Nedd9/Hef1/Cas-L mediates the effects of environmental pollutants on cell migration and plasticity.

Aryl hydrocarbon receptor (AhR), or dioxin receptor, is a transcription factor that induces adaptive metabolic pathways in response to environmental pollutants. Recently, other pathways were found to be altered by AhR and its ligands. Indeed, developmental defects elicited by AhR ligands suggest that additional cellular functions may be targeted by this receptor, including cell migration and plasticity. Here, we show that dioxin-mediated activation of Ahr induces Nedd9/Hef1/Cas-L, a member of the Cas protein family recently identified as a metastasis marker. The Hef1 gene induction is mediated by two xenobiotic responsive elements present in this gene promoter. Moreover, using RNA interference, we show that Nedd9/Hef1/Cas-L mediates the dioxin-elicited changes related to cell plasticity, including alterations of cellular adhesion and shape, cytoskeleton reorganization, and increased cell migration. Furthermore, we show that both E-cadherin repression and Jun N-terminal kinases activation by dioxin and AhR also depend on the expression of Nedd9/Hef1/Cas-L. Our study unveils, for the first time, a link between pollutants exposure and the induced expression of a metastasis marker and shows that cellular migration and plasticity markers are regulated by AhR and its toxic ligands.

Activation of the dioxin/aryl hydrocarbon receptor (AhR) modulates cell plasticity through a JNK-dependent mechanism.

Environmental chemicals such as dioxin adversely affect immune, neurological and reproductive functions and have been implicated in cancer development. However, the mechanisms responsible for dioxin toxicity are still poorly understood. Here, we show that dioxin and related pollutants trigger a marked morphological change in epithelial cells that remodel their cytoskeleton to increase interaction with extra cellular matrix while loosening cell-cell contacts. Furthermore, dioxin-treated cells show increased motility. These dioxin-mediated effects are mimicked by constitutive expression and activation of the intracellular dioxin receptor (aryl hydrocarbon receptor (AhR)). They correlate with activation of the Jun NH2-terminal kinase (JNK) and are reverted by treatment with a JNK inhibitor. Dioxin-induced effects occur 48 h post-treatment initiation, a time scale, which argues for a genomic effect of the AhR, linked to induction of target genes. This novel Ahr action on cell plasticity points to a role in cancer progression.

UVDDB p127-binding modulates activities and intracellular distribution of hepatitis B virus X protein.

Mammalian hepatitis B viruses encode a unique regulatory protein termed X, which is essential for infection and likely plays a role in the carcinogenic process associated with hepadnaviral infection. Among the numerous properties ascribed to X protein, two have been widely documented: promiscuous transcriptional transactivation and proapoptosis. However, full understanding of the mechanisms underlying these activities requires the identification of the genuine X partners among the multiple X-binding host proteins. Here we show that (i) mutations in X protein, which markedly alter affinity for the host protein UVDDBp127, inactivate both transactivation and proapoptosis; (ii) ectopic fusion of a functional UVDDB-binding domain to a deficient binding X mutant restored its activity; (iii) in contrast to the loss-of-binding mutants, a mutant with a strong gain-of-binding exerted trans-dominant negative effects on wt X activity and localized in the nucleus and (iv) increase in intracellular UVDDB concentration enhanced both wt X-mediated transactivation and apoptosis. Taken together, our data provide strong evidence for a common upstream step in X mode of action, consisting of its productive interaction with UVDDB, via a structurally and functionally autonomous module. In addition, they underscore a nuclear location step of the viral protein that depends on its ability to bind UVDDB.

Correct binding of viral X protein to UVDDB-p127 cellular protein is critical for efficient infection by hepatitis B viruses.

A fully effective treatment of chronic human hepatitis B virus (HBV) infection is still missing and HBV remains the first etiological agent of liver cancer. Although the viral regulatory X protein is essential for infection, its mode of action remains obscure, due the lack of an in vitro infection system. In the accompanying study, we showed the functional importance of interaction between X and the host protein UVDDB-p127, in the transactivation and apoptotic properties of the viral protein. Here, we addressed the biological role of X-UVDDB interaction in the infectious process using a genetic approach in the woodchuck virus closely related to HBV. We show that (i) mutations in X, which markedly affect UVDDB-binding, also abolished productive infection in woodchucks, (ii) in the few cases where mutant viruses led to infection, compensatory mutations had occurred in the X gene of the viral progeny, which restored correct UVDDB-binding. We conclude that efficient viral replication in vivo requires proper X-UVDDB interaction. The interaction may thus provide a novel therapeutic target for the treatment of hepatitis

A genomic approach of the hepatitis C virus generates a protein interaction map.

The hepatitis C virus (HCV) causes severe liver disease, including liver cancer. A vaccine preventing HCV infection has not yet been developed, and, given the increasing number of infected people, this virus is now considered a major public-health problem. The HCV genome is a plus-stranded RNA that encodes a single polyprotein processed into at least 10 mature polypeptides. So far, only the interaction between the protease NS3 and its cofactor, NS4A, which is involved in the processing of the non-structural region, has been extensively studied. Our work was aimed at constructing a protein interaction map of HCV. A classical two-hybrid system failed to detect any interactions between mature HCV polypeptides, suggesting incorrect folding, expression or targetting of these proteins. We therefore developed a two-hybrid strategy, based on exhaustive screens of a random genomic HCV library. Using this method, we found known interactions, such as the capsid homodimer and the protease dimer, NS3-NS4A, as well as several novel interactions such as NS4A-NS2. Thus, our results are consistent with the idea that the use of a random genomic HCV library allows the selection of correctly folded viral protein fragments. Interacting domains of the viral polyprotein are identified, opening the possibility of developing specific anti-viral agents, based on their ability to modulate these interactions.

The proapoptotic effect of hepatitis B virus HBx protein correlates with its transactivation activity in stably transfected cell lines.

The role of hepatitis B virus HBx protein in the carcinogenesis associated with chronic viral infection remains ill-defined. Indeed, pleiotropic effects have been ascribed to HBx: in addition to its well-documented ability to indirectly stimulate transcription, the protein has been reported to affect cell growth, signal transduction, DNA repair and apoptosis. In this work, we generated Chang (CCL-13)-derived cell lines constitutively expressing wild type or mutant HBx, as a model of HBx-host cell interaction closer to the chronic infection setting, than the classically used transient expression systems. We document the potentiation by HBx of the apoptotic cell death pathway in the recipient cells. This effect is unlikely to rely on p53 activity since the protein is functionally inactivated in CCL-13. In addition, antioxidants and cyclosporin A failed to reduce the apoptotic response back to the normal level, suggesting that production of reactive oxygen species and calcineurin activation are not directly involved in the proapoptotic effect of HBx. In contrast, our data show that transactivation and stimulation of apoptosis are tightly linked HBx activities. Finally, expression of transactivation-active protein did not result in detectable change in the pattern of MAP kinases phosphorylation nor did it affect the ability of the host cell to repair in vitro irradiated plasmid DNA.

Infection of WHV/c-myc transgenic mice with Moloney murine leukaemia virus and proviral insertion near the syndecan-4 gene in an early liver tumour.

The capacity of Moloney murine leukaemia virus (MoMLV) to infect neonatal hepatocytes and to accelerate liver carcinogenesis was examined in a transgenic mouse model. WHV/c-myc mice which are highly susceptible to the development of liver tumours were infected with MoMLV shortly after birth, when expression of the murine ecotropic retroviral receptor gene was still detectable in the neonatal liver. All MoMLV-infected transgenic mice and non-transgenic littermates succumbed to T-cell lymphomas within 2-9 months; during this period of time, three infected transgenic animals developed primary hepatocellular carcinomas. Remarkably, one of these liver tumours arose significantly faster than tumours from uninfected WHV/c-myc controls, and it harboured a unique MoMLV provirus. The provirus integration site was located 5.5 kb upstream of the first exon of the syndecan-4 gene, which encodes a heparan sulphate proteoglycan implicated in growth factor activation and protein kinase C distribution in focal adhesions. Our data provide evidence for clonal MoMLV provirus integration in a hepatocellular carcinoma, and indicate that parenchymal liver cells may be susceptible to MoMLV infection following neonatal inoculation.

Interaction of SP100 with HP1 proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment.

The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RARalpha hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.

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.

The bifunctional DCOH protein binds to HNF1 independently of its 4-alpha-carbinolamine dehydratase activity.

HNF1 is a liver enriched atypical homeoprotein isolated from vertebrates which is involved in the transcriptional activation of liver, kidney, intestine and pancreas specific genes. HNF1 contains an N-terminal dimerisation and a POU-like domain both essential together with the homeodomain for DNA specific recognition. Using the yeast two-hybrid system we searched for proteins interacting with HNF1. We repeatedly obtained cDNA clones encoding DCOH/4-alpha-carbinolamine dehydratase, an enzyme involved in the oxidation of aromatic amino acids that was shown to bind to and stabilise HNF1 dimers. Using the yeast system, we show that the enzymatic activity of DCOH is not essential for HNF1 binding and that the HNF1 dimerisation domain is sufficient for DCOH binding. Furthermore we demonstrate that both proteins co-localise in co-transfected cells.

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.

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.

Analysis of integrated ground squirrel hepatitis virus and flanking host DNA in two hepatocellular carcinomas.

We cloned the integrated ground squirrel hepatitis B virus (GSHV) sequences from two hepatomas showing a single viral insertion. The GSHV inserts shared structural features with integrated DNAs of other hepadnaviruses. Insertional activation of a cellular gene appears unlikely: the integrated GSHV sequences lacked the known viral enhancers and were not expressed in the tumors, and we found no evidence for the presence of a gene at the integration site. Our results, together with those earlier studies, suggest that GSHV does not behave as an extensive insertional mutagen, in sharp contrast with the closely related woodchuck hepatitis virus. GSHV may thus cause carcinogenesis by more indirect mechanisms, as does the human hepatitis B virus.

Expression of the woodchuck N-myc2 retroposon in brain and in liver tumors is driven by a cryptic N-myc promoter.

The woodchuck intronless proto-oncogene N-myc2 was initially discovered as a frequent target site for hepadnavirus integration in hepatocellular carcinoma. N-myc2 possesses characteristics of a functional retroposon derived from the woodchuck N-myc gene. We have investigated the regulatory signals governing N-myc2 expression and found that a short promoter, including a variant TATA box and potential binding sites for several transcription factors, is localized in the N-myc2 sequences homologous to the 5' untranslated region of the second N-myc exon. The corresponding region in the intron-containing woodchuck N-myc gene also exhibited promoter activity in transient transfection assays. The high evolutionary conservation of these sequences in mammalian N-myc genes suggests that they contain a cryptic N-myc promoter which may be unmasked in the particular context provided by the N-myc2 retroposon. Although N-myc2, like the woodchuck N-myc gene, contributes to an extended CpG island and was found constitutively hypomethylated, it presents a highly restricted expression pattern in adult animals. Whereas the intron-containing N-myc gene is expressed at low levels in different tissues, N-myc2 mRNA was detected only in brain tissue, raising questions about the functional significance of the maintenance of a second N-myc gene in the woodchuck genome.

Frequent amplification of c-myc in ground squirrel liver tumors associated with past or ongoing infection with a hepadnavirus.

Persistent infection with hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC) in humans. HCC has also been observed in animals chronically infected with two other hepadnaviruses: ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV). A distinctive feature of WHV is the early onset of woodchuck tumors, which may be correlated with a direct role of the virus as an insertional mutagen of myc genes: c-myc, N-myc, and predominantly the woodchuck N-myc2 retroposon. In the present study, we searched for integrated GSHV DNA and genetic alterations of myc genes in ground squirrel HCCs. Viral integration into host DNA was detected in only 3/14 squirrel tumors and did not result in insertional activation of myc genes, despite the presence of a squirrel locus homologous to the woodchuck N-myc2 gene. This suggests that GSHV may differ from WHV in its reduced ability to induce mutagenic integration events. However, the high frequency of c-myc amplification (6/14) observed in ground squirrel HCCs indicates that myc genes might be preferential effectors in the tumorigenic processes associated with rodent hepadnaviruses, a feature not reported so far in HBV-induced carcinogenesis. Together with previous observations, our results suggest that hepadnaviruses, despite close genetic and biological properties, may use different pathways in the genesis of liver cancer.

Antibody-induced mitogenicity mediated by a chimeric CD2-c-fms receptor.

A chimeric receptor composed of the extracellular domain of the human T-cell antigen CD2 (T11) joined to the membrane-spanning segment and the intracellular tyrosine kinase domain of the human colony-stimulating factor 1 receptor (CSF-1R) was expressed in murine NIH 3T3 fibroblasts. Stimulation of these cells with monoclonal antibodies to CD2 induced phosphorylation of the chimeric glycoprotein on tyrosine, receptor downmodulation, and mitogenesis. In contrast, neither human CSF-1R nor the chimeric receptor was able to function in interleukin-2-dependent murine T cells. In fibroblasts, then, CSF-1 per se is not required for activation of the receptor kinase or for a biological response, whereas in T cells, CSF-1R may be unable to engage the downstream signal transduction machinery.

A new cell surface molecule closely related to mouse class I transplantation antigens.

The "37" H-2 class I mouse gene, located in the Tla complex, is poorly polymorphic and is transcribed in a wide variety of cells and tissues. Using antisera directed against peptides deduced from the gene sequence, we demonstrate that the 37 gene encodes a 45.5-kDa cell surface glycoprotein. This protein, initially identified on the surface of mouse L cells transfected with an overexpressing 37 gene, is present on cells such as splenocytes of various mouse strains except those of A/J mice. Thus, mouse cells may express not only the major transplantation antigens encoded by the K and D region genes, but at least one additional poorly polymorphic class I molecule encoded by the 37 gene.

Deletion of the cytoplasmic region of the CD3 epsilon subunit does not prevent assembly of a functional T-cell receptor.

The T-cell receptor (TCR) is a molecular complex comprised of a clonally restricted, immunoglobulin-like heterodimer (Ti), responsible for specific antigen recognition, and a set of monomorphic polypeptide CD3 subunits, termed gamma, delta, epsilon, zeta, and eta, presumed to be involved in transmembrane signaling events. To investigate the role of the CD3 epsilon subunit in signal transduction, we have transfected a murine hybridoma T-cell line with either wild-type or variant human CD3 epsilon cDNA that encodes a protein lacking 49 of the 55 cytoplasmic amino acid residues. Both wild-type and truncated CD3 epsilon human proteins assemble with endogenous murine CD3/Ti subunits to form functional surface TCRs: Anti-human CD3 epsilon monoclonal antibodies bind exclusively to these chimeric TCRs and trigger interleukin 2 production from the murine cells. Thus, the CD3 epsilon cytoplasmic domain is not required for assembly of the multimeric TCR. Furthermore, it is dispensable for the transduction of a stimulus delivered to the external part of the molecule, suggesting that interaction between the transmembrane and/or external regions of the other TCR chains is a prerequisite for transmembrane signaling.