Epigenetic determinants of resistance to etoposide regulation of Bcl- x(L) and Bax by tumor microenvironmental factors

Abstract

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 μM) and then cultured in the presence of an activating anti-CD40 antibody, vascular cellular adhesion molecule-1 (VCAM-1) - to activate VLA-4 (α4β1) 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-κB 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