Caspases are the main executioners of Fas-mediated apoptosis, irrespective of the ceramide signalling pathway

Tumor necrosis factor alpha (TNF) or cytotoxic anti-fas antibodies lead to the activation of apoptotic proteases (caspases) and to sphingomyelinase-mediated ceramide generation. Caspases and ceramide are both known to induce apoptosis on its own, but their relative contribution to Fas-and TNF-induced cell death is not well established. We report here that rapid apoptosis induced by TNF in U937 cells or anti-fas in Jurkat cells, in the presence of cycloheximide, induced only a very low increase (<20%) in the cell ceramide content. Neither treatment with inhibitors of sphingomyelinases nor incubation of cells with fumonisin B-1, which inhibits de novo ceramide synthesis, prevented TNF and Fas-mediated apoptosis. Increasing or depleting the cell ceramide content by prolonged culture in the presence of monensin or fumonisin B1, respectively, did not prevent TNF and Fas-mediated apoptosis. Treatment of cells with sphingomyelinase inhibitors did not affect to the activation of CPP32 (caspase-3) induced by TNF or anti-fas antibodies, Chromatin condensation and fragmentation in cells treated with anti-fas or TNF was abrogated by peptide inhibitors of caspases, which also inhibited Fas-, but not TNF-induced cell death, These results indicate that while ceramide does not seem to act as a critical mediator of TNF and Fas-induced apoptosis, it is generated as a consequence of CPP32 activation and could contribute to the spread of the intracellular death signal.