Cooperation of tumor‐derived HBx mutants and p53‐249ser 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.