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The Role of Folic Acid in Maintaining Colorectal Cancer Cell DNA Methylation Patterns and Cancer Stem Cell Phenotype in Vitro

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The Role of Folic Acid in Maintaining Colorectal Cancer Cell DNA Methylation Patterns and Cancer Stem Cell Phenotype in Vitro The role of folic acid in maintaining colorectal cancer cell DNA methylation patterns and cancer stem cell phenotype in vitro By Nathan Farias A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Biomedical Sciences Guelph, Ontario, Canada © Nathan Farias December, 2013 ABSTRACT The role of folic acid in maintaining colorectal cancer cell DNA methylation patterns and cancer stem cell phenotype in vitro Nathan Farias Advisor: University of Guelph, 2013 Dr. B. L. Coomber Folic acid is a B vitamin involved in DNA CpG methylation. Mandated dietary fortification has led to a subsequent increase in blood folate concentration which has been correlated to a simultaneous spike in colorectal cancer incidence in Canada and the US. Several human colorectal cancer cell lines were cultivated under low (0 mg/L), standard (4 mg/L), and high (16 mg/L) folate conditions for seven days, then assessed for DNA methyltransferase1 protein expression, changes in DNA methylation, and ability to generate colonospheres in culture. Low folic acid levels generally led to reduced DNMT1 protein expression, CpG hypomethylation, and reduced colonosphere yield. High folic acid levels led to increased DNMT1 protein expression, CpG hypermethylation, and maintained colonosphere yield. This data demonstrates that varying levels of folic acid in vitro can influence the methylation status and cancer stem cell self-renewal ability of human colorectal cancer cells. ACKNOWLEDGEMENTS I would like to take this opportunity to acknowledge and thank everyone who contributed and helped me throughout the course of my masters degree. Firstly, I would like to acknowledge and thank Dr. Brenda Coomber for providing me with the opportunity to contribute to such an exciting and rewarding field as cancer research. Her passion and willingness to educate made her an invaluable resource, while her determination and commitment to research were admirable qualities that motivated me throughout my degree. Brenda’s persistence in challenging my ideas and concepts not only ensured that my work was accurate, but it contributed to the development of my critical thinking skills that I will carry on in my future endeavors. I would also like to thank my committee members Dr. Terry Van Raay and Dr. Marica Bakovic for being involved in my project and providing me with guidance and instruction throughout the course of my work. Dr. Van Raay assisted me in understanding the Wnt pathway activation in my cell model, while Dr. Bakovic assisted me in understanding the folate cycle in regards to DNA methylation. I would also like to acknowledge Dr. Bekim Sadikovic for undertaking the data analysis of the Illumina Human Methylation 450k Array. The results that he generated were important contributions to my thesis Next I would like to thank everyone involved with teaching me how to use equipment or new protocols. I would like to thank Amanda Barber for being my first instructor in a lab setting and teaching me essential techniques and protocols, such as cell culture and western blotting. Amanda gave me a running start on my project and without her it wouldn’t have been possible. Next I would like to acknowledge Jodi Morrison for not only being a fantastic technician but also a vital resource throughout the course of my work. Her substantial technical knowledge and iii patience make her and essential asset in our lab and it would be difficult to picture a smooth running operation without her. In addition to this, I would like to thank Helen Coates, Monica Antenos, Richard Gilbert and Leanne Delaney for helping me with understanding new protocols and equipment. I would like to thank all my fellow colleagues and lab mates for providing me with a fun and positive atmosphere both in and outside the lab. I would like to acknowledge Amanda Barber, Nelson Ho, Amy Richard, Sonja Zours, Richard Gilbert, Fiasal Alibhai, Peter Podobed, Lindsay Robinson, Stacey Butler, Mai Jarad, Meghan Doerr, Jonathan Asling, and Sean Masson. I would like to especially acknowledge Leanne Delaney, someone who I could always count on for assistance and reinforcement. Finally, I would like to acknowledge my friends and family from far and wide for providing me with the support and encouragement that I needed to be successful in my degree. I would like to thank Vovo (Grandma), Anthony Piccolo, Alban Vuktilaj, Alessia Piccolo, Sandra Piccolo, Jack Piccolo and the entire Delaney Family. Most importantly, I would like to thank my mother, Elvira Farias, for always being there to support and motivate me. Thank you for always showing an interest and listening to me even though you don’t really understand what I do. My mother is the inspiration for everything that I accomplish and I owe all my success to her for raising me into who I am today. I can never thank you enough. iv DECLARATION OF WORK PERFORMED I declare that all work reported in this thesis was performed by me, with the exception of the items indicated below. Illumina HumanMethylation450K BeadChip array was done at the Genetic & Molecular Epidemiology Laboratory, Hamilton General Hospital, Hamilton, ON, and analyzed by Dr. Bekim Sadikovic (Assistant Professor Pathology and Molecular Medicine, McMaster University). v TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................ iii DECLARATION OF WORK PERFORMED ...........................................................................v TABLE OF CONTENTS ........................................................................................................... vi LIST OF FIGURES ................................................................................................................... viii LIST OF ABBREVIATIONS ..................................................................................................... ix INTRODUCTION ........................................................................................................................1 LITERATURE REVIEW .............................................................................................................4 Colorectal cancer ....................................................................................................................................4 Normal Colon Homeostasis .....................................................................................................................6 Hierarchical crypt organization ...........................................................................................................6 Identifying normal ISC .........................................................................................................................7 Normal colon stem cell maintenance ...................................................................................................8 Wnt ........................................................................................................................................................8 Notch ....................................................................................................................................................9 BMP ....................................................................................................................................................10 Hedgehog ............................................................................................................................................10 Cancer Stem Cells .................................................................................................................................11 CSC assays .........................................................................................................................................12 Colon CSCs .........................................................................................................................................14 CSC niche ...........................................................................................................................................14 CSC markers .......................................................................................................................................16 DNA Methylation ..................................................................................................................................18 DNA methylation associated genetic regulation ................................................................................18 Aberrant DNA methylation in cancer .................................................................................................20 Hypomethylation in CRC .....................................................................................................................21 Hypermethylation in CRC ..................................................................................................................22 Folate, DNA Methylation, and Cancer ...............................................................................................23 Folate cycle .........................................................................................................................................24 Folate and CRC incidence ..................................................................................................................25 RATIONALE ..............................................................................................................................29 MATERIALS
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