Definition of Mechanisms of Mutation Generation in Tissues and Embryonic Stem Cells of the Constitutive Fhit Knockout Mouse
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Definition of mechanisms of mutation generation in tissues and embryonic stem cells of the constitutive Fhit knockout mouse DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Carolyn Anne Paisie, H.BSc. Biomedical Sciences Graduate Program The Ohio State University 2015 Dissertation Committee: Kay Huebner, PhD, Advisor Vincenzo Coppola, MD Sissy Jhiang, PhD Yuri Pekarsky, PhD Copyright by Carolyn Anne Paisie, H.BSc. 2015 Abstract Genome instability, which can be defined as an increase in changes at both the nucleotide and chromosomal level (e.g. point mutations, chromosomal translocations), results from errors in normal biological processes that function to repair, replicate, and segregate the genome during cell division. Genomic instability is a hallmark of human neoplasia, present in varying degrees in all stages of cancer, from precancerous to advanced cancer. Genome instability is initiated due to loss of expression of the FHIT gene, located at 3p14.2; the murine Fhit gene is located at 14A2. Fhit loss occurs early in human cancer development and is frequently observed in preneoplastic lesions. The Fhit protein is a tumor suppressor and genome caretaker that modulates genome stability and level of DNA damage that accumulates beginning in precancerous lesions. To investigate the contribution of loss of Fhit expression to the generation of mutations and to define mechanisms that underlie these genome alterations, we performed an analysis of whole exome sequences from cell lines and tissues, from wildtype and Fhit-/- mice. -/- cells and tissues demonstrated increased numbers of C>T and T>C mutations and Fhit-deficient kidney cell cultures that survived dimethylbenz(a)anthracene treatment exhibited increased numbers of T>A mutations. Following determination of trinucleotide contexts of mutations, a Fhit-loss signature was ii proposed to consist of C>T and T>C mutations that may be due to respective increased spontaneous deamination (C>T mutations) and deoxyribonucleotide triphosphates pool imbalance (T>C mutations), a signature similar to the 'age at diagnosis' signature identified in human cancers. Increased T>C mutations in -/- exomes may be due to the imbalance in deoxyribonucleotide triphosphates, particularly thymidine triphosphate, resulting from decreased expression of Thymidine Kinase 1 in Fhit-deficient cells and tissues. Fhit-deficient kidney cell cultures that survived in vitro dimethylbenz(a)anthracene treatment additionally exhibited an increase in T>A mutations, a signature known to be generated by treatment with carcinogens such as dimethylbenz(a)anthracene, a consequence of inefficient nucleotide excision repair. We continued our examination of mutations in genomes of short-term cultured embryonic stem cells and derived differentiated cells. These studies revealed an increase in mutations in -/- embryonic stem and derived differentiated cells vs +/+ embryonic stem and derived differentiated cells. From this analysis we cannot separate mutations due to genetic drift, relative to the B6 reference, from possible accumulation of mutations in the -/- germline. In addition to point mutations, -/- kidney tissue contained more insertions and particularly more deletions compared to +/+ liver tissue; loci at regions of insertions and deletions in -/- kidney tissue contained sequence motifs (e.g. poly(C) stretches) and sequences that might participate in insertion/deletion through microhomology directed repair. In summary, the results revealed that multiple types of mutations occurred more frequently in the Fhit-/- environment, with multiple mechanisms of damage and repair involved in generating mutations. A future goal will be to follow the initiation and iii accumulation of mutations through tissue development and through the germline in a conditional Fhit-/- strain on pure background, after deleting Fhit in the germline and in specific organs through recombinase technology. iv Dedication To my family, for their love and support. In loving memory of Martha Paisie and Dorothy Walsh. v Acknowledgments I am especially grateful to my advisor, Dr. Kay Huebner, for mentoring and supporting me through my graduate research years. I would like to thank my thesis committee members, Drs. Vincenzo Coppola, Sissy Jhiang, and Yuri Pekarsky, for their helpful comments and constructive critiques during committee meetings, for their suggestions on my current and future research, and for ensuring my timely progression through the program. I would like to thank the Biomedical Science Graduate Program for offering me a position in the program in 2009 and a special thank you to Dr. Ginny Sanders, the program director at the time, for her support and encouragement. I thank members of the Huebner lab, both past and present, for making the lab an enjoyable environment to work in on a daily basis. I would like to thank Dr. Satoshi Miuma for performing the initial experiments which generated the raw data that served as the starting point for the work described in this dissertation and Dr. Jie Zhang for assistance with the initial bioinformatics analysis. I would like to thank Catherine Waters, Morgan Schrock, and Jenna Karras for many insightful discussions, critical reading of abstracts and manuscripts, and troubleshooting assistance and additional thanks to Morgan Schrock for her maintenance of our mouse colony. Special thanks to vi Teresa Druck for her assistance in preparing figures for publication, including those found in this dissertation, technical expertise, and assistance in performing important experiments that are included in this dissertation. I would also like to thank Dr. Sigrid Eckardt and Dr. John McLaughlin for the establishment of the mouse embryonic stem cell lines that were crucial for providing the exome sequencing data that was described in this dissertation. I greatly appreciate the financial support provided by The Ohio State University Wexner Medical Center and the National Institutes of Health. vii Vita June 2003 .................................................... Southeast Raleigh High School June 2007 .................................................... H.BSc. Developmental Biology, University of Toronto August 2009 to present ............................... Graduate Research Associate, Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Publications Paisie CA, Schrock MS, Karras JR*, Zhang J*, Miuma S*, Ouda IM, Waters CE, et al. Exome-wide single-base substitutions in tissues and derived cell lines of the constitutive Fhit knockout mouse. BMC Genomics. In review. *These authors contributed equally to this work. Karras JR*, Paisie CA*, Huebner K. Replicative Stress and the FHIT Gene: Roles in Tumor Suppression, Genome Stability and Prevention of Carcinogenesis. Cancers (Basel). 2014 Jun 4;6(2):1208-19. * These authors contributed equally to this work. Gasparini P, Fassan M, Cascione L, Guler G, Balci S, Irkkan C, Paisie C, et al. Androgen receptor status is a prognostic marker in non-Basal triple negative breast cancers and determines novel therapeutic options. PLoS One. 2014 Feb 5;9(2):e88525. Miuma S, Saldivar JC, Karras JR, Waters CE, Paisie CA, et al. Fhit deficiency-induced global genome instability promotes mutation and clonal expansion. PLoS One. 2013 Nov 14;8(11):e80730. viii Neviani P, Harb JG, Oaks JJ, Santhanam R, Walker CJ, Ellis JJ, Ferenchak G, Dorrance AM, Paisie CA, et al. PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells. J Clin Invest. 2013 Oct 1;123(10):4144-57. Wu Y, Feng X, Jin Y, Wu Z, Hankey W, Paisie C, et al. A novel mechanism of indole- 3-carbinol effects on breast carcinogenesis involves induction of Cdc25A degradation. Cancer Prev Res (Phila). 2010 Jul;3(7):818-28. Fields of Study Major Field: Biomedical Science Minor Field: Cancer Biology ix Table of Contents Abstract .......................................................................................................................... ii Dedication .......................................................................................................................v Acknowledgments ........................................................................................................ vii Vita ........................................................................................................................... viiiii List of Tables.............................................................................................................. xivv List of Figures ............................................................................................................... xv Abbreviations...................................................................................................................xvii Chapter 1: Introduction....................................................................................................1 I. Genome instability..........................................................................................................1 I.1 Chromosomal instability............................................................................................2 I.2 Microsatellite instability............................................................................................3 I.3 Insertions and deletions.............................................................................................4 II. The FHIT gene..............................................................................................................6