REACTIVE OXYGEN SPECIES-MEDIATED REGULATION OF THE Na+/H+ EXCHANGER, NHE-1 GENE EXPRESSION: A NEW MECHANISM FOR TUMOR CELLS’ RESISTANCE TO APOPTOTIC CELL DEATH SUFYAN AKRAM (MBBS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHYLOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS I wish to acknowledge my deepest gratitude and appreciation to my supervisor, Dr Marie-Véronique Clément, Associate Professor at Department of Biochemistry, National University of Singapore Without her continuous encouragement and advice this dissertation would have been far short of what it is today No less I would like to thank Associate Professor Shazib Pervaiz for the excellent cooperation, interaction and guidance throughout the course of this work I am very grateful to all my colleagues, who have helped me in one way or another during my stay in the lab My appreciation also goes to my parents for their persistent support and love throughout my life Special thanks belong to my wife and two sons The knowledge of them being there has been of great encouragement and importance for me Without their support, this work could never have been accomplished i TABLE OF CONTENTS Acknowledgments i Table of contents ii Abstract vi List of figures vii List of abbreviations x Chapter I Introduction I.1 Cell Death I.1.a Types of Cell Death I.1.a Programmed cell death or apoptosis I.1.b Apoptotic machinery I.2 Reactive oxygen species and apoptosis I.2.a Pro and Anti-Apoptotic functions of ROS I.2.b Rac1 and superoxide anion production 11 I.2.c Superoxide anion and inhibition of apoptosis 13 I.3 14 Intracellular milieu I.3.a Intracellular pH (pHi) 16 I.3.b pHi and apoptosis 17 I.4 18 Intracellular pH regulation I.4.a Plasma membrane pHi regulators I.5 19 Na+-H+ Exchanger (NHE) 21 I.5.a NHE1: Basic structure 22 I.5.b NHE1: Major Functions 24 I.5.b.i pHi and cell volume regulation ii 24 I.5.b.ii Cell proliferation and differentiation 24 I.5.b.iii Cell motility 26 I.5.b.iv As a plasma membrane scaffold 26 I.5.b.v Cell injury 26 I.5.c NHE1: Regulation 27 I.5.d NHE1: Regulation of gene expression 28 I.5.e NHE1: Pathological Functions 31 I.5.e.i NHE1: Role in myocardium 31 I.5.e.ii NHE1: Role in tumorigenesis 32 I.6 Rac subfamily and NHE1 34 I.7 Conclusion 35 Chapter II Materials and methods 37 II.1 Chemicals 37 II.2 Cells 37 II.3 cDNA Plasmids and transfections 40 II.4 β-Galactosidase Survival Assay 41 II.4a Measurement of β-Galactosidase activity 41 II.5 siRNA transfection 42 II.6 Luciferase reporter gene assay 43 II.7 CAT (Chloramphenicol acetyltransferase) ELISA 43 II.8 Measurement of steady state pHi 44 II.9 Measurement of acid load and pHi recovery (NHE-1 activity) 45 II.10 Western blot analysis of NHE-1 protein 45 II.11 Measurement of intracellular superoxide 46 iii II.12 Crystal Violet Assay 46 II.13 Caspase assay 47 II.14 Statistical Analysis 47 Chapter III Results 48 III.1.a Regulation of NHE-1 gene expression regulates cells’ response 48 to death triggers in NIH3T3 cells III.1.b Regulation of NHE-1 gene expression regulates cells’ response 52 to death triggers in Tumor cells III.2.a Superoxide mediated cell survival is NHE-1-dependant 55 III.2.b Role of superoxide in NHE1-dependent cell survival in tumor cells 59 III.3 NHE-1 gene expression is growth factor regulated 64 III.4 Intracellular superoxide activates NHE-1 promoter activity 69 III.5.a Small GTPase Rac1-mediated survival is dependent upon NHE-1 71 protein expression III.5.b Rac1-mediated NHE-1 protein expression is a function of its 74 ability to produce superoxide III.5.c Serum-induced NHE-1 expression might involve activation of Rac1 79 III.6 H2O2 inhibits NHE-1 promoter activity 85 III.7 H2O2 leads to decreased NHE-1 expression and increased 87 susceptibility to cell death III.8 Regulation of intracellular pH as one of the mechanisms of 89 NHE-1-mediated cell survival III.9 Region of NHE-1 promoter involved in O2. mediated activation iv 94 Chapter IV Discussion and conclusions 102 IV.1 How tumors develop 102 IV.2 Permissive intracellular milieu 102 IV.3 Alkaline pHi and role of NHE-1 103 IV.4 Regulation of NHE-1 105 IV.5 Rationale of our study 105 IV.6 Regulation of NHE-1 gene expression regulates cells’ response 106 to death triggers IV.7 Superoxide mediated cell survival is NHE-1-dependant 107 IV.8 NHE-1 gene expression is growth factor regulated 108 IV.9 Intracellular superoxide activates NHE-1 promoter activity 109 IV.10 Small GTPase Rac1-mediated survival is dependent upon 110 NHE-1 protein expression IV.11 H2O2 inhibits NHE-1 promoter activity and leads to increased 112 susceptibility to cell death IV.12 Regulation of intracellular pH as one of the mechanisms of 113 NHE-1-mediated cell survival IV.13 Region of NHE-1 promoter involved in O2. mediated activation 115 IV.14 Conclusions 116 IV.15 Prospective Studies 119 References 120 Publication and presentations 143 v Abstract Reactive Oxygen Species have long been known to cause cellular stress and damage But recently, ROS have been implicated as signaling molecules Tumor cells display an altered redox status Our lab has recently shown that expression of a constitutively active form of Rac1 (RacV12) inhibits tumor cell death by apoptosis through intracellular production of superoxide anion (O2-) (Pervaiz et al 2001, Oncogene) Another characteristic of transformed cells is a shift towards alkaline intracellular pH NHE-1, one of the major pHi regulators, has been shown to be of particular importance in tumor cells Current study was designed to study the effect of ROS on NHE1 regulation and the role it may play in modulating apoptosis Our data shows that production of intracellular O2•- induces transcription of NHE-1 while increase in H2O2 inhibited it Using Rac mutants, which have differential ability to produce O2•in the cell, and drugs that affect the intracellular ROS levels, we were able to show that NHE1 gene is redox-responsive Changes in NHE1 gene expression were translated into NHE1 protein expression By over-expressing or silencing NHE-1 gene we show that cell response to apoptotic triggers such as staurosporin and etoposide correlates with the amount of NHE-1 protein expression on the cell surface Moreover, down-regulation of NHE-1 gene expression in tumor cell lines tested reverted their resistant phenotype These results support a critical role for NHE-1 expression in tumor cells’ response to anticancer therapy and provide a possible mechanism for Rac1-mediated survival in tumor cells Pervaiz S., Cao J., Chao OSP, Chin YY Clément M-V Activation of the RacGTPase inhibits apoptosis in human tumor cells Oncogene 20: 6263-6268, 2001 vi List of Figures Fig A Major differences between Apoptotic and Necrotic types of Cell Death Fig B Death-Receptor mediated Apoptosis Fig C Mitochondial pathway of Apoptosis Fig D Wide array of functions attributed to Reactive Oxygen Species Fig E A model of ROS-mediated regulation of Apoptosis 15 Fig F Various plasma membrane-bound pHi regulators 20 Fig G Structure of mammalian NHE-1 23 Fig H Physiological functions of NHE-1 25 Fig I Basic structure of proximal part of NHE1 promoter 30 Table Various cell lines used in this project 38 Fig Increased NHE-1 expression leads to inhibition of staurosporine-induced cell death in NIH3T3 cells 49 Fig Silencing of NHE-1 gene leads to increased susceptibility to cell death in NIH3T3 cells 51 Fig Level of NHE-1 expression correlates with NIH3T3 cells’ sensitivity to staurosporine-induced cell death 51 Fig Silencing of NHE-1 gene leads to increased susceptibility to 54 cell death in U87 cells Fig Time-dependent Caspase (DEVDase) activity in NHE-1 silenced U87 cells treated with etoposide 54 Fig Silencing of NHE-1 gene leads to increased susceptibility to cell death in LNCaP cells 56 Fig Caspase (DEVDase) activity in NHE-1 silenced LNCaP cells upon treatment with etoposide and staurosporine 56 Fig Manipulation of NHE-1 expression does not affect intracellular 58 vii superoxide levels Fig DDC leads to significant increase in intracellular superoxide levels in NIH3T3 cells 58 Fig 10 DDC-mediated inhibition of cell death is dependent on NHE-1 gene expression 60 Fig 11 Inhibition of intracellular superoxide production prevents NHE-1 protein expression in U87 cells 63 Fig 12 Inhibition of intracellular superoxide production prevents NHE-1 expression in U87 cells and increases their susceptibility to etoposide-induced cell death 65 Fig 13A NIH3T3 1A8 cells stably transfected with proximal 1.1 kb fragment of NHE-1 promoter/enhancer upstream of a luciferase gene 67 Fig 13B Basic Principle of Luciferase Reporter Assay 67 Fig 14 NHE-1 gene expression is growth factor regulated 68 Fig 15 Serum-induced activation of NHE-1 is dependant upon intracellular production of superoxide 70 Fig 16 Superoxide levels in NIH3T3 1A8 cells in response to different drugs 72 Fig 17 Superoxide is a signal for NHE-1 promoter activity 73 Fig 18 Expression of RacV12 induces NHE-1 promoter activity in a variety of cells 75 Fig 19 Rac loss-of-function mutants 77 Fig 20 NADPH oxidase interaction domain of Rac1 is required for Rac1-induced NHE-1 promoter activity 78 Fig 21 Rac1-mediated cell survival is dependent on its ability to produce superoxide 80 Fig 22 Expression of RacN17 inhibits serum-induced NHE-1 promoter activity 81 Fig 23 Manipulation of NHE-1 protein expression in M14pIRES and M14pIRES-RacV12 cells by NHE-1 siRNA transfection 83 Fig 24 Rac-induced cell survival is dependant upon NHE-1 expression 84 viii Fig 25 H2O2 inhibits NHE-1 promoter activity in NIH3T3 cells 86 Fig 26 H2O2 treatment of NIH3T3 cells results in increased susceptibility to etoposide-induced killing 88 Fig 27 Increased NHE1 protein expression level leads to an increase in pHi 90 Fig 28 Silencing of NHE1 gene results in a drop in intracellular pH in NIH3T3 cells 90 Fig 29 Increase in intracellular pH correlates with the ability of Rac mutants to 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Wu KL, Khan S, Lakhe-Reddy S, Wang L, Jarad G and Miller RT (2003) Renal tubular epithelial cell apoptosis is associated with caspase cleavage of the NHE1 Na+/H+ exchanger Am J Physiol Renal Physiol 284(4): F829-39 141 Wu LK, Khan S, Reddy SL, Jarad G, Mukherjee A, Obejero-Paz CA, Konieczkowski M, Sedor JR and Schelling JR (2004) The NHE1 Na+/H+ Exchanger Recruits Ezrin/ Radixin/ Moesin Proteins to Regulate Akt-dependent Cell Survival J Biol Chem 279(25): 26280-26286 Wyllie AH, Kerr JF, and Currie AR (1980) Cell death: the significance of apoptosis Int Rev Cytol 68: 251-306 Yang W, Dyck JR and Fliegel L (1996) Regulation of NHE1 expression in L6 muscle cells Biochim Biophys Acta 1306: 107-113 Zhaoyu Jin, Wafik S El-Deiry (2005) Overview of Cell Death Signaling Pathways Cancer Biology & Therapy 4(2), 139-163 142 PUBLICATION S Akram, Huey Fern, Larry Fleigel, Shazib Pervaiz, M-V Clement Reactive oxygen species-mediated regulation of the Na+-H+ exchanger gene expression connects intracellular redox status with cells’ sensitivity to death triggers Published online 23rd September 2005 in Cell Death and Differentiation (doi: 10.1038/sj.cdd.4401775) POSTERS & ABSTRACTS PRESENTED S Akram, Lim Chin Aeng, M-V Clement Redox regulation of the Na+/H+ Exchanger , NHE1 gene expression: A new mechanism involved in tumor cell response to Apoptosis Abstract presented at 5th Combined Annual Scientific Meeting (SSMB, SSBMB, BRETSS and GSS-FOM) held at NUS, Singapore in May 2004 S Akram, M-V Clement Intracellular superoxide production and transcription of the Na+/H+ Exchanger leading to tumor cells alkalinity may represent a new therapeutic target for conventional chemotherapeutic agents Abstract presented at Postgraduate conference on Immunology and Cancer Biology at City University of Hong Kong in February 2003 S Akram, M-V Clement Regulation of intracellular pH by superoxide anion production: a new mechanism for tumor cells’ resistance to apoptosis Poster presented at 10th Euro-conference on Apoptosis held at Pasteur Institute, Paris, France in October 2002 S Akram, M-V Clement Regulation of intracellular pH by superoxide anion production: a new mechanism for tumor cells’ resistance to apoptosis Abstract presented at 6th NUS-NUH Annual Scientific Meeting held in conjunction with Johns Hopkins Singapore and Institute of Molecular and Cell Biology, Singapore in August 2002 S Akram, M-V Clement Intracellular superoxide production and transcription of the Na+/H+ Exchanger induced by the small GTP-binding protein Rac1 and the PKB/Akt kinase: a new mechanism for tumor cell resistance to apoptosis Abstract accepted for poster presentation at 93rd Annual Meeting of AACR (American Association for Cancer Research) held in San Francisco, California, USA in April 2002 S Akram, M-V Clement Regulation of the Na+/H+ Exchanger Gene Expression by the Small GTP-binding Protein Rac1: Role in the Inhibition of Apoptosis in Tumor Cells Poster presented at 3rd Combined Annual Scientific Meeting (SSMB, SSBMB, BRETSS) held at NUS, Singapore in November 2000 143 AWARDS AND SCHOLARSHIPS NUS-NUH/NMRC Young Scientist Award (awarded at the 6th NUS-NUH Annual Scientific Meeting) August 2002 National University of Singapore Research Scholarship Jun 2000 - Jun 2004 144 ... I.5 Na+- H+ Exchanger (NHE) The Na+- H+ exchangers (NHEs) are a family of membrane glycoproteins which transport H+ out of the cell in exchange for Na+ with a stoichiometry of 1: 1 In mammalian cells, ... increased 11 2 susceptibility to cell death IV .12 Regulation of intracellular pH as one of the mechanisms of 11 3 NHE- 1 -mediated cell survival IV .13 Region of NHE- 1 promoter involved in O2. mediated activation... List of abbreviations x Chapter I Introduction I .1 Cell Death I .1. a Types of Cell Death I .1. a Programmed cell death or apoptosis I .1. b Apoptotic machinery I.2 Reactive oxygen species and apoptosis