Thesis (MSc.(Biochemistry)) -- University of Limpopo, 2021 / Drug discovery from medicinal plants continues to play an important role in the development of anticancer agents, this is because medicinal plants are reservoirs of bioactive compounds that exert a plethora of pharmacological effects on human beings. This study aimed to analyse the phytochemical constituents of the Momordica cardiospermoides crude acetone and methanol leaf extracts as well as investigate their potential anti-metastatic effects on the MDA-MB-231 breast cancer cell line. Momordica cardiospermoides leaves were extracted with absolute methanol or acetone to produce crude methanol and acetone extracts, respectively. The extracts were then screened and analysed for phytochemicals using thin layer chromatography, qualitative and quantitative phytochemical tests, and their antioxidant activity was determined using the quantitative 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity assay. The fingerprint profiles of the M. cardiospermoides leaf extracts revealed that compounds of the acetone extracts were optimally separated in the nonpolar mobile phase (TAE), whereas those of the methanol extract separated best in the polar mobile phase (EMW), thereby suggesting that the crude acetone and methanol extracts had more non-polar and polar compounds present, respectively. Furthermore, the qualitative phytochemical analysis indicated the presence of various phytochemicals such as flavonoids, steroids, coumarins, and tannins in both plant extracts, however, saponins were found present in the methanol extract and not in the acetone extract. Moreover, quantification of major phytochemicals revealed that the acetone extract had the highest total phenolic content (23.0683 mg GAE/g), total tannin content (22.0442 mg GAE/g) and total flavonoid (32.6933 mg QE/g) content as compared to the methanol extract (14.2349 mg GAE/g, 11.3164 mg GAE/g and 7.692 mg QE/g respectively). The DPPH free radical scavenging activity assay revealed that the extracts exhibited an increase in percentage inhibition/ DPPH scavenging effect, with an increase in extract concentration. The results also revealed that the acetone extract possessed a higher radical scavenging activity as compared to the methanol extract. These results are in correlation with the quantitative analysis of the extracts, as all the major phytochemicals found in higher amounts in the acetone extract have antioxidant properties. The extracts were then assessed in vitro for their cytotoxic effects on MDA MB-231 breast cancer cells and HEK 293 cells using the cell count and viability assay and the results obtained revealed a concentration-dependent decrease in the viability of MDA-MB-231 cells at 24 hours of treatment with either the acetone or methanol extract. Comparatively, treatment of HEK 293 cells with the acetone extract resulted in a significant decrease in the percentage of viable cells, whereas treatment with the methanol extract had no significant effect on the viability of HEK 293 cells, as the percentage of viable cells was maintained at 85–98% at 24 hours of treatment. These results also revealed that the methanol extract is more selective to cancer cells in comparison to the acetone extract, suggesting that the methanol extract is a better antineoplastic candidate. The mode of cell death induced by the methanol or acetone extracts was assessed using the acridine orange and ethidium bromide dual staining assay and the annexin V and dead cell kit. The results from the acridine orange/ethidium bromide dual staining assay showed that both extracts induced nuclei and cellular morphological changes in a concentration-depended manner, at 24 hours of treatment. Moreover, the annexin V and dead assay kit results revealed that the acetone extract induced necrotic cell death, while the methanol extract induced apoptotic cell death. Since the acetone extract was shown to be non-selective towards normal cells and induced necrotic cell death, it was discontinued for further assays. The effect of the methanol extract on MDA-MB-231 cell migration and attachment was determined using the wound healing assay and the adhesion assay. The results revealed that treatment with 150 or 300 µg/ml significantly suppressed MDA-MB-231 cell migration, associated with serpin E1 downregulation and TIMP-1 upregulation, at 24 hours of treatment. Moreover, treatment with the methanol extract also significant inhibited MDA-MB-231 cell adhesion in a concentration-dependent manner, as evident by the decrease in the number of crystal violet stained cells. The effect of the methanol extract on the expression of matrix metalloproteinase-2 and -9 was assessed using western blotting, and the results revealed that the extract significantly downregulated the expression of both MMP-2 and -9, suggesting that the methanol extract has inhibitory effects on MDA-MB-231 cell invasion. The human angiogenesis antibody array kit was then used to determine the effect of the extract on the expression of angiogenesis-related proteins. Treatment with 150 or 300 µg/ml of the extract significantly upregulated the expression levels of tissue inhibitor of metalloproteinases (TIMP) -1 and thrombospondin-1 in a concentration-dependent manner. The results also revealed a significant downregulation in the expression of serpin E1, in a concentration-dependent manner, in comparison to the untreated control. However, the expression of uPA, VEGF, and IGFBP-1, 2 and -3 was upregulated following treatment with 150 and 300 µg/ml of the extract. In conclusion, the current study demonstrated the potential of M. cardiospermoides crude methanol extract as an effective anti-metastatic agent or a source of compounds with anti-metastatic properties / South African Medical Research Council (SAMRC) Research Capacity Development Initiative and National Research Foundation (NRF)

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

Gene expression profiles obtained by means of semi-quantitative RT-PCR showed that both PPP1R1B and PPP1R3C were frequently silenced in multiple carcinomas. Bisulfite treated tumor DNA was subjected to Methylation-specific PCR (MSP) using primers flanking across the ∼130bp CpG island of the promoter of the particular gene of interest. It was revealed that PPP1R1B and PPP1R3C gene silencing in the carcinoma cell lines were due to promoter CpG island hypermethylation. Such claim was further confirmed by bisulfite genomic sequencing (BGS). Treatment with 5' azacytidine and TSA restored PPP1R1B and PPP1R3C expression in carcinoma cells through demethylating the hypermethylated promoter. In terms of cancer growth inhibition, ectopic expression of PPP1R1B and PPP1R3C could significantly inhibit the proliferation of carcinoma cell lines by 40--50% and 50--60%, respectively, according to the result of anchorage-dependent colony formation assay. / Overall, we believed that PPP1R1B and PPP1R3C are the putative tumor suppressor genes in which their expression silencing through promoter CpG island hypermethylation may be strongly linked to the development of cancer. / Protein Phosphatase 1 regulatory subunits are a family of small molecules which define the substrate specificity and subcellular localization of protein phosphatase-1 upon their interactions. Downregulation of Protein Phosphatase 1 regulatory subunits were often associated with tumor initiation and progression, for example, ASPP family (PPP1R13A and PPP1R13B). In the present study, PPP1R1B and PPP1R3C were identified in which their tumor suppressor functions had been investigated. / Reduction in the level of p-ser473 Akt and p-ser552 beta-catenin could be observed when PPP1R1B expression was restored in respective carcinoma cells. In addition, the transcription activity of AP-1 decreased in the presence of full-length PPP1R1B expression as determined by Dual-Luciferase reporter assay system. Ectopic expression of PPP1R3C increased the amount of inactive pSer9-GSK-3beta as shown in the western blot analysis and a concomitant increased in p53 level was observed in colorectal carcinoma HCT116 cells. Transcription activity of NF-kappaB in HCT116 cells was increased but decreased in KYSE150 cells (ESCC) in the presence of PPP1R3C expression. Subcellular localization study using the GFP-fusion protein revealed that PPP1R1B protein was distributed throughout the cytoplasm while PPP1R3C protein was mainly localized around the nuclear membrane. / Leung, Ching Hei. / Adviser: Tak Cheung Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 160-183). / 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.

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.

Liu, Xin. / "December 2009." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 119-126). / Abstracts in English and Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.vi / List of abbreviations --- p.vii / List of Tables List of Figures --- p.X xii / List of Publications --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Gastric cancer epidemiology and etiology --- p.1 / Chapter 1.2 --- Molecular carcinogenesis --- p.4 / Chapter 1.3 --- Tumor suppressor gene and the modes of tumor suppressor gene inactivation --- p.4 / Chapter 1.4 --- DNA methylation and carcinogenesis --- p.8 / Chapter 1.5 --- Identification of tumor suppressor genes --- p.15 / Chapter 1.6 --- "Vitamins, vitamin B complex, thiamine transporters and diseases" --- p.18 / Chapter 1.7 --- "Glucose metabolism, glycolysis and carcinogenesis" --- p.22 / Chapter 1.8 --- Clinical implications of DNA methylation --- p.28 / Chapter Chapter 2 --- Research Aim and Procedure --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Cell lines and human tissue samples --- p.35 / Chapter 3.2 --- Cell culture --- p.35 / Chapter 3.3 --- Total RNA extraction --- p.36 / Chapter 3.4 --- Genomic DNA extraction --- p.37 / Chapter 3.5 --- Reverse transcription PCR (RT-PCR) --- p.38 / Chapter 3.5.1 --- Reverse transcription (RT) --- p.38 / Chapter 3.5.2 --- Semi-quantitative RT-PCR --- p.40 / Chapter 3.5.3 --- Real time RT-PCR --- p.42 / Chapter 3.6 --- General techniques --- p.44 / Chapter 3.6.1 --- DNA and RNA quantification --- p.44 / Chapter 3.6.2 --- Gel electrophoresis --- p.44 / Chapter 3.6.3 --- LB medium and LB plate preparation --- p.44 / Chapter 3.6.4 --- Plasmid DNA extraction --- p.45 / Chapter 3.6.4a --- Plasmid DNA mini extraction --- p.45 / Chapter 3.6.4b --- Plasmid DNA midi extraction --- p.46 / Chapter 3.6.5 --- DNA sequencing --- p.46 / Chapter 3.7 --- Methylation status analysis --- p.49 / Chapter 3.7.1 --- CpG island analysis --- p.49 / Chapter 3.7.2 --- Sodium bisulfite modification of DNA --- p.49 / Chapter 3.7.3 --- Methylation-specific PCR (MSP) --- p.50 / Chapter 3.7.4 --- Bisulfite genomic sequencing (BGS) --- p.53 / Chapter 3.8 --- Construction of expression plasmid DNA --- p.55 / Chapter 3.8.1 --- Construction of the SLC19A3-expressing vector --- p.55 / Chapter 3.8.2 --- Construction of the FBP1-expressing vector --- p.57 / Chapter 3.9 --- Functional analyses --- p.58 / Chapter 3.9.1 --- Monolayer colony formation assay --- p.58 / Chapter 3.9.2 --- Cancer cell growth curve analysis --- p.59 / Chapter 3.9.3 --- Lactate assay --- p.60 / Chapter 3.10 --- Statistical analysis --- p.61 / Chapter Chapter 4 --- Results --- p.62 / Chapter 4.1 --- Identification of novel candidate tumor suppressor genes downregulated by DNA methylation --- p.62 / Chapter 4.2 --- Selection of genes for further study --- p.62 / Chapter 4.3 --- Identification of SLC19A3 as a novel candidate tumor suppressor gene in gastric cancer --- p.64 / Chapter 4.3.1 --- Pharmacological restoration of SLC 19A3 downregulation in gastric cancer --- p.64 / Chapter 4.3.2 --- Methylation analysis of SLC 19A3 promoter region --- p.66 / Chapter 4.3.3 --- Functional analysis of SLC 19A3 in gastric cancer --- p.72 / Chapter 4.3.4 --- Clinicopathologic characteristics of SLC 19A3 promoter methylation in gastric cancer --- p.75 / Chapter 4.3.5 --- Discussion --- p.78 / Chapter 4.4 --- Identification of FBP1 as a novel candidate tumor suppressor gene regulated by NF-kB in gastric cancer --- p.85 / Chapter 4.4.1 --- Pharmacological restoration of FBP1 downregulation in gastric cancer --- p.85 / Chapter 4.4.2 --- Methylation analysis of FBP 1 promoter region --- p.87 / Chapter 4.4.3 --- Functional analysis of FBP 1 in gastric cancer --- p.93 / Chapter 4.4.4 --- Reduction of lactate generation under FBP1 expression --- p.95 / Chapter 4.4.5 --- Clinicopathologic characteristics of FBP 1 promoter methylation in gastric cancer --- p.98 / Chapter 4.4.6 --- NF-kB mediated FBP1 promoter hypermethylation in gastric cancer --- p.104 / Chapter 4.4.7 --- Discussion --- p.106 / Chapter Chapter 5 --- General discussion --- p.112 / Chapter Chapter 6 --- Summary --- p.117 / Reference list --- p.119

Cancer is the leading cause of death in Hong Kong (21,300 new cases and 11,500 deaths in 2003), with nasopharyngeal carcinoma (NPC), esophageal cancer (ESCC), and colorectal cancer (CRC) among the common ones. For these tumors, most patients present with advanced stage disease and poor treatment outcome, with an urge of early detection. Epigenetic inactivation of tumor suppressor genes (TSG) by CpG methylation represents an important mechanism of tumorigenesis, in addition to genetic abnormalities. Tumor-specific methylation can also be used as biomarkers for the identification of novel TSGs and for cancer early diagnosis and prognosis prediction. / Finally, for the purpose of development of epigenetic biomarker for cancer molecular diagnosis, I screened gene methylation in the serum samples. Aberrant methylation of PCDH10 and DLC1 was detected in serum samples (2/14 (14%) and 4/14 (29%) respectively) from tumor patients but not in normal controls. It suggests that screening for PCDH10 and DLC1 methylation in sera could be a tumor-specific and non-invasive epigenetic biomarker for molecular diagnosis and prognostics. (Abstract shortened by UMI.) / In the second approach, 1-Mb array-based comparative genomic hybridization (aCGH) was carried out to detect DNA copy number aberrations, which contain potential TSG loci, in a panel of NPC and ESCC cell lines. Frequent deletions include: 1p36.3, 3p14-11, 4p16-15, 5p13-q12, 6p21-12, 8p22-cent, 9p, 9q22-31, 10p, 13q12, 14q32, 16q23-24, 17q11.2, 18q in NPC, and 1p21, 4q21, 7p21, 7q35, 8p22-23, 8q11, 10p11, 11q22, 13q31, 14q32, 18q11-23 in ESCC. Several deletions (3p14-11 and 16q23) were further investigated in detail in this study. More than 12 genes were identified to be frequently silenced by methylation in tumors, including FHIT (3p14), WNT5A (3p14), ADAMTS9 (3p14), FEZF2 (3p14), ROBO (3p12), CADM2 (3p12), EPHA3 (3p11), RAB (11q22), ADAMTS18 (16q23), and TUSC8 (16q23), while homozygous deletion of these genes was infrequently detected. Aberrant methylation of these genes was also frequently detected in primary tumors in a tumor-specific manner. The tumor suppressor functions of TUSC8, WNT5A, CADM2 and ROBO were further investigated and validated. Further experiment indicated that induction of tumor cell apoptosis may contribute to the tumor suppressor function of TUSC8. / Modified genomic methylation subtractive approaches using uracil-DNA glycosylase or combined with pharmacological demethylation were developed. GADD45G, PCDH10, ROR2, DLC1L1 were among a series of novel methylated targets identified by these approaches. Methylation-associated silencing of these genes was frequently detected in various types of tumor cell lines and primary tumors including NPC, ESCC and CRC, in a tumor-specific manner. Ectopic expression of these genes strongly suppressed tumor cell growth and colony formation of silenced tumor cells. Epigenetic inactivation of GADD45G is the major mechanism for the loss of its response to environmental stresses. Reintroduction of PCDH10 strongly suppressed tumor cell migration and invasion. Ectopic expression of DLC1L1 in silenced tumor cells resulted in a remarkable suppression of tumor cell clonogenicity, which depends on its GAP activity. Furthermore, DLC1L1, but not its inactivating mutants, inhibited Ras mediated oncogenic transformation. Thus, these identified genes are functional TSGs. / Ying Jianming. / "July 2007." / Adviser: Qian Tao. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0083. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 147-173). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

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.

Background & aims. DNA methylation induced tumor suppressor gene silencing plays an important role in carcinogenesis. By using methylation-sensitive representational difference analysis, we identified paired box gene 5 (PAX5) being methylated in human cancer. PAX5 locates at human chromosome 9p13.2 and encodes a 391 amino acids transcription factor. However, the role of PAX5 in gastric cancer is still unclear. Hence, we analyzed its epigenetic inactivation, biological functions, and clinical implications in gastric cancer. / Conclusions. Our results demonstrated that PAX5 promoter methylation directly mediates its transcriptional silence and commonly occurs in gastric cancer. PAX5 gene can act as a functional tumor suppressor in gastric carcinogenesis by playing an important role in suppression of cell proliferation, migration, invasion, and induction of cell apoptosis. Detection of methylated PAX5 may be utilized as a biomarker for the prognosis of gastric cancer patients. / Methods. Methylation status of PAX5 promoter in gastric cancer cell lines and clinical samples was evaluated by methylation specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS). The effects of PAX5 re-expression in cancer cell lines were determined in proliferation, cell cycle, apoptosis, migration and invasion assays. Its in vivo tumorigenicity was investigated by injecting cancer cells with PAX5 expression vector subcutaneously into the dorsal flank of nude mice. Chromosome Immunoprecipitation (ChIP) and cDNA expression array were performed to reveal the molecular mechanism of the biological function of PAX5. / Results. PAX5 was silenced or down-regulated in seven out of eight of gastric cancer cell lines examined. A significant down-regulation was also detected in paired gastric tumors compared with their adjacent non-cancer tissues (n = 18, P = 0.0196). In contrast, PAX5 is broadly expressed in all kinds of normal adult and fetal tissues. The gene expression of PAX5 in the gastric cancer cell line is closely linked to the promoter hypermethylation status. In addition, the expression levels could be restored by exposure to demethylating agents 5-aza-21-deoxycytidine. Re-expression of PAX5 in AGS, BGC823 and HCT116 cancer cells reduced colony formation (P < 0.01) and cell viability (P < 0.05), arrested cell cycle in G0/G1 phase (P = 0.0055), induced cell apoptosis (P < 0.05), repressed cell migration and invasion (P = 0.0218) in vitro. It also inhibited tumor growth in nude mice (P < 0.05). The molecular basis of its function were investigated by cDNA expression array and demonstrated that ectopic expression of PAX5 up-regulated tumor suppressor gene P53, anti-proliferation gene P21, pro-apoptosis gene BAX, anti-invasion gene MTSS1 and TIMP1; and down-regulated anti-apoptosis gene BCL2, cell cycle regulator cyclinD1, migration related gene MET and MMP1. ChIP assay indicated that P53 and MET are direct transcriptional target of PAX5. Moreover, PAX5 hypermethylation was detected in 90% (145 of 161) of primary gastric cancers compared with 16% (3 of 19) of non-cancer tissues (P < 0.0001). After a median follow-up period of 15.4 months, multivariate analysis revealed that gastric cancer patients with PAX5 methylation had a significant poor overall survival compared with the unmethylated cases (P = 0.0201). / Li, Xiaoxing. / Advisers: Hsiang Fu Kung; Jun Yu. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-159). / 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.

Les tumeurs gastro-intestinales stromales (Gastro-Intestinal Stromal Tumours - GIST en Anglais) sont les sarcomes les plus fréquents du tube digestif. Sur base de leur profil d'expression génique et de similitudes morphologiques, il a été établi que les GIST dérivent des cellules interstitielles de Cajal (Interstitial Cells of Cajal - ICC en Anglais) ou d'un précurseur commun. Le développement et le maintient des ICC sont dépendant de voies de signalisation du récepteur tyrosine kinase KIT. Des mutations oncogéniques de KIT, conduisant indépendamment du ligand à l'activation des voies de signalisation en aval, sont présentes dans environ 85% des GIST. Depuis une dizaine d'années, des molécules de synthèse qui inhibent la phosphorylation -et donc l'activation - de KIT ont été introduites avec succès dans le traitement clinique des GIST. Cependant des résistances, souvent causées par des mutations secondaires, apparaissent fréquemment et environ 50% des patients traités rechutent dans les 2 ans. Le développement de nouvelles stratégies diagnostiques et thérapeutiques pour les GIST demeure donc essentiel. Ces dernières années, de nouveaux marqueurs diagnostiques ou cibles thérapeutiques potentielles ont été rapportés dans la littérature (p.ex. Discovered on GIST-1 (DOG1, anoctamin 1), Protein kinase C theta (PKC theta), Carbonic anhydrase II (CAII)) .Il faut relever que tous ces gènes sont aussi exprimés par les ICC KIT+ du tube digestif normal et que leur présence dans les GIST reflète donc vraissemblablement essentiellement leur parenté avec les ICC.<p>Dans la présente étude, nous nous sommes attachés à identifier de nouveaux marqueurs diagnostiques ou cibles thérapeutiques potentielles exprimés par les GIST mais absent des ICC KIT+ normales.<p>Pour ce faire, nous avons comparé le profile d'expression géniques de l'antre gastrique de souris porteuses de la mutation oncogénique Kit K641E et de souris contrôles (wild type WT en Anglais) par la technique de cDNA microarray. Les différences d'expression génique ont été ensuite confirmées par réactions de PCR quantitative (qPCR) en temps réel et l'immunoréactivité (-ir) pour les candidats les plus prometteurs a été localisée par immunofluorescence (IF) dans la muscularis propria du tube digestif, avec une attention spéciale pour les cellules KIT+.<p>Plusieurs gènes identifiés appartenaient tant au profil d'expression génique des GIST qu'au profil d'expression des ICC Kit+ de l'intestin grêle murin, validant ainsi la pertinence du modèle murin KitK641E pour l'approche choisie (Chapitre 3). D'autre part, trois gènes identifiés (Neurotensin receptor 1 (Ntsr1), Trophoblast glycoprotein (Tpbg/5T) et Sprouty homolog 4 (Spry4)) étaient quant à eux présents dans la couche hypertrophié de cellules Kit+ de l'antre des souris KitK641E mais absentes des ICC Kit+ chez les souris WT (Chapitres 3, 4, 5). Ces gènes représentant donc de nouveaux candidats potentiels comme marqueurs spécifiques et/ou comme cibles thérapeutiques dans les GIST, nous avons, dans la seconde partie de notre travail, approfondi l'étude de leur expression et de leur régulation en utilisant des modèles cellulaires et tissulaires murins, ainsi que du matériel anatomopathologique de GIST humains.<p>Dans le tube digestif normal, NTSR1 et TPBG/5T4 ir ont été identifiés dans les neurones myentériques mais pas dans les ICCKIT+. Deux "tissue arrays" indépendants, totalisants 97 spécimens humains de GIST, ont révélés la présence de NTSR1-ir dans tous les GIST, en ce compris les cas négatifs pour KIT, tandis que TPBG/5T4-ir était présente dans 36/49 GIST. Un fort immunomarquage pour TPBG/5T4 était statistiquement associée aux tumeurs malignes et de haut risque (Chapitre 5; Annexe 1).<p>L'expression différentielle de membres de la famille des "Sprouty homologues" (Spry) dans l'antre des souris KitK641E a aussi été identifiée. Spry4-ir n'était pas détectable dans les ICC KIT+ des souris WT alors que Spry4-ir était présente dans la couche hyperplasique des cellules Kit+ chez les souris KitK641E. A l'opposé, l'ARN messager de Spry2 présentait un niveau d'expression similaire et Spry2-ir était détectée dans les cellules musculaires lisses - mais pas dans les cellules Kit+ - dans tous les génotypes (Chapitre 3). Pour sa part, l'expression de Spry1 apparaissait réprimée par le mutant oncogénique KitK641E, tant in vivo qu'in vitro, conduisant à la dérégulation de la boucle de rétrocontrôle négatif de la voie Ras/Erk (Chapitre 4). <p>Dans la dernière partie de cette thèse, nous avons étudié l'expression Endoglin (ENG) - aussi connues sous le nom de CD105 – dans le modèle murin de GIST KitK641E, dans les GIST humains et dans le modèle cellulaire murin Ba/F3 in vitro. ENG est une glycoprotéine transmembraire et un composant auxiliaire du complexe du récepteur au TGF-& / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Poon, Fan Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-124). / Abstract also in 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.6 / Chapter Chapter 2 --- Literature Reviews --- p.8 / Chapter 2.1 --- Cancer genetics --- p.8 / Chapter 2.1.1 --- Oncogenes and TSGs --- p.8 / Chapter 2.1.2 --- Kundsońةs two-hit event of cancer gene --- p.9 / Chapter 2.2 --- Cancer Epigenetics --- p.9 / Chapter 2.2.1 --- Types of Epigenetic regulation --- p.10 / Chapter 2.2.2 --- DNA methylation in TSGs --- p.10 / Chapter 2.2.2.1 --- Promoter CpG island in DNA methylation --- p.10 / Chapter 2.2.2.2 --- Protection system in DNA methylation --- p.11 / Chapter 2.2.2.3 --- Transcriptional silencing by DNA methylation --- p.11 / Chapter 2.2.2.4 --- DNA methylation of TSG silencing in cancers --- p.13 / Chapter 2.2.3 --- Hypomethylation of the cancer genome --- p.14 / Chapter 2.2.4 --- Clinical relevance of cancer epigenetic --- p.14 / Chapter 2.3 --- EGFR/STAT/SOCS pathway --- p.15 / Chapter 2.3.1 --- General Introduction of the EGFR pathway --- p.15 / Chapter 2.3.2 --- EGFR survival signaling pathways --- p.16 / Chapter 2.3.3 --- EGFR/STAT/SOCS signaling --- p.17 / Chapter 2.3.4 --- EGFR/STAT/SOCS signaling and cancers --- p.18 / Chapter 2.3.4.1 --- EGF and cancers --- p.18 / Chapter 2.3.4.2 --- EGFR/STAT/SOCS pathway and cancers --- p.18 / Chapter 2.3.4.3 --- EGF survival signaling as a target for cancer therapy --- p.19 / Chapter 2.4 --- TSGs in the EGFR/STAT/SOCS pathway --- p.20 / Chapter 2.4.1 --- Suppressors of cytokine signaling (SOCS) family --- p.20 / Chapter 2.4.2 --- Signal transducers and activators of transcription (STATs) family --- p.22 / Chapter 2.4.3 --- Sprouty (SPRY) family --- p.23 / Chapter 2.4.4 --- Protein Inhibitor of Activated STAT (PIASs) family --- p.25 / Chapter 2.4.5 --- Ras and Rab Interactor (RIN) family --- p.26 / Chapter 2.4.6 --- Ras-association domain family (RASSF) --- p.26 / Chapter 2.4.7 --- Glycine N-methyltransferase (GNMT) --- p.28 / Chapter 2.5 --- Nasopharyngeal carcinoma (NPC) --- p.30 / Chapter 2.5.1 --- Epidemiology of NPC --- p.30 / Chapter 2.5.2 --- Histopathology of NPC --- p.30 / Chapter 2.5.3 --- Genetic and epigenetic alteration in NPC --- p.31 / Chapter 2.5.4 --- EGFR signaling in NPC --- p.32 / Chapter 2.6 --- Esophageal squamous cell carcinoma (ESCC) --- p.33 / Chapter 2.6.1 --- Epidemiology of ESCC --- p.34 / Chapter 2.6.2 --- Histopathology of ESCC --- p.34 / Chapter 2.6.3 --- Genetic and epigenetic alteration in ESCC --- p.35 / Chapter 2.6.4 --- EGFR signaling in ESCC --- p.36 / Chapter Chapter 3 --- Materials and Methods --- p.38 / Chapter 3.1 --- General Materials --- p.38 / Chapter 3.1.1 --- "Cell lines, tumor and normal tissue samples" --- p.38 / Chapter 3.1.2 --- Maintenance of cell lines --- p.38 / Chapter 3.1.3 --- Drugs treatment of cell lines --- p.39 / Chapter 3.1.4 --- Total RNA extraction --- p.39 / Chapter 3.1.5 --- Genomic DNA extraction --- p.40 / Chapter 3.2 --- General techniques --- p.40 / Chapter 3.2.1 --- Agarose gel electrophoresis of DNA --- p.40 / Chapter 3.2.2 --- TA cloning and blunt end cloning of PCR product --- p.40 / Chapter 3.2.3 --- Transformation of cloning products to E. coli competent cells --- p.41 / Chapter 3.2.4 --- Preparation of plasmid DNA --- p.41 / Chapter 3.2.4.1 --- Mini-prep plasmid DNA extraction --- p.41 / Chapter 3.2.4.2 --- Midi-prep of plasmid DNA --- p.42 / Chapter 3.2.5 --- Measurement of DNA or RNA concentrations --- p.42 / Chapter 3.2.6 --- DNA sequencing of plasmid DNA and PCR products --- p.42 / Chapter 3.3 --- Preparation of reagents and medium --- p.43 / Chapter 3.4 --- Semi-quatitative Reverse-Transcription (RT) PCR expression analysis --- p.44 / Chapter 3.4.1 --- Reverse transcriptin reaction --- p.44 / Chapter 3.4.2 --- Semi-quantitative RT-PCR --- p.44 / Chapter 3.4.2.1 --- Primers design --- p.44 / Chapter 3.4.2.2 --- PCR reaction --- p.46 / Chapter 3.5 --- Methylation analysis of candidate genes --- p.47 / Chapter 3.5.1 --- Bisulfite treatment of genomic DNA --- p.47 / Chapter 3.5.2 --- Methylation-specific PCR (MSP) --- p.48 / Chapter 3.5.2.1 --- Bioinformatics prediction of CpG island --- p.48 / Chapter 3.5.2.2 --- Primers design --- p.48 / Chapter 3.5.2.3 --- PCR reaction --- p.49 / Chapter 3.5.3 --- Bisulfite Genomic Sequencing (BGS) --- p.50 / Chapter 3.6 --- Construction of expression vectors of candidate genes --- p.51 / Chapter 3.6.1 --- Sub-cloning of expression vector of candidate genes --- p.51 / Chapter 3.6.1.1 --- Mouse Socsl expression vector --- p.51 / Chapter 3.6.1.2 --- SPRY1 expression vector --- p.51 / Chapter 3.6.1.3 --- GNMT expression vector --- p.52 / Chapter 3.6.2 --- Restriction digestion of cloning vectors and expression --- p.52 / Chapter 3.6.3 --- Ligation of cloning fragments --- p.53 / Chapter 3.6.4 --- Colony formation assay on monolayer culture --- p.53 / Chapter 3.6.5 --- Statistical analysis --- p.54 / Chapter Chapter 4 --- Screening of candidate TSGs in EGFR pathway --- p.55 / Chapter 5.3.3 --- Restoration of GNMT expression by pharmacological demethylation --- p.89 / Chapter 5.3.4 --- Confirmation of the methylation status of GNMT promoter by BGS --- p.90 / Chapter 5.3.5 --- Methylation status of GNMT in ESCC and NPC primary tumors --- p.90 / Chapter 5.3.6 --- GNMT inhibited the growth of tumor cells in-vitro --- p.90 / Chapter 5.3.7 --- Discussion --- p.95 / Chapter Chapter 6 --- General Discussion --- p.100 / Chapter Chapter 7 --- Summary --- p.105 / Chapter Chapter 8 --- Future Study --- p.107 / Reference --- p.109