The purpose of the present studies was to examine the role and regulation of Fas-associated death domain-like IL-1- converting enzyme-like inhibitory protein [FLIP; long (FLIP(L)) and short (FLIP(S)) forms] in human ovarian epithelial cancer cells by TNFalpha and their significance in the resistance of the cells to the proapoptotic action of the cytokine. OV2008, A2780-s, and OVCAR-3 cells were cultured in serum-free media with or without cycloheximide (CHX, 10 micro g/ml) +/- TNFalpha (5, 10, 20 ng/ml) or transfected with a mammalian expression vector containing either a dominant negative inhibitor kappaB (IkappaB), FLIP(S) sense or antisense cDNA and cultured with or without TNFalpha. In the presence of CHX, TNFalpha increased caspase-8 and -3 cleavage and apoptosis. It also induced IkappaB phosphorylation, nuclear factor kappaB activation, and the expression of FLIP(S) but not of FLIP(L). Overexpression of dominant negative IkappaB attenuated TNFalpha-induced FLIP(S) expression and enhanced TNFalpha-induced apoptosis. Apoptosis induced by TNFalpha and CHX was facilitated by FLIP(S) antisense expression but attenuated by sense transfection. This study demonstrates that TNFalpha up-regulates FLIP(S) expression, and this effect is mediated by the activation of nuclear factor kappaB. The induction of FLIP(S) expression by TNFalpha might contribute to the resistance of ovarian epithelial cancer cells to the proapoptotic action of the cytokine.