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Mechanisms of Angiostatin Formation by Tumour Cells Mechanisms of Angiostatin Formation by Tumour Cells ANGELINA JAP LAY A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of New South Wales Australia, 2001 TABLE OF CONTENTS TABLE OF CONTENTS LIST OF FIGURES viii LIST OF TABLES xi LIST OF ABBREVIATIONS xii LIST OF PUBLICATIONS xiv STATEMENT xv ACKNOWLEDGMENTS xvi DEDICATION xvii SUMMARY xviii CHAPTER 1 LITERATURE REVIEW 1.1 ANGIOGENESIS 1.1.1 Introduction. 1 1 . .21 Role of endothelial cells in normal physiology . 4 1.1.3 Angiogenesis a cascade of events . 5 1.1.3.1 The extracellular matrix remodelling. 6 1.1.3.1.1 Metalloproteinases in angiogenesis.................. 6 1.1.3.1.2 Plasminogen activator (PA)-system in angiogenesis... 7 1.1.3.2 Initiation of angiogenic cascade. 9 1.1.3.3 Endothelial cell proliferation and migration . .10 1.1.3.4 Maturation and stabilisation of the neovasculature......... 11 1. 1.4 Control of angiogenesis the balance hypothesis . .12 1.1.4.1 Angiogenic stimulators.................................. 14 1.1.4.1.1 Direct angiogenic inducers.......................... 14 1.1.4.1.2 Indirect angiogenic inducers . .15 . 1.1.4.2 Angiogenic inhibitors. .16 . 1.1.5 Triggers of angiogenic response . .17 . 1.1.5.1 Hypoxia................................................ 18 1.1.5.2 Mechanical injury. 19. 1.1.5.3 Inflammation . .19 . 1.1.5.4 Genetic factors . .19 . 1.1.6 Tumour angiogenesis . 20. 1.1.7 Antiangiogenic therapy...................................... 22 1.2 PLASMINOGEN/PLASMIN SYSTEM 1.2.1 Introduction................................................. 24 1.2.2 Structure . .24 . 1.2.3 Kring le domains of plasminogen . 27. 1.2.4 Variants of plasminogen..................................... 28 1.2.4.1 Glu-plasminogen . .28 . 1.2.4.2 Lys-plasminogen . .28 . 1.2.5 Plasminogen activation. 30. 1.2.5.1 Plasminogen activators . .31 . 1.2.5.1.1 Tissue-type plasminogen activator (tPA) . 32 1.2.5.1.2 Urokinase plasminogen activator (uPA) . .33 1.2.5.1.3 Urokinase plasminogen activator receptor (uPAR) . 35 1.2.5.2 Plasminogen activator inhibitors (PAI) . .36 . 1.2.5.2.1 Plasminogen activator inhibitor 1 (PAl1).............. 36 1.2.5.2.2 Plasminogen activator inhibitor 2 (PAl2). 37 1.2.5.2.3 Protease-nexin 1 (PN-1) . .38 . 1.2.5.2.4 Alpha 2-antiplasmin . .38 . 1.2.5.3 Plasminogen receptors.................................. 40 1.3 ANGIOSTATIN 1.3.1 Introduction................................................. 42 1.3.2 Structure.................................................. 43 1.3.3 Angiostatin converting activity of various enzymes............. 43 1.3.4 Function. .45 . 1.3.5 Inhibitory activity of kringle domains of angiostatin . .46 1.3.6 Role of kringle 5 plasminogen................................ 48 1.3.7 Mechanism of action . 48. 1.4 PHOSPHOGL YCERATE KINASE (PGK) 1.4.1 Introduction................................................. 51 1.4.2 Role of PGK in glycolysis . 51. 1.4.3 Structure of PGK. .53 . 1.4.3.1 The 3-Dimensional structure of PGK . .54 . 1.4.4 Domain movement in PGK. 56. ii 1.4.5 PGK isoenzyme . 57. 1.4.6 Phosphoglycerate kinase in diseases. 58. CHAPTER 2 HYPOTHESIS AND AIMS 2.1 HYPOTHESISANDAIMS ....................................... 61 CHAPTER 3-CHARACTERISATION OF PLASMIN REDUCTASE 3.1 INTRODUCTION. .59 . 3.2 MATERIALS AND METHODS.................................... 62 3.2.1 Cell culture . .62 . 3.2.1.1 HT1080 cell culture . .. .. ... .. 62 3.2.1.2 Breast carcinoma cell culture . 63. 3.2.1.3 Preparation of conditioned media (CM) . .63 3.2.2 Assay for plasmin reductase activity . .63 . 3.2.2.1 Angiostatin generation from HT1080 CM . .64 3.2.2.2 MPB blot assay.. ... .. ... .65 3.2.2.3 Microtitre plate assay . 65. 3.2.3 Endogenous glutathione in HT1080CM . 66. 3.2.4 Competitive inhibition of plasmin reductase activity by NADH . 67 3.2.4.1 Binding of plasmin reductase to Cibachron Blue-Sepharose 67 3.2.5 Alkylation of thiol groups in plasmin reductase. 67 3.2.5.1 Binding of plasmin reductase to thiol activated sepharose.. 68 3.3 RESULTS .............................. _. .69 . 3.3.1 Elisa for determining plasmin reductase activity . 69 3.3.2 Streptavidin-HRP blot for MPS-labelled angiostatin . 71 3.3.3 Plasmin reductase activity secreted by various cultured cells . 72 3.3.4 Effect of NADH on plasmin reductase activity . .73 3.3.4.1 Plasmin reductase bound to Cibachron Blue-Sepharose... 74 3.3.5 Thiol modifying reagents reduced plasmin reductase activity . 75 3.3.6 Plasmin reductase bound to thiol activated sepharose . 76 3.4 DISCUSSION. 77. iii CHAPTER 4 PLASMIN REDUCTASE PURIFICATION 4.1 INTRODUCTION. 80. 4.2 MATERIALS AND METHODS.................................... 81 4.2.1 Large scale production of CM................................ 81 4.2.1.1 Cell factory............................................. 81 4.2.2 Quantitative analysis of protein............................... 81 4.2.3 ELISA and Streptavidin-HRP blot for plasmin reductase activity. 82 4.2.4 Concentration of HT1080 CM................................ 82 4.2.5 8Iue-Sepharose CL-68 column chromatography . 82 4.2.6 Thiopropyl-Sepharose 68 chromatography. 83. 4.2. 7 Gel filtration (Sephacryl S-200 HR) . 84. 4.2.8 NHi-terminal sequence analysis . 84. 4.3 RESULTS . 86. 4.3.1 Cibachron 8Iue-Sepharose 48 chromatography . .86 4.3.2 Thiopropyl-Sepharose 68 chromatography. 88. 4.3.3 Sephacryl S-200 HR gel filtration. .89 . 4.3.4 Protein microsequencing . .91 . 4.3.4.1 Amino acid sequence of human PGK. 92. 4.3.5 Stages of plasmin reductase purification . .93 . 4.4 DISCUSSION. .94 . CHAPTER 5 CLONING AND EXPRESSION OF RECOMBINANT HUMAN PGK (rhPGK) 5.1 INTRODUCTION. .96 . 5.2 MATERIALS AND METHODS. .97 . 5.2.1 Enzymes and reagents ..................... : . .97 5.2.2 Cellular mRNA extraction. .97 . 5.2.3 Construction of human PGK cDNA . 98. 5.2.3.1 PCR reaction. .98 . iv 5.2.3.2 cDNA and plasmid vector digestion . 99 5.2.3.2.1 cDNA vector digestion . 99 5.2.3.2.2 Plasmid vector digestion . .. .. 99 5.2.3.3 Ligation reaction. 99 5.2.3.4 Transformation . 100 5.2.4 Expression of rhPGK. 100 5.2.4.1 Analysis of rhPGK expression . .. .. .. .. 100 5.2.4.2 Large scale production of rhPGK. 101 5.2.5 Purification of rhPGK. .. .. .. .. .. .. 101 5.2.5.1 Ammonium sulfate precipitation. 101 5.2.5.2 Cibachron Blue-Sepharose . .. .. .. .. 102 5.2.5.3 S-Sepharose chromatography . 102 5.2.6 Mass spectrometry. 102 5.2.7 ELISA for reductase activity of rhPGK ........................ 103 5.3 RESULTS . 102 5.3.1 cDNA sequences of rhPGK. 102 5.3.2 rhPGK purification . .. .. .. .. .. 103 5.3.3 Mass spectrometry of recombinant human PGK. 104 5.3.4 Plasmin reductase activity of rhPGK. 105 5. 3.5 Comparison of plasmin reductase activity of various PGK . 106 5.4 DISCUSSION. 107 CHAPTER 6 IN VIVO STUDY OF rhPGK 6.1 INTRODUCTION. 109 6.2 MATERIALS AND METHODS .................................... 110 6.2.1 Cell culture . 110.
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