The role of the Kinin-Kallikrein System in chronic lymphocytic leukaemia

ii Table of Content.iv List of Tables.xvii List of Figures.xix List of Appendices.xxv List of Publications.xxvi Acknowledgements.xxvii Declaration . xxviii List of Abbreviations.xxix Chapter 1. Understanding Chronic Lymphocytic Leukaemia.2 1.1 General Introduction to Chronic Lymphocytic Leukaemia . 2 1.2 Epidemiology of Chronic Lymphocytic Leukaemia . 2 1.3 An Introduction to Clinical Aspects of Chronic Lymphocytic Leukaemia. 5 1.3.1 Clinical Presentation of Chronic Lymphocytic Leukaemia . 5 1.3.2 Criteria for diagnosis . 6 1.3.3 Morphology of CLL B-lymphocytes . 8 1.3.4 Clinical staging in Chronic Lymphocytic Leukaemia . 10 1.3.5 Treatment of Chronic Lymphocytic Leukaemia . 12 1.3.5.1 Traditional Therapeutic Strategies in CLL Outlined in the 2008 IWCLL Guidelines . 12 1.3.5.2 Novel Therapeutic Agents . 18 The Role of the Kinin-Kallikrein System in Chronic Lymphocytic Leukaemia. April 2014 Elena Kashuba Page v 1.4 Aetiology of Chronic Lymphocytic Leukaemia . 21 1.5 Pathogenesis of Chronic Lymphocytic Leukaemia . 23 1.5.1 The Origin and Development of Chronic Lymphocytic Leukaemia. 23 1.5.1.1 B-Cell Receptor Complex . 25 1.5.1.2 Somatic Hypermutation and Class Switch Recombination Processes During Normal B cell Development and Their Contribution to B-CLL pathogenesis . 27 1.5.1.2.1 Normal B cell Development. 27 1.5.1.2.2 Somatic Hypermutation . 31 1.5.1.2.3 Class Switch Recombination . 32 1.5.1.2.4 The Contribution to B-CLL Pathogenesis . 33 1.5.2 BCR-Mediated Signalling in Normal B-lymphocyte . 36 1.5. 3 Molecular and Cellular Biology of CLL . 40 1.5. 3.1 The Role of the Microenvironment in Pathophysiology of Chronic Lymphocytic Leukaemia . 40 1.5. 3.1.1 Tumour-Microenvironment Proliferative Events . 40 1.5. 3.1.2 Microenvironmental anti-Apoptotic and pro-Survival Signalling . 41 1.5. 3.1.3 Cell Trafficking . 43 1.5. 3.2 BCR-associated signalling in Chronic Lymphocytic Leukaemia . 45 Chapter 2. Predicting and Defining Chronic Lymphocytic Leukaemia Clinical Outcomes. 50 2.1 Prognostic and Predictive Factors in Chronic Lymphocytic Leukaemia. 51 2.1.1 Traditional Clinical Prognostic Markers . 51 The Role of the Kinin-Kallikrein System in Chronic Lymphocytic Leukaemia. April 2014 Elena Kashuba Page vi 2.1.2 New Prognostic Biomarkers . 52 2.1.2.1 Response to Therapy and Duration of Response . 53 2.1.2.2 Minimal Residual Disease . 55 2.1.2.3 Performance Status (Section 1.3.5.1) . 55 2.1.2.4 Chromosome and Gene Associated Biomarkers . 56 2.1.2.4.1 Immunoglobulin Gene Mutation Status (IgVH Status). 56 2.1.2.4.2 Genetic Aberrations and Cytogenetic Parameters . 59 2.1.2.5 Cytokines and Soluble Molecules . 62 2.1.2.5.1 Beta2-Microglobulin (β2M). 62 2.1.2.6 Cellular Expression-Associated Biomarkers . 63 2.1.2.6. 1 CD38 as a Prognostic Marker . 63 2.1.2.6. 2 ZAP-70 as a Prognostic Factor . 64 2.2 The identification of novel candidate biomarkers . 66 2.2.1 Biomarker Discovery . 67 2.2.2 The study of the proteome . 69 2.2.2.1 Proteomics Pipeline for Biomarker Discovery. 69 2.2.3 Applying Proteomics as a Tool to Identify Novel Prognostic Biomarkers in Chronic Lymphocytic Leukaemia . 71 2.2.3.1 A Proteomic Approach to Investigate Protein Targets Associated with B Cell Receptor Signalling in Good and Poor Prognosis Chronic Lymphocytic Leukaemia Samples . 72 The Role of the Kinin-Kallikrein System in Chronic Lymphocytic Leukaemia. April 2014 Elena Kashuba Page vii Chapter 3. The Kinin-Kallikrein System: Physiological Roles, Pathophysiology and Its Relationship to Cancer.75 3.1 The Kinin-Kallikrein System: The Introduction . 75 3.2 The Contact System and the Mechanism of Activation . 77 3.2.1 Factor XII and Factor XI . 77 3.3 The Structure and Organisation of the Kinin-Kallikrein System . 81 3.3.1 Enzymes . 82 3.3.1.1 Kinin Forming Enzymes . 82 3.3.1.2 Carboxypeptidases . 84 3.3.1.3 Angiotensin Converting Enzyme . 86 3.3.1.4 Neutral Endopeptidase . 87 3.3.2 Large Proteins. 87 3.3.2.1 Kininogens. 87 3.3.2.1.1 Protein Structure and Function . 89 3.3.3 Polypeptides . 96 3.3.3.1 Kinins . 96 3.3.3.2 Kinin Receptors . 97 3.3.4 Assembly and Activation of KKS . 98 3.4 KKS: Pathophysiology and Dysregulation . 101 3.4.1 Physiology and Pathophysiology of KKS . 101 The Role of the Kinin-Kallikrein System in Chronic Lymphocytic Leukaemia. April 2014 Elena Kashuba Page viii 3.4.2 Kinin-Kallikrein System: The Implications in Carcinogenesis . 107 3.4.2.1 Kallikreins: The Role and Association with Carcinogenesis . 108 3.4.2.2 Kininogens: Implications in Cancer . 110 3.4.2.3 The Involvement of Kinins and Kinin Receptors in Tumourigenesis . 112 3.5 Conclusions . 114 3.6 Project Aims and Objectives . 114 Chapter 4. Materials and Methods.119 4.1 Cell Culture . 119 4.2 Thawing of Cryopreserved Cells . 119 4.2.1 Cells Growth and Culturing . 121 4.2.2 Manual Cell Counting . 122 4.2.3 Cryopreservation of cultured cells . 123 4.3 Clinical Samples Collections and Characteristics . 124 4.3.1 Isolation of Leukocytes from whole blood samples by density gradient centrifugation .