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Apoptosis, also known as programmed cell death, is an evolutionarily conserved mechanism by which the organism removes unwanted cells. The process is common to somatic as well as germ cells. It is actively involved in development and morphogenesis, cell number control and removal of infected, mutated or damaged cells. Many insights on the activation of final triggers and pathways in apoptosis came from genetic studies of apoptosis using the worm, C.elegans This work showed that the worm Ced-3 is a protease of pivotal role in apoptosis of C.elegans. The mammalian counterparts of CED-3 have been identified as members of a family of intracellular proteases that form the core of the apoptotic machinery. Since these are cysteine proteases that cleave cellular substrates at specific aspartate residues, they were all termed caspases. In the last ten years natural and synthetic inhibitors and genetic approaches have been used to study elucidate specific caspase function. An alternative system is gene disruption in the chicken DT40 cell line. These cells have a high homologous recombination rate that facilitates the isolation of knockout alleles. The aim of my project is the genetic analysis of caspase-6 and -7 function. My work started with an ongoing caspase-6 knockout project along with a senior postdoctoral scientist in our lab, Dr Sandrine Ruchaud. Subsequently, I concentrated my work on the caspase-7 knockout poject. I generated DT40 cell line where both alleles of caspase-6 and -7 were disrupted independently. Caspase-6: No obvious morphological differences were observed in the apoptotic process in caspase-6 deficient cells compared to the wild type. However, examination of apoptosis in a cell free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A-transfected Jurkat cells were incubated in caspase-6 deficient apoptotic extracts. Transfection of exogenous caspase-6 into the clone reversed this phenotype. Lamins A and C, which are caspase-6-only substrates, were cleaved by the wild type and heterozygous apoptotic extracts but not by the extracts lacking caspase-6. Furthermore, the caspase-6 inhibitor zVEID-fmk mimicked the effects of caspase-6 deficiency and prevented the cleavage of lamin A. Taken together these observations indicate that caspase-6 activity is essential for lamin A cleavage and that when lamin A is present, it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution. Caspase-7: Viability assays showed that caspase-7' clones are more resistant to common apoptosis-inducing drugs such as etoposide and staurosporine. Further examination of the apoptotic process revealed that caspase-7 cells show a delay in phosphatidylserine externalization and DNA fragmentation as well as cleavage of the caspase substrates PARP and lamins BI and B2. Caspase affinity labeling and activity assays indicated that deficient cells exhibit a delay in caspase activation when compared to wild type DT40 cells, providing an explanation for the differences in apoptotic execution between caspase-7 null and wild type DT40 cells. These results strongly suggest that caspase-7 is involved earlier than other effector caspases in the apoptotic execution process in DT40 B lymphocytes. My results have provided important new insights into the function of caspase-6 and caspase-7 during apoptotic execution.

Apoptosis is a form of cell death in which cells die in a controlled fashion. It is characterised by a distinct series of morphological changes and its incidence during embryogenesis, tissue homeostasis, T-cell maturation and following exposure to a number of toxins suggests that a pathway common to many diverse stimuli brings about the changes typical of apoptosis. The discovery that genes required for apoptosis in Caenorhabditis elegans are functionally conserved in man and the observation that some biochemical events recur in different examples of apoptosis supports the existence of a common pathway. In this thesis, rat thymocytes have been used to identify early events within a common apoptotic pathway. Initially a previously identified early apoptotic population of thymocytes was further characterised and shown to be committed to progressing to a fully apoptotic phenotype. The hypothesis that proteins required for mitosis are also required for apoptosis and that apoptosis can be considered an aberrant mitosis was then tested. No correlation between cdc2 kinase activity and the incidence of mitosis was found showing that apoptosis cannot be considered an aberrant mitosis. The effects of a number of protease inhibitors on different stages of thymocyte apoptosis were also investigated. Na-tosyl-lysinyl chloromethyl ketone (TLCK), an inhibitor of trypsin-like proteases and benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone, an inhibitor of interleukin-1beta-converting enzyme-like proteases prevented apoptosis induced by diverse stimuli by all criteria used. The TLCK target was pre-existing and not synthesised in response to apoptotic stimuli. N-tosyl- phenylalaninyl chloromethyl ketone, a chymotrypsin-like protease inhibitor prevented only late changes of apoptosis. This work clearly that there are distinct stages within the apoptotic process and that a common apoptotic pathway contains a hierarchy of proteases.

Apoptosis is a form of cell death in which the cell actively participates. Apoptosis was induced in two human leukaemic cell lines, U937 and HL-60, by incubation with a diverse array of chemical agents. Cell death was assessed by gel electrophoresis, light microscopy and flow cytometry. It was demonstrated that apoptosis involved the formation of large kilobase pair DNA fragments (20-50, 145-245 and 580 kilobase pairs) prior to, or accompanying, internucleosomal cleavage. Degradation of DNA to large kilobase pair sizes also occurred in some forms of necrosis. These fragments were similar, but not identical, to those generated during apoptosis. The identity of the endonuclease(s) responsible for DNA cleavage to large kilobase pair fragments is as yet unknown. One suggestion is that topoisomerase II might be involved. Using an HL-60 subclone with reduced topoisomerase II expression, it was shown that topoisomerase II was not necessary for the formation of large kilobase pair DNA fragments and oligonucleosomal fragments, except where topoisomerase II inhibition was the stimulus for apoptosis. However, topoisomerase II did appear to facilitate 20-50 kilobase pair-to-oligonucleosome conversion, and in one case, it was an absolute requirement for this conversion. Specific proteases have been implicated in the process of apoptosis. The effect of three protease inhibitors was investigated in U937 and HL-60 cells. Aproposed inhibitor of the protease, calpain, had little effect. In contrast, two serine protease inhibitors inhibited the formation of oligonucleosomal fragments. One of these also inhibited large kilobase pair fragments. The other induced apoptotic fragmentation of DNA to large kilobase pair fragments. The results suggest that different proteases may suppress or promote apoptosis within the same cell.

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