The role of Bcl-2 expression in EGF inhibition of TNF-alpha/IFN-gamma-induced villous trophoblast apoptosis

The inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and imm une interferon gamma (IFN-gamma) stimulate villous cytotrophoblast apoptosi s while epidermal growth factor (EGF) protects. We hypothesize that TNF-alp ha, IFN-gamma and EGF regulate apoptosis in part by modulating cellular exp ression levels of the anti-death gene bcl-2. While Bcl-2 is reported to be strongly expressed in villous syncytiotrophoblasts, it is not known whether the protein is expressed in cultured villous cytotrophoblasts (CT) and, if so, whether it is functional. We show by Northern blot analysis that bcl-2 mRNA is expressed in cultured CT and by immunoblot analysis that the prote in is strongly expressed in highly purified first trimester and term villou s cytotrophoblasts. The expression levels of Bcl-2 protein were the same in first trimester and term cytotrophoblasts. Culture with TNF-alpha/IFN-gamm a and EGF did not alter expression of either Bcl-2 protein or of the pro-ap optotic Bcl-2 family member Bak. Double label flow cytometric analysis that measured apoptosis and Bcl-2 content simultaneously showed that cells expr essing low levels of Bcl-2 underwent TNF-alpha/IFN-gamma-induced apoptosis at a higher frequency than cells expressing lower levels. We conclude that Bcl-2 is expressed in cytotrophoblasts, that its expression is constitutive and that modulation of its expression levels does not mediate cytokine and growth factor regulation of apoptosis in these cells. (C) 1999 W. B. Saund ers Company Ltd.