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

Results. Though there was no recognizable decrease of cell number in
KG-1 through broad range of 17-AAG concentrations (0.02 to 2.5 uM),
dose-dependent decrease of cell number was prominent in K562. Cell
death in KG-1 and K562 was mainly mediated by apoptosis and the pattern
of annexin V/PI staining at 72 hours, not 24 and 48 hours, was consistent
with the degree of decrease in cell proliferation measured at 96
hours. AKT and p-AKT level in K562 were reversely correlated with the
concentration of 17-AAG, but not in KG-1. To verify the role of AKT
in KG-1 which was not sensitive to 17-AAG, cells were cultured with
PI3K inhibitor LY294002 or ERK1/2 inhibitor PD98059. Not PD98059
but LY294002 caused severe apoptotic cell death accompanied with the
decrease of AKT and p-AKT. Summary/conclusions. HSP 90 inhibitor 17-
AAG affected on apoptotic cell death and AKT according to the type
and origin of leukemic cells differently and treatment of leukemia with
HSP 90 inhibitor should be selectively applied with caution. Inhibition
of AKT could be a main target when leukemia is not sensitive to HSP
90 inhibitor.
1358
ANALYSIS OF GENE EXPRESSION PATTERN IN BONE MARROW AND LIVER OF MICE
DEVELOPED MYELOPROLIFERATIVE DISEASE AFTER LONG-TERM G-CSF TREATMENT
A. Bigildeev, A.L. Grishchuk, N.V. Saz, I.N. Nifontova,
D.A. Svinareva, N.J. Drize
National Hematology Research Centre, MOSCOW, Russian Federation
Background. At the present time there is a strong need for animal models
to study the mechanisms of leukemogenesis. We obtained transplantable
leukemia after treatment of (CBAxC57Bl6) F1 12-16 week-old
mice with low (25 mcg/kg) G-CSF doses. G-CSF was injected 4 days
successively once a month. MPD-like myeloid leukemia with histiocytic
sarcoma occurred in one out of 20 mice after 3d G-CSF course. Bone
marrow and liver cells of the mouse are fully transplantable, recipients
become moribund within 17-32 days since cells injection. All ill animals
had enlarged liver (M 4,1±0,1 g versus normal 1,44±0,03 g) and spleen
(M 304,9±15,5 mg versus normal 93,1±1,8 mg). The developed
leukemia was not of virus origin that was proved by three independent
methods. Aims. To analyze gene expression pattern in leukemic
mice for elucidation of mechanisms that have led to the development
of leukemia. Methods. Subtracted cDNA library of differentially
expressed sequences was constructed from bone marrow cells of normal
and leukemic mice using Suppression Subtractive Hybridization
(SSH). Leukemia- and normal-specific cDNAs were sequenced and associated
with known genes. Expression level of several leukemia-specific
genes was further studied by RT (reverse transcription)-PCR in bone
marrow and liver of leukemic mice. Results. 3 out of 48 leukemia-specific
sequences turned out to be subunits of ATP synthase, each of the
following up-regulated genes: IL1-r2 (CD121b), CathK and ARF-BP1,
ATPase subunits of 26S protease was identified twice. Up-regulated
expression of these genes was proved by RT-PCR. Clinically ill mice
showed a moderate extent of anemia and reticulocytosis accompanied
by extensive suppression of ‚-globin expression (8 out of 10 down-regulated
genes). The expression level of c-Abl and G-CSF doubled in bone
marrow of leukemic mice compared to the normal bone marrow. The
expression level of genes regulating cell proliferation did not change
dramatically-only C-Myc expression had increased 3-fold, however
concentration of early hematopoietic precursor cells (LTC-IC) had
decreased about 5-fold (0,75 versus 3.32 per 105 cells in healthy mice).
The pronounced changes had been revealed in expression of Mpo gene
(3,4-fold increase). The liver of ill mice consisted of undifferentiated
cells. Increasing of CD45 expression up to 11-fold simultaneously with
substitution of liver parenchyma by tumor cells points to hematopoietic
origin of invading cells. Oligopotent hematopoietic precursor cells
(CFU-C) were also revealed in affected liver (52,5±7,7 per 105
cells).There were mild changes in G-CSF expression in the liver cells of
leukemic mice, whereas the expression of Csf3r (CD114) increased 18fold
compared to the normal liver cells. The expression of Csf1r
(CD115) increased 20-fold, fibronectin and granulin increased 5-fold
and Ly6E increased 3-fold compared to the leukemic bone marrow indicating
highly malignant phenotype of cells invading liver. The expression
of antiapoptotic genes was elevated up to 4-fold for bcl-2 and 2fold
for cIAP2. Summary/Conclusions. The G-CSF treatment may lead to
the development of myeloid leukemia with high ability to invade liver
tissue and dramatically changed gene expression pattern.
12 th Congress of the European Hematology Association
1359
APOPTOSIS REGULATION AND THE ROLE OF VEGF IN B-CELL CHRONIC LYMPHOCYTIC
LEUKEMIA
B. Power, 1 J. Harmey, 1 P. Murphy, 2
1 2 Royal College of Surgeons in Ireland, DUBLIN, Ireland; Beaumont Hospital,
DUBLIN, Ireland
B-cell chronic lymphocytic leukaemia (CLL) is the most common
leukaemia in the western world with an incidence of 3.36/100,000 in
European males. It is characterised by a clonal growth of long lived,
slowly proliferating mature B lymphoid cells in the bone marrow (BM),
peripheral blood (PB) and lymphoid tissues. CLL has an extremely variable
prognosis at presentation: a third of cases require chemotherapy
from the start, a third of cases have an extremely indolent course and
never require active therapy and a third of cases progress over time and
eventually require some form of chemotherapy. Although CLL generally
responds to initial chemotherapy, it remains an incurable disease.
B-CLL cells have a long survival owing to alterations in the normal pathways
of apoptosis. It has been shown that B-CLL cells rapidly undergo
apoptosis during in-vitro culture indicating that signals from the microenvironment
are of vital importance in maintaining resistance to apoptosis.
Recent studies indicate that vascular endothelial growth factor
(VEGF) and it’s receptors may have an important role to play in CLL cell
survival. The cytokine IL-4 is also known to have a pro-survival role for
CLL cells. The aim of our study was to analyse survival of purified CLL
cells cultured in serum free and serum supplemented conditions with
the addition of VEGF and IL-4 and also analyse VEGF patient plasma
levels. Survival of purified CLL cells cultured in serum free and serum
supplemented conditions was assessed by Annexin V/Propidium Iodide
staining followed by flow cytometry analysis. Patient plasma VEGF
levels were assessed by ELISA. Informed consent was obtained from
patients. Addition of VEGF to serum free media did not result in
increased cell viability whereas IL-4 consistently increased cell viability.
Addition of VEGF to serum supplemented media resulted in a significant
increase in cell viability in a subset of patients (6 out of 17).
Analysis of VEGF levels in patient plasma samples (23) showed a correlation
between increased levels of VEGF and high white blood
cell/lymphocyte counts (p