HBV integrants of hepatocellular carcinoma cell lines contain an active enhancer

Hepatitis B virus (HBV) infection is a major risk factor worldwide for the development of hepatocellular carcinoma (HCC). Integrated HBV DNA fragments , often highly rearranged, are frequently detected in HCC. In woodchuck, th e viral enhancer plays a central role in hepatocarcinogenesis, but in human s the mechanism of HBV oncogenesis has not been established. In this study we investigated the status of the viral enhancer in two human HCC cell line s, Hep3B and PLC/PRF/5 each containing one or more integrated HBV DNA fragm ents. Active enhancer was defined by virtue of its protein occupancy as det ermined by genomic in vivo DMS footprinting. In PLC/ PRF/5 cells, the HBV D NA was integrated in a cellular gene at chromosome 11q13, at a locus report ed to be amplified in many tumors. We show here that in both cell lines, th e integrated HBV DNA fragments contain an active enhancer-I. In particular, the occupation of the two previously defined basic enhancer elements, E an d EP, was prominent. While in both cell lines the same protein binds to the EP elements, the E element, however, is occupied in a cell-line specific m anner. In PLC/PRF/5 but not Hep3B, the prominent binding of an undefined pr otein was detected. Our data suggest that this protein is likely to be the fetoprotein transcription factor (FTF). The finding that enhancer sequences are conserved and functional in different cell lines suggests a selection pressure for their long-term maintenance. We therefore propose that the HBV enhancer-I might play a role in hepatocellular carcinogenesis.