by Po Chu, Leung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-169). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abbreviation --- p.iii / Abstract --- p.vii / Chinese Abstract --- p.x / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- The Development of Hematopoietic Progenitor Cells --- p.1 / Chapter 1.1.2 --- Hematopoietic Growth Factors --- p.3 / Chapter 1.1.3 --- Transcriptionl Factors Involved in Lineage Commitment of Hematopoietic Progenitor Cells --- p.5 / Chapter 1.2 --- Leukemia --- p.7 / Chapter 1.2.1 --- Occurrence and Classification of Leukemia --- p.7 / Chapter 1.2.2 --- The Pathological Features and Etiology of Leukemia --- p.10 / Chapter 1.2.3 --- The Molecular Basis of Leukemia --- p.13 / Chapter 1.2.4 --- Current Therapeutic Strategies --- p.14 / Chapter 1.2.4.1 --- Conventional Therapies for Leukemia --- p.14 / Chapter 1.2.4.2 --- Induction of Cell Differentiation and Apoptosis for Treatment of Leukemia --- p.16 / Chapter 1.2.5 --- The Use of Murine Myelomonocytic Leukemia WEHI-3B JCS Cells As a Model for the Study of Leukemia Cell Proliferation， Differentiation and Survival --- p.22 / Chapter 1.3 --- Taxol: A Novel Anti-cancer Agent --- p.23 / Chapter 1.3.1 --- Discovery and Action Mechanism --- p.23 / Chapter 1.3.2 --- Metabolism and Toxicity of Taxol --- p.27 / Chapter 1.3.3 --- The Biological Activities of Taxol --- p.28 / Chapter 1.3.4 --- The Anti-tumor Effects of Taxol --- p.30 / Chapter 1.3.5 --- The Effects of Taxol on Leukemia --- p.31 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.32 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Mice --- p.35 / Chapter 2.1.3 --- "Culture Media,Buffer and Other Solutions" --- p.37 / Chapter 2.1.4 --- Radioisotope and Scintillation Fluid --- p.39 / Chapter 2.1.5 --- Taxol --- p.40 / Chapter 2.1.6 --- Recombinant Cytokines --- p.40 / Chapter 2.1.7 --- Vitamin Analogs --- p.42 / Chapter 2.1.8 --- Various Signal Transduction Pathway Activators and Inhibitors --- p.42 / Chapter 2.1.9 --- Monoclonal Antibodies and Buffers for Flow Cytometry --- p.43 / Chapter 2.1.10 --- Reagents and Chemicals for Gene Expression Study --- p.45 / Chapter 2.1.11 --- Chemical Solutions and Buffers for Western Blot --- p.50 / Chapter 2.1.12 --- Reagents for Colony Assay --- p.54 / Chapter 2.2 --- Methods --- p.55 / Chapter 2.2.1 --- Culture of Leukemia Cell Lines --- p.55 / Chapter 2.2.2 --- Treatment of Leukemia Cells with Various Drugs and Cytokines --- p.55 / Chapter 2.2.3 --- Cell Morphological Study --- p.55 / Chapter 2.2.4 --- Determination of Leukemia Cell Survival and Proliferation --- p.56 / Chapter 2.2.5 --- Colony Assay --- p.56 / Chapter 2.2.6 --- Flow Cytometry Analysis --- p.57 / Chapter 2.2.6.1 --- Surface Antigen Immunophenotyping --- p.57 / Chapter 2.2.6.2 --- Assay of Endocytic Activity --- p.58 / Chapter 2.2.6.3 --- Cell Cycle /DNA Content Evaluation --- p.58 / Chapter 2.2.7 --- Gene Expression Study --- p.59 / Chapter 2.2.7.1 --- Preparation of Total Cellular RNA --- p.59 / Chapter 2.2.7.2 --- Reverse Transcription --- p.60 / Chapter 2.2.7.3 --- Polymerase Chain Reaction (PCR) --- p.60 / Chapter 2.2.7.4 --- Agarose Gel Electrophoresis --- p.61 / Chapter 2.2.8 --- DNA Fragmentation Analysis --- p.61 / Chapter 2.2.9 --- Protein Expression Study --- p.62 / Chapter 2.2.9.1 --- Protein Extraction --- p.62 / Chapter 2.2.9.2 --- Quantification of the Protein --- p.62 / Chapter 2.2.9.3 --- Western Blot Analysis --- p.63 / Chapter 2.2.10 --- Statistical Analysis --- p.64 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Effects of Taxol on the Proliferation and Apoptosis of the Murine Myeloid Leukemia Cells --- p.65 / Chapter 3.1.1 --- Growth-Inhibitory Effects of Taxol on Murine Myeloid Leukemia WEHI-3B JCS cells --- p.65 / Chapter 3.1.2 --- Cytotoxic Effects of Taxol on Murine Bone Marrow Cells and Myeloid Leukemia WEHI-3B JCS Cells --- p.69 / Chapter 3.1.3 --- Anti-proliferative Effect of Taxol on Different Leukemia Cell Lines --- p.72 / Chapter 3.1.4 --- Effects of Taxol on the Cell Cycle Kinetics of WEHI-3B JCS Cells --- p.81 / Chapter 3.1.5 --- Induction of DNA Fragmentation of WEHI-3B JCS cells by Taxol --- p.83 / Chapter 3.1.6 --- Effect of Taxol on the Clonogenicity of WEHI-3B JCS Cells In Vitro and Tumorigenicity In Vivo --- p.86 / Chapter 3.2 --- Effects of Taxol on the Induction of Monocytic Cell Differentiation in Murine Myeloid Leukemia Cells --- p.88 / Chapter 3.2.1 --- Morphological Changes in Taxol-Treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.88 / Chapter 3.2.2 --- Surface Antigen Immunophenotyping of Taxol-treated WE HI-3B cells --- p.91 / Chapter 3.2.3 --- Endocytic Activity of Taxol-treated WEHI-3B JCS cells --- p.95 / Chapter 3.3 --- Modulatory Effect of Taxol and Cytokines on the Proliferation of WEHI- 3B JCS Cells --- p.96 / Chapter 3.4 --- Modulatory Effect of Taxol and Physiological Differentiation Inducers on the Proliferation of WEHI-3B JCS cells --- p.103 / Chapter 3.5 --- The Possible Involvement of Protein Kinase C in the Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.106 / Chapter 3.6 --- Modulation of Apoptotic Gene Expression in Taxol-treated WEHI-3B JCS cells --- p.113 / Chapter 3.7 --- Modulatory Effects of Taxol on the Protein Expression of WEHI-3B JCS Cells --- p.119 / Chapter Chapter 4: --- Discussion and Conclusions / Chapter 4.1 --- "Effects of Taxol on the Proliferation,Differentiation and Apoptosis of the Murine Myeloid Leukemia Cells" --- p.126 / Chapter 4.2 --- "Modulatory Effects of Taxol, Cytokines and Physiological Differentiation Inducers on the Proliferation of the Myelomonocytic Leukemia WEHI-3B JCS Cells" --- p.132 / Chapter 4.3 --- The Possible Involvement of Protein Kinase C in Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.136 / Chapter 4.4 --- The Modulation of Apoptosis Gene Expression in Taxol-treated WEHI-3B JCS Cells --- p.137 / Chapter 4.5 --- The Modulation of Protein Expression in Taxol-treated WEHI-3B JCS Cells --- p.138 / Chapter 4.6 --- Conclusions and Future Perspectives --- p.139 / References --- p.141

Leung Pui-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 226-266). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.viii / 撮要 --- p.xii / TABLE OF CONTENTS --- p.xvi / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis & Leukemia --- p.1 / Chapter 1.1.1 --- Introduction to Hematopoiesis and its Regulation --- p.1 / Chapter 1.1.2 --- Leukemia --- p.7 / Chapter 1.1.2.1 --- Classification and Epidemiology of Leukemia --- p.7 / Chapter 1.1.2.2 --- Conventional Approaches to Leukemia Therapy --- p.13 / Chapter 1.1.2.3 --- New Approaches to Leukemia Therapy --- p.16 / Chapter 1.2 --- Coumarins --- p.18 / Chapter 1.2.1 --- Introduction to Coumarins --- p.18 / Chapter 1.2.1.1 --- "Historical Development, Occurrence of Coumarins and their Functions in Plants" --- p.18 / Chapter 1.2.1.2 --- Beneficial Effects of Coumarins for Human Use --- p.20 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Coumarins --- p.22 / Chapter 1.2.2.1 --- Chemical Structures of Coumarins --- p.22 / Chapter 1.2.2.2 --- Biosynthesis of Coumarins --- p.24 / Chapter 1.2.2.3 --- Toxicology of Coumarins --- p.24 / Chapter 1.2.2.4 --- Mode of Entry of Coumarins --- p.25 / Chapter 1.2.2.5 --- Metabolic Pathways of Coumarins --- p.27 / Chapter 1.2.3 --- Pharmacological Activities of Coumarins --- p.30 / Chapter 1.2.3.1 --- Anti-edema and Anti-inflammatory Activities --- p.30 / Chapter 1.2.3.2 --- Cardiovascular Protective Function --- p.32 / Chapter 1.2.3.3 --- Anti-tumor Activity --- p.33 / Chapter 1.2.3.3.1 --- Anti-tumor Activity In Vitro --- p.33 / Chapter 1.2.3.3.2 --- Anti-tumor Activity In Vivo and Clinical Applications --- p.34 / Chapter 1.2.3.4 --- Immunomodulatory Activity --- p.36 / Chapter 1.2.3.4.1 --- Immunological Considerations --- p.36 / Chapter 1.2.3.4.2 --- Immunomodulation In Vitro --- p.37 / Chapter 1.2.3.4.3 --- Immunomodulation In Vivo --- p.38 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.41 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.43 / Chapter 2.1.1 --- Animals --- p.43 / Chapter 2.1.2 --- Cell Lines --- p.43 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.44 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.49 / Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.49 / Chapter 2.1.6 --- Reagents for DNA Extraction --- p.52 / Chapter 2.1.7 --- Cell Death Detection ELISAplus Kit --- p.54 / Chapter 2.1.8 --- Reagents for Total RNA Isolation --- p.55 / Chapter 2.1.9 --- Reagents and Buffers for RT-PCR --- p.56 / Chapter 2.1.10 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Aicds --- p.60 / Chapter 2.1.11 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.61 / Chapter 2.1.12 --- Reagents for Measuring Caspase Activity --- p.67 / Chapter 2.1.13 --- Reagents for Neutral Red Assay --- p.70 / Chapter 2.2 --- Methods --- p.71 / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.11 / Chapter 2.2.2 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.71 / Chapter 2.2.3 --- Determination of Cell Viability --- p.72 / Chapter 2.2.4 --- In Vivo Anti-tumor Study --- p.73 / Chapter 2.2.5 --- Analysis of Cell Cycle Profile/DNA Content by Flow Cytometry --- p.74 / Chapter 2.2.6 --- Measurement of Apoptosis --- p.74 / Chapter 2.2.7 --- "Islation ,Preparation and Culture of Mouse Peritoneal Macrophages" --- p.76 / Chapter 2.2.8 --- Gene Expression Study --- p.77 / Chapter 2.2.9 --- Protein Expression Study --- p.80 / Chapter 2.2.10 --- Determination of the Mitochondrial Membrane Potential --- p.84 / Chapter 2.2.11 --- Measurement of Caspase Activity --- p.84 / Chapter 2.2.12 --- In Vivo Macrophage Migration Assay --- p.85 / Chapter 2.2.13 --- Study of Endocytic Activity of Macrophages --- p.85 / Chapter 2.2.14 --- Determination of Nitric Oxide Production by Macrophages --- p.86 / Chapter 2.2.15 --- Study of Mitogenesis of Murine Splenocytes --- p.87 / Chapter 2.2.16 --- Analysis of T Cell Population and its Sub-populations --- p.88 / Chapter 2.2.17 --- Measurement of Lymphokine Activated Killer (LAK) Cell Activity by Neutral Red Assay --- p.90 / Chapter 2.2.18 --- Statistical Analysis --- p.91 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI PROLIFERATIVE EFFECT OF COUMARINS ON MACROPHAGE-LIKE LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Anti-proliferative Effect of Coumarins on Various Macrophage-like Leukemia Cell Lines In Vitro --- p.94 / Chapter 3.2.2 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Coumarins on Macrophage-like Leukemia PU5-1.8 Cells --- p.105 / Chapter 3.2.3 --- Cytotoxic Effect of Coumarins on Macrophage-like Leukemia PU5-1.8 Cells In Vitro --- p.110 / Chapter 3.2.4 --- Effect of Coumarins on the Cell Cycle Kinetics of the Macrophage-like Leukemia PU5-1.8 Cells In Vitro --- p.113 / Chapter 3.2.5 --- Effect of Coumarins on the Expression of Cell Cycle- and Growth-regulatory Genes in the Macrophage-like Leukemia PU5-1.8 Cells --- p.119 / Chapter 3.2.6 --- Effect of Coumarins on the Expression of Cell Cycle-regulatory Proteins in the Macrophage-like Leukemia PU5-1.8 Cells --- p.123 / Chapter 3.2.7 --- Effect of Coumarins on the In Vivo Tumorigenicity of the Macrophage-like Leukemia PU5-1.8 Cells --- p.127 / Chapter 3.2.8 --- Effect of Coumarins on the In Vivo Growth of the Macrophage- like Leukemia PU5-1.8 Cells in Syngeneic BALB/c Mice --- p.131 / Chapter 3.3 --- Discussion --- p.133 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF COUMARINS ON MACROPHAGE-LIKE LEUKEMIA PU5- CELLS --- p.1.8 / Chapter 4.1 --- Introduction --- p.143 / Chapter 4.2 --- Results --- p.152 / Chapter 4.2.1 --- Induction of Apoptosis in the Murine Macrophage-like Leukemia PU5-1.8 Cells by Coumarins --- p.152 / Chapter 4.2.2 --- Modulatory Effect of Coumarins on the Expression of Apoptosis-regulatory Genes in the Macrophage-like Leukemia PU5-1.8 Cells --- p.159 / Chapter 4.2.3 --- Modulatory Effect of Coumarins on the Expression of Apoptosis-regulatory Proteins in the Macrophage-like Leukemia PU5-1.8 Cells --- p.166 / Chapter 4.2.4 --- Effect of Coumarins on the Mitochondrial Membrane Depolarization of the Macrophage-like Leukemia PU5-1.8 Cells --- p.169 / Chapter 4.2.5 --- Effect of Coumarins on the Induction of Caspase Activity in the Macrophage-like Leukemia PU5-1.8 Cells --- p.172 / Chapter 4.2 --- Discussion --- p.177 / Chapter CHAPTER 5: --- STUDIES ON THE IMMUNOMODULATORY EFFECT OF COUMARINS ON MURINE MACROPHAGES AND OTHER IMMUNE CELLS / Chapter 5.1 --- Introduction --- p.184 / Chapter 5.2 --- Results --- p.187 / Chapter 5.2.1 --- Effect of Coumarins on the Viability of Macrophages In Vitro --- p.187 / Chapter 5.2.2 --- Effect of Coumarins on the In Vivo Migration of Macrophages --- p.190 / Chapter 5.2.3 --- Effect of Coumarins on the Endocytic Ability of Murine Macrophages --- p.192 / Chapter 5.2.4 --- Effect of Coumarins on the Nitric Oxide Production by Murine Macrophages --- p.194 / Chapter 5.2.5 --- Effect of Coumarins on the Viability of Murine Splenocytes --- p.199 / Chapter 5.2.6 --- Effect of Coumarins on the Mitogenesis of Murine Splenocytes --- p.201 / Chapter 5.2.7 --- Effect of Coumarins on T-cell Population and its Sub-populations --- p.205 / Chapter 5.2.8 --- Effect of Coumarins on the Induction of Murine LAK Activity --- p.208 / Chapter 5.3 --- Discussion --- p.210 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.218 / REFERENCES --- p.226

Chan Wai Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 199-210). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / CHINESE ABSTRACT --- p.x / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- What is hematopoiesis? --- p.1 / Chapter 1.1.1 --- The development of hematopoietic progenitor cells --- p.1 / Chapter 1.1.2 --- The regulation of hematopoiesis by environmental factors --- p.4 / Chapter 1.1.3 --- The regulation of hematopoiesis by transcription factors --- p.7 / Chapter 1.2 --- Leukemia --- p.9 / Chapter 1.2.1 --- Classification of leukemia --- p.11 / Chapter 1.2.2 --- Pathology and etiology of leukemia --- p.15 / Chapter 1.2.2.1 --- Inheritance --- p.15 / Chapter 1.2.2.2 --- Environmental factors --- p.16 / Chapter 1.2.2.3 --- Virus infection --- p.17 / Chapter 1.2.3 --- Genetics of leukemia --- p.17 / Chapter 1.2.3.1 --- Point mutations --- p.17 / Chapter 1.2.3.2 --- Translocations --- p.18 / Chapter 1.2.3.3 --- Gene and chromosomal deletions --- p.18 / Chapter 1.2.3.4 --- Chromosomal duplication or gene amplification --- p.18 / Chapter 1.2.4 --- Current therapeutic strategies for leukemia --- p.20 / Chapter 1.2.4.1 --- Chemotherapy --- p.20 / Chapter 1.2.4.2 --- Stem cell transplantation --- p.21 / Chapter 1.2.4.3 --- Immunotherapy --- p.22 / Chapter 1.2.4.4 --- Gene therapy --- p.23 / Chapter 1.2.5 --- Novel approaches for the treatment of leukemia --- p.23 / Chapter 1.2.5.1 --- Differentiation therapy of leukemia --- p.24 / Chapter 1.2.5.2 --- Induction of apoptosis in the treatment of leukemia --- p.25 / Chapter 1.3 --- Topoisomerase-targeting agents --- p.27 / Chapter 1.3.1 --- What is topoisomerase? --- p.27 / Chapter 1.3.2 --- Structures and action mechanisms of Top I- and Top II-targeting agents --- p.28 / Chapter 1.3.3 --- Anti-tumor activities of topoisomerase-targeting agents --- p.37 / Chapter 1.4 --- Aims and scopes of this investigation --- p.39 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Mice --- p.42 / Chapter 2.1.2 --- Cell lines --- p.42 / Chapter 2.1.3 --- "Cell culture medium, buffers and other reagents" --- p.43 / Chapter 2.1.4 --- Radioisotope and scintillation fluid --- p.47 / Chapter 2.1.5 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.6 --- Antibodies for flow cytometry --- p.50 / Chapter 2.1.7 --- Recombinant cytokines --- p.51 / Chapter 2.1.8 --- Reagents for DNA extraction --- p.52 / Chapter 2.1.9 --- Reagents for total RNA isolation --- p.53 / Chapter 2.1.10 --- Reagents and buffers for RT-PCR --- p.54 / Chapter 2.1.11 --- Reagents and buffers for gel electrophoresis --- p.58 / Chapter 2.1.12 --- Reagents and buffers for Western blot analysis --- p.59 / Chapter 2.1.13 --- Reagents for measuring caspase activity --- p.64 / Chapter 2.2 --- Methods --- p.67 / Chapter 2.2.1 --- Culture of the leukemia cell lines --- p.67 / Chapter 2.2.2 --- Isolation and preparation of normal hematopoietic cells --- p.67 / Chapter 2.2.3 --- [3 H]-TdR proliferation assay --- p.68 / Chapter 2.2.4 --- Determination of cell viability --- p.69 / Chapter 2.2.5 --- Assay for anti-leukemic activity in vivo --- p.70 / Chapter 2.2.6 --- Assessment of differentiation-associated characteristics --- p.71 / Chapter 2.2.7 --- Assays for apoptosis --- p.73 / Chapter 2.2.8 --- Cell cycle analysis (DNA content evaluation) --- p.75 / Chapter 2.2.9 --- Gene expression study --- p.75 / Chapter 2.2.10 --- Protein expression study --- p.79 / Chapter 2.2.11 --- Measurement of caspase activity --- p.82 / Chapter 2.2.12 --- Statistical analysis 一 --- p.83 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-PROLIFERATIVE EFFECT OF TOPOISOMERASE-TARGETING AGENTS ON LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.84 / Chapter 3.2 --- Results --- p.86 / Chapter 3.2.1 --- The anti-proliferative effect of topoisomerase-targeting agents on human and murine leukemia cells in vitro --- p.86 / Chapter 3.2.2 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the clonogenicity of the murine myeloid leukemia WEHI-3B JCS cells in vitro --- p.105 / Chapter 3.2.3 --- Effects of 10-hydroxy-camptothecin and etoposide phosphate on the tumorigenicity and proliferation of the murine myeloid leukemia WEHI-3B JCS cells invivo --- p.106 / Chapter 3.2.4 --- Cytotoxic effect of 10-hydroxy-camptothecin and etoposide phosphate on normal hematopoietic cells and WEHI-3B JCS cells in vitro --- p.109 / Chapter 3.2.5 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the cell cycle kinetics of WEHI-3B JCS cells --- p.114 / Chapter 3.2.6 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the expression of cell cycle-regulatory genes in the murine myeloid leukemia WEHI-3B JCS cells --- p.116 / Chapter 3.2.7 --- Combination effect of 10-hydroxy-camptothecin or etoposide phosphate with cytokines on the proliferation of the murine myeloid leukemia WEHI-3B JCS cells --- p.123 / Chapter 3.3 --- Discussion --- p.127 / Chapter CHAPTER 4: --- STUDIES ON THE DIFFERENTIATION-INDUCING EFFECT OF 10-HYDROXY-CAMPTOTHECIN AND ETOPOSIDE PHOSPHATE ON THE MURINE MYELOID LEUKEMIA WEHI-3B JCS CELLS / Chapter 4.1 --- Introduction --- p.132 / Chapter 4.2 --- Results --- p.134 / Chapter 4.2.1 --- Morphological changes in the murine myeloid leukemia WEHI-3B JCS cells treated with 10-hydroxy-camptothecin and etoposide phosphate --- p.134 / Chapter 4.2.2 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the size and granularity of the murine myeloid leukemia WEHI-3B JCS cells --- p.138 / Chapter 4.2.3 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the plastic adhering property of the murine myeloid leukemia WEHI-3B JCS cells --- p.140 / Chapter 4.2.4 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the NBT-reducing activity of the murine myeloid leukemia WEHI-3B JCS cells --- p.142 / Chapter 4.2.5 --- Surface antigen immunophenotyping of the murine myeloid leukemia WEHI-3B JCS cells treated with 10-hydroxy- camptothecin and etoposide phosphate --- p.145 / Chapter 4.2.6 --- Induction of non-specific esterase activity in the murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy- camptothecin and etoposide phosphate --- p.152 / Chapter 4.3 --- Discussion --- p.154 / Chapter CHAPTER 5: --- STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF 10-HYDROXY-CAMPTOTHECIN AND ETOPOSIDE PHOSPHATE ON THE MURINE MYELOID LEUKEMIA WEHI-3B JCS CELLS / Chapter 5.1 --- Introduction --- p.157 / Chapter 5.2 --- Results --- p.160 / Chapter 5.2.1 --- Induction of nuclear disintegration in the murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy-camptothecin and etoposide phosphate --- p.160 / Chapter 5.2.2 --- Induction of DNA fragmentation in the murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy-camptothecin and etoposide phosphate --- p.162 / Chapter 5.2.3 --- Induction of phosphatidylserine translocation in murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy- camptothecin and etoposide phosphate --- p.167 / Chapter 5.2.4 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the expression of apoptosis-regulatory genes in the murine myeloid leukemia WEHI-3B JCS cells --- p.171 / Chapter 5.2.5 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the expression of apoptosis-regulatory proteins in the murine myeloid leukemia WEHI-3B JCS cells --- p.177 / Chapter 5.2.6 --- Induction of mitochondrial membrane depolarization in the murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy- camptothecin and etoposide phosphate --- p.179 / Chapter 5.2.7 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on caspase activity in the murine myeloid leukemia WEHI-3B JCS cells --- p.181 / Chapter 5.2.8 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate intracellular Ca2+ level in the murine myeloid leukemia WEHI-3B JCS cells --- p.186 / Chapter 5.3 --- Discussion --- p.189 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.192 / REFERENCES --- p.199

白血病由血液或骨髓中的癌細胞所形成，基於其造血幹細胞（HSC）的增殖和分化出現偶聯或不平衡的情況而產生的結果。白血病是香港最常見的兒童癌症，報告指出，於2005年至2009年期間，平均每年約有270名患者死於該病。傳統治療白血病的方法包括化療，放射性治療，骨髓或外周血幹細胞移植，至於採用哪種療法，則要視乎白血病的類型和階段。然而，這些療法會為患者帶來各種副作用，因此，在過去十年間，研發新型治療白血病的藥物引起了越來越多人的關注。 / 共軛脂肪酸（CFA），是指一群位置及幾何異構體的多元不飽和脂肪酸（PUFA），於它們的化學結構中，最少有一組共軛雙鍵。天然的共軛脂肪酸包括，存在於反芻動物的肌肉及乳製品中的共軛雙烯酸（CLA），存在於植物種子油中的共軛三烯酸（CLN），以及存在於海藻中的共軛四烯酸，共軛五烯酸（CEPA）和共軛六烯酸（CDHA）。過往的研究證實了共軛雙烯酸擁有各種生理及醫藥功效，包括抗脂肪分化，抗動脈硬化，抗糖尿病，免疫調節和抗腫瘤作用。根據體外實驗報告指出，共軛三烯酸和共軛四烯酸對多種腫瘤細胞株皆具有生長抑制作用，然而，它們對小鼠巨噬細胞樣的白血病細胞之調節作用和機制仍有待研究。因此，在這篇論文中，共軛三烯酸和共軛四烯酸對小鼠巨噬細胞樣的白血病細胞的抗增殖作用，以及它們引起的相關機制將會被探討。 / 本實驗計劃研究了九個不同的多元不飽和脂肪酸異構體對小鼠巨噬細胞樣的白血病細胞PU5-1.8細胞的抗增殖能力，當中包括三烯酸、四烯酸、共軛雙烯酸、共軛三烯酸和共軛四烯酸的異構體。結果清楚地表明，共軛三烯酸和共軛四烯酸異構體皆能對白血病細胞表現出劑量依賴性的生長抑制作用。於十個異構體當中，順式-8，反式-10，順式-12共軛三烯酸(蘭花酸)和順式-9，反式-11，反式-13，順式-15共軛四烯酸(杷茬酸)較其他共軛脂肪酸異構體更有效抑制白血病細胞的生長，因此，他們被選定為主要的研究對象，以便對它們所引起的相關機制作進一步了解。此外，蘭花酸和杷茬酸對其他小鼠巨噬細胞樣的白血病細胞，包括J774 A.1細胞和P388D1細胞，也具備抗增殖作用，表現其生長抑制作用並不是純粹針對單一種腫瘤細胞株的。有趣的是，蘭花酸和杷茬酸對PU5-1.8細胞的生長抑制作用是可以局部逆轉的，但只限以低濃度的共軛脂肪酸培養白血病細胞以及培養的時間不多於24小時，否則，隨著培養的時間增加或共軛脂肪酸的濃度增加時，該生長抑制作用幾乎是不可逆轉的。另一方面，結果也表明蘭花酸和杷茬酸在其抑制白血病細胞增殖率為五十百分比之濃度下，它們對腫瘤細胞以及小鼠正常細胞的毒性作用是很少的。除了在體外研究，預先以蘭花酸處理的PU5-1.8細胞於BALB/c小鼠內導致白血病細胞生長的能力也以劑量依賴方式被抑制。 / 幾種不同的機制也能解釋蘭花酸和杷茬酸對PU5-1.8細胞的生長抑制能力，當中包括阻礙腫瘤細胞週期的前進，增加腫瘤細胞內活性氧（ROS）的生產或誘導腫瘤細胞的凋亡。研究結果指出，蘭花酸和杷茬酸可以抑制PU5-1.8細胞週期的進程並將其停留在G₀/G₁時相，換來的是減少處於S時相的細胞的比例。此外，透過西方蛋白質印跡分析，細胞週期蛋白E的表達有所下調，同時，幾個細胞週期調控蛋白的表達，包括p21，p27及p53蛋白則被上調，跟上述的實驗結果吻合。此外，經蘭花酸和杷茬酸處理後，PU5-1.8細胞內的ROS濃度和線粒體質量也有所增加，而它們對白血病細胞的生長抑制作用則會被抗氧化劑所減弱，這一點說明了蘭花酸和杷茬酸對PU5-1.8細胞的抗增殖作用可能與細胞內的脂質過氧化物濃度和線粒體質量有關。最後，通過各種測試細胞凋亡的實驗，包括利用細胞死亡檢測的ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40}試劑盒，Annexin-V和JC-1染色等方法，清楚地表明了蘭花酸和杷茬酸能誘導PU5-1.8細胞的凋亡。加上西方蛋白質印跡分析，PU5-1.8細胞內Bcl-2和Bcl-XL的蛋白的表達水平有所下降，而相反Bax蛋白的表達水平則有所提升，足以證明蘭花酸和杷茬酸能引發PU5-1.8細胞的凋亡。 / 總括來說，蘭花酸和杷茬酸對PU5-1.8細胞的抗增殖作用呈現時間和劑量依賴性，該作用可能基於阻礙腫瘤細胞週期的前進，增加腫瘤細胞內ROS的生產或誘導腫瘤細胞的凋亡。由於蘭花酸和杷茬酸分別在植物種子油和海藻的含量相當高，再加上它們對正常細胞無直接毒性，若果能夠對它們的抗腫瘤作用以及其分子機制有更透徹的理解，它們有望發展成為未來治療白血病的藥物。 / Leukemia is a cancer of the blood or bone marrow which is the result of uncoupling or imbalance of the proliferation and differentiation of hematopoietic stem cells (HSC). It is the most common childhood cancer in Hong Kong and it claims the lives of around 270 patients per year from 2005 to 2009 in average. Conventional approaches to leukemia therapy include chemotherapy, radiotherapy and bone marrow or peripheral blood stem cell transplantation, depending on the types and stages of leukemia. Nevertheless, these therapies are accompanied by a number of undesirable effects to the patients, hence, the development and research in novel treatments of leukemia are attracting increasing attention in the past decades. / Conjugated fatty acids (CFA) refer to the positional and geometric isomers of polyunsaturated fatty acids (PUFA) with conjugated double bonds. Naturally-occurring CFA include conjugated linoleic acids (CLA) from meat and dairy products of ruminant animals, conjugated linolenic acids (CLN) from plant seed oils, conjugated tetraenoic acids, conjugated eicosapentaenoic acids (CEPA) and conjugated docosahexaenoic acids (CDHA) from seaweeds. CLA have been shown to possess various biological and pharmacological activities, including anti-adipogenic, anti-atherogenic, anti-diabetogenic, immunomodulatory and anti-tumor effects. Furthermore, previous researches have demonstrated the growth-inhibitory effects of CLN and conjugated tetraenoic acids on a wide variety of cancer cell lines in vitro, however, their modulatory effects and action mechanisms on murine macrophage-like leukemia cells remain poorly understood. In this thesis project, the anti-proliferative effects of CLN and conjugated tetraenoic acids on the murine macrophage-like leukemia cells, as well as their action mechanisms will be elucidated. / Nine different PUFA isomers, including linolenic acid, tetraenoic acid, CLA, CLN and conjugated tetraenoic acids were examined for their anti-proliferative effects on the murine macrophage-like leukemia PU5-1.8 cells. The results clearly showed that all CLN isomers and cis-parinaric acid could exhibit growth-inhibitory effects on the leukemia cells in a dose-dependent manner. It was found that jacaric acid and cis-parinaric acid were relatively more potent than the other isomers used in the present study, hence, they were chosen to be the specific targets for more in-depth mechanistic studies. In addition, the anti-proliferative effects of jacaric acid and cis-parinaric acid were observed in other murine macrophage-like leukemia cell lines, including J774 A.1 cells and P388D1 cells, suggesting that the effects were not cell-line specific. Interestingly, the growth-inhibitory effects were partially reversible at lower concentrations of CFA used within 24 hours of incubation, but the effects were almost irreversible when either the incubation time or the concentration of CFA used was increased. Furthermore, the results showed that both jacaric acid and cis-parinaric acid at their IC₅₀ growth-inhibitory concentrations on PU5-1.8 cells exerted minimal, if any, direct cytotoxic effects on the tumor cells as well as the murine normal cells. Apart from the in vitro studies, it was also demonstrated that pre-treatment of PU5-1.8 cells with jacaric acid could significantly decrease the leukemic cell growth in syngeneic BALB/c mice in a dose-dependent manner. / Several mechanisms were proposed for the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells, including the triggering of cell cycle arrest, increasing the production of intracellular reactive oxygen species (ROS) or induction of apoptosis in the tumor cells. The results showed that jacaric acid and cis-parinaric acid could inhibit the cell cycle progression since an accumulation of PU5-1.8 cells at the G₀/G₁ phase was observed, together with a decrease in the cell population at the S phase. This finding was supported by the down-regulation of cyclin E protein and up-regulation of several cell cycle regulatory proteins, including the p21, p27 and p53 proteins. Apart from that, the intracellular ROS concentration and the mitochondrial mass were found to be increased in jacaric acid- or cis-parinaric acid-treated PU5-1.8 cells, and their growth-inhibitory effects were alleviated after the addition of antioxidants. Therefore, the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells might be correlated with the intracellular concentration of lipid peroxides and the mitochondrial mass. Furthermore, the results clearly demonstrated that both jacaric acid and cis-parinaric acid exhibited dose-dependent apoptosis-inducing effects on PU5-1.8 cells, as revealed by the Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit, annexin V assay and JC-1 dye staining method. In addition, it was found that the expression levels of Bcl-2 and Bcl-xL proteins were decreased, whereas the expression level of Bax protein was increased in PU5-1.8 cells, further confirming that apoptosis occurred in PU5-1.8 cells after treatment with jacaric acid and cis-parinaric acid. / Collectively, the results showed that jacaric acid and cis-parinaric acid could exhibit their anti-proliferative effects on PU5-1.8 cells in a time- and dose-dependent manner, through the triggering of cell cycle arrest, increasing the production of intracellular ROS or induction of apoptosis in the tumor cells. Owing to their high abundance in plant seed oils and seaweeds, and being relatively non-cytotoxic, they might be potential candidates for the treatment of leukemia. Further investigations are required in order to develop a better understanding on the molecular action mechanisms underlying the anti-tumor effects of jacaric acid and cis-parinaric acid on leukemia cells before they could be developed as the therapeutic drugs for leukemia. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Wai Nam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 169-182). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Acknowledgements --- p.viii / List of Abbreviations --- p.ix / List of Figures and Tables --- p.xiii / Publications --- p.xvii / Chapter Chapter 1 --- General introduction / Chapter 1.1 --- Introduction to hematopoiesis and leukemia --- p.1 / Chapter 1.1.1 --- Introduction to hematopoiesis --- p.1 / Chapter 1.1.2 --- Introduction to leukemia --- p.7 / Chapter 1.1.2.1 --- Classification of leukemia --- p.7 / Chapter 1.1.2.2 --- Epidemiology of leukemia --- p.10 / Chapter 1.1.2.3 --- Conventional approaches to leukemia therapy --- p.12 / Chapter 1.1.2.4 --- Alternative approaches to leukemia therapy --- p.17 / Chapter 1.2 --- Introduction to conjugated fatty acids --- p.19 / Chapter 1.2.1 --- An overview of polyunsaturated fatty acids and conjugated fatty acids --- p.19 / Chapter 1.2.2 --- Chemical structures and physical properties of CLN and conjugated tetraenoic acids --- p.21 / Chapter 1.2.3 --- Natural occurrence of CLN and conjugated tetraenoic acids --- p.26 / Chapter 1.2.4 --- Synthesis of CLN and conjugated tetraenoic acids --- p.28 / Chapter 1.2.5 --- Metabolism of CLN --- p.30 / Chapter 1.2.6 --- Major biological and pharmacological activities of CLN and conjugated tetraenoic acids --- p.30 / Chapter 1.2.6.1 --- Anti-obese and hypolipidemic property --- p.31 / Chapter 1.2.6.2 --- Anti-carcinogenic property --- p.32 / Chapter 1.2.6.2.1 --- Anti-proliferative effect --- p.32 / Chapter 1.2.6.2.2 --- Apoptosis-inducing effect --- p.33 / Chapter 1.3 --- Aims and scopes of this thesis --- p.36 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Animals --- p.39 / Chapter 2.1.2 --- Cell lines --- p.39 / Chapter 2.1.3 --- Cell culture media and reagents --- p.40 / Chapter 2.1.4 --- Fatty acids --- p.44 / Chapter 2.1.5 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.6 --- Reagents and buffers for Western blotting --- p.51 / Chapter 2.1.7 --- Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit --- p.60 / Chapter 2.2. --- Methods --- p.62 / Chapter 2.2.1 --- Culture of tumor cell lines --- p.62 / Chapter 2.2.2 --- Isolation and culture of murine normal cells --- p.63 / Chapter 2.2.3 --- Determination of cell proliferation by CyQUANT® NF cell proliferation assay --- p.65 / Chapter 2.2.4 --- Determination of cell viability --- p.66 / Chapter 2.2.5 --- Cytotoxicity test of CFA on normal cells --- p.67 / Chapter 2.2.6 --- In vivo tumorigenicity assay --- p.68 / Chapter 2.2.7 --- Analysis of cell cycle profile --- p.69 / Chapter 2.2.8 --- Measurement of DNA fragmentation by Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit --- p.70 / Chapter 2.2.9 --- Analysis of Annexin V-GFP/PI dual staining profile --- p.71 / Chapter 2.2.10 --- Determination of mitochondrial membrane potential by JC-1 staining --- p.72 / Chapter 2.2.11 --- Determination of intracellular reactive oxygen species generation --- p.72 / Chapter 2.2.12 --- Determination of mitochondrial mass --- p.73 / Chapter 2.2.13 --- Protein expression study --- p.74 / Chapter 2.2.14 --- Statistical analysis --- p.78 / Chapter Chapter 3 --- Studies on the anti-proliferative effects of jacaric acid and cis-parinaric acid on murine macrophage-like leukemia cells / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Results --- p.82 / Chapter 3.2.1 --- Anti-proliferative effects of CFA isomers on murine macrophage-like leukemia PU5-1.8 cells in vitro --- p.82 / Chapter 3.2.2 --- Kinetic and reversibility studies of the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells --- p.92 / Chapter 3.2.3 --- Cytotoxic effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells --- p.97 / Chapter 3.2.4 --- Cytotoxic effects of jacaric acid and cis-parinaric acid on murine normal cells in vitro --- p.99 / Chapter 3.2.5 --- Effect of jacaric acid on the in vivo tumorigenicity of PU5-1.8 cells --- p.102 / Chapter 3.3 --- Discussion --- p.104 / Chapter Chapter 4 --- Mechanistic studies on the anti-tumor effects of jacaric acid and cis-parinaric acid on murine macrophage-like leukemia cells / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Results --- p.117 / Chapter 4.2.1 --- Effects of jacaric acid and cis-parinaric acid on the cell cycle profile of murine macrophage-like leukemia PU5-1.8 cells --- p.117 / Chapter 4.2.2 --- Effects of jacaric acid and cis-parinaric acid on the expression of cell cycle regulatory proteins in murine macrophage-like leukemia PU5-1.8 cells --- p.121 / Chapter 4.2.3 --- Effects of jacaric acid and cis-parinaric acid on the generation of reactive oxygen species in murine macrophage-like leukemia PU5-1.8 cells --- p.124 / Chapter 4.2.4 --- Effects of antioxidants on the anti-proliferative effects of jacaric acid and cis-parinaric acid on the murine macrophage-like leukemia PU5-1.8 cells --- p.128 / Chapter 4.2.5 --- Effects of jacaric acid and cis-parinaric acid on the mitochondrial mass in murine macrophage-like leukemia PU5-1.8 cells --- p.131 / Chapter 4.2.6 --- Effects of jacaric acid and cis-parinaric acid on the induction of apoptosis in murine macrophage-like leukemia PU5-1.8 cells --- p.135 / Chapter 4.2.7 --- Effects of jacaric acid and cis-parinaric acid on the induction of phosphatidylserine externalization in murine macrophage-like leukemia PU5-1.8 cells --- p.139 / Chapter 4.2.8 --- Effects of jacaric acid and cis-parinaric acid on the mitochondrial membrane potential in murine macrophage-like leukemia PU5-1.8 cells --- p.144 / Chapter 4.2.9 --- Effects of jacaric acid and cis-parinaric acid on the expression of apoptosis-regulatory proteins in murine macrophage-like leukemia PU5-1.8 cells --- p.149 / Chapter 4.3 --- Discussion --- p.152 / Chapter Chapter 5 --- Conclusions and future perspectives / Chapter 5.1 --- Conclusions --- p.161 / Chapter 5.2 --- Future perspectives --- p.164 / References --- p.169

Liu, Xiaozhuo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 156-169). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iv / Acknowledgments --- p.vi / List of Abbreviations --- p.vii / Table of Contents --- p.xiv / Chapter Chapter One: --- General Introduction / Chapter 1.1 --- Hematopoiesis and Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.2 --- Leukemia --- p.6 / Chapter 1.1.2.1 --- An Overview of Leukemia --- p.6 / Chapter 1.1.2.2 --- Classification and Epidemiology of Leukemia --- p.8 / Chapter 1.1.2.3 --- Conventional Approaches to Leukemia Therapy --- p.12 / Chapter 1.1.2.4 --- Novel Approaches to Leukemia Therapy --- p.15 / Chapter 1.2 --- Sophoraflavanone G: A Bioactive Compound Isolated from Kushen --- p.18 / Chapter 1.2.1 --- An Overview of Kushen: A Traditional Chinese Medicine --- p.19 / Chapter 1.2.2 --- An Overview of Lavandulyl Flavanones --- p.22 / Chapter 1.2.3 --- Historical Development and Occurrence of Sophoraflavanone G --- p.24 / Chapter 1.2.4 --- Biological Activities of Sophoraflavanone G --- p.25 / Chapter 1.2.4.1 --- Anti-microbial and Insecticidal Activities --- p.25 / Chapter 1.2.4.2 --- Anti-tumor Activities --- p.26 / Chapter 1.2.4.3 --- Pharmacodynamics of Sophoraflavanone G --- p.27 / Chapter 1.3 --- Objectives and Scopes of the Present Study --- p.30 / Chapter Chapter Two: --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Animals --- p.32 / Chapter 2.1.2 --- Cell lines --- p.32 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.34 / Chapter 2.1.4 --- Reagents and Buffers for Flow Cytometry --- p.37 / Chapter 2.1.5 --- Reagents for DNA Extraction --- p.39 / Chapter 2.1.6 --- Reagents for Measuring Caspase Activity --- p.40 / Chapter 2.1.7 --- "Reagents, Buffers and Materials for Western Blotting" --- p.43 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Extraction and Isolation of Sophoraflavanone G from Kushen --- p.48 / Chapter 2.2.2 --- Culture of Tumor Cell Lines --- p.49 / Chapter 2.2.3 --- "Isolation, Preparation and Culturing of Human Peripheral Blood Leukocytes and Murine Bone Marrow Cells" --- p.50 / Chapter 2.2.4 --- Assays for Anti-proliferation and Cytotoxicity --- p.51 / Chapter 2.2.5 --- Determination of Anti-leukemic Activity In Vivo (In Vivo Tumorigenicity Assay) --- p.52 / Chapter 2.2.6 --- Cell Cycle Analysis by Flow Cytometry --- p.53 / Chapter 2.2.7 --- Measurement of Apoptosis-induced Activities --- p.54 / Chapter 2.2.8 --- Protein Expression Study --- p.59 / Chapter 2.2.9 --- Assessment of Differentiation-associated Characteristics --- p.64 / Chapter 2.2.10 --- Statistical Analysis --- p.65 / Chapter Chapter Three: --- Studies on the Anti-proliferative Effect of Sophoraflavanone G on Human Myeloid Leukemia Cells / Chapter 3.1 --- Introduction --- p.66 / Chapter 3.2 --- Results --- p.69 / Chapter 3.2.1 --- Structure Identification of Sophoraflavanone G Isolated from Sophora flavescens --- p.69 / Chapter 3.2.2 --- Anti-proliferative Activity of Sophoraflavanone G on Various Myeloid Leukemia Cell Lines --- p.72 / Chapter 3.2.3 --- Effect of Sophoraflavanone G on the Viability of the Human Promyelocytic Leukemia HL-60 Cells --- p.80 / Chapter 3.2.4 --- Cytotoxic Effect of Sophoraflavanone G on Primary Normal Cells In Vitro --- p.83 / Chapter 3.2.5 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Sophoraflavanone G on the Human Promyelocytic Leukemia HL-60 Cells --- p.85 / Chapter 3.2.6 --- Effect of Sophoraflavanone G on the In Vivo Tumorigenicity of the HL-60 Cells --- p.88 / Chapter 3.2.7 --- Effect of Sophoraflavanone G on the Cell Cycle Profile of the HL-60 cells In Vitro --- p.90 / Chapter 3.2.8 --- Effect of Sophoraflavanone G on the Expression of Cell Cycle-regulatory Proteins in the HL-60 Cells --- p.93 / Chapter 3.2.9 --- Anti-proliferative Effect of Sophoraflavanone G on Multidrug-resistant (MDR) Leukemia Cell Line HL-60/MX2 Cells --- p.95 / Chapter 3.3 --- Discussion --- p.101 / Chapter Chapter Four: --- Studies on the Apoptosis- and Differentiation-inducing Activities of Sophoraflavanone G on Human Myeloid Leukemia Cells / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.2 --- Results --- p.114 / Chapter 4.2.1 --- Induction of DNA Fragmentation in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G --- p.114 / Chapter 4.2.2 --- Induction of Phosphatidylserine Externalization in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G as Detected by Annexin V-GFP and PI Double Staining Method --- p.116 / Chapter 4.2.3 --- Effects of Sophoraflavanone G on the Caspase Activities in the Human Promyelocytic Leukemia HL-60 Cells --- p.119 / Chapter 4.2.4 --- Induction of Mitochondrial Membrane Depolarization in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G --- p.124 / Chapter 4.2.5 --- Involvement of Bcl-2 Family Members in Sophoraflavanone G-induced Apoptosis in the Human Promyelocytic Leukemia HL-60 Cells --- p.128 / Chapter 4.2.6 --- Effects of Sophoraflavanone G on the Induction of Reactive Oxygen Species in the Human Promyelocytic Leukemia HL-60 Cells --- p.131 / Chapter 4.2.7 --- Effect of Sophoraflavanone G on the Intracellular Ca2+ Level in the Human Promyelocytic Leukemia HL-60 Cells --- p.134 / Chapter 4.2.8 --- Morphological Studies on the Sophoraflavanone G-treated Human Promyelocytic Leukemia HL-60 Cells --- p.136 / Chapter 4.2.9 --- Effect of Sophoraflavanone G on the NBT Reducing Activity of the Human Promyelocytic Leukemia HL-60 Cells --- p.138 / Chapter 4.3 --- Discussion --- p.140 / Chapter Chapter Five: --- Conclusions and Future Perspectives --- p.148 / References --- p.156

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 144-172).

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1991. / Vita. Includes bibliographical references (leaves [218]-228).

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on May 29, 2009). Includes bibliographical references.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted. Includes bibliographical references.