Inhibition of hepatocellular carcinoma development in hepatitis B virus transfected mice by low dietary casein

In a comprehensive human ecological study, primary liver cancer has been shown to be highly significantly associated with 1) the prevalence of persistent infection with hepatitis B virus (HBV) and 2) plasma cholesterol concentrations that are, in turn, associated with the consumption of animal based foods. In rat studies, aflatoxin-induced hepatocellular carcinoma is substantially prevented by decreasing the intake of animal based protein (casein), a hypercholesterolemic nutrient. Thus the development of primary liver cancer associated with persistent HBV infection or with aflatoxin exposure may be controlled by reduced intake of animal-based proteins. Transgenic mice transfected with an HBV gene fragment containing the viral transactivator of hepatis B virus, HBx, which induces the formation of hepatocellular carcinoma, were used to examine the ability of dietary casein to modify tumor formation. Reducing the concentration of dietary casein to 6% from the traditional level of 22% markedly inhibited (by 75%) hepatic tumor formation in these transgenic mice. Tumor development also was substantially altered by interchanging dietary casein concentration well after tumor development had begun (at 8 months), increasing by 173% from the expected yield when casein intake was increased and decreasing by 99% when casein was reduced. These findings suggest that the development of liver tumor formation among individuals persistently infected with HBV may be controlled by minimizing or eliminating the intake of animal protein-based foods.     

High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice

We studied the development of liver tumors in male HBx gene transgenic mice. Of two lineages studied, in the lineage with the lowest HBx gene expression liver tumors developed only in an incidence comparable with that in normal CD-1 strain, whereas 84% of male mice with a high level of the HBx gene product succumbed to liver neoplasia, indicating that continued HBx gene expression higher than a certain threshold level may be necessary for the development of hepatic neoplasia. Sixty-five mice from a lineage with a high level of HBx expression were then followed throughout their 24-mo lifespan. The livers of transgenic mice showed foci of cellular alteration with cytoplasmic vacuolations around the central veins from the age of 2 mo, but these foci did not expand progressively by the age of 12 mo. Immunostaining demonstrated such hepatocytes had higher expression of HBx protein than surrounding cells. Neoplastic lesions including liver cell adenomas and hepatocellular carcinomas developed from the age of 13 mo. By bromodeoxyuridine labeling analysis, hepatocytes in altered foci were found to have increased DNA synthesis, whereas no labeling was observed in age- and sex-matched nontransgenic littermate controls. Furthermore, DNA content analysis revealed the existence of several small aneuploid peaks in the transgenic liver before the age of tumor development. These results suggest that the continued expression of HBx gene may initiate a complex process to hepatocellular carcinoma by inducing DNA synthesis and placing large numbers of hepatocytes subjective to secondary events for transformation.     

In vitro transformation by hepatitis B virus DNA

There is strong epidemiological evidence that the hepatitis B virus (HBV) contributes to the development of hepatocellular carcinoma (HCC). In several immortalized cell lines, an in vitro transforming activity of HBV DNA and expression vectors for the viral protein X (HBx) has now been demonstrated. Furthermore, it appears as if still unknown parts of the HBV genome other than HBx contribute to the transforming activity of HBV DNA in vitro. Only one of several studies found that HBx-transgenic mouse lines develop HCC. A mouse line transgenic for the large surface protein of HBV develops HCC due to concomitant necroinflammatory infection. Growing evidence shows the importance of recombination of integrated viral DNA and cellular DNA for HCC development. A direct transforming potential of one of these viral integrates has been demonstrated. Chemical carcinogens are more effective in HBV-containing cell lines or transgenic mice.     

Regulation of hepatitis B virus mRNA expression in a hepatitis B surface antigen transgenic mouse model by IFN-gamma-secreting T cells after DNA-based immunization

The immunotherapeutic effect of DNA-mediated immunization against chronic hepatitis B virus (HBV) infection has been evaluated in transgenic mice expressing the sequences that code for the envelope proteins of HBV in the liver. In this model of HBV chronic carriers, a single i.m. injection of plasmid DNA encoding HBV envelope proteins is sufficient to generate specific immune responses leading to the clearance of the transgene expression product and the control of HBV mRNA. The relative contributions of the T cell subpopulations induced by DNA immunization were examined using adoptive transfer experiments. It was shown that either CD8+ or CD4+ T lymphocytes from immunocompetent DNA-immunized animals were sufficient to control viral gene expression in the livers of the recipient transgenic mice. This effect was mediated by a cytokine-dependent mechanism common to both T cell subpopulations; this mechanism did not require cell lysis, but did involve the production of IFN-gamma by the activated T cells.     

Molecular analysis of the major MHC recombinational hot spot located within the G7c gene of the murine class III region that is involved in disease susceptibility

The hepatitis B virus X protein (HBx) is a broadly acting transactivator implicated in the development of liver cancer. Recently, HBx has been reported to interact with several different cellular proteins, including our report of its binding to XAP-1, the human homolog of the simian repair protein UVDDB. In the present study, several HBx mutants were used to localize the minimal domain of HBx required for binding to XAP-1/UVDDB to amino acids 55 to 101. The normal function of XAP-1/UVDDB is thought to involve binding to damaged DNA, the first step in nucleotide excision repair (NER); therefore, we hypothesized that this interaction may affect the cell's capacity to correct lesions in the genome. When tested in two independent assays that measure NER (unscheduled DNA synthesis and host cell reactivation), the expression of HBx significantly inhibited the ability of cells to repair damaged DNA. Under the assay conditions, HBx was expressed at a level similar to that previously observed during natural viral infection and was able to transactivate several target reporter genes. These results are consistent with a model in which HBx acts as a cofactor in hepatocarcinogenesis by preventing the cell from efficiently repairing damaged DNA, thus leading to an accumulation of DNA mutations and, eventually, cancer. An adverse effect on cellular DNA repair processes suggests a new mechanism by which a tumor-associated virus might contribute to carcinogenesis.     

Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase

Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.     

Transactivation of human hepatitis B virus X protein, HBx, operates through a mechanism distinct from protein kinase C and okadaic acid activation pathways

Human hepatitis B virus (HBV) X protein, HBx, transactivates virus and host genes through a wide variety of cis-elements. Expression of HBx is controlled by HBV enhancer 1 (Enh1). Both Enh1 and the core sequence of Enh1, which consists of an AP-1 related site (cFAP1) and a C stretch, respond to HBx and a phorbol ester (TPA). Biochemical pathways of the responses to HBx and TPA are still controversial. We therefore asked whether HBx and TPA stimulate Enh1 core activity through a common process. Protein kinase C (PKC) inhibitors, H-7 and staurosporin, did not inhibit HBx transactivation at concentrations sufficient to abolish the TPA effects in HepG2 cells. Although HBx transactivation synergized independently with TPA or a phosphoprotein phosphatase inhibitor, okadaic acid (OA), the PKC inhibitors eliminated only the TPA contribution. HBx transactivation required both the cFAP1 and the C stretch of the Enh1 core region; however, mutations in either or both of the two cis-elements demonstrated that TPA augmentation required only cFAP1. These results imply that HBx transactivation operates through a mechanism distinct from the PKC and OA activation pathways.     

Pathogenetic effector function of CD4-positive T helper 1 cells in hepatitis B virus transgenic mice

The pathogenetic effector functions of hepatitis B virus (HBV)-specific CD4+, Th1 cells were analyzed in two inbred lineages of HBV transgenic mice, one of which overexpresses the HBV large envelope protein rendering the hepatocytes hypersensitive to the cytopathic effects of IFN-gamma, and another that expresses all of the HBV proteins and replicates the virus in the liver. Transfer of HBV envelope-specific Th1 cells resulted in recognition of viral Ag expressed by hepatic nonparenchymal cells, cytokine release, and a transient necroinflammatory liver disease in both lineages. The liver disease was very severe in the IFN-gamma-sensitive lineage, and it was less severe in the lineage that replicates the HBV genome; nonetheless, in this lineage the Th1 cytokines produced by these cells suppressed viral replication in the liver. These results demonstrate that CD4+ T cells with a Th1 functional phenotype can perform pathogenetic and antiviral effector functions in vivo. This suggests that CD4+ T cells can contribute directly to disease pathogenesis and inhibit viral replication during HBV infection.     

乙肝病毒X蛋白对肝细胞生物钟基因的影响及其意义

Objective: The aim of this study was to investigate the influence of hepatitis B virus X protein (HBx) on the clock genes in LO2 cells and its significance. Methods: A cell line LO2-HBx, Stably transfected with HBx gene, was established. The levels of mRNA and protein expression of CLOCK and BMAL1 were detected by real-time PCR and western blot. Results: The expression of CLOCK mRNA and protein were increased in cell line LO2-HBx (P < 0.05), while the expression of BMAL1 mRNA and protein were decreased in cell line LO2-HBx (P < 0.05). Conclusion: The expressions of core clock gene CLOCK and BMAL1 have been changed by HBx, which breaks down the previous circadian rhythm of liver cells. This maybe one of the reasons leads to the formation of liver cancer.     

Choledochal cyst: review of 74 pediatric cases

Cytokine-mediated apoptotic destruction of viral-infected cells, downregulation of virus production and inhibition of anchorage dependent (clonal) cell growth were evaluated using virus-transfected human hepatoblastoma (HepG2) cells. The cytokines evaluated were interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha) and thymosin alpha 1 (T alpha 1), all of which have previously been implicated in control of various viral infections. The viruses evaluated were Hepatitis B (HBV) and the transforming virus, SV-40. TNF-alpha-induced apoptosis in the HBV-transfected cell line and the control HepG2 cells but not the HepG2 cells transfected with SV-40 virus. IFN-alpha and T alpha 1 had no effect on apoptosis. TNF-alpha also prevented the clonal growth of the HBV-HepG2 and control HepG2 but enhanced the growth of the SV-40-transfected HepG2 cells. IFN-alpha inhibited the clonal growth of all three cell lines in contrast to T alpha 1 which inhibited the clonal growth of only the HBV-transfected cells. Although TNF-alpha, IFN-alpha, and T alpha 1 when given alone did not significantly inhibit HBV-DNA production in the culture supernatant from HBV-HepG2 cells, the combination of T alpha 1 and IFN-alpha resulted in a statistically significant inhibition of virus production. These studies demonstrate that HepG2 cells transfected with HBV and SV-40 are useful for defining the mechanisms of cytokine activity. The HBV-transfected cells are especially useful in defining possible in vivo differences in responses to cytokines with respect to HBV production, apoptosis and clonal cell growth. Multiple mechanisms through which different cytokines can influence HBV infection and hepatoblastoma growth were identified and the importance of defining effective combinations to improve therapy in vivo demonstrated.     

Clinical features associated with correction of T-cell senescence: increased acute-phase response, amyloidosis and arthritis

Two prominent features associated with immunosenescence are thymic involution and altered T-cell phenotype and responsiveness. We have shown previously that in CD2-fas transgenic mice, in which the Fas apoptosis molecule is constituatively expressed on T cells, T-cell senescence is greatly reduced. Using a different experimental approach, the relationship between T-cell senescence and apoptosis was analyzed on human PBMCs. The results indicate that there was increased apoptosis of CD45RO- (CD45RA+) T cells upon activation. We propose that this could account for the increase in CD45RO+ 'memory' T cells with aging in humans. Together these results are consistent with the notion that T-cell senescence is associated with altered apoptosis and decreased T-cell responsiveness. T-cell responsiveness remained high in CD2-fas transgenic aged mice, but there was no increase in overall life span of the mice. Increased T-cell responsiveness was associated with an increased acute-phase response and amyloid A deposition in the glomerulus of these mice. These data suggest that restoration of the T-cell immune function in aged individuals must be carried out in concert with correction of other immune factors that down modulate the acute-phase response to prevent undesirable side-effects.     

Hepatitis B injury, male gender, aflatoxin, and p53 expression each contribute to hepatocarcinogenesis in transgenic mice

The major risk factors for human liver cancer: hepatitis B virus (HBV) related liver injury, male gender, aflatoxin exposure, and p53 expression, are evaluated and compared in experimental transgenic mouse models. Transgenic mice that express hepatitis B surface antigen (HBsAg) in their liver and develop liver tumors at 18 months of age (HBV+ mice) were bred to p53 null mice (p53-/-) to produce mice p53+/-, HBV+ mice. These mice and control littermates ([p53+/+, HBV+], [p53+/-, HBV-], and [p53+/+, HBV-) were divided into groups that did or did not receive an injection of aflatoxin at 1 week of age. At sacrifice at 13 months of age, 100% (7/7) of male mice with each of the three risk factors (p53+/-, HBV+, AFB1+) developed high-grade hepatocellular carcinomas (HCC). If any one of the risk factors was absent, the incidence drops: if both p53 alleles are present, 62% (10/16); if HBsAg is not expressed, 14% (1/7); if AFB1 is not given, 25% (2/8). If only one of the risk factors is present no tumors above grade I are found. Similar results were observed in female mice except that HCC incidence in each group is less than in male mice. Some of the tumors in mice with more than one risk factor are of unusual histological types, such as hepatocholangio-carcinomas, adenocarcinomas and undifferentiated carcinomas that are not usually seen in HBV transgenic C57BL/6 mice. No loss or mutation of the p53 gene is detected in any of the tumors. Possibilities of how the four major risk factors for HCC interact to produce malignant liver tumors in these transgenic mouse models of hepatocarcinogenesis are discussed.