Hepatitis C and Tumor Suppressors in Hepatocellular

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This project seeks to determine the clinical relevance and potential role in hepatic carcinogenesis of interactions between host cell and hepatitis C virus (HCV) proteins that have been identified in previous cell culture-based experiments. The retinoblastoma susceptibility protein (pRb) and the cellular DEAD-box RNA helicase DDXS play critical roles in regulating the cell cycle. Our previous work indicates that the HCV RNA polymerase, NS5B, fc^nns a complex with pRb, targeting it for ubiquitination and proteasome-dependent degradation, activating E2F-responsive promoters, and stimulating cell proliferation. Similarly, HCV cofe protein expression alters the cellular abundance and localization of DDX3. Our hypothesis is that these and other interactions with host proteins (p53 and DDX5) promote proliferation of hepatocytes and impair DNA damage responses, tljiereby contributing to HCV carcinogenesis. In Aim 1, we will develop and validate fluorescent quantum dot probes capable of sensitive detection of HCV proteins expressed in virus-infected cells. We will also determine whether HCV protein expression conrelates with altered tumor suppressor expression in transgenic mice. Aim 2 will utilize these novel probes in laser scanning confocal and multi photon microscopy studies of liver tissue from patients with chronic hepatitis C, and ask whether the abundance and cellular localization of pRb, p53, DDXS, or DDXS is altered in infected hepatocytes. In Aim 3, we will determine whether HCV infection is associated on a single-cell basis with increased expression of the proliferation markers Ki-67 and PCNA. We will also determine whether the proportion of cells displaying proliferation markers is reduced after standard-of-care peg-IFN/ribavirin therapy. These results will be correlated with oligonucleotide microarray and quantitative RT-PCR assays to determine whether liver specimens with a high proportion of HCV-infected cells display transcriptional patterns indicative of hepatocellular proliferation and/or E2F transcription factor activation. RELEVANCE TO PUBLIC HEATH:,' Liver cancer is one qf the most rapidly increasing types of cancers in the United States, reflecting an increased prevalence ^nd risk of liver cancer in persons with chronic hepatitis C. This project seeks a better understanding of the interaction of HCV proteins with important regulators of cell proliferation (pRb, p53, DDXS, and DDXS) anc( the role of such interactions in liver cancer caused by HCV infection. PERFORMANCE SITE