The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2012 Understanding the roles of Non-homologous end joining and p53 after DNA damage Omid Tavana Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Medicine and Health Sciences Commons Recommended Citation Tavana, Omid, "Understanding the roles of Non-homologous end joining and p53 after DNA damage" (2012). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 227. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/227 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. Understanding the roles of Non-homologous end joining and p53 after DNA damage by Omid Tavana, BS, Ph.D. APPROVED: ______________________________ Chengming Zhu, Ph.D., Supervisory Professor ______________________________ Hui-Kuan Lin, Ph.D. ______________________________ Randy Legerski, Ph.D. ______________________________ Bin Wang, Ph.D. ______________________________ Junjie Chen, Ph.D. APPROVED: ____________________________ Dean, The University of Texas Graduate School of Biomedical Sciences at Houston [Type text] i [Type text] Understanding the roles of Non-homologous end joining and p53 after DNA damage A DISSERTATION Presented to the Faculty of The University of Texas Health Science Center at Houston And The University of Texas M. D. Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY By Omid Tavana, B.S. Houston, Texas May, 2012 ii DEDICATION Trying to express in words my gratefulness to having such loving, understanding, and encouraging parents does not do justice to their contribution in my life, so I humbly dedicate this work to them—I would be nothing without you guys. To my sister, thank you for leading the way; you have always been my example of hard work, strength and excellence. To my best friend and twin brother, I am lucky to have such a role model; genius is often misused, but you are truly a genius. My favorite times have always been spent around the dinner table. iii ACKNOWLEDGEMENTS This work could not have been accomplished without the help of many. First, I owe my deepest thanks to my mentor Chengming (Ben) Zhu, who agreed to take me under his wing as his student. Led by example, you have molded me into the mature scientist I am today and always with a smile on your face. You were the perfect match for my personality, putting my needs/concerns as a top priority. My lab members, both former and current, taught me many different scientific techniques making this work possible. I am grateful to Dr. Melanie Dujka who, as a senior student, eased my transition as well as to Dr. Nahum Puebla for technical help and guidance. To our lab technician and manager, Mei Sang, thank you for always placing orders and taking care of anything needed without question. To Stella Jang for, at times, diverting Ben’s attention away from me. To Dr. Cara Benjamin, although we ended in disagreement, I thank you for allowing me to complete and publish your unfinished project. All my committee members: Drs. Jianping Jin, Phil Carpenter, Randy Legerski, Phoebus Lin, Bin Wang, Steve Ullrich, Junjie Chen, and Hui-Kwan Lin. Thank you for taking time out of your busy schedules traveling down to south campus and offering invaluable advice for my projects. iv My auntie, Dr. Khandan Keyomarsi has always been an excellent role model and quasi co-mentor. Although we disagree sometimes, I know you always have my best interests in mind. I am so lucky to have you on my side. To all my fourth floor family, your help with techniques, reagents, and willingness to help has been fantastic. Dr. Ma Ying, thank you for being my neighbor; your presence every morning starts my day off with a smile. Dr. Hongbo Hu, you are a great friend and lifting partner. Dr. Huiyuan Zhang, thank you for all your advice, interest in my projects, and always being so trendy. Dr. Nam Nguyen-Jackson, you are my greatest scientific buddy. Dr. Grzegorz Chodaczek, thanks for all the music and fun; sorry I still cannot pronounce your name properly. To Venna Papanna, thank you for all the stories and fun times we shared in our common space. To Mrs. Janis Johnson, thank you for being a presence in my life. Your smile is heartwarming and compassion is addictive. Lastly, I would like to thank all my graduate school friends who shared this same experience with me. I would be terribly bored on Friday and Saturday nights without you guys. To my good college friends and my dearest of friends the ‘wolf pack’ from back home in Alabama. v ABSTRACT The inability to maintain genomic stability and control proliferation are hallmarks of many cancers, which become exacerbated in the presence of unrepaired DNA damage. Such genotoxic stresses trigger the p53 tumor suppressor network to activate transient cell cycle arrest allowing for DNA repair; if the damage is excessive or irreparable, apoptosis or cellular senescence is triggered. One of the major DNA repair pathway that mends DNA double strand breaks is non-homologous end joining (NHEJ). Abrogating the NHEJ pathway leads to an accumulation of DNA damage in the lymphoid system that triggers p53-mediated apoptosis; complete deletion of p53 in this system leads to aggressive lymphomagenesis. Therefore, to study the effect of p53- dependent cell cycle arrest, we utilized a hypomorphic, separation-of-function mutant, p53p/p, which completely abrogates apoptosis yet retains partial cell cycle arrest ability. We crossed DNA ligase IV deficiency, a downstream ligase crucial in mending breaks during NHEJ, into the p53p/p background (Lig4-/-p53p/p). The accumulation of DNA damage activated the p53/p21 axis to trigger cellular senescence in developing lymphoid cells, which absolutely suppressed tumorigenesis. Interestingly, these mice progressively succumb to severe diabetes. Mechanistic analysis revealed that spontaneous DNA damage accumulated in the pancreatic -cells, a unique subset of endocrine cells solely responsible for insulin production to regulate glucose homeostasis. The genesis of adult -cells predominantly occurs through self-replication, therefore modulating cellular proliferation is an essential component for renewal. The progressive accumulation of DNA damage, caused by Lig4-/-, activated p53/p21-dependent cellular senescence in mutant pancreatic -cells that lead to islet involution. Insulin levels subsequently vi decreased, deregulating glucose homeostasis driving overt diabetes. Our Lig4-/-p53p/p model aptly depicts the dichotomous role of cellular senescence—in the lymphoid system prevents tumorigenesis yet in the endocrine system leads to the decrease of insulin-producing cells causing diabetes. To further delineate the function of NHEJ in pancreatic -cells, we analyzed mice deficient in another component of the NHEJ pathway, Ku70. Although most notable for its role in DNA damage recognition and repair within the NHEJ pathway, Ku70 has NHEJ-independent functions in telomere maintenance, apoptosis, and transcriptional regulation/repression. To our surprise, Ku70-/-p53p/p mutant mice displayed a stark increase in -cell proliferation, resulting in islet expansion, heightened insulin levels and hypoglycemia. Augmented -cell proliferation was accompanied with the stabilization of the canonical Wnt pathway, responsible for this phenotype. Interestingly, the progressive onset of cellular senescence prevented islet tumorigenesis. This study highlights Ku70 as an important modulator in not only maintaining genomic stability through NHEJ-dependent functions, but also reveals a novel NHEJ-independent function through regulation of pancreatic -cell proliferation. Taken in aggregate, these studies underscore the importance for NHEJ to maintain genomic stability in -cells as well as introduces a novel regulator for pancreatic -cell proliferation. vii TABLE OF CONTENTS DEDICATION -------------------------------------------------------------------------------------------------------------------iii ACKNOWLEDGEMENTS -------------------------------------------------------------------------------------------------------iv ABSTRACT -------------------------------------------------------------------------------------------------------------------vi TABLE OF CONTENTS -----------------------------------------------------------------------------------------------------viii LIST OF FIGURES -------------------------------------------------------------------------------------------------------xi LIST OF TABLES -----------------------------------------------------------------------------------------------------xiii CHAPTER 1. INTRODUCTION --------------------------------------------------------------------------------------------1 1.1