Biomedical subjects

P Tiollais

Publications and source records attributed to P Tiollais.

At least 37 records · Page 2Linked to original sources

PubMed1998

In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen.

DNA immunization offers a novel means to induce humoral and cellular immunity in inbred or in outbred animals. Here we have tested the efficiency of genetic immunization with hepatitis B virus (HBV) envelope-based vectors. In naive primates, injection of a plasmid DNA encoding HBV envelope proteins induced an HBV-specific cytotoxic response and appearance of potentially protective anti-HBs antibodies. Moreover, intramuscular and intradermal injections of a DNA expression vector encoding an epitope of the human immunodeficiency virus envelope fused to the surface protein of the hepatitis B virus (HBsAg) induced strong humoral and cytotoxic responses to antigenic determinants of both viruses in mice and nonhuman primates alike. In addition, in protein-primed Rhesus monkeys B-cell memory was successfully boosted by DNA injection of hybrid vectors and animals subsequently developed a multispecific cellular response. This suggests that DNA-based immunization could be used to boost efficiently and broaden the immune response in individuals immunized with conventional vaccines, regardless of their genetic variability. These results also indicate that it might be possible to rationally design HBsAg-based expression vectors to induce multispecific immune responses for vaccination against hepatitis B and other pathogens.

Animals↗

PubMed1998

Extensive analysis of duplicated-inverted hepatitis B virus integrations in human hepatocellular carcinoma.

Hepatitis B virus (HBV) DNA is found chromosomally integrated into the genome of the majority of hepatocellular carcinomas (HCC) arising in chronic HBV carriers suggesting that, in some instances, viral sequences may be directly responsible for oncogenic conversion. In an attempt to clarify the oncogenic potential of integrated HBV sequences, we performed an extensive analysis of two single integrations present in HCC which developed in non-cirrhotic livers from HBsAg-positive Korean patients. In both cases, integrated viral sequences were characterized by a duplicated-inverted configuration involving the flanking cellular sequences, a pattern consistently found in many amplicons isolated from mammalian cells. Integration sites are characterized by an AT-rich content and the presence of topoisomerase I and II cleavage target sequences as well as other recombination-prone motifs. The chromosomal locations of the integration sites were determined as 8q13 and 10q22 in the human genome, two regions known to harbour genes involved in tumorigenesis. The cis-activating potential of the integrations in their original configuration was also investigated in a transient transfection assay in HepG2 cells. Integrated sequences, rather than activating heterologous promoters, show either no activity or a weak tendency to inhibit activation of neighbouring reporter genes. The implications of our findings for the understanding of primary liver cancer development are discussed.

Adult↗

PubMed1998

Functional analysis of ground squirrel hepatitis virus enhancer II.

We have characterized a major regulatory element of ground squirrel hepatitis virus (GSHV) located within a 90-nucleotide fragment of the core promoter upstream sequences and have compared its organization to that of woodchuck hepatitis virus (WHV) enhancer II (We2). The GSHV element (Ge2) stimulates transcription from the viral core promoter and heterologous promoters in an orientation-independent manner but displays a lower level of activity than We2 in transient transfection assays in human hepatoma cells. The general organization of Ge2 into binding sites for the liver-enriched HNF-1 and HNF-4 proteins and for ubiquitous factors of the NF1 and Oct families was found to be mostly conserved with respect to the homologous We2 region. Accordingly, transactivation by HNF-1 and HNF-4 plays an essential role in the liver-specific transcriptional activity of both the GSHV and WHV core promoters. Distinctive features of the GSHV enhancer consist of its ability to bind C/EBP family factors in a central motif that overlaps with one of the two HNF-4 sites and its differential binding affinities for HNF-4.

Animals↗

PubMed1998

Woodchuck hepatitis virus enhancer I and enhancer II are both involved in N-myc2 activation in woodchuck liver tumors.

Direct activation of the N-myc2 oncogene by insertion of woodchuck hepatitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatocarcinogenesis. We previously reported that WHV enhancer II (We2), which controls expression of the core/pregenome RNA, can also activate the N-myc2 promoter in hepatoma cell lines. To better define the integrated WHV regulatory sequences responsible for N-myc2 promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the win locus in tumor 2260T1 and mapping approximately 175 kb 3' of N-myc2. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with We2 sequences and 1 with WHV sequence homologous to that of hepatitis B virus enhancer I (We1). In transient transfection assays in hepatoma-derived cells, the We2 activator was found to be fully effective only when inserted in close proximity to the N-myc2 promoter whereas the We1 element by itself was apparently devoid of activity. In contrast, the 2260T1 viral insert exhibited a potent enhancer capacity that depended both on multimerized We2 and on We1 sequences. In a survey of different woodchuck hepatomas, both elements were commonly found within integrated viral sequences involved in long-range N-myc2 activation.

Animals↗

PubMed1998

[Hepatitis B virus and hepatocellular carcinoma].

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.

Animals↗

PubMed1997

Cellular and viral trans-acting factors modulate N-myc2 promoter activity in woodchuck liver tumors.

Activation of the N-myc2 oncogene by integration of woodchuck hepatitis virus (WHV) DNA is a central event in woodchuck liver oncogenesis. In this study, we have evaluated the influence of several cellular and viral trans-acting factors and mediators of inflammation on N-myc2 promoter activity in hepatoma cell lines. Ets oncoproteins, including Ets1, Ets2 and PEA3 efficiently activated a chimeric N-myc2 promoter/luciferase reporter gene. By electrophoretic mobility shift assays, we show that Etsl and Ets2 proteins can efficiently bind two consensus Ets sites located within a 59 bp sequence upstream of the N-myc2 transcription start site. Site-directed mutagenesis of these Ets-binding motifs abolished transactivation of the N-myc2 promoter by Ets proteins. Addition of interleukin-6 (IL-6) induced a weak but reproducible activation of the N-myc2 promoter, while IL-1 was ineffective. We further show that the N-myc2 promoter can be transactivated by the hepadna-virus X protein, and that distal promoter sequences are required for both IL-6 and X responsiveness. Similar effects of these factors were observed in the context of the N-myc2 promoter activated by WHV cis-regulatory elements. In view of the high-level expression of the N-myc2 oncogene in most woodchuck liver tumors, the Ets oncoproteins, inflammation-associated cytokine IL-6 and the viral X transactivator might play important roles in hepadnavirus-associated tumorigenesis.

Animals↗

PubMed1997

Comprehensive allelotyping of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world, however the molecular mechanisms underlying liver cell transformation remain obscure. A genome-wide scan of loss of heterozygosity (LOH) in tumors provides a powerful tool to search for genes involved in neoplastic processes. To identify recurrent genetic alterations in liver tumors, we examined DNAs isolated from 120 HCCs and their adjacent non tumorous parts for LOH using a collection of 195 microsatellite markers located roughly every 20 cM throughout 39 autosomal arms. The mean heterozygosity was 73%. Our findings provide additional support that LOH for loci on chromosomal arms 1p, 4q, 6q, 8p, 13q and 16p is significantly elevated in HCC. The highest percentage of LOH is found for a locus in 8p23 (42% of informative csaes). This corresponds to one of the most common genetic abnormalities reported to date in these tumors. In addition, high ratio of LOH (> or = 35%) is observed on chromosome arms which had not been implicated in previous studies, notably on 1q, 2q and 9q. No correlation was found between LOH of specific chromosomal regions and etiologic factors such as chronic infections with hepatitis B or C viruses. This first report of an extensive allelotypic analysis of HCC should help in identifying new genes whose loss of function contributes to the development of liver cancer.

Alleles↗

PubMed1997

The RAR alpha-PLZF chimera associated with Acute Promyelocytic Leukemia has retained a sequence-specific DNA-binding domain.

In most cases, Acute Promyelocytic Leukemia (APL) is associated with t(15;17) translocation which juxtaposes sequences from PML and retinoic acid receptor alpha (RAR alpha) genes. The generated PML-RAR alpha fusion interferes with wild type RAR alpha-mediated transcription and disrupts subnuclear compartments, known as PML bodies. Both defects are corrected by all trans retinoic acid (ATRA) therapy which induces differentiation of leukemic cells and clinical remission. In a rare APL syndrome associated with t(11;17), fusion of the RAR alpha gene with the PLZF gene, encoding a Zinc-finger protein produces two reciprocal RAR alpha chimeras. Although PLZF-RAR alpha and PML-RAR alpha are similar in their apparent dominant negative effects, t(11;17)-associated APL is refractory to ATRA therapy. In a yeast two-hybrid genetic screening, we isolated clones encoding the GAL4 transactivation domain fused to various parts of PLZF. Using these autonomously transactivating hybrids, similar in structure to the RAR alpha-PLZF fusion, we mapped the DNA-binding domain of PLZF to the last five Zinc-fingers, a region retained in RAR alpha-PLZF chimera and characterized a specific PLZF target sequence. Our data support the hypothesis that RAR alpha-PLZF chimera is not an inert product of reciprocal translocation and may thus contribute to ATRA unresponsiveness of t(11;17)-associated APL.

Base Sequence↗

PubMed1997

DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice.

Hepatitis B virus (HBV) remains a serious worldwide health problem and the possibility to control it will depend on the availability of safe, effective and affordable vaccines. Recombinant protein or plasma-derived vaccines containing HBV surface antigen (HBsAg) are safe and generally efficacious, however, they are too expensive for widespread use in areas of HBV endemicity and are only partially effective for treatment of HBV chronic carriers. Immunization of mice by injection of HBsAg-expressing plasmid DNA results in rapid induction of strong and long-lasting humoral and cell-mediated immune responses. Here we report optimization of the humoral response with the use of necrotizing agents, co-expression of cytokines or co-stimulatory molecules and formulation of the DNA with cationic liposomes. DNA-based immunization of HBsAg-transgenic mice can also overcome non-response to HBsAg. Thus, DNA vaccines against HBV may be useful for both prophylactic and therapeutic purposes.

Animals↗

PubMed1997

Interaction of the UV-damaged DNA-binding protein with hepatitis B virus X protein is conserved among mammalian hepadnaviruses and restricted to transactivation-proficient X-insertion mutants.

We carried out a comparative analysis of several proposed host protein partners of the human hepatitis B virus X protein (HBx) using both the GAL4- and the LexA-based yeast two-hybrid system. We showed that the interaction of HBx with the UV-damaged DNA-binding protein (UVDDB) is positive in both yeast systems, detectable in cotransfected human cells, conserved by rodent hepadnavirus X proteins (known to transactivate in human cells), and tightly correlated with the transactivation proficiency of X-insertion mutants. Taken together, our results strongly suggest that UVDDB is involved in X-mediated transactivation.

Binding Sites↗

PubMed1997

Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to evaluate and map genomic aberrations in 50 hepatocellular carcinomas (HCCs) from patients chronically infected with hepatitis B virus (HBV). CGH clearly detected nonrandom genomic imbalances. Losses were most prevalent on chromosome regions 4q (70%), 8p (65%), 16q (54%), 17p (51%), 13q and 6q (37% each), and lp (30%). The most frequent gains occurred on 8q (60%), 1q (58%), and 6p and 17q (33% each). In a few cases, sequence amplifications were detected that were mapped to bands 11q12, 12p11, 14q12, and 19q13.1. This study represents the first analysis of primary liver cancers by CGH, and it confirms the presence of previously known chromosomal aberrations in HCC and highlights new quantitative abnormalities and sequence amplifications. These findings should lead to the characterization of new loci involved in liver cancer pathogenesis.

Carcinoma, Hepatocellular↗

PubMed1996

DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.

Animals↗

PubMed1996

A functional N-myc2 retroposon in ground squirrels: implications for hepadnavirus-associated carcinogenesis.

Three hepatitis B viruses infecting humans, woodchucks and ground squirrels increase the risk of hepatocellular carcinoma in their respective hosts. The woodchuck hepatitis B virus (WHV), unlike the two other viruses, induces a rapid carcinogenic process characterized by direct activation of myc proto-oncogenes by insertion of viral DNA. The highly preferred target of insertional mutagenesis in woodchucks is N-myc2, an intronless N-myc gene. Strikingly, N-myc2 has no human homolog and the homologous N-myc2 locus previously detected in the ground squirrel genome, remains silent during hepatocarcinogenesis. Therefore, N-myc2 may represent a critical host determinant in the evolution of the disease associated with hepadnavirus infection. To address this question, we performed a structural and functional analysis of the ground squirrel N-myc2 locus. We show that ground squirrel N-myc2 is highly homologous to its woodchuck counterpart and is a functional proto-oncogene. Existence of a functional N-myc2 gene as a potential target for insertional activation by viral DNA is therefore not restricted to the woodchuck species. This suggests that viral rather than host factors determine the higher oncogenic phenotype of WHV as compared to the two other mammalian hepadnaviruses.

Amino Acid Sequence↗

PubMed1996

DNA-based immunization against the envelope proteins of the hepatitis B virus.

Intramuscular injection in mice of DNA expression vectors encoding the envelope proteins of the hepatitis B virus induced humoral responses specific to several antigenic determinants of the viral envelope. The use of different promoter elements in the plasmid vectors influenced the kinetics and specificity of antibodies produced to the envelope proteins. The first antibodies appeared within 1-2 weeks after injection of DNA and included antibodies of the IgM isotype. Over the following weeks, an IgM-to-IgG class switch occurred, indicating helper T-lymphocyte activity. Peak IgG titers were reached by 4 weeks after a single DNA injection and were maintained for at least 6 months without further DNA injections. The antibodies to the envelope proteins reacted with both group- and subtype-specific antigenic determinants of the HBV surface antigen (HBsAg). The nature of the immune response to the envelope proteins provides indirect evidence that the proteins have adopted a native conformation and have probably been assembled into particles after intramuscular expression from the plasmid vectors. These results indicate that it may be possible to rationally design DNA expression vectors to induce a particular type of immune response for vaccination against hepatitis B or other pathogens.

Animals↗

PubMed1996

Unusual activation of the integrated preS1 promoter of woodchuck hepatitis virus in a liver tumour.

We have analysed abnormal virus RNAs produced from integrated woodchuck hepatitis virus (WHV) sequences in two woodchuck liver tumours. Analysis of cDNA clones revealed that these transcripts consisted of rearranged, virus-specific RNAs encoding the WHV surface antigens. In one tumour, transcription was driven by the major preS2/S promoter and terminated at a cryptic poly(A) signal in the 5' end of the P gene, giving rise to a truncated version of the normal viral S message. In contrast, the integrated preS2/S promoter remained silent in the second tumour. The start sites of two abundant WHV transcripts encoding the large and middle surface proteins were localized about 100 bp upstream and 300 bp downstream of the preS1 translation initiation codon, corresponding to minor start sites of the normal surface protein mRNAs in chronically infected liver. Thus, the preS1 promoter, a weak promoter in episomal replicative forms of the virus, was activated in the integrated state in this tumour. Our results indicate that alternative usage of the preS1 or the preS2/S promoter in the integrated state may yield differential production of the three virus surface proteins in woodchuck liver tumours.

Animals↗

PubMed1996

A t(3;8) chromosomal translocation associated with hepatitis B virus intergration involves the carboxypeptidase N locus.

Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocation present in a hepatocellular carcinoma developed in noncirrhotic liver tissue. One side of the translocation, in 8p23, is shown to be in the vicinity of the carboxypeptidase N gene, a locus that is heavily transcribed in liver tissue and frequently deleted in hepatocellular carcinomas and other epithelial tumors. The other side of the translocation, in 3q27-29, is widely implicated in several types of translocations occurring in different malignancies, such as large-cell lymphomas. The present data strongly support a model in which HBV-induced chromosomal rearrangements play a key role during multistep liver oncogenesis.

Adult↗

PubMed1996

The HNF1/HNF4-dependent We2 element of woodchuck hepatitis virus controls viral replication and can activate the N-myc2 promoter.

Transcriptional activation of myc family proto-oncogenes through the insertion of viral sequences is the predominant mechanism by which woodchuck hepatitis virus (WHV) induces liver tumors in chronically infected animals. The main target is N-myc2, a functional retroposon of the N-myc gene, but c-myc and N-myc are also marginally involved. Here we identify a major, liver-specific regulatory element in the WHV genome (We2) which efficiently activates the N-myc2 promoter in cultured hepatoma cells. In the context of the episomal viral genome, We2 governs the production of pregenomic RNA and thus plays a central role in the control of viral replication. We2 activity is primarily controlled by the liver-enriched HNF1 and HNF4 transcription factors, although NF1 and Oct proteins were also shown to bind in a central region. The expression of HNF1 and HNF4 appears to be maintained in woodchuck tumors. Thus, We2 is a prime candidate for controlling myc gene cis activation during WHV-induced hepatocarcinogenesis.

Animals↗

PubMed1995

DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects of the humoral response mimic hepatitis B viral infection in humans.

Intramuscular injection of plasmid DNA expression vectors encoding the three envelope proteins of the hepatitis B virus (HBV) induced humoral responses in C57BL/6 mice specific to several antigenic determinants of the viral envelope. The first antibodies appeared within 1-2 weeks after injection of DNA and included antibodies of the IgM isotype. Over the next few weeks, an IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. Peak IgG titers were reached by 4-8 weeks after a single DNA injection and were maintained for at least 6 months without further DNA injections. The antibodies to the envelope proteins reacted with group- and subtype-specific antigenic determinants of the HBV surface antigen (HBsAg). Expression vectors encoding the major (S) and middle (preS2 plus S) envelope proteins induced antibodies specific to the S protein and preS2 domain, and preS2 antibodies were prominent at early time points. In general, the expression vectors induced humoral responses in mice that mimic those observed in humans during the course of natural HBV infection.

Animals↗