Epigenetic repression of E-cadherin expression by hepatitis B virus x antigen in liver cancer.

Loss of E-cadherin is associated with acquisition of metastatic capacity. Numerous studies suggest that histone deacetylation and/or hypermethylation of CpG islands in E-cadherin gene (CDH1) are major mechanisms responsible for E-cadherin silencing in different tumors and cancer cell lines. The hepatitis B virus (HBV)-encoded X antigen, HBx, contributes importantly to the development of hepatocellular carcinoma using multiple mechanisms. Experiments were designed to test if in addition to CDH1 hypermethylation HBx promotes epigenetic modulation of E-cadherin transcriptional activity through histone deacetylation and miR-373. The relationships between HBx, E-cadherin, mSin3A, Snail-1 and miR-373 were evaluated in HBx expressing (HepG2X) and control (HepG2CAT) cells by western blotting, immunoprecipitation (IP), chromatin IP as well as by immunohistochemical staining of liver and tumor tissue sections from HBV-infected patients. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A and Snail-1 were detected. Reciprocal IP with anti-HBx and anti-mSin3A demonstrated mutual binding. Furthermore, HBx-mSin3A colocalization was detected by immunofluorescent staining. HBx downregulated E-cadherin expression by the recruitment of the mSin3A/histone deacetylase complex to the Snail-binding sites in human CDH1. Histone deacetylation inhibition by Trichostatin-A treatment restored E-cadherin expression. Mir-373, a positive regulator of E-cadherin expression, was downregulated by HBx in HepG2X cells and tissue sections from HBV-infected patients. Thus, histone deacetylation of CDH1 and downregulation of miR-373, together with the previously demonstrated hypermethylation of CDH1 by HBx, may be important for the understanding of HBV-related carcinogenesis.

RNA interference-mediated URG4 gene silencing diminishes cyclin D1 mRNA expression in HepG2 cells.

Up-regulated gene 4 (URG4), stimulated by HBxAg, is a novel gene located on chromosome 7 (7p13). The full-length URG4 clone is 3.607 kb and encodes a polypeptide of 922 amino acids, with a molecular weight of 104 kDa (GeneID: 55665). It promotes cell growth, growth factor-independent survival, and anchorage-independent growth in HepG2 cells, and it accelerates tumor formation in nude mice. Hence, URG4 may be a natural effector of HBxAg and a putative oncogene that contributes to multi-step hepatocarcinogenesis. Cyclin D1 is frequently over-expressed in hepatocellular carcinoma, exhibiting a number of malignant phenotypes. We found that down-regulation of URG4 through RNA interference-mediated silencing suppressed cell proliferation in HepG2 cells. Over-expression of URG4 up-regulated cyclin D1 mRNA expression, whereas RNA interference-mediated URG4 silencing diminished cyclin D1 mRNA expression in HepG2 cells. The data suggest that URG4 may play an important role in the development of hepatocellular carcinoma by partially regulating the expression of cyclin D1 and has potential for use as a therapeutic target for hepatocellular carcinoma.

Downregulation of E-cadherin by hepatitis B virus X antigen in hepatocellullar carcinoma.

Hepatitis B virus (HBV)-encoded X antigen (HBxAg) contributes to the development of hepatocellular carcinoma (HCC). A frequent characteristic of HCC is reduced or absent expression of the cell adhesion protein, E-cadherin, although it is not known whether HBxAg plays a role. To address this, the levels of E-cadherin were determined in HBxAg-positive and -negative HepG2 cells in culture, and in tumor and surrounding nontumor liver from a panel of HBV carriers. The results showed an inverse relationship between HBxAg and E-cadherin expression both in tissue culture and in vivo. In HBxAg-positive cells, E-cadherin was suppressed at both the mRNA and protein levels. This was associated with hypermethylation of the E-cadherin promoter. Depressed E-cadherin correlated with HBxAg trans-activation function, as did the migration of HepG2 cells in vitro. Decreased expression of E-cadherin was also associated with the accumulation of beta-catenin in the cytoplasm and/or nuclei in tissues and cell lines, which is characteristic of activated beta-catenin. Additional work showed that HBxAg-activated beta-catenin. Together, these results suggest that the HBxAg is associated with decreased expression of E-cadherin, accumulation of beta-catenin in the cytoplasm and nucleus, and increased cell migration, which may contribute importantly to hepatocarcinogenesis.

Activation of fibronectin gene expression by hepatitis B virus x antigen.

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 kappa B (NF-kappa B). To identify NF-kappa B responsive genes that are differentially expressed in HBxAg-positive cells, HepG2 cells were stably transfected with HBxAg, and then with pZeoSV2 or pZeoSV2-I kappa B alpha. When RNAs from each culture were compared by PCR-select cDNA subtraction, fibronectin (FN) mRNA was shown to be strongly down-regulated by I kappa B alpha. 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-kappa B-dependent. 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-kappa B and inactivation of p53, thereby contributing to the accumulation of FN in the liver over the course of chronic HBV infection.

Chronic ethanol consumption stimulates hepatitis B virus gene expression and replication in transgenic mice.

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.

A cellular gene up-regulated by hepatitis B virus-encoded X antigen promotes hepatocellular growth and survival.

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 hybridization 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 kappaB (IkappaBalpha) into HBxAg-positive HepG2 cells decreased the levels of messenger RNA (mRNA) and protein, suggesting that its up-regulation is nuclear factor kappaB (NF-kappaB) dependent. Hence, HBxAg activation of NF-kappaB may result in the up-regulation of a cellular protein that promotes growth factor-independent 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 hepatocellular carcinoma (HCC).

New animal models of hepatitis B and C.

The narrow host range of infection and lack of suitable tissue culture systems for the propagation of hepatitis B and C viruses are limitations that have prevented a more thorough understanding of persistent infection and the pathogenesis of chronic liver disease. With hepatitis B virus (HBV), this lack of knowledge has been partially overcome by the discovery and characterization of HBV-like viruses in wild animals. With hepatitis C virus (HCV), related flaviviruses have been used as surrogate systems for such studies. Other laboratories have developed transgenic mice that express virus gene products and/or support virus replication. Some HBV transgenic mouse models develop fulminant hepatitis, acute hepatitis, or chronic liver disease after adoptive transfer, and others spontaneously develop hepatocellular carcinoma (HCC), as in human infections. Among HCV transgenic mice, most develop no disease, but acute hepatitis has been observed in one model, and HCC in another. Although mice are not susceptible to HBV and HCV, their ability to replicate these viruses and to develop liver diseases characteristic of human infections provides new opportunities to study pathogenesis and develop novel therapeutics.

Hepatitis B virus in hepatocarcinogenesis.

Hepatitis B virus (HBV) is an important etiologic agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Although the mechanism whereby HBV causes HCC is not fully understood, it is likely that there are many relevant molecular pathways that contribute to the development of HBV-associated HCC. This review provides an overview of some of these proposed pathways and their relative importance. It also raises questions on basic and translational research that will signficantly contribute to the better understanding of underlying mechanisms, prevention, and treatment of this tumor type.

Hepatitis B virus transgenic mouse model of chronic liver disease.

A model for hepatitis B virus-associated chronic liver disease has been made using cloned hepatitis B virus DNA as a transgene in a severe combined immunodeficient host. These mice consistently support virus gene expression and replication. After adoptive transfer of unprimed, syngeneic splenocytes, these mice cleared virus from liver and serum, and developed chronic liver disease. This model will permit identification of the host and virus contributions to chronic liver disease in the absence of tolerance.

The translation initiation factor, hu-Sui1 may be a target of hepatitis B X antigen in hepatocarcinogenesis.

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 differences in gene expression that distinguish X positive from X negative cells by subtractive PCR. One differentially 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. Introduction 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 hepatocellular carcinoma.

Use of SV40 to immunize against hepatitis B surface antigen: implications for the use of SV40 for gene transduction and its use as an immunizing agent.

We have described a novel gene transfer system, in which replication-incompetent, T antigen-deleted simian virus-40 (SV40) is used as the transduction vehicle. We report here successful immunization using such an SV40-derived viral vector. Hepatitis B surface antigen (HBsAg) cDNA was cloned downstream of two tandem SV40 early promoters to yield a T antigen-deficient SV40 derivative, SV(HBS). Cultured TC7 cells were exposed to SV(HBS), and expression of HBsAg was detected 24 h later by Northern blot and RT-PCR analysis. Immunochemistry and Western blot analysis were also performed 24 h after infection to detect expression of HBsAg. Once it was ascertained that we could express HBsAg in this way, we used SV(HBS) to elicit anti-HBs. SV(HBS) was injected intraperitoneally or subcutaneously into mice every 4 weeks. These mice were bled every 2 weeks and their sera assayed for antibody activity against HBsAg and SV40. Production of anti-HBs was measured by ELISA and confirmed by Western blot analysis, both of which demonstrated significant levels of anti-HBs after the second injection. We also tested production of anti-SV40 antibodies by the ability of sera to neutralize SV(HBS) infectivity. We found no evidence of neutralization of SV(HBS) infectivity even after eight inoculations. Thus, replication-incompetent SV40 is itself not a strong antigen. Our data suggest that SV40-based transduction systems may be a useful vehicle for immunization and for other gene transfer applications when a need for multiple inoculations is anticipated.

4-Aminobiphenyl DNA damage in liver tissue of hepatocellular carcinoma patients and controls.

Epidemiologic studies have suggested that cigarette smoking is a risk factor for the development of hepatocellular carcinoma (HCC). To further investigate this relation, the authors measured levels of 4-aminobiphenyl-DNA adducts by an immunoperoxidase method in surgical liver tissues obtained between 1984 and 1995 from 105 Taiwanese patients with histologically confirmed HCC and 37 Taiwanese patients with metastatic liver tumors or intrahepatic stones. Information on clinicopathologic characteristics, cigarette smoking, and alcohol drinking was abstracted from hospital charts. Mean relative staining intensity for 4-aminobiphenyl-DNA was slightly higher in tumor tissues than in nontumor tissues obtained from HCC patients. Both mean intensities were significantly higher than the mean intensity of control tissues taken from non-HCC patients. However, no difference in mean relative staining intensity was found between smokers and nonsmokers in tissues obtained from non-HCC patients, or in tumor or nontumor tissues taken from HCC cases. After stratification of the relative staining intensities of 4-aminobiphenyl-DNA adduct levels into tertiles according to the total numbers of control tissues analyzed, there was a monotonically increasing risk of HCC. Odds ratios were 4.14 (95% confidence interval (CI) 1.15-15.50) and 9.71 (95% CI 2.82-34.86) for medium and high adduct levels compared with low adduct levels, respectively. The linear relation between adduct levels in liver tissue and HCC risk was also significant after adjustment for covariates, including hepatitis B surface antigen (HBsAg) status. The multivariate adjusted odds ratios were 3.41 (95% CI 0.82-14.25) and 6.48 (95% CI 1.59-26.50) for medium and high adduct levels, respectively. Moreover, there were monotonically increasing HCC risks for higher adduct levels in both HBsAg carriers and noncarriers. The increased risk ratios were more pronounced in noncarriers than in carriers. However, because of the small numbers of subjects, especially controls positive for HBsAg, the interaction between HBsAg status and 4-aminobiphenyl-DNA adduct level was not significant. Among HCC cases, none of the clinicopathologic characteristics were associated with relative staining intensity. These results indicate that 4-aminobiphenyl exposure, which is primarily a result of cigarette smoking, plays a role in the development of HCC in humans.

Identification of a protein isolated from senescent human cells that binds to hepatitis B virus X antigen.

Hepatitis B virus-encoded X antigen contributes to the development of hepatocellular carcinoma. Given that X antigen functions by binding to other proteins, additional X-binding proteins were sought from an adult human liver cDNA library in a yeast two-hybrid system. The results yielded a clone encoding a 55-kd protein that is associated with replicative senescence (p55sen). Binding of p55sen to X antigen was confirmed in vitro by immunoprecipitation and affinity chromatography. The expression of endogenous p55sen inversely correlated with cell growth. Transient transfection of X antigen or p55sen into HepG2 cells stimulated DNA synthesis by twofold to threefold, whereas cotransfection did not, suggesting that these molecules functionally interact. The detection of p55sen in embryonic mouse liver, its absence in adult mouse and human livers, and its reappearance in livers from carriers with chronic liver disease, suggest that it may play important roles in the regulation of liver cell growth. The similarity between p55sen and a notch ligand, which is involved in cell fate determinations during embryogenesis, implies that the binding of p55sen by X antigen may also contribute to an alteration in cell fate, which is characteristic of carcinogenesis.

Hepatitis B x antigen and p53 in the development of hepatocellular carcinoma.

Hepatitis B virus (HBV) is one of the major etiological agents responsible for the appearance of chronic liver diseases, including hepatocellular carcinoma (HCC). There is increasing evidence that the HBV excoded x antigen (HBxAg) is involved in one or more steps that contribute to multistep hepatocarcinogenesis. Recent work has now defined one of these steps as the physical binding and functional inactivation of the tumor suppressor protein, p53, by HBxAg. The centrality of p53 to genomic stability, cell cycle arrest, induction of apoptosis, and in senescence related pathways, suggests that its disruption by HBxAg will result in genomic instability, loss of cell cycle control, a lower apoptotic rate, and an extension in the life span of HBV-infected cells. It is proposed that HBxAg/p53 complex formation represents one of several steps whereby HBV contributes to the development of HCC.

Partial characterization of the woodchuck tumor suppressor, p53, and its interaction with woodchuck hepatitis virus X antigen in hepatocarcinogenesis.

Full length cDNAs for p53 were made by reverse transcription-polymerase chain reaction of total RNA from two normal woodchuck livers. Two randomly chosen clones from each liver were sequenced and shown to be identical. This sequence revealed 80% or more identity with p53 sequences from human, monkey, and mouse. The cDNA was translated into a 55 kD protein in vitro that was immunoprecipitated by antibodies to p53. Cotranslation of woodchuck p53 with woodchuck hepatitis virus X antigen, followed by immunoprecipitation suggested X/p53 complex formation. Similar complexes were also immunoprecipitated from extracts of infected liver, but not from uninfected liver. The finding of X/p53 complexes in vivo and in vitro in the woodchuck hepadnavirus system, combined with analogous data with hepatitis B, suggests a common mechanism by which these viruses contribute to hepatocellular transformation.

Direct synthesis of full length p53 cDNA from frozen liver.

RNA secondary structures can inhibit efficient first strand synthesis in the construction of cDNA clones. Here we report a procedure that incorporates an initial computer analysis of potential secondary structures in related mRNAs. Then a modified, direct reverse transcription-polymerase chain reaction (RT-PCR) procedure optimized for obtaining a full length cDNA starting from a small amount of frozen tissue, without the need for a library construction, is performed. This modified approach is compared to the gene-specific first strand primer method. In addition, the first strand obtained by each of these methods is amplified using two different types of PCR. The utility of this procedure is demonstrated by the successful synthesis of a full length 1.2 kb p53 cDNA by both methods of PCR, from total RNA isolated from woodchuck liver.

Hepatitis B virus X antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma.

Chronic infection with hepatitis B virus is associated with a high incidence of liver diseases, including hepatocellular carcinoma. Hepatitis-B-virus-encoded X antigen (HBxAg) stimulates virus gene expression and replication, which may be important for the establishment and maintenance of the chronic carrier state. Integration of viral DNA encoding HBxAg during chronic infection results in increased X antigen expression. HBxAg overexpression may alter signal transduction pathways important for the regulation of cell growth during hepatocellular regeneration. The finding that HBxAg binds to and inactivates negative growth-regulatory molecules, such as the tumor suppressor p53, suggests additional ways that HBxAg may act in hepatocarcinogenesis. HBxAg may also stimulate the expression of positive growth regulators, such as insulin-like growth factor II and the insulin-like growth factor I receptor. The finding that HBxAg may compromise DNA repair and that it may effect the normal turnover of growth-regulatory molecules in the proteasome may also contribute to its carcinogenic properties. Hence, HBxAg may contribute to the pathogenesis of chronic infection and development of hepatocellular carcinoma in a variety of ways.

Integrity of p53 in hepatitis B x antigen-positive and -negative hepatocellular carcinomas.

Inactivation of the tumor suppressor p53 seems to be important to the pathogenesis of hepatocellular carcinoma (HCC) associated with chronic hepatitis B virus infection. Although this inactivation may be due to mutations in the p53 gene, recent evidence suggests that the hepatitis B virus-encoded X antigen (HBxAg) binds to and inactivates wild-type p53. Hence, experiments were designed to test the hypothesis that there is a low frequency of p53 mutations in HBxAg-positive HCC. HBxAg and p53 were assayed by immunohistochemistry (IHC) in HCC and nontumor liver from 16 Chinese patients, half of whom were hepatitis B surface antigen carriers. HBxAg was detectable in tumor and/or nontumor cells from all patients by IHC; six of these samples also had detectable p53. To determine whether p53 detection by IHC, and hence stabilization, is associated with mutation, sequencing of p53 exons 5-8 was performed with each patient sample. Wild-type sequences were found in 13 of 16 HBxAg-positive cases (81%). Hence, HBxAg is a common marker of HCC that correlates with the persistence of wild-type p53 among both carriers and noncarriers. The low frequency of p53 mutations in HCC in these patients implies that p53 inactivation may occur predominantly by complex formation with HBxAg.

Interactions between alcohol and hepatitis viruses in the liver.

There is a high frequency of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections among individuals with alcoholic liver disease (ALD) and alcohol-associated hepatocellular carcinoma (HCC), suggesting that these viruses are implicated in the pathogenesis of these diseases. Alcohol may act synergistically by promoting the development and progression of liver disease. The interactions of alcohol with infected hepatocytes and with antiviral immunity may result in altered patterns of virus gene expression and replication, making diagnosis difficult in some cases of ALD and HCC. The potential association of ALD and alcoholic HCC with serologically negative virus variants raises major difficulties in the epidemiologic assessment of cause and effect, providing major challenges for the future.

Hepatocellular injury in hepatitis B and C virus infections.

Most of the liver cell injury in hepatitis B and C infections is likely to be immune-mediated. Variation in the pathogenesis of these infections likely is contributed by a variety of host and virus factors. Host factors include the human leukocyte antigen (HLA) haplotype as well as the ability of the host to both recognize antigen on virus-infected cells and to receive the appropriate co-stimulatory signals in a timely fashion during infection. Virus factors include the genetic variation, direct cytopathic effects, and the alteration of infected hepatocytes to cytotoxic cytokines. The lack of suitable tissue culture systems and animal models limits the ability to understand the pathogenesis fully but provides challenges for their future development so that the basis for liver cell damage can be elucidated and approaches for therapeutic intervention can be achieved.