Haematologica 2003 - Supplements

locked the translocation of Ras to the plasma membrane, Ras
activation and ERK1,2 phosphorylation in both cell lines and also
in the INA-6/BMSC co-culture model. Treatment with FTI
induced apoptosis of MM cells (cell lines and primary cells) and
was equally effective in the presence or absence of BMSCs.
Conversely, the human Burkitt lymphoma cell line Namalwa,
which lacks constitutive ERK1,2 activity, was resistant against
FTI-induced apoptosis.
Conclusions: Treatment of MM cells with FPT III blocks the
Ras/MAPK pathway and induces apoptosis in the absence and in
the presence of BMSCs. Our experiments support the hypothesis
that the sensitivity of MM cells to FTIs depends on the activation
status of the Ras/MAPK pathway rather than on the mutation
status of Ras. These findings underscore the potential of FTIs for
the development of novel therapeutic strategies for the treatment
of MM.
383
Effect of farnesyl transferase inhibitor R115777 alone
or in combination with incadronate on the growth of
fresh and cloned myeloma cells in vitro
Naoya Ochiai, Ryo Uchida, Shin-ichi Fuchida, Akira Okano,
Masashi Okamoto, Hideyo Hirai, Eishi Ashihara, Tohru
Inaba, Naohisa Fujita, Masao Nakagawa and Chihiro
Shimazaki
Second Department of Medicine, Kyoto Prefectural University of
Medicine, Kyoto, Japan
Multiple myeloma (MM) is a malignancy of B cells characterized
by the monoclonal proliferation of malignant plasma cells.
Several chemotherapeutic regimens and high-dose chemotherapy
supported by autologous stem cell transplantation (auto-SCT)
have been employed to improve the survival of patients with MM
but many patients relapsed for a long time follow up period.
Therefore there is an obvious need for new therapeutic strategies.
Ras gene mutations occur in 30% to 40% of multiple myeloma
(MM), and farnesylation is the first step in the posttranslational
modification of Ras proteins. Zarnestra TM (R115777) is one of
the potent farnesyl transferase inhibitors (FTIs), but is less
effective to K-Ras mutation, which is one of the most common
mutations in MM, than N- and H-Ras mutations. It has recently
been reported that bisphosphonates have direct antitumor effect in
vitro and in vivo, and nitrogen-containing bisphosphonates, such
as incadronate, inhibit the mevalonate pathway and prevent posttranslational
prenylation of GTP-binding proteins. Mevalonate
pathway is essential for the biosynthesis of sterols and long chain
isoprenoid lipids, including farnesylpyrophosphate (FPP) and
geranylgeranylpylophosphate (GGPP) which are substrates for
the FTase and geranylgelanyl transferase (GGTase) 30. Based on
these observations, we examined the effect of R115777 alone or
in combination with incadronate on the growth of fresh and
cloned myeloma cells in vitro. R115777 inhibited the growth of
fresh and cloned myeloma cells dose-dependently. Flow
cytometric analysis using annexin V and 7AAD showed that
R115777 induced apoptosis of two of three myeloma cell lines at
the concentration of 1.0×10-8 mol/L. The inhibition of cell
growth was intensified when R115777 was combined with
incadronate at 1.0×10-6 mol/L and 1.0×10-5 mol/L. R115777
appears to be a potent inducer of apoptosis in fresh and cloned
myeloma cells, and the effect was intensified in combination with
incadronate. R115777 alone or in combination with incadronate
might have some benefit in the treatment of myeloma patients.
384
MOLECULAR CHARACTERISTICS OF APOPTOSIS
INDUCED BY THE FARNESYLTRANSFERASE
INHIBITOR (FTI) BMS-214662 IN HUMAN MYELOMA
CELLS
María Gómez-Benito, Pilar Giraldo, Daniel Rubio-Félix,
Alberto Anel, Javier Naval & Isabel Marzo
Departamento de Bioquimica y Biologia Molecular y Celular,
Universidad de Zaragoza, and Servicio de Hematologia, Hospital
Miguel Servet Zaragoza (Spain).
We have studied the molecular mechanism of apoptosis induced
in vitro, by the FTI BMS-214662, in several human multiple
myeloma (MM) cell lines (RPMI-8226, NCI-H929, U266 and
IM-9). In MM cells treated with BMS-214662 farnesylation of
cytosolic chaperone HDJ-2 and of lamin A was clearly observed.
BMS-214662 treatment induced the typical morphology of
apoptosis, including condensation and fragmentation of
chromatin, in all MM cell lines at low doses (0.3-1 µM) after 24 h
incubation. Cell death was not prevented by co-treatment with the
protein synthesis inhibitor cycloheximide. Apoptosis was
accompanied by cell shrinking, phosphatidylserine exposure in
plasma membrane, loss of mitochondrial membrane potential
(m), activation of caspases 9 and 3 and traslocation of
Apoptosis-Inducing Factor (AIF) from mitochondria to nucleus.
Cotreatment of cells with the pan-caspase inhibitor Z-VAD-fmk
prevented development of apoptotic morphology and death in
RPMI8226 and U266 cells. Z-VAD-fmk attenuated some
morphological and biochemical characteristics of apoptosis in
NCI-H929, but did not prevent cell death. Selective inhibitors of
caspase-3 or caspase-9 did not block cell death in any cell line
tested. Levels of antiapoptotic proteins Bcl-2 and Mcl-1
decreased in IM-9, RPMI-8226 and U266 cells upon treatment
with BMS-214662. This FTI caused a concomitant increase in
levels of pro-apoptotic BH3-only proteins Bik and Bim during
treatment. Multidomain, proapoptotic Bax protein levels also
increased in U266 treated with BMS-214662. These results
support the potential usefulness of FTIs as new therapeutic agents
for the therapy of MM.
385
Rituximab-mediated signaling and sensitization of
8226/S and 8226/Dox40 MM cell lines to paclitaxelmediated
apoptosis.
Ali Jazirehi, Gary Schiller, Alan Lichtenstein and Benjamin
Bonavida.
Departments of Microbiology, Immunology and Molecular
Genetics, and Medicine, University of California at Los Angeles,
CA 90095, USA.
Rituximab is a chimeric mouse anti-human CD20 monoclonal
Antibody that has been approved by the FDA for the treatment of
patients with low grade and follicular NHL. The response rate is
approximately 50%. Following initial treatment, patients with
MM develop chemo-resistance. Thus, there is a need for
alternative therapeutic approaches. Previous findings from our
laboratory have demonstrated that Rituximab sensitizes NHL cell
lines to various drug-mediated apoptosis (Clinical Cancer
Research, 7: 709, 2001, Cancer Research, 61: 5137, 2001). The
objective of this study was to investigate whether Rituximab has
an effect on MM. We have used the drug sensitive 8226/S
parental and the doxorubicin-resistant variant 8226/Dox40 cell
lines as a model system. A fraction of the cells (>20%) expressed
CD20 as determined by flow cytometry. Treatment of the 8226/S
and 8226/Dox40 cells with Rituximab (20g/ml-24h) resulted in
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