ESTABLISHMENT AND CHARACTERIZATION OF CELL-LINES CONSTITUTIVELY EXPRESSING HEPATITIS-B VIRUS X-PROTEIN

We prepared human hepatoma cell lines, which expressed the human hepat itis B virus-X gene product. The plasmid pMAMneo-X, containing an HBV- X gene promoter, an enhancer and a structural gene was constructed. Tr ansfected HBV-X gene integration and expression were detected by South ern and Northern blotting, as well as by chloramphenicol acetylase tra nsferase (CAT) assay using various kinds of promoter-CAT reporter syst ems. HBV-X protein expression in stable transfectants was confirmed by immunofluorescence microscopy. Transfected cell lines showed permanen t expression of HBV-X proteins. The HBV-X transfectant activated its t arget promoters in promoter-CAT constructs as reporters. The HBV-X tra nsfectant enhanced AP-1 transcription factor binding to its target DNA . Therefore, X-transfectants are not only stable, but also have specif ic biological functions. Cell cycle analysis by flow cytometry showed that the majority of the transfectant cells are arrested in the G1 or G2 phase of the cell cycle. These cell lines may be useful in analyzin g the biological functions of HBV-X and its functional role in the for mation of hepatocellular carcinomas. (C) 1998 Elsevier Science B.V.