Epigenetic determinants of resistance to etoposide regulation of Bcl-X(L) and Bax by tumor microenvironmental factors.
BACKGROUND
Epigenetic factors (i.e., alterations of gene activity not involving mutations), as well as genetic changes in surviving cancer cells, may play an important role in drug resistance following cancer chemotherapy-a common cause of tumor relapse. Bcl-2 family proteins are central to the regulation of apoptotic cell death and modulate drug sensitivity. We investigated how survival signals in the cellular microenvironment affect the expression, protein conformation, and protein-protein interactions of the Bcl-2 family proteins Bax and Bcl-x(L) and how changes in response to microenvironmental signals alter the response of cancer cells to the drug etoposide.


METHODS
JLP119 human B-lymphoma cells were treated with etoposide (40 microM) and then cultured in the presence of an activating anti-CD40 antibody, vascular cellular adhesion molecule-1 (VCAM-1)-to activate VLA-4 (alpha4beta1) integrin, and interleukin 4. Cell fate was monitored after etoposide treatment with or without these microenvironmental signals. Bcl-x(L) gene transcription and protein levels of Bcl-x(L) and Bax were measured by northern and western blotting, respectively. Nuclear translocation of transcription factor NF-kappaB was monitored by immunofluorescence and inhibited by (E)-capsaicin. Bax conformation and Bax-Bcl-x(L) interactions were monitored by immunofluorescence and immunoprecipitation, respectively.


RESULTS
Microenvironmental survival signals produced statistically significant reductions in etoposide-induced apoptotic cell death, from 84.6% (95% confidence interval [CI] = 76.7%-92.4%) to 21.3% (95% CI = 19.5%-23.0%); P<.001. Activation of surface protein CD40 increased Bcl-x(L) protein levels via an (E)-capsaicin-inhibitable activation of NF-kappaB; i.e. , (E)-capsaicin restored etoposide sensitivity. Interleukin 4 had no effect on Bcl-x(L) protein levels but accelerated the increase in Bcl-x(L) protein associated with CD40 activation. VCAM-1- and interleukin 4-mediated signals diminished conformational changes in Bax protein and prevented the etoposide-induced disruption of constitutive Bax-Bcl-x(L) binding.


CONCLUSIONS
Microenvironmental factors reduce the sensitivity of a B-cell lymphoma to etoposide in vitro by modulating the expression and functions of Bax and Bcl-x(L). This interaction may provide a paradigm for epigenetically induced drug resistance in other tumors.
Taylor
S T
ST
Cancer Research Campaign Molecular and Cellular Pharmacology Group, School of Biological Sciences, University of Manchester, U.K.
Hickman
J A
JA
Dive
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C
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002051:Burkitt Lymphoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D000972:Antineoplastic Agents, Phytogenic; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D039041:Integrin alpha4beta1; D016023:Integrins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016201:Receptors, Lymphocyte Homing; D019010:Vascular Cell Adhesion Molecule-1; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D015847:Interleukin-4; D005047:Etoposide