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Luxembourg,<br />
January 26th to 28th, 2011<br />
New Conference Center Kirchberg<br />
NCCK - Luxembourg<br />
Cell Signal-omics 2011<br />
Integrated cellular pathology<br />
Systems biology of human disease<br />
Proceedings<br />
<strong>and</strong><br />
Program<br />
Marc Diederich <strong>and</strong> Guido Kroemer<br />
Organized by<br />
Recherches Scientifiques Luxembourg asbl<br />
Under the auspices of<br />
the European Research Institute for Integrated Cellular Pathology
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Luxembourg,<br />
January 26th to 28th, 2011<br />
New Conference Center Kirchberg<br />
NCCK - Luxembourg<br />
Cell Signal-omics 2011<br />
Integrated cellular pathology - Systems biology of human disease<br />
Proceedings <strong>and</strong> Program<br />
Editor<br />
Marc Diederich<br />
This meeting is organized <strong>and</strong> financed by<br />
Recherches Scientifiques Luxembourg asbl<br />
Under the auspices of<br />
the European Research Institute for Integrated Cellular Pathology<br />
This meeting is co-financed by<br />
the Fonds National de la Recherche, Luxembourg<br />
Printing of the Proceedings sponsored by<br />
the Fondation de Recherche Cancer et Sang (Luxembourg)
Table of content<br />
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Preface<br />
In 1998, we organized the first specialized meeting in the field of signal transduction <strong>and</strong> gene<br />
expression in Luxembourg. This type of meeting was originally thought to teach doctoral<br />
students of the molecular <strong>and</strong> cellular biology master training program of the University<br />
of Nancy I (France).<br />
Since then, more than 6000 fundamental, clinical <strong>and</strong> industrial researchers were<br />
gathering in Luxembourg for eight different meetings in order to discuss therapeutic<br />
applications in the field of signal transduction, transcription <strong>and</strong> translation related to novel<br />
therapeutic applications. These meetings allow new insights into this rapidly moving field.<br />
Novel antibodies against receptors, protein kinase inhibitors, antisense oligonucleotides <strong>and</strong><br />
siRNA targeting both signal transduction <strong>and</strong> gene expression will certainly enhance<br />
therapeutic approaches for the next century.<br />
For the 2011 edition of meeting, with 350 participants, I am convinced that this meeting will<br />
be a great success.<br />
Welcome to Luxembourg!<br />
Marc Diederich<br />
1
Acknowledgments<br />
This meeting has been realized under the aegis of:<br />
Recherches Scientifiques asbl<br />
The Fondation de "Recherche Cancer et Sang"<br />
The Fonds National de la Recherche, Luxembourg<br />
Under the auspices of :<br />
the European Research Institute for Integrated Cellular Pathology<br />
We have the pleasure to acknowledge support from:<br />
The Fonds National de la Recherche, Luxembourg<br />
Recherches Scientifiques asbl<br />
Kuwait Petroleum SA - Luxembourg<br />
The City of Luxembourg<br />
The Fondation de Recherche "Cancer et Sang"<br />
Computer Home, Luxembourg<br />
Canon Luxembourg<br />
Your onsite organization team:<br />
Claudia Cerella, Sebastien Chateauvieux, Marc Diederich, Cristina Florean, Elodie Frenger,<br />
Francois Gaascht, Anthoula Gaigneaux, Stefania Gonfloni, Cindy Gr<strong>and</strong>jenette, Christina<br />
Grigorakaki, Sheherazade Hajjouli, Estelle Henry, Liliane Hermes, Monika Jain, Tommy<br />
Karius, Mareike Kelkel, Noemie Legr<strong>and</strong>, Fabienne Mack, Franck Morceau, Florian Muller,<br />
Karoline Noworyta, Marie-Anne Olinger, Barbora Orlikova, Michael Schnekenburger,<br />
Christiane Schuld, Marc Schumacher, Carole Seidel, Cyril Sobolewski, Marie-Helene Teiten,<br />
Anne Trecul, Elodie Viry.<br />
3
European Research Institute for Integrated Cellular Pathology (ERI-ICP)<br />
About ERI-ICP<br />
ERI-ICP is a professional organization with some 100 Founding Members including many of<br />
the most prominent Europea researchers (<strong>and</strong> several Nobel laureates) working on integrated<br />
cellular pathology. The principal objective of ERI-ICP, a virtual research Institute, will be to<br />
promote cell biology research applied to major human diseases in Europe, including (but not<br />
limited to) neurodegenerative diseases, myopathies, mitochondriopathies, infectious diseases,<br />
cancer <strong>and</strong> pathological aging. ERI-ICP will promote the implementation <strong>and</strong> utilization of<br />
systems biology (or integrated biology) by establishing a continuum between fundamental,<br />
translational <strong>and</strong> clinical research. ERI-ICP will strive for excellence, independence,<br />
leadership, diversity <strong>and</strong> flexibility. Throughout its work, ERI-ICP will provide independent,<br />
authoritative <strong>and</strong> evidence-based advice to underpin policy for stimulating the implementation<br />
of systems biology applicable to the investigation, classification, diagnosis <strong>and</strong> therapeutic<br />
management of major human diseases.<br />
ERI-ICP will host a website <strong>and</strong> will disseminate “official” (but ameliorable) methods <strong>and</strong><br />
protocols applicable to integrated cellular pathology. ERI-ICP will also participate in efforts<br />
of data integration, provide a web forum for researchers working in the area of systems<br />
biology applied to human disease, <strong>and</strong> organize annual conferences. By the conjunction of<br />
these activities, ERI-ICP should be able to set up the st<strong>and</strong>ards for data quality, for data<br />
exchange <strong>and</strong> storage that are crucial for systems biology, as applied to human disease.<br />
Therefore, ERI-ICP will serve as an instrument to crystallize <strong>and</strong> streamline all efforts within<br />
this area in the European Research Area. The Founding Members of ERI-ICP have been<br />
selected among highly renowned scientists from all areas of disease-oriented fundamental <strong>and</strong><br />
clinical research. The Founding Group will be responsible for defining the overall objectives<br />
of the Institute, addressing issues such as credibility of the institution, membership <strong>and</strong><br />
governance. To steer the process, a ‘Governing Body’ consisting of a President, a Vice-<br />
President, a Secretary-General <strong>and</strong> five fellows representing all disciplines Europe-wide, will<br />
be established. To address issues as they arise, ad hoc taskforces will be organized amongst<br />
members of the Institute, specific to their area of expertise. As a Founding Member of<br />
ERIICP you can anticipate contributing to the preparation of position papers, interacting with<br />
EU <strong>and</strong> national institutions to facilitate proposals <strong>and</strong> the implementation of new policies<br />
that ensure that disease-oriented systems biology remain at the top of the European research<br />
agenda.<br />
The official launch of ERI-ICP will be one of the highlights at the opening of the 1st<br />
Conference on Integrated Cellular Pathology, at the Pasteur Institute, in Paris, on April 22-24,<br />
2010. The second annual conference is planned for January 26-29, 2011, in Luxembourg.<br />
http://kroemerlab.com/ERI-ICP_About.html<br />
5
General Information<br />
Meeting Venue<br />
All meeting sessions are held at the New Congress Center Kirchberg<br />
4, Place de l’Europe<br />
L-1499 Luxembourg-Kirchberg<br />
This new building is hidden located behind the Philharmonie Concert Hall (left side) <strong>and</strong> is in<br />
fact integrated into the basis of the white Tower building (see map with photos for details).<br />
Registration will take place in the at the registration desk open daily (9h00-19h00).<br />
Coffee breaks will be served in the exhibit area daily (See general program for details).<br />
Lunch<br />
For the participants that pre-paid lunch, lunch is served from 13h00 - 15h00. Additional<br />
tickets are NOT available at the registration desk.<br />
Exhibits are open daily form January 26 th to 28 th , 2011.<br />
The Exhibit opens on Wednesday January 26 th (Coffee break 10h30) <strong>and</strong> ends on Friday<br />
January 28 st , 2011 after the afternoon coffee break.<br />
Posters<br />
All posters will be up from Wednesday January 26 th until Friday January 28 th :<br />
Wednesday January 26 th 14h00 – 16h00: Posters with even numbers<br />
Thursday January 27 th 14h00 – 16h00: Posters with odd numbers<br />
Friday January 28 th 14h00 – 16h00: All posters<br />
On Friday January 28 th 16h00, all poster presenters are requested to recover their<br />
posters (the boards will be removed).<br />
Welcome reception offered by the City of Luxembourg (expo area)<br />
On Wednesday January 26, 2011 from 19h30-20h30 at the expo surface.<br />
Gala Dinner<br />
The Gala Dinner will take place at the Hôtel Le Royal 12, boulevard Royal L-2449<br />
Luxembourg Tel: + 352 24 16 16-1, Fax: + 352 22 59 48 on Thursday, January 27th starting<br />
at 20h30. Additional tickets are NOT available at the registration desk.<br />
Transportation<br />
Our bus shuttles will bring the participants from the hotels to the meeting center in the<br />
morning <strong>and</strong> back to the hotels or city center in the evening. Please note that the busses leave<br />
from your hotel depending on the distance to the meeting center (Check timetable for details).<br />
The busses are red, orange <strong>and</strong> yellow <strong>and</strong> are from the bus company "Demy Cars".<br />
Taxis can be called from the registration desk.<br />
7
How to reach the congress center?<br />
- By taxi:<br />
from the airport (Luxembourg - Findel) in about 15 minutes.<br />
from the railroad station (about 15 minutes)<br />
- By bus: take bus line number 16 (every 20 minutes from the city center)<br />
- By car:<br />
From France: Highway A4 from Metz, take Highway A1 (E44) direction Trier Plateau<br />
de Kirchberg Aéroport, choose exit number "8", orient towards "Quartier Europeen Sud<br />
Luxembourg-Centre"<br />
From Belgium: Highway A411 from Brussels, take Highway A6 (E44) direction Trier<br />
Plateau de Kirchberg Aéroport, choose exit number "8", orient towards "Quartier Europeen<br />
Sud Luxembourg Centre"<br />
From Germany: Highway A6 (E44) from Trier, direction Luxembourg, choose exit<br />
number "8", orient towards "Quartier Europeen Sud Luxembourg Centre".<br />
8
9<br />
Exhibition
Many thanks to all exhibitors<br />
Please visit our Expo !<br />
10
11<br />
Scientific Program
Wednesday January 26th, 2011<br />
8h00 - 19h00: Registration<br />
Keynote session:<br />
Chair: Marc Diederich (LBMCC, Luxembourg)<br />
9h30 - 10h30 Mario Capecchi (University of Utah School of Medicine, Salt Lake City, USA):<br />
Gene Targeting into the 21st Century: Mouse Models of Human Disease from Cancer to<br />
Neuropsychiatric Disorders<br />
10h30 - 11h00 Coffee break <strong>and</strong> visit of the expo<br />
Session 1: Cell death<br />
Chair: Marc Diederich (LBMCC, Luxembourg)<br />
11h00 - 11h30<br />
11h30 - 12h00<br />
12h00 - 12h30<br />
12h30 - 12h45<br />
Eileen White (Rutgers University, New Brunswick, NJ, USA):<br />
Therapeutic targeting of death pathways in cancer: mechanisms for activating cell death in<br />
cancer cells.<br />
Gerry Melino (University of Rome Tor Vergata, Italy):<br />
Why we need the p53 family? The Guardian of maternal reproduction<br />
Stefania Gonfloni (University of Rome Tor Vergata, Dept of Biology, Italy):<br />
DNA-damage stress response in female germ cells: is Abl a fine tuner or a dangerous<br />
amplifier?<br />
Cinzia Di Pietro (Università degli Studi di Catania, Italy):<br />
Altered expression of Apoptotic Machinery genes explains the decrease of oocytes<br />
competence with aging<br />
12h45 – 13h00<br />
Inna N. Lavrik (DKFZ, Heidelberg, Germany):<br />
Towards underst<strong>and</strong>ing of life <strong>and</strong> death regulation at CD95/Fas<br />
13h00 - 14h00 Lunch <strong>and</strong> visit of the expo<br />
Workshops 1 <strong>and</strong> Poster Session 1<br />
14h00 - 15h00<br />
15h00 - 16h00<br />
Géraldine Guérin-Peyrou (Polyplus-transfection, France)<br />
Winfried van Eyndhoven (Agilent Technologies, The Netherl<strong>and</strong>s)<br />
Karoly Szuhai (Leiden University Medical Center Dept, The Netherl<strong>and</strong>s)<br />
Ian Majewski (Netherl<strong>and</strong>s Cancer Institute, The Netherl<strong>and</strong>s)<br />
14h00 - 15h30 Poster session (Even numbers) <strong>and</strong> visit of the expo<br />
15h30 – 16h00 Coffee break <strong>and</strong> visit of the expo<br />
13
Session 2: Cell signaling<br />
Chair: Ilya Shmulevich (Institute for Systems Biology, Seattle, USA)<br />
16h00 - 16h30<br />
Ilya Shmulevich (Institute for Systems Biology, Seattle, USA):<br />
Computational Systems Biology in Cancer Research<br />
Varadharajan Sundaramurthy (Max Planck Institute of Molecular Cell Biology <strong>and</strong><br />
16h30 - 17h00<br />
Genetics, Dresden, Germany):<br />
Systems Analysis of Endocytosis by Quantitative Image Analysis: Insights into Pathogenetic<br />
Mechanisms<br />
17h00 - 17h30<br />
Anne Grosse-Wilde (Institute for Systems Biology, Seattle, USA):<br />
Using single cell analysis tools to search for human breast cancer stem cells<br />
17h30 - 18h00<br />
Josef Penninger (Institute of Molecular Biotechnology, Vienna, Austria):<br />
Whole genome scans in drosophila to model human disease<br />
18h00 - 18h15<br />
Sébastien Chateauvieux (LBMCC, Luxembourg):<br />
Valproic acid perturbs hematopoietic differentiation networks<br />
18h15 - 18h30<br />
Ivana Scovassi (Istituto di Genetica Molecolare CNR, Pavia, Italy):<br />
Search for the effects of anticancer drugs: A look to apoptosis but also to autophagy<br />
18h30 - 18h45<br />
Vladimir L. Katanaev (University of Konstanz, Germany):<br />
Signaling by the Frizzled family of receptors: G protein-coupled mechanisms <strong>and</strong> targets<br />
19h00 - 19h30 Official talks<br />
19h30 - 20h30 Reception offered by the City of Luxembourg <strong>and</strong> visit of the expo<br />
20h30 - 21h00 Shuttles leave to the hotels from the meeting center<br />
Thursday January 27th, 2011<br />
Shuttle bus from the hotels to the meeting center (see map for details)<br />
8h00 - 19h00: Registration<br />
Session 3: Transcriptional control<br />
Chair: Young-Joon Surh (Seoul National University, South Korea)<br />
9h00 - 9h30<br />
Young-Joon Surh (Seoul National University, South Korea):<br />
<strong>Redox</strong> regulation of transcription factors by electrophilic lipid mediators<br />
9h30 - 10h00<br />
Luciano Di Croce (Center for Genomic Regulation, Barcelona, Spain):<br />
Novel mechanisms of transcriptional control in differentiation <strong>and</strong> cancer<br />
10h00 - 10h30<br />
Thomas Luft (University of Heidelberg, Germany):<br />
A dual role of JAK1: regulation of IFN-! dependent <strong>and</strong> independent IL-12p70 production<br />
10h30 - 11h00 Coffee break <strong>and</strong> visit of the expo<br />
Session 4: Immunology<br />
Chair: Eileen White (Rutgers University, New Brunswick, NJ, USA)<br />
11h00 - 11h30<br />
Bali Pulendran (Emory University, Atlanta, USA):<br />
Learning immunology from successful vaccines: innate immunity to systems vaccinology<br />
11h30 - 12h00<br />
Marie-Lise Gougeon (Institut Pasteur, Paris, France):<br />
Immunity <strong>and</strong> death in chronic viral infections<br />
Kasper Hoebe (University of Cincinnati College of Medicine, USA):<br />
12h00 - 12h30 Reduced T cell survival leads to loss of T cell quiescence <strong>and</strong> subsequent immunopathology<br />
in Gimap5-deficient mice<br />
12h30 - 13h00<br />
Claude Condé (Virology <strong>and</strong> immunology unit, GIGA-research, University of Liège,<br />
Belgium): Study of the anti-apoptotic role of SHIP-1 in T cells<br />
13h00 - 16h00 Lunch <strong>and</strong> visit of the expo<br />
14
Workshops 2 <strong>and</strong> Posters Session 2<br />
14h00 - 15h00 Sarah Payne (GE Healthcare Europe GmbH)<br />
15h00 - 16h00 Alex Sim (AMSBIO)<br />
14h00 - 15h30 Poster session (Odd numbers) <strong>and</strong> visit of the expo<br />
15h30 - 16h00 Coffee break <strong>and</strong> visit of the expo<br />
Session 5: Proteomics<br />
Chair: Gerry Melino (University of Rome Tor Vergata, Italy)<br />
16h00 - 16h30<br />
Antonio del Sol (Université de Luxembourg, Gr<strong>and</strong>-Duchy of Luxembourg):<br />
Protein population shift leading to disease-related changes in cellular networks<br />
16h30 - 17h00<br />
Bernd Wollscheid (Institute of Molecular Systems Biology, Zürich, Switzerl<strong>and</strong>):<br />
Proteomic phenotyping of the cell surface information gateway<br />
17h00 - 17h30<br />
Pascale Cossart (Institut Pasteur, Paris, France):<br />
De-SUMOylation: a new strategy used by Listeria to counteract the host defense<br />
17h30 - 18h00<br />
Christopher Overall (The University of British Columbia, Canada):<br />
To the Ends of the Proteome World: Multiplex Quantitative N <strong>and</strong> C-Terminomics<br />
18h00 - 18h15<br />
Margarida Fardilha (University of Aveiro, Portugal):<br />
Identification of the Human Testis Protein Phosphatase 1 Interactome<br />
Alfred C. O. Vertegaal (LUMC, Leiden, The Netherl<strong>and</strong>s):<br />
18h15 - 18h30 Site-Specific Identification of SUMO-2 Conjugation Sites in Cells Reveals Novel<br />
SUMOylation Motifs<br />
Angela Brieger (Goethe-University, Frankfurt a.M., Germany):<br />
18h30 - 18h45 Regulation of Lynch syndrome-related DNA mismatch repair heterodimer MutLalpha by<br />
phosphorylation?<br />
19h00 Shuttles leave to the hotels from the meeting center<br />
Friday January 28th, 2011<br />
Shuttle bus from the hotels to the meeting center (see map for details)<br />
8h00 - 19h00: Registration<br />
Session 6: Epigenetics<br />
Chair: Luciano Di Croce (Center for Genomic Regulation, Barcelona, Spain)<br />
9h00 - 9h30<br />
François Fuks (ULB, Brussels, Belgium):<br />
Mechanisms of Epigenetics in Health <strong>and</strong> Disease<br />
9h30 - 10h00<br />
Michael Bots (Peter MacCallum Cancer Centre, Victoria, Australia):<br />
Treating Cancer with drugs that target the epigenome<br />
10h00 - 10h30<br />
Roberto Gambari (University of Ferrara, Italy):<br />
MicroRNA <strong>and</strong> erythroid differentiation<br />
10h30 - 11h00 Coffee break <strong>and</strong> visit of the expo<br />
15
Session 7: Cancer signaling networks<br />
Chair: Dean W. Felsher (Stanford University, USA)<br />
11h00 - 11h30<br />
Andrei Zinovyev (Institut Curie, Paris, France):<br />
Computational Systems Biology of Cancer: modeling cell fate decision <strong>and</strong> disease outcome<br />
11h30 - 12h00<br />
Dean W. Felsher (Stanford University, USA):<br />
A Systems Approach to Modeling <strong>and</strong> Predicting Oncogene Addiction<br />
12h00 - 12h30<br />
Alex<strong>and</strong>er R. A. Anderson (H. Lee Moffitt Cancer Center, USA):<br />
Microenvironmental Independence In Tumor Progression: An Integrated Approach<br />
Evan Keller (University of Michigan, USA):<br />
12h30 - 13h00 Targeting the invasive phenotype using systemic evolution of lig<strong>and</strong>s by exponential<br />
enrichment creates anti-metastatic aptamers<br />
13h00 - 14h00 Lunch <strong>and</strong> visit of the expo<br />
Session 8: Gene expression networks in health <strong>and</strong> disease<br />
Chair: Guido Kroemer (Institut Gustave Roussy, Paris France)<br />
14h00 - 14h30 Guido Kroemer (Institut Gustave Roussy, Paris France):<br />
Autophagy - suicidal self-cannibalism or homeostatic recycling?<br />
14h30 - 15h00 Yong Sang Song (Cancer Research Institute, Seoul National University, Korea):<br />
Genes related to energy metabolism differ the visceral adipose-derived stem cells from the<br />
subcutaneous ones<br />
15h00 – 15h15 Andrew Koff (Memorial Sloan-Kettering Cancer Center, NY, USA):<br />
A bioinformatic approach to illuminate why a biomarker, p27, has prognostic value in<br />
human tumors<br />
15h15 – 15h30 Stefanie Kaempf (NonWoTecc Medical GmbH, Cologne, Germany):<br />
Pathological aspects of vascular aging <strong>and</strong> strategies for new biomimetic <strong>and</strong> biological<br />
active NonWoTecc biomaterials<br />
15h30 – 16h00 Coffee break <strong>and</strong> visit of the expo<br />
Session 9: Neurodegenerative diseases<br />
Chair: Vittorio Calabrese (University of Catania, Catania, Italy)<br />
16h00 - 16h30<br />
16h30 - 17h00<br />
17h00 - 17h30<br />
17h30 - 18h00<br />
Mark P. Mattson (Laboratory of Neurosciences, National Institute on Aging Intramural<br />
Research Program, Baltimore, MD. USA):<br />
Signaling Pathways that Help Neurons Help Themselves<br />
Junying Yuan (Harvard Medical School, USA):<br />
Genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging <strong>and</strong><br />
in Alzheimer's disease<br />
Vittorio Calabrese (University of Catania, Catania, Italy):<br />
Vitagenes, metabolic stress <strong>and</strong> Hormesis in aging <strong>and</strong> Neurodegenerative disorders<br />
Xin-Fu Zhou (Flinders University, Adelaide, South Australia):<br />
p75NTR regulates Ab deposition by increasing Ab production but inhibiting Ab aggregation<br />
with its extracellular domain<br />
18h00 - 18h30<br />
Paul R. Fisher (La Trobe University, Melbourne, Australia):<br />
A “no-brainer” – mitochondrial <strong>and</strong> neurodegenerative disease in Dictyostelium.<br />
18h30 End of the meeting<br />
18h30 Shuttles leave to the hotels from the meeting center<br />
16
Oral presentations<br />
(In chronological order)<br />
17
Gene Targeting into the 21 st Century: Mouse Models of Human Disease from Cancer to<br />
Neuropsychiatric Disorders<br />
Mario Capecchi<br />
University of Utah School of Medicine, Salt Lake City, USA<br />
Gene targeting allows the designed modification of any gene in the mouse genome. Since<br />
genes impact all biological phenomena this methodology can be used to study any biological<br />
phenomena common to mammals in the mouse. We are using it to model human disease in<br />
the mouse. The models can be used to analyze the pathology of the disease at a level not<br />
feasible in humans <strong>and</strong> as a platform for the development of new therapeutic protocols. I will<br />
discuss modeling of a human cancer, synovial sarcoma, <strong>and</strong> a neuropsychiatric disorder,<br />
obsessive compulsive (OCD) spectrum disorder in the mouse. The former provides insight<br />
into the molecular biology of cancer. The latter provides the unexpected conclusion that<br />
microglia derived from bone marrow may be controlling behavior.<br />
18
Activated Ras Requires Autophagy to Maintain Oxidative Metabolism <strong>and</strong><br />
Tumorigenesis<br />
Eileen White<br />
The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ<br />
08903<br />
Autophagy is a catabolic pathway utilized by cells to support metabolism in response to<br />
starvation <strong>and</strong> to clear damaged proteins <strong>and</strong> organelles in response to stress. We report here<br />
that expression of a H-ras V12 or K-ras V12 oncogene upregulates basal autophagy, which is<br />
required for tumor cell survival in starvation <strong>and</strong> in tumorigenesis. In Ras-expressing cells,<br />
defective autophagosome formation or cargo delivery causes accumulation of abnormal<br />
mitochondria <strong>and</strong> reduced oxygen consumption. Autophagy defects also lead to tricarboxylic<br />
acid (TCA) cycle metabolite <strong>and</strong> energy depletion in starvation. As mitochondria sustain<br />
viability of Ras-expressing cells in starvation, autophagy is required to maintain the pool of<br />
functional mitochondria necessary to support growth of Ras-driven tumors. Human cancer<br />
cell lines bearing activating mutations in Ras commonly have high levels of basal autophagy,<br />
<strong>and</strong> in a subset of these, down-regulating the expression of essential autophagy proteins<br />
impaired cell growth. As cancers with Ras mutations have a poor prognosis, this “autophagy<br />
addiction” suggests that targeting autophagy <strong>and</strong> mitochondrial metabolism are valuable new<br />
approaches to treat these aggressive cancers.<br />
19
p53 family, its involvement in neuronal development via miR-34a:<br />
Why we need them?<br />
Melino G,<br />
University Tor Vergata, Rome, Italy; Medical Research Council, Toxicology Unit,<br />
Leicester, UK<br />
In the last ten years, p63 <strong>and</strong> p73 have been identified as the ancestral members of the p53<br />
family. Despite the high sequence <strong>and</strong> structural similarity, the mouse knockouts revealed a<br />
crucial role in neural development for p73 <strong>and</strong> in epidermal formation for p63. We identified<br />
several transcriptional targets, the mechanisms of regulation of cell death, <strong>and</strong> the p63<br />
isoform involved in epithelial development. Both genes are involved in female infertility <strong>and</strong><br />
maternal reproduction as well as in cancer formation, although with distinct mechanisms.<br />
p73 steady state protein levels are kept low under normal physiological conditions through<br />
degradation by the 26S proteasome, mediated by the HECT-containing E3 ubiquitin ligase<br />
ITCH, for which we are developing an inhibitor. We have also described additional<br />
mechanisms of degradation: (1) the orphan F-box protein FBXO45 ; (2) the ring finger<br />
domain ubiquitin ligase PIR2 (p73-induced Ring Finger 2), <strong>and</strong> (3) the antizyme ubiquitinindependent,<br />
proteasome-dependent pathway, both specific for the !Np73 isoforms<br />
Here, we describe the involvement of p73 in neuronal development. TAp73 knockout mice<br />
(TW Mak G&D 2008) show hippocampal dysegensis. Conversely, !Np73 knockout mice<br />
(TW Mak G&D 2010) show sign of moderate neurodegeneration with a significant loss of<br />
cellularity in the cortex. TAp73 is able to drive the expression of miR-34a, acting on specific<br />
binding sites present on the miR-34a promoter. In agreement with these in vitro data, miR-<br />
34a transcript expression is significantly reduced in vivo both in the cortex <strong>and</strong> hippocampus<br />
of p73-/- mice. In keeping, we show a role for miR-34a, in parallel to TAp73 expression,<br />
during in vitro differentiation of ES cells. Expression of miR-34a increases during in vitro<br />
neuronal terminal differentiation, of ex vivo primary cortical neuronal cultures, in parallel<br />
with the expression of TAp73. Moreover, we also detect an increase ex vivo of miR-34a<br />
steady state expression during postnatal development of the brain <strong>and</strong> cerebellum, when<br />
synaptogenesis occurs. We further confirm a role for miR-34a in synaptogenesis, as<br />
overexpression or silencing of miR-34a results in an inverse expression of a number of<br />
synaptic genes, via their 3’-UTR. In particular, miR-34a overexpression decreases<br />
synaptotagmin I <strong>and</strong> syntaxin-1A expression, <strong>and</strong> the endogenous levels of miR-34a are able<br />
to regulate only synaptotagmin I expression. Our findings show that p73 drives the<br />
expression of miR-34a during terminal, synaptic differentiation.<br />
Finally, we will speculate on why we need the p53 family at evolutionary level: it is the<br />
guardian of maternal reproduction.<br />
20
DNA-damage stress response in female germ cells: is Abl a fine tuner or a dangerous<br />
amplifier?<br />
Claudia Di Bartolomeo 1 , Emiliano Maiani 1 , Flavio Maina § , Francesca Sacco 1 , Marc<br />
Diederich # , Gianni Cesareni 1 <strong>and</strong> Stefania Gonfloni 1<br />
1 Department of Biology, University of Rome, “Tor Vergata”, via della Ricerca<br />
Scientifica, I-00133 Rome, Italy; § Developmental Biology Institute of Marseille-Luminy<br />
(IBDML) CNRS UMR 6216- Campus de Luminy – case 907, F-13288 Marseille cedex 09<br />
- France; # Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Fondation de<br />
Recherche Cancer et Sang, Hôpital Kirchberg, 9 rue Edward Steichen, L-2540<br />
Luxembourg, Luxembourg<br />
Cells experiencing DNA damage undergo a complex response entailing cell cycle arrest,<br />
DNA repair <strong>and</strong> apoptosis, the relative importance of the three being modulated by the extent<br />
of the lesions. The observation that Abl interacts in the nucleus with several proteins involved<br />
in different aspects of DNA repair has lead to propose that this kinase is part of the damagesensing<br />
mechanism. However, the mechanistic details underlying the role of Abl in DNA<br />
repair are largely unclear. Here, we will discussed the evidences supporting our current<br />
underst<strong>and</strong>ing of Abl activation following DNA insults while focusing on the relevance of<br />
such mechanisms in protecting DNA injured germ cells. Recent findings have implicated Abl<br />
in a cisplatin-induced signaling pathway eliciting death of immature oocytes 1 . A p53-related<br />
protein, TAp63, is an important immediate downstream effector of this pathway.<br />
Pharmacological inhibition of Abl by Imatinib (STI571) protects the ovarian reserve from the<br />
toxic effect of cisplatin <strong>and</strong> has a long-term effect on fertility. Taken together these<br />
observations indicate that imatinib may act as a fertoprotective drug by counteracting the<br />
effects of chemotherapy on the follicle reserve 2 . Although the mechanistic details involved in<br />
such a process are not fully understood, it remains beyond doubt, that a fine-tuning of nuclear<br />
outcomes by pharmacological inhibition of c-Abl will provide the basis for the development<br />
of effective fertoprotective adjuvants.<br />
References<br />
1) Gonfloni S. et al Nat. Med 2009; 15:1179-1185.<br />
2) Woodruff T. Nat. Med. 2009; 15:1124-1125.<br />
21
Altered expression of Apoptotic Machinery genes explains the decrease of oocytes<br />
competence with aging.<br />
MR Guglielmino 1 , M Vento 2 , M Santonocito 1 , P Borzì 2 , M Ragusa 1 , D Barbagallo 1 , I<br />
Casciano 3 , L Statello 1 , P Scollo 2 , M Romani 3 , M Purrello 1 , C Di Pietro 1 .<br />
1<br />
Dipartimento Gian Filippo Ingrassia, Sezione di Biologia, Genetica, Genomica<br />
Cellulare e Molecolare Giovanni Sichel, Università degli Studi di Catania; E-mail:<br />
dipietro@unict.it<br />
2<br />
Servizio di PMA/Azienda Ospedaliera Cannizzaro, Catania; E-mail:<br />
mrln.vento@gmail.com<br />
3<br />
Tumor Genetics <strong>and</strong> Epigenetics, Istituto Nazionale per la Ricerca sul Cancro - IST<br />
Genova; E-mail: massimo.romani@istge.it<br />
To identify the biomolecular bases of reduced oocyte competence with aging, we compared<br />
the Apoptotic Machinery (AM) transcriptome of pooled MII oocytes from two cohorts of<br />
women, respectively younger than 35 (young) <strong>and</strong> older than 38 years (old). We found that<br />
the expression pro<strong>file</strong> of AM genes is strongly dependent on maternal age. In fact, 36 out of<br />
the 84 AM genes analyzed showed a specific age-dependent expression pro<strong>file</strong>: 16<br />
proapoptotic genes were considerably upregulated <strong>and</strong> 7 antiapoptotic genes were<br />
downregulated in oocytes from older women. Through Real-Time PCR on single MII oocytes<br />
(6 from young <strong>and</strong> 5 from older women), we confirmed that the mRNAs encoding prosurvival<br />
proteins Bcl2 <strong>and</strong> cFLAR are more abundant in oocytes from younger women (p
Towards underst<strong>and</strong>ing of life <strong>and</strong> death regulation at CD95/Fas<br />
Nicolai Fricker(1,3), Carina Pforr(1), Rol<strong>and</strong> Eils(2,3,4), Peter H. Krammer(1),<br />
Inna N. Lavrik(1,3)<br />
1Division of Immunogenetics, German Cancer Research Center (DKFZ), 69120<br />
Heidelberg, Germany<br />
2Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ),<br />
69120 Heidelberg, Germany<br />
3Bioquant, University of Heidelberg, 69120 Heidelberg, Germany<br />
4Department for Bioinformatics <strong>and</strong> Functional Genomics, Institute for Pharmacy <strong>and</strong><br />
Molecular Biotechnology, University of Heidelberg, 69120 Heidelberg, Germany<br />
CD95 (APO-1/Fas) is a member of the death receptor family. CD95 stimulation leads to the<br />
induction of apoptotic as well as non-apoptotic pathways. CD95 signaling starts with the<br />
formation of the death-inducing signaling complex (DISC). The DISC consists of CD95, the<br />
adaptor molecule FADD (Fas-Associated Death Domain), procaspase-8, procaspase-10 <strong>and</strong> c-<br />
FLIPL/S/R (the cellular FLICE-inhibitory proteins). The regulation of life <strong>and</strong> death decisions<br />
at the DISC is largely influenced by the ratio between pro- <strong>and</strong> anti-apoptotic DED (death<br />
effector domain)–containing proteins <strong>and</strong> is poorly understood. We have applied a systems<br />
biology approach to underst<strong>and</strong> the regulation of life/death decisions in CD95 signaling<br />
pathway in quantitative terms. In our experiments we have used HeLa cells overexpressing<br />
various components of the CD95 system. Stimulation of HeLa cells has led to the induction of<br />
both apoptotic <strong>and</strong> non-apoptotic pathways depending on the concentration of the CD95<br />
DISC components. We have quantitatively analyzed apoptotic <strong>and</strong> non-apoptotic responses<br />
applying single-cell <strong>and</strong> population-based measurements. Based on these experimental data a<br />
mathematical model of the CD95 DISC has been constructed. The model takes into account<br />
the complex interplay between various DED-containing proteins of the DISC, e.g.<br />
procaspase-8 <strong>and</strong> c-FLIP isoforms as well as their cleavage products. The application of<br />
systems biology allowed to identify new mechanisms of regulation of CD95 signaling on the<br />
quantitative level <strong>and</strong> has demonstrated how exact concentrations of c-FLIP <strong>and</strong> procaspase-8<br />
<strong>and</strong> their cleavage products at the DISC can determine life/death decisions.<br />
23
Computational Systems Biology in Cancer Research<br />
Ilya Shmulevich<br />
Shmulevich Group, Institute for Systems Biology, 1441 North 34th Street , Seattle 98103,<br />
USA<br />
Cancer is a complex disease of the genes caused by genetic aberrations that disrupt the normal<br />
functioning of molecular networks in cells. I will discuss our efforts within The Cancer<br />
Genome Atlas (TCGA) project to carry out integrated analyses of TCGA-derived highthroughput<br />
experimental data sets in order to: (a) identify mechanisms of genomictranscriptomic<br />
regulation associated with specific cancers <strong>and</strong> clinical parameters; (b) infer<br />
networks that explain cancer type-specific transcriptional pro<strong>file</strong>s, <strong>and</strong> identify molecules that<br />
may be important control nodes in these networks as a means to prioritize drug targets for<br />
therapeutic intervention; <strong>and</strong> (c) compare cancer-associated features across multiple cancer<br />
types within TCGA, to gain insights into the regulatory basis for cancer progression.<br />
24
Systems Analysis of Endocytosis by Quantitative Image Analysis: Insights into<br />
Pathogenetic Mechanisms<br />
Varadharajan Sundaramurthy, Claudio Collinet, Martin Stöter, Charles Bradshaw,<br />
Nikolay Samusik, Jochen C. Rink, Bianca Habermann, Eugenio Fava, Yannis<br />
Kalaidzidis <strong>and</strong> Marino Zerial<br />
Max Planck Institute of Molecular Cell Biology <strong>and</strong> Genetics MPI-CBG, Dresden,<br />
Germany<br />
Endocytosis is an essential process serving multiple key cellular functions, such as nutrient<br />
uptake, signal transduction, <strong>and</strong> defence against pathogens. We have undertaken a broad<br />
systems biology analysis of endocytosis. We systematically pro<strong>file</strong>d the activity of human<br />
genes with respect to Transferrin <strong>and</strong> EGF endocytosis by performing an image-based RNAi<br />
screening of HeLa cells in cooperation with the HT-TDS, the screening facility of the MPI-<br />
CBG. The genes were identified on the basis of a multi-parametric analysis quantitatively<br />
measuring uptake <strong>and</strong> intracellular cargo distribution. We uncovered novel regulators of<br />
endocytosis <strong>and</strong> endosome trafficking, including many signalling pathways (e.g. Wnt,<br />
Integrin, TGF-!, <strong>and</strong> Notch). A systems analysis by Bayesian networks further uncovered<br />
design principles regulating the number, size, concentration of cargo <strong>and</strong> intracellular position<br />
of endosomes. Mathematical modelling revealed reverse engineering principles governing the<br />
organization <strong>and</strong> transport properties of the endocytic pathway. These results have profound<br />
implications for our underst<strong>and</strong>ing of the mechanisms regulating organelle biogenesis <strong>and</strong><br />
signalling at the cellular, tissue <strong>and</strong> organism level. They further provide insights into the<br />
pathogenetic mechanisms underlying a spectrum of human diseases (e.g. infectious diseases,<br />
metabolic disorders, cancer <strong>and</strong> metastasis, neurodegeneration).<br />
25
Using single cell analysis tools to search for human breast cancer stem cells<br />
Anne Grosse-Wilde, Rolf E Kuestner, <strong>and</strong> Adrian Ozinsky<br />
Institute for Systems Biology, Seattle, WA, 98103 USA<br />
Studies suggest that chemotherapy resistance <strong>and</strong> tumor metastatic potential are dependent on<br />
a small sub-population of cancer stem cells. These cells seem to mimic many of the<br />
characteristics of embryonic or induced pluripotent stem cells, <strong>and</strong> are presumed to be<br />
regulated by stem cell-specific transcription factors. We have been examining whether these<br />
genes actually are co-expressed within individual cancer cells. We have focussed on<br />
measuring gene expression during cell lineage transitions between epithelial <strong>and</strong><br />
mesenchymal cells that are induced by in vitro assays that enrich for aggressive <strong>and</strong><br />
metastatic cell phenotypes.<br />
Grant acknowledgments: NIH (NIGMS P50GM076547) <strong>and</strong> the Luxembourg Centre for<br />
Systems Biomedicine <strong>and</strong> the University of Luxembourg<br />
26
Whole genome scans in drosophila to model human disease<br />
Josef Penninger<br />
IMBA<br />
A plethora of genes have been correlated with human diseases. Genetic animal models have<br />
proven to be extremely valuable to elucidate the essential functions of genes in normal<br />
physiology <strong>and</strong> the pathogenesis of disease. During the last years, my laboratory have done<br />
large scale Drosophila screens to map pain perception (thermal avoidance, Neely et al. Cell<br />
2010), stress induced heart failure (Neely et al. Cell, 2010), obesity (Pospisilik et al. Cell<br />
2010), <strong>and</strong> bacterial infections <strong>and</strong> gut immunity (Cronin et al. Science 2009). The idea is to<br />
mine the genome of one organism using systems genetics <strong>and</strong> then link <strong>and</strong> compare our<br />
results to large scale genome association studies in human disease populations. Based on such<br />
comparisons, we then choose new genes/pathways to generate knock-out mice for validation<br />
in mammals.<br />
27
Valproic Acid perturbs hematopoietic homeostasis by inhibition of erythroid<br />
differentiation <strong>and</strong> activation of the myelo-monocytic pathway<br />
Sébastien Chateauvieux 1,3 , Serge Eifes 1,3 , Franck Morceau 1 , Christina Grigorakaki 1 ,<br />
Michael Schnekenburger 1 , Estelle Henry 1 , Mario Dicato 2 , Marc Diederich 1 .<br />
1 Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, L-<br />
2540 Luxembourg; 2 Luxembourg Medical Center, Luxembourg, L-1210 Luxembourg;<br />
3 These authors contributed equally<br />
As a histone deacetylase inhibitor, Valproic acid (VPA) is a c<strong>and</strong>idate for anticancer therapy.<br />
Besides, VPA exhibits various mechanisms of action <strong>and</strong> its effects on the molecular basis of<br />
hematopoiesis remain unclear. To study the effects of VPA on the hematopoietic system, we<br />
performed microarray analysis using K562 cells treated with 1 mM VPA over a 72 h time<br />
course. The association between Gene Ontology (GO) terms <strong>and</strong> the lists of differentially<br />
expressed genes was tested using the Bioconductor package GOstats. Enrichment analysis for<br />
cellular differentiation pathways was performed based on manually curated gene lists. Results<br />
from microarray analysis were confirmed by studying cell differentiation features at the<br />
molecular <strong>and</strong> cellular levels using other hematopoietic cell lines as well as hematopoietic<br />
stem/progenitor CD34 + cells. Microarray analysis revealed 3440 modulated genes in the<br />
presence of VPA. Genes involved in the granulo-monocytic differentiation pathway were upregulated<br />
while genes of the erythroid pathway were down-regulated. This was confirmed by<br />
analyzing erythrocytic <strong>and</strong> myeloid membrane markers <strong>and</strong> lineage-related gene expression in<br />
HEL, MEG01, HL60 as well as CD34 + cells. Moreover, GATA-1 <strong>and</strong> its co-factors (FOG1,<br />
SP1) were down-regulated, while myelopoiesis activator PU.1 was up-regulated, in agreement<br />
with an inhibition of erythropoiesis. Our functional profiling <strong>and</strong> cell phenotyping approach<br />
demonstrates that VPA is able to alter hematopoietic homeostasis by modifying the cell<br />
population balance in the myeloid compartment. This may lead to a potential failure of<br />
erythropoiesis in patients with cancer or chronic inflammatory diseases having a welldescribed<br />
propensity to anemia.<br />
28
Search for the effects of anticancer drugs: A look to apoptosis but also to autophagy!<br />
Micol Tillhon 1 , Francesca Donà 1 , Michele Parks 1 , Vincenzo Giansanti 1 , Giuliano<br />
Mazzini 1 , Ennio Prosperi 1 , Paolo Lombardi 2 , A. Ivana Scovassi 1<br />
1 Istituto di Genetica Molecolare CNR, Via Abbiategrasso 207, I-27100 Pavia;<br />
2 Naxospharma, Novate Milanese, Italy scovassi@igm.cnr.it<br />
Drug resistance of cancer cells is often correlated to apoptosis evasion; however, an active<br />
involvement of autophagy in this scenario has been recently proposed, based on the evidence<br />
that this process could exert a protective role towards the activation of apoptosis in cancer<br />
cells. We addressed this question by analysing the effects of 2-methoxyestradiol (2-ME),<br />
which inhibits the growth of cancer cells, on cell proliferation <strong>and</strong> cell cycle of HeLa <strong>and</strong><br />
colon carcinoma cell lines, namely HCT116 <strong>and</strong> SW613-B3. We showed a net time- <strong>and</strong><br />
dose-dependent anti-proliferative effect of 2-ME on both cell lines, which is accompanied by<br />
cell cycle arrest leading to the accumulation of cells in G2/M. We also demonstrated that 2-<br />
ME induces caspase-dependent apoptosis characterized by mitochondria impairment. Of note,<br />
we detected autophagic markers in 2-ME-treated cells (Parks et al. 2-Methoxyestradiol: New<br />
perspectives in colon carcinoma treatment. Mol. Cell Endocrinol. 331:119-128 (2011). The<br />
simultaneous detection of apoptosis <strong>and</strong> autophagy prompted us to investigate in the same cell<br />
lines the effect of the naturally-occurring alkaloid berberine <strong>and</strong> of structurally related novel<br />
derivatives. In cells treated with berberine <strong>and</strong> analogues, we detected several apoptotic<br />
hallmarks (nuclear shrinkage, DNA degradation, caspase activation, PARP-1 proteolysis) as<br />
well as the evidence for autophagy markers (LC3 conversion, increased beclin-1 expression).<br />
On the whole, our results could contribute to the underst<strong>and</strong>ing of the role of autophagy in<br />
cancer treatment.<br />
29
Signaling by the Frizzled family of receptors: G protein-coupled mechanisms <strong>and</strong><br />
targets<br />
Vladimir L. Katanaev<br />
Department of Biology, University of Konstanz. Universitätsstrasse 10, Box 643.<br />
Konstanz 78457, Germany. E-mail vladimir.katanaev@uni-konstanz.de<br />
The Frizzled family of unconventional GPCRs transmits the signals from the developmentally<br />
<strong>and</strong> medically important group of Wnt-type growth factors. Despite the GPCR topology of<br />
Frizzled proteins, their functional belonging to the G protein-coupled type of receptors has<br />
been long questioned. In the recent years, the involvement of heterotrimeric G proteins in<br />
Frizzled signaling has been established in a number of model systems, as was the biochemical<br />
activity of Frizzled proteins as GPCRs. Frizzled proteins from flies to humans have been<br />
shown to couple to the Go/i type of heterotrimeric G proteins. A number of interesting targets<br />
of G proteins in Frizzled signaling, including the scaffolding protein Axin <strong>and</strong> the small<br />
GTPase Rab5 have been identified. These advances shed light on the mechanisms of this<br />
intricate signal transduction cascade <strong>and</strong> identify new approaches for the anti-cancer drug<br />
development.<br />
Recent papers:<br />
1. Purvanov V, Koval A, Katanaev VL. (2010) A direct <strong>and</strong> functional interaction between<br />
the trimeric G protein Go <strong>and</strong> Rab5 in G protein-coupled receptor signaling. Sci. Signal. 3:<br />
ra65.<br />
2. Koval A, Katanaev VL. (2010) Wnt3a Stimulation Elicits G Protein-Coupled Receptor<br />
Properties of Mammalian Frizzled Proteins. Biochem. J. DOI 10.1042/BJ20101878<br />
3. Egger-Adam D, Katanaev VL. (2010) The trimeric G protein Go inflicts a double impact<br />
on Axin in the Wnt/Frizzled signaling pathway. Dev. Dyn. 239:168–83.<br />
4. Kopein D, Katanaev VL. (2009) Drosophila GoLoco-protein Pins is a target of G!omediated<br />
G protein-coupled receptor signaling. Mol. Biol. Cell 20:3865-77.<br />
30
<strong>Redox</strong> regulation of transcription factors by electrophilic lipid mediators<br />
Young-Joon Surh<br />
WCU Department of Biopharmaceutical Sciences <strong>and</strong> Molecular Medicine,<br />
College of Pharmacy, Seoul National University, Seoul 151-742, South Korea<br />
The implication of inflammatory cell/tissue damage in carcinogenesis has been under intense<br />
investigation both at the research level <strong>and</strong> in clinical practice. Numerous studies have been<br />
reported with the global biochemical profiling technologies, such as DNA microarray,<br />
proteomics, metabolomics, lipidomics, etc., to identify <strong>and</strong> characterize a series of critical<br />
molecules/changes in the inflammatory signaling. It is by gaining this type of mechanistic<br />
underst<strong>and</strong>ing of a disease that researchers will unlock the keys to discovering new<br />
diagnostics as well as preventive <strong>and</strong> therapeutic strategies for the management of<br />
inflammation-associated cancer. Cyclopentenone prostagl<strong>and</strong>ins of J series as products of<br />
cyclooxygenase-2 (COX-2)-mediated arachidonic acid cascades have been reported to possess<br />
multifaceted cellular functions, including anti-inflammatory <strong>and</strong> cytoprotective effects. A<br />
typical example is 15-deoxy-! 12,14 -prostagl<strong>and</strong>in J2 (15d-PGJ2) that modulates the activities<br />
of various intracellular signaling molecules. Because of the electrophilic ",#-unsaturated<br />
carbonyl moiety present in its cyclopentenone ring structure, 15d-PGJ2 acts as a Michael<br />
addition acceptor <strong>and</strong> can readily interact with critical cellular nucleophiles, such as cysteine<br />
thiol groups of proteins. Many of the biological effects induced by 15d-PGJ2 are mediated by<br />
targeting redox-sensitive transcription factors <strong>and</strong> their regulators, including I$B kinase-NF-<br />
$B, AP-1, Nrf2-Keap1, HIF-1!, STAT3 <strong>and</strong> p53 tumor suppressor. We have reported that<br />
15d-PGJ2 treatment rescues PC12 cells from peroxynitrite- <strong>and</strong> hydrogen peroxide-induced<br />
apoptosis by upregulating the Nrf2-driven expression of glutamate cysteine ligase or heme<br />
oxygenase-1. Elevated production of 15d-PGJ2 has also been implicated in tumorigenesis<br />
through induction of angiogenesis <strong>and</strong> metastasis. Cysteine thiols present in various<br />
transcription factors <strong>and</strong> their regulators function as redox sensors in fine-tuning of<br />
transcriptional regulation of many genes essential for maintaining cellular homeostasis. Thus,<br />
oxidation or covalent modification of thiol groups present in some redox-sensitive<br />
transcription factors <strong>and</strong> their regulating molecules can provide a unique strategy for<br />
molecular target-based chemoprevention <strong>and</strong> cytoprotection.<br />
31
Novel mechanisms of transcriptional control in differentiation <strong>and</strong> cancer<br />
Luciano Di Croce<br />
Epigenetic events in Cancer, CRG/ICREA, Dr. Aiguader 88, Barcelona<br />
Underst<strong>and</strong>ing the genetic basis of cancers has been a topic of intense research, <strong>and</strong> hundreds<br />
of gene mutations have been identified that can cause carcenogenesis. However, in the past<br />
few years, increasing evidence has suggested that mutations are not the only genetic changes<br />
that lead to cancer. Indeed, perturbations of chromatin structure <strong>and</strong> of other epigenetic<br />
mechanisms can cause inappropriate gene expression <strong>and</strong> genomic instability, resulting in<br />
cellular transformation <strong>and</strong> malignant outgrowth.<br />
I will discuss the role of several protein complexes that are involved in chromatin dynamics<br />
<strong>and</strong> metabolism, which when altered could participate in the establishment <strong>and</strong> maintenance<br />
of the aberrant silencing of tumor suppressor genes during transformation. Our data suggested<br />
that the ZRF1, Polycomb group of proteins (PcG) <strong>and</strong> the histone variant macroH2A are -<br />
with different timing <strong>and</strong> kinetics - involved in setting up an altered chromatin structure with<br />
aberrant gene silencing in cancer cells. Furthermore, the role of ubiquitylated H2A, <strong>and</strong> its<br />
localization in the human genome will also be discussed.<br />
32
A dual role of JAK1: regulation of IFN-! dependent <strong>and</strong> independent IL-12p70<br />
production<br />
Andreas H. Wagner $ , Michael Conzelmann* # , Sascha Dietrich* # , Claudia Schäfer,<br />
Oliver Krämer, Michael Hess* # , Annika Zota*, Christine S. Falk ! , Markus Hecker $ , <strong>and</strong><br />
Thomas Luft* #<br />
*From the German Cancer Research Center, Dept. of Molecular Oncology/Hematology,<br />
Heidelberg, # Dept. of Medicine V, University of Heidelberg, $ Institute of Physiology <strong>and</strong><br />
Pathophysiology, University of Heidelberg, <strong>and</strong> ! National Center for Tumor diseases<br />
<strong>and</strong> Institute of Immunology, University of Heidelberg, Germany.<br />
Inhibition of JAK1 is an emerging clinical concept resulting in spleen size reduction due to<br />
cytokine inhibition in human myelofibrosis. Therefore, JAK1 inhibition might become a<br />
promising concept for a variety of autoimmune diseases, but similarly raises concerns<br />
regarding immunosuppressive side effects.<br />
At the example of IL-12p70 production we demonstrate that JAK1 has a dual role in<br />
differentially regulating the effects of weak <strong>and</strong> strong activation stimuli.<br />
Weak NF-"B-activating stimuli such as CD40L or LPS require complementary JAK1inducing<br />
cytokines such as IFN-! to induce IL-12p70. This pathway involves RELA, cREL,<br />
JAK1 <strong>and</strong>/or JAK2, STAT1, IRF1 <strong>and</strong> IRF8 <strong>and</strong> is inhibited by RELB <strong>and</strong> TYK2.<br />
Here we show evidence that an alternative pathway exists depending on strong NF-"B<br />
induction (e.g. via intact E.coli or LPS plus IL-1#) that is inhibited by JAK1, STAT3 <strong>and</strong><br />
RELB. STAT3 directly binds to a combined STAT/NF-"B site at the IL-12p35 promoter<br />
without altering access of RELA <strong>and</strong> cREL. Furthermore, a direct role of JAK1/STAT3 is<br />
demonstrated for RELB expression using siRNA. This pathway is independent of IFN-!,<br />
STAT1, IRF1 <strong>and</strong> IRF8.<br />
Our study suggests that in vivo blockade of JAK1 specifically inhibits pro-inflammatory<br />
effects of weak, IFN-! dependent, NF-"B activation stimuli (such as CD40L or LPS) whilst<br />
preserving or enhancing IL-12p70 induced by strong, IFN-! independent activation stimuli.<br />
33
Learning immunology from successful vaccines: innate immunity to systems<br />
Vaccinology<br />
Bali Pulendran<br />
Emory Vaccine Center, Emory University, Atlanta, GA 30322, USA.<br />
bpulend@emory.edu<br />
Despite their great success, we underst<strong>and</strong> little about how effective vaccines stimulate<br />
protective immune responses. Two recent developments promise to yield such underst<strong>and</strong>ing:<br />
the appreciation of the crucial role of the innate immune system in sensing microorganisms<br />
<strong>and</strong> tuning immune responses, <strong>and</strong> advances in systems biology. In this presentation, I will<br />
discuss how these developments are yielding insights into the mechanism of some of the most<br />
successful vaccines ever developed. Furthermore, such developments promise to address a<br />
major challenge in vaccinology: that the efficacy of a vaccine can only be ascertained<br />
retrospectively, upon infection. The identification of molecular signatures induced rapidly<br />
after vaccination, which correlate with <strong>and</strong> predict the later development of protective<br />
immune responses, would represent a strategy to prospectively determine vaccine efficacy.<br />
Such a strategy would be particularly useful when evaluating the efficacy or immunogenicity<br />
of untested vaccines, or in identifying individuals with sub-optimal responses amongst high<br />
risk populations, such as infants or the elderly. We have recently used a systems biology<br />
approach to identify early gene signatures that correlate with, <strong>and</strong> predict the later immune<br />
responses in humans vaccinated with the live attenuated yellow fever vaccine YF-17D, or<br />
with the influenza vaccines. I will review these studies, <strong>and</strong> discuss their broader implications<br />
for vaccinology.<br />
34
Immunity <strong>and</strong> death in chronic viral infections<br />
Marie-Lise Gougeon<br />
Antiviral immunity, Biotherapy <strong>and</strong> Vaccine Unit,<br />
Institut Pasteur, 28 rue du Dr. Roux, 75015 Paris, France<br />
Persistent viruses are able to subvert innate host antiviral strategies. For example, HIV-1 has<br />
evolved ways to exploit dendritic cells (DCs), thereby facilitating viral dissemination.<br />
Dendritic cells (DCs) are the first targets for HIV upon primary mucosal infection. The fate of<br />
DCs is extremely dependent on the interaction with autologous natural killer (NK) cells <strong>and</strong><br />
once infected, they should be rapidly eliminated by these innate effectors. NK-DC interaction<br />
is bidirectional <strong>and</strong> may lead to DC maturation <strong>and</strong> priming for Th1 responses, or may<br />
enhance NK-cell cytotoxicity through IL-12 release. NK-mediated killing of infected DCs is<br />
believed to be an essential step for early control of viral replication <strong>and</strong> dissemination. We<br />
discovered that, once infected with HIV, DCs become resistant to NK-dependent lysis.<br />
Apoptosis resistance of infected DCs is dependent upon a crosstalk with NK cells that leads to<br />
the dramatic upregulation of two key inhibitors of apoptosis, cIAP-2 <strong>and</strong> c-FLIP, resulting in<br />
a lack of response to TRAIL released by NK cells. During this crosstalk, NK cells trigger HIV<br />
replication in DCs, thus contributing to the constitution of reservoirs in infected DCs.<br />
The molecules involved in DC resistance to NK-dependent apoptosis were identified <strong>and</strong> the<br />
essential role of HMGB1, an alarmin expressed at NK-DC synapse, was discovered. Indeed,<br />
HMGB1 is responsible for the upregulation of the two apoptosis inhibitors c-FLIP <strong>and</strong> c-IAP2<br />
in infected DCs, <strong>and</strong> blocking HMGB1 activity by glycyrrhizin or specific antibodies restored<br />
the susceptibility of HIV-infected DC to NK cell killing, <strong>and</strong> abrogated HIV replication in<br />
DCs. The pivotal role of HMGB1 was confirmed by the ability of exogenous rhHMGB1 both<br />
to upregulate the two apoptosis inhibitors <strong>and</strong> to trigger HIV-1 replication in infected DCs<br />
Overall, these observations provide new insights into how HIV hijacks DCs to promote viral<br />
dissemination <strong>and</strong> uses the NK-DC interaction to maintain viability of long-term reservoirs.<br />
In addition, they challenge the question of the in vivo involvement of HMGB1 in the<br />
establishment of viral persistence <strong>and</strong> identify potential therapeutic targets to eliminate viral<br />
reservoirs.<br />
35
Gimap5: a critical mediator of lymphocyte homeostasis <strong>and</strong> determinant of autoinflammatory<br />
disease.<br />
Halil Aksoylar, Michael J Barnes, <strong>and</strong> Kasper Hoebe<br />
Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio, USA<br />
Homeostatic control of the immune system involves mechanisms that ensure the selftolerance,<br />
survival <strong>and</strong> quiescence of hematopoietic-derived cells. Previous reports linked<br />
genetic aberrancies in the GTPase the immunity-associated protein 5 (Gimap5) to autoimmune<br />
disorders in both humans <strong>and</strong> rats, suggesting that Gimap5 is an important mediator<br />
of immune homeostasis. Our laboratory recently identified a recessive ENU-induced germline<br />
mutation that destabilizes Gimap5. Homozygote mutant mice develop lymphopenia, hepatic<br />
extramedullary hematopoiesis, weight loss <strong>and</strong> intestinal inflammation. Although Gimap5deficient<br />
CD4 + T <strong>and</strong> B cells undergo normal development, they fail to proliferate upon<br />
antigen-receptor stimulation even though NF-!B, MAP kinase <strong>and</strong> Akt activation occur<br />
normally. Activation of CD4 + T cells results in normal cytokine secretion predominantly<br />
polarized towards TH17. In addition mutant mice contain reduced numbers of peripheral Treg<br />
cells that also exhibit a reduced suppressive capacity compared to wildtype Treg cells.<br />
Together, these data establish Gimap5 as a key regulator of hematopoietic integrity <strong>and</strong><br />
lymphocyte homeostasis.<br />
36
Study of the anti-apoptotic role of SHIP-1 in T cells<br />
Claude Condé 1 , Xavier Rambout 2 , Franck Dequiedt 2 , Geoffrey Gloire 1 <strong>and</strong> Jacques<br />
Piette 1<br />
1 Virology <strong>and</strong> immunology unit, GIGA-research (+2), University of Liège<br />
1, avenue de l’hôpital Bât B34 4000 Liège – claude.conde@ulg.ac.be<br />
2 Cellular <strong>and</strong> Molecular Biology Unit, Gembloux Agro Bio-Tech, University of Liège<br />
13, Avenue Maréchal Juin Bât 92 5030 Gembloux<br />
Background :<br />
SHIP-1 is an inositol 5’-phosphatase, principally expressed in hematopoietic cells. It acts by<br />
dephosphorylating phosphatidylinostol-3-phosphate thereby down-modulating PI3K pathway<br />
<strong>and</strong> cellular proliferation. From studies with SHIP-1 KO mice, it becomes apparent that<br />
SHIP-1 plays a dual role: in one h<strong>and</strong>, it has been demonstrated that SHIP-1 has a<br />
proapoptotic activity in myeloid <strong>and</strong> B cells, this properties is directly linked to its catalytic<br />
activity. In the other h<strong>and</strong>, SHIP-1 plays an anti-apoptotic role in T cells that is mediated by it<br />
adaptator function. For a better underst<strong>and</strong>ing of the anti-apoptotic activity of SHIP-1 in T<br />
cells, we searched for new protein interaction partners of SHIP-1.<br />
Methods:<br />
To identify new protein interaction partners of SHIP-1, we carried out a yeast two hybrid<br />
screening using different constructs of SHIP-1 as a bait <strong>and</strong> the hORFeome v5.1 pooled in<br />
preys. The advantage of this method is that the preys are not restricted to a specific tissue or a<br />
specific cDNA library.<br />
Results :<br />
Among the partner of SHIP-1, we identified two members of the IAP family : cIAP-1 <strong>and</strong><br />
XIAP. These proteins are well known for their anti-apoptotic activity but also for playing an<br />
important role in many cellular pathways. Our goal is first to demonstrate the interaction<br />
between SHIP-1 <strong>and</strong> XIAP or cIAP-1 in vivo, <strong>and</strong> then to characterise the role of this<br />
interaction.<br />
37
Protein population shift leading to disease-related changes in cellular networks<br />
Antonio del Sol 1 , Wiktor Jurkowski 1 , Kirsten Roomp 1 , Hiroaki Kitano 2<br />
1<br />
Luxembourg Center for Systems Biomedicine (LCSB), University of Luxembourg,<br />
Luxembourg<br />
2<br />
The Systems Biology Institute, Shinjuku-ku, Tokyo, Japan<br />
Proteins exist as dynamic ensembles of different structural <strong>and</strong> dynamic states including<br />
functionally relevant conformers as the most populated states, as well as malfunctioning<br />
conformers as less populated states. Despite a large number of cellular <strong>and</strong> environmental<br />
changes that proteins experience under normal physiological conditions, robustness of protein<br />
structures guarantees performance of their functional activities. Yet, specific perturbations<br />
such as mutations or binding to metabolites are capable of shifting a protein population<br />
towards a malfunctioning state. These protein population shifts may lead to distinct<br />
perturbations of cellular networks, <strong>and</strong> thus the population of cells shifts towards disease<br />
states as their internal networks are perturbed. Here we study prion disease as an illustrative<br />
example of protein population shift leading to disease-related network perturbations.<br />
Conversion of benign forms of prion protein (PrPC) to disease-specific isoforms (PrPSc),<br />
yields to the activation of bi-stable circuits enabling the transition of a steady stable healthy<br />
state of a cellular network towards a disease-related steady stable state. In addition we carry<br />
out a structural analysis of the effect of disease-causing mutations on proteins associated to<br />
different diseases. Furthermore, in each case we analyze the impact of the particular protein<br />
malfunction on the relevant cellular pathways that are associated with the disease outcome.<br />
Thus, this study shows that a molecular analysis of cellular network components can provide<br />
important insights into disease-related network perturbations, with potentially important<br />
consequences for molecular therapeutic strategies.<br />
38
Proteomic phenotyping of the cell surface information gateway<br />
Bernd Wollscheid<br />
Institute of Molecular Systems Biology (IMSB), NCCR Neuro Center for Proteomics,<br />
ETH Hönggerberg, HPT D77, Wolfgang Pauli-Str. 16, CH-8093 Zürich, Switzerl<strong>and</strong><br />
The classification of cell types has relied on the identification of cell surface proteins as<br />
differentiation markers. Currently, flow cytometry allows for the detection of up to a dozen<br />
differentiation markers in a single measurement. We have developed the cell surface<br />
capturing (CSC) technology for the multiplexed mass-spectrometric identification of several<br />
hundred N-glycosites specifically from cell surface exposed glycoproteins, which can be used<br />
to phenotype cells without antibodies in an unbiased fashion <strong>and</strong> without a priori knowledge.<br />
We show that the CSC technology allows for the parallel detection <strong>and</strong> relative quantitative<br />
comparison of the cell surface N-glycoproteome in various cell types, as well as for the<br />
monitoring of emerging <strong>and</strong> declining ES cell surface N-glycoprotein markers during their<br />
controlled differentiation towards the neuronal lineage. CSC technology enables a snapshot<br />
view of the cell surface N-glycoprotein l<strong>and</strong>scape <strong>and</strong> can detect panels of N-glycoproteins as<br />
potential differentiation markers that are currently not accessible by other means.<br />
39
De-SUMOylation: a new strategy used by Listeria to counteract the host defense<br />
David Ribet 1,2,3 <strong>and</strong> Pascale Cossart 1,2,3<br />
1 Institut Pasteur, Unité des Interactions Bactéries-Cellules, F-75015 Paris, France.<br />
2 Inserm, U604, F-75015 Paris, France.<br />
3 INRA, USC2020, F-75015 Paris, France.<br />
The effects of pathogenic bacteria on SUMOylation, a fundamental post-translational<br />
modification in eukaryotic cells, remain largely unknown. We have examined SUMOylation<br />
during infection by the intracellular bacterial pathogen Listeria monocytogenes. Our data<br />
reveal that Listeria monocytogenes infection leads to a decrease in the levels of host SUMOconjugated<br />
proteins. This event is triggered by the bacterial virulence factor listeriolysin O<br />
(LLO) which induces a degradation of Ubc9, an essential enzyme of the SUMOylation<br />
machinery, <strong>and</strong> a degradation of some SUMOylated proteins. The effect of LLO on Ubc9 is<br />
dependent on the pore-forming capacity of the toxin <strong>and</strong> is shared by other bacterial poreforming<br />
toxins like perfringolysin O (PFO) <strong>and</strong> pneumolysin (PLY). Ubc9 degradation was<br />
also observed in vivo in infected mice. Furthermore, we showed that SUMO overexpression<br />
impairs bacterial infection. Together, our results reveal that Listeria, <strong>and</strong> probably other<br />
pathogens, dampen the host response to infection by preventing SUMOylation of key<br />
regulatory proteins [1].<br />
1. Ribet, D., Hamon, M., Gouin, E., Nahori, M.-A., Impens, F., Neyret-Kahn, H.,<br />
Gevaert, K., V<strong>and</strong>ekerckhove, J., Dejean, A. <strong>and</strong> Cossart, P. Listeria monocytogenes impairs<br />
SUMOylation for efficient infection. Nature 2010; 464 :1192-5.<br />
40
Traveling to the Ends of the Proteome World. Positional N-terminal <strong>and</strong> C-terminal<br />
proteomics deciphers protein terminal <strong>and</strong> proteolytic post-translational modifications<br />
in complex proteomes in vivo.<br />
Christopher M. Overall.<br />
UBC Centre for Blood Research, University of British Columbia, 2350 Health Sciences<br />
Mall, Vancouver, B.C. V6T 1Z3 Canada. chris.overall@ubc.ca http://www.clip.ubc.ca.<br />
Specific degradomics techniques are needed to rapidly identify <strong>and</strong> quantify the N- <strong>and</strong> Cterminomes<br />
in order to reveal the extent of post-translational modifications of protein termini<br />
<strong>and</strong> therefore the functional state of key molecules, the extent of proteolysis in a system, <strong>and</strong><br />
to identify new protease substrates. Using positional proteomics we find that ~50% of<br />
proteins in skin are present as stable cleavage products <strong>and</strong> in E Coli ~40% of proteins have a<br />
truncated C-terminus. Broad coverage N-terminome analysis necessitates a negative selection<br />
procedure as the variety of original mature protein N-terminal blocked peptides each present<br />
individual chemical hurdles for their enrichment by positive selection strategies. We<br />
developed two combined terminomics <strong>and</strong> protease substrate discovery degradomics<br />
platforms for the simultaneous quantitative analysis of the N-terminome <strong>and</strong> proteolysis on a<br />
proteome-wide scale. A specific C-terminal analysis procedure was recently reported<br />
(Schilling et al 2010 Nature Methods 7, 508-511) as well as a N terminal procedure termed<br />
Terminal Amino Isotopic Labelling of Substrates (TAILS, Kleifeld et al Nature<br />
Biotechnology 28, 281-288; Prudova et al 2010 Mol Cell Proteomics; auf dem Keller et al<br />
2010 Mol Cell Proteomics). By using a novel polymer to deplete the internal tryptic peptides,<br />
TAILS suffers little from sample loss <strong>and</strong> low yields, so requiring only 100 microgram of<br />
sample <strong>and</strong> one MS/MS analysis per sample. By a two-day procedure with flexible labelling<br />
options, TAILS can be adapted to a variety of experimental situations including cell culture<br />
<strong>and</strong> complex biological sample analysis. Incorporating iTRAQ labelling iTRAQ-TAILS also<br />
provides wide coverage of all forms of naturally blocked N-terminal peptides <strong>and</strong> allows for<br />
their quantification through labelling of lysine side-chains in up to 8 samples. TAILS permits<br />
exploitation of the acetylated <strong>and</strong> other blocked mature protein N-terminal peptides as a<br />
statistical classifier that is then used to set isotope ratio cut offs that reveal protease activity.<br />
Being a quantitative procedure, TAILS can analyse the substrate degradome of a broad<br />
specificity protease or one with no known specificity without manual data parsing, in the<br />
same experiment, <strong>and</strong> also do this in vivo. We have applied TAILS to a variety of<br />
metalloproteases <strong>and</strong> compared protease knock out mice, analysed inflamed skin, breast<br />
cancer <strong>and</strong> arthritic tissues. Typical analyses identify over 3000 N-terminal peptides from<br />
which we found that the removal of the N-terminal methionine is dependent upon the amino<br />
acid at position 2 with distinct preferences found for valine, glycine, alanine <strong>and</strong> serine. In<br />
one experiment, acetylation occurred on 731 original mature protein N-terminal peptides but<br />
at the initiator methionine in only 153 of these instances. In 578 cases, acetylation was at<br />
position 2 in the protein after removal of 1 Met, with alanine, serine <strong>and</strong> methionine being the<br />
preferred acetylated residues. Finally N-terminal positional proteomics enables MS sample<br />
simplification with proteins identified in bronchoalvelar fluid having abundances spanning a<br />
range greater than six orders of magnitude.<br />
41
Identification of the Human Testis Protein Phosphatase 1 Interactome<br />
Margarida Fardilha 1 , Sara L.C. Esteves 1 , Luís Korrodi-Gregório 1 , Ana Paula Vintém 1 ,<br />
Sara C.T.S. Domingues 2 , S<strong>and</strong>ra Rebelo 2 , Nick Morrice 3 , Patricia T. W. Cohen 3 , Odete<br />
A.B. da Cruz e Silva 2 <strong>and</strong> Edgar F. da Cruz e Silva 1†<br />
1 Signal Transduction Laboratory, Centre for Cell Biology, Biology Department,<br />
University of Aveiro; <strong>and</strong> 2 Neuroscience Laboratory, Centre for Cell Biology, Health<br />
Sciences Department, University of Aveiro; <strong>and</strong> 3 Medical Research Council Protein<br />
Phosphorylation Unit, School of Life Sciences, University of Dundee; † deceased on the<br />
2 nd March 2010<br />
Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end,<br />
PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in<br />
turn, depend on complexes it forms with PP1 Interacting Proteins – PIPs. The importance of<br />
the testis/sperm-enriched variant, PP1!2, in sperm motility <strong>and</strong> spermatogenesis has<br />
previously been shown. Given the key role of PIPs, it is imperative to identify the<br />
physiologically relevant PIPs in testis <strong>and</strong> sperm. Hence, we performed Yeast Two-Hybrid<br />
screens of a human testis cDNA library using as baits the different PP1 isoforms <strong>and</strong> also a<br />
proteomic approach aimed at identifying PP1!2 binding proteins. To the best of our<br />
knowledge this is the largest data set of the human testis PP1 interactome. We report the<br />
identification of 77 proteins in human testis <strong>and</strong> 7 proteins in human sperm that bind PP1. The<br />
data obtained increased the known PP1 interactome by reporting 72 novel interactions.<br />
Confirmation of the interaction of PP1 with 5 different proteins was also further validated by<br />
co-immunoprecipitation or protein overlays. The data here presented provides important<br />
insights towards the function of these proteins <strong>and</strong> opens new possibilities for future research.<br />
In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological<br />
intervention.<br />
Related publications:<br />
!" Fardilha M, Esteves SL, Korrodi LMG, da Cruz e Silva OA <strong>and</strong> da Cruz e Silva<br />
FF (2010) The physiological relevance of Protein Phosphatase 1 <strong>and</strong> its interacting<br />
proteins to health <strong>and</strong> disease. Current Medicinal Chemistry Oct 13. [Epub ahead of<br />
print]#<br />
2. Gareth J. Browne*, Margarida Fardilha*, Senga Oxenham*, Wenjuan Wu, Nick<br />
Helps, Odete A. B. da Cruz e Silva, Patricia T.W. Cohen <strong>and</strong> Edgar F. da Cruz e Silva<br />
(2007). SARP, a new alternatively spliced protein phosphatase 1 <strong>and</strong> DNA interacting<br />
protein. Biochemical Journal 402, 187-196. *, These authors contributed equally to this<br />
work.<br />
3. Wu W, Baxter JE, Wattam SL, Hayward DG, Fardilha M, Knebel A, Ford EM,<br />
da Cruz E Silva E, Fry AM (2007). Alternative splicing controls nuclear translocation<br />
of the cell cycle regulated nek2 kinase. J Biol Chem. Sep 7;282(36):26431-26440 (Jul 11;<br />
Epub ahead of print).<br />
42
Site-Specific Identification of SUMO-2 Conjugation Sites in Cells Reveals Novel<br />
SUMOylation Motifs<br />
Joost Schimmel 1 , Ivo A. Hendriks 1 , Ivan Matic 2 , Matthias Mann 2 <strong>and</strong> Alfred C.O.<br />
Vertegaal 1<br />
1 Molecular Cell Biology, Leiden University Medical Center, Leiden, 2300 RC, the<br />
Netherl<strong>and</strong>s. 2 Max-Planck Institute for Biochemistry, Martinsried, D-82152, Germany.<br />
Email: vertegaal@lumc.nl<br />
Post-translational modification by SUMO is essential for eukaryotic viability. Mass<br />
spectrometry-based proteomics projects have uncovered hundreds of potential SUMOylated<br />
proteins. However, direct identification by mass spectrometry of the SUMOylated lysines in<br />
endogenous target proteins is still very challenging. We have developed a novel method for<br />
selective enrichment of SUMOylated peptides from complex cellular proteomes. We have<br />
used this method to map 103 SUMO modification sites in endogenous target proteins purified<br />
from cell lysates. Our method is generic <strong>and</strong> can be adapted to study other ubiquitin-like<br />
proteins. Our results provide the following novel insights into protein SUMOylation:<br />
1. We have discovered an inverted SUMOylation consensus site E/DxKpsi.<br />
2. Several alternative amino acids were identified that precede the SUMOylated lysines in<br />
KxE type SUMOylation sites.<br />
3. We have identified SUMO attachment sites in sixteen proteins on lysines that were<br />
preceded by hydrophobic clusters of at least three hydrophobic residues <strong>and</strong> named this novel<br />
type of SUMOylation site the Hydrophobic Cluster SUMOylation Motif (HCSM). Follow up<br />
studies established the remarkable stoichiometric SUMOylation of target proteins via this<br />
HCSM.<br />
4. Virtually all identified SUMOylation sites that fit the classical SUMOylation consensus site<br />
psiKxE/D contain a glutamic acid instead of an aspartic acid.<br />
5. We have uncovered crosstalk between SUMOylation <strong>and</strong> phosphorylation with a preferred<br />
spacer of four amino acids between the SUMOylated lysine <strong>and</strong> the phosphorylated serine.<br />
6. SUMO-acceptor lysines in four proteins were previously identified as acetylated lysines.<br />
Since SUMOylation <strong>and</strong> acetylation are mutually exclusive, this indicates competition<br />
between acetylation <strong>and</strong> SUMOylation for the same lysines.<br />
43
Regulation of Lynch syndrome-related DNA mismatch repair heterodimer MutLalpha<br />
by phosphorylation?<br />
Angela Brieger, S<strong>and</strong>ra Passmann, Stefan Zeuzem <strong>and</strong> Jörg Trojan<br />
Medical Clinic I, Biomedical Research Laboratory, Goethe-University, Frankfurt a.M.,<br />
Germany<br />
Lynch syndrome, a hereditary disease associated with many different tumor types, is caused<br />
by mutations in DNA mismatch repair (MMR)-genes. 50% of these mutations are detected in<br />
the MLH1 protein. The heterodimer MutLalpha, which is formed by MLH1 <strong>and</strong> PMS2,<br />
coordinates a series of key events in the MMR mechanism. In addition to MMR, MutLalpha<br />
is involved in many other cellular processes as the regulation of cell cycle checkpoints <strong>and</strong><br />
apoptosis most likely signaling DNA damage to downstream pathways. A sophisticated<br />
regulation of the different MutLalpha functions might be important as well as reasonable. In<br />
order to focus on MutLalpha modulation, we analyzed MLH1 <strong>and</strong> PMS2 for phosphorylation<br />
sites using different computational systems <strong>and</strong> detected multiple potential loci <strong>and</strong><br />
corresponding kinases for both genes. Using phospho-specific purification columns we were<br />
able to isolate unphosphorylated <strong>and</strong> phosphorylated MLH1. In addition to this, Westernblot<br />
analysis of 2-D gels resolved MLH1 into several different distinct forms detected in lysates<br />
from HEK293 cells. Furthermore, we detected phosphorylation of purified MLH1 by<br />
PKCdelta, one of the computer-supported most probable MutLalpha kinases. Ongoing<br />
projects are directed towards the underst<strong>and</strong>ing how phosphorylation might be capable to<br />
switch MutLalpha function between MMR <strong>and</strong> DNA damage signaling.<br />
44
Mechanisms of Epigenetics in Health <strong>and</strong> Disease<br />
François Fuks<br />
Laboratory of Cancer Epigenetics, Free University of Brussels (ULB), 808 route de<br />
Lennik, 1070 Brussels, Belgium.<br />
!"#$%&'()*+',-.$/*+)0$+.$,%/-1'+.'$1-*&$,.$2+.2&1$+.3$,0$+00-2,+'&3$4,'($5&.&$0,*&.2,.5$<br />
-6$ '7%-71$ 07//1&00-1$ 5&.&08$ 9(&$ %&'()*+',-.$ -6$ :/;$ 0,'&0$ ,0$ &0'+
Molecular characterization of HDAC inhibitor-induced death in a mouse model of acute<br />
myeloid leukemia<br />
Michael Bots 1 , Inge Verbrugge 1 , Ben Martin 1 , Jessica Salmon 1 , Kym Stanley 1 , Johannes<br />
Zuber 2 , Scott Lowe 2 <strong>and</strong> Ricky Johnstone 1<br />
1 Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC,<br />
Australia; 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, United States<br />
Underst<strong>and</strong>ing the molecular pathways targeted by chemotherapeutic drugs is essential<br />
information to decide on treatment modalities for patients with malignant disease. Histone<br />
deacetylase inhibitors (HDACi) are an interesting <strong>and</strong> encouraging group of anti-cancer<br />
drugs. However, the specific mechanism behind their anti-cancer effect remains largely<br />
unknown <strong>and</strong> may depend on the specific type of tumor <strong>and</strong>/or the oncogenic lesions that<br />
drive tumorigenesis.<br />
We have examined the therapeutic effect of HDACi in a mouse model of acute myeloid<br />
leukemia (AML). This model is based on the expression of both mutant Nras (co-expressed<br />
with luciferase) <strong>and</strong> the fusion protein AML1/ETO9a (co-expressed with GFP), in<br />
hematopoietic stem cells to drive tumorigenesis. Recruitment of HDAC-containing repressor<br />
complexes by this fusion protein is important for the onset <strong>and</strong> progression of AML1/ETOexpressing<br />
AML. This provides a strong rationale for using HDACi in the treatment of<br />
AML1/ETO9a-driven AML.<br />
Upon transplantation of primary leukemias into recipient mice, we found that treatment with<br />
the HDACi, panobinostat, indeed results in a good response, with clearance of leukemic blasts<br />
from the periphery. Importantly, this in vivo response translates into a significant overall<br />
survival benefit. In contrast to conventional chemotherapy, mice bearing p53 -/- leukemias still<br />
display survival benefit from treatment with panobinostat. To further characterize the<br />
molecular pathways utilized by panobinostat, we generated AML1/ETO9a leukemias with<br />
defined genetic alterations in the intrinsic as well as extrinsic apoptotic pathways. Similar to<br />
non-manipulated leukemias, we found that loss of TRAIL or DR5 <strong>and</strong> over-expression of Bcl-<br />
2 does not change the response to panobinostat. All these leukemias still displayed an overall<br />
survival benefit. This data suggest that induction of apoptosis is not required for therapeutic<br />
efficacy of panobinostat. Further mechanistic studies using additional genetically manipulated<br />
AML1/ETO9a tumors are currently ongoing.<br />
Our studies provide an important insight into the mechanism of HDACi-induced anti-tumor<br />
responses <strong>and</strong> demonstrate that HDACi are a rational choice for the treatment of human AML<br />
driven by expression of AML1/ETO.<br />
46
MicroRNAs <strong>and</strong> erythroid differentiation<br />
Roberto Gambari<br />
BioPharmaNet/ThalLab, Department of Biochemistry <strong>and</strong> Molecular Biology, Ferrara<br />
University, via Fossato di Mortara 74, 44121, Ferrara, Italy; e-mail:gam@unife.it.<br />
MicroRNAs (miRNAs, miRs) are a family of small noncoding RNAs that regulate gene<br />
expression by sequence-selective targeting of mRNAs, leading to translational repression or<br />
mRNA degradation. Considering that a single miRNA can target several mRNAs <strong>and</strong> the<br />
3’UTR sequence of a single mRNA might contain several signals for miRNA recognition, at<br />
least 10-40% of human mRNAs are potential targets of microRNAs, leading to control of<br />
highly regulated biological functions. In our laboratory we have analyzed by microarray the<br />
miR-pro<strong>file</strong> in erythroid precursor cells (ErPC) from normal <strong>and</strong> thalassemic patients<br />
expressing different levels of foetal haemoglobin (including patients exhibiting a HPFH<br />
phenotype). The microarray data were confirmed by RT-PCR analysis, <strong>and</strong> allowed us to<br />
identify miR-210 as highly expressed in the erythroid precursor cells from HPFH patients.<br />
When RT-PCR was performed on mithramycin (MTH)-induced K562 cells <strong>and</strong> erythroid<br />
precursor cells, we demonstrated that miR-210 is induced in time-dependent <strong>and</strong> dosedependent<br />
fashion, together with induction of !-globin genes. Molecular biology studies<br />
allowed to identify raptor mRNA <strong>and</strong> mTORC1 as putative miR-210 targets. As far as<br />
modulation of miR-210, peptide nucleic acids (PNAs) might be of interest. We have studied a<br />
PNA conjugated to a polyarginine peptide which (a) is efficiently internalized within target<br />
cells; (b) strongly inhibits miR-210 activity; (c) deeply alters the expression of raptor <strong>and</strong> !globin<br />
genes.<br />
Supported by Telethon (GGP10124), Fondazione CARIPARO <strong>and</strong> MIUR-PRIN-2007.<br />
47
MATHEMATICAL MODELLING OF CELLULAR DECISIONS BETWEEN LIFE<br />
AND DEATH IN CANCER<br />
Andrei Zinovyev 1 , Laurence Calzone 1 , Laurent Tournier 1 , Simon Fourquet 1 , Emmanuel<br />
Barillot 1 , Denis Thieffry 2 , Boris Zhivotovsky 3<br />
1 Institut Curie, INSERM U900, Paris, France<br />
2 Ecole Normale Superieur, Paris, France<br />
3 Karolinska Institutet, Stockholm, Sweden<br />
E-mail: <strong>and</strong>rei.zinovyev@curie.fr<br />
Compromised balance between cellular decisions towards survival <strong>and</strong> various modalities of<br />
programmed cell death is responsible for uncontrolled cell proliferation in tumors. In normal<br />
cells this balance is tightly controlled by a complex systems of molecular switches that<br />
irreversibly trigger a particular cellular fate. The complexity of this mechanism <strong>and</strong> the level<br />
of crosstalk between survival <strong>and</strong> cell death pathways are such that systems approach is<br />
needed to decipher its main features <strong>and</strong> predict possible intervention strategies to restore the<br />
violated balance.<br />
In order to formalize the existing knowledge in the field <strong>and</strong> to make it accessible for<br />
computer analysis, we reconstructed a comprehensive map of molecular interactions of the<br />
cell death machinery. For the moment this map contains 1587 chemical species involved in<br />
1133 reactions. This formal description is annotated by 563 relevant publications in the field.<br />
Formal analysis of the map together with high-throughput data allows to make general<br />
conclusions about involvement of particular molecular players in concrete cancers.<br />
In order to better underst<strong>and</strong> the main features of cell fate decision machinery, we have<br />
constructed a mathematical model of cell fate decision in response to triggering death<br />
receptors, based on logical (discrete) formalism. The model provides a generic high-level<br />
view of the interplays between NF!B pro-survival pathway, RIP1-dependent necrosis <strong>and</strong> the<br />
apoptosis pathway <strong>and</strong> constitutes a valuable reasoning tool that can suggest experiments for<br />
novel biological insights.<br />
48
A Systems Approach to Modeling <strong>and</strong> Predicting Oncogene Addiction<br />
Dean W Felsher<br />
Division of Oncology, Departments of Medicine <strong>and</strong> Pathology, Stanford, Medicine-<br />
Oncology, Stanford, CA, 94305<br />
The targeted inactivation of oncogenes can elicit sustained tumor regression, associated with<br />
the phenomenon that has been described as oncogene addiction. Utilizing conditional<br />
transgenic mouse models, we have gleaned some insight into the mechanisms of oncogene<br />
addiction illustrating that upon oncogene inactivation tumors undergo proliferative arrest,<br />
apoptosis, differentiation <strong>and</strong>/or senescence. The specific consequences of oncogene<br />
addiction appear to depend upon both cellular <strong>and</strong> genetic context <strong>and</strong> both tumor cell<br />
intrinsic <strong>and</strong> host-dependent mechanisms appear to be critical. Recently, we have utilized a<br />
conditional mouse model of MYC induced T-acute lymphoblastic lymphoma to dissect the<br />
role of proliferative <strong>and</strong> death signaling in tumor regression associated with MYC<br />
inactivation. By multi-scale modeling, we were able to show that characteristic changes in<br />
survival <strong>and</strong> death signaling can be used to predict oncogene addiction. We were able to<br />
directly test this model by interrogating the consequences of the mutation of specific key<br />
regulators of cellular signaling <strong>and</strong> demonstrating that using our model they had a predictable<br />
outcome on tumor regression. Finally, we were able to use our model to predict clinical<br />
outcome to erlotinib therapy in patients with lung cancer. Our results illustrate that using a<br />
systems approach we are able to derive models that may be useful to predict oncogene<br />
addition <strong>and</strong> thereby the clinical response to targeted oncogene inactivation.<br />
49
Microenvironmental Independence In Tumor Progression: An Integrated Approach<br />
Alex<strong>and</strong>er R. A. Anderson<br />
Integrated Mathematical Oncology (IMO), H. Lee Moffitt Cancer Center <strong>and</strong> Research<br />
Institute,Tampa, FL 33612.<br />
Mathematical <strong>and</strong> computational models are an ideal compliment to experimentation as they<br />
allow for the integration of multiple experimental results across a range of spatial <strong>and</strong><br />
temporal scales. In application to cancer they hold great promise for prediction of tumor<br />
outcomes; however their application has been limited by the lack of experimental data<br />
integration. Even though mathematical modeling of cancer is not new, in fact it goes back<br />
over half a century, it has largely been ignored - mostly due to insufficient communication<br />
between the mathematical <strong>and</strong> biological communities. These facts highlight the need for a<br />
new integrated approach to underst<strong>and</strong>ing cancer <strong>and</strong> biological systems in general. In this<br />
approach the dialogue between the modeler <strong>and</strong> the experimentalist drives the definition <strong>and</strong><br />
direction of both the model <strong>and</strong> the experiments that ultimately leads to fundamental insights<br />
that could not have been achieved independently. In this talk we discuss our recent efforts<br />
using mathematical modeling, computation <strong>and</strong> experimentation to study cancer progression,<br />
with a special emphasis on the role of the tumor microenvironment. Specifically, to<br />
underst<strong>and</strong> the competitive dynamics of tumor cells in diverse microenvironments, we<br />
experimentally parameterized a hybrid discrete-continuum mathematical model with<br />
phenotypic trait data from a set of related mammary cell lines with normal, transformed, or<br />
tumorigenic properties. Surprisingly, in a resource-rich microenvironment, with few<br />
limitations on proliferation or migration, transformed but not tumorigenic cells were most<br />
successful <strong>and</strong> outcompeted other cell types in heterogeneous tumor simulations. Conversely,<br />
constrained microenvironments with limitations on space <strong>and</strong>/or growth factors gave a<br />
selective advantage to phenotypes derived from tumorigenic cell lines. Analysis of the<br />
relative performance of each phenotype in constrained versus unconstrained<br />
microenvironments revealed that, although all cell types grew more slowly in resourceconstrained<br />
microenvironments, the most aggressive cells were least affected by<br />
microenvironmental constraints. Our results suggest that a critical feature of the process of<br />
tumor progression is selection of cells that can escape from resource limitations by achieving<br />
a relative microenvironmental independence.<br />
50
Targeting the invasive phenotype using systemic evolution of lig<strong>and</strong>s by exponential<br />
enrichment creates anti-metastatic aptamers.<br />
Greg Shelley, Xiaohua Zhang, Jinlu Dai, Chunyan Yu, Elisabeth A. Pedersen, Yusuke<br />
Shiozawa, June Escara-Wilke, Jill Keller, Jian Zhang, Russell S. Taichman, Evan T.<br />
Keller.<br />
University of Michigan, USA<br />
Multiple methods (e.g., small molecules <strong>and</strong> antibodies) have been designed to target specific<br />
proteins <strong>and</strong> signaling pathways in cancer. However, many mediators of the cancer<br />
phenotype are unknown <strong>and</strong> the ability to target these phenotypes would help mitigate cancer.<br />
Aptamers are small DNA or RNA molecules that are designed for therapeutic use. Design of<br />
aptamers to target cancers can be challenging. Accordingly, we used a modification of<br />
systemic evolution of lig<strong>and</strong>s by exponential enrichment (SELEX) to target a known<br />
phenotype of cancer metastasis, i.e. invasion. We call this method pheno-SELEX. A highly<br />
invasive prostate cancer (PCa) cell line was established <strong>and</strong> used to identify aptamers that<br />
bound to it with high affinity as opposed to a less invasive variant to the cell line. The antiinvasive<br />
aptamer (AIA1) was found to inhibit in vitro invasion of the original PCa cell line, as<br />
well as an additional PCa cell line <strong>and</strong> an osteosarcoma cell line. AIA also inhibited in vivo<br />
development of metastasis in both a PCa <strong>and</strong> osteosarcoma model of metastasis. These<br />
results indicate that pheno-SELEX can be successfully used to identify aptamers without<br />
knowledge of underlying molecular targets. This study establishes a new paradigm for<br />
identification of functional aptamers.<br />
51
Autophagy: suicidal self-cannibalism or homeostatic recycling?<br />
Guido Kroemer<br />
INSERM, U848, Villejuif, France; Metabolomics Platform, Institut Gustave Roussy, Villejuif,<br />
France; Centre de Recherche des Cordeliers, Paris, France; Pôle de Biologie, Hôpital Européen<br />
Georges Pompidou, AP-HP, Paris, France; Université Paris Descartes, Paris 5, Paris, France.<br />
Autophagy has been considered for some time as a mechanism of cellular self-destruction leading to<br />
cell death. We have tackled the question whether autophagy is cytoprotective or cytotoxic in two<br />
ways.<br />
First we have screened more than 1000 established or experimental anticancer agents for their capacity<br />
to induce autophagic LC3 puncta in human cancer cells, finding that some 100 were able to do so, but<br />
that none among these “autophagy inducers” killed tumor cells in an autophagy-dependent fashion.<br />
Rather autophagy inhibition sensitized the tumor cells to cell death induction.<br />
Second, we determined the impact of whole-body autophagy induction in experimental animals. We<br />
found that autophagy-inducing pharmacological agents such as resveratrol <strong>and</strong> spermidine enhanced<br />
life span in an autophagy-dependent fashion. Similarly genetic manipulations that increase longevity<br />
(such as inactivation of the C. elegans p53 ortholog Cep1 or overexpression of the SIRT1 ortholog<br />
sirt2.1) induce autophagy, <strong>and</strong> inhibition of autophagy abolishes their lifespan-extending effect. We<br />
found that induction of autophagy by resveratrol requires the NAD + -dependent deacetylase sirtuin 1<br />
(SIRT1). The acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human<br />
<strong>and</strong> yeast cells as well as in nematodes. Although resveratrol <strong>and</strong> spermidine ignite autophagy through<br />
distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant<br />
modifications of the acetylproteome. Both agents favor convergent deacetylation <strong>and</strong> acetylation<br />
reactions in the cytosol <strong>and</strong> in the nucleus, respectively. At doses at which neither resveratrol nor<br />
spermidine did stimulate autophagy alone, these agents synergistically induced autophagy, both in<br />
vitro <strong>and</strong> in vivo. Altogether, these data underscore the importance of an autophagy-regulatory<br />
network of antagonistic deacetylases <strong>and</strong> acetylases that can be pharmacologically manipulated.<br />
Moreover, our data indicate that autophagy is mostly (always?) a cytoprotective event.<br />
References:<br />
Madeo F, Tavernarakis N, Kroemer G. Can autophagy promote longevity? Nat Cell Biol 2010 Sep;12(9):842-6<br />
Criollo A, Senovilla L, Kepp O, Authier H, Shen S, Maiuri MC, Tasdemir E, Tallier M, Morselli E, Galluzzi L, Delahaye N,<br />
Tesniere A, Commo F, Harper F, Vicencio JM, Ben Younes A, Pierron G, Lav<strong>and</strong>ero S, Zitvogel L, Israel A, Baud V,<br />
Kroemer G. The IKK complex contributes to the induction of autophagy. EMBO J. 2010 Feb 3;29(3):619-31<br />
Kroemer G, Marino G, Levine B. Autophagy <strong>and</strong> the integrated stress response. Mol Cell. 2010 Oct 22;40(2):280-93<br />
Morselli E, Maiuri MC, Markai M, Megalou E, Pasparaki A, Palikaras K, Galluzzi L, Criollo A, Malik SA, Madeo F,<br />
Tavernarakis N, Kroemer G. Caloric restriction <strong>and</strong> resveratrol prolong longevity via the sirtuin-1 dependent induction of<br />
autophagy. Cell Death Disease 1, e10<br />
Morselli E, Marino G, Bennetzen M, Eisenberg T, Megalou E, Schroeder S, Carbrera S, Bénit P, Rustin P, Criollo A, Shen S,<br />
Kepp O, Miauri C, Horio Y, Lopez-Otin C, Andersen JS, Tavernarakis N, Madeo F, Kroemer G. Spermidine <strong>and</strong> resveratrol<br />
induce autophagy by distinct yet convergent pathways affecting the acetylproteome. J Cell Biol in press<br />
Eisenberg T, Knauer H, Schauer A, Büttner S, Ruckenstuhl C, Carmona-Gutierrez D, Ring J, Schroeder S, Magnes C,<br />
Antonacci L, Fussi H, Deszcz L, Hartl R, Schraml E, Criollo A, Megalou E, Weiskopf D, Laun P, Heeren G, Breitenbach M,<br />
Grubeck-Loebenstein B, Herker E, Fahrenkrog B, Fröhlich KU, Sinner F, Tavernarakis N, Minois N, Kroemer G*, Madeo F.<br />
(*Co-corresponding author). Induction of autophagy by spermidine promotes longevity. Nat Cell Biol. 2009<br />
Nov;11(11):1305-14<br />
Tavernarakis N, Pasparaki A, Tasdemir E, Maiuri MC, Kroemer G. The effects of p53 on whole organism longevity are<br />
mediated by autophagy. Autophagy. 2008 Oct 1;4(7):870-3.<br />
Tasdemir E, Maiuri MC, Galluzzi L, Vitale I, Djavaheri-Mergny M, D'Amelio M, Criollo A, Morselli E, Zhu C, Harper F,<br />
Nannmark U, Samara C, Pinton P, Vicencio JM, Carnuccio R, Moll UM, Madeo F, Paterlini-Brechot P, Rizzuto R,<br />
Szabadkai G, Pierron G, Blomgren K, Tavernarakis N, Codogno P, Cecconi F, Kroemer G. Regulation of autophagy by<br />
cytoplasmic p53. Nat Cell Biol. 2008 Jun;10(6):676-87<br />
Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008 Jan11; 132(1): 27-42<br />
Maiuri MC, Le Toumelin G, Criollo A, Rain JC, Gautier F, Juin P, Tasdemir E, Pierron G, Troulinaki K, Tavernarakis N,<br />
Hickman JA, Geneste O, Kroemer G. Functional <strong>and</strong> physical interaction between Bcl-X(L) <strong>and</strong> a BH3-like domain in<br />
Beclin-1. EMBO J. 2007 May 16;26(10):2527-39.<br />
52
Genes related to energy metabolism differentiate the visceral adipose-derived stem cells<br />
from the subcutaneous ones<br />
Yong Sang Song 1, 2, 3 , Boram Lee 1 , Seung Pyo Gong 4 , Aekyung Park 5 , Hee Seung Kim 2 ,<br />
Hyun Hoon Chung 2 , Sangchul Lee 6 , Woong-Yang Park 5 , Jeong Mook Lim 3, 7 , Noh Hyun<br />
Park 2 , Eunsik Lee 6<br />
1 Cancer Research Institute, College of Medicine, Seoul National University, Seoul 110-<br />
799, Republic of Korea (Song YS: yssong@snu.ac.kr; Lee B: kjkjmi@snu.ac.kr)<br />
2 Department of Obstetrics <strong>and</strong> Gynecology, Seoul National University College of<br />
Medicine, Seoul 110-744, Republic of Korea (Song YS: yssong@snu.ac.kr; Park NH:<br />
pnhkhr@snu.ac.kr; Chung HH; chhkmj@gmail.com; Kim HS; bboddi0311@snu.ac.kr)<br />
3 Major in Biomodulation, World Class University, Seoul National University, Seoul 151-<br />
921, Republic of Korea (Song YS: yssong@snu.ac.kr; Lim JM: limjm@snu.ac.kr)<br />
4 Department of Marine Bio-materials & Aquaculture, College of Fisheries Science,<br />
Pukyong National University, Busan 608-737, Republic of Korea (Gong SP:<br />
kongpyo2@snu.ac.kr)<br />
5 Department of Biomedical Sciences, Seoul National University College of Medicine,<br />
Seoul 110-744, Republic of Korea (Park WY: wypark@snu.ac.kr; Park A:<br />
parkak11@snu.ac.kr)<br />
6 Department of Urology, Seoul National University College of Medicine, Seoul 110-744,<br />
Republic of Korea (Lee E: eslee@snu.ac.kr; Lee S: uromedi@naver.com)<br />
7 Department of Agricultural Biotechnology, Research Institute for Agriculture <strong>and</strong> Life<br />
Science, Seoul National University, Seoul 151-742, Republic of Korea (Lim JM:<br />
limjm@snu.ac.kr)<br />
Central obesity is more strongly implicated in the pathogenesis of metabolic syndrome than<br />
peripheral obesity. The obesity, central or peripheral, has been presumed to be determined<br />
genetically. We investigated the expression pro<strong>file</strong>s of adipose-derived stem cells (ASC) from<br />
human subcutaneous (SC) <strong>and</strong> visceral (VIS) adipose tissues. First, we confirmed the<br />
characteristics of adipose derived stem cells (ASC) of isolated cells from SC <strong>and</strong> VIS fat<br />
depots by FACS using CD31, CD45, CD34, CD73 <strong>and</strong> CD105 antibody, <strong>and</strong> the<br />
differentiation of ASC into adipocyte, osteocyte <strong>and</strong> chondrocyte. Differential expression<br />
pattern was shown between S-ASC <strong>and</strong> V-ASC in more than 500 of 23700 genes. Genes<br />
related to lipid synthesis <strong>and</strong> oxidative phosphorylation were significantly up-regulated in V-<br />
ASC, while genes associated with insulin signaling were up-regulated in S-ASC. The<br />
expression patterns of adipocytes from subcutaneous <strong>and</strong> visceral fat tissue were similar. In<br />
inbred mice, the immunohistochemistry using antibody against each depot specific antigen<br />
showed the same pattern as shown in human samples. These data suggest that S-ASC may be<br />
different from V-ASC <strong>and</strong> this difference may explain the different risk of metabolic<br />
syndrome between central <strong>and</strong> peripheral obesity.<br />
53
A bioinformatic approach to illuminate why a biomarker, p27, has prognostic value in<br />
human tumors.<br />
Jeffrey P. Miller, Doruk Keskin, Andrew S. Goldstein, David Cobrinik, <strong>and</strong> Andrew<br />
Koff.<br />
Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.<br />
Bioinformatics hold the promise to revolutionize discovery in basic science by constructing<br />
robust molecular networks. Thus, we decided to use a bioinformatic approach to address a<br />
problem originally raised by studies on cell transformation carried out by Hahn <strong>and</strong> his<br />
colleagues. Those investigators showed that SV40 small t-antigen (ST) or activated alleles of<br />
myc, akt, rac or ral could collaborate with SV40 large T-antigen (LT) <strong>and</strong> activated ras v12 to<br />
promote transformation in a variety of immortalized human cells. However, precisely what<br />
the relationship between these four signaling pathways was is unclear.<br />
Thus, we examined the connectivity of those oncogenes in silico. We found that the cdk<br />
inhibitor, p27, was connected either directly or through a single intermediary to every<br />
oncogene that would substitute for ST. p27 plays a role regulating the choice between<br />
proliferation <strong>and</strong> exit from the cell cycle, <strong>and</strong> a low p27 staining index is associated with poor<br />
outcome in a wide assortment of intermediate grade human cancers. Consistent with this, the<br />
loss of p27 will also accelerate tumor progression in a variety of mouse models regardless of<br />
the tissue of origin or the initiating oncogenic stimulus.<br />
Given that the oncogenes that replace ST were all linked to p27, <strong>and</strong> p27 loss cooperates with<br />
the expression of LT to accelerate tumor progression in mice, we tested the genetic<br />
relationship between ST <strong>and</strong> p27 as predicted by the bioinformatic model. We determined that<br />
p27 loss could substitute for the expression of ST during transformation of both rodent <strong>and</strong><br />
human cells. ST expression reduces p27 accumulation by accelerating phosphorylationdependent<br />
p27 turnover. ST mutants that lack the ability to inhibit PP2A activity do not<br />
cooperate with LT, nor was ST able to cooperate with LT to transform phosphorylationresistant<br />
S10A <strong>and</strong> T187A mutant cells to anchorage-independent growth. Thus, in silico<br />
pathway analysis developed a network that suggested that p27 levels were a direct reflection<br />
of the activation state of many oncogenic signaling pathways, which may account for its<br />
broad utility in human cancer prognosis.<br />
54
Pathological aspects of vascular aging <strong>and</strong> strategies for new biomimetic <strong>and</strong> biological<br />
active NonWoTecc biomaterials<br />
Stefanie Kaempf , Christoph Classen, Andreas Henseler, Frank Willems<br />
NonWoTecc Medical GmbH, Nattermannallee 1, Cologne 50829, Germany,<br />
s.kaempf@nonwotecc.de, c.classen@nonwotecc.de, a.henseler@nonwotecc.de,<br />
f.willens@nonwotecc.de, Tel: 0049 (0)221 992247-0, Fax: 0049 (0)221 992247-29<br />
The rising aging of the population <strong>and</strong> the typical lifestyle in the industrial nations are reasons<br />
for the increasing number of diabetes, arteriosclerosis <strong>and</strong> cardiovascular diseases. Hence<br />
there is a high medical need for new cardiovascular drugs <strong>and</strong> biomaterials in combination<br />
with stem/progenitor cell repair like endothelial progenitor cells (EPCs). Current vascular<br />
materials provide tolerable biocompatibility <strong>and</strong> average patency, except for small diameter<br />
coronary vessels. Especially for arteriosclerotic patients this situation often is a dilemma since<br />
there is no suitable autologous arteries or veins available for transplantation. In contrast to<br />
current bypasses the NonWoTecc graft is based on an entirely new biomimetic technology<br />
that mimics biomechanical properties <strong>and</strong> biological structures of arterial wall layers <strong>and</strong><br />
allows a long-term patency by autologous endothelial seeding via EPCs, adventitia sourced<br />
progenitor cells <strong>and</strong> cellular vascular wall reconstruction especially in aged patients. The EPC<br />
cell number is slightly reduced in individual aging patients but will sometimes increase<br />
because of disease stimuli like arteriosclerosis. Despite the age dependent changes of EPCs in<br />
older patients these cells will retain regenerative potential for vascular repair.<br />
As a result of the new cellular based autologous stem/progenitor cell concept, a significant<br />
reduced thrombosis <strong>and</strong> hyperplasia there is a need for new anti-inflammatory drugs for the<br />
NonWoTecc-technology which enable the endothel regeneration. COX-1 <strong>and</strong> COX-2<br />
inhibition by Aspirin is a good concept to inhibit thrombosis as well as neointimal<br />
hyperplasia, whereas Clopidogrel prevents thrombosis via P2Y12-receptor inhibition.<br />
The in vivo results in animals <strong>and</strong> clinial results in the first 30 humans after 10 months<br />
demonstrate the biomimetic concept <strong>and</strong> outst<strong>and</strong>ing biocompatibility of the of the<br />
NonWoTecc grafts by a reconstruction of a functional arterial wall scaffold with<br />
Neoendothel, -media <strong>and</strong> –adventitia, promising a long-term patency of NonWoTecc vascular<br />
grafts <strong>and</strong> an improvement of quality of life in aging, vascular diseased patients.<br />
55
Signaling Pathways that Help Neurons Help Themselves<br />
Mark P. Mattson<br />
Laboratory of Neurosciences, National Institute on Aging Intramural Research<br />
Program, Baltimore, MD. USA.<br />
It is important that neurons be stimulated regularly to activate adaptive stress response<br />
signaling pathways involving transcription factors that induce the expression of neurotrophic<br />
factors, protein chaperones <strong>and</strong> antioxidant enzymes. Such stimulation can be accomplished<br />
by exercise (both physical <strong>and</strong> cognitive), dietary energy restriction <strong>and</strong> ingestion of certain<br />
phytochemicals <strong>and</strong> drugs. The results of studies of animal models suggest that dietary<br />
energy restriction <strong>and</strong> exercise can enhance neural plasticity <strong>and</strong> reduce the vulnerability of<br />
the brain to disorders such as Alzheimer’s, Parkinson’s diseases, <strong>and</strong> stroke. Diabetes <strong>and</strong><br />
obesity may compromise the function of adaptive cellular stress response pathways <strong>and</strong><br />
thereby render neurons vulnerable to aging, injury <strong>and</strong> disease. Energy state-sensitive factors<br />
that are proving particularly important in regulating energy balance <strong>and</strong> improving or<br />
preserving cognitive function are brain-derived neurotrophic factor <strong>and</strong> glucagon-like peptide<br />
1. Alternate day calorie restriction, novel insulin-sensitizing <strong>and</strong> neuroprotective agents, <strong>and</strong><br />
drugs that activate adaptive stress response pathways, are examples of approaches for<br />
preserving brain function that show promise in preclinical studies.<br />
56
Control of class III PI3 kinase in autophagy <strong>and</strong> cancers<br />
Marta M. Lipinski, Minsu Kim, Greg Hoffman, Aylwin Ng, John Blenis, Ramnik J.<br />
Xavier <strong>and</strong> Junying Yuan. .<br />
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston,<br />
MA 02115; Center for Computational <strong>and</strong> Integrative Biology, Massachusetts General<br />
Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114<br />
Autophagy, a lysosome-dependent catabolic process involved in the turnover of cellular<br />
components, mediates normal homeostasis during development <strong>and</strong> protects multicellular<br />
eukaryotes from neurodegeneration, cancer <strong>and</strong> other diseases. However, the mechanisms<br />
regulating autophagy under normal nutritional conditions most frequently encountered by<br />
cells in organisms under physiological conditions remain unknown. Our recent studies<br />
highlight an important role of class PI3 kinase in regulating autophagy. First, we show that<br />
phosphorylation of Vps34 by Cdk1 <strong>and</strong> Cdk5 negatively regulate type III PI3 kinase <strong>and</strong><br />
production of PI3P in mitotic cells <strong>and</strong> in neurodegeneration. Second, we show that multiple<br />
growth factors negatively regulate the activity of type III PI3 kinase. In an image-based<br />
genome-wide human siRNA screen, we show that up regulation of autophagy by only 14<br />
(6.4%) of the identified hit genes was accompanied by down regulation of activity of<br />
mTORC1, an essential regulator of starvation induced autophagy. On the other h<strong>and</strong>, we<br />
found that 110 (50.2%) of the hit genes, including a group of growth factors <strong>and</strong> cytokines,<br />
regulate autophagy by targeting the type III PI3 kinase. These cytokines inhibit the type III<br />
PI3 kinase through multiple pathways. Third, we show that ROS may have a general signaling<br />
function in autophagy. We uncovered a large group of genes (117, or 54% of all genes tested)<br />
whose knock-down led to vesicular translocation of LC3-GFP in the absence but not the<br />
presence of anti-oxidant, suggesting that ROS are required for the induction of autophagy.<br />
Finally, using a small molecule inhibitor that promotes the degradation of class III PI3 kinase,<br />
we show that successful targeting this pathway can lead to selective cancer cell death. Our<br />
study suggests that the type III PI3 kinase integrates diverse signals to regulate cellular levels<br />
of autophagy. Thus, autophagy <strong>and</strong> cell proliferation may represent two alternative cell fates<br />
that are regulated in a mutually exclusive manner, implicating autophagy as a common tumor<br />
suppression mechanism downstream of multiple signaling pathways.<br />
57
VITAGENES, METABOLIC STRESS AND HORMESIS IN AGING AND<br />
NEURODEGENERATIVE DISORDERS<br />
Vittorio Calabrese<br />
Chairman <strong>and</strong> Professor of Clinical Biochemistry, Faculty of Medicine, University of<br />
Catania, Italy;<br />
Protein quality control is a critical feature of intracellular homeostasis. In particular, unfolded or<br />
misfolded proteins resulting from environmental stresses or free radicals are rapidly degraded via the<br />
ubiquitin–proteasome pathway 1 . In addition, modulation of endogenous cellular defense mechanisms<br />
via the stress response signaling represents an innovative approach to therapeutic intervention in<br />
diseases causing chronic tissue damage, such as neurodegeneration <strong>and</strong> cancer 1 . Protein thiols play a<br />
key role in redox sensing, <strong>and</strong> regulation of cellular redox state is crucial mediator of multiple<br />
metabolic, signalling <strong>and</strong> transcriptional processes. Under optimal conditions long-term health<br />
protection is accomplished by protein homeostasis, a highly complex network of molecular<br />
interactions that balances protein biosynthesis, folding, translocation, assembly/disassembly, <strong>and</strong><br />
clearance 2,3 . Efficient functioning of maintenance <strong>and</strong> repair processes is crucial for both survival <strong>and</strong><br />
physical quality of life. This is accomplished by a complex network of the so-called longevity<br />
assurance processes, which are composed of several genes termed vitagenes 4-8 . The term vitagenes<br />
refers to a group of genes which are strictly involved in preserving cellular homeostasis during<br />
stressful conditions. The vitagene family is actually composed of the heat shock proteins (Hsp) Hsp32,<br />
Hsp70, the thioredoxin system <strong>and</strong> the sirtuin system 9 . Dietary antioxidants, such as polyphenols <strong>and</strong><br />
L-carnitine/acetyl-L-carnitine, have recently been demonstrated in vitro to be neuroprotective through<br />
the activation of hormetic pathways, including vitagenes 10-12 . Over the past decade there has been a<br />
remarkable increase of interest in hormesis as a result of more significance being given to low dose<br />
effects <strong>and</strong> the use of more powerful study designs which have enabled to identify rational approaches<br />
to detect hormetic biphasic dose responses in the low dose zone. The hormetic dose–response,<br />
challenging long-st<strong>and</strong>ing beliefs about the nature of the dose–response in a lowdose zone, has the<br />
potential to affect significantly the design of pre-clinical studies <strong>and</strong> clinical trials as well as strategies<br />
for optimal patient dosing in the treatment of numerous diseases, including oxidant disorders. Given<br />
the broad cytoprotective properties of the heat shock response there is now strong interest in<br />
discovering <strong>and</strong> developing pharmacological agents capable of inducing stress responses. We have<br />
recently focused our research on the role of acetylcarnitine in the defense mechanisms against cellular<br />
stress <strong>and</strong> neurodegeneration. In addition, with a redox proteomics approach, we identified<br />
mitochondrial oxidatively modified proteins as a function of brain aging, specifically in those brain<br />
regions, such as cortex <strong>and</strong> hippocampus, that are commonly affected by the aging process. In all brain<br />
regions examined, many of the identified proteins were energy-related, such as pyruvate kinase, ATP<br />
synthase, aldolase, creatine kinase, <strong>and</strong> a-enolase. These alterations were associated with increased<br />
expression of Hsps, as well as carnosinase <strong>and</strong> thioredoxin reductase <strong>and</strong> with significant changes in<br />
both cytosolic <strong>and</strong> mitochondrial redox status in all brain regions analyzed. This findings are relevant<br />
to potential pharmacological interventions in healthy medicine strategy, pointing to maximize cellular<br />
stress resistance of the brain thus providing neuroprotection 9-14 , <strong>and</strong> will be extended to other systemic<br />
oxidant disorders such as diabetic nephropathy or cancer.<br />
References:<br />
1 Halliwell B. (2008) Arch Bioch Biophys 476:107; 2 Calabrese V. (2007) Nature Neurosci 8,766;<br />
3 Gutteridge JM (2010) BBRC 393:561; 4 Calabrese V. (2010) J Neurosi Res 88:3498;<br />
5 Calabrese V. (2010) Antiox <strong>Redox</strong> Signal 13:1763; 6 Calabrese V. (2009) Front Biosci. 14:376;<br />
7 Calabrese V. (2009) Antiox <strong>Redox</strong> Signal 11:2717; 8 Calabrese V. (2010) Neurochem Res 35:1880;<br />
9 Calabrese V. (2009) Antiox <strong>Redox</strong> Signal 11:2759; 10 Calabrese EJ (2010) Hum Exp Toxicol. 29:1034;<br />
11 Calabrese EJ (2010) Hum Exp Toxicol. 29:980; 12 Calabrese V. (2010) Neurochem Res 35:2184;<br />
13 Mattson MP. (2010) Neuron 67:900; 14 Min SW. (2010) Neuron 67:953.<br />
58
p75NTR regulates A! deposition by increasing A! production but inhibiting A!<br />
aggregation with its extracellular domain<br />
Xin-Fu Zhou, Xin Wang, Jian-Jun Lu, Qiao-Xin Li, Chang-Yue Gao, Xiao-Hong Liu,<br />
Yin Sun, Miao Yang, Yoon Lim, Genevieve Evin, Jin-Hua Zhong, Hua-Dong Zhou, Yan-<br />
Jiang Wang<br />
Department of Human Physiology <strong>and</strong> Centre for Neuroscience, Flinders University<br />
GPO Box 2100, Adelaide, South Australia<br />
Accumulation of toxic amyloid-beta (A!) in the cerebral cortex <strong>and</strong> hippocampus is a major<br />
pathological feature of Alzheimer’s disease (AD). The neurotrophin receptor, p75NTR, has<br />
been proposed to mediate A!- induced neurotoxicity; however its role in the development of<br />
AD remains to be clarified. The p75NTR/ExonIII-/- mice <strong>and</strong> APPSwe/PS1dE9 mice were<br />
crossed to generate transgenic AD mice with deletion of p75NTR gene. Study of p75NTR<br />
immunoreactivity in the AD model APPSwe/PS1dE9 transgenic mice, indicates its<br />
localization to the basal forebrain neurons <strong>and</strong> degenerative neurites in amyloid plaques of<br />
neocortex, increase with aging, <strong>and</strong> further activation by A! accumulation. Deletion of the<br />
p75NTR gene by crossing APPSwe/PS1dE9 mice with p75NTR knockout mice reduced<br />
soluble A! levels in the brain <strong>and</strong> plasma, but increased the accumulation of insoluble A! <strong>and</strong><br />
A! plaque formation. There was no change in the levels of amyloid precursor protein (APP)<br />
<strong>and</strong> its proteolytic derivatives, or ", ! <strong>and</strong> # secretase activities, or in levels of BACE1,<br />
neprilysin (NEP) <strong>and</strong> insulin degrading enzyme (IDE) proteins. Recombinant extracellular<br />
domain of p75NTR attenuated the oligomerization <strong>and</strong> fibrillation of synthetic A!42 peptide<br />
in vitro, <strong>and</strong> reduced local A! plaques after hippocampus injection in vivo. In addition,<br />
deletion of p75NTR attenuated microgliosis but increased the microhemorrhage pro<strong>file</strong>s in<br />
the brain. The deletion of p75NTR did not significantly change the cognitive function of the<br />
mice up to the age of 9 months. Our data suggest that p75NTR plays a critical role in<br />
regulating A! levels by both increasing A! production <strong>and</strong> attenuating its aggregation <strong>and</strong><br />
they caution that a therapeutic intervention simply reducing p75NTR may exacerbate AD<br />
pathology.<br />
59
A “no-brainer” – mitochondrial <strong>and</strong> neurodegenerative disease in Dictyostelium.<br />
Paul R. Fisher, Lisa M. Francione, Claire Y. Allan, Paige K. Smith, Sanjanie Fern<strong>and</strong>o,<br />
Shawn de Piazza, Jasmine Ilievska, Oana Sanislav, Anita Chavan, Suwei Chen, William<br />
J. Burrage <strong>and</strong> Sarah J. Annesley.<br />
Department of Microbiology, La Trobe University, Melbourne, AUSTRALIA.<br />
P.Fisher@latrobe.edu.au<br />
The major intracellular signalling pathways of eukaryotic cells were already present in the<br />
primeval ancestor of animals, fungi <strong>and</strong> Amoebozoa. These ancient regulatory networks are<br />
deranged in many human diseases, including mitochondrial <strong>and</strong> neurodegenerative diseases.<br />
The social amoebozoan Dictyostelium discoideum is an experimentally tractable<br />
microorganism whose unique life cycle offers diverse phenotypic “readouts” of conserved<br />
signalling pathways. We have created, in Dictyostelium, sublethal genetic disease models for<br />
mitochondrial disorders <strong>and</strong> various neurodegenerative diseases (Batten Disease,<br />
Mucolipidosis, Parkinson’s Disease). In all cases the disease leads to a chronic dysregulation<br />
of intracellular signalling networks, sublethal at the cellular level but causing whole organism<br />
pathology. We have begun to dissect these pathways genetically <strong>and</strong> to study the resulting<br />
disease phenotypes in growth <strong>and</strong> cell cycle progression, photo- <strong>and</strong> thermosensory signal<br />
transduction, multicellular development (including programmed autophagic cell death),<br />
endocytic nutrient uptake <strong>and</strong> susceptibility to the intracellular bacterial pathogen, Legionella<br />
pneumophila. In mitochondrial disease ATP generation is compromised, leading to chronic<br />
hyperactivity of an energy-sensing protein kinase, AMPK (AMP-activated protein kinase) <strong>and</strong><br />
thence to diverse downstream cytopathologies. As well as AMPK-mediated defects,<br />
additional disease pathways are activated in a specific Complex I deficiency caused by<br />
knocking out MidA, a novel Complex I assembly factor. AMPK is not involved at all in<br />
Neuronal Ceroid Lipofuscinosis (Batten Disease). Instead, one of its downstream targets,<br />
TOR Complex I (TORC1) is inhibited via the Rheb GTPase. Rheb also acts on TOR Complex<br />
2 (TORC2) so that lysosomal dysfunction in Batten Disease also misregulates TORC2 targets.<br />
In Mucolipidosis, decreased function of the lysosomal Ca 2+ channel, mucolipin, causes<br />
defects in intracellular Ca 2+ signalling that are superimposed on <strong>and</strong> modify the phenotypic<br />
outcomes of lysosomal dysfunction. Both wild type <strong>and</strong> disease-causing point mutant forms<br />
of the Parkinson’s Disease protein alpha-synuclein are cytotoxic but the pathways are distinct<br />
from those of mitochondrial <strong>and</strong> lysosomal disorders. However a disease-causing truncated<br />
form of the protein is mislocalized in the cell, forms cytoplasmic aggregates that associate<br />
with the mitochondria <strong>and</strong> causes additional phenotypes characteristic of mitochondrial<br />
dysfunction.<br />
60
Workshop presentations<br />
61
Underst<strong>and</strong>ing <strong>and</strong> performing DNA <strong>and</strong> siRNA transfection<br />
Geraldine Guerin-Peyrou<br />
Polyplus Transfection<br />
Transfection is widely used as a tool in research. It consists in introducing nucleic acids into<br />
mammalian cells. If inadequate, transfection can become a limiting step for any project. Thus<br />
it is critical to underst<strong>and</strong> the mechanism of transfection to choose the most appropriate<br />
method. This tutorial will present the various transfection mechanisms as well as currently<br />
available methods <strong>and</strong> reagents for DNA <strong>and</strong> siRNA transfection. We will explain why some<br />
methods are more effective at transporting <strong>and</strong> releasing DNA while others are better for<br />
siRNA. In addition the critical parameters for transfection <strong>and</strong> their optimisation will be<br />
described. Our goal is to give a solid basis in transfection in order to obtain the best results in<br />
the shortest timeframe.<br />
62
Agilent Technologies<br />
New enrichment tools with high flexibility for targeted sequencing of exones <strong>and</strong> cancer related<br />
regions.<br />
Winfried van Eyndhoven<br />
Agilent Technologies, Amstelveen, The Netherl<strong>and</strong>s.<br />
While next-generation sequencing has revolutionized the way genomes are sequenced, this technology<br />
possesses a fundamental weakness—the inability to easily target specific regions of a genome. To<br />
address this, Agilent has developed the SureSelect platform that allows next-generation sequencing<br />
researchers to focus their sequencing efforts on genomic targets of interest, greatly improving nextgeneration<br />
sequencing process efficiencies. The SureSelect platform of products allows nextgeneration<br />
sequencing researchers to increase study sample size <strong>and</strong> reduce overall experimental cost.<br />
During this presentation information will be given about the enrichment technology <strong>and</strong> its<br />
performance for capturing whole exomes, kinases or RNA transcripts.<br />
Opening the archives for state of the art tumor genetic research – FFPE based sample<br />
processing for array-CGH.<br />
Karoly Szuhai<br />
Leiden University Medical Center dept. Molecular Cell Biology, Leiden, The Netherl<strong>and</strong>s.<br />
Molecular genetic studies on rare tumor entities, such as bone tumors, often require the use of<br />
decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which<br />
decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the<br />
possibility of further molecular genetic testing. We set out to establish a robust protocol that would<br />
overcome these intrinsic hurdles for bone tumor research. This presentation will discuss the findings<br />
of these developments.<br />
Developing a kinome sequencing platform to identify key signaling pathways in breast cancer<br />
Ian J. Majewski PhD<br />
The Netherl<strong>and</strong>s Cancer Institute (NKI-AVL), Department of Molecular Carcinogenesis<br />
email: i.majewski@nki.nl<br />
Developing a kinome sequencing platform to identify key signaling pathways in breast cancer Breast<br />
cancer is a heterogeneous disease. Our work focuses on two very different types of breast cancer,<br />
invasive lobular carcinoma (ILC) <strong>and</strong> triple negative breast cancer (TN), which together represent<br />
25% of all breast cancers. New therapies are desperately required for the treatment of these cancers.<br />
Kinases are important mediators of cell signaling that are considered to be good therapeutic targets.<br />
We have developed an exon capture system for high-throughput mutation analysis that will allow us to<br />
identify kinases that are mutated in ILC <strong>and</strong> TN breast cancer. I will discuss our work to validate the<br />
kinome sequencing platform in cell lines models <strong>and</strong> xenografts. We aim to extend this work to survey<br />
several hundred cancers from patients with ILC <strong>and</strong> TN breast cancer.<br />
Speaker:<br />
63
An Introduction to High Content Screening of Intracellular Signaling Assays using<br />
the IN Cell Analyzer 2000<br />
Sarah Payne<br />
Modality Manager IN Cell <strong>and</strong> Cell Factory, GE Healthcare<br />
The IN Cell Analyzer 2000 from GE Healthcare represents a high performance, automated<br />
imaging system which is capable of managing the entire high content analysis workflow, from<br />
automated image acquisition through to data quantification, data mining <strong>and</strong> data<br />
visualisation. Designed with flexibility in mind, the IN Cell Analyzer 2000 is equipped<br />
to h<strong>and</strong>le a diverse range of research requirements, from investigative microscopy to<br />
automated high throughput screening. The scope of biological assays that can be performed<br />
using this system range from those on a sub-cellular <strong>and</strong> cellular level, through to the imaging<br />
of tissue sections <strong>and</strong> small organisms. This modular system may be equipped with<br />
transmitted light options for label-free analysis of cellular responses, plus environmental<br />
control (CO2, temperature control, humidity) <strong>and</strong> liquid h<strong>and</strong>ling for the monitoring of fast<br />
<strong>and</strong> slow kinetic effects within live cells.<br />
This presentation will provide an overview of the features <strong>and</strong> benefits of the IN Cell<br />
Analyzer 2000 for monitoring intracellular signalling events.<br />
64
MAPTrix TM biomimetics from amsbio: Innovation in cell culture for cancer, stem cell<br />
<strong>and</strong> regenerative medicine research<br />
Alex Sim<br />
AMSBIO<br />
The extracellular matrix (ECM) is a natural substrate/scaffold that has been widely used in cell<br />
culture to enhance cellular adhesion. Cellular adhesion to ECM proteins is a fundamental feature<br />
of cell development, maintenance of tissue organization, <strong>and</strong> many pathologicalb conditions. It<br />
is, therefore, crucial to underst<strong>and</strong> the adhesion process in order to study cellular functions or<br />
tissue engineering applications.<br />
Most experiments require cells to proliferate, differentiate <strong>and</strong> migrate on the substrate. Cell<br />
adhesion to ECM lig<strong>and</strong>s is of growing importance in studying invasion, migration <strong>and</strong> wound<br />
healing.<br />
The immobilization of short peptides such as RGD on synthetic or natural materials may<br />
produce biofunctional surfaces that bind adhesion receptors <strong>and</strong> promote cell adhesion,<br />
however, this conventional chemistry approach is time-consuming, inconvenient, <strong>and</strong> lacks<br />
reproducibility.<br />
To overcome these problems, AMSBIO will present MAPTrix TM , a series of “ECM-like”<br />
biomimetic surfactant polymers that exhibit quantitative control over the proliferation <strong>and</strong><br />
migrational properties of various human cells.<br />
MAPTrix TM technology has been used to produce:<br />
- Direct ECM substitutes,<br />
- A basement membrane non-animal protein incorporating peptides that mimic the biological<br />
activity of the large, natural growth factors,<br />
- A user-defined <strong>and</strong> in situ formable hydrogel system called MAPTrix HyGel TM which<br />
generates three-dimensional (3D) extracellular matrix environments for 3D cell culture<br />
applications.<br />
The Mussel Adhesive Protein based matrix (MAPTrix TM ) line of products are coating<br />
reagents with genetically incorporated bioactive peptides. They mimic the extracellular matrix<br />
(ECM) activity of collagen, fibronectin, laminin, <strong>and</strong> vitronectin proteins <strong>and</strong> promote cell<br />
attachment, spreading <strong>and</strong> growth. A MAPTrix TM surface coating offers an animal-free,<br />
chemically defined attachment <strong>and</strong> growth surface that provides a highly controlled environment<br />
for cell culture <strong>and</strong> tissue engineering applications.<br />
The presentation will focus on 3D cell culture without the need for Matrigel <strong>and</strong> similar animalderived<br />
substrates in cell culture experiments. Changing the way we consider cell culture is<br />
crucial to underst<strong>and</strong>ing the adhesion process in order to study cellular functions or tissue<br />
engineering applications. In particular, promotion of attachment <strong>and</strong> spreading of many normal<br />
<strong>and</strong> stem cells including epithelial, endothelial, hepatocyte <strong>and</strong> mesenchymal stem cells will be<br />
covered.<br />
65
j_A5p_11:BJ 17/1/11 11:14 Page 1<br />
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BUSINESS<br />
CENTER<br />
FIERSCHTER-<br />
HAUS<br />
NEIDUERF-<br />
KAPELL<br />
HUESE-<br />
GRËNDCHEN<br />
Neudorf<br />
FRANCISCAINES<br />
MERL-<br />
PARC<br />
PLACE DE<br />
FRANCE<br />
LAANG<br />
HECK<br />
GRUEF<br />
PLAAK<br />
BARTRÉNG-<br />
FLEURI<br />
BARTRÉNG-<br />
MONTEREALE<br />
BARTRÉNG-<br />
GEMENG<br />
CITÉ<br />
AÉROPORT<br />
KALCHES-<br />
BRÜCK<br />
LAANGE-<br />
GRONN<br />
RUMM<br />
CRÉCY<br />
16<br />
CHARLES DE<br />
TORNACO<br />
NEIDUERF-<br />
BRASSERIE<br />
JULES<br />
SALENTINY<br />
HIERZEKRËPP FIXMER NEIDUERF-<br />
KIERCH<br />
MALAKOFF<br />
Centre<br />
17<br />
RHEINSHEIM<br />
BRAGANCE<br />
GUILLAUME<br />
ORVAL<br />
ST HUBERT<br />
OP DER<br />
MILLEN<br />
BARTRÉNG-<br />
RICHTERWEE<br />
BARTRÉNG-<br />
KIERFECHT<br />
CATHÉDRALE<br />
7<br />
6<br />
AÉROPORT<br />
PAUL<br />
ALBRECHT<br />
FORT<br />
DUMOULIN<br />
KÉIBIERG-<br />
INS<br />
HELFENTERBRÜCK<br />
GABRIEL DE<br />
MAIRIE<br />
ARTHUR<br />
KNAFF<br />
TAWIOUN<br />
STADGRONN-<br />
BRÉCK<br />
HÄLLEPULL<br />
CENTS-<br />
WAASSERTUERM<br />
CENTS-<br />
KIERCH<br />
23<br />
MARTYRS<br />
STE ZITHE<br />
13<br />
F. D. ROOSEVELT<br />
NASSAU<br />
HELFENTERBRÜCK-PLETZER<br />
HELFENTERBRÜCK-<br />
CITY CONCORDE<br />
NIC<br />
ROLLINGER CENTS-HALTE CFL<br />
PARIS/ZITHA<br />
14<br />
TRÈVES CARMEL CAMILLE<br />
POLFER<br />
ROBERT<br />
BRUCH<br />
ÄPPELWEE-INS<br />
Grund<br />
MICHEL<br />
RODANGE<br />
Merl<br />
HELFENTERBRÜCK-<br />
AUTOMOBILE CLUB<br />
BARTRÉNG-<br />
DICKS<br />
POMMIERS<br />
GÉNÉRAL<br />
PATTON<br />
AL AVENUE<br />
Cents<br />
LABORATOIRE<br />
MARIE-<br />
ADÉLAÏDE<br />
ATHÉNÉE<br />
PULVERMÜHL-<br />
HAMMERDÄLLCHEN<br />
LAVOISIER<br />
Gare<br />
HEENTZE PARK<br />
WALLIS<br />
Hamm<br />
HUESE VIC<br />
GARE<br />
CENTRALE<br />
8<br />
15<br />
Pulvermühl<br />
VIRCHOW<br />
23 20<br />
9<br />
10 11<br />
COMMERCE<br />
Hol lerich<br />
CONSERVATOIRE<br />
NANCY<br />
RUE DE BITBOURG<br />
ETATS-UNIS<br />
RUE<br />
HAUTE<br />
HAMM-<br />
SCHOUL<br />
ENGLEBERT<br />
NEVEU<br />
KOFFER-<br />
FABRIK<br />
FRATERNITÉ<br />
POINCARE<br />
P+R<br />
ROTONDE<br />
JEAN-BAPT.<br />
MERKELS<br />
FONDERIE<br />
SALZHAFF<br />
P+R<br />
BARRIÈRE BOUILLON<br />
SCHLUECHTHAUS<br />
15<br />
KÄSCHTEWEE<br />
HAMM-<br />
KIERCH<br />
MONTMÉDY HAMM-<br />
COLONIE<br />
GABRIEL<br />
LIPPMANN<br />
CELTES<br />
MARCEL<br />
CAHEN<br />
JEAN<br />
JACOBY<br />
WISESTROOSS<br />
LÉON XIII<br />
DERNIER SOL<br />
ANATOLE<br />
FRANCE<br />
FORT<br />
WEDELL<br />
PIERRE &<br />
PAUL<br />
ASSURANCES SOCIALES<br />
FISCHERHAFF<br />
Bonnevoie<br />
DEMY<br />
SCHLECHTER<br />
ALSACE<br />
ARTISANS BARRÈS<br />
JEANNE<br />
D'ARC<br />
LASCOMBES<br />
HOLLERICH-GARE<br />
IZEGERKNUPP<br />
WILLIBRORD<br />
HIPPODROME<br />
DÉPORTATION<br />
12<br />
17<br />
1<br />
24<br />
5 6<br />
PONT REMY<br />
LYCÉE TECHNIQUE DE BONNEVOIE<br />
NEUFCHÂTEAU/LTB<br />
MILLEWEE<br />
ED. GRENIER<br />
AL<br />
GASPERICH<br />
KLENSCH<br />
GAASPERECHERBIERG<br />
CLEMENCEAU<br />
HOWALD-CIPA<br />
RANGWEE<br />
BEI DER<br />
AUER<br />
LOUIS DE FROMENT<br />
CESSANGE-KIERCH<br />
HOWALD-JHANGELI<br />
Cessange<br />
KALTREIS<br />
HOWALD-<br />
MOURESCHANZ<br />
NIC<br />
MARTHA<br />
JULES<br />
FISCHER<br />
RICHARD<br />
WAGNER<br />
RUE VERTE<br />
CESSANGE-DUERF<br />
BOY KONEN<br />
HOWALD-BEI DER KIERCH<br />
HOWALD-<br />
RONNEBËSCH<br />
Numéro de ligne<br />
10<br />
AM ECK<br />
4<br />
2<br />
HOWALD-MINSBËSCH<br />
HOWALD-<br />
OP DER STIRZEL<br />
Gasperich<br />
BEETHOVEN<br />
Terminus<br />
LUXEXPO<br />
KUELEBIERG<br />
TUBISHAFF<br />
ANTOINE<br />
HOWALD-WAASSERTUERM<br />
HESPER-<br />
CITÉ UM SCHLASS<br />
P+R LUX-SUD<br />
RUE DE<br />
LEUDELANGE<br />
Arrêt desservi en deux directions<br />
AM ECK<br />
3<br />
16<br />
22<br />
P+R<br />
14<br />
FRANÇOIS<br />
HOGENBERG<br />
STUMPER<br />
RAIFFEISEN<br />
PLANTIN<br />
Cloche d'Or<br />
Arrêt desservi uniquement dans<br />
le sens de la flèche<br />
MILLEWEE<br />
Howald<br />
CLOCHE D'OR<br />
24<br />
GUILLAUME KROLL<br />
Tronçon non desservi en permanence<br />
RUPPERT<br />
Kockelscheuer<br />
21<br />
P+R<br />
18<br />
Park <strong>and</strong> Ride<br />
P+R<br />
BIAN<br />
KOCKELSCHEUER-PATINOIRE<br />
SCHARFEN ECK<br />
KOCKELSCHEUER-<br />
CAMPING<br />
Gare CFL<br />
Aéroport<br />
Édition novembre 2009
Luxembourg,<br />
January 25th to 28th, 2012<br />
Luxembourg<br />
Natural compounds -<br />
Regulators of cell signaling<br />
pathways <strong>and</strong> novel therapeutic<br />
tools<br />
Contact : Marc Diederich<br />
Laboratoire de Biologie Moléculaire<br />
et Cellulaire du Cancer (LBMCC)<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
Fax: + 352 2468-4060<br />
Email: marc.diederich@lbmcc.lu<br />
Please register at:<br />
http://www.transduction-meeting.lu
A heart for cancer sick children<br />
Together we can make difference in the lives of children<br />
<strong>and</strong> their families living with childhood cancer
About geneXplain<br />
GeneXplain’s mission is to provide a comprehensive<br />
platform for bioinformatic, systems<br />
biological <strong>and</strong> cheminformatic tools. The raison<br />
d'être of this platform is to assist translational<br />
research in the life sciences, mainly in the context<br />
of personalized medicine <strong>and</strong> pharmacogenomics.<br />
We intend to make our expertise available to<br />
academic <strong>and</strong> commercial partners in collaborative<br />
research projects.<br />
Statistics<br />
Input: High-throughput<br />
data from patients<br />
(genomics,<br />
transcriptomics, ChIPseq,<br />
proteomics, etc.)<br />
Output: List of relevant<br />
genes or proteins<br />
The geneXplain platform<br />
Based on the BioUML technology created by<br />
the Institute of Systems Biology, a platform<br />
has been developed that allows to integrate a<br />
number of individual modules, “bricks”, each<br />
of which provides a well-defined function in<br />
the field of bioinformatics, systems biology or<br />
cheminformatics. Altogether, the whole<br />
system will provide a toolbox suitable to<br />
establish complete pipelines from<br />
experimental high-throughput (“-OMICS”)<br />
data to new drugs.<br />
The central platform is going to be made<br />
freely available, so that any community<br />
member can contribute own modules.<br />
In addition, geneXplain GmbH provides a<br />
number of state-of-the-art bricks on a<br />
commercial basis, implying a thorough<br />
quality control <strong>and</strong> maintenance guarantee.<br />
The workflow<br />
The incorporated statistical<br />
analyses help to identify relevant genes or<br />
proteins in the raw dataa,<br />
e.g. those that are differentially expressed.<br />
The Bioinformatics block<br />
allows to reveal potential regulation of genes by<br />
transcription factors or miRNAs.<br />
Directors: E. Wingender, A. Kel �Coommercial<br />
register:<br />
HRB 202564, Amtsger. Braunschweeig<br />
�VAT No.: DE271983408<br />
To achieve this, geneXplain offers:<br />
• The X-Platform to provide an integrated <strong>and</strong><br />
comprehensive workflow management of a large<br />
number of “bricks”, each providing a specific function<br />
in analyzing biological data, in particular OMICS<br />
results<br />
• In Silico Molecular Cloning (IMC) for h<strong>and</strong>ling largescale<br />
genome data<br />
• PASS <strong>and</strong>d Ph PharmaExpert E t ffor predicting di ti bi biological l i l<br />
activities of potential drugs<br />
Bioinformatics<br />
Search for regulatory<br />
Systems biology approaches analyze networks of<br />
molecular events, in particular gene regulatory <strong>and</strong><br />
signaling pathways, <strong>and</strong> suggest promising drug<br />
target molecules <strong>and</strong> their mechanisms of action.<br />
modules in any genomic Th The cheminformatics h i f i tools l enable bl to di direct<br />
regions<br />
Output: List of transcription<br />
factors potentially<br />
compound screening by pre-selection of chemicals<br />
with desirable <strong>and</strong> without adverse or toxic effects.<br />
responsible for the observed<br />
(co-)regulation of genes<br />
Systems Biology<br />
Toopological<br />
analysis of the<br />
networks upstream of<br />
transcription factors,<br />
simulation<br />
of the network<br />
behavior, patient<br />
stratification<br />
OOutput:<br />
List of potential<br />
master regulators<br />
Cheminformatics<br />
Prediction of biological<br />
activities of the<br />
compounds, d selection l ti of f<br />
compounds with required<br />
effects <strong>and</strong> without<br />
adverse or toxic effects.<br />
Output: List of potential<br />
lead structures for<br />
validation<br />
geneXplain GmbH<br />
Am Exer 10b<br />
D-38302 Wolfenbüttel, Germany<br />
info@genexplain.com<br />
www.genexplain.com
Wednesday January 26th<br />
Keynote session:<br />
9h30 - 10h30 Mario Capecchi<br />
10h30 - 11h00 Coffee break/expo<br />
Session 1: Cell death<br />
11h00 - 11h30 Eileen White<br />
11h30 - 12h00 Gerry Melino<br />
12h00 - 12h30 Stefania Gonfloni<br />
12h30 - 12h45 Cinzia Di Pietro<br />
12h45 – 13h00 Inna N. Lavrik<br />
13h00 - 14h00 Lunch/expo<br />
Workshops 1 <strong>and</strong> Poster Session 1<br />
14h00 - 15h00 Polyplus-transfection<br />
15h00 - 16h00 Agilent Technologies, Belgium<br />
14h00 - 15h30 Poster (Even numbers)/expo<br />
15h30 – 16h00 Coffee break/expo<br />
Session 2: Cell signaling<br />
16h00 - 16h30 Ilya Shmulevich<br />
16h30 - 17h00 Varadharajan Sundaramurthy<br />
17h00 - 17h30 Anne Grosse-Wilde<br />
17h30 - 18h00 Josef Penninger<br />
18h00 - 18h15 Sébastien Chateauvieux<br />
18h15 - 18h30 Ivana Scovassi<br />
18h30 - 18h45 Vladimir L. Katanaev<br />
19h00 - 19h30 Official talks<br />
19h30 - 20h30 Reception/expo<br />
20h30 - 21h00 Shuttles to the hotels<br />
------------------------------------------------<br />
Thursday January 27th<br />
Session 3: Transcriptional control<br />
9h00 - 9h30 Young-Joon Surh<br />
9h30 - 10h00 Luciano Di Croce<br />
10h00 - 10h30 Thomas Luft<br />
10h30 - 11h00 Coffee break/expo<br />
Session 4: Immunology<br />
11h00 - 11h30 Bali Pulendran<br />
11h30 - 12h00 Marie-Lise Gougeon<br />
12h00 - 12h30 Kasper Hoebe<br />
12h30 - 13h00 Claude Condé<br />
13h00 - 16h00 Lunch/expo<br />
Workshops 2 <strong>and</strong> Posters Session 2<br />
14h00 - 15h00 GE Healthcare Europe GmbH<br />
15h00 - 16h00 AMSBIO<br />
14h00 - 15h30 Posters (Odd numbers)/expo<br />
15h30 - 16h00 Coffee break/expo<br />
Session 5: Proteomics<br />
Cell Signal-omics 2011 on one page<br />
16h00 - 16h30 Antonio del Sol<br />
16h30 - 17h00 Bernd Wollscheid<br />
17h00 - 17h30 Pascale Cossart<br />
17h30 - 18h00 Christopher Overall<br />
18h00 - 18h15 Margarida Fardilha<br />
18h15 - 18h30 Alfred C. O. Vertegaal<br />
18h30 - 18h45 Angela Brieger<br />
19h00 Shuttles to the hotels<br />
------------------------------------------------<br />
Friday January 28th<br />
Session 6: Epigenetics<br />
9h00 - 9h30 François Fuks<br />
9h30 - 10h00 Michael Bots<br />
10h00 - 10h30 Roberto Gambari<br />
10h30 - 11h00 Coffee break/expo<br />
Session 7: Cancer signaling networks<br />
11h00 - 11h30 Andrei Zinovyev<br />
11h30 - 12h00 Dean W. Felsher<br />
12h00 - 12h30 Alex<strong>and</strong>er R. A. Anderson<br />
12h30 - 13h00 Evan Keller<br />
13h00 - 14h00 Lunch/expo<br />
Session 8: Gene expression networks<br />
14h00 - 14h30 Guido Kroemer<br />
14h30 - 15h00 Yong Sang Song<br />
15h00 – 15h15 Andrew Koff<br />
15h15 – 15h30 Stefanie Kaempf<br />
15h30 – 16h00 Coffee break/expo<br />
Session 9: Neurodegenerative diseases<br />
16h00 - 16h30 Mark P. Mattson<br />
16h30 - 17h00 Junying Yuan<br />
17h00 - 17h30 Vittorio Calabrese<br />
17h30 - 18h00 Xin-Fu Zhou<br />
18h00 - 18h30 Paul R. Fisher<br />
18h30 End of the meeting<br />
18h30 Shuttles to the hotels<br />
!<br />
!
Poster presentations<br />
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 1: Cell death<br />
66
Session 1: Cell death Poster 1<br />
The molecular chaperone HSP90 is cleaved by oxidative stress at a highly conserved Nterminal<br />
amino acid motif.<br />
Pedro Buc Calderon a , Raphael Beck a , Nicolas Dejeans a , Christophe Glorieux a , Mélanie<br />
Creton a , Edouard Delaive b , Marc Dieu b , Martine Raes b , Philippe Levêque c , Bernard<br />
Gallez c , Matthieu Depuydt d , Jean-François Collet d , <strong>and</strong> Julien Verrax a .<br />
a Toxicology <strong>and</strong> Cancer Biology Research Group, Louvain Drug Research Institute,<br />
Université Catholique de Louvain, Belgium; b Unité de Recherche en Biologie Cellulaire,<br />
FUNDP, Belgium; c Biomedical Magnetic Resonance Unit, Louvain Drug Research<br />
Institute, Université Catholique de Louvain, Belgium; d de Duve Institute, Université<br />
Catholique de Louvain, Belgium. Email: pedro.buccalderon@uclouvain.be<br />
Hsp90 is an essential chaperone which is necessary for the folding, stability <strong>and</strong> activity of<br />
numerous proteins. Here, it is shown that free radicals formed during oxidative stress<br />
conditions can cleave Hsp90. The cleavage is observed in intact cells, in cell lysates <strong>and</strong> in<br />
purified Hsp90 protein demonstrating that proteases are not required. In cells, Hsp90 cleavage<br />
leads to the degradation of its client proteins like Bcr-Abl, RIP, c-Raf, IKK! (NEMO) <strong>and</strong><br />
hTert. As this cleavage is dependent on ROS production <strong>and</strong> the availability of free redoxactive<br />
iron, we hypothesized that it occurs through a Fenton-type reaction. The formation of<br />
protein radicals upon the exposure of Hsp90 to oxidative stress was confirmed by immunospin<br />
trapping <strong>and</strong> EPR experiments. Using a proteomic analysis, we determined that the<br />
cleavage occurs in a conserved motif of the N-terminal nucleotide binding site, between Ile-<br />
126 <strong>and</strong> Gly-127 in Hsp90". Our results indicate that in situ formation of ROS by a Fentontype<br />
reaction at the N-terminal nucleotide binding pocket of Hsp90 forms a protein radical,<br />
which, by rearrangement, causes the rupture of the peptide backbone <strong>and</strong> leads to the<br />
degradation of client proteins that are critical for cells.<br />
67
Session 1: Cell death Poster 2<br />
Tumor GRP94 overexpression: a hallmark of aggressiveness <strong>and</strong> recurrence in breast<br />
cancer.<br />
Nicolas Dejeans a , Samuel Guenin b , Christophe Glorieux a , Raphael Beck a , Julien<br />
Verrax a , Brice Sid a , Bettina Bisig b , Philippe Delvenne b , Pedro Buc Calderon a .<br />
a Université Catholique de Louvain, Louvain Drug Research Institute, Toxicology <strong>and</strong><br />
Cancer Biology Research Group, PMNT Unit, Belgium. b Department of Pathology,<br />
GIGA Cancer, University of Liege, B23 CHU Sart Tilman, 4000 Liège. Email:<br />
nicolas.dejeans@uclouvain.be<br />
Targeting the altered redox status of cancer cells is emerging as an interesting approach to<br />
potentiate chemotherapy. However, to favor the effectiveness of this strategy <strong>and</strong> define the<br />
proper chemotherapeutic association, it is important to underst<strong>and</strong> the biological<br />
consequences of chronically exposing cancer cells to ROS. Using an H2O2 generating system,<br />
we have selected a ROS-resistant MCF-7 cell line, namely the Resox cells. By exploring<br />
different survival pathways that usually are induced during an oxidative stress, we identified<br />
in these cells a constitutive overexpression of the endoplasmic reticulum chaperone GRP94,<br />
while levels of either its cytoplasmic homologue HSP90 or GRP78 are not modified. Such an<br />
overexpression is not mediated by constitutive unfolded protein response (UPR) activation.<br />
The increase of GRP94 is tightly associated to an increase in proliferation <strong>and</strong> migration<br />
capacities, as shown by GRP94 silencing experiments. By doing immunohistochemistry on<br />
breast tumor biopsies, we showed that GRP94 expression was elevated in recurrent human<br />
breast cancers compared to their paired primary tissues. Likewise in the Resox cells, this<br />
increase was not associated with a higher phosphorylation of eIf2, demonstrating that it was<br />
not linked to an increase in UPR activation in recurrent tumors. In conclusion, this study<br />
suggests that GRP94 overexpression may be a hallmark of aggressiveness <strong>and</strong> recurrence in<br />
breast cancers.<br />
68
Session 1: Cell death Poster 3<br />
In vitro characterization of bovine neutrophil cell death following Escherichia coli<br />
phagocytosis<br />
Sofie Notebaert 1* , Kristel Demeyere 1 *, Dieter Demon 1 , Filip Boyen 2 , Chris Guerin 3 ,<br />
Peter V<strong>and</strong>enabeele 4 , Evelyne Meyer 1<br />
1 Laboratory of Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan<br />
133, 9820 Merelbeke, Belgium – sofie.notebaert@hotmail.com; kristel.demeyere@ugent.be;<br />
dieter.demon@ugent.be; evelyne.meyer@ugent.be<br />
2 Department of Pathology, Bacteriology <strong>and</strong> Avian Diseases, Faculty of Veterinary Medicine,<br />
Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium – filip.boyen@ugent.be<br />
3 Microscopy Core Facility, Department for Molecular Biomedical Research, VIB, Ghent<br />
University, 9052 Zwijnaarde, Belgium - chris.guerin@dmbr.vib-ugent.be<br />
4 Molecular Signaling <strong>and</strong> Cell Death Unit, Department for Molecular Biomedical Research,<br />
VIB, Ghent University, 9052 Zwijnaarde, Belgium - peter.v<strong>and</strong>enabeele@dmbr.vib-ugent.be<br />
* These authors contributed equally to this work.<br />
Neutrophils are known to play a key role in the early host defense towards coliform mastitis. The<br />
molecular events during phagocytosis-induced cell death (PICD) are rarely described, especially for<br />
bovine neutrophils. Therefore, our objective was to elucidate the type of cell death of bovine<br />
neutrophils after in vitro phagocytosis of Escherichia coli.<br />
Peripheral blood samples were collected from clinically healthy heifers. Neutrophils were isolated by<br />
density gradient centrifugation, resuspended in RPMI with 10 % fcs <strong>and</strong> incubated with E. coli strain<br />
P4:O32 (moi 5:1) at 37°C. PS exposure <strong>and</strong> cell membrane integrity loss was determined flow<br />
cytometrically by Annexin-V-FITC/PI. ROS were measured as luminol-amplified chemiluminescence.<br />
Equal amounts of total cell lysates were analyzed by western blotting using anti caspase-1 (C-1),<br />
cleaved C-3 <strong>and</strong> C-7 <strong>and</strong> X-IAP. C-3/-7 activity was determined using the caspase-Glo ® assay <strong>and</strong><br />
bovine IL-1! was quantified with ELISA.<br />
The higher rate of PS exposure in activated than in control neutrophils undergoing spontaneous<br />
apoptosis, indicates that bovine neutrophil cell death is accelerated following phagocytosis of E. coli,<br />
as recently described. In contrast to control cells, PICD occurred with significant ROS production but<br />
independent from C-3/-7 activation. The latter finding is in line with a recent report describing<br />
caspase-independent cell death of human neutrophils incubated with E. coli, although no ROS<br />
production was seen. In both human <strong>and</strong> mouse neutrophils, NADPH oxidase prevents caspase<br />
activation following phagocytosis of S. aureus. Nevertheless, all these data do not corroborate a study<br />
with human neutrophils incubated with E. coli where C-3 activation was demonstrated. This<br />
discrepancy could be either species <strong>and</strong>/or micro-organism related. Interestingly, the well-described<br />
X-IAP inhibitor for C-3 <strong>and</strong> C-7, was only present in control samples, suggesting that X-IAP may<br />
control C-3/-7 activity in spontaneous bovine neutrophil apoptosis but is superfluous in E. coli PICD<br />
in vitro. Life cell imaging using GFP-labeled E. coli <strong>and</strong> PI staining showed no clear morphological<br />
features of either netosis or autophagy, but late PI positivity <strong>and</strong> membrane blebbing without apoptotic<br />
bodies. Complementing these features with C-1 activation <strong>and</strong> intracellular IL-1! suggests that the<br />
PICD in our setup is executed by pyroptosis.<br />
Taken together, our in vitro data indicate that bovine neutrophils undergo C-3/-7-assisted spontaneous<br />
apoptosis <strong>and</strong> that E. coli stimulated PICD most likely can be characterized as C-3/-7-independent, but<br />
C-1- <strong>and</strong> ROS-dependent pyroptosis.<br />
69
Session 1: Cell death Poster 4<br />
The in vivo role of executioner Caspase-3 <strong>and</strong> -7 in the Escherichia coli-infected<br />
mammary gl<strong>and</strong><br />
Koen BREYNE 1,! , Dieter DEMON 1,! , Tom VANDEN BERGHE 2 , Peter<br />
VANDENABEELE 2 , <strong>and</strong> Evelyne MEYER 1<br />
1 Laboratory of Biochemistry, Department of Pharmacology, Toxicology <strong>and</strong><br />
Biochemistry, Ghent University, 9820 Merelbeke, Belgium, Koen.Breyne@Ugent.be,<br />
Dieter.Demon@Ugent.be, Evelyne.Meyer@Ugent.be<br />
2 Molecular Signaling <strong>and</strong> Cell Death Unit, Department for Molecular Biomedical<br />
Research, Technologiepark 927, 9052 Ghent, Belgium, Tom.V<strong>and</strong>enberghe@dmbr.vib-<br />
Ugent.be, Peter.V<strong>and</strong>enabeele@dmbr.vib-Ugent.be<br />
! These authors contributed equally to this work<br />
Bacterial infection of the mammary gl<strong>and</strong> can disturb lactation <strong>and</strong> involution through a process called<br />
mastitis. The pathogenesis of mastitis in humans <strong>and</strong> animals is insufficiently understood, resulting in<br />
excessive use of antibiotics. To reduce bacterial resistance due to the use of antibiotics, alternative<br />
therapeutic strategies are desirable. Mastitis is characterized by apoptosis <strong>and</strong> inflammation, two<br />
processes in which caspases are involved. Executioner Caspase-3 (C-3) <strong>and</strong> C-7 play central roles in<br />
coordinating the stereotypical events during apoptosis. Moreover, C-7 is also involved in<br />
inflammatory signaling. Underst<strong>and</strong>ing the pathways involving C-3 <strong>and</strong> -7 may identify targets<br />
towards a faster <strong>and</strong> more accurate diagnosis or treatment of mastitis. Therefore, we evaluated C-3 -/-<br />
<strong>and</strong> -7 -/- single knockout mice in the acute Escherichia coli (E. coli)-induced mastitis model.<br />
Lactating wild type (wt; n=10), C-3 -/- (n=5) <strong>and</strong> C-7 -/- (n=5) mice were intraductally inoculated with<br />
the bovine mastitis isolate E. coli P4:O32 in the right mammary gl<strong>and</strong> of the 4 th pair <strong>and</strong> with<br />
phosphate buffered saline (PBS) in the contralateral gl<strong>and</strong>. Serum was collected at 18h postinoculation<br />
<strong>and</strong> the inoculated gl<strong>and</strong>s were isolated, homogenized <strong>and</strong> plated to quantify colony<br />
forming units (CFU). Homogenates were lysed in 1% NP40 buffer for western blot <strong>and</strong> cytokine<br />
analysis. Two gl<strong>and</strong> pairs/group were paraffin-embedded for histopathological evaluation.<br />
Our data show significantly more proteolysis of C-7 in E. coli-infected than in control mammary<br />
gl<strong>and</strong>s. Interestingly, next to the classically detected p20 fragment of active C-7 we observed in both<br />
gl<strong>and</strong>s a 23 kDa fragment with unknown function thus far. E .coli infection also results in the loss of a<br />
C-3 cleavage fragment, a phenomenon which is typically seen with inhibition by X-IAP, the welldescribed<br />
inhibitor of C-3 <strong>and</strong> -7. Interestingly, more intense fragments of X-IAP are seen in E. coliinoculated<br />
gl<strong>and</strong>s, suggesting that it may indeed regulate C-3 <strong>and</strong> -7 activity in mastitis. However, the<br />
absence of C-3, which is typically linked with apoptosis, does not affect bacterial growth nor local <strong>and</strong><br />
systemic cytokine release during E. coli-induced intramammary infection, indicating no critical role<br />
for this caspase in mastitis. Ablation of C-7 seems to sensitize mice slightly to intramammary E. coli<br />
infection, as highlighted by the higher final CFU/g gl<strong>and</strong>, <strong>and</strong> local <strong>and</strong> systemic levels of the proinflammatory<br />
cytokines IL-1! <strong>and</strong> IL-6, respectively. Prototype inflammatory C-1 is also activated in<br />
E. coli-infected gl<strong>and</strong>s <strong>and</strong> could thus be the upstream activator of C-7. However, the PARP cleavage<br />
pattern suggests the functional contribution of calpain, cathepsin-B <strong>and</strong> granzyme-B, which have all<br />
been shown to activate C-7 in vitro. Analysis of the separate cell populations (macrophage, neutrophil<br />
or epithelial cell), instead of total homogenates will help to further elucidate the specific role of C-7 in<br />
mastitis.<br />
70
Session 1: Cell death Poster 5<br />
Functional crosstalk between p53 <strong>and</strong> HtrA2/Omi<br />
Eric Duplan, Frédéric Checler <strong>and</strong> Cristine Alves da Costa<br />
Institut de Pharmacologie Moléculaire et Cellulaire <strong>and</strong> Institut de NeuroMédecine<br />
Moléculaire, UMR 6097 CNRS/UNSA, 660 route des Lucioles, 06560 Valbonne, France.<br />
duplan@ipmc.cnrs.fr<br />
p53 is the product of a well known tumor-suppressor gene, mutations in which lead to its<br />
inactivation <strong>and</strong> increased risk to cancer. It is well established that p53 is a key regulator of<br />
the intrinsic apoptotic pathway via its transcriptional factor dependent <strong>and</strong> independent<br />
properties. It has been recently shown that p53 induces the transcription of HtrA2/Omi, a<br />
serine protease that is known to cleave (among other substrates) <strong>and</strong> inactivate the Inhibitor of<br />
Apoptosis Proteins, XIAP. Thus, p53 can regulate cell death processes via transcriptional<br />
activation of the protease Omi. Omi activity is also compromised in several cancer types <strong>and</strong><br />
recently it has been shown that following apoptotic stimuli, Omi accumulates in the nucleus<br />
where it cleaves the p53 family member p73alpha. The processed p73 fragment can then<br />
activate the apoptotic gene bax. These results suggest that the serine protease properties of<br />
Omi accounts for its pro-apoptotic function. Since p73 <strong>and</strong> p53 contain homologous<br />
transactivation, DNA-binding, <strong>and</strong> oligomerization domains, we thus decided to examine if<br />
Omi was capable to regulate the levels of p53 <strong>and</strong>, as a corollary, its pro-apoptotic function.<br />
We found that Omi controls the transcriptional activity, protein <strong>and</strong> mRNA levels of p53. The<br />
precise mechanism of this regulation will be discussed.<br />
71
Session 1: Cell death Poster 6<br />
Alpha-Synuclein cytotoxicity in Dictyostelium.<br />
Sanjanie G. Fern<strong>and</strong>o 1 , William A. Burrage 2 , Peter L. Beech 2 , Sarah J. Annesley 1 , Paul<br />
R. Fisher 1<br />
1 Department of Microbiology, Latrobe University, VIC 3086, Australia. 2 Centre for<br />
Cellular <strong>and</strong> Molecular Biology, Deakin University, Burwood, VIC 3125, Australia.<br />
Alpha-synuclein is implicated in the pathogenesis of Parkinson’s disease (PD) which is a<br />
neurodegenerative disorder. Hyperexpression or mutations in the alpha-synuclein gene are<br />
believed to be associated with mitochondrial abnormalities <strong>and</strong> onset of familial PD. To<br />
establish a Dictyostelium model for PD <strong>and</strong> to investigate the role of mitochondrial<br />
dysfunction in its cytopathology, we created stable, clonal Dictyostelium transformants<br />
hyperexpressing these normal <strong>and</strong> mutant forms of alpha-synuclein. All three forms were<br />
cytotoxic <strong>and</strong> impaired both phagocytosis <strong>and</strong> growth on bacterial lawns. We have also used<br />
immunofluorescence <strong>and</strong> confocal microscopy to show that alpha-synuclein is concentrated in<br />
the membrane particularly at the leading edge of the cell (resembling the presynaptic<br />
membrane in neurons) which is the same subcellular location as in human cells. However, it<br />
did not colocalize or associate with mitochondria. Further phenotypic characterisation showed<br />
that the aberrant phenotypes caused by alpha-synuclein are distinct from those observed in<br />
mitochondrial disorders in Dictyostelium. This indicates that there are distinct alphasynuclein<br />
cytotoxicity pathways that differ from those associated with mitochondrial<br />
dysfunction.<br />
72
Session 1: Cell death Poster 7<br />
Model-based dissection of CD95 signaling dynamics reveals both a pro- <strong>and</strong><br />
antiapoptotic role of c-FLIPL.<br />
Fricker N, Beaudouin J, Richter P, Eils R, Krammer PH, Lavrik IN.<br />
C-FLIP proteins (isoforms: c-FLIPL, c-FLIPS <strong>and</strong> c-FLIPR) regulate caspase-8 activation <strong>and</strong><br />
death receptor-induced apoptosis. The function of the two short c-FLIP isoforms, c-FLIPS <strong>and</strong><br />
c-FLIPR, has been well described. However there are contradictory reports with respect to the<br />
pro- or anti-apoptotic role of the long isoform, c-FLIPL. Here, using a combination of<br />
mathematical modeling, imaging <strong>and</strong> quantitative western blots we determine conditions<br />
leading to pro- or anti-apoptotic function of c-FLIPL. We present a mathematical model<br />
describing the regulation of caspase-8 activation by c-FLIP at the DISC (death inducing<br />
signalling complex). We show that c-FLIPL has a pro-apoptotic role only upon moderate<br />
expression in combination with strong receptor stimulation or in the presence of high amounts<br />
of one of the short c-FLIP isoforms: c-FLIPS or c-FLIPR. Our findings resolve the present<br />
controversial discussion on the function of c-FLIPL as a pro- or anti-apoptotic protein in death<br />
receptor-mediated apoptosis.<br />
73
Session 1: Cell death Poster 8<br />
Reverse effects of AMP-activated protein kinase on pro-apoptotic extracellular signalregulated<br />
kinase activation by inducing dual-specificity protein phosphatases under<br />
glucose deprivation in human colon carcinoma.<br />
Sooho Lee <strong>and</strong> Joohun Ha<br />
Department of Biochemistry <strong>and</strong> Molecular Biology, Medical Research Center <strong>and</strong><br />
Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul 130-701,<br />
Republic of Korea. E-mail: sooholee0827@gmail.com<br />
Mitogen-activated protein kinase (MAPK) pathways are involved in the regulation of cellular<br />
responses, including cell proliferation, differentiation, cell growth, <strong>and</strong> apoptosis. Because<br />
these responses are tightly related to cellular energy level, AMP-activated protein kinase<br />
(AMPK), which plays an essential role in energy homeostasis, has emerged as another key<br />
regulator. In the present study, we demonstrate a novel signal network between AMPK <strong>and</strong><br />
MAPK in HCT116 human colon carcinoma. Glucose deprivation activated AMPK <strong>and</strong> three<br />
MAPK subfamilies, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase<br />
(JNK), <strong>and</strong> p38 MAPK. Under these conditions, inhibition of endogenous AMPK by<br />
expressing a dominant-negative form significantly potentiated ERK activation, indicating that<br />
glucose deprivation-induced AMPK is specifically antagonizing ERK activity in HCT116<br />
cells. Moreover, we provide novel evidence that AMPK activity is critical for p53-dependent<br />
expression of dual-specificity phosphatase (DUSP) 1 <strong>and</strong> 2, which are negative regulators of<br />
ERK. Notably, ERK exhibits pro-apoptotic effects in HCT116 cells under glucose<br />
deprivation. Collectively, our data suggest that AMPK protects HCT116 cancer cells from<br />
glucose deprivation, in part, via inducing DUSPs, which suppresses pro-apoptotic ERK,<br />
further implying that a signal network between AMPK <strong>and</strong> ERK is a critical regulatory point<br />
in coupling the energy status of the cell to the regulation of cell survival.<br />
74
Session 1: Cell death Poster 9<br />
Protective Effects of Marine Natural Products against UVB-induced Oxidative Damage<br />
<strong>and</strong> Cell Death in Human Skin Fibroblast<br />
Chan Lee, En-Joo Lee, <strong>and</strong> Chan-Ik Park<br />
Department of Cosmeceutical Science, Daegu Haany University, Gyeongsangbuk-do<br />
712-715, S. Korea (E-mail : cipark@dhu.ac.kr)<br />
Ultraviolet is the one of the main environmental factors promoting aging process via<br />
increased intracellular accumulation of reactive oxygen species (ROS) <strong>and</strong> decreased<br />
expression of endogenous antioxidant molecules. Therefore, in this study, we have tried to<br />
search for skin-protective antioxidant marine natural products (Porphyra thalli, Laminariae<br />
japonicae thallus, Ostreae concha, Sargassum thallus, Undaria thallus, Haliotidis concha,<br />
Codium thalli, Syngnathoides biaculeatus, Hippocampus, Stichopus stichopus, Thalli, Hizikia<br />
fusiforme thalli) which can protect against UVB-induced oxidative cell death in HS86 cells.<br />
Free radical scavenging activity was shown in the order of Undaria thallus, Sargassum thallus,<br />
Laminariae japonicae thallus, Hippocampus, Haliotidis concha, Ostreae concha, <strong>and</strong><br />
Syngnathoides biaculeatus as assessed by DPPH assay. In another experiment, UVB-induced<br />
cell death in HS68 cells were effectively suppressed by treatment with Sargassum thallus,<br />
Haliotidis concha, Codium thalli, or Hippocampus water extract by inhibition of intracellular<br />
accumulation of ROS. The protective effects of marine natural products against UVB-induced<br />
oxidative cell death seemed to be mediated by increased expression of antioxidant enzymes<br />
such as catalase, superoxide dismutase, <strong>and</strong> heme oxygenase-1. These results suggest that<br />
Sargassum thallus, Haliotidis concha, Codium thalli, <strong>and</strong> Hippocampus may have preventive<br />
<strong>and</strong>/or protective potentials against oxidative stress-mediated skin damages <strong>and</strong> aging with<br />
antioxidant properties.<br />
Keywords: Ultraviolet, marine natural products, antioxidant, oxidative stress, skin fibroblast<br />
75
Session 1: Cell death Poster 10<br />
Different response to UV-B irradiation of Niemann-Pick B-Lymphocytes cell line<br />
Barbara Canonico 1 , Erica Cesarini 1 , Marcella Arcangeletti 1 , Fulvio Palma 1 , Loris<br />
Zamai 1 , Stefano Papa 1 <strong>and</strong> Francesca Luchetti 1<br />
1<br />
Department of Earth, Life <strong>and</strong> Environmental Sciences. University of Urbino “ Carlo<br />
Bo”<br />
Via Cà le Suore 2 61029 Urbino. barbara.canonico@uniurb.it; erica.cesarini@uniurb.it;<br />
marcella.arcangeletti@uniurb.it; fulvio.palma@uniurb.it; loris.zamai@uniurb.it;<br />
stefano.papa@uniurb.it; francesca.luchetti@uniurb.it<br />
Autophagy is an intracellular lysosomal degradation process, characterized by the formation<br />
of double membrane-bounded vesicle (autophagosome). Recently, the autophagic pathway<br />
was coupled with lysosomal storage disorders (LSD), on the hypothesis that accumulation of<br />
undegraded substrates in lysosomes may impair the autophagic process. Niemann-Pick<br />
disease (NPD) type A <strong>and</strong> B are lysosomal storage disorders (LSD) resulting from the<br />
sphingomyelin accumulation in lysosomes relying on reduced or absent acid<br />
sphingomyelinase (ASM). The purpose of this study is to examine the autophagic pathway in<br />
a NPD B-Lymphocytes cell line, in response to UV-B irradiation, by means of flow cytometry<br />
<strong>and</strong> microscopic analysis. We evaluated cell viability, caspase activation, various<br />
mitochondrial parameter, acidic compartment (by Lysotracker-LT <strong>and</strong> Acridine Orange–AO<br />
fluorescence) <strong>and</strong> we performed preliminary test on endosomal network (by CD63 staining).<br />
Microscopic analysis was applied to evaluate morphological aspects. Cells were UVirradiated<br />
for 5 <strong>and</strong> 10 minutes <strong>and</strong> treated with nocodazole (NZ) <strong>and</strong> wortmannin (WM), two<br />
autophagy inhibitors. Furthermore we also used rapamicyn (RM), an autophagic inductor. Our<br />
preliminary data show a different trend between short (5’) <strong>and</strong> prolonged (10’) stress: a<br />
predominance of apoptotic <strong>and</strong> autophagic trend, respectively. A mitochondrial involvement<br />
is evident in both times of irradiation of cells, with a progressive decrease in mitochondrial<br />
integrity (NAO) <strong>and</strong> function (TMRE). Lymphocytes treated by NZ <strong>and</strong> WM highlighted<br />
immunofluorescence values (IF) for AO higher than control lymphocytes (+19% <strong>and</strong> +40%,<br />
respectively). We found a decrease in CD63+ cells, strong for RM (55%), WM+UV10’ (54%)<br />
<strong>and</strong> NZ+UV5’ (85%), weaker for WM+UV5’ (12%). Similarly UV irradiated cells highlight<br />
decrease in CD63+ events. On the contrary IF for CD63 exhibited an opposite trend: we<br />
found the highest values in RM samples <strong>and</strong> UV10’ treated samples with or without pretreatment<br />
with both inhibitors. Our preliminary results suggest a possible autophagy<br />
involvement in NPB cells oxidative stress-response.<br />
76
Session 1: Cell death Poster 11<br />
MAPK/ERK targeting by Sorafenib down-regulates Doxorubicin-induced death<br />
signaling in hepatocellular carcinoma Hep3B cells: the role of autophagy.<br />
Yulia Pollak, Rinata Broneshter, Theodore C. Iancu <strong>and</strong> Irena Manov<br />
Pediatric Research <strong>and</strong> Electron Microscopy Unit, Faculty of Medicine, Technion-Israel<br />
Institute of Technology, Haifa, Israel, e-mail: irmanov@tx.technion.ac.il<br />
A multikinase inhibitor of Raf/MEK/ERK pathway, sorafenib is increasingly used in the<br />
management of hepatocellular carcinoma (HCC) <strong>and</strong> its combination with doxorubicin<br />
(DOX) stimulates particular interest. Although the MEK/ERK pathway was identified as<br />
survival signaling in tumors, activation of these kinases mediates DOX-triggered death<br />
signaling. In the present study, we investigated the effect of sorafenib on DOX efficacy in<br />
HCC cells <strong>and</strong> possible mechanisms of drug interaction. Treatment of Hep3B cells with DOX<br />
arrested proliferation <strong>and</strong> reduced cell viability. Electron microscopy revealed intensive<br />
vacuolization of cytoplasm <strong>and</strong> formation of autophagosomes, while apoptotic changes were<br />
not conspicuous. Immunofluorescence demonstrated induction of LC3B in DOX-exposed<br />
Hep3B cells. Sorafenib alone affected viability <strong>and</strong> cell cycle progression <strong>and</strong> induced a<br />
rapid, massive mitochondrial degradation. In contrast, sorafenib combined with DOX<br />
increased survival, facilitated cell cycle progression <strong>and</strong> reduced autophagy. To elucidate the<br />
mechanisms by which sorafenib reduced DOX efficacy, we examined the implication of<br />
ERK1/2 <strong>and</strong> AKT, as well as molecular sensors of autophagy (MCL-1) <strong>and</strong> cell cycle<br />
progression (cyclin D1). The increased phospho-ERK in DOX-exposed cells was reduced by<br />
sorafenib. Similarly to sorafenib, MEK/ERK targeting by U0126 or PD98059 deregulated<br />
DOX-induced death signaling <strong>and</strong> increased survival. DOX had no effect on phospho-Akt,<br />
whereas combined treatment with sorafenib resulted in AKT activation. Sorafenib-induced<br />
degradations of Cyclin D1 <strong>and</strong> MCL1 were amplified by co-treatments with DOX. Thus,<br />
MEK/ERK counter-effective regulation <strong>and</strong> stimulation of cell survival via AKT, may<br />
produce the paradoxical effect of DOX plus sorafenib co-treatment. The escape from<br />
autophagy through MCL1 <strong>and</strong> cyclin D1 down regulation is discussed. The use of<br />
MEK/ERK inhibitors in combination with chemotherapeutics, meant to enhance anti-cancer<br />
efficacy, requires considering possible antagonistic effects.<br />
77
Session 1: Cell death Poster 12<br />
MOUSE MODELS OF ODONTOGENESIS – FOCUSED ON CELL DEATH<br />
Eva Matalová (1,2), Tom V<strong>and</strong>en Berghe (3), Ivana Chlastáková (1,2), Eva !v<strong>and</strong>ová<br />
(1), Ivan Mí"ek (1), Jaroslav Doubek (2), Abigail S. Tucker (4)<br />
(1) Department of Physiology, University of Veterinary <strong>and</strong> Pharmaceutical Sciences,<br />
Palackého 1-3, 612 42, Brno, Czech Republic, (2) Institute of Physiology <strong>and</strong> Genetics<br />
CAS, v.v.i, Veve#í 97, 602 00 Brno, Czech Republic, (3) Department for Molecular<br />
Biomedical Research, Ghent University, Technologiepark 927B, 9052 Ghent, (4)<br />
Department of Craniofacial Development, King´s College London, Guy´s Hospital, SE1<br />
9RT London, UK<br />
matalova@iach.cz, Tom.V<strong>and</strong>enberghe@dmbr.VIB-UGent.be,<br />
ivanachlastakova@seznam.cz, 184576@mail.muni.cz, misek@iach.cz, doubekj@vfu.cz,<br />
abigail.tucker@kcl.ac.uk<br />
Abnormal number or shape of teeth is the most common congenital malformation in humans.<br />
Dentition defects may occur as isolated disorder, or as components of many hereditary syndromes.<br />
Molecular networks underlying tooth formation are mostly studied in the mouse, the classical<br />
experimental model is the mouse first molar (M1). Knowledge obtained in the mouse has been<br />
successfully extrapolated to human odontogenesis, <strong>and</strong> related disorders connected with altered tooth<br />
number, size, structure <strong>and</strong> shape found in human dentistry (1). Over 20 % of human population miss<br />
one or more third molars; 5 % lack other permanent tooth.<br />
Here, we studied morphogenesis of the mouse third molar (M3) with a special interest in signalling<br />
centres, the enamel knots. These specific populations are known to release mitotic signals related to<br />
tooth shaping, particularly transition from the bud to the cap stage (primary enamel knot, PEK). The<br />
PEK cells do not proliferate <strong>and</strong> undergo apoptosis after fulfilling their missing. PEK in M3 was found<br />
as a small cluster of concentrically arranged cells in the posterior part of the enamel organ at the<br />
postnatal day 3. The presence of the PEK was confirmed by in situ hybridisation for Fgf-4 <strong>and</strong> Shh.<br />
Apoptotic bodies were evident, accompanying the gradual elimination of the PEK. PEK cells were<br />
PCNA (proliferating cell nuclear antigen) negative.<br />
PEK apoptosis was shown to be caspase dependent. In our previous studies (2,3), caspase-9 (along<br />
with Apaf-1) <strong>and</strong> caspase-3 were demonstrated to be essential for PEK apoptosis. In this work, another<br />
member of so called death trio of execution caspases (3-6-7), caspase-7, was investigated.<br />
Immunohistochemistry (IHC) of active caspase-7 was used in serial histological sections of mouse<br />
heads of the caspase-7 knock-out <strong>and</strong> wild type mice. Caspase-7 activation was evaluated along with<br />
morphology (apoptotic bodies), TUNEL test (apoptotic DNA breaks), caspase-3 activation (IHC) <strong>and</strong><br />
cell proliferation (IHC of PCNA). Even when caspase-3 <strong>and</strong> caspase-7 are often considered as<br />
functionally redundant, this does not seem to apply for PEK apoptosis. As caspase-3 deficiency<br />
inhibited PEK apoptosis, lack of caspase-7 did not cause any alterations. However, interestingly,<br />
active caspase-7 was found in osteoclasts in areas forming the bony socket of developing molar tooth,<br />
moreover, also in osteoclasts not undergoing apoptosis (TUNEL negative). These findings suggest<br />
possible involvement of caspase-7 in osteoclast differentiation as reported for caspase-3 (4).<br />
(1) Fleischmannova et al. (2008) Eur J Oral Sci 116, 1-10.<br />
(2) Setkova et al. (2007) Arch Oral Biol 52: 15-19<br />
(3) Matalová et al. (2006) Int J Dev Biol 50: 491-497<br />
(4) Szymczyk et al. (2006) J Cell Physiol 209: 836-844<br />
The research was funded by the GAAV project IAA600450904.<br />
78
Session 1: Cell death Poster 13<br />
Nitric oxide, P-glycoprotein <strong>and</strong> calreticulin: three mediators of<br />
chemoimmunoresistance in human tumors.<br />
Chiara Riganti, Joanna Kopecka, Dario Ghigo, Amalia Bosia<br />
Dep. of Genetics, Biology <strong>and</strong> Biochemistry, University of Turin, via Santena 5/bis,<br />
10126, Turin, Italy; emails: chiara.riganti@unito.it; joanna.kopecka@unito.it;<br />
dario.ghigo@unito.it; amalia.bosia@unito.it<br />
A great obstacle in the successful treatment of tumors is multidrug resistance (MDR), due to<br />
the overexpression of membrane pumps, like P-glycoprotein (Pgp) <strong>and</strong> MDR-related proteins<br />
(MRPs), which mediate the efflux of several anticancer drugs. Doxorubicin is a versatile<br />
chemotherapeutic agent, because exerts both direct cytotoxic <strong>and</strong> indirect pro-immunogenic<br />
cell death; unfortunately it is a substrate of Pgp <strong>and</strong> MRPs.<br />
We show that in chemosensitive cancer cells doxorubicin up-regulates the inducible nitric<br />
oxide (NO) synthase (iNOS) gene <strong>and</strong> the synthesis of NO. The latter mediates part of the<br />
doxorubicin cytotoxicity; induces chemosensitization (by reducing the efflux activity of Pgp<br />
<strong>and</strong> MRP3, through a direct nitration of critical tyrosines on the transporters); plays a role in<br />
the doxorubicin pro-immunogenic effects, promoting - via the cGMP/protein kinase G<br />
pathway <strong>and</strong> the actin cytoskeleton remodelling - the surface translocation of calreticulin<br />
(CRT) <strong>and</strong> triggering the tumor cells phagocytosis by dendritic cells (DCs). The lack of NO<br />
synthesis is responsible for a chemo-immunoresistant phenotype: in drug-resistant cells, as<br />
well as in drug-sensitive cells stably silenced for iNOS, doxorubicin is extruded <strong>and</strong> does not<br />
induce direct tumor cell death nor translocation of CRT. These events however are restored<br />
by using NO donors, iNOS inducers other than doxorubicin or doxorubicins synthetically<br />
conjugated with NO-releasing groups. Unexpectedly, despite high levels of surface CRT, the<br />
DCs-mediated phagocytosis does not occur in chemoresistant cells, where CRT is bound by<br />
Pgp in endoplasmic reticulum <strong>and</strong> translocates with it on plasma-membrane. Since the<br />
phagocytosis is restored only after silencing Pgp, we hypothesize that CRT-Pgp interaction<br />
impairs the functional activity of CRT <strong>and</strong> that Pgp may represent not only a marker of<br />
chemoresistance, but also an immunosuppressive signal.<br />
Our findings show that chemo- <strong>and</strong> immunoresistance are associated in human tumors <strong>and</strong><br />
could rely on low levels of NO, low levels of CRT <strong>and</strong> high levels of Pgp. The modulation of<br />
their amounts may lead to new strategies in chemo-immunotherapy protocols.<br />
79
Session 1: Cell death Poster 14<br />
Pathways of cell death induced by nanoparticles<br />
Fengjuan Wang, Nikolai Slavov, Iseult Lynch, , Anna Salvati, Kenneth A. Dawson<br />
Centre for BioNano Interactions, University College Dublin, Belfield, Dublin 4, Irel<strong>and</strong><br />
Fengjuan.Wang@cbni.ucd.ie, nslavov.princeton@gmail.com, Iseult.Lynch@cbni.ucd.ie,<br />
Anna.Salvati@cbni.ucd.ie, Kenneth.A.Dawson@cbni.ucd.ie<br />
Nanoparticles composed of very different materials can enter most cells easily, <strong>and</strong> those<br />
without a targeting moiety are often seen to accumulate in lysosomes, with no clear evidence<br />
of their subsequent export or degradation. 1, 2 Very different outcomes for the cell can be<br />
observed, depending on the nature of the material: in many cases no alteration of cellular<br />
functions can be detected, whereas in other specific cases, nanoparticles can induce<br />
programmed cell death.<br />
The capability of activating very different pathways, including cell death, together with their<br />
small size which allows them to enter almost everywhere in living organisms <strong>and</strong> inside the<br />
cells, makes nanoparticles a powerful tool for targeted therapeutics.<br />
Our hypothesis is that nanoparticle uptake, trafficking <strong>and</strong> impact on cellular functions are<br />
governed by the properties of the nanoparticles (such as surface charge) together with the<br />
nature of the adsorbed proteins, as once in contact with biological fluids, such as serum,<br />
nanoparticles get coated with a very specific layer of proteins, the so-called protein corona,<br />
which constitutes a new biological identity interacting with the cellular machinery.<br />
Where cell death is induced following uptake of specific nanoparticles, a time resolved study<br />
of the pathways <strong>and</strong> signals activated by the nanoparticles as they travel to the lysosomes has<br />
been obtained, which includes mitochondrial <strong>and</strong> lysosomal damage, with activation of<br />
apoptosis <strong>and</strong> autophagy. Gene arrays over the same time frame allow us to begin to untangle<br />
<strong>and</strong> describe the activated pathways at system level.<br />
1. Shapero K, Fenaroli F, Lynch I, Cottell DC, Salvati A, Dawson KA. Time <strong>and</strong> space<br />
resolved uptake study of silica nanoparticles by human cells. Molecul BioSyst 2010; DOI:<br />
10.1039/C0MB00109K:<br />
2. Lesniak A, Campbell A, Monopoli M, Lynch I, Salvati A, Dawson KA. Serum Heat<br />
Inactivation Affects Protein Corona Composition And Nanoparticle Uptake. Biomater 2010;<br />
31: 9511-9518.<br />
3. Cedervall T, Lynch I, Lindman S, Berggard T, Thulin E, Nilsson H, Dawson KA,<br />
Linse S. Underst<strong>and</strong>ing the nanoparticle-protein corona using methods to quantify exchange<br />
rates <strong>and</strong> affinities of proteins for nanoparticles. Proc Natl Acad Sci USA 2007; 104: 2050-<br />
2055.<br />
4. Lynch I, Salvati A, Dawson KA. Protein-nanoparticle interactions: What does the cell<br />
see? Nat Nanotech 2009; 4: 546-547.<br />
80<br />
3, 4
Session 1: Cell death Poster 15<br />
�<br />
ATP regulated Apoptosis versus Autophagy in Cultured Dendritic Cells.<br />
Sarah Flacke 1 , Patricia Schilling 1 , Fengguang Liu 1 , Karl J. Foehr 1 , Manfred Weiss, Paul<br />
Walther 2 , E. Marion Schneider 1<br />
1 Sektion Experimentelle Anaesthesiologie, University Hospital Ulm, Steinhoevelstr. 9, 89075<br />
Ulm, Germany; 2 Electron Microscopy Facility, Ulm University, Ulm, Germany<br />
Cell stress may lead to the release of nucleotides in many tissues. Specifically, fluid shear, cell<br />
stretching, hypoxia, osmotic swelling, but also temperature-related stimuli induce ATP release in<br />
addition to other nucleotides. Extracellular nucleotides bind to cell surface P2 receptors, either P2X,<br />
which are ATP-gated nonselective cation channels, or P2Y, which are G-protein-coupled receptors.<br />
There are seven subtypes of P2X (P2X1–7) <strong>and</strong> eight subtypes of P2Y (P2Y1, -2, -4, -6, <strong>and</strong> -11–14)<br />
receptors currently identified in mammals. A major effect occurs by ATP following stimulation of the<br />
P2RX7 receptor leading to extensive cell blebbing in addition to pore formation, facilitating the<br />
transport of molecules as large as 900 kD. Amongst cells of the hematopoietic system, macrophages<br />
<strong>and</strong> dendritic cells express P2RX7 receptors at high density. The expression of P2RX7 as well as its<br />
functional activity can be further increased by in vitro culture in the absence of exogenous growth<br />
factors <strong>and</strong> cytokines. Using cell biology methods, patch clamping, caspase 3/7 <strong>and</strong> caspase 8<br />
measurements in lysates, <strong>and</strong> or LC-3 staining by flow cytometry, we determined the effect of<br />
extracellular ATP on cultured dendritic cells from patients with macrophage activation syndromes<br />
(MAS). In contrast to healthy donors, MAS patients were unique by their increased precursor<br />
frequencies of P2RX7 high expressing dendritic cells in peripheral blood. We found two types of<br />
ATP- responses in selected donors: One which induced apoptosis following ATP-stimulation <strong>and</strong><br />
another mediated increased LC-3 expression <strong>and</strong> autophagy by the same ATP stimulation protocol.<br />
These differential results corresponded to a donor-specifically smaller (about 10pA/pF) or larger<br />
(about 100pA/pF) electrophysiological response as determined by patch clamping. Pyrosequencing<br />
was applied to study functional single nucleotide polymorphisms of the P2RX7 receptor in different<br />
donors. Out of 5 SNPs investigated*, the P2RX7 signal intensity as well as pro- vs. anti-apoptosis <strong>and</strong><br />
the corresponding induction of autophagy in these dendritic cells, was largely related to a 1513A!C<br />
polymorphism, responsible for a Glu496!Ala exchange in the intracellular C-terminus of the P2RX7<br />
receptor. We therefore conclude that functional SNPs of the P2RX7 receptor are responsible for<br />
dichotomous stress-induced <strong>and</strong> P2RX7-mediated response leading to either apoptosis or increased<br />
survival of immature dendritic cells. Moreover, this observation may explain a differential traumainduced<br />
stress response in vivo.<br />
*Geistlinger et al. Clin Chim Acta, in press.<br />
81
Session 1: Cell death Poster 16<br />
Involvement of AMPK signaling in liver xenobiotic-metabolizing enzymes regulation.<br />
Brice SID, Nicolas DEJEAN, Christophe GLORIEUX, Raphael BECK, Julien<br />
VERRAX <strong>and</strong> Pedro Buc CALDERON<br />
Toxicology <strong>and</strong> Cancer Biology Research Group, Louvain Drug Research Institute,<br />
Université catholique de Louvain, 1200-Brussels. BELGIUM. Email:<br />
brice.sid@uclouvain.be<br />
AMP-activated protein kinase (AMPK) is a phylogenetically conserved serine/threonine<br />
protein kinase, which has been proposed as a metabolic master switch mediating the cellular<br />
adaption to environmental or nutritional stress factors. AMPK responds to any cellular stress<br />
that threatens to lower ATP levels by arresting nonessential ATP-using functions <strong>and</strong><br />
stimulating ATP-generating pathways. Cytochrome P450 (CYPs) <strong>and</strong> Transferases are<br />
responsible for metabolism of most xenobiotics <strong>and</strong> required for the efficient elimination of<br />
foreign chemicals from the body. Paradoxically, theses enzymes may activate biologically<br />
inert compounds to eletrophilic derivatives that can cause toxicity, cell death <strong>and</strong> sometimes<br />
cellular transformation resulting in cancer. Because the regulation of theses enzymes can be<br />
affected by metabolic <strong>and</strong> nutritional stress <strong>and</strong> some studies showed that AMPK is involved<br />
in the induction of some CYPs in human hepatoma-derived cells <strong>and</strong> in primary cultures of<br />
human <strong>and</strong> mouse hepatocytes, we investigated the role of AMPK in rat liver xenobiotic<br />
metabolism regulation. In our study, we used rat precision-cut liver slices (PCLS), which are<br />
described as valuable tools for in vitro metabolic studies of drug c<strong>and</strong>idates. As compared to<br />
isolated hepatocytes, this model offers the advantages of preserving the tissue architecture <strong>and</strong><br />
the proportion of the different cell types. We showed that treatment of the slices with<br />
A769662 AMPK activator during 6h induces AMPK phosphorylation on Thr172 residue,<br />
which is essential for its activity as well as downstream Ser79 phosphorylation of acetyl-<br />
CoA-carboxylase (ACC), a metabolic target of AMPK. In parallel, A769662 treatment<br />
significantly decreases CYP2E1 <strong>and</strong> glucuronosyltransferases (UGT) activities without<br />
altering ATP content <strong>and</strong> lactate deshydrogenase leakage. These data suggest an essential role<br />
of AMPK in the liver xenobiotic metabolism regulation.<br />
82
Session 1: Cell death Poster 17<br />
Increase in p21 CDKN1A protein levels by protesome inhibitors does not affect the<br />
apoptotic response after DNA damage<br />
Micol Tillhon 1 , Nicoletta Pieri 1 , Ornella Cazzalini 2 , Lucia A. Stivala 2 , A. Ivana Scovassi 1 ,<br />
Ennio Prosperi 1<br />
1 Istituto di Genetica Molecolare CNR, Pavia; 2 Dipartimento di Medicina Sperimentale,<br />
Sez. Patologia generale, Università di Pavia, Via Ferrata 1, 27100 Pavia, Italy -<br />
tillhon@igm.cnr.it<br />
p21 CDKN1A is a well known cell cycle inhibitor playing also important roles in transcription<br />
regulation, apoptosis, <strong>and</strong> DNA repair. Pharmacological approaches have been developed to<br />
contrast tumor cell proliferation, by exploiting the p21 ability to induce cell cycle arrest. In<br />
this regard, both inhibitors of histone deacetylases (HDAC) <strong>and</strong> of the proteasomal<br />
machinery, have been shown to increase p21 protein levels, thereby impairing tumor cell<br />
growth. However, this type of intervention has been regarded as potentially dangerous, when<br />
applied in concomitance with typical anticancer drugs inducing DNA damage. In fact, high<br />
p21 levels have been shown to inhibit the apoptotic program <strong>and</strong> to allow the repair of DNA<br />
damage, thereby rendering tumor cells less susceptible to the killing activity of the latter<br />
drugs. Thus, p21 protein levels may greatly influence the outcome of chemotherapy. We have<br />
previously found that HDAC inhibitors did not significantly affect initiation of nucleotide<br />
excision repair (NER) in the presence of p21 protein. In contrast, no clear information is<br />
available on the effect of proteasome inhibitors on DNA repair. Here, we have investigated<br />
whether the presence of elevated p21 protein levels induced by the proteasome inhibitor<br />
MG132, may affect the NER <strong>and</strong> the apoptotic process. We have analyzed the recruitment of<br />
NER proteins <strong>and</strong> p21 to localized DNA damage sites, <strong>and</strong> determined NER efficiency in<br />
normal, as well as in p21-null human fibroblasts. The results have shown that MG132 induced<br />
the persistence of XPC, PCNA <strong>and</strong> p21 protein, at local DNA damage sites. The persistence<br />
of p21 at DNA damage sites did not significantly affect the recruitment of other PCNAinteracting<br />
NER factors, like DNA polymerase delta. However, NER efficiency was reduced<br />
<strong>and</strong> cell death was increased by the proteasome inhibitor. These results suggest that MG132<br />
affects NER efficiency <strong>and</strong> induce apototic cell death, independently of the presence of p21<br />
protein. [Work supported by AIRC].<br />
83
Session 1: Cell death Poster 18<br />
Mechanistic study on protective activity of humanins.<br />
Zapa!a B 1 , Góralska J 1 , Knapp A 1 , "liwa A 1 , Awsiuk M 1 , Tjelle TE 2 , Wybra#ska I 1 ,<br />
Polus A 1 ; Rustan AC 3 , Blomhoff R 2 , Berge RK 4 , Dembi#ska-Kie$ A 1 .<br />
1<br />
Department of Clinical Biochemistry Jagiellonian University Medical College, Krakow,<br />
Pol<strong>and</strong><br />
2<br />
Department of Nutrition Institute of Basic Medical Sciences University of Oslo, Norway<br />
3<br />
Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo,<br />
Norway<br />
4<br />
Institute of Medicine, Section of Medical Biochemistry, University of Bergen, Norway<br />
Humanin (HN), the 24-amino acid peptide, known as a potent intracellular inhibitor of<br />
apoptosis, interacts with Bax, the member of the Bcl2 proapoptotic protein family. Increased<br />
expression of HN peptide observed in the AD brain <strong>and</strong> skeletal muscles cells from patients<br />
with mitochondrial disorders suggests that HN plays a role in modification of pathological<br />
events such as neurodegeneration, inflammation, or energy deficiency.<br />
The aim of the study was to examine the influence of exogenous HN <strong>and</strong> its derivatives:<br />
HNG, 13ThrHN10b <strong>and</strong> 13IleHN10b on mitochondria-dependent antiapoptotic mechanisms<br />
in cellular (LN-18, Daoy, C8-D1A HUVEC,) as well as animal transgenic models of<br />
inflammatory response (NFkß-luciferase reporter mice <strong>and</strong> the human TNF-! overexpressed<br />
mice).<br />
HN <strong>and</strong> its synthetic isoforms did not influence directly the changes in mitochondrial<br />
permeability (BD Bioimager 855 microscopy <strong>and</strong> Canto II flow cytometry), oxygen<br />
consumption (Oxygraph-2k, OROBOROS), mitochondrial ATP generation (ATPliteTM<br />
Luminescence ATP Detection Assay System, Perkin Elmer) or oxidation of energy substrates<br />
( 14 C-glucose <strong>and</strong> 14 C-oleic acid) (Microbeta, PerkinElmer) in cells. The mitochondrial<br />
megachannel activation (Hewlett Packard 8452A) <strong>and</strong> calcium retention capacity<br />
(extramitochondrial calcium ([Ca 2+ ]e) of isolated rat liver mitochondria was also not<br />
influenced by HN peptides.<br />
The apoptotic gene expression (TaqMan Applied Microarray system, TLDA) in the brain cell<br />
lines pointed to the higher than endothelial cells antiapoptotic potential. HN peptides<br />
modulated the antiapoptotic potential not only by influencing Bcl/Bax genes expression ratio.<br />
Results from microarray analyses in brain from mice treated with HNG <strong>and</strong> challenged with<br />
LPS showed that HNG down regulated numerous gene sets associated with apoptosis <strong>and</strong><br />
neurogenerative processes (GSEA analyses). In NFkß-luciferase reporter mouse model there<br />
were a tendency that HNG inhibited NFkB-regulated luciferase activity, although not<br />
significant, in heart <strong>and</strong> brain. Moreover in TNF-! overexpressed mice the reduction of<br />
arthritis development (confirmed by forelimb grip test) pointed to the possible<br />
antiinflammatory activity of HN. However, the observed effects seem to be stressordependent,<br />
cell-dependent as well as dependent on humanin peptide structure.<br />
The study was supported by Polish-Norwegian grant no. PNRF-104-AI-1/07 <strong>and</strong> Polish grant<br />
no. k/pbp/000318<br />
84
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 2: Cell signaling<br />
85
Session 2: Cell signaling Poster 1<br />
NT-S100A8 INHIBITS INSULIN RELEASE AND ACTIVATES AKT AND NF-KB<br />
CELL SIGNALLING IN PANCREATIC CANCER (PC) CELLS<br />
Daniela Basso, Paola Fogar, Andrea Padoan,Stefania Moz, Dania Bozzato, Elisa Fadi,<br />
Eliana Greco, Filippo Navaglia, Carlo-Federico Zambon <strong>and</strong> Mario Plebani.<br />
Department of Laboratory Medicine, University of Padua, Via Giustiniani 2, 35128<br />
Padova, Italy. E-mail: biolmol@unipd.it<br />
Objectives: to verify whether NT-S100A8, a peptide isolated from PC tissue of diabetic<br />
patients:1) alters Akt <strong>and</strong> NFkB signalling in PC cells, 2) interferes with insulin (Ins)<br />
signalling, 3) alters Ins response to glucose stimulation.<br />
Methods: PC cell lines (BxPC3,Capan1,MiaPaCa2) remained unstimulated or were stimulated<br />
with 50 mU Ins for 10 min with/without 50 <strong>and</strong> 500 nM NT-S100A8 for 5,10,15 <strong>and</strong> 30 min.<br />
Cell lysates immunoblots were performed with pIkBa, pAkt (Ser 473 ,Thr 308 ), Akt, pPDK1<br />
(Ser 241 ), p-mTOR (Ser 2448 ), b-actin antibodies. bTC6 rat insulinoma cells were untreated or<br />
treated 30 min daily for 1 week with 50, 200 <strong>and</strong> 500 nM NT-S100A8 plus 20 mM glucose or<br />
with 20 mM glucose alone. Cells were then stimulated with 20 mM glucose <strong>and</strong> Ins was<br />
measured at 2,3,5,10,15,30 min.<br />
Results: both Ins <strong>and</strong> NT-S100A8 independently induced Akt Ser 473 phosphorylation in<br />
BxPC3 <strong>and</strong> MiaPaCa2, not in Capan1. Akt Thr 308 phosphorylation in all PC cell lines was<br />
induced by Ins, not by NT-S100A8. NT-S100A8 time <strong>and</strong> dose-dependently induced pmTOR<br />
in BxPC3, but it did not induce pPDK1. To study NFkB signalling we assessed the<br />
phosphorylation of its cytoplasmic inhibitor IkBa. In all cell lines IkBa was constitutively<br />
phosphorylated. Ins determined a significant reduction of pIkBa in Capan1 <strong>and</strong> MiaPaCa2<br />
<strong>and</strong> an enhancement in BxPC3, effects not counteracted by NT-S100A8. In BxPC3 <strong>and</strong><br />
Capan1, NT-S100A8 caused an increase in pIkBa after 5min, followed by a reduction at 10<br />
<strong>and</strong> 15min <strong>and</strong> a recovery at 30 min. pIkBa in MiaPaCa2 cells was not modified. Glucose<br />
induced early (2min) <strong>and</strong> late (15-30min) Ins release in control bTC6 cells. The amount of Ins<br />
release was progressively reduced when cells were stimulated with glucose for one week <strong>and</strong><br />
almost completely abolished when glucose was given with NT-S100A8 (Repeated measures<br />
ANOVA: p
Session 2: Cell signaling Poster 2<br />
NPHS2 GENE MUTATION ANALYSIS RESULTS OF 631 TURKISH PATIENTS<br />
WITH STEROID RESISTANT NEPHROTIC SYNDROME<br />
AFIG BERDELI 1 , SINEM NALBANTOGLU 1 , DEMET TIGLI 1 , MERVE ATAN 1 ,<br />
PERVIN TOPARLAK 1 , AYBEN PASOLAR 1 , ILKAY DEMIREL 1 , FATMA<br />
AKYIGIT 1 , SEVGI MIR 2<br />
1. Ege University Faculty of Medicine, Child Hospital Molecular Medicine Laboratory,<br />
35100, Bornova, izmir, Turkey.<br />
2. Ege University Faculty of Medicine, Department of Pediatrics, Pediatric Nephrology<br />
Division, 35100, Bornova, izmir, Turkey.<br />
NPHS2 gene, encoding the 24-KDa slit diaphragm localized glomerular protein podocin, is<br />
mutated in autosomal recessive steroid-resistant nephrotic syndrome (SRNS) which is<br />
characterized by the presence of the central findings of proteinuria, hypoalbuminemia, <strong>and</strong><br />
edema. Mutations in NPHS2 encoding podocin are found as the prevalent cause of steroidresistant<br />
nephrotic syndrome.<br />
In this respect, we performed mutation analysis by DNA sequencing analysis of all coding<br />
exons <strong>and</strong> intron/exon boundaries of the NPHS2 gene in a total of 631 children patient group<br />
clinically diagnosed with SRNS (of which 66 of them were familial <strong>and</strong> the remaining 565<br />
were sporadic cases). A total of 100 healthy children were involved in the mutation scanning<br />
analysis for healthy control group.<br />
According to the mutation analysis results of NPHS2 gene; 18% of the patients were<br />
evaluated as mutation positive NPHS2 group (n=112) in which this rate corresponds to 31%<br />
of the familial cases, <strong>and</strong> to 17% of the sporadic cases group. In the mutation positive NPHS2<br />
group, most of the mutations were found out to harbor in exons 1, 4, <strong>and</strong> 5 while no mutation<br />
were found in exon 6 of the respective gene. A total of 46 mutations were determined in the<br />
NPHS2 gene, <strong>and</strong> 32 of which were characterized as a novel mutation. The mutation rate of<br />
R138Q was determined as 3.7% in our study group contrary to the findings of Europe <strong>and</strong><br />
Israil populations in which the observed frequency was 50%. Additionally, P118L, R138X,<br />
R168H, R138Q, IVS7+5G>A, A212T, c.460-467insT, c.503delG, H228D, S211A, <strong>and</strong><br />
V218G mutations were typically found as the cause of end-stage renal disease (ESRD). For<br />
the remaining 519 mutation negative patients, effects of possible modifier loci involving other<br />
podocin interacting proteins may be considered.<br />
In brief, as a causative SRNS gene, we should consider NPHS2 gene mutation<br />
screening in early diagnosis <strong>and</strong> the follow-up of the clinical course. In relation to<br />
homozygous or compound heterozygous NPHS2 mutated patients who have the lack of<br />
response to st<strong>and</strong>ard steroid therapy we suggest to perform NPHS2 gene mutation analysis for<br />
every child (if consent can be obtained) soon after the first episode of SRNS. For the newly<br />
diagnosed patients, the crucial certain determination of the causative disease gene mutation<br />
will enable clinicians to avoid redundant immunosuppressive therapeutic trials.<br />
87
Session 2: Cell signaling Poster 3<br />
Laminin 5 promotes epithelial mesenchymal transition (EMT) through phosphorylation<br />
– dependent integrin linked kinase activation.<br />
Dorota Gil, Joanna Dulinska-Litewka <strong>and</strong> Piotr Laidler<br />
Chair of Medical Biochemistry Jagiellonian University Medical College, Kraków,<br />
Pol<strong>and</strong>; ul.Kopernika 7, 31-034 Kraków, e-mail: dorotabeata.gil@uj.edu.pl<br />
Integrin linked kinase (ILK) is a serine/threonine protein kinase that regulates physiological<br />
number of cellular function. ILK is a binding partner of the beta1 <strong>and</strong> beta3 integrin subunit<br />
as a cytoplasmic effector of integrin receptors. Upon activation by different stimuli ILK<br />
phosphorylates Akt <strong>and</strong> GSK-3beta <strong>and</strong> triggers in the related signaling cascades. One of their<br />
major roles is to regulate beta-catenin transcriptional activity. In addition Akt <strong>and</strong> GSK-3<br />
have been reported to activate the NF-kB p65 subunit, increasing the binding of the NF-kB<br />
complex to DNA or inactivates the NF-kB signaling, respectively. We postulate that ILK is a<br />
major signaling mediator involved in EMT, the critical event in the process of cancer<br />
progression. Integrin-mediated cell adhesion is known to regulate the expression of number of<br />
genes. There is no integrin alpha chain associated with beta1 that is as yet known to be<br />
specifically involved in EMT <strong>and</strong> the molecular mechanism through which ECM regulates<br />
EMT are undefined. Some studies suggests however, that alpha3beta1 intergin (laminin 5<br />
receptor) is required for accumulation of beta-catenin in the nucleus of cells undergoing EMT.<br />
We demonstrated that ILK is in human melanoma cells involved in signal transduction from<br />
integrin alpha3beta1 as its activation by laminin5 induced translocation of beta-catenin <strong>and</strong><br />
NF-kB (p65) to nucleus <strong>and</strong> this phenomenon was mediated through ILK phosporylation at S-<br />
343 <strong>and</strong> T -173 <strong>and</strong> by phosphorylation of IKKB. We were also able to show that silencing<br />
of ILK expression by siRNA significantly abolished the presence of molecular <strong>and</strong><br />
phenotypic markers for EMT. In addition NF-kB (p65) nuclear translocation <strong>and</strong> beta-catenin<br />
activation were completely abolished upon ILK silencing in melanoma cells.<br />
It is a first report that shows regulation of ILK by direct phosphorylation.<br />
This work is supported by: MNiSW through Jagiellonian University Medical College<br />
K/ZDS/001464<br />
88
Session 2: Cell signaling Poster 4<br />
Establishing a pipeline for the determination of cell line <strong>and</strong> experiment specific gene<br />
regulatory networks<br />
Thorben Kaetzel, Fabrice Tolle, Jean-Luc Bueb, Thomas Sauter.<br />
<strong>Inflammation</strong> group/Systems Biology group; Life Sciences Research Unit; Faculty of<br />
Science, Technology <strong>and</strong> Communication; University of Luxembourg, 162A, Avenue de<br />
la Faïencerie, L-1511 Luxembourg. Contact: thorben.kaetzel@uni.lu<br />
A bioinformatics pipeline was established for the fast <strong>and</strong> convenient extraction of cell line-<br />
<strong>and</strong> experiment specific c<strong>and</strong>idate networks based on public <strong>and</strong> own microarray data sets <strong>and</strong><br />
by integration of additional available interaction data. This automated workflow allows for<br />
obtaining such networks in a few steps <strong>and</strong> helps to identify nodes, respectively genes which<br />
play significant roles in signalling pathways of interest. Two filtering steps are applied<br />
therefore: 1) cell type specific array data available from public databases is used for coexpression<br />
analysis (assuming that co-expressed genes are also co-regulated) to identify cell<br />
type specific clusters <strong>and</strong> interactions in general; 2) additional (own) stimulation data of<br />
specific expression pro<strong>file</strong>s is applied then to further filter the co-expression network. Only<br />
genes with a fold change over <strong>and</strong> under a certain threshold were depicted therefore <strong>and</strong><br />
included in the c<strong>and</strong>idate networks. To avoid the exclusion of important central nodes of the<br />
network during this filtering step, IPA Knowledge Base (Ingenuity) was used to ensure the<br />
significance <strong>and</strong> completeness of the networks. The curated <strong>and</strong> filtered c<strong>and</strong>idate networks<br />
can then be subjected to mathematical modelling <strong>and</strong> further analysis using Probabilistic<br />
Boolean Modelling. The pipeline was applied to atherosclerosis related microarray data from<br />
two cell lines (THP-1 monocytes <strong>and</strong> HUVEC endothelial cells) which were chosen to mimic<br />
monocytic <strong>and</strong> endothelial behaviour during the process of development of atherosclerosis.<br />
Both cells were stimulated during 1, 4 <strong>and</strong> 24 hours with the cytokine TNF-alpha <strong>and</strong>/or the<br />
micronutrient 1alpha,25(OH)2 vitamin D3, looking for interactions between stress-sensing<br />
nuclear factors <strong>and</strong> nutrient-sensing nuclear receptors which could, by a lifetime exposure, be<br />
part of a misbalanced molecular process towards atherosclerosis.<br />
In a first attempt, two co-expression networks could be extracted for THP-1 <strong>and</strong> HUVEC. In<br />
case of HUVEC, a 1500 node co-expression network could be filtered as described above <strong>and</strong><br />
resulted in a final core network of 84 genes.<br />
89
Session 2: Cell signaling Poster 5<br />
Cooperativity of the MUC1 <strong>and</strong> HGF/c-Met Pathway in HCC<br />
Giray BOZKAYA 1* , Peyda KORHAN 1* , Murat COKAKLI 1 , Esra ERDAL 1 , Ozgul<br />
SAGOL 2 , Sedat KARADEMIR 3 , Cristopher KORCH 4 , Nese ATABEY 1**<br />
1 Dokuz Eylul University, School of Medicine, Department of Medical Biology <strong>and</strong> Genetics, 35340,<br />
Balcova-Izmir, Turkey, korhanpeyda@hotmail.com<br />
2 Dokuz Eylul University, School of Medicine, Department of Pathology, 35340, Balcova-Izmir, Turkey<br />
3 Dokuz Eylul University, School of Medicine, Department of Surgery, 35340, Balcova-Izmir, Turkey<br />
4 University of Colorado Cancer Center-DNA Sequencing <strong>and</strong> Analysis Core Aurora, CO, USA<br />
* Equal contribution **Corresponding author<br />
Hepatocyte growth factor (HGF) induced c-Met activation is known as the main stimulus for<br />
hepatocyte proliferation <strong>and</strong> essential for liver development <strong>and</strong> regeneration. Inappropriate<br />
HGF/c-Met signaling has been correlated with aggressive phenotype <strong>and</strong> poor prognosis in<br />
hepatocellular carcinoma (HCC). MUC1 is a transmembrane mucin whose overexpression is<br />
also correlated with poor prognosis in HCC. However, involvement of MUC1 in HGF/c-Met<br />
signaling is unknown in HCC. In this study we aimed to elucidate possible cooperation of<br />
MUC1 <strong>and</strong> c-Met expressions <strong>and</strong> their association in HCC. We first analyzed the expression<br />
of MUC1 <strong>and</strong> c-Met receptor in poorly differentiated (SNU-475, SNU-449, Mahlavu) <strong>and</strong><br />
well differentiated (HuH-7, Hep3B, Hep G2) HCC cell lines by immunoblotting. We showed<br />
that poorly differentiated HCC cell lines had high expression of both MUC1 <strong>and</strong> c-Met,<br />
whereas well differentiated ones revealed no MUC1 expression <strong>and</strong> lower level of c-Met<br />
expression compared to poorly differentiated ones. We then demonstrated a physical<br />
interaction between c-Met <strong>and</strong> MUC1 in Mahlavu cells by immunoprecipitation.<br />
Interestingly, c-Met activation reduced MUC1 protein level which resulted in decreased<br />
MUC1/c-Met interaction in a time dependent manner. Using SU11274, we restored HGF<br />
induced MUC1 downregulation <strong>and</strong> established that MUC1 downregulation was mediated by<br />
c-Met activation in our experimental system. Intriguingly, siRNA silencing of MUC1 resulted<br />
in enhanced c-Met activation. Lastly we demonstrated that c-Met inhibited tumor cells<br />
exhibited poor migrative <strong>and</strong> invasive ability in response to HGF, whereas MUC1 silencing<br />
increased HGF induced cell motility <strong>and</strong> invasion. Overall, these results suggest that coexpression<br />
<strong>and</strong>/or association of MUC1 <strong>and</strong> c-Met might affect c-Met mediated signaling in<br />
HCC.<br />
90
Session 2: Cell signaling Poster 6<br />
LGR5 has been regulated by Wnt/!-catenin signaling in HCC cells<br />
Imge Kunter 1 , Ne"e Atabey 1 , Esra Erdal 1<br />
1 Dokuz Eylul University, Faculty of Medicine, Department of Medical Biology <strong>and</strong><br />
Genetics, #zmir, Turkey.<br />
Leucine-rich repeat containing G-protein coupled receptor 5 (LGR5) recently has been<br />
described as a stem cell marker in gastrointestinal organs <strong>and</strong> it has been defined as a Wnt/!catenin<br />
target in colon carcinoma. Although overexpression of LGR5 has been observed in<br />
the hepatocellular carcinoma containing !-catenin mutations, there is no enough data<br />
to elucidate the role of LGR5 in liver cancer. In this study expression of LGR5 has<br />
been examined in HCC cell lines (Huh7, Hep3B, HepG2, SNU398, PLC, SNU 475, SNU449,<br />
SKHep1, SNU387, SNU 182) at both transcriptional <strong>and</strong> protein levels. Out of ten cell lines,<br />
HepG2 <strong>and</strong> SNU398 only have overexpression for LGR5 at protein level as expected since<br />
both cell lines contain distinct !-catenin overexpression mutations. When wnt/ !-catenin<br />
signaling has been activated by lithium treatment, expression of LGR5 has been increased<br />
significantly in Huh7, Hep3B <strong>and</strong> PLC cell lines. Additionally, the induction of the Wnt/!catenin<br />
signaling through transfection of the BCAT overexpression plasmid vector leads to<br />
increase in the LGR5 expression, contrarily, diminishing !-catenin activity on Tcf/Lef<br />
transcription through transfection of "NTCF plasmid vector leads to decrease in the LGR5<br />
expression in Huh7 cells. This data provides us evidence that LGR5 may be a target gene of<br />
Wnt/!-catenin signaling pathway in HCC. LGR5 might be very important gene to describe the<br />
initiation mechanisms in the development of HCC.<br />
91
Session 2: Cell signaling Poster 7<br />
Double suppression of the Galpha activity by RGS proteins<br />
Chen Lin 1, 2 1, 3<br />
<strong>and</strong> Vladimir.Katanaev<br />
1 University of Konstanz, Department of Biology, Universitätsstrasse 10, Box 643, 78457<br />
Konstanz, Germany.<br />
konstanz.de<br />
2 Chen.Lin@uni-konstanz.de<br />
92<br />
3 Vladimir.Katanaev@uni-<br />
Signaling molecules such as neurotransmitters, hormones <strong>and</strong> other extracellular lig<strong>and</strong>s act<br />
via G-protein-coupled receptors to regulate crucial functions in animal physiology <strong>and</strong><br />
development. Regulator of G-protein signaling (RGS) proteins are a large <strong>and</strong> diverse<br />
superfamily characteristic by the conserved RGS domain which possesses the GAP (GTPase<br />
activating protein) activity towards G!-subunits of heterotrimeric G proteins, resulting in<br />
acceleration of the GTP hydrolysis <strong>and</strong> faster deactivation of G protein signaling. By using<br />
the yeast two-hybrid screening, we found CG5036, a Drosophila member of the RGS protein<br />
RZ subgroup, as the binding partner of Drosophila G!o. Surprisingly, both in the yeast twohybrid<br />
system as well as in the biochemical pull-down experiments, CG5036 interacted with<br />
both the GTP - <strong>and</strong> GDP - loaded forms of G!o. Furthermore, in addition to GAP activity,<br />
CG5036 possessed the GDI (guanine nucleotide dissociation inhibitor) activity towards G!o<br />
in vitro. These findings were recapitulated using mammalian G!o <strong>and</strong> G!i, as well as RGS19<br />
proteins. To confirm the biological significance of this unexpected GDI activity, we began to<br />
downregulate CG5036 function during the asymmetric cell divisions leading to the<br />
development of the peripheral sensory bristles of Drosophila - the developmental program<br />
coordinately regulated by the G!o <strong>and</strong> G!i proteins. We found that downregulation of<br />
CG5036 could enhance the dominant phenotypes induced not only by expression of the wildtype<br />
form of the G proteins, but also by the GTPase-deficient mutant form of G!o. Thus, our<br />
results provide the first in vitro <strong>and</strong> in vivo data for the double mechanism of action of an<br />
RGS protein towards G!-subunits, involving both inhibition of GTP incorporation by G! <strong>and</strong><br />
the acceleration of GTP hydrolysis by it.
Session 2: Cell signaling Poster 8<br />
The trimeric Go protein transduces the Wingless-Frizzled2 signal <strong>and</strong> interacts with<br />
Anykrin2 to regulate microtubule stability in Drosophila neuromuscular junction<br />
Anne-Marie Lüchtenborg 1,2 , Diane Egger-Adam 1,3 , &Vladimir L. Katanaev 1,4<br />
1<br />
University of Konstanz, Department of Biology, Universitätsstrasse 10, Box 643, 78457<br />
2 3<br />
Konstanz, Germany. anne-marie.luechtenborg@uni-konstanz.de, diane.eggeradam@uni-konstanz.de,<br />
4 vladimir.katanaev@uni-konstanz.de<br />
The Drosophila neuromuscular junction is a powerful model system to investigate<br />
glutamatergic synapse formation <strong>and</strong> stability. In the fruit fly the correct formation of the<br />
neuromuscular junction depends among others on components of the Wingless signal<br />
transduction pathway which acts in the transcription-independent manner in the synapse.<br />
Giant isoforms of Ankyrin2, a neuronal protein that links cytoskeleton <strong>and</strong> the membrane,<br />
have also been shown important for synapse formation. We could show that the trimeric G<br />
protein Go is heavily expressed in the neuromuscular junction <strong>and</strong> its presynaptic expression<br />
is crucial for formation of this structure. We demonstrate that Go is a transducer of the<br />
Wingless signal acting downstream from Frizzled2. In addition, the alpha subunit of Go<br />
physically <strong>and</strong> genetically interacts with Ankyrin2. Our genetic studies suggest that the<br />
Wingless signal bifurcates downstream from Frizzled2 into the Ankyrin2-dependent branch<br />
<strong>and</strong> the signaling branch involving the Axin-based destruction complex. We provide evidence<br />
that both pathways converge on the microtubule binding protein Futsch, the Drosophila<br />
homolog of the MAP1B. This elaborate mechanism results in coordinated regulation of<br />
neuromuscular junction formation.<br />
93
Session 2: Cell signaling Poster 9<br />
Microvesicles Harbouring Glycosylphosphatidylinositol-Anchored Proteins <strong>and</strong> Derived<br />
From Rat Adipocytes <strong>and</strong> Serum Transfer RNA Coding For Lipid Synthesis Into<br />
Adipocytes<br />
Günter Müller 1 , Eva-Maria Wetekam 1 , Susanne Wied 1 <strong>and</strong> Elisabeth-Ann Dearey 1<br />
1 Sanofi-Aventis Deutschl<strong>and</strong> GmbH, Research & Development, Diabetes Division,<br />
Industrial Park Höchst, Bldg. H821, 65926 Frankfurt am Main, Germany.<br />
guenter.mueller@sanofi-aventis.com<br />
Small vesicles, such as microvesicles <strong>and</strong> exosomes, have been amply documented to transfer<br />
proteins <strong>and</strong> nucleic acids from a variety of donor to acceptor cells with corresponding<br />
physiological <strong>and</strong> pathological consequences. Recently the in vitro transfer of<br />
glycosylphosphatidylinositol-anchored proteins (GPI-proteins) from vesicles released from<br />
large rodent adipocytes to intracellular lipid droplets of small adipocytes has been shown to<br />
be stimulated by certain physiological (palmitate, H2O2) <strong>and</strong> pharmacological stimuli (antidiabetic<br />
sulfonylurea drug glimepiride) <strong>and</strong> to induce the stimulation of esterification into <strong>and</strong><br />
inhibition of the release of fatty acids from triacylglycerol. Here the analysis of small vesicles<br />
derived from rat adipocytes or plasma revealed that those harbouring the GPI-proteins, Gce1<br />
<strong>and</strong> CD73, contain RNA species which are transferred into acceptor adipocytes <strong>and</strong> trigger<br />
upregulation of fatty acid esterification. The transferred RNA species are specific for fatty<br />
acid esterification (e.g. glycerol-3-phosphate acyltransferase) <strong>and</strong> lipid droplet biogenesis<br />
(perilipin-A, caveolin-1). The transfer is more efficient for small rather than large adipocytes<br />
<strong>and</strong> is significantly upregulated by palmitate, glimepiride <strong>and</strong> H2O2. Together the data<br />
suggest, that vesicles released from large adipocytes stimulate triacylglycerol storage in small<br />
adipocytes by mediating the transfer of the required information encoded by either relevant<br />
RNA <strong>and</strong> GPI-protein species. Paracrine <strong>and</strong> endocrine regulation of triacylglycerol storage<br />
<strong>and</strong> in parallel cell size between large <strong>and</strong> small adipocytes by RNA- <strong>and</strong> GPI-proteinharbouring<br />
vesicles may represent a novel target for interference with metabolic diseases,<br />
such as obesity <strong>and</strong> type II diabetes.<br />
94
Session 2: Cell signaling Poster 10<br />
MAPK signaling pathway is involved in the proliferative stimulus triggered by early<br />
weaning in the gastric mucosa<br />
Luciana H Osaki, Patricia Gama<br />
Laboratory of Gastrointestinal Epithelium Biology, Department of Cell <strong>and</strong><br />
Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Av.<br />
Prof. Lineu Prestes 1524, 05508-900, Sao Paulo, SP, Brazil, luciana.osaki@usp.br<br />
Epidermal Growth Factor Receptor (EGFR) regulates cell proliferation <strong>and</strong> differentiation by<br />
activating several signaling pathways, such as MAPK <strong>and</strong> Src. The dietary pattern is an<br />
important regulator of the growth <strong>and</strong> maturation of the stomach. Early weaning (EW), which<br />
means an abrupt change from milk to solid food ingestion, stimulates gastric cell proliferation<br />
<strong>and</strong> differentiation, in parallel with the overexpression of EGFR. ERK <strong>and</strong> Src are also more<br />
activated in EW animals, suggesting their involvement in the modifications of the gastric<br />
growth triggered by EGFR. Here, we investigated whether ERK pathway is involved in<br />
gastric cell proliferation stimulus after EW. Early weaning was performed by separating 15-dold<br />
Wistar rat pups from the dam <strong>and</strong> feeding them with powdered chow. One group of<br />
animals was injected with PD98059 at 300 !g/kg to test the effects of MAPK inhibition <strong>and</strong><br />
the control group received 0.5% DMSO. Samples were collected 2 <strong>and</strong> 3 days after the onset<br />
of treatment. We confirmed that p-ERK was decreased in the gastric mucosa after PD98059<br />
treatment (p
Session 2: Cell signaling Poster 11<br />
The Role of MT1-MMP in FGF Signaling<br />
Hoi Leong Xavier Wong, Kui Ming Chan, Zhongjun Zhou<br />
Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong<br />
Kong, Hong Kong SAR, China. xavier _1110@hotmail.com<br />
Craniosynostosis is a craniofacial defect resulting from the premature fusion of cranial<br />
sutures. It is often linked to the deregulated Fibroblast Growth Factor (FGF) Receptor<br />
signaling. Membrane Type 1-Matrix Metalloproteinase, MT1-MMP, is a membrane tethered<br />
enzyme capable of remodeling extracellular matrix. MT1-MMP knockout mice display severe<br />
craniofacial phenotypes including delayed suture closure which resembles FGF signaling<br />
mutant mice. To underst<strong>and</strong> the defects in craniofacial development in MT1-MMP deficient<br />
mice <strong>and</strong> to elucidate the molecular mechanism behind, we have examined the embryonic<br />
development of calvarial bone in MT1-MMP deficient mice. We found that MT1-MMP<br />
deficient mice exhibit defects in calvarial bone development, initiating at 15.5dpc. MT1-<br />
MMP co-expresses with FGF lig<strong>and</strong>s <strong>and</strong> FGF receptors during parietal bone development at<br />
the embryonic stage. Moreover, proliferation rate of primary calvarial osteoblast deficient in<br />
MT1-MMP is significantly reduced <strong>and</strong> FGF2 induced accelerated bone growth is also<br />
reduced in MT1-MMP -/- calvaria explants in ex vivo culture. More importantly, MT1-<br />
MMP/FGFR1 compound heterozygous mice exhibit abnormal bone phenotypes, such as<br />
shortened long bone <strong>and</strong> reduced body length, which clearly indicates the genetic interaction<br />
between mmp14 <strong>and</strong> fgfr1. Our results suggest the importance of MT1-MMP in calvarial<br />
osteogenesis via regulation of FGF signaling.<br />
96
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 3: Transcriptional control<br />
97
Session 3: Transcriptional control Poster 1<br />
Mithramycin-mediated regulation of raptor gene transcription during erythroid<br />
differentiation of K562 cells<br />
Alessia Finotti 1 , Nicoletta Bianchi 1 , Cristina Zuccato 1 , Enrica Fabbri 1 ,<br />
Monica Borgatti 1 <strong>and</strong> Roberto Gambari 1<br />
1 Laboratory for the Development of Pharmacological <strong>and</strong> Pharmacogenomic Therapy<br />
of Thalassemia, Biotechnology Center, Ferrara University, via Fossato di Mortara 64,<br />
44121, Ferrara, Italy; e-mail:gam@unife.it.<br />
The mTOR pathway might be associated with erythroid differentiation, as rapamycin (a<br />
strong <strong>and</strong> highly selective mTOR inhibitor) is able to induce erythroid differentiation of<br />
K562 cells. Here we have analyzed the effects of erythroid-inducer mithramycin (MTH) on<br />
raptor gene transcription, in consideration of the fact that raptor is an important companion of<br />
mTOR. We have found that raptor gene promoter contains at least 10 binding sites for the<br />
transcription factor Sp1. By gel retardation assay, we have demonstrated that three of these<br />
sites are bound very efficiently by Sp1. Interestingly, MTH interferes with these Sp1/DNA<br />
interactions. Moreover, chromatin immunoprecipitation assays fully support the concept that<br />
MTH inhibits in intact K562 cells the recruitment of Sp1 to the raptor gene promoter. This is<br />
associated with a strong effect of MTH on raptor gene expression. The results obtained by<br />
quantitative RT-PCR clearly indicate that content of raptor mRNA decreases during erythroid<br />
induction of human leukemia K562 cells. These data have been confirmed by western blotting<br />
also using erythroid precursor cells (ErPC) isolated from beta-thalassemic patients <strong>and</strong> treated<br />
with MTH. In this case, we always observed a reduction of raptor mRNA accumulation <strong>and</strong><br />
raptor production. Accordingly, inhibition of phosphorilated p70S6K <strong>and</strong> of ribosomal S6<br />
protein phosphorilation were found following MTH treatment.<br />
Roberto Gambari has received grants from Fondazione CARIPARO (Cassa di Risparmio di<br />
Padova e Rovigo) <strong>and</strong> Telethon (contract GGP10124). This research is also supported by The<br />
Associazione Veneta per la Lotta alla Talassemia, Rovigo.<br />
98
Session 3: Transcriptional control Poster 2<br />
Regulation of the catalase expression in a model of breast cancer MCF-7 resistant to<br />
oxidative stress.<br />
Christophe Glorieux, Julien Verrax, Nicolas Dejeans, Raphaël Beck, Brice Sid <strong>and</strong><br />
Pedro Buc Calderon.<br />
Toxicology <strong>and</strong> Cancer Biology research group, Louvain Drug Research Institute,<br />
Université catholique de Louvain, Brussels, Belgium. Email :<br />
christophe.glorieux@uclouvain.be<br />
Cancer cells are more sensitive to oxidative stress due a low level of antioxidant enzymes<br />
(catalase, glutathion peroxidase, superoxide dismutase,...). Given the increasing interest to<br />
develop pro-oxidant therapies in clinical studies by targeting intracellular redox alteration, we<br />
decided to study the molecular consequences of chronically exposing cancer cells to oxidative<br />
stress. To this end, we used an H2O2-generating system, namely the association<br />
ascorbate/menadione (asc/men) that generates an oxidative stress which ultimately causes<br />
cancer cell death. We generate breast cancer MCF-7 cells resistant to oxidative stress<br />
(asc/men) in order to underst<strong>and</strong> the mechanism of resistance acquisition. In these resistant<br />
cells we founded an increased expression of catalase. We decided to explore how the catalase<br />
gene is regulated in these resistant cell lines. We showed a two fold increase of the catalase<br />
mRNA in the resistant cell lines <strong>and</strong> transcriptional activity by using a reporter plasmid. At<br />
present time, no evidence of signalling pathways (Foxo3a, PPAR gamma,...) were identified.<br />
Interestingly, we found a 2,5 fold increase of the protein level <strong>and</strong> the enzymatic activity of<br />
the catalase but the post-translational modifications appeared to be unchanged as compared to<br />
control MCF-7 cells. Finally, we observed an effect of both HDACs <strong>and</strong> DNMTs inhibitors<br />
suggesting a possible role of epigenetic events to explain the overexpression of catalase level<br />
in resistant cells.<br />
99
Session 3: Transcriptional control Poster 3<br />
The NF-kappaB signaling pathway is inhibited by heat shock independently of active<br />
transcription factor HSF1 <strong>and</strong> increased levels of HSP70i.<br />
Patryk Janus 1 , Magdalena Kalinowska-Herok 1 , Ma!gorzta Paku!a 2 , Katarzyna<br />
Szo!tysek 1 , Wojciech Pig!owski 1 , Marek Kimmel 3 , Piotr Wid!ak 1<br />
1) Maria Sklodowska-Curie Memorial Cancer Center <strong>and</strong> Institute of Oncology; Gliwice,<br />
Pol<strong>and</strong>; e-mail: patrykjanus@gmail.com, mkalinowska@io.gliwice.pl, kszoltysek@io.<br />
gliwice.pl, wpiglowski@io.gliwice.pl, widlak@io.gliwice.pl; 2) University of Aarhus;<br />
Aarhus, Denmark; e-mail: mpakula@inano.dk; 3) Silesian University of Technology;<br />
Gliwice, Pol<strong>and</strong>; e-mail: mkimmel@polsl.pl<br />
NF-!B transcription factor regulates numerous genes important for inflammation, immune<br />
response <strong>and</strong> cell survival. HSF1 is the primary transcription factor activated under stress<br />
conditions that is responsible for induction of genes encoding heat shock proteins. The NF-<br />
!B activation pathway is blocked by heat shock. Here we investigated whether active<br />
HSF1 inhibited this pathway in the absence of stress conditions. Activation of the NF-!B<br />
pathway <strong>and</strong> expression of NF-!B-dependent genes was analyzed in TNF"-stimulated U-2<br />
OS human osteosarcoma cells that were either preconditioned with hyperthermia or<br />
engineered to express a constitutively active form of HSF1 in the absence of heat shock. The<br />
hyperthermia resulted in a general blockade in the degradation of the I!B" inhibitor, nuclear<br />
translocation of NF-!B <strong>and</strong> expression of NF-!B-dependent target genes. In marked contrast,<br />
the presence of constitutively active HSF1 did not block TNF"-induced activation of the NF-<br />
!B pathway or general expression of the NF-!B-dependent genes. We concluded that in the<br />
absence of heat shock the NF-!B activation pathway is not inhibited by active HSF1<br />
transcription factor or by increased level of HSF1-induced HSP70i.<br />
100
Session 3: Transcriptional control Poster 4<br />
Leflunomide overcomes CD40L/IL-4-mediated resistance to fludarabine-induced<br />
apoptosis <strong>and</strong> effectively blocks proliferation of CLL cells<br />
S. Dietrich, A. Zota, M. Hess, M. Rieger, C. Schweizer, A.D. Ho, P. Dreger, T. Luft,<br />
Dept. Medicine V, University of Heidelberg (Heidelberg, DE)<br />
BACKGOUND AND AIM: Chronic lymphocytic leukemia (CLL) has a poor prognosis if resistance<br />
to purine analogues, such as fludarabine, occurs. Therefore strategies to overcome fludarabine<br />
resistance in poor-risk CLL are highly warranted. Proliferation <strong>and</strong> resistance to fludarabine-induced<br />
apoptosis of CLL cells can be induced in vitro with CD40L <strong>and</strong> IL-4. This experimental setting may<br />
mimic the anti-apoptotic environment protecting CLL cells in lymph nodes in vivo. CD40 signalling<br />
involves NF-kB activation, whereas IL-4 acts through the JAK/STAT pathway. Leflunomide is an oral<br />
drug approved for treatment of rheumatoid <strong>and</strong> psoriasis arthritis. Leflunomide exerts its<br />
immunosuppressive effects by directly inhibiting dihydroorotate dehydrogenase (DHODH), an<br />
enzyme involved in pyrimidine synthesis, but has also been reported to interfere with NF-kB. Serum<br />
concentrations of more than 100 !g/ml of the active metabolite (A771726) can be achieved in patients<br />
with a 20 mg tablet per day.<br />
METHODS: We established a long-term culture system in order to study proliferation <strong>and</strong> apoptosis<br />
resistance of human CLL cells in vitro. This system involves stimulation of CLL cells with a CD40Ltransfected<br />
baby hamster kidney cell line (BHK) <strong>and</strong> IL-4 (50 U/ml). Thymidine incorporation assays<br />
<strong>and</strong> intracellular KI-67 FACS analyses were used to measure proliferation. Apoptosis was quantified<br />
using AnnexinV/7-AAD FACS analyses. Intracellular nuclear FACS analyses for phospho-p65,<br />
phospho-STAT1, phospho-STAT3, survivin <strong>and</strong> bcl-xL as well as quantitative Western Blot analyses<br />
were used to examine the mechanism of apoptosis resistance in CLL cells. Fludarabine (10!g/ml), the<br />
pan-JAK inhibitor Pyridone 6 (0.15 !g/ml) <strong>and</strong>/or Leflunomide (A771726: 0-100 !g/ml) were<br />
investigated in the context of a CD40L/IL-4 signal.<br />
RESULTS: Proliferation of CLL cells required a complementary CD40 <strong>and</strong> JAK/STAT signal <strong>and</strong><br />
could be blocked by the JAK inhibitor. In contrast, resistance to fludarabine-mediated apoptosis could<br />
be induced by CD40 activation alone. This coincided with persistently high levels of intracellular<br />
phospho-p65, survivin <strong>and</strong> bcl-xL. Apoptosis resistance was further enhanced by a complementary<br />
JAK/STAT signal but could not be blocked by the JAK inhibitor. Leflunomide inhibited proliferation<br />
at very low concentrations (IC50: 4 !g/ml), but induced apoptosis of CD40L+IL-4-activated<br />
(“resistant”) CLL cells only at higher concentrations (IC50: 72 !g/ml). Furthermore leflunomide in<br />
combination with fludarabine showed synergistic effects on induction of apoptosis of CLL cells (IC50:<br />
50 !g/ml). The anti-proliferative effect of low Leflunomide concentrations was likely due to inhibition<br />
of DHODH, as inhibition of STAT1/3 phosphorylation was observed at higher concentrations only<br />
(50-100 !g/ml). Similarly, in concentrations which induced apoptosis of CD40L/IL4-activated CLL<br />
cells, leflunomide efficiently inhibited phosphorylation of RelA resulting in low expression levels of<br />
the anti-apoptotic proteins bcl-xL <strong>and</strong> survivin. In contrast, fludarabine alone was inefficient to block<br />
bcl-xL <strong>and</strong> survivin expression in CD40L/IL-4 stimulated CLL cells.<br />
CONCLUSIONS: Leflunomide can overcome CD40L/IL4-mediated fludarabine resistance at<br />
clinically achievable concentrations by inhibiting phosphorylation of STAT1/3 <strong>and</strong> NF-kB-induced<br />
expression of survivin <strong>and</strong> bcl-xL. Furthermore leflunomide has a strong, JAK/STAT-independent,<br />
anti-proliferative effect on CD40L/IL4-activated CLL cells already at very low concentrations.<br />
Therefore leflunomide might be a promising c<strong>and</strong>idate drug to attack CLL cells in chemoresistant<br />
niches.<br />
101
Session 3: Transcriptional control Poster 5<br />
Role of MDC1 in NF-kappaB activation by DNA double-str<strong>and</strong> breaks.<br />
Helene Sabatel, Jacques Piette <strong>and</strong> Yvette Habraken.<br />
Laboratory of Virology <strong>and</strong> Immunology, Signal Transduction Unit, GIGA-R,<br />
University of Liège, 4000 Liège, Belgium. helene.sabatel@student.ulg.ac.be,<br />
jpiette@ulg.ac.be, yvette.habraken@ulg.ac.be<br />
Many anticancer treatments, such as ionizing radiation (IR) <strong>and</strong> DNA topoisomerase I <strong>and</strong> II,<br />
induce apoptosis in tumor cells by generating DNA double str<strong>and</strong> breaks (DSB). ATM kinase,<br />
rapidly activated by DSB, orchestrates different aspects of the DNA Damage Response: cell<br />
cycle arrest, DSB repair <strong>and</strong> transcription factors activation. Both NF-kappaB <strong>and</strong> p53 are<br />
activated by DSB, controlling anti- <strong>and</strong> pro-apoptotic signals <strong>and</strong> therefore affecting cell<br />
survival <strong>and</strong> the outcome of the cancer treatment.<br />
In this work, the importance of MDC1 (Mediator DNA damage checkpoint 1) in the DNA<br />
damage signalling to NF-!B is studied. As expected, MDC1 reduction by siRNAs resulted in<br />
a smaller number of nuclear foci after IR <strong>and</strong> Camptothecin treatments. We observed that<br />
reduced MDC1 level leads to a reduction of NF-!B nuclear translocation after both<br />
treatments. NF-!B transactivation potential was assessed with two reporter systems; either a<br />
transiently transfected !B-Luc reporter plasmid or a stably integrated !B-GFP reporter. In<br />
both cases, we noticed a large reduction of the transcriptionnal activity in cells treated with<br />
siRNA-MDC1. The reduction was however less important than the one observed in cells<br />
treated with an siRNA-ATM. Mefs WT <strong>and</strong> KO for MDC1 were tested for NF-!B activation<br />
by IR <strong>and</strong> Camptothecin. However, as Mefs WT did not activate NF-!B following DNA<br />
damage we could not use this model. The effect of the deletion of different domains of MDC1<br />
is also analysed to determine by which way this protein interferes with NF-kappaB signalling<br />
pathway (foci dependent or independent manner).<br />
In summary, our data indicate that deletion of MDC1 has an impact on NF-!B activation after<br />
both IR <strong>and</strong> Camptothecin treatments contrarily to the disruption of NBS/Mre/Rad50 complex<br />
that affects only IR signalling under the tested conditions.<br />
Financial supports: IAP 6/18, Centre anti-cancéreux de l’ULg, FRS-FNRS, F.R.S.M<br />
102
Session 3: Transcriptional control Poster 6<br />
Regulation of NFkappaB-dependent <strong>and</strong> p53-dependent genes – crosstalk between<br />
signaling pathways.<br />
Katarzyna Szo!tysek 1 , Patryk Janus 1 , Adam Makuchowski 2 , Marek Kimmel 2 , Piotr<br />
Wid!ak 1<br />
1) Maria Sklodowska-Curie Memorial Cancer Center <strong>and</strong> Institute of Oncology; Gliwice,<br />
Pol<strong>and</strong>; e-mail: kszoltysek@io.gliwice.pl, patrykjanus@gmail.com,<br />
widlak@io.gliwice.pl;<br />
2) Silesian University of Technology; Gliwice, Pol<strong>and</strong>; e-mail:<br />
adam.makuchowski@polsl.pl, mkimmel@polsl.pl;<br />
Background: Signaling pathways that depend on p53 or NFkappaB transcription factors are<br />
essential components of cellular responses to stress. Both proteins participate in regulation of<br />
the expression of numerous genes that are involved in cell cycle arrest, DNA repair,<br />
apoptosis, immune response <strong>and</strong> inflammation. Here we analyzed the interference between<br />
both signaling pathways at the level of expression of p53- <strong>and</strong> NFkappaB-dependent genes.<br />
Methods: Colon carcinoma HCT116 cell line was used in two congenic variants either<br />
containing or lacking transcriptionally competent p53. Cells were stimulated with TNF-alpha<br />
cytokine to activate the NFkappaB pathway, <strong>and</strong>/or irradiated with UV to activate the p53<br />
pathway; both stimuli were used in two different combinations – cells were treated with TNF<br />
either 3 hrs before or 6 hrs after irradiation. Activation of the NFkappaB <strong>and</strong> p53 pathways<br />
was monitored by Western-blotting. Expression levels of selected p53-dependent genes<br />
(MDM2, p21/WAF1, PTEN, NOXA) were assessed by QRT-PCR at different time points<br />
after irradiation. Expression pro<strong>file</strong>s of 84 NFkappaB dependent genes were analyzed by the<br />
Human NFkappaB Signaling Pathway RT!Pro<strong>file</strong>r PCR Array at 1 hr time point after the<br />
TNF stimulation.<br />
Results: We observed that radiation-induced activation of p53-dependent genes was affected<br />
in cells stimulated with TNF: UV-induced expression of MDM2, p21/WAF1 <strong>and</strong> NOXA<br />
genes was further up-regulated by either type of TNF treatment. Analysis of NFkappaB<br />
dependent genes revealed 7 genes, namely BCL3, NFKBIA, REL, IL1A, IL8, TNFA,<br />
TNFAIP3, which expression levels differed between cells with different status of p53.<br />
Notably, irradiation of p53-competent cells before activation of the NFkappaB pathway<br />
resulted in down-regulation of all these 7 genes. The data indicated crosstalk between<br />
activation of NFkappaB or p53 pathways <strong>and</strong> expression of genes dependent on the opposite<br />
factor.<br />
103
Session 3: Transcriptional control Poster 7<br />
The NAD dependent deacetylase Sirt1 suppresses pituitary GH synthesis through a<br />
GSK3beta/PP1/ CREB pathway.<br />
Marily Theodoropoulou, Maria A. Tichomirowa, Jose Luis Monteserin Garcia, Günter<br />
K. Stalla<br />
The energy sensor silent information regulator 2 (Sir2) /Sirtuin 1 (Sirt1) plays a pivotal role in<br />
regulating organism metabolic homeostasis <strong>and</strong> lifespan in response to nutrient availability,<br />
by sensing perturbations in NAD + /NADH ratio. Sirt1 activation (by overexpression or<br />
resveratrol treatment) <strong>and</strong> inhibition (by siRNA, sirtinol or nicotinamide treatment)<br />
respectively suppressed <strong>and</strong> upregulated pituitary GH synthesis. GH transcription is primarily<br />
regulated by the cAMP/CREB pathway. Sirt1 significantly suppressed CREB DNA binding<br />
<strong>and</strong> transcriptional activity. CREB transcription <strong>and</strong> protein stability were not altered, but its<br />
dephosphorylation rate was accelerated in pituitary cells overexpressing Sirt1 or treated with<br />
resveratrol. In contrast, overexpression of the deacetylase dead Sirt1-H363Y completely<br />
abolished this effect; in these cells the physiological attenuation phase of CREB<br />
phosphorylation normally observed after three hours of forskolin treatment did not take place<br />
<strong>and</strong> CREB remained phosphorylated. Sirt1 was found to physically associate with CREB <strong>and</strong><br />
to decrease its acetylation levels, indicating a link between CREB acetylation <strong>and</strong><br />
phosphorylation status. CREB dephosphorylation is dependent on the protein phosphatase 1<br />
(PP1), which we found to physically associate with <strong>and</strong> be activated by Sirt1. PP1 is activated<br />
when its inhibitor I-2 gets phosphorylated at threonine residues <strong>and</strong> dissociates from PP1<br />
leaving it in an active form. Resveratrol treatment increased I-2 threonine phosphorylation at<br />
the site phosphorylated by GSK3beta <strong>and</strong> decreased the phosphorylated GSK3beta-Ser9<br />
levels indicative of kinase activation. All these events needed a deacetylase intact Sirt1. Their<br />
importance is evident by the fact that treatment with phosphatase inhibitor okadaic acid <strong>and</strong><br />
with the GSK3beta inhibitor SB-415286 abolished resveratrol’s suppressive action on<br />
forskolin-induced CREB phosphorylation levels. In summary, the present study shows that<br />
the energy sensor Sirt1 regulates GH synthesis by activating GSK3beta <strong>and</strong> PP1 <strong>and</strong><br />
suppressing CREB transcriptional activity. This may be a novel mechanism through which<br />
pituitary somatotroph cells sense changes in energy levels <strong>and</strong> subsequently adjust GH<br />
synthesis to meet the metabolic needs of the organism.<br />
104
Session 3: Transcriptional control Poster 8<br />
Stress- <strong>and</strong> nutrition-sensing transcription factors: looking for biomarkers in<br />
atherosclerosis<br />
Fabrice Tolle, Amit Kumar, Thomas Sauter, Jean-Luc Bueb.<br />
<strong>Inflammation</strong> group/Systems biology group; Life Sciences Research Unit; Faculty of<br />
Science, Technology <strong>and</strong> Communication; University of Luxembourg, 162A, Avenue de<br />
la Faïencerie,<br />
L-1511 Luxembourg - Contact: fabrice.tolle@uni.lu<br />
A misbalance of functional interactions between nutrient-sensing nuclear receptors (e.g.<br />
Vitamin D receptor, VDR) <strong>and</strong> stress-sensing transcription factors (e.g. NF-kappaB) induced<br />
by lifetime exposure to micronutrients <strong>and</strong> cytokines, may be a central molecular process<br />
towards atherosclerosis. Our objectives are to highlight potential interactions between VDR<br />
<strong>and</strong><br />
NF-kappaB signalling in vascular endothelial cells, the first cell population implicated in this<br />
disease. Microarray analyses were done on total RNA which was extracted from EA.hy926<br />
cells (human umbilical vein endothelial cells line) after stimulation with TNFalpha <strong>and</strong>/or<br />
1alpha25(OH)2 vitamin D3 (VD) up to 24 hours. The raw data were transformed <strong>and</strong><br />
normalised using Bioconductor software <strong>and</strong> then analysed using IPA Knowledge Base<br />
(Ingenuity) with a special focus on chemokines molecules. As expected, most of the CCL <strong>and</strong><br />
CXCL chemokine families are up-regulated by the TNFalpha treatment <strong>and</strong> interestingly,<br />
these up-regulations are decreased by VD. To confirm these results, we focused our attention<br />
on a group of genes that appear to be implicated in macrophage chemo-attraction (Chemokine<br />
(C-C motif) lig<strong>and</strong> 2, 4 <strong>and</strong> 5 (CCL2, 4 <strong>and</strong> 5)). The microarray data were verified <strong>and</strong><br />
confirmed by real time RT-PCR for CCL2, 4 <strong>and</strong> 5. Moreover, the effect of VD was<br />
abolished when the VDR expression was switched off by siRNA. The same observation was<br />
also done when cycloheximide was added to the TNFalpha <strong>and</strong> VD treatment. We confirmed<br />
on EA.hy926 cells that CCL2, 4 <strong>and</strong> 5 genes are up-regulated by TNFalpha. We showed that<br />
these up-regulations are decreased in presence of VD <strong>and</strong> dependent on VDR expression. The<br />
implication of VDR seems however to be indirect <strong>and</strong> to involve a de novo synthesis of<br />
protein(s), which we are now trying to identify, as well as their way of interaction with NFkappaB<br />
signalling <strong>and</strong> CCL2, 4 <strong>and</strong> 5 genes.<br />
105
Session 3: Transcriptional control Poster 9<br />
Control of the response to chemotherapy by the Nrf2-antioxidant response element<br />
signaling pathway<br />
Julien Verrax, Nicolas Dejeans, Raphaël Beck, Christophe Glorieux <strong>and</strong> Pedro Buc<br />
Calderon.<br />
Toxicology <strong>and</strong> Cancer Biology research group, Louvain Drug Research Institute,<br />
Université catholique de Louvain, Brussels, Belgium. Email :<br />
julien.verrax@uclouvain.be<br />
The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a major regulator<br />
of the cellular antioxidant response. Nrf2, which is activated in response to either electrophilic<br />
or oxidative stress, controls detoxifying enzymes <strong>and</strong> transporters that protect cells from<br />
reactive oxygen species (ROS) <strong>and</strong> toxic chemicals.<br />
Although Nrf2 plays an important cytoprotective role in healthy individuals, mutations of the<br />
genes encoding for the components of its regulatory pathway can lead to constitutive<br />
activation of Nrf2, a phenomenon that is suspected to promote the resistance of cancer cells to<br />
chemotherapy.<br />
We have therefore explored the putative impact of Nrf2 activation on the efficacy of different<br />
chemotherapies. Our results show that Nrf2 activation (achieved by either using chemical<br />
inducers or Keap1 silencing) leads to the up-regulation of several antioxidant genes <strong>and</strong> a<br />
two-fold increase in glutathione content. Cancer cells in which Nrf2 was activated were also<br />
more resistant to both alkylating <strong>and</strong> intercalating agents. Taken together, our results support<br />
the view that Nrf2 can be either a friend or a foe, depending on the context.<br />
J.Verrax is a FNRS postdoctoral researcher.<br />
106
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 4: Immunology<br />
107
Session 4: Immunology Poster 1<br />
Detection of extracellular matrix proteins <strong>and</strong> apoptotic cells in varicose veins wall of<br />
patients of different age groups<br />
Marina Aunapuu, Andres Arend<br />
Department of Anatomy, University of Tartu Ravila 19, 50411 Tartu, Estonia; e-mail:<br />
marina.aunapuu@ut.ee<br />
Varicose veins of lower extremities represent the most common pathology of peripheral blood<br />
vessels. Recently, in connection with the introduction of new immunological, molecular<br />
biological <strong>and</strong> histological methods, the underst<strong>and</strong>ing of the mechanism of progression of<br />
varicose vein disease has significantly improved. The aim of our study was to investigate<br />
morphological changes <strong>and</strong> differences in the protein expression of collagen I, II, IV <strong>and</strong><br />
laminin in the wall of great saphenous vein from male <strong>and</strong> female patients of different age. In<br />
addition, apoptotic endothelial <strong>and</strong> smooth muscle cells were detected. The patients were<br />
divided into 3 age groups – younger than 35 years (Group I, 8 males <strong>and</strong> 28 females), from 36<br />
to 50 years (Group II; 12 males <strong>and</strong> 35 females) <strong>and</strong> older than 50 years (Group III; 5 males<br />
<strong>and</strong> 31 females). Collagen I, II, IV <strong>and</strong> laminin were determined by means of<br />
immunohistochemistry, apoptotic cells were detected by the transferase-mediated dUTP nickend<br />
labeling (TUNEL) method using In Situ Cell Death Detection Kit (Roche Diagnostics,<br />
Roche, Germany). In most patients veins were stretched out with highly varying wall<br />
thickness. The amount of connective tissue was increased. The immunohistochemistry<br />
showed that expression of laminin was increased in varicose veins media <strong>and</strong> adventitia,<br />
especially in III group female patients. The expression of collagen IV in all layers of varicose<br />
veins wall <strong>and</strong> collagen II in the adventitia of older female patients was higher compared with<br />
younger patients. In the expression of collagen I differences between male <strong>and</strong> female patients<br />
groups were found. The percentage of apoptotic endothelial <strong>and</strong> smooth muscle cells<br />
increased in the female group III compared with younger patients, but in case of male patients<br />
no differences between age groups were found. The results demonstrate that morphological<br />
alterations in varicose veins become more pronounced with advancing age, especially in the<br />
female patients.<br />
108
Session 4: Immunology Poster 2<br />
Immunophenotyping of mammary gl<strong>and</strong> cells in an acute mouse mastitis model<br />
Koen BREYNE 1, Dieter DEMON 1, Kristel DEMEYERE 1, <strong>and</strong> Evelyne MEYER 1<br />
1 Laboratory of Biochemistry, Department of Pharmacology, Toxicology <strong>and</strong><br />
Biochemistry, Ghent University, 9820 Merelbeke, Belgium, Koen.Breyne@Ugent.be,<br />
Dieter.Demon@Ugent.be, Kristel.Demeyere@Ugent.be, Evelyne.Meyer@Ugent.be<br />
Mammary gl<strong>and</strong> infection (mastitis) is one of the major concerns for the dairy industry. A<br />
variety of bacteria can colonize the milk-producing alveoli <strong>and</strong> provoke local <strong>and</strong> systemic<br />
inflammation. Different immune <strong>and</strong> non-immune mammary cells participate in the innate<br />
defense against invading pathogens by the secretion of specific cytokines <strong>and</strong> bactericidal<br />
molecules. However, the contribution of each cell type in clearance of the bacteria remains<br />
largely unknown. To address the participation of the key cell types in mastitis, we propose<br />
flow cytometric identification of neutrophils, macrophages <strong>and</strong> epithelial cells. As an initial<br />
approach, we compare Escherichia coli (E.coli)-infected mouse mammary gl<strong>and</strong>s with<br />
lactating <strong>and</strong> involuting gl<strong>and</strong>s.<br />
For the acute E. coli-induced mouse mastitis model, 12 to 14 days lactating wild type<br />
C57BL/6 mice were intraductally inoculated with bovine mastitis isolate E. coli P4:O32 in the<br />
right mammary gl<strong>and</strong> of the 4 th pair (mastitis gl<strong>and</strong>) <strong>and</strong> with phosphate buffered saline (PBS)<br />
in the contralateral gl<strong>and</strong> (involuting control gl<strong>and</strong>). Mammary gl<strong>and</strong>s were isolated either<br />
from 12 to 14 days lactating females (lactating gl<strong>and</strong>) or at about 20h post-infection <strong>and</strong><br />
manually minced in small pieces. The remaining clumps were enzymatically digested <strong>and</strong><br />
filtered to obtain a single cell suspension. For multi-parametric flow cytometric analysis, cells<br />
were stained with a panel of fluorochrome-conjugated monoclonal antibodies. In addition to<br />
their forward <strong>and</strong> side scatter characteristics, leukocytes <strong>and</strong> epithelial cells were identified by<br />
the expression of the cell surface antigens CD45 <strong>and</strong> CD24, respectively. Leukocytes were<br />
subdivided in alveolar neutrophils <strong>and</strong> macrophages based on the expression of ly6G or<br />
F4/80, respectively. Myoepithelial <strong>and</strong> luminal epithelial cells were gated according to their<br />
difference in CD24 expression, as previously described.<br />
Our results indicate a significantly higher influx of neutrophils (Ly6G + /CD45 + ) in the mastitis<br />
gl<strong>and</strong> than in both the involuting control <strong>and</strong> fully lactating gl<strong>and</strong>. As expected, the lactating<br />
gl<strong>and</strong>s contain the most epithelial cells (CD24 + /CD45 - ). The proportion of macrophages<br />
(F4/80 + /CD45 + ) in the three different conditions is not significantly altered. Microscopic<br />
evaluation after sorting of CD45 + /Ly6G + <strong>and</strong> CD45 + /F4/80 + -cells confirmed that these sorted<br />
populations only consists of neutrophils <strong>and</strong> macrophages, respectively.<br />
In conclusion, a set of cell type-specific markers enabled the immunophenotyping of different<br />
cell populations in the infected murine mammary gl<strong>and</strong>. We confirmed the diapedesis of<br />
neutrophils in response to an intramammary E. coli invasion <strong>and</strong> the reduction of epithelial<br />
cells in the mammary gl<strong>and</strong> during early involution to decrease the milk production. In the<br />
future, this elegant tool will be used to elucidate the participation of each cell type in relevant<br />
knock-out mice or infections with diverse bacterial strains.<br />
109
Session 4: Immunology Poster 3<br />
The adaptor Lnk (SH2B3) is a key regulator of endothelial cell signaling <strong>and</strong> a potential<br />
molecular target to control vascular injury<br />
Julie DEVALLIERE, Juliette FITAU, Mathias CHATELAIS, Nathalie GERARD <strong>and</strong><br />
Béatrice CHARREAU<br />
INSERM UMR 643 <strong>and</strong> ITUN, Nantes University Hospital, University of Nantes, France<br />
Lnk (SH2B3) belongs to a family of SH2-containing proteins <strong>and</strong> is a signaling adaptor<br />
molecule that plays regulatory functions in the homeostasis <strong>and</strong> proliferation of immune cells.<br />
We previously showed that Lnk is expressed in endothelial cells (EC) <strong>and</strong> phosphorylated in<br />
response to TNF (1, 2). We further demonstrated that, in human EC, Lnk is a negative<br />
regulator of TNF signaling that inhibits CAM (E-selectin <strong>and</strong> VCAM-1) expression in<br />
response to TNF (3). Mechanistically, NFkappaB/p65 <strong>and</strong> IkappaBalpha phosphorylation<br />
were unchanged, suggesting that Lnk may not modulate canonical NF!B activity. However,<br />
Lnk activates PI3-kinase through Akt phosphorylation. Sustained Lnk-mediated activation of<br />
PI3-kinase in TNF"-activated ECs correlated with the inhibition of ERK1/2 phosphorylation,<br />
while phosphorylation of p38 <strong>and</strong> JNK MAPKs was unchanged. ERK1/2 inhibition decreases<br />
VCAM-1 expression in TNF"-treated ECs. In the present study we show that integrin<br />
ligation, using anti-CD29 Abs, also promotes Lnk activation <strong>and</strong> regulation in EC whereas<br />
integrin-mediated signaling (Akt <strong>and</strong> GSK3# phosphorylation) consecutive to CD29 ligation<br />
is abrogated in EC from Lnk-/- mice. We found that Lnk binds to ILK <strong>and</strong> activates FAK <strong>and</strong><br />
paxillin. Overall, Lnk causes F-actin stress fibers, cytoskeleton reorganization <strong>and</strong> increases<br />
both the number <strong>and</strong> density of focal adhesions. Functionally, Lnk expression dramatically<br />
increases EC adhesion, slows down EC migration <strong>and</strong> inhibits apoptosis induced by anoïkis.<br />
Transfection of mutants <strong>and</strong> SiRNA identified alpha-parvin as a molecular partner of Lnk<br />
mediating the negative regulation of EC migration (4). Thus, the adaptor Lnk is an effective<br />
key regulator of inflammatory- <strong>and</strong> integrin-signaling controlling EC activation, migration<br />
<strong>and</strong> death, all critical in vascular remodeling <strong>and</strong> regeneration. Our current work in animal<br />
models also proposes to modulate Lnk in graft EC to provide a cytoprotective signal in organ<br />
transplantation (5). (1) Boulday G. et al., Circ. Res. (2001) 2;88:430; (2) Boulday et al.<br />
Transplantation (2002) 4(9):1352; (3) Fitau J. et al., J. Biol. Chem. (2006), 281:20148; (4)<br />
Devallière J. et al. (2010) Submitted ; (5) Chatelais M. et al. (2010) in revision.<br />
110
Session 4: Immunology Poster 4<br />
Immunostimulatory <strong>and</strong> anti-tumor effects of 22-nt double str<strong>and</strong>ed RNA<br />
L.V.Kovtonyuk, T.O.Kabilova, E.I. Ryabchikova, N.A. Popova, V.P. Nikolin, V.I.<br />
Kaledin, M.A. Zenkova, V.V. Vlassov, E.L. Chernolovskaya<br />
Institute of Chemical Biology <strong>and</strong> Fundamental Medicine, SB RAS, Novosibirsk, Russia.<br />
Institute of Cytology <strong>and</strong> Genetics, SB RAS, Novosibirsk, Russia.<br />
We identified a set of immunostimulatory 22 b.p. double-str<strong>and</strong>ed RNAs (isRNAs) with<br />
original sequence, which has no specific homology with human mRNAs. In vitro experiments<br />
showed that enzymatically synthesized isRNA effectively inhibit growth of human cancer<br />
cells (KB-3-1, SK-N-MC, HEK 293). We found that the pretreatment of KB-3-1 with 2aminopurine<br />
(inhibitor of dsRNA-binding protein kinase R (PKR) ) abolished the<br />
antiproliferative activity of isRNA-U/A. These results indicate the involvement of PKRmediated<br />
pathway in isRNA intracellular signaling in carcinoma KB-3-1 cells. Transfection<br />
of isRNAs induce synthesis of IFN-! <strong>and</strong> pro-inflammatory cytokines IL-6 <strong>and</strong> TNF-! in<br />
adherent mononuclear cells from human peripheral blood. To determine the<br />
immunostimulatory consensus motif in the studied isRNA we investigated antiproliferative<br />
<strong>and</strong> immunostimulatory activities of isRNA with different substitutions in the sequence. We<br />
have found that introduction of mismatches in the middle part of isRNA sequence (10 – 16<br />
positions) do not decrease significantly its antiproliferative activity, while mismatches at the<br />
3’-end of isRNA sequence substantially reduce antiproliferative action of investigated<br />
isRNAs.<br />
In vivo investigation of immunostimulatory properties of the studied isRNAs revealed that<br />
single intravenous injection of isRNA in complex with transfection reagent (Lipofectamine TM<br />
2000) increased the level of IFN-!, but did not change significantly the level of proinflammatory<br />
cytokines IL-6 <strong>and</strong> TNF-! in mice blood serum.<br />
We found, that isRNAs (3 injections, 10 "g per each mouse) effectively inhibited metastasis<br />
spreading of melanoma in C57BL/6-mice: the average number of the metastasis in the lungs<br />
of treated animals was 5 times lower than those in untreated animals. The data demonstrate<br />
that isRNAs caused reliable 25% inhibition of tumor growth of hepatoma in CBA-mice.<br />
Microscopic analysis of liver cross sections also revealed antimetastatic effects of isRNAs on<br />
hepatoma: the number of the metastasis found in the livers of treated animals <strong>and</strong> their<br />
average size were significantly reduced.<br />
The obtained results revealed the pronounced immunostimulatory, anti-tumor <strong>and</strong><br />
antimetastatic properties of the studied isRNAs. This short double str<strong>and</strong>ed isRNAs can be<br />
evaluated as potential agents for the immunotherapy of oncological <strong>and</strong> viral diseases.<br />
This work was supported by RAS programs “Molecular <strong>and</strong> Cellular Biology” <strong>and</strong> “Basic<br />
sciences for medicine”, grant from SB RAS No. 41.<br />
111
Session 4: Immunology Poster 5<br />
Identification of new partners of the protein Nod2 <strong>and</strong> evaluation of their potential role.<br />
Aurore Lecat 1 , E. Di Valentin 1 , M. Fillet 2 , J. Piette 1 , Sylvie Legr<strong>and</strong> 1<br />
1. GIGA Research, Unit of Virology <strong>and</strong> Immunology, B34, University of Liege, Belgium<br />
– aurore.lecat@ulg.ac.be 2. GIGA Research, Unit of Chemical Chemistry, B34,<br />
University of Liege, Belgium<br />
The mammalian innate immune system has evolved to detect pathogens-associated<br />
molecular patterns (PAMPS). Recognition of PAMPS involves membrane-spanning proteins<br />
like the Toll-like receptors (TLR) <strong>and</strong> cytosolic proteins such as the recently identified NODlike<br />
receptors (NLR). Best studied are mutations in Nod2 that are linked to Crohn’disease<br />
(CD) <strong>and</strong> Blau syndrome. Nod2 was shown to sense the bacterial peptidoglycan subunit<br />
muramyl-dipeptide (MDP) <strong>and</strong> to subsequently mediate inflammatory responses in cells by<br />
activating the NF-kB <strong>and</strong> MAPKS signalling pathways.<br />
This project consists in the identification of new Nod2 partners <strong>and</strong> in the evaluation<br />
of their role in the Nod2 signalling pathway. We chose a proteomic strategy consisting in the<br />
purification of Nod2-containing complexes in stably Nod2-expressing HEK293 cells after<br />
MDP treatment or infection by Listeria monocytogenes followed by Nod2 partners<br />
identification by mass spectrometry.<br />
Among the c<strong>and</strong>idates, we focused on a Jun N-terminal kinase-binding protein, termed<br />
JNKBP1. This protein is ubiquitously expressed <strong>and</strong> contains twelve WD40 repeats. We<br />
checked interaction between Nod2 <strong>and</strong> JNKBP1 <strong>and</strong> showed that both the N-terminal CARDs<br />
<strong>and</strong> C-terminal LRRs cooperate in the wild type protein for efficient interaction with<br />
JNKBP1. SiRNA-mediated knockdown of endogenous JNKBP1 significantly upregulated the<br />
phosphorylation of IkB-alpha induced by MDP in stably Nod2-expressing HEK293 cells.<br />
These preliminary results suggest that JNKBP1, through Nod2 interaction, could act as a<br />
suppressor of Nod2-mediated NF-kB activation. In the context of the CD, where loss-offunction<br />
variants of Nod2 have been shown to play a causative role, a suppressor of Nod2<br />
signalling could be of special interest.<br />
We plan to determine the modulator effect of JNKBP1 on Nod2-mediated MAPKs activation<br />
as well as on target gene expression. The molecular mechanism by which JNKBP1<br />
modulates Nod2 signalling could be investigated. All of these experiments should be also<br />
performed in a more relevant cellular model expressing both endogenous proteins such as<br />
intestinal epithelial cells. Finally, we will determine whether JNKBP1 expression is<br />
modulated in pro-inflammatory conditions such as in the mucosa of CD patients.<br />
112
Session 4: Immunology Poster 6<br />
Identification of new partners of the protein Nod2 <strong>and</strong> evaluation of their potential role.<br />
Aurore Lecat 1 , E. Di Valentin 1 , M. Fillet 2 , J. Piette 1 , Sylvie Legr<strong>and</strong> 1<br />
1. GIGA Research, Unit of Virology <strong>and</strong> Immunology, B34, University of Liege, Belgium<br />
– aurore.lecat@ulg.ac.be 2. GIGA Research, Unit of Chemical Chemistry, B34,<br />
University of Liege, Belgium<br />
The mammalian innate immune system has evolved to detect pathogens-associated<br />
molecular patterns (PAMPS). Recognition of PAMPS involves membrane-spanning proteins<br />
like the Toll-like receptors (TLR) <strong>and</strong> cytosolic proteins such as the recently identified NODlike<br />
receptors (NLR). Best studied are mutations in Nod2 that are linked to Crohn’disease<br />
(CD) <strong>and</strong> Blau syndrome. Nod2 was shown to sense the bacterial peptidoglycan subunit<br />
muramyl-dipeptide (MDP) <strong>and</strong> to subsequently mediate inflammatory responses in cells by<br />
activating the NF-kB <strong>and</strong> MAPKS signalling pathways.<br />
This project consists in the identification of new Nod2 partners <strong>and</strong> in the evaluation<br />
of their role in the Nod2 signalling pathway. We chose a proteomic strategy consisting in the<br />
purification of Nod2-containing complexes in stably Nod2-expressing HEK293 cells after<br />
MDP treatment or infection by Listeria monocytogenes followed by Nod2 partners<br />
identification by mass spectrometry.<br />
Among the c<strong>and</strong>idates, we focused on a Jun N-terminal kinase-binding protein, termed<br />
JNKBP1. This protein is ubiquitously expressed <strong>and</strong> contains twelve WD40 repeats. We<br />
checked interaction between Nod2 <strong>and</strong> JNKBP1 <strong>and</strong> showed that both the N-terminal CARDs<br />
<strong>and</strong> C-terminal LRRs cooperate in the wild type protein for efficient interaction with<br />
JNKBP1. SiRNA-mediated knockdown of endogenous JNKBP1 significantly upregulated the<br />
phosphorylation of IkB-alpha induced by MDP in stably Nod2-expressing HEK293 cells.<br />
These preliminary results suggest that JNKBP1, through Nod2 interaction, could act as a<br />
suppressor of Nod2-mediated NF-kB activation. In the context of the CD, where loss-offunction<br />
variants of Nod2 have been shown to play a causative role, a suppressor of Nod2<br />
signalling could be of special interest.<br />
We plan to determine the modulator effect of JNKBP1 on Nod2-mediated MAPKs activation<br />
as well as on target gene expression. The molecular mechanism by which JNKBP1<br />
modulates Nod2 signalling could be investigated. All of these experiments should be also<br />
performed in a more relevant cellular model expressing both endogenous proteins such as<br />
intestinal epithelial cells. Finally, we will determine whether JNKBP1 expression is<br />
modulated in pro-inflammatory conditions such as in the mucosa of CD patients.<br />
113
Session 4: Immunology Poster 7<br />
Protective Effects of Korean Medicinal Herbs against Lipopolysaccharide- <strong>and</strong><br />
Dinitrochlorobenzene-induced Pro-inflammatory Responses in HaCaT Cells <strong>and</strong><br />
NC/Nga Mice<br />
Chan Lee, En-Joo Lee, <strong>and</strong> Chan-Ik Park<br />
Department of Cosmeceutical Science, Daegu Haany University, Gyeongsangbuk-do<br />
712-715, S. Korea (E-mail : cipark@dhu.ac.kr)<br />
Atopic dermatitis (AD) is a complex skin disorder accompanied by severe itching <strong>and</strong><br />
inflammation with frequently repeated episodes. The purpose of this study is to search for<br />
naturally occurring medicinal herbs which can protect against lipopolysaccharide (LPS)induced<br />
pro-inflammatory responses in HaCaT cells <strong>and</strong> improve dinitrochlorobenzene<br />
(DNCB)-induced AD-like skin lesions in NC/Nga Mice. Houttuynia Cordata (HC), Scutella<br />
Baicalensis (SB), <strong>and</strong> Coptis Chinensis (CC) extracts inhibited LPS-induced expression of<br />
inducible nitric oxide synthase (iNOS) <strong>and</strong> cyclooxygenase-2 (COX-2) <strong>and</strong> subsequent<br />
generation of nitric oxide (NO) <strong>and</strong> prostagl<strong>and</strong>in E2 (PGE2). In another experiments, topical<br />
application of HC, SB, <strong>and</strong> CC extracts to NC/Nga mice suppressed the development of<br />
scratching <strong>and</strong> AD-like dermatitis. Moreover, expression of inflammatory enzymes,<br />
production of pro-inflammatory mediators, activation of mast cells, <strong>and</strong> elevated serum levels<br />
of IgE were effectively attenuated by HC, SB, <strong>and</strong> CC extracts. These findings suggest that<br />
Korean medicinal herbs including HC, SB, <strong>and</strong> CC may have preventive <strong>and</strong> therapeutic<br />
potential against inflammatory skin damages in AD.<br />
Keywords : Houttuynia Cordata, Scutella Baicalensis, Coptis Chinensis,<br />
atopic dermatitis, inflammation<br />
114
Session 4: Immunology Poster 8<br />
Small GTPase RJL promotes breast cancer progression by inducing expansion <strong>and</strong><br />
accumulation of MDSCs via IL-6 <strong>and</strong> PGE2<br />
Qiuyan Liu, Taoyong Chen, Chaoxiong Zhang, <strong>and</strong> Xuetao Cao<br />
Institute of Immunology <strong>and</strong> National Key Laboratory of Medical Immunology, Second<br />
Military Medical University, Shanghai 200433, China, Email:lqy1969@yahoo.com.cn<br />
The expansion <strong>and</strong> accumulation of myeloid-derived suppressor cells (MDSCs) have been<br />
found in cancer patients <strong>and</strong> tumor-bearing mice, which are potent suppressors of adaptive<br />
<strong>and</strong> innate immunity <strong>and</strong> can promote tumor progression. RJLs are a family of Ras-related<br />
GTP-binding proteins characterized by the N-terminal GTP-binding domain (small GTPase<br />
domain) <strong>and</strong> the C-terminal J domain with unknown functions. Here, we report that RJL is<br />
overexpressed in most of the tumor cells <strong>and</strong> tumor tissues, <strong>and</strong> RJL overexpression promotes<br />
breast cancer growth, invasion <strong>and</strong> metastasis in vitro <strong>and</strong> in vivo. More expansion <strong>and</strong><br />
accumulation of MDSCs is found in RJL-overexpressing 4T1 tumor-bearing mice.<br />
Furthermore, RJL overexpression induces 4T1 cancer cells to produce more proinflammatory<br />
factor IL-6 <strong>and</strong> PGE2 by activating p38 MAPK, PI-3K/Akt, <strong>and</strong> NF!B signal pathways.<br />
Accordingly, silencing of RJL expression significantly decreases breast cancer progression<br />
<strong>and</strong> reduces MDSCs expansion <strong>and</strong> accumulation. Administration of COX2 specific inhibitor<br />
SC58125 can significantly reduce MDSCs expansion <strong>and</strong> accumulation in spleen, <strong>and</strong> tumor<br />
tissues, <strong>and</strong> administration of anti-IL-6 neutralization antibody reduces MDSCs accumulation<br />
in tumor tissue. Therefore, our results demonstrate that RJL can induce IL-6 <strong>and</strong> PGE2<br />
production, which contributes to the RJL-induced MDSCs expansion <strong>and</strong> recruitment<br />
promoting breast cancer progression. The results suggest that RJL maybe a novel oncogenic<br />
therapeutic target.<br />
Keywords:RJL; Myeloid-derived suppressor cells; IL-6; PGE2; immune escape<br />
This work was supported by grants from the National Natural Science Foundation of China<br />
(30771984, 30972688)<br />
115
Session 4: Immunology Poster 9<br />
Premature aging of immune system: regulatory T cells as a therapeutic target<br />
Alex<strong>and</strong>er Pukhalsky 1,2 , Galina Shmarina 1,2 <strong>and</strong> Vladimir Alioshkin 2<br />
1 Research Centre for Medical Genetics, Moscow 115478, 2 Gabrichevsky Institute of<br />
Epidemilogy <strong>and</strong> Microbilogy, Moscow 125212, Russia. E-mail: osugariver@yahoo.com<br />
It is well known that the brain <strong>and</strong> immune system are the two principal adaptive systems in the body,<br />
which permanently swap for the signals. Two major pathways are involved in such interaction:<br />
hypothalamic-pituitary-adrenal (HPA) axis <strong>and</strong> the sympathetic nervous system. During the immune<br />
response glucocorticoids (GCs) <strong>and</strong> catecholamines, the major stress hormones stimulate a negative<br />
feedback mechanism, which protects the organism from a surplus activity of pro-inflammatory<br />
cytokines <strong>and</strong> other products with tissue-damaging potential. Simultaneously regulatory T cells<br />
(Tregs) lose their suppressive activity. As immune response develops Treg number increases <strong>and</strong> their<br />
activity is restored. Augmentation of active Treg number results in gradual attenuation of the immune<br />
response. Normally, both mechanisms of inflammation control prompt (GCs <strong>and</strong> catecholamines) <strong>and</strong><br />
delayed (Tregs), are well equalized. However, the balance may be disturbed by age due to repeated<br />
episodes of stress <strong>and</strong> HPA axis activation. In time HPA axis activation replaces by its depletion <strong>and</strong><br />
Tregs become a principal mechanism of anti-inflammatory machinery. This mode of regulation of<br />
inflammatory reaction being more slow <strong>and</strong> rough does not allow to fine tune immune system <strong>and</strong><br />
each new environmental challenge promotes further Treg accumulation that leads to stable<br />
immunosuppression. Permanent stress <strong>and</strong>/or severe systemic disease may accelerate such aging<br />
process. Different methods of Treg depletion have been proposed. In our opinion alkylating drugs<br />
(ADs) are most promising among them. ADs belonging to nitrogen mustard family are commonly<br />
used as cytostatic <strong>and</strong> immunosuppressive agents. The effect of the drugs is mainly associated with<br />
cross-linking of DNA double str<strong>and</strong>s <strong>and</strong>, at higher concentrations, with induction of DNA str<strong>and</strong><br />
breaks. Although DNA is not a unique target for alkylation in the cell the others do not play any role<br />
in the cytostatic effect realization if the drug is used at a DNA-altering dose. However, when the dose<br />
is gradually decreased, the number of targets for alkylation will also be reduced. Studies in in vitro<br />
lymphocyte proliferation model show that the scenario varies with the ADs concentration decrease. If<br />
the concentration of ADs is high (100 µg/ml or more), cells die within few hours due to irreversible<br />
DNA damage. If the concentrations of ADs vary in the range from 30 to 100 µg/ml numerous sites of<br />
DNA are also alkylated, but DNA damaged segments restored during DNA repair. Nevertheless, the<br />
affected cells are died due to apoptosis induction. Moderate concentrations of ADs (1-10 µg/ml) do<br />
not kill the cells but prevent IL-2 production making the cells resistant to proliferative stimuli. Ultralow<br />
concentrations of ADs (0.3 µg/ml <strong>and</strong> lower) selectively inhibit Tregs due to disruption of the<br />
signal transduction by IL-2R. Among a large variety of T cell subsets only Tregs constitutively<br />
express high affinity receptor for IL-2, the cytokine, which is the factor of their growth <strong>and</strong> survival.<br />
IL-2R is not a unique receptor, which may be blocked with ADs. In addition similar effect has been<br />
shown for at least two other surface receptors: TNFR <strong>and</strong> Fas. So, ADs have a unique capacity for<br />
selective elimination of Tregs. Such elimination may be achieved with low doses of the drug, which<br />
do not affect other cell subsets. Optimal effect may be obtained when ADs are used in the form of<br />
pulse-therapy: repeated dose after one month recovery. The concept of such therapy may be<br />
formulated as “hit <strong>and</strong> run away”.<br />
116
Session 4: Immunology Poster 10<br />
INJURY INDUCED INFLAMMASOME ACTIVATION LEADING TO IL-1b AND IL-<br />
17 MEDIATED LUNG INFLAMMATION IS IL-22 INDEPENDENT<br />
Valérie Quesniaux, Isabelle Couillin, François Erard, Bernhard Ryffel<br />
UMR6218, CNRS <strong>and</strong> University, UMR6218 Orléans, France<br />
To underst<strong>and</strong> the molecular mechanisms of acute lung injury induced inflammation <strong>and</strong><br />
fibrosis we investigated <strong>and</strong> found a critical role for signaling though the IL-1 receptor 1 (IL-<br />
1R1) (Gasse et al 2007, 2009). Here we show that bleomycin induced injury results in<br />
expression of IL-1b, IL-23, IL-17 <strong>and</strong> IL-22 expression in the lung which depends on the<br />
activation of the NLPR3 inflammasome complex. To test the role of IL-17 <strong>and</strong> IL-22 in the<br />
pathogenesis we used neutralizing antibodies <strong>and</strong> gene deficient mice. We report here that IL-<br />
17A, but not IL-22, is critical to develop lung inflammation <strong>and</strong> fibrosis. The source of IL-17<br />
<strong>and</strong> IL-22 in the lung <strong>and</strong> the molecular pathways are under investigations.<br />
Therefore the data suggest that injury induced inflammasome activation leads to the activation<br />
the activation ofIL-17 while IL-22 is not involved in this process.<br />
117
Session 4: Immunology Poster 11<br />
T cell signaling in the development <strong>and</strong> treatment of cognitive disorders in mice<br />
Galina Shmarina 1,2 , Alex<strong>and</strong>er Pukhalsky 1,2 , <strong>and</strong> Vladimir Alioshkin 2<br />
1 Research Centre for Medical Genetics, Moscow 115478, 2 Gabrichevsky Institute of<br />
Epidemilogy <strong>and</strong> Microbilogy, Moscow 125212, Russia. E-mail: osugariver@yahoo.com<br />
Surplus accumulation of regulatory T cells (Tregs) is known to be at the bottom of many<br />
morbid conditions among them being neuropsychiatric diseases. In particular, Tregs may<br />
inhibit Th1 cells, including brain autoimmune lymphocytes, controlling the local microglial<br />
response <strong>and</strong> brain tissue homeostasis. CNS-specific T cells were also found to be required<br />
for special learning <strong>and</strong> memory <strong>and</strong> for the expression of brain-derived neurotrophic factors.<br />
Malfunction of these cells leads to the appearance of neurodegenerative foci. The present<br />
study was undertaken in an attempt to suggest a novel approach for the treatment of<br />
maladaptation to mental stress associated with excessive Treg accumulation. Recently it was<br />
shown that alkylating agents in the dose 100 fold lower than cytostatic one are capable to<br />
disturb signal transduction by various cell surface receptors, such as IL-2R, TNFR <strong>and</strong> Fas.<br />
Molecular mechanisms of the receptor blockage were investigated on the model of TNF<br />
signaling. We succeeded in demonstration that low concentrations of alkylating agent<br />
melphalan (Mp) protect murine fibroblastoid cells against TNF!-induced cytotoxicity. The<br />
protection did not depend on de novo protein synthesis. Moreover, no increase in NF-kB in<br />
nuclear extracts of Mp-treated cells was observed. At the same time, 1-hour treatment with<br />
Mp markedly reduced NF-kB activity in nuclear extracts of the cells challenged with TNF!.<br />
These data support the suggestion that specific alkylation of components in the cytoplasm or<br />
cell membrane by Mp interferes with surface receptor signaling pathway. As Tregs are a<br />
unique lymphocyte subset, which permanently expresses high affinity receptor for IL-2 (the<br />
cytokine, which is the factor of their growth <strong>and</strong> survival), low Mp concentrations might<br />
selectively eliminate these cells without affecting other T cell subsets. The animal model of<br />
Treg accumulation has been used. BALB/c mice were chronically treated with dexamethasone<br />
<strong>and</strong> IL-2. Spatial learning/memory was assessed in the Morris Water Maze Behavioral Test. It<br />
was shown that the pharmacologically-induced Treg accumulation leads to cognitive <strong>and</strong><br />
behavioral abnormalities, which may be prevented by Mp administration. Indeed, already on<br />
the 2 nd day of training the percentage of Mp-treated mice, which were able to find a<br />
submerged platform, was significantly higher (p=0.04; Wilcoxon paired test) than in untreated<br />
animals after Treg induction. In conclusion, disturbance of IL-2 signaling with alkylating<br />
agents is promised to be a new safe method for the treatment of cognitive disorders.<br />
118
Session 4: Immunology Poster 12<br />
The level of inducible nitric oxide (NO) synthesis in immune system of mice as the aging<br />
risk marker.<br />
Svetlana V. Vasilieva<br />
Institute of Biochemical Physics RAS, Russia 119334, Moscow, 4 Kosygin str.<br />
svasilieva@polymer.chph.ras.ru<br />
Nitric oxide (NO) which generates in mammals by NO- synthases (NOSs) has been<br />
proven to possess cyto- <strong>and</strong> genotoxicity associated with the immune response, thus<br />
decreasing resistance of bacteria, viruses <strong>and</strong> other invasive organisms. The present study<br />
examines changes in NO levels induced in two organs of mice by LPS intra abdominal<br />
injection. The experiments have been performed using a pair of isogenic lines of mice of the<br />
same age of 5 months, differ by the single mutation in the “hairless” gene (hr mutation): B10<br />
(wild type) <strong>and</strong> "Rhino" mutant line with hr mutation. The mutant animals had some defects<br />
in immune system due to over expression of the hr gene. They characterized by a specific<br />
phenotype, suffered from erythroleukaemia <strong>and</strong> died normally at the age of 6-8 months. There<br />
is the information that the hr mutation impairs a DNA-repair capacity, as well. Additionally<br />
we studied mice of NZB line at the ages of 5 <strong>and</strong> 8,5 months which are used frequently as the<br />
model of autoimmune hemolytic anemia, developing with aging. To control the levels of NO<br />
generated the EPR spectroscopy has been used. Diethyl dithiocarbamate (DDC, “Sigma”)<br />
formed [Fe 2+ -DDC] complex with endogenous or exogenous iron when injected into the<br />
animals. Acting as NO scavenger this complex bound NO <strong>and</strong> gave rise to paramagnetic<br />
mononitrosyl iron complex (MNIC-DDC). This process is accompanied by the appearance of<br />
protein-bound mononuclear dinitrosyl -iron complexes with SH groups of proteins, which are<br />
characterized by the EPR signal with g!=0,35 <strong>and</strong> g"=2,02. While initially, until 2 months of<br />
age, the experimental mice resembled each other in phenotype <strong>and</strong> the levels of inducible NO<br />
were more or less the same. During aging the animals diverged significantly in phenotype <strong>and</strong><br />
differed greatly in NO levels from organ to organ. This was the case for the animals of NZB<br />
line at the ages of 5 <strong>and</strong> 8, 5 months when we observed a significant decreasing in NO levels<br />
in liver <strong>and</strong> intestines of the elder suffering animals. NO was found to accumulate in the<br />
organs of B10 mice (w.t.) - 4 <strong>and</strong> 10 fold over the initial levels in liver <strong>and</strong> intestines,<br />
respectively. But this was not the case for the isogenic Rhino line: only 0 <strong>and</strong> 3, 5 fold over<br />
the initial NO level in liver <strong>and</strong> intestines, respectively. These differences in NO inducible<br />
levels in liver <strong>and</strong> intestines were due to the single hr mutation. So, the level of inducible<br />
nitric oxide synthesis is a specific "aging risk marker" in mice.<br />
119
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 5: Proteomics<br />
120
Session 5: Proteomics Poster 1<br />
PhosphoSitePlus ® : a Resource for the Proteome-wide Study of Protein Modifications in<br />
Cancer<br />
Peter V. Hornbeck, Elzbieta Skrzypek, Bin Zhang, Jon M. Kornhauser, Beth L.<br />
Murray, Vaughan M. Latham, <strong>and</strong> Sasha Tkachev.<br />
Cell Signaling Technology, Danvers, MA 01923 phornbeck@cellsignal.com,<br />
eskrzypek@cellsignal.com, bzhang@cellsignal.com, jornhauser@cellsignal.com,<br />
bmurray@cellsignal.com, vlatham@cellsignal.com, <strong>and</strong> stkachev@cellsignal.com.<br />
PhosphoSitePlus ® (PSP) is an open, dynamic, continuously curated, <strong>and</strong> highly interactive<br />
systems biology resource for studying experimentally observed post-translational<br />
modifications (PTMs). PSP has recently exp<strong>and</strong>ed its content <strong>and</strong> interfaces to enable the<br />
systematic investigation of protein modifications associated with various cancers. PSP<br />
includes critical structural <strong>and</strong> functional information about the topology, biological function<br />
<strong>and</strong> regulatory significance of specific modification sites, <strong>and</strong> powerful tools for mining <strong>and</strong><br />
interpreting this data in the context of diseases, tissues, <strong>and</strong> cell lines. PSP integrates both<br />
low- <strong>and</strong> high-throughput (LTP <strong>and</strong> HTP) data sources into a single reliable <strong>and</strong><br />
comprehensive resource. PSP has recently exp<strong>and</strong>ed. Information in PSP is shared with the<br />
research community in multiple ways. For example, users can access specific information<br />
about a single protein <strong>and</strong> each of its modification sites, or browse <strong>and</strong> <strong>download</strong><br />
comprehensive datasets of modification sites observed in specified diseases, cell lines, <strong>and</strong><br />
tissues. Metaanalyses of protein modifications in non-small cell lung cancer <strong>and</strong> chronic<br />
myelogenous leukemia suggest that distinct molecular signatures of different cancers may be<br />
identified using PSP. It is our hope that PSP, by providing a reliable <strong>and</strong> powerful resource<br />
focused on the roles of protein modifications in biological control, will accelerate the pace of<br />
discovery of basic mechanisms of cellular signaling, further our underst<strong>and</strong>ing of cellular<br />
regulation in health <strong>and</strong> disease, <strong>and</strong> facilitate the discovery of critical disease biomarkers <strong>and</strong><br />
potential drug targets.<br />
121
Session 5: Proteomics Poster 2<br />
Automated identification of Bcl-2 homologues using structure-aided HMM framework<br />
Valentine Rech de Laval 1 , Christophe Combet 1 , Gilbert Deléage 1 , Abdel Aouacheria 2<br />
1 Unité Bases Moléculaires et Structurales des Systèmes Infectieux; UMR 5086 CNRS –<br />
UCBL – IBCP, IFR128 Biosciences Lyon-Gerl<strong>and</strong>, 7 passage du Vercors, 69367 Lyon<br />
Cedex 07, France ; FR 3302 ; e-mail: c.combet@ibcp.fr<br />
2 Laboratoire de Biologie Moléculaire de la Cellule of Ecole Normale Supérieure de<br />
Lyon, UMR 5239 CNRS – UCBL – ENS Lyon, IFR128 Biosciences Lyon-Gerl<strong>and</strong>, 46<br />
Allée d’Italie, 69364 Lyon Cedex 07, France ; e-mail: a.aouacheria@ens-lyon.fr<br />
The Bcl-2 family controls induction of apoptosis (programmed cell death) at the mitochondria<br />
via opposing functions of prosurvival <strong>and</strong> proapoptotic regulators. Members of this family are<br />
classified, based on the presence of one or more Bcl-2 Homology (BH) domains, as<br />
antiapoptotic molecules (four BH domains: BH1–4) (such as Bcl-2), proapoptotic homologs<br />
(two or three BH domains: BH1-3) (such as Bax) or BH3-only death proteins (only the BH3<br />
domain) (including Bid or Bim). However, recent data highlighted the extent of sequence<br />
variation between Bcl-2 family proteins, including at the level of the BH domains, pointing to<br />
the need for a computational redefinition of the family. This goal can be achieved by the<br />
development of alignment pro<strong>file</strong>s combining sequence <strong>and</strong> structure information, namely<br />
structure-based hidden Markov models (HMMs). Here, we present a set of HMM-pro<strong>file</strong>s<br />
specific for cellular <strong>and</strong>/or viral-coded proteins sharing the same structural fold as Bcl-2.<br />
These models were checked for sensitivity <strong>and</strong> compared to st<strong>and</strong>ard domain signatures for<br />
Bcl-2 family recognition. Our results indicate that these novel pro<strong>file</strong>s are useful in<br />
identifying cellular <strong>and</strong> viral Bcl-2 homologs, including those with low sequence similarity.<br />
Indeed, we cover all known homology groups <strong>and</strong> we extend them by finding proteins<br />
belonging to new homology groups. Analysis of the full set of known <strong>and</strong> novel protein<br />
sequences retrieved by the HMM pro<strong>file</strong>s will be used for automatic update of the Bcl-2<br />
Family Database (http://bcl2db.ibcp.fr/) <strong>and</strong> for integrated systems biology <strong>and</strong> phylogenetic<br />
approaches.<br />
122
Session 5: Proteomics Poster 3<br />
Proteomic Profiling of Human Melanoma Metastatic Cell Line Secretomes<br />
Micaela Rocco 1* , Livia Malorni 2* , Giuseppe Palmieri 3 , Carla Rozzo 3 , Augusto Parente 1<br />
<strong>and</strong> Angela Chambery 1 .<br />
1<br />
Dipartimento di Scienze della Vita, Via Vivaldi 43, Seconda Università di Napoli, I-<br />
81100 Caserta, Italy<br />
micaela.rocco@unina2.it; augusto.parente@unina2.it; angela.chambery@unina2.it<br />
2<br />
Proteomic <strong>and</strong> Biomolecular Mass Spectrometry Center, Institute of Food Science <strong>and</strong><br />
Technology, National Research Council (CNR), Via Roma 64, I-83100 Avellino, Italy<br />
lmalorni@isa.cnr.it<br />
3<br />
Unit of Cancer Genetics, Institute of Biomolecular Chemistry, National Research<br />
Council (CNR), Traversa La Crucca 3, Baldinca Li Punti, I-07100 Sassari, Italy<br />
palmierig@icb.cnr.it; carla.rozzo@icb.cnr.it<br />
* These Authors contributed equally to this work.<br />
During the last years the incidence <strong>and</strong> mortality of melanoma have rapidly increased.<br />
Metastatic spread of malignant melanoma is often associated to cancer progression with poor<br />
prognosis <strong>and</strong> survival. These processes are controlled by dynamic interactions between<br />
tumor melanocytes <strong>and</strong> neighbouring stromal cells, whose deregulation leads to the<br />
acquisition of cell proliferation capabilities <strong>and</strong> invasiveness. It is increasingly clear that a key<br />
role in carcinogenesis is played by secreted molecules either by tumor <strong>and</strong> surrounding<br />
stromal cells. To address the issue of the proteins secreted by different metastases of the same<br />
patient, the proteomic profiling of secretomes of human metastatic cell lines (i.e. PES 41 <strong>and</strong><br />
PES 47 from two subcutaneous metastases <strong>and</strong> PES 43 from a lung metastasis) was<br />
performed by applying a shotgun LC-MS/MS-based approach. The results provide a list of<br />
c<strong>and</strong>idates associated to the metastatic potential of PES human melanoma cell lines. Among<br />
them, several matricellular proteins previously reported as involved in melanoma<br />
aggressiveness were identified (i.e. SPARC, osteopontin, galectins). In addition, the<br />
extracellular matrix protein 1 that stimulates the proliferation <strong>and</strong> angiogenesis of endothelial<br />
cells <strong>and</strong> fibronectin that is involved in cell adhesion <strong>and</strong> motility were identified. The<br />
presented work provides the basis to clarify the complex extracellular protein networks<br />
implicated in human melanoma cell invasion, migration <strong>and</strong> motility.<br />
123
Session 5: Proteomics Poster 4<br />
Proteomic analysis of signaling processes induced in cultured glioblastoma cells after<br />
sub-lethal photodynamic treatment<br />
A. Uzdensky 1 , A. Juzeniene 2 , J. Moan 2<br />
1<br />
Southern Federal University, 194/1, Stachky ave., NIINK, Rostov-on-Don, 344090,<br />
Russia, auzd@y<strong>and</strong>ex.ru<br />
2<br />
Institute for Cancer Research, Montebello, N-0310, Oslo, Norway<br />
Photodynamic therapy (PDT) is currently used for killing of malignant cells in oncology. To<br />
characterize initial signaling processes involved in response of glioblastoma D54Mg cells to<br />
PDT treatment with 5-aminolevulinic acid, we used small PDT doses that killed
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 6: Epigenetics<br />
125
Session 6: Epigenetics Poster 1<br />
Effects of Lithium-Induced Cell Cycle Arrest on The Histone 3 Lysine 27 Methylation in<br />
Huh7, Hepatocellular Carcinoma Cell Line.<br />
Sanem TERCAN AVCI 1 , Ne!e ATABEY 1 , Esra ERDAL 1<br />
1 Dokuz Eylul University School of Medicine, Department of Medical Biology <strong>and</strong><br />
Genetics, 35340-Inciralti, Izmir/TURKEY<br />
Exposure of normal liver cell with a viral or chemical agent triggers telomere shortening<br />
associated with repetitive necrosis/proliferation rotation. It causes changes in<br />
microenvironment of liver, activation of stem cells <strong>and</strong>/or satellite cells, after that, activation<br />
of cirrhosis, displastic nodule <strong>and</strong> hepatocellular carcinoma (HCC) depending on the aberrant<br />
cell division <strong>and</strong> resistance to apoptosis. It was demonstrated that during<br />
hepatocarcinogenesis most of genes silenced by methylation on their promoter, even though<br />
the roles of histone code alterations have not been known yet.<br />
In our previous study, it has been shown that lithium cause G1 cell cycle arrest on Huh7 cells,<br />
HCC cell line <strong>and</strong> microarray analysis further demonstrated that transcripts of histone<br />
modifier enzymes changed significantly in lithium treated cells in compared to untreated ones<br />
(Erdal E., 2005). So, we hypothesized that histone modifications on the chromatin might<br />
control cell cycle of HCC cells. In this study, it has been first shown that expression of EZH2<br />
histone methyl transferase decreased in both transcript <strong>and</strong> protein level under the effect of<br />
lithium treatment in Huh7 cells <strong>and</strong> expression of JMJD3 histone demethylase increased in<br />
transcript level as expected. Since both enzymes are known as responsible for the regulation<br />
of histone 3 lysine 27 methylation, we inquired whether H3K27 is affected in lithium treated<br />
Huh7. Unexpectedly, level of H3K27me3 modification has been shown as significant<br />
increase. This result makes us thinking lithium affects by a different mechanism. Recently, it<br />
was shown that GSK3! phosphorylates EZH2 <strong>and</strong> phosphorylated form of EZH2 in<br />
Polycomb Repressive Complex 2 (PRC2) do not catalyzed H3K27 methylation. Since lithium<br />
is very well known inhibitor of GSK3!, we can explain the unexpected result regarding<br />
decline in H3K27me3 although EZH2 decreases in protein level. To test this hypothesis, we<br />
analyzed level of phosphorylated form of EZH2 under the treatment of lithium. It has been<br />
found that lithium causes decrease on the phosphorylated form of EZH2 at 2nd <strong>and</strong> 6th hours<br />
after treatment.<br />
As a conclusion, these analyses showed us the alterations of histone modification patterns,<br />
especially H3K27me3 modification, might affect cell proliferation on HCC.<br />
Key Words: HCC, histone modifications, EZH2<br />
126
Session 6: Epigenetics Poster 2<br />
Reexpression of the hic1 tumorsuppressor gene after pharmacological unmasking<br />
results in increased radio-sensitivity of head <strong>and</strong> neck squamous cell carcinoma<br />
Jürgen Brieger, Sylvia Mann, Wolf Mann<br />
Laboratory of Molecular Tumor Biology, Department of Otolaryngology, Head <strong>and</strong><br />
Neck Surgery, University Medical Center of the Johannes Gutenberg University, 55101<br />
Mainz, Germany, eMail: juergen.brieger@unimedizin-mainz.de<br />
Hypermethylation of the genome <strong>and</strong> especially of tumor suppressor genes have been<br />
recognized as causative for the generation of several types of tumours. Here we analysed 5-<br />
Azacytidin (5-Aza) for demethylation treatment of the tumor suppressor hic1<br />
(hypermethylated in cancer 1) <strong>and</strong> as a radio-sensitizing agent in head <strong>and</strong> neck squamous cell<br />
carcinoma (HNSCC). To this end we treated an established HNSCC cell line with different<br />
concentrations of 5-Aza for 72h, followed by an single irradiation with 4 or 50 Gy.<br />
Methylation status <strong>and</strong> re-expression of hic1 were analysed by methylation specific PCR,<br />
quantitative PCR <strong>and</strong> Western blot before <strong>and</strong> after treatment. Survival, apoptosis,<br />
proliferation, <strong>and</strong> migration were analysed as functional parameters. We found hic1<br />
frequently hypermethylated in HNSCC. After 5-Aza treatment hic1 expression was restored<br />
<strong>and</strong> cellular survival reduced. The combined application of 5-Aza <strong>and</strong> irradiation further<br />
decreased survival of the cells. Additionally, migration was impaired as well as apoptosis<br />
increased.<br />
The data show the relevance of hypermethylation for radiation resistance <strong>and</strong> suggest that the<br />
reactivation of relevant tumor suppressor genes by demethylating active drugs might be<br />
beneficial to overcome irradiation resistance.<br />
127
Session 6: Epigenetics Poster 3<br />
Polyarginine peptide nucleic acids inibit biological activity of microRNA 210<br />
Enrica Fabbri 1 , Nicoletta Bianchi 1 , Eleonora Brognara 1 ,Alessia Finotti 1 , Giulia<br />
Breveglieri 1 , Monica Borgatti 1 , Alex Manicardi 2 , Roberto Corradini 2 ,<br />
Rosangela Marchelli 2 <strong>and</strong> Roberto Gambari 1<br />
1 BioPharmaNet, Department of Biochemistry <strong>and</strong> Molecular Biology, Ferrara<br />
University, via Fossato di Mortara, 74, 44121, Ferrara, Italy, e-mail:gam@unife.it;<br />
2 Department of Organic Chemistry, Parma University, Parco Area delle Scienze, 17/A,<br />
43124 Parma, Italy.<br />
Peptide nucleic acids are DNA mimics extensively used for pharmacological regulation of<br />
gene expression in antisense <strong>and</strong> anti-gene therapies. At present, very few data are available<br />
on the use of PNAs as molecules targeting microRNAs. MicroRNAs are a family of small (19<br />
to 25 nucleotide in length) noncoding RNAs that regulate gene expression by sequenceselective<br />
targeting of mRNAs, leading to a translational repression or mRNA degradation,<br />
depending on the degree of complementarity between microRNAs <strong>and</strong> the target sequences.<br />
MicroRNAs are deeply involved in the control of highly regulated biological functions, such<br />
as differentiation, cell cycle <strong>and</strong> apoptosis. The aim of the present study was to determine the<br />
activity of a PNA conjugated to polyarginine peptide <strong>and</strong> designed against microRNA 210, a<br />
microRNA associated to hypoxia <strong>and</strong> involved in the erythroid differentiation modulation.<br />
Our studies demonstrated that this PNA is efficiently internalized within target cells <strong>and</strong><br />
strongly inhibits microRNA 210 activity with an alteration of the raptor <strong>and</strong> !-globin gene<br />
expression. Unlike commercially available antagomiRs, which need continous<br />
administrations, a single administration of this PNA (without using transfection reagents like<br />
lipofectin or lipofectamine) is sufficient to obtain these biological effects. Our results<br />
demostrate PNA-based molecules are very promising reagents to modulate the biological<br />
activity of micro RNAs.<br />
128
Session 6: Epigenetics Poster 4<br />
Dataset integration identifies transcriptional regulation of microRNA genes by PPARG<br />
in mouse adipogenesis<br />
Elisabeth John 1 , Anke Wienecke-Baldacchino 1 , Merja Heinäniemi 1 , Carsten Carlberg 1,2#<br />
<strong>and</strong> Lasse Sinkkonen 1<br />
1<br />
Life Sciences Research Unit, University of Luxembourg, L-1511 Luxembourg,<br />
Luxembourg<br />
2<br />
Department of Biosciences, University of Eastern Finl<strong>and</strong>, FIN-70211 Kuopio, Finl<strong>and</strong><br />
E-mail: elisabeth.john@uni.lu, anke.wienecke@uni.lu, merja.heinäniemi@uni.lu,<br />
carsten.carlberg@uni.lu, lasse.sinkkonen@uni.lu<br />
Peroxisome proliferator-activated receptor G (PPARG) is a key transcription factor in<br />
mammalian adipogenesis. Genome-wide approaches identified thous<strong>and</strong>s of PPARG binding<br />
sites in mature mouse 3T3-L1 adipocytes <strong>and</strong> PPARG up-regulates hundreds of proteincoding<br />
genes during adipogenesis. However, so far no microRNA (miRNA) genes were<br />
identified as primary PPARG targets. By integration of four separate datasets of genome-wide<br />
PPARG binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPARG<br />
binding sites within 50 kb from miRNA gene transcription start sites. Nineteen of these<br />
putative PPARG-regulated mature miRNAs were up-regulated above 2-fold during 3T3-L1<br />
adipogenesis. Focusing on miRNA loci with PPARG binding sites confirmed by at least three<br />
datasets resulted in six high confidence miRNA target loci. The up-regulation of five miRNA<br />
genes miR-103-1 (host gene Pank3), miR-148b (Copz1), miR-182/96/183, miR-205 <strong>and</strong> miR-<br />
378 (Ppargc1b) followed that of Pparg. The PPARG dependence of four of these miRNA loci<br />
was demonstrated by PPARG knock-down. The loci of miR-103-1 (Pank3) <strong>and</strong> miR-378<br />
(Ppargc1b) were also responsive to the PPARG lig<strong>and</strong> rosiglitazone. Finally, chromatin<br />
immunoprecipitation analysis validated three in silico predicted PPARG binding sites at the<br />
miR-103-1 locus <strong>and</strong> two at the miR-378 locus. In conclusion, we identified 22 putative<br />
PPARG target miRNAs genes, showed the PPARG dependence of four of these genes <strong>and</strong><br />
demonstrated two as functional PPARG target genes in mouse adipogenesis.<br />
129
Session 6: Epigenetics Poster 5<br />
Histone deacetylases 1, 6 <strong>and</strong> 8 are critical for invasion in breast cancer<br />
Soon Young Park, Ji Ae Jun, Kang Jin Jeong, Hoi Jeong Heo, Jang Sihn Sohn, Hoi<br />
Young Lee, Chang Gyo Park, <strong>and</strong> Jaeku Kang<br />
Myunggok Medical Research Institute, College of Medicine, Konyang University,<br />
Daejeon 302-718, Republic of Korea; E-mail: jaeku@konyang.ac.kr<br />
Histone deacetylases (HDACs) are associated with the development <strong>and</strong> progression of<br />
cancer, but which HDAC isoforms play important roles in breast cancer metastasis is not<br />
known. Here, we identify the specific HDAC isoforms that are necessary for invasion <strong>and</strong>/or<br />
migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more<br />
invasive <strong>and</strong> expressed higher levels of matrix metalloproteinase-9 (MMP-9) than MCF-7<br />
cells. We compared the expression of HDAC isoforms between MCF-7 <strong>and</strong> MDA-MB-231<br />
cells <strong>and</strong> found greater expression of HDAC4, 6 <strong>and</strong> 8 in MDA-MB-231 by RT-PCR <strong>and</strong><br />
Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to<br />
attenuate the invasion, migration <strong>and</strong> MMP-9 expression in MDA-MB-231 cells. Using<br />
specific siRNAs directed against HDAC1, 4, 6 <strong>and</strong> 8, we show that inhibition of HDAC1, 6<br />
<strong>and</strong> 8, but not HDAC4, are responsible for invasion <strong>and</strong> MMP-9 expression in MDA-MB-231<br />
cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 <strong>and</strong><br />
found that overexpression of HDAC1, 6 or 8 increased invasion <strong>and</strong> MMP-9 expression. By<br />
developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present<br />
study can be extended to developing therapies for breast cancer invasion.<br />
130
Session 6: Epigenetics Poster 6<br />
Functional studies of CBP/TDG complexes reveal a close link between DNA repair,<br />
transcription, <strong>and</strong> epigenetic signaling.<br />
Hélène Léger1, Caroline Smet-Nocca2, Sebastian Eilebrecht1, <strong>and</strong> Arndt Benecke1<br />
1 Institut des Hautes Études Scientifiques & CNRS USR3078, 35 route de Chartres,<br />
91440 Bures sur Yvette, France<br />
2 Unité de glycobiologie structurale et fonctionnelle CNRS UMR8576, Université des<br />
Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France<br />
The packaging of eukaryotic DNA into chromatin represents an essential organizational <strong>and</strong><br />
an important regulatory event. Chromatin structure can specifically contribute positively or<br />
negatively to the correct assembly of transcription factors (TFs) <strong>and</strong> their activity. A local<br />
<strong>and</strong>/or global loss of epigenetic information is often a significant factor in genetic disorders<br />
<strong>and</strong> cancers because it leads to a deregulation of gene expression. Unfortunately the<br />
modifications occurring at the level of the chromosome, are ample <strong>and</strong> only in part<br />
understood. We have highlighted direct coupling between mechanisms of transcription by<br />
retinoic acid receptor (RAR) <strong>and</strong> its coregulator, the CREB Binding Protein (CBP) <strong>and</strong><br />
mechanisms of base excision repair (BER) by the Thymine DNA Glycosylase (TDG). TDG<br />
also acts as a coactivator of the family of RAR transcription factors <strong>and</strong> thus plays an<br />
essential role during differentiation <strong>and</strong> development. Furthermore, TDG has the ability to<br />
repair G: T mismatches produced by deamination of G: meC (methylated cytosine) in CpG<br />
isl<strong>and</strong>s in order to restore a G: C pair. Thus TDG deregulation might be an important<br />
contributor to demethylation of DNA. The CBP-RAR-TDG complex that we have shown to<br />
exist in living cells is the first link between DNA repair by base excision, transcription <strong>and</strong><br />
epigenetics <strong>and</strong> thus is a new way of regulating the integrity of the epigenome <strong>and</strong> gene<br />
expression. The aim of our work was to characterize functional CBP-TDG-RAR complexes in<br />
transcriptional regulation <strong>and</strong> epigenetics. The ultimate aim of this work is the underst<strong>and</strong>ing<br />
of genomic plasticity induced by the CBP-TDG complex <strong>and</strong> its role in oncogenesis.<br />
Transcriptome analyses with different mutant forms of TDG demonstrate altered levels of<br />
expression of transcription factors, developmental genes, migration factors, genes with<br />
functions in cell localization <strong>and</strong> cell proliferation. Mutations of the sumoylation site stabilize<br />
the CBP-TDG complex, whereas a mutation of TDG, which is not capable for acetylation by<br />
CBP prevent formation of CBP-TDG complex. We have also carried out transcriptome<br />
analyses of CBP/TDG complexes in the presence of different chemical agents to induce<br />
nuclear receptor driven transcription with retinoic acid <strong>and</strong> beta-estradiol, or to inhibit de<br />
novo methylation using 5-aza-2 deoxycytidine, <strong>and</strong> finally to massively induce DNA<br />
mismatches in vivo using 5-fluorouracil. In parallel, we have characterized in vitro the<br />
dynamics of the CBP-TDG complex analyzing the kinetics of the cleavage of<br />
oligonucleotides containing G / T or G / U, G / FU mismatches in the presence or absence of<br />
CBP <strong>and</strong> / or SUMO-1 for various forms of TDG. The results of these studies will be<br />
discussed here. Keywords : CREB binding protein (CBP), Thymine DNA glycosylase (TDG),<br />
Epigenetic regulation, Transcriptional regulation, Base Excision Repair (BER).<br />
131
Session 6: Epigenetics Poster 7<br />
Combinatorial microRNA regulation during adipogenesis: a systems biology approach.<br />
Maria Liivr<strong>and</strong> 1 , Merja Heinäniemi 2 , Carsten Carlberg 2 , Lasse Sinkkonen 2 <strong>and</strong> Thomas<br />
Sauter 1 .<br />
1 University of Luxembourg, LSRU, Systems Biology Group<br />
2 University of Luxembourg, LSRU, Computational Biology Group<br />
maria.liivr<strong>and</strong>@uni.lu; merja.heinäniemi@uni.lu; carsten.carlberg@uni.lu;<br />
lasse.sinkkonen@uni.lu; thomas.sauter@uni.lu.<br />
http://wwwen.uni.lu/research/fstc/life_sciences_research_unit/systems_biology<br />
Integration of knowledge <strong>and</strong> data between various fields such as biology, informatics <strong>and</strong><br />
medicine is the key to successful research. This thesis aims to create a new holistic<br />
perspective on adipogenesis by connecting molecular biology, computational modelling <strong>and</strong><br />
high-throughput datasets. The main focus will be on identifying miRNA families regulating<br />
single target genes in a synergistic, combinatorial manner. Relying on our previous modelling<br />
experience of a small adipogenic miRNA-target network as well as using new experimental<br />
data <strong>and</strong> computational approaches we aim first at constructing <strong>and</strong> validating a small detailed<br />
model of one core transcription factor (e.g. PPARG), two or three microRNAs (miRNAs) <strong>and</strong><br />
their shared targets’ interactions in an interconnected network. This will allow studying<br />
endogenous real-life examples of combinatorial regulation <strong>and</strong> help to draw general rules<br />
about miRNA function.<br />
The kinetic reconstruction will provide predictions on in silico stimulated changes to<br />
investigate the intrinsic stability <strong>and</strong> concentrational boundaries of molecular constituents of<br />
that system. In the next step, a larger network of molecular interactions will be created using<br />
computational databases <strong>and</strong> applications. We aim to identify more genes that are coregulated<br />
by multiple miRNAs from different families. Both networks will also be<br />
investigated for network motifs, common components of genetic interconnected circuits. This<br />
information will be further incorporated into an integrated model reflecting ratios between<br />
miRNAs <strong>and</strong> their targeted mRNAs. Finally, all these distinct relations will be organized into<br />
a larger model reflecting relations between core transcription factors, miRNAs <strong>and</strong> their target<br />
mRNAs.<br />
In this conceptual project we aim to reflect the effect of singular <strong>and</strong> combined molecular<br />
changes on a significant biological process – pre-adipocyte differentiation; find balancing<br />
scenarios in environmentally or genetically perturbated situations during adipogenesis <strong>and</strong><br />
provide significant insight into combinatorial miRNA regulation.<br />
132
Session 6: Epigenetics 8<br />
Switching Genes Promote Novel Pathway Response to Porphyromonas gingivalis<br />
Infection in Lean <strong>and</strong> Obese Macrophages<br />
Salomon Amar* x , Niraj S. Trivedi* <strong>and</strong> Calin Belta*.<br />
*Department of Bioinformatics, x Department of Periodontology <strong>and</strong> Oral Biology,<br />
Boston University, Boston, MA.<br />
!<br />
Obesity is a world epidemic problem affecting approximately 33% of US <strong>and</strong> 15% of the EU<br />
adult population. Obesity increases the risk of several chronic diseases including a diminished<br />
immune response to infection. To determine the underlying molecular dysfunction involved in<br />
the response of obese subjects to infection, we extracted Bone Marrow Macrophages (BMM)<br />
from lean <strong>and</strong> obese mice. BMM were exposed to Porphyromonas gingivalis (P. g.) for three<br />
incubation times (1h, 4h <strong>and</strong> 24h). Using a novel computational approach in conjunction with<br />
microarray data, we identified a special kind of differentially expressed genes, that we named<br />
switching genes. Our computation suggested that these genes controlled the behavior of<br />
different pathways by activating them in one condition, e.g. the lean macrophage, <strong>and</strong><br />
deactivating them in the second condition, e.g. the obese macrophage, <strong>and</strong> vice versa. The<br />
two most prominent switching genes that we found were proinflammatory factors:<br />
thrombospondin 1 <strong>and</strong> arginase 1. Our data, validated by PCR, showed that the inflammatory<br />
response was repressed in obese macrophage compared to lean counterparts. Our data suggest<br />
that, in order to produce a response to the P. g. infection, obese macrophages use switching<br />
genes to promote pathways associated with macrophage proliferation of <strong>and</strong> angiogenesis.<br />
The recruitment of these pathways is suggested to be the cause of the delay in the response to<br />
infection of obese subjects.<br />
133
Session 6: Epigenetics Poster 9<br />
Genome-wide DNA methylation Signatures of Cholangiocarcinoma<br />
Ruethairat Sriraksa 1,3 , Constanze Zeller 2 , Wei Dai 2 , Afshan Siddiq 4 , Andrew J Walley 5 ,<br />
Siobhan C McKay 7 , Naoya Kobayashi 6 , Geraldine Thomas 7 , Temduang Limpaiboon 3 ,<br />
Robert Brown 2 .<br />
1<br />
Graduate School, Khon Kaen University, Khon Kaen 40002, Thail<strong>and</strong>;<br />
r.sriraksa@gmail.com;<br />
2<br />
Epigenetics Unit, Department of Surgery <strong>and</strong> Oncology, Hammersmith Hospital,<br />
Imperial College, Du Cane Road, London W12 0NN, UK; c.zeller@imperial.ac.uk;<br />
w.dai@imperial.ac.uk; b.brown@imperial.ac.uk;<br />
3<br />
Centre for Research <strong>and</strong> Development of Medical Diagnostic Laboratories, Faculty of<br />
Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thail<strong>and</strong>;<br />
temduang@kku.ac.th;<br />
4<br />
Department of Epidemiology <strong>and</strong> Biostatistics, School of Public Health, Burlington-<br />
Danes Building, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK;<br />
a.siddiq@imperial.ac.uk;<br />
5<br />
Department of Genomics of Common Disease, School of Public Health, Burlington-<br />
Danes Building, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK;<br />
a.walley@imperial.ac.uk;<br />
6<br />
Department of Gastroenterological Surgery, Transplant <strong>and</strong> Surgical Oncology,<br />
Okayama University Graduate School of Medicine, Dentistry <strong>and</strong> Pharmaceutical<br />
Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan; immortal@md.okayamau.ac.jp;<br />
7<br />
Molecular Pathology, Hammersmith Hospital, Imperial College, Du Cane Road,<br />
London W12 0NN, UK; siobhan.mckay08@imperial.ac.uk;<br />
geraldine.thomas@imperial.ac.uk<br />
To address genome-wide DNA methylation changes in cholangiocarcinoma (CCA), we performed<br />
genome-wide DNA methylation profiling of 32 primary CCA, 6 matched adjacent normal samples, 5<br />
CCA cell lines <strong>and</strong> a normal biliary cell line using HumanMethylation27 BeadChips. We identified<br />
718 <strong>and</strong> 594 CG sites as being hypermethylated <strong>and</strong> hypomethylated in CCA compared to adjacent<br />
normal samples, respectively (False Discovery Rate; FDR85% of primary CCA as compared to adjacent normal samples using bisulfite<br />
pyrosequencing. Moreover, detailed gene set enrichment analysis (GSEA) showed overrepresentation<br />
of hypermethylated CpG sites associated with homeobox genes, PRC2 targets, EED targets, SUZ12<br />
targets <strong>and</strong> H3K27 targets. In contrast, hypomethylation of NOS targets <strong>and</strong> OCT4 targets was<br />
significantly enriched in CCA. Epigenetic aberrations of these genes have been proposed to play roles<br />
in the stem cell origin of cancers <strong>and</strong> they might provide new therapeutic targets for CCA. To our<br />
knowledge, this is the first report of genome-wide DNA methylation study in cholangiocarcinoma<br />
which provides a useful resource of epigenetic signature that could serve as the biomarkers. However,<br />
the biological functions <strong>and</strong> clinical application of these c<strong>and</strong>idate DNA methylation changes should<br />
be further clarified.<br />
134
Session 6: Epigenetics Poster 10<br />
Epigenetic regulation of proMMP-1, -2 <strong>and</strong> -9 expression in HT1080 human<br />
fibrosarcoma cell line.<br />
M. Poplineau 1 , J. Dufer 1 , F. Antonicelli 1 <strong>and</strong> A. Trussardi-Regnier 1 .<br />
1 Facultés de Médecine et Pharmacie, Unité MEDyC, UMR CNRS/URCA n°6237 :1,<br />
avenue du Maréchal Juin, 51096 REIMS Cedex, France.<br />
E-mail address: aurelie.trussardi@univ-reims.fr, frank.antonicelli@univ-reims.fr,<br />
jean.dufer@univ-reims.fr, mathilde.poplineau@etudiant.univ-reims.fr.<br />
The matrix metalloproteinase (MMP) family members play an important role in various<br />
physiological <strong>and</strong> pathological processes. The collagenase MMP-1 <strong>and</strong> the gelatinases MMP-<br />
2 <strong>and</strong> MMP-9 are involved in tumor invasiveness but the regulation of their expression is not<br />
fully elucidated <strong>and</strong> could implicate epigenetic mechanisms (DNA methylation <strong>and</strong>/or histone<br />
posttranslational modifications). The aim of this study was to analyze the effects of the<br />
histone deacetylase inhibitor trichostatine A (TSA) <strong>and</strong> the inhibitor of DNA methylation 5aza-2’-deoxycytidine<br />
(5-azadC) on the MMP-1,-2 <strong>and</strong> -9 expressions in human HT1080<br />
fibrosarcoma cell line. Real time RT-PCR revealed that 5-azadC or 5-azadC + TSA but not<br />
TSA alone (despite histone H4 hyperacetylation) increase mRNA levels. This transcription<br />
activation is correlated with chromatin decondensation observed by nuclear texture image<br />
analysis. Western blot <strong>and</strong> gelatin zymography analysis of cell culture media revealed<br />
significant secretion of proMMP-1 <strong>and</strong> high activity of MMP-2 <strong>and</strong> MMP-9, after 5-azadC or<br />
5-azadC + TSA treatment. These results suggest that epigenetic mechanism could be involved<br />
in MMP-1, MMP-2, MMP-9 gene <strong>and</strong> protein expression <strong>and</strong> could thereby modulate the<br />
invasive behavior of tumor cells.<br />
135
Session 6: Epigenetics Poster 11<br />
Epigenetic regulation of CIITA expression in T cells<br />
M.C.J.A. van Eggermond a , R. J. Wierda a , T. M. Holling a <strong>and</strong> P.J. van den Elsen a,b .<br />
a<br />
Department of Immunohematology <strong>and</strong> Blood Transfusion, LUMC, Leiden, The<br />
Netherl<strong>and</strong>s.<br />
b<br />
Department of Pathology, VU University medical center, Amsterdam, The<br />
Netherl<strong>and</strong>s.<br />
Previously we have shown that activated T cells accomplish expression of MHC-II molecules<br />
through induction of the class II transactivator (CIITA) by employment of CIITA promoter III<br />
(CIITA-PIII). In this study we show that epigenetic regulation controls CIITA-PIII promoter<br />
accessibility in normal T cells during activation. In unstimulated T cells, lacking CIITA <strong>and</strong><br />
MHC-II molecule expression, CIITA-PIII chromatin displays relative high levels of<br />
repressive histone methylation marks (3Me-K27-H3 <strong>and</strong> 3Me-K20-H4) <strong>and</strong> low levels of<br />
acetylated histones H3 (Ac-H3) <strong>and</strong> H4 (Ac-H4). These repressive histone marks are<br />
downregulated <strong>and</strong> replaced by histone methylation marks (3Me-K4-H3) associated with<br />
transcriptional active genes <strong>and</strong> high levels of Ac-H3 <strong>and</strong> Ac-H4 in activated T cells<br />
expressing CIITA. This is associated with concomitant recruitment of RNA polymerase II.<br />
Furthermore, both unstimulated <strong>and</strong> activated T cells lacked DNA methylation of CIITA-PIII.<br />
In T leukemia cells, devoid of CIITA expression, similar repressive histone methylation<br />
marks <strong>and</strong> low levels of acetylated histone H3 correlated with lack of CIITA expression. This<br />
in contrast to CIITA expressing T lymphoma cells, which display high levels of Ac-H3 <strong>and</strong><br />
3Me-K4-H3, <strong>and</strong> relative low levels of the repressive 3Me-K27-H3 <strong>and</strong> 3Me-K20-H4 marks.<br />
Of interest was the observation that the levels of histone acetylation <strong>and</strong> methylation<br />
modifications in histones H3 <strong>and</strong> H4 were also noted in chromatin of the downstream CIITA-<br />
PIV promoter as well as the upstream CIITA-PI <strong>and</strong> CIITA-PII promoters both in normal T<br />
cells <strong>and</strong> in malignant T cells. Together our data show that CIITA-PIII chromatin in activated<br />
T cells <strong>and</strong> in T lymphoma cells expressing CIITA display similar histone acetylation <strong>and</strong><br />
methylation characteristics associated with an open chromatin structure allowing RNA<br />
polymerase II recruitment. The opposite is true for unstimulated T cells <strong>and</strong> in T-leukemia<br />
cells lacking CIITA expression, which display histone modifications characteristic of<br />
condensed chromatin affecting RNA polymerase II recruitment.<br />
136
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 7: Cancer signaling networks<br />
137
Session 7: Cancer signaling networks Poster 1<br />
Retardation of human cancer cells proliferation by siRNAs targeted to the c-myc,<br />
N-myc, Her2, Cyclin B1 <strong>and</strong> PKC genes<br />
Ivan A. Akimov, Tatyana O. Kabilova, Marina A. Zenkova, Valentin V. Vlassov, Elena<br />
L. Chernolovskaya.<br />
Institute of Chemical Biology <strong>and</strong> Fundamental Medicine SB RAS,<br />
Novosibirsk 630090, Russia. E-mail: akimov@niboch.nsc.ru<br />
Elevated expression of genes encoding cell cycle regulatory proteins is a frequent cause of<br />
uncontrolled human cells proliferation leading to cancer. The aim of the research was to<br />
design effective inhibitors of proliferation of human cancer cells of different origin <strong>and</strong> to<br />
identify the effective therapeutic targets in each cell line. We investigated the impact of<br />
specific inhibition of c-myc, N-myc, Her2, Cyclin B1 <strong>and</strong> PKC genes expression by siRNAs<br />
(50-200 nM) on proliferation rate of KB-3-1, SK-N-MC, IMR-32, MCF-7 <strong>and</strong> HL-60 human<br />
cancer cells. The average level of gene expression was reduced to 20% compared to control.<br />
The maximum inhibition effect was achieved 72h after transfection <strong>and</strong> the initial targeting<br />
RNA level restored to 7th day. The obtained data demonstrate that inhibition of this gene<br />
expression reduces of proliferation rate differently depending on the cell type. The most<br />
effective reduction of proliferation rate induced in !"-3-1 cells (down to 10%) by inhibition<br />
of c-myc gene expression, in SK-N-MC cells (down to 4%) by inhibition of Cyclin B1 gene<br />
expression, in IMR-32 cells (down to 30%) by inhibition of N-myc gene expression <strong>and</strong> in<br />
MCF-7 cells (down to 10%) by inhibition of PKC gene expression. We investigated the<br />
proliferation of the cells after recovery of the baseline expression of target genes. We found<br />
that the proliferation rate restored after reactivation of Her2, Cyclin B1 <strong>and</strong> PKC genes in<br />
KB-3-1 <strong>and</strong> MCF-7 cells, while proliferation rate of neuroblastoma cells SK-N-MC remained<br />
reduced (to 35% <strong>and</strong> 15% respectively) after restoration of Her2 <strong>and</strong> Cyclin B1 genes<br />
expression. A selective staining of live, dead <strong>and</strong> cells in apoptotic stage showed that there are<br />
no increase in the percentage of dead <strong>and</strong> apoptotic cells in population, therefore retardation<br />
of cell division but not cell death is the reason of observed antiproliferative action. The<br />
mechanisms of cell differentiation may also be involved. Thus the obtained data suggest that<br />
Her2 <strong>and</strong> Cyclin B1 genes are the promising targets for the treatment of neuroblastomas. This<br />
work was supported by RAS programs “Molecular <strong>and</strong> cellular biology” <strong>and</strong> “Fundamental<br />
science for medicine”, president`s program SS-7101.2010.4 <strong>and</strong> SB RAS gr<strong>and</strong> No.41.<br />
138
Session 7: Cancer signaling networks Poster 2<br />
Control of CDC25 phosphatases splicing by genotoxic agents in MCF-7 breast cancer<br />
cell line.<br />
Hélène Albert 1 , Eric Battaglia 1 , Susana Santos 2 , Carolino Monteiro 2 , Denyse Bagrel 1<br />
1 Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, EA 3940, FR<br />
CNRS 2843, Université Paul Verlaine-Metz, Rue du Général Delestraint, 57070, Metz,<br />
France.<br />
2 Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003<br />
Lisboa, Portugal.<br />
Email : bagrel@univ-metz.fr<br />
CDC25 phosphatases play a crucial role in cell cycle progression <strong>and</strong> are overexpressed in a<br />
large number of cancers. CDC25 are represented by three isoforms: CDC25A, CDC25B <strong>and</strong><br />
CDC25C, encoded by three different genes, all of them subjected to an alternative splicing<br />
mechanism. Alternative splicing is an essential process concerning 75 % of human genes <strong>and</strong><br />
thus contributes to proteomic diversity. Many studies are currently focused on alternative<br />
splicing regulation in cellular stress conditions. However, none of them have been conducted<br />
so far on CDC25. Using semi-quantitative <strong>and</strong> quantitative RT-PCR, we showed that<br />
treatment of MCF-7 human mammary adenocarcinoma cells with doxorubicin (1 !M) does<br />
not affect CDC25A <strong>and</strong> CDC25B splicing, while a modification of CDC25C splicing pro<strong>file</strong><br />
was observed after 6-12 hours. CDC25C5 splice variant proportion was increased 10-fold in<br />
comparison to that of CDC25C1 variant at both mRNA <strong>and</strong> protein levels. A similar<br />
alteration of CDC25C alternative splicing was observed with other genotoxic agents,<br />
including topoisomerases inhibitors camptothecin <strong>and</strong> etoposide, the electrophilic agent<br />
cisplatin <strong>and</strong> the reactive oxygen species generating agent tert-butyl hydroperoxide, but not<br />
with cyclophosphamide <strong>and</strong> vinblastine. These observations suggest that CDC25C alternative<br />
splicing, but not that of CDC25A <strong>and</strong> CDC25B, is modulated under conditions associated to<br />
DNA damage. Further studies are needed to elucidate the regulatory pathways involved in this<br />
mechanism.<br />
139
Session 7: Cancer signaling networks Poster 3<br />
Novel CDC25 small coumarin-based inhibitors: biological evaluation.<br />
E. Bana, S. Valente, E. Viry, G. Kirsch, D. Bagrel<br />
Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, LIMBP, avenue<br />
du général Delestraint, 57070 Metz, France - bana@univ-metz.fr<br />
Cell division cycle 25 (CDC25) phosphatases (A, B, <strong>and</strong> C) are key cell cycle control proteins<br />
in eukaryotic cells <strong>and</strong> numerous studies rise up the correlation between their overexpression<br />
<strong>and</strong> cancer aggressiveness, high grade tumors <strong>and</strong> low vital prognosis. CDC25s are attractive<br />
c<strong>and</strong>idates for new cancer therapy targets as their inhibition could be able to slow down tumor<br />
growth. To date, about one hundred chemical compounds have been reported in literature as<br />
inhibitors of CDC25s. Most are quinonoid, phosphate surrogates or electrophilic inhibitors.<br />
Several silybin derivatives showed a cell cycle arrest <strong>and</strong> induced level variations of the three<br />
CDC25s in PC3 cells, <strong>and</strong> several flavones are able to inhibit CDC-25.1 phosphatase activity<br />
in C. elegans. These findings prompted us to project the design, synthesis <strong>and</strong> biological<br />
validation of novel coumarin-based (like flavon isomer) derivatives. Our study started with<br />
insertion of 4-methoxy(hydroxy)phenyl <strong>and</strong> styryl groups in C3 position of the coumarin<br />
nucleus. We then explored the C4 position with the chalcone-coumarin (benzoylvinyl)<br />
derivatives <strong>and</strong> the reverse chalcone-coumarin (cinnamoyl). At last we investigated the<br />
insertion of a sulfur atom in the spacer-chain between coumarin <strong>and</strong> benzoyl moiety,<br />
obtaining final thio-analogs.<br />
The CDC25 inhibitory potential of 16 new compounds was tested using the three human<br />
(glutathion-S-transferase)-CDC25s recombinant enzymes. This screening highlighted two<br />
chalcon-coumarin derivatives, namely 6a <strong>and</strong> 6d, which showed an IC50 value in the level of<br />
25 !M against CDC25A <strong>and</strong> C. 6a behaves as an irreversible inhibitor of CDC25A. Using<br />
breast cancer cell lines (MCF7 <strong>and</strong> derivatives), the study of a potential correlation between<br />
in vitro <strong>and</strong> in cellulo CDC25 inhibitions as well as cytostatic effect <strong>and</strong> cell cycle blocking is<br />
going on.<br />
We consider 6a <strong>and</strong> 6d as two new lead compounds worthy of note for models in developing<br />
new CDC25 inhibitors, <strong>and</strong> thus new potential anticancer drugs.<br />
140
Session 7: Cancer signaling networks Poster 4<br />
The inhibition of poly(ADP-ribosylation) affects cancer cell proliferation <strong>and</strong> telomere<br />
elongation<br />
Cristina Belgiovine, Francesca Donà, Ilaria Chiodi, Tatiana Raineri, Roberta Ricotti,<br />
Chiara Mondello <strong>and</strong> A. Ivana Scovassi<br />
Istituto di Genetica Molecolare CNR, Via Abbiategrasso 207, I-27100 Pavia, Italy<br />
belgiovine@igm.cnr.it<br />
Poly(ADP-ribosylation) plays a central role in Base Excision Repair (BER) <strong>and</strong> also in<br />
neoplastic transformation <strong>and</strong> telomere length regulation. It is catalyzed by a family of<br />
poly(ADP-ribose) polymerases (PARPs), of which the best characterized are PARP-1, PARP-<br />
2, TANK-1 <strong>and</strong> TANK-2, which convert NAD into ADP-ribose further used to form<br />
polymers. We investigated their expression levels during neoplastic transformation of cen3tel<br />
cells, which derive from human fibroblasts immortalized by ectopic hTERT expression <strong>and</strong>,<br />
during propagation in vitro, acquired the ability to grow in agar <strong>and</strong> subsequently to form<br />
tumors in nude mice. Upon further propagation, tumorigenic cells became more aggressive,<br />
showing a reduction in the time required to induce tumors; moreover, they reached telomere<br />
lengths >100 Kb <strong>and</strong> increased telomerase activity. We found a notable increase of PARP-1<br />
protein (<strong>and</strong> mRNA) in parallel to the acquisition of the tumorigenic potential; PARP-2 levels<br />
showed the same trend, while tankyrase expression did not show significant changes. As<br />
supported by poly(ADP-ribose) accumulation, overexpressed PARP-1 <strong>and</strong> -2 were very<br />
active, thus suggesting that poly(ADP-ribosylation) is modulated during neoplastic<br />
transformation <strong>and</strong> could be a target for contrasting tumor development. To address this point,<br />
we analyzed the effect of the known PARP inhibitor, 3-aminobenzamide (3-AB), on cell<br />
proliferation <strong>and</strong> telomere length. Increasing concentrations of 3-AB inhibited the<br />
proliferation of neoplastic cells in a dose-dependent manner. Remarkably, prolonged 3-AB<br />
administration to early tumorigenic cells also affected telomere length. On the whole, our data<br />
support an active involvement of poly(ADP-ribosylation) in neoplastic transformation <strong>and</strong><br />
telomere length maintenance. Thus, our work provides additional evidence in favor of the use<br />
of PARP inhibitors for the treatment of human cancer [Donà et al., Curr. Pharmaceut.<br />
Biotechnol. (2010), in press].<br />
141
Session 7: Cancer signaling networks Poster 5<br />
Development of MIMOKINES, a New Class of Short CXCR4 Antagonists for Tumor<br />
<strong>and</strong> HIV-1 Treatment.<br />
Andy Chevigné1, Virginie Fievez1, Julie Mathu1, Jean-Claude Schmit1,2 <strong>and</strong> Sabrina<br />
Deroo1<br />
1Laboratory of Retrovirology, Centre de Recherche Public-Santé, 84, Val Fleuri, L-1526<br />
Luxembourg. 2Service National des Maladies Infectieuses, Centre Hospitalier<br />
Luxembourg, 4, Rue E. Barblé, L-1210 Luxembourg. <strong>and</strong>y.chevigne@crp-sante.lu<br />
The chemokine receptor CXCR4 is a G protein coupled receptors (GPCR) playing a crucial<br />
role in cancer <strong>and</strong> tumor cell biology by interacting with its unique lig<strong>and</strong> the chemokine<br />
CXCL12. CXCR4 also acts as an HIV-1 coreceptor for T cell infection. To date, no approved<br />
drugs targeting metastasis development or HIV-1 entry via CXCR4 are yet available.<br />
Therefore, the development of new molecules blocking the interactions between CXCR4 <strong>and</strong><br />
CXCL12 or viral gp120 protein is of great importance. In this study, we developed a new<br />
class of short CXCR4 antagonists based on peptides mimicking the N-terminus of CXCL12.<br />
We minimized the size of the chemokine to a peptide level (17-mer) <strong>and</strong> generated a large<br />
number of variants called Mimokines. Five phage displayed Mimokines libraries of high<br />
complexity (107 to 108 clones), characterized by complementary <strong>and</strong> progressive<br />
r<strong>and</strong>omization were engineered. Ten selection campaigns using either competitive or acid<br />
elution were performed on a simplified CXCR4 model. For acid elution campaigns<br />
enrichments up to 10 were obtained <strong>and</strong> 11 different positive clones with higher affinity (ratio<br />
1.3 to 8.5) for CXCR4 than the wild type peptide (wt) were identified. For competitive elution<br />
higher enrichments (up to 76) were obtained including clones with affinity ratio’s ranging<br />
from 3 to 14 compared to wt peptide. Sequence analysis revealed the presence of clones<br />
isolated at various frequencies (1/22 to 6/22). These peptides will be further analyzed for their<br />
antagonist properties towards CXCR4-CXCL12 axis <strong>and</strong> HIV-1 entry inhibition <strong>and</strong> may<br />
potentially provide new anti-metastasis <strong>and</strong> anti-HIV-1 lead molecules.<br />
142
Session 7: Cancer signaling networks Poster 6<br />
Study of the combined effect of 5-ALA-based photodynamic therapy <strong>and</strong> NF-kappaB<br />
inhibition on human glioblastoma cell survival<br />
Isabelle Coupienne, Grégory Fettweis <strong>and</strong> Jacques Piette<br />
University of Liege, GIGA-Research, Virology <strong>and</strong> Immunology Unit, B34, 1 avenue de<br />
l’Hopital, 4000 Liege, Belgium<br />
icoupienne@ulg.ac.be<br />
Glioblastoma constitute the most frequent <strong>and</strong> deadliest type of brain tumors in human adults.<br />
They are very resistant to all current therapies <strong>and</strong> are associated with a huge rate of<br />
recurrence. In most cases, this type of tumor is characterized by a constitutive activation of<br />
the nuclear factor-kappaB (NF-kappaB). This factor is known to be a key regulator of various<br />
physiological processes such as inflammation, immune response, cell growth or apoptosis. In<br />
the present study, we explored the role of NF-kappaB activation in the sensitivity of human<br />
glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)–based photodynamic<br />
therapy (PDT). Our results show that inhibition of NF-kappaB improves glioblastoma cell<br />
death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the<br />
cell death induced by PDT combined or not with NF-kappaB inhibition. We found that PDT<br />
mainly induced necrosis in glioblastoma cells <strong>and</strong> NF-kappaB was found to have anti-necrotic<br />
functions in this context. In the second part of this study, we examined the role of the kinase<br />
RIP3, recently identified as a key effector of the necrotic pathway, in 5-ALA-PDT-induced<br />
necrosis <strong>and</strong> studied whether NF-kappaB interfered in RIP3-dependent necrosis induction.<br />
143
Session 7: Cancer signaling networks Poster 7<br />
The Anti Proliferative Effect of Aloin <strong>and</strong> Aloe Emodin on MCF-7 <strong>and</strong> MDA-MB-231<br />
Breast Cancer Cells<br />
Melor M. Daud, Muhammad J. Ibrahim, Gabriele R. A. Froemming <strong>and</strong> Narimah A.<br />
H. Hasani<br />
Institute of Medical Molecular Biotechnology, Universiti Teknologi MARA Sungai<br />
Buloh Medical Campus, Jalan Hospital, Sungai Buloh, 47 000 Selangor, Malaysia<br />
- impaxmelor@gmail.com<br />
Aloin <strong>and</strong> Aloe Emodin are active components in Aloe Barbadensis Miller, commonly known<br />
as Aloe vera. Earlier studies had shown both anthraquinones display anti cancer activity. We<br />
were investigating their possible anti tumor properties, upon oestrogen receptor positive (ER + )<br />
MCF-7 cells <strong>and</strong> oestrogen receptor negative (ER - ) MDA-MB-231 breast cancer cells.<br />
Normal breast cell line, MCF-10A represented as negative control <strong>and</strong> Tamoxifen as positive<br />
control. Cell proliferation was measured using WST-1 Reagent at 450nm between 0!M to<br />
500!M.<br />
For MCF-7, we found IC50 of Tamoxifen at 64!M, 78!M for Aloe Emodin <strong>and</strong> no inhibitory<br />
concentration by Aloin up to 150!M. In MDA-MB-231, Tamoxifen induced anti proliferative<br />
effect at IC50 of 62!M, whereas below 150!M both Aloin <strong>and</strong> Aloe Emodin does not reach<br />
the IC50 level respectively. For MCF-10A, IC50 for Tamoxifen is 38!M, 410!M for Aloin <strong>and</strong><br />
no IC50 by Aloe Emodin up to 150!M.<br />
These results indicated that Aloe Emodin can be an effective inhibitor to the hormonal<br />
dependence MCF-7 breast cancer cells at low concentration without being cytotoxic to normal<br />
breast cells. The therapeutic properties can further be investigated for apoptotic effect <strong>and</strong><br />
signaling pathways. However, Aloe Emodin does not inhibit proliferation of MDA-MB-231<br />
cancer cells. Aloin shows no effect to both cancer <strong>and</strong> normal mammary cells up to 500!M,<br />
suggesting that it might not have anti carcinogenic properties. In conclusion, Aloe Emodin<br />
shows anti proliferative effect selectively to breast cancer cells with significant probability to<br />
demonstrate chemotherapeutic activities in breast cancer in vitro studies.<br />
144
Session 7: Cancer signaling networks Poster 8<br />
Phosphatidylinositol 3-kinase inhibitor - LY294002 in Regulation of Androgen<br />
Receptors in LNCaP prostate cancer cells.<br />
Joanna Duli!ska-Litewka, Dorota Gil, Paulina Dudzik <strong>and</strong> Piotr Laidler<br />
Chair of Medical Biochemistry, Jagiellonian University Medical College, 31-034<br />
Kraków, ul. Kopernika 7, Pol<strong>and</strong>; e-mail: mblitewk@cyf-kr.edu.pl<br />
Prostate cancer is one of males’ most serious health problem <strong>and</strong> has a high prevalence to<br />
metastasize making it a very difficult to treat if not detected early. Androgen receptor (AR)<br />
controls the expression of genes involved in cell proliferation, migration, differentiation, <strong>and</strong><br />
cell death. Increasing level of the AR not only intensify <strong>and</strong>rogen-induced cell proliferation<br />
but also increases the sensitivity to non<strong>and</strong>rogenous mediators, allowing prostate cancer cells<br />
to grow in a low <strong>and</strong>rogen environment. We observed that knockout of AR functions by<br />
siRNA leads to meaningful inhibition of LNCaP cells’ proliferation. In addition we noticed<br />
that under normal growth conditions, inhibition of PI3K/Akt signaling by inhibitor of PI3K -<br />
LY294002 - causes LNCaP cell arrest in G1 phase rather than apoptosis. Akt can modulate<br />
<strong>and</strong>rogen signaling since beta-catenin acts as AR coactivator, however how these two<br />
regulators cooperate to control prostate cancer cell growth <strong>and</strong> death remains as yet unclear.<br />
Inactivation of PI3K in LNCaP cell has been demonstrated to lead to the decreased<br />
phosphorylation of Akt in prostate cancer cell lines. In parallel treatment of LNCaP cells with<br />
LY294002 results in the increase of prostate-specific antigen (PSA) <strong>and</strong> prostatic acid<br />
phosphates (PAP) mRNA expression without testosterone or DHT treatment. This increase<br />
suggests that blockade of PI3K/Akt signaling pathway enhances AR transactivation. To find<br />
out the mechanism that explains their interaction we treated LNCaP cells with LY294002 or<br />
knocked out their AR <strong>and</strong> studied mRNA (RT-PCR) <strong>and</strong> protein (Western blot) expression of<br />
Akt/(pAkt), PSA, PAP, beta-catenin, cyclin D1, bax, bcl-2 <strong>and</strong> AR. pAkt was inhibited by<br />
LY294002 treatment but PSA, PAP, beta-catenin <strong>and</strong> AR protein levels were increased<br />
significantly, suggesting that blockade of PI3K/Akt pathway may enhance AR activity by the<br />
increase of AR protein expression. Activation of Akt in effect of AR silencing has been<br />
implicated in protection of cells from apoptosis throughout stimulation of c-Myc, cyclinD1<br />
expression <strong>and</strong> phosphorylation of the Bcl-2, thereby promoting cell survival. This important<br />
finding implies that combined inhibition of both PI3K/Akt <strong>and</strong> AR signaling may become a<br />
powerful approach to treat the prostate cancer.<br />
This work was supported by MNiSzW grants: K/ZDS/001003 <strong>and</strong> K/PBW/ 000561 -<br />
Jagiellonian University Medical College, Krakow, Pol<strong>and</strong><br />
145
Session 7: Cancer signaling networks Poster 9<br />
HGF/c-Met signaling pathway regulates TXNIP, major actor of oxidative stress, in SK-<br />
Hep-1, HCC cell line.<br />
Gozukizil A., Ozen E., Erdal E., Atabey N.<br />
Dokuz Eylul University, School of Medicine Depertmant of Medical Biology <strong>and</strong><br />
Genetics, 35340, Inciralti/Izmir, Turkey a.gkizil@hotmail.com<br />
In the development of Hepatocellular carcinoma (HCC), imbalance between oxygen <strong>and</strong><br />
glucose homeostasis contributes to the production of the reactive oxygen species following<br />
induction of oxidative stress. It is very well known that chronic inflammation, one of the<br />
hallmarks of development of HCC, has been triggered by oxidative stress after occurring<br />
continuous rounds of hepatocyte injury <strong>and</strong> following regeneration. Additionally, TXNIP,<br />
transcription factor, regulates cellular responses under stress conditions, such as hypoxia,<br />
serum starvation <strong>and</strong> heat-shock. It is recently shown that over-expression of TXNIP results<br />
in activation of oxidative stress <strong>and</strong> apoptotic responses of cells <strong>and</strong> it behaves as tumor<br />
suppressor. However, the role of TXNIP in HCC is not defined properly.<br />
In this study, expression levels of TXNIP have been analysed 12 HCC cell lines (Huh7,<br />
Hep3B, HepG2, PLC/PRF/5, SNU398, SNU387, SNU423, SNU449, Snu475, SNU182, SK-<br />
Hep-1, Mahlavu) by using western blotting <strong>and</strong> RT-PCR. Interestingly TXNIP expression<br />
was significantly high in HCC cell lines which have mesenchymal-like phenotype <strong>and</strong> high<br />
basal motility ability such as SNU182, SNU387 <strong>and</strong> SNU423. In contrast there was no<br />
expression of TXNIP in HCC cell lines that have epithelial-like phenotype such as Huh7,<br />
Hep3B <strong>and</strong> PLC/PRF/5. To underst<strong>and</strong> the molecular mechanism of TXNIP up-regulation in<br />
HCC, we analyzed the regulation of its expression by HGF/c-Met signaling which is known<br />
as important protector against to oxidative stress during development of HCC. Here, we have<br />
demonstrated for the first time, TXNIP expression is up-regulated by HGF treatment via<br />
activation of the both HGF/c-Met <strong>and</strong> Erk1/2 mediated pathways in SK-Hep-1 cell line. In<br />
addition, when activated form of c-Met was inhibited by SU11274, TXNIP expression was<br />
blocked, in parallel, with the decreased in activation of Erk1/2. Also, TXNIP up-regulation<br />
has been observed as in parallel with activation of HGF/c-Met signaling under stress<br />
conditions induced by high glucose <strong>and</strong> hypoxia. When we classified these HCC cells based<br />
on basal HGF/c-Met activation levels, it is notably important that invasive <strong>and</strong> metastatic<br />
HCC cell lines which have also c-Met tyrosine kinase over-expression, becomes TXNIP upregulation.<br />
As a conclusion, HGF/c-Met signaling regulates TXNIP expression at both transcript <strong>and</strong><br />
protein levels, in parallel, it protects cells against oxidative stress regulated by elevated<br />
TXNIP expression in the first steps of hepatocarcinogenesis. Following, these cells become<br />
more motile <strong>and</strong> aggressive in time. Thus, we may propose TXNIP behaves as an oncogene in<br />
development of HCC.<br />
Key words: TXNIP, HCC, HGF/c-Met<br />
146
Session 7: Cancer signaling networks Poster 10<br />
A new Ca 2+ /calmodulin antagonist HBC exhibits anti-angiogenic effect by suppressing<br />
signal transduction to hypoxia-inducible factor<br />
Hye Jin Jung, Jong Hyeon Kim, Joong Sup Shim, <strong>and</strong> Ho Jeong Kwon*<br />
Chemical Genomics National Research Laboratory, Department of Biotechnology,<br />
Translational Research Center for Protein Function Control, College of Life Science <strong>and</strong><br />
Biotechnology, Yonsei University, Seoul 120-749, Korea<br />
* Corresponding author: kwonhj@yonsei.ac.kr<br />
Targeting hypoxia-inducible factor-1 (HIF-1) signaling is a promising strategy to treat cancer<br />
growth <strong>and</strong> metastasis through an anti-angiogenic effect. Recent reports have shown that<br />
Ca 2+ /calmodulin (Ca 2+ /CaM) activates HIF-1 <strong>and</strong> consequently induces the expression of proangiogenic<br />
factors such as vascular endothelial growth factor (VEGF). Here, we demonstrated<br />
the anti-angiogenic effect of a new Ca 2+ /CaM antagonist HBC (4-{3,5-bis-[2-(4-hydroxy-3methoxyphenyl)ethyl]-4,5-dihydropyrazol-1-yl}benzoic<br />
acid). HBC potently inhibited the<br />
proliferation of human umbilical vascular endothelial cells with no cytotoxicity. Furthermore,<br />
HBC blocked in vitro angiogenesis such as tube formation <strong>and</strong> chemoinvasion as well as<br />
neovascularization of the chorioallantoic membrane of growing chick embryo in vivo. We<br />
also evaluated the effect of HBC on the activity of HIF-1. The compound markedly inhibited<br />
HIF-1! expression at the translational level during hypoxia, thereby reducing HIF-1<br />
transcriptional activity <strong>and</strong> expression of its major target gene VEGF. In addition,<br />
combination treatment with HBC <strong>and</strong> various HIF-1 inhibitors had greater anti-angiogenic<br />
activity than treatment with each single agent. Collectively, HBC is a promising antiangiogenic<br />
agent targeting HIF <strong>and</strong> might be applied in combination therapy to overcome the<br />
chemoresistance of solid tumors.<br />
147
Session 7: Cancer signaling networks Poster 11<br />
Increased expression of delta ex2TFF2 mRNA associates with prolonged survival of<br />
cholangiocarcinoma patients<br />
Surasee Kamlua 1,2,3 , Siriporn Patrakitkomjorn 2,3 , Patcharee Jearanaikoon 2,3 , Trevelyan<br />
R. Menheniott 4 , Andrew S. Giraud 4 , Temduang Limpaiboon 2,3 .<br />
1 Graduate School, Khon Kaen University, Khon Kaen 40002, Thail<strong>and</strong>; !<br />
2 Department of Clinical Chemistry, Centre for Research <strong>and</strong> Development of Medical<br />
Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University,<br />
Khon Kaen 40002, Thail<strong>and</strong>;<br />
3 Liver Fluke <strong>and</strong> Cholangiocarcinoma Research Center, Faculty of Medicine, Khon<br />
Kaen University, Khon Kaen 40002, Thail<strong>and</strong>;<br />
4 Murdoch Children’s Research Institute, Royal children’s Hospital, Parkville, VIC<br />
3052, Australia.<br />
E-mail: kamikaze_me@hotmail.com<br />
Trefoil factor 2 (TFF2) is a member of trefoil factor family that plays the role in epithelial<br />
restitution in the gastrointestinal tract. Recently, we demonstrated the overexpression of TFF2<br />
in liver fluke-related cholangiocarcinoma (CCA). Moreover, we found a novel alternative<br />
splice variant of TFF2 mRNA, delta ex2TFF2, in CCA cell lines. Delta ex2TFF2 mRNA<br />
sequence revealed an out-of-frame deletion of exon 2 of TFF2 mRNA resulting in a stop<br />
codon (TAG) at exon 1 resulting in the translation of a truncated protein lacking the paired<br />
trefoil domains of the canonical TFF2 protein. The clinical significance of delta ex2TFF2 is<br />
unclear. Here, we studied the expression of delta ex2TFF2 mRNA <strong>and</strong> its association with<br />
clinical parameters of CCA patients. Seventy eight tumors <strong>and</strong> 15 normal adjacent tissues<br />
were quantified for the expression of delta ex2TFF2 <strong>and</strong> wild type TFF2 (wtTFF2) mRNA<br />
against the internal reference gene GAPDH. The relative expression of delta ex2TFF2 mRNA<br />
<strong>and</strong> delta ex2TFF2/wtTFF2 ratio in tumor tissues were significantly higher than in normal<br />
(P
Session 7: Cancer signaling networks Poster 12<br />
Collagen I Regulates Cell Motility through Integrin Alpha 4 in Hepatocellular<br />
Carcinoma Cell Lines<br />
Emine K<strong>and</strong>emis 1 , Nese Atabey 1 , Esra Erdal 1<br />
1 Dokuz Eylul University, Faculty of Medicine, Department of Medical Biology <strong>and</strong><br />
Genetics, 35340-Inciralti, Izmir, Turkey. eminecelik@gmail.com<br />
Hepatocellular carcinoma (HCC) abundantly arises on the viral <strong>and</strong>/or chemicalinduced<br />
cirrhosis in liver. One of the most prominent feature of liver cirrhosis is the<br />
uncontrolled collagen I production. It is also known that increasing in collagen I affects cell<br />
behavior such as cell motility <strong>and</strong> invasion through activation of gene expression in key<br />
regulators on the preneoplastic stage of HCC such as TNF-alpha, integrins, MMP2 <strong>and</strong> MT1-<br />
MMP. However, there is no enough information about in which mechanisms collagen I<br />
affects the cell motility in HCC cells. In our study we investigated the effect of collagen I on<br />
HCC cell lines (PLC/PRF/5, Hep3B, SNU-423, Sk-Hep-1 <strong>and</strong> SNU-449) by using wound<br />
assay <strong>and</strong>/or boyden-chamber motility methods. Cellular motility of PLC/PRF/5, Hep3B,<br />
SNU-423, SNU-449 <strong>and</strong> Sk-Hep-1 cells grown on the collagen I significantly decreased in<br />
different levels. Further we analyzed expression of integrins (alpha 1, alpha 2, alpha 3, alpha<br />
4, alpha 5, alpha 6, alpha v, beta 1 <strong>and</strong> beta 4) on the effect of collagen I by using reverse<br />
transcriptase PCR in SNU-449 cells. Unexpectedly, integrin alpha 1, alpha 2 <strong>and</strong> alpha 3<br />
which are collagen I receptors did not significantly change in the presence of collagen I,<br />
contrarily, the expression of integrin alpha 4 decreased in both mRNA <strong>and</strong> protein levels in<br />
SNU-449. Interestingly, we observed that HCC cell lines, SNU-423, SNU-449 <strong>and</strong> Sk-Hep-1,<br />
having high ability in cell motility expressed integrin alpha 4 in a much more level than HCC<br />
cell lines, Hep3B <strong>and</strong> PLC/PRF/5, which have low basal motility. These data suggest that<br />
reducing effect of collagen I on HCC cell motility might be regulated by integrin alpha 4<br />
expression. It may be an unique mechanism to explain the role of collagen I induced<br />
mechanisms during hepatocarcinogenesis.<br />
149
Session 7: Cancer signaling networks Poster 13<br />
Butein sensitizes human hepatoma cells to TRAIL-induced apoptosis via ERK/Sp1dependent<br />
DR5 up-regulation <strong>and</strong> NF-!B inactivation<br />
Chang-Hee Kang, 1 Jang-Yeon Jeong, 1 R.G.P.T. Jayasooriya, 1 Sang-Hyuck Kang, 1 Yung<br />
Hyun Choi, 2 <strong>and</strong> Gi-Young Kim 1,*<br />
1 Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National<br />
University, Jeju 690-756, Republic of Korea<br />
2 Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan<br />
614-051, Republic of Korea<br />
Tumor necrosis factor (TNF)-related apoptosis-inducing lig<strong>and</strong> (TRAIL) induces cell death in<br />
various types of cancer cells but has little or no effect on normal cells. Human hepatoma cells<br />
are resistant to TRAIL-induced apoptosis. Although butein is known to mediate anti-cancer,<br />
anti-inflammatory, <strong>and</strong> anti-oxidant activities, little is known about the mechanism of butein<br />
in terms of TRAIL-induced apoptosis of human hepatoma cells. In this study, we determined<br />
that butein enhances TRAIL-induced apoptosis in hepatoma cells through up-regulation of<br />
DR5. Luciferase analysis showed that a 5’-flanking region containing four Sp1 binding sites<br />
within the DR5 promoter was enhanced by butein (-305/-300). Electrophoretic mobility shift<br />
assays <strong>and</strong> chromatin immunoprecipitation studies were used to analyze the elevation of Sp1<br />
binding to DR5 promoter sites by butein. Point mutations of the Sp1 binding site also<br />
attenuated promoter activity. Furthermore, pre-treatment of the blocking chimeric antibody<br />
<strong>and</strong> small interfering RNS for DR5 significantly suppressed TRAIL-mediated apoptosis by<br />
butein in Hep3B cells. Butein also stimulated ERK activation, <strong>and</strong> the ERK inhibitor<br />
PD98059 blocked butein-induced DR5 expression <strong>and</strong> suppressed binding of Sp1 to the DR5<br />
promoter. Additionally, generation of reactive oxygen species (ROS) had no effect on cell<br />
viability, although pre-treatment with N-acetyl-L-cystein or glutathione inhibited combined<br />
treatment-induced ROS. Indeed, butein repressed the TRAIL-mediated activation of NF-!B<br />
<strong>and</strong> decreased its transcriptional activity. Our results suggest that butein could sensitize<br />
certain human hepatoma cells to TRAIL-induced apoptosis through stimulating its death<br />
signaling <strong>and</strong> by repressing the survival function in these cells.<br />
150
Session 7: Cancer signaling networks Poster 14<br />
Sulforaphane sensitizes tumor necrosis factor-related apoptosis inducing lig<strong>and</strong><br />
(TRAIL)-mediated apoptosis through downregulation of ERK <strong>and</strong> Akt in lung<br />
adenocarcinoma A549 cells<br />
Chang-Hee Kang, 1 Yeon-Jeong Jang, 1 R.G.P.T. Jayasooriya, 1 Sang-Hyuck Kang, 1<br />
Cheung-Yun Jin, 2 Yung Hyun Choi, 2 <strong>and</strong> Gi-Young Kim 1,*<br />
1 Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National<br />
University, Jeju 690-756, Republic of Korea<br />
2 Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan<br />
614-051, Republic of Korea<br />
The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing lig<strong>and</strong> (TRAIL)<br />
is limited in some cancer cells, including A549 lung adenocarcinoma cells. However,<br />
treatment with TRAIL in combination with subtoxic concentrations of sulforaphane (SFN)<br />
sensitizes TRAIL-resistant A549 cells to TRAIL-mediated apoptosis. Combined treatment<br />
with SFN <strong>and</strong> TRAIL induced chromatin condensation, DNA fragmentation, annexin V<br />
staining <strong>and</strong> sub-G1 phase DNA content. These indicators of apoptosis correlate with the<br />
induction of caspase-3 activity, which results in the cleavage of poly(ADP-ribose)polymerase<br />
(PARP) <strong>and</strong> the release of lactate dehydrogenase (LDH). Both the cytotoxic effect <strong>and</strong><br />
apoptotic characteristics induced by combined treatment were significantly inhibited by z-<br />
DEVD-fmk, a caspase-3 inhibitor, demonstrating the important role of caspase-3 in the<br />
observed cytotoxic effect. Combined treatment also triggered the activation of p38 MAPK<br />
<strong>and</strong> JNK, <strong>and</strong> downregulation of ERK <strong>and</strong> Akt. Inhibitors of ERK (PD98059) or Akt<br />
(LY294002), but not p38 MAPK, resulted in significantly decreased cell viability. Although<br />
the activation of JNK was increased in response to combined treatment, inhibition of the JNK<br />
pathway significantly attenuated cell viability. These results indicate that caspase-3 is a key<br />
regulator of apoptosis in response to combined SFN <strong>and</strong> TRAIL in human lung<br />
adenocarcinoma A549 cells through downregulation of ERK <strong>and</strong> Akt.<br />
151
Session 7: Cancer signaling networks Poster 15<br />
Heat shock proteins in oncology: Diagnostic biomarkers <strong>and</strong> therapeutic targets<br />
Ashraf A. Khalil 1 , Nihal F. Kabapy 1 , Sahar F. Deraz 1 , Christopher Smith 2<br />
1 Department of Protein Technology, Institute of Genetic Engineering <strong>and</strong><br />
Biotechnology, Mubarak City for Scientific Research, New Borg Elarab, Alex<strong>and</strong>ria,<br />
Egypt.<br />
2 Department of Biological Sciences, University of Chester, Parkgate Road Chester, UK<br />
Heat shock proteins (HSP) are a family of proteins induced in cells exposed to different<br />
insults allowing cells to survive stress conditions. Mammalian HSPs have been classified into<br />
six families according to their molecular size: HSP100, HSP90, HSP70, HSP60, HSP40 <strong>and</strong><br />
small HSPs (15 to 30 kDa) including HSP27. These proteins act as molecular chaperones<br />
either helping in the refolding of misfolded proteins or assisting in their elimination if they<br />
become irreversibly damaged. In recent years, proteomic studies have characterized several<br />
different HSPs in various tumour types which may be putative clinical biomarkers or<br />
molecular targets for cancer therapy. This has led to the development of a series of molecules<br />
capable of inhibiting HSPs. Furthermore, protein profiling of several models involving<br />
administered HSP90 inhibitors have revealed new biomarkers that can help in the evaluation<br />
of the degree of HSP90 inhibition as well as predicting the therapeutic response. Numerous<br />
studies speculated that over expression of HSP is in part responsible for resistance to many<br />
anti-tumours <strong>and</strong> chemotherapeutics. Hence, from a pharmacological point of view, the coadministration<br />
of HSP inhibitors along with other anti-tumour agents is of major importance<br />
to overcome therapeutic resistance. We provide an overview of the current status of the HSPs<br />
in diseases with special emphasis on cancer.<br />
152
Session 7: Cancer signaling networks Poster 16<br />
Endometriosis-related Ovarian Cancer Risk <strong>and</strong> Prognosis: A Meta-analysis<br />
Yong Beom Kim 1 , Hee Seung Kim 2 , Chae Hyeong Lee 3 , Yong-Tark Jeon 1 , Yong Sang<br />
Song 2<br />
1 Department of Obstetrics <strong>and</strong> Gynecology, Seoul National University Bundang<br />
Hospital, Seoungnam 463-707, Republic of Korea (Kim YB, ybkimlh@snubh.org; Jeon<br />
YT, asidof@snubh.org)<br />
2 Department of Obstetrics <strong>and</strong> Gynecology, Seoul National University College of<br />
Medicine, Seoul 110-744, Republic of Korea (Kim HS, bboddi0311@snu.ac.kr; Song YS,<br />
yssong@snu.ac.kr)<br />
3 Department of Obstetrics <strong>and</strong> Gynecology, Dongguk University Medical Center,<br />
Goyang 410-773, Republic of Korea (Lee CH, gynelee@dumc.or.kr)<br />
Background: The association between ovarian cancer <strong>and</strong> endometriosis has not been well<br />
established in previous studies. We investigated the impact of endometriosis on the<br />
development of ovarian cancer (OC) <strong>and</strong> prognosis in patients with OC using a meta-analysis.<br />
Methods: After we performed a MEDLINE search to identify all relevant studies between<br />
March 1945 <strong>and</strong> December 2010, we found 4 studies (3 case-control, 1 cohort; n=157,292)<br />
related with OC risk <strong>and</strong> 4 studies (1 case-control, 3 cohort; n=46,716) associated with<br />
prognosis in patients with OC among 2,390 potentially relevant studies. This meta-analysis<br />
was performed using the fixed-effect model because of no heterogeneity.<br />
Results: Endometriosis increased the risk of OC (OR, 1.42; 95% CI, 1.22-1.65). Although<br />
endometriosis did not influence the risk of OC in one cohort study (OR, 0.78; 95% CI, 0.25-<br />
2.44), 3 case-control studies showed that endometriosis was associated with the increased of<br />
OC (OR, 1.43; 95% CI, 1.23-1.67). On the other h<strong>and</strong>, endometriosis-associated OC (EAOC)<br />
showed improved overall survival (OS) when compared with non-EAOC (HR, 0.77; 95% CI,<br />
0.64-0.92). In sub-analyses based on the design of study, one case-control study demonstrated<br />
that EAOC was associated with improved OS (HR, 0.56; 95% CI, 0.36-0.87), <strong>and</strong> EAOC was<br />
also related with improved OS with marginal significance when compared with non-EAOC<br />
(HR, 0.82; 95% CI, 0.67-1.00) in 3 cohort studies.<br />
Conclusions: These findings suggest that women with endometriosis may have a higher risk<br />
of OC than those without endometriosis, whereas coexistent endometriosis may be associated<br />
with improved OS in patients with OC.<br />
153
Session 7: Cancer signaling networks Poster 17<br />
Impact of Body Mass Index on Overall Survival in Endometrial or Ovarian Cancer<br />
Survivors: A Meta-analysis<br />
Hee Seung Kim 1 , Chae Hyeong Lee 2 , Yong-Tark Jeon 3 , Yong Beom Kim 3 , Yong Sang<br />
Song 1<br />
1 Department of Obstetrics <strong>and</strong> Gynecology, Seoul National University College of<br />
Medicine, Seoul 110-744, Republic of Korea (Kim HS, bboddi0311@snu.ac.kr; Song YS,<br />
yssong@snu.ac.kr)<br />
2 Department of Obstetrics <strong>and</strong> Gynecology, Dongguk University Medical Center,<br />
Goyang 410-773, Republic of Korea (Lee CH, gynelee@dumc.or.kr)<br />
3 Department of Obstetrics <strong>and</strong> Gynecology, Seoul National University Bundang<br />
Hospital, Seoungnam 463-707, Republic of Korea (Kim YB, ybkimlh@snubh.org; Jeon<br />
YT, asidof@snubh.org)<br />
Background: The role of body mass index (BMI) has not been well-established for overall<br />
survival (OS) in patients with endometrial or ovarian cancer. To evaluated the impact of BMI<br />
in the patients, we performed a meta-analysis for comparing OS between (1) BMI
Session 7: Cancer signaling networks Poster 18<br />
Metformin inhibits gynaecological cancer cell growth through reduction of Dvl3 in<br />
Wnt/b-catenin signalling<br />
Virginia HT Kwan, David W Chan, Vincent WS Liu <strong>and</strong> Hextan YS Ngan<br />
Department of Obs. & Gyn., The University of Hong Kong, h0660139@hkusua.hku.hk<br />
Aberrant activation of Wnt/!-catenin signaling pathway is frequently associated with many<br />
human cancers. Dishevelled 3 (Dvl 3) is a key mediator of this pathway <strong>and</strong> functions as<br />
oncogenic factors in promoting cell proliferation.<br />
In this study, we found that the protein but not the mRNA level of Dvl3 is up-regulated in<br />
cervical cancer cell lines. Interestingly, treatment of Metformin can remarkably reduce the<br />
aberrant upregulation of Dvl3 in gynaecological cancer cell lines. Based on such discrepancy<br />
between basal mRNA <strong>and</strong> protein level, we hypothesized that the reduction of Dvl3 by<br />
metformin is due to the inhibition of protein synthesis through changes in AMPK/mTOR<br />
signaling pathway <strong>and</strong>/or promotion of proteasomal degradation machinery in these<br />
gynaecological cancer cells. On the other h<strong>and</strong>, treatment of proteosomal inhibitor (AM114)<br />
in cervical cancer <strong>and</strong> endometrial cancer cell lines can alleviate the metformin-mediated<br />
down-regulation of Dvl3, which strongly shows the involvement of proteasome degradation<br />
in Dvl3 down-regulation. Taken together, these data indicate that the upregulation of Dvl3 in<br />
gynaecological cancer cells is due to abnormal translational control <strong>and</strong> protein turnover.<br />
Meanwhile, metformin is also able to inhibit gynaecological cancer cell growth. In Colony<br />
formation <strong>and</strong> XTT assay, there was significant reduction of cell viability under the treatment<br />
of metformin in a dose-dependent manner. However, further biochemical studies on the<br />
responses of these pathways to Metformin in gynaecological cancer cells are warranted.<br />
155
Session 7: Cancer signaling networks Poster 19<br />
The effect of hCaMKIINalpha on TLR4-triggered cytokine production of colon cancer<br />
cells<br />
Nan Li, Chunmei Wang, Xingguang Liu, Qinghua, Zhang, Xuetao Cao<br />
National Key Laboratory of Medical Immunology <strong>and</strong> Institute of Immunology, Second<br />
Military Medical University, Shanghai 200433, P.R.China. Email: linan@immunol.org<br />
Increasing evidences suggest that chronic inflammation contributes to cancer development<br />
<strong>and</strong> progression. One of the underlying mechanisms is proposed that tumor cell-derived<br />
inflammatory <strong>and</strong> immunosuppressive cytokines contribute to tumor immune escape <strong>and</strong><br />
resistance to immunotherapy. Toll-like receptors (TLRs) have been implicated in tumor<br />
progression <strong>and</strong> metastasis. Our previous study showed that calcium/calmodulin-dependent<br />
protein kinase II (CaMKII)� promoted TLR-triggered proinflammatory cytokine in<br />
macrophages. hCaMKIINalpha, a novel CaMKII inhibitory protein identified by us,<br />
suppressed the growth of colon cancer cell by inducing cell cycle arrest in vitro <strong>and</strong> in vivo.<br />
Thus we wonder whether hCaMKIINalpha-mediated CaMKII inhibition affects TLR4triggered<br />
cytokine production of colon cancer cells for immune escape. In this study, we<br />
demonstrate that TLR4 is expressed on human colon cancer cell lines. TLR4 ligation<br />
promotes production of immunosuppressive cytokines IL-8 <strong>and</strong> VEGF. Overexpression of<br />
hCaMKIINalpha inhibits TLR4-triggered production of IL-8 <strong>and</strong> VEGF; H282R, constitutive<br />
activated CaMKII, significantly promotes TLR4-triggered IL-8 <strong>and</strong> VEGF secretion. In<br />
addition, we also observe that hCaMKIINalpha inhibits LPS-mediated activation of p-<br />
ERK1/2 <strong>and</strong> LPS-mediated TLR4 expression in SW620 cells. Furthermore, hCaMKIINalphamediated<br />
inhibition of ERK1/2 is necessary for suppression of TLR4-triggered IL-8 <strong>and</strong><br />
VEGF secretion. These results suggest that hCaMKIINalpha-mediated CaMKII inhibition<br />
might play important roles in the suppression TLR4-triggered metastasis <strong>and</strong> immune escape<br />
of human colon cancer cells by inhibiting immunosuppressive cytokine production.<br />
156
Session 7: Cancer signaling networks Poster 20<br />
Mechanism of action <strong>and</strong> anti-cancer activity of novel derivative of glycyrrhetic acid<br />
Logashenko E.B. 1 , Markov A.V. 1 , Salomatina O.V. 2 , Salakhutdinov N.F. 2 , Popova N.A. 3 ,<br />
Kaledin V.I. 3 , Nikolin V.P. 3 , Zenkova M.A. 1 .<br />
1 Institute of Chemical Biology <strong>and</strong> Fundamental Medicine SB RAS,<br />
Novosibirsk, Russian Federation, evg_log@niboch.nsc.ru<br />
2 Novosibirsk Institute of Organic Chemistry SB RAS, Novosibirsk, Russian Federation,<br />
3 Institute of Cytology <strong>and</strong> Genetics SB RAS, Novosibirsk, Russian Federation<br />
Glycyrrhetinic acid, aglycon of glycyrrhizin is abundant component of licorice root.<br />
Glycyrrhizin content in the triterpene extract of licorice root amounts to 90%. Recent reviews<br />
described the wide spectrum of glycyrrhetinic acid bioactivities, such as anti-inflammatory,<br />
antiviral, hepatoprotective, antitumor <strong>and</strong> immunomodulatory activity. Several studies<br />
reported that glycyrrhizin <strong>and</strong> glycyrrhetinic acid cause moderate cytotoxic <strong>and</strong> apoptotic<br />
effects on cancer cells, most of the results confirmed only moderate or low potency of the<br />
compounds. In attempt to prepare more potent analog we synthesized by directed<br />
modification of A, C <strong>and</strong> E rings of glycyrrhetic acid the methyl 2–cyano–3,12–dioxo–11–<br />
deoxo–18!H–glycyrrhet–1(2),9(11)–dienoate derivative (SG).<br />
The antitumor activity of the novel derivative was determined in vitro for different cancer cell<br />
lines by effects on cell growth by MTT assay. It was shown that SG is cytotoxic for cancer<br />
cells. IC50 varied from 5"10 -5 M (MCF-7 cell line) to 0,6x10 -6 M (#$-3-1cell line). Detailed<br />
study of mechanism of cells death showed that SG induces caspase–dependent apoptosis with<br />
disruption of mitochondrial potential. Exposure of the cells to glutathione that is an<br />
antioxidant completely prevents activity of SG.<br />
The antitumor activity in vivo was evaluated using Ehrlich's ascite tumor developed in<br />
%%57BR mice. Mice were twice intraperitoneally injected with SG (20 mg/kg). It was shown<br />
that SG administration leads to two fold decreasing of number of cancer cells in ascite as<br />
compared to control. Intraperitoneal injections of SG in mice with lymphosarcoma increase<br />
their live duration to 24 %.<br />
To increase solubility of SG we used Tween-80 <strong>and</strong> Cremophore–EL. Intraperitoneal<br />
injections of SG emulsions (50 mg/kg) with 10% Tween-80 <strong>and</strong> Cremophore–EL in mice<br />
with ascite tumor Krebs-2 supress tumor growth to 76 <strong>and</strong> 57%, respectively.<br />
Obtained data indicated that novel derivative of glycyrrhetic acid SG exhibits pronounced<br />
anti-cancer activity in vitro <strong>and</strong> in vivo.<br />
This work was supported by RAS programs “Molecular <strong>and</strong> cellular biology” <strong>and</strong><br />
“Fundamental sciences to medicine”, <strong>and</strong> by SB RAS (Interdisciplinary grant & 104).<br />
157
Session 7: Cancer signaling networks Poster 21<br />
Dendritic cells-based antitumor vaccines: opposite effects depending on application<br />
scheme<br />
Mironova N.L. 1 , Markov O.A. 1 , Popova N.A. 2 , Kaledin V.I. 2 , Nikolin V.P. 2 , Vlassov<br />
V.V. 1 , Zenkova M.A. 1<br />
1 Institute of Chemical Biology <strong>and</strong> Fundamental Medicine SB RAS, Lavrentieva ave. 8,<br />
Novosibirsk, 630090, mironova@niboch.nsc.ru<br />
2 Institute of Cytology <strong>and</strong> Genetics SB RAS, Lavrentieva ave. 11, Novosibirsk, 630090<br />
Dendritic cells (DC) are specialized antigen presenting cells that acquire, process, <strong>and</strong> present<br />
tumor-associated antigens to T cells for the induction of antigen-specific tumor immune<br />
responses. Triggering of effective antitumor immune response by ex vivo generated mature<br />
DC pulsed with tumor-associated antigens is the perspective approach to enhance the<br />
efficiency of anticancer therapy.<br />
Here the tumoricidal activity of DC depending on single or multiple antigen sources was<br />
studied in vitro <strong>and</strong> in vivo. We show that the efficacy of cytotoxic T lymphocytes (CTL)<br />
activated by DC pulsed with source of multiple antigens (further DC-MA) is 1.5-times higher<br />
than the efficacy of CTL generated by DC pulsed with source of a single antigen (further, DC<br />
SA).<br />
Anticancer potential of DC pulsed with tumor antigens was evaluated in mice model with<br />
Krebs-2 tumor. Administration of DC-MA as a vaccine prior to tumor transplantation resulted<br />
in the drastic suppression of CD8+ <strong>and</strong> CD4+ T cells proliferation <strong>and</strong> total absence of any<br />
antitumor effects. Administration of the same DC-MA after tumor transplantation resulted in<br />
2-fold retardation of primary tumor growth <strong>and</strong> was accompanied by enhanced proliferation<br />
of CD8+ T cells as well as significant increase of IFN-gamma production by factor 2.8. Thus,<br />
we revealed that DC can cause the opposite therapeutic effects depending on the time of DC<br />
administration. Attempts of DC application as a pre-exposure vaccine resulted in depletion of<br />
T-cell pool followed by break-off the cytotoxic immune response during tumor progression<br />
whereas therapy with DC upon tumor development had considerable antitumor effect.<br />
This work was supported by RAS programs “Molecular <strong>and</strong> cellular biology” <strong>and</strong><br />
“Fundamental sciences to medicine”, by RFBR grant 08-04-00753 <strong>and</strong> by Interdisciplinary<br />
grant of SB RAS (No. 15).<br />
158
Session 7: Cancer signaling networks Poster 22<br />
Glucose transport in Saccharomyces cerevisiae reveals heterogeneity of cell population<br />
Maria Nardelli 1 , Malkhey Verma 1 , Kathryn Blount 2 , Hans V. Westerhoff 1,2<br />
1<br />
Manchester Centre for Integrative Systems Biology, EPS, University of Manchester,<br />
131 Princess Street, M1 7DN, Manchester (UK)<br />
2<br />
DTC for Integrative Systems Biology, EPS, University of Manchester, 131 Princess<br />
Street, M1 7DN, Manchester (UK)<br />
Maria.Nardelli@manchester.ac.uk, Malkhey.Verma@manchester.ac.uk,<br />
Kathryn.Blount@postgrad.manchester.ac.uk, Hans.Westerhoff@manchester.ac.uk<br />
Saccharomyces cerevisiae is able to adjust to a wide range of glucose availability. Yeasts can<br />
modulate the expression of 17 different transporters to adapt <strong>and</strong> optimize the uptake of<br />
glucose. In our study we tested the ability of CEN-PK strain to express specific subsets of<br />
glucose transporters in response to the availability of glucose in their medium. To follow the<br />
uptake of glucose a fluorescent analogue, the 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)amino)-2-deoxyglucose<br />
(2NBDG), has been used. Yeast adapted to different amount of<br />
glucose <strong>and</strong> exposed to different concentrations of 2NBDG were analysed.<br />
The uptake of the molecule was followed measuring the fluorescence associated with the<br />
cells. The analysis of yeast cells via flow cytometry revealed the presence of heterogeneity in<br />
the yeast population depending on the growth condition <strong>and</strong> concentration of 2NBDG used.<br />
The presence of different yeast subpopulations indicates that the glucose transporter<br />
expression <strong>and</strong>/or activity require fine tuning. Underst<strong>and</strong>ing this aspect of the glucose uptake<br />
in yeast may provide further information to target the glucose transporters for cancer<br />
therapeutics.<br />
159
Session 7: Cancer signaling networks Poster 23<br />
Cinnamic acid effects on cultured human melanocytes <strong>and</strong> melanoma derived cells:<br />
evaluation of the specificity of its antitumor potential<br />
Ev<strong>and</strong>ro L O Niero, Camila Lau<strong>and</strong>, Gláucia M Machado-Santelli<br />
Laboratory of Cell <strong>and</strong> Molecular Biology, Department of Cell <strong>and</strong> Developmental<br />
Biology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu<br />
Prestes 1524, 05508-900, São Paulo, SP, Brazil, eloniero@usp.br<br />
Phenolic acids, commonly found in plants, <strong>and</strong> their derivatives have been largely studied<br />
because of their antitumor activities <strong>and</strong> low cytotoxicity in normal cells. Thus, the present<br />
work aimed to evaluate the specificity of the cinnamic acid (CA) (a phenolic acid)<br />
cytotoxicity in human melanocytes of blue nevus (NGM) <strong>and</strong> human melanoma derived cells<br />
(HT-144) just following cell cycle arrest, cell death <strong>and</strong> formation of nuclear aberration. CA<br />
was very efficient in inhibiting cell growth of melanoma cells compared to melanocytes<br />
according to MTT assay. The inhibition was probably associated with DNA damage leading<br />
to DNA synthesis inhibition as showed by BrdU incorporation assay, induction of nuclear<br />
aberrations (micronucleation, binucleation <strong>and</strong> multinucleation) leading to apoptosis in both<br />
cell lines. BrdU incorporation assay indicates cell cycle arrest in both cell lines induced by<br />
0.4mM of CA treatment. We also identified induction of cell death after 24 hours of<br />
treatment, especially in HT-144 cells, according to activated caspase-9 assay. CA at 3.2 mM<br />
also induced formation of nuclear buds <strong>and</strong> micronuclei in NGM cells but the effects were<br />
milder than in melanoma cells. This data could be associated with higher genetic instability of<br />
HT-144 cells. We identified, for example, many copies of chromosome 9 as showed by the<br />
presence of several positive signals to centromeric regions in melanoma cells. Nevertheless,<br />
we observed two or less signals to p16 in HT-144 cell line while NGM cells showed signals<br />
to two copies of both regions. High concentration of CA (3.2 mM) induced formation of<br />
nuclear buds <strong>and</strong> micronuclei in NGM cells. Together, our results indicate a high cytotoxic<br />
potential of CA to HT-144 cells compared to NGM. The genotoxic effects showed by both<br />
cells after exposure to 3.2 mM of CA indicates that the mechanisms of action of CA must be<br />
further investigated, but it could be related to its antioxidant activity <strong>and</strong> capacity to induce<br />
DNA damage.<br />
160
Session 7: Cancer signaling networks Poster 24<br />
Regulation of HGF-induced cell invasion through EGR1 in Hepatocellular Carcinoma<br />
Ozen E., Gozukizil A., Erdal E., Atabey N.<br />
Dokuz Eylul University School of Medicine Depertmant of Medical Biology <strong>and</strong><br />
Genetics, Inciralti Izmir , Turkey evinozen@hotmail.com<br />
Hepatocyte growth factor (HGF) is a mesenchyme-derived pleotrophic growth factor<br />
that elicits mitogenic, morphogenic <strong>and</strong> motogenic responses in wide variety of cells by<br />
binding to its receptor, c-Met receptor tyrosine kinase. HGF/cMet pathway induces activation<br />
of several downstream signaling cascades, such as mitogen-activated protein kinase (MAPK)<br />
<strong>and</strong> phosphatidyl-inositol 3-kinase (PI3K) pathways then regulates tissue regeneration as well<br />
as invasion <strong>and</strong> metastasis in hepatocellular carcinoma (HCC). Furthermore, it is known that<br />
matrix metalloproteinases (MMP) activation is important step in HGF-induced, invasion <strong>and</strong><br />
metastasis in different models. However, the molecular mechanism behind the regulation of<br />
MMP activation on the HGF-induced invasion has not been well understood yet in HCC.<br />
Recently, it has been reported that Early Growth Response Factor 1 (EGR1) plays a key role<br />
in HGF-induced angiogenesis <strong>and</strong> cell scattering in HCC. So we hypothesized that EGR1<br />
might be a downstream transcription factor that regulates HGF-induced invasion through<br />
activation of MMPs during hepatocarcinogenesis.<br />
In this study it has been first shown that activation of HGF/c-Met signaling by treatment of<br />
HGF induces significantly transcriptional activity of EGR1 in a time dependent manner <strong>and</strong><br />
inhibition of HGF/c-Met signaling by treatment of SU11274 decreases expression level of<br />
EGR1 in SKHep1, HCC cell line. Also, it has been demonstrated that activation of HGF/c-<br />
Met signaling activates MAPK signaling molecules <strong>and</strong> cell invasion in this cells as expected.<br />
Expression of EGR1 has been further stably knock-down by using inducible pSUPERIOR<br />
system in SKHep1. Silencing of EGR1 caused significant decrease in HGF-mediated<br />
invasion. It also resulted in decrease in HGF-mediated activation of both MMP-2 <strong>and</strong> MMP-<br />
9, in parallel, decrease in expression of MT1-MMP in SKHep1 cell.<br />
As a conclusion, we found that EGR1 activates HGF-induced cell invasion possibly through<br />
the regulation of MMPs in HCC. In future, this mechanism may provide a way to develop<br />
new drug targets in the treatment of HCC.<br />
In this study, we found that HGF induces invasion of SK-Hep-1 cell lines by activation of<br />
MMP’s through EGR1.<br />
Key words: HGF, invasion, EGR1, MMPs, HCC.<br />
161
Session 7: Cancer signaling networks Poster 25<br />
Tumor infiltrating lymphocytes as a prognostic factor after the radical surgery of<br />
colorectal carcinoma<br />
Authors: Pitule P.(1,2), Vycital O.(1), Cedikova M.(2), Bruha J.(1), Liska V(1), Treska<br />
V.(1), Daum O.(3)<br />
(1) Department of Surgery, (2) Department of Histology <strong>and</strong> Embryology, (3)<br />
Department of Pathological Anathomy, Faculty of Medicine in Pilsen, Charles<br />
University<br />
email adress of the first author: pitulep@seznam.cz<br />
Introduction: Tumor infiltrating lymphocytes (TIL) are important factor which corresponds<br />
with the ability of the organism to attenuate the development of the malignity.<br />
Aim of the study: We correlated the broadly used clinical <strong>and</strong> histopathological factors with<br />
the presence of TIL in the tumor. The goal of the study was to identify patients with the high<br />
risk of the poor overall survival (OS) <strong>and</strong> early reccurence (short disease-free interval, DFI)<br />
of the malignity after the radical surgical treatment.<br />
Methods: The set of 150 tumor samples was analysed for several factors – pre-operational<br />
leukocytosis, radicality of the surgery, post-operational complications, oncological treatment,<br />
tumor grading <strong>and</strong> staging, morphological features of the tumor, characterisation <strong>and</strong><br />
quantification of the TIL <strong>and</strong> the reactive changes in the lymp nodes (LN). All of these factors<br />
were statistically correlated with the OS <strong>and</strong> DFI of the patients. We have also evaluated<br />
immunohistochemical positivity of TIL (CD4 + ,CD8 + ).<br />
Results: We have included in the study 93 men <strong>and</strong> 57 women, with the 1, 3 <strong>and</strong> 5 years OS<br />
92,2%, 76,5% <strong>and</strong> 70,2% respectivelly, <strong>and</strong> 1, 3 <strong>and</strong> 5 years DFI 85,3%, 64,3% <strong>and</strong> 49,4%<br />
respectivelly. Endolyphatic infiltration <strong>and</strong> metastatic inflitration of the LN were confirmed as<br />
a negative prognostic factor for OS. N2 stage descrease the OS 9,3 times <strong>and</strong> DFI 5 times.<br />
Crohn-like peritumoral lymphocytic infiltration <strong>and</strong> follicular hyperplasia were confirmed as<br />
a protective factor. CD8 + <strong>and</strong> CD4 + TIL were confirmed as a positive prognostic factor <strong>and</strong><br />
their presence leads to the increase in OS <strong>and</strong> DFI. The CD8 + /CD4 + ratio was not significantly<br />
confirmed as a prognostic factor. Perineural infiltration was confirmed as a prognostic factor<br />
of early reccurence. Higher infiltration by CD8 + TIL was found to be positive prognostic<br />
factor which increase the DFI.<br />
The study was supported by grants VZ MSM 0021620819 <strong>and</strong> IGA MZ CR 10230.<br />
162
Session 7: Cancer signaling networks Poster 26<br />
Selective cytotoxic activity of a bifunctional chimeric protein containing a ribosome<br />
inactivating protein (PD-L4) <strong>and</strong> a serine protease inhibitor (WSCI).<br />
Rachele Tamburino 1 , Elio Pizzo 2 , Elia Poerio 3 <strong>and</strong> Antimo Di Maro 1<br />
1<br />
Dipartimento di Scienze della Vita, Seconda Università di Napoli, via Vivaldi, 43, I-<br />
81100 Caserta, Italy<br />
rachele.tamburino@unina2.it; antimo.dimaro@unina2.it<br />
2<br />
Dipartimento di Biologia Strutturale e Funzionale, Università “Federico II”, via<br />
Cinthia, I-80126 Napoli, Italy elipizzo@unina.it<br />
3<br />
Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia, via S. Camillo de<br />
Lellis, I-01100 Viterbo, Italy poerio@unitus.it<br />
A bifunctional chimeric protein, potentially able to biotechnological applications, has been<br />
designed <strong>and</strong> expressed in E. coli cells. The first domain corresponds to the toxic/antiviral<br />
protein PD-L4 type 1 RIP, firstly isolated from P. dioica L. leaves. The second domain<br />
corresponds to the wheat protein inhibitor WSCI which is able to interfere with subtilisin,<br />
pancreatic chymotrypsin <strong>and</strong> chymotrypsin-like activities isolated from the midgut of a<br />
number phytophagous insect larvae. The chimeric construct, pd-l4-cDNA-oligonucleotide<br />
linker-wsci-cDNA, was cloned in the expression vector pET22b <strong>and</strong> employed in trasforming<br />
E. coli cells (strain BL21-DE3). The chimeric protein PD-L4/WSCI was expressed in E. coli<br />
<strong>and</strong> recovered from the inclusion bodies. Both t<strong>and</strong>em domains (PD-L4 <strong>and</strong> WSCI) retained<br />
their original activities. Cytotoxicity assays on SVT2 cells revealed that the chimeric protein<br />
strongly affected their viability; the recorded levels of toxicity were greater than those<br />
observed for PD-L4. Somehow, the presence of the non-toxic C-terminal domain WSCI<br />
contributed to enhance cytotoxicity of the bifunctional chimeric product. Surprisingly,<br />
cytotoxicity assays revealed that the chimera did not affect 3T3 cells viability.<br />
163
Session 7: Cancer signaling networks Poster 27<br />
New benzophenone polyphenols with potential chemoprotective effects<br />
Tzvetomira Tzanova a,c , Mariana Gerova b , Ognyan Petrov b , Margarita Karaivanova a ,<br />
Denyse Bagrel c<br />
a Medical University of Sofia, Faculty of Pharmacy, Department of Pharmacology <strong>and</strong><br />
Toxicology, 2 Dunav street, Sofia 1000, Bulgaria, tztzanova@gmail.com,<br />
mkaraivanova2001@yahoo.com<br />
b University of Sofia St. Kliment Ohridski, Faculty of Chemistry, Department of Applied<br />
Organic Chemistry, 1 James Bourchier Avenue, 1164 Sofia, Bulgaria,<br />
OPetrov@chem.uni-sofia.bg, ohtmg@chem.uni-sofia.bg<br />
c Laboratoire d'Ingénierie Moléculaire et Biochimie Pharmacologique, Université Paul<br />
Verlaine – Metz, FR CNRS 2843, UFR SciFA, Campus Bridoux, rue du Général<br />
Delestraint, 57070 Metz, France<br />
bagrel@univ-metz.fr<br />
Among phenolics, natural hydroxybenzophenones are biologically active metabolites present<br />
in plants <strong>and</strong> especially in Guttiferae family. Therefore we tested the antioxidant properties of<br />
novel natural benzophenones isolated from Hypericum annulatum. One of them –<br />
hypericophenonoside (Hd19) displayed a high in vitro antioxidant <strong>and</strong> antiradical activity.<br />
The cytoprotective effects of Hd19 <strong>and</strong> silymarine, our natural reference compound, were<br />
tested on breast cell lines – ie the cancerous MCF7 <strong>and</strong> the non-cancerous hTERT-HME1.<br />
The hypericophenonoside decreases the intracellular oxidative stress induced by cisplatin <strong>and</strong><br />
adriamycin, without affecting viability, apoptosis, necrosis <strong>and</strong> cell cycle progression<br />
modulated by these chemotherapeutic agents. A very promising fact was Hd19 <strong>and</strong> silymarine<br />
protection on the proximal renal tubular cells against cisplatin toxicity, its chief dose-limiting<br />
side effect.<br />
These interesting biological effects led us to design <strong>and</strong> synthesize novel benzophenones<br />
containing a benzothiazolone moiety which could adjoin important pharmacological<br />
activities. The protective potential of three novel synthetic benzophenones was characterized.<br />
One of them named 6c exerted a higher antioxidant activity than Trolox, our synthetic<br />
reference compound. An important increase in cell viability <strong>and</strong> GSH level was observed in<br />
hTERT-HME1 cells. 6c showed high protective potential against oxidative stress <strong>and</strong> cell<br />
mortality induced by tBHP. Moreover, we observed a significant protection in<br />
cardiomyoblastic H9c2 cells, which may be of great interest in the frame of novel antioxidant<br />
strategies for the limitation of anthracycline cardiac toxicity.<br />
164
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 8: Gene expression networks in health<br />
<strong>and</strong> disease<br />
166
Session 8: Gene expression networks in health <strong>and</strong> disease Poster 1<br />
Dictyostelium discoideum as a model for Mucolipidosis Type IV<br />
Claire Y Allan 1 , Paul R Fisher 1<br />
Department of Microbiology, La Trobe University, Melbourne, AUSTRALIA<br />
cyallan@students.latrobe.edu.au<br />
Mucolipidosis Type IV (ML4) is a neurological lysosomal disease, which results in severe<br />
mental retardation, retinal degeneration <strong>and</strong> achlorhydria. Loss of function mutations in<br />
Mucolipin-1 (TRPML1), are responsible for ML4. TRPML1 is a member of the Transient<br />
Receptor Potential family of cation channels, <strong>and</strong> is involved in membrane trafficking along<br />
the endocytic pathway. The phenotypes associated with ML4 could result from<br />
endosomal/lysosomal dysfunction, as enlarged acidic compartments are formed as a result of<br />
defective lysosomal biogenesis <strong>and</strong> trafficking. Altered Ca2+ signalling associated with lack<br />
of function of TRPML1 may contribute to the pathogenesis of the disease, however the<br />
mechanisms of TRPML’s function in the endocytic pathway are not well understood. For this<br />
study, a model for ML4 has been created in the eukaryotic slime mould Dictyostelium<br />
discoideum. The D. discoideum mucolipin homologue was cloned to over-express <strong>and</strong> knock<br />
down the protein, <strong>and</strong> the resulting phenotypes showed similarities to those of ML4 patients.<br />
Immunofluorescence indicates that the protein is localised to acidic compartments <strong>and</strong> when<br />
the protein was both over- <strong>and</strong> underexpressed a build up of acidic compartments was<br />
observed. Observation of fruiting body morphologies showed a thickening of the basal disk<br />
<strong>and</strong> stalk. Since the stalk <strong>and</strong> basal disk in Dictyostelium are the endpoints of a form of<br />
programmed, autophagic cell death, this increased stalk <strong>and</strong> disk differentiation suggests<br />
increased autophagy, another phenotype associated with the human disease. The involvement<br />
of mucolipin in the endocytic pathway was demonstrated, as uptake of nutrients via<br />
phagocytosis, <strong>and</strong> subsequently growth rates, are increased in strains both overexpressing <strong>and</strong><br />
underexpressing mucolipin. Rates of pinocytosis in knockdown strains were also increased,<br />
however, interestingly, the growth in liquid medium was slightly slower, indicating<br />
differences in mucolipin involvement in the processing of macropinosomes <strong>and</strong> phagosomes.<br />
Alterations in Ca2+ signalling, have been observed in both over <strong>and</strong> underexpressing strains.<br />
These variations in signalling may be associated with the presenting phenotypes, as Ca2+<br />
signalling is known to be involved in fusion <strong>and</strong> biogenesis of endocytic vesicles, <strong>and</strong><br />
especially with formation of the phagocytic cup. Combined, all observations suggest that D.<br />
discoideum may be a useful model to study potential therapies for ML4.<br />
167
Session 8: Gene expression networks in health <strong>and</strong> disease Poster 3<br />
Mediterranean Fever Gene Mutation Profiling of 3051 Turkish Patients with Periodic<br />
Fever<br />
AFIG BERDELI 1 , SINEM NALBANTOGLU 1 , DEMET TIGLI 1 , MERVE ATAN 1 ,<br />
PERVIN TOPARLAK 1 , AYBEN PASOLAR 1 , ILKAY DEMIREL 1 , FATMA AKYIGIT 1<br />
1. Ege University Faculty of Medicine, Child Hospital Molecular Medicine Laboratory,<br />
35100, Bornova, izmir, Turkey.<br />
Familial Mediterranean Fever (FMF, MIM249100) is an autoinflammatory disorder<br />
characterized by seemingly unprovoked episodes of inflammation in the absence of high-titer<br />
autoantibodies or antigen-specific T cells. 16p13.3 chromosomally located MEFV<br />
(Mediterranean Fever) gene encoding the 781 amino acid protein “pyrin” is the responsible<br />
gene for this monogenic Mendelian disease. Pyrin is a component of inflammasome complex<br />
whose exact function on inflammatory stimuli is still unknown. To date, mutations were<br />
found in exons 1, 2, 3, 5, 9 <strong>and</strong> 10 of the MEFV gene, <strong>and</strong> exons 2 <strong>and</strong> 10 constitutes the socalled<br />
mutational `hot spots'. The objective of this study was to perform the MEFV<br />
(Mediterranean Fever) gene sequencing <strong>and</strong> to establish the genotype-phenotype relation in<br />
Turkish patients with definitive, posssible <strong>and</strong> suspicious FMF diagnosis. Mutation analysis<br />
of exons 2, 3, 5, <strong>and</strong> 10 of MEFV gene was carried out in 3051 patients <strong>and</strong> relatives of<br />
unrelated families by DNA sequencing analysis. Additionally, for patients who do not have<br />
mutations in exons 2, 3, 5, <strong>and</strong> 10; DNA sequencing was performed in exons 1, 7, 8 <strong>and</strong> 9, as<br />
well. In the DNA sequencing analysis, 67 different genotypes involving 10 novel mutations<br />
<strong>and</strong> 1 novel SNP in exons 2, 3, 5, 9, <strong>and</strong> 10 including R151S, S166L, G340R, P350R, G456A,<br />
Y471X, S503C, I506V, K695N, L709R, <strong>and</strong> P588P, G219G, were characterized <strong>and</strong><br />
registered in INFEVERS (database of hereditary autoinflammatory disorders mutations).<br />
Missense/nonsense <strong>and</strong> synonymous amino acid variations accounted for 37% <strong>and</strong> 55.8% of<br />
MEFV chromosomes (7.1% without any mutation or polymorphism), respectively. Through<br />
the missense mutations; 114 were homozygous, 257 were compound <strong>and</strong> complex<br />
heterozygous, <strong>and</strong> 760 were heterozygous for one mutation. Allelic frequencies of M694V,<br />
E148Q, V726A, <strong>and</strong> M680I accounted for 38,3; 21,3; 11,1; <strong>and</strong> 10,9%, respectively; followed<br />
by P369S, R408Q, K695R, M694I, R761H accounting for 4,3; 3,5; 2,8; <strong>and</strong> 1,6%.<br />
Significantly, 760 patients of the 1131 mutation positive group (67%), have only one single<br />
mutation (only one mutated MEFV) receiving colchicine therapy with whom several SNPs<br />
including D102D, D103D, P124P, G138G, A165A, P180P, R202Q, R314R, S363S, E474E,<br />
Q476Q, D510D, R501R, I506I, P588P, S683S, <strong>and</strong> P706P accompany.<br />
In conclusion, if screening for the most common mutations does not reveal any known<br />
common disease causing mutations <strong>and</strong> for asymptomatic individuals in particularly for the at<br />
risk populations, spesific DNA sequence mutation screening covering entire coding exons<br />
should need to be considered.<br />
169
Session 8: Gene expression networks in health <strong>and</strong> disease Poster 4<br />
Regulation of Lynch syndrome-related DNA mismatch repair heterodimer MutLalpha<br />
by phosphorylation?<br />
Angela Brieger, S<strong>and</strong>ra Passmann, Stefan Zeuzem <strong>and</strong> Jörg Trojan<br />
Medical Clinic I, Biomedical Research Laboratory, Goethe-University, Frankfurt a.M.,<br />
Germany<br />
Lynch syndrome, a hereditary disease associated with many different tumor types, is caused<br />
by mutations in DNA mismatch repair (MMR)-genes. 50% of these mutations are detected in<br />
the MLH1 protein. The heterodimer MutLalpha, which is formed by MLH1 <strong>and</strong> PMS2,<br />
coordinates a series of key events in the MMR mechanism. In addition to MMR, MutLalpha<br />
is involved in many other cellular processes as the regulation of cell cycle checkpoints <strong>and</strong><br />
apoptosis most likely signaling DNA damage to downstream pathways. A sophisticated<br />
regulation of the different MutLalpha functions might be important as well as reasonable. In<br />
order to focus on MutLalpha modulation, we analyzed MLH1 <strong>and</strong> PMS2 for phosphorylation<br />
sites using different computational systems <strong>and</strong> detected multiple potential loci <strong>and</strong><br />
corresponding kinases for both genes. Using phospho-specific purification columns we were<br />
able to isolate unphosphorylated <strong>and</strong> phosphorylated MLH1. In addition to this, Westernblot<br />
analysis of 2-D gels resolved MLH1 into several different distinct forms detected in lysates<br />
from HEK293 cells. Furthermore, we detected phosphorylation of purified MLH1 by<br />
PKCdelta, one of the computer-supported most probable MutLalpha kinases. Ongoing<br />
projects are directed towards the underst<strong>and</strong>ing how phosphorylation might be capable to<br />
switch MutLalpha function between MMR <strong>and</strong> DNA damage signaling.<br />
170
Session 8: Gene expression networks in health <strong>and</strong> disease Poster 5<br />
Interferences between Protein S <strong>and</strong> angiogenic factors signalling pathway in<br />
endothelial cells<br />
Sylvain Fraineau, Julie Talbot, Michel Philippe, Arnaud Monvoisin <strong>and</strong> Omar<br />
Benzakour<br />
Institut de Physiologie et de Biologie Cellulaires UMR CNRS 6187, Université de<br />
Poitiers, Poitiers, France<br />
Protein S is a plasma vitamin K-dependent glycoprotein, mainly known as a negative<br />
regulator of blood coagulation. Protein S heterozygous mice have been shown to display<br />
defects in vessel development <strong>and</strong> function 1 . Protein S secretion by endothelial cells <strong>and</strong> its<br />
substantial contribution to circulating protein S has been previously described. Expression of<br />
the putative tyrosine kinase receptors for both protein S <strong>and</strong> its structural homolog Gas6,<br />
termed TAM 2 (Tyro3, Axl <strong>and</strong> Mer) in endothelial cells was reported 3 . TAM receptors are<br />
involved in many cellular processes such as endothelial cells survival <strong>and</strong> morphogenesis 4 .<br />
Inhibition of VEGF/VEGFR-2 pathway by Axl/Gas6 signalling, involving the tyrosine<br />
phosphatase SHP-2 (SH-2 domain-containing tyrosine phosphatase), has been described 5 The<br />
major aim of our study was to assess if Gas6 homolog, protein S may also be involved in<br />
inhibiting VEGF/VEGFR-2-induced endothelial cells activation.<br />
We show that Mer stimulation by protein S inhibits vascular endothelial growth factor<br />
(VEGF)- <strong>and</strong> fibroblast growth factor 2 (FGF-2)-induced endothelial cells proliferation.<br />
Protein S also inhibits VEGF-inducedVEGFR-2. Proliferation assays using NSC 87877, a<br />
pharmacological inhibitor of tyrosine phosphatases SHP-1 <strong>and</strong> 2, suggested their implication<br />
in the inhibition by protein S/Mer complex of VEGFR-2 activation. Protein S also inhibited<br />
VEGF <strong>and</strong> FGF-2-activated downstream signalling pathways including inhibition of VEGF<br />
<strong>and</strong> FGF-2-induced MAP kinase Erk 1/2 phosphorylation. Interestingly, on its own, Protein S<br />
induced in HUVECs MAP kinase Erk 1/2 activation <strong>and</strong> cell proliferation which was<br />
suppressed in the presence of Erk 1/2 pathway inhibitors.<br />
Altogether, these data argue for new roles for the plasma vitamin K-dependent factor,<br />
protein S in regulating endothelial cells proliferation occurring during angiogenesis.<br />
1 Burstyn-Cohen T. et al., J Clin Invest (2009)<br />
2 Stitt T.N. et al., Cell (1995)<br />
3 Burnier L. et al., Blood (2010)<br />
4 O'Donnell K. et al., Am J Pathol (1999)<br />
5 Gallicchio M. et al., Blood (2005)<br />
171
Session 8: Gene expression networks in health <strong>and</strong> disease Poster 6<br />
Tungsten carbide cobalt nanoparticles exert hypoxia-like effects on the gene expression<br />
level in human keratinocytes<br />
Wibke Busch 1 , Dana Kühnel 1 , Kristin Schirmer 2 , Stefan Scholz 1<br />
1 Helmholtz-Centre for Environmental Research Leipzig - UFZ, Department<br />
Bioanalytical Ecotoxicology, Permoserstr. 15, 04318 Leipzig, Germany<br />
2 Eawag, Swiss Federal Institute of Aquatic Science <strong>and</strong> Technology, 8600 Dübendorf,<br />
Switzerl<strong>and</strong><br />
Tungsten carbide (WC) <strong>and</strong> tungsten carbide cobalt (WC-Co) nanoparticles are of<br />
occupational health relevance because of the increasing usage in hard metal industries. Earlier<br />
studies showed an enhanced toxic potential for WC-Co compared to WC or cobalt ions alone.<br />
Therefore, we investigated the impact of these particles on the global gene expression level in<br />
a human keratinocyte cell line (HaCaT) <strong>and</strong> compared the effects to those of cobalt ions<br />
(CoCl2). Changes in gene expression patterns were analysed after 3 hours <strong>and</strong> 3 days of<br />
exposure using whole genome microarrays <strong>and</strong> were confirmed by qPCR. WC nanoparticles<br />
exerted very little effects on transcription patterns. In contrast, WC-Co nanoparticles caused<br />
significant transcriptional changes that were similar to those provoked by CoCl2. However,<br />
the transcriptional response - both in terms of the number of affected genes as well as with<br />
respect to the relative fold changes - appeared to be more pronounced for CoCl2. Gene set<br />
enrichment analyses revealed that the differentially expressed genes were related to hypoxia<br />
response, carbohydrate metabolism <strong>and</strong> endocrine pathways. Furthermore, the identified<br />
genes were targets of several transcription factors whereas a majority of effects was found to<br />
be mediated by the transcription factor HIF1 (hypoxia inducible factor 1) which is known to<br />
be stabilised by cobalt ions. In conclusion, we show that WC nanoparticles caused low<br />
transcriptional responses while WC-Co nanoparticles are able to exert responses similar to<br />
that of free cobalt ions, particularly the induction of hypoxia-like effects via interactions with<br />
HIF1! in human keratinocytes. Since almost no particle specific expression patterns were<br />
found, this study shows that particle uptake is hardly detectable on the gene expression level<br />
<strong>and</strong> that leached ions are of great relevance in nanoparticle toxicity.<br />
!"#$%&'(&)*+&!,-&.'/012$#&34546&55789<br />
172
Session 8: Gene expression networks in health <strong>and</strong> disease Poster 7<br />
Thalassemia mouse model for human !-globin gene IVS-I-6 mutation<br />
Giulia Breveglieri, 1 Alessia Finotti, 2 Irene Mancini, 2 Nicoletta Bianchi, 2 Ilaria<br />
Lampronti, 2 Francesca Salvatori, 1 Giordana Feriotto, 2 Cristina Zuccato, 2 Monica<br />
Borgatti, 2 Gianni Car<strong>and</strong>ina, 3 Claudia Mel<strong>and</strong>ri, 3 Fiorella Altruda, 4 Sharmila<br />
Fagoonee, 4 Maddalena Iannicella, 4 Laura Breda, 5 Stefano Rivella 5 <strong>and</strong> Roberto<br />
Gambari 1,2<br />
1 Laboratory for the Development of Pharmacological <strong>and</strong> Pharmacogenomic Therapy<br />
of Thalassemia, Biotechnology Center, Ferrara University, Italy; e-mail:gam@unife.it;<br />
2 BioPharmaNet, Department of Biochemistry <strong>and</strong> Molecular Biology, Ferrara<br />
University, Italy; 3 Laboratory for Chemical <strong>and</strong> Clinical Analysis <strong>and</strong> Microbiology,<br />
University Hospital, Ferrara, Italy; 4 Molecular Biotechnology Center, University of<br />
Turin, Italy; 5 Division of Hematology-Oncology, Department of Pediatrics, Weill Cornell<br />
Medical College, New York, NY, USA<br />
In !-thalassemias, mutations of the !-globin gene or its regulatory regions cause absence (!°)<br />
or reduced (! + ) synthesis of !-globin chains, associated with a corresponding excess of the<br />
complementary "-globins. Mouse models for the different mutations (>200) of the !-globin<br />
gene causing thalassemia are very important to test in vivo the activity of novel mutationspecific<br />
therapeutic approaches. The IVS-I-6 mutation is the most frequent in the middle-east<br />
region <strong>and</strong> recurrent in Italy <strong>and</strong> Greece. This mutation leads to the activation of three criptic<br />
splicing sites, which generate three aberrantly spliced RNAs. In this study we report the<br />
production <strong>and</strong> characterization of a transgenic mouse line carrying a human !-globin gene<br />
containing the IVS-I-6 thalassemia point mutation. Our TG-!-IVS-I-6 mouse model displays<br />
a tissue specific expression of the transgene (fully overlapping with that of the endogenous<br />
murine !-globin gene) <strong>and</strong> produces normally spliced human !-globin mRNA (giving rise to<br />
!-globin production <strong>and</strong> formation of a human-mouse tetrameric chimeric hemoglobin " m 2! h )<br />
<strong>and</strong> unspliced aberrant !-globin-IVS-I-6 RNA in blood. Therefore, the TG!-IVS-I-6 mouse<br />
might be used as an in vivo model to characterize the effects of antisense ODN targeting the<br />
criptic site responsible for the generation of aberrantly spliced human !-globin RNA<br />
sequences.<br />
Supported by Telethon, UE Ithanet Project, Fondazione Cassa di Risparmio di Padova e<br />
Rovigo, AVLT (Rovigo Association for the Fight Against Thalassemia).<br />
173
Session 8: Gene expression networks in health <strong>and</strong> disease Poster 8<br />
Digoxin <strong>and</strong> ouabain increase the synthesis of cholesterol in human liver cells.<br />
Joanna Kopecka, Ivana Campia, Amalia Bosia <strong>and</strong> Chiara Riganti.<br />
Department of Genetics, Biology <strong>and</strong> Biochemistry, University of Torino <strong>and</strong> Research<br />
Center on Experimental Medicine (CeRMS), Via Santena, 5/bis, 10126, Torino, Italy.<br />
joanna.kopecka@unito.it, ivana.campia@unito.it, amalia.bosia@unito.it,<br />
chiara.riganti@unito.it.<br />
Cardioactive glycosides, like digoxin <strong>and</strong> ouabain inhibit the Na + /K + -ATPase: forcing the<br />
Na + /Ca 2+ exchanger to extrude Na + in exchange with Ca 2+ <strong>and</strong> thus increasing the [Ca 2+ ]i<br />
concentration, they exert a strong inotropic effect on heart. For this reason they are used in the<br />
treatment of congestive heart failure <strong>and</strong> cardiac arrhythmias. Recent findings have shown<br />
that cardioactive glycosides exert pleiotropic effects on cells, independently from the<br />
inhibition of Na + /K + -ATPase: for instance, they affect the cell response to hypoxia, modulate<br />
several signalling pathways involved in cell death <strong>and</strong> proliferation, regulate the transcription<br />
of different genes. Moreover, having a sterol-like chemical structure, cardioactive glycosides<br />
can modulate the metabolism of steroid hormones in vivo, probably taking part into fine feedback<br />
mechanisms. Until now no evidence has been provided about the effects of glycosides<br />
on the synthesis of the steroids hormones precursor, that is cholesterol.<br />
Here we report that digoxin <strong>and</strong> ouabain increase the synthesis of cholesterol in human liver<br />
HepG2 cells, enhancing the activity <strong>and</strong> the expression of the 3-hydroxy-3-methylglutarylcoenzyme<br />
A reductase (HMGCR), the rate-limiting enzyme of sterol biosynthesis. This effect<br />
was mediated by the binding of the sterol regulatory element binding protein-2 (SREBP-2) to<br />
the HMGCR promoter, <strong>and</strong> was lost in cells silenced for SREBP-2 or loaded with increasing<br />
amounts of cholesterol. Digoxin <strong>and</strong> ouabain competed with cholesterol for the binding on the<br />
sterol sensor domain of SREBP-cleavage-activating protein (SCAP), thus appearing as new<br />
critical regulators of cholesterol synthesis in human liver cells.<br />
Our results might have physiopathological significance: for instance it is known that different<br />
levels of cholesterol in plasma membranes may modify the activity of Na + /K + -ATPase <strong>and</strong> its<br />
affinity for inhibitors, thus affecting the therapeutic efficacy of glycosides. Since alterations<br />
of cholesterol levels <strong>and</strong> heart diseases are frequently associated, the potential cross-talk<br />
between digoxin <strong>and</strong> endogenous synthesis of cholesterol should be taken into consideration,<br />
because it may critically modulate the clinical response to cardioactive glycosides.<br />
174
Session 8: Gene expression networks in health <strong>and</strong> disease Poster 9<br />
Towards domino systems biology <strong>and</strong> an integrated ATP-centric model of regulation in<br />
Saccharomyces cerevisiae<br />
Malkhey Verma 1 , Maria Nardelli 1 <strong>and</strong> Hans V Westerhoff 1,2<br />
1 Manchester Interdisciplinary Biocentre, The University of Manchester, UK<br />
2 MCISB, The University of Manchester, UK<br />
Email: malkhey.verma@manchester.ac.uk; hans.westerhoff@manchester.ac.uk<br />
The domino systems biology approach was developed <strong>and</strong> implemented in the resolution of<br />
three paradoxical observations. These concern the response of ATP levels to perturbations in<br />
glycolysis, drug efflux pumping <strong>and</strong> DNA repair in S. cerevisiae. The analysis reinforced that<br />
the energetics of these three aspects of cell function cannot be understood in terms of these<br />
functions alone, but depend on the dynamic integration of the multiple cell functions that<br />
operate in the corresponding conditions. Even for underst<strong>and</strong>ing a functional component<br />
process of the cell, one must therefore implement systems biology. We developed domino<br />
systems biology shown to be an effective way of doing this.<br />
175
Posters are classified by session<br />
<strong>and</strong> then in alphabetical order (PRESENTING AUTHOR)<br />
(Late breaking abstracts are at the end of the <strong>book</strong>)<br />
Session 9: Neurodegenerative Diseases<br />
176
Session 9: Neurodegenerative Diseases Poster 1<br />
Parkin-mediated transcriptional function links p53 to autosomal recessive Parkinson’s<br />
disease.<br />
Cristine A. Costa <strong>and</strong> Frédéric Checler<br />
Institut de Pharmacologie Moléculaire et Cellulaire <strong>and</strong> Institut de NeuroMédecine<br />
Moléculaire, UMR6097 CNRS/UNSA, 660 route des Lucioles, 06560 Valbonne, France,<br />
acosta@ipmc.cnrs.fr<br />
Parkinson’s disease (PD) is the second cause of age-related neurodegenerescence in human<br />
beings. Although its etiology is mainly sporadic, a subset of genetic cases that is characterized<br />
by early onset <strong>and</strong> exacerbated loss of dopaminergic neurons of the substantia nigra pars<br />
compacta have been identified. Familial cases of PD can be associated with a dominant or<br />
recessive inheritance. Parkin is responsible for most of the autosomal recessive cases of PD.<br />
The main function of this protein appeared to be supported by its associated ubiquitin-ligase<br />
enzymatic activity <strong>and</strong> several lines of evidence indicated that impairment of this activity<br />
could contribute to the pathology.<br />
Although never demonstrated, a putative parkin-associated transcription factor activity could<br />
have been suspected from several observations including parkin localization in the nucleus,<br />
control of several genes activated in stress conditions <strong>and</strong> a Ring-IBR-Ring structure that<br />
predicts possible transcription factor properties. Based on these arguments we have<br />
documented a novel function of parkin as a transcription factor capable of transrepressing<br />
p53. We have demonstrated that parkin reduces p53 promoter transactivation, mRNA <strong>and</strong><br />
protein levels. The parkin-associated function was fully prevented by p53 depletion. We have<br />
described a physical interaction between parkin <strong>and</strong> the p53 promoter by both gel shift <strong>and</strong><br />
chromatin immunoprecipitation assays <strong>and</strong> we established that parkin binds to p53 promoter<br />
via its Ring1 domain. Importantly, the influence of parkin on p53 was also observed in PDaffected<br />
brains. Interestingly, parkin-associated repression of p53 promoter transactivation<br />
was prevented by all familial mutations studied in our work, independently of their ability to<br />
abolish or preserve parkin ubiquitin-ligase activity. This data pinpoints the fact that some<br />
functions harbored by parkin may be independent of its canonic ubiquitin-ligase activity.<br />
177
Session 9: Neurodegenerative Diseases Poster 2<br />
The TOR Signalling Pathway Mediates Batten Disease Phenotypes in Dictyostelium<br />
discoideum<br />
Paige K. Smith 1 , Anita Chavan 1 , Bethany S<strong>and</strong>s 1 , Gemma Shanley 1 , Paul R. Fisher 1 ,<br />
Sarah J. Annesley 1<br />
1 Department of Microbiology, La Trobe University, Bundoora, VIC, Australia<br />
Batten Disease is the common name given to a group of lysosomal storage disorders known<br />
as the Neuronal Ceroid Lipofuscinoses (NCLs). Eight forms of the disease have been<br />
identified <strong>and</strong> are characterised by their age of onset <strong>and</strong> an accumulation of a fluorescent<br />
lipofuscin-like pigment. Whilst each is genetically different, symptoms are similar in the<br />
different forms of the disease. We have used a simple eukaryotic organism, Dictyostelium<br />
discoideum to create models of Batten disease. We selected five different genetic forms of<br />
Batten’s disease, those resulting from mutant alleles at the CLN1, 2, 3, 5 <strong>and</strong> 7 loci. Multiple<br />
Dictyostelium mutant cell lines (strains) were created, in each of which the expression of the<br />
Dictyostelium homologue of one of these proteins was antisense-inhibited. Phenotypic<br />
analysis of the mutant strains revealed a consistent pattern of aberrant phenotypes – impaired<br />
aggregation <strong>and</strong> defective growth accompanied by increased rates of nutrient uptake. We<br />
show the results of Batten disease created through down regulation of the CLN2 protein as<br />
one example. Comparison of the phenotypes produced by Batten disease <strong>and</strong> those predicted<br />
to be caused by defective TOR signalling implicated the TOR signalling pathway as a<br />
mediator of the Batten disease phenotypes. This was confirmed by rapamycin treatment of<br />
wild type cells <strong>and</strong> antisense-inhibition of expression of Rheb, an upstream activator of TOR<br />
signalling, both of which caused the same phenotypes as Batten disease. The results could<br />
reveal possible drug targets for treatment in mammalian systems.<br />
178
Session 9: Neurodegenerative diseases Poster 3<br />
The comparison of the survival of P19-derived neuroprogenitors <strong>and</strong> P19 naive cells<br />
after intracerebellar application in a mice model with <strong>and</strong> without neurodegeneration.<br />
Cedikova M. (1), Pitule P. (1, 2), Houdek Z. (3, 5), Cendelin J. (3), Kulda V. (4),<br />
Kralickova M. (1, 5), Vozeh F. (3), Babuska V. (5), Pachernik J. (6), Zech N. (5, 7), Uher<br />
P. (5).<br />
(1) Department of Histology <strong>and</strong> Embryology, (2) Department of Surgery, (3)<br />
Department of Pathophysiology, (4) Department of Medical Chemistry <strong>and</strong><br />
Biochemistry, Faculty of Medicine, Charles University, Plzen, (5) IVF Centers Prof.<br />
Zech - Pilsen, Plzen,<br />
(6) Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno,<br />
(7) Departments for Obstetrics <strong>and</strong> Gynecology, Unit of Gynecological Endocrinology<br />
<strong>and</strong> Reproductive Medicine, University of Graz, Austria.<br />
Email adress of the first author: mirka.cedikova@seznam.cz<br />
Mouse embryonal carcinoma (EC) cells (P19 line) were studied for their survival <strong>and</strong><br />
developmental potential in the intact cerebellum of B6CBA mice <strong>and</strong> a model of<br />
neurodegeneration, in Lurcher mutant mice. Lurcher (Lc) mutant mice represent a model of<br />
olivocerebellar degeneration.<br />
The P19 cells were labeled with a green fluorescent protein <strong>and</strong> either cultured without<br />
differentiation or differentiated into neuroprogenitors using the retinoic acid. The<br />
intracerebellar application was performed in 73 mice - group A consisted of wt mice that<br />
received neuroprogenitors (n=21), group B of wt mice that received naive P19 cells (n=22),<br />
group C consisted of Lc mice that received neuroprogenitors (n=15) <strong>and</strong> group D of Lc mice<br />
that received naive P19 cells (n=15). The morphology of transplanted cells in the context of<br />
surrounding cerebellar tissue was evaluated after three weeks. The groups were compared for<br />
statistically significant difference using the Fisher’s test.<br />
Transplantations resulted in survival <strong>and</strong> further neurodifferentiation of cells in 13 cases in<br />
group A, in 7 cases in group B, in 3 cases in group C <strong>and</strong> in 3 cases in group D which means<br />
that the survival rates are 62%, 32%, 20% <strong>and</strong> 20%. Statistically, there is a significant<br />
difference between the groups of Wt (group A <strong>and</strong> B) <strong>and</strong> Lc mice (group C <strong>and</strong> D) (P=2,77).<br />
This is a pilot study comparing the fate of transplanted EC cells in the cerebellum with <strong>and</strong><br />
without a presence of neurodegenerative process. Our results confirm that EC cells can further<br />
differentiate in the host tissue. It seems that the differentiated elements do not survive less<br />
than undifferentiated <strong>and</strong> that the neurodegeneration affects the survival of EC derived<br />
elements. We plan to follow the impact of such transplantations on restoration of cerebellar<br />
functions. With further validation, the EC cells could become a valuable model for cell<br />
therapy of neurodegenerative diseases. This work has been supported by grants VZ MSM<br />
021620816, COST B30/2007 OC 152 <strong>and</strong> COST BM0603 No. OC10038, by the grant from<br />
the Czech Science Foundation no. 301/08/0717 <strong>and</strong> by SVV-2010- 260 806.<br />
179
Session 9: Neurodegenerative diseases Poster 4<br />
Alteration of MAP Kinase activity mediates the neurotoxic effects of the prion protein<br />
recombinant fragment hPrP90-231: reversal by minocycline.<br />
Aless<strong>and</strong>ro Corsaro 1 , Valentina Villa 1 , Stefano Thellung 1 , Mario Nizzari 1 , Antonio<br />
Aceto* <strong>and</strong> Tullio Florio 1<br />
1 Laboratory of Pharmacology, Dept. Oncology Biology <strong>and</strong> Genetics, University of<br />
Genova, Genova, Italy. E-mail Tullio.Florio@unige.it<br />
2 Department of Biomedical Sciences, Section of Biochemistry, University G.<br />
D’Annunzio, Chieti, Italy<br />
The recombinant peptide corresponding to the protease-resistant core of the human prion<br />
protein (hPrP90-231) can be converted in a PrP SC -like conformation (Corsaro, Paludi et al.<br />
2006) representing a useful model to study the molecular mechanism of neurotoxicity<br />
occurring during prion diseases. SH-SY5Y human neuroblastoma cells, in the absence of the<br />
neurotrophic factors present in fetal calf serum (FCS), undergo to marked activation of the<br />
apopotic pathway due to the loss of phosphorylation/activation of ERK1/2. Apoptosis is a<br />
tightly regulated process, where molecular cascades, triggered by both pro-survival <strong>and</strong> proapoptotic<br />
stimuli, elicit modifications of MAP kinase activity. In particular, the activation of<br />
ERK1/2 <strong>and</strong> p38 was reported to act in a mutually antagonistic way to induce cell survival or<br />
commitment to apoptosis. Interestingly, SH-SY5Y treatment with hPrP90–231 significantly<br />
affects the ability of FCS-contained neurotrophic factors to activate ERK1/2. The abolishment<br />
of ERK1/2 activity in these cells caused the removal of a tonic inhibition of p38 MAP kinase<br />
that is activated <strong>and</strong>, in turn, causes caspase 3 activation. This observation suggests that<br />
hPrP90-231 causes cell death inhibiting the pro-survival ERK1/2 activity of neurotrophic<br />
factors, sustaining the activity of the pro-apoptotic p38 MAP kinase <strong>and</strong> the subsequent<br />
caspase 3 activation. We also demonstrate that minocycline treatment causes a significant<br />
inhibition of hPrP90-231 induced apoptosis. In our experimental model minocycline prevents<br />
the down-regulation of the ERK1/2 pro-survival pathway induced by hPrP90-231, <strong>and</strong> the<br />
increased ERK1/2 activity keeps p38 silenced, preventing caspase 3 activation. In<br />
conclusion, we demonstrated that neurotoxic hPrP90-231 induces apoptosis unbalancing<br />
ERK1/2 <strong>and</strong> p38 MAP kinase activities <strong>and</strong> minocycline may inhibit this effect restoring<br />
ERK1/2 neurotrophic trasductional signalling.<br />
Reference<br />
Corsaro, A., D. Paludi, et al. (2006). "Conformation dependent pro-apoptotic activity of the<br />
recombinant human prion protein fragment 90-231." Int J Immunopathol Pharmacol 19(2):<br />
339-56.<br />
180
Session 9: Neurodegenerative diseases Poster 5<br />
Implication of the Notch pathway in the adult CNS during neurodegenerative processes<br />
Gauthier Dorban, Paul Felten, Claire Hoenen, Paul Heuschling <strong>and</strong> Eleonora Morga<br />
Life Sciences RU, FSTC, University of Luxembourg, 162a avenue de la Faïencerie, L-<br />
1511 Luxembourg. gauthier.dorban@uni.lu<br />
In the CNS, the Notch pathway is implicated in many aspects of cellular development <strong>and</strong><br />
functions. Recent studies have highlighted the role of Notch pathway in neurodegenerative<br />
diseases. It is now established that Notch plays a critical role in steering an immune response<br />
toward inflammation by regulating expression of various cytokines <strong>and</strong> proinflammatory<br />
compounds. Inappropriate or chronic deployment of the inflammation can become a<br />
destructive force in neuropathogenic processes such as multiple sclerosis (MS) <strong>and</strong><br />
Alzheimer’s disease (AD). The reexpression of Notch pathway components, especially the<br />
lig<strong>and</strong> Jagged1, is induced during neurodegenerative diseases <strong>and</strong> seems to create an<br />
environment that induces neuronal death.<br />
The aim of our study is to underst<strong>and</strong> the implication of the Notch pathway in the adult CNS<br />
during neurodegenerative processes. Using a mouse model, we have, identified Jagged1expressing<br />
cells in the forebrain before <strong>and</strong> after a stab wound. The gliosis was induced in the<br />
motorcortex of B6 mice by a mechanical lesion. A kinetic study has been realized to analyze<br />
the modulation of Jagged1 expression in the lesion until 20 days after the stab wound injury.<br />
Next, we have validated two methods to modulate in vivo Jagged1 expression. In our first<br />
model, cholesterol-labelled siRNA directed against Jagged1 has been injected within the<br />
lesion <strong>and</strong> allowed to spread around the stab wound. In the second model, mice bearing<br />
homozygously floxed alleles for Jagged1 were crossed to transgenic mice expressing a<br />
tamoxifen inducible Cre-recombinase. These in vivo strategies enabled us to study the<br />
induced modulations of the inflammatory reaction in the CNS. Jagged1 inhibition<br />
significantly decreases microglial activation <strong>and</strong> astrogliosis within the lesion.<br />
Our results show that Jagged1 plays an important role in the control of inflammatory reactions<br />
<strong>and</strong> could thus allow development of new therapeutic strategies helping to reduce the gliosis<br />
<strong>and</strong> the inflammatory response in the CNS.<br />
181
Session 9: Neurodegenerative diseases Poster 6<br />
Reduced p53 transcriptional activity in long term hypoxia increases chemoresistance in<br />
Medulloblastoma<br />
Yuen Ngan Fan, Daniel Meley, Barry Pizer, Violaine Sée<br />
Medulloblastoma (MB) is the most common malignant paediatric brain tumour. We have<br />
previously shown the key role of p53 signalling in medulloblastoma sensititvity to<br />
chemotherapeutic drugs (Meley et al, Cell Death <strong>and</strong> Disease 2010). Here, we have explored<br />
how hypoxia affects p53 activity <strong>and</strong> cell sensitivity upon chemotherapeutic treatment. We<br />
have shown that the miRNA miR34a is a p53 target <strong>and</strong> is up-regulated upon treatment with<br />
the chemotherapeutic drug etoposide. We also demonstrated that a miR34a mimic is able to<br />
induce a death program independent on p53 status. This suggests that we can bypass p53dependent<br />
drug resistance by directly targeting miR34a. We have found that MB cells (D283)<br />
become less sensitive to etoposide after long-term hypoxia exposure, while short-term<br />
hypoxia did not alter their sensitivity. We have further demonstrated that the increased drug<br />
resistance was concomitant with the inhibition of etoposide-induced p53 transcriptional<br />
activity <strong>and</strong> hence mir34a transcription. In conclusion, miR34a mimic can constitute a new<br />
molecular target for tumour treatment especially in p53 mutated cells or hypoxic tumour cells.<br />
182
Session 9: Neurodegenerative diseases Poster 7<br />
<strong>Inflammation</strong> induces transitory dedifferentiation of astrocytes<br />
Sebastien Gabel, Tony Heurtaux, Paul Felten, Eleonora Morga, Paul Heuschling <strong>and</strong><br />
Luc Gr<strong>and</strong>barbe<br />
Life Sciences Research Unit, FSTC, University of Luxembourg, 162A, avenue de la<br />
Faïencerie, L-1511 Luxembourg, (e-mail: luc.gr<strong>and</strong>barbe@uni.lu)<br />
Brain inflammation, currently described in some neurodegenerative diseases, has been<br />
recognized as a complex phenomenon that generates negative consequences, like<br />
neurotoxicity. However, several studies suggest that inflammatory signals could also exert a<br />
potentially positive influence on neural cell proliferation, survival, migration <strong>and</strong><br />
differentiation. In the adult CNS, astrocytes with neural stem cell capacity exist in specific<br />
neurogenic niches but adult parenchymal astrocytes are widely considered as terminally<br />
differentiated cells with no neurogenic capacity. This orthodoxy has been challenged by<br />
several observations that have shown that both immature parenchymal <strong>and</strong> reactive astrocytes<br />
demonstrate multipotency. Recently correlative studies have suggested that in injured<br />
conditions astrocytes undergo a process of dedifferentiation, <strong>and</strong> may acquire the potential of<br />
neural stem cells (NSC). However this process of dedifferentiation following injury remains<br />
unclear. Here, we report that during early response of reactive gliosis, inflammation induces a<br />
conversion of mature astrocytes into neural progenitors. After 24 h of TNF treatment most of<br />
the astrocytes lose the expression of GFAP <strong>and</strong> some of them re-express markers of the<br />
stemness state, such as CD133 <strong>and</strong> Musashi-1 (Msi-1). Thus TNF treatment results in the<br />
appearance of cells with neural progenitor phenotype, which are able to proliferate <strong>and</strong><br />
differentiate into neurons or astrocytes. To confirm that inflammatory signaling pathway<br />
activation directs the dedifferentiation of astrocytes in vivo, we realized a cortical lesion by a<br />
stab wound injury in adult mice brains. The in vivo data obtained, suggest that inflammation<br />
induces a decrease of GFAP expression in astrocytes <strong>and</strong> the re-expression of stemness<br />
markers in the area of the lesion after 24 h. The data presented here reveal the plasticity of<br />
astrocytes, which could be a potential target for the therapeutic strategies in injured CNS<br />
regeneration.<br />
183
Session 9: Neurodegenerative diseases Poster 8<br />
Abeta disaggregating proteins counteract Abeta induced neurotoxic responses<br />
Ana Gabriela Henriques, Bárbara Gomes, Raquel Ruivo, Edgar F. da Cruz e Silva* <strong>and</strong><br />
da Odete A. B. Cruz e Silva<br />
Laboratório de Neurociências, Centro de Biologia Celular, Universidade de Aveiro,<br />
3810-193 Aveiro, Portugal.<br />
*Laboratório de Transdução de Sinais, Centro de Biologia Celular, Universidade de<br />
Aveiro, 3810-193 Aveiro, Portugal.<br />
odetecs@ua.pt<br />
Abeta peptide is recognized as a key player in Alzheimer´s disease pathology, being<br />
responsible for the induction of several cellular responses associated with neurotoxicity <strong>and</strong><br />
neurodegeneration. Of note, it was shown that the fibrillar/aggregated species of Abeta are<br />
highly toxic. Thus, several therapeutic strategies aimed at depolymerizing Abeta provide<br />
attractive avenues of intervention. Laminin <strong>and</strong> gelsolin provide such a model, given that they<br />
both prevent Abeta aggregation by forming Abeta-laminin <strong>and</strong> Abeta-gelsolin complexes.<br />
Further, these complexes attenuate the neurotoxic effects of Abeta. Additionally, we have<br />
recently shown that Abeta can lead to intracellular accumulation of the neurotrophic <strong>and</strong><br />
neuroprotector secreted Alzheimer´s amyloid precursor protein fragment (sAPP) <strong>and</strong> preincubation<br />
of Abeta with laminin <strong>and</strong> gelsolin also renders it less potent in this respect. In<br />
conclusion, our data indicates that Abeta-laminin <strong>and</strong> Abeta-gelsolin complexes are less<br />
apoptotic <strong>and</strong> less potent at inducing intracellular sAPP retention than fibrillar Abeta. Hence,<br />
these data validate the potential of both proteins as therapeutic targets to prevent Abetainduced<br />
effects both at the neurotoxic <strong>and</strong> at the APP metabolic levels.<br />
184
Session 9: Neurodegenerative diseases Poster 9<br />
Microglia in astroglial culture activation: an underestimated role<br />
Tony Heurtaux, Sophie Losciuto, Gauthier Dorban, Sebastien Gabel, Luc Gr<strong>and</strong>barbe,<br />
Eleonora Morga, Paul Heuschling<br />
Life Sciences Research Unit, FSTC, University of Luxembourg, 162A, avenue de la<br />
Faïencerie, L-1511 Luxembourg (e-mail: tony.heurtaux@uni.lu)<br />
Development <strong>and</strong> progression of neurodegenerative diseases is associated with a chronic<br />
inflammatory state. <strong>Inflammation</strong> is probably not a primary pathogenic event in most forms of<br />
neurodegenerative diseases, however, there is substantial evidence that inflammatory<br />
responses of microglia, astrocytes <strong>and</strong> other immune cells contribute to disease severity.<br />
In accordance with different ontogenic origins of astrocytes <strong>and</strong> microglial cells, their<br />
physiological roles <strong>and</strong> many of their responses upon activation are also different. The<br />
purpose of this work is to evaluate the consequences of the presence of microglia in the<br />
activation of astroglial cultures. Indeed, the presence <strong>and</strong> role that microglial cells play in<br />
astroglial cultures are often underestimated. Thus, we have compared the impact of proinflammatory<br />
events on different glial cultures. We have stimulated murine microglia, mixed<br />
glial cultures, astrocyte-enriched cultures <strong>and</strong> neurosphere-derived astrocytes with proinflammatory<br />
compounds (lipopolysaccharide LPS <strong>and</strong> interferon-gamma IFNg). Cell<br />
cultures have been characterized by immunocytochemistry. mRNA <strong>and</strong> protein expression<br />
levels have been determined by PCR <strong>and</strong> ELISA assays respectively.<br />
Microglial cultures were strongly activated in presence of LPS <strong>and</strong> IFNg. Compared to<br />
microglia, the same treatments induced a lower activation of mixed glial cultures whereas a<br />
weak activation was observed on astrocyte-enriched cultures <strong>and</strong> neurosphere-derived<br />
astrocytes. In conclusions, we clearly show that microglia-devoided cultures (astrocyteenriched<br />
cultures <strong>and</strong> neurosphere-derived astrocytes) are poorly activated by proinflammatory<br />
agents. These results support the hypothesis that activation of astrocytes during<br />
neuroinflammatory states may be due to prior microglial activation inducing finally a vicious<br />
circle <strong>and</strong> a chronic inflammatory state. We present evidence that the magnitude of the proinflammatory<br />
response is proportional to the percentage of microglial cells present in glial<br />
cultures.<br />
185
Session 9: Neurodegenerative diseases Poster 10<br />
The role of the ESCRT-II complex in Dictyostelium discoideum<br />
Jasmina Ilievska 1 , Paul R Fisher 1 , Naomi E Bishop 1 <strong>and</strong> Sarah J Annesley 1<br />
1 Department of Microbiology, La Trobe University, Bundoora 3086, Victoria, Australia.<br />
Email: p.fisher@latrobe.edu.au<br />
The ESCRT (endosomal sorting complexes required for transport) machinery is composed of<br />
four complexes (0, I, II, III) integral to the degradation of cargo through the endocytic <strong>and</strong><br />
autophagic pathway. Mutations of ESCRT genes are associated with neurodegenerative<br />
diseases such as Alzheimer’s, Parkinson’s <strong>and</strong> Huntington’s. These diseases are characterised<br />
by accumulation of intracellular protein aggregates <strong>and</strong> defective autophagic compartments.<br />
The ESCRT complexes are involved in autophagy however their role in autophagic cell death<br />
(ACD) is not well known. Research of the autophagic pathway is often complicated by the<br />
additional cell death pathway of apoptosis. Both pathways result in the same cell death<br />
morphology making the two processes morphologically indistinguishable. The NIH<br />
recognised model eukaryotic system Dictyostelium discoideum provides a unique way to<br />
study autophagic cell death as it lacks an apoptosis pathway.<br />
The aim of the research is to alter the expression of the ESCRT-II proteins (Vps22, 25 <strong>and</strong> 36)<br />
in Dictyostelium discoideum <strong>and</strong> determine their roles in the autophagic cell death pathway.<br />
Preliminary results suggest that antisense inhibition of Vps22, 25 <strong>and</strong> 36 causes defects in cell<br />
growth, development, endocytosis <strong>and</strong> autophagic cell death. Here we present results for two<br />
of the members of the ESCRT-II complex Vps22 <strong>and</strong> Vps25.<br />
186
Session 9: Neurodegenerative diseases Poster 11<br />
A Possible Role of GSK-3beta-Nrf2 Signaling Pathway for the Prevention <strong>and</strong>/or<br />
Treatment of Alzheimer’s Disease<br />
Chan Lee 1 , Young-Joon Surh 2 , <strong>and</strong> Jung-Hee Jang 1<br />
1 College of Oriental Medicine, Daegu Haany University, Daegu 706-828, 2 College of<br />
Pharmacy, Seoul National University, Seoul 151-742, South Korea<br />
beta-Amyloid peptide (Abeta) is the major component of senile plaques accumulated in the<br />
brains of patients with Alzheimer’s disease (AD) <strong>and</strong> has been reported to cause neuronal cell<br />
death via oxidative stress. Therefore, attention has been focused on identifying redoxsensitive<br />
transcription factors <strong>and</strong> their target genes protecting against Abeta-induced<br />
oxidative cell death. Nrf2 plays a pivotal role in the transcriptional regulation of antioxidant<br />
proteins <strong>and</strong> detoxification enzymes <strong>and</strong> blocks apoptosis caused by a wide array of death<br />
signals. Ectopic expression of Nrf2 rescued cells from Abeta-induced cytotoxicity, apoptosis,<br />
intracellular accumulation of reactive oxygen species <strong>and</strong> oxidative damages. Moreover, Nrf2<br />
overexpression increased the expression of gamma-glutamylcysteine ligase (GCL), a ratelimiting<br />
enzyme in cellular glutathione biosynthesis. Conversely, knockdown of Nrf2 gene<br />
expression with siRNA or dominant negative mutant Nrf2 exacerbated Abeta-induced<br />
oxidative cell death. To further elucidate the upstream regulator for Nrf2 activation, we have<br />
focused on glycogen synthase kinase-3beta (GSK-3beta). Inhibition of Abeta-induced GSK-<br />
3beta activation by pharmacological inhibitors such as LiCl led to nuclear accumulation Nrf2<br />
<strong>and</strong> transcriptional activation of Nrf2 downstream target genes <strong>and</strong> protected against Abetamediated<br />
oxidative cell death. In another experiment, some dietary <strong>and</strong> medicinal<br />
phytochemicals attenuated A!-induced oxidative cell death via suppression of GSK-3beta <strong>and</strong><br />
subsequent activation of Nrf2. Taken together, these findings suggest that GSK-3beta-Nrf2<br />
signaling pathway may act as a survival mediator against AD.<br />
187
Session 9: Neurodegenerative diseases Poster 12<br />
Neuroprotective <strong>and</strong> Memory Enhancing Effects of Cypress-containing Essential Oils on<br />
Scopolamine-induced Amnesia in C57BL/6 Mice<br />
Gil-Yong Lee <strong>and</strong> Chan-Ik Park<br />
Department of Cosmeceutical Science, Daegu Haany University, Gyeongsangbuk-do<br />
712-715, S. Korea (E-mail : cipark@dhu.ac.kr)<br />
This study was performed to investigate the memory enhancing effects of essential oils from<br />
rosemary, thyme red, bergamot, pine needle, lavender <strong>and</strong> cypress against scopolamineinduced<br />
amnesia in C57BL/6 mice. To induce amnesia, scopolamine (1mg/kg) was<br />
intraperitonically injected into C57BL/6 mice 30 min before starting behavior tests. We have<br />
conducted Y-maze, Morris water-maze, passive avoidance <strong>and</strong> fear conditioning tests to<br />
monitor learning <strong>and</strong> memory functions. Essential oils of bergamot, pine needle, lavender <strong>and</strong><br />
cypress effectively reversed scopolamine-induced memory impairment in C57BL/6 mice<br />
which was represented by improvement of spontaneous alterations in Y-maze test, mean<br />
escape time in water-maze test, step-through latency in passive avoidance test <strong>and</strong> freezing<br />
response in fear conditioning tests. Particularly, combination of cypress with other essential<br />
oils (cypress + lavender > cypress + pine needle > cypress + bergamot) exhibited enhanced<br />
memory enhancing effects. However, compared with aforementioned natural essential oils,<br />
synthetic perfume mixtures showed relatively lower activities under the same experimental<br />
condition. These results suggest that cypress-containing essential oils may have therapeutic<br />
potentials <strong>and</strong> be applied for the prevention <strong>and</strong>/or treatment of amnesia-related neurological<br />
disorders.<br />
Keywords : amnesia, scopolamine, learning <strong>and</strong> memory, essential oil, cypress<br />
188
Session 9: Neurodegenerative diseases Poster 13<br />
Development of Natural Resources for the Prevention <strong>and</strong> Treatment of Alzheimer's<br />
Disease via Activating Nrf2-HO-1 Signaling Pathway<br />
Chan Lee <strong>and</strong> Jung-Hee Jang<br />
Department of Pathology, College of Oriental Medicine, Daegu Haany University,<br />
Daegu 706-828, Korea<br />
beta-Amyloid peptide (Abeta), the major component of senile plaques is considered to have a<br />
causal role in the development <strong>and</strong> progression of Alzheimer's disease (AD). Increasing<br />
evidence supports that Abeta-induced cell death is mediated by oxidative stress. Induction of<br />
heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation process, has been<br />
associated with adaptive survival response to oxidative insults. Treatment of SH-SY5Y cells<br />
with Abeta time-dependently increased protein expression of HO-1. Furthermore pretreatment<br />
of these cells with ZnPP, an inhibitor of HO-1 activity, aggravated Abeta-induced apoptotic<br />
cell death. Abeta treatment resulted in a transient activation of upstream redox-sensitive<br />
transcription factor, NF-E2-related factor 2 (Nrf2). Conversely, knockdown of Nrf2 gene<br />
expression abolished Abeta-induced HO-1 expression. In another experiment, we have<br />
searched for phytochemicals which can attenuate Abeta-induced apoptosis <strong>and</strong> memory<br />
impairment via Nrf2-HO-1 signaling pathway. We have investigated the neuroprotective<br />
effect of luteolin, 3´,4´,5,7-tetrahydroxyflavone present in the perilla leaf <strong>and</strong> seed, against<br />
Abeta-induced neuronal cell death <strong>and</strong> cognitive deficit in Sprague-Dawley (SD) rats. Abetacaused<br />
learning <strong>and</strong> memory deficits were effectively improved by oral administration of<br />
luteolin as assessed by several behavior tests such Morris water-maze, passive avoidance, fear<br />
conditioning tests through up-regulation of HO-1 by Nrf2 activation. Under the same<br />
experimental condition, luteolin attenuated Abeta-induced pro-apoptotic signals in the cortex<br />
<strong>and</strong> hippocampus such as activation of JNK <strong>and</strong> increased ratio of Bax to Bcl-2. Taken<br />
together, these findings suggest that up-regulation of HO-1 via activation of Nrf2 may act as a<br />
survival signal <strong>and</strong> have therapeutic potentials for the prevention <strong>and</strong> treatment of AD.<br />
Keywords: beta-amyloid, glycogen synthase kinase-3beta, NF-E2-related factor-2, oxidative<br />
stress, phytochemicals<br />
189
Session 9: Neurodegenerative diseases Poster 14<br />
REGULATION OF INFLAMMATORY PHENOTYPES OF MICROGLIA AND<br />
MICROGLIA-ASTROCYTE COMMUNICATION BY LIVER X RECEPTORS.<br />
Sophie Losciuto, Xavier C. Hever, Tony Heurtaux, Eleonora Morga <strong>and</strong> Paul<br />
Heuschling<br />
Life Sciences Research Unit, FSTC, University of Luxembourg, 162A, avenue de la<br />
Faïencerie, L-1511 Luxembourg (e-mail: xavier.hever@uni.lu)<br />
Alzheimer’s Disease is a neurodegenerative disorder characterized by the accumulation <strong>and</strong><br />
aggregation of Amyloid beta (Ab) peptide. In this pathology, microglia are frequently<br />
activated by a local inflammation, in part caused by the presence of Ab. Pro-inflammatory<br />
compounds secreted by activated microglia attract <strong>and</strong> activate astrocytes. These astrocytes<br />
also produce pro-inflammatory molecules leading to an inflammatory vicious circle in the<br />
brain accelerating neuronal death. It is known that activated microglia can differentiate<br />
towards a continuous spectrum of phenotypes of which the two extreme states are called M1<br />
for the pro-inflammatory state <strong>and</strong> M2 for the anti-inflammatory state. The Liver X Receptors<br />
(LXRs) are lig<strong>and</strong>-activated nuclear receptors known to regulate inflammatory responses by<br />
repressing pro-inflammatory gene expressions in activated microglia.<br />
In this study, Ab was used to induce the M1 pro-inflammatory state of microglia. We first<br />
showed that activated LXR was able to decrease the M1 state of activated microglia but was<br />
not able to induce the M2 anti-inflammatory state. These results suggest that LXR activation<br />
induces in microglia an intermediary state between M1 <strong>and</strong> M2. Since activated LXR reduces<br />
microglial activation, we analysed whether this effect could have an impact on the cellular<br />
communication between microglia <strong>and</strong> astrocytes. We showed that LXR activation has no<br />
direct effect on astrocyte activation. In co-culture experiments, preliminary observations<br />
showed that microglia, treated with an LXR agonist, were able to down-regulate astrocytic<br />
activation. Activated LXR appears to be indirectly able to modulate the phenotype of<br />
astrocytes through its action on microglia.<br />
This work emphasizes the role of activated LXR in cellular communication between<br />
microglia <strong>and</strong> astrocytes. Thus, LXR activation could reduce brain inflammation <strong>and</strong> may<br />
protect from neuronal death.<br />
190
Session 9: Neurodegenerative diseases Poster 15<br />
Cellular Antioxidant Adaptive Survival Response to 6-Hydroxydopamine-induced<br />
Nitrosative Cell Death in C6 Glioma Cells<br />
Chan Lee 1 , Gyu Hwan Park 2 , <strong>and</strong> Jung-Hee Jang 1*<br />
1 College of Oriental Medicine, Daegu Haany University, Daegu 706-828, South Korea,<br />
2 Department of Neurology, Columbia University, New York, NY 10032, USA<br />
Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized<br />
by selective loss of dopaminergic neurons in the substantia nigra. 6-Hydorxydopamine (6-<br />
OHDA) is a catecholaminergic neurotoxin widely used to produce experimental models of PD<br />
<strong>and</strong> has been reported to cause oxidative <strong>and</strong>/or nitrosative stress. In this study, we have<br />
investigated 6-OHDA-induced nitrosative cell death <strong>and</strong> its self-defense mechanism in C6<br />
glioma cells. Treatment of C6 cells with 6-OHDA increased expression of inducible nitric<br />
oxide synthase (iNOS) <strong>and</strong> subsequent production of nitric oxide (NO). Furthermore 6-<br />
OHDA treatment led to peroxynitrite generation <strong>and</strong> nitrotyrosine formation. 6-OHDAinduced<br />
nitrosative stress ultimately caused apoptotic cell death as determined by decreased<br />
Bcl-2/Bax ratio, activation of c-Jun N-termianl kinase (JNK), <strong>and</strong> cleavage of caspase-3 <strong>and</strong><br />
poly(ADP-ribose)polymerase (PARP), which were attenuated by peroxynitrite decomposition<br />
catalyst, FeTPPS. In another experiment, exposure of C6 glioma cells to 6-OHDA resulted in<br />
an increased expression of HO-1 <strong>and</strong> 6-OHDA-induced cytotoxicity was effectively<br />
suppressed by the HO-1 inducer SnCl2, supporting the cytoprotective role of HO-1. To<br />
elucidate the molecular mechanism underlying 6-OHDA-mediated HO-1 induction, we have<br />
examined the possible involvement of NF-E2-related factor 2 (Nrf2), which plays an<br />
important role in the transcriptional regulation of phase II detoxification <strong>and</strong> antioxidant<br />
enzymes. 6-OHDA treatment increased nuclear translocation <strong>and</strong> transcriptional activity of<br />
Nrf2, which seemed to be mediated by activation of Akt/ protein kinase B. Taken together<br />
these findings suggest that HO-1 up-regulation via Nrf2 activation may mediate cellular<br />
adaptive survival response to 6-OHDA-induced nitrosative cell death in C6 glioma cells.<br />
191
Session 9: Neurodegenerative diseases Poster 16<br />
Influence of free fatty acids on mitochondrial function in human brain cell line exposed<br />
to amyloid-beta.<br />
Agnieszka !liwa, Joanna Góralska, Barbara Zapa"a, Anna Gruca, Urszula Czech, Anna<br />
Knapp, Magdalena Awsiuk, Aldona Dembi#ska-Kie$<br />
Department of Clinical Biochemistry, Collegium Medicum, Jagiellonian University,<br />
Kopernika 15a, 31-501 Krakow, Pol<strong>and</strong><br />
Background:<br />
The cellular effects of nonestrified fatty acids (FFA) are complex <strong>and</strong> related in between to<br />
their role as substrates for energy production, as ion channel modulators, mitochondrial<br />
uncoupling agents <strong>and</strong> modulators of genes expression.<br />
The nutrient-induced changes in cellular metabolism is the recent target in the preventive<br />
medicine.<br />
Aim: The aim of the study was to investigate the effect of dietary FFA on mitochondrial<br />
function of the neuronal origin cells.<br />
Methods: LN-18 cells (glioblastoma) were preincubated with FFA: PA, OA, AA, EPA <strong>and</strong><br />
TTA (30µM) for 24h <strong>and</strong> exposed to !-amyloid (25 µM) for the last 18 hours. Measurement<br />
of mitochondrial membrane potential ("#) was performed by using flow cytometry <strong>and</strong> BD<br />
Bioimager 855 microscopy.Mitochondrial metabolic activity was monitored by measurements<br />
of the mitochondrial oxygen consumption rates (OROBOROS ® Oxygraph-2k) <strong>and</strong> ATP<br />
generation (ATP Lite Parkin Elmer).<br />
Results:<br />
In LN-18 cells PA significantly decreased mitochondrial membrane potential, mitochondrial<br />
respiration <strong>and</strong> ATP level. ATP generation was also diminished by OA, <strong>and</strong> the effect was<br />
equal to effect of !-amyloid alone. Preincubation of LN-18 cells with EPA prevent the<br />
negative effect of !-amyloid on mitochondrial respiration, though did not prevent the decrease<br />
in oxidative phosphorylation capacity.<br />
Conclusion:<br />
Our data suggest negative effect of PA on mitochondrial functions of the CNS-origin cells.<br />
Potentially neuroprotective action of EPA may be related to preventing !-amyloid induced<br />
mitochondrial dysfunction.<br />
Acknowledgments: this study was supported by Polish-Norwegian grant no. PNRF-104-AI-<br />
1/07, K/ZDS/000935 as well as the EU COST Action FA0602.<br />
192
Session 9: Neurodegenerative diseases Poster 17<br />
Study of the role of the Parkinson’s disease gene, Park2, in the development of gliomas.<br />
Julien Viotti, Eric Duplan, Frédéric Checler <strong>and</strong> Cristine Alves da Costa<br />
Institut de Pharmacologie Moléculaire et Cellulaire <strong>and</strong> Institut de NeuroMédecine<br />
Moléculaire, UMR6097 CNRS/UNSA, 660 route des Lucioles, 06560 Valbonne, France,<br />
viotti@ipmc.cnrs.fr<br />
Cancer is a huge age-related problem of public health that is mainly characterized by cell<br />
death control impairment. In contrast, Parkinson’s disease (PD) is a neurodegenerative<br />
disorder that is due to a massive loss of dopaminergic neurons of the substantia nigra by<br />
exacerbated apoptosis. Interestingly, several epidemiological data evidenced a negative<br />
correlation between PD <strong>and</strong> cancer. This negative correlation raises the question of whether<br />
despite phenotypically distinct, these two diseases could share common protein effectors. In<br />
this context, it is worth noting that most of familial PD-associated proteins are implicated in<br />
cell cycle control <strong>and</strong> often abnormally expressed in tumors. We have just ascribed a new<br />
function to one of them, parkin, as a transcriptional factor capable of trans-repressing p53. Of<br />
most interest several works pinpoint the role of parkin as a potential tumor suppressor protein,<br />
the mutations of which have been associated to glioblastoma generation. The main goal of the<br />
present work was to explore this newly identified transcription factor function of parkin in the<br />
etiology of human brain tumors. Our first results show that the overexpression of parkin can<br />
increase cell viability, decrease both the number of pre-apoptotic nuclei <strong>and</strong> caspase-3 activity<br />
<strong>and</strong> modulate cell cycle kinetics. In addition, we have observed a modulation of both parkin<br />
protein <strong>and</strong> mRNA levels according to the grade of gliomas. Our data suggest that the<br />
putative utilization of parkin as a brain tumor biomarker could be used in replacement of the<br />
subjective histological analysis performed in routine to date <strong>and</strong> perhaps allow an earlier <strong>and</strong><br />
bona fide diagnosis of this devastating <strong>and</strong> incurable type of cerebral cancer.<br />
193
Notes<br />
194
Notes<br />
195
Notes<br />
196
List of participants<br />
(In alphabetical order)<br />
(Updated January 13 th , 2011)<br />
197
Miss Siti Aminah Abdul Rahim<br />
norlux neuro oncology laboratory<br />
crp santé<br />
84 val fleuri<br />
L-1526 luxembourg<br />
LUX<br />
Email: sitiaminah.abdulrahim@crpsante.lu<br />
Mr. Soheil Akbari<br />
molecular biology <strong>and</strong> cancer research lab<br />
Dokuz Eylul University school of<br />
medicine<br />
inciralti /balcova<br />
35350 Izmir<br />
TUR<br />
Email: s.akbari84@gmail.com<br />
Mr. Rolf Altenburger<br />
Dept Bioanalytical Ecotoxicology<br />
UFZ Helmholtz Centre for Environmental<br />
Research<br />
Permoserstr. 15<br />
4318 Leipzig<br />
DEU<br />
Email: rolf.altenburger@ufz.de<br />
Dr. Salomon Amar<br />
The Center for Anti-Inflammatory<br />
Therapeutics<br />
Boston University<br />
650 Albany St., X-343<br />
MA Boston<br />
USA<br />
Email: samar@bu.edu<br />
Pr. Frank Antonicelli<br />
Dermatology<br />
URCA<br />
51 Rue Cognacq-jay<br />
51420 Reims<br />
FRA<br />
Email: frank.antonicelli@univ-reims.fr<br />
Dr. Aicha Ba<br />
Bioorganische Chemie-School of<br />
Pharmacy<br />
Saarl<strong>and</strong> University<br />
Universitaet Camous<br />
66123 Saarbruecken<br />
DEU<br />
Email: la.ba-bernardi@mx.unisaarl<strong>and</strong>.de<br />
Mr. Khalil Abouelaradat<br />
Radiobiology Unit<br />
SCK-CEN<br />
Boeretang 200<br />
B-2400 Mol<br />
BEL<br />
Email: kabouela@sckcen.be<br />
Miss Hélène Albert<br />
LIMBP<br />
Université Paul Verlaine Metz<br />
Rue du Général Delestraint<br />
57070 METZ<br />
FRA<br />
Email: albert2@univ-metz.fr<br />
Dr. Cristine Alves Da Costa<br />
IPMC<br />
CNRS<br />
660 ROUTE DES LUCIOLES<br />
6560 VALBONNE<br />
FRA<br />
Email: acosta@ipmc.cnrs.fr<br />
Mr. Alex<strong>and</strong>er Anderson<br />
Integrated Mathematical Oncology<br />
H. Lee Moffitt Cancer Center<br />
12901 Magnolia Drive<br />
33612 Tampa<br />
USA<br />
Email: alex<strong>and</strong>er.<strong>and</strong>erson@moffitt.org<br />
Pr. Safiye Nese Atabey<br />
Department of Medical Biology <strong>and</strong><br />
Genetics<br />
DEU Medical School<br />
Mithatpasa caddesi, Temel Bilimler<br />
Binasi, Kat 3<br />
35340 izmir<br />
TUR<br />
Email: nese.atabey@deu.edu.tr<br />
Mrs. Jaehee Bae<br />
Pharmacology<br />
Konyang University<br />
685, Gasuwondong, Seogu<br />
302-718 Daejeon<br />
KOR<br />
Email: jaeku@skku.edu<br />
198<br />
Mr. Adebayo Adewunmi Ebenezer<br />
UNIVERSITY COLLEGE HOSPITAL<br />
NO 27 AYORINDE STREET EKOTEDO,<br />
OPP SHOP 142 ADAMASINGBA<br />
SHOPPING COMPLEX<br />
2234 IBADAN<br />
NGA<br />
MASTERLARYYCHO@YAHOO.COM<br />
Miss Claire Allan<br />
Microbial Cell Biology<br />
La Trobe University<br />
Kingsbury Drive<br />
3086 Bundoora<br />
AUS<br />
Email: c_y_a_001@hotmail.com<br />
Dr. Sarah Annesley<br />
Micorbial Cell Biology<br />
La Trobe University<br />
Plenty Rd<br />
3086 Bundoora<br />
AUS<br />
Email: S.Annesley@latrobe.edu.au<br />
Pr. Marina Aunapuu<br />
Histology<br />
University of Tartu<br />
Ravila 19<br />
50411 Tartu<br />
EST<br />
Email: marina.aunapuu@ut.ee<br />
Pr. Denyse Bagrel<br />
LIMBP<br />
Université Paul Verlaine-Metz<br />
Rue du Général Delestraint<br />
57070 Metz<br />
FRA<br />
Email: bagrel@univ-metz.fr
Mrs. Emilie Bana<br />
Laboratoire d'Ingénierie Moléculaire et<br />
Biochimie Pharmacologique<br />
Université Paul Verlaine - Metz<br />
Avenue du Général Delestraint<br />
57070 METZ<br />
FRA<br />
Email: emilie.bana@free.fr<br />
Dr. Henry Bayele<br />
Structural <strong>and</strong> Molecular Biology<br />
University College London<br />
Gower Street<br />
WC1E 6BT London<br />
GBR<br />
Email: h.bayele@medsch.ucl.ac.uk<br />
Miss Cristina Belgiovine<br />
IGM-CNR<br />
Via Abbiategrasso 207<br />
27100 Pavia<br />
ITA<br />
Email: belgiovine@igm.cnr.it<br />
Dr. Rafi Benotmane<br />
Radiobiology Unit<br />
SCK-CEN<br />
Boeretang 200<br />
B-2400 Mol<br />
BEL<br />
Email: abenotma@sckcen.be<br />
Pr. Afig Berdeli<br />
MOLECULAR MEDICINE<br />
LABORATORY<br />
EGE UNIVERSITY FACULTY OF<br />
MEDICINE CHILD HOSPITAL<br />
BORNOVA<br />
35100 IZMIR<br />
TUR<br />
Email: afigberdeli@yahoo.com<br />
Miss Kim BoAe<br />
Formulation Lab. of New Cosmeceuticals<br />
Daegu Haany University<br />
290, Yugok-dong<br />
712-715 Gyeongsan-si, Gyeongsangbukdo<br />
KOR<br />
Email: gajette@hanmail.net<br />
Miss Burcin Baran<br />
molecular biology <strong>and</strong> cancer research lab<br />
dokuz eylul school of medicine<br />
Inciralti/Balcova<br />
35350 Izmir<br />
TUR<br />
Email: burcinbrn@yahoo.com<br />
Dr. Juergen Becker<br />
PromoCell GmbH<br />
PromoCell GmbH<br />
Sickingenstrasse 63/65<br />
69126 Heidelberg<br />
DEU<br />
Email: info@promocell.com<br />
Miss Houda Benboubker<br />
LIMBP<br />
Campus Bridoux<br />
57000 Metz<br />
FRA<br />
Email: houda@benboubker.com<br />
Mrs. Lynda Benyoub<br />
AUREUS PHARMA<br />
174 Quai de Jemmapes<br />
75010 PARIS<br />
FRA<br />
Email: sabine.clement@aureuspharma.com<br />
Mrs. Geert Bienvenue<br />
GE Healthcare<br />
koterveldstraat 2<br />
1831 Diegem<br />
BLZ<br />
Email: geert.bienvenue@ge.com<br />
Mr. William Booth<br />
AMSBIO<br />
184 Milton Park<br />
OX14 4SE Abingdon<br />
GBR<br />
Email: stephanieg@amsbio.com<br />
199<br />
Dr. Daniela Basso<br />
Department of Laboratory Medicine<br />
University Hospital of Padova<br />
Via Giustiniani 2<br />
35128 Padova<br />
ITA<br />
Email: daniela.basso@sanita.padova.it<br />
Mrs. Adeline Beillerot<br />
LIMBP<br />
UPV-Metz<br />
rue du général Delestraint<br />
57070 Metz<br />
FRA<br />
Email: beillero@univ-metz.fr<br />
Dr. Aline Bennasroune<br />
Laboratoire d'Ingénierie Moléculaire et<br />
Biochimie Pharmacologique<br />
Rue du general Delestraint<br />
57070 Metz<br />
FRA<br />
Email: aline.bennasroune@univ-metz.fr<br />
Mr. Omar Benzakour<br />
Institut de Physiologie et Biologie<br />
Cellulaires<br />
Université de Poitiers<br />
1, rue Georges Bonnet<br />
86022 Poitiers<br />
FRA<br />
Email: omar.benzakour@univ-poitiers.fr<br />
Dr. Romain Blasius<br />
prophac<br />
Prophac<br />
5 rangwee<br />
2412 luxembourg<br />
LUX<br />
Email: romain.blasius@prophac.lu<br />
Dr. Monica Borgatti<br />
BIOCHEMISTRY AND MOLECULAR<br />
BIOLOGY DEPARTMENT<br />
FERRARA UNIVERSITY<br />
VIA FOSSATO DI MORTARA 74<br />
44121 FERRARA<br />
ITA<br />
Email: brgmnc@unife.it
Dr. Pieter Borger<br />
Departement Biomedizin Pulmonology<br />
University Hospital Basel<br />
Hebelstrasse 20<br />
4031 Basel<br />
CHE<br />
Email: pieter.borger@unibas.ch<br />
Dr. Angela Brieger<br />
Biomedical Research Laboratory<br />
JWG University Frankfurt a.M.<br />
Theodor-Stern-Kai 7<br />
60590 Frankfurt<br />
DEU<br />
Email: a.brieger@em.uni-frankfurt.de<br />
Pr. Jean-Luc Bueb<br />
Life Sciences Research Unit<br />
Université du Luxembourg<br />
162a, avenue de la Faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: jean-luc.bueb@uni.lu<br />
Dr. Mario Capecchi<br />
Department of Human Genetics<br />
University of Utah <strong>and</strong> The Howard<br />
Hughes Medical Institute<br />
15 North 2030 East #5440<br />
84112 Salt LakeCity<br />
USA<br />
Email: mario.capecchi@genetics.utah.edu<br />
Pr. Park ChanIk<br />
Formulation Lab. of New Cosmeceuticals<br />
Daegu Haany University<br />
290, Yugok-dong<br />
712-715 Gyeongsan-si, Gyeongsangbukdo<br />
KOR<br />
Email: gajette@hanmail.net<br />
Dr. Elena Chernolovskaya<br />
Laboratory of Nucleic Acids<br />
Biochemistry<br />
Institute of Chemical Biology <strong>and</strong><br />
Fundamental Medicine SB RAS<br />
Lavrentiev ave., 8<br />
630090 Novosibirsk<br />
RUS<br />
Email: elena_ch@niboch.nsc.ru<br />
Mr. Michael Bots<br />
Lab for Exp. Oncology <strong>and</strong> Radiobiology<br />
Meibergdreef 9<br />
1105 AZ Amsterdam<br />
NLD<br />
Email: m.bots@amc.uva.nl<br />
Dr. Juergen Brieger<br />
Molecular Tumor Biology<br />
University Medical Center of the<br />
Johannes Gutenberg University Mainz<br />
Department of Otolaryngology,<br />
55131 Mainz<br />
DEU<br />
Email: juergen.brieger@unimedizinmainz.de<br />
Mrs. Wibke Busch<br />
Department Bioanalytical Ecotoxicology<br />
Helmholtz-Centre for Environmental<br />
Research - UFZ<br />
Permoser Str. 15<br />
4318 Leipzig<br />
DEU<br />
Email: wibke.busch@ufz.de<br />
Dr. Miroslava Cedikova<br />
Department of Histology <strong>and</strong> Embryology<br />
Karlovarska 48<br />
301 00 Plzen<br />
CZE<br />
Email: mirka.cedikova@seznam.cz<br />
Dr. Beatrice Charreau<br />
INSERM UMR643<br />
30 bd Jean Monnet<br />
44093 Nantes<br />
FRA<br />
Email: Beatrice.Charreau@univ-nantes.fr<br />
Dr. Andy Chevigné<br />
Retrovirology<br />
CRP-Santé<br />
Val Fleuri<br />
L-1526 Luxembourg<br />
LUX<br />
Email: <strong>and</strong>y.chevigne@crp-sante.lu<br />
200<br />
Mr. Koen Breyne<br />
Biochemistry - Fac. Veterinary Sciences<br />
Ghent University<br />
Salisburylaan 133<br />
9820 Merelbeke (Ghent)<br />
BEL<br />
Email: koen.breyne@ugent.be<br />
Pr. Pedro Buc Calderon<br />
PMNT 7369<br />
université catholique de Louvain<br />
73, avenue E. Mounier<br />
1200 Bruxelles<br />
BEL<br />
Email: pedro.buccalderon@uclouvain.be<br />
Pr. Vittorio Calabrese<br />
Clinical Biochemistry <strong>and</strong> clinical<br />
molecular Biology<br />
University of Catania<br />
Viale Andrea Doria 6<br />
95125 CATANIA<br />
ITA<br />
Email: calabres@unict.it<br />
Dr Claudia Cerella<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: claudia.cerella@lbmcc.lu<br />
Dr Sebastien Chateauvieux<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: sebastien.chateauvieux@lbmcc.lu<br />
Miss Claude Condé<br />
labotory of fundamental virology <strong>and</strong><br />
immunology<br />
Ulg<br />
1, avenue de ll'hôpital Bat B34<br />
4000 Liège<br />
BEL<br />
Email: claude_conde@hotmail.com
Dr. Aless<strong>and</strong>ro Corsaro<br />
Laboratory of Pharmacology, Dept.<br />
Oncology Biology <strong>and</strong> Genetics,<br />
University of Genova<br />
Viale benedetto XV<br />
16132 Genova<br />
ITA<br />
Email: ale.corsaro@unige.it<br />
Miss Brigitte Czepukojc<br />
Bioorganische Chemie-School of<br />
Pharmacy<br />
Saarl<strong>and</strong> University<br />
Universitaet Campous<br />
66123 Saarbruecken<br />
DEU<br />
Email: b.czepukojc@mx.uni-saarl<strong>and</strong>.de<br />
Mrs. Saskia De Meyer<br />
LSCA<br />
Agilent Technologies<br />
Pegasus Park - De Kleetlaan 5 Bus 9<br />
1831 Diegem<br />
BEL<br />
Email: godelieve_minet@agilent.com<br />
Mrs. Debora Dell'Era Dosch<br />
Europe<br />
Enzo Life Sciences<br />
Industriestrasse 17<br />
CH-4415 Lausen<br />
CHE<br />
Email: agrainger@enzolifesciences.com<br />
Dr. Dieter Demon<br />
Biochemistry - Fac. Veterinary Sciences<br />
Ghent University<br />
Salisburylaan 133<br />
9820 Merelbeke (Ghent)<br />
BEL<br />
Email: dieter.demon@ugent.be<br />
Mr. Luciano Di Croce<br />
Epigenetic events in Cancer<br />
CRG/ICREA<br />
Dr. Aiguader 88<br />
8003 Barcelona<br />
ESP<br />
Email: luciano.dicroce@crg.es<br />
Pr. Pascale Cossart<br />
Interactions Bactéries Cellules<br />
Institut Pasteur<br />
25 rue du Docteur Roux<br />
75015 Paris<br />
FRA<br />
Email: pcossart@pasteur.fr<br />
Mr. Incarnato Danny<br />
NEUROSCIENCE DEPARTMENT<br />
SIENA BIOTECH SPA<br />
STRADA DEL PETRICCIO E<br />
BELRIGUARDO 35<br />
53100 SIENA<br />
ITA<br />
Email: dincarnato@sienabiotech.it<br />
Mr. Nicolas Dejeans<br />
PMNT 7369<br />
université catholique de Louvain<br />
73, avenue E. Mounier<br />
1200 Bruxelles<br />
BEL<br />
Email: nicolas.dejeans@uclouvain.be<br />
Pr. Aldona Dembinska-Kiec<br />
Department of Clinical Biochemistry<br />
Jagiellonian University<br />
Kopernika 15 a<br />
31-501 Cracow<br />
POL<br />
Email: mbkiec@cyf-kr.edu.pl<br />
Mr. Harald Desch<br />
Leica Microsystems<br />
3, rue des Joncs<br />
L-1818 Howald<br />
LUX<br />
Email: steve.engel@lecuit.lu<br />
Dr. Maria Di Girolamo<br />
G-Protein-Mediated Signalling<br />
Laboratory<br />
Consorzio Mario Negri Sud<br />
Via Nazionale 8/A<br />
66030 S. Maria Imbaro<br />
ITA<br />
Email: mdigirolamo@negrisud.it<br />
201<br />
Miss Isabelle Coupienne<br />
Virology <strong>and</strong> Immunology<br />
1, avenue de l'Hopital<br />
4000 Liege<br />
BEL<br />
Email: icoupienne@ulg.ac.be<br />
Mrs. Odete de Cruz e Silvia<br />
Laboratorio Neurociencias<br />
Centro Biologia Celular, Universidade de<br />
Aveiro<br />
Campus Santiago<br />
3810-193 Aveiro<br />
PRT<br />
Email: aghenriques@ua.pt<br />
Pr. Antonio del Sol<br />
Computational Biology Unti<br />
Luxembourg Center for Systems<br />
Biomedicine-University of Luxembourg<br />
162a, avenue de la faiencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: antonio.delsol@uni.lu<br />
Mrs. Kristel Demeyere<br />
Biochemistry-Fac veterinary sciences<br />
Ghent University<br />
Salisburylaan 133<br />
9820 MERELBEKE (GHENT)<br />
BEL<br />
Email: Kristel.demeyere@ugent.be<br />
Mr. Brian DeWitt<br />
Biorefinery<br />
IBBL<br />
6, rue Nicolas Barblé<br />
1210 Luxembourg<br />
LUX<br />
Email: brian.dewitt@ibbl.lu<br />
Pr. Cinzia Di Pietro<br />
biology <strong>and</strong> genetics<br />
university of catania<br />
via s. sofia 87<br />
95124 catania<br />
ITA<br />
Email: dipietro@unict.it
Dr Marc Diederich<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: marc.diederich@lbmcc.lu<br />
Dr. Catherine Dostert<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: catherine.dostert@uni.lu<br />
Dr. Joanna Dulinska-Litewka<br />
Chair of Medical Biochemistry<br />
Jagiellonian University Medical College<br />
Kopernika 7<br />
31-034 Krakow<br />
POL<br />
Email: mblitewk@cyf-kr.edu.pl<br />
Mr. Steve Engel<br />
Lecuit Opto-Technical / Leica<br />
Microsystems<br />
3, rue des Joncs<br />
L-1818 Howald<br />
LUX<br />
Email: steve.engel@lecuit.lu<br />
Miss Yuen Ngan Fan<br />
university of Liverpool<br />
Crown street<br />
L69 7ZB Liverpool<br />
GBR<br />
Email: carolfan@liverpool.ac.uk<br />
Pr. Dean Felsher<br />
Felsher Laboratory<br />
Stanford University<br />
269 Campus Drive<br />
94305-5151 Stanford<br />
USA<br />
Email: dfelsher@stanford.edu<br />
Dr. Sascha Dietrich<br />
Adaptive Immunregulation (G331)<br />
Im Neuenheimer Feld 580<br />
69120 He<br />
Im Neuenheimer Feld 410<br />
69120 Heidelberg<br />
DEU<br />
Email: sascha.dietrich@med.uniheidelberg.de<br />
Mr. Peng Du<br />
Bioorganische Chemie-School of<br />
Pharmacy<br />
Saarl<strong>and</strong> University<br />
Universitaet Campous<br />
66123 Saarbruecken<br />
DEU<br />
Email: du-peng@live.com<br />
Dr. Eric Duplan<br />
IPMC<br />
CNRS<br />
660 Route des lucioles<br />
6560 Valbonne<br />
FRA<br />
Email: duplan@ipmc.cnrs.fr<br />
Dr. Esra Erdal<br />
Department of Medical Biology <strong>and</strong><br />
Genetics<br />
DEU Medical School<br />
Mithatpasa caddesi, Temel Bilimler<br />
Binasi, Kat 3<br />
34350 izmir<br />
TUR<br />
Email: esra.erdal@deu.edu.tr<br />
Dr. Margarida Fardilha<br />
Signal Transduction<br />
Campus de Santiago<br />
3810-193 Aveiro<br />
PRT<br />
Email: mfardilha@ua.pt<br />
Mr. Paul Felten<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: paul.felten@uni.lu<br />
202<br />
Dr. Gauthier Dorban<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: gauthier.dorban@uni.lu<br />
Mrs. Elodie Dubus<br />
AUREUS PHARMA<br />
174 Quai de Jemmapes<br />
75010 PARIS<br />
FRA<br />
Email: elodie.dubus@aureus-pharma.com<br />
Mrs. Bessem Cecilia Elango<br />
Laboratory Techniciant<br />
Institude Of Biomedical Research<br />
Rose Garden Muea-Buea po box 485<br />
south west region Cameroon<br />
237 Buea<br />
CMR<br />
Email: kimbifuen@yahoo.co.uk<br />
Dr. Zsolt Fabian<br />
Regenerative Medicine Institute<br />
University Road<br />
Co. Galway Galway<br />
IRL<br />
Email: zsolt.fabian@nuigalway.ie<br />
Pr. Dean Felsher<br />
Felsher laboratory<br />
Stanford<br />
269 Campus Dr<br />
94305-5151 Stanford<br />
USA<br />
Email: dfelsher@stanford.edu<br />
Mrs. Sanjanie Fern<strong>and</strong>o<br />
PRF lab, Department of Microbiology<br />
Latrobe University<br />
Kingsbury Drive<br />
VIC3086 Bundoora<br />
AUS<br />
Email:<br />
sgfern<strong>and</strong>o@students.latrobe.edu.au
Pr. Paul Fisher<br />
Microbial Cell Biology<br />
Kingsbury Drive<br />
VIC 3086 Melbourne<br />
AUS<br />
Email: P.Fisher@latrobe.edu.au<br />
Mr. Sylvain Fraineau<br />
Institut de Physiologie et Biologie<br />
Cellulaires<br />
Université de Poitiers<br />
1 rue Georges Bonnet<br />
86022 Poitiers<br />
FRA<br />
Email: sylvain.fraineau@univ-poitiers.fr<br />
Mr Francois Gaascht<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: francois.gaascht@lbmcc.lu<br />
Dr. Michael Gale<br />
STRIDE Center<br />
University of Washington<br />
Box 357650<br />
98195-7650 Seattle<br />
USA<br />
Email: mgale@uw.edu<br />
Dr. Dorota Gil<br />
Chair of Medical Biochemistry<br />
Jagiellonian University Medical College<br />
Kopernika 7<br />
31-034 Krakow<br />
POL<br />
Email: dgil@poczta.fm<br />
Dr. Bernhard Goetz<br />
Prophac<br />
5 rangwee<br />
2412 luxembourg<br />
LUX<br />
Email: bernhard.goetz@prophac.lu<br />
Dr Cristina Florean<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: cristina.florean@lbmcc.lu<br />
Mrs Elodie Frenger<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: elodie.frenger@lbmcc.lu<br />
Miss Karoline Gäbler<br />
Signal Transduction Laboratory<br />
162a, avenue de la Faincerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: karoline.gaebler@uni.lu<br />
Pr. Roberto Gambari<br />
BIOCHEMISTRY AND MOLECULAR<br />
BIOLOGY DEPARTMENT<br />
FERRARA UNIVERSITY<br />
VIA FOSSATO DI MORTARA 74<br />
44121 FERRARA<br />
ITA<br />
Email: gam@unife.it<br />
Mrs. Agnes Giot<br />
GE Healthcare<br />
koterveldstraat 2<br />
1831 Diegem<br />
BEL<br />
Email: agnes.giot@ge.com<br />
Dr Stefania Gonfloni<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: stefania.gonfloni@lbmcc.lu<br />
203<br />
Dr. Paola Fogar<br />
Department of Laboratory Medicine<br />
University-Hospital of Padova<br />
Via Giustiniani 2<br />
35128 Padova<br />
MRT<br />
Email: paola.fogar@unipd.it<br />
Dr. Nicolai Fricker<br />
Department of Immunogenetics, DKFZ<br />
Im Neuenheimer Feld 267<br />
69120 Heidelberg<br />
DEU<br />
Email: n.fricker@dkfz.de<br />
Dr Anthoula Gaigneaux<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: anthoula.gaigneaux@lbmcc.lu<br />
Dr. Alex Gamma<br />
Zurich University Psychiatric Hospital<br />
Lenggstrasse 31<br />
8032 Zurich<br />
CHE<br />
Email: agamma@ethz.ch<br />
Mr. Christophe Glorieux<br />
PMNT 7369<br />
université catholique de Louvain<br />
73, avenue E. Mounier<br />
1200 Bruxelles<br />
BEL<br />
Email: christophe.glorieux@uclouvain.be<br />
Dr. Stefania Gonfloni<br />
Molecular Genetics<br />
University of Rome 'Tor Vergata<br />
via della Ricerca Scientifica<br />
133 Rome<br />
ITA<br />
Email: Stefania.Gonfloni@uniroma2.it
Dr. Marie-Lise Gougeon<br />
Antiviral Immunity, Biotherapy <strong>and</strong><br />
Vaccine Unit<br />
INSTITUT PASTEUR<br />
25-28 rue du Dr. Roux<br />
75015 Paris<br />
FRA<br />
Email: mlgougeo@pasteur.fr<br />
Dr. Luc Gr<strong>and</strong>barbe<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: luc.gr<strong>and</strong>barbe@uni.lu<br />
Mr. Anne Grosse-Wilde<br />
Ozinsky Lab<br />
Institute for Systems Biology<br />
1441 N 34th St<br />
98103 Seattle<br />
USA<br />
Email:<br />
AGWILDE@SYSTEMSBIOLOGY.ORG<br />
Dr. Yvette Habraken<br />
Virology <strong>and</strong> Immunology<br />
University of Liège<br />
Allée de l'Hopital 1, Bât B34<br />
4000 Liège<br />
BEL<br />
Email: yvette.habraken@ulg.ac.be<br />
Miss Luciana Harumi Osaki<br />
Gastrointestinal Epithelium Biology<br />
Av. Prof. Lineu Prestes<br />
5508900 Sao Paulo<br />
BRA<br />
Email: luciana.osaki@usp.br<br />
Mrs Liliane Hermes<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: liliane.hermes@lbmcc.lu<br />
Miss Aysim Gozukizil<br />
Molecular biology research labratory<br />
Dokuz Eylul University<br />
inciralti<br />
35330 izmir<br />
TUR<br />
Email: a.gkizil@hotmail.com<br />
Dr Cindy Gr<strong>and</strong>jenette<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: cindy.gr<strong>and</strong>jenette@lbmcc.lu<br />
Miss Geraldine Guerin-Peyrou<br />
Technical Support<br />
Polyplus-transfection<br />
Bioparc, Bd S. Brant<br />
67401 Illkirch<br />
FRA<br />
Email: ggp@polyplus-transfection.com<br />
Mrs Sheherazade Hajjouli<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: sheherazade.hajjouli@lbmcc.lu<br />
Mrs Estelle Henry<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: estelle.henry@lbmcc.lu<br />
Dr. Tony Heurtaux<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: tony.heurtaux@uni.lu<br />
204<br />
Miss Astrid Grainger<br />
Benelux<br />
Enzo Life Sciences<br />
Melkerijweg 3<br />
BE-2240 Z<strong>and</strong>hoven<br />
BEL<br />
Email: agrainger@enzolifesciences.com<br />
Mrs Christina Grigorakaki<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: christina.grigorakaki@lbmcc.lu<br />
Dr. Claude Haan<br />
Signal Transduction Laboratory<br />
University of Luxembourg<br />
162A avenue de la faiencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: claude.haan@uni.lu<br />
Pr. Pirkko Härkonen<br />
Institute of Biomedicine<br />
University of Turku<br />
Kiinamyllynkatu 10<br />
20720 Turku<br />
FIN<br />
Email: harkonen@utu.fi<br />
Miss Ivana Hermanova<br />
CLIP Charles University in Prague<br />
V Uvalu 84<br />
15006 Prague<br />
CZE<br />
Email: ivana.hermanova@lfmotol.cuni.cz<br />
Dr. Xavier Hever<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: xavier.hever@uni.lu
Mr. Kasper Hoebe<br />
Cincinnati Children's Hospital<br />
3333 Burnet Avenue<br />
45229 Cincinnati<br />
USA<br />
Email: kasper.hoebe@cchmc.org<br />
Dr. Andrew Hufton<br />
Molecular Systems Biology<br />
Meyerhofstr. 1<br />
69117 Heidelberg<br />
DEU<br />
Email: <strong>and</strong>rew.hufton@embo.org<br />
Pr. Claus Jacob<br />
Bioorganische Chemie-School of<br />
Pharmacy<br />
Saarl<strong>and</strong> University<br />
Universitaet Camous<br />
66123 Saarbruecken<br />
DEU<br />
Email: c.jacob@mx.uni-saarl<strong>and</strong>.de<br />
Mr. Patryk Janus<br />
Center for Translational Research <strong>and</strong><br />
Molecular Biology of Cancer<br />
Wybrzeze Armii Krajowej 15<br />
44-101 Gliwice<br />
POL<br />
Email: patrykjanus@gmail.com<br />
Mr. Jang-Yeon Jeong<br />
Immunobiology<br />
Jeju National University<br />
66 jejudaehakno<br />
690756 Jeju<br />
KOR<br />
Email: ckdgml3735@hanmail.net<br />
Pr. Jang Jung-Hee<br />
College of Oriental Medicine<br />
Daegu Haany University<br />
165 Sang-dong, Suseong-gu<br />
706-828 Daegu<br />
KOR<br />
Email: pamy202@paran.com<br />
Miss Claire Hoenen<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: claire.hoenen@uni.lu<br />
Miss Jasmina Ilievska<br />
Microbial Cell Biology<br />
La Trobe University<br />
Plenty Rd<br />
3086 Bundoora<br />
AUS<br />
Email: jilievska@students.latrobe.edu.au<br />
Dr Monika Jain<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: monika.jain@lbmcc.lu<br />
Mr. R.G.P.T. Jayasooriya<br />
Immunobiology<br />
Jeju National University<br />
66 jejudaehakno<br />
690756 Jeju<br />
KOR<br />
Email: www.jayasamanthas@gmail.com<br />
Mrs. Elisabeth John<br />
Life Sciences Research Unit,<br />
FSTCUniveristy of Luxembourg<br />
Avenue de la Faiencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: elisabeth.john@uni.lu<br />
Dr. Stefanie Kaempf<br />
Medical Manager<br />
NonWoTecc Medical GmbH<br />
Nattermannallee 1<br />
50829 Cologne<br />
DEU<br />
Email: s.kaempf@nonwotecc.de<br />
205<br />
Mr. Jean Marie Hoornaert<br />
EUROGENTEC<br />
5 rue du Bois Saint Jean<br />
4102 Seraing<br />
BEL<br />
Email: j.m.hoornaert@eurogentec.com<br />
Dr. Ute Inegbenebor<br />
Medical <strong>and</strong> Research Laboratory<br />
Ambrose Alli University<br />
Benin Auchi Way<br />
23401 Ekpoma<br />
NGA<br />
Email: druteinegbenebor@yahoo.com<br />
Mr. Yeon-Jeong Jang<br />
Immunobiology<br />
Jeju National University<br />
66 jejudaehakno<br />
690756 Jeju<br />
KOR<br />
Email: ckdgml3735@hanmail.net<br />
Dr. Yongtark Jeon<br />
Seoul National University Bundang<br />
Hospital<br />
300 Gumidong<br />
463-707 Sungnam<br />
KOR<br />
Email: asidof1@snu.ac.kr<br />
Dr. Hye Jin Jung<br />
Department of Biotechnology<br />
Yonsei University<br />
262 Seongsanno<br />
120-749 Seoul<br />
KOR<br />
Email: hjjung96@gmail.com<br />
Mr. Thorben Kaetzel<br />
lsru<br />
Université du Luxembourg<br />
162a, avenue de la Faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: thorben.kaetzel@uni.lu
Mr. Surasee Kamlua<br />
Centre for Research <strong>and</strong> Development of<br />
Medical Diagnostic Laboratories<br />
Khon Kaen University<br />
Mitrapap<br />
40002 Khon Kaen<br />
THA<br />
Email: kamikaze_me@hotmail.com<br />
Mr. Chang-Hee Kang<br />
Immunobiology<br />
Jeju National University<br />
66 jejudaehakno<br />
690756 Jeju<br />
KOR<br />
Email: ckdgml3735@hanmail.net<br />
Dr. Michael Katze<br />
STRIDE Center<br />
University of Washington<br />
Box 358070<br />
WA Seattle<br />
USA<br />
Email: honey@uw.edu<br />
Dr Mareike Kelkel<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: mareike.kelkel@lbmcc.lu<br />
Pr. Yong Beom Kim<br />
Department of OB&GYN, Seoul National<br />
University Bundang Hospital<br />
166 Gumi-ro, Bundang-gu<br />
463-707 SEONGNAM<br />
KOR<br />
Email: ybkimlh@snubh.org<br />
Dr. Hee Seung Kim<br />
Seoul National University<br />
Unavailable<br />
110-744 Seoul<br />
KOR<br />
Email: bboddi0311@snu.ac.kr<br />
Mrs. Emine K<strong>and</strong>emis<br />
molecular biology <strong>and</strong> cancer research lab<br />
dokuz eylul school of medicine<br />
Inciralti/Balcova<br />
35350 Izmir<br />
TUR<br />
Email: eminecelik@gmail.com<br />
Mr Tommy Karius<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: tommy.karius@lbmcc.lu<br />
Dr. Alex<strong>and</strong>er Kel<br />
Chief Scientific Officer<br />
geneXplain GmbH<br />
Am Exer 10b<br />
D-38302 Wolfenbuettel<br />
DEU<br />
Email: alex<strong>and</strong>er.kel@genexplain.com<br />
Mr. Khairan Khairan<br />
Bioorganische Chemie-School of<br />
Pharmacy<br />
Saarl<strong>and</strong> University<br />
Universitaet Campous<br />
66123 Saarbruecken<br />
DEU<br />
Email: khannazia-yusuf@yahoo.com<br />
Mr. Gi-Young Kim<br />
Immunobiology<br />
Jeju National University<br />
66 jejudaehakno<br />
690756 Jeju<br />
KOR<br />
Email: immunkim@jejunu.ac.kr<br />
Dr. Olga Kofanova<br />
Biorefinery<br />
IBBL<br />
6, rue Nicolas Barblé<br />
1210 Luxembourg<br />
LUX<br />
Email: olga.kofanova@ibbl.lu<br />
206<br />
Pr. Jaeku Kang<br />
Department of Pharmacology<br />
College of Medicine, Konyang University<br />
685, Gasuwon-dong, Seo-gu,<br />
302-718 Daejeon<br />
KOR<br />
Email: jaeku@konyang.ac.kr<br />
Dr. Vladimir Katanaev<br />
Katanaev<br />
University of Konstanz<br />
Universitätsstrasse 10, Box 643<br />
78457 Konstanz<br />
DEU<br />
Email: vladimir.katanaev@unikonstanz.de<br />
Dr. Olga Kel-Margoulis<br />
Applied Life Science Informatics<br />
geneXplain GmbH<br />
Am Exer 10b<br />
D-38302 Wolfenbuettel<br />
DEU<br />
Email: olga.kelmargoulis@genexplain.com<br />
Miss Kiyoon Kim<br />
Department of Biochemistry <strong>and</strong><br />
Molecular Biology, Rm 419<br />
School of Medicine, Kyung Hee<br />
University<br />
1 Hoegidong, Dongdaemungu<br />
130-701 Seoul<br />
KOR<br />
Email: navy07i@naver.com<br />
Dr. Hyun-Duck Kim<br />
Preventive <strong>and</strong> social dentistry<br />
Seoul Ntional University School of<br />
Dentistry<br />
28 Yeonkeon-Dong Jongno-Ku<br />
110-749 Seoul<br />
KOR<br />
Email: hyundkim@snu.ac.kr<br />
Pr. Andrew Koff<br />
Cell Cycle Regulation<br />
Memorial Sloan-Kettering Cancer Center<br />
1275 York Avenue<br />
10021 New York, New York<br />
USA<br />
Email: koffa@mskcc.org
Mr. Hendrik Koopmans<br />
Cell Biology<br />
Westburg BV<br />
PO Box 214<br />
3830 AE Leusden<br />
NLD<br />
Email: F.v<strong>and</strong>erLoop@westburg.eu<br />
Miss Karolina Kramarzova<br />
CLIP, Charles University in Prague<br />
V Uvalu 84<br />
15006 Prague<br />
CZE<br />
Email: karolina.kramarzova@fnmotol.cz<br />
Miss Imge Kunter<br />
Molecular biology <strong>and</strong> cancer research<br />
lab<br />
Dokuz Eylul University School of<br />
Medicine<br />
inciralti/ balcova<br />
35350 Izmir<br />
TUR<br />
Email: imge_kunter@yahoo.com<br />
Mr. An<strong>and</strong> Lachmansingh<br />
Sales<br />
Bio-Connect B.V.<br />
Begonialaan 3a<br />
NL-6851 TE Huissen<br />
NLD<br />
Email: peters@bio-connect.nl<br />
Mr. Chae Hyeong Lee<br />
Dongguk University Ilsan Hospital<br />
Siksa-dong 814<br />
410-773 Goyang<br />
KOR<br />
Email: gynelee@paran.com<br />
Mrs. Elisabeth Letellier<br />
Signal Transduction Laboratory<br />
University of Luxembourg<br />
162 A avenue de la Faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: elisabeth.letellier@uni.lu<br />
Mr. Joanna Kopecka<br />
Department of Genetics, Biology <strong>and</strong><br />
Biochemistry, University of Torino<br />
Via Santena 5/bis<br />
10126 Torino<br />
ITA<br />
Email: joanna.kopecka@unito.it<br />
Dr. Stephanie Kreis<br />
Signal Transduction Laboratory<br />
University fo Luxembourg<br />
162A Ave de la Faiencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: stephanie.kreis@uni.lu<br />
Miss Hoi Tung Kwan<br />
NGAN HEXTAN<br />
DEPARTMENT OF O & G, LKS<br />
FACULTY OF MEDICINE, THE<br />
UNIVERSITY OF HONG KONG<br />
21 SASSON ROAD<br />
0 HONG KONG<br />
HKG<br />
Email: virg1234@hotmail.com<br />
Dr. Inna Lavrik<br />
DKFZ/Bioquant<br />
Im Neuenheimer Feld 280<br />
69120 Heidelberg<br />
DEU<br />
Email: i.lavrik@dkfz.de<br />
Miss Helene Leger<br />
IHES<br />
35 route de chartres<br />
91440 Bures-sur-Yvette<br />
FRA<br />
Email: leger@ihes.fr<br />
Mrs. Nan Li<br />
Institute of Immunology<br />
Second Military Medical University<br />
800 Xiangyin Road<br />
200433 Shanghai<br />
CHN<br />
Email: linan@immunol.org<br />
207<br />
Mrs. Peyda Korhan<br />
Molecular Biology <strong>and</strong> Cancer Research<br />
Lab<br />
Dokuz Eylul University School of<br />
Medicine<br />
Inciralti, Balcova<br />
35350 Izmir<br />
TCA<br />
Email: korhanpeyda@hotmail.com<br />
Pr. Guido Kroemer<br />
INSERM U848<br />
UniversitÈ Paris DÈscartes<br />
15 rue del l\'Ecole de MÈdecine<br />
75006 Paris<br />
FRA<br />
Email: kroemer@orange.fr<br />
Pr. Ho Jeong Kwon<br />
Department of Biotechnology<br />
Yonsei University<br />
262 Seongsanno<br />
120-749 Seoul<br />
KOR<br />
Email: kwonhj@yonsei.ac.kr<br />
Miss Aurore Lecat<br />
Virology <strong>and</strong> Immunology (GIGA-R)<br />
avuenue de l'hopital<br />
4000 Liège<br />
BEL<br />
Email: aurore.lecat@ulg.ac.be<br />
Mrs Noemie Legr<strong>and</strong><br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: noemie.legr<strong>and</strong>@lbmcc.lu<br />
Miss Maria Liivr<strong>and</strong><br />
Systems Biology, LSRU, FSTC<br />
University of Luxembourg<br />
162a, avenue de la Faiencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: marialiivr<strong>and</strong>@gmail.com
Mrs. Chen Lin<br />
AG Katanaev<br />
Universitaet Konstanz<br />
Universitaetsstrasse 10<br />
78464 Konstanz<br />
DEU<br />
Email: Chen.Lin@uni-konstanz.de<br />
Mrs. Evgeniya Logashenko<br />
Laboratory of biochemistry of nucleic<br />
acids<br />
8, Lavrentev ave<br />
630090 Novosibirsk<br />
RUS<br />
Email: evg_log@niboch.nsc.ru<br />
Mr. Ev<strong>and</strong>ro Luis de Oliveira Niero<br />
Cell <strong>and</strong> Molecular Biology<br />
Av. Prof. Lineu Prestes<br />
5508900 Sao Paulo<br />
BRA<br />
Email: eloniero@usp.br<br />
Dr. Thomas Luft<br />
Adaptive Immune Regulation<br />
University of Heidelberg<br />
Im Neuenheimer Feld 410<br />
69120 Heidelberg<br />
DEU<br />
Email: t.luft@dkfz.de<br />
Dr. Eva Matalova<br />
Department of Physiology<br />
University of Veterinary <strong>and</strong><br />
Pharmaceutical Sciences<br />
Palackeho 1-3<br />
612 42 Brno<br />
CZE<br />
Email: matalova@iach.cz<br />
Pr. Gerry Melino<br />
Dip Experimental Medicine<br />
University Rome Tor Vergata<br />
via Montpellier 1<br />
133 Rome<br />
ITA<br />
Email: melino@uniroma2.it<br />
Mrs. Qiuyan Liu<br />
Institute of Immunology<br />
Second Military Medical University<br />
800 Xiangyin Road<br />
200433 Shanghai<br />
CHN<br />
Email: lqy1969@yahoo.com.cn<br />
Miss Axelle Loriaux<br />
Promega Benelux<br />
Schipholweg 1<br />
2316XB LEIDEN<br />
NLD<br />
Email: axelle.loriaux@promega.com<br />
Dr. Francesca Luchetti<br />
Cytometry<br />
University<br />
Via Ca' le Suore 2<br />
61029 Urbino<br />
ITA<br />
Email: francesca.luchetti@uniurb.it<br />
Mrs Fabienne Mack<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: fabienne.mack@lbmcc.lu<br />
Dr. Conny Mathay<br />
Biorefinery<br />
IBBL<br />
6, rue Nicolas Barblé<br />
1210 Luxembourg<br />
LUX<br />
Email: conny.mathay@ibbl.lu<br />
208<br />
Miss Julia Lochead<br />
PromoCell GmbH<br />
PromoCell GmbH<br />
Sickingenstrasse 63/65<br />
69126 Heidelberg<br />
DEU<br />
Email: info@promocell.com<br />
Miss Sophie Losciuto<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: sophie.losciuto@uni.lu<br />
Miss Anne-Marie Lüchtenborg<br />
AG Katanaev<br />
Universität Konstanz<br />
Universitätsstrasse 10<br />
78457 Konstanz<br />
DEU<br />
Email: anne-marie.luechtenborg@unikonstanz.de<br />
Dr. Irena Manov<br />
Pediatric Research <strong>and</strong> Electron<br />
Microscopy Unit<br />
Technion-Israel Institute of Technology<br />
Efron 1<br />
31096 Haifa<br />
ISR<br />
Email: irmanov@tx.technion.ac.il<br />
Dr. Mark Mattson<br />
Laboratory of Neurosciences<br />
National Institute on Aging<br />
251 Bayview Boulevard - BRC 5C214<br />
21224 Baltimore<br />
USA<br />
Email: mattsonm@grc.nia.nih.gov<br />
Miss Mohd Daud Melor<br />
INSTITUTE OF MEDICAL<br />
MOLECULAR BIOTECHNOLOGY<br />
UITM SUNGAI BULOH CAMPUS<br />
JALAN HOSPITAL<br />
47000 SUNGAI BULOH<br />
MYS<br />
Email: impaxmelor@gmail.com
Mr. Romuald Menth<br />
Technical Support<br />
Polyplus-transfection<br />
Bioparc, Bd S. Brant<br />
67401 Illkirch<br />
FRA<br />
Email: rmenth@polyplustransfection.com<br />
Dr. Nicola Miglino<br />
Departement Biomedizin Pulmonology<br />
University Hospital Basel<br />
Hebelstrasse 20<br />
4031 Basel<br />
CHE<br />
Email: nicola.miglino@unibas.ch<br />
Dr Franck Morceau<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: franck.morceau@lbmcc.lu<br />
Dr. Günter Müller<br />
R & D Diabetes Division<br />
Sanofi-Aventis Deutschl<strong>and</strong> GmbH<br />
Industrial Park Hochst Bldg. H821<br />
65926 Frankfurt am Main<br />
DEU<br />
Email: guenter.mueller@sanofiaventis.com<br />
Mr. Olaf Nijst<br />
Unit Vaccinology<br />
RIVM<br />
Anthonie van Leeuwenhoeklaan 9<br />
3721 MA Bilthoven<br />
NLD<br />
Email: Olaf.Nijst@rivm.nl<br />
Mrs Barbora Orlikova<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: barbora.orlikova@lbmcc.lu<br />
Dr. Bernard Metz<br />
Unit Vaccinology<br />
RIVM<br />
A. van Leeuwenhoeklaan 9<br />
3721 MA Bilthoven<br />
NLD<br />
Email: Bernard.Metz@rivm.nl<br />
Dr. Nadezda Mironova<br />
Institute of Chemical Biology <strong>and</strong><br />
Fundamental Medicine SB RAS<br />
Lavrentiev ave. 8<br />
630090 Novosibirsk<br />
RUS<br />
Email: mironova@niboch.nsc.ru<br />
Dr. Eleonora Morga<br />
Laboratoire de Neurobiologie<br />
Université du Luxembourg<br />
162A, avenue de la faïencerie<br />
L-1511 Luxembourg<br />
LUX<br />
Email: eleonora.morga@uni.lu<br />
Dr. Sinem Nalbantoglu<br />
MOLECULAR MEDICINE<br />
LABORATORY<br />
EGE UNIVERSITY FACULTY OF<br />
MEDICINE CHILD HOSPITAL<br />
BORNOVA<br />
35100 IZMIR<br />
TUR<br />
Email: nalbantoglusinem@gmail.com<br />
Mrs Karoline Noworyta<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: karoline.noworyta@lbmcc.lu<br />
Pr. Chris Overall<br />
Centre for Blood Research<br />
University of British Columbia<br />
4.401 Life Sciences Institute, 2350 Health<br />
Sciences Mall<br />
V6T 1Z3 Vancouver<br />
CAN<br />
Email: chris.overall@ubc.ca<br />
209<br />
Pr. Evelyne Meyer<br />
Biochemistry-Fac veterinary sciences<br />
Ghent University<br />
Salisburylaan 133<br />
9820 MERELBEKE (GHENT)<br />
BEL<br />
Email: evelyne.meyer@ugent.be<br />
Miss Brenda Molenberghs<br />
Exhibitor<br />
Cell Signaling Technology Europe<br />
Plesmanlaan 1d<br />
2333 BZ Leiden<br />
NLD<br />
Email: b.molenberghs@bioke.com<br />
Mr Florian Muller<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: florian.muller@lbmcc.lu<br />
Dr. Maria Nardelli<br />
Hans Westerhoff's Research Group,<br />
University of Manchester (UK)<br />
University of Manchester<br />
131 Princess Street<br />
M1 7DN Manchester<br />
GBR<br />
Email: maria.nardelli@manchester.ac.uk<br />
Mrs Marie-Anne Olinger<br />
LBMCC<br />
Hôpital Kirchberg<br />
9, rue Edward Steichen<br />
L-2540 Luxembourg<br />
LUX<br />
Email: marie_anne.olinger@lbmcc.lu<br />
Miss Evin Ozen<br />
Molecular Biology <strong>and</strong> Cancer Research<br />
Lab<br />
Dokuz Eylül Universty Medicine School<br />
Institute of Health Science<br />
Inciralti<br />
35350 Izmir<br />
TUR<br />
Email: evinozen@hotmail.com
Mr. Stefano Papa<br />
Cytometry<br />
University<br />
Via Ca' le Suore 2<br />
61029 Urbino<br />
ITA<br />
Email: stefano.papa@uniurb.it<br />
Mrs. Stéphanie Philippot<br />
LIMBP<br />
Université Paul Verlaine - Metz<br />
Campus Bridoux rue Général Delestraint<br />
57070 METZ<br />
FRA<br />
Email: stephanie.philippot@univ-metz.fr<br />
Mr. Pavel Pitule<br />
Department of Histology <strong>and</strong> Embryology<br />
Karlovarska 48<br />
301 00 Plzen<br />
CZE<br />
Email: pitulep@seznam.cz<br />
Dr. Bali Pulendran<br />
Emory VaccineCenter at Yerkes<br />
Emory University<br />
954 Gatewood Road<br />
30329 Atlanta<br />
USA<br />
Email: pbackma@emory.edu<br />
Dr. Chiara Riganti<br />
Biochemistry Unit, Department of<br />
Genetics, Biology <strong>and</strong> Biochemistry<br />
University of Turin<br />
via Santena 5/bis<br />
10126 Turin<br />
ITA<br />
Email: chiara.riganti@unito.it<br />
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Dr. Sarah Payne<br />
Cell Technologies<br />
GE Healthcare<br />
Amersham Place<br />
HP7 9NA Little Chalfont<br />
GBR<br />
Email: sarah.payne@ge.com<br />
Dr. Bob Phillips<br />
IBBL<br />
IBBL<br />
6, rue Nicolas Barblé<br />
1210 Luxembourg<br />
LUX<br />
Email: marie-paule.hoffmann@ibbl.lu<br />
Dr. Yulia Pollak<br />
Pediatric Research <strong>and</strong> Electron<br />
Microscopy Unit<br />
Technion-Israel Institute of Technology<br />
Efron 1<br />
31096 Haifa<br />
ISR<br />
Email: mdyulia@tx.technion.ac.il<br />
Mr. Roel Quintens<br />
Radiobiology Unit<br />
SCK-CEN<br />
Boeretang 200<br />
B-2400 Mol<br />
BEL<br />
Email: roel.quintens@sckcen.be<br />
Miss Micaela Rocco<br />
Biochemistry, Department of Life<br />
Sciences<br />
Via Vivaldi 43<br />
81100 Caserta<br />
ITA<br />
Email: micaela.rocco@unina2.it<br />
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imba<br />
dr. bohrgasse 3<br />
1030 vienna<br />
AUT<br />
Email: josef.penninger@imba.oeaw.ac.at<br />
Dr. Jacques Piette<br />
Virology <strong>and</strong> Immunology, GIGA-R<br />
University of Liège<br />
Allée de l'Hopital 1, Bât B34<br />
4000 Liège<br />
BEL<br />
Email: jpiette@ulg.ac.be<br />
Pr. Alex<strong>and</strong>er Pukhalsky<br />
Department of Cystic Fibrosis, Research<br />
Centre for Medical Genetics<br />
1 Moskvorechie Street<br />
115478 Moscow<br />
RUS<br />
Email: osugariver@yahoo.com<br />
Miss Valentine Rech De Laval<br />
IBCP<br />
CNRS<br />
7 passage du Vercors<br />
69367 Lyon Cedex 07<br />
FRA<br />
Email: valentine.rech-de-laval@ibcp.fr<br />
Pr. Bernhard Ryffel<br />
Immunology<br />
CNRS<br />
3B ure de la Ferollerie<br />
45071 Orleans<br />
FRA<br />
Email: bryffel@cnrs-orleans.fr<br />
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Berthold Detection Systems<br />
new! ELISA Workstation<br />
new! Sirius L Tube Luminometer<br />
new! Zoom HT Microplate Washer<br />
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Berthold Detection Systems GmbH<br />
Bleichstraße 56–68<br />
75173 Pforzheim<br />
Phone: +49 (0)7231. 9206-0<br />
www.berthold-ds.com<br />
Luminometers<br />
Choose From the Widest Range of Luminometers<br />
for Research <strong>and</strong> Diagnostics<br />
· Single tube luminometers with up to 2 injectors<br />
· Microplate luminometers with up to 4 injectors<br />
· State-of-the-art PC software for Windows 7<br />
ELISA Workstation<br />
The Walk-away Solution for Immunoassays in Microplate Format<br />
· Combines 5 functions: washer, shaker, dispenser, incubator <strong>and</strong> reader<br />
· Walk-away system: microplate in, results out!<br />
· Ideal for low to medium size labs<br />
· Compact size: only 30 cm wide!<br />
Assay Automation<br />
A Range of Assay Processors for Reliable Start-to-finish Automation<br />
· Scalable stacker magazines<br />
· High processing speed <strong>and</strong> excellent accuracy<br />
· Walk-away functionality<br />
Microplate Washing<br />
Robust, Rapid <strong>and</strong> Easy-to-use Instruments for Automated Plate Washing<br />
· Intuitive protocol-based operation<br />
· Flexible add-on dispensers<br />
· From fast ELISA processing to gentle cell washing<br />
· Integrated stacker option<br />
Microplate Coating<br />
by<br />
The Economical Solution for Your Plate Coating Needs<br />
· Ideal for medium throughput (< 800 plates per day)<br />
· High precision <strong>and</strong> reproducibility<br />
· Robust <strong>and</strong> reliable<br />
by<br />
by
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