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Regulation of Cancer Cell Proliferation and Mitosis by NF-Κb

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Regulation of Cancer Cell Proliferation and Mitosis by NF-Κb Regulation of cancer cell proliferation and mitosis by NF-κB Adeline LEDOUX Thesis submitted for the degree of Doctor of Philosophy Newcastle University Faculty of Medical Sciences Institute for Cell and Molecular Biosciences September 2012 Table of Contents List of Illustrations ...................................................................................... vi Acknowledgements .................................................................................... ix Abstract .......................................................................................................... xi Abbreviations .............................................................................................. xii 1 - Introduction .............................................................................. 1 1.1 The transcription factor NF-κB ................................................................ 1 1.1.1 Transcription and transcription factors ............................................. 1 1.1.2 Overview of the NF-κB pathway ....................................................... 2 1.1.3 The IκB family .................................................................................. 6 1.1.4 The IKK family .................................................................................. 9 1.1.5 Canonical pathway of NF-κB activation ......................................... 11 1.1.6 Activation of the non-canonical NF-κB pathway ............................. 13 1.1.7 Processing of p100 in p52 .............................................................. 16 1.1.8 Other NF-κB activation pathways ................................................... 17 1.1.9 DNA binding ................................................................................... 19 1.1.10 Post transcriptional modifications of NF-κB ................................. 20 1.1.11 NF-κB target genes ...................................................................... 23 1.1.12 Opposing roles for NF-κB subunits .............................................. 24 1.2 The cell cycle ........................................................................................ 26 1.2.1 The regulation of the cell cycle ....................................................... 28 1.2.2 NF-κB and the regulation of the cell cycle ...................................... 30 1.3 The centrosome .................................................................................... 31 1.3.1 Centrosome duplication cycle ........................................................ 31 1.3.2 The spindle checkpoint ................................................................... 35 1.3.3 The PLK family ............................................................................... 37 i 1.3.4 NF-κB and regulation of centrosome duplication ........................... 41 1.4 Cancer .................................................................................................. 43 1.4.1 The Hallmarks of cancer ................................................................ 43 1.4.2 NF-κB in cancer ............................................................................. 46 1.4.3 NF-κB knockout mice ..................................................................... 48 1.5 Preliminary data .................................................................................... 49 1.6 Aims of the thesis .................................................................................. 51 2 Materials and Methods ............................................................. 52 2.1 Cell culture ............................................................................................ 52 2.1.1 Tissue culture cell lines and growth condition ................................ 52 2.1.2 Frozen cell storage ......................................................................... 52 2.1.3 RNAi transfection ........................................................................... 53 2.1.4 DNA transfection ............................................................................ 54 2.1.5 CDK1 inhibitor ................................................................................ 54 2.1.6 Cell synchronization ....................................................................... 54 2.1.7 Cell proliferation assay ................................................................... 55 2.1.8 Immunofluorescence ...................................................................... 55 2.1.9 Fluorescence Activated Cell Sorting (FACS) ................................. 56 2.1.10 Reporter gene assay .................................................................... 56 2.1.11 Chromatin ImmunoPrecipitation (ChIP) ........................................ 56 2.2 Protein and RNA extracts ..................................................................... 58 2.2.1 Protein extracts .............................................................................. 58 2.2.2 Western Blotting ............................................................................. 58 2.2.3 Antibodies ....................................................................................... 59 2.2.4 RNA extracts .................................................................................. 61 2.2.5 Reverse transcription ..................................................................... 61 2.2.6 Quantitative Polymerase Chain Reaction (PCR) ........................... 61 2.3 Molecular Biology techniques ............................................................... 63 ii 2.3.1 Escherichia coli growth conditions ................................................. 63 2.3.2 Transformation of competent E. coli cells ...................................... 63 2.3.3 Minipreparation of plasmid DNA ..................................................... 63 2.3.4 Maxipreparation of plasmid DNA ................................................... 64 2.3.5 Quantification of DNA and RNA ..................................................... 64 2.3.6 Agarose Gel electrophoresis .......................................................... 64 2.3.7 Purification of DNA fragments from agarose gels .......................... 65 2.3.8 Digestion of DNA with restriction endonucleases .......................... 65 2.3.9 Phosphatase treatment of linearized DNA ..................................... 65 2.3.10 Ligations ....................................................................................... 65 2.3.11 DNA sequencing .......................................................................... 66 2.3.12 Plasmids ....................................................................................... 66 3 Results Chapter 1: Characterization of the p52 effect .......... 69 3.1 Introduction ........................................................................................... 69 3.2 Results .................................................................................................. 70 3.2.1 Co-localization of p100/p52 with the centrosome .......................... 70 3.2.2 p52 affects cyclin and CDK gene expression ................................. 73 3.2.3 Genes involved in the centrosome duplication ............................... 75 3.2.4 NF-κB affects PLK4 expression ..................................................... 77 3.2.5 p52 and PLK4 affect cell proliferation ............................................ 82 3.3 Discussion ............................................................................................. 82 3.4 Conclusion ............................................................................................ 88 4 – Results Chapter 2: PLK4 regulation .................................... 89 4.1 Introduction ........................................................................................... 89 4.2 Results .................................................................................................. 90 4.2.1 The PLK4 promoter ........................................................................ 90 4.2.2 PLK4 is a direct NF-κB target gene ................................................ 91 4.2.3 p52 binding on the PLK4 promoter is reversible ............................ 95 iii 4.2.4 PLK4 is a target for exogenously expressed p52 ........................... 98 4.2.5 PLK4 is a NF-κB target gene in mouse .......................................... 98 4.2.6 Luciferase constructs of the PLK4 promoter ................................ 102 4.2.7 NF-κB subunit regulation of the PLK4 promoter .......................... 104 4.2.8 Cooperation between NF-κB subunits ......................................... 106 4.2.9 Depletion of multiple NF-κB subunits ........................................... 106 4.2.10 Differential effect of RelB depletion with PLK4 promoter deletions 109 4.2.11 NF-κB subunit effects on the PLK4 promoter deletion constructs 109 4.3 Discussion ........................................................................................... 112 4.4 Conclusion .......................................................................................... 117 5 – Results Chapter 3: Cell cycle regulation ........................... 118 5.1 Introduction ......................................................................................... 118 5.2 Results ................................................................................................ 119 5.2.1 Cell synchronization ..................................................................... 119 5.2.2 PLK4 and p52 levels during the cell cycle ...................................
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