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The Regulation of P53 Transcriptional Activity by Hnrnpul-1 and the DNA Damage Response Induced by a Novel

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The Regulation of P53 Transcriptional Activity by Hnrnpul-1 and the DNA Damage Response Induced by a Novel The regulation of p53 transcriptional activity by hnRNPUL-1 and the DNA damage response induced by a novel chemotherapeutic agent, ALX By Anoushka Thomas A thesis presented to the University of Birmingham, for the degree of Doctor of Philosophy School of Cancer Sciences School of Medical and Dental Sciences University of Birmingham University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Summary The tumour suppressor, p53, is a vital DNA damage response protein and its means of transcriptional regulation are vast. hnRNPUL-1 is a multifunctional protein and previous studies have implicated it in the modulation of p53 transcriptional activity, although this has been rather poorly understood. Results presented here demonstrate that hnRNPUL-1 represses p53 transcriptional activity and negatively regulates p21 levels. This is consistent with the depletion of hnRNPUL-1 leading to an increase in cells arrested in G1/S. Together these results confirm that hnRNPUL-1 acts as a p53 co-repressor with specific cellular targets. Mutations in p53 and other DNA damage response proteins not only often contribute to the onset of tumourigenesis but can also be the cause of drug resistance during treatment. The development of a novel chemotherapeutic agent, Alchemix (ALX), was based on the requirement for effective treatment in the face of resistance mechanisms. Little has been known about the mechanism of action of ALX up to now. Findings here demonstrate that ALX treatment primarily induces an ATR-dependent DNA damage response that occurs specifically in cycling cells and culminates in cell death via mitotic catastrophe. Results also show that the response elicited by ALX requires TOP2α and TOP2β, as well as its alkylating ability, but does not involve ATM, p53, or components of the MMR and FA pathway. Therefore, ALX has the potential to treat tumours that have developed resistance to conventional chemotherapeutic drugs. i To my parents ii Acknowledgements I would like to acknowledge my two supervisors, Dr Roger Grand and Dr Grant Stewart. I would like to thank Grant for his help with the ALX work. Special thanks must go to Rog who has helped me so much throughout my entire PhD. He has always supported and motivated me, especially during the most frustrating times. I would also like to thank Klaus Pors for enabling me to work on a novel chemotherapeutic agent. I would like to thank Natasha and Rachel, particularly during my last few fraught months in the lab! I would also like to thank Helen for all her help and advice, and Ed for being such a good friend and for always making me laugh. I would especially like to thank Kelly for her constant support and friendship, which has meant so much. I would like to thank Angelo for his help with the qRT-PCR, as well as Tracey for her expertise in the mouse work. A big thank you goes to Sarah for all the help with the FACS. I would also like to thank Malcolm for funding me for the last few months, and the Medical School who supported my research. Thank you to all my friends for their encouragement. I would like to say thank you to Dave for his friendship and help. Special thanks must go to Jen for always being there in good times and bad, and to Eish for her constant support all the way through. I would like to say a massive thank you to Stu for the most incredible support and patience during this time. He has (almost!) always managed to calm me down and words cannot express how much his help has meant to me. Finally, I would like to thank my family. To Ellie who kept me company during the writing process. To my two sisters, thank you for always supporting and encouraging me, no matter what I do. And to my parents, to whom this is dedicated, thank you for your undivided, unquestioning love and support, always. iii Abbreviations 53BP1 p53 binding protein 1 3meA N3-methyl adenine 7meG N7-methyl guanine 9-1-1 RAD9-RAD1-HUS1 ALKBH2/3 alpha-ketoglutarate-dependent dioxygenase alkB homologues 2/3 Alt-NHEJ alternative NHEJ ALX Alchemix AP apurinic/apyrimidinic site APS ammonium persulphate ASPP apoptosis-stimulating proteins of p53 A-T ataxia-telangiectasia ATLD ataxia-telangiectasia-like disorder ATM ataxia-telangiectasia mutated ATR ATM and Rad3-related ATRIP ATR interacting protein Bak BCL2 antagonist killer Bax BCL2-associated X Bcl2 B-cell lymphoma 2 BBS BRD7 binding site BER base excision repair BRCA breast cancer susceptibility gene iv BRD7 bromodomain-containing 7 BSA bovine serum albumin CBP CREB binding protein cDNA complementary DNA CDK cyclin-dependent kinase Chk1/2 checkpoint protein 1/2 CIN chromosomal instability CKI cyclin-dependent kinase inhibitor CLL chronic lymphocytic leukaemia COG4639 clusters of orthologous groups of proteins 4639 CPT camptothecin CtIP C-terminal binding protein interacting protein DAPI 4’, 6-diamidino-2-phenylindole DBD DNA binding domain DDR DNA damage response DISC death-inducing signalling complex DMSO dimethyl Sulphoxide DNA deoxyribonucleic acid DNA-PK DNA-dependent protein kinase DNA-PKcs DNA-dependent protein kinase catalytic subunit DSB double-strand break DSBR double-strand break repair dsDNA double-stranded DNA v ECL enhanced chemiluminescence EXO-1 exonuclease-1 FA Fanconi anaemia FACS fluorescence-activated cell sorting FCS foetal calf serum GADD45 growth arrest and DNA damage-inducible 45 GG-NER global genome NER γH2AX phosphorylated histone H2AX H3 histone variant H3 H3 S10-P phosphorylated histone H3 HJ holliday junction hnRNPUL-1/2 heterogeneous nuclear ribonucleoprotein U-like-1/2 HR homologous recombination ICL interstrand crosslink IR ionising radiation kDa kilodalton LB Luria broth MDC1 mediator of DNA damage checkpoint protein 1 MDM2 murine double minute 2 MGMT O6-methylguanine-DNA methyltransferase MMEJ microhomology mediated end-joining MMR mismatch repair mRNA messenger RNA vi MSI microsatellite instability NBS nijmegen breakage syndrome NBSLD nijmegen breakage syndrome-like disorder NER nucleotide excision repair NHEJ non-homologous end-joining O6Cl-ethylG O6–chloroethyl guanine O6meG O6–methyl guanine PARP poly(ADP-ribose) polymerase PBS phosphate buffered solution PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PI propidium iodide PIKK phosphatidylinositol 3-kinase-like protein kinase pRB retinoblastoma protein PTM post-translational modification PUMA p53 upregulated modulator of apoptosis qRT-PCR quantitative reverse transcriptase polymerase chain reaction RFC RAD17-replication factor C RGG arginine/glycine-rich region RNA ribonucleic acid RPA replication protein A rpm revolutions per minute SAF scaffold attachment factor vii siRNA small interfering RNA SPRY spore lysis A/ryanodine receptor SSA single-strand annealing ssDNA single-stranded DNA SDSA synthesis-dependent strand annealing SMC1 structural maintenance of chromosomes-1 SSB single-strand break SSBR single-strand break repair TAD transactivation domain TC-NER transcription-coupled NER TOP1/2/3 topoisomerase I/II/III TOPBP1 topoisomerase-binding protein-1 TOPcc topoisomerase cleavage complex TLS translesion synthesis UV ultraviolet light WT wildtype XP xeroderma pigmentosum XRCC X-ray repair cross-complementary protein viii Table of Contents CHAPTER 1 INTRODUCTION ........................................................................................ 2 1.1 HALLMARKS OF CANCER .................................................................................................. 2 1.2 THE DNA DAMAGE RESPONSE ......................................................................................... 3 1.2.1 ATM-mediated DNA damage response .................................................................................. 4 1.2.2 ATR-mediated DNA damage response ................................................................................... 7 1.2.3 ATM and ATR cross-talk ......................................................................................................... 8 1.2.4 Cell cycle checkpoints ............................................................................................................. 10 1.2.4.1 The G1 phase checkpoint .......................................................................................................... 11 1.2.4.2 The S phase checkpoint ............................................................................................................ 12 1.2.4.3 The G2/M phase checkpoint ..................................................................................................... 15 1.3 DNA DAMAGE REPAIR ...................................................................................................... 15 1.3.1 Repair of double strand breaks ............................................................................................. 15 1.3.1.1 Homologous Recombination .................................................................................................... 16 1.3.1.2 Non-homologous end-joining ..................................................................................................
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