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(Sarm) Tissue-Selectivity

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(Sarm) Tissue-Selectivity A COMPREHENSIVE INVESTIGATION INTO THE MOLECULAR MECHANISM RESPONSIBLE FOR SELECTIVE ANDROGEN RECEPTOR MODULATOR (SARM) TISSUE-SELECTIVITY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Natalie Elizabeth Goldberger, M.S. ***** The Ohio State University 2008 Dissertation Committee: Approved by: Dr. James T. Dalton, Ph.D., Advisor Dr. Charles E. Bell, Ph.D. ____________________________ Dr. Mamuka Kvaratskhelia, Ph.D. Advisor Dr. Charles Brooks, Ph.D. Biophysics Graduate Program i ABSTRACT The androgen receptor (AR) is a member of the superfamily of nuclear receptors (NRs) that regulate gene expression in a ligand-dependent manner. Selective androgen receptor modulators (SARMs) are ligands that activate AR in a tissue-selective manner to produce unique physiological effects. To investigate the mechanism responsible for SARM tissue-selectivity, the conformation of AR was analyzed using the NHS-biotin labeling technique. Briefly, ligand-bound AR was affinity purified from Sf9 cells and incubated with NHS-biotin to biotinylate surface accessible lysine residues. Unfortunately, no biotinylation was observed for any of the AR samples, thus the possibility that AR chaperone proteins and cofactors were blocking NHS-biotin access to AR was subsequently investigated using native gels, immunoprecipitation (IP) and liquid chromatography coupled with ion trap mass spectroscopy (MS). IP and native gel analysis subsequently revealed multiple ligand-specific bands. The largest native gel protein bands (~ 690 kDa) for both the dihydrotestosterone- and SARM- treated samples were analyzed using tandem MS, and ligand-specific cofactors were identified. Most interesting was the identification of nuclear receptor corepressor (NCoR) in only the SARM-treated sample. This exciting result was subsequently confirmed in LNCaP when a higher degree of AR-SARM/NCoR versus AR-DHT/NCoR association was observed. Lastly, reduced cytoplasmic-nuclear transport and nuclear foci formation were observed by Western blot and immunofluorescence microscopy for AR-SARM versus AR-DHT. In summary, by using a combination of proteomics and immunofluorescence, support for the presence of functionally distinct AR-SARM protein complexes, in terms of AR cellular transport and transactivation, was generated. Most of all, the structural and functional differences revealed herein between AR-DHT and AR-SARM highlight potential avenues of research which may achieve a more comprehensive and detailed picture of the SARM tissue-selective mechanism. ii Dedicated to the sweetest dog in the world (aka Casper) and my loving and supportive parents Virginia and Lawrence Goldberger iii ACKNOWLEDGMENTS First of all, I would like to thank my advisor, James Dalton, for his support and confidence in my research. In particular, for allowing me to independently investigate novel and relatively risky research directions. Most of all, I thoroughly appreciated his friendship and support during many challenging periods throughout my graduate career. I am also extremely grateful to Mitch Phelps for his unwavering support and guidance over many years, and for finally convincing me to stop doing experiments and start writing my thesis. I also appreciated the selfless manner in which he helped me prepare for my candidacy exam, future experiments, and job interviews. I will never forget his generosity and kindness, mostly because it wasn’t required, but done simply because he was being “Mitch”. I would also like to thank Zengru Wu for running all of my MS samples. Without her help, none of the MS results listed herein would have been possible. I also appreciated all of the stimulating and informative MS discussions I had with Chien-ming Li. And, I am grateful to all of my other lab mates who provided helpful research discussions and assistance throughout the years. In addition, I want to thank all the friends and mentors who have made a meaningful and positive impact during my life. I would not be who I am today without the support and enthusiasm of many individuals who impact my life every day. I also need to thank my sweet dog Casper for his steadfast loyalty, companionship and uncanny ability to make me laugh, even during the gloomiest days. Lastly, I am extremely grateful to family members who have cheered for my success and happiness throughout my life. I know my late grandfather John Patterson would have been extremely proud. And, I would especially like to thank my grandparents Henry and Dorie Goldberger for their unwavering love and interest in my life. Most of all, I would not be the person I am today without the selfless sacrifice and deep love displayed by my parents, Virginia and Lawrence Goldberger, who always put their family first. iv VITA October 1, 1973…………………..Born – Ithaca, NY 1998………………………………..B.S. Biochemistry, University of Delaware 1998-1999…………………………Research Assistant, Georgetown University, Institute for Molecular and Human Genetics 1999-2002…………………………Biologist, National Human Genome Research Institute, Cancer Genetics Branch 2002-present………………………Graduate Research Associate, The Ohio State University PUBLICATIONS 1. Bisson WH, Cheltsov AV, Bruey-Sedano N, Lin B, Chen J, Goldberger N , May LT, Christopoulos A, Dalton JT, Sexton PM, Zhang X-K, and Abagyan R. Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs. PNAS 2S-4; 104:11927-11932. 2. Mousses S, Bubendorf L, Wagner U, Hostetter G, Kononen J, Cornelison R, Goldberger N, Elkahloun AG, Willi N, Koivisto P, Ferhle W, Raffeld M, Sauter G, and Kallioniemi O-P. Clinical validation of candidate genes associated with prostate cancer progression in the CWR22 model system using tissue microarrays . Cancer Research 2002; 62:1256-60. 3. Rozenblum E, Vahteristo P, Sandberg T, Bergthorsson JT, Syrjakoski K, Weaver D, Haraldsson K, Johannsdottir HK, Vehmanen P, Nigam S, Goldberger N, Robbins C, Pak E, Dutra A, Gillander E, Stephan DA, Bailey-Wison J, Hank Juo S-H, Kainu T, Arason A, Barkardottir RB, Nevanlinna H, Borg A, and Kallioiemi O-P. A genomic map of a 6-Mb region at 13q21-q22 implicated in cancer development: identification and characterization of candidate genes . Human Genetics 2002; 110:111-21. 4. Allander SV, Nupponen N, Ringner M, Hostetter G, Maher GW, Goldberger N , Chen Y, Carpten J, Elkahloun AG, and Meltzer PS. Gastrointestinal stromal tumors with v KIT mutations exhibit a remarkably homogeneous gene expression profile . Cancer Research 2001; 61:8624-8. FIELDS OF STUDY Major Field: Biophysics vi TABLE OF CONTENTS ABSTRACT .....................................................................................................................ii DEDICATION….................................................................................................................iii ACKNOWLEDGMENTS .................................................................................................iv VITA................................................................................................................................v LIST OF FIGURES.........................................................................................................xi LIST OF TABLES ..........................................................................................................xv ABBREVIATIONS ....................................................................................................... xvii CHAPTER 1 ................................................................................................................... 1 1.1. Androgen Receptor............................................................................................. 1 1.2. Androgen Response Elements............................................................................ 1 1.3. AR N-Terminal Domain Structure and Function. ................................................. 2 1.4. AR N-Terminal and C-Terminal Interaction ......................................................... 2 1.5. Dynamic NR Transactivation Process ................................................................. 3 1.6. AR Cytoplasmic-Nuclear Transport and Cellular Distribution Patterns................. 3 1.7. Discovery of Selective Androgen Receptor Modulators....................................... 4 1.8. Therapeutic Promise of SARMs .......................................................................... 4 1.9. SARM Tissue-Selectivity Hypothesis................................................................... 5 1.10. Statement of Problem ........................................................................................ 6 1.11. Methods Applied to Solve Problem .................................................................... 6 CHAPTER 2 ..................................................................................................................18 ANALYSIS OF LIGAND-SPECIFIC AR PURITY, YIELD AND STABILITY ....................18 2.1. INTRODUCTION ................................................................................................18 2.2. MATERIALS AND METHOD...............................................................................20 2.2.1. Cell Culture...................................................................................................20 2.2.2. Generation of High-Titer Stocks....................................................................20 2.2.3. AR Expression and Affinity Purification.........................................................20
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