Thesis (Ph.D.). / Written for the Dept. of Pharmacology and Therapeutics. Title from title page of PDF (viewed 2008/07/23). Includes bibliographical references.

Gastric cancer is the second leading cause of cancer death worldwide and in China. The mechanism of gastric carcinogenesis is not fully understood. Epigenetic studies indicated that inactivation of tumor suppressor genes by DNA hypermethylation plays a crucial role in the progression of gastric cancer. Epigenetic inactivation of secreted frizzled-related protein (SFRP 1) by methylation plays a pivotal role on the development of various cancers. However, the role of SFRP family genes in gastric cancer remains largely unknown. We aimed to characterize the epigenetic abnormalities and discover novel biomarkers for early detection of gastric cancer. We investigated the epigenetic alterations in gastric adenocarcinoma by microarray based analysis and gene promoter hypermethylation. Based of the microarray data, we determined the functional significance and frequency of SFRP family genes hypermethylation in human gastric cancer. We screened the mRNA expression and methylation status of the SFRP family members in human gastric cancer cell lines and primary gastric cancer samples. Demethylation study of SFRP family genes were done by treating gastric cancer cell lines with 5'Aza. The biological effects of SFRP were analyzed by flow cytometry, cell viability assay and tumor growth in nude mice. SFRP1, 2, 4 and 5 were undetectable in 100% (7/7), 100% (7/7), 42.8% (3/7) and 85.7% (6/7) of gastric cancer cell lines, respectively. However, only SFRP2 showed significant down-regulation in gastric cancer compared with adjacent non-cancer samples (P<0.01). Treatment with demethylation agent, 5'-Aza, restored the expression of SFRP2 in all 7 cancer cell lines. Promoter hypermethylation of SFRP2 was detected in 73.3% of primary gastric cancer samples and 20% of adjacent non-cancer tissue (P<0.01). Bisulfite sequencing confirmed the density of promoter methylation in cell line, primary gastric cancer tissue and their adjacent non-cancer tissue. Transfection of SFRP2 induced cell apoptosis, inhibited proliferation in vitro and suppressed tumor growth in vivo. Furthermore, SFRP2 methylation was detected in 37.5% of samples showing intestinal metaplasia. Methylated SFRP2 was also detected in 66.7% of serum samples from cancer patients but not in normal controls. Epigenetic inactivation of SFRP2, but not SFRP1, SFRP4 and SFRP5 is a common and early event of carcinogenesis. Hence, detection of SFRP2 methylation in serum may have diagnostic value in gastric cancer patients. / by Cheng, Yuen Yee. / Adviser: FKL Chan. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0803. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 165-179). / 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 also in Chinese. / School code: 1307.

Orientador: Maria Luiza Silveira Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T21:08:10Z (GMT). No. of bitstreams: 1 Moraes_AlbertodaSilva_D.pdf: 18240611 bytes, checksum: 0c094268046690a939c185449986f74f (MD5) Previous issue date: 2008 / Resumo: Envelhecimento pode ser definido como as mudanças sofridas por um organismo ao longo do tempo. Esse processo, em biologia, é denominado senescência. A senescência celular é um fenômeno observado em células isoladas, e tem sido estudada tipicamente em células em cultura. Sua ocorrência in vivo foi observada em alguns tecidos de mamíferos. As mudanças na estrutura e organização da cromatina que ocorrem em células senescentes incluem, aumento na resistência da cromatina à digestão por nucleases e acúmulo de modificações de histonas e proteínas associadas à heterocromatina. Embora nem todas as células em um organismo envelhecido estejam em estado de senescência, espera-se que mudanças na estrutura e organização da cromatina ocorram. A restrição calórica é a única intervenção conhecida que tem a capacidade de estender o tempo de vida em mamíferos. Após uma dieta de restrição calórica ou jejum muitos genes, cuja expressão encontra-se alterada em animais idosos, têm sua expressão restabelecida aos níveis observados em animais jovens. Acredita-se que mudanças na cromatina também possam ocorrer durante o jejum, e que induzam mudanças no nível de expressão de diversos genes. No presente trabalho, buscando-se alterações na organização da cromatina em hepatócitos de camundongo ao longo do envelhecimento ou submetidos ao jejum, observou-se um aumento das propriedades viscoelásticas da cromatina ao longo do envelhecimento, de acordo com as mudanças na habilidade dessa cromatina em formar fibras estendidas de cromatina. Essas diferenças foram acompanhadas por um desempacotamento da cromatina. Observou-se também que essa viscoelasticidade da cromatina era dependente principalmente de interações desta com a matriz nuclear, e que cópias de genes cuja atividade transcricional não é mais requerida, ou requerida em um nível menor em animais idosos, podem desligar-se temporariamente da matriz nuclear. Mudanças nas propriedades viscoelásticas da cromatina e no seu grau de compactação já haviam sido observadas previamente em animais em jejum. Apesar disso, no presente trabalho, nenhuma diferença com relação à interação dos genes rDNA com a matriz nuclear foi encontrada em animais em jejum. Contudo, independente da condição fisiológica, o DNA aderido à matriz nuclear parece ser rico em genes, enquanto as seqüências heterocromáticas, pobres em genes, geralmente são encontradas tanto associadas com a matriz nuclear quanto dissociadas desta (cuidado com essa conclusão. Está forte). Em hepatócitos de animais idosos foi observado acúmulo de marcadores heterocromáticos (modificações de histonas) e de outras proteínas (proteínas formadoras de heterocromatina e glicoproteínas presentes principalmente nos cromocentros), assim como diminuição das modificações de histonas associadas com transcrição ativa. Todas essas modificações estão relacionadas com alterações na síntese de RNA já relatadas para animais idosos, e são uma evidência de que o controle da expressão gênica, a organização e a composição da cromatina estão intimamente relacionados. Em um outro tipo celular como espermatozóides de camundongo, uma diferente organização nuclear levou a propriedades diferenciadas de sua cromatina com relação às suas propriedades viscoelásticas (aumentadas). Tais diferenças possivelmente estejam relacionadas com um padrão modificado de expressão gênica, uma vez que em espermatozóides, a atividade transcricional é nula ou quase ausente / Abstract: Aging may be defined as the changes that take place in an organism with time. This process, in biology, is called senescence. Cellular senescence is observed in isolated cells, and has been studied typically in cultured cells, but its occurrence in vivo has been shown only in some mammalian tissues. Chromatin changes that take place with cellular senescence include increase in the resistance of chromatin to nuclease digestion and accumulation of histone modifications and non-histone proteins associated with heterochromatin. Although not all cells in an aged organism are subjected to cellular senescence, it is expected that changes in the chromatin structure and organization still occur. Caloric restriction is the only intervention known to extend life span in mammals. It has been shown that many genes whose expression pattern is altered in aged animals can be reverted to the levels observed in young animals after a caloric restriction diet or complete food withdrawal. Changes in chromatin structure may occur during the starvation period to induce changes in the expression level of several genes. With the aim of screening for alterations in the chromatin organization in mouse hepatocyte nuclei with aging or following starvation, we observed an increase in the viscoelastic properties of chromatin with aging, in terms of changes in the ability of this chromatin to form extended chromatin fibers after a lysis treatment in liver imprints on histological slides. These differences were accompanied by chromatin unpackage. Most of the viscoelasticity of the chromatin were dependent on its interactions with the nuclear matrix, and copies of genes whose transcription are no longer required in aged animals, tended to detach from the nuclear matrix. Changes in the viscoelastic properties and packing degree of chromatin had been shown previously in starved animals. However, no differences regarding this feature were seen in the present work. Nevertheless, regardless the physiological condition, DNA attached to the nuclear matrix seems to be gene-rich, while heterochromatic gene-poor regions were found both attached and detached from the nuclear matrix. We observed accumulation of heterochromatic marks (histone modifications) and non-histone proteins (heterochromatin proteins and glycoproteins present mainly in the chromocenters), as well as decreased histone modifications associated with transcription in hepatocyte nuclei of aged mice. All these changes are related to altered RNA synthesis observed in aged animals and are an evidence of the strong relationship between chromatin organization, composition, and control of gene expression. In another cell type, mouse sperm cells, its nuclear organization lead to different chromatin properties regarding its viscoelastic properties (increased). These differences are possibly related to a modified pattern of gene expression since gene transcription is almost or completely absent in sperm cells / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientador: Marcelo Rocha Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba. / Made available in DSpace on 2018-08-20T06:47:39Z (GMT). No. of bitstreams: 1 Camargo_Glauciade1985-_M.pdf: 1041410 bytes, checksum: aba09e2e49414c9f9f0be4938f591a70 (MD5) Previous issue date: 2012 / Resumo: A periodontite crônica é uma doença inflamatória que leva à perda de inserção de elementos dentários, e é desencadeada e mantida por um biofilme subgengival periodontopatogênico. A presença de alguns tipos de lipopolissacarídeos (LPS), derivados de bactérias no sítio periodontal doente, pode iniciar uma sinalização por meio das células do tecido gengival, que culminará com um microambiente com diferentes células do sistema imune e com uma alteração no padrão de expressão de citocinas inflamatórias. Já foi evidenciado que no tecido gengival de pacientes com periodontite crônica, genes que codificam receptores celulares para o LPS, podem sofrer alterações epigenéticas. O objetivo deste estudo foi avaliar se a periodontite crônica e o LPS bacteriano derivado de P. gingivalis podem modular a expressão gênica de alguns fatores reguladores de eventos epigenéticos. Biópsias de tecido gengival inflamado e sem inflamação foram coleados de pacientes com periodontite crônica e de pacientes saudáveis respectivamente, o RNA total foi extraído e a expressão dos genes DNMT1 (DNA metiltransferase 1), DNAMT3a (DNA metiltransferase 3a), histona demetilase JMJD3 e histona demetilase UTX foram analisadas por meio de RT-PCR quantitativo. Fibroblastos gengivais humanos derivados de cultura primária, e queratinócitos (HaCaT) foram expostos a LPS de P. gingivalis ou ao veículo do LPS, e foram avaliadas a viabilidade celular por meio do teste MTT e a expressão gênica de DNMT1, DNMT3a, JMJD3 e UTX por meio de RT-PCR quantitativo. As análises dos resultados demonstraram que nem a periodontite e nem o LPS exposto a fibroblastos gengivais foram capazes de modular a expressão dos genes estudados. Contudo, o LPS promoveu a diminuição da expressão de DNMT1, DNMT3a e JMJD3 nas células HaCaT. Pode-se concluir que LPS derivado P. gingivalis pode modular, em queratinócitos, a expressão gênica de algumas enzimas promotoras de eventos epigenéticos / Abstract: The aim of this study was to assess whether P. gingivalis LPS can modulate, in culture of the human keratinocytes and human gingival fibroblasts, gene expression levels of the some enzymes that promote epigenetic events. In addition, the same enzymes were evaluated in sample from healthy and periodontitis affected individuals. Primary gingival fibroblast culture and keratinocytes (HaCaT) were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24hs. After this period, cell viability were assessed by MTT test, and total RNA were extracted to evaluate gene expression levels of the enzymes: DNMT1 (DNA methyltransferase 1), DNMT3a (DNA methyltransferase 3a), histone demethylases JMJD3 and UTX, by qRT-PCR. To evaluate the gene expression in healthy and periodontitis affected individuals, total RNA was extracted from biopsies of gingival tissue from sites with (periodontitis) or without periodontitis (healthy), and gene expression of DNMT1, DNAMT3a, JMJD3 and UTX were evaluated by qRT-PCR. No significant differences were found in the gene expression analysis between healthy gingival tissues and gingival tissue from periodontitis sites. The results showed that LPS downregulated DNMT1 (p<0.05), DNMT3a (p<0.05) and JMJD3 (p<0.01) gene expression in HaCaT cells, but no modulation was found to gingival fibroblasts. P. gingivalis LPS exposure to keratinocytes, downregulates gene expression of the enzymes that promote epigenetic events / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Kou Ying Chuck. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 186-199). / 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.

Lam, Yuk Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / CONTRIBUTORS --- p.viii / PUBLICATIONS --- p.ix / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xi / LIST OF ABBREVIATIONS --- p.xiii / TABLE OF CONTENTS --- p.xiv / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL ANEUPLOIDIES --- p.2 / Chapter 1.1 --- The need for prenatal screening and diagnosis --- p.2 / Chapter 1.2 --- Patau Syndrome (Trisomy 13) --- p.2 / Chapter 1.3 --- Current methods for fetal aneuploidy detection --- p.4 / Chapter 1.3.1 --- Routine prenatal screening tests --- p.4 / Chapter 1.3.2 --- Definitive prenatal diagnosis by invasive procedures --- p.7 / Chapter 1.4 --- New approach for noninvasive prenatal diagnosis --- p.11 / Chapter 1.4.1 --- Circulating fetal cells --- p.11 / Chapter 1.4.2 --- Cell-free fetal nucleic acids in maternal circulation --- p.12 / Chapter 1.4.3 --- Diagnostic applications of cell-free fetal nucleic acids in maternal plasma --- p.12 / Chapter CHAPTER 2: --- DEVELOPMENT OF FETAL EPIGENETIC MARKERS IN MATERNAL PLASMA --- p.17 / Chapter 2.1 --- Limitations of fetal DNA markers --- p.17 / Chapter 2.2 --- DNA methylation is an actively-researched area under the field of epigenetics --- p.18 / Chapter 2.3 --- Genome-scale DNA methylation analysis brings new insight into epigenetics --- p.20 / Chapter 2.4 --- The first demonstration of using an epigenetic method for detecting maternally-inherited fetal DNA in maternal plasma --- p.22 / Chapter 2.5 --- The first universal marker for fetal DNA in maternal plasma --- p.24 / Chapter 2.6 --- Discovery of more fetal epigenetic markers --- p.25 / Chapter 2.6.1 --- Methylated fetal epigenetic markers are more desirable --- p.25 / Chapter 2.6.2 --- Discovery of hypermethylated fetal epigenetic markers by studying tumor suppressor genes --- p.26 / Chapter 2.6.3 --- Discovery of hypermethylated fetal epigenetic markers on chromosome 21 --- p.28 / Chapter 2.7 --- Noninvasive detection of fetal aneuploidies using fetal epigenetic markers --- p.29 / Chapter 2.7.1 --- Noninvasive detection of fetal trisomy 18 by the epigenetic allelic ratio (EAR) approach --- p.29 / Chapter 2.7.2 --- Noninvasive detection of fetal trisomy 21 by the epigenetic-genetic (EGG) approach --- p.30 / Chapter 2.8 --- Aim of thesis --- p.32 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.34 / Chapter CHAPTER 3: --- METHODS FOR QUANTITATIVE ANALYSIS OF DNA METHYLATION --- p.35 / Chapter 3.1 --- Subject recruitment and sample collection --- p.35 / Chapter 3.2 --- Sample processing --- p.38 / Chapter 3.3 --- DNA extraction --- p.38 / Chapter 3.3.1 --- Placental tissues --- p.38 / Chapter 3.3.2 --- Maternal blood cells --- p.39 / Chapter 3.3.3 --- Maternal plasma --- p.40 / Chapter 3.4 --- Methylated DNA immunoprecipitation and tiling array analysis (MeDIP-chip) --- p.41 / Chapter 3.4.1 --- Principles --- p.41 / Chapter 3.4.2 --- DNA sample and array processing --- p.43 / Chapter 3.4.2.1 --- DNA preparation and target hybridization --- p.43 / Chapter 3.4.2.2 --- Data analysis --- p.44 / Chapter 3.5 --- DNA methylation analysis on randomly-chosen regions on chromosome / Chapter 3.6 --- Bisulfite conversion --- p.46 / Chapter 3.6.1 --- Principles of bisulfite conversion --- p.46 / Chapter 3.6.2 --- Procedures of bisulfite conversion --- p.46 / Chapter 3.7 --- Quantitative analysis of DNA methylation --- p.47 / Chapter 3.7.1 --- Bisulfite PCR and genomic sequencing --- p.47 / Chapter 3.7.1.1 --- Primer design for bisulfite polymerase chain reaction (PCR) --- p.47 / Chapter 3.7.1.2 --- Bisulfite PCR --- p.49 / Chapter 3.7.1.3 --- Cloning --- p.50 / Chapter 3.7.1.4 --- Bisulfite genomic sequencing --- p.52 / Chapter 3.7.1.5 --- Data acquisition and interpretation --- p.53 / Chapter 3.7.2 --- EpiTYPER，a mass-spectrometry-based method --- p.54 / Chapter 3.7.2.1 --- Principles of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.54 / Chapter 3.7.2.2 --- Primer design of the EpiTYPER assay --- p.55 / Chapter 3.7.2.3 --- The EpiTYPER assay and its principle --- p.56 / Chapter 3.8 --- Methylation-sensitive restriction enzyme (MSRE)-mediated real-time quantitative PCR (qPCR) --- p.61 / Chapter 3.9 --- Digital PCR --- p.66 / Chapter 3.9.1 --- Principles of digital PCR --- p.66 / Chapter 3.9.2 --- Poisson distribution --- p.68 / Chapter 3.10 --- Statistical analyses --- p.69 / Chapter SECTION III: --- SYSTEMATIC IDENTIFICATION OF A FETAL DNA METHYLATION MARKER ON CHROMOSOME 13 FOR DETECTION OF FETAL TRISOMY 13 --- p.70 / Chapter CHAPTER 4: --- SYSTEMATIC IDENTIFICATION OF POTENTIAL FETAL EPIGENETIC MARKERS BY MEDIP-CHIP ANALYSIS --- p.71 / Chapter 4.1 --- Systematic discovery of fetal epigenetic markers on chromosome 13 by MeDIP-chip analysis --- p.71 / Chapter 4.2 --- Experimental design --- p.73 / Chapter 4.3 --- Results --- p.76 / Chapter 4.3.1 --- Identification of differentially methylated DNA regions by MeDIP-chip or non-MeDIP-chip approaches followed by EpiTYPER analysis --- p.76 / Chapter 4.3.2 --- Confirmation of differential methylation patterns and exclusion of regions with high inter-individual variations by EpiTYPER analysis --- p.82 / Chapter 4.3.3 --- Confirmation of differential DNA methylation patterns with higher resolution by bisulfite sequencing --- p.85 / Chapter 4.4 --- Discussion --- p.95 / Chapter CHAPTER 5: --- THE APPLICATION OF FETAL EPIGENETIC MARKER ON CHROMSOME 13 FOR DETECTION OF FETAL TRISOMY 13 --- p.98 / Chapter 5.1 --- Identification of a fetal epigenetic marker on chromosome 13 for the detection of fetal trisomy 13 by the epigenetic-genetic (EGG) chromosome dosage approach --- p.98 / Chapter 5.2 --- Experimental design --- p.101 / Chapter 5.3 --- Results --- p.105 / Chapter 5.3.1 --- Optimization of the digestion protocol --- p.105 / Chapter 5.3.2 --- Detection of digestion-resistant EFNB2-3'UTR moleculesin maternal plasma --- p.109 / Chapter 5.3.3 --- Evaluation of the fetal specificity of digestion-resistant EFNB2´ؤ3 'UTR DNA molecules in maternal plasma --- p.111 / Chapter 5.3.4 --- Comparison of EFNB2-3'UTR methylation profiles between the euploid and trisomy 13 placental tissue samples --- p.115 / Chapter 5.3.5 --- Chromosome dosage analysis by the EGG analysis using placental tissue samples --- p.118 / Chapter 5.4 --- Discussion --- p.122 / Chapter SECTION IV: --- CONCLUDING REMARKS --- p.125 / Chapter CHAPTER 6: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.126 / Chapter 6.1 --- Development of fetal epigenetic markers for noninvasive prenatal diagnosis --- p.126 / Chapter 6.2 --- Systematic identification of fetal epigenetic markers on chromosome13 --- p.127 / Chapter 6.3 --- Detection of fetal trisomy 13 by the epigenetic-genetic (EGG) relative chromosome dosage analysis --- p.129 / Chapter 6.4 --- Future perspectives --- p.132 / Appendix I --- p.134 / Appendix II --- p.136 / REFERENCES --- p.142

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines. Most cells proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line MeWo. The MeWo-LC1 cell line has been shown to have a cellular phenotype similar to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin dependent enzymes methionine synthase and methylmalonyl-CoA mutase. Inability of cblC cells to complement the defect in cobalamin metabolism in MeWo-LC1 suggested that the defect was caused by decreased activity of the MMACHC gene product. However, no potentially disease causing mutations could be detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression could be detected in MeWo-LC1, and there was virtually complete methylation of a CpG island at the 5' end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation; the CpG island was partially methylated in MeWo and only lightly methylated in control fibroblasts. Transfection of MeWo-LC1 with wild type MMACHC with a constitutive promoter resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.

No description available.

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.

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 &lt; 0.01) and cell viability (P &lt; 0.05), arrested cell cycle in G0/G1 phase (P = 0.0055), induced cell apoptosis (P &lt; 0.05), repressed cell migration and invasion (P = 0.0218) in vitro. It also inhibited tumor growth in nude mice (P &lt; 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 &lt; 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.