Molecular events occurring during the induction and execution of apoptosis

Apoptosis is vital in maintaining homeostasis, and its deregulation can have dire pathological consequences. Recent years have seen a dramatic increase in interest in the biochemical mechanisms of this phenomenon.;During the later stages of apoptosis there is extensive internucleosomal cleavage of DNA. In the first part of the work, eighteen dinucleosomal fragments from apoptotic mouse thymocytes were sequenced to ascertain whether there were any trends in the sequence of the DNA which could influence its likelihood to be cleaved. No such patterns could be identified.;The second part of this thesis deals with the participation of Ca2+ in the signalling mechanisms that induce apoptosis in rat thymocytes. A comparison of apoptosis induced by thapsigargin, cyclopiazonic acid and 2,5-di-(t-butyl)-1, 4-benzohydroquinone, three inhibitors of the microsomal Ca2+-ATPase, with that caused by the steroid dexamethasone, the chemotherapeutic drug etoposide and the protein kinase inhibitor staurosporine established that the three Ca2-ATPase inhibitors use the pathway of an early elevation in intracellular free Ca2+ concentration to trigger apoptosis.;The discovery of the homology between the nematode death gene ced-3 and the human interleukin-1 converting enzyme (ICE) has established that enzymes such as these are ubiquitous effectors of apoptosis. The final section of work involves the compound benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.FMK), which inhibits apoptosis in many cell models. Using an in vitro system incorporating the radiolabelled protein substrates [35S]PARP and [35S]proIL-1, it was found that Z-VAD.FMK inhibits the proteolytic activation of the ICE/Ced-3 homologue CPP32 induced by both Fas-mediated apoptosis and apoptosis induced by chemical stimuli. Furthermore, it was deduced that ICE is not involved in Fas-mediated apoptosis in Jurkat T cells.