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


CONCLUSIONS
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.
Zhang
Wei-ying
WY
Department of Cancer Research, Key Laboratory of Molecular Microbiology and Technology of Ministry of Education, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.
Cai
Na
N
Ye
Li-hong
LH
Zhang
Xiao-dong
XD
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D018385:Centrosome / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008099:Liver / Q000166:cytology*; D008113:Liver Neoplasms / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D041681:NIH 3T3 Cells; D000077022:Survivin; D019098:Telomerase / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D016328:NF-kappa B; D000077022:Survivin; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D019098:Telomerase