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published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Only discovered about 20 years ago, the structure of the eukaryotic vault particle has been studied extensively, but the function has yet to be determined. Vault numbers are up regulated in many types of cancer cells that are treated with chemotherapy agents and it is thought that they may act to transport chemotherapy drugs out of such cells, leading to multi-drug resistance (MDR). To determine a possible role of the vault particle in MDR, the goal of this research was to examine one of the functional vault proteins, vault poly(ADP)ribose (VPARP) for interactions with other proteins. Two forms of small cell lung cancer cells were used; GLC4/S which do not exhibit MDR and the MDR cells GLC4/ADR, which are cultured with the chemotherapy drug doxorubicin. Both cell cultures were subjected to a subcellular fractionation followed by gentle immunoprecipitation with an antibody to VPARP. Immunoprecipitated proteins interacting with VPARP were only observed in GLC4/ADR cells, as seen on a PAGE gel. This sample was taken to Monarch Life Sciences and analyzed by mass spectrometry. One interacting protein was found to be NALP1 pyrin domain (PYD), a member of the death domain family of proteins which is involved in inflammation and apoptosis. The interaction of VPARP with NALP1, which only occurred in MDR cells, suggests an exciting, previously unreported possibility – that VPARP binding may inhibit NALP 1-stimulated apoptosis when MDR is occurring. Future studies are needed to examine if levels of NALP1 vary in GLC4 cells with and without treatment with doxorubicin and in normal lung cells. The cellular location (nucleus or cytoplasm) of the interactions should also be identified. Furthermore, immunoprecipitation of proteins interacting with NALP1 should include VPARP and perhaps identify other proteins interacting in the signaling pathways under MDR and normal culture conditions. This information may contribute insight into the function of VPARP and vaults within the cell. / Department of Biology

[Truncated abstract] Hepatic progenitor cells (HPC) are becoming increasingly recognized as facultative stem cells capable of regenerating the liver during chronic liver injury and also as targets of malignant transformation. Similar markers are expressed by hepatocellular carcinoma (HCC) and HPC, and a precursor-product relationship is well established. This thesis focuses on the ways in which the HPC population can be controlled under circumstances of chronic liver injury, and in this manner, reduce the risk of progression to HCC reduced. The major aim of Chapters 3 to 5 was to elucidate the effect of interferon α (IFNα) therapy on HPC. Chronic hepatitis C affects approximately 250 million individuals world wide. Approximately 80% of infections progress to chronicity, which places the individuals at greater risk of developing HCC. The gold standard of treatment of chronic hepatitis C is a combination of pegylated IFNα and ribavirin. ...The results were surprising. While IFNγ exerted a pro-apoptotic and antiproliferative effect on HPC in vitro, administration of IFNγ to CDE-fed mice for 14 days increased fibrosis, enhanced inflammatory infiltration and exacerbated the HPC response, with concurrent hepatocyte cell death. In addition, increased morbidity and mortality were observed in the IFNγ-treated mice compared to control. IFNγ treatment was found to prime the liver for the HPC response by recruiting inflammatory cells and altering the hepatic cytokine profile, both of which may facilitate an increased HPC response. Numbers of activated HSC were also increased in the IFNγ-treated, CDE-fed mice, correlating with the increased fibrosis seen in these animals. This data contradicts the current experimental use of IFNγ for treatment of fibrosis. Based on our results, we suggest that IFNγ promotes HPC proliferation in the CDE model, by encouraging inflammatory infiltration and hepatocyte damage and this initiates pro-fibrotic events. Concurrent proliferation of HPC and activated HSC further supports the view that there is a close relationship between the two cell types, and thus, a link between the HPC response and fibrosis. In conclusion, findings documented in this thesis suggest that administration of IFNα and IFNγ can contribute to shaping the HPC response. IFNα therapy may reduce HCC risk in chronic hepatitis C patients by bringing the HPC population under control. In contrast, IFNγ treatment can exacerbate the HPC response, liver fibrosis and parenchymal damage, illustrating the need to approach this method of fibrosis treatment with caution.