by Chan Hoi Tak. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 129-137). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Abbreviations --- p.vi / Contents --- p.viii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Aflatoxins --- p.1 / Chapter 1.1.1 --- AFB1 Metabolism --- p.5 / Chapter 1.1.2 --- Bioactivation --- p.5 / Chapter 1.1.3 --- Detoxification --- p.8 / Chapter 1.1.4 --- Toxicity of AFB1 --- p.9 / Chapter 1.2 --- Licorice Plants --- p.11 / Chapter 1.2.1 --- Glycyrrhizic Acid (GA) --- p.13 / Chapter 1.2.2 --- Metabolism of GA --- p.16 / Chapter 1.2.3 --- Adverse Effects of GA --- p.19 / Chapter 1.3 --- Aim of Research --- p.19 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.21 / Chapter 2.1 --- Cell cultures --- p.21 / Chapter 2.1.1 --- Isolation of splenocytes --- p.21 / Chapter 2.1.2 --- Culture of cell lines --- p.22 / Chapter 2.1.3 --- Trypsinziation of cells --- p.22 / Chapter 2.2 --- Preparation of drugs --- p.23 / Chapter 2.2.1 --- Preparation of commercially available chemicals for cell culture --- p.23 / Chapter 2.2.2 --- Preparation of Licorice extracts for cell culture --- p.23 / Chapter 2.2.3 --- Preparation of chemicals for enzymatic reaction --- p.24 / Chapter 2.3 --- Cytotoxicity assay Methods --- p.24 / Chapter 2.3.1 --- MTT assay --- p.25 / Chapter 2.3.2 --- Neutral red assay --- p.25 / Chapter 2.4 --- Cytotoxicity Assay --- p.32 / Chapter 2.5 --- Chemoprotection assay --- p.32 / Chapter 2.5.1 --- Direct addition of drugs --- p.33 / Chapter 2.5.2 --- Pretreatment of cells --- p.33 / Chapter 2.5.3 --- Culture of cells with drugs --- p.34 / Chapter 2.6 --- Preparation of enzymes --- p.36 / Chapter 2.6.1 --- Preparation of cells --- p.36 / Chapter 2.6.2 --- Pretreatment of rat microsomes --- p.36 / Chapter 2.7 --- Bradford Assay --- p.37 / Chapter 2.8 --- Alkoxyresorufin O-Dealkylase assay --- p.39 / Chapter 2.8.1 --- Effects of AFB1 on P450 --- p.44 / Chapter 2.8.2 --- Effects of drugs on P450 --- p.44 / Chapter 2.9 --- Glutathione-S-transferase assay --- p.44 / Chapter 2.9.1 --- GST assay in rat microsomal fractions --- p.48 / Chapter 2.9.1.1 --- Effects of AFB1 on GST --- p.48 / Chapter 2.9.1.2 --- Effects of drugs on GST --- p.48 / Chapter 2.9.2 --- GST assay in crude fractions from cells --- p.48 / Chapter 2.9.2.1 --- Direct addition effects of drugs on GST --- p.48 / Chapter 2.9.2.2 --- Effects of drug-containing medium on GST --- p.49 / RESULTS / Chapter CHAPTER THREE --- CELL CULTURES --- p.50 / Chapter 3.1 --- Cytotoxicity assay --- p.51 / Chapter 3.1.1 --- Cytotoxicity of AFB1 --- p.51 / Chapter 3.1.1.1 --- Cytotoxicity of AFB1 to splenocytes --- p.51 / Chapter 3.1.1.2 --- Cytotoxicity of AFB1 to cell lines --- p.57 / Chapter 3.1.2 --- Cytotoxicity of drugs to cell lines --- p.62 / Chapter 3.2 --- Chemoprotection assay --- p.73 / Chapter 3.2.1 --- Direct addition of drugs --- p.73 / Chapter 3.2.2 --- Pretreatment of cells with drugs --- p.78 / Chapter 3.2.3 --- Culture of cells with drugs --- p.83 / Chapter CHAPTER FOUR --- ENZYMATIC ASSAYS --- p.87 / Chapter 4.1 --- Alkoxyresorufin O-dealkylase assay --- p.88 / Chapter 4.1.1 --- Effects of AFB1 on P450s --- p.88 / Chapter 4.1.2 --- Effects of GA on P450s --- p.92 / Chapter 4.1.3 --- Effects of EX on P450s --- p.95 / Chapter 4.2 --- Glutathione -S- transferase assay --- p.98 / Chapter 4.2.1 --- Effects of drugs on GST in cells --- p.100 / Chapter 4.2.2 --- Effects of AFB1 and drugs on GST from rats --- p.108 / Chapter 4.2.2.1 --- Effects of AFB1 on GST from rats --- p.108 / Chapter 4.2.2.2 --- Effects of drugs on GST from rats --- p.112 / Chapter CHAPTER FIVE --- DISCUSSION --- p.115 / Chapter 5.1 --- Cytotoxicity of AFB1 --- p.118 / Chapter 5.2 --- "Cytotoxicity of EX, GA and AA" --- p.119 / Chapter 5.3 --- Chemoprotection of GA and EX on AFB1 cytotoxicity --- p.121 / Chapter 5.4 --- Effects of GA and EX on enzymes involved in AFB1 metabolism --- p.123 / REFERENCES --- p.129

Tsang Yuen-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 200-218). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.vii / CHINESE ABSTRACT --- p.xi / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.2 --- Role of Cytokines in the Control of Hematopoiesis --- p.4 / Chapter 1.2 --- Leukemia --- p.5 / Chapter 1.2.1 --- Abnormalities in Hematopoietic Cell Development --- p.5 / Chapter 1.2.2 --- Classification of Leukemia --- p.7 / Chapter 1.2.3 --- Etiology and Symptoms of Leukemia --- p.9 / Chapter 1.2.4 --- Therapeutic Strategies for Leukemia --- p.10 / Chapter 1.2.4.1 --- Conventional Therapies --- p.10 / Chapter 1.2.4.2 --- Differentiation Therapy and Induction of Apoptosis in Leukemia --- p.11 / Chapter 1.2.5 --- Regulation of Apoptosis and Cell Cycle Progression --- p.12 / Chapter 1.2.5.1 --- Apoptosis --- p.12 / Chapter 1.2.5.2 --- Cell Cycle --- p.13 / Chapter 1.2.5.3 --- Disregulation of Apoptosis and Cell Cycle Contribute to the Development of Leukemia --- p.14 / Chapter 1.3 --- Licorice --- p.16 / Chapter 1.3.1 --- Chemistry of Licorice --- p.16 / Chapter 1.3.2 --- Pharmacological Activities of Glycyrrhizin and 18-β Glycyrrhetinic Acid --- p.22 / Chapter 1.3.2.1 --- Mineralocorticoid Activity --- p.22 / Chapter 1.3.2.2 --- Anti-inflammatory Effect --- p.23 / Chapter 1.3.2.3 --- Anti-allergic Effect --- p.24 / Chapter 1.3.2.4 --- Enhancement of Immune Response --- p.24 / Chapter 1.3.2.5 --- Anti-hepatotoxic Effects --- p.26 / Chapter 1.3.2.6 --- Anti-viral Activity --- p.27 / Chapter 1.3.2.7 --- Anti-carcinogenic and Anti-tumor Effects --- p.28 / Chapter 1.3.3 --- Other Biological Activities of Licorice --- p.30 / Chapter 1.3.4 --- A 96-kDa Glycyrrhizin-Binding Protein (gb96) --- p.31 / Chapter 1.3.5 --- Evaluation of Health Hazard --- p.32 / Chapter 1.4 --- Aims and Scopes of This Research --- p.34 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- Animals --- p.37 / Chapter 2.1.2 --- Cell Lines --- p.37 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Reagents" --- p.39 / Chapter 2.1.4 --- Recombinant Cytokines --- p.42 / Chapter 2.1.5 --- [methyl-3H] Thymidine (3H-TdR) --- p.43 / Chapter 2.1.6 --- Liquid Scintillation Cocktail --- p.44 / Chapter 2.1.7 --- Reagents and Buffers for Flow Cytometry --- p.44 / Chapter 2.1.8 --- Monoclonal Antibodies --- p.45 / Chapter 2.1.9 --- Reagents for DNA Extraction --- p.47 / Chapter 2.1.10 --- Reagents for Total RNA Isolation --- p.48 / Chapter 2.1.11 --- Reagents and Buffers for RT-PCR Study --- p.49 / Chapter 2.1.12 --- Reagents and Buffers for Gel Electrophoresis --- p.55 / Chapter 2.1.13 --- Reagents and Buffers for Western Blot Analysis --- p.56 / Chapter 2.2 --- Methods --- p.63 / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.63 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Primary Mouse Cells" --- p.63 / Chapter 2.2.3 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.64 / Chapter 2.2.4 --- Determination of Cell Viability --- p.65 / Chapter 2.2.5 --- Cell Morphology Study --- p.66 / Chapter 2.2.6 --- Apoptosis Study by DNA Fragmentation --- p.66 / Chapter 2.2.7 --- Flow Cytometric Analysis --- p.67 / Chapter 2.2.8 --- Cell Cycle/DNA Content Evaluation --- p.68 / Chapter 2.2.9 --- Gene Expression Study --- p.69 / Chapter 2.2.10 --- Protein Expression Study --- p.72 / Chapter 2.2.11 --- Statistical Analysis --- p.75 / Chapter CHAPTER 3: --- THE ANTI-TUMOR EFFECTS OF GLYCYRRHIZIN AND 18-β GLYCYRRHETINIC ACID ON VARIOUS LEUKEMIC CELL LINES / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Results --- p.78 / Chapter 3.2.1 --- The Growth Inhibitory Effects of Glycyrrhizin on Various Leukemic Cell Lines --- p.78 / Chapter 3.2.1.1 --- Differential Anti-proliferative Effects of Glycyrrhizin on Various Leukemic Cell Lines In Vitro --- p.78 / Chapter 3.2.1.2 --- Effects of Glycyrrhizin on the Viability of Various Leukemic Cell Lines and Normal Hematopoietic Cells In Vitro --- p.89 / Chapter 3.2.1.3 --- Induction of DNA Fragmentation in Leukemia Cells by Glycyrrhizin --- p.94 / Chapter 3.2.1.4 --- Effect of Glycyrrhizin on the Cell Cycle Kinetics of HL-60 Cells In Vitro --- p.97 / Chapter 3.2.1.5 --- Effect of Glycyrrhizin on the Cell Cycle Kinetics of JCS Cells In Vitro --- p.100 / Chapter 3.2.1.6 --- Effect of Glycyrrhizin on the In Vivo Tumorigenicity of the Murine Myeloid Leukemia JCS Cells --- p.103 / Chapter 3.2.2 --- The Growth Inhibitory Effects of 18-β Glycyrrhetinic Acid on Various Leukemic Cells Lines --- p.105 / Chapter 3.2.2.1 --- Differential Anti-proliferative Effect of 18-β Glycyrrhetinic Acid on Various Leukemic Cell Lines In Vitro --- p.105 / Chapter 3.2.2.2 --- Effects of 18-β Glycyrrhetinic Acid on the Viability of Various Leukemic Cell Lines and Normal Hematopoietic Cells In Vitro --- p.115 / Chapter 3.2.2.3 --- Induction of DNA Fragmentation in Leukemia Cells by 18-β Glycyrrhetinic Acid --- p.120 / Chapter 3.2.2.4 --- Effect of 18-β Glycyrrhetinic Acid on the Cell Cycle Kinetics of HL-60 Cells In Vitro --- p.123 / Chapter 3.2.2.5 --- Effect of 18-β Glycyrrhetinic Acid on the Cell Cycle Kinetics of JCS Cells In Vitro --- p.126 / Chapter 3.2.2.6 --- Effect of 18-β Glycyrrhetinic acid on the In Vivo Tumorigenicity of the Murine Myeloid Leukemia JCS Cells --- p.129 / Chapter 3.3 --- Discussion --- p.131 / Chapter CHAPTER 4: --- THE DIFFERENTIATION-INDUCING EFFECTS OF GLYCYRRHIZIN AND 18-β GLYCYRRHETINIC ACID ON MURINE MYELOID LEUKEMIA CELLS / Chapter 4.1 --- Introduction --- p.135 / Chapter 4.2 --- Results --- p.138 / Chapter 4.2.1 --- Morphological Changes in Glycyrrhizin or 18-β Glycyrrhetinic Acid-treated JCS Cells --- p.138 / Chapter 4.2.2 --- Surface Antigen Immunophenotyping of Glycyrrhizin or 18-β Glycyrrhetinic Acid-treated JCS Cells --- p.141 / Chapter 4.2.3 --- Endocytic Activity of Glycyrrhizin or 18-β Glycyrrhetinic Acid-treated JCS Cells --- p.155 / Chapter 4.3 --- Discussion --- p.158 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI LEUKEMIC ACTIVITIES OF GLYCYRRHIZIN AND 18-P GLYCYRRHETINIC ACID / Chapter 5.1 --- Introduction --- p.161 / Chapter 5.2 --- Results --- p.164 / Chapter 5.2.1 --- Combining Effect of Glycyrrhizin or 18-β Glycyrrhetinic Acid with Hematopoietic Cytokines in Modulating the Proliferation of the Murine Myeloid Leukemia JCS Cells --- p.164 / Chapter 5.2.1.1 --- Combining Effect of Glycyrrhizin and IL-lα on the Proliferation of JCS Cells --- p.164 / Chapter 5.2.1.2 --- Combining Effects of Glycyrrhizin with IFN-γ or TNF-α on the Proliferation of JCS Cells --- p.166 / Chapter 5.2.1.3 --- Combining Effect of 18-β Glycyrrhetinic Acid and IL-lα on the Proliferation of JCS Cells --- p.169 / Chapter 5.2.1.4 --- Combining Effects of 18-β Glycyrrhetinic Acid with IFN-γ or TNF-α on the Proliferation of JCS Cells --- p.169 / Chapter 5.2.2 --- Elucidation of the Molecular Mechanisms of Glycyrrhizin or 18-β Glycyrrhetinic Acid on Leukemic Cell Differentiation and Growth Arrest --- p.173 / Chapter 5.2.2.1 --- Modulatory Effects of Glycyrrhizin and 18-β Glycyrrhetinic Acid on the Expression of Cytokine Genes in the Leukemia JCS Cells --- p.173 / Chapter 5.2.2.2 --- Modulatory Effects of Glycyrrhizin and 18-β Glycyrrhetinic Acid on the Expression of PKC Isoforms in the Leukemia JCS Cells --- p.176 / Chapter 5.2.2.3 --- Modulatory Effects of Glycyrrhizin and 18-β Glycyrrhetinic Acid on the Expression of Growth- regulatory Genes in the Leukemia JCS Cells --- p.180 / Chapter 5.2.2.4 --- Modulatory Effects of 18-β Glycyrrhetinic Acid on the Expression of Apoptosis-related Genes in the Leukemia JCS Cells --- p.183 / Chapter 5.2.2.5 --- Modulatory Effects of 18-β Glycyrrhetinic Acid on the Expression of Growth-regulatory and Apoptosis-related Proteins in JCS Cells --- p.185 / Chapter 5.3 --- Discussion --- p.187 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.194 / REFERENCES --- p.200