Multiple lines of evidence indicate that chronic infection with hepatitis B virus (HBV) is causally related to hepatocellular carcinoma (HCC). Furthermore, there is increasing evidence that the recently identified hepatitis C virus (HCV) also plays a role in the development of HCC. A high percentage of the HBV-positive or HBV-negative patients with HCC is seropositive for anti-HCV antibodies indicating previous or current HCV infection. Premalignant cytologic changes of hepatocytes have been observed in patients with chronic hepatitis C. In recent studies using the polymerase chain reaction (PCR) we detected HCV RNA sequences in liver and tumor tissues from patients with HCC. These observations support the hypothesis that HCV may persist in hepatocytes as they progress from preneoplasia to neoplasia. It is the long term goal of our research to elucidate whether this RNA virus or subgenomic viral sequences, particularly in combination with HBV or other events such as p53 alterations, mediate malignant transformation. In this application we propose to determine by sensitive and specific techniques (PCR, ribonuclease protection assay, and in situ hybridization) the presence of HCV RNA sequences in non-neoplastic liver tissue, preneoplastic liver tissue, and tumor tissue of patients with or without chronic HCV and/or HBV infection. Next, we will investigate whether different levels or specific sequences of HCV are associated with defined preneoplastic and neoplastic liver lesions. These viral sequences will be cloned and sequenced and their oncogenic potential will be determined by transfection or retrovirus mediated gene transfer and transformation studies of several non-tumorigenic cell lines such as human fetal hepatocytes, immortalized transgenic mouse hepatocytes (FMH202) and HBV genome transfected cells. It is expected that these molecular studies in correlation with the histopathologic progression from normal hepatocytes to HCC will provide new insights into the pathogenesis of human HCC.