Biomedical subjects

Arsène Burny

Publications and source records attributed to Arsène Burny.

At least 19 recordsLinked to original sources

PubMed2007

Cell dynamics and immune response to BLV infection: a unifying model.

Bovine Leukemia virus (BLV) is the natural etiological agent of a lymphoproliferative disease in cattle. BLV can also be transmitted experimentally to a related ruminant species, sheep, in which the pathogenesis is more acute. Although both susceptible species develop a strong anti-viral immune response, the virus persists indefinitely throughout life, apparently at a transcriptionally silent stage, at least in a proportion of infected cells. Soon after infection, these humoral and cytotoxic activities very efficiently abolish the viral replicative cycle, permitting only mitotic expansion of provirus-carrying cells. Short term cultures of these infected cells initially indicated that viral expression protects against spontaneous apoptosis, suggesting that leukemia is a process of accumulation of long-lived cells. This conclusion was recently reconsidered following in vivo dynamic studies based on perfusions of nucleoside (bromodeoxyuridine) or fluorescent protein markers (CFSE). In sheep, the turnover rate of infected cells is increased, suggesting that a permanent clearance process is exerted by the immune system. Lymphocyte trafficking from and to the secondary lymphoid organs is a key component in the maintenance of cell homeostasis. The net outcome of the immune selective pressure is that only cells in which the virus is transcriptionally silenced survive and accumulate, ultimately leading to lymphocytosis. Activation of viral and/or cellular expression in this silent reservoir with deacetylase inhibitors causes the collapse of the proviral loads. In other words, modulation of viral expression appears to be curative in lymphocytic sheep, an approach that might also be efficient in patients infected with the related Human T-lymphotropic virus type 1. In summary, a dynamic interplay between BLV and the host immune response modulates a complex equilibrium between (i) viral expression driving (or) favoring proliferation and (ii) viral silencing preventing apoptosis. As conclusion, we propose a hypothetical model unifying all these mechanisms.

Animals↗

PubMed2006

Bortezomib (PS-341, Velcade) increases the efficacy of trastuzumab (Herceptin) in HER-2-positive breast cancer cells in a synergistic manner.

BACKGROUND: Preclinical and clinical studies have shown that the proteasome inhibitor bortezomib (PS341, Velcade) is highly effective when combined with chemotherapeutic agents. The value of trastuzumab (Herceptin) in HER-2-positive (3+ score by immunohistochemistry or fluorescence in situ hybridization positive) breast cancer is also known; however, the response rate is <40% for metastatic breast cancer. These two pharmacologic agents prevent nuclear factor-kappaB (NF-kappaB) activation and induce nuclear accumulation of the cyclin-dependent kinase inhibitor p27(kip1), suggesting that combining bortezomib with trastuzumab could increase trastuzumab efficacy. METHODS: Drug cytotoxicity, both individually and together, and drug effects on p27 localization and NF-kappaB activation were investigated on four breast cancer cell lines: SKBR-3 (HER-2+++), MDA-MB-453 (HER-2++), HER-2-transfected MCF-7 (HER-2+++), and MCF-7 (HER-2-). RESULTS: Bortezomib induced apoptosis in HER-2-positive and HER-2-negative breast cancer cells in a dose- and time-dependent manner. Together, these drugs induced apoptosis of HER-2++/+++ cells at low concentrations, which had no effect when used alone, indicating there was a synergistic effect. Sequential treatment (trastuzumab then bortezomib) induced either necrosis or apoptosis, depending on the trastuzumab preincubation time. Susceptibility to bortezomib alone and the drug combination correlated with NF-kappaB activity and p27 localization. CONCLUSIONS: The addition of bortezomib to trastuzumab increases the effect of trastuzumab in HER-2+++/++ cell lines in a synergistic way. This effect likely results from the ability of these two drugs to target the NF-kappaB and p27 pathways. The potential clinical application of this drug combination is under current evaluation by our group in a phase 1 clinical trial.

Antibodies, Monoclonal↗

PubMed2006

Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep.

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.

Animals↗

PubMed2006

Human T-cell leukemia virus type-1 Tax oncoprotein regulates G-protein signaling.

Human T-cell leukemia virus type-1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and neurological syndromes. HTLV-1 encodes the oncoprotein Tax-1, which modulates viral and cellular gene expression leading to T-cell transformation. Guanine nucleotide-binding proteins (G proteins) and G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins known and are involved in the regulation of most biological functions. Here, we report an interaction between HTLV-1 Tax oncoprotein and the G-protein beta subunit. Interestingly, though the G-protein beta subunit inhibits Tax-mediated viral transcription, Tax-1 perturbs G-protein beta subcellular localization. Functional evidence for these observations was obtained using conditional Tax-1-expressing transformed T-lymphocytes, where Tax expression correlated with activation of the SDF-1/CXCR4 axis. Our data indicated that HTLV-1 developed a strategy based on the activation of the SDF-1/CXCR4 axis in the infected cell; this could have tremendous implications for new therapeutic strategies.

Cells, Cultured↗

PubMed2006

Insights into gene expression changes impacting B-cell transformation: cross-species microarray analysis of bovine leukemia virus tax-responsive genes in ovine B cells.

Large-animal models for leukemia have the potential to aid in the understanding of networks that contribute to oncogenesis. Infection of cattle and sheep with bovine leukemia virus (BLV), a complex retrovirus related to human T-cell leukemia virus type 1 (HTLV-1), is associated with the development of B-cell leukemia. Whereas the natural disease in cattle is characterized by a low tumor incidence, experimental infection of sheep leads to overt leukemia in the majority of infected animals, providing a model for studying the pathogenesis associated with BLV and HTLV-1. Tax(BLV), the major oncoprotein, initiates a cascade of events leading toward malignancy, although the basis of transformation is not fully understood. We have taken a cross-species ovine-to-human microarray approach to identify Tax(BLV)-responsive transcriptional changes in two sets of cultured ovine B cells following retroviral vector-mediated delivery of Tax(BLV). Using cDNA-spotted microarrays comprising 10,336 human genes/expressed sequence tags, we identified a cohort of differentially expressed genes, including genes related to apoptosis, DNA transcription, and repair; proto-oncogenes; cell cycle regulators; transcription factors; small Rho GTPases/GTPase-binding proteins; and previously reported Tax(HTLV-1)-responsive genes. Interestingly, genes known to be associated with human neoplasia, especially B-cell malignancies, were extensively represented. Others were novel or unexpected. The results suggest that Tax(BLV) deregulates a broad network of interrelated pathways rather than a single B-lineage-specific regulatory process. Although cross-species approaches do not permit a comprehensive analysis of gene expression patterns, they can provide initial clues for the functional roles of genes that participate in B-cell transformation and pinpoint molecular targets not identified using other methods in animal models.

Animals↗

PubMed2005

Reduced cell turnover in lymphocytic monkeys infected by human T-lymphotropic virus type 1.

Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is particularly important for rare and aggressive forms of human cancers, such as adult T-cell leukemia. For this purpose, we have measured the death and proliferation rates of the CD4+ T lymphocyte population in squirrel monkeys (Saimiri sciureus) infected by human T-lymphotropic virus type 1 (HTLV-1). The kinetics of in vivo bromodeoxyuridine labeling revealed no modulation of the cell turnover in HTLV-1-infected monkeys with normal CD4 cell counts. In contrast, a substantial decrease in the proliferation rate of the CD4+ T population was observed in lymphocytic monkeys (e.g. characterized by excessive proportions of CD4+ T lymphocytes and by the presence of abnormal flower-like cells). Unexpectedly, onset of HTLV-associated leukemia thus occurs in the absence of increased CD4+ T-cell proliferation. This dynamics significantly differs from the generalized activation of the T-cell turnover induced by other primate lymphotropic viruses like HIV and SIV.

Animals↗

PubMed2005

Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity.

We have previously identified in the pol gene of human immunodeficiency virus type 1 (HIV-1) a new positive transcriptional regulatory element (nt 4481-4982) containing recognition sites for nuclear proteins (sites B, C, D and a GC-box) [C. Van Lint, J. Ghysdael, P. Paras, Jr, A. Burny and E. Verdin (1994) J. Virol. 68, 2632-2648]. In this study, we have further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated in the context of chromatin that Sp1, Sp3, Oct-1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately (i.e. in the absence of the other sites) have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity.

Animals↗

PubMed2005

Valproate activates bovine leukemia virus gene expression, triggers apoptosis, and induces leukemia/lymphoma regression in vivo.

Leukemogenic viruses like human T-lymphotropic virus and bovine leukemia virus (BLV) presumably persist in the host partly by latent integration of the provirus in a fraction of infected cells, leading to accumulative increase in the outgrowth of transformed cells. Furthermore, viral infection also correlates with a blockade of the apoptotic mechanisms concomitant with an apparent latency of the host cell. Conceptually, induction of viral or cellular gene expression could thus also be used as a therapeutic strategy against retroviral-associated leukemia. Here, we provide evidence that valproate, an inhibitor of deacetylases, activates BLV gene expression in transient transfection experiments and in short-term cultures of primary B-lymphocytes. In vivo, valproate injection into newly BLV-inoculated sheep did not abrogate primary infection. However, valproate treatment, in the absence of any other cytotoxic drug, was efficient for leukemia/lymphoma therapy in the sheep model leading to decreased lymphocyte numbers (respectively from 25.6, 35.7, and 46.5 x 10(3) cells per mm3 to 1.0, 10.6, and 24.3 x 10(3) cells per mm3 in three leukemic sheep) and tumor regression (from >700 cm3 to undetectable). The concept of a therapy that targets the expression of viral and cellular genes might be a promising treatment of adult T cell leukemia or tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy, diseases for which no satisfactory treatment exists so far.

Animals↗

PubMed2005

The homeobox protein MSX2 interacts with tax oncoproteins and represses their transactivation activity.

Bovine leukemia virus (BLV) tax is an essential gene involved in the transcriptional activation of viral expression. Tax is also believed to be implicated in leukemogenesis because of its ability to immortalize primary cells in vitro. To gain insight into the molecular pathways mediating the activities of this important gene, we identified cellular proteins interacting with Tax. By means of a two-hybrid approach, we show that Tax specifically interacts with MSX2, a general repressor of gene expression. GST pull-down experiments and co-immunoprecipitation assays further confirmed binding specificity. Furthermore, the N-terminal residues 1-79 of MSX2 are required for binding, whereas the C-terminal residues 201-267 of MSX2 do not play a critical role. Whereas the oncogenic potential of Tax in primary cells was only slightly affected by overexpression of MSX2, the other function of Tax, namely LTR-dependent transcriptional activation, was inhibited by MSX2 in human HeLa and bovine B-lymphoblastoid (BL3) cell lines. This MSX2 repression function can be counteracted by overexpression of transcription factors CREB2 and RAP74. The Tax/MSX2 interplay thus results in repression of viral transcriptional activation possibly acting as a regulatory feedback loop. Importantly, this viral gene silencing is not strictly associated with a concomitant loss of Tax oncogenicity as measured by its ability to immortalize primary cells. And interestingly, MSX2 also interacts with and inhibits the transactivation function of the related Tax1 protein encoded by the Human T-cell leukemia virus type 1 (HTLV-1).

Activating Transcription Factor 4↗

PubMed2005

Transcriptional regulation of the human CD3 gamma gene: the TATA-less CD3 gamma promoter functions via an initiator and contiguous Sp-binding elements.

Growing evidence that the CD3gamma gene is specifically targeted in some T cell diseases focused our attention on the need to identify and characterize the elusive elements involved in CD3gamma transcriptional control. In this study, we show that while the human CD3gamma and CD3delta genes are oriented head-to-head and separated by only 1.6 kb, the CD3gamma gene is transcribed from an independent but weak, lymphoid-specific TATA-less proximal promoter. Using RNA ligase-mediated rapid amplification of cDNA ends, we demonstrate that a cluster of transcription initiation sites is present in the vicinity of the primary core promoter, and the major start site is situated in a classical initiator sequence. A GT box immediately upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these adjacent elements enhanced by the presence of a second GC box 10 nt further upstream. The primary core promoter is limited to a sequence that extends upstream to -15 and contains the initiator and GT box. An identical GT box located approximately 50 nt from the initiator functions as a weak secondary core promoter and likely generates transcripts originating upstream from the +1. Finally, we show that two previously identified NFAT motifs in the proximal promoter positively (NFATgamma(1)) or negatively (NFATgamma(1) and NFATgamma(2)) regulate expression of the human CD3gamma gene by their differential binding of NFATc1 plus NF-kappaB p50 or NFATc2 containing complexes, respectively. These data elucidate some of the mechanisms controlling expression of the CD3gamma gene as a step toward furthering our understanding of how its transcription is targeted in human disease.

Base Sequence↗

PubMed2005

Defective CD3gamma gene transcription is associated with NFATc2 overexpression in the lymphocytic variant of hypereosinophilic syndrome.

OBJECTIVE: Determine the molecular defects underlying the CD3(-)CD4(+) T-cell phenotype and persistence of this clonal population in patients with hypereosinophilic syndrome. PATIENTS AND METHODS: Patients in this study suffer from the lymphocytic variant of hypereosinophilic syndrome distinguished by a CD3(-)CD4(+) T-cell clone that overexpresses Th2 cytokines upon activation and thereby provokes the eosinophilia. Interleukin-2-dependent CD3(-)CD4(+) T-cell lines were derived from patient blood at various disease stages and used to investigate the molecular modifications correlated with their abnormal phenotype. RESULTS: We demonstrate that the CD3(-)CD4(+) T cells, characterized by a clonal TCRbeta gene rearrangement, maintained the same immunophenotype over the 6-year period of our study, during which one patient progressed from premalignant disease to CD3(-)CD4(+) T-cell lymphoma. We show that a specific loss of CD3gamma gene transcripts is responsible for the defect in TCR/CD3 surface expression. In addition, the level of NFATc2 binding to NFAT motifs in the CD3gamma gene promoter was greatly increased in the abnormal T cells. Our studies indicate that CD3gamma promoter activity can be positively influenced by NFATc1 plus NF-kappaB p50 and negatively regulated by NFATc2 containing complexes. We show that in patients' CD3(-)CD4(+) T cells, an increase in nuclear NFATc2 occurs in parallel with a decrease in NFATc1 and NF-kappaB gene expression. CONCLUSION: Hypereosinophilic syndrome joins the growing number of pathological conditions where a defect in surface expression and/or function of the TCR/CD3 complex results from altered regulation of CD3gamma gene expression.

Adult↗

PubMed2005

Exclusive ubiquitination and sumoylation on overlapping lysine residues mediate NF-kappaB activation by the human T-cell leukemia virus tax oncoprotein.

The transcription factor NF-kappaB is critical for the induction of cancer, including adult T-cell leukemia, which is linked to infection by human T-cell leukemia virus type 1 and the expression of its regulatory protein Tax. Although activation of the NF-kappaB pathway by Tax involves its interaction with the regulatory subunit of the IkappaB kinase (IKK) complex, NEMO/IKKgamma, the mechanism by which Tax activates specific cellular genes in the nucleus remains unknown. Here, we demonstrate that the attachment of SUMO-1 to Tax regulates its localization in nuclear bodies and the recruitment of both the RelA subunit of NF-kappaB and free IKKgamma in these nuclear structures. However, this sumoylation step is not sufficient for the activation of the NF-kappaB pathway by Tax. This activity requires the prior ubiquitination and colocalization of ubiquitinated Tax with IKK complexes in the cytoplasm and the subsequent migration of the RelA subunit of NF-kappaB to the nucleus. Thus, the ubiquitination and sumoylation of Tax function in concert to result in the migration of RelA to the nucleus and its accumulation with IKKgamma in nuclear bodies for activation of gene expression. These modifications may result in targets for the treatment of adult T-cell leukemia.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Deacetylase inhibitors and the viral transactivator TaxBLV synergistically activate bovine leukemia virus gene expression via a cAMP-responsive element- and cAMP-responsive element-binding protein-dependent mechanism.

Efficient bovine leukemia virus (BLV) transcription requires the virus-encoded transactivator Tax(BLV), which acts through three Tax(BLV)-responsive elements located in the 5' long terminal repeat. It has been proposed that the binding of the CRE-binding protein (CREB) and the activating transcription factor (ATF) to the three imperfect cAMP-responsive elements (CREs) located in each Tax(BLV)-responsive element mediates Tax(BLV) transactivation. Here we demonstrated that deacetylase inhibitors (HDACis) synergistically enhanced the transcriptional activation of the BLV promoter by Tax(BLV) in a CRE-dependent manner. Tax(BLV) was acetylated in vivo at its N(alpha) terminus but not at internal lysine residues. Rather, HDACi potentiation of Tax(BLV) transactivation was mediated by an HDACi indirect action that requires new protein synthesis. Mechanistically, using a dominant-negative form of CREB, we showed that Tax(BLV) and HDACi synergistically activated BLV gene expression via a CREB-dependent mechanism. Moreover, electrophoretic mobility shift assay and Western blot experiments revealed that HDACi increased the in vitro DNA binding activity of CREB/ATF but did not alter CREB/ATF intranuclear presence. Remarkably, chromatin immunoprecipitation assays demonstrated that HDACi treatment increased the level of CREB bound to the BLV promoter in vivo. Our results together suggest that an increase in CREB/ATF occupancy of the viral CREs in response to HDACi potentiates Tax(BLV) transactivation of the BLV promoter.

Animals↗

PubMed2004

Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein-based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia.

PURPOSE: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). METHODS: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. RESULTS: Some patients had preexisting systemic IFN-gamma CD4+ (1/10) and CD8+ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN-gamma CD8+ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)-directed CD4+ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. CONCLUSIONS: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients.

Adult↗

PubMed2004

Gene activation and gene silencing: a subtle equilibrium.

The genetic make-up of a cell resides entirely in its DNA. Now that the nucleotide sequence of several genomes has been determined, the major challenging problem is to understand how cell differentiation, proliferation or death are controlled. Major steps include analysis of the determinants of the cell cycle, the unravelling of RNAs and proteins involved in the control of gene expression and the dissection of the protein-destruction machinery. The successive steps to be considered are transcription of RNA on the DNA template, mRNA stabilization or degradation, and mRNA translation and protein localization in the right cell compartment. Gene expression or gene silencing is the result of many DNA-RNA-protein interactions and chromatin is among the key regulators of gene expression. Open chromatin (euchromatin) allows expression of the DNA message. This chromatin structure is generally characterized by the presence on the gene promoters of transcription complexes associated with histone acetyltransferases (HATs). On the contrary, closed chromatin (heterochromatin) is poorly acetylated and more condensed. It contains histone deacetylases (HDACs), potentially associated with DNA methyltransferases (DNMTs). DNMT activity leads to methylation and silencing of the DNA. Thus, a major problem in the field of gene regulation resides in understanding chromatin structure at each promoter, a formidable task for the years to come.

Acetylation↗

PubMed2004

Overlapping CRE and E box motifs in the enhancer sequences of the bovine leukemia virus 5' long terminal repeat are critical for basal and acetylation-dependent transcriptional activity of the viral promoter: implications for viral latency.

Bovine leukemia virus (BLV) infection is characterized by viral latency in a large proportion of cells containing an integrated provirus. In this study, we postulated that mechanisms directing the recruitment of deacetylases to the BLV 5' long terminal repeat (LTR) could explain the transcriptional repression of viral expression in vivo. Accordingly, we showed that BLV promoter activity was induced by several deacetylase inhibitors (such as trichostatin A [TSA]) in the context of episomal LTR constructs and in the context of an integrated BLV provirus. Moreover, treatment of BLV-infected cells with TSA increased H4 acetylation at the viral promoter, showing a close correlation between the level of histone acetylation and transcriptional activation of the BLV LTR. Among the known cis-regulatory DNA elements located in the 5' LTR, three E box motifs overlapping cyclic AMP responsive elements (CREs) in U3 were shown to be involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutations of these three E box motifs markedly reduced the inducibility of the BLV promoter by TSA. E boxes are susceptible to recognition by transcriptional repressors such as Max-Mad-mSin3 complexes that repress transcription by recruiting deacetylases. However, our in vitro binding studies failed to reveal the presence of Mad-Max proteins in the BLV LTR E box-specific complexes. Remarkably, TSA increased the occupancy of the CREs by CREB/ATF. Therefore, we postulated that the E box-specific complexes exerted their negative cooperative effect on BLV transcription by steric hindrance with the activators CREB/ATF and/or their transcriptional coactivators possessing acetyltransferase activities. Our results thus suggest that the overlapping CRE and E box elements in the BLV LTR were selected during evolution as a novel strategy for BLV to allow better silencing of viral transcription and to escape from the host immune response.

5' Untranslated Regions↗

PubMed2003

Interaction of retroviral Tax oncoproteins with tristetraprolin and regulation of tumor necrosis factor-alpha expression.

BACKGROUND: The Tax oncoproteins are transcriptional regulators of viral expression involved in pathogenesis induced by complex leukemogenic retroviruses (or delta-retroviruses, i.e., primate T-cell leukemia viruses and bovine leukemia virus). To better understand the molecular pathways leading to cell transformation, we aimed to identify cellular proteins interacting with Tax. METHODS: We used a yeast two-hybrid system to identify interacting cellular proteins. Interactions between Tax and candidate interacting cellular proteins were confirmed by glutathione S-transferase (GST) pulldown assays, co-immunoprecipitation, and confocal microscopy. Functional interactions between Tax and one interacting protein, tristetraprolin (TTP), were assessed by analyzing the expression of tumor necrosis factor-alpha (TNF-alpha), which is regulated by TTP, in mammalian cells (HeLa, D17, HEK 293, and RAW 264.7) transiently transfected with combinations of intact and mutant Tax and TTP. RESULTS: We obtained seven interacting cellular proteins, of which one, TTP, was further characterized. Tax and TTP were found to interact specifically through their respective carboxyl-terminal domains. The proteins colocalized in the cytoplasm in a region surrounding the nucleus of HeLa cells. Furthermore, coexpression of Tax was associated with nuclear accumulation of TTP. TTP is an immediate-early protein that inhibits expression of TNF-alpha at the post-transcriptional level. Expression of Tax reverted this inhibition, both in transient transfection experiments and in stably transfected macrophage cell lines. CONCLUSION: Tax, through its interactions with the TTP repressor, indirectly increases TNF-alpha expression. This observation is of importance for the cell transformation process induced by leukemogenic retroviruses, because TNF-alpha overexpression plays a central role in pathogenesis.

Animals↗

PubMed2003

Expression in a RabGAP yeast mutant of two human homologues, one of which is an oncogene.

The yeast proteins Msb3p and Msb4p are two Ypt/Rab-specific GTPase-activating proteins (GAPs) involved in cell growth polarization. Both proteins share with a wide variety of other proteins the highly conserved TBC domain forming the catalytically active RabGAP domain. In particular, Msb3p and Msb4p are similar to the human proteins oncTre210p (the 786-amino-acid product of the human Tre2 oncogene, implicated in Ewing's sarcoma) and RN-tre (a Rab5-GAP controlling endocytosis of the EGFR). To further understand the biochemical function of Tre2 oncogene, we expressed its cDNA and, as a control, the RN-tre cDNA, in an msb3 msb4 double mutant yeast strain. Complementation data show that RN-tre can, unlike Tre2, replace the function of the MSB3 and MSB4 genes. As two highly conserved amino acids, including the catalytic arginine, are mutated in the oncTre210p TBC domain, we restored these two amino acids and expressed the modified Tre2 cDNA in the yeast mutant.

Adaptor Proteins, Signal Transducing↗