Differential Immediate Early Gene Expression in Conditional Hepatitis B Virus pX-transforming VersusNontransforming Hepatocyte Cell Lines

Abstract

We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1␣ and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1␤ and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation.Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. Hepatitis B virus (HBV)1 infection causes acute hepatitis in humans, 10% of cases resulting in chronic hepatitis (1), which is linked to development of hepatocellular carcinoma (HCC) (1); integrated HBV DNA is present in virtually all HBV-mediated liver cancers (2, 3). However, the mechanism of HBV-mediated hepatocarcinogenesis remains elusive.HBV encodes a 16.5-kDa protein termed the X antigen (pX) (4). pX is expressed during viral infection (5-7), is required for the viral life cycle (7), and is highly conserved in all oncogenic mammalian hepadnaviruses (8, 9). In contrast, the avian hepatitis virus, which lacks oncogenic potential, is devoid of an X open reading frame (10). pX promotes liver tumor formation in transgenic mice expressing high levels of pX (11), potentiates c-Myc-induced hepatocarcinogenesis in c-myc/pX bitransgenics (12), and acts as a tumor promoter in hepatocarcinogenesis (13). While the published data clearly implicate pX in HCC, the direct oncogenic effect of pX in hepatocyte transformation has not been demonstrated. Herein, we provide evidence demonstrating the causal link of pX expression to the transformation of immortalized hepatocytes and describe a conditional pX expression system that is amenable to the study of the mechanism of pX-mediated transformation.pX is a multifunctional protein, with reported activities affecting transcription (14), cell growth (15, 16), and apoptotic cell death (17, 18). Although pX does not directly bind doublestranded DNA, pX acts as a promiscuous transactivator (reviewed in Ref. 14), via interaction with several components of the transcriptional apparatus (19 -23). In addition, specific pXresponsive cis-acting elements have been identified, e.g. NF-B (24 -28), AP-1 (29 -34), AP-2 (29), and CRE sites (35-37). Transcriptional activation of the AP-1 and NF-B sites by pX is via cytoplasmic signaling pathways, including the protein kinase C pathway (31, 32) a...

Activated ras oncogene collaborates with HBx gene of hepatitis B virus to transform cells by suppressing HBx-mediated apoptosis

“…In this mouse model, HBx alone has no direct pathological e ect, but has accelerated tumor formation induced by c-myc. Likewise, HBx expression has been shown to lead to anchorage-independent growth of AML12 cell line, mouse hepatocyte cell line derived from TGF-a transgenic mouse (Tarn et al, 1999). In these cells, it is presumed that either c-myc or TGF-a collaborated with HBx to exhibit the transforming phenotype.…”

“…Numerous attempts have been made to examine the oncogenic potential of HBx in cell culture (Hohne et al, 1990;Oguey et al, 1996;Shirakata et al, 1989;Tarn et al, 1999). However, its transforming ability was barely measurable only when cells were immortalized by other oncogenes, such as SV40 T-antigen (Hohne et al, 1990) or TGF-a (Oguey et al, 1996;Tarn et al, 1999).…”

“…However, its transforming ability was barely measurable only when cells were immortalized by other oncogenes, such as SV40 T-antigen (Hohne et al, 1990) or TGF-a (Oguey et al, 1996;Tarn et al, 1999). Further, most transgenic mouse harboring the HBx gene did not result in serious liver diseases or tumors (Billet et al, 1995;Guidotti et al, 1995;Koike et al, 1994a;Lee et al, 1990).…”

The hepatitis B virus HBx protein induces adherens junction disruption in a src-dependent manner

“…To further con®rm the e ect of HBx on adherens junctions, we studied the distribution of cadherin in the 3pX and 4pX stable transfectants, derived from the murine hepatocytic cell line AML12, which express HBx in a tetracyclin-repressible manner (Tarn et al, 1999). The 3pX clone shows the features of a transformed di erentiated hepatocyte, with low HBx expression, whereas the 4pX clone displays a more dedi erentiated phenotype and higher expression of HBx (Tarn et al, 1999).…”

Stabilized β-catenin promotes hepatocyte proliferation and inhibits TNFα-induced apoptosis

“…A murine hepatocyte cell line AML12, established by Wu et al from a human transforming growth factor a (TGFa) transgenic mouse, 32 has been used as a cell model to study the tumorigenic activity of hepatitis B viral pX protein. 33 In this study, the investigators demonstrated that the pX protein was capable of transforming the immortalized hepatocytes as determined by anchorage-independent cell growth assay. In another study, Dumenco et al 34 showed that introduction of mutant TP53 conferred AML12 cell growth advantage rather than cell transformation.…”