Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) Spring 3-19-2014 Molecular Mechanisms of Programmed Necrotic Death Initiated by Intrinsic Death Signals Gary Xiaoshi Wang Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Recommended Citation Wang, Gary Xiaoshi, "Molecular Mechanisms of Programmed Necrotic Death Initiated by Intrinsic Death Signals" (2014). All Theses and Dissertations (ETDs). 1267. https://openscholarship.wustl.edu/etd/1267 This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has been accepted for inclusion in All Theses and Dissertations (ETDs) by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Molecular Cell Biology Dissertation Examination Committee: Emily H.-Y. Cheng, Chair Kendall J. Blumer, Co-Chair Ron Bose Shin-ichiro Imai Gerald P. Linette Jason C. Mills Zhongsheng You Molecular Mechanisms of Programmed Necrotic Death Initiated by Intrinsic Death Signals by Gary Xiaoshi Wang A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2014 Saint Louis, Missouri 1 © 2014, Gary Xiaoshi Wang TABLE OF CONTENTS List of Figures……………………………………………………………………………………………..iv Acknowledgements………………………………………………………………………………………vi Abstract………………………………………………………………………………………………….....ix Chapter One: Introduction………………………………………………………………………………1 1.1 Introduction……………………………………………………………………………………………..2 1.2 Apoptosis……………………………………………………………………………………………….3 1.3 Autophagy…………………………………………………………………………………………….16 1.4 Necrosis……………………………………………………………………………………………….23 1.5 Significance…………………………………………………………………………………………...34 1.6 Figure Legends……………………………………………………………………………………….35 1.7 Figures………………………………………………………………………………………………...36 1.8 References……………………………………………………………………………………………38 Chapter Two: p63 Inhibits Oxidative Stress-Induced Cell Death by Orchestrating Glutathione Metabolism………………………………………………………………………………..60 2.1 Abstract………………………………………………………………………………………………..61 2.2 Introduction……………………………………………………………………………………………61 2.3 Results………………………………………………………………………………………………...62 2.4 Discussion…………………………………………………………………………………………….67 2.5 Materials and Methods………………………………………………………………………………68 2.6 Figure Legends……………………………………………………………………………………….73 2.7 Figures………………………………………………………………………………………………...77 2.8 References……………………………………………………………………………………………89 Chapter Three: DNA Damage Induces Mitochondrial ROS Production to Execute Programmed Necrotic Death…………………………………………………………………………..93 ii 3.1 Introduction……………………………………………………………………………………………94 3.2 Results………………………………………………………………………………………………...95 3.3 Materials and Methods………………………………………………………………………………98 3.4 Discussion……………………………………………………………………………….................101 3.5 Figure Legends………………………………………………………………………………..........103 3.6 Figures……………………………………………………………………………….......................108 3.7 References…………………………………………………………………………………………..113 Chapter Four: Conclusions, Discussion, and Future Directions..…………………………….117 5.1 Conclusions………………………………………………………………………………………….118 5.2 Discussion and Future Directions…………………………………………………………………119 5.3 Concluding Remarks………………………………………………………………………………..123 5.4 References…………………………………………………………………………………………..124 Curriculum Vitae.………………………………………………………………………………………127 iii LIST OF FIGURES Chapter One Figure 1.1 Morphological features of apoptosis, autophagy, and necrosis…………………………36 Figure 1.2 Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies ………………………………………………………………………………………………………………37 Figure 1.3 Schematic depiction of the autophagy pathway and its core molecular machinery in mammalian cells…………………………………………………………………………………..………37 Chapter Two Figure 2.1 ∆Np63 abrogates DNA damage-induced oxidative stress and enhances clonogenic survival of transformed p53-/-Bax-/-Bak-/- mouse embryonic fibroblasts……………………………..77 Figure 2.2 p63 dictates the homeostasis of intracellular redox through transcriptional controls of glutathione metabolism genes.………………………………………………………………………….78 Figure 2.3 Deficiency of ∆Np63α generates a more oxidized intracellular redox state and sensitizes cells to oxidative stress………………………………………………………………………79 Figure 2.4 ΔNp63α mitigates anoikis-induced oxidative stress and cooperates with anti-apoptotic BCL-2 to disrupt the luminal clearance of acini in three-dimensional culture of mammary epithelial cells.………………………………………………………………………………………………………..80 Supplementary Figures Figure S2.1 DNA damage-induced cell death in Bax-/-Bak-/- DKO MEFs does not require RIP1...81 Figure S2.2. p53-/-Bax-/-Bak-/- TKO MEFs are more resistant to DNA damage-induced cell death than Bax-/-Bak-/- DKO MEFs……………………………………………………………………………...81 Figure S2.3 p53-/-Bax-/-Bak-/- TKO MEFs are protected by antioxidants against DNA damage- induced cell death…………………………………………………………………………………………82 -/- -/- -/- Figure S2.4 ΔNp63α inhibits DNA damage-induced ROS in p53 Bax Bak TKO MEFs……….82 Figure S2.5 Loss of endogenous p63 does not protect Bax-/-Bak-/- DKO or p53-/-Bax-/-Bak-/- TKO MEFs from DNA damage-induced death……………………………………………………………….83 iv Figure S2.6. A heatmap representation of glutathione metabolism genes differentially regulated by ΔNp63α…………………………………………………………………………………………………84 Figure S2.7. Immunoblot or qRT-PCR analysis of siRNA-mediated knockdown………………….85 Figure S2.8 Knockdown of p63 by siRNA and mir-30-based shRNA induces ROS in human cancer cell line…………………………………………………………………………………………….86 Figure S2.9 Deficiency of ΔNp63α sensitizes cells to chemotherapeutic agent-induced cell death………………………………………………………………………………………………………..87 Figure S2.10. Upregulation of GCLC by ΔNp63α is Nrf2-independent……………………………..88 Chapter Three Figure 3.1 Induction of mitochondrial matrix ROS by DNA damage is an early event that precedes cell death and ATP depletion……..…………………………………………………………………....108 Figure 3.2 ETC complex III mediates DNA double strand break-induced ROS and cell death…109 Figure 3.3 Figure 3.3 ATM-mediated DNA damage response pathway induces ROS, cell death, and ETC complex III activity following double strand breaks……………………………………….111 v ACKNOWLEDGEMENTS I would like to sincerely thank my mentor and thesis advisor, Dr. Emily Cheng. Over the five-odd years that I spent in her laboratory, first in St. Louis then in New York City, she has always pointed me towards important and novel scientific questions to address, rescued me when I struggled with my project, helped me remember how much I enjoy scientific discovery, and showed faith in me when I lacked it myself. She taught me that there are always ways to improve my techniques, my writing and communications skills, my way of thinking, my thesis project; in short, she showed me by example what it means to constantly pursue perfection. Without a doubt, completing this Ph.D. thesis is the hardest thing I have ever done, and likely one of the hardest things I will ever do. I would not be here without the lessons she has taught me, and the guidance and encouragement she has given me throughout the past five years. Her lessons have changed me for the better; it has made me more capable, confident, and goal-oriented; and it has helped set the trajectory for the rest of my life. On a similar note, I would also like to thank Dr. James Hsieh, for all the time he took to guide me through personal and professional hurdles and insecurities. Ultimately, through their efforts, Dr. Cheng and Dr. Hsieh showed me a better version of myself than the one I had known before I worked with them, and helped me on my way to becoming that person. For that, I will always be deeply grateful. I am also deeply grateful to Drs. Ho-Chou Tu, Hyungjin Kim, Decheng Ren, David Chen, Satoru Sasagawa, Shugaku Takeda, and Han Liu, as well as Todd Westergard and Hsiu-Fang Chen. They were my introduction to Dr. Cheng and Dr. Hsieh’s labs, and it was them as much as my mentor and my rotation project that sealed my decision to carry out my thesis work in Dr. Cheng’s lab. Hsiu-Fang I especially thank for taking care of my car in St. Louis while I was in New York City trying to make science work; and for welcoming me to her house and cooking a truly wonderful meal for me when I returned to St. Louis for my final thesis update. I also wish to thank Mr. Hoson Chao and Ms. Smrutiben Mehta, my wonderful laboratory managers in New York. vi I particularly wish to thank Drs. Ho-Chou Tu, Shugaku Takeda, Han Liu, Yiyu Dong, Anders Jacobsen, and Gregory Bean, and Mr. Yogesh Ganesan for their incredible enthusiasm and willingness to help me with many critical experiments during my thesis research. I wish to thank the Medical Scientist Training Program staff at the Washington University in St. Louis School of Medicine for all of their wonderful support to me through the years, and for their patience and guidance. I also would like to acknowledge the financial support I received for my M.D. and Ph.D. training, and for my thesis work, from the MSTP Training Grant, the Washington University in St. Louis School of Medicine, and the National Institutes of Health. My work was also supported by grants to Dr. Cheng from the NIH and the American Cancer Society. Finally, I want to thank my parents, Chenghong and Lu Wang, for all their patience,