Biomedical subjects

P Tiollais

Publications and source records attributed to P Tiollais.

At least 19 recordsLinked to original sources

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

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↗

PubMed1994

Evidence for long-range oncogene activation by hepadnavirus insertion.

Insertional mutagenesis of host genes, a common oncogenic strategy of slow transforming retroviruses, has recently been described for a DNA virus of the hepadnavirus group: the woodchuck hepatitis virus. This virus causes insertional activation of myc genes, mainly the intronless N-myc2 oncogene, in > 50% of woodchuck liver tumours. In most remaining tumours, N-myc2 is overexpressed without any apparent genetic alteration. To elucidate the role of the virus in such cases, we have cloned and analysed single integration sites in four woodchuck tumours carrying wild-type myc alleles. All sites were clustered within < 20 kb in a single locus, in which scarce unique sequences showed no detectable transcriptional activity. By fluorescent in situ hybridization, N-myc2 and the new locus (win) were localized to the same region of the long arm of the woodchuck X chromosome, and a 150-180 kb intervening distance was deduced from pulse-field gel analysis. The detection of viral integrations in win in additional tumours that produced abundant N-myc2 transcripts further substantiates the link between these two loci in woodchuck tumorigenesis. We propose that efficient activation of the N-myc2 promoter by the hepadnavirus enhancer acting over a long distance might operate in liver cell transformation.

Animals↗

PubMed1994

Inhibition of hepatitis B virus surface antigen gene expression in carcinogen-induced liver tumors from transgenic mice.

We previously showed that hepatitis B surface antigen (HBsAg)-producing transgenic mice were more sensitive to hepatocarcinogens than their normal littermates were. We have now investigated the regulation of hepatitis B virus (HBV) gene expression in carcinogen-induced liver tumors of HBV-carrier transgenic mice and in three cell lines derived from tumor samples. Transcription of the S gene was repressed in 17 tumors even though they had normal levels of liver-specific mRNAs such as albumin and transferrin. Three hepatoma cell lines, derived from independent tumor samples, were analyzed for their capacity to express the S gene after transfection of cloned DNA. Although they no longer expressed the endogenous S gene, they were still able to express it from transfected viral DNA both transiently and stably. The loss of HBsAg expression in tumors and in the cell lines was accompanied by de novo methylation of the S region, which is a way to permanently repress gene expression. Our data confirm in an animal model previous observations of S-gene expression in human hepatocarcinoma and suggest a role for its downregulation in tumor progression.

Animals↗

PubMed1994

Characterization and mapping of a B-cell immunogenic domain in hepatitis C virus E2 glycoprotein using a yeast peptide library.

To identify conserved humoral antigenic determinants within the hepatitis C virus (HCV) envelope protein E2, we expressed a peptide library containing random short fragments of the HCV envelope in yeast. Clones were identified using a monospecific rabbit antibody to a region downstream of the E2 hypervariable region. The clones define the limits of two original antigenic domains: a major one (aa 493-576) and a minor one (aa 535-606). The major antigenic domain maps in a region that displays a high degree of homology within a (HCV) subtype (92-97.6% identity). Yeast-encoded determinants were characterized by Western blot analysis, N-glycosidase F digestion, and using a panel of synthetic peptides. The data suggest that the major antigenic domain contains at least two determinants, one of them mimicked by an 18-mer peptide (aa 514-531). ELISA and competitive inhibition assays demonstrated that: (1) the determinants appear subtype 1a-specific, (2) seroprevalence of antibody to the determinants is rather low (20.6%), and (3) donors show a heterologous response to the different determinants. Antibody response to the E2 determinants was studied in HCV-infected chimpanzees and post-transfusion-associated NANB hepatitis cases. The antibody response was found during chronic infection and may not be effective for virus clearance.

Amino Acid Sequence↗

PubMed1994

Vaccine-induced neutralizing antibodies directed in part to the simian immunodeficiency virus (SIV) V2 domain were unable to protect rhesus monkeys from SIV experimental challenge.

The potential of the simian immunodeficiency virus (SIV) variable 2 (V2) domain as an effective region to boost SIV-neutralizing antibodies and to protect against live SIV challenge was tested in rhesus macaques. In this study, two rhesus macaques were primed with vaccinia virus recombinants expressing the surface glycoprotein gp140 of SIVmac and were given booster injections with the SIVmac V2 domain presented by a highly immunogenic carrier, the hepatitis B surface antigen (HBsAg). The two vaccinated macaques exhibited SIV-neutralizing antibodies after primer injections that were enhanced by the V2/HBsAg injections. Part of these SIV-neutralizing antibodies were directed specifically to the V2 region, as shown by neutralization-blocking experiments. However, despite having consistent SIV-neutralizing antibody titers, animals were not protected against homologous challenge with BK28, the molecular clone of SIVmac251. No SIV envelope-specific cellular cytotoxic response was detected throughout the immunization protocol, suggesting that neutralizing antibodies directed to SIV envelope gp140 and especially to the V2 domain were unable on their own to protect against SIV challenge. Furthermore, the vaccinees seemed to have higher viral loads than control animals after challenge, raising the question of whether neutralizing antibodies induced by vaccination and directed to the SIV envelope selected viral escape mutants, as shown previously in SIV-infected macaques. This mechanism is certainly worthy of intensive investigation and raises some concern for SIV envelope-targeted immunization.

Amino Acid Sequence↗

PubMed1994

Immunogenicity of hybrid hepatitis B surface antigen particles.

Hepatitis B surface antigen (HBsAg) produced by recombinant DNA technology is now widely and safely used worldwide for hepatitis B vaccination. We used the HBsAg particle as a carrier molecule for presentation of selected human immunodeficiency virus type 1 (HIV-1) determinants to the immune system. Immunization studies carried out in primates showed that the particles elicit HIV specific virus neutralizing antibodies as well as T cell proliferative responses to both pathogens. As an experimental approach to active immunotherapy for hepatitis B virus (HBV) chronic carriers, we evaluated the efficiency of such hybrid antigen to overcome B cell tolerance in HBV transgenic mice.

AIDS Vaccines↗

PubMed1994

Liver-specific expression and high oncogenic efficiency of a c-myc transgene activated by woodchuck hepatitis virus insertion.

The high oncogenic efficiency of woodchuck hepatitis virus (WHV) has been correlated with the ability of this virus to provoke insertional activation of myc family genes. To assess the impact of viral integration on liver cell transformation, we have generated transgenic mice carrying the mutated c-myc gene and adjacent viral DNA from a woodchuck tumor, in original configuration. Virtually all mice from two different strains developed hepatocellular carcinoma with a mean latency period of 8-12 months. The c-myc transgene was expressed transiently in neonatal livers, and re-expressed at preneoplastic and neoplastic stages in adult livers. Woodchuck c-myc mRNA driven by the normal P1 and P2 promoters and WHV-specific transcripts encoding viral surface antigens were produced in a strictly co-regulated fashion during development and tumorigenesis, indicating a predominant regulatory influence of the viral enhancer. Furthermore, the activity of the viral enhancer in response to various biological stimuli was apparently modulated by glucose uptake and glucagon/insulin balance in differentiated hepatocytes. In this model, a viral integration event selected from a naturally occurring tumor proved to be determinant for induction of hepatocarcinogenesis, although enforced, liver-specific expression of c-myc was limited to a particular developmental stage.

Animals↗

PubMed1993

A hepatitis B virus pre-S-retinoic acid receptor beta chimera transforms erythrocytic progenitor cells in vitro.

In this report, we investigated the transforming properties of retinoic acid receptor beta (RAR beta). The v-erbA protein, which is the viral oncogenic homologue of the thyroid hormone receptor, was replaced by either the complete RAR beta (beta R) or a hepatitis B virus pre-S-RAR beta (H beta R) hybrid product in an avian erythroblastosis virus-based vector. In chicken hematopoietic cells, the H beta R protein was able to transform erythroid progenitor cells, whereas no such transformation was observed with the wild-type beta R protein. Moreover, the fully transformed phenotype was observed even in the absence of v-erbB, and H beta R-transformed erythroid cells grew independently of growth factors and transforming growth factor alpha. The analysis of erythrocytic-specific proteins revealed that the transformed cells were blocked at the colony-forming unit-erythroid stage and that the expression of the carbonic anhydrase II gene, a gene normally regulated by thyroid hormones, was repressed by the H beta R protein. Finally, hepatocarcinomas rapidly developed in some chickens infected in ovo with viruses encoding either the normal or the hybrid H beta R, suggesting that an inappropriate expression of the RAR beta gene may represent an important event in oncogenesis.

Amino Acid Sequence↗

PubMed1993

Induction of anti-hepatitis B surface antigen (HBsAg) antibodies in HBsAg producing transgenic mice: a possible way of circumventing "nonresponse" to HBsAg.

As a model for studying possible mechanisms of nonresponse toward hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV) carriers, we used transgenic (Tg) mice which constitutively express this antigen in the liver from before birth. The mice secrete large amounts of HBsAg particles into the sera without producing antibodies. Tg and control mice were immunized with either recombinant HBsAg particles of a different subtype, or with recombinant hybrid HBsAg particles carrying a human immunodeficiency virus (HIV) envelope determinant. The presence of determinants to which the mice are tolerant on the injected particles does not hamper the response to the foreign epitope. Moreover, a weak but significant anti-HBs response is clearly detectable in Tg mice immunized with these particles. Antibodies to epitopes carried by the transgenic antigen are made even after injection of homologous antigen, and a concomitant decrease in circulating HBsAg is observed. This immune response does not induce any liver damage. It was demonstrated that in these Tg mice, B cell self-tolerance toward HBsAg can be overcome by immunization. This phenomenon raises the possibility of designing more effective methods of immunotherapy for HBV carriers.

Animals↗

PubMed1993

Expression of HTLV-I Env and Tax recombinant peptides in yeast: identification of immunogenic domains.

A peptide library of HTLV-I Env and Tax proteins was constructed in yeast in order to characterize which domains of these proteins are immunogenic in HTLV-I-infected individuals. Five yeast colonies (A to E) were selected using HTLV-I positive plasma, and one yeast colony (F) was selected using rabbit anti-Tax sera. Plasmid DNA present in each positive clone was recovered and sequenced. Overlapping clones A to E covered the C-terminal part of the gp46 exterior glycoprotein (aa 197 to 305) and were all glycosylated. Clone F encoded the C-terminal 25 aa of Tax (aa 329-353). Recombinant peptides were recognized by more than 40% of the HTLV-I positive human sera, confirming that they are major immunodominant domains. We studied the antibody response to each recombinant peptide in patients with TSP/HAM and asymptomatic carriers. Higher absorbance values were obtained with sera from TSP/HAM patients than from asymptomatic carriers, but the difference between the two groups was not statistically significant. Our study confirms that the COOH-terminal region of gp46 is highly immunogenic in HTLV-I-infected individuals and demonstrates a new immunogenic epitope of the Tax protein.

Amino Acid Sequence↗