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Tracking Profiles of Genomic Instability in Spontaneous Transformation and Tumorigenesis Lesley Lawrenson Wayne State University

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Tracking Profiles of Genomic Instability in Spontaneous Transformation and Tumorigenesis Lesley Lawrenson Wayne State University Wayne State University Wayne State University Dissertations 1-1-2010 Tracking profiles of genomic instability in spontaneous transformation and tumorigenesis Lesley Lawrenson Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Bioinformatics Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Lawrenson, Lesley, "Tracking profiles of genomic instability in spontaneous transformation and tumorigenesis" (2010). Wayne State University Dissertations. Paper 492. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. TRACKING PROFILES OF GENOMIC INSTABILITY IN SPONTANEOUS TRANSFORMATION AND TUMORIGENESIS by LESLEY EILEEN LAWRENSON DISSERTATION Submitted to the Graduate School of Wayne State University Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2010 MAJOR: MOLECULAR MEDICINE AND GENETICS Approved by: Advisor Date © COPYRIGHT BY LESLEY LAWRENSON 2010 All Rights Reserved DEDICATION This work is dedicated to my husband, family, friends, mentors, and colleagues with deepest gratitude for their guidance and support. ii ACKNOWLEDGEMENTS The submission of this dissertation brings to an end a wonderful period in which I was a graduate student in Molecular Medicine and Genetics at Wayne State University School of Medicine. Along this path, my mentors, friends and family have helped me grow in sharing with me the many joyous moments as well as the challenges presented throughout the development of this work. I am forever indebted to those who encouraged me to continue to pursue my education. I am deeply grateful to my advisors, Prof. Henry Heng and Prof. Wayne Lancaster for their guidance, encouragement, and faith. Both have served as wonderful role models for me. Their knowledge, reasoning, and experience will continue to serve me well in my future career. I wish to thank the members of my committee for their patience, wisdom, and good humor. I am extremely grateful for the contributions of Dr. Lucie Gregoire for sharing her knowledge of cell culture of this model and for her guidance throughout this time; Dr. Raja Rabah for evaluations of the tumor histopathology for the animal portion of this work; Dr. Josh Stevens for his technical assistance, his willingness to help, and for our numerous discussions in the development of this manuscript; and Guo Liu for his contributions with cell culture and SKY analysis. Additionally, I am grateful to Jayson Field, MD for his clinical expertise and for providing me the opportunity to participate in the surgical and clinical care of ovarian cancer patients. I would like to thank my family. Thank you to my loving parents, for instilling in me the desire for knowledge and the determination to succeed. Finally, I thank my husband, Gavin, for his unending support and for always believing in me. iii TABLE OF CONTENTS Dedication……………………….……………………………………………………………...…….ii Acknowledgments………………………………..…………………………………………...…….iii List of Tables…………………………………….…………...…...………….....….…………….....v List of Figures………………………...…….…………………….………………………………....vi CHAPTER 1. TRACKING PROFILES OF GENOMIC INSTABILITY IN SPONTANEOUS TRANSFORMATION AND TUMORIGENESIS Background and Significance …….……………………........………………………...…… 1 Methods…………………………………………………………………………….……......…13 Results…………………………………….…………………………………………………… 26 Discussion……………………………………………………………..........………………… 81 Theoretical Considerations………………………………………………...…………...........105 References……………………………………………………..……………………………………. 129 Abstract……………………………………………………………………………………………….155 Autobiographical Statement………………….………………………………………………….. 157 iv LIST OF TABLES Table 1. Phenotype and characteristics of mouse ovarian surface epithelial cell transformation in vitro ……………….……………………............................................................. 34 Table 2. Karyotype data and Shannon variability indices at key transformative stages and of cells from harvested tumors ………………….………………...................……………….............. 37 Table 3. Population karyotype characteristics for mouse ovarian surface epithelial cell stages and lines………………….…………………………......................................……………................43 Table 4. Gene list and expression profiles by cluster for 599 differentially expressed genes during transformation ……………………………............................................................….…..... 48 Table 5. Significant genes between consecutive stages by paired comparisons analysis........ 55 Table 6. Functional enrichment analysis for genes discovered by paired comparisons …........63 Table 7. In vivo tumorigenicity analyses of mouse ovarian surface epithelial cells ….............. 80 v LIST OF FIGURES Figure 1. Experimental overview and analyses performed on key stages of spontaneous mouse ovarian surface epithelial cell transformation……………….…………………………......… 27 Figure 2. Chromosome count data for replicate and non-viable primary cell lines, each seeded from a single C57BL6 mouse ovary………………….………………………………......................... 28 Figure 3. Morphologic and behavioral characteristics of mouse ovarian surface epithelial cell senescent and mitotic sub-populations……………….…………………………………….............…30 Figure 4. Spectral karyotype analysis of nuclear and cytogenetic abnormalities from primary mouse ovarian surface epithelial cells before day 40………………………………....................…. 32 Figure 5. Cellular morphology and phenotype of key transformative stages during spontaneous transformation in vitro………………………………………………………………..............................35 Figure 6. Karyographs showing the extent of karyotype diversity throughout the spontaneous transformation of mouse ovarian surface epithelial cells………………....................................…..40 Figure 7. Violin plots and Shannon Indices characterizing karyotype diversity throughout the spontaneous transformation of mouse ovarian surface epithelial cells…............................…….. 42 Figure 8. Karyotypic variability in day 450 subpopulations selected on the basis of presence or absence of 4;3 clonal translocation…............……………………………………............................. 45 Figure 9. Time course profiling, cluster analysis and biological significance for each of eight significant temporal expression profiles during tumorigenesis…...................................................47 Figure 10. Area proportional diagrams of the relationships among genes with significant differential expression during tumorigenesis…………………………………..………......…….…… 71 Figure 11. Relationships between chromosome counts and average mRNA transcript abundance for key transitional stages and between consecutive time points…….………......….. 73 Figure 12. Histopathology of subcutaneous and intraperitoneal tumors in vivo for day 245 versus day 528 allografts………....................................................………….…………………....... 75 Figure 13. Karyograph analysis and key karyotypic features of late state tumorigenic cells versus and harvested tumors from C57BL6 mice…………................…....………………….......... 77 Figure 14. Whole chromosome count data for array competitive genomic hybridization…...........79 vi 1 1. BACKGROUND AND SIGNIFICANCE 1.1 Ovarian Cancer Etiology Ovarian cancer is the most prevalent and deadly gynecologic malignancy in the United States, and, as the fifth most common cause of cancer death in women, accounts for approximately 16,000 deaths per year [1]. Nearly all ovarian cancers originate from the epithelial cells comprising the outermost surface of the ovary. There are four major subtypes of epithelial ovarian cancers (EOC) which are categorized by their histopathologic characteristics [2]. EOC variability is present not only in the morphology of histopathologic samples, but also in the wide variety of unique karyotypes, and the numerous low frequency genetic abnormalities associated with this disease. Specific genetic alterations tend to be inconsistent between studies, but include changes on many levels including DNA sequence, copy number, methylation status, and miRNA levels [3-4]. Features on each level have been correlated with resistance to chemotherapy and with differing patient survival rates [5]. Despite an increased knowledge of the etiology of this disease, improvements in surgical techniques, advancements in chemotherapeutic treatments, and the characterization of ovarian cancer genomes at many levels [5-6], the morbidity and mortality associated with EOC overall has remained largely unchanged (0.39 in 1980–1989 to 0.43 in 1990–1997) [7]. EOC is often asymptomatic in its earliest stages and strategies with sufficient sensitivity and specificity to detect early-stage disease are currently not available. As a result, most patients are diagnosed with late-stage disseminated EOC which typically becomes resistant to both standard and combination chemotherapies [7-9] and carries a prognosis of only 20% survival over five years [7]. Therefore, the specific challenges presented by the heterogeneous and clinically insidious nature of EOC underlie the rationale for continued focus on these areas of research to improve upon the high morbidity and mortality currently associated with this disease. Due to the paucity of early-stage clinical samples, the initiating events in ovarian cancer transformation are not well
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