Acta Pharmacologica Sinica (2009) 30: 1153-1161; doi: 10.1038/aps.2009.99; published online 6 July 2009

Aim: To explore the mechanism of hepatocarcinogenesis associated with the hepatitis B virus X protein (HBx), we investigated the role of HBx in transformation using human liver L-O2 cells stably transfected with HBx as a model.

Methods: Plasmids encoding HBx were stably transfected into immortalized human liver L-O2 cells and rodent fibroblast NIH/3T3 cells.  The expression of alfa-fetoprotein (AFP), c-Myc, HBx, and survivin in the engineered cells was examined by Western blotting.  The malignant phenotype of the cells was demonstrated by anchorage-independent colony formation and tumor formation in nude mice.  RNA interference assays, Western blotting, luciferase reporter gene assays and flow cytometry analysis were performed.  The number of centrosomes in the L-O2-X cells was determined by γ-tubulin immunostaining.  The effect of HBx on the transcriptional activity of human telomerase reverse transcriptase (hTERT) and hTERT activity in L-O2-X cells and/or 3T3-X cells was detected by the luciferase reporter gene assay and telomerase repeat amplification protocol (TRAP). 

Results: Stable HBx transfection resulted in a malignant phenotype in the engineered cells in vivo and in vitro.  Meanwhile, HBx was able to increase the transcription of the NF-κB, AP-1, and survivin genes and to upregulate the expression levels of c-Myc and survivin.  Abnormal centrosome duplication and activated hTERT were responsible for the transformation. 

Conclusion: Stable HBx transfection leads to genomic instability of host cells, which is responsible for hepatocarcinogenesis; meanwhile, transactivation by the HBx protein contributes to the development of hepatocellular carcinoma (HCC).  The L-O2-X cell line is an ideal model for investigating the mechanism of HBx-mediated transformation. 

Keywords: HBx; transformation; hepatoma; genomic instability; stable transfection