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Dissecting the Telomere-Independent Pathways Underlying Human Cellular Senescence

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Dissecting the Telomere-Independent Pathways Underlying Human Cellular Senescence Dissecting the Telomere-Independent Pathways Underlying Human Cellular Senescence By Emilie Marie Isabelle Rovillain A thesis submitted to the University College of London for the degree of Doctor of Philosophy Department of Neurodegenerative diseases Institute of Neurology UCL Queen Square London WC1 3BG 2010 ABSTRACT Cellular senescence is an irreversible program of cell cycle arrest triggered in normal somatic cells in response to a variety of intrinsic and extrinsic stimuli including telomere attrition, DNA damage, physiological stress and oncogene activation. Finding that inactivation of the pRB and p53 pathways by SV40-LT antigen cooperates with hTERT to immortalize cells has allowed us to use a thermolabile mutant of SV40- LT to develop human fibroblasts where the cells are immortal if grown at 34oC but undergo an irreversible growth arrest within 5 days at 38oC. When these cells cease dividing, senescence-associated-β-galactosidase (SA-β-Gal) activity is induced and the growth-arrested cells have many features of senescent cells. Since these cells growth-arrest in a synchronous manner, I have used Affymetrix expression profiling to identify the genes differentially expressed upon senescence. This identified 816 up- and 961 down-regulated genes whose expression was reversed when growth arrest was abrogated. I have shown that senescence was associated with activation of the NF-B pathway and up-regulation of a number of senescence-associated-secretory- proteins including IL6. Perturbation of NF-κB signalling either by direct silencing of NF- B subunits or by upstream modulation overcame growth-arrest indicating that activation of NF-B signalling has a causal role in promoting senescence. I also applied a retroviral shRNA screen covering ~10,000 genes to the same cell model. Overlapping with the microarray data revealed particularly interesting targets, such as LTBP3 and Layilin. Finally, I profiled micro-rna expression. 15 of the top micro-rnas down-regulated upon senescence were chosen to express in the HMF3A system. 6 of them were able to bypass the growth-arrest. In conclusion, my work has uncovered novel markers involved in senescence as well as identifying that both activation of p53 and pRb pathway result in activation of NF-B signalling which promotes senescence. Both results lead to a better understanding of senescence and its pathways. 2 TABLE OF CONTENTS ABSTRACT .................................................................................................................... 2 TABLE OF CONTENTS ................................................................................................ 3 LIST OF FIGURES ....................................................................................................... 14 LIST OF TABLES ........................................................................................................ 17 LIST OF ABBREVIATIONS ........................................................................................ 18 ENCLOSED UNBOUND MATERIAL ......................................................................... 22 STATEMENT CONCERNING COLLABORATIONS ................................................. 22 ACKNOWLEDGEMENTS ........................................................................................... 22 1 INTRODUCTION ................................................................................................. 24 1.1 REPLICATIVE SENESCENCE DISCOVERY ............................................. 24 1.2 DEFINITION OF SENESCENCE ................................................................. 25 1.3 TELOMERE INDUCED SENESCENCE ...................................................... 25 1.3.1 Telomeres .................................................................................................. 25 1.3.2 Telomeres and DDR ................................................................................... 27 1.3.3 hTERT ....................................................................................................... 29 1.3.4 Telomerase and tumourigenesis.................................................................. 30 1.4 DNA DAMAGE INITIATED SENESCENCE ............................................... 30 1.5 ONCOGENE-INDUCED SENESCENCE ..................................................... 32 1.6 CANCER AND SENESCENCE .................................................................... 33 1.7 AGEING AND SENESCENCE ..................................................................... 33 1.8 PATHWAYS OF SENESCENCE .................................................................. 34 1.8.1 The p53 pathway ........................................................................................ 34 1.8.1.1 TP53 gene and p53 protein ................................................................. 35 1.8.1.2 Functions of the p53 protein ............................................................... 35 1.8.1.3 Regulation of p53 activity................................................................... 36 1.8.1.4 p19Arf protein ...................................................................................... 38 1.8.1.5 Oncogenic Ras ................................................................................... 40 1.8.1.6 p21WAF1/Cip1/Sdi1 ................................................................................... 40 1.8.1.7 p53 family: p63 and p73 proteins ........................................................ 41 1.8.2 The pRb pathway ....................................................................................... 42 1.8.2.1 Cell cycle, cyclins and CDKs ............................................................. 42 1.8.2.2 CDK inhibitors ................................................................................... 42 1.8.2.3 CDKs and E2F ................................................................................... 43 1.8.2.4 Rb family of proteins .......................................................................... 43 1.8.2.5 pRb gene ............................................................................................ 44 1.8.2.6 pRb discovery .................................................................................... 44 1.8.2.7 pRb function ....................................................................................... 45 1.8.2.8 E2F .................................................................................................... 47 1.8.3 Common pathways ..................................................................................... 48 1.8.3.1 INK4A Locus ..................................................................................... 48 1.8.3.2 p16INK4a .............................................................................................. 49 1.8.3.3 p14ARF ................................................................................................ 50 1.9 DNA TUMOUR VIRUSES ........................................................................... 52 1.9.1 SV40 .......................................................................................................... 52 1.9.2 LT .............................................................................................................. 53 1.9.3 Adenovirus Type 5 ..................................................................................... 55 1.9.3.1 E1A .................................................................................................... 55 1.9.3.2 E1B .................................................................................................... 56 1.9.4 HPV Type 16 ............................................................................................. 56 1.9.4.1 E7 ...................................................................................................... 57 1.9.4.2 E6 ...................................................................................................... 58 1.10 SASP: SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE AND ROS: REACTIVE OXYGEN SPECIES .................................................................... 58 1.11 NF-B PATHWAY ....................................................................................... 60 1.11.1 Introduction ............................................................................................... 60 1.11.2 NF-κB family ............................................................................................. 61 1.11.3 Activation .................................................................................................. 62 1.11.4 Inhibition ................................................................................................... 63 1.11.4.1 The IκB family ............................................................................... 63 1.11.4.2 IκB kinase: IKK............................................................................. 63 1.11.5 Canonical NF-B pathway ......................................................................... 65 1.11.6 Non-canonical pathway .............................................................................. 66 1.11.7 NF-B and cancer ...................................................................................... 68 1.11.8 NF-B, senescence and ageing ................................................................... 68 1.12 MICRO-RNAS .............................................................................................. 69 4 1.12.1 Introduction ..............................................................................................
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