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UNIVERSITY of CALIFORNIA RIVERSIDE Quantitative Proteomic UNIVERSITY OF CALIFORNIA RIVERSIDE Quantitative Proteomic Analysis for Assessing the Mechanisms of Action of Anti-Cancer Drugs and Arsenite A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry by Fan Zhang December 2013 Dissertation Committee: Dr. Yinsheng Wang, Chairperson Dr. Cynthia Larive Dr. Pingyun Feng Copyright by Fan Zhang 2013 The Dissertation of Fan Zhang is approved: Committee Chairperson University of California, Riverside ACKNOWLEDGEMENTS This dissertation can never be completed without the help and support from many people. I owe my appreciation to all those who have made this dissertation possible and because of whom my graduate experience has been one that I will cherish forever. First and foremost, I would like to give my deepest gratitude to my research adviser, Professor Yinsheng Wang, for his valuable guidance and consistent encouragement on all my research projects during my PhD study at UCR. Professor Wang encouraged me not only to grow as a solid chemist and experimentalist, but also as an independent thinker. His diligent and serious research attitude has impressive impact on the maturity of my personality. His patience and support helped me to overcome the obstacles and desperate situations during these past five years. I would say that I could never finish the PhD study without Professor Wang‟s great mentorship. For everything you have done for me, Professor Wang, thank you. I would like to give my appreciation to my committees: Professor Cynthia Larive and Professor Pingyun Feng, for reading my dissertation and providing me their helpful comments; Professor Quan (Jason) Cheng, for teaching me the knowledge in his class; and Professor Jeff Bachant, for some wise advice he gave me on my research. I would give my sincere thanks to some staff members in Chemistry Department and Center of Plant Cell Biology and Core Instrumentation Facility at UCR. I want to thank Dr. David Carter, for his kind assistance and advice on confocal microscopy; and iv Ms. Holly Eckelhoefer for her training and help with flow cytometry. I also want to thank Dr. Dan Borchardt for the training on circular dichroism, Dr. Songqin Pan and Mr. Ronald New for their assistance on MALDI mass spectrometers. I would express my thanks to all my friends in the Wang group. Thanks go to Dr. Haibo Qiu, Dr. Lei Xiong, and Dr. Xiaoli Dong for their generous help and patience when I started work in this group. I would give my gratitude to Dr. Bifeng Yuan who is always helpful and nice; my experimental techniques improved a lot thanks to all the assistance he offered. Thanks also go to Dr. Xiaoxia Dai, Qian Cai, Lijuan Fu, Pengcheng Wang and Yongsheng Xiao for their help in completing my research projects; and Dr. John Prins for his discussion on my research. Special thanks go to Candace Guerrero for her precious friendship, and the other members in the Wang group for their support. I would always appreciate and memorize the time in this laboratory. Finally, and the most importantly, I would like to deeply thank my parents whose support, encouragement, patience and unwavering love are always the most valuable gifts at every stage of my personal and academic life. No matter what happened, they always stand behind me and offer their faith in me. In addition, I give special thanks to my boyfriend Dr. Qichi Yang whose understanding and accompany mean a lot to me. He is always the first person to encourage me in every possible way during the past four years. I would cherish the great time we had together and your continuous love and support. v COPYRIGHT ACKNOWLEDGEMENTS The text and figures in Chapter 2, in part or in full, is a reprint of the material as it appears in Mol. Cell. Proteomics 2012, 11, M111 016915. The co-author (Dr. Yinsheng Wang) in that publication directed and supervised the research which forms the basis for this chapter. The co-author (Xiaoxia Dai) listed in that publication performed siRNA knockdown and real-time PCR experiments. vi ABSTRACT OF THE DISSERTATION Quantitative Proteomic Analysis for Assessing the Mechanisms of Action of Anti-Cancer Drugs and Arsenite by Fan Zhang Doctor of Philosophy, Graduate Program in Chemistry University of California, Riverside, December 2013 Dr. Yinsheng Wang, Chairperson Mass spectrometry is a powerful tool used in proteomic analysis. By combining with different isotope labeling methods, protein quantification at the entire proteome scale can be achieved. In this dissertation, I employed an LC-MS/MS coupled with stable isotope labeling by amino acid in cell culture (SILAC) strategy, to explore the molecular mechanisms of action of anti-cancer drugs and an environmental toxicant, arsenic. In Chapters 2 and 3, I performed a quantitative assessment of global proteome changes upon treatment with two anti-cancer drugs, both are nucleoside analogues, i.e. 5- aza-2‟-deoxytidine (5-Aza-CdR) and 6-thioguanine (SG). We found, for the first time, that 5-Aza-CdR exerts its cytotoxic effects in leukemia and melanoma cells through epigenetic reactivation of DPP4 gene and the resultant inhibition of cholesterol biosynthesis. In addition, proteome-wide analysis suggested that SG may exert its cytotoxic effect by inducing mitochondrial dysfunction and reactive oxygen species formation in acute lymphoblastic leukemia cells. vii In Chapters 4 and 5, we utilized the same MS-based quantitative proteomic method, to study the mechanisms underlying trivalent arsenic-induced carcinogenic effect. Our results confirmed the previous findings and provided a more complete picture about the biological pathways that are altered upon arsenite treatment in human skin fibroblast cells. To further understand how arsenic affects RING-finger proteins that are known to be important in DNA damage repair, we performed in vitro and in vivo arsenite binding experiments. Our studies revealed, for the first time, that arsenite may exert its carcinogenic effect by targeting cysteine residues in the RING finger domains of the RNF20-RNF40 histone E3 ubiquitin ligase, thereby altering histone epigenetic mark and compromising DNA DSB repair. viii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... iv ABSTRACT OF THE DISSERTATION ......................................................................... vii TABLE OF CONTENTS ................................................................................................... ix LIST OF FIGURES ......................................................................................................... xvi LIST OF TABLES ........................................................................................................ xxvii CHAPTER 1. General Overview ..................................................................................... 1 1.1 Introduction ................................................................................................................... 1 1.2 MS-based quantitative proteomics ................................................................................ 3 1.2.1 Chemical labeling strategies ................................................................................... 6 1.2.1.1 Isotope-coded affinity tag (ICAT) ................................................................... 6 1.2.1.2 Isobaric tag for relative and absolute quantitation (iTRAQTM) ....................... 8 1.2.1.3 Other chemical labeling strategies ................................................................. 10 1.2.2 Metabolic labeling strategies ................................................................................ 10 1.2.2.1 Stable isotope labeling by amino acids in cell culture (SILAC) ................... 12 1.2.2.2 15N labeling .................................................................................................... 14 1.3 Label-free quantification ............................................................................................. 15 1.4 Nucleoside analogue-based anti-cancer drugs ............................................................ 16 1.4.1 Thiopurines........................................................................................................... 17 1.4.2 5-aza-2‟-deoxytidine and 5-azacytidine ............................................................... 19 ix 1.5 Arsenite ....................................................................................................................... 22 1.6 Scope of the dissertation ............................................................................................. 24 1.7 References ................................................................................................................... 27 x CHAPTER 2. 5-Aza-2’-deoxycytidine Induced Growth Inhibition of Leukemia Cells through Modulating Endogenous Cholesterol Biosynthesis ....................................... 34 Introduction ....................................................................................................................... 34 Materials and methods ...................................................................................................... 36 Cell Culture ................................................................................................................... 36 5-Aza-CdR treatment and sample preparation .............................................................. 37 SDS-PAGE separation and in-gel digestion.................................................................
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