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PubMed1991

[Hepatitis B virus and hepatocellular carcinoma].

Hepatocellular carcinoma in woodchuck were characterized for woodchuck hepatitis virus integration nea c-myc oncogene. In one tumor, viral integration resulted in overexpression of a c-myc viral cotranscript. In a second tumor, viral insertion, 600 bp upstream of c-myc exon 1, was associated with increased levels of normal c-myc mRNA. These results demonstrate that integration of woodchuck hepatitis virus near a proto-oncogene can contribute to the genesis of liver tumors. From a comparison of a single hepatitis B virus (HBV) integration site in a human hepatoma with the corresponding unoccupied site have shown HBV DNA insertion in a putative cellular exon. This exon presented striking similarity to the DNA-binding domain of the thyroid/steriod hormones receptors. The corresponding cDNA has been isolated (hap gene) as shown to encode the retinoic acid receptor. It is most probable that consequent to HBV insertion, hap gene became inappropriately expressed as an altered chimaeric gene retinoic acid receptor, thus contributing to the cell transformation. As for woodchuck these results strongly support the possibility that HBV, may play a direct role in liver carcinogenesis by insertional mutagenesis.

Amino Acid Sequence↗

PubMed2001

Analysis of integrated hepatitis B virus DNA and flanking cellular sequence by inverse polymerase chain reaction.

Although hepatitis B virus (HBV) DNA has been detected in the human hepatoma cell line, HAGS 2.1, viral and cellular junction sequences have not been investigated fully. To facilitate the analysis of HBV DNA integration sites in HAGS 2.1 cells, a combination of conventional polymerase chain reaction (PCR) and inverse PCR (IPCR) was carried out to identify the junction between the viral and the cellular gene. The HBV integrant and its cellular counterpart sequence were cloned and analyzed. The sequencing data indicated that the breakpoints on the HBV integrant are at nucleotide 2111 of the C gene and nucleotide 1558 of the X gene. The length of the integrated HBV DNA in HAGS 2.1 was approximately 2.6 kb, which includes partial C, P, and X genes and an intact S gene. The cellular sequence flanking the integrated HBV gene was very similar to a human satellite III repetitive sequence with 43 and 56 of GGAAT repeats on the left- and right-hand side, respectively. Although the findings on the viral-cellular junction in HAGS 2.1 cells cannot explain the liver tumorigenesis, the current study shows that by choosing the nearest restriction site, which can be determined by conventional PCR rather than using a unique site within the integrated viral sequence to do IPCR, gives a higher successful rate for cloning and the subsequent analysis of the viral-cellular junctions.

Cloning, Molecular↗

PubMed1988

Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome.

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication.

Animals↗

PubMedDate not supplied

[IMPACT OF THE IMMUNIZATION PROGRAM INTEGRATED TO THE EXPANDED IMMUNIZATION PROGRAM(EPI) IN HUANTA,1994-1997]

INTRODUCTION: Huanta is an interandean valley at 2,400 meters above sea level in the peruvian highlands. It is hyperendemic for HBV, and deaths related to HBV such a fulminant hepatitis, cirrhosis and hepatic carcinoma make up 8% of the total mortality. A pilot program of inmunization against HBV integrated with the Expanded Immunization Program (EPI) was established in 1994, so as to limit the incidence if HBV-HDV, and as a strategy to improve EPI coverages.MATERIALS AND METHODS: A total of 1,412 children under 1 year old and 5,175 children from 1 to 4 years old were scheduled for vaccination. Three doses of the recombinant DNA vaccine agains HBV were used for each child. The schedule was adapted to the EPI vaccination calendar. In children under a year the schedule was: newborns: BCG, Polio, HBVI; 2 months: Poliol DPTI, HBV2; 3 months: Polio 2, DPT2; 4 months: polio 3, DPT3, HBV3; 9 months: Measles. In the group of children from 1 to 4 years old, the schedule was: HBVI at child recruitment; HBV2: after 2 months of the first one, HBV3: after 6 month of the first one.RESULTS: One year after starting, 3 dose immunizations have been made in 1,386 (98.1%) children under one year old and 4,353 (84.1%) in children from 1 to 4 years old. No important side effects related to the HB vaccine have been recorded; one case of HAV and two of HBV occurred in children who were beginning their immunization schedule. The objective of improving vaccination coverage by the EPI was achieved; the coverage in children under one year old for DPT were 76% (1991), 64.5% (1992), 55.2% (1993), and as a result of the strategy the coverage was improved to 98.1%. The program efficacy is demonstrated by the significative reduction of the infection rates of children 3-4 years old in 1994 (24.4-30.4%) compared with the children infection rates of the same age in 1997 (2.3-5.1 %).CONCLUSION: Including the HBV vaccine within the EPI program in a hyperendemic area for HBV-HDV has improved the EPI coverages; the vaccination compaign strategy has shown its effectiveness and safety, showing impact in the reduction of infection rates.

Journal Article↗

PubMed1991

[Hepatitis B virus and hepatocellular carcinoma].

Hepatocellular carcinomas in woodchuck were characterized for woodchuck hepatitis virus integration near c-myc oncogene. In one tumor, viral integration resulted in overexpression of a c-myc viral cotranscript. In a second tumor, viral insertion, 600 bp upstream of c-myc exon 1, was associated with increased levels of normal c-myc mRNA. These results demonstrate that integration of woodchuck hepatitis virus near a proto-oncogene can contribute to the genesis of liver tumors. From a comparison of a single hepatitis B virus (HBV) integration site in a human hepatoma with the corresponding unoccupied site have shown HBV DNA insertion in a putative cellular exon. This exon presented striking similarity to the DNA-binding domain of the thyroid/steroid hormones receptors. The corresponding cDNA has been isolated (hap gene) a shown to encode the retinoic acid receptor. It is most probable that consequent to HBV insertion, has became inappropriately expressed as an altered chimaeric gene retinoic acid receptor, thus contributing to the cell transformation. As for woodchuck these results strongly support the possibility that HBV may play a direct role in liver carcinogenesis by insertional mutagenesis.

Amino Acid Sequence↗

PubMed1988

Effect of recombinant gamma interferon on hepatocellular carcinoma cell lines.

The effects of recombinant gamma interferon (rIFN-gamma) were tested on 4 different hepatocellular carcinoma (HCC) cell lines PLC/PRF/5, HepG2, Hep3B and SK-Hep1. Exposure of these HCC cells to 100/ml of rIFN-gamma induced high levels of major histocompatibility complex (MHC) class I expression on their membrane, but not class II antigens. The enhancement of class I antigen synthesis occurs without a corresponding increase in synthesis of HBsAg by HBV genome positive PLC/PRF/5 and Hep3B cells. A 5 log dose of rIFN-gamma also did not trigger the integrated HBV genome into active replication as indicated by a lack of HBcAg and HBeAg synthesis. These results point to a possible role for the use of rIFN-gamma to enhance tumour cell recognition by the host immune system via the increased MHC class I expression.

Carcinoma, Hepatocellular↗

PubMed2004

Cyclooxygenase-2 pathway correlates with vascular endothelial growth factor expression and tumor angiogenesis in hepatitis B virus-associated hepatocellular carcinoma.

Evidence indicates that cyclooxygenase (COX)-2-derived prostaglandins (PGs) contribute to tumor growth by inducing angiogenesis. We investigated the role of COX-2 in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). COX-2 and vascular endothelial growth factor (VEGF) expressions were examined by immunohistochemistry in 24 HBV-associated HCC. Tumor micro-vessel density (MVD) was assessed using CD34 immunohistochemistry. Hep3B HCC cell line, which carries integrated HBV genome, was stably transfected with human COX-2 cDNA. COX-2 and VEGF expressions were determined by Western blot while PG level was determined by ELISA. The effects of PGs on VEGF expression were also investigated. Expression of COX-2 and VEGF in HCC cells were observed in 19 (79%) and 16 (67%) cases, respectively. Well-differentiated HCC expressed COX-2 more strongly than less-differentiated HCC (p<0.001). COX-2 expression was found to correlate with VEGF expression and MVD (p=0.003 and 0.004, respectively). COX-2 overexpressing Hep3B clone had higher VEGF expression as compared to non-COX-2 expressing clone and parental cells. Treatment of the COX-2 overexpressing cells with a COX-2-selective inhibitor, NS-398 (10 microM), decreased PGE2 level and attenuated VEGF expression. Addition of PGE2 (10 microM) and the stable analog of PGI2, carbaprostacyclin (5 microM), to Hep3B cells also increased VEGF expression. Up-regulation of COX-2 correlates with VEGF expression and tumor angiogenesis in HBV-associated HCC. Moreover, COX-2 up-regulates VEGF expression in HCC cells, possibly via PGs production. Selective inhibition of COX-2 may block HCC associated angiogenesis and thus provides a rational approach for treatment of this malignancy.

Adolescent↗

PubMed1994

Hepatitis B virus infection, the immune response and hepatocellular carcinoma.

More than 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and have a 200-fold increased risk of developing hepatocellular carcinoma (HCC). This is one of the most common cancers in the world with a geographical distribution highest in areas where HBV is endemic. A number of molecular mechanisms have been proposed to explain this correlation including an acutely transforming viral oncogene; chromosomal aberrations due to HBV integration (i.e. deletions, translocations, duplications); activation of cellular proto-oncogenes; inactivation of cellular anti-oncogenes; and transactivation of cellular genes by HBV gene products. HCC usually develops only after 20-30 years of persistent HBV infection accompanied by hepatocyte necrosis, inflammation and regenerative hyperplasia. Because HBV is not directly cytopathic, liver injury must be immune mediated. Factors that predispose HBV-infected individuals to develop HCC are chronicity, an immune response and liver injury rather than a direct genetic event. Hepatic injury and continuous hepatocyte regeneration may allow an accumulation of multiple mutational events sufficient for the emergence of HCC. Pathways which lead to chronicity, the immune response during HBV infection, mechanisms of pathogenesis and methods to prevent HBV infection are all relevant to the development of HCC. Recent studies characterizing the humoral and cellular immune responses in patients chronically infected with HBV and transgenic mouse models of HBV-specific immune tolerance and pathogenesis are providing new insights into the complex association between HBV infection and HCC.

Animals↗

PubMedDate not supplied

The significance of detailed hepatitis B virus serology in chronic liver diseases.

Hepatitis B virus (HBV) markers were studied with Sorin RIA kits in serum samples from 390 patients suffering from histologically confirmed chronic liver disease. On the basis of negative HBsAg, anti-HBs, anti-HBc tests, HBV infection was excluded in 235 of the cases. The diagnosis was fatty liver and/or alcoholic hepatitis in 52%, while chronic active hepatitis and/or liver cirrhosis only in 21.7%. Part or present HBV infection was proven in 155. In 53% of these cases the diagnosis was chronic active hepatitis and/or liver cirrhosis, whereas fatty liver and alcoholic hepatitis occurred in 27.7%. Detailed HBV marker analysis was performed in 76 patients. Previous infection without replication (positive anti-HBs and/or anti-HBc and/or anti-HBe) was proven in 48 cases, 12 patients had active HBV infection (positive HBsAg, HBe, IgM anti-HBc), while in 16 cases HBV integration (positive HBsAg, anti-HBc, anti-HBe) was proven. HBsAg-IgM complex seropositivity was shown in every case with active HBV replication. Because of therapeutic, prognostic and epidemiologic reasons, the significance of detailed HBV serology in chronic liver diseases is stressed.

Adolescent↗

PubMed1996

Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus.

Three well differentiated SV40-immortalized rat hepatocyte cell lines, CWSV1, CWSV2, and CWSV14, and Hepatitis B Virus (HBV)-producing cell lines derived from them were examined for sensitivity to tumor necrosis factor (TNF)-alpha. CWSV1, CWSV2, and CWSV14 cells were co-transfected with a DNA construct containing a dimer of the HBV genome and the neo gene and selected in G418 to generate stable cell lines. Characterization of these cell lines indicated that they contain integrated HBV DNA, contain low molecular weight HBV DNA compatible with the presence of HBV replication intermediates, express HBV transcripts, and produce HBV proteins. The viability of CWSV1, CWSV2, and CWSV2 cells was not significantly altered when they were treated with TNF-alpha at concentrations as high as 20,000 U/ml. The HBV-expressing CWSV1 cell line, SV1di36, and the HBV-expressing CWSV14 cell line, SV14di208, were also not killed when treated with TNF-alpha. However, the HBV-expressing CWSV2 cell line, SV2di366, was extensively killed when treated with TNF-alpha at concentrations ranging from 200 to 20,000 U/ml. Analysis of several different HBV-producing CWSV2 cell lines indicated that TNF-alpha killing depended upon the level of HBV expression. The TNF-alpha-induced cell killing in high HBV-producing CWSV2 cell lines was accompanied by the presence of an oligonucleosomal DNA ladder characteristic of apoptosis.

Animals↗

PubMed1989

Demethylation by 5-azacytidine results in the expression of hepatitis B virus surface antigen in transgenic mice.

In 14p3HB transgenic mice, which carry three tandem copies of hepatitis B virus (HBV) DNA, the HBV DNA was significantly methylated and no viral proteins were produced. To analyze the causal relationship between hypermethylation and gene inactivity, 5-azacytidine was injected into the mice to demethylate HBV DNA. When postnatal 14p3HB mice were treated with the drug, hepatitis virus surface antigen was produced in these mice by 3 weeks of age, and the integrated HBV DNA of the liver was less heavily methylated. Our results suggest that injection of 5-azacytidine can be used to efficiently activate a silent transgene such as HBV DNA in transgenic mice.

Animals↗

PubMed2003

[Activating effect of hepatitis B virus preS/S protein on proliferating cell nuclear antigen gene promoter].

BACKGROUND: PreS/S gene was derived from hepatitis B virus (HBV) integration fragment of human hepatocellular carcinoma genome, containing the promoter of preS/S and the C terminal truncated preS/S open reading frame. PreS/S protein may have important roles in processing hepatoma in some HBV-infected patients. The aim of the study was to study the activity of HBV preS/S protein on proliferating cell nuclear antigen (PCNA) promoter and to localize compartment of the preS/S protein in the liver cell line L02. METHODS: The authors studied the effect of the 3 -truncated preS/S on human PCNA promoter by co-transfecting the expression plasmids of luciferase reporter gene, used the immunohistochemical method to localize the preS/S protein. RESULTS: The expression product of the plasmid, pKSH7C-HpaI which contained the 3 -truncated preS/S and the flanking cellular sequences, stimulated the expression of PCNA promoter dose dependently,and its effect was 0.5 folds higher than control. Immunohistochemistry showed that the preS/S protein located in the cytosolic region of the liver cell line L02. CONCLUSIONS: The HBV preS/S protein could stimulate the PCNA promoter of the liver cell, its effect was not direct, which suggests that the effect of preS/S protein on PCNA promoter was probably through the cell signal transduction pathway.

Carcinoma, Hepatocellular↗

PubMed1991

Analysis of serum pre-S antigens in chronic hepatitis B virus infection in relation to the expression pattern of hepatitis B virus DNA in the liver.

Pre-S antigens have been analyzed in the serum of patients with chronic hepatitis B virus (HBV) infection according to the expression pattern of HBV DNA in the liver. Pre-S1 and pre-S2 have been identified (1) in all viremic cases with free replicative forms of viral DNA irrespective of the simultaneous detection of integrated sequences; (2) in 2 out of 3 patients with only integrated HBV DNA, and (3) in 19 patients who lacked viral DNA sequences detectable in the host genome. The amounts of hepatitis B surface and pre-S antigens were significantly higher in high-viremic versus low-viremic patients and correlated with the hepatocellular expression of HBV DNA. Conversely, the pre-S-to-hepatitis B surface antigen ratios were lower in the presence of viral DNA sequences in the liver. In summary, detection and level of pre-S antigens are closely related to the hepatocellular expression of viral DNA and seem to reflect reliably different stages of the virus life cycle during the course of HBV infection.

Adult↗

PubMed1990

[Characteristics of hepatocellular carcinoma in the non-cirrhotic liver].

Out of 99 histologically verified carcinomas (HCC), 73 (73.7%) belonged to HCC with associated cirrhosis and 26 (26.2%) belonged to HCC without cirrhosis. The relatively high incidence of HBsAg in the serum of patients with non-cirrhotic HCC (57.6%) shows that HBV infection persistence could be, one of the very important bases for HCC development, more than cirrhosis itself. The negative finding of HBsAg in the serum couldn't exclude the possibility of an integrated HBV genome in the cirrhotic tissue. There is an alternative possibility of an induced infection with hepatitis NANB (transfusions). A non-significant inclination towards younger age groups exists in non-cirrhotic HCC. Males are predominant in both groups, but females are more present in non-cirrhotic HCC than in cirrhotic HCC. The clinical course of non-cirrhotic HCC is usually slower; often there are no signs of portal hypertension or surrounding organ involvement, which offers better prognostic prospects than cirrhotic HCC. In relation to cirrhotic HCC, patients with non-cirrhotic HCC had a less frequent history of hepatitis (19.2%), a higher albumin-globulin ratio (1.5) and lower values in serum alpha-1 fetoprotein elevation (53.8%). Ultrasonographically, HCC without cirrhosis is presented with a mono-nodular shape in the majority of cases, or as a primary and dominant form, with surrounding satellite lesions even within the other lobe. Pathoanatomic findings usually show massive, compact, solid tissue formation alone, or accompanied by satellite lesions. Histologically, there is low-degree evolution with minimal necrosis and mild mitotic activity with a presence of minimal fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMedDate not supplied

Molecular aspects of persistent woodchuck hepatitis virus and hepatitis B virus infection and hepatocellular carcinoma.

It seems evident that the development of fully malignant HCC is a multistage process with many variables. One possible mechanism by which many of these variables may interact is as follows. During chronic active hepatitis, viral DNA integration occurs randomly and at a low frequency in hepatocytes. Integration may be stimulated by the increased rate of hepatocyte cell division resulting from liver necrosis and regeneration during chronic disease. The presence of viral integrations in the cellular genome provides focal points for the generation of chromosomal aberrations. One pathway by which these aberrations may be generated involves rearrangement of integrated viral and cellular sequences following viral DNA integration. The rearrangements which occur may include deletion, translocation, transposition or amplification of specific viral and cellular DNA sequences. We and others have directly demonstrated that all of these events are associated with different HBV integrations. The presence of viral integrations in chromosomes may also, by some unknown mechanism, destabilize those chromosomes such that whole chromosomes fail to segregate and are lost from particular cells. Preliminary studies we have conducted using restriction fragment length polymorphisms have revealed the loss of Chromosome 11 alleles in several HCC, indicating that chromosome loss may be a common occurrence in HCC. Our studies with restriction fragment length polymorphisms support such a mechanism involving Chromosome 11 in HCC. Specific chromosomal aberrations associated with all HCCs have not yet been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Persistence of hepatitis B virus and establishment of delta virus infection.

A typical feature of an infection with the hepatitis B virus (HBV) is the development of a persistent infection manifested by a chronic carriership of hepatitis B antigen (HBsAg). There is a marked geographical variation in the frequency of the chronic HBsAg carrier state. The replication of the HBV is unique for a DNA virus and has functional characteristics of a retrovirus, i.e. replication of viral DNA through an RNA intermediate. In addition to episomal viral DNA involved in virus replication other episomal forms and integrated viral DNA have been found in infected cells. The integrated HBV DNA in liver cells is an essential factor in the development of hepatocellular carcinoma. Many epidemiological studies have made it clear that age, sex and immunological status are important factors predisposing to the development of chronic infection. Hepatitis D virus (HDV) is a distinct infectious agent but it is defective as it requires the helper function of HBV for its replication. There are two possible modes of infection, either a coinfection, i.e. simultaneous infection with HBV and HDV, or a superinfection, i.e. a chronic HBsAG carrier who becomes infected with HDV. The majority (70-90%) of all superinfections take a chronic course.

Carrier State↗

PubMed1990

Malignant transformation of immortalized transgenic hepatocytes after transfection with hepatitis B virus DNA.

Persistent infection by hepatitis B virus (HBV) is epidemiologically correlated with the prevalence of hepatocellular carcinoma, but its role in tumor development is not yet understood. To study the putative oncogenic potential of HBV, a non-malignant immortal mouse hepatocyte line FMH202 harboring metallothionein promoter-driven simian virus 40 large tumor antigen was transfected with HBV DNA. All stably transfected clones which replicated HBV displayed malignant growth characteristics in soft agar and were tumorigenic upon inoculation in nude mice. The nude mice tumors were histologically classified as differentiated or anaplastic hepatocellular carcinomas. As with human liver carcinomas, rearrangements of in vitro integrated HBV sequences were observed in the nude mouse tumors, and in tumor-derived cell lines. In one case, expression of viral core and surface antigens was blocked in the tumors, correlating with hypermethylation of the HBV genome. However, the expression of X gene was maintained in most tumors and tumor-derived cell lines. X protein was detected in nuclei by immune fluorescence and by immune blot. These results provide the first demonstration that HBV displays oncogenic potential in an experimental system. This system could be useful to functionally identify HBV genes which convey a tumorigenic phenotype.

Animals↗

PubMed2000

Hepatitis B virus surface (S) transactivator with DNA-binding properties.

Chronic infection with hepatitis B virus (HBV) in humans is strongly linked to the development of hepatocellular carcinoma (HCC). Activation of growth-regulatory genes may play a crucial role in carcinogenesis. Proto-oncogene expression has been shown to be higher in HCC tissue with integrated HBV DNA than in the normal liver. Earlier, we showed that the 3' end of the HBV major surface gene (S) (426-855 nucleotides of the S region) is a transactivator of the X promoter-enhancer regulatory element in co-transfection experiments. This region expresses a truncated carboxy terminal S protein extending from amino acid residues 102 to 226. In this study, the truncated S protein (trc-S) was examined for its enhancing activity on several viral and cellular regulatory elements. The results indicate that trc-S activates rous sarcoma virus long terminal repeat (LTR), human T-lymphotropic virus 2 LTR, human immunodeficiency virus 1 LTR, and the c-jun and c-fos promoters. Electrophoretic mobility shift assays carried out to investigate its DNA-binding properties established that trc-S binds to HBV X promoter and oligonucleotides representing binding sites for the AP1 and TFIID transcription factors. The specificity of this interaction was confirmed by using competition experiments and supershift assays. These experiments suggest that trc-S is a transactivator of several cellular and viral promoters and that this activity is mediated by direct interaction with DNA.

Avian Sarcoma Viruses↗