G2 Phase Cell Cycle Regulation by E2F4 Following Genotoxic Stress
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G2 Phase Cell Cycle Regulation by E2F4 Following Genotoxic Stress by MEREDITH ELLEN CROSBY Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Thesis Advisor: Dr. Alex Almasan Department of Environmental Health Sciences CASE WESTERN RESERVE UNIVERSITY May, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS TABLE OF CONTENTS………………………………………………………………….1 LIST OF FIGURES……………………………………………………………………….5 LIST OF TABLES………………………………………………………………………...7 ACKNOWLEDGEMENTS……………………………………………………………….8 LIST OF ABBREVIATIONS……………………………………………………………10 ABSTRACT……………………………………………………………………………...15 CHAPTER 1. INTRODUCTION 1.1. CELL CYCLE REGULATION: HISTORICAL OVERVIEW………………...17 1.2. THE E2F FAMILY OF TRANSCRIPTION FACTORS……………………….22 1.3. E2F AND CELL CYCLE CONTROL 1.3.1. G0/G1 Phase Transition………………………………………………….28 1.3.2. S Phase…………………………………………………………………...28 1.3.3. G2/M Phase Transition…………………………………………………..30 1.4. CELL CYCLE MISREGULATION BY GENOTOXIC STRESS: IONIZING RADIATION 1.4.1. The cellular response to ionizing radiation (IR) ….…………………….32 1.4.2. DNA damage response: cell cycle arrest versus apoptosis……………...35 1.4.3. Cell cycle specific arrest mechanisms………………………………......37 1.5. OBJECTIVES AND THESIS OVERVIEW……………………………………38 1 CHAPTER 2. PHYSIOLOGIC TARGETS OF P53 IDENTIFIED THROUGH CHROMATIN IMMUNOPRECIPITATION (CHIP) 2.1. ABSTRACT……………………………………………………………………..42 2.2. INTRODUCTION………………………………………………………………43 2.3. MATERIALS AND METHODS 2.3.1. Cell culture and treatment…………………………………………………45 2.3.2. MOLT-4 expression profiling with cDNA arrays………………………...46 2.3.3. Chromatin Immunoprecipitation Assay (ChIP) …………………………..48 2.4. RESULTS 2.4.1. Expression profiling identifies radiation responsive genes………………..52 2.4.2. Identification of p53 binding sites in radiation-responsive genes implicated in apoptosis and cell cycle control………………………55 2.4.3. Chromatin immunoprecipitation (ChIP) of p53 with regulatory regions of radiation-responsive genes implicated in apoptosis and cell cycle control………………………………………………………57 2.5. DISCUSSION…………………………………………………………………...64 CHAPTER 3. BEYOND P53: THE OPPOSING ROLES OF E2FS IN CELL CYCLE PROLIFERATION AND DEATH 3.1. ABSTRACT……………………………………………………………………..68 3.2. INTRODUCTION………………………………………………………………68 2 3.3. E2F1: A MULTITASKING MEDIATOR………………………………………70 3.4. E2F4: A REMARKABLE REPRESSOR……………………………………….72 3.5. CONCLUSIONS……………………………………………………………..…75 CHAPTER 4. THE ROLE OF E2F4 IN PROMOTING THE G2 ARREST RESPONSE TO IR IN PROSTATE CARCINOMA 4.1. ABSTRACT……………………………………………………………………..77 4.2. INTRODUCTION………………………………………………………………78 4.3. MATERIALS AND METHODS 4.3.1. Cell culture and treatment…..……………………………………………..81 4.3.2. Flow cytometry 4.3.2.1. Propidium Iodide staining……………………………………….82 4.3.2.2. Multiparametric staining ………………………………………..82 4.3.2.3. BrdU/PI labeling………………………………………………...83 4.3.2.4. Caspase activation assay………………………………………...83 4.3.3. siRNA……………………………………………………………………..84 4.3.4. Clonogenic assay …………………………………………………………84 4.3.5. Determination of surviving fraction……………………………………….85 4.3.6. Western Blot detection…………………………………………………….85 4.3.7. Confocal microscopy ……………………………………………………..86 4.3.8. Comet assay……………………………………………………………….86 4.3.9. C4-2 expression profiling oligonucleotide arrays…………………………87 3 4.3.10. RTQ-PCR………………………………………………………………...88 4.3.11. Chromatin Immunoprecipitation Assay (ChIP) …………………………88 4.4. RESULTS 4.4.1. C4-2 prostate carcinoma cells undergo a G2/M arrest following IR……………………………………………………………….90 4.4.2. E2F4/p130 complexes are formed following IR…………………………..98 4.4.3. A physiological role for E2F4 following IR in the G2/M phase control of the cell cycle……………………………………………102 4.4.4. Identification of putative E2F4 target genes involved in the IR response….………………………………………………………..113 4.5. DISCUSSION………………………………………………………………….119 CHAPTER 5. FUTURE DIRECTIONS: MANIPULATING CELL CYCLE CHECKPOINTS FOR TARGETED THERAPIES FOR CANCER TREATMENT 5.1. INTRODUCTION……………………………………………………………..125 5.2. E2FS AS MOLECULAR TARGETS IN CANCER THERAPY……………...126 5.3. SUMMARY AND FUTURE DIRECTIONS………………………………….127 BIBLIOGRAPHY………………………………………………………………………129 4 LIST OF FIGURES CHAPTER 1 Fig. 1-1. Phases of the cell cycle…………………………………………………………18 Fig. 1-2. Temporal expression of cyclins………………………………………………...21 Fig. 1-3. Members of the E2F family of transcription factors…………………………...24 Fig. 1-4. The electromagnetic spectrum…………………………………………………33 CHAPTER 2 Fig. 2-1. Model for p53-dependent regulation of cell cycle arrest and apoptosis following IR.…………………………………………………………53 Fig. 2.2. General scheme for performing chromatin immunoprecipitation experiments in MOLT-4 cells …………………………………………………59 Fig. 2-3. p53 binding is differentially regulated following IR…………………………...61 CHAPTER 4 Fig. 4-1. Radiation induces a G2/M cell cycle arrest independently of p53 function in prostate carcinoma C4-2 cells……………………………………..91 Fig. 4-2. The radiation-induced arrest is specific to the G2 phase of the cell cycle…………………………………………………………………...95 Fig. 4-3. E2F4 levels are sustained after IR and E2F4 co-localized with p130 at the time of G2 arrest……………………………………………………99 Fig. 4-4. E2F4 downregulation causes an inappropriate G2 cell cycle arrest.…………103 5 Fig. 4-5. Lack of BrdU incorporation indicates that formerly arrested G2 cells are not recycling…………………………………………………….107 Fig. 4-6. E2F4 knock-down by siRNA initiates caspase activation and DNA strand breakage…………………………………………………………110 Fig. 4-7. Clonogenic assays indicated that E2F4 knock-down by siRNA can radiosensitize cells………………………………………………..112 Fig. 4-8. IR induces the downregulation of E2F4 targets………………………………116 6 LIST OF TABLES CHAPTER 2 Table 2-1. ChIP sense (S) and antisense (AS) primers used for detecting p53 bound to chromatin …………..………………………………….…………..51 Table 2-2. Expression profiling reveals radiation responsive genes containing putative p53 binding sites in their regulatory regions………………………..54 Table 2-3. Basal levels of p53 binding validate putative binding sites in regulatory regions within individual genes……………………………………………...63 CHAPTER 4 Table 4-1. E2F4 target genes identified by HU-95 Affymetrix array…………………..114 Table 4-2. Putative E2F4 target genes and their respective primer sequences…………115 7 ACKNOWLEDGEMENTS Life moves us in ways that are unpredictable and gives us challenges that demand coping strategies, which in turn promote growth in character. To steer through moments of joy and self-doubt, one ultimately needs to adapt to new situations, accept uncontrollable events, and take time to reflect upon experiences. Meeting challenges directly and growing from them pervade every facet of life. In this experience, we arrive at a true sense of humility and curiosity, which are cultured through evaluating ideas and learning to accept the criticism of others. The pursuit of obtaining the Ph.D. represents a culmination of maturity on multiple of levels. Likewise, a mentor accepting students shares in this responsibility by enabling the student to mature as a scientist by providing valuable experiential knowledge and guidance. From this vantage point, I thank my advisor, Dr. Alex Almasan, whom has encouraged my creativity and scientific independence. I have enjoyed working and studying in his laboratory. The support of people in the laboratory is an important element of succeeding in science, as their input and criticism have been very useful to my work. On a personal level, these people have also nurtured my understanding of what a world citizen truly is. I am grateful to the members of the Almasan laboratory for their support on a professional and personal level. I thank my labmates Dr. Erica DuPree, Dr. Damodar Gupta, Dr. Suparna Mazumder, Marcela Oancea, Dragos Plesca, and Dr. Subrata Ray for their scientific insight and companionship through my graduate education. My thanks also go to my thesis committee members Drs. Jacobberger, Silverman, Stavnezer, and Veigl, for their constructive criticism. I thank the Departments of Environmental Health Sciences 8 and Cancer Biology for giving me the opportunity to pursue a Ph.D. and for supporting my scientific growth at conferences. Finally, I thank my parents, Walter and Ethel, my sister, Melissa, my brother, Jonathan, and my dearest Alex for their sustained encouragement. Because they believed in me, they gave me the strength and the ability to face difficulties. I am where I am today because of their endless love and support. 9 LIST OF ABBREVIATIONS 14-3-3σ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (sigma isoform) APAF-1 apoptotic protease activating factor 1 APC Anaphase Promoting Complex AS antisense (primer direction) ATM ataxia-telangiectasia mutated ATP adenosine triphosphate ATR ataxia-telangiectasia and rad3 related Bax Bcl-2-associated X protein Bcl-2 B-cell CLL/lymphoma 2 BER Base Excision Repair Bik Bcl-2-interacting killer Bim Bcl-2-interacting protein bp base