Evidence against an early signalling role for ceramide in Fas-mediated apoptosis.
We have investigated whether the increases in ceramide levels that occur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depend on the activation of caspases. Using cells prelabelled to equilibrium with [14C]acetate, it was shown that the amount of ceramide approximately doubled after 24 h incubation with anti-Fas, but the time course of ceramide changes was slower than that of anti-Fas-induced cell death. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added together with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a structurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, but in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentation. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence of processes downstream of the activation of caspases and increases in cytosolic calcium concentration.
Sillence
D J
DJ
Department of Physiology, University College London, Rockefeller Building, University Street, London WC1E 6JJ, U.K.
Allan
D
D
D000085:Acetates / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002518:Ceramides / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D016393:Lymphoma, B-Cell; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000276:immunology / Q000502:physiology*
D000085:Acetates; D000590:Amino Acid Chloromethyl Ketones; D000906:Antibodies; D002518:Ceramides; D015853:Cysteine Proteinase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D000001:Calcimycin; D003546:Cysteine Endopeptidases; D002118:Calcium