Haematologica 2003 - Supplements

inhibition of phosphorylation of MAPK/ERK1,2, or Akt, in either
the presence or absence of BMSCs.
Conclusions: These experiments show that the apoptosis-inducing
effect of AG490 is not mediated through inhibition of the STAT3
pathway or other pathways commonly associated with the
malignant growth of MM. Thus, the precise mechanism of action
of AG490 in MM cells remains elusive. Although our data
indicates that tyrphostins are of potential interest for the treatment
of MM, adverse side effects - in particular on the hematopoietic
system - may be expected.
125
Bone morphogenetic protein (BMP) family members
BMP-4, -5, -6 and 7 induce apoptosis in human
myeloma cells.
Torstein Baade Ro, Anne-Tove Brenne, Magne Rekvig,
Anders Waage, Magne Borset and Anders Sundan
Dept. of Cancer Research and Molecular Medicine, Faculty of
Medicine, Norwegian University of Science and Technology,
Trondheim, Norway
BMPs are members of the TGFβ superfamily, originally
identified as molecules capable of inducing bone and cartilage
formation. More recent knowledge shows that BMPs regulate a
broad spectrum of biological responses like proliferation,
differentiation and apoptosis in a wide variety of cell types,
including hematopoietic cells. Recently, we have discovered that
several members of the bone morphogenetic protein (BMP)
family, BMP-4,-5,-6 and -7, are able to induce apoptosis in
myeloma cell lines as well as in primary myeloma cells.
We found a dose-dependent induction of apoptosis in the
majority of cell lines and primary myeloma cell samples
examined. BMPs reduced viability in more than 70% of purified
primary myeloma cell samples. Myeloma cells resistant to one
type of BMP (e.g. BMP-4) were still sensitive to other BMPs
(e.g. BMP-6). In a panel of 5 different myeloma cell lines this
diversity was explained by different expression patterns of BMP
receptors. The induction of apoptosis seemed dependent on
synthesis of new proteins, as the signs of apoptosis appeared
relatively late compared to FAS- and TRAIL- receptor-induced
apoptosis (24-48 vs. 1-6 hours), and as the induction of apoptosis
was inhibited by cycloheximide. Myeloma cells were protected
from apoptosis by a general caspase inhibitor, indicating
involvement of caspases in the development of apoptosis. The
intracellular mechanisms of BMP- induced apoptosis in myeloma
cells are under futher investigation.
BMP-induced apoptosis was influenced by interaction with other
factors. The BMP antagonist noggin was able to abolish BMP-4-
induced apoptosis, but it did not influence BMP-6 or -7-induced
apoptosis. Whereas growth induced by IL-6, IL-21, TNF or IGF-
1 were almost completely inhibited by BMPs, IL-15 was able to
partially counteract the growth-inhibiting effect of BMPs.
Heparin in doses of 10-100 µg/ml was able to fully abolish BMP-
5 and BMP-6-induced apoptosis, and partially inhibit BMP-4 and
BMP-7-induced apoptosis. Thus, interaction between BMPs and
heparan sulfate chains of syndecan-1 may be of importance in
vivo.
BMPs have intriguing anti-tumor effects in vitro. BMP-7 is
currently in use as local treatment against fracture non-unions.
Intravenously, BMP-7 has shown promising results as an agent
reducing ischemic damage following stroke in rat brains. It is
possible that therapeutic use of BMP or BMP analogues could
have impact on both myeloma bone disease and myeloma cell
growth.
126
Both mitochondrial (Type 2) and non-mitochondrial
(Type 1) apoptotic pathways are activated in authentic
multiple myeloma cells by Apo2L/TRAIL.
Sung Lin Yeh, Karly Koutrouvelis, Cindy Baulch-Brown,
Andrew Spencer
The Alfred Hospital
TNF-alpha related apoptosis inducing ligand (TRAIL/Apo2L) is
a member of the TNF family and has unique potential as an antitumour
agent. We have demonstrated previously that TRAIL
induces apoptosis of primary multiple myeloma (MM) tumour
cells from MM patients but does not appear to be harmful to
normal haemopoietic cells. Furthermore, we have demonstrated
that one or both of the effector receptors for TRAIL (R1 and R2)
is present on 90% of primary MM tumour populations and that
TRAIL resistance is not due to expression of TRAIL decoy
receptors (R3 and R4). To develop a rational approach to
overcoming TRAIL resistance and the development of synergistic
drug combinations with anti-MM potential we have endeavoured
to define the mechanism(s) by which TRAIL induces apoptosis.
Five authentic MM cell lines expressing both R1 and R2 were
used as targets for TRAIL evaluation. At 1 hour post-TRAIL
treatment 3 lines OPM-2, RPMI 8226 and LP-1 demonstrated
sensitivity to TRAIL with 39%, 37% and 34% apoptosis,
respectively, whereas NCI H929 and U266 were resistant with
only 8% and 5% apoptosis, respectively. The 3 sensitive lines all
demonstrated efficient cleavage of Pro-caspase 8 within 15
minutes of TRAIL treatment but this was delayed and reduced in
the resistant lines. All 5 lines exhibited similar cytosolic
accumulation of both Cytochrome C and SMAC confirming
activation of the Type 2 pathway. To clarify the relative
contributions of the Type 2 pathway it was inhibited by preincubation
with a Caspase 9 inhibitor and the effect on TRAILinduced
apoptosis determined. This revealed a negative
correlation between uninhibited TRAIL-induced apoptosis and
the effect of Type 2 pathway inhibition with a reduction in
apoptosis of 7.1%, 20.3%, 48.8% and 64.3% for the lines
RPMI8226, LP-1, NCI H929 and U266, respectively. The latter is
consistent with a greater reliance on the Type 2 pathway in MM
cells with low TRAIL-induced Procaspase 8 activation efficiency.
In contrast, there is significant redundancy in cells with efficient
TRAIL-induced Procaspase 8 activation. Based on this we
hypothesised that enhancing Type 2 pathway activation in cells
relatively resistant to TRAIL may enhance TRAIL-induced
apoptosis. Preliminary experiments with NCI H929 have
demonstrated significant synergy between TRAIL and
dexamethasone consistent with this hypothesis. We conclude that
both mitochondrial and non-mitochondrial apoptotic pathways
are activated in MM cells by TRAIL and this observation
provides a basis for a rational approach for the evaluation of
synergistic anti-MM drug combinations with TRAIL.
127
Both CD45 and PTEN phosphatase expression are
critical for Akt/PI-3Kinase signaling in Multiple
myeloma
Géraldine Descamps, Régis Bataille and Martine Amiot
INSERM U463 Institut de Biologie44035 Nantes
In Multiple Myeloma, the Akt/PI-3 Kinase pathway is involved in the
proliferation of myeloma cells. In the current study, we have
investigated the mechanisms that control Akt/PI 3-kinase activation.
We show that this activation is restricted to IGF-1 but not to IL-6
stimulation. Its level in response to IGF-1 is highly variable from one
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