HepG2 cells stably transfected with a full-length, infectious hepatitis C virus (HCV) cDNA demonstrated consistent replication of HCV for more than 3 years. Intracellular minus strand HCV RNA was present. Minus strand synthesis was NS5B dependent, and was sensitive to interferon alpha (IFNa) treatment. NS5B and HCV core protein were detectable. HCV stimulated HepG2 cell growth and survival in culture, in soft agar, and accelerated tumor growth in SCID mice. These mice becameHCVRNApositive in blood, where the virus was also sensitive to IFNa. The RNA banded at the density of HCV, and was resistant to RNase prior to extraction. Hence,HCVstably replicates inHepG2cells, stimulates epatocellular growth and tumorigenesis, and is susceptible to IFNa both in vitro and in vivo. J. Cell. Physiol. 201: 447-458, 2004.

The development of hepatocellular carcinoma (HCC) is a multistep process associated with changes in host gene expression, some of which correlate with the appearance and progression of tumor. reneoplastic changes in gene expression result from altered DNA methylation, the actions of hepatitis B and C viruses, and point mutations or loss of heterozygosity (LOH) in selected cellular genes. Tumor progression is characterized by LOH involving tumor suppressor genes on many chromosomes and by gene ampli

The role of hepatitis B virus X antigen in the development of hepatocellular carcinoma was explored by stably transfecting HepG2 cells with an X antigen expression vector, and identifying the ierences in gene expression that distinguish X positive from X negative cells by subtractive PCR. One ierentially expressed gene, the human homolog of sui1 (hu-sui1), encodes a translation initiation factor whose expression was suppressed by X antigen in HepG2 cells. Hu-Sui1 was also expressed in nontumor liver but not in tumor cells from patients with hepatocellular carcinoma. ntroduc-tion of hu-sui1 into HepG2 cells inhibited cell growth in culture, in soft agar, and partially inhibited tumor formation in nude mice. Hence, the suppression of hu-sui1 by X antigen may result in the abrogation of negative growth regulation and contribute to the development of epatocellular carcinoma.

Hepatitis B and related viruses that infect mammalian hosts encode the "X" protein that has been shown to contribute importantly to the pathogenesis of chronic liver disease (CLD) and to the evelopment of hepatocellular carcinoma (HCC). In a variety of tissue culture systems, hepatitis B virus (HBV) X antigen, or HBxAg, has been shown to trigger apoptosis, while other evidence suggests that HBxAg inhibits apoptosis and stimulates the cell cycle by constitutively activating a number of signaling pathways that are important for hepatocellular growth and survival. These apparently contrasting properties of HBxAg may be associated with differences in the X protein itself, since carboxy-terminaltruncated forms of HBxAg appear to be associated with HCC lesions. Alternatively, or in addition, these differences may be due to the cell type, state of cell differentiation, and whether expression occurs in resting or dividing cells. Further, the association between HBxAg expression and chromosomal instability, may also contribute to the apparently contrasting fates of HBxAg positive cells. It is proposed that in many of these systems, the different outcomes of HBxAg expression may be due to the nature of the cellular response to HBxAg, and not due to differences in the fundamental properties of HBxAg, the latter of which promote cell survival, cell cycle rogression, and the development of HCC.

The hepatitis B virus (HBV)-encoded X antigen (HBxAg) may contribute to the development of epatocellular carcinoma (HCC) through the upregulated expression of selected cellular genes. To identify these genes, RNAs isolated from HBxAg-positive and -negative HepG2 cells were compared by PCR select cDNA subtraction. One gene overexpressed in HBxAg-positive cells by Northern and Western blotting is the ribosomal protein S15a. The S15a mRNA is 535 base pairs, encoding a protein 130 amino acids long with a molecular weight of 14.3kDa. S15a expression was upregulated in HBV-infected livers, where it costained with HBxAg. Overexpression of S15a stimulated cell growth, colony formation in soft agar, and tumor formation in SCID mice. Hence, HBxAg upregulated the expression of S15a, the latter of which participates in the development of HCC, perhaps by altering the integrity of translation.

Epidemiologic observations show a higher frequency of hepatitis B virus (HBV) serologic markers in chronic alcoholics compared with the general population. This may be the result of an increased susceptibility of alcoholics to infection and/or to an ethanol-mediated stimulation of HBV gene expression and replication. To test the latter hypothesis, HBV transgenic SCID mice, which support consistent levels of virus replication, were fed with a standard Lieber-DiCarli or isocaloric diet for 5 weeks. In ethanol-fed mice, the levels of hepatitis B surface antigen (HBsAg) and viral DNA in serum increased by up to 7-fold compared with mice fed the control diet. Ethanol-treated mice also had elevated HBV-RNA levels, and increased expression of surface, core, and X antigens in the liver, especially in the pericentral regions. None of these changes were observed in transgenic mice fed isocaloric diets. Thus, chronic alcohol consumption alters the patterns of HBV gene expression and replication in the serum and liver of HBV transgenic SCID mice, and may provide a partial explanation for the increased frequency of HBV markers among alcoholics. (HEPATOLOGY 2001; 34: 792-797.)

Hepatitis B virus encoded X antigen (HBxAg) may contribute to the development of hepatocelIular carcin-oma (HCC) by up-or downregulating the expression of cellular genes that promote cell growth and urvival. To test this hypothesis, HBxAg-positive and-negative HepG2 cells were constructed. and the patterns of cellular gene expression compared by polymerase chain reaction select cDNA subtraction. The full-length clone of one of these upregulated genes (URG), URG4, encoded a rotein of about 104kDa. URG4 was strongly expressed jn hepatitis B-infected Iiver and in HCC cells. where it costained with HBxAg, and was weakly ex

Polymerase chain reaction (PCR) select complementary DNA (cDNA) subtraction of hepatitis B x antigen (HBxAg)-positive compared with -negative HepG2 cells resulted in the up-regulated expression of a cellular gene that encodes a transcript of 745 bases and a polypeptide 99 amino acids long. GenBank analysis revealed extensive homology with the amino terminal domain of cellular multidrug resistant proteins (MRP), although overexpression of this gene did not confer an MRP phenotype. In situ ybridization and immunostaining showed colocalized expression with HBxAg in the liver of hepatitis B carriers. Overexpression of this protein stimulated the growth of HepG2 cells in serum-free medium, and partially protected cells from anti-Fas-mediated killing, but did not promote growth in soft agar or tumor formation in nude mice. Introduction of the dominant negative inhibitor of nuclear factor B (I B) into HBxAgpositive HepG2 cells decreased the levels of messenger RNA (mRNA) and protein, uggesting that its up-regulation is nuclear factor B (NF-B) dependent. Hence, HBxAg activation of NF-B may result in the up-regulation of a cellular protein that promotes growth factor-ndependent survival and protects against Fas-mediated killing. This factor may contribute to the persistence of infected hepatocytes during chronic infection, which is important for the later development of epatocellular carcinoma (HCC). (HEPATOLOGY 2001; 34: 146-157.)

SUMMARY. The development of fibrosis and cirrhosis during chronic hepatitis B virus (HBV) infection correlates with the persistent expression of HBV x antigen (HBxAg), which acts in part, by stimulating selected signal transduction pathways, including nuclear factor jB (NF-jB). To identify NF-jB responsive genes that are differentially expressed in HBxAgpositive cells, HepG2 cells were stably transfected with HBxAg, and then with pZeoSV2 or pZeoSV2-IjBa. When RNAs from each culture were compared by PCR-select cDNA subtraction, fibronectin (FN) mRNA was shown to be strongly down-regulated by IjBa. Up-regulated expression of FN and co-expression between FN and HBxAg were observed in liver sections from HBV carriers that were stained for HBxAg and analysed for FN mRNA by in situ hybridization (ISH). In liver cell cultures, HBxAg increased the levels of FN mRNA and protein. This was because of the HBxAg-mediated trans-activation of the FN promoter, which was NF-jBdependent. HBxAg also antagonized the repression of the FN promoter by the tumour suppressor, p53. Hence, the FN gene may be a natural target for HBxAg trans-activation, perhaps through activation of NF-jB and inactivation of p53, thereby contributing to the accumulation of FN in the liver over the course of chronic HBV infection.