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

Abstract

To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets ®rst observable after 1 h at 328C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was ®rst observed 4 h after p53 induction. Using peptide inhibitors we identi®ed activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 speci®c inhibitor z-IETD.fmk did not aect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the Nterminus 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. However, the VP16 ± p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process. Oncogene (2000) 19, 4096 ± 4107.

Basic fibroblast growth factor (bFGF)-induced cell death is mediated through a caspase-dependent and p53-independent cell death receptor pathway

“…Therefore p53 status does not distinguish between ESFT cell lines susceptible to bFGF-induced cell death, and expression of wild-type p53 protein is not essential for bFGFinduced cell death. These data are consistent with that of Kovar et al (2000) establishing that the transcriptional activity of p53 is not su cient to initiate a caspase cascade and activate cell death. Although mutations of p53 are extremely rare in ESFT (Kovar et al, 1993;Radig et al, 1998), they would not signi®cantly impact on the induction of cell death by bFGF.…”

Apoptosis caused by p53-induced protein with death domain (PIDD) depends on the death adapter protein RAIDD

“…Despite the overwhelming evidence indicating that p53 promotes apoptosis through the mitochondrial pathway (4,5), compelling evidence supports a relationship between p53 and the death receptor pathway of apoptosis (19). Interestingly, p53-mediated cell death can be attenuated by overexpression of c-Flip-s, a specific inhibitor of caspase-8, and by pharmacological inhibition of caspase-8, at least in some cell types (20,21). Our findings indicate that enforced PIDD expression promotes cell death through the activation of both the RAIDD͞caspase-2 and FADD͞caspase-8 pathways.…”

p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation

“…This contrasts reports implying the activation of Caspase 8 as an upstream event in p53-dependent apoptosis in different experimental systems. [61][62][63] p53 deficiency or expression of Mdm-2, which destabilizes p53 by targeting for proteasomic degradation, 64 prevented mitochondrial accumulation of Bax and Cytochrome c release. Again, no Cytochrome c release was observed in bax À/À MEF under the present experimental conditions ( Figure 3).…”