RUNX3是RUNX轉錄因子家族的其中一位成員。RUNX轉錄因子家族是負責調控細胞的增殖和分化。最近研究表明RUNX3可能在造血過程中扮演其中一個角色。可是，它在髓系細胞中的調節角色依然未明。此前，我們發現在核心結合因子急性骨髓性白血病中的融合蛋白RUNX1-ETO和CBFB-MYH11會抑制RUNX3基因表達，並且RUNX3表達水平對兒童急性骨髓性白血病的預後有顯著影響。本研究的目的是要調查RUNX3在成人急性骨髓性白血病的預後價值，並透過闡明RUNX3的轉錄調節去了解其在髓系細胞分化扮演的角色。 / 首先，我們透過實時定量聚合鏈反應去量化在174個成人急性骨髓性白血病的患者骨髓中的RUNX3表達，從而調查RUNX3表達與成人急性骨髓性白血病預後的關係。我們發現低RUNX3表達與較好預後的核型(P=0.045)，NPM1基因突變(P=0.014) 和較年青患者(P=0.084) 有關聯。在存活分析中，我們把有完整生存數據的非急性前骨髓性白血病病人分成高RUNX3表達和低RUNX3表達兩組。在成人急性骨髓性白血病中，高RUNX3表達和較差整體存活率(OS) (P=0.011)和無事件存活率(EFS) (P=0.003)有顯著的關聯，這和我們在兒童急性骨髓性白血病所觀察的一致。高RUNX3表達和較差存活率的關係在有野生型FLT3基因的病人中更為明顯(OS, P=0.004; EFS, P=0.001)。由於低RUNX3表達和較好預後核型有關聯，我們進一步只對擁有較差預後核型的病人作將存活分析，發現RUNX3表達仍是影響EFS的一個顯著因素(P=0.017)。在多元分析中，高RUNX3表達在所有病人(EFS, P=0.026, HR=2.433, 95%CI = 1.114-5.356)，野生v 型FLT3基因的病人(OS, P=0.016, HR=4.830, 95%CI = 1.335-17.481; EFS, P=0.007, HR=4.103, 95%CI = 1.480-11.372)和較差預後核型的病人(EFS, P=0.024,HR=2.339, 95%CI = 1.117-4.896) 中都是一個獨立的不利預後因素。 / 接著，我們研究RUNX3基因的表達調控。我們鑒定出一個最小啟動子區對於在髓系細胞的基因表達有關鍵作用。透過預測啟動子區和轉錄因子結合位點的分析，顯示這個活性區域含有PU.1，AP-1和Sp1轉錄因子結合位點。我們透過報告基因系統研究，染色質免疫沈澱技術及電泳遷移率改變分析去闡明PU.1，c-Jun及Sp1和相對的轉錄因子結合位點參與RUNX3基因的表達調控。我們進一步透過PU.1基因剔除去證實RUNX3是PU.1的直接下遊靶基因並發現PU.1與RUNX3表達在急性骨髓性白血病人中呈正相關性。 / 由於RUNX3基因表達受到PU.1, c-Jun及Sp1的控制，我們繼續研究RUNX3在髓系細胞分化的功用。我們透過實時定量聚合鏈反應及流式細胞儀檢測發現RUNX3過度表達誘導K562細胞株作單核細胞及粒細胞分化。RUNX3能激活髓系基因的啟動子。它在成熟髓系細胞的表達水平明顯比血幹細胞為高。根據以上結果，RUNX3也許在單核細胞及粒細胞分化中有一定功能。但是，有別於其他癌細胞，RUNNX3不能在髓系細胞誘導細胞凋亡和周期阻滯。 / 總括而言，RUNX3表達在成人急性骨髓性白血病中是一個獨立的預後因素。除此之外，本研究表明RUNX3受到PU.1，c-Jun及Sp1的表達調控並在單核細胞及粒細胞分化中有一定功能。 / RUNX3 is a member of Runt-related domain (RUNX) transcription factor family, which regulates cell proliferation and differentiation. Recent studies have suggested a role of RUNX3 in hematopoiesis. However, its regulatory function in myeloid cells remains unclear. Our group previously showed that RUNX3 expression was repressed by the fusion proteins RUNX1-ETO and CBFB-MYH11 in core-binding factor acute myeloid leukemia (CBF-AML) and had prognostic implication in childhood AML patients. The aim of this study is to investigate the prognostic value of RUNX3 in adult AML patients and its role in myeloid differentiation by elucidating its transcriptional control. / To investigate the relationship between RUNX3 expression and prognosis of adult AML, RUNX3 expression in the diagnostic bone marrow samples from 174 adult AML patients were quantified by real time quantitative PCR (RQ-PCR). Low RUNX3 expression was found to be associated with favorable cytogenetic group (P=0.045), NPM1 mutations (P=0.014) and younger age (P=0.084). For the survival analysis, 110 non-acute promyelocytic leukemia (non-APL) patients with complete survival data were dichotomized into high and low expression groups. Concordant with our previous observation in childhood AML, a significant association between high RUNX3 expression and poorer overall survival (OS) (P=0.011) and event-free survival (EFS) (P=0.003) was observed. The association between high RUNX3 expression and poorer survival was further strengthened in patients with wild-type FLT3 (P=0.004 and 0.001 for OS and EFS respectively). Since low RUNX3 expression was associated with favorable cytogenetics, the analysis was next restricted to patients with non-favorable cytogenetics and RUNX3 expression remained as a significant factor for EFS (P=0.017). In multivariate analysis, high RUNX3 expression was an independent adverse prognostic factor in the whole cohort (EFS, P=0.026, HR=2.433, 95%CI = 1.114-5.356), patients with wild-type FLT3 (OS, P=0.016, HR=4.830, 95%CI = 1.335-17.481; EFS, P=0.007, HR=4.103, 95%CI = 1.480-11.372) and patients with non-favorable genetics (EFS, P=0.024,HR=2.339, 95%CI = 1.117-4.896). / Next, the transcriptional regulation of RUNX3 in myeloid cells was investigated. A minimal promoter region was identified to be critical for myeloid-specific promoter activity. Sequence analysis of the fragment revealed potential transcription factor binding sites for PU.1, AP-1 and Sp1.The involvement of these putative binding sites and corresponding transcription factors in transcriptional regulation of RUNX3 was demonstrated by promoter reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA).Furthermore, PU.1 knockdown in U937 cells confirmed RUNX3 was a direct downstream target of PU.1 and a positive correlation between PU.1 and RUNX3 expression was observed in AML patient samples. / As RUNX3 was shown to be transcriptionally regulated by PU.1, c-Jun and Sp1, a role of RUNX3 in myeloid differentiation was postulated. Overexpression of RUNX3 induced both monocytic and granulocytic markers in K562 myeloid cells as detected by flow cytometry and RQ-PCR. RUNX3 was also found to activate myeloid-specific gene promoters and its expression was significantly higher in mature myeloid cells than in hematopoietic stem cells. This suggested a role of RUNX3 in both monocytic and granulocytic differentiation. However, unlike in other solid tumors, RUNX3 did not induce apoptosis and cell cycle arrest in myeloid cells. / In conclusion, RUNX3 expression was an independent prognostic factor in adult AML. Furthermore, our findings showed that RUNX3 was transcriptionally regulated by the master myeloid regulator PU.1 along with c-Jun and Sp1 and implicated a role in monocytic and granulocytic differentiation. / 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. / Kwan, Tsz Ki. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 171-202). / Abstracts also in Chinese.

La thrombopénie familiale avec prédisposition à la leucémie aiguë myéloïde (FPD/AML) est une pathologie rare caractérisée par une thrombocytopénie. La FPD/AML est causée par des mutations germinales dans le gène codant le facteur de transcription RUNX1. Ces mutations sont de type dominant négatif (DN), associées à un risque plus élevé de développer une leucémie, ou de type haploinsuffisance (HI) induisant une thrombocytopénie seule. Nous avons démontré une diminution presque complète de l’expression du répresseur transcriptionnel ZBTB1 dans les progéniteurs hématopoïétiques des patients porteurs de mutations DN. Le gène ZBTB1 pourrait être une cible directe de RUNX1, et pourrait contribuer à la dérégulation de la lymphopoïèse T conduisant à une prédisposition à la LAL-T.Nous avons identifié dans les cellules lymphocytaires murines le site de fixation de RUNX1 sur un enhanceur localisé à 270 kb en amont du promoteur de Zbtb1, et ce aux stades doubles négatifs pour les marqueurs CD4/CD8. Dans les stades plus matures (CD4+CD48+), cette fixation n’est pas observée. En utilisant des lignées lymphocytaires humaines représentant les différents stades doubles négatifs CD4/CD8 de la différenciation lymphocytaire, nous ne sommes pas arrivés à démontrer cette liaison suggérant qu’elle a lieu à un stade très précis et transitoire difficilement identifiable. Les souris KO Runx1 et KO Zbtb1 montrent un blocage de la lymphopoïèse T dès les stades les plus précoces de la maturation thymique. Nous voulions démontrer que la surexpression de Zbtb1 dans un contexte KO Runx1 aboutirait à un sauvetage du phénotype lymphocytaire. Pour cela, nous avons utilisé un modèle in vitro (culture des progéniteurs hématopoïétiques sur des lobes thymiques) et in vivo reposant sur la greffe de souris irradiées par des progéniteurs hématopoïétiques de souris KO Runx1 surexprimant Zbtb1. Le KO Runx1 incomplet et une quasi-absence de la prise de greffe dans les conditions de KO Runx1 ne nous ont pas permis de valider notre hypothèse. Cependant nous avons pu observer que ZBTB1 régule négativement le compartiment des cellules souches et la prise de greffe.Nous nous sommes aussi intéressés au phénotype mégacaryocytaire des souris KO Zbtb1. De manière intéressante, ces souris montrent in vivo un défaut du cycle cellulaire des mégacaryocytes, tandis que in vitro une diminution drastique de la différenciation mégacaryocytaire est observée suggérant ainsi une compensation du microenvironnement in vivo. De plus, nous avons montré une régulation négative directe de ZBTB1 par RUNX1 dans les mégacaryocytes humains. Dans la deuxième partie de ma thèse nous nous sommes intéressés au mécanisme d’induction de la leucémie chez un patient FPD/AML porteur de mutation de type DN (RUNX1R174Q), nous avons identifié une mutation additionnelle à une fréquence de 1% dans le gène TET2 ayant contribué à l’amplification du clone pré-leucémique. Actuellement nous étudions la coopération entre la mutation de RUNX1R174Q et le shTET2 in vivo en greffant des souris NSG avec des cellules progénitrices humaines CD34+ portant la mutation RUNX1 et le shTET2 qui mime la mutation perte de fonction de TET2P1962T observée chez le patient. Des résultats prometteurs montrent une prise de greffe primaire et secondaire plus importante dans les conditions RUNX1R174Q/shTET2 et shTET2 seul. Les expériences in vitro réalisées en parallèle montrent que la mutation de RUNX1R174Q induit des dommages à l’ADN alors que la diminution de l’expression de TET2 par shARN induit une prolifération augmentée des progéniteurs hématopoïétiques. L’addition des deux mutations pourrait ainsi conduire à l’acquisition de mutations additionnelles et à une transformation leucémique. / Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is a rare condition characterized by thrombocytopenia. FPD/AML is caused by germline mutations in the gene coding for the transcription factor RUNX1. These mutations are devided on dominant-negative (DN) mutations associated with a higher risk of developing leukaemia or haploinsufficiency (HI) mutations inducing thrombocytopenia alone.We have demonstrated an almost complete decrease in the expression of the transcriptional repressor ZBTB1 in hematopoietic progenitors of patients with DN-type mutations. ZBTB1 could be a direct target of RUNX1, and could contribute to deregulation of T lymphopoiesis, leading to a predisposition to T-ALL.In murine immature T lymphocytes (CD4-CD8- stages), we demonstrated a fixation of RUNX1 on an enhancer at 270 kb upstream of Zbtb1 promoter. This fixation is no longer observed in the more mature stages (CD4+CD8+). Using human lymphocyte cell lines representing the different CD4-CD8- differentiation stages, we have not been able to demonstrate this binding suggesting that it takes place at a very precise and transitory stage that is difficult to identify.The KO Runx1 and KO Zbtb1 mice show a blockade of T lymphopoiesis in the earliest stages of thymic maturation. We wanted to demonstrate that the overexpression of Zbtb1 in a KO Runx1 context would result at least in a partial rescue of the lymphocyte phenotype. For this we used an in vitro model (culture of hematopoietic progenitors on thymic lobes) and in vivo based on the grafting of irradiated mice with hematopoietic progenitors of KO Runx1 overexpressing Zbtb1. The incomplete KO Runx1 and the almost complete absence of engraftment in the KO Runx1 conditions did not allow us to validate our hypothesis. However, we observed that ZBTB1 negatively regulates the stem cell compartment and the engraftment capacity.We also studied the megakaryocytic phenotype of KO Zbtb1 mice. Interestingly, these mice show, in vivo, a megakaryocyte cell cycle defect; while in vitro a drastic decrease in megakaryocytic differentiation is observed suggesting an in vivo micro-environmental compensation. We also showed a direct negative regulation of ZBTB1 by RUNX1 in human megakaryoycytes.In the second part of my thesis, we investigated the mechanism of induction of leukemia in an FPD/AML patient with a DN-type mutation (RUNX1R174Q). We demonstrated an additional mutation at a frequency of 1% in TET2 gene, which contribute to the amplification of a preleucemic clone.Currently we are studying the cooperation between the RUNX1R174Q mutation and the shTET2 in vivo by grafting NSG mice with human CD34+ progenitor cells carrying RUNX1R174Q mutation and an shTET2, which mimics the loss of function of TET2 observed in the patient. Promising results show greater primary and secondary graft under RUNX1R174Q /shTET2 and shTET2 conditions. The in vitro experiments carried out, show that the mutation of RUNX1R174Q induces DNA damages, whereas the decrease in the expression of TET2 by shRNA induces an increased proliferation of hematopoietic progenitors. The addition of the two mutations could thus lead to the acquisition of additional mutations and to a leukemic transformation.

A mathematical model of the interaction between chronic lymphocytic leukemia (CLL) and CD4+ (helper) T cells was developed to study the role of T cells in cancer survival. In particular, a system of four nonlinear advection diffusion reaction partial differential equations were used to simulate spatial effects such as chemical diffusion and chemotaxis on CLL survival and proliferation.

The purpose of the study was to assess psycho-social differences between two groups. A review of the literature suggested psychological and social factors may affect the onset and progression of malignant disease. Comparisons were made between a group of 23 experimental families with a leukemic child, and a group of control families with a normal child matched for child's sex, age, and number of siblings. Data. was gathered on three quantifiable measures; Coddington's Social Readjustment Rating Questionnaire; a specially constructed Child's Questionnaire, and The Minnesota Multiphasic Personality Inventory. The leukemic children and their matched normal controls had approximately equal numbers, and similar types, of social events that had occurred within their present life span. This suggests that the occurrence of a specific stressful life event is not, in itself, a major precipitating factor in onset of leukemia. The leukemic children and their normal controls were likewise similar in their responses concerning self-reported aggressive behavior and attitudes toward expression of aggressive feelings. This implies that, other than the presence of disease, the leukemic children respond to specific stimuli in a manner typical of normal children. The parents of the leukemic children however, were differentiated from the parents of normal children, through the statistical method of stepwise discriminant analysis of MMPI responses. A combination of five variables, for each pair of parents, father's Sc 0 (Si) score, mother's Sc I (Hs) score, and father's Sc 9 (Ma), F scale, and Sc 3 (Hy) scores, had a level of significance. This finding presents evidence that parents of leukemic children differ from parents of normal children on personality characteristics assessed by the MMPI. Some possible interpretations of these results, and suggestions for treatment and additional research, were offered.

The ability of low, clinically relevant levels of the chemotherapeutic drugs epirubicin and vinblastine to induce drug resistance was examined in the K562. U937, KG-la and HEL human leukaemia cell lines. Treatment with epirubicin and vinblastine induced the MDR phenotype and P-glycoprotein expression in K562 and U937 cells. However this treatment had no effect on drug resistance in the P-glycoprotein expressing KG-la and HEL cells. In the U937 cells, drug resistant cells were not only MDR but were also resistant to other drugs including cisplatinum and chlorambucil which are not normally associated with MDR. The drug resistant U937 sublines were also sensitised to doxorubicin, cisplatinum and chlorambucil by buthionine sulphoximine (BSO), suggesting that glutathione-related mechanisms also contributed to resistance in these sublines. The U937 sublines also had an increased DNA content and an increased ability to recover from DNA damage, as determined by cell cycle analysis, indicating that the broad cross-resistance exhibited by these cells was due to the co-existence of multiple resistance mechanisms. Drug treatment induced changes in expression of differentiation associated antigens in all four cell lines. Treatment with inducers of differentiation (TPA, sodium butyrate, granulocyte-macrophage colony-stimulating factor; GM-CSF). Treatment of K562 and K562/E15B cells with TPA induced megakaryocytic differentiation, with increases in drug resistance, and increased P-glycoprotein expression in the K562/E15B subline. TPA induced monocytic differentiation in the U937 cells but not the U937/EIS subline, with increased P-glycoprotein expression and function in the U937/E15 cells but not the U937 cells. Staurosporine, an inhibitor of PKC, inhibited differentiation in these cell lines, but did not inhibit increases in P-glycoprotein expression, suggesting drug resistance was not mediated by PKC. Sodium butyrate induced erythroid differentiation, and increased P-glycoprotein expression in the K562/E15B cells. However at a higher concentration (15 mM) this was not accompanied by increased drug resistance. Granulocyte monocyte colony stimulating factor (GM-CSF) did not induce differentiation in the K562 cells or K562/E15B subline, although the K562/E15B cells became more drug resistant after treatment with GM-CSF. Treatment with GM-CSF induced differentiation in the U937/E15 subline but did not change drug resistance in either the U937 cells or the U937/EI5 subline. Therefore the P-glycoprotein expressing K562/E15B and U937/E15 sublines were more responsive to inducers of differentiation than the parental cell lines. Induction of differentiation therefore induced increases in P-glycoprotein expression and drug resistance, suggesting that expression of P-glycoprotein or a multidrug resistance phenotype was associated with differentiation.

B cell chronic lymphocytic leukemia (B-CLL) is a hematologic neoplasm characterised by the proliferation and accumulation of sIgM+/D+ B cells that fail to progress to the final stages of B cell development. The malignant cells in B-CLL also express the pan-T cell antigen CD5, suggesting that CLL is a malignancy of the CD5+ subset of B cells. Additional characteristics of the malignant clone include a low proliferative index, enhanced in vivo survival and constitutive expression of the anti-apoptosis oncoprotein bcl-2. The behaviour of leukemic CD5 B cells in vitro contrasts their arrested in vivo state. That is, despite the majority of cells being arrested in the G0 phase of the cell cycle, the leukemic B cells are not irreversibly frozen as they can be induced to differentiate to Ig-secreting cells under appropriate in vitro conditions. Furthermore, leukemic CD5 B cells rapidly undergo death by apoptosis following in vitro culture. This thesis describes the requirements for in vitro activation of leukemic CD5+ B cells, the characterisation of the events involved in apoptosis of these cells as well as the identification of various growth factors capable of modulating these events. Stimulation of unfractionated peripheral blood lymphocytes (PBLs) from three patients with B-CLL with the phorbol ester PMA and the mitogens PHA and PWM resulted in significant increases in cell proliferation, RNA synthesis and 1gM secretion when compared to unstimulated cell populations. PMA was the most potent inducer of 1gM secretion and this occurred irrespective of the presence of residual T cells. PMA-induced proliferation and RNA synthesis were also independent of T cells. However, in the presence of T cells, these parameters of cellular activation were enhanced during in vitro culture. Thus, the inductive ability of PMA on leukemic CD5 B cells was independent of T cells. In contrast, activation and differentiation of the leukemic CD5 B cells into 1gM-secreting cells following culture with mitogens did not occur in the absence of T cells. Interestingly, co-stimulation of leukemic CD5+ B cells with PMA and anti-Ig induced cellular responses that exceeded those induced by either activator alone. Thus, leukemic CD5+ B cells from patients with B-CLL can be activated in vitro and differentiate in response to stimulation via both T cell-dependent and T cell-independent mechanisms. Apoptotic cell death was characterised in purified leukemic CD5 B cells obtained from six B-CLL patients. All leukemic CD5 B cell populations entered an apoptotic pathway in vitro as evidenced by a reduction in cell size, loss of cell viability and fragmentation of DNA into multimers of -180 base pairs. Following 24 hours of in vitro culture 24.0±16% of DNA was fragmented. After 8 days, the majority of DNA was fragmented, and fewer than 10% of cultured cells were viable. Examination of bcl-2 expression in the malignant B cells by flow cytometry revealed a unimodal pattern of expression in greater than 85% of cells from each B-CLL patient prior to culture. During in vitro culture, bcl-2 expression became bimodal such that the B cells displayed a bcl-2hjgh and bcl-2iow phenotype. The level of expression by the bCl2hjgh cells was similar to that observed prior to in vitro culture, indicating that bcl-2 is down-regulated in apoptosing cells. Interestingly, despite this downregulation, the overall number of cells positive for bcl-2 remained constant. This suggests that the enhanced survival of leukemic CD5+ B cells in vivo is mediated by the sustained expression of bcl-2 and that additional mechanisms exist capable of overriding the protective effect of bcl-2 when bcl-2 is present at reduced levels. Leukemic B cell apoptosis has previously been reported to be delayed or prevented by IL-4, IFN-y and IFN-a. These results were confirmed in this study where it was found that culture of leukemic CD5 B cells with IL-4 or IFN-y enhanced cell viability and delayed apoptosis in 6/6 and 5/6 populations of leukemic B cells, respectively. This function was also found to be shared by IL-2, IL-6, IL-13 and TNF-a as these cytokines enhanced cell viability and delayed apoptosis in some of the cell populations examined at a level similar to that observed for IL-4 and IFN-y. These cytokines may mediate their effect via the expression of bcl2 as culture in the presence of IL-2, IL-4, IL-6, IL-13, IFN-y or TNF-a resulted in a higher percentage of cells displaying the bcl-2high phenotype, compared to unstimulated cells. Taken together, these results suggest that autocrine and/or paracrine growth loops may play a role in the pathogenesis of B-CLL and that cytokines that prevent apoptosis in vitro may be targets for treatment of this B cell malignancy.

<p>Healthy cells require input from growth factor signaling pathways to maintain cell metabolism and survival. Growth factor deprivation induces a loss of glucose metabolism that contributes to cell death in this context, and we have previously shown that maintenance of glycolysis after growth factor deprivation suppresses the activation of p53 and the induction of the pro-apoptotic protein Puma to prevent cell death. However, it has remained unclear how cell metabolism regulates p53 activation and whether this increased glycolysis promotes cell survival in the face of additional types of cell stress. To examine these questions, we have utilized a system in which stable overexpression of the glucose transporter Glut1 and hexokinase 1 in hematopoietic cells drives growth-factor independent glycolysis. This system allows us to examine the effects of glucose metabolism in the absence of other signaling events activated downstream of growth factor receptors. Here, we demonstrate that elevated glucose metabolism, characteristic of cancer cells, can suppress PKC&#948;-dependent p53 activation to maintain cell survival after growth factor withdrawal. In contrast, DNA damage-induced p53 activation was PKC&#948;-independent and was not metabolically sensitive. Both stresses required p53 serine 18 phosphorylation for maximal activity but led to unique patterns of p53 target gene expression, demonstrating distinct activation and response pathways for p53 that were differentially regulated by metabolism.</p><p>Unlike the growth factor-dependence of normal cells, cancer cells can maintain growth factor-independent glycolysis and survival and often demonstrate dramatically increased rates of glucose uptake and glycolysis, in part to meet the metabolic demands associated with cell proliferation. Given the ability of elevated glucose metabolism to suppress p53 activity in the context of metabolic stress, we examined the effect of increased glucose uptake on leukemogenesis using a mAkt-driven model of leukemia and adoptive transfer experiments. We show here that elevated glucose uptake promoted leukemogenesis in vivo, perhaps through suppression of p53 transcriptional activity. During the process of leukemogenesis, cancerous cells can acquire growth factor independent control over metabolism and survival through expression of oncogenic kinases, such as BCR-Abl. While targeted kinase inhibition can promote cancer cell death, therapeutic resistance develops frequently and further mechanistic understanding regarding these therapies is needed. Kinase inhibition targets the necessary survival signals within cancerous cells and may activate similar cell death pathways to those initiated by growth factor deprivation. As we have demonstrated that loss of metabolism promotes cell death after growth factor withdrawal, we investigated whether cell metabolism played a role in the induction of apoptosis after treatment of BCR-Abl-expressing cells with the tyrosine kinase inhibitor imatinib. Consistent with oncogenic kinases acting to replace growth factors, treatment of BCR-Abl-expressing cells with imatinib led to reduced metabolism and p53- and Puma-dependent cell death. Accordingly, maintenance of glucose uptake inhibited p53 activation and promoted imatinib resistance, while inhibition of glycolysis enhanced imatinib sensitivity in BCR-Abl-expressing cells with wild type p53 but had little effect on p53 null cells. Together, these data demonstrate that distinct pathways regulate p53 after DNA damage and metabolic stress and that inhibition of glucose metabolism may enhance the efficacy of and overcome resistance to targeted molecular cancer therapies.</p> / Dissertation

No description available.

B-cell chronic lymphocytic leukemia is the most common leukemia in the adult population of Western developed countries. In 2005, an estimated 9,730 adults in the United States will be diagnosed with B-CLL and an estimated 4,600 deaths will occur. B-CLL is a common heterogeneous malignant disease with variable outcome. B-CLL is divided into two groups based on whether somatic hypermutation is observed in the variable region of the immunoglobulin heavy-chain locus (IgVH). The two distinct groups are named mutated and unmutated. The B-CLL mutated group has a more favorable prognosis than the unmutated group. Gene expression profiling has been used successfully to decipher the biological and clinical diversity of many leukemias and lymphomas. Recently, other small RNAs (microRNAs) have been shown to be important in hematopoiesis. MicroRNAs are small 20-28 nucleotide RNAs that are believed to control many important cellular and developmental processes by posttranscriptional gene silencing, translational repression, and modulating epigenetic events. We are interested in whether microRNA expression correlates with the mutational status of IgVH. This study is significant in the following ways: (1) microRNAs may become surrogate markers for the mutational status of IgVH of B-CLL, which implies a more rapid diagnostic means as compared to the current practice, and (2) microRNAs, in the particular context of B-CLL, may play some significant roles in a gene regulatory network that is further responsible for chromosomal abnormalities found in B-CLL. This thesis presents a study comparing microRNA expression in mutated and unmutated B-CLL groups. Instead of using a genome-wide expression profiling strategy, we selected a specific set of microRNAs based on their chromosome locations and mRNA targets. Specifically, we chose the following eight microRNAs (with their chromosomal abnormalities): mir16-1 (deletion 13), let-7i (trisomy 12), mir196-2 (trisomy 12), mir26a-2 (trisomy 12), mir-34b (deletion 11), mir-125b (deletion 11), mir-181C (trisomy 19), mir-125a (trisomy 19). We used solution hybridization assays to monitor the expression of microRNAs. We successfully characterized the microRNA expression in twelve B-CLL patient samples (eight mutated and four unmutated). Among the eight microRNAs examined, three (mir196-2, mir-125a, mir-125b) are not expressed in the two B-CLL groups, four (mir16-1, mir26a-2, let-7i, mir-34b) have significant differences in expressions over the two groups, and one (mir-181c) has no significant difference in expressions over the two groups.

Feline leukemia viruses (FeLVs) are pathogenic retroviruses of the domestic cat. FeLV transmission and emergence of pathogenic variants show striking similarity to HIV pathogenesis. The emergence of pathogenic subgroup-C FeLV from the transmitted subgroup-A FeLV coincides with a switch in host receptor used for infection as a result of mutations in the viral envelope protein (Env). I have characterized a novel FeLV Env that may represent an evolutionary intermediate between FeLV-A and FeLV-C. I have also reported evidence suggesting that FeLVs may use co-factors/co-receptors for infection. I have found that FeLVs inefficiently infect murine NIH3T3 cells overexpressing FeLV receptors (NIH3T3/Receptor). I have provided evidence that the low infection is caused by a block at a post-binding but pre-entry stage of FeLV infection. Furthermore, fusion of NIH3T3/Receptor cells with highly susceptible cells rescues inhibition to infection suggesting that FeLVs, like HIV, may also use co-receptors for infection.