Thesis advisor: Thomas N. Seyfried / Mitochondria are the key regulators of the bioenergetic state of the cell. Damage to mitochondrial protein, DNA, or membrane lipids can result as the cause or affect of disease pathology. Regardless, this damage can impair mitochondrial function resulting in a decreased ability to produce ATP to support cellular viability. This thesis research examined the mitochondrial lipidome by shotgun lipidomics in different populations of C57BL/6J (B6) brain mitochondria (non-synaptic and synaptic) and correlated lipid changes to differences in electron transport chain (ETC) activities. Furthermore, a comparison was made for non-synaptic mitochondria between the B6 and the VM mouse strain. The VM strain has a 1.5% incidence of spontaneous brain tumors, which is 210 fold greater than the B6 strain. I determined that differences in the brain mitochondrial lipidome existed in the VM strain compared to the B6 strain, likely corresponding to an increased rate of spontaneous brain tumor formation. Analysis of the mitochondrial genome in the CT-2A, EPEN, VM-NM1, and VM-M3 brain tumors compared to their syngeneic controls mouse strains, C57BL/6J (B6) and VM mice, was examined to determine if mutations existed in experimental brain cancer models. No pathogenic mtDNA mutations were discovered that would likely cause a decrease in the mitochondrial functionality. A novel hypothesis was devised to examine the tumor mitochondrial lipidome to determine if quantitative or molecular species differences existed that could potentially alter the functionality of the ETC. Brain tumor mitochondria were examined from tumors grown in vivo as well as in vitro. Numerous lipid differences were found in the mitochondria of brain tumors, of which the most interesting involved the unique molecular speciation of cardiolipin. ETC activities were significantly decreased in the primary ETC complexes which contribute protons to the gradient as well as the linked complexes of brain tumor mitochondria compared to controls. Taken together, it is likely that differences in the mitochondrial lipidome of brain tumors results in severe impairment of the mitochondria’s ability to produce ATP through the ETC. This research has provided a new understanding of the role of mitochondrial lipids in brain as well as brain cancer and offers an alternative explanation for metabolic dysfunction in cancer. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

O Adenoma adrenocortical é frequente em adultos, já o carcinoma é raro e agressivo. Mesmo com critérios padronizados, ainda há dificuldade para diferenciar esses tumores, sendo necessário o estudo de marcadores eficientes na detecção e diferenciação. Por serem raros e com diversas manifestações clínicas, culturas in vitro pode ser uma ferramenta para o estudo de processos que envolvem a doença. Foi realizada a caracterização molecular e funcional de culturas de células de tumores de pacientes. Resultados de PCR Array não mostraram um padrão que diferenciasse as culturas em função dos diagnósticos. Desta análise, 7 oncogenes apresentaram maior expressão e 9 supressores de tumor apresentaram baixa expressão nas culturas. WWOX, FHIT e TP73 foram validados por qPCR e a sugestiva interação entre esses fatores nos tumores adrenocorticais merecem futuras investigações. O potencial funcional das culturas T83-ACC, T36-REC e T7-ACA(P) foram evidenciados, e mostraram que podem ser bons modelos para estudo da ação de hormônios e seus mecanismos. / The adrenocortical adenoma is common in adults, since carcinoma is rare and aggressive. Even with standardized scores, it is still difficult to differentiate these tumors, the study of efficient markers in the detection and differentiation is necessary. Because they are rare and diverse clinical manifestations in vitro cultures can be a tool for the study of disease processes that involve. Molecular and functional characterization of cultured tumor cells of patients was conducted. PCR Array results did not show a pattern that differentiates cultures on the basis of diagnoses. This analysis showed higher expression 7 oncogenes and tumor suppressors 9 showed low expression in cultures. WWOX, FHIT and TP73 were validated by qPCR and suggestive interaction between these factors in adrenocortical tumors deserve further investigation. The functional potential of T83-ACC, T36-REC and T7-ACA(P) cell cultures were found, and shown confirm that they can be good models for studying the action of hormones and their mechanisms.

University of Macau / Institute of Chinese Medical Sciences

Tesis para optar al título de Doctor en Farmacología / Helicobacter pylori (H. pylori) es un bacilo Gram negativo que infecta en algún momento de la vida a la mitad de la población mundial. Se ha demostrado que la interacción de H. pylori con las células del epitelio gástrico provocan cambios en la expresión de genes. Sin embargo, sólo se han realizado estudios en poblaciones adultas que presentan una infección persistente y sintomática por H. pylori o un cáncer gástrico definido. Estudios realizados en nuestro laboratorio permitieron la identificación de genes diferencialmente expresados en niños infectados con H. pylori comparado con niños no infectados, involucrados en la regulación del ciclo celular, apoptosis, migración celular o la activación del sistema inmune, destacando dentro de ellos el gen SLC5A8. Este es un gen caracterizado como un supresor de cáncer, debido a esta cualidad nuestro trabajo se focalizó en la caracterización de este gen. SLC5A8 es expresado en la membrana apical de las células del epitelio gástrico y es un transportador de ácidos grasos de cadena corta como butirato, permitiendo el ingreso de este sustrato dentro de la célula y favoreciendo su rol como supresor tumoral. La hipótesis planteada en esta tesis es la siguiente “La infección persistente con H. pylori se relaciona con una reducción de expresión de SLC5A8 asociada a hipermetilación en la región promotora de SLC5A8”. En primer lugar, se demostró mediante ensayos de qPCR en muestras de sangre y tejido epitelial gástrico, que la infección por H. pylori se asoció a una expresión disminuida de SLC5A8 en niños infectados cuando se comparó con niños no infectados. (2) Para identificar una posible hipermetilación de la región promotora de SLC5A8, que diera cuenta de la reducción de expresión, en tejido epitelial gástrico se realizaron experimentos de secuenciación con bisulfito. Los resultados obtenidos mostraron una tendencia a la hipermetilación del gen en niños infectados, pero estos resultados no son aún concluyentes. (3) Por otro lado, se comprobó una menor expresión del mRNA SLC5A8 en líneas celulares gástricas infectadas con H. pylori comparado con células no infectadas. (4) Finalmente se evaluó el efecto de la infección de H. pylori en efectores de SLC5A8 como: p53, p21, survivina, y COX2, comprobándose un aumento de COX2, pero una disminución de p21. En conclusión, se demostró que la infección por H. pylori se asocia con una disminución de expresión del gen SLC5A8, tanto in vivo en niños sintomáticos y asintomáticos, como in vitro en líneas celulares. El mecanismo epigenético podría involucrar la hipermetilación de la región promotora de este gen, aunque más experimentos son necesarios para comprobarlo con mayor certeza / Helicobacter pylori (H. pylori) is a gram-negative bacillus that infects half of the population worldwide at some point in life. Several studies have shown that the interaction with H. pylori causes many changes in gene expression in gastric epithelial cells. However, to date all these studies have been performed in adult populations with persistent, symptomatic H. pylori infection or gastric cancer. Research in our laboratory lead to the identification of differential expression of genes in children infected with H. pylori, compared to non-infected children, involved in cell cycle regulation, apoptosis, cell migration and/or activation of the immune system. Of the genes identified, we selected SLC5A8 gene as our focus of attention for this thesis project because of its predicted role as a tumor suppressor and potential connection with cancer genesis upon expression reduction. This transporter is present in the apical membrane of gastric epithelial cells, where it facilitates uptake of short-chain fatty acids, such as butyrate, modulating histone deacetylase function, the potential mechanism for tumor suppression. The hypothesis of this work is “The SLC5A8 expression is diminished during H. pylori infection in children due to hyper-methylation in the promoter region of SLC5A8”. Our main results are (1) H. pylori infection in children is associated with a decrease in SLC5A8 expression, as assessed by qPCR analysis of blood and tissue samples, compared to uninfected children. (2) We observed a trend in the association between reduced expression and hyper-methylation of the promoter region in gastric epithelial tissue. However, more experiments are required to confirm these results. (3) We verified in vitro, in gastric cell lines that SLC5A8 expression is decreased upon infection with H. pylori. (4) Finally, we evaluated in these cells the effect of H. pylori infection on the expression of SLC5A8 effector genes such: p53, p21, COX2, and surviving, showing increased expression for COX2, but an decreased expression in p21. In conclusion, we demonstrated an association between H. pylori infection and decrease expression of SLC5A8 gene, both in vivo, among symptomatic and asymptomatic children, and in vitro, in gastric cell lines. The epigenetic mechanism involved can be a hyper-methylation in the gene promoter of SLC5A8 although more experiments are needed to demonstrate this for sure / Conicyt; Fondecyt; Senescyt

Leung Siu Wai. / Bibliography: leaves 135-150 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

by Chun-kwok Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 181-196. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.iii / ABBREVIATIONS --- p.iv / CHAPTER / Chapter 1. --- INTRODUCTION : An overview of Tumor Necrosis Factor ( TNF) / Chapter 1. --- The discovery of tumor necrosis factor (TNF) --- p.1 / Chapter 2. --- Production of tumor necrosis factor --- p.3 / Chapter 3. --- Physiochemical properties of TNF --- p.5 / Chapter 4. --- Biological activities of TNF on various cells in the mammal --- p.8 / Chapter 5. --- Mechanisms of anti-tumor action of TNF --- p.12 / Chapter 6. --- Clinical studies of Hr-TNF --- p.19 / Chapter 2. --- AIM OF INVESTIGATION --- p.23 / Chapter 3. --- MATERIALS AND METHODS / Chapter A. --- MATERIALS --- p.26 / Chapter B. --- METHODS / Chapter 1. --- Preparation of Reagents --- p.30 / Chapter 2. --- Cell Culture --- p.31 / Chapter 3. --- Lymphocytes proliferation --- p.32 / Chapter 4. --- In vitro production of tumor necrosis factor (TNF) by peritoneal macrophages of ICR mice --- p.33 / Chapter 5. --- Production of TNF in animals --- p.34 / Chapter 6. --- Determination of TNF titre --- p.35 / Chapter 7. --- Determination of TNF containing serum titre on EAT in vitro --- p.35 / Chapter 8. --- Mortality determination of mice --- p.36 / Chapter 9. --- "3H-Thymidine, 3H-uridine, 14C-leucine incorporation" --- p.36 / Chapter 10. --- Glucose uptake determination --- p.37 / Chapter 11. --- Whole body hyperthermic treatment of EAT bearing mice --- p.37 / Chapter 12. --- Lipolysis assay --- p.38 / Chapter 13. --- Statistical analysis --- p.39 / Chapter 4. --- IN VIVO PRODUCTION OF TUMOR NECROSIS FACTOR USING ZYMOSAN AND LIPOPOLYSACCHARIDE / INTRODUCTION --- p.40 / EXPERIMENTAL --- p.43 / RESULTS --- p.45 / DISCUSSION --- p.65 / Chapter 5. --- SIDE EFFECTS DURING IN VIVO PRODUCTION OF TUMOR NECROSIS FACTOR IN EHRLICH ASCITES TUMOR BEARING MICE / INTRODUCTION --- p.70 / EXPERIMENTAL --- p.72 / RESULTS --- p.74 / DISCUSSION --- p.93 / Chapter 6. --- MODIFIED PROCEDURE FOR THE IN VIVO PRODUCTION OF TUMOR NECROSIS FACTOR FOR THE TREATMENT OF EHRLICH ASCITES TUMOR BEARING MICE / INTRODUCTION --- p.98 / EXPERIMENTAL --- p.99 / RESULTS --- p.100 / DISCUSSION --- p.108 / Chapter 7. --- "COMBINED TREATMENTS OF IN VIVO PRODUCTION OF TUMOR NECROSIS FACTOR (TNF) WITH HYPERTHERMIA, METHOTREXATE (MTX), POLYRIBOINOSINIC-POLYRIBOCYTIDYLIC ACID (POLY I.C), N-(PHOSPHONACETYL)-L-ASPARTATE (PALA) ON EAT BEARING MICE" / INTRODUCTION --- p.111 / EXPERIMENTAL --- p.116 / RESULTS --- p.118 / DISCUSSION --- p.133 / Chapter 8. --- EFFECTS OF IN VIVO PRODUCTION OF TUMOR NECROSIS FACTOR ON EHRLICH ASCITES TUMOR CELLS CYTOTOXICITY / INTRODUCTION --- p.138 / EXPERIMENTAL --- p.140 / RESULTS --- p.142 / DISCUSSION --- p.151 / Chapter 9. --- SIDE EFFECTS OF TUMOR NECROSIS FACTOR AND LIPOPOLYSACCHARIDE ON RAT IN VITRO AND IN VIVO / INTRODUCTION --- p.154 / EXPERIMENTAL --- p.157 / RESULTS --- p.159 / DISCUSSION --- p.170 / Chapter 10. --- CONCLUSION AND OUTLOOK --- p.174 / BIBLIOGRAPHY --- p.181

by Kin-mang Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 148-167). / Chapter I --- ABSTRACT --- p.1 / Chapter II --- INTRODUCTION --- p.3 / Chapter II. 1 --- Epidemiology of prostate cancer --- p.3 / Chapter II.2 --- Anatomy of the human prostate --- p.13 / Chapter II.3 --- Pathology of prostate diseases --- p.14 / Chapter II.3.1 --- Prostatic Hyperplasia / Chapter II.3.2 --- Atypical Hyperplasia / Chapter II.3.3 --- Prostatic Carcinoma / Chapter II.4 --- Tumour Suppressor Gene - Human p53 --- p.19 / Chapter II.4.1 --- General aspects / Chapter II.4.2 --- Human p53 gene - Historical perspectives / Chapter II.4.3 --- Human p53 gene - Structure / Chapter II.4.4 --- Human p53 protein - Structure / Chapter II.4.5 --- Wild-type p53 protein - Biochemical functions / Chapter II.4.6 --- Wild-type p53 protein - Biological function / Chapter II.4.7 --- Regulation of p53 function / Chapter II.4.8 --- p53 mutations in Human cancers / Chapter II.4.9 --- Properties of mutant p53 protein / Chapter II.5 --- Human Papillomavirus (HPV) --- p.38 / Chapter II.5.1. --- Virion Structure / Chapter II.5.2 --- Classification / Chapter II.5.3. --- Papillomaviruses in Human Cancers / Chapter II.5.4. --- Relationship between p53 alteration and HPV infection / Chapter II.6 --- p53 alteration and Prostate cancers --- p.42 / Chapter II.6.1. --- Cytogenetic studies / Chapter II.6.2. --- Hybridization analysis / Chapter II.6.3. --- p53 alterations and Prostatic cell lines / Chapter III. --- OBJECTIVES OF STUDY --- p.46 / Chapter IV --- MATERIALS & METHODS --- p.47 / Chapter IV.1 --- Patients and Materials --- p.47 / Chapter IV.2 --- Histological Grading --- p.47 / Chapter IV.2.1 --- Gleason grading / Chapter IV.2.2 --- W.H.O. grading (Mostofi) / Chapter IV.3 --- Staging of Prostatic carcinoma --- p.48 / Chapter IV.4 --- Collection of Blood and Tissue samples --- p.49 / Chapter IV.5 --- Immunohistochemical studies of Prostatic lesions --- p.50 / Chapter IV.5.1 --- Antibodies used / Chapter IV.5.2 --- Methods in frozen sections / Chapter IV.5.3 --- Methods in paraffin sections / Chapter IV.5.4 --- Controls / Chapter IV.5.5 --- Immunohistochemical evaluation / Chapter IV.6 --- Extraction of DNA from tissues and blood samples --- p.52 / Chapter IV.6.1 --- Extraction of genomic DNA from blood / Chapter IV.6.2 --- Extraction of genomic DNA from tissue / Chapter IV.7 --- Hybridization analysis --- p.54 / Chapter IV.7.1 --- Preparation of Cloned Probe DNA / Chapter IV.7.2 --- Transformation of CaCl2-treated competent cell / Chapter IV.7.3 --- Cultures of Transformants / Chapter IV.7.4 --- Isolationof plasmid DNA from transformant cultures / Chapter IV.7.5 --- Purification of DNA Probe by electroelution / Chapter IV.7.6 --- Radioactive labelling of DNA Probes / Chapter IV.7.7 --- Purification of radioactive labelled DNA Probes / Chapter IV.7.8 --- Southern Blotting Technique / Chapter IV.7.9 --- Hybridization of DNA Blots with labelled DNA Probe / Chapter IV.8 --- Polymerase Chain Reaction - Single Stranded Conformational Polymorphism (PCR-SSCP) --- p.63 / Chapter IV.8.1 --- 5'-radioactive labelling of primer / Chapter IV.8.2 --- Amplification of target sequence by PCR / Chapter IV.8.3 --- Nondenaturing Polyacrylamide Gel Electrophoresis / Chapter IV.8.4 --- Direct DNA sequencing of PCR products with p53 mutation / Chapter IV.8.5 --- Controls / Chapter IV.9 --- PCR method for detection of Human Papillomavirus (HPV) --- p.71 / Chapter IV.9.1 --- PCR-Amplification / Chapter IV.9.2 --- DNA alkali Blotting Technique / Chapter IV.9.3 --- Preparation of Radioactive labelled Oligoprobes / Chapter IV.9.4 --- Hybridization of DNA Blots with radioactive labelled Oligoprobes / Chapter IV.9.5 --- Controls / Chapter IV.9.6 --- Sensitivity of HPV 18 detection by PCR / Chapter V --- RESULTS --- p.76 / Chapter V.1 --- Grading and Staging of patients with prostatic carcinoma --- p.76 / Chapter V.2 --- Immunohistochemistry in prostatic lesions --- p.80 / Chapter V.3 --- Results of hybridization analysis --- p.81 / Chapter V.4 --- PCR-SSCP findings in prostatic hyperplasia and carcinoma --- p.97 / Chapter V.5 --- PCR detection of HPV in human prostate --- p.110 / Chapter VI --- DISCUSSION --- p.125 / Chapter VII --- CONCLUSION --- p.146 / Chapter VIII --- REFERENCES --- p.148 / Chapter IX --- APPENDIX --- p.168 / Chapter X --- ACKNOWLEDGEMENT --- p.172

No description available.

腫瘤發生過程中，遺傳和/或表觀遺傳異常都可導致抑癌基因(TSG)的失活。新TSG的鑒定和研究對理解癌症發展至關重要，并能提供潛在的治療靶點和腫瘤標誌物。本研究的目標是在人類腫瘤中鑒定新的被表觀遺傳異常沉默的TSG并進一步研究其抑癌的分子機理。 / 表觀遺傳修飾因子是重要的腫瘤相關基因。這裡，我研究了兩個作為功能性TSG的表觀遺傳修飾基因。首先，我發現一個在腫瘤中被甲基化和下調的候選TSG, PRDM5, 可通過抑制TCF/LEF依賴的轉錄和引起多個癌基因的表觀遺傳下調而抑制癌細胞增殖。其次，通過檢測24個表觀遺傳修飾基因在癌細胞系中的表達，我發現一個頻繁下調的新候選TSG，TUSC12。TUSC12在正常組織中普遍表達，但在腫瘤細胞系中被啟動子區甲基化下調，且原發腫瘤中也存在高頻TUSC12甲基化。TUSC12顯著抑制癌細胞克隆形成能力，但這種抑制效果因其PHD功能域的失活被部份削弱。TUSC12與轉錄抑制因子KAP1在細胞核中共定位，并可能通過招募HDAC相關複合體而抑制基因轉錄。 / 另外，我研究了一個通過以前鼻咽癌(NPC) aCGH鑒定得到的新3p14.2 TSG，ZNF312。啟動子區甲基化導致ZNF312在NPC細胞系和原發癌中的沉默。恢復ZNF312表達可抑制NPC細胞生長，并引起細胞週期阻滯和凋亡。作為一個轉錄抑制因子，ZNF312靶向EZH2和MDM2的啟動子區并下調它們的表達。 / 之前的基因數字表達分析已篩選出一些可能在腫瘤中低表達的基因。我研究了一個通過這種方法分離出的新TSG，TUSC45。相比正常組織，TUSC45表達在癌組織中顯著下降，而且TUSC45低表達與病人的低存活相關。TUSC45在多個腫瘤細胞系中也被下調，但它的基因組缺失卻不多見。啟動子區甲基化導致TUSC45在多數細胞系中的沉默，而且藥物或遺傳去甲基化能顯著激活它的表達。特別的，TUSC45在腫瘤組織而不在正常組織中被高頻率甲基化。誘導TUSC45表達可抑制細胞增殖，引起細胞凋亡、週期阻滯、和衰老，並上調一個關鍵抑癌基因p53。TUSC45以p53依賴的方式激活p53的靶基因，而TUSC45對p53缺失的H1299和HCT116/p53KO細胞沒有生長抑制作用。TUSC45與p53/MDM2複合物結合并正向調節p53蛋白穩定性。 TUSC45表達導致MDM2蛋白半衰期縮短，提示一種可能的TUSC45調節p53通路的機制。 / 本研究表明癌變過程中四個抑癌基因腫瘤特異的甲基化和沉默引起了多個細胞信號通路紊亂，並可作為潛在的腫瘤檢測標誌物。 / Tumor suppressor genes (TSGs) can be inactivated by genetic and/or epigenetic alterations during carcinogenesis. Identification and characterization of novel TSGs are critical for the understanding of cancer development, and provide potential targets for clinical treatment and biomarkers for tumor diagnosis. In this study, I aimed to identify novel TSGs epigenetically silenced in human cancers and further characterize the molecular basis of their anti-tumorigenic functions. / Emerging evidence highlights the importance of epigenetic modifiers as cancer genes. Here, I characterized two epigenetic modifier genes as functional TSGs. First, I found PRDM5, a candidate TSG methylated and downregulated in multiple cancers, suppressed tumor cell proliferation through inhibiting TCF/LEF-dependent transcription and inducing epigenetic repression of multiple oncogenes. Second, through expression profiling of 24 epigenetic modifiers, I identified a novel candidate TSG, TUSC12, showing frequent silencing in tumor cell lines. TUSC12 was broadly expressed in human normal tissues, but downregulated by promoter CpG methylation in tumor cell lines. Frequent TUSC12 methylation was detected in primary tumors as well. TUSC12 dramatically inhibited tumor cell clonogenicity, but this growth inhibitory effect was partially impaired by disrupting its PHD domain. TUSC12 colocalized with the transcription corepressor KAP1 in the nucleus and is likely to repress gene transcription through recruit HDAC-associated complex. / I also studied a novel 3p14.2 TSG, ZNF312, identified from previous aCGH profiling of nasopharyngeal carcinoma (NPC). Frequent promoter methylation silenced ZNF312 in NPC cell lines and tissues. Restored ZNF312 expression strongly suppressed NPC cell growth through inducing cell cycle arrest and apoptosis. Further, ZNF312 acted as a transcription repressor targeting the promoter regions of EZH2 and MDM2 and downregulating their expression. / Moreover, previous digital gene expression subtraction from cDNA libraries revealed a list of genes possibly downregulated in tumors compared to normal tissues. I characterized a novel TSG, TUSC45, initially isolated from this strategy. The expression of TUSC45 was significantly reduced in tumor tissues compared to normal tissues, with lower TUSC45 expression associated with poorer patient survival. Downregulation of TUSC45 in multiple tumor cell lines was also observed, while only infrequent genomic deletion was detected. In contrast, promoter methylation was responsible for TUSC45 silencing in most cell lines, in which pharmacologic or genetic demethylation can dramatically restore its expression. Remarkably, TUSC45 was frequently methylated in primary tumors but not in normal tissues. Further, TUSC45 suppressed anchorage-dependent and -independent tumor cell growth. Induced TUSC45 expression inhibited cell proliferation, induced apoptosis, cell cycle arrest and senescence, and lead to the upregulation of a key tumor suppressor p53. Moreover, TUSC45 activated p53 target genes in a p53-dependent manner. Forced TUSC45 expression in p53-null H1299 and HCT116/p53KO (knock out) cells showed no inhibitory effect on cell growth. Finally, TUSC45 interacted with p53/MDM2 complex and positively regulated p53 protein stability. The protein half-life of MDM2 was shortened by TUSC45, indicating a possible mechanism for TUSC45 modulation on p53 signaling. / In conclusion, this study showed the tumor-specific methylation and silencing of the four TSGs lead to the epigenetic disruption of multiple cell signalings during tumorigenesis and could potentially be used as biomarkers for cancer detection. / 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. / Shu, Xingsheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 121-140). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Publications --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction and Literature Reviews --- p.1 / Chapter 1.1 --- Tumor suppressor genes (TSGs) and the pathways they control --- p.2 / Chapter 1.1.1 --- Basic concepts about TSG --- p.2 / Chapter 1.1.1.1 --- The discovery of TSG --- p.2 / Chapter 1.1.1.2 --- Knudson’s “two-hit“ hypothesis --- p.3 / Chapter 1.1.1.3 --- New insights of the “two-hit“ model --- p.4 / Chapter 1.1.2 --- Key TSGs and their cellular pathways --- p.5 / Chapter 1.1.2.1 --- p53 pathway --- p.6 / Chapter 1.1.2.2 --- Rb pathway --- p.9 / Chapter 1.1.2.3 --- APC and Wnt/β-catenin pathway --- p.10 / Chapter 1.1.2.4 --- Chromatin regulators with tumor suppressive properties --- p.12 / Chapter 1.1.3 --- Methods for TSG identification --- p.17 / Chapter 1.2 --- The epigenetic abnormalities of TSGs in cancer --- p.19 / Chapter 1.2.1 --- Promoter CpG methylation --- p.20 / Chapter 1.2.1.1 --- Molecular basis of DNA methylation --- p.20 / Chapter 1.2.1.2 --- Silencing of TSGs by promoter methylation --- p.22 / Chapter 1.2.1.3 --- Mechanism of methylation-induced transcription repression --- p.23 / Chapter 1.2.1.4 --- Abnormal DNA methylation contributes to early stages of tumorigenesis --- p.25 / Chapter 1.2.2 --- Aberrant histone modification and chromatin remodeling --- p.26 / Chapter 1.2.3 --- Clinical applications of epigenetic biomarkers and therapeutic targets --- p.28 / Chapter 1.2.3.1 --- DNA methylation and histone modification as biomarkers for cancer diagnosis and prognosis --- p.28 / Chapter 1.2.3.2 --- Epigenetic targets for cancer treatment --- p.29 / Chapter Chapter 2 --- Aims and Design of This Study --- p.32 / Chapter Chapter 3 --- Materials and Methods --- p.34 / Chapter 3.1 --- Cell lines --- p.34 / Chapter 3.2 --- Human normal and cancer tissues --- p.35 / Chapter 3.3 --- DNA and RNA extraction --- p.35 / Chapter 3.4 --- Semi-quantitative and quantitative RT-PCR --- p.36 / Chapter 3.5 --- DNA methylation analysis --- p.37 / Chapter 3.5.1 --- Bisulfite modification of genomic DNA --- p.37 / Chapter 3.5.2 --- CpG island analysis --- p.37 / Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.38 / Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.38 / Chapter 3.5.5 --- Pharmacologic demethylation treatment of cell lines --- p.38 / Chapter 3.6 --- Luciferase assay of gene promoter activity --- p.39 / Chapter 3.7 --- Multiplex genomic-DNA PCR --- p.39 / Chapter 3.8 --- Construction of expression plasmids and PCR-mediated mutagenesis --- p.40 / Chapter 3.9 --- Plasmid mini- and midi-preparation --- p.41 / Chapter 3.10 --- Creation of stable cell line for inducible gene expression --- p.42 / Chapter 3.11 --- Monolayer-culture and soft-agar colony formation assay --- p.42 / Chapter 3.12 --- Cell proliferation assay --- p.43 / Chapter 3.13 --- Flow cytometry analysis of cell cycle --- p.43 / Chapter 3.14 --- Apoptosis assay --- p.44 / Chapter 3.15 --- Senescence cell staining --- p.44 / Chapter 3.16 --- Western blotting --- p.45 / Chapter 3.17 --- Co-immunoprecipitation (Co-IP) --- p.46 / Chapter 3.18 --- Transcription factor activity assay --- p.46 / Chapter 3.19 --- Immunofluorescence microscopy --- p.47 / Chapter 3.20 --- Chromatin Immunoprecipitation (ChIP) --- p.47 / Chapter 3.21 --- Gene expression and copy number analysis using Oncomine database --- p.48 / Chapter 3.22 --- Protein half-life assay --- p.48 / Chapter 3.23 --- In vivo ubiquitination assay --- p.48 / Chapter 3.24 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Two Epigenetic Modifying Genes, PRDM5 and TUSC12, Suppress Tumor Cell Growth and are Silenced by Promoter Methylation in Human Cancers --- p.50 / Chapter 4.1 --- PRDM5 --- p.50 / Chapter 4.1.1 --- PRDM5 is a candidate TSG downregulated and methylated in multiple carcinomas --- p.50 / Chapter 4.1.2 --- PRDM5 is a stress responsive gene but its response is disrupted by promoter methylation --- p.53 / Chapter 4.1.3 --- PRDM5 suppresses cancer cell growth and proliferation --- p.54 / Chapter 4.1.4 --- PRDM5 inhibits TCF/LEF-dependent transcription and induced epigenetic repression of multiple oncogenes --- p.55 / Chapter 4.2 --- TUSC12 --- p.59 / Chapter 4.2.1 --- mRNA expression profiling of epigenetic modifying genes in tumor cell lines --- p.59 / Chapter 4.2.2 --- Promoter CpG methylation contributes to TUSC12 silencing in tumor cells --- p.61 / Chapter 4.2.3 --- Demethylation of TUSC12 promoter restores its expression --- p.63 / Chapter 4.2.4 --- TUSC12 methylation in primary tumor tissues --- p.65 / Chapter 4.2.5 --- TUSC12 expression inhibits anchorage-dependent and -independent tumor cell growth --- p.66 / Chapter 4.2.6 --- TUSC12 is an epigenetic modifier repressing transcription --- p.68 / Chapter 4.3 --- Discussion --- p.69 / Chapter Chapter 5 --- The Human Zinc Finger Protein 312 is a Novel Tumor Suppressor for Nasopharyngeal Carcinoma --- p.73 / Chapter 5.1 --- Identification of ZNF312 as a candidate 3p14.2 TSG --- p.74 / Chapter 5.2 --- Silencing of ZNF312 by promoter methylation in NPC cell lines --- p.75 / Chapter 5.3 --- ZNF312 is frequently downregulated and methylated in primary NPC tissues --- p.78 / Chapter 5.4 --- ZNF312 suppresses tumor cell clonogenicity --- p.79 / Chapter 5.5 --- ZNF312 is a transcription repressor --- p.80 / Chapter 5.6 --- ZNF312 regulates cell cycle progression, induces apoptosis, and inhibits cell stemness --- p.83 / Chapter 5.7 --- ZNF312 represses oncogene expression --- p.85 / Chapter 5.8 --- Discussion --- p.87 / Chapter Chapter 6 --- Identification of a Novel Tumor Suppressor Regulating p53 Signaling and Frequently Methylated in Multiple Tumors --- p.91 / Chapter 6.1 --- TUSC45 is broadly expressed in human normal tissues but frequently downregulated in tumor cell lines --- p.91 / Chapter 6.2 --- Reduced TUSC45 expression in primary tumors is associated with poor survival of patients --- p.94 / Chapter 6.3 --- TUSC45 is mainly silenced by promoter CpG methylation in tumor cell lines --- p.96 / Chapter 6.4 --- Pharmacologic or genetic demethylation reactivates TUSC45 in silenced cell lines --- p.98 / Chapter 6.5 --- TUSC45 is frequently methylated in primary tumors --- p.99 / Chapter 6.6 --- TUSC45 suppresses anchorage-dependent and -independent tumor cell growth --- p.101 / Chapter 6.7 --- Induction of TUSC45 expression inhibits tumor cell growth through inducing apoptosis, cell cycle arrest and senescence --- p.104 / Chapter 6.8 --- TUSC45 tumor-suppressive function is dependent on p53 signaling --- p.108 / Chapter 6.9 --- TUSC45 positively regulates p53 protein stability --- p.110 / Chapter 6.10 --- Discussion --- p.112 / Chapter Chapter 7 --- General Discussion --- p.116 / Chapter 7.1 --- General discussion --- p.116 / Chapter 7.2 --- Future perspectives --- p.118 / Reference List --- p.121

Ko Samuel. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 230-252). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.