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

Abstract

Chronic infection with hepatitis B virus (HBV) is associated with the development of human hepatocellular carcinoma (HCC). One of the HBV genes, HBx, may have transforming potential, but this issue is still the subject of controversy. One of the major difficulties in addressing this question is the lack of a suitable in vitro model. We used a nontransformed, differentiated murine hepatocyte cell line (AML12) to transfect the HBx gene and examine its transforming capabilities. Because mutations of the p53 gene, in particular at codon 249, have been implicated in HCC development in geographical areas with high incidence of the tumor, we also studied the putative cooperative role in transformation between HBx and mutated p53 by cotransfecting HBx with a murine p53 mutant equivalent to human ser249 (ser246p53). Transfection with HBx plasmids containing the HBx gene under the control of two different promoters resulted in fewer colonies than in control plasmids. The toxic effect of HBx on colony formation was abolished by cotransfection with 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 their ability to grow in soft agar and form tumors in nude mice. At passages 19-27 after transfection, one of four HBx-expressing lines showed the capacity for anchorage-independent growth in soft agar and produced poorly differentiated hepatocellular carcinomas in 8 of 13 sites of injection in nude mice. HBX lines as well as clonal cell lines of cells transfected with 246p53 (246 cell lines), cotransfected with HBx and 246p53 (246x lines) or transfected with control plasmids, were analyzed by flow cytometry to determine the fraction of cells in S phase (SPF). 246 and 246X lines had similar SPFs that were approximately twofold greater than control or HBX lines. 246x lines showed morphological changes in culture such as marked cellular heterogeneity, cell crowding, and the presence of multinucleated giant cells, but their tumorigenicity was not increased compared with the HBX 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 increase tumorigenicity in AML12 cells.

Hepatitis B virus X mutants, present in hepatocellular carcinoma tissue abrogate both the antiproliferative and transactivation effects of HBx

“…Furthermore, ongoing HBx overexpression led to chromatin condensation and fragmentation, disruption of the nuclear membrane, and the appearance of hypoploid cells, thus demonstrating that HBx can, at least under certain experimental conditions, induce apoptosis. Our results agree with recent reports on the reduction of cell colonies Oguey et al, 1996), cell cycle arrest , inhibition of transformation as well as induction of (Chirillo et al, 1997;Kim et al, 1998;Terradillos et al, 1998) or sensitization (Su and Schneider, 1997) to apoptosis by HBx. On the other hand, they di er from those which showed cell cycle progression upon HBx expression (Benn and Schneider, 1995;Koike et al, 1994), inhibition of p53-dependent apoptosis (Elmore et al, 1997).…”

“…To study the e ects of HBx on cell growth, we performed colony formation assay in both differentiated murine hepatocytes from mice transgenic for transforming growth factor a(a-ML) (Oguey et al, 1996) and undi erentiated human Chang cells. Cells were transfected with HBx, p53 or empty parental vector (pcDNA3) carrying the neomycin gene.…”

“…a-ML (Oguey et al, 1996), Saos-2, NIH3T3, primary REF (Bio-Whittaker Bioproducts), and Chang cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. Transfections were carried out using the calcium phosphate co-precipitation technique.…”

“…Finally, previous studies point to a direct interaction of HBx with p53 (Feitelson et al, 1993;Truant et al, 1995;Wang et al, 1994) as well as DNA repair factor involved in nucleotide excision repair (Lee et al, 1995), suggesting a direct link to cellular DNA repair, cell growth and apoptosis. In fact, several reports have provided evidence that HBx does modulate apoptosis (Chirillo et al, 1997;Kim et al, 1998;Oguey et al, 1996;Terradillos et al, 1998) although the ®nal outcome of the pleiotropic e ects of HBx on cell cycle and viability is still highly debated. These di culties are thought to be largely due to its heterologous and ectopic expression in various cellular environments.…”

p53-independent apoptotic effects of the hepatitis B virus HBx protein in vivo and in vitro

“…Evidence that HBx expression is detrimental to cell viability was provided in earlier studies by the di culty in generating stable cell clones that express the X protein (Kim et al, 1998;Oguey et al, 1996). We succeeded in establishing permanent cell lines which stably expressed HBx by isolating hepatocytes from HBx/p53-null and HBx/AT-cyto-MET bitransgenic animals.…”