ANALYSIS OF THE TUMORIGENICITY OF THE X-GENE OF HEPATITIS-B VIRUS IN A NONTRANSFORMED HEPATOCYTE CELL-LINE AND THE EFFECTS OF COTRANSFECTION WITH A MURINE P53 MUTANT EQUIVALENT TO HUMAN CODON-249

Chronic infection with hepatitis B virus (HBV) is associated with the development of human hepatocellular carcinoma (HCC), One of the HBV ge nes, HBx, may have transforming potential, but this issue is still the subject of controversy, One of the major difficulties in addressing t his question is the lack of a suitable in vitro model, We used a nontr ansformed, differentiated murine hepatocyte cell Line (AML12) to trans fect the HBx gene and examine its transforming capabilities, Because m utations of the p53 gene, in particular at codon 249, have been implic ated in HCC development in geographical areas with high incidence of t he tumor, we also studied the putative cooperative role in transformat ion between HBx and mutated p53 by cotransfecting HBx with a murine p5 3 mutant equivalent to human ser249 (ser246p53), Transfection with HBx plasmids containing the HBx gene under the control of two different p romoters resulted in fewer colonies than in control plasmids, The toxi c effect of HBx on colony formation was abolished by cotransfection wi th 246p53, suggesting that the inhibitory effect requires functionally intact p53, Clonal cell lines that stably expressed HBx messenger RNA (mRNA) (HBX lines) were tested for their growth characteristics and t heir ability to grow in soft agar and form tumors in nude mice, At pas sages 19-27 after transfection, one of four HBx-expressing lines showe d the capacity for anchorage-independent growth in soft agar and produ ced poorly differentiated hepatocellular carcinomas in 8 of 13 sites o f injection in nude mice, HEX lines as well as clonal cell lines of ce lls transfected with 246p53 (246 cell lines), cotransfected with HBx a nd 246p53 (246x lines) or transfected with control plasmids, were anal yzed by now cytometry to determine the fraction of cells in S phase (S PF), 246 and 246X Lines had similar SPFs that were approximately twofo ld greater than control or HEX Lines, 246x lines showed morphological changes in culture such as marked cellular hetero-geneity, cell crowdi ng, and the presence of multinucleated giant cells, but their tumorige nicity was not increased compared with the HEX lines, These data show that HBx has a weak tumorigenicity in murine hepatocytes and that the addition of mutation of p53 at codon 249 to HBx expression does not in crease tumorigenicity in AML12 cells.