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1. Consistent high concentration of the viral microRNA BART17 in plasma samples from nasopharyngeal carcinoma patients - evidence of non-exosomal transport

Author: Ana Barat, Arij Ben Chaaben, François-Régis Ferrand, Aurore Gelin, Charles-Henry Gattolliat, Claire Gourzones, Fethi Guemira, Corinne Amiel, Joël Guigay, Anne-Sophie Jimenez-Pailhes, Philippe Lang, Jihène Klibi, Maryse Guerin, Véronique Schneider, Benjamin Verillaud, Philippe Busson, Interactions moléculaires et cancer ( IMC (UMR 8126) ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Centre National de la Recherche Scientifique ( CNRS ), École du Val de Grâce ( EVDG ), Service de Santé des Armées, Service de virologie, Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Tenon [APHP], Service ORL, Assistance publique - Hôpitaux de Paris (AP-HP)-Hôpital Lariboisière, Dyslipidémies, inflammation et athérosclérose dans les maladies métaboliques, Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Clinical Biology Department, Institut Salah Azaiz, Département de cancérologie cervico-faciale [Gustave Roussy] ( CCF ), Institut Gustave Roussy ( IGR ), Service de Radiothérapie [CHU Pitié-Salpêtrière], Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Pitié-Salpêtrière [APHP], This study was supported by grants from the Gustave Roussy Foundation (Head and Neck tumors - 2011), the Institut National du Cancer (INCa-DHOS translational grant) and the Ligue Nationale contre le Cancer (comité du Val de Marne). CG was supported by the Association pour la Recherche sur le Cancer., BMC, Ed., Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), École du Val de Grâce (EVDG), CHU Tenon [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Lariboisière-Fernand-Widal [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Département de cancérologie cervico-faciale [Gustave Roussy] (CCF), Institut Gustave Roussy (IGR), Service d'Oncologie Radiothérapie [CHU Pitié Salpétrière], CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)
Subjects: Male, Herpesvirus 4, Human, medicine.disease_cause, Exosomes, Plasma, 0302 clinical medicine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, 0303 health sciences, microRNA, Middle Aged, 3. Good health, [ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Real-time polymerase chain reaction, Infectious Diseases, 030220 oncology & carcinogenesis, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, RNA, Viral, Female, Adult, Lipoproteins, Nasopharyngeal neoplasm, miR-BART, Biology, Real-Time Polymerase Chain Reaction, Exosome, 03 medical and health sciences, Virology, Head and Neck carcinomas, medicine, Carcinoma, Nasopharyngeal carcinoma, Humans, Epstein-Barr virus, 030304 developmental biology, Aged, Research, Biological Transport, Nasopharyngeal Neoplasms, Biomarker, medicine.disease, Epstein–Barr virus, Head and neck squamous-cell carcinoma, Microvesicles, MicroRNAs, DNA, Viral, Biomarkers, DNA load
Abstract: Background Because latent Epstein Barr (EBV)-infection is a specific characteristic of malignant nasopharyngeal carcinoma (NPC), various molecules of viral origin are obvious candidate biomarkers in this disease. In a previous study, we could show in a few clinical samples that it was possible to detect a category of EBV microRNAs called miR-BARTs in the plasma of at least a fraction of NPC patients. The first aim of the present study was to investigate the status of circulating miR-BART17-5p (one of the miR-BARTs hereafter called miR-BART17) and EBV DNA in a larger series of NPC plasma samples. The second aim was to determine whether or not circulating miR-BART17 was carried by plasma exosomes. Patients and methods Plasma samples were collected from 26 NPC patients and 10 control donors, including 9 patients with non-NPC Head and Neck squamous cell carcinoma and one healthy EBV carrier. Concentrations of miR-BART17 and two cellular microRNAs (hsa-miR-16 and -146a) were assessed by real-time quantitative PCR with spike-in normalization and absolute quantification. In addition, for 2 patients, exosome distributions of miR-BART17 and miR-16 were investigated following plasma lipoprotein fractionation by isopycnic density gradient ultrcentrifugation. Results The miR-BART17 was significantly more abundant in plasma samples from NPC patients compared to non-NPC donors. Above a threshold of 506 copies/mL, detection of miR-BART17 was highly specific for NPC patients (ROC curve analysis: AUC=0.87 with true positive rate = 0.77, false positive rate = 0.10). In this relatively small series, the concentration of plasma miR-BART17 and the plasma EBV DNA load were not correlated. When plasma samples were fractionated, miR-BART17 co-purified with a protein-rich fraction but not with exosomes. Conclusions Detection of high concentrations of plasma miR-BART17 is consistent in NPC patients. This parameter is, at least in part, independent of the viral DNA load. Circulating miR-BART17 does not co-purify with exosomes.
Published: 2013

2. Kinome expression profiling to target new therapeutic avenues in multiple myeloma

Author: Jérôme Moreaux, Claire Gourzones, Nicolas Robert, Anke Maes, Michel Jourdan, Philippe Pasero, Elke De Bruyne, Guillaume Cartron, Angelique Bruyer, Anja Seckinger, Alboukadel Kassambara, Laure Vincent, Dirk Hose, Hugues de Boussac, Génétique, Reproduction et Développement (GReD ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020]), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Cellules souches normales et cancéreuses, Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Institut de recherche en biothérapie (IRB), Université Montpellier 1 (UM1)-Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Vrije Universiteit Brussel (VUB), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Heidelberg University Hospital [Heidelberg], Dynamique des interactions membranaires normales et pathologiques (DIMNP), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), ANR-17-CE15-0024,TIE-Skip,Impact des immunoglobulines tronquées produites par saut d'exon dans les plasmocytes : vers des approches thérapeutiques utilisant des oligonucléotides antisens(2017), Vrije Universiteit Brussel, Clinical sciences, Faculty of Medicine and Pharmacy, Basic (bio-) Medical Sciences, and Hematology
Subjects: Melphalan, CHK1, SRPK1, Cell Cycle Proteins, [SDV.CAN]Life Sciences [q-bio]/Cancer, Protein Serine-Threonine Kinases, Article, Plasma Cell Disorders, 03 medical and health sciences, 0302 clinical medicine, immune system diseases, hemic and lymphatic diseases, MELK, medicine, Humans, Immunologic Factors, Kinome, CHEK1, Multiple myeloma (MM), Lenalidomide, Multiple myeloma, 030304 developmental biology, 0303 health sciences, Kinase, business.industry, MPS1/TTK), [SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology, Hematology, medicine.disease, 3. Good health, Gene expression profiling, 030220 oncology & carcinogenesis, Cancer research, CDC7-DBF4, PLK4, Neoplasm Recurrence, Local, Multiple Myeloma, business, medicine.drug, Protein kinas-es (PBK
Abstract: International audience; Multiple myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Kinases inhibitors are widely used and their efficiency for the treatment of cancers has been demonstrated. Here, in order to identify kinases of potential therapeutic interest for the treatment of MM, we investigated the prognostic impact of the kinome expression profile in large cohorts of patients. We identified 36 kinome-related genes significantly linked with a prognostic value to MM, and built a kinome index based on their expression. The Kinome Index (KI) is linked to prognosis, proliferation, differentiation, and relapse in MM. We then tested inhibitors targeting seven of the identified protein kinas-es (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK) in human myeloma cell lines. All tested inhibitors significantly reduced the viability of myeloma cell lines, and we confirmed the potential clinical interest of three of them on primary myeloma cells from patients. In addition, we demonstrated their ability to potentialize the toxicity of conventional treatments, including Melphalan and Lenalidomide. This highlights their potential beneficial effect in myeloma therapy. Three kinases inhibitors (CHK1i, MELKi and PBKi) overcome resistance to Lenalidomide, while CHK1, PBK and DBF4 inhibitors re-sensitize Melphalan resistant cell line to this conventional therapeutic agent. Altogether, we demonstrate that kinase inhibitors could be of therapeutic interest especially in high-risk myeloma patients defined by the KI. CHEK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK inhibitors could represent new treatment options either alone or in combination with Melphalan or IMiD for refractory/relapsing myeloma patients.
Published: 2020

3. GALNT9 Gene Expression Is a Prognostic Marker in Neuroblastoma Patients

Author: Charles-Henry Gattolliat, Sétha Douc-Rasy, Laura Capandeguy, Nora Berois, Enrique Barrios, Dominique Valteau-Couanet, Eduardo Osinaga, Jean Bénard, Tumor Immunology and Glycobiology / Glicobiología e Inmunobiología Tumoral [Montevideo], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Departamento de Metodos Cuantitativos, Universidad de la República [Montevideo] (UCUR), Département de Pédiatrie, Institut Gustave Roussy (IGR), Département de biologie et pathologie médicales [Gustave Roussy], Departamento de inmunobiologia, Comision Honoraria de Lucha Contra el Cancer, Montevideo, Uruguay, Programa Grupos de Investigacion CSIC, Montevideo, Uruguay, and Societe Française des Cancers de l'Enfant, La Ligue contre le Cancer Comite Oise., and Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Cell Line, Tumor, [SDV]Life Sciences [q-bio], Clinical Biochemistry, MESH: N-Acetylgalactosaminyltransferases, Biology, Bioinformatics, MESH: Prognosis, Neuroblastoma, 03 medical and health sciences, 0302 clinical medicine, Cell Line, Tumor, MESH: Reverse Transcriptase Polymerase Chain Reaction, Gene expression, Biomarkers, Tumor, medicine, Humans, Gene family, Gene, 030304 developmental biology, 0303 health sciences, MESH: Humans, Reverse Transcriptase Polymerase Chain Reaction, Proportional hazards model, Biochemistry (medical), Infant, Prognosis, medicine.disease, MESH: Infant, MESH: Neuroblastoma, Pediatric cancer, 3. Good health, medicine.anatomical_structure, Tumor progression, 030220 oncology & carcinogenesis, MESH: Tumor Markers, Biological, Cancer research, N-Acetylgalactosaminyltransferases, Bone marrow
Abstract: BACKGROUND The enzymes encoded by the GALNT [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GALNAC-T)] gene family catalyze the first step of O-glycosylation. Little is known about the link between expression of the genes encoding GALNAC-T enzymes and tumor progression in neuroblastoma, a pediatric cancer that can be classified as either low or high risk. We assessed the expression of genes in the GALNT family in a large cohort of neuroblastoma patients and characterized members of this family that might be used as new prognostic markers. METHODS Reverse-transcription PCR analysis of 14 GALNT genes with a panel of neuroblastoma cell lines identified the GALNT9 gene as playing a potential role in disease progression. We used the log-rank test and the multivariable Cox proportional hazards model with a cohort of 122 neuroblastoma patients to analyze the relationship between GALNT9 expression and overall survival or disease-free survival. RESULTS In the high-risk neuroblastoma experimental model IGR-N-91, GALNT9 expression was present in neuroblasts derived from primary tumors but not in neuroblasts from metastatic bone marrow. Moreover, GALNT9 in neuroblastoma cell lines was expressed in substrate adherent (S)-type cell lines but not in neuronal (N)-type lines. In the tumor cohort, GALNT9 expression was associated with high overall survival, independent of the standard risk-stratification covariates. GALNT9 expression was significantly associated with disease-free survival for patients currently classified as at low risk (P < 0.0007). CONCLUSIONS GALNT9 expression correlates with both improved overall survival in low- and high-risk groups and an improved clinical outcome (overall and disease-free survival) in low-risk patients. Thus, the GALNT9 expression may be a prognostic marker for personalized therapy.
Published: 2013

4. Neurotrophin-3 production promotes human neuroblastoma cell survival by inhibiting TrkC-induced apoptosis

Author: Céline Delloye-Bourgeois, Marie-Anne Raquin, Jean-Guy Delcros, Valérie Combaret, Servane Tauszig-Delamasure, Jimena Bouzas-Rodriguez, Raphael Rousseau, Patrick Mehlen, Jean Bénard, Jorge Ruben Cabrera, Gabriel Ichim, Apoptose Cancer et Développement, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), Département de Pédiatrie, Institut Gustave Roussy (IGR), Centre Léon Bérard [Lyon], Laboratoire d'Oncologie Moléculaire, Département de biologie et pathologie médicales [Gustave Roussy], Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Apoptosis, Cancer, and Development Laboratory, Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)
Subjects: MESH: Neoplasm Proteins, Apoptosis, Dependence receptor, Tropomyosin receptor kinase C, Mice, Neuroblastoma, 0302 clinical medicine, Neurotrophin 3, MESH: Up-Regulation, MESH: Animals, Receptor, 0303 health sciences, biology, MESH: Receptor, trkC, MESH: Chickens, MESH: Gene Expression Regulation, Neoplastic, General Medicine, Neoplasm Proteins, Up-Regulation, Gene Expression Regulation, Neoplastic, Autocrine Communication, MESH: Cell Survival, Gene Knockdown Techniques, 030220 oncology & carcinogenesis, Tyrosine kinase, Research Article, Neurotrophin, Programmed cell death, MESH: Cell Line, Tumor, animal structures, Cell Survival, Transplantation, Heterologous, Mice, Nude, Neurotrophin-3, 03 medical and health sciences, Cell Line, Tumor, MESH: Mice, Nude, Animals, Humans, Receptor, trkC, Autocrine signalling, MESH: Transplantation, Heterologous, MESH: Mice, 030304 developmental biology, MESH: Humans, MESH: Apoptosis, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Neuroblastoma, MESH: Gene Knockdown Techniques, MESH: Neurotrophin 3, nervous system, biology.protein, Cancer research, MESH: Autocrine Communication, Chickens, Neoplasm Transplantation, MESH: Neoplasm Transplantation
Abstract: International audience; Tropomyosin-related kinase receptor C (TrkC) is a neurotrophin receptor with tyrosine kinase activity that was expected to be oncogenic. However, it has several characteristics of a tumor suppressor: its expression in tumors has often been associated with good prognosis; and it was recently demonstrated to be a dependence receptor, transducing different positive signals in the presence of ligand but inducing apoptosis in the absence of ligand. Here we show that the TrkC ligand neurotrophin-3 (NT-3) is upregulated in a large fraction of aggressive human neuroblastomas (NBs) and that it blocks TrkC-induced apoptosis of human NB cell lines, consistent with the idea that TrkC is a dependence receptor. Functionally, both siRNA knockdown of NT-3 expression and incubation with a TrkC-specific blocking antibody triggered apoptosis in human NB cell lines. Importantly, disruption of the NT-3 autocrine loop in malignant human neuroblasts triggered in vitro NB cell death and inhibited tumor growth and metastasis in both a chick and a mouse xenograft model. Thus, we believe that our data suggest that NT-3/TrkC disruption is a putative alternative targeted therapeutic strategy for the treatment of NB.
Published: 2010

5. A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed RNA to Vpr-bridged DNA

Author: Robert J. Gorelick, Eric Le Cam, Vincent Parissi, Tobias Restle, Sébastien Lyonnais, José M. Gatell, Jean-François Mouscadet, Gilles Mirambeau, Serge Bouaziz, Fatima Heniche-Boukhalfa, Laboratoire d'Electronique Moléculaire (LEM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), School of Life Sciences, Carleton University, Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Fondamentale et Pathogénicité (MFP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Bouaziz, Serge, AIDS Vaccine Program, NCI at Frederick, Unité de Pharmacologie Chimique et Génétique (UPCG - UMR_S 640/UMR 8151), Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut des sciences du Médicament -Toxicologie - Chimie - Environnement (IFR71), Institut de Recherche pour le Développement (IRD)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut des sciences du Médicament -Toxicologie - Chimie - Environnement (IFR71), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)
Subjects: Cancer Research, [SDV]Life Sciences [q-bio], viruses, MESH: Reverse Transcription, HIV Integrase, MESH: vpr Gene Products, Human Immunodeficiency Virus, gag Gene Products, Human Immunodeficiency Virus, Pre-integration complex, MESH: HIV-1, chemistry.chemical_compound, Nuclear pore, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030302 biochemistry & molecular biology, vpr Gene Products, Human Immunodeficiency Virus, MESH: gag Gene Products, Human Immunodeficiency Virus, HIV Reverse Transcriptase, 3. Good health, Integrase, Cell biology, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: RNA, Viral, RNA, Viral, MESH: Genome, Viral, Base pair, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: HIV Reverse Transcriptase, Genome, Viral, Biology, Article, 03 medical and health sciences, Virology, DNA Packaging, Humans, MESH: DNA Packaging, 030304 developmental biology, MESH: Humans, RNA, Reverse Transcription, Molecular biology, Reverse transcriptase, Nucleoprotein, MESH: DNA, Viral, chemistry, DNA, Viral, biology.protein, HIV-1, MESH: HIV Integrase, DNA
Abstract: International audience; HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing pre-integration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin.
Published: 2013

6. Differences in Transcription Patterns between Induced Pluripotent Stem Cells Produced from the Same Germ Layer Are Erased upon Differentiation

Author: Iryna Pirozhkova, Thomas Robert, Jörg Tost, Yegor S. Vassetzky, Gilles Carnac, Marc Lipinski, Elena S. Kim, Ana Barat, Dalila Laoudj-Chenivesse, Florence Busato, Petr Dmitriev, Justine Guegan, Chrystèle Bilhou-Nabera, Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Interactions moléculaires et cancer (IMC (UMR 8126)), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Laboratoire de Biologie Intégrative des Modèles Marins (LBI2M), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Station biologique de Roscoff (SBR), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Centre National de Génotypage (CNG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre d'Etude du Polymorphisme Humain (CEPH), Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Fondation Jean Dausset-Université de Paris (UP), Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Fondation Jean Dausset-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Passerieux, Emilie
Subjects: Cellular differentiation, Embryoid body, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Muscle Development, Myoblasts, Mice, 0302 clinical medicine, Molecular cell biology, Transduction, Genetic, Induced pluripotent stem cell, Cells, Cultured, Oligonucleotide Array Sequence Analysis, 0303 health sciences, Multidisciplinary, Stem Cells, Cell Differentiation, Medicine, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Stem cell, Cellular Types, Germ Layers, Research Article, Homeobox protein NANOG, KOSR, Science, Induced Pluripotent Stem Cells, DNA transcription, Kruppel-Like Transcription Factors, Biology, Proto-Oncogene Proteins c-myc, 03 medical and health sciences, Kruppel-Like Factor 4, SOX2, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Animals, Humans, Embryoid Bodies, Embryonic Stem Cells, 030304 developmental biology, Gene Expression Profiling, SOXB1 Transcription Factors, Mesenchymal Stem Cells, Fibroblasts, Molecular biology, Embryonic stem cell, Retroviridae, Gene expression, Octamer Transcription Factor-3, 030217 neurology & neurosurgery, Developmental Biology
Abstract: International audience; Little is known about differences between induced pluripotent stem cells produced from tissues originating from the same germ layer. We have generated human myoblast-derived iPS cells by retroviral transduction of human primary myoblasts with the OCT3/4, SOX2, KLF4 and MYC coding sequences and compared them to iPS produced from human primary fibroblasts. When cultivated in vitro, these iPS cells proved similar to human embryonic stem cells in terms of morphology, expression of embryonic stemness markers and gene promoter methylation patterns. Embryonic bodies were derived that expressed endodermal, mesodermal as well as ectodermal markers. A comparative analysis of transcription patterns revealed significant differences in the gene expression pattern between myoblast-and fibroblast-derived iPS cells. However, these differences were reduced in the mesenchymal stem cells derived from the two iPS cell types were compared. Citation: Pirozhkova I, Barat A, Dmitriev P, Kim E, Robert T, et al. (2013) Differences in Transcription Patterns between Induced Pluripotent Stem Cells Produced from the Same Germ Layer Are Erased upon Differentiation.
Published: 2013

7. Defective Regulation of MicroRNA Target Genes in Myoblasts from Facioscapulohumeral Dystrophy Patients

Author: Thomas Robert, Andrei Petrov, Philippe Dessen, Ana Barat, Eugénie Ansseau, Luiza Stankevicins, Petr Dmitriev, Ahmed Turki, Gilles Carnac, Alexandra Belayew, Yegor S. Vassetzky, Emmanuel Labourer, Dalila Laoudj-Chenivesse, Marc Lipinski, Vladimir Lazar, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Laboratoire de Biologie Intégrative des Modèles Marins (LBI2M), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Station biologique de Roscoff (SBR), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Intégrée en cancérologie à Villejuif (IRCIV), Université de Mons (UMons), Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Passerieux, Emilie, and Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, [SDV.MHEP.PHY] Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Cellular differentiation, Muscle Development, Biochemistry, Transcriptome, 0302 clinical medicine, Facioscapulohumeral muscular dystrophy, Muscular Dystrophy, Muscular dystrophy, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, Genetics, 0303 health sciences, Facioscapulohumeral Dystrophy, Molecular Bases of Disease, Cell Differentiation, MicroRNA, Middle Aged, Muscular Dystrophy, Facioscapulohumeral, Up-Regulation, Genetic Diseases, embryonic structures, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Female, Transcription, musculoskeletal diseases, Adult, congenital, hereditary, and neonatal diseases and abnormalities, Myoblasts, Skeletal, Down-Regulation, Biology, 03 medical and health sciences, Young Adult, DUX4, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Humans, Transcription Regulation, Molecular Biology, Gene, 030304 developmental biology, Homeodomain Proteins, Gene Expression Profiling, Cell Biology, medicine.disease, nervous system diseases, Gene expression profiling, MicroRNAs, Homeobox, 030217 neurology & neurosurgery
Abstract: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder linked to the deletion of an integral number of 3.3-kb-long macrosatellite repeats (D4Z4) within the subtelomeric region of chromosome 4q. Most genes identified in this region are overexpressed in FSHD myoblasts, including the double homeobox genes DUX4 and DUX4c. We have carried out a simultaneous miRNome/transcriptome analysis of FSHD and control primary myoblasts. Of 365 microRNAs (miRNAs) analyzed in this study, 29 were found to be differentially expressed between FSHD and normal myoblasts. Twenty-one microRNAs (miR-1, miR-7, miR-15a, miR-22, miR-30e, miR-32, miR-107, miR-133a, miR-133b, miR-139, miR-152, miR-206, miR-223, miR-302b, miR-331, miR-362, miR-365, miR-382, miR-496, miR-532, miR-654, and miR-660) were up-regulated, and eight were down-regulated (miR-15b, miR-20b, miR-21, miR-25, miR-100, miR-155, miR-345, and miR-594). Twelve of the miRNAs up-regulated in FHSD were also up-regulated in the cells ectopically expressing DUX4c, suggesting that this gene could regulate miRNA gene transcription. The myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206 were highly expressed in FSHD myoblasts, which nonetheless did not prematurely enter myogenic differentiation. This could be accounted for by the fact that in FSHD myoblasts, functionally important target genes, including cell cycle, DNA damage, and ubiquitination-related genes, escape myogenic microRNA-induced repression.
Published: 2013

8. Functional muscle impairment in facioscapulohumeral muscular dystrophy is correlated with oxidative stress and mitochondrial dysfunction

Author: Sébastien Bommart, Emilie Passerieux, Ahmed Turki, Raul Juntas Morales, Gilles Carnac, Alexandra Belayew, Dalila Laoudj-Chenivesse, Sylvia Pietri, Karen Lambert, Joël Pincemail, Fabien Pillard, Gérald Hugon, Maurice Hayot, Eric Raynaud de Mauverger, Jacques Mercier, Yegor S. Vassetzky, Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Centre Hospitalier Régional Universitaire de Toulouse (CHRU Toulouse), CHU, Liège, Institut de Chimie Radicalaire (ICR), Aix Marseille Université (AMU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Service de Biologie Moléculaire, Université de Mons, Belgique, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Université Montpellier 1 (UM1), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), and Pietri, Sylvia
Subjects: Male, MnSOD, Cu-SOD, DUX4, [SDV]Life Sciences [q-bio], Glutathione reductase, Mitochondria dysfunction, thiobarbituric acid-reactive substances, medicine.disease_cause, Biochemistry, Antioxidants, chemistry.chemical_compound, 0302 clinical medicine, Facioscapulohumeral muscular dystrophy, endurance limit time, double homebox 4 gene, Muscular dystrophy, ComputingMilieux_MISCELLANEOUS, 2 minute walking test n Correspondence to: INSERM, reactive oxygen species, 0303 health sciences, 2-MWT, Chemistry, T Lim, Autosomal dominant trait, ROS, oxidized glutathione, Muscular Dystrophy, Facioscapulohumeral, Mitochondria, [SDV] Life Sciences [q-bio], GR, medicine.anatomical_structure, Radical oxygen species, MVC, copper-zinc-dependent superoxide dismutase, Female, GSSG, musculoskeletal diseases, Adult, medicine.medical_specialty, 4-hydroxy-2-nonenal, TBARS, Lipofuscin, Facioscapulohumeral muscular dystrophy (FSHD), 03 medical and health sciences, Physiology (medical), Internal medicine, medicine, GSH, Humans, Facioscapulohumeral muscular dystrophy (FSHD) 4-Hydroxy-2-nonenal Thiobarbituric acid-reactive substances Lipofuscin Oxidative stress Protein oxidation Radical oxygen species Mitochondria dysfunction Antioxidants Abbreviations: FSHD, reduced glutathione, glutathione reductase, 030304 developmental biology, manganese-dependent superoxide dismutase, Skeletal muscle, facioscapulohumeral dystrophy, Glutathione, Bâtiment Crastes de Paulet, medicine.disease, Endocrinology, HNE, Oxidative stress, maximal voluntary contraction, Protein oxidation, 030217 neurology & neurosurgery, U1046
Abstract: Facioscapulohumeral muscular dystrophy (FSHD), the most frequent muscular dystrophy, is an autosomal dominant disease. In most individuals with FSHD, symptoms are restricted to muscles of the face, arms, legs, and trunk. FSHD is genetically linked to contractions of the D4Z4 repeat array causing activation of several genes. One of these maps in the repeat itself and expresses the DUX4 (the double homeobox 4) transcription factor causing a gene deregulation cascade. In addition, analyses of the RNA or protein expression profiles in muscle have indicated deregulations in the oxidative stress response. Since oxidative stress affects peripheral muscle function, we investigated mitochondrial function and oxidative stress in skeletal muscle biopsies and blood samples from patients with FSHD and age-matched healthy controls, and evaluated their association with physical performances. We show that specifically, oxidative stress (lipid peroxidation and protein carbonylation), oxidative damage (lipofuscin accumulation), and antioxidant enzymes (catalase, copper-zinc-dependent superoxide dismutase, and glutathione reductase) were higher in FSHD than in control muscles. FSHD muscles also presented abnormal mitochondrial function (decreased cytochrome c oxidase activity and reduced ATP synthesis). In addition, the ratio between reduced (GSH) and oxidized glutathione (GSSG) was strongly decreased in all FSHD blood samples as a consequence of GSSG accumulation. Patients with FSHD also had reduced systemic antioxidative response molecules, such as low levels of zinc (a SOD cofactor), selenium (a GPx cofactor involved in the elimination of lipid peroxides), and vitamin C. Half of them had a low ratio of gamma/alpha tocopherol and higher ferritin concentrations. Both systemic oxidative stress and mitochondrial dysfunction were correlated with functional muscle impairment. Mitochondrial ATP production was significantly correlated with both quadriceps endurance (T(LimQ)) and maximal voluntary contraction (MVC(Q)) values (rho=0.79, P=0.003; rho=0.62, P=0.05, respectively). The plasma concentration of oxidized glutathione was negatively correlated with the T(LimQ), MVC(Q) values, and the 2-min walk distance (MWT) values (rho=-0.60, P=0.03; rho=-0.56, P=0.04; rho=-0.93, P0.0001, respectively). Our data characterized oxidative stress in patients with FSHD and demonstrated a correlation with their peripheral skeletal muscle dysfunction. They suggest that antioxidants that might modulate or delay oxidative insult may be useful in maintaining FSHD muscle functions.
Published: 2012

9. 3'-5' phosphoadenosine phosphate is an inhibitor of PARP-1 and a potential mediator of the lithium-dependent inhibition of PARP-1 in vivo

Author: Antoine Danchin, Vasily Ogryzko, Daniel Ladant, Undine Mechold, Elie Toledano, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), AMAbiotics SAS, This work was supported by the Centre National de la Recherche Scientifique and Institut Pasteur. E.T. was supported by the Université Pierre et Marie Curie. V.O. was supported by Association pour la Recherche sur la Cancer [grant numbers 5950 and 7659]., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV]Life Sciences [q-bio], Poly (ADP-Ribose) Polymerase-1, MESH: Poly (ADP-Ribose) Polymerase-1, Biochemistry, Poly (ADP-Ribose) Polymerase Inhibitor, Substrate Specificity, Histones, chemistry.chemical_compound, 0302 clinical medicine, Polymerase, chemistry.chemical_classification, bipolar disorder, MESH: Histones, 0303 health sciences, biology, MESH: Kinetics, 3′-5′ phosphoadenosine phosphate (pAp), 3. Good health, Adenosine Diphosphate, Sfn/Orn, small fragment nuclease/oligoribonuclease, pAp, 3′-5′ phosphoadenosine phosphate, Poly(ADP-ribose) Polymerases, PARP-1, poly(ADP-ribose) polymerase 1, Research Article, poly(ADP-ribose) polymerase 1 (PARP-1), Poly ADP ribose polymerase, NDK, nucleoside diphosphate kinase, Lithium, Poly(ADP-ribose) Polymerase Inhibitors, MESH: Poly(ADP-ribose) Polymerase Inhibitors, 03 medical and health sciences, FBS, fetal bovine serum, In vivo, PAPS, phosphoadenosine 5′ phosphosulfate, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, 030304 developmental biology, MESH: Humans, MESH: Adenosine Diphosphate, MESH: Lithium, MESH: Poly(ADP-ribose) Polymerases, Lipid metabolism, Cell Biology, Molecular biology, In vitro, Adenosine diphosphate, Kinetics, Enzyme, chemistry, MESH: HeLa Cells, biology.protein, MESH: Substrate Specificity, 030217 neurology & neurosurgery, HeLa Cells
Abstract: International audience; pAp (3'-5' phosphoadenosine phosphate) is a by-product of sulfur and lipid metabolism and has been shown to have strong inhibitory properties on RNA catabolism. In the present paper we report a new target of pAp, PARP-1 [poly(ADP-ribose) polymerase 1], a key enzyme in the detection of DNA single-strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulated both the Km and the Vmax of PARP-1. In addition, we showed that upon treatment with lithium, a very potent inhibitor of the enzyme responsible for pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.
Published: 2012

10. Multiple binding of repressed mRNAs by the P-body protein Rck/p54

Author: Sylvie Souquere, Michèle Ernoult-Lange, Philippe Andrey, Thomas Boudier, Maryannick Harper, Nancy Standart, Michel Kress, Sonia Baconnais, Dominique Weil, Nicola Minshall, Eric Le Cam, Gérard Pierron, Marianne Bénard, Biologie de l'ARN (RnBi), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Dept Biochem, Université de Cambridge, Rétrovirus endogènes et éléments rétroides des eucaryotes supérieurs (REERES (UMR 8122)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative (IFR-BI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Jean-Pierre Bourgin (IJPB), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Ligue Nationale Contre le Cancer, Association pour la Recherche sur le Cancer, Royal Society/CNRS, CNRS, University Pierre et Marie Curie, IFR83, Université Paris 06, Compartimentation et trafic intracellulaire des mRNP = Compartmentation and intracellular traffic of mRNPs (LBD-E14), Laboratoire de Biologie du Développement [Paris] (LBD), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Interfaces biomatériaux/tissus hôtes, Université de Reims Champagne-Ardenne (URCA)-IFR53-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Gustave Roussy (IGR), University of Cambridge [UK] (CAM), Génétique moléculaire et intégration des fonctions cellulaires (GMIFC), Centre National de la Recherche Scientifique (CNRS), Ligue nationale contre le cancer Fondation ARC pour la Recherche sur le CancerAustralian Research CouncilEuropean Commission Royal Society of LondonCentre National de la Recherche Scientifique (CNRS) Centre National de la Recherche Scientifique (CNRS)European Commission University Pierre et Marie Curie UK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC)BB/C514766/1, Laboratoire de Biologie du Développement (LBD), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), CNRS FRE 3402, Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Biologie intégrative (FRBI), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)
Subjects: Untranslated region, [SDV.SA]Life Sciences [q-bio]/Agricultural sciences, [SDV]Life Sciences [q-bio], DEAD-box RNA Helicases, STORED MRNP PARTICLES, Adenosine Triphosphate, mRNA decay, MESH: Adenosine Triphosphate, [PHYS]Physics [physics], 0303 health sciences, CPEB, biology, MESH: Protein Multimerization, DEAD-box, 030302 biochemistry & molecular biology, GRANULES, LOCALIZATION, Cell biology, Protein Binding, DEAD box, Repressor, Models, Biological, Article, 03 medical and health sciences, mRNA storage, DEAD BOX HELICASE, Proto-Oncogene Proteins, DDX6, TRANSLATIONAL REPRESSION, Humans, MESH: DEAD-box RNA Helicases, MESH: Protein Binding, Protein Interaction Domains and Motifs, RNA, Messenger, Molecular Biology, MESH: RNA, Messenger, 030304 developmental biology, MESH: Protein Interaction Domains and Motifs, Messenger RNA, C-TERMINAL DOMAIN, MESH: Humans, COMPLEX, P54, C-terminus, MESH: Models, Biological, RNA, masking, Molecular biology, MESH: Proto-Oncogene Proteins, Decapping complex, MESH: HeLa Cells, biology.protein, Protein Multimerization, HeLa Cells
Abstract: International audience; Translational repression is achieved by protein complexes that typically bind 3′ UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5′ extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation.
Published: 2012

11. The Krüppel-like Factor 15 as a Molecular Link between Myogenic Factors and a Chromosome 4q Transcriptional Enhancer Implicated in Facioscapulohumeral Dystrophy

Author: Yegor S. Vassetzky, Dalila Laoudj, Marc Lipinski, Sébastien Charron, Ahmed Turki, Frédérique Coppée, Andrei Petrov, Thomas Robert, Luiza Stankevicins, Alexandra Belayew, Vladimir Lazar, Petr Dmitriev, Tomas Jan Bos, Elena S. Kim, Gilles Carnac, Eugénie Ansseau, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Université de Mons (UMons), Vrije Universiteit Brussel (VUB), Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Passerieux, Emilie, Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV.MHEP.PHY] Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Enhancer RNAs, MyoD, Muscle Development, Biochemistry, Mice, 0302 clinical medicine, Cricetinae, Transcriptional regulation, Facioscapulohumeral muscular dystrophy, Muscular Dystrophy, ComputingMilieux_MISCELLANEOUS, 2. Zero hunger, 0303 health sciences, Myogenesis, Nuclear Proteins, Molecular Bases of Disease, Muscular Dystrophy, Facioscapulohumeral, DNA-Binding Proteins, Enhancer Elements, Genetic, Chromosomes, Human, Pair 4, Myocyte-specific enhancer factor 2A, musculoskeletal diseases, congenital, hereditary, and neonatal diseases and abnormalities, Kruppel-Like Transcription Factors, Biology, 03 medical and health sciences, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Enhancer, Muscle, Skeletal, Transcription Regulation, Molecular Biology, Transcription factor, Chromatin Structure, 030304 developmental biology, MyoD Protein, Transcription Enhancers, FSHD, D4Z4, Cell Biology, medicine.disease, Molecular biology, nervous system diseases, Gene Expression Regulation, 030217 neurology & neurosurgery, HeLa Cells, Transcription Factors
Abstract: Facioscapulohumeral muscular dystrophy (FSHD), a dominant hereditary disease with a prevalence of 7 per 100,000 individuals, is associated with a partial deletion in the subtelomeric D4Z4 repeat array on chromosome 4q. The D4Z4 repeat contains a strong transcriptional enhancer that activates promoters of several FSHD-related genes. We report here that the enhancer within the D4Z4 repeat binds the Kruppel-like factor KLF15. KLF15 was found to be up-regulated during myogenic differentiation induced by serum starvation or by overexpression of the myogenic differentiation factor MYOD. When overexpressed, KLF15 activated the D4Z4 enhancer and led to overexpression of DUX4c (Double homeobox 4, centromeric) and FRG2 (FSHD region gene 2) genes, whereas its silencing caused inactivation of the D4Z4 enhancer. In immortalized human myoblasts, the D4Z4 enhancer was activated by the myogenic factor MYOD, an effect that was abolished upon KLF15 silencing or when the KLF15-binding sites within the D4Z4 enhancer were mutated, indicating that the myogenesis-related activation of the D4Z4 enhancer was mediated by KLF15. KLF15 and several myogenesis-related factors were found to be expressed at higher levels in myoblasts, myotubes, and muscle biopsies from FSHD patients than in healthy controls. We propose that KLF15 serves as a molecular link between myogenic factors and the activity of the D4Z4 enhancer, and it thus contributes to the overexpression of the DUX4c and FRG2 genes during normal myogenic differentiation and in FSHD.
Published: 2011

12. Ring-like distribution of constitutive heterochromatin in bovine senescent cells

Author: Andrey Pichugin, Yegor S. Vassetzky, Xavier Vignon, Nathalie Beaujean, Biologie du développement et reproduction (BDR), Centre National de la Recherche Scientifique (CNRS)-École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), This research project was supported by a Marie Curie Early Stage Research Training Fellowship of the European Community's Sixth Framework Program under contract number MEST-CT-2004-504854, by the 'PluriSys' Collaborative Project from the European Community's under contract number HEALTH-F4-2009-223485, and INRA 'Young Team' funding. The research in YV's laboratory was funded by the Fondation de France and Institut National de Cancer (INCa). AP was a recipient of the postdoctoral fellowship from the Fondation de France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Biologie du Développement et Reproduction (BDR), Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), and Vassetzky, Yegor
Subjects: multidisciplinary sciences, Aging, bovin, Anatomy and Physiology, Nucleolus, lcsh:Medicine, Hydroxamic Acids, chemistry.chemical_compound, 0302 clinical medicine, Molecular Cell Biology, lcsh:Science, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Cellular Senescence, Centromeres, 0303 health sciences, Multidisciplinary, culture cellulaire, DNA methylation, Chromosome Biology, Biologie du développement, Development Biology, Chromatin, 030220 oncology & carcinogenesis, Epigenetics, DNA modification, Histone modification, Cell aging, Research Article, Cell Physiology, science and technology, Heterochromatin, cellule sénescente, microscopie confocale, Centromere, Biology, Cell Line, 03 medical and health sciences, Histone H3, hybridation fluorescente in situ, Genetics, Constitutive heterochromatin, immunomarquage, Animals, DAPI, immunodétection, 030304 developmental biology, lcsh:R, nuclear architecture, heterochromatin, bovine senescent, fungi, Fibroblasts, Molecular biology, hétérochromatine, chemistry, cellule somatique, lcsh:Q, Cattle, Physiological Processes
Abstract: BACKGROUND: Cells that reach "Hayflick limit" of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. METHODS: We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). RESULTS: Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli. CONCLUSIONS: Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.
Published: 2011

13. A brain-specific isoform of mitochondrial apoptosis-inducing factor: AIF2

Author: Pierre Rustin, Klas Blomgren, Sylvie Lachkar, Guido Kroemer, A de Andrade, Carsten Culmsee, Nazanine Modjtahedi, Günter U. Höglinger, Gérard Pierron, Gian Maria Fimia, Vladimir Lazar, Matteo Bordi, Emilie Hangen, Francis Harper, Jose Miguel Vicencio, Jean Bénard, Paule Bénit, Mauro Piacentini, J-L Perfettini, Changlian Zhu, Philippe Dessen, D De Zio, F Caffin, Francesco Cecconi, Laboratoire de Biochimie de l'Ecole polytechnique (BIOC), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Service de pneumologie, oncologie thoracique et soins intensifs respiratoires [Rouen], Hôpital Charles Nicolle [Rouen]-CHU Rouen, Normandie Université (NU)-Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU), Laboratory of Cell Biology, National Institute for Infectious Diseases IRCCS 'L. Spallanzani', Neuroprotection du Cerveau en Développement / Promoting Research Oriented Towards Early Cns Therapies (PROTECT), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Robert Debré-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Geoservices, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Apoptose, cancer et immunité (U848), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Radiothérapie moléculaire (UMR 1030), Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Laboratory of Molecular Neuroembryology, Clinical and Behavioral Neurology - Neuroscienze e riabilitazione, IRCCS Fondazione Santa Lucia [Roma]-Università degli Studi di Roma Tor Vergata [Roma]-Dulbecco Telethon Institute, Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology [Göteborg]-University of Gothenburg (GU), Génomes et cancer (GC (FRE2939)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Génomique Fonctionnelle et Biologie Systémique pour la Santé, Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), National Institute for Infectious Diseases, Réplication de l'ADN - Ultrastructure de la cellule, Institut André Lwoff-Centre National de la Recherche Scientifique (CNRS), Physiopathologie, conséquences fonctionnelles et neuroprotection des atteintes du cerveau en développement, Université Paris Diderot - Paris 7 (UPD7)-IFR2-Institut National de la Santé et de la Recherche Médicale (INSERM), Experimental Neurology, Philipps University, Department of Pharmacy, Philipps Universität Marburg = Philipps University of Marburg -Institute for Pharmacology and Toxicology, Peer, Hal, University of Rome 'Tor Vergeta'-Clinical and Behavioral Neurology - Neuroscienze e riabilitazione, IRCCS Fondazione Santa Lucia [Roma]-Dulbecco Telethon Institute, Philipps Universität Marburg-Institute for Pharmacology and Toxicology, Hangen, E, De Zio, D, Bordi, M, Zhu, C, Dessen, P, Caffin, F, Lachkar, S, Perfettini, J. L, Lazar, V, Benard, J, Fimia, Gian Maria, Piacentini, M, Harper, F, Pierron, G, Vicencio, J. M, Bénit, P, de Andrade, A, Höglinger, G, Culmsee, C, Rustin, P, Blomgren, K, Cecconi, F, Kroemer, G, and Modjtahedi, N.
Subjects: neural differentiation, Cellular differentiation, [SDV]Life Sciences [q-bio], brain development, Mitochondrion, Mice, 0302 clinical medicine, Protein Isoforms, Inner mitochondrial membrane, ComputingMilieux_MISCELLANEOUS, Neurons, 0303 health sciences, Apoptosis Regulatory Protein, Tumor, Amino Acid Sequence, Animals, Apoptosis Inducing Factor, Apoptosis Regulatory Proteins, Brain, Cell Differentiation, Cell Line, Humans, Mitochondria, Mitochondrial Proteins, Molecular Sequence Data, Sequence Alignment, Cell biology, Apoptosis-inducing factor, Intermembrane space, Human, Gene isoform, Programmed cell death, Settore BIO/06, oxidative phosphorylation, Biology, 03 medical and health sciences, neuroblastoma, Cell Line, Tumor, Mitochondrial Protein, Inner membrane, Molecular Biology, 030304 developmental biology, Animal, Protein Isoform, Cell Biology, Neuron, gene expression, 030217 neurology & neurosurgery
Abstract: International audience; Apoptosis-Inducing factor (AIF) plays important supportive as well as potentially lethal roles in neurons. Under normal physiological conditions, AIF is a vital redox-active mitochondrial enzyme, whereas in pathological situations, it translocates from mitochondria to the nuclei of injured neurons and mediates apoptotic chromatin condensation and cell death. Here, we reveal the existence of a brain-specific isoform of AIF, AIF2, whose expression increases as neuronal precursor cells differentiate. AIF2 arises from the utilization of the alternative exon 2b, yet uses the same remaining 15 exons as the ubiquitous AIF1 isoform. AIF1 and AIF2 are similarly imported to mitochondria where they anchor to the inner membrane facing the intermembrane space. However, the mitochondrial inner membrane sorting signal (IMSS) encoded in the exon 2b of AIF2 is more hydrophobic than that of AIF1, indicating a stronger membrane anchorage of AIF2 than AIF1. AIF2 is more difficult to be desorbed from mitochondria than AIF1 upon exposure to non-ionic detergents or basic pH. Furthermore, AIF2 dimerizes with AIF1, thereby preventing its release from mitochondria. Conversely, it is conceivable that a neuron-specific AIF isoform, AIF2, may have been "designed" to be retained in mitochondria and to minimize its potential neurotoxic activity.
Published: 2010

14. The epigenetic landscape of mammary gland development and functional differentiation

Author: Elena B. Kabotyanski, Yegor S. Vassetzky, Jeffrey M. Rosen, Mohamad B. Montazer-Torbati, Monique Rijnkels, Eve Devinoy, C. Hue Beauvais, Baylor College of Medicine (BCM), Department of Molecular Cellular Biology, Baylor University-Baylor University, University of Birjand, Unité de recherche génomique et physiologie de la lactation (GPL), Institut National de la Recherche Agronomique (INRA), Université Paris-Sud - Paris 11 (UP11), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), and Institut Gustave Roussy (IGR)
Subjects: CHROMATIN, Cancer Research, Epigenetic regulation of neurogenesis, RNA, Untranslated, Transcription, Genetic, Cellular differentiation, [SDV]Life Sciences [q-bio], Breast Neoplasms, Biology, EPIGENETIQUE, Chromatin remodeling, Article, Epigenesis, Genetic, Histones, 03 medical and health sciences, DEVELOPMENT, 0302 clinical medicine, Epigenetics of physical exercise, Mammary Glands, Animal, Animals, Humans, [INFO]Computer Science [cs], Epigenetics, Mammary Glands, Human, ChIA-PET, 030304 developmental biology, Epigenomics, Genetics, EPIGENETIC, 0303 health sciences, Stem Cells, MAMMARY GLAND, Cell Differentiation, Chromatin Assembly and Disassembly, Chromatin, Gene Expression Regulation, Neoplastic, Oncology, 030220 oncology & carcinogenesis, MILK PROTEIN GENES, Female, Transcription Factors
Abstract: International audience; Most of the development and functional differentiation in the mammary gland occur after birth. Epigenetics is defined as the stable alterations in gene expression potential that arise during development and proliferation. Epigenetic changes are mediated at the biochemical level by the chromatin conformation initiated by DNA methylation, histone variants, post-translational modifications of histones, non-histone chromatin proteins, and non-coding RNAs. Epigenetics plays a key role in development. However, very little is known about its role in the developing mammary gland or how it might integrate the many signalling pathways involved in mammary gland development and function that have been discovered during the past few decades. An inverse relationship between marks of closed (DNA methylation) or open chromatin (DnaseI hypersensitivity, certain histone modifications) and milk protein gene expression has been documented. Recent studies have shown that during development and functional differentiation, both global and local chromatin changes occur. Locally, chromatin at distal regulatory elements and promoters of milk protein genes gains a more open conformation. Furthermore, changes occur both in looping between regulatory elements and attachment to nuclear matrix. These changes are induced by developmental signals and environmental conditions. Additionally, distinct epigenetic patterns have been identified in mammary gland stem and progenitor cell sub-populations. Together, these findings suggest that epigenetics plays a role in mammary development and function. With the new tools for epigenomics developed in recent years, we now can begin to establish a framework for the role of epigenetics in mammary gland development and disease.
Published: 2010

15. Netrin-1 acts as a survival factor for aggressive neuroblastoma

Author: Dwayne G. Stupack, Agnès Bernet, Akira Nakagawara, Alain Puisieux, Dominique Valteau-Couanet, Marie-Anne Raquin, Raphael Rousseau, Valérie Combaret, Patrick Mehlen, Julien Fitamant, Andrea Paradisi, Céline Delloye-Bourgeois, Sétha Douc-Rasy, Jean Bénard, David Cappellen, Apoptose Cancer et Développement, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Département de Pédiatrie, Institut Gustave Roussy (IGR), departement of Pathology, University of California [San Diego] (UC San Diego), University of California-University of California, Division od Biochemistry, Chiba Center Center Research Institute, Centre Léon Bérard [Lyon], Laboratoire d'Oncologie Moléculaire, equipe 2, Oncogénèse et progression tumorale, Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Département de biologie et pathologie médicales [Gustave Roussy], Apoptosis, Cancer, and Development Laboratory, Bissardon, Bérangère, and University of California (UC)-University of California (UC)
Subjects: MESH: Cell Death, Dependence receptor, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Metastasis, Neuroblastoma, 0302 clinical medicine, MESH: Reverse Transcriptase Polymerase Chain Reaction, Netrin, MESH: RNA, Small Interfering, MESH: Up-Regulation, Immunology and Allergy, Survivors, RNA, Small Interfering, Receptor, MESH: Survivors, 0303 health sciences, Cell Death, Reverse Transcriptase Polymerase Chain Reaction, MESH: Infant, Newborn, MESH: Gene Expression Regulation, Neoplastic, MESH: Neoplasm Staging, Netrin-1, Prognosis, MESH: Infant, Up-Regulation, 3. Good health, Gene Expression Regulation, Neoplastic, MESH: Cell Survival, 030220 oncology & carcinogenesis, Programmed cell death, animal structures, MESH: Cell Line, Tumor, Cell Survival, Immunology, Biology, Disease-Free Survival, Article, MESH: Prognosis, 03 medical and health sciences, Downregulation and upregulation, Cell Line, Tumor, medicine, Humans, MESH: Tumor Suppressor Proteins, Nerve Growth Factors, Autocrine signalling, Neoplasm Staging, 030304 developmental biology, MESH: Humans, MESH: Nerve Growth Factors, Tumor Suppressor Proteins, fungi, Infant, Newborn, Infant, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease, MESH: Neuroblastoma, nervous system, MESH: Disease-Free Survival, Cancer research
Abstract: International audience; Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in >60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression. We show that autocrine production of netrin-1, a multifunctional laminin-related molecule, conveys a selective advantage in tumor growth and dissemination in aggressive NB, as it blocks the proapoptotic activity of the UNC5H netrin-1 dependence receptors. We show that such netrin-1 up-regulation is a potential marker for poor prognosis in stage 4S and, more generally, in NB stage 4 diagnosed infants. Moreover, we propose that interference with the netrin-1 autocrine loop in malignant neuroblasts could represent an alternative therapeutic strategy, as disruption of this loop triggers in vitro NB cell death and inhibits NB metastasis in avian and mouse models.
Published: 2009

16. The universal Kae1 protein and the associated Bud32 kinase (PRPK), a mysterious protein couple probably essential for genome maintenance in Archaea and Eukarya

Author: Marc Graille, Arnaud Hecker, Edwige Madec, Herman van Tilbergh, Patrick Forterre, Eric Le Cam, Danièle Gadelle, Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie de l'Ecole polytechnique (BIOC), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), and Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)
Subjects: Pyrococcus abyssi, Archaeal Proteins, [SDV]Life Sciences [q-bio], Saccharomyces cerevisiae, Molecular Sequence Data, Biology, Biochemistry, Protein Structure, Secondary, 03 medical and health sciences, Genome, Archaeal, DNA-(Apurinic or Apyrimidinic Site) Lyase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Kinase activity, Lyase activity, Protein kinase A, 030304 developmental biology, Comparative genomics, Genetics, 0303 health sciences, 030302 biochemistry & molecular biology, Methanocaldococcus jannaschii, biology.organism_classification, Fusion protein, Archaea, DNA-Binding Proteins, Eukaryotic Cells, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Protein Kinases
Abstract: International audience; The similarities between essential molecular mechanisms in Archaea and Eukarya make it possible to discover, using comparative genomics, new fundamental mechanisms conserved between these two domains. We are studying a complex of two proteins conserved in Archaea and Eukarya whose precise biological role and biochemical function remain unknown. One of them is a universal protein known as Kae1 (kinase-asociated endopeptidase 1). The second protein is a serine/threonine kinase corresponding to the proteins Bud32 in Saccharomyces cerevisiae and PRPK (p53-related protein kinase) in humans. The genes encoding the archaeal orthologues of Kae1 and PRPK are either contiguous or even fused in many archaeal genomes. In S. cerevisiae, Kae1 and Bud32 (PRPK) belong to a chromatin-associated complex [KEOPS (kinase, endopeptidase and other proteins of small size)/EKC (endopeptidase-like kinase chromatin-associated)] that is essential for telomere elongation and transcription of essential genes. Although Kae1 is annotated as O-sialoglycoprotein endopeptidase in most genomes, we found that the Kae1 protein from Pyrococcus abyssi has no protease activity, but is an atypical DNA-binding protein with an AP (apurinic) lyase activity. The structure of the fusion protein from Methanocaldococcus jannaschii revealed that Kae1 maintains the ATP-binding site of Kae1 in an inactive configuration. We have in fact found that Kae1 inhibits the kinase activity of Bud32 (PRPK) in vitro. Understanding the precise biochemical function and biological role of these two proteins (which are probably essential for genome maintenance) remains a major challenge.
Published: 2009

17. Degradation of nanoRNA is performed by multiple redundant RNases in Bacillus subtilis

Author: Ciarán Condon, Undine Mechold, Antoine Danchin, Vasily Ogryzko, Ming Fang, Wencke-Maria Zeisberg, Génétique des Génomes Bactériens, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), European Union Network of Excellence BioSapiens[LSHG CT-2003-503265 to A.D.], the French ResearchAgency (Agence nationale de la recherche) [BLAN06-3_135068 toC.C.], La Ligue Contre le Cancer[9ADO1217/1B1-BIOCE to V.O.], Institut national duCancer [247343/1B1-BIOCE to V.O.]. Funding for openaccess charge: Institut Pasteur., Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Ribonucleases, MESH: Mutation, RNase P, Mutant, Bacillus subtilis, Biology, medicine.disease_cause, 03 medical and health sciences, Ribonucleases, MESH: RNA, Genetics, medicine, Genomic library, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Phylogeny, Escherichia coli, Phylogeny, 030304 developmental biology, 0303 health sciences, MESH: Genetic Complementation Test, Oligoribonucleotides, Nucleic Acid Enzymes, 030306 microbiology, Genetic Complementation Test, fungi, MESH: DNA, RNA, DNA, respiratory system, MESH: Bacillus subtilis, biology.organism_classification, Molecular biology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Complementation, Biochemistry, Mutation, MESH: Oligoribonucleotides, sense organs, Exoribonuclease activity
Abstract: International audience; Escherichia coli possesses only one essential oligoribonuclease (Orn), an enzyme that can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). Firmicutes including Bacillus subtilis do not have an Orn homolog. We had previously identified YtqI (NrnA) as functional analog of Orn in B. subtilis. Screening a genomic library from B. subtilis for genes that can complement a conditional orn mutant, we identify here YngD (NrnB) as a second nanoRNase in B. subtilis. Like NrnA, NrnB is a member of the DHH/DHHA1 protein family of phosphoesterases. NrnB degrades nanoRNA 5-mers in vitro similarily to Orn. Low expression levels of NrnB are sufficient for orn complementation. YhaM, a known RNase present in B. subtilis, degrades nanoRNA efficiently in vitro but requires high levels of expression for only partial complementation of the orn(-) strain. A triple mutant (nrnA(-), nrnB(-), yhaM(-)) in B. subtilis is viable and shows almost no impairment in growth. Lastly, RNase J1 seems also to have some 5'-to-3' exoribonuclease activity on nanoRNA and thus can potentially finish degradation of RNA. We conclude that, unlike in E. coli, degradation of nanoRNA is performed in a redundant fashion in B. subtilis.
Published: 2009

18. YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

Author: N. A. Shanina, Anne Tarrade, Alain Mechulam, V.D. Vasiliev, Nadezhda V. Popova, Olga V. Skabkina, Lev P. Ovchinnikov, Flavio Toma, Patrick A. Curmi, Vandana Joshi, Elena S. Nadezhdina, David Pastré, Sonia Baconnais, Konstantin G. Chernov, Judith Melki, Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institute of Protein Research, Russian Academy of Sciences, Pushchino, Lomonosov Moscow State University (MSU), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Institute of Protein Research, Russian Academy of Sciences [Moscow] (RAS), Hadassah University Hospital, and Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Microtubule-associated protein, [SDV]Life Sciences [q-bio], lcsh:Animal biochemistry, RNA-binding protein, Microscopy, Atomic Force, Microtubules, Biochemistry, Chromatography, Affinity, lcsh:Biochemistry, 03 medical and health sciences, Tubulin, Microtubule, Transcription (biology), Gene expression, Animals, Humans, lcsh:QD415-436, RNA, Messenger, Molecular Biology, lcsh:QP501-801, 030304 developmental biology, 0303 health sciences, biology, Tissue Extracts, 030302 biochemistry & molecular biology, Nuclear Proteins, RNA-Binding Proteins, RNA, Translation (biology), Peptide Fragments, 3. Good health, Cell biology, DNA-Binding Proteins, Ribonucleoproteins, biology.protein, Rabbits, Y-Box-Binding Protein 1, Microtubule-Associated Proteins, Research Article
Abstract: Background YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.
Published: 2008

19. Canstatin gene electrotransfer combined with radiotherapy: preclinical trials for cancer treatment

Author: Lluis M. Mir, Claire Magnon, Michel Perricaudet, D Martel-Renoir, Elisabeth Connault, Paule Opolon, Vectorologie et transfert de gènes (VTG / UMR8121), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), and Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Collagen Type IV, Male, Pathology, medicine.medical_specialty, Angiogenesis, medicine.medical_treatment, Genetic enhancement, Recombinant Fusion Proteins, Mice, Nude, Gene electrotransfer, Apoptosis, Breast Neoplasms, 03 medical and health sciences, Prostate cancer, Mice, 0302 clinical medicine, Radioresistance, Cell Line, Tumor, Genetics, medicine, Animals, Humans, Muscle, Skeletal, Molecular Biology, Serum Albumin, 030304 developmental biology, 0303 health sciences, business.industry, Cancer, Prostatic Neoplasms, Radiotherapy Dosage, Genetic Therapy, medicine.disease, Combined Modality Therapy, Xenograft Model Antitumor Assays, Peptide Fragments, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Radiation therapy, Mice, Inbred C57BL, Electroporation, 030220 oncology & carcinogenesis, Cancer research, Molecular Medicine, Syngenic, Female, business
Abstract: Given as a prophylactic treatment, a single muscle electrogene transfer of plasmid coding canstatin fused to human serum albumin (CanHSA), slowed down the development of two xenografted human carcinomas from mammary (MDA-MB-231) and prostate origin (PC-3) in nude mice and delayed lung metastatic spreading of B16F10 melanoma cells in syngenic mice. No effect was observed with unfused canstatin. The long lasting circulating blood level of CanHSA (20 ng ml(-1)) resulted in a profound disorganization of the tumor blood vessel network. However, when used as a curative treatment, on well-established tumors, CanHSA electrogenetherapy was ineffective in reducing tumor growth. As radiation is known to increase the alphavbeta3 and alphavbeta5 integrins, which are canstatin receptors, to extend the use of CanHSA electrogenetherapy, as a curative treatment, we explored the combination of CanHSA and ionizing radiation. We demonstrated a better efficacy (P=0.01) of the bitherapy over irradiation alone, as a result of strong vessel disorganization and dramatic increase of tumor cells apoptosis. This extremely simple virus free curative protocol could open the door to potential clinical applications, especially for prostate cancer that often develops radioresistance.Gene Therapy advance online publication, 12 June 2008; doi:10.1038/gt.2008.100.
Published: 2008

20. Lobular and ductal carcinomas of the breast have distinct genomic and expression profiles

Author: Jocelyne Jacquemier, Isabelle Treilleux, Agnès Martinec, Charles Theillet, Frédéric Bibeau, Alain Puisieux, Vincent Nègre, François Bertucci, Lisa Ursule, Carole Rougé, Pascal Finetti, Daniel Birnbaum, Marie-Christine Mathieu, Jean-Charles Ahomadegbe, Qing Wang, Jean Bénard, Béatrice Orsetti, Cancérologie (Inserm U599/IPC), Université de la Méditerranée - Aix-Marseille 2-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de recherche en cancérologie de Montpellier (IRCM - U896 Inserm - UM1), Université Montpellier 1 (UM1)-CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Pharmacologie et nouveaux traitements des cancers, Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR), Laboratoire d'anatomopathologie, CRLC Val d'Aurelle-Paul Larmarque, Département de biologie et pathologie médicales [Gustave Roussy], Institut Gustave Roussy (IGR), Département d'anatomopathologie, biopathologie, Centre Léon Bérard [Lyon], Ipsogen S.A., Laboratoire d'Oncologie Moléculaire, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), equipe 2, Oncogénèse et progression tumorale, Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Laboratoire d'Oncologie Moléculaire, This study was developped as part of a joint program « Développement d'outils de diagnostic moléculaire en Cancérologie : Applications aux cancers du sein » Ministère de l'Enseignement Supérieur, de la Recherche et de la Technologie and Fédération Nationale des Centres de Lutte Contre le Cancer and was supported by funds from INSERM, the Association de Recherche sur le Cancer (ARC), grant 5102, Institut National du Cancer, Cancéropoles PACA and Grand Sud Ouest. The help of the Génopole Montpellier Languedoc-Roussillon is gratefully acknowledged. The authors thank Pr Dominique Maraninchi and Dr Claude Mawas for setting up this work and Mrs Sophie Tourpin for technical help., and Le Ster, Yves
Subjects: Cancer Research, Chromosomes, Artificial, Bacterial, Microarray, medicine.disease_cause, MESH: Cadherins, MESH: Nucleic Acid Hybridization, 0302 clinical medicine, MESH: Carcinoma, Lobular, MESH: Reverse Transcriptase Polymerase Chain Reaction, RNA, Neoplasm, MESH: Tumor Suppressor Protein p53, skin and connective tissue diseases, Oligonucleotide Array Sequence Analysis, Genetics, Regulation of gene expression, 0303 health sciences, Reverse Transcriptase Polymerase Chain Reaction, Carcinoma, Ductal, Breast, Nucleic Acid Hybridization, MESH: Gene Expression Regulation, Neoplastic, Cadherins, 3. Good health, Gene Expression Regulation, Neoplastic, 030220 oncology & carcinogenesis, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Female, Breast disease, DNA microarray, MESH: Chromosomes, Artificial, Bacterial, MESH: Mutation, genetic profiles, [SDV.CAN]Life Sciences [q-bio]/Cancer, Breast Neoplasms, Biology, Article, 03 medical and health sciences, MESH: Gene Expression Profiling, breast cancer, [SDV.CAN] Life Sciences [q-bio]/Cancer, Antigens, CD, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Humans, RNA, Messenger, Molecular Biology, neoplasms, 030304 developmental biology, MESH: RNA, Messenger, MESH: Humans, Cadherin, Gene Expression Profiling, Ductal carcinoma, medicine.disease, MESH: RNA, Neoplasm, Gene expression profiling, MESH: Carcinoma, Ductal, Breast, body regions, Carcinoma, Lobular, array-CGH, MESH: Oligonucleotide Array Sequence Analysis, Mutation, Cancer research, Tumor Suppressor Protein p53, Carcinogenesis, MESH: Female, MESH: Breast Neoplasms
Abstract: International audience; Invasive ductal carcinomas (IDCs) and invasive lobular carcinomas (ILCs) are the two major pathological types of breast cancer. Epidemiological and histoclinical data suggest biological differences, but little is known about the molecular alterations involved in ILCs. We undertook a comparative large-scale study by both array-compared genomic hybridization and cDNA microarray of a set of 50 breast tumors (21 classic ILCs and 29 IDCs) selected on homogeneous histoclinical criteria. Results were validated on independent tumor sets, as well as by quantitative RT-PCR. ILCs and IDCs presented differences at both the genomic and expression levels with ILCs being less rearranged and heterogeneous than IDCs. Supervised analysis defined a 75-BACs signature discriminating accurately ILCs from IDCs. Expression profiles identified two subgroups of ILCs: typical ILCs ( approximately 50%), which were homogeneous and displayed a normal-like molecular pattern, and atypical ILCs, more heterogeneous with features intermediate between ILCs and IDCs. Supervised analysis identified a 75-gene expression signature that discriminated ILCs from IDCs, with many genes involved in cell adhesion, motility, apoptosis, protein folding, extracellular matrix and protein phosphorylation. Although ILCs and IDCs share common alterations, our data show that ILCs and IDCs could be distinguished on the basis of their genomic and expression profiles suggesting that they evolve along distinct genetic pathways.
Published: 2008

21. Multiple displacement amplification for complex mixtures of DNA fragments

Author: Sonia Baconnais, Eric Le Cam, Muhammad Shoaib, Undine Mechold, Marc Lipinski, Vasily Ogryzko, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Institut Gustave Roussy (IGR), Génétique des Génomes Bactériens, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Chromatin Immunoprecipitation, lcsh:QH426-470, [SDV]Life Sciences [q-bio], lcsh:Biotechnology, Biology, Genetic analysis, 03 medical and health sciences, chemistry.chemical_compound, lcsh:TP248.13-248.65, Genetics, Humans, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Whole Genome Amplification, 0303 health sciences, Methodology Article, 030302 biochemistry & molecular biology, Multiple displacement amplification, Nucleic acid amplification technique, DNA, Molecular biology, Chromatin, lcsh:Genetics, chemistry, Rolling circle replication, Biophysics, DNA microarray, Nucleic Acid Amplification Techniques, Biotechnology, HeLa Cells
Abstract: Background A fundamental requirement for genomic studies is the availability of genetic material of good quality and quantity. The desired quantity and quality are often hard to obtain when target DNA is composed of complex mixtures of relatively short DNA fragments. Here, we sought to develop a method to representatively amplify such complex mixtures by converting them to long linear and circular concatamers, from minute amounts of starting material, followed by phi29-based multiple displacement amplification. Results We report here proportional amplification of DNA fragments that were first converted into concatamers starting from DNA amounts as low as 1 pg. Religations at low concentration (< 1 ng/μ L) preferentially lead to fragment self-circularization, which are then amplified independently, and result in non-uniform amplification. To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/μ L), which helped in the formation of long concatamers and hence resulted in uniform amplification. To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter. The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so. Conclusion We believe that this method allows for generation of abundant amounts of good quality genetic material from a complex mixture of short DNA fragments, which can be further used in high throughput genetic analysis.
Published: 2008

22. The Srs2 Helicase Activity Is Stimulated by Rad51 Filaments on dsDNA: Implications for Crossover Incidence during Mitotic Recombination

Author: Francis Fabre, Eric Le Cam, Serge Gangloff, Cyrille Le Breton, Xavier Veaute, Pauline Dupaigne, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Laboratoire d'Etudes de la Réparation de l'ADN (LERA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Radiobiologie moléculaire et cellulaire (RMC), and Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)
Subjects: Saccharomyces cerevisiae Proteins, Mitotic crossover, [SDV]Life Sciences [q-bio], Saccharomyces cerevisiae, RAD51, DNA, Single-Stranded, Mitosis, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Crossing Over, Genetic, DNA, Fungal, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, DNA Helicases, Fungal genetics, Helicase, Cell Biology, biology.organism_classification, RNA Helicase A, Molecular biology, Cell biology, chemistry, biology.protein, Rad51 Recombinase, 030217 neurology & neurosurgery, DNA
Abstract: International audience; Saccharomyces cerevisiae Srs2 helicase was shown to displace Rad51 in vitro upon translocation on single-stranded DNA. This activity is sufficient to account for its antirecombination effect and for the elimination of otherwise dead-end recombination intermediates. Roles for the helicase activity are yet unknown. Because cells lacking Srs2 show increased incidence of mitotic crossovers, it was postulated that Srs2 promotes synthesis-dependent strand annealing (SDSA) by unwinding the elongating invading strand from the donor strand. We report here that synthetic DNA structures that mimic D loops are good substrates for the Srs2 helicase activity, that Srs2 translocates on RPA-coated ssDNA, and, furthermore, that the helicase activity is largely stimulated by the presence of Rad51 nucleoprotein filaments on double-stranded DNA. These properties strongly support the idea that Srs2 actively prevents crossovers by promoting SDSA.
Published: 2008

23. Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver

Author: Frédéric Vigant, Betsy Jullienne, Paule Opolon, Stéphanie Esselin, Karim Benihoud, Emmanuelle Vigne, Michel Perricaudet, Thierry Tordjmann, Elisabeth Connault, Delphyne Descamps, Perricaudet, Michel, Vectorologie et transfert de gènes (VTG / UMR8121), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Centre d'Etudes Lasers Intenses et Applications (CELIA), Université de Bordeaux (UB)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Service de microbiologie, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Signalisation calcique et interactions cellulaires dans le foie, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Bordeaux (UB), and Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)
Subjects: Genetic enhancement, Genetic Vectors, Mice, Nude, Biology, Polymerase Chain Reaction, Adenoviridae, 03 medical and health sciences, Transduction (genetics), Carcinoma, Lewis Lung, Leukocyte Count, Mice, 0302 clinical medicine, In vivo, Transduction, Genetic, Cell Line, Tumor, Drug Discovery, medicine, Genetics, Animals, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Tropism, 030304 developmental biology, Pharmacology, 0303 health sciences, Mice, Inbred BALB C, Mice, Inbred C3H, Interleukin-6, Platelet Count, Alanine Transaminase, Virology, 3. Good health, Hypervariable region, Mice, Inbred C57BL, medicine.anatomical_structure, Capsid, Liver, Cell culture, 030220 oncology & carcinogenesis, Hepatocyte, Molecular Medicine, Capsid Proteins
Abstract: Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.
Published: 2008

24. Srs2 removes deadly recombination intemediates independently of its interaction with SUMO-modified PCNA

Author: Eric Le Cam, Thomas Robert, Francis Fabre, Serge Gangloff, Cyrille Le Breton, Pauline Dupaigne, Xavier Veaute, Laboratoire d'Etudes de la Réparation de l'ADN (LERA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Radiobiologie moléculaire et cellulaire (RMC), Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Université de Liège, Laboratoire d'Hydrologie et de Géochimie de Strasbourg (LHyGeS), Ecole et Observatoire des Sciences de la Terre (EOST), Université de Strasbourg (UNISTRA)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Rouergue Auvergne Gévaudan Tarnais, Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Université de Strasbourg (UNISTRA)-Institut national des sciences de l'Univers (INSU - CNRS)-Ecole et Observatoire des Sciences de la Terre (EOST), Institut national des sciences de l'Univers (INSU - CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)
Subjects: Saccharomyces cerevisiae Proteins, DNA Repair, Ultraviolet Rays, DNA repair, [SDV]Life Sciences [q-bio], SUMO-1 Protein, Saccharomyces cerevisiae, RAD51, medicine.disease_cause, DNA-binding protein, 03 medical and health sciences, chemistry.chemical_compound, Suppression, Genetic, Proliferating Cell Nuclear Antigen, Genetics, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Recombination, Genetic, 0303 health sciences, Mutation, RecQ Helicases, biology, 030302 biochemistry & molecular biology, DNA Helicases, Helicase, DNA, biology.organism_classification, Proliferating cell nuclear antigen, DNA-Binding Proteins, DNA Repair Enzymes, chemistry, Biochemistry, biology.protein, Rad51 Recombinase, Gene Deletion
Abstract: International audience; Saccharomyces cerevisiae Srs2 helicase plays at least two distinct functions. One is to prevent recombinational repair through its recruitment by sumoylated Proliferating Cell Nuclear Antigen (PCNA), evidenced in postreplication-repair deficient cells, and a second one is to eliminate potentially lethal intermediates formed by recombination proteins. Both actions are believed to involve the capacity of Srs2 to displace Rad51 upon translocation on single-stranded DNA (ssDNA), though a role of its helicase activity may be important to remove some toxic recombination structures. Here, we described two new mutants, srs2R1 and srs2R3, that have lost the ability to hinder recombinational repair in postreplication-repair mutants, but are still able to remove toxic recombination structures. Although the mutants present very similar phenotypes, the mutated proteins are differently affected in their biochemical activities. Srs2R1 has lost its capacity to interact with sumoylated PCNA while the biochemical activities of Srs2R3 are attenuated (ATPase, helicase, DNA binding and ability to displace Rad51 from ssDNA). In addition, crossover (CO) frequencies are increased in both mutants. The different roles of Srs2, in relation to its eventual recruitment by sumoylated PCNA, are discussed.
Published: 2008

25. The rolling-circle plasmid pTN1 from the hyperthermophilic archaeon Thermococcus nautilus

Author: Nicolas Soler, Sophie Quevillon-Cheruel, Eric Le Cam, Patrick Forterre, Evelyne Marguet, Florence Lorieux, Anthony Justome, Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Pyrococcus abyssi, Archaeal Proteins, Molecular Sequence Data, Electrophoretic Mobility Shift Assay, Biology, Microscopy, Atomic Force, Microbiology, 03 medical and health sciences, Plasmid, Microscopy, Electron, Transmission, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Insertion sequence, Molecular Biology, Peptide sequence, 030304 developmental biology, Zinc finger, 0303 health sciences, Models, Genetic, Sequence Homology, Amino Acid, 030306 microbiology, DNA Helicases, biology.organism_classification, Molecular biology, DNA-Binding Proteins, Thermococcus, Biochemistry, Rolling circle replication, Trans-Activators, Domain of unknown function, Plasmids
Abstract: The hyperthermophilic archaeon Thermococcus nautilus carries a plasmid, pTN1, which encodes a rolling-circle (RC) replication initiator protein of 74 kDa (Rep74) and an orphan protein of 24 kDa (p24). The Rep74 protein is homologous to the Rep75 protein encoded by the RC plasmid pGT5 from Pyrococcus abyssi. Comparative analysis of Rep74 and Rep75 sequences shows that these proteins correspond to a new family of RC initiators formed by the fusion of a Rep domain with an N-terminal domain of unknown function. Surprisingly, the Rep domain of Rep74/75 is more closely related to transposases encoded by IS elements than to Rep proteins of other RC plasmids. The p24 protein contains a hydrophobic segment, a highly charged region and a zinc finger motif. A recombinant p24 protein lacking the hydrophobic segment binds and condenses both single- and double-stranded DNA, and forms DNA aggregates with extreme compaction at high protein to DNA ratio. In addition to encoding proteins of significant interest, pTN1 is remarkable by being the only characterized plasmid isolated from a Thermococcus strain, thus being useful to develop genetic tools in Thermococcus kodakaraensis for which gene disruption methods became recently available.
Published: 2007

26. HIRIP3 is a nuclear phosphoprotein interacting with and phosphorylated by the serine-threonine kinase CK2

Author: Frédéric Galisson, Nadine Assrir, Odile Filhol, Marc Lipinski, Laboratoire de génétique et biologie cellulaire (LGBC), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Département Réponse et Dynamique Cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Epigenetique et Cancer, Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Dambo, Marie-Annie, Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), and Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-École Pratique des Hautes Études (EPHE)
Subjects: Clinical Biochemistry, MESH: Rabbits, Cell Cycle Proteins, MESH: Casein Kinase II, MESH: Amino Acid Sequence, Mitogen-activated protein kinase kinase, Biochemistry, 0302 clinical medicine, Protein Interaction Mapping, MESH: Animals, Phosphorylation, Nuclear protein, Casein Kinase II, 0303 health sciences, Chemistry, Nuclear Proteins, Chromatin, Cell biology, 030220 oncology & carcinogenesis, Rabbits, Casein kinase 2, DNA, Complementary, Molecular Sequence Data, MESH: Carrier Proteins, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Two-Hybrid System Techniques, 03 medical and health sciences, Two-Hybrid System Techniques, Animals, Humans, Amino Acid Sequence, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, 030304 developmental biology, MAPK14, Serine/threonine-specific protein kinase, MESH: K562 Cells, MESH: Humans, MESH: Molecular Sequence Data, MESH: Phosphorylation, fungi, MESH: Protein Interaction Mapping, MESH: DNA, Complementary, MESH: Hela Cells, Phosphoprotein, Cyclin-dependent kinase 9, Carrier Proteins, K562 Cells, MESH: Nuclear Proteins, HeLa Cells
Abstract: The HIRIP3 protein had been identified from its interaction with the HIRA histone chaperone. Experiments using anti-peptide antisera indicated that this 556-aa protein is nuclear throughout the cell cycle and excluded from condensed chromatin during mitosis. Based on its electrophoretic migration and sensitivity to phosphatase treatment, endogenous HIRIP3 was found to be heavily phosphorylated. HIRIP3 can be phosphorylated in vitro by a recombinant form of the serine-threonine kinase CK2. Moreover, HIRIP3 protein was found to co-purify with a CK2 activity. Together, these data prompt us to propose HIRIP3 as a new member of the growing list of CK2 substrates with a possible role in chromatin metabolism.
Published: 2007

27. Nucleosome chiral transition under positive torsional stress in single chromatin fibers

Author: Julien Mozziconacci, Andrei Sivolob, Natalia Conde e Silva, Jean-Louis Viovy, Gaudeline Wagner, Liliane Mouawad, Aurélien Bancaud, Maria Barbi, Jean-Marc Victor, Christophe Lavelle, Ariel Prunell, Eric Le Cam, Hua Wong, Physico-Chimie-Curie (PCC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), Department of General and Molecular Genetics, Taras Shevchenko National University, Taras Shevchenko National University of Kyiv, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Imagerie intégrative de la molécule à l'organisme, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Gustave Roussy (IGR), Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Pierre et Marie Curie - Paris 6 (UPMC), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), and Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Torsion Abnormality, Magnetic tweezers, Time Factors, Rotation, [SDV]Life Sciences [q-bio], Biology, Models, Biological, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, MESH: Rotation, Tetramer, MESH: Nucleosomes, RNA polymerase, Nucleosome, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, ComputingMilieux_MISCELLANEOUS, MESH: Biomechanics, 030304 developmental biology, 0303 health sciences, MESH: Time Factors, MESH: Models, Biological, Membrane Proteins, Biomolecules (q-bio.BM), Cell Biology, Linker DNA, Molecular biology, Biomechanical Phenomena, Nucleosomes, Chromatin, chemistry, Quantitative Biology - Biomolecules, FOS: Biological sciences, Biophysics, MESH: Membrane Proteins, Elongation, 030217 neurology & neurosurgery, DNA, MESH: Torsion
Abstract: Using magnetic tweezers to investigate the mechanical response of single chromatin fibers, we show that fibers submitted to large positive torsion transiently trap positive turns, at a rate of one turn per nucleosome. A comparison with the response of fibers of tetrasomes (the (H3-H4)2 tetramer bound with ~50 bp of DNA) obtained by depletion of H2A-H2B dimers, suggests that the trapping reflects a nucleosome chiral transition to a metastable form built on the previously documented righthanded tetrasome. In view of its low energy, 33 pages (double spacing), 7 figures
Published: 2007

28. Membrane-Bound Interleukin (IL)-15 on Renal Tumor Cells Rescues Natural Killer Cells from IL-2 Starvation-Induced Apoptosis

Author: Salem Chouaib, Bruno Azzarone, Anne Caignard, Paule Opolon, Sylvie Da Rocha, Krystel Khawam, Pierre Validire, Julien Giron-Michel, Sebastian Wittnebel, Bernard Escudier, Abdelali Jalil, Vectorologie et transfert de gènes (VTG / UMR8121), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Département d'immunothérapie, and Institut Gustave Roussy (IGR)
Subjects: Cancer Research, medicine.medical_specialty, Lung Neoplasms, Apoptosis, Biology, Natural killer cell, 03 medical and health sciences, Interleukin 21, 0302 clinical medicine, Lymphocytes, Tumor-Infiltrating, NK-92, Interleukin-15 Receptor alpha Subunit, Internal medicine, medicine, Humans, RNA, Small Interfering, Carcinoma, Renal Cell, 030304 developmental biology, Interleukin-15, 0303 health sciences, Lymphokine-activated killer cell, Microscopy, Confocal, Reverse Transcriptase Polymerase Chain Reaction, Cell Membrane, Receptor Cross-Talk, Natural killer T cell, Flow Cytometry, Immunohistochemistry, Kidney Neoplasms, Lymphocyte Subsets, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Killer Cells, Natural, medicine.anatomical_structure, Endocrinology, Oncology, Interleukin 15, 030220 oncology & carcinogenesis, Interleukin 12, Cancer research, Myeloid-derived Suppressor Cell, Interleukin-2
Abstract: Renal cell carcinoma primary tumors and lung metastases are infiltrated by activated natural killer (NK) cells. Interleukin (IL)-15, a major cytokine involved in cross-talk between accessory cells (dendritic cells and macrophages) and NK cells, is produced by epithelial renal cells. We show that renal cell carcinoma cells and normal renal cells express IL-15 mRNA and membrane-bound IL-15 (MbIL-15). These cells also express IL-15 receptor α (IL-15Rα). Silencing of IL-15Rα by specific small interfering RNA in renal cell carcinoma had no effect on MbIL-15 production, indicating that the cytokine is not cross-presented by IL-15Rα in renal cell carcinoma cells but anchored to the membrane. Furthermore, we show that MbIL-15 from renal cell carcinoma cells is functional and involved in rapid nuclear translocation of phosphorylated signal transducers and activators of transcription 3 in IL-2–starved NK cells. MbIL-15 on the target did not interfere with resting NK cell activation and target cell cytolysis but rescued NK cells from IL-2 starvation-induced apoptosis through contact-dependent interaction. Masking of MbIL-15 with soluble IL-15Rα molecules restored NK cell apoptosis. These findings suggest that IL-15 produced by renal tumor cells is involved in the maintenance of active NK cells at the tumor site. [Cancer Res 2007;67(12):5594–9]
Published: 2007

29. High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein--DNA complexes

Author: Pauline Dupaigne, Cyrille Le Breton, Loic Hamon, Eric Le Cam, David Pastré, Olivier Piétrement, Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Département de Radiobiologie et Radiopathologie (CEA/DSV/DRR), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut Gustave Roussy (IGR), and Maciejak, Olek
Subjects: DNA repair, Spermidine, [SDV]Life Sciences [q-bio], Ionic bonding, DNA, Single-Stranded, Cooperativity, 02 engineering and technology, Biology, Microscopy, Atomic Force, Divalent, Single-stranded binding protein, 03 medical and health sciences, chemistry.chemical_compound, Genetics, 030304 developmental biology, chemistry.chemical_classification, Electrophoresis, Agar Gel, 0303 health sciences, Escherichia coli Proteins, 021001 nanoscience & nanotechnology, Molecular biology, [SDV] Life Sciences [q-bio], DNA-Binding Proteins, stomatognathic diseases, chemistry, Biophysics, biology.protein, Methods Online, Aluminum Silicates, Mica, 0210 nano-technology, DNA
Abstract: International audience; DNA in living cells is generally processed via the generation and the protection of single-stranded DNA involving the binding of ssDNA-binding proteins (SSBs). The studies of SSB-binding mode transition and cooperativity are therefore critical to many cellular processes like DNA repair and replication. However, only a few atomic force microscopy (AFM) investigations of ssDNA nucleoprotein filaments have been conducted so far. The point is that adsorption of ssDN A–SSB complexes on mica, necessary for AFM imaging, is not an easy task. Here, we addressed this issue by using spermidine as a binding agent. This trivalent cation induces a stronger adsorption on mica than divalent cations, which are commonly used by AFM users but are ineffective in the adsorption of ssDNA–SSB complexes. At low spermidine concentration (
Published: 2007

30. Induction of glutathione synthesis explains pharmacodynamics of high-dose busulfan in mice and highlights putative mechanisms of drug interaction

Author: Micheline Re, Jérôme Bouligand, Estelle Daudigeos, Alain Deroussent, Paule Opolon, Gilles Vassal, Jackie Morizet, Nicolas Simonnard, Elisabeth Connault, Angelo Paci, Vectorologie et transfert de gènes (VTG / UMR8121), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Groupe d'étude de l'atmosphère météorologique (CNRM-GAME), Institut national des sciences de l'Univers (INSU - CNRS)-Météo France-Centre National de la Recherche Scientifique (CNRS), Centre national de recherches météorologiques (CNRM), Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire Midi-Pyrénées (OMP), and Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National d'Études Spatiales [Toulouse] (CNES)-Centre National de la Recherche Scientifique (CNRS)-Université Fédérale Toulouse Midi-Pyrénées-Météo-France -Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National d'Études Spatiales [Toulouse] (CNES)-Centre National de la Recherche Scientifique (CNRS)-Université Fédérale Toulouse Midi-Pyrénées-Météo-France -Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, MESH: Hepatic Veno-Occlusive Disease, Metabolic Clearance Rate, MESH: Busulfan, MESH: Drug Interactions, Hepatic Veno-Occlusive Disease, Pharmaceutical Science, Pharmacology, 03 medical and health sciences, chemistry.chemical_compound, Mice, 0302 clinical medicine, Pharmacokinetics, In vivo, MESH: Mice, Inbred C57BL, medicine, Animals, Drug Interactions, MESH: Animals, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Bone Marrow Transplantation, Busulfan, MESH: Mice, 030304 developmental biology, Bone Marrow Transplantation, Glutathione Transferase, MESH: Glutathione, MESH: Metabolic Clearance Rate, 0303 health sciences, MESH: Glutathione Transferase, Glutathione, Drug interaction, Acute toxicity, MESH: Male, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Mice, Inbred C57BL, chemistry, Liver, 030220 oncology & carcinogenesis, Pharmacodynamics, Toxicity, medicine.drug, MESH: Liver
Abstract: Busulfan is an example of a drug eliminated through glutathione S-transferase (GST)-catalyzed conjugation with reduced glutathione (GSH). We studied the pharmacokinetics and toxicity of busulfan in C57BL6 mice in correlation with liver GST activity and GSH synthesis by accurate determination of precursors, namely, gamma-glutamyl-cysteine and cysteine. A significantly lower incidence of acute toxicity was observed in mice receiving busulfan 16.5 mg/kg twice a day compared with animals receiving 33 mg/kg once a day. In both cases, a total dose of 132 mg/kg was administered over 4 days. The difference in toxicity was explained by pharmacokinetics since a strong induction of clearance was observed only in animals treated twice daily. Induction of metabolism was correlated with an increase in liver cysteine content and enhanced glutathione synthesis rate, whereas GST activity was unchanged. To our knowledge, this is the first time that in vivo flux of GSH synthesis has been shown to be closely related to a drug plasma clearance and toxicity. These results allow hypothesizing that GSH liver synthesis may directly influence busulfan clearance in humans with possible implications in the occurrence of hepatic veno-occlusive disease.
Published: 2007

31. An archaeal orthologue of the universal protein Kae1 is an iron metalloprotein which exhibits atypical DNA-binding properties and apurinic-endonuclease activity in vitro

Author: Danièle Gadelle, Herman van Tilbeurgh, Sophie Quevillon-Cheruel, Eric Le Cam, Nicolas Leulliot, Anthony Justome, Pierre Dorlet, Céline Brochier, Arnaud Hecker, Marc Graille, Patrick Forterre, Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale et microbiologie (IBSM), Université de la Méditerranée - Aix-Marseille 2-Université Paul Cézanne - Aix-Marseille 3-Université de Provence - Aix-Marseille 1-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biologie Moléculaire du Gène chez les Extrêmophiles (BMGE), Institut Pasteur [Paris] (IP), This work was supported by the Centre National de la Recherche Scientifique, by the Human Frontier Science Program (HSFP), the Association pour la Recherche sur le Cancer (ARC), the Agence Nationale de la Recherche (ANR) and by the European 3D-repertoire program (LSHG-CT-2005-512028). Funding to pay the Open Access publication charges for this article was provided by Institut Pasteur., Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Université de Provence - Aix-Marseille 1, Institut Pasteur [Paris], Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), and Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Pyrococcus abyssi, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Archaeal Proteins, [SDV]Life Sciences [q-bio], RAD51, MESH: Metalloendopeptidases, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, DNA-binding protein, 03 medical and health sciences, Endonuclease, chemistry.chemical_compound, Endopeptidase activity, Adenosine Triphosphate, Iron-Binding Proteins, MESH: Adenosine Triphosphate, Genetics, DNA-(Apurinic or Apyrimidinic Site) Lyase, AP site, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: DNA-(Apurinic or Apyrimidinic Site) Lyase, MESH: Iron-Binding Proteins, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Phylogeny, MESH: Pyrococcus abyssi, Phylogeny, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, Nucleic Acid Enzymes, 030302 biochemistry & molecular biology, MESH: DNA, Metalloendopeptidases, DNA, MESH: Archaeal Proteins, biology.organism_classification, DNA-(apurinic or apyrimidinic site) lyase, DNA-Binding Proteins, chemistry, Biochemistry, biology.protein, MESH: DNA-Binding Proteins, MESH: Models, Molecular
Abstract: The Kae1 (Kinase-associated endopeptidase 1) protein is a member of the recently identified transcription complex EKC and telomeres maintenance complex KEOPS in yeast. Kae1 homologues are encoded by all sequenced genomes in the three domains of life. Although annotated as putative endopeptidases, the actual functions of these universal proteins are unknown. Here we show that the purified Kae1 protein (Pa-Kae1) from Pyrococcus abyssi is an iron-protein with a novel type of ATP-binding site. Surprisingly, this protein did not exhibit endopeptidase activity in vitro but binds cooperatively to single and double-stranded DNA and induces unusual DNA conformational change. Furthermore, Pa-Kae1 exhibits a class I apurinic (AP)-endonuclease activity (AP-lyase). Both DNA binding and AP-endonuclease activity are inhibited by ATP. Kae1 is thus a novel and atypical universal DNA interacting protein whose importance could rival those of RecA (RadA/Rad51) in the maintenance of genome integrity in all living cells.
Published: 2007

32. EBV-associated nasopharyngeal carcinomas: from epidemiology to virus-targeting strategies

Author: Philippe Busson, Cécile Keryer, Tadamassa Ooka, Marilys Corbex, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Virologie et pathogenèse virale (VPV), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Genetic Epidemiology Unit, International Agency for Cancer Research (IACR), and I gratefully acknowledge the contributions of Becky Duerst in revision of the manuscript, and the thoughtful comments of Pat Stuart. I also extend an apology to many colleagues whose contributions to the research described here could not be cited owing to space constraints.
Subjects: Microbiology (medical), medicine.medical_specialty, Herpesvirus 4, Human, viruses, Nasopharyngeal Neoplasms/epidemiology/*virology, Nasopharyngeal neoplasm, Biology, Malignancy, Microbiology, Virus, 03 medical and health sciences, 0302 clinical medicine, Virology, Epidemiology, Genetic predisposition, medicine, Gene silencing, Humans, OCIS 000.1430, Carcinogen, Herpesviridae Infections/*complications/epidemiology, 030304 developmental biology, 0303 health sciences, Herpesvirus 4, Nasopharyngeal Neoplasms, Herpesviridae Infections, medicine.disease, 3. Good health, Infectious Diseases, Nasopharyngeal carcinoma, 030220 oncology & carcinogenesis, Immunology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Human
Abstract: Nasopharyngeal carcinoma is a human malignancy consistently associated with the Epstein-Barr virus. Exposure to non-viral carcinogens and genetic predisposition are other crucial etiologic factors. Tumor development appears to require the expression of a small subset of transforming viral RNAs and proteins with concomitant silencing of most other viral genes. Impairment of the interactions of viral proteins with cellular partners or disruption of viral latency might prove to be useful for novel therapeutic strategies.
Published: 2006

33. Transmission Electron Microscopy Reveals an Optimal HIV-1 Nucleocapsid Aggregation with Single-stranded Nucleic Acids and the Mature HIV-1 Nucleocapsid Protein

Author: Etienne Delain, Laurence Hameau, Robert J. Gorelick, Gilles Mirambeau, Sébastien Lyonnais, Anthony Justome, Josette Jeusset, Sophie Lafosse, Eric Le Cam, Dominique Coulaud, Institut Gustave Roussy (IGR), Laboratoire d'Electronique Moléculaire (LEM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Eco-Anthropologie et Ethnobiologie (EAE), Muséum national d'Histoire naturelle (MNHN)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), AIDS Vaccine Program, NCI at Frederick, Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), and Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)
Subjects: viruses, [SDV]Life Sciences [q-bio], DNA, Single-Stranded, Gene Products, gag, Context (language use), Biology, gag Gene Products, Human Immunodeficiency Virus, Viral Proteins, 03 medical and health sciences, chemistry.chemical_compound, Protein structure, Microscopy, Electron, Transmission, Structural Biology, Magnesium, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Zinc finger, 0303 health sciences, 030302 biochemistry & molecular biology, RNA, DNA, Nucleocapsid Proteins, Molecular biology, Reverse transcriptase, Protein Structure, Tertiary, Capsid, chemistry, HIV-1, Nucleic acid, Biophysics, Capsid Proteins
Abstract: HIV-1 nucleocapsid protein (NCp7) condenses the viral RNA within the mature capsid. In a capsid-free system, NCp7 promotes an efficient mechanism of aggregation with both RNA and DNA. Here, we show an analysis of these macromolecular complexes by dark-field imaging using transmission electron microscopy. Thousands of mature NCp7 proteins co-aggregate with hundreds of single-stranded circular DNA molecules (ssDNA) within minutes, as observed with poly(rA). These co-aggregates are highly stable but dynamic structures, as they dissociate under harsh conditions, and after addition of potent ssDNA or NCp7 competitive ligands. The N-terminal domain and zinc fingers of NCp7 are both required for efficient association. Addition of magnesium slightly increases the avidity of NCp7 for ssDNA, while it strongly inhibits co-aggregation with relaxed circular double-stranded DNA (dsDNA). This DNA selectivity is restricted to mature NCp7, compared to its precursors NCp15 and NCp9. Moreover, for NCp15, the linkage of NCp7 with the Gag C-terminal p6-peptide provokes a deficiency in ssDNA aggregation, but results in DNA spreading similar to prototypical SSB proteins. Finally, this co-aggregation is discussed in a dynamic architectural context with regard to the mature HIV-1 nucleocapsid. On the basis of the present data, we propose that condensation of encapsidated RNA requires the C-terminal processing of NCp. Subsequently, disassembly of the nucleocapsid should be favoured once dsDNA is produced by HIV-1 reverse transcriptase.
Published: 2006

34. Anionic Polyelectrolyte Adsorption on Mica Mediated by Multivalent Cations: A Solution to DNA Imaging by Atomic Force Microscopy under High Ionic Strengths

Author: David Pastré, Alain Zozime, Fabrice Landousy, Loic Hamon, Olivier Piétrement, Marie-Odile David, Eric Le Cam, Isabelle Sorel, Laboratoire Structure et Reconnaissance des Biomolécules, EA3637, Université Evry-Val d’Essonne (LSRB), Université d'Évry-Val-d'Essonne (UEVE), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), and Le Cam, Eric
Subjects: Anions, Polymers, Surface Properties, [SDV]Life Sciences [q-bio], Inorganic chemistry, Ionic bonding, 02 engineering and technology, Sodium Chloride, Microscopy, Atomic Force, Sensitivity and Specificity, Divalent, Electrolytes, 03 medical and health sciences, chemistry.chemical_compound, Adsorption, Cations, Electrochemistry, Molecule, General Materials Science, Particle Size, Spectroscopy, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Atomic force microscopy, Osmolar Concentration, DNA, Surfaces and Interfaces, 021001 nanoscience & nanotechnology, Condensed Matter Physics, Solutions, [SDV] Life Sciences [q-bio], stomatognathic diseases, Crystallography, chemistry, Polyelectrolyte adsorption, Aluminum Silicates, Mica, 0210 nano-technology
Abstract: Adsorption of DNA molecules on mica, a highly negatively charged surface, mediated by divalent or trivalent cations is considered. By analyzing atomic force microscope (AFM) images of DNA molecules adsorbed on mica, phase diagrams of DNA molecules interacting with a mica surface are established in terms of concentrations of monovalent salt (NaCl) and divalent (MgCl2) or multivalent (spermidine, cobalt hexamine) salts. These diagrams show two transitions between nonadsorption and adsorption. The first one arises when the concentration of multivalent counterions is larger than a limit value, which is not sensitive to the monovalent salt concentration. The second transition is due to the binding competition between monovalent and multivalent counterions. In addition, we develop a model of polyelectrolyte adsorption on like-charged surfaces with multivalent counterions. This model shows that the correlations of the multivalent counterions at the interface between DNA and mica play a critical role. Furthermore, it appears that DNA adsorption takes place when the energy gain in counterion correlations overcomes an energy barrier. This barrier is induced by the entropy loss in confining DNA in a thin adsorbed layer, the entropy loss in the interpenetration of the clouds of mica and DNA counterions, and the electrostatic repulsion between DNA and mica. The analysis of the experimental results provides an estimation of this energy barrier. We then discuss some important issues, including DNA adsorption under physiological conditions.
Published: 2006

35. Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles

Author: Patrick Tauc, Jean-Claude Brochon, Eric Deprez, Andrei Maksimenko, Alexander A. Ishchenko, Murat Saparbaev, Stabilité Génétique et Oncogenèse (UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Vectorologie et transfert de gènes (VTG / UMR8121), Laboratoire de Biotechnologie et Pharmacogénétique Appliquée (LBPA), Réparation de l’ADN (CNRS UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Bioalliance Pharma SA, Bioalliance Pharma, and Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11)
Subjects: MESH: Oxidation-Reduction, DNA Repair, MESH: Escherichia coli Proteins, MESH: Chelating Agents, MESH: Zinc, AP endonuclease, Substrate Specificity, Mice, MESH: Oxidants, DNA-(Apurinic or Apyrimidinic Site) Lyase, MESH: Animals, MESH: DNA-(Apurinic or Apyrimidinic Site) Lyase, Cells, Cultured, Chelating Agents, MESH: DNA Repair, 0303 health sciences, Multidisciplinary, Deoxyadenosines, MESH: Escherichia coli, Escherichia coli Proteins, 030302 biochemistry & molecular biology, MESH: DNA, Base excision repair, Biological Sciences, Oxidants, Deoxyribonuclease IV (Phage T4-Induced), [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Zinc, Biochemistry, Oxidation-Reduction, MESH: Cells, Cultured, MESH: Mutation, DNA repair, DNA damage, Biology, 03 medical and health sciences, MESH: Deoxyribonuclease IV (Phage T4-Induced), Drug Resistance, Bacterial, MESH: Drug Resistance, Bacterial, Escherichia coli, Animals, Humans, AP site, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Mice, 030304 developmental biology, MESH: DNA Damage, MESH: Humans, DNA, MESH: Deoxyadenosines, DNA-(apurinic or apyrimidinic site) lyase, DNA glycosylase, Mutation, biology.protein, MESH: Substrate Specificity, Nucleotide excision repair, DNA Damage
Abstract: The multifunctional DNA repair enzymes apurinic/apyrimidinic (AP) endonucleases cleave DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases in the base excision repair pathway. Alternatively, in the nucleotide incision repair (NIR) pathway, the same AP endonucleases incise DNA 5′ of a number of oxidatively damaged bases. At present, the physiological relevance of latter function remains unclear. Here, we report genetic dissection of AP endonuclease functions in base excision repair and NIR pathways. Three mutants of Escherichia coli endonuclease IV (Nfo), carrying amino acid substitutions H69A, H109A, and G149D have been isolated. All mutants were proficient in the AP endonuclease and 3′-repair diesterase activities but deficient in the NIR. Analysis of metal content reveals that all three mutant proteins have lost one of their intrinsic zinc atoms. Expression of the nfo mutants in a repair-deficient strain of E. coli complemented its hypersensitivity to alkylation but not to oxidative DNA damage. The differential drug sensitivity of the mutants suggests that the NIR pathway removes lethal DNA lesions generated by oxidizing agents. To address the physiological relevance of the NIR pathway in human cells, we used the fluorescence quenching mechanism of molecular beacons. We show that in living cells a major human AP endonuclease, Ape1, incises DNA containing α-anomeric 2′-deoxyadenosine, indicating that the intracellular environment supports NIR activity. Our data establish that NIR is a distinct and separable function of AP endonucleases essential for handling lethal oxidative DNA lesions.
Published: 2006

36. A new approach to DNA bending by polyamines and its implication in DNA condensation

Author: David Pastré, Isabelle Sorel, Alain Zozime, Fabrice Landousy, Eric Le Cam, Loic Hamon, Marie-Odile David, Etienne Delain, Olivier Piétrement, Laboratoire d'Etude des milieux Nanométriques (LMN), Université d'Évry-Val-d'Essonne (UEVE), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Interactions moléculaires et cancer (IMC (UMR 8126)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'étude des milieux nanométriques (LEMN), and Le Cam, Eric
Subjects: Models, Molecular, Spermidine, Base pair, Entropy, [SDV]Life Sciences [q-bio], Biophysics, DNA Solutions, Microscopy, Atomic Force, Nucleic Acid Denaturation, 010402 general chemistry, DNA condensation, 01 natural sciences, DNA-binding protein, Bleomycin, 03 medical and health sciences, chemistry.chemical_compound, Electrochemistry, Polyamines, Molecule, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Binding Sites, DNA, General Medicine, Polyamine binding, 0104 chemical sciences, [SDV] Life Sciences [q-bio], Solutions, Crystallography, Models, Chemical, chemistry, DNA, Viral, Nucleic Acid Conformation, Polyamine
Abstract: Polyamines are known to induce dynamical bending of DNA molecules. This mechanism is very important since many DNA binding proteins (DNAse, transcription factor, etc.) exert their action by their ability to bend DNA. We propose an analytical model which describes the dynamical bending of DNA by polyamine ions in highly diluted DNA solutions. The bending probability depends on the entropy loss of polyamines due to their localization. This localization is facilitated by the electrostatic repulsion between multivalent counterions condensed on DNA, which reduces the entropy loss in counterion localization. Therefore DNA bending by polyamines depends on the competition between monovalent counterions and polyamines. We find that the bending probability is weak for a low binding ratio of polyamines (i.e. number of bound polyamines per base pair), whereas a high bending probability can be reached at large polyamine binding ratio. In addition, we describe a new mechanism of DNA bending. It occurs with the help of thermal agitation, which initiates the bending and favours the polyamine localization. This model provides further insights into DNA bending by polyamines and its implication in DNA condensation. A qualitative estimation of the DNA bending probability is obtained by measuring the cleavage efficiency of DNA by bleomycin versus spermidine concentration. Indeed, a local helix distortion by polyamines results in an amplification of the double-strand cleavage by bleomycin. The measurement of the bleomycin amplification is performed by analysing images of DNA molecules with atomic force microscope. Some features of the dynamical bending indicate that condensation and bending are interrelated.
Published: 2006

37. Studying the effect of a charged surface on the interaction of bleomycin with DNA using an atomic force microscope

Author: Marie-Odile David, Stéphane Fusil, David Pastré, Fabrice Landousy, Loic Hamon, Olivier Piétrement, Eric Le Cam, Alain Zozime, Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Etude des milieux Nanométriques (LMN), Université d'Évry-Val-d'Essonne (UEVE), Interactions moléculaires et cancer (IMC (UMR 8126)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Unité mixte de physique CNRS/Thales (UMPhy CNRS/THALES), THALES-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Laboratoire d'étude des milieux nanométriques (LEMN), THALES [France]-Centre National de la Recherche Scientifique (CNRS), and Le Cam, Eric
Subjects: Surface Properties, DNA damage, [SDV]Life Sciences [q-bio], Static Electricity, Biophysics, Microscopy, Atomic Force, Cleavage (embryo), Bleomycin, 03 medical and health sciences, chemistry.chemical_compound, Adsorption, Coated Materials, Biocompatible, Image Interpretation, Computer-Assisted, Materials Testing, Static electricity, Microscopy, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Atomic force microscopy, 030302 biochemistry & molecular biology, DNA, General Medicine, [SDV] Life Sciences [q-bio], Crystallography, chemistry, Aluminum Silicates, DNA Damage
Abstract: The cleavage of DNA caused by the antitumoral drug bleomycin has been investigated using atomic force microscopy (AFM). This work deals with the effect that adsorbing DNA onto a positively- or negatively-charged surface has on the double-strand cleavage of DNA by Fe(III)/bleomycin. Quantitative analysis of the number of breaks per DNA molecule, in bulk and at the surface of the mica substrate, has been performed by analyzing AFM images. It turns out that the cleavage of DNA is strongly inhibited by a positively-charged surface. Our experiments can be interpreted using a simple electrostatic model. This paper is a first step in the study of DNA accessibility to ligand such as bleomycin, using AFM in liquids.
Published: 2005

38. Reversible Binding of DNA on NiCl 2 -Treated Mica by Varying the Ionic Strength

Author: Stéphane Fusil, and Alain Zozime, Eric Le Cam, Fabrice Landousy, Josette Jeusset, Loic Hamon, Olivier Piétrement, David Pastré, Marie-Odile David, Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Etude des milieux Nanométriques (LMN), Université d'Évry-Val-d'Essonne (UEVE), Unité mixte de physique CNRS/Thales (UMPhy CNRS/THALES), THALES-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Interactions moléculaires et cancer (IMC (UMR 8126)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'étude des milieux nanométriques (LEMN), THALES [France]-Centre National de la Recherche Scientifique (CNRS), and Le Cam, Eric
Subjects: [SDV]Life Sciences [q-bio], Nanotechnology, 010402 general chemistry, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Electrochemistry, Molecule, General Materials Science, Computer Science::Databases, Spectroscopy, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Quantitative Biology::Biomolecules, 0303 health sciences, Atomic force microscopy, food and beverages, Surfaces and Interfaces, Condensed Matter Physics, Quantitative Biology::Genomics, 0104 chemical sciences, [SDV] Life Sciences [q-bio], chemistry, Chemical engineering, Ionic strength, Mica, DNA
Abstract: The atomic force microscope is a key tool for investigating DNA conformation and DNA−protein interactions in liquid. The main advantage of this technique is that moving molecules can be studied in ...
Published: 2003

39. Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism

Author: Olivier Piétrement, Christophe Lavelle, Pauline Dupaigne, Gilles Mirambeau, Eric Le Cam, Anthony Justome, Marc Lipinski, Sophie Lafosse, Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Eco-Anthropologie et Ethnobiologie (EAE), Muséum national d'Histoire naturelle (MNHN)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Institut Gustave Roussy (IGR)
Subjects: Saccharomyces cerevisiae Proteins, [SDV]Life Sciences [q-bio], RAD51, lcsh:Medicine, Saccharomyces cerevisiae, macromolecular substances, Biology, Microscopy, Atomic Force, Chromatin remodeling, Protein filament, 03 medical and health sciences, Adenosine Triphosphate, 0302 clinical medicine, Biophysics/Macromolecular Assemblies and Machines, Recombinase, Nucleosome, Molecular Biology/Chromatin Structure, lcsh:Science, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Molecular Biology/DNA Repair, Multidisciplinary, lcsh:R, Chromatin Assembly and Disassembly, Chromatin, Nucleosomes, Cell biology, Nucleoprotein, Rec A Recombinases, enzymes and coenzymes (carbohydrates), Biochemistry/Macromolecular Assemblies and Machines, lcsh:Q, Rad51 Recombinase, biological phenomena, cell phenomena, and immunity, Homologous recombination, 030217 neurology & neurosurgery, Research Article
Abstract: Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics.
Published: 2008

40. Apoptosis and TRAF-1 cleavage in Epstein-Barr virus-positive nasopharyngeal carcinoma cells treated with doxorubicin combined with a farnesyl-transferase inhibitor

Author: Hector Ardila-Osorio, Abdelmajid Khabir, Rachid Jlidi, Marie C. Brezak, Philip G. Kasprzyk, Tadamassa Ooka, Isabelle Viossat, Jean Michel Vicat, Gregoire Prevost, Paule Opolon, Philippe Busson, Dolly P. Huang, Interactions moléculaires et cancer, Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Service d'Anatomie et de Cytologie Pathologique, Hopital Habib Bourguiba - Habib Bourguiba Hospital [Sfax], Institut Henri Beaufour, Biomeasure, Milford, Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Virologie et pathogenèse virale (VPV), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, and This work was supported by the association contre le cancer (grant 5238) , the institute Gustave Roussy (grant 2000.13) and by a joint grant from the INSERM (France) and the DGRST (Tunisia). Jean Michel Vicat was the recipient of the fellowship from the centre nationale de la recherche scientifique and from the ligue nationale contre le cancer (région centre). We thanks to Nouafal Zamzami for helpful discussion. Yann Lecluze and Elizabeth Connault for technical assistance.
Subjects: Herpesvirus 4, Human, Doxorubicin/*pharmacology, Apoptosis, medicine.disease_cause, Biochemistry, Nitriles/*pharmacology, Proteins/*metabolism, 0302 clinical medicine, Heterocyclic Compounds, Tumor Cells, Cultured, Cytotoxic T cell, Enzyme Inhibitors, Non-U.S. Gov't, OCIS 000.1430, 0303 health sciences, Cultured, Alkyl and Aryl Transferases/antagonists & inhibitors, 3. Good health, Tumor Cells, Drug Combinations, 030220 oncology & carcinogenesis, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Tumor necrosis factor alpha, Female, Heterocyclic Compounds, 3-Ring, Cell Division, medicine.drug, Programmed cell death, Cell Division/drug effects, 3-Ring/*pharmacology, Biology, Research Support, 03 medical and health sciences, Nitriles, medicine, otorhinolaryngologic diseases, Farnesyltranstransferase, Humans, Doxorubicin, 030304 developmental biology, Pharmacology, Alkyl and Aryl Transferases, Enzyme Inhibitors/*pharmacology, Human/isolation & purification, Herpesvirus 4, Proteins, Nasopharyngeal Neoplasms, medicine.disease, Epstein–Barr virus, TNF Receptor-Associated Factor 1, Nasopharyngeal Neoplasms/metabolism/*pathology/virology, stomatognathic diseases, Nasopharyngeal carcinoma, Cell culture, Cancer research
Abstract: Epstein-Barr virus (EBV)-associated nasopharyngeal carcinomas (NPC) are much more sensitive to chemotherapy than other head and neck carcinomas. Spectacular regressions are frequently observed after induction chemotherapy. However, these favorable responses are difficult to predict and often of short duration. So far there have been only few experiments to investigate the mechanisms which underline the cytotoxic effects of anti-neoplastic drugs against NPC cells. In addition, these studies were performed almost entirely on EBV-negative cell lines therefore not truly representative of NPC cells. For the first time, we have used two EBV-positive NPC tumor lines derived from a North African (C15) and a Chinese (C666-1) patient as in vitro targets for a panel of anti-neoplastic agents. Doxorubicin, taxol and in a lesser extent cis-platinum efficiently inhibited NPC cell proliferation at clinically relevant concentrations, but all three agents failed to induce apoptosis. However, massive apoptosis of C15 cells was achieved when doxorubicin (1 microM) was combined with a farnesyl-transferase inhibitor, BIM 2001 (5 microM). Moreover, this apoptotic process was associated with a caspase-dependent early cleavage of the TNF-receptor associated factor 1 (TRAF-1) molecule, a signaling adaptor which is specifically expressed in latently EBV-infected cells. TRAF-1 cleavage might become a useful indicator of chemo-induced apoptosis in EBV-associated NPCs.

41. Simultaneous miRNA and mRNA transcriptome profiling of human myoblasts reveals a novel set of myogenic differentiation-associated miRNAs and their target genes

Author: Ana Barat, Petr Dmitriev, Ahmed Turki, Thomas A. Walsh, Yegor S. Vassetzky, Dalila Laoudj-Chenivesse, Mary J. O'Connell, Philippe Dessen, Anna Polesskaya, Gilles Carnac, Marc Lipinski, Vladimir Lazar, Thomas Robert, Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046] (PhyMedExp), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Interactions moléculaires et cancer (IMC (UMR 8126)), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Génétique moléculaire et destin cellulaire (FRE 3377), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Bioinformatics and Molecular Evolution Group, Dublin City University [Dublin] (DCU)-School of Biotechnology, Institut Gustave Roussy (IGR), This research was supported by grants from the Association Française contre les Myopathies (AFM) to YSV and DL PD is co-funded by the University Montpellier I and the association 'Amis FSH'., Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), and BMC, Ed.
Subjects: Myoblast, Cellular differentiation, Down-Regulation, Skeletal muscle, Biology, Muscle Development, Proteomics, Myoblasts, 03 medical and health sciences, 0302 clinical medicine, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], microRNA, Genetics, Humans, RNA, Messenger, Muscle, Skeletal, Gene, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Messenger RNA, Myogenesis, Gene Expression Profiling, Methodology Article, Cell Differentiation, Molecular biology, CD56 Antigen, Up-Regulation, Cell biology, Gene expression profiling, MicroRNAs, 030220 oncology & carcinogenesis, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], DNA microarray, Biotechnology
Abstract: BackgroundmiRNA profiling performed in myogenic cells and biopsies from skeletal muscles has previously identified miRNAs involved in myogenesis.ResultsHere, we have performed miRNA transcriptome profiling in human affinity-purified CD56+ myoblasts induced to differentiatein vitro. In total, we have identified 60 miRNAs differentially expressed during myogenic differentiation. Many were not known for being differentially expressed during myogenic differentiation. Of these, 14 (miR-23b, miR-28, miR-98, miR-103, miR-107, miR-193a, miR-210, miR-324-5p, miR-324-3p, miR-331, miR-374, miR-432, miR-502, and miR-660) were upregulated and 6 (miR-31, miR-451, miR-452, miR-565, miR-594 and miR-659) were downregulated. mRNA transcriptome profiling performed in parallel resulted in identification of 6,616 genes differentially expressed during myogenic differentiation.ConclusionsThis simultaneous miRNA/mRNA transcriptome profiling allowed us to predict with high accuracy target genes of myogenesis-related microRNAs and to deduce their functions.
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