NF-kappaB signaling mediates the induction of MTA1 by hepatitis B virus transactivator protein HBx.

Metastasis-associated protein 1 (MTA1), a master chromatin modifier, has been shown to regulate cancer progression and is widely upregulated in human cancer, including hepatitis B virus-associated hepatocellular carcinomas (HCCs). Here we provide evidence that hepatitis B virus transactivator protein HBx stimulates the expression of MTA1 but not of MTA2 or MTA3. The underlying mechanism of HBx stimulation of MTA1 involves HBx targeting of transcription factor nuclear factor (NF)-kappaB and the recruitment of HBx/p65 complex to the NF-kappaB consensus motif on the relaxed MTA1 gene chromatin. We also discovered that MTA1 depletion in HBx-expressing cells severely impairs the ability of HBx to stimulate NF-kappaB signaling and the expression of target proinflammatory molecules. Furthermore, the presence of HBx in HBx-infected HCCs correlated well with increased MTA1 and NF-kappaB-p65. Collectively, these findings revealed a previously unrecognized integral role of MTA1 in HBx stimulation of NF-kappaB signaling and consequently, the expression of NF-kappaB targets gene products with functions in inflammation and tumorigenesis.

DDB2 induces nuclear accumulation of the hepatitis B virus X protein independently of binding to DDB1.

The hepatitis B virus (HBV) X protein (HBx) is critical for the life cycle of the virus. HBx associates with several host cell proteins including the DDB1 subunit of the damaged-DNA binding protein DDB. Recent studies on the X protein encoded by the woodchuck hepadnavirus have provided correlative evidence indicating that the interaction with DDB1 is important for establishment of infection by the virus. In addition, the interaction with DDB1 has been implicated in the nuclear localization of HBx. Because the DDB2 subunit of DDB is required for the nuclear accumulation of DDB1, we investigated the role of DDB2 in the nuclear accumulation of HBx. Here we show that expression of DDB2 increases the nuclear levels of HBx. Several C-terminal deletion mutants of DDB2 that fail to bind DDB1 are able to associate with HBx, suggesting that DDB2 may associate with HBx independently of binding to DDB1. We also show that DDB2 enhances the nuclear accumulation of HBx independently of binding to DDB1, since a mutant that does not bind DDB1 is able to enhance the nuclear accumulation of HBx. HBV infection is associated with liver pathogenesis. We show that the nuclear levels of DDB1 and DDB2 are tightly regulated in hepatocytes. Studies with regenerating mouse liver indicate that during late G1 phase the nuclear levels of both subunits of DDB are transiently increased, followed by a sharp decrease in S phase. Taken together, these results suggest that DDB1 and DDB2 would participate in the nuclear functions of HBx effectively only during the late-G1 phase of the cell cycle.

Hepatitis B virus X protein acts as a tumor promoter in development of diethylnitrosamine-induced preneoplastic lesions.

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens. Although the mechanism for this cofactor role remains unknown, the ability of HBx to inhibit DNA repair and to influence cell cycle progression suggests two possible pathways. To investigate these possibilities in vivo, we treated double-transgenic mice that both express HBx (ATX mice) and possess a bacteriophage lambda transgene with the hepatocarcinogen diethylnitrosamine (DEN). Histological examination of liver tissue confirmed that DEN-treated ATX mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Treatment of mice with DEN resulted in a six- to eightfold increase in the mutation frequency (MF), as measured by a functional analysis of the lambda transgene. HBx expression was confirmed by immunoprecipitation and Western blotting and was associated with a modest 23% increase in the MF. Importantly, the extent of hepatocellular proliferation in 14-day-old mice, as measured by the detection of proliferating cell nuclear antigen and by the incorporation of 5-bromo-2'-deoxyuridine, was determined to be approximately twofold higher in ATX livers than in wild-type livers. These results are consistent with a model in which HBx expression contributes to the development of DEN-mediated carcinogenesis by promoting the proliferation of altered hepatocytes rather than by directly interfering with the repair of DNA lesions.

Transcriptional regulation of the TFIIH transcription repair components XPB and XPD by the hepatitis B virus x protein in liver cells and transgenic liver tissue.

Human hepatitis B virus is a risk factor for the development of hepatocellular carcinoma. The hepatitis B virus x protein (HBx) has been shown to inactivate the p53 tumor suppressor protein and impair DNA repair, cell cycle, and apoptosis mechanisms. Herein we report that HBx represses two components of the transcription-repair factor TFIIH, XPB (p89), and XPD (p80), both in p53-proficient and p53-deficient liver cells. This inhibition is observed while HBx maintains its transactivation function. Expression of HBx in liver cells results in down-regulation of endogenous XPB and XPD mRNAs and proteins; this inhibition is not observed with other TFIIH subunits, XPA or PCNA. In liver tissue from HBx transgenics, XPB and XPD proteins are down-regulated in comparison to matched normal liver tissue. HBx has been shown to interact with Sp1 transcription factor and affects its DNA binding activity. Sp1 is essential for the basal promoter activity of XPB in liver cells and Drosophila SL2 cells. In the Sp1-deficient SL2 cells, HBx-induced XPB and XPD inhibition is Sp1-dependent. In summary, our results provide evidence that HBx represses the expression of key TFIIH proteins at least in part through Sp1 elements; this repression may impair TFIIH function in DNA repair mechanisms.

Stimulation of cellular proliferation by hepatitis B virus X protein.

Chronic infection with the hepatitis B virus (HBV) is a known risk factor in the development of human hepatocellular carcinoma (HCC). The HBV-encoded X protein, HBx, has been investigated for properties that may explain its cancer cofactor role in transgenic mouse lines. We discuss here recent data showing that HBx is able to induce hepatocellular proliferation in vitro and in vivo. This property of HBx is predicted to sensitize hepatocytes to other HCC cofactors, including exposure to carcinogens and to other hepatitis viruses. Cellular proliferation is intimately linked to the mechanism(s) by which most tumor-associated viruses transform virus-infected cells. The HBx alteration of the cell cycle provides an additional mechanism by which chronic HBV infection may contribute to HCC.

Dissociation of DDB1-binding and transactivation properties of the hepatitis B virus X protein.

The hepatitis B virus (HBV) X protein (HBx) is a transactivator encoded by mammalian hepadnaviruses, and is thought to stimulate transcription by interacting with one or more host cell factors. Numerous cellular proteins have been reported to interact with HBx including a component of the nucleotide excision repair complex called ultraviolet damaged DNA binding (UV-DDB, or DDB1) protein. Recent studies have identified a role for DDB1 in transcription, raising the possibility that HBx may acquire its broad transcriptional properties by interacting with DDB1. A panel of HBx mutant proteins, some of which no longer bind to DDB1, was used to test this hypothesis. Plasmid DNAs encoding HBx wildtype and mutant derivatives were transfected into HepG2 cells, and their ability to transactivate a cotransfected reporter plasmid tested. Results from the transactivation assays in HepG2 cells were then compared with data obtained from HBx-DDB1 binding studies performed in yeast. Several HBx mutant proteins unable to bind DDB1 remained competent for transactivation, indicating that HBx binding to DDB1 is not required for HBx transactivation of the ETS1 promoter. It remains possible that a subset of HBx transactivation function targets an as yet undefined DDB1-specific pathway.

Expression of hepatitis B virus X protein does not alter the accumulation of spontaneous mutations in transgenic mice.

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens, although the mechanism for this cofactor role remains unknown. The ability of HBx to inhibit DNA repair in transiently transfected cell lines suggests one possible pathway. In the present study, primary hepatocytes isolated from transgenic mice that possess the HBV X gene under the control of the human alpha-1-antitrypsin regulatory region (ATX mice) were found to be deficient in their ability to conduct unscheduled DNA synthesis in response to UV-induced DNA damage. In order to measure the impact of HBx expression on DNA repair in vivo, double-transgenic mice that express HBx and possess a bacteriophage lambda transgene were sacrificed at 30, 90, and 240 days of age. Mutation frequency was determined for high-molecular-weight liver DNA of ATX and control mice by functional analysis of the lambda transgene. Expression of HBx did not significantly increase the accumulation of spontaneous mutations. These results are consistent with previous studies of HBx transgenic mice in which no effect of HBx on liver histology was apparent. This new animal model provides a powerful system in which to investigate the in vivo cooperation between HBx expression and environmental carcinogens.

The naturally occurring mutants of DDB are impaired in stimulating nuclear import of the p125 subunit and E2F1-activated transcription.

The human UV-damaged-DNA binding protein DDB has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E), because a subset of XP-E patients lack the damaged-DNA binding function of DDB. Moreover, the microinjection of purified DDB complements the repair deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E mutations of DDB, 82TO and 2RO, have been characterized. They have single amino acid substitutions (K244E and R273H) within the WD motif of the p48 subunit of DDB, and the mutated proteins lack the damaged-DNA binding activity. In this report, we describe a new function of the p48 subunit of DDB, which reveals additional defects in the function of the XP-E mutants. We show that when the subunits of DDB were expressed individually, p48 localized in the nucleus and p125 localized in the cytoplasm. The coexpression of p125 with p48 resulted in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The mutant forms of p48, 2RO and 82TO, are deficient in stimulating the nuclear accumulation of the p125 subunit of DDB. In addition, the mutant 2RO fails to form a stable complex with the p125 subunit of DDB. Our previous studies indicated that DDB can associate with the transcription factor E2F1 and can function as a transcriptional partner of E2F1. Here we show that the two mutants, while they associate with E2F1 as efficiently as wild-type p48, are severely impaired in stimulating E2F1-activated transcription. This is consistent with our observation that both subunits of DDB are required to stimulate E2F1-activated transcription. The results provide insights into the functions of the subunits of DDB and suggest a possible link between the role of DDB in E2F1-activated transcription and the repair deficiency disease XP-E.

Expression of ras, c-myc, and p53 proteins in cervical intraepithelial neoplasia.

BACKGROUND: The development of cervical carcinoma is influenced by multiple factors, including the presence of certain high risk types of human papillomavirus. The purpose of the current study was to investigate possible cooperating genetic changes by examining the expression of p53, p62 myc, and p21 ras in cervical biopsy specimens. METHODS: Three hundred and ninety-five cervical biopsy specimens representing normal through high grade cervical intraepithelial neoplasia (CIN) were screened by immunohistochemistry for expression of p53, p62myc, and p21ras. RESULTS: Neither the proportion of tissues staining positive for a given protein nor the staining patterns within the epithelial layers differed significantly among normal or CIN biopsy samples. However, grade specific nuclear staining of p21ras was found in the cells of 10 lesions that were classified as CIN I by histology. CONCLUSIONS: These results established the normal distribution and expression patterns of p53, p62myc, and p21ras within 395 cervical biopsy samples representing normal through CIN III histology. The expression of these proteins (e.g., staining intensity and layer of epithelium staining positive) is similar in normal tissues and those demonstrating all grades of CIN.

Hepatitis B virus X protein interferes with cellular DNA repair.

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.

Hepatitis B virus-induced liver injury and altered expression of carcinogen metabolising enzymes: the role of the HBx protein.

Hepatitis B virus (HBV) and aflatoxins are major risk factors for hepatocellular carcinoma (HCC) exhibiting a synergistic interaction in the development of this disease. The molecular mechanisms of this interaction remain to be elucidated but an altered carcinogen metabolism in the presence of hepatitis-induced liver injury is one hypothesis. The availability of biomarkers of aflatoxin exposure and metabolism permits this hypothesis to be examined in human populations whilst animal models, such as HBV transgenic mice permit parallel studies in an experimental setting. The hepatitis B virus X protein (HBx) is suspected to play a role in the hepatocarcinogenic process by virtue of its capacity to transactivate oncogenes and several other cellular genes via cis-acting elements. In previous studies in HBV transgenic mice expressing the HB surface antigen and X genes we observed a marked induction of specific cytochrome P450s (CYP) (Kirby et al., 1994a). In the current study we investigated the status of CYP, glutathione S-transferases (GST) and antioxidant enzymes in mice carrying only the X gene under the control of the alpha-1 antitrypsin regulatory elements (ATX mice). Livers of ATX mice showed no major pathological alterations compared to age-matched non-transgenic control mice. Immunohistochemical staining for CYP1A, 2A5 and GST expression and determination of related enzymatic activities (7-ethoxyresorufin O-deethylation, 7-methoxyresorufin O-deethylation, coumarin 7-hydroxylation and GST activities) revealed no differences between control and ATX mice. In addition, no differences in antioxidant enzymes were observed. Overall, these results support the conclusion that HBx expression alone is insufficient to induce transactivation of CYP and GST genes or to alter the antioxidant system and that the induction in other HBV models is a result of inflammatory injury in the liver, a feature absent in ATX mice. These data are compared to biomarker studies of enzyme activities in aflatoxin-exposed human populations with and without HBV infection.

Frequent loss of chromosome 8p in hepatitis B virus-positive hepatocellular carcinomas from China.

The majority of hepatocellular carcinomas (HCCs) from hepatitis B virus (HBV)-endemic areas contain integrated viral sequences. To better understand the role of HBV DNA insertion in tumorigenesis, we examined the integration site of a HCC harboring a single insert. Cellular DNAs flanking the viral sequences were mapped to chromosomes 17 and 8, indicating a translocation had occurred at the site of viral integration. Regional mapping of chromosome 17 demonstrated that HBV had integrated in 17p12-pter, a region that harbors the p53 tumor suppressor gene. Many studies have shown that chromosome 17p allele loss occurs frequently in HCCs from certain geographical areas. To investigate the chromosome 8 allele status in Chinese HCCs, a panel of 37 matched normal and HCC DNAs from Qidong, China was analyzed for tumor-specific allele loss with RFLP probes from both arms of chromosome 8. Tumor-specific loss of heterozygosity was highest on the short arm with 71.4% (10/14) and 85.0% (17/20) of the informative patients missing an allele for 8p23 (YNM3) or 8p21 (NEFL), respectively. Allele loss from the long arm of chromosome 8 was also observed with 30.0% (6/20) and 33.3% (7/21) of the samples informative for 8q22 (CA2) and 8q24 (MCT128.2), respectively. The high allele loss on 8p correlates with recent studies of other human cancers and is interpreted to indicate that a tumor suppressor gene(s) whose loss is important for carcinogenesis lies within this region. These findings also support a model in which HBV insertions associated with gross chromosomal changes can identify genomic regions where alteration is important for development of some HCCs.

Activation of the HTLV-I long terminal repeat by the hepatitis B virus X protein.

The human T-cell leukemia virus type I (HTLV-I) Tax protein and the hepatitis B virus (HBV) X protein have each been shown to activate transcription of their respective viral promoters as well as a subset of cellular gene promoters. Here we show that the HTLV-I long terminal repeat (LTR) is responsive to HBV X transactivation. Maximum levels of X-mediated transactivation of the LTR were 8-fold. An X-responsive-region (XRR) of the LTR is located between nucleotides -355 and -276 and contains an AP-2 binding site, a previously recognized X-responsive element. We demonstrated that Tax and X synergize to activate transcription from the HTLV-I LTR, although the AP-2 binding site was not required for this synergy. These results raise the possibility that the HBV X protein may affect the level of HTLV-I gene expression in co-infected individuals.

Increased sensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus X gene.

The role of the hepatitis B virus (HBV) X protein in liver tumorigenesis is unresolved. Transgenic mice harboring the X gene (nt 1376-1840 under the control of the human alpha-1-antitrypsin regulatory elements) (ATX mice) display only minor histopathologic alterations of the liver. To determine if ATX mice are more susceptible to the effects of hepatocarcinogens, 12- to 15-d-old male ATX and control littermate mice were injected with a single dose (2 microgram/g body weight) of diethylnitrosamine (DEN). The animals were killed 6-10 mo after exposure and were analyzed for histological changes in the liver. One hundred percent of the DEN-treated AXT mice developed abnormal liver lesions. Then their liver tissues were compared by stereological analysis with those of non-transgenic animals, the ATX mice had a relative twofold increase in the total number of focal lesion and a twofold increase in the incidence of hepatocellular carcinoma. Elevated levels of X protein and p53 protein were not detected in carcinogen-induced nodules or tumors. These results are consistent with a model in which the expression of the HBV X protein potentiates the induction of DEN-mediated liver disease.

Induction of senescent cell-derived inhibitor of DNA synthesis gene, SDI1, in hepatoblastoma (HepG2) cells arrested in the G2-phase of the cell cycle by 9-nitrocamptothecin.

BACKGROUND: Recent studies have demonstrated that the plant-derived alkaloid camptothecin (CPT) and its derivative, 9-nitro-CPT (9NC), are cytotoxic in tumorigenic cells but cytostatic in nontumorigenic cells in vitro and in vivo. Also, CPT induces differentiation of human leukemia cells in vitro along specific lineages. In this study, we have investigated the effects of 9NC on nontumorigenic HepG2 cells derived from human hepatoblastoma. A newly discovered senescent cell-derived inhibitor (SDI1) plays a critical role in the cell cycle, so we evaluated the effect of 9NC on the expression of the SDI1 gene. EXPERIMENTAL DESIGN: The effects of 9NC on HepG2 cells were evaluated by monitoring DNA synthesis, morphologic and ultrastructural changes of cells, and perturbation in the cell cycle and by assessing the levels of p53 protein and SDI1 mRNA. RESULTS: Treatment of HepG2 cells with 9NC results in a dose-dependent inhibition of cell proliferation and DNA synthesis. Flow cytometric analysis of DNA content showed that 9NC-treated HepG2 cells are arrested in the G2-phase of the cell cycle. Light and electron microscopic examination revealed that 9NC at low concentrations induces morphologic and growth features that resemble properties highly differentiated or senescent cells, i.e., increased cell size and decreased nuclear/cytoplasmic ratio, as well as enlarged numbers of lysosomes, mitochondria, and lipid in the cytoplasm. No significant alteration in the p53 protein level was noted in 9NC-treated cells. In contrast to untreated, logarithmically grown HepG2 cells, 9NC-treated cells arrested at the G2-phase of the cell cycle and contained increased levels of SDI1 mRNA. Kinetic studies revealed gradual increases in SDI1 mRNA synthesis. CONCLUSIONS: Induction of SDI1 mRNA by 9NC represents the first documentation that the SDI1 gene can be overexpressed in the G2-phase of the cell cycle and provides a valuable cell culture system to dissect the events controlling the G2 checkpoint. In addition, this finding corroborates the hypothesis that genes up-regulated in senescent cells can also be induced in tumor-derived immortalized cells.

Viral co-factors in liver cancer: lessons from hepatitis B virus.

Hepatitis B virus (HBV) is a co-factor in some hepatocellular carcinomas (HCC). Chronic infection with HBV is a risk factor for tumor development, suggesting the accumulation of cellular genetic changes. HBV DNA is frequently found integrated at random sites in HCC, with chromosomal deletions and rearrangements being common at the sites of viral integration. Tumor suppressor gene p53 is frequently altered in HCC. Environmental carcinogens are factors in HCC development in certain geographic locations. HBV encodes a protein (X) known to transactivate viral and cellular genes; the X gene is often retained in HCC. To learn more about X gene function. We employed the yeast two-hybrid genetic system to seek X-interactive proteins. A cellular protein, designated XAP-1, was recovered that interacts specifically with the X protein. XAP-1 is the human homologue of the monkey UV-damaged DNA-binding protein (UV-DDB); the UV-DDB protein functions in DNA repair and is defective in some xeroderma pigmentosum group E patients. The interaction between XAP-1 and HBV X protein was confirmed by several independent methods. This suggests that cellular DNA repair processes may be affected by HBV and that the resulting genetic instability may contribute to hepatocellular carcinogenesis. A unifying model of the molecular basis of HBV involvement in HCC development is presented. Fundamental components of the model are chronic infection by HBV and viral effects on cellular DNA repair. This model has implications for the possible role of HCV infection in the induction of HCV-associated HCC.

Allelotype and loss of heterozygosity of p53 in primary and recurrent hepatocellular carcinomas. A study of 150 patients.

BACKGROUND: The allelotype and loss of heterozygosity (LOH) of the p53 gene in human hepatocellular carcinoma (HCC) were studied in 150 patients with resected primary HCC and 18 with recurrent HCC. METHODS: DNA samples of paired HCC and livers were cut with BanII enzyme for the study of p53 allelotype and allele loss. The medical records of the patients were carefully reviewed. RESULTS: Sixty-four (42.7%) patients were heterozygous for the p53 gene, 69 (46%) were homozygous for the 1.5/1.4 kb small (S) allele, and 17 (11.3%) were homozygous for the 2.9 kb large (L) allele. The frequencies of the minor L allele (0.323) and of the major S allele (0.677) in this population of Chinese patients differed from the frequencies previously reported for North American Caucasians (0.13 and 0.87, respectively). The heterozygous patients tended to have lower serum hepatitis B surface- and e antigens (HBsAg and HBeAg) and higher diabetes mellitus (DM) than did homozygous patients (SS and LL). Thirty-seven (57.8%) of the 64 heterozygous patients had a tumor-specific p53 allele LOH, being two times more common in HCC tumors larger than 8 cm than in HCC tumors 2 cm or smaller. The frequency of DM was four times higher in the heterozygous patients who had p53 LOH than in those who retained both alleles. LOH of p53 did not correlate with tumor invasiveness or differentiation, hepatitis B or C virus infection, or prognosis. CONCLUSION: The allelotype of p53 gene in HCC correlates with HBsAg and HBeAg seropositivities and DM. LOH of the p53 gene is a common event in HCC, correlates with DM, and occurs less often in familial HCC. LOH can identify the clonal origin of recurrent HCC but is not a critical prognostic factor.

Deletion of the E-cadherin gene in hepatitis B virus-positive Chinese hepatocellular carcinomas.

Frequent allele loss from chromosome 16q was recently described for human tumors of the breast, prostate gland and liver, indicating the possible presence of a tumor-suppressor gene on that chromosome arm. In this study, the chromosome 16 allele status of 38 hepatocellular carcinomas in Chinese patients was determined with restriction-fragment-length polymorphism analysis. Tumor-specific allele loss was detected in 14 (74%) of 19 patients informative for 16p markers and in 22 (85%) of 26 patients informative for 16q markers. Quantitative densitometric analysis revealed reduction to hemizygosity of the E-cadherin cell adhesion gene (localized to 16q22.1) in 18 (64%) of the 28 patients for whom quantitative data were available. Reduced expression of E-cadherin has been associated with invasion and metastasis in several human cell lines and primary tumors, and our results suggest that one mechanism of reduced E-cadherin expression is the loss of one copy of the E-cadherin gene.