CASPASES ARE THE MAIN EXECUTIONERS OF FAS-MEDIATED APOPTOSIS, IRRESPECTIVE OF THE CERAMIDE SIGNALING PATHWAY

Tumor necrosis factor alpha (TNF) or cytotoxic anti-fas antibodies lea d to the activation of apoptotic proteases (caspases) and to sphingomy elinase-mediated ceramide generation. Caspases and ceramide are both k nown to induce apoptosis on its own, but their relative contribution t o Fas-and TNF-induced cell death is not well established. We report he re that rapid apoptosis induced by TNF in U937 cells or anti-fas in Ju rkat cells, in the presence of cycloheximide, induced only a very low increase (<20%) in the cell ceramide content. Neither treatment with i nhibitors 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, resp ectively, did not prevent TNF and Fas-mediated apoptosis. Treatment of cells with sphingomyelinase inhibitors did not affect to the activati on of CPP32 (caspase-3) induced by TNF or anti-fas antibodies, Chromat in condensation and fragmentation in cells treated with anti-fas or TN F was abrogated by peptide inhibitors of caspases, which also inhibite d Fas-, but not TNF-induced cell death, These results indicate that wh ile ceramide does not seem to act as a critical mediator of TNF and Fa s-induced apoptosis, it is generated as a consequence of CPP32 activat ion and could contribute to the spread of the intracellular death sign al.