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.
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.
Jiang
Weidong
W
Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Wang
Xin Wei
XW
Unger
Tamar
T
Forgues
Marshonna
M
Kim
Jin Woo
JW
Hussain
S Perwez
SP
Bowman
Elise
E
Spillare
Elisa A
EA
Lipsky
Michael M
MM
Meck
Jeanne M
JM
Cavalli
Luciane R
LR
Haddad
Bassem R
BR
Harris
Curtis C
CC
D000782:Aneuploidy; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002871:Chromosome Banding; D003114:Colony-Forming Units Assay; D020869:Gene Expression Profiling; D006515:Hepatitis B virus; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009154:Mutation / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D032681:Spectral Karyotyping; D019098:Telomerase / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
D015415:Biomarkers; D012333:RNA, Messenger; C495901:TP53 protein, human; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D019098:Telomerase