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Molecular Mechanisms and Outcomes of Arsenic-Induced Histone Acetylation and Microrna Regulation in Cellular Transformation

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Molecular Mechanisms and Outcomes of Arsenic-Induced Histone Acetylation and Microrna Regulation in Cellular Transformation Molecular Mechanisms and Outcomes of Arsenic-Induced Histone Acetylation and MicroRNA Regulation in Cellular Transformation. By Sunniyat Rahman - A thesis submitted for the degree of Doctor of Philosophy (PhD). - Imperial College London Centre for Pharmacology and Therapeutics Department of Medicine - 2015 - 1 Abstract Chronic exposure to arsenic causes negative health outcomes, particularly malignant neoplasms of the skin, lung and bladder. Although epidemiological data has associated arsenic exposure to cancer, a clear molecular mechanism has remained elusive. This thesis studied the impact of As2O3 exposure on histone acetylation and microRNA expression at both tolerated and toxic levels in vitro to determine an epigenetic-based mechanism of carcinogenesis. This thesis outlines a framework for identifying tolerated and toxic As2O3 exposures, as a prerequisite to epigenetic characterisation. Tolerated As2O3 exposure increased cellular survival, anchorage-independent colony formation, cell-cycle progression and proliferation in HEK293T cells. HEK293T and UROtsa cells treated with tolerated As2O3 exhibited global H3K9 hyperacetylation at 3 hours and global H3K9 hypoacetylation at 72 hours. This was mediated by an imbalance in the intracellular HDAC2 to PCAF mRNA expression ratio. Global H3K9 hypoacetylation occurred for both tolerated and toxic exposures, giving poor mechanistic differentiation between these separated cellular outcomes. Chromatin immunoprecipitation identified PCAF recruitment, E2F1 binding and H3K9 acetylation at the FOS proto-oncogenic promoter leading to an elevation in FOS mRNA levels at the tolerated concentration only. This thesis also reports As2O3 induced chromatin relaxation in HEK293T cells followed by a return to nominal levels for the tolerated concentration. This is in contrast to the toxic exposure, which leads to clear chromatin condensation and apoptosis. This thesis postulates that arsenic-induced global H3K9 hypoacetylation is caused by a miR-372 -mediated attenuation mechanism targeting PCAF mRNA, as predicted through bioinformatic analysis. In summary, tolerated As2O3 exposure resulted in measurable perturbations in both global and promoter-specific histone acetylation in addition to the aberrant expression of microRNAs, which led to cellular transformation over toxicity. Sunniyat Rahman | Imperial College London 2 Declaration of Originality I, Sunniyat Rahman hereby declare that the work submitted in this thesis is my own and entirely original. This thesis has not and will not be submitted for any other degree at any other institution. Where figures, graphics, data and materials have been provided from an external source, a citation has been made to acknowledge such a contribution. A full reference list is provided at the end of this thesis with regards to in-text citations. Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Sunniyat Rahman | Imperial College London 3 Contents ABSTRACT ............................................................................................................. 2 DECLARATION OF ORIGINALITY ................................................................... 3 COPYRIGHT DECLARATION ............................................................................ 3 CONTENTS ........................................................................................................... 4 FIGURES & TABLES ............................................................................................. 9 DEDICATION ....................................................................................................... 13 ACKNOWLEDGEMENTS ................................................................................... 14 PUBLICATIONS, CONFERENCES & GRANTS .............................................. 15 Publications related to this thesis: .................................................................................................... 15 Publications via collaborations: ........................................................................................................ 15 Conferences ...................................................................................................................................... 16 Grants ................................................................................................................................................ 16 ABBREVIATIONS ................................................................................................ 17 INTRODUCTION ................................................................................................ 22 1.1 – Chemistry and Behaviour of Arsenic in Brief. .......................................................................... 22 1.2 – Sources of Human Exposure to Arsenic. ................................................................................. 25 1.3 – Epidemiological Evidence of Arsenic-Induced Negative Health Outcomes ......................... 26 1.4 – Metabolism of Arsenic .............................................................................................................. 30 1.5 – Possible Mechanisms of Arsenic-induced Toxicity and Carcinogenicity ............................... 34 1.6 – Epigenetic Mechanisms ........................................................................................................... 37 1.6.1 – DNA methylation ..................................................................................................................................... 37 1.6.2 – Histone post-translational modifications .............................................................................................. 39 1.6.3 – microRNAs ................................................................................................................................................ 44 Sunniyat Rahman | Imperial College London 4 1.7 – Epigenetics: A Suitable and Challenging Approach to Interrogate Arsenic-induced Carcinogenesis .................................................................................................................................. 47 1.8 – Arsenic-induced Perturbations of DNA methylation and Associated Outcomes. .................. 51 1.8.1 – Global DNA methylation levels are affected by arsenic exposure. .................................................. 51 1.8.2 – Promoter-specific DNA methylation effects following arsenic exposure. ...................................... 53 1.8.3 – DNA methylation effects following in utero arsenic exposure. .......................................................... 58 1.9 – Arsenic-induced Changes of Histone Post-translational Modifications. ................................ 60 1.9.1 – Arsenic-induced effects on global histone post-translational modifications. .................................. 60 1.9.2 – Arsenic-induced histone post-translational modification effects at promoter loci. ....................... 62 1.9.3 – Arsenic-induced interference of histone modifying enzymes and proteins that regulate chromatin structure. .................................................................................................................................................................. 63 1.10 – Disruptions in MicroRNA expression Following Arsenic Exposure and Implications. ....... 64 1.11 – Combined Epigenetic Effects Following Arsenic Exposure. ................................................. 67 1.12 – Arsenic Exposure in a Therapeutic Context and Associated Epigenetic Effects. ................. 69 HYPOTHESIS AND AIMS ................................................................................... 72 MATERIALS AND METHODS ........................................................................... 74 3.1 – Cells, Culture Conditions and Harvesting ............................................................................... 74 3.2 – Toxicants and Cell Treatments ................................................................................................ 76 3.3 – Protein Extraction .................................................................................................................... 76 3.4 – Histone Extraction ................................................................................................................... 76 3.5 – Bradford Assay of Protein ........................................................................................................ 77 3.6 – Immunoblotting of Histones and Other Proteins ................................................................... 77 3.7 – Real-Time Quantitative Reverse Transcription PCR .............................................................. 79 3.8 – Clonogenic Assay ..................................................................................................................... 83 3.9 – Cell Proliferation Assay ............................................................................................................ 84 3.10 – Flow Cytometry Assays: Quantification of Mitochondrial ROS, Mitochondrial Membrane Potential (∆ψm), Cell-Cycle analysis and Caspase Activation .........................................................
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