MECHANISMS OF EXTRACELLULAR NUCLEOTIDE ACCUMULATION DURING REGULATED CELL DEATH IN TUMOR CELLS By ANDREA MICHELLE BOYD TRESSLER Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: George R Dubyak, PhD Department of Pharmacology Case Western Reserve University May 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Andrea Michelle Boyd Tressler candidate for the degree of Doctor of Philosophy*. Ruth Keri (Committee Chair) George Dubyak (Dissertation Advisor) Vera Moiseenkova-Bell (Committee Member) Clark Distelhorst (Committee Member) Thomas Kelley (Committee Member) Date of Defense March 22, 2016 *We also certify that written approval has been obtained for any proprietary material contained therein. i Dedication I would like to dedicate this to my parents, Nik and Debbie, who have been my biggest supporters my entire life. From ballet lessons to my PhD, they have always challenged and encouraged me, sacrificing time and money to make sure they were front and center at everything I did. I would not be the woman I am today without them. Also, to my niece, Taylor, who is my favorite person and a big reason I’ve never given up. ii Table of Contents List of Figures ………………….……………………..…………...…………….......vi Acknowledgements …………………………………………………………………x List of Abbreviations………………………………………………………………...xii Abstract………………………………………………………………………………….1 Chapter 1: Introduction and Background…………………………………………3 1.1 Tumor immunotherapy………………….………………………………………….3 1.2 Programmed Cell Death………………….………………………………………..8 1.2.1 Apoptosis………………………………………………………………………….8 1.2.1.1 Extrinsic…………………………………………………….……………………8 1.2.1.2 Intrinsic…………………………………………………………………………12 1.2.2 Necroptosis………………………………………………………………………13 1.3 Chemotherapeutic Drugs…………………………………………..………….....18 1.3.1 Doxorubicin……………………………………...…………………..…………..18 1.3.2 Etoposide……………………………………………………………..………….19 1.3.3 Staurosporine…………………………………………………………..………..20 1.4 Release of Intracellular ATP…………………………………………….……….21 1.4.1 Purinergic Signaling…………………………………………………….………21 1.4.2 Mechanisms of ATP Release…………………………………………..……...22 1.4.2.1 Exocytosis …………………………………………………………………….23 1.4.2.2 Plasma Membrane Permeabilization……………………………………….23 1.4.2.3 Regulated efflux via channels……………………………………………….23 iii 1.5 Pannexin 1 Channel……………………………………………………………....26 1.6 ATP metabolism…………………………………………………………………...31 1.6.1 Ecto-Nucleotide triphosphate diphosphohydrolase………………………...32 1.6.2 Ecto-nucleotide pyrophosphatase/phosphodiesterase…………………….32 1.6.3 CD73……………………………………………………………………………..32 1.7 Statement of Purpose…………………………………………………………….33 Chapter 2: Materials and Methods………………………………………………...36 Chapter 3: Chemotherapeutic Drugs Induce ATP Release via Caspase- gated Pannexin-1 Channels and a Caspase/Pannexin-1-independent Mechanism………………………………………………………………………….....47 3.1 Abstract…………………………………………………………………………….47 3.2 Introduction………………………………………………………………………..48 3.3 Results……………………………………………………………………………..51 3.4 Discussion………………………………………………………………………....67 3.5 Acknowledgements……………………………………………………………….77 Chapter 4: Upregulated ectonucleotidases in FADD- and RIP1-deficient Jurkat leukemia cells counteracts extracellular ATP/AMP accumulation via pannexin-1 channels during chemotherapeutic drug-induced apoptosis ...…………………..……………...........................................................................101 4.1 Abstract…………………………………………………………………………...101 4.2 Introduction……………………………………………………………………....102 4.3 Results………………………………………………………………………..…..106 4.5 Discussion…………………………………………………………………..……121 iv 4.5 Acknowledgements……………………………………………………………...130 Chapter 5: Regulation of Adenine Nucleotide release through Pannexin 1 channels in EG7 Murine Lymphoma Cells…………………………………......147 Chapter 6: Discussion and Future Directions……………………………..…..157 6.1 Mechanisms of Programmed Cell Death Induced Adenine Nucleotide Release……………………………………………………………………..………...159 6.2 Role of FADD and RIP1 in ectonucleotidase activity……………..………….160 6.3 Decrease in immune cell activation………………………………..…………..163 6.4 Post-translational modification of Panx1………………………………………164 6.5 Concluding Remarks…………………………………………………………….166 Appendix……………………………………………………………………………..170 References………………………………………………………………………...…185 v List of Figures Figure 1.1: Activation of the purinergic anti-tumor immune response after treatment of chemotherapy………………………………………………………..…...5 Figure 1.2: Intrinsic and Extrinsic Activation of Apoptosis…………………….........9 Figure 1.3: TNF signaling……………………………………………………………..15 Figure 1.4: Mechanisms of ATP Release…………………………………………...24 Figure 1.5: Pannexin 1 Channel……………………………………………………..27 Figure 3.1: Comparative time courses for accumulation of active caspase-3 and loss of viability in Jurkat leukemic T cells treated with different chemotherapeutic agents…………………………………………………………………………………..78 Figure 3.2: Chemotherapeutic drugs induce caspase-3 mediated cleavage of the pannexin-1 C-terminal autoinhibitory domain………………………………………80 Figure 3.3: Chemotherapeutic drugs induce accumulation of active pannexin-1 channels via a caspase dependent activation mechanism………………………..82 Figure 3.4: Efflux of both ATP and ATP metabolites is triggered during chemotherapeutic drug-induced apoptosis of Jurkat cells………………………...84 Figure 3.5: Caspase-activated pannexin-1 channels mediate the efflux of ATP and ATP metabolites during chemotherapeutic drug-induced apoptosis but an alternative ATP release mechanism is engaged in the context of suppressed caspase activity………………………………………………………………………..87 Figure 3.6: Carbenoxolone blocks the efflux of ATP and ATP metabolites during chemotherapeutic drug-induced apoptosis…………………………………………89 vi Figure 3.7: Caspase-insensitive ATP release stimulated by chemotherapeutic drugs is resistant to carbenoxolone blockade but suppressed by intracellular Ca2+ buffering………………………………………………………………………….91 Figure 3.8: Caspase-insensitive ATP release stimulated by staurosporine does not involve direct Ca2+mobilization by staurosporine or activation of phosphatidyl inositol phospholipase C signaling…………………………………………………..94 Figure 3.9: Proteosome inhibition induces caspase-3-mediated cleavage of the pannexin-1 C-terminal autoinhibitory domain and pannexin-1-mediated release of adenine nucleotides…………………………………………………………………...96 Figure 3.10: Pannexin-1 is more highly expressed in human leukemic leukocytes than in normal human T cells…………………………………………………………99 Figure 4.1: TNFα-induction of necroptosis or extrinsic apoptosis induces release of adenine nucleotides from Jurkat cancer cells via mechanistically distinct pathways………………………………………………………………………..…….131 Figure 4.2: Intrinsic apoptotic signaling and Panx1 channel cleavage in FADD- deficient Jurkat cancer cells is uncoupled from accumulation of extracellular adenine nucleotides………………………………………………………………….134 Figure 4.3: Caspase-3-cleaved Panx1 channels are functionally active in FADD- deficient Jurkat cancer cells during intrinsic apoptosis………………………......136 Figure 4.4: CD73 ecto-nucleotidase activity is upregulated in FADD-deficient Jurkat cancer cells and counteracts Panx1 channel-mediated efflux of ATP/AMP during intrinsic apopotosis…………………………………………………………..138 vii Figure 4.5: Apoptotic signaling and Panx1 channel activation in RIP1-deficient Jurkat cancer cells is also uncoupled from accumulation of extracellular adenine nucleotides……………………………………………………………………………140 Figure 4.6: RIP1 deficient cells have increased CD73 activity………………….143 Figure 4.7: Increased expression of CD39 ecto-ATPase in RIP1-deficient Jurkat cells relative to FADD-deficient or wildtype Jurkat cells………………………….145 Figure 5.1: Comparative activation of apoptosis in Jurkat T cells and EG7 cells treated with chemotherapeutic agent ……..……………………………………….151 Figure 5.2: Activation of apoptosis in EG7 cells by chemotherapies does not lead to Panx1 mediated Adenine Nucleotide accumulation…………………………..153 Figure 5.3: Cleavage of Panx1 in EG7 cells leads to increased YO-Pro dye influx…………………………………………………………………………………...155 Appendix 1: Full Western Blot of Panx1 in Anti-Fas-treated Jurkat T cells in the presence of absence of zVAD………………………………………………………170 Appendix 2: Full Western Blot of Panx1 in STS-treated Jurkat T cells in the presence of absence of zVAD………………………………………………………171 Appendix 3: Full Western Blot of Panx1 in Dox-treated Jurkat T cells in the presence of absence of zVAD………………………………………………………172 Appendix 4: Full Western Blot of Panx1 in Etop-treated Jurkat T cells in the presence of absence of zVAD………………………………………………………173 Appendix 5: Full Western Blot of Panx1 in TS- or Anti-Fas-treated WT Jurkat T cells…………………………………………………………………………………….174 viii Appendix 6: Full Western Blot of Panx1 in TS- or TS+zVAD-treated FADD-def Jurkat T cells………………………………………………………………………….175 Appendix 7: Full Western Blot of Panx1 in STS-, Etop- or Anti-Fas-treated WT Jurkat T cells………………………………………………………………………….176 Appendix 8: Full Western Blot of Panx1 in STS-, Etop- or Anti-Fas-treated FADD- def Jurkat T cells……………………………………………………………………..177 Appendix 9: Full Western Blot of Panx1 in STS-, or Etop-treated WT and RIP1- def Jurkat T cells…………………………………………………………………….178 Appendix 10: Full Western Blot of Panx1 in STS- or Dox-treated EG7 Lymphoma cells…………………………………………………………………………………….179 Appendix 11: Copy right permission forms………………………………………..180 ix Acknowledgements I would first like to thank my advisor, Dr. George Dubyak for giving me the amazing opportunity to work in his lab. When I