Methotrexate, a folic acid antagonist, has been used in the clinical treatment of a wide variety of malignant neoplasms for over 20 years, either as a single agent or in combination with other antineoplastic agents. It is a cell cycle specific inhibitor and kills cells only in the S phase of growth. MTX is a potent inhibitor of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3.), which catalyses the NADPH dependent reduction of dihydrofolic acid and folic acid to tetrahydrofolic acid: the metabolically active coenzyme form of folic acid essential in the biosynthesis of dTMP from dUMP by thymidylate synthetase. Inhibition of DHFR therefore leads to the inhibition of DNA synthesis and cell death. Methotrexate has many favourable properties; for instance, it interacts directly with intracellular sites without the need for prior metabolic transformation. It can be administered in large doses because toxicity to normal cells can be minimized by the administration of folinic acid (N5 formyl tetrahydrofolic acid) shortly after the administration of MTX. However, the effectiveness of MTX is inevitably compromised by the emergence of drug resistance, which can be either intrinsic, i.e. the tumour cells are resistant to MTX at the outset, or the tumour cells acquire resistance after exposure to MTX. An understanding of the mechanisms of resistance to MTX is therefore very important if treatment with this potent antineoplastic agent is to be improved. Three mechanisms of resistance to MTX have been determined from studies with experimental tumour systems: impaired uptake of MTX; increased levels of dihydrofolate reductase; and appearance of altered dihydrofolate reductase with a lower affinity for MTX. Impaired uptake of MTX and increased levels of OHFR can both theoretically be overcome by sustaining increased concentrations of free intracellular MTX. This can be achieved by exposing the cells to higher concentrations of MTX, and many chemotherapeutic regimens now use 'high-dose' MTX which can achieve plasma concentrations of MTX as high as 10⁻³M. However, resistance to MTX is still a major clinical problem and the use of 'high-dose' MTX has not significantly increased the therapeutic index of MTX treatment. Appearance of DHFR with a lower affinity for MTX suggests as an alternative the synthesis of an agent which would be a potent inhibitor of the altered enzyme, and requires the detailed characterization of the properties of this enzyme. If the altered enzyme retains some affinity for MTX, the administration of MTX and the more potent agent would result in better growth inhibition of the resistant tumour. In this thesis, a mouse leukemia cell line (L5178Y) grown in suspension culture was used to isolate two MTX-resistant cell lines and these were used to study the mechanisms leading to MTX resistance. Both resistant cell lines exhibited impaired MTX uptake when exposed to 10⁻⁶M MTX but not when exposed to 10⁻⁴M MTX, Both lines also had elevated DHFR levels (7 to 9 fold). A variant form of DHFR present in small amounts in both cell lines was isolated by MTX-sepharose affinity chromatography. The altered DHFR differed from the major form of reductase present in these cells in its markedly lower affinity (100,000 fold) for MTX. The two forms of the enzyme were purified from the most resistant cell line and their properties compared. They were found to differ moderately in their Km for substrates, however, the Ki of MTX differed by a factor of 100,000 for the two forms. In addition there were marked differences in their heat stability, isoelectric points and sensitivity to p-chloromercuriphenyl-sulphonate, and a minor difference in their molecular weights. It is concluded that the presence of a highly resistant form of DHFR in these cell lines represents an important mechanism in conferring a high degree of resistance to these cells. The importance of this form of DHFR in MTX resistance is discussed in relation to impaired transport and elevated DHFR levels. Experiments to determine the amino acid sequence of the altered enzyme are underway and once determined should facilitate the synthesis of specific inhibitors of its activity. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Acute leukemias are the main type of malignancy affecting children. They are defined by their precursor cell lineage: myeloid lineage for acute myeloid leukemia (AML) and lymphoid lineage for acute lymphoblastic leukemia (ALL). In this thesis, we use systems biology approaches to characterize transcription factor (TF) programs that define novel AML subtypes. We combine this approach with machine learning methods to group patients sharing similar TF programs and risk-stratify them. We identify a 9-cluster solution with statistically significant survival differences ranging from 84% for the best group to 41% for the worst. Each of the clusters is composed of patients with various cytogenetic aberrations that would not necessarily have been classified together. We identify top aberrantly activated TFs and potential master regulators or drug targets in each cluster. We also propose a novel stratification for FLT3-ITD patients with no other cytogenetic abnormalities. These patients are currently all classified as high-risk; however, we find a low-risk subtype and identify a TF signature that is predictive of risk in this subtype. Finally, we develop a binary classifier that is able to stratify the patients into two risk groups. We find that the activity of a large cluster of HOXA TFs is highly correlated with poor prognosis. In the second part, we characterize some mechanisms of relapse in B-ALL at a single-cell resolution focusing again on the patterns of activation and deactivation of TF activity in the course of the disease in matched trios of samples (diagnosis, remission and relapse). After a discussion on some of the technical aspects of differentiating normal cells and leukemic cells at a single-cell RNA sequencing resolution, we perform computational pseudo-lineage reconstruction based on groups of TFs whose activities rise and fall together through pseudotime. We find that each patient has unique mechanisms at the earliest pseudotimes but they seem to converge at the later pseudotimes into signatures in which the B-cell identity (in the case of B-ALL) gradually fades away. We also identify small populations of cells isolated at diagnosis in the later pseudotimes which is consistent with the view that many of the persistent cells in ALL pre-exist the malignancy and are selected by the treatment. This novel systems biology approach for characterizing clinical outcome in patients and defining lineage reconstruction identifies biochemical mechanisms and signaling pathways that are responsible for the development and maintenance of the malignancy and identifies potential therapeutic targets. The results exposed in this thesis will lead to a better understanding of some of the inner workings of pediatric acute leukemias and may lead to the development of improved targeted therapies.

Currently there is no curative therapy for Chronic Myelogenous Leukemia (CML), and patients must remain on the current prescribed treatment, tyrosine kinase inhibitors (TKI), indefinitely. Although many patients can survive in the chronic phase of the disease under TKI treatment, some patients do progress to the terminal blast crisis phase of the disease. Patients in this terminal phase do not respond to TKI treatment. We evaluated the therapeutic benefit of targeting the oncogene c-MYC in CML, using the CML cell line K562. This was achieved by inhibiting the enzyme O-linked β-N-acetylglucosamine Transferase (OGT), using two indirect inhibitors 2-deoxyglucose and Azaserine, and the direct inhibitor ST078925. Treatment with these inhibitors resulted in decreased half-life of c-MYC protein in K562, reduced c-MYC protein in K562 cells, and reduced K562 cell growth. Together these results suggest that targeting c-MYC through OGT may be a potential therapeutic option for patients with CML.

Lam Kin Bong Hubert. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 139-161). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / (in chinese) --- p.iv / Acknowledgements --- p.vi / Publications --- p.ix / Table of Contents --- p.x / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Abbreviations --- p.xvi / Chapter CHAPTER1： --- General Introduction --- p.1 / Chapter Section 1.1 --- Historical Background and Application of Arsenic Trioxide as an Anti-cancer Agent --- p.1 / Chapter Section 1.2 --- Arsenic Trioxide Induces Apoptosis in Cancer Cells --- p.3 / Chapter 1.2.1 --- The Intrinsic and Extrinsic Pathways of Apoptosis Initiation --- p.4 / Chapter 1.2.2 --- The Convergence of Pathways --- p.8 / Chapter 1.2.3 --- Induction of Apoptosis by Arsenic Trioxide --- p.9 / Chapter 1.2.3.1 --- Controversies in the Involvement of the Extrinsic Pathway --- p.9 / Chapter 1.2.3.2 --- "Arsenic Trioxide, Oxidative Stress and the Mitochondria" --- p.10 / Chapter 1.2.3.3 --- Caspase-3 Activation in Arsenic Trioxide-mediated Apoptosis --- p.12 / Chapter Section 1.3 --- Arsenic Trioxide Perturbs the Cell Division Cycle --- p.13 / Chapter 1.3.1 --- The Cell Cycle Oscillator --- p.14 / Chapter 1.3.2 --- DNADamage and Cell Cycle Checkpoints --- p.15 / Chapter 1.3.3 --- Induction of Cell Cycle Arrest by Arsenic Trioxide and its Association with Apoptosis --- p.17 / Chapter Section 1.4 --- Acute Megakaryocytic Leukemia and Arsenic Trioxide --- p.20 / Chapter CHAPTER 2： --- Objectives --- p.28 / Chapter CHAPTER 3： --- Methodology --- p.30 / Chapter Section 3.1 --- Materials --- p.30 / Chapter Section 3.2 --- Methods --- p.39 / Chapter 3.2.1 --- Culture of Megakaryocytic Cells and Their Treatment with Arsenic Trioxide --- p.39 / Chapter 3.2.1.1 --- Maintenance of Cell Lines --- p.39 / Chapter 3.2.1.2 --- Treatment with Arsenic Trioxide --- p.39 / Chapter 3.2.2 --- "Effects of Arsenic Trioxide on Cell Proliferation, Apoptosis, Mitochondrial Integrity and Cell Division Cycle Profiles of Human Megakaryocytic Leukemia Cell Lines" --- p.40 / Chapter 3.2.2.1 --- Trypan Blue Exclusion Assay --- p.40 / Chapter 3.2.2.2 --- Quantitation of Externalized Phosphatidylserine --- p.41 / Chapter 3.2.2.3 --- Quantitation of Active Caspase-3 Expression --- p.42 / Chapter 3.2.2.4 --- Assessment of Mitochondrial Intensity --- p.42 / Chapter 3.2.2.5 --- Analysis of Cell Division Cycle Profile --- p.43 / Chapter 3.2.2.6 --- Analysis of Cell Cycle Kinetics by BrdU Labeling --- p.43 / Chapter 3.2.2.7 --- Identification of Cell Cycle Specificity of Caspase-3 Expression --- p.45 / Chapter 3.2.3 --- Effects of Arsenic Trioxide on the Expression of Apoptotic Signals in Human Megakaryocytic Leukemia Cell Lines --- p.45 / Chapter 3.2.3.1 --- Effects of Arsenic Trioxide on mRNA Expression Levels of Apoptotic Regulators --- p.45 / Chapter 3.2.3.2 --- Effects of Arsenic Trioxide on Protein Expression Levels of Apoptotic Regulators --- p.50 / Chapter 3.2.3.2.1 --- Flow Cytometric Analysis --- p.50 / Chapter 3.2.3.2.2 --- Western Blot Analysis --- p.51 / Chapter 3.2.4 --- Effects of Arsenic Trioxide on Gene Expression Profiles of Human Megakaryocytic Leukemia Cell Lines By Microarray Analysis --- p.54 / Chapter 3.2.5 --- Statistical Analysis --- p.57 / Chapter CHAPTER 4： --- "Effects of Arsenic Trioxide on Cell Proliferation, Apoptosis, Mitochondrial Integrity and Cell Division Cycle Profiles of Human Megakaryocytic Leukemia Cell Lines" --- p.62 / Chapter Section 4.1 --- Introduction --- p.62 / Chapter Section 4.2 --- Results --- p.63 / Chapter 4.2.1 --- Effects of Arsenic Trioxide on Proliferation Kinetics --- p.63 / Chapter 4.2.2 --- Effects of Arsenic Trioxide on Cell Viability --- p.64 / Chapter 4.2.3 --- Apoptosis-inducing Capability of Arsenic Trioxide --- p.65 / Chapter 4.2.3.1 --- Quantitation of Externalized Phosphatidylserine --- p.65 / Chapter 4.2.3.2 --- Quantitation of Active Caspase-3 Expression --- p.66 / Chapter 4.2.4 --- Effects of Arsenic Trioxide on Mitochondrial Integrity --- p.67 / Chapter 4.2.5 --- Effects of Arsenic Trioxide on Cell Division Cycle Profiles --- p.69 / Chapter 4.2.6 --- Effects of Arsenic Trioxide on Cell Cycle Kinetics by Bromodeoxyuridine Labeling --- p.69 / Chapter 4.2.7 --- Identification of Cell Cycle Specificity of Arsenic Trioxide-Induced Caspase-3 Activation --- p.71 / Chapter Section 4.3 --- Discussion --- p.72 / Chapter CHAPTER 5： --- Effects of Arsenic Trioxide on Apoptotic Signal Expression in Human Megakaryocytic Leukemia Cell Lines --- p.91 / Chapter Section 5.1 --- Introduction --- p.91 / Chapter Section 5.2 --- Results --- p.92 / Chapter 5.2.1 --- Effects of Arsenic Trioxide on mRNA Expression Levels of Apoptotic Regulators --- p.92 / Chapter 5.2.2 --- Effects of Arsenic Trioxide on Protein Expression Levels of Apoptotic Regulators --- p.94 / Chapter 5.2.2.1 --- Flow Cytometric Analysis --- p.94 / Chapter 5.2.2.2 --- Western Blot Analysis --- p.96 / Chapter Section 5.3 --- Discussion --- p.96 / Chapter CHAPTER 6： --- Effects of Arsenic Trioxide on Gene Expression Profiles of Human Megakaryocytic Leukemia Cell Lines by Microarray Analysis --- p.119 / Chapter Section 6.1 --- Introduction --- p.119 / Chapter Section 6.2 --- Results --- p.119 / Chapter Section 6.3 --- Discussion --- p.122 / Chapter CHAPTER 7： --- General Discussion and Conclusions --- p.135 / BIblography --- p.139

Addendum attached to back facing leaves. Includes bibliographical references (leaves 172-199). The findings establish leukemia inhibitory factor, and possibly oncostatin M, as new members of a small but growing class of cytokines produced in an intracellularly active form and also suggest that the production of alternate transcripts and intercellularly-retained proteins may be a common and important feature of cytokines of the IL-6 and other families.

No description available.

Addendum attached to back facing leaves. / Includes bibliographical references (leaves 172-199). / 199 leaves, 5 photographic plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The findings establish leukemia inhibitory factor, and possibly oncostatin M, as new members of a small but growing class of cytokines produced in an intracellularly active form and also suggest that the production of alternate transcripts and intercellularly-retained proteins may be a common and important feature of cytokines of the IL-6 and other families. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 2000

Thesis (M.D.)--University of Adelaide, 1985. / Includes bibliographical references (leaves 1-14 of section Reference).

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 89-99). Also available on the World Wide Web.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 2000. / Addendum attached to back facing leaves. Includes bibliographical references (leaves 172-199).