Note:

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

The tumour microenvironment consists of an immunosuppressive niche created by the complex interactions between cancer cells and surrounding stromal cells. A critical component of this environment are myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immature myeloid cells arrested at different stages of differentiation and expanded in response to a variety of tumour factors. MDSCs exert diverse effects in modulating the interactions between immune effector cells and malignant cells. An increased presence of MDSCs is associated with tumour progression, poorer outcomes, and decreased effectiveness of immunotherapeutic strategies. In this project, we sought to quantify and characterise MDSC populations in patients with Acute Myeloid Leukaemia (AML) and delineate the mechanisms underlying their expansion. We have demonstrated that immune suppressive MDSCs are expanded in the peripheral blood and bone marrow of patients with AML. Furthermore, AML cells secrete extra-cellular vesicles (EVs) that skew the tumour microenvironment from antigen-presentation to a tumour tolerogenic environment, through the expansion of MDSCs. We then demonstrated that MDSC expansion is dependent on tumour and EV expression of the oncoproteins MUC1 and c-Myc. Furthermore, we determined that MUC1 signalling promotes c-MYC expression in a microRNA (miRNA) dependent mechanism. This observation lead us to elucidate the critical role of MUC1 in suppressing microRNA-genesis in AML, via the down-regulation of the DICER protein, a key component of miRNA processing machinery. Finally, exploiting this critical pathway, we showed that MDSCs can be targeted by MUC1 inhibition or by the use of a novel hypomethylating agent SGI-110.

Previous studies had demonstrated the presence of the p70 (CAMAL) molecule in human myeloid leukemia cells and the promyelocytic leukemia cell line HL60, but not in equivalent preparations of normal cells (Malcolm et al., 1982, 1984; Shipman et al., 1983; Logan et al., 1984). Subsequent studies demonstrated that the p70 (CAMAL) protein was detectable and expressed in human myeloid leukemia cells and the leukemic cell lines HL60, KG1, K562 and U937. The association of p70 (CAMAL) expression with human myeloid leukemia cells prompted its consideration as a candidate leukemia-associated antigen. The demonstration, following CAMAL purification and peptide sequencing, that two tryptic peptides (tp27, tp31) displayed significant homology to sequences present in human serum albumin (HSA) and human alpha-1-fetoprotein (AFP), while one tryptic peptide (tp20) displayed unique peptide sequence, suggested that CAMAL might represent a protein that was structurally and functionally related to the albumins. Consequently, a comparative biochemical analysis of CAMAL and HSA was initiated. The results of the comparative studies demonstrated that although CAMAL and HSA shared conformational antigenic determinants, both proteins were also shown to be distinct molecules by a number of other criteria. The possibility that the CAMAL preparation, used for protein sequencing and comparative studies, was contaminated with HSA was thought likely, in view of the HSA/AFP-related peptide sequences from the CAMAL tryptic peptide sequence analysis. However, other results, particularly the antibody reactivity and ligand binding studies, showed that the CAMAL preparation was not contaminated with HSA. The unique CAMAL tryptic peptide (tp20) sequence supported further the contention that CAMAL was a distinct protein with regions homologous to HSA and AFP. Further analysis of the CAMAL molecule, through extensive protein sequencing, will be, in all likelihood, the only means by which to establish the degree of relatedness between CAMAL, HSA and AFP. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

No description available.

No description available.

No description available.

Leung Po-Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 204-235). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / 撮要 --- p.x / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis & Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.2 --- Leukemia - Aberrant Hematopoiesis --- p.4 / Chapter 1.1.2.1 --- Classification and Epidemiology of Leukemia --- p.4 / Chapter 1.1.2.2 --- Pathophysiology and Etiology of Leukemia --- p.7 / Chapter 1.1.2.3 --- Conventional Treatments for Leukemia --- p.9 / Chapter 1.1.2.4 --- New Avenues for Leukemia Therapy --- p.11 / Chapter 1.2 --- Coumarins: General Properties and Pharmacological Activities --- p.13 / Chapter 1.2.1 --- Introduction to Coumarins --- p.13 / Chapter 1.2.1.1 --- Historical Development of Coumarins --- p.13 / Chapter 1.2.1.2 --- Occurrence and Functions of Coumarins in Plants --- p.13 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Coumarins --- p.14 / Chapter 1.2.2.1 --- Chemical Structures of Coumarins --- p.14 / Chapter 1.2.2.2 --- Biosynthesis of Coumarins --- p.18 / Chapter 1.2.2.3 --- Toxicology of Coumarins --- p.18 / Chapter 1.2.2.4 --- Metabolic Pathways and Pharmacokinetics of Coumarins --- p.19 / Chapter 1.2.3 --- Pharmacological Activities of Coumarins --- p.22 / Chapter 1.2.3.1 --- Anti-edema and Anti-inflammatory Activities --- p.22 / Chapter 1.2.3.2 --- Immunomodulatory Activity --- p.23 / Chapter 1.2.3.3 --- Anti-tumor Activity --- p.23 / Chapter 1.2.3.3.1 --- Mode of Entry of Coumarins into Tumor Cells --- p.23 / Chapter 1.2.3.3.2 --- Anti-carcinogenic Effect --- p.24 / Chapter 1.2.3.3.3 --- Anti-proliferative Activity --- p.25 / Chapter 1.2.3.3.4 --- Induction of Cell Differentiation --- p.26 / Chapter 1.2.3.3.5 --- Other Biological Activities --- p.26 / Chapter 1.2.4 --- Clinical Applications of Coumarins --- p.27 / Chapter 1.2.4.1 --- Treatment of Lymphoedema and Other High-protein Edemas --- p.27 / Chapter 1.2.4.2 --- Treatment of Thermal Injuries --- p.27 / Chapter 1.2.4.3 --- Therapeutic Agent for Renal Cell Carcinoma --- p.28 / Chapter 1.2.4.4 --- Therapy of Prostate Cancer --- p.29 / Chapter 1.3 --- Tumor Models Used in This Study --- p.30 / Chapter 1.3.1 --- Myeloid Leukemias --- p.30 / Chapter 1.3.1.1 --- HL-60 --- p.30 / Chapter 1.3.1.2 --- K562 --- p.30 / Chapter 1.3.1.3 --- EoL-1 --- p.30 / Chapter 1.3.1.4 --- WEHI-3B JCS --- p.31 / Chapter 1.3.2 --- Neuroblastoma - Neuro-2a BU-1 --- p.31 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.33 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Animals --- p.36 / Chapter 2.1.2 --- Cell Lines --- p.36 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.37 / Chapter 2.1.4 --- [methyl-3H] Thymidine (3H-TdR) --- p.40 / Chapter 2.1.5 --- Methylthiazoletetrazolium (MTT) --- p.41 / Chapter 2.1.6 --- Nitro Blue Tetrazolium (NBT) --- p.41 / Chapter 2.1.7 --- Liquid Scintillation Cocktail --- p.42 / Chapter 2.1.8 --- Reagents and Buffers for Flow Cytometry --- p.42 / Chapter 2.1.9 --- Mouse Anti-MAP-2 Monoclonal Antibody --- p.44 / Chapter 2.1.10 --- Reagents for DNA Extraction --- p.44 / Chapter 2.1.11 --- Reagents for Total RNA Isolation --- p.45 / Chapter 2.1.12 --- Reagents and Buffers for RT-PCR --- p.46 / Chapter 2.1.13 --- Reagents and Buffers for Gel Electrophoresis --- p.49 / Chapter 2.1.14 --- Reagents and Buffers for Western Blot Analysis --- p.50 / Chapter 2.1.15 --- Reagents for Measuring Caspase Activity --- p.58 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.61 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.61 / Chapter 2.2.3 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.62 / Chapter 2.2.4 --- Determination of Cell Viability --- p.62 / Chapter 2.2.5 --- Cell Morphology Study --- p.63 / Chapter 2.2.6 --- Immunocytochemistry --- p.64 / Chapter 2.2.7 --- Confocal Microscopy --- p.64 / Chapter 2.2.8 --- NBT Reduction Assay --- p.65 / Chapter 2.2.9 --- In vivo Tumorigenicity Assay --- p.65 / Chapter 2.2.10 --- In vivo Anti-tumor Study --- p.65 / Chapter 2.2.11 --- Measurement of In vivo Macrophage Migration --- p.66 / Chapter 2.2.12 --- Measurement of Cytokine Production by ELISA --- p.66 / Chapter 2.2.13 --- Measurement of Apoptosis by DNA Fragmentation Analysis --- p.67 / Chapter 2.2.14 --- Determination of the Mitochondrial Membrane Potential --- p.67 / Chapter 2.2.15 --- Cell Cycle/DNA Content Evaluation --- p.68 / Chapter 2.2.16 --- Nitric oxide/Annexin V-PE Dual Sensor Assay --- p.68 / Chapter 2.2.17 --- Gene Expression Study --- p.68 / Chapter 2.2.18 --- Protein Expression Study --- p.71 / Chapter 2.2.19 --- Measurement of Caspase Activity --- p.74 / Chapter 2.2.20 --- Statistical Analysis --- p.75 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-TUMOR ACTIVITIES OF COUMARINS ON MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Results --- p.18 / Chapter 3.2.1 --- Differential Anti-proliferative Effect of Coumarins on Various Leukemic Cell Lines In Vitro --- p.78 / Chapter 3.2.2 --- Cytotoxic Effect of Coumarins on Various Leukemic Cell Lines In Vitro --- p.91 / Chapter 3.2.3 --- "Kinetic, Reversibility and Stability Studies of the Anti- proliferative Effect of Coumarins on the Leukemia JCS cells" --- p.94 / Chapter 3.2.4 --- Induction of DNA Fragmentation in Myeloid Leukemia Cells by Coumarins --- p.100 / Chapter 3.2.5 --- Effect of Coumarins on the Cell Cycle Kinetics of the Leukemia JCS Cells In Vitro --- p.107 / Chapter 3.2.6 --- Effect of Coumarins on the In Vivo Tumorigenicity of the Leukemia JCS Cells --- p.112 / Chapter 3.2.7 --- Effect of Esculetin on the In Vivo Growth of the Leukemia JCS cells in Syngeneic Mice --- p.115 / Chapter 3.3 --- Discussion --- p.117 / Chapter CHAPTER 4: --- AN INVESTIGATION ON THE DIFFERENTIATION- INDUCING EFFECT OF COUMARINS / Chapter 4.1 --- Introduction --- p.122 / Chapter 4.2 --- Results --- p.124 / Chapter 4.2.1 --- The Differentiation-inducing Effect of Coumarins on Myeloid Leukemia Cells --- p.124 / Chapter 4.2.1.1 --- Morphological Changes in Coumarin-treated HL-60 Cells --- p.124 / Chapter 4.2.1.2 --- NBT Reduction of HL-60 Cells --- p.127 / Chapter 4.2.1.3 --- Effects of Coumarins on the Cell Size and Granularity of HL-60 Cells --- p.129 / Chapter 4.2.2 --- The Anti-proliferative and Differentiation-inducing Effects of Coumarins on Neuroblastoma Cells --- p.131 / Chapter 4.2.2.1 --- Anti-proliferative Effect of Coumarins on the BU-1 Cell Line In Vitro --- p.131 / Chapter 4.2.2.2 --- Morphological Changes in Coumarin-treated BU-1 Cells --- p.134 / Chapter 4.2.2.3 --- Immunocytochemistry of Coumarin-treated BU-1 Cells --- p.137 / Chapter 4.3 --- Discussion --- p.139 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI-LEUKEMIC ACTIVITIES OF COUMARINS / Chapter 5.1 --- Introduction --- p.142 / Chapter 5.2 --- Results --- p.147 / Chapter 5.2.1 --- Modulatory Effects of Coumarins on the Expression of Apoptosis-regulatory Genes in the Leukemia JCS Cells --- p.147 / Chapter 5.2.2 --- Modulatory Effects of Coumarins on the Expression of Growth-related Genes in the Leukemia JCS Cells --- p.151 / Chapter 5.2.3 --- Modulatory Effects of Coumarins on the Expression of Apoptosis-regulatory Proteins in Leukemia JCS Cells --- p.157 / Chapter 5.2.4 --- Modulatory Effects of Coumarins on the Expression of Growth-related Proteins in Leukemia JCS Cells --- p.162 / Chapter 5.2.5 --- Effect of Coumarins on the Mitochondrial Membrane Depolarization of the Leukemia JCS cells --- p.165 / Chapter 5.2.6 --- Induction of Apoptosis and Nitric Oxide Production in Leukemia JCS Cells by Coumarins --- p.168 / Chapter 5.2.7 --- Effects of Coumarins on the Caspase Activity in the Leukemia JCS cells --- p.172 / Chapter 5.3 --- Discussion --- p.177 / Chapter CHAPTER 6: --- STUDIES ON THE IMMUNOMODULATORY EFFECT OF COUMARINS ON MURINE MACROPHAGES / Chapter 6.1 --- Introduction --- p.185 / Chapter 6.2 --- Results --- p.188 / Chapter 6.2.1 --- Effect of Coumarins on the Viability of Macrophages In vitro --- p.188 / Chapter 6.2.2 --- Effect of Coumarins on the In vivo Migration of Macrophages --- p.190 / Chapter 6.2.3 --- Effect of Coumarins on Cytokine Production by Macrophages --- p.192 / Chapter 6.3 --- Discussion --- p.194 / Chapter CHAPTER 7: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.197 / REFERENCES --- p.204

Myelodysplasia (MDS) is characterised by inefficient haematopoiesis with dysplastic features of blood and bone marrow, reduction of mature blood cells and continuous bone marrow failure (BMF). Acute myeloid leukaemia (AML) is characterised by the accumulation of immature myeloid blasts in the bone marrow. MDS and AML are mostly sporadic clonal disorders affecting older patients. Familial occurrence of MDS/AML is rare, and most of these cases occur in the setting of genetic syndromes. However, it has also been reported to be caused by germline heterozygous mutations in genes including RUNX1, CEBPA, TERC, TERT, GATA2, SRP72, and ANKRD26. Our group has collected 115 families that have two or more individuals with BMF with at least one of whom has MDS or AML. The aim of this project was to identify disease causing gene variants that can lead to familial MDS/AML. Identification of predisposing variants to familial MDS/AML is critical for effective management in these families. This will also provide new insights into the biology of MDS/AML in general. Herein, we have characterised a subset of families with MDS/AML as well as identified candidate disease genes using a range of genetic studies. Specifically, we have: i. Identified new genetic variants in some of the known disease genes such as RUNX1 and GATA2. ii. Our studies have substantiated the discovery of DDX41 as a disease gene as we have identified several families harbouring novel heterozygous loss of function (LoF) DDX41 variants. iii. Identified germline heterozygous LoF RTEL1 variants in a subset of families with myelodysplasia and liver disease. This defines a new disease group in this field, RTEL1 can now be added to the list of familial MDS/AML disease genes. iv. We have identified nine new candidate disease genes which are involved in RNA splicing, transcription factor, DNA modification, cell signalling and intracellular transport.