Haematologica 2003 - Supplements

morphologically or using Annexin V/PI staining. For induction of
ER stress, Thapsigargin (Tg) was utilized. Activation of XBP–1 and
cleavage of caspase–12 in two MM cell lines, U266 and KMS–12–
PE, were also analyzed using western blot.
Results: Activation of XBP–1 was present in 25 fresh MM cases,
suggesting the existence of ER stress in vivo. Tg stimulation
caused significant apoptosis in purified MM cells but not in
normal peripheral blood mononuclear cells. The Tg-induced
apoptosis in MM cell lines accompanied XBP–1 activation and
caspase–12 cleavage. When MM cells were placed under an
overgrowth state, apoptosis also occurred with caspase–12
cleavage, suggesting that ER stress is inducible by either
exogenous stimulation or nutrition deficiency.
Conclusion: The present results suggest that MM cells undergo
apoptosis through ER stress. Induction of apoptosis via ER stress
could open a new avenue for the development of therapy of MM.
This work is supported by International Myeloma Research
Foundation.
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CD28 Mediates Multiple Myeloma Cell Proliferation and
Survival: Role of Cellular Interaction with Dendritic
Cells
Nizar J. Bahlis, Anne M. King, Louise M. Carlson, Despina
Kolonias, Lawrence H. Boise, Kelvin P. Lee
Department of Microbiology and Immunology, and the Sylvester
Comprehensive Cancer Center, University of Miami
The interactions between the malignant plasma cells of multiple
myeloma (MM) and the bone marrow stroma are essential for
myeloma cell proliferation and survival. Therefore these interactions
critically influence the response or resistance to treatment, and thus
represent attractive therapeutic targets. However, within the complex
bone marrow microenvironment it is largely unknown which specific
cellular elements are involved in these interactions. One approach to
this question is to identify cell surface receptors that are
characteristically expressed on myeloma cells, and then identify cells
that express the ligands for these receptors. Of particular interest is
the costimulatory receptor CD28, which is expressed predominantly
on T cells but also on both normal plasma cells and myeloma cells.
For T cells, it has been clearly shown that CD28 is activated through
binding to its ligands CD80 (B7-1) and CD86 (B7-2) expressed on
dendritic cells (the predominant antigen presenting cell). CD28
activation delivers an essential costimulatory signal (signal 2), that in
conjunction with the antigen-specific signal delivered through the T
cell receptor (signal 1) results in T cell activation, proliferation,
cytokine secretion and survival. In contrast, activation and function of
CD28 in plasma/myeloma cells is largely undefined. For example,
whether the equivalent of the T cell signal 1 is also required for CD28
to function in myeloma cells (and what that signal 1 is) is not known.
However, the observation that CD28 expression directly correlates
with disease progression and relapse suggests an important
contribution of CD28 signaling to myeloma cell survival. We
propose that CD28 can transduce survival signals to plasma/myeloma
cells, and that CD28 (along with whatever receptor delivers signal 1)
on MM cells is activated through cell-cell contact with bone marrow
dendritic cells (DC). We have found that direct activation of CD28
can protect myeloma cell lines against serum starvation and
dexamethasone-induced cell death. This CD28 activation
upregulates NFκB activity in myeloma cells. We have also found
that myeloma cell proliferation is downmodulated (without induction
of cell death) when cocultured with dendritic cells. This can be
blocked by CTLA4Ig (which binds to CD80 and CD86, preventing
CD28 activation). These findings suggest a previously undescribed
myeloma:DC cell-cell interaction involving CD28 that may play an
important role in myeloma cell survival within the bone marrow
stroma.
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Possible role of LeuCAM, VLA-6 and VCAM-1 adhesion
molecules in multiple myeloma clinical progression
Raya Sanchez JM, Hernández Nieto L, Brito Barroso ML,
Fernández Martín R, González Brito G.
Servicio de Hematología y Hemoterapia. Hospital Universitario de
Canarias. Universidad de La Laguna. Tenerife. Spain.
BACKGROUND: Normal plasma cells express a wide repertory
of cell-adhesion molecules (CAM) on their membranes, which
anchor them to the tissues where they usually stay. The loss or
underexpression of some of these CAM and the acquisition of
others, could be related with migration processes to bone marrow
or other extramedullary tissues. In myeloma patients, these
phenotypic changes could contribute to tumour spreading and
clinical progression of the disease. Our aim was to analyze the
differential expression of some CAM on myeloma plasma cells,
in a group of myeloma patients distributed by Durie & Salmon
clinical staging system. PATIENTS AND METHODS: We have
studied 18 myeloma patients distributed according to Durie &
Salmon staging classification. Expression of sixteen different
CAM (VLA-1 to VLA-6, LFA-1, CR3, CR4, LeuCAM beta-2
subunit, HCAM, ICAM-1, NCAM, L-selectin, VCAM-1, and
PECAM-1) was analyzed by flow cytometry (double-color
protocols) in bone marrow myeloma plasma cells. Identification
and analysis of plasma cells was performed by gating CD138
(syndecan-1) positive cells. Investigated parameters were
percentage of CAM-positive cells and mean fluorescence
intensity (MFI) for each monoclonal antibody, which explores the
antigenic density of CAM on plasma cell membrane.
RESULTS: The percentage of CR3 (CD11b) positive plasma
cells was significantly low in stage III myeloma patients, when
compared with stages I and II (see Table). There were no
differences in percentages of positivity between the three stages
for the remain analyzed CAM. The value of MFI for LeuCAM
beta-2 subunit (CD18), VLA-6 (CD49f) and VCAM-1 (CD106)
decreased significantly from stage I to stage III, while the other
studied CAM did not show differences.
MoAb Stage I Stage II Stage III p value
(CAM) (n=6) (n=7) (n=5)
CD11b 50.7 +/- 52.5 +/- 20.6 +/- 0.038
(%) 37.7 39.9 19.3
CD18 4.06 +/- 1.78 +/- 1.44 +/- 0.044
(MFI) 3.61 0.64 0.41
CD49f 2.39 +/- 1.35 +/- 1.20 +/- 0.013
(MFI) 1.29 0.55 0.65
CD106 1.69 +/- 1.15 +/- 0.88 +/- 0.035
(MFI) 0.44 0.17 0.35
CONCLUSIONS: Our findings suggest that clinical progression
in myeloma patients could be related with the loss of certain
membrane CAM in myeloma plasma cells. Underexpression of
beta-2 integrins, VLA-6 and VCAM-1 may play a role in tumor
spreading and progression of the disease. Inmunophenotypic
changes include a decrease in antigenic density for these
molecules on myeloma plasma cells membranes. If confirmed by
further studies, these preliminary results could contribute to
design new therapeutic strategies.
This work was supported in part by a grant of the Consejería de
Educación, Cultura y Deportes del Gobierno Autónomo de
Canarias (PI-1997/028).
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