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Title: Activation and subcellular localisation of caspases in wild-type p53-induced apoptosis in vitro and fas-induced apoptosis in vivo
Author: Chandler, Julia M.
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1998
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available