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Antiviral and Antitumor Functions of Rnase L

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Antiviral and Antitumor Functions of Rnase L Antiviral and Antitumor Functions of RNase L By Geqiang Li Submitted in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy Thesis Advisor: Dr. Robert H Silverman Department of Genentics Case Western Reserve University January 2005 Case Western Reserve University School of graduate studies We hereby approve the thesis/dissertation of __________________________Geqiang Li________________________ candidate for the _____________________Doctorate_____________ degree. ( Signed ) ______________Robert H. Siverman_____________________ (Chair of the Committee) ______________Bryan G Williams_______________________ _____________ David Samols___________________________ ______________Ganes C. Sen___________________________ ______________Alexandtru Almasan_____________________ ( Date )___________10-11-2004____ 1 TABLE OF CONTENTS TABLE OF CONTENTS……………………………………………… ……………2 LIST OF FIGURES…………………………………………………………………..7 LIST OF ABBREVIATIONS……………………………………………..………….9 ABSTRACT………………………………………………………………………...11 CHAPTER 1. INTRODUCTION 1.1. Overview of the interferon (IFN) system………………………………….…...14 1.2. 2-5A / RNase L system………………………………….…….…………..…..15 1.2.1. Properties of RNase L…………………………………………………...17 1.2.2. Antiviral activity of RNase L…………………………………..………..20 1.2.3. Apoptotic activity of RNase L……………………………………..……22 1.2.4 Involvement of RNase L in the biology of prostate cancer………….…23 CHAPTER 2. AN APOPTOTIC SIGNALING PATHWAY IN THE INTERFERON ANTIVIRAL RESPONSE MEDIATED BY RNASE L AND C-JUN NH2- TERMINAL KINASE ABSTRACT…………………………………………….…………………….……29 2.1 INTRODUCTION…………………………………..………..……………...…31 2.2 MATERIALS AND METHODS………………….…..………….……......…..35 2.2.1 Cell culture………………………………….…………………………….35 2.2.2 Transfections……………………………………………….……...……..36 2.2.3 Viral Infections.….………………...……………...……………………...36 2.2.4 Measuring Protein Synthesis in Intact Cells..…………..……...…………36 2.2.5 RNase L Activity in Intact Cells……………………….…………………36 2.2.6 RNase L Activity in a Cell-free System……………………………….….37 2.2.7 2-5A Binding Assay for RNase L…………....…………………………...37 2.2.8 Cell Viability Assay………………………………………………………38 2 2.2.9 Western Blots………………………..………...………………….……...38 2.2.10 TUNEL Assay………………………………………………………….38 2.2.11 JNK Kinase Assay…………………….………………………………..39 2.3 RESULTS………………….…………………………………………………....41 2.3.1 Viral Activation of JNK and Apoptosis Are Deficient in Cells Lacking RNaseL…………………………………………………...….41 2.3.2 2-5A Activation of RNase L Results in Stimulation of JNK and Apoptosis ……………………………………………………….…42 2.3.3 Inhibition of JNK Impairs RNase L-induced Apoptosis………………...45 2.3.4 Ablation of JNK Suppresses Apoptosis Induced by 2-5A Activation of RNase L…………………………………………...…….…...46 2.4 DISCUSSION………………..………………...……… …………………..….49 2.4.1 Essential Role of RNase L in Viral Activation of JNK…….…………...49 2.4.2 JNK Participation in RNase L-mediated Apoptosis………………….…51 2.4.3 The RNase L Apoptotic Signaling Pathway………………………….....52 CHAPTER 3. RNASE L IS A NEGATIVE REGULATOR OF CELL MIGRATION ABSTRACT…………………………….…………………………………….…..73 3.1 INTRODUCTION………………………………...………………….……….75 3.2 METERIALS AND METHODS…………………………..…………….........80 3.2.1 Cell culture and treatment…………..…………………….…….……...…80 3.2.2 Cell Adhesion assay………………………………………….….………80 3.2.2 Migration assays……………………………………..……………........80 3.2.3 In vitro kinase assays…………………………………….…….…….....81 3.2.4 Detection of protein-protein interactions…………………….…………82 3.2.5 Immunoprecipitations and immunoblot analysis…………….…………83 3.2.6 Immunofluorescence Microscopy……..………………………..……....83 3.3 RESULTS…………………………………………………………….….….…85 3 3.3.1 RNase L inhibits cell migration but not adhesion……………………....85 3.3.2 Integrin β1 mediated cell migration is inhibited by RNase L…….……86 3.3.3 RNase L inhibited adhesion dependent FAK activation……….……….87 3.3.4 Adhesion dependent of JNK activation and C-Jun phopshorylation is increased in RNase L-/- MEF cells…………………………………...87 3.3.5 The JNK inhibitor SP600125 suppresses cell migration more significantly in RNase L-/- than in RNase L+/+ MEF cells…………....…88 3.3.6 RNase L is able to interacts with integrin β1, and FAK………...……..89 3.3.7 RNase L can be phosphorylated by FAK in vitro……………………..91 3.3.8Activation of RNase L by 2-5A inhibits cell migration……...………....91 3.3.9 2-5A promote integrin migrating to cell membrane………….………...92 3.4 DISCUSSION……………..…………………………………………………..93 3.4.1 RNase L is a negative regulator of cell migration………….....………..93 3.4.2 JNK is involved in RNase L mediated inhibition of cell migration…....94 3.4.3 RNase L interact with integrin β1 and FAK…………………...……....95 3.4.4 2-5A induce inhibition of cell movement……………………………..97 CHAPTER 4 PMA INDUCED RNASE L PHOSPHORYLATION AND PREVENT 2-5A INDUCED APOPTOSIS 4.1 INTRODUCTION……………………………………………………...…...112 4.2 MATERIALS AND METHODS………………………………………...….116 4.2.1 Protein interaction assays……………...……………….……………..116 4.2.2 RNase L immnopricipitation………………………….………………116 4.2.3 2D gel electrophoresis…………...…………………….………….…..116 4.2.4 Silver Staining…………………………………………………...……117 4.2.5 Western blot analysis……………………………………………...….118 4.3 RESULTS AND DISCUSSION……………………………………….……119 4.3.1 PMA stimulate RNase L phosphorylation……………………………119 4.3.2 PMA blocked 2-5A induced apoptosis………………………………..119 4 4.3.3 Interaction of RNase L with PKCα……………………………………….120 CHAPTER 5. SUMMARY AND FUTURE DIRECTIONS 5.1 SUMMARY………………………………………………..…………..……125 5.2 FUTURE DIRECTIONS…………………………………..……………..…125 5.2.1. Investigate the mechanism of 2-5A induced JNK activation……..…127 5.2.2. Investigate involvement of RNase L in PKC signaling pathways......128 5.2.2.1 To determine if PKCα is the only protein responsible for phosphorylating RNase L in response to PMA………….129 5.2.2.2 Examine the effect of PMA treatment on RNase L interaction with cytoskeleton……………………..……..129 5.2.2.3 To determine the PKC signaling pathway affected by RNase L……………………………………………………...130 5.2.2.4 PMA induced gene expression file in RNase L+/+ and RNase L-/- fibroblasts………………………………....130 5.2.3. Investigate the role of RNase L in cell migration……..……………...131 5.2.3.1 Expression of DN-JNK in RNase L-/- cells and examine its effect on migration………………………..….….131 -/- 5.2.3.2 Expression of RNase L and RNase L∆EN in RNase L fibroblasts, and then examine the effect on cell migration…...132 5.2.3.3 Investigate the effect of RNase L on FAK autophosphorylation..............................................….132 5.2.3.4 Examine the effect of RNase L in inhibition of tumor metastasis in nude mice…………………………………….. ...132 REFERERENCE…………………………………………………………………134 5 LIST OF FIGURES CHAPTER 1 Figure 1.1 Antiviral mechanism of interferon action………………………….....…..15 Figure 1.2 The IFN-regulated 2-5A system, a RNA decay pathway with antiviral and apoptotic activities…………………………………..……..16 Figure 1.3 The structure of RNase L…………………………...………………..…..18 Figure 1.4 Functional model for the activation of RNase L by 2-5A…………..…..19 Figure 1.5 Comparison of the domains and motifs in human RNase L and yeast Ire1p………………….……..……...........................20 Figure 1.6 RNase L mutations in different populations of prostate cancer cases aligned to the domain structure of RNase L………………....…….23 Figure 1.7 Progression of prostate cancer and steps where RNase L could interfere……………………………………………..…....28 CHAPTER 2 Figure 2.1 Apoptosis and JNK activation are deficient in virus-infected. RNase L-/- cells……………………………………………….…….55-56 Figure 2.2. Activation of RNase L with 2-5A induces phosphorylation of c-Jun and JNK but not p38 and inhibits protein synthesis……....…57-58 Figure 2. 3. Apoptosis requires functional RNase L……………………………..…59 Figure 2.4 Bcl-2 overexpression blocks apoptosis in response to 2-5A activation of RNase L…………………………………………….....…60 Figure 2.5 Kinetics of c-Jun phosphorylation and rRNA cleavage in Hey1b cells……………………………………………..………...61-62 Figure 2.6 2-5A induces c-Jun phosphorylation in Hey1b cells pretreated with cycloheximide……………………………………….63 Figure 2.7 RNase L is involved in dsRNA mediated JNK activation………….…..64 Figure 2.8. The JNK inhibitor, SP600125, suppresses c-Jun phosphorylation 6 and apoptosis in response to 2-5A activation of RNase L………..…65-68 Figure 2.9 Suppression of c-Jun phosphorylation and apoptosis by transfecting cells with siRNA against JNK1 and JNK2………….…….69 Figure 2.10.Apoptosis in response to IFN and 2-5A is inhibited in Jnk1-/- Jnk2-/- MEF cells………………………….………………...70-71 Figure 2.11. Apoptotic signaling pathway in virus-infected cells mediated by RNase L and JNK……………………………………….…………….72 CHAPTER 3 Figure 3.1 RNase L inhibits cell haptotactic migration……………………...….98-99 Figure 3.2. RNase L doesn’t affect cell adhesion on fibronectin and laminin……100 Figure 3.3 RNase L inhibit β1 integrin mediated haptotaxis………………….….101 Figure 3.4 RNase L inhibit adhesion dependent FAK tyrosine phosphorylation….102 Figure 3.5. RNase L inhibit adhesion dependent JNK activation and c-Jun phophorylation……………………………….………..…….103 Figure 3.6. The JNK inhibitor, SP600125 suppresses cell migration in RNase L deficient cells………………………………………….……104 Figure 3.7. RNase L interacts with integrin β1 and FAK……….…….……..105-106 Figure 3.8 RNase L could be tyrosine phosphorylated by FAK in vitro…………107 Figure 3. 9 Activation of RNase L by 2-5A abolish cell haptotaxis………….108-109 Figure 3.10 2-5A transfection promote integrins migrate to plasma membrane…..110 Figure 3.11 RNase L suppressed integrin induced FAN and JNK activation……..111 CHAPTER 4 Figure 4.1 PMA stimulated RNase L phosphorylation………………………..….122 Figure
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