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The Effects of Leptomycin B on Hpv-Infected Cells

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The Effects of Leptomycin B on Hpv-Infected Cells THE EFFECTS OF LEPTOMYCIN B ON HPV-INFECTED CELLS Carol Elizabeth Jolly A Thesis Submitted for the Degree of PhD at the University of St Andrews 2008 Full metadata for this item is available in St Andrews Research Repository at: http://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/900 This item is protected by original copyright This item is licensed under a Creative Commons Licence The Effects of Leptomycin B on HPV-Infected Cells By Carol Elizabeth Jolly A thesis submitted to the University of St Andrews in partial fulfilment of the requirement of the degree of Doctor of Philosophy Bute Medical School October 2008 Contents Declaration ii Copyright declaration iii Acknowledgements iv – v Abbreviations vi - viii Abstract ix Table of Contents x - xiii List of Figures xiv - xvi List of Tables xvi i Declaration I, Carol Elizabeth Jolly, hereby certify that this thesis, which is approximately 58,000 words in length, has been written by me, that it is the record of work carried out by me and that it has not been submitted in any previous application for a higher degree. I was admitted as a research student in September, 2004 and as a candidate for the degree of Doctor of Philosophy Medicine in October, 2005; the higher study for which this is a record was carried out in the University of St Andrews between 2004 and 2008. Date I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of Doctor of Philosophy Medicine in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. Date ii Copyright Declaration In submitting this thesis to the University of St Andrews we understand that we are giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. We also understand that the title and the abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker, that my thesis will be electronically accessible for personal or research use unless exempt by award of an embargo as requested below, and that the library has the right to migrate my thesis into new electronic forms as required to ensure continued access to the thesis. We have obtained any third-party copyright permissions that may be required in order to allow such access and migration, or have requested the appropriate embargo below. The following is an agreed request by candidate and supervisor regarding the electronic publication of this thesis: Embargo on both all or part of printed copy and electronic copy for the same fixed period of 5 years on the following ground: publication would preclude future publication; Date Signature of candidate Signature of supervisor iii Acknowledgements I would like to express my deepest thanks to my supervisor Prof. Simon Herrington. Without his support, guidance, and unrelenting kindness and patience the completion of my research and subsequent thesis would never have been possible. Many thanks must also go to Dr Sonia Lain for providing endless practical advice and materials. I would also like to thank the members of the Bute past and present, particularly the members of E-floor who have, over the years, provided a wealth of advice, assistance and support, whilst also proving to be a brilliant group of friends. In particular I would like to thank Dr Lindsey Gray for guidance during my research, Drs Katherine Feeney and Lissa Herron for their patience and help with my thesis and many other things, and Drs Keeley Brookes, Yimin Ren, Fleur Davey and Mrs Tina Briscoe for answering lots of questions and providing great banter and good times. No research within the Bute could possibly have taken place without the efficiency, assistance and professionalism of the Bute stores team: Iain Grieve, Alex Taylor and Graeme Russell. Many thanks must also go to the University Dyslexia Adviser, Janice McGregor and the student support team. Janice’s practical assistance, support and guidance, in combination with the natural friendliness of the whole team, have made coping with my dyslexia a much more manageable task (even although I still can’t spell!!!). iv After spending so many years in St Andrews, I would like to thank all those who have made it such a great place to be. In particular, Dawn Rimmer, a great friend through bad times and good. Finally thanks must also go to my whole family, especially my Mum and Dad for their support, encouragement and love. Their faith in me throughout my academic career has allowed me to pursue the subject I love. P.S. I haven’t cured cancer yet!! v List of Abbreviations Act-casp-3 Activated caspase-3 AEC 3-amino-9-ethylcarbazole AIF Apoptosis-inducing factor AMV Avian Myeloblastosis Virus ANT Adenine nucleotide translocator APAF1 Apoptotic protease activating factor-1 ATP Adenosine triphosphate AU Arbitrary units B2M Beta-2-microglobulin BAD Bcl-2-antagonist of cell death BAK Bcl-2 homologous antagonist/killer BAX Bcl-2 associated X Bcl-2 B-cell leukaemia/lymphoma 2 BH Bcl-2 homology BID Bcl-2 Interacting Domain BIM Bcl-2-interacting mediator of cell death BOK Bcl-2-related ovarian killer BSA Bovine serum albumen C/EBPα CCAAT enhancer-binding protein α CAD Caspase-activated deoxyribonuclease CBP CREB binding protein cdk Cyclin dependent kinase cDNA Reverse transcribed mRNA CFS Common fragile sites Ci Curie CIAP Calf intestinal alkaline phosphatase CIN Cervical intraepithelial neoplasia CK18 Cytokeratin 18 CKI Cyclin-dependent kinase inhibitor CKI Cyclin dependent kinase inhibitors CMV Cytomegalovirus COSHH Control of Substances Hazardous to Health CRE cis regulatory element CRIME CRM1, importin β, etc CRM1 Chromosome region maintenance 1 CRPV Cottontail Rabbit papillomavirus dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate DIABLO Direct IAP binding protein with low pI DISC Death –inducing signalling complex DMEM Dulbecco’s modified essential medium DNA Deoxyribonucleic acid DNA Deoxyribonucleic acid Dnp53 Dominant negative p53 plasmid pBabe-R248W dTTP Deoxythymidine triphosphate EDTA Ethylenediaminetetraacetic acid vi EGF Epidermal growth factor EGFR Epidermal growth factor receptor EV Epidermodysplasia verruciformis FACS Fluorescence-Activated Cell Sorting FADD Fas-associated death domain protein Fas-L Fas ligand FLIP FADD-like interleukin-1 -converting enzyme-like protease gDNA Genomic DNA GDP Guanidine diphosphate GFP Green fluorescent protein GTP Guanidine Triphosphate h Hours HBSS Hanks’ balanced salts solution HCL Hydrochloric acid hDLD Drosophila discs large HECT Homologous to the E6-AP carboxyl terminal HIV Human immunodeficiency virus HPV Human papillomavirus HRP Horse radish peroxidase HSIL high grade squamous intraepithelial lesion HSV-1 Herpes simplex virus type 1 hTERT Human reverse transcriptase IAP Inhibitor of apoptosis proteins ICAD Caspase-activated deoxyribonuclease inhibitor IGF Insulin-like growth factor IMM Inner mitochondrial membrane KGM2 Keratinocyte growth medium 2 LCR Long control region LLETZ Large loop excision of the transformation zone LMB Leptomycin B LSIL Low grade squamous intraepithelial lesion MAPK Mitogen-activated protein kinase MHC I Major histocompatibility complex class I Mo-MuLV Moloney murine leukaemia virus mPBST PBST with milk Mtc Mitomycin C NaCl Sodium chloride NaOH Sodium NES Nuclear export signal NF-κB nuclear factor kappa B NLS Nuclear localisation signal NNK Nitrosamine 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone nt Nucleotides OMM Outer mitochondrial membrane ORF Open reading frame PAC1 Phosphatase of activated cells 1 Pap Papanicolaou PBS Phosphate buffered saline PBST PBS with Tween-20 PBT 1xPBS with BSA vii PCNA Proliferating cell nuclear antigen PCR Polymerase Chain Reaction PDGF Platelet derived growth factor PDVF Polyvinylidene Fluoride PDZ PSD95/Discs Large/ZO-1 PHK Primary human keratinocyte PI3K Phosphatidylinositol 3-kinase PKB Protein kinase B POD Promyelocytic leukaemia oncogenic domains pRb Retinoblastoma protein PTEN Phosphatase and tensin homolog PTPC Permeability transition pore complex RAN-GDP Ran associated GDP RAN-GTP Ran associated GTP ROCK1 Rho kinase 1 RSB Reducing sample buffer RT Reverse transcription RT-PCR Reverse transcription PCR SAPK Stress-activated protein kinase SDS Sodium Dodecyl Sulphate SDS-PAGE Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis SF2/ASF Splicing Factor 2/Alternative Splicing Factor SIL Squamous intraepithelial lesion siRNA Small interfering RNA SMAC Second mitochondria-derived activator of caspase snRNPs Small nuclear ribonucleoprotein particles SR Serine/arginine SSC Saline-Sodium Citrate TBE Tris buffered EDTA TBP TATA-box-binding protein TBS Tris buffered saline TE Trypsin-EDTA TGF-β Transforming growth factor-β TNF-R Tumour necrosis factor receptor TNF-α Tumour necrosis factor-α TNS Trypsin neutralising solution TopBP1 DNA topoisomerase II-beta-binding protein 1 TRAIL- R1 TNF-related apoptosis-inducing ligand-R1 TSA Trichostain A URR Upstream regulatory region UVC Ultraviolet C v/v Volume to volume v/w Volume to weight VLP Virus-like particles vp pBabe-puro vector only wt Wild type viii Abstract Cervical cancer is a major cause of death in women and is strongly associated with infection by human papillomavirus (HPV). Integration of HPV is thought to form a key step in the formation of cancer, and is thought to involve the upregulation of HPV E6 and E7 due to the loss of E2 transcriptional control. Leptomycin B (LMB), a nuclear export inhibitor, has previously been shown to induce apoptosis in HPV-containing cancer cell lines and HPV 16 E7 or E6/E7 transduced primary keratinocytes, but not in normal cells. This thesis shows that LMB can induce apoptosis and a reduction in the colony survival of derivatives of the W12 cell line that contain HPV 16 in either episomal or integrated form.
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