Lafferty-Whyte, Kyle (2010) Senescence signaling, regulation and bypass by telomere maintenance. PhD thesis. http://theses.gla.ac.uk/2212/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Senescence signaling, regulation and bypass by telomere maintenance Kyle Lafferty-Whyte BSc (Hons) Cancer Research UK Centre for Oncology and Applied Pharmacology University of Glasgow A thesis submitted to the University of Glasgow in partial fulfilment of the requirements for the Degree of Doctor of Philosophy April 2010 ii Abstract The permanent cell cycle arrest known as cellular senescence is a major block to tumorigenesis. Currently the effects of latent senescence signaling on disease progression, response to therapy and outcome are poorly understood. Furthermore, the role of microRNAs in the regulation of senescence remains to be fully elucidated. For immortalisation to occur replicative senescence must be bypassed usually by activating a telomere maintenance mechanism (TMM). However, the expression differences between TMMs are also poorly understood. To address these questions a combination of gene expression and miRNA microarray profiling, virtual drug and siRNA kinase screening were utilised. These findings highlight the distinct roles of secretory and damage associated senescence pathways in disease progression and in response to therapy. Examination of the differentially expressed genes between TMMs also highlighted a differentially expressed gene expression network surrounding TERT, regulated by c-Myc and TCEAL7 in TMMs. These findings give further insight into the complex regulation network surrounding senescence signaling during tumorigenesis. iii Table of Contents ABSTRACT II TABLE OF CONTENTS III LIST OF TABLES VI LIST OF FIGURES VII ABBREVIATIONS VIII ACKNOWLEDGEMENTS IX AUTHOR’S DECLARATION X 1 INTRODUCTION ...................................................................................................... 1 1.1 Cellular Senescence .............................................................................................................................. 1 1.1.1 Replicative Senescence ............................................................................................................................................... 3 1.1.2 Oncogene induced senescence ............................................................................................................................... 8 1.1.3 Stress induced senescence .................................................................................................................................... 11 1.2 Senescence pathways ......................................................................................................................... 11 1.2.1 Damage associated senescence ........................................................................................................................... 12 1.2.2 Secretory senescence ............................................................................................................................................... 12 1.2.3 Autophagy and senescence ................................................................................................................................... 16 1.3 Physiological Senescence: Senescence in vivo ............................................................................. 18 1.3.1 Senescence and Aging .............................................................................................................................................. 19 1.3.2 Senescence and Wound Healing ......................................................................................................................... 20 1.3.3 Senescence as a block to tumour progression. ............................................................................................ 20 1.4 Senescence bypass by telomere maintenance ............................................................................ 22 1.4.1 Telomere maintenance by telomerase ............................................................................................................ 22 1.4.2 Telomere maintenance by alternative lengthening of telomeres ....................................................... 26 1.5 Aims of this Study ................................................................................................................................ 32 2 MATERIALS AND METHODS .................................................................................. 33 2.1 Materials ................................................................................................................................................ 33 2.1.1 Tissue Culture Reagents and Consumables ................................................................................................... 33 2.1.2 Cell Lines ........................................................................................................................................................................ 34 2.1.3 Primary Cell Cultures ............................................................................................................................................... 35 2.1.4 Luciferase Reporter Plasmid Vectors ............................................................................................................... 36 2.1.5 Antibodies ...................................................................................................................................................................... 36 2.1.6 Kits, Reagents and Enzymes ................................................................................................................................. 36 2.1.7 Chemicals ....................................................................................................................................................................... 38 2.1.8 General laboratory supplies and reagents ..................................................................................................... 38 2.1.9 Oligonucleotide Primers for qPCR ..................................................................................................................... 39 2.1.10 Oligonucleotides for siRNA ............................................................................................................................. 40 2.1.11 Equipment ............................................................................................................................................................... 40 2.2 RNA Methods......................................................................................................................................... 41 2.2.1 RNA extraction ............................................................................................................................................................ 41 2.2.2 RNA quality assessment.......................................................................................................................................... 42 2.2.3 Gene expression microarray hybridisation, washing and scanning.................................................. 43 2.2.4 miRNA expression microarray hybridisation, washing and scanning ............................................. 43 2.2.5 cDNA synthesis by reverse transcriptase PCR ............................................................................................. 43 2.2.6 Polymerase Chain reaction .................................................................................................................................... 44 2.2.7 Quantitative polymerase chain reaction (qPCR) ........................................................................................ 44 2.2.8 qPCR validation of miRNA expression in mesenchymal tumours ...................................................... 46 iv 2.3 DNA techniques .................................................................................................................................... 46 2.3.1 Transformation of cells with Plasmid DNA ................................................................................................... 46 2.3.2 Glycerol stocks ............................................................................................................................................................. 47 2.3.3 Small scale preparation of plasmid DNA ........................................................................................................ 47 2.3.4 DNA sequencing .......................................................................................................................................................... 47 2.3.5 Large scale preparation of plasmid DNA ........................................................................................................ 47 2.3.6 Restriction Digests....................................................................................................................................................