Haematologica 2003 - Supplements

integrin in IGF-1-triggered MM cell adhesion and migration. A
functional blocking antibody against β1 integrin and a RGD
peptide, as well as cytochalasin D (cyt D: a cytoskeleton
inhibitory agent), significantly reduced IGF-1-induced cell
adhesion. Immunoprecipitation studies demonstrated that IGF-1
rapidly and transiently induces association of IGF-1R and 1
integrin, which is associated with phosphorylation of IGF-1R,
IRS-1, and p85/PI3K. IGF-1 also triggers phosphorylation of
AKT and ERK. We next demonstrated that IGF-1R and 1
integrin comigrate to cholesterol-rich microdomains on plasma
membrane (membrane rafts) following IGF-1 stimulation, using
western blotting for the Triton-insoluble and Triton-soluble
fraction of the cells and dual immunofluorescence staining. Using
methyl-β-cyclodextrin to disrupt membrane raft structure, we
further show that β1 integrin-mediated IGF-1-stimulated
adhesion requires intact lipid rafts. In addition, IGF-1 (100 ng/ml
and 40 ng/ml for MM.1S and OPM6, respectively) triggers
polymerization of F-actin, indicating focal adhesion formation,
and immunoprecipitation experiments confirm that IGF-1 induces
interaction of β1 integrin with cytoskeletal and signaling proteins
localized at focal adhesions, including focal adhesion kinase
(p125FAK), α-actinin, and paxillin. Pretreatment of cyt D
abrogates activation of p125FAK and paxillin induced by IGF-1.
Importantly, IGF-1-induced MM cell adhesion to FN is achieved
only when 1 integrin and PI3K/AKT are activated. In a 96-well
transmigration assay, IGF-1 induces a 2-3 (MM.1S) and 3-4-fold
(OPM6) increase in migration. Conversely, IR3 and a blocking
anti-β1 integrin mAb, as well as Cyt D, abrogate MM cell
transmigration. Interestingly, IGF-1 induces MM cell migration
independent of de no vo protein synthesis. Finally, primary
CD138+ MM cells isolated from 3 patients were also responsive
to IGF-1-induced adhesion on FN-coated plates and BM stromal
cells. Together, these studies demonstrate that IGF-1 induces MM
cell adhesion and migration, suggesting a role of IGF-1 in the
trafficking and localization of these cells in the BM
microenvironment. Morover, these results identify a functional
cooperation between IGF-1R and β1 integrin in MM homing,
supporting blockade of IGF-1/IGF1R system as a novel treatment
strategy of MM.
113
HHV-8 viral IL-6 homologue is as active as human IL-6
on human myeloma cells
Andreas Günther1, Frank Bakker1, Wolfgang Baum1,
Renate Burger1, Frank Neipel2 and Martin Gramatzki1
1Department of Medicine III / Div. of Hematology/Oncology and
2Institute for Clinical and Molecular Virology, University of
Erlangen, Germany
Kaposi-Sarcoma-associated Virus/ Human-Herpesvirus-8
(KSHV/HHV-8) is associated with at least three human
lymphoproliferative disorders: primary effusion lymphomas
(PEL), Castleman’s disease (CD) and plasmablastic lymphoma.
However, the role of HHV-8 in multiple myeloma (MM) is still
controversial. Some groups detected HHV-8-DNA in bone
marrow samples of MM patients whereas other groups failed to
confirm these data. An important issue is the potential functional
role of HHV-8 which encodes for several viral cytokines. Most
interest was given to the viral homologue of IL-6 (vIL-6) since
human IL-6 (huIL-6) is not only an essential growth and survival
factor for malignant plasma cells but plays an important role in
PEL, CD and plasmablastic lymphoma. We could show that
prokaryotically expressed vIL-6 (pro-v-IL-6) has biological
activity on human myeloma cells in vitro (Burger et al., Blood
1998). However, the vIL-6 concentrations needed for a maximum
stimulatory growth effect on the strictly IL-6 dependent human
myeloma cell line INA-6 were dramatically higher (4000 ng/ml)
than those needed for recombinant huIL-6 (1.25 ng/ml) This
requirement for very high amounts of vIL-6 argued against a
causative role of vIL-6 in human diseases. However, this study
was performed with prokaryotically expressed recombinant vIL-6
which could be less active than vIL-6 expressed in human cells.
Now, we could overcome these limitations. First we used
conditioned medium (CM) of the HHV-8 infected PEL cell line
BCBL-1, containing only small amounts of hu IL-6 (45 pg/ml).
The addition of 50% CM was able to induce a proliferative
response of the INA-6 plasma cell line which would have
required 1 ng/ml of recombinant huIL-6. Anti-gp130 antibody
(Ab) or the combination of anti-IL-6 receptor and anti-gp130
Abs, but not anti-IL-6R Ab alone, were effective in inhibiting
BCBL-1 CM activity on INA-6. These results are similar to those
obtained with vIL-6 and indicate that the growth induction of
BCBL-1 CM is mainly caused by vIL-6. In a second approach we
used a baculovirus vector expression system in eukaryotic cells.
Eukaryotically expressed vIL-6 (eu-v-IL-6) had a significantly
higher activity than pro-v-IL-6 and induced a maximum
stimulatory growth response in INA-6 cells at the same
concentration as huIL-6 (1.25 ng/ml). Similar to the results
published previously for pro-vIL-6 and obtained for BCBL-1
CM. The growth effects induced by eu-v-IL-6 could be blocked
by anti-gp 130 Ab or a combination of anti-IL-6R and anti-gp130
Abs, but not anti-IL-6R Ab alone. Thus, our data indicate that
the HHV-8 viral homologue of IL-6 is as active on human cells as
hu IL-6. HHV-8 vIL-6 may therefore act as a substitute for the
human cytokine in multiple myeloma and HHV-8 associated
diseases and could play a causative role in their development.
114
Macrophage inflammatory protein 1-alpha (MIP-1α)
triggers migration and signaling cascades mediating
survival and proliferation in multiple myeloma cells
Suzanne Lentzsch, Margarete Gries, Martin Janz, Ralf
Bargou, Bernd Dorken, and Markus Y. Mapara.
Humboldt University of Berlin, Charite Campus Buch, Robert-
Roessle-Klinik, 13125 Berlin, Germany, Tel.: +49-30-94171370,
FAX:+49-30-94171209, e-mail: lentzsch@rrk-berlin.de
Recently, several studies have suggested that MIP-1 contributes
to the pathophysiology of multiple myeloma (MM). Thus, it has
been demonstrated that MIP-1α is an osteoclast stimulatory
factor and that MIP-1α neutralizing antibody (ab) or antisense
inhibit bone destruction and reduce tumor load in a SCID-mice
model of MM. Furthermore, MM patients have significantly
higher bone marrow plasma levels of MIP-1α than healthy
controls.
The current study was designed to determine the direct effects of
MIP-1α on MM cells. We found expression of very high levels of
MIP-1α and its receptor CCR5 in MM cell lines and patientderived
primary cells. Furthermore, we were able to demonstrate
that MIP-1α significantly increased proliferation of different MM
cell lines (MM1.S, H929, OPM-2, INA-6) and patient-derived
primary MM cells. MIP-1α specific migration could be observed
in a dose-dependent fashion up to 21 fold compared to control. In
MM cell line and patient MM cells, MIP-1α induces
phosphorylation of p44/42 mitogen-activated protein kinase
(MAPK) as well as AKT and its downstream target FKHR.
STAT3 was not activated by MIP-1α. In addition, inhibition of
AKT activation by the PI3-Kinase (PI3-K) inhibitors wortmannin
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