CERAMIDE-INDUCED APOPTOSIS IS MEDIATED BY CASPASE ACTIVATION INDEPENDENTLY FROM RETINOBLASTOMA PROTEIN POSTTRANSLATIONAL MODIFICATION

Recent evidence suggests that untimely retinoblastoma protein (RE) dep hosphorylation and/or proteolytic degradation might provide key events downstream cysteine protease (caspase) activation in apoptosis induct ion. We have dealt with this issue by studying apoptosis induced by N- hexanoylsphingosine (C-6-Cer) in CHP-100 human neuroepithelioma cells, maintained in complete growth medium. We report that C-6-Cer-induced apoptosis occurred predominantly in G(1)/S phases of the cycle and was associated with RE dephosphorylation, in the setting of negligible Bc l-2 expression. Apoptosis was also associated with poly(ADP-ribose) po lymerase (PARP) cleavage, thus indicating activation of CPP32/Yama/apo pain (caspase-3); however, while the tripeptide caspase inhibitor Z-Va l-Ala-DL-Asp-fluoromethhylketone was able to prevent both C-6-Cer-indu ced PARP cleavage and apoptosis, it was ineffective in preventing RE d ephosphorylation. Moreover proteolytic RE cleavage occurred only to a marginal extent after C-6-Cer treatment. These results indicate that a poptosis induced by ceramide in CHP-100 cells is caspase-mediated, but RE post-translational modification does not provide a key step, downs tream caspase activation, in apoptosis execution. (C) 1998 Academic Pr ess.