CD95 (FAS APO-1) INDUCES CERAMIDE FORMATION AND APOPTOSIS IN THE ABSENCE OF A FUNCTIONAL ACID SPHINGOMYELINASE/

CD95 is a potent inducer of apoptosis. It activates the caspase cascad e, but also induces ceramide (Cer) production, reportedly involving ac id sphingomyelinase (aSMase) activity. A role for Cer as a second mess enger for apoptosis induction was proposed, based on the finding that synthetic Cer analogues can induce cell death. We have tested whether aSMase is required for 1) apo ptosis induction and 2) Cer production b y CD95. For this purpose, we have used cultured Niemann-Pick disease ( NPD) lymphoid cells with a defined mutation (R600H) in the aSMase prot ein. Despite their inherited deficiency of aSMase, we found that these cells readily undergo apoptosis upon CD95 stimulation. After retrovir us-mediated gene transfer of the aSMase cDNA, the transduced (i.e. ''c orrected'') NPD cells showed neither increased levels of apoptosis nor altered kinetics of caspase-8 and caspase-3 activation and apoptosis induction as compared with empty vector-transduced cells. The slow sus tained elevation of Cer levels in response to CD95, which we have prev iously documented for Jurkat T cells (Tepper, A. D., Boesen-de Cock, J . G. R., de Vries, E., Borst, J., and van Blitterswijk, W. J. (1997) J . Biol. Chem. 272, 24308-24312), was similarly found in NPD cells. Mor eover, the kinetics of Cer formation remained unaffected after aSMase transduction. These results indicate that this Cer does not result fro m aSMase activity. We conclude that aSMase is not required for and doe s not facilitate CD95-mediated apoptosis and that it is not responsibl e for the late Cer response.