EVIDENCE AGAINST AN EARLY SIGNALING ROLE FOR CERAMIDE IN FAS-MEDIATEDAPOPTOSIS

We have investigated whether the increases in ceramide levels that occ ur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depe nd on the activation of caspases. Using cells prelabelled to equilibri um with [C-14]acetate, it was shown that the amount of ceramide approx imately doubled after 24 h incubation with anti-Fas, but the time cour se of ceramide changes was slower than that of anti-Fas-induced cell d eath. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor zyloxy carbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added toge ther with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a s tructurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, bu t in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentatio n. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence o f processes downstream of the activation of caspases and increases in cytosolic calcium concentration.