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And Double-Strand Break Repair INVOLVEMENT OF SINGLE- AND DOUBLE-STRAND BREAK REPAIR PROCESSES IN BETA-LAPACHONE-INDUCED CELL DEATH by MELISSA SROUGI BENTLE Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. David A. Boothman Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY August, 2007 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. ii For Noline, Mom and the memory of my Father iii TABLE OF CONTENTS Title Page i Signature Sheet ii Dedication iii Table of Contents 1 List of Tables 4 List of Figures 5 Preface 7 Acknowledgements 8 List of Abbreviations 9 Abstract 13 CHAPTER 1: Introduction New Tricks for Old Drugs: The Anticarcinogenic Potential of DNA Repair Inhibitors Introduction to DNA Repair 15 Targeting DNA DSB Response Pathways for the Prevention and Treatment of Cancer 19 Mechanisms of SSB Response Pathways and Utility as Targets for Chemotherapy 25 Targeting Direct Repair for Chemotherapy 27 Poly(ADP-ribosyl)ation, PARP-1, and DNA Repair 28 PARP-1 Intervention as a Means of Inhibiting Multiple DNA Repair 1 Pathways and Transformation 32 β-Lap: an Anticarcinogenic DNA Repair Inhibitor 35 Mechanism of β-Lap Toxicity in Cancer Cells 39 β-Lap and DNA Repair Inhibition 46 Concluding Remarks 47 CHAPTER 2: Calcium-Dependent Modulation of Poly(ADP-ribose) Polymerase-1 Alters Cellular Metabolism and DNA Repair Abstract 49 Introduction 50 Experimental Procedures 52 Results 58 Discussion 89 CHAPTER 3: Non-Homologous End Joining is Essential for Cellular Resistance to the Novel Antitumor Agent, β-Lapachone Abstract 98 Introduction 99 Experimental Procedures 102 Results 105 Discussion 126 CHAPTER 4: Discussion and Future Directions 134 A.1 Conclusions: PARP-1 Mediates β-Lap-Induced Cell Death 135 A.2 Future Directions β-Lap’s Cytotoxic Properties Resemble Ischemia-Reperfusion 137 2 Metabolic Implications of β-Lap Usage for Cancer Treatment 141 The Role of PARG in Modulating β-Lap-Induced Cell Death 142 B.1 Conclusions: Targeting DSB Repair to Enhance β-Lap-Induced Lethality 144 B.2 Future Directions LMDS as Potential Critical Lesions Formed After β-Lap Exposure 145 Model of β-Lap-Induced DNA Damage Responses 147 Interplay Between PARP-1 and DNA-PK in Improving the Efficacy of β-Lap 147 C. Summary 148 BIBLIOGRAPHY 150 3 LIST OF TABLES Table 2.1. Addition of BAPTA-AM allows DNA repair after β-lap exposure 94 Table 4.1. Comparison of the mechanism(s) of cell death induced by ischemia- reperfusion and β-lap 138 4 LIST OF FIGURES Figure 1.1. Model of anticarcinogenic potential of DNA repair 17 inhibitors Figure 1.2. Mechanism of β-lap-induced cell death 44 Figure 2.1. β-Lap-induced cell death is time- and Ca2+ -dependent 60 Figure 2.2. Ca2+ chelation prevents β-lap-induced nuclear morphological changes and atypical PARP-1 and p53 proteolysis, but not STS- induced PARP-1 cleavage 62 Figure 2.3. β-Lap induces NQO1- and Ca2+-dependent PARP-1 hyperactivation 66 Figure 2.4. NQO1- and Ca2+-dependent PARP-1 hyperactivation after β-lap exposure is not cell type specific 68 Figure 2.5. PARP-1-dependent NAD+ and ATP pool depletion leads to cell death after β-lap exposure in MCF-7 cells 72 Figure 2.6. PARP-1 plays an essential role in β-lap-induced apoptotic cell death as monitored by TUNEL 75 Figure 2.7. β-Lap-induced γ-H2AX foci formation is abrogated by BAPTA-AM pre-treatment 78 Figure 2.8. Ca2+ modulates DNA repair in β-lap-treated cells 81 Figure 2.9. Ca2+ chelation modulates DNA repair after β-lap treatment 84 2+ Figure 2.10. H2O2 causes Ca -dependent PARP-1 hyperactivation and cell death 86 5 Figure 2.11. H2O2 causes time-dependent ATP depletion in NQO1 expressing cells 87 2+ Figure 2.12. H2O2 causes Ca -dependent, presumably µ-calpain-mediated, atypical PARP-1 and p53 proteolysis 88 Figure 3.1. The MRN complex is activated upon β-lap treatment 107 Figure 3.2. Perinuclear localization of DNA repair proteins following β-lap treatment 108 Figure 3.3. Dose-dependent ATM and DNA-PK activation after β-lap administration 111 Figure 3.4. Loss of DNA-PKcs activity potentiates β-lap-induced cell death 116 Figure 3.5. β-Lap-induced cell death is not dependent on ATM 119 Figure 3.6. β-Lap causes ATR activation and SSBs 123 Figure 3.7. β-Lap exposure induces DNA damage 125 Figure 3.8. Model of β-lap-induced cell death after lethal and sub-lethal doses 127 Figure 3.9. β-Lap-induced DNA damage is NQO1-mediated 129 6 PREFACE As research intensifies on the search for agents that are effective against cancer, more attention has focused on the use of naturally occurring compounds. One such agent, β-lapachone, is currently under Phase I/Phase II clinical trials for the treatment of pancreatic and other cancers. β-Lapachone has a rich history stemming from its use as a folk medicine centuries past. It is a metabolite of lapachol, a main constituent of the inner bark of the Lapacho tree (Tabebuia heptaphylla, T. impetiginosa, or T. avellanedae). Natives would use the bark in a variety of preparations for the treatment of bacterial, viral, and fungal diseases to cancer. This thesis describes in more mechanistic detail the cancer chemotherapeutic properties of β-lapachone and its future as an effective and selective treatment for human cancers. 7 ACKNOWLEDGMENTS The scientific world is an opened-door community. As such, this body of work is not solely the result of an individual effort; rather it is the melding of a number of ideas and perspectives. I would like to express my gratitude to my advisor, David A. Boothman, Ph.D. and the members of my thesis committee Monica Montano, Ph.D., George Dubyak, Ph.D, Clark Distelhorst, M.D., Anna-Liisa Nieminen, Ph.D and Paul MacDonald, Ph.D. I am especially indebted to the following individuals for their guidance, technical assistance and support: John J. Pink, Ph.D., Erik A. Bey, Ph.D., Kathryn E. Reinicke, Ph.D., Minh Lam, Ph.D., John-Paul Lavik, as well as the members of the Case Comprehensive Cancer Center on the 3rd floor of the Wolstein Research Building. Thank you. 8 LIST OF ABBREVIATIONS AGT O6-alkylguanine-DNA-alkyltransferase AIF Apoptosis-inducing factor AP Apurinic or apyrimidinic AT Ataxia telangiectasia ATM Ataxia telangiectasia mutated ATP Adenosine 5’-triphosphate ATR Ataxia telangiectasia and Rad3-related ARE Antioxidant response element BAPTA-AM 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra-(acetoxymethyl ester) BER Base excision repair β-Lap β-Lapachone BRCA1 Breast cancer 1, early onset Ca2+ Calcium Chk2 Checkpoint 2 DCF 6-carboxy-2′7′-dichlorodihydrofluorescin diacetate, di(acetoxymethyl ester) DIC Dicoumarol DNA Deoxyribonucleic acid DNA-PK DNA-dependent protein kinase DNA-PKcs DNA-dependent protein kinase catalytic subunit 9 DPQ (3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)- isoquinoline DSB Double-strand break ER Endoplasmic reticulum γ-H2AX Phosphorylated histone H2AX GGR Global genome repair HR Homologous recombination H2O2 Hydrogen peroxide HQ Hydroquinone iCAD Inhibitor of caspase-activated DNAse IR Ionizing radiation KU-55933 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one LDH-A Lactate dehydrogenase A MGMT O6-methyltransferase-DNA methyltransferase MMP Mitochondrial membrane depolarization MNNG N-methyl-N′-nitro-N-nitroguanidine MOMP Mitochondrial outer membrane permeabilization MMP Mitochondrial member permeabilization MRN Mre11-Rad50-Nbs-1 NAD+ Nicotinamide adenine dinucleotide NER Nucleotide excision repair NHEJ Non-homologous end joining NSCLC Non-small cell lung carcinoma 10 ns-shRNA Non-silencing short hairpin RNA NQO1 NAD(P)H quinone oxidoreductase-1, DT-diaphorase O6-BG O6-benzylguanine OXPHOS Oxidative phosphorylation PAR Poly(ADP-ribose) PARG Poly(ADP-ribose) glycohydrolase PARP-1 Poly(ADP-ribose) polymerase-1 PIKKs Phosphatidylinositol 3-kinase related kinases PI3Ks Phosphoinositide-3 kinases PLDR Potentially lethal DNA damage recovery PMCA Plasma membrane Ca2+-ATPase PP2A Protein phosphatase 2A ROS Reactive oxygen species RPA Replication protein A UV Ultraviolet radiation SERCA Sarcoplasmic/endoplasmic reticulum ATPase SSB Single-strand break ss Single-stranded STS Staurosporine SQ Semiquinone TCR Transcription coupled repair TMZ Temozolomide Topo Topoisomerase 11 TRPM Transient receptor potential-melastatin-like TUNEL Terminal deoxynucleotidyl transferease-mediated dUTP nick-end labeling U1-Mel Human malignant melanoma cells XRE Xenobiotic response element 3-AB 3-aminobenzamide 231 MDA-MB-231 231-NQ- MDA-MB-231-NQO1-negative 231-NQ+ MDA-MB-231-NQO1-positive 12 Involvement of Single- and Double-Strand Break Repair Processes in Beta- Lapachone-Induced Cell Death Abstract by MELISSA SROUGI BENTLE β-Lapachone (β-Lap; a.k.a. ARQ 501) is a novel antitumor quinone currently in Phase II clinical trials for the treatment of pancreatic and head/neck cancers. β-Lap has been shown to be an effective cancer chemotherapeutic agent both in vitro and in vivo against a number of human cancers that express NAD(P)H:quinone oxidoreductase-1 (NQO1). Bioactivation of β-lap by NQO1 caused a futile oxidoreduction, leading to reactive oxygen species generation (ROS). Unique to this compound was its ability to kill cancer cells regardless of abnormalities in commonly altered apoptosis-related proteins, such as p53, Bcl-2, and Bax/Bak. This thesis describes in more mechanistic detail the cell death pathway activated after β-lap treatment, with particular focus on its involvement with, and resistance by, DNA repair.
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