Na leucemia mielóide crônica (LMC) a proteína Bcr-Abl possui atividade tirosina-quinase constitutivamente ativada, induzindo a mieloproliferação e a resistência das células à apoptose. A maioria dos pacientes na fase crônica da LMC tratados com inibidores de tirosina-quinase (TKIs), como o mesilato de imatinibe, apresenta remissão citogenética completa da doença, mas uma parcela desses pacientes tem se mostrado resistente à terapia. A fisiopatologia da LMC e os mecanismos celulares e moleculares envolvidos na resistência à terapia com TKIs são diversos e precisam ser melhor estudados. Nesse contexto, o objetivo do presente estudo foi quantificar os níveis de expressão do gene DIDO, incluindo suas diferentes isoformas (DIDO 1, 2 e 3) e promotores (DIDO PP e PD) em controles e em pacientes com LMC nas diferentes fases da doença, tratados ou não com TKIs, bem como em linhagens celulares BCR-ABL1+ sensíveis (S) e resistentes (R) ao mesilato de imatinibe (MI). A literatura relata que DIDO 1 participa do processo de apoptose e que alterações na expressão de DIDO 2 e DIDO 3 podem estar associadas com o desenvolvimento de neoplasias mielóides. Dessa forma, foram estudados 60 pacientes com LMC e 57 controles, assim como as linhagens celulares HL-60, HL-60.Bcr-Abl+, LAMA 84 S, LAMA 84 R, KCL 22 S e KCL 22 R. Foram separadas as células mononucleares de sangue periférico dos pacientes e controles, com posterior extração de RNA e síntese de cDNA, que foi então empregado nas reações de PCR em tempo real (qPCR) para quantificação das expressões gênicas de DIDO 1, 2, 3, PP, PD e ORF. As linhagens celulares foram tratadas por 4h com TKIs e então a expressão das diferentes isoformas de DIDO foi também quantificada por qPCR. A avaliação da expressão proteica de Bcr-Abl, c-Abl e proteínas fosforiladas nas linhagens foi realizada por Western-blotting. A expressão de DIDO 1 e 2 foi maior nos pacientes nas fases avançadas da LMC e nos pacientes na fase crônica da doença tratados com TKIs (mesilato de imatinibe ou dasatinibe) do que nos controles. Os pacientes na fase crônica da LMC tratados com MI expressaram mais DIDO 1 e 3 do que os pacientes na fase crônica sem tratamento. Houve uma correlação positiva entre expressão de BCR-ABL1 e de DIDO 2 e entre índice de Sokal dos pacientes e expressão de DIDO 2. Na linhagem HL60.Bcr-Abl+, a expressão de proteínas fosforiladas reduziu após tratamento de 4h com TKIs, mas não houve alteração na expressão gênica de DIDO. Nas linhagens celulares S e R, houve aumento da expressão de DIDO 1 após o tratamento de 4h com MI. Conclui-se, portanto, que as diferentes isoformas de DIDO parecem exercer funções distintas na leucemia mielóide crônica; que o tratamento de pacientes e linhagens BCR-ABL1 positivas com inibidores de tirosina-quinase aumenta expressão de DIDO 1; e que a expressão de DIDO 2 correlaciona-se positivamente à expressão de BCR-ABL1 e ao índice de Sokal dos pacientes. / In chronic myeloid leukemia (CML) the Bcr-Abl protein has constitutively activated tyrosine kinase activity, that induces to myeloproliferation and apoptosis resistance of the cells. Most patients in chronic phase of CML treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate have a complete cytogenetic remission, but a portion of these patients have been shown to be resistant to therapy. The pathophysiology of CML and the cellular and molecular mechanisms involved in resistance to TKIs therapy are diverse and require further study. In this sense, the aim of this study was to quantify the expression levels of the DIDO gene, including its isoforms (DIDO 1, 2 and 3) and promoters (DIDO PP and PD) in controls and in patients with CML in different phases of the disease treated or not treated with TKIs, as well as in cell lines BCR-ABL1+ sensitive (S) and resistant (R) to imatinib mesylate (IM). The literature reports that DIDO 1 is involved in apoptosis process and that alterations of DIDO 2 and DIDO 3 expression may be associated with the development of myeloid neoplasms. Thus, 60 CML patients, 57 control individuals and the cell lines HL-60, HL-60.Bcr-Abl+, LAMA 84 S, LAMA 84 R, KCL 22 S and KCL 22 R were studied. Peripheral blood mononuclear cells of patients and controls were isolated and RNA extraction and cDNA synthesis were performed. The cDNA samples were used in Real-Time PCR reactions (qPCR) to quantify the DIDO 1, 2, 3, PP, PD and ORF gene expression. The cell lines were treated during 4h with TKIs and then the expression of DIDO different isoforms was also quantified by qPCR. The assessment of protein expression of Bcr-Abl, c-Abl and phosphorylated proteins in this cell lines was performed by Western blotting. The DIDO 1 and 2 expression was higher in advanced phases patients and in chronic phase patients CML treated with TKIs (imatinib mesylate and dasatinib) than in controls. Chronic phase CML Patients treated with IM expressed more DIDO 1 and 3 than chronic phase untreated patients. There was a positive correlation between BCR-ABL1 expression and DIDO 2 expression and between Sokal score prognostic and DIDO 2 expression. In HL60.Bcr-Abl+ cells the expression of phosphorylated proteins was lower after treatment during 4h with TKIs, but there was no change in DIDO gene expression. There were an increase of DIDO 1 expression in all S and R cell lines after treatment during 4h with IM. Therefore we conclude that the DIDO different isoforms may have different functions in chronic myeloid leukemia; the treatment of patients and BCR-ABL1+ cell lines with TKIs increases DIDO 1 expression; and that the DIDO 2 expression is positively correlated to the BCR-ABL1 expression and Sokal score prognostic of CML patients.

Lui Oi Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [216]-240). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vii / 撮要 --- p.x / PUBLICATIONS --- p.xiii / TABLE OF CONTENTS --- p.xiv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis and Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoietic Development --- p.1 / Chapter 1.1.2 --- Leukemia --- p.8 / Chapter 1.1.2.1 --- General Diagnostic Tests for Leukemia --- p.9 / Chapter 1.1.2.2 --- Classification and Epidemiology of Leukemia --- p.10 / Chapter 1.1.2.3 --- Conventional Approaches to Leukemia Therapy --- p.17 / Chapter 1.1.2.4 --- Novel Approaches to Leukemia Therapy --- p.20 / Chapter 1.2 --- Conjugated Linoleic Acid --- p.23 / Chapter 1.2.1 --- Introduction: Historical Development and Occurrence of Conjugated Linoleic Acid --- p.23 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Conjugated Linoleic Acid --- p.24 / Chapter 1.2.2.1 --- Chemical Structures of Conjugated Linoleic Acid Isomers --- p.24 / Chapter 1.2.2.2 --- Biosynthesis of Conjugated Linoleic Acid --- p.26 / Chapter 1.2.2.3 --- Metabolism of Conjugated Linoleic Acid --- p.30 / Chapter 1.2.2.4 --- Mode of Entry and Tissue Incorporation of Conjugated Linoleic Acid --- p.33 / Chapter 1.2.2.5 --- Toxicology of Conjugated Linoleic Acid --- p.33 / Chapter 1.2.3 --- Physiological Activities of Conjugated Linoleic Acid: Reported Health Benefits --- p.35 / Chapter 1.2.3.1 --- Anti-adipogenesis / Chapter 1.2.3.2 --- Anti-diabetogenesis --- p.36 / Chapter 1.2.3.3 --- Anti-atherosclerosis --- p.38 / Chapter 1.2.3.4 --- Anti-carcinogenesis --- p.39 / Chapter 1.2.3.5 --- Anti-tumor Activity --- p.40 / Chapter 1.2.3.6 --- Effects of Conjugated Linoleic Acid on Lipid Metabolism --- p.44 / Chapter 1.2.3.6.1 --- Actions on Phospholipids by Conjugated Linoleic Acid --- p.45 / Chapter 1.2.3.6.2 --- Conjugated Linoleic Acid as a Ligand for the PPAR System --- p.47 / Chapter 1.2.3.7 --- Immunomodulation --- p.47 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.50 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Animals --- p.52 / Chapter 2.1.2 --- Cell Lines --- p.52 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.52 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.54 / Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.58 / Chapter 2.1.6 --- Reagents for DNA Extraction --- p.59 / Chapter 2.1.7 --- Cell Death Detection ELISAPLUS Kit --- p.63 / Chapter 2.1.8 --- Reagents for Measuring Caspase Activity --- p.65 / Chapter 2.1.9 --- Reagents for Total RNA Isolation --- p.66 / Chapter 2.1.10 --- Reagents and Buffers for RT-PCR --- p.69 / Chapter 2.1.11 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Acids --- p.74 / Chapter 2.1.12 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.75 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.80 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.81 / Chapter 2.2.3 --- Determination of Cell Viability --- p.82 / Chapter 2.2.4 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.83 / Chapter 2.2.5 --- In Vivo Tumorigenicity Study --- p.83 / Chapter 2.2.6 --- Analysis of Cell Cycle Profile / DNA Content by Flow Cytometry --- p.83 / Chapter 2.2.7 --- Measurement of Apoptosis --- p.84 / Chapter 2.2.8 --- Determination of the Mitochondrial Membrane Potential --- p.86 / Chapter 2.2.9 --- Measurement of Caspase Activity --- p.87 / Chapter 2.2.10 --- Study of Intracellular Accumulation of Reactive Oxygen Species (ROS) --- p.88 / Chapter 2.2.11 --- Study of the Scavenging Activity of Antioxidants --- p.88 / Chapter 2.2.12 --- Gene Expression Study --- p.89 / Chapter 2.2.13 --- Protein Expression Study --- p.92 / Chapter 2.2.14 --- Measurement of Cell Differentiation --- p.95 / Chapter 2.2.15 --- Statistical Analysis --- p.98 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-TUMOR ACTICITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction / Chapter 3.2 --- Results --- p.99 / Chapter 3.2.1 --- Anti-proliferative Activity of CLA-mix and CLA Isomers on Various Myeloid Leukemia Cell Lines In Vitro --- p.101 / Chapter 3.2.2 --- Cytotoxic Effect of CLA-mix on the WEHI-3B JCS Cells In Vitro --- p.109 / Chapter 3.2.3 --- Cytotoxic Effect of CLA-mix on Primary Murine Myeloid Cells In Vitro --- p.111 / Chapter 3.2.4 --- Kinetic and Reversibility Studies of the Anti-proliferative Activity of CLA-mix on the WEHI-3B JCS Cells --- p.113 / Chapter 3.2.5 --- Effect of CLA-mix and its isomers on the Cell Cycle Profiles of the WEHI-3B JCS Cells In Vitro --- p.116 / Chapter 3.2.6 --- Effect of CLA-mix and its isomer on the Expression of Cell Cycle-regulatory Genes in the WEHI-3B JCS Cells --- p.123 / Chapter 3.2.7 --- Effect of CLA-mix and its isomer on the In V Tumorigenicity of the WEHI-3B JCS Cells ivo --- p.128 / Chapter 3.3 --- Discussion --- p.131 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING ACTIVITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 4.1 --- Introduction --- p.141 / Chapter 4.2 --- Results --- p.141 / Chapter 4.2.1 --- Induction of Apoptosis in Both Murine and Human Myeloid Leukemia Cells by CLA --- p.144 / Chapter 4.2.2 --- Effect of CLA and its Isomer on the Mitochondrial Membrane Potential of the WEHI-3B JCS Cells --- p.151 / Chapter 4.2.3 --- Effect of CLA-mix and its Isomer on the Expression of Apoptosis-regulatory Genes of the Bcl-2 Family in the WEHI-3B JCS Cells --- p.154 / Chapter 4.2.4 --- Effect of CLA-mix and its Isomer on the Expression of Apoptosis-regulatory Proteins in the WEHI-3B JCS Cells --- p.158 / Chapter 4.2.5 --- Effect of CLA-mix and its Isomer on the Induction of Caspase Activity in the WEHI-3B JCS Cells --- p.161 / Chapter 4.2.6 --- Effect of CLA-mix and its Isomer on the Induction of ROS in the WEHI-3B JCS Cells --- p.170 / Chapter 4.2.7 --- Effect of Antioxidants on the Induction of ROS by CLA-mix and its Isomer in the WEHI-3B JCS Cells --- p.173 / Chapter 4.2.8 --- Effect of Antioxidants on the Induction of Apoptosis by CLA-mix and its Isomer in the WEHI-3B JCS Cells --- p.176 / Chapter 4.2 --- Discussion / Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ACTIVITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 5.1 --- Introduction --- p.187 / Chapter 5.2 --- Results --- p.190 / Chapter 5.2.1 --- Morphological Alterations in CLA-mix- and CLA isomer-treated WEHI-3B JCS Cells --- p.190 / Chapter 5.2.2 --- Effects of CLA-mix on the Cell Size and Granularity of WEHI-3B JCS Cells --- p.196 / Chapter 5.2.3 --- Studies of the Surface Phenotypic Changes in the CLA-mix-treated WEHI-3B JCS cells --- p.198 / Chapter 5.2.4 --- Studies on the Induction of Monocytic Serine Esterase (MSE) Activity in the CLA-mix-treated WEHI-3B JCS Cells --- p.200 / Chapter 5.2.5 --- Studies on the Induction of Endocytic Activity in the CLA-mix-treated WEHI-3B JCS Cells --- p.201 / Chapter 5.2.6 --- Studies on the Expression of the Differentiation-regulatory Cytokine Genes in the CLA-mix-treated WEHI-3B JCS Cells --- p.202 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES REFERENCES --- p.208 / REFERENCES --- p.217

The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and produce all mature blood cells throughout the life of an organism. This is tightly regulated to maintain a balance between survival, proliferation, and differentiation. The HSCs are located in specialized niches in the bone marrow thought to be low in oxygen, which is suggested to be involved in the regulation of HSC maintenance, proliferation, and migration. However, the importance of hypoxia in the stem cell niche and the molecular mechanisms involved remain fairly undefined. Another important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3, which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause constitutively active signaling. This leads to uncontrolled survival and proliferation, which can result in development of acute myeloid leukemia (AML). One of the purposes with this thesis is to investigate how survival, proliferation and self-renewal in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse bone marrow. We found that hypoxia maintained an immature phenotype. In addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the signature of HSCs with long term multipotential capacity. A dormant state of HSCs is suggested to be critical for protecting and preventing depletion of the stem cell pool. Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced cell death, implicating that hypoxia is important in the bone marrow niche to limit reactive oxidative species (ROS) production and give life-long protection of HSCs. Another focus in this thesis is to investigate downstream pathways involved in tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic cells expressing mutated FLT3. / <p>On the day of the defence date the title of article II was "Hypoxia, via hypoxia-inducible factor (HIF)-1, mediates low cell cycle activity and preserves the engraftment potential of mouse hematopoietic stem cells" and one of the authors is no longer included in the article.</p>

Chin, Chi Hou. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [165]-178). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iv / Acknowledgements --- p.vi / Abbreviations --- p.vii / List of Figures and Tables --- p.xi / Publications --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Hematopoiesis and Leukemia --- p.2 / Chapter 1.1.1. --- Hematopoiesis --- p.2 / Chapter 1.1.2. --- Leukemia --- p.8 / Chapter 1.1.2.1. --- Overview of leukemia --- p.8 / Chapter 1.1.2.2. --- Symptoms and diagnosis of leukemia --- p.9 / Chapter 1.1.2.3. --- Classification of leukemia --- p.9 / Chapter 1.1.2.4. --- Epidemiology of leukemia --- p.13 / Chapter 1.1.2.5. --- Conventional treatments for leukemia --- p.15 / Chapter 1.1.2.6. --- Novel approaches to leukemia treatment --- p.18 / Chapter 1.2. --- Statins --- p.22 / Chapter 1.2.1. --- Overview of statins --- p.22 / Chapter 1.2.2. --- Chemical structures of statins --- p.24 / Chapter 1.2.3. --- Pharmacokinetics of statins --- p.26 / Chapter 1.2.4. --- Pleiotropic effects of statins --- p.29 / Chapter 1.2.4.1. --- Anti-inflammatory and immunomodulatory effects of statins --- p.29 / Chapter 1.2.4.2. --- Anti-angiogenic effects of statins --- p.30 / Chapter 1.2.4.3. --- Anti-tumor effects of statins --- p.31 / Chapter 1.3. --- Objectives and scope of the present study --- 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 media, buffers and other reagents" --- p.37 / Chapter 2.1.3.1. --- Cell culture media and reagents --- p.37 / Chapter 2.1.3.2. --- Drugs and chemicals --- p.40 / Chapter 2.1.3.3. --- Reagents and buffers for primary culture --- p.42 / Chapter 2.1.3.4. --- Dye solutions --- p.43 / Chapter 2.1.3.5. --- Reagents for cell proliferation assays --- p.44 / Chapter 2.1.3.6. --- Reagents and buffers for flow cytometry --- p.44 / Chapter 2.1.3.7. --- Reagents for Hoechst staining --- p.45 / Chapter 2.1.3.8. --- Reagents and buffers for DNA isolation --- p.46 / Chapter 2.1.3.9. --- Reagents and buffers for DNA agarose gel electrophoresis --- p.48 / Chapter 2.1.3.10. --- Reagents and buffers for Cell Death ELISA --- p.50 / Chapter 2.1.3.11. --- Reagents and buffers for measuring caspase activity --- p.51 / Chapter 2.1.3.12. --- Reagents and buffers for Western blotting --- p.55 / Chapter 2.1.3.13. --- Reagents for determining nitric oxide production --- p.63 / Chapter 2.2. --- Methods --- p.64 / Chapter 2.2.1. --- Culture of tumor cell lines --- p.64 / Chapter 2.2.2. --- "Isolation, preparation and culture of murine peritoneal macrophages" --- p.64 / Chapter 2.2.3. --- Cell proliferation and cytotoxicity studies --- p.66 / Chapter 2.2.4. --- In vivo tumorigenicity study --- p.68 / Chapter 2.2.5. --- Cell cycle profile and flow cytometric analysis --- p.69 / Chapter 2.2.6. --- Hoechst staining --- p.69 / Chapter 2.2.7. --- DNA fragmentation analysis --- p.70 / Chapter 2.2.8. --- Cell Death ELISA --- p.71 / Chapter 2.2.9. --- Mitochondrial membrane potential analysis --- p.73 / Chapter 2.2.10. --- Measurement of caspase activity --- p.73 / Chapter 2.2.11. --- Protein expression study --- p.75 / Chapter 2.2.12. --- Cell morphological staining --- p.80 / Chapter 2.2.13. --- Cell size and granularity analysis by flow cytometry --- p.81 / Chapter 2.2.14. --- Determination of nitric oxide production by macrophages --- p.81 / Chapter 2.2.15. --- Statistical analysis --- p.82 / Chapter Chapter 3 --- Anti-Proliferative Effect of Statins on Myeloid Leukemia Cells / Chapter 3.1. --- Introduction --- p.84 / Chapter 3.2. --- Results --- p.86 / Chapter 3.2.1. --- Anti-proliferative effect of statins on various murine and human myeloid leukemia cells --- p.86 / Chapter 3.2.2. --- Cytotoxicity of fluvastatin on murine myelomonocytic leukemia WEHI-3B JCS cells --- p.93 / Chapter 3.2.3. --- Cytotoxicity of fluvastatin on primary murine myeloid cells --- p.96 / Chapter 3.2.4. --- Kinetic and reversibility studies on the anti-proliferative effect of fluvastatin on WEHI-3B JCS cells --- p.98 / Chapter 3.2.5. --- Relationship between the anti-proliferative effect of fluvastatin and the cholesterol biosynthesis pathway in WEHI-3B JCS cells --- p.102 / Chapter 3.2.6. --- Effect of fluvastatin on the in vivo tumorigenicity of WEHI-3B JCS cells --- p.106 / Chapter 3.2.7. --- Effect of fluvastatin on the cell cycle profile of WEHI-3B JCS cells --- p.108 / Chapter 3.2.8. --- Effect of fluvastatin on the expression of cell cycle regulatory proteins inWEHI-3B JCS cells --- p.113 / Chapter 3.3. --- Discussion --- p.116 / Chapter Chapter 4 --- Apoptosis- and Differentiation-inducing Effects of Fluvastatin on Murine Myelomonocytic Leukemia WEHI-3B JCS Cells / Chapter 4.1. --- Introduction --- p.124 / Chapter 4.2. --- Results --- p.128 / Chapter 4.2.1. --- Induction of chromatin condensation in WEHI-3B JCS cells by fluvastatin --- p.128 / Chapter 4.2.2. --- Induction of DNA fragmentation in WEHI-3B JCS cells by fluvastatin --- p.130 / Chapter 4.2.3. --- Effect of fluvastatin on the mitochondrial membrane potential in WEHI-3B JCS cells --- p.134 / Chapter 4.2.4. --- Effect of fluvastatin on the caspase activities in WEHI-3B JCS cells --- p.138 / Chapter 4.2.5. --- Effect of fluvastatin on the expression of pro-apoptotic protein AIF in WEHI-3B JCS cells --- p.144 / Chapter 4.2.6. --- Effect of fluvastatin on the morphology of WEHI-3B JCS cells --- p.147 / Chapter 4.2.7. --- Effect of fluvastatin on the cell size and granularity of WEHI-3B JCS cells --- p.149 / Chapter 4.2.8. --- Immunomodulation of murine peritoneal macrophages by fluvastatin --- p.151 / Chapter 4.3. --- Discussion --- p.153 / Chapter Chapter 5 --- Conclusions and Future Perspectives --- p.160 / References --- p.165

BMI1 est une protéine appartenant à la famille des polycombs impliquée dans la régulation épigénétique de la transcription. Il a été montré que cette protéine est essentielle à la régulation de la prolifération, de la sénescence et du métabolisme ainsi qu’à l’auto-Renouvellement des cellules souches hématopoïétiques et cancéreuses. Ce répresseur transcriptionnel au fort potentiel oncogénique est retrouvé surexprimé dans de nombreux types de cancer ; dans le cas de la Leucémie Myéloïde Chronique (LMC) le niveau d’expression de BMI1 augmente avec l’aggravation de la pathologie. Cependant, les voies de signalisation impliquées dans sa surexpression et le rôle qu’il joue au sein de cette maladie demeurent méconnus. En réprimant l’expression de BMI1 par ARN interférence nous avons pu mettre en évidence que ce polycomb était essentiel à la prolifération cellulaire ainsi qu’au potentiel clonogénique des cellules de LMC. Nous avons également démontré pour la première fois que BMI1 soutenait la croissance tumorale à travers la répression d’un processus autophagique délétère pour la cellule cancéreuse. Une approche transcriptomique nous a permis d’identifier la cible transcriptionnelle impliquée dans ce processus, la Cycline G2. Nous avons, pour finir, trouvé une molécule, via une approche bioinformatique, capable de réinduire l’expression de la Cycline G2 dans les cellules de LMC, l’alexidine dihydrochloride. Cette molécule induit une forte autophagie dans les cellules cancéreuses ainsi que de l’apoptose. Elle s’est également montrée capable de resensibiliser à l’imatinib (un inhibiteur de BCR-ABL) une lignée pourtant résistante. / The polycomb protein Bmi1 is a major epigenetic regulator. It has been shown that this protein is essential for the regulation of cell proliferation, senescence and metabolism but also self-Renewal of hematopoïetic and cancer stem cells. This transcriptional repressor, with a strong oncogenic potential, is overexpressed in many types of cancer. In case of Chronic Myeloid Leukemia (CML) the expression level of BMI1 is associated with worsening prognosis. However, the signaling pathways involved in its overexpression and its role in this disease remains unclear. By using RNAi to repress BMI1 expression we highlighted that this polycomb was essential for proliferation and clonogenicity of CML cells. We also demonstrated, for the first time, that BMI1 supported tumor growth through repression of deleterious cancer cell autophagy. A transcriptomic approach allowed us to identify a transcriptional target involved in this process: the Cyclin G2. Through a bioinformatic approach, we finally found a molecule capable of expression re-Induction of Cyclin G2 in CML cells : alexidine dihydrochloride. This molecule induced a high level of autophagy as well as apopotosis in cancer cells. It had also been able to re-Sensitize to imatinib a resistant cell line. In conclusion, our results revealed a new role for the polycomb BMI1 in supporting the CML pathology. Moreover, our work allowed the identification of two new approaches for therapeutically targeting this oncogene functions.

Ces travaux de thèse ont porté sur le développement de formulations innovantes et nanoparticulaire, destinées à améliorer la prise en charge des patients atteints de leucémie aigüe myéloïdes (LAM). Cette amélioration de la qualité de vie peut passer par le développement d’une formulation orale de décitabine.Trois stratégies de formulations différentes ont été développées : deux formulations de nanocapsules lipides (LNCs) avec encapsulation ou de décitabine, ou d’une prodrogue de décitabine (décitabine(C12)2) . La troisième stratégie a été le développement de particules de type liposomal, dans lesquelles la décitabine a été encapsulée. Après avoir été caractérisée sur des critères physicochimiques, chacune des stratégies basées sur les LNCs a été évaluée par des essais in vitro pour évaluer la perméabilité intestinale de la décitabine lorsqu’elle a été encapsulée. Une des stratégies a permis d’accroitre la perméabilité, in vitro, de la décitabine. L’activité sur la prolifération cellulaire a ensuite été évaluée sur des cellules humaines de LAM. Il a été démontré que l’encapsulation dans les LNCs améliore l’activité de la décitabine et de la décitabine (C12)2. Après l’ensemble de ces essais, en vue d’évaluer le potentiel avantages de ces formulations pour augmenter la demi-vie plasmatique de la décitabine, leurs stabilités dans du plasma humain a été évaluée. La décitabine (C12)2 libre et encapsulée permettent de limiter la dégradation rapide de la décitabine. Finalement, une étude de pharmacocinétique a été mise en place. L’encapsulation de la décitabine, en synthétisant au préalable une prodrogue permet d’augmenter les concentrations maximales atteintes. / The aim of this phD work was to develop nanoparticle formulations to improve patients’quality of life in case of acute myeloid leukemia (AML). These formulations could, for example, allow an oral administration of decitabine. Three different formulations were developed: two were based on lipid nanocapsules (LNCs) with an encapsulation of decitabine or a decitabine prodrug (decitabine(C12)2). The third strategy was aliposomal formulation with a decitabine encapsulation. After being characterized on physico-chemical parameters, in vitro intestinal permeability studies were performed on LNCs strategies. One strategy was able to enhance decitabine permeability. Cell proliferation studies performed on human AMLcell lines showed that encapsulations into LNCs improve decitabine and decitabine(C12)2 activities. In order to evaluate the potential of these formulations to enhance decitabine plasma half-life, their stabilities in human plasma were then assayed. Free decitabine(C12)2 or encapsulated into LNCs has been shown to limit the rapid decitabine degradation. Finally, pharmacokinetic studies were performed. Decitabine encapsulation into LNCs with a previous decitabine prodrug synthesis was able to increase maximal plasma concentrations.

<p>The aims of the studies described in the thesis were to investigate different treatment strategies in chronic myeloid leukemia (CML) patients. Furthermore, activity of imatinib was investigated by <i>in vitro</i> cytotoxicity assay, and the gene expression pattern in interferon treated patients.</p><p>In a randomized prospective national study, we examined the influence of busulphan (n=89) versus hydroxyurea (n=90) treatment on time to blast crisis, and survival. There was no significant difference in survival between hydroxyurea and busulphan treated patients; median survival was 3.5 and 3.2 years, respectively. The 26 patients who underwent allogeneic stem cell transplantation had a significantly longer median survival (4.7 years) than those who were not transplanted.</p><p>We investigated the feasibility of mobilizing Philadelphia chromosome negative blood stem cells with intensive chemotherapy and lenograstim in CML patients. Twenty-three patients (62%) were successfully mobilized. Twenty-one of these patients underwent autologous stem cell transplantation later on, with a 5-year overall survival at 68%.</p><p>Fluorometric Microculture Cytotoxicity Assay was used to analyze 32 tumor cell samples from CML patients, (26 chronic phase and 6 blast crisis). Imatinib showed a higher <i>in vitro</i> activity and more positive drug interactions in cells from blast crisis than from chronic phase. Interferon, daunorubicin and arsenic trioxide had the greatest benefit from a combination with imatinib.</p><p>Microarray-based gene expression analyses were performed on diagnostic CML samples prior to interferon treatment. We identified six genes that were differentially expressed in responders and non-responders to interferon. It might prove possible to use gene expression analysis to predict future response to interferon.</p>

The aims of the studies described in the thesis were to investigate different treatment strategies in chronic myeloid leukemia (CML) patients. Furthermore, activity of imatinib was investigated by in vitro cytotoxicity assay, and the gene expression pattern in interferon treated patients. In a randomized prospective national study, we examined the influence of busulphan (n=89) versus hydroxyurea (n=90) treatment on time to blast crisis, and survival. There was no significant difference in survival between hydroxyurea and busulphan treated patients; median survival was 3.5 and 3.2 years, respectively. The 26 patients who underwent allogeneic stem cell transplantation had a significantly longer median survival (4.7 years) than those who were not transplanted. We investigated the feasibility of mobilizing Philadelphia chromosome negative blood stem cells with intensive chemotherapy and lenograstim in CML patients. Twenty-three patients (62%) were successfully mobilized. Twenty-one of these patients underwent autologous stem cell transplantation later on, with a 5-year overall survival at 68%. Fluorometric Microculture Cytotoxicity Assay was used to analyze 32 tumor cell samples from CML patients, (26 chronic phase and 6 blast crisis). Imatinib showed a higher in vitro activity and more positive drug interactions in cells from blast crisis than from chronic phase. Interferon, daunorubicin and arsenic trioxide had the greatest benefit from a combination with imatinib. Microarray-based gene expression analyses were performed on diagnostic CML samples prior to interferon treatment. We identified six genes that were differentially expressed in responders and non-responders to interferon. It might prove possible to use gene expression analysis to predict future response to interferon.

Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %. In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML. The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved. Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS. Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL). Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.

The diagnosis and monitoring of AML relies predominantly on the identification of blast cells in the bone marrow and peripheral blood. While at the time of diagnosis the identification of leukemic cells is relatively easy, during remission the identification of small numbers of blasts is problematic. This is most evident by the fact that patients who achieve complete remission frequently relapse, despite pathologic examination indicating a marked reduction in leukemic cell burden. In this thesis I have explored the potential of using serum proteins secreted by leukemic cells as a means of monitoring disease in patients. To identify proteins that might be useful for monitoring, I took advantage of published gene expression arrays and looked into online bioinformatics databases. Using specific characteristics, I was able to identify approximately 107 candidate proteins secreted by AML cells. RT-PCR analysis and ELISA assays were performed to evaluate the variability of expressions and serum level differences of twelve different proteins in the list.