Detection of integrated hepatitis B virus DNA in hepatocellular carcinoma cell lines by nonradioactive in situ hybridization

Abstract

A sensitive and specific nonradioactive in situ hybridization method capable of detecting single-copy integrated hepatitis B virus (HBV) DNA sequences in hepatocellular carcinoma (HCC) cell lines was developed. In situ hybridization of biotinylated HBV (adr, adw) DNA probes with nine different human HCC cell lines were carried out in 96-well microtiter plates. Integration was detected in HCC cell lines HCCM, Hep3B, huH-1, huH-4, and PLC/PRF/5. Detection of single-copy HBV DNA sequences was also achieved in Hep3B and huH-4. HCC cell lines HepG2, HUH-6, HUH-7, Mahlavu, and the non-HCC control MCF-7, gave clear negative results. These results show a 100% correlation with those obtained by Southern blot hybridization assay. The results demonstrate that nonradioactive detection of single-copy integrated HBV DNA sequences in HCC cell lines is possible by the method described, which has potential application for viral genome analysis requiring in situ hybridization.