Wang, Yajun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 118-126). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / Abbreviations and Symbols --- p.xiii / List of Publications and Sequence Submissions during the Study --- p.xv / Chapter Chapter One: --- General Introduction --- p.1 / Chapter Chapter Two: --- Literature Review --- p.8 / Chapter 2.1 --- DNA methylation --- p.8 / Chapter 2.1.1 --- Epigenetic changes --- p.8 / Chapter 2.1.2 --- Differential methylation pattern in normal and tumor cells --- p.10 / Chapter 2.2 --- TSGs --- p.13 / Chapter 2.2.1 --- "Cancer initiation, progression and cancer genes" --- p.13 / Chapter 2.2.2 --- TSGs could be inactivated through promoter hypermethylation --- p.14 / Chapter 2.3 --- NPC --- p.17 / Chapter 2.3.1 --- Epidemiology ofNPC --- p.18 / Chapter 2.3.2 --- Molecular genetic and epigenetic studies ofNPC --- p.19 / Chapter 2.3.3 --- NPC and chromosome 11q --- p.21 / Chapter 2.4 --- ESCC --- p.21 / Chapter 2.4.1 --- Epidemiology of ESCC --- p.22 / Chapter 2.4.2 --- Genetic and epigenetic studies of ESCC --- p.23 / Chapter 2.4.3 --- ESCC and chromosome 11q --- p.24 / Chapter 2.5 --- Chromosome 11q and other carcinomas --- p.24 / Chapter 2.5.1 --- Breast cancer --- p.24 / Chapter 2.5.2 --- Ovarian cancer --- p.25 / Chapter 2.5.3 --- Neuroblastoma --- p.26 / Chapter 2.5.4 --- Melanoma --- p.27 / Chapter 2.5.5 --- Multiple myeloma --- p.27 / Chapter 2.5.6 --- Lung Cancer --- p.27 / Chapter 2.6 --- Important candidate genes located at the project study 1 lq region --- p.28 / Chapter 2.6.1 --- ETS1 --- p.28 / Chapter 2.6.2 --- FLI1 --- p.29 / Chapter 2.6.3 --- P53AIP1 --- p.30 / Chapter 2.6.4 --- RICS --- p.30 / Chapter 2.6.5 --- BARX2 --- p.30 / Chapter 2.6.6 --- ST14 --- p.32 / Chapter 2.6.7 --- ADAMTS8 --- p.33 / Chapter 2.6.8 --- ADAMTS15 --- p.35 / Chapter 2.6.9 --- HNT --- p.36 / Chapter 2.6.10 --- OPCML --- p.36 / Chapter Chapter Three: --- Materials and Methods --- p.37 / Chapter 3.1 --- Cell lines and primary tumor samples --- p.37 / Chapter 3.2 --- Cell line demethylation treatment --- p.38 / Chapter 3.3 --- DNA and RNA extraction from cell lines and tissues --- p.39 / Chapter 3.4 --- Semiquantitative RT-PCR --- p.41 / Chapter 3.5 --- DNA bisulfite treatment --- p.42 / Chapter 3.6 --- Promoter analysis and identification of 5' CpG islands of target genes --- p.45 / Chapter 3.7 --- Methylation-Specific PCR (MSP) --- p.45 / Chapter 3.8 --- Bisulfite Genomic Sequencing (BGS) --- p.46 / Chapter 3.8.1 --- BGS PCR reaction --- p.46 / Chapter 3.8.2 --- TA cloning of the PCR products into the sequencing vector --- p.47 / Chapter 3.8.3 --- Plasmid mini-preparation on 96-well plate --- p.48 / Chapter 3.8.4 --- Plasmid sequencing --- p.49 / Chapter 3.9 --- Homozygous deletion detection --- p.50 / Chapter 3.10 --- Construction of expression plasmids --- p.51 / Chapter 3.10.1 --- The strategy of full length cDNA cloning --- p.51 / Chapter 3.10.2 --- Obtaining of full length covered cDNA by cloning PCR --- p.53 / Chapter 3.10.3 --- Ligation and transformation --- p.54 / Chapter 3.10.4 --- Mini preparation of plasmid in Eppendorf tubes --- p.54 / Chapter 3.10.5 --- Verification of correct inserts in the plasmid --- p.55 / Chapter 3.10.6 --- Subcloning --- p.55 / Chapter 3.10.7 --- Bacteria storage --- p.57 / Chapter 3.11 --- Colony formation assays (CFA) --- p.57 / Chapter 3.11.1 --- Midiprep of the transfection grade plasmid --- p.57 / Chapter 3.11.2 --- Transfection --- p.58 / Chapter 3.11.3 --- Selection of the transfected cells with G418 --- p.59 / Chapter 3.11.4 --- Colony staining --- p.60 / Chapter 3.12 --- Statistical analysis --- p.60 / Chapter Chapter Four: --- Results --- p.61 / Chapter 4.1 --- Narrow down the candidate genes for further study --- p.61 / Chapter 4.1.1 --- Define the study chromosome region --- p.61 / Chapter 4.1.2 --- Database search of all candidate genes --- p.61 / Chapter 4.1.3 --- Transcriptional expression analysis of the candidate genes --- p.63 / Chapter 4.1.4 --- Selection of the genes with tumor specific expression downregulation for further intensive study --- p.64 / Chapter 4.2 --- Further characterization of ADAMTS8 --- p.69 / Chapter 4.2.1 --- Tissue transcriptional expression panel --- p.69 / Chapter 4.2.2 --- Semiquantitative RT-PCR results in tumor cell lines --- p.70 / Chapter 4.2.3 --- Promoter CpG island identification and promoter methylation study --- p.70 / Chapter 4.2.4 --- Transcription reactivation by demethylation treatment --- p.72 / Chapter 4.2.5 --- High resolution promoter methylation analysis by BGS --- p.72 / Chapter 4.2.6 --- Detection of homozygous deletion --- p.73 / Chapter 4.2.7 --- Analysis of ADAMTS8 promoter methylation in clinical samples --- p.74 / Chapter 4.2.8 --- ADAMTS8 full length cDNA cloning --- p.74 / Chapter 4.2.9 --- Colony formation assay --- p.75 / Chapter 4.3 --- Further characterization of HNT --- p.80 / Chapter 4.3.1 --- Tissue transcriptional expression panel --- p.80 / Chapter 4.3.2 --- Semiquantitative RT-PCR results in tumor cell lines --- p.80 / Chapter 4.3.3 --- Promoter CpG island identification and promoter methylation study --- p.81 / Chapter 4.3.4 --- Transcription reactivation by demethylation treatment --- p.82 / Chapter 4.3.5 --- HNT full length cDNA cloning --- p.82 / Chapter 4.4 --- Further characterization of BARX2 --- p.87 / Chapter 4.4.1 --- Tissue transcriptional expression panel --- p.87 / Chapter 4.4.2 --- Semiquantitative RT-PCR results in tumor cell lines --- p.87 / Chapter 4.4.3 --- Promoter CpG island identification and promoter methylation study --- p.88 / Chapter 4.4.4 --- Transcription reactivation by demethylation treatment --- p.89 / Chapter 4.4.5 --- BARX2 full length cDNA cloning --- p.89 / Chapter 4.5 --- Further study of other downregulated genes --- p.92 / Chapter 4.5.1 --- FLII --- p.92 / Chapter 4.5.2 --- ADAMTS15 --- p.94 / Chapter 4.5.3 --- P53AIP1 --- p.97 / Chapter Chapter Five: --- Discussion --- p.100 / Reference List --- p.118 / Appendix I: Reagents Preparation Recipe --- p.127 / Appendix II: PCR Primers for cDNA Cloning --- p.129

In brief, mRNA expression profiling of candidate genes in each locus was performed using semi-quantitative RT-PCR in a panel of ESCC and NPC cell lines, normal tissues and immortalized epithelial cell lines. Genes downregulated in cancer cells but with high expression in normal tissues and immortalized epithelial cells were subjected to promoter methylation analysis using methylation-specific PCR (MSP), bisulfite genomic sequencing (BGS) and pharmacological demethylation treatment. Genes with tumor-specific downregulation and methylation were further selected as candidates and their tumor suppressive roles were verified via functional studies. / In conclusion, RAB39 and WDRX, epigenetically silenced in multiple cancer cell lines, were identified as novel TSG candidates in this study. Meanwhile, the tumor suppressive functions of ADAMTS8 were further validated, proving the efficiency of this integrative approach. Further study on these novel TSG candidates may help to elucidate the detailed molecular mechanisms for ESCC and NPC, and provide novel therapeutic targets and biomarkers. / In this study, RAB39 and WDRX were identified as candidate TSGs in 11q22.3 and 15q21.3, respectively. Both genes were broadly expressed in normal tissues and immortalized epithelial cell lines, but significantly downregulated and methylated in multiple cancer cell lines. Demethylation treatment with 5-Aza-2'-deoxycytidine restored their mRNA expression, indicating that CpG methylation directly contributed to their transcriptional inactivation. Methylation of RAB39 and WDRX was detected in primary ESCC and NPC, but rarely observed in normal tissues, implicating that their tumor-specific methylation might be used as biomarkers. Ectopic expression of both genes significantly inhibited the clonogenicity of multiple cancer cell lines, supporting their potential roles as functional TSGs. Moreover, WDRX repressed WNT/beta-catenin signaling, underscoring a possible anti-tumorigenic mechanism for it. In addition, ADAMTS8 was revealed to inhibit clonogenicity of NPC and ESCC cell lines, acting as a negative modulator for ERK pathway and a potential pro-apoptotic metalloprotease. / Inactivation of tumor suppressor genes (TSGs) contributes to the genesis of cancers including esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC), two prevalent causes of death in Hong Kong. Apart from genetic abnormalities, epigenetic disruptions including CpG methylation represent another major mechanism for TSG inactivation. Promoter methylation of multiple TSGs was detected in different cancer types, suggesting that it could be utilized as therapeutic target or biomarker for disease diagnosis and prognosis. / TSGs are often located at frequently deleted chromosomal regions and subjected to tumor-specific methylation, making it possible to use an integrative epigenetic and genomic approach combining array comparative genomic hybridization (aCGH) with epigenetic profiling to screen for novel TSGs. Previous aCGH revealed that several loci in 11822.3, 15q14, 15q21.1 and 15q21.3 underwent frequent copy number loss in ESCC cell lines. Loss of heterozygosity (LOH) of these regions was also reported in other cancers, indicating that TSGs might reside within them. The aim of this study was thus to identify the candidate TSGs in these loci and study their anti-tumorigenic roles. In addition, the tumor suppressive function of ADAMTS8, a silenced 11q25 candidate TSG previously identified in our lab via this approach, was also studied. / Li, Jisheng. / Adviser: Qian Tao. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 136-159). / 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. / Abstract also in Chinese.

First, expression profiling of ZNFs with CpG islands at 10 clusters of Chr19 was examined in a panel of NPC and ESCC cell lines by semi-quantitative RT-PCR, with adult normal tissues - larynx and esophagus as controls. Several down-regulated genes were identified, and I further focused on 5 candidates: ZNF382, ZNF545, ZFP30, ZNFT1 and ZNFT2. These genes were frequently downregulated in NPC, ESCC, lung, gastric, colon and breast carcinomas. Their promoters were frequently methylated in multiple downregulated cell lines but less in non-tumor cell lines as revealed by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS). Their expression could be restored by pharmacologic or genetic demethylation, suggesting that DNA methylation was directly involved in their silencing. The frequent methylation of these genes indicated they could act as potential biomarkers. / In conclusion, several novel candidate TSGs epigenetically silenced in tumor cells were identified in this study. Their downregulation by promoter methylation was tumor-specific, which could be use as epigenetic biomarkers for diagnosis. / More functional studies were done for ZNF382 and ZNF545, I found that ectopic expression of ZNF382 and ZNF545 in tumor cells lacking endogenous expression could inhibit tumor cell clonogenicity, proliferation and induce apoptosis. I found that ZNF382 suppressed tumorigenesis through mediating heterochromatin formation, as ZNF382 was revealed to be co-localized and interacts with heterochromatin protein. For ZNF545, I found that it is a transcriptional repressor. I further showed that ZNF545 was located in the nucleus and sequestered in the nucleolus. ZNF545 could inhibit tumorigenesis at least partially through downregulating the transcription of target genes or regulating nucleolus function such as ribosome biogenesis. / The development of a tumor from a normal cell is a complex and multi-step process. A large number of oncogenes, tumor suppressor genes (TSGs) and signal transduction pathways are involved in this process. Tumor-specific methylation of TSGs in multiple tumors indicated that it could be used as epigenetic biomarker for molecular diagnosis and therapeutics. / The functions of KRAB-containing proteins are critical to cell differentiation, proliferation, apoptosis and neoplastic transformation. A large number of ZNF genes are located in 10 clusters at chromosome 19. Some of the KRAB-ZNF may function as potential TSGs with epigenetic alterations. Thus, I try to identify silenced novel KRAB-ZNF candidate TSGs through screening chromosome 19. / Cheng, yingduan. / Adviser: Tao Qian. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 110-136). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.