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

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

by Xin-xia Tian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 195-212). / 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.

by Nellie Yuk Fei Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 111-120). / Abstract also in Chinese. / Abstract --- p.i / List of Abbreviations --- p.iii / Acknowledgements --- p.iv / Publication of this study --- p.vi / Table of Contents --- p.vii / Chapter Chapter1: --- Introduction --- p.1 / Chapter 1.1 --- Multiple Myeloma --- p.2 / Chapter 1.2 --- The Problem --- p.2 / Chapter Chapter2: --- Literature Review --- p.5 / Chapter 2.1 --- Molecular Genetics of Multiple Myeloma --- p.6 / Chapter 2.1.1 --- Cytogenetics --- p.6 / Chapter 2.2 --- Alterations of Proto-Oncogenes --- p.9 / Chapter 2.2.1 --- c-myc --- p.9 / Chapter 2.2.2 --- Ras --- p.10 / Chapter 2.2.3 --- Bcl-2 and Related Protein --- p.10 / Chapter 2.3 --- Alteration of Tumor-Suppressor genes --- p.11 / Chapter 2.3.1 --- p53 Gene Mutations --- p.11 / Chapter 2.3.2 --- Retinoblastoma (Rb) Gene --- p.11 / Chapter 2.3.3 --- p16 and p15 Genes --- p.13 / Chapter Chapter3: --- DNA Methylation and Cancers --- p.14 / Chapter 3.1 --- Role of DNA Methylation --- p.15 / Chapter 3.2 --- CpG Islands --- p.15 / Chapter 3.3 --- Abnormalities of DNA Methylation in Neoplasia --- p.16 / Chapter 3.3.1 --- DNA Hypomethylation in Cancer --- p.16 / Chapter 3.3.2 --- DNA Methyltransferase Activity in Cancer --- p.17 / Chapter 3.4 --- Regional DNA Hypermethylation in Cancer --- p.17 / Chapter 3.4.1 --- p16 and p15 Genes in Solid Tumors --- p.18 / Chapter 3.4.2 --- The p16 and p15 Genes in Leukemia and other Hematopoietic Malignancies --- p.19 / Chapter 3.4.3 --- Retinoblastoma Gene --- p.20 / Chapter 3.5 --- Mechanism Underlying the DNA Methylation Changes --- p.21 / Chapter Chapter4: --- Background of Study --- p.23 / Chapter 4.1 --- Background of Study --- p.24 / Chapter 4.2 --- Project Objectives --- p.27 / Chapter Chapter5: --- Materials and Methods --- p.29 / Chapter 5.1 --- Patients Samples --- p.30 / Chapter 5.2 --- Normal Controls --- p.30 / Chapter 5.3 --- Storage of the Samples --- p.32 / Chapter 5.4 --- Materials --- p.32 / Chapter 5.4.1 --- Chemicals --- p.32 / Chapter 5.4.2 --- Primers --- p.33 / Chapter 5.4.3 --- Enzymes --- p.35 / Chapter 5.5 --- Methods --- p.35 / Chapter 5.5.1 --- Cloning of p16 and p15 Exon 1 Probes for Southern Analysis --- p.35 / Chapter 5.5.1.1 --- PCR Amplification of p16 and p15 exon1 Probes from Normal Blood DNA --- p.35 / Chapter 5.5.1.2 --- Recovery and Purification of p16 and p15 Exon 1 DNA Fragment --- p.36 / Chapter 5.5.1.3 --- Ligation --- p.37 / Chapter 5.5.1.4 --- Transformation --- p.37 / Chapter 5.5.1.5 --- Plating --- p.38 / Chapter 5.5.1.6 --- Screening of Recombinant Plasmid --- p.38 / Chapter 5.5.1.7 --- Confirmation of Cloned DNA by Sequencing --- p.42 / Chapter 5.5.2 --- DNA Extraction and Purification --- p.45 / Chapter 5.5.2.1 --- DNA Extraction from Bone Marrow Aspirate and Peripheral Blood --- p.45 / Chapter 5.5.2.2 --- Isolation of Plasmid DNA from Transformant Cutures --- p.46 / Chapter 5.5.2.3 --- Qualification and Quantification of DNA --- p.49 / Chapter 5.5.3 --- Detection of Hypermethylation by Southern Analysis --- p.50 / Chapter 5.5.3.1 --- Restriction Enzyme Digestion --- p.50 / Chapter 5.5.3.2 --- Agarose Gel Electrophoresis --- p.51 / Chapter 5.5.3.3 --- Southern Transfer --- p.51 / Chapter 5.5.3.4 --- Membrane Fixation --- p.51 / Chapter 5.5.3.5 --- Recovery and Purification of p16 and p15 Exon 1 Probes from Plasmid --- p.52 / Chapter 5.5.3.6 --- Probe Labeling --- p.54 / Chapter 5.5.3.7 --- Purification of Radioactive labeled DNA --- p.54 / Chapter 5.5.3.8 --- Southern Hybridization --- p.55 / Chapter 5.5.3.9 --- Post Hybridization --- p.55 / Chapter 5.5.3.10 --- Autoradiography --- p.56 / Chapter 5.5.4 --- Polymerase Chain Reaction-Single Strand Conformational Polymorphism Analysis (PCR-SSCP) --- p.56 / Chapter 5.5.4.1 --- 5'- end Radioactive Labeling of Primer --- p.56 / Chapter 5.5.4.2 --- Amplification of Target Sequence by PCR --- p.57 / Chapter 5.5.4.3 --- Non-denaturing Polyacrylamide Gel Electrophresis --- p.57 / Chapter 5.5.4.4 --- Direct DNA Sequence of PCR Products --- p.58 / Chapter 5.5.5 --- Prevention of Overall Contamination in PCR --- p.60 / Chapter 5.5.6 --- "Sensitivity, Specificity Controls" --- p.62 / Chapter Chapter6: --- Results --- p.64 / Chapter 6.1 --- Patient Characteristics --- p.65 / Chapter 6.1.1 --- General Patient Characteristics --- p.65 / Chapter 6.1.2 --- Clinical and Laboratory Features --- p.65 / Chapter 6.2 --- Southern Blot Analysis of p16/p15 and Rb --- p.79 / Chapter 6.2.1 --- Absence of Deletions or hypermethylationin Normal Controls --- p.79 / Chapter 6.2.2 --- Absence of Homozygous Deletions or Mutationsin p16/15 and Rb among all MM Patients --- p.79 / Chapter 6.2.3 --- Hypermethylation of p16 --- p.89 / Chapter 6.2.4 --- Hypermethylation of p15 --- p.92 / Chapter 6.3 --- Hypermethylation of p16/p15 and Clinico-pathologic Correlation --- p.94 / Chapter Chapter7: --- Discussion --- p.97 / Chapter 7.1 --- "Absence of Homozygous Deletions, Gene Rearrangements and Mutations in p16/p15 and Rb" --- p.98 / Chapter 7.2 --- Hypermethylation of p16/p15-An Alternative Way for Gene Inactivation --- p.100 / Chapter 7.2.1 --- Methylation of p15 Gene --- p.101 / Chapter 7.2.2 --- Methylation of 5'-CpG Island of p16/p15 and Lack of Gene Expression --- p.102 / Chapter 7.2.3 --- Comparison of Methylation Status of Primary Samples and Cell Lines in MM --- p.103 / Chapter 7.2.4 --- Progressive Gene Inactivation by Random Methylation Errors --- p.104 / Chapter 7.2.5 --- The Lack of Correlation of Tumor Contents Revealed by the Southern Analysis and Morphologic Assessment --- p.105 / Chapter 7.3 --- Knudson's Two-hit Model of Tumorigenesis --- p.106 / Chapter 7.4 --- Inverse Relationship of p16 and Rb --- p.107 / Chapter 7.5 --- Implications of Our Findings --- p.109 / Chapter 7.6 --- Future Studies --- p.109 / References --- p.111

Xiong, Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 79-97). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

抑制腫瘤的基因（又稱抑癌基因）之表達失活，是導致癌變的重要機制之一。除了基因突變之外，越來越多研究證明抑癌基因的關閉轉錄，是由於抑癌基因啟動子區的CpG島甲基化所致。本論文的研究確定了兩個候選抑癌基因ADAMTS8和TUSC14，在多種腫瘤細胞株中經常因動子區的CpG島甲基化而下調或停止表達，這有別於它們在正常組織中廣泛表達的情況。沉默細胞株在脫氧核糖核酸甲基轉移酶的抑製劑5-氮-2'-脫氧胞苷(5-aza-2′-deoxycytidine; Aza) 或與組蛋白去乙酰化酶抑製劑曲古抑菌素A (trichostatin A, TSA)的去甲基化作用下，能恢復這兩個抑癌基因的表達，因而證明了啟動子甲基化是直接導致其表達下調及沉默的機制。 / 論文的第一部分，主要調查ADAMTS8啟動子區在原發腫瘤樣本被甲基化的比率，並研究其腫瘤抑制功能。含血小板凝血酶敏感蛋白基序的解整聯蛋白金屬蛋白酶 (ADAMTSs) ，在各種癌症中的表達異常已有報導。然而，它們在腫瘤的職能作用仍然模糊不清。本研究發現，異位表達ADAMTS8誘導細胞凋亡，因而顯著抑制腫瘤細胞克隆形成的能力,。這些都突顯其抑制腫瘤的功能。此外，作為分泌蛋白酶的ADAMTS8，能夠透過減少表皮生長因子受體(EGFR) 蛋白的磷酸化，抑制EGFR / MEK / ERK信號通路，並進一步破壞肌動蛋白應力纖維的組織，抑制腫瘤細胞的遷移性。 / 論文的第二部分，集中於研究一個未知功能的基因TUSC14，這基因的蛋白質編碼具有氨基末端蛋白質相互作用域 (BTB/POZ domain)及C₂H₂乙炔鋅指結構。TUSC14的異位表達能抑制腫瘤細胞克隆的形成，但這種抑制作用會在删除蛋白中的BTB/POZ或C₂H₂乙炔鋅指結構功能域後消失。因此證實了TUSC14蛋白同時需要BTB / POZ和C₂H₂乙炔鋅指結構兩個功能域來抑制腫瘤生長。此外，TUSC14具有抑制NF-kB轉錄的功能，其功能不但依賴於组蛋白去乙酰基酶(HDAC)，並且與c-MYC和cIAP-2等NF-κB靶基因下調表達相關。TUSC14的抑癌功能，包括抑制腫瘤生長與增加細胞凋亡，與其減少c-MYC及抗凋亡基因cIAP-2的表達，效果一致。進一步的分析發現，TUSC14與HDAC1和P65於蛋白質複雜免疫共沉澱實驗中，有物理相互作用。此外，染色質免疫沉澱實驗顯示TUSC14透過與c-MYC和cIAP-2的相互作用，抑制其基因啟動子區的轉錄功能。結果表明，TUSC14是通過招募HDAC至NF-κB靶基因的啟動子區這機制，來抑制NF-kB靶基因的轉錄，以達至抑制癌細胞生長和誘導癌細胞凋亡的效果。因此，TUSC14的沉默是破壞癌細胞中NF-kB信號通路負調控(negative regulation)的重要因素。 / 綜上所述，本研究鑒定了兩個在多種腫瘤細胞因表觀遺傳沉默效應而表達下調或沉默的抑癌基因ADAMTS8和TUSC14，並證實它們具有抑癌功能。 / Inactivation of tumor suppressor genes (TSGs) is one of the critical mechanisms leading to carcinogenesis. Apart from genetic mutations, a growing number of TSG has been shown to be silenced through promoter CpG methylation. In this thesis, we identified two candidate TSGs: ADAMTS8 and TUSC14 that are frequently downregulated or silenced in multiple carcinoma cell lines by promoter methylation while broadly expressed in normal tissues. Expression of these two genes was restored after treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) or in combination with a histone deacetylase inhibitor trichostatin A (TSA), suggesting promoter-methylation directly contributes to their silencing. / In the first part of the thesis, prevalence silencing of ADAMTS8 was detected in primary tumor samples. Expression of many disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) was reported to be dysregulated in various cancers. However, their functional roles in tumorigenesis remain obscure. This study revealed that ectopic expression of ADAMTS8 markedly inhibits tumor cell clonogenicity by inducing apoptosis, underscoring its function as a tumor suppressor. Furthermore, ADAMTS8, as a secreted protease, inhibits EGFR/MEK/ERK signaling pathway by reducing their phosphorylation, further resulting in the disruption of actin stress fiber organization and suppression of tumor cell motility. / The second part of the thesis focused on a novel gene TUSC14 which encodes a protein with BTB/POZ domain and C₂H₂zinc-fingers. Ectopic expression of TUSC14 suppresses colony formation of cancer cells but this inhibitory effect is abolished with the deletion of BTB/POZ domain or C₂H₂ zinc-fingers. This suggested that both BTB/POZ domain and C₂H₂ zinc-fingers are required for inhibiting tumor cell clonogenecity. In addition, TUSC14 functions as a transcriptional repressor of NF-kB pathway that is dependent on HDAC. Suppression of NF-κB transcriptional activity by TUSC14 expression correlates with the downregulation of NF-κB target genes including c-MYC and cIAP-2. Reduction of c-MYC and anti-apoptotic cIAP-2 agrees well with the consequent growth suppression and enhanced apoptosis following the ectopic expression of TUSC14. Further analyses showed TUSC14 physically interacts with HDAC1 and p65 via co-immunoprecipitation assay. Preliminary ChIP assay showed that TUSC14 associates with gene promoters of c-MYC and cIAP-2 for their transcription repressions. These results revealed that TUSC14 represses NF-kB activity through recruiting HDAC to the NF-kB target genes; and transcription repression of NF-kB represents a mechanism for TUSC14 to mediate its growth inhibitory and apoptosis-inducing effects in cancer. Hence, silencing of TUSC14 contributes to the lost of negative regulation on NF-kB signaling in cancer. / In summary, this study demonstrated that ADAMTS8 and TUSC14 are functional tumor suppressors that are epigenetically silenced in multiple tumors. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Choi, Ching Gee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 140-153). / Abstract also in Chinese. / Abstract --- p.i / Chinese abstract --- p.iv / AcknowledgEments --- p.vii / List of Figures --- p.ix / List of Tables --- p.xi / LIST OF ABBREVIATIONS --- p.xii / List of PUBLICATIONs --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of cancer epigenetics --- p.1 / Chapter 1.2 --- Epigenetic events --- p.2 / Chapter 1.2.1 --- DNA methylation --- p.2 / Chapter 1.2.2 --- Histone modifications --- p.5 / Chapter 1.2.3 --- The interdependence of DNA methylation and histone modifications --- p.8 / Chapter 1.3 --- Epigenetic alterations in cancer --- p.9 / Chapter 1.3.1 --- Genome-wide DNA hypomethylation --- p.9 / Chapter 1.3.2 --- CpG island promoter hypermethylation silencing of tumour suppressor genes in tumorigenesis --- p.10 / Chapter 1.3.3 --- Aberrations of histone modifications --- p.11 / Chapter 1.4 --- Causes for epigenetic deregulation in cancer --- p.14 / Chapter 1.5 --- The interplay of genetic and epigenetic aberration in cancer progression --- p.21 / Chapter 1.6 --- Epigenetic inactivation of tumor suppressor genes in cancer --- p.23 / Chapter 1.7 --- Clinical implications of epigenetic research --- p.27 / Chapter 1.7.1 --- Epigenetic modifications as biomarker for cancer diagnosis --- p.27 / Chapter 1.7.2 --- Targeting epigenetic modifications as therapeutics towards cancers --- p.29 / Chapter 1.8 --- Roles of ADAMTS proteins in cancer --- p.32 / Chapter 1.8.1 --- Introduction on ADAMTS metalloproteases --- p.32 / Chapter 1.8.2 --- Deregulation of ADAMTS protein in cancer --- p.34 / Chapter 1.9 --- Roles of BTB/POZ-ZF family of transcription factors in cancer --- p.36 / Chapter 1.9.1 --- Introduction on BTB/POZ-ZF Family --- p.36 / Chapter 1.9.2 --- BTB/POZ-ZF functions as transcription repressors --- p.37 / Chapter 1.9.3 --- Many BTB/POZ-ZF proteins are important players in tumorigenesis --- p.39 / Chapter 1.9.4 --- The role of BTB/POZ-ZF in tumor initiation and progression --- p.40 / Chapter CHAPTER 2 --- Aims of this study --- p.44 / Chapter CHAPTER 3 --- General Methodology --- p.46 / Chapter 3.1 --- Cell Culture --- p.46 / Chapter 3.1.1 --- Growth and maintenance of cells --- p.46 / Chapter 3.1.2 --- Mammalian cell transfection --- p.46 / Chapter 3.1.3 --- Drug and stress treatments --- p.47 / Chapter 3.2 --- DNA and RNA extraction --- p.47 / Chapter 3.3 --- Semi-quantitative RT-PCR and Real-time PCR --- p.48 / Chapter 3.4 --- CpG island and Transcription factor binding sites analysis --- p.49 / Chapter 3.5 --- Methylation-specific PCR (MSP) and Bisulfite genomic sequencing (BGS) --- p.49 / Chapter 3.6 --- Bacterial transformation and Plasmid extraction --- p.50 / Chapter 3.6.1 --- Heat-shock transformation --- p.50 / Chapter 3.6.2 --- Mini-scale preparation of plasmid DNA --- p.51 / Chapter 3.6.3 --- Preparation of endotoxin-free plasmids --- p.52 / Chapter 3.7 --- DNA cycle sequencing --- p.52 / Chapter 3.8 --- Indirect immunofluorescence for subcellular localization study --- p.54 / Chapter 3.9 --- Colony formation assay --- p.54 / Chapter 3.10 --- Cell cycle analysis --- p.55 / Chapter 3.11 --- Apoptosis assay --- p.56 / Chapter 3.12 --- Co-immunoprecipitation and Western blot --- p.56 / Chapter 3.13 --- Chromatin immunoprecipitation (ChIP) --- p.58 / Chapter 3.14 --- Dual Firefly and Renilla luciferase reporter gene assay --- p.59 / Chapter 3.15 --- Statistical analysis --- p.60 / Chapter CHAPTER 4: --- Characterization of the Tumor Suppressive Functions of ADAMTS8 --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Materials and Methods --- p.64 / Chapter 4.2.1 --- Tumor samples --- p.64 / Chapter 4.2.2 --- Expression of ADAMTS8 --- p.64 / Chapter 4.2.3 --- Immunofluorescence staining of ADAMTS8 --- p.64 / Chapter 4.2.4 --- Detection of secreted ADAMTS8 in culture medium --- p.65 / Chapter 4.2.5 --- Collection of conditioned medium and Western Blotting --- p.66 / Chapter 4.2.6 --- Wound healing assay --- p.66 / Chapter 4.3 --- Result and Discussion --- p.69 / Chapter 4.3.1 --- Frequent ADAMTS8 methylation in primary carcinomas --- p.69 / Chapter 4.3.2 --- ADAMTS8 is a secreted protease --- p.70 / Chapter 4.3.3 --- ADAMTS8 inhibits phosphorylation of pEGFR --- p.73 / Chapter 4.3.4 --- ADAMTS8 suppresses cell migration --- p.77 / Chapter 4.4 --- Summary --- p.81 / Chapter CHAPTER 5: --- Epigenetic Alterations of TUSC14 Gene in multiple carcinomas --- p.83 / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.2 --- Materials and Methods --- p.84 / Chapter 5.2.1 --- Cell lines --- p.84 / Chapter 5.2.2 --- Normal and primary tumor tissues --- p.85 / Chapter 5.3 --- Results and Discussion --- p.86 / Chapter 5.3.1 --- Expression profiling of TUSC14 in normal tissues and tumor cell lines --- p.86 / Chapter 5.3.2 --- Frequent inactivation of TUSC14 by promoter CpG methylation --- p.90 / Chapter 5.3.3 --- Pharmacologic and genetic demethylation restores TUSC14 expression --- p.94 / Chapter 5.3.4 --- Frequent TUSC14 methylation in primary tumors --- p.96 / Chapter 5.4 --- Summary --- p.98 / Chapter CHAPTER 6 --- Characterization of the Tumor Suppressive Functions of TUSC14 --- p.99 / Chapter 6.1 --- Introduction --- p.91 / Chapter 6.2 --- Materials and Methods --- p.100 / Chapter 6.2.1 --- Gene cloning and plasmids construction of TUSC14 --- p.100 / Chapter 6.2.2 --- Drug and stress treatments of cells --- p.100 / Chapter 6.3 --- Results and Discussion --- p.102 / Chapter 6.3.1 --- TUSC14 localizes to nuclear speckles --- p.102 / Chapter 6.3.2 --- TUSC14 inhibits clonogenicity --- p.107 / Chapter 6.3.3 --- Expression of TUSC14 induces apoptosis in tumor cells --- p.110 / Chapter 6.3.4 --- TUSC14 alters cell cycle progression --- p.112 / Chapter 6.3.5 --- TUSC14 acts as a transcriptional repressor of multiple genes --- p.114 / Chapter 6.3.6 --- TUSC14 represses NF-кB activity through an HDAC-dependent mechanism --- p.118 / Chapter 6.3.7 --- The effect of TUSC14 on the expression of downstream targets of NF-κB Signaling --- p.120 / Chapter 6.3.8 --- TUSC14 co-immunoprecipitates with HDAC1 and p65 --- p.124 / Chapter 6.3.9 --- ChIP analysis of promoters of TUSC14-regulated genes --- p.127 / Chapter 6.4 --- Summary --- p.130 / Chapter CHAPTER 7 --- General Discussion --- p.133 / Chapter CHAPTER 8 --- Conclusions --- p.138 / References --- p.140

Zheng Ping-pin. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 118-159). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT(ENGLISH/CHINESE) --- p.iii / CONTENTS --- p.viii / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xii / PUBLICATION --- p.xiii / Chapter CHAPTER I --- INTRODUCTION / Chapter I.1. --- Preface --- p.1 / Chapter I.2. --- Overview of Carcinogenesis --- p.2 / Chapter I.3. --- Oncogene --- p.5 / Chapter I.4. --- Tumor Suppressor Genes (TSGs) --- p.6 / Chapter I.5 --- Detection of Oncogene and Tumor Suppressor Genes --- p.9 / Chapter I.5.1 --- Detaction of Oncogene --- p.9 / Chapter I.5.2. --- Detection of Tumor Suppressor Genes --- p.11 / Chapter I.6. --- Profiles of Oncogenes/TSGs and Molecular Subtype about Astrocytic Tumors --- p.17 / Chapter I.7. --- Intratumoral Heterogeneity and Microsatellite Instability --- p.20 / Chapter I.8. --- Outline of Ependymoma --- p.20 / Chapter I.9. --- Clinicopathological Factors and Prognosis --- p.22 / Chapter I.9.1. --- Histology and Grading (2000) --- p.22 / Chapter I.9.2. --- Prognosis Factors --- p.23 / Chapter I.9.2.1. --- Age/Sex/Location --- p.23 / Chapter I.9.2.2. --- Extent of Resection --- p.25 / Chapter I.9.2.3. --- Radiotherapy and Chemotherapy --- p.25 / Chapter I.9.2.4. --- Histology --- p.26 / Chapter I.10. --- "Cytogenetic, Molecular Genetic and Molecular Studies" --- p.27 / Chapter I.11. --- Advantages and Disadvantages of The Research Methods --- p.34 / Chapter CHAPTER II --- AIM OF STUDY --- p.36 / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.37 / Chapter III.1. --- Tumor Samples and DNA Preparations --- p.37 / Chapter III.1.1. --- Tumor Samples --- p.38 / Chapter III.1.2. --- DNA Preparation --- p.38 / Chapter III.2. --- Comparative Genomic Hybridization --- p.42 / Chapter III.2.1. --- Metaphase Preparation --- p.42 / Chapter III.2.2. --- "DNA Labeling, Hybridization, and Detection" --- p.43 / Chapter III.2.3. --- Digital Image Analysis --- p.45 / Chapter III.3 --- High-Resolution Allelotying (Microsatellite Analysis) --- p.46 / Chapter III.3.1 --- General Outline --- p.46 / Chapter III.3.2 --- Multiplex PCR --- p.47 / Chapter III.3.3 --- Pooling of PCR Products --- p.49 / Chapter III.3.4 --- Electrophoresis --- p.50 / Chapter III.3.5. --- Assessment of Allelic Imbalance by Calculating Allelic Ratio --- p.52 / Chapter III.3.6 --- Standards of Evalution --- p.53 / Chapter III.3.7 --- Separating Allelic Loss from Allelic Duplication --- p.54 / Chapter III.3.8 --- Statistical Analyses --- p.54 / Chapter CHAPTER IV --- RESULTS --- p.54 / Chapter IV.1. --- CGH Study --- p.54 / Chapter IV.1.1 --- Overview --- p.54 / Chapter IV.1.2 --- Common Deletion Regions --- p.58 / Chapter IV.1.3 --- Common duplication Regions --- p.60 / Chapter IV.2. --- High-Resolution Allelotyping (Microsatellite Analysis) --- p.60 / Chapter IV.2.1. --- Overview of Results --- p.60 / Chapter IV.2.2. --- LOH profile of Individual Chromosome --- p.93 / Chapter IV.2.3. --- Overlapping Small Deletion Regions --- p.95 / Chapter CHAPTER V --- DISCUSSION --- p.97 / Chapter V.1. --- . General Outline --- p.98 / Chapter V.2. --- Chromosome 22 --- p.99 / Chapter V.3. --- Chromosome 17 --- p.102 / Chapter V.4. --- Chromosome 6 --- p.104 / Chapter V.5. --- Chromosome 16 --- p.105 / Chapter V.6. --- Chromosome 19 --- p.107 / Chapter V.7. --- Chromosome 20 --- p.108 / Chapter V.8. --- Chromosome 7 --- p.109 / Chapter V.9. --- Chromosome 12 --- p.110 / Chapter V.10. --- Chromosome 9 --- p.111 / Chapter V.11. --- Chromosome 5 --- p.112 / Chapter V.12. --- Chromosome 4 --- p.112 / Chapter V.13. --- Correlation of CGH with Allelotyping in the Study --- p.112 / Chapter V.14. --- Conclusion --- p.114 / Chapter CHAPTER VI --- LIMITATIONS OF THE STUDY --- p.115 / Chapter CHAPTER VII --- FUTURE STUDY --- p.116 / REFERENCES --- p.118

Lee, Kwan Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-113). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Content --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / List of papers published during the study --- p.xvi / Chapter Chapter 1 --- Introduction and Aim of Study --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Project objective and potential significances --- p.5 / Chapter Chapter 2 --- Literatures Review --- p.6 / Chapter 2.1 --- Cancer genetics and Tumor suppressor genes --- p.6 / Chapter 2.2 --- Epigenetic --- p.7 / Chapter 2.2.1 --- DNA methylation and promoter CpG island --- p.8 / Chapter 2.2.2 --- Establishment and maintenance of DNA methylation --- p.9 / Chapter 2.2.3 --- Transcriptional silencing by DNA hypermethylation --- p.9 / Chapter 2.3 --- Cancer epigenetic --- p.11 / Chapter 2.3.1 --- Hypomethylation of the cancer genome --- p.12 / Chapter 2.3.2 --- Hypermethylation in cancers --- p.12 / Chapter 2.3.3 --- Clinical relevance of cancer epigenetic --- p.13 / Chapter 2.4 --- Nasopharyngeal carcinoma --- p.14 / Chapter 2.4.1 --- NPC genetic and epigenetic --- p.15 / Chapter 2.5 --- 12p as a putative tumor suppressor locus --- p.16 / Chapter 2.5.1 --- Hematological malignancies associated with 12p loss --- p.17 / Chapter 2.5.2 --- Prostate cancer associated with 12p loss --- p.20 / Chapter 2.5.3 --- Lung cancer associated with 12p loss --- p.22 / Chapter 2.5.4 --- 12p deletion in other cancers --- p.23 / Chapter 2.6 --- 16q as a tumor suppressor locus --- p.24 / Chapter 2.6.1 --- Breast cancer and 16q --- p.25 / Chapter 2.6.2 --- Loss of 16q and prostate cancer --- p.26 / Chapter 2.6.3 --- Loss of 16q and hepatocellular carcinoma --- p.28 / Chapter 2.6.4 --- 16q deletion associated with other cancers --- p.29 / Chapter Chapter 3 --- Materials and Methods --- p.30 / Chapter 3.1 --- Cell lines and tissue samples --- p.30 / Chapter 3.1.1 --- Cell lines --- p.30 / Chapter 3.1.2 --- Maintenance of cell lines --- p.31 / Chapter 3.1.3 --- Drugs treatment of cell lines --- p.31 / Chapter 3.1.4 --- Normal tissues --- p.32 / Chapter 3.1.5 --- Total RNA extraction --- p.32 / Chapter 3.1.6 --- Genomic DNA extraction --- p.32 / Chapter 3.2 --- General techniques --- p.33 / Chapter 3.2.2 --- TA cloning and blunt end cloning of PCR product --- p.33 / Chapter 3.2.3 --- Transformation of cloning products to E. coli competent cells --- p.34 / Chapter 3.2.4 --- Preparation of plasmid DNA --- p.34 / Chapter 3.2.4.1 --- Mini-prep plasmid DNA extraction --- p.34 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.35 / Chapter 3.2.5 --- Measurement of DNA or RNA concentrations --- p.36 / Chapter 3.2.6 --- DNA sequencing of plasmid DNA and PCR products --- p.36 / Chapter 3.3 --- Preparation of reagents and medium --- p.37 / Chapter 3.4 --- Semi-quantitative Reverse-Transcription (RT) PCR expression analysis --- p.38 / Chapter 3.4.1 --- Reverse transcription reaction --- p.38 / Chapter 3.4.2 --- Semi-quantitative RT-PCR --- p.39 / Chapter 3.4.2.1 --- Primers design --- p.39 / Chapter 3.4.2.2 --- PCR reaction --- p.39 / Chapter 3.5 --- Methylation analysis of candidate genes --- p.40 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.41 / Chapter 3.5.2 --- Methylation-specific PCR (MSP) --- p.42 / Chapter 3.5.2.1 --- Bioinformatics prediction of CpG island --- p.42 / Chapter 3.5.2.2 --- Primers design --- p.42 / Chapter 3.5.2.3 --- PCR reaction --- p.42 / Chapter 3.5.3 --- Bisulfite Genomic Sequencing (BGS) --- p.43 / Chapter 3.5.3.1 --- Primers design --- p.43 / Chapter 3.5.3.2 --- PCR reaction --- p.44 / Chapter 3.6 --- Construction of expression vectors of candidate genes --- p.44 / Chapter 3.6.1 --- Construction of IRF8 expression vector --- p.44 / Chapter 3.6.2 --- Construction of PTPRO expression vector --- p.44 / Chapter 3.6.2.1 --- Experimental design --- p.44 / Chapter 3.6.2.2 --- PCR and cloning of PCR products --- p.46 / Chapter 3.6.2.3 --- Restriction digestion of cloning vectors and expression vector --- p.48 / Chapter 3.6.2.4 --- Ligation of cloning fragments --- p.48 / Chapter 3.7 --- Colony formation assay on monolayer culture --- p.48 / Chapter 3.8 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Identification of candidate TSGs in deleted regions --- p.50 / Chapter 4.1 --- Research plan --- p.50 / Chapter 4.2 --- Results --- p.50 / Chapter 4.2.1 --- Mapping of the deleted B AC clones on their chromosomal locations --- p.50 / Chapter 4.2.2 --- Identification of down-regulated genes in NPC by semi-quantitative RT-PCR analysis --- p.51 / Chapter 4.3 --- Discussion --- p.55 / Chapter Chapter 5 --- Tumor suppressor function studies of candidate TSGs --- p.60 / Chapter 5.1 --- Research plan --- p.60 / Chapter 5.2. --- IRF8 is the 16q candidate TSG --- p.60 / Chapter 5.2.1 --- Frequent silencing of IRF8 mRNA expression in multiple carcinomas --- p.60 / Chapter 5.2.2 --- Methylation status of IRF8 promoter region correlated with its transcriptional silencing --- p.62 / Chapter 5.2.3 --- Restoration of IRF8 expression by pharmacological and genetic demethylation --- p.65 / Chapter 5.2.4 --- IRF8 inhibited the anchorage dependent growth of tumor cells on monolayer culture --- p.67 / Chapter 5.2.5 --- Discussion --- p.68 / Chapter 5.3 --- PTPRO is the down-regulated target at 12pl3.2-12.3 tumor suppressor locus --- p.73 / Chapter 5.3.1 --- Frequent silencing of PTPRO in multiple carcinoma cell lines --- p.73 / Chapter 5.3.2 --- Frequent methylation of PTPRO promoter CpG island in multiple carcinoma cell lines correlated with its reduced expression --- p.74 / Chapter 5.3.3 --- Re-expression of PTPRO by pharmacological and genetic demethylation --- p.77 / Chapter 5.3.4 --- PTPRO inhibited the growth of tumor cells in vitro --- p.79 / Chapter 5.3.5 --- Discussion --- p.81 / Chapter 5.4 --- RERG is another candidate TSG in 12pl3.2 - 12.3 region --- p.87 / Chapter 5.4.1 --- Down-regulation of RERG mRNA expression in carcinoma cell line --- p.87 / Chapter 5.4.2 --- Hypermethylation of RERG promoter is a frequent event in multiple carcinomas --- p.88 / Chapter 5.4.3 --- Re-expression of RERG mRNA following pharmacological and genetic demethylation --- p.90 / Chapter 5.4.4 --- Discussion --- p.92 / Chapter Chapter 6 --- General discussion --- p.96 / Chapter Chapter 7 --- Summary --- p.101 / Reference --- p.103

Ng, Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-113). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of abbreviations --- p.ii / List of Tables --- p.iv / List of Figures --- p.v / List of Publications --- p.viii / Abstract in English --- p.ix / Abstract in Chinese --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Tumor suppressor genes (TSGs) and the modes of TSG inactivation during carcinogenesis --- p.1 / Chapter 1.2 --- Epigenetic modifications --- p.3 / Chapter 1.2.1 --- DNA methylation --- p.4 / Chapter 1.2.1a --- Establishment of DNA methylation patterns and DNA methyltransferases --- p.5 / Chapter 1.2.1b --- DNA hypermethylation and carcinogenesis --- p.6 / Chapter 1.2.1c --- Mechanism for gene silencing by CpG methylation --- p.6 / Chapter 1.2.1d --- DNA hypomethylation and carcinogenesis --- p.10 / Chapter 1.2.1e --- Loss of imprinting and carcinogenesis --- p.11 / Chapter 1.2.1f --- Potential factors leading to aberrant methylation patterns in cancers --- p.12 / Chapter 1.2.2 --- Deregulation of histone modifications and carcinogenesis --- p.14 / Chapter 1.2.3 --- Interplay between chromatin modifications and DNA methylation --- p.15 / Chapter 1.3 --- Identification of tumor suppressor genes (TSGs) --- p.17 / Chapter 1.4 --- Nasopharyngeal carcinoma as a cancer model of the current project --- p.18 / Chapter 1.5 --- Genetic and epigenetic changes in NPC --- p.19 / Chapter 1.6 --- Involvement of 5qll-ql2 and 14q32 in carcinogenesis --- p.22 / Chapter 1.6.1 --- Chromosome 5ql l-ql2 and carcinogenesis --- p.22 / Chapter 1.6.2 --- Chromosome 14q32 and carcinogenesis --- p.24 / Chapter 1.7 --- Clinical implications of epigenetics in cancers --- p.27 / Chapter Chapter 2 --- Aims of study and Research plan --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.34 / Chapter 3.1 --- Cell lines and Normal Tissues --- p.35 / Chapter 3.2 --- Routine cell line maintenance --- p.35 / Chapter 3.3 --- Drug treatments --- p.35 / Chapter 3.4 --- Total RNA extraction --- p.35 / Chapter 3.5 --- Genomic DNA extraction --- p.36 / Chapter 3.6 --- General techniques --- p.37 / Chapter 3.6.1 --- Gel electrophoresis --- p.37 / Chapter 3.6.2 --- DNA and RNA quantification --- p.37 / Chapter 3.6.3 --- LB medium and LB plate preparation --- p.38 / Chapter 3.6.4 --- Plasmid extraction --- p.38 / Chapter 3.6.4a --- Mini-scale preparation of plasmid DNA --- p.38 / Chapter 3.6.4b --- Large-scale preparation of endotoxin-free plasmid DNA --- p.39 / Chapter 3.6.5 --- DNA sequencing --- p.39 / Chapter 3.7 --- Reverse transcription-PCR (RT-PCR) --- p.40 / Chapter 3.7.1 --- Reverse transcription (RT) --- p.40 / Chapter 3.7.2 --- Semi-quantitative RT-PCR --- p.41 / Chapter 3.8 --- Methylation analysis --- p.42 / Chapter 3.8.1 --- Sodium bisulfite modification of DNA --- p.42 / Chapter 3.8.2 --- CpG island analysis --- p.42 / Chapter 3.8.3 --- Methylation-specific PCR (MSP) --- p.43 / Chapter 3.8.4 --- Bisulfite genomic sequencing (BGS) --- p.44 / Chapter 3.9 --- Construction of expression plasmids --- p.45 / Chapter 3.9.1 --- Construction of the MGC80-expressing vector --- p.45 / Chapter 3.9.2 --- Construction of the TUSC14-expressing vector --- p.46 / Chapter 3.10 --- Functional analyses --- p.47 / Chapter 3.10.1 --- Monolayer colony formation assay --- p.47 / Chapter 3.10.2 --- Soft agar assay --- p.48 / Chapter 3.11 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Results --- p.50 / Chapter 4.1 --- Identification of 5qll-ql2 and 14q32.2-q32.32 as frequently deleted regions in NPC by aCGH --- p.50 / Chapter 4.2 --- Identification of novel candidate TSGs at chromosome 5qll-ql2 through integrative genomics and epigenetics --- p.51 / Chapter 4.2.1 --- Expression profiling of the candidate genes at 5ql l-ql2 in NPC cell lines --- p.51 / Chapter 4.2.2 --- MGC80 as a target of study at 5ql2 --- p.54 / Chapter 4.2.2a --- Ubiquitous expression in normal human tissues and frequent down-regulation of MGC80 in multiple tumor cell lines --- p.54 / Chapter 4.2.2b --- Methylation analysis of MGC80 --- p.56 / Chapter 4.2.2c --- Restoration of MGC80 expression after pharmacologic and genetic demethylation --- p.59 / Chapter 4.2.2d --- Functional study of MGC80 in multiple carcinomas --- p.61 / Chapter 4.2.2e --- Discussion --- p.63 / Chapter 4.2.3 --- TUSC14 as a target of study at 5ql2 --- p.67 / Chapter 4.2.3a --- TUSC14 was broadly expressed in normal human tissues and frequently down-regulated in multiple tumor cell lines --- p.67 / Chapter 4.2.3b --- Methylation analysis of TUSCI4 --- p.69 / Chapter 4.2.3c --- Pharmacologic and genetic demethylation reactivated TUSC14 expression --- p.72 / Chapter 4.2.3d --- Functional study ofTUSC14 in multiple carcinomas --- p.74 / Chapter 4.2.3e --- Discussion --- p.76 / Chapter 4.3 --- Identification of candidate TSGs at chromosome 14q32 through integrative genomics and epigenetics --- p.80 / Chapter 4.3.1 --- Expression profiling of the candidate genes at 14q32 in NPC cell lines --- p.80 / Chapter 4.3.2 --- DLK1 as a target of study at 14q32 --- p.82 / Chapter 4.3.2a --- Expression analysis of DLK1 in normal tissues and NPC cell lines --- p.82 / Chapter 4.3.2b --- Methylation analysis ofDLKl in NPC --- p.83 / Chapter 4.3.2c --- Restoration of DLK1 expression after pharmacologic demethylation --- p.84 / Chapter 4.3.2d --- Functional study ofDLKl in NPC --- p.85 / Chapter 4.3.2e --- Discussion --- p.87 / Chapter Chapter 5 --- General discussion --- p.92 / Chapter Chapter 6 --- Summary --- p.99 / Chapter Chapter 7 --- Future study --- p.101 / Reference list --- p.103

Epithelial ovarian cancer (EOC) is difficult to detect in early stage disease, resulting in a high mortality rate. The molecular events underlying EOC development remain largely unknown. Chromosome 3 exhibits frequent deletions and rearrangements in EOC by cytogenetic analysis. In addition, loss of heterozygosity (LOH) mapping of matched ovarian tumour and constitutional DNA samples exhibits specific regions of chromosome 3 loss involving distinct regions: 3p25-p26, 3p24 and a region proximal to 3p14. Thus, chromosome 3p loss points to the location of tumour suppressor genes (TSG) implicated in tumourigenesis, based on Knudson's 'two-hit' model and the paradigm of the classical TSG. The dissertation hypothesis states at least one TSG implicated in EOC is located on chromosome 3p. A novel complementation approach based on the transfer of normal chromosome 3 fragments into OV-90, a tumourigenic EOC cell line harbouring LOH of the 3p arm, was used to generate functional evidence for chromosome 3p TSGs. Three hybrids exhibited complete suppression of tumourigenic potential based on the inability to form colonies in soft agarose, spheroids in cell culture, and tumours in nude mice xenograft models. While all hybrids had acquired various chromosome 3 regions, they all shared in common a 3p12-pcen interval, suggesting at least one common gene may have affected the suppression of tumourigenicity in the OV-90-derived hybrids. Twelve known/hypothetical genes mapping to 3p12-pcen region were characterized based on gene expression and mutation analysis following a classical model for TSG inactivation. To establish the relevance to EOC, gene expression of candidates was investigated in primary cultures of normal ovarian surface epithelial cells and both malignant serous and benign serous tumour samples. The gene expression and genetic analysis identified seven TSG candidates, none of which appeared to be mutated or transcriptionally silenced based on classical mechanisms of TSG inactivation in OV-90, thus suppression of tumourigenicity may have resulted from the functional complementation of one more haploinsufficient 3p12-pcen genes. Several genes (GBE1, VGLL3, ZNF654 ) appeared underexpressed in malignant tumours and these findings suggest the intriguing possibility that more than one 3p12-pcen gene was involved in the suppression of tumourigenicity in OV-90, and by extension, EOC.