Acute Myeloid Leukaemia (AML) is a disease normally found in elderly patients, with the median age of presentation at about 68 years. At this age, many of the patients are frail and unlikely to respond well to intensive chemotherapy treatments. Conventional chemotherapy eradicates the proliferating leukaemic progenitors while leaving the quiescent leukaemic stem cells undisturbed. These quiescent LSCs are able to then bring about leukaemic relapse. Fumagillin is a natural metabolite from Asperigillus fumigatus that is generally used as an anti-microbial agent but it is also known to bind to intracellular MetAP-II and inhibit endothelial cell growth. Many cancers are found to have an over expression of MetAP-II. In the past, MetAP-II inhibitors have been tested and shown success in angiogenesis inhibition and tumor reduction. The aim of this study is to observe whether methionine aminopeptidase-2 inhibitors can be used in the treatment of acute myeloid leukemia. The investigation included a dose response comparison of various AML cell lines to fumagillin treatment, cell proliferation assay, a colony forming unit assay, and cell cycle analysis of KG-1 cells following three days of fumagillin treatment. I have determined that fumagillin does indeed decrease the cellular proliferation of KG-1 in vivo and at 10μM, prevents colony formation in methylcellulose plating. There is an increase in cells found in the sub-G1 phase with fumagillin treatment, as analyzed by flow cytometry. It is interpolated that fumagillin treatment increases AML cell apoptosis, in addition to hindering its ability to grow in culture. / published_or_final_version / Medicine / Master / Master of Medical Sciences

The current work was undertaken to test the ability of avidin to affect the course of leukemia when administered intraperitoneally.

Background: Chronic myeloid leukaemia (CML) is a haematological malignant disease involving haematopoietic stem cells. It is caused by a known reciprocal chromosomal t(9;22)(q34;q11) translocation, or known as Philadelphia chromosome. The translocation results in the formation of a chimeric BCR-ABL fusion gene. In the most recent guidelines published by NCCN and European LeukemiaNet in 2013, tyrosine kinase inhibitors (TKI) specifically inhibiting the Bcr-Abl tyrosine kinase, are the first-line therapy for patients with chronic phase CML. Imatinib is the oldest among the 3 TKI, and is the most commonly prescribed. Despite its proven therapeutic role in CML, imatinib is a drug of extreme high cost. Estimated annual drug cost is HKD$223,380for a standard 400mg adult daily dose. Therefore, this study aims to survey on the prescribing pattern of imatinib in CML patients, its funding status, response; and estimate its economic burden on the Hong Kong population. Methodology: This is a retrospective patient chart review study. All patients who were diagnosed with CML from 2003 to 2012 and were managed in QMH or QEH were reviewed. Electronic records were retrieved to see whether imatinib was started as first-line treatment within 6 months of diagnosis. The reasons for not initiating imatinib were also investigated. Patients’ response to imatinib, and funding source for the drug, were documented. Annual drug cost of imatinib was estimated from all CML patients who attended all Hospital Authority institutions in 2012 who were prescribed with the drug. Results: Total 153 patients from the 2 institutions were reviewed. One hundred twenty four (81%) of them started imatinib as first-line therapy within 6 months of diagnosis. Nine patients started second generation TKI as first-line. Among those who did not start TKI, the most common reasons are patient preference (3.9%) and financial difficulties (3.3%). Twelve paediatric patients are identified, and all but one of them started imatinib. Seventy one% patients on imatinib experienced side effects. Most frequently reported adverse reactions are thrombocytopenia, oedema and neutropenia. Twenty eight% switched to second generation TKI due to suboptimal response or intoleranceto imatinib. During their course of treatment, 46.3% patients on imatinib require social subsidy from Samaritan Fund. From the dispensing records, the average drug cost per patient per year is HK$113,902. The estimated annual cost burden on the whole Hong Kong population is HK$43,425,878. Conclusion: The prescribing rate of imatinib in chronic phase CML patients in Hong Kong is comparable to overseas prescribing rate. The drug has become a significant financial burden to patients’ family and the society as a whole. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Medical Sciences

Progress in understanding cancer progression has been hampered over the years by the different types of mutations present and irregular changes of gene regulation associated with any given cancer. In this work, molecular interactions between TALI, LMO1, and decorin were investigated. Numerous studies have shown that ectopic expression of decorin protein induced growth suppression in neoplastic cells of various histogenic origins. Furthermore, ectopic expression of TAL1 and LMOI oncoproteins has been shown to occur in approximately 50% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). It was of interest then to determine the preventive or interactive role decorin played with the oncogenic activity of TAL I and LMO1. In this investigation, decorin was introduced into a murine T-cell line (AKR-DP-603) through the use of the mammalian expression vector pcDNA3.1 (-). This particular cell line was previously engineered to express TALI and LMO1. Protein expression patterns in all cell populations were analyzed using the Western blot technique and a proteoglycan with a molecular weight of 100 kDa before chondroitinase ABC treatment and a core protein of55 kDa after treatment with chondroitinase ABC was seen. This finding is significant since it implies that the pcDNA3. 1(-) vector containing decorin cDNA was present, and the corresponding decorin peptides were expressed in both cytoplasmic and nuclear extracts. Furthermore, Northern blot analysis was performed on total RNA extracts to determine the transcriptional state of endogenous decorin rRNA, as well as exogenously introduced decorin. Northern blot analysis revealed no decorin-specific mRNA transcripts from the various cell populations. This result did not imply a lack of possible regulatory effect on protein and mRNA levels of TALL and LMOI by decorin. Finally, cell growth assays were performed on all cell populations and cell counts were used to assess the growth pattern of each population after serum withdrawal. The results show possible growth suppressive effects of decorin on TAL1 and LMOI expressing cells. Results obtained from this study shed further light on the molecular interactions influencing T-ALL and may also help in the design of potentially beneficial cancer treatments using decorin. / Department of Biology

It is the purpose of this paper to attempt to confirm the work of Schlagenhauf and Ingebrigsten and to show the effects of repeated injections of guinea pig serum immunized with various strains of mouse leukemia.

白血病由血液或骨髓中的癌細胞所形成，基於其造血幹細胞（HSC）的增殖和分化出現偶聯或不平衡的情況而產生的結果。白血病是香港最常見的兒童癌症，報告指出，於2005年至2009年期間，平均每年約有270名患者死於該病。傳統治療白血病的方法包括化療，放射性治療，骨髓或外周血幹細胞移植，至於採用哪種療法，則要視乎白血病的類型和階段。然而，這些療法會為患者帶來各種副作用，因此，在過去十年間，研發新型治療白血病的藥物引起了越來越多人的關注。 / 共軛脂肪酸（CFA），是指一群位置及幾何異構體的多元不飽和脂肪酸（PUFA），於它們的化學結構中，最少有一組共軛雙鍵。天然的共軛脂肪酸包括，存在於反芻動物的肌肉及乳製品中的共軛雙烯酸（CLA），存在於植物種子油中的共軛三烯酸（CLN），以及存在於海藻中的共軛四烯酸，共軛五烯酸（CEPA）和共軛六烯酸（CDHA）。過往的研究證實了共軛雙烯酸擁有各種生理及醫藥功效，包括抗脂肪分化，抗動脈硬化，抗糖尿病，免疫調節和抗腫瘤作用。根據體外實驗報告指出，共軛三烯酸和共軛四烯酸對多種腫瘤細胞株皆具有生長抑制作用，然而，它們對小鼠巨噬細胞樣的白血病細胞之調節作用和機制仍有待研究。因此，在這篇論文中，共軛三烯酸和共軛四烯酸對小鼠巨噬細胞樣的白血病細胞的抗增殖作用，以及它們引起的相關機制將會被探討。 / 本實驗計劃研究了九個不同的多元不飽和脂肪酸異構體對小鼠巨噬細胞樣的白血病細胞PU5-1.8細胞的抗增殖能力，當中包括三烯酸、四烯酸、共軛雙烯酸、共軛三烯酸和共軛四烯酸的異構體。結果清楚地表明，共軛三烯酸和共軛四烯酸異構體皆能對白血病細胞表現出劑量依賴性的生長抑制作用。於十個異構體當中，順式-8，反式-10，順式-12共軛三烯酸(蘭花酸)和順式-9，反式-11，反式-13，順式-15共軛四烯酸(杷茬酸)較其他共軛脂肪酸異構體更有效抑制白血病細胞的生長，因此，他們被選定為主要的研究對象，以便對它們所引起的相關機制作進一步了解。此外，蘭花酸和杷茬酸對其他小鼠巨噬細胞樣的白血病細胞，包括J774 A.1細胞和P388D1細胞，也具備抗增殖作用，表現其生長抑制作用並不是純粹針對單一種腫瘤細胞株的。有趣的是，蘭花酸和杷茬酸對PU5-1.8細胞的生長抑制作用是可以局部逆轉的，但只限以低濃度的共軛脂肪酸培養白血病細胞以及培養的時間不多於24小時，否則，隨著培養的時間增加或共軛脂肪酸的濃度增加時，該生長抑制作用幾乎是不可逆轉的。另一方面，結果也表明蘭花酸和杷茬酸在其抑制白血病細胞增殖率為五十百分比之濃度下，它們對腫瘤細胞以及小鼠正常細胞的毒性作用是很少的。除了在體外研究，預先以蘭花酸處理的PU5-1.8細胞於BALB/c小鼠內導致白血病細胞生長的能力也以劑量依賴方式被抑制。 / 幾種不同的機制也能解釋蘭花酸和杷茬酸對PU5-1.8細胞的生長抑制能力，當中包括阻礙腫瘤細胞週期的前進，增加腫瘤細胞內活性氧（ROS）的生產或誘導腫瘤細胞的凋亡。研究結果指出，蘭花酸和杷茬酸可以抑制PU5-1.8細胞週期的進程並將其停留在G₀/G₁時相，換來的是減少處於S時相的細胞的比例。此外，透過西方蛋白質印跡分析，細胞週期蛋白E的表達有所下調，同時，幾個細胞週期調控蛋白的表達，包括p21，p27及p53蛋白則被上調，跟上述的實驗結果吻合。此外，經蘭花酸和杷茬酸處理後，PU5-1.8細胞內的ROS濃度和線粒體質量也有所增加，而它們對白血病細胞的生長抑制作用則會被抗氧化劑所減弱，這一點說明了蘭花酸和杷茬酸對PU5-1.8細胞的抗增殖作用可能與細胞內的脂質過氧化物濃度和線粒體質量有關。最後，通過各種測試細胞凋亡的實驗，包括利用細胞死亡檢測的ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40}試劑盒，Annexin-V和JC-1染色等方法，清楚地表明了蘭花酸和杷茬酸能誘導PU5-1.8細胞的凋亡。加上西方蛋白質印跡分析，PU5-1.8細胞內Bcl-2和Bcl-XL的蛋白的表達水平有所下降，而相反Bax蛋白的表達水平則有所提升，足以證明蘭花酸和杷茬酸能引發PU5-1.8細胞的凋亡。 / 總括來說，蘭花酸和杷茬酸對PU5-1.8細胞的抗增殖作用呈現時間和劑量依賴性，該作用可能基於阻礙腫瘤細胞週期的前進，增加腫瘤細胞內ROS的生產或誘導腫瘤細胞的凋亡。由於蘭花酸和杷茬酸分別在植物種子油和海藻的含量相當高，再加上它們對正常細胞無直接毒性，若果能夠對它們的抗腫瘤作用以及其分子機制有更透徹的理解，它們有望發展成為未來治療白血病的藥物。 / Leukemia is a cancer of the blood or bone marrow which is the result of uncoupling or imbalance of the proliferation and differentiation of hematopoietic stem cells (HSC). It is the most common childhood cancer in Hong Kong and it claims the lives of around 270 patients per year from 2005 to 2009 in average. Conventional approaches to leukemia therapy include chemotherapy, radiotherapy and bone marrow or peripheral blood stem cell transplantation, depending on the types and stages of leukemia. Nevertheless, these therapies are accompanied by a number of undesirable effects to the patients, hence, the development and research in novel treatments of leukemia are attracting increasing attention in the past decades. / Conjugated fatty acids (CFA) refer to the positional and geometric isomers of polyunsaturated fatty acids (PUFA) with conjugated double bonds. Naturally-occurring CFA include conjugated linoleic acids (CLA) from meat and dairy products of ruminant animals, conjugated linolenic acids (CLN) from plant seed oils, conjugated tetraenoic acids, conjugated eicosapentaenoic acids (CEPA) and conjugated docosahexaenoic acids (CDHA) from seaweeds. CLA have been shown to possess various biological and pharmacological activities, including anti-adipogenic, anti-atherogenic, anti-diabetogenic, immunomodulatory and anti-tumor effects. Furthermore, previous researches have demonstrated the growth-inhibitory effects of CLN and conjugated tetraenoic acids on a wide variety of cancer cell lines in vitro, however, their modulatory effects and action mechanisms on murine macrophage-like leukemia cells remain poorly understood. In this thesis project, the anti-proliferative effects of CLN and conjugated tetraenoic acids on the murine macrophage-like leukemia cells, as well as their action mechanisms will be elucidated. / Nine different PUFA isomers, including linolenic acid, tetraenoic acid, CLA, CLN and conjugated tetraenoic acids were examined for their anti-proliferative effects on the murine macrophage-like leukemia PU5-1.8 cells. The results clearly showed that all CLN isomers and cis-parinaric acid could exhibit growth-inhibitory effects on the leukemia cells in a dose-dependent manner. It was found that jacaric acid and cis-parinaric acid were relatively more potent than the other isomers used in the present study, hence, they were chosen to be the specific targets for more in-depth mechanistic studies. In addition, the anti-proliferative effects of jacaric acid and cis-parinaric acid were observed in other murine macrophage-like leukemia cell lines, including J774 A.1 cells and P388D1 cells, suggesting that the effects were not cell-line specific. Interestingly, the growth-inhibitory effects were partially reversible at lower concentrations of CFA used within 24 hours of incubation, but the effects were almost irreversible when either the incubation time or the concentration of CFA used was increased. Furthermore, the results showed that both jacaric acid and cis-parinaric acid at their IC₅₀ growth-inhibitory concentrations on PU5-1.8 cells exerted minimal, if any, direct cytotoxic effects on the tumor cells as well as the murine normal cells. Apart from the in vitro studies, it was also demonstrated that pre-treatment of PU5-1.8 cells with jacaric acid could significantly decrease the leukemic cell growth in syngeneic BALB/c mice in a dose-dependent manner. / Several mechanisms were proposed for the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells, including the triggering of cell cycle arrest, increasing the production of intracellular reactive oxygen species (ROS) or induction of apoptosis in the tumor cells. The results showed that jacaric acid and cis-parinaric acid could inhibit the cell cycle progression since an accumulation of PU5-1.8 cells at the G₀/G₁ phase was observed, together with a decrease in the cell population at the S phase. This finding was supported by the down-regulation of cyclin E protein and up-regulation of several cell cycle regulatory proteins, including the p21, p27 and p53 proteins. Apart from that, the intracellular ROS concentration and the mitochondrial mass were found to be increased in jacaric acid- or cis-parinaric acid-treated PU5-1.8 cells, and their growth-inhibitory effects were alleviated after the addition of antioxidants. Therefore, the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells might be correlated with the intracellular concentration of lipid peroxides and the mitochondrial mass. Furthermore, the results clearly demonstrated that both jacaric acid and cis-parinaric acid exhibited dose-dependent apoptosis-inducing effects on PU5-1.8 cells, as revealed by the Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit, annexin V assay and JC-1 dye staining method. In addition, it was found that the expression levels of Bcl-2 and Bcl-xL proteins were decreased, whereas the expression level of Bax protein was increased in PU5-1.8 cells, further confirming that apoptosis occurred in PU5-1.8 cells after treatment with jacaric acid and cis-parinaric acid. / Collectively, the results showed that jacaric acid and cis-parinaric acid could exhibit their anti-proliferative effects on PU5-1.8 cells in a time- and dose-dependent manner, through the triggering of cell cycle arrest, increasing the production of intracellular ROS or induction of apoptosis in the tumor cells. Owing to their high abundance in plant seed oils and seaweeds, and being relatively non-cytotoxic, they might be potential candidates for the treatment of leukemia. Further investigations are required in order to develop a better understanding on the molecular action mechanisms underlying the anti-tumor effects of jacaric acid and cis-parinaric acid on leukemia cells before they could be developed as the therapeutic drugs for leukemia. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Wai Nam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 169-182). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Acknowledgements --- p.viii / List of Abbreviations --- p.ix / List of Figures and Tables --- p.xiii / Publications --- p.xvii / Chapter Chapter 1 --- General introduction / Chapter 1.1 --- Introduction to hematopoiesis and leukemia --- p.1 / Chapter 1.1.1 --- Introduction to hematopoiesis --- p.1 / Chapter 1.1.2 --- Introduction to leukemia --- p.7 / Chapter 1.1.2.1 --- Classification of leukemia --- p.7 / Chapter 1.1.2.2 --- Epidemiology of leukemia --- p.10 / Chapter 1.1.2.3 --- Conventional approaches to leukemia therapy --- p.12 / Chapter 1.1.2.4 --- Alternative approaches to leukemia therapy --- p.17 / Chapter 1.2 --- Introduction to conjugated fatty acids --- p.19 / Chapter 1.2.1 --- An overview of polyunsaturated fatty acids and conjugated fatty acids --- p.19 / Chapter 1.2.2 --- Chemical structures and physical properties of CLN and conjugated tetraenoic acids --- p.21 / Chapter 1.2.3 --- Natural occurrence of CLN and conjugated tetraenoic acids --- p.26 / Chapter 1.2.4 --- Synthesis of CLN and conjugated tetraenoic acids --- p.28 / Chapter 1.2.5 --- Metabolism of CLN --- p.30 / Chapter 1.2.6 --- Major biological and pharmacological activities of CLN and conjugated tetraenoic acids --- p.30 / Chapter 1.2.6.1 --- Anti-obese and hypolipidemic property --- p.31 / Chapter 1.2.6.2 --- Anti-carcinogenic property --- p.32 / Chapter 1.2.6.2.1 --- Anti-proliferative effect --- p.32 / Chapter 1.2.6.2.2 --- Apoptosis-inducing effect --- p.33 / Chapter 1.3 --- Aims and scopes of this thesis --- p.36 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Animals --- p.39 / Chapter 2.1.2 --- Cell lines --- p.39 / Chapter 2.1.3 --- Cell culture media and reagents --- p.40 / Chapter 2.1.4 --- Fatty acids --- p.44 / Chapter 2.1.5 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.6 --- Reagents and buffers for Western blotting --- p.51 / Chapter 2.1.7 --- Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit --- p.60 / Chapter 2.2. --- Methods --- p.62 / Chapter 2.2.1 --- Culture of tumor cell lines --- p.62 / Chapter 2.2.2 --- Isolation and culture of murine normal cells --- p.63 / Chapter 2.2.3 --- Determination of cell proliferation by CyQUANT® NF cell proliferation assay --- p.65 / Chapter 2.2.4 --- Determination of cell viability --- p.66 / Chapter 2.2.5 --- Cytotoxicity test of CFA on normal cells --- p.67 / Chapter 2.2.6 --- In vivo tumorigenicity assay --- p.68 / Chapter 2.2.7 --- Analysis of cell cycle profile --- p.69 / Chapter 2.2.8 --- Measurement of DNA fragmentation by Cell Death Detection ELISA{U+1D3E}{U+1D38}{U+1D41}{U+1D40} kit --- p.70 / Chapter 2.2.9 --- Analysis of Annexin V-GFP/PI dual staining profile --- p.71 / Chapter 2.2.10 --- Determination of mitochondrial membrane potential by JC-1 staining --- p.72 / Chapter 2.2.11 --- Determination of intracellular reactive oxygen species generation --- p.72 / Chapter 2.2.12 --- Determination of mitochondrial mass --- p.73 / Chapter 2.2.13 --- Protein expression study --- p.74 / Chapter 2.2.14 --- Statistical analysis --- p.78 / Chapter Chapter 3 --- Studies on the anti-proliferative effects of jacaric acid and cis-parinaric acid on murine macrophage-like leukemia cells / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Results --- p.82 / Chapter 3.2.1 --- Anti-proliferative effects of CFA isomers on murine macrophage-like leukemia PU5-1.8 cells in vitro --- p.82 / Chapter 3.2.2 --- Kinetic and reversibility studies of the anti-proliferative effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells --- p.92 / Chapter 3.2.3 --- Cytotoxic effects of jacaric acid and cis-parinaric acid on PU5-1.8 cells --- p.97 / Chapter 3.2.4 --- Cytotoxic effects of jacaric acid and cis-parinaric acid on murine normal cells in vitro --- p.99 / Chapter 3.2.5 --- Effect of jacaric acid on the in vivo tumorigenicity of PU5-1.8 cells --- p.102 / Chapter 3.3 --- Discussion --- p.104 / Chapter Chapter 4 --- Mechanistic studies on the anti-tumor effects of jacaric acid and cis-parinaric acid on murine macrophage-like leukemia cells / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Results --- p.117 / Chapter 4.2.1 --- Effects of jacaric acid and cis-parinaric acid on the cell cycle profile of murine macrophage-like leukemia PU5-1.8 cells --- p.117 / Chapter 4.2.2 --- Effects of jacaric acid and cis-parinaric acid on the expression of cell cycle regulatory proteins in murine macrophage-like leukemia PU5-1.8 cells --- p.121 / Chapter 4.2.3 --- Effects of jacaric acid and cis-parinaric acid on the generation of reactive oxygen species in murine macrophage-like leukemia PU5-1.8 cells --- p.124 / Chapter 4.2.4 --- Effects of antioxidants on the anti-proliferative effects of jacaric acid and cis-parinaric acid on the murine macrophage-like leukemia PU5-1.8 cells --- p.128 / Chapter 4.2.5 --- Effects of jacaric acid and cis-parinaric acid on the mitochondrial mass in murine macrophage-like leukemia PU5-1.8 cells --- p.131 / Chapter 4.2.6 --- Effects of jacaric acid and cis-parinaric acid on the induction of apoptosis in murine macrophage-like leukemia PU5-1.8 cells --- p.135 / Chapter 4.2.7 --- Effects of jacaric acid and cis-parinaric acid on the induction of phosphatidylserine externalization in murine macrophage-like leukemia PU5-1.8 cells --- p.139 / Chapter 4.2.8 --- Effects of jacaric acid and cis-parinaric acid on the mitochondrial membrane potential in murine macrophage-like leukemia PU5-1.8 cells --- p.144 / Chapter 4.2.9 --- Effects of jacaric acid and cis-parinaric acid on the expression of apoptosis-regulatory proteins in murine macrophage-like leukemia PU5-1.8 cells --- p.149 / Chapter 4.3 --- Discussion --- p.152 / Chapter Chapter 5 --- Conclusions and future perspectives / Chapter 5.1 --- Conclusions --- p.161 / Chapter 5.2 --- Future perspectives --- p.164 / References --- p.169

Acute Myeloid Leukemia (AML), the most common form of acute leukemia in adults, is only curable in approximately 30% of all cases. Despite prognostic risk stratification using sub-typing and cytogenetic analysis to direct therapy, the mortality and relapse rate remains high. AML patients with normal karyotypes are defined as intermediate risk and are the most challenging to treat. Somatic mutations may be the key in refining prognostic stratification and providing useful therapeutic targets. The FMS-like tyrosine kinase 3 (FLT3) and Nucleophosmin (NPM1) genes have common mutated forms that are associated with overall survival and response to therapy. We assessed mutations in the FLT3 and NPM1 genes and their levels of expression in twenty eight AML patients in the presence and absence of HIV and their response to induction therapy. Furthermore, we used a novel technique, High Resolution Melting (HRM) Analysis to detect FLT3 Internal Tandem Duplications (ITD) and NPM1 exon 12 mutations. Five of the patients in this study were HIV positive, three of whom did not survive post-induction therapy. Of the AML patients, 17.9% were positive for the NPM1 mutation and 21% had mutated FLT3. Interestingly, the presence of the FLT3 and NPM1 mutations were coupled with an increase in expression levels of FLT3 and NPM1 from presentation to post-induction respectively and the loss of these mutations were coupled with a decrease in levels of expression from presentation to post-induction. However, an increase/decrease from presentation to post-induction did not necessarily denote the presence/absence of a mutation. Therefore, while mutational status of genes may generally confer mRNA levels, our results showed that there existed no definitive trend between mRNA levels of NPM1 and FLT3 expression and mutational status. We found that the HRM method was definitive for the simpler NPM1 mutation however detection of the FLT3-ITD mutation was challenging. There isn’t a clear distinction between mutated and non-mutated FLT3 due to the formation of hetero-duplexes during analysis, making detection highly subjective and error-prone. Sequencing allowed confirmation of mutated FLT3 and non-mutated FLT3 which were not in all instances in concordance with HRM analysis. The prognostic value in terms of overall survival of NPM1 and FLT3 mutations in this study is indefinite. Furthermore, the analysis of the HIV positive AML patients revealed no clear correlation between NPM1 and FLT3 levels of mRNA expression and mutational status. Also, the small number of HIV positive AML patients did not allow for conclusions to be made regarding HIV status and survival when affected with AML. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Ng, Ka Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 118-131). / Abstracts in English and Chinese. / Thesis Abstract --- p.i / 論文摘要 --- p.vi / Acknowledgements --- p.x / Abbreviations --- p.xii / Thesis Content --- p.xv / List of Figures --- p.xix / List of Tables --- p.xxi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Haematopoiesis --- p.1 / Chapter 1.2 --- Leukemia --- p.2 / Chapter 1.3 --- Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) --- p.3 / Chapter 1.3.1 --- Epidemiology --- p.4 / Chapter 1.3.2 --- Causes and risk factors --- p.4 / Chapter 1.3.3 --- Clinical features --- p.6 / Chapter 1.3.4 --- Morphology --- p.6 / Chapter 1.4 --- Classification of BCP-ALL --- p.7 / Chapter 1.4.1 --- Immunophenotyping --- p.7 / Chapter 1.4.2 --- Cytogenetics and molecular genetics --- p.9 / Chapter 1.5 --- Prognostic factors --- p.13 / Chapter 1.6 --- Current treatments of BCP-ALL --- p.15 / Chapter Chapter 2 --- Literature Review --- p.18 / Chapter 2.1 --- Cytogenetics abnormalities in BCP-ALL --- p.18 / Chapter 2.1.1 --- Chromosomal translocation --- p.18 / Chapter 2.1.2 --- Aneuploidy --- p.21 / Chapter 2.2 --- Epigenetic aberrations --- p.21 / Chapter 2.2.1 --- DNA methylation --- p.22 / Chapter 2.2.2 --- Mechanism of DNA Methylation in Transcription Repression --- p.23 / Chapter 2.3 --- DNA Methylation in Normal Haematopoiesis --- p.25 / Chapter 2.4 --- DNA Methylation in Haematological Malignancies --- p.26 / Chapter 2.4.1 --- DNA methylation in ALL --- p.26 / Chapter 2.4.2 --- DNA methylation in BCP-ALL --- p.29 / Chapter 2.5 --- Angiogenesis in pathogenesis of acute leukemias --- p.30 / Chapter 2.6 --- Thrombospondin-1 (TSP-1) --- p.32 / Chapter 2.6.1 --- Structure of TSP-1 --- p.33 / Chapter 2.6.2 --- The role of TSP-1 in tumorigenesis --- p.34 / Chapter 2.6.3 --- TSP-1 mediates the activation of TGFβ --- p.36 / Chapter 2.6.4 --- TSP-1 mediates TGFβ-induced Apoptosis --- p.37 / Chapter 2.6.5 --- Association of TGFβ with normal haematopoiesis and haematological malignancies progression --- p.40 / Chapter 2.6.6 --- TSP-1 Induced Apoptosis via its Receptor CD36 --- p.42 / Chapter 2.6.7 --- THBS1 promoter hypermethylation and its association with tumorigenesis --- p.43 / Chapter 2.6.8 --- Effect of THBS1 aberrant methylation on TGFp --- p.45 / Chapter Chapter 3 --- Rationale of Study --- p.47 / Chapter Chapter 4 --- Materials and Methods --- p.52 / Chapter 4.1 --- Patient sample --- p.52 / Chapter 4.2 --- Cell lines --- p.52 / Chapter 4.3 --- Mononuclear cells isolation --- p.53 / Chapter 4.4 --- THBS1 promoter hypermethylation analysis --- p.54 / Chapter 4.4.1 --- DNA extraction from mononuclear cells and cell lines --- p.54 / Chapter 4.4.2 --- Bisulfite conversion --- p.55 / Chapter 4.4.3 --- Methylation specific PCR (MSP) --- p.55 / Chapter 4.5 --- Quantification of THBS1 mRNA expression --- p.57 / Chapter 4.5.1 --- RNA extraction --- p.57 / Chapter 4.5.2 --- Reverse transcription PCR --- p.58 / Chapter 4.5.3 --- Real-time RT-PCR --- p.58 / Chapter 4.6 --- Determination of plasma TSP-1 level --- p.59 / Chapter 4.7 --- TSP-1 treatment --- p.60 / Chapter 4.8 --- Flow cytometry analysis --- p.60 / Chapter 4.8.1 --- Annexin-V analysis --- p.60 / Chapter 4.8.2 --- Cell fixation --- p.61 / Chapter 4.8.3 --- Analysis of Caspase-3 activation --- p.62 / Chapter 4.8.4 --- "Analysis of TGFβ downstream pathway activation: Phosphorylation of Smad2/3, JNK and p38" --- p.62 / Chapter 4.9 --- Determination ofTGF-β expression --- p.63 / Chapter 4.10 --- Statistical analysis methods --- p.64 / Chapter Chapter 5 --- Results / Chapter 5.1 --- THBS1 methylation statuses in BCP-ALL patients and cell lines --- p.66 / Chapter 5.2 --- Correlation of THBS1 methylation statuses and clinico- pathological features in BCP-ALL patients --- p.68 / Chapter 5.3 --- Association of THBS1 methylation and THBS1 mRNA expression --- p.69 / Chapter 5.4 --- Effect of TSP-1 treatment on apoptosis level of BCP-ALL cells --- p.72 / Chapter 5.4.1 --- Annexin-V assay --- p.72 / Chapter 5.4.2 --- Caspase-3 activation assay --- p.75 / Chapter 5.5 --- THBS1 methylation and activation of secreted TGFβ --- p.78 / Chapter 5.6 --- Effect of TSP-1 treatment on activation of TGFβ --- p.80 / Chapter 5.7 --- The involvement ofTGFβ activation in TSP-1 induced apoptosis in BCP-ALL --- p.82 / Chapter 5.8 --- The association of TGFβ signaling pathway activities with THBS1 methylation --- p.86 / Chapter Chapter 6 --- Discussion --- p.91 / Chapter 6.1 --- THBS-1 promoter hypermethylation in BCP-ALL cell lines and patients: Correlation with expression and clinico-pathological profile --- p.93 / Chapter 6.1.1 --- THBS1 promoter hypermethylation status in childhood BCP-ALL --- p.93 / Chapter 6.1.2 --- THBS1 methylation as prognostic markers --- p.94 / Chapter 6.1.3 --- "Association of THBS1 methylation status, mRNA expression and TSP-1 protein expression in childhood BCP-ALL" --- p.96 / Chapter 6.2 --- Study of the correlation of TSP-1 induced apoptosis with the THBS1 promoter methylation status --- p.99 / Chapter 6.3 --- Elucidation of the molecular mechanisms of TSP-1 induced apoptosis: study of the involvement of TGFβ activation --- p.103 / Chapter 6.3.1 --- Latent TGFβ activation by TSP-1 in BCP-ALL and association with THBS1 methylation status --- p.103 / Chapter 6.3.2 --- TSP-1 induced cell death through activation ofTGFβ --- p.105 / Chapter 6.3.3 --- TSP-1 induced apoptotic signals via TGFβ signaling pathway --- p.107 / Chapter 6.4 --- Limitation of study --- p.113 / Chapter 6.5 --- Future studies --- p.114 / Chapter 6.5.1 --- Continuation study in TSP-1 induced TGFβ-mediated pathways --- p.114 / Chapter 6.5.2 --- Microarray analysis --- p.115 / Chapter 6.6 --- TSP-1 in treatment of childhood BCP-ALL --- p.115 / Chapter Chapter 7 --- Conclusion --- p.117 / Reference --- p.118

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

No direct stimulation of human peripheral blood mononuclear cells proliferation could be detected with incubation of various AB extracts including JAB80E70 in vitro. However, ex vivo assay performed using BALB/c mice orally fed with either water (control) or JAB80E70 for 28 days indicated for the first time that extract-treated groups significantly lowered the ex vivo mitogen-stimulated lymphocyte proliferation. / Our study started with the preparations of different AB extracts using various solvent systems with or without heating. Among all AB extracts tested, the extract JAB80E70 (extracted with 70% v/v ethanol at 80°C) showed the strongest selective suppression on the growth of human leukemia NB-4 and K-562 cells. The anti-leukemia effect of JAB80E70 was also confirmed in vivo using xenografted NB-4 bearing athymic nude mice model. / Phytochemical studies suggested that the selective anti-leukemia activity of JAB80E70-W-B-1 was retained in a relatively polar sub-fraction. One compound was isolated and identified as adenosine from JAB80E70-W-B-1. To the best of our knowledge, this is the first report of the presence of adenosine in AB, even though it exhibited no anti-leukemia activity in vitro. / The mushroom Agaricus blazei (AB) is traditionally used as a remedy against various cancers, infections and immune-related diseases. In the present study, the underlying mechanisms of the anti-tumor and immunomodulatory effects of AB extracts on human leukemia cells were systematically investigated. / With bioassay-guided fractionation, a sub-fraction (JAB80E70-W-B-1) with almost 5-fold improved selective cytotoxicity towards NB-4 cells was obtained. Both JAB80E70 and JAB80E70-W-B-1 could induce apoptosis characteristic DNA fragmentation and nucleosomes enrichment in NB-4 cells. An increase in the pro-apoptotic and a decrease in the anti-apoptotic Bcl-2 family proteins expression were observed in NB-4 cells treated with JAB80E70-W-B-1. JAB80E70-W-B-1 was also found to enhance activities of caspases 3, 8 and 9 in NB-4 cells. However, the activation of caspases 3 and 9 was not affected by the inhibition of caspase 8. Furthermore, JAB80E70-W-B-1 induced apoptosis in NB-4 cells was accompanied by a significant reduction in mitochondria membrane potential and telomerase activity. These results demonstrated that JAB80E70-W-B-1 induced apoptosis in NB-4 cells was dependent on caspase activity, and involved multiple molecular pathways. / Jiang, Jingjing. / Adviser: Lau Bik San Clara. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3454. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 224-250). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.