Cell cycle modulations during Hepatitis C Virus associated disease progression
Date of Award
Doctor of Philosophy in Health Science (PhD)
Biological and Biomedical Sciences
The disease burden caused by hepatitis C virus (HCV) infection entails a serious public health problem. Chronic infection causes liver inflammation and fibrosis, which often leads to liver cirrhosis and hepatocellular carcinoma (HCC). Increasing scientific evidence has suggested an impaired proliferation of hepatocytes during the course of HCV-associated pathogenesis. Considering the fundamental role played by cell cycle proteins in controlling cell proliferation and survival, altered regulation of these proteins could significantly contribute to HCV disease progression and subsequently, HCC. The aim of the work described in this thesis was to identify cell cycle modulations in early and advanced stages (pre-cancerous stages) of HCV-associated disease. In particular we wanted to explore the contribution of cell cycle genes in disease progression. Genome wide expression profiling was conducted on pooled RNA samples from HCV infected liver specimens with early (fibrosis stage 0-1) and advanced (fibrosis stage 3-4) disease stages. The results of this analysis reflect the changes taking place during the transition from early to advanced liver fibrosis, when the liver function becomes impaired and extracellular matrix deposition increases. In addition, it also showed altered expression of genes with functions in cancer development, cell growth and proliferation and cell death. Investigation of cell cycle alterations was the next target of this study. For this purpose, a comprehensive analysis of cell cycle pathway was performed on pooled RNA samples using Q RT-PCR. The results characterized early HCV (E-HCV) disease with increased expression of proliferation genes accompanied by elevated levels of cell cycle inhibitors possibly as a response to DNA damage or other cellular injuries. In advanced HCV (AHCV), DNA damage response genes and CDK inhibitors were found up-regulated. This was also accompanied by the down-regulation of genes involved in DNA repair and chromosomal stability. These results are suggestive of aberrant DNA replication and cell division that increases the risk of transformation and subsequent development of HCC. Post transcriptional regulation plays an important role in cell cycle proteins function. We further evaluated the protein expression of seven differentially expressed genes that were selected on the basis of their crucial role in cell cycle checkpoints. Consistent with the mRNA levels elevated expression of proliferation marker Mcm-2 and CDK inhibitors p27 and p27 were observed in hepatocytes of chronic hepatitis C patients. Moreover, expression of Mcm-2, p2l and p27 significantly associate with the progression of liver fibrosis. Expression of p53 was also elevated with no association to p2l expression. Among the mitotic checkpoint regulators, increased expression of KNTC1 and MAD2L1 proteins were found in E-HCV while in A-HCV KNTC1 expression significantly reduced and MAD2LI showed a non-significant decrease. This might suggest loss of their function in A-HCV. The analysis of apoptotic protein Caspase-3 revealed that its expression was mostly confined to sinusoidal lining cells and little in hepatocytes. Expression of anti-apoptotic protein Bcl-2 was negligible in hepatocytes and principally detected in infiltrating lymphocytes suggesting that the reduced apoptosis in hepatocytes might not be due to the anti-apoptotic activity of Bcl-2. Together these results are suggestive of an altered expression of cell cycle regulators in hepatocytes of HCV infected patients that could not only impair the regenerative potential of liver but could also make these cells susceptible to acquire mutations resulting in cell transformation and development of HCC.
Sarfraz, S. (2008). Cell cycle modulations during Hepatitis C Virus associated disease progression (Unpublished doctoral thesis). Aga Khan University, Karachi, Pakistan.