University of Dundee DOCTOR OF MEDICINE The Non-Wnt Functions of APC Unravelling the Link between APC and Apoptosis Cuddihy, Jane Award date: 2016 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 27. Sep. 2021 The Non-Wnt Functions of APC: Unravelling the Link between APC and Apoptosis Jane Cuddihy Doctor of Philosophy Supervisors: Kevin Hiom and Inke Näthke, April, 2016 Table of Contents List of Figures ................................................................................................................... 8 List of Tables .................................................................................................................. 11 Acknowledgements ......................................................................................................... 12 Declaration ..................................................................................................................... 13 Abstract .......................................................................................................................... 14 Abbreviations .................................................................................................................. 15 Chapter 1 – Introduction 1.1 Background on colorectal cancer .............................................................................. 21 1.2 Development of colorectal cancer ............................................................................. 21 1.3 Physiology of the human intestine ............................................................................. 24 1.4 Adenomatous polyposis coli (APC) ........................................................................... 24 1.4.1 APC and Wnt signalling .............................................................................. 28 1.4.2 APC and microtubules……………………………………………………………29 1.4.2.1 Microtubule poisons ..................................................................... 30 1.4.2.2 Taxol mechanism of action .......................................................... 31 1.4.2.3 Microtubules and the spindle assembly checkpoint ...................... 31 1.5 Cell death ................................................................................................................. 32 1.5.1 Apoptosis…………………………………………………………………………..32 1.5.1.1 The extrinsic pathway .................................................................. 33 1.5.1.2 Bcl-2 family proteins…………………………………………………...33 1.5.1.3 The intrinsic pathway ................................................................... 36 1.5.1.4 Crosstalk between the intrinsic and extrinsic pathways ................ 37 1.5.1.5 Small molecule inhibitors against pro-life proteins ........................ 37 1.5.1.5.1 ABT-737 ............................................................ 38 1 1.5.1.5.2 ABT-263 (Navitoclax) ........................................ 39 1.5.1.5.3 ABT-199 ............................................................ 39 1.5.1.5.4 GX-015-070 (obatoclax) .................................... 39 1.6 Cellular stress responses .......................................................................................... 40 1.7 Overall aims of the thesis .......................................................................................... 43 Chapter 2 - Materials and Methods 2.1 Materials 2.1.1 Antibiotics .............................................................................................................. 45 2.1.2 Drugs ..................................................................................................................... 45 2.1.3 Buffers and Stains .................................................................................................. 45 2.1.4 Antibodies .............................................................................................................. 50 2.1.5 Bacterial strains ..................................................................................................... 52 2.1.6 Plasmids ................................................................................................................ 52 2.1.7 Oligonucleotides .................................................................................................... 54 2.1.8 Matrigel .................................................................................................................. 55 2.2 Cell Culture Methods 2.2.1 Cell lines ................................................................................................................ 55 2.2.1.1 Suspension cells (DT40) .......................................................................... 59 2.2.1.2 Adherent cells .......................................................................................... 59 2.2.2 Organoid culture .................................................................................................... 60 2.2.2.1 Intestinal crypt isolatiion .......................................................................... 60 2.2.2.2 Culturing and passaging .......................................................................... 60 2.2.3 Transfection procedures ........................................................................................ 61 2 2.2.3.1 Transient transfection in suspension cells by electroporation .................. 61 2.2.3.2 Stable transfection for targeted insertion in DT40 Cells ........................... 61 2.2.3.3 Transient transfection in adherent cells by chemical methods ................. 62 2.2.3.4 Excision of floxed sequences by transient expression of Cre ................... 62 2.2.4 Subcloning by limiting dilution to derive clonally related cells from a heterogeneous population (DT40)................................................................................... 62 2.2.5 MTS cell viability assay .......................................................................................... 63 2.2.6 Organoid cell viability assay ................................................................................... 63 2.2.7 Hypoxia treatment .................................................................................................. 64 2.2.8 Mitochondrial staining ............................................................................................ 64 2.2.9 JC-1 Mitochondrial potential sensor ....................................................................... 64 2.3 Protein Methods 2.3.1 Cell lysis................................................................................................................. 65 2.3.2 Bradford assay ....................................................................................................... 65 2.3.3 Western blot ........................................................................................................... 65 2.3.4 Co-immunoprecipitation (CoIP) .............................................................................. 67 2.3.5 Proteasome inhibition............................................................................................. 68 2.3.6 The auxin-inducible degron system ........................................................................ 68 2.3.7 Mass spectrometry ................................................................................................. 68 2.3.8 Subcellular fractionation ......................................................................................... 69 2.3.9 Mitochondrial fractionation ..................................................................................... 70 2.3.10 Endoplasmic reticulum isolation ........................................................................... 71 2.3.11 Bcl-2 chemical inhibition using ABT-737 .............................................................. 71 3 2.4 DNA/RNA Methods 2.4.1 Genomic DNA extraction from DT40 cells .............................................................. 72 2.4.2 Screening for targeted insertion by gPCR .............................................................. 72 2.4.3 PCR product Blunt-end ligation and subsequent PCR product sequencing ............. 73 2.4.4 Restriction digest to linearize targeting vectors ...................................................... 74 2.4.5 Ethanol precipitation of targeting vectors................................................................ 74 2.4.6 DNA gel electrophoresis ........................................................................................ 75 2.4.7 Reverse-Transcription PCR (RT-PCR) in DT40 cells ............................................. 75 2.4.8 Quantitative Reverse-Transcription PCR................................................................ 76 2.4.9 RNAi .....................................................................................................................