O gene humano lysine methyltransferase 2E (KMT2E) pertence ao grupo Trithorax (TrxG) e age como proteína modificadora de histonas envolvida no controle transcricional de genes relacionados a hematopoiese. Foi previamente identificado como supressor tumoral, atuando sobre a diferenciação, proliferação e ciclo celular. DAMM et al. (2011) e LUCENA-ARAÚJO et al. (2014) descreveram a associação entre baixos níveis de expressão do gene KMT2E e desfechos desfavoráveis do tratamento de pacientes com leucemia mielóide aguda (LMA) e leucemia promielocítica aguda (LPA), respectivamente. O objetivo desse trabalho foi estudar os efeitos do aumento da expressão do gene KMT2E na leucemia mielóide aguda (LMA). Foi utilizada a linhagem celular U937, reconhecida como modelo de LMA, e o aumento da expressão do gene de interesse foi obtido por meio da transfecção das células com um vetor lentiviral contendo o cDNA codificante para a isoforma longa do gene (pCDH-MSCV-MCS-EF1-GFP+Puro, aqui chamado pMEG). As partículas lentivirais foram geradas por co-transfecção em células da linhagem HEK 293T, e posteriormente, titulados com a linhagem celular HT 1080. A expressão do gene e a presença da proteína foram confirmadas por qPCR e western blotting, respectivamente. Foram realizados ensaios funcionais de ciclo celular, proliferação, viabilidade, apoptose espontânea e induzida por trióxido de arsênico e luz ultravioleta e diferenciação celular induzida por 12-miristato 13-acetato de forbol (TPA), com as amostras U937 wild type (WT), U937 pMEG (U937 transduzidas com o vetor vazio) e U937 pMEG-KMT2E. Também foram realizadas mensurações da massa tumoral das células inoculadas em camundongos NSG. A expressão relativa do gene KMT2E na célula U937 pMEG-KMT2E foi 1000 vezes mais alta que na célula U937 sem a modificação genética. Os ensaios de diferenciação celular demonstraram que as células U937 pMEG-KMT2E apresentaram maior diferenciação em monócitos/macrófagos que as células controles, quando levada em consideração a marcação para o antígeno CD11c. A expressão induzida de KMT2E em células U937 não alterou a proliferação, viabilidade, ciclo celular, apoptose, ix espontânea ou induzida e o aspecto clonogênico in vitro, porém, foi associado a um maior crescimento tumoral em modelo animal. Nossa hipótese para justificar as diferenças entre os achados in vitro e in vivo é que o aumento da expressão de KMT2E, talvez por meio do aumento de CD11c, facilitou a interação entre as células e o microambiente, estimulando assim o crescimento tumoral in vivo. / The human lysine methyltransferase 2E (KMT2E) gene belongs to the Trithorax (TrxG) group and acts as a histone modifying protein participating in the transcriptional regulation of hematopoiesis-related genes. KMT2E has been previously described as a tumor suppressor, involved in cellular differentiation, proliferation and cell cycle progression. DAMM et al. (2011) and LUCENA-ARAÚJO et al. (2014) described the association between low levels of KMT2E gene expression and poor treatment outcomes in patients with acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL), respectively. The aim of this project was to study the effects of high levels of KMT2E expression in acute myeloid leukemia (AML). For this purpose, the U937 AML cell line was used and an high expression of the gene was obtained by transfecting the cells with a lentiviral vector containing the cDNA encoding the long isoform of the gene (pCDH-MSCV-MCS-EF1- GFP + Pure, here called pMEG). The lentiviral particles were transfected into HEK 293T cells and the viral concentration was determined by titration using HT 1080 cells. The gene expression and the protein presence were confirmed by qPCR and western blotting, respectively. All experiments to determine the biological function of overexpressed KMT2E were conducted with U937 wild type, U937 pMEG (U937 transduced with the empty vector) and U937 pMEG-KMT2E cells. In-vitro the impact of overexpressed KMT2E was studied on cell cycle progression, proliferation and cell viability, spontaneous and induced apoptosis by arsenic trioxide and ultraviolet light and cell differentiation induced by 12-myristate 13-phorbol acetate (TPA). In vivo, the effect of overexpressed KMT2E was detected by comparing the tumor mass growth in NSG mice when inoculating U937 pMEG and pMEG-KMT2E cells in each flank of the same mouse. The relative expression level of the KMT2E gene in pMEG-KMT2E U937 cells was 1000 higher than in the wild type U937 strain. The cell differentiation assay revealed that U937 pMEG-KMT2E cells presented an increased monocyte/macrophage differentiation, when analyzing the CD11c antigen. Induced xi overexpression of KMT2E in U937 cells did not alter cell proliferation, cell viability, cell cycle progression, spontaneous or induced apoptosis or clonogenic appearance in vitro. However, the overexpression of KMT2E resulted in an increased tumor mass formation in vivo. Taking our discrepant in vitro and in vivo results into account, we could hypothesize that the increased expression of KMT2E, possibly caused by the enhanced expression of CD11c, favored the interaction between U937 pMEGKMT2E cells and their microenvironment, thereby stimulating tumor growth in vivo.

In Western Europe and North America, acute myeloid leukaemia (AML) is predominantly a disease of the elderly, with a median age at the time of presentation in excess of 60 years. However, many clinical trials in AML fail to recruit elderly adults due to a combination of strict entry criteria, or physician or patient bias. Thus, clinical outcome data from many trials may not be readily applicable to older patients with the disease. Furthermore, because the clinical outcome for many older patients with AML is frequently poor, elderly patients who receive intensive chemotherapy with curative intent are frequently selected for treatment on clinical criteria rather than on objective prognostic criteria that may define clinical outcome. The karyotype at the time of presentation may be considered one of the most important prognostic factors in adult AML. Therefore, the aim of this thesis were firstly to analyse the clinical outcome data from a cohort of elderly patients managed at a single centre in order to document the cytogenetic features of AML in an elderly population, to define the prognostic importance of presentation karyotype in the elderly, and to identify other prognostic factors. Retrospective analysis clearly demonstrated improved clinical outcome for older patients with AML over time, primarily as a consequence of improved supportive care and the delivery of more intensive chemotherapy. In addition, 'unfavourable' presentation karyotype, increasing age and raised serum LDH were found to correlate with poor clinical outcome Molecular cytogenetic techniques based upon fluorescence in-situ hybridisation technology offer the chance to detect and analyse cytogenetic aberrations at a higher resolution than can be achieved with conventional techniques. The cytogenetic data provided by comparative genomic hybridisation and mulitplex fluorescence in-situ hybridisation when used in the analysis of elderly patients with AML were found to correlate well with results obtained by conventional methods. Importantly, additive cytogenetic data were more likely to be provided if multiplex-fluorescence in-situ hybridisation was used in the analysis of cases with marker chromosomes or in cases with complex karyotype, although the technique was limited by an inability to reliably detect telomeric translocations. In addition, although both techniques can be used to complement conventional G-banding analysis, conventional FISH methods are often required to confirm the results.

Recent developments in single-cell measurement technologies have yielded dramatic increases in throughput (measured cells per experiment) and dimensionality (measured features per cell). In particular, the introduction of mass cytometry has made possible the simultaneous quantification of dozens of protein species in millions of individual cells in a single experiment. The raw data produced by such high-dimensional single-cell measurements provide unprecedented potential to reveal the phenotypic heterogeneity of cellular systems. In order to realize this potential, novel computational techniques are required to extract knowledge from these complex data. Analysis of single-cell data is a new challenge for computational biology, as early development in the field was tailored to technologies that sacrifice single-cell resolution, such as DNA microarrays. The challenges for single-cell data are quite distinct and require multidimensional modeling of complex population structure. Particular challenges include nonlinear relationships between measured features and non-convex subpopulations. This thesis integrates methods from computational geometry and network analysis to develop a framework for identifying the population structure in high-dimensional single-cell data. At the center of this framework is PhenoGraph, and algorithmic approach to defining subpopulations, which when applied to healthy bone marrow data was shown to reconstruct known immune cell types automatically without prior information. PhenoGraph demonstrated superior accuracy, robustness, and efficiency, compared to other methods. The data-driven approach becomes truly powerful when applied to less characterized systems, such as malignancies, in which the tissue diverges from its healthy population composition. Applying PhenoGraph to bone marrow samples from a cohort of acute myeloid leukemia (AML) patients, the thesis presents several insights into the pathophysiology of AML, which were extracted by virtue of the computational isolation of leukemic subpopulations. For example, it is shown that leukemic subpopulations diverge from healthy bone marrow but not without bound: Leukemic cells are apparently free to explore only a restricted phenotypic space that mimics normal myeloid development. Further, the phenotypic composition of a sample is associated with its cytogenetics, demonstrating a genetic influence on the population structure of leukemic bone marrow. The thesis goes on to show that functional heterogeneity of leukemic samples can be computationally inferred from molecular perturbation data. Using a variety of methods that build on PhenoGraph's foundations, the thesis presents a characterization of leukemic subpopulations based on an inferred stem-like signaling pattern. Through this analysis, it is shown that surface phenotypes often fail to reflect the true underlying functional state of the subpopulation, and that this functional stem-like state is in fact a powerful predictor of survival in large, independent cohorts. Altogether, the thesis takes the existence and importance of cellular heterogeneity as its starting point and presents a mathematical framework and computational toolkit for analyzing samples from this perspective. It is shown that phenotypic and functional heterogeneity are robust characteristics of acute myeloid leukemia with clinically significant ramifications.

Chan Sin-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 154-169). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iv / Abstract (Chinese Version) --- p.vi / Contents --- p.viii / Abbreviations --- p.xiii / List of Figures and Tables --- p.xvi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- Ontogeny of hematopoiesis --- p.1 / Chapter 1.1.2 --- Hiercharay of hematopoiesis --- p.2 / Chapter 1.2 --- Regulation of hematopoiesis --- p.5 / Chapter 1.2.1 --- Bone marrow stromal cell --- p.5 / Chapter 1.2.2 --- Hematopoietic growth factor --- p.6 / Chapter 1.2.3 --- Hematopoietic growth factor receptors and signal transduction --- p.10 / Chapter 1.2.4 --- Transcriptional regulation of myeloid cell development --- p.11 / Chapter 1.3 --- Deregulated hematopoiesis - Leukemia --- p.20 / Chapter 1.3.1 --- Classification of leukemia --- p.20 / Chapter 1.3.2 --- Molecular basis of leukemia --- p.20 / Chapter 1.4 --- Prostaglandin E2 induced WEHI-3B JCS and JCS-14 cell differentiation --- p.22 / Chapter 1.4.1 --- Induced leukemia cell differentiation --- p.22 / Chapter 1.4.2 --- Inducer of cell differentiation - Prostaglandin E2 --- p.22 / Chapter 1.4.3 --- WEHI-3B JCS and subline JCS-14 cells --- p.24 / Chapter 1.5 --- The aims of study --- p.26 / Chapter Chapter Two --- Identification of differentially expressed genes during PGE2-induced WEHI-3B JCS-14 cell differentiation / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.1.1 --- Strategy for studying PGE2-induced JCS-14 cell differentiation --- p.28 / Chapter 2.1.2 --- Method for studying differential gene expression: Microarry Technology --- p.29 / Chapter 2.2 --- Materials --- p.32 / Chapter 2.2.1 --- Cell line --- p.32 / Chapter 2.2.2 --- AtlasT M Mouse cDNA Expression Array --- p.32 / Chapter 2.2.3 --- Chemicals --- p.32 / Chapter 2.2.4 --- Solutions and buffers --- p.33 / Chapter 2.2.5 --- Reagents --- p.34 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Morphological study of PGE2-induced JCS-14 cell differentiation --- p.35 / Chapter 2.3.2 --- Preparation of total RNA from PGE2-induced JCS-14 cells --- p.35 / Chapter 2.3.2.1 --- Preparation of cell lysates --- p.35 / Chapter 2.3.2.2 --- Isolation of total RNA --- p.35 / Chapter 2.3.3 --- Preparation of cDNA probes --- p.36 / Chapter 2.3.3.1 --- Probe synthesis from total RNA --- p.36 / Chapter 2.3.3.2 --- Purification of the labeled cDNA probes --- p.37 / Chapter 2.3.4 --- Hybridization cDNA probes to the Atlas Array and stringency wash --- p.37 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Morphological changes in PGE2-treated JCS-14 cells --- p.39 / Chapter 2.4.2 --- Analysis of total RNA from PGE2-induced JCS-14 cells --- p.43 / Chapter 2.4.3 --- Hybridization of cDNA probes to AtlasT M cDNA Expression Array --- p.45 / Chapter 2.5 --- Discussion --- p.73 / Chapter 2.5.1 --- Morphological study of JCS-14 cell differentiation --- p.73 / Chapter 2.5.2 --- Differentiation commitment of JCS-14 cell under PGE2 induction --- p.73 / Chapter 2.5.3 --- Gene expression profile by microarray --- p.74 / Chapter 2.5.4 --- Gene expression profile of 5 hours PGE2-induced JCS-14 cells --- p.74 / Chapter 2.5.5 --- Further analysis of regulatory genes in PGE2-induced JCS-14 cell differentiation --- p.77 / Chapter Chapter Three --- Expression profile of identified genes in WEHI-3B JCS-14 and JCS cell differentiation / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.1.1 --- Quantitation of mRNA by Real time RT-PCR --- p.80 / Chapter 3.1.2 --- Relative quantitation of gene expression --- p.83 / Chapter 3.2 --- Materials --- p.85 / Chapter 3.2.1 --- Cell lines --- p.85 / Chapter 3.2.2 --- SYBR® Green PCR core kit --- p.85 / Chapter 3.2.3 --- Chemicals --- p.85 / Chapter 3.2.4 --- Solutions and buffers --- p.86 / Chapter 3.2.5 --- Enzymes and nucleic acids --- p.86 / Chapter 3.3 --- Methods --- p.88 / Chapter 3.3.1 --- Preparation of total RNA from PGE2-induced JCS-14 and JCS cells --- p.88 / Chapter 3.3.1.1 --- Preparation of cell lysates --- p.88 / Chapter 3.3.1.2 --- Isolation of total RNA --- p.88 / Chapter 3.3.2 --- Reverse transcription (RT) --- p.88 / Chapter 3.3.3 --- Design of real-time PCR primers --- p.88 / Chapter 3.3.4 --- Determination of relative efficiency of target and reference amplification by validation experiment --- p.89 / Chapter 3.3.5 --- Confirmation of expression profile of identified genes in JCS-14 and JCS cells by comparative CT method in real-time PCR --- p.90 / Chapter 3.4 --- Results --- p.91 / Chapter 3.4.1 --- Analysis of total RNA from PGE2-induced JCS-14 and JCS cells --- p.91 / Chapter 3.4.2 --- Validation experiment of real-time PCR primers --- p.93 / Chapter 3.4.3 --- Expression profile of specific genes in JCS-14 and JCS cells by comparative CT method --- p.101 / Chapter 3.5 --- Discussion --- p.114 / Chapter 3.5.1 --- Study of gene expression profiles in JCS-14 and JCS cell differentiation --- p.114 / Chapter 3.5.2 --- Transcription analysis by real-time PCR --- p.114 / Chapter 3.5.3 --- Gene expression profiles during PGE2-induced JCS-14 and JCS cell differentiation --- p.115 / Chapter Chapter Four --- Inhibition of specific gene expression in WEHI-3B JCS-14 and JCS cells using antisense blocking technique / Chapter 4.1 --- Introduction --- p.121 / Chapter 4.1.1 --- Antisense technique --- p.122 / Chapter 4.1.2 --- Design of antisense oligonucleotides --- p.125 / Chapter 4.1.3 --- Transfer of oligonucleotides to cells --- p.128 / Chapter 4.2 --- Materials --- p.129 / Chapter 4.2.1 --- Cell lines --- p.129 / Chapter 4.2.2 --- Chemicals --- p.129 / Chapter 4.2.3 --- Reagents --- p.129 / Chapter 4.2.4 --- Solutions --- p.129 / Chapter 4.3 --- Methods --- p.131 / Chapter 4.3.1 --- Design of antisense oligonucleotides --- p.131 / Chapter 4.3.2 --- Transfection of oligonucleotides into cells --- p.134 / Chapter 4.3.3 --- Morphological study of PGE2-induced JCS-14 and JCS cells --- p.134 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- Effect of antisense oligonucleotides on JCS-14 cell differentiation --- p.135 / Chapter 4.4.2 --- Effect of antisense oligonucleotides on JCS cell differentiation --- p.136 / Chapter 4.5 --- Discussion --- p.146 / Chapter 4.5.1 --- Effects of antisense B-myb on JCS-14 and JCS cell differentiation --- p.146 / Chapter 4.5.2 --- Effects of antisense thyroid hormone receptor (c-erbA) and transcription terminator factor (TTF-1) on JCS-14 and JCS cell differentiation --- p.147 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Introduction --- p.148 / Chapter 5.2 --- Differentiation program triggered by Prostaglandin E2 --- p.148 / Chapter 5.2.1 --- Lineage preference during differentiation --- p.148 / Chapter 5.2.2 --- Differentially expressed genes during PGE2-induced JCS-14 cell differentiation --- p.149 / Chapter 5.2.3 --- Expression patterns of the three differentially expressed genes in PGE2-induced JCS-14 and JCS cells --- p.149 / Chapter 5.2.4 --- Antisense blocking during differentiation --- p.151 / Chapter 5.3 --- Further studies --- p.152 / References --- p.154

by Lau Kwok Wing, Wilson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 166-188). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.viii / Chapter CHAPTER 1 : --- GENERAL INTRODUCTION --- p.1 / Chapter (1.1) --- Hematopoiesis : An Overview --- p.2 / Chapter (1.1.1) --- Development of Blood Cells and Sites of Hematopoiesis --- p.2 / Chapter (1.1.2) --- Hematopoietic Cytokine Network --- p.4 / Chapter (1.1.3) --- Molecular Control of Hematopoietic Cell Development --- p.5 / Chapter (1.2) --- Leukemia : An Overview --- p.8 / Chapter (1.2.1) --- Leukemia : Abnormalities in Blood Cell Formation --- p.8 / Chapter (1.2.2) --- Pathophysiology and Etiology of Leukemia --- p.10 / Chapter (1.2.3) --- New Avenues for Therapy : Induction of Differentiation and Apoptosis --- p.12 / Chapter (1.3) --- Induction of Differentiation in Myeloid Leukemia Cells --- p.14 / Chapter (1.3.1) --- Inducers of Leukemic Cell Differentiation --- p.14 / Chapter (1.3.2) --- Cytokines as Inducers of Myeloid Leukemic Cell Differentiation --- p.17 / Chapter (1.3.3) --- Phenotypic Changes and Functional Characterizations --- p.20 / Chapter (1.3.4) --- Modulation of Gene Expression in Myeloid Leukemic Cell Differentiation --- p.22 / Chapter (1.4) --- Apoptosis and Leukemic Cell Death --- p.23 / Chapter (1.4.1) --- Apoptosis : An Overview --- p.23 / Chapter (1.4.2) --- Cytokines and Apoptosis in Myeloid Leukemia --- p.26 / Chapter (1.5) --- Objectives and Research Strategy --- p.27 / Chapter (1.5.1) --- The Murine Myeloid Leukemia Cell Line (Ml) as an Experimental Cell Model for Acute Myeloid Leukemia --- p.27 / Chapter (1.5.2) --- Leukemia Inhibitory Factor (LIF) as a Differentiation Inducer --- p.28 / Chapter (1.5.3) --- Aims and Scopes of This Investigation --- p.31 / Chapter CHAPTER 2 ： --- MATERIALS AND METHODS --- p.33 / Chapter (2.1) --- Materials --- p.34 / Chapter (2.1.1) --- Mice --- p.34 / Chapter (2.1.2) --- Cell Lines --- p.34 / Chapter (2.1.3) --- Recombinant Cytokines --- p.34 / Chapter (2.1.4) --- Monoclonal Antibodies --- p.36 / Chapter (2.1.5) --- Oligonucleotide Primers and Internal Probes --- p.37 / Chapter (2.1.6) --- "Buffers, Culture Medium and Other Reagents" --- p.39 / Chapter (2.1.7) --- Reagents and Solutions for Gene Expression Study --- p.41 / Chapter (2.2) --- Methods --- p.46 / Chapter (2.2.1) --- Culture of Myeloid Leukemia Cell Lines --- p.46 / Chapter (2.2.2) --- Induction of Leukemic Cell Differentiation --- p.46 / Chapter (2.2.3) --- Determination of Cell Growth and Proliferation --- p.46 / Chapter (2.2.4) --- Cell Morphological Study --- p.47 / Chapter (2.2.5) --- Assessment of Differentiation-Associated Characteristics --- p.48 / Chapter (2.2.5.1) --- Nitroblue Tetrazolium (NBT) Reduction Assay --- p.48 / Chapter (2.2.5.2) --- Phagocytosis Assay --- p.48 / Chapter (2.2.5.3) --- Assay of Plastic Adherence --- p.49 / Chapter (2.2.6) --- Flow Cytometric Analysis --- p.49 / Chapter (2.2.6.1) --- Surface Antigen Immunophenotyping --- p.49 / Chapter (2.2.6.2) --- Assay of Endocytic Activity --- p.50 / Chapter (2.2.6.3) --- Assay of Non-specific Esterase Activity --- p.50 / Chapter (2.2.6.4) --- Cell Cycle / DNA Content Evaluation --- p.51 / Chapter (2.2.7) --- Gene Expression Analysis --- p.52 / Chapter (2.2.7.1) --- Preparation of Cell Lysate --- p.52 / Chapter (2.2.7.2) --- RNA Isolation --- p.52 / Chapter (2.2.7.3) --- Reverse Transcription --- p.53 / Chapter (2.2.7.4) --- Polymerase Chain Reaction (PGR) --- p.54 / Chapter (2.2.7.5) --- Agarose Gel Electrophoresis --- p.55 / Chapter (2.2.7.6) --- 3' End Labelling of Oligonucleotide Probes --- p.56 / Chapter (2.2.7.7) --- Dot Blot Hybridization --- p.56 / Chapter (2.2.7.8) --- Digoxigenin (DIG) Chemiluminescent Detection --- p.57 / Chapter (2.2.8) --- DNA Fragmentation Analysis --- p.58 / Chapter (2.2.9) --- Statistical Analysis --- p.59 / Chapter CHAPTER 3 ： --- "EFFECTS OF LEUKEMIA INHIBITORY FACTOR ON THE PROLIFERATION, DIFFERENTIATION, AND APOPTOSIS OF MURINE MYELOID LEUKEMIA Ml CELLS" --- p.60 / Chapter (3.1) --- Introduction --- p.61 / Chapter (3.2) --- Results --- p.63 / Chapter (3.2.1) --- Induction of Growth Arrest in rmLIF-Treated Ml Cells --- p.63 / Chapter (3.2.2) --- Induction of Monocytic Differentiation of Ml cells by rmLIF --- p.66 / Chapter (3.2.2.1) --- Morphological Changes --- p.66 / Chapter (3.2.2.2) --- Induction of Plastic Adherence --- p.70 / Chapter (3.2.2.3) --- Surface Antigen Immunophenotyping --- p.70 / Chapter (3.2.2.4) --- NBT-Reducing Activity of rmLIF-Treated Ml Cells --- p.76 / Chapter (3.2.2.5) --- Non-specific Esterase Activity of rmLIF-Treated Ml Cells --- p.77 / Chapter (3.2.2.6) --- Endocytic Activity of rmLIF-Treated Ml Cells --- p.78 / Chapter (3.2.2.7) --- Phagocytic Activity of rmLIF-Treated Ml Cells --- p.79 / Chapter (3.2.3) --- Induction of Differentiation-Associated DNA Fragmentation --- p.80 / Chapter (3.2.4) --- Production of Differentiation-Inducing Factors --- p.84 / Chapter (3.3) --- Discussion --- p.88 / Chapter CHAPTER 4 : --- CYTOKINE INTERACTIONS IN REGULATING THE PROLIFERATION AND DIFFERENTIATION OF MURINE MYELOID LEUKEMIA Ml CELLS --- p.94 / Chapter (4.1) --- Introduction --- p.95 / Chapter (4.2) --- Results --- p.97 / Chapter (4.2.1) --- Synergistic Effect of LIF and IL-6 on the Proliferation and Differentiation of Ml Cells --- p.97 / Chapter (4.2.2) --- Regulation of Proliferation and Differentiation of Ml Cells by LIF and OSM --- p.101 / Chapter (4.2.3) --- Effects of LIF and TNF-α on the Proliferation and Differentiation of Ml Cells --- p.104 / Chapter (4.2.4) --- Synergistic Effect of LIF and IL-1 on the Proliferation and Differentiation of Ml Cells --- p.107 / Chapter (4.3) --- Discussion --- p.115 / Chapter CHAPTER 5 ： --- MODULATION OF CYTOKINE AND CYTOKINE RECEPTOR GENE EXPRESSION IN LIF- INDUCED DIFFERENTIATION OF MURINE MYELOID LEUKEMIA Ml CELLS --- p.120 / Chapter (5.1) --- Introduction --- p.121 / Chapter (5.2) --- Results --- p.123 / Chapter (5.3) --- Discussion --- p.152 / Chapter CHAPTER 6 ： --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.158 / REFERENCES --- p.166

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

Myeloid Leukemia Factor 1 (Mlf1) is the murine homolog of MLF1, which was identified as a fusion gene with Nucleophosmin (NPM) resulting from the (3;5)(q25.1;q34) translocation associated with acute myeloid leukemia and myelodysplastic syndrome (Yoneda-Kato et al., 1996). Mlf1 was independently isolated using cDNA representational difference to identify genes up-regulated when an erythroleukemic cell line underwent a lineage switch to display a monoblastoid phenotype (Williams et al., 1999). Mlf1 has been shown to enhance myeloid differentiation and suppress erythroid differentiation; however, its mechanism of action is unknown. A yeast two hybrid screen was employed to identify Mlf1-interacting proteins. This screen isolated a number of known protein, as well as several novel molecules, that bound Mlf1. One of these was 14-3-3ξ, a member of a family of molecules that bind phosphoserine motifs and regulate the subcellular localization of partner proteins. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and associated with 14-3-3ξ; via this phosphorylated motif (Lim et al., 2002). The aim of this thesis was to characterise a novel Mlf1-interacting protein that had some homology to protein kinases and was named Mlf1 Adaptor Molecule (Madm). Adaptor proteins are molecules that possess no enzymatic or transcriptional activity, but instead mediate protein-protein interactions. Madm is encoded by a gene consisting of 18 exons and promoter analysis suggested Madm expression might be widespread; indeed Northern blotting of adult tissues and in situ hybridization of embryos demonstrated ubiquitous Madm expression. Significantly, the Madm protein sequence is highly conserved across diverse species. / Madm formed dimers and although it contains a kinase-like domain, the protein lacks several critical residues required for catalytic activity, including an ATP-binding site. Purification of recombinant Madm revealed that the protein was not a kinase; however, studies in mammalian cells showed that Madm associated with a kinase and that Madm was phosphorylated on serine residues in vivo and in vitro. Madm also contains a nuclear localization sequence and nuclear export sequence and was shown to localise to both cytoplasm and nucleus by subcellular fractionation and confocal microscopy. The presence of two nuclear receptor binding motifs (consensus MILL) suggests that Madm may have a functional role in the nucleus. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, the Madm-associated kinase phosphorylated Mlf1 on serine residues, including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein NPM-MLF1 did not bind 14-3-3i; and localized exclusively in the nucleus. Although Madm co-immunoprecipitated with NPM-MLF1 the binding mechanism was altered. As Mlf1 is able to reprogram erythroleukemic cells to display a monoblastoid phenotype and potentiate myeloid maturation (Williams et al., 1999), the effects of Madm on myeloid differentiation was investigated. However, unlike Mlf1, ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation. / In summary, the data presented in this thesis reports on the cloning and characterization of a novel adaptor protein that is involved in the phosphorylation of the proto-oncoprotein MIM. Phosphorylation of Mlf1 is likely to affect its interaction with other proteins, such as 14-3-3~. Complex formation, therefore, may well alter the localization of Mlf1 and Madm, and influence hematopoietic differentiation.

Understanding the factors that regulate the growth and differentiation of haemopoietic stem cells (HSC) remains a major challenge. In this study, the proliferation and differentiation of CD34+ cells from normal donors and chronic myeloid leukaemia (CML) patients was compared. The proliferation and entry of CML cells into the cell cycle was decreased relative to cells from normal donors, and greater heterogeneity in the phenotype of CML cells at the initiation of culture was observed. Analysis of phenotype concomitant with cell division also demonstrated that the differentiation of normal CD34+ cells was consistent between donors, while marked variability was observed in the differentiation of CD34+ cells from CML patients. This included expression of CD13, CD33, CD38 and HLA-DR, which were linked to cell division in normal but not CML cells. The tyrosine kinase inhibitor, imatinib, is a novel drug displaying promising results in the treatment of CML by specifically inhibiting the growth of leukaemic cells. To examine whether myelosuppression observed in patients treated with imatinib may arise from inhibition of normal haemopoiesis, imatinib was added to colony assays established using cells from normal bone marrow. Suppression of monocyte/macrophage growth, but not that of eosinophils or neutrophils, was observed at therapeutic concentrations of imatinib. Inhibition of monocytic differentiation to macrophages was also observed and was associated with decreased functional capacity such as altered antigen uptake, production of proinflammatory cytokines and stimulation of responder cells. The specific suppression of monocyte/macrophage differentiation and function was not due to blockade of tyrosine kinases known to be inhibited by imatinib and was consistent with an inhibition of the M-CSF/c-fms signalling pathway. This hypothesis was tested using a cell line that was dependent on M-CSF for growth and survival. Cell proliferation and phosphorylation of c-fms were inhibited at an IC50 of 1.9μM and 1.4μM imatinib respectively and this was not attributable to decreased c-fms expression. These important findings therefore identify c-fms as a further target of imatinib, and suggest that imatinib should be considered for treatment of diseases where c-fms is implicated. This includes breast and ovarian cancer and inflammatory conditions such as rheumatoid arthritis. Potential side effects resulting from imatinib treatment must also be considered. / Thesis (Ph.D.)--School of Medicine, 2004.

The transmembrane isoform of mucin 1 (MUC1/TM) is a well recognized tumor antigen, contributing to tumorigenesis and immune evasion. While MUC1/TM has been correlated with malignancy, it appears that a secreted splice variant of MUC1 (MUC1/sec) has antitumor properties and prevents tumor development. It was discovered that MUC1/sec expressing tumor cells (DA-3/sec) have a significant reduction in expression of urokinase plasminogen activator (uPA) relative to the parental tumor line, and tumor cells expressing MUC1/TM (DA-3/TM). The serine protease uPA, has been found to be involved in growth promoting signaling, angiogenesis, and induction of matrix remodeling leading to metastasis. Furthermore, the tumor suppressive and interferon responsive Stat1 transcription factor is dramatically upregulated in DA-3/sec cells. In addition, treatment of various murine and human cell lines with conditioned media containing MUC1/sec results in up-regulation of Stat1. DA-3/sec tumor cells are also sensitized to the anti-proliferative effects of IFN-g. Furthermore, transfection of the Stat1 gene into DA-3 tumor cells leads to a downregulation of uPA, and delays tumor progression. Since myeloid-derived suppressor cells (MDSC) play a critical role in tumor-induced immunosuppression, we investigated their recruitment by DA-3/sec and DA-3/TM cells. DA-3/sec tumor cells recruit dramatically lower levels of MDSC, relative to DA-3/TM cells. Since MUC1/sec down-regulates tumor expression of uPA, its potential role in MDSC recruitment was investigated. Tumor-derived uPA is capable of recruiting MDSC, and correlates with tumor development. In addition to diminishing recruitment of MDSC, the effect of MUC1/sec on MDSC suppressive mechanisms was investigated. MUC1/sec, or its unique immunoenhancing peptide (IEP), is capable of blocking expression of arginase 1 and production of reactive oxygen species (ROS) in MDSC, implicated in the suppression of T cells. These findings demonstrate a new mechanism of MDSC recruitment, and provide evidence that MUC1/sec has antitumor properties affecting both tumor cells and MDSC. Furthermore, it was discovered that MDSC home to the liver in addition to the tumor, bone marrow, blood, and spleen of tumor bearers, as previously described. The liver is thus an organ where MDSC accumulate and can contribute to immunosuppression directly and indirectly, via interactions with a variety of immune cells.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.