by Tung Kai Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 152-166). / Abstracts in English and Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABBREVIATIONS --- p.iv / ABSTRACT --- p.vii / 撮要 --- p.x / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- AN OVERVIEW OF THE IMMUNE SYSTEM --- p.1 / Chapter 1.1.1 --- Innate Immunity --- p.1 / Chapter 1.1.2 --- Specific Acquired Immunity --- p.4 / Chapter 1.2 --- IMMUNE RESPONSE TO TUMOURS --- p.7 / Chapter 1.2.1 --- Specific Mechanisms --- p.7 / Chapter 1.2.1.1 --- Cellular Immune Response --- p.7 / Chapter 1.2.1.2 --- Humoral Immune Response --- p.9 / Chapter 1.2.2 --- Non-specific Mechanism --- p.10 / Chapter 1.2.2.1 --- Natural Killer Cells --- p.10 / Chapter 1.2.2.2 --- Lymphokine-activated Cells --- p.11 / Chapter 1.2.2.3 --- Tumour Infiltrating Lymphocytes --- p.12 / Chapter 1.2.2.4 --- Macrophages --- p.13 / Chapter 1.2.2.5 --- Other Cell Types Mediating Non-specific Immunity --- p.14 / Chapter 1.3 --- PHYTOCHEMICALS AS POTENTIAL IMMUNOMODULATORS and anti-tumour agents --- p.15 / Chapter 1.3.1 --- Modulation of the Immune System --- p.15 / Chapter 1.3.2 --- Induction of Cancer Cell Differentiation --- p.16 / Chapter 1.3.3 --- Stimulation of Apoptosis --- p.17 / Chapter 1.3.4 --- Other Anti-tumour Mechanisms --- p.18 / Chapter 1.4 --- GREEN TEA: GENERAL PROPERTIES AND PHARMACO- logical activities --- p.19 / Chapter 1.4.1 --- General Introduction --- p.19 / Chapter 1.4.2 --- Chemistry of Tea --- p.21 / Chapter 1.4.3 --- Physiological and Pharmacological Effects of Green Tea Catechins --- p.26 / Chapter 1.4.3.1 --- Anti-oxidative Activity --- p.26 / Chapter 1.4.3.2 --- Anti-mutagenic Activity --- p.27 / Chapter 1.4.3.3 --- Anti-carcinogenic Activity --- p.27 / Chapter 1.4.3.4 --- Anti-inflammatory Activity --- p.32 / Chapter 1.4.3.5 --- Hypocholesterolemic and Hypolipidemic Activities --- p.32 / Chapter 1.4.3.6 --- Anti-microbial Activity --- p.33 / Chapter 1.5 --- aims and scopes of this investigation --- p.34 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- MATERIALS --- p.36 / Chapter 2.1.1 --- Animals --- p.36 / Chapter 2.1.2 --- Cell lines --- p.36 / Chapter 2.1.3 --- Green Tea Catechins and Epicatechin Isomers --- p.38 / Chapter 2.1.4 --- Recombinant Murine Interleukin-2 (rmIL-2) --- p.38 / Chapter 2.1.5 --- Antibodies --- p.39 / Chapter 2.1.6 --- "Buffers, Culture Medium and Other Reagents" --- p.40 / Chapter 2.1.7 --- Reagents for Gel Electrophoresis --- p.47 / Chapter 2.1.8 --- Radioisotopes --- p.48 / Chapter 2.2 --- METHODS --- p.49 / Chapter 2.2.1 --- "Isolation, Purification and Characterization of Green Tea Catechins and Epicatechin Isomers" --- p.49 / Chapter 2.2.1.1 --- Extraction of Green Tea Catechins --- p.49 / Chapter 2.2.1.2 --- Analysis of Epicatechin Isomers by HPLC --- p.49 / Chapter 2.2.1.3 --- Determination of the Bio-toxicity of Green Tea Catechins --- p.50 / Chapter 2.2.1.4 --- Assay of In Vitro Cytotoxicity of Green Tea Catechins on Splenocytes --- p.51 / Chapter 2.2.2 --- Assays for the Immunomodulatory Activities of Green Tea Catechins --- p.52 / Chapter 2.2.2.1 --- Isolation and Preparation of Cells --- p.52 / Chapter 2.2.2.2 --- In Vitro Lymphocyte Transformation Assay --- p.53 / Chapter 2.2.2.3 --- Mixed Lymphocyte Culture --- p.53 / Chapter 2.2.2.4 --- Colorimetric Assay of Alloreactive Cytotoxic T Lymphocytes --- p.54 / Chapter 2.2.2.5 --- Foodpad Swelling Assay of Delayed-type Hypersensitivity --- p.55 / Chapter 2.2.2.6 --- Haemagglutination Assay of In Vivo Antibody Formation --- p.56 / Chapter 2.2.2.7 --- Characterization of the Lymphocyte Subpopulations from Spleen by Flow Cytometry --- p.56 / Chapter 2.2.2.8 --- In Vivo Migration of Macrophages --- p.57 / Chapter 2.2.2.9 --- In Vitro Assay of Phagocytic Activity of PEC --- p.58 / Chapter 2.2.2.10 --- In Vitro Assay of Macrophage-mediated Cytostatic Activity --- p.58 / Chapter 2.2.3 --- Assays for the Anti-tumour Activities of Green Tea Catechins --- p.60 / Chapter 2.2.3.1 --- Assay of In Vivo Anti-tumour Activity --- p.60 / Chapter 2.2.3.2 --- Induction and Assay of Natural Killer Cell Activity --- p.60 / Chapter 2.2.3.3 --- Induction and Assay of Lymphokine-activated Killer Cell Activity --- p.61 / Chapter 2.2.3.4 --- Assay of In Vitro Tumour Cell Proliferation --- p.62 / Chapter 2.2.3.5 --- In Vitro Tumour Clonogenicity Assay --- p.63 / Chapter 2.2.3.6 --- Induction of Myeloid Leukemic Cell Differentiation --- p.63 / Chapter 2.2.3.7 --- DNA Fragmentation Analysis --- p.64 / Chapter 2.2.3.8 --- Cell Cycle and DNA Content Evaluation --- p.66 / Chapter 2.2.4 --- Statistical Analysis --- p.66 / Chapter CHAPTER 3: --- "Extraction, Purification and Characterization of GTCs" / Chapter 3.1 --- INTRODUCTION --- p.67 / Chapter 3.2 --- RESULTS --- p.69 / Chapter 3.2.1 --- "Extraction of Catechins from the Chinese Green Tea, Ji Pin Long Jing" --- p.69 / Chapter 3.2.2 --- Analysis of Epicatechin Isomers by HPLC --- p.69 / Chapter 3.2.3 --- Bio-toxicity Determination by Brine Shrimp Bioassay --- p.71 / Chapter 3.2.4 --- Effect of GTCs on the Viability of Murine Splenocytes --- p.72 / Chapter 3.3 --- DISCUSSION --- p.74 / Chapter CHAPTER 4: --- The Immunomodulatory Activities of GTCs / Chapter 4.1 --- INTRODUCTION --- p.75 / Chapter 4.2 --- RESULTS --- p.77 / Chapter 4.2.1 --- Effects of GTCs on the In Vitro Proliferation of Murine Splenocytes --- p.77 / Chapter 4.2.2 --- In Vitro Co-mitogenic Effect of GTCs on Murine Splenocytes --- p.77 / Chapter 4.2.3 --- Stimulation of Lymphocyte Proliferation In Vitro by Intraperitoneal Administration of GTCs --- p.81 / Chapter 4.2.4 --- Effect of In Vivo Treatment of GTCs on In Vitro Mixed Lymphocyte Reaction --- p.81 / Chapter 4.2.5 --- Effect of In Vivo Administration of GTCs on Splenic Lymphocytes Subpopulations --- p.87 / Chapter 4.2.6 --- Effect of In Vivo Administration of GTCs on the Induction of Alloreactive Cytotoxic T Lymphocytes --- p.87 / Chapter 4.2.7 --- In Vivo Induction of Delayed-type Hypersensitivity (DTH) Response to Sheep Red Blood Cells (SRBC) by GTCs --- p.91 / Chapter 4.2.8 --- Primary Humoral Immune Response to SRBC in GTCs- treated Mice --- p.91 / Chapter 4.2.9 --- Effect of GTCs on In Vivo Migration of Macrophages --- p.95 / Chapter 4.2.10 --- Effect of GTCs on the Phagocytic Activity of Thioglycollate- elicited Macrophages In Vitro --- p.95 / Chapter 4.2.11 --- Effect of In Vivo Administration of GTCs on Phagocytic Activity of Thioglycollate-elicited Macrophages In Vitro --- p.98 / Chapter 4.2.12 --- Effect of GTCs on In Vitro Cytostatic Activity of Picolinic Acid-activated Macrophages --- p.98 / Chapter 4.2.13 --- Effect of In Vivo Administration of GTCs on the Cytostatic Activity of Picolinic Acid-activated Macrophages --- p.101 / Chapter 4.3 --- DISCUSSION --- p.103 / Chapter CHAPTER 5: --- THE ANTI-TUMOUR ACTIVITIES OF GTCs / Chapter 5.1 --- introduction --- p.107 / Chapter 5.2 --- RESULTS --- p.109 / Chapter 5.2.1 --- Effects of GTCs on the Growth of Transplantable Tumour Cells In Vivo --- p.109 / Chapter 5.2.2 --- Effect of GTCs on In Vivo Induction of Natural Killer Cells --- p.109 / Chapter 5.2.3 --- Effect of GTCs on In Vitro Induction of Lymphokine- activated Killer Cell Activity --- p.113 / Chapter 5.2.4 --- In Vitro Cytotoxicity of Green Tea Epicatechin Isomers on Murine and Human Tumour Cell Lines --- p.115 / Chapter 5.2.5 --- In Vitro Cytostatic Effect of EGCG on Various Tumour Cell Lines --- p.118 / Chapter 5.2.6 --- Effect of EGCG on the In Vitro Clonogenicity of Myeloid Leukemia Cells --- p.127 / Chapter 5.2.7 --- Induction of Apoptosis of Myeloid Leukemia HL-60 Cells In Vitro --- p.130 / Chapter 5.2.8 --- Effect of EGCG on the Cell Cycle Kinetics of Myeloid Leukemia HL-60 Cells In Vitro --- p.131 / Chapter 5.2.9 --- Effect of EGCG on the Morphological Changes of Myeloid Leukemia Cells --- p.136 / Chapter 5.2.10 --- Effect of EGCG on the Endocytic Activity of Myeloid Leukemia Cells --- p.136 / Chapter 5.3 --- discussion --- p.141 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPEC- TIVES --- p.145 / REFERENCES --- p.152

by Ip Wai-Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 213-233). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / 撮要 --- p.x / TABLE OF CONTENTS --- p.xiv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- Introduction to Hematopoiesis --- p.1 / Chapter 1.1.2 --- Cytokines in Hematopoiesis --- p.4 / Chapter 1.2 --- Leukemia --- p.6 / Chapter 1.2.1 --- Leukemia: Abnormalities in Blood Cell Formation --- p.6 / Chapter 1.2.2 --- Classification of Leukemia --- p.8 / Chapter 1.2.3 --- The Causes and Molecular Basis of Leukemia --- p.8 / Chapter 1.2.4 --- Therapy of Leukemia --- p.11 / Chapter 1.2.5 --- Control of Leukemia by Hematopoietic Growth Factors and Other Compounds --- p.12 / Chapter 1.2.6 --- Molecular Control of Apoptosis and Cell Cycle in Leukemia --- p.13 / Chapter 1.2.6.1 --- Regulation of Cell Cycle and Apoptosis by Genes and Regulatory Proteins --- p.14 / Chapter 1.2.6.1.1 --- Cell Cycle --- p.14 / Chapter 1.2.6.1.2 --- Apoptosis --- p.15 / Chapter 1.2.6.2 --- Role of Apoptosis and Cell Cycle in the Development of Leukemia --- p.17 / Chapter 1.3 --- Green Tea --- p.19 / Chapter 1.3.1 --- Origin and Cultivation of Tea Plants --- p.19 / Chapter 1.3.2 --- Classification and Manufacturing of Tea --- p.21 / Chapter 1.3.3 --- The Chemistry of Tea --- p.22 / Chapter 1.3.3.1 --- Chemical Composition of Tea --- p.22 / Chapter 1.3.3.2 --- Separation and Purification of Green Tea Polyphenols --- p.27 / Chapter 1.3.3.3 --- The Chemical Properties of Green Tea Polyphenols --- p.28 / Chapter 1.3.4 --- Bioavailability and Pharmacokinetic of Green Tea Epicatechins --- p.28 / Chapter 1.3.4.1 --- Human Studies --- p.29 / Chapter 1.3.4.2 --- Animal Studies --- p.30 / Chapter 1.3.5 --- Physiological and Pharmacological Activities of Green Tea Catechins --- p.31 / Chapter 1.3.5.1 --- Anti-oxidative Activity --- p.32 / Chapter 1.3.5.2 --- Hypocholesterolemic and Hypolipidemic Activity --- p.33 / Chapter 1.3.5.3 --- Anti-inflammatory Activity --- p.34 / Chapter 1.3.5.4 --- Anti-microbial Activity --- p.35 / Chapter 1.3.5.5 --- Anti-mutagenic Activity --- p.36 / Chapter 1.3.5.6 --- Anti-carcinogenesis --- p.37 / Chapter 1.3.5.7 --- Direct Anti-tumor Activity --- p.41 / Chapter 1.3.5.8 --- Modulating Activity in Endocrine System --- p.43 / Chapter 1.3.5.9 --- Other Biological Activities --- p.43 / Chapter 1.3.6 --- Possible Anti-cancer Mechanisms of Green Tea Epicatechins --- p.44 / Chapter 1.3.6.1 --- Modulation of Anti-tumor Immunity --- p.44 / Chapter 1.3.6.2 --- Direct Growth Inhibition by Controlling the Signal Transduction Pathways --- p.45 / Chapter 1.3.6.3 --- Induction of Apoptosis and Cell Cycle Arrest --- p.46 / Chapter 1.3.6.4 --- Inhibition of Tumor Metastasis --- p.47 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.48 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.50 / Chapter 2.1.1 --- Animals --- p.50 / Chapter 2.1.2 --- Cell Lines --- p.50 / Chapter 2.1.3 --- Sheep Red Blood Cells (SRBC) --- p.52 / Chapter 2.1.4 --- "Cell Culture Medium, Buffers and Reagents" --- p.52 / Chapter 2.1.5 --- Tea Extracts and Green Tea Epicatechins --- p.56 / Chapter 2.1.6 --- Recombinant Cytokines --- p.57 / Chapter 2.1.7 --- Vitamin Analogs --- p.59 / Chapter 2.1.8 --- Taxol (Baccatin III N-benzoyl-β-phenyllisoserine ester) --- p.59 / Chapter 2.1.9 --- 18β-Glycyrrhetinic Acid (18β-GA) --- p.60 / Chapter 2.1.10 --- [methyl-3H] Thymidine (3H-TdR) --- p.60 / Chapter 2.1.11 --- Liquid Scintillation Cocktail --- p.60 / Chapter 2.1.12 --- Reagents and Buffers for Flow Cytometery --- p.61 / Chapter 2.1.13 --- Reagents for DNA Extraction --- p.62 / Chapter 2.1.14 --- Reagents for Total RNA Isolation --- p.63 / Chapter 2.1.15 --- Reagents and Buffers for RT-PCR Study --- p.64 / Chapter 2.1.16 --- Reagents and Buffers for Gel Electrophoresis --- p.67 / Chapter 2.1.17 --- Reagents and Buffers for Western Blot Analysis --- p.68 / Chapter 2.2 --- Methods --- p.77 / Chapter 2.2.1 --- Culture of the Leukemic Cell Lines --- p.77 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Primary Mouse Cells" --- p.77 / Chapter 2.2.3 --- Determination of Cell Viability --- p.78 / Chapter 2.2.4 --- [3H]-TdR Incorporation Assay --- p.79 / Chapter 2.2.5 --- Cell Morphology Study --- p.79 / Chapter 2.2.6 --- Apoptosis Study --- p.80 / Chapter 2.2.7 --- Animal Studies --- p.81 / Chapter 2.2.8 --- Gene Expression Study --- p.82 / Chapter 2.2.9 --- Protein Expression Study --- p.85 / Chapter 2.2.10 --- Statistical Analysis --- p.88 / Chapter CHAPTER 3: --- THE ANTI-TUMOR ACTIVITIES OF TEA EXTRACTS AND PURIFIED GREEN TEA EPICATECHIN ISOMERS ON VARIOUS LEUKEMIC CELL LINES / Chapter 3.1 --- Introduction --- p.89 / Chapter 3.2 --- Results --- p.91 / Chapter 3.2.1 --- The Effects of Tea Extracts on Various Leukemia Cells --- p.91 / Chapter 3.2.1.1 --- Differential Anti-proliferative Effect of Different Tea Extracts on Various Leukemic Cell Lines In Vitro --- p.91 / Chapter 3.2.1.2 --- Differential Cytotoxic Effect of Different Tea Extracts on the Murine Lymphocytic Leukemia L1210 Cells In Vitro --- p.92 / Chapter 3.2.1.3 --- Induction of Apoptosis in HL-60 Cells by Different Tea Extracts In Vitro --- p.92 / Chapter 3.2.2 --- The Effects of Purified Green Tea Epicatechin Isomers on Various Leukemic Cell Lines --- p.101 / Chapter 3.2.2.1 --- In Vitro Anti-proliferative Effect of Green Tea Epicatechin Isomers on Various Human and Murine Leukemic Cell Lines --- p.101 / Chapter 3.2.2.2 --- In Vitro Cytotoxic Effect of Green Tea Epicatechin Isomers on Various Human and Murine Leukemic Cell Lines --- p.117 / Chapter 3.2.2.3 --- Effects of Green Tea Epicatechin Isomers on the Differentiation of Myeloid Leukemia Cells --- p.131 / Chapter 3.2.2.4 --- Apoptosis-Inducing Effect of Different Green Tea Epicatechin Isomers on HL-60 and JCS Cells --- p.134 / Chapter 3.2.2.5 --- Effect of EGCG on the In Vivo Tumorigenicity of Leukemia JCS and L1210 Cells --- p.142 / Chapter 3.3 --- Discussion --- p.144 / Chapter CHAPTER 4: --- MECHANISTIC STUDIES ON THE ANTI PROLIFERATIVE AND APOPTOSIS-INDUCING ACTIVITIES OF GREEN TEA EPICATECHIN ISOMERS ON LEUKEMIA CELLS / Chapter 4.1 --- Introduction --- p.149 / Chapter 4.2 --- Results --- p.152 / Chapter 4.2.1 --- Combining Effect of EGCG and Physiological Differentiation Inducers on the Proliferation of HL-60 and JCS Cells --- p.152 / Chapter 4.2.2 --- Combining Effect of EGCG and Cytokines on the Proliferation of JCS Cells --- p.155 / Chapter 4.2.3 --- Combining Effect ofEGCG and Other Phytochemicals on the Proliferation of HL-60 and JCS Cells --- p.161 / Chapter 4.2.4 --- Modulatory Effect of EGCG on the Expression of Apoptosis-regulatory Genes in HL-60 Cells --- p.168 / Chapter 4.2.5 --- Modulatory Effect of EGCG on the Expression of Growth-related and Apoptosis-regulatory Proteins in HL-60 Cells --- p.170 / Chapter 4.3 --- Discussion --- p.177 / Chapter CHAPTER 5: --- EFFECTS OF GREEN TEA EPICATECHIN ISOMERS ON THE GROWTH AND DIFFERENTIATION OF MURINE HEMATOPOIETIC CELLS / Chapter 5.1 --- Introduction --- p.184 / Chapter 5.2 --- Results --- p.186 / Chapter 5.2.1 --- In Vitro Effects of EGCG on Murine Lymphocytes --- p.186 / Chapter 5.2.1.1 --- In Vitro Effect of EGCG on the Proliferation of Murine Splenocytes --- p.186 / Chapter 5.2.1.2 --- In Vitro Effect of EGCG on the Mitogen-induced Proliferation of Murine Splenocytes --- p.186 / Chapter 5.2.1.3 --- Cytotoxic Effect of EGCG on Murine Lymphocytes --- p.189 / Chapter 5.2.2 --- Primary Humoral Immune Response to SRBCin EGCG-treated Mice --- p.191 / Chapter 5.2.3 --- In Vitro Studies of the Effects of EGCG on Murine Bone Marrow Cells --- p.192 / Chapter 5.2.3.1 --- Effects of EGCG on the In Vitro Proliferation of Murine Bone Marrow Cells --- p.192 / Chapter 5.2.3.2 --- The Combining Effect of EGCG and Growth Factors on the In Vitro Proliferation of Murine Bone Marrow Cells --- p.192 / Chapter 5.2.3.3 --- In Vitro Cytotoxic Effect of EGCG on Murine Bone Marrow Cells --- p.196 / Chapter 5.2.4 --- Effect of EGCG on the Differentiation of Murine Bone Marrow Cells --- p.199 / Chapter 5.2.5 --- Combining Effects of EGCG and Growth Factors on the Morphology of Murine Bone Marrow Cells --- p.199 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.207 / REFERENCES --- p.213