf,
Germany, nadine@wcv-mail.de
Objective: Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL/APO2L)
induces programmed cell death in a variety of neoplastic cell types, but only in a few nonneoplastic
cells. In this study, we report on the tumor progression dependent expression of
two novel alternative splice variants of TRAIL – identified by our group – in neoplastic and
non-neoplastic human cells lacking either exon 3 (TRAIL-beta) or exons 2 and 3 (TRAILgamma).
Moreover, we analyzed the expression of the TRAIL receptor DR5 (TRICK2) and
its alternative splice variants TRICK2a and TRICK2b during tumor progression of renal cell
carcinoma (RCC). Methods: Determination of tumour grading and staging by
histopathological examination, total RNA-isolation, RT-PCR, statistical analysis by the
Mann-Whitney and Wilcoxon test. Results: 1. TRAIL-alpha, -beta and -gamma are
significantly (p=0.029, 0.024, 0.008) higher expressed in non-neoplastic than in neoplastic
cells. 2. The expression ratio of TRAIL-beta and –gamma shows a significant (p=0.002)
difference in normal vs. tumor cells. 3. All TRAIL variants were not regulated during tumor
progression from pT1/2 to advanced pT3 stage. 4. The expression levels of the DR5 receptor
variants TRICK2a and TRICK2b were not affected in normal vs. tumor cells. Interestingly,
the expression levels of TRICK2a significantly (p= 0.012) increased in tumor stage pT1/2 vs.
non-neoplastic cells. 5. The expression of TRICK2b in pT1/2 stages significantly (p=0.037)
decreased in advanced tumor stages (pT3). Conclusions: Our data suggest that all three
TRAIL variants - mainly TRAIL-gamma - and TRICK2b are significantly decreased in tumor
cells, indicating their potential role in RCCs. Alternative splicing of the immature TRAIL-
RNA as well as the TRICK2-RNA might be involved in fine tuning of TRAIL-induced
apoptosis and underlines the complexity of the TRAIL-system in renal cell carcinoma.
- 365 -

Session X : Cell death in cancer Poster X, 33
Membrane fluidity changes are associated with benzo[a]pyrene-induced apoptosis in
F258 cells: protection by exogenous cholesterol
1Morgane Gorria, 1Xavier Tekpli, 2Odile Sergent, 1Laurence Huc, 3François Gaboriau,
1Mary Rissel, 1Marie-Thérèse Dimanche-Boitrel, 1Dominique Lagadic-Gossmann
1Inserm U620, Université de Rennes 1; 2UPRES EA 3891, Université de Rennes 1;
3Inserm U522, Hôpital Ponchaillou, 35000 Rennes, FRANCE. E-mail:
morgane.gorria@univ-rennes1.fr
Polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants, to which
humans are largely exposed. Besides their well-known carcinogenic effects, PAHs also
induce apoptosis in different cell types such as hepatic cells (Huc et al., Faseb J, 2004;
Solhaug et al., Carcinogenesis, 2004). Although activation of p53 plays a primordial role in
this apoptosis, permissive pathways might also occur; indeed, we have recently evidenced a
parallel activation of Na+/H+ exchanger (NHE1) with consequences on apoptosis occurrence.
Such an activation might result from changes in membrane characteristics, especially as
PAHs are highly lipophilic molecules and have been shown to interact with biological
membranes (Jimenez et al., Environ Toxicol Chem, 2002). This study was therefore to
investigate the effects of benzo[a]pyrene (B[a]P) on the membrane fluidity of F258 cells and
to test the role of these changes in the related apoptosis.
Using the fluorescence polarization technique and the DPH probe, we first demonstrated that
B[a]P (50 nM) induced an increase in bulk membrane fluidity following 48 h of exposure.
This result was confirmed using EPR technique and the 12-DSA spin label. Using cariporide
(30 µM) to inhibit NHE1 activation observed at 48 h (Huc et al., Faseb J, 2004), we further
showed that this change in fluidity was partly related to this transporter. Exogenous
application of cholesterol (30 mg/mL), a well known membrane stabilizer, was then used to
test the involvement of membrane fluidization in B[a]P-induced apoptosis. Our data
demonstrated that a co-treatment with this compound, besides inhibiting any membrane
fluidization, significantly reduced apoptosis, as evidenced by a decrease of cell population
exhibiting nuclear fragmentation and of caspase 3/7 activity. We further showed that the
protective effect of cholesterol was mainly through inhibition of B[a]P-induced iron uptake,
leading to subsequent reduction of oxidative stress, and hence apoptosis.
In conclusion, this work suggest a role for membrane fluidity in early events of B[a]P-induced
apoptosis and an implication of NHE1 activation in B[a]P-induced membrane fluidization.
- 366 -

Session X : Cell death in cancer Poster X, 34
INVOLVEMENT OF TRACE ELEMENT CONTAINING-PROTEINS IN THE
APOPTOSIS AND REDOX PROCESSES IN THE PROSTATE AND HUMAN
PROSTATE CELLS
I. Grbavac, C. Wolf, N. Wenda, D. Alber, M. Kühbacher, D. Behne, A. Kyriakopoulos
Hahn-Meitner-Institut Department for molecular trace element research in the life
science Glienicker Str. 100, 14109 Berlin, Germany
It is well known that trace elements play a significant role in many functions of the human
body. Most of them are bound to proteins and have an important function like selenium and
zinc in the prostate. Trace elements like copper and zinc are known for their role in the
apoptosis of different cells. In order to obtain more information about several trace elements
in this organ, analytical methods such as instrumental neutron activation analysis (INAA) and
ICP-MS, which are appropriate procedures to determine trace elements, were used. To find
out, whether some of these elements are bound to proteins of the prostate, the gland was
homogenized and the proteins in the homogenate were separated by electrophoretic
techniques. Several antibodies against specific prostate proteins were tested. By means of
these antibodies several trace element containing-proteins such as selenoproteins and Cu- Znproteins
could be identified which are involved in the apoptosis and redox processes. By
combining of SEC and ICP-MS some trace elements, which are bound to proteins were
detected in the prostate cytosol and human prostate cell lines. The spectra of the SEC-ICP-MS
let assume that trace elements like copper, zinc, manganese and others have a strong affinity
to several proteins in the prostate. The finding of some new metals- and metalloids containing
proteins in the micro-organelles of the prostate, let assume the existence of metalloproteins
and metalloidproteins in this organ which might be play a role in the apoptosis and redox
system. In this paper some characteristics are present with regard to the newly found trace
element -containing proteins and their relationship to the redox processes.
- 367 -

Session X : Cell death in cancer Poster X, 35
A Bench-to-Bed-Side Approach for the Identification and Validation of Novel
Compounds to Induce Apoptosis of Cancer Cells
Maria Hägg1, Takayuki Ueno1, Maria Berndtsson1, Aleksandra M. Havelka1, Frank
Neumann2, Heiko van der Kuip3, Thomas E. Murdter3, Maria C. Shoshan1, Masakazu
Toi4 and Stig Linder1
1Cancer Center Karolinska, Department of Oncology and Pathology, R8:03, Karolinska
Institute and Hospital, S-171 76 Stockholm, Sweden. 2BIOAXXESS Technology, Moor
Court Farm, Upper Pendock WR13 6JW, UK, fneumann@bioaxxess.com, 3Dr
Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, D-
70376 Stuttgart, Germany, 4Department of Surgery and Breast Oncology, Tokyo
Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, 113-8677,
Tokyo, Japan
Cell based screening offers the advantage that active, cell permeable, agents such as those that
induce tumor apoptosis can directly be identified. Two-dimensional tissue culture conditions
are, however, very different from those of the human tumor environment and it is difficult to
decide which compounds should be developed for further pre-clinical and clinical studies.
We here describe an approach based on the assessment of a caspase-cleaved protein fragment
of cytokeratin-18 released from apoptotic cells that can be used to identify apoptosis-inducing
drugs, assess their potency to induce apoptosis in organ cultures of human tumors, and finally
monitor carcinoma cell apoptosis in vivo by using plasma samples from drug-treated mice
bearing human tumor xenografts. Finally, this assay can be used to address nearly in real time
the drug-induced tumor cell apoptosis by using patient blood samples.
This method should be useful to facilitate the drug development process by bridging the gap
between screening of drugs to clinical phase I studies.
[1] Linder S, Havelka AM, Ueno T, Shoshan MC. Determining tumor apoptosis and necrosis
in patient serum using cytokeratin 18 as a biomarker.
Cancer Lett. (2004) 214: 1-9
- 368 -

Session X : Cell death in cancer Poster X, 36
Antigens and cytokine gene in antitumoral vaccines: the importance of the temporal
delivery sequence in the antitumor signals
Maria Jose Herrero, Rafael Botella, Francisco Dasi, Rosa Algas, Maria Sanchez,
Salvador F. Aliño.
Laboratory of Gene Therapy, Department of Pharmacology, Faculty of Medicine,
University of Valencia. Avda Blasco Ibáñez, 15. 46010 Valencia-España. E-mail:
mariajoseherrero@ono.com
Different studies against cancer in the last years, including clinical trials, have shown that a
correct activation of the immune system can lead to tumor rejection but also, on the other
hand, an incorrect signalling results in no positive effects or even anergy. How the different
signals must be given to the immune system in order to get one or the other response is not
clear yet. Knowing that the genetic cell vaccines with cytokine genes as gm-csf (granulocyte
and macrophage colony stimulating factor) are the best to mediate efficient antitumoral
responses, we have worked assuming that both signals, GM-CSF and tumor antigens, are
crucial. In order to study which is the ideal temporal sequence for their administration we
have worked in a murine model of antimelanoma vaccine, with B16 cells and C57/BL6 mice,
using tumor membrane protein antigens (TMP) or whole tumor cells in combination with gmcsf
transfer before or after the antigen delivery. B16 cells were transfected using PEI
(polyethyleneimine) polyplexes (0.02 mg/ml). Tisular transfection in TMP experiments was
performed by sc. injection of 0.01 or
0.05 mg m-gmcsf in 0.5 ml saline solution. Our results show that: 1) when gmcsf tisular
transfection is performed before TMP delivery, a good dose-dependent correlation in tumor
volume decrease is observed, but with a limit effect; in contrast, when higher doses of antigen
were administered before DNA transfection, more antitumor efficacy was obtained. 2) A
similar behaviour, but with stronger positive results, was observed employing whole tumor
cells as antigens. Thus, an inefficient or efficient (total survival of treated mice) antitumoral
response was observed when DNA was administered before or after the cell vaccine,
respectively. We conclude that optimal antitumoral response can be obtained when a high
antigen signal is given before (or simultaneously) to GMCSF production, while the inversion
of the signals could result in the non desired inhibition or anergy of the immune response.
- 369 -

Session X : Cell death in cancer Poster X, 37
Epigallocatechin Gallate Dose-Dependently Induces Apoptosis or Necrosis in Human
MCF-7 Cells
Yan-Der Hsuuw1 and Wen-Hsiung Chan2
1. Department of Animal Science, National Pingtung University of Science and
Technology, Neipu, Pingtung, Taiwan 91201. E-mail: hsuuw@yahoo.com.tw
2. Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan
Christian University, Chung Li, Taiwan 32023. E-mail: whchan@cycu.edu.tw
The catechins, a family of polyphenols found in tea, can evoke various responses, including
cell death. However, the precise molecular mechanisms of these effects are unknown. Here,
we demonstrate that treatment of human MCF-7 cells with 50 µM (-)-Epigallocatechin-3gallate
(EGCG), a catechin that is highly abundant in green tea, can induce apoptotic changes,
including mitochondrial membrane potential changes and activation of c-Jun N-terminal
kinase (JNK), caspase-9 and caspase-3. In contrast, higher concentrations of EGCG (100-400
µM) do not induce apoptosis, but rather trigger necrotic cell death in MCF-7 cells.
Investigations of the possible mechanisms underlying these differences revealed that
treatment with lower concentrations of EGCG (10-50 µM) directly increased intracellular
oxidative stress, while higher concentrations (100-400 µM) did not. Immunoblotting revealed
that treatment of MCF-7 cells with 10-50 µM EGCG caused increases in Bax protein levels
and decreases in Bcl-2 protein levels, shifting the Bax/Bcl-2 ratio to favor apoptosis, while
treatment with 100-400 µM EGCG had no such effect. Moreover, we observed a dosedependent
decrease in intracellular ATP levels in cells treated with high-dose EGCG.
Blockade of reactive oxygen species (ROS) generation and ATP synthesis using antioxidants
and ATP synthesis inhibitors revealed that ROS and ATP play important roles to switch cell
death types with apoptosis or necrosis. Collectively, these results indicate for the first time
that EGCG treatment has a dose-dependent effect on ROS generation and intracellular ATP
levels in MCF-7 cells, leading to either apoptosis or necrosis, and that the apoptotic cascade
involves JNK activation, Bax expression, mitochondrial membrane potential changes, and
activation of caspase-9 and caspase-3.
- 370 -

Session X : Cell death in cancer Poster X, 38
Cooperation of Amphiregulin and IGF1 Inhibits Bax- and Bad-mediated Apoptosis via a
PKC-dependent pathway in non-small cell lung cancer cells
Amandine HURBIN, Carole NIANG, Jean-Luc COLL and Marie C. FAVROT
Lung Cancer Research Group, INSERM U578, Institut Albert Bonniot, 38706 La
Tronche Cedex, France. E-mail : Amandine.Hurbin@ujf-grenoble.fr
Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to
promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1)
AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322
NSCLC cells from serum-starved apoptosis; 2) H358 cells resist to Bax-induced apoptosis
through an inhibition of Bax conformational change. We show here that the anti-apoptotic
activity of AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C
and staurosporine, but not by the MAPK and PI3K inhibitors PD98059 and wortmannin,
suggesting the involvement of a PKC-dependent, MAPK- and PI3K-independent survival
pathway. The PKC% inhibitor rottlerin restores apoptosis induced by serum deprivation. In
addition, phosphorylation of PKC and PKC / , but not of PKCalpha/#II, increases in
serum-starved H358 cells and in H322 cells treated with AR/IGF1 combination and is
blocked by calphostin C. Combination of AR and IGF1 increases p90Rsk and Bad
phosphorylation as well as it inhibits the conformational change of Bax by a PKC-dependent
mechanism. Finally, PKC , PKC or p90Rsk siRNAs block the anti-apoptotic activity of
AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each
factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced
apoptosis, whereas a catalytically inactive form of p90Rsk restores it. Thus, AR and IGF1
cooperate to prevent apoptosis by activating a specific PKC-p90Rsk-dependent pathway,
which leads to Bad and Bax inactivation. This signalling pathway is different to that used by
single factor.
- 371 -

Session X : Cell death in cancer Poster X, 39
Non-autonomous p53/p21 mediated signaling in tumor – stroma interactions
Hippokratis Kiaris, George Trimis & Ioulia Chatzistamou
Department of Biochemistry, University of Athens Medical School, M. Asias 75, 115 27
Athens, Greece. E-mail: hkiaris@med.uoa.gr
During carcinogenesis stromal fibroblasts, in parallel with the neoplastic cells, undergo
certain changes and progressively enter a cancer-associated state. Despite however recent
progress in understanding the role of fibroblasts in tumor biology, how this process is being
regulated remains obscure. The finding that tumor fibroblasts frequently harbor p53 mutations
in breast and other cancers prompted us to address the role of fibroblasts’ p53 in
tumorigenesis. Tumor transplantation experiments showed that that indeed, p53 ablation in
the hosts induced reduced the latency for the development of MCF7 human breast tumors.
Co-innoculation of MCF7 cells with fibroblasts differing in the status of p53 showed that this
finding is at least in part due to the stromal fibroblasts. Subsequently we asked if these effects
of fibroblasts’ p53 are mediated by a p21waf1/Cip1 (p21)/dependent mechanism. Therefore,
analogous tumor co-inoculation experiments have been performed in SCID mice with MCF7
cells and fibroblasts isolated from wild type and p21 deficient animals. Our results show that
p21 deletion in stromal fibroblasts accelerates tumorigenesis by non-autonomous
mechanisms. This non-autonomous stimulation of MCF7 growth by p21 ablation in
fibroblasts was confirmed in vitro in co-culture experiments and also showed that at least in
part it might be due to the increased expression of transforming growth factor-b and stromal
cell derived factor-1. Apparently, these findings were biologically relevant because in human
benign fibroadenomas p21 expression is clustered proximal to the mammary epithelial cells
while in invasive breast cancers stromal fibroblasts express p21 randomly, in a mosaic
pattern. Collectively our results imply that stromal p53/p21 play important rolesin
tumorigenesis and that modulation of endogenous p21 expression in stromal fibroblasts of
primary tumors might be implicated, in the regulation of neoplastic growth.
- 372 -

Session X : Cell death in cancer Poster X, 40
ER stress induces Jpk, which inhibits cell cycle progression in F9 teratocarcinoma cell
Hye Sun Kim, Kyoung-Ah Kong, Hyunjoo Chung, Sungdo Park, and Myoung Hee Kim
Department of Anatomy, Embryology Lab., Brain Korea 21 Project for Medical
Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
E-mail: goldfish79@hanmail.net; mhkim1@yumc.yonsei.ac.kr
A Jopock (Jpk), a trans-acting factor associating with the position-specific regulatory element
of murine Hoxa-7, has shown to induce cell death in both prokaryotic and eukaryotic cells
when introduced and overexpressed. Since Jpk protein harbors a transmembrane domain
(TM) and a putative ER-retension signal at the N-terminus, a subcellular localization of the
protein was analyzed after fusing it into the green fluorescent protein (GFP). Both N-term
(Jpk-EGFP) and C-term fused-Jpk (EGFP-Jpk) showed to be localized in the endoplasmic
reticulum (ER) when analyzed under the fluorescence microscopy after staining the cells with
ER- and MitoTracker. Through deletion analysis TM turned out to be important for ER
localization of Jpk. When flow cytometric analysis was performed, both cells expressing Jpk-
EGFP and EGFP-Jpk led cell cycle arrest and subsequent apoptotic cell death. In order to see
whether Jpk is expressed during ER-stress mediated apoptosis, F9 cells were treated with
DTT, an ER-stress inducer. In the presence of 4 mM of DTT, about 50% of cells died
strongly expressing (7 fold) Jpk as well as Grp78, a molecular chaperone and Chop-10, a well
known protein arresting cells at the G0/G1 phase and apoptosis. In summery, excess ER stress
inducing apoptosis upregulated the expression of Jpk, which seemed to inhibit the cell cycle
progression. These results altogether suggest that Jpk could be a useful cell death triggering
molecule applicable for cancer therapy.
- 373 -

Session X : Cell death in cancer Poster X, 41
Buddlejasaponin IV and Pleurospermum kamtschaticum extract induce apoptosis by
reducing alpha(2)beta(1) integrin-mediated adhesion in HT-29 human colon cancer cells
Jin-Eun Kim1,2,3, Won-Yoon Chung1,2, Hee-Juhn Park4, Hyun-Ju Jung4 and Kwang-
Kyun Park1,2,3
1Department of Oral Biology, 2Oral Science Research Center, College of Dentistry,
3Brain Korea 21 Project for Medical Science, Yonsei University, Seoul 120-752,
4Department of Botanical Resources, Sangji University, Wonju 220-702, E-mail:
judyjean@hanmail.net
Buddlejasaponin " (BS-") is one of the active components of
Pleurospermum kamtschaticum (Umbelliferae). The aerial part of P. kamtschaticum is used
traditionally to treat cold, atherosclerosis, arthritis and to overcome fatigue in Korea.
However, the antitumor effect of the aerial part of P. kamtschaticum extract (Pl-K) and BS-"
has not been studied. Integrins are cell surface adhesion receptors that regulate cell survival
and cell growth in response to cues derived from the extracellular matrix. Alpha(2)beta(1)
integrin downregulation induces apoptosis in various colon cancer cell lines. We investigated
whether apoptosis induced by Pl-K and BS-" was associated with alterations in integrin
signaling. Pl-K and BS-" induced apoptosis in HT-29 cells through mitochondrial dependent
pathway. Additionally, Pl-K and BS-" decreased the cell attachment to type# and "
collagen, resulting from the downregulation of alpha(2)beta(1) integrin. Furthermore, Pl-K
and BS-" inhibited expression and phosphorylation of FAK, Akt, and ERK. These results
suggest that Pl-K and BS-" induce apoptosis and inhibit survival by reducing alpha(2)beta(1)
integrin-mediated adhesion in HT-29 cells.
- 374 -

Session X : Cell death in cancer Poster X, 42
Caspase activation and extracelluar signal regulated kinase inhibition were involved in
luteolin induced apoptosis in Lewis lung carcinoma cells
Jin-Hyung Kim, Eun-Ok Lee, Hyo-Jung Lee, Sung-Hoon Kim
Department of Oncology, Graduate School of East-West Medical Science, KyungHee
University, Yongin 449-701, Republic of Korea. E-mail:sungkim7@khu.ac.kr
Luteolin, a naturally occurring flavonoid, was reported to inhibit angiogenesis, DNA
topoisomerase I and induce apoptosis in different cancer cells. However, the apoptotic
mechanism and in vivo efficacy of luteolin still remains unclear. Thus, in the present study,
we examined the underlying molecular mechanism of luteolin and its effect on in vivo tumor
growth of Lewis lung carcinoma (LLC) cells. Luteolin exhibited anti-proliferative activity
against LLC cells with IC50 of 12 µ). Luteolin effectively increased Annexin V positive
cells as well as sub G1 DNA peaks by flow cytometric analysis. Western blotting has revealed
that luteolin effectively activates the expression of caspase 9 and 3, cleaves poly(ADP-ribose)
polymerase (PARP) and increases the ratio of Bax/Bcl-2. Furthermore, mitochondrial
membrane potential was reduced in a concentration dependent manner by luteolin. However,
it did not affect the expression of protein kinase c (PKC) "/#, PKC & and pan PKC, while it
down-regulated the expression of extracellular signal-regulated kinase (ERK). In addition,
luteolin significantly inhibited the growth of LLC cells implanted on the flank of mice to 40
% and 60% of untreated control group at 2 mg/kg and 10 mg/kg, respectively. Similarly,
luteolin significantly reduced the expression of proliferating cell nuclear antigen (PCNA) as
well as increased the expression of Terminal deoxynucleotidyl transferase biotin-dUTP nick
end labeling (TUNEL) in tumor section of LLC bearing mice by immunohistochemistry.
Taken together, these results suggest that luteolin exerts anti-tumor activity by caspase
activation and ERK inhibition.
- 375 -

Session X : Cell death in cancer Poster X, 43
The anti-cancer activity of decursin and decursinol isolated from Angelica gigas Nakai
Mi-Jeong Kim1,2, Won-Yoon Chung1, Jin-Eun Kim1,2, Seung Hwa Son1, Sang Kook
Lee3, Kwang-Kyun Park1,2
1Department of Oral Biology, Oral Science Research Institute, Oral Cancer Research
Institute, Yonsei university College of Dentistry, Seoul 120-752, Republic of Korea,
2Brain Korea 21 Project for Medical Science, Yonsei University, Seoul Republic of
Korea 120-752, Republic of Korea, 3College of Pharmacy , Ewha Womans University,
Seoul Republic of Korea 120-750 E-mail: kimmi780507@hanmail.net
Angiogenesis, the process of vascular growth by sprouting of preexisting vessels, plays a
crucial role in tumor growth and metastasis. Vascular endothelial cell proliferation and
migration are critical steps in angiogenesis and are regulated by various growth factors such
as vascular endothelial growth factor (VEGF). Angelica gigas Nakai root has been
traditionally used in Korean folk medicine as a tonic and for treating anemia and other
common disease. In the present study, we observed that decursin and decursinol isolated from
A. gigas inhibited VEGF-induced proliferation and migration of human umbilical vein
endothelial cells (HUVECs). Also, these compounds showed antiangiogenic activity in a
mouse Matrigel plug assay and CAM assay. In addition, decursin and decursinol inhibited the
formation of tumor nodules in spontaneous lung metastasis induced with murine colon cancer
CT-26 cells in BALB/c mice. Our results suggest that decursin and decursinol possess an
antimetastatic potential by suppressing VEGF-induced angiogenesis. Furthermore, to explain
the mechanism for how decursin and decursinol block VEGF-induced angiogenesis, we
investigated their effect on PI3k/Akt/eNOS signaling in HUVECs.
- 376 -

Session X : Cell death in cancer Poster X, 44
ER stress induces Jpk, which inhibits cell cycle progression in F9 teratocarcinoma cell
Hye Sun Kim, Kyoung-Ah Kong, Hyunjoo Chung, Sungdo Park, and Myoung Hee Kim
Department of Anatomy, Embryology Lab., Brain Korea 21 Project for Medical
Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
E-mail: goldfish79@hanmail.net; mhkim1@yumc.yonsei.ac.kr
A Jopock (Jpk), a trans-acting factor associating with the position-specific regulatory element
of murine Hoxa-7, has shown to induce cell death in both prokaryotic and eukaryotic cells
when introduced and overexpressed. Since Jpk protein harbors a transmembrane domain
(TM) and a putative ER-retension signal at the N-terminus, a subcellular localization of the
protein was analyzed after fusing it into the green fluorescent protein (GFP). Both N-term
(Jpk-EGFP) and C-term fused-Jpk (EGFP-Jpk) showed to be localized in the endoplasmic
reticulum (ER) when analyzed under the fluorescence microscopy after staining the cells with
ER- and MitoTracker. Through deletion analysis TM turned out to be important for ER
localization of Jpk. When flow cytometric analysis was performed, both cells expressing Jpk-
EGFP and EGFP-Jpk led cell cycle arrest and subsequent apoptotic cell death. In order to see
whether Jpk is expressed during ER-stress mediated apoptosis, F9 cells were treated with
DTT, an ER-stress inducer. In the presence of 4 mM of DTT, about 50% of cells died
strongly expressing (7 fold) Jpk as well as Grp78, a molecular chaperone and Chop-10, a well
known protein arresting cells at the G0/G1 phase and apoptosis. In summery, excess ER stress
inducing apoptosis upregulated the expression of Jpk, which seemed to inhibit the cell cycle
progression. These results altogether suggest that Jpk could be a useful cell death triggering
molecule applicable for cancer therapy.
- 377 -

Session X : Cell death in cancer Poster X, 45
Akt involvement in paclitaxel chemoresistance of human ovarian cancer cells
Su-Hyeong Kim 1, Yong-Sung Juhnn 1,2, Yong-Sang Song1,3
1Cancer Research Institute, 2Department of Biochemistry and Molecular Biology,
3Department of Obstetrics and Gynecology, Seoul National University, College of
Medicine Seoul, 110-799, Korea, E-mail:yssong@snu.ac.kr
Paclitaxel (taxol) is extensively used clinically for chemotherapy of various cancers including
ovarian cancer. Although paclitaxel induces apoptosis in cancer cells, its exact mechanism of
action still remains to be determined. Akt mediates survival signals that might protect cancer
cells from apoptosis and thus is a potentially important therapeutic target. Here, we
demonstrated that inhibition of Akt increases the efficacy of the paclitaxel-induced apoptosis
using SKOV3 and PA-1 human ovarian cancer cells.
The sensitivity to paclitaxel of SK0V3 and PA-1 cells was examined using the MTT assay. At
a concentration of 30 µM, PA-1 cells were more sensitive to paclitaxel than SKOV3 cells.
Apoptosis was accompanied by release of cytochrome c into the cytoplasm and cleavage of
poly (ADP-ribose) polymerase (PARP). To further elucidate the mechanism of sensitization
by paclitaxel, we explored the difference in the Akt phosphorylation between paclitaxelresistant
SKOV3 cells and paclitaxel–sensitive PA-1 cells. The higher level of phosphorylated
Akt was shown in SKOV3 cells than in PA-1 cells in response to paclitaxel.
Inhibition of Akt by specific phosphatidyinostiol-3-kinase (PI3K)-Akt (Wortmannin, and
LY294002) increased the efficacy of the paclitaxel-induced apoptosis in both cells.
These results suggest that combination therapy of paciltaxel with the Akt inhibitor may
increase the therapeutic efficacy of paclitaxel.
- 378 -

Session X : Cell death in cancer Poster X, 46
Cytoplasmic fraction of Lactococcus latic ssp. Lactis induces apoptosis and G0/G1 cell
cycle arrest in SNU1 human stomach cancer cells
Seo Young Kim, Ki Won Lee, Min A Jeong, Ji Yeon Kim, and Hyong Joo Lee*
Department of Food Biotechnology, School of Agricultural Biotechnology, and Center
for
Agricultural Biomaterials, Seoul National University, Seoul 151-742, Republic of Korea
Lactic acid bacteria are known to have antitumor activity, but the underlying mechanisms
remain unclear. The present study investigated antiproliferative activity of cytoplasmic
fraction of Lactococcus latic ssp. (L.lac CF) on SNU-1 human stomach cancer cell line (SNU-
1 cells) and underlying molecular mechanisms. The proliferation of SNU-1 cells was
inhibited by the treatment of L.lac CF in a time- and dose-dependent. In addition, L.lac CF
showed a significantly more relevant G0/G1 cell cycle arrest, which associated increase of
p21 and reduction of cyclin D1 and phosphorylation level of Rb. Furthermore, L.lac CF
induced apoptosis of SNU-1 cells, but not SNU-C2A human colon cancer cells, as evidenced
by nuclei condensation, increase of sub-G1 peak, and DNA fragmentation. In particular, p53
and bcl-2 appears to play a critical role in the apoptosis of SNU-1 cells by L.lac CF. We
further investigated if arginine deiminase (ADI) is involved in the antiproliferative activity of
L.lac CF, since arginine is known as essential amino acid for growing cells. Surprisingly, only
L.lac CF, which showed significantly strong antiproliferative activity and ADI activity among
tested six LAB. The concentration of L.lac CF showed linear relationship with both ADI
activity and antiproliferative activity. Furthermore, addition of L-arginine recover the
antiproliferative activity of L.lac CF. We purified ADI from L.lac CF by subsequent
fractionations, and the IC50 value of final ADI fraction was 2 µg/ml. The ADI fraction also
exerted apoptosis as evidenced by nuclei condensation in cells. These results, taken together,
indicate that the antiproliferative activity of L.lac CF on SNU-1 is attributable to induction of
G0/G1 cell cycle arrest and apoptosis, particularly the latter one by ADI.
- 379 -

Session X : Cell death in cancer Poster X, 47
Involvement of AMPK signaling cascade in capsaicin-induced apoptosis of
HT-29 colon cancer cells
Young Min Kim 1 Jin-Taek Hwang 2 , Dong Wook Kwak 1 , Yun Kyung Lee 1 and
Ock Jin Park 3
1 Department of Biological Sciences, Hannam University, Daejeon 306-791, Korea
2 Department of Biochemistry and Molecular Biology, Medical Research Center for
Bioreaction to Reactive Oxygen Species, Kyung Hee University College of Medicine,
Seoul 130-791, Korea
3 Department of Food and Nutrition, Hannam University, 133 Ojeong-dong Daedeok-gu,
Daejeon 306-791, Korea, E-mail: ojpark@hannam.ac.kr
Capsaicin has been used in food additives and drugs. In animal studies there are conflicting
reports of the effects of capsaicin on carcinogenesis. Capsaicin itself was mutagenic and
promoted tumor formation, whereas it showed anticarcingenic and antimutagenic properties.
Capsaicin induces apoptosis in certain types of cancers, but the molecular mechanism of
apoptosis caused by capsaicin has not been elucidated. In this study, we have investigated the
effects of capsaicin on apoptosis in relation to AMPK (AMP-activated protein kinase)
activation in colon cancer cell. Capsaicin-induced apoptosis was revealed by presence of
nucleobodies in the capsaicin-treated HT-29 colon cancer cells. Concomitantly, the activation
of AMPK and the increased expression of the inactive form of acetyl-CoA carboxylase
(ACC) were detected in capsaicin-treated colon cancer cells. We showed that both capsaicin
and AICAR, an AMPK activator possess the AMPK activating capacity as well as apopotosisinducing
properties. Also, compared to genistein or EGCG, capsaicin exhibited the similar
AMPK activating capacity. We suggest that AMPK is an important component of apoptosis
induced by capsaicin in colon cancer cells further implying AMPK as a possible target of
cancer control.
- 380 -

Session X : Cell death in cancer Poster X, 48
Ceramide–modulation of antigen-triggered Ca2+-signals and cell fate: a diversity in the
responses by different lymphoid and myeloid cells.
Endre Kiss, Gabriella Sármay and János Matkó
Eötvös Lorand University, Institute of Biology, Department of Immunology, Budapest,
Hungary
Release of ceramide (Cer) from plasma membrane sphingomyelin upon cell death, stress and
inflammation stimuli is a signal mediating the mitochondrial cell death pathway in various
cell types. We have shown recently that Cer-accumulation differentially affects T-cell fate
(apoptosis vs. survival) depending on its strength and duration [Detre et al, Cellular Signalling
2006. 18:294-306]. Moderate, non-apoptotic Cer stimuli suppressed the early and late events
of both antigen-specific or polyclonal T-cell activation signalling. K+ channels (Kv1.3, KCa)
as regulators and Ca2+ channels (CRAC, VDCC) as executors of the calcium influx were
proposed so far as potential targets of Cer-mediated inhibition. The molecular background of
the ceramides’ modulatory effect, however, still remained unresolved. Here we analyzed how
general is this modulation among further antigen-dependent cells of the immune system, with
attention to their maturation or differentiation stage, as well. The antigen-dependent calcium
signals of murine (IP12-7) and human (Jurkat) T cells, as well as of mast cells (RBL-2H3)
were remarkably inhibited by short (10 min) C2-Cer stimuli, in a concentration dependent
manner. Similar, but much less inhibition was observed in murine B splenocytes and
immature or mature B cell lines, 38C13 and A20, respectively. In contrast, in two human
Burkitt’s lymphoma B cell lines (BL41 and ST486) or the X16C murine B cell line of
marginal zone origin the magnitude of the antigen-BCR mediated calcium response was
uneffected or enhanced by Cer compared to the untreated cells. Interestingly, the sensitivity of
the above cells to Cer-mediated apoptosis (long duration C2-Cer), monitored through
mitochodrial depolarization and DNA-fragmentation, showed a similar diversity. The
Burkitt’s lymphoma cells were highly resistant, in contrast to the high and Cer dosedependent
apoptotic rate of T cells. The murine immature and mature B cells did not display
any apoptotic marker either, but became PI-positive (necrotic), depending on the ceramide
dose. Our data together suggest that the antigen-dependent immune cells have differential
sensitivity to ceramide-generating death or stress signals. Understanding the mechanisms
underlying these differences needs further investigations, particularly on Cer-induced
reorganization of the plasma membrane and identification of further molecular targets for
ceramide action involved in modulation of antigen-induced activation signals.
- 381 -

Session X : Cell death in cancer Poster X, 49
Sensitivity of human glioma cells to pro-apoptotic cannabinoid treatment is due to
specific expression of cannabinoid receptors
Liliana Konarska 1, Aleksandra Ellert-Miklaszewska 1, Bozena Kaminska 2, Wieslawa
A. Grajkowska 3, Konrad Gabrusiewicz 2, Malgorzata Danilkiewicz 2
1Dept. of Biochemistry and Clinical Chemistry, Medical University, Banacha 1 str., 02-
091 Warsaw, Poland, Email: konarska@nencki.gov.pl 2Lab. of Transcription
Regulation, Nencki Institute, Pasteura 3 str., 02-093 Warsaw, Poland, 3Dept. of
Pathology, Children’s Memorial Health Institute, Dzieci Polskich 20 str., 04-730
Warsaw, Poland
Cannabinoids, originally derived from Cannabis sativa, have been recently extensively
studied as potential antitumoral agents. In the current study we examined whether synthetic
cannabinoids with different receptor specificity are able to induce apoptosis in human glioma
cells and whether selective CB2 receptor activation is sufficient to effectively kill tumor cells.
Human glioma cell lines derived from highly malignant brain tumors, including T98G,
U373MG, U87MG and LN229 cells as well as 3 primary human glioma cell lines T3, T10
and T1Y1 were exposed to WIN55,212-2 (non-selective CB1/CB2 agonist) and JWH133
(CB2-selective agonist). The expression of CB1 and CB2 cannabinoid receptors was
investigated by RT-PCR and immunocytochemistry. WIN-55,212-2 decreased cell viability in
all examined cell lines and induced severe changes in cell morphology. Susceptibility of the
cells to JWH133 treatment correlated with the CB2 cannabinoid receptor expression. Both
cannabinoids, once effective, triggered a decrease of mitochondrial membrane potential,
cleavage of caspase-9 and effector caspases. Using immunohistochemistry we evaluated the
CB2 receptor expression in paraffin sections from various histopathological types of human
gliomas. Most of the analyzed tumors expressed significant levels of CB2 receptor. The
extent of CB2 expression in the tumour specimens was related to tumour malignancy, which
was in line with our RT-PCR and immunocytochemistry studies. We conclude that
cannabinoids are efficient inhibitors of human glioma cells growth, once the cells express
specific type of cannabinoid receptor. Due to the presence of the CB1 receptors on normal
cells, the use of a selective CB2 receptor agonist, such as JWH133, seems to be a better
choice for in vivo studies and in terms of potential therapeutic approaches. Supported by the
grant No. 06/2002 from the Polish Pharmacy and Medicine Development Foundation by
Polpharma S.A.
- 382 -

Session X : Cell death in cancer Poster X, 50
Role of MAPK kinase signalling pathways in photoinduced apoptosis in
cancer cells
Jarmila Kralova1, Michal Dvorak1, and Vladimir Kral2
1 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,
Flemingovo nám 2, 166 37 Prague 6, E-mail: kralova@img.cas.cz, mdvorak@img.cas.cz;
2 Department of Analytical Chemistry, Institute of Chemical Technology, Technická 5,
166 28 Prague 6, Czech Republic. E-mail: Vladimir.Kral@vscht.cz
Oxidative stress, such as photodynamic therapy (PDT), can act as an apoptosis inducer. In the
present study we investigated apoptotic pathways activated by novel photosensitizer
tetrakis(p-oligoethylenglycol) pentafluorophenyl porphyrin-mediated PDT in human
promyelocytic leukemia HL-60 and mouse mammary carcinoma 4T1. We show that p38 and
JNK MAP kinases are markedly activated during this process. Blocking the p38 MAPK
activation by specific inhibitor SB203580 or PD169316 resulted in the suppression of
apoptosis contrary to JNK blocking by specific inhibitor SP600125 that rather led to its
enhancement. The onset of apoptotic events + cytochrome c release, caspase activation and
dramatic changes in the expression of some Bcl-2 family members (down-regulation of Bcl-2
and Bak, cleavage of Bad and Mcl-1, activation of pro-apoptotic Bid protein) appeared
concurrently within 30 min following PDT treatment in a time-response manner. Apoptosis
could also be partly inhibited by overexpression of Bcl-2. Pre-incubation of cells with caspase
family inhibitor, fluoromethylketone (Z-VAD-FMK), significantly reduced apoptosis, while
individual caspase-specific inhibitors had only a partial effect. These observations indicate
that multiple signalling pathways, which play diverse roles in the apoptotic process, are
activated during PDT in a cell type-specific manner. The p38 MAPK signalling pathway
seems to be directly involved in the activation, while the Akt – JNK1 pathway in the cellular
resistance against PDT-induced apoptosis. Multi-functional signalling network enables
additional augmentation of the apoptotic process e.g. through activation of pro-apoptotic Bcl-
2 family protein Bid and caspase 8.
- 383 -

Session X : Cell death in cancer Poster X, 51
Analysis of interleukin-24-induced signalling in melanoma cells
Stephanie Kreis*, Georg A. Munz**, Laure Dumoutier***, Claude Haan*, Waraporn
Komyod**, Jean-Christophe Renauld***, Peter C. Heinrich** and Iris Behrmann*
*Laboratoire de Biologie et Physiologie Integrée, Université du Luxembourg, 162a,
avenue de la Faïencerie, L-1511 Luxemburg, ** Dept. of Biochemistry, RWTH Aachen
Medical School, D-52074 Aachen, Germany, ***Ludwig Institute for Cancer Research,
University Catholique de Louvain, B-1200 Brussels, Belgium
Interleukin (IL)-24, which was initially named “melanoma differentiation-associated gene 7”
(mda-7), is an IL-10-type cytokine that has been discovered by means of differentiation
therapy and subtraction hybridization in melanoma cells. Via two receptor complexes (IL-
20R1/IL-20R2 and IL-22R/IL-20R2), IL-24 can activate tyrosine kinases of the Janus family
(Jaks) and STAT transcription factors in target cells. In addition to this „classical“ signalling
pathway, IL-24 has the unique property to induce apoptosis in many different cancer cells, but
not in normal cells, when applied via an adenoviral vector or as GST fusion protein.
Moreover, it promotes anti-oncogenic bystander activity, it inhibits angiogenesis, synergises
with radiation, and modulates immune responses. However, the mechanisms of cancer cell
selectivity as well as the receptor-independent signalling events leading to IL-24-induced
apoptosis remain unclear.
A panel of 20 melanoma cell lines as well as normal human keratinocytes and primary human
melanocytes were analysed with respect to their Jak-STAT signalling capacity in response to
IL-24 stimulation. Although all cells are RT-PCR-positive for expression of specific cytokine
receptor subunits, only keratinocytes responded to IL-24 stimulation by phosphorylation of
STAT3. Neither the primary melanocytes nor the 20 tested melanoma lines reacted to IL-24
stimulation. Of note, we did not observe STAT3 phosphorylation in
unstimulated melanoma cells.
To further investigate the cancer apoptosis-inducing function of IL-24, we have generated and
expressed a GST-IL-24 fusion protein and variants thereof, based on differential splicing and
on a structure/function comparison of IL-10 like cytokines. Furthermore, inducible stable
melanoma cell lines expressing IL-24 are being generated and first resulting data will be
discussed.
- 384 -

Session X : Cell death in cancer Poster X, 52
Caspase 3 activation, Bcl-2 contents and soluble Fas-ligand are appear to be
independent of the inflammatory marker profile in patients with sepsis and septic shock
Fabian Kriebel1, Silke Wittemann2, Hsin-Yun Hsiu2, Thomas Joos2, Manfred Weiss3,
E. Marion Schneider1
Manfred.weiss.ulm@online.de
1Sektion Experimentelle Anaesthesiologie, Universitaetsklinikum Ulm, Ulm, Germany;
2NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen
, Germany; 3Abteilung Klinische Anaesthesiologie, Universitaetsklinikum Ulm, Ulm,
Germany.
Objective: In order to extend treatment and diagnostics in intensive care patients suffering
from systemic inflammatory response syndrome (SIRS), sepsis and/or septic shock, we asked
whether apoptosis plays a role in hyperinflammation.
Patients: Twenty intensive care unit (ICU) patients were analyzed daily: 2 had systemic
inflammatory response syndrome (SIRS), 5 suffered from sepsis (2 died), and 13 had septic
shock (5 died).
Methods: EDTA-blood was lysed on ice with Cell Lysis Buffer (BD-Germany; 10mM Tris-
Hcl, 10mM NaH2PO4/NaHPO4, 130mM NaCl, 1% Triton X-100, 10mM PPi, protease
inhibitor cocktail of 800 µg/ml benzamidine- HCL, 500 µg/ml o-phenanthroline, 500 µg/ml
aprotinin, 500 µg/ml leupeptin, 500 µg/ml pepstatin A, 50nM PMSF). The lysate was
centrifuged at 14,000rpm for 10min and stored at –20˚C. Active caspase-3 was measured with
the R&D Quantikine Active Caspase-3 Immunoassay (R&D Systems, Hamburg, Germany).
Cytokines and active metalloproteinases (MMPs) were quantified by the Beadlyte(
Multiplex Cytokine Detection System (Upstate USA, Lake Placid, NY, USA).
Metalloproteinases (MMPs) were quantified using Luminex assisted Beadlyte Assays (Qiagen
LiquiChip, Hilden, Germany). Soluble FAS-Ligand (sCD178) was determined by ELISA
(MBL, purchased via Beckman-Coulter, Krefeld, Germany).
Results: Laboratory data obtained by plasma and cell lysate analysis demonstrate that active
caspase-3 was identified in defined samples of whole blood lysates, (but never in EDTAplasma)
covering (e.g. 5/7, 8/18, 6/11) consecutive days during the patients’ stay on the ICU.
Caspase-3 antigen contents were not found to be related to any of the inflammatory markers,
including the inflammatory cytokines (IL-1, IL-6, IL-8, TNF-a, etc.) the metalloproteinases
(MMPs 1, 3, 7, 9, 13) and C-reactive protein (CRP). However, when comparing the kinetics
of active caspase 3 and Bcl-2 protein quantified in whole blood lysates with plasma
concentrations of soluble Fas-ligand (sCD178-L), all 3 parameters were found to be elevated
either simultaneously or in close time window.
Conclusions: We conclude that the activation of apoptosis can be determined in whole blood
by active caspase-3 and by Bcl-2. Interestingly, the upregulation of caspase 3 coincides with
high Bcl-2 contents and a consecutive peak of plasma soluble FAS-ligand in a patient with a
successful reconstitution after septic shock. We therefore conclude that pro- and antiapoptotic
effects during sepsis may not be related to inflammatory markers may indicate
modelling of leukocyte subpopulations and may play an important role in immune
reconstitution.
- 385 -

Session X : Cell death in cancer Poster X, 53
Cyclosporine A induced cytotoxicity, cell cycle arrest and mitochondrial apoptosis in a
human monocytic leukaemia cell line
Molay Kumar Roy*1, Makiko Takenaka1, Masuko Kobori1, Kazuhiko Nakahara2,
Seiichiro Isobe1, Tojiro Tsushida1.
1National Food Research Institute, 2-1-9, Kannondai, Tsukuba, Ibaraki, 305-0051,
Japan.
2Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Owashi,
Tsukuba, Ibaraki 305-8686, Japan.
The immunosuppressive drug cyclosporine A (CsA) has been used in organ transplantation
and in the treatment of autoimmune disorders. However, the drug causes adverse effects in
kidney, liver and nervous system characterized by cellular loss in the affected area. Apoptosis
was found to play a role in CsA induced cytotoxicity. Because mitochondrial membrane
permeabilization is a common criterion in the most setting of apoptosis in vertebrate cells, the
aim of this study was to evaluate whether CsA induces mitochondrial function loss in the
pathway leading to cytotoxicity in a cell line. In this study we found that CsA caused a
concentration and time dependent cell viability loss in U937 cell line. CsA treatment of cells
at 10 µM dose resulted in G0/G1 arrest with concurrent decrease of cells in S and G2/M
phases. In mechanistic studies related to its viability lost we found that treatments of cells
with 10 µM CsA for 24 h resulted in DNA fragmentation and in the increase of annexin V
positive cells. CsA also increased the activity of a cysteine protease caspase-3. In other
studies, we observed that CsA treatment decreased mitochondrial membrane potential and
increased cytochrome c release into cytosol. Furthermore, CsA treatment increased the
number of cells in sub-G0/G1 peak, an indicative of reduced DNA, however this effect was
not observed, when cells were pre-treated with a broad caspase inhibitor. In the study, we
also found that a higher dose of CsA induces LDH release when the cells were incubated for a
longer period. The overall result suggests that the mode of cell death is dose and time
dependent. Short-term incubation with lower doses of CsA arrests cell growth, this overlaps
with the occurrence of apoptosis and then necrosis after longer treatment periods with higher
doses.
- 386 -

Session X : Cell death in cancer Poster X, 54
Synergistic apoptosis induction of breast cancer cells by tamoxifen and TRAIL
Lagadec C, Chopin V, Anderiaenssens E, Hondermarck H, Le Bourhis X
ERI-8 INSERM "Signalisation des facteurs de croissance dans le cancer du sein.
Proteomique fonctionnelle" UPRES-EA 1033, IFR-118 Université des Sciences et
Technologies de Lille, France
Although tamoxifen is widely used in the treatment of breast cancer expressing ostrogen
receptors (ER); it has been recently reported that tamoxifen can induce apoptosis of ERnegative
breast cancer cells. On the other hand, the TNF-related apoptosis-inducing ligand
(TRAIL), also called Apo2L, has been the subject of recent research as a potential new
anticancer agent. We investigated the effect of tamoxifen alone or in combination with
TRAIL in breast cancer cells. We showed that TRAIL-induced apoptosis was potentiated by
tamoxifen in both ER-positive and ER-negative breast cancer cells. From a mechanistic
standpoint, combination treatment with tamoxifen and TRAIL resulted in increased cleavage
of procasepases-8, 9 and Bid. Tamoxifen and TRAIL cooperated also to increase the
expression of FADD, procaspase-8, Bax and Bid and to decrease the expression of FLIP and
Bcl-2. These modifications could constitute multiple amplification loops to result from the
final synergistic induction of apoptosis. Importantly, the combination treatment with
tamoxifen and TRAIL did not affect the viability of cultured normal breast epithelial cells.
Moreover, tamoxifen and TRAIL were well tolerated in mice and the combination of
tamoxifen and TRAIL dramatically reduced tumor burden in in vivo MDA-MB-231 tumor
xenograf model. These data suggest that combination of tamoxifen and TRAIL may be useful
in the treatment of ER negative breast cancers.
- 387 -

Session X : Cell death in cancer Poster X, 55
Forced expression of the novel heat shock protein H11 triggers cancer cell apoptosis
through TAK1 activation providing a molecular target for cancer therapy
Jennifer Laing, Baiquan Li, Cynthia Smith, and Laure Aurelian.
Pharmacology and Experimental Therapeutics, University of Maryland School of
Medicine, Baltimore, MD, 21201, U.S.A. E-mail: laurelia@umaryland.edu
H11 is a small heat shock protein (Hsp) recently cloned in our laboratory. It retains the alphacrystallin
motif, but differs from canonical family members in that its expression is inhibited
in some cancer cells (notably melanoma, prostate and breast) relative to matched normal
tissues. Inhibition is by aberrant DNA methylation, and can be forced by treatment with
demethylating agents. The current studies were designed to examine the role of H11 overload
in tumor cell fate determination. Melanoma was used as a model, because it is a prevalent
cancer, which is resistant to most types of chemotherapy. To control specificity of gene
upregulation, human melanoma cells were stably transfected with a retrovirus that expresses
H11 under the control of a tetracycline inducible promoter. They were examined for H11
expression and apoptosis at 1-3 days after doxycycline (Dox) treatment. H11 was rapidly (1
day) expressed and expression was associated with activation (cleavage) of caspases-9 and -3
and p38MAPK, as well as a significant increase in the % TUNEL+ cells.
Immunopercipitation/immunoblotting assays indicated that H11, but not its apoptosis
dominant negative mutant W51C, binds TAK1 resulting in the activation of its kinase activity
(determined by immunocomplex PK assays with MKK6 as the phosphorylation substrate).
Activated TAK1, in turn, activates p38MAPK, which is responsible for caspase-3 cleavage, as
evidenced by inhibition with the TAK1 dominant negative mutant K63Wor the
pharmacologic inhibitor SB203580. In addition, the H11-TAK1 complex binds and
phosphorylates #-catenin, thereby inhibiting its transcriptional activity and resulting in the
inhibition of MITF and CDK2, both of which are required for melanoma cell proliferation.
Preliminary data using animal xenograft models indicate that H11 overload inhibits tumor cell
growth and triggers apoptosis, also in vivo. The data indicate that modulation of signaling
pathways by H11 overload is a promising novel paradigm for cancer therapy.
- 388 -

Session X : Cell death in cancer Poster X, 56
Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during
nutrient starvation*
Grégory Lavieu†, Francesca Scarlatti§, Giusy Sala§, Thierry Levade‡, Riccardo
Ghidoni§, Joëlle Botti† and Patrice Codogno†
†INSERM U504, Institut André Lwoff, 16 avenue Paul-Vaillant-Couturier, 94807
Villejuif Cedex France, §Laboratory of Biochemistry and Molecular Biology, San Paolo
Medical School, via A. di Rudinì 8, 20142 Milan Italy, ‡INSERM U466, Institut Louis
Bugnard, Centre Hospitalier Universitaire de Rangueil, BP 84225, 31432 Toulouse
Cedex 4 France..
The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death
with autophagic features in cancer cells (1, 2). Here we show that overexpression of
sphingosine kinase 1 (SK1), an enzyme responible for the production of sphingosine 1phosphate
(S1P), stimulates autophagy by increasing the formation of LC3 positive
autophagosomes and the rate of proteolysis sentitive to the autophagy inhibitor 3methyladenine.
Furthermore, autophagy was blocked in the presence of dimethylsphingosine,
an inhibitor of SK activity and in cells expressing a catalytically inactive form of SK1. In
contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy was characterized : 1by
the inhibition of mTOR signaling independently of the Akt/PKB signaling arm, 2-the lack
of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation
induced both the stimulation of autophagy and SK activity. Silencing the expression of the
autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and
exacerbated cell death with apoptotic hallmarks. In conclusion, these results show that
SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient
deprivation.
- 389 -

Session X : Cell death in cancer Poster X, 57
Wogonin, a plant flavone, potentiates etoposide-induced apoptosis in cancer cells
Eibai Lee 1 , Riyo Enomoto 1 , Chie Suzuki 1 , Masataka Ohno 1 , Toshinori Ohashi 1 , Azusa
Miyauchi 1 , Eriko Tanimoto 1 , Kaori Maeda 1 , Hiroyuki Hirano 2 , Toshio Yokoi 2 and
Chiyoko Sugahara 1
1 Department of Pharmacology and 2 Department of Pharmaceutical Chemistry, Faculty
of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe
651-2180 Japan. E-mail : elee@pharm.kobegakuin.ac.jp
Etoposide, a podophylotoxin anticancer agent, induces apoptotic cell death in normal and
cancer cells. Etoposide-induced apoptosis plays a role in not only anticancer effect but also
adverse reaction such as myelosuppression. Since we have found that wogonin, a flavone
found in Scutellaria baicalensis, suppresses thymocyte apoptosis induced by various
compounds including etoposide, we examined the effect of this flavone on etoposide-induced
apoptosis in cancer cells. Although wogonin itself hardly affected biochemical and
morphological features of apoptosis in leukemia cells such as Jurkat and HL-60 cells, this
flavone significantly potentiated etoposide-induced apoptosis in these cells. Similarly,
wogonin accelerated etoposide-induced cell death in lung cancer cells. Since wogonin had no
effect on the action of other anticancer agents such as 5-FU and cisplatin, this flavone seems
to accelerate only apoptotic cell death induced by etoposide in cancer cells. These results
suggest that the modification of etoposide-induced apoptosis by wogonin may be available to
reduce the adverse reaction of this agent.
- 390 -

Session X : Cell death in cancer Poster X, 58
Jpk, a novel cell death inducer, regulates the expression of Hoxa7 in F9 teratocarcinoma
cells, but not during apoptosis
Eun Young Lee and Myoung Hee Kim
Department of Anatomy, Embryology Lab., Brain Korea 21 Project for Medical
Science, Yonsei University College of Medicine, Sodaemoongu Shinchondong 134, Seoul,
120-752, Korea. E-mail: nannaya1210@paran.com ; mhkim1@yumc.yonsei.ac.kr
Increased expression of both Meis1 and Hox genes, such as Hoxa7 and –a9 has been
characterized in several acute myeloid leukemia. Although the overexpression of Meis1 alone
was shown to induce massive apoptosis, coexpression of Meis1 and Hox was reported to
suppress the apoptosis, suggesting that Hox protein might participate in the apoptotic process.
Jpk was isolated as a putative regulatory factor associating with the upstream regulatory
sequence of murine Hoxa7. Since overexpression of Jpk caused apoptosis in bacteria as well
as in eukaryotic cells, we tried to analyze the relationship between Jpk and Hoxa7 during
apoptosis after confirming the regulatory effect of Jpk on the expression of Hoxa7 in F9
teratocarcinoma cells. For that purpose, an effector (pEGFP-Jpk) and reporter (pGL2-NM307)
plasmid containing a luciferase gene under the 307 bp (NM307) of Hoxa7 upstream
regulatory sequence were constructed. In the presence of Jpk (effector), luciferase activity
was increased and this enhancement was decreased by siRNA against Jpk, suggesting that Jpk
is a regulatory factor of Hoxa7. In order to see whether Jpk still regulate the expression of
Hoxa7 during apoptosis, F9 cells were transiently transfected with pcDNA-Jpk, and analyzed
the expression of Jpk, Hoxa7, and CHOP-10 using RT-PCR. Hoxa7 was not upregulated in
the presence of Jpk whereas CHOP-10 was upregulated, indicating that the regulatory
mechanism of Jpk on the expression of Hoxa7 might be different depending on the cell status;
i. e., apoptotic or proliferative condition.
- 391 -

Session X : Cell death in cancer Poster X, 59
Isoliquiritigenin inhibits osteolytic bone metastasis through reduction of COX-
2/RANKL expression
Sun Kyoung Lee 1,2,3, Kwang Kyun Park 1,2,3, Jung Han Yoon Park 4, Soon Sung Lim
4, Won Yoon Chung 1,2
1Department of Oral Biology, 2Oral Science Research Center, College of Dentistry,
3Brain Korea 21 Project for Medical Science Yonsei University, 120-752, Seoul,
Republic of Korea, 4Division of Life Sciences and Silver Biotechnology Research Center,
Hallym University, Chunchon, Republic of Korea, E-mail : l-pluto@hanmail.net
Breast cancer metastasis to the bone occurs frequently, causing numerous complications
including fracture and hypercalcemia. The bone destruction is mediated by the osteoclasts
rather than directly by tumor cells. The interaction between tumor cells, tumor-derived
humoral factors and bone cells, called to ‘vicious cycle’, is crucial for the initiation and
promotion of skeletal malignancies. We examined the effect of isoliquiritigenin(ISL) on
vicious cycle of osteolytic bone metastasis by human breast cancer cells. ISL reduced the cell
growth and the mRNA expression of IL-1beta, -6 and PTHrP in MDA-MB-231 cells. ISL
also inhibited the expression of COX-2 and receptor activator of nuclear factor-kappa B
ligand(RANKL), but induced the expression of osteoprotegerin(OPG) in human osteoblast
cells, hFOB1.19 treated with conditioned medium of MDA-MB-231 cells. Consequently, the
ratio of RANKL/OPG was decreased by ISL. Moreover, ISL inhibited RANKL-induced
osteoclastogenesis in mouse bone marrow monocyte(BMM) and the formation of resorption
pit. Taken together, our results suggest that ISL may block osteolytic bone metastasis by
inhibiting RANKL expression through COX-2 expression.
- 392 -

Session X : Cell death in cancer Poster X, 60
Involvement of MAPKs and NF-kB in diosgenin-induced megakaryocytic differentiation
and subsequent apoptosis in HEL cells
David Y. Léger, Bertrand Liagre, Jeanne Cook-Moreau and Jean-L. Beneytout
Laboratoire de Biochimie, UPRES EA 1085, Faculté de Pharmacie, Limoges, France. Email:
bertrand.liagre@unilim.fr
Recent reports demonstrated that NF-kB activation participated in megakaryocytic
differentiation. A growing number of studies demonstrated the key role of the MAPK
pathway during megakaryocytic differentiation. After differentiation, the fate of mature
megakaryocytes is to produce platelets. Platelet shedding results from cytoplasmic
fragmentation, which leads to the formation of denuded megakaryocytes constituted of the
megakaryocyte nucleus, its envelope, and a ring of cytoplasm. These senescent
megakaryocytes have been identified as apoptotic cells. Furthermore, platelet formation has
recently been shown to require precise caspase activation. These data emphasized the
involvement of apoptotic-related phenomena in thrombopoiesis.
The treatment of HEL cells with 10 µM diosgenin caused an initial activation of ERK1/2
within 5 min (5-fold over untreated cells), which was sustained up to 12h. Furthermore,
diosgenin induced a rapid and sustained de-activation of p38. With diosgenin stimulation, we
found an early slight activation of NF-!B at 24h. Then, diosgenin progressivly inhibited NF-
!B translocation after 48-96h of treatment leading to a complete inhibition at 192h of
treatment. During diosgenin treatment, two distinct bursts of caspase-3 activation were
observed. At 48h stimulation, we observed a rapid and strong increase in active caspase-3.
Then, active caspase-3 levels rapidly decreased at 96h but remained higher than controls.
Afterwards, caspase-3 activity increased again and once more reached high levels by the end
of the treatment. In addition, the intensity of the PARP cleaved 85 Kd fragment followed the
kinetics of caspase-3 activation. Diosgenin treatment also led to the fragmentation of
differentiated cells. Cellular fragmentation started at 96h post-stimulation and at the end of
the treatment (at 192h), most of the cells were fragmenting or already fragmented.
In conclusion, diosgenin induced the megakaryocytic differentiation of HEL cells through a
combined activation of the ERK signaling pathway and inhibition of the p38 MAPK pathway.
Afterwards, differentiated cells showed a marked inhibition of NF-kB nuclear translocation
and an activation of caspase-3 together with PARP cleavage.
- 393 -

Session X : Cell death in cancer Poster X, 61
HGF/SF regulates expression of apoptotic genes in human mammary epithelial cells
Catherine LEROY, Julien DEHEUNINCK, Sylvie REVENEAU, Bénédicte FOVEAU,
Zongling JI, David TULASNE, Céline Villenet*, Sabine Quief*, and Jean-Pierre
Kerckaert* and Véronique FAFEUR.
CNRS UMR 8117, Institut de Biologie de Lille, Institut Pasteur de Lille, B.P.447, 59021
Lille, FRANCE. E-mail : veronique.fafeur@ibl.fr
* Plate-forme Biopuces, Université de Lille 2, Faculté de Médecine, Place de Verdun,
59045, Lille, FRANCE
Hepatocyte growth factor/scatter factor (HGF/SF) induces scattering, morphogenesis and
survival of epithelial cells through the activation of the MET tyrosine kinase receptor.
HGF/SF and MET are involved in normal and tumoral development of many tissues and
organs, including the mammary gland. At present, little is known of the target genes of
HGF/SF implicated in mediating its biological responses.
We searched for HGF/SF target genes using the human epithelial mammary cell line (MCF-
10A), which is sensitive to its scattering and survival effect. We used a DNA microarray
spotted with 60-mer oligonucleotide sets from Sigma-Genosys that include 1920 different
genes. MCF-10A cells were treated or not with HGF/SF for 2 h. Total RNA was then
extracted and purified, followed by reverse transcription to cDNA, with concomitant
incorporation of fluorescent dCTP (Cy3 or Cy5). The probes were mixed and hybridized onto
the microarray, and the fluorescent signals were detected using a GMS confocal scanner
(Affymetrix). The ratios of Cy5/Cy3 were analyzed using Jaguar Software (Screensaver). The
expression of 38 genes was found to be modified by HGF/SF. Among them, only three genes
were clearly classified in apoptosis, with A20 being up-regulated by HGF/SF and DAXX and
SMAC being down-regulated by HGF/SF. Changes in expression of A20, DAXX and SMAC
were confirmed by real time quantitative PCR using a Light Cycler (Roche). According to
published data, A20 is anti-apoptotic, SMAC is pro-apoptotic, while a pro- or anti- apoptotic
role of DAXX is still controversial. The fact that HGF/SF up-regulates an anti-apoptotic gene
(A20) and down-regulates a pro-apoptotic gene (SMAC) is in agreement with its survival
effect in MCF10A cells. This study identified novel target genes of HGF/SF that can
contribute to its anti-apoptotic effect.
- 394 -

Session X : Cell death in cancer Poster X, 62
The nucleoside analogue cidofovir suppresses lung metastasis and induces apoptosis in
FGF2-overexpressing endothelial cells.
Sandra Liekens*, Sofie Gijsbers*, Erik De Clercq*, Erik Verbeken§, and Sigrid Hatse*
*Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven,
Belgium and §Division of Histopathology, K.U.Leuven. E-mail:
Sandra.liekens@rega.kuleuven.be
Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, (S)-HPMPC] is an
antiviral drug that is clinically used for the treatment of cytomegalovirus retinitis in AIDS
patients (1). Cidofovir also possesses potent activity against human papillomavirus-induced
tumors in animal models and patients (1). We have recently shown that cidofovir inhibits the
development of vascular tumors induced by basic fibroblast growth factor (FGF2)overexpressing
endothelial (3F2T) cells in mice (2). Here, we demonstrate that the cytotoxic
activity of cidofovir in 3F2T cells may result from the specific induction of apoptosis. Cell
cycle analysis revealed that cidofovir induces accumulation of cells in the S phase and, upon
prolonged treatment, a significant increase in sub-G1 cells, exhibiting a sub-diploid DNA
content. Moreover, annexin V binding, an early event in apoptosis induction, was increased in
cidofovir-treated 3F2T cells. Cidofovir also caused nuclear fragmentation and the activation
of caspase-3-like proteases in 3F2T cells, as evidenced by the cleavage of poly(ADPribose)polymerase.
In addition, cidofovir treatment resulted in a pronounced up-regulation of
p53 protein. Protein kinase B/Akt is recognized as a principal mediator of survival signals that
protect cells from undergoing apoptosis. Cidofovir did not suppress the phosphorylation of
Akt nor its downstream regulator Bad, indicating that the Akt pathway is not affected by
cidofovir treatment of 3F2T cells. However, cidofovir inhibited the expression of FGF2 and
FGF2 signaling through Erk42/44, as shown by Western blot analysis. In vivo, cidofovir
markedly suppressed the development of experimental lung metastasis, induced by
inoculation of 3F2T cells in the tail vein of SCID mice. Our results indicate that cidofovir
may inhibit the growth of 3F2T cells via inhibition of FGF2 expression and signaling, and via
the induction of apoptosis through the caspase-3 pathway.
1. De Clercq and Holy (2005) Nat Rev Drug Discov. 4: 928-940.
2. Liekens et al. (2001) Cancer Res. 61: 5057-5064.
- 395 -

Session X : Cell death in cancer Poster X, 63
DMNQ S64 induces apoptosis via caspase activation and cyclooxygenase-2 inhibition in
human non-small cell A549 lung cancer cells
Eu-Soo Lim, Yun-Hee Rhee, Eun-Ok Lee, Eun-Young Lee, Sung-Hoon Kim
Department of Oncology, Graduate School of East-West Medical Science, KyungHee
University, Yongin 449-701, Republic of Korea. E-mail:sungkim7@khu.ac.kr
Shikonin has been shown to induce apoptosis and inhibit angiogenesis in vivo and in vitro.
However, it still has drawbacks of solubility and toxicity. Thus, in the present study, the
underlying apoptotic mechanism of 6-ppim (1-propoxyiminoalkyl)-DMNQ-S64 (DMNQ
S64), a shikonin derivative, was examined. DMNQ S64 exerted cytotoxicity against human
A549 lung carcinoma cells with IC50 of 30 µM. Apoptotic bodies were observed in DMNQ
S64 treated A549 cells. DMNQ S64 also increased sub G1 DNA peaks in a concentration
dependent manner by flow cytometric analysis. Western blotting has revealed that DMNQ
S64 effectively activates the expression of caspase 8, 9 and 3, cleaves poly(ADP-ribose)
polymerase and increases the ratio of Bax/Bcl-2. Furthermore, mitochondrial membrane
potential was reduced in a concentration dependent manner by DMNQ S64. It significantly
inhibited the level of prostaglandin E2 by enzyme linked immunosorbent assay and
downregulated the expression of cyclooxygenase-2 (COX-2) in a concentration dependent
manner by Western blotting. Taken together, DMNQ S64 may exhibit cytotoxicity against
A549 cells via caspase activation and COX-2 inhibition.
- 396 -

Session X : Cell death in cancer Poster X, 64
The effect on cell death in drug and CD95-mediated apoptosis by the apoptosis enhancer
Daxx in renal cell carcinoma
C Mahotka, N Wethkamp, P Reinecke, and HE Gabbert
Institute of Pathology, Heinrich Heine-University, Duesseldorf, Germany; e-mail:
mahotka@med.uni-duesseldorf.de
Introduction: Deregulation of apoptosis is involved in the development of cancer. Expression
of Daxx is implicated in apoptosis but whether its function is pro- or anti-apoptotic is still
discussed controversial. Daxx is involved in the regulation of p53 dependent transcription and
associates with the promyelocytic leukemia protein (PML) in speckled subnuclear structures
so called PML oncogenic domains (PODs). Overexpression-based experiments in renal cell
carcinoma (RCC) were used to explore the impact of Daxx on the CD95-dependent apoptosis
and cell death induced by different anticancer agents such as Etoposide, Topotecan, Taxol and
Doxorubicine. Results: 1. Constitutive overexpression of Daxx in the CD95-ligand sensitive
human RCC cell line clearCa-6 (clear-Ca-6/GFP-Daxx) has no influence on cell growth. 2.
Treatment of clearCa-6/GFP-Daxx with the CD95-receptor agonistic antibody CH11 leads to
no further enhancement of the CD95-dependent apoptosis compared to control cell line clear-
Ca-6/GFP as analyzed by MTT Assay and Caspase-8 and PARP Western Blot, respectively.
3. Daxx overexpression in the CD95-ligand resistent RCC cell line clearCa-2 (clearCa-2/GFP-
Daxx) also causes no sensitization to the CD95-dependent apoptosis. 4. Although Daxx is
known to trigger CD95-mediated apoptosis via the activation of JNK by a Caspase-8
independent mechanism, even under Caspase-8 inhibition no Daxx-mediated modulation of
the CD95-mediated apoptosis was detectable and moreover, no differences in CD95-mediated
JNK aktivation were obvious as shown by in vitro kinase assays. 5. Treatment of both GFP-
Daxx transduced cell lines with various anticancer drugs points to a protective role of Daxx
during Taxol-dependent apoptosis of clearCa-2/GFP-Daxx, which is probably mediated by an
enhanced activation of the p38/JNK pathway as shown by in vitro kinase assays.
Conclusions: Our study suggests that the CD95-mediated apoptosis in renal cell carcinoma is
not effected by the overexpression of the apoptosis enhancer Daxx. In contrast, the data
indicate a rather anti-apoptotic than pro-apoptotic role for Daxx in human RCC as shown by
the Daxx overexpression-mediated protective effect during Taxol-dependent apoptosis in the
RCC cell line clearCa-2.
- 397 -

Session X : Cell death in cancer Poster X, 65
Increased expression of high mobility group box 1 (HMGB1) protein is associated with
an elevated level of iNOS in lymphocytic thyroiditis and papillary thyroid cancer.
Stefania Mardente, Gianluca Maiani, Emanuela Mari, Alessandra Zicari, Massimo
Realacci, Stefania Natalizi, Fabrizio Consorti*, Carlo Della Rocca and Alfredo
Antonaci*.
Department of Experimental Medicine and Pathology and * Department of Surgical
Sciences and Applied Technologies, University “La Sapienza”, Viale Regina Elena 324,
Rome Italy: email: stefania.mardente@uniroma1.it
Some recent works have shown that autoimmune thyroiditis, although considered a benign
condition often harbours a genetic rearrangement that is highly specific for papillary
carcinoma. Submicroscopic foci of papillary carcinoma exist in autoimmune thyroiditis
although its behaviour is still benign. We have recently demonstrated that peripheral
lymphocytes (type Tc1 and Tc2) infiltrate thyroids in autoimmune thyroiditis and in most
papillary carcinomas. Direct cytotoxicity, secretion of cytokines, presence of intrathyroidal B
secreting autoantibodies, would altoghether work as a defect in suppressing the immune
process. The local inflammation would lead to secretion of NO that could exacerbate the
autoimmune response and hypothyroidism. It has been demonstrated that high concentrations
of NO are genotoxic in the sense of promoting mutations that could lead to cancer. Local
hypoxia and high concentrations of NO may induce apoptosis of thyreocytes in chronic
inflammation and cancer but it may also lead to necrotic death. HMGB1 is a non histone
chromosomal protein implicated in a variety of processes including transcription,
differentiation and development. An increased expression of this protein has been reported for
several tumor types. Its overexpression inhibits apoptosis. According to our western blot
analysis of primary cultures of thyreocytes from patients with thyroiditis and papillary cancer
and immunohistochemistry of iNOS, we identified a molecular pathway triggered by HMGB1
that could inhibit apoptosis of thyreocytes in a microenvironment rich of NO and other
proinflammatory cytokines.
- 398 -

Session X : Cell death in cancer Poster X, 66
Arsenic trioxide induces a p53-independent cell death in Ewing sarcoma cells.
Julie Mathieu, Michel Lanotte and Françoise Besançon
INSERM Unité 685, Hopital St-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France.
E-mail : julie.mathieu@stlouis.inserm.fr; francoise.besancon@stlouis.inserm.fr
Ewing sarcoma (ES), a highly malignant pediatric tumor, is consistently associated with
translocations that fuse the EWS gene with a member of the ETS family gene, most
commonly FLI-1. Despite significant advances with multi-agent chemotherapy, surgery and
radiotherapy, about 40% of ES patients still die from the disease. It is therefore necessary to
explore novel agents for possible treatment of this tumor. Here we investigated the sensitivity
of ES cells to arsenic trioxide (As2O3), a compound known to induce differentiation and
apoptosis of other types of malignant cells. We report that low doses of As2O3 (1-4
micromolar) uniformly inhibited growth of six ES-derived cell lines irrespective of their p53
status. As2O3 resulted in caspase activation, loss of mitochondrial potential and an apoptotic
phenotype which was inhibited by the broad-spectrum caspase inhibitor ZVADfmk. These
effects correlated with prolonged JNK activation, which is a signal for apoptosis in ES cells.
As2O3 also decreased basal and cytokine-induced NF-kappa B activity, which was previously
shown to protect ES cells from apoptosis induced by various stimuli. Together, our results
demonstrate that clinically tolerable concentrations of As2O3 trigger p53-independent
apoptosis of ES cells. They further suggest that inclusion of this compound in future trials of
novel treatment strategies for Ewing sarcoma may be beneficial.
- 399 -

Session X : Cell death in cancer Poster X, 67
Functional Analysis of the Anti-Apoptotic Livin Protein
Christina Mensger, Irena Crnkovic-Mertens, Claire Cullmann, Karin Butz and Felix
Hoppe-Seyler
Infection and Cancer, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242,
D-69120 Heidelberg, Germany. E-mail: c.mensger@dkfz.de
Cancer cells are typically characterized by increased resistance to apoptosis, which enables
their survival under abnormal growth stimulation. Moreover, deficiency in apoptosis is
considered to be a major cause for therapeutic resistance of tumours in the clinic, since many
chemo- and radiotherapeutic agents act through the induction of apoptosis. The functional
inhibition of specific anti-apoptotic factors should therefore provide a rational basis for the
development of novel therapeutic strategies.
Livin is a relatively new member of the "Inhibitors of Apoptosis Protein" (IAP) family.
Initially associated with malignant melanoma, it recently has been found that Livin is also
expressed in many additional cancers, such as lung cancer. Notably, livin gene expression is
typically detectable in the tumor cells but not, or to substantially lower levels, in the
corresponding normal tissue.
We found that the targeted inhibition of Livin by RNA interference (RNAi) led to a proapoptotic
sensitization towards different pro-apoptotic stimuli, which was specific for Livinexpressing
tumor cells. Moreover, long-term inhibition of Livin expression in clonogenic
survival assays led to the elimination of Livin-expressing tumor cells, even in the absence of
additional pro-apoptotic stimuli. Isoform-specific RNAi showed that the biological
significance of the two known Livin isoforms, Livin " and Livin #, can strongly differ,
possibly in a cell-dependent manner. Ongoing work concentrates on the elucidation of the
critical intracellular pathways and natural interaction partners which mediate the antiapoptotic
activity of Livin.
Based on its cancer-associated expression pattern, Livin may serve as a novel diagnostic and
prognostic tumor marker. In addition, the targeted inhibition of Livin could represent a new
antitumor strategy.
- 400 -

Session X : Cell death in cancer Poster X, 68
EXTRACELLULAR SURVIVIN UPREGULATES ADHESION MOLECULES ON
THE SURFACE OF LEUKOCYTES CHANGING THEIR REACTIVITY PATTERN
Simona Mera, Mattias Magnusson, Andrej Tarkowski, and Maria Bokarewa
Department of Rheumatology and Inflammation Research, Sahlgrenska University
Hospital, Göteborg, Sweden
Background. Rheumatoid arthritis (RA) is an autoimmune disease with joints as a principal
target of inflammation. We have recently shown that the extracellular presence of antiapoptotic
protein survivin was associated with unfavourable, i.e. destructive course of RA.
Here we addressed the effects of extracellular survivin on peripheral leukocytes.
Methods. Human peripheral blood leukocytes were cultured with and without recombinant
survivin and assessed for the surface binding of survivin and the expression of adhesion
molecules. The intracellular expression of survivin in non-activated human leukocytes was
determined. Finally, the expression of adhesion molecules on leukocytes as a function of
circulating survivin was analysed in blood of 24 patients with RA and compared to healthy
individuals.
Results. We found that the anti-apoptotic protein survivin expresses immuno-modulatory
properties when released extracellularly. It binds to a 47% of neutrophils inducing the
activation of #2-integrins and their ligand, ICAM-1. Survivin also binds to lymphocytes
inducing the expression of L-selectin. Survivin-induced expression of #2-integrins could be
abolished by the inhibitors of NFkB (parthenolide), PI3-kinase (LY294002), but not by MAPkinase
inhibitors (PD98059, SB203580). Clinical relevance of our findings is proved by the in
vivo association of extracellular survivin with an increased expression of CD11c on blood
monocytes and neutrophils in RA patients.
Conclusion. The results of our study demonstrate that extracellular survivin affects the
function of leukocytes having possible impact on inflammatory responses during arthritis.
- 401 -

Session X : Cell death in cancer Poster X, 69
A new method to assess drug sensitivity on breast tumor acute slices preparation
Pedro Mestres1, Andrea Morguet1, Werner Schmidt2, Axel Kob3, Ralf Ehret3
Department of Anatomy and Cell Biology1, Clinic for Gynecology2, University of
Saarland, D-66421 Homburg, Bionas Inc. 3, D-18119 Rostock. Germany
The method described is based on: 1) preparation of tissue slices and 2) use of silicon chips
equipped with electrochemical sensors (multisensor array). The slices (200-300 µm thick) are
prepared after surgery and incubated in a medium for recovery after slicing. The advantage,
compared to other preparations, is that the original three-dimensional structure is retained.
Multisensor arrays measure: 1) pericellular acidification (anaerobic metabolism) and 2)
oxygen consumption (respiration). The innovative aspect is that such measurements can be
made online, as opposed to the large battery of endpoint tests on cell vitality and proliferation.
Electron microscopy of slices serves to determine cell density, structure and induction of
apoptosis/necrosis.
Over 200 breast tumors were used. Inhibition of metabolic activities was performed with
sodium fluoride, which dose-dependently reduces glycolysis, and potassium cyanide which
inhibits respiration. In other experiments cytostatics were applied, providing results obtained
with Taxol, an anti-cytoskeleton agent. In addition, the application of beta-adrenergic receptor
agonists, creates a situation in which metabolic patterns associated with ligand receptor
interaction can be observed all the time. In conclusion, the methodology presented here, is
able to provide information on drug sensitivity of a tumor, of assistance in designing
individualized therapy and for drug-screening. (supported by BMBF to PM)
- 402 -

Session X : Cell death in cancer Poster X, 70
Cytotoxicity of TRAIL/anticancer agent combinations in human primary cells
Olivier Meurette1, Anne Fontaine1, Amélie Rebillard1, Thierry Lamy2, Dominique
Lagadic-Gossmann1 and Marie-Thérèse Dimanche-Boitrel1
1 INSERM Unit 620, University of Rennes 1, Faculty of Pharmacy, 2 Av du Pr. Leon
Bernard 35043 Rennes cedex France; 2 Department of Hematology, Pontchaillou
Hospital, Rennes, France.
TRAIL (TNF-"-Related Apoptosis Inducing Ligand) is a potential anticancer agent that
induces apoptosis by binding to its death receptors TRAIL-R1 and/or -R2 in cancer cells but
not in most normal cells. TRAIL also possesses two decoy receptors (TRAIL-R3/-R4). Some
cancer cells are resistant to TRAIL-induced apoptosis. Combining TRAIL with a wide range
of anticancer agents can restore cancer cell sensitivity to cell death, and combination of
TRAIL with anticancer drugs is now tested in clinical trials. At present, the cytotoxic effect of
such combinations in normal human primary cells is not well known.
We studied here the sensitivity of several human primary cells: hepatocytes, lymphocytes and
neutrophils to combination of TRAIL with cisplatin or 5-fluorouracil in relation with TRAIL
receptors membrane expression. We show that human primary hepatocytes express
exclusively TRAIL-R4 decoy receptor on the cell membrane. In contrast to Fas ligand,
TRAIL is not cytotoxic towards human primary hepatocytes. However, these normal cells
become only sensitive to TRAIL/cisplatin but not to TRAIL/5-fluorouracil. Cisplatin or 5fluorouracil
treatment has no effect on TRAIL receptors membrane expression in human
primary hepatocytes. Freshly isolated lymphocytes express solely TRAIL-R4 at the cell
membrane and are resistant to TRAIL. However, as human hepatocytes, lymphocytes are only
sensitive to TRAIL/cisplatin and resistant to TRAIL/5-fluorouracil. Interestingly, these both
combinations are cytotoxic towards PHA+IL-2 activated lymphocytes which express a little
amount of TRAIL-R1 and TRAIL-R2 on the cell membrane. Finally, freshly isolated
neutrophils express exclusively TRAIL-R3 on the plasma membrane and are resistant to
TRAIL and to TRAIL/anticancer agent combinations.
Altogether, these data demonstrate that human primary cells express exclusively TRAIL
decoy receptors (TRAIL-R3 or TRAIL-R4) and are resistant to TRAIL. However, the
cytotoxicity of certain TRAIL/anticancer drugs combinations towards human primary cells
should be taken into account for the development of TRAIL-based anticancer therapy.
- 403 -

Session X : Cell death in cancer Poster X, 71
TRAIL induces a RIP (receptor interacting protein) dependent necrosis-like cell death
at acidic extracellular pH (pH=6.5).
Olivier Meurette1, Laurence Huc1, Amélie Rebillard1, Gwenaelle Le Moigne1, Olivier
Micheau2, Delphine Merino2, Dominique Lagadic-Gossmann1 and Marie-Thérèse
Dimanche-Boitrel1.
1 INSERM Unit 620, University of Rennes 1, Faculty of Pharmacy 2 Av, du Pr. Leon
Bernard 35043 Rennes cedex, France. 2 INSERM U517, Faculty of Medicine, University
of Burgundy, 7 boulevard Jeanne d’Arc, 21033 Dijon Cedex, France.
TRAIL (TNF-alpha-Related Apoptosis Inducing Ligand) is a potential anticancer agent that
induces apoptosis in cancer cells but not in most normal cells. TRAIL is known to induce
apoptosis via the extrinsic death pathway. In certain conditions a death signal amplification
via the intrinsic death pathway is necessary. TRAIL has also been shown to induce necrosis
independently of caspase activation. At present, necrosis-like programmed cell death
signalisation is poorly understood. The Death Domain containing serine/threonine kinase RIP
(Receptor Interacting Protein) has been involved both in necrosis-like cell death and in NFkB
activation after death receptor ligation.
We have recently shown that under acidic extracellular pH (6.5), TRAIL induced a cell death
sharing some apoptotic and necrotic characteristics in human HT29 colon carcinoma and
HepG2 hepatocarcinoma cell lines. We demonstrate here, that in spite of a necrosis-like cell
death phenotype, caspases are activated and are necessary in this cell death process. By using
a geldanamycin pretreatment or a small interference RNA approach, we show that RIP is
necessary for TRAIL-induced necrosis-like cell death in HT29 cells at acidic extracellular pH
(6.5). Furthermore, the expression of RIP protein is increased in HT29 cells after TRAIL
treatment at both physiological and acidic extracellular pH. Whereas RIP is cleaved after
TRAIL treatment at physiological extracellular pH (7.4), it is not cleaved at acidic
extracellular pH (6.5) despite high level of caspase activation. Finally, at acidic extracellular
pH (6.5), TRAIL strongly activates NF-kB in HT29 cells, whose activation is dispensable for
necrosis-like cell death induction.
In conclusion, these data demonstrate that under acidic extracellular pH conditions TRAIL
induces in HT29 cells a necrosis-like cell death which depends on caspase activation and on
RIP expression.
- 404 -

Session X : Cell death in cancer Poster X, 72
Oxidative stress response in telomerized human fibroblasts from a centenarian
Chiara Mondello1, Maria Grazia Bottone1,2, Sakon Noriki3, Cristiana Soldani2, Carlo
Pellicciari2, A. Ivana Scovassi1
1 Istituto di Genetica Molecolare del CNR, Via Abbiategrasso 207, 27100 Pavia, Italy, Email:
mondello@igm.cnr.it, scovassi@igm.cnr.it; 2 Dipartimento di Biologia Animale,
Piazza Botta 10, 27100, Pavia, Italy. E-mail: bottone@unipv.it, soldani@unipv.it,
pelli@unipv.it; 3 Department of Oncological Pathology, Faculty of Medicine, Matsuoka,
Yoshida-Gun, 9l0-ll93, Fukui, Japan. E-mail: noriki@fmsrsa.fukui-med.ac.jp
It has been reported that cells with ectopic expression of telomerase (so-called telomerized)
are more resistant to apoptotic cell death than their normal counterpart. However,
controversial results have been obtained as to the response of telomerized cells to the
oxidative stress. In the present research, we investigated the effects of the treatment with
either tert-butylhydroperoxide (tBOOH) or 2-deoxy-D-ribose (D-ribose) on human fibroblasts
from a centenarian individual (cen3) and on their counterpart with reactivated telomerase
(cen3tel). Our results suggest that the effects of these drugs are modulated by telomerase
reactivation. In fact, after drug treatment the cell number of both cell lines was lower than in
controls, indicating that both agents impaired cell proliferation; remarkably, proliferation was
always inhibited at a larger extent in cen3tel than in cen3 cells. Different parameters of
apoptosis were also studied in situ (i.e., chromatin condensation, phosphatidylserine
externalization, and DNA fragmentation) and showed that the percentage of apoptotic cells
was lower in telomerized cell cultures, although apoptosis could not completely account for
the observed cell loss in treated cultures. The evidence of active phagocytosis of apoptotic
cells occurring in our fibroblast cultures provides an explanation for these contradictory
results.
- 405 -

Session X : Cell death in cancer Poster X, 73
Met acts on Mdm2 through mTOR to signal cell survival in vivo
Anice Moumen1, Almudena Porras2, Armand Tasmadjian1, Rosanna Dono1 and Flavio
Maina1
1IBDML, Campus de Luminy-Case 907, 13288 Marseille Cedex 09, France - 2Dpto.
Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense,
Ciudad Universitaria, 28040 Madrid, Spain.
E-mail: maina@ibdm.univ-mrs.fr
Coordination of cell death and survival is crucial during embryogenesis and adulthood and
alterations to this balance can result in degeneration or cancer. Growth factor receptors such
as Met can activate phosphatidyl-inositol-3’ kinase (PI3K), a major intracellular mediator of
growth and survival. PI3K can antagonise p53-triggered cell death but the underlying
mechanisms are not fully understood. Using genetic and pharmacological approaches, we
demonstrated that PI3K acts through mTOR to regulate p53 activity both in vitro and in vivo.
mTOR inhibits p53 by promoting translation of Mdm2, the negative regulator of p53.
Increased Mdm2 protein levels require the mTOR effector p70s6k/S6 ribosomal protein.
Unexpectedly, although it is required for Mdm2 nuclear translocation, Akt is dispensable for
Met-triggered mTOR activation and Mdm2 up-regulation. Inhibition of mTOR is sufficient to
block cell survival induced by HGF/Met in vitro, to down-regulate Mdm2 protein levels and
to induce p53-dependent apoptosis in vivo. Our studies identify a novel mechanism for cell
survival, involving translational regulation of Mdm2 by mTOR, thus reinforcing mTOR as a
potential drug target in cancer.
- 406 -

Session X : Cell death in cancer Poster X, 74
HGF supports embryonic hepatocyte survival by suppressing apoptotic pathways
involving p53 and TNFalpha/FasL
Anice Moumen1, Alessandro Ieraci1 and Flavio Maina1
1IBDML, Campus de Luminy-Case 907, 13288 Marseille Cedex 09, France - 2Dpto.
Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense,
Ciudad Universitaria, 28040 Madrid, Spain.
E-mail: moumen@ibdm.univ-mrs.fr
Survival of embryonic hepatocytes is controlled by a number of anti- and pro-apoptotic
signals. To date, the only ligand/receptor couple known to induce hepatocyte survival in vivo
is HGF and its cognate receptor tyrosine kinase Met. However, their downstream effectors
involved in this process remain to be elucidated. We previously showed that met specificityswitch
mutants are not able to mediate hepatocyte survival, thus resulting in massive
apoptosis in developing livers as observed in met loss-of-function mutants. Taking advantage
of the incomplete signalling of these mutant receptors, we asked which combination of
signalling effectors is sufficient for proper hepatocyte survival. We found that Met makes use
of distinct networks involving Flip and Mdm2 to block the pro-apoptotic signals TNF/FasL
and p53, respectively. In contrast, modulators of cell survival such as CREB, Gab1 and Jun,
are not sufficient. Thus, our results clearly show that during liver development, Met controls
hepatocyte survival by modulating the cross-talk of pro- and anti-apoptotic signals.
Furthermore, our use of defined mouse signalling mutants resolves some of the complexity
underlying the mechanisms that regulate the survival of hepatocytes and their susceptibility to
apoptotic signals.
- 407 -

Session X : Cell death in cancer Poster X, 75
The essential role of the mitochondria-dependent death signaling cascade in
chemotherapy-induced potentiation of Apo2L/TRAIL cytotoxicity in cultured thoracic
cancer cells: Amplified caspase 8 is indispensable for combination-mediated massive
cell death.
Dao M. Nguyen, Wen-Shuz Yeow, Rishindra M. Reddy, M. Firdos Ziauddin, Wilson
Tsai, David S. Schrump.
Section of Thoracic Oncology, Surgery Branch, Center for Cancer Research, National
Cancer Institute, National Institutes of Health, Bethesda, MD, USA. Email address:
dao_nguyen@nih.gov
Chemotherapeutic drugs, regardless of their mechanism of action, sensitize cancer cells to
Apo2L/TRAIL cytotoxicity in vitro and in vivo animal model. The molecular basis of this
effect is diverse, ranging from modulation of the DISC activity to phenotypic alteration of the
levels of anti-/pro-apoptotic proteins. We hypothesized that chemotherapeutic drugs, by
altering the pro-apoptotic threshold of mitochondria, synergize with Apo2L/TRAIL to
promote cytotoxicity via recruitment of the type II caspase activation cascade. Cisplatin,
Paclitaxel, histone deacetylase inhibitors Trichostatin A or Valproic acid all sensitize multiple
cultured thoracic cancer cells to Apo2L/TRAIL. Combining individual chemotherapeutics (at
sublethal concentrations) with Apo2L/TRAIL results in 2- to >20-fold enhancement of
cellular sensitivity to this ligand and massive synergistic induction of apoptosis (

Session X : Cell death in cancer Poster X, 76
Modulation of p53 transcriptional activity by PRIMA-1 and Pifithrin-alpha : effects on
staurosporine-induced apoptosis of wild-type and mutated p53 epithelial cells.
Magali Nicolier, Jean-François Charlot, Jean-Luc Prétet and Christiane Mougin.
IFR133, EA 3181, Carcinogenèse épithéliale, UFR Médecine et Pharmacie, 19 rue
Ambroise Paré, 25000 Besançon, France. Email : magali.nicolier@univ-fcomte.fr
The p53, a specific DNA-binding transcription factor, triggers multiple biological
responses including cell cycle arrest and apoptosis. Inactivation of p53 functions by mutations
or by viral oncoproteins such as E6 from high risk papillomavirus (HPV) is a common event
in many human cancers. New chemotherapeutic approaches consist in the restoration of
apoptosis through refolding p53 mutants by PRIMA-1 (p53 reactivation and induction of
massive apoptosis). As a result, PRIMA-1 restores the transcriptional activity of mutated p53
(p53mt). Anti-cancer therapies also damage surrounding normal tissues which highly express
wild-type p53 (p53wt). That is why Pifithrin-alpha (p-fifty three inhibitor), a chemical
inhibitor of p53wt, has been proposed as an adjunct to protect normal cells from otherwise
lethal doses of chemo/radiotherapy.
Recently, we argued for a major role of p53 in staurosporine (ST)-induced apoptosis of
immortalized epithelial cells, according to p53 status (wt or mt) [Charlot et al., Apoptosis
2004]. Here, we investigated the effects of PRIMA-1 and Pifithrin-alpha on ST-induced
apoptosis of 5 different immortalized cell lines with variable p53 status (p53wt HeLa HPV
18+ and CaSki HPV 16+ cells ; p53mt C33-A cells; p53-/- SaOs-2 cells) [Charlot, Nicolier et
al., Apoptosis, in press]. As expected, PRIMA-1 has no effect on p53wt or p53-/- cells. On the
other hand, it increases p21 and Bax expression, mitochondrial membrane potential
dissipation and DNA fragmentation of p53mt cells. By contrast, Pifithrin-alpha does not
modify ST-induced apoptosis of p53mt and p53-/- cells but readily decreases the apoptosis of
cells harbouring a transcriptionnally active p53.
These data strength the evidence that PRIMA-1 could increase ST efficiency in tumors with
mutant p53. Moreover, Pifithrin-alpha could be used in combination with ST and PRIMA-1 to
prevent side effects of anti-tumor therapies on healthy tissues.
- 409 -

Session X : Cell death in cancer Poster X, 77
Reciprocal neutralization of the antiapoptotic effects of magnetic fields and melatonin
due to interference between the two respective effectors, NO synthase and lipoxygenase
Silvia Nuccitelli, Flavia Radogna, Claudia Cerella, Milena De Nicola, Maria D’Alessio,
Andrea Magrini*, Antonio Bergamaschi*, Lina Ghibelli
Dipartimento di Biologia;*Cattedra di Medicina del Lavoro, Università di Roma Tor
Vergata, Via Ricerca Scientifica 1, 00133 Rome Italy. E-mail: silvia.nuccitelli@libero.it
Chemical/physical agents able to prevent apoptosis are receiving much attention for their
potential health hazard as tumor promoters and for their potential therapeutic role against
tissue degenerations. Magnetic fields (MFs), which have been shown to increase the
development of some tumors, reduce damage-induced apoptosis by a mechanism involving
Ca2+ entry into cells [Fanelli et al., FASEB J. 13, 95-102, 1999]. The neuro-hormone
melatonin, which is giving great expectations as therapeutic agent, also reduce damageinduced
apoptosis, again by a mechanism involving Ca2+ entry. The pathways responsible for
the two anti-apoptotic effects are complex and different (see accompanying presentations),
involving NO synthase (NOS) as one effector of MFs protection; and lipoxygenase (LOX) as
one of the effectors of melatonin protection. We wanted to know if the anti-apoptotic effects
of MFs and melatonin were synergic. Thus, we induced apoptosis in the presence of both
agents. Surprisingly we found that MFs and melatonin are not only not synergic, but abrogate
their respective anti-apoptotic effects. In order to verify whether this was due to an
interference between the effectors of the two different anti-apoptotic pathways, we inhibited
one or the other effectors (NOS with L-NAME and LOX with AA861) and checked whether
this would restore the protection mechanisms of melatonin or MFs, respectively. We found
that in U937 induced to apoptosis in the presence of both MFs and melatonin, L-NAME
abrogates MFs action and restores the anti-apoptotic effect of melatonin; vice versa, AA861
abrogates melatonin action and restores the anti-apoptotic effects of MFs. These results
indicate that the signal transduction pathways involving NOS and LOX interfere and
neutralise each other. The reciprocal interference implies that no direct inhibition is exerted
between the two enzymes, but rather the products of each interfere with the downstream
effectors of the other. No reports of effects of LOX or its products on NOS are available to
the best of our knowledge whereas. it is well known that NO inactivates lipoxygenase
[BBRC 219, 128-133, 1996]. These results recommend to explore the potential use of
melatonin as a mean to contrast the harmful effects of unwanted MFs exposure.
- 410 -

Session X : Cell death in cancer Poster X, 78
Let’s flip the FLIP idea
Beata Pajak, Arkadiusz Orzechowski
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw
Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland, e-mail:
bepaj@wp.pl
The resistance of cancer cells to elimination by death ligands (TNF-alpha, TRAIL, FasL) in
the extrinsic apoptotic pathway allows a variety of tumors to grow and develop. The
elucidation of the antiapoptotic mechanisms responsible for this phenomenon is the major
challenge of the contemporary medicine. The last decade revealed several strategies of cancer
cells (including colon adenocarcinomas) of how to avoid cell death and to maintain cell
survival. Colorectal cancer cells inhibit death ligand-induced apoptosis by the expression of
several antiapoptotic proteins, such as FLIP (FLICE-inhibitory protein), ,cIAPs (cellular
inhibitors of apoptosis), and others which inhibit either signal transduction or execution of
apoptosis. In colorectal cancer COLO 205 cell line the immunoprecipitation of FADD (Fasassociated
death domain-containing protein) showed its assembly with FLIP. Usually, the
presence of FLIP in the TNF-alpha signalosome inhibits the caspase 8 (FLICE - FADD-like
interleukin-1#-converting enzyme) autoactivation, and blocks TNF-alpha-induced apoptosis.
Several studies demonstrated that the reduction of FLIP level by the protein synthesis
inhibitors, such as cycloheximide or bis-indolylmaleimide could sensitize cells to apoptotic
stimuli. Similarly, actinomycin D, the inhibitor of DNA transcription exerts cell deathpromoting
properties. In COLO 205 cell line the Western-blot and immunocytochemistry
analyses demonstrated, that the use of cycloheximide decreases the FLIP protein level in a
time-dependent manner, what correlates positively with the progression of cell death. In
contrast, the use of siRNA-FLIP did not lead to cell death upon TNF-alpha treatment. It is
concluded that protein(s) other than FLIP inhibited TNF-alpha-dependent cell death
irrespective to the interaction of FLIP with the TNF-RI signalosome. Thus, unknown
antiapoptotic protein(s) blocked apoptosis downstream to the TNF-R1 signalosome. This
undefined protein(s) is essential for “immune escape” of COLO 205 cells. It is also assumed,
that susceptibility of colorectal cancer cells to TNF-alpha-induced cell death upon
concomitant cycloheximide treatment could be a consequence of oxidative stress and changes
in the intracellular milieu. The latter is currently under detailed scrutiny.
- 411 -

Session X : Cell death in cancer Poster X, 79
Mitochondria role in the control of the activity of p53 and Rb
Ioana Pârvu-Ferecatu, Nelly Godefroy, Vincent Rincheval, Nathalie Leleu, Bernard
Mignotte and Jean-Luc Vayssière
Laboratoire de Génétique et Biologie Cellulaire (FRE CNRS 2445), Université de
Versailles Saint-Quentin-en-Yvelines et Laboratoire de Génétique Moléculaire et
Physiologique,
Ecole Pratique des Hautes Etudes, 45 avenue des Etats-Unis 78035 Versailles Cedex
We previously showed in rat embryo fibroblasts that the p53 protein can activate two distinct
two apoptotic pathways (1, 2 and 3). The first one involves p53’s targets gene transactivation,
whose products trigger a mitochondrial apoptosis that is sensitive to Bcl-2 inhibition. The
second pathway is mediated by the p53’s transcriptional repression activity, initiating a
caspase-independent and Bcl-2-insensitive massive cell death. We also reported that the Rb
tumour suppressor protein contributes to the outcome of p53 activation. Indeed, we observed
that the full length form of Rb, named p110Rb, supports the transrepressional activity of p53,
whereas its cleaved p76Rb form, generated by caspase-9, allows cell survival (3).
Here, we describe a new role for mitochondria in regulating the activity of both the p53 and
Rb proteins. Indeed, in most of the cell types we tested, the inactive p53 protein displays a
mitochondrial location under normal conditions (i.e. proliferating cells), suggesting that the
docking of p53 to mitochondria contributes to its inactivation. After stress, posttranslational
modifications (e.g.: phosphorylation) of p53 promotes its translocation towards the nucleus,
followed by either apoptosis or growth arrest. Equally, we showed for the first time a
mitochondrial location of the Rb protein which would be correlated to its ability to antagonize
p53-induced apoptosis. Thereby, these data support a model in which mitochondria play a
regulatory role of the activity of both p53 and Rb by trapping these proteins, to prevent at
least their nuclear activity.
- 412 -

Session X : Cell death in cancer Poster X, 80
Over-expression of microRNA-21 modulates phosphoinositide-3 kinase and PTEN
signaling and promotes tumor cell survival.
Tushar Patel, Fanyin Meng, Roger Henson, Hania Wehbe, Molly Lang
Scott and White Clinic, Texas A&M University System Health Science Center College of
Medicine, 2401 South 31st Street, Temple, TX 76508, U.S.A. E-mail:
tpatel@swmail.sw.org
MicroRNAs (miRNAs) are endogenous RNA molecules that can regulate gene expression.
Altered expression of miRNA such as miR-21 has been reported for several different cancers.
However the role of miR-21 in tumor progression and the mechanisms involved remain
unknown. Our aims were to assess the effect of miR-21 on critical survival signaling
pathways. Methods: A miRNA microarray was used to profile the miRNA expression in
malignant (KMCH, Mz-ChA-1 and TFK) and non-malignant (H69) cholangiocyte cell lines.
Expression of miR-21 was confirmed by Northern blot and real-time PCR. miR-21
expression was altered using mir-21 specific antisense oligonulceotides or mir-21 precursor
miRNAs. Expression of PTEN and p85 was assessed by Western blots. For in vivo studies,
homogenates were obtained from xenografts of Mz-ChA-1 cells in nude mice under basal
conditions or following treatment with gemcitabine. Results: miR-21 expression was
increased in all malignant cell lines compared to non-malignant H69 cholangiocytes (2.74 ±
0.11 fold in Mz-ChA-1 cells, 4.35 ± 0.14 fold in TFK cells, and 1.84 ± 0.03 fold in KMCH-1
cells; all p

Session X : Cell death in cancer Poster X, 81
Induction of osteoclasts apoptosis by using a decoy oligodeoxynucleotide in vivo
mimicking a region of distal promoter C of ER alpha gene
Letizia Penolazzi, Margherita Zennaro, Ercolina Bianchini, Eros Magri, Elisabetta
Lambertini, Elisa Tavanti, Roberto Gambari and Roberta Piva.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy. Email:
piv@unife.it
The function of osteoclasts (OCs), the multinucleated cells responsible for the resorption of
extracellular bone matrix, are regulated by different kinds of hormones, cytokines,
inflammatory and transcription factors. Of particular interest is the role of estrogen receptor
alpha (ER) protein that mediates the action of estrogen. In particular, it suppresses OCs
formation and activity and inhibits OCs-mediated bone resorption in part by stimulating OCs
to undergo apoptosis through a receptor-mediated genomic action. In order to study
therapeutic strategies to control bone formation, we investigated the possibility to inhibit
osteoclastogenesis through a specific decoy strategy. We obtained an increase of ER
expression by interfering with the activity of a negative transcription factor and by removing
it with a decoy oligonucleotide (RA4-3’) mimicking a region of distal promoter C of ER gene.
We demonstrated that this decoy was able to induce apoptosis in human primary OCs, but not
in osteoblasts, in an estrogen dependent manner, increasing also Caspase 3 and Fas receptor
levels. These findings may be of relevance for a possible therapeutical approach for tumors,
bone metastasis and osteopenic diseases. At this purpose,the clinical orthodontic offers a good
opportunity to study in vivo the efficacy of our approach. In fact, the increased number and
activity of OCs is involved in the regulation of alveolar bone resorption during orthodontic
tooth movement, and also in the origin of dental problems in diseases of OCs activation
affecting the maxillo-mandibular bone. We designed in vivo experiments aimed at regulating
alveolar bone resorption. Six Wistar male rats were subjected to orthodontic forces, in
combination or not with RA4-3’ decoy treatment by using a split-mouth design. Examination
of paraffin sections of the excised molars showed that orthodontic forces caused a percentage
of apoptotic OCs that appears to be highly potentiated by RA4-3’, but not by scramble ODN.
These data confirm the results obtained in vitro with human OCs from peripheral blood,
suggesting an important correlation between ER and the possibility to modulate OCs
functions.
- 414 -

Session X : Cell death in cancer Poster X, 82
Bcl-2 expession in oral squamous cell carcinoma
Branka Popovi'1, Zvezdana Tepav(evi', Vladimir Juri)i'2 and Jelena. Mila)in1
1Institute of Biology and Human Genetics, School of Stomatology, Belgrade, Serbia and
Montenegro, E-mail: jelena_milasin@yahoo.com
2Institute of Pathophysiology, School of Medicine, Kragujevac, Serbia and Montenegro
One of the mechanisms involved in the pathogenesis of oral squamous cell carcinoma
(OSCC) is deregulation of the programmed cell death-apoptosis. Apoptosis is a genetically
determined process controlled by bcl-2 gene family, with different genes promoting or
inhibiting cell death depending on the ratio of proteins with opposite function in apoptotic
athway. Bcl-2 gene belongs to antiapoptotic factors which overexpression may lead to
tumour progression.
The aim of this study was to estimate the bcl-2 immunoreactivity in OSSC and to assess its
clinicopathological implication. A series of the 26 OSCC formalin fixed, paraffin embedded
samples was analyzed for bcl-2 expression. The immunohistochemical staining was scored
according to the percentage of positive stained tumour cells. Using TNM classification there
were 7 tumours in stage II, 14 in stage III and 5 in stage IV. The positive staining was present
in all samples, with mean values and standard deviations 8.3±2.5, 7.5±1.1 and 8.4±5.8 within
stages II, III and IV, respectively. All tumours independently of the stage did not show
statistically significant difference in positive cytoplasmic staining against bcl-2, but its lower
expression was significantly associated with higher overall survival. Our results suggest that
bcl-2 expression could be a valuable biological marker in predicting disease behaviour.
- 415 -

Session X : Cell death in cancer Poster X, 83
Histone deacetylase inhibitor LAQ824 downregulates beta-catenin, inhibits cell
proliferation and induces apoptosis in colon carcinoma cell lines.
Ignacio Portero-Robles, Martine Pape, Sina Droll, Dieter Holzer, Ulrich Böcker, Martin
Ruthard, Oliver G. Ottman and Noriko Koyama.
Universitäts-Klinikum Frankfurt. ZIM II Hämatologie. Theodor-Stern-Kai 7 60590
Frankfurt am Main. Germany. E-Mail: robles@em.uni-frankfurt.de
Histone deacetylase (HDAC) inhibitors represent a class of anticancer agents that act by
promoting acetylation of histones, activating genes implicated in proliferation and apoptosis.
Mutations in the Wnt signaling pathway result in a continuous translocation of beta-catenin to
the nucleus acting as cofactor of TCF/LEF. Targets genes of these complex are constitutively
expressed inhibiting apoptosis and promoting tumor cells proliferation.
The aim of this study is to analyze the effect of LAQ824 (Novartis, Basel), a HDAC inhibitor,
in colon carcinoma cell lines and the contribution of the Wnt-signaling pathway to HDAC
inhibitor-induced apoptosis and inhibition of cellular proliferation.
After treatment with 10 to 200nM of LAQ824, we show acetylation of histones -3 and -4 and
induction of p21Waf/Cip1. Under the same condition cell proliferation is inhibited, annexin-V
expressing cells are increased, caspase -3, -9, -8, -2 as well as PARP cleavage is observed.
Similar assays with HCT116 p53(-/-) cells reveal no significant differences with the parental
HCT116.
Treatment of HCT116 cells with 100nM LAQ824 leads to decrease the protein level of betacatenin
and to downregulate consequently its target genes, c-jun, c-myc and cyclin D1.
Similar effects are also observed when cells are treated with 0,5µM Trichostatin A and 5mM
Valproic acid. HDAC2, an indirect target of beta-catenin which is overexpressed in colon
cancer tissues, is downregulated by VPA but not by LAQ and TSA.
Our results demonstrate that LAQ824 inhibit cell proliferation and induces apoptosis in a
caspase-dependent and p53-independent manner and indicate that Wnt signaling could be
involved in the anti-tumor effects of LAQ824.
- 416 -

Session X : Cell death in cancer Poster X, 84
APOPTOTIC PROPERTIES OF HYPERFORIN ON HUMAN B-CLL LEUKEMIC
CELLS
Claire Quiney, Christian Billard, Pezhman Mirshahi, Anne-Marie Faussat, Jean-
Dominique Fourneron *, and Jean-Pierre Kolb.
INSERM U736, Centre Biomédical des Cordeliers, Paris and * UMR6171, Faculté des
Sciences St Jérôme, Marseille
The natural compound hyperforin (HF), purified from Saint John’s wort, was tested on
leukemic cells from B cell lymphocytic leukemia (B-CLL) patients and on the ESKOL cell
line derived from a patient with hairy cell leukemia. HF was found to inhibit the proliferation
of ESKOL cells (IC50 around 1 µg/ml) and to elicit their apoptosis. Similarly, HF triggered a
time- and dose-dependent induction of apoptosis in B-CLL leukemic cells that was detected
by the increase in the percentage of annexinV-labelled cells and the augmentation of
internucleosomal DNA fragmentation. This was accompanied by the activation of caspase 3,
the disruption of the mitochondrial transmembrane potential &*m and the inhibition of nitric
oxide (NO) production. HF-induced apoptosis was reverted in the presence of the general
caspases inhibitor Z-VAD-fmk.
Incubation of B-CLL cells with HF also resulted in a marked suppression of their capacity to
release matrix metalloprotease 9 (MMP-9), an essential component in the degradation of the
extra cellular matrix. In addition, HF prevented the formation of tubules by human bone
marrow endothelial cells (HBMEC) cultured on matrigel, emphasizing its potential interest as
an anti neo-vasculogenesis drug.
Moreover, HF was found to impair the activity of several ABC pumps that are involved in the
multidrug resistance of the leukemic cells against chemotherapeutic reagents.
- 417 -

Session X : Cell death in cancer Poster X, 85
Multiple, independent melatonin-engaged signal transduction pathways are required for
melatonin anti-apoptotic effect.
Flavia Radogna, Laura Paternoster, Silvia Nuccitelli, Maria D’Alessio, Claudia Cerella,
Milena De Nicola, Antonio Bergamaschi*, Lina Ghibelli
Dipartimento di Biologia; *Cattedra Medicina del Lavoro, Università di Roma Tor
Vergata, Via Ricerca Scientifica 1, 00133 Rome Italy. E-mail: flavia.radogna@libero.it
Melatonin, in addition to its main role as regulator of circadian rhythms, has recently been
shown to modulate immune functions by controlling the behaviour of white blood cells
(WBC), which are indeed able to synthesize melatonin and possess the specific high affinity
(1nM) plasma membrane receptors (MT1 and MT2). Great interest is receiving the ability of
melatonin to contrast apoptosis, a well accepted fact whose mechanisms however are still
quite controversial. In this study, we analyze the mechanisms involved in the anti-apoptotic
effect of melatonin in normal and tumor WBC. We have shown that this effect is due to two
different, cooperating mechanisms, involving two primary targets melatonin directly interacts
with, i.e., MT1/MT2 receptors; and calmodulin, a known melatonin low affinity (63uM)
target. Receptor engagement and calmodulin binding give rise to two parallel signal
transduction pathways, consisting of a canonical signal transduction on the one side, and
phospholipase A2 (a known calmodulin interactor) and consequent 5-lipoxygenase (LOX)
activation and 5-HETE production, on the other. The two pathways converge in the inhibition
of Bax dimerisation and activation, key event of the intrinsic apoptotic pathway. The antiapoptotic
effect is completely abrogated if one or the other pathway is inhibited. The
necessity of the low affinity calmodulin binding explains the requirement of high melatonin
doses (>100uM), thus answering to the many perplexities as to whether melatonin antiapoptotic
effect was indeed due to MT1/MT2 receptor stimulation. The involvement of 5-
LOX in the anti-apoptotic effect of melatonin is particularly intriguing since 5-LOX has been
reported to be involved in tumor progression by inhibiting apoptosis in some tumor types. In
addition, the recruitment of a key enzyme of the inflammatory response may shed new lights
on the role melatonin plays in the regulation of the immune response. Moreover, LOX
activation implies a burst of free radicals that immediately (

Session X : Cell death in cancer Poster X, 86
Effect of roscovitine on wt p53 protein in human MCF-7 breast cancer cells: uncoupling
of p53 nuclear accumulation and its activation by site-specific phosphorylation
Carmen Ranftler, Marieta Gueorguieva and Józefa W*sierska-G+dek
Cell Cycle Regulation Group, Division: Institute of Cancer Research, Department of
Medicine I, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.
We have recently observed activation of wt p53 protein in human MCF-7 breast cancer cells
upon treatment with roscovitine (ROSC), a potent cyclin-dependent inhibitor. ROSC is
known to inhibit the activating phosphorylation of the carboxy-terminal domain of RNA
polymerase II, thereby blocking the global transcription. It has been previously suggested that
ROSC-induced upregulation of p53 protein is attributable to the inhibition of transcription of
Mdm2, a negative p53 regulator. However, the analysis of the expression of p53-responsive
genes revealed that Mdm2 was upregulated in ROSC-treated MCF-7 cells. The kinetics of the
Mdm2 increase was slightly delayed as compared with that for p21waf1. Exposure of human
MCF-7 cells to different proteosome inhibitors resulted in the time-dependent increase of p53
levels. However, unlike ROSC, they failed to modify p53 protein at Ser46 and to induce
p53AIP1 protein. Moreover, whereas ROSC arrested MCF-7 cells in the G2 phase of the cell
cycle, proteosome inhibitors blocked cells in the S-phase, presumably due to the prevention of
cyclin degradation. Our results indicate that prevention of p53 degradation by proteasome
inhibitors does not mimic the action of ROSC.
- 419 -

Session X : Cell death in cancer Poster X, 87
Role of plasma membrane fluidity in cisplatin-induced apoptosis in HT29 human colon
cancer cells
Amélie Rebillard1, Odile Sergent2, Olivier Meurette1, Gwénaëlle Le Moigne1, Morgane
Gorria1, Laurent Vernhet1, Dominique Lagadic-Gossmann1 & Marie-Thérèse
Dimanche-Boitrel1*
1INSERM UMR 620 & 2Laboratoire de Biologie Cellulaire et Végétale, Faculté de
Pharmacie, Université Rennes 1, 2 av du Prof Léon Bernard, 35043 Rennes cedex,
France.
Most current anticancer therapies induce tumor cell death through the induction of apoptosis.
However, the biochemical lesions leading to cell death are not always understood. The plasma
membrane has been considered as the most important target, other than DNA, for many
anticancer drugs. From a structural viewpoint, the plasma membrane is an heterogeneous
structure composed of distinct microdomains termed lipid rafts. These lipid rafts are enriched
in cholesterol and sphingolipids (sphingomyelin and glycosphingolipids) thus defining more
ordered area in the plasma membrane. These lipid microdomains actively participate in
several signaling transduction pathways, particularly in the Fas death receptor pathway, and
also in drug-induced apoptosis. We have recently shown that an early and transient increase in
plasma membrane fluidity, determined by a spin-labeling method using EPR (Electron
Paramagnetic Resonance), has been associated to agregation and relocalisation of Fas receptor
into plasma membrane lipid rafts during cisplatin-induced apoptosis in HT29 human colon
cancer cells (Lacour et al., Cancer Res. 2004, 64 (10): 3593-3598).
In order to study the role of plasma membrane fluidity, the effects of membrane stabilizing
agents (ursodeoxycholic acid (UDCA), cholesterol (Chol) and monosyaloganglioside 1
(GM1)) have been investigated in cisplatin-induced apoptosis in HT29 cells.
UDCA, Chol and GM1 inhibit the early increase in plasma membrane fluidity following
cisplatin treatment, and about 30 % the apoptosis induced by cisplatin, without modifying the
entry of cisplatin into HT29 cells (measured by atomic absorption spectrometry). Moreover,
we show that a pretreatment with stabilizing agents inhibits Fas agregation as well as lipid
rafts agregation evidenced by a FITC-cholera toxin labeling on the cell surface of HT29 cells
treated with cisplatin. Altogether, these data suggest that the transient increase in plasma
membrane fluidity contribute to cisplatin-induced early plasma membrane events and
subsequently to cisplatin-induced apoptosis.
- 420 -

Session X : Cell death in cancer Poster X, 88
In T lymphocytes, ablation of STAT3 expression reinstates STAT1-dependent apoptosis
by both IFNg and IL-6
Gabriella Regis1,2, Laura Icardi1,2, Laura Conti1,2, Roberto Chiarle1, Paola
Bernabei1, Valeria Poli1 and Francesco Novelli1,2
1Center for Experimental Research and Medical Studies (CERMS), San Giovanni
Battista Hospital, Via Santena 5, 10126, Turin, Italy and 2Department of Medicine and
Experimental Oncology, University of Turin, Corso Raffaello 30, 10125, Turin, Italy.
The Interferon gamma (IFNg)/Signal Transducer and Activator of Transcription (STAT) 1
pathway is mainly involved in the control of T lymphocytes homeostasis. However, these
cells downregulate IFNgR2 signaling chain by both ligand-dependent and ligand-independent
mechanisms and become refractory to the IFNg-mediated control of their expansion. Here, we
investigate the role of STAT3, a transcriptional factor activated by many cytokines including
IFNg, in the negative regulation of the IFNg/STAT1 signaling pathway. The expression of
STAT3 in two human malignant T cell lines, PF383 and ST4, was almost completely
inhibited by lentivirus that deliver small interfering (si) RNA against STAT3. The survival of
many neoplastic cells is dependent on STAT3 expression, whereas the viability of malignant
T cell lines we used was not influenced by STAT3 expression silencing. In T cells expressing
normal STAT3 amounts, either IFNg or IL-6 induced a weak activation of STAT1. By
contrast, in the absence of STAT3 both IFNg and IL-6 dependent STAT1 activation was
enhanced. This increased activation of STAT1 by IL-6 confirms previous data that showed
that this cytokine activates STAT1 and mediates an IFNg-like response in cells lacking
STAT3. As STAT1 regulates the expression of many genes involved in the control of cellular
proliferation and/or apoptosis we checked the proliferative responses to IFNg or IL-6 in T cell
lines where STAT3 is silenced or not, but we didn’t observe any alteration. On the contrary,
both IFNg and IL-6 switched on the apoptotic program in T cells devoid of STAT3
expression, whereas they were ineffective in cells expressing normal amounts of STAT3. The
apoptotic pathways are currently under investigation in our Laboratory. However, these data
clearly show that the combined inhibition of STAT3 synthesis and the treatment with IFNg or
IL-6 reinstate STAT1-dependent apoptosis in human T lymphocytes.
- 421 -

Session X : Cell death in cancer Poster X, 89
Overexpression of Lifeguard (LFG) suppresses TRAIL-induced apoptosis and activates
NF-kB
Kerstin Reimers, Claudia YU Choi, Susanne Kall, Peter M Vogt
Department of Plastic, Hand and Reconstructive Surgery, Medical School Hannover,
Podbielskistr. 380, 30659 Hannover, Germany, E-mail: Reimers.Kerstin@MH-
Hannover.de
TRAIL is a promising cytokine for cancer therapies, which preferentially induces apoptosis in
cancer cells by binding to death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5. Other tumor
cells, however, are resistant against TRAIL and require aditional treatment with radio- or
chemotherapy. Lifeguard (LFG) is an anti-apoptotic protein related to the Bax-inhibitory
protein 1 (BI-1). Cells expressing LFG are resistant against TRAIL- and Fas-induced cell
death. We were able to show that this protective effect can be transmitted to non-LFGexpressing
Fas-sensitive cell lines using transient transfection of LFG. In order to characterize
the functional mechanism of this antiapoptotic protein we determined the DNA-binding
activity of the NF-kB p65 subunit in cells overexpressing LFG. The cells showed an increased
nuclear translocation of the p65 subunit. The resulting activation of NF-kB depends on IkBa
degradation. An increased NF-kB activity is correlated with a sensitization of the treated cells
against induction of apoptosis by TRAIL. Many cancer cells show constitutive activation of
NF-kB leading to insensitiviy against therapeutically induced apoptosis. High expression of
LFG might contribute to cell death prevention by activation of NF-kB.
- 422 -

Session X : Cell death in cancer Poster X, 90
THE INFLUENCE OF ECHINACEA PURPUREA L. MOENCH TO THE TOXICITY
OF CADMIUM
Nijole Savickiene3, Alina Smalinskiene1, Stanislovas Ryselis1, Oleg Abdrakhmanov1,
Rima Kregzdyte1, Vaiva Lesauskaite2, Ilona Sadauskiene1, Leonid Ivanov1, , Virgilijus
Zitkevi(ius3, Arunas Savickas4
1Institute for Biomedical Research, Kaunas University of Medicine, 2Institute of
Cardiology, Kaunas University of Medicine, 3Department of Pharmaceutical Chemistry
and Pharmacognosy, Kaunas University of Medicine, 4Department of Drug Technology
and Pharmaceutical Management, Kaunas University of Medicine, Kaunas, Lithuania
E-mail : savickienenijole@takas.lt
Cadmium has a diversity of toxic effects including nephrotoxicity, carcinogenicity,
teratogenicity, endocrine toxicity. The aim of this study was to investigate the accumulation
of various levels of Cd2+ in tissues, and the effects of Echinacea purpurea L. Moench liquid
extract on Cd2+ induced changes in mice.
Materials and methods. Experiments were carried out on the white laboratory mice (n=47).
Chronic experiments were performed by giving to drink solution of different Cadmium and
EP concentrations: Cd2+ 25 mg/l, Cd2+ 250 mg/l, EP – 3.5 ml/l, daily for 8 weeks. Control
group mice was giving to drink equal amount of deionisated water. Cd concentration was
determined by atomic absorption spectrophotometer Perkin-Elmer/Zeeman 3030. The number
of mitotic liver cells was counted in 10 randomly selected reference areas (0.04 mm2).
Apoptosis of liver cells was histochemically detected by the TUNEL assay using AP (Roche)
in situ cell death detection kit. Preparation of extract from herb of Echinacea purpurea (L.)
Moench was made according British herbal pharmacopoeia.
Results. Cd2+ concentration in all explored organs and blood of mice, which were giving to
drink CdCl2 was statistically significantly higher than that of control group mice. After 8
weeks of experiment with mice giving to drink CdCl2 250 mg/l and EP extract solutions we
estimated, that number of apoptotic liver cells statistically significantly (p

Session X : Cell death in cancer Poster X, 91
Functional analysis of the tumorsuppressor Ras Association Family Domain 1
(RASSF1A) reveals an important role of the SARAH-domain
Claudia Seidel, Antje Klagge, Reinhard Dammann
AWG Tumor Genetics of the Medical Faculty, Martin-Luther-University Halle-
Wittenberg, 06097 Halle/Saale, Germany
INTRODUCTION: RASSF1A is a tumor suppressor gene, which is often epigenetically
inactivated in several cancer types. In lung cancer, a rate of between 30 to 45% of
hypermethylation of the RASSF1A promoter has been found. In several cancer types a
correlation between hypermethylation of RASSF1A and an impaired prognosis for cancer
patients was reported. The promoter of the RASSF1C isoform is never hypermethylated. The
two isoforms RASSF1A and RASSF1C only differ in the first domain, which is a
diacylglycerol binding site and lacks in RASSF1C. Moreover RASSF1A encodes a Ras
association domain and a SARAH domain, which is a protein-protein interaction domain.
METHODS: In order to investigate the function of RASSF1A, we created mutations and
deletions of these different domains and performed functional experiments in the lung cancer
cell line A549. Proliferation analyses as well as soft agar experiments were performed to
investigate the influence of the aberrant RASSF1A forms. The ability of the different forms of
RASSF1A to induce apoptosis was investigated. To analyse the localisation of RASSF1A and
its altered forms the cells were transient transfected using Yellow Fluorescent Protein (YFP)
constructs. Additionally, the interaction of RASSF1A with the proapoptotic kinase MST1 was
determined in precipitation experiments with protein extract from human cells.
RESULTS: The reduced proliferation of A549 cells that express RASSF1A could be
confirmed. Analysis of cells stable transfected with RASSF1A with a deletion of the SARAH
domain (RASSF1ADelSARAH) showed a even lower proliferation rate in comparison to
RASSF1A. A higher rate of apoptosis in cells with RASSF1ADelSARAH could be observed
using transient transfected cells. RASSF1A is when overexpressed as a YFP-fusion
colocalised to the cytoskeleton and during mitosis to the spindles and spindle poles. Using
RASSF1ADelSARAH the mitoses show strong spindles no spindle poles and the
chromosomes are not divided equally. In precipitation experiments MST1 interacts with
RASSF1A but not with RASSF1ADelSARAH.
CONCLUTIONS: The SARAH domain is necessary for the function of the tumor suppressor
RASSF1A. It is responsible for the interaction with MST1 and plays an important role in
regulation of mitotic processes as well as in induction of apoptosis.
This study was supported by BMBF grant 01ZZ0104.
- 424 -

Session X : Cell death in cancer Poster X, 92
ASSESSMENT OF EFFECT OF ECHINACEA PURPUREA ON APOPTOTIC AND
MITOTIC ACTIVITY OF LIVER CELLS DURING INTOXICATION BY CADMIUM
Alina Smalinskiene1, Stanislovas Ryselis1, Oleg Abdrakhmanov1, Rima Kregzdyte1,
Vaiva Lesauskaite2, Ilona Sadauskiene1, Leonid Ivanov1, Nijole Savickiene3, Virgilijus
Zitkevi(ius3, Arunas Savickas4
1Institute for Biomedical Research, Kaunas University of Medicine, 2Institute of
Cardiology, Kaunas University of Medicine, 3Department of Pharmaceutical Chemistry
and Pharmacognosy, Kaunas University of Medicine, 4Department of Drug Technology
and Pharmaceutical Management, Kaunas University of Medicine, Kaunas, Lithuania
E-mail : smalina@vector.kmu.lt
Cadmium (Cd) is a very toxic and carcinogenic element. It disturbs the activity of
biochemical systems of cells. Echinacea purpurea (L.) Moench (EP) can modify its influence.
The aim of the study was to evaluate the effect of EP to accumulation of Cd in blood, liver,
and kidney, mitotic and apoptotic activity of liver cells after the chronic intoxication by Cd2+.
Materials and methods. Experiments were carried out on the white laboratory mice. Mice
(n=57) were periodically i.p. injected for 6 weeks with CdCl2 (0.05 LD50 Cd2+) and EP
extract solutions of two different concentrations (0.05 LD50 and 0.1 LD50) and their
combinations. Control mice were injected with 0.9% saline. Cd concentration in blood and
tissue specimens was determined by atomic absorption spectrophotometer Perkin-
Elmer/Zeeman 3030. The number of mitotic liver cells was counted in 10 randomly selected
reference areas (0.04 mm2). Apoptosis of liver cells was histochemically detected by the
TUNEL assay using AP (Roche) in situ cell death detection kit. Preparation of extract from
herb of Echinacea purpurea (L.) Moench was made according British herbal pharmacopoeia.
Results. Cd2+ concentration in blood of mice in group EP(0.05)+Cd was 6.31-fold higher,
therefore in group EP(0.1)+Cd was 8.25-fold higher comparing to Cd group, in liver that was
1.78 fold and 2.11-fold, respectively, in kidney 2.1-fold and 1.92-fold, respectively. Higher
concentration (0.1LD50) of EP extract and Cd+EP 0.1LD50 arise apoptotic activity of liver
cells to compare with control group. The mitotic activity of liver cells induced by Cd2+ after
injection of EP extract was the same as in control group.
Conclusion. Long-term injections of extract of EP (0.1 LD50) combined with CdCl2 (0.05
LD50) leads to the significant increase of cadmium concentration in blood and all
investigated organs of experimental mice. EP decreases the mitotic activity of liver cells
induced by cadmium and increases apoptotic activity of the liver cells.
- 425 -

Session X : Cell death in cancer Poster X, 93
Antiangiogenic effect of Isoliquiritigenin through down-regulation of vascular
endothelial growth factor (VEGF) in breast cancer cells
Seung Hwa Son1,2,3, Kwang Kyun Park1,2,3, Mi Jeong Kim1,2,3, Jung Han Yoon
Park4, Soon Sung Lim4, Won Yoon Chung1,2,3
1Department of Oral Biology, 2Oral Science Research Center, 3Oral Cancer Research
Center, College of Dentistry, Yonsei University, Seoul 120-752, 4Division of Life Sciences
and Silver Biotechnology Research Center, Hallym University, Chunchon, Republic of
Korea, E-mail : hwaa7@msn.com
Vascular endothelial growth factor (VEGF) is an intensively studied molecule that has
significant potential, both in stimulating angiogenesis and as a target for antiangiogenic
approaches. VEGF achieves its multiple functions by activating two receptor tyrosine kinases,
Flt-1 and KDR, both of which are selectively expressed on primary vascular endothelium.
The interaction of VEGF and its receptors regulates tumor angiogenesis. In the current study,
we found that isoliquiritigenin (ISL) reduced VEGF and Flt-1 expression in two human breast
cancer cells, MCF-7 and MDA-MB-231. ISL also inhibited dose- and time- dependently the
proliferation of breast cancer cells in the presence and absence of VEGF. ISL completely
reduced VEGF-stimulated expression of two receptor tyrosine kinases in human umbilical
vein endothelial cells (HUVECs). Moreover ISL suppressed VEGF-induced cell proliferation,
migration and tube formation in HUVECs. Antiangiogenic activity of ISL was confirmed in
mouse Matrigel plug assay. In conclusion, these results suggest that ISL can be a candidate
agent for the prevention and treatment of breast cancer by blocking angiogenesis.
- 426 -

Session X : Cell death in cancer Poster X, 94
Apoptotic effect of celecoxib dependent upon p53 status in human ovarian carcinoma
cells
Yoo Cheol Song1, Su-Hyeong Kim1, Yong Sang Song1,2
1Cancer Research Institute, 2Department of Obstetrics and Gynecology, Seoul National
University, College of Medicine Seoul, Korea, E-mail: yssong@snu.ac.kr
Celecoxib, a selective cyclooxygenase-2 inhibitor, induces the apoptosis in various cancers in
COX-2 dependent and/or independent manners. The p53 protein is mutated in 50% of all
human tumors and plays a key role in apoptosis, cell cycle, and the expression of several
proteins. In ovarian cancer, the rate of p53 mutation has been shown to be very high and
associated with poor prognosis. To explore the importance of functional status of p53 in
apoptosis by celecoxib in ovarian carcinoma cells, the cellular response to celecoxib was
determined in SK-OV3 ovarian carcinoma cells with null type p53 and PA-1 with wild type
p53. Our results showed that celecoxib inhibited cell growth more in PA-1 than in SK-OV3.
The underlying antiproliferative mechanism may differ between these two cell types
dependent upon the functional status of p53, which plays integral roles in regulating cell cycle
and survival. Higher subG1 was shown in PA-1 than in SK-OV3 in response to celecoxib.
Caspase -8, -9, and -3 were activated more in PA-1 cells than in SK-OV3. These results
suggest that death receptor and mitochondria-mediated apoptotic pathways may be involved
in celecoxib induced apoptosis irrespective of the functional status of p53. Upregulation of
p21 were shown only in PA-1 cells. In summary, our study demonstrated that the celecoxib
effectively inhibited cell growth and induced apoptosis in human ovarian carcinoma cells.
However, apoptotic effect by celecoxib seemed to be different dependent upon the functional
status of p53.
- 427 -

Session X : Cell death in cancer Poster X, 95
Effect of paclitaxel on intracellular localization of c-Myc and P-c-Myc in prostate
carcinoma cell lines
Rosanna Supino1, Giuditta Cuccuru1, Enrica Favini1, Franco Zunino1, A. Ivana
Scovassi2
1Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milano,
Italy;, E-mail: rosanna.supino@istitutotumori.mi.it; 2Istituto di Genetica Molecolare del
CNR, Via Abbiategrasso 207, 27100 Pavia, Italy, E-mail: scovassi@igm.cnr.it
The proto-oncogene c-myc is involved in multiple cell pathways with opposite effects on cell
outcome of death or of proliferation. It has been proposed that these different roles depend on
its sequestration in cellular compartments and/or its phosphorylation. We speculated that
subcellular localization of c-Myc protein and of its phosphorylated form (P-c-Myc) could
have a role in the different response to PTX in two prostate carcinoma cell lines, PC3 and
DU45 cells, which undergo multinucleation or c-myc-dependent apoptosis, respectively. cmyc
is amplified only in PC3, but a similar extent of c-Myc phosphorylation was observed in
both cell lines after PTX treatment. By immunofluorescence we found that PTX-induced
upregulation of c-myc in DU145 cells, not occurring in PC3 cells, cannot be ascribed to
different protein localization, and that similar c-Myc and P-c-Myc nuclear translocation
occurs in both cell lines after drug treatment. Thus, subcellular localization of c-Myc and P-c-
Myc is not crucial in determining the mode of cell death in these prostate carcinoma cell lines.
- 428 -

Session X : Cell death in cancer Poster X, 96
Breast cancer cells response to the antineoplastic agents cisplatin, carboplatin and
doxorubicin, at the mRNA expression levels of distinct apoptosis-related genes,
including the new member, BCL2L12.
Hellinida Thomadaki and Andreas Scorilas
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of
Athens, Panepistimioupolis, Zografou, 15 701, Athens, Greece, Email:
elthoma@biol.uoa.gr, ascorilas@biol.uoa.gr
The drugs cisplatin, carboplatin and doxorubicin exhibit anticancer activity, the mechanism of
which is not yet completely clarified, although these agents are known to modulate the
expression of several genes including apoptosis-related genes, such as members of the BCL2
family. Recently, BCL2L12, a new member of the BCL2 family, was cloned by our group. In
order to define unequivocally the significance of the expression patterns of such genes as a
response to anticancer drug cytotoxic activity, we studied the possible alterations in the
mRNA expression levels of various apoptosis-related genes (BCL2, BAX, BCL2L12,
CASPASE-9, FAS), after cell treatment with distinct anticancer drugs (cisplatin, carboplatin
and doxorubicin), in the breast cancer cell line, MCF-7. The kinetics of cell toxicity were
evaluated by the MTT method, whereas the expression levels of distinct apoptosis-related
genes were analysed by RT-PCR, using gene specific primers. The percentage of non-viable
cells was up regulated with increasing concentrations and cell exposure time to the different
anticancer drugs. Distinct modulations of apoptosis-related genes, at the mRNA level, were
also observed. Further work is required in order to ascertain whether the mRNA expression
levels of distinct apoptosis-related genes may serve as a new prognostic factor predicting
chemotherapy outcome in breast cancer. Acknowledgements: The present research was
supported by an "EPEAEK II" grant, under the act “PYTHAGORAS I – SUPPORT OF
UNIVERSITY RESEARCH GROUPS”, with co-funding of 75% from the European Social
Fund and 25% from National Funds.
- 429 -

Session X : Cell death in cancer Poster X, 97
Effects of gamma and proton irradiation on human HTB140 melanoma cell growth,
induction of apoptosis and cell cycle redistribution
Danijela V. Todorovic1, Ivan M. Petrovic1, Lela B. Koricanac1, Lucia M. Valastro2,
Luigi Raffaele2, Giacomo Cuttone2, Aleksandra M. Ristic-Fira1
1 Vin(a Institute of Nuclear Sciences, Belgrade, Serbia and Montenegro
2 Istituto Nazionale di Fisica Nucleare, LNS, Catania, Italy
E-mail: aristic@vin.bg.ac.yu
Human HTB140 melanoma cells were irradiated with either gamma rays or protons, over the
dose range of 8 to 24 Gy. Cell growth was investigated using microtetrasolium (MTT) and
sulforhodamine B (SRB) assays 7 days after irradiation, time needed for cell survival
evaluation. With the increase of dose significant growth inhibition for both irradiation
qualities was detected. The same dose level of protons induced stronger inactivation of
HTB140 cells than single irradiation with gamma rays. Cell proliferation, detected by 5bromo-2
-deoxyuridine (BrdU) incorporation assay 7 days after proton and gamma
irradiation, has shown highly significant (p

Session X : Cell death in cancer Poster X, 98
Inhibition of cell-free apoptosis in Xenopus oocyte extracts by Src kinase
Alexander A. Tokmakov1, Mikako Shirouzu1, Shigeyuki Yokoyama1,2,3
1Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi, Yokohama, 230-0045
Japan, 2RIKEN Harima Institute at Spring-8, 3University of Tokyo, Japan. E-mail:
tokmak@gsc.riken.go.jp
Oncoproteins can induce both cell proliferation and apoptosis, depending on cellular context.
v-Src oncoprotein has been shown to activate several kinases, which can inhibit apoptosis,
including ERK and PI3-K. To further characterize the involvement of Src kinase in apoptosis,
we investigated the effect of Src overexpresion in the cell-free extracts prepared from oocytes
and eggs of the African clawed frog, Xenopus laevis. These extracts can recapitulate a range
of apoptotic events, including release of cytochrom C, caspase activation, and nuclear
fragmentation (Newmeyer et al., 1994). Notably, interphase-arrested and metaphase-arrested
extracts have different susceptibility to undergo apoptosis. The MAPK pathway active in
metaphase-arrested extracts was shown to protect them from apoptosis (Tashker et al., 2002).
In the present study we measured the caspase 3/7 activation and cytochrom C release in
apoptotic cell-free Xenopus oocyte and egg extracts, overexpressing constitutively active (v-
Src) and kinase negative (Src KN) mutants of Src kinase. We found that interphase-arrested
oocyte extracts were highly apoptotic. They released cytochrom C and displayed elevated
levels of caspase activity after several hours’-long incubation at room temperature. Caspase
activation in these extracts could be prevented by overexpression of either v-Src or Src KN
protein kinase mutants. No or little effect of Src overexpression on cytochrom C release was
detected. On the other hand, metaphase arrested egg extracts were resistant to apoptosis and
they could not be regulated by Src kinase. Our data suggest that the inhibition of apoptosis in
Xenopus oocyte extracts by Src kinase is not mediated by activation of the MAPK pathway.
- 431 -

Session X : Cell death in cancer Poster X, 99
Hypoxia-induced cell death in human malignant glioma cells: the role of metabolic
pathways
Felix Tritschler, Michael W. Ronellenfitsch, Michael Weller and Joachim P. Steinbach
Laboratory of Molecular Neuro-Oncology, Department of General Neurology, Hertie
Institute for Clinical Brain Research, University of Tuebingen, Medical School, Otfried-
Mueller-Str. 27, 72076 Tuebingen, Germany
Presenting author: Tel. +49 7071 2981969, FAX: +49 7071 295260,
E-mail: f.tritschler@gmx.de
Adaptive responses to hypoxia are important determinants of tumor cell survival under the
adverse conditions of the tumor microenvironment. We have previously established a
paradigm of hypoxia-induced cell death of human malignant glioma cells. In this model,
epidermal growth factor receptor (EGFR) inhibition and cycloheximide protect glioma cells
from hypoxia by reducing energy consumption and maintaining ATP levels and
mitochondrial integrity. Here, we have examined the impact of glucose and amino acid
restriction and withdrawal on cellular energy homeostasis and cell death.
Restriction of glucose to 2 mM or less was necessary for cell death. Amino acid withdrawal
had a smaller but definite independent influence. Notably, EGFR inhibition and
cycloheximide were only protective when glucose was present, suggesting that they exert
their protective effect exclusively via reducing glucose consumption. Then, signaling
cascades downstream of EGFR were examined. Hypoxia suppressed the phosphorylation of
protein kinase B (PKB)/Akt and the mammalian target of rapamycin (mTOR) effectors
ribosomal protein S6 (RPS6) and eukaryotic initiation factor 4E-binding protein 1. Further,
hypoxia induced phosphorylation of AMP-activated protein kinase (AMPK), a crucial
metabolic regulator downstream of the tumor suppressor kinase LKB-1. The AMPK target
acetyl-CoA carboxylase was also phosphorylated. Activation of AMPK by the synthetic
compound 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAr), mimicking a
starvation signal, protected glioma cells from hypoxia.
These data delineate how glioma cells generate adaptive metabolic responses and suggest
targets for therapeutic interventions.
- 432 -

Session X : Cell death in cancer Poster X, 100
Potential cytoprotective effects of new synthesised sulphur containing compounds
Tzvetomira A. Tzanova1, 3, Ognyan Petrov2, Laurent Brault3, Denyse Bagrel3,
Margarita H. Karaivanova1
1. Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str, 1000 Sofia,
Bulgaria; e-mail: tzvete_tzanova@abv.bg
2. Faculty of Chemistry, University of Sofia"St. Kliment Ohriski", 1 James
Bourchier Blvd, Sofia 1164, Bulgaria; e-mail: OPetrov@chem.uni-sofia.bg
3. Laboratoire d'Ingénierie Moléculaire et Biochimie Pharmacologique, UFR
SciFA, Université Paul Verlaine - Metz, Campus Bridoux, rue du Général Delestraint,
57070 Metz, France; e-mail: bagrel@univ-metz.fr
Cancer pathogenic mechanisms are associated with DNA damage, oxidative stress, and
chronic inflammation. Therapies goal to maximizing tumour death while minimizing damage
to normal tissues. The delivery of high doses of chemotherapy will increase survival though it
is often limited by the development of severe adverse effects. Numerous studies have
demonstrated that sulphur-containing nucleophiles can antagonize the dose–limiting effects of
alkylating agents. The aim of this study is to assess the chemoprotective potential of seven
new 1-(4-Bromo-2-mercaptophenyl)-3, 4-dimethyl-1, 3-dihydroimidazol-2-one derivatives.
The chemoprotective effect of these synthesized sulphur-containing compounds was
evaluated on HL-60, HL-60/Dox, SKW-3, K-562, EJ and MCF-7 malignant cell lines (MTT
assay). Four of the tested molecules present an interesting effect since they did not display
cytotoxic effects on cells at concentrations up to 100 µM when used as pre-treatment. They
limit the cell mortality induced by cisplatin on MCF-7 breast carcinoma cell line. As expected
cisplatin treatments resulted in accumulation of cells in S phase. Two compounds, containing
mercaptoacetyl group, did not influence cisplatin effect on the cell cycle (DNA
quantification) indicating that these molecules did not seem to interact directly with cisplatin.
According to these results the oxidative stress was evaluated. A pre-treatment of the cells with
these compounds followed by administration of cisplatin did not affect the level of GSH, the
major intracellular antioxidant (HPLC method). As reactive oxygen species are important
mediators of antineoplastic drugs, we actually focus on the evaluation of antiradical and
antioxidant activities of the most interesting compounds as well as on their implication in
apoptosis induction.
- 433 -

Session X : Cell death in cancer Poster X, 101
Glycolysis inhibition in cancer cells by redox-cycling compounds
Julien Verrax, Henrik S. Taper and Pedro Buc Calderon
Pharmacokinetic, Metabolism, Nutrition and Toxicology Unit, School of Pharmacy,
Université Catholique de Louvain, e-mail:julien.verrax@pmnt.ucl.ac.be
Among all the different features of cancer cells, two of them are of particular interest: their
nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress, both
resulting presumably from the combination between high anabolic needs and environmental
growth conditions lacking O2. Therefore, we took advantage of these features to develop an
experimental approach by exposing cancer cells to an oxidant insult induced by the
association of menadione and ascorbate. This association develop a synergistic antitumor
activity in vivo as well as in vitro, based on the potentiation of the menadione redox cycling
by ascorbate. This leads to the intracellular generation of reactive oxygen species among them
H2O2 appears to be the main responsible of the cytolytic effect. Since lactate and ATP are
severely and rapidly depressed following ascorbate/menadione exposure, we suggest that the
major intracellular event is related to the impairment of glycolysis [Verrax et al, 2005].
Indeed, NAD+ is rapidly consumed, related with a strong Poly(ADP-ribose) polymerase
activation, thus explaining the glycolysis inhibition. The profile of cell death do not
correspond to apoptosis (no caspase-3 nor PARP processing, DNA strand breaks with spread
pattern) but is rather close to necrosis [Verrax et al, 2004]. Strong morphological events occur
during the cytolytic process including the formation of cytoplasmic pieces, organelles-free.
On these morphological basis, such a cell death has been called autoschizis, which appears as
a new kind of necrotic cell death [Jamison et al, 2002]. Due to the high energetic dependence
of cancer cells towards glycolysis, the impairment of such an essential pathway may explain
the effectiveness of this association on many tumor cells.
This work was financed by grant from the Belgian Fonds National de la Recherche
Scientifique (FNRS-FRSM Grant 3.4.594.04.F)
- 434 -

Session X : Cell death in cancer Poster X, 102
Influence on Caspase-3 and –9 activity by Survivin splice variants in vitro
J Wagener, HE Gabbert, C Mahotka
Institute of Pathology, Heinrich Heine-University, Moorenstr.5, 40225 Düsseldorf,
Germany, jessica.wagener@gmx.de
Objective: One important hallmark of cancer cells is the resistance to apoptosis. Survivin is a
structurally and functional unique member of the inhibitor of apoptosis protein (IAP) family
that acts at the interface between apoptosis and cell cycle control. In many human cancers
aberrant expression of Survivin correlates with poor prognosis and resistance to
chemotherapy. Moreover, one of the main impacts of Survivin is the inhibition of caspases.
Therefore, we analysed the inhibitory potential of the three Survivin splice variants, i.e.
Survivin-deltaEx3, Survivin-2B, and Survivin-3B in vitro.
Methods: Transfection of HEK293 cells (with GFP-Survivin, GFP-Survivin-deltaEx3, GFP-
Survivin-2B and GFP-Survivin-3B), Western Blotting, Caspase-Assays with purified
recombinant Survivin, XIAP, and Caspase-3 and –9, and cell extracts of transfected HEK293
cells.
Results: 1. Survivin inhibits recombinant Caspase-3 as well as Caspase-9 in a concentration
dependent manner. 2. XIAP enhances the ability of Survivin to inhibit Caspase-3 activity. 3.
In contrast, Survivin does not increase the potential of XIAP to suppress Caspase-9 activity.
4. After transfection with GFP-tagged Survivin, HEK293 cells extracts inhibit recombinant
Caspase-9 but not recombinant Caspase-3. 5. HEK293 cell extracts transfected with GFP-
Survivin-deltaEx3, GFP-Survivin-2B and GFP-Survivin-3B fail to inhibit both Caspase-3 and
-9 activity.
Conclusions: IAPs contribute to the lack of ability of many tumors to undergo apoptosis. Our
findings show that – in contrast to Survivin and XIAP – the mainly carboxy-terminal
modified splice variants Survivin-deltaEx3, Survivin-2B, and Survivin-3B fail to inhibit
caspases-3 and -9 activity in vitro. Therefore, there is a striking evidence for the
carboxyterminal region of Survivin to be involved in caspase inhibition.
- 435 -

Session X : Cell death in cancer Poster X, 103
Dual action of roscovitine on human MCF-7 breast cancer cells: induction of G2 arrest
via inhibition of CDKs and initiation of apoptosis by activation of site-specific
phosphorylation of wt p53 protein
Józefa W*sierska-G+dek, Carmen Ranftler and Marieta Gueorguieva
Cell Cycle Regulation Group, Division: Institute of Cancer Research, Department of
Medicine I, Medical University of Vienna, Borschkegasse 8 a, A-1090 Vienna, Austria.
E-mail: Jozefa.Gadek-Wesierski@meduniwien.ac.at
Exposure of MCF-7 cells to roscovitine (ROSC), a potent CDK inhibitor, resulted in a strong
inhibition of cell proliferation. Detailed analysis revealed that ROSC arrested MCF-7 cells in
G2 phase of the cell cycle and induced apoptosis. Cell cycle arrest preceded the main wave of
apoptosis. The cell cycle block was attributable to the inactivation of CDK2 and CDK1. Postincubation
of G2 arrested cells for 24h in a drug-free medium did not diminish the number of
the G2 cell population indicating that ROSC-mediated cell cycle arrest was prolonged. ROSC
induced wt p53 tumor suppressor protein in MCF-7 cells in a time- and dose-dependent
manner. ROSC increased p53 steady-state. The half-life of wt p53 protein increased
approximately forty-fold after exposure of MCF-7 cells to ROSC for 15h. At 4h after ROSC
administration a strong phosphorylation of serine at position 46 was observed. The onset of
Ser-46-p53 phosphorylation preceded the induction of apoptosis. P-Ser-46-p53
transcriptionally upregulated p53AIP1 protein, a component of mitochondria. Surprisingly,
the reconstitution with human caspase-3 did not sensitize MCF-7 cells to the action of ROSC.
Our results unequivocally show that ROSC simultaneously inhibits CDK2 and CDK1 and
activates a kinase catalyzing the phosphorylation of p53 protein at Ser46.
- 436 -

Session X : Cell death in cancer Poster X, 104
Protein profiling of different human breast cancer cells lines in response to cisplatin
treatment and validation of anti-apoptotic Bcl-2 as a target for enhancing cisplatin
sensitivity
Christina Westmose Yde and Olaf-Georg Issinger
Institute of Biochemistry and Molecular Biology, University of Southern Denmark,
Campusvej 55, 5230 Odense M, Denmark. E-mail: chriswy@bmb.sdu.dk
Clinical problems in the current treatment of breast cancer patients include severe dosedependent
side effects of the compounds used and development of resistance to the treatment.
Therefore, in prospect of improving the current therapy of breast cancer patients it is
important to study the mechanisms behind drug responsiveness. Five different human breast
carcinoma cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-435, HCC-1937 and CAL-
148, were analyzed in respect to sensitivity to the chemotherapeutic compound cisplatin and
dose-dependent drug-induced cell death was measured by reduction in cell viability (WST-1
test) and by poly(ADP)ribose polymerase (PARP) cleavage. While high doses of cisplatin
were required in order to induce cell death in MCF-7 and MDA-MB-231 the cell lines, MDA-
MB-435, HCC-1937 and CAL-148, were sensitive to cisplatin at much lower doses. In order
to identify proteins involved in sensitizing the cells to cisplatin the breast cancer cell lines
were profiled in respect to protein expression levels of a variety signaling proteins. In all cell
lines an increase in cyclin E and a decrease in cyclin D1 was found after cisplatin treatment.
Furthermore, the mitogen-activated protein kinases, ERK and p38, were phosphorylated at
their activating residues when cell were treated with cisplatin. Neither expression of wild type
p53, estrogen receptor status nor activated Akt seemed to be of primary importance in respect
to cisplatin sensitivity, however, an inverse correlation was found between expression of the
anti-apoptotic protein Bcl-2 in the breast cancer cell lines and cisplatin sensitivity. Therefore,
RNA interference was used in order to knock down Bcl-2 protein in MCF-7, which expressed
the highest amount of Bcl-2, and it was found that Bcl-2 siRNA significantly sensitized MCF-
7 to cisplatin.
Hence, our results confirm the anti-apoptotic Bcl-2 as a possible target for increasing the
effect of chemotherapeutic compounds in breast cancer.
- 437 -

Session X : Cell death in cancer Poster X, 105
Significant co-expression of GLUT-1, Bcl-xl and Bax in colorectal cancer.
Andrzej Wincewicz, Mariola Sulkowska, Mariusz Koda, Luiza Kanczuga-Koda,
Stanislaw Sulkowski.
Department of Pathology, Medical University of Bialystok, Waszyngtona 13, 15-269
Bialystok, Poland. E-mail: sulek@zeus.amb.edu.pl
Hypoxic cancer cells overexpress GLUT-1 (Glucose transporter 1) to accelerate glucose
intake mainly for low effective, anaerobic respiration, so that they wouldn’t die of oxygen
deficiency. Ischemic cell injury triggers apoptosis. Regulators of cell suicide like Bax and
Bcl-xl combine their functions to cause apoptosis or to rescue cells from death. GLUT-1, Bax
and Bcl-xl are of prognostic significance in colorectal cancer but they have not been
compared, yet. Thus, we aimed to determine eventual correlations between GLUT-1, Bax and
Bcl-xl in association with different clinicopathological features of colorectal cancer patients.
Expressions of the proteins were evaluated in specimens of 150 colorectal patients by
immunohistochemistry with appliance of specific antibodies. The levels of tissue expressions
were statistically analyzed with Spearman’s correlation test. Similarly to all the patients,
GLUT-1 matched Bcl-xl and Bax in statistically significant manner regardless node
involvement, grade of histological differentiation, histopathological type, tumor site, sex and
age of patients. GLUT-1 correlated highly with Bcl-xl in both T2 and T3 tumors
(p

Session X : Cell death in cancer Poster X, 106
Impact of Rb knock down on the modulation erufosine signal transduction in human
leukemia cells
Maya M. Zaharieva1,2, Milen Kirilov2, Minqiang Chai2, Stefan Berger3, Spiro M.
Konstantinov1,2, Martin R. Berger2
1Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str, 1000 Sofia, Bulgaria
2German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany
3Central Institute for Mental Help, Department of Molecular Biology, J5, 68 159
Mannheim
The retinoblastoma gene Rb1 is a prototypical tumour supressor. Consistent with its tomoursupressor
function Rb is known to inhibit proliferation. Recent studies indicate its role in
suppressing apoptosis, too. Loss of Rb sensitizes cells to apoptosis, but loss of Rb can only
contribute to tumor development under conditions in which apoptosis response is already
compromised. The new alkylphosphocholine erufosine is an inductor of Rb in leukemic cells.
Therefore, we decided to suppress the expression of RB using conventional phosphorotioate
antisense oligonucleotides and siRNAs. Unfortunately, both types of oligonucleotides used
didn’t cause stable and reproducible knock-down and we initiated viral transfection
experiments for stable Rb knock-down in SKW-3, BV-173 and K-562 cells. The antisense
sequence was firstly cloned in the pSUPER vector and tested on HEK 293T cells. For
normalizing the transfection efficiency an EGFP-expressing vector was used in addition. To
realize the transfer of the H1-shRNA cassette directed to the Rb-mRNA, the cells were
infected with LentiLox virus which co-express an EGFP-protein. The resulting levels of target
mRNA were measured by real-time RT-PCR after excluding of non-fluorescent cells by flow
cytometry and cell sorting. The highest decrease of the Rb-mRNA (about 86%) was found in
SKW-3 cells. A concomitent knock-down at the protein level was evidenced by Western
blotting. The proliferation activity of the infected cells was estimated by colorimetric MTTdye
reduction assay before and after erufosine treatment. Comparison of Rb knock-down and
wild type leukemic cells revealed the presence of significant differences in cell cycle duration
and sensitivity to erufosine, thus confirming the impact of Rb modulation for the mode of
action of alkylphosphocholines which are known as promising antineoplastic signal
transduction modulators.
- 439 -

Session X : Cell death in cancer Poster X, 107
Upregulation and phosphorylation of p53 and its related proteins after treatment with
valproic acid in MOLT-4 leukemia cells
Darina Zá)kodová1, Martina ,ezá(ová1, Ji-ina Vávrová J2, Doris Vokurková3, Ale)
Tich.2
1Inst. of Medical Biochemistry and 3Inst. of Clin. Immunology and Allergology, Univ.
Hospital and Faculty of Medicine in Hradec Králové, Charles Univ. in Prague, Czech
Republic, 2Dept. of Radiobiology, Faculty of Military Health Sciences Hradec Králové,
Univ. of Defense Brno, Czech Republic, E-mail: zaskodovad@lfhk.cuni.cz
Objective: Our work consists in evaluating changes of protein expression in MOLT-4
leukemia cells after treatment with valproic acid, a histone deacetylases inhibitor. Inhibitors
of HDAC significantly affect transcription of genes involved in the regulation of cell cycle,
apoptosis and DNA synthesis by changes of histone acetylation and therefore by changes of
tertiary structure of DNA. The cell cycle arrest or apoptosis can be induced by tumor supresor
protein p53. Activated p53 binds to DNA and induces transcription of appropriates genes
including p21, bax, MDM2 and cyclin G. Materials and methods: Apoptosis has been
measured by flow-cytometry. Western blotting has been used for determination of histone
acetylation and expression of p53, MDM2 and p21. Results: VA in low concentrations (2 and
4 mM) causes elevated expression of p53 and p53 phosphorylation at serin 392 in a period
from 2 to 4 hours of treatment. This is followed (in the case of 2mM VA) by the expression of
protein p21. Concurrently with p53 upregulation occurs the MDM2 phosphorylation at serine
166. The cleavege of lamin B as an apoptosis control proceeds from 24 hours after 4mM VA
treatment. When the cells were incubated three days with VA, EC50 determined by
clonogenic survival assay was 1.76 mM. In the case of continuous exposure (14 days) to VA
EC50 decreased to 0.625 mM. The percentage of apoptic cells determined with flowcytometric
analysis (subG1 peak) increased to 37% after 72 hours of treatment with 4 mM
VA. Conclusion: Our results show that after treatement by valproic acid the amount of active
p53 increases, which is related to increased inactivation of MDM2. VA in 2 mM
concentration causes primarily the cell cycle arrest via p21, but in 4 mM concentration leads
the cells directly to apoptosis. This work was supported by The Ministry of Education of
Czech Republic, project MSM 0021620820
- 440 -

Session X : Cell death in cancer Poster X, 108
Targeting the cell cycle and the PI3K pathway: a universal strategy to reactivate innate
tumor suppressor programmes in cancer cells?
Thérèse David-Pfeuty1*, Michel Legraverend2 , Odile Ludwig2, and David S. Grierson2
Institut Curie-Recherche, 1UMR 146 and 2UMR 176 du CNRS, Bâtiment 110, Centre
Universitaire, 91405 Orsay Cedex, France
A rationale for why corruption of the Rb and p53 modules facilitates tumorigenesis is because
it lends oncogene-bearing cells a surfeit of Cdk activity and growth enabling them to
elaborate strategies to evade tumor suppressor mechanisms and inappropriately divide. Thus,
a potentially universal means to palliate their deficiency in cancer cells would be to target
both the activity of Cdks and their PI3K module that is a central regulator of cell growth. This
hypothesis is supported by our observation that the killing efficacy of Cdk inhibitors,
roscovitine and 16 other purine derivatives, decreased with increasing corruption of the Rb
and p53 modules. Notably, the two most resistant tumor cell lines were (Rb-/p53-) Saos-2,
followed by HBL100 (in which Rb and p53 are inactivated by binding to the large T antigen
of SV40); conversely, the most sensitive cell lines were (Rb+/p53*) HaCaT and A431 that
display normal differentiation features in culture, suggestive of a robust Rb pathway, followed
by (Rb+/p53+) MCF-7 and SCC15 tumor cell lines that carry a faulty Rb pathway. A further
case has been provided by the observation that the potentiation of roscovitine-induced
apoptosis by the PI3K inhibitor, LY294002, also decreased with increasing corruption of the
Rb and p53 modules. Another important finding of our work is that purines differing by a
single substitution, which exerted little lethal effects on distant cell types (differing by their
Rb/p53 status and tumorigenic potential) grown on a rich medium, could display widelydiffering
cytotoxicity profiles toward the same cell types grown on a poor medium. This
highlights that structurally related compounds targeting the same Cdks may also target unique
proteins or Cdks that may control unique biological functions or compete for the interaction
between the compounds and their therapeutically relevant Cdk targets. The range and
concentration of such crossreacting targets would depend on the cell translational capacity
that, in turn, would depend on the cell genotype. In the perspective of clinical development in
association with inhibitors of the PI3K pathway, it might be advised, then, to select tumor cell
type-selective Cdk inhibitors on the basis of their cytotoxicity in cell-based assays performed
at a limiting serum concentration, sufficient to suppress their interaction with undesirable
crossreacting targets.
- 441 -

Notes
- 442 -

Notes
Notes
- 443 -

- 444 -

Session XI : Cell death and cardiovascular diseases
- 445 -

Session XI : Cell death and cardiovascular diseases Poster XI, 1
Role of phospho-p42/44 MAPK in the altered reactivity of vascular smooth muscle cells
during experimental cirrhosis.
Antonia Alcaraz, David Iyu, Noemí M. Atucha, Joaquín García-Estañ, María C. Ortiz.
Dpto. Fisiología, Fac. Medicina, Universidad de Murcia, Spain. E-mail: clara@um.es
High levels of nitric oxide (NO) and oxidative stress (OS) appear as important contributors in
the altered vascular function of liver cirrhosis. In fact, administration of NO synthesis
inhibitors or antioxidants improves or corrects this alteration although the underlying
mechanisms by which that happen are not yet clear. NO and OS derived products are
regulatory molecules in signaling pathways influencing the contractile function of vascular
smooth muscle cells (VSMC). OS can activate the MAPK (mitogen-activated protein kinase)
signaling pathway, which can be interdependent with other pathways (PKB, NF-kB)
implicated in the intracellular regulation of NO levels. Thus, in this study we evaluated the
role of phospho-p42/44 MAPK in the altered reactivity of VSMC in cirrhotic rats by chronic
bile duct ligation (CBDL; 3 weeks). For that, we examined the effect of the antioxidant
vitamin E (VitE; 5000 IU/kg diet) or/and the NO synthesis inhibitor, L-NAME (10-4 M,
acutely), on the vasoconstrictor responses to phenylephrine (PE) in aortic rings of CBDL rats.
Besides, we studied the activation of p42/44 MAPK in primary culture of aortic VSMC of
CBDL and control rats (3-5 pass), before or after adding a NO-donor with sodium
nitroprusside (SNP; 200-1000 µM) or stimulating OS with deoxycholic acid (DOXCA; 50-
200 µM). To assess the role of NO and OS on MAPK activation, we pre-incubated the cells
with L-NAME or VitE (300-1000 µM). Results: aortic rings of CBDL rats (treated or not with
VitE) responded less to PE than controls and the addition of L-NAME abolished this
hyporesponsiveness. Aortic phospho-p42/44 MAPK expression tended to increase in CBDLs
(100±10, 117±7 and 133±23 %, in control, CBDL and CBDL+VitE, respectively) without
difference between groups. Cultured VSMC from CBDLs showed higher activation of p42/44
MAPK than controls and L-NAME and VitE significantly decreased it. SNP and DOXCA
increased dose-dependently the activation of p42/44 MAPK in both groups of cells, but at
higher degree in controls at lower doses. In conclusion, VSMC from CBDL rats showed a
MAPK activation sensitive to NO and OS. This altered intracellular signaling might
contribute to specific functional changes in VSMC and thus to the abnormal vascular
response in cirrhosis.
- 446 -

Session XI: Cell death and cardiovascular diseases Poster XI, 2
Greater storage calcium capacity but lower capacitative calcium entry in platelets of
rats with biliary cirrhosis.
Noemí M. Atucha, F. Ramírez, Esther García, Dolores Robles, David Iyú, Antonia
Alcaraz, María C. Ortiz, Joaquín García-Estañ.
Dpto de Fisiología, Facultad de Medicina, 30100 Murcia, Spain. E-mail: ntma@um.es
It is known that liver cirrhosis shows a tendency to bleeding, which is in part due to a
functional alteration of platelets. Platelet stimulation with physiological agonists such as ADP
produces an increase in cytoplasmic calcium levels ([Ca 2+ ] c ), due to the release from internal
stores and to the entry stimulated by its depletion (capacitative entry). In platelets there are
two types of calcium stores and they can be separetely studied by analyzing their sensitivity to
calcium pump inhibitors, thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone
(TBHQ). In this work, we have analyzed the role of these two types of stores in the mediation
of the changes in [Ca 2+ ] c produced by capacitative calcium entry. Methods: the experiments
have been performed in washed platelets, loaded with fura-2, obtained from cirrhotic (bile
duct ligation) or control rats. Fluorescence has been measured in a spectrofluorimeter,
following a well established method. Capacitative calcium entry was studied, after the
depletion of internal stores in zero-calcium conditions, by adding 0.5 mM external calcium or
by fura-2 quenching in the presence of extracellular Mn 2+ , measured at 360 nm. Results: the
elevations in extracellular calcium were greater with TBHQ than with TG, which indicates
that the stores sensitive to TBHQ are bigger than those sensitive to TG. In both cases,
platelets of BDL rats showed greater calcium elevations than those of the controls, suggesting
a greater storage capacity in these rats. A bigger calcium storage was also confirmed with
ADP or thrombin which, in the absence of external calcium, produced greater calcium
elevations in platelets from BDL rats. After partial depletion with TBHQ or total depletion
with TBHQ+TG, capacitative calcium entry was always significantly lower in platelets from
BDL rats. Platelet aggregation in response to ADP was also lower in BDL rats. These results
indicate that capacitative calcium entry, in response to store depletion, is altered in platelets
from rats with experimental cirrhosis. This altered response can produce lower calcium levels
in response to agonists acting through the depletion of internal calcium stores. This alteration
can be related to the aggregation defects showed by the platelets of the cirrhotic rats.
- 447 -

Session XI: Cell death and cardiovascular diseases Poster XI, 3
Role of urocortin in early myocardial pre- and postconditioning
Barbara Cserepes, Boglarka Racz, Balazs Gasz, Andrea Ferencz, Maria Kurthy, Janos
Lantos, Gabor Jancso, Erzsebet Roth
Department of Surgical Research and Techniques, Medical Faculty, University of Pecs,
Hungary. E-mail: barbicska@yahoo.co.uk
Pre- and postconditioning are powerful endogenous adaptive phenomenon of the organism
whereby different stimuli (hypoxia, drugs, hypotermia) enhance the tolerance against various
types of stress. Urocortin, member of the corticotrophin-releasing factor family has potent
effects on the cardiovascular system including vasodilation, increases in coronary blood flow
and cardiac contractility, and induces protection against ischaemia/reperfusion damage.
Recent studies reported that urocortin has similar cardioprotective effects as the ischaemic
preconditioning in both early and delayed phases. The aim of this study was to investigate the
effect of urocortin on cultured cardiomyocytes in the process of pre– and postconditioning.
Isolated neonatal rat ventricular myocytes were preconditioned with adenosine, simulated
ischaemia and urocortin (10 minutes treatment followed by 10 minutes
reperfusion/resolution). For detecting the effect of alternative types of preconditioning,
necrosis enzyme (LDH) release, vital staining (trypan blue) and ratio of apoptosis/necrosis
were examined after cardiac cells were exposed to 3 hours sustained ischaemia and 2 hours
reperfusion. Same parameters were measured in the postconditioned groups (30 or 60 minutes
ischaemia followed by postconditioning with 10 min ischaemic stimulus or urocortin and 2
hours reperfusion). Cells exposed to 3 h ischaemia followed by 2 h reperfusion were shown as
control.
Our results show, that LDH release and the ratio of necrotised cells was decreased, but
proportion of alive/dead myocytes was increased in all preconditioned groups compared with
control group. In postconditioned groups LDH content of culture medium, number of trypan
blue-stained cells was reduced, rate of apoptotic/necrotic cells was raised contrasted with non
postconditioned group, and the difference was more expressed in urocortin treated group after
60 min ischaemia.
We can conclude that urocortin might play an important role in myocardial cytoprotection.
Preconditioning with urocortin induced such a powerful cell protective effect as adenosine
and ischaemia. Furthermore, postconditioning with urocortin after 60 min ischaemia was
more cardioprotective than ischaemic postconditioning. Supported by the Hungarian
Scientific Research Foundation (OTKA) T-048851, T-038035, F046593.
- 448 -

Session XI: Cell death and cardiovascular diseases Poster XI, 4
MAPKs are activated via cPLA2 during ischaemia/reperfusion induced injury in
neonatal cardiomyocytes.
Anna-Mart Engelbrecht and Amanda Lochner.
Departments of Physiological Sciences and Medical Physiology, University of
Stellenbosch, Stellenbosch, 7600, South Africa. E-mail: ame@sun.ac.za
Myocardial ischaemia/reperfusion (I/R) injury induces death of a significant number of
cardiomyocytes and contributes to mortality. Although it has become increasingly clear that
myocardial I/R result in the death of myocytes through apoptosis, the molecular basis of this
process remains to be elucidated. Therefore, the aim of this study was to investigate the role
of cPLA2 in MAPK phosphorylation and caspase-3 cleavage in I/R-induced apoptosis in
neonatal cardiomyocytes.
Cultured neonatal cardiomyocytes were exposed to various inhibitors for 30 minutes before
the onset of 60 minutes of simulated ischaemia (induced with deoxyglucose and KCN)
followed by 30 minutes of reperfusion. Samples were analysed by Western blotting with
phospho-specific antibodies recognizing total- and phosphorylated p38 MAPK, ERK and
cPLA2. Apoptosis was determined by poly(ADP-ribose)polymerase cleavage and caspase-3
activation. Hoechst 33342 stain was used to view the morphological features of apoptosis by
fluorescence microscopy. Cell viability was measured by the MTT assay.
Inhibition of cPLA2 with AACOCF3 significantly improved cell viability during SI/R (60.17
± 1.77 to 80.17 ± 1.97%, p

Session XI: Cell death and cardiovascular diseases Poster XI, 5
The effect of different angiotensin converting enzyme inhibitors on subcellular toxicity
of paraquat in isolated rat liver mitochondria
Mahmoud Ghazi-Khansari, Mir Jamal Hossieni , Ali Mohammadi- bardbori
Department of Pharmacology, School of Medicine, Tehran University of Medical
Sciences, Tehran, I.R. Iran. Email: ghazikha@sina.tums.ac.ir
Widespread use of the herbicide paraquat in the recent years has necessitated further studies
on the mechanisms of its toxicity and metabolism. The defect in electron transfer chain of
mitochondria by paraquat is linked to free radical formation. In the present study we
compared the abilities of different angiotensin converting enzyme inhibitor, captopril (a thiol
ACEi), enalapril, and lisinopril (two nonthiol ACEi) on mitochondria toxicity due to paraquat.
The rat liver mitochondria were first isolated by centrifuge (at 4°C at speed of 7000g) in a
mixture of 0.25M saccharose solution and 0.05M Triss buffer. Various concentrations of
paraquat (1, 5, 10µM), enalapril (0.05, 0.5, 0.0.25µM), lisinopril (0.05, 0.01, 0.1µM) and
captopril (0.5, 0.08, 0.1µM) on the mitochondria isolated from the liver with respect to time
were investigated. Paraquat at concentration of 5µM was determined to be significantly
different compared to control (p

Session XI: Cell death and cardiovascular diseases Poster XI, 6
SMAD and AP1 are crucial for apoptosis induction in cardiomyocytes
J. Heger, D. Schneiders, HM Piper, G Euler-Taimor
Physiologisches Institut, Justus-Liebig Universität, Gießen, Germany E-mail:
jacqueline.heger@physiologie.med.uni-giessen.de
In adult cardiomyocytes both apoptosis and hypertrophy are mediated by the transcription
factor AP-1. SMAD proteins that are only activated by TGF# under apoptotic conditions are
able to interact with AP-1 and may be candidates which direct AP-1 activation towards
apoptosis. To test this hypothesis we infected isolated ventricular cardiomyocytes (CM) with
adenovirus encoding Smad4 (AdSmad4) and analyzed its impact on apoptosis induction in
comparison to TGF#.
TGF# enlarged apoptosis (11,3 ± 0,9 % vs. controls 7,3 ± 0,7 % (n=10, p

Session XI: Cell death and cardiovascular diseases Poster XI, 7
Atrial appendages transcriptional profile in atrial fibrillation patients with structural
heart diseases.
Maria S. Kharlap, Angelica V.Timofeeva, Lyudmila E. Goryunova, George L.
Khaspekov, Andrey V. Skamrov, Sergey L. Dzemeshkevich, Renat S. Akchurin, Sergey
P.Golitsyn and Robert Sh. Beabealashvilli.
Department of Clinical Electrophysiology/Laboratory of Genetic Engineering, Russian
Cardiology Research and Development Complex, 3rd Cherepkovskaya str. 15a, 121552,
Moscow, Russia. E-mail: kharlapmaria@yahoo.com
During the last few years several investigations devoted to gene expression analysis in human
atria in patients with atrial fibrillation (AF) have been performed. The subject of those studies
was atrial tissue from the patients undergoing open heart surgery with sinus rhythm and with
AF. Such kind of investigations were limited by the presence of structural changes associated
with underlying heart disease in control atrial tissue samples.
In this regard, it seemed to be reasonable to compare the atrial tissue samples from AF
patients with those from subjects without structural heart diseases. For this purpose, we
employed cDNA microarray technique. Tissue samples of right atrial appendages (RAA)
were obtained from 12 AF patients undergoing open heart surgery with valve disease,
myxoma and coronary artery disease. Control group included 11 autopsy RAA tissue samples
from people with no signs of cardiovascular diseases died during the accidents. Gene
expression was analyzed using Human cDNA Expression Arrays from Clontech (total 4704
genes). The results were verified by reverse transcription polymerase chain reaction. 15 genes
were found to be down-regulated in AF patients independently of the type of the structural
heart disease and ranged from 1.2 to 28 fold (p

Session XI: Cell death and cardiovascular diseases Poster XI, 8
Effects of selenium deficiency on the protein expression of tissues of the cardiovascular
system
A. Kyriakopoulos., A. Richter., C. Wolf., A. Plotnikov., I. Grbavac., D. Behne
Hahn-Meitner-Institut, Dept. “Molecular Trace Element Research in the Life Sciences”
Glienicker Str. 100, D-14109 Berlin, Germany.
Organs of the cardiovascular system such as Heart and aorta contain a lot of mitochondria
(powerhouse of the cell) in which the reactive oxygen species (ROS) are generated. ROS are
damaging the cell. Antioxidantive system protects the cell against the aggressive ROS and
inflammation. Some components of the antioxidative defence system are also metalloenzymes
and selenoenzymes. In order to examine the expression of the heart and aorta proteins (part of
them) in the homogenate of selenium deficient (Se-) and selenium control rats (Se+), 2 D
electrophoresis was used by means of giga gels (30x35 cm). After the electrophoresis the
protein-expression pattern of the (Se-)-gel and (Se+)-gel were compared. The evaluation of
the protein difference was implemented by means of a computer program suitable for the
analysis of protein separated by the two-dimensional electrophoresis. In this way more than
2000 proteins a gel (heart) were detected and more than 1900 protein spots were detected in
the aorta fraction. Ten significant differences were found between the gel of (Se+) and (Se-)-
heart of the rat and more than 15 significant differences between the gel of (Se+) and (Se-) of
the aorta. By means of MALDI-MS/ESI-MS some of these proteins with the different
expression level were determined until now. Of those three proteins were detected as the
alpha myosin heavy chain (alpha-MHC), myosin light chain 1 and 2 (MLC 1 and 2) and the
mitochondrial enzyme creatinin Kinase. First results suggest that selenium deficiency effects
myocardial energy metabolism and contractile proteins.
- 453 -

Session XI: Cell death and cardiovascular diseases Poster XI, 9
Transient and sustained oxidative stress cause differential JNK/SAPK and c-Jun
activation in rat cardiac myocytes
+nastasia Pechtelidou, Catherine Gaitanaki, and Isidoros Beis
Department of Animal and Human Physiology, School of Biology, Faculty of Sciences,
University of Athens, Panepistimioupoli, Athens, 157 84, Greece
email: ibeis@biol.uoa.gr
The aim of the present study was to examine the hypothesis that oxidative stress induces cell
death in H9c2 cell line (rat embryonic cardiac myocytes), and to determine whether signalling
via JNKs/SAPKs may be involved. We examined the activation of JNKs/SAPKs in response
to increasing concentrations of H2O2 (0.04-1 mM), both for the transient and for the
sustained oxidative stress. Sustained H2O2 stimulation (0.4 mM) significantly induces
maximal phosphorylation of JNKs at 2h, while transient H2O2 stimulation for 5 min induces
a maximum phosphorylation of JNKs at 15min after withdrawal. At 2 hours of sustained
stimulation peak phosphorylation of the transcription factor c-Jun is also observed, while at
the same time significant levels of nuclear activated JNKs are detected. SP600125, the JNK
inhibitor II (0.025 mM), abolishes JNK activation induced by 0.4 mM H2O2, as shown by the
absence of phosphorylated c-Jun at 2 hours of exposure. For the purpose of measuring cell
viability, we used the MTT method. Our results reveal that sustained H2O2 stimulation
significantly decreases cell viability, while transient stress does not. Moreover, from the
antioxidants tested, only catalase inhibits cell death induced by oxidative stress. Preliminary
experiments showed increased Hoechst staining after 24 hours of sustained exposure, but
normal nuclear morphology after 5min of stress and 24h withdrawal. In addition, the same
pattern of cell morphology is observed under the optical microscope. These results
demonstrate an apoptotic type of death under oxidative stress. Based on the above results, we
conclude that in H9c2 cells the transient and sustained activation of the JNKs/SAPKs
pathway may be differentially involved in cellular signalling during oxidative stress-induced
apoptosis.
(This study was funded by O.P.E.I.V.T II)
- 454 -

Session XI: Cell death and cardiovascular diseases Poster XI, 10
RoY a Novel Synthetic Peptide with Angiogenic and anti Apoptotic properties
1Annat Raiter, 1 Chana Weiss, 1 Orly Kudasi, 2Alexander Battler and 1 Britta Hardy.
1Felsenstein Medical Research Center, Tel-Aviv University School of Medicine,
2Cardiology Dept., Rabin Medical Center, Petah-Tikva, Israel.
Email: bhardy@post.tau.ac.il
Background: We have identified a novel 12 amino-acid synthetic peptide, RoY, selected
from a phage display peptide library by screening against endothelial cells under hypoxia.
RoY peptide specifically binds endothelial cells, induces angiogenesis in vitro manifested by
proliferation, migration and tube formation.
Objectives: to evaluate the therapeutic potential of the synthetic RoY peptide in restoring
perfusion in a mouse hind limb ischemic model and to elucidate the mechanism of RoY
peptide angiogenic activity under hypoxia by gene expression.
Results: Ischemia was created by ligation and excision of the femoral artery in one hind limb
of C57BL mice. Seven days after operation, perfusion in the ischemic leg was reduced to
50% in control PBS group, to 65% in RoY peptide group and to 70% in the VEGF group.
However, while RoY peptide increased perfusion to 99% at 21 days, perfusion was reduced to
80% with VEGF and remained unchanged with PBS. Gene array, confirmed by RT-PCR,
revealed up-regulation of angiogenesis growth factors Angiopoitin-2 and bFGF, Upregulation
of chemokine CCL20 and of the anti-apoptotic gene TNF-RSF10D. In contrast,
VEGF gene expression in endothelial cells incubated with RoY peptide for 3 or 24 hours was
not up-regulated. The percent of apoptosis, measured by FACS analysis, of endothelial cells
incubated under hypoxic conditions was inhibited by the addition of RoY peptide from 62%
to 33%.
Conclusion: RoY is a synthetic peptide that exhibits angiogenic and anti-apoptotic properties
under hypoxia by up regulation of growth factors, cytokines and anti apoptotic genes. RoY
peptide activity is VEGF independent thus suggests a way to bypass classical VEGF
angiogenic system. Treatment of ischemic leg with RoY peptide improved perfusion and
walking and climbing function in the mouse. This novel peptide with pro-angiogenic
properties may be used as a new therapeutic modality to hypo-vascular diseases.
- 455 -

Session XI: Cell death and cardiovascular diseases Poster XI, 11
Gene expression profiling in peripheral blood leukocytes from patients with arterial
hypertension
Angelica V.Timofeeva, Lyudmila E. Goryunova, George L. Khaspekov, Dmitrii A.
Kovalevskii, Andrey V. Skamrov, Olga S. Bulkina and Robert Sh. Beabealashvilli.
Russian Cardiology Reseach and Development Complex, 3rd Cherepkovskaya str. 15a,
121552, Moscow, Russia. E-mail: Angelica_T@cardio.ru
The pathogenesis of hypertension is a multifactorial process that involves the interaction of
genetic and environmental factors. In varying degrees, abnormalities of volume regulation,
enhanced vasoconstriction, and remodeling of the arterial wall contribute to the development
of hypertension. A wide variety of interdependent physiologic systems have been found to
influence blood pressure. Among these systems are baroreceptors, natriuretic peptides, the
renin-angiotensin-aldosterone system, the kinin-kallikrein system, the adrenergic receptor
system, and factors produced by blood vessels that cause vasodilation or contraction. As these
physiologic systems interact in complex fashion, it is difficult to establish the primary
abnormalities underlying blood pressure elevation for each studied patient. Since blood can be
considered as a connecting-link between all these systems, we hypothesized that circulating
blood cells may carry disease-specific information because of alterations in their local
environment. In this regard, we employed cDNA microarray technology to reveal differences
in gene expression in peripheral blood leukocytes from patients with arterial hypertension
(AH) comparing with normal individuals. Microarray results were confirmed by semiquantitative
RT-PCR. We have identified a gene expression pattern that accurately
distinguished AH patients from normal volunteers and was the same among AH patients
independently of the AH stage and the presence or absence of pharmacotherapy. We could
identify 13 AH-specific genes significantly activated compared with control group and 3
genes expressed at reduced level in AH. These genes are reffered to the cystein protease,
tyrosine phosphatase, thiol-disulfide oxidoreductase and chemokine receptor families,
transcription factors and heat shock proteins. Unfortunately, we cannot conclude from our
findings whether the changes in peripheral leukocytes gene expression are cause or
consequence of the pressure elevation in the AH, but we can speculate about the possible
implications of selected group of genes in vascular pathophysiology occurring in AH.
- 456 -

Notes
- 457 -

Session XII : Cell death and neurodegenerative diseases
- 458 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 1
MDMX is a Critical Regulator of Neuronal Apoptosis induced by APP Stimulat Session
Samir Benosman1, Koji Okamoto2, Christian Gaiddon3 and Jean-Philippe Loeffler4
1,3,4 INSERM U692, School of Medicine, Louis Pasteur University; 11 Humann St.,
Strasbourg, France.
2 National Cancer Center; Tokyo, Japan
Emails: 1 benosmans@neurochem.u-strasbg.fr ; 3 gaiddon@neurochem.u-strasbg.fr ;
4 loeffler@neurochem.u-strasbg.fr
MDMX is an analogue of MDM2 that has been shown to modulate p53 function in various
cell lines. In the case of neurons, data regarding the implication of MDMX in the p53
apoptotic pathway are still lacking. We investigated the role of MDMX in primary immature
and mature Cerebellar Granule Neurons undergoing different stresses leading to cell death. To
that aim, we used DNA damaging agents including Neocarzinostatin (NCS) and Cisplatin
(CIS), and neuron-specific stresses such as low K+ (LK) and APP stimulation. In All
conditions we obtained a caspase-activating apoptosis. p53 protein was stabilized in all
conditions except LK. Furthermore, apoptosis caused by DNA damage, but not by LK, was
suppressed by the dominant-negative mutant of p53, indicating that cell death caused by LK
is p53-independent. In contrast, E2F-1, another pro-apoptotic factor known to interact with
MDMX, was stabilized in response to LK and poorly in the remaining conditions.
Concomitantly, MDMX protein levels decreased in all conditions, LK included. The loss of
MDMX was not due to transcriptional repression since MDMX mRNA levels remained stable
in the former conditions. On the other hand, protease inhibitors reversed the loss of MDMX
suggesting a regulation of protein stability during neuronal insults. Furthermore, overexpression
of MDMX inhibited the transcriptional activity of both p53 and E2F-1, and
partially restored neuronal viability following apoptotic treatments. Taken together, our data
show that MDMX is an antiapoptotic factor in neurons in which MDMX degradation is
induced by multiple stress signals, allowing activation of p53 and E2F-1 during neuronal
apoptosis.
Supported by Association Francaise contre les Myopathies (AFM) and Association pour la
Recherche sur le Cancer (ARC)
- 459 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 2
Age-related decline of synaptic turnover as an early event in neurodegeneration
Carlo Bertoni-Freddari, Patrizia Fattoretti, Belinda Giorgetti, Yessica Grossi, Marta
Balietti, Tiziana Casoli, Giuseppina Di Stefano, *Gemma Perretta
Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8,
60121 Ancona, Italy Email: c.bertoni@inrca.it and *Istituto di Neurobiologia e
Medicina Molecolare-CNR, S.p Anguillarese km 1.3, 00060 Roma, Italy
The changes of synaptic ultrastructure in the frontal (FC) and temporal (TC) cortex were
investigated in adult and aged monkeys (Macaca fascicularis), by means of quantitative
morphometry, in order to assess the potential role of age-related synaptic deterioration in
neurodegeneration. The average synaptic size (S), the synaptic numeric density (Nv: number
of synapses/cubic micron of tissue), the synaptic surface density (Sv: overall area of synaptic
junctional zones/cubic micron of tissue) and the number of synapses/neuron (Syn/Neur) were
semiautomatically measured by a computer-assisted image analysis system. The results of FC
evaluation revealed no significant differences of Nv and Sv between adult and old monkeys,
while S was significantly increased in the aged animals. In TC, Sv did not show changes due
to age, while Nv was significantly decreased and S was significantly increased in aged
monkeys. The fraction (%) of perforated junctions was similar in FC and TC of the adult
animals, while it was decreased by 22.6% in TC vs. FC in the old monkeys. A percent
distribution of S showed that the fraction of enlarged synapses (>0.20 square micron) was
higher in TC than in FC regardless of the age of the animals (21.3% vs.16.9% in adult and
33.9% vs. 26.0% in aged monkeys, respectively). The comparison between data from adult
and old TC revealed that the increase was markedly higher in aged monkeys (i.e. 33.9% vs.
21.3%). In these animals, Syn/Neur was significantly decreased by 14.1% in TC, whereas it
was not significantly reduced in FC (- 4.4%). Sv, Nv, S and Syn/Neur are morphometric
parameters currently adopted to estimate the ongoing rearrangements of synaptic
ultrastructure to react to environmental stimuli. Our findings provide evidence of an agerelated
decline of synaptic plasticity in the brain of aged monkeys that is significant in TC.
According to current literature data on synaptic structural dynamics, this decay might
represent an early and subtle alteration able to trigger the development of senile plaques and
neurodegenerative events.
- 460 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 3
Early ultrastructural alterations of synaptic mitochondria in ischemic delayed neuronal
death
Carlo Bertoni-Freddari, Patrizia Fattoretti, Tiziana Casoli, Giuseppina Di Stefano,
Moreno Solazzi, *Elisa Perna, *Clara De Angelis
Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8,
60121 Ancona, Italy. Email: c.bertoni@inrca.it, *Morphometry Laboratory, Sigma-Tau,
00040 Pomezia, Italy
The effect of transient global ischemia (20 minutes) on the ultrastructural features of synaptic
mitochondria at the distal dendrites of CA1 hippocampal neurons was investigated in 3month-old
rats. Sham surgery was carried out on animals of the same age used as controls.
The number of mitochondria/cubic micron of neuropil (Nv: numeric density) was measured
by means of the disector counting method. On the same organelles sampled by the disector we
measured: the mitochondrial average size (average volume: V), the mitochondrial longer
diameter (Fmax) and the overall fraction of neuropil occupied by mitochondria (volume
density: Vv). In ischemic rats, a 10% not significant decrease of Nv was found, V increased
not significantly by 11% and Fmax increased not significantly by 5% vs. controls. No
significant difference was found between the two groups of rats as regards Vv. The percent
distribution of V showed that in ischemic animals the population of CA1 synaptic
mitochondria was composed by an increased fraction of oversized organelles while the
percent distribution of Fmax showed that this enlargement was due to an increased percent of
elongated organelles. According to current literature data, the increase in mitochondrial size
may represent a physiological compensatory response to balance the numeric loss of
organelles, however, it remains to be proven whether this compensation is of functional
significance. Although not significant, because of the high plasticity of mitochondrial
ultrastructure in the young CNS, the alterations found by us may play an early and subtle role
in triggering necrotic/apoptotic processes reported to affect selectively CA1 neurons
following ischemia. Mild metabolic impairments and the peculiar rake-like vasculature of the
hippocampal formation may be reasonably supposed to be responsible for the marked
vulnerability of these neurons that are also characterized by extended dendritic trees.
- 461 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 4
Preservation of mitochondrial volume homeostasis at the early stages of age-related
synaptic deterioration
Carlo Bertoni-Freddari, Patrizia Fattoretti, Belinda Giorgetti, Yessica Grossi, Marta
Balietti, Tiziana Casoli, Giuseppina Di Stefano, *Gemma Perretta
Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8,
60121 Ancona, Italy Email: c.bertoni@inrca.it and *Istituto di Neurobiologia e
Medicina Molecolare-CNR, S.p Anguillarese km 1.3, 00060 Roma, Italy
A morphometric study on synaptic mitochondria was performed in the frontal (FC) and
temporal (TC) cortex of adult (mean age: 10.6 years) and aged (mean age: 20.8 years)
monkeys (Macaca fascicularis). In order to identify ultrastructural alterations due to age, the
average mitochondrial size (average volume: V), the overall volume covered by mitochondria
(volume density: Vv) and the number of mitochondria per cubic micron of tissue (numeric
density: Nv) were measured. Either in FC and TC no significant age-related differences were
revealed for any of the above mentioned morphometric parameters. Namely, in FC of aged
monkeys a decrease of Nv (6%) and Vv (2%) was observed, whereas V showed an increase
by 5%. In TC of aged animals, both Nv and Vv increased by 7% and V was decreased by 2%.
While the observed changes in FC are in general agreement with data previously reported on
age-related changes in mitochondrial ultrastructure with reference to TC an opposite trend
was shown. Mitochondria are very plastic organelles capable of consistent rearrangements of
their morphology in reaction to different environmental stimuli. Accordingly, V, Nv and Vv
account for changes in single aspects of mitochondrial ultrastructure, nonetheless, when
considered together per experimental group, they might represent an index of the structural
rearrangements occurring on discrete pools of organelles (in this study, those located at the
synaptic terminals). On the basis of these assumptions, the present findings document a
preservation of the mitocondrial volume homeostasis in the brain of aged monkeys. Since our
data from a previous investigation on the same animals showed early signs of synaptic
deterioration in FC and TC during aging, this seems to be in contrast with the results obtained
in the present study. However, an age-related preservation of the mitochondrial potential for
structural dynamics may be interpreted as a reactive response to an altered synaptic function.
We suggest that mitochondria might represent sensitive targets for therapeutic interventions
aimed at counteracting early events of synaptic critical alterations able to trigger the
pathogenesis of neurodegenerative diseases.
- 462 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 5
Immunohistochemical evidence for impaired neuregulin-1 signaling in the prefrontal
cortex in schizophrenia but not in affective disorders
Iris Bertram1, Hans-Gert Bernstein1, Uwe Lendeckel2, Alicja Bukowska2, Henrik
Dobrowolny1, Gerburg Keilhoff3, Christian Mawrin4 ,Peter Falkai5 and Bernhard
Bogerts1
1 Exp. Psychiatry Lab., Psychiatry, University of Magdeburg, Magdeburg, Germany;
2 Exp. Int. Med. lab., Internal Medicine, University of Magdeburg, Magdeburg,
Germany; 3 Cell Tissue Lab., Med. Neurobiology, University of Magdeburg,
Magdeburg, Germany; 4 Exp. Neuropathology Lab., Neuropathology, University of
Magdeburg, Magdeburg, Germany; 5 Department of Psychiatry, University of
Saarland, Homburg; E-mail: Hans-Gert.Bernstein@medizin.uni-magdeburg.de
In the CNS neuregulin-1 (NRG-1) proteins function in neuronal migration, differentiation and
survival of oligodendrocytes. The NRG-1 gene codes for at least 15 different isoforms, which
may be classified on the basis of their molecular structure. At least two different haplotypes of
the NRG-1 gene may be associated with schizophrenia. An abnormal expression pattern of
NRG-1 mRNA was found in the prefrontal cortex of schizophrenic patients in comparison to
controls. We here show that the NRG-1alpha isoform is significantly reduced in white matter
of the prefrontal cortex in schizophrenia but not in affective disorders. Brains were obtained
from pathologists in accordance rules outlined by German law and the local ethics
commission. We studied brains of 22 schizophrenics, 12 patients with affective disorders (7
unipolar and 5 bipolar) and 22 matched controls. NRG-1alpha immunoreactive material was
detected with a polyclonal antiserum against the synthetic peptide from alpha-type EGF-like
domain of human neuregulin (Ab-3, Neomarkers). When stereologically analysing the
number of NRG-1alpha expressing neurons we found a statistically significant reduction of
these cells in the white, but not in the gray, cortical matter in schizophrenia but not in
depression. The demonstrated decreased number of NRG-1alpha immunoreactive neurons in
brains of schizophrenics point to an important role of this NRG-1 splice variant in
neuropsychiatric disorders. The diminished expression of NRG-1alpha in interstitial white
matter neurons strongly supports an early neurodevelopmental component to schizophrenia.
Supported by NBL-3 of the Ministry of Research of Germany, and Stanley Foundation.
- 463 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 6
Contribution of RhoGTPases to calcium dependent activation of p38 and excitotoxic cell
death
Jiong Cao, Maria M. Semenova, Anu M.J. Mäki-Hokkonen and Michael J. Courtney/
Molecular Signalling Lab, A. I. Virtanen Institute, University of Kuopio, Kuopio FIN
70211, Finland. E-mail : courtney@messi.uku.fi /
Excitotoxic neuronal death contributes to many neurological disorders. It results from
excessive stimulation of glutamate receptors and involves both calcium influx and stressactivated
protein kinases (SAPKs), though the relationship between these events is poorly
defined. RhoGTPases are major regulators of SAPK activation but their contribution to
excitotoxic cell death has not been reported. Indirect evidence implicates Rac/cdc42
RhoGTPases in neuronal death subsequent to NGF withdrawal, whereas RhoA is involved in
inhibition of neurite regeneration and release of amyloidogenic A#42 peptide. We find that
stimulation of glutamate receptors leads to activation of RhoGTPases in primary cultured
neurons cultured neurons. This activation is calcium-dependent and contributes to the rapid
glutamate-induced activation of p38 and the ensuing neuronal death that we find is dependent
on this kinase. However, RhoGTPases alone are not sufficient to induced cell death, revealing
that requirements in addition to the GTPase-mediated p38 activation exist for excitotoxic cell
death to proceed. These observations reveal RhoGTPases as novel and essential components
of the excitotoxic cell death pathway.
- 464 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 7
Protein domain interactions as a target for inhibition of excitotoxic p38 stress-activated
protein kinase activation and neuronal cell death: the PSD95–nNOS interface./
Jiong Cao, Lotta E. Parviainen, Anu M.J. Mäki-Hokkonen, Jenni I. Viholainen and
Michael J. Courtney/
Molecular Signalling Lab, A. I. Virtanen Institute, University of Kuopio, Kuopio FIN
70211, Finland. E-mail : courtney@messi.uku.fi /
The stress-activated protein kinase p38 and nitric oxide (NO) are proposed downstream
mediators of excitotoxicity, a mechanism of neuronal cell death common to both acute
conditions such as cerebral ischaemia and chronic neurodegenerative diseases. The
postsynaptic density protein PSD95 can recruit a wide range of effectors, such as the calciumdependent
neuronal NO synthase (nNOS), to the calcium-permeable channel of the NMDA
subtype of glutamate receptor. Depletion and displacement of PSD95 from NMDA receptors
reportedly inhibits excitotoxicity, but the multifunctional nature of the PSD95 interactome
makes it hard to deduce from this the precise mechanism of neuroprotection provided by
targeting PSD95. The possibility that selective uncoupling of nNOS from PSD95 might be
neuroprotective has not been previously explored. The relationship between excitotoxic
stress–generated NO and activation of p38, and the significance of the PSD95–nNOS
interaction to p38 activation also remain unclear. We find that NOS inhibitors reduce both
glutamate-induced p38 activation and the resulting neuronal death, whereas NO donor has
effects consistent with NO as an upstream regulator of p38 in glutamate-induced cell death.
Experiments using a panel of decoy constructs targeting interactions between NMDA
receptors, PSD95 and nNOS suggest that the PSD95–nNOS interaction and subsequent NO
production are critical for glutamate-induced p38 activation and the ensuing cell death, and
demonstrate that the PSD95–nNOS interface provides a genuine possibility for design of
neuroprotective drugs with increased selectivity.
- 465 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 8
Release of ß-amyloid from high-density platelets: implications for Alzheimer’s disease
pathology
Tiziana Casoli, Giuseppina Di Stefano, Belinda Giorgetti, Yessica Grossi, Marta Balietti,
Patrizia Fattoretti, Carlo Bertoni-Freddari
Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8,
60121 Ancona, Italy. Email: t.casoli@inrca.it
The main component of Alzheimer’s disease senile plaques in the brain is amyloid-beta
peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP). Platelets
contain both APP and amyloid-beta peptide and many evidence suggest that these cells may
represent a useful tool to study both amyloidogenic and non-amyloidogenic pathways of APP
processing. It has been demonstrated that platelets activated by physiological agonists such as
thrombin and collagen specifically secrete amyloid-beta peptide ending at residue 40. To
verify whether APP beta-processing could be observed also in an in vitro system of highly
concentrated platelets, we measured the Abeta released in the incubation media of 5x109
platelets/ml by enzyme-linked immunosorbent assay. The activation status of platelets was
investigated by ultrastructural analysis. We found that Abeta40 levels were significantly
higher in incubation media of 5x109/ml platelets in comparison with 108/ml platelets
(normalized values), while Abeta42 levels were not affected by cell density. The
ultrastructural analysis showed platelets at different phases of activation: some platelets were
at earlier stage, characterized by granule swelling and dilution, others had granules
concentrated in a compact mass in the cell centres within constricted rings of circumferential
microtubules (later stage). Normally concentrated cells had the characteristic morphology of
resting platelets. Our data suggest that high-density platelets undergo activation likely by
increased frequency of platelet-platelet collisions. This, in turn, determines the activation of
APP beta-processing with consequent release of Abeta40. Investigating the biochemical
pathways triggering amyloid-beta peptide secretion in platelets could provide important
informations for developing tools to modulate this phenomenon in AD brains.
- 466 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 9
c-Jun N-terminal protein kinase signalling mediates lovastatin-induced neuroblasts
apoptosis.
María I. Cerezo-Guisado1, Luis J. García-Marín2, Alberto Álvarez-Barrientos3, Eva
Pérez-Lorenzo1, Ricardo Argent1, María J. Bragado1 and María J. Lorenzo1.
1Departamento de Bioquímica y Biología Molecular y Genética, 3Departamento de
Fisiología, Facultad de Veterinaria, Universidad de Extremadura, Cáceres. 2Centro
Nacional de Investigaciones Cardiovasculares, Madrid. Spain. E-mail:
mjlorenzo@unex.es
We have previously shown that lovastatin, a competitive HMG-CoA reductase inhibitor,
induces apoptosis in spontaneously immortalized rat brain neuroblasts. Many studies have
implicated the c-Jun N-terminal protein kinase (JNK) pathway as a key regulator of apoptosis.
The aim of this study was to investigate the role for JNK in neuroblasts apoptosis induced by
lovastatin. Treatment of neuroblasts with lovastatin increased JNK activity in a concentration-
and time-dependent manner. The activation of JNK preceded the induction of apoptosis, and
JNK activity remained elevated for at least 24 hr. Lovastatin also increased c-Jun
phosphorylation and AP-1 DNA-binding activity in a concentration- and time-dependent
manner. Lovastatin effects were fully prevented by mevalonate, the final product of HMG-
CoA reductase. To examine whether the activation of JNK is involved in the process of
lovastatin-induced neuroblasts apoptosis, we utilized JNK inhibitor I, a specific inhibitor for
JNK. JNK inhibition prevented the morphological changes, the appearance of
internucleosomal DNA fragmentation, the increase in the percentage of apoptotic neuroblasts
and the amount of the activated form of caspase-3 elicited by lovastatin. In all the
experiments tested, JNK inhibitor effects were concentration-dependent. Taken together,
these data suggest that lovastatin may induce neuroblasts apoptosis by activating the JNKdependent
cell death pathway.
This work has been supported by Grants, SAF 2001-0154 (MCYT) and 2PR01B007 (JUEX).
M. I. Cerezo-Guisado is supported by a doctoral fellowship from Junta de Extremadura.
- 467 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 10
Attenuation of A#-induced apoptosis of plant extract (Shaengshik) mediated by the
inhibition of mitochondrial dysfunction and anti-oxidative effect
Chu-Yue Chen 1 , Jung-Hee Jang 1 , Mi Hyun Park 2 , Sung Joo Hwang 2 , Young-Joon Surh 1 ,
Ock Jin Park 3
1 College of Pharmacy, Seoul National University, Seoul, Korea
2 Eromlife R &D center Eromlife Cooporation, Korea
3 Department of Food and Nutrition, Hannam University, 133 Ojeong-dong Daedeok-gu,
Daejeon 306-791, Korea, E-mail: ojpark@hannam.ac.kr
Recently, considerable attention has been focused on dietary manipulation of oxidative and/or
nitrosative damage on neuronal cells. In this study, a neuroprotective effect of plant
(Shaengshik) extracts was investigated. Rat pheochromocytoma (PC12), cells treated with
A#-amyloid underwent apoptotic death as determined by positive in situ terminal endlabeling
(TUNEL staining), decreased mitochondrial transmembrane potential, and elevated
caspase-3 activity co-occurring with enhanced MDA accumulation and the reduction of GSH
levels. Shaengshik pretreatment attenuated A#-amyloid-induced apoptosis in PC12 cells
possibly by inhibiting mitochondrial dysfunction and exerting antioxidant properties.
Shaengshik pretreatment inhibited the loss of mitochondrial membrane potentials and reduced
the activation of caspase-3. The in vitro antioxidant activities of Shaengshik extracts were
verified by the DPPH method and superoxide dismutase (SOD) mimetic activity. In A#amyloid-challenged
PC 12 cells, Shaengshik prevented the production of ROS, decreased the
level of MDA, and elevated GSH. The potential of Shaengshik as one of the neuroprotective
regimens has been suggested through this study, and the combination with defined
pharmaceuticals or other dietary antioxidants may provide a better therapeutic or preventive
advantage for the management of Alzheimer diseases.
- 468 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 11
Selenium supplementation acting through the induction of specific selenoproteins
protects microglial cells from damage by hydrogen peroxide
Lisa Dalla Puppa1, Nicolai E. Savaskan2, Anja U. Bräuer3, Dietrich Behne1 and
Antonios Kyriakopoulos1.
1Hahn-Meitner-Institute, Department of Molecular Trace Element Research in the Life
Sciences, Glienicker Str. 100, 14109 Berlin, Germany (e-mail: dallapuppa@hmi.de)
2Division of Cellular Biochemistry, The Netherlands Cancer Institute (NKI),
Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
3Institute of Cell Biology and Neurobiology, Center for Anatomy Charité-University
Medical School Berlin, Phillipstr. 12, 10115 Berlin, Germany
Activation of glia has been observed in numerous central nervous system pathologies, like
brain infections, neurodegenerative diseases, inflammation, ischemia and normal aging. A
hallmark of such pathologies is the activation of microglia cells (the resident macrophage of
the brain) that produce neurotoxic factors such as cytokines, reactive oxygen species (ROS),
nitric oxide, which contribute to neuronal injury.
In the present study, we demonstrate that the trace element selenium, added as sodium
selenite, repressed the damage induced by hydrogen peroxide (H2O2) on microglial BV2
cells. Selenium is known to play a critical role in the central nervous system in addition to
acting as an essential nutrient for general body functions. Selenium is specifically
incorporated as the amino acid selenocysteine into numerous selenoproteins which are mainly
involved in antioxidant processes and redox status. It could therefore be assumed that
selenium, added as selenite, inhibits microglial activation via the expression of specific
selenoproteins. Here we report that selenium has a protective effect on cell viability of BV2
treated with H2O2. It reduces also the intracellular ROS production and inhibits the induced
apoptosis. We further applied 75Se-selenite in order to assess the expression of selenoproteins
and we used siRNA technology to identify protective selenoproteins in microglia.
This project was supported by the German Research Council DFG: Priority Program
Selenoproteins 1087.
- 469 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 12
Altered subcellular distribution of the Alzheimer’s Amyloid Precursor Protein under
stress conditions
Sara C. T. S. Domingues, Ana Gabriela Henriques, Edgar F. da Cruz e Silva and Odete
A. B. da Cruz e Silva
Center for Cell Biology, University of Aveiro, 3810-193 Aveiro, Portugal (E-mail:
odetecs@bio.ua.pt)
Altered metabolism of the Alzheimer’s Amyloid Precursor Protein (APP) appears to be a key
event in the pathogenesis of Alzheimer’s Disease and both altered phosphorylation and
oxidative stress appear to affect the production of the toxic Abeta fragment. Our results show
that altered processing of APP was observed under conditions of stress induced by sodium
azide in the presence of 2-deoxy-D-glucose (2DG). As previously reported, the production of
sAPP (the secreted fragment of APP) was inhibited. Using APP-GFP fusion proteins, we
show that 2DG causes the accumulation/delay of APP in the ER/Golgi. This effect was
augmented in the presence of sodium azide. APP subcellular distribution was also affected
and changes could also be monitored at the plasma membrane. The involvement of protein
phosphorylation in APP subcellular localization was reinforced by the effect of drugs such as
PMA (phorbol 12-myristate 13-acetate), since in the presence of 2DG and PMA APP was
completely absent from the membrane. Thus, the hypothesis that APP is processed in a
phosphorylation-dependent manner and that this may be of clinical relevance appears to hold
true even under stress conditions. Our results provide evidence for a role of protein
phosphorylation in APP trafficking under stress conditions and contribute to the
understanding of the molecular basis of Alzheimer’s Disease.
Supported by the EU VI Framework Program, FCT and CBC.
- 470 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 13
Mitochondrial metabolic competence in the early stages of neuronal cell death
Patrizia Fattoretti, Carlo Bertoni-Freddari, *Rina Recchioni, Belinda Giorgetti, Marta
Balietti, Yessica Grossi, Moreno Solazzi, Tiziana Casoli, Giuseppina Di Stefano,
*Fiorella Marcheselli
Neurobiology of Aging Laboratory and *Centre of Cytology, INRCA Research
Department, Via Birarelli 8, 60121 Ancona, Italy Email: p.fattoretti@inrca.it
A quantitative morphometric study has been carried out in human neuroblastoma SKNBE
cells to evaluate the ultrastructural features and the metabolic efficiency of mitochondria
involved in the early steps of apoptosis. In mitochondria from control and apoptotic cells
cytochrome oxidase (COX) activity was estimated by preferential cytochemistry. Number of
mitochondria (numeric density: Nv) and volume fraction occupied by mitochondria/cubic
micron of cytoplasm (volume density: Vv) and average mitochondrial volume (V) were
calculated on both COX positive and negative organelles. The ratio (R) of the cytochemical
precipitate area (CPA) to the overall area of each mitochondrion (MA) was evaluated on COX
positive organelles to estimate the inner mitochondrial membrane fraction actively involved
in cellular respiration. Following apoptotic stimulus, the whole mitochondrial population
showed a significant increase of Nv and Vv, while V was significantly decreased. In COX
positive organelles higher values of Nv were found, V appeared significantly reduced and Vv
was unchanged. R was increased at a not significant extent in apoptotic cells. COX positive
mitochondria accounted for 21% and 35% of the whole population in control and in apoptotic
cells, respectively. These findings document that in the early stages of apoptosis the increased
numeric density of mitochondria of small size provides an adequate amount of ATP for
progression of the programmed cell death process. In turn, an increased fraction of small and
more efficient mitochondria appears to represent a reactive response to the loss of
metabolically-impaired organelles. A better understanding of the mitochondrial role in
neuronal apoptosis may suggest potential interventions to prevent the extensive nerve cell
death typical of neurodegenerative diseases.
- 471 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 14
The role and regulation of BH3-only proteins in arsenite-induced apoptosis of cortical
neurons – a dominant role for Puma
Michael Fricker, Hon K. Wong, Aviva M. Tolkovsky
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge,
CB2 1QW, UK
Email: mf309@cam.ac.uk; anduswong@brain.riken.jp; amt@mole.bio.cam.ac.uk
Increasing evidence points towards apoptosis as a major mechanism of developmental and
neurodegenerative cell death in the central nervous system. Proapoptotic BH3-only Bcl-2
family members are thought to act as sentinels of the cell, with different BH3-only proteins
recruited by discrete signalling pathways. We previously demonstrated that sodium arsenite
caused apoptosis of cortical neurons and induced expression of Bim - via a JNK/cJundependent
mechanism - as well as Puma and Noxa - via a partially p53-dependent
mechanism.
Using neurons cultured from knockout (KO) mice we investigated the relative importance of
three BH3-only proteins, Bim, Noxa and Puma, in causing arsenite-induced apoptosis. Whilst
genetic ablation of Bim and Noxa provided little protection against arsenite-induced
apoptosis, deletion of Puma imparted complete resistance to arsenite treatment despite
continued upregulation of Bim and Noxa in these neurons. Puma deletion also protected
cortical neurons from numerous DNA damaging agents. As Puma has been previously shown
to be regulated at the transcriptional level by p53, we examined the effect of p53 deletion on
arsenite-induced death and Puma upregulation. Although p53 KO neurons were completely
protected against DNA damaging agents, the ability of arsenite to induce apoptosis was only
partially impaired in the absence of p53, as was its ability to cause upregulation of both Puma
mRNA and protein levels. Potential candidates for p53-independent Puma upregulation
include the p53 homologue p73. A number of p53/p73 target genes were upregulated in the
absence of p53 including CHOP/gadd153, a proapoptotic transcription factor that has also
been linked to Puma regulation. In summary, our data establish Puma as an important and
dominant inducer of apoptosis in cortical neurons, and we show that at least in the case of
arsenite, p53-independent mechanisms of Puma upregulation are active.
- 472 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 15
Involvement of cathepsin S in amyloid precursor protein-mediated neuronal cell death
Carole HONIGMANN, Corinne MBEBI, Luc DUPUIS, Jean-Philippe LOEFFLER and
Yves LARMET.
INSERM U-692, Laboratoire de Signalisations Moléculaires et Neurodégénérescence,
Université Louis Pasteur, Faculté de Médecine, Strasbourg, France
E-mails : honigmann@neurochem.u-strasbg.fr, mbebi@neurochem.u-strasbg.fr,
larmet@neurochem.u-strasbg.fr, loeffler@neurochem.u-strasbg.fr,
The Amyloid Precursor Protein (APP), the precursor of beta-amyloid, plays a central role in
Alzheimer's disease (AD) since mutations in the APP gene are associated with early onset
AD. The biological function of APP is however still not elucidated. APP is a transmembrane
proteine and there is now accumulating evidence that APP operates as a transduction unit. We
have previously reported that antibody binding to the cell surface APP (APP-Ab) causes
neuronal cell death via a GTP binding protein G0 that forms complexes with the APP
cytoplasmic domain (Mbebi et al., 2002). Anatomopathological studies revealed increased
cathepsins levels in Alzheimer brain tissues and preliminary studies from our laboratory
showed that APP-Ab are sufficient to increase cathepsins in primary culture of cortical
neurones.
In the study presented here, we delineate the relationship between the APP cytotoxic
functions and cathepsin S expression. We provide evidence that acting directly and
specifically upon neuronal APP through APP-Ab binding, stimulates the synthesis of
cathepsin S. In addition, a Jun-Kinase inhibitor could reverse cathepsin S activation. Finally,
following APP-Ab treatment, the addition of a specific inhibitor of cathepsin S reverse APP-
Ab dependent cell death. Taken together, our results show that cathepsin S is a crucial
executor of APP neurotoxicity. These findings raise the possibility that the lysosomal
pathway may contribute to neuronal cell death in AD.
Supported by Alsace Alzheimer 68.
- 473 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 16
Transduction pathway in oligodendrocyte death induced by TNF
Anna Jurewicz, Mariola Matysiak, Krzysztof Tybor and Krzysztof Selmaj
Department of Neurology and Department of Neurosurgery, Medical University of
Lodz, Lodz, Poland. E-mail: ajur@afazja.am.lodz.pl
Tumor necrosis factor (TNF) induces apoptosis-like death of oligodendrocytes (OLs), the
putative cell target in multiple sclerosis. We defined that the intracellular transduction
pathway involved in TNF-induced death of human OLs (hOLs) is dependent on c-jun NH2terminal
kinase-3 (JNK3) activation, change of mitochondrial membrane permeability and
intranuclear apoptosis inducing factor (AIF) translocation. TNF-induced death of hOLs is
non-caspase dependent, as evidenced by: lack of generation of caspase-8, -1 and -3 active
subunits; lack of cleavage of caspase-1 and -3 fluorogenic substrates; and lack of hOL death
inhibition by the general caspase inhibitor, ZVAD.FMK. JNK activation, measured by c-jun
phosphorylation and induction of the phosphorylated form of JNK, was enhanced, prolonged
and correlated with cell death in hOLs exposed to TNF. Comparative autoradiographic
analysis revealed that JNK-3, but not JNK-1 or JNK-2, is responsible for prolonged JNK
activation in TNF exposed hOLs. Expression of a dominant-negative mutant of JNK upstream
kinase, MKK4/SEK1, inhibited apoptosis induced by TNF, whereas expression of a
constitutive active mutant of MEKK1, an upstream kinase to JNK, accelerates TNF-induced
apoptosis. JNK activation occurred prior to changes of mitochondrial membrane potential in
hOLs exposed to TNF. The co-localization experiments showed that upon exposure to TNF
AIF translocated into the nucleus and electrophoresis of TNF-exposed hOLs DNA revealed
large scale DNA fragmentation characteristic of apoptosis-inducing factor (AIF)-mediated
cell death. AIF depletion by an antisense strategy prevented TNF-induced hOL death. These
results demonstrate that TNF-induced death in adult hOLs depends on prolonged JNK-3
activation, requires the mitochondrial dysfunction that occurs after JNK activation and
requires AIF translocation into nucleus. This is the first evidence that a JNK-3 isoform and
AIF are involved in oligodendrocyte death and might have significant importance in
designing new molecules to protect hOLs demise in multiple sclerosis.
- 474 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 17
GALANTAMINE PREVENTS THE NEUROTOXICITY INDUCED BY BETA-
AMYLOID PEPTIDE
Joana B Melo 1,2, Carla Sousa 1, Pedro Garção1, Paula Agostinho1,3, Catarina R
Oliveira1,3
1Center for Neurosciences and Cell Biology, 2Institute of Medical Biology and 3Institute
of Biochemistry, Faculty of Medicine, University of Coimbra, Portugal
jbmelo@cnc.cj.uc.pt
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the formation of
senile plaques in patients’ brain, composed by amyloid #-peptide (A#). Several reports
indicate that the degree of #-aggregation seems to be particularly important for the
neurotoxicity of this amyloidogenic peptide. Cholinergic abnormalities, such as severe loss of
nicotinic acetylcholine receptors (nAChRs) are also found in AD brains. Several studies on
brains displaying AD lesions have shown changes in the expression and distribution of
acetylcholinesterase (AChE). Although AChE activity is lost in specific regions of the AD
brain, an increase of AChE activity has also been found to co-localize with A# deposits.
Galantamine, a drug currently approved for the symptomatic treatment of AD, is an inhibitor
of AChE and has an additional activity as an allosteric modulator of nAChRs.
The goal of the current study, using cortical cells as a neuronal model, was to evaluate the
effect of several concentrations of galantamine in the neurotoxicity induced by the synthetic
peptide A#1-40 correlating these results with the state of aggregation of this peptide. We
observed that galantamine prevents, in a concentration dependent manner, the increase of
AChE activity and the neuronal injury induced by A#1-40. As expected, amyloid deposition,
evaluated by Congo Red dye, that positively stains #-sheet conformation and Thioflavin S
dye, is increased in the presence of A#1-40 and the treatment of cells with galantamine
decreases this deposition. However, in a cell-free system, galantamine did not revert amyloid
fibril formation. These results suggest that the neuroprotective role of galantamine could also
be correlated with an effect on the aggregation state of amyloid beta-peptide present in the
patients’ brains.
(Supported by Janssen -Cilag and GAI-Faculty Medicine of University of Coimbra)
- 475 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 18
Intensive remodelling of Purkinje cells spines after climbing fibres deafferentation does
not involve MAPKs and AKT activation
Jelena M. Mila)in1,2, Annalisa Buffo1, Daniela Carulli1, Piergiorgio Strata 1,3
1 Rita Levi Montalcini Brain Repair Center, University of Turin, Turin, Italy
2 School of Dentistry, University of Belgrade, Belgrade, Serbia and Montenegro, E-mail:
jelena_milasin@yahoo.com
3 Fondazione Santa Lucia, Rome, Italy
Subtotal lesion of the inferior olive (IO) achieved by treating experimental animals with 3
acetylpyridine (3AP) induces partial Purkinje cells (PC) deafferentation that leads to PC
hyperactivity and new spine formation. Coincidently, the olivary terminals belonging to the
few survived olivary neurons undergo an extensive collateral sprouting resulting in
reinnervation of the neighbouring denervated PCs. We obtained chemical deafferentation of
PCs in adult rats (body weight, 120-170 gm; age, 35-40 d) by a single intraperitoneal injection
of 3-AP (65 mg/kg body weight) and as early as three days after 3AP treatment, important
morphological changes could be observed on PCs. MAPK cascades and more specifically
ERK1/2 play a critical role in the signaling events underlying synaptic plasticity. For instance
LTD in the adult hippocampus and LTP in cerebellum both involve ERK activation. Since
PCs deprived of their CF afferents initiate an intensive remodelling of the spines and rapid
recall of the remaining CFs, it prompted us to see whether the observed phenomena correlated
with ERK activation. Immunohistochemistry and western blotting were done at various time
points after 3AP application (from 24h to 6 days), as the exact dynamics of CF loss is not
precisely known. As judged by western-blotting, there was no increase of activated ERK in
the cerebellum. However, immunohistochemistry revealed increased ERK phosphorylation in
the „pinceaux“ of basket cells in 3AP animals. Similarly, SAPK/JNK, p38 MAPK and Akt
activation were also studied by means of western-blotting and immunohistochemistry. In the
course of IO destruction, PCs and CFs sprouting following 3AP treatment no changes in
phosphorylation status could be seen in the different kinases subjected to analysis. Our results
suggest that activation of MAPK and Akt cascades is not essential in this model of neuronal
plasticity.
- 476 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 19
Amyloid precursor protein and Presenilin 1 interaction in human H4cells studied by
FLIM and FRET.
Mario Nizzari1, Valentina Venezia1, Paolo Bianchini2, Valentina Caorsi2, Alberto
Diaspro2, Emanuela Repetto1, Stefano Thellung1, Alessandro Corsaro1, Gennaro
Schettini1, Pia Carlo1, Tullio Florio1 and Claudio Russo3.
1Pharmacology, Dept. Oncology, Biology and Genetics, 2LAMBS-MicroscoBio, Dept
Physics, Univ Genova, Italy, 3Dept. of Health Sciences Univ Molise. e-mail:
mario.nizzari@unige.it
The pathologic hallmarks of Alzheimer’s disease (AD) are senile plaque and neurofibrillary
tangles. Senile plaque are primilary made up of deposits of amyloid-beta protein, a proteolytic
product derived from the amyloid precursor protein (APP). APP is a transmembrane protein
inserted into the endoplasmic reticulum, transported to the Golgi apparatus, to the cell surface,
and recycled by endocytosis to endosomes. Proteolytic processing, lead at the formation of
amyloid-beta protein, and a C-terminal fragments (CTFs). It’s not fully understood where
amyloid-beta is generated. However the identification of presenilins (PS), a component of
gamma secretase complex that cleave CTFs, leaving 40 or 42 amino acids amyloid-beta
peptides and 58 or 56 amino acids intracellular domains (AICD), provides a opportunity to
study APP-Presenilin interaction in specific cell compartments. In our study we used two
biophysical assays of protein proximity: fluorescence resonance energy transfer (FRET), and
fluorescence lifetime immaging microscopy (FLIM), that can provide information about
molecular interactions when two proteins are in the close proximity (of

Session XII : Cell death and neurodegenerative diseases Poster XII, 20
Study of staurosporine-induced apoptotic response in a cybrid cell line carrying the
A8344G MERRF mutation in the mitochondrial DNA.
Guillaume Rommelaere, Ludovic Mercy, Andrée Houbion, Catherine Demazy, Noelle
Ninane, José Remacle, Martine Raes, Thierry Arnould and Patricia Renard
Laboratory of Biochemistry and Cellular Biology, University of Namur (F.U.N.D.P.),
Rue de Bruxelles, 61, 5000 Namur, Belgium. Email : patsy.renard@fundp.ac.be
The Myoclonic Epilepsy with Ragged Red Fibers (MERRF) is a neurodegenerative
mitochondrial disease characterized by ataxia, myoclonic epilepsy and progressive muscular
weakness. It is caused by the single base pair substitution A8344G in the mitochondrial
tRNALys gene. This mutation causes a 70% decrease in the mitochondrial protein synthesis,
leading to impaired respiratory complexes activities and a strong decrease in ATP synthesis
by OXPHOS. In high energy consuming tissues like brain and muscles, apoptotic features
have been reported in biopsies (Mirabella et al, Brain (2000), 123, 93). Hypothesizing an
apoptotic origin of the degenerative aspect of the pathology, we are studying the apoptotic
response of cybrid cells carrying the A8344G mutation .
In a preliminary study, we have shown that a cybrid cell line derived from 143B osteosarcoma
cells and carrying the A8344G mutation (mutated cybrids) is more sensitive to staurosporineinduced
apoptosis than a parental cybrid cell line carrying the wild type mtDNA (wild type
cybrids). Indeed the staurosporine-induced DNA fragmentation and caspase-3 activity, the
main protease involved in programmed cell death, are higher in the mutated cybrids.
Using a DNA microarray dedicated to apoptosis studies, we analysed the gene expression
variation in the two cybrid cell lines in response to staurosporine treatment. Among others,
two genes, BIRC2 and BIRC3 are upregulated in the wild type cybrids in response to
staurosporine, as compared to mutated cybrids. As they code for cIAP-1 and cIAP-2, two
antiapoptotic proteins, this upregulation could explain the resistance of wild type cybrids to
staurosporine-induced apoptosis. This hypothesis is still under investigation.
This study will help to reveal the molecular bases for an enhanced apoptotic response of cells
presenting a strong mitochondrial impairment due to a defective mitochondrial tRNALys
gene.
G. Rommelaere is a fellow of the FRIA (Fonds pour la Recherche dans l’Industrie et
l’Agriculture, Belgium). T. Arnould is a Research Assistant of FNRS (Fonds National de la
Recherche Scientifique, Belgium).
- 478 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 21
ERK1/2 phosphorylation, I-NOS induction and chemokine secretion are three events
involved in the complex signaling of prion protein fragment 90-231 in microglial cells.
1Stefano Thellung, 1Alessandro Corsaro, 1Valentina Villa, 1Valentina Venezia, 1Mario
Nizzari, 1Michela Bisaglia, 2Claudio Russo, 1Gennaro Schettini, 3Antonio Aceto and
1Tullio Florio
1Pharmacology Dept. of Oncology, Biology & Genetics, University of Genova, Italy.
2Dept. Health Sciences, University of Molise, Campobasso Italy. 3Section of
Biochemistry, Dept. Biomedical Sciences, University G. D’Annunzio of Chieti, Italy.
E-mail: thellung@yahoo.com.
We show that PrP90-231, a neurotoxic prion protein fragment, activates the murine microglial
cell line N9 and describe the transduction mechanisms involved. PrP90-231 induced
phosphorylation of MAP kinase ERK1/2 and the expression of the inducible isoform of nitric
oxide synthase (I-NOS). Time course experiments, show that ERK1/2 activation is detectable
after 15 minutes of treatment, reaches a maximum at 25 minutes and is sustained up to 60
minutes of cell exposure to the peptide. Importantly, late onset and sustained ERK1/2
activation are typical events involved in cell differentiation rather than proliferation. Indeed,
we have observed by both MTT and [3H]-tymidine uptake assays, that PrP90-231 blocked N9
cell proliferation without inducing cell death. I-NOS induction was detectable after 6 hours of
cell exposure to PrP90-231 and reached its maximal value after 24 hours of treatment. We
excluded that I-NOS expression was dependent on ERK1/2 activation, since cell pretreatment
with the MEK inhibitor PD98059 did not prevent I-NOS activation by PrP90-231. Beside
nitric oxide, microglial cells produce and release several cytokines in response to
proinflammatory inputs. We observed that after 24 hours of stimulation with PrP90-231,
RANTES release is enhanced. By culturing N9 in chambers separated by a microporous
barrier we have observed that ERK1/2 activation and I-NOS expression were elicited by
PrP90-231 only if cells were directly exposed to the peptide. We conclude that these events
are not dependent on autocrine/paracrine loop triggered by the peptide and sustained by
diffusible mediators Rather, we suggest that a sustained physical interaction of PrP90-231
with the cell is required to keep microglia in an activated state.
- 479 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 22
Cas III-ia Induces Autophagy and Apoptotic Death in Glioma C6 Cells In Vitro and In
Vivo.
Cristina Trejo-Solís1, Guadalupe Palencia1, Lucrecia Márquez-Rosado2, Dolores
Jiménez-Farfán3, Aurora Sánchez1, Arturo Cruz-Salgado1, Isabel Gracia-Mora4, Lena
Ruiz-Ramírez4 and Julio Sotelo1.
1Departamento de Neuroinmunología, Instituto Nacional de Neurología y Neurocirugía
“MVS”, México, DF. 2Departamento de Biología Celular, Centro de investigación y
Estudios Avanzados del IPN, México, DF. 3 Departamento de Neuroinmunología,
Instituto de Odontología, UNAM, México, DF. 4Departamento de Química y Medicina
Nuclear, UNAM, México, DF.
In this reaserch, we investigated the effects of casiopeina III-ia (Cas III-ia)- a new copper
compound that exhibits antineoplastic activity- on glioma C6 cells under both in vitro and in
vivo conditions, in an attempt to identify potential therapeutic agents against maliganant
glioma. The exposure of C6 to Cas III-ia significantly inhibited cell proliferation, increased
reactive oxygen species (ROS) formation, and induced cell death. In cultured C6 cells, Cas
III-ia caused the accumulation of autophagic vacuoles, formation of LC3-II, and nuclear
translocation of AIF and Endonuclease G in all tested concentrations. At higher concentration
were observed, loss of mitochondrial transmembrane potential, over-expression of Beclin 1,
LAMP3, Bax and Bid, phosphorylation of JNK and ERK, and nuclear translocation of AP-1.
Administration of N-acetyl-L—cystein (an antioxidant) resulted in significative inhibition of
anti-neoplastic effect of Cas III-ia on C6 glioma cells. These results suggest that Cas III-ia
induced death cell by autophagy and caspase independient apoptosis. In addition, treatment
of glioma C6- positive rats with Cas III-ia reduced tumor volumen as well as mitotic and cell
proliferation indexes, while it increased the apoptotic index. Our findings support the use of
cas III-ia for the treatment of malignant gliomas.
This work was supported by CONACYT grant U41997-MA1.
- 480 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 23
Targeting the IGF-IR for Therapy in Metastatic Human Neuroblastoma
Cynthia M. van Golen, Ph.D. 1, Tracy S. Schwab, Ph.D. 1, Bhumsoo Kim, Ph.D.1, Mary
Soules1, Sang Su Oh1, Kevin Fung, M.D. 2, Kenneth L van Golen, Ph.D. 3 and Eva L
Feldman, M.D., Ph.D. 1.
1Neurology, University of Michigan, Ann Arbor, MI, 48109, 2Otorhinolaryngology,
University of Michigan, Ann Arbor, MI, 48109, and 3Internal Medicine, Hematology
and Oncology Division, University of Michigan, Ann Arbor, MI, 48109.
Neuroblastoma (NBL), the second most common pediatric extracranial tumor, produces
metastases in bone resulting in a survival rate of less than 7%. Therefore, understanding how
bony metastases form is critical for improving patient survival. Our laboratory has shown
that Type I insulin-like growth factor receptor (IGF-IR) expression and activation are present
in advanced stage NBL tumors and regulate NBL cell proliferation, motility, invasion, and
survival. Bone expresses large amounts of IGF ligands, and the IGF system is required for
normal bone physiology. Therefore, we addressed in the current study the role of the IGF
system in NBL metastasis to bone. Upon reaching the bone marrow through the circulation,
NBL cells must adhere to the bone marrow endothelium, extravasate into the bone
microenvironment, and destroy bone tissue to allow for tumor growth. We show high-IGF-
IR-expressing NBL cells migrate across a human bone marrow endothelial cell layer toward
bone stromal cells, which produce IGF-I, and then adhere tightly to these stromal cells.
Transendothelial migration is blocked by both a small molecule inhibitor and a monoclonal
neutralizing antibody against the IGF-IR. Intratibial injection of human NBL cells expressing
high levels of the IGF-IR, either endogenously or through transfection of an IGF-IR
expression construct, leads to tumor formation, osteolytic lesions, and additional secondary
tumors in other organs. Intraventricular injection of high-IGF-IR-expressing NBL cells also
leads to tumor formation within bone and osteolysis. Osteolytic lesions form when
osteoclasts differentiate and become activated in response to tumor cells. When high IGF-IR
expressing NBL cells are injected intratibially, increased numbers of multinucleated tartrateresistant
acid phosphatase (TRAP) positive cells are detected, suggesting an increase in
osteoclast differentiation. In vivo, micro-CT analysis of the bone demonstrates that high-IGF-
IR expressing NBL cells promote osteolysis of 81% of trabecular bone by 3 weeks and over
97% of trabecular bone by 4 weeks, with resorption pits also evident within the cortical bone.
Conditioned media from high IGF-IR-expressing NBL cells are also capable of supporting
osteoclast differentiation in vitro within 10 d. These data suggest that IGF-IR expression in
NBL cells in part determines their ability to form osteolytic metastatic tumors within bone;
therefore, targeted treatment against the receptor may prove efficacious in treating advanced
stage NBL. Targeting the IGF-IR for therapy has recently gained attention in numerous
tumors, leading to pharmaceutical production of a number of inhibitors. We are currently
testing several of these inhibitors for both in vitro and in vivo efficacy. This work was
supported by the Program for Understanding Neurological Diseases (PFUND), the Juvenile
Diabetes Research Foundation Center for the Study of Complications in Diabetes, NIH RO1
NS36778, and NIH RO1 NS38849.
- 481 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 24
Amyloid precursor protein modulates ERK1,2 signaling in glial cells.
Valentina Venezia1, Mario Nizzari1, Emanuela Repetto1, Elisabetta Violani1,
Alessandro Corsaro1, Stefano Thellung1, Valentina Villa1, Pia Carlo1, Gennaro
Schettini1, Tullio Florio1 and Claudio Russo2.
1Section of Pharmacology, Dept. Oncology, Biology and Genetics, University of Genova,
Italy. 2Dept. Health Sciences, University of Molise, Campobasso, Italy. e-mail:
venezia@unige.it
The amyloid precursor protein (APP) is a transmembrane protein with a short cytoplasmic
tail. Its location and structural features are characteristics of a receptor for extracellular
ligand. Yet, the physiological function of APP is unclear, although it is well documented that
APP’s proteolytic processing, could influence the development of Alzheimer’s disease (AD),
through the formation of membrane-bound C-terminal fragments (CTFs) and of beta-amyloid
peptides (Ab). We have recently shown that tyrosine-phosphorylated APP and CTFs may
interact with Grb2 and ShcA adaptor proteins and that this coupling occurs at a higher extent
in AD subjects only.
To study the interaction between APP or CTFs and ShcA/Grb2 and to investigate their
molecular target we have used as experimental model two different glial cell lines: H4 human
neuroglioma cells and APP/APLP null mouse embryonal fibroblast cells (MEF). Here we
show that in H4 cells APP interacts with Grb2; conversely in APP/APLP null MEF cells this
interaction is possible only after the reintroduction of human APP by transfection. We have
also shown that in MEF cells the transfection of a plasmid encoding for human APP wt
enhances the phosphorylation of ERK 1,2 as revealed by western blotting and
immunofluorescence experiments. Finally, also in H4 cells the overexpression of APP
upregulates the levels of phospho-ERK1,2.
In summary our data suggest that APP may influence phospho-ERK 1,2 signaling through its
binding with Grb2 and ShcA adaptors. The meaning of this event is not clear, but APP
interaction with these adaptors could be relevant to regulate either the physiological function
of APP or to sustain glial proliferation in Alzheimer Disease pathology.
- 482 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 25
Characterization of the pro-apoptotic intracellular mechanisms induced by a toxic
conformer of the recombinant human prion protein fragment 90-231
Valentina Villa1, Alessandro Corsaro1, Stefano Thellung1, Domenico Paludi2, Katia
Chiovitti3, Valentina Venezia1, Mario Nizzari1, Claudio Russo4, Gennaro Schettini1,
Antonio Aceto3 and Tullio Florio1
1Section of Pharmacology, Dept. Oncology Biology and Genetics, University of Genova,
Italy. 2Dept. Scienze degli Alimenti, Veterinary School, University of Teramo, Italy.
3Section of Biochemistry, Dept. Biomedical Sciences, University G. D’Annunzio of
Chieti, Italy. 4Dept. Health Sciences, University of Molise, Campobasso Italy.
Prion diseases comprise a group of fatal neurodegenerative disorders that affect both animals
and humans. The transition of the prion protein (PrP) from a mainly alpha structured isoform
(PrPC) to a prevalent beta sheet-containing protein (PrPSc) is believed to represent a major
pathogenetic mechanism in prion diseases. To investigate the linkage between PrP
neurotoxicity and its conformation, we used a recombinant prion protein fragment
corresponding to the amino acidic sequence 90-231 of human prion protein (hPrP90-231).
Using thermal denaturation, we set up an experimental model to induce the process of
conversion from PrPC to PrPSc .We report that partial thermal denaturation converts hPrP90-
231 into a beta sheet-rich isoform, displaying a temperature and time-dependent conversion
into oligomeric structures that share some physico-chemical characteristics with brain PrPSc.
SH-SY5Y cells were chosen to characterize the potential neurotoxic effect of hPrP90-231 in
its different structural conformations. We demonstrated that hPrP90-231 in beta
conformation, but not when alpha structured, powerfully affected the survival of this cells.
hPrP90-231 beta structured caused DNA fragmentation and a significant increase in caspase-3
proteolytic activity (maximal effects +170%), suggesting the occurrence of apoptotic cell
death. Finally we investigated the involvement of MAP kinases in the regulation of beta
hPrP90-231 dependent apoptosis. We observed that the p38 MAP kinase blocker SB203580
prevented the apoptotic cell death evoked by hPrP90-231 and Western blot analysis revealed
that the exposure of the cells to the peptide induced p38 phosphorylation. In conclusion, we
demonstrate that the hPrP90-231 pro-apoptotic activity is mediated by p38 and caspase-3
activation. (grants by MIUR PRIN 2004 and FIRB 2001 to TF)
- 483 -

Session XII : Cell death and neurodegenerative diseases Poster XII, 26
MEKK1 controls neurite regrowth after injury via ERK1/2 and JNK2 signaling
Vicki Waetzig and Thomas Herdegen
Institute of Pharmacology, Hospitalstrasse 4, UKSH, 24106 Kiel, Germany, E-mail:
vicki.waetzig@pharmakologie.uni-kiel.de
After injury, peripheral neuronal cells initiate complex signaling cascades to promote survival
and regeneration. We have identified the mitogen-activated protein kinase (MAPK) isoforms
which are necessary for nerve growth factor (NGF)-induced neurite regrowth after injury of
differentiated PC12 cells. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the
otherwise apoptotic c-Jun N-terminal kinase 2 (JNK2) are crucial for neurite regrowth, while
p38 plays no role in this context. Surprisingly, the MEK1 inhibitors PD98059 and U0126
blocked ERK1/2 and JNK phosphorylation, indicating a novel form of balancing MAPK
cascade crosstalk. We identified the upstream kinase MEKK1 as an activator of both the
ERK1/2 and JNK2 pathways, whereby the ERK1/2 kinase MEK1 and the JNK kinase MKK7
bound to MEKK1 in a competing fashion. The use of siRNAs against ERK1/2 excluded a
direct interaction between ERK1/2 and JNKs. In JNK knockout mice, we have confirmed the
importance of JNKs for facial nerve regeneration.
- 484 -

Notes
- 485 -

Notes
Notes
- 486 -

- 487 -

Session XIII : Cell signaling pathways leading to regulated
chromatin modifications
- 488 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 1
ICBP90, a methyl binding protein, as potential gene suppressor for p73 expression in
Jurkat cells
Abdul-Qader Abbady, Michaël Jeanblanc, Marie Schöller-Guinard, Jean-Paul Klein,
Ermanno Candolfi and Marc Mousli.
Institut National de la Santé et de la Recherche Médicale, UMR-S 392/E.A. 3950, Faculté
de Médecine, 3 rue Koeberlé, 67000 Strasbourg, France. E-mail:
marc.mousli@medecine.u-strasbg.fr
T cell activation involves induction of genes mediating antigen-induced cell death (AICD)
through T cell receptor activation. AICD controls the expansion of antigen-activated T cells
after an immune response and deletes self-reactive T cells by negative selection. This occurs
from late G1 cell cycle checkpoint that is dependent on E2F-1 and p73. Methylation at CpG
dinucleotides, the most abundant epigenetic modification in vertebrate genomes, plays an
essential part in the control of gene expression. Inverted-CCAAT-box-binding protein of 90
kDa (ICBP90) has been recently identified as a novel methyl-CpG-binding protein. Since
methylation of p73 gene plays an important role in its transcriptional activity, we
hypothesized that ICBP90 might be involved in the regulation of p73 gene expression. In the
present study, we analyzed the expression of ICBP90 in Jurkat and CCRF-CEM lymphocytes
and in THP-1 monocytes as control after triggering by phytohemagglutinin (PHA). We found
that activation of Jurkat and CCRF-CEM cells with PHA decreased the number of living cells
and increased the number of apoptotic cells in a significant manner while these remained
unchanged in THP-1 cells. We also observed a significant decrease in ICBP90 expression
when compared to unstimulated lymphocytes and THP-1 control cells. Moreover, the
promoter activity of ICBP90 gene, measured using the luciferase reporter gene, was also
significantly inhibited after triggering Jurkat cells by PHA whereas no change was observed
in THP-1 cells. Finally, ICBP90 down expression was accompanied with an increase of p73
expression in activated Jurkat cells whereas p73 was almost undetectable in THP-1 cells. In
conclusion, our results suggest that down regulation of ICBP90 as a methyl binding protein,
may play a key role in p73 gene activation in AICD and T cell clonal expansion.
- 489 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 2
Interdependency or Exclusion? Investigation of the phosphorylation state of histone H3
at serine 10 and threonine 11 during mitosis
Anne Conradi and Karl Heinz Scheidtmann
Institute of Genetics, University of Bonn, Roemerstr. 164, D-53117 Bonn, Germany
E-mail: kh.scheidtmann@uni-bonn.de
Histones are subject to numerous post-translational modifications. During mitosis histone H3
becomes heavily phosphorylated at serine 10 by Aurora-B kinase, as well as at threonine 11
probably by Dlk/ZIP kinase. Phosphorylation at serine 10 occurs along the entire
chormosomal arms, but not at centromeres. It coincides with chromatin condensation and may
be involved in initiation of this process. In contrast, phosphorylation at threonine 11 appears
to be centromere-specific and may be involved in kinetochore assembly and/or function.
These phosphorylation events seem to occur in an exclusive manner which is also supported
by in vitro phosphorylation data. We investigated a possible interrelationship between
Aurora-B and Dlk/ZIP kinase. In vitro kinase reactions with purified Aurora-B and Dlk/ZIP
kinase revealed no crossphosphorylation between the two kinases. In vivo Aurora-B-specific
kinase inhibitors revealed loss of H3 phosphorylation at serine 10 but not at threonine 11. Our
data suggest that both phosphorylations occur independently.
Acknowledgements:
This work was supported by Deutsche Forschungsgemeinschaft grant Sche 246/16-1.
- 490 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 3
Pharmacological restoration of ROS impaired steroid function: the role of HDAC-2
Paul A. Kirkham, Koremu Meja, Gillian Spooner and John A. Marwick.
Novartis Institutes for Biomedical Research, Horsham, West Sussex, RH12 5AB, UK.
Email: paul.kirkham@novartis.com
HDAC-2 activity plays a major role in regulating steroid function and inflammation. COPD
patients, severe asthmatics and mild asthmatics who smoke are all insensitive to steroid
treatment, as a result of exposure to oxidative stress. Moreover, oxidative stress has a direct
inhibitory effect on HDAC-2 activity through post-translational modification. We
hypothesised that upregulating HDAC activity after exposure to oxidative stress should
restore steroid function. Oxidative stress reduced HDAC-2 but not HDAC-1 activity. Both
theophylline and curcumin specifically restored HDAC-2 activity in pre-ROS stressed cells
only, in both a time and dose dependant manner. Similar effects on restoring HDAC activity
after ROS stress were observed using either JNK or Akt inhibitors, but not p38, cAMP or
calcium modulators. ROS induced reduction of HDAC-2 activity correlated with a decrease
in steroid responsiveness. Co-incubation or theophylline or curcumin with budesonide (1nM)
before stimulation with LPS (10ng/ml) for 16 hours restored steroid responsiveness in ROS
treated cells as assessed by TNFalpha release. Interestingly, neither theophyline or curcumin
alone (upto 10uM) were anti-inflammatory in LPS stimulated pre-ROS stressed U397 cells.
Moreover, theophylline (1µM) also restored the ability of dexamethasone to reduce
inflammatory gene associated histone acetylation in pre-ROS stressed U937 cells as assessed
by chromatin immunoprecipitation (ChIP) assay. Serine phosphorylation on HDAC-2 is
reduced by oxidative stress. However, treatment with curcumin/theophyline (1µM) post ROS
stress increases HDAC-2 serine phosphorylation. In conclusion, our data suggests that
restoring/upregulating HDAC activity and thereby steroid function after prior reduction by
oxidative stress can be achieved through pharmacological intervention. The mechanism of
action for theophyline and curcumin in this process is unclear at present. However, inhibition
of JNK or Akt signaling pathways may play a role. It is therefore likely that both theophyline
and curcumin act on signalling pathways that regulate HDAC protein post-translational status
and hence activity.
- 491 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 4
Two states of the nuclear matrix-bound p53 in HEK293 cells
Maria A. Lapshina, Igor I. Parkhomenko and Alexei A. Terentiev.
Molecular Biology Laboratory, Department of Kinetics of Chemical and Biological
Processes, Institute of Problems of Chemical Physics RAS, Chernogolovka, Moscow
Region, 142432, Russia. E-mail: lapshina@iap.ac.ru
The tumor suppressor protein p53 is a transcription factor playing important roles in
regulation of the cell cycle progression and the cell death in response to a vide variety of
stressors, such as DNA damaging factors, oncogene activation, oxidation, etc. Like many
other transcription factors, p53 protein is found to associate with the nuclear matrix (NM), the
filamentous protein network of the cell nucleus that contributes to the higher order chromatin
structures and to most of nuclear processes including transcription, replication, DNA repair,
RNA processing and molecular transport. We studied the intranuclear distribution of the p53
protein in HEK293 cells with use of various techniques of NM preparation. P53 is found to be
associated with NM independently on the methods used, but its appearance in non-matrix
fractions depends strongly on the order of the extraction steps: approximately 40% of nuclear
p53 is observed in high salt fraction before DNase I digestion, and almost no p53 is extracted
in high salt buffer after nuclease treatment. The matrix-bound p53 is found to be modified,
most probably ubiquitinated, as it is observed as higher molecular weight bands. Further
extraction of matrix-bound proteins in alkaline buffers causes partial release of p53 in a
soluble fraction, and the higher molecular weight forms of p53 are extracted completely under
alkaline conditions. In acidic buffers, p53 protein is also partially extracted from the matrix,
but the higher molecular weight forms of p53 retain their association with NM. To study if
these two forms of matrix-bound p53 could be functionally different, we activated p53 in
MCF-7 cells with actinomycin D and carried out the same extractions of NM proteins. The
ActD-activated p53 is found to represent only one of two forms observed in HEK293 cells: it
is completely extracted from NM under alkaline conditions and resistant to acidic extractions.
Thus, p53 protein is found in the nuclear matrix in different states (forms). These forms of
matrix-bound p53 are differentially sensitive to alkaline and acidic extractions and might
differ in either protein-protein interactions (with separate NM proteins) or charges (e.g. as the
consequence of differential phosphorylation and/or ubiquitination).
- 492 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 5
MARK/Par-1 kinases, EMK and C-TAK1 regulate localization and activity of class IIa
HDACs through hierarchical phosphorylation
Maud Martin, Nathalie Mari, Julia Von Blume, Didier Vertommen, Emily Lecomte,
Sabine Ruidant, Marie-France Heinen, Malte Bachmann, Jean-Claude Twizere, Chris
Huang, Mark H. Rider, Helen Piwnica-Worms, Thomas Seufferlein, Richard Kettmann
and Franck Dequiedt
Cellular and Molecular Biology Unit, Faculty of Agronomy, B-5030, Gembloux,
Belgium. E-mail : dequiedt.f@fsagx.ac.be
Class IIa histone deacetylases (HDACs) are found both in the cytoplasm and in the nucleus
where they repress genes involved in several developmental programs. In response to specific
signals, the repressive activity of class IIa HDACs is neutralized through their
phosphorylation on multiple N-terminal serine residues and 14-3-3 mediated nuclear
exclusion. Here, we demonstrate that class IIa HDACs are subjected to signal-independent
nuclear export that relies on their constitutive phosphorylation. We identify EMK and C-
TAK1, two members of the MARK/Par-1 family, as regulator of this process. We further
show that EMK and C-TAK1 phophorylate class IIa HDACs on one of their multiple 14-3-3
binding sites and alter their subcellular localization and repressive function. Using HDAC7 as
a paradigm, we extended these findings by demonstrating that signal-independent
phosphorylation of class IIa HDACS conforms to a hierarchical pattern in which
phosphorylation of the MARK/Par-1 site is a prerequisite for the phosphorylation of the other
14-3-3 sites. We propose that this multisite hierarchical phosphorylation by a variety of
kinases allows for sophisticated regulation of class IIa HDACs function.
- 493 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 6
Combinatorial histone deacetylase inhibitors effect to the differentiation of human
promyelocytic leukemia HL-60 cell
Rasa Merzvinskyte1, Grazina Treigyte1, Jurate Savickiene1, Karl-Eric Magnusson2
and Ruta Navakauskiene1
1Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius,
Lithuania. E-mail: rasa_merzvinskyte@yahoo.com
grazina.treigyte@bchi.lt savickiene@bchi.lt ruta.navakauskiene@bchi.lt
2Division of Medical Microbiology, Department of Molecular and Clinical Medicine,
Linköping University, SE-581 85 Linköping, Sweden. E-mail: karma@imk.liu.se
Recently, a novel strategy for the treatment of leukemias through the modulation of chromatin
structure has been applied. In this study, we conducted an analysis of anti-leukemic efects of
the histone deacetylase (HDAC) inhibitors, phenyl butyrate (PB) and vitamin B3, and in
combination with the differentiation agent, retinoic acid (RA), on the promyelocytic leukemia
cell line HL-60. We found that the HDACI combinations exert different effects on cell cycle
arrest, differentiation and apoptosis. We also assessed the expression of the early granulocytic
differentiation marker CD11b on the cells treated with different combination of HDACI and
RA. The most promising treatment for differentiation therapy was defined using a 6-h
pretreatment with phenyl butyrate and vitamin B3 before the combined exposition to RA with
vitamin B3. This significantly accelerated and increased cell differentiation (up to 95%)
during the 48-h treatment. The examined HDAC inhibitors, in combination with the
differentiation agent caused rapid histone H3 and H4 modifications in HL-60 cells. The
increased level of histone H4 acetylation was associated with the initiation and maturation
stages of HL-60 cell differentiation. Our results suggest that the chromatin remodeling using
HDAC inhibitors provides a rationale for the treatment of acute promyelocytic leukemia.
- 494 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 7
Histone acetylation-targeted differential responses of leukemia cells HL-60 and NB4 to
histone deacetylase inhibitor FK228
Jurate Savickiene1, Grazina Treigyte1, Ruta Navakauskiene1 and Karl-Eric
Magnusson2
1Department of Developmental Biology, Institute of Biochemistry, LT-2600 Vilnius,
Lithuania. E-mail: grazina.treigyte@bchi.lt savickiene@bchi.lt
ruta.navakauskiene@bchi.lt
2Division of Medical Microbiology, Department of Molecular and Clinical Medicine,
Linköping University, SE-58185 Linköping, Sweden. E-mail: karma@imk.liu.se
The histone deacetylase inhibitor (HDACI), depsipeptide (FK228), was shown previously as a
chemopreventive agent for the treatment of adult T-cell lymphomas. In this report, we
investigated the in vitro antileukemic activity of FK228 alone, and in combination with alltrans
retinoic acid (RA), on the human leukemia cell lines, NB4 and HL-60. The results show
that FK228 induced a dose-dependent (0.2-1 ng/ml) cell growth arrest and death by apoptosis
in NB4 and HL-60. The combined treatment with RA accelerated and enhanced granulocytic
differentiation in both cell lines distinctly; the 6 h - pretreatment with HDACI had an additive
differentiating effect in HL-60 cells only. These effects were accompanied by a time- and
dose-dependent histone H4 hyper-acetylation and histone H3 dephosphorylation, occuring
after 2-8 h exposure to HDACI. FK228 up-regulated NF-!B binding to the FasL promoter,
and in combination with RA it altered the DNA binding of NF-!B linking the findings to cell
death and differentiation. Pifithrin-" (PFT), an inhibitor of p53 transcriptional activity,
protected from apoptosis only in NB4 cells with functional p53, i.e. when used 4 h before
treatment with FK228 only or together with HDACI. In NB4 cells, PFT inhibited p53 binding
to the p21 (Waf1/Cip1) promoter and induced DNA binding of NF-!B leading to enhanced
cell survival. Thus, FK228 may be a promising antileukemic agent in combination with RA,
since it exerts antiproliferative, differentiating and apoptotic effects in leukemia cells without
and with functional p53, acting via histone modifications and selective involvement of
transcription factors, like NF-!B and p53.
- 495 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 8
A role for Class I histone deacetylases in proliferation of tumor cells
Silvia Senese 1, Katrin Zaragoza-Dorr 1, Simone Minardi 2, Loris Bernard 2, Giulio F.
Draetta 3, Myriam Alcalay 2, Christian Seiser 4 and Susanna Chiocca 1*
1European Institute of Oncology, Department of Experimental Oncology, 20141 Milan,
Italy,
2 IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy, 3 Cancer Research, Merck
Research Laboratories, Basic Research, 33 Avenue Louis Pasteur, Boston, MA 02115,
USA, 4 Department of Medical Biochemistry, Division of Molecular Biology,Medical
University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria
* Corresponding author. Mailing address: European Institute of Oncology, Department
of Experimental Oncology, Via Ripamonti, 435, 20141 Milan, Italy.
Histone deacetylases (HDACs) inhibitors are currently tested in clinical trials as anti-cancer
drugs. Previous studies point towards HDAC1 as one of the possible targets for these tumor
drugs. Therefore, the role of individual Class I HDACs in the regulation of cancer cell
proliferation was investigated using RNAi-mediated protein knockdown. We show here that
ablating HDAC1 and HDAC3 protein expression results in the inhibition of U2OS cell
proliferation and an increase in the percentage of apoptotic cells. On the contrary HDAC2
knockdown shows no effect, unless we concurrently knockdown both HDAC1 and HDAC2.
Moreover, RNAi against HDAC1 alone or in combination with HDAC2 increases the
expression of p21 protein, a cyclin-dependent kinase inhibitor. We also observe that only in
the absence of both HDAC1 and HDAC2 histones H3 and H4 are hyperacetylated. In
addition, HDAC1 knockdown abolishes the ability of cells to reach M phase. Our results
demonstrate that HDAC1 and HDAC3 play a major role in proliferation and survival of tumor
cells and that these HDACs might impair cell cycle progression not only by affecting the
transcription of specific target genes (p21) but also through effects on other important
biological processes.
- 496 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 9
Modulation of epigenetic processes in HRAS-transformed cells
Christine Sers1, Karen Weißhaupt1, Thomas Mikeska2, Diana Jammas2, Xiaohua
Chen1, Ralf-Jürgen Kuban3, Ute Ungethüm3, Ulf Krapfenbauer1, Hans-Peter Herzel4,
Reinhold Schäfer1,3, Jörn Walter2, Per Lund1.
1Laboratory of Molecular Tumor Pathology, Institute of Pathology, Charité, Berlin,
Schumannstr. 20/21, D-10117 Berlin, Germany; 2Department of Natural Sciences –
Technical Faculty III FR 8.3, Biological Sciences, Institute of Genetics/Epigenetics,
University of Saarland, Saarbrücken, Germany; 3Laboratory of Functional Genome
Research, Charité, Berlin, Schumannstr. 20/21, D-10117 Berlin, Germany; 4Institute for
Theoretical Biology, Humboldt University Berlin, Invalidenstrasse 43, D-10115 Berlin,
Germany;
Silencing of tumour suppressor genes is often caused by promoter hypermethylation, but little
is known about the impact of oncogenic signalling pathways on methylation. The aim of this
study was to investigate the role of signalling pathways downstream of RAS involved in
methylation-dependent down-regulation of genes. Immortalised 208F rat fibroblasts and
HRAS(V12)-transformed rat fibroblasts (FE-8) were treated with the de-methylating agent 5aza-deoxycytide
(5-aza-CdR) and with the HDAC-inhibitor Trichostatin A (TSA). The effect
of 5-aza-CdR on the expression of genes down-regulated in the HRAS-transformed cells was
investigated by conventional northern blot analysis (74 genes) and by microarray
hybridisation (7186 sequences). Fifty-three genes analysed by Northern blot analysis and 10
genes analysed by microarray hybridsation were re-expressed in FE-8 cells after treatment
with 5-aza-CdR. Among these genes, Clusterin, Mama, MMP2, TIMP2 and
Thrombospondin-1 were also re-expressed after inhibition of the MEK/ERK pathway.
Hypermethylation of putative regulatory elements in two independent HRAS-transforemd cell
lines and in cells harbouring an inducible HRAS, was detected within a CpG-island 14.5 kb
upstream of clusterin, within the clusterin promoter and within a CpG-island of the Mmp2promoter
by bisulphite sequencing. No significant hypermethylation was detected within the
promoter regions of Timp2 and syndecan 4. Our study shows that RAS oncogene-dependent
suppression and methylation-dependent silencing of genes are connected and impinge in part
on the same target genes.
- 497 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 10
Signaling pathways converting extra-cellular cues into chromatin modifications at
muscle specific loci during myogenesis
Cristiano Simone1,2 , Sonia V. Forcales3,
Lucia Latella3, Pier Lorenzo Puri3
1 Sbarro Institute for Cancer Research and Molecular Medicine, Department of
Biology, College of Science and Technology Temple University, Philadelphia PA 19122,
USA. 2 Division of Medical Genetics, Department of Biomedicine in Childhood,
University of Bari, Bari 70124, Italy. 3 DTI at Fondazione A. Cisalpino, ICBTE, Rome
00128, Italy; and The Burnham Institute, La Jolla, San Diego, CA 92093, USA
Myogenic differentiation is a stepwise process influenced by external cues, which are
converted by cytoplasmic cascades into specific chromatin modifications, to coordinate the
transcription of muscle specific genes. For instance, two distinct cytoplasmic cascades, the
p38 and IGF1-mediated PI3K/Akt pathways, transmit to the nucleus pro-differentiation cues
to activate the myogenic program.
We have investigated the individual contribution of these pathways in the regulation of the
assembly of the chromatin-modifying enzymes on muscle-specific promoters. Sequential
recruitment of muscle regulating factors (MRFs), histone acetyltransferases (HATs) and
remodeling complexes (SWI/SNFs) on muscle enhancers/promoters was differently affected
by specific inhibition of PI3K (by LY294002) and p38 (by SB203580), resulting in distinct
patterns of chromatin modifications. While pharmacological blockade of PI3K inhibited
HATs recruitment, thereby preventing acetylation of both histones and MyoD and reducing
the levels of chromatin-bound MyoD, SB203580 (SB) selectively affected SWI/SNF
chromatin binding and remodeling. In both cases, transcription of muscle-specific genes was
inhibited.
This evidence establishes a functional interdependence between IGF-1/PI3K/Akt and p38
pathways in coordinating the assembly of the myogenic transcriptosome, and suggests that
hyperacetylation of muscle enhancers/promoters in myoblasts exposed to IGF-1 precedes
chromatin remodeling, which is induced by p38. The dissection of the events leading to
muscle-specific transcription by pharmacological targeting of individual signaling cascades
could reveal the rationale for manipulating skeletal myogenesis in vivo.
- 498 -

Session XIII : Cell signaling pathways leading to regulated chromatin modifications
Poster XIII, 11
The role of p300/CBP complex in the differential regulation of apoptosis induced by
diverse types of cytotoxic stress
G. Xenaki1, M. Krstic-Demonacos2, C. Demonacos1
University of Manchester 1. School of Pharmacy, Coupland 3 building, and
2. Faculty of Life Sciences, Michael Smith building, Oxford Road, Manchester, M13
9PL
The underlying mechanisms triggering apoptosis have been unravelled in recent years and it
is accepted that diverse apoptotic stimuli (anti-cancer drugs, $- or UV-irradiation, cytokines,
hypoxia, deprivation of survival factors) converge on a common cascade of events leading to
apoptosis. The Bcl-2 family members play a central role in these apoptotic events. Protein
complexes that include transcription factors and coactivators (p300, P/CAF HATs) or
repressors (HDACs) frequently govern the expression of this specific set of genes in order to
delicately balance the protein levels of anti- and pro-apoptotic members of this family and
eventually determine the cell fate. The main focus of our work is to dissect the regulatory
pathways determining the cellular fate under diverse stress conditions. We have observed that
differential composition of the transcription co-activator complexes under diverse stress
conditions fine tune the protein levels of pro- and anti apoptotic members of the Bcl-2 family
determining whether the cell will survive or die through apoptosis
. In particular, we have studied the expression of Bid, a pro-apoptotic member of the Bcl-2
family, under conditions known to activate the p53 and/or Hif-1" transcription factors
(binding sites for both transcription factors have been identified in the Bid promoter region).
We have identified this way the cellular components regulating the mRNA synthesis of Bid
under diverse stress conditions. In this context our studies have provided evidence that two
components of the p300/CBP complex namely Strap and P/CAF directly or indirectly mediate
critical signalling events that regulate both p53 and Hif-1" transcriptional activities under
hypoxia.
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Notes
- 500 -

Notes
Notes
- 501 -

- 502 -

Session XIV : Transcriptional and translational control
- 503 -

Session XIV : Transcriptional and translational control Poster XIV, 1
MAPKs signaling cascades-mediated transcriptional regulation in H9c2 cardiac cells
exposed to oxidative stress
Ioanna-Katerina Aggeli, Catherine Gaitanaki and Isidoros Beis
Department of Animal and Human Physiology, School of Biology, Faculty of Sciences,
University of Athens, Panepistimioupolis, Athens 157 84, GREECE, E-mail :
iageli@hotmail.com
One of the most important insults that cardiomyocytes experience in vivo is an increase in the
levels of reactive oxygen species (ROS) i.e. during ischemia, reperfusion, as well as, in the
failing myocardium. MAPKs have been found to play a pivotal role in transducing the
oxidative stress-signal in cardiomyocytes. Accordingly, we found that all three subfamilies
(ERKs, JNKs and p38) were phosphorylated thus activated maximally at 5-15 min of H2O2
treatment (200µ)), declining thereafter (immunoblotting). In an effort to define possible
substrates of the MAPKs activated, the phosphorylation of ATF2 as well as c-Jun was studied
(immunoblotting). Both transcription factors were equally induced after 30min and 1h,
respectively, a result which indicates their involvement in the observed response. In order to
decipher the signaling mechanism triggered under these conditions a number of
pharmacological inhibitors of kinase pathways was used. What is more, since ATF2 and c-Jun
are known AP1 components, the transcriptional regulation of possible target genes with a
CRE/AP-1 element in their promoter region was examined (RT-PCR). Further studies are
however required so as to establish the physiological role of these cascades’ components
promoting either cell survival or apoptosis.
This work was funded by PYTHAGORAS 1 grant (70/3/7399).
- 504 -

Session XIV : Transcriptional and translational control Poster XIV, 2
Bile acids modulate p53 transcriptional targeting in primary rat hepatocytes
Joana D. Amaral, Rui E. Castro, Susana Solá, Cecília M.P. Rodrigues
Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, 1600-083
Lisbon, Portugal. E-mail: jamaral@ff.ul.pt
Ursodeoxycholic acid (UDCA) is a potent inhibitor of classical mitochondrial pathways of
apoptosis. Further, UDCA prevents transforming growth factor beta1 (TGF-beta1)-induced
hepatocyte apoptosis by modulating the E2F-1/p53/Bax pathway. However, the
mechanism(s) by which UDCA regulates gene transcription remains to be clarified. The aim
of this study was to evaluate the modulation of a specific pro-apoptotic transcriptional target
of p53 by several bile acids. Primary rat hepatocytes were cotransfected with plasmid DNA
encoding wild-type or mutant p53 and a reporter gene construct that utilized the bax gene
promoter to drive transcription of chloramphenicol acetyltransferase (CAT). A luciferase
reporter construct was used to assess transfection efficiencies. Hepatocytes were transfected
and simultaneously treated with either vehicle or 100 µM of several bile acids. Twelve hours
later, 1 nM TGF-beta1 was included in the cultures. After an additional 36 h, hepatocytes
were harvested for CAT ELISA and luciferase assays. Total protein extracts were also
prepared and evaluated by immunoblotting. Cultures were scored for apoptotic cells by
Hoechst staining. Electrophoretic mobility shift assays (EMSA) were performed in nuclear
extracts from COS-7 cells, producing high levels of endogenous p53. Our results confirmed
that UDCA abrogated TGF-beta1- and p53-induced hepatocyte apoptosis. DNA-binding
activity of p53 was not altered by the bile acid. Nevertheless, in functional studies,
hepatocytes transfected with wild-type p53 and treated with UDCA showed a marked
decrease in bax transcriptional activation. When the mutant form of p53 was used, UDCA no
longer exerted its protective effect. Similar results were obtained after incubation of cells
with both taurine- and glycine-conjugated analogues of UDCA. However, when cells were
treated with lithocholic, deoxycholic, or chenodeoxycholic acids, the pro-apoptotic gene was
further activated at the transcription level, revealing that bile acids are able to differentially
modulate transcription of p53-driven bax. Taken together, these data suggest that proapoptotic
p53 is a specific molecular target of UDCA, further clarifying the role of bile acids
at modulating apoptosis. (Supported by POCTI/SAU-FCF/62479/04 from FCT, Portugal).
- 505 -

Session XIV : Transcriptional and translational control Poster XIV, 3
Liver steatosis alters the expression of nuclear receptors and genes involved in bile acid
metabolism
Patricia Aspichueta, Oscar Briz, Lourdes Palacios, Ainara Cano, Xabier Buqué, Jose J.
Marín, Begoña Ochoa
Department of Physiology, University of the Basque Country, Faculty of Medicine and
Dentistry, Leioa, Spain. Email: patricia.aspichueta@ehu.es
Accumulation of fat within hepatocytes is a simple steatosis and a sign of lipotoxicity that is
frequently associated with changes in bile acid metabolism and cholestasis. The aim of this
work was to analyze whether transport proteins and cytochromes involved in bile acid
synthesis and secretion as well as some regulatory nuclear receptors are altered in obesityrelated
steatosis at the mRNA expression level. For this purpose, we i) isolated hepatocytes
from obese Zucker rats, an established model for non-alcoholic fatty liver disease, of 6, 9 and
12 weeks of age -hence, in three stages of steatosis- and from their lean littermates, ii)
extracted the RNA from hepatocytes, and iii) determined specific transcript levels by realtime
PCR. Our results showed that the mRNA levels of Cyp7a1 were 60% and 40% decreased
in the 6- and 12-week old steatotic animal while those of Cyp27a1 were similar to lean rat
hepatocytes. There was a dramatic decrease (of ,98%) in ABCG8 mRNA levels in the three
steatotic groups, accompanied by an important, though less marked decrease in ABCG5
expression. No changes were found in the mRNA levels of the transporters Bsep, mdr2, mrp2
and Ntcp. Discordant changes were observed between several major nuclear receptors and
their regulated target genes. Particularly, the transcription factors studied involved in Cyp7a1
expression regulation do not seem to be responsible for the changes observed in this study.
The mRNA levels of farnesoid X receptor (FXR) were modified in none of the obesity-related
steatotic groups, of the small heterodimer partner (SHP) were doubled only in 9 week-old
obese rat hepatocytes and of the alpha-fetoprotein transcription factor (FTF) were increased in
the three stages of liver steatosis. In conclusion, our results suggest that repression of Cyp7a1
in obesity-related steatosis may be linked to down-regulation of ABCG5, ABCG8 and LXR,
rather than to the mediation of mechanisms controlled by the FXR/SHP/FTF cascade.
Supported by Grant G03/015 from the Ministerio de Sanidad y Consumo
- 506 -

Session XIV : Transcriptional and translational control Poster XIV, 4
Proteomic analysis for Helicobacter pylori - infected human gastric mucosa: oxidative
stress - related proteome changes
Hye Yeon BAEK, Joo Weon LIM, and Hyeyoung KIM*
Department of Pharmacology and Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea, E-mail: ehrehrwh@hanmail.net
Helicobacter pylori (H. pylori) infection leads to gastroduodenal inflammation, peptic
ulceration and gastric carcinoma. Proteomic analysis for human gastric mucosa of the patients
with erosive gastritis or peptic ulcer, which were either infected or non-infected with H.
pylori, was used to determine the differentially expressed proteins by H. pylori in human
gastric mucosa to investigate the pathogenic mechanism of H. pylori-induced gastric diseases.
Mass spectrometry analysis (MALDI-TOF) of tryptic fragment and data search allowed
identification of increased four proteins (78kD glucose-regulated protein precursor,
endoplasmin precursor, aldehyde dehydrogenase 2, L-lactate dehydrogenase B chain) and
decreased four proteins (intracellular chloride channel protein 1, glutathione-s-transferase,
heat shock protein 60, cytokeratin 8) caused by H. pylori infection in gastric mucosa. These
proteins are related to cell proliferaton, carcinogenesis, cytoskeletal function, and cellular
defensive mechanism. Common feature is that these proteins are related to oxidative stressmediated
cell damage. In conclusion, the established gastric mucosal proteome map could be
useful for the detection of diseases-related protein changes. H. pylori-induced alterations in
protein expression demonstrates the involvement of oxidative stress in the pathogenesis of H.
pylori-induced gastric diseases including inflammation.
- 507 -

Session XIV : Transcriptional and translational control Poster XIV, 5
Gene expression modulation in A549 human lung cells in response to combustiongenerated
nano-sized particles.
Christa Baumstark-Khan, Andrea Arenz, Christine E. Hellweg and Horst-Henning
Grotheer.
Cellular Biodiagnostics, Department of Radiobiology, Institute of Aerospace Medicine,
German Aerospace Center, 51170 Köln, Germany. E-mail : christa.baumstarkkhan@dlr.de
Cell response to different kinds of environmental pollution is complex and involves the
participation of different classes of genes for different cellular outcomes (DNA repair, cell
cycle control, signal transduction, inflammation, apoptosis and oncogenesis). Ambient
particulate matter (PM) is a complex mixture of chemicals and particles that may be
compositionally diverse depending on geography and season. High levels of ambient air
pollution are associated with aggravation of asthma, respiratory morbidity, and
cardiopulmonary morbidity; long-term exposures to PM have been linked to possible
increases in lung cancer risk, chronic respiratory disease, and death rates. Nanometer-sized
particles (diameter 1.5 to 5 nm, mass numbers ranging from 1000 to 40.000 amu) which are
generated by combustion processes as soot precursors show surprising features, such as water
solubility and transparency, in contrast to the better known soot particles. The damaging
effects of nanoparticles are attributed to the fact that they are respirable and water soluble and
can penetrate lung mucosa. These particles are less readily cleared than fine particles, thereby
prolonging interaction with the lung epithelium and potentiating cellular damage. Particulate
matter has been shown to invoke inflammatory responses after exposure in animal models. Invitro
experiments have revealed that diesel exhaust and PM10 are capable to induce the
release of proinflammatory cytokines such as IL-6 and IL-8 from bronchial epithelial cells
mediated by the transcription factor NF-!B through a mechanism partially involving TNF- ".
Thus, an important aspect of our work was to determine whether nanoparticles cause
activation of NF-!B and expression of NF-!B dependent genes in pulmonary epithelial cells.
Recombinant A549 lung cells (A549-NF-!B-EGFP) were incubated with different
concentrations of condensation water samples collected from model flames (combustion of
gaseous fuels propane and ethylene under laboratory conditions). TNF- " treated cells were
used for comparison. RNA was extracted from exposed cells after various recovery times and
a real-time QRT-PCR assay was applied, which employs relative quantification of candidate
mRNA biomarkers. The expressions of different DNA damage inducible genes (GADD45#,
p21) and NF-kB dependent genes (NFkBIA, IL-6, “!B-EGFP”) were analysed. The results
show a reproducible up-regulation for the NF-kB dependent genes IL-6 (32x) and NF!BIA
(13x) with maximal values for 1 and 2 hrs treatment with TNF-", while the DNA damage
inducible genes are not induced. For nanoparticle treated cells, a down-regulation of NF-!B
dependent genes, especially for IL-6, could be shown for high particle concentrations
(Dilution ratios 1:8 to 1:20). For intermediate particle concentrations (Dilution ratio 1:100),
the DNA damage inducible gene GADD45 # is up-regulated (~5x) for up to 4 hrs and for
lower particle concentrations (Dilution ratio 1:200) the maximal induction value for
GADD45# is about 3. NF-!B dependent gene expression is down-regulated for the first hours
of nanoparticle incubation for NF!BIA (-3x, 4 hrs). For IL-6 a first down-regulation (-6.5, 30
min) is followed by a significant up-regulation for incubation times of 2 and 4 hrs.
- 508 -

Session XIV : Transcriptional and translational control Poster XIV, 6
p38 Mitogen-Activated Protein Kinase Pathway Regulates the Transcription of Skeletal
Muscle Late Genes .
Eyal Bengal1, Bennett H Penn2 and Stephen J Tapscott2.
1Department of Biochemistry, Rappaport Institute for Research in the Medical
Sciences,Faculty of Medicine. Technion-Israel Institute of Technology. P.O. Box 9649.
Haifa 31096, Israel. email: bengal@tx.technion.ac.il and 2 Division of Human Biology,
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024 USA.
The p38 mitogen-activated protein kinase (MAPK) pathway is induced during the
differentiation of proliferating myoblasts to multinucleated myotubes and is absolutely
essential for this process to occur in cell cultures.
Previous work showed that the p38 pathway augmented the activity of transcription factors
from the MEF2 and MyoD families. Here, we present data suggesting that only a small
subgroup of muscle-specific genes is regulated by p38 MAPK. A DNA microarray analysis
performed on muscle cells expressing an activated MKK6 protein revealed that the p38
MAPK pathway was involved in the expression of several muscle-structural genes.
Expression of these late-activated genes was shifted to the early stages of differentiation by
precocious activation of p38 and expression of Mef2D.
To understand how p38 MAPK regulates the transcription of these genes, we performed a
chromatin immunoprecipitation (ChIP) analysis. We show that p38 activity facilitates MyoD
and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits
Pol II.
This demonstrates that a MyoD-generated feed-forward regulatory circuit, wherein factors
induced by MyoD feed-forward to regulate MyoD activity at subsequent target genes, acts to
temporally pattern the relative timing of gene expression during skeletal myogenesis.
- 509 -

Session XIV : Transcriptional and translational control Poster XIV, 7
Holger Bertelsmann a, Harald Sieme b, Dietrich Behne a and Antonios Kyriakopoulos a
Selenium and zinc in equine spermatozoa: Correlations between the element
concentrations in sperm nuclei and spermatozoa
a Hahn- Meitner Institut, Department “Trace Element Research in the Life Science”,
Glienickerstr.100, 14109 Berlin, Germany
E- mail: bertelsmann@hmi.de
b Hanoverian State Stud Celle, Spoerckenstr. 10, D-29221 Celle, Germany
E- mail: stallions.celle@t-online.de
In the sperm nuclei of mammalian species selenium has been found only in the form of sperm
nuclei glutathione peroxidase (snGPx) where it is most likely bound to the chromatin of
spermatozoa. Over 80 % of selenium in sperm is bound to the selenoprotein phospholipid
hydroperoxide glutathione (PHGPx) peroxidase in the mid- piece of sperm. Zinc in sperm is
mainly contained in the outer dense fiber (ODF) proteins of the flagella of spermatozoa. In the
sperm nuclei zinc is predominately located in the chromatin to the protamine proteins. In
order to investigate if the insertion of zinc and selenium in sperm chromatin is regulated the
element concentrations were determined in equine spermatozoa and purified sperm nuclei.
We found a significant positive correlation between the selenium concentration in
spermatozoa and sperm nuclei. The same finding was obtained for the zinc concentration in
spermatozoa and sperm nuclei. The results assume that the distribution of selenium and zinc
in equine spermatozoa is regulated by a signal transduction pathway and in this way
determining the selenium and zinc amount in the chromatin of spermatozoa.
- 510 -

Session XIV : Transcriptional and translational control Poster XIV, 8
Protein folding information in nucleic acids which is not present in the genetic code
Jan C. Biro
Homulus Foundation; 88 Howard, #1205; San Francisco, 94195 CA, USA.
jan.biro@sbcglobal.net; www.janbiro,com
Background: All the information necessary for protein folding is supposed to be present in the
amino acid sequence. It is still not possible to provide specific ab initio structure predictions
by bioinformatical methods. It is suspected that additional folding information is present in
protein coding nucleic acid sequences, which is not represented by the known genetic code.
Results: Nucleic acid subsequences comprising the 1st and/or 3rd codon residues in mRNAs
express significantly higher free folding energy (FFE) than the subsequence containing only
the 2nd residues (p

Session XIV : Transcriptional and translational control Poster XIV, 9
Reconstitution of human hypoxia inducible factor HIF-1 in yeast cells: a simple in vivo
system for identification of HIF-1 inhibitors.
Georgia Braliou, Emmanouel Venieris, Alkmini Kalousi and George Simos
Laboratory of Biochemistry, School of Medicine, University of Thessaly, Larissa,
Greece. E-mail: simos@med.uth.gr
Hypoxia inducible factor 1 (HIF-1) is the master regulator of the genes that are activated
under hypoxic conditions. HIF-1 is also involved in many diseases, including cancer, and is
an important target for drug development. The control of HIF-1 itself is complex and involves
many post-transcriptional events triggered by hypoxia and by various signaling and oncogenic
pathways that operate in metazoan cells. In order to study the basic function of human HIF-1
in a simple and genetically tractable in vivo system we attempted to express it in the yeast S.
cerevisiae that lacks the components of the mammalian hypoxia response pathway. We show
here that inducible expression of both human HIF-1 subunits (HIF-1alpha and ARNT) in
yeast is possible and leads to the formation of a transcriptional active heterodimer, as shown
by hypoxia response element (HRE) - dependent production of a reporter enzyme. The
activity of HIF-1 in yeast is impaired by two Hsp90 inhibitors, geldanamycin A and radicicol,
and by mutations in Hsp90 co-chaperones, suggesting that the Hsp90 chaperone system is
important for HIF-1 integrity. We conclude that the expression of human HIF-1 in yeast
provides a model system for both functional studies and identification of compounds that can
act as direct HIF-1 inhibitors.
- 512 -

Session XIV : Transcriptional and translational control Poster XIV, 10
Translational control of pancreatic beta cell survival : PKB-activation inhibits apoptosis
through stimulation of mTOR
Ying Cai, Qidi Wang, Zhidong Ling, Daniel Pipeleers, Harry Heimberg and Mark Van
de Casteele
(Diabetes Research Center, Brussels Free University - VUB. Laarbeeklaan 103, B-1090
Brussels, Belgium. E-mail : mvdcaste@vub.ac.be).
Prolonged exposure of primary beta cells to low glucose levels induces their apoptosis [1].
This effect has been attributed to: a) reduced synthesis of anti-apoptotic proteins [1], b)
sustained activation of AMP-activated protein kinase (AMPK) [2]. Since AMPK activators
AICA-riboside and Metformin inhibit protein biosynthesis before inducing apoptosis of the
cells [3], we investigated whether this inhibition could be responsible for the apoptotic effect.
It has been reported that AMPK negatively regulates mTOR signaling in various cell types
and thus inhibits protein synthesis [4]. We therefore examined whether AMPK activation
decreases mTOR signaling in beta cells, and whether PKB, an upstream activator of mTOR,
can protect the cells against apoptosis.
Our data demonstrate that culture in low glucose, AICAR, or metformin, decreases
phosphorylation of both ribosomal S6 protein and 4EBP protein, but not of PKB, which is
similar to the effect of the specific mTOR inhibitor rapamycin. Expression of constitutively
active AMPK mimicked these effects. Adenoviruses expressing constitutively active PKB
(CA-PKB) decreased both low glucose and AICAR induced apoptosis, while increasing
mTOR-activity in beta cells. In AICAR or low glucose, CA-PKB restored partially the mTOR
signaling without influencing the phosporylation of AMPK. Inhibition of mTOR activity by
rapamycin, curtailed the antiapoptotic effect of PKB. We conclude that sustained AMPKactivation
decreases mTOR activity in beta cells leading to inactivation of translation factors ;
this effect can be counteracted by activated PKB resulting in rescue of beta cells from
apoptosis by mTOR-stimulation.
- 513 -

Session XIV : Transcriptional and translational control Poster XIV, 11
JAK-STAT - mediated cytokine expression in Helicobacter pylori-infected gastric
epithelial cells
BORAM CHA, KYUNG HWAN KIM, and HYEYOUNG KIM
Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea. Email:
chaboram@yumc.yonsei.ac.kr
The JAK-STAT(Janus kinase-signal transducers and activators of transcription) cascade is
known as an essential inflammatory signaling pathway. Peroxisome proliferators-activated
receptor gamma (PPAR gamma) ligand, 15Deoxy- 12,14 Prosta-glandin J2 (15d-PGJ2)is
reported as an anti-inflammatory agent in rheumatoid arthritis. We previously demonstrated
that H. pylori in a Korean isolate (HP99) induced proinflammatory cytokines in gastric
epithelial cells. The purpose of the present study is to determine whether 1) HP99 induces
proinflammatory cytokines, IL-8 and RANTES, and the activation of JAK-STAT in human
gastric epithelial AGS cells, 2) 15d-PGJ2 inhibits HP99-induced IL-8 and RANTES
expression in AGS cells, and 3) 15d-PGJ2 inhibits the phosphorylation of STAT-JAK in
HP99-infected AGS cells. As a result, HP99 induced the expression of IL-8 and RANTES as
well as the phosphorylation of JAK and STAT3 in AGS cells. PPAR gamma ligand, 15d-
PGJ2 attenuated HP99-induced IL-8 and RANTES expression by inhibiting the activation of
JAK-STAT in gastric epithelial AGS cells. In conclusion, PPAR gamma ligand, 15d-PGJ2
might be beneficial for treatment of Helicobacter pylori- induced gastric inflammation.
- 514 -

Session XIV : Transcriptional and translational control Poster XIV, 12
Mechanisms of hypoxia inducible factor HIF-1alpha regulation in airway smooth muscle
cells
Georgia Chachami1, 2, George Simos2, Apostolia Hatziefthimiou1, Panagiotis Liakos2,
Sophia Bonanou2, Paschalis-Adam Molyvdas1 and Efrosyni Paraskeva1
1Laboratory of Physiology and 2Laboratory of Biochemistry, Department of Medicine,
University of Thessaly, Larissa, Greece. E-mail:fparaskeva@med.uth.gr
Hypoxia inducible factor 1alpha (HIF-1alpha) is the regulatory subunit of HIF-1, the key
mediator of the cellular response to hypoxia. HIF-1alpha is induced by hypoxia but also at
normoxic conditions by exposure to heavy metals, treatment with growth factors or oncogenic
transformation. We investigate the role of HIF-1 in the physiology of the respiratory tract by
the use of primary cultures of airway smooth muscle (ASM) cells derived from rabbit trachea.
Smooth muscle cells are not terminally differentiated and dynamically exhibit distinct
contractile and proliferative phenotypes with unique morphological, biochemical, functional
and gene expression characteristics. ASM cells in primary cultures maintained in full growth
medium that contains fetal bovine serum (FBS) have the tendency to acquire the proliferative
phenotype. However, prolonged serum deprivation allows a subset of cells to re-acquire the
morphological and functional characteristics of contractile cells within intact tissues.
Exposure of “proliferative” ASM cells to low oxygen concentration as well as to cobalt can
cause a rapid increase of the intracellular levels of HIF-1alpha. The use of specific inhibitors
reveals that induction of HIF-1alpha by cobalt is due to an increase of both HIF-1alpha
stabilization and active protein synthesis and involves the phosphatidylinositol 3-kinase
(PI3K) pathway and the production of reactive oxygen species.
We are currently investigating the induction of HIF-1alpha in serum-deprived quiescent
“contractile” cells and the cellular pathways involved. Cobalt induces HIF-1alpha in
“contractile” cells by affecting predominantly its protein stability. On the other hand, readdition
of serum to these cells also causes HIF-1alpha induction, which is not due to
increased protein stability, but rather requires on-going transcription, active protein synthesis
and is PI3K-dependent. Interestingly, the simultaneous incubation of “contractile” cells with
cobalt and serum results in an additive induction of HIF-1alpha protein levels and more
efficient nuclear localization. Moreover, simultaneous incubation of rabbit trachea strips with
cobalt and serum changes their contractile properties by increasing their responsiveness to
acetylcholine.
- 515 -

Session XIV : Transcriptional and translational control Poster XIV, 13
Transcriptional regulation of cyclooxygenase-2 in response to proteasome inhibitors
involves reactive oxygen species-mediated signaling pathway and recruitment of
CCAAT/enhancer binding protein-delta and CREB-binding protein
Jun-Jie Chen, Wei-Chien Huang and Ching-Chow Chen
Department of Pharmacology, College of Medicine, National Taiwan University,
Taipei 10018, Taiwan. E-Mail: ccchen@ha.mc.ntu.edu.tw
Inhibition of ubiquitin-proteasome pathway has been shown to be a promising strategy for the
treatment of inflammation and cancer. Here, we show that proteasome inhibitors MG132,
PSI-1 and lactacystin induce COX-2 expression via enhancing gene transcription rather than
preventing protein degradation in the human alveolar NCI-H292 and A549, and gastric AGS
epithelial cells. NF-IL6 and CRE, but not NF-!B elements on the COX-2 promoter were
involved in the gene transcription event. The binding of CCAAT/enhancer binding protein
(C/EBP)-beta and C/EBP-delta to the CRE and NF-IL6 elements, as well as the recruitment of
CBP and the enhancement of histone H3 and H4 acetylation on the COX-2 promoter was
enhanced by MG132. However, it did not affect the total protein levels of C/EBP-beta and
C/EBP-delta. MG132-induced DNA-binding activity of C/EBP-delta but not C/EBP beta
was regulated by p38, PI3K, Src and PKC. Small interfering RNA of C/EBP-delta suppressed
COX-2 expression, further strengthening the role of C/EBP-delta in COX-2 gene
transcription. In addition, the generation of intracellular reactive oxygen species (ROS) in
response to MG132 contributed to the activation of MAPKs and Akt. These findings reveal
that the induction of COX-2 transcription induced by proteasome inhibitors requires ROSdependent
protein kinases activation and the subsequent recruitments of C/EBP-delta and
CBP.
- 516 -

Session XIV : Transcriptional and translational control Poster XIV, 14
Signal transduction on the expression of inflammatory enzymes in Helicobacter pylori -
infected gastric epithelial cells
Soon Ok Cho, Hyeyoung Kim, Kyung Hwan Kim
Department of Pharmacology and Brain Korea 21 Project for Medical Science
Yonsei University College of Medicine, Seoul 120-752, Korea
E-mail:ehrehrwh@hanmail.net
Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are important
enzymes that mediate inflammatory processes. Oxygen radicals are important regulators in
Helicobacter pylori (H. pylori)-induced gastric ulceration and carcinogenesis, COX-2 and
iNOS may be regulated by oxidant-sensitive transcription factors, NF-kB and AP-1. In
addition, the binding sites for NF-kB and AP-1 are found in the promoter region of COX-2
and iNOS gene. Present study aims to investigate whether H. pylori-induced expressions of
COX-2 and iNOS are regulated by NF-kB and AP-1 in gastric epithelial AGS cells, and
whether the transcriptional regulation of COX-2 and iNOS are inhibited by transfection with
mutant genes for Ras (ras N-17), c-jun (TAM67), and IkBa (MAD3). As a result, H. pylori
induced the expression of mRNA and protein for COX-2 and iNOS via activation of NF-kB
and AP-1. Transfection with mutant genes for Ras (ras N-17), c-jun (TAM67), and IkBa
(MAD3) inhibited H. pylori-induced COX-2 and iNOS expression in AGS cells. In
conclusion, H. pylori induced activation NF-kB and AP-1 and thus COX-2 and iNOS
expression in gastric epithelial cells. Either suppression of NF-kB or AP-1 may inhibit H.
pylori-induced COX-2 and iNOS expression in gastric epithelial cells.
- 517 -

Session XIV : Transcriptional and translational control Poster XIV, 15
Serine/threonine protein phosphatases and MCP-1 in Helicobacter pylori–infected
gastric epithelial cells
Hae-Yun Chung, Boram Cha, Ji Hye Seo, Kyung Hwan Kim, and Hyeyoung Kim
Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea. E-mail :
hchung02@yumc.yonsei.ac.kr
The phosphorylation of proteins controlled by protein kinases and phosphatases is a major
mechanism that regulates cellular processes such as inflammation. It has been reported that
the activity of at least 30% of all proteins can be regulated by phosphorylation in eukaryotes.
Among these proteins, MAPK and some transcription factors play a pivotal role in
inflammation. We previously demonstrated that Helicobacter pylori in a Korean isolate
(HP99) induced chemokine expression by activating MAPK and transcription factors, NF-kB
and AP-1 in gastric epithelial AGS cells. To determine the role of phosphorylation /
dephosphorylation in HP99-induced inflammation, we analyzed the expression of
phosphatases, the activation of MAPK and AP-1 and the expression of chemokine, MCP-1 in
AGS cells stimulated with HP99 and cultured in the presence or absence of a serine/threonine
phosphatase inhibitor, okadaic acid (OA). As a result, HP99 induced the expression of protein
phosphatases, PP1 and PP2A as well as the activation of MAPK and AP-1, and the induction
of MCP-1 in AGS cells, which was augmented by OA. In conclusion, gastric epithelial cells
induced the expression of PP1 and PP2A in response to HP99 as a defense mechanism against
inflammatory chemokine expression by inhibiting the activation of MAPK and AP-1.
- 518 -

Session XIV : Transcriptional and translational control Poster XIV, 16
Regulation of Vascular Endothelial Growth Factor (VEGF) expression by Human
Papillomavirus 18 E6 oncoprotein in HeLa cells
Nicolas Clere1, Laurent Bermont1, Sylvie Fauconnet1, Isabelle Lascombe1, Evelyne
Chezy2 and Christiane Mougin1,2
(1) University of Franche-Comté, EA 3181–IFR133, UFR des Sciences Médicales et
Pharmaceutiques, Besançon, France, (2) Laboratoire de Biologie Cellulaire et
Moléculaire, Centre Hospitalier Universitaire, Besançon, France
Human papillomaviruses (HPVs) are a large group of small DNA viruses which infect
cutaneous or mucosal epithelia. High risk HPVs are the primary cause of cervical cancer, the
second most prevalent cancer in women worldwide, with the continuous expression of early
viral oncogene E6 secondary to viral DNA integration into the host genome. E6 is able to
immortalize cells and induces malignant transformation by inactivating p53 protein. However,
this expression is not sufficient for the progression of solid tumor. Tumor growth and
metastasis are often influenced by the production of VEGF, a key mediator of angiogenesis.
In solid tumor, angiogenesis is associated with VEGF expression by tumor cells. VEGF exists
as five isoforms of 121, 145, 165, 189 and 206 amino-acids. In cervical cancer, regulation of
VEGF expression is complex and poorly described. Thus, we addressed the question of
whether E6 oncoprotein could modulate the expression of VEGF in HPV 18 cervical derived
cancer cells. We performed a quantitation of VEGF by real time RT-PCR from HeLa cells
harboring HPV18 and a wild type p53. Three transcripts encoding VEGF 121, 165 and 189
were expressed. Synthetic siRNA targeting E6 were also applied to inhibit oncogene
expression and to evaluate the role of the E6 oncoprotein on VEGF expression. Among the
three VEGF transcripts expressed, those encoding VEGF121 were majority and represented
90% of all isoforms. The silencing of E6 reduced by 50% the level of these transcripts when
compared to cells transfected with control siRNA. In the same way, a decrease of
approximatively 30% of secreted VEGF was observed in transfected cells. According to our
results, we may raise the hypothesis that E6 induced VEGF expression in a p53-independent
manner. Additionnal data will be presented at the meeting.
- 519 -

Session XIV : Transcriptional and translational control Poster XIV, 17
Lipid metabolism alterations induced by mitochondrial dysfunction in preadipocytes: a
new role for the transcription factor CREB
Aurélia De Pauw 1 , Sebastien Vankoningsloo 1 , Andrée Houbion 1 , Silvia Tejerina 1 ,
Catherine Demazy 1 , Françoise de Longueville 2 , Vincent Bertholet 2 , Patricia Renard 1 ,
José Remacle 1,2 , Martine Raes 1 and Thierry Arnould 1
1
Laboratory of Biochemistry and Cellular Biology, University of Namur (F.U.N.D.P.),
Rue de Bruxelles, 61, 5000 Namur, Belgium. Email : HYPERLINK
"mailto:thierry.arnould@fundp.ac.be" thierry.arnould@fundp.ac.be,
2
Eppendorf
Array Technologies, Rue du Séminaire, 12, 5000 Namur, Belgium.
The role of mitochondria in cellular lipid homeostasis, adipogenesis and insulin sensitivity is
now evidenced in various pathologies resulting from genetic alterations or chronical exposure
to molecules that impair mitochondrial activity. However, the molecular mechanisms
underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly
understood. In this study, we showed that respiratory complex III inhibition by antimycin A
as well as mitochondrial protein synthesis inhibition trigger the accumulation of triglyceride
vesicles in 3T3-L1 fibroblasts. We also found that antimycin A treatment triggers CREB
activation in these cells. To delineate how mitochondrial dysfunction induces triglyceride
accumulation in preadipocytes, we developed a low-density DNA microarray that contains 89
probes allowing gene expression analysis for major effectors and/or markers of adipogenesis.
We thus determined gene expression profiles in 3T3-L1 incubated with antimycin A and
compared the patterns obtained with differentially expressed genes during the course of in
vitro adipogenesis induced by a standard proadipogenic cocktail. After a 8-day treatment, a
set of 39 genes was found to be differentially expressed in antimycin A-treated cells among
which CHOP-10 (C/EBP homologous protein-10), GPDmit (mitochondrial glycerol-
3phosphate dehydrogenase), and SCD1 (stearoyl-CoA desaturase 1). We also demonstrated
that the overexpression of dominant negative mutants of CREB (K-CREB and M1-CREB) as
well as siRNA transfection that respectively disrupt the factor activity and expression inhibit
antimycin A-induced triglyceride accumulation. These results highlight a new role for CREB
in the control of triglyceride metabolism during the adaptative response of preadipocytes to
mitochondrial dysfunction.
T. Arnould and A. De Pauw are respectively Research Associate and Research Assistant of
FNRS (Fonds National de la Recherche Scientifique, Brussels, Belgium).S. Tejerina is a
recipient of a CUD (Coopération Universitaire au Développement) fellowship.
- 520 -

Session XIV : Transcriptional and translational control Poster XIV, 18
Transcriptional regulation of cell proliferation signalling pathways by the transcription
factor E2F2 during liver regeneration
Igotz Delgado, Yuri Rueda, Olatz Fresnedo and Begoña Ochoa.
Department of Physiology, University of the Basque Country Medical School, PO Box
699, Bilbao, Spain. E-mail: igotzdelgado@yahoo.es
E2F transcription factors are key regulators of cell cycle progression; they are fundamental
for crossing the G1/S checkpoint as they regulate the expression of genes required for DNA
replication. During the G1 phase, pocket proteins such as pRb (retinoblastoma protein) are
blocking E2Fs activity. Cyclin dependent kinases phosphorylate pRbs so that E2Fs are
released. When associated with pRb family members, the E2Fs function as transcriptional
repressors, whereas free E2F activates transcription. The pRb-E2F pathway is a downstream
target of mitogenic signalling pathways. E2F2 transcription factors are activator members of
E2F family as they induce cell cycle entry in quiescent cells. Liver regeneration is considered
an appropriate model to analyse factors related to cell cycle because, following an hepatic
tissue resection, 95% of hepatic cells (which normally stay quiescent) enter cell cycle to start
regeneration while maintaining their metabolic functions. We have addressed the role of E2F2
in liver regeneration using the well characterised paradigm of 70% partial hepatectomy (PH)
in wild-type and E2F2-/- mice and high-density oligonucleotide arrays to identify genes in
which the change in expression in response to hepatectomy differed. Measurements were
performed at 48 hours post-hepatectomy, as hepatocyte replication peaked at this time in the
regenerating mouse. E2F2-/- mice exhibited a delayed liver regeneration, showing the
importance of this transcription factor in the regenerative process. The microarray analysis
showed that genes belonging to antiproliferative signaling pathways such as Rras, dual
specific phosphatases 6 and 16 or Map kinase-activated protein kinase 2 were upregulated in
E2F2-/- mice during the regenerative process, this explaining, at least in fact, that liver
regeneration is retarded in the absence of E2F2. In conclusion, regardless of its action on
proliferation, the transcription factor E2F2 enhances the expression of some genes involved in
antiproliferative signalling pathways of the liver.
Supported by the Basque Government (Etortek02, IE019)
- 521 -

Session XIV : Transcriptional and translational control Poster XIV, 19
Transcriptional Regulation of Human Plasminogen Activator Inhibitor-1 Gene
Expression by IGF-1 and Insulin: Role of HIF-1 and Forkhead transcription factor
Elitsa Y. Dimova, Daniela Fluegel and Thomas Kietzmann
Department of Biochemistry, Faculty of Chemistry, University of Kaiserslautern, D-
67663 Kaiserslautern, Germany. E-mail: edimova@gwdg.de
One risk factor for pathological conditions associated with hypoxia or hyperinsulinemia is
plasminogen activator inhibitor-1 (PAI-1). IGF-1 and insulin have been shown to induce PAI-
1 expression but the molecular mechanisms behind these effects have not been fully
elucidated. Therefore, we searched for putative IGF-1- and insulin-responsive element(s),
transcription factor(s) and signalling components mediating the IGF-1 and insulin-dependent
human PAI-1 expression, using HepG2 cells as a model system. IGF-1 and insulin treatment
enhanced PAI-1 expression and promoter activity. Mutation of the hypoxia responsive
element (HRE), which could bind hypoxia-inducible factor-1 (HIF-1), nearly abolished the
induction by IGF-1 and insulin. Mutation of E-boxes E4 and E5 did not affect the IGF-1- and
insulin-dependent activation of the PAI-1 promoter constructs under normoxia but abolished
the effects of IGF-1 and insulin under hypoxia. Additionally, mutation of a putative Forkhead
binding site abolished the insulin-dependent activation under normoxia, but not under
hypoxia. Overexpression of Forkhead transcription factor resulted in suppression of the
hypoxia–mediated response of the human PAI-1 promoter constructs, but not in abolishment
of the insulin effect. Further the IGF-1 and insulin-induced up-regulation of PAI-1 was
associated with activation of HIF-1alpha IGF-1 enhanced HIF-1alpha protein levels and
HIF-1 DNA-binding to each HRE, E4 and E5 as shown by EMSAs. Inhibition of the PI(3)K
and MAPK pathways by specific inhibitors or by overexpression of the key enzymes e.g.
dominant-negative PDK1, TRB3 which inhibits Akt/PKB or dominant-negative Raf-1
revealed that IGF-1 activates human PAI-1 gene expression through activation of PI(3)K and
ERK1/2 whereas insulin-induced PAI-1 gene expression occurred mainly via the MAPK
pathway.
- 522 -

Session XIV : Transcriptional and translational control Poster XIV, 20
Daily swimming diminishes the isolation-induced changes on the level of glucocorticoid
receptor and heat shock protein 70 in rat brain
Filipovi' M. Dragana, Gavrilovi' Ljubica, Dronjak Sladjana and Radoj(i' B. Marija
Institute of Nuclear Sciences «Vin(a», Laboratory of Molecular Biology and
Endocrinology,11000 Belgrade, Serbia and Montenegro. E-mail : dragana@vin.bg.ac.yu
Animals chronically exposed to a particular stressor display an exaggerated response of
hypothalamo-pituitary adrenal axis to various novel stressors, as judged by plasma level of
glucocorticoids and adrenocorticotropic hormone (ACTH). The molecular mechanism of such
observation is believed to involve the action of glucocorticoid receptor (GR) and its nuclear
transporter heat shock protein 70 (Hsp70). The main aim of the present study was to define
the change on the level of GR and Hsp70 in a cytosol of hippocampus and brain cortex of
Wistar rat males exposed to 21 daily isolation or isolation plus 15 minute/daily swimming as
chronic stressors, sole or in combination with 2h acute stress of immobilization or cold (40C).
The level of GR and Hsp70 were quantified by Western immunoblotting. Plasma ACTH and
corticosterone (CORT) were measured by chemiluminescent method and RIA, respectively. A
significant decrease of both proteins was observed in acutely stressed rats. Chronic stress
conditions led to moderate decreases in both cytosol GR and Hsp70 level. Isolation as chronic
stressor caused reduced responsiveness to a novel acute stressors, judged by the cytosol GR
and Hsp70. This was not observed when isolation plus swimming preceeded the application
of the acute stressors. The only exeption was GR level when isolation plus swimming were
followed by immobilization. The obtained results led to the conclusion that 15 minute/daily
swimming may diminish chronic social isolation-induced changes on the levels of
glucocorticoid receptor and heat shock protein Hsp70 in cytosol of hippocampus and brain
cortex of rats.
- 523 -

Session XIV : Transcriptional and translational control Poster XIV, 21
Nemo-Like Kinase Inhibits Androgen Receptor Transcriptional Activity in Prostate
Cancer Cells
Katayoon H. Emami, Lisha G. Brown, Tiffany EM Pitts, and Eva Corey
GU Cancer Research Laboratory, Department of Urology, University of Washington,
Seattle, WA 98195, USA. Email: ecorey@u.washington.edu
Androgen receptor (AR) signaling and its interactions with other signaling pathways play
important roles in growth and differentiation of prostate and prostate cancer (CaP) cells.
Nemo-like kinase (NLK), a MAP kinase, has been reported to affect the activity of various
transcriptional factors via its interactions with co-activators #-catenin and CBP/p300. Because
MAP Kinases have been implicated in regulation of the AR signaling pathway and AR
signaling utilizes both #-catenin and CBP/p300 as coactivators, y we examined the effects of
NLK on AR signaling in CaP cells.
Our data show that inhibition of NLK expression by siRNA upregulates AR promoter
activity, while overexpression of NLK dramatically inhibits the activation of both an artificial
ARE and the PSA promoter in LNCaP prostate cancer cells. A kinase-dead NLK(K155M)
inhibited only ~50% of the AR transcriptional activity in LNCaP cells, implying that the
kinase activity of NLK is important for this inhibition but that other mechanisms are also
involved. Our data show that the mechanisms involved in the reduced transcriptional activity
of AR include disruption of interactions of AR with #-catenin and p300, decreased AR-DNA
binding, and alteration of the subcellular localization of AR. We undertook further studies to
investigate the biological effects associated with the inhibitory role of NLK in AR-directed
transcription and showed that NLK increases the rate of apoptosis in these cells.
Further studies of the roles of NLK in regulation of prostate cancer are warranted, since
understanding the molecular mechanism of regulation of AR transcriptional activity in CaP
cells is essential for identification of new targets to control this disease and may have a
dramatic impact on the therapeutic strategies adopted for the prevention and treatment of CaP.
- 524 -

Session XIV : Transcriptional and translational control Poster XIV, 22
The effects of dexamethasone on the expression of alpha- and beta- isoforms of
glucocorticoid receptor in cultured human myoblasts and myotubes
Dragana M. Filipovi'1, Katarina Mi)2, Toma0 Mar)2 and Zoran Grubi( 2.
1Institute of Nuclear Sciences “Vin(a”, Laboratory of Molecular Biology and
Endocrinology, 11000 Belgrade, Serbia and Montenegro, 2Institute of Pathophysiology,
School of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia. E-mail :
dragana@vin.bg.ac.yu
Critical illness myopathy is one of the major problems in intensive care units. High doses of
glucocorticoids (GCs) received by such patients are assumed to play essential role in the
etiology of this disorder, however, the precise pathophysiologic mechanisms underlying this
GC action are mostly unknown. Recovery is often incomplete in these patients and the final
outcome depends at least partly on muscle regeneration which might also be affected by
glucocorticoids. In order to approach this problem systematically, we first studied the
expression of glucocorticoid receptor (GR) isoforms, GR alpha and GR beta differentially in
human myoblasts and myotubes which are the precursors of muscle regeneration. We also
studied the effects of synthetic GC Dexamethasone (Dex) on the GR alpha and GR beta
expression since this is the major regulatory mechanism modulating GC sensitivity in
peripheral tissues including muscle. Cultured human myoblasts and myotubes were treated by
Dex (0.2µM and 1µM) for 24h. Expression pattern of the GR isoforms in control and Dextreated
cells were followed at both, mRNA and protein levels using RT-PCR and Western
blot, respectively. Up to now we found both receptor subtypes expressed already at the
earliest (myoblast) as well as at the myotube stage, however we found no differential response
between these two stages with regard to the Dex – regulated expression of GR subtypes. This
finding suggests that response to GCs at the receptor level is established very early in the
myogenesis of the human muscle and remains practically unchanged afterwards. We will
continue this study by approaching other mechanisms underlying GC effects on muscle
regeneration.
- 525 -

Session XIV : Transcriptional and translational control Poster XIV, 23
Antagonistic function of S100 proteins regulates AP-1 dependent gene expression during
tumor development
Christoffer Gebhardt,1,4 Michiel van der Sanden,1 Lars Hummerich,2 Kai Breuhahn,3
Julia Németh,1 Meinhard Hahn,2 Alexander Enk,4 Gerhard Fuerstenberger,5 Cornelia
Mauch,6 Peter Schirmacher,3 Peter Lichter,2 Peter Angel1 and Jochen Hess1
1Division of Signal Transduction and Growth Control; 2Division of Molecular Genetics;
5Division of Eicosanoids and Tumor Development, Deutsches Krebsforschungszentrum,
D-69120 Heidelberg, Germany; 3Department of Pathology; 4Department of
Dermatology, University Hospital Heidelberg, 69120 Heidelberg, 6Department of
Dermatology, University of Cologne, Joseph-Stelzmann-Str. 9, D-50924 Köln, Germany
Email: c.gebhardt@dkfz.de
Despite compelling data demonstrating a direct link between altered expression of S100
proteins located on human chromosome 1q21 and common epithelial malignancies, the
knowledge of their function and mode of action in epithelial cells and in the evolution or
progression of cancer is largely unknown. Here, we have identified a novel signaling pathway
in epithelial cells initiated by extracellular S100A8/A9 heterodimers resulting in the
activation of AP-1-dependent gene expression. Importantly, co-expression of S100A3 inhibits
S100A8/A9 mediated AP-1 activation, which is in line with repression of this gene during
chemically induced skin carcinogenesis in mice suggesting a negative role for S100A3 in
epithelial malignancy. We found elevated levels of MMP2 and MMP9, two well-known AP-1
regulated genes, and identified S100A6 as an additional target gene of S100A8/A9 signaling
in epithelial cells. Using tissue-microarrays, significant co-expression of S100A8 and S100A9
in concert with phosphorylation of c-Jun and elevated levels of S100A6 protein was evident
in cutaneous squamous cell carcinomas of patients. Altogether, our data suggest that targeting
the net activity of S100 induced signaling represents an auspicious strategy for innovative
cancer prevention and/or therapy.
- 526 -

Session XIV : Transcriptional and translational control Poster XIV, 24
Study of a molecular retrograde communication that involves SREBP-1 and SREBP-2 in
143 B cell responding to a lysosomal sucrose accumulation
Sophie Goblet1, Andrée Houbion2, Isabelle Hamer1, Laetitia Payen2, Marie-Jeanne
Vertez1, Michel Jadot1 and Thierry Arnould2
1. URPhyM : Unité de Recherche en Physiologie Moléculaire, Faculté de Médecine
(F.U.N.D.P.)
2. URBC : Unité de Recherche en Biologie Cellulaire, Faculté des Sciences (F.U.N.D.P)
61 rue de Bruxelles, 5000 Namur, Belgium. e-mail : thierry.arnould@fundp.ac.be
While numerous lysosomal diseases have been described to result from mutations in genes
encoding lysosomal hydrolases leading to lysosomal enzyme activity deficiency and/or
accumulation of non-hydrolysable substrates, cell signaling initiated by lysosomal storage
disorder is still poorly understood. We thus intended to identify new signaling pathway(s)
activated by a lysosomal storage of sucrose in human osteosarcoma cells (143B).
We found that these cells overexpress lysosomal markers in response to sucrose and we
characterized some aspects of the cell phenotype during sucrose accumulation. Among
several transcription factors studied, we next showed that members of SREBP (sterolresponsive
element binding protein) transcription factors are activated in response to a sucrose
lysosomal storage as luciferase reporter constructs driven by the FAS (fatty acid synthase),
HMG-CoA reductase, LDLR (low density lipoprotein receptor) promoters are found to be
activated in response to a sucrose incubation. We found that both SREBP-1 and SREBP-2
protein accumulate in the nucleus of the sucrose-treated cells. These data are supported by
findings showing that SREBP activation is correlated with a modification in cholesterol
distribution in the sucrose-treated cells as observed after a filipin staining. Furthermore, 25hydroxycholesterol
completely inhibits SREBP activation and dramatically reduces SREBP-2
abundance in the nucleus of sucrose-treated cells. We also investigated signaling pathways
that could lead to the activation or nuclear translocation of SREBP and found that SREBP
activation is sensitive to PI3-K and MEK1/2 inhibitors. Taken together, these data suggest
that lysosomal accumulation of a non-hydrolysable substrate such as sucrose, triggers the
activation of a cell signaling pathway leading to SREBP activation.
T. Arnould is a Research Associate of FNRS (Fonds National de la Recherche Scientifique,
Brussels, Belgium). S. Goblet is a recipient of a FRIA fellowship. We thank the governement
of the french-speaking community (Belgium) for financial support through an ARC
(Action de Recherche Concertée) programme.
- 527 -

Session XIV : Transcriptional and translational control Poster XIV, 25
Effect of NF-kB inhibition on the development of endometriosis in a nude mouse model
Reinaldo González Ramos1, Anne Van Langendonckt1, Sylvie Defrère1, Marcel
Mettlen2 and Jacques Donnez1
1 Gynecology Unit, 2 Cell Unit, Université Catholique de Louvain, 1200 Brussels,
Belgium. E-mail: donnez@gyne.ucl.ac.be
Introduction: Endometriosis is a gynecological disorder characterized by the ectopic growth
of endometrial tissue, in which inflammation plays an important role. The transcription factor
nuclear factor-kappaB (NF-kB), has a key function in the transduction of proinflammatory
signals. The NF-kB pathway is activated in response to TNF" in human endometrial and
endometriotic stromal cells.
The purpose of this study is to test BAY 11-7085 (an inhibitor of IkB phosphorylation) and
SN-50 (inhibitor of NF-kB nuclear translocation) on the development of endometriosis and
the proliferation activity of ectopic endometrium in a murine model of endometriosis.
Material and Methods: Endometriosis was induced in nude mice by direct intraperitoneal
injection of minced human menstrual endometrium (200µl), labelled with CFDA-SE (a
fluorescent tracker). Mice were injected on days 1, 3 and 5, either with 200 µl PBS (control
mice), BAY 11-7085 (5 mg/kg in 200 µl of PBS) or SN-50 (5 mg/kg in 200 µl of PBS).
Five days after endometrial injection, lesions were identified and dissected under a
fluorescence microscope. Endometriosis development was evaluated using three methods:
weight, fluorimetry and morphometry. Wet weight was measured for each lesion and the
results were pooled per mouse. Fluorimetry involves the quantification of the fluorescence of
lesions by means of a Kodak 2000MM image station. Morphometry measures the surface of
endometriotic tissue on the largest section of a lesion immunolabelled with CK-22 (an
epithelial marker) and CD-10 (a stromal marker) antibodies.
NF-kB activation and epithelial cell proliferation were assessed after immunostaining with
NF-kB and Ki-67 antibodies, respectively.
Results: Six series of experiments were performed for BAY 11-7085 versus control mice and
three series for SN-50 versus control mice. 25 lesions were recovered in a total of 15 mice
operated. There was a significant reduction in lesion weight, fluorimetry (p

Session XIV : Transcriptional and translational control Poster XIV, 26
Heavy metals and thermal stress induce pro- or anti-apoptotic events via the p38-MAPK
signal transduction pathway in Mytilus galloprovincialis
Eleni Gourgou, Eirini Kefaloyianni, Catherine Gaitanaki and Isidoros Beis
Department of Animal & Human Physiology, School of Biology, Faculty of Sciences,
University of Athens, Panepistimioupolis, Athens 15784, Greece
In the present study, a number of heavy metals (copper, zinc and cadmium) as well as thermal
stress was used, in order to investigate their possible effect on the p38-MAPK signaling
pathway, in Mytilus galloprovincialis mantle and gill tissues. All the stressful stimuli were
applied to the whole organism and both mantle and gill tissues of the same animal were used
for protein detection. In the mantle tissue, 1 uM Cu2+ induced a sustained p38-MAPK
activation, 50 uM Zn2+ induced a transient activation and 1 uM Cd2+ a biphasic profile with
maximal values at 15 and 120 min of treatment, respectively. Furthermore, 1 uM SB203580
abolished the Cu2+-induced kinase phosphorylation. In gills, both Cu2+ and Zn2+, induced a
considerably higher p38-MAPK activation which sustained for at least 2 hours, whereas,
Cd2+ induced a maximal kinase activation within 60 min of treatment. Hypothermia (4oC)
induced a moderate p38-MAPK phosphorylation (maximised at 60 min), whereas
hyperthermia (30°C) induced a rapid (within 15 min) kinase phosphorylation that sustained
considerably above basal levels for at least 2 hours. As far as it concerns the possible
synergistic effects of hyperthermia and Cu2+, it was revealed that these two stressful stimuli
act co-operatively, inducing an almost double p38-MAPK activation. Furthermore,
investigating the possibility of pro-apoptotic or anti-apoptotic events occurring as a result of
the p38-MAPK activation, it was revealed that identical stimuli may lead to pro-apoptotic
events in the mantle tissue via the caspase-3 activation and to anti-apoptotic events possibly
via the induction of Hsp70 over-expression in the gill tissue, indicating a tissue-specific
physiological reaction.
Acknowledgements: This study was funded by the Empeirikion Foundation of Athens and by
G.S.R.T. (PENED 01ED5).
- 529 -

Session XIV : Transcriptional and translational control Poster XIV, 27
Arylhydrocarbon Receptor Repressor (AhRR) controls AhR regulated genes: clues for
recruitment of HDACs
Thomas Haarmann-Stemmann, Hanno Bothe, Ellen Fritsche, Josef Abel
Institut für umweltmedizinische Forschung, Auf’m Hennekamp 50, 40225 Düsseldorf,
Germany; E-mail: haarmann@uni-duesseldorf.de
The AhRR is a recently discovered new member of the bHLH-PAS superfamily of
transcription factors, closely related to the arylhydrocarbon receptor (AhR) and ARNT. These
proteins are key regulators of drug metabolizing enzymes in response to xenobiotics like
benzo(a)pyrene (B(a)P) or 3-methylcholanthrene (3-MC). Here we report about the regulation
and function of the AhRR in different human cell lines, varying in expression and inducibility
of the AhRR: the responsive HepG2 cells, the low-responsive A549 cells, and the nonresponsive
human primary fibroblast and HeLa cells. Expression analyses revealed a high
expression of the AhRR in fibroblast and HeLa cells and low expression in HepG2 and A549
cells. Short term treatment with 10µM B(a)P enhanced the AhRR mRNA only in HepG2
cells. No significant increases were found in A549, fibroblast and HeLa cells. In order to test
whether methylation or acetylation status of the AhRR promoter account for the cell-specific
response of the AhRR, non-responsive cell lines were treated with either Azacytidine (Aza) or
Na-butyrate with or without B(a)P. Aza-treatment did not change the constitutive expression
of the AhRR, whereas co-treatment with Aza and B(a)P enhanced the AhRR mRNA only in
A549 cells. Bisulfit sequencing revealed no remarkable differences in methylation status of
AhRR promoter between responsive and non-responsive cell lines. Studies with the HDACinhibitor
indicate the importance of acetylation status in the regulation of AhRR and CYP1A1
expression. Chromatin immunoprecipitation (ChIP) revealed a strong XRE-binding of AhRR
in the non-responsive cell lines which disappears after Na-butyrate treatment. Coimmunoprecpitation
analyses imply an AhRR-HDAC interaction. These findings indicate that
the AhRR recruits HDACs and thereby controls expression of AhR-regulated genes. This
model is confirmed by siRNA analyses showing that with decreasing amount of AhRR the
expression of CYP1A1 increased. In summary, our data provide evidence that the AhRR is an
important regulator of transcription of AhR regulated genes via its ability to recruit HDACs.
From our study we suggest that the AhRR exerts a nuclear co-repressor function, but details
of this mechanism remain to be elucidated.
- 530 -

Session XIV : Transcriptional and translational control Poster XIV, 28
Activation of NF-kappaB by different agents – influence of culture conditions.
Christine E. Hellweg, Andrea Arenz, Susanne Bogner, Claudia Schmitz and Christa
Baumstark-Khan.
Cellular Biodiagnostics, Department of Radiobiology, Institute of Aerospace Medicine,
German Aerospace Center, 51170 Köln, Germany. E-mail : christine.hellweg@dlr.de
The transcription factor NF-kappaB or other components of this pathway have been identified
as possible therapeutic targets in inflammatory processes, cancer and autoimmune diseases. In
order to clarify the role of NF-kappaB in epithelial cells in the response to different stressors,
a cell-based screening assay for activation of NF-kappaB dependent gene transcription in
human embryonic kidney cells (HEK/293) was developed (J. Biomol. Screen. 2003, 8, 511-
521). This assay allows detection of NF-kappaB activation by measurement of the
fluorescence of the reporter protein destabilized Enhanced Green Fluorescent Protein
(d2EGFP). HEK/293 cells are stably transfected with a vector carrying the d2EGFP gene
under control of a synthetic promoter consisting of four kappa B elements and the minimal
thymidine kinase promoter. Treatment of the recombinant HEK-pNF-kB-d2EGFP/Neo cells
with the known NF-kappaB activator tumour necrosis factor alpha (TNF-alpha) results in
strong induction of NF-kappa B dependent gene expression in up to 90 % of the cells.
Involvement of the p65 subunit in this response was shown using an oligonucleotide-assay.
For a better characterisation of the cell-based assay, activation of the pathway by other agents,
e.g. interleukin-1beta (IL-1beta), lipopolysaccharide (LPS), camptothecin, and phorbol ester
(PMA), the influence of the culture conditions on NF-kappa activation by TNF-alpha, and the
expression of possible target genes were examined. NF-kappa B was activated by TNF-alpha,
IL-1beta and PMA in a dose-dependent manner, but not by camptothecin or LPS. TNF-alpha
results in the strongest induction of NF-kappa B dependent gene expression. However, this
response fluctuated from 30 to 90 %. Activation of NF-kappaB by TNF-alpha was maximal
within 48 hours after seeding of cells. With increasing confluence of the cells, the activation
potential decreased. In a confluent cell layer, only 30-50 % of the cell population showed
d2EGFP expression. This effect was also observed when cells were seeded in different cell
densities and treated at the same time point; the higher the cell density, the lower the d2EGFP
expression was after 20 hours incubation with TNF-alpha. The underlying mechanism to this
phenomenon can be the production of soluble factors by the cells inhibiting the NF-kappaB
activation or the direct communication via gap junctions in the cell layer diminishing the
TNF-alpha response. The quantification of the transcripts of several possible NF-kappaB
target genes by quantitative PCR after treatment of cells with TNF-alpha revealed a strong
induction of IkappaB alpha and IL-6. In conclusion, the sequence of events from liberation of
NF-kappa B subunit p65 and its binding to DNA, the initiation of transcription and translation
in response to the NF-kappaB binding have been shown for the cell-based NF-kappaB assay.
The experiment conditions of this assay have to be very well controlled since cell density and
growth duration influence the NF-kappa B activation.
- 531 -

Session XIV : Transcriptional and translational control Poster XIV, 29
The transcription factor Nrf2 controls the expression of heme oxygenase-1 and Phase IIrelated
genes in cells exposed to aqueous extracts of cigarette smoke
Arnd Hengstermann1, Constanze Knörr-Wittmann1, Stephan Gebel1, Jawed Alam2,
Thomas Müller1.
1PHILIP MORRIS Research Laboratories GmbH, Cologne, Germany. 2Department of
Molecular Genetics, Alton Ochsner Clinic Foundation, New Orleans, LA, U.S.A. E-mail:
Arnd.Hengstermann@pmintl.com
Exposure of cells and tissues to cigarette smoke (CS) triggers a pronounced anti-oxidant
response, which is hallmarked by the transcriptional up-regulation of heme oxygenase-1
(hmox1), initiating a self-protection mechanism resulting in the formation of endogenous
antioxidant molecules, i.e., biliverdin and bilirubin from intracellular heme moieties. To
characterize the regulatory elements involved in CS-mediated hmox1 expression, we studied
the expression of various hmox1 promoter/luciferase reporter constructs in NIH3T3 cells
exposed to aqueous extracts of CS. The results showed that the CS-dependent expression of
hmox1 is governed primarily by the distal enhancers 1 and 2, both of which contain three
canonical anti-oxidant responsive element (ARE)-like stress responsive elements. These sites
are potentially addressed by the transcription-factor Nrf2, a principal inducer of antioxidant
and Phase II-related genes. As shown by Western-blot analysis, Nrf2 was strongly stabilized
and became detectable in nuclear extracts in cells exposed to aqueous extracts of CS.
Furthermore, nuclear localization of Nrf2 coincided with increased DNA binding of a putative
Nrf2/MafK heterodimer to ARE, as determined by EMSA. Notably, siRNA-mediated knockdown
of Nrf2 expression significantly compromised both CS-induced hmox1 promoter
activation and HO-1 expression. Finally, by using this approach CS-induced expression of
Phase II-related genes NAD(P)H:quinone oxidoreductase and glutamate-cysteine ligase,
catalytic subunit was shown to be completely abrogated, as determined by Real Time
quantitative PCR.
Taken together, these results add to the understanding of the central role of Nrf2 in the
context of CS-mediated oxidative stress by orchestrating an efficient transcriptional response
aimed at resolving the stressing conditions.
- 532 -

Session XIV : Transcriptional and translational control Poster XIV, 30
Expression and Regulation of various tumor suppressor genes in Gastrointestinal
Stromal Tumor
Keun Hur, Hyuk-Joon Lee, Han-Kwang Yang.
Cancer Research Institute Seoul National University College of Medicine 28 Yongon-
Dong, Chongno-Gu, Seoul, Korea 110-744. E-mail: blue2001@snu.ac.kr (alternate email:
longcoat@hanmail.net )
Gastrointestinal stromal tumors (GISTs) are Kit/CD117 expressing mesenchymal tumors.
Aberrant hypermethylation of promoter CpG islands is an important mechanism for the
inactivation of tumor suppressor genes. CpG island hypermethylation occurs in relation to
tumorigenesis in many human tumors. Moreover, we have previously reported that
transcriptional repression of Cox-2 is caused by hyper-methylation of the Cox-2 promoter
region CpG island in human gastric carcinoma (BBRC., 310(3), 844-851, 2003). The
pathogenesis of GISTs involves a gain-of-function mutation in the KIT proto-oncogene,
leading to ligand-independent constitutive activation of the KIT receptor. KIT-wild-type
GISTs have shown mutually exclusive platelet-derived growth factor receptor (PDGFR)
mutation and activation. However, the data on the methylation status of various tumor
suppressor genes and mutation status of KIT and PDGFR in GISTs has been very limited. We
attempted to determine the methylation status of 12 genes (APC, COX-2, GSTP1, MGMT,
p14, p16, RUNX-3, THBS-1, TIMP-3, DAP-kinase, E-cadherin, RASSF1A), in 30 human
gastrointestinal stromal tumor tissues. In addition, we confirmed the mutation status of KIT
and PDGFR genes in 30 human gastrointestinal stromal tumor tissues. Thirty c-kit-positive
Gastrointestinal stromal tumors were selected for this study. Genomic DNAs and RNAs from
human Gastrointestinal stromal tumors were analyzed for the relationship between expression
and methylation status by cDNA microarray and methylation specific PCR (MSP). CpG
island hyper-methylation was found in 40% for APC, 46% for COX-2, 16% for GSTP1, 26%
for MGMT, 60% for p14, 30% for p16, 16% for RUNX-3, 23% for THBS-1, 10% for TIMP-
3, 20% for DAP-kinase, 36% for E-cadherin, 10% for RASSF1A. Our results suggest that the
DNA methylation-mediated transcriptional silencing of various tumor suppressor genes is a
predominant mechanism for the down-regulation of various tumor suppressor genes
expression in human gastrointestinal stromal tumors.
- 533 -

Session XIV : Transcriptional and translational control Poster XIV, 31
Multiple isoforms of delta TA p73 transcripts accumulate in liver fluke related
cholangiocarcinoma
Patcharee Jearanaikoon1,5, Cheuypratoom P 1 Temduang Limpaiboon1,5 Banchob
Sripa2,5 Leelayuwat C,3,5,Vajarabhongsa Bhudhisawasdi4,5
1Department of Clinical Chemistry, Centre for Research and Development of Medical
Diagnostic Laboratories, Faculty of Associated Medical Sciences, 2Department of
clinical imunology , Faculty of Associated Medical Sciences , 3Pathology Department of
Pathology, 4Department of Surgery, 5Liver Fluke and Cholangiocarcinoma Research
Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
E-mail: patjea@kku.acth
Intrahepatic cholangiocarcinoma (CCA) is usually a fatal malignant neoplasm originating
from bile duct epithelium. It is the highest incidence cancer in Northeast Thailand which is
caused by liver fluke (Opisthorchis viverrrini) infection. No data describing the presence of
oncogenic delta transactivation p73 transcripts( &TAp73) which lacking N terminal in
correlation to the clinicopathological parameter has been reported in liver fluke related CCA.
We examined the p73 expression in 88 tumors using IHC staining with two monoclonal
antibodies specific to N and C terminal of TP73 in combination. Nearly to 80% (79/88cases)
reveal no p73 expression indicating down regulation during tumor development. About 15%
(13/88cases) of the IHC positive cases , demonstrate oncogenic( &TAp73) protein. To
improve the lower sensitivity for the oncogenic p73detection , Nested RT –PCR was firstly
established to investigate five TP73 (TAp73, p73 &ex2 , p73 &ex2/3, &Np73 and &N’p73)
in 23 frozen tumor specimen. The frequency of four p73 transcripts can be observed in wild
type TA p73, &Np73,p73 &ex2 and p73 &ex2/3 with 74%,74 %,26 %and 26% ,respectively.
The &Np73 oncogenic transcript is generated from the second promoter usage of p73 gene
whereas p73 &ex2 and p73 &ex2/3 created by the alternate splicing of the common promoter.
Our results demonstrate the accumulation of oncogenic isoforms simultaneously generating
from both p73 promoters in 4/23 cases . Although, no statistical significance has been shown
between the oncogenic transcript and patient survival. This might be depended on the reason
that most of patients were in advanced stage.
- 534 -

Session XIV : Transcriptional and translational control Poster XIV, 32
Aromatase expression was not detected by immunohistochemistry in endometrial cancer
Yong-Tark Jeon, Jae Weon Kim, Noh-Hyun Park, Soon-Beom Kang, Hyo-Pyo Lee,
Yong-Sang Song
Department of Obstetrics and Gynecology, Cancer Research Institute, College of
Medicine, Seoul National University, Seoul, Korea, E-mail: yssong@snu.ac.kr
Several studies suggested that aromatase could play an important role in tumor progression
and prognosis in endometrial cancer because androstenedione is converted to estrogen by the
enzyme. For better understanding of the aromatase expression in endometrial cancer and its
relation to diverse clinicopathological parameters, we conducted this study. This study was
carried out with 141 endometrial cancer patients, all of whom had undergone operations in
our institution from 1993 to 2002. Paraffin-embedded tissue blocks were sectioned and
immunostained with monoclonal anti-aromatase antibody using human placental tissue as
positive control. Clinicopathological variables of all patients were also reviewed. Despite of
quite high aromatase expression in positive control, there was no endometrial cancer
specimen showing the enzyme expression. Our result, although need further investigation on
the cause of the difference from other studies, suggested that aromatase might not have
important role in endometrial cancer.
- 535 -

Session XIV : Transcriptional and translational control Poster XIV, 33
NF-kB, AP-1 and mitogen-activated protein kinase in chemokine expression in
pancreatic Acinar cells.
Kyung Don Ju, Ji Hoon Yu, Kyung Hwan Kim and Hyeyoung Kim*
Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea,E-mail:clock1375@paran.com
Cholecystokine(CCK) analogue cerulein causes pathophysiological, morphological and
biochemical similarities to various aspects of human pancreatitis. Doses of CCK or cerulein
beyond those that cause the maximum pancreatic secretion of amylase and lipase results in
pancreatitis, which is characterized by a dysregulation of the digestive enzyme production and
cytoplasmic vacuolization and the death of acinar cells, edema formation, and an infiltration
of inflammatory cells into the pancreas. Present study aims to investigate whether cerulein
induced NF-kB and AP-1 activation, MAPK phosphorylation and the expression of MCP-1 in
pancreatic acinar cells, and whether these alterations were inhibited by transfection of I-kB
mutant, c-jun dominant negative and inhibitors of MAPK. As a result, cerulein induced NFkB,
AP-1 activation, MAPK phosphorylation and induction of chemokines(IL-8, MCP-1)
expression in the cells. Both transfection of I-kB mutant, c-jun dominant negative and
inhibitors of MAPK inhibited cerulein-induced chemokines in acinar cells. In conclusion,
cerulein activates NF-kB, AP-1 and MAPK resulting in upregulation of inflammatory MCP-1
expression in acinar cells. Inhibition of NF-kB, AP-1 and MAPK might alleviate the
inflammatory response in pancreatic acinar cells by suppressing chemokine expression.
- 536 -

Session XIV : Transcriptional and translational control Poster XIV, 34
NF!B and MAPK signalling in response to oxidative stress in skeletal myoblasts
Eirini Kefaloyianni, Catherine Gaitanaki and Isidoros Beis
Department of Animal and Human Physiology, School of Biology, Faculty of Sciences,
University of Athens, Panepistimioupolis, Athens 15784, Greece
e-mail: ibeis@biol.uoa.gr
Skeletal muscle cells are continuously subjected to oxidative stress during exercise due to an
increased production of reactive oxygen species (ROS). Oxidative stress has been also
associated with skeletal muscle atrophy and damage in many diseases, as well as, during
aging. However, skeletal muscle cells are capable of modifying their function in order to limit
ROS and protect themselves and the surrounding tissues. Main signalling pathways that are
activated during ROS accumulation are the MAPKs and NFkB ones, as it has been
demonstrated in various cell types. Nevertheless, the precise mechanisms involved in their
activation and/or function remain obscure. In this study, we examined whether NFkB
participates in the response of skeletal myoblasts to oxidative stress as exemplified by H2O2
and whether there is a cross talk with the MAPK signal transduction pathways. H2O2 induced
a mild translocation of the transcription factor to the nucleus, in a dose and time dependent
profile, as well as a moderate phosphorylation of its endogenous cytoplasmic inhibitor IkB (at
Ser32/36), without any significant decrease in IkB total levels. Moreover, oxidative stress
induced a strong phosphorylation of the p65 subunit of NFkB by a mechanism involving the
Src kinases and, at least in part, EGFR. The Src pathway also mediated the activation of
ERKs and JNKs by H2O2. Nevertheless, inhibition of these two MAPK pathways by
selective inhibitors did not appear to affect this H2O2-induced phosphorylation of p65. In
addition, a strong phosphorylation of p65 at Ser276 was also induced by H2O2. This
phosphorylation was found to be mediated by MSK1, a known substrate of ERKs and p38-
MAPK and to be Src-dependent. In conclusion, it seems that IkB degradation is crucial for
NFkB translocation to the nucleus and probably these two procedures are not related with the
activation of MAPKs. On the other hand, p65 phosphorylations, which are in part mediated
by MAPKs pathways, are probably related to signal specificity, possibly through diverse
interaction with other signalling factors.
This study was funded by G.S.R.T. (PENED 01ED5).
- 537 -

Session XIV : Transcriptional and translational control Poster XIV, 35
Finding crossroads of signal transduction pathways targeting promoters of “disease”
genes involved in pathological calcification.
Alexander Kel1, Nico Voss1, Olga Kel-Margoulis1, Evelin Katona3, Gerd Schmitz3 and
Edgar Wingender1,4
1 BIOBASE GmbH, Halchtersche Str. 33, D-38304 Wolfenbüttel, Germany; 3 Institute
for Clinical Chemistry and Laboratory Medicine University Hospital Regensburg
Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany; 4 Dept. Bioinformatics,
UKG/Univ. Göttingen, Goldschmidstr. 1, 37077 Göttigne, Germany
E-mail : alexander.kel@biobase-international.com
Different signal transduction pathways converge at key molecules that master the regulation
of certain cellular processes. Such crossroads of signal networks are often their “Achilles
Heels” causing a disease when not functioning properly. We have developed a computational
method for analysis microarray gene expression data and identification the key transcription
factors and “upstream” signaling molecules that might be the causes of the observed change
in gene expression. There are two steps of analysis (using TRANSFAC® and
TRANSPATH® databases): 1) CMFinder analyzes 5’-upstream regions of coexpressed
genes and applies a genetic algorithm to reveal composite modules (CMs) consisting of cooccurring
single TF binding sites and composite elements; 2) ArrayAnalyzer is a fast
network search engine that analyzes signal transduction networks controlling the activities of
the corresponding TFs and finds the key signaling molecules. The method was applied to
micrarray data on Pseudoxanthoma elasticum (PXE) (a disease linked to the monogenic
defects of ABCC6 transporter and characterized by alterations of elastic fibers and dystrophic
calcification). We provided evidence that the SOX9-related transcriptional pathway and the
IL-1#-linked signaling route are defective in PXE. We have identified PKAc kinase and
TRAF-6 as important key nodes in the signaling pathways that are pathologically altered.
They can be considered as prospective drug targets. We hypothesized that impaired activity of
the transporter may change a lipid balance in extracellular matrix resulting in activation of
certain signal transduction mechanism leading to the disease symptoms.
- 538 -

Session XIV : Transcriptional and translational control Poster XIV, 36
15-Deoxy-&12,14-prostaglandin J2-mediated upregulation of heme oxygenase-1 and
vascular endothelial growth factor in human breast cancer cells : a potential role of Nrf2
Eun-Hee Kim, Hye-Kyung Na and Young-Joon Surh
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
Elevated expression or activity of heme oxygenase-1 (HO-1), a ubiquitous stress-responsive
enzyme, has been reported to stimulate proliferation and to accelerate angiogenesis in several
types of tumor cells. 15-Deoxy-&12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand
of peroxisome proliferator-activated receptor $, has been known to induce HO-1 in some cell
lines. In the present work, we found that treatment of human breast cancer (MCF-7) cells
with nontoxic doses of 15d-PGJ2 led to concentration- and time-dependent increases in the
expression and activity of HO-1. The induction of HO-1 expression preceded the
upregulation of vascular endothelial growth factor (VEGF) in MCF-7 cells stimulated with
15d-PGJ2. The upregulation of VEGF production by 15d-PGJ2 was abrogated by the HO-1
inhibitor, ZnPP. Moreover, ZnPP decreased the in vitro capillary formation induced by 15d-
PGJ2 in human umbilical vein endothelial cells. Likewise, ZnPP treatment mitigated the
manifestation of migrative phenotype of 15d-PGJ2-treated MCF-7 cells as determined by the
wound migration assay. Nrf2, a basic-leucine zipper transcription factor, plays a key role in
regulating the antioxidant response element (ARE)-mediated expression of HO-1 and other
antioxidant enzymes. 15d-PGJ2 increased nuclear translocation and subsequent ARE binding
of Nrf2. MCF-7 cells transfected with dominant negative Nrf2 exhibited reduced expression
of HO-1 and VEGF in response to 15d-PGJ2 treatment. Taken together, these results suggest
that 15d-PGJ2-induced expression of HO-1 via the Nrf2 signaling is implicated in
angiogenesis in human breast cancer.
- 539 -

Session XIV : Transcriptional and translational control Poster XIV, 37
Inhibition of leptin receptor signaling by SOCS proteins
Holger Knobelspies, Julia Zeidler, Simone Bamberg-Lemper and Walter Becker
Department of Pharmacology, RWTH Aachen University, Wendlingweg 2, 52074
Aachen, Germany; E-mail: hknobelspies@ukaachen.de
The murine leptin receptor (LR) comprises three conserved intracellular tyrosine residues
(Y985, Y1077, Y1138) that are phosphorylated upon ligand binding. Phosphorylated (p)
Y1138 and pY1077 have been shown to form binding sites for the signal transducers and
activators of transcription (STAT)3 and STAT5. pY985 recruits suppressor of cytokine
signaling (SOCS)3, a negative feedback regulator of LR signal transduction. We used point
mutants of the LR, lacking either Y985 (designated FYY), Y1077 (YFY) or both tyrosines
(FFY) to elucidate interactions between the intracellular tyrosines of the LR and SOCS1 or
SOCS3. A pancreatic beta-cell line from rat (RINm5F) stably expressing the LR showed
desensitization during leptin stimulation of 2h or longer, as we assessed by Western blotting
using specific antibodies against pY-STAT3. Binding studies revealed that this effect was not
due to internalization of LR, since the surface expression was reduced by only 22±16%
(mean±S.D.;n=8) after 2h of stimulation. The time-course of leptin-induced pY-STAT3 levels
was similar for the wild-type (WT) LR and for the FYY mutant, approaching background
levels after 4h of stimulation. In contrast, the FFY double mutant showed a decrease by only
14%. This suggests the involvement of Y1077 and not only Y985 in the desensitization.
Reporter gene assays in HepG2 cells with a STAT3-responsive promoter showed that
overexpressed SOCS3 decreased the signaling by any YF mutant, but the receptors with
functional Y985 (WT, YFY) were stronger affected than the mutants lacking Y985 (decrease
to 6%, 59%, 4% and 23% for WT, FYY, YFY and FFY receptors, respectively). In SOCS3
knockdown experiments, the FFY mutant showed no effect, whereas the WT receptor
increased the reporter gene expression to 508 % and the Y985F to 278%. This supports our
hypothesis of SOCS3 being able to bind to Y985 or Y1077 and thereby inhibiting LR
signaling. Overexpression of SOCS1, which is not induced by leptin in RINm5F LR cells,
dose-dependently inhibited either receptor mutant. Again, the receptors with functional Y985
showed stronger effects (WT 14%, FYY: 55%, YFY: 14%, FFY: 60%). Knockdown of
SOCS1 showed an increase in signaling of WT and either single mutant (WT: 58%, FYY:
47%, YFY: 50%) whereas the knockdown had no effect on the FFY mutant. We conclude
hereof that SOCS1 inhibits LR signaling by interaction with Y985 or Y1077 in our system.
- 540 -

Session XIV : Transcriptional and translational control Poster XIV, 38
SOX3 expression is driven by multiple CCAAT control elements
Aleksandar Krstic, Milena Stevanovic
Laboratory for human molecular genetics, Institute of Molecular Genetics and Genetic
Engineering, Vojvode Stepe 444a, 11010 Belgrade, Serbia and Montenegro,
E-mail : hmgbox@eunet.yu
Early neurogenesis and neuronal differentiation are precisely controlled by a series of genes.
Sox3 is expressed in the brain from initial stages of development and it is considered to be
one of the earliest markers in vertebrates playing the role in specifying neuronal fate. In order
to elucidate molecular mechanisms underlying the regulation of the human SOX3 gene
expression, computer prediction software was used to search the matrix database and to
identify potential transcription binding sites within the human SOX3 promoter. Among the
number of putative consensus binding sites three evolutionary conserved CCAAT boxes,
representing the putative binding sites for the general transcription factor NF-Y, were
identified at positions –101 bp to –105 bp, -246 bp to -250 bp and -326 bp to -330 bp. EMSA
and “supershift” experiments, for fragments containing each of the identified CCAAT boxes,
were performed to prove the specificity of the NF-Y binding. To examine whether the
putative CCAAT boxes are functional, site directed mutagenesis was performed. The ability
of the single, double and triple site mutants and its wild-type counterpart to drive expression
of the cat reporter gene was examined in stem and RA induced NT2/D1 cells. We have
shown that mutagenesis of all three CCAAT boxes affects the expression of the reporter gene,
compared to the wild-type counterpart. Results obtained with double NF-Y site mutants
suggest functional/spatial interaction of these regulatory elements. Triple NF-Y site mutant
reduced reporter construct activity by almost 20 fold. All results strongly suggest -that all
three CCAAT box motifs present in the SOX3 promoter play functional role as transcriptional
activators of this gene.
- 541 -

Session XIV : Transcriptional and translational control Poster XIV, 39
Runx2 Transcription Factor Controls F promoter Activity of Human ER alpha Gene
in Osteoblasts
Elisabetta Lambertini, Elisa Tavanti, Letizia Penolazzi, Roberto Gambari and Roberta
Piva.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy. Email:
piv@unife.it
Bone growth, development and maintenance is a highly regulated process. The balance
between bone formation and resorption involves two cell types: bone forming osteoblasts
(OBs) and bone-resorbing osteoclasts (OCs). OBs-dependent bone formation varies with age,
metabolic disease states or with pharmacological intervention. OBs formation and maturation
are under control of a specific transcription factor, Runx2, that regulates the expression of
bone-specific genes. Another important transcription factor playing a central role in bone is
the estrogen receptor alpha (ER) that mediates estrogen-induced effects on gene expression.
Considering the potent gene regulatory activity of Runx2 and ER we investigated the role of
Runx2 on the regulation of ER expression valuating its interaction with F promoter of ER
gene. F promoter is one of the multiple promoters of human ER gene and is the only active
promoter in bone. In this promoter three Runx2 sites (A), (B), and (C) are present. To
understand if they are involved in influencing F promoter activity, different promoter-reporter
deletion and mutation constructs were assayed for function after transient transfection into the
Runx2-producing SaOS-2 human osteoblastic cells. The comparison of luciferase activities
allowed the identification of a negative role of a sequence context, within the F promoter
containing the three Runx2 binding sites. In addition, by using electrophoretic mobility shift
assay (EMSA) in combination with supershift we demonstrated that a significant supershifted
complex was formed only with the Runx2 (A) site. In order to demonstrate the
possible association of the Runx2 protein to the F promoter “ in vivo” chromatin
immunoprecipitation assay (ChIP) was performed. The results demonstrate that Runx2
interacts “ in vivo” with the region of the F promoter within the sequence context containing
the Runx2 binding sites in SaOS-2 cells indicating that Runx2 may be considered one of the
factors controlling specific expression of human ER alpha gene in osteoblasts.
- 542 -

Session XIV : Transcriptional and translational control Poster XIV, 40
Differential regulation of vascular endothelial growth factor expression by peroxisome
proliferator-activated receptors via adipocyte-fatty acid binding protein in bladder
cancer cells
Isabelle Lascombe, Guillaume Boiteux, Hugues Bittard and Sylvie Fauconnet
Université de Franche-Comté, EA 3181 “Carcinogenèse épithéliale”, IFR133, UFR
Médecine et Pharmacie, 19 rue Ambroise Paré, 25000 Besançon, France. E-mail :
isabelle.lascombe@voila.fr
Vascular endothelial growth factor (VEGF) plays a prominent role in vesical tumor
angiogenesis regulation (Lascombe et al., 2005, PBCR review). Peroxisome proliferatoractivated
receptors (PPAR), which are transcription factors, are involved in angiogenesis
process. Recently, we reported for the first time that, in two different human bladder cancer
cell lines RT4 (derived from grade I tumor) and T24 (derived from grade III tumor), VEGF is
differentially up-regulated by the three PPAR isotypes. Its expression is increased by
PPARalpha, # and in RT4 cells and only by PPAR# in T24 cells via a transcriptional
activation of the VEGF promoter through an indirect mechanism (Fauconnet et al., J Biol
Chem, 2002). Our purpose was to elucidate the molecular mechanism involved in PPARinduced
VEGF expression. We hypothesized that in high grade T24 cells, the inactivity of
PPARalpha and $ could be due to the absence of adipocyte-fatty acid binding protein (A-
FABP) expression or to the loss of the protein functionality. Indeed, loss of A-FABP is
associated with progression in bladder transitional cell carcinomas. Furthermore, recent
studies establish that FABP govern the transcriptional activities of their ligands by targeting
them to PPAR in the nucleus, thereby enabling PPAR to exert their biological functions. Our
first results suggest that A-FABP is present in cytoplasm of both RT4 and T24 cells but does
not locate to the nucleus of the T24 cells. Preliminary data obtained with siRNA targeting A-
FABP seem to confirm the role of this protein in PPAR-induced VEGF expression. These
data contribute to a better understanding of the mechanisms by which PPARs regulate VEGF
expression in non aggressive bladder tumors. This could lead to a new therapeutic approach
for human bladder cancer in which excessive angiogenesis is a negative prognostic factor.
- 543 -

Session XIV : Transcriptional and translational control Poster XIV, 41
Role of VEGF-D and AT2 receptor on cytokine expression in pancreatic acinar cells
JANGWON LEE, SHIN YOUNG YUN, JOO WEON LIM, KYUNG HWAN KIM, and
HYEYOUNG KIM
Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea. E-mail :honorjw@hotmail.com
Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the
digestive enzyme production, the death of acinar cells, and an infiltration of inflammatory
cells into the pancreas. Our previous proteomic analysis showed several differentially
expressed proteins in cerulein-treated pancreatic acinar cells, which are related to cellular
stress such as reactive oxygen species. We hypothesized that membrane proteins may be
altered as the early event during the induction of acute pancreatitis. The present study was
designed to elucidate firstly whether or not VEGF receptor and AT2 receptor be involved in
controlling cytokine expression. Secondly, the role of VEGF-D and AT2 receptor in
pancreatic acinar cells and their regulation by acute pancreatitis are also investigated. The
differentially expressed membrane proteins by cerulein will provide valuable information to
understand pathophysiologic mechanism of acute pancreatitis and may be useful for
prognostic indices of acute pancreatitis. AP-1 and NF-kB regulate cytokine gene expression
in pancreatic acinar cells treated with cerulein. AT2 receptor is associated with VEGF-D to
induce IL-6 expression in AR42J cells. Cerulein-induced VEGF-D expression and AP-1/NFkB
activation were inhibited in the cells transfected with VEGF-D antisense oligonucleotide,
indicating the involvement of VEGF-D in regulating the cytokine expression during acute
pancreatitis.
- 544 -

Session XIV : Transcriptional and translational control Poster XIV, 42
Nuclear loss of Ku DNA repair protein in cells exposed to oxidative stress
Jong Hwa Lee, Ji Yeon Song, Ji Hoon Yu, Kyung Hwan Kim, Hyeyoung Kim*
Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea. E-mail : pico798@naver.com
Recent studies show that NF-kB plays a critical role in protecting the cells from apoptotic
cell death. We previously demonstrated that oxidative stress – induced cell death is caused by
nuclear loss of DNA repair protein Ku proteins in pancreatic acinar AR42J cells. This study
aims to investigate the role of NF- kB on nuclear translocation of Ku proteins and apoptotic
cell death, induced by oxidative stress in AR42J cells. We examined Ku expression, cell
viability, and apoptosis of the cells treated with or without H2O2, which is continuously
generated by G/GO and in the caspase-3 inhibitor. Wild-type cells as well as the cells
transfected with Ku dominant negative gene or I-kB alpha mutant gene was used. As a result,
G/GO induced decrease in nuclear Ku70 and Ku80 time-and concentration-dependently.
G/GO induced alterations in Ku expression were inhibited, in part, by caspase-3 inhibitor.
G/GO induced apoptosis, which was in parallel with loss of nuclear Ku70 and Ku80 in the
cells transfected with the control pcDNA3 vector. G/GO increased apoptosis in those
transfected with Ku dominant negative mutant or I-kB alpha mutant gene. Nuclear level of Ku
proteins were decreased in the cells transfected with I-kB alpha mutant gene. Conclusively,
nuclear translocation of Ku proteins may be mediated by NF-kB in the cells. Decrease in
nuclear translocation of Ku proteins may result in apoptotic cell death in AR42J cells.
- 545 -

Session XIV : Transcriptional and translational control Poster XIV, 43
DNA hypomethylation of CAGE Promoters in Squamous Cell Carcinoma of Uterine
Cervix
Taek Sang Lee a,b, Jae Weon Kima,b,c,*, Gyeong Hoon Kangb,d Noh Hyun Parka,b,
Yong Sang Songa,b, Soon Beom Kanga,b, Hyo Pyo Leea,b
aDepartment of Obstetrics and Gynecology, bCancer Research Institute, and cHuman
Genome Research Institute, dDepartment of Pathology, College of Medicine, Seoul
National University, Seoul, 110-744, Korea
E-mail: kjwksh@snu.ac.kr
This study was performed to determine whether promoter hypomethylation of CAGE is
involved in cervical carcinogenesis. The surgical specimens from 40 cervical squamous cell
carcinoma patients who treated at Seoul National University Hospital and from 48 healthy
controls were used with informed consent. By methylation specific PCR (MSP) using
unmethylation specific primer, the promoter hypomethylation status of CAGE was
investigated. We found that hypomethylation of CAGE promoter was present at frequencies
of nearly one hundred percent in cervical squamous cell carcinomas (38/40, 95%), but less
than 4% in controls (p

Session XIV : Transcriptional and translational control Poster XIV, 44
Identification in rat of a yeast She3 homolog as interaction partner of AATF
Peter Leister, Sven Burgdorf, Andrea Felten, Lutz Uhlmann, and Karl Heinz
Scheidtmann
Institute of Genetics, University of Bonn
Roemerstr. 164, D-53117 Bonn
peterleister@uni-bonn.de
AATF, or Che-1, is a coactivator of several transcription factors including steroid hormone
receptors, E2F, and SP1. In search of novel interaction partners of AATF we identified a so
far unknown protein from rat with high degree of homology to She3p from budding yeast.
She3p is an adapter protein for myosinV type motor protein Myo4p and is involved in
selective transport of mRNA and ER vesicles. The novel protein was termed SHIA for She3
Homolog Interacting with AATF. A single transcript of SHIA mRNA of 1050 bp was highly
expressed in brain, heart, spleen, liver, testis and kidney and to a lower extent in lung,
whereas it was undetectable in skeletal muscle. However, sequence alignments with database
sequences revealed that human, mouse and rat orthologs exist in two or three isoforms
generated by alternative splicing or alternative usage of start codons, giving rize to
polypeptides of 142 and 99 residues, respectively. Strikingly, besides some variations at their
N-termini SHIA proteins are extremely conserved. A core sequence between residues 20 and
140 is strictly conserved within all vertebrates represented in the database, and certain
sequence motifs are found even in insects and C. elegans and yeast. This high degree of
conservation suggests that SHIA plays an essential role in all eukaryotes.
The rat SHIA clone isolated appears to contain an extra exon (termed exon 2b) resulting in a
protein of 168 amino acids with MW of 20 kDa. SHIA contains a She3p homology region at
its N-terminus and a leucine zipper at its C-terminus, which mediates interaction with AATF.
When expressed as GFP fusion protein, GFP-SHIA was localized in the cytoplasm,
preferentially nuclear near structures and the actin cytoskeleton. A small fraction of GFP-
SHIA was also observed within the nucleus. Overexpression of SHIA resulted in disturbed
actin fibers, and coexpression of SHIA and AATF resulted in partial relocalisation of AATF
from the nucleus to the cytoplasm. Interestingly, SHIA enhanced reporter gene activty in
androgen receptor based transactivation assays, thus, SHIA behaved like a transcriptional coactivator.
- 547 -

Session XIV : Transcriptional and translational control Poster XIV, 45
The MECP2 gene mutation screening in Rett syndrome patients from Croatia
Tanja Matijevi'1, Jelena Kne0evi'1, Ingeborg Bari)i'2, Vida 1uli'3 and Jasminka
Paveli'1.
1 Laboratory of Molecular Oncology, Division of Molecular Medicine, Rudjer Bo)kovi'
Institute, 10002 Zagreb, Croatia. E-mail : jpavelic@irb.hr
2 Department of Pediatrics, Children’s Hospital Zagreb, University of Zagreb, Medical
School, 10000 Zagreb, Croatia
3 Department of Medical Genetics, Pediatric Clinic, Clinical Hospital Split, 21000 Split,
Croatia
Rett syndrome is an X-linked dominant neurodevelopmental disorder almost exclusively
affecting females and is usually sporadic. Mutations in MECP2 gene have been found in more
than 80% of females with typical features of Rett syndrome. In this study, we analyzed 14
sporadic cases of Rett syndrome. In 6 of 14 patients (43%), we detected pathogenic
mutations in the coding parts of MECP2. We found one missense (T158M), two nonsense
(R168X, R270X), two frameshift mutations (P217fs and a double deletion of 28-bp at 1132-
1159 and 10-bp at 1167-1176) and one in-frame deletion (L383_E392del10). According to
our knowledge, the last two mutations have not been reported yet. We also detected one
previously described polymorphism (S194S). In conclusion, these results show that fourth
exon should be the first one analyzed because it harbors most of the known mutations.
Moreover, mutation-negative cases should be further analyzed for gross rearrangements. This
is the first study of this kind in Croatia and it enabled us to give the patients an early
confirmation of Rett syndrome diagnosis.
- 548 -

Session XIV : Transcriptional and translational control Poster XIV, 46
Identification of a prostaglandin-responsive element in the Na,K-ATPase beta1 subunit
promoter which is regulated by cAMP and Ca2+ : Evidence for an interactive role of
CREB and Sp1
Keikantse Matlhagela, Maryann Borsick, Trivikram Rajkhowa, and Mary Taub*
Biochemistry Dept, 140 Farber Hall, State University of New York at Buffalo, Buffalo,
NY 14214, U.S.A., Email: *biochtau@buffalo.edu
The Na,K-ATPase, is a transmembrane protein responsible for maintaining electrochemical
gradients across the plasma membrane in all mammalian cells, a process which is subject to
regulation at the transcriptional and post-transcriptional levels. Included amongst physiologic
regulators in the kidney are prostaglandins. Previously, we presented evidence that
transcription of the Na,K-ATPase ß1 subunit is stimulated by PGE1, an effect mediated
through both cAMP and Ca2+ pathways. Transient transfection studies using 5’ deletion
mutants in the human ß1 subunit promoter indicate that a region located between –92 to –100
containing the sequence AGTCCCTGC (a Prostaglandin Response Element, or PGRE) is
required for eliciting the stimulatory effects of PGE1, 8Br-cAMP, Phorbol 12-myristate 13acetate
(TPA) and okadaic acid. Electrophoretic Mobility Shift Assays (EMSAs) indicate that
both the cAMP Regulatory Element Binding Protein (CREB) and Sp1 bind to this PGRE. The
involvement of the PGRE and Sp1 sites in regulation by PGE1 was further confirmed by
studies with a heterologous, and mutant promoters. The PGE1 stimulation was reduced when
the 2 GC boxes adjacent to the PGRE were translocated further upstream from the PGRE.
Also consistent with an interaction between CREB and Sp1 are our immunoprecipitation and
GST-pull down studies. In summary our results indicate the PGE1 induction involves
interactions between Sp1 and CREB which occur during the binding of these transcription
factors to a novel regulatory element, a PGRE, and an adjacent Sp1 site on the Na,K-ATPase
ß1 subunit promoter.
- 549 -

Session XIV : Transcriptional and translational control Poster XIV, 47
Process simulation in a mechatronic bioreactor device with speed regulated motors for
growing of three-dimensional cell cultures
M. Mihailova1, V. Trenev2, P. Genova2, S. Konstantinov3
1IPF of TU-Sofia, 8800 Sliven, 59 Bourgasko chaussee Str, Bulgaria;
e-mail: mina-todorova@abv.bg
2CLMI, Bulgarian Academy of Sciences, 1 Acad. G. Bonchev Str, 1113 Sofia, Bulgaria;
e-mail: vtrenev@clmi.bas.bg; Pgen@TU-Sofia.bg
3Lab for Experimental Chemotherapy, Dept. of Pharmacology, Faculty of Pharmacy,
Medical University of Sofia, 1000 Sofia, 2 Dunav Str, Bulgaria;
e-mail: Konstantinov.spiromihaylov@gmail.com
Tissue engineering is a new scientific research field that allows the establishment of tissue
equivalents rising from isolated cells in combination with biocompatible materials and
cultivation in more or less sophisticated bioreactor systems. Such systems gave the unique
opportunity to perform in vitro investigations of transcription and translation, cell growth,
biochemistry and mechanics of healthy normal organs as well as those affected by malignant
tumours, infections and immune deficiency under controlled conditions. In rotating vessel
bioreactors under microgravity and defined medium content cells proliferate, stay abundant to
each other and form three-dimensional structures, assigned as spheroids. Such spheroids
might be grown on micro carriers. A wide spectrum of different cell culture experiments
involving normal and transformed human cells indicate that: in a rotating bioreactor system
miniPERM no complete lack of gravity could be reached; a great part of the seeded cell
material do not proliferate at the beginning and the appearance of bigger spheroids is rather
random. We describe the acquisition of spheroids in HD-MY-Z and Neuro-2A tumour cell
lines as well as in bone marrow fibroblasts from acute myeloid leukaemia patients. Spheroids
of 100 and more cells were obtained from HD-MY-Z and Neuro-2A cells. Interestingly,
chronic myeloid leukaemia LAMA-84 cells did not form any cell clumps and they kept a
completely undifferentiated phenotype despite their semi-adherent growth manner under
conventional conditions. A detailed theoretical and virtual simulation study of the influence of
every component of gravitation, inertia and hydrodynamic force fields was performed.
Thereby, a new concept for mechatronic bioreactor device with active electronic control was
developed and virtually tested.
- 550 -

Session XIV : Transcriptional and translational control Poster XIV, 48
Estrogen receptor-mediated transcriptional regulation may involve different cross-talk
interaction between estrogen receptor and xenobiotic nuclear receptors depending on
the estrogen target cell types
Gyesik Min
Department of Microbiological Engineering, Jinju National University, Jinju,
Gyeongsangnam-Do, 660-758 South Korea E-mail: g-min@jinju.ac.kr
The purpose of this study was to examine the effects of xenobiotic nuclear receptors, CAR,
SXR, and PPARgamma on the transcriptional activity of estrogen receptor in human breast
cancer cell lines and compare with those in human hepatoma cell line. Three different breast
cancer cell lines, MCF-7 with ER expression, MDA-MB-231 without ER expression, and
MCF-7K3 with estrogen-independent growth were cultured and effects of CAR, SXR, and
PPARgamma on the ER-mediated transcriptional activation of synthetic (4ERE)-tk-luciferase
reporter gene were analyzed. Consistent with the previous report, CAR significantly inhibited
ER-mediated transactivation and SXR repressed modestly whereas the PPARgamma did not
repress the ER-mediated transactivation in Hep G2 cells. However, in breast cancer cells
neither of the xenobiotic receptors repressed the ER-mediated transactivation. Instead, they
tend to increase the transactivation depending on the cell type and xenobiotic nuclear
receptors. In MCF-7, SXR but not CAR or PPARgamma slightly increased ER-mediated
transactivation whereas in MDA-MB-231, CAR and PPARgamma but not SXR tend to
increase the transactivation of the reporter gene. In addition, in MCF-7K3 only high dose of
CAR slightly increased estrogen dependent ER transactivation. These results indicate that the
effects of cross-talk in ER-mediated transactivation between estrogen and the xenobiotic
nuclear receptors, CAR, SXR, PPARgamma, are different in breast cancer cells from
hepatoma cells. Different responses to each xenobiotic receptor from the three breast cancer
cell lines may suggest diverse signal transduction pathways for the receptors, CAR, SXR,
PPARgamma, present in different breast cancer cell lines. In conclusion, the transcriptional
regulation by estrogen in ER-mediated transactivation can involve different cross-talk
interaction between estrogen receptor and xenobiotic nuclear receptors depending on the
estrogen target cell types.
- 551 -

Session XIV : Transcriptional and translational control Poster XIV, 49
Differential transactivation by constitutive androstane receptor of Phenobarbital
Responsive Unit(PBRU) of CYP2B gene between human hepatoma cell line Hep G2 and
monkey kidney epithelial derived cell line COS-7
Gyesik Min
Department of Microbiological Engineering, Jinju National University, Jinju,
Gyeongsangnam-Do, 660-758 South Korea E-mail: g-min@jinju.ac.kr
The objective of this study was to examine if transient transfection of CAR can transactivate
CYP2B1 PBRU reporter gene in COS-7 cells in which the endogenous CYP2B1 gene is not
induced by PB. In non-transfeced cells of both Hep G2 and COS-7, the endogenous
expression of CAR was not detected by antibody against CAR. When cultured cells were
transfected with CAR expression plasmid, mCAR1-GFP, both cell types expressed high
levels of CAR protein and could allow to examine the effect of CAR in PBRU transactivation.
Both cell types expressed endogenous RXR and transfection of RXR expression plasmid
dramatically increased its protein expression. Whereas CAR transactivated
PBRU2C1Luciferase about 12 fold as compared to 2C1Luciferase in Hep G2 cells, it did not
stimulate the luciferase activity of the PBRU reporter gene in COS-7 cells. These results
indicate that Hep G2 cells can respond to CAR differently from COS-7 cells, and suggest that
factors other than CAR and RXR may be required in inducing PBRU activation and the
expression of these factors may be different between liver and kidney.
- 552 -

Session XIV : Transcriptional and translational control Poster XIV, 50
Regulation of 2-Deoxy Glucose Transport, Lactate Metabolism and MMP-2 Secretion by
the Hypoxia Mimetic Cobalt Chloride in Articular Chondrocytes
Ali Mobasheri1 Nicola Platt1, Colin Thorpe1 and Mehdi Shakibaei2
1Faculty of Veterinary Science, University of Liverpool, Liverpool, L69 7ZJ, United
Kingdom; 2Musculoskeletal Research Group, Institute of Anatomy, Ludwig-
Maximilian-University Munich, Pettenkoferstrasse 11, 80336 Munich, Germany.
E-mail: a.mobasheri@liverpool.ac.uk, mehdi.shakibaei@med.uni-muenchen.de
Articular cartilage is avascular and this significantly reduces the availability of oxygen and
nutrients. Therefore, chondrocyte survival and cartilage homeostasis require effective
mechanisms for oxygen and nutrient signaling. To gain a better understanding of the
mechanisms responsible for oxygen and nutrient sensing in chondrocytes we investigated the
effects of hypoxic simulation induced by cobalt chloride treatment (a hypoxia mimetic) on
glucose uptake and lactate production in chondrocytes. We also studied the effects of cobalt
chloride and glucose deprivation on the expression and secretion of active MMP-2.
Primary cultures of articular chondrocytes were either maintained in 20% O2 (normoxia) or
exposed to the hypoxia mimetic cobalt chloride for up to 24 h at the following concentrations:
15 µM, 37.5, µM and 75 µM. Glucose transport was determined by measuring the net uptake
of non-metabolizable 2-deoxy-D-[2, 6-3H] glucose into chondrocytes. Expression of GLUT1
and GLUT3 was determined by western blotting and immunohistochemistry. Active MMP-2
secretion was assayed by gelatin zymography. Lactic acid production was assayed using a
lactate kit.
The hypoxia-responsive GLUT1 and GLUT3 proteins were expressed in chondrocytes.
Exposure to cobalt chloride significantly increased the uptake of 2-deoxy-D-[2, 6-3H] glucose
and the production of lactate. Glucose deprivation and cobalt chloride treatment increased
levels of active MMP-2 in the culture medium. Our results suggest that these metabolic
alterations are important events during adaptation to hypoxia. Upregulation of MMP-2 and
the build-up of lactic acid will have detrimental effects on the extracellular matrix and may
contribute to the pathogenesis and progression of osteoarthritis.
- 553 -

Session XIV : Transcriptional and translational control Poster XIV, 51
Microtubule interfering agents affect signaling pathways involved in the regulation of
cytochrome P450 genes expression.
Martin Modriansk., Jitka Ulrichová, and Zden2k Dvo-ák
Institute of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacky
University, Hnevotinska 3, 775 15 Olomouc, Czech Republic. Email:
oregon@tunw.upol.cz
Microtubule interfering agents, such as vincristine, vinblastine, taxol (paclitaxel) etc., are used
in human pharmacotherapy as anti-neoplastic drugs. Use of colchicine, a substance displaying
the same microtubule disrupting properties, is limited to the treatment of acute gout attacks
and familial mediterranean fever. Besides the common feature of tubulin binding these
substances share metabolism by cytochromes P450 (CYP). This is the basis for investigations
into potential drug-drug interactions on the level of CYP genes expression. All of these
microtubule interfering agents restrict the expression of CYP genes regulated by
glucocorticoid (GR) and aryl hydrocarbon (AhR) receptors. While this activity may be
associated with cell cycle phase, we show that it is independent of the cell cycle status.
Therefore microtubule integrity is important for GR and AhR transcriptional activity.
ACKNOWLEDGEMENT
This research is supported by grant MSM 6198959216 from the Ministry of Education, Youth
and Sports of the Czech Republic and grant GACR 303/04/P074 from the Grant Agency of
the Czech Republic.
- 554 -

Session XIV : Transcriptional and translational control Poster XIV, 52
Immunomorphological analysis of RAGE-receptor expression and NFkB-activation in
tissue samples from normal and degenerated intervertebral discs of various ages
A. Nerlich (1), B. Bachmeier (2), E. Schleicher (3), H. Rohrbach (1), G. Paesold (4), N.
Boos (4)
Dept. of Pathology, Academic Hospital Munich-Bogenhausen, Germany,
andreas.nerlich@extern.lrz-muenchen.de
Dept. of Clinical Chemistry Clin. Biochemistry, Univ. Munich, Germany
Dept. Pathobiochemistry, Univ. Tübingen, Germany
Spinal Surgery Unit, Orthopaedic Clinic, Univ. Zürich, Switzerland
Recent studies provided circumstantial evidence that the activation of the RAGE-receptor by
the oxidation-mediated amino acid modification carboxymethyl-lysine (CML) leads to NFkBactivation
and translocation into the nucleus. This leads to the up-regulation of the synthesis
of extracellular matrix molecules along with matrix degrading enzymes. In consequence, the
structure and function of tissue changes. Recent extensive analyses provided good evidence
that the age-associated degeneration of the intervertebral disc is closely associated with both
the alteration of the extracellular disc matrix and the accumulation of oxidation-mediated
CML modification of proteins. Accordingly, pervious own studies have shown significantly
enhanced levels of CML associated with disc degeneration. We therefore investigated the
pattern of RAGE-expression and NFkB-translocation into the nucleus.
For this study, 43 complete cross-sections of complete human lumbar intervertebral discs had
been prepared for the immunohistochemical localization of the RAGE-receptor and NFkBexpression
(both antibodies: Santa Cruz Biotech., CA, USA). The samples covered an age
between fetal (35th weeks of gestation) to senile age (85 years) with well defined macro- and
micromorphological features. The amount of positively labelled cells (RAGE) or nuclei
(activated NFkB) were evaluated morphometrically by light microscopy in the inner and outer
anterior and posterior annulus fibrosus and nucleus pulposus.
No significant expression of RAGE and no obvious activation of NFkB (by translocation into
the nucleus) were seen in fetal, juvenile and young adolescent discs (until age 13). In senile
discs (age 77 – 85 years) RAGE-expression remained elevated, but NFkB activation was
absent. In between, variably high numbers of labelled cells/ nuclei were seen with highest
values ranging between 25 – c. 50% of cells in the nucleus pulposus for RAGE, and 15 – 60
% of nuclei for NFkB with significant correlation between both parameters. In the anterior
and posterior annulus significantly lower values were seen again for both parameters with
more pronounced levels in the inner than the outer annulus.
This study is the first that described activation of the NFkB system in human klumbar
intervertebral discs in vivo. The close correlation between the expression of RAGE and
NFkB-activation suggests that the stimulation of RAGE (e.g. by CML) may induce
transcription factor activation in this system. The association between expression rates for
both parameters and increasing age indicates a potential link between transcription activation
and disc degeneration. This may represent an important mechanism inducing changes in both
cellular and extracellular structures of the disc, potentially prone to therapeutic intervention.
- 555 -

Session XIV : Transcriptional and translational control Poster XIV, 53
The nuclear scaffold protein NIPP1 directly binds to EZH2 and is essential for the
trimethylation of Histone H3 on Lysine 27
Mieke Nuytten1, Nivedita Roy1, Aleyde Van Eynde1, Lijs Beke1, Monique Buellens1,
Gerald Thiel2, and Mathieu Bollen1
1Division of Biochemistry, Department of Molecular Cell Biology, Faculty of Medecine,
KULeuven, B-3000 Leuven, Belgium and 2Department of Medical Biochemistry and
Molecular Biology, University of Saarland Medical Center, Homburg, Germany.
NIPP1 is a nuclear protein that is ubiquitously expressed in metazoans and plants but not in
yeast. The knockout of NIPP1 in mice is embryonic lethal before gastrulation and NIPP1-/-
cell lines are not viable. NIPP1 binds to protein Ser/Thr kinase MELK and protein Ser/Thr
phosphatase-1 but also interacts with nucleic acids and the essential pre-mRNA splicing
factors CDC5L and SAP155.
EZH2 is part of the Polycomb Repressive complex 2 (PRC2), which also contains EED and
SUZ12 as core elements. PRC2 is involved in the initiation of gene silencing. EZH2 is a
methyltransferase that methylates Lys27 of Histone H3 (H3K27). Trimethylated H3K27
serves as a docking site for the chromodomain-containing Polycomb protein, a component of
PRC1. The recruitment of PRC1 maintains gene silencing by mechanisms that are not yet
completely understood.
We show that NIPP1 is associated with H3K27-trimethylated chromatin and interacts directly
with both EED and EZH2. Moreover, NIPP1 acts as a transcriptional repressor of a reporter
gene and this activity requires both EED and catalytically active EZH2. NIPP1-/-mouse
blastocyst outgrowths and cultured cells with a siRNA-induced knockdown of NIPP1 are
severely deficient in trimethylation of histone H3 on Lys27 but are normally trimethylated on
Lys9. Our data show that the spliceosomal protein NIPP1 is required for trimethylation of
histone H3 by EZH2, possibly by its ability to recruit PRC2 to its target loci.
- 556 -

Session XIV : Transcriptional and translational control Poster XIV, 54
A dynamic equilibrium of IkB metabolism confers NF-kB responsiveness to UV
irradiation
Ellen O’Dea and Alexander Hoffmann
Signaling Systems Laboratory, Department of Chemistry and Biochemistry, University
of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0375, USA
Email: eodea@ucsd.edu; ahoffmann@ucsd.edu
The transcription factor NF-kB (nuclear factor-kappaB) is activated by a variety of stimuli,
the majority of which signal to the IKK (IkappaB kinase) complex that triggers stimulusresponsive
degradation of IkB proteins. Ultraviolet (UV) irradiation activates NF-kB through
a distinctly different mechanism. Previous work reveals that UV-induced NF-kB activation
involves translational inhibition through an ER-stress mediated pathway that induces
phosphorylation of the eukaryotic initiation factor eIF2a.
Even a modest suppression of protein synthesis may result in a disruption of the homeostasis
of the cell and its signaling components. In the context of the NF-kB signaling module, we
utilize a mathematical model to guide our experimental studies of the mechanisms that allow
translational inhibition to result in NF-kB activation. Interestingly, the protein half-lives of
IkB-bound to NF-kB and of the free form are regulated by distinct degradation pathways. We
find that even in unstimulated cells, bound IkB is degraded by the well-characterized IKKmediated
mechanism that is dependent on constitutive IKK activity. Indeed, computational
simulations and experimental results demonstrate that genetic alterations in constitutive IKK
activity or in IkB-responsiveness to IKK alter UV inducibility of NF-kB. In contrast, free
IkB exists as a small pool in the cell that undergoes rapid degradation by a poorly understood
IKK-independent mechanism. Similarly integrated computational/experimental studies reveal
that short half-life control of free IkB is critical to UV responsiveness. Finally, we present
evidence that cells are capable of altering this dynamic equilibrium of rapid IkB degradation
balanced by constitutive synthesis thereby either attenuating or enhancing UV-induced NF-kB
activation. Potential signaling crosstalk mechanisms will be discussed.
- 557 -

Session XIV : Transcriptional and translational control Poster XIV, 55
Identification and characterization of signaling pathways to NF-kB in lymphocytes
Andrea Oeckinghaus1, Elmar Wegener1, Alexander Beck2, Claus Scheidereit1, Daniel
Krappmann1,3
1 Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin,
Germany
2 University Hospital Tübingen, Internal Medicine IV, Otfried-Müller-Str. 10, 72076
Tübingen
3 GSF - Research Center for Environment and Health, Institute of Toxicology,
Ingolstädter
Landstr.1, 85764 Neuherberg, Germany
Activation, differentiation and clonal expansion of T-lymphocytes are initiated upon the
concerted action of the T-cell receptor (TCR) and the CD28 co-stimulatory receptor.
TCR/CD28 engagement initiates canonical NF-kB signaling, which involves IKK (IkB
kinase) dependent phosphorylation and degradation of inhibitors of NF-kB (IkBs) enabling
nuclear translocation of NF-kB. Genetic ablation in mice has identified PKCq, Carma1, Malt1
and Bcl10 as essential mediators of antigen receptor induced IKK activation. Formation of a
Carma1/Bcl10/Malt1 (CBM) complex represents a crucial step. But the composition of the
regulatory CBM complex, that forms after T cell stimulation in vivo, and the mechanism of
signal transduction from the TCR to the IKK complex are not understood to date.
Using gel filtration analysis, GST pull down experiments, tandem affinity purifications and
antibody immunoprecipitations of signaling mediators, we have analyzed the composition and
the dynamics of the cellular CBM complex. We find that Bcl10-Malt1 associates with IKK#
in unstimulated T cells and is rapidly recruited to Carma1 in response to TCR signaling.
Sustained TCR/CD28 signaling leads to a disengagement of the CBM complex and
abrogation of downstream signaling. Further, we find that antigen induced phosphorylation of
Bcl10 requires the putative downstream kinase IKKb. Mapping and functional studies of
phospho-acceptor sites on Bcl10 reveal, that IKKb triggered Bcl10 phosphorylation serves to
balance TCR induced lymphocyte activation. Our studies indicate that the formation of large
signaling clusters upon T cell activation initiates a network of protein interactions and
modifications and thereby facilitates a non-linear order of signaling to IKK/NF-kB.
- 558 -

Session XIV : Transcriptional and translational control Poster XIV, 56
Characterization and functional analysis of human SOX18 promoter
Isidora Petrovic, Milena Stevanovic.
Laboratory for human molecular genetics, Institute of Molecular Genetics and Genetic
Engineering, Vojvode Stepe 444a, 11010 Belgrade, Serbia and Montenegro,
E-mail: hmgbox@eunet.yu
Sox/SOX constitute a large family of genes encoding transcription factors, involved in the
decision of cell fates during development and implicated in the control of diverse
development processes. Human SOX18 gene is expressed in fetal brain as well as in a wide
range of fetal and adult tissues indicating its function is not restricted to early development.
Now it is clear that human SOX18 gene is involved in vascular cell growth and suggest that
this transcription factor may play role in atherosclerosis. The aim of our study has been to
determine and characterize the promoter of the human SOX18 gene and to elucidate
molecular mechanisms underlying the regulation of its expression. We have isolated and
performed the first characterization of human SOX18 promoter. We have identified the
transcription start point and carried out the structural and functional analysis of the regulatory
region responsible for SOX18 expression in HeLa cell line. Using promoter-reporter
constructs, we have determined the minimal SOX18 promoter region that shows basal
promoter activity. Further, we have investigated functional properties of GC-rich motif within
the core promoter sequence. In silico analysis has shown that this motif has several putative
binding sites for transcription factor Sp1 (specificity protein 1). SP1 is characteristic
transcription factor that regulates gene expression within TATA-less promoters, and SOX18
promoter has no TATA box or any other conserved element commonly present in TATA-less
promoter. Our experiments confirmed binding of Sp1 transcription factor to proximal GC-rich
motif. Finally, functional analysis revealed that over expression of Sp1 transcription factor in
HeLa cell line positively regulates the activity of SOX18 promoter.
- 559 -

Session XIV : Transcriptional and translational control Poster XIV, 57
Expression of the E2F family of transcription factors and its clinical relevance in a test
set of 78 ovarian cancer patients.
Daniel Reimer, Susann Sadr, Annemarie Wiedemair, Nicole Concin, Gerda Hofstetter,
Christian Marth and Alain G. Zeimet
Department of Obstetrics and Gynaecology, Medical University Innsbruck, Austria
Email: daniel.reimer@uibk.ac.at
The E2F family of transcription factors, firstly discovered as a cellular activity that could bind
to and activate the adenoviral E2 gene promoter, plays a pivotal role in the regulation of
cellular proliferation by controlling the expression of genes required for both cell cycle
progression and apoptosis. Based upon sequence homology and function, eight distinct
members of E2F transcription factors have been distinguished until to date. E2F family is
roughly subdivided into proliferation-promoting (E2F1, E2F2, E2F3a) and –inhibiting
(E2F3b, E2F4, E2F5, E2F6) transcription factors. E2F7 and the recently described E2F8,
represent a distinct subgroup through their unique structure predominantly inhibiting E2Fdriven
gene promoters. Whereas, E2F family members generally require binding to its
dimerization partner DP for transcriptional activity, E2F7 and E2F8 lack the dimerization
domain.
The regulation of E2F transcription factors is closely associated with the function of the
retinoblastoma family of tumour suppressors, the retinoblastoma binding protein (pRB), p107
and p130. In the last decade various alterations of distinct components of the pRB-E2F
pathway were found to be associated with tumour progression through increase of
proliferation or inhibition of apoptosis. However, no data on the role of E2F family members
are available in tumour biology of ovarian cancer. Here we describe an expression study of
E2F transcription factors in various human ovarian cancer cell lines and its clinical relevance
was examined in a training set of 78 ovarian cancer patients.
Expression levels of E2F1, E2F2 and E2F8 were elevated in all the ovarian cancer cell lines
studied when compared with peritoneal mesothelial cells (PMC) and ovarian surface
epithelial cells (OSE). Interestingly, interferon-gamma treatment showed a time-dependent
reduction of the activating transcription factors E2F1 and E2F2 in HTB-77 cells, indicating
that the antiproliferative effect of interferon-gamma is mediated at least in part through downregulation
of proliferation-promoting E2F members. High expression of E2F1, E2F2 and
E2F8 was found to be associated with poor overall survival and large residual disease after
initial debulking surgery in ovarian cancer patients, whereas high expression levels of E2F4, a
repressive transcription factor, was associated with favourable overall survival.
Taken together, these data suggest that E2F transcription factors, mainly E2F1, E2F2, E2F8
and E2F4, play a pivotal role in tumour biology of ovarian cancer and may be candidates for
specific therapeutic targets.
- 560 -

Session XIV : Transcriptional and translational control Poster XIV, 58
Identification and Functional Analysis of genes related to the malignant transformation
induced by the Polyomavirus Middle T oncoprotein
Leonardo de O. Rodrigues.; Fernanda Festa.; Sheila Maria B. Winnischofer; Karin
Krogh; Christian Colin; Mari Cleide Sogayar
University of São Paulo, Chemistry Institute, Biochemistry Department, CP26077, São
Paulo, SP, Brazil, E-mail: leonardo@iq.usp.br
Polyomavirus (Py), a virus associated with the development of animal tumors, has been
widely used as a model for studies of malignant transformation, cell signaling pathways and
cell proliferation control. Polyomavirus Middle T antigen (MT) plays a central role in cell
transformation by binding to and activating several cytoplasmic proteins, which participate in
the transduction of growth factor-induced mitogenic signals to the nucleus. To uncover the
molecular basis of cell transformation induced by the MT antigen we used three different
approaches. Firstly, commercially available cDNA arrays were hybridized with cDNA probes
synthesized from mRNA extracted from Balb/c 3T3 cell lines transformed with a retroviral
vector containing the wild type MT-antigen (MTWT) or with the empty vector (PLJ). In the
second approach, cDNA libraries for MT-induced or repressed genes were constructed using
the RDA (Representational Difference Analysis) cDNA subtraction methodology, followed
by immobilization onto nylon membranes to generate DNA macroarrays, which were
screened for induced or repressed genes. The third approach involved a commercially
available gene array (CodeLink bioarrays) consisting of 10,000 mouse genes. Upon
identification of differentially expressed cDNA candidates, their differential expression was
confirmed by quantitative Real-Time PCR analysis. After confirmation of their differential
expression, two genes displaying a complete inhibition upon MT overexpression were
selected for further functional analysis. Isolation and characterization of Py-MT-regulated
genes may contribute to better understanding of the molecular mechanisms underlying not
only malignant transformation, but also the cell cycle control. Support: FAPESP, CNPq,
FINEP and PRP-USP
- 561 -

Session XIV : Transcriptional and translational control Poster XIV, 59
Endoglin modify wound healing related dermal fibroblasts properties
Alicia Rodríguez-Barbero, Miguel Pericacho, Marta Prieto, José M. López-Novoa
Instituto Reina Sofía de Investigación Nefrológica. Departamento de Fisiología y
Farmacología. Universidad de Salamanca. Campus Unamuno, Edificio Departamental.
Avda. Campo Charro s/n. 37007. Salamanca. Spain. E-mail: barberoa@usal.es
Endoglin is a transmembrane glycoprotein involved in pathophysiological processes such a
vascular remodeling, angiogenesis and fibrosis, but its contribution in wound healing has not
been study. Endoglin is an accessory receptor of the transforming growth factor beta (TGF-#).
As TGF-# is a cytokine involve in wound healing and endoglin modulate some cellular
responses to TGF-#, we hypothesize that endoglin could modulate processes involved in
wound healing. The aim of the present study was to evaluate the role of endoglin on cellular
properties implicated in wound healing such as cell proliferation, migration and extracellular
matrix synthesis. We generated an in vitro model of primary cultured dermal fibroblasts from
Eng+/- C57BL/6 and wild type mice. Western blot analysis revealed that dermal fibroblasts
expressed endoglin and that its expression is reduced by 50% in Eng+/- fibroblasts compared
to control cells. The rate of cell growth was faster in Eng+/- than in control fibroblasts. Cell
motility was measured by a multi-channel wounding system such that repair into the denuded
area was due entirely to cell migration and by using the Boyden camera. Migration was higher
in Eng+/- fibroblasts than in control cells. In addition, fibronectin synthesis, assessed by
Western blot, was higher in Eng+/- fibroblasts than in control cells. These data reveal the
importance of endoglin in modulating proliferation, migration, and extracellular matrix
synthesis in dermal fibroblasts.
- 562 -

Session XIV : Transcriptional and translational control Poster XIV, 60
Isolation and characterization of the rat SND p102 gene promoter region
Lorena Rodríguez, Nerea Bartolomé, Itsaso García-Arcos, Begoña Ochoa, María J.
Martínez
Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque
Country, P. Box 669, Bilbao, Bizkaia, Spain.
ofbronol@lg.ehu.es
In this work we describe the isolation and characterization of the promoter region of the rat
endoplasmic reticulum cholesteryl ester hydrolase gene, named as SND p102 in GenBank
according to the structural properties and molecular weight of the protein. A region of 1900
bases upstream the ATG start codon has been isolated by a double PCR-based method. The
transcription initiation site has been localised 216 bases upstream the ATG codon by RLM-
RACE. Bioinformatic analysis of the isolated sequence has revealed the presence of putative
binding sites for many transcription factors implicated in both basal and regulated processes,
although no TATA box has been found. Electrophoretic mobility shift and supershift assays
(EMSA) using nuclear extracts from HepG2 cells have demonstrated the specific binding of
the nuclear factor-Y (NF-Y), a heterotrimeric transcriptional activator which binds to CCAAT
boxes, to regions [-257, -253], [-290, -286] and [-370, -366] of the promoter sequence. The
absence of TATA box and the situation of the binding sites for NF-Y suggest a role for this
transcription factor in the transcriptional regulation of the rat SND p102 gene.
This work was supported by DGICYT (BMC2001-0067).
- 563 -

Session XIV : Transcriptional and translational control Poster XIV, 61
Synergy of p38 MAPK and Stat1 in Transcription
Iwona Sadzak, Barbara Schaljo and Pavel Kovarik
Max F.Perutz Laboratories, Department of Microbiology and Immunobiology,
University of Vienna, A-1030 Vienna, Austria
Both interferon (IFN) and stress signals are required for full activation of immune responses.
We and others have shown that the stress-activated p38 MAPK increases transcription by
Stat1, the key transcription factor of interferon signaling. Thus, the two signals synergistically
increase transcriptional responses under conditions of inflammation. We show here that
synergy of p38 MAPK with Stat1 generates unique gene expression patterns that are specific
for inflammatory conditions and allow identification of novel interferon-regulated genes. The
molecular basis for the synergy is unknown, but it does not depend on phosphorylation of
Stat1 by p38 MAPK.
We propose a working model for the synergy and describe the proteomic approach that we
employ to unravel the molecular mechanisms. We focus on the analysis of Stat1-containing
transcriptional complexes and p38-mediated modifications of their composition and/or
activity. We show that the amount of Stat1 protein on the promoters of target genes does not
change upon synchronous activation of IFNg and p38 MAPK pathway.Further we observe
that the association of CBP/p300 with Stat1 is unaffected by p38 MAPK activation.
- 564 -

Session XIV : Transcriptional and translational control Poster XIV, 62
JUNB-DEPENDENT VEGF TRANSCRIPTION AND TUMOR ANGIOGENESIS
Dirk Schmidt1, Oliver Pein1, Björn Textor1, Alexander Licht1, Norbert E. Fusenig2,
Peter Angel1 and Marina Schorpp-Kistner1
1,2Deutsches Krebsforschungszentrum (DKFZ), 1Division of Signal Transduction and
Growth Control and 2Division of Differentiation and Carcinogenesis, Im Neuenheimer
Feld 280, D-69120 Heidelberg, Germany. E-mail: marina.schorpp@dkfz.de
The JunB subunit of the AP-1 transcription factor complex mediates gene regulation in
response to a plethora of extracellular stimuli. Previously, we showed that the complete
ablation of JunB resulted in an early embryonic lethal phenotype due to vascular defects. In
order to elucidate the underlying molecular mechanisms we analyzed various JunB-deficient
cell systems and targeted deletion of the junB gene to endothelial cells. Endothelial-cell
specific ablation of junB resulted in a similar embryonic lethal phenotype and confirmed the
absolute requirement of JunB for vasculogenic and angiogenic processes in the developing
embryo.
Analysis of JunB-deficient embryonic stem cells, endothelioma cells and fibroblasts revealed
that JunB itself is upregulated in response to hypoxia. This induction is independent of HIF
and MAPK signaling but requires NF-kB, as junB mRNA induction is lost in fibroblasts with
suppressed NF-kB activity. JunB, in turn, is directly required for basal and hypoxia-induced
transcription of VEGF as demonstrated by independent experimental approaches such as
QRT-PCR, EMSA and coexpression studies. VEGF regulation by JunB is also of
physiological relevance in tumor angiogenesis, as teratocarcinomas derived from junB-/- ES
cells exhibit a pale and growth retarded phenotype with significantly reduced amounts of
midsize and large blood vessels. The failure of host-derived vessels to recolonise the tumor
tissue correlates with a reduced capacity of JunB null tumor cells to release the angiogenic
factor VEGF due to the absence of JunB. Yet, other typically hypoxia-induced genes as such
of the glycolytic pathway are not governed by JunB. Consistently, JunB is dispensable for the
early maximal induction of VEGF in response to hypoglycemia which requires the JunB
sibling c-Jun.
- 565 -

Session XIV : Transcriptional and translational control Poster XIV, 63
IKKalphalpha controls NF-kappaB at the skp2 gene promoter to regulate G1 to S phase
progression in pancreatic cancer cells
Günter Schneider, Dieter Saur, Jens T. Siveke, Ralph Fritsch and Roland M. Schmid
II. Department of Internal Medicine, Technical University of Munich, Ismaningerstr.
22, 81675 Munich, Germany. E-mail: guenter.schneider@lrz.tum.de
The IkappaB inducing kinase (IKK) consists of the catalytical subunits IKKalphalpha and
IKK#eta and the regulatory subunit IKK amma. IKK regulated signaling pathways are
believed to promote proliferation of normal cells and aberrant proliferation of cancer cells.
The molecular mechanisms linking IKK components to the cell cycle machinery are not
entirely understood. To study the functions of the catalytical subunits of the IKK complex we
used pancreatic cancer cells, because of their known constitutive IKK activity. We show that
the G1 Phase of the cell cycle is selectively regulated by the IKKalphalpha subunit.
IKKalphalpha regulates protein stability of the cyclin-dependent kinase inhibitor p27Kip1.
Increased levels of p27Kip1 after the transfection of IKKalphalpha specific siRNAs are due to
the downregulation of the F-box protein S-phase-kinase associated protein 2 (skp2). We
further demonstrate, that IKKalphalpha signaling regulates transcription of the skp2 gene by
controlling a NF-kappaB complex at the skp2 gene promoter. Together, this work defines a
novel IKKalphalpha regulated growth pathway important for pancreatic cancer cell biology.
- 566 -

Session XIV : Transcriptional and translational control Poster XIV, 64
The cAMP Responsive Unit of the human Insulin-like Growth Factor Binding Protein-1
(hIGFBP-1) gene promoter comprises a functional Insulin Response Element
Ghislaine Schweizer-Groyer 1, Guillaume Fallot 3, Françoise Cadepond 1 and André
Groyer 2
1 Inserm U.488, Lab hormones, 94276, Le Kremlin-Bicêtre Cédex, France ; 2 Inserm
U.683 and 3 Inserm U.481, Faculté de Médecine Xavier Bichat, 75870, Paris Cédex 18,
France. E-mail: groyer@bichat.inserm.fr
Insulin-like Growth Factor-Binding Protein-1 (IGFBP-1) is one of the genes involved in
glucose homeostasis. In vivo, its level is increased by glucocorticoids and glucagon (via
cAMP) and decreased by insulin, these variations being primarily correlated with IGFBP-1
gene transcription. A functional Insulin Response Element (IRE), localised immediately 5’ to
the Glucocorticoid Response Element (GRE), has already been shown to mediate insulin
inhibition of basal and glucocorticoid-induced stimulation of IGFBP-1 promoter activity.
In this work, using the human hepatoma HepG2 cells as a model system and transient
transfection, we showed : 1) that both basal and cAMP-induced hIGFBP-1 promoter (nt-1 to -
341) activity are inhibited by insulin; 2) that Forkhead Box class O (Foxo) transcription
factors enhance constitutive hIGFBP-1 promoter activity without interfering with stimulatory
effect of cAMP; 3) that PI-3’ kinase signaling is involved in the inhibition of both constitutive
and cAMP-induced promoter activity by insulin; 4) that the Foxos mediate the inhibitory
effect of insulin on transcription from the whole IGFBP-1 promoter; 5) that the cAMP
Responsive Unit (CRU) composed of a cAMP Responsive Element (CRE) and of a putative
IRE, is functional in mediating both cAMP stimulation and insulin inhibition of a
heterologous promoter and 6) that the inhibitory effects of insulin on the isolated CRU are
mediated by the Foxos.
- 567 -

Session XIV : Transcriptional and translational control Poster XIV, 65
Role of Proteinase-Activated Receptor-2 on Cyclooxygenase-2 induction in H. pylori -
Infected Gastric Epithelial Cells.
Ji Hye Seo, Joo Weon Lim, Kyung Hwan Kim and Hyeyoung Kim*
Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei
University College of Medicine, Seoul 120-752, Korea. E-mail:jhseo@yumc.yonsei.ac.kr
Proteinase-activated receptor-2(PAR2) belongs to a novel subfamily of G-protein-coupled
receptors with seven-transmembrane domains. PAR2 is activated by serine proteases such as
trypsin, mast cell tryptase, and allergic or bacterial proteases. The presence of trypsin has
been shown in human stomach. Cyclooxygenase-2(COX-2) is induced by inflammatory
cytokines, growth factors, gastrin and reactive oxygen species in gastric epithelial cells which
may led to mutagenesis and subsequent metaplasia, dysplasia, and cancer formation. We
investigated whether PAR-2 is activated in H. pylori-infected cells, which is related to
induction of COX-2 in gastric epithelial cells. After treatment of H. pylori to AGS cells at the
ratio of 100:1, we determine the expression and activation of PAR-2 and induction of COX-2.
The same experiments were performed in the cells treated with PAR2 agonist peptide or
PAR2 antisense oligonucleotide. mRNA and protein expression were determined by RT-PCR
and Western blotting. PAR-2 activation was assessed by increase in intracellular calcium
level. As a result, H. pylori induced the activation and expression of PAR-2 as well as COX-2
expression, which were inhibited in the cells treated with PAR2 antisense oligonucleotide.
PAR2 agonist peptide increased the expression of COX-2 in H. pylori-infected AGS cells. In
conclusion, PAR-2 mediates H. pylori- induced COX-2 expression in gastric epithelial cells.
- 568 -

Session XIV : Transcriptional and translational control Poster XIV, 66
IRF-7: new role in the regulation of genes involved in the adaptive immunity
Marco Sgarbanti, Giulia Marsili, Anna Lisa Remoli, Roberto Orsatti
and Angela Battistini
Department of infectious, Parasitic and immunomediated Diseases, Istituto Superiore di
Sanità, viale Regina Elena, 299-Rome 00161, Italy. E-mail: battist@iss.it
The interferon regulatory factor 7 (IRF-7) a member of the IRF family of transcription factors
is a key player in the innate immune response against viral infections. Constitutive expression
of IRF-7 is limited to peripheral blood lymphocytes and dendritic cells while in other cell
types its expression can be induced by type I Interferon. IRF-7 is sequestered in the cytoplasm
of uninfected cells and following viral infection, double stranded RNA (dsRNA) or Toll-like
receptor (TLR) signalling it becames phosphorylated by TBK and IKK-i kinases.
Phosphorylated IRF-7 migrates in the nucleus where it can activate IFN-alpha genes and other
Interferon stimulated genes (ISGs). Here we report that the over-expression of a constitutively
active form of IRF-7 positively regulates the promoter of Interferon regulatory factor 1 (IRF-
1) and of the latent membrane protein 2 (LMP-2), two proteins, which play an important role
in adaptive immunity. We previously showed that in T cells, the HIV-1 trans-activator Tat
protein is able to modulate the expression of LMP2 through regulation of IRF-1 expression.
Experiments are, therefore, ongoing to determine whether the up-regulated expression of IRF-
1 and LMP-2 is mediated by the Tat-induced IRF-7 activation through the engagement of
TBK-1 and IKK-i kinases. Our data point to IRF-7 as a mediator that bridges innate and
adaptive immunity and a potential target of the HIV-1 Tat-mediated immune-modulation.
- 569 -

Session XIV : Transcriptional and translational control Poster XIV, 67
Hodgkin/Reed-Sternberg cells as a Model System for Studying the Canonical and Novel
NF-kB pathways
Michael Stillmann, Yoshiaki Sunami, Michael Hinz, Meike Brömer, Jan Ebert and
Claus Scheidereit.
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin,
Germany. E-mail : y.sunami@mdc-berlin.de
Proteins of the NF-kB/Rel transcription factor family are involved in the regulation of a
variety of physiological processes including adaptive and innate immune responses,
inflammation, cellular survival and proliferation. Constitutive NF-kB activity is hallmark of
malignant Hodgkin/Reed-Sternberg cells (H/RS) in Hodgkin lymphoma. In some H/RS cell
types aberrant NF-!B activation could be traced back to inactivating mutations in the IkBa
gene. Moreover, H/RS cell types exhibit enduring activation of the canonical pathway, due to
a not yet identified molecular aberration. Employing a combination of classical biochemical
techniques and RNAi we want to analyze the signal transduction through canonical and novel
signaling pathways in H/RS cells.
We have observed an enhanced kinase activity of the IKK complex and high expression of
p100/p52 resulting in a highly increased NF-kB DNA-binding and hence target gene
activation. Moreover, phosphorylation of p100 which is prerequisite for further processing to
p52, suggests ongoing signaling through the novel pathway. Pulse chase analysis in H/RS
cells demonstrated direct generation of p52 from de novo translated endogenous p100,
suggesting a co-translational mechanism.
Several groups proposed the aberrant overexpression of TNF family receptors CD40 and
CD30 in H/RS cells as initial cause for constitutive NF-kB activation. However, silencing of
CD40 and CD30 expression by RNAi neither affected NF-kB activity nor p100 processing. At
the moment, a possible contribution of a range of NF-kB regulators is analyzed using this
approach. Alternatively, we are applying comparative proteomic methods to define
differential protein-protein interaction patterns of NF-kB key regulators in Hodgkin and Non-
Hodgkin cells.
- 570 -

Session XIV : Transcriptional and translational control Poster XIV, 68
Induction of PAI-1 Expression by TNFalpha in Endothelial Cells is Mediated by its
Responsive Element Located in the 4G/5G Site.
Maria Swiatkowska1, Janusz Szemraj 2, Czeslaw S. Cierniewski1,3
Department of Molecular and Medical Biophysics1, Department of Biochemistry2,
Medical University in Lodz, Center of Medical Biology3, Polish Academy of Sciences,
Lodz, Poland
Plasminogen activator inhibitor type 1 (PAI-1) is induced by many proinflammatory and prooxidant
factors. Among them, tumor necrosis factor alpha (TNFalpha), a pivotal early
mediator that regulates and amplifies the development of inflammation, is one of the strongest
PAI-1 synthesis activators. Location of the TNFalpha response element in the PAI-1 promoter
is still ambiguous. In this study, we attempted to evaluate the significance of the element
located in the 4G/5G site of the PAI-1 promoter in the TNFalpha stimulation of PAI-1
expression in endothelial cells. PAI-1 expression was monitored at: (a) the level of mRNA
using real-time PCR, (b) PAI-1 gene transcription by transfection reporter assays, and (c)
protein synthesis using the enzyme immunoassay. NF-kB activity was monitored using the
electrophoretic mobility shift assay. Its activity was modified by either antisense
oligonucleotides or transfection of endothelial cells with the wild-type or mutated IkBalpha.
We have shown that TNFalpha-induced expression and gene transcription of PAI-1 involves a
regulatory region present in segment -664 ⁄-680 of the PAI-1 promoter. This reaction involves
the TNFalpha-induced generation of superoxide leading to activation of NF-kB, and can be
abolished by antioxidants and by overexpression of a super-suppressor phosphorylationresistant
IkBalpha. Stimulation of PAI-1 under these conditions involves the motif of the PAI-
1 promoter adjacent to the 4G⁄5G site, which can directly interact with NF-kB. We show that
activation of PAI-1 gene by TNFalpha and reactive oxygen species is mediated by interaction
of NF-kB with the cis-acting element located in the -675 4G⁄5G insertion ⁄ deletion in the
PAI-1 promoter.
- 571 -

Session XIV : Transcriptional and translational control Poster XIV, 69
Monitoring of dynamics NF-kB activation and correlation with mitochondrial and RE
Ca2+ in cftr-deficient airway cells
Olivier Tabary1,2, Emilie Boncoeur1, Rainer de Martin3, Rainer Pepperkok2, Annick
Clément1, Carsten Schultz2, Jacky Jacquot1
Inserm, U719, Université Pierre et Marie Curie, Hôpital Saint-Antoine, 75012, Paris,
France ; Cell Biology/Cell Biophysics and Gene Expression Program, EMBL,
Meyerhosfstr. 1,D-69117, Heidelberg, Germany;
Department of Vascular Biology and Thrombosis Research, Medical University of
Vienna, Austria.
Dysregulation of nuclear factor kappa B (NF-kB) and increased Ca2+ signals have been
reported in airway epithelial cells of CF patients. The hypothesis that Ca2+ signalling may
regulate NF-kB activation was tested in a CF bronchial epithelial cell line (IB3-1, CFTR
genotype DF508/W1282X) and compared to the CFTR-corrected epithelial cell line S9 using
fluorescence microscopy to monitored in situ dynamics of NF-kB activation at the single cell
level. We examined the change of [Ca2+]i levels using Fluo-3, Rhod-FF and NF-kB
activation was monitored by fluorescence microscopy (FRET) with YFP-p65 and IkBalpha-
CFP fluorescent fusion proteins in response to stimulation with 20 ng/ml of IL-1b. Upon
stimulation with IL-1b, we first observed, a slow but prolonged [Ca2+]i increase (up to 10
min) in IB3-1 cells compared to S9 cells. During the same time we observed a rapid and large
discharge of mitochondrial Ca2+ in CFTR-corrected S9 cells whereas in CFTR-deficient IB3-
1 cells we observed a relative increase to baseline followed by a slow decrease. The IL-1binduced
[Ca2+]i response was further accompanied by an activation of NF-kB in IB3-1 but
not in S9 cells. Pretreatment of IB3-1 cells with the ER Ca2+ pump inhibitor thapsigargin
inhibited the IL-1b-induced [Ca2+]i response. Treatment with either the calcium chelator
BAPTA or an inhibitor of IkBalpha phosphorylation (digitoxin) led to a drastic [Ca2+]i
decrease accompanied by an inhibition of NF-kB activation of IL-1beta-stimulated IB3-1
cells in comparison to untreated cells. In IB3-1 cells cultured at low temperature (26°C)
during 16h, the IL-1b-induced [Ca2+]i response was inhibited and no significant NF-kB
activation was observed. To our knowledge, this is the first report of visualization of the
Ca2+-mediated activation of NF-kB in individual living airway epithelial cells. Our results
support the concept that [Ca2+]i is a key regulator of NF-kB activation in CF airway
epithelial cells.
Tabary O, Boncoeur E, de Martin R, Pepperkok R, Clement A, Schultz C, Jacquot J. Calciumdependent
regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells. Cell
Signal. 2005
- 572 -

Session XIV : Transcriptional and translational control Poster XIV, 70
Characterization of 3T3-L1 adipocytes dedifferentiation induced by a mitochondrial
dysfunction
Silvia Tejerina1, Aurélia De Pauw1, Sebastien Vankoningsloo1, Andrée Houbion1,
Catherine Demazy1, Françoise de Longueville2, Vincent Bertholet2, Patricia Renard1,
José Remacle1,2, Martine Raes1 and Thierry Arnould1
1Laboratory of Biochemistry and Cellular Biology, University of Namur (F.U.N.D.P.),
Rue de Bruxelles, 61, 5000 Namur, Belgium. Email : thierry.arnould@fundp.ac.be,
2Eppendorf Array Technologies, Rue du Séminaire, 12, 5000 Namur, Belgium.
Studies of several lipid disorders have evidenced a strong link between mitochondrial
dysfunction and fat storage abnormalities and it is now well accepted that a mitochondrial
dysfunction affects lipid-metabolizing tissues. However, molecular mechanisms involved in
the adipocyte dedifferentiation programme are still poorly understood. In this study, we set up
an experimental model to characterize the dedifferentiation of 3T3-L1 adipocytes induced by
a mitochondrial dysfunction triggered by FCCP, an uncoupling molecule. Differentiating
adipocytes incubated for several days with FCCP show modifications in their phenotype and
are characterized by a decrease in the triglyceride content accompanied by an increase in
glycerol release (a marker of fatty acid mobilization). However, mechanisms and cell
signaling seem to be totally different than the ones triggered by TNFalpha! a cytokine
known to stimulate adipocyte dedifferentiation, as PKA and MEK1/2 inhibitors do not modify
the triglyceride content in FCCP-treated adipocytes. Using a low density cDNA microarray
allowing gene expression analysis for numerous adipogenic markers, we also found a
significant decrease in the expression of genes encoding key transcription factors involved in
adipogenesis (PPARgamma and C/EBPalpha) in adipocytes with impaired mitochondrial
activity. Decrease in the activity of these transcriptional regulators has also been confirmed by
DNA-binding activity assays. Finally, among the genes found to be differentially expressed in
differentiating adipocytes, CHOP-10/GADD153, a natural dominant negative mutant of
C/EBPbeta is specifically up-regulated in adipocytes with uncoupled mitochondria. These
results highlight some new effectors potentially involved in adipocyte dedifferentiation in
response to a mitochondrial dysfunction.
T. Arnould and A. De Pauw are respectively Research Associate and Research Assistant of
FNRS (Fonds National de la Recherche Scientifique, Brussels, Belgium).S. Tejerina is a
recipient of a CUD (Coopération Universitaire au Développement) fellowship.
- 573 -

Session XIV : Transcriptional and translational control Poster XIV, 71
Ferric citrate abolishes the induction of the hypoxia inducible factor HIF-1alpha
expression and restores the inhibition of cell proliferation produced by the flavonoid
quercetin
Anastasia Triantafyllou1, Panagiotis Liakos1, Andreas Tsakalof1, Georgia
Chachami1,2, Efrosyni Paraskeva2, Ilias Athanasiadis1, Paschalis-Adam Molyvdas2,
Eleni Georgatsou1, George Simos1 and Sophia Bonanou1
1Laboratory of Biochemistry and 2Laboratory of Physiology, Department of Medicine,
School of Health Sciences, University of Thessaly, Larissa, Greece.
E-mail: atsakal@med.uth.gr
Hypoxia induces the expression of the regulatory alpha subunit of the Hypoxia-Inducible
Factor-1 (HIF-1), by preventing its hydroxylation by O2 - and Fe2+-dependent prolyl
hydroxylases (PHDs), which target it for proteosomal degradation. Quercetin, a bioflavonoid
with anti-oxidant, metal-chelating, kinase-modulating and anti-proliferative properties, can
induce the expression of HIF-1alpha in normoxia, but its mechanism of action has not been
determined. In this study we characterized the induction of HIF-1alpha expression and
inhibition of proliferation in HeLa and ASM (airway smooth muscle) cells by quercetin and
examined the effect of iron on these processes. We also investigated the cellular uptake and
biotransformations of quercetin and its relevance for HIF-1alpha induction and growth
inhibition. Our data demonstrate that addition of excess Fe(III) can abrogate the quercetininduced
stabilization of HIF-1alpha in both cell systems. Moreover, the inhibition of DNA
synthesis, cell proliferation and cycle progression caused by quercetin can also be reversed by
excess iron. We propose that iron chelation is the key event in the induction of HIF-1alpha as
well as in the inhibition of cell proliferation produced by quercetin.
- 574 -

Session XIV : Transcriptional and translational control Poster XIV, 72
Glutamate-induced synapse-to-nucleus shuttling of ERK and the transcription factor
Elk-1 in striatal neurons requires vesicular transport.
Trifilieff P1,2; Lavaur J1 ; Brami-Cherrier K1 ; Pagès C1; Micheau J2 ; Caboche J1
and Vanhoutte P1
1Lab. Signalisation Neuronale et Régulations Géniques-Univ. Pierre et Marie Curie
Paris 6-CNRS-UMR7102.
2 Lab. Neurosciences Cognitives-Univ. Bordeaux I-CNRS-UMR5106.
MAP kinases of the ERK subtype (Extracellular signal-Regulated Kinase) play a critical role
in long term neuronal adaptations, learning and memory. These long term events require gene
regulations that depend on the presence of activated ERKs in the nucleus. ERKs are expressed
and activated in neurites, at considerable distance from the nucleus, and shuttle towards the
nucleus upon activation by a mechanism that is not clearly defined yet. Furthermore, we have
previously shown that one characteristic of neuronal cells is that the transcription factor Elk-1,
one of the major ERKs’ target, has the same expression pattern as ERK and is expressed in
both cytoplasmic and nuclear compartments.
In the present study, we analysed the sub-cellular fate of ERK and Elk-1 in a model of ERKdependent
Immediate Early Gene (IEG) induction on striatal neurons. We show that ERK and
Elk-1 rapidly translocate to the nucleus upon stimulation with the same kinetics. Confocal
analysis of ERK and Elk-1 staining reveal a “clustered” pattern with a high degree of colocalization
with clathrin-coated vesicle (CCV) markers. Co-immunoprecipitation assays
show an interaction of ERK and Elk-1 with markers of CCV, which is transiently increased
upon stimulation. Despite the fact that glutamate stimulates endocytosis of both ionotropic
and metabotropic glutamate receptors, we establish that ERK interact specifically with
AMPA-containing vesicles. Interestingly, blockade of endocytosis inhibits glutamatedependent
ERK and Elk-1 nuclear translocation without alteration of neither ERK activation
nor activation of other MAP kinases. In our model, where inhibition of vesicular transport
restricts ERK activation to the cytoplasm, we measured the consequences of the absence of
nuclear translocation of activated ERKs. We underline the multiple roles of ERKs in this
compartment and show their functions as key regulators of IEG expression as well as
modulators of chromatin structure via the control of histone phosphorylation. This study
underlies the key role of the vesicular transport in the synase-to-nucleus shuttling of ERK. We
are currently analyzing the functional relevance of the vesicular transport-mediated ERK
nuclear translocation in the establishment of plasticity in the striatum in vivo.
- 575 -

Session XIV : Transcriptional and translational control Poster XIV, 73
A novel human glucocorticoid receptor transcript splice variant: Identification and
tissue distribution of hGR[delta]313-338, an alternative exon 2 transactivation domain
isoform.
Jonathan D. Turner 1 , and Claude P. Muller 1,2
1
Institute of Immunology, Laboratoire National de Santé, 20A rue Auguste Lumière, L-
1011, Luxembourg
2
Department of Immunology, Graduate School of Psychobiology, University of Trier, D-
54290, Germany
Email: jonathan.turner@LNS.ETAT.LU and claude.muller@LNS.ETAT.LU
All human glucocorticoid receptor isoforms (hGR) are encoded by the NR3C1 gene. NR3C1
consists of 7 core exons (exons 2-8) common to all the known protein isoforms, has two
major exon 8-9 splice variants, and a 5’ UTR consisting of 11 alternative splice variants.
Here, we report the existence of a novel splice variant, hGR[delta]313-338, containing a 26
residue (78 bp) deletion in the N-terminal region (encoded by exon 2) between amino acids
313 and 338. This N-terminal region has previously been shown to include the 1
transactivation domain, that is thought to make contact with proteins in the basal
transcriptional apparatus, including the TATA box-binding protein (TBP). The
hGR[delta]313-338 observed at the mRNA level represents a transcript variant encoding a
protein isoform with a deletion between the tau 1 domain and the DNA binding domain
(DBD) encoded by exons 3 and 4. Interestingly, the deleted residues show a relatively high
abundance of potential phosphorylation sites (4 serines, 2 threonines, and 2 tyrosines, 28% of
deleted residues) including serine 317 that has been shown to be phosphorylated. It is thought
that phosphorylation plays an important role in transactivation action of hGR. It is also known
from knockout mice that removal of the entire exon 2 covering both the tau 1 transactivation
domain and our deleted region does not result in the expression of a transcriptionally inactive
receptor; however, it produces an altered glucocorticoid- induced transcription pattern. Here
we report the first observation of hGR[delta]313-338, and its sequence and its expression in a
range of human tissues. We hypothesise that hGR[delta]313-338 represents an isoform of the
hGR with an altered glucocorticoid induced transactivation profile.
- 576 -

Session XIV : Transcriptional and translational control Poster XIV, 74
Importance of the coordinate expression of PKC" and Ets1 for breast cancer cells
Martina Vetter 1 , Dario Schunke 1 , Paul N. Span 2 , Fred Sweep 2 , Henrike Ronneburg 1 ,
H.-J. Holzhausen 3 , Angela Dittmer 1 , Christoph Thomssen 1 and Jürgen Dittmer 1
1 Clinic for Gynecology, University of Halle, Germany, 2 Department of Chemical
Endocrinology, Radboud University Nijmegen Medical Centre, Nijmegen, Germany,
3 Institute for Pathology, University of Halle, Germany
Protein kinase C" (PKC") and the transcription factor Ets1 are often associated with
advanced tumor progression. We have previously shown that PKC" is able to
phosphorylate Ets1 and to increase its transcriptional activity. By using RNA interference
we could now show that PKC" also positively influences Ets1 expression in a variety of
cancer cell lines. One major way by which PKC" regulates Ets1 expression is by
increasing Ets1 protein stability. The relationship between PKC" and Ets1 was confirmed
by the finding that, in primary breast cancers, Ets1 expression correlates with that of
PKC". In searching for similar effects of PKC" and Ets1 we found that the response of
MDA-MB-231 cells to mithramycin or UV-light were similarly affected by PKC"- and
Ets1-specific siRNAs, whereas only PKC"-specific siRNA modulated cell morphology
and anchorage-independent growth. In an effort to identify PKC" and Ets1-responsive
genes by microarray analysis we found that the RNA levels of thirty-two genes were
similarly affected by both the PKC"- and Ets1-specific siRNAs. Among those genes were
MMP1 and MMP9 known to be regulated by Ets1 as well as the Ets1 co-activators SP100
and AML-1. Another responder gene was Rho-GDI#, an inhibitor of Rho-GTPases some
of which are important regulators of cellular migration. In MDA-MB-231 cells, PKC"- or
Ets1-specific siRNA downregulated both the RNA- as well as the protein level of Rho-
GDI#. In primary breast cancer, expression of Rho-GDI#, but not of Rho-GDI" and Rho-
GDI$, correlated with the levels of PKC" and Ets1 suggesting that there is a general link
between PKC"/Ets1 and Rho-GDI# in breast cancer cells. Interestingly, expression of
Vav1, another Rho-regulating protein which cooperates with Rho-GDI# in T-cells to
activate NFAT, was found to correlate with Rho-GDI#, PKC" and Ets1 expression. The
importance of Rho-GDI# and Vav1 in breast cancer cells is currently under investigation.
- 577 -

Session XIV : Transcriptional and translational control Poster XIV, 75
c-Jun as a sensor of DNA damage
Vinciguerra M.1-2, Esposito I.2, Cosentino C.2, Maggiolini M.1 and Musti A.M.1-2
1. Dipart. Farmaco-Biologico Università della Calabria, Rende, Italy
2. Dipart. Biol. Patol. Cell. Mol. I Università di Napoli “Federico II”, Italy
DNA damage elicits a cellular response resulting in the onset of cell cycle delay, DNA repair
or apoptosis. Central to this process is the DNA-damage dependent activation of three IP3
kinase family members ATM, ATR and DNA-PK, which in turn initiate phosphorylation of
proteins involved in the DNA damage pathway. The inability to respond properly to DNA
damage leads to genetic instability, which in turn may enhance the rate of cancer
development. Emerging evidence suggest that, in early stages of oncogenesis, the surviving
transformed cells can be reprogrammed to cell death trough the stress signalling activated by
DNA-damaging anticancer chemotherapies. However advance tumors are frequently resistant
to these treatments, a response that in certain cancer cells seems to be mediated by DNA-PK
over-activity, or by active c-Jun/AP1 transcription factor. c-Jun represents the intersection of
multiple pathways eliciting quite opposite programs, as cellular survival or apoptosis. So far
the regulatory signals leading to this divergence have not been identified. We have
characterized a novel c-Jun consensus phosphorylation site for ATM/ATR kinases. Our
results indicate that DNA-damage dependent phosphorylation of c-Jun at the ATM/ATR
consensus site in turn switches on c-Jun pro-apoptotic phosphorylation at JNK-specific sites.
Furthermore, our results suggest that differential regulation of c-Jun phosphorylation by
apical DNA-sensor kinases may play an important role in the chemoresponsiveness of tumor
cells exposed to DNA-damaging agents such as the topoisomerase
II inhibitor etoposide.
- 578 -

Session XIV : Transcriptional and translational control Poster XIV, 76
Blocking NF-kB activation by engineering proteins targeted to the minimal
oligomerization domain of NEMO
Emanuel Wyler*, Monika Kaminska, Andreas Plückthun, Michel Veron, and Fabrice
Agou
The Unit of Enzymatic Regulation of Cell Activities, Pasteur Institut, Paris, France,
*Present address: Institut of Biochemistry, ETH, Zurich, Switzerland
wyler@bc.biol.ethz.ch,kaminska@pasteur.fr,plueckthun@bioc.unizh.ch
mveron@pasteur.fr, fagou@pasteur.fr
The link between NF-kB signal transduction pathway and cancer is now well established.
Thus, inhibiting this pathway is a promising clinical approach through a pro-apoptotic effect
in the treatment of certains cancers. It has already proved to be efficient in therapy, in
combination with chemotherapy and radiation for a variety of cancers. The IKK complex is a
priviledged target for designing inhibitors due to its central role of the pathway. We
previously defined a minimal oligomerization domain of NEMO and showed that
oligomerization was necessary for NEMO function. This allowed us to develop new
inhibitory peptides of the NF-kB pathway targeting NEMO oligomerization (1).
Ankyrins constitute an attractive class of stable and small repeat proteins that provide variable
and modular binding surfaces to a target protein. We used the ribosom display method to
select ankyrins binding to the NEMO minimal oligomerization domain from a native ankyrin
library. After four rounds of selection, several ankyrins with affinity in the low nanomolar
range were isolated.When expressed in 293T cells, the selected ankyrins strongly inhibit
TNF-a-mediated NF-kB activation while having no effect on the basal activity. Controls with
native ankyrin or null plasmid were without effect. Furthermore, we could show that this NFkB
inhibition occurs through a specific interaction between ankyrin binders and the
endogenous NEMO, resulting in the IKK inhibition. Our findings indicate that high-affinity
binders selected from peptide or chemical libraries against the minimal oligomerization
domain of NEMO can be a promising strategy to search for specific NF-kB inhibitors. Our
binders might also be used for structural studies for both NEMO and the minimal
oligomerization domain.
F. Agou et al. (2004) J.Biol.Chem. 279, 54248-54257
- 579 -

Session XIV : Transcriptional and translational control Poster XIV, 77
Diphenyleneiodonium inhibits cerulein-induced apoptosis in pancreatic acinar cells
Ji Hoon Yu, Joo Weon Lim, Kyung Hwan Kim, and Hyeyoung Kim*
Department of Pharmacology, Brain Korea 21 Project for Medical Science, Yonsei
University, College of Medicine, Seoul 120-752, Korea. E-mail: jihoonyu@hotmail.com
Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently we
demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox and p67phox are
constitutively expressed in pancreatic acinar cells. Diphenyleneiodonium (DPI) is widely used
as an inhibitor of flavoenzymes, particularly NADPH oxidase. We investigated whether
inhibition of NADPH oxidase with treatment of DPI suppresses ROS production and the
activation of NADPH oxidase in pancreatic acinar AR42J cells stimulated with cerulein, and
whether cerulien induces the expression of apoptosis-inducing factor (AIF), caspase-3
activation and apoptosis, which is inhibited by DPI. To determine the effect of DPI on
cerulein-evoked Ca++ influx, intracellular Ca2++ level was monitored in the cells treated
with or without DPI and cultured in the presence of cerulein. As a result, cerulein induced the
activation of NADPH oxidase, determined by ROS production and translocation of cytosolic
subunits p47phox and p67phox to the membrane, which is in parallel with increase in
apoptotic indices, including the expression of AIF, caspase-3 activation, TUNEL staining,
DNA fragmentation and decrease in cell viability. Treatment with DPI inhibited ceruleininduced
activation of NADPH oxidase and apoptosis, but not Ca++ influx in pancreatic acinar
cells. These results demonstrate that inhibition of NADPH oxidase by DPI may alleviate
ROS-mediated apoptosis in pancreatic acinar cells.
- 580 -

Session XIV : Transcriptional and translational control Poster XIV, 78
NFAT and bone disease: a possible “decoy” therapeutic approach
Margherita Zennaro, Elisabetta Lambertini, Roberto Gambari, Letizia Penolazzi and
Roberta Piva.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy. Email:
piv@unife.it
Bone is a dynamic tissue, under constant remodelling all life long. This process results from
the balance between the activity of osteoblasts (OBs) and osteoclasts (OCs), that deposit and
resorb bone respectively. Any modification of this delicated equilibrium can affect bone
integrity, leading to patologies such as osteopenic disorders or osteopetrosis. A central role in
the control of OCs functions and in some OBs activities too, was recently described for
Nuclear Factor of Activated T-cells (NFAT) c1. In this study, we suggest a possible
involvment of NFATc1 in the transcriptional regulation of human estrogen receptor alpha
(ER) gene in both kind of cells. It is well known that estrogen influences OBs and OCs
activities, playing a fundamental role in protection of bone mass, as indicated, for example, by
association of osteoporosis and menopause.
We previously demonstrated that a specific oligonucleotide (RA4-3’) used as decoy molecule
can increase ER expression in bone cells, by interfering with the activity of an unidentified
negative transcription factor. Interestingly, RA4-3’ sequence contains a putative binding site
(TGAAAA) for NFAT that is the aim of these investigations. First of all we detected a high
level of NFATc1 expression in OCs and in SaOS-2 osteoblastic cells, treated with PMA and
ionomycin. We then verified the specificities of the interaction between NFAT protein and
our sequence, by using cross competition in electrophoretic mobility shift assay. Preliminary
results indicat that a specific interaction between NFAT and our sequence occurs, probably
mediated by some NFAT co-transcription factors, such as AP-1. Next, we verified NFAT
functionality in OCs and SaOS-2 through the transfection of pNFAT-TA luc cis-reporter
vector, containing three tandem copies of the NFAT-consensus sequence upstream of the
luciferase gene, in combination with RA4-3’. This caused a decrease of Luc activity, due to
the recruitment of NFAT proteins by RA4-3’. In addition, we demonstrated that RA4-3’
selectively induces apoptosis in OCs but not in OBs. In view of a possible therapeutic
application of NFAT decoy, the role of NFAT in this issue was also investigated.
- 581 -

Notes
- 582 -

Notes
Notes
- 583 -

- 584 -

Session XV : Reactive oxygen species and cell signaling
- 585 -

Session XV : Reactive oxygen species and cell signaling Poster XV, 1
Evidence for a defective lung NF-kB activation by oxidative stress in cystic fibrosis cell
culture and mouse models
Emilie Boncoeur, Olivier Tabary, Elise Bonvin, Annick Clément, Alexandra Henrion-
Caude, Jacky Jacquot.
Inserm U 719, Hôpital Saint-Antoine, UPMC ParisVI, 75571, Paris, France
Lung in patients with cystic fibrosis (CF) is characterized by structural damage and altered
repair due to oxidative stress. To gain insights into the oxidative stress-related damage in CF
lung, we studied the effects of hyperoxia (95 % O2) in CF and normal mouse lung and cell
culture models. In normal lung epithelium, protection against hyperoxic injury has been
shown to be related to an increased expression of the cell cycle inhibitor p21WAF1/CIP1 thus
confering a survival advantage to damaged cells and to increased NF-kB activity by involving
a cytoplasm-to-nucleus translocation after degradation of the inhibitor protein IkB alpha by
the proteasome machinery. Here we demonstrate in CF in vivo and in vitro models that
hyperoxia did not induce neither increased p21WAF1/CIP1 expression nor lung NF-kB
activation IkB alpha degradation, nor induction of caspase 3 and subsequent apoptosis but,
unexpectedly caused enhanced proteasome activity, in contrast to that observed in normal in
vivo and in vitro controls. Interestingly, ectopic expression of p21WAF1/CIP1 in hyperoxiaexposed
CF lung epithelial cells or treatment with the selective proteasome inhibitor MG132
restored the NF-kB activation and IkB alpha degradation which was associated with an
increased caspase-3 activity and apoptotic cell death. Our data suggest that the use of
proteasome inhibitors or related substances modulating p21WAF1/CIP1 degradation,
promoting the activation of NF-kB in hyperoxic conditions could be a valuable approach to
protect lung epithelial cells from oxidative stress in CF patients.
- 586 -

Session XV : Reactive oxygen species and cell signaling Poster XV, 2
Production of DNA strand breaks by metal-induced oxygen radicals
Ezzatollah Keyhani(1,2), Farnoosh Attar(1), Fatemeh Abdi-Oskoui(1), Jacqueline
Keyhani(2)
(1)Institute of Biochemistry and Biophysics, University of Tehran, 13145 Tehran, and
(2)Laboratory for Life Sciences, Tehran 19979, Iran. E-mail: keyhanie@ibb.ut.ac.ir
Elevated levels of reactive oxygen species (ROS) have been implicated in the etiology of
cancer, neurodegenerative and cardiovascular diseases, as well as in the aging process and as
a condition promoting breast cancer metastases. H2O2, a product of the cell metabolism, is a
potential source of ROS. In small concentrations (10-6 M), H2O2 is a signaling molecule
capable of inducing chemotactic activity, stimulating the synthesis of cytoskeleton elements,
and causing changes in cytosolic calcium concentrations. H2O2 is normally disposed of by
specialized enzymes such as catalases and peroxidases. However, various physiologic
perturbations of cellular homeostasis may lead to a dramatic increase in the amount of H2O2
within a cell. In the presence of transition metals, H2O2 is rapidly converted to the highly
reactive and highly toxic hydroxyl radical. The latter is responsible for lipid peroxidation,
oxidative damage to proteins and breakage of DNA strands. In this study, the extent of DNA
damage caused by H2O2 in the presence of various transition metals was evaluated in vitro.
Purified Salmonella typhimurium DNA was exposed to 0.1 to 100 mM H2O2 and either
Fe2+, Fe3+, Cu2+, Ni2+, or Cd2+ in increasing concentrations from 0.01 to 0.1 mM.
Damage to DNA was assessed by electrophoresis of the DNA preparations in 1% agarose gel.
Breakage of the DNA strands would produce a series of DNA fragments of various sizes
resulting in a smear in the gel after electrophoresis while intact DNA would produce a single
band. Results showed that all of the metals investigated triggered DNA breakage in the
presence of H2O2. The extent of breakage depended on the metal ion, its concentration, and
H2O2 concentration. Addition of either EDTA or catalase to the reaction mixture completely
inhibited the DNA degradation, confirming the involvement of both the metal ion and the
H2O2 in the breakage of DNA strands. Production of the hydroxyl radical when H2O2 and a
metal ion were both present in the reaction mixture was verified by the thiobarbituric acid
method. The extent of DNA breakage caused by the metal ions was as follows:
Cu2+>Fe2+>Fe3+>Ni2+>Cd2+.
- 587 -

Session XV : Reactive oxygen species and cell signaling Poster XV, 3
Antioxidant enzymes during hypoxia-anoxia signaling events in Crocus sativus L. corm
Ezzatollah Keyhani(1,2), Jacqueline Keyhani(2), Mahnaz Hadizadeh(1), Lila
Ghamsari(1)
(1)Institute of Biochemistry and Biophysics, Univ. of Tehran, 13145 Tehran, Iran, and
(2)Laboratory for Life Sciences, 19979 Tehran, Iran. E-mail: keyhanie@ibb.ut.ac.ir
Saffron has been shown to have therapeutic and preventive effects for various cancers and
other ailments. The saffron plant (Crocus sativus L.) is propagated only via its corms. Thus a
better knowledge of the corm biochemistry and of its response to stresses is mandatory.
Reactive oxygen species (ROS) are involved in the response to hypoxia/anoxia (H/A), and it
was also shown that anoxia stress led to hydrogen peroxide formation in plants. H/A was
produced in Crocus sativus L. corms, either dormant or cultivated for 3 days, by flooding
them. Flooded corms were withdrawn after 1, 2, 3, 4, 8, 10 and 14 days under water and used
immediately, as well as control corms cultivated under normoxic conditions, for extract
preparation. ROS scavenging enzymes activity was studied in the obtained extracts. Catalase
activity was 2.5 times the control value in dormant corms flooded for 8 days, but exhibited a
four-fold increase compared to control in corms cultivated for 3 days and then flooded for just
1 day. The activities of o-dianisidine and ascorbate peroxidases decreased in dormant corms
maintained in H/A, while they consistently increased in corms cultivated for 3 days prior to
H/A. Superoxide dismutase (SOD) activity exhibited a two-fold increase in dormant corms
flooded for 2 to 4 days, then returned to the control value. In corms cultivated for 3 days
prior to H/A, SOD activity remained at the control level except for a two-fold increase at day
8. Glutathione peroxidase activity was consistently higher than the control, both in dormant
corm and in corm cultivated for 3 days prior to H/A. Data showed that 1) H/A stimulated
catalase, SOD and gluthatione peroxidase activities in dormant corms; 2) H/A stimulated all
five enzymes studied in corms cultivated under normoxic conditions prior to flooding with a
maximum stimulation for catalase, followed, in decreasing order, by o-dianisidine peroxidase,
ascorbate peroxidase, glutathione peroxidase and SOD; 3) there is a fine regulation of ROS
scavenging enzymes response depending on whether H/A occured in dormant corms, or in
corms grown for 3 days under normoxic conditions prior to H/A.
- 588 -

Session XV : Reactive oxygen species and cell signaling Poster XV, 4
4-Hydroxyestradiol Promotes Neoplastic Transformation of Human Breast Epithelial
Cells through Generation of Reactive Oxygen Species
Sin-Aye Park, Eun-Hee Kim, Hye-Kyung Na, and Young-Joon Surh
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
Epidemiological studies and animal experiments have revealed that estrogens are
carcinogenic. Catechol estrogens are considered to be critical intermediates in estrogeninduced
carcinogenesis. It has been suggested that further oxidation of catechol estrogens to
quinones through redox cycling produces reactive oxygen species (ROS) which can cause
oxidative DNA damage in cells. In the present study, we have examined whether
carcinogenic potential of catechol estrogens is associated with production of ROS and
subsequent activation of cellular signaling pathways. 4-Hydroxyestradiol (4-OHE2), a
representative catechol estrogen, significantly enhanced colony formation in human
mammary epithelial cells (MCF10A) initiated with 9,10-dimethyl-1,2-benz(a)anthracene
(DMBA). 4-OHE2-treated MCF10A cells also accumulated the intracellular ROS, which was
abolished by the antioxidant Trolox. N-acetyl-L-cysteine (NAC) significantly inhibited the 4-
OHE2-promoted anchorage-independent colony formation initiated with DMBA. NF-!B, a
representative redox sensitive transcription factor, has been known to be involved in
carcinogenesis. 4-OHE2 induced NF-!B DNA binding and transcription activity, but did not
affect AP-1 DNA binding. 4-OHE2 induced expression of cyclooxygenase-2 (COX-2) and
inducible nitric oxide synthase in MCF10A cells. 4-OHE2 also activated ERK1/2 and AKT
which are up-stream target molecules of NF-!B. To determine whether 4-OHE2-enhanced
ROS production is associated with phosphorylation of ERK and AKT, MCF10A cells were
treated with NAC in the presence of 4-OHE2. NAC inhibited the phosphorylation of ERK1/2
and AKT induced by 4OHE2. NAC and pharmacological inhibitors of ERK and AKT
attenuated the 4-OHE2-induced NF-!B DNA binding activity and also COX-2 expression in
MCF10A cells. Taken together, above findings suggest that 4-OHE2-induced ROS
production contributes to neoplastic transformation of MCF10A cells via activation of
AKT/ERK/NF-!B signaling.
- 589 -

Session XV : Reactive oxygen species and cell signaling Poster XV, 5
Functional Genetic Screens to Identify Genes in Adaptative Responses to Hypoxia
Celine Rofel1, Iris Partouns1, Raymond Hessing1, Elena Bardina1, Marianne
Koritzinsky2, Brad Wouters2, Jan Willem Voncken1
1) Department of Molecular Genetics
2) Department of Radiation Oncology; Research Institute Growth & Development,
Maastricht University, Universiteitssingel 50 6229 ER; Maastricht, The Netherlands
Tumors need oxygen and nutrient to grow, but these two factors are rapidly depleted, due to a
rapid growth, leading to hypoxic tissue in the tumor. Hypoxia remains a constant feature of
these tumors and contributes to tumor progression by triggering adaptative mechanisms.
HIF-1 is one of most important transcription factor up-regulated during hypoxia.
HIF-1 alpha appear to be important for tumor vascularization and metabolic adaptation to
hypoxia, which are essential for tumor progression.
To further investigate genes involved in mechanism of adaptation to hypoxia we screened a
placenta retroviral cDNA library for genes capable to bypass a growth arrest induced by
hypoxic conditions.
The HCT116 colon carcinoma cell line were selected to screen the library based on their
capacity to form colonies, to be genetically stable and sensitive to hypoxia.
The HCT116 were infected by the cDNA retroviral library 2 days before applying the hypoxic
treatment of 0% during 5 days, and then placed under normoxia (21%) for 10 days. The DNA
from the cells was isolated and a PCR was performed to amplify the integrated cDNA. Two
intense bands of 1kb and 4kb were observed. The 1kb band was isolated, subcloned into a
PCR4 topo plasmid and sequenced. 2 clones were identified as placental lactogen cDNA. The
cDNA of placental lactogen was cloned into a retroviral plasmid to validate the gene function
and elucidate the mechanism. To confirm that placental lactogen increase the survival of the
cells submitted to hypoxia reoxygenation we will analyze HCT116 expressing placental
lactogen survival by colony formation assay. To evaluate the capacity of placental lactogen to
give a growth advantage under hypoxia we will submit the cells to 0% during 2 and 3 days to
achieve a growth curve.
Based on the literature this gene plays a role in suppression of apoptosis pathway, thus it
could be interesting to investigate whether it also plays a role in hypoxia-induced apotosis.
Therefore we will assess the number of apoptotic cells submitted to hypoxic condition
expressing or not placental lactogen.
- 590 -

Notes
- 591 -

Session XVI : Chemopreventive agents
- 592 -

Session XVI : Chemopreventive agents Poster XVI, 1
Anti-neoplastic and anti-clastogenic properties of curcumin
Tzvetan Alaikov1, Spiro M. Konstantinov2,4, Tzvetomira Tzanova2, Kyril Dinev2,
Margarita Topashka-Ancheva3, Martin R. Berger4
1University Hospital St. Anna, Medical University of Sofia, Department of Internal
Medicine, 1 D. Mollov Str, 1000 Sofia, Bulgaria; e-mail: Alaikov@abv.bg
2Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str, 1000 Sofia, Bulgaria; email:
Pgen@TU-Sofia.bg
3Inst. of Zoology, Bulgarian Academy of Sciences, 1 Tzar Osvoboditel Blvd., 1000 Sofia,
Bulgaria; e-mail: Topashka.mn@lycos.com
4Unit of Toxicology and Chemotherapy; German Cancer Research Center, Im
Neuenheimer Feld 280, 69120 Heidelberg, Germany; e-mail: M.Berger@DKFZ.de
Curcumin is natural pigment of turmeric and is reported as signal transduction modulator and
inhibitor of transcription factors, e.g. NF-kB. In our study we found a concentrationdependent
cytotoxic activity of curcumin in a panel of leukemic cell lines: SKW-3, CEM, U-
937, HL-60, HL-60/Dox, K-562, LAMA-84 and AR-230. Additive to synergistic interactions
were recorded for combinations with bendamustine and idarubicine in SKW-3 and LAMA-84
cells. Noteworthy, a strong potentiation of the efficacy of bendamustine by concomitant
curcumin application was found in multiple myeloma (MM) cells (RPMI-8226 and U-266).
Moreover, curcumin increased the bendamustine cytotoxicity in primary cultures, isolated
from the bone marrow of patients with MM. This interaction could be explained by NF-kB
inhibition, because NF-kB is activated in many human cancers, especially leukaemia and
MM. Curcumin is characterized by low toxicity in vivo and was described to have
chemoprotective activity. Therefore, the intracellular level of reduced glutathione (GSH) was
measured and a concentration dependent increase of GSH level was recorded in AR-230 and
SKW-3 cells for concentrations ranging from 5 to 25 mcM. Experiments with mice showed
significant protection against cisplatin induced chromosomal aberrations (clastogenic effect)
and inhibition of mitoses in bone marrow cells. Curcumin alone caused significant reduction
of the mitotic index, but ameliorated this parameter when combined with cisplatin. Our
experimental data indicate that curcumin has pleyotropic effects on signal transduction by
inhibiting transcription cascades and thus exerting direct antitumor activity and by enhancing
the free radical scavenging that can explain the protective and anti-clastogenic activity.
- 593 -

Session XVI : Chemopreventive agents Poster XVI, 2
In vivo effects of the chemopreventive agent curcumin in the 5T33MM myeloma model.
Jo Caers, Els Van Valckenborgh, Benjamin Van Camp, Karin Vanderkerken.
Laboratory of hematology & immunology. Vrije Universiteit Brussel, Brussels, Belgium
Multiple Myeloma (MM) is a plasma cell malignancy characterised by the accumulation of
monoclonal plasma cells in the bone marrow. The disease is usually preceded by an agedependent
pre-malignant condition called monoclonal gammopathy of undetermined
significance (MGUS). Since these MGUS patients are observed off therapy, we believe that
these patients might benefit from a preventive treatment. In this study, we wanted to
investigate the effects of such a chemopreventive agent, namely curcumin, on MM disease
progression by using the murine 5T33MM myeloma model. In vitro, cell proliferation was
assessed by 3H thymidine uptake. The IC50 for curcumin was about 7,5 µM. Compared to
dexamethasone, a classic drug in MM therapy, curcumin was 50 times more effective. After
overnight, curcumin increased the apoptosis rate of myeloma cells about twofold. In addition,
an inihibitory effect could be seen on neo-vascularisation using a rat aortic ring assay with a
reduction in total number, length and branching of newly created vessels by curcumin
treatment. In vivo analysis of tumor burden was analyzed by injecting two groups of 10 mice
with 5T33MM cells, one group of 10 naive mice was included as negative control. Intraperitoneal
treatment with curcumin (50 mg/kg) was started two days before tumor
inoculation. At week 3, when the vehicle controls showed signs of morbidity, the mice were
sacrificed and tumor load was analyzed by determining bone marrow invasion, liver and
spleen mass and serum paraprotein concentrations. In treated mice, a 47 % reduction in serum
paraprotein concentrations and a 30% reduction in the percentage of 5T33MM idiotype
positive cells in the BM were monitored. Treatment further reduced the splenomegaly by 42%
and the hepatomegaly with 30 %. In conclusion, these are the first in vivo data on the effects
of curcumin on myeloma development. In human myeloma disease, curcumin was earlier
described as inhibitor of NF-kappa b and the JAK-STAT pathway. The inhibitory effects of
curcumin on these pathways will be further assessed on murine 5T33MM cells.
- 594 -

Session XVI : Chemopreventive agents Poster XVI, 3
A fully dissociated compound of plant origin for inflammatory gene repression.
Karolien De Bosscher*, Wim Vanden Berghe*, Ilse M.E. Beck*, Wim Van Molle#,
Nathalie Hennuyer§, Janet Hapgood‡, Claude Libert#, Bart Staels§, Ann Louw‡ and
Guy Haegeman*.
* Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST),
Department of Molecular Biology, Ghent University, K. L. Ledeganckstraat 35, B-9000
Gent, Belgium. E-mail: Guy.Haegeman@Ugent.be. # Department for Molecular
Biomedical Research, Flanders Interuniversity Institute for Biotechnology (V.I.B.) and
Ghent University (UGent), 'Fiers-Schell-Van Montagu' building,Technologiepark 927,
B-9052 Gent (Zwijnaarde), Belgium. § Département d’Arthérosclérose – U.545, Institut
National de la Santé et de la Recherche Médicale, Institut Pasteur de Lille, 1 Rue
Calmette BP245, 59019 Lille Cedex, France .‡ Department of Biochemistry, University
of Stellenbosch, Matieland 7602, Stellenbosch, Rep. of South Africa.
The identification of selective glucocorticoid receptor (GR) modifiers, which separate
transactivation and transrepression properties, represents an important goal for steroid
pharmacology. While the gene-activating properties of GR are mainly associated with
undesirable side effects, its negative interference with the activity of transcription factors,
such as NF-kB, greatly contributes to its anti-inflammatory and immune-suppressive
capacities. We found that Compound A (CpdA), a plant-derived phenyl aziridine precursor,
although not belonging to the steroidal class of GR-binding ligands, does mediate geneinhibitory
effects by activating the glucocorticoid receptor (GR). We demonstrated that CpdA
exerts an anti-inflammatory potential by down-modulating TNF-induced pro-inflammatory
gene expression, but interestingly, does not at all enhance GRE-driven genes or induces GR
binding to GRE-dependent genes in vivo. Furthermore, we have shown that the specific
gene-repressive effect of CpdA depends on the presence of functional GR, displaying a
differential phosphorylation status with CpdA as compared to DEX treatment. The antiinflammatory
mechanism involves both a reduction of the in vivo DNA-binding activity of
p65 as an interference with the transactivation potential of NF-kB. Finally, we present
evidence that CpdA is as effective as DEX in counteracting acute inflammation in vivo, and
does not cause a hyperglycemic side effect. Taken together, this compound may be a lead
compound of a novel class of anti-inflammatory agents with fully dissociated properties and
might thus hold great potential for therapeutic use.
- 595 -

Session XVI : Chemopreventive agents Poster XVI, 4
Inhibition of TNF-alpha induced NF-kappa B activation by kava (Piper methysticum)
derivatives
Folmer Florence1, Marc Diederich2, Marcel Jaspars1, Romain Blasius2, Jioji
Tabudravu1
1Marine Natural Products Laboratory, Department of Chemistry, University of
Aberdeen, Aberdeen AB24 3UE, Scotland. E-mail: f.folmer@abdn.ac.uk
2Laboratoire de biologie moleculaire et cellulaire du cancer (LBMCC), Hopital du
Kirchberg, 9, rue Edward Steichen, L-2540 Luxembourg, Luxembourg. Email :
marc.diederich@lbmcc.lu
The inducible transcription factor NF-kappa B plays a central role in the regulation of
immune, inflammatory, and carcinogenic responses. While normal activation of NF-kappa B
is required for cell survival and immunity, its deregulated expression is characteristic for
inflammatory and infectious diseases and for cancer. NF-kappa B has hence become a major
target in anti-inflammatory and anti-cancer drug discovery. In the present study, we
investigated molecular mechanisms induced by lactones and chalcones isolated from Fijian
kava (Piper methysticum) used in traditional medicine against urinary tract infections and
asthma. In order to understand underlying regulatory mechanisms, inhibition of both NFkappa
B-driven reporter gene expression and TNF-alpha induced NF-kappa B binding to a
consensus response element were assayed. Total inhibition was achieved at the concentrations
of 320 microM (flavokavain A), 175 microM (flavokavain B), and 870 microM (kavain and
dihydrokavain). Moreover, treatment with either kavain, flavokavain A, or flavokavain B led
to the inhibition of both the degradation of the inhibitor of kappa B (IkappaB) and the
subsequent translocation of the p50 and p65 subunits of NF-kappa B from the cytoplasm to
the nucleus, as shown by western blot analysis. Additionally, kinase specific screening and
co-transfections with IKK (kinase of IkappaB) and IKK dominant negative expression genes
showed that flavokavain A, but not kavain, nor flavokavain B, inhibits IKK. Flavokavain A
was also shown to inhibit PRAK (p38 regulated/activated kinase), MAPKAP-K3 (MAPKactivated
protein kinase 3), DYRK1A (dual-specificity tyrosine-phosphorylated and regulated
kinase 1A), and Aurora B. Altogether, these results give a first insight into anti-inflammatory
mechanisms triggered by traditionally used chemopreventive kava derivatives.
- 596 -

Session XVI : Chemopreventive agents Poster XVI, 5
Resveratrol induces apoptosis in chemo-resistant cancer cells via modulation of AMPK
signaling pathway
Jin-Taek Hwang 1 , Dong Wook Kwak 2 , Sun Kyo Lin 2 , Hye Min Kim 2 , Young Min Kim 2
and Ock Jin Park
Department of Food and Nutrition, Hannam University, 133 Ojeong-dong Daedeok-gu,
Daejeon 306-791, Korea, E-mail: ojpark@hannam.ac.kr
1 Department of Biochemistry and Molecular Biology, Medical Research Center for
Bioreaction to Reactive Oxygen Species, Kyung Hee University College of Medicine,
Seoul 130-791, Korea
2 Department of Biological Sciences, Hannam University, Daejeon 306-791, Korea
Resveratrol has been reported to possess therapeutic effects for various cancers including
colon cancers. In the present study, the molecular basis of resveratrol with emphasis on its
ability to control intracellular signaling cascades of AMP-activated kinase (AMPK)
responsible for inducing apoptosis in cancer drug-resistant cells was investigated. Recently,
the evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target
molecule of cancer control. We have investigated the effects of resveratrol on apoptosis in
relation to AMPK in HT-29 cells made chemo-resistant by the treating a cancer drug
etoposide. Resveratrol exhibited a variety of molecular events in etoposide-based
combination therapy in HT-29 colon cancer cells including the AMPK activation, inhibition
of cell growth, induction of apoptosis and ROS generation. The involvement of AMPK
signaling cascade in resveratrol based cancer therapy was clearly shown by comparing the
conditions of AMPK activated states and inactivated states. We have identified ROS as an
upstream regulator of AMPK.
- 597 -

Session XVI : Chemopreventive agents Poster XVI, 6
Combined effects of gallic acid and resveratrol on gap junction intercellular
communication and matrix metalloproteinase
Jong Hoon Kim, Ki Won Lee, Hyo Jin Kim, and Hyong Joo Lee*
Department of Food Biotechnology, School of Agricultural Biotechnology, and Center
for
Agricultural Biomaterials, Seoul National University, Seoul 151-742, Republic of Korea
Dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity has been
considered one of the plausible ways to prevent reactive oxygen species (ROS)-mediated
human diseases and aging, but antioxidant therapy has been at best equivocal. The present
study investigated the possible combined effect of gallic acid and resveratrol, which is major
antioxidants in red wine, on gap junction intercellular communication (GJIC) and on matrix
metalloproteinase-2 (MMP-2) closely related to carcinogenesis. Resveratrol, but not gallic
acid, prevented inhibition of GJIC and hyperphosphorylation of connexin 43 (Cx43) induced
by hydrogen peroxide (H2O2) in normal rat liver epithelial cells. The sustained production of
H2O2 by phenazine methosulfate (PMS), a chemical generating intracellular ROS, induced
activation of MMP-2 in HT1080 human fibrosarcoma cells. Resveratrol prevented activation
of pro-MMP-2 by PMS, but gallic acid did not exert these effects. Gallic acid rather induced
inhibition of GJIC and activation of MMP-2 without H2O2 in a dose-dependent manner. Both
GA and resveratrol generated H2O2 in the media without cells, and GA generated almost 5
times more H2O2 than resveratrol in a dose- and time-dependant manner. The inhibition of
GJIC by gallic acid was mediated by hyperphosphorylation of connexin 43 (Cx43) and
activation of extracellular signal-regulated kinase 1/2 (ERK1/2) through generation of ROS,
while catalase and resveratrol significantly reversed gallic acid-induced inhibition of GJIC.
The activation of MMP-2 and increase of motility by gallic acid was also partly abolished by
catalase and resveratol. These results indicate potential prooxidant activity of gallic acid and
the combined effect of red wine phenolics on carcingenesis. Taken together, above findings
suggest that antioxidants may act differently in ROS-mediated carcinogenesis depending on
the conditions and structure.
- 598 -

Session XVI : Chemopreventive agents Poster XVI, 7
Jaceosidin, a pharmacologically active flavone derived from Artemisia plants, induces
apoptosis in ras-transformed human breast epithelial (MCF10A-ras) cells
Min-Jung Kim1, Do-Hee Kim1, Ki Won Lee1, Do-Young Yoon2, and Young-Joon Surh1
1National Research Laboratory of Molecular Carcinogenisis & Chemoprevention,
College of Pharmacy, Seoul National University, Seoul 151-742 and 2Department of
Molecular Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
Extracts of Artemisia plants possess anti-inflammatory and anti-oxidative activities. Eupatilin
(5,7-dihydroxy-3,4,6-tri-methoxy-flavone), a pharmacologically active flavone derived from
Artemisia asiatica, was shown to inhibit phorbol ester-induced cyclooxygenase-2 induction
and NF-kB activation in mouse skin (H.J. Seo et al., Int. J. Cancer, 100: 456-62, 2002), and to
induce cell cycle arrest in ras-transformed human mammary epithelial (MCF10A-ras) cells
(D.H. Kim et al., Biochem Pharmacol., 68: 1081-7, 2004). In the present study, we examined
the ability of jaceosidin (4 ,5,7-trihydroxy-3 ,6-dimethoxyflavone) isolated from Artemisia
argyi to induce apoptosis in MCF10A-ras cells. Jaceosidin inhibited the growth of MCF10Aras
cells to a greater extent than eupatilin. Jaceosidin-induced apoptosis was mediated by
intracellular ROS accumulation in MCF10A-ras cells, which was blocked by the antioxidant
N-acetylcysteine (NAC). Jaceosidin has an additional hydroxyl moiety at the 4 -position
which was replaced by the methoxy group in eupatilin. To better assess the pro-apoptotic
effects of jaceosidin, we analyzed the treated cells by the flow cytometry. MCF10A-ras cells
treated with jaceosidin (100 µM) exhibited the increased proportion of hypodiploid or
apoptotic cells (48.72% as composed to 7.78% in control cells). Jaceosidin treatment also
decreased the ratio of pro-apoptotic Bax to the anti-apoptotic Bcl-2 and induced the cleavage
of caspase-3 and poly(ADP-ribose)polymerase (PARP). Moreover, jaceosidin elevated
expression of p53 and p21, while inhibited the activation of ERK1/2 which is an important
component of cell survival pathways. In conclusion, the pro-apoptotic activity of jaceosidin
is associated with ROS accumulation and inhibition of ERK1/2 activation in MCF10A-ras
cells.
- 599 -

Session XVI : Chemopreventive agents Poster XVI, 8
Epigallocatechin gallate inhibits phorbol ester-induced expression of COX-2 and
activation of NF-kappaB and CREB in mouse skin in vivo
Joydeb Kumar Kundu and Young-Joon Surh
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
Despite accumulating evidence supporting the cancer chemopreventive properties of green
tea, the underlying molecular mechanisms are not fully clarified. The representative green tea
polyphenol epigallocatechin gallate (EGCG) has been reported to inhibit mouse skin tumor
promotion. Since an inappropriate expression of cyclooxygenase-2 (COX-2) has been
frequently implicated in the pathogenesis of cancer, we attempted to determine the effects of
EGCG on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in mouse
skin. EGCG administered by gavage inhibited COX-2 expression in TPA-treated mouse skin.
To further elucidate the underlying mechanisms of COX-2 inhibition, we examined the
effects of EGCG on the activation of different transcription factors that regulate COX-2
expression. While EGCG inhibited TPA-induced DNA binding of NF-kappaB and cyclic
AMP response element binding protein (CREB), the compound failed to block the activation
of AP-1 and CCAAT/enhancer binding protein (C/EBP) in TPA treated-mouse skin. EGCG
diminished TPA-induced phosphorylation and degradation of IkappaBalpha and nuclear
translocation of p65. Moreover, TPA-induced activation of extracellular signal-regulated
protein kinase and p38 mitogen-activated protein (MAP) kinase was suppressed by EGCG.
Pretreatment of mouse skin with pharmacological inhibitors of MAP kinases revealed that
SB203580, but not U0126, inhibited TPA-induced CREB DNA binding. Taken together, our
study indicates that EGCG inhibits TPA-induced COX-2 expression and activation of NF-!B
and CREB in mouse skin by downregulation of p38 MAP kinase.
- 600 -

Session XVI : Chemopreventive agents Poster XVI, 9
Wogonin, a plant flavone, potentiates etoposide-induced apoptosis in tumor cells
Eibai Lee1, Riyo Enomoto1, Chie Suzuki1, Masataka Ohno1, Toshinori Ohashi1, Azusa
Miyauchi1, Eriko Tanimoto1, Kaori Maeda1, Hiroyuki Hirano2, Toshio Yokoi2 and
Chiyoko Sugahara1
1Department of Pharmacology and 2Department of Pharmaceutical Chemistry, Faculty
of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe
651-2180 Japan. E-mail : elee@pharm.kobegakuin.ac.jp
Etoposide, a podophylotoxin anticancer agent, induces apoptotic cell death in normal and
cancer cells. Etoposide-induced apoptosis plays a role in not only anticancer effect but also
adverse reaction such as myelosuppression. Since we have found that wogonin, a flavone
found in Scutellaria baicalensis, suppresses thymocyte apoptosis induced by various
compounds including etoposide, we examined the effect of this flavone on etoposide-induced
apoptosis in cancer cells. Although wogonin itself hardly affected biochemical and
morphological features of apoptosis in leukemia cells such as Jurkat and HL-60 cells, this
flavone significantly potentiated etoposide-induced apoptosis in these cells. Similarly,
wogonin accelerated etoposide-induced cell death in lung cancer cells. Since wogonin had no
effect on the action of other anticancer agents such as 5-FU and cisplatin, this flavone seems
to accelerate only apoptotic cell death induced by etoposide in cancer cells. These results
suggest that the modification of etoposide-induced apoptosis by wogonin may be available to
reduce the adverse reaction of this agent.
- 601 -

Session XVI : Chemopreventive agents Poster XVI, 10
Resveratrol inhibits the growth of PA-1 ovarian cancer cells by down-regulating
eEF1A2 expression
Mee-Hyun Lee1, Bu-Young Choi2 and Young-Joon Surh1
1National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Seoul 151-742 and 2C&C Research
Labs, Hwasung City, Gyeonggi-do 445-970, South Korea
The eukaryotic elongation factor 1A2 (eEF1A2), a subtype of eEF1A that is a key factor in
the translational process of protein synthesis, promotes the transfer of animoacylated tRNAs
to the A site of the ribosome. In human, eEF1A2 expression is restricted to the heart, brain,
and skeletal muscle. Recently, eEF1A2 has been shown to be a potential oncogene that is
over-expressed in ovarian cancer. Therefore, the regulation of inappropriate expression of
eEF1A2 is considered as a rational strategy for ovarian cancer therapy. Resveratrol (3,4’,5trihydroxy
stilbene), a phytoalexin present in grapes, has been reported to possess antioxidant,
anti-inflammatory, and anti-carcinogenic activities. In the present study, we examined the
anti-carcinogenic effect of resveratrol in PA-1 ovarian cancer cells considering eEF1A2 as a
potential target. Resveratrol inhibited the growth of PA-1 cells. Treatment of PA-1 cells with
insulin (10 µg/ml) or FBS (1%) resulted in the induction of eEF1A2. Pretreatment of PA-1
cells with resveratrol suppressed insulin- or FBS-induced expression of eEF1A2. Previous
studies have demonstrated that eEF1A2 confers anti-apoptotic effect by down-regulating
caspase-3 activity. Resveratrol was found to induce caspase-3 activity and cause apoptosis in
eEF1A2 over-expressing cells, suggesting that the induction of apoptosis by resveratrol in
PA-1 cells is mediated, at least in part, via down-regulation of eEF1A2 expression. Taken
together, these results suggest that oncogenic eEF1A2 is a potential target for ovarian cancer
chemoprevention with resveratrol.
- 602 -

Session XVI : Chemopreventive agents Poster XVI, 11
Resveratrol Inhibits IL-1#-Induced Stimulation of Caspase 3 and Apoptosis in Human
Articular Chondrocytes in vitro
Shakibaei M1, John T2, Seifarth C1, Mobasheri A3
1Musculoskeletal Research Group, Institute of Anatomy, Ludwig-Maximilian-
University Munich, Pettenkoferstrasse 11, 80336 Munich; 2Charité Medicine University
Berlin, Campus Benjamin Franklin, Department for Trauma Surgery, Berlin,
Germany; 3Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of
Veterinary Science, University of Liverpool, United Kingdom.
Resveratrol (trans-3,4´-trihydroxystilbene) is a polyphenolic phytoalexin that is present in
various fruits, in the skin of red grapes, peanuts and root extracts. Recent studies have shown
that resveratrol exhibits potent antioxidant properties and is able to exert anti-inflammatory
and anti-catabolic properties in several cell types. The pro-inflammatory cytokine interleukin
1 # (IL-1#) plays a pivotal role in the pathogenesis of osteoarthritis (OA) in humans and
animals. In this study we investigated whether resveratrol is able to block the proinflammatory
effects of IL-1#, specifically the activation of caspase 3 and subsequent
induction of apoptosis in human articular chondrocytes.
Cultures of human articular chondrocytes were pre-stimulated with 10 ng/ml IL-1# for 1, 12
and 24 h before being co-treated with IL-1# and 100 µM/ml resveratrol or 50µM the caspase
inhibitor Z-DEVD-FMK for 1, 12 and 24 h respectively in vitro.
Resveratrol significantly increased the IL-1#-induced inhibition the attenuated expression of
cartilage specific collagen type II and signal transduction receptor integrin #1 in a time
dependent manner. Incubation of chondrocytes with IL-1# resulted in activation of the
cysteine protease caspase 3, PARP cleavage and apoptotic cell death. In this report we present
evidence from ultrastructural studies that mitochondria play a major role in IL-1#-induced
apoptosis. The treatment of human chondrocytes with IL-1# induced mitochondrial swelling
in a time-dependent manner. These changes were observed as early as 1 hour after treatment
of cells with IL-1#. These effects (activation of caspase 3, cleavage of PARP and
mitochondrial changes) were abolished through the co-treatment with resveratrol.
Furthermore, co-treatment of the IL-1#–stimulated cells with the caspase inhibitor Z-DEVD-
FMK blocked apoptosis as shown by electron microscopy and Western blotting, suggesting
that this process is a caspase-dependent pathway.
In summary, our results confirm that resveratrol is an effective in vitro inhibitor of caspase 3
and apoptosis in human chondrocytes. These findings suggest that this dietary polyphenolic
compound may have future applications in the nutraceutical based therapy of human and
animal OA.
- 603 -

Session XVI : Chemopreventive agents Poster XVI, 12
Possible link between NO concentrations and COX-2 expression in systems treated with
soy-isoflavones
Jang-In Shin 1 , Yoon-Kyung Lee 1 , Young Min Kim 2 and Ock Jin Park 1
1 Department of Food and Nutrition, Hannam University, 133 Ojeong-dong Daedeok-gu,
Daejeon 306-791, Korea, E-mail: ojpark@hannam.ac.kr
2 Department of Biological Sciences, Hannam University, 133 Ojeong-dong Daedeok-gu,
Daejeon 306-791, Korea,
The production of nitric oxide (NO) emerges as an essential determinant in auto- and
paracrine signaling. NO is known to be generated under inflammatory conditions,
carcinogenesis and circulatory shock. The large amount of NO produced in response to
cytokines plays an important role in inflammatory conditions. Cyclooxygenase (COX), the
central enzyme in prostanoid biosynthesis, is involved in the first step of prostanoid synthesis
from arachidonic acid. The reported studies to evaluate the relationship between NO and
COX-2 have revealed both inhibitory and stimulatory effects of NO on COX-2 expression.
Genistein, one of soy-isoflavones, is a polyphenolic flavonoid and a potent antioxidant and
anti-inflammatory agent. In the present study, the effect of soy-isoflavones on NO production
and COX-2 gene expression was examined. NO production by soy-isoflvaones was greatly
increased even though eNOS and iNOS expression were not different from non-treated
control. The increment of NO was accompanied with the elevated expression of COX-2 and
the concentrations of PGE 2. The COX-2 stimulatory effect of soy-isoflavones appeared to be
modulated by ERK1/2 and p38.
- 604 -

Session XVI : Chemopreventive agents Poster XVI, 13
Antiinflammatory, Antioxidative, and Chemopreventive Effects of Zerumbone, a
Sesquiterpene Derived from Tropical Ginger
Jun-Wan Shin1, Yoshimasa Nakamura2, Akira Murakami3, Hajime Ohigashi3 and
Young-Joon Surh1
1College of Pharmacy, Seoul National University, Seoul 151-742, South Korea,
2Graduate School of Natural Science and Technology, Okayama University, Okayama
700-8530 & 3Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Zerumbone (ZER), a sesquiterpene derived from tropical ginger Zingiber zerumbet Smith
(Zingiberaceae), was found to suppress chemically-induced colon and skin carcinogenesis.
Dextran sulfate sodium-induced colitis as well as production of proinflammatory cytokines
(TNF-alpha, interleukin-1 alpha and interleukin-1 beta) and prostaglandin E2 was attenuated
in mice fed ZER. In another experiment, topical application of ZER inhibited phorbol esterinduced
COX-2 expression and tumpor promption in mouse skin. Treatment of mouse
epidermal JB-6 cells with zerumbone induced the expression of heme oxygenase-1 (HO-1), a
respresentative antioxidant enxyme that degrades heme to produce iron, carbon monoxide and
biliverdin. Zerumbone-induced expression of HO-1 in JB-6 cells appears to be mediated
through antioxidant response elements (ARE)-driven activation of the redox-sensitive
transcription factor Nrf2. Likewise, zerumbone upregulated HO-1 and several other
antioxidant enzymes via ARE-Nrf2 siganling in the rat hepatic epithelial cell line. These
findings suggest that chemopreventive effects of zerumbone is attributable to its
antiinflammatory and antioxidant properties.
- 605 -

Notes
- 606 -

Session XVII : Cell signaling in health and disease
- 607 -

Session XVII : Cell signaling in health and disease Poster XVII, 1
Mitochondrial “Movement” and Lens Optics Following Oxidative Stress from UV-B
Radiation: Cultured Bovine Lens and Cultured Human Retinal Pigment Epithelial Cells
(ARPE-19) as Examples
Vladimir Bantseev and Hyun-Yi Youn
School of Optometry/Department of Biology, University of Waterloo, Waterloo, ON
N2L 3G1, Canada. E-mail: vbantsee@uwaterloo.ca
Mitochondria provide energy generated by oxidative phosphorylation and at the same time
play a central role in apoptosis and aging. As a by-product of respiration, the electron
transport chain is known to be the major intracellular site for the generation of reactive
oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and
thus production of ROS and subsequent cell death, has been implicated in a large spectrum of
skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis
generates photoreceptor dysfunction and ultimately visual impairment.
The purpose of this study was to characterize in vitro changes following oxidative stress with
UV-B radiation in 1) ocular lens optics and cellular function in terms of mitochondrial
dynamics of bovine lens epithelium and superficial cortical fiber cells and 2) human retinal
pigment epithelial (ARPE-19) cells. Cultured bovine lenses and confluent cultures of ARPE-
19 cells were irradiated with broadband UV-B radiations at energy levels of 0.5 and 1.0
J/cm2. Lens optical function (spherical aberration) was monitored daily up to 14 days using
an automated laser scanning system that was developed at the University of Waterloo. This
system consists of a single collimated scanning helium-neon laser source that projects a thin
(0.05mm) laser beam onto a plain mirror mounted at 450 on a carriage assembly. This mirror
reflects the laser beam directly up through the scanner table surface and through the lens
under examination. A digital camera captures the actual position and slope of the laser beam
at each step. When all steps have been made, the captured data for each step position is used
to calculate the back vertex distance for each position and the difference in that measurement
between beams. To investigate mitochondrial movement, the mitochondria-specific
fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510
(configuration Meta 18) confocal laser scanning microscope (Carl Zeiss Inc., Toronto
Canada) equipped with an inverted Axiovert 200M microscope and 40-x water-immersion C-
Apochromat objective (NA 1.2).
The optical analysis showed energy level-dependent increases in back vertex distance
variability (loss of sharp focus) from 0.39±0.04mm (control, n=11) to 1.63±0.33mm (1.0
J/cm2, n=10) and 0.63±0.13mm (0.5 J/cm2, n=9). Confocal laser scanning microscopy
analysis of both bovine lenses and ARPE-19 cells showed that following treatment at 0.5
J/cm2 the mitochondria stopped moving immediately whereas at 1.0 J/cm2 not only did the
mitochondria stop moving, but fragmentation and swelling was seen. Untreated control tissue
exhibited up to 15µm/min of movement of the mitochondria. This could represent normal
morphological change, presumably allowing energy transmission across the cell from regions
of low to regions of high ATP demand. Lack of mitochondrial movement, fragmentation and
swelling of mitochondria may represent early morphological changes following oxidative
stress that may lead to activation of caspase-mediated apoptotic pathways.
- 608 -

Session XVII : Cell signaling in health and disease Poster XVII, 2
2-Methoxyestradiol inhibits superoxide anion generation while enhaces superoxide
dismutase activity in swine granulosa cells
Giuseppina Basini, Sujen E. Santini, Francesca Grasselli
Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualità e Sicurezza
degli Alimenti, Sezione di Fisiologia Veterinaria, Università di Parma, 43100 Parma,
Italy. E-mail: basini@unipr.it
Angiogenesis and antiangiogenesis physiologically take place during ovarian follicular
growth and regression. Antiangiogenesis has been the subject of intense interest because of its
implications for restricting tumor growth. 2-Methoxyestradiol (2-ME) is an estradiol
metabolite with antiangiogenic and antitumor activity. It is formed by granulosa cell and is
present in the normal follicle at high concentrations. In this unique microenvironment, it may
regulate selected cell types via autocrine and/or paracrine action. Identification of reactive
oxygen specie role in the molecular pharmacology of 2-ME is an active area of research. To
this purpose we evaluated the effect of 2-ME on both superoxide anion (O2-) production
(WST-1 test, Roche, Mannheim, Germany) and on superoxide dismutase activity (SOD Assay
kit Dojndo Molecular Technologies, Japan) in swine granulosa cells. Cells were obtained
from follicles > 5 mm, seeded in 200 ul M199 in 96well plates and treated for 48 h with 1 uM
2-ME. O2- assay was performed on 104 cells/well and 20 ul WST-1 were added during the
last 4 h of incubation. SOD assay was performed on 2'105 cells/well; after treatment, cells
were lysed and assayed for enzyme activity. In both assays the absorbance was read at 450
nm against 620 nm. 2-ME inhibited (p

Session XVII : Cell signaling in health and disease Poster XVII, 3
Hypoxic responses in acute myeloid leukaemia (AML)
Siv Lise Bedringaas, Kimberley Hatfield, Gry Sjøholt, Lars Helgeland#, Jørn Skavland,
Olav Dahl§, Øystein Bruserud, Bjørn Tore Gjertsen
Hematology section, Institute of Medicine, University of Bergen and Department of
Internal Medicine, Haukeland University Hospital, N-5021 Bergen. #Department of
Pathology, The Gade Institute, University of Bergen, Haukeland University Hospital.
§Section of Oncology, Institute of Medicine and Department of Oncology, Haukeland
University Hospital, Bergen, Norway.
Acute myeloid leukaemia (AML) is a haematological malignancy characterized with block in
differentiation and numerous, but also recurrent, chromosomal translocations. The most
common recognized mutation (approx. 30%) is in the class III receptor FMS-related tyrosine
kinase 3 (Flt3/Stk1/Flk2). Even though AML blasts are in a normoxic environment in the
peripheral blood, our hypotheses is that excessive growth of malignant blasts in the bone
marrow create a hypoxic environment. Tumour hypoxia is known to promote chemo
resistance and aggressive disease.
In this study we have investigated the hypoxic effect in cryopreserved primary AML cells and
AML cell lines (HL-60, NB4) by Western blot, ELISA, flow cytometry, two-dimensional gel
electrophoresis (2DE) and mass spectrometric identification of proteins, as well as the twodimensional
differential gel electrophoresis (DIGE). In particular the protein levels of
Hypoxia Inducible Factor-1" (HIF-1"), vascular epithelial growth factor (VEGF),
osteopontin (OPN), cyclin-dependent kinase inhibitor 1A (CDKN1A/p21/CIP1), and Flt3
were also investigated in the patient samples, and HDM2 in cell lines were examined.
We investigated AML cells in hypoxia (1% O2) and Co2+ that mimics hypoxic conditions.
Preliminary results show that 30% of patients (10/30) and both cell lines responded to
hypoxia with increased protein level of HIF-1". More than 50% of the patients responded
with VEGF production (17/30), but a significant fraction of the VEGF responders were not
HIF-1" responders (7/17). The VEGF non-responders had non-detectable levels of VEGF
before and during hypoxia. Approximately 50% of the patients responded with OPN increase.
CDKN1A/p21 and Flt3 showed no response to hypoxia in any patients. The level of HDM2
was reduced in both cell lines tested during hypoxia.
Preliminary results suggest that DIGE is more sensitive than conventional 2DE in detection of
protein modulation induced by hypoxia (1% O2) and Co2+. Ongoing experiments are aimed
to map the protein networks that differ between hypoxic HIF-1" responsive and nonresponsive
AML.
- 610 -

Session XVII : Cell signaling in health and disease Poster XVII, 4
Strongly reduced density of parvalbumin-immunoreactive projection neurons in the
mammillary bodies in schizophrenia: further evidence for limbic neuropathology
Hans-Gert Bernstein1, Stephanie Krause2, Dieter Krell1, Henrik Dobrowolny1, Renate
Stauch1, Karin Ranft1, Peter Danos2 and Bernhard Bogerts1
1Department of Psychiatry, University of Magdeburg, Leipziger Str. 44, D-39120
Magdeburg and 2Department of Psychiatry, 2University of Giessen, Germany; E-mail:
Hans-Gert.Bernstein@medizin.uni-magdeburg.de
The mammillary bodies are important relay nuclei within limbic and extralimbic connections.
They receive information from the hippocampus and are reciprocally connected to the anterior
thalamic nuclei, the septum and the midbrain. The mammillary bodies are known to play roles
in memory formation and are affected in alcoholism and vitamin B1 deficiency. Their
strategic position linking temporolimbic to frontothalamic brains structures make the
mammillary bodies candidates for alterations in schizophrenia. We studied 15 postmortem
brains of schizoprenics and 15 matched control brains from the Magdeburg Brain Collection.
A demographic and clinical history was available for all cases. Brains were fixed in formalin,
embedded in paraffin and cut into 20 µm thick coronal sections. Brain sections were stained
either for Heidenhain-Woelcke, calretinin or parvalbumin. We determined the volumes of the
mammillary bodies and performed cell countings using stereological principles and a
computerized image analysis system. Our volumetric calculations revealed that the volumes
of mammillary bodies do not differ between schizophrenics and controls. However, in
schizophrenia the neuronal densities were significantly reduced on both sides (on left side by
35.9%, on right side by 22 %). No changes were seen in the density of calretinin-containing
neurons, whereas the number of parvalbumin-immunoreactive mammillary neurons was
profoundly reduced (by more than 50%). This cell loss (as a result of developmental
malformation and/or neurodegeneration) points to a prominent involvement of the
mammillary bodies in the pathomorphology of schizophrenia. Parvalbumin-immunoreactive,
GABAergic interneurons have repeatedly been reported to be diminished in several brain
regions in schizophrenia. However, in the mammillary bodies parvalbumin labels a
subpopulation of glutamate/aspartate-containing neurons projecting to the anterior thalamus.
Thus, our data provide new evidence for impaired limbic circuits in schizophrenia.
Supported by NBL-3 and Stanley-Foundation.
- 611 -

Session XVII : Cell signaling in health and disease Poster XVII, 5
Sustained gene transfer with photochemical internalisation in breast cancer cells.
Sanae Bouali, Jihane Mirouah, Jean L. Merlin
Laboratoire de recherche en oncologie, EA 3452, Centre Alexis Vautrin, Avenue de
Bourgogne, 54511 Vandoeuvre Les Nancy cedex, France. E-MAIL :
s.bouali@nancy.fnclcc.fr
Although the vectors used to deliver therapeutic genes to targeted cells have traditionally been
modified viruses, non viral synthetic vectors are also receiving much attention as an
alternative method. Most non viral gene therapy vectors deliver transgene into cells through
the endocytic pathway. Lack of escape from endocytic vesicles in many cases constitutes a
barrier for delivery of the transgene. A new biotechnology method was developed named
photochemical internalisation (PCI). PCI has been described as an innovative procedure for
releasing macromolecules from endocytic vesicles to the cytosol of target cells. This
technology is based on light induced delivery of macromolecules such us DNA, proteins, and
other therapeutic molecules in a cytosol.
METHODS: Human breast cancer cells were preincubated with the photosensitiser mesotetraphenylporphine
(TPPS2a) followed by treatment with plasmid encoding enhanced Green
Fluorescent Protein complexed with Polyethylenimine or Eudragit RL or transfectin and light
exposure. The expression of the GFP gene was scored by flow cytometry.
RESULTS: The photochemical using light doses improve the efficiency of transfection
mediated by polyethylenimine , but not by Eudragit RL and transfectin. This study was
designed to optimise the PCI of Polyethylenimine, Eudragit RL and tranfectin-DNA
complexes gene transfer in vitro.
CONCLUSION: In the present study, results confirm the potency of combining the PCI with
Polyethylenimine mediated gene transfer.
- 612 -

Session XVII : Cell signaling in health and disease Poster XVII, 6
GENETIC AND EPIGENETIC CONTROL OF HEMATOPOIETIC STEM CELLS
PROLIFERATION.
Leonid V. Bystrykh, Alice Gerrits, Leonie M. Kamminga, Ronald van Os, Albertina
Ausema, Ellen Weersing, Bert Dontje, Gerald de Haan.
Department of Cell Biology, Section Stem Cell biology. University Medical Center
Groningen, University of Groningen, 9713 AV Ant.Deusinglaan 1, The Netherlands.
Previously, we identified two major loci controlling HSC behaviour, namely Scp2 at ch11
(55-85 Mb) affecting proliferation, and a locus at ch18 (12-32 Mb) affecting HSC pool size.
Genome-wide microarray-based profiling of gene expressions using mRNA isolated from
murine hematopoietic stem cells was deposited in the www.genenetwork.org database [1].
Having used a set of 30 recombinant BXD inbred lines we were able to map controlling loci
(QTLs) for thousands of transcripts. From a list of 12422 probe sets on the Affymetrix Murine
Genome U74Av2 GeneChip we identified 3615 transcripts with heritable variation in
expression levels. Of these, 399 were highly significantly cis acting (cutoff -10logP 2.93)
and 3216 trans-acting transcripts. Our studies with BXDs suggest that the HSC controlling
locus on ch18 is likely to be regulated by chromatin remodeling genes.
It is known that serial transplantation of mouse HSC causes gradual deterioration of stem cell
function, reminiscent with senescence phenotype. Our microarray analysis of serially
transplanted HSC showed 300 genes to be either up- or downregulated. Comparison of these
data with recently published microarray data, allowed us to identify essential components of
a genomic network operating in HSCs. These include components of PcG and TrxG
epigenetic complexes, which appear to cross talk with Shh, Wnt ant Notch pathways.
Collectively, these genes are largely responsible for maintenance and proliferation of HSC.
Currently we are verifying our network by selective retroviral overexpression of some
essential genes (Ezh2, Msi1h).
Literature
[1] Bystrykh L, Weersing E, Dontje B, Sutton S, Pletcher MT, Wiltshire T, Su AI, Vellenga
E, Wang J, Manly KF, Lu L, Chesler EJ, Alberts R, Jansen RC, Williams RW, Cooke MP, de
Haan G. Uncovering regulatory pathways that affect hematopoietic stem cell function using
'genetical genomics'. Nat Genet. 2005;37(3):225-32.
- 613 -

Session XVII : Cell signaling in health and disease Poster XVII, 7
Expression and role of phosphatidylcholine-specific phospholipase C (PC-PLC) in
human NK cells and its relationship with CD16 receptor.
Serena Cecchetti, Francesca Spadaro, Franca Podo and Carlo Ramoni.
Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale
Regina Elena 299, 00161, Rome Italy.
Phosphatidylcholine hydrolysis by a specific phospholipase C (PC-PLC) is a widespread
response elicited by most growth factors, cytokines, neurotrasmitters, hormones and other
extra-cellular signals. Our previous studies showed that PC-PLC is involved in the functional
role of NK-mediated cell killing and in the lytic granules exocytosis process. PC-PLC was
detected both on specific cytoplasmic compartments and on the outer membrane surface, in
which the enzyme translocated upon cell activation and lytic ability maturation.
Interestingly, PC-PLC expression on NK membrane surface was directly proportional to that
of CD16, the low-affinity receptor for the Fc fragment of IgG, suggesting a possible
correlation between the enzyme and NK cell maturation.
In the present work we analyzed the trafficking from the plasma membrane to cytoplasmic
regions of CD16 receptor and PC-PLC enzyme. Both proteins were internalized upon
antibody engagement, degraded and newly synthetized, moreover, they needed an integral and
functional actin cytoskeleton during their trafficking. Pre-incubation of NK cells with the PC-
PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) determined a dramatic decrease
of PC-PLC fraction expressed on NK plasma membrane. Surprisingly, D609 also downmodulated
the membrane expression of CD16 receptor. Among phenotype markers of
peripheral blood lymphocytes analyzed after D609 treatment, only CD16 was deeply downmodulated,
thus suggesting a possible specific role of PC-PLC enzyme in regulating CD16
receptor expression. Moreover, we demonstrated that PC-PLC was activated, within 5
minutes, upon CD16 stimulation, thus suggesting an involvement of the enzyme in CD16
signal transduction. We also evidenced that in antibody-dependent cellular cytotoxicity
process PC-PLC played a double functional role both in regulating CD16 membrane
expression and in transducing CD16 receptor signals, since D609 totally abolished this
important lytic mechanism.
- 614 -

Session XVII : Cell signaling in health and disease Poster XVII, 8
Different ways to arrest proliferation of cancer cells by dsRNA
Elena L.Chernolovsksya, Tatyana O. Kabilova, Al`bina V. Vladimirova, Valentin V.
Vlassov
Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiav ave., 8,
Novosibirsk 630090, Russia, E-mail: elena_ch@niboch.nsc.ru
Deregulation of genes encoding components of signaling pathways are considered as a key
factor in the development of different types of tumors in humans. We investigated inhibition
of MYC genes by dsRNAs in KB-3-1, SC-N-MC and IMR-32 cells. Sequence-specific
siRNA (si3ex) targeted to the third exon of c-myc gene was found to decrease the level of cmyc,
but not N-myc mRNA. si3ex decreased the rate or even arrested the proliferation of cmyc
overexpressing cell lines KB-3-1 and SC-N-MC, but did not affect the proliferation of
IMR-32 (which overexpress N-myc). si2ex homological to the conservative region of the
second exon of both c- and N-myc was able to downregulate both genes and to reduce
proliferation of KB-3-1, SC-N-MC and IMR-32 cells. PKR or/and OAS1 mRNA levels were
not effected. Long double stranded RNAs and some short dsRNA can stimulate innate
cytokine responses in mammals, which can induce proliferation blockage, differentiation or
apoptosis in cancer cells. Long double stranded RNAs: dsMyc homologous to the 3 exon of cmyc
gene, dsGFP homologous to mRNA of EGFP gene and GU-rich siRNA (siI),
homologous to the intron sequence of human MDR1 gene were found to inhibit proliferation
and to decrease the mRNA level of interferon-sensitive genes: c-myc and beta-actin when
delivered into cancer human cells. The level of downregulation was march higher than that of
synthetic interferon inducer poly(I:C) and is likely depends on the properties of dsRNA: the
thermodynamic stability, nuclease resistance and affinity to dsRNA-binding proteins.
Antiproliferation activity of long dsRNA, displayed in the absence of transfection reagent,
was cell line specific and correlated with the ability of dsRNA to inhibit the expression of cmyc
and beta-actin genes. The elevation of PKR or/and OAS1 mRNA levels was detected in
all cells affected by long dsRNA and does not correlates with proliferation blockage. The data
suggest, that double stranded RNAs can serve as antiproliferative agents, acting sequencespecifically
or activating innate immunity response.
This work was supported by RFBR (grant No. 06-04-49480- ), RAS programs “Molecular
and Cellular Biology” and “Fundamental science for medicine”, Interdisciplinary grant from
SB RAS, FCSTP RI-012/001/254.
- 615 -

Session XVII : Cell signaling in health and disease Poster XVII, 9
Progressive Factors in Uterine Cervical Cancer associated with High-risk Human
Papillomavirus
Yoon Pyo Choi*, Suki Kang, Owen Ding*, Nam Hoon Cho*
Dept of Pathology, Yonsei University College of Medicine
Brain Korea 21 Project for Medical Science, Yonsei University*
Human papillomavirus (HPV) infections play a crucial role in the progress of cervical cancer.
In the present study, we aimed to establish the proteomic profiles and characterization of the
tumor related proteins by using 2-DE and MALDI-TOF MS in 6 cervical swab samples,
which were obtained from the patients with infection of HPV 16 and 18. We compared
nuclear protein and cytoplasmic protein, separately, by using the subcellular fraction in the
proteomic analysis. On comparison of differential protein spots between cervical cancer
tissues with HPV 16 or HPV 18 and HaCaT cell lines as the control, we confirmed that
proteins, such as PCNA, 14-3-3 sigma, CDC25A, Mdm2, Mdm4, minimal chromosomal
maintenance (MCM) 3, MCM4 and MCM8 according to surrogate endpoint biomarkers
(SEB), were upregulated in nuclear fractions of cervical cancer tissues and that also CDC25B
was downregulated in nuclear fractions, whereas being upregulated in cytoplasmic fractions
of the cervical cancer tissues. This subcellular shuttling protein, CDC25B, indicates that it is
probably associated with G2 arrest in the carcinogenesis of cervical cancer by HPV infection.
And then, for validation of proteomic analysis, on application of candidates to have been
screened in the proteomic analysis on 201 archival samples of cervical neoplasia associated
with various HPV types infection (75 CIN 1, 28 CIN II, 82 CIN III and 16 cancer) by
immunohistochemistry, PCNA, MDM2, CDC25A, MCM4 and MCM7, were found to be
significantly correlated to the disease progression. MCM8 also revealed one of the most
specific and correlative markers to the disease progression and even to the poor outcome of
the cervical cancer. In conclusion, we have demonstrated that HPV can cause the unstable
regulation of a lot of carcinogenesis-related proteins, such as proliferation factors, cell cycle
regulatory factors, cell signaling factors, oncogenes etc. without the significant difference of
any specific molecules being found according to the HPV genotypes, in the ongoing process
of cervical cancer. MCM8 appears to be another novel marker in addition to well-known
biomarkers to predict poor outcome in uterine cervical cancer. Until a marker of such
versatility is found, knowing everything possible about the markers that we have is crucial,
and this study has shown some new findings about these known markers.
- 616 -

Session XVII : Cell signaling in health and disease Poster XVII, 10
Androgens induce while PPAR-ligands inhibit prostate cancer cells migration.
Joanna Duli"ska-Litewka, Dorota Gil and Piotr Laidler
Department of Medical Biochemistry, Jagiellonian University Medical College, ul.
Kopernika 7, 31-034 Kraków, POLAND; e-mail: mblitewk@kinga.cyf-kr.edu.pl,
tel/fax:+48(12)4223272
Purpose: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes which
degrade the extracellular matrix or components of the basement membrane. MMP have been
found to be associated with prostate cancer metastasis. Prostate growth, survival and
pathology is generally dependent on androgen stimulation mediated by the androgen receptor
(AR). Several ligands of peroxisome proliferators activated receptors (PPARs) were shown to
decrease the proliferation and enhance apoptosis in various cancers, also in prostate cancer
thus somehow opposing androgen effects. Therefore we sought to determine if and how
androgens and PPAR-ligands influence MMPs expression and prostate cancer cells migration.
Methods: Studies were carried out on human prostate cancer cell lines: LNCaP, Du-145 and
PC-3. We studied the effect of testosterone and PPAR$ ligands (ciglitazone, arachidonic acid)
on cells’ migration (Boyden’s chamber), expression and activity of MMPs (zymography) and
selected proteins engaged in cell signaling (RT-PCR, Western blot). The antagonist of PPAR$
– GW9662 and inhibitor of 5"-reductase – finasteride were also included.
Results: Treatment of LNCaP cells with testosterone resulted in an increase of pro-MMP-2
expression and MMP-2 activity and led to the increased proliferation and decreased
expression of PPAR$. Finasteride clearly suppressed this effects. Selective PPAR$ ligands
significantly decreased proliferation, migration and the level of active MMPs. RT-PCR and
Western blot analyses basically indicated the opposite effects of testosterone and ciglitazone
on expression of AR, PPAR$, c-myc and Akt. Inhibition of the expression of phosphorylated
Akt by ciglitazone did not affect total Akt levels, indicating the functional effect of PPAR$
ligand on Akt signal transduction.
Conclusions: The results suggest that the expression and regulation of the activity of AR and
PPAR$ significantly and clearly in opposite manner affect prostate cancer cell proliferation,
migration and MMP expression. It seems that important signaling step in androgen-induced
migration might be inhibited by PPAR$ ligands. Moreover, Akt signal transduction seems to
be linked to AR and PPAR ligands modulation of these basic cell properties through as yet
not fully recognized mechanism. This results may provide a novel target of PPAR$ activators
and indicate their potential as useful therapeutic agents in the treatment of prostate cancer.
This work was financially supported by the KBN: Jagiellonian University Medical College –
grant W,/253/P/L and the State Committee for Scientific Research - grant No 6 PO5A 074 21,
Poland
- 617 -

Session XVII : Cell signaling in health and disease Poster XVII, 11
The orphan nuclear receptor RORalpha activity is regulated by post-translational
modifications : the role of phorbol esters and protein kinase C.
E. DUPLUS, V. SOUBEYRE, Y. LEMAIGRE-DUBREUIL, B. BRUGG
Nuclear receptor superfamily includes ligand-dependant transcription factors that control
numerous genes involved in differentiation, proliferation and cell homeostasis. Some of these
receptors have also been described to be regulated by post-translational modifications like
phosphorylations. For a long time, Retinoid-related orphan receptor alpha (RORalpha) has
been considered as a constitutively active transcription factor with no known ligand nor
effector. However, recent new data suggest that this nuclear receptor is controled by ligand
binding. It is expressed in a wide range of organs, particularly in the cerebellum where it
seems to be implicated in neuronal survival and differentiation. No regulatory pathways of
these biological functions have been described to control RORalpha activity so far. The aim
of our work is to identify different signaling protein kinases, usually described to regulate
nuclear receptor, to directly modulate RORalpha transcriptionnal activity. Among these
proteins, some of protein kinase C isoforms (PKCs) are of great interest considering their
involvement, as RORalpha, in neurone survival and differentiation. In this study, we show
that PKC activator, Phorbol 12-Myristate 13-Acetate (PMA) inhibits RORalpha activity in
COS-7 cell line. This effect is totally prevented by PKC inhibitor Bisindolylmaleimide 1
suggesting that PKCs are involved in PMA effect. Western blot experiments in COS-7 cells
demonstrated that PKC are also implicated in post-translational modifications of RORalpha
leading to an electrophoretic migration shift. In order to identify theses changes, we carried
out metabolic labeling experiments which suggested that 1) RORalpha is a phosphoprotein, 2)
Its phosphorylation state is enhanced by PKC activation in COS-7 cells
Thus, these data constitute the first evidence of PKC-dependent RORalpha phosphorylation
and inhibition of its transcriptional activity. They open important therapeutic prospects,
especially for neurodegenerative diseases.
- 618 -

Session XVII : Cell signaling in health and disease Poster XVII, 12
Are key factors of signalling intracellular pathways involved in the regulation of
neuromuscular changes after disuse conditions ?
Erwan Dupont, Marie-Hélène Canu, Maurice Falempin, Yvonne Mounier and Laurence
Stevens
Laboratoire de Plasticité Neuromusculaire, EA 1032, IFR 118, Unité de Neurosciences et
Physiologie Adaptatives, Bât SN4, Université des Sciences et Technologies de Lille,
59655 Villeneuve d’Ascq cedex, France
The performance of the neuromuscular system is highly dependent on activity. A situation of
disuse induced by hypodynamia-hypokinesia (HH) in rats induces profound changes in
skeletal muscles like phenotype transformations, and a marked atrophy resulting from a loss
in mass and protein content. HH is also a model of hindpaw sensory deprivation since in these
conditions, the cutaneous receptors located on the foot sole are deactivated. HH produces a
cortical reorganisation of the somatosensory cortex (SmI), indicating the existence of a
neuronal plasticity.
The factors responsible for these impairments are still unknown and the cellular and
molecular processes sustaining this “plasticity” are not well described. The major aims here
were to better understand the mechanisms i) that characterized muscular plasticity,
phenotypical transitions and atrophy, and ii) involved in cortical plasticity (sensory
conduction and/or cortical excitability changes, regulation of neurotransmitter and
neurotrophin expression). Among multiple intracellular signalling pathways, one major is
implied in controlling more specifically muscle and/or nervous plasticity : the MAPKinase
(Mitogen-Activated Protein Kinase) cascade. We induced modulations of transformations
using different periods of disuse (from 7 to 28 days of HH) in rats. Key markers of
MAPKinase cascade were followed : ERK, JNK and p38. Their protein expressions were
evaluated by western blots (using specific antibodies) performed on all the animal groups, as
well at the muscular as at the cortical levels. For the muscles, the expression of all MAPK
markers was increased in a time-dependent manner of disuse, excepted for 14 days of HH, the
increase being lower than for 7 and 28 days. At the cortical level, the increase was less
pronounced and not disuse-dependent.
Taken together, these results may help to develop strategies that aim at attenuating the effects
of pathologies, like long-term immobilizations, on the neuromuscular system.
- 619 -

Session XVII : Cell signaling in health and disease Poster XVII, 13
#1 - INTEGRIN REGULATION OF MECHANOTRANSDUCTION IN
OSTEOARTHRITIC HUMAN ARTICULAR CHONDROCYTES.
Kerry J. Elliot*, Tina T. Chowdhury** and Donald M. Salter*.
* The Queen’s Medical Research Institute, The University of Edinburgh, 47 Little
France Crescent, Edinburgh, UK, EH16 4TJ. E-mail: kelliot@staffmail.ed.ac.uk
** Queen Mary, University of London, Department of Engineering, Mile End Road,
London, UK, E1 4NS.
Introduction: Normal and osteoarthritic (OA) human articular chondrocytes (HAC) respond
to 0.33 Hz cyclical mechanical stimulation (MS) via a "5#1 integrin mediated
mechanotransduction pathway although downstream signalling pathways are different. In
both normal and OA cells, the pathway involves tyrosine kinase activity and protein kinase C
(PKC). However, while normal HAC respond to 0.33 Hz mechanical stimulation with
increases in aggrecan gene expression, aggrecan mRNA levels of OA HAC are unchanged.
Experiments were performed utilising a panel of function-modifying antibodies to investigate
whether modulation of #1 integrin function influenced OA chondrocyte mechanotransduction.
Methods: OA HAC were incubated with function modifying #1 integrin receptor antibodies
JB1a, P4C10 and TS2/16 for 30 min prior to 0.33 Hz MS. RNA was extracted 24 hours post-
MS and relative levels of aggrecan mRNA were assessed by semi-quantitative RT-PCR. PKC
translocation and FAK phosphorylation in response to MS were assessed by western blotting.
Results: Treatment with anti #1 integrin antibodies for 30 mins did not alter aggrecan mRNA
levels, however following 24 hours antibody incubation significant decreases in aggrecan
mRNA levels were seen with the #1 integrin antibody P4C10 but not JB1a. Following MS,
there was no alteration in aggrecan mRNA levels in untreated OA cells and MS had no effects
on P4C10 down-regulation of aggrecan mRNA levels. Cells treated with JB1a showed a
significant increase in aggrecan mRNA levels following MS. 30 seconds MS of chondrocytes
resulted in translocation of PKC- and PKC. to the cell membrane which is inhibited by
P4C10 but not by JB1a or TS2/16. 1 minute MS induced FAK phosphorylation which was
unaffected by P4C10 or TS2/16. In the presence of JB1a a second enhanced phosphorylation
of FAK was seen following 5 minutes MS.
Conclusions: The results suggest that HAC mechanotransduction can be manipulated by
function modifying #1 integrin receptor antibodies. The results also suggest that JB1a, in
conjunction with MS acts in a positive manner raising the possibility that anti #1 integrin
therapy may be used to alter the balance between catabolic and anabolic activity in
osteoarthritis.
- 620 -

Session XVII : Cell signaling in health and disease Poster XVII, 14
Menin protracts Chk1 phosphorylation and increases the ratio of homology-directed
DNA repair
Esposito I.1, Gallo A.2, Terrazzano G.1 and Musti A.M.1,3
1 Dip.Biol.Patol.Cell.Mol., University of Naples “FedericoII, Italy
2 I.E.O.S. of CNR c/o University of Naples “FedericoII”, Italy
3 Dip. Farmaco-biologico, University of Calabria, Rende (Cs), Italy
Multiple Endocrine Neoplasia type 1 (MEN1) is an inherited tumor disease characterised by
pancreatic, parathyroid and anterior pituitary adenomas Besides, many MEN-1 patients also
develop tumors in a range of non-endocrine tissues, suggesting that MEN1 may control tumor
development in different tissues, depending on genetic interactions with other cancerassociated
genes. Menin, the nuclear protein encoded by the MEN1 gene, interacts with a
large number of proteins, among which nuclear proteins involved in chromatin modification
or DNA repair, as Tritorax, RPA and FANCD2. Besides, loss of menin expression leads to
increased sensitivity to DNA damage, both in embryonic mouse fibroblasts and Drosophila
larvae.
We have investigated the effect of Menin over-expression on the cellular response to DNA
damage. We found that menin protracts the ATM/ATR-dependent phosphorylation of the Sphase
checkpoint kinase Chk1, in response to etoposide or UV radiation, without affecting the
rate of DNA damage. However, menin-induced persistence of Chk1 activation had no effect
on either etoposide-induced S-phase arrest or apoptosis, but increased the ratio of
homologous-directed DNA repair. These results provide novel insights into the
oncosuppressor activity of MEN-1 by linking menin to DNA-repair pathways.
- 621 -

Session XVII : Cell signaling in health and disease Poster XVII, 15
Nogo-A promotes denervation in an amyotrophic lateral sclerosis model
Anissa Fergani1, Natasa Jokic1, Jose-Luis Gonzalez De Aguilar, Leda Dimou2, Shuo
Lin3, Markus A. Ruegg3, Martin E. Schwab2, Luc Dupuis1 & Jean-Philippe Loeffler1
1Laboratoire de Signalisations Moléculaires et Neurodégénérescence, INSERM U-692,
Université Louis Pasteur, Faculté de Médecine, 11 rue Humann, 67000 Strasbourg,
France,
e-mail: anissa.fergani@neurochem.u-strasbg.fr, jnataly@yahoo.com,
loeffler@neurochem.u-strasbg.fr, 2Brain Research Institute, University of Zurich and
Department of Biology, ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich,
Switzerland, schwab@hifo.unizh.ch, 3Biozentrum, University of Basel,
Klingelbergstrasse 70, CH-4056 Basel, Switzerland,
e-mail: shuo.lin@unibas.ch, markus-a.ruegg@unibas.ch.
The pathogenesis of ALS still remains unclear. Growing evidence suggests that initial
alterations in skeletal muscle, preceding the onset of disease symptoms, are related to a loss of
neuromuscular junction integrity, axonal degeneration and muscle denervation, rather than
motor neuron death. Our previous studies showed a characteristic expression pattern of the
three major isoforms of the neurite outgrowth inhibitor Nogo (including Nogo-A, -B and -C)
in skeletal muscles of ALS patients and SOD1(G86R) mice. We found that the increased
levels of Nogo-A and Nogo-B, which had been barely detectable in muscles of control
subjects, correlate significantly with the severity of motor impairment of ALS patients, as
determined by the clinically validated ALS functional rating scale. We wished to determine
the impact of knocking down Nogo-A on the progression of ALS pathology in SOD1(G86R)
mice and gain insight into the role of Nogo up-regulation in skeletal muscle. We crossbred
mice knockout for Nogo-A with mice overexpressing the ALS-related mutation G86R, and
followed the survival time. We also performed in vivo skeletal muscle transfection of a vector
expressing Nogo-A in Thy-1/YFP mice, and looked at the morphology of the neuromuscular
junction (NMJ). Double-transgenic G86R/Nogo-A(-/-) mice survived longer than
G86R/Nogo-A(+/+) mice. Transient expression of Nogo-A in skeletal muscle fibers was
sufficient to injury the NMJ by inducing dismantlement of the post-synaptic structures and
loss of pre-synaptic terminals. The ectopic expression of Nogo-A in skeletal muscle initiates a
cascade of events leading to loss of NMJs and denervation. This deletereous effect may be
relevant to ALS since mice suffering from an ALS-like pathology and lacking Nogo-A live
longer. Supported by AFM and ARS.
- 622 -

Session XVII : Cell signaling in health and disease Poster XVII, 16
Molecular regulation of the expression of human A#H-J-J Locus, encoding Aspartyl-#hydroxylase,
Junctin and Junctate
Giordana Feriotto1, Alessia Finotti1, Giulia Breveglieri1, Pompeo Volpe3, Susan
Treves2, Francesco Zorzato2, and Roberto Gambari1
Dept of Biochemistry and Molecular Biology1, Dept of Experimental and Diagnostic
Medicine2, Ferrara University, Italy; Dept of Biomedical Experimental Sciences3,
Padova University, Italy; E-mail: gam@unife.it
We have recently cloned and characterised the A#H-J-J locus, a genomic sequence that
generates three functionally distinct proteins, the enzyme aspartyl #-hydroxylase (A#H),
junctin, a structural protein of sarcoplasmic reticulum membrane, and the membrane-bound
protein junctate (1, 2). The expression of the A#H-J-J locus is regulated by (a) transcription
directed by the P1 and the P2 promoters (located upstream of exon 1 and exon 2 respectively),
and (b) alternative splicing. In order to functionally characterise the two promoter sequences,
we first demonstrate that mRNAs from P1 promoter are actively transcribed in all the human
tissues and cell lines analysed, while the expression directed from the P2 promoter is tissue
specific, in particular restricted to skeletal muscle, heart and brain. The P1 region responsible
for maximal transcription contains at least twelve GC-box homologous to Sp1 consensus
binding sequence; by EMSA we identified three GC-rich elements which bind Sp family
nuclear factors with high efficiency (3). On the contrary, the P2 promoter contains sequences
which bind to different transcription factors, including MEF-2 and MEF-3 (4). The
transcription directed by the P2 promoter is induced by high expression of MEF-2 and
inhibited by HDAC4. ChIP analysis showed that MEF-2 efficiently binds chromatin of the P2
promoter in C2C12 myotubes (4). In addition, we found that A#H and junctate transcripts
exhibit single or multiple exons skipping in the region coding for the Ca++ binding domain
leading to a number of A#H and junctate mRNA variants.
1. Treves S et al. (2000) J. Biol. Chem. 275, 39555; 2. Treves S et al. (2004) J. Cell. Biol.
166, 537; 3. Mischiati C et al. (1999) J. Biol. Chem. 274, 33114; 4. Feriotto G et al. (2005)
Mol. Cell. Biol. 25, 3261.
- 623 -

Session XVII : Cell signaling in health and disease Poster XVII, 17
Combined supplementation of folic acid and vitamin E diminishes diabetes-induced
dysmorphogenesis in rat embryos
Mattias Gäreskog, Ulf J Eriksson and Parri Wentzel
Department of Medical Cell Biology, Uppsala University, Sweden
Husargatan 3, Box 571, 751 23 Uppsala
Mattias.Gareskog@medcellbiol.uu.se
Diabetic embryopathy and growth disturbances are 3-5 fold more common in infants of
diabetic mothers than in offspring of non-diabetic pregnancy. The mechanisms causing these
disturbances are likely to be multifactorial.
It has been suggested that oxidative stress and apoptosis plays an important roles during
organogenesis. This notion may also be involved in the induction of embryonic
dysmorphogenesis in diabetic pregnancy. We aimed to investigate the effect of administration
of folic acid alone or combined with vitamin E on embryonic malformations and different
markers of apoptosis, such as NFkB activity, Bcl-2, Bax protein levels and p-53 protein and
mRNA expression.
Diabetes was induced by a single injection of streptozotocin. Pregnant, non-diabetic and
diabetic, rats were treated with daily injections of folic acid or treated with both folic acid and
5% vitamin E in the diet. Untreated rats from both groups served as controls. Embryos were
collected on gestational day 10 or 11 and were evaluated with regard to malformations and
growth retardation. Some embryos were used for NFkB assay, western blot and cDNA
synthesis.
We found increased malformations and growth retardation in a diabetic milieu compared to
normal environment. Supplementation of folic acid alone and combined with vitamin E
normalized these parameters
In addition, we found decreased NFkB activity and Bcl-2 protein, increased expression of Bax
and p53 protein and mRNA in embryos of diabetic rats compared to normal rats.
Administration of folic acid to the diabetic rats increased NFkB activity and Bcl-2 protein.
Combined administration of folic acid and vitamin E normalized Bcl-2 expression in the
diabetic environment.
Combined folic acid and vitamin E supplementation to pregnant diabetic rats diminished
diabetes-induced dysmorphogenesis and had several beneficial effects on embryonic
apoptotic rate.
- 624 -

Session XVII : Cell signaling in health and disease Poster XVII, 18
Alterations in salivary antioxidants, nitric oxide, and transforming growth factor-# in
relation to disease activity in Crohn’s disease patients
1Fakhteh Ghorbani, 1Azadeh Eshghtork, 2Ali Rezaie, 1Mohammad J. Zamani,
1Shekoufeh Nikfar, 1Gholamreza Dehghan, 1Bardia Taghavi, 3Nasser E. Daryani, and
1Mohammad Abdollahi
2Department of Community Health Sciences, Faculty of Medicine, University of
Calgary, Canada, 1Department of Toxicology and Pharmacology, Faculty of Pharmacy,
and Pharmaceutical Sciences Research Center, and 3Department of Gastrointestinal
Diseases, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran,
Email: mohammad.abdollahi@utoronto.ca
It has been postulated that oxidative stress, nitric oxide (NO) and transforming growth factor
#1 (TGF- #1) have major roles in the pathophysiology of Crohn’s disease (CD). The aim of
this study was to determine the salivary levels of total antioxidant capacity (TAC), specific
antioxidants (i.e. uric acid, albumin, transferrin and thiol molecules), lipid peroxidation
(LPO), NO and TGF- #1in CD patients and control subjects and also investigate their
correlation with activity of the disease. Twenty-eight patients with confirmed CD diagnosis
were enrolled and whole saliva samples were obtained. Smokers, diabetics, those who
suffered from periodontitis, and those who were consuming antioxidant supplements were
excluded from the study. Crohn’s Disease Activity Index (CDAI) was used to determine the
severity of the disease. Twenty healthy subjects were also recruited. In CD patients significant
reductions in salivary levels of TAC (0.248±0.145 vs. 0.342±0.110 mmol/L), albumin
(1.79±0.42 vs. 2.3±0.2 µg/ml) and uric acid (3.1±1.4 vs. 4.1±2.0 mg/dl) were found. TGF-#1
was significantly increased in CD patients compared to healthy subjects (3.02±1.54 vs.
2.36±0.52 ng/ml). A four-fold increase in NO levels (198.8±39.9 vs. 50.2±21.3 µmol/L) along
with a five-fold increase in LPO concentration (0.146±0.064 vs. 0.027±0.019 µmol/L) was
documented in CD patients in comparison to control group. CDAI significantly correlated
with the TAC, LPO and the interaction between TAC and LPO (r2=0.625, r2=0.8, F test’s
P

Session XVII : Cell signaling in health and disease Poster XVII, 19
TAT fusion proteins as a drug delivery system
Mira Grdi)a, Ana-Matea Mikecin and Miroslav Pozni',
Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, 10 000 Zagreb,
Croatia Email: grdisa@irb.hr
TAT protein, a basic domain of human immunodeficiency virus type 1 (HIV-1), possess the
ability to traverse biological membranes efficiently in a process termed «protein
transduction». Protein transduction can be described as the direct uptake by the cell of
exogenous proteins/peptides, as a result of a specific property of the protein/peptide
component. The mechanism is unknown. Transduction occurs in receptor-transporter-
independent manner that appears to target the lipid bilayer directly. Thus, HIV-1 Tat proteins
have tramendous potential to deliver large-sized compounds (drugs) into the cells.
In the rpesent study, transduction of full-length Tat-p27, pt-mutated Tat-p27 and N'- Tat-p27
(truncated p27 on the C-terminal end) fusion proteins into human tumor cell lines was
examined and whether these transduced proteins induced apoptosis in the cells.
The proteins (p27, p23, Mp27) were transdused into different cell lines (NALM, MOLT, Raji,
SuDHL, K562, RKO and MiaPaCa2) and their effect on proliferation of the cells were
measured. The fusion proteins influenced moderately on the proliferation of different cell
lines and varied among the proteins and type of the cells. A transduced p27 did not
remarkable influence on proliferation of examined cell lines. Mutated p27 inhibited the
proliferation of examined cell lines up to 30 %. On the other hand, a transduction of p23
protein, truncated form of p27, inhibited the proliferation all of examined cell lines 30-60 %.
Also the effects on expression of host p27 protein were examined, as well as an influence on
induction of apoptosis.
The expression of cell cycle regulatory proteins (cycD2, D3, cycE) were not remarkable
affected with transduced proteins. It seems that transduced proteins induced apoptosis in
examined cell lines. According the results, different apoptotic pathways were induced,
depending on the type of cells.
- 626 -

Session XVII : Cell signaling in health and disease Poster XVII, 20
Apoptotic cell signaling in lymphocytes from HIV+ patients during successful therapy
Sandro Grelli,1 Emanuela Balestrieri,2 Claudia Matteucci,1 Antonella Minutolo,1
Gabriella d’Ettorre,3 Fiorella Di Sora,4 Francesco Montella,4 Vincenzo Vullo,3 Stefano
Vella,5 Cartesio Favalli,1 Antonio Mastino,6 Beatrice Macchi,2,7
1Dept. of Exp. Med. and Bioch. Sci. and 2Dept. of Neuroscience, Univ. of Rome ‘Tor
Vergata’, Via Montpellier 1, 00133 Rome, Italy. E-mail: grelli@med.uniroma2.it 3Dept
of Infect. and Trop. Dis., Univ. of Rome ‘La Sapienza’, Rome;4Clin. Immunol. Unit, S.
Giovanni Hospital, Rome; 5Istituto Superiore di Sanità, Rome; 6Dept. of Microbiol.,
Genet. and Mol. Sci., Univ. of Messina, Messina; 7IRCCS S. Lucia, Rome. Italy
The simultaneous expression of 15 genes involved in apoptotic cell signalling was analyzed
by RNA-protection assay in peripheral blood mononuclear cells of HIV-infected patients
before and during highly active antiretroviral therapy. After 12 months of successful therapy,
characterized by an increase in CD4+ T cells and a decrease in viral load and in spontaneous
apoptosis of lymphocytes, the expression of the pro-apoptotic genes FAS, FASL, FAF1,
FADD, CASPASE 8, DR3, TRAIL, TNFR1, TRADD and BAX was significantly downregulated
with respect to time 0, while that of BCL-2, BCL-XL and MCL-1 was up-regulated.
Moreover, non-parametric bivariate Spearman’s analysis of correlation showed that apoptosis
was directly correlated significantly with mRNA levels for TRAIL, FASL and CASPASE 8.
Conversely, apoptosis levels were significantly inversely correlated with those of mRNA for
BCL-2, BCL-XL and MCL-1. These results contribute to better define the signalling
pathways that control apoptosis during HIV infection, showing for the first time in vivo the
possible involvement of some death signalling mediators, such as FAF1,FADD, DR3 and
TRADD, or regulators, such as MCL-1 or BAX. Overall our data suggest that inhibition of
apoptosis in HIV+ patients under successful therapy is the results of a complex network of
signalling, involving both death and survival of lymphocytes.
- 627 -

Session XVII : Cell signaling in health and disease Poster XVII, 21
Selection of non-apoptotic human spermatozoa provides superior tyrosine
phosphorylation
during capacitation and improved acrosome reaction
Sonja Grunewald, Thomas Baumann, Uwe Paasch, Hans-Juergen Glander
EAA Center University of Leipzig, Philipp-Rosenthal-Str. 23-25, 04103 Leipzig,
Germany
sonja.grunewald@medizin.uni-leipzig.de
Introduction: Capacitation and acrosome reaction (AR) of human spermatozoa are
prerequisites for fertilization. Major changes during capacitation result from tyrosine
phosphorylations, a process regulated by a cAMP-dependent pathway involving protein
kinase A. Annexin-V-MACS is able to separate apoptotic from non-apoptotic sperm based on
their externalization of phosphatidylserine (EPS). The non-apoptotic (EPS-) fraction is
characterized by lowest amounts of caspase activation, disrupted mitochondrial potential and
DNA fragmentation. The aim of our study was to investigate the separation effect of
Annexin-V-MACS on capacitation-related tyrosine phosphorylations and acrosome reaction.
Methods: Semen specimens from 10 healthy donors were separated into 2 samples each, one
was left untreated (control) and the second was subjected to Annexin-V-MACS. Two aliquots
of both, the control as well as the EPS negative fraction after Annexin-V-MACS were
incubated in BWW at 37°C, 5% CO2 for 3h either with 3% BSA (capacitation) or without
additives. Capacitation (CAP) was monitored by tyrosine phosphorylation (TyrP, western
blot). AR was determined by labeling with mab CD46-FITC before and after stimulation with
calcium ionophore A23187, followed by flow cytometric evaluation of the percentage of
CD46+ sperm.
Results: Densitometric analyses of the 105kDa and 80 kDa bands of the TyrP western blots
demonstrated significantly higher TyrP in the capacitated aliquots compared to the noncapacitated
aliquots. Furthermore, EPS- samples presented with significant more TyrP
compared to the non-separated semen samples (TyrP: Control 100%, Control-CAP 136±30%,
EPS- 115±13%. EPS--CAP 165±34%). There was no difference in spontaneous AR in all
groups (CD46+ sperm: Control 3.5±0.7%, Control-CAP 4.8±1.2%, EPS- 3.4±1.6%, EPS--
CAP 4.9±1.9%). In contrast, after induction of AR the capacitated as well as EPS- aliquots
showed significantly increased amounts of CD46 positive sperm. AR was best inducible in
EPS- sperm after capacitation (CD46+ sperm: control 25.9±11.4%, control-CAP 44.3±9.3%,
EPS- 37.4±8.0%, EPS--CAP 55.7±15.1%).
Conclusions: Non-apoptotic human spermatozoa are characterized by superior ability to
capacitate and consequently by maximum potential to perform acrosome reaction after
stimulation. Selection of EPS negative sperm by Annexin-V-MACS may be of advantage for
assistant reproduction in order to prepare the sperm subpopulation with the highest fertilizing
potential.
- 628 -

Session XVII : Cell signaling in health and disease Poster XVII, 22
The Skeletal Muscle Transcriptome In Amyotrophic Lateral Sclerosis.
Benoît HALTER1, Luc DUPUIS1, Marc DE TAPIA1, Franck DI SCALA1, Christa
Niederhause-Wiederkehr2, Philippe Demougin2, Michael Primig2, Jean-Philippe
LOEFFLER1 & Jose-Luis GONZALEZ de AGUILAR1
1Laboratoire de Signalisation Moléculaire et Neurodégénérescence – INSERM U692 -
Faculté de Médecine - 11, rue Humann - 67085 Strasbourg Cedex FRANCE. Tel.: (+33)
390 24 30 8! Fax: (+33) 390 24 30 65. Mail: halter@neurochem.u-strasbg.fr;
gonzalez@neurochem.u-strasbg.fr; loeffler@neurochem.u-strasbg.fr
2Biozentrum and Swiss Institute of Bioinformatics, University of Basel Switzerland
The mechanism underlying ALS pathogenesis is still unclear but growing evidence suggests
that initial alterations in skeletal muscle, preceding the onset of disease symptoms, may
contribute to the disease process. We compared the muscle transcriptome of ALS-related
SOD1(G86R) mice with that of sciatic nerve-axotomized mice. We used a high-density
oligonucleotide microarray and applied advanced bio-informatics protocols to provide a high
throughput and accurate analysis of gene expression on a large scale. An unsupervised
clustering algorithm identified genes that are highly regulated in SOD1(G86R) mice. While
some of these genes represented the characteristic denervation process occurring in ALS,
others were specifically regulated under the pathological condition but not after experimental
denervation. Skeletal muscle provides an additional source of information for the
comprehension of the pathological mechanisms acting in ALS. The systematic approach at
the gene level presented herein may serve to identify new targets for therapeutic intervention
and diagnosis.
Supported by AFM (Association Française contre les Myopathies) and ARS (Association
pour la Recherche sur la Sclérose Latérale Amyotrophique).
- 629 -

Session XVII : Cell signaling in health and disease Poster XVII, 23
THE ANALGESIC AND ULCEROGENIC EFFECTS OF TRIBULUS TERRESTRIS
EXTRACTS IN ANIMAL MODELS
Mahmoud Reza Heidari, Jalal, Vafazadeh, Sayed Ahmad Hoseini , Mohammad Ghazi
Khansari and Masood Yakhchali
Dept. of Toxicology and Pharmacology, Faculty of Pharmacy, Neuroscience and
Physiology Research Center, Kerman, Iran, Heidarimr@yahoo.com
Tribulus terrestris has been used in traditional medicine as releiving reuhmatic pain and
analgesic plant from long ago. Extraction of the fruits of plant was done by two different
methods(suxheletion and perculation) with methanol 80%. The perculated extract with
different doses 50, 100, 200, 400 and 800 mg/Kg were injected intaperitoneally to mice. The
analgesic effects of the methanolic extract of this plant were evaluated by formalin and tailflick
test in male albino mice. Ulcerogenicity test was done by Azuumi method using J Score.
The dose of 100 mg/kg perculated extract had the highest analgesic effect in Formalin and
Tail-flick test. The ulcerogenicity of plant extract is lower than the indomethacin. The extracts
have had less pathologic effect in rat,s stomach.
- 630 -

Session XVII : Cell signaling in health and disease Poster XVII, 24
Expression and protective role of heme oxygenase-1 in the delayed myocardial
preconditioning
Gabor Jancso, Barbara Cserepes, Balazs Borsiczky, Andrea Ferenc, Boglarka Racz,
Janos Lantos, E Roth
Department of Surgical Research and Techniques, Faculty of Medicine, University of
Pécs, Pécs, Hungary ; e-mail: jancsogabor@hotmail.com
Ischaemic preconditioning (PC) is an endogenous adaptation response of the myocardium to
stress, whereby short ischaemic-reperfusion periods increase the myocardium’s tolerance to a
longer ischaemic attack. Heme oxygenase (HO)-1, that degrades the pro-oxidant heme to
carbon monoxide and to the antioxidant bilirubin, has been shown to protect cardiomyocytes
against oxidative injury. We aimed to demonstrate the expression and protective effect of HO-
1 in the delayed PC. Neonatal rat cardiac myocytes were exposed to ischaemic PC (ischaemic
medium for 20 min) and pharmacological (adenosine, norepinephrine, opioid)
preconditioning (group 1). 24 hours later cells were subjected to a test ischaemia (TI) –
culturing for 3 hours in ischaemic medium, following with a 2 h reperfusion in normal
medium – and then lactate dehydrogenase (LDH), live/death ratio, and apoptosis were
measured. In group 2. HO-1 enzymatic activity was competitively inhibited by administration
of zinc protoporphyrin (ZnPPIX) after PC. In group 3. HO-1 synthesis was blocked with HO-
1 siRNA before PC. In group 4. HO-1 expression was induced by administration of cobalt
protoporphyrin (CoPPIX). In the group 5. (control) we made only test ischaemia without
preconditioning. Furthermore we demonstrated HO-1 expression in the various groups with
immuno-staining. Our results showed a significant decrease of LDH release in PC groups vs.
control group, that has been risen in ZnPPIX and HO-1 siRNA treated groups. Increased
apoptosis and cell death were seen in PC groups which were treated with ZnPPIX and HO-1
siRNA. CoPPIX pretreatment resulted in decreased LDH level, apoptosis and cell death,
which was comparable to PC groups. HO-1 immuno-staining showed an appreciable HO-1
expression in PC groups, which was abolished with HO-1 siRNA administration, but not in
ZnPPIX group. Our results suggest that HO-1 expression increases in both ischaemic and
pharmacological PC, and HO-1 has cellular protective effect against cell death and apoptosis
in ischaemia-reperfusion induced oxidative injury. Supported by OTKA T-048851; OTKA
F046593; OTKA F046504; OTKA T 038035.
- 631 -

Session XVII : Cell signaling in health and disease Poster XVII, 25
Array and conventional comparative genomic hybridization reveal genomic copy
number changes are closely linked to outcome of salivary gland carcinomas.
Kowan J. Jee1 Kristiina Heikinheimo2, Silvana DiPalma3, Antti Mäkitie4, Sakari
Knuutila1 and Ilmo Leivo*1
1 Department of Pathology, Haartman Institute and Helsinki University Central
Hospital, FIN-00014 University of Helsinki, Finland
2Department of Oral and Maxillofacial Surgery, Institute of Dentistry, University of
Turku, FIN-20520 Turku, Finland
3Department of Histopathology, Royal Surrey County Hospital, Surrey, United
Kingdom (SDP)
4Department of Otorhinolaryngology, Helsinki University Central Hospital, FIN-00290
HUS (AM), Finland
Genetic abnormalities at the subchromosomal level including whole genome survey which is
capable to providing a precise abnormal localization on chromosomes are not well
documented in salivary gland carcinomas. We employed both conventional comparative
genomic hybridization (cCGH) and oligonucleotide based array CGH (aCGH) technologies to
identify genes for amplifications and deletions in salivary glands. We used eleven cases of
mucoepidermoid carcinoma (MEC), nine cases of adenoid cystic carcinoma (AdCC) and eight
cases of salivary duct carcinoma (SDC) of paraffin embedded tissues.
In cCGH, the DNA copy number changes were detected in 19 of 28 carcinomas (67.9%). The
most common gains were found on 1q and chromosome X, and the most common losses were
whole chromosome 18, on 18q only, and minimum common regions on 18q21-qter.
Interestingly,
low-grade MEC showed almost normal DNA copy number (in average 0.33 changes per
tumor) and clear contrast of the number of aberrations showed in high-grade carcinomas.
Analysis of aCGH showed a detail genomic aberration in a case of high-grade MEC in which
we compared both results. The results provided more precise genomic localization and target
genes. Additional gains of 8q21.3-qter, 19q13.12 and losses of 5q13.2-q22.2 were identified
in this case. Even in a pooled low-grade MEC, and AdCC which were shown be normal
cCGH pattern, we found an agreement results with cCGH in MEC, however, interestingly a
restricted amplification of 6p22 in AdCC was identified.
In conclusions, aCGH is a useful method to identify fine resolution of chromosomal
aberrations by using paraffin materials and the results are indicating in both methods, the
degree of DNA copy number change is directly associated with rapidly aggressive behavior of
salivary gland carcinomas.
Furthermore, the target genes may useful for developments for new drugs.
- 632 -

Session XVII : Cell signaling in health and disease Poster XVII, 26
Antioxidative effects of plant polyphenols: from protection of G-protein signaling to
prevention of age-related diseases
Viktor Jefremov1,2, Mihkel Zilmer1, Kersti Zilmer1, Nenad Bogdanovic3 and Ello
Karelson1
1Department of Biochemistry, Tartu University, 50411 Tartu, Estonia. E-mail:
ello.karelson@ut.ee; mihkel.zilmer@ut.ee; 2Department of Neurology and
Neurosurgery, Tartu University Hospital, 50411 Tartu, Estonia. E-mail:
viktor.jefremov@kliinikum.ee 3Geriatric Department, Neurotec, Karolinska Institute,
Karolinska University Hospital, S-14186 Huddinge, Stockholm, Sweden. E-mail:
nenad.bogdanovic@neurotec.ki.se
The oxidative stress hypothesis of aging and age-related diseases suggests beneficial effects
of antioxidative plant polyphenols in preventing and suppressing the neurodegeneration and
atherogenesis. Recent data reported that certain plant polyphenols, incl. phytoestrogens, can
improve cognitive function in aging and in Alzheimer’s disease (AD) and slow
atherosclerosis development (Joseph et al., 2005; Manach et al., 2005). However, the
mechanisms behind these protective actions of polyphenols remain to be elucidated. This
study was undertaken to elucidate the antioxidant potency of three polyphenols (resveratrol,
curcumin, genistein) by using the two human models: 1) down-regulation of pro-oxidant
stimulated G-proteins in the postmortem brain membranes of aging and AD subjects; 2)
elevation of oxy-resistance (lag-phase) of low-density lipoproteins (LDL) in the human
serum. We used [35S]-GTP$S binding to investigate the effects of polyphenols on the activity
of the stimulated G-proteins in the human brain membranes (Mahlapuu et al., 2003). The
effect of the agents on duration of lag-phase and maximal rate of the Cu2+-induced LDL
oxidation was determined by formation of conjugated dienes at 234 nm. In aging and AD
frontocortical (FC) membranes, 3-10 µM of investigated polyphenols dose-dependently
depressed the G-protein stimulation induced by 10 µM hydrogen peroxide or 500 µM
homocysteine (an approximate 25% stimulation by both pro-oxidants was observed). In aging
FC, resveratrol revealed higher antioxidant effects towards the stimulated G-proteins than
genistein whereas the effect of curcumin was lowest. In AD, the antioxidant potency for
curcumin showed significant decline from the value in normal aging whereas the antioxidant
effects for resveratrol and genistein did not reduce remarkably. In both, aging and AD brain
FC, the antioxidant effect of 3-10 µM 17-#-estradiol on the stimulated G-proteins showed
moderate values, similarly to genistein.
In the plasma concentration (1 µM), resveratrol, curcumin and genistein significantly
increased the resistance of LDL to oxidation, prolonging the oxidation lag-phase 4.5, 2.8 and
1.5 fold, respectively. The ability of curcumin and genistein to significantly reduce the
maximal rate of LDL oxidation (compared with control) might represent as a specific
mechanism enabling the moderate antioxidant (antiatherogenic) activity for these compounds.
Our results suggest that plant polyphenols have structure-dependent antioxidant
(antiatherogenic) potency which might realize via protection of G-proteins from oxidative
damage in normal aging and neurodegeneration as well as via prevention of LDL from
abnormal (excessive) oxidation in atherogenesis.
- 633 -

Session XVII : Cell signaling in health and disease Poster XVII, 27
Dissecting the mTOR Pathway
Manel Joaquin1, Takahiro Nobukini1, Marta Roccio2, Stephen G. Dann3, So Young
Kim1, Pawan Gulati3, Maya P. Byfield4, Jonthan M. Backer4, François Natt5, Johannes
L. Boss2, Fried J.T. Zwartkruis2 and George Thomas1
1Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel
Switzerland; 2Department of Physiological Chemistry and Centre for Biomedical
Genetics, University Medical Center Utrech, Universiteitsweg 100, 3584 CG, Utrech,
The
Netherlands; 3Genome Research Institute, University of Cincinnati, 2180E Galbraith
Road, Cincinnati, OH 45237; 4Department of Molecular Pharmacology, Albert Eisntein
College of Medicine, NY 10461; 5Novartis Institue for Biomedical Research, Lichstrasse
35, 4002 Basel, Switzerland. manel.joaquin@upf.edu
During the evolution of metazoans and the rise of systemic hormonal regulation, the
insulincontrolled
class 1 phosphatidyl-inositide-3OH-kinase (PI3K) pathway was merged with the
primordial amino acid driven mammalian Target of Rapamycin (mTOR) pathway to control
the growth and development of the organism. Insulin regulates mTOR function through a
recently-described canonical signaling pathway, which is initiated by the sequential activation
of class 1 PI3K and Protein Kinase B (PKB) and subsequent repression of the Tuberous
Sclerosis tumor suppressor complex proteins (TSC1/2), culminating in the activation of the
Ras homologue enriched in brain (Rheb). However, how the amino acid input is integrated
with that of insulin signaling pathway is unclear. Here we employed a number of molecular,
biochemical and pharmacological approaches to address this issue. Unexpectedly, we found
that the major pathway by which amino acids control mTOR signaling is distinct from that of
insulin and that instead of signaling through components of the insulin/class 1 PI3K pathway,
amino acids mediate mTOR activation by signaling through the class 3 PI3K, hVps34.
- 634 -

Session XVII : Cell signaling in health and disease Poster XVII, 28
C-MIP: A NOVEL REGULATORY PROTEIN IMPLICATED IN T CELL
SIGNALING.
Maud Kamal , Karine Dahan, Marina Candelier, Philippe Lang, Georges Guellaen,
Djillali Sahali.
Nephrology department and INSERM U 581-équipe Avenir, Hôpital Henri Mondor,
Créteil 94010, France. e-mail : kamal@im3.inserm.fr.
It is believed that the pathogenesis of idiopathic nephrotic syndrome involves T cell
dysfunction. Using subtractive cloning and differential screening, we isolated a new
transcript, c-mip (c-maf inducing protein), which is up-regulated during the active phase of
the disease. The transcript encodes an 85 kDa protein characterized by a pleckstrin homology
(PH) domain and a Leucine rich repeat (LRR) domain. The localization of c-mip is restricted
to the thymus, fetal liver, T cells and kidneys. We have demonstrated that the stimulation of T
cells with Phytohemagglutinin (PHA) and Concanavalin A (Con A) downregulates c-mip.
The expression of c-mip in stimulated T cells is further downregulated by Calphostin C, a
PKC inhibitor. On the other hand, the inhibition or the activation of the MAPKs ERK1/2, by
PD 98059 and IFNalpha respectively, does not affect the level of expression of c-mip in these
stimulated T cells. These results suggest that PKC is involved in c-mip regulation
independently of RAS-GRP. Moreover, we have demonstrated that the overexpression of cmip,
in Jurkat cells, activates the ERK1/2 pathway and increases the expression of the
regulatory subunit of the PI3 kinase, p85alpha. Overexpression of c-mip inhibits NFkB
activation without any direct interference with NFkB p65. Altogether, these results suggest
that c-mip recruits p85alpha and plays a negative regulatory role on NFkB signaling via
mechanisms that are still under investigation.
- 635 -

Session XVII : Cell signaling in health and disease Poster XVII, 29
Over-expression of transforming growth factor and nitric oxide in saliva of ulcerative
colitis patients with various disease activities
1Sara Khalaj, 2Ali Rezaie, 1Maryam Shabihkhani, 1Mohammad J. Zamani,
1Shekoufeh Nikfar, 3Naser E. Daryani, and 1Mohammad Abdollahi
2Department of Community Health Sciences, Faculty of Medicine, University of
Calgary, Canada, 1Department of Toxicology and Pharmacology, Faculty of Pharmacy,
and Pharmaceutical Sciences Research Center, and 3Department of Gastrointestinal
Diseases, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Email: mohammad.abdollahi@utoronto.ca
Growth factors and nitric oxide (NO) play a major role in dysregulated immune response in
ulcerative colitis (UC). Recent evidence showed increased levels of transforming growth
factor-#1 (TGF-#1) in UC and suggested an anti-inflammatory effect for this factor. Based on
our recent study, dysfunctional immunoregulation is present in saliva of UC patients, we
hypothesized that salivary level of NO and TGF-#1 may differ by severity of UC and be
useful to determine the activity of the disease. Thirty-seven consecutive patients with
confirmed UC were enrolled and saliva samples were obtained. Fifteen healthy control
subjects were also recruited. Truelove-Witts severity index and modified Truelove-Witts
severity index were used to determine the severity of the disease. NO and TGF-#1 levels were
detected in saliva of all patients and control subjects using Enzyme-Linked Immunosorbent
Assay. Twenty-one patients had mild disease while 8 had moderate and 8 had severe colitis.
Adjusted for baseline characteristics, the levels of NO and TGF-#1 in different groups were
compared. Salivary NO and TGF-#1 levels were higher in UC patients comparing to controls
(P

Session XVII : Cell signaling in health and disease Poster XVII, 30
15-Deoxy-&12,14-prostaglandin J2 covalently modifies and functionally inactivates p53
in human breast cancer (MCF-7) cells
Do-Hee Kim, Hye-Kyung Na and Young-Joon Surh
National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
Cyclopentenone prostaglandins (cyPGs) exert diverse cellular functions, such as antiinflammatory
and cytoprotective effects. 15-Deoxy- &12,14-prostaglandin J2 (15d-PGJ2), a
representative cyPG of the J series, has been reported to exert biphasic effects on proliferation
and apoptosis. In the present study, we found that MCF-7 human breast cancer cells treated
with 15d-PGJ2 accumulated the tumor suppressor gene product p53 in both cytosolic and
nuclear fractions. Under the same experimental conditions, however, the mRNA level of p53
remained unchanged. This prompted us to investigate the molecular mechanisms responsible
for the 15d-PGJ2-mediated accumulation of p53 protein. Murine double-minute 2 (MDM2), a
zinc finger protein, is known to negatively regulate p53. 15d-PGJ2 treatment caused failure of
proper p53-MDM2 interaction in MCF-7 cells which could stabilize p53 protein by blocking
MDM2-mediated degradation. We observed that 15d-PGJ2 covalently modify cysteine
residues of p53 in MCF-7 cells. Covalent modification of p53 by 15d-PGJ2 is considered to
interfere with DNA binding of p53. In support of this assumption, 15d-PGJ2 inhibited the
DNA binding ability of both recombinant p53 and MCF-7 nuclear extracts in a concentrationrelated
manner. Likewise, p53 conjugation with 15d-PGJ2 may hamper its interaction with
MDM2, and subsequent degradation. The majority of accumulated p53 in 15d-PGJ2-treated
MCF-7 cells represents a functionally inactive or mutant form as assessed by
immunoprecipitation and immunofluorescence analysis. It is anticipated that the electrophilic
carbon center located in the alpha,#-unsaturated carbonyl moiety of the cyclopentenone ring
might be critical for the 15d-PGJ2-induced covalent modification of p53, cysteine thiol,
which retarded its degradation. MCF-7 cells treated with 9,10-dihydro-15-deoxy-&12,14prostaglandin
J2 that lacks the electrophilic alpha #-unsaturated functionality failed to
accumulate p53 protein, lending further support to the above assumption. In summary, 15d-
PGJ2 directly conjugates with cysteine thiol residues of p53, interfering its interaction with
MDM2, thereby stabilizing p53. Covalent modification of p53 by 15d-PGJ2 also hampers p53
DNA binding, leading to functional inactivation of this tumor suppressor.
- 637 -

Session XVII : Cell signaling in health and disease Poster XVII, 31
EFFECT OF ENDOTHELIN ON THE SODIUM/HYDROGEN EXCHANGER (NHE)
ACTIVITY OF HUMAN MONOCYTES AND ATHEROSCLEROSIS-RELATED
FUNCTIONS
Koliakos G1., Befani Chr. 2, Paletas K.3, Kaloyianni M.2
1Laboratory of Biological Chemistry, Medical School, Aristotle University of
Thessaloniki, 54124
2Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle
University of Thessaloniki, 54124
3 B’ Medical Clinic, Medical School, Aristotle University of Thessaloniki, 54124
Aims: The aim of the present study is to investigate the influence of endothelin on human
monocyte sodium/hydrogen exchanger (NHE-1) activity and on the atherosclerosis-related
monocyte functions. NHE-1 is an integral membrane protein that exchanges one intracellular
H+ ion for an extracellular Na+ ion. It plays an essential role in all cell types regulating the
internal pH (pHi), cell growth, differentiation and apoptosis. Endothelin is a potent
vasoconstrictive and mitogenic 21-amino-acid peptide that has been implicated in the
pathogenesis of atherosclerosis. Monocytes are key contributors to the atherosclerotic process.
Methods: The effect of endothelin (10pg/ml) on monocyte NHE-1 activity was
studied by means of pHi (using the fluorescent indicator BCECF-AM) and 22Na uptake.
Specific inhibitors were used in order to avoid interference from other sodium-exchanging
pumps. In an attempt to investigate the signal transduction pathway from endothelin to NHE 1
we used PD 98059 MEK1 (50µ)) (MAPKp42/44 inhibitor), GF-109203X (10µ)) (inhibitor
of all the isoforms of PKC) and Gö 6976 (500nM) (specific inhibitor of the isoform PKCs). In
addition, the effect of endothelin (10pg/ml) on the ability of monocytes to bind on the
basement membrane glycoprotein laminin and to migrate on trans-well culture inserts was
estimated. We also measured the density of CD36 receptor using a fluorescein isothiocyanate
(FITC)-linked anti CD36 monoclonal antibody.
Results: Endothelin caused an increase in pHi (p

Session XVII : Cell signaling in health and disease Poster XVII, 32
Implication of TRIP6, a novel molecular partner of the MAGI-1b scaffolding molecule
in invasiveness
Larissa Kotelevets1, Erik Bruyneel3, Alexei Kruglov1,4, Marc Bracke3, Frans van Roy2
and Eric Chastre1
1 INSERM U683, Faculté de Médecine X. Bichat, Paris, France
2 Department for Molecular Biomedical Research, VIB-Ghent University, B-9052 Ghent
3 Laboratory of Experimental Cancerology, Ghent University Hospital, B-9000 Ghent,
Belgium
4 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences,
Pushchino, Russia.
We recently established the critical role of the PTEN/MAGI-1b signalosome in the
stabilization of cell-cell contacts and the suppression of invasiveness. The PTEN tumor
suppressor is recruited to E-cadherin junctional complexes through the binding to the 2nd
PDZ domain of the MAGI-1b scaffolding molecule, whereas #-catenin interacts with the 5th
PDZ domain of MAGI-1b (Kotelevets et al, FASEB J 19,115,2005). To identify additional
effector molecules that might regulate the activity and the composition of the PTEN / MAGI-
1b complexes, we used yeast-two hybrid screening. Among the clones identified, we focused
on TRIP6 (Thyroid Receptor Interacting Protein 6)/ZRP-1 (Zyxin-related Protein 1). TRIP6
has been reported to associate with p130Cas in focal adhesion complexes. We demonstrated
that TRIP6 interacted directly with MAGI-1b through binding to the 5th PDZ-binding motif.
Ectopic expression of TRIP6 induced invasiveness in the epithelial kidney MDCK and
MDCKts-src cells in a PI3-kinase dependant manner, and reverted the E-cadherin-dependent
cell-cell aggregation. In this connection, we observed a slight increase in AKT activity in
MDCKts-src derivatives transfected with TRIP6 expression vectors, compared to parental
cells. The TRIP6Stop473 mutant, lacking the PDZ binding motif was unable to promote cell
invasiveness and did not interfere with cell-cell aggregation. The competing peptides
corresponding to the C-terminus of TRIP6 or b-catenin promoted invasiveness in TRIP6
Stop473-transfected MDCKts-src cells but not in the parental cell line. These results suggest
that TRIP6-induced invasiveness in epithelial cells involves the induction of cell motility by
the TRIP6 core molecule, whereas the C-terminus is required to destabilizes E-cadherin
junctional complexes by competing with #-catenin for the interaction with MAGI-1b.
- 639 -

Session XVII : Cell signaling in health and disease Poster XVII, 33
Microgravity signal ensnarls cell adhesion and cytoskeleton of rat osteoblasts: osteopontin,
CD44, osteonectin, and "-tubulin
Yasuhiro Kumei 1 , Sadao Morita 1 , Hitoyata Shimokawa 1 , Kei-ichi Ohya 1 , Hisako Katano 2 ,
Hideo Akiyama 3 , Masahiko Hirano 3
1 Graduate School of Tokyo Medical and Dental University, Tokyo, Japan
2 University of Tokyo Medical Science Institute, Tokyo, Japan
3 Toray Research Center, Kamakura, Japan
We examined gene expression of osteopontin (OP), osteonectin (ON), CD44, and "-tubulin in
rat osteoblasts that were cultured aboard Space Shuttle for 4 days. During the last 24 hrs, all
the cultures were treated with 1",25 dihydroxyvitamin D3, and solubilized by guanidine
solution. The relative mRNA levels were determined by quantitative RT-PCR, followed by
normalization to glyceraldehyde 3-phosphate dehydrogenase. Microgravity decreased the OP
mRNA levels by 40%, while increased the CD44 mRNA levels by 280%, as compared to the
ground controls. OP is a major extracellular matrix protein in bone, and important for
hydroxyapatite crystal formation and osteoclast attachment to the bone surface. The
hyaluronic acid receptor CD44 interacts with OP and FN, however, fibronectin gene
expression was not altered by microgravity. Microgravity also increased the ON mRNA
levels by 40%, but decreased the "-tubulin mRNA levels by 40%, as compared to ground
control levels. ON is another matrix protein associated with tubulin. Association/dissociation
of these molecules may mediate cell adhesion, motility, and signaling via rearrangement of
cytoskeletal structure under microgravity conditions.
- 640 -

Session XVII : Cell signaling in health and disease Poster XVII, 34
Induction of accumulation of $-globin mRNA in human erythroid cells treated with
psoralens
Ilaria Lampronti1, Nicoletta Bianchi1, Cristina Zuccato1, Francesco Dall’Acqua2,
Margherita Zennaro1, Daniela Vedaldi2, Giampietro Viola2 and Roberto Gambari1,3.
1Department of Biochemistry and Molecular Biology, University of Ferrara,
2Department of Pharmaceutical Sciences, University of Padova, 3GenTech-for-Thal,
Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy of
Thalassemia, Ferrara
The identification of potential therapeutic agents for treatment of hematological diseases,
including #-thalassemia and sickle cell anemia (SCA), can be based on pharmacologicallymediated
regulation of the expression of human $-globin genes, leading to an increase of the
production of fetal hemoglobin (HbF). DNA-binding drugs appear to be of great interest and
represent a promising approach to control gene expression. Among molecules able to interact
with DNA, psoralens and related compounds could be relevant. In this study, we analysed
several linear and angular psoralens. To verify the activity of these furano-cumarins, we
employed two experimental cell systems, the human leukemic K562 cell line for preliminary
studies and the two-phase liquid culture of human erythroid progenitors isolated from normal
donors for further analysis on the most active compounds. Erythroid differentiation was
analysed by benzidine-staining; expression of $-globin genes by quantitative RT-PCR assays
using the Perkin-Elmer 7700 Sequence Detector. The results of our investigation suggest that
angelicin, bergapten and several structurally-related compounds (trimethylangelicin, psoralen,
8-methoxy-psoralen, 4-methyl-psoralen and 5’-methyl-psoralen) are powerful inducers of
erythroid differentiation and $-globin mRNA accumulation both in K562 cells and in human
erythroid precursors. The activity of trimethylangelicin and 8-methoxy-psoralen were found
higher than that displayed by hydroxyurea, commonly used as HbF inducers in $-thalassemia
and SCA patients. Our results could have practical impact, as it is well know that an increase
in $-globin mRNA and fetal hemoglobin production could ameliorate the clinical status of
patients with $-thalassemia and sickle cell anemia.
Work supported by CIB, AVLT (Associazione Veneta per la Lotta alla Talassemia), EU
(Fondi Strutturali Obiettivo 2), Fondazione CARIPARO (Cassa di Risparmio Padova-
Rovigo), ER-SPINNER and PRRIITT.
- 641 -

Session XVII : Cell signaling in health and disease Poster XVII, 35
Regulation of fibroblasts adhesion and migration by protease nexin-1
Xiaobiao Li
Friedrich Miescher Institute, Basel
Protease Nexin-1 (PN-1), a 43 KDa glycoprotein inhibits extracellular serine proteases
activity. It also triggers signal transduction by interacting with members of distinct surface
receptor families, such as low-density lipoprotein protein receptors (e.g. LRP1), and heparan
sulfate proteoglycans (e.g. syndecan-1). These interactions lead to activation of either PKA or
ERK signaling, and the final outcome depends on the cross talk between these two pathways.
In wild type mouse embryonic fibroblasts, PN-1 activates PKA via interaction with LRP1,
leading to inhibition of Ras-ERK signaling. In cells deficient in LRP1, interaction between
PN-1 and syndecan-1 activates ERK signaling (Li et al., submitted).
In this study, we observed that PN-1 activated ERK signaling and its down stream effector
Rac1, causing lamellipodia formation and enhanced cell migration in LRP1-/- MEF cells.
This effect was mediated by the interaction between PN-1 and syndecan-1. Overexpression of
PN-1 activated ERK signaling constitutively and increased cell adhesion and migration on
vitronectin. The effect was even more significant when LRP1-/- MEF cells were
overexpressing syndecan-1. We also observed that stimulated LRP1-/- MEF cell migration
was inhibited by preincubation with antibodies against syndecan-1, and integrin #3, which is
functionally coupled to this proteoglycan. Furthermore PN-1 was specifically coimmunoprecipitated
with the integrin #3 subunit. Consequently our data indicate that PN-1
affects ERK signaling and cell migration through its interactions with integrin #3 and
syndecan-1.
- 642 -

Session XVII : Cell signaling in health and disease Poster XVII, 36
Differential activation of Akt and VEGF in tumor-derived microvascular endothelial
cells: a novel mechanism for acquired chemoresistance in liver cancers
Fanyin Meng, Roger Henson, Hania Wehbe, Molly Lang, and Tushar Patel
Scott and White Clinic, Texas A&M University System Health Science Center College of
Medicine, 2401 South 31st Street, Temple, TX 76508, U.S.A. E-mail:
fmeng@swmail.sw.org
Targeting endothelial cells that line tumor blood vessels is a promising new strategy for the
treatment of cancers such as liver cancer. The transcription factor Nuclear Factor Kappa B
(NF-kB) can regulate the expression of key mediators of angiogenesis and cell survival such
as protein kinase B (PKB/Akt) and vascular endothelial growth factor (VEGF). However, the
involvement and role of these mechanisms in tumor derived endothelial cells is unknown.
Methods: Hepatocellular carcinoma was induced in BALB/c mice using Ndiethylnitrosamine.
Microvascular endothelial cells (MVEC) were isolated from liver tumors
(T-MVECs) as well as normal liver tissues (N-MVECs) using a magnetic bead
immunoaffinity technique. In vitro angiogenesis was quantitated using a commercial assay kit
(Chemicon). Results: In both types of MVEC, transfection with NF-kB increased Akt
phosphorylation and VEGF expression. However, the increased VEGF expression was
blocked by dominant negative Akt in T-MVECs, but not N-MVECs. Moreover, overexpression
of Akt directly increased VEGF expression and NF-kB dependent angiogenesis in
T-MVECs. Incubation with gemcitabine to induce chemotherapeutic stress dramatically
increased NF-kB activation in both T-MVECs and N-MVECs, but increased Akt and VEGF
expression only in T-MVECs. In addition, Akt activation and VEGF expression was
dependent on NF-kB in T-MVECs. Over-expression of Akt decreased gemcitabine-induced
apoptosis in T-MVECs, but not in N-MVECs. In vitro angiogenesis was increased by
chemotherapeutic stress in T-MVECs, not in N-MVECs, and was abolished by inhibition of
either NF-kB or Akt. Conclusions: Akt is selectively activated in tumor derived endothelial
cells and may contribute to NF-kB dependent VEGF expression and angiogenesis and
survival during chemotherapeutic stress. Targeting NF-kB dependent up-regulation of Akt
and VEGF may be useful to decrease chemoresistance and tumor progression in liver cancers.
- 643 -

Session XVII : Cell signaling in health and disease Poster XVII, 37
$-Glutamyltransferase is upregulated after oxidative stress through the Ras signal
transduction pathway in colon carcinoma cell lines
Seila Møller, Serhyi Pankiv, Ugo Moens, Nils-Erik Huseby
Department of Medical Biochemistry, Faculty of Medicine, University of Tromsø,
Norway. email: nilseh@fagmed.uit.no
Gamma-glutamyltransferase (GGT) has a central role in glutathione (GSH) homeostasis.
After oxidative stress and depletion of GSH, upregulation of GGT has been reported. As
oxidative stress may activate various Ras signal transduction pathways, we examined whether
the regulation of GGT is mediated through these pathways. Acute exposure of rat colon
carcinoma cells to menadione resulted in elevated GGT activity and protein levels detectable
after 24 h. Quantitative PCR showed increased total GGT mRNA levels 6 h after the
exposure. Actinomycin D and cycloheximide reduced the elevation. When the oxidative stress
exposures were performed in the presence of protein kinase inhibitors that block downstream
targets p38, PI-3K and MEK1/2 of Ras, the upregulation of GGT was attenuated. GGT is
transcribed from five promoters into multiple mRNAs. Using semiquantitative RT-PCR,
promoter II was found increased after acute oxidative stress. Transient cotransfection studies
using this promoter II region in a luciferase plasmid together with an active Ras plasmid,
resulted in increased luciferase activity, whereas no activity was measured with dominant
negative Ras. This promoter contains putative binding sites for AP1, AP2, NFkB, SRE and
SP1. Using plasmids with binding sites for the mentioned trancription factors we found that
SRE and AP1 were active in transcribing the enzyme.
The present study shows an upregulation of GGT after exposure to oxidative stress through a
de novo transcription of the GGT gene being mediated through several Ras signal
transduction pathways. The cells will benefit from the enzyme activity in maintaining the
intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor
cells during oxidative stress.
- 644 -

Session XVII : Cell signaling in health and disease Poster XVII, 38
HYPOXIA IS RESPONSIBLE OF sVEGF-R1 INDUCTION IN VILLOUS
TROPHOBLAST EXPLANTS CULTURE
Carine Munaut , Sarah Berndt , Sophie Lorquet, Christel Pequeux, Francis Frankenne,
Van den Brûle Frédérique and Jean-Michel Foidart.
Laboratory of Tumor and Development Biology, CRCE, CHU, GIGA, EMBIC partners.
University of Liège, Tour de Pathologie (B23), Sart Tilman, B-4000 Liège, Belgium.
Preeclampsia is a disorder unique to human pregnancy characterized by a generalized
systemic maternal inflammatory response, associated with diffuse endothelial cell
dysfunction. It is one of the leading causes of maternal and perinatal morbidity and mortality,
affecting 5% to 7% of all pregnancies, yet the etiology and pathogenesis have not been fully
defined.
Oxygen plays a central role in this pathology. Recently, preeclampsia has been described as a
state of imbalance between pro-angiogenic and anti-angiogenic factors. Main pro-angiogenic
factors that promote angiogenesis in the placenta belong to VEGF family.
We have previously shown that preeclampsia was associated with low levels of circulating
PlGF and increased levels of total VEGF-A and soluble VEGF-R1 (Tsatsaris et al, 2003,
J.Clin.Endocrinol.Metab). Here, our study was undertaken to test the hypothesis that high
levels of those angiogenic factors could be related to hypoxic status of placenta in
preeclampsia.
Small fragments of placental villi from first trimester (11-14 weeks) voluntary interrupted
pregnancies were used for explant culture under normoxia (20% O2, and 5% CO2) or hypoxia
(1% O2, 5% CO2 and 94% N2). Under hypoxia, villous trophoblast explants expressed higher
levels of VEGF-A, VEGF-R1, sVEGF-R1 and VEGF-R2 mRNAs than under normoxia
culture. By contrast, PlGF mRNA was decreased in hypoxia. Protein secretion of VEGF-A
and sVEGF-R1, determined by ELISA, were also found elevated in hypoxic explant cultures.
No histological or morphological modifications were observed under hypoxia as compared to
normoxia.
Our data show that villous trophoblast is the likely source of increased plasma levels of
VEGF-A and VEGF-R1 in pregnant woman. Our results also support the hypothesis that
overproduction of sVEGF-R1 by villous trophoblast submitted to extended hypoxia in
preeclampsia could account for the depletion of maternal blood free VEGF.
- 645 -

Session XVII : Cell signaling in health and disease Poster XVII, 39
Mechanisms of selenium cytotoxicity in a malignant mesothelioma model – targeting
Thioredoxin Reductase-1
Gustav Nilsonne, Branka Kocic, Aristi Potamitou Fernandes, Agnes Stein, Anna-Klara
Rundlöf, Mikael Björnstedt, Anders Hjerpe, and Katalin Dobra.
All authors: Karolinska Institutet, Institution for Laboratory Medicine, Dept. of
Pathology. Karolinska Univ Hosp Huddinge F-46, S-141 86 Huddinge, Sweden.
Malignant mesothelioma (MM) is an aggressive tumour arising from the serous cavities. MM
cells may differentiate into an epithelioid or a sarcomatoid phenotype, and the latter is more
chemoresistant and associated with a worse prognosis. The thioredoxin (Trx) system is
upregulated in many tumors, and the highest reported levels have been found in MM. The
system comprises Trx, Thioredoxin Reductase (TrxR1) and NADPH. It functions mainly to
maintain intracellular redox balance, but also counteracts apoptosis. Sodium selenite causes
oxidative stress.
The objectives of this study were to investigate whether selenite could induce apoptosis in a
sarcomatoid and an epithelioid MM cell line, and what mechanisms would mediate the
cytotoxic effects.
Apoptosis was measured by three independent methods and was induced in the sarcomatoid
cell line at low concentrations. The IC 50 was 7,5 µM. IC 50 of the epithelioid cell line, with
greater expression of the Trx system, was 21 µM. The DCF probe revealed ROS formation
following selenite treatment, analysed with confocal microscopy. Selenite concentrations of
10 µM or more inhibited TrxR1 effectively.
ICC revealed p53 activation in both cell lines. Immunostaining with FACS analysis revealed
that Bax was activated by selenite. The DioC6 marker analysed by FACS showed loss of
mitochondrial membrane potential. Inhibition of JNK using SP600125 had no effect in either
cell line. Inhibition of p38 decreased the activation of Bax. No caspase activity was detected
after selenite treatment. The pan-caspase inhibitor z-VAD-fmk did not attenuate apoptosis.
In conclusion, selenite generated ROS, inhibited TrxR1 and caused apoptosis in two MM cell
lines. The therapy resistant sarcomatoid cells were more sensitive. The effects were mediated
through p53 and Bax, but appear to be independent of both caspases and JNK. This is a
promising new treatment option for an aggressive and chemoresistant tumor.
- 646 -

Session XVII : Cell signaling in health and disease Poster XVII, 40
Signaling pathways regulating production of hyaluronic acid in pig oocyte-cumulus cell-
complexes
Radek Procházka and Eva Nagyová
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech
Republic, Lib2chov, 277 21 Czech Republic. E-mail: prochazka@iapg.cas.cz
Cumulus cells undergo an extensive expansion following the preovulatory surge of
gonadotrophins due to increased production of hyaluronic acid (HA) and its deposition in
extracellular spaces. In vitro, the expansion can be stimulated with FSH in all mammalian
species. The insulin like-growth factor-I (IGF-I) can modify the action of FSH on cumulus
cells. The objective of this study was to characterize mechanisms by which the IGF-I affects
the FSH-stimulated production and deposition of HA in pig oocyte-cumulus complexes
(OCCs). For this purpose, signaling pathways activated in cumulus cells by IGF-I were
studied and their importance for production of HA was assessed. OCCs isolated from 3-5 mm
follicles were cultured in M-199 medium supplemented with 3 mg/ml polyvinyl pyrrolidone
under an atmosphere of 5% CO2 in air for 24 h. To quantify production of HA, OCCs were
cultured in the presence of 2.5 µCi of tritiated glucosamine hydrochloride and the
incorporated label was measured by a liquid scintillation counter in medium containing OCCs
(total HA) or within the complexes alone (retained HA). We found that production and
retention of HA was significantly higher in OCCs cultured in medium with FSH (10 ng/ml)
than in control group cultured without FSH (P

Session XVII : Cell signaling in health and disease Poster XVII, 41
Inactivation of human melanoma cells induced by alkylating agents and/or highly
ionizing radiation
Aleksandra M. Risti'-Fira1, Ivan M. Petrovi'1, Lela B. Kori'anac1, Danijela V.
Todorovi'1, Lucia M. Valastro2, Giacomo Cuttone2, Giuseppe Privitera3
1 Vin(a Institute of Nuclear Sciences, Belgrade, Serbia and Montenegro
2 Istituto Nazionale di Fisica Nucleare, LNS, Catania, Italy
3 Institute of Radiology and Radiation Oncology, University of Catania, Italy
E-mail: aristic@vin.bg.ac.yu
The response of human HTB140 melanoma cells to chemotherapeutic drugs fotemustine
(FM) and dacarbazine (DTIC) as well as of proton irradiation were studied. Viability of cells
treated with 100 and 250 micromolar drugs was assessed after incubation of 6, 24, 48, 72 and
96 h. Proton irradiations of exponentially growing cells were performed at the CATANA
(Centro di Adro Terapia e Applicazzioni Nucleari Avanzati) facility for treatment of eye
melanoma at INFN, LNS – Catania, delivering to the cell monolayer single doses of 2, 4, 8,
12 and 16 Gy. Cell viability was evaluated 7 days after irradiation. For all treatments,
inactivation level was estimated using microtetrasolium (MTT) and sulforhodamine B (SRB)
assays. The combined effects of each drug and protons, were carried out using the same drug
concentrations, while proton doses applyed were those used in therapy, i.e. 12 and 16 Gy. In
this case viability was estimated using only SRB staining since it was shown to be more
reliable than MTT assay for the incubation of 48 h. With the increase of the drug
concentration or irradiation dose, the level of cell inactivation was more pronounced, reaching
approximately 60 %, 48 h after drug treatment or 7 days after irradiation at 16 Gy.
Considering the rate of drug concentrations used, as well as the level of doses applied, it
appears that HTB140 cells are more resistant to proton irradiation than to alkylating agents
tested. The combined treatment with FM or DTIC and protons did not show significant
changes of cell viability as compared to the effects of single agents. Slightly better cell
inactivation was obtained for 250 micromolar DTIC combined with 16 Gy proton irradiation.
Taking into account the fact that the chosen time point for measuring cumulative effects of
drug and irradiation was 48 h post-irradiation, it seams that the obtained level of viability
could be attributed almost only to the effects of drugs.
- 648 -

Session XVII : Cell signaling in health and disease Poster XVII, 42
GSK-3 mediates differentiation and activation of pro-inflammatory dendritic cells
Elena Rodionova, Eugene Maraskovsky, Michael Hess, Michael Kirsch, Anthony D. Ho,
and Thomas Luft
Glycogen synthase kinase (GSK)-3 is a multifunctional enzyme critical for cellular
differentiation, apoptosis, self renewal and motility, and dysregulation of GSK-3 is linked to
immune deviations associated with diabetes, cancer, schizophrenia and Alzheimer’s disease.
We demonstrate that GSK-3 is constitutively active in human monocytes. During
differentiation of monocyte-derived dendritic cells (MoDC), GSK-3 inhibits macrophage
development and allows DC to accumulate in an immature stage by inhibiting spontaneous
maturation.
However, in the context of DC activation GSK-3 acquires a pro-inflammatory function
mediating high levels of IL-12p70, IL-6 and TNF-alpha without influencing migration or
secretion of IL-10.
These paradoxical inhibitory and pro-inflammatory effects of GSK-3 highlight changes in
intracellular “primed” GSK-3 targets due to the activity of other kinases. The proinflammatory
effect of GSK-3 in the context of DC activation is immediately counterbalanced
by Akt-1. Akt-1 is phosphorylated during DC activation and partially inhibits GSK-3 activity
by Ser21/9 phosphorylation. Thus, IL-12p70 secretion results from the competition for the
phosphate-binding pocket of GSK-3 between GSK-3 target proteins (primed by other kinases)
and the inhibitory Ser9/21 moiety (phosphorylated by Akt-1).
Our results demonstrate that GSK-3 is a crucial enzyme mediating the differentiation and
activation of pro-inflammatory DC.
- 649 -

Session XVII : Cell signaling in health and disease Poster XVII, 43
Adaptive functional differentiation of dendritic cells – integrating the network of extra-
and intracellular signals
Elena Rodionova1, Eugene Maraskovsky3,4, Michael Kirsch2, Michael Hess2 and
Thomas Luft1,2.
Phenotypic maturation, cytokine secretion and migration are distinct functional characteristics
of dendritic cells (DCs). These functions are independently regulated by a number of
extracellular variables, such as type, strength and persistence of an array of soluble and
membrane-bound mediators. Since the exact composition of these variables in response to
infection may differ between individuals, the intracellular signalling pathways activated by
these extracellular networks may more closely correlate with DC function and predict the
course of adaptive immunity. We found that activation of p38K, ERK1/2 and PC-PLC
enhanced cytokine secretion, whereas p38K, cAMP and PC-PLC enhanced migration. In
contrast, PI3K/Akt-1 and cAMP inhibited cytokine secretion whilst ERK1/2 inhibited
migration. Migration and cytokine secretion further differed in their sensitivity to inhibition
over time. However, although DCs could be manipulated to express migration, cytokine
secretion or both, the level of activation or persistence of intracellular pathway signalling was
not predictive. Our results suggest a modular organization of function. We hypothesize that
the expression of specific DC functions integrates a large variety of activating and inhibitory
variables, and is represented by the formation of a functional unit of molecular networks - the
signal response module (SRM). The combined activities of these modules define the
functional outcome of DC activation.
- 650 -

Session XVII : Cell signaling in health and disease Poster XVII, 44
Alterations in cell signaling in the model organism Saccharomyces cerevisiae caused by
heterologous expression of enterobacterial virulence factors
Isabel Rodríguez-Escudero,1 Ainel Alemán,1 Philip R. Hardwidge,2 B. Brett Finlay, 2
Rafael Rotger, 1 Víctor J. Cid1 and María Molina1
1Dpto. de Microbiología II, Fac. de Farmacia, Universidad Complutense, Pza. de
Ramón y Cajal s/n, 28040 Madrid, Spain. E-mail: isabelre@farm.ucm.es
2Biotechnology Laboratory, Room 237, Wesbrook Building, 6174 University Boulevard,
University of British Columbia, Vancouver, BC, V6T 1Z3. Canada.
Certain proteins from pathogenic bacteria, injected into the cell via a specialized type III
secretion system (TTSS), account for the induction of local actin polymerization by
interfering with host cell signaling. Enteropathogenic E. coli (EPEC) strains cause severe
diarrhea by inducing characteristic “effacing” lesions in enterocytes through the development
of actin-supported pedestals at the site of bacterial adhesion. Pathogenesis requires a
functional TTSS, which injects into the host cell the intimin receptor, Tir, as well as other
effectors. We have made use of the model organism Saccharomyces cerevisiae to probe the
functions of several EPEC effector proteins, studying their effects on cell growth, cytoskeletal
function and signaling pathways. We found that some EPEC effectors were able to interfere
with cytoskeletal function and to activate MAPK pathways when expressed in yeast, offering
a feasible host cell model for molecular studies on these proteins. On the same trend, we have
used this model to study the function of the SigD/SopB TTSS effector from Salmonella, a
bacteria that invades epithelial cells by modifying epithelial cell signaling to induce its own
internalization. SigD/SopB shares homology with mammalian phosphatidylinositol 4phosphatases.
By expressing this protein in S. cerevisiae we have been able to dissect two
functional regions into SigD, the catalytic C-terminal region and an N-terminal extension that
is able to disrupt actin. We have isolated yeast Cdc42 as a suppressor by overexpression of
the actin-depolarization phenotype caused by heterologous SigDR468A, a novel catalytically
inactive version of this protein. This provides evidence that small GTPases within the host
cell are putative targets for this bacterial virulence factor.
- 651 -

Session XVII : Cell signaling in health and disease Poster XVII, 45
Delta-opioid receptor function is regulated by phosducin-like protein long (PhLPL) in
cultured astroglyal cells
Pilar Sánchez-Blázquez, Carlos Montero and Javier Garzón.
Laboratory of Neuropharmacology, Instituto Cajal, Avenida Doctor Arce 37, Madrid
28002. Spain. Email: psb@cajal.csic.es
Affinity-purified IgGs raised against different PhLPL sequences recognised two major bands
of about 38 (non-glycosylated) and 55 kDa (N-glycosylated) in Western blots of proteins
from resting astroglial cells. While in resting astrocytes little PhLPL was found in the nucleus,
stimulation of delta-opioid receptors with the selective agonist DPDPE, induces a
redistribution of PhLPL, and a large fraction of the cell membrane protein pool translocated to
the nucleus. Although the mechanism by which PhLPL reaches the nucleus remains unknown,
nuclear phosphorylation of PhLPL might facilitate its binding to 14-3-3, allowing the Cterminal
domain of PhLPL to act as an activator of transcription. In our work, Gbeta coimmunoprecipitated
with the PhLPL, and the association increased in presence of the delta
opioid agonist DPDPE. At longer intervals (6 and 24 h) the association diminished while that
of PhLPL with the P-Ser-binding protein 14-3-3 augmented. This observation suggests that
the stimulation of delta opioid-receptors markedly increase phosphorylation of PhLPL over
the very modest observed in resting astrocytes. We next examine the kinases involved in
PhLPL phosphorylation analyzing the effects of different cell-permeable inhibitors (0.1 to 100
nM): the PKA inhibitor peptide-fragment 6-22 amide; the CaMKII inhibitors, KN-93 and
autoclamide 2-related inhibitory peptide; the selective inhibitor of PKC, chelerythrine; and the
CK2 inhibitor, DRB. At the concentrations and exposure times used, none of these kinase
inhibitors interfered with the shape or viability of the astroglial cells. In the whole astrocyte
we found that PKC, CaMKII and to a lesser extent CK2, were all important kinases in PhLPL
phosphorylation. However, differences were observed between the N-glycosylated and nonglycosylated
forms of PhLPL. Thus, G protein signalling in astroglial cells induces the
cellular redistribution of PhLPL and its compartment-dependent phosphorylation by different
kinases.
(This work was supported by grants MCYT SAF2003-0112 and BMC2002-03228.)
- 652 -

Session XVII : Cell signaling in health and disease Poster XVII, 46
Modification of GSTP1-1 by cyclopentenone prostaglandins
Fancisco J. Sánchez-Gómez, Javier Gayarre, Konstantinos Stamatakis and Dolores
Pérez-Sala
Departamento de Estructura y Función de Proteínas. Centro de Investigaciones
Biológicas, C.S.I.C., 28040 Madrid, Spain. Email: dperezsala@cib.csic.es
Glutathione-S-transferases (GST) constitute a family of enzymes which play a key role in the
detoxification of electrophilic compounds by catalyzing their conjugation with glutathione.
Therefore, these enzymes play an important role in cell defense mechanisms, including the
development of resistance of tumor cells towards anticancer drugs. Cyclopentenone
prostaglandins are electrophilic compounds that have shown antiproliferative activity against
a variety of cancer cell lines, which is related to their ability to modify cellular proteins by
Michael addition. It is known that cyclopentenone PG can inhibit the activity of several GST
isoforms, both in vitro and in intact cells. The molecular basis for this effect appears to be
complex, but to date, covalent interactions between GST and cyclopentenone PG have not
been documented. Here we show that the cyclopentenone PG 15-deoxy-Delta-12,14-PGJ2
covalently binds to GSTP1-1 in vitro. This binding can be demonstrated by MALDI-TOF
mass spectrometry, as well as by the use of a biotinylated 15d-PGJ2 analog. Using this analog
we have evidenced the binding of 15d-PGJ2 to GSTP1-1 in intact human T-cell leukemia
Jurkat cells. In addition, treatment of Jurkat cells with cyclopentenone PG induces the
oligomerization of GSTP1-1 and a concomitant increase in the phosphorylation of JNK and of
c-Jun. This effect is associated with the induction of apoptosis. Monomeric, but not
oligomeric GST has been shown to act as an endogenous inhibitor of JNK. In the light of this
evidence our results provide a basis to explore the involvement of the GST-JNK pathway in
the apoptotic process and put forward a potential mechanism for the activation of JNK by
cyclopentenone PG in Jurkat cells.
- 653 -

Session XVII : Cell signaling in health and disease Poster XVII, 47
M-CSF-induced proliferation and LPS-dependent activation of macrophages requires
Raf-1 phosphorylation to induce MKP-1 (DUSP1) expression
Ester Sánchez-Tilló, Monica Comalada, Consol Farrera, Annabel F. Valledor, Jorge
Lloberas and Antonio Celada
Macrophage Biology Group, Institute of Biomedical Research, Barcelona Science Park,
University of Barcelona, Barcelona, Spain. E-mail: acelada@ub.edu
Macrophages are key regulators of immune responses. Bone marrow-derived macrophages
undergo proliferation in response to their specific growth factor, M-CSF. The addition of
activating agents, such as lipopolysaccharide (LPS), results in macrophage growth arrest and
engagement in a pro-inflammatory response, which produces nitric oxide (NO) and cytokines
including tumor necrosis alpha (TNF-"), IL-1 and IL-6. Although activation of ERK is
required for both macrophage proliferation and activation, the kinetics of these two processes
differs. In our cellular model, early peak of ERK activation (5 min) correlates with cellular
proliferation whereas a later peak (15 min) is associated with the activation program. M-CSF
and LPS also induce with different time-course the dual specificity MAP kinase phosphatase
MKP1 (DUSP1), which correlates with the dephosphorylation of ERK. Therefore, MKP1 is a
key regulator of the time-course of ERK activity. Using RNA interference and
pharmacological inhibitors, here we provide evidence that macrophagic MKP1 expression
induced by M-CSF and LPS is dependent on Raf-1 activation. In addition, Raf-1 interacts
with PKC', which is also involved in MKP-1 induction. Concordantly, the distinct timecourse
of Raf-1 and MEK-1/2 activation correlates with that of ERK-1/2 induced by M-CSF
or LPS. ERK activation in response to M-CSF is Raf-1-dependent, but an alternative pathway
induces ERK activation in response to LPS. Blockage of Raf-1 activity results in increased
expression of cyclin dependent kinase inhibitors p21Waf-1 and p27Kip-1 and macrophage
growth arrest even in the presence of M-CSF. In contrast, LPS stimulation has no effect on
the expression of pro-inflammatory cytokines and inducible nitric oxide synthase.
- 654 -

Session XVII : Cell signaling in health and disease Poster XVII, 48
Role of ATP in trauma associated cytokine release by P2X7 ion channel stimulation
E. Marion Schneider1, Katrin Vorlaender1, Xueling Ma1, Weidong Du1, Manfred
Weiss2,
1Sektion Experimentelle Anaesthesiologie, Klinische Anaesthesiologie2,
Universitaetsklinikum Ulm, Ulm, Germany;
Marion.schneider@uni-ulm.de
Objective: Trauma and massive tissue damage contribute to immediate cytokine release and systemic
inflammatory response syndromes (SIRS) which may eventually lead to life threatening sepsis and
septic shock. Knowledge about mechanisms explaining hyperinflammation in the absence of
infectious complications would improve preventive therapeutic options. We asked whether P2X7 ion
channel activation may be involved.
Background: The P2X7 receptor is ubiquitiously expressed and belongs to a family of ligand-gated
channels activated by extracellular ATP. Binding of extracellular ATP activates multimerization of
P2X7 subunits into trimers and hexamers, allowing transmembrane passage of cations and small
molecules. Short stimulation with extracellular ATP upregulates cytokine release. Prolonged or
repeated exposure to ATP induces the formation of a cytolytic pore and triggers cell death. An A to C
single nucleotide polymorphism (SNP) at position 1513 (1513A_C) of the Glu-496 to Ala residue in
the intracellular C-terminal tail leads to nonfunctional P2X7, whereas heterozygous individuals have a
lower response.
Methods: Cell isolates were plated with 2x105 cell/ml and ATP stimulation was performed with 1 and
5 mM ATP for 6h and 24h. Cytokines released were determined by a chemiluminescence assay
(Immulite, DPC-Biermann, Bad Nauheim, Germany). P2X7 SNPs were determined by
pyrosequencing (Biotage, Uppsala, Sweden). Ca++ signalling was studied by Fluo-4 (Ca++ dye)
assisted confocal laser scanning microscopy using a Leica-TCS NT microscope and argon/krypton
laser using semi-confluent HeLa, microvascular endothelium and macrophages.
Results: Effects of P2X7 stimulation on the release of inflammatory cytokines in spontaneous B cell
lines as well as in whole blood and in Ficoll isolated blood mononuclear cells (PBMC) were as
follows: ATP induced solely IL-8 but neither IL-1ß, nor IL-6 or IL-10 in B cells. By contrast, PBMC
of healthy donors responded with positive IL-1ß in addition to IL-8, no TNF-a was induced. Thus in
addition to the effect on caspase 1 leading to IL-1ß secretion, ATP activates the release of IL-8 as an
universal oxidative stress chemokine. Responses were stronger in wildtype P2X7 as compared to the
1513A_C heterozygous cells. P2X7 signalling involves a Ca++ release response generated by
phosphatidylinositol-3-phosphate (IP3) from intracellular stores. A number of inhibitors applied
(Ryanodine, Paxilline: 1µM, Xestospongin C: 10µM, oxATP: 1µM) clearly demonstrated that the
calcium signal involves both, intracellular Ca++ stores and extracellular Ca++ influx as well. In
addition to the involvement of phospholipase C (PLC) to generate IP3-induced intracellular Ca++
release, the blockade by paxilline further suggests a role by large conductance Ca++ -activated K+
channels (BK). Upon decline of the Ca++ response, strong fluorescent labelling was transiently
detected in swollen mitochondria. Apoptosis was associated with impressive blebbing of the plasma
membrane.
Conclusions: ATP stimulation of the P2X7 receptor induces the release of IL-1ß and IL-8 in
monocytic cells and IL-8 as a universal stress marker in B cells and in other, also non hematopoietic
cells. In vivo, P2X7 stimulation may well occur in severe trauma, may lead to neutrophil recruitment
via IL-8-induced chemotaxis as well as cell damage in endothelial and epithelial cells. Via P2X7,
ATP-induced apoptosis is dependent on PLC activation, intracellular Ca++ release and the activation
of large conductance potassium channels as well.
- 655 -

Session XVII : Cell signaling in health and disease Poster XVII, 49
Control of Human Herpesvirus Type 8-associated diseases by NK cells
1Maria C. Sirianni, 1Massimo Campagna, 1Donato Scaramuzzi, 1Maurizio Carbonari,
2Elena Toschi, 2Ilaria Bacigalupo, 2Paolo Monini and 2Barbara Ensoli
1Clinical Immunology, Department of Clinical Medicine, University of Rome “La
Sapienza”, 00161 Rome and 2National AIDS Center, Istituto Superiore di Sanita’,
00161 Rome, Italy. E-mail: mariacaterina.sirianni@uniroma1.it
The ‘natural killer’ (NK) cells, a first line defense mechanism against tumors and viruses,
preferentially kill target cells lacking surface Major Histocompatibility Complex class I
(MHC-I) molecule expression. NK cells recognize these targets through membrane receptors,
which can trigger either activating or inhibitory signals for killing. Several tumor or virus
infected cells down-regulate MHC-I expression as a mechanism to evade recognition and
killing by cytotoxic T lymphocytes (CTL). They, however, become targets for NK cells
cytotoxic activity.
NK cell activity is reduced during disease progression in Human Immunodeficiency Virus
(HIV) infection, and in associated tumors latently infected by the oncogenic Human
Herpesvirus Type 8 (HHV8) such as Kaposi’s sarcoma (KS) and primary effusion lymphomas
(PEL). We have demonstrated that HIV-related KS (AIDS-KS) is characterized by an
increased expression on T lymphocytes of inhibitory receptors recognizing HLA-C molecules
(1), whereas non-HIV infected patients with KS (classic KS, C-KS) have a substantial number
of peripheral blood NK cells bearing these receptors (2). NK cells from patients with C-KS,
are equipped with cytolytic molecules including granzyme A and perforin. However, the
cytotoxic activity of NK cells is reduced in patients with C-KS, AIDS-KS or PEL patients,
which are all infected by the HHV8, and this correlates with disease severity (3-5). Moreover,
we have found that HHV8 infected cell lines established from PELs have a reduced surface
expression of MHC-I molecules and are sensitive to the lysis mediated by NK cells (4, 5).
Since PEL cells express the same HHV8 latency program as KS cells, these data point to
MHC-I downregulation by HHV8 as a primary immune evasion mechanism against CTL
responses, further reinforced by up-regulation of inhibitory receptors on T and NK cells in the
setting of HIV and HHV8 infection. Thus, studies on killing receptor regulation and signaling
in T and NK cells may shed light on HHV8-associated tumors both in HIV-infected or non
infected patients.
- 656 -

Session XVII : Cell signaling in health and disease Poster XVII, 50
Proteomic identification of cellular targets for posttranslational modification by antiinflammatory
cyclopentenone prostaglandins
Konstantinos Stamatakis, Javier Gayarre, Francisco J. Sánchez-Gómez and Dolores
Pérez-Sala
Departamento de Estructura y Función de Proteínas. Centro de Investigaciones
Biológicas, C.S.I.C., 28040 Madrid, Spain. Email: kostas@cib.csic.es
Prostaglandins with cyclopentenone structure have been shown to display protective effects in
numerous cellular and animal models of inflammation and injury. These effects have been
related to their ability to block the inflammatory response and to elicit a cellular heat shock
response. An important determinant in the beneficial effects of cyclopentenone PG is their
ability to form covalent adducts with thiol groups in proteins by Michael addition. Here we
have explored the ability of biotinylated analogs of the cyclopentenone PG, 15d-PGJ2 and
PGA1, to mimic the effect of their parent PG. Biotinylated 15d-PGJ2 inhibited the response
of renal mesangial cells to proinflammatory stimuli and elicited a cellular stress response. In
addition, both biotinylated analogs induced the activation of MAPK in murine fibroblasts.
Therefore, we have used these biotinylated PG to identify, through a proteomic approach,
some of the protein targets the modification of which may be involved in the biological
effects of cyclopentenone PG. Extracts from cells incubated in the presence of biotinylated
PG were analyzed by 2D electrophoresis and the spots showing positive biotin staining were
analyzed by tryptic digestion and MALDI-TOF mass spectrometry. In biotinylated 15d-PGJ2treated
cells we have identified several proteins involved in the regulation of cellular
architecture and dynamics, such as actin, tubulin, vimentin and tropomyosin. Modification of
vimentin was associated with a collapse of the intermediate filament network in mesangial
cells, which was reminiscent of a heat shock response. In addition, several proteins known to
be regulated by oxidative stress are also targets for the addition of 15d-PGJ2, including
Hsp90, nucleoside diphosphate kinase and methyl thioadenosine phosphorylase. The
structural and functional implications of the modification of these various targets and their
potential involvement in the biological effects of cyclopentenone PG will contribute to a
better understanding of the selectivity and therapeutic potential of these prostanoids.
- 657 -

Session XVII : Cell signaling in health and disease Poster XVII, 51
Primer Design and Polymerase Chain Reaction (PCR) Amplification of the Mutation
Cluster Regon (MCR) of the Adenomatous Polyposis Coli (APC) Gene
Mary Eloisa Torres-Rowshangah and Filipinas F. Natividad
Department of Biology, College of Science, University of the Philippines Baguio,
Governor Pack Road, Baguio City 2600, Philippines E-mail :metorres@eudoramail.com
Research and Biotechnology Division, St. Luke’s Medical Center, 279 E. Rodriguez
Senior Boulevard, Quezon City 1102 Phlippines E-mail :ffnatividad@st luke.com.ph
Colorectal cancer is a major health concern worldwide. The Adenomatous Polyposis Coli
(APC) gene, a tumor suppressor gene, controls colonocyte epithelial proliferation.. Mutation
in the APC gene is also implicated in cell migration, cell-cell adhesion, chromosomal
stability, cell cycle control and apoptosis. The APC gene is a suitable marker for colorectal
cancer detection because APC gene mutation is an initiating event in colorectal tumorigenesis
for both sporadic and hereditary colorectal cancers.Germline mutations are scattered
throughout the 5’half of the APC gene while somatic mutations are found in a Mutation
Cluster Region (MCR) in exon 15 that spans codons 1250 to 1500 with hotspots at codons
1309 and 1450. A pair of primers encompassing the MCR of the APC gene was designed
using OLIGO software and the Polymerase Chain Reaction (PCR) mix and conditions were
determined using DNAsis software. After obtaining informed consent from 109 patients
clinically diagnosed with sporadic colorectal cancer with no prior treatment at the Institute of
Digestive Diseases of the St. Luke’s Medical Center (SLMC) included in the study,
Deoxyribonucleic acid (DNA was extracted from a portion of the surgical tissue samples and
the MCR of the APC gene was amplified using U3612 and L4500 and the PCR mix and
conditions determined earlier. The obtained PCR product was run in 1.5% agarose gel
yielding positive results.This new protocol offers an effective way of amplifying the MCR of
the APC gene. The advantages of this procedure includes lower cost, reduced hands-on time,
greater simplicity and reliability that is suitable for research and clinical management.
- 658 -

Session XVII : Cell signaling in health and disease Poster XVII, 52
N-(4-hydroxyphenyl)retinamide interferes with functionally active IGF-1-mediated
survival pathways in human retinoblastoma cells.
Roberta Venè1, Giuseppe Arena1, Douglas M. Noonan2, Adriana Albini,1 and
Francesca Tosetti1
1Experimental Oncology A Laboratory, National Cancer Research Institute (IST), L.go
R. Benzi 10, 16132 Genova, Italy. 2Università degli Studi dell'Insubria, Varese, Italy. Email:
francesca.tosetti@istge.it , roberta.vene@istge.it
The cancer chemopreventive retinoid N-(4-hydroxyphenyl)retinamide (4HPR) kills Y79
retinoblastoma cells through a complex cell death pathway involving ROS elevation,
lysosomal damage and irreversible mitochondrial dysfunction. The Insulin-like growth factor
I (IGF-I) sustains the survival of tumor cells by different mechanisms including protection
from cell death and resistance to chemotherapeutic agents. Since IGF-I is also a survival
factor in the human retina, we asked whether IGF-I was able to protect Y79 cells against
4HPR cytotoxicity. IGF-I increased Y79 cell proliferation, in contrast, IGF-I could not
prevent 4HPR-induced DNA fragmentation, loss of MTT reduction, LDH release, and late
ATP depletion. IGF-I induced a rapid phosphorylation of AKT at Ser473 and mTOR at
Ser2448 that were sustained over time. Phosphorylation of AKT, but not of mTOR , was
almost completely suppressed by 4HPR at 24h. To confirm AKT inactivation by 4HPR, we
considered GSK3beta, a downstream substrate of AKT that is inactivated by phosphorylation
at Ser9 and is involved in the survival response induced by a number of growth factors and
chemical inhibitors of mitochondrial membrane depolarization. Surprisingly, we found that
not only 4HPR sustains IGF-I-induced GSK3beta phosphorylation at Ser9, but that 4HPR
itself is able to induce GSK3beta phosphorylation. The antioxidant NAC reversed this effect,
suggesting the involvement of a ROS-mediated mechanism in stimulation of GSK3 beta
phosphorylation by 4HPR.
- 659 -

Session XVII : Cell signaling in health and disease Poster XVII, 53
HSP90 INHIBITION BY GELDANAMYCIN UPREGULATES EXPRESSION OF
HEAT SHOCK PROTEINS IN PC-12 CELLS.
Christina B. Wiegand 1, Madhavi J. Rane3, Leroy R. Sachleben Jr 2, Rachel Wu 3 and
Evelyne Gozal 1,2
Depts of Pharm. & Tox.1, Pediatrics2, Medicine3, University of Louisville, Louisville,
KY
Heat shock proteins (Hsps) play a role in cellular injury, proteins repair and ubiquitination, or
induction of apoptosis when cells are beyond repair. Hsp90 regulates the function of its client
proteins and can bind Akt kinase or heat shock factor (HSF). Upon cellular stress HSF is
released to the nucleus to induce Hsps transcription. Geldanamycin (GA) binds Hsp90 ATPbinding
site, inhibiting its ATPase function and disrupting protein associations. We
previously showed that 6h sustained hypoxia (SH; 0.1% O2) increased Akt-Hsp90 binding
and Akt phosphorylation in PC-12 cells, without altering cell survival. However, 24h SH
decreased Akt phosphorylation, coinciding with the onset of cell death. GA induced cell death
and decreased Akt expression in a dose dependent manner, with higher toxicity in normoxia
(RA; 21% O2) than in SH, and disrupted Hsp90-Akt association without affecting SHinduced
Akt phosphorylation. Because of Hsps dual function in cellular repair and apoptosis,
we hypothesized that GA may affect cell survival by disrupting Hsp90 binding with
additional Hsps or with HSF. To determine whether GA modulates Hsps expression, GAtreated
cells were exposed to 6h, 12h, and 24h RA or SH. GA increased Hsp25, Hsp70 and
Hsp90 expression independently from FiO2, while Akt/Hsp90 binding was differentially
altered in SH and RA. Thus, increased Hsps expression observed in GA-treated PC-12 cells
may result from the dissociation of HSF from Hsp90 and its subsequent translocation
independently from the hypoxic stress. Increased Hsps expression may initially protect cells
from cytotoxic effects of low GA concentrations, as suggested by GA dose-dependent effect
on cell survival. However, longer GA exposures may increase cell death resulting from
accumulating damaged proteins or SH-related energy depletion. We are currently
investigating changes in HSF association with the Hsp90 complex in SH and RA GA-treated
cells. These findings suggest that disruption of Hsp90 chaperone functions by GA leads to
inactivation of survival pathways, alterations in the protein associations of Hsp90 complexes,
and induction of stress proteins targeting cells to apoptosis. NIH F30-NS051998-01, HL-
074296, 2 P20 RR15576-06, and AHA 0335278N.
- 660 -

Session XVII : Cell signaling in health and disease Poster XVII, 54
The effect of UV-irradiation on telomerase activity and other stress-related proteins in
lens epithelial cells
WU Zhi-hong,(!"!) ZHANG Jin-shong#"#$%
Department of Ophthalmology, the Forth Affiliated Hospital, China Medical University.
Shenyang 110001, China
Corresponding author: WU Zhi-hong
Email: fswuzhihong@sina.com&bjwuzhihong@yahoo.com
Background To investigate the change and role of telomerase activity and other stressrelated
proteins UV-induced DNA damage and repair in lens epithelial cells. Methods Human
lens epithelial cells were irradiated at UV-doses 0.0(control group) and
0.5'1.5'2.5'3.5'5.0'7.5'10.0 mJ/cm2(treated 1~7group). telomerase activity was
determined by Telomerase Repeat Amplification Protocol-Enzyme Linked Immunosorbent
Assay#TRAP-ELISA%(P53'P21'growth arrest and DNA damage inducible
(GADD45)'proliferating cell nuclear antigen(PCNA) and P16 protein levels were analyzed
by Western blotting.
Results Telomerase activity in control group and treated 1~7group showed increased
tendency, compairing in 8 groups, there are significant difference (F=45.65,P

Session XVII : Cell signaling in health and disease Poster XVII, 55
PI3 kinase inhibition provides a novel mechanism for the action of valproic acid
Xuehua Xu1, Helmut Kae2, Annette Müller-Taubenberger3, Kathryn Adley4, Nadine
Pawolleck5, Vivian Lee6, Talvinder Sihra6, Claudia Wiedemann7, Gerry Weeks2, Jan
Faix8, Markus Maniak5, Tian Jin1 and Robin S.B. Williams4
1. Chemotaxis Signal Sect, NIAID, Rockville, MD20852, USA. TJIN@niaid.nih.gov.
2. Microbiol. Immunol. UBC, Vancouver, V6T1Z3, Canada. weeks@ciml.univ-mrs.fr.
3. M-Plank Biochem, LRZ, Munich, 80336, Germany. amueller@lrz.uni-muenchen.de.
4. Dept Biol and WIBR, UCL, London, WC1E 6BT, UK. robin.williams@ucl.ac.uk.
5. Inst Cell Bio, University of Kasse, Kassel, 34132, Germany. maniak@uni-kassel.de.
6. Dept Pharmacol., UCL, London, WC1E6BT, UK. t.sihra@ucl.ac.uk.
7. Mol Cell Bio, UCL Med. School, NW32PF, UK. c.wiedemann@medsch.ucl.ac.uk.
8. Hanover Med School, Hanover, D-30623, Germany. hans@imap.bpc.mh-hannover.de.
Valproic acid (VPA, 2 propyl pentanoic acid) is a short chain fatty acid that was accidentally
found to be an effective treatment for epilepsy in 1962, and is now also used to treat bipolar
disorder and migraine, and is undergoing trials for cancer therapy. Despite its wide use, the
therapeutic targets of VPA are not clear. We have identified an effect of VPA in blocking
chemotaxis, a process controlled by phosphoinositide 3-kinase (PI3K) signalling, using the
social amoeba Dictyostelium discoideum. In this model, we show that VPA attenuates PI3K
activity by inhibiting signal-induced PIP3 production, that both VPA and a PI3K inhibitor
reverse the phenotype of activated Rac1A, reduce Ras activation and VPA also inhibits
vesicle trafficking. These results suggest that VPA functions through the reduction of PIP3
production to modulate downstream effectors including small GTPases and vesicle release.
To examine the therapeutic potential of this effect, we show that the human PI3Kgamma
enzyme is weakly but directly inhibited by VPA and that both VPA and a PI3K inhibitor
suppress depolarization-dependent neurotransmitter release in purified rat nerve terminals.
These results thus suggest that VPA may function to reduce PI3K activity as a therapeutic
mechanism for the treatment of epilepsy, bipolar disorder, migraine, and cancer.
- 662 -

Session XVII : Cell signaling in health and disease Poster XVII, 56
Determination of oxidative stress status and concentration of TGF-B1 in the blood and
saliva of osteoporotic patients
1Gholamreza Yousefzadeh, 1Bagher Larijani, 2Azadeh Mohammadirad, 1Ramin
Heshmat, 2Roja Rahimi, and 2Mohammad Abdollahi
1Endocrinology and Metabolism Research Centre, and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran, Iran
Email: mohammad.abdollahi@utoronto.ca
Introduction: The maintenance of bone mass is influenced by genetic, race, hormonal,
mechanical and nutritional factors which modulate the local and systemic mechanisms
regulating bone turnover. It seems that oxidative stress and interleukins particularly TGF-#1
also influences bone mass.
Objectives: to determine if oxidative stress and TGF-#1 have any role in the bone mass
density.
Methods: Blood and saliva samples of twenty two osteoporotic patients were collected.
TBARS and FRAP assay respectively were used to determine levels of lipid peroxidation and
antioxidant status in these samples. The concentration of TGF-#1 antigen was measured by
using the capture sandwich ELISA.
Results: Total antioxidant power in blood and saliva of osteoporotic patients was lower
(p

Session XVII : Cell signaling in health and disease Poster XVII, 57
Erufosine: a membrane targeting antineoplastic agent with signal transduction
modulating effects
Maya M. Zaharieva1,2, Spiro M. Konstantinov1,2, Martin R. Berger2
1Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str, 1000 Sofia, Bulgaria
2German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany
The ether lipid analogue erufosine (erucylphospho-N,N,N,-trimethyl-propylammonium,
ErPC3) has high activity against leukaemic cells without affecting the normal hematopoiesis.
It belongs to the group of alkylphosphocholines that are inhibitors of protein kinase C and of
phospholipase C. However, the mechanism of action of erufosine remains rather unclear. We
focused on combination effects with the tyrosine kinase inhibitor imatinib mesylate (gleevec,
former STI-571 or CGP-57148) against chronic myeloid leukemia (CML) derived cell lines.
The influence of erufosine on proteins involved in the phosphatidylinositol-3-phosphate
pathway and on the expression of the retinoblastoma protein Rb as key component for the cell
cycle entry and progression in mammalian cells was studied. A panel of three cell lines:
SKW-3 (chronic T-cell leukemia), BV-173 and K-562 (CML) were treated with erufosine (at
concentrations ranging from 2 to 50 mcM). Whole cell lysates were prepared for Western
Blot analysis. The combination effect of erufosine with imatinib on BV-173 and K-562 was
estimated after the treatment using MTT-dye reduction assay. The consecutive treatment of
K-562 and BV-173 cells with erufosine (2.5, 5, 15, 30 mcM) and imatinib (0.05, 0.1 mcM)
led to synergism and therefore such combinations could be beneficial for relapsed patients
with drug resistant disease. Erufosine caused decrease of pAkt and BCR-ABL protein
expression, while it induced the Rb in K-562 cells. These alterations in the signal
transduction could be an explanation for the drug interaction found. Furthermore, Rb is a
substrate of caspases and it is cleaved during apoptosis. Such Rb degradation was observed in
SKW-3 and BV-173 cells after incubation with erufosine for 14 h. Our experimental findings
suggest that erufosine acts through induction of changes in protein signaling and especially
through Rb induction. This unique mode of action makes it an attractive partner for
combination therapies, e.g. with imatinib-based therapy for CML.
- 664 -

Session XVII : Cell signaling in health and disease Poster XVII, 58
Impact of Rb knock down on the modulation erufosine signal transduction in human
leukemia cells
Maya M. Zaharieva1,2, Milen Kirilov2, Minqiang Chai2, Stefan Berger3, Spiro M.
Konstantinov1,2, Martin R. Berger2
1Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str, 1000 Sofia, Bulgaria
2German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany
3Central Institute for Mental Help, Department of Molecular Biology, J5, 68 159
Mannheim
The retinoblastoma gene Rb1 is a prototypical tumour supressor. Consistent with its tomoursupressor
function Rb is known to inhibit proliferation. Recent studies indicate its role in
suppressing apoptosis, too. Loss of Rb sensitizes cells to apoptosis, but loss of Rb can only
contribute to tumor development under conditions in which apoptosis response is already
compromised. The new alkylphosphocholine erufosine is an inductor of Rb in leukemic cells.
Therefore, we decided to suppress the expression of RB using conventional phosphorotioate
antisense oligonucleotides and siRNAs. Unfortunately, both types of oligonucleotides used
didn’t cause stable and reproducible knock-down and we initiated viral transfection
experiments for stable Rb knock-down in SKW-3, BV-173 and K-562 cells. The antisense
sequence was firstly cloned in the pSUPER vector and tested on HEK 293T cells. For
normalizing the transfection efficiency an EGFP-expressing vector was used in addition. To
realize the transfer of the H1-shRNA cassette directed to the Rb-mRNA, the cells were
infected with LentiLox virus which co-express an EGFP-protein. The resulting levels of target
mRNA were measured by real-time RT-PCR after excluding of non-fluorescent cells by flow
cytometry and cell sorting. The highest decrease of the Rb-mRNA (about 86%) was found in
SKW-3 cells. A concomitent knock-down at the protein level was evidenced by Western
blotting. The proliferation activity of the infected cells was estimated by colorimetric MTTdye
reduction assay before and after erufosine treatment. Comparison of Rb knock-down and
wild type leukemic cells revealed the presence of significant differences in cell cycle duration
and sensitivity to erufosine, thus confirming the impact of Rb modulation for the mode of
action of alkylphosphocholines which are known as promising antineoplastic signal
transduction modulators.
- 665 -

Session XVII : Cell signaling in health and disease Poster XVII, 59
Labile zinc and zinc transporter-4 (ZnT4) expression in mast cells and airway
epithelium
1Zalewski, P., 1Grosser, D., 2Murgia, C., 1Lang, C., 1Ho, L., 1Truong-Tran, A.,
3Danscher, G., 3Stoltenberg, M and 1Ruffin, R.
1Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital,
Woodville, South Australia 5011. 2Istituto Nazionale Ricerca per gli Alimenti e la
Nutrizione, 00178, Rome, Italy. 3Department of Anatomy, University of Aarhus,
Denmark.
In addition to tightly-bound Zn(II) in metalloenzymes, zinc fingers and other metalloproteins,
cells contain more dynamic, exchangeable pools of labile Zn(II). Labile Zn(II) is involved in
processes as diverse as mast cell secretion, caspase-regulation and neurotransmission. Our
previous studies, using the Zn(II)-specific fluorophore Zinquin, have shown localization of
abundant pools of labile Zn(II) in secretory granules of mast cells and islet cells and in apical
vesicles of human and animal airway epithelium (AE) [1-3]. Using autometallography to
detect labile Zn(II) by electron microscopy, we have demonstrated the presence of large,
vesicular-like aggregates of Zn(II) in the apical cytoplasm of rat AE. How Zn is incorporated
into these organelles is unclear, but a role for specific intracellular Zn(II) transporters appears
likely. Here we show abundant expression (mRNA and protein) for the vesicular Zn
transporter ZnT4 in human and rodent mast cells and AE. Immunofluorescence staining for
ZnT4 in AE was strikingly similar to that of Zinquin fluorescence of Zn(II), both showing
vesicular and apical cytoplasmic distributions. In addition, some of the AE cells had a rim of
fluorescence at the basolateral membrane and occasionally around the entire plasma
membrane, suggesting a role for this Zn(II) transporter in membrane uptake of Zn(II), as well
as redistribution to the apical vesicles. Since apical AE Zn(II) is markedly decreased in a
murine model of allergic airway inflammation, we studied the effects of airway inflammation
on expression of ZnT4. Balb/c mice were sensitized to ovalbumin (OVA) and challenged with
aerosolized OVA. There was a significant decrease in whole lung ZnT4 mRNA expression
(by Real-time PCR) as well as ZnT4 immunofluorescence in the bronchial epithelium of the
OVA-treated mice when compared to saline-treated controls. We propose that ZnT4 plays an
important role in vesicular/granular uptake of Zn(II) in mast cells and AE.
1. Carter JE, Truong-Tran AQ, Grosser D, Ho L, Ruffin RE and Zalewski PD. 2002
Involvement of Redox events in caspase activation in Zn-depleted airway epithelial cells.
Biochem Biophys Res Commun 279, 1062-70.
2. Zalewski, P. D., Millard, S. H., Forbes, I. J., Kapaniris, O., Slavotinek, A. and Betts, W. H.
(1994) Video image analysis of labile zinc in viable pancreatic islet cells using a specific
fluorescent probe for zinc. J. Histochem. Cytochem. 42, 877-884.
3. Ho LH, Ruffin RE, Murgia C, Li L, Krilis SA, Zalewski PD. 2004. Labile zinc and zinc
transporter ZnT4 in mast cell granules: Role in regulation of caspase activation and NF-kB
translocation J Immunol 172, 7750-60.
- 666 -

Session XVII : Cell signaling in health and disease Poster XVII, 60
Animal model of tumor progression: a study of combined therapy to overcome the
multiple drug resistance syndromes of malignant cells
Marina Zenkova, Nadezda Mironova, Olga Shklyaeva, Ekaterina Andreeva, and
Valentin Vlassov
Institute of Chemical Biology and Fundamental Medicine SB RAS
8, Lavrentiev ave., 630090, Novisibirsk, Russian Federation E-mail:
marzen@niboch.nsc.ru
In this work we developed experimental animal model of tumor progression on the base of
mice lymphosarcomas LS and RLS. LS tumor displays high sensitivity to cyclophosphamide,
which is widely used in anticancer therapy to initiate apoptosis. RLS was derived from LS by
passaging at low concentration of cyclophosphamide in mice (10-20 mg/kg) and characterized
by resistance to high dose of cyclophosphamide (50-150 mg/kg).
The primary cultures (LS and RLS)) of these tumors were obtained and characterized by
differences in expression of the genes involved in formation of multiple drug resistant
phenotype. We show that RLS tumors are characterized by high level of mdr1b and bcl-2
genes expression as compared with LS and low level of p53 gene expression. The study of
RLS sensitivity to cytostatics revealed that about 10% of cells display MDR phenotype and
survive even at high dose of cytostatics. By cultivation of RLS on medium with increasing
vinblastine concentrations cell line RLS40 was obtained. RLS40 exhibited high levels of
mdr1a/1b genes expression as compared to RLS and 20-folds increase of resistance to
cytostatics. Drug resistant RLS40 cells were transplanted into CBA mice and sensitivity of
formed tumors (solid and ascetic) to anticancer drugs was tested. We show that RLS40
tumors were resistant to a number of cytostatics usually used in standard protocols of
antitumor therapy (cyclophosphamide, cysplatine, adriamicine, rubomicine and TNF): these
therapeutics have no effect on tumor growth. Thus, RLS40 tumor can be used as an animal
model of tumors which display high drug resistance and poor prognosis of treatment. This
model corresponds to tumor status observed in patients after one or several courses of
chemotherapy and can be used for testing combined conventional therapy and/or developing
gene-targeted therapeutics (siRNAs, antisense oligonucleotides, ribozymes) affecting
expression of mdr1a/1b and bcl-2 genes.
This work was supported by RAS programs Molecular and cellular biology and Science to
medicine, FCNTP grant RI-012/001/254.
- 667 -

Session XVII : Cell signaling in health and disease Poster XVII, 61
Intracrine angiotensin II induces intracellular signaling in rabbit kidney convoluted
proximal tubule cells
Jia L. Zhuo, Oscar A. Carretero and Xiao C. Li.
Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, MI
48202, USA. E-mail: jzhuo1@hfhs.org
Although the role of extracellular Ang II and the underlying mechanisms mediated by cell
surface AT1 receptors have been well studied in the kidney, whether intracellular Ang II
plays a physiological role in renal proximal tubule cells remains poorly understood. We tested
the hypothesis that extracellular Ang II is taken up by proximal tubule cells through AT1
receptor-mediated endocytosis, and that internalized Ang II induces intracellular calcium
mobilization, activates NF!B, and increases RNA transcription by stimulating cytoplasmic
and nuclear AT1 receptors. When proximal tubule cells were exposed to extracellular Ang II
(1 nM), intracellular Ang II levels were increased by 60% (161 ± 6 vs. 98 ± 5 pg/mg protein;
p < 0.001). The increases in intracellular Ang II were blocked by the endocytotic inhibitors
colchicine (10-6 M) (p < 0.01), phenylarsine oxide (PAO) (10-6 M), which inhibits tyrosine
phosphatase (p < 0.01), and the AT1 receptor blocker losartan (10-5 M) (p < 0.01), but not by
the AT2 receptor antagonist PD 123319 (10-5 M). To determine whether intracellular Ang II
has a physiological role, Ang II was microinjected into single proximal tubule cells (1 nM),
while cell surface AT1 receptors were blocked with extracellular losartan (10-5 M).
Intracellular Ang II induced a robust increase in intracellular calcium, which peaked at 15 to
30 s. When Ang II was microinjected together with losartan, the calcium responses were
blocked while concurrent microinjection of Ang II and PD 123319 had no effect. Moreover,
blockade of AT1 receptor-mediated Ang II internalization by colchicine or losartan inhibited
Ang II-induced activation of NF!B (p < 0.01). Ang II (1 nM) also significantly increased
RNA transcription in freshly isolated proximal tubule cell nuclei (p < 0.01) and the effect was
inhibited by losartan. We conclude that extracellular Ang II is internalized in proximal tubule
cells through AT1 receptor-mediated endocytosis and intracellular Ang II may exert
important intracrine effects by activating cytoplasmic and nuclear AT1 receptors. This work
is supported by grants from National Institutes of Health (RO1DK067299), American Heart
Association Greater Miwest Affiliate (0355551Z), and National Kidney Foundation of
Michigan.
- 668 -

Notes
- 669 -

Notes
Notes
- 670 -

- 671 -

List of participants
(by alphabetical order)
- 672 -

- 673 -

Pr. Mohammad Abdollahi
14155-6451 Tehran IRN
Email: mohammad@tums.ac.ir
Mr. Indra Aerts
Cellular Biochemistry
2610 Antwerp BEL
Email: indra.aerts@ua.ac.be
Mrs. Pia Agren
6228 CV Maastricht Maastricht
NLD
Email:
pia.agren@farmaco.unimaas.nl
Dr. Maria Cristina Albertini
61029 Urbino ITA
Email: cemetbio@uniurb.it
Pr. Dario Alessi
MRC Protein Phosphorylation
Unit
DD1 5EH Dundee GBR
Email: d.r.alessi@dundee.ac.uk
Dr. Alexandra Andreeva
Department of Pharmacology
60612 Chicago, IL USA
Email: aandreev@uic.edu
Mrs. Adrienn Angyal
Dept. of Immunology
1117 Budapest HUN
Email: sarmayg@cerberus.elte.hu
Dr. Josef Abel
Toxicology
40225 Düsseldorf DEU
Email: josef.abel@uniduesseldorf.de
Pr. Bharat Aggarwal
Cytokine Research
TX 77030 Houston USA
Email: aggarwal@mdanderson.org
Pr. Hee Jung Ahn
Pathology
463-712 Sungnam KOR
Email: hjahn@cha.ac.kr
Dr. Veronica Albertini
Laboratory of Experimental
Oncology
6500 Bellinzona CHE
Email:
veronica.albertini@irb.unisi.ch
Mrs. Asude Alpman
Ege University Medical Faculty
Medical Genetics
35100 Izmir TUR
Email: asudealpman@gmail.com
Dr. Allison-Lynn Andrews
Brroke Laboratory
SO16 6YD Southampton GBR
Email: ala@soton.ac.uk
Mr. Sébastien ANTHERIEU
Equipe de Toxicologie Cellulaire
et Moléculaire et Génomique
6903 Sophia-Antipolis FRA
Email: antherieu@antibes.inra.fr
Mr. ERIC ADRIAENSSENS
INSERM ERI-8
59655 Villeneuve d'Ascq FRA
Email: eric.adriaenssens@univlille1.fr
Dr. Ioanna-Katerina Aggeli
Animal & Human Physiology
157 84 Athens GRC
Email: iageli@hotmail.com
Mrs. Cano Ainara
Biochemistry
48940 Leioa ESP
Email: ainaroco@hotmail.com
Dr. Alise Alesenko
Molecular mechanisms of cell
proliferation
154 Moscow RUS
Email: ales@sky.chph.ras.ru
Mr. Joana Amaral
1600-083 Lisbon PRT
Email: jamaral@ff.ul.pt
Dr. Peter Angel
Division Signal Transduction and
Growth Control
69120 Heidelberg DEU
Email: p.angel@dkfz.de
Dr. Frank Antonicelli
Biochemistry-Dermatology
51095 Reims FRA
Email: frank.antonicelli@univreims.fr

Dr. Nassera Aouali
L-1526 Luxembourg LUX
Email: nassera.aouali@crpsante.lu
Mr. Thierry Arnould
URBC
5000 Namur BEL
Email:
thierry.arnould@fundp.ac.be
Dr. Beatrice Bachmeier
Inst. of Pathology
D-81925 Munich DEU
Email: bachmeier@med.unimuenchen.de
Dr. Marek Baltaziak
Department of Pathology
15-269 Bialystok POL
Email: sulek@zeus.amb.edu.pl
Dr. Joana Barbosa Melo
Institute Medical Biology and
Center for Neurosciences, Faculty
of Medicine, University of
Coimbra
3004-504 Coimbra PRT
Email: jbmelo@cnc.cj.uc.pt
Mrs. Montserrat Barragan
Unitat de senyalitzacio cel!lular
E-08003 Barcelona ESP
Email: montse.barragan@upf.edu
Dr. Christa Baumstark-Khan
Cellular Biodiagnostics,
Department of Radiobiology,
Institute of Aerospace Medicine
51170 Köln DEU
Email: christa.baumstarkkhan@dlr.de
Dr. Jose Aramburu
Department of Experimental and
Health Sciences
8003 Barcelona ESP
Email: jaramburu@imim.es
Pr. Ami Aronheim
31096 Haifa ISR
Email: aronheim@tx.technion.ac.il
Mrs. Hye Yeon BAEK
Department of Pharmacology
120-752 Seoul KOR
Email: ehrehrwh@hanmail.net
Pr. Vladimir Bantseev
School of Optometry/Biology
N2L 3G1 Waterloo CAN
Email: vbantsee@uwaterloo.ca
Mr. Fawzia Bardag-gorce
90502 Torrance USA
Email: fgorce@labiomed.org
Dr. Giuseppina Basini
Dipartimento di Produzioni
Animali-Sezione di Fisiologia
43100 Parma ITA
Email: basini@unipr.it
Mrs. Ilse Beck
LEGEST
B-9000 Gent BEL
Email: IlseME.Beck@Ugent.be
Dr. Vivienne Armbruester
Department of Medicine
10029 New York City USA
Email:
vivienne.armbruester@mssm.edu
Dr. Sigridur Asgeirsdottir
Dept Pathology & Laboratory
Medicine, Medical Biology
Section
9713 GZ Groningen NLD
Email:
s.asgeirsdottir@med.umcg.nl
Mrs. Denyse BAGREL
Laboratoire d'Ingénierie
Moléculaire et Biochimie
Pharmacologique
57070 Metz FRA
Email: bagrel@univ-metz.fr
Dr. Federica Barbieri
pharmacology-department of
oncology biology and genetics
16132 Genova ITA
Email: federica.barbieri@unige.it
Mrs. Elena Bardina
Molecular Genetics
6229 ER Maastricht NLD
Email: e.bardina@gen.unimaas.nl
Mr. Eric BATTAGLIA
LIMBP
57070 Metz FRA
Email: battaglia@univ-metz.fr
Mrs. Siv Lise Bedringaas
Hematology section
5021 BERGEN NOR
Email: siv.bedringaas@med.uib.no

Dr. Jeffrey Beekman
Dept. of Immunology
3584 EA Utrecht NLD
Email: j.beekman@azu.nl
Dr. Isidoros Beis
Animal & Human Physiology
157 84 Athens GRC
Email: ibeis@biol.uoa.gr
Dr. Antonino Belfiore
Clinical and Experimental
Department
88100 Catanzaro ITA
Email: belfiore@unicz.it
Dr. Yinon Ben-Neriah
Lautenberg Center for
Immunology
91120 Jerusalem ISR
Email: yinonbn@yahoo.com
Pr. Hans-Gert Bernstein
Experimental Psychiatry
D-39120 Magdeburg DEU
Email: Hans-
Gert.Bernstein@medizin.unimagdeburg.de
Mr. Alexandre Berthaut
75006 Paris FRA
Email: alexberthaut@aol.com
Mrs. Françoise Besançon
U685 INSERM
75010 Paris FRA
Email:
Francoise.Besancon@stlouis.inser
m.fr
Pr. Iris Behrmann
Laboratoire de Biologie et
Physiologie Intégrée
1511 Luxembourg LUX
Email: iris.behrmann@uni.lu
Mr. Lijs Beke
department of biochemistry
3000 Leuven BEL
Email:
Lijs.Beke@med.kuleuven.be
Pr. Jean Louis Beneytout
Biochimie
87025 Limoges FRA
Email: beneytout@unilim.fr
Mr. Samir Benosman
INSERM U692
67000 Strasbourg FRA
Email:
samirbenosman@gmail.com
Mrs. Saoussen BERRAHMOUNE
Equipe Projet "Photobiologie en
Cancérologie"
54511 Vandoeuvre les Nancy FRA
Email:
s.berrahmoune@nancy.fnclcc.fr
Dr. Carlo Bertoni-Freddari
Neurobiology of Aging Laboratory
60121 Ancona ITA
Email: c.bertoni@inrca.it
Dr. CHARLES BESTWICK
MOLECULAR NUTRITION
AB21 9SB ABERDEEN GBR
Email: V.Smith@rowett.ac.uk
Mrs. Adeline BEILLEROT
LIMBP
57070 Metz FRA
Email: beillerotadeline@yahoo.fr
Mrs. Blanco Belen
Janssen Cilag
28042 Madrid ESP
Email: amex.c.viajes@aexp.com
Dr. Eyal Bengal
Department of Biochemistry
31096 Haifa ISR
Email: bengal@tx.technion.ac.il
Mrs. Sarah Berndt
Laboratoire de Biologie des
Tumeurs et du Développement
4000 Liége BEL
Email: sarah.berndt@ulg.ac.be
Dr. Holger Bertelsmann
14109 Berlin DEU
Email: bertelsmann@hmi.de
Mrs. Iris Bertram
Exp. Psychiatry Lab.
39120 Magdeburg DEU
Email: iris.bertram@gmx.de
Mr. Arnaud Bianchi
UMR 7561 LPPA
54505 Vandoeuvre FRA
Email:
arnaud.bianchi@medecine.uhpnancy.fr

Dr. Giordano Bianchi
Clinic of internal medicine I
16132 Genoa ITA
Email:
giordano.bianchi@katamail.com
Pr. Jan C. Biro
Informatics
94105 San Francisco USA
Email: jan.biro@sbcglobal.net
Dr. Romain Blasius
LBMCC
L-2540 Luxembourg LUX
Email: romain.blasius@lbmcc.lu
Pr. Mathieu Bollen
Cell signaling laboratory, Division
of Biochemistry, Department of
Molecular Cell Biology
3000 Leuven BEL
Email:
Mathieu.Bollen@med.kuleuven.be
Dr. Emilie Boncoeur
Insem U719
75012 Paris FRA
Email: emilie.boncoeur@stantoine.inserm.fr
Mrs. Meike Boosen
Institute of Genetics
53117 Bonn DEU
Email: meike.boosen@unibonn.de
Mr. Pedro Borralho
1600-083 Lisbon PRT
Email: borralho@ff.ul.pt
Dr. Hendrik Bielau
Department of Psychiatry
39120 Magdeburg DEU
Email:
Hendrik.Bielau@Medizin.Uni-
Magdeburg.de
Dr. Konstantin Birukov
IL 60637 Chicago USA
Email:
kbirukov@medicine.bsd.uchicago.
edu
Mrs. Rikke Bogebo
2600 Glostrup DNK
Email: rb@kvl.dk
Dr. Lina BOLOTINE
Equipe Projet "Photobiologie en
Cancérologie"
54511 Vandoeuvre les Nancy FRA
Email: l.bolotine@nancy.fnclcc.fr
Mrs. SARA BONDIONI
ISTITUTO DI SCIENZE
ENDOCRINE
20122 MILANO ITA
Email: sara.bondioni@unimi.it
Dr. Jean-Paul Borg
Molecular Pharmacology
13009 Marseille FRA
Email: borg@marseille.inserm.fr
Mrs. Veronika Borutinskaite
Division of Medical Microbiology
SE-581 85 Linkoping SWE
Email: verbu@imk.liu.se
Dr. Walter Birchmeier
13125 Berlin DEU
Email: wbirch@mdc-berlin.de
Dr. Anna Birukova
IL 60637 Chicago USA
Email:
abirukov@medicine.bsd.uchicago.
edu
Dr. Maria Bokarewa
Dep of Rheumatology and
Inflammation Research
S-41346 Goteborg SWE
Email:
maria.bokarewa@rheuma.gu.se
Dr. Sophia Bonanou
Biochemistry, School of Medicine
41222 Larissa GRC
Email: sbonanou@med.uth.gr
Mr. Guillaume Bonnot
HP2
38700 La Tronche FRA
Email: Guillaume.Bonnot@e.ujfgrenoble.fr
Dr. Jürgen Borlak
Pharmaforschung und
medizinische Biotechnologie
30625 Hannover DEU
Email: borlak@item.fraunhofer.de
Pr. Johannes L. Bos
Physiological Chemistry
3584 CG Utrecht NLD
Email: m.c.arpesella@med.uu.nl

Dr. Daniela Boselli
Tumor Immunology Laboratory
10126 Turin ITA
Email: daniela.boselli@unito.it
Mrs. Joelle BOTTI
U 504
94807 VILLEJUIF FRA
Email: botti@vjf.inserm.fr
Mrs. Sylvina Bouleau
Laboratoire de génétique et
biologie cellulaire
78035 Versailles FRA
Email: bouleau@genetique.uvsq.fr
Pr. Elisabeth BRAMBILLA
Lung Research Group
38706 LA TRONCHE FRA
Email: EBrambilla@chugrenoble.fr
Mr. Michael Bright
Cancer Cell Biology
W1W 7BS London GBR
Email: michael@ludwig.ucl.ac.uk
Mr. Bernard BRUGG
UMR7102 Neurobiologie des des
Processus Adaptatifs
75005 Paris FRA
Email:
bernard.brugg@snv.jussieu.fr
Mrs. Isabelle Buck
LBMCC
L-2540 Luxembourg LUX
Email: isabelle.buck@lbmcc.lu
Dr. Herbert Bosshart
Innate Immunity Research
Laboratory
CH-8091 Zurich CHE
Email:
herbert.bosshart@puk.zh.ch
Mrs. Sanae BOUALI
Laboratoire de Recherche
54511 Vandoeuvre les Nancy FRA
Email: s.bouali@nancy.fnclcc.fr
Mrs. Lana Bozulic
Brian Hemmings lab/Friedrich
Miescher Institute
4058 Basel CHE
Email: lana.bozulic@fmi.ch
Mrs. Malene Brandt
2600 Glostrup DNK
Email: mabr@kvl.dk
Mr. Christophe Broca
Equipe Avenir INSERM (U661)
34094 Montpellier FRA
Email:
christophe.broca@igf.cnrs.fr
Mr. Justin Brumbaugh
Schultz
69117 Heidelberg DEU
Email: brumbaug@embl.de
Dr. Miranda Buitenhuis
Dept. of Immunology
3584 EA Utrecht NLD
Email: m.buitenhuis@azu.nl
Pr. Serge Bottari
HP2
38700 La Tronche FRA
Email: Serge.Bottari@ujfgrenoble.fr
Mrs. Nadia Bougarne
LEGEST
9000 Ghent BEL
Email: nadia.bougarne@UGent.be
Pr. Marcantonio Bragadin
Dept. Environmental Sciences
30123 Venice ITA
Email: bragadin@unive.it
Mr. Laurent Brault
LIMBP
57070 Metz FRA
Email: brault@univ-metz.fr
Mr. Elisabeth Brouwer
9713 GZ Groningen NLD
Email: E.Brouwer@int.umcg.nl
Pr. Pedro Buc Calderon
Pharmacokinetic, Metabolism,
Nutrition and Toxicology
1200 Bruxelles BEL
Email: calderon@pmnt.ucl.ac.be
Mrs. Katarzyna Bukalis
14109 Berlin DEU
Email: k.bukalis@hmi.de

Mrs. Isabel Burghardt
Laboratory of Molecular Neuro-
Oncology, Department of General
Neurology
72076 Tuebingen DEU
Email: isabel.burghardt@gmx.net
Mr. Leonid Bystrykh
Stem Cell
9713 AV Groningen NLD
Email: bystrykh@med.umcg.nl
Mrs. Ying Cai
Diabetes Research Center
1090 Brussels BEL
Email: ying.cai@vub.ac.be
Dr. Serena Cecchetti
Dept. Cell Biology and
Neurosciences
162 Rome ITA
Email: ramoni@iss.it
Dr. Eva M Cernuda-Morollon
Cancer Cell Biology
W1W 7BS London GBR
Email: eva@ludwig.ucl.ac.uk
Pr. Benjamin Chain
W1T4JF London GBR
Email: b.chain@ucl.ac.uk
Dr. Wen-Hsiung Chan
Department of Bioscience
Technology
32023 Chung-Li TWN
Email: whchan@cycu.edu.tw
Dr. Hauke Busch
Theoretical Bioinformatics
69120 Heidelberg DEU
Email: h.busch@dkfz.de
Dr. Jocelyne Caboche
Signalisation neuronale et
Régulations géniques
75005 Paris FRA
Email:
jocelyne.caboche@svn.jussieu.fr
Mrs. Isabelle CARLAVAN
Research
06902 cedex Sophia-
Antipolis/Valbonne FRA
Email:
isabelle.carlavan@galderma.com
Dr. Claudia Cerella
133 Rome ITA
Email: cerella@uniroma2.it
Mrs. BORAM CHA
department of Pharmacology
120-752 Seoul KOR
Email:
chaboram@yumc.yonsei.ac.kr
Mrs. Florence Chainiaux
URBC
5000 Namur BEL
Email:
florence.chainiaux@fundp.ac.be
Mr. Alain Chariot
Medical Chemistry, CTCM
4000 Liége BEL
Email: alain.chariot@ulg.ac.be
Mrs. Helen Bye
Biochemistry
CB 2 1QW Cambridge GBR
Email: hlc24@mole.bio.cam.ac.uk
Dr. Jo Caers
Laboratory of Hematology &
Immunology
1090 Brussels BEL
Email: jcaers@vub.ac.be
Dr. Anje CAUWELS
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email:
Anje.Cauwels@dmbr.UGent.be
Mrs. Maria I. Cerezo-Guisado
Departamento de Bioquimica y
Biologia Molecular y Genética
10071 Caceres ESP
Email: mbcerezo@unex.es
Mrs. Georgia Chachami
Lab. of Biochemistry, School of
Medicine
41222 Larissa GRC
Email: ghah@med.uth.gr
Dr. Rachel Chambers
Centre for Respiratory Research
WC1E 6JJ London GBR
Email: r.chambers@ucl.ac.uk
Mr. Jean-François Charlot
Laboratoire de Biologie Cellulaire
et Moléculaire
25000 BESANCON FRA
Email: jfcharlot@hotmail.com

Mr. Eric CHASTRE
75018 Paris FRA
Email: chastre@bichat.inserm.fr
Mr. Jun-Jie Chen
Deparmemt
10018 Taipei TWN
Email: d91443001@ntu.edu.tw
Dr. Elena Chernolovskaya
630090 Novosibirsk RUS
Email: elena_ch@niboch.nsc.ru
Mrs. Soon Ok CHO
Department of Pharmacology
120-752 Seoul KOR
Email: ehrehrwh@hanmail.net
Pr. Kang-Yell Choi
Molecular Complex Control
120-749 Seoul KOR
Email: kychoi@yonsei.ac.kr
Dr. Tina Chowdhury
Cell and Tissue Engineering, Dept
of Engineering
E1 4NS London GBR
Email: t.t.chowdhury@qmul.ac.uk
Dr. Victor Cid
Dpto. Microbiologia II
28040 Madrid ESP
Email: vicjcid@farm.ucm.es
Dr. Ioulia Chatzistamou
Biochemistry
115 27 Athens GRC
Email: ihatzist@med.uoa.gr
Mrs. Miao-Der Chen
Animal Science
912-01 Ping Tung County TWN
Email:
miaoder124@yahoo.com.tw
Dr. Susanna Chiocca
Dept. of Experimental Oncology
20141 Milan ITA
Email: susanna.chiocca@ifom-ieocampus.it
Dr. Claudia Choi
30659 Hannover DEU
Email: Choi.Claudia@MH-
Hannover.de
Mr. Yoon Pyo Choi
Pathology
120-752 Seoul KOR
Email:
yoonpyo@yumc.yonsei.ac.kr
Mrs. Gitte Christensen
2600 Glostrup DNK
Email: glc@dcb-glostrup.dk
Dr. Iwona Ciechomska
Department of Biochemistry
CB2 1QW Cambridge GBR
Email: iac23@mole.bio.cam.ac.uk
Pr. Ching-Chow Chen
10018 Taipei TWN
Email: ccchen@ha.mc.ntu.edu.tw
Dr. Steven Cheng
21746 Ijamsville, MD USA
Email: scheng@cellogenetics.com
Pr. Nam Hoon Cho
Pathology
120-752 Seoul KOR
Email:
cho1988@yumc.yonsei.ac.kr
Mr. Jang Hyun Choi
Signaling Network Lab.
790-784 Pohang KOR
Email: janghyun@postech.ac.kr
Mr. Rasheduzzaman Chowdhury
C. J. Schofield
Ox1 3TA Oxford GBR
Email:
rasheduzzaman.chowdhury@chem
.ox.ac.uk
Dr. Hae-Yun Chung
Department of Pharmacology
120-752 Seoul KOR
Email:
hchung02@yumc.yonsei.ac.kr
Dr. Aleksandra Cierniewska-
Cieslak
92-215 Lodz POL
Email: ola_cier@poczta.onet.pl

Pr. Czeslaw Cierniewski
92-215 Lodz POL
Email: cciern@zdn.am.lodz.pl
Mr. Nicolas CLERE
Laboratoire de Biologie Cellulaire
et Moléculaire
25040 BESANCON FRA
Email: clere.nicolas@wanadoo.fr
Pr. Paul Coffer
Dept. of Immunology
3584 EA Utrecht NLD
Email: p.j.coffer@umcutrecht.nl
Mrs. Anne Conradi
Institute of Genetics
53117 Bonn DEU
Email: anne.conradi@uni-bonn.de
Dr. Eva Corey
GU Cancer Research Laboratory
98008 Seattle USA
Email: ecorey@u.washington.edu
Dr. Evelyne Crapez
Research Cancer Center
34298 Montpellier FRA
Email: ecrapez@valdorel.fnclcc.fr
Mrs. Tania Cruz
Division of Clinical Immunology
and Rheumatology
1100 DE Amsterdam NLD
Email: k.a.reedquist@amc.uva.nl
Mrs. Saskia Cillessen
Pathology
1081 HV Amsterdam NLD
Email: sagm.cillessen@vumc.nl
Dr. Daniel Closa
Experimental Pathology
8036 Barcelona ESP
Email: dcabam@iibb.csic.es
Dr. Eleanor Coffey
Neuronal Signalling lab
Fin-20521 Turku FIN
Email: ecoffey@btk.fi
Dr. Laura Conti
Tumor Immunology Laboratory
10126 Turin ITA
Email: laura.conti@unito.it
Dr. Sigrid CORNELIS
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email: Sigrid@dmbr.UGent.be
Dr. Pascale Crépieux
Physiologie de la Reproduction et
des Comportements
37380 Nouzilly FRA
Email: crepieux@tours.inra.fr
Dr. Barbara Cserepes
Department of Surgical Research
and Techniques
7624 Pécs HUN
Email: barbicska@yahoo.co.uk
Mr. Thomas Clahsen
Institut für Biochemie
52074 Aachen DEU
Email: thomas.clahsen@post.rwthaachen.de
Dr. Laura Cobeno
Dept. Pharmacology
28040 Madrid ESP
Email: acogolludo@ift.csic.es
Dr. Angel Cogolludo
Dept. Pharmacology
28040 Madrid ESP
Email: acogolludo@ift.csic.es
Mrs. Elisabeth-Anne CORCELLE
UMR 65 48
6107 Nice FRA
Email: ecorcell@unice.fr
Dr. Michael Courtney
Molecular Signalling Lab
FIN 70211 KUOPIO FIN
Email: courtney@messi.uku.fi
Mrs. Silvia Cristofanon
LBMCC
L-2540 Luxembourg LUX
Email:
silvia.cristofanon@lbmcc.lu
Mr. Alfredo Csibi
HP2
38700 La Tronche FRA
Email: AlfredoCsibi@e.ujfgrenoble.fr

Mrs. Claire Cullmann
69115 Heidelberg DEU
Email: c.cullmann@dkfz.de
Mrs. Lisa Dalla Puppa
14109 Berlin DEU
Email: dallapuppa@hmi.de
Dr. Ben Davidson
Department of Pathology
N-0310 Oslo NOR
Email:
ben.davidson@radiumhospitalet.n
o
Mr. Maite De los Frailes
Screening and Compound Profile
28760 Tres Cantos- Madrid ESP
Email:
maite_de_los_frailes@gsk.com
Dr. Maarten de Smit
Biological Chemistry, Leiden
University
2333 CC Leiden NLD
Email:
m.smit@chem.leidenuniv.nl
Mrs. Nicole DEFER
INSERM U.581
94010 Créteil FRA
Email: defer@im3.inserm.fr
Mr. Pim Dekker
Corporate Research
MK44 1LQ Sharnbrook GBR
Email: pim.dekker@unilever.com
Pr. Edgar F. da Cruz e Silva
LaboratÛrio de TransduÁ„o de
Sinais
3810-193 Aveiro Aveiro PRT
Email: dacruze@bio.ua.pt
Mr. Avais DAULAT
75014 Paris FRA
Email: daulat@cochin.inserm.fr
Pr. Roger Davis
1605 Worcester USA
Email:
Roger.Davis@Umassmed.edu
Dr. Milena De Nicola
133 Rome ITA
Email:
milena.de.nicola@uniroma2.it
Mrs. Fabienne DE TONI
INSERM Unit 563
31024 TOULOUSE FRA
Email:
fadetoni@toulouse.inserm.fr
Mrs. Sylvie Defrére
Gynecology
1200 Bruxelles BEL
Email:
sylvie.defrere@gyne.ucl.ac.be
Mrs. Anne-Isabelle Delattre
URBC
5000 Namur BEL
Email: anneisabelle.delattre@fundp.ac.be
Pr. Odete A. B. da Cruz e Silva
LaboratÛrio de NeurociÍncias
3810-193 Aveiro Aveiro PRT
Email: odetecs@bio.ua.pt
Dr. Frank Dautzenberg
CNS Research
2340 Beerse BEL
Email: fdautzen@prdbe.jnj.com
Dr. Xander de Haan
Virology
3584 CL Utrecht NLD
Email: x.haan@vet.uu.nl
Mrs. Aurélia De Pauw
URBC
5000 Namur BEL
Email:
aurelia.depauw@fundp.ac.be
Dr. Wim DECLERCQ
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email:
Wim.Declercq@dmbr.UGent.be
Mr. Julien Deheuninck
CNRS UMR 8117
59270 LILLE FRA
Email: julien.deheuninck@ibl.fr
Mrs. Daphne deLaunay
Division of Clinical Immunology
and Rheumatology
1100 DE Amsterdam NLD
Email: k.a.reedquist@amc.uva.nl

Mr. Igotz Delgado
Biochemistry
48940 Leioa ESP
Email: ofbdebai@lg.ehu.es
Mrs. Xiaoling Deng
Centre for Respiratory Research
WC1E 6JJ London GBR
Email: caseydxl@yahoo.com
Dr. Valentina Di Felice
Molecular Anatomy
90127 Palermo ITA
Email: valentina.difelice@unipa.it
Dr. Elitsa Dimova
Biochemistry
67663 Kaiserslautern DEU
Email: dimova@chemie.uni-kl.de
Dr. Katalin Dobra
Department of Laboratory
Medicine
SE-14186 Stockholm SWE
Email:
katalin.dobra@labmed.ki.se
Mrs. Sofia Dos Santos
URBC
5000 Namur BEL
Email:
sofia.dossantos@fundp.ac.be
Mrs. Joe-Lin du Toit
Dept of Physiological Sciences
7600 Stellenbosch ZAF
Email: jl@sun.ac.za
Mrs. Raffaella Dell'Eva
Experimental Oncology A
16132 Genoa ITA
Email: raffaelladelleva@yahoo.it
Dr. Anne Devin
IBGC
33077 BORDEAUX FRA
Email: anne.devin@ibgc.ubordeaux2.fr
Dr. Marc Diederich
LBMCC
L-2540 Luxembourg LUX
Email: marc.diederich@lbmcc.lu
Pr. Caroline Dive
Clinical and Experimental
Pharmacology Group
M20 4BX Manchester GBR
Email: cdive@picr.man.ac.uk
Mrs. Sara C. T. S. Domingues
LaboratÛrio de NeurociÍncias
3810-193 Aveiro Aveiro PRT
Email: a15922@alunos.bio.ua.pt
Dr. Alexandra Dreuw
Institut für Biochemie
52074 Aachen DEU
Email: serge.haan@rwthaachen.de
Dr. Lenka Dubska
Department of Laboratory
Medicine
Msaryk Memorial Cancer Institute
Brno 656 53 Czech Republic
Email: dubska@mou.cz
Dr. Maurice Dematteis
40202 Louisville, KY USA
Email:
maurice.dematteis@louisville.edu
Dr. Luciano Di Croce
Epigenetic events in cancer
8003 Barcelona ESP
Email: luciano.dicroce@crg.es
Mrs. Nathalie Dijsselbloem
LEGEST
9000 Gent BEL
Email:
nathalie.dijsselbloem@ugent.be
Dr. Mojgan Djavaheri-Mergny
Glycobiologie et signalisation
cellulaire
94807 Villejuif FRA
Email: mergny@vjf.inserm.fr
Dr. Rosanna DONO
CNRS
13288 Marseille FRA
Email: dono@ibdm.univ-mrs.fr
Dr. Sophia V. Drouva
Interactions Cellulaires
Neuroendocriniennes
13916 Marseille FRA
Email: drouva.sv@jeanroche.univ-mrs.fr
Dr. Joanna Dulinska
Department of Medical
Biochemistry
31-034 Krakow POL
Email: mblitewk@cyf-kr.edu.pl

Mr. JACQUES DUMONT
IRIBHM
1070 BRUSSELS BEL
Email: jedumont@ulb.ac.be
Dr. Zdenek Dvorak
Institute of Medical Chemistry and
Biochemistry
77515 Olomouc CZE
Email: moulin@email.cz
Dr. Kerry Elliot
E1 4NS London GBR
Email: kelliot@staffmail.ed.ac.uk
Dr. Anna-Mart Engelbrecht
Dept of Physiological Sciences
7600 Stellenbosch ZAF
Email: ame@sun.ac.za
Dr. Ilaria Esposito
Prof. Anna Maria Musti
80131 Napoli ITA
Email: ila_esposito@libero.it
Dr. Susanna Fagerholm
Molecular studies on leukocyte
adhesion, Carl G. Gahmberg
14 Helsinki FIN
Email:
susanna.fagerholm@helsinki.fi
Mrs. Consol Farrera-Sinfreu
Group of Macrophage Biology
8028 Barcelona ESP
Email: cfarresi7@qui.ub.edu
Mr. Eric DUPLUS
UMR7102 Neurobiologie des des
Processus Adaptatifs
75005 Paris FRA
Email: eric.duplus@snv.jussieu.fr
Mrs. Souhayla El Maadidi
LIMBP
57070 Metz FRA
Email: elnaadid@univ-metz.fr
Mr. YALIN EMRE
CNRS UPR 9078
75730 CEDEX 15 PARIS FRA
Email: emre@necker.fr
Dr. Riyo Enomoto
Department of Pharmacology
651-2180 Kobe JPN
Email:
enomoto@pharm.kobegakuin.ac.jp
Pr. Alfred Esser
Cell Biology & Biophysics
64110 Kansas City, Missouri USA
Email: essera@umkc.edu
Dr. Alexander Faltusz
65824 Schwalbach/Ts DEU
Email:
alexander.faltusz@merckbioscienc
es.de
Dr. Patrizia Fattoretti
Neurobiology of Aging Laboratory
60121 Ancona ITA
Email: p.fattoretti@inrca.it
Dr. Erwan Dupont
Lab Neuromuscular Plasticity
59655 Villeneuve d'Ascq FRA
Email: Erwan.Dupont@univlille1.fr
Mrs. Aleksandra Ellert-
Miklaszewska
Dept. of Biochemistry and Clinical
Chemistry
02-097 Warsaw POL
Email: aellert@nencki.gov.pl
Dr. Takayuki Endoh
Department of Physiology
2618502 Chiba JPN
Email: tendoh@tdc.ac.jp
Mr. Morten Eskildsen
2600 Glostrup DNK
Email: me@dcb-glostrup.dk
Dr. Beatrice EYMIN
Lung Research Group
38706 LA TRONCHE FRA
Email: Beatrice.Eymin@ujfgrenoble.fr
Dr. Margarida Fardilha
LaboratÛrio de TransduÁ„o de
Sinais
3810-193 Aveiro Aveiro PRT
Email: mfardilha@bio.ua.pt
Mrs. Sylvie Fauconnet
Laboratoire de Biologie Cellulaire
et Moléculaire
25000 BESANCON FRA
Email: s.fauconnet@libertysurf.fr

Mrs. Anissa Fergani
INSERM U-692
67000 Strasbourg FRA
Email: anissafergani@yahoo.fr
Mrs. Nele FESTJENS
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email: Nele@dmbr.UGent.be
Mrs. Katherine G Finegan
Faculty of Life Sciences
M13 9PT Manchester GBR
Email:
K.G.Finegan@postgrad.mancheste
r.ac.uk
Dr. Daniela Flügel
Biochemistry
67663 Kaiserslautern DEU
Email: dfluege@gwdg.de
Mrs. Bénédicte Foveau
CNRS UMR8117
59021 Lille FRA
Email: benedicte.foveau@ibl.fr
Mrs. Giovanna Frazziano
Dept. Pharmacology
28040 Madrid ESP
Email: acogolludo@ift.csic.es
Mr. Michael Fricker
Department of Biochemistry
CB2 1QW Cambridge GBR
Email: mf309@cam.ac.uk
Mrs. Anne FERNANDEZ VIDAL
INSERM Unit 563
31024 TOULOUSE FRA
Email:
fervidal@toulouse.inserm.fr
Mrs. Dragana Filipovic
Laboratory for molecular biology
and endocrinology 090
11000 Belgrade YUG
Email: dragana@vin.bg.ac.yu
Dr. Alessia Finotti
Dept of Biochemistry and
Molecular Biology
44100 Ferrara ITA
Email: gam@unife.it
Dr. Emma Folch-Puy
Experimental Pathology
8036 Barcelona ESP
Email: efpbam@iibb.csic.es
Dr. Thierry Franck
Centre de l'Oxygéne, Recherche et
Développement
4000 Liége BEL
Email: T.franck@ulg.ac.be
Dr. Samuel W. French
90502 Torrance USA
Email: sfrench@labiomed.org
Mr. Hans Friedrichsen
Department Biochemistry
CB2 1QW Cambridge GBR
Email: hfriedrichsen@gmail.com
Mrs. Laura Ferrero-Miliani
Lab. of Gastroenterology C 5403,
Herlev Hospital
2730 Herlev DNK
Email: lali@stud.ku.dk
Dr. Carmela Fimognari
Pharmacology
40126 Bologna ITA
Email:
carmela.fimognari@unibo.it
Mr. KONSTANTINOS FLOROS
BIOCHEMISTRY AND
MOLECULAR BIOLOGY
15701 ATHENS GRC
Email: florksgr@yahoo.gr
Mrs. Florence Folmer
AB25 2WN Aberdeen GBR
Email:
florence_folmer@yahoo.com
Mrs. Aurélie FRANCOIS
Equipe Projet "Photobiologie en
Cancérologie"
54511 Vandoeuvre les Nancy FRA
Email: a.francois@nancy.fnclcc.fr
Dr. Véronique FREUND-
MICHEL
EA3771
67401 ILLKIRCH FRA
Email: vfreund@pharma.ustrasbg.fr
Dr. Ellen Fritsche
Toxicology
40225 Düsseldorf DEU
Email: ellen.fritsche@uniduesseldorf.de

Mr. Thomas Frogne
DK-2100 Copenhagen DNK
Email: tfr@cancer.dk
Mr. Gérald GAIBELET
IPBS
31077 TOULOUSE FRA
Email: gerald.gaibelet@ipbs.fr
Mr. Mattias Gäreskog
Inst. Medical Cell Biology
751 23 Uppsala SWE
Email:
Mattias.Gareskog@medcellbiol.uu
.se
Mr. Javier Gayarre
28040 Madrid ESP
Email: jgayarre@cib.csic.es
Pr. Penka Genova
CLMP
1113 Sofia BGR
Email: Pgen@TU-Sofia.bg
Mrs. Lieke Gerris
Xtrack
3572GA Utrecht NLD
Email: L.M.Gerris@students.uu.nl
Dr. Lina Ghibelli
133 Roma ITA
Email: ghibelli@uniroma2.it
Dr. François Fuks
1070 Brussels BEL
Email: ffuks@ulb.ac.be
Dr. Catherine Gaitanaki
Animal & Human Physiology
157 84 Athens GRC
Email: cgaitan@biol.uoa.gr
Dr. Clare Garvey
N1 9XW London GBR
Email: c.garvey@nature.com
Dr. Christoffer Gebhardt
Division of Signal Transduction
and Growth Control
69120 Heidelberg DEU
Email: c.gebhardt@dkfz.de
Mr. Thomas Gerken
C. J. Schofield
Ox1 3TA Oxford GBR
Email:
thomas.gerken@bioch.ox.ac.uk
Dr. Arieh Gertler
The Hebrew University of
Jerusalem
76100 Rehovot ISR
Email: gertler@agri.huji.ac.il
Pr. Sankar Ghosh
Section of Immunobiology
6519 New Haven USA
Email: sankar.ghosh@yale.edu
Dr. Jozefa Gadek-Wesierski
Dept. of Medicine I, Div.: Inst. of
Cancer Research
A-1090 Vienna AUT
Email: jozefa.gadekwesierski@meduniwien.ac.at
Pr. Roberto Gambari
Dept of Biochemistry and
Molecular Biology
44100 Ferrara ITA
Email: gam@unife.it
Dr. Jean-Stéphane Gatot
Chimie médicale
4000 Liége BEL
Email: jsgatot@ulg.ac.be
Mr. Christian Geest
Dept. of Immunology
3584 EA Utrecht NLD
Email: C.Geest@azu.nl
Dr. Sarah Gerlo
LEGEST
9000 Gent BEL
Email: sarah.gerlo@ugent.be
Dr. Mahmoud Ghazi-Khansari
Tehran University of Medical
Sciences
13145 - 784 Tehran PCN
Email: ghazikha@sina.tums.ac.ir
Dr. Dorota Gil
Department of Medical
Biochemistry
31-034 Krakow POL
Email: dgil@poczta.fm

Dr. Mark Ginsberg
Mail code 0726
92093-0726 La Jolla USA
Email: mhginsberg@ucsd.edu
Mrs. Nadine Goebel
40225 Duesseldorf DEU
Email: nadine@wcv-mail.de
Mr. Arnaud Goolaerts
Physiopathology
1070 Brussels BEL
Email: agoolaer@ulb.ac.be
Mrs. Delphine Goubau
Dr J. Hiscott
H3T 1E2 Montreal CAN
Email:
delphine.goubau@mcgill.ca
Dr. Evelyne Gozal
40202 Louisville, KY USA
Email:
evelyne.gozal@louisville.edu
Dr. Sandro Grelli
Dept. Exp.Med.Bioch.Sci.
133 Rome ITA
Email: grelli@med.uniroma2.it
Dr. André Groyer
Inserm U.683
75870 Paris Cédex 18 FRA
Email: groyer@bichat.inserm.fr
Pr. Hans-Juergen Glander
EAA Training Center
4103 Leipzig DEU
Email: glah@medizin.unileipzig.de
Dr. Christoph Goemans
Department of Biochemistry
CB2 1QW Cambridge GBR
Email: cgg20@mole.bio.cam.ac.uk
Mrs. Morgane GORRIA
INSERM U620
35043 Rennes FRA
Email: morgane.gorria@univrennes1.fr
Mrs. Eleni Gourgou
Animal and Human Physiology
15784 Athens GRC
Email: elgour@biol.uoa.gr
Mr. Igor Grbavac
14109 Berlin DEU
Email: grbavac@hmi.de
Mrs. Femke Groeneweg
Xtrack
3572 GA Utrecht NLD
Email:
F.L.Groeneweg@students.uu.nl
Dr. Sonja Grunewald
EAA Training Center
4103 Leipzig DEU
Email:
sonja.grunewald@medizin.unileipzig.de
Mrs. Sophie Goblet
URBC
5000 Namur BEL
Email: sophie.goblet@fundp.ac.be
Dr. Reinaldo Gonzalez Ramos
Gynecology
1200 Bruxelles BEL
Email:
reinaldogonzalezr@yahoo.com
Mrs. Valerie Gossye
LEGEST
9000 Gent BEL
Email: valerie_gossye@ugent.be
Mr. Ganjam Goutham Kumar
Biochemistry
67663 Kaiserslautern DEU
Email: ganjam@chemie.uni-kl.de
Dr. Mira Grdisa
of molecular oncology
10000 Zagreb HRV
Email: grdisa@irb.hr
Pr. Bernd Groner
Institute for Biomedical Research
D-60596 Frankfurt am Main DEU
Email: groner@em.unifrankfurt.de
Mrs. Barbara Guerra
Institute of Biochemistry and
Molecular Biology
5230 Odense DNK
Email: bag@bmb.sdu.dk

Mr. Thierry GUILLAUDEUX
UPRES-EA 3889
35043 Rennes FRA
Email: thierry.guillaudeux@univrennes1.fr
Dr. Deepak Gupta
All India Institue of Medical
Sciences, New Delhi, India
110029 Delhi IDN
Email:
drdeepakkumargupta@yahoo.com
Dr. Claude Haan
LBPI
L-1511 Luxemburg LUX
Email: claude.haan@uni.lu
Dr. Yvette HABRAKEN
Virology and Immunology
4000 LIEGE BEL
Email: Yvette.habraken@ulg.ac.be
Mr. Eric Hajduch
Unité 671
75006 Paris FRA
Email:
eric.hajduch@bhdc.jussieu.fr
Mr. Adam Hardy
Schofield
OX1 3TA Oxford GBR
Email:
adam.hardy@chem.ox.ac.uk
Dr. Bettina Hartenstein
Division of Signal Transduction
and Growth Control
69120 Heidelberg DEU
Email: b.hartenstein@dkfz.de
Dr. Nursel Gul
Ankara Universitesi, Fen Fakultesi
Biyoloji Bolumu
Degol
6100 TCA
Email: ngul@science.ankara.edu.tr
Mr. Eric Gutknecht
CNS Research
2340 Beerse BEL
Email: egutknec@prdbe.jnj.com
Dr. Serge Haan
Institut für Biochemie
52074 Aachen DEU
Email: serge.haan@rwthaachen.de
Pr. Guy HAEGEMAN
Laboratory for Eukaryotic Gene
Expression and Signal
Transduction (LEGEST)
9000 GENT BEL
Email: guy.haegeman@ugent.be
Dr. Reza Haji Hosseini
Biology Group
19569 Tehran IRN
Email: hosseini@pnu.ac.ir
Dr. Britta HARDY
Felsenstein Medical Research
Center
49100 Petah-Tikva ISR
Email: bhardy@post.tau.ac.il
Mrs. Michal Hayun
Prof. Benjamin Sredni
52900 Ramat-Gan ISR
Email: michalbgnm@netscape.net
Mrs. Laura Gumy
Department Biochemistry
CB2 1QW Cambridge GBR
Email: lfg23@cam.ac.uk
Dr. Joohun Ha
Biochemistry & Molecular
Biology
130-701 Seoul KOR
Email: hajh@khu.ac.kr
Mr. Thomas Haarmann-Stemmann
Toxicology
40225 Düsseldorf DEU
Email: haarmann@uniduesseldorf.de
Dr. Jody HAIGH
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email:
Jody.Haigh@dmbr.UGent.be
Mr. Benoit Halter
INSERM U692
67000 Strasbourg FRA
Email: ben_halter@yahoo.fr
Dr. Shannon Harper
Johannes L. Bos
3584 CG Utrecht NLD
Email: s.harper@med.uu.nl
Mr. Rami Hayun
Prof. Benjamin Sredni
52900 Ramat-Gan ISR
Email: rami_hayun@walla.co.il

Dr. Jochen Hefl
Division of Signal Transduction
and Growth Control
69120 Heidelberg DEU
Email: j.hess@dkfz.de
Mr. Mahmoud Reza Heidari
Toxicology and Pharmacology
98 Kerman IRN
Email: Heidarimr@yahoo.com
Dr. Arnd Hengstermann
Molecular Biology
50939 Cologne DEU
Email:
arnd.hengstermann@pmintl.com
Mr. Estelle Henry
LBMCC
L-2540 Luxembourg LUX
Email: estelle.henry@lbmcc.lu
Dr. Andreas Herrmann
52074 Aachen DEU
Email: aherrmann@ukaachen.de
Mrs. Majbrit Hjerrild
2600 Glostrup DNK
Email: mh@dcb-glostrup.dk
Mr. CAROLE HONIGMANN
inserm U-692
67000 Strasbourg FRA
Email: honigmann_c@yahoo.fr
Mrs. Csilla Hegedüs
Signal Transduction
H-1113 Budapest HUN
Email: hecsillu@yahoo.com
Dr. Christine Hellweg
Radiobiology
51170 Koeln DEU
Email: christine.hellweg@dlr.de
Mrs. Rachel Henkens
Anatomie et Cytologie
Pathologiques
4000 Liége BEL
Email:
R.Henkens@student.ulg.ac.be
Dr. Thomas Herdegen
24105 Kiel DEU
Email:
herdegen.office@pharmakologie.u
ni-kiel.de
Dr. Stefan Hippenstiel
Dept. of Infectious Diseases
13353 Berlin DEU
Email:
Stefan.Hippenstiel@charite.de
Dr. Tjadine M. Holling
Division of Molecular Biology,
Department of
Immunohematology and Blood
Transfusion
2333 ZA Leiden NLD
Email: T.M.Holling@lumc.nl
Mrs. Diana Hoogeboom
Physiological Chemistry
3584 CG Utrecht NLD
Email: d.hoogeboom@med.uu.nl
Dr. Jacqueline Heger
Institute of Physiology
35392 Gieflen DEU
Email:
Jacqueline.Heger@physiologie.me
d.uni-giessen.de
Dr. Brian Hemmings
CH 4058 Basel CHE
Email: brian.hemmings@fmi.ch
Dr. Hanjo Hennemann
53175 Bonn DEU
Email: hennemann@caesar.de
Mrs. Maria José Herrero
Lab. Terapia Génica, Dpt.
Farmacologia
46010 Valencia ESP
Email: mariajoseherrero@ono.com
Pr. John Hiscott
Molecular Oncology
H3T 1E2 Montreal Quebec CAN
Email: john.hiscott@mcgill.ca
Dr. Olaf Holtkoetter
Skin / Hair Physiology - Molecular
Biology
D-40191 Duesseldorf DEU
Email:
olaf.holtkoetter@henkel.com
Mrs. Zuzana Horejsi
Molecular Virology
166 37 Prague CZE
Email: zhora@img.cas.cz

Mr. Ruben Hoya-Arias
LEGEST - Department Molecular
Biology -
9000 Gent BEL
Email:
Ruben.HoyaArias@UGent.be
Mr. Haeng-Jeon Hur
Department of food biotechnology
151-742 Seoul KOR
Email: snufst_hj@hotmail.com
Dr. Britta Husse
Julius Bernstein Institute of
Physiology
D-06097 Halle/Saale DEU
Email: britta.husse@medizin.unihalle.de
Mr. Aman Iqbal
Schofield
OX1 3TA Oxford GBR
Email: aman.iqbal@chem.ox.ac.uk
Mrs. Andrea Ivascu
Cell Biology
82377 Penzberg DEU
Email: andrea.ivascu@roche.com
Dr. Kowan Ja Jee
Hartman Instutute, Department of
Pathology
FIN-00014, PO Box 21 Helsinki
FIN
Email: kowan.jee@helsinki.fi
Mr. Namkyeong Jeon
Department of oral pathlogy
120-752 Seoul KOR
Email: op1@yumc.yonsei.ac.kr
Pr. Yan-Der Hsuuw
Animal Science
912-01 Ping Tung County TWN
Email: hsuuw@yahoo.com.tw
Dr. Amandine Hurbin
Lung Research Group
38706 LA TRONCHE FRA
Email: Amandine.Hurbin@ujfgrenoble.fr
Mr. Jin-Taek Hwang
Biochemistry & Molecular
Biology
130-701 Seoul KOR
Email: hjt153@hanmail.net
Dr. Tarik Issad
Institut Cochin
75014 Paris FRA
Email: issad@cochin.inserm.fr
Dr. Gabor Jancso
Department of Surgical Research
and Techniques
7624 Pécs HUN
Email: jancsogabor@hotmail.com
Mr. Viktor Jefremov
Department of Biochemistry
50411 Tartu EST
Email:
viktor.jefremov@kliinikum.ee
Mr. Yong-Tark Jeon
Laboratory of Gynecological
Oncology
110-799 Seoul KOR
Email: rbssh9@snu.ac.kr
Pr. Nam-ho Huh
Cell Biology
700-8558 Okayama JPN
Email: namu@md.okayamau.ac.jp
Pr. Nils-Erik Huseby
Medical Biochemistry
9037 Tromso Tromso UMI
Email: nilseh@fagmed.uit.no
Mr. Jin-Taek Hwang
Dept of Food and Nutrition
306-791 Daejeon KOR
Email: ojpark@hannam.ac.kr
Dr. Tarik Issad
Institut Cochin
75014 Paris FRA
Email: issad@cochin.inserm.fr
Dr. PATCHAREE
JEARANAIKOON
CLINICAL
CHEMISTRY,FACULTY OF
ASSOCIATED MED SCIENCES
40002 KHON KAEN THA
Email: patjea@kku.ac.th
Mrs. Nam K Jeon
depatrment of oral pathlogy
120-752 seoul KOR
Email: aroma990@hanmail.net
Mrs. Min-A Jeong
Department of food biotechnology
151-742 Seoul KOR
Email: accamy@hanmail.net

Mr. Jan Jepsen
2100 Copenhagen DNK
Email: jsj@cancer.dk
Mr. Claus Johansen
Department of Dermatology
8000 Aarhus DNK
Email: claus.johansen@ki.au.dk
Mr. Kyung Don JU
Department of Pharmacology
120-752 Seoul KOR
Email: clock1375@paran.com
Dr. Magdalena Kacprzak
Molecular Signalling Lab
FIN 70211 KUOPIO FIN
Email:
Magdalena.Kacprzak@uku.fi
Dr. Martha Kaloyianni
Animal Physiology
54124 Thessaloniki GRC
Email: kaloyian@bio.auth.gr
Pr. GAOUSSOU KANOUTE
LABORATOIRE NATIONAL
DU MALI
BP 232 BAMAKO MLI
Email:
kanoutelaboratoire@yahoo.fr
Dr. Lena Karlsson
Clinical and Experimental
Research Laboratory
S-41346 Goteborg SWE
Email: Lena.Karlsson@hjl.gu.se
Mrs. Hanna Jerczynska
Department of Mol. & Med.
Biophysics
92-215 Lodz POL
Email: hanuka@zdn.am.lodz.pl
Mrs. Anne-Laure Joly
LBMCC
2540 Luxembourg LUX
Email: anne-laure.joly@edu.univfcomte.fr
Pr. Yong-Sung Juhnn
Department of Biochemistry and
Molecular Biology
110-799 Seoul KOR
Email: juhnn@snu.ac.kr
Mrs. PANAGIOTA KAKATSOU
BIOCHEMISTRY AND
MOLECULAR BIOLOGY
15701 ATHENS GRC
Email: florksgr@yahoo.gr
Mrs. MAUD KAMAL
INSERM U581 Equipe Avenir
94010 CRETEIL FRA
Email:
maud.kamal@creteil.inserm.fr
Dr. Elodie Kara
Physiologie de la Reproduction et
des Comportements
37380 Nouzilly FRA
Email: kara@tours.inra.fr
Mr. Atsufumi Kawabata
Div. Physiol. Pathophysiol., Sch.
Pharm. Sci.
577-8502 Higashi-Osaka JPN
Email:
kawabata@phar.kindai.ac.jp
Mr. Manel Joaquin
Cell Signaling Unit
8002 Barcelona ESP
Email: manel.joaquin@upf.edu
Dr. RHIANNON JONES
ANTIONCOGENE
SW3 6JB LONDON GBR
Email: rhiannon.jones@icr.ac.uk
Dr. Anna Jurewicz
Neurology
90-153 Lodz POL
Email: ajur@afazja.am.lodz.pl
Dr. Tuula Kallunki
Apoptosis Department
2100 Copenhagen DNK
Email: tk@cancer.dk
Mrs. Monika Kaminska
Enzymatic Regulation of Cell
Activities
75724 Paris FRA
Email: kaminska@pasteur.fr
Mr. Emin Karaca
Ege University Medical Faculty
Medical Genetics
35100 Izmir TUR
Email: asudealpman@gmail.com
Dr. Shirakawa Kazuo
Saitama medical center
350-8550 Kawagoe JPN
Email: shirak@saitama-med.ac.jp

Mrs. Eirini Kefaloyianni
Animal and Human Physiology
15784 Athens GRC
Email: ikefalog@biol.uoa.gr
Pr. Ezzatollah Keyhani
University of Tehran
13145 Tehran IRN
Email: keyhanie@ibb.ut.ac.ir
Dr. Maria Kharlap
Clinical
Electrophysiology/Genetic
Engineering
121552 Moscow RUS
Email: shadow1@mail.cnt.ru
Mrs. Do-Hee Kim
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: calladohee@yahoo.co.kr
Pr. Hyeyoung Kim
Department of Pharmacology
120-752 Seoul KOR
Email:
kim626@yumc.yonsei.ac.kr
Mrs. Mi Jeong Kim
Oral Biology
120-752 Seoul KOR
Email:
kimmi780507@hanmail.net
Mrs. Seo-Young Kim
Department of food biotechnology
151-742 Seoul KOR
Email: ssoy5200@hanmail.net
Mrs. Lilian Kessels
6200 MD Maastricht NLD
Email:
l.vanwylick@farmaco.unimaas.nl
Dr. Jacqueline Keyhani
University of Tehran
13145 Tehran IRN
Email: keyhanie@ibb.ut.ac.ir
Dr. Hippokratis Kiaris
Biochemistry
115 27 Athens GRC
Email: hkiaris@med.uoa.gr
Mrs. Eun-Hee Kim
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: idi@dreamwiz.com
Mrs. Hyo-jin Kim
Department of food biotechnology
151-742 Seoul KOR
Email: ipkhj@hanmail.net
Mrs. Min-Jung Kim
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: bluebeer99@hanmail.net
Mrs. Su-Hyeong Kim
110-799 Seoul KOR
Email: rbssh9@snu.ac.kr
Mr. HUR KEUN
Cancer Research Institute
110-744 Seoul PRK
Email: blue2001@snu.ac.kr
Dr. Khalid Khabar
Program in BioMolecular
Research
11211 Riyadh SAU
Email: khabar@kfshrc.edu.sa
Dr. Simone Kiermayer
Internal Medicine II
66421 Homburg/Saar DEU
Email: inskie@unikliniksaarland.de
Mrs. Hye Sun Kim
Dept. of Anatomy, Embryology
Lab.
120-752 Seoul KOR
Email: goldfish79@hanmail.net
Mrs. Jin Eun Kim
Department of Oral Biology
120-752 Seoul KOR
Email: judyjean@hanmail.net
Pr. Myoung Hee Kim
Dept. of Anatomy, Embryology
Lab.
170-752 Seoul KOR
Email:
mhkim1@yumc.yonsei.ac.kr
Pr. SUNG KIM
ANGIOGENESIS AND
CHEMOPREVENTION
449701 YONGIN KOR
Email: sungkim7@khu.ac.kr

Pr. Young Min Kim
Division of Biological Sciences
306-791 Daejeon KOR
Email: kym@hannam.ac.kr
Mrs. Mélanie KIRCHMEYER
Physiopathologie et
Pharmacologie Articulaires
54500 Vandoeuvre lËs Nancy
FRA
Email: melkirch2002@yahoo.fr
Mrs. Rasmus Boye Kjellerup
Department of Dermatology
8000 Aarhus DNK
Email: rbk@studmed.au.dk
Mrs. Anna Klemm
Biophysic Group
91052 Erlangen DEU
Email: aklemm@biomed.unierlangen.de
Mr. Holger Knobelspies
Institute of Pharmacology and
Toxicology
52074 Aachen DEU
Email: hknobelspies@ukaachen.de
Dr. George Koliakos
Biological Chemistry
54124 Thessaloniki GRC
Email: koliakos@med.auth.gr
Pr. Liliana Konarska
Dept. of Biochemistry and Clinical
Chemistry
02-091 Warsaw POL
Email: konarska@nencki.gov.pl
Dr. Young Min Kim
Dept of Food and Nutrition
306-791 Daejeon KOR
Email: ojpark@hannam.ac.kr
Mr. Milen Ivanov Kirilov
Experimental Chemotherapy
1000 Sofia BGR
Email: m.kirilov@dkfz.de
Dr. Jean-Paul Klein
Inserm UMR-S 392/E.A. 3950
ULP
67000 Strasbourg FRA
Email: jeanpaul.klein@medecine.u-strasbg.fr
Dr. Stefan Knapp
Structural Genomics Consortium
OX3 7LD Oxford GBR
Email: stefan.knapp@sgc.ox.ac.uk
Dr. Mariusz Koda
Department of Pathology
15-269 Bialystok POL
Email: sulek@zeus.amb.edu.pl
Dr. Natalia Koltovaya
Laboratory of Radiation Biology
141980 Dubna RUS
Email: koltovaya@jinr.ru
Dr. Paul Kong Thoo Lin
School of Life Sciences
AB25 1HG Aberdeen GBR
Email: p.kong@rgu.ac.uk
Mr. Matthew King
Department of Biochemistry
CB2 1QW Cambridge GBR
Email:
mak45@mole.bio.cam.ac.uk
Dr. Paul Kirkham
RH12 5AB Horsham GBR
Email:
paul.kirkham@novartis.com
Mrs. Lilach Kleinberg
Department of Pathology
N-0310 Oslo NOR
Email: lilachkl@odont.uio.no
Mrs. Jelena Knezevic
Laboratory for molecular oncology
Institute
10000 Zagreb HRV
Email: jknezev@irb.hr
Dr. Kersti Kokk
50411 Tartu EST
Email: kersti.kokk@ut.ee
Dr. Radko Komers
140 21 Prague CZE
Email: rako@medicon.cz
Pr. Spiro Konstantinov
Experimental Chemotherapy
1000 Sofia BGR
Email: Pgen@TU-Sofia.bg

Mr. Daniel Korr
Functional Genomics
13342 Berlin DEU
Email: daniel.korr@schering.de
Pr. Peter H Krammer
Tumorimmunology Program
D-69120 Heidelberg DEU
Email: p.krammer@dkfz.de
Dr. Anna Krichevsky
2115 Boston USA
Email:
akrichevsky@rics.bwh.harvard.ed
u
Mr. Aleksandar Krstic
Laboratory for human molecular
genetics
11010 Belgrade YUG
Email: hmgbox@eunet.yu
Mrs. Joanna Kuldo
9713 GZ Groningen NLD
Email: j.m.kuldo@med.umcg.nl
Mrs. Young E. Kwak
department of oral pathlogy
120-752 Seoul KOR
Email: op1@yumc.yonsei.ac.kr
Dr. joerg Labahn
IBI-2
52435 Juelich DEU
Email: j.labahn@fz-juelich.de
Mr. Larissa Kotelevets
inserm u683
18 Paris FRA
Email: koteleve@bichat.inserm.fr
Dr. Bertolt Kreft
Functional Genomics
13342 Berlin DEU
Email: bertolt.kreft@schering.de
Mr. Fabian Kriebel
Sektion Experimentelle
Anaesthesiologie
89075 Ulm DEU
Email: fabiankriebel@web.de
Dr. Jan K Kubicek
IBI-2
52425 Juelich DEU
Email: j.kubicek@fz-juelich.de
Mr. Joydeb Kumar Kundu
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: kundujk@yahoo.com
Mr. Jung-Yeon Kwon
Department of food biotechnology
151-742 Seoul KOR
Email: nararav@hanmail.net
Mrs. Marianne LABUSSIERE
Histopathologie Expérimentale et
Moléculaire
54505 Vandoeuvre les Nancy FRA
Email:
marianne.labussiere@voila.fr
Dr. Jarmila Kralova
Molecular Virology
166 37 Prague CZE
Email: kralova@img.cas.cz
Dr. Stephanie Kreis
LBPI
L-1511 Luxembourg LUX
Email: stephanie.kreis@uni.lu
Mr. Guido Kroemer
CNRS-UMR8125
94805 Villejuif FRA
Email: kroemer@igr.fr
Mrs. Satoko Kubo
Div. Physiol. Pathophysiol., Sch.
Pharm. Sci.
577-8502 Higashi-Osaka JPN
Email: 0544410001t@kindai.ac.jp
Dr. Mikhail Kutuzov
Department of Pharmacology
60607 Chicago, IL USA
Email: kutuzov@uic.edu
Pr. Antonios Kyriakopoulos
Molecular Trace Elements
Research in the Life Science
14109 Berlin DEU
Email: Kyriakopoulos@hmi.de
Mrs. Piret Laht
molecular biology
12618 Tallinn EST
Email: piret.laht@ttu.ee

Mr. Christoph Lahtz
AG Tumorgenetik
6097 Halle (Saale) DEU
Email: Christoph_Lahtz@gmx.de
Mrs. Elise Langenkamp
Medical Biology
9713 GZ Groningen NLD
Email:
elise.langenkamp@gmail.com
Mrs. Isabelle Lascombe
Laboratoire de Biologie Cellulaire
et Moléculaire
25000 BESANCON FRA
Email: isabelle.lascombe@voila.fr
Mrs. Xuefen Le Bourhis
INSERM ERI-8
59655 Villeneuve d'Ascq FRA
Email: xuefen.lebourhis@univlille1.fr
Dr. Eibai Lee
Department of Pharmacology
651-2180 Kobe JPN
Email:
elee@pharm.kobegakuin.ac.jp
Mrs. Eun Young Lee
Dept. of Anatomy, Embryology
Lab.
120-752 Seoul KOR
Email: nannaya1210@paran.com
Mr. Jeong-Sang Lee
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: jslee11@yahoo.com
Mrs. Jennifer Laing
Virology and Immunology
21201 Baltimore USA
Email: jlain001@umaryland.edu
Dr. Sylvie LANTUEJOUL
Lung Research Group
38706 LA TRONCHE FRA
Email: SLantuejoul@chugrenoble.fr
Dr. Yves Laumonnier
Department of Pharmacology of
Natural Products & Clinical
Pharmacology
89081 Ulm DEU
Email: yves.laumonnier@uniulm.de
Mr. Chantal LEBRUN
IPBS
31077 TOULOUSE FRA
Email: chantal.lebrun@ipbs.fr
Mrs. EUN LEE
ANGIOGENESIS AND
CHEMOPREVENTION
449701 YONGIN KOR
Email: sungkim7@khu.ac.kr
Pr. EunJu Lee
department of oral pathlogy
120-752 Seoul KOR
Email: e16lee@yumc.yonsei.ac.kr
Mr. Jong Hwa Lee
Department of Pharmacology
120-752 Seoul KOR
Email: pico798@naver.com
Dr. Elisabetta Lambertini
Dept of Biochemistry and
Molecular Biology
44100 Ferrara ITA
Email: elambertini@yahoo.it
Mrs. Maria Lapshina
Molecular biology laboratory
142432 Chernogolovka RUS
Email: lapshina@icp.ac.ru
Dr. Antigone Lazou
Animal Physiology
54124 Thessaloniki GRC
Email: lazou@bio.auth.gr
Mrs. Marielle Lebrun
Virologie et Immunologie
4000 Liege BEL
Email: mlebrun@student.ulg.ac.be
Pr. Eun Ju Lee
Oral Cancer Research Institute,
Department of Oral pathology
120752 Seoul KOR
Email: e16lee@yumc.yonsei.ac.kr
Mr. Jangwon Lee
Department of Pharmacology
120-752 Seoul KOR
Email: honorjw@hotmail.com
Dr. Ki-won Lee
Department of food biotechnology
151-742 Seoul KOR
Email: opening2@snu.ac.kr

Mrs. Mi-Hyun Lee
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: mhyun_lee@yahoo.co.kr
Mrs. Eija Lehtomaki
Molecular Signalling Lab
FIN 70211 KUOPIO FIN
Email: elehtoma@messi.uku.fi
Dr. Nathalie Leleu
Laboratoire de génétique et
biologie cellulaire
78035 Versailles FRA
Email:
nathalie.leleu@genetique.uvsq.fr
Mrs. Min Li
1748 Hopkinton USA
Email: minl@biosource.com
Dr. Bertrand Liagre
Biochimie
87025 Limoges FRA
Email: bertrand.liagre@unilim.fr
Mrs. Yi-Chu Lin
10018 Taipei TWN
Email: f92443016@ntu.edu.tw
Dr. Metoda Lipnik-ätangelj
Department of Pharmacology and
Experimental Toxicology
SI-1000 Ljubljana SVN
Email: metoda.lipnikstangelj@mf.uni-lj.si
Mrs. Sun Kyoung Lee
Department of Oral Biology
120-752 Seoul KOR
Email: l-pluto@hanmail.net
Mr. Peter Leister
Institute of Genetics
53117 Bonn DEU
Email: peterleister@uni-bonn.de
Dr. Dina Lepik
Institute of Molecular and Cell
Biology
51010 Tartu EST
Email: dlepik@ebc.ee
Dr. Xiao Li
Receptor and Signal Transduction
48202 Detroit USA
Email: xcli1@hfhs.org
Dr. Sandra Liekens
virology and experimental
chemotherapy
3000 Leuven BEL
Email:
sandra.liekens@rega.kuleuven.be
Mrs. Asa Lindgren
medical microbiology and
immunology
41390 Goteborg SWE
Email:
asa.lindgren@microbio.gu.se
Dr. David Litchfield
Department of Biochemistry
N6A 5C1 London CAN
Email: litchfi@uwo.ca
Mr. Taek-Sang Lee
Laboratory of Gynecological
Oncology
110-799 Seoul KOR
Email: rbssh9@snu.ac.kr
Dr. Marilena E Lekka
Biochemistry
451 10 Ioannina GRC
Email: mlekka@cc.uoi.gr
Mrs. Mei-Hua Li
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: maylee3737@yahoo.com
Mrs. Xiaobiao Li
Prof. Denis Monard
4058 Basel CHE
Email: xiaobiao@fmi.ch
Dr. Temduang Limpaiboon
40002 Khon Kaen THA
Email: temduang@kku.ac.th
Dr. Maria Maddalena Lino
Prof. Monard
4058 Basel CHE
Email: linom@fmi.ch
Mr. Yan-Zhi Liu
Animal Science
912-01 Ping Tung County TWN
Email: f58725128@yahoo.com.tw

Pr. Etta Livneh
84105 Beer Sheva ISR
Email: etta@bgu.ac.il
Dr. Maria J. Lorenzo
Departamento de Bioquimica y
Biologia Molecular y Genetica
10071 Caceres ESP
Email: mjlorenzo@unex.es
Dr. Karen Lyle
Johannes L. Bos
3584 CG Utrecht NLD
Email: k.s.lyle@med.uu.nl
Dr. Shyamala Maheswaran
Surgical Oncology
2114 Boston USA
Email:
maheswaran@helix.mgh.harvard.e
du
Dr. Flavio Maina
CNRS
13288 Marseille FRA
Email: maina@ibdm.univ-mrs.fr
Pr. Livio Mallucci
Pharmaceutical Sciences Research
Division
SE1 9NH London GBR
Email: livio.mallucci@kcl.ac.uk
Mrs. Sophie MARCHAL
Equipe Projet "Photobiologie en
Cancérologie"
54511 Vandoeuvre les Nancy FRA
Email: s.marchal@nancy.fnclcc.fr
Mrs. Elisabeth LONGAVENNE
Laboratoire de Recherche
54511 Vandoeuvre les Nancy FRA
Email: s.bouali@nancy.fnclcc.fr
Dr. Sophie Lorquet
Tumour and Development Biology
Laboratory, CRCE
4000 Liége, Sart-Tilman BEL
Email:
S.Lorquet@student.ulg.ac.be
Mrs. Delphine Magard
LBMCC
L-2540 Luxembourg LUX
Email:
delphine.magard@laposte.net
Dr. Csaba Mahotka
40225 Duesseldorf DEU
Email: mahotka@med.uniduesseldorf.de
Mrs. Adva Maissel
prof Etta Livneh
84105 Beer sheva ISR
Email: advak@bgumail.bgu.ac.il
Mr. Steve Manderscheid
Institute of Genetics
53117 Bonn DEU
Email: stevemander@web.de
Dr. Stefania Mardente
Dep Experimental Medicine
162 Rome ITA
Email:
stefania.mardente@uniroma1.it
Mr. Francisco Lopez Hernandez
Toxicologia
37007 Salamanca ESP
Email: flopezher@usal.es
Mr. Thomas Luft
Molecular Oncology and
Hematology
69120 Heidelberg DEU
Email: t.luft@dkfz.de
Dr. Maria Magocsi
Signal Transduction
H-1113 Budapest HUN
Email:
m.magocsi@biomembrane.hu
Mr. Allan Maillis
Laboratoire de recherche
CardioVasculaire
L-1150 Luxembourg LUX
Email: allan.maillis@crpsante.healthnet.lu
Mrs. Anu Maki-Hokkonen
Molecular Signalling Lab
FIN 70211 KUOPIO FIN
Email: makihokk@hytti.uku.fi
Mrs. Barbara Mänz
Pharmakologie und Toxikologie
52075 Aachen DEU
Email: Barbara.Maenz@gmx.de
Dr. Christiane Margue
LBPI
1511 Luxembourg LUX
Email: christiane.margue@uni.lu

Mrs. Nathalie Mari
Cellular and Molecular Biology
Unit
5030 Gembloux BEL
Email: mari.n@fsagx.ac.be
Pr. Alberto Maria Martelli
Cell Signalling
40126 Bologna ITA
Email: alberto.martelli@unibo.it
Dr. Carlos Martinez-Salgado
Unidad de Investigacion
37007 Salamanca ESP
Email: carlosms@usal.es
Dr. Antonio Mastino
Dept. Microbiol., Gen., Mol. Sci.
98166 Messina ITA
Email: antonio.mastino@unime.it
Dr. Ray Mattingly
Department of Pharmacology
MI 48201 Detroit USA
Email: r.mattingly@wayne.edu
Mr. Laurent MEIJER
Station Biologique de Roscoff
29680 ROSCOFF FRA
Email: meijer@sb-roscoff.fr
Dr. Paul Mercer
Centre for Respiratory Research
WC1E 6JJ London GBR
Email: paul.mercer@ucl.ac.uk
Mr. Jean-Claude Marie
75018 Paris FRA
Email: ugh@bichat.inserm.fr
Mrs. Maud Martin
Cellular and Molecular Biology
Unit
5030 Gembloux BEL
Email: martin.m@fsagx.ac.be
Dr. John Marwick
RH12 5AB Horsham GBR
Email:
john.marwick@novartis.com
Mrs. Julie Mathieu
INSERM U685
75010 Paris FRA
Email:
Julie.Mathieu@stlouis.inserm.fr
Dr. Mariola Matysiak
Neurology
90-153 Lodz POL
Email: ajur@afazja.am.lodz.pl
Dr. Fanyin Meng
76504 Temple USA
Email: fmeng@swmail.sw.org
Pr. Ofer Merimsky
Dept. of Cell and Developmental
Biology
69978 Tel-Aviv ISR
Email: histol3@post.tau.ac.il
Mrs. Giulia Marsili
Department of Infectious, Parasitic
and Immunomediated Diseases
161 roma ITA
Email: gmarsili@iss.it
Mrs. Natalia Martinez
Institute of Immunology
L-1950 Luxembourg LUX
Email:
natalia.martinez@LNS.ETAT.LU
Dr. Alessandro Massa
pharmacology-department of
oncology biology and genetics
16132 Genova ITA
Email: alemassa74@hotmail.com
Dr. Janos Matko
Immunology
1117 Budapest HUN
Email: matko@cerberus.elte.hu
Dr. Ilya Mazo
20850 Rockville USA
Email:
mazo@ariadnegenomics.com
Mrs. Christina Mensger
69115 Heidelberg DEU
Email: c.mensger@dkfz.de
Dr. Marie-Paule MERVILLE
Medical Chemistry
4000 Liége BEL
Email: mpmerville@ulg.ac.be

Mrs. Rasa Merzvinskyte
Department of Developmental
Biology
LT-08662 Vilnius LTU
Email: sypssena@one.lt
Mr. Olivier Meurette
INSERM U620 UPRES EA 38-89
35043 Rennes FRA
Email: olivier.meurette@univrennes1.fr
Pr. Jelena Milasin
Human Genetics
11000 Belgrade YUG
Email: jelena_milasin@yahoo.com
Dr. Martin Modriansky
Institute of Medical Chemistry and
Biochemistry
77515 Olomouc CZE
Email: oregon23@email.cz
Dr. Fabrizio Montecucco
Clinic of internal medicine I
16132 Genoa ITA
Email:
fabriziomontecucco@tiscali.it
Dr. Franck Morceau
LBMCC
L-2540 Luxembourg LUX
Email: franck.morceau@lbmcc.lu
Dr. Sophie Mouillet-Richard
Laboratoire de différenciation
cellulaire et prions
94801 VILLEJUIF FRA
Email: mouillet@vjf.cnrs.fr
Dr. Pedro Mestres
Anatomy and Cell Biology
D-66421 Homburg/Saar DEU
Email: anpmes@uniklinikumsaarland.de
Dr. Uwe Michelsen
LSA R&D
64293 Darmstadt DEU
Email: uwe.michelsen@merck.de
Dr. Gyesik Min
Department of Microbiological
Engineering
660-758 Jinju KOR
Email: g-min@jinju.ac.kr
Mrs. Michele Moes
Laboratoire de Biologie et de
Physiologe intégrée
L-1511 Luxembourg LUX
Email: michele.moes@uni.lu
Mrs. Michaela Moors
Toxicology
40225 Düsseldorf DEU
Email: moors@uni-duesseldorf.de
Mrs. Laura Moreno
Dept. Pharmacology
28040 Madrid ESP
Email: acogolludo@ift.csic.es
Dr. Ange Mouithys-Mickalad
Centre de l'Oxygéne, Recherche et
Développement
4000 Liége BEL
Email: amouithys@ulg.ac.be
Dr. Emmanuelle Meuillet
85721 Tucson USA
Email:
emeuillet@azcc.arizona.edu
Mrs. Frederique Mies
physiopathologie
1070 Bruxelles BEL
Email: fmies@ulb.ac.be
Dr. Marie-Christine Miquel
UMR 7102 Neurobiologie des
Processus Adaptatifs
75005 Paris FRA
Email: mariechristine.miquel@snv.jussieu.fr
Mrs. Antonella Montalbano
Dipartimento Medicina
Sperimentale-Sezione Anatomia
Umana
90127 Palermo ITA
Email: a.montalbano@unipa.it
Dr. Ana Isabel Morales Martin
Toxicologia
37007 Salamanca ESP
Email: amorales@usal.es
Mrs. Andrea Morguet
Anatomy and Cell Biology
D-66421 Homburg/Saar DEU
Email: anpmes@uniklinikumsaarland.de
Mr. Anice MOUMEN
CNRS
13288 Marseille FRA
Email: moumen@ibdm.univmrs.fr

Dr. Marc Mousli
Inserm UMR-S 392/E.A. 3950
ULP
67000 Strasbourg FRA
Email: marc.mousli@medecine.ustrasbg.fr
Dr. Thomas Mueller
51149 Cologne DEU
Email:
thomas.mueller@pmintl.com
Dr. Gerhard Müller-Newen
Institut für Biochemie
52074 Aachen DEU
Email: mueller-newen@rwthaachen.de
Dr. Anna Maria Musti
Dipartimento Farmaco-Biologico
87036 Rende ITA
Email: ammusti@yahoo.it
Dr. Elena Navolotskaya
Laboratory of Peptide
Bioregulators
142290 Pushchino, Moscow
Region RUS
Email:
navolots@fibkh.serpukhov.su
Dr. Frank Neumann
WR 13 6JW Upper Pendock GBR
Email: fneumann@bioaxxess.com
Mrs. Christina Nielsen
Apoptosis Department
2100 Copenhagen DNK
Email: cn@cancer.dk
Dr. Marc Mousli
Inserm UMR-S 392/E.A. 3950
ULP
67000 Strasbourg FRA
Email: marc.mousli@medecine.ustrasbg.fr
Dr. Claude P. Muller
Institute of Immunology
L-1950 Luxembourg LUX
Email:
claude.muller@LNS.ETAT.LU
Dr. Carine Munaut
Laboratoire de Biologie des
Tumeurs et du Développement
4000 Sart Tilman (Liége) BEL
Email: c.munaut@ulg.ac.be
Dr. ILIAS MYLONIS
LABORATORY OF
BIOCHEMISTRY, SCHOOL OF
MEDICINE
41222 LARISSA GRC
Email: mylchem3@in.gr
Mrs. Bartolomé Nerea
Biochemistry
48940 Leioa ESP
Email: ofbbaesn@lg.ehu.es
Dr. Dao Nguyen
Section of Thoracic Oncology,
Surgery Branch
20892 Bethesda. Maryland USA
Email: dao_nguyen@nih.gov
Mrs. Lotte B Nielsen
2600 Glostrup DNK
Email: lbn@dcb-glostrup.dk
Mrs. Jihanne MRIOUAH
Laboratoire de Recherche
54511 Vandoeuvre les Nancy FRA
Email: d.leprince@nancy.fnclcc.fr
Dr. Susanne Muller-Knapp
Structural Genomics Consortium
OX3 7LD Oxford GBR
Email: susanne.mullerknapp@sgc.ox.ac.uk
Dr. Georg Munz
Institut für Biochemie
52074 Aachen DEU
Email: georg_munz@yahoo.com
Mr. GEORGE NAKOS
10563 ATHENS GRC
Email:
k.despotopoulou@heronia.com
Pr. Andreas Nerlich
Inst. of Pathology
D-81925 Munich DEU
Email: andreas.nerlich@extern.lrzmuenchen.de
Mrs. Magali Nicolier
Laboratoire de Biologie Cellulaire
et Moléculaire
25000 BESANCON FRA
Email: magali_nicolier@yahoo.fr
Dr. Shekoufeh Nikfar
Drug Evaluation Commitee
14155-6451 Tehran IRN
Email: s_nikfar@yahoo.com

Mr. Gustav Nilsonne
Department of Laboratory
Medicine
SE-14186 Stockholm SWE
Email: gustav.nilsonne@ki.se
Dr. Jill Norman
Renal Cell Biology
NW3 2PF London GBR
Email:
j.norman@medsch.ucl.ac.uk
Dr. Silvia Nuccitelli
133 Rome ITA
Email: silvia.nuccitelli@libero.it
Mrs. Ellen O'Dea
Signaling Systems Laboratory
92093 La Jolla, California USA
Email: eodea@ucsd.edu
Mrs. Sandra Olsson
Macrophage Signaling
22184 Lund SWE
Email:
sandra.olsson@medkem.lu.se
Pr. Moshe Oren
Molecular Cell Biology
76100 Rehovot ISR
Email:
moshe.oren@weizmann.ac.il
Dr. Joost Oudejans
Pathology
1117HV Amsterdam NLD
Email: jj.oudejans@vumc.nl
Dr. Mario Nizzari
Pharmacology-Department of
oncology biologi and genetics
16132 Genova ITA
Email: mario.nizzari@unige.it
Mr. Jiri Novotny
Laboratory of Biochemistry of
Membrane Receptors
142 20 Prague CZE
Email: novjiri@biomed.cas.cz
Mrs. Susanna Nurmi
Molecular studies on leukocyte
adhesion
14 (Helsinki) University of
Helsinki FIN
Email: Susanna.Nurmi@helsinki.fi
Mrs. Andrea Oeckinghaus
AG Scheidereit
13125 Berlin DEU
Email: an.oecki@gmx.de
Mrs. Tove Olsson
Department of Odontology
SE-141 04 Huddinge SWE
Email: tove.olsson@ki.se
Dr. MarÌa Clara Ortiz Ruiz
30100 Murcia ESP
Email: clara@um.es
Mrs. Ferda Ozkinay
Ege University Medical Faculty
Medical Genetics
35100 Izmir TUR
Email: ferdafo@yahoo.com
Dr. Sylvia Nome Kvam
7006 Trondheim NOR
Email:
sylvia.nome.kvam@stolav.no
Mr. TOBIAS NUBEL
CNRS UPR 9078
75730 PARIS FRA
Email: NUEBEL@NECKER.FR
Mrs. Mieke Nuytten
Department of Biochemistry
3000 Leuven BEL
Email:
mieke.nuytten@med.kuleuven.be
Mrs. Jung min Oh
signal transduction
110-799 Seoul KOR
Email: snow99@snu.ac.kr
Mr. Huseyin Onay
Ege University Medical Faculty
Medical Genetics
35100 Izmir TUR
Email: ferdafo@yahoo.com
Mr. Pavel Ostasov
Laboratory of Biochemistry of
Membrane Receptors
142 20 Prague CZE
Email: ostasov@biomed.cas.cz
Dr. Nesrin Ozsoy
6100 Tandogan / ANKARA TUR
Email:
ozsoy@science.ankara.edu.tr

Dr. Elisabeth Paczoska-
Eliasiewicz
The Hebrew University of
Jerusalem
76100 Rehovot ISR
Email: kozlicka103@yahoo.pl
Dr. Valérie Palissot
laboratoire d'hémato-cancérologie
expérimentale
L-1526 Luxembourg LUX
Email: lhce@crp-sante.lu
Mrs. In-Ja Park
Dept of Food and Nutrition
306-791 Daejeon KOR
Email: ojpark@hannam.ac.kr
Pr. Ock Jin Park
Dept of Food and Nutrition
306-791 Daejeon KOR
Email: ojpark@hannam.ac.kr
Mrs. Ioana Parvu-Ferecatu
Laboratoire de génétique et
biologie cellulaire
78035 Versailles FRA
Email: Ioana-
Costina.Parvu@genetique.uvsq.fr
Pr. Jasminka Pavelic
Rudjer Boökovic
10000 Zagreb HRV
Email: jpavelic@irb.hr
Dr. Zofia Pawlowska
Department of Mol. & Med.
Biophysics
92-215 Lodz POL
Email: pawlow@zdn.am.lodz.pl
Mr. Artur Padzik
Neuronal Signalling Laboratory
20521 Turku FIN
Email: apadzik@btk.fi
Mrs. Cornelieke Pals
Dept. of Immunology
3584 EA Utrecht NLD
Email: c.e.g.m.pals@azu.nl
Pr. Kwang-Kyun Park
120-749 Seoul KOR
Email: kkp304@hanmail.net
Mrs. Sin-Aye Park
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: qorwh7@empal.com
Dr. Tushar Patel
TX 76508 Temple USA
Email: tpatel@swmail.sw.org
Mrs. Catherine PAVOINE
INSERM U.581
94010 CRETEIL FRA
Email: pavoine@im3.inserm.fr
Mrs. Leticia Yamila Peche
National Laboratory
34012 Trieste ITA
Email: cib@lncib.it
Mrs. Beata Pajak
02-776 Warsaw POL
Email: bepaj@wp.pl
Dr. Efrosyni Paraskeva
Lab. of Biochemistry, School of
Medicine
41222 Larissa GRC
Email: fparaskeva@med.uth.gr
Pr. Ock Jin Park
Division of Biological Sciences
306-791 Daejeon KOR
Email: ojpark@hannam.ac.kr
Mrs. Lotta Parviainen
Molecular Signalling Lab
FIN 70211 KUOPIO FIN
Email: leparvia@hytti.uku.fi
Dr. Réjane Paumelle
INSERM/U545
59019 Lille FRA
Email: rejane.paumelle@pasteurlille.fr
Mrs. Patrycja Pawlikowska
02-776 Warsaw POL
Email: pmuras@wp.pl
Mrs. Anastasia Pechtelidou
Animal and Human Physiology
15784 Athens GRC
Email: apechtel@biol.uoa.gr

Mrs. Christel Péqueux
Tumour and Development Biology
Laboratory, CRCE
4000 Liége, Sart-Tilman BEL
Email: C.Pequeux@ulg.ac.be
Dr. Richard Pestell
Lombardi Comprehensive Cancer
Center
20057-1468 Washington, D.C.
USA
Email: randp@georgetown.edu,
pestell@georgetown.edu
Mrs. Marlene Pickl
Cell Biology
82372 Penzberg DEU
Email: marlene.pickl@roche.com
Mrs. Kaie Pill
molecular biology
12618 Tallinn EST
Email: kaie.pill@ttu.ee
Dr. Sébastien PLANCON
LBPI
L-1511 Luxembourg LUX
Email: sebastien.plancon@uni.lu
Mrs. Branka Popovic
Human Genetics
11000 Belgrade YUG
Email: brankapo@verat.net
Dr. Jacques Pouyssegur
Institute of Signaling,
Developmental Biology & Cancer
Research
6189 Nice FRA
Email: pouysseg@unice.fr
Dr. Dolores Pérez-Sala
28040 Madrid ESP
Email: dperezsala@cib.csic.es
Mrs. Isidora Petrovic
Laboratory for human molecular
genetics
11010 Belgrade YUG
Email: hmgbox@eunet.yu
Pr. Jacques Piette
Virology & Immunology
B-4000 Liége BEL
Email: jpiette@ulg.ac.be
Mrs. Sophie PINEL
Histopathologie Expérimentale et
Moléculaire
54505 Vandoeuvre les Nancy FRA
Email: pinel.sophie@tiscali.fr
Dr. Krzysztof Pluta
Molecular Signalling Lab
FIN 70211 KUOPIO FIN
Email: Krzysztof.Pluta@uku.fi
Mr. Ignacio Portero-Robles
Zim II Hämatologie/Oncologie
60590 Frankfurt am Main DEU
Email: robles@em.uni-frankfurt.de
Dr. Radek Prochazka
Laboratory of Developmental
Biology
277 21 Libechov CZE
Email: prochazka@iapg.cas.cz
Dr. Francisco Pérez-Vizcaino
Dept. Pharmacology
28040 Madrid ESP
Email: acogolludo@ift.csic.es
Mrs. ERIKA PEVERELLI
ISTITUTO DI SCIENZE
ENDOCRINE
20122 MILANO ITA
Email: erika.peverelli@unimi.it
Dr. Albrecht Piiper
Department of Internal Medicine II
D-66421 Homburg/Saar DEU
Email: inapii@uniklinikumsaarland.de
Dr. Michael PINKOSKI
Email: mp191@leicester.ac.uk
Pr. Alfonso Pompella
Dept. of Experimental PAthology
56126 Pisa ITA
Email: apompella@biomed.unipi.it
Mr. Benoit POURCET
UR 545
59000 LILLE FRA
Email: benoit.pourcet@pasteurlille.fr
Dr. Florian Puehler
Experimental Oncology
13342 Berlin DEU
Email:
Florian.Puehler@Schering.de

Pr. Alexander Pukhalsky
Laboratory of Immunogenetics
115478 Moscow RUS
Email: osugariver@medgen.ru
Mr. Thibaut QUILLARD
UMR 643
44093 Nantes FRA
Email: thibaut.quillard@univnantes.fr
Pr. Parvaneh Rafiee
53226 Milwaukee USA
Email: prafiee@mcw.edu
Dr. Annat Raiter
Felsenstein Medical Research
Center
49100 Petah Tikva ISR
Email: araiter@post.tau.ac.il
Dr. Madhavi Rane
Dept of Medicine
40202 Louisville, KY USA
Email: mrane@louisville.edu
Mrs. Jane Rasaiyaah
Dept Immunology and Molecular
Pathology
W1Y 4JF London GBR
Email: rekajra@ucl.ac.uk
Mr. Laurent REBER
EA3771
67401 ILLKIRCH FRA
Email: lreber@pharma.u-strasbg.fr
Mrs. Emma Quigley
EC2A 4LQ London GBR
Email: p.garner@ashley-pub.com
Mrs. Claire Quiney
75006 Paris FRA
Email:
claire.quiney@bhdc.jussieu.fr
Dr. Irfan Rahman
14620 Rochester, NY USA
Email:
Irfan_Rahman@urmc.rochester.ed
u
Mrs. Carole RAMACCI
Laboratoire de Recherche
54511 Vandoeuvre les Nancy FRA
Email: c.ramacci@nancy.fnclcc.fr
Mrs. Carmen Ranftler
Dept. of Medicine 1,Div.: Inst. of
Cancer Research
A-1090 VIENNA AUT
Email: cfr1978@yahoo.de
Dr. SID D. RAY
MOLECULAR TOXICOLOGY,
PHARM. SCI. DIV.
11201 BROOKLYN, NY USA
Email: sray@liu.edu
Mr. Amélie REBILLARD
INSERM U620
35043 Rennes FRA
Email: amelie.rebillard@univrennes1.fr
Dr. Frédéric Quignon
LIMBP
F-57070 Metz FRA
Email: frederic.quignon@univmetz.fr
Mrs. Flavia Radogna
133 Rome ITA
Email: flavia.radogna@libero.it
Dr. Souad Rahmouni
Pathology
4000 Liege BEL
Email: srahmouni@ulg.ac.be
Dr. Carlo Ramoni
Dept. Cell Biology and
Neurosciences
162 Rome ITA
Email: ramoni@iss.it
Pr. Ulf R. Rapp
Institut für Medizinische
Strahlenkunde und Zellforschung
97078 Würzburg DEU
Email: rappur@mail.uniwuerzburg.de
Dr. Massimo Realacci
Molecular biology
161 Rome ITA
Email:
massimo.realacci@uniroma1.it
Dr. Kris Reedquist
Division of Clinical Immunology
and Rheumatology
1100 DE Amsterdam NLD
Email: k.a.reedquist@amc.uva.nl

Dr. Gabriella Regis
Tumor Immunology Laboratory
10126 Turin ITA
Email: gabriella.regis@unito.it
Mrs. Patsy Renard
URBC
5000 Namur BEL
Email: patsy.renard@fundp.ac.be
Mr. Simone Reuter
LBMCC
L-2540 Luxembourg LUX
Email: simone.reuter@lbmcc.lu
Mrs. Elena Rodionova
Molecular Oncology and
Hematology
69120 Heidelberg DEU
Email: e.rodionova@dkfz.de
Mrs. Lorena Rodriguez
Biochemistry
48940 Leioa ESP
Email: ofbronol@lg.ehu.es
Dr. Maria Rodriguez-Munoz
Neuropharmacology
28002 Madrid ESP
Email: mrodriguez@cajal.csic.es
Mr. David Romano
Interactions Cellulaires
Neuroendocriniennes (ICNE)
13916 cedex 20 Marseille FRA
Email: romano.d@nord.univmrs.fr
Dr. Daniel U. Reimer
6020 Innsbruck AUT
Email: daniel.reimer@uibk.ac.at
Mrs. Flore Renaud
Laboratoire de Génétique et
Biologie cellulaire
78035 Versailles FRA
Email: frenaud@genetique.uvsq.fr
Pr. Michel Rigoulet
IBGC
33077 BORDEAUX FRA
Email: michel.rigoulet@ibgc.ubordeaux2.fr
Dr. Sophie Rodius
LBPI
L-1511 Luxembourg LUX
Email: sophie.rodius@uni.lu
Dr. Alicia Rodriguez-Barbero
Deparrtamento de Fisiologia y
Farmacologia
37007 Salamanca ESP
Email: barberoa@usal.es
Mrs. Celine Rofel
Molecular Genetics
6229 ER Maastricht NLD
Email: c.rofel@gen.unimaas.nl
Mr. Salvatore Romeo
Pathology
2333 ZA Leiden NLD
Email: S.Romeo@lumc.nl
Dr. Kerstin Reimers
30659 Hannover DEU
Email: Reimers.Kerstin@MH-
Hannover.de
Pr. Saverio Francesco Retta
Department of Genetics, Biology
and Biochemistry
10126 Torino ITA
Email: francesco.retta@unito.it
Dr. Aleksandra Ristic-Fira
Laboratory for Molecular Biology
and Endocrinology
11001 Belgrade YUG
Email: aristic@vin.bg.ac.yu
Mr. Leonardo Rodrigues
Laboratory of Cellular and
Molecular Biology
05508-900 Sao Paulo BRA
Email: leonardo@cecm.usp.br
Dr. Isabel Rodriguez-Escudero
Dpto. Microbiologia II
28040 Madrid ESP
Email: isabelre@farm.ucm.es
Mrs. Catherine Rolvering
LBPI
L-1511 Luxembourg LUX
Email: catherine.rolvering@uni.lu
Mr. Christian Rommel
1228 Plan-les-Ouates, Geneva
CHE
Email:
christian.rommel@serono.com

Mr. Guillaume Rommelaere
URBC
5000 Namur BEL
Email:
guillaume.rommelaere@student.fu
ndp.ac.be
Mr. Vladimir Rudajev
Laboratory of Biochemistry of
Membrane Receptors
142 20 Prague CZE
Email: rudajev@biomed.cas.cz
Mrs. Iwona Sadzak
Department of Microbiology and
Immunbiology
A-1030 Vienna AUT
Email: iwona.sadzak@univie.ac.at
Mr. Carlos Salazar
Theoretical Biophysics / Institute
of Biology
10115 Berlin DEU
Email: carlos.salazar@rz.huberlin.de
Dr. Pilar Sanchez-Blazquez
Neuropharmacology
28002 Madrid ESP
Email: psb@cajal.csic.es
Dr. Nijole Savickiene
Department of Pharmaceutical
Chemistry and Pharmacognosy
3000 Kaunas LTU
Email: savickienenijole@takas.lt
Dr. Elisabeth Schaffner-Reckinger
Laboratoire de Biologie et
Physiologie Intégrée
L-1511 Luxembourg LUX
Email: elisabeth.schaffner@uni.lu
Mrs. Caroline Rouaux
INSERM U-692
67000 Strasbourg FRA
Email: carolinerouaux@yahoo.fr
Dr. M. Begona Ruiz-Larrea
Dep. Physiology, Medicine School
48080 Leioa ESP
Email: mbego.ruizlarrea@ehu.es
Dr. Xavier SAELENS
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email:
Xavier.Saelens@dmbr.UGent.be
Mr. ALEX SALSMANN
LBPI
1134 LUXEMBOURG LUX
Email: salsmann@cu.lu
Dr. Sujen Eleonora Santini
Dipartimento di Produzioni
Animali-Sezione di Fisiologia
43100 Parma ITA
Email: basini@unipr.it
Mrs. Claudia Schäfer
Toxicology
40225 Düsseldorf DEU
Email: claudia.schaefer@uniduesseldorf.de
Mrs. Barbara Schaljo
Department of Microbiology and
Immunbiology
A-1030 Vienna AUT
Email:
barbara.schaljo@univie.ac.at
Dr. Molay Roy
305-8642 Tsukuba JPN
Email: mkroy@affrc.go.jp
Dr. José Ignacio Ruiz-Sanz
Dep. Physiology, Medicine School
48080 Leioa ESP
Email: joseignacio.ruizs@ehu.es
Pr. Ronit Sagi-Eisenberg
Chair, Dept. of Cell and
Developmental Biology
69978 Tel-Aviv ISR
Email: histol3@post.tau.ac.il
Mrs. Ester Sanchez Tillo
Group of Macrophage Biology
8028 Barcelona ESP
Email: esanchez@ub.edu
Mr. Gabriella Sarmay
Dept. of Immunology
1117 Budapest HUN
Email: sarmayg@cerberus.elte.hu
Pr. Reinhold Schäfer
Molecular Tumor Pathology
D-10117 Berlin DEU
Email:
reinhold.schaefer@charite.de
Pr. Gert Jan Scheffer
Dept Anesthesiology
6500 HB Nijmegen NLD
Email: g.scheffer@anes.umcn.nl

Dr. Carsten Scheller
97078 Wuerzburg DEU
Email: scheller@vim.uniwuerzburg.de
Mrs. Martine Schmitz
LBPI
1511 luxembourg LUX
Email: martine.schmitz@uni.lu
Mr. Günter Schneider
Technical University Munich, II.
Department of Internal Medicine
D-81675 Munich DEU
Email:
guenter.schneider@lrz.tum.de
Mrs. Yvonne Schrage
2333 ZA Leiden NLD
Email: y.m.schrage@lumc.nl
Mr. Marc Schumacher
Laboratoire de chimie organique
synthétique
L-2162 Luxembourg LUX
Email: reenert@hotmail.com
Dr. Anna Ivana Scovassi
Istituto di Genetica Molecolare
27100 Pavia ITA
Email: scovassi@igm.cnr.it
Dr. RITA SELVATICI
44100 FERRARA ITA
Email: svr@unife.it
Dr. Bernd Schmeck
Dept. of Infectious Diseases
13353 Berlin DEU
Email: Bernd.Schmeck@charite.de
Dr. Benoït Schneider
Laboratoire de différenciation
cellulaire et prions
94801 VILLEJUIF FRA
Email: bschneid@vjf.cnrs.fr
Dr. Marina Schorpp-Kistner
Division of Signal Transduction
and Growth Control
69120 Heidelberg DEU
Email: marina.schorpp@dkfz.de
Dr. Jutta Schueller
MO
51149 Koeln DEU
Email: jutta.schueller@pmintl.com
Dr. Chantal Schwartz
Hémato-Cancérologie
expérimentale
1526 Luxemburg LUX
Email: chantal.schwartz@crpsante.lu
Mrs. Claudia Seidel
AG Tumorgenetik
6097 Halle DEU
Email:
Claudia.Seidel@medizin.unihalle.de
Dr. Gregg Semenza
Institute for Cell Engineering
21205 Baltimore, MD USA
Email: gsemenza@jhmi.edu
Mrs. Debora Schmitz
Brian Hemmings lab/Friedrich
Miescher Institute
4058 Basel CHE
Email: debora.schmitz@fmi.ch
Pr. E. Marion Schneider
Sektion Experimentelle
Anaesthesiologie
89075 Ulm DEU
Email: marion.schneider@uniulm.de
Mrs. Andrea Schote
Institute of Immunology
L-1950 Luxembourg LUX
Email:
andrea.schote@LNS.ETAT.LU
Dr. Wolfgang Schulz
Urology
40225 Duesseldorf DEU
Email: wolfgang.schulz@uniduesseldorf.de
Mr. Massimiliano Scolz
National Laboratory
34012 Trieste ITA
Email: cib@lncib.it
Mrs. Chantal Sellier
Laboratoire de Régulation des
signaux de division
59655 Villeneuve d'Ascq FRA
Email: chantal_sellier@yahoo.fr
Mrs. Ji Hye Seo
department of Pharmacology
120-752 Seoul KOR
Email: jhseo@yumc.yonsei.ac.kr

Mr. Christine Sers
Laboratory of Molecular Tumor
Pathology
10117 Berlin DEU
Email: christine.sers@charite.de
Pr. Mehdi Shakibaei
Institute of Anatomy
80336 Munich DEU
Email: mehdi.shakibaei@med.unimuenchen.de
Dr. Galina Shmarina
Laboratory of Immunogenetics
115478 Moscow RUS
Email: osugariver@medgen.ru
Pr. Anna Siniscalchi
44100 FERRARA ITA
Email: snn@unife.it
Dr. Asa Sjoling
medical microbiology and
immunology
413 90 Goteborg SWE
Email: asa.sjoling@microbio.gu.se
Mr. Gary Robert Smith
Chemistry Department
MK7 6AA Milton Keynes GBR
Email:
gary.smith@persescomms.com
Dr. Ulrike Sommer
Institut für Biochemie
52074 Aachen DEU
Email: ulrike.sommer@rwthaachen.de
Dr. Jan Seyfried
Faculty of Life Sciences
M13 9PT Manchester GBR
Email:
jan.seyfried@manchester.ac.uk
Dr. Yu Shaoqing
Department of of
Otorhinolaryngology
250031 Jinan City, Shandong
province CHN
Email: yu_shaoqing@hotmail.com
Dr. Georgios Simos
Lab. of Biochemistry, School of
Medicine
41222 Larissa GRC
Email: simos@med.uth.gr
Pr. ANNA SINISCALCHI
DEPT. CLINICAL EXPTL.
MEDICINE, SECT.
PHARMACOLOGY
44100 FERRARA ITA
Email: snn@unife.it
Mrs. Iben Holst Eriksen Skjoth
Biomedical Research and
Molecular Cell Biology Group
5230 Odense M DNK
Email: ibens@bmb.sdu.dk
Dr. Dik Snijdelaar
Department of Anesthesiology
(550)
6500 HB Nijmegen NLD
Email: d.snijdelaar@anes.umcn.nl
Mrs. Seung Wha Son
120-749 Seoul KOR
Email: spritlove@nate.com
Mr. Marco Sgarbanti
Department of Infectious, Parasitic
and Immunomediated Diseases
161 Roma ITA
Email: marco.sgarbanti@iss.it
Mr. Jun-Wan Shin
National Research Laboratory of
Molecular Carcinogenesis and
Chemoprevention
151-742 Seoul KOR
Email: senjon@hotmail.com
Dr. Helle-Evi Simovart
Institute of Anatomy
50411 Tartu EST
Email: helle-evi.simovart@ut.ee
Pr. Maria Caterina Sirianni
Clinical Immunology, Dept. of
Clin. Medicine
185 Rome ITA
Email:
mariacaterina.sirianni@uniroma1.i
t
Dr. Alina Smalinskiene
Environmental and Health
LT- 5009 Kaunas LTU
Email: smalina@vector.kmu.lt
Mrs. Mayra Solis
Dr J. Hiscott
H3T 1E2 Montreal CAN
Email: somayr@hotmail.com
Pr. Yong-Sang Song
Laboratory of Gynecological
Oncology
110-799 Seoul KOR
Email: rbssh9@snu.ac.kr

Mr. Yoo-Cheol Song
Laboratory of Gynecological
Oncology
110-799 Seoul KOR
Email: rbssh9@snu.ac.kr
Dr. Christina Spiropoulou
Special Pathogens Branch
30333 Atlanta USA
Email: ccs8@cdc.gov
Pr. Dominique Stéhelin
Email: Dominique.Stehelin@ibl.fr
Dr. Simon Stockwell
Anti-oncogene
SW3 6JB London GBR
Email: simons@icr.ac.uk
Pr. Pann-Ghill Suh
Signaling Network Lab.
790-784 Pohang KOR
Email: pgs@postech.ac.kr
Dr. Rosanna Supino
Unit of Chemotherapy and
Antitumor Preclinical
Pharmacology
20133 Milano ITA
Email:
rosanna.supino@istitutotumori.mi.
it
Mr. Anna Szypowska
Physiological Chemistry
3584 CG Utrecht NLD
Email: a.a.szypowska@med.uu.nl
Mr. Tavera Soraya
Janssen Cilag
28042 Madrid ESP
Email: amex.c.viajes@aexp.com
Mr. Konstantinos Stamatakis
28040 Madrid ESP
Email: kostas@cib.csic.es
Pr. Laurence Stevens
Lab Neuromuscular Plasticity
59655 Villeneuve d'Ascq FRA
Email: Laurence.Stevens@univlille1.fr
Mr. Jiri Stohr
Laboratory of Biochemistry of
Membrane Receptors
142 20 Prague CZE
Email: stohr@biomed.cas.cz
Dr. Stanislaw Sulkowski
Department of Pathology
15-269 Bialystok POL
Email: sulek@zeus.amb.edu.pl
Pr. Young-Joon Surh
College of Pharmacy
151-742 Seoul KOR
Email: surh@plaza.snu.ac.kr
Mr. Olivier Tabary
Insem U719
75012 Paris FRA
Email: olivier.tabary@stantoine.inserm.fr
Mrs. Tia Sorsa
2600 Glostrup DNK
Email: ts@dcb-glostrup.dk
Mrs. Konstantina Stathopoulou
157 84 Athens GRC
Email: kstath@biol.uoa.gr
Mr. Michael Stilmann
AG Scheidereit
13125 Berlin DEU
Email: Stilmann@gmx.de
Mr. Markus Streiff
CH-4002 Basel CHE
Email:
markus.streiff@novartis.com
Mr. Yoshiaki Sunami
13092 Berlin DEU
Email: y.sunami@mdc-berlin.de
Mrs. Chie Suzuki
Department of Pharmacology
651-2180 Kobe JPN
Email:
suzuki@pharm.kobegakuin.ac.jp
Mrs. Fatiha Tabet
Ottawa Health Research Institute
K1H 8M5 Ottawa CAN
Email: ftabet@uottawa.ca

Mrs. Aurélie Tacheny
URBC
5000 Namur BEL
Email:
aurelie.tacheny@fundp.ac.be
Mr. Marie-Helene Teiten
LBMCC
L-2540 Luxembourg LUX
Email:
marie_helene.teiten@lbmcc.lu
Dr. Stefano Thellung
Pharmacology-Departmento of
biology oncology and genetics
16132 Genova ITA
Email: thellung@yahoo.com
Mr. Lionel Tintignac
Brian Hemmings lab/Friedrich
Miescher Institute
4058 Basel CHE
Email: lionel.tintignac@fmi.ch
Dr. Alexandra Tomova
Laboratory of Genetic Engineering
of Pathogenic Microorganisms
123098 Moscow RUS
Email: altomova@abv.bg
Mr. Felix Tritschler
Laboratory of Molecular Neuro-
Oncology, Department of General
Neurology
72076 Tuebingen DEU
Email: f.tritschler@gmx.de
Dr. SOPHIE TURBAN-
RAJAONAH
Molecular Physiology
DD1 4HN Dundee GBR
Email: s.turban@dundee.ac.uk
Dr. Tetsuya Tanaka
Department of Applied Genetics,
Institut de Biologie et de Médecine
Moléculaires
B-6041 Gosselies BEL
Email: tetsuyat2003@hotmail.com
Mrs. Silvia Tejerina
URBC
5000 Namur BEL
Email: silvia.tejerina@fundp.ac.be
Dr. Marily Theodoropoulou
D80804 Munich DEU
Email: marily@mpipsykl.mpg.de
Dr. Alexander Tokmakov
Genomic Sciences Center
230-0045 Yokohama JPN
Email: tokmak@gsc.riken.go.jp
Dr. Grazina Treigyte
Department of Developmental
Biology
LT-08662 Vilnius LTU
Email: grazina.treigyte@bchi.lt
Dr. Andreas Tsakalof
Lab. of Biochemistry, School of
Medicine
41222 Larissa GRC
Email: atsakal@med.uth.gr
Dr. Jonathan Turner
Institute of Immunology
L-1950 Luxembourg LUX
Email:
jonathan.turner@LNS.ETAT.LU
Pr. Mary Taub
Taub Laboratory, Biochemistry
Dept. 140 Farber Hall
14214 Buffalo, NY USA
Email: biochtau@buffalo.edu
Mr. Bjorn Textor
Division of Signal Transduction
and Growth Control
Heidelberg Heidelberg DEU
Email: b.textor@dkfz.de
Mrs. Angelica Timofeeva
Genetic Engineering
121552 Moscow RUS
Email: Angelica_T@cardio.ru
Dr. Aviva Tolkovsky
Biochemistry
CB 2 1QW Cambridge GBR
Email: amt@mole.bio.cam.ac.uk
Dr. Cristina Trejo-Solis
Neuroimmunology
14269 Mexico City MEX
Email: trejosolis@yahoo.com.mx
Dr. Jurg Tschopp
Dept. Biochemistry
1066 Epalinges CHE
Email: jurg.tschopp@unil.ch
Mrs. Tzvetomira Tzanova
LIMBP
57070 Metz FRA
Email: tzvete_tzanova@abv.bg

Dr. Lucia Maria Valatro
Laboratori Nazionali del Sud
95100 Catania ITA
Email: valastro@lns.infn.it
Dr. Michiel van den Brekel
1066 CX Amsterdam NLD
Email: m.vd.brekel@nki.nl
Mr. Edwin van der Linden
Dept. of Immunology
3584 EA Utrecht NLD
Email: E.vanderlinden-6@azu.nl
Pr. Aleyde Van Eynde
Afdeling Biochemie
3000 Leuven BEL
Email:
Aleyde.VanEynde@med.kuleuven
.be
Dr. Kenneth van Golen
48109 Ann Arbor, Michigan USA
Email: kgolen@umich.edu
Dr. Anne Van Langendonckt
Gynaecology
1200 Brussels BEL
Email:
anne.vanlangendonckt@gyne.ucl.a
c.be
Dr. Tom VANDEN Berghe
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email:
Tom.Vandenberghe@dmbr.UGent
.be
Mrs. Marjan Van Cleemput
LEGEST
9000 Ghent BEL
Email:
marjan.vancleemput@ugent.be
Pr. Peter J. Van den Elsen
Division of Molecular Biology,
Department of
Immunohematology and Blood
Transfusion
2333 ZA Leiden NLD
Email: pjvdelsen@lumc.nl
Mrs. Kristan van der Vos
Dept. of Immunology
3584 EA Utrecht NLD
Email: K.vandervos-
3@umcutrecht.nl
Mr. Joost van Galen
Dept. Pathology
1081 HV Amsterdam NLD
Email: j.vangalen@vumc.nl
Mrs. Tanja van Harn
Xtrack
3572GA Utrecht NLD
Email: T.vanHarn@students.uu.nl
Mr. Jorg van Loosdregt
Dept. of Immunology
3584 EA Utrecht NLD
Email: J.vanloosdregt@azu.nl
Dr. Wim Vanden Berghe
LEGEST
9000 Gent BEL
Email: w.vandenberghe@ugent.be
Dr. Mark Van de Casteele
Diabetes Research Center
1090 Brussels BEL
Email: mvdcaste@vub.ac.be
Dr. Patrick van der Holst
LBPI
l-1511 Luxembourg LUX
Email: patrick.vanderholst@ist.lu
Mr. Dave van Ditmarsch
Xtrack
3572GA Utrecht NLD
Email:
D.vanDitmarsch@students.uu.nl
Dr. Cynthia van Golen
48109 Ann Arbor, Michigan USA
Email: kgolen@umich.edu
Mrs. Marijn van Jaarsveld
Xtrack
3572GA Utrecht NLD
Email:
M.T.M.vanJaarsveld@students.uu.
nl
Dr. André van Puijenbroek
Target Discovery Unit
5340 BH Oss NLD
Email:
andre.vanpuijenbroek@organon.co
m
Pr. Peter VANDENABEELE
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email:
Peter.Vandenabeele@dmbr.UGent
.be

Dr. Peter Vanhoutte
Signalisation neuronale et
Régulations géniques
75005 Paris FRA
Email:
peter.vanhoutte@svn.jussieu.fr
Mrs. Roberta Vené
Experimental Oncology A
16132 Genoa ITA
Email: robertavene@yahoo.it
Mrs. Elisabeth VERCAMMEN
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email: Elisav@dmbr.UGent.be
Mr. Julien Verrax
Pharmacokinetic, Metabolism,
Nutrition and Toxicology
1200 Bruxelles BEL
Email:
julien.verrax@pmnt.ucl.ac.be
Mr. Eduardo Villamor
6202 AZ Maastricht NLD
Email: eiv@paed.azm.nl
Pr. Athanase Visvikis
MAEM UMR 7567
54500 Nancy FRA
Email: visvikis@maem.uhpnancy.fr
Mr. J Willem Voncken
Div. Molecular Genetics
6229 ER Maastricht NLD
Email:
w.voncken@gen.unimaas.nl
Mrs. Nele VANLANGENAKKER
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email: Nelev@dmbr.UGent.be
Dr. Valentina Venezia
Pharmacology-Department of
oncology biologi and genetics
16132 Genova ITA
Email: venezia@unige.it
Mrs. Liesbeth Verhagen
Dept. of Immunology
3584 EA Utrecht NLD
Email: L.verhagen@azu.nl
Mrs. Lynn VERSTREPEN
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email: Lynnv@dmbr.UGent.be
Dr. Maria Vinciguerra
Prof. Anna Maria Musti
80131 Napoli ITA
Email: kachera@libero.it
Mr. Michael Vogt
52074 Aachen DEU
Email: michaelvogt1@gmx.de
Dr. James Wachira
department of biology
21251 baltimore USA
Email: jwachir@jewel.morgan.edu
Mrs. Emilie VELOT
Laboratoire de Recherche Cardio-
Vasculaire
1150 LUXEMBOURG LUX
Email: emilie.velot@crp-sante.lu
Dr. Donatella Verbanac
Program Office
10 000 Zagreb HRV
Email: dverbanac@pliva.hr
Dr. Helene Verheije
Virology
3584 CL Utrecht NLD
Email: h.verheije@vet.uu.nl
Dr. Valentina Villa
pharmacology-department of
oncology biology and genetics
16132 Genova ITA
Email: valentina.villa@unige.it
Mrs. Elodie VIRY
LIMBP
57070 Metz FRA
Email: elodieviry@hotmail.fr
Mr. Joerg von Hagen
LSA R&D
64271 Darmstadt DEU
Email: joerg.von.hagen@merck.de
Dr. Vicki Waetzig
24105 Kiel DEU
Email:
vicki.waetzig@pharmakologie.uni
-kiel.de

Mrs. Jessica Wagener
40225 Duesseldorf DEU
Email: jessica.wagener@gmx.de
Mr. Piotr Wardega
751 24 Uppsala SWE
Email:
Piotr.Wardega@LICR.uu.se
Dr. Thomas Welss
Skin / Hair Physiology -
Development Screening Systems
D-40191 Duesseldorf DEU
Email: thomas.welss@henkel.com
Dr. Johanna Westra
dept Rheumatology
9713 GZ Groningen NLD
Email: j.westra@med.umcg.nl
Dr. Robin SB Williams
Department of Biology
WC1 E6BT London GBR
Email: robin.williams@ucl.ac.uk
Mrs. Ellen WIRAWAN
Dept. of Molecular Biomedical
Research
B-9052 Zwijnaarde BEL
Email: Ellenw@dmbr.UGent.be
Dr. Verena Wünsche
65824 Schwalbach/Ts DEU
Email:
verena.wuensche@merckbioscienc
es.de
Mrs. Samantha Wales
Virology and Immunology
21201 Baltimore USA
Email: sqwales@yahoo.com
Pr. Manfred Weiss
Abteilung Klinische
Anaesthesiologie
89075 Ulm DEU
Email:
manfred.weiss.ulm@online.de
Dr. Sibylle Wenzel
Institute of Physiology
35392 Giessen DEU
Email: sibylle.wenzel@gmx.de
Pr. Linda R. White
Department of Neurology
N-7006 Trondheim NOR
Email: linda.white@ntnu.no
Pr. Pat Wilson
Renal Cell Biology
NW3 2PF London GBR
Email: pat.wilson@mssm.edu
Mr. Jiong Wu
1915 Beverly, Massachusetts USA
Email: mdolan@cellsignal.com
Mrs. Georgia Xenaki
Molecular Pharmacology, Dr. C.
Demonacos
M13 9PL Manchester GBR
Email:
georgiaxenaki@hotmail.com
Dr. Yihua Wang
Laboratory of Cell and Molecular
Biology
100021 Beijing CHN
Email: yihua_wang@hotmail.com
Dr. Valerie Wells
Cell Signalling laboratory,
Pharmaceutical Sciences Research
Division
SE1 9NH London GBR
Email: valerie.wells@kcl.ac.uk
Mrs. Shannon Werner
Signaling Systems Laboratory
92093-0375 La Jolla, CA USA
Email: slwerner@ucsd.edu
Mrs. Christina Wiegand
40202 Louisville, KY USA
Email:
cbwieg01@gwise.louisville.edu
Dr. Andrzej Wincewicz
Department of Pathology
15-269 Bialystok POL
Email: sulek@zeus.amb.edu.pl
Mrs. Kun Wu
Nutrition and Food Hygiene
Department
150086 Harbin CHN
Email: wukun@public.hr.hl.cn
Dr. Ying Xia
Department of Environmental
Health
45267 Cincinnati USA
Email: xiay@email.uc.edu

Pr. Ningzhi Xu
Laboratory of Cell and Molecular
Biology
100021 Beijing CHN
Email: xningzhi@public.bta.net.cn
Dr. Katsuji Yoshioka
Cancer Research Institute
920-0934 Kanazawa JPN
Email: ysok-f01@siren.ocn.ne.jp
Mrs. Maya Margaritova Zaharieva
Experimental Chemotherapy
1000 Sofia BGR
Email: zaharieva26@yahoo.com
Dr. Jana Zdychova
VÌde&#328
14021 Prague CZE
Email:
jana.zdychova@medicon.cz
Dr. Margherita Zennaro
Dept of Biochemistry and
Molecular Biology
44100 Ferrara ITA
Email:
margheritazennaro@yahoo.it
Mrs. Jun Zhao
Physiological Chemistry
3584 CG Utrecht NLD
Email: j.zhao@med.uu.nl
Pr. Alessandra Zicari
IMMUNO_BIOLOGY
161 Rome ITA
Email:
alessandra.zicari@uniroma1.it
Dr. Michael B. Yaffe
Center for Cancer Research
2139 Cambridge, MA USA
Email: myaffe@mit.edu
Mr. Ji Hoon YU
Department of Pharmacology
120-752 Seoul KOR
Email: jihoonyu@hotmail.com
Dr. Peter Zalewski
Department of Medicine
5011 Woodville AUS
Email:
peter.zalewski@adelaide.edu.au
Dr. Alain G. Zeimet
6020 Innsbruck AUT
Email: alain.zeimet@uibk.ac.at
Dr. Ying Zhang
Cellular And Molecular Biology
20892-4256 Bethesda USA
Email: yingz@helix.nih.gov
Dr. Yuan Zhu
Center of Reproductive Medicine,
430030 Wuhan city, Hubei
province CHN
Email:
huyuan0528@yahoo.com.cn
Mrs. Rhyenne Zimmerman
Biological Chemistry, Leiden
University
2333 CC Leiden NLD
Email:
r.zimmerman@chem.leidenuniv.nl
Mrs. Christina Westmose Yde
Biomedical Research and
Molecular Cell Biology Group
5230 Odense M DNK
Email: chriswy@bmb.sdu.dk
Dr. Tülay Yucel-Lindberg
141 04 Huddinge SWE
Email: tulay.lindberg@ki.se
Mrs. Darina Zaskodova
Department of Medical
Biochemistry
500 38 Hradec Kralove CZE
Email: zaskodovad@lfhk.cuni.cz
Dr. Marina Zenkova
630090 Novosibirsk RUS
Email: marzen@niboch.nsc.ru
Mr. Zhongchun Zhang
Physiological Chemistry
3584 CG Utrecht NLD
Email: Z.Zhang@med.uu.nl
Dr. Jia Zhuo
Receptor and Signal Transduction
48202 Detroit USA
Email: jzhuo1@hfhs.org
Mrs. Bea Zoer
6200 MD Maastricht NLD
Email:
bea.zoer@farmaco.unimaas.nl

Pr. Moncef ZOUALI
Centre Viggo Petersen
75475 Paris Cedex 10 Paris FRA
Email: moncef.zouali@wanadoo.fr