Integrative Modelling of Glucocorticoid Induced Apoptosis with a Systems Biology Approach A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2012 Daphne Wei-Chen Chen 1 Table of Contents List of Abbreviations ..................................................................................................................... 7 List of Tables ................................................................................................................................ 11 List of Figures .............................................................................................................................. 12 Abstract ........................................................................................................................................ 14 Short Abstract ............................................................................................................................. 15 Declaration ................................................................................................................................... 15 Copyright Statement ................................................................................................................... 15 Acknowledgements ...................................................................................................................... 16 1 CHAPTER 1: Introduction .......................................................................................... 17 1.1 Glucocorticoids (GCs) .................................................................................................. 17 1.1.1 Structure............................................................................................................... 17 1.1.2 Physiological role of glucocorticoids .................................................................. 18 1.1.3 Risks of treatment with glucocorticoids side effects ........................................... 19 1.1.4 Acute Lymphoblastic Leukaemia (ALL) ............................................................ 20 1.2 The Glucocorticoid Receptor (GR) .............................................................................. 21 1.2.1 Expression of human glucocorticoid receptor gene ............................................ 21 1.2.2 GR Protein Structure ........................................................................................... 22 1.2.3 Mode of action of GR .......................................................................................... 25 1.3 GR: the transcription factor .......................................................................................... 27 1.3.1 General transcription ........................................................................................... 27 1.3.2 Transactivation .................................................................................................... 28 1.3.3 Transrepression .................................................................................................... 29 1.4 Post translational modifications .................................................................................... 31 1.5 GR cofactors ................................................................................................................. 33 1.5.1 Coactivators ......................................................................................................... 35 1.5.2 Corepressors ........................................................................................................ 37 1.6 GC induced apoptosis and resistance ........................................................................... 38 1.6.1 GC resistance ....................................................................................................... 38 1.6.2 GC induced apoptosis: extrinsic and intrinsic pathways ..................................... 39 1.7 Mechanism of GR induced apoptosis ........................................................................... 41 1.7.1 The Bcl-2 family: the pro- and anti-survival members ....................................... 41 1.7.2 Post translational regulation of the Bcl-2 family in apoptosis............................. 43 1.7.3 GR: Transcriptional regulator of the Bcl-2 family .............................................. 45 1.7.4 Bcl-XL .................................................................................................................. 47 2 1.7.5 Bim ...................................................................................................................... 48 1.7.6 Bmf ...................................................................................................................... 49 1.8 The AP-1 complex: Jun and Fos ................................................................................... 50 1.8.1 Jun ........................................................................................................................ 50 1.8.2 Fos ....................................................................................................................... 51 1.8.3 Crosstalk between AP-1 and GR ......................................................................... 52 1.9 The Ets proteins: Erg and its relevance to GR in ALL ................................................. 53 1.10 Systems Biology: The integrated approach to study gene network .............................. 55 1.10.1 Microarray, clustering and the identification of biomarkers ............................... 56 1.10.2 Overview: Gene network modelling.................................................................... 58 1.10.3 Systems biology approach and the kinetic modelling of GR .............................. 61 1.10.4 Modelling and Parameter Estimation tools ......................................................... 62 1.11 Hypothesis and Aims .................................................................................................... 63 1.12 References ..................................................................................................................... 65 2 CHAPTER 2: Quantitative analysis of glucocorticoid controlled genes ................. 80 2.1 Abstract ......................................................................................................................... 80 2.2 Introduction ................................................................................................................... 80 2.3 Material and Methods ................................................................................................... 83 2.3.1 Cell culture & treatments ..................................................................................... 83 2.3.2 Immunoblotting ................................................................................................... 83 2.3.3 Quantitative real-time PCR ................................................................................. 84 2.3.4 Statistical analysis................................................................................................ 84 2.3.5 Model development ............................................................................................. 85 2.3.6 Simulations .......................................................................................................... 86 2.4 Results........................................................................................................................... 86 2.4.1 Analysis of GR-mediated gene expression in GC-sensitive cell line CEM-C7-14 . ............................................................................................................................. 86 2.4.2 Analysis of GR-mediated gene expression in the GC-resistant cell line CEM-C1- 15 ............................................................................................................................. 90 2.4.3 Model simulations in CEM-C7-14 ...................................................................... 92 2.4.4 Model simulations in CEM-C1-15 ...................................................................... 99 2.5 Discussion ................................................................................................................... 101 2.6 Conclusion .................................................................................................................. 104 2.7 Supplementary data .................................................................................................... 106 2.7.1 Supplementary Tables ....................................................................................... 106 3 2.7.2 Supplementary figures ....................................................................................... 107 2.8 References ................................................................................................................... 110 CHAPTER 3: Erg and AP-1 as determinants of glucocorticoid response in acute lymphoblastic leukaemia ....................................................................................................... 115 3.1 Abstract ....................................................................................................................... 115 3.2 Introduction ................................................................................................................. 116 3.3 Material and Methods ................................................................................................. 117 3.3.1 Cell culture & treatments ..................................................................................