Investigations into the molecular mechanism of apoptosis

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Gilmore, K. J.
Faculty of Science
thesis / dissertation description
A detailed understanding of the chronology of cellular events in apoptosis is required order to gain insight into the mechanisms involved and to identify the critical components responsible for the execution of cell death. The results presented describe detailed chronology of the cellular events that occur in HL60, U-937 and Jurkat cells response to the apoptosis-inducing agents etoposide, staurosporine, anti-Fas antibody and the combination of TNFα and cycloheximide. The chronology of apoptotic events was found to be specific for the cell type and inducing agent. The role of permeability transition in apoptotic cell execution was investigated, the results suggesting that permeability transition is not always a critical triggering event in apoptosis and is always the mechanism for release of cytochrome c from mitochondria. The pinocytic loading of cytochrome c into Jurkat and U-937 cells showed that the presence of cytosolic cytochrome c is sufficient to trigger activation of caspases-3 and -9, exposure of phosphatidylserine, loss of mitochondrial transmembrane potential and DNA degradation. Evidence for the existence of a caspase-dependent feedback loop which would result in amplification of the apoptotic cascade is presented. It was demonstrated during etoposide-induced apoptosis of HL60 cells that rapid endocytosis occurred after cell shrinkage and prior to changes in membrane permeability, providing a possible mechanism for the dramatic reduction in cell surface area that occurs during apoptosis.