Wolf-Bernhard Offensperger

Hepatocellular carcinoma and hepatitis B virus infection: molecular evidence for monoclonal origin and expansion of malignantly transformed hepatocytes

SummaryThe clonality of tumor cells was studied in a patient with metastasizing hepatocellular carcinoma (HCC). Using hepatitis B virus (HBV) DNA as a genetic marker, the pattern of integration of viral DNA into the tumor cell genome was determined by Southern blot analyses of DNAs extracted from different HCC lesions in the liver and both lungs. All tumor tissues examined were found to have viral DNA integrated into the same site(s) of the cellular genome. This finding provides direct molecular evidence for a monoclonal origin and expansion of malignantly transformed hepatocytes during tumor growth and metastasis. This characteristic is similar to other human cancers associated with viral infections, such as adult T-cell leukemia, Burkitts lymphoma, or cervical cancer, and is important für our understanding of viral oncogenesis in man.

Inhibition of duck hepatitis B virus infection by lysosomotropic agents.

The early phases of hepadnaviral infection were studied in primary duck hepatocyte cultures. Incubation of duck hepatocytes in vitro with duck hepatitis B virus (DHBV) resulted in infection with high levels of viral replication. The lysosomotropic agents ammonium chloride and chloroquine effectively inhibited viral infection, indicating that DHBV infection, similar to infection with other enveloped viruses, depends on receptor-mediated endocytosis and involves membrane fusion triggered by low pH.

Antisense therapy of hepatitis B virus infection.

Chronic infection with the hepatitis B virus (HBV) is a major health problem worldwide. The only established therapy is interferon-a with an efficacy of only 30–40% in highly selected patients. The discovery of animal viruses closely related to the HBV has contributed to active research on antiviral therapy of chronic hepatitis B. The animal model tested and described in this article are Peking ducks infected with the duck hepatitis B virus (DHBV). Molecular therapeutic strategies aimed at blocking gene expression include antisense DNA. An antisense oligodeoxynucleotide directed against the 5′-region of the preS gene of DHBV inhibited viral replication and gene expression in vitro in primary duck hepatocytes and in vivo in Peking ducks. These results demonstrate the potential clinical use of antisense DNA as antiviral therapeutics.

Duck hepatitis B virus: DNA polymerase and reverse transcriptase activities of replicative complexes isolated from liver and their inhibition in vitro

The duck hepatitis B virus (DHBV)-associated activities of reverse transcriptase and DNA polymerase and their inhibition in vitro were studied. Replicative complexes (RCs) were isolated from DHBV-infected liver by gel chromatography followed by sucrose gradient centrifugation. The RCs were detected by dot blot hybridization, using radiolabeled cloned DHBV DNA as a probe, and by the incorporation of 32P-TTP in the presence of dATP, dCTP, dGTP, and Mg2+ (endogenous DNA polymerase activity). The endogenous DNA polymerase activity associated with RCs was further studied using exogenous templates: reverse transcriptase and DNA polymerase activities were demonstrated using as substrates 32P-TTP and poly(rA) p(dT)12 or poly(dA) p(dT)12-18, respectively. Both activities were biochemically characterized. Their inhibition by various antiviral agents was studied in vitro: actinomycin D, ara-ATP, aphidicolin, suramin, chloroquin, and phosphonoformate. Among these, suramin, chloroquin, phosphonoformate, and ara-ATP were shown to be potent inhibitors of viral reverse transcriptase and DNA polymerase. Studies are now in progress to establish their antiviral activity in vivo.

Molecular therapeutic strategies in hepatitis B virus infection

Chronic infection with the hepatitis B virus is a major health problem worldwide. The only established therapy is interferon-α, with an efficacy of only 30–40% in highly selected patients. Nucleoside analogues do not show a significant clinical benefit. Molecular therapeutic strategies aimed at blocking gene expression include antisense DNA/RNA and ribozymes acting at the posttranscriptional level and triple helix formation blocking at the transcriptional level. In vitro, antisense oligodeoxynucleotides inhibit viral replication and gene expression in human hepatoma cell lines. In vivo, an antisense oligodeoxynucleotide directed against the 5′-region of the pre-S gene of the duck hepatitis B virus inhibited viral replication and gene expression in ducks. In vitro, ribozymes accurately cleave HBV substrate RNA. Triple helix formation is another very promising molecular approach. Results in hepadnaviral infection are not yet available, however.

Human non-hepatocytes support hepadnaviral replication and virion production

The competence of non-hepatocytes to support hepatitis B virus (HBV) gene expression and replication was studied by transient transfection of various human cell lines with a head-to-tail dimer of HBV DNA. Independent of their neuroectodermal, mesenchymal or epithelial origin, all non-hepatocyte cell lines tested synthesized and secreted hepatitis B surface antigen (HBsAg) and hepatitis B core/e antigen (HBc/eAg). Further analyses of two of these cell lines (LS 180 and COLO 320) identified the two major HBV transcripts of 3.6 and 2.2/2.4 kb length, respectively. LS 180 cells were permissive for HBV and duck hepatitis B virus (DHBV) DNA replication and secretion of infectious virions. COLO 320 cells also supported HBV DNA replication, but did not appear to export complete viral particles. These findings provide direct evidence that both HBV and DHBV can replicate in non-hepatic tumour cell lines, one of which is shown also to produce infectious virions.

Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides

Phosphorothioate-modified antisense oligodeoxynucleotides (ASOs) are used to suppress gene expression by inducing RNase H-mediated cleavage with subsequent degradation of the target mRNA. However, previous observations suggest that ASO/RNase H can also result in the generation of stable mRNA cleavage fragments and expression of truncated proteins. Here, we addressed the underlying translational mechanisms in more detail using hepadnavirus-transfected hepatoma cells as a model system of antisense therapy. Generation of stable mRNA cleavage fragments was restricted to the ASO/RNase H pathway and not observed upon cotransfection of isosequential small interfering RNA or RNase H-incompetent oligonucleotides. Furthermore, direct evidence for translation of mRNA fragments was established by polysome analysis. Polysome-associated RNA contained cleavage fragments devoid of a 5′ cap structure indicating that translation was, at least in part, cap-independent. Further analysis of the uncapped cleavage fragments revealed that their 5′ terminus and initiation codon were only separated by a few nucleotides suggesting a 5′ end-dependent mode of translation, whereas internal initiation could be ruled out. However, the efficiency of translation was moderate compared to uncleaved mRNA and amounted to 13–24% depending on the ASO used. These findings provide a rationale for understanding the translation of mRNA fragments generated by ASO/RNase H mechanistically.

Ligand-mediated retargeting of recombinant adenovirus for gene transfer in vivo

The development of efficient and safe methods for in vivo gene transfer is central to the success of gene therapy. Recombinant adenoviral vectors, although highly efficient, are limited by the host immune response, potential safety hazards due to obligatory cotransfer of viral proteins, and their broad tissue tropism. Here, we demonstrate in an animal model that host range and tissue tropism of a recombinant adenovirus from a distant species can be modified by complexing adenovirus with a cell-specific ligand. Thus, a replication-deficient lacZ recombinant human adenovirus, which naturally does not infect avian cells, allowed highly efficient and specific gene transfer to the liver of ducks in vivo when complexed with N-acetylglucosamine, a ligand for the chicken hepatic lectin. This combination of ligand-mediated receptor targeting with adenoviral uptake and intracellular processing of a given gene represents a novel approach to gene therapy of inherited and acquired liver diseases.

Antiviral therapy of hepatitis B virus infection: Blocking viral gene expression

Abstract Hepatitis B virus (HBV) infection is a major cause of chronic viral hepatitis and liver cirrhosis worldwide. HBV has been characterized in great detail and can be specifically identified by serological and molecular techniques. Chronic hepatitis B frequently progresses to liver cirrhosis with its clinical sequelae and is associated with the development of hepatocellular carcinoma (HCC). Strategies aimed at the prevention of liver cirrhosis include primary prevention of HBV infection by various measures as well as secondary prevention by therapy of acute or chronic hepatitis B as precursors of liver cirrhosis and HCC development. For the treatment of chronic hepatitis B interferon-α or -β are the only drugs currently available for clinical use in selected patients. Given their limited efficacy, combination therapies of interferon-α or -β with synthetic antiviral agents or other drugs as well as gene therapy strategies are presently being explored. These molecular strategies are designed to specifically deliver antiviral nucleic acids to infected cells, thereby improving therapeutic efficacy and reducing extrahepatic side-effects.

Biological efficacy and signal transduction through STAT proteins of recombinant duck interferon in duck hepatitis B virus infection

The aim of this study was to characterize the interferon induced intracellular signals in duck hepatocytes and to investigate the effects of duck interferon on virus replication in duck hepatitis B virus (DHBV) infected ducks. Interestingly, duck interferon was found to activate intracellular signal transduction pathways similar to those of its mammalian counterparts. An interferon stimulated gel shift activity like that of gene factor 3 is induced, as well as serum inducible element binding factors homologous to serum inducible factor A (SIF-A), SIF-B and SIF-C. Duck interferon induced signal transducer and activator of transcription activation is not inhibited by DHBV infection of hepatocytes. DHBV infected ducks treated for 10 days with recombinant duck interferon show a decrease in viral DNA in hepatocytes, and in many cases disappearance of viraemia. These findings confirm the usefulness of the DHBV infection model for the study of human hepatitis B virus infection.