Haematologica 2003 - Supplements

5. Signal transduction pathways and
cytokine networks
5.1 Surface reception.
108
ATYPICAL RECEPTOR INTERACTIONS IN MYELOMA
CELLS: USE OF RNA INTERFERENCE (RNAi) TO
INVESTIGATE FUNCTIONAL SIGNIFICANCE
Denise K. Walters and Diane F. Jelinek
Dept. of Immunol. and the Tumor Biology Graduate Program,
Mayo Graduate School, Mayo Clinic, Rochester, MN, USA, 55905.
Our laboratory has had a long-standing interest in the growth
regulation of myeloma cells. Furthermore, we have had a
particular interest in multiple myeloma (MM) cell responsiveness
to interferon-alpha (IFN-α because of its ability to modulate
myeloma cell growth in a variable manner. Thus, IFN-α
typically results in growth inhibition of myeloma cells, however
some MM cells, such as the KAS-6/1 MM cell line, display an
atypical growth response. Moreover, during the course of other
studies, we serendipitously and unexpectedly discovered that the
IFN-α growth responsive KAS-6/1 MM cell line expresses
ErbB3, whereas MM cell lines that are growth arrested by IFN-
do not. In this regard, there is a growing awareness of the
complications introduced by unexpected receptor interactions that
result in synergistic down-stream cellular effects, e.g., enhanced
growth or resistance to apoptosis of cancer cells. We
hypothesized, therefore, that ErbB3 expression may play a role in
the atypical IFN-KAS-6/1 response. Indeed, we were initially
able to demonstrate that IFN-α stimulation resulted not only in
the activation of the IFN-α receptor, but also in the
transactivation of ErbB3, as revealed by tyrosine
phosphorylation. To address the significance of these
observations, we employed two strategies. First, we transfected
ErbB3 negative DP-6 MM cells with ErbB3 and analyzed
whether IFN-α stimulation could transactivate ectopically
expressed ErbB3. Although IFN-α transactivated ErbB3 in the
DP-6 transfectants, it did not confer growth responsiveness to
IFN-α. Second, we wished to use RNA inhibition (RNAi) to
assess the functional significance of atypical expression of ErbB3
in the KAS-6/1 cells. RNA interference (RNAi) is a recently
discovered natural response to double-stranded RNA (dsRNA)
that can result in sequence specific gene silencing. In ancillary
studies aimed at adapting this methodology to myeloma cells, we
demonstrated the striking dependence of dsRNA-mediated gene
silencing in some cells on the methods of dsRNA transfection.
Thus, we were able to optimize the inhibitory effects of ErbB3
siRNA in the KAS-6/1 cells by utilizing electroporation, rather
than conventional transfection agents, to transfect the cells.
Using Western blot analysis, we were able to demonstrate that
ErbB3 siRNA treatment resulted in the loss of this receptor from
the KAS-6/1 cells. Of great interest, silencing ErbB3 expression
in KAS-6/1 cells decreased the overall growth response to both
IFN-α and IL-6. Taken together these two lines of investigation
suggest that ErbB3 expression alone does not uniquely confer
IFN-α growth responsiveness but instead, may amplify
proliferation rates in MM cells that have acquired atypical
expression of this receptor. In conclusion, we believe that novel
receptor interactions have the potential to allow dysregulated
tumor cell function. In this regard, atypical expression of ErbB3
in multiple myeloma may provide a mechanism whereby the
tumor cell is afforded with either an ability to grow or to survive.
We are currently investigating this possibility, along with
analyzing primary myeloma cells for ErbB3 expression.
109
DOWN-MODULATION OF ERK PROTEIN KINASE
ACTIVITY SUPPRESSES HUMAN MYELOMA CELL
PROLIFERATION AND MYELOMA-INDUCED
ANGIOGENESIS BY VEGF INHIBITION
Nicola Giuliani2, Paolo Lunghi1, Francesca Morandi,
Magda Hojden2, Sabrina Bonomini2, Simona Colla2,
Giovanni Roti2 , Regis Bataille3, Vittorio Rizzoli2, Antonio
Bonati1
1Insitute of Medical Pathology and 2Hemaology, University of
Parma, Italy, 3INSERM U463, Nantes, France.
The mitogen-activated protein (MAP) cascade leading to the
activation of ERK (Extracellular signal-regulated kinase) is
critical for regulating myeloma cell growth, however the
relationships of ERK1/2 protein kinases activity with vascular
endothelial growth factor (VEGF) production and the effects of
their down-modulation in myeloma cells are not completely
investigated. In order to elucidate this issue we first studied the
expression and the activation of ERK1/2 protein kinases in
RPMI-8226, XG6, U266 and OPM-2 human myeloma cell lines
(HMCL) (5x106) growing in 5ml of complete RPMI medium
with or without IL-6 (20ng/ml). By immunoenzymatic MAP
kinase assay using Elk1 as substrate (Elk1-p) or dualphosphorylation-ERK1/2
specific antisera (p-ERK1/2) we found
that steady-state levels of phosphorylated ERK 1/2 (p-ERK) were
stronger in U266 than RPMI, and XG6. The total amounts of
ERK1 and ERK2 proteins were comparable in U266, XG6 and
OPM-2 whereas in RPMI the expression of ERK1 was
significantly higher. Basal VEGF secretion, detected by ELISA
was similar in OPM-2 (Mean±SD: 835±10 pg/ml), XG-6 (668±15
pg/ml), U266 (739±12 pg/ml) and higher in RPMI-8226 (1381.6±
20pg/ml). IL-6 stimulation significantly increased ERK1/2
activity and VEGF secretion in XG-6, U266, RPMI-8226.
Therefore, we used the specific inhibitor of MEK, PD98059, in
order to down-modulate activated ERK1/2 (p-ERK1/2). The
treatment for 2 hours of myeloma cells with 40mM of MEK-1
inhibitor PD98059 produced a reduction of p-ERK1/2 levels of
more than 80% and prevented the increase of p-ERK1/2 induced
by IL-6 treatment. PD98059 induced a significant inhibition of
HMCL proliferation (-33±5%) and blunted the stimulatory effect
induced by IL-6. A more potent inhibition on HMCL
proliferation was observed with the ERK inhibitor PD184352 at
2mM (-70%±4%). PD184352 but not PD98059 induced HMCL
apoptosis in combination with anti CD95 mAb or Arsenic
Trioxide 2m. A significant dose and time dependent inhibition
on basal VEGF secretion by HMCL was induced by PD98059
treatment, more evident in RPMI-8226 (771±12 vs 1350±18
pg/ml, -43%; p