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Mechanistic Study of Fragile Site Instability by Investigating Ret/Ptc Rearrangements, a Common Cause of Papillary Thyroid Carcinoma

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Mechanistic Study of Fragile Site Instability by Investigating Ret/Ptc Rearrangements, a Common Cause of Papillary Thyroid Carcinoma MECHANISTIC STUDY OF FRAGILE SITE INSTABILITY BY INVESTIGATING RET/PTC REARRANGEMENTS, A COMMON CAUSE OF PAPILLARY THYROID CARCINOMA BY LAURA WILLIAMS DILLON A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Biochemistry and Molecular Biology May 2012 Winston-Salem, North Carolina Approved By: Yuh-Hwa Wang, Ph.D., Advisor David A. Ornelles, Ph.D., Chair Peter A. Antinozzi, Ph.D. Thomas Hollis, Ph.D. Alan J. Townsend, Ph.D. ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor, Dr. Yuh-Hwa Wang. She has provided my support and guidance over the years and I couldn’t have accomplished any of this without her. Throughout my graduate career under her supervision, she has always put education and scientific growth as a priority. I thank her for all the knowledge she has imparted to me, shaping me into the researcher I am today. I would also like to thank my committee members, Dr. David Ornelles, Dr. Peter Antinozzi, Dr. Thomas Hollis, and Dr. Alan Townsend, for their scientific guidance and encouragement. Thank you to all the current and past members of the Wang laboratory for providing support and discussing scientific thoughts and problems. I would like to especially thank Christine Lehman, for her work and contribution to the analysis of double-strand DNA breaks in patient thyroid tissue samples, and Allison Weckerle, for being a great friend and always available for advice. Additionally, I would like to thank our collaborators, without whom much of this work would not have been possible. Dr. Yuri Nikiforov and his colleague Dr. Manoj Gandhi from the University of Pittsburgh for collecting the data on chromosomal breakage at RET/PTC genes and detection of RET/PTC rearrangements in thyroid cells, and providing thyroid tissue samples for the DNA breakage analysis studies. Dr. Jennifer Cannon from Wake Forest School of Medicine for also providing patient thyroid tissue samples. ii Finally, I would like to thank my family and friends for all their love and support. My parents, David and Susan Williams, who have always encouraged me to do anything I put my mind to and been by my side every step of the way. From an early age, they provided a model for me of what it takes to be a great scientist, and I hope to always lead by their example. Thank you to my husband, Stephen, who has supported me in all my endeavors and always knows how to brighten my day. iii TABLE OF CONTENTS Page LIST OF ABBREVIATIONS…………………………………………………………………………………………….…..v LIST OF FIGURES……………………………………………………………………………………………………………..x LIST OF TABLES……………………………………………………………………………………………………………..xii ABSTRACT…………………………………………………………………………………………………………….………xiv Chapter I. INTRODUCTION…………………………………………………………………………………………………..1 II. DNA BREAKS AT FRAGILE SITES GENERATE ONCOGENIC RET/PTC REARRANGEMENTS IN HUMAN THYROID CELLS…………………………………25 Published in Oncogene, April 2010 III. DNA TOPOISOMERASES PARTICIPATE IN ONCOGENE RET FRAGILITY…………………………………………………………………………………………………..47 Manuscript in Preparation IV. DNA SECONDARY STRUCTURES INVOLVED IN FRAGILE SITE BREAKAGE FROM THE STUDY OF HUMAN CHROMOSOME 10………………………….78 Manuscript Submitted V. DEVELOPMENT OF A DNA BREAKAGE ASSAY TO DETECT SUSCEPTIBILITY TO RET/PTC REARRANGEMENT FORMATION AND POTENTIAL EXPOSURE TO ENVIRONMENTAL FRAGILE SITE-INDUCING CHEMICALS…………………………………………………………………………….117 VI. CONCLUSIONS…………………………………………………………………………………………………137 APPENDIX…………………………………………………………………………………………………………………..162 SCHOLASTIC VITA……………………………………………………………………………………………………….191 iv LIST OF ABBREVIATIONS °C Degrees Celcius 2-AP 2-Aminopurine ADD3 Adductin 3 (gamma) APH Aphidicolin ATM Ataxia telangiectasia mutated ATP Adenosine triphosphate ATR Ataxia telangiectasia and Rad3 related ATRIP ATR interacting protein BA Betulinic acid BAC Bacteria artificial chromosome BCL-2 B-cell CLL/lymphoma 2 bp Base pair BRCA1 Breast cancer 1 BRCA2 Breast cancer 2 BrdU Bromodeoxyuridine c-MYC V-myc myelocytomatosis viral oncogene homolog (avian) CA California CCDC6 Coiled-coil domain containing 6 cDNA Complementary deoxyribonucleic acid CFS Common fragile site CHK1 Checkpoint kinase 1 v CHK2 Checkpoint kinase 2 Chr Chromosome cm Centimeter CPT Camptothecin CPT-11 Camptothecin-11 DEN Diethylnitrosamine DNA Deoxyribonucleic acid DNA-PKcs DNA-dependent protein kinase, catalytic subunit dsDNA double-strand DNA dUTP Deoxyuridine triphosphate EDTA Ethylenediaminetetraacetic acid FA Fanconi amenia FANCD2 Fanconi anemia, complementation group D2 FANCI Fanconi anemia, complementation group I FHIT Fragile histidine triad FISH Fluorescence in situ hybridization FUdR Fluorodeoxyuridine G1-phase Gap 1 phase G2-phase Gap 2 phase G6PD Glucose-6-phosphate dehydrogenase H2AX H2A histone family, member X HR Homologous recombination vi HUS1 HUS1 checkpoint homolog (S. pombe) IGH@ Immunoglobulin heavy locus INA Internexin neuronal intermediate filament protein, alpha Kb Kilobase kcal Kilocalorie KCl Potassium Chloride L Liter LM-PCR Ligation-mediated Polymerase chain reaction M Molar Mb Megabase mg Milligram min Minute mL Milliliter mM Millimolar mol Mole mRNA Messenger ribonucleic acid Na Sodium NaOH Sodium hydroxide NC North Carolina NCBI National Center for Biotechnology Information NCOA4 Nuclear receptor co-activator 4 NFKB2 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 vii ng Nanogram NHEJ Non-homologous end joining nM Nanomolar NSC Non-small cell nt Nucleotide NUP98 Nucleoporin 98 kDa P Phosphorous PA Pennsylvania PAC P1 artificial chromosome PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PC Positive control PCNA Proliferating cell nuclear antigen PCR Polymerase chain reaction PI Propidium iodide PTC Papillary thyroid carcinoma PTEN Phosphatase and tensin homolog RAD51 RAD51 homolog (S. cerevisiae) RET Rearranged during transfection proto-oncogene RFS Rare fragile site RNA Ribonucleic acid RNase Ribonuclease viii RPMI Roswell Park Memorial Institite Medium RT-PCR Reverse transcription polymerase chain reaction S-phase Synthesis phase SD Standard deviation SDS Sodium dodecyl sulfate sec Second SMC1 Structural maintenance of chromosomes 1 SV40 Simian virus 40 TBE Tris/Borate/EDTA TBXAS1 Thromboxane A synthase 1 (platelet) TE Tris/EDTA TopBP1 Topoisomerase II binding protein 1 Tris Tris(hydroxymethyl)aminomethane USA United States of America V Volt VP-16 VePesid-16, etoposide WRN Werner syndrome, RecQ helicase-like WWOX WW domain containing oxidoreductase ZMIZ1 Zinc finger, MIZ-type containing 1 μg Microgram μL Microliter μM Micromolar ix LIST OF FIGURES Page 2.1: Fluorescence in situ hybridization on metaphase chromosomes of HTori-3 cells after treatment with fragile site-inducing chemicals…………………………………………….31 2.2: DNA breaksite mapping by LM-PCR……………………………………………………………………….34 2.3: LM-PCR detection of breaks formed in HTori-3 cells after treatment with APH……..35 2.4: Location of breakpoints within intron 11 of RET induced by treatment with APH….36 2.5: LM-PCR detection of breaks formed in HTori-3 cells after treatment with APH……..37 2.6: Detection of RET/PTC rearrangements in HTori-3 cells after treatment with fragile site-inducing chemicals……………………………………………………………………………………..39 3.1: Location of APH-induced DNA breakpoints within intron 11 of RET detected by LM-PCR……………………………………………………………………………………………………………………54 3.2: Location of APH-induced breakpoints within intron 11 of RET relative to known patient breakpoints…………………………………………………………………………………………..57 3.3: Comparison of APH-induced DNA breakpoints to predicted DNA topoisomerase I and II cleavage sites……………………………………………………………………………58 3.4: Comparison of CPT-11 and VP-16 induced topoisomerase I and II cleavage to predicted cleavage sites……………………………………………………………………………………………60 3.5: Location of APH-induced RET intron 11 breakpoints on predicted DNA secondary structures……………………………………………………………………………………………………62 3.6: Cell survival of HTori-3 cells following drug treatment…………………………………………..65 3.7: The effect of DNA topoisomerase catalytic inhibitors on the APH-induced common fragile site breakage………………………………………………………………………………………66 3.8: Frequency of APH-induced DNA breakage in combination with CPT-11 treatment…………………………………………………………………………………………………………70 4.1: Free energy values for predicted DNA secondary structures on chromosome 10….90 4.2: Division of the chromosome 10 sequence into non-fragile and fragile regions………91 x 4.3: Density of secondary structure forming potential on chromosome 10………………….95 4.4: Establishment and validation of a threshold for Mfold prediction of chromosomal fragility…………………………………………………………………………………………………..98 4.5: Regions predicted to exhibit fragile site breakage within APH-induced common fragile site FRA10G………………………………………………………………………………………101 4.6: DNA secondary structure prediction and in vitro detection within regions predicted to exhibit fragile site instability…………………………………………………………………..104 4.7: The most stable Mfold predicted DNA secondary structures and free energy values for DNA fragments analyzed by in vitro reduplexing assays……………………………..106 4.8: Location of regions predicted to exhibit fragile site instability and correlation with cancer-associated chromosomal aberrations…………………………………………..…………109 5.1: Induction of fragile site breakage by environmental and dietary agents, benzene and DEN……………………………………………………………………………………………………….125 5.2: The effect of APH, benzene, and DEN treatments on the cell cycle……………………..127 5.3: Frequency
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