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Abstract

TNF-alpha activated caspase 8 and caspase 3 in PC12 cells, leading to cell death by apoptosis (DNA fragmentation). TNF-alpha caspase activation and cell killing were blocked by transfection and overexpression of the viral protein CrmA, which specifically inhibits caspase 8. CrmA was also able to block the TNF-alpha-induced increase in ceramide formation in PC12 cells. Conversely, if caspase 8 was activated by light-activated Rose Bengal, there was an increase in both ceramide and caspase 3-mediated apoptosis, which was blocked by CrmA overexpression. This suggested that caspase 8 increases ceramide either by increasing its synthesis or by activating sphingomyelinase. Since fumonisin B1 did not block and sphingomyelin decreased when ceramide increased, we concluded that activation of sphingomyelinase is the most likely mechanism. The Rose Bengal activation of caspase 8 and increased ceramide formation was blocked with IETD-CHO, to show that reactive oxygen species (also generated by Rose Bengal) were not responsible for the observed increase in ceramide. Thus in PC12 pheochromocytoma cells, ceramide appears to amplify the death signal and there appears to be a sequence of events: TNF; TRADD, pro-caspase 8, caspase 8, sphingomyelinase, ceramide, caspase 3, apoptosis.