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Cyclin A/CDK2 Regulates Multiple G2 Phase Pathways

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Cyclin A/CDK2 Regulates Multiple G2 Phase Pathways Cyclin A/CDK2 regulates multiple G2 phase pathways Vanessa Oakes BSc (Hons) A thesis submitted to Queensland University of Technology in fulfilment of the requirements for the degree of Doctor of Philosophy AUGUST 2012 Diamantina Institute for Cancer, Immunology and Metabolic Medicine University of Queensland, Princess Alexandra Hospital, Woolloongabba, QLD, Australia and Queensland University of Technology, School of Biomedical Sciences, Faculty of Health, Brisbane, QLD, Australia i TABLE OF CONTENTS Statement of originality ............................................................................. Error! Bookmark not defined. Contribution by other colleagues .............................................................. Error! Bookmark not defined. Acknowledgements .................................................................................................................................. vi Abstract .................................................................................................................................................. viii Publications ............................................................................................................................................. xii List of Abbreviations .............................................................................................................................. xiii List of Figures ........................................................................................................................................ xvii List of Tables ......................................................................................................................................... xixx Chapter 1 Summary of the literature. ................................................................................................. 1-54 1.1 The cell cycle, structure and function. ......................................................................................... 2 1.1.1 Stages of mitosis ................................................................................................................... 2 1.2 Cancer and the cell cycle .............................................................................................................. 6 1.3 Regulation of cell cycle................................................................................................................. 7 1.3.1 The cyclin/CDK complexes .................................................................................................... 8 1.3.2 Cell cycle checkpoints ......................................................................................................... 13 1.4 Exploiting the DNA damage checkpoints, a cancer therapy. ..................................................... 17 1.5 DNA damage pathways .............................................................................................................. 18 1.5.1 ATM/ATR ............................................................................................................................. 20 1.5.2 Chk1 and Chk2..................................................................................................................... 24 1.5.3 p53 ...................................................................................................................................... 27 1.5.4 Cdc25 ................................................................................................................................... 28 1.6 Other important cell cycle regulators ........................................................................................ 32 1.6.1 p38 MAPK ............................................................................................................................ 32 1.6.2 Claspin ................................................................................................................................. 35 1.6.3 Wee1 ................................................................................................................................... 37 1.6.4 Cdh1 .................................................................................................................................... 38 1.6.5 Plk1 ...................................................................................................................................... 40 ii 1.7 Checkpoint recovery ................................................................................................................... 42 1.8 G2/M regulation by cyclin A/CDK2 and cyclin B/CDK1 .............................................................. 44 1.9 Chk1 regulation .......................................................................................................................... 47 1.9.1 The role of Chk1 during an unperturbed cell cycle ............................................................. 50 1.9.2 Chk1 inactivation ................................................................................................................. 52 1.10 Hypothesis ................................................................................................................................ 53 1.11 Significance ............................................................................................................................... 54 1.12 Aims of the study ...................................................................................................................... 54 Chapter 2 Materials and Methods .................................................................................................... 55-82 2.1 Antibodies ................................................................................................................................... 56 2.2 Cell Biology Methods. ................................................................................................................. 58 2.2.1 Cell culture ........................................................................................................................... 58 2.2.2 Cell Synchronisation. ........................................................................................................... 59 2.2.3 Cell Treatments. .................................................................................................................. 59 2.2.4 Transfection Treatments. .................................................................................................... 60 2.2.5 Cell fixation and immunofluorescent labelling. ................................................................... 62 2.2.6 Microscopy and image acquisition ...................................................................................... 65 2.2.7 FACS Analysis ....................................................................................................................... 66 2.2.8 Cellomics .............................................................................................................................. 68 2.3 Molecular Biology Methods ....................................................................................................... 69 2.3.1 Plasmid list ........................................................................................................................... 69 2.3.2 Plasmid construct cloning........................................................................................................ 74 2.3.2.1. Generation of Cherry-Chk1 and GST-Chk1 constructs. ................................................... 74 2.3.2.1 Generation of PBD-GFP .................................................................................................... 75 2.4 Protein Biochemistry Methods. .................................................................................................. 76 2.4.1 Immunoblot Analysis ........................................................................................................... 76 2.4.2 Immunoprecipitation ........................................................................................................... 77 2.4.3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses ........................................................................................................................................ 79 2.4.4 Cyclin A/CDK2 kinase assay ................................................................................................. 80 Chapter 3 Active Chk1 is present in unperturbed G2 phase ........................................................... 83-111 3.1 INTRODUCTION .......................................................................................................................... 84 iii 3.2 RESULTS...................................................................................................................................... 87 3.2.1 Activated Chk1 is present during an unperturbed cell cycle. ............................................. 87 3.2.2 ATR is responsible for Chk1 phosphorylation in an unperturbed cell cycle during G2 phase. ..................................................................................................................................................... 97 3.2.3 pChk1 localises to the centrosomes during mitosis. ......................................................... 101 3.3 DISCUSSION .............................................................................................................................. 104 Chapter 4 Characterising the cyclin A/CDK2 dependent G2 phase delay
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