Activation of caspase-9 and-3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation

The treatment of PC12 cells with H2O2 (100-500 mu M) resulted in typical ap optotic changes including fragmentation and condensation of nuclei, and DNA fragmentation observed as DNA ladder. H2O2-induced apoptosis was associate d with activation of caspase-3 as assessed by cleavage of specific fluoroge nic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) p olymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibi ted activation of caspase-3 and apoptotic cell death. However, the relative ly specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activ ation of caspase-3, induction of cytochrome c release followed by processin g of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PC12 cell s overexpressing Bcl-2, the number of apoptotic cells Mas markedly decrease d and activation of both caspases-9 and -3 was prevented. These results sug gest that cytochrome c and caspase-9 initiate the activation of executor ca spase-3 in H2O2-treated PC12 cells, and that Bcl-2 inhibits H2O2-induced re lease of cytochrome c from mitochondria and then proteolytic processing of procaspase-9.