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Cyclin E Provides a Link Between Cell Cycle, Dna CYCLIN E PROVIDES A LINK BETWEEN CELL CYCLE, DNA REPAIR AND APOPTOSIS A dissertation submitted to Kent State University in collaboration with the Lerner Research Institute, Cleveland Clinic in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Dragos Costin Plesca May, 2008 Dissertation written by Dragos Costin Plesca Pharm.D., University of Medicine and Pharmacy “Carol Davila”, Romania, 2002 Ph.D., Kent State University, 2008 Approved by ____________________, Chair, Doctoral Dissertation Committee Alexandru Almasan, Ph.D ____________________, Member, Doctoral Dissertation Committee James Blank, Ph.D ____________________, Member, Doctoral Dissertation Committee Gail Fraizer, Ph.D ____________________, Member, Doctoral Dissertation Committee Olena Piontkivska, Ph.D ____________________, Graduate Faculty Representative Jennifer Marcinkiewicz, Ph.D Accepted by ____________________, Director, School of Biomedical Sciences Robert V. Dorman, Ph.D ____________________, Dean, College of Arts and Sciences John R. D. Stalvey, Ph.D ii TABLE OF CONTENTS List of Figures.................................................................................................................vi List of Tables ..................................................................................................................ix Acknowledgments ............................................................................................................x Chapter I. Introduction ................................................................................................1 The Cell Cycle .....................................................................................3 Cyclin E and cell cycle regulation ...........................................8 Transcriptional regulation of Cyclin E ..................................14 Post-translational regulation of Cyclin E...............................20 Deregulated expression of Cyclin E in cancer.......................27 Therapeutic approaches involving cell cycle inhibitors.........32 Apoptosis ...........................................................................................38 TRAIL, APO2L .....................................................................40 Adaptor proteins.....................................................................41 Cytochrome c.........................................................................42 Bcl-2 family members............................................................42 Caspases.................................................................................43 Other apoptosis-regulatory proteins.......................................45 Apoptotic pathways ...............................................................46 iii DNA damage and repair mechanisms................................................51 Homologous recombination...................................................55 Ku70/Ku80 heterodimer ........................................................56 DNA-PKcs .............................................................................60 Artemis...................................................................................62 XRCC4...................................................................................62 Ligase IV................................................................................63 XLF........................................................................................64 Non-Homologous End Joining ..............................................66 Other DNA double-strand break repair pathways .................66 Generation of p18-Cyclin E and its role in modulating cellular response to genotoxic stress...............................................................70 Chapter II. p18-Cyclin E regulates Non-Homologous End Joining by preventing the recruitment of the XLF/XRCC4/Ligase IV heterocomplex to the DNA repair complex ................................................................................73 Abstract..............................................................................................73 Introduction........................................................................................74 Materials and Methods.......................................................................78 Results................................................................................................82 Discussion..........................................................................................95 iv Chapter III. Two new mechanisms for proteasome-mediated Cyclin E degradation uncovered in hematopoietic tumor cells undergoing apoptosis .........102 Abstract............................................................................................102 Introduction......................................................................................103 Materials and Methods.....................................................................106 Results..............................................................................................110 Discussion........................................................................................137 Chapter IV. Summary and future directions ............................................................145 Summary..........................................................................................145 Future directions ..............................................................................148 References.....................................................................................................................157 Appendix. Abbreviations..........................................................................................193 v LIST OF FIGURES Chapter I Fig 1.1 Cellular response to DNA damage ....................................................................2 Fig 1.2 Schematic representation of the cell cycle.........................................................4 Fig 1.3 Periodic oscillation in the expression levels of cyclins ...................................10 Fig 1.4 Schematic representation of the progression through the different cell cycle phases...............................................................................................................19 Fig 1.5 Schematic representation of the Cyclin E turnover mechanisms.....................23 Fig 1.6 Schematic representation of the cell cycle and the different therapeutic approaches undertaken.....................................................................................37 Fig 1.7 Schematic representation of the extrinsic and intrinsic apoptotic pathways ...49 Fig 1.8 Three-dimensional structure of the Ku70/Ku80 heterodimer..........................59 Fig 1.9 Schematic representation of the XLF/XRCC4/Ligase IV heterocomplex.......65 Fig 1.10 Schematic representation of the NHEJ pathway..............................................69 Fig 1.11 Schematic representation of Cyclin E domains and cleavage sites .................71 vi Chapter II Fig 2.1 Non-toxic levels of p18-Cyclin E sensitize cells to VP-16 and IR treatment..85 Fig 2.2 p18-Cyclin E inhibits in vitro DNA ligation and plasmid reactivation ...........88 Fig 2.3 p18-Cyclin E prevents efficient repair of the DNA damage induced by IR and CPT-11.............................................................................................................91 Fig 2.4 p18-Cyclin E impairs the recruitment of XLF, XRCC4, and Ligase IV to the DNA repair complex........................................................................................94 Fig 2.5 Schematic representation of the NHEJ DNA repair pathway and the way in which p18-Cyclin E affects the assembly of repair factors ...........................101 Chapter III Fig 3.1 p18-Cyclin E is a short-lived protein that is stabilized by proteasome inhibition........................................................................................................112 Fig 3.2 p18-Cyclin E is effectively ubiquitinated ......................................................115 Fig 3.3 Fbw7 interacts with p18-Cyclin E independently of phosphorylation at the C- terminal phosphodegron ................................................................................120 Fig 3.4 p18-Cyclin E is regulated by the SCFFbw7 complex and independently of Cdk2 binding ...........................................................................................................125 Fig 3.5 Ku70 but not Cdk2 binding regulates p18-Cyclin E stability........................130 Fig 3.6 p18-cyclin E sensitizes cells to Bortezomib treatment through enhanced association with Ku70 and release of Bax from Ku70 ..................................135 Fig. 3.7 Chemical structure of the proteasome inhibitor, Bortezomib........................141 vii Chapter IV Fig 4.1 Dual regulation of apoptosis and DNA repair by p18-Cyclin E....................147 Fig 4.2 Localization of p18-Cyclin E-GFP, Cyclin E-GFP, and Cyclin ER130A-GFP 151 Fig 4.3 Cyclin E sequence alignment across human, mouse, rat, rabbit, chicken, and frog.................................................................................................................153 viii LIST OF TABLES Table 1. Critical protein substrates of Caspase-3...........................................................45 ix ACKNOWLEDGMENTS None of the scientific results or achievements that I have had during my last five years would have been possible without the sustained professional and moral support that my adviser, Dr. Alex Almasan
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