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Hepatitis b Viruses and Hepatocellular Carcinoma
Abstract
This chapter explores and compares different possible mechanisms by which hepadnaviruses maytrigger liver cell proliferation and transformation, and considers the factors that may influence the primacy of some oncogenic pathways over others in tumors induced by different viruses of the same family. Primary hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world and is also one of the rare human cancers showing seroepidemiologic association with a viral infection. The role of hepatitis B virus (HBV) as a causal agent of HCC is established and the increased risks of developing HCC are estimated. Productive HBV infections potentiate the action of exogenous carcinogenic factors like aflatoxins and alcohol. The clinical and immunological aspects of HBV infections and their modes of transmission are also discussed as are genetic organization of the HBV genome, genome structure and replication, and regulated expression of viral genes. HBV DNA integration patterns into host-cell DNA have led to a different hypothesis on the contribution of HBV to hepatocarcinogenesis. Hepatitis B is rendered highly contagious by the unusual stability of infectious HBV virions present in the blood and other body fluids like saliva, urine, and semen.
... One is that HBV may activate cellular proto-oncogenes by insertional mutagenesis. This has been observed with woodchuck hepatitis virus and the myc genes in HCC (Buendia, 1992), but human HBV insertion takes place at multiple sites, and the vast majority of HBV DNA insertion sites are not associated with any known human proto-oncogene or tumour suppressor gene. Another suggestion is that of the transactivation of cellular genes by the HBV gene product HBx as well as the repression of transactivation by p53 due to binding of HBx to p53 (Buendia, 1992;Truant et al., 1995). ...
... This has been observed with woodchuck hepatitis virus and the myc genes in HCC (Buendia, 1992), but human HBV insertion takes place at multiple sites, and the vast majority of HBV DNA insertion sites are not associated with any known human proto-oncogene or tumour suppressor gene. Another suggestion is that of the transactivation of cellular genes by the HBV gene product HBx as well as the repression of transactivation by p53 due to binding of HBx to p53 (Buendia, 1992;Truant et al., 1995). Alternatively, HBV could have an indirect role in the induction of liver cirrhosis and cell regeneration eventually leading to HCC; similar observations have been made in woodchucks chronically infected with woodchuck hepatitis virus and in HBV transgenic mice overexpressing the viral large envelope polypeptide (Buendia, 1992). ...
... Another suggestion is that of the transactivation of cellular genes by the HBV gene product HBx as well as the repression of transactivation by p53 due to binding of HBx to p53 (Buendia, 1992;Truant et al., 1995). Alternatively, HBV could have an indirect role in the induction of liver cirrhosis and cell regeneration eventually leading to HCC; similar observations have been made in woodchucks chronically infected with woodchuck hepatitis virus and in HBV transgenic mice overexpressing the viral large envelope polypeptide (Buendia, 1992). Finally, a possible role for HBV in inducing chromosomal alterations in hepatocytes has been proposed in view of the frequent loss of heterozygosity (LOH) at specific loci in HCC (Kitagawa et al., 1995). ...
Primary hepatocellular carcinoma (HCC) is one of the most common cancers in Thailand; chronic infection with hepatitis B virus (HBV) is endemic and represents a major risk factor for the development of this cancer. Several mechanisms for HBV‐related hepatocarcinogenesis have been proposed, among them a direct role of HBV in the promotion of genetic recombination leading to chromosomal alterations. Minisatellite DNA sequences are hypervariable regions dispersed throughout the genome which are susceptible to genetic recombination events. In the present study, somatic rearrangements affecting minisatellite sequences were examined in a total of 26 HCC from Thai patients. Multilocus DNA fingerprinting using probes 33.15 and 33.6 detected rearrangements in 11 and 12 HCC, respectively, all of them carrying integrated HBV DNA. The frequency of rearranged bands was calculated for each probe based on the total number of rearrangements observed in the 26 tumours and the total number of bands revealed by DNA fingerprinting in the non‐tumour DNA. With each probe a total of 23 rearrangements was observed, yielding rearrangement frequencies of 3.7% and 4.2% for the 33.15 and 33.6 minisatellite families, respectively. To test for possible clustering of these rearrangements at specific loci, we used minisatellite locus‐specific probes previously cloned from 33.15 and 33.6. Minisatellites located at 1p33‐35, 7q36‐ter and 12q24.3‐ter were shown to be frequently affected by rearrangement events in this series of HBV‐positive HCC. Frequent rearrangements at minisatellite locus D7S22 (7q36‐ter) in HBV‐positive human HCC have not been reported so far. Int. J. Cancer 72:248–254, 1997. © 1997 Wiley‐Liss, Inc.
... Hepatocellular carcinoma (HCC) is one of the world's most common cancers, occurring especially in Africa and South-East Asia (Beasley, 1982;Parkin et al., 1988). Epidemiological studies indicate that contamination of food with aflatoxin B 1 ( A F B 1 ) and chronic infection with hepatitis B virus (HBV) are the major risk factors for human liver cancer (Buendia, 1992;Ross et al., 1992;Yeh et al. , 1989). Several investigations in different species of experimental animals have demonstrated synergistic effects of A F B 1 and HBV in hepatocarcinogenesis (Sell et al., 1991;Cova et al., 1994;Bannasch et al., 1995). ...
... For example, DHBV differs from HHBV such that is lacks the X gene and it induces only mild liver tissue disease in its host (Mandart et al., 1984). H o w e v e r, WHV appears to be more oncogenic than HBV (Popper et al., 1987;Buendia, 1992). There may be additional differences in infectivity and oncogenicity among these hepadnaviruses. ...
Using tree shrew as an animal model, our previous studies have demonstrated synergistic effects of aflatoxin B-1 (AFB(1)) and human hepatitis B virus (HHBV) in the induction of hepatocellular carcinoma (HCC). In the present study, we have examined expression of p53 gene in HCCs induced by AFB(1) with or without HHBV infection in tree shrews. Avidin-biotin-peroxidase complex immunohistochemical method with human p53-CM1 polyclonal antibody has been used to detect p53 expression in serial sections of paraffin-embedded liver and HCC tissues. Five out of 9 animals with HCCs (55.6%) induced by AFB(1) with HHBV infection and 2/3 animals with HCCs (66.7%) induced by AFB(1) alone expressed the p53 protein. Out of 18 HCCs examined, expression of p53 protein was observed in 9/10 moderately and poorly differentiated HCCs (0/8). None of the well differentiated HCCs (0/8) expressed p53 (0%). Similarly, no p53 expression was observed in either non-tumorous or hyperplastic liver tissues or nodules. These results suggest that p53 expression associated with p53 mutation is a late event occurring probably during tumor progression in AFB(1) and HHBV induced hepatocarcinogenesis in the tree shrew. This report is the first example of an experimental animal model where combination of human HBV and AFB(1)-induced HCCs demonstrate p53 expression.
... However, frequent amplification of cmyc genes has been described in ground squirrel HCCs associated with GSHV infection (13). Elevated expression of several other protooncogenes c-fos, c-fins, c-jun, H-ras, N-ras as well as tumor growth factor (TGF)-a and insulin-like growth factor (IGF)-U has been reported in hepadnavirus-associated HCCs of humans and woodchucks (reviewed in 2, 3,14). It is not clear so far how the enhanced expression of these genes is related to viral infection. ...
... Normal functions of the p53 gene might be affected by interaction with hepatitis virus proteins in a manner similar to that of SV40 large T antigen or adenovirus E1B and papillomavirus E6 proteins (reviewed in 18,23). A frequently proposed candidate for such a role is hepatitis B virus X protein (HBx), which can transactivate various cellular and viral promoters in different cell types (2,3,51). Several research groups have reported direct interaction of HBx with p53 in vitro. ...
Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high
rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently
related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks
in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4–9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were
treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4–9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated
a low rate of p53 mutations in HCCs of AFB1treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks
not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development
of p53 mutations less than in humans.
... Early preneoplastic lesions consist of altered hepatic foci that overexpress N-myc and IGF-II [22]. At tumoral stage, almost all tumors carry integrated viral sequences that may be detected by Southern blotting, reflecting selection by clonal outgrowth of a transformed cell targeted by the integration event [14]. ...
... The human hepatitis B virus is the prototype of the hepadnavirus family, which includes closely related viruses infecting a limited number of primates, mammals and birds [13]. Mammalian hepadnavirus models have been extensively used for studying the molecular mechanisms leading to liver cancer [14], as well as for experimenting potential therapeutical approaches in the management of HBV infection [15]. In particular, woodchucks chronically infected with the woodchuck hepatitis virus (WHV) have provided a unique model in which viral DNA integration into the host genome plays a pivotal role in the tumoral process. ...
Epidemiological studies have provided overwhelming evidence for a causal role of chronic HBV infection in the development of hepatocellular carcinoma (HCC), but the molecular mechanisms underlying virally-induced tumorigenesis remain largely debated. In the absence of a dominant oncogene encoded by the HBV genome, indirect roles have been proposed, including insertional activation of cellular oncogenes by HBV DNA integration, induction of genetic instability by viral integration or by the regulatory protein HBx, and long term effects of viral proteins in enhancing immune-mediated liver disease. In this chapter, we discuss different models of HBV-mediated liver cell transformation based on animal systems of hepadnavirus infection as well as functional studies in hepatocyte and hepatoma cell lines. These studies might help identifying the cellular effectors connecting HBV infection and liver cell transformation.
... Chronic HBV infection is a major risk factor for the development of HCC, accounting for 60% of cases worldwide [3]. Integrated viral DNA has been reported in approximately 85% of HBV-related HCC cases [4][5][6]. The HBV X gene is often integrated into a chromosome in HCC cases [7] and correlated with HCC development and progression [8][9][10][11]. ...
Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.
... Apart from this, another mechanism that involves viral response towards the chronic inflammation to produce HCCs [30,31]. Also, the process of integration of virus into the host genome also facilitates the replication HBV in the host cell genome [32]. Upon integration, the viral assemblies undergo fragmentation and rearrangement to produce the infected viral particles [33]. ...
The applications of gene therapy-based treatment of cancers were started almost two decades back as a boon over the chemotherapeutic treatment strategies. Gene therapy helps in correcting the genetic sequences for treatment of cancers, thus also acts like a vaccine to induce the cellular and humoral immunity. However, the cancer vaccines typically suffer from a series of biopharmaceutical challenges due to poor solubility, low systemic availability and lack of targeting ability. Owing to these challenges, the physicians and pharmaceutical scientists have explored the applications of nanocarriers as quite promising systems for effective treatment against the tumors. A series of nanotherapeutic systems are available to date for diverse drug therapy applications. Systematic understanding on the preparation, evaluation and application of nanomedicines as a carrier system for delivering the cancer vaccines is highly important. The present review article provides an in-depth understanding on the challenges associated with cancer vaccine delivery and current opportunities with diverse nanomedicinal carriers being available for treatment of cancers.
... Although an effective preventive vaccine has been available for more than 30 years, more than 350 million individuals worldwide still have CHB (Curtis et al., 2005). Chronically infected individuals are at high risk for cirrhosis and hepatocellular carcinoma (Buendia, 1992). The HBV vaccine elicits almost no production of protective antibodies in patients with CHB (Trepo et al., 2014). ...
Hepatitis B virus (HBV) infection is endemic in Asia and chronic hepatitis B (CHB) is a major public health issue worldwide. Current treatment strategies for CHB are not satisfactory as they induce a low rate of hepatitis B surface antigen (HBsAg) loss. Extracts were prepared from lettuce hydroponically cultivated in solutions containing glycine or nitrate as nitrogen sources. The lettuce extracts exerted potent anti-HBV effects in HepG2 cell lines in vitro, including significant HBsAg inhibition, HBV replication and transcription inhibition, without exerting cytotoxic effects. When used in combination interferon-alpha 2b (IFNα-2b) or lamivudine (3TC), the lettuce extracts synergistically inhibited HBsAg expression and HBV replication. By using differential metabolomics analysis, Luteolin-7-O-glucoside was identified and confirmed as a functional component of the lettuce extracts and exhibited similar anti-HBV activity as the lettuce extracts in vitro. The inhibition rate on HBsAg was up to 77.4%. Moreover, both the lettuce extracts and luteolin-7-O-glucoside functioned as organic antioxidants and, significantly attenuated HBV-induced intracellular reactive oxygen species (ROS) accumulation. Luteolin-7-O-glucoside also normalized ROS-induced mitochondrial membrane potential damage, which suggests luteolin-7-O-glucoside inhibits HBsAg and HBV replication via a mechanism involving the mitochondria. Our findings suggest luteolin-7-O-glucoside may have potential value for clinical application in CHB and may enhance HBsAg and HBV clearance when used as a combination therapy.
... The normal replication cycle of HBV does not include integration into the host cell genome. However, the virus has been found integrated into the host cell genome in around 80% of HCC cases associated with HBV [283] . The integrated virus is fragmented and rearranged, so that it cannot produce infectious particles [284286] . ...
In the early 1900s, numerous seminal publications reported that high rates of cancer occurred in certain occupations. During this period, work with infectious agents produced only meager results which seemed irrelevant to humans. Then in the 1980s ground breaking evidence began to emerge that a variety of viruses also cause cancer in humans. There is now sufficient evidence of carcinogenicity in humans for human T-cell lymphotrophic virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, human papillomavirus, Epstein-Barr virus, and human herpes virus 8 according to the International Agency for Research on Cancer (IARC). Many other causes of cancer have also been identified by the IARC, which include: Sunlight, tobacco, pharmaceuticals, hormones, alcohol, parasites, fungi, bacteria, salted fish, wood dust, and herbs. The World Cancer Research Fund and the American Institute for Cancer Research have determined additional causes of cancer, which include beta carotene, red meat, processed meats, low fibre diets, not breast feeding, obesity, increased adult height and sedentary lifestyles. In brief, a historical review of the discoveries of the causes of human cancer is presented with extended discussions of the difficulties encountered in identifying viral causes of cancer.
... Hepatitis B virus (HBV) belongs to the family Hepadnaviridae, a group of small, hepatotropic DNA viruses that also includes related animal viruses, such as the duck HBV (DHBV) and the woodchuck hepatitis virus (31). Chronic HBV infection remains a major public health problem worldwide, with over 350 million chronic HBV carriers who are at serious risk of developing liver cirrhosis and hepatocellular carcinoma (5,7,12). At present, the immunomodulatory and antiviral cytokine alpha interferon and several nucleoside analogs represent two distinct classes of therapies for chronic HBV infections (14). ...
... Integrated viral DNA is found in 85%-90% of HBVrelated HCCs and its presence in tumors from non-cirrhotic livers of children or young adults further supports the role of viral DNA integration in hepatocarcinogenesis [17,18] . A significant feature of chronic HBV infection is that HBV DNA fragments are integrated into different locations within the host DNA [19][20][21][22][23] . Tumor progression is often associated with rearrangement and partial gain or loss of both viral and cellular sequences [24][25][26] . ...
Liver cancer ranks sixth in cancer incidence, and is the third leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, which arises from hepatocytes and accounts for approximately 70%-85% of cases. Hepatitis B virus (HBV) frequently causes liver inflammation, hepatic damage and subsequent cirrhosis. Integrated viral DNA is found in 85%-90% of HBV-related HCCs. Its presence in tumors from non-cirrhotic livers of children or young adults further supports the role of viral DNA integration in hepatocarcinogenesis. Integration of subgenomic HBV DNA fragments into different locations within the host DNA is a significant feature of chronic HBV infection. Integration has two potential consequences: (1) the host genome becomes altered ("cis" effect); and (2) the HBV genome becomes altered ("trans" effect). The cis effect includes insertional mutagenesis, which can potentially disrupt host gene function or alter host gene regulation. Tumor progression is frequently associated with rearrangement and partial gain or loss of both viral and host sequences. However, the role of integrated HBV DNA in hepatocarcinogenesis remains controversial. Modern technology has provided a new paradigm to further our understanding of disease mechanisms. This review summarizes the role of HBV DNA integration in human carcinogenesis.
... Hepatitis B virus, together with hepatitis C virus infection is the two primary causes for liver cancer and other liver diseases. HBV infection, due to the high prevalence compared with HCV, outweighs HCV as the most clinico-epidemiologically important risk factor of HCC [2]. Hepatitis B virus genome is circular and partially double stranded DNA. ...
Proteomics study was performed to investigate the specific protein expression profiles of HepG2 cells transfected with mutant HBV compared with wildtype HBV genome, aiming to identify the specific functions of SH3 binding domain (proline rich region) located in HBx. In addition to the cell movement and kinetics changes due to the expression of HBV genome we have observed previously, here we further targeted to explore the specific changes of cellular proteins and potential intracellular protein interactions, which might provide more information of the potential cellular mechanism of the differentiated cell movements. Specific changes of a number of proteins were shown in global protein profiling in HepG2 cells expressing wildtype HBV, including cell migration related proteins, and interestingly the changes were found recovered by SH3 binding domain mutated HBV. The distinctive expressions of proteins were validated by Western blot analysis.
... 514 Challenge with virus in neonates leads to a chronic infection, while adults only develop the acute phase of disease. 515 A closely related species to the woodchuck is the Marmota Himalayan. This animal is also susceptible to the woodchuck hepadna virus upon IV injection. ...
... When using proposed cell cocktail alternatives, scientists should indicate the rationale for including or excluding particular cell line(s) from their cocktail and ensure that they have a follow-up strategy in place if they attempt to harvest the tumor tissue from the in vivo study to assess which cell line(s) used in the cell cocktail did not respond to treatment. Alternatively, human biopsies instead of tumor cell lines can be used to create xenograft or orthotopic models, but caveats to this approach are the high cost of procedures and transfer of biopsies along with safety issues due to a high rate of hepatitis C (HCV) and hepatitis B (HBV) infections in HCC patients19202122. In addition, the use of biomarkers such as AFP or IVIS imaging in studies using human biopsies will not always be possible. ...
The overwhelming need to improve preclinical models in oncology has stimulated research efforts to refine and validate robust orthotopic models that closely mimic the disease population and therefore have the potential to better predict clinical outcome with novel therapies. Sophisticated technologies including bioluminescence, contrast enhanced ultrasound imaging, positron emission tomography, computed tomography and magnetic resonance imaging have been added to existing serum- and histology-based biomarkers to assist with patient selection and the design of clinical trials. The rationale for the use of human hepatocellular carcinoma (HCC) cell lines, implementation of xenograft and orthotopic animal models and utilization of available biomarkers have been discussed, providing guidelines to facilitate preclinical research for the development of treatments for HCC patients.
... Different mechanisms are explained for viral carcinogenesis, including immunosuppression, oncogene activation, and tumour suppressor gene inactivation [9][10][11]. The association of Epstein-Barr virus (EBV), Human T-cell Lymphotropic virus (HTLV1), human herpes virus 6 (HHV6), hepatitis B virus, and hepatitis C virus with tumours has been reported [7,[12][13][14][15][16]. ...
Congenital tumours are a group of distinct infrequent disorders whose exact aetiologies have not clearly been understood so far. Viral infection seems to be one of the key factors involved in the carcinogenesis of certain tumours. This study was performed to assess whether viral DNAs are present in the congenital tumours or not. Nucleic acid from 31 congenital tumours was extracted. Detection of Epstein-Barr virus, Cytomegalovirus (CMV), adenovirus, Herpes simplex virus 1 (HSV1) and 2, Human herpes virus 6 (HHV6), and BK virus was performed using polymerase chain reaction. Viral nucleic acid was detected in eight subjects (25.8%), mostly adenovirus, CMV, and HHV6. Despite their low frequencies, a possible role could be identified for viral infections in tumour development or progression.
... 10 -12 Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, exposure to aflatoxin B 1 , and excessive intake of alcohol have been identified as major risk factors. 10,13,14 HCCs associated with HBV infection are most frequent in Southeast Asia and sub-Saharan Africa, 15 whereas HCCs associated with HCV are most prevalent in southern Europe and Japan. In Italy, Spain, and Japan, 50 -75% of cases of HCCs are associated with HCV infection. ...
Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide. Hepatitis B virus and hepatitis C virus infections, exposure to aflatoxin, and excessive intake of alcohol have been identified as major risk factors. However, the molecular mechanisms underlying their development are still poorly understood. Recently, β-catenin, one of the key components of the Wnt signaling pathway, has been found to be mutated in about 20% of HCCs, suggesting a role of the Wnt pathway in their development. In this study, we examined β-catenin and APC mutations in 22 HCCs associated with HCV infection, using single-strand conformation polymorphism (SSCP) followed by direct DNA sequencing. β-Catenin mutations were found in nine (41%) cases, but no APC mutations were found. β-Catenin immunohistochemistry revealed nuclear accumulation of β-catenin protein in all nine tumors with a β-catenin mutation and two additional tumors without a mutation. These results suggest that activation of the Wnt signaling pathway by β-catenin mutation contributes significantly to the hepatocellular carcinogenesis associated with HCV infection.
... Primary HCC represents 0.5-2% of all of the cancers in most Western countries, but its incidence is extremely high in certain endemic areas of Southeast Asia and Southern Africa. Although major environmental risk factors have been identified (chronic infections with hepatitis-B virus and hepatitis-C virus, exposure to aflatoxin B1, alcohol consumption, estrogenic steroids, rare genetic disorders, etc.), the molecular mechanisms of hepatic tumorigenesis remain poorly understood (1)(2)(3)(4). Genetic alterations have been reported for p53, K-ras, N-ras, Rb, and the insulinlike growth factor II receptor in liver tumors, as well as frequent overexpression of c-myc and cyclin D1 (5-9). In addition, LOH at chromosomes 1p, 4q, 6q, 8p, 9p, 13q, 16p, 16q, and 17p has also been identified in a high percentage of HCCs, suggesting that several genes are potentially involved in the multistep process of hepatocarcinogenesis (3, 10-12). ...
Mutations affecting phosphorylation sites in the b-catenin gene have been implicated in the development of human and rodent hepatocellular carcinomas (HCCs). To further investigate the involvement of this gene in hepatocarcinogenesis, we used several transgenic mouse models of hepatic tumors induced by overexpression of c-myc in the liver either alone or in combination with transforming growth factor (TGF) a or TGF-b1. Acti- vation of b-catenin, as judged by the presence of mutations and/or nuclear translocation of the protein, was most frequent in liver tumors from c-myc (4/17; 23.5%) and c-myc/TGF-b1 (6/18; 33.3%) transgenic mice. How- ever, it was very rare in faster growing and histologically more aggressive HCCs developed in c-myc/TGF-a mice (1/20; 5%). Administration of diethylnitrosamine, phenobarbital, or 2-amino-3,8-diethylimidazo(4,5- f)quinoxaline did not significantly affect the occurrence of b-catenin mutations. Notably, nuclear accumulation of b-catenin was observed only in adenomas and highly differentiated carcinomas with eosinophilic phe- notype. Furthermore, preneoplastic lesions with eosinophilic phenotype frequently displayed focal nuclear positivity, colocalized with areas of high proliferation. In contrast, basophilic and clear-cell foci, as well as pseudo- glandular and poorly differentiated HCCs, exhibited a normal or reduced membranous immunoreactivity for b-catenin. These studies suggest that nuclear translocation of b-catenin and activation of Wingless/Wnt signal- ing may represent an early event in liver carcinogenesis, providing a growth advantage in a subset of hepatic tumors with a more differentiated phenotype.
... A significant feature of chronic HBV infection is the integration of subgenomic HBV DNA fragments into different locations within the host DNA (10)(11)(12)(13)(14). Tumor progression is often associated with the rearrangement and partial gain or loss of both viral and cellular sequences (15)(16)(17). ...
Chronic hepatitis B virus (HBV) infection is epidemiologically associated with hepatocellular carcinoma (HCC), but its role
in HCC remains poorly understood due to technological limitations. In this study, we systematically characterize HBV in HCC
patients. HBV sequences were enriched from 48 HCC patients using an oligo-bead-based strategy, pooled together and sequenced
using the FLX-Genome-Sequencer. In the tumors, preferential integration of HBV into promoters of genes (P < 0.001) and significant enrichment of integration into chromosome 10 (P < 0.01) were observed. Integration into chromosome 10 was significantly associated with poorly differentiated tumors (P < 0.05). Notably, in the tumors, recurrent integration into the promoter of the human telomerase reverse transcriptase (TERT) gene was found to correlate with increased TERT expression. The preferred region within the HBV genome involved in integration
and viral structural alteration is at the 3ʹ-end of hepatitis B virus X protein (HBx), where viral replication/transcription initiates. Upon integration, the 3ʹ-end of the HBx is often deleted. HBx–human chimeric transcripts, the most common type of chimeric transcripts, can be expressed as chimeric
proteins. Sequence variation resulting in non-conservative amino acid substitutions are commonly observed in HBV genome. This
study highlights HBV as highly mutable in HCC patients with preferential regions within the host and virus genome for HBV
integration/structural alterations.
... Alternatively, there may be different molecular mechanisms underlying the development of HCC with and without cirrhosis. For instance, HBV viral genome can directly integrate into the host human genome and act as an oncogenic factor, a process that is independent of the chronic inflammation that commonly characterizes cirrhosis [32]. In line with this notion, recent in vitro and in vivo studies suggested that the HBx protein, encoded by the HBV viral genome, increases both the expression of telomere reverse transcriptase and telomerase activity, the enzyme responsible for the maintenance of telomere length, thus prolonging the lifespan of hepatocytes and contributing to malignant transformation [33,34]. ...
Telomere length has emerged as a promising risk predictor of various cancers including hepatocellular carcinoma (HCC). However, the majority of studies in this area measured telomere length in hepatocytes and one in lymphocytes with conflicting results. Moreover, no studies have been reported on using circulating DNA telomere length as a non-invasive HCC biomarker.
We conducted a nested case-control study to determine the relative telomere length (RTL) in serum DNA from 140 hepatitis B virus (HBV)-related HCC cases and 280 frequency-matched cancer-free HBV controls.
Cases had a significantly longer RTL (median, 0.31; range, 0.02-2.31) than controls (median, 0.20; range, 0.01-1.60) (P = 0.003). Consistently, longer RTLs conferred a significantly increased HCC risk compared to short RTLs in a univariate logistic regression analysis (odds ratio [OR] = 1.55, 95% confidence interval [CI] = 1.02-2.33, P = 0.038). This association attenuated after multivariate adjustment (OR = 1.40, 95% CI = 0.90-2.19, P = 0.132). In a quartile analysis, a significant dose-response relationship was noted in univariate analysis (P(trend) = 0.017) which was again attenuated in multivariate analysis (P(trend) = 0.079). Further analyses revealed that the significant association between serum RTL and HCC risk was evident in non-cirrhotic (OR = 3.54, 95% CI 1.58-7.93 P = 0.002), but not cirrhotic (OR = 0.95, 95% CI 0.55-1.64, P = 0.860) HBV patients. Moreover, the significantly increased HCC risk conferred by cirrhosis was modulated by RTL with a significant interaction effect (P(interaction) = 0.013).
RTL in circulating cell-free serum DNA could potentially be used as a novel non-invasive biomarker for non-cirrhotic HCC. Prospective cohort studies are warranted to validate this finding and assess its clinical significance in HCC prevention.
... 2. Alcohol lifestyle dependent + Hakulinen et al. 1974; Adelstein and White 1976; Hirayama 1981; IARC 1988 3. Hepatitis B virus blood transfusion + + Snyder et al. 1982; Buendia 1992 ...
... Hepatocellular carcinoma (HCC) ranks among the most common and deadly cancers worldwide [1]. HBV and HCV infection, alcohol abuse and exposure to aflatoxin B have been identified as major risk factors [2][3][4]. HCC associated with HCV infections evolves after many years of chronic infection and is generally preceded by the development of cirrhosis. However, the mechanisms underlying HCV-associated hepatocarcinogenesis are not fully understood. ...
The Hepatitis C virus (HCV) core protein has been implicated as a potential oncogene or a cofactor in HCV-related hepatocellular carcinoma (HCC), but the underlying mechanisms are unknown. Overactivation of the Wnt/β-catenin signaling is a major factor in oncogenesis of HCC. However, the pathogenesis of HCV core-associated Wnt/β-catenin activation remains to be further characterized. Therefore, we attempted to determine whether HCV core protein plays an important role in regulating Wnt/β-catenin signaling in HCC cells.
Wnt/β-catenin signaling activity was investigated in core-expressing hepatoma cells. Protein and gene expression were examined by Western blot, immunofluorescence staining, RT-qPCR, and reporter assay.
HCV core protein significantly enhances Tcf-dependent transcriptional activity induced by Wnt3A in HCC cell lines. Additionally, core protein increases and stabilizes β-catenin levels in hepatoma cell line Huh7 through inactivation of GSK-3β, which contributes to the up-regulation of downstream target genes, such as c-Myc, cyclin D1, WISP2 and CTGF. Also, core protein increases cell proliferation rate and promotes Wnt3A-induced tumor growth in the xenograft tumor model of human HCC.
HCV core protein enhances Wnt/β-catenin signaling activity, hence playing an important role in HCV-associated carcinogenesis.
این کتاب که در سال 1382 پیرو آغاز پروژه واکسن های ژنی در ایران در سال 1380 توسط دکتر علی کرمی شروع شد منتشر گردید و به توضیح نسل چهارم واکسن ها یعنی استفاده از مواد ژنتیکی شامل
DNA یا RNA بجای میکرب یا پروتئین نوترکیب بعنوان واکسن برای القا سیستم ایمنی در اثر بیان ژن در داخل سلول ها می پردازد
تا کنون واکسن ها شامل میکرب های کشته یا ضعیف شده یا ساب یونیتی یعنی بخشی از میکرب یا سم بودند ولی در این روش از ماده ژنتیکی رمز کننده پروتئین مورد نظر استفاده می شود در این فناوری که با استفاده از بیوتکنولوژی و مهندسی ژنتیک ژن رمز کننده پروتئین ایمنی زا به یک حامل یا پلاسمنید خاص کلون می شود این پلاسمید دارای ویژگی بیان ژن در داخل سلول است بنابر این پروتئین نوترکیب تولید شده در داخل سلول سبب القا سیستم ایمنی هومورال و سلولار شده و هم آنتی بادی ضد آنتی ژن تولید می شود و هم ایمنی دراز مدت سلولی علیه میکرب یا سم ایجاد شده و مزایای بسیاری نسبت به واکسن های نسل اوب تا سوم را دارد. دکتر کرمی در این پروژه که بنیان آن را در ایران نهادند این فرضیه را مطرح کردند که از ژن رمز کننده پروتئین های حفاظت دهنده یا ایمنی زا می توان بجای میکرب کشته یا ضعیف شده یا پروتئین نوترکیب استفاده کرد. دکتر کرمی این گون واکسن ها را تهیه و آزمایشات بسیاری بروی حیوانات آزمایشگاهی انجام دادند و مقالات بسیاری نیز منتشر کردند ولی بنا به احتیاط های ایمنی هر گز این واکسن ها در انسان ها آزمایش نشدند چون نیاز به بررسی های بیشتری برای اطمینان کامل از ایمنی آنها در تزریق به جمعیت انسانی وجود دارد. به هر حال بنیان گزار واکسن های ژنی در ایران دکتر کرمی بودند. برای اطلاع دانشمندان بخش هایی از این کتاب به شکا رایگان در اینجا منتشر می شود. اصل کتاب منتشر شده توسط انتشارات دانشگاه دانشگاه امام حسین ع می باشد.
Chronic infection by hepatitis B virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (covalently closed circular DNA [cccDNA]), integration of HBV DNA into the host cell genome is regularly observed in the liver in infected patients. While reported as a prooncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well understood, chiefly due to the lack of in vitro infection models that have detectable integration events. In this study, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of > 1 per 10,000 cells, with the most consistent detection in Huh7-NTCP cells. The integration rate remained stable between 3 and 9 days postinfection. HBV DNA integration was efficiently blocked by treatment with a 200 nM concentration of the HBV entry inhibitor Myrcludex B, but not with 10 μM tenofovir, 100 U of interferon alpha, or a 1 μM concentration of the capsid assembly inhibitor GLS4. This suggests that integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV genome replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration.
واکسن های ژنی یا نسل چهارم واکسن ها عبارت است از استفاده از پلاسمید های حاوی ژن رمز کننده پروتئین حفاظت دهنده ای که می تواند حضور ان در بدن سیستم ایمنی بر علیه عامل بیماری تحریک و سبب ایمنی بر علیه عامل می شود. این فناوری با کلون کردن ژن مورد نظز در یک پلاسمید بیانی با پروموتر مناسب و تخلیص پلاسیمید در حدی که فاقد هر گونه آلودگی به مواد وراٍتی و پروتئین باشد صورت می گیرد.
As the threat of exposure to emerging and reemerging viruses within a naïve population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. Recent outbreaks of Middle East respiratory syndrome corona virus, Ebola virus, Chikungunya virus, and Zika virus illustrate the emerging threats that are encountered. By utilizing animal models in this endeavor, the host response to viruses can be studied in a more complex and integrated context to identify novel drug targets, and assess the efficacy and safety of new products rapidly. This is especially true in the advent and implementation of the FDA animal rule. Although no one animal model is able to recapitulate all aspects of human disease, understanding the current limitations allows for a more targeted experimental design. Important facets to consider prior to an animal study are route of viral exposure, species of animal, biomarkers of disease, and a humane endpoint. This chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist.
Schon vor etwa 100 Jahren wurden in verschiedenen experimentellen Untersuchungen Hinweise für eine infektiöse Ursache von Tumorerkrankungen gewonnen. Durch Ellermann und Bang wurden Leukämien mit Hilfe zellfreier Extrakte auf Vögel übertragen. Auch bei Mäusen gelang es etwas später, Leukämien durch Extrakte aus leukämischen Zellen anderer Vögel hervorzurufen. Analog konnte Peyton Rous Sarkome bei Hühnern und McFadyan und Hobday Papillome bei Hunden überimpfen. Zu Beginn der 30er Jahre untersuchte Sir Richard Shope große hornartige Papillome, die bei einer bestimmten Kaninchenart, den sog. „cottain tail rabbits“ (Abb. 3.1). Es gelang ihm ebenfalls, diese Läsionen von einem Tier auf ein anderes und auch auf Hauskaninchen zu übertragen. Bei einigen der Kaninchen kam es nach Monaten zur spontanen Rückbildung der Läsion. Bei einigen persistierten die Papillome über einen langen Zeitraum, und bei einer weiteren Gruppe der Kaninchen entwickelten sich aus den übertragenen Papillomen invasiv wachsende metastasierende Plattenepithelkarzinome. Zusammengenommen zeigten diese frühen Arbeiten, daß eine Vielzahl von Tumorerkrankungen zumindest bei Tieren durch filtrierbare infektiöse Erreger mit hervorgerufen werden.
Although several important studies have pointed out the general and versatile role of calcium in cell growth and differentiation (Berridge et al., 1998), evidence that Ca2+-governing genes may be selectively mutated and involved in cell transformation is, as yet, lacking.
In woodchuck hepatomas induced by woodchuck hepatitis virus (WHV) infection, the predominant role of viral integration has been pointed out. In more than 50% of cases, integrated viral sequences have been shown to interrupt myc family genes including c-myc, N-myc and a woodchuck specific retroposon called N-mycl. In a manner similar to that observed in retroviral insertions into myc genes in murine T lymphoma, activated expression from the myc promoters resulted from nearby integration of the viral enhancer. In ground squirrel hepatomas related to past or ongoing ground squirrel hepatitis virus (GSHV) infection, c-myc is amplified in about 50% of the tumors analyzed. In these tumors, integration of viral DNA in the host genome was not apparently implicated. Transgenic mice carrying a mutated c-myc gene and adjacent WHV sequences were constructed. Virtually all mice from two different strains developed hepatocellular carcinoma with a mean latency period of 8–12 months. A direct correlation was established between the level of c-myc expression in the liver and the onset of liver tumors. Woodchuck c-myc mRNA driven by the normal P1 and P2 promoters and WHV-specific transcript encoding viral surface antigens were produced in a strictly coregulated fashion during development and tumorigenesis, indicating a predominant regulatory influence of the viral enhancer.
Rearrangements of both integrated viral and associated chromosomal DNA during chronic infection appear to be key events in malignant transformation of the hepatocyte. Probably the most relevant of such alterations involve the HBV X gene and the preS + S gene, both of which encode functionally modified transactivator proteins that stimulate a variety of cellular promoters via NFкB and other transcription factors. In addition, integration of viral DNA can physically subvert normal control of a nearby cellular gene, sometimes leading to unregulated expression of cellular growth-related genes as suggested by a number of recent examples. Because of the diversity of viral DNA integration sties in tumors, several more such genes should potentially be identifiable.
The review by Dr Johnson clearly indicates the strength of the epidemiological association between chronic infection by HBV and hepatocellular carcinoma (HCC), the most frequent histological form of primary liver cancer (PLC). However, the mechanisms involved in liver carcinogenesis still remain a matter of debate. Cirrhosis, which is present in around 90% of patients with HCC tumors in most areas, has long been recognized as an important risk factor. Early clinical observations in Africa showed the evolution of acute hepatitis (AH) to chronic active hepatitis (CAH), cirrhosis and, eventually, liver cancer [1, 2]. The importance of this association has since been confirmed but its prevalence is considered to be lower in Africa (around 50% of cases) than in Asia, Europe, and America (80–100%). The molecular basis of the promoting effect of cirrhosis is far from clear but generally related to “liver cell regeneration”.
The mechanism of action of hepatitis B virus (HBV) X protein in transcriptional transactivation and in tumorigenesis remains obscure, We have used the yeast two-hybrid system to identify a cellular protein that can interact with HBV X protein. This protein, designated X-associated protein 1 (XAP-1), is a human homolog of the UV-damaged DNA-binding protein (UV-DDB) recovered from a monkey cell cDNA library. UV-DDB is presumed to be involved in DNA repair. The interaction between X protein and XAP-1 protein was verified by immunoprecipitation of yeast cell lysates expressing both proteins and by in vitro mixing with X protein expressed as a glutathione S-transferase fusion protein and XAP-1 protein either in HeLa cell extracts or synthesized by in vitro translation. We speculate that the interaction of X protein with a DNA repair protein may recruit cellular proteins to repair the partially double-stranded HBV genome or may modify cellular transcription processes. An effect on the cellular DNA repair system may explain a cofactor role for HBV in liver cancer development.
HBV genomes have been described even in HBsAg-negative patients with hepatocellular carcinoma (HCC), but the role of hepatitis B virus (HBV) in liver transformation is still unclear. We assessed the rate of persistence, the structure, and the RNA expression of HBV genomes in HBsAg-negative HCC using HBV- DNA PCR and HBV-RNA RT-PCR. HBV DNA genomes have been detected in 37 (59%) of 63 HBsAg-negative HCC. Defective genomes have been found more frequently in tumorous than nontumorous tissues (19/29 vs 1/9) and in seronegative than seroconverted patients (9/12 vs 1/7). A significant accumulation of X RNA was shown in 7/9 tumorous and 7/8 nontumorous tissues. By contrast, S and C RNAs were barely expressed in both tumorous and nontumorous tissues. These results suggest a role for the potentially transforming, transactivating X protein in the HBV-related liver carcinogenesis in HBsAg-negative patients.
The role of hepatitis B virus (HBV) as a risk factor for hepatocellular carcinoma (HCC) in man is well attested to by epidemiological and experimental evidence. There are, however, a number of outstanding questions that deserve further attention because of their relevance to cancer prevention. This paper highlights these issues relating to HBV transmission, determinants of persistent viral infection, risk factors for cancer and vaccination. In addition, a number of mechanistic questions are reviewed, including the possible hepatocarcinogenic mode of action of the virus and interactions with the dietary carcinogens, the aflatoxins which often co- occur with HBV in areas of high HCC incidence.
As the threat of exposure to emerging and reemerging viruses within a naive population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. By using animal models in this endeavor, the response to viruses can be studied in a more natural context to identify novel drug targets, and assess the efficacy and safety of new products. This is especially true in the advent of the Food and Drug Administration's animal rule. Although no one animal model is able to recapitulate all the aspects of human disease, understanding the current limitations allows for a more targeted experimental design. Important facets to be considered before an animal study are the route of challenge, species of animals, biomarkers of disease, and a humane endpoint. This chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist.
Viral hepatitis in humans has been causally related to a variety of agents, which belong to different virus groups and differ markedly in their structural and biological properties. As many as five viruses associated with human hepatitis (the hepatitis A, B, C, D, and E viruses) have been isolated and fully characterized,(1–5) and the question of whether additional, uncharacterized viruses may be responsible for liver disease in some cases is still debated. Among the known human hepatitis viruses, two—the hepatitis B and C viruses—appear to be more pathogenic for humans because of their ability to induce persistent infections in the infected host. Chronic, long-lasting infections with both agents frequently result in the development of liver cirrhosis and ultimately evolve to primary liver cancer in a significant proportion of virus carriers.
About 80% of hepatocellular carcinoma (HCC) is caused by hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections especially in the setting of established cirrhosis or advanced fibrosis, making HCC prevention a major goal of antiviral therapy. HCC tumors are highly complex and heterogeneous resulting from the aberrant function of multiple molecular pathways. The roles of HCV or HBV in promoting HCC development are still either directly or indirectly are still speculative, but the evidence for both effects is compelling. In patients with chronic hepatitis viral infection, cirrhosis is not a prerequisite for tumorigenesis.
There are very few studies on the incidence and risk factors of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) in the absence of advanced fibrosis. Our objective was to identify the clinical-pathological features of these patients.
We retrospectively reviewed 162 patients admitted to our hospital for HCV-related HCC between 2000 and 2010. Patients with hepatitis of other aetiologies, human immunodeficiency virus co-infection, or treated with interferon were excluded. We compared demographic, laboratory, clinical and outcome parameters of patients with and without advanced fibrosis.
137 patients had advanced fibrosis (85%). Median age was higher in the advanced fibrosis vs. the non-advanced fibrosis group (62 vs. 65 years, respectively; p=0.025). Steatosis was significantly more frequent in patients with advanced fibrosis compared to those without advanced fibrosis (43% vs. 20%, respectively; p=0.032). Independent predictors associated to the occurrence of HCC in patients without advanced fibrosis were hepatitis B core antigen (odds ratio: 3.86; p=0.044) and duration of hepatitis C infection (odds ratio: 1.21; p=0.003).
Risk factors such as steatosis or diabetes were not frequent in patients without advanced fibrosis. Further studies are needed to evaluate the role of occult hepatitis B and the duration of hepatitis infection in patients with HCC and chronic hepatitis C without advanced fibrosis.
Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
The hepatitis vi ruses are a diverse collection which, for the most part, have little in common except their name and the disease they cause in a susceptible host. This article outlines what is currently known about each of the hepatitis viruses A - E and summarises the methods which are available for treatment. The most recent developments that have been published in the patent literature during 1994 are also outlined. Emphasis is placed on hepatitis B and C as these are currently perceived to be the largest areas of unmet need for antiviral therapy.
Chronic hepatitis B virus (HBV) infection is epidemiologi-cally associated with hepatocellular carcinoma (HCC), but its role in HCC remains poorly understood due to technological limitations. In this study, we systematically characterize HBV in HCC patients. HBV sequences were enriched from 48 HCC patients using an oligo-bead-based strategy, pooled together and sequenced using the FLX-Genome-Sequencer. In the tumors, preferential integration of HBV into promoters of genes (P < 0.001) and significant enrichment of integration into chromo-some 10 (P < 0.01) were observed. Integration into chromosome 10 was significantly associated with poorly differentiated tumors (P < 0.05). Notably, in the tumors, recurrent integration into the promoter of the human telomerase reverse transcriptase (TERT) gene was found to correlate with increased TERT expression. The preferred region within the HBV genome involved in integration and viral structural alteration is at the 3ʹ-end of hepatitis B virus X protein (HBx), where viral replication/transcription initiates. Upon integration, the 3ʹ-end of the HBx is often deleted. HBx– human chimeric transcripts, the most common type of chimeric transcripts, can be expressed as chimeric proteins. Sequence variation resulting in non-conservative amino acid substitutions are commonly observed in HBV genome. This study highlights HBV as highly mutable in HCC patients with preferential regions within the host and virus genome for HBV integration/structural alterations.
RNA of recently reported, putative non-A to E hepatitis virus designated GB virus C (GBV-C) was determined by reverse-transcription polymerase chain reaction in sera from 231 Japanese patients with primary hepatocellular carcinoma. GBV-C RNA was detected in 21 patients (9% of the total), including two of the 23 patients (8%) with markers of hepatitis B virus infection, 11 of the 114 patients (10%) with markers of hepatitis C virus infection, seven of the 86 patients (8%) with markers of both hepatitis B and C virus infections, and one of the eight patients (13%) without such markers. These results indicate that the contribution of GBV-C infection to the development of hepatocellular carcinoma would be small either by itself or in cooperation with hepatitis B and C viruses.
Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading
to liver cell transformation remain largely unknown. The Wnt-β-catenin pathway is activated in colon cancers and some melanoma
cell lines, but has not yet been investigated in HCC. We have examined the status of the β-catenin gene in different transgenic
mouse lines of HCC obtained with the oncogenes c-myc or H-ras. Fifty percent of the hepatic tumors in these transgenic mice had activating somatic mutations within the β-catenin gene
similar to those found in colon cancers and melanomas. These alterations in the β-catenin gene (point mutations or deletions)
lead to a disregulation of the signaling function of β-catenin and thus to carcinogenesis. We then analyzed human HCCs and
found similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations
led to the accumulation of β-catenin in the nucleus. Thus alterations in the β-catenin gene frequently are selected for during
liver tumorigenesis and suggest that disregulation of the Wnt-β-catenin pathway is a major event in the development of HCC
in humans and mice.
This chapter discusses the role of hepatitis B surface antigen (HBsAg) in hepatocarcinogenesis. Hepatocellular carcinoma (HCC) is one of the most common human malignant neoplasms. It is the fifth most common cancer and the third cause of cancer death in the world. The prognosis of HCC is very poor with or without therapeutic intervention. The overall 5 years survival rate worldwide is estimated at only ∼3% because of late diagnosis. Although the molecular pathogenesis of HCC remains elusive, the etiologic association between hepatitis B virus (HBV) infection and hepatocarcinogenesis has been well established. There are an estimated 387 million HBV carriers worldwide, and geographic distribution of HBV carriers match well with HCC incidences. Epidemiologic studies have demonstrated that the risk of developing HCC among HBV carriers is 100 fold higher than among noncarriers and the death rate from HCC is between 10% and 25%. The HBsAg polypeptides are important for HBV infection and are an important target for diagnosis and vaccine development.
AIM: Growing evidence demonstrates that hepatitis B virus (HBV) integration and re-sultant de novo genetic mutations play an important role on tumorigenesis and malig-nant progression of hepatocellular carcinomas (HCCs). In the present study, we sought to address the impact of HBV integration on the genome of the host cell. METHODS: We employed genome-wide oligo-array comparative genomic hybridiza-tion (CGH) to profile genetic copy number alterations (CNAs), including deletion or am-plification, within genomes of parental HepG2 cells and the HBV-transfected variant, HepG2215. To determine whether or not the selection pressure during establishing stable transfectant contributed to genetic alteration, another HepG2 variant, HepG2.2 cells, previously transfected with a HBV-unrelated gene followed by G418 selection was also included in our experiments. Results were validated by dye-swap experiments. RESULTS: Extensive de novo genetic aberrations have been detected In HepG2215 cells but not present in HepG2 cells and HepG2.2 cells, such as gains on chromosome arms 8q, 9q, 11p, 12q, 14q, 15q, 19p, 21q, 22q and losses on 8p, 8q, 9p, 9q, 11p, 11q, 12p, 12q, 14q, 15q, 18p, 18q, 19p, 19q, 21q, 22q. It is noteworthy that chromosome 8 harbored several de novo CNAs in comparison with those of HepG2. In addition, genes implicated in cell cycle control, apoptosis, tumorigenesis and malignant progression have been assigned to these altered regions. CONCLUSION: Our results demonstrate that HepG2215 cells harbored de novo CNAs at several sites in comparison with parental HepG2 cells. There was no significant dif-ference between the genetic profile of HepG2 and HepG2.2 cells, indicating the new ge-netic aberrations were not generated by G418 selection. The genome instability of HepG2215 cells could be a result of HBV DNA integration or arised from the trans-acting of viral gene product. These findings provide important information on application of HepG2215 cells and support the hypothesis that HBV infection associates with devel-opment of some HCCs by interfering with cellular processes responsible for the insta-bility of genome. Journal of Cancer Molecules 1(2): 93-98, 2005.
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 α-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 μg/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 ATX mice developed abnormal liver lesions. When 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 lesions 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. © 1996 Wiley-Liss, Inc.
Hepatocellular carcinoma (HCC) is among the most common cancers in the world and one of the few human
cancers caused by viral infections. The hepatitis B virus (HBV) is a major risk factor of HCC development, but the underlying oncogenic mechanisms have not been fully elucidated. Malignant transformation occurs after a long period of chronic liver disease, which is frequently associated with cirrhosis, suggesting a nonspecific mechanism triggered by the host immune response. However, the virus might play a direct role as an insertional mutagen through viral DNA integration into the host genome. Viral proteins including the transcriptional activator HBx and the large and middle surface proteins can act as cofactors by interfering with regulatory processes. Because HCC carries a dismal prognosis, there is urgent need to develop early diagnostic markers of HCC and effective therapies against chronic hepatitis B. This chapter reviews various mechanisms linking chronic HBV infection to malignant transformation, with reference to recent advances provided by genomewide analysis of HBV-related HCC.
Objective: In order to investigate the relationship between the expression of ras gene and the development of hepatocellular carcinoma (HCC). Materials and Methods: The experimental tree shrews were divided
into four groups: group A, infected human hepatitis B virus (HBV) and exposed to aflatoxin B1 (AFB1); group B, infected human
HBV alone; group C, only exposed to AFB1; group D, use as controls. The serial bioptic liver tissues were detected for ras p21 protein using immunohistochemical method. Results: The total p21 protein positive rates in group A, B, C and D were 35.3%, 5.3%, 13.3%, 0, respectively, thus the significant difference were
showed between group A and group B (P<0.05); The HCC incidences in group A, B, C and D were 47.1%, 0, 13.3%, 0, respectively, and there was a significant difference
between group A and C (P<0.05). The incidences of HCC in the animals with and without p21 protein positive in group A were 100% and 18.2%, respectively, and there was a significant difference among them (P<0.01). Conclusion: HBV and AFB1 play a remarkable synergistic role in the development of HCC; they can enhance the expression
of ras gene. The over-expression of ras gene is closely related to pathogenesis of HCC in tree shrews.
DNA sequences of specific human papillomavirus (HPV) types are found integrated in the cell genome in most invasive genital carcinomas. We have determined the chromosomal localization of integrated HPV type 16 (HPV-16) or HPV-18 genomes in genital cancers by in situ hybridization experiments. In three cancers, HPV sequences were localized in chromosome band 8q24.1, in which the c-myc gene is mapped, and in one cancer HPV sequences were localized in chromosome band 2p24, which contains the N-myc gene. In three of the four cases, the proto-oncogene located near integrated viral sequences was found to be structurally altered and/or overexpressed. These data indicate that HPV genomes are preferentially integrated near myc genes in invasive genital cancers and support the hypothesis that integration plays a part in tumor progression via an activation of cellular oncogenes.
Camptothecin (CPT), a topoisomerase I-specific inhibitor, was found in this study to inhibit the replication of equine infectious anemia virus (EIAV) in chronically infected CF2Th cells (designated CF2Th/EIAV). By measuring viral reverse transcriptase activity in the culture medium, we demonstrated that treatment for 1 h with noncytotoxic doses of this drug inhibited production by 32 to 52%, whereas continuous exposure to this drug resulted in an 85 to 92% inhibition. No effect on the viability or growth rate of the cells was detected in any of these treatments. Indirect immunofluorescence analysis of the CPT-treated CF2Th/EIAV cells with anti-p26 capsid protein antibodies showed 60 to 85% reduction in the immunofluorescence-positive cells following drug treatment, and radioimmunoprecipitation analysis of these cells showed a comparable decrease of the pr55gag precursor protein. These data suggest that CPT acts as an anti-EIAV agent to block virus replication in the chronically infected cells.
A detailed mutational analysis of the regulatory DNA sequence elements that control expression of the hepatitis B virus major surface antigen gene was performed in the human hepatoma cell lines HepG2.1 and Huh7, using transient transfection assays. Seven regions (A to G) of the major surface antigen promoter located within 200 nucleotides of the RNA initiation site have been identified which influence the level of transcription from this promoter. The three distal regions (A to C), located between -188 and -68, appear to possess a level of redundancy in their ability to influence the transcriptional activity from the major surface antigen promoter. The simultaneous deletion of regions A, B, and C resulted in an approximately fourfold reduction in transcription from the major surface antigen promoter. Region D, located between -67 and -49, is an essential element of the major surface antigen promoter. The three proximal regions (E to G) are located within 45 nucleotides of the major transcription initiation site. Region E prevents the negative influence of region F and can compensate for the effect of mutation of region G on transcription from the major surface antigen promoter. Region G can compensate for the effect of the loss of a functional region E sequence on the transcriptional activity of the major surface antigen promoter only in the absence of a functional region F sequence. These results imply that the level of expression of the major surface antigen gene is controlled by the complex interplay between a minimum of six transcription factors which activate and one transcription factor which represses transcription from this gene.
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.
In Taiwan, hepatocellular carcinoma is one of the major malignancies in children between 5 and 14 yr of age. We studied the status of hepatitis B virus DNA in the hepatocellular carcinoma and nontumorous liver tissues of eight children with positive serum HBsAg and maternal HBsAg. The hepatocellular carcinoma tissues from five of the eight children showed integration of hepatitis B virus DNA into host cellular DNA sequences. A pattern of single‐site integration in four children and a multiple‐site integration pattern in one child were demonstrated. In the remaining three children, hepatitis B virus DNA could not be demonstrated in the tumor tissues. Using subgenomic fragments of the hepatitis B virus genome as probes, we found that the X gene fragment and the surface antigen gene fragment were the most conserved sequences. The single‐site integration of hepatitis B virus DNA in childhood hepatocellular carcinoma may have hit the critical region, resulting in insertional mutagenesis and early development of hepatocellular carcinoma. With a short incubation period and less exposure to environmental carcinogens during early life, childhood hepatocellular carcinoma may provide a good model to study the carcinogenic potential of hepatitis B virus. (HEPATOLOGY 1991;13:316–320).
To determine the clonal evolution of hepatocellular carcinoma, the integrated hepatitis B virus DNA patterns of the main tumor, satellites and/or metastatic lesions were analyzed by Southern‐blot hybridization in 28 hepatocellular carcinomas, including three HBsAg‐seronegative cases. Unicentric or multicentric hepatocellular carcinoma was confirmed by histopathological criteria in 89% of the cases. Among 17 unicentric hepatocellular carcinomas, minor changes of the integration pattern–including partial loss or addition of the integration sites or both–were detected in the metastatic lesions in 29% of the cases. Furthermore, none of five cases with free‐form hepatitis B virus DNA in the primary tumor had detectable free hepatitis B virus DNA in the metastatic lesions. These results suggest that the alteration of integrated hepatitis B virus DNA pattern during the course of tumor growth and metastasis may occur more often than previously perceived and that the switch‐off of virus replication may be related to tumor metastatic potential. In eight cases with unilateral, multicentric hepatocellular carcinoma, two clones were detected in six cases, three were seen in another and four were seen in one. One case of note was a 9‐yr‐old boy with two histological types and two different integration patterns, one associated with vascular invasion and lung metastasis. Three patients with bilateral hepatocellular carcinoma were confirmed to have bicentric or tricentric hepatocellular carcinoma rather than intrahepatic dissemination and had survival rates similar to those in unicentric hepatocellular carcinoma. Three invasive HBsAg‐seronegative hepatocellular carcinomas were found to have hepatitis B virus DNA integration and were of unicentric origin. These results suggest that Southern‐blot analysis is not only a valuable tool for the study of tumor clonal origin and evolution of hepatitis B virus‐related hepatocellular carcinoma, but it also provides valuable information to better understand its biological behavior. (HEPATOLOGY 1991;923–928.)
The C gene of hepatitis B virus (HBV) codes for a nucleocapsid protein made of 183 amino acid residues and is preceded in phase by the precore (pre-C) region, encoding 29 residues. The pre-C-region product is required for the synthesis and secretion of hepatitis B e antigen (HBeAg), which is made of the C-terminal 10 amino acid residues of the pre-C-region product and the N-terminal 149 residues of the C-gene product. HBV mutants with pre-C-region defects prevailed in the circulation of three asymptomatic carriers as they seroconverted from HBeAg to the corresponding antibody (anti-HBe), and these mutants finally replaced nondefective HBV. HBV DNA clones were propagated from sera of an additional 15 carriers with anti-HBe and sequenced for the pre-C region. Essentially all HBV DNA clones (56 of 57 [98%]) revealed mutations that prohibited the translation of a functional pre-C-region product. A point mutation from G to A at nucleotide 83, converting Trp-28 (TGG) to a stop codon (TAG), was by far the commonest and was observed in HBV DNA clones from 16 (89%) of 18 carriers seropositive for anti-HBe. In addition, there were point mutations involving ATG codon to abort the translation initiation of the pre-C region, as well as deletion and insertion to induce frameshifts. Such mutations leading to pre-C-region defects were rarely observed in persistently infected individuals positive for HBeAg or in patients with type B acute hepatitis after they had seroconverted to anti-HBe. These results would indicate a selection of pre-C-defective mutants in persistently infected hosts, along with seroconversion to anti-HBe, by immune elimination of hepatocytes harboring nondefective HBV with the expression of HBeAg.
The entire genome of human hepatitis B virus (HBV) occurring in Latvia was sequenced. This sequence, which is 3182 nucleotides long, was compared with the other previously published HBV genomes and was shown to share maximum homology with HBV subtype ayw DNA. The coordinates of 4 main open reading frames as well as hairpin structures are very well conserved in the two genomes. The distribution of nucleotide substitutions among different HBV genomes suggests that the open reading frames P and X can fulfil a coding function. On the basis of primary stucture comparison for hepadnaviral DNAs several evolutionary conclusions can be drawn.
Patients who receive large numbers of transfusions for anemia and other causes may develop precipitins in their blood. These precipitins may react in agar gel double diffusion experiments with specific human serum lipoprotein found in the blood of other individuals. Since these precipitins were found only in patients who had received transfusions they were thought to be antibodies against serum lipoproteins which developed in the patients as a result of the repeated transfusions. The precipitin is referred to as an isoprecipitin since it develops against a specificity found in an individual from the same species. The antilipoprotein isoprecipitin1,2 developed in approximately 30% of 47 patients with thalassemia who had received transfusions. Isoprecipitins also developed in smaller number of transfused patients with other diseases. All precipitins stained with sudan black, a dye specific for lipid. Immunoelectrophoretic and ultracentrifugal studies showed that the protein with which the isoprecipitins reacted was a
Mutations of the p53 gene are found in hepatocellular carcinoma (HCC), the most common form of primary liver cancer. Specific mutations might reflect exposure to specific carcinogens and we have screened HCC samples from patients in 14 different countries to determine the frequency of a hotspot mutation at codon 249 of the tumour suppressor p53 gene. We detected mutations in 17% of tumours (12/72) from four countries in south Africa and the southeast coast of Asia. There was no codon 249 mutation in 95 specimens of HCC from other geographical locations including North America, Europe, Middle East, and Japan. Worldwide, the presence of the codon 249 mutation in HCCs correlated with high risk of exposure to aflatoxins and the hepatitis B virus (HBV). Further studies were completed in two groups of HBV-infected patients at different risks of exposure to aflatoxins. 53% of patients (8/15) from Mozambique at high risk of aflatoxin exposure had a tumour with a codon 249 mutation, in contrast with 8% of patients from Transkei (1/12) who were at low risk. HCC is an endemic disease in Mozambique and accounts for up to two thirds of all tumours in men. A codon 249 mutation of the p53 gene identifies an endemic form of HCC strongly associated with dietary aflatoxin intake.
In Europe, only ≈1% of the general population are chronic carriers of the hepatitis B surface antigen (HBsAg) but hepatitis B is unevenly distributed in the region. Based on the prevalence of HBsAg, the region may be divided into three hepatitis B epidemiological patterns: the UK and the Scandinavian countries (>0.1%); most countries in Western Europe (0.1–0.5%); and countries situated along the Mediterranean Sea and in Eatern Europe (1–5%). Existing screening and vaccination programmes depend on such factors as the carrier rate of the indigenous population and the influx of immigrants from highly endemic areas. Vaccination of health care workers is, in general, advised but not required. The accent has been placed, in most countries, on the screening of pregnant women for the presence of HBsAg and the vaccination of newborns of carrier mothers. Educational programmes are needed to enhance awareness of general practitioners regarding these risk groups. The institution of mass vaccination will depend upon the cost of vaccine, although the cost factor is less important in Europe than in developing countries.
Hepatitis derived from hepatitis B virus (HBV) infection is endemic throughout the world, but it is particularly prevalent in Asia and Africa1. In these areas, demographic studies show a strong coincidence between HBV infection (assayed by HBV antigenic markers) and the incidence of primary liver cancer. On these grounds, a causal link between HBV infection and primary hepatocellular cancer has been proposed2–5. Recently, a human hepatoma cell line (PLC/PRF/5; Alexander cells) has been shown to produce hepatitis B surface antigen (HBsAg)6. We show here that the Alexander cell line contains at least six (four complete and two partial) hepatitis B viral genomes integrated into high molecular weight host DNA. An analysis using specific probes to fragments of the HBV genome suggests that integration of the virus in most cases occurs at the nicked cohesive end region of the virus. Expression of viral sequences using Northern blots demonstrates the presence of RNA transcripts specific for the surface antigen sequences of HBV DNA and the absence of detectable transcripts corresponding to the hepatitis B core antigen.
The hepatitis B virus (HBV) is a small, circular, double stranded DNA virus that causes self limited (acute) or prolonged (chronic) infection, with or without associated liver cell injury (hepatitis). Longstanding chronic hepatitis B carries a 200-fold or greater increased risk of development of hepatocellular carcinoma.1 Although much has been learned in recent years about the structure and organization of the HBV genome, its gene products and its replication strategy, the mechanisms responsible for viral clearance and persistence, hepatocellular injury and malignant transformation are not well understood.HBV is a member of the Hepadnavirus family which consists of several related hepatotropic DNA viruses that share many genetic and biological features (hepatitis B virus; woodchuck hepatitis virus; duck hepatitis B virus, ground squirrel hepatitis virus, and heron hepatitis virus). Because HBV does not infect cells in tissue culture and does not infect common laboratory animals, investigators have relied heavily on these animal models to explore the biological and pathogenetic properties of HBV and to map and manipulate the viral genome.
The polymerase chain reaction was evaluated as a diagnostic tool in 72 chronic hepatitis B virus carriers. Hepatitis B virus DNA was detectable in the serum of HBsAg—positive virus carriers using aliquots as small as 100 al. The detection limit for cloned hepatitis B virus DNA was 100 ag. Primer pairs for different regions of the HBV genome resulted in different sensitivity. Detection of the amplified hepatitis B virus DNA by Southern blotting and subsequent scintillation counting or densitometry allowed a semiquantitative assay. Using several primer pairs in parallel for optimal detection, all HBeAg-positive HBsAg carriers, 80% of HBe antibody—positive symptomatic HBsAg carriers and 57% of asymptomatic HBe antibody—positive HBsAg carriers were found to have hepatitis B virus DNA in the serum. During antiviral therapy hepatitis B virus DNA disappeared by the polymerase chain reaction assay in patients who became HBeAg negative, but polymerase chain reaction detected a relapse earlier than did the conventional dot blot. Pre-S antigens were assayed in serum and liver samples from most chronic carriers by enzyme-linked immunosorbent assay and/or immunoblot. Although most viremic carriers were strongly positive for pre-S1 and pre-S2 antigens, some hepatitis B virus DNA—positive HBsAg carriers did not have detectable pre-S antigens, and vice versa. Our data show that assay of hepatitis B virus DNA in the serum by polymerase chain reaction is by far more proficient than by dot blot and that it cannot be replaced by serological assays of HBeAg or pre-S antigen. (HEPATOLOGY 1991;13:158–166).
The X protein can act on the enhancer of hepatitis B virus in an in vitro system and elevate the transcriptional level of hepatitis B virus. However, because no relationship had been reported between X protein expression and hepatitis B virus replication in patients with chronic hepatitis B, we focused on its expression in the liver in comparison with markers of hepatitis B virus replication. Liver biopsy samples and sera from 59 carriers with HBsAg were examined immunohistochemically for X protein using rabbit IgG against recombinant X protein. There was a significant difference in the serum hepatitis B virus DNA level between X protein–positive and –negative patients (p < 0.001). Serum pre-S1 and pre-S2 antigens were also measured quantitatively by enzyme immunoassay using monoclonal antibodies specific against each antigen. The titers of pre-S1 antigen in patients positive for X protein were significantly higher (p < 0.001) than those of the X protein–negative patients (3.02 ± 0.99 vs. 2.00 ± 0.59, respectively). Similarly, the titers of pre-S2 antigen were 2.98 ± 0.91 vs. 1.94 ± 0.54, respectively (p < 0.001). The rate of positivity of the X protein was higher (38 of 49; 77.6%) in the replicative group (serum HBeAg, serum hepatitis B virus DNA or HBcAg in liver positive) compared with that observed in the nonreplicative group (3 of 10; 30% – serum HBeAg, serum hepatitis B virus DNA and HBcAg in liver negative) (p < 0.01). Our findings indicate that the X protein is closely correlated with hepatitis B virus replication and may have an important role in viral replication in chronic hepatitis B virus infection. (HEPATOLOGY 1991;13:417–421.)
Summer's discovery in 1978 of a DNA virus, very close to human Hepatitis B virus in a woodchuck population in the U.S.A. (Pennsylvania) was a confirmation of the first description made by Snyder at Penrose Research Laboratory (Philadelphia). It was the first animal model of human B hepatitis infection. The comparative study of morphological, ecological and ethological characteristics of the marmot (Marmota marmota) and the woodchuck (Marmota monax) enables an easy distinction between these two species. The natural infection of M. monax by the WHV shows that the woodchuck is a good model for human B hepatitis and should be extended to M. marmota. A sample of 24 marmots caught in the Alpes of Haute-Provence has not revealed any spontaneous infection in these animals by the woodchuck virus. The failure of experimental inoculation of the marmot (24 animals) with the WHV confirms the refractory status of this species (no viremia and very low and short serological response with or without an immunosuppressive treatment). These preliminary results require a confirmation in other animals of different age and geographical region and also by using more specific tests such as molecular hybridation, research on DNA polymerase and direct transfection trials.
The livers of 33 captive woodchucks were examined histologically in 30 biopsy and 10 autopsy specimens and the findings were correlated with serum determinations for woodchuck hepatitis virus (WHV), surface antigen (WHsAg) and antibody (anti-WHs), and WHV DNA and DNA polymerase. The liver appeared normal in all 3 serum-negative animals, 7 of 16 with indeterminate WHV status, and 1 of 4 with anti-WHs, but not in 10 animals with WHsAg, WHV DNA, and DNA polymerase. Mild hepatic inflammation was found in 7 woodchucks with indeterminate status, 4 with anti-WHs, and 2 with each marker of WHV infection. Significant inflammation was found in 2 of indeterminate status and 4 with every marker, whereas more severe lesions (2 of chronic active type) occurred, almost always in autopsy specimens, in 8 animals with every marker. Eight of 10 animals with all markers had orcein-positive inclusions (Shikata's technique) and 6 had hepatocellular carcinoma associated with acute and chronic hepatic inflammation and, usually, neoplastic nodules in the noncarcinomatous parenchyma. Features distinguishing the woodchuck lesion from human hepatitis B disease were: association of carcinoma with acute hepatic inflammation (but not with cirrhosis) and DNA polymerase in the serum; transition to carcinoma from neoplastic nodules; conspicuous plasma-cellular reaction of hepatic inflammation, and hematopoietic cells in the tumor. Significant hepatic lesions in the woodchucks were regularly associated with serum WHsAg, WHV DNA, and DNA polymerase. In contrast to man, hepatocellular carcinoma in woodchucks was regularly associated with these markers of active viral replication. The nature of the orcein-positive inclusions requires elucidation, although they may assist in screening for similar viruses in other species. The woodchuck may help in the study of the relation between hepatocellular carcinoma and hepatitis B, including the possibility of cocarcinogenic factors.
We made a prospective study on the development of hepatocellular carcinoma (HCC) in patients with liver cirrhosis with hepatitis B virus infection from April, 1973 to December, 1977. Seven out of 30 patients (23%) with hepatitis B surface antigen (HBsAg)-positive cirrhosis developed HCC. On the other hand, only 5.9% of the patients with HBsAg-negative liver cirrhosis developed HCC. These patients were classified into three groups according to their anti-HB core (anti-HBc) titers. When the anti-HBc titer, expressed as a dilution of serum, was 210 or more (Group I),20-24 % of the liver cirrhosis patients developed HCC either with or without a detectable amount of HBs Ag present in the sera. When the anti-HBc titer was 29 or less (Group II), only 0–5.7% developed HCC. There was no significant difference between this and the anti-HBc and HBsAg-negative group (Group III), which was 4.4%. In five individual cases from group I, HBsAg was detected in serum, and in biopsies of liver cells, before HCC could be detected by angiography and/or rising levels of alphafetoprotein (AFP).In all of these cases, the anti-HBc titer was higher than 210 throughout the observation period, even before the development of HCC. These findings indicate that active virus proliferation in chronic hepatitis B virus infection precedes the development of HCC as indicated by a higher anti-HBc titer. Therefore we have prepared these studies to show the pathogenic role of hepatitis B virus in the development of hepatocellular carcinoma.
A total of 33 hepatocellular carcinomas, induced in woodchucks by chronic infection with woodchuck hepatitis virus (WHV),
a virus closely related to the human hepatitis B virus, were analyzed for the state of viral DNA, the expression of viral
genes and of different cellular proto-oncogenes. Low levels of viral replication and presence of integrated viral forms including
sequences of the enhancer element, appeared as a general rule in these tumors. Enhanced expression of one or more of the nuclear
protooncogenes: c-myc, N-myc, c-fos, c-jun and jun-B was frequently observed. In two hepatomas, elevated expression and allelic
alterations of c-myc were subsequent to integration of WHV DNA near the c-myc coding domain. The viral strategy for insertional
activation of c-myc in these tumors appeared basically identical to that of mammalian retroviruses in T-cell lymphomas of
mice and rats. Whether insertional mutagenesis of different oncogenes may be more generally linked to liver oncogenesis induced
by WHV and hepatitis B viruses remains to be determined.
A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3′ end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR− Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process.
In the absence of an inbred animal model of hepatitis B virus (HBV) infection, several laboratories have chosen to study the murine immune response to HBV-encoded proteins as immunogens as opposed to an infectious agent. In this article, David Milich reviews the immunogenicity and the fine specificity of T- and B-cell recognition of HBV antigens, and the genetic influences that regulate these responses. This approach should increase our understanding of immune-mediated viral clearance mechanisms during HBV infection, and may provide the framework for the design of second and third generation HBV vaccines.
Two hepatocellular carcinomas, induced in woodchucks chronically infected with woodchuck hepatitis virus, were characterized for viral integration near c-myc and alterations of c-myc expression. In one tumor, viral integration within the untranslated region of c-myc exon 3 resulted in overexpression of a long c-myc viral cotranscript. In the second tumor, a single insertion of highly rearranged viral sequences 600 bp upstream of c-myc exon 1 was associated with increased levels of normal c-myc mRNA. In both cases, viral enhancer insertion and disruption of normal c-myc transcriptional or posttranscriptional control appear to be involved in c-myc activation. These results demonstrate that integration of woodchuck hepatitis virus near a cellular proto-oncogene, as in several retroviral models, can contribute to the genesis of liver tumors.
In the US in 1988, 54011 cases of viral hepatitis were reported to the Center for Disease Control (CDC), 42% of which were categorized as hepatitis B. Because of incomplete reporting and subclinical disease, the CDC estimates that ≈300 000 acute cases of hepatitis B are occuring in the US each year for an annual estimated incidence of 125 cases per 100 000 population. Since 1985, a gradual decrease in the reported incidence of hepatitis B has been observed which may be attributed partly to the availability of new hepatiti B vaccines. Comprehensive control of hepatitis B infection in North America has, however, been impeded by the diversity and mobility of high-risk groups who often are unaware of their potential to infect other individuals. Surveillance studies have indicated that the modes of transmission of hepatitis B may be changing in the US. A larger proportion of cases are seen in parenteral drug abusers and in active heterosexuals. It is believed that hepatitis B vaccine has been administered to only >% of the population who are at risk of acquiring hepatitis B virus (HBV). To achieve maximum control, universal immunization of infants will therefore be necessary. A first programme, already inaugurated in the US, involves screening of all pregnant women and providing appropriate prophylaxis to neonates of hepatitis B surface antigen-positive mothers and to susceptible household contacts of these individuals. Along with mandatory guidelines, collaboration between the hepatitis community, vaccine manufacturers, the CDC and appropriate medical, legislative and health care organization should be fostered to acheive successful control of HBV in North America.
Hepatitis B e antigen (HBeAg) constitutes the nucleocapsid of hepatitis B virus (HBV) and occurs in association with plasma proteins, particularly with IgG, in the serum of persons infected with the virus. A polypeptide with an approximate m.w. of 15,500 (P15.5) is obtained either from HBeAg in the serum or from the nucleocapsid of HBV. P15.5 preparations from serum and virus resembled closely each other in the amino acid composition. The C-terminus amino acid sequence of P15.5 from serum was determined to be -Thr-Thr-Val-Val, whereas that from the virus ended with -Thr-Thr. The same sequence of four amino acid residues was found on the gene coding for the nucleopeptide of hepatitis B virus with a molecular size of 19,000 daltons (P19). P15.5 identified on the nucleotide sequence of P19 was composed of 149 amino acid residues with a calculated molecular size of 16,770 daltons. The gene coding for P19 had two -Asp-Pro-connections. By splitting these connections in P19 and P15.5 preparations with formic acid, smaller polypeptides were obtained with sizes predicted from the nucleotide sequence and with the N-terminus amino acid of proline as expected. One of two monoclonal antibodies raised against the core of Dane particles (HBcAg) bound with P15.5 preparations purified from serum and HBV. The IgG fraction from a human serum containing antibodies to HBcAg but not to HBeAg bound with P15.5 also. On the basis of the results obtained, the IgG molecules associated with P15.5 in the serum of persons infected with HBV may well represent the antibodies against HBcAg with limited specificities.
The recent finding of c-myc activation by insertion of woodchuck hepatitis virus DNA in two independent hepatocellular carcinoma has given support to the hypothesis that integration of hepatitis B viruses into the host genome, observed in most human and woodchuck liver tumours, might contribute to oncogenesis. We report here high frequency of woodchuck hepatitis virus DNA integrations in two newly identified N-myc genes: N-myc1, the homologue of known mammalian N-myc genes, and N-myc2, an intronless 'complementary DNA gene' or 'retroposon' that has retained extensive coding and transforming homology with N-myc. N-myc2 is totally silent in normal liver, but is overexpressed without genetic rearrangements in most liver tumours. Moreover, viral integrations occur within either N-myc1 or N-myc2 in about 20% of the tumours, giving rise to chimaeric messenger RNAs in which the 3' untranslated region of N-myc was replaced by woodchuck hepatitis virus sequences encompassing the viral enhancer. Insertion sites were clustered in a short sequence of the third exon that coincides with a retroviral integration hotspot within the murine N-myc gene, recently described in T-cell lymphomas induced by murine leukaemia virus. Thus, comparable mechanisms, leading to deregulated expression of N-myc genes, may operate in the development of tumours induced either by hepatitis virus or by nonacute retroviruses in rodents. Activation of myc genes by insertion of hepadnavirus DNA now emerges as a common event in the genesis of woodchuck hepatocellular carcinoma.
The complete nucleotide sequences of two different subtypes (adr and adw) of hepatitis B virus (HBV) DNA cloned in E. coli were determined. The sequence of the viral genome of the adr clone was 3188 nucleotides long, and that of the adw clone was
3200 nucleotides long. The adr and adw clones differed from the reported cloned ayw HBV DNA (3182 nucleotides long) in 11.2%
and 10.0% of nucleotides, respectively. Heterogeneity of the HBV genome in the clones with the same subtype was observed.
Hepatitis B virus DNA made fully double stranded by a virion DNA polymerase reaction could be converted from circular to linear molecules by heating in 10 mM NaCl at 77 degrees C or in 100 mM NaCl at 90 degrees C for 15 min. Heat-generated linear hepatitis B virus DNA was reannealed to circular molecules by incubating in higher salt concentrations. The identity of the molecular forms was established by their electrophoretic mobility and appearance in electron micrographs. Recircularization was blocked by reacting linear molecules with nuclease S1 or avian myeloblastosis virus reverse transcriptase. These results suggest that the heated linear DNA had single-stranded ends with complementary nucleotide sequences. It also suggests that a discontinuity or nick is present in each strand of the circular DNA molecule after the single-stranded region is made double stranded by the virion DNA polymerase reaction. The difference in contour length by electron microscopy of circular and linear molecules spread under aqueous conditions suggested that the discontinuities in the two strands were about 270 base pairs apart. The amount of nucleotide incorporated into the ends of heat-generated linear hepatitis B virus DNA by reverse transcriptase suggested that the single-stranded ends were about 305 bases in length. This fully double-stranded linear DNA was cleaved with EcoRI or HpaI restriction endonuclease. The sum of the two fragments generated by each totaled 3,510 base pairs, 310 base pairs greater than the contour length of circular hepatitis B virus DNA which represents a third estimate of the distance between the discontinuities in the two DNA strands of circular DNA. Restriction endonuclease cleavage also indicated that the ends of heated linear DNA which correspond to the discontinuities in the two strands of the circular DNA are at unique sites in the DNA with respect to the restriction sites.
Particles with properties similar to those associated with human hepatitis B were found in serum from woodchucks with chronic hepatitis and hepatocellular carcinoma. It is suggested that woodchuck hepatitis virus is a second member of a novel class of viruses represented by the human hepatitis B virus.
A composite DNA sequence of regions of hepatitis B virus, determined from a series of recombinant plasmids, reveals the genes for the surface antigen and the core antigen of the virus. The sequence of the core antigen shows it to be a DNA binding protein. The core antigen gene is expressed in Escherichia coli and when injected into rabbits the bacterial product induces antibodies which react with core antigen isolated from human sources.
The complete nucleotide sequence of hepatitis B virus genome (subtype ayw) cloned in Escherichia coli has been determined using the Maxam and Gilbert method and the dideoxynucleotide method. This sequence is 3,182 nucleotides long. Location of the nonsense codons shows that the coding capacity of the L chain is larger than the coding capacity of the S chain. Eight open regions, able to code for polypeptide chains larger than 100 amino acids, have been located. Region 6, which is the largest, covers more than 80% of the genome. The gene S which codes for polypeptide I of the Hbs Ag and was previously located between coordinates 95.1 and 73.6 is contained in region 7.
DNA isolated from the hepatitis B antigen form known as the Dane particle was examined by electron microscopy before and after the endogenous Dane particle DNA polymerase reaction. The most frequently occurring form was an untwisted circular double-stranded DNA molecule approximately 1 mum in length. Less frequently occurring forms included circular DNA of approximately unit length and having one or more small single-stranded regions, similar circular molecules with one or more tails either shorter or longer than 1 mum in length, and very small circular molecules with tails. There was no increase in frequency or length of tails after a DNA polymerase reaction, suggesting that tails were not formed during this reaction. The mean length of circular molecules increased by 23% when DNA was spread in formamide compared with aqueous spreading, suggesting that single-stranded regions are present in most of the molecules. The mean length of circular molecules obtained from aqueous spreading increased by 27% after a Dane particle DNA polymerase reaction. This indicates that single-stranded regions were converted to double-stranded DNA during the reaction.
The relationship of e antigen (eAg) and its antibody (anti-e) to vertical transmission of hepatitis B surface antigen (HBsAg) from chronic asymptomatic HBsAg carrier women to their children was investigated in Taiwan. Sera from 20 of the 62 women studied were positive for eAg (32%); serum from only one woman was positive for anti-e (2%). A total of 85% of the babies born to eAg positive mothers became HBsAg carriers, while only 31% of the babies became carriers when the mother was eAg negative. Maternal e antigenemia correlated with a high HBsAg titer, and both parameters were equally good predictors of vertical transmission.
Testing of serum samples of 23 pregnant women who were asymptomatic carriers of hepatitis B surface antigen for e antigen and antibody to e with an immunodiffusion technic identified 10 mothers with e antigen and seven with e antibody. Their babies were tested for hepatitis B surface antigen in serum at intervals for more than 12 months. In all 10 babies born to e-antigen-positive mothers hepatitis B surface antigen developed and persisted through the observation period, and all 10 elder siblings of these newborn babies were found to be asymptomatic carriers. In remarkable contrast, all seven babies born to mothers positive for antibody to e escaped antigenemia, and none of their three elder siblings carried surface antigen. On the basis of these results, e antigen may be used as an indicator of transmission, and antibody to e as that of absence of transmission of hepatitis B virus from carrier mothers to children.
DNA extracted from Dane particles has been characterized by gel electrophoresis and restriction enzyme cleavage with endonuclease R-HaeIII (from Hemophilus aegyptius). Dane particle DNA is proposed to be a double-stranded circular DNA approximately 3600 nucleotides in length containing a single-stranded gap of 600-2100 nucleotides. The endogenous DNA polymerase (DNA nucleotidyl-transferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) reaction appears to repair this single-stranded gap.
Cytogenetic analysis of eight human hepatoma-derived cell lines and one primary hepatocellular carcinoma biopsy revealed multiple chromosome abnormalities; however, only chromosome 1 was consistently affected by rearrangements. Pseudopolysomy 1 as well as chromosome 1 deletions and/or translocations that resulted in loss of the distal 1p region from at least one copy of chromosome 1 were observed in all but one of the cell lines analysed. Molecular analyses of tumor-derived and normal genomic DNA from six cases of hepatocellular carcinoma and from two of hepatoblastoma, using a panel of chromosome 1p-specific DNA probes indicated allelic loss in the distal 1p region in five of the six hepatocellular carcinomas but not in either hepatoblastoma. These results suggest the location of a gene in the distal 1p region whose functional loss may be involved in hepatocellular carcinogenesis.
Epstein-Barr virus (EBV) not only induces growth transformation in human B lymphocytes, but has more recently been shown to enhance B cell survival under suboptimal conditions where growth is inhibited; both effects are mediated through the coordinate action of eight virus-coded latent proteins. The effect upon cell survival is best recognized in EBV-positive Burkitt's lymphoma cell lines where activation of full virus latent gene expression protects the cells from programmed cell death (apoptosis). Here we show by DNA transfection into human B cells that protection from apoptosis is conferred through expression of a single EBV latent protein, the latent membrane protein LMP 1. Furthermore, we demonstrate that LMP 1 mediates this effect by up-regulating expression of the cellular oncogene bcl-2. The interplay between EBV infection and expression of this cellular oncogene has important implications for virus persistence and for the pathogenesis of virus-associated malignant disease.
RNA was isolated from tissue of two patients with hepatocellular carcinoma developed on the background of a chronic hepatitis B virus infection. For identification and characterization of 3' ends of X gene open reading frame (ORF)-related transcripts, RNA was reverse transcribed into cDNA and subjected to polymerase chain reaction. Cloned amplification products from tumor tissue of one patient represented an approximately even distribution of transcripts terminating at the established poly(A) signal (standard transcripts) and of truncated transcripts terminating at a CATAAA poly(A) signal within the 3' end region of X gene ORF (truncated transcripts). Amplified cDNA from tumor tissue of the second patient could be attributed mainly to the standard type of transcripts, whereas cDNA from the nontumor tissue of the same patient could be assigned to four groups of transcripts: (i) standard transcripts, (ii) transcripts with internal deletions affecting the 3' end of the X gene, (iii) truncated transcripts, and (iv) hybrid transcripts displaying 5' sequences from the X gene ORF fused to cellular sequences.
We have examined a series of squamous cell carcinomas (SCC) of the anus, anal intraepithelial neoplasia grade III (AINII) lesions and haemorrhoids for the presence of sequences from transforming human papillomavirus (HPV) types by polymerase chain reaction (PCR)/Southern blotting. In addition, the same DNAs have been analysed for abnormalities in the c-myc, p53 and retinoblastoma (Rb-1) gene loci by Southern blotting. HPV16 sequences were detected in a total of 38 of 50 (76%) and HPV18 sequences in 4 of the 50 cancers (8%). Of 12 haemorrhoids examined, none contained HPV16 or HPV18 sequences. Amplification of c-myc was demonstrated in 15 of the 50 cancers (30%), of which 13 were HPV16 positive, and one also positive for HPV18. Amplification of c-myc was not observed in the 5 AINIII or any of the 41 haemorrhoid DNAs analysed. Rearrangement of c-myc was not seen in any of the DNAs. Gross rearrangement, or loss of p53 or Rb-1 loci was not observed in any normal or tumor tissue. However, in preliminary analysis of p53 sequence, three tumours negative for HPV were heterozygous for p53 point mutation whereas six HPV positive tumours and two haemorrhoids were wild-type sequence throughout exons four to ten.