Cooperation of tumor-derived HBx mutants and p53-249(ser) mutant in regulating cell proliferation, anchorage-independent growth and aneuploidy in a telomerase-immortalized normal human hepatocyte-derived cell line

Abstract

Hepatocellular carcinoma (HCC) is a common cancer, and hepatitis B virus (HBV) is a major etiological agent. Convincing epidemiological and experimental evidence also links HCC to aflatoxin, a naturally occurring mycotoxin that produces a signature p53-249(ser) mutation. Recently, we have reported that tumor-derived HBx variants encoded by HBV exhibited attenuated transactivation and proapoptotic functions but retained their ability to block p53-mediated apoptosis. These results indicate that mutations in HBx may contribute to the development of HCC. In this study, we determined whether tumor-derived HBx mutants along, or in cooperation with p53-249(ser), could alter cell proliferation and chromosome stability of normal human hepatocytes. To test this hypothesis, we established a telomerase immortalized normal human hepatocycte line HHT4 that exhibited a near diploid karyotype and expressed many hepatocyte-specific genes. We found that overexpression one of the tumor-derived HBx mutants, CT, significantly increased colony forming efficiency (CFE) while its corresponding wild-type allele CNT significantly decreased CFE in HHT4 cells. p53-249(ser) rescued CNT-mediated inhibition of colony formation. Although HHT4 cells lacked an anchorage independent growth capability as they did not form any colonies in soft agar, the CT-expressing HHT4 cells could form colonies, which could be significantly enhanced by p53-249(ser). Induction of aneuploidy could be observed in HHT4 cells expressing CT, but additionally recurring chromosome abnormalities could only be detected in cells coexpressing CT and p53-249(ser). Our results are consistent with the hypothesis that certain mutations in HBx and p53 at codon 249 may cooperate in contributing to liver carcinogenesis.

Figure 1

Establishment of hTERT-immortalized human hepatocyte-derived cell lines. (A) Morphological characteristics of primary cultured human hepatocytes and their hTERT-immortalized derivatives HHT3 and HHT4 at different passages. (B) Telomerase activities of HHT3 and HHT4 cells determined by the telomeric repeat amplification protocol (TRAP) assay. (C) Representative amplification products, as analyzed by electrophoresis in a non-denaturing 10% polyacrylamide gel, from (B) are shown. The primary human hepatocytes (HH1249) are included as a negative control, and hTERT-immortalized NHF cells (NHF-hTERT) as a positive control.

Figure 2

Characterization of HHT3 and HHT4 cells. (A) Representative phase-contrast cell image (a) and electron micrographs (b, c) of HHT4 cells are shown. Representative images of a mitotic HHT3 cell (d-f) and a mitotic HHT4 cell (g-i) analyzed by fluorescence in situ hybridization (FISH) with a whole chromosome 16 painting probe or whole chromosome 1 painting probe, respectively. (B) Expression profiles of hepatocyte-specific genes in HHT3 and HHT4 cells analyzed by cDNA microarray. The expression ratios of HHT3 or HHT4 cells over hTERT-immortalized normal human fibroblasts (NHF) are shown. Hepatocyte-specific genes were selected based on a SAGE transcript profile of freshly isolated and cultured normal primary human hepatocytes (33).

Figure 3

Effect of p53-249ser mutant or various tumor-derived HBx mutants on HHT4 cell growths. (A) Representative cultured plates with HHT4 cell colonies expressing various recombinant genes. (B) Colony formation efficiency of HHT4 cells expressing CT, CNT, MT, MNT or p53-259ser mutants. (C) Effect of p53-249ser on colony formation of HHT4 cells expressing CNT or CT.

Figure 4

Anchorage-independent growth of HHT4 cells stably expressing CT, MT or MNT with or without expression of p53-249ser mutant. (A) Representative colonies of a CT stably expressing HHT4 clone CT2-4 in soft agar or coexpressing p53-249ser. (B) Six CT-transduced clones, 3 MT-transduced clones and 2 MNT-transduced clones from HHT4 cells alone with 3 vector control clones were evaluated for colony formation in soft agar.

Figure 5

Expression of HBx in HHT4-derived clones stably transducing various HBx mutants analyzed by western blot (A) or qRT-PCR (B).

Figure 6

Aneuploidy in hTERT-immortalized normal human hepatocytes expressing a tumor-derived HBx mutant and p53-249ser hot-spot mutant. (A, B) Percent of metaphase cells derived from HHT4, CT2-4 (CT) and CT2-4-1 (CT+p53-249ser) cells with abnormal chromosome numbers. (C) Spectral karyotyping of a representative metaphase from CT-2-4-1 cells.