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

In this study, a novel compound was isolated and purified from the solid culture medium (potato dextrose agar) of shiitake 1358 strain through series of methods, such as ethanol precipitation, macroporous resin column separation, semi-preparative high performance liquid chromatography separation and preparative thin-layer chromatography separation. Analyzing spectra from fourier transform infra-red spectroscopy, gas chromatography-mass spectrometry, 1-dimension and 2-dimension nuclear magnetic resonance, the chemical structure of the novel compound was determined and named as 4-amino-5,6-dihydrobenzo[d]oxonine-2,7(1H,4H)-dione. It could inhibit the proliferation of HL-60 leukemia cells significantly and with an IC50 of 1.56 mug/ml (7.123 mumol/L) in the 72-hour treatment. From the results, it is suggested that this compound could activate the G2 phase checkpoint control of the cell cycle to arrest the cell cycle in G2 phase. In addition, it could suppress the replicative DNA synthesis to inhibit the proliferation of HL-60 leukemia cells. The more important is that this compound can induce the apoptosis of HL-60 leukemia cells significantly through intrinsic and extrinsic apoptotic pathways. The compound could induce intrinsic and extrinsic apoptosis through the regulation of the apoptosis-related proteins, such as Fas ligand, Bax, Bcl-2, Caspase 8, Caspase 9, and Caspase 3. For intrinsic pathway, the compound might upregulate Bax, downregulated Bcl-2, activated the Caspase 9, subsequently activated Capase 3, and ultimately led to cell death. For extrinsic pathway, the compound upregulated the Fas ligand, cleaved and activated Procaspase 8 to active Caspase 8, further cleaved and activated Procaspase 3 to active Caspase 3 to commit the cells to apoptosis. / Leukemia is a malignant cancer that involves the bone marrow and blood circulation systems. Leukemia results in the uncontrolled growth of abnormal (leukemic) white blood cells and may also invade other organs, including the liver, spleen, lymph nodes, testes, and brain. In 2007, about 44,240 new cases of leukemia were diagnosed and 21,790 patients died from all types of leukemias in USA. / Shiitake was first cultivated in China more than 800 years ago. It is the second most commonly cultivated edible mushrooms in the world nowadays. For a long time, shiitake has been valued for its unique taste and flavor and as a medicinal invigorant. According to ancient Chinese medicinal theory, consumption of shiitake was in favor of long life and good health. In China and Japan, shiitake has been used as both a food and a medicinal herb for thousands of years. It is the source of several well-studied preparations with proven pharmacological properties, especially the polysaccharide lentinan. Currently, most researches concentrate on the anticancer activities of the extracts from the fruiting body of shiitake, especially polysaccharides. Report about the anti-cancer effects of other components from the shiitake mushroom is scarce. The objectives of this investigations were: (1) to study the anticancer activities of brownish substances obtained during the solid medium culture of shiitake on specific cancer cell unes, especially HL60 cancer cell line; (2) to isolate and characterize the active compound(s) in the brown mushroom exudates; and (3) to propose the possible mechanism of actions, especially the function of the bcl-2 family genes and proteins. / by Guo, Yuming. / Adviser: Chung Hale Yin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3314. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 188-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Chiu Lai-Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 137-152). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / Chapter Chapter One --- General introduction / Chapter 1.1 --- Haemopoiesis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Regulation --- p.2 / Chapter 1.1.2.1 --- Stromal cells --- p.2 / Chapter 1.1.2.2 --- Haemopoietic regulator --- p.3 / Chapter 1.1.2.3 --- Haemopoietic regulator receptors and signal transduction --- p.5 / Chapter 1.2 --- Disorder of haemopoiesis --- p.9 / Chapter 1.2.1 --- Causes --- p.9 / Chapter 1.2.2 --- Types of leukemia --- p.9 / Chapter 1.2.3 --- Treatment of leukemia --- p.10 / Chapter 1.3 --- Prostaglandins --- p.13 / Chapter 1.3.1 --- Introduction --- p.13 / Chapter 1.3.2 --- Types and biosynthesis --- p.14 / Chapter 1.3.3 --- Prostaglandin receptors --- p.15 / Chapter 1.3.4 --- Prostaglandins and cell differentiation --- p.17 / Chapter 1.3.4.1 --- PGD2 and cell differentiation --- p.19 / Chapter 1.3.4.2 --- PGE2 and cell differentiation --- p.20 / Chapter 1.3.4.3 --- PGJ2 and cell differentiation --- p.22 / Chapter 1.4 --- WEHI-3B JCS cells --- p.25 / Chapter 1.5 --- Aims of study --- p.27 / Chapter Chapter Two --- Roles of Prostaglandin D2，E2 and J2 in WEHI-3B JCS myeloid leukemia cell differentiation / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Morphological studies of JCS cells --- p.28 / Chapter 2.1.2 --- Methods in determining cell proliferation --- p.29 / Chapter 2.1.3 --- Methods in determining differentiated cells --- p.31 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Cell line --- p.33 / Chapter 2.2.2 --- Chemicals --- p.33 / Chapter 2.2.3 --- Solutions and buffers --- p.34 / Chapter 2.3 --- Methods --- p.36 / Chapter 2.3.1 --- Microscopic studies of the JCS cells --- p.36 / Chapter 2.3.1.1 --- Histochemical staining of JCS --- p.36 / Chapter 2.3.1.2 --- Transmission electronic microscopic --- p.36 / Chapter 2.3.2 --- [3H]-thymidine incorporation assay --- p.37 / Chapter 2.3.3 --- MTT assay --- p.37 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- Histochemical staining of JCS cells --- p.38 / Chapter 2.4.2 --- Electron microscopy --- p.40 / Chapter 2.4.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.44 / Chapter 2.4.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.48 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- Morphological differentiation of JCS cells --- p.53 / Chapter 2.5.2 --- The ultra-structures of JCS cells --- p.53 / Chapter 2.5.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.54 / Chapter 2.5.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.55 / Chapter Chapter Three --- Roles of Prostaglandin E2 in normal haemopoiesis and the detection of PGE2 receptors expression in JCS and bone marrow cells / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.1.1 --- Colony assay --- p.57 / Chapter 3.1.2 --- The use of RT-PCR --- p.58 / Chapter 3.1.3 --- Prostaglandin E receptors --- p.59 / Chapter 3.2 --- Materials --- p.62 / Chapter 3.2.1 --- Bone marrow cells --- p.62 / Chapter 3.2.2 --- Cell line --- p.62 / Chapter 3.2.3 --- Chemicals --- p.62 / Chapter 3.2.4 --- Primers --- p.63 / Chapter 3.2.5 --- Solutions and buffers --- p.64 / Chapter 3.2.6 --- Enzymes and reagents --- p.65 / Chapter 3.3 --- Methods --- p.66 / Chapter 3.3.1 --- Titration of mouse IL-3 --- p.66 / Chapter 3.3.2 --- Determination of suitable IL-3 concentration for growth of bone marrow cells in colony assay --- p.66 / Chapter 3.3.2.1 --- Preparation of bone marrow cells --- p.66 / Chapter 3.3.2.2 --- Preparation of culture medium for colony assay --- p.67 / Chapter 3.3.3 --- Investigation of the effect of PGE2 on normal haemopoiesis by colony assay --- p.68 / Chapter 3.3.4 --- Detection of PGE2 receptors expression on JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.1 --- Preparation of cell lysates --- p.68 / Chapter 3.3.4.2 --- Preparation of total RNA of JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.3 --- RT-PCR --- p.69 / Chapter 3.4 --- Results --- p.71 / Chapter 3.4.1 --- Titration of mouse IL-3 --- p.71 / Chapter 3.4.2 --- Effect of mouse IL-3 on normal haemopoiesis --- p.73 / Chapter 3.4.3 --- Effect of PGE2 on mouse IL-3 driven normal bone marrow cell differentiation --- p.76 / Chapter 3.4.4 --- Analysis of total RNA prepared from uninduced JCS cells and bone marrow cells --- p.79 / Chapter 3.4.5 --- "Expression of gapdh in heart, liver, spleen, JCS and bone marrow cells" --- p.81 / Chapter 3.4.6 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.82 / Chapter 3.5 --- Discussion --- p.84 / Chapter 3.5.1 --- Effect of PGE2 on IL-3 driven normal bone marrow cells differentiation --- p.84 / Chapter 3.5.2 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.85 / Chapter Chapter Four --- Gene expression profile of JCS cells under 5 hours of PGE2 induction / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.1.1 --- Review of methods studying differential gene expression --- p.88 / Chapter 4.1.2 --- The choice of method studying differential gene expression --- p.92 / Chapter 4.1.3 --- The microarray --- p.93 / Chapter 4.2 --- Materials --- p.95 / Chapter 4.2.1 --- Cell line --- p.95 / Chapter 4.2.2 --- Kits --- p.95 / Chapter 4.2.3 --- Chemicals --- p.95 / Chapter 4.2.4 --- Solutions and buffers --- p.96 / Chapter 4.2.5 --- Reagents --- p.97 / Chapter 4.3 --- Methods --- p.98 / Chapter 4.3.1 --- Preparation of total RNA from PGE2 induced JCS cells --- p.98 / Chapter 4.3.2 --- Preparation of cDNA probes --- p.98 / Chapter 4.3.2.1 --- Probe synthesis from total RNA --- p.98 / Chapter 4.3.2.2 --- Column chromatography --- p.99 / Chapter 4.3.3 --- Hybridizing cDNA probes to the Atlas Array --- p.99 / Chapter 4.4 --- Results --- p.101 / Chapter 4.4.1 --- Spectrophotometric analysis of total RNA after ethanol precipitation --- p.101 / Chapter 4.4.2 --- Hybridization of cDNA probes to Atlas Array --- p.102 / Chapter 4.5 --- Discussion --- p.121 / Chapter 4.5.1 --- Genes with increased expression --- p.121 / Chapter 4.5.2 --- Genes with decrease expression --- p.127 / Chapter 4.5.3 --- Study of gene expression profile by microarray --- p.128 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.2 --- Roles of PGE2 in JCS cells differentiation --- p.131 / Chapter 5.3 --- Roles of PGE2 in normal haemopoiesis --- p.134 / Chapter 5.4 --- Further studies --- p.135 / References --- p.137

Wong, Wing Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-105). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Abstract (Chinese Version) --- p.v / Table of Contents --- p.vi / List of Figures --- p.ix / List of Abbreviations --- p.xi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of cancer --- p.1 / Chapter 1.2 --- Acute lymphoblastic leukemia --- p.3 / Chapter 1.2.1 --- T-cell acute lymphoblastic leukemia --- p.5 / Chapter 1.2.1.1 --- Chemotherapy / Chapter 1.2.1.2 --- Induction therapy / Chapter 1.2.1.3 --- Intensification therapy / Chapter 1.2.1.4 --- Maintenance therapy --- p.6 / Chapter 1.2.2 --- Chemoresistance in T-ALL / Chapter 1.3 --- Apoptosis and cancer --- p.7 / Chapter 1.3.1 --- Chemoresistance --- p.9 / Chapter 1.4 --- Caspase-dependent apoptosis --- p.10 / Chapter 1.4.1 --- Regulation of caspase-dependent apoptosis / Chapter 1.4.2 --- Initiation of apoptosis --- p.11 / Chapter 1.4.3 --- Exrtinsic pathway / Chapter 1.4.4 --- Intrinsic mitochondrial pathway --- p.15 / Chapter 1.4.4.1 --- Regulation of apoptosis by Bcl-2 family proteins --- p.16 / Chapter 1.4.4.2 --- Reactive Oxygen Species --- p.19 / Chapter 1.5 --- Selenium --- p.23 / Chapter 1.5.1 --- Importance of Se to human health --- p.25 / Chapter 1.5.2 --- Cancer chemoprevention by Se --- p.27 / Chapter 1.5.3 --- Preclinical studies --- p.28 / Chapter 1.5.4 --- Clinical investigations / Chapter 1.5.5 --- Mechanisms of action by selenocompounds --- p.29 / Chapter 1.6 --- Aims of current study --- p.31 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell culture --- p.32 / Chapter 2.2 --- Measurement of growth and survival of T-ALL cell lines / Chapter 2.3 --- Induction and quantification of apoptosis --- p.34 / Chapter 2.4 --- Western blotting / Chapter 2.4.1 --- Protein extraction and determination of protein concentration / Chapter 2.4.2 --- SDS-PAGE and immunodetection --- p.35 / Chapter 2.5 --- Analysis of mitochondrial membrane potential --- p.36 / Chapter 2.6 --- Measurement of ROS generation --- p.37 / Chapter 2.7 --- Verification of ROS generation via the addition of N-Acetyl-L-cysteine and glutathione / Chapter 2.8 --- Statistical analysis --- p.38 / Chapter Chapter 3 --- Results / Chapter 3.1 --- SeC induces prominent growth inhibition on Sup-T1 --- p.39 / Chapter 3.2 --- SeC induces S-phase arrest in cell cycle and triggers apoptosis in Sup-T1 --- p.44 / Chapter 3.3 --- SeC triggers DNA fragmentation in Sup-T1 --- p.48 / Chapter 3.4 --- SeC induces PARP cleavage in Sup-T1 --- p.52 / Chapter 3.5 --- SeC activates caspases in Sup-T1 --- p.53 / Chapter 3.6 --- SeC abrogates mitochondrial membrane potential in Sup-T1 cells --- p.56 / Chapter 3.7 --- SeC modulates expressions of Bcl-2 members and activates Bim and Bid in Sup-T1 --- p.61 / Chapter 3.8 --- SeC induces ROS production in Sup-T1 --- p.64 / Chapter 3.9 --- Antioxidants protect Sup-T1 cells from SeC-induced growth inhibition --- p.66 / Chapter 3.10 --- Antioxidants protect Sup-T1 cells from SeC-induced apoptosis --- p.69 / Chapter 3.11 --- Antioxidants effectively block SeC-induced ROS generation in Sup-T1 cells --- p.72 / Chapter 3.12 --- SeC induces mitochondrial membrane permeabilization via ROS-mediated mechanisms Sup-T1 cells --- p.75 / Chapter Chapter 4 --- Discussion --- p.79 / Conclusion --- p.87 / References --- p.90

A leucemia linfóide aguda nos adultos apresenta prognóstico reservado. Os objetivos deste estudo são descrição e análise de parâmetros clínicos, laboratoriais e fatores prognósticos em 102 pacientes tratados com diferentes protocolos de quimioterapia no período de 1990 a 2005, no Serviço de Hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Em estudo de coorte retrospectivo, com exclusão de LLA subtipo L3 (FAB) ou B-IV (EGIL), foram analisadas a taxa de remissão completa (RC), sobrevida global (SG) e sobrevida livre de doença (SLD) para a população geral e para os dois principais protocolos de tratamento. A análise estatística foi feita pelo programa SPSS 10.0. Associação entre variáveis, fatores prognósticos e resposta foram observados através do teste ?2 de Person. Curvas de SG e SLD foram construídas pelo método de Kaplan-Meier e as diferenças analisadas pelo teste de log-rank. A idade média foi de 30,6 anos (12 a 82 anos) e predominou o sexo masculino (55,9%). Ao diagnóstico, os achados clínicos foram: fadiga (58,2%), esplenomegalia (59,7%), hepatomegalia (54,6%), linfadenopatia (52,6), febre (38,8%), dor óssea(28,6%), sangramento (27,5%) e cefaléia (15,3%). Envolvimento do sistema nervoso central (SNC) foi detectado em 11 (11,8%) pacientes, enquanto envolvimento testicular acometeu um paciente. O valor médio de hemoglobina, leucócitos e plaquetas foram 8,5g/dl, 84.341/mm3 e 76.275/mm3, respectivamente. 98,7% dos pacientes apresentaram linfoblastos no sangue periférico. A classificação FAB foi igualmente observada entre os tipos L1 e L2. As LLA B e T foram observadas em 69,7% e 30,2%, respectivamente. O cariótipo foi realizado em 40 pacientes, e t (9;22) foi identificada em 20% (8/40) dos casos. Os pacientes foram tratados com quatro diferentes protocolos: BFM 86 modificado (BFM 86M) em 47,15% (48/102), Linker et al em 39,2% (40/102), Lister et al em 5,9% (6/102) e CHOP em 7,8% (8/102). Na análise para a população geral, na fase de indução, 70,6% (65/92) dos pacientes entraram em RC. Idade inferior a 18 anos e ausência de infiltração de SNC foram fatores preditores positivos de resposta em análise multivariada (p=0,03). Com mediana de seguimento de 49 meses, observamos taxa de 30,5% e 27% para SG e SLD em 4 anos. Ausência de sangramento e hepatomegalia, ao diagnóstico, e idade < 35 anos estiveram associados à maior SG através de análise multivariada (p=0,01). Os dois protocolos com maior número de pacientes, apresentaram distribuição semelhante de parâmetros clínicos e laboratoriais, a exceção da variável FAB. RC foi obtida em 76,7% e 63,9% dos pacientes tratados respectivamente com os protocolos BFM 86M e Linker (p=0,21). A SG foi de 49,5% com o BFM 86M em 4 anos Vs 16% com o protocolo Linker (p=0,004). Observou-se que o protocolo BFM86M teve melhor SG para pacientes com idade <35 anos (p=0,01), sem sangramento e hepatomegalia ao diagnóstico (p=0,03 e p=0,01) e sem leucocitose (B <30.000mm3 e T <100.000mm) (p=0,04); enquanto que pacientes com LLA T tratados com o protocolo Linker apresentaram SG inferior (p=0,05). A diferença de SLD entre os dois protocolos não foi significativa (p=0,58), entretanto na faixa etária entre 21-35 anos, o protocolo BFM se mostrou superior (p=0,03). Verificamos que o BFM 86M é superior ao Linker et al, sendo um bom protocolo para tratamento de LLA em pacientes adolescentes e adultos jovens sem fatores de risco. / Acute lymphoblastic leukemia in adults has a poor outcome. The aim of this study is to describe and evaluate clinical, laboratory and prognostic factors in 102 patients reated with different protocols of chemotherapy from 1990 to 2005. Adult ALLsubtype L3 (FAB) or B-IV (EGIL) was excluded. We evaluated complete remission (CR), overall survival (OS) and disease free survival (DFS) rates for the whole population and for the two principal treatment protocols. This retrospective cohort was done in hematology department of the FMUSP. Statistical analysis was done by SPSS 10.0. The association of features and prognosis was assessed by Person\'s chi-square. OS and DFS curves were constructed by Kaplan-Meier method and the differences were calculated by the log-rank test. Mean age was 30,6 (12 to 82) years and 55,9% was male. Clinical findings, at diagnosis, were fatigue (58,2%), splenomegaly (59,7%), hepatomegaly (54,6%), ymphadenopathy (52,6%), fever (38,8%), bone pain (28,6%), bleeding (27,5%) and headache (15,3%). Involvement of central nervous system (CNS) was detected in 11 (11,8%) patients and testicular involvement was observed in one patient. Mean blood values were 8,5g/dl, 84.341/mm3 and 76.275/mm3 for hemoglobin, leucocytes and platelets respectively. 98,7% of the patients presented with lymphoblasts in peripheral blood. FAB classification was equally observed between L1 and L2. B and T ALL was noted in 69,7% and 30,2% respectively. Karyotype analysis was performed in 40 cases, where Philadelphia chromosome (ph) was identified in 20% (8/40) of them. Patients were treated with four different protocols: BFM 86 modified (BFM 86M) in 47,1% (48/102), Linker et al in 39,2% (40/102), Lister et al in 5,9% (6/102) and CHOP in 7,8% (8/102) of the patients. In the judgment for the entire population, in induction treatment, 70,6% (65/92) of the patients had CR. Age below 18 years and no infiltration in CNS were positive factors for CR in multivariate analyses (p=0,03). In a median follow up of 49 months, we have observed a 4 years OS and DFS of 30,5% and 27% respectively. No bleeding and hepatomegaly, at diagnosis, and age less than 35 years were factors associated a better OS in multivariate analyses (p=0, 01). Protocols with highest number of patients (BFM and Linker) showed the same distribution of clinical and laboratory factors; exception FAB classification. CR were seen in 76,7% and 63,9% of the patients treated with BFM 86M and Linker respectively. (p=0,21). OS was 49,5% with BFM protocol in 4 years Vs 16% with Linker (p=0,004). We observed a better OS for patients with age below 35 years (p=0,01), no bleeding and no hepatomegaly at diagnosis (p=0,03 ; p=0,01) and no leucocytosis ( B < 30000/mm3 and T < 100000/mm3) treated with BFM 86M; however ALL - T treated with Linker protocols had inferior OS (p=0,05). DFS between protocols wasn\'t significant (p=0,58), but with age between 21 and 35 years BFM was better (p=0,03). We conclude that BFM 86M is superior than Linker et al and it is a good treatment for childhood / young adults without risk factors

Tang Sze-Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknownledgement --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Table of Contents --- p.xi / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- Trichosanthes spp --- p.1 / Chapter 1.1.1 --- Use of Trichosanthes --- p.2 / Chapter 1.1.2 --- Trichosanthin --- p.2 / Chapter 1.1.3 --- Karasurin --- p.5 / Chapter 1.1.4 --- Ribosome Inactivating Proteins --- p.6 / Chapter 1.1.5 --- Immunosuppresion --- p.7 / Chapter 1.1.6 --- Anti-Cancer Activity --- p.8 / Chapter 1.1.7 --- Miscellaneous Uses --- p.8 / Chapter 1.2 --- Cancer --- p.9 / Chapter 1.2.1 --- Oncogenes --- p.10 / Chapter 1.2.2 --- Tumor-Suppressor Genes --- p.11 / Chapter 1.2.3 --- Stability Genes --- p.12 / Chapter 1.2.4 --- Types of Cancer --- p.13 / Chapter 1.2.5 --- Cancer Therapy --- p.13 / Chapter 1.2.6 --- Apoptosis --- p.14 / Chapter 1.3 --- Chronic Myelogenous Leukemia (CML) --- p.17 / Chapter 1.3.1 --- Philadelphia Chromosome and BCR-ABL gene --- p.18 / Chapter 1.3.2 --- Treatment of CML --- p.20 / Chapter 1.4 --- Dendritic Differentiation of LC976 on K-562 --- p.20 / Chapter 1.4.1 --- Dendritic Cells --- p.21 / Chapter 1.4.2 --- Cancer Vaccine Development of Leukemia --- p.22 / Chapter 1.4.3 --- Dendritic differentiation of K-562 cells --- p.23 / Chapter 1.5 --- Perspective of the Project --- p.23 / Chapter Chapter 2 - --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Chemicals and Reagents --- p.25 / Chapter 2.1.2 --- Bioassay Kits --- p.26 / Chapter 2.1.3 --- Human Cell Lines --- p.26 / Chapter 2.1.4 --- Lab Wares and Equipments --- p.28 / Chapter 2.2 --- Extraction of LC9 --- p.76 / Chapter 2.2.1 --- Chemical Properties of the Lead Compound --- p.28 / Chapter 2.2.2 --- Crude Extraction of Trichosanthes sp --- p.29 / Chapter 2.2.3 --- Purification by Reversed-Phase Column --- p.29 / Chapter 2.2.4 --- Lyophilization and Preparation of LC976 --- p.31 / Chapter 2.3 --- Anti-Proliferation Effect of LC976 on Human Cell Lines / Chapter 2.3.1 --- Maintenance of Cell Lines --- p.32 / Chapter 2.3.2 --- MTT Assay --- p.32 / Chapter 2.3.3 --- BrdU Cell Proliferation ELISA --- p.34 / Chapter 2.4 --- Apoptosis Induction on K-5 --- p.62 / Chapter 2.4.1 --- PI Staining --- p.35 / Chapter 2.4.2 --- Annexin V-FITC FACS Analysis --- p.36 / Chapter 2.4.3 --- Caspase Activation --- p.37 / Chapter 2.5 --- Effect on Normal Human Lymphocytes / Chapter 2.5.1 --- Preparation of Human Normal Lymphocytes --- p.38 / Chapter 2.5.2 --- MTT Cell Viability Assay --- p.38 / Chapter 2.5.3 --- PI Staining --- p.39 / Chapter 2.5.4 --- Annexin V-FITC FACS Analysis --- p.39 / Chapter Chapter 3 - --- Results / Chapter 3.1 --- Extraction of LC976 --- p.40 / Chapter 3.2 --- LC976 Inhibited Proliferation of Human Cell Lines / Chapter 3.2.1 --- MTT Assay --- p.41 / Chapter 3.2.2 --- BrdU Cell Proliferation ELISA --- p.52 / Chapter 3.3 --- LC976 Induced Apoptosis in K-562 Cells / Chapter 3.3.1 --- PI Staining --- p.63 / Chapter 3.3.2 --- Annexin V-FITC FACS Analysis --- p.70 / Chapter 3.3.3 --- Caspase Activation --- p.73 / Chapter 3.4 --- Effect on Normal Human Lymphocytes / Chapter 3.4.1 --- MTT Cell Viability Assay --- p.76 / Chapter 3.4.2 --- PI Staining --- p.78 / Chapter 3.4.3 --- Annexin V-FITC FACS Analysis --- p.82 / Chapter Chapter 4 - --- Discussion / Chapter 4.1 --- Extraction of LC976 --- p.85 / Chapter 4.2 --- LC976 Inhibited Proliferation of Human Cell Lines / Chapter 4.2.1 --- MTT Assay --- p.86 / Chapter 4.2.2 --- BrdU Cell Proliferation ELISA --- p.88 / Chapter 4.3 --- LC976 induced Apoptosis in K-562 Cells / Chapter 4.3.1 --- PI Staining --- p.90 / Chapter 4.3.2 --- Annexin V-FITC Analysis --- p.95 / Chapter 4.3.3 --- Caspase Activation --- p.96 / Chapter 4.4 --- Effect on Normal Human Lymphocytes / Chapter 4.4.1 --- MTT Cell Viability Assay --- p.98 / Chapter 4.4.2 --- PI Staining --- p.99 / Chapter 4.4.3 --- Annexin V-FITC FACS Analysis --- p.100 / Chapter 4.5 --- Conclusion --- p.103 / Reference --- p.104

A leucemia linfóide aguda nos adultos apresenta prognóstico reservado. Os objetivos deste estudo são descrição e análise de parâmetros clínicos, laboratoriais e fatores prognósticos em 102 pacientes tratados com diferentes protocolos de quimioterapia no período de 1990 a 2005, no Serviço de Hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Em estudo de coorte retrospectivo, com exclusão de LLA subtipo L3 (FAB) ou B-IV (EGIL), foram analisadas a taxa de remissão completa (RC), sobrevida global (SG) e sobrevida livre de doença (SLD) para a população geral e para os dois principais protocolos de tratamento. A análise estatística foi feita pelo programa SPSS 10.0. Associação entre variáveis, fatores prognósticos e resposta foram observados através do teste ?2 de Person. Curvas de SG e SLD foram construídas pelo método de Kaplan-Meier e as diferenças analisadas pelo teste de log-rank. A idade média foi de 30,6 anos (12 a 82 anos) e predominou o sexo masculino (55,9%). Ao diagnóstico, os achados clínicos foram: fadiga (58,2%), esplenomegalia (59,7%), hepatomegalia (54,6%), linfadenopatia (52,6), febre (38,8%), dor óssea(28,6%), sangramento (27,5%) e cefaléia (15,3%). Envolvimento do sistema nervoso central (SNC) foi detectado em 11 (11,8%) pacientes, enquanto envolvimento testicular acometeu um paciente. O valor médio de hemoglobina, leucócitos e plaquetas foram 8,5g/dl, 84.341/mm3 e 76.275/mm3, respectivamente. 98,7% dos pacientes apresentaram linfoblastos no sangue periférico. A classificação FAB foi igualmente observada entre os tipos L1 e L2. As LLA B e T foram observadas em 69,7% e 30,2%, respectivamente. O cariótipo foi realizado em 40 pacientes, e t (9;22) foi identificada em 20% (8/40) dos casos. Os pacientes foram tratados com quatro diferentes protocolos: BFM 86 modificado (BFM 86M) em 47,15% (48/102), Linker et al em 39,2% (40/102), Lister et al em 5,9% (6/102) e CHOP em 7,8% (8/102). Na análise para a população geral, na fase de indução, 70,6% (65/92) dos pacientes entraram em RC. Idade inferior a 18 anos e ausência de infiltração de SNC foram fatores preditores positivos de resposta em análise multivariada (p=0,03). Com mediana de seguimento de 49 meses, observamos taxa de 30,5% e 27% para SG e SLD em 4 anos. Ausência de sangramento e hepatomegalia, ao diagnóstico, e idade < 35 anos estiveram associados à maior SG através de análise multivariada (p=0,01). Os dois protocolos com maior número de pacientes, apresentaram distribuição semelhante de parâmetros clínicos e laboratoriais, a exceção da variável FAB. RC foi obtida em 76,7% e 63,9% dos pacientes tratados respectivamente com os protocolos BFM 86M e Linker (p=0,21). A SG foi de 49,5% com o BFM 86M em 4 anos Vs 16% com o protocolo Linker (p=0,004). Observou-se que o protocolo BFM86M teve melhor SG para pacientes com idade <35 anos (p=0,01), sem sangramento e hepatomegalia ao diagnóstico (p=0,03 e p=0,01) e sem leucocitose (B <30.000mm3 e T <100.000mm) (p=0,04); enquanto que pacientes com LLA T tratados com o protocolo Linker apresentaram SG inferior (p=0,05). A diferença de SLD entre os dois protocolos não foi significativa (p=0,58), entretanto na faixa etária entre 21-35 anos, o protocolo BFM se mostrou superior (p=0,03). Verificamos que o BFM 86M é superior ao Linker et al, sendo um bom protocolo para tratamento de LLA em pacientes adolescentes e adultos jovens sem fatores de risco. / Acute lymphoblastic leukemia in adults has a poor outcome. The aim of this study is to describe and evaluate clinical, laboratory and prognostic factors in 102 patients reated with different protocols of chemotherapy from 1990 to 2005. Adult ALLsubtype L3 (FAB) or B-IV (EGIL) was excluded. We evaluated complete remission (CR), overall survival (OS) and disease free survival (DFS) rates for the whole population and for the two principal treatment protocols. This retrospective cohort was done in hematology department of the FMUSP. Statistical analysis was done by SPSS 10.0. The association of features and prognosis was assessed by Person\'s chi-square. OS and DFS curves were constructed by Kaplan-Meier method and the differences were calculated by the log-rank test. Mean age was 30,6 (12 to 82) years and 55,9% was male. Clinical findings, at diagnosis, were fatigue (58,2%), splenomegaly (59,7%), hepatomegaly (54,6%), ymphadenopathy (52,6%), fever (38,8%), bone pain (28,6%), bleeding (27,5%) and headache (15,3%). Involvement of central nervous system (CNS) was detected in 11 (11,8%) patients and testicular involvement was observed in one patient. Mean blood values were 8,5g/dl, 84.341/mm3 and 76.275/mm3 for hemoglobin, leucocytes and platelets respectively. 98,7% of the patients presented with lymphoblasts in peripheral blood. FAB classification was equally observed between L1 and L2. B and T ALL was noted in 69,7% and 30,2% respectively. Karyotype analysis was performed in 40 cases, where Philadelphia chromosome (ph) was identified in 20% (8/40) of them. Patients were treated with four different protocols: BFM 86 modified (BFM 86M) in 47,1% (48/102), Linker et al in 39,2% (40/102), Lister et al in 5,9% (6/102) and CHOP in 7,8% (8/102) of the patients. In the judgment for the entire population, in induction treatment, 70,6% (65/92) of the patients had CR. Age below 18 years and no infiltration in CNS were positive factors for CR in multivariate analyses (p=0,03). In a median follow up of 49 months, we have observed a 4 years OS and DFS of 30,5% and 27% respectively. No bleeding and hepatomegaly, at diagnosis, and age less than 35 years were factors associated a better OS in multivariate analyses (p=0, 01). Protocols with highest number of patients (BFM and Linker) showed the same distribution of clinical and laboratory factors; exception FAB classification. CR were seen in 76,7% and 63,9% of the patients treated with BFM 86M and Linker respectively. (p=0,21). OS was 49,5% with BFM protocol in 4 years Vs 16% with Linker (p=0,004). We observed a better OS for patients with age below 35 years (p=0,01), no bleeding and no hepatomegaly at diagnosis (p=0,03 ; p=0,01) and no leucocytosis ( B < 30000/mm3 and T < 100000/mm3) treated with BFM 86M; however ALL - T treated with Linker protocols had inferior OS (p=0,05). DFS between protocols wasn\'t significant (p=0,58), but with age between 21 and 35 years BFM was better (p=0,03). We conclude that BFM 86M is superior than Linker et al and it is a good treatment for childhood / young adults without risk factors