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Title: IFNγ induced apoptosis in the murine hepatocyte
Author: Tura, Benjamin James
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Specifically we set out to identify the upstream caspases that were activated by IFNγ and the role of the mitochondria in apoptosis. We selected an in vitro primary murine hepatocytes system to investigate this pathway. Despite experimental difficulties and questions about the relevance of animal research to human disease, this system was chosen as it provided a practical model of cell injury. We demonstrate that apoptosis occurs in a significant proportion of the population 72 hrs after administration of IFNγ and that this apoptosis is subject to modulation. Serum was shown to have an effect on the kinetics of apoptosis. An interaction of the IFNγ and Fas apoptosis pathways is demonstrated with the use of the Jo2 anti-Fas antibody. Using caspase inhibitors and western blot analysis it was demonstrated that both caspase 8 and caspase 9 are crucially involved in hepatocytes apoptosis. Following 48 hours of IFNγ treatment evidence of Bid cleavage is seen. Following 72 hours of IFNγ treatment cleavage and activation of caspase 9 is seen. These results demonstrate for the first time that in IFNγ treated hepatocytes caspase 8 is activated followed by caspase 9. We hypothesised that a type II membrane death receptor pathway was operating. This hypothesis was tested with the FasL blocking antibody MFL3. IFNγ-induced apoptosis was successfully blocked with this agent. Our results lead us to conclude that IFNγ acts through the Fas pathway to achieve hepatocytes apoptosis. This finding suggests manipulation of components in the Fas pathway may be a beneficial treatment strategy in hepatitis. To investigate the involvement of the mitochondria in the pathway we administered the drug CsA to IFNγ treated cells. CsA was effective in reducing the percentage of cells showing apoptotic morphology at 72 hours. Studies of the mitochondrial membrane potential revealed that a large proportion of the population (~80%) undergo mitochondrial depolarisation. Membrane depolarisation was found to occur despite the presence of apoptosis inhibitors.  Both CsA and the caspase 8 inhibitor z-IETD-fmk, were unable to prevent mitochondrial depolarisation indicating that it is not dependent on the mitochondrial permeability transition pore or the presence of tBid.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available