12th Congress of the European Hematology ... - Haematologica

0146
ARSENIC TRIOXIDE INDUCES ACCUMULATION OF CYTOTOXIC LEVELS OF CERAMIDE IN
ACUTE PROMYELOCYTIC LEUKEMIA AND ADULT T-CELL LEUKEMIA/LYMPHOMA CELLS
THROUGH DE NOVO CERAMIDE SYNTHESIS AND INHIBITION OF GLUCOSYLCERAMIDE
SYNTHASE ACTIVITY
A. Bazarbachi, 1 G. Dbaibo, 1 Y . Kfoury, 1 N. Darwiche, 1 S. Panjarian, 1
L. Kozhaya, 1 R. Nasr, 1 M. Abdallah, 1 O. Hermine, 2 M. El-Sabban, 1
H. De Thé 3
1 American University of Beirut, BEIRUT, Lebanon; 2 Necker Hospital, PARIS,
France; 3 UMR 7151 CNRS, PARIS, France
Background. Arsenic trioxide (As) is an effective treatment for acute
promyelocytic leukemia (APL) and potentially for HTLV-I associated
adult T cell leukemia/lymphoma (ATL). Many cytotoxic drugs induce
apoptosis through the generation and accumulation of the sphingolipid
breakdown product, ceramide, a coordinator of the cellular response to
stress. Aims. We investigated the contribution of ceramide to the mechanism
of action of As in APL and ATL. Results. Treatment of APL and ATL
derived cells with clinically achievable concentration of As induced accumulation
of cytotoxic levels of ceramide. Arsenic effects on ceramide levels
in APL cells were more potent compared to all trans retinoic acid
(ATRA) effects. We also show that As downregulated neutral sphingomyelinase
activity and that, in contrast to ATRA, As-induced ceramide
accumulation was not due to induction of acidic sphingomyelinase, but
rather resulted from both de novo ceramide synthesis and inhibition of
glucosylceramide synthase activity. Interestingly, As effects on de novo
ceramide synthesis were similar in APL and ATL derived cells despite the
defective pathway in ATL cells. Summary and Conclusions. These results
indicate that As-induced ceramide accumulation may represent a general
mediator of As effects, which paves the way for new therapeutic interventions
that target the metabolic pathway of this important sphingolipid
secondary messenger.
0147
INSULIN-LIKE GROWTH FACTOR II: A NOVEL AUTOCRINE GROWTH FACTOR IN THE A
POPTOSIS, PROLIFERATION, AND MATURATION OF ERYTHROID PROGENITORS IN
UMBILICAL CORD BLOOD
T. Nagatomo, 1 K. Muta2 1 2 Ehime prefectural Central Hospital, MATSUYAMA, Kyushu University,
FUKUOKA, Japan
Human umbilical cord blood (CB) contains a higher proportion of erythroblasts
compared with adult peripheral blood (PB). The increased
number of erythroid progenitor cells (EPCs) in CB could compensate for
intrauterine hypoxia. On the other hand, it remains unclear how the
abundant EPCs contribute to the fetal hematopoiesis by themselves. To
search the novel function of EPCs, CB-derived CD36 + EPCs were subjected
to cDNA microarray. It revealed 124-fold higher levels of insulinlike
growth factor II (IGF-II) gene expression in CB-EPCs than those in
stimulated lymphocytes of adult PB. Then, the following experiments
were conducted to determine whether IGF-II exerts as an autocrine erythropoietic
factor, which suggests a novel function of EPCs. Methods/
Results. The real-time PCR verified that IGF-II mRNA levels were highest
in CB-EPCs of all other CB- or adult PB-fractionated cells studied.
When CB-EPCs were cultured with erythropoietin (EPO) in serum-free
medium, the addition of anti-IGF-II-antibody (Ab) reduced the number
of erythroid colonies. To reveal the role of IGF-II in erythropoiesis via
an autocrine mechanism erythroid colony-forming cells (ECFCs) were
purified from CB and adult PB for further analysis. In brief, CB samples
were obtained from normal full-term deliveries. Mononuclear cells
(MNCs) were separated by density gradient centrifugation. After depletion
of adherent cells, nonadherent cells were collected and negative
selection was performed using anti-CD3, CD11b, CD15, and CD45RA
antibodies and immunomagnetic beads. The remaining cells were cultured
with FCS, human AB serum, stem cell factor (SCF), interleukin-3
(IL-3), and EPO (day0). Day3 ECFCs were collected and incubated under
serum-free condition without IL-3. Day7 ECFCs were collected and used
in the following experiments. When CB- and adult PB-ECFCs were cultured
with IL-3, SCF and EPO, the mRNA levels of IGF-II and type 1 or
type 2 IGF receptor increased with the maturation of both ECFCs. The
increasing rate of ECFC maturation by IGF-II was higher in CB-ECFCs
than in adult PB-ECFCs. Immunocytochemistry demonstrated IGF-II
protein in the majority of CB-ECFCs. The addition of anti-IGF-II Ab, but
not anti-IGF-I Ab, reduced the number of ECFCs in the liquid culture
with EPO. Anti-IGF-II Ab effectively inhibited the proliferation, and
accelerated the apoptosis of ECFCs, assessed by MTT and BrdU assays,
12 th Congress of the European Hematology Association
and flow cytometry. The addition of anti-IGF-II Ab reduced the proportion
of glycophorin-A+ cells in ECFCs, which showed a larger in size
with a less nuclear condensation, indicating immature erythroid cells.
Summary. The microarray analysis and quantitative PCR demonstrated
the high expressions of IGF-II mRNA in EPCs. The expression levels of
IGF-II, type 1 or 2 IGF receptor in the mature erythroid cells were higher
than those in the immature erythroid cells. We confirmed that IGF-II
is produced by EPCs by themselves, and has a crucial role in fetal erythropoiesis
by modulating the apoptosis, proliferation and maturation
in an autocrine fashion.
0148
BCL11B KNOCKDOWN LEADS TO APOPTOSIS OF MALIGNANT T-CELLS
K. Dittmann, 1 P. Grabarczyk, 2 K. Dittmann, 2 , G.K. Przybylski, 3
M. Depke, 2 U. Völker, 2 J. Bahr, 2 K. Assmus, 2 B. Bröcker, 2 R. Walther, 2
C.A. Schmidt2 1 Universität Greifswald, GREIFSWALD, Germany; 2 University of Greifswald,
GREIFSWALD, Germany; 3 Insitute of Human Genetics, POZNAN, Poland
Background. The B-cell CLL/lymphoma 11B gene (BCL11B) encodes a
Krüppel-like zinc finger protein which plays a crucial role in thymopoiesis
and has been associated with hematopoietic malignancies. A
tumor suppressor function of BCL11B has been hypothesized, but the
precise protein function has not yet been elucidated. Aims. To get more
insights in the biological function of BCL11B we analyzed the effects of
BCL11B knock down in Jurkat and HUT-78 T-cell lines, Raji B-cell line
and in normal T-lymphocytes. Methods. Upon BCL11B downregulation
using siRNA the DNA content was analyzed by propidium iodide incorporation
assay, apoptosis was determined by Annexin V binding assay
and Caspase 3 activation was measured by FACS. The genes regulated
by Bcl11b were screened by comparing the expression profile of the siR-
NA and mock transfected T-cell lines using the Affymetrix microarray
Genome U133 Plus 2.0. and subsequently confirmed by quantitative
real-time PCR. Results. We demonstrated that the survival of human Tcell
leukemia and lymphoma cell lines depends critically on Bcl11b. Suppression
of Bcl11b by RNA interference selectively induced apoptosis in
transformed T-cells while normal mature T-cells remained unaffected.
The apoptosis was executed by simultaneous activation of death receptor-mediated
and intrinsic apoptotic pathways, most likely as a result of
TRAIL up-regulation and suppression of the Bcl-xL antiapoptotic protein.
Our data indicate an antiapoptotic function of Bcl11b. The resistance of
normal mature T-lymphocytes to Bcl11b suppression-induced apoptosis
and restricted expression pattern make it an attractive therapeutic
target in T-cell malignancies. Summary and Conclusions. Our study provides
for the first time clear evidence for Bcl11b being an anti-apoptotic
protein in T-cell malignancies. The resistance of normal mature Tlymphocytes
to Bcl11b suppression-induced apoptosis and restricted
expression pattern make it an attractive therapeutic target in T-cell malignancies.
0149
P19 (CDKN2D) PLAYS AN IMPORTANT ROLE IN THE ENDOMITOTIC ARREST LINKED TO
THE ACCELERATION OF THE MEGAKARYOCYTE MATURATION
D. Bluteau, R. Guièze, L. Gendre, V. Cordette-Lagarde,
D. Baudry-Bluteau, R. Favier, N. Debili, W. Vainchenker, H. Raslova
Inserm U790, VILLEJUIF, France
Ploidization, which appears during normal megakaryocyte differentiation,
is linked to a modification in gene expression profile. One of the
genes which transcription is positively regulated during ploidization, is
a cell cycle inhibitor, p19(CDKN2D). p19, p18, p16 and p15 are members
of INK4 family of cyclin dependent kinase (CDK) inhibitors that
function in G1 to block the activity of CDKs 4 and 6. Except the role of
p19 in cell cycle, this protein can regulate neuronal and macrophage differentiation
and has been found to be expressed in erythroid cells. p19
acts also as a protector against apoptotic cell death. In this work, for the
first time we have evidenced the role of p19 in megakaryopoiesis. Here
we demonstrated that the expression level of p19 in megakaryocyte lineage
is 10-fold higher than in the erythroid lineage. Moreover, the
expression of p19 increases not only during ploidization after sorting
the MKs according to their DNA content but also during MK differentiation
(in diploid CD34 + CD41 + CD42 – , CD34 + CD41 + CD42 + and
CD34 – CD41 + CD42 + populations) both at the mRNA and protein level.
Transduction of CD34 + cells at day 1 and 2 of culture by two different
shRNAs p19 leads to a moderate increase (31.7±5%) in mean ploidy level
suggesting a role of p19 in the arrest of endomitosis as well as mito-
haematologica/the hematology journal | 2007; 92(s1) | 53