Protease inhibitors act at different stages of thymocyte apoptosis.

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.