Lee Y, Bong Y, Poo H, Lee Y, Park J, Oh S, Sohn M, Lee S, Park U, Kim N, Hyun S
Molecular Cell Biology Research Division, Korea Research Institute of Bioscience and Biotechnology, Korea Institute of Science and Technology, Taejon, South Korea.
Gene. 1998 Jan 30;207(2):111-8. doi: 10.1016/s0378-1119(97)00603-3.
We prepared human hepatoma cell lines, which expressed the human hepatitis B virus-X gene product. The plasmid pMAMneo-X, containing an HBV-X gene promoter, an enhancer and a structural gene was constructed. Transfected HBV-X gene integration and expression were detected by Southern and Northern blotting, as well as by chloramphenicol acetylase transferase (CAT) assay using various kinds of promoter-CAT reporter systems. HBV-X protein expression in stable transfectants was confirmed by immunofluorescence microscopy. Transfected cell lines showed permanent expression of HBV-X proteins. The HBV-X transfectant activated its target promoters in promoter-CAT constructs as reporters. The HBV-X transfectant enhanced AP-1 transcription factor binding to its target DNA. Therefore, X-transfectants are not only stable, but also have specific 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 analyzing the biological functions of HBV-X and its functional role in the formation of hepatocellular carcinomas.
我们制备了表达人乙型肝炎病毒X基因产物的人肝癌细胞系。构建了含有乙肝病毒X基因启动子、增强子和结构基因的质粒pMAMneo-X。通过Southern和Northern印迹法，以及使用各种启动子-CAT报告系统的氯霉素乙酰转移酶（CAT）测定法，检测转染的乙肝病毒X基因的整合和表达。通过免疫荧光显微镜证实稳定转染子中乙肝病毒X蛋白的表达。转染细胞系显示出乙肝病毒X蛋白的永久表达。乙肝病毒X转染子在作为报告基因的启动子-CAT构建体中激活其靶启动子。乙肝病毒X转染子增强了AP-1转录因子与其靶DNA的结合。因此，X转染子不仅稳定，而且具有特定的生物学功能。通过流式细胞术进行的细胞周期分析表明，大多数转染细胞停滞在细胞周期的G1或G2期。这些细胞系可能有助于分析乙肝病毒X的生物学功能及其在肝细胞癌形成中的作用。
