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

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.

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.

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.)

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.