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A Study of Photodynamic Damage to the Dna Replication System

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A STUDY OF PHOTODYNAMIC DAMAGE TO THE DNA REPLICATION SYSTEM DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ran Zhao, M.S. Ohio State Biochemistry Program The Ohio State University 2009 Dissertation Committee Dr. Robert M. Snapka, Advisor Dr. Marshall V. Williams Dr. Donald H. Dean Dr. Charles E. Bell Copyright by Ran Zhao 2009 ABSTRACT Photodynamic therapy (PDT) is a promising clinical modality for killing unwanted cells, especially cancer cells by the combined use of light, photosensitizers and molecular oxygen. In the process of PDT, energy flows from light-activated triplet state photosensitizers to the triplet ground state molecular oxygen by the type II pathway, converting it to singlet oxygen, which can damage cancer cells by directly reacting with nearby biomolecules, or indirectly by destroying tumor vasculature and invoking systemic immune responses. To improve the efficiency of PDT in clinical practice, we studied photodynamic damage to important nuclear proteins involved in DNA replication/repair, and identified the [Ru(tpy)(pydppn)]2+complex as a very promising photosensitizer. PCNA, p53, SV40 large T-antigen, topoisomerase I and lamin B were identified as cellular protein targets for photodynamic damage caused by proflavine and visible light. Following photodynamic damage, p53 was detected in a distinct covalent crosslinking profile, with p53 tetramers and dimers being the predominant forms; SV40 large T-antigen was mainly crosslinked into a hexamer while lamin B was crosslinked to dimers, trimers and tetramers. 2+ [Ru(tpy)n(pydppn)2-n] (n = 0, 1) complexes have been reported to be capable of photosensitizing the generation of singlet oxygen with near 100% efficiency in vitro. In ii our study, [Ru(tpy)(pydppn)]2+ caused efficient covalent oligomerization of cellular 2+ 2+ PCNA and p53 in light. In the cell lysates, both [Ru(tpy)(pydppn)] and [Ru(pydppn)2] were able to produce efficient PCNA and p53 photodynamic crosslinking. Cellular PCNA photocrosslinking caused by [Ru(tpy)(pydppn)]2+ increased linearly with [Ru(tpy)(pydppn)]2+ concentration, time of uptake, or visible light exposure. The inclusion of azide, a singlet oxygen quencher, led to significant suppression of p53 photocrosslinking, suggesting that singlet oxygen is the reactive agent causing p53 photocrosslinking. [Ru(tpy)(pydppn)]2+ caused efficient photodynamic protein-DNA crosslinking in cells, which increased with increasing levels of photodynamic damage. Photodynamic damage by [Ru(tpy)(pydppn)]2+ resulted in inhibition of DNA replication in a biphasic manner in mammalian cells. Another ruthenium containing complex, [Ru(bpy)2(CH3CN)2]Cl2 inhibited SV40 viral DNA replication to a great extent in a light- dependent manner, but no protein damage of the types found for photodynamic ruthenium complexes (protein-protein crosslinking, protein-DNA crosslinking) was detected by Western blotting. We also studied the contribution of photodynamic properties of certain topoisomerase poisons in improving their cytotoxicity. It was shown that the topoisomerase II poison ellipticine reduced cell survival to a significantly greater extent when irradiated with visible light than in the dark conditions. However, the topoisomerase-DNA-ellipticine cleavable complex appears not to be the relevant cytotoxic target. iii Dedicated to my parents iv ACKNOWLEDGMENTS I owe my deepest gratitude to all people who have helped and encouraged me during my graduate study in the Ohio State University. I would like to give sincere thanks to my advisor, Dr. Robert M. Snapka, for his guidance and inspiration, for teaching me how to think critically and deeply on scientific questions, as well as for his encouragement during my research and study. His perpetual enthusiasm in research had motivated all the advisees, including me. Special thanks are given to my current committee members, Dr. Marshall V. Williams, Dr. Donald H. Dean and Dr. Charles E. Bell, and two of my former committee members, Dr. Duxin Sun and Dr. Gary E. Means for their valuable suggestions and comments. I want to thank Mrs. Edie Yamasaki for her great help in both research and personal matters. I want to thank the former and current members in Snapka laboratory for their company and help. I am particularly grateful to Dr. SooIn Bae and Dr. Ragu Kanagasabai for being a constant source of encouragement and support for my study and life. The discussions and interactions with you had inspired me deeply both in my research and personal life, and made the long hours spent in the lab fruitful and memorable. Thanks are also given to the members of Radiobiology Division including Dr. Qianzheng Zhu, Dr. Qien Wang and Dr. Haiming Ding for their help, kindness and valuable suggestions. v I thank Dr. Randolph P. Thummel (University of Houston) and Dr. Claudia Turro (Department of Chemistry, the Ohio State University) for providing ruthenium complexes and help with the revision and submission of the ruthenium paper, Dr. Altaf A. Wani (Radiology Department, the Ohio State University) for providing mouse anti-p53 antibodies, ts 85 cell lines, Dr. M. Yoshida (University of Tokyo, Japan) for providing Leptomycin B. Last but not least, I want to thank my family for their unflagging love and support. I am deeply grateful to my husband, Xiaoqiang Liu, for his encouragement, understanding and support. I am deeply indebted to my dad for his support and encouragement. I can’t find suitable words to express my gratitude for my mom, whose unconditional love has been my greatest strength all along. The constant love and support from my sister is sincerely acknowledged. Without all the support and help, I could not be able to finish this degree at the Ohio State University. vi VITA 1998-2002 ............................................. B.S., Biotechnology Shandong Agricultural University, China 2002-2005 ............................................. M.S., Cell Biology Institute of Botany the Chinese Academy of Sciences, China 2005-2009 ............................................. PhD Graduate Student, Biochemistry The Ohio State University PUBLICATIONS 1. Ran Zhao, Richard Hammitt, Randolph P. Thummel, Yao Liu, Claudia Turro, Robert M. Snapka. (2009). Nuclear targets of photodynamic tridentate ruthenium complexes. Dalton Trans, DOI: 10.1039/b913959a 2. SooIn Bae, Ran Zhao, Robert. M. Snapka. (2008). PCNA damage caused by antineoplastic drugs. Biochem Pharmacol, 76, 1653-1668 3. Xiaoqiu Du, Qiying Xiao, Ran Zhao, Feng Wu, Qijiang Xu, Kang Chong, Zheng Meng. (2008). TrMADS3, a new MADS-box gene, from a perennial species Taihangia rupestris (Rosaceae) is up-regulated by cold and experiences seasonal fluctuation in expression level. Dev Genes Evol, 218, 281-292 FIELDS OF STUDY Major Field: Biochemistry vii TABLE OF CONTENTS Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita……………. ............................................................................................................... vii List of Tables ...................................................................................................................... x List of Schemes .................................................................................................................. xi List of Figures ................................................................................................................... xii Chapters: 1.Introduction 1.1 Photodynamic damage ......................................................................................... 1 1.2 Proteins important for DNA replication ............................................................ 10 1.3 Transition metal complexes as photosensitizers(PSs) ....................................... 14 1.4 Simian virus 40 (SV40) as a model system for mechanistic studies of DNA replication inhibiting drugs ................................................................................ 19 2. Identification of protein targets of photodynamic damage in cell culture system 2.1 Introduction ........................................................................................................ 26 2.2 Materials and methods ....................................................................................... 30 viii 2.3 Results ................................................................................................................ 33 2.4 Discussion .......................................................................................................... 41 2+ 3. A noval singlet oxygen producer:[Ru(tpy)(pydppn)] 3.1 Introduction ........................................................................................................ 70 3.2 Materials and methods ....................................................................................... 71 3.3 Results ................................................................................................................ 76 3.4 Discussion .......................................................................................................... 84 4. Analysis of the inhibitory effect on DNA replication
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