Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induce d apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10-100 nM, Apoptosis was largely, albeit not c ompletely, dependent on cystein protease (caspase) activation. CPP32 proces sing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluorom ethylketone (Z-VAD.fmk) (100 mu M) had negligible effect on apoptosis induc ed by In nM OA, but rescued from death an increasing cell fraction as OA co ncentration was raised from 20-100 nM, Cell treatment for 24 h with OA indu ced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50-100 nM OA, In cells exposed to 50 nM OA, cer amide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 mu M) had no effect on cer amide elevation induced by 50 nM OA within 5 h, but markedly reduced cerami de accumulation as the incubation was prolonged to 24 h, The latter phenome non was accompanied by elevation of glucosylceramide levels, thus suggestin g that a caspase-dependent reduction of glucosylceramide synthesis might co ntribute to late ceramide accumulation, Short-chain ceramide (30 mu M) indu ced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10-20 nM), These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibiti on induces caspase activation and apoptosis in CHP-100 cells.