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 invo lving 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 reg ulation of apoptotic cell death and modulate drug sensitivity, We investiga ted how survival signals in the cellular microenvironment affect the expres sion, protein conformation, and protein-protein interactions of the Bcl-2 f amily proteins Bar and Bcl-x(L) and how changes in response to microenviron mental signals alter the response of cancer cells to the drug etoposide, Me thods: JLP119 human a-lymphoma cells were treated with etoposide (40 mu M) and then cultured in the presence of an activating anti-CD40 antibody, vasc ular cellular adhesion molecule-1 (VCAM-1)-to activate VLA-4 (alpha 4 beta 1) integrin, and interleukin 4, Cell fate was monitored after etoposide tre atment with or without these microenvironmental signals, Bcl-x(L) gene tran scription and protein levels of Bcl-x(L) and Bax were measured by northern and western blotting, respectively, Nuclear translocation of transcription factor NF-kappa B was monitored by immunofluorescence and inhibited by (E)- capsaicin, Bar conformation and Bax-Bcl-x(L) interactions were monitored by immunofluorescence and immunoprecipitation, respectively. Results: Microen vironmental survival signals produced statistically significant reductions in etoposide-induced apoptotic cell death, from 84.6% (95% confidence inter val [CI] = 76.7%-92.4%) to 21.3%(95% CI = 19.5%-23.0%); P < .001. Activatio n of surface protein CD40 increased Bcl-x(L) protein levels via an (E)-caps aicin-inhibitable activation of NF-KB; i.e, (E)-capsaicin restored etoposid e sensitivity, Interleukin 4 had no effect on Bcl-x(L) protein levels but a ccelerated the increase in Bcl-x(L) protein associated with CD40 activation , VCAM-1- and interleukin 4-mediated signals diminished conformational chan ges in Bar protein and prevented the etoposide-induced disruption of consti tutive Bax-Bcl-x(L) binding, Conclusions: Microenvironmental factors reduce the sensitivity of a B-cell lymphoma to etoposide in vitro by modulating t he expression and functions of pax and Bcl-x(L), This interaction may provi de a paradigm for epigenetically induced drug resistance in other tumors.