Haematologica 2003 - Supplements

survive initial drug exposure and allows for the ultimate
acquistion of resistance to drugs, including melphalan.
References:
Catlett-Falcone R, Landowski T H, Oshiro M M, Turkson J,
Levitzki A, Savino R, Ciliberto G, Moscinski L, Fernandez-Luna
J L, Nunez G, Dalton W S and Jove R (1999) Constitutive
Activation of Stat3 Signaling Confers Resistance to Apoptosis in
Human U266 Myeloma Cells. Immunity 10: pp 105-115.
Damiano JS, Cress A E, Hazlehurst L A, Shtil A A and Dalton W
S (1999) Cell Adhesion Mediated Drug Resistance (CAM-DR):
Role of Integrins and Resistance to Apoptosis in Human
Myeloma Cell Lines. Blood 93: pp 1658-1667.
Nefedova Y, Landowski TH, Dalton WS (2003) Bone marrow
stroma derived soluble factors and direct cell contact contribute
to de novo drug resistance in myeloma cells by distinct
mechanisms. Leukemia (in press).
Shain K Landowski TH, and Dalton WS (2002) Adhesion
mediated intracellular redistribution of FLIP-L confers resistance
to CD95-induced apoptosis in hematopoietic cell lines. Journal
of Immunology 168: 2544-2553.
P6.2
CELL ADHESION ALTERS GENE EXPRESSION AND
ENHANCES CELL SURVIVAL IN MM
Terry H. Landowski, Nancy E. Olashaw, D. Agrawal, and
William S. Dalton
H. Lee Moffitt Cancer Center and Research Institute, University of
South Florida, Tampa, FL, and University of Arizona Cancer
Center, University of Arizona, Tucson, AZ.
Resistance to chemotherapeutic drugs is the primary obstacle to
the successful treatment of multiple myeloma. A large number of
studies have demonstrated that mechanisms of resistance to cell
death are frequently induced in response to drug exposure,
however, more recent work has documented the contribution of
the tumor microenvironment to the anti-apoptotic phenotype.
The interaction between tumor cell and environment may explain
how some tumor cells survive the initial drug exposure and
acquire classical mechanisms of drug resistance. We have
previously described the phenomenon of cell adhesion mediated
resistance (CAM-DR) to programmed cell death in hematopoietic
cell lines (1;2). This phenotype is characterized by increased
resistance to physiological stimuli, such as CD95 ligation, and to
a wide variety of chemotherapeutic drugs following adhesion to
fibronectin through β1 integrins (3;4). While extensive research
has identified a number of individual mechanisms contributing to
CAM-DR, the paramount question remaining is how do
malignant cells coordinate global changes in gene expression to
alter the phenotypic state of the tumor from that of pro-apoptotic,
to that of pro-survival.
To identify signal transduction pathways and gene products that
may contribute to CAM-DR, we have utilized oligonucleotide
microarray analysis of 8226 myeloma cells adhered to FN
compared to cells maintained in suspension (5). Cells maintained
in suspension were designated as the reference population, and
genes with altered expression in cells adhered to FN ranked in
numerical order based on fold change. We found that in 8226
myeloma cells, FN adhesion repressed the expression of 469 gene
products, while 53 genes with known coding sequences were
increased by two fold or more. Of the 53 known genes induced
by adhesion to FN, 11 have been documented to be regulated by
the transcription factor NF-κB (Table I).
Table I. NF-κB regulated genes induced by adhesion to fibronectin
GenBank
Description
Fold change
Accession
M92357 tumor necrosis factor, alpha-induced protein 2 6.5
Y00081 interleukin 6 (interferon, beta 2) 4.9
X56692 C-reactive protein, pentraxin-related 4.4
D79206 cIg protein, syndecan-4 3.9
M69043 nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor,
2.9
alpha
X53683 small inducible cytokine A4 (homologous to mouse Mip-1b) 2.8
Z11697 CD83 antigen (activated B lymphocytes, immunoglobulin superfamily) 2.2
U37546 apoptosis inhibitor 2 2.1
X66867 MAX protein 2.1
Y00787 interleukin 8 2.0
X66365 cyclin-dependent kinase 6 2.0
Table I. The cDNA expression profile of 8226 myeloma cells adhered to fibronectin for 8 hours was compared to
cells in suspension and ranked by fold increase. Of the top 53 genes induced by FN, 11 are known to be
regulated by NF-κB.
EMSA analysis demonstrated NF-κB binding activity
significantly increased in cells adhered to fibronectin compared to
cells in suspension. Supershift analysis with antibodies specific
for NF-κB family members demonstrates the majority of the
DNA binding activity in FN adhered cells is composed of
RelB/p50 heterodimers with very low levels of p65. This activity
was distinct from that seen following treatment with TNFα,
which induces p65/p50 within 30 minutes of exposure.
Several of the NF-κB gene products identified by microarray
analysis in our study are well-characterized survival factors in
multiple myeloma, including interleukin 6, cIAP-2. Rnase
protection and Western Blot analysis confirmed a 2 fold
induction of the anti-apoptotic molecule cIAP-2 following B1
integrin-mediated adhesion to FN. We propose that activation of
RelB/NF-κB in myeloma cells by adhesion to fibronectin in the
bone marrow microenvironment may contribute to the CAM-DR
phenotype.
References:
(1) Hazlehurst LA, Dalton WS. Mechanisms associated
with cell adhesion mediated drug resistance (CAM-DR) in
hematopoietic malignancies. Cancer Metastasis Rev 2001; 20(1-
2):43-50.
(2) Shain KH, Landowski TH, Dalton WS. The tumor
microenvironment as a determinant of cancer cell survival: a
possible mechanism for de novo drug resistance. Curr Opin
Oncol 2000; 12(6):557-563.
(3) Hazlehurst LA, Valkov N, Wisner L, Storey JA,
Boulware D, Sullivan DM et al. Reduction in drug-induced DNA
double-strand breaks associated with beta1 integrin-mediated
adhesion correlates with drug resistance in U937 cells. Blood
2001; 98(6):1897-1903.
Shain KH, Landowski TH, Dalton WS. Adhesion-mediated
intracellular redistribution of c-Fas-associated death domain-like
IL-1-converting enzyme-like inhibitory protein-long confers
resistance to CD95-induced apoptosis in hematopoietic cancer
cell lines. J Immunol 2002; 168(5):2544-2553.
Landowski TH, Olashaw NE, Agrawal D., and Dalton WS Cell
Adhesion Mediated Drug Resistance (CAM-DR) is Associated
with Activation of NF-κB (RelB/p50) in Myeloma cells.
Oncogene 2003, in press.
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