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Copper Transporter 2 (CTR2) As a Regulator of Cisplatin Accumulation and Sensitivity

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Copper Transporter 2 (CTR2) As a Regulator of Cisplatin Accumulation and Sensitivity UNIVERSITY OF CALIFORNIA SAN DIEGO Copper Transporter 2 (CTR2) as a Regulator of Cisplatin Accumulation and Sensitivity A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Brian G. Blair Committee in charge: Professor Stephen B. Howell, Chair Professor Daniel J. Donoghue Professor Paul A. Insel Professor Stanley J. Opella Professor Nicholas J. Webster 2009 Copyright Brian G. Blair, 2009 All rights reserved. The dissertation of Brian G. Blair is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2009 iii DEDICATION To my parents and grandparents for their hard work and sacrifices that made my education possible, and for teaching me to follow my passions. To Erin, my loving and supportive wife, for encouraging me to be the best that I can be in everything that I do. iv TABLE OF CONTENTS Signature Page ……………………………………………………………… iii Dedication ……………………………..……………………………….…. iv Table of Contents …………………………………………………………. v List of Figures ……………………………………………………………... viii List of Tables ………………………………………………………………. xi Acknowledgements ………………………………………………………… xii Curriculum Vitae …………………………………………………………… xx Abstract of the Dissertation………………………………………………….. xxiii Chapter 1: Introduction ……………………………………………………… 1 Platinum Based Chemotherapy ……………………………………… 1 Resistance to Platinum-Drugs ……………………………………….. 4 Platinum-Drug Transport ...………………………………………….. 5 Copper Homeostasis ...……………………………………………….. 7 Copper Transporter 2 ..…………….…………………………………. 16 Summary ...…………………………………………………………… 19 Hypothesis ...…………………………………………………………. 19 Acknowledgements ………………………………………………….. 20 Chapter 2: Effect of the Loss of CTR2 on Pt-drug Sensitivity and Accumulation in Vitro ……………………………………………. 21 Introduction …………………………………………………………... 21 Results ..…………….……………………………………………….... 21 Discussion ..……………….…………………………………………... 32 Materials and Methods ..………….…………………………………… 34 Acknowledgements ..…………………………………………………. 39 Chapter 3: Effect of the Loss of CTR2 on Pt-drug Sensitivity and Accumulation in Vivo ……………………………………………. 41 Introduction …………………………………………………………... 41 v Results ..…………….……………………………………………….... 42 Discussion ..……………….…………………………………………... 50 Materials and Methods ..………….…………………………………… 53 Acknowledgements …………………………………………………… 56 Chapter 4: The Regulation of CTR2 Expression and Degradation By Copper and Platinum .....……………………………………… 57 Introduction …………………………………………………………... 57 Results ..…………….……………………………………………….... 58 Discussion ..……………….…………………………………………... 70 Materials and Methods ..………….…………………………………… 76 Acknowledgements …………………………………………………… 81 Chapter 5: CTR2 is Partially Localized in the Nucleus ………………………. 82 Introduction …………………………………………………………... 82 Results ..…………….……………………………………………….... 83 Discussion ..……………….…………………………………………... 88 Materials and Methods ..………….…………………………………… 90 Acknowledgements …………………………………………………… 92 Chapter 6: CTR2 Limits CTR2 Accumulation Through the Inhibition of Endocytosis ……………………………………………………. 93 Introduction …………………………………………………………... 93 Results ..…………….……………………………………………….... 95 Discussion ..……………….…………………………………………... 105 Materials and Methods ..………….…………………………………… 108 Acknowledgements …………………………………………………… 114 Chapter 7: Conclusions and Future Directions ..……………………………… 115 Summary ..………………………………………………………......... 115 Effect of decreased CTR2 levels on accumulation and sensitivity of DDP, CBDCA and Cu ..…………………………………………… 116 In Vivo Studies ..…………………………………………………........ 118 Regulation of CTR2 by Copper and Cisplatin ..……………………… 120 Regulation of CTR2 by Atox1 ..……………………………………… 121 Nuclear Localization of CTR2 ………………………..……………… 123 vi CTR2 as a Regulator of Endocytosis …..…………………………….. 125 Clinical Implications ...……………………………………………….. 127 Conclusions …..……………………………………………………..... 131 Acknowledgements …………………………………………………… 130 References ……………………………………………………………………. 132 vii LIST OF FIGURES Chapter 1 Figure 1-1. Chemical structures of cisplatin, carboplatin, and oxaliplatin …… 3 Figure 1-2. The Cu transport pathway ………………………………………. 10 Figure 1-3. Structure of hCTR1 protein ……………………………………... 12 Figure 1-4. Solution structure of Atox1 ……………………………………... 14 Figure 1-5. Structure of hCTR2 ……………………………………………… 18 Chapter 2 Figure 2-1. Relative CTR2 mRNA and protein levels in parental and knockdown cells measured by qRT-PCR and Western blot analysis ….…….. 23 Figure 2-2. Inhibition of growth as a function of concentration …………….. 26 Figure 2-3. Cell accumulation of DDP in CTR1 +/+ , CTR1 +/+ CTR1kd , CTR1 -/-, CTR1 -/- CTR1 kd cells ……………………………………………. 27 Figure 2-4. Cell accumulation of [ 14 CBDCA] and 64 Cu in CTR1 +/+ , CTR1 +/+ CTR1 kd , CTR1 -/-, CTR1 -/- CTR1 kd cells ………………. 28 Figure 2-5. Pt accumulation in DNA following 1 h exposure to 30 µM DDP as measured by ICP-MS …………………………………………. 30 Chapter 3 Figure 3-1. Innoculation into mice …………………………………………… 43 Figure 3-2. Immunohistochemical characterization of proliferation and apoptosis in CTR1 -/- and CTR1 -/- CTR1 kd cells …………………………..... 44 Figure 3-3. Immunohistochemical characterization of angiogenesis in CTR1 -/- and CTR1 -/- CTR1 kd cells …………………………..... 45 viii Figure 3-4. Steady state copper levels in CTR1 -/- and CTR1 -/- CTR1 kd grown in vitro or in vivo ………………………………………………… 46 Figure 3-5. Tumor accumulation of DDP in CTR1 -/- and CTR1 -/- CTR1 kd tumors 1 h following a single intraperitoneal injection of 10 mg/kg DDP …………………………………………………... 47 Figure 3-6. Effect of knocking down CTR2 on responsiveness to DDP in vivo ……………………………………………………… 48 Figure 3-7. Relationship between CTR2 expression and DDP sensitivity in ovarian carcinoma cell lines …………………………………….. 50 Chapter 4 Figure 4-1. Measurement of CTR2 in 2008 cells following 1h pre-treatment with either 200 µM CuSO 4, 100 µM BCS, 30 µM DDP or drug-free media ………………………………………………….. 59 Figure 4-2. Representative deconvolution micrographs of 2008 cells stains for CTR2 following 1 h pre-treatment with either 200 µM CuSO 4, 100 µM BCS, 30 µM DDP or drug-free media ………… 60 Figure 4-3. Measurement of CTR2 half-life by Western blot ……………….. 62 Figure 4-4. Deconvolution microscopy of 2008 cells following exposure to drug-free control media or 50 mM bortezomib in the presence or absence of 100 µM BCS ………………………………………… 63 Figure 4-5. Effect of Cu and BCS on DDP uptake and sensitivity ………….. 65 Figure 4-6. Deconvolution microscopic images of Atox1 +/+ and Atox1 -/- cells following 1 h pre-treatment of either 200 µM CuSO 4, 100 µM BCS, 30 µM DDP or drug-free media ………………………………… 68 Figure 4-7. CTR2 expression in Atox1 +/+ and Atox1 -/- cells ………………… 70 Figure 4-8. Model of CTR2 regulation by Atox1 …………………………… 75 Chapter 5 Figure 5-1. Deconvolution microscopic images of CTR2 in 2008 cells …….. 83 ix Figure 5-2. Subcutaneous mouse tumor section at 20x and 40x focal planes .. 84 Figure 5-3. Effect of Cu and BCS on nuclear CTR2 …………………………. 86 Figure 5-4. Western blot analysis of CTR2 in the cytosolic and nuclear fractions of 2008 cells following 1 h pre-treatment with either 200 µM CuSO 4, 100 µM BCS, 30 µM DDP or drug-free media ………………….. 87 Chapter 6 Figure 6-1. Pt accumulation in DNA and vesicles following exposure to DDP. 96 Figure 6-2. Pt content as a function of efflux time ………………………….. 97 Figure 6-3. Whole cell accumulation of Texas red labeled dextran ………… 99 Figure 6-4. Dextran content as a function of efflux time ……………………. 100 Figure 6-5. Whole cell Pt accumulation in CTR1+/+, CTR1+/+ CTR2kd, CTR1-/- and CTR1-/- CTR2kd cells ……………………………. 102 Figure 6-6. 1 h accumulation of 30 µM DDP with and without PDGF pre-treatment …………………………………………….. 103 Figure 6-7. CTR2 activates the GTPases that control macropinocytosis ……. 104 Chapter 7 Figure 7-1. Stabilization of macropinocytosis by activated Rac1 and cdc42 .. 126 x LIST OF TABLES Chapter 2 Table 2-1. Accumulation of DDP, CBDCA, and Cu ……………………… 31 xi ACKNOWLEDGEMENTS I would like to thank and acknowledge the following people: Jesse Bertinato for providing personal knowledge on CTR2 as well as the antibody used in these experiments; Sandra Schmid for providing discourse and input with regards to the study of endocytosis; Martin Hetzer and Jessica Tsalmas for providing the NPC antibody; Matt Niederst for providing Akt reagents and antibodies; Minji Jo for input and resources for the study of GTPases; Kersi Pestonjamasp for providing training and assistance in collecting microscopy data; Nissi Varki for providing assistance in this analysis of pathology samples; Laarni Gapuz and the UCSD Cancer Center Histology and Immunohistochemistry Shared Resource for their technical assistance; xii Gina Butcher and Leanne Nordeman of the Program in Biomedical Sciences for their assistance and support; Ben Ho Park and Kurt Bachman for setting me off on this crazy career called science. Thank you for taking an inept college grad and turning him into a scientist. You truly are two kings; Drs. Daniel J. Donoghue, Paul A. Insel, Stanley J Opella and Nicholas J. Webster for taking the time to serve on my dissertation committee. Your valuable input, guidance and support is greatly appreciated and served to assist my growth as a scientist; All the members of the Howell Laboratory, past and present, who have assisted in the composition of this dissertation; Alison Holzer and Goli Samimi for laying the groundwork that made this work possible; Christopher Larson for assistance and contributions made through the entirety of these studies; xiii Preston Adams for assistance
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