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Survivin and Aurora B Kinase, Two Targets in the Search for Anti - Mitotic Drugs; Identification of a New Class of Aurora Kinase Inhibitors

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Survivin and Aurora B Kinase, Two Targets in the Search for Anti - Mitotic Drugs; Identification of a New Class of Aurora Kinase Inhibitors Survivin and Aurora B kinase, two targets in the search for anti - mitotic drugs; identification of a new class of Aurora kinase inhibitors. Thi My-Nhung Hoang To cite this version: Thi My-Nhung Hoang. Survivin and Aurora B kinase, two targets in the search for anti - mitotic drugs; identification of a new class of Aurora kinase inhibitors.. Life Sciences [q-bio]. Université Joseph-Fourier - Grenoble I, 2008. English. tel-00398008v1 HAL Id: tel-00398008 https://tel.archives-ouvertes.fr/tel-00398008v1 Submitted on 24 Jun 2009 (v1), last revised 13 Jul 2009 (v2) HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Université Joseph Fourier-Grenoble 1 Chimie et Science du Vivant (Arrêtés ministériels du 5 juillet 1984 et du 30 mars 1992) THÈSE pour obtenir le titre de Docteur de l’Université Joseph Fourier Discipline Biologie Présentée et soutenue publiquement par HOANG Thi My Nhung Le 31 Janvier 2008 SURVIVIN AND AURORA B KINASE, TWO TARGETS IN THE SEARCH FOR ANTI-MITOTIC DRUGS; IDENTIFICATION OF A NEW CLASS OF AURORA KINASE INHIBITORS. COMPOSITION DU JURY Véronique Baldin Rapportrice Claude Prigent Raporteur Dinh Duy Khang Examinateur Marylin Vantard Présidente Stéfan Dimitrov Examinateur Tran Cong Yen Directeur de thèse (VNU) Annie Molla Directrice de thèse (UJF) Thèse préparée au sein du Centre de Recherche INSERM Mécanismes d’assemblage et de régulation de l’appareil génétique Unité INSERM U823 Institut Albert Bonniot – Université Joseph Fourier – Grenoble 1 Acknowledgements First of all, I would like to express my deepest gratitude to Dr. Annie Molla for her supervision, patient guidance, and encouragement from the beginning of my research in France as well as giving me excellent advice throughout this study. I also want to thank her for assistances in preparing and writing this thesis. I would like to warmly thank to my thesis co-supervisor, Dr. Tran Cong Yen, for his invaluable support and enthusiasm to my graduate experience. I could never have embarked and started all of this without his prior teachings in biology and thus opened up unknown areas to me. My special thanks to Dr. Stefan Dimitrov for giving me the opportunity to work on his equipment and for his assistances, suggestions in my research. I gratefully thank Dr. Véronique Baldin and Dr. Claude Prigent for reviewing my thesis and for their constructive comments on this manuscript. I would like to thank Dr. Marylin Vantard and Dr. Dinh Duy Khang for accepting to be members of the committee. My stay and my PhD in France is financial supported by a Scholarship from Sandwich Programme, a project initiated and managed from Ministry of Education and Training of Vietnam and partly from French. I also thank to ARC organization which supported for the last year of my thesis. It was a pleasure for me to work with all wonderful people in our lab here in IAB. I would like to give many thanks to the fellow graduated students before me, Gaelh, Marlene, Flore, and Fabienne Sirot, for sharing experiences and knowledge during the time they were here. My thanks are given to another Fabienne for “science” discussions and the pleasure working together in the lab. I would also acknowledgment Véro for helping me in protein extraction and purification technique, thank for her technical assistance. I am grateful to my colleagues: Andre, Thierry, Eric, Sophie, Fang, Jean, Amina… I would like to thank to the members of INSERM U823 for their support and their comradeship. Thank Aude for her indispensable help dealing with administration and bureaucratic matter during my stay in IAB. I am very grateful to members of platform, Alexei and Jack, whose expertise, understanding and patience in microscope are very helpful for my work. I also want to thank Catherine Souchier for helps in using confocor microscope. A great thank to my department, Faculty of Biology, Hanoi University of Science, Hanoi National University (VNU) for supporting and creating good conditions for me to do my PhD. To my best French friend, Cedric Badowski, I gratefully acknowledge him for his kindness and useful advices during the lifetime I spent in France. He provides me unflinching encouragement and support in various ways. I have much benefited by advice and guidance from him who always kindly grants me his time even for answering some of my unintelligent question about microscope and molecular technique…. I am much indebted to him! My years as a PhD student would not have been as much fun without my friends: Khoat, Nam, Dung, Hung, Vi Thuy, Thai, Chi, Lien…with whom I shared the unforgettable moments. Thanks! It is pleasure to express my gratitude wholeheartedly to Annie’s family for their kindness hospitality during my stay in Grenoble. Thank Thierry for his assistance on editing my thesis writing. Finally, I take this opportunity to express my profound gratitude to my grand family: my beloved parent, parent in law, my brothers, sisters for their inseparable support and patience during my study in France. Especially, I wish to express all my warmest thanks and deepest appreciations to my dear husband and my little daughter for their love and sacrifices for me. Without my two loves, I would not finish my thesis!!! Grenoble, December 2007 Abstract The chromosomal passenger complex (CPC) plays a key role in mitosis : controlling both chromosome segregation, spindle tension, anaphase onset and cytokinesis. The complex is composed of four proteins: INCENP, Aurora B kinase, Survivin and Borealin. Taking into account that Survivin is phosphorylated by Aurora B and has a pivotal role in the complex, we have studied the phosphomimetic mutant SurvivinT117E. Survivin phosphorylation is required for anaphase onset and the phospho-mutant is poorly linked to centromere. Moreover it exhibits a dominant negative function in cytokinesis, preventing abscission. In a search for Aurora kinase inhibitors we have identified a new class of Aurora B kinase inhibitors that prevents Histone H3 phosphorylation, impairs mitotic spindle checkpoint. Moreover these molecules prevent tumor cell proliferation. These inhibitors are interesting tools for understanding CPC function and represent a new lead for the development of anti-cancer drugs. Survivin and Aurora B kinase, which are expressed exclusively in mitosis, are thus two druggable targets for new anti-mitotic therapies. Key words: Mitosis, chromosomal passenger complex, Aurora kinase, Survivin, mitotic checkpoint. Résumé Le complexe passager joue un rôle clé en mitose: contrôlant à la fois la ségrégation des chromosomes, la tension du fuseau, l’entrée en anaphase et la cytodirèse. Le complexe est composé de quatre protéines: INCENP, la kinase Aurora B, Survivine et Boréaline. Sachant que la protéine Survivine est phosphorylée par Aurora B et qu’elle a un role pivot au sein du complexe, nous avons étudié un mutant mimant sa phosphorylation: Survivine T117E. La phosphorylation de Survivine est nécessaire à la transition Métaphase/ Anaphase. Le mutant Survivine T117E est faiblement lié aux centromères en métaphase et agit comme un dominant négatif de la cytodirèse, empêchant la séparation des deux cellules filles. Lors de la recherche d’inhibiteurs des Aurora kinases, nous avons identifié une nouvelle classe de molécules qui inhibent la phosphorylation de l’histone H3 et le point de contrôle du fuseau. Ces molécules préviennent la prolifération des cellules tumorales. Ces composés sont des outils intéressants pour étudier la fonction du complexe passager et représentent un nouveau motif moléculaire pour le développement de drogues anti- cancéreuses. Survivine et Aurora B kinase dont l’expression est restreinte à la mitose sont deux cibles pour de nouvelles thérapies anti-mitotiques. Mots clé : Mitose, complexe passager, Aurora kinase, Survivine, point de contrôle du fuseau. ABBREVIATIONS APC/C : Anaphase promoting complex/Cyclosome ATP: Adenosine 5'-triphosphate BIR: Baculovirus IAP Repeat CDE/CHR: Cell cycle Dependent Element/ Cell cycle genes Homology Region; Cdks: Cyclin-dependent kinases CENP-A: Centromere protein A CENP-E: Centromere protein E CIN: Chromosomal INstability CPC: Chromosomal Passenger Complex CPP: Chromosomal Passenger Protein DAPI: 4',6-diamidino-2-phenylindole DMSO: Dimethyl Sulfoxide DNA: Deoxyribo Nucleic Acid DTT: Dithiothreitol EDTA: Ethylene Diamine Tetraacetic Acid FACS: Fluorescence Activated Cell Sorting FRAP: Fluorescent Recovery After Photobleaching GDP: Guanosine Di-Phosphate GFP: Green Fluorescent Protein GST: Glutathione S-Transferase GTP: Guanosine Tri-Phosphate HP1: Heterochromatin Protein 1 HRP: Horse Radish Peroxidase IAPs: Inhibitors of apoptosis proteins INCENP: INner CENtromere Protein IPTG: Isopropyl-ß-D-1-thiogalactopyranoside kDa: kilo Dalton MCAK: Mitotic Centromere Associated Kinesin MCC: Mitotic Checkpoint Complex MPF : M-phase promoting factor MKLP1: Mitotic Kinesin-Like Protein 1 OD600: Optical Density at 600 nm PBS: Phosphate Buffer Saline PFA: ParaFormAldehyde PP1: Protein Phosphatase 1 RNA: Ribo Nucleic Acid rpm: round per minute SAC: Spindle Assembly
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