Tura, Benjamin James

2018-03-29T12:20:46Z

2018-03-29T12:20:46Z

2005

Interferon gamma (IFNγ) is a key mediator of liver injury and hepatitis. The major fashion in which IFNy brings about injury is through apoptosis of the parenchymal cells ofthe liver, the hepatocytes. In the primary murine hepatocyte IFNγ-induced hepatocyte apoptosis is dependent on the anti-tumourigenic transcription factor IRF1 and involves caspase 3 like enzymes. Liver injury leads to a pre-malignant state that can lead to carcinogenesis. Inappropriately controlled apoptosis is thought to be one ofthe factors that contribute to cancer formation.

The aim ofthese investigations was to investigate the pathway to apoptosis between the IRF-1 transcription factor and caspase 3. These areas may be affected by environment, viruses, drugs or by the cell itself in favour of life or death. Specifically we set out to identify the upstream caspases that were activated by IFNγ and the role ofthe mitochondria in apoptosis.

We selected an in vitro primary murine hepatocyte 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 ofthe population 72hrs after administration of IFNy and that this apoptosis is subject to modulation. Serum was shown to have an effect on the kinetics of apoptosis. An interaction ofthe IFNy and Fas apoptosis pathways is demonstrated with the use ofthe Jo2 anti-Fas antibody. This pathway is sensitive to inhibition by Cyclosporin A (CsA) and media changes

Using caspase inhibitors and western blot analysis it was demonstrated that both caspase 8 and caspase 9 are involved in hepatocyte 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 hepatocyte 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. This model is relevant to hepatocarcinogenesis where both IFNγ and Fas play leading roles.

To summarise, the major findings ofthis thesis show for the first time activation of caspases 8 and 9 following IFNγ treatment, an effect of IFNγ on the hepatocyte mitochondria and a direct link between IFNγ and the Fas pathway in the hepatocyte

http://hdl.handle.net/1842/29406

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 17

Already catalogued

IFNγ induced apoptosis in the murine hepatocyte

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy