Characterization of distinct consecutive phases in non-genotoxic p53-induced apoptosis of Ewing tumor cells and the rate-limiting role of caspase 8

To dissect the p53-dependent apoptotic pathway events following induction o f temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell l ine. Transcriptional deregulation of p53 targets first observable after 1 h at 32 degrees C preceded activation of caspases and the break-down of mito chondrial respiratory activity. Activation of caspases was first observed 4 h after p53 induction. Using peptide inhibitors we identified activation o f caspase 8 upstream of caspases-9 and -3. Although the caspase 8 specific inhibitor z-IETD.fmk did not affect translocation of BAX to the mitochondri al membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforci ng mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signal ing. Expression of BCL2 only partially suppressed caspase activation but bl ocked apoptosis, Replacement of the N-terminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. How ever, the VP16-p53 fusion failed to trigger caspases and subsequent inducti on of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregul ation, triggering of the caspase cascade and the mitochondrial break-down o ccur in a timely ordered sequence coordinated by the genuine p53 amino term inus and suggest caspase 8 and PIG3 as key regulatory elements in this proc ess.