Activation and subcellular localisation of caspases in wild-type p53-induced apoptosis in vitro and fas-induced apoptosis in vivo

Despite previous studies demonstrating caspase-3 processing following treatment with DNA-damaging agents, there has been no direct evidence of p53WT-induced caspase activation. In this thesis an in vitro temperature-sensitive cell system was employed to study the effects of p53WT. Consistent with previous studies, p53WT induced morphological and biochemical changes characteristic of apoptosis.;p53WT-induced activation of caspase-3 and caspase-7, supported by the cleavage of PARP and the fluorogenic substrate z-DEVD.afc, together with activation of caspase-8, supported by cleavage of the fluorogenic substrate z-IETD.amc, were directly demonstrated for the first time. In addition, indirect evidence for p53WT-induced activation of caspase-6 was gained through the cleavage of lamin B1 and supported by the cleavage of the fluorogenic substrate Ac-cleavage of lamin B1 and supported by the cleavage of the fluorogenic substrate Ac-VEID.amc. Abrogation of p53WT-induced apoptotic morphology, caspase activation. PARP and lamin B1 proteolysis and cleavage of the fluorogenic substrates was achieved with the caspase inhibitor z-VAD.fmk. Evidence was also obtained to support the translocation of caspase-3 and caspase-7 from the cytosol to another subcellular compartment.;The issue of the different subcellular localisation of the substrates and the caspases which cleave them was addressed in the in vivo model of Fas-induced apoptosis in mouse liver. An agonistic Fas antibody induced apoptosis in mouse hepatocytes, which was associated with z-DEVD.afc cleavage activity. Both apoptotic morphology and z-DEVD.afc cleavage were blocked by z-VAD.fmk. Subcellular fractionation of the mouse livers demonstrated that whereas active caspase-3 remained in the cytosol, active caspase-7 was translocated to the mitochondrial and microsomal fractions. The observed cleavage of the endoplasmic reticular-specific substrate SREBP-1 following Fas-induced apoptosis is consistent with SREBP-1 being a substrate for the colocalised caspase-7, in vivo. Thus, differential distribution of the caspases may partly explain the existence of caspase homologues with similar substrate specificities.