The role of hepatitis B virus-related chronic hepatitis

Chronic active hepatitis (CAH) is recognized as an important risk factor for hepatocellular carcinoma (HCC). The mechanisms involved in chronic active hepatitis include a combination of several, complementary effects involved in liver cell necrosis, inflammation and thus, cytokine synthesis and fibrosis. This was addressed and developed in several recent reviews (Hui and Sung, 2005; McMahon, 2005; Moradpour and Blum, 2005; Roberts and Gores, 2005). Cirrhosis is the histological end point of this chronic inflammatory and fibrotic process, and liver cell DNA synthesis is indeed increased in cirrhotic, as compared to normal, livers. Clonal analysis of some cirrhotic nodules clearly indicates monoclonal cell expansion (Paradis et al., 1998; Kojiro and Roskams, 2005); however, the underlying chronic active hepatitis is the main driving carcinogenic factor and this accounts for the higher risk of tumor occurrence in patients with active cirrhosis. Animal models for Hepadna virus infection have provided fundamentally important information on these issues. Woodchucks and ground squirrels chronically infected by the woodchuck hepatitis virus (WHV) and ground squirrel hepatitis virus, respectively, develop HCC against a background of CAH without cirrhosis; this observation emphasizes the absence of a strict requirement for cirrhosis in liver carcinogenesis (see review in Buendia, 1994; Tennant et al., 2004). The availability of transgenic mice containing different hepatitis B virus (HBV) DNA inserts has also been extremely valuable to demonstrating the importance of liver cell regeneration secondary to hepatocyte necrosis, in the absence of a direct mutagenic agent (Chisari, 1995; Larkin et al., 1999). In particular, targeting in the liver, using an albumin promoter, of an accumulation of the large PreS1/S2/S HBV envelope protein induces a direct toxic effect on hepatocytes, independent of the immune response to the viral protein (Toshkov et al., 1994). In another context, the immune response to HBV-expressing hepatocytes in transgenic mice can also trigger such liver cell regeneration and give rise to HCC (Nakamoto et al., 1998; Chen et al., 2005b; Wang et al., 2005). Furthermore, HBV-positive, transgenic mice exhibit extensive oxidative DNA damage, possibly related to cytokine synthesis (Hagen et al., 1994; Hsieh et al., 2004); this observation may explain their increased sensitivity to chemical carcinogens and is probably relevant in humans exposed to both hepatitis viruses and chemical carcinogens (Bannasch et al., 1989; Sell, 1993). Finally, it is also noteworthy that, besides triggering liver cell proliferation, an accumulation of viral proteins, such as HBsAg in so-called ‘ground glass’ hepatocytes, may modify detoxification pathways such as those implicating cytochrome P450; this effect may enhance the metabolism of chemical carcinogens.

The role of hepatitis B virus in liver cell carcinogenesis

It is clear that HBV-induced CAH and cirrhosis constitute major factors in liver carcinogenesis. However, evidence is now available concerning direct effects of HBV in this process; indeed, recent data demonstrate that serum HBV DNA level is a strong risk predictor of HCC independent of HBeAg, serum alanine aminotransferase level and liver cirrhosis (Chen et al., 2006). Thus, HBV should be considered as having synergistic effects with chronic inflammation (Figure 1).

Figure 1
figure 1

Schematic representation of the mechanisms involved in HBV-related chronic liver lesions and hepatocellular carcinoma. The figure underlines the dual effects of HBV. Expression of the viral proteins stimulates the host immune response and triggers liver inflammation; some viral proteins as well as insertion of the viral DNA into liver cell genome also directly interfere with cell proliferation and viability and induce genetic alterations.

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A combination of immunohistochemical and in situ hybridization procedures has shown that HBV DNA replication, identified by HBV DNA replicative molecules, and HBsAg synthesis are generally detected in different hepatocytes; in contrast, HBV DNA replication is generally, but not systematically, shown in hepatocytes expressing HBcAg (see review in Han et al., 1993). At the time of HCC development, tumor cells no longer allow viral DNA replication and do not express HBcAg, although HBsAg can be detected in approximately 20% of cases (Wang et al., 1991). Consistent with these observations, induction of HCCs by chemical carcinogens in HBsAg-expressing transgenic mice leads to decreased expression of HBsAg (Farza et al., 1994). Serum HBV DNA levels generally decline markedly by the time HCC develops. Downregulation of HBV genome expression is likely owing to a combination of several molecular mechanisms: it has been demonstrated in vivo in transgenic mice that the immune response to HBV antigen will trigger the secretion of cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN) and interleukin (IL)-2, which can downregulate the accumulation of HBV RNAs (Guidotti et al., 1999a, 1999b; Koziel, 1999). In vitro studies have also shown downregulation of the core promoter by TNF-α (Romero and Lavine, 1996). Under these conditions, cytokines released by the immune response to the virus will in turn lower the level of HBV antigen synthesis in liver cells and thus prevent elimination of the infected cells (Guidotti and Chisari, 2000). In contrast, some investigators have demonstrated the persistence of replicative intermediates in tumoral liver cells from patients with HCC, and suggested that they may not normally be encapsidated (Raimondo et al., 1988). In contrast, reports from Taiwan have shown that HBV multiplication persists frequently when HCC occurs (Chen et al., 1986; Loncarevic et al., 1990).

Involvement of mutations in HBV sequences in the risk of HCC evolution is an open question. Only a few studies have addressed the questions of the rate of HBV mutations in tumor tissues (Pontisso et al., 1984; Raimondo et al., 1988; Clementi et al., 1993). However, our group and others have shown that mutations in basal core promoter, PreC/C, envelope and X sequences can be identified in HCC tissues (Minami et al., 1996; Fan et al., 2000, 2001; Tu et al., 2001; Oon et al., 2002; Iavarone et al., 2003; Kao et al., 2003; Kuang et al., 2004). We have observed that in the Core gene, most of the non-synonymous mutations (i.e. leading to amino-acid changes) have been shown in HBV genomes detected in either tumoral or non-tumoral tissues; in contrast, most synonymous mutations have been identified in both tumoral and non-tumoral tissues. These observations are consistent with the hypothesis concerning the selection of some HBV molecules during long-term persistence and HCC development (Minami et al., 1996). In addition, preS mutant may induce oxidative DNA damage and mutagenesis of the host genome and thus plays an important role in HCC (Hsieh et al., 2004). Furthermore, mutations have been identified on HBV X (HBx) encoding sequences that might reduce viral DNA replication, HBV gene expression or enhance transformation (see next section on HBx).

The important issue of a distinct impact of the various HBV genotypes in HCC has so far not been directly addressed (see review in Orito and Mizokami, 2003; Chan et al., 2004; Pujol and Devesa, 2005). A recent study showed a high prevalence of preS mutant in HCC patients infected with genotypes B and C (Huy et al., 2003). Furthermore, it has also been suggested that genotype C has a higher rate of mutation in the core promoter regions (Orito et al., 2001).

Altogether, this combination of genomic mutations and/or deletions, together with transcriptional and post-transcriptional regulations, will therefore allow the establishment of viral persistence and the ongoing synthesis of HBV antigens.

Integration of hepatitis B virus DNA allows persistence of the virus and induces genetic alterations

Hepatitis B virus DNA is a small, circular DNA with a highly compact genetic organization and overlapping open reading frames; it shares with retroviruses the use of reverse transcription during its replication. In contrast, viral DNA integration into the cellular DNA is not necessary for viral replication (see reviews in Feitelson, 2005) but allows persistence of the viral genome in the cell. Viral DNA insertion as well as cellular DNA replication occurs during liver cell proliferation, secondary to the necrosis/apoptosis of adjacent hepatocytes.

Hepatitis B virus DNA sequences are integrated into cellular DNA in HCC tumor samples but HBV genome integration has also been shown in patients with chronic hepatitis (Brechot et al., 2000; Minami et al., 2005); moreover, ancient observations based on Southern blotting have been recently confirmed by PCR-based approaches, demonstrating that, at least in some cases, integration of the viral DNA may occur during the acute stage of infection (Lugassy et al., 1987; Murakami et al., 2004). Integration therefore precedes development of the tumor, and comparative analyses of the various restriction profiles at different times during the course of HBV infection suggest progressive clonal expansion of certain infected cells.

It is most important, when investigating the functional consequences of HBV DNA integration, to realize that it should be viewed as a dynamic process. Long-term chronic inflammation, associated with increased liver cell proliferation, induces several rearrangements of the integrated viral sequences (Hessein et al., 2005). Deletions of part of the viral genome as well as more complex rearrangements are frequently observed (Wang et al., 2004b).

Hepatitis B virus insertion can induce chromosomal deletions at the HBV integration sites. Moreover, transpositions of the viral sequences, together with the flanking cellular sequences from one chromosome to another, have been recently confirmed (Ziemer et al., 1985; Wang et al., 2004b). Thus, integrated HBV can generate chromosomal instability and it has been suggested that viral DNA sequences encompassing the encapsidation signal may exhibit intrinsic recombinogenic activity via binding to a putative ‘recombinogenic’ cellular protein (review in Aoki et al., 1996; Hino et al., 2002).

The insertion of viral DNA into a cellular gene, as well as modification of its expression (i.e. cis-activation), is another potential consequence of HBV DNA integration. This has been for years a very unclear issue, two very different situations being observed in HCCs developed in woodchucks infected by WHV and in humans infected by HBV: in WHV-related HCCs, the integration of WHV DNA exhibits common insertion or activation of proto-oncogenes of the myc family, predominantly the N-myc2 oncogen; then, it was reported that N-myc2 could be activated by WHV integration in win and b3n locus (Buendia, 1994; Bruni et al., 1999, 2004); in contrast, in HBV-related HCCs, integration of the viral genome in cellular gene has been viewed as a rare event. We have recently entirely revisited this concept by using a PCR-based approach, allowing a much faster analysis of the flanking cellular sequences and thus the investigation of a much larger number of HBV insertion sites (review in Paterlini-Brechot et al., 2003; Murakami et al., 2005). With this approach, we have demonstrated that (1) HBV insertion into cellular genes is in fact frequent (around 70%) and (2) HBV integration can occur in genes encoding for proteins with a fundamental role in the control of cell signaling, proliferation and viability (Figure 2) (Horikawa and Barrett, 2001; Ferber et al., 2003; Paterlini-Brechot et al., 2003; Murakami et al., 2005; Tamori et al., 2005). Most importantly, our work, combined with recent investigations from other groups, has also led to demonstrate that the telomerase and mixed lineage leukemia encoding genes are in fact targeted by HBV in different HBV-related HCCs, thus suggesting common pathways in HBV-related carcinogenesis (Horikawa and Barrett, 2001; Ferber et al., 2003; Paterlini-Brechot et al., 2003; Murakami et al., 2005; Tamori et al., 2005).

Figure 2
figure 2

The insertion of HBV genome in cellular genes frequently targets genes that regulate key cellular pathways. The figure illustrates that HBV targets a variety of genes controlling various steps of cellular signaling, cell proliferation and viability (see references cited in the text for details).

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Thus, altogether, we would submit that HBV DNA might be viewed as a ‘proviral tag’ (Li et al., 1999) capable of identifying new cellular genes or unravel new mechanisms controlling known gene expression (Sanchez-Prieto et al., 1999; Collier et al., 2005).

Expression of HBV X and the newly discovered hepatitis B spliced proteins can modulate cell proliferation and viability

As stated above, it is clear that the immune response to the viral proteins associated with the antiviral response plays a major role in the liver disease outcome. Although HBV is not considered as a cytopathic virus, there is now solid evidence for some HBV proteins directly participating in chronic hepatitis and HCC development. A clearer understanding of immune and cytopathogenic mechanisms during the expression of certain viral proteins is vital to the development of novel diagnostic or therapeutic strategies.

Examination of the viral DNA sequences present in tumor cells has shown that, in a large proportion of them, sequences encoding for the HBx and/or truncated envelope PreS2/S viral proteins are retained (Schluter et al., 1994; Anthony, 2001; Wang et al., 2004b). Moreover, we have described a novel viral protein, hepatitis B spliced protein (HBSP), which is encoded by spliced HBV RNA and is expressed during the course of chronic HBV infection. We will focus in this review on HBx and HBSP.

HBx

The HBx protein is 154 amino acid in size, with a molecular mass of about 17 kDa (Diao et al., 2001). The HBx gene is highly conserved among all mammalian hepadna viruses. The highest conservation was observed in the helical domains located in the amino- and caboxy-terminal region and a coiled-coil region. HBx is expressed at low levels during acute and chronic hepatitis and induces a humoral and cellular immune response (Chung et al., 1999; Hwang et al., 2002; Chun et al., 2003; Malmassari et al., 2005). In some studies, anti-HBx antibodies were more frequent among patients with HCC than those with chronic hepatitis B without cancer (Hess et al., 1988; Levrero et al., 1991; Hwang et al., 2003). In most integrated subviral DNA, the HBx gene is maintained and transcribed and several studies have now demonstrated both HBx RNA and protein expression in human HCC tumor cells in the absence of HBV replication (Su et al., 1998; Peng et al., 2005). The available in vivo data suggest that HBx expression in transgenic mice likely induces HCC by sensitization of the animals to chemical carcinogens or by altering cellular oncogenes such as c-myc (Terradillos et al., 1997; Madden et al., 2001; Lakhtakia et al., 2003; Hung and Kumar, 2004; Wang et al., 2004a; Zhu et al., 2004; Koo et al., 2005).

HBx appears to function as a regulatory protein for viral replication and is required for efficient infectivity of woodchucks with WHV, a member of hepadna virus family (Chen et al., 1993; Zoulim et al., 1994). In transgenic mice, HBx is not essential for HBV replication, but it can enhance viral replication (Reifenberg et al., 2002; Xu et al., 2002). Recently, in vitro experiments suggest that HBx stimulates HBV replication via its transactivation function (Melegari et al., 2005; Tang et al., 2005). It has been demonstrated that HBx action on viral replication may be dependent to modulation of calcium homeostasis, which provides novel clues for HBx biology (Bouchard et al., 2001, 2003; Choi et al., 2005). In addition, HBx interactions with the proteasome complex or DDB1 and HBx activation of Src tyrosine kinases have also been reported to participate in the regulation of HBV replication (Klein et al., 1999; Zhang et al., 2004; Leupin et al., 2005).

HBx is mainly located in the cytoplasm and can be detectable in the nucleus and exhibits pleiotropic effects that modulate cell responses to genotoxic stress, protein degradation and signaling pathways (Figure 3) (for review, see Bouchard and Schneider, 2004). HBx protein does not bind directly to DNA but rather acts on cellular promoters by protein–protein interactions by modulating cytoplasmic pathways. HBx also appears to act as a paracrine factor (Tralhao et al., 2002). These various biologic effects have overlapping effects on cell proliferation and viability (Arbuthnot et al., 2000; Ahn et al., 2002). The results of studies varied, depending on the amount of HBx expressed in cells, the cell type utilized and their status of differentiation. HBx transactivates a number of cellular promoters and enhancers containing binding sites for NF-κB, activator protein-1, activator protein-2, c-EBP, activating transcription factor/c-AMP-responsive element binding protein, RNA polymerase and NF-AT, cellular promoter of genes associated with cell proliferation such as IL-8, TNF, transforming growth factor (TGF)-β and early growth response factor and cytosolic signal transduction pathways such as Ras/Raf mitogen-activated protein kinase, Src kinases, cjun N-terminal kinase, Jak1/STAT and PK (Andrisani and Barnabas, 1999; Bouchard and Schneider, 2004; Pang et al., 2005).

Figure 3
figure 3

HBx an enigmatic regulatory protein. The figure illustrates the complexity of the biological actions of HBx.

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Numerous studies have identified cellular proteins that interact with HBx (Figure 4). However, relevance of these interactions in an HBV infection model remained to be evaluated. In fact, according to the experimental conditions, HBx may act as a pro- or antiapoptotic protein (Arbuthnot et al., 2000). HBx has been shown to interact with p53, thereby inactivating several critical p53-dependent activities, including p53-mediated apoptosis (Feitelson et al., 1993; Lin et al., 1997), transactivation properties of p53 (Elmore et al., 1997; Lin et al., 1997; Bergametti et al., 1999), regulation of cell cycle (Ahn et al., 2002), DNA repair genes (Mathonnet et al., 2004; Lee et al., 2005a) and tumor suppressor genes (Chun et al., 2003). HBx can also exert antiapoptotic functions independently of p53 via modulation of activities of the serine protease hepsin (Zhang et al., 2005a) and upregulation of survivin (Li et al., 2003). HBx protein may also promote apoptosis dependent on p53 (Chirillo et al., 1997) or by regulating the expressions of Fas/FasL (Terradillos et al., 1998; Shin et al., 1999; Lee et al., 2002; Yoo and Lee, 2004), inactive procaspase-8, cFLICE (Kim and Seong, 2003), Bax/Bcl-2 (Miao et al., 2005), HSP60 (Tanaka et al., 2004), UV-DDB1 (Sitterlin et al., 2000; Bergametti et al., 2002; Leupin et al., 2003) and c-myc gene (Kalra and Kumar, 2004). HBx negatively regulates proteasome function and, thus, controls degradation of cellular and viral proteins (Sirma et al., 1998; Hu et al., 1999; Kim et al., 2003; Zhang et al., 2004). In addition, it was reported that inhibition of cellular proteasome activity could block the antiviral effect of alpha/beta IFN (Robek et al., 2002). In contrast, a recent report based on proteomic analysis revealed that proteasome subunits were upregulated in tumor liver tissues in HBx p21 knockin transgenic mice (Cui et al., 2006). HBx exerts powerful effects on mitochondria either directly owing to their channel-forming activity or indirectly through interactions with endogenous channels (Takada et al., 1999; Henkler et al., 2001; Lee et al., 2004). HBx reportedly interacts with at least two mitochondrial proteins, namely heat-shock protein 60 and 70 (Tanaka et al., 2004; Zhang et al., 2005b) and the VDAC isoform VDAC3 (Rahmani et al., 2000; Kim, 2005).

Figure 4
figure 4

Schematic representation of the different targets of HBx. The figure shows the cytoplasmic, mitochondrial and nuclear transduction cascades activated by HBx.

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HBx may also contribute to tumorigenesis in HCC through modulation of angiogenesis pathway. HBx expression could induce upregulation of the potent angiogenic factor vascular endothelial growth factor transcription or by stabilization of hypoxia inducible factor HIF-1 (Lee et al., 2000; Yoo et al., 2003, 2004; Moon et al., 2004). HBx could activate Wnt/beta-catenin signaling by upregulating the cytoplasmic beta-catenin (Cha et al., 2004; Ding et al., 2005). An alternative mechanism for Wnt activation is the repression of E-cadherin transcription through hypermethylation of E-cadherin promoter by activation of DNA methyltranferase 1 (Kanai et al., 1997; Lee et al., 2005b). Recently, studies have shown that HBx may induce cellular migration through activation of matrix metalloproteinase 3 and 9 (Chung et al., 2004; Yu et al., 2005).

Natural mutants of HBx have been described in the liver and serum of patients with different clinical conditions (Sirma et al., 1999; Yeh et al., 2000; Tu et al., 2001; Iavarone et al., 2003; Kwun and Jang, 2004; Chen et al., 2005a; Lin et al., 2005). We have refined these observations by using laser-based microdissection and shown that HBx sequences deleted in their C-terminal portion are mostly detected in the HCC tumor cells (Iavarone et al., 2003). These findings can be used to address the effects of HBx and its mutants from human HCC on cell growth and viability. Thus, deletion of the C-terminal region of HBx, observed in several HBx mutants isolated from HCC tissue, leads to abrogation of HBx-dependent transactivation, cell cycle arrest and apoptosis inhibition; the results also suggest that such deletions may enhance HBx-transforming capacity (Sirma et al., 1999; Tu et al., 2001). Overall, these results are consistent with selection of HBx sequences in clonally expanding liver cells that favor cell proliferation and transformation (Figure 4). Other HBx mutations have been described in patients with or without HCC. HBx with mutations in amino acids 130 and 131 have been frequently reported and may be associated with the severity of chronic hepatitis; these mutations have also been detected in HCC (Yotsuyanagi et al., 2002; Iavarone et al., 2003; Leon et al., 2005), and a recent study of serum samples indicated that these mutations arise before the development of HCC (Kuang et al., 2004). However, the functional consequences of these mutated HBx proteins are still unclear.

Finally, HBx appears to facilitate the capacity of cells to acquire increased activity and provide a permanent growth-promoting signal, and thus, raise the frequency of immortalization. Elucidating the biological significance of HBx will help us to gain further insight into the oncogenic role of this protein, and assist in the development of therapeutic strategies to treat chronically infected patients predisposed to HCC.

Defective viral particles and hepatisis B virus spliced protein

Data in the literature have shown that HBV pregenomic RNA, the matrix for viral replication, may undergo simple or multiple splicing (Günther et al., 1997). These RNAs are differentiated by the site of splicing and may represent up to 30% of total HBV pregenomic RNA. However, the role of these spliced RNAs in viral replication has not yet been established. A previous study showed that these spliced RNA might be encapsidated and retrotranscribed and give rise to defective particles (Terré et al., 1991; Günther et al., 1997). We showed that, in vivo, at least one of these defective particles was detected in the sera of patients with chronic hepatitis or acute hepatitis developing towards chronicity, but not in patients who had recovered from their acute hepatitis (Rosmorduc et al., 1995) (Figure 5). We also showed that the expression of this defective HBV DNA (unlike wild-type HBV DNA) inhibited the expression of the IFN-α-inducible MxA protein and that accumulation of viral capsid protein, observed during expression of defective HBV DNA, was responsible for this inhibition (Rosmorduc et al., 1999). The antiviral activity of this protein versus a certain number of RNA viruses has clearly been demonstrated (Haller et al., 1998). These results show that defective forms were associated with viral multiplication during chronic evolution of the disease and may participate in viral resistance to treatment.

Figure 5
figure 5

Hepatitis B virus defective viral particle formation. Hepatitis B virus pregenomic RNA, the matrix for viral replication, may undergo simple or multiple splicing. These spliced RNA might be encapsidated and retrotranscribed and give rise to defective particles.

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A new HBV protein, referred to as HBSP, is encoded by one of the spliced RNAs of HBV (Soussan et al., 2000) (Figure 6). This protein is translated from the AUG of polymerase and because of splicing, is endowed in its C-terminal moiety with a sequence that differs from that of other viral proteins. In vivo, HBSP protein has been detected in liver biopsy specimens from patients with active chronic hepatitis. In addition, antibodies directed against this protein have been found in the serum of approximately 45% of patients with chronic hepatitis (Soussan et al., 2000, 2003). In vitro, HBSP expression induces apoptosis without cell-cycle block (Soussan et al., 2000). More recently, HBSP protein expression was correlated with viral replication and with the onset of hepatic fibrosis (Soussan et al., 2003). Furthermore, the correlation between the detection of anti-HBSP antibodies and fibrosis was independent of viral replication. Recent data based on quantitative PCR of wild-type and defective particles have confirmed the direct association between HBSP and the severity of fibrosis. This suggests a direct involvement of HBSP protein in the pathogenesis of liver disease. Indeed, hepatic fibrosis occurs in reaction to hepatocyte cytolysis, which is observed during viral hepatitis. In addition, in vitro findings suggest that HBSP can induce apoptosis and may modulate TGF-β-dependent signaling. Thus, these results offer a novel mechanism for the direct role of HBV in inducing liver fibrosis.

Figure 6
figure 6

The HBSP, a new HBV protein. The HBSP protein is encoded by one of the spliced pregenomic RNAs of HBV. This protein is translated from the AUG of polymerase and because of splicing, is endowed in its C-terminal moiety with a sequence, that differs from that of other viral proteins.

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Collectively, these studies show that some HBV proteins can both trigger immune responses and directly interfere with different steps of liver carcinogenesis. These observations further substantiate the importance to treat patients chronically infected with HBV, even when cirrhosis has already developed, and decrease viral multiplication.

Hepatitis B virus chronic infection may also be a risk factor in HBsAg-negative hepatocellular carcinoma

The issue of persisting HBV infections in patients without detectable HBsAg detection in the serum has been a matter of debate for many years (see recent reviews in Brechot et al., 2000, 2001). Although there are still many discrepancies among the studies, one can summarize the findings as follows: (1) Occult HBV infections do exist. (2) Their prevalence vary from one geographical area to another but is often high in patients with HCC. In particular, there is now more direct evidence for an impact of occult HBV infections in hepatitis C virus (HCV)-related HCC occurrence (Pollicino et al., 2004); in fact, concomitant infection by HBV and HCV is a major emerging issue. (3) Occult HBV infections however can also be identified in resolved acute infections; thus, as for HBsAg-positive infections, there is likely a ‘spectrum’ of liver diseases associated with persisting HBV infections, whose severity depends on other environmental as well as genetic factors. (4) There are several mechanisms that account for absence of serum HBsAg detection in these patients: progressive decline in HBV replication is a major factor, and genetic mutations and environmental factors might also be involved in some cases. (5) HBx protein expression and cellular gene cis-activation can be identified in HCC tumor cells and the mechanisms we have described above for HBsAg-positive HCCs may also apply to HBsAg-negative subjects.

With regard to the state of HBV DNA, its low copy number per cell has hampered the interpretation of the results of Southern blotting. However, using PCR tests and distinct primers distributed on the S, PreS/S, C and X HBV genes, we and others have been able to demonstrate the presence of defective HBV DNA in the tumor cells; interestingly, defective HBV genomes have been identified more frequently in tumors than in non-tumor tissues and the PCR profiles were consistent with the clonal expansion of cells containing defective and integrated HBV DNA (Paterlini et al., 1993; Bréchot, 1998). In this view, it is interesting that some cases of HBV-related cis-activation, such as that involving the SERCA1 encoding gene (see sections above), has been in fact shown in an HBsAg-negative, anti-HBc-positive individual (Chami et al., 2000). Finally, our group has identified a high rate of mutations in the HBx ORF gene in the tumor tissue from such HBsAg-negative, HBV DNA-positive patients; this high rate of amino-acid changes contrasted with a lower rate of mutations in PreC:C sequences (Poussin et al., 1999). In addition, PCR has allowed us to detect the expression of the HBx RNA in such tumors (Paterlini et al., 1995; Poussin et al., 1999). Similar results were reported in Japan (Tamori et al., 1999). Our study also enabled demonstration of the expression of HBx protein in both tumor and non-tumor cells from such liver tissues (Poussin et al., 1999).

Taken together, the results demonstrate a high rate of persistent HBV infection in patients with HBsAg-negative HCC, many of whom also lack detectable antibodies to the virus. They also show clonal expansion of the tumor cells containing the integrated viral DNA, and the preferential transcription in these cells of HBV RNA sequences encoding the viral protein. The involvement of HBV in HBsAg-negative liver cancers is also reinforced by two pieces of evidence. In the woodchuck HCC model, 17% of animals infected by WHV developed primary liver cancers, despite negativation of the assay for WHV surface antigen in the serum and the appearance of antibodies to surface and capsid viral antigens; furthermore, WHV DNA was detected in tumor tissue from these animals, with a much lower copy per cell number than in WHsAg-positive woodchucks (approximately 1 and 1000 molecules per cell, respectively) (Korba et al., 1989; Gerin et al., 1991), an observation fairly similar to that made in human HCCs. Ground squirrel may also prove to be an interesting model, as GSHV DNA sequences have been identified in HCCs developing in completely seronegative animals (Transy et al., 1992). Another piece of evidence for a direct role of HBV in liver cancer arises from the detection of HBV DNA in HCCs in HBsAg-negative patients, developing on non-cirrhotic tissue, histologically very similar to normal liver; thus, cirrhosis alone cannot account for the induction of cancer in these cases (Bréchot, 1998). Although these findings argue strongly in favor of a role for HBV in the development of these tumors, one paradoxical result still has to be explained: why is the copy number of HBV DNA per cell so low if there is clonal expansion of the infected cells? It is possible to hypothesize that, after it has triggered the cascade of events that will lead to liver cell transformation, the persistence of HBV DNA is no longer necessary. Furthermore, as discussed above, there is little or no HBV replication in tumor cells and thus no generation of new integrants from free viral genomes. Finally, chromosomal rearrangements may eliminate integrated viral DNA sequences from tumor clones. This explanation has also been put forward to account for human and bovine papillomavirus- and some retrovirus-related tumors (Galloway and McDougall, 1983; Smith and Campo, 1988; Morgan et al., 1990). Interestingly, recent evidence has been found in the Duck Hepatitis model, which supports these hypotheses (Gong et al., 1996).

Thus, the overall information point to persistence of HBV DNA as a risk factor for HCC development, a finding quite consistent with that reported in woodchucks with WHsAg-negative persistent WHV infections. This is a major issue for epidemiological studies on HCC, which should include detection of the viral genomes using sensitive, PCR-based assays.

Interactions between hepatitis B virus, hepatitis C virus and alcohol

Hepatitis B virus and HCV can interact with chronic alcohol consumption, and, as extensively discussed in the review by Donato et al., (1998), there is circumstantial evidence for a synergistic effect of HBV and HCV infections as well as of chronic alcohol consumption in liver carcinogenesis. We have substantiated these epidemiological investigations by detection of persistent HBV infection through HBV DNA detection in alcoholics with HCC (see review in Brechot et al., 2000, 2001).

These studies have also revealed that concomitant infection by HBV and HCV is a major emerging issue. The discovery of HCV has led us to demonstrate that a high proportion of patients with HBsAg-negative, yet HBV DNA-positive, chronic liver disease were in fact coinfected by HBV and HCV. Similary, studies of HCV-infected subjects have confirmed the high incidence of HBV DNA persistence in the liver and/or samples. Some studies have demonstrated the clinical impact of such coinfections on the severity of chronic hepatitis (Pollicino et al., 2004) and, possibly, the efficacy of IFN. The high prevalence of HBV–HCV co-infections among patients with HCC has also been emphasized in Europe (Bréchot, 1998) and Japan. Further molecular studies are still needed to investigate how viral proteins might have a synergistic impact on liver cell transformation.

Conclusions

The data we present here illustrate that HBV constitutes a major environmental etiological factor for primary liver cancer in humans. Furthermore, detection of the viral genomes using sensitive, PCR-based assays is mandatory to enable an accurate appraisal of their prevalence. The evidence available demonstrates that HBV, both by synthetizing some of its own proteins (HBx, PreS2/S, HBSP) and by inducing genetic alterations, can deregulate the proliferation, differentiation and viability of liver cells. Recent major advances in the genetics of HCC will now make it possible to combine studies analysing the genetics of these tumors, independently of HBV and HCV, with those directly focused on HBV- and HCV-related carcinogenesis. Thus, the analysis of HBV-related carcinogenesis should be viewed as a tool enabling the identification of new, liver-specific, regulatory mechanisms.