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1. First detection and genome sequencing of SARS‐CoV‐2 in an infected cat in France

Author

Loic Comtet, Bernard Klonjkowski, Marine Dumarest, Valérie Caro, Stéphan Zientara, Philippe Pourquier, Jessica Vanhomwegen, Manon Delaplace, Aurélia Kwasiborski, Corinne Sailleau, Jean-Claude Manuguerra, Véronique Hourdel, Sophie Le Poder, Alix Barbarino, Patrick Chevaillier, Virologie UMR1161 (VIRO), École nationale vétérinaire - Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris] (IP), Centre Collaborateur de l'OIE de Détection et identification chez l’homme des pathogènes animaux émergents et développement d’outils pour leur diagnostic / Collaborating Center for the Detection and identification in humans of emerging animal pathogens and development of tools for their diagnoses (CCOIE-OIECC), Institut Pasteur [Paris] (IP)-Organisation Mondiale de la Santé Animale / World Organisation Animal Health [Paris] (OIE), Clinique Vétérinaire de la place Davout [Savigny-sur-Orge], Centre Hospitalier Universitaire Vétérinaire d'Alfort [Maison Alfort] (CHUVA), École nationale vétérinaire - Alfort (ENVA)-PRES Université Paris-Est, IDvet [Grabels], Manon Delaplace is supported by a PhD fellowship from the DIM 1Health (Region Ile de France) and the Direction Générale de l’Enseignement et la Recherche from the French ministry of Agriculture (DGER)., The authors thank the veterinary practitioners for contributing to this study, the AFVAC (Association Française des Animaux de Compagnie) and the SNVEL of region Ile de France for their help. We are also grateful to Dr Meriadeg Le Gouil and Pr Astrid Vabret for helpful discussions. We thank Damien Hoinard for the technical support provided for the serological analysis., École nationale vétérinaire d'Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Pasteur [Paris], Organisation Mondiale de la Santé Animale / World Animal Health Information System (OIE-WAHIS)-Institut Pasteur [Paris], École nationale vétérinaire d'Alfort (ENVA)-PRES Université Paris-Est, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de la Recherche Agronomique (INRA)-École nationale vétérinaire d'Alfort (ENVA), Cellule d'Intervention Biologique d'Urgence (CIBU), Université Pierre et Marie Curie - Paris 6 (UPMC), Spectrométrie de Masse structurale et protéomique, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), IDvet, Signalisation et Mecanismes Moleculaires de l'Apoptose, and Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Coronavirus disease 2019 (COVID-19), 040301 veterinary sciences, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), viruses, next‐generation sequencing, Cat Diseases, DNA sequencing, SARS‐CoV‐2, Serology, 0403 veterinary science, 03 medical and health sciences, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Clade, skin and connective tissue diseases, Gene, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, CATS, biology, General Veterinary, General Immunology and Microbiology, SARS-CoV-2, fungi, cats, COVID-19, virus diseases, 04 agricultural and veterinary sciences, General Medicine, Virology, 3. Good health, body regions, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, biology.protein, luminex, Female, ELISA, next-generation sequencing, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, France, Antibody, pets, Rapid Communication
Abstract

International audience; After its first description in Wuhan (China), SARS-CoV-2 the agent of coronavirus disease 2019 (COVID-19) rapidly spread worldwide. Previous studies suggested that pets could be susceptible to SARS-CoV-2. Here, we investigated the putative infection by SARS-CoV-2 in 22 cats and 11 dogs from owners previously infected or suspected of being infected by SARS-CoV-2. For each animal, rectal, nasopharyngeal swabs and serum were taken. Swabs were submitted to RT-qPCR assays targeting 2 genes of SARS-CoV-2. All dogs were tested SARS-CoV-2 negative. One cat was tested positive by RT-qPCR on rectal swab. Nasopharyngeal swabs from this animal were tested negative. This cat showed mild respiratory and digestive signs. Serological analysis confirms the presence of antibodies against the SARS-CoV-2 in both serum samples taken 10 days apart. Genome sequence analysis revealed that the cat SARS-CoV-2 belongs to the phylogenetic clade A2a like most of the French human SARS-CoV-2. This study reports for the first time the natural infection of a cat in France (near Paris) probably through their owners. There is currently no evidence that cats can spread COVID-19 and owners should not abandon their pets or compromise their welfare.

Published
2020
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2. Prospective assessment of the predictive value of the BRCA1 gene status in sarcoma patients treated with trabectedin: an updated analysis of the EORTC 62091 trial

Author

F. Collin, Philippe Pourquier, Antoine Italiano, Saskia Litière, Nathan Touati, Alessandro Gronchi, Laboratory of Solid Tumors Genetics, Nice University Hospital, European Organization for Research and Treatment of Cancer (EORTC), EORTC, Centre Régional de Lutte contre le cancer Georges-François Leclerc [Dijon] (UNICANCER/CRLCC-CGFL), UNICANCER, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), CRLCC Val d'Aurelle - Paul Lamarque, Institut Bergonié [Bordeaux], Actions for OnCogenesis understanding and Target Identification in ONcology (ACTION), UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Fondazione IRCCS Istituto Nazionale Tumori - National Cancer Institute [Milan], and Herrada, Anthony

Subjects
0301 basic medicine, Oncology, Cancer Research, medicine.medical_specialty, [SDV]Life Sciences [q-bio], education, [SDV.CAN]Life Sciences [q-bio]/Cancer, law.invention, 03 medical and health sciences, 0302 clinical medicine, [SDV.CAN] Life Sciences [q-bio]/Cancer, Randomized controlled trial, law, Internal medicine, medicine, Radiology, Nuclear Medicine and imaging, Prospective cohort study, ComputingMilieux_MISCELLANEOUS, Trabectedin, Brca1 gene, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, business.industry, Clinical Cancer Research, medicine.disease, Predictive value, 3. Good health, Clinical trial, Editorial, 030104 developmental biology, 030220 oncology & carcinogenesis, Predictive value of tests, Sarcoma, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, medicine.drug
Abstract

International audience; We describe the predictive value of BRCA1 gene status on trabectedin efficacy and found no correlation despite the mechanisms of action of this drug that rely on DNA repair systems.

Published
2018

3. Carboxylate-functionalized foldamer inhibitors of HIV-1 integrase and Topoisomerase 1: artificial analogues of DNA mimic proteins

Author

Victor Dos Santos, Ivan Huc, Krzysztof Ziach, Valentina Corvaglia, Panchami Prabhakaran, Vincent Parissi, Pradeep K. Mandal, Carole Legeay, Daniel Carbajo, Philippe Pourquier, Rachel Isaksson Vogel, European Research Council, Herrada, Anthony, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Ludwig-Maximilians-Universität München (LMU), Sanofi-Aventis R&D, SANOFI Recherche, Microbiologie Fondamentale et Pathogénicité (MFP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Université Sciences et Technologies - Bordeaux 1-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Centre National de la Recherche Scientifique (CNRS), CNRS UMR 5097, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Chimie supramoléculaire biomimétique et bio-organique - Institut Européen de Chimie et Biologie, and Université Bordeaux Segalen - Bordeaux 2-Université Sciences et Technologies - Bordeaux 1

Subjects
Stereochemistry, Protein Conformation, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV]Life Sciences [q-bio], [CHIM.THER] Chemical Sciences/Medicinal Chemistry, HIV Integrase, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Crystallography, X-Ray, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Solid-phase synthesis, Chemical Biology and Nucleic Acid Chemistry, Biomimetic Materials, Genetics, Humans, HIV Integrase Inhibitors, ComputingMilieux_MISCELLANEOUS, Solid-Phase Synthesis Techniques, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, Molecular Structure, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Peptide stapling, Topoisomerase, Foldamer, Proteins, Phosphonate, Amides, 3. Good health, Integrase, Monomer, Enzyme, chemistry, DNA Topoisomerases, Type I, biology.protein, Biocatalysis, HIV-1, DNA, B-Form, Peptides, 030217 neurology & neurosurgery, DNA
Abstract

Inspired by DNA mimic proteins, we have introduced aromatic foldamers bearing phosphonate groups as synthetic mimics of the charge surface of B-DNA and competitive inhibitors of some therapeutically relevant DNA-binding enzymes: the human DNA Topoisomerase 1 (Top1) and the human HIV-1 integrase (HIV-1 IN). We now report on variants of these anionic foldamers bearing carboxylates instead of phosphonates. Several new monomers have been synthesized with protecting groups suitable for solid phase synthesis (SPS). Six hexadecaamides have been prepared using SPS. Proof of their resemblance to B-DNA was brought by the first crystal structure of one of these DNA-mimic foldamers in its polyanionic form. While some of the foldamers were found to be as active as, or even more active than, the original phosphonate oligomers, others had no activity at all or could even stimulate enzyme activity in vitro. Some foldamers were found to have differential inhibitory effects on the two enzymes. These results demonstrate a strong dependence of inhibitory activity on foldamer structure and charge distribution. They open broad avenues for the development of new classes of derivatives that could inhibit the interaction of specific proteins with their DNA target thereby influencing the cellular pathways in which they are involved. © The Author(s) 2019., Agence Nationale de la Recherche [ANR-11-BS07-013-01]; European Research Council under the European Union’s Seventh Framework Programme [ERC-2012-AdG-320892 and PEOPLE-2011-IEF-300948]; Ligue Contre Le Cancer (Comité Languedoc-Roussillon); SIRIC Montpellier Cancer [INCa Inserm DGOS 12553]; SIDACTION (AO2016, VIH2016721002). Funding for open access charge: University of Munich fund.

Published
2019

4. Les cellules progénitrices du système nerveux central sont à l’origine de la neurogenèse intratumorale

Author

Philippe Pourquier, Hadjer Dellal, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, and Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
[SDV] Life Sciences [q-bio], Cancer Research, Oncology, [SDV]Life Sciences [q-bio], Radiology, Nuclear Medicine and imaging, Hematology, General Medicine, Biology, Molecular biology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2019

5. Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

Author

Frédéric Godde, Jean-Marie Schmitter, Vincent Parissi, Valentina Corvaglia, Philippe Pourquier, Stéphane Chaignepain, Mathieu Marchivie, Céline Chollet, Ivan Huc, Panchami Prabhakaran, Krzysztof Ziach, Partha Pratim Bose, Katta Laxmi-Reddy, Toulin, Stéphane, BLANC - Mimes oligoamides aromatiques de l'ADN double brin - - ARYNAMICS2011 - ANR-11-BS07-0013 - BLANC - VALID, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Fondamentale et Pathogénicité (MFP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de la Matière Condensée de Bordeaux (ICMCB), Université de Bordeaux (UB)-Institut Polytechnique de Bordeaux-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), ANR-11-BS07-0013,ARYNAMICS,Mimes oligoamides aromatiques de l'ADN double brin(2011), Université Sciences et Technologies - Bordeaux 1-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Centre National de la Recherche Scientifique (CNRS), Service de Chimie Bio-Organique et de Marquage (SCBM), Médicaments et Technologies pour la Santé (MTS), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay, CNRS UMR 5097, Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB), Institut Européen de Chimie et Biologie, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Chimie supramoléculaire biomimétique et bio-organique - Institut Européen de Chimie et Biologie, Université Bordeaux Segalen - Bordeaux 2-Université Sciences et Technologies - Bordeaux 1, École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), and Microbiologie cellulaire et moléculaire et pathogénicité (MCMP)

Subjects
Surface Properties, [SDV]Life Sciences [q-bio], General Chemical Engineering, 010402 general chemistry, 01 natural sciences, DNA-binding protein, chemistry.chemical_compound, Molecule, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, [CHIM.MATE] Chemical Sciences/Material chemistry, biology, 010405 organic chemistry, Chemistry, Topoisomerase, Chemical tools, DNA-binding proteins, Nucleic acids, [CHIM.MATE]Chemical Sciences/Material chemistry, General Chemistry, Amides, 3. Good health, 0104 chemical sciences, Integrase, Enzyme, biology.protein, Nucleic acid, Biophysics, Nucleic Acid Conformation, DNA, B-Form, Double stranded, DNA
Abstract

This work was supported by the Agence Nationale de la Recherche (project no. ANR-11-BS07-013-01 and project RETROSelect, jcjc2011 program), by the French National Research Agency against AIDS (ANRS, AO2016), by SIDACTION (AO2016, VIH20160721002), by the European Union under the Seventh Framework Programme (grant agreements nos. ERC-2012-AdG-320892 and PEOPLE-2011-IEF-300948) and by the Ligue contre le Cancer (Comité Languedoc Roussillon).; International audience; Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein–DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer–protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein–DNA interactions.

Published
2018

6. LINE-1 as a therapeutic target for castration-resistant prostate cancer

Author

Philippe Pourquier, Pier Vincenzo Piazza, Nadine Houede, Pourquier, Philippe, Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), Physiopathologie du système nerveux central - Institut François Magendie, Université Bordeaux Segalen - Bordeaux 2-IFR8-Institut National de la Santé et de la Recherche Médicale (INSERM), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), and CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)

Subjects
0106 biological sciences, Oncology, Cyclopropanes, Male, medicine.medical_specialty, Efavirenz, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Disease, 01 natural sciences, 03 medical and health sciences, Prostate cancer, chemistry.chemical_compound, 0302 clinical medicine, Clinical Trials, Phase II as Topic, Prostate, Internal medicine, medicine, Humans, Cause of death, Chemotherapy, business.industry, Cancer, medicine.disease, 3. Good health, Benzoxazines, Clinical trial, [SDV] Life Sciences [q-bio], Gene Expression Regulation, Neoplastic, Prostatic Neoplasms, Castration-Resistant, medicine.anatomical_structure, Long Interspersed Nucleotide Elements, Treatment Outcome, chemistry, 030220 oncology & carcinogenesis, Alkynes, Reverse Transcriptase Inhibitors, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, 010606 plant biology & botany
Abstract

International audience; Prostate cancer is the third leading cause of death by cancer in men. Surgery or hormone deprivation usually contains the progression of the local forms of the disease. In metastatic situations, chemotherapy or second generation hormone therapies are used with an overall survival that never exceeds 36 months when tumors become resistant to castration. In the search for new alternatives, clinical trials with various classes of anticancer drugs have been performed, including chemotherapies, targeted therapies with kinase inhibitors, radium-223, or immunotherapies with somehow limited efficacy. Targeting LINE-1 with reverse transcriptase inhibitors was also proposed as an attractive strategy as retrotransposons may play a role in the initiation and the progression of prostate cancers. After reviewing the biological rational to use RT inhibitors in the treatment of prostate cancers, we will discuss the results of the phase II trial evaluating the efficacy of Efavirenz in the treatment of castration-resistant prostate cancers with a particular emphasis on pharmacokinetics data that were obtained. We will also discuss the positioning of other RT inhibitors in the current therapeutic armamentarium.

Published
2017

7. Association of NR1I2, CYP3A5 and ABCB1 genetic polymorphisms with variability of temsirolimus pharmacokinetics and toxicity in patients with metastatic bladder cancer

Author

Matthieu Gassiot, Philippe Pourquier, Nadine Houédé, Alejando Goberna, Litaty Mbatchi, Hakim Mahammedi, Loic Mourey, Serge Lumbroso, Alexandre Evrard, Florence Joly, Laboratoire de Biochimie [CHRU Nîmes], Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Institut Bergonié [Bordeaux], UNICANCER, Centre Jean Perrin [Clermont-Ferrand] (UNICANCER/CJP), Institut Universitaire du Cancer de Toulouse - Oncopole (IUCT Oncopole - UMR 1037), CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Régional de Lutte contre le Cancer François Baclesse [Caen] (UNICANCER/CRLC), UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU), Institut des Neurosciences de Montpellier - Déficits sensoriels et moteurs (INM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Medical oncology department [Toulouse], Institut Claudius Regaud, Cancers et préventions, Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Herrada, Anthony, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut National de la Santé et de la Recherche Médicale (INSERM), Normandie Université (NU)-UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN), and Institut des Neurosciences de Montpellier (INM)

Subjects
Male, 0301 basic medicine, Cancer Research, Temsirolimus, CYP3A5, Genotype, [SDV]Life Sciences [q-bio], Antineoplastic Agents, Single-nucleotide polymorphism, [SDV.CAN]Life Sciences [q-bio]/Cancer, Pharmacology, Toxicology, Polymorphism, Single Nucleotide, 03 medical and health sciences, 0302 clinical medicine, NR1I2, [SDV.CAN] Life Sciences [q-bio]/Cancer, Pharmacokinetics, Cytochrome P-450 CYP3A, Humans, Medicine, Pharmacology (medical), ComputingMilieux_MISCELLANEOUS, Aged, Sirolimus, Polymorphism, Genetic, Bladder cancer, business.industry, ABCB1, Middle Aged, medicine.disease, 3. Good health, 030104 developmental biology, Urinary Bladder Neoplasms, Oncology, Pharmacogenetics, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, 030220 oncology & carcinogenesis, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Female, Mantle cell lymphoma, business, medicine.drug
Abstract

International audience; PURPOSE:Temsirolimus is a mammalian target of rapamycin (mTOR) inhibitor that exhibits antitumor activity in renal cell carcinoma and mantle cell lymphoma. The metabolism of temsirolimus and its active metabolite sirolimus mainly depends on cytochrome P450 3A4/5 (CYP3A4/A5) and the ABCB1 transporter. Differently from sirolimus, no pharmacogenetic study on temsirolimus has been conducted. Therefore, the aim of this pilot study was to identify genetic determinants of the inter-individual variability in temsirolimus pharmacokinetics and toxicity.METHODS:Pharmacokinetic profiles were obtained for 16 patients with bladder cancer after intravenous infusion of 25 mg temsirolimus. Non-compartmental analysis was performed to calculate the pharmacokinetic parameters of temsirolimus and sirolimus, its main metabolite. The presence of single nucleotide polymorphisms (SNPs) in CYP3A5, ABCB1 and in their transcriptional regulator NR1I2 (PXR) was assessed by genotyping. Non-parametric statistical tests were used to assess associations between candidate SNPs and temsirolimus pharmacokinetics and toxicity.RESULTS:The ratio between sirolimus AUC and temsirolimus AUC was 1.6-fold higher in patients who experienced serious toxic events (p = 0.034). The frequency of adverse events was significantly higher in patients homozygous for the NR1I2-rs6785049 A allele (OR = 0.065, p = 0.04) or NR1I2-rs3814055 C allele (OR = 0.032, p = 0.006). These NR1I2 SNPs were also predictive of temsirolimus half-life and global exposure to temsirolimus and sirolimus. Finally, the effect of the ABCB1-rs1128503, ABCB1-rs2032582 and CYP3A5*3 SNPs on sirolimus pharmacokinetics was confirmed.CONCLUSIONS:Our findings suggest that SNPs of NR1I2 and its target genes CYP3A5 and ABCB1 are genetic determinants of temsirolimus pharmacokinetics and toxicity in patients with bladder cancer.

Published
2017

8. Polymorphisms in SLCO1B3 and NR1I2 as genetic determinants of hematotoxicity of carboplatin and paclitaxel combination

Author

Philippe Pourquier, Litaty Mbatchi, Fabienne Thomas, Alexandre Evrard, Yoann Cazaubon, Jacques Robert, Etienne Chatelut, Serge Lumbroso, Jean-Paul Brouillet, Antonin Schmitt, Jean-Christophe Boyer, Herrada, Anthony, Laboratoire de Biochimie [CHRU Nîmes], Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Individualisation des traitements des cancers ovariens (ITCO), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut Universitaire du Cancer Toulouse - Oncopôle (IUCT), Université de Montpellier (UM), Validation et identification de nouvelles cibles en oncologie (VINCO), Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut des Neurosciences de Montpellier (INM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2, Institut des Neurosciences de Montpellier - Déficits sensoriels et moteurs (INM), Centre Hospitalier Régional Universitaire de Nîmes (CHRU Nîmes), Centre d'épidémiologie des populations (CEP), Université de Bourgogne (UB)-Centre Régional de Lutte contre le cancer - Centre Georges-François Leclerc (CRLCC - CGFL), Evaluation et modélisation des effets thérapeutiques, Département biostatistiques et modélisation pour la santé et l'environnement [LBBE], 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), 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 Aimé Cotton (LAC), École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Institut de Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), EA3035, Institut Claudius Regaud, Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université Bordeaux Segalen - Bordeaux 2-Institut Bergonié - CRLCC Bordeaux-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, Oncology, Receptors, Steroid, [SDV]Life Sciences [q-bio], Organic Anion Transporters, Sodium-Independent, Pharmacology, 030226 pharmacology & pharmacy, Linkage Disequilibrium, Cohort Studies, chemistry.chemical_compound, paclitaxel, pregnane X receptor, 0302 clinical medicine, Gene Frequency, hemic and lymphatic diseases, ComputingMilieux_MISCELLANEOUS, Aged, 80 and over, Middle Aged, 3. Good health, Paclitaxel, 030220 oncology & carcinogenesis, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, carboplatin, Molecular Medicine, Female, Adult, medicine.medical_specialty, PXR, SNP, Antineoplastic Agents, Single-nucleotide polymorphism, Neutropenia, Polymorphism, Single Nucleotide, Solute Carrier Organic Anion Transporter Family Member 1B3, Young Adult, 03 medical and health sciences, Internal medicine, Genetics, medicine, Humans, Allele, Alleles, Genetic Association Studies, Aged, hematotoxicity, business.industry, Haplotype, SLCO1B3, medicine.disease, Antineoplastic Agents, Phytogenic, Thrombocytopenia, Carboplatin, drug metabolism, Haplotypes, chemistry, Pharmacodynamics, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, business
Abstract

Aim: The goal of our study was to assess the impact of patients’ genetic background on their sensitivity to carboplatin/paclitaxel hematotoxicity. Patients & methods: Parameters describing sensitivity to neutropenia and to thrombocytopenia of 201 patients were extracted from a previous pharmacokinetic/pharmacodynamics analysis, in order to assess their association with 52 candidates SNPs in 18 genes. Results: Carriers of a T allele of SLCO1B3-rs4149117 were 19% less sensitive to thrombocytopenia than the homozygotes for the G allele (p = 0.00279). Carriers of two copies of the ATG haplotypes of NR1I2-rs1523130, rs3814055 and rs1523127 were 19% less sensitive to thrombocytopenia than those harboring other haplotypes (p = 0.025). Conclusion: Our results revealed the importance of SLCO1B3 and NR1I2 in the sensitivity to carboplatin/paclitaxel thrombocytopenia.

Published
2015

9. Early objective response may not be a prognostic factor of survival for patients with metastatic urothelial carcinoma: from a retrospective analysis of a cohort of 113 patients

Author

P. Beuzeboc, Philippe Pourquier, Stéphane Culine, Nadine Houede, Stéphane Oudard, Amandine Quivy, Véronique Brouste, Christine Chevreau, Diego Tosi, Sandrine Lavau-Denes, Aude Flechon, Guilhem Roubaud, BMC, BMC, Service d'Oncologie Médicale [Bordeaux], Institut Bergonié [Bordeaux], UNICANCER-UNICANCER, UNICANCER, Service d'Oncologie médicale [CHU Pitié-Salpêtrière], 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), Service d'Oncologie Médicale, Centre Léon Bérard [Lyon], Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Service d'Oncologie médicale [CHU Limoges], CHU Limoges, Institut Claudius Regaud, Service d'oncologie médicale, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Service d'oncologie médicale [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), Service d'oncologie médicale [CHU Pitié-Salpêtrière], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université, Service d'Oncologie Médicale [Centre hospitalier Lyon Sud - HCL], Centre Hospitalier Lyon Sud [CHU - HCL] (CHLS), Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL), Institut Universitaire du Cancer de Toulouse - Oncopole (IUCT Oncopole - UMR 1037), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Saint-Louis, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), UNICANCER - Institut Bergonié [Bordeaux], Institut Bergonié Bordeaux, Institut Bergonié - CRLCC Bordeaux, Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Pitié-Salpêtrière [APHP], Institut de Recherche en Cancérologie de Montpellier ( IRCM - U1194 Inserm - UM ), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université de Montpellier ( UM ), CRLCC Institut Claudius Regaud, Assistance publique - Hôpitaux de Paris (AP-HP)-Université Paris Diderot - Paris 7 ( UPD7 ) -Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP)-Hôpital Européen Georges Pompidou [APHP] ( HEGP ), Centre Hospitalier Régional Universitaire de Nîmes ( CHRU Nîmes ), and Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)

Subjects
Male, Oncology, Multivariate analysis, Survival, [SDV]Life Sciences [q-bio], Cohort Studies, Pharmacology, Toxicology and Pharmaceutics(all), Objective response, Prospective Studies, General Pharmacology, Toxicology and Pharmaceutics, Prospective cohort study, ComputingMilieux_MISCELLANEOUS, Medicine(all), General Medicine, Middle Aged, Prognosis, Metastatic urothelial carcinoma, 3. Good health, Survival Rate, [SDV] Life Sciences [q-bio], Treatment Outcome, Cohort, Female, France, Cohort study, Adult, Urologic Neoplasms, medicine.medical_specialty, Metastatic Urothelial Carcinoma, Antineoplastic Agents, Prognostic factors, General Biochemistry, Genetics and Molecular Biology, Internal medicine, medicine, Humans, Survival rate, Aged, Retrospective Studies, [ SDV ] Life Sciences [q-bio], business.industry, Biochemistry, Genetics and Molecular Biology(all), Research, Cancer, Retrospective cohort study, medicine.disease, Surgery, Cisplatin, business, Cisplatin-based regimen, Follow-Up Studies
Abstract

Background This study aims to better define prognostic factors for patients with metastatic urothelial carcinoma (mUC), and to identify patients who will benefit from first-line cisplatin-based chemotherapy. We test the hypothesis that early objective response (EOR), defined as the occurrence of an objective response following 2 or 3 courses of chemotherapy, could be a prognostic factor for overall survival (OS) and thus be used to guide treatment decisions. Data from 113 patients with evaluable mUC receiving first-line cisplatin-based treatment between January 2004 and December 2006 was collected retrospectively from prospectively-maintained databases across seven French cancer centers. Clinical factors potentially associated with survival and EOR were analyzed in univariate and multivariate analysis. Results One hundred three patient records were complete and available for inclusion in the multivariate model. Four factors were independently associated with OS: Performance status 1 and 2 (HR 2.3 [95 % CI 1.3–3.9], p = 0.002; HR 3.4 [95 % CI 1.6–7.2], p = 0.001 respectively); presence of visceral metastases (HR 2.2 [95 % CI 1.3–3.9], p = 0.004); abnormal hemoglobin levels (HR 1.7 [95 % CI 1.01–2.8], p = 0.045); disease progression (HR 10.1 [95 % CI 4.2–24.1], p < 0.001). Conclusions This study confirms the prognostic factors previously reported in first-line chemotherapy for mUC. However, we failed to demonstrate that EOR was an independent predictive factor of OS. Nevertheless, an early response evaluation is recommended since early progression is an important parameter that can be used to decide whether treatment should be interrupted and changed for alternative strategies integrating the concept of personalized medicine or new immune therapies.

Published
2015

10. Apoptotic Topoisomerase I-DNA Complexes Induced by Staurosporine-mediated Oxygen Radicals

Author

Isabelle Plo, Akira Yoshida, Yoshimasa Urasaki, Murat Saparbaev, Smitha Antony, Qasim A. Khan, Glenda Kohlhagen, Yves Pommier, Philippe Pourquier, Eric Solary, Jacques Laval, Olivier Sordet, Pourquier, Philippe, Hematopoïèse et Cellules Souches (U362), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hématopoïèse normale et pathologique (U1170 Inserm), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Lipides - Nutrition - Cancer (U866) (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA), 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), Laboratory of Molecular Pharmacology, National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), and National Institutes of Health [Bethesda] (NIH)

Subjects
[SDV]Life Sciences [q-bio], Apoptosis, DNA Fragmentation, Cleavage (embryo), Biochemistry, law.invention, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, law, Tumor Cells, Cultured, medicine, Humans, Staurosporine, Enzyme Inhibitors, Molecular Biology, 030304 developmental biology, 0303 health sciences, biology, Topoisomerase, Apoptotic DNA fragmentation, DNA, Cell Biology, Cell cycle, Molecular biology, Cell biology, [SDV] Life Sciences [q-bio], DNA Topoisomerases, Type I, chemistry, 030220 oncology & carcinogenesis, biology.protein, Recombinant DNA, DNA supercoil, Reactive Oxygen Species, medicine.drug
Abstract

International audience; Topoisomerase I (Top1), an abundant nuclear enzyme expressed throughout the cell cycle, relaxes DNA supercoiling by forming transient covalent DNA cleavage complexes. We show here that staurosporine, a ubiquitous inducer of apoptosis in mammalian cells, stabilizes cellular Top1 cleavage complexes. These complexes are formed indirectly as staurosporine cannot induce Top1 cleavage complexes in normal DNA with recombinant Top1 or nuclear extract from normal cells. In treated cells, staurosporine produces oxidative DNA lesions and generates reactive oxygen species (ROS). Quenching of these ROS by the antioxidant N-acetyl-l-cysteine or inhibition of the mitochondrial dependent production of ROS by the caspase inhibitor benzyloxycarbonyl-VAD prevents staurosporine-induced Top1 cleavage complexes. Down-regulation of Top1 by small interfering RNA decreases staurosporine-induced apoptotic DNA fragmentation. We propose that Top1 cleavage complexes resulting from oxidative DNA lesions generated by ROS in staurosporine-treated cells contribute to the full apoptotic response.

Published
2004

11. Structural Basis for Topoisomerase I Inhibition by Nucleoside Analogs

Author

Yves Pommier, Shuyuan Yu, Philippe Pourquier, Richard T. Pon, William H. Gmeiner, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Molecular Pharmacology, National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), and National Institutes of Health [Bethesda] (NIH)

Subjects
Antimetabolites, Antineoplastic, DNA polymerase, [SDV]Life Sciences [q-bio], Topoisomerase-I Inhibitor, Cleavage (embryo), Deoxycytidine, Biochemistry, chemistry.chemical_compound, Genetics, medicine, Enzyme Inhibitors, biology, Nucleoside analogue, Chemistry, Topoisomerase, Cytarabine, DNA replication, Nucleosides, Cytidine, General Medicine, Gemcitabine, [SDV] Life Sciences [q-bio], biology.protein, Molecular Medicine, Topoisomerase I Inhibitors, DNA, medicine.drug
Abstract

International audience; Nucleoside analogs such as 1-beta-D-arabinofuranosyl cytidine (AraC) and 2',2'-difluoro deoxycytidine (dFdC) are important components of the anticancer chemotherapeutic arsenal and are among the most effective anticancer drugs currently available. Although both AraCTP and dFdCTP impede DNA replication through pausing of DNA polymerases, both nucleoside analogs are ultimately incorporated into replicated DNA and interfere in DNA-mediated processes. Our laboratories are investigating the structural basis for the poisoning of topoisomerase I (top1) due to antipyrimidine incorporation into duplex DNA. We recently reported that both AraC and dFdC induce formation of top1 cleavage complexes, and poisoning of top1 contributes to the anticancer activities of both these drugs. Recent NMR and thermodynamic studies from our laboratories provide insight into the mechanism by which AraC and dFdC poison top1. NMR studies from our laboratories have revealed that the arabinosyl sugar of AraC adopted a C2'-endo conformation. Although this is a B-type sugar pucker characteristic of duplex DNA, the conformation is rigid, and this lack of flexibility probably contributes to inhibition of the religation step of the top1 reaction. In contrast to AraC, NMR studies revealed dFdC adopted a C3' endo sugar pucker characteristic of RNA, rather than DNA duplexes. dFdC substitution enhanced formation of top1 cleavage complexes, but did not inhibit religation. The enhancement of top1 cleavage complexes most likely results from a combination of conformational and electrostatic effects. The structural effects of dFdC and AraC are being further investigated in duplex DNA with well-defined top1 cleavage sites to analyze more specifically how these structural perturbations lead to enzyme poisoning.

Published
2003

12. Targeting the genetic alterations of the PI3K-AKT-mTOR pathway: Its potential use in the treatment of bladder cancers

Author

Philippe Pourquier, Nadine Houede, Service d'Oncologie Médicale, Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Le Ster, Yves, 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), Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, and Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
PTEN, [SDV]Life Sciences [q-bio], Urothelial bladder cancer, [SDV.CAN]Life Sciences [q-bio]/Cancer, PI3K, Phosphatidylinositol 3-Kinases, chemistry.chemical_compound, [SDV.CAN] Life Sciences [q-bio]/Cancer, medicine, Animals, Humans, Pharmacology (medical), Protein kinase B, ComputingMilieux_MISCELLANEOUS, PI3K/AKT/mTOR pathway, Phosphoinositide-3 Kinase Inhibitors, Pharmacology, Everolimus, Bladder cancer, Vinflunine, biology, TOR Serine-Threonine Kinases, AKT, Cancer, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, medicine.disease, Gemcitabine, 3. Good health, TSC1, [SDV] Life Sciences [q-bio], [SDV.SP] Life Sciences [q-bio]/Pharmaceutical sciences, Urinary Bladder Neoplasms, chemistry, Cancer research, biology.protein, mTOR, Proto-Oncogene Proteins c-akt, medicine.drug
Abstract

International audience; : Urothelial carcinoma of the bladder is the most frequent tumor of the urinary tract and represents the fifth cause of death by cancer worldwide. The current first line chemotherapy is a combination of cisplatin and gemcitabine with median survival not exceeding 15months. Vinflunine is the only drug approved by EMEA as second-line treatment and few progresses have been made for the past 20years to increase the survival of metastatic patients, especially those who are not eligible for cisplatin-based regimen. The recent studies characterizing the genetic background of urothelial cancers of the bladder, revealed chromosomal alterations that are not seen at the same level in other types of cancers. This is especially the case for mutations of genes involved in the PI3K/AKT/mTOR signaling pathway that occupies a major place in the etiology of these tumors. Here, we describe the mutations leading to constitutive activation of the PI3K/AKT/mTOR pathway and discuss the potential use of the different classes of PI3K/AKT/mTOR inhibitors in the treatment of urothelial bladder cancers. Despite the recent pivotal study evidencing specific mutations of TSC1 in bladder cancer patients responding to everolimus and the encouraging results obtained with other derivatives than rapalogs, few clinical trials are ongoing in bladder cancers. Because of the genetic complexity of these tumors, the cross-talks of the PI3K/AKT/mTOR pathway with other pathways, and the small number of eligible patients, it will be of utmost importance to carefully choose the drugs or drug combinations to be further tested in the clinic.

Published
2014

13. From old alkylating agents to new minor groove binders

Author

Philippe Pourquier, Danièle Montaudon, Stéphane Puyo, Pourquier, Philippe, IHU-LIRYC, CHU Bordeaux [Bordeaux]-Université Bordeaux Segalen - Bordeaux 2, Signalisation et Mecanismes Moleculaires de l'Apoptose, and Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Genetics, Alkylating Agents, O6-methylguanine, Alkylation, business.industry, [SDV]Life Sciences [q-bio], Hematology, DNA, Bioinformatics, Tumor response, [SDV] Life Sciences [q-bio], Oncology, Drug Resistance, Neoplasm, Neoplasms, Medicine, Animals, Humans, business, Antineoplastic Agents, Alkylating, ComputingMilieux_MISCELLANEOUS, Minor groove, Signal Transduction
Abstract

Alkylating agents represent the oldest class of anticancer agents with the approval of mechloretamine by the FDA in 1949. Even though their clinical use is far beyond the use of new targeted therapies, they still occupy a major place in the treatment of specific malignancies, sometimes representing the unique option for the treatment of refractory tumors. Here, we are reviewing the major classes of alkylating agents, with a particular focus on the latest generations of compounds that specifically target the minor groove of the DNA. These naturally occurring derivatives have a unique mechanism of action that explains the recent regain of interest in developing new classes of alkylating agents that could be used in combination with other anticancer drugs to enhance tumor response in the clinic.

Published
2014

14. Major Efficacy of Trabectedin in 2 Metastatic Osteosarcoma Patients with Wild-Type Asp1104 ERCC5 Tumor Status

Author

Philippe Pourquier, Antoine Thyss, Valérie Le Morvan, Esma Saâda-Bouzid, Lauris Gastaud, Juliette Thariat, Antoine Ianessi, Antoine Italiano, Centre de Lutte contre le Cancer Antoine Lacassagne [Nice] (UNICANCER/CAL), UNICANCER-Université Côte d'Azur (UCA), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Régional de Lutte contre le Cancer François Baclesse [Caen] (UNICANCER/CRLC), UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU), Laboratory of Solid Tumors Genetics, Nice University Hospital, Haematology, Centre Hospitalier Universitaire de Nice (CHU Nice), Normandie Université (NU)-UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN), and Pourquier, Philippe

Subjects
Oncology, Male, Cancer Research, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Tumor Status, 0302 clinical medicine, Tetrahydroisoquinolines, Trabectedin, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Osteosarcoma, Nuclear Proteins, Hematology, 3. Good health, [SDV] Life Sciences [q-bio], DNA-Binding Proteins, Treatment Outcome, 030220 oncology & carcinogenesis, Metastatic osteosarcoma, Female, medicine.drug, musculoskeletal diseases, Surgical resection, Genetic Markers, medicine.medical_specialty, Adolescent, Bone Neoplasms, Dioxoles, Polymorphism, Single Nucleotide, 03 medical and health sciences, Young Adult, Internal medicine, medicine, Biomarkers, Tumor, Humans, neoplasms, 030304 developmental biology, Chemotherapy, business.industry, Wild type, medicine.disease, Endonucleases, Cancer research, Methotrexate, business, Transcription Factors
Abstract

Background: Treatment of osteosarcoma of the extremities consists of surgical resection preceded and followed by chemotherapy, including high-dose methotrexate or adriamycin-based protocols. When distant relapse occurs, therapeutic options are scarce. Trabectedin, a DNA-binding agent, is indicated for the treatment of patients with advanced soft tissue sarcomas after failure of anthracyclines and ifosfamide. In this indication, the 6-month progression-free survival is about 35-40%. Recent reports showed that some specific single nucleotide polymorphisms (SNPs) from DNA repair genes could be associated with sensitivity to trabectedin in soft tissue sarcomas. Case Reports: We report our experience of 2 metastatic, heavily pre-treated osteosarcoma patients who were treated with trabectedin. Pyrosequencing analyses of tumors from both patients for several SNPs of the ERCC1, ERCC5 and BRAC1 genes were performed. Both patients showed major response to trabectedin, which was interestingly related with homozygoty of the common guanine allele of ERCC5 (G/G genotype; Asp/Asp) after pyrosenquencing analysis of tumors from both patients. This polymorphism was previously shown to be associated with better outcome in soft tissue sarcoma patients treated with trabectedin. Conclusion: Homozygoty for the wild-type Asp1104 SNP of the ERCC5 gene was found in 2 cases of relapsed osteosarcoma, who responded to trabectedin.

Published
2013

15. Gene Expression Signature Predicting High-Grade Prostate Cancer Responses to Oxaliplatin

Author

Stéphane Puyo, Philippe Pourquier, Audrey Kauffmann, Nadine Houede, Pierre Richaud, Jacques Robert, IHU-LIRYC, Université Bordeaux Segalen - Bordeaux 2-CHU Bordeaux [Bordeaux], Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), Bioénergie et Microalgues (EBM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Aimé Cotton (LAC), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-École normale supérieure - Cachan (ENS Cachan), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), and Pourquier, Philippe

Subjects
Male, Organoplatinum Compounds, [SDV]Life Sciences [q-bio], Antineoplastic Agents, Bioinformatics, 03 medical and health sciences, Prostate cancer, 0302 clinical medicine, DU145, Prostate, Cell Line, Tumor, LNCaP, medicine, Humans, Gene silencing, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Pharmacology, Cisplatin, 0303 health sciences, business.industry, Prostatic Neoplasms, Cancer, medicine.disease, 3. Good health, Oxaliplatin, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, 030220 oncology & carcinogenesis, Cancer research, Molecular Medicine, Neoplasm Grading, Transcriptome, business, medicine.drug
Abstract

Prostate cancer is one of the leading causes of cancer-related deaths among men. Several prognostic factors allow differentiation of low-grade tumors from high-grade tumors with high metastatic potential. High-grade tumors are currently treated with hormone therapy, to which taxanes are added when the tumors become resistant to castration. Clinical trials with other anticancer agents did not take into account the genetic backgrounds of the tumors, and most trials demonstrated low response rates. Here we used an in silico approach to screen for drug candidates that might be used as alternatives to taxanes, on the basis of a published expression signature involving 86 genes that could distinguish high-grade and low-grade tumors (Proc Natl Acad Sci USA 103:10991-10996, 2006). We explored the National Cancer Institute databases, which include data on the gene expression profiles of 60 human tumor cell lines and the in vitro sensitivities of the cell lines to anticancer drugs, and we identified several genes in the signature for which expression levels were correlated with chemosensitivity. As an example of the validation of this in silico approach, we identified a set of six genes for which expression levels could predict cell sensitivity to oxaliplatin but not cisplatin. This signature was validated in vitro through silencing of the genes in DU145, LNCaP, and C4-2B prostate cancer cells, which was accompanied by changes in oxaliplatin but not cisplatin cytotoxicity. These results demonstrate the relevance of our approach for the identification of both alternative treatments for high-grade prostate cancers and new biomarkers to predict clinical tumor responses.

Published
2012

16. The Polyphenolic Ellagitannin Vescalagin Acts As a Preferential Catalytic Inhibitor of the α Isoform of Human DNA Topoisomerase II

Author

Rémi Jacquet, Céline Auzanneau, Danièle Montaudon, Francesca De Giorgi, Denis Deffieux, Philippe Pourquier, Stéphane Puyo, Laurent Pouységu, Assia Elkaoukabi-Chaibi, Stéphane Quideau, François Ichas, Pourquier, Philippe, Validation et identification de nouvelles cibles en oncologie (VINCO), Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut des Sciences Moléculaires (ISM), Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1 (UB)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), IHU-LIRYC, Université Bordeaux Segalen - Bordeaux 2-CHU Bordeaux [Bordeaux], Plateforme de génétique moléculaire des cancers d'Aquitaine, UNICANCER-UNICANCER, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Université Sciences et Technologies - Bordeaux 1-Université Montesquieu - Bordeaux 4-Institut de Chimie du CNRS (INC)

Subjects
Gene isoform, [SDV]Life Sciences [q-bio], Down-Regulation, Antineoplastic Agents, Biology, 01 natural sciences, Catalysis, 03 medical and health sciences, chemistry.chemical_compound, Epipodophyllotoxin, Antigens, Neoplasm, medicine, Tumor Cells, Cultured, Humans, Protein Isoforms, Doxorubicin, DNA Breaks, Double-Stranded, Cytotoxicity, Poly-ADP-Ribose Binding Proteins, Etoposide, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Cell Proliferation, Pharmacology, chemistry.chemical_classification, 0303 health sciences, 010405 organic chemistry, Topoisomerase, DNA, Kinetoplast, Hydrolyzable Tannins, 3. Good health, 0104 chemical sciences, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], Enzyme, DNA Topoisomerases, Type II, chemistry, Biochemistry, biology.protein, Molecular Medicine, Oxidation-Reduction, DNA, medicine.drug
Abstract

Polyphenolic ellagitannins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models by a mechanism that remains unclear. Potential targets have been proposed, including DNA topoisomerases II (Top2). Top2α and Top2β, the two isoforms of the human Top2, play a crucial role in the regulation of replication, transcription, and chromosome segregation. They are the target of anticancer agents used in the clinic such as anthracyclines (e.g., doxorubicin) or the epipodophyllotoxin etoposide. It was recently shown that the antitumor activity of etoposide was due primarily to the inhibition of Top2α, whereas inhibition of Top2β was responsible for the development of secondary malignancies, pointing to the need for more selective Top2α inhibitors. Here, we show that the polyphenolic ellagitannin vescalagin preferentially inhibits the decatenation activity of Top2α in vitro, by a redox-independent mechanism. In CEM cells, we also show that transient small interfering RNA-mediated down-regulation of Top2α but not of Top2β conferred a resistance to vescalagin, indicating that the α isoform is a preferential target. We further confirmed that Top2α inhibition was due to a catalytic inhibition of the enzyme because it did not induce DNA double-strand breaks in CEM-treated cells but prevented the formation of Top2α- rather than Top2β-DNA covalent complexes induced by etoposide. To our knowledge, vescalagin is the first example of a catalytic inhibitor for which cytotoxicity is due, at least in part, to the preferential inhibition of Top2α.

Published
2012

17. [Alkylating agents]

Author

Pourquier, Philippe, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, and Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
[SDV] Life Sciences [q-bio], Poly Adenosine Diphosphate Ribose, DNA Repair Enzymes, DNA Repair, [SDV]Life Sciences [q-bio], Neoplasms, Tumor Suppressor Proteins, Humans, Proteins, Drug Synergism, DNA, Neoplasm, Antineoplastic Agents, Alkylating, DNA Modification Methylases
Abstract

International audience; With the approval of mechlorethamine by the FDA in 1949 for the treatment of hematologic malignancies, alkylating agents are the oldest class of anticancer agents. Even though their clinical use is far beyond the use of new targeted therapies, they still occupy a major place in specific indications and sometimes represent the unique option for the treatment of refractory diseases. Here, we are reviewing the major classes of alkylating agents and their mechanism of action, with a particular emphasis for the new generations of alkylating agents. As for most of the chemotherapeutic agents used in the clinic, these compounds are derived from natural sources. With a complex but original mechanism of action, they represent new interesting alternatives for the clinicians, especially for tumors that are resistant to conventional DNA damaging agents. We also briefly describe the different strategies that have been or are currently developed to potentiate the use of classical alkylating agents, especially the inhibition of pathways that are involved in the repair of DNA lesions induced by these agents. In this line, the development of PARP inhibitors is a striking example of the recent regain of interest towards the "old" alkylating agents.

Published
2011

18. ERCC5/XPG, ERCC1, and BRCA1 gene status and clinical benefit of trabectedin in patients with soft tissue sarcoma

Author

Juan Carlos Tercero, Roberta Sanfilippo, Isabelle Ray-Coquard, Antoine Italiano, Audrey Laroche, Jean-Yves Blay, Armelle Laurand, Axel Le Cesne, Binh Bui, Jean Michel Coindre, Paolo G. Casali, Jacques Robert, Ian Judson, Antonio Nieto, Philippe Pourquier, José Enrique Garzón Jimeno, Laboratory of Solid Tumors Genetics, Nice University Hospital, Virologie Structurale, Institut Pasteur [Paris], Département de médecine oncologique [Gustave Roussy], Institut Gustave Roussy (IGR), Equipe 11, Centre de Recherche en Cancérologie de Lyon (CRCL), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Service d'Oncologie Médicale, Centre Léon Bérard [Lyon], Département d'oncologie médicale, Institut Bergonié - CRLCC Bordeaux, Service de Pathologie, Institut Bergonié, Children’s Hospital La Fe, Laboratoire Aimé Cotton (LAC), École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Scanella, Marie-Pierre, Institut Bergonié [Bordeaux], UNICANCER-UNICANCER, Institut de signalisation, biologie du développement et cancer (ISBDC), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Institut Pasteur [Paris] (IP), Service d'Oncologie Médicale, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), 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 National de la Recherche Scientifique (CNRS), Pediatric Allergy and Pneumology Unit, Children's Hospital La Fe, HEC Montréal (HEC Montréal), Validation et identification de nouvelles cibles en oncologie (VINCO), UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Service d'Oncologie Médicale

Subjects
Male, Cancer Research, DNA Repair, [SDV]Life Sciences [q-bio], Genes, BRCA1, MESH: Flow Cytometry, Apoptosis, MESH: Antineoplastic Agents, Alkylating, MESH: Base Sequence, MESH: Tetrahydroisoquinolines, 0302 clinical medicine, MESH: Endonucleases, Tetrahydroisoquinolines, MESH: Dioxoles, skin and connective tissue diseases, Trabectedin, Cell Line, Transformed, MESH: Aged, MESH: DNA Repair, Recombination, Genetic, 0303 health sciences, MESH: Middle Aged, Soft tissue sarcoma, MESH: Polymorphism, Single Nucleotide, Nuclear Proteins, Sarcoma, MESH: Transcription Factors, Middle Aged, Flow Cytometry, 3. Good health, DNA-Binding Proteins, Oncology, 030220 oncology & carcinogenesis, MESH: Recombination, Genetic, Female, medicine.drug, MESH: DNA Primers, Adult, Xeroderma pigmentosum, Adolescent, DNA repair, Single-nucleotide polymorphism, [SDV.CAN]Life Sciences [q-bio]/Cancer, Dioxoles, Polymorphism, Single Nucleotide, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, medicine, Humans, MESH: Cell Line, Transformed, RNA, Messenger, Antineoplastic Agents, Alkylating, 030304 developmental biology, MESH: RNA, Messenger, Aged, DNA Primers, MESH: Adolescent, MESH: Humans, Base Sequence, business.industry, MESH: Apoptosis, MESH: Adult, medicine.disease, Endonucleases, MESH: Male, MESH: Sarcoma, Cancer research, ERCC1, business, MESH: Female, MESH: Genes, BRCA1, MESH: Nuclear Proteins, MESH: DNA-Binding Proteins, Nucleotide excision repair, Transcription Factors
Abstract

BACKGROUND: The objective of this study was to determine whether specific single nucleotide polymorphisms (SNPs) from nucleotide excision repair (NER) and homologous recombination (HR) DNA repair pathways are associated with sensitivity to trabectedin in patients with soft tissue sarcoma (STS). METHODS: The authors analyzed excision repair cross-complementation group 5/xeroderma pigmentosum group G (ERCC5/XPG) (NER), excision repair cross-complementation group 1 (ERCC1) (NER), and breast cancer 1 (BRCA1) (HR) SNPs and messenger RNA expression levels in tumor specimens from 113 patients with advanced STS who were enrolled in previously published phase 2 trials or in a compassionate-use program. The 6-month progression-free rate (PFR), progression-free survival (PFS), and overall survival (OS) were analyzed according to ERCC5, ERCC1, and BRCA1 status using log-rank tests. RESULTS: High expression of the common allele (aspartic acid at codon 1104) of ERCC5, high expression of ERCC1, and BRCA1 haplotype were associated significantly with improved PFR, PFS, and OS. The ERCC1 thymine-to-cytosine (T -> C) SNP at codon 19007 and BRCA1 expression were not associated with outcome. On univariate analysis, tumor histology, favorable NER status (high expression of common ERCC5 and/or high ERCC1 expression status), and favorable BRCA1 haplotype (at least 1 triple-adenine plus guanine [AAAG] allele) were the sole variables associated significantly with PFS and OS. CONCLUSIONS: In the current study, ERCC5, ERCC1, and BRCA1 status represented a potential DNA repair signature that could be used for the prediction of clinical response to trabectedin in patients with STS. Cancer 2011;117:3445-56. (C) 2011 American Cancer Society.

Published
2011

19. New Topoisomerase I mutations are associated with resistance to camptothecin

Author

Sandie Tuduri, Vincent Denis, Nadia Vezzio-Vie, Arnaud Coquelle, Philippe Pourquier, Annick Causse, Philippe Pasero, Céline Auzanneau, Maguy Del Rio, Gwenaëlle Collod-Béroud, Pierre Martineau, Céline Gongora, Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Validation et identification de nouvelles cibles en oncologie (VINCO), Université Bordeaux Segalen - Bordeaux 2-Institut Bergonié - CRLCC Bordeaux-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de génétique des maladies rares. Pathologie moleculaire, etudes fonctionnelles et banque de données génétiques (LGMR), Université Montpellier 1 (UM1)-IFR3, Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de recherche en cancérologie de Montpellier (IRCM - U896 Inserm - UM1), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 1 (UM1), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2, This work was supported by funds from Sanofi Aventis and from the INSERM, 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), IFR3, Université Montpellier 1 (UM1)-Université Montpellier 1 (UM1)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de physiologie et biologie cellulaires (IPBC), Université de Poitiers-Centre National de la Recherche Scientifique (CNRS), BMC, Ed., Institut Bergonié [Bordeaux], and UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Cancer Research, [SDV]Life Sciences [q-bio], Protein Structure, Secondary, 0302 clinical medicine, Transcription (biology), DNA Breaks, Double-Stranded, heterocyclic compounds, 0303 health sciences, biology, Cell Cycle, Cell cycle, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, 3. Good health, Oncology, DNA Topoisomerases, Type I, 030220 oncology & carcinogenesis, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], DNA supercoil, Molecular Medicine, Colorectal Neoplasms, medicine.drug, DNA Replication, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Topoisomerase-I Inhibitor, lcsh:RC254-282, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Cell Line, Tumor, medicine, Humans, neoplasms, 030304 developmental biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, Base Sequence, Topoisomerase, Research, DNA replication, HCT116 Cells, Molecular biology, Antineoplastic Agents, Phytogenic, digestive system diseases, Clone Cells, Drug Resistance, Neoplasm, Mutation, biology.protein, Camptothecin, Topoisomerase I Inhibitors, Linker
Abstract

Background Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. Methods We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing. Results In SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells. Conclusions These results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.

Published
2011

20. Targeting the p38 MAPK Pathway Inhibits Irinotecan Resistance in Colon Adenocarcinoma

Author

Paillas, Salome, Bibeau, Frédéric, Denouël, Amélie, Mollevi, Caroline, Causse, Annick, Denis, Vincent, Vezzio-Vié, Nadia, Marzi, Laetitia, Cortijo, Cédric, Ait-Arsa, Imade, Askari, Nadav, Pourquier, Philippe, Martineau, Pierre, Del Rio, Maguy, Gongora, Celine, Boissière, Florence, Vezzio-Vie, N., 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), Département de Pathologie [CHU Caen], Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), EndoFrance [CHRU Montpellier], Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cyclotron Réunion Océan Indien (CYROI), Université de La Réunion (UR)-Centre Hospitalier Universitaire de La Réunion (CHU La Réunion), Sanford-Burnham Medical Research Institute, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'anatomo-pathologie, CRLCC Val d'Aurelle - Paul Lamarque, Unité de biostatistiques, Validation et identification de nouvelles cibles en oncologie (VINCO), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2, Centre Régional de Lutte contre le Cancer François Baclesse (CRLC François Baclesse ), Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN), IDvet, Laboratoire d'Informatique, de Modélisation et d'optimisation des Systèmes (LIMOS), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Sigma CLERMONT (Sigma CLERMONT)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure des Mines de St Etienne, CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 1 (UM1), Institut Bergonié [Bordeaux], and UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Cancer Research, Pyridines, Colorectal cancer, [SDV]Life Sciences [q-bio], Cell, Leucovorin, Drug resistance, p38 Mitogen-Activated Protein Kinases, MESH: Drug Synergism, Mice, 0302 clinical medicine, Antineoplastic Combined Chemotherapy Protocols, MAPK p38, MESH: Protein Kinase Inhibitors, MESH: Animals, Phosphorylation, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Imidazoles, Drug Synergism, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, Immunohistochemistry, MESH: Drug Resistance, Neoplasm, 3. Good health, Isoenzymes, MESH: Antineoplastic Combined Chemotherapy Protocols, medicine.anatomical_structure, Oncology, 030220 oncology & carcinogenesis, Colonic Neoplasms, MESH: Isoenzymes, MESH: Camptothecin, Adenocarcinoma, Female, Fluorouracil, MESH: Imidazoles, medicine.drug, MESH: Xenograft Model Antitumor Assays, MAP Kinase Signaling System, MESH: HCT116 Cells, Mice, Nude, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Irinotecan, Article, 03 medical and health sciences, Downregulation and upregulation, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, MESH: Mice, Nude, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Kinase Inhibitors, MESH: Mice, 030304 developmental biology, MESH: Colonic Neoplasms, MESH: Humans, MESH: Phosphorylation, MESH: MAP Kinase Signaling System, MESH: Adenocarcinoma, MESH: Pyridines, Cancer, MESH: Immunohistochemistry, HCT116 Cells, medicine.disease, Xenograft Model Antitumor Assays, MESH: p38 Mitogen-Activated Protein Kinases, SN38, Drug Resistance, Neoplasm, Immunology, Cancer research, Camptothecin, MESH: Leucovorin, MESH: Female, MESH: Fluorouracil
Abstract

Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. To better elucidate the molecular mechanisms involved in resistance to irinotecan (and its active metabolite SN38), we established SN38-resistant clones derived from HCT-116 and SW48 cell lines. These clones show various levels (6- to 60-fold) of resistance to SN-38 and display enhanced levels of activated MAPK p38 as compared with the corresponding parental cells. Because four different isoforms of p38 have been described, we then studied the effect of p38 overexpression or downregulation of each isoform on cell sensivity to SN38 and found that both α and β isoforms are involved in the development of resistance to SN38. In this line, we show that cell treatment with SB202190, which inhibits p38α and p38β, enhanced the cytotoxic activity of SN38. Moreover, p38 inhibition sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Finally, we detected less phosphorylated p38 in primary colon cancer of patients sensitive to irinotecan-based treatment, compared with nonresponder patients. This indicates that enhanced level of phosphorylated p38 could predict the absence of clinical response to irinotecan. Altogether, our results show that the p38 MAPK pathway is involved in irinotecan sensitivity and suggest that phosphorylated p38 expression level could be used as a marker of clinical resistance to irinotecan. They further suggest that targeting the p38 pathway may be a potential strategy to overcome resistance to irinotecan-based chemotherapies in colorectal cancer. Cancer Res; 71(3); 1041–9. ©2010 AACR.

Published
2011

21. Molecular determinants of response to topoisomerase I inhibitors

Author

Philippe, Pourquier, Amélie, Lansiaux, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Génétique moléculaire et approches thérapeutiques des hémopathies malignes, and IRCL-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
[SDV] Life Sciences [q-bio], DNA Repair, DNA Topoisomerases, Type I, Drug Resistance, Neoplasm, [SDV]Life Sciences [q-bio], Humans, Camptothecin, Topoisomerase I Inhibitors, Irinotecan, Topotecan, DNA Damage
Abstract

International audience; Nuclear topoisomerase I (Top1) is involved in the relaxation of DNA supercoiling and plays a pivotal role in the coordination of essential DNA processes such as transcription, replication, DNA recombination and DNA damage signalling. For all these reasons, Top1 has been an attractive target for the development of anticancer drugs, which poison Top1 by trapping the enzyme on its DNA cleavage sites, which results in irreversible DNA lesions that are responsible for their cytotoxicity. They derive from the natural compound camptothecin and two derivatives are approved in the clinic, topotecan and irinotecan; other compounds such as indolocarbazoles and indenoisoquinolines are in development. However, the efficacy of these drugs is often limited by the problem of resistance, which involves various mechanisms at different steps of drug action, from drug transport and/or metabolism to the signalling and/or repair of the DNA lesions that are generated. A better understanding of these mechanisms is a major concern for the future development of new Top1 inhibitors and the identification of biomarkers that could be used to predict tumour response to these drugs in the clinic and to adapt the treatment to each patient.

Published
2011

22. Molecular determinants of response to topoisomerase II inhibitors

Author

Amélie, Lansiaux, Philippe, Pourquier, Pourquier, Philippe, Génétique moléculaire et approches thérapeutiques des hémopathies malignes, IRCL-Institut National de la Santé et de la Recherche Médicale (INSERM), Signalisation et Mecanismes Moleculaires de l'Apoptose, and Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
DNA Replication, DNA Repair, Transcription, Genetic, [SDV]Life Sciences [q-bio], Cell Cycle Checkpoints, [SDV] Life Sciences [q-bio], DNA Adducts, DNA Topoisomerases, Type II, Drug Resistance, Neoplasm, Humans, Topoisomerase II Inhibitors, ATP-Binding Cassette Transporters, Anthracyclines, DNA Damage, Podophyllotoxin
Abstract

International audience; Human nuclear topoisomerases II (Top2) are involved in the relaxation of DNA supercoiling during transcription and replication but also play a pivotal role in the segregation of newly replicated chromosomes and in chromatin remodelling. Top2 have been used as targets for the development of anticancer drugs. These inhibitors include anthracyclines (doxorubcin, daunorubicin, epirubicin) and epipodophyllotoxins (etoposide), which are widely used in the clinic. These drugs poison Top2 by trapping the enzyme on its DNA cleavage sites, which results in irreversible double-strand breaks that are responsible for cell death. They also include Top2 catalytic inhibitors such as bisdioxopiperazines (ICRF-187 and merbarone), which inhibit Top2 binding to its substrate. Efficacy of Top2 inhibitors is still limited by the problem of resistance, which involves various mechanisms from drug transport and/or metabolism to the signalling and/or repair of Top2-mediated DNA lesions. Secondary malignancies induced by the poisoning of Top2β are also a major clinical issue. A better understanding of these mechanisms is critical for the future development of new Top2 inhibitors and the identification of biomarkers that could be used to predict tumour response to these drugs in the clinic and to adapt the treatment to each patient.

Published
2011

23. General overview on DNA repair

Author

Philippe, Pourquier, Jacques, Robert, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Aimé Cotton (LAC), and École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV] Life Sciences [q-bio], Cell Death, DNA Repair, Chromosomal Instability, Neoplasms, [SDV]Life Sciences [q-bio], Antineoplastic Agents, Poly(ADP-ribose) Polymerases, DNA Mismatch Repair, DNA Damage
Abstract

International audience; DNA repair is implemented through a large variety of mechanisms, each of them being adapted to a specific type of lesion: direct repair, mismatch repair for the errors occurring during the replication process, base-excision repair, nucleotide-excision repair, double-strand breaks DNA repair by homologous or non-homologous recombination. Each of these mechanisms involves numerous proteins associated as supramolecular functional complexes. Some anticancer drugs are able to generate DNA lesions which may overflow the repair mechanisms. The impossibility to repair DNA damage usually leads to cell death, but alterations of repair mechanisms may favour genetic instability and hence contribute to oncogenesis.

Published
2011

24. The necrotic signal induced by mycophenolic acid overcomes apoptosis-resistance in tumor cells

Author

Marie-Sarah Dilhuydy, Benjamin Chaigne-Delalande, Patrick Legembre, Benoît Pinson, Francois-Xavier Mahon, Philippe Pourquier, Gwendaline Guidicelli, Walid Mahfouf, Jean-Max Pasquet, Jean-François Moreau, Composantes innées de la réponse immunitaire et différenciation (CIRID), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Laboratory of Immunology, National Institutes of Health (NIH)-National Institutes of Allergy and Infectious Diseases, Service des maladies du sang, CHU Bordeaux [Bordeaux]-Hôpital Haut-Lévêque [CHU Bordeaux], CHU Bordeaux [Bordeaux]-Groupe Hospitalier Sud, Institut de biochimie et génétique cellulaires (IBGC), Biothérapies des maladies génétiques et cancers, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Transfert de Genes a Visee Therapeutique Dans les Cellules Souches, Institut Bergonié [Bordeaux], UNICANCER, Signalisation et Mecanismes Moleculaires de l'Apoptose, Biogéosciences [UMR 6282] [Dijon] (BGS), Centre National de la Recherche Scientifique (CNRS)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Chemistry, Oncogenesis, Stress and Signaling (COSS), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-CRLCC Eugène Marquis (CRLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Pourquier, Philippe, Biogéosciences [UMR 6282] (BGS), Université de Bourgogne (UB)-Centre National de la Recherche Scientifique (CNRS), and Université de Rennes (UR)-CRLCC Eugène Marquis (CRLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
[SDV]Life Sciences [q-bio], lcsh:Medicine, Apoptosis, Jurkat cells, Cell Biology/Cell Signaling, Jurkat Cells, IMP Dehydrogenase, 0302 clinical medicine, IMP dehydrogenase, Tumor Cells, Cultured, Lymphocytes, RNA, Small Interfering, Oncology/Hematological Malignancies, cdc42 GTP-Binding Protein, lcsh:Science, 0303 health sciences, Multidisciplinary, Cell Biology/Cellular Death and Stress Responses, Transfection, 3. Good health, [SDV] Life Sciences [q-bio], Cdc42 GTP-Binding Protein, 030220 oncology & carcinogenesis, Guanosine Triphosphate, Signal transduction, Research Article, Signal Transduction, Blotting, Western, Green Fluorescent Proteins, Biology, Guanosine Diphosphate, Cell Line, Necrosis, 03 medical and health sciences, LYN, Cell Line, Tumor, Humans, 030304 developmental biology, Dose-Response Relationship, Drug, lcsh:R, Mycophenolic Acid, Leukemia, Lymphocytic, Chronic, B-Cell, Molecular biology, Microscopy, Electron, Drug Resistance, Neoplasm, Mutation, Cancer research, lcsh:Q, K562 Cells, K562 cells
Abstract

Background The amount of inosine monophosphate dehydrogenase (IMPDH), a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP), is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA) are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42. Methodology/Principal Findings Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA–mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL–overexpressing cells). All tested cells remained sensitive to MPA–mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers. Conclusions/Significance These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells.

Published
2009

25. Protein arginine ( N )-methyl transferase 7 (PRMT7) as a potential target for the sensitization of tumor cells to camptothecins

Author

Vincent Verbiest, Judith Markovits, Jacques Robert, Philippe Pourquier, Michel T. Tautu, Joyce Moukarzel, François Ichas, Danièle Montaudon, Jean-Pierre Portail, Pourquier, Philippe, Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Aimé Cotton (LAC), École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Plateforme de génétique moléculaire des cancers d'Aquitaine, Institut Bergonié [Bordeaux], UNICANCER-UNICANCER, Signalisation et Mecanismes Moleculaires de l'Apoptose, and Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Gene isoform, Protein-Arginine N-Methyltransferases, Methyltransferase, Arginine, [SDV]Life Sciences [q-bio], Resistance, Biophysics, Drug response, Hamster, Down-Regulation, Apoptosis, Antineoplastic Agents, Biology, Biochemistry, HeLa, 03 medical and health sciences, Downregulation and upregulation, Structural Biology, Cell Line, Tumor, Cricetinae, Neoplasms, Genetics, medicine, Animals, Humans, heterocyclic compounds, Molecular Biology, neoplasms, 030304 developmental biology, 0303 health sciences, 030302 biochemistry & molecular biology, Cell Biology, Methyltransferases, Oligonucleotides, Antisense, biology.organism_classification, Molecular biology, Topoisomerase I, Isoenzymes, [SDV] Life Sciences [q-bio], Protein arginine-methyl transferase (PRMT), Drug Resistance, Neoplasm, Camptothecin, medicine.drug, HeLa Cells
Abstract

International audience; PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.

Published
2008

26. Inhibition of Topoisomerase I Cleavage Activity by Thiol-reactive Compounds

Author

Stéphane Quideau, Komaraiah Palle, Philippe Pourquier, Laurent P. Rivory, Danièle Montaudon, Jacques Robert, Céline Douat-Casassus, Mary-Ann Bjornsti, Pourquier, Philippe, Laboratoire Aimé Cotton (LAC), École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physico -& Toxico Chimie des systèmes naturels (LPTC), Université Sciences et Technologies - Bordeaux 1 (UB)-Centre National de la Recherche Scientifique (CNRS), Institut des Sciences Moléculaires (ISM), Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1 (UB)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-École normale supérieure - Cachan (ENS Cachan), Université Sciences et Technologies - Bordeaux 1-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Université Sciences et Technologies - Bordeaux 1-Université Montesquieu - Bordeaux 4-Institut de Chimie du CNRS (INC)

Subjects
Stereochemistry, [SDV]Life Sciences [q-bio], Mutant, Cleavage (embryo), Biochemistry, Dithiothreitol, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, medicine, Phenylarsine oxide, Molecular Biology, 030304 developmental biology, 0303 health sciences, biology, Topoisomerase, Cell Biology, [SDV] Life Sciences [q-bio], chemistry, 030220 oncology & carcinogenesis, biology.protein, DNA supercoil, Camptothecin, medicine.drug, Cysteine
Abstract

International audience; DNA topoisomerase I (Top1) is a nuclear enzyme that plays a crucial role in the removal of DNA supercoiling associated with replication and transcription. It is also the target of the anticancer agent, camptothecin (CPT). Top1 contains eight cysteines, including two vicinal residues (504 and 505), which are highly conserved across species. In this study, we show that thiol-reactive compounds such as N-ethylmaleimide and phenylarsine oxide can impair Top1 catalytic activity. We demonstrate that in contrast to CPT, which inhibits Top1-catalyzed religation, thiolation of Top1 inhibited the DNA cleavage step of the reaction. This inhibition was more pronounced when Top1 was preincubated with the thiol-reactive compound and could be reversed in the presence of dithiothreitol. We also established that phenylarsine oxide-mediated inhibition of Top1 cleavage involved the two vicinal cysteines 504 and 505, as this effect was suppressed when cysteines were mutated to alanines. Interestingly, mutation of Cys-505 also altered Top1 sensitivity to CPT, even in the context of the double Cys-504 to Cys-505 mutant, which relaxed supercoiled DNA with a comparable efficiency to that of wild-type Top1. This indicates that cysteine 505, which is located in the lower Lip domain of human Top1, is critical for optimal poisoning of the enzyme by CPT and its analogs. Altogether, our results suggest that conserved vicinal cysteines 504 and 505 of human Top1 play a critical role in enzyme catalytic activity and are the target of thiol-reactive compounds, which may be developed as efficient Top1 catalytic inhibitors.

Published
2007

27. Genetic polymorphisms of the XPG and XPD nucleotide excision repair genes in sarcoma patients

Author

Nadine Houede, Philippe Pourquier, Michel Longy, Binh Bui, Catherine Bonaïti-Pellié, Valérie Le Morvan, Jacques Robert, Jean-Michel Coindre, Validation et identification de nouvelles cibles en oncologie (VINCO), Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Département de pathologie, UNICANCER-UNICANCER, Troubles du comportement alimentaire de l'adolescent (UMR_S 669), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Sud - Paris 11 (UP11), Département d'oncologie médicale, Centre Hospitalier Universitaire de Nîmes (CHU Nîmes), Service de Pathologie, Laboratoire Aimé Cotton (LAC), École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), UNICANCER, Laboratoire de pharmacologie des agents anticancéreux (LPAA), Université Bordeaux Segalen - Bordeaux 2-Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Centre National de la Recherche Scientifique (CNRS), Génétique humaine, développement et cancer, Université Bordeaux Segalen - Bordeaux 2-EA3669, Génétique épidémiologique et structures des populations humaines (Inserm U535), Epidémiologie, sciences sociales, santé publique (IFR 69), Université Paris 1 Panthéon-Sorbonne (UP1)-Université Paris-Sud - Paris 11 (UP11)-École des hautes études en sciences sociales (EHESS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-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 1 Panthéon-Sorbonne (UP1)-Université Paris-Sud - Paris 11 (UP11)-École des hautes études en sciences sociales (EHESS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-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 National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'anatomopathologie, UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2, Pourquier, Philippe, Université Paris-Sud - Paris 11 (UP11)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), and Kaniewski, Nadine

Subjects
Male, Cancer Research, DNA Repair, [SDV]Life Sciences [q-bio], Cockayne syndrome, Loss of heterozygosity, MESH: Genotype, 0302 clinical medicine, MESH: Aged, 80 and over, MESH: Child, MESH: Endonucleases, Genotype, Child, Aged, 80 and over, MESH: Aged, MESH: DNA Repair, 0303 health sciences, MESH: Middle Aged, MESH: DNA, Nuclear Proteins, Sarcoma, Exons, MESH: Transcription Factors, Middle Aged, MESH: Xeroderma Pigmentosum Gro, Xeroderma Pigmentosum Group A Protein, 3. Good health, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], Oncology, 030220 oncology & carcinogenesis, Female, Adult, Xeroderma pigmentosum, Adolescent, DNA repair, Biology, 03 medical and health sciences, MESH: Polymorphism, Genetic, medicine, Humans, Allele, Aged, 030304 developmental biology, MESH: Adolescent, Polymorphism, Genetic, MESH: Humans, MESH: Adult, DNA, Endonucleases, medicine.disease, Molecular biology, MESH: Male, [SDV.SPEE] Life Sciences [q-bio]/Santé publique et épidémiologie, MESH: Sarcoma, Cancer research, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, MESH: Exons, MESH: Female, MESH: Nuclear Proteins, MESH: DNA-Binding Proteins, Transcription Factors, Nucleotide excision repair
Abstract

International audience; There are more than 50 subtypes of soft tissue sarcomas, among which 30% are associated with specific genetic alterations, including translocations. Several studies have reported associations between cancer risk and polymorphisms of DNA repair genes from the nucleotide excision repair (NER) pathway. NER involves more than 20 proteins whose inactivation leads to xeroderma pigmentosum (XP) or cockayne syndrome (CS), among which XPD, a helicase allowing DNA strand excision by the endonuclease XPG. DNA from 93 patients with synovial sarcomas, myxoid liposarcomas, dermatofibrosarcomas protuberans (DFSP), malignant fibrous histiocytomas and leiomyosarcomas were genotyped for both XPD Lys751Gln and XPG Asp1104His polymorphisms. Departure from Hardy-Weinberg was highly significant for the XPG polymorphism with an excess of heterozygotes in synovial sarcomas (p = 1.5 x 10(-5)), myxoid liposarcomas (p = 1.5 x 10(-4)) and to a lesser extent in DFSP (p = 0.028). In the case of XPD, a significant deviation was observed in synovial sarcomas (p = 3 x 10(-6)) and DFSP (p = 0.0014). When tumors were pooled according to their genetic alterations, the proportion of carriers of the variant XPG allele was significantly increased in sarcomas with specific translocations as compared to sarcomas with complex genetics (p < 10(-9)). No difference was found for XPD. Genotyping of the tumor samples in synovial sarcomas and myxoid liposarcomas revealed frequent loss of heterozygosity for XPG, mostly due to the loss of the frequent allele. For XPD, both alleles were lost with a similar frequency. Our results raise the potential implication of the XPG Asp1104His polymorphism in the occurrence of chromosomal translocations associated with specific subtypes of sarcomas.

Published
2006

28. The DNA polymerase is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

Author

Yvan Canitrot, Audrey Laroche-Clary, François Boudsocq, Philippe Pourquier, Pascale Bertrand, Christophe Cazaux, Bernard S. Lopez, Jean-Pascal Capp, Jean-Sébastien Hoffmann, Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Laboratoire d'étude des Mécanismes de la recombinaison (LMR), Centre National de la Recherche Scientifique (CNRS), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Laboratoire de microbiologie et génétique moléculaires (LMGM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Toulouse (CRCT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-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 Toulouse III - Paul Sabatier (UT3), and Pourquier, Philippe

Subjects
Genome instability, Saccharomyces cerevisiae Proteins, DNA Repair, DNA damage, DNA polymerase, DNA repair, [SDV]Life Sciences [q-bio], Molecular Sequence Data, DNA polymerase beta, DNA-Directed DNA Polymerase, Genomic Instability, Article, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Radiation, Ionizing, Genetics, Animals, Deoxyribonucleases, Type II Site-Specific, DNA Polymerase beta, 030304 developmental biology, Recombination, Genetic, 0303 health sciences, Base Sequence, biology, fungi, DNA, Molecular biology, DNA polymerase lambda, [SDV] Life Sciences [q-bio], genomic DNA, enzymes and coenzymes (carbohydrates), chemistry, 030220 oncology & carcinogenesis, Mutation, biology.protein, Camptothecin, DNA Damage
Abstract

International audience; DNA polymerase lambda (pollambda) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that pollambda participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of pollambda (pollambdaDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polbeta and polmu do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, pollambdaDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for pollambda in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway.

Published
2006

29. The Chemistry of Wine PolyphenolicC-Glycosidic Ellagitannins Targeting Human Topoisomerase II

Author

Cédric Saucier, Yves Glories, Patrick Pardon, Michael Jourdes, Dorothée Lefeuvre, Philippe Pourquier, Stéphane Quideau, Danièle Montaudon, Institut des Sciences Moléculaires (ISM), Centre National de la Recherche Scientifique (CNRS)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Université Sciences et Technologies - Bordeaux 1-Université Montesquieu - Bordeaux 4-Institut de Chimie du CNRS (INC), Unité de Recherche Oenologie [Villenave d'Ornon], Institut National de la Recherche Agronomique (INRA)-Université de Bordeaux (UB)-Institut des Sciences de la Vigne et du Vin (ISVV), University of British Columbia (UBC), Institut des Sciences de la Vigne et du Vin [Villenave d'Ornon] (ISVV), Université de Bordeaux (UB), Laboratoire de Physico -& Toxico Chimie des systèmes naturels (LPTC), Université Sciences et Technologies - Bordeaux 1-Centre National de la Recherche Scientifique (CNRS), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Pourquier, Philippe, Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1 (UB)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Œnologie, Institut National de la Recherche Agronomique (INRA)-Université Victor Segalen - Bordeaux 2, Université Sciences et Technologies - Bordeaux 1 (UB)-Centre National de la Recherche Scientifique (CNRS), Institut des Sciences Moléculaires (CNRS-UMR 5255), Institut Européen de Chimie et Biologie, and Université Bordeaux Segalen - Bordeaux 2

Subjects
Models, Molecular, Stereochemistry, [SDV]Life Sciences [q-bio], Catechols, Molecular Conformation, Wine, 010402 general chemistry, Crystallography, X-Ray, 01 natural sciences, Catalysis, chemistry.chemical_compound, Structure-Activity Relationship, Ellagitannin, Phenols, Organic chemistry, Humans, Topoisomerase II Inhibitors, Glycosides, chemistry.chemical_classification, 010405 organic chemistry, Chemistry, Hydrolysis, Organic Chemistry, Biphenyl Compounds, Catechin, Glycosidic bond, General Chemistry, Oenin, Hydrolyzable Tannins, 0104 chemical sciences, [SDV] Life Sciences [q-bio], Polyphenol, Epimer, Castalagin
Abstract

International audience; Polyphenolic nonahydroxyterphenoyl-containing C-glycosidic oak ellagitannins are found in wine as a result of the aging of this beverage in oak-made barrels. Once in the slightly acidic wine (pH approximately 3-4), some of these complex natural products such as (-)-vescalagin (1), but not its C-1 epimer (-)-castalagin (2), can capture grape-derived nucleophilic entities such as ethanol, the flavanols catechin (10a) and epicatechin (10b), the anthocyanin oenin (13b), and the thiolic glutathione (16) to furnish condensation products with retention of configuration at the C-1 locus. A computer-aided rationale of this high diastereoselectivity is given. These condensation products can contribute to the modulation of organoleptic properties of the wine, as evidenced by the 23 nm bathochromic shift color absorbance observed with the novel oenin-based anthocyano-ellagitannin (15b). Hydrolysis of 1 under solvolytic conditions furnished another novel compound that we refer to as vescalene (21), in addition to the known (-)-vescalin (18). Of pharmacological importance is the fact that most of these found-in-wine water-soluble ellagitannin derivatives are much more potent than etoposide (VP-16) at inhibiting top2-mediated DNA decatenation in vitro (top2=topoisomerase II)). The known (-)-vescalin (18) and the novel vescalene (21) fully inhibited top2 at 10 microM concentration!

Published
2005

30. Hepatitis C virus core triggers apoptosis in liver cells by inducing ER stress and ER calcium depletion

Author

Francesca De Giorgi, Naoual L Benali-Furet, François Ichas, Patrizia Paterlini-Bréchot, Rosario Rizzuto, Ludivine Houel, David Lagorce, Mounia Chami, Ralf Bartenschlager, Fabienne Vernejoul, Louis Buscail, CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Carcinogenèse Hépatique et Virologie Moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM), Università degli Studi di Ferrara (UniFE), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Biologie et Pathologie Digestive, IFR 31 Louis Bugnard (IFR 31), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Toulouse [Toulouse]-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Molecular Virology, and Universität Heidelberg [Heidelberg]

Subjects
Cancer Research, [SDV]Life Sciences [q-bio], aequorin, Hepacivirus, Endoplasmic Reticulum, Membrane Potentials, Mice, 0302 clinical medicine, LIVER-CANCER, Promoter Regions, Genetic, Endoplasmic Reticulum Chaperone BiP, Calcium signaling, 0303 health sciences, 3. Good health, Cell biology, Mitochondria, APOPTOSIS, cell death, Liver, 030220 oncology & carcinogenesis, HCV, ER stress, Liver cancer, Programmed cell death, HVC Core, chemistry.chemical_element, Mice, Transgenic, Calcium, Biology, Transfection, Models, Biological, 03 medical and health sciences, HCV core, Cell Line, Tumor, Genetics, In Situ Nick-End Labeling, Animals, Humans, Molecular Biology, 030304 developmental biology, Calcium metabolism, Apoptosis, Calcium signalling, Endoplasmic reticulum, Intracellular Membranes, Virology, CALCIUM SIGNALLING, Calcium ATPase, Mice, Inbred C57BL, chemistry, Unfolded protein response, endoplasmic reticulum, mitochondria
Abstract

International audience; Hepatitis C virus (HCV) core, known to be involved in liver carcinogenesis, is processed in the endoplasmic reticulum (ER). We thus investigated the impact of three HCV core isolates on ER stress, ER calcium signalling and apoptosis. We show that HCV core constructs trigger hyperexpression of Grp78/BiP, Grp 94, calreticulin and sarco/endoplasmic reticulum calcium ATPase, inducing ER stress. By using the ER-targeted aequorin calcium probe, we found that ER calcium depletion follows ER stress in core-expressing cells. HCV core induces apoptosis through overexpression of the CHOP/GADD153 proapoptotic factor, Bax translocation to mitochondria, mitochondrial membrane depolarization, cytochrome c release, caspase-3 and PARP cleavage. Furthermore, reversion of HCV core-induced ER calcium depletion (by transfection of SERCA2) completely abolished mitochondrial membrane depolarization, suggesting that both ER stress (through CHOP overexpression) and calcium signalling play a major role in the HCV core-mediated control of apoptosis. ER stress and apoptosis were also found in a proportion of HCV-full-length replicon-expressing cells and in the liver of HCV core transgenic mice. In conclusion, our data demonstrate that HCV core deregulates the control of apoptosis by inducing ER stress and ER calcium depletion providing new elements to understand the mechanisms involved in HCV-related liver chronic diseases.

Published
2005

31. Predicting drug response and toxicity based on gene polymorphisms

Author

Jacques Bonnet, Valérie Le Morvan, Denis Smith, Philippe Pourquier, Jacques Robert, Pourquier, Philippe, Laboratoire Aimé Cotton (LAC), École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Service d'Hépato-gastro-entérologie et oncologie digestive (CHU de Bordeaux), CHU Bordeaux [Bordeaux]-Hôpital Haut-Lévêque [CHU Bordeaux], CHU Bordeaux [Bordeaux], Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Socio-économie des Entreprises et des ORganisations (ISEOR), Institut de socio-économie des entreprises et des organisations, Hôpital Haut-Lévêque [CHU Bordeaux], and CHU Bordeaux [Bordeaux]-CHU Bordeaux [Bordeaux]

Subjects
Drug, Drug-Related Side Effects and Adverse Reactions, media_common.quotation_subject, [SDV]Life Sciences [q-bio], Single-nucleotide polymorphism, Drug action, Pharmacology, Efficacy, 03 medical and health sciences, 0302 clinical medicine, Cytochrome P-450 Enzyme System, Predictive Value of Tests, Neoplasms, Dihydropyrimidine dehydrogenase, Medicine, Humans, 030304 developmental biology, media_common, 0303 health sciences, Polymorphism, Genetic, Thiopurine methyltransferase, biology, business.industry, Hematology, 3. Good health, Enzymes, [SDV] Life Sciences [q-bio], Treatment Outcome, Oncology, Drug Resistance, Neoplasm, 030220 oncology & carcinogenesis, Toxicity, biology.protein, business, Drug metabolism
Abstract

International audience; The sequencing of the human genome has allowed the identification of thousands of gene polymorphisms, most often single nucleotide polymorphims (SNP), which may play an important role in the expression level and activity of the corresponding proteins. When these polymorphisms occur at the level of drug metabolising enzymes or transporters, the disposition of the drug may be altered and, consequently, its efficacy may be compromised or its toxicity enhanced. Polymorphisms can also occur at the level of proteins directly involved in drug action, either when the protein is the target of the drug or when the protein is involved in the repair of drug-induced lesions. There again, these polymorphisms may lead to alterations in drug efficacy and/or toxicity. The identification of functional polymorphisms in patients undergoing chemotherapy may help the clinician prescribe the optimal drug combination or schedule and predict with more accuracy the response to these prescriptions. We have recorded in this review the polymorphisms that have been identified up till now in genes involved in anticancer drug activity. Some of them appear especially important in predicting drug toxicity and should be determined in routine before drug administration; this is the case of the most common variations of thiopurine methyltransferase for 6-mercaptopurine and of dihydropyrimidine dehydrogenase for fluorouracil. Other appear determinant for drug response, such as the common SNPs found in glutathione S-transferase P1 or xereoderma pigmentosum group D enzyme for the activity of oxaliplatin. However, confusion factors may exist between the role of gene polymorphisms in cancer risk or overall prognosis and their role in drug response.

Published
2004

32. Predicting drug response based on gene expression

Author

Jacques Robert, Jacques Bonnet, Antoine Vekris, Philippe Pourquier, Laboratoire Aimé Cotton (LAC), École normale supérieure - Cachan (ENS Cachan)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Neuroinflammation: imagerie et thérapie de la sclérose en plaques, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Signalisation et Mecanismes Moleculaires de l'Apoptose, Institut de Socio-économie des Entreprises et des ORganisations (ISEOR), Institut de socio-économie des entreprises et des organisations, and Pourquier, Philippe

Subjects
Drug, DNA repair, media_common.quotation_subject, [SDV]Life Sciences [q-bio], Antineoplastic Agents, Drug resistance, Biology, Bioinformatics, 03 medical and health sciences, 0302 clinical medicine, Neoplasms, Gene expression, medicine, Animals, Humans, Genes, Tumor Suppressor, Gene, 030304 developmental biology, media_common, Regulation of gene expression, 0303 health sciences, Clinical Trials as Topic, Cancer, Hematology, medicine.disease, Prognosis, 3. Good health, Gene expression profiling, Gene Expression Regulation, Neoplastic, [SDV] Life Sciences [q-bio], Oncology, Drug Resistance, Neoplasm, 030220 oncology & carcinogenesis
Abstract

International audience; Predicting drug response is a challenging problem in oncology. In the 1975-1985 decade, important efforts were devoted to the generation of cellular assays able to predict, on an individual basis, the in vitro response of tumour cells to chemotherapeutic agents, but such methods could not be adopted in routine. Numerous mechanisms of resistance to anticancer agents have been identified in cultured cell lines selected for growth in the presence of infratoxic, increasing doses of anticancer agents. They mainly concern drug transport, drug activation or detoxification, target quantitative or qualitative alterations, DNA repair efficiency, and alterations in signalling and/or execution of cell death programmes. New molecular biology techniques have been developed in order to identify the genes involved in drug resistance; they mainly involve differential expression techniques, but functional approaches may also prove informative. The availability of techniques of gene expression profiling has allowed to establish correlations between gene expression and drug sensitivity of tumour cells or human cancers. This type of approach has been initiated on in vitro systems by the National Cancer Institute (NCI) in the USA and is pursued by a growing number of public and private laboratories around the world. In the clinical setting, a number of genes or proteins have been identified as potential predictive markers of drug activity and their use could be progressively implemented for drug selection in patients receiving chemotherapy, allowing thus more rational and individualised treatments.

Published
2004

33. Mechanisms of Camptothecin Resistance by Human Topoisomerase I Mutations

Author

Bart L. Staker, Matthew R. Redinbo, Yves Pommier, Alex B. Burgin, Jill E. Chrencik, Philippe Pourquier, L. Stewart, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Molecular Pharmacology, National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH), University of North Carolina [Chapel Hill] (UNC), University of North Carolina System (UNC), and Pourquier, Philippe

Subjects
Models, Molecular, [SDV]Life Sciences [q-bio], Protein-DNA complex, Camptothecin Analogue, Biology, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Structural Biology, Transcription (biology), medicine, Humans, Molecular Biology, 030304 developmental biology, 0303 health sciences, Base Sequence, Topoisomerase, DNA replication, DNA, Antineoplastic Agents, Phytogenic, 3. Good health, [SDV] Life Sciences [q-bio], DNA Topoisomerases, Type I, chemistry, Biochemistry, 030220 oncology & carcinogenesis, Mutation, biology.protein, Camptothecin, Topotecan, medicine.drug
Abstract

International audience; Human topoisomerase I relaxes superhelical tension associated with DNA replication, transcription and recombination by reversibly nicking one strand of duplex DNA and forming a covalent 3'-phosphotyrosine linkage. This enzyme is the sole target of the camptothecin family of anticancer compounds, which acts by stabilizing the covalent protein-DNA complex and enhancing apoptosis through blocking the advancement of replication forks. Mutations that impart resistance to camptothecin have been identified in several regions of human topoisomerase I. We present the crystal structures of two camptothecin-resistant forms of human topoisomerase I (Phe361Ser at 2.6A resolution and Asn722Ser at 2.3A resolution) in ternary complexes with DNA and topotecan (Hycamtin), a camptothecin analogue currently in widespread clinical use. While the alteration of Asn722 to Ser leads to the elimination of a water-mediated contact between the enzyme and topotecan, we were surprised to find that a well-ordered water molecule replaces the hydrophobic phenylalanine side-chain in the Phe361Ser structure. We further consider camptothecin-resistant mutations at seven additional sites in human topoisomerase I and present structural evidence explaining their possible impact on drug binding. These results advance our understanding of the mechanism of cell poisoning by camptothecin and suggest specific modifications to the drug that may improve efficacy.

Published
2004

34. Elongation of oligonucleotide primers forming a triple helix on double-stranded DNA templates by purified DNA polymerases

Author

Patrick Lestienne, Jacques Bonnet, Philippe Pourquier, Physiopathologie du cancer du foie, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Signalisation et Mecanismes Moleculaires de l'Apoptose, Institut de Socio-économie des Entreprises et des ORganisations (ISEOR), Institut de socio-économie des entreprises et des organisations, and Pourquier, Philippe

Subjects
DNA Replication, Macromolecular Substances, Base pair, DNA polymerase, DNA polymerase II, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Biophysics, DNA-Directed DNA Polymerase, Biochemistry, 03 medical and health sciences, Molecular Biology, DNA Primers, 030304 developmental biology, 0303 health sciences, Binding Sites, DNA clamp, Base Sequence, biology, 030302 biochemistry & molecular biology, DNA replication, DNA, Sequence Analysis, DNA, Cell Biology, [SDV] Life Sciences [q-bio], biology.protein, Nucleic Acid Conformation, DNA supercoil, Primase, In vitro recombination, Protein Binding
Abstract

International audience; Current knowledge on the replication of DNA involves enzymatic steps of DNA strand separation upon helicase activity, thus enabling the exposed bases of the single-stranded DNA to direct the polymerization of complementary nucleotides through Watson-Crick base pairing rules by DNA-dependent DNA polymerases. Here, we report that oligonucleotide primers (9-11 nt long) bound to the double-stranded DNA, can be elongated by the T7 and by the Thermus thermophilus DNA polymerases and by the Escherichia coli Klenow fragment. A perfectly base-paired DNA cannot be used as a template, but a single A/A mismatch located even 5 bp upstream from the theoretical 3'-end of the oligonucleotide primer is sufficient for DNA elongation by these first two enzymes, while five are required for the Klenow fragment. Elongation products from templates containing A/A mismatches at different positions revealed similar patterns independently of the positions of the A/A mismatches. The sequencing of the elongated products revealed that both purine and pyrimidine are incorporated at the pyrimidine-purine-pyrimidine transitions of the template strands, probably because of a shift of the primers on the double helix template. These data confirm that prokaryotic DNA polymerases may accommodate transiently three DNA strands in their catalytic centre. They also show for the first time that replication can occur on double-stranded DNA in the absence of DNA helicase, when mismatches are present in the vicinity of the triple helix initiation complex.

Published
2003

35. Human Apurinic/Apyrimidinic Endonuclease (Ape1) and Its N-terminal Truncated Form (AN34) Are Involved in DNA Fragmentation during Apoptosis

Author

Ettore Appella, Manabu Inuzuka, Dominic G. Rothwell, Philippe Pourquier, John N. Weinstein, Yves Pommier, Takanori Ueda, Mark Waltham, Ann Charlotte Bergman, Ian D. Hickson, Akira Yoshida, Yoshimasa Urasaki, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), The University of Texas M.D. Anderson Cancer Center [Houston], Laboratory of Molecular Pharmacology, National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), and National Institutes of Health [Bethesda] (NIH)

Subjects
Exonucleases, Exonuclease, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Apoptosis, DNA Fragmentation, Biochemistry, AP endonuclease, Endonuclease, DNA-(Apurinic or Apyrimidinic Site) Lyase, AP site, Amino Acid Sequence, Gene Silencing, Fragmentation (cell biology), Molecular Biology, Endodeoxyribonucleases, biology, Caspase 3, Apoptotic DNA fragmentation, Cell Biology, Staurosporine, Molecular biology, Chromatin, [SDV] Life Sciences [q-bio], Caspases, biology.protein, DNA fragmentation
Abstract

International audience; We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.

Published
2003

36. Gemcitabine (2',2'-difluoro-2'-deoxycytidine), an antimetabolite that poisons topoisomerase I

Author

Pourquier, Philippe, Gioffre, Christopher, Kohlhagen, Glenda, Urasaki, Yoshimasa, Goldwasser, François, Hertel, Lary, Yu, Shuyuan, Pon, Richard, Gmeiner, William, Pommier, Yves, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de Cancérologie Médicale [AP-HP Cochin], Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Laboratory of Molecular Pharmacology, National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH), and Pourquier, Philippe

Subjects
Antimetabolites, Antineoplastic, Time Factors, Dose-Response Relationship, Drug, [SDV]Life Sciences [q-bio], Oligonucleotides, Deoxycytidine, Gemcitabine, [SDV] Life Sciences [q-bio], Mice, Models, Chemical, Tumor Cells, Cultured, Animals, Humans, Enzyme Inhibitors, Topoisomerase I Inhibitors
Abstract

Gemcitabine-containing regimens are among standard therapies for the treatment of advanced non-small cell lung,pancreatic, or bladder cancers. Gemcitabine is a nucleoside analogue and its cytotoxicity is correlated with incorporation into genomic DNA and concomitant inhibition of DNA synthesis. However, it is still unclear by which mechanism(s) gemcitabine incorporation leads to cell death.We used purified oligodeoxynucleotides to study the effects of gemcitabine incorporation on topoisomerase I (top1) activity and tested the role of top1 poisoning in gemcitabine-induced cytotoxicity in cancer cells.We found that top1-mediated DNA cleavage was enhanced when gemcitabine was incorporated immediately 3' from a top1 cleavage site on the nonscissile strand. This position-specific enhancement was attributable to an increased DNA cleavage by top1 and was likely to have resulted from a combination of gemcitabine-induced conformational and electrostatic effects. Gemcitabine also enhanced camptothecin-induced cleavage complexes. We also detected top1 cleavage complexes in human leukemia CEM cells treated with gemcitabine and a 5-fold resistance of P388/CPT45 top1-deficient cells to gemcitabine, indicating that poisoning of top1 can contribute to the antitumor activity of gemcitabine.The present results extend our recent finding that incorporation of 1-beta-D-arabinofuranosylcytosine into DNA can induce top1 cleavage complexes [P. Pourquier et al. Proc. Natl. Acad. Sci. USA, 97: 1885-1890, 2000]. The enhancement of camptothecin-induced top1 cleavage complexes may, at least in part, contribute to the synergistic or additive effects of gemcitabine in combination with topotecan and irinotecan in human breast or lung cancer cells.

Published
2002

37. Interaction of human nuclear topoisomerase I with guanosine quartet-forming and guanosine-rich single-stranded DNA and RNA oligonucleotides

Author

Gary S. Laco, Christophe Marchand, Yves Pommier, Philippe Pourquier, Naijie Jing, Laboratory of Molecular Pharmacology, National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH)-National Institutes of Health [Bethesda] (NIH), Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH), and Pourquier, Philippe

Subjects
DNA polymerase, Base pair, DNA polymerase II, [SDV]Life Sciences [q-bio], DNA, Single-Stranded, Biochemistry, chemistry.chemical_compound, Humans, Molecular Biology, Cell Nucleus, DNA clamp, Oligoribonucleotides, biology, Base Sequence, Guanosine, Oligonucleotide, DNA replication, Cell Biology, Molecular biology, [SDV] Life Sciences [q-bio], chemistry, DNA Topoisomerases, Type I, Oligodeoxyribonucleotides, biology.protein, Camptothecin, Primase, DNA
Abstract

International audience; Human nuclear DNA topoisomerase I (top1) plays a crucial role in DNA replication, transcription, and chromosome condensation. In this study, we show that intra- and intermolecular guanosine quartets (G-quartets) can inhibit top1-mediated DNA cleavage at a high affinity site. Top1-mediated DNA cleavage was also inhibited by a 16-mer single-stranded oligodeoxynucleotide (ODN) containing a G-rich sequence (G(2)T(2)G(5)TG(2)TG(3)) and by its RNA equivalent, neither of which form G-quartet structures. A comparison of various single-stranded ODN for their ability to inhibit top1-mediated DNA cleavage indicated that G-rich sequences containing repeats of 2 or 3 consecutive guanines interspaced with thymines specifically inhibited top1. We also found that both single-stranded and G-quartet-forming ODNs bind to top1 without being cleaved by the enzyme. These results demonstrate that either DNA or RNA G-rich single-stranded and G-quartet-forming oligonucleotides can bind to top1 and prevent cleavage of duplex DNA.

Published
2002

38. Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair

Author

Yuji Takebayashi, Kenneth H. Kraemer, Philippe Pourquier, Drazen B. Zimonjic, Takahiro Ueda, Shin-ichi Akiyama, Kentaro Nakayama, Steffen Emmert, Yoshimasa Urasaki, Atsuko Kanzaki, Yves Pommier, Nicholas C. Popescu, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Molecular Pharmacology, National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), and National Institutes of Health [Bethesda] (NIH)

Subjects
congenital, hereditary, and neonatal diseases and abnormalities, Xeroderma pigmentosum, DNA Repair, Transcription, Genetic, [SDV]Life Sciences [q-bio], Blotting, Western, Loss of Heterozygosity, Antineoplastic Agents, Dioxoles, Ecteinascidia turbinata, Polymerase Chain Reaction, General Biochemistry, Genetics and Molecular Biology, Cockayne syndrome, Cell Line, chemistry.chemical_compound, Tetrahydroisoquinolines, medicine, Animals, Urochordata, skin and connective tissue diseases, Gene, Trabectedin, Genetics, biology, Genetic Complementation Test, nutritional and metabolic diseases, Nuclear Proteins, General Medicine, biology.organism_classification, medicine.disease, Endonucleases, Isoquinolines, Molecular biology, Complementation, [SDV] Life Sciences [q-bio], DNA-Binding Proteins, chemistry, DNA, Cell Division, Nucleotide excision repair, medicine.drug, DNA Damage, Transcription Factors
Abstract

International audience; While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Published
2001

39. Topoisomerase I-mediated DNA damage

Author

Philippe Pourquier, Yves Pommier, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Molecular Pharmacology, National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), and National Institutes of Health [Bethesda] (NIH)

Subjects
Genetics, DNA Repair, DNA repair, DNA damage, Topoisomerase, [SDV]Life Sciences [q-bio], DNA replication, Antineoplastic Agents, Eukaryotic DNA replication, DNA, Biology, Models, Biological, Catalysis, Protein Structure, Tertiary, Cell biology, [SDV] Life Sciences [q-bio], DNA Topoisomerases, Type I, biology.protein, Animals, Humans, DNA supercoil, DNA mismatch repair, Replication protein A, DNA Damage, Protein Binding
Abstract

International audience; Topoisomerase I is a ubiquitous and essential enzyme in multicellular organisms. It is involved in multiple DNA transactions including DNA replication, transcription, chromosome condensation and decondensation, and probably DNA recombination. Besides its activity of DNA relaxation necessary to eliminate torsional stresses associated with these processes, topoisomerase I may have other functions related to its interaction with other cellular proteins. Topoisomerase I is the target of the novel anticancer drugs, the camptothecins. Recently a broad range of physiological and environmentally-induced DNA modifications have also been shown to poison topoisomerases. This review summarizes the various factors that enhance or suppress top1 cleavage complexes and discusses the significance of such effects. We also review the different mechanisms that have been proposed for the repair of topoisomerase I-mediated DNA lesions.

Published
2001

40. Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate

Author

Jolanta Fertala, Mary-Ann Bjornsti, Yves Pommier, Philippe Pourquier, John R. Vance, Pourquier, Philippe, Signalisation et Mecanismes Moleculaires de l'Apoptose, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratory of Molecular Pharmacology, National Institutes of Health [Bethesda] (NIH)-National Cancer Institute [Bethesda] (NCI-NIH), and National Institutes of Health [Bethesda] (NIH)

Subjects
[SDV]Life Sciences [q-bio], Mutant, Eukaryotic DNA replication, Saccharomyces cerevisiae, Biochemistry, Substrate Specificity, chemistry.chemical_compound, Enzyme Stability, medicine, Molecular Biology, Binding Sites, biology, Base Sequence, Topoisomerase, Active site, Cell Biology, Enzyme structure, [SDV] Life Sciences [q-bio], Enzyme binding, chemistry, Amino Acid Substitution, DNA Topoisomerases, Type I, biology.protein, Mutagenesis, Site-Directed, Nucleic Acid Conformation, Camptothecin, Asparagine, DNA, medicine.drug, Plasmids
Abstract

International audience; Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin. Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin. To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p. Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity. Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity. However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA. Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin. These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle.

Published
2000

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