Transformation of human liver L-O2 cells mediated by stable HBx transfection

Acta Pharmacol Sin. 2009 Aug;30(8):1153-61. doi: 10.1038/aps.2009.99. Epub 2009 Jul 6.


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 gamma-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-kappaB, 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carcinoma, Hepatocellular / genetics*
  • Cell Line
  • Cell Proliferation
  • Cell Transformation, Neoplastic*
  • Centrosome / pathology
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Inhibitor of Apoptosis Proteins
  • Liver / cytology*
  • Liver Neoplasms / genetics*
  • Mice
  • Mice, Nude
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • NIH 3T3 Cells
  • Survivin
  • Telomerase / metabolism
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism
  • Transcription Factor AP-1 / genetics
  • Transcription Factor AP-1 / metabolism
  • Transfection*
  • Viral Regulatory and Accessory Proteins


  • BIRC5 protein, human
  • Inhibitor of Apoptosis Proteins
  • Microtubule-Associated Proteins
  • NF-kappa B
  • Survivin
  • Trans-Activators
  • Transcription Factor AP-1
  • Viral Regulatory and Accessory Proteins
  • hepatitis B virus X protein
  • Telomerase