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The Role of DNA Methylation in the Development of Colorectal Neoplasia

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The Role of DNA Methylation in the Development of Colorectal Neoplasia The role of DNA methylation in the development of colorectal neoplasia Justin Jong Leong Wong BSc (Hons) July 2008 A thesis submitted for the degree of Doctor of Philosophy in the School of Medical Sciences, University of New South Wales, Australia Acknowledgements The completion of this PhD thesis would not have been possible without the support and assistance of the following individuals. First of all, I would like to thank my principal supervisor Professor Nicholas John Hawkins for his consistent support and constructive criticisms throughout my candidature. I truly appreciate his efforts in organising regular meetings with me to ensure that I am always on the right track. I am also grateful to my co-supervisor Dr Megan Hitchins for her continuous encouragement and technical advice. A special thank you also goes out to Professor Robyn Ward for providing me the facilities to undergo my research and her intellectual input. I am also grateful to the past and present members of the Molecular and Cellular Oncology group for their constant support, assistance, company and friendship. I would like to express my special thanks to the past and present students in the laboratory; Andrew, Nerida, Chau-To, Joice and James for being such a good company especially during the odd hours. Finally, I am indebted to my family, especially my mom and dad for their continuous encouragement. A big thank you also goes out to my special friend, Katherine for her cherished love, patience and friendship. Contents Contents………………………………………………………………………….………...i List of Tables……………………………………………………………………….…….ix List of Figures………………………………………………………………….……......xiii Abbreviations…………………………………………………………………………..xviii Abstract………………………………………………………………………………....1 CHAPTER 1: Literature review………………………………………….......................3 1.1 Colorectal neoplasia............................................................................................ 4 1.1.1 Epidemiology of the disease............................................................................ 4 1.1.2 Aetiology of the disease................................................................................... 5 1.1.3 Pathology of the disease .................................................................................. 5 1.1.3.1 Non-invasive colorectal neoplasms......................................................... 6 1.1.3.1.1 Aberrant crypt foci ............................................................................ 6 1.1.3.1.2 Hyperplastic polyps........................................................................... 8 1.1.3.1.3 Serrated adenomas ............................................................................ 8 1.1.3.1.4 Colorectal adenomas ......................................................................... 9 1.1.3.2 Colorectal carcinomas........................................................................... 10 1.1.3.3 The adenoma-carcinoma sequence ....................................................... 11 1.1.3.4 The serrated neoplasia pathway ............................................................ 12 1.1.3.5 Pathological staging of colorectal cancer.............................................. 13 1.2 Genetic mechanisms of colorectal neoplasia .................................................... 15 1.2.1 Chromosomal instability................................................................................ 16 1.2.2 Microsatellite instability................................................................................ 16 1.2.2.1 Diagnosis and features of microsatellite instability .............................. 17 1.3 Genetic basis of colorectal neoplasia ................................................................ 18 1.3.1 Familial cancer syndromes ............................................................................ 18 1.3.1.1 Familial adenomatous polyposis (FAP) syndrome ............................... 19 1.3.1.2 Other FAP variants................................................................................ 20 1.3.1.3 MYH-associated Polyposis (MAP)........................................................ 21 1.3.1.4 HNPCC syndrome................................................................................. 22 1.3.2 Sporadic colorectal cancer............................................................................. 23 i 1.4 Genetic alterations in cancer development ....................................................... 24 1.4.1 Adenomatous polyposis coli (APC) .............................................................. 24 1.4.2 KRAS.............................................................................................................. 26 1.4.3 Loss of 18q .................................................................................................... 26 1.4.4 p53 ................................................................................................................. 27 1.4.5 BRAF ............................................................................................................. 28 1.5 The “Epigenetic Code” and its changes in colorectal neoplasia....................... 29 1.5.1 Epigenetic events in normal human cells ...................................................... 30 1.5.1.1 Chromatin modifications....................................................................... 30 1.5.1.2 DNA methylation.................................................................................. 32 1.5.1.3 RNA modification................................................................................. 34 1.5.2 Epigenetic basis of colorectal neoplasia........................................................ 34 1.5.2.1 Global DNA hypomethylation in colorectal cancer.............................. 35 1.5.2.2 Hypermethylation of CpG islands in colorectal cancer ........................ 36 1.5.2.3 MLH1 methylation in MSI colorectal cancer as a classic example of promoter hypermethylation in colorectal cancer ............... 39 1.5.2.4 Dysregulation of chromatin modifications ........................................... 40 1.6 Patterns of gene methylation in colorectal cancer............................................. 41 1.6.1 CpG island methylator phenotype (CIMP) and the serrated neoplasia pathway.......................................................................................................... 41 1.6.2 Long-range epigenetic silencing.................................................................... 44 1.7 CpG island methylation as a marker for the predisposition of colorectal neoplasia........................................................................................................... 46 1.8 Germline epimutation and colorectal cancer..................................................... 47 1.9 Methods for detection of DNA methylation at candidate gene promoters ....... 49 1.9.1 Traditional methods....................................................................................... 49 1.9.2 Quantitative methods..................................................................................... 55 1.10 Research aims and outline................................................................................. 56 CHAPTER 2 : Materials and methods………….....………………………………….59 2.1 Materials............................................................................................................ 60 2.1.1 Suppliers ........................................................................................................ 60 2.1.2 Oligonucleotide primers ................................................................................ 60 ii 2.1.3 Reagents......................................................................................................... 62 2.1.4 Buffers and solutions ..................................................................................... 63 2.1.5 Kits................................................................................................................. 64 2.1.6 Restriction enzyme ........................................................................................ 64 2.1.7 DNA modification enzyme and substract...................................................... 64 2.1.8 Growth media for cell line............................................................................. 65 2.2 Methods............................................................................................................. 65 2.2.1 Sample collection.......................................................................................... 65 2.2.1.1 Handling of tissue samples ................................................................... 65 2.2.1.2 Handling of blood samples ................................................................... 66 2.2.2 Patient clinical details.................................................................................... 66 2.2.3 Histopathological data of patients ................................................................. 66 2.2.4 Cell lines........................................................................................................ 67 2.2.5 Cell culture.................................................................................................... 67 2.2.6 Extraction and quantification of DNA........................................................... 68 2.2.6.1 DNA extraction from blood samples .................................................... 68 2.2.6.2 DNA extraction from tissue and cell lines...........................................
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