The Post-Transcriptional Regulation of Antioxidant Enzymes BY EMILY N. REINKE B.Sc., University of St. Andrews, 2006 M.S., Washington State University, 2008 THESIS Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pathology in the Graduate College of the University of Illinois at Chicago, 2013 Chicago, IL Defense Committee: Alan Diamond, Chair and Advisor Larisa Nonn Bao-Shiang Lee, Research Resources Center Rajendra Mehta, Illinois Institute of Technology Chiayeng Wang, Oral Sciences ACKNOWLEDGEMENTS The assistance of several people was necessary for the completion of this dissertation, without which I might never have made it. Firstly, I would like to thank my advisor Dr. Alan Diamond, whose patience, guidance and support throughout my dissertation project provided me with the means to go beyond what I thought I was capable of academically. He has taken the time to provide instruction in a broad range of academic and research aspects, which have greatly aided in my professional development and my readiness to pursue the next stage of my career. I would also like to thank my committee, Drs. Larisa Nonn, Wancai Yang, Chiayeng Wang, Bao- Shiang Lee and Rajendra Mehta, whose guidance and suggestions have helped to complete this project. Great appreciation goes out to the current and former members of the Diamond lab, for insightful conversation, technical suggestions and most importantly the support to make it through, especially Marci Gutt, Dr. Anita Jerome-Morais, Dr. Frank Weinberg and Dr. Soumen Bera. Thank you to the other Diamond lab grad students Dede Ekoue and Emmanuel Ansong, for providing extra hands when two just weren’t enough. I’d also like to acknowledge my parents Donn and Kathi Terry and my siblings Kristin, Armando and the Larios family, who have supported me in all of my pursuits and instilled a never ceasing love of learning and curiosity. Finally, I would like to thank my husband, Adam Reinke, for his love, extreme patience, and support throughout this entire pursuit and into the future. ii TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION .............................................................................................................. 1 A. Generation of reactive oxygen species ................................................................... 1 1. Superoxide is generated primarily by cellular metabolism......................... 1 2. Superoxide dismutase enzymes protect against superoxide-induced cell damage ........................................................................................................ 2 3. Hydrogen peroxide is produced in multiple cellular compartments ........... 4 4. Hydrogen peroxide is a vital component in pathogen defense and in signal transduction ................................................................................................. 5 5. Excess reactive oxygen species levels can be detrimental to cell function and DNA fidelity......................................................................................... 6 B. Multiple antioxidant proteins regulate hydrogen peroxide levels ........................... 7 1. Catalase reduces hydrogen peroxide under oxidative stress ....................... 8 2. Thioredoxin and thioredoxin reductase maintain antioxidant reductive capacity ....................................................................................................... 9 3. Thioredoxin reductase is an antioxidant selenoprotein ............................... 9 4. Thioredoxin reductase 1 has more than antioxidant properties ................ 11 C. The glutathione peroxidase family: antioxidant selenoproteins ........................... 12 1. Glutathione peroxidase activity is dependent on the antioxidant substrate glutathione................................................................................................. 12 2. Glutathione peroxidase enzymes detoxify hydrogen peroxide and lipid peroxides ................................................................................................... 13 3. Glutathione peroxidase selenoprotein expression is dependent on selenium status ......................................................................................................... 13 4. The glutathione peroxidase selenoprotein family members ..................... 14 5. The antioxidant glutathione peroxidase 4 regulates fertility, cell viability, and inflammation ...................................................................................... 15 6. Cytosolic glutathione peroxidase 1 is a ubiquitous antioxidant protein ... 16 7. Properties of glutathione peroxidase that affect cell growth and proliferation............................................................................................... 17 8. Glutathione peroxidase is associated with risk or progression of multiple diseases ..................................................................................................... 19 a. Glutathione peroxidase is deleted in multiple cancer types .......... 19 b. Allelic variations in glutathione peroxidase enzyme activity ....... 19 c. Cancer risk is increased by glutathione peroxidase 1 polymorphisms .............................................................................. 21 d. Obesity and increased reactive oxygen species increase diabetes risk via glutathione peroxidase 1 .................................................. 22 e. Glutathione peroxidase 1 and selenium intake affect onset of cardiomyopathies .......................................................................... 23 iii TABLE OF CONTENTS (continued) CHAPTER PAGE 9. Regulation of glutathione peroxidase 1 .................................................... 23 10. Glutathione peroxidase 1 transcription is dependent on reactive oxygen species levels ............................................................................................. 24 11. Glutathione peroxidase 1 translation is increased in a selenium-dependent manner....................................................................................................... 25 12. Post-translational modifications of glutathione peroxidase 1 affect enzyme activity....................................................................................................... 27 D. Understanding the regulation of glutathione peroxidase 1 ................................... 28 II. CHANGES IN THE ACTIVITY OF THE GLUTATHIONE PEROXIDASE 1 ANTIOXIDANT SELENOENZYME IN MONONUCLEAR CELLS FOLLOWING IMATINIB TREATMENT ............................................................................................... 28 A. Introduction ........................................................................................................... 28 B. Materials and methods .......................................................................................... 28 C. Results ............................................................................................................................... 30 1. Assay reliability ........................................................................................ 30 2. Changes in glutathione peroxidase activity following imatinib therapy .. 32 D. Discussion ......................................................................................................................... 34 III. THE IN VITRO EFFECTS OF IMATINIB AND RAPAMYCIN ON ANTIOXIDANT PROTEIN LEVELS AND ACTIVITY ............................................................................ 36 A. Introduction ........................................................................................................... 36 B. Materials and methods .......................................................................................... 36 1. Tissue culture ............................................................................................ 36 2. Cell lysis, protein and RNA isolation, and cDNA synthesis .................... 38 3. Protein analysis and enzyme activity ........................................................ 39 4. Analysis of transcript levels ...................................................................... 41 5. Retroviral SECIS reporter construct synthesis and analysis of reporter signal ......................................................................................................... 41 6. Statistical analysis ..................................................................................... 43 C. Results ................................................................................................................... 43 1. Treatment of chronic myelogenous leukemia cell lines with imatinib increases antioxidant enzymes .................................................................. 43 a. Subcloning KU812 and determining a cytostatic imatinib dose ... 43 b. Imatinib treatment increases glutathione peroxidase 1 protein and enzyme levels ................................................................................ 44 c. The increase in glutathione peroxidase 1 protein levels is not from altered transcript levels or protein decay ...................................... 45 d. Imatinib also increases glutathione peroxidase 1 in the chronic myelogenous leukemia cell line MEG-01 ..................................... 45 iv TABLE OF CONTENTS (continued) CHAPTER PAGE e. Imatinib treatment does not increase glutathione peroxidase 1 in non-Bcr-Abl-expressing cell lines ................................................ 46 f. Ectopic Bcr-Abl expression in LNCaP decreases glutathione peroxidase 1 levels ........................................................................ 46 g. Imatinib treatment increases antioxidant proteins in addition to glutathione peroxidase 1 ............................................................... 47 2. Rapamycin and mTOR inhibition regulates selenoprotein levels............. 57 a. Selenoprotein levels are increased by inhibition of mTOR .......... 57 b. UGA readthrough by the SECIS is not enhanced by rapamycin treatment ....................................................................................... 58 3. Inhibition of PI3K decreases gluthathione peroxidase activity ................ 64 D. Discussion ............................................................................................................. 66 1. Inhibition of Bcr-Abl by imatinib increases protein and activity levels of multiple antioxidant proteins .................................................................... 66 a. Steady-state mRNA levels of glutathione peroxidase 1 and manganese superoxide dismutase are not altered by Bcr-Abl inhibition ....................................................................................... 67 b. Inhibition of Bcr-Abl by imatinib increases antioxidant protein levels post-transcriptionally .......................................................... 68 c. Increased glutathione peroxidase 1 levels are not a result of decreased rates of protein decay ................................................... 71 d. Evidence that post-translational modifications also affect glutathione peroxidase 1 and manganese superoxide dismutase activity........................................................................................... 71 e. Increased antioxidant protein levels following imatinib treatment are Bcr-Abl dependent .................................................................. 72 2. Inhibition of mTOR by rapamycin increases the protein levels of specific selenoproteins ........................................................................................... 74 a. Increased selenoprotein levels following mTOR inhibition are independent of Bcr-Abl................................................................. 74 b. Increased glutathione peroxidase 1 levels by inhibition of mTOR may be independent of PI3K regulation. ...................................... 75 c. Increased glutathione peroxidase 1 levels following inhibition of mTOR are not dependent on the selenocysteine insertion sequence element for UGA recoding............................................................ 77 d. Mammalian target of rapamycin may alter levels of glutathione peroxidase 1 and other selenoproteins by altering translation initiation or elongation .................................................................. 78 IV. CONCLUSIONS AND FUTURE DIRECTIONS............................................................ 80 APPENDICES .............................................................................................................................. 85 APPENDIX A ................................................................................................................... 85 v TABLE OF CONTENTS (continued) CHAPTER PAGE APPENDIX B ................................................................................................................... 95 APPENDIX C ................................................................................................................... 97 APPENDIX D ................................................................................................................. 104 CITED LITERATURE ............................................................................................................... 108 VITA……………………………………………………………………………………………141 vi LIST OF TABLES TABLE PAGE TABLE I. PATIENT INFORMATION AND DEGREE OF CHANGE IN GPX ACTIVITY FOLLOWING IMATINIB TREATMENT ............................................................... 33 TABLE II. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF IMATINIB TREATMENT OF KU812A ON SELECTED ANTIOXIDANT PROTEINS ................................................................................... 85 TABLE III. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF CYCLOHEXIMIDE ± IMATINIB TREATMENT OF KU812A PROTEIN DECAY RATE OF GPX1 ....................................................................... 86 TABLE IV. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF IMATINIB TREATMENT OF MEG-01 ON SELECTED ANTIOXIDANT PROTEINS ................................................................................... 87 TABLE V. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF IMATINIB TREATMENT OF GM10832 ON SELECTED ANTIOXIDANT PROTEINS ................................................................................... 88 TABLE VI. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF IMATINIB TREATMENT OF LNCAP ON SELECTED ANTIOXIDANT PROTEINS ................................................................................... 89 TABLE VII. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE TRANSFECTION OF BCR-ABL INTO LNCAP ON GPX1 .......................... 90 TABLE VIII. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF RAPAMYCIN TREATMENT OF KU812A ON SELECTED ANTIOXIDANT PROTEINS ................................................................................... 91 TABLE IX. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF RAPAMYCIN TREATMENT OF MEG-01 ON SELECTED ANTIOXIDANT PROTEINS ................................................................................... 92 TABLE X. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF RAPAMYCIN TREATMENT OF GM10832 ON SELECTED ANTIOXIDANT PROTEINS ................................................................................... 93 TABLE XI. BAND INTENSITY AS DETERMINED BY DENSITOMETRY INDICATING THE EFFECT OF RAPAMYCIN TREATMENT OF LNCAP ON SELECTED ANTIOXIDANT PROTEINS ................................................................................... 94 vii LIST OF FIGURES FIGURE PAGE Figure 1. Reproducibility of the GPx enzyme assay. ................................................................ 31 Figure 2. GPx activity in MNC extracts derived from patients before and after imatinib therapy. ...................................................................................................................... 33 Figure 3. No difference in growth between parental and clonal KU812 cell lines following imatinib treatment. ..................................................................................................... 48 Figure 4. GPx1 protein and activity levels are enhanced by 7-day treatment of KU812a CML cells with 150 nM imatinib. ....................................................................................... 49 Figure 5. GPx1 protein and activity levels are enhanced in a dose- and time-dependent manner following imatinib treatment of KU812a cells. ......................................................... 50 Figure 6. Increases in GPx1 protein are not a result of increased steady-state transcript levels or increased protein decay rates. .................................................................................... 51 Figure 7. GPx1 protein and activity levels are increased in MEG-01 following 300 nM imatinib treatment. ..................................................................................................... 52 Figure 8. GPx1 protein and activity levels are unaffected by imatinib treatment of LNCaP and GM10832 cells. .......................................................................................................... 53 Figure 9. GPx1 protein and activity levels are decreased by exogenous Bcr-Abl in LNCaP. .. 54 Figure 10. Imatinib increases MnSOD protein and activity levels and TrxR1 protein levels following treatment with imatinib in CML cell lines. ............................................... 55 Figure 11. Imatinib does not affect catalase or GPx4 protein levels in KU812a or MEG-01 cell lines. ........................................................................................................................... 56 Figure 12. Imatinib does not affect catalase, GPx4 or TrxR1 protein levels in GM10832 or LNCaP cell lines. ....................................................................................................... 56 Figure 13. Overexpression of Bcr-Abl in LNCaP does not result in decreased expression of MnSOD and TrxR1 protein levels. ............................................................................ 57 Figure 14. Rapamycin enhances GPx protein and enzyme activity levels in KU812a and MEG- 01. .............................................................................................................................. 59 Figure 15. Rapamycin treatment did not affect mRNA transcript levels for GPx1 and MnSOD following rapamycin treatment in KU812a. .............................................................. 60 viii LIST OF FIGURES (continued) FIGURE PAGE Figure 16. Rapamycin treatment in non-CML cell lines significantly increases GPx1 protein and activity levels. ............................................................................................................ 61 Figure 17. Rapamycin increases levels of GPx4 and TrxR1 protein levels in cell lines. ........... 62 Figure 18. MnSOD protein and activity levels are not increased following rapamycin treatment. ................................................................................................................................... 63 Figure 19. The translational efficiency of UGA readthrough by the GPx1 SECIS is not enhanced by rapamycin treatment. ............................................................................ 64 Figure 20. Treatment of KU812a by 20 µM LY294002 for 4 days inhibits PI3K phosphorylation activity and decreases GPx activity levels. ..................................... 65 Figure 21. Proposed signaling cascade for the regulation of GPx1 transcription and translation. ................................................................................................................................... 82 ix LIST OF ABBREVIATIONS 4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1 ADP Adenosine diphosphate AP-1 Activator protein-1 ATP Adenosine triphosphate Bax Bcl-2-associated X protein Bcl-2 B-cell lymphoma-2 protein Bcr Breakpoint cluster region CD95 Cluster of differentiation 95 Cdk Cyclin-dependent kinase CHO Chinese hamster ovary CML Chronic myelogenous leukemia Ct Cycle threshold Cys Cysteine DM Diabetes mellitus dNTP Deoxyribonucleotide triphosphates ECL Enhanced chemiluminescence eIF Eukaryotic initiation factor EJC Exon-junction complex Erk Extracellular signal-regulated kinase FAD Flavin adenine dinucleotide FBS Fetal bovine serum x