Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

Bik-Yu Hui. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 187-203). / 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.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 4 uppsatser.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, February 2015 / Androgen Insensitivity Syndrome (AIS) is an X-linked disorder caused by mutations in the androgen receptor (AR) gene. The phenotype is variable and ranges from a complete feminine syndrome to simple gynecomastia. The phenotypes are described in terms of complete, partial and mild forms (CAIS, PAIS and MAIS). We describe novel and previously reported (recurrent) mutations in the AR gene for a family in which segregation of breast cancer (BC) and gynecomastia/MAIS is present. Methods: We studied a family of 16 members spanning four generations. Based on the presentation of symptoms, the family was divided into affected, unaffected, and control groups. Seven patients (six males diagnosed with MAIS and one female diagnosed with BC) formed the affected group, four genetically related individuals (two males and two females) formed the unaffected group and five genetically unrelated family members (one male and four females) served as controls. In each of these individuals, PCR amplification, cloning and the sequencing of exon 1 were carried out. Exons 2-8 were sequenced directly after PCR amplification. Exon 1 (CAG)n and (GGN)n repeats were classified according to their length: short (S) (n<23), long (L) (n>23) and wild type (WT) (n=23). Results: Part 1-The (CAG)n repeats varied among individuals and generations. In the 2nd generation, the unaffected male was S and the control female was WT. In the 3rd generation, three affected males were S, 2 of the controls were WT, one control was L and the other S. In the 4th generation, the 4 affected individuals were L, 1 of the unaffected was WT and the other 2 unaffected were L. Part 2- The (GGN)n variations also differed among individuals and generations. In the 2nd generation, the unaffected male and the control were S. In the 3rd generation, all three affected family members were S and among the controls, 1 was WT, 1 was L and 2 were S. In the 4th generation, 3 of the affected were S and one was WT and among the 3 unaffected, 2 were S and one was WT. Part 3- 30 unreported (novel) mutations as well as 13 recurrent (previously reported) mutations in exon 1 of the AR gene were identified. 17 novel and 5 reported mutations were identified in the affected group, 8 novel and 5 reported mutations, including one premature stop codon mutation, were identified in the related unaffected group and 7 novel and 4 reported mutations were found in the controls. Of the above-mentioned mutations, four mutations were identified in the activation function-1 (AF-1) domain of exon 1 in 4 members (3 affected: M-2, F-1 and 1 unaffected: F-1) of the family. All the point mutations identified were somatic in nature and were present in heterogeneous form i.e wild and mutant (mixture) as determined by cloning. The analysis of exons 2 through 8 revealed completely WT sequences. Conclusions: The (CAG)n and (GGN)n repeat analysis showed an indeterminate association with MAIS and BC in the family. Generation specific patterns of (CAG)n were detected and suggest generation specific modulation of the AR. Novel mutations including AF-1 region mutations were identified in exon 1. The disruption of the AF-1 domain may affect the transactivation activity of the AR.

by Kwong Joseph. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 142-164). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prostatic Secretory Proteins --- p.1 / Chapter 1.2 --- Rat Prostatic Secretory Proteins --- p.1 / Chapter 1.2.1 --- Prostatic Secretory Proteins in Ventral Prostate --- p.2 / Chapter 1.2.1.1 --- Prostatic Binding Protein (PBP) --- p.2 / Chapter 1.2.1.2 --- Androgen-Suppressed Proteins of Rat Ventral Prostate --- p.6 / Chapter 1.2.1.3 --- The 20-kDa Protein --- p.8 / Chapter 1.2.1.4 --- Spermine-Binding Proteins --- p.9 / Chapter 1.2.1.5 --- Prostatic Acid Phosphatase (PAP) --- p.10 / Chapter 1.2.2 --- Prostatic Secretory Proteins in Dorsal Prostate --- p.12 / Chapter 1.2.2.1 --- Dorsal Proteins I and II (DP I and DPII) --- p.12 / Chapter 1.2.2.2 --- Seminal Vesicle Secretion II (SVSII) --- p.14 / Chapter 1.2.2.3 --- Probasin --- p.16 / Chapter 1.2.3 --- Prostatic Secretory Proteins in Lateral Prostate --- p.18 / Chapter 1.3 --- Human Prostatic Secretion --- p.18 / Chapter 1.4 --- Human Prostatic Secretory Proteins --- p.18 / Chapter 1.4.1 --- Prostatic Acid Phosphatase (PAP) --- p.19 / Chapter 1.4.2 --- Prostate Specific Antigen (PSA) --- p.22 / Chapter 1.4.2.1 --- Molecular Biology of PSA --- p.22 / Chapter 1.4.2.2 --- Synthesis of PSA --- p.23 / Chapter 1.4.2.3 --- Kallikrein Gene Family --- p.23 / Chapter 1.4.2.4 --- Physiological Function of PSA --- p.24 / Chapter 1.4.2.5 --- PSA as an Immunohistochemical Marker --- p.25 / Chapter 1.4.2.6 --- PSA is not a Prostate-Specific Molecule --- p.26 / Chapter 1.4.3 --- Prostatic Secretory Protein of 94 Amino Acids (PSP94) --- p.27 / Chapter 1.4.3.1 --- Nucleotide Sequence of the PSP94 cDNA --- p.28 / Chapter 1.4.3.2 --- Amino Acid sequence of PSP94 --- p.28 / Chapter 1.4.3.3 --- Biological Properties of PSP94 --- p.29 / Chapter 1.4.3.4 --- Physiological Roles of PSP94 --- p.31 / Chapter 1.4.3.5 --- PSP94 and Its mRNA in Other Non-Prostatic Tissue --- p.31 / Chapter 1.4.3.6 --- PSP94 as a Tumor Marker of Prostate Cancer --- p.32 / Chapter 1.4.3.7 --- Homologous Proteins of PSP94 --- p.34 / Chapter 1.5 --- Aim of Study --- p.35 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and Supply of Noble Rat --- p.37 / Chapter 2.2 --- Chemicals --- p.37 / Chapter 2.3 --- Bilateral Ochidectomy of Animals --- p.37 / Chapter 2.4 --- Androgen Replacement --- p.38 / Chapter 2.5 --- Hormonal and Drug Treatments on Castrated Animals --- p.38 / Chapter 2.6 --- Induction of Prostatic Intraepithelial Neoplasia in Noble Rat Prostate Gland by Long-Term Treatment with Steroids --- p.39 / Chapter 2.6.1 --- Preparation of Steroid Hormone-Filled Silastic® Tubings --- p.39 / Chapter 2.6.2 --- Surgical Implantation of Silastic® Tubings --- p.39 / Chapter 2.6.3 --- Protocols of Hormonal Treatments --- p.40 / Chapter 2.7 --- Androgen-Dependent Rat Dunning Prostatic Adenocarcinoma --- p.40 / Chapter 2.8 --- Androgen-Independent Prostatic Carcinoma Line (AIT) of Noble Rat --- p.41 / Chapter 2.9 --- Plasmids --- p.41 / Chapter 2.10 --- Restriction Enzyme Digestions of pLvB10 and cM-403 --- p.42 / Chapter 2.11 --- Amplification of Rat SVSII cDNA Fragment by RT-PCR and Subcloning --- p.42 / Chapter 2.12 --- Purification of DNA Fragment from Agarose Gel --- p.43 / Chapter 2.13 --- Subcloning of DNA into Vector --- p.44 / Chapter 2.14 --- Tissue Preparation for In-situ Hybridization --- p.47 / Chapter 2.15 --- Synthesis of Digoxigenin (DIG)-Labeled RNA Probe --- p.47 / Chapter 2.16 --- In-situ Hybridization --- p.48 / Chapter 2.17 --- Total RNA Extraction --- p.50 / Chapter 2.18 --- Northern Blotting Analysis --- p.51 / Chapter 2.19 --- Primers and Cycling Conditions --- p.53 / Chapter 2.20 --- Reverse Transcription Polymerase Chain Reaction (RT-PCR) --- p.54 / Chapter 2.21 --- Southern Blotting Analysis --- p.56 / Chapter 2.21.1 --- Southern Blotting --- p.56 / Chapter 2.21.2 --- Preparation of DIG-dUTP Labeled Rat PSP94 cDNA Probe --- p.56 / Chapter 2.21.3 --- Hybridization --- p.57 / Chapter 2.22 --- Restriction Mapping --- p.58 / Chapter 2.23 --- Semi-Quantitative RT-PCR --- p.59 / Chapter 2.24 --- Statistical Analysis --- p.59 / Chapter 2.25 --- "Protein Extraction, SDS-PAGE and Western Blotting Analysis" --- p.60 / Chapter 2.26 --- Immunohistochemistry --- p.63 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Subcloning of DNAs into Vector --- p.65 / Chapter 3.1.1 --- Subcloning of 18s Ribosomal RNA cDNA Fragment --- p.65 / Chapter 3.1.2 --- Subcloning of Probasin cDNA Fragment --- p.66 / Chapter 3.1.3 --- Subcloning of SVSII cDNA Fragment --- p.66 / Chapter 3.1.4 --- Restriction Enzyme Mapping for PCR Product of SVSII --- p.67 / Chapter 3.2 --- Detection of mRNA and Protein Expression of PSP94 in Normal Rat Prostates --- p.68 / Chapter 3.2.1 --- In-situ Hybridization --- p.68 / Chapter 3.2.2 --- Northern Blotting --- p.68 / Chapter 3.2.3 --- RT-PCR Amplification --- p.69 / Chapter 3.2.4 --- Immunohistochemistry --- p.69 / Chapter 3.2.5 --- Western Blotting --- p.70 / Chapter 3.3 --- Detection of mRNA Expression of Probasin and SVSII in Normal Rat Prostates --- p.71 / Chapter 3.3.1 --- In-situ Hybridization --- p.71 / Chapter 3.3.2 --- RT-PCR Amplification --- p.71 / Chapter 3.4 --- "Androgen Regulation of PSP94,Probasin and SVSII mRNA Expression" --- p.72 / Chapter 3.4.1 --- In-situ Hybridization --- p.72 / Chapter 3.4.2 --- "Relative Expression Levels of PSP94, Probasin and SVSII mRNA in Normal, Castrated and Androgen Replaced Rat Lateral Prostates as Measured by a Semiquantitative RT-PCR Method" --- p.73 / Chapter 3.4.2.1 --- Determination of Exponential Range of PCR --- p.73 / Chapter 3.4.2.2 --- Semi-Quantitative RT-PCR --- p.74 / Chapter 3.4.3 --- Western Blot Analysis --- p.75 / Chapter 3.5 --- "Effect of Steroid Hormones and Zinc on the PSP94, Probasin and SVSII Expressions in Castrated Rat Prostates" --- p.76 / Chapter 3.5.1 --- Semi-Quantitative RT-PCR --- p.76 / Chapter 3.5.2 --- Western Blot Analysis --- p.77 / Chapter 3.6 --- "Detection of PSP94, Probasin and SVSII mRNA Expression in Dysplastic and Neoplastic Rat Prostates" --- p.78 / Chapter 3.6.1 --- "Detection of PSP94, Probasin and SVSII mRNA Expression in T+E2-Induced Prostatic Intraepithelial Neoplasia (PIN) of the Lateral Prostate of Noble Rats by In-situ Hybridization" --- p.78 / Chapter 3.6.2 --- "Detection of PSP94,Probasin and SVSII mRNA Expression in Dunning Tumor and AIT Prostatic Tumor" --- p.79 / Chapter 3.6.2.1 --- In-situ Hybridization --- p.79 / Chapter 3.6.2.2 --- RT-PCR Amplification --- p.79 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Specific Expression of PSP94 in the Lateral Lobe of Rat Prostate --- p.114 / Chapter 4.2 --- Androgen Regulation of PSP94 --- p.118 / Chapter 4.2.1 --- Molecular Mechanism of Androgen Action --- p.118 / Chapter 4.2.2 --- Androgen Regulation of PSP94 in Rat Lateral Prostate --- p.121 / Chapter 4.3 --- "Effect of Steroid Hormones and Zinc on the PSP94, Probasin and SVSII Expressions in Castrated Rat Lateral Prostate" --- p.126 / Chapter 4.4 --- "Detection of PSP94, Probasin, SVSII mRNA Expression in Dysplastic and Neoplastic Rat Prostates" --- p.133 / Chapter 4.5 --- Gene Therapy --- p.139 / Chapter Chapter 5 --- Conclusions --- p.141 / References --- p.142 / Appendixes --- p.165

by Ng Mei Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 173-183). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abbreviations --- p.3 / Abstracts --- p.4 / List of figures and tables --- p.7 / Contents Page No / General Introduction --- p.13 / Chapter CHAPTER ONE --- Biological Characterization of A431 Cells & SiHa Cells Under Different Microenvironments --- p.16 / Chapter 1.1 --- Introduction --- p.17 / Chapter 1.1.1 --- Microenvironment Surrounding Tumor Cells --- p.18 / Chapter 1.1.2 --- Hypoxic Environment --- p.20 / Chapter 1.1.2.1 --- The Hypoxic Chamber --- p.20 / Chapter 1.1.3 --- Reoxygenation --- p.22 / Chapter 1.1.4 --- Acidic Environment --- p.23 / Chapter 1.1.5 --- Glucose Depletion --- p.24 / Chapter 1.1.6 --- Irradiation --- p.25 / Chapter 1.2 --- Objectives --- p.26 / Chapter 1.3 --- Materials and Methods --- p.27 / Chapter 1.3.1 --- Materials --- p.27 / Chapter 1.3.2 --- Methods --- p.29 / Chapter 1.3.2.1 --- Cell Lines --- p.29 / Chapter 1.3.2.2 --- The Working Procedure for the Hypoxic Chamber --- p.30 / Chapter 1.3.2.3 --- "Aerobic, Hypoxic and Reoxygenated Conditions" --- p.33 / Chapter 1.3.2.4 --- Acidic Condition --- p.35 / Chapter 1.3.2.5 --- Glucose Depleted Condition --- p.36 / Chapter 1.3.2.6 --- Gamma-Irradiation --- p.37 / Chapter 1.3.2.7 --- Analysis of the Growth Pattern by MTT Assay and Cell Counting --- p.38 / Chapter 1.3.2.8 --- Cell Cycle Analysis --- p.39 / Chapter 1.3.2.9 --- Western Blot Analysis --- p.40 / Chapter 1.3.2.10 --- DNA Fragmentation Analysis --- p.42 / Chapter 1.4 --- Results --- p.43 / Chapter 1.4.1 --- Cell Proliferation Profile by MTT Assay --- p.43 / Chapter 1.4.1.1 --- Proliferation of cells under hypoxia --- p.43 / Chapter 1.4.1.2 --- Proliferation of cells under acidic pH environments --- p.49 / Chapter 1.4.1.3 --- Proliferation of cells under glucose depleted environment --- p.52 / Chapter 1.4.2 --- Distribution of cell cycles under different micro environments --- p.54 / Chapter 1.4.3 --- General Protein Expression Pattern by Western Blot Analysis --- p.57 / Chapter 1.4.4 --- Detection of Apoptosis by DNA Fragmentation Assay --- p.59 / Chapter 1.5 --- Discussion --- p.58 / Chapter CHAPTER TWO --- REACTION KINETICS OF A431 CELLS AND SiHa CELLS INDUCED BY EGF --- p.71 / Chapter 2.1 --- Introduction --- p.72 / Chapter 2.1.1 --- Structure of EGF and EGFR --- p.74 / Chapter 2.1.2 --- EGF Signaling Pathway --- p.76 / Chapter 2.2 --- Objectives --- p.79 / Chapter 2.3 --- Materials and Methods --- p.80 / Chapter 2.3.1 --- Materials --- p.80 / Chapter 2.3.2 --- Methods --- p.82 / Chapter 2.3.2.1 --- Cell Lines --- p.82 / Chapter 2.3.2.2 --- EGF Sensitivity Assay --- p.83 / Chapter 2.3.2.3 --- Combination Effect of Hypoxia and EGF --- p.83 / Chapter 2.3.2.4 --- Early Kinetics Analysis by Low EGF Concentration Treatment --- p.84 / Chapter 2.3.2.5 --- Late Kinetics Analysis by High EGF Concentration Treatment --- p.85 / Chapter 2.4 --- Results --- p.86 / Chapter 2.4.1 --- Sensitivity of A431 cells and SiHa cells to EGF by MTT Assay --- p.86 / Chapter 2.4.2 --- Early/Late Kinetics of EGF induced protein tyrosine phosphorylation Pattern --- p.90 / Chapter 2.4.3 --- Raf protein expression --- p.96 / Chapter 2.4.4 --- EGFR expression level --- p.100 / Chapter 2.5 --- Discussions --- p.104 / Chapter CHAPTER THREE --- IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN A431 CELLS BY DIFFERENTIAL DISPLAY UNDER DIFFERENT TUMOR MICROENVIRONMENTS --- p.107 / Chapter 3.1 --- Introduction --- p.108 / Chapter 3.2 --- Materials and Methods --- p.112 / Chapter 3.2.1 --- Materials --- p.112 / Chapter 3.2.2 --- Methods --- p.114 / Chapter 3.2.2.1 --- Spheroid Cells --- p.114 / Chapter 3.2.2.2 --- Identification of Differentally Expressed Genes by RT-PCR --- p.117 / Chapter 3.2.2.3 --- Ligation and Cloning of Differentially Expressed cDNA --- p.120 / Chapter 3.2.2.4 --- Screening and Sequencing of the cDNA Inserts --- p.121 / Chapter 3.2.2.5 --- Northern Blot Analysis --- p.123 / Chapter 3.3 --- Results --- p.124 / Chapter 3.4 --- Discussions --- p.161 / GENERAL CONCLUSION --- p.165 / REFERENCES --- p.167

by Choy Kwong Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 120-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

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

Lee Tin Lap. / "January 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 161-185). / 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.