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Hepatitis B infection is associated with an increased risk of hepatocellular carcinoma development. Hepatitis B proteins, such as the hepatitis B x protein, the large hepatitis B surface protein, or truncated middle hepatitis B surface proteins, regulate transcription of many candidate genes for liver carcinogenesis by trans-mechanisms. They also alter mechanisms of apoptosis and interfere with nucleotide excision repair of damaged DNA. Together with an influence on cellular signaling, these mechanisms may favor the cell's clonal expansion., (Copyright 2002 S. Karger AG, Basel)

Hepatitis B infection is strongly linked epidemiologically to hepatocellular carcinoma development. This article reviews the molecular mechanisms by which hepatitis B encoded proteins such as hepatitis B x and hepatitis B surface transactivators may interact with gene transcription, tumor suppression, apoptosis, and signalling pathways of the liver cell with the possible consequence of tumor induction. Data on the interaction between hepatitis B proteins and cellular processes are often conflicting indicating a non-specific simultaneous interaction with antagonistic cellular processes that result in the formation of escape mutants that are not subject to these selective pressures.

We have previously reported a case of integration of HBV sequences in the 5'-noncoding region of the gene for mevalonate kinase (Mk) (EC.2.7.1.36) in the human hepatoma cell line PLC/PRF/5, resulting in overexpression of viral-cellular fusion transcripts and enhanced intracellular levels of the enzyme. Here, we present an evaluation of the functionalities of Mk and HBV/Mk fusion proteins derived from viral-cellular fusion- and Mk-transcripts, some of which lack 156 bp in the Mk coding region as a result of a differential splicing process. cDNA clones with a full-length Mk-ORF produce proteins which can metabolize mevalonate to its monophosphorylated form. Our results suggest that the enhanced and inappropriate expression of Mk may lead to increased metabolism of mevalonate and phosphorylation of hitherto unknown cellular proteins. This consequence of HBV-DNA insertion could thus be related to the activation of proteins that may be relevant in oncogenesis.

Epidemiologic data indicate the crucial role of chronic hepatitis B virus (HBV) infection in hepatocellular carcinoma (HCC) development. On the molecular level, HBV sequences are frequently integrated in hepatocellular DNA. However, in contrast to the woodchuck model, in which specific HBV-DNA integration is detectable in most cases, insertional (in-) activation of cellular genes seems to be a rare event in man. The recent discovery of transactivating functions exerted by HBx and truncated HBs(urface) proteins supports the notion that transactivation of cellular gene expression could be relevant to hepatocarcinogenesis. HBV transactivator sequences are present in 81% (21/26) of HCC tissues or hepatoma-derived cell lines. At least one transactivator protein was functional in all cases investigated so far. The 16.5-kDa HBx transactivator has been shown to stimulate gene expression from various cellular target sequences. In vitro, HBx displays oncogenic potential. A second type of transactivator is encoded in the preS/S region of HBV. In contrast to HBx, HBs transactivators require carboxyterminal truncation to gain their transactivating function. Unlike full-length M(iddle)HBs, the truncated MHBst is retained in the endoplasmic reticulum and not secreted into the surrounding medium. Cellular gene expression is stimulated by regulatory elements of the human proto-oncogenes c-fos and c-myc, as well as by the hepatic acute-phase interleukin-6 gene. Synthetic binding sites for the transcription factors NF-kappa B, AP-1, AP-2, SRE, and Sp1 render minimal promoters activatable. NF-kappa B-mediated transactivation by MHBst can be suppressed by radical scavenging antioxidants, indirectly suggesting that reactive oxygen intermediates are involved.(ABSTRACT TRUNCATED AT 250 WORDS)

The hepatitis B virus (HBV) frequently integrates into hepatocellular genomic DNA during viral infection. Transcriptional transactivators encoded by integrated HBV X and 3' truncated preS/S sequences are known to stimulate gene expression from homologous and heterologous promoters. Here we demonstrate that 21 of 26 (81%) hepatocellular carcinoma tissues/cell lines contain coding sequences for at least one of the two known transactivators. Four integrated X and three preS/S transactivator sequences contained in five isolates from three hepatoma primary tissues or cell lines were used as examples to prove functionality of the encoded transactivators. In one case, where both X and preS/S sequences were present, dissection of X and preS/S transactivator sequences showed independent functionality. The investigation of X- and preS/S-specific RNA and protein expression revealed the existence of carboxyterminally truncated viral-cellular fusion proteins that were able to stimulate gene expression from the c-fos proto-oncogene promoter five- to ten-fold. These results demonstrate that structurally intact HBV transactivator sequences are integrated in the majority of HBV-associated HCCs/hepatoma cell lines. In all tested examples integrated DNAs had retained functionality as transactivators. This data thereby support indirectly the hypothesis of a possible involvement of HBV transactivators in liver cell proliferation and hepatocarcinogenesis.

The use of HBV and HDV experimental models has significantly contributed to understand the viral life cycle and to systematically test antiviral effects of various drugs on a pre-clinical level. Similar replication strategies of related hepadna viruses permit the use of chimpanzees (Pan troglodytes), woodchucks (Marmota monax), ground and tree squirrels (Spermophilus beecheyi) or Pekin ducks (Anas domesticus) as appropriate animal models. Cell culture systems for in vitro infection or transfection using both primary cultures of human and non-human hepatocytes and non-hepatocytes and cell lines have recently been identified. The advantages and restrictions of these experimental models with respect to evaluation of interferon effects on viral and hepatocellular gene expression are discussed.

Insertional mutagenesis of growth related genes by hepatitis B virus (HBV) DNA is presumed to play a role in hepatocarcinogenesis. Here, we report on insertional activation of the mevalonate kinase (MK) gene in the human hepatoma cell line PLC/PRF/5. Integration of HBV DNA dissociated the promoter and upstream regulatory elements of the gene from its coding sequences. This led to the over-expression of hybrid transcripts arising from an HBV promoter and the consequent over-production of functionally active mevalonate kinase. MK phosphorylates mevalonate, a major intermediate in the branched cholesterol/isoprenoid biosynthetic pathway. Isoprenylation is crucial to the functions of cellular proteins related to growth control, including the proto-oncogene ras. As the enzymes of these biosynthetic pathways are regulated at multiple points by negative feedback, both transcriptionally and at the protein level, the results discussed here support the idea that aberrant growth could result from deregulated overexpression of MK and, perhaps, other enzymes in the cholesterol pathway. These results invoke novel mechanisms by which cell transformation might occur.

A hepatoblastoma cell line transfected with hepatitis B virus (HBV) DNA (Hep G2.2.15) was used to investigate the effects of interferons (IFNs) on HBV replication and hepatocellular gene expression. IFN-alpha 2b or -beta inhibited HBV replication transiently. In parallel, there was a decrease in the amount of HBV mRNA. Hepatitis B surface antigen and early antigen secretion were not influenced; however, their intracellular levels diminished during treatment. The cellular 2',5'-oligoadenylate synthetase activity was increased 9- to 18-fold during treatment of cells with IFN-gamma, -alpha, or -beta. The number of IFN-alpha and -beta receptors was down-regulated, while the number of IFN-gamma receptors remained constant. The expression of major histocompatibility complex class I antigens was stimulated by addition of IFN-alpha or -beta. These data show that both IFN-alpha and -beta can effectively inhibit HBV replication and induce a cellular IFN response in Hep G2.2.15 cells similar to that seen in humans.

C-terminal truncation of the middle surface antigen from hepatitis B virus (MHBs) gives rise to a novel transactivating protein, called MHBst. In this study we show that MHBst like the HBx protein of HBV, can cause nuclear appearance of NF-kappa B DNA binding activity and induce various kappa B-controlled reporter genes. While an inhibitor of protein kinase C could not block gene induction by MHBst, the antioxidants N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) could potently suppress transactivation at mM and microM concentrations, respectively. Also, kappa B-dependent gene induction by the transactivator HBx was blocked. The effects were selective because PDTC did not interfere with MHBst and HBx-induced activation of the c-fos promoter/enhancer, nor with the basal activity of several other reporter genes lacking functional NF-kappa B binding motifs. Our data suggest that induction of a prooxidant state is crucial for the activation of NF-kappa B by MHBst and HBx and might be related to the hepatocarcinogenic potential of the viral proteins. MHBst had a subcellular localization unusual for a viral transactivator: it appeared to be an integral membrane protein of the endoplasmic reticulum.

Chronic hepatitis B virus infection is often associated with major structural rearrangements of both the integrated viral DNA and the associated cellular sequences. We present here the structure of a single-copy hepatitis B virus insert cloned from human hepatocellular carcinoma DNA recently reported to encode a novel transcriptional trans-activator function. The hepatitis B virus portion of the clone consists of two colinear fragments covering the X gene with its promoter and enhancer (nucleotides 717 to 1796) and a 3' truncated pre-S/S gene (nucleotides 2703 to 423). The lack of the entire pre-C/C gene caused a fusion of the 3' end of the X gene with sequences upstream from the pre-S gene. The structure of the integrated viral DNA fragments suggests insertion of hepatitis B virus replication intermediates into cellular DNA and subsequent recombination between these primary integrations to generate the final structure of the clone. The 5' and 3' cellular flanking sequences mapped to the centromeric alpha-satellite DNA of chromosome 17 and to the short arm of chromosome 7 (p14-pter), respectively, indicating that chromosomal translocation was associated with the hepatitis B virus DNA integration. Because this is the fourth case reported in which hepatitis B virus-associated rearrangements have affected chromosome 17, it is conceivable that a loss of important cellular genes (such as the p53 antioncogene on chromosome 17) may be a crucial step in hepatitis B virus-related hepatocarcinogenesis.

The X gene of wild-type hepatitis B virus or integrated DNA has recently been shown to stimulate transcription of a variety of enhancers and promoters. To further delineate the viral sequences responsible for trans-activation in hepatomas, we cloned the single hepatitis B virus insert from human hepatocellular carcinoma DNA M1. The plasmid pM1 contains 2004 base pairs of hepatitis B virus DNA subtype adr, including truncated preS/S sequences and the enhancer element. The X promoter and 422 nucleotides of the X coding region are present. The entire preC/C gene is deleted. In transient cotransfection assays using Chang liver cells (CCL 13), pM1 DNA exerts a 6- to 10-fold trans-activating effect on the expression of the pSV2CAT reporter plasmid. The trans-activation occurs by stimulation of transcription and is dependent on the simian virus 40 enhancer in the reporter plasmid. Deletion analysis of pM1 subclones reveals that the trans-activator is encoded by preS/S and not by X sequences. A frameshift mutation within the preS2 open reading frame shows that this portion is indispensable for the trans-activating function. Initiation of transcription has been mapped to the S1 promoter. A comparable trans-activating effect is also observed with cloned wild-type hepatitis B virus sequences similarly truncated. These results show that a transcriptional trans-activator function not present in the intact gene is generated by 3' truncation of integrated hepatitis B virus DNA preS/S sequences.

Hepatitis B virus (HBV) is regarded as the main aetiologic factor in the development of human hepatocellular carcinoma (HCC), one of the most frequent fatal malignancies worldwide. Detection of integrated HBV sequences in the cellular DNA of almost all HCCs studied, and the recent finding that the integrated HBV open reading frame (orf) X encodes a transactivating activity, supports the notion that integrated HBV DNA could contribute to liver carcinogenesis by activation of cellular genes in trans. But not all HCCs seem to harbour a functional orf X. We report here that 3'-truncated preS2/S sequences in integrated HBV DNA of liver cell carcinomas encode a so far unidentified transcriptional trans-activation activity. This activity is also produced by an artificially 3'-truncated preS2/S gene of the wild-type HBV genome. Besides the simian virus 40 promoter of the reporter plasmid pSV2CAT, the promoter of the human c-myc oncogene can also be activated. These results, taken together with the fact that preS/S is the only HBV gene found to be integrated in almost every HBV-related HCC analysed so far, indicate that trans-activation by integrated preS2/S sequences is a possible mechanism for HBV-associated oncogenesis.