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Nuclear Receptor Co-Repressor Actions in Bladder Cancer

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Nuclear Receptor Co-Repressor Actions in Bladder Cancer Nuclear receptor co-repressor actions in bladder cancer By Syed Asad Abedin A thesis presented to the School of Clinical and Experimental Medicine, University of Birmingham, For the degree of Doctor of Medicine (MD). School of Clinical and Experimental Medicine College of Medical and Dental Sciences University of Birmingham September 2009 1 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Nuclear receptors are ligand dependent transcription factors and have defined expression patterns in classical tissues for example the Vitamin D receptor is expressed in small bowel, kidney and skin, as related to its function in maintaining serum calcium levels. However, nuclear receptor expression has also been demonstrated in non classical tissues for example VDR expression in the prostate and breast. Differential expression of a panel of nuclear receptors in bladder cancer cell lines (RT-4, RT-112, HT1376 and EJ-28 cells) at mRNA level and VDR and FXR expression at protein level has ben demonstrated. These cell lines demonstrate a range of anti-proliferative responses upon treatment with ligands to the panel of nuclear receptors (1α,25(OH)2D3, 9 cis RA, EPA, ETYA, CDA, LCA, 22HC, GW3965 and DHA). EJ-28 cells are the least sensitive to the anti-proliferative effects of 5 of these ligands (1α,25(OH)2D3, LCA, EPA, ETYA, CDA). EJ-28 cells also have the highest expression of the co-repressor NCoR1; this may be partly responsible for the reduced sensitivity displayed by EJ-28 cells by maintaining a closed chromatin structure around the nuclear receptor response elements. To test this, NCoR1 has been stably over-expressed in RT-4 cells which have the lowest relative expression of this co-repressor. This led to a statistically significant reduction in anti-proliferative response to CDA, LCA and the histone deacetylase inhibitor SAHA. To further test the hypothesis that raised NCoR1 expression and hence a predominance of the co- repressor complex in EJ-28 cells was affecting the sensitivity to NR ligands, the four bladder cancer cell lines were co-treated with the NR ligands and the HDAC inhibitor SAHA. This demonstrated a strongly additive anti-proliferative response in RT-112 and E-28 cells which have raised NCoR1 expression. This may be due increased HDAC association with NRs at their respective response elements which may make these particular cells more susceptible to co-treatment with ligand and SAHA. The possible mechanism of the anti-proliferative response within EJ-28 cells is demonstrated to be a G1/S phase cell cycle arrest upon treatment with LCA +/- SAHA. The expression of putative target genes was investigated using Q-RT-PCR, Q-RT-PCRm and Affymetrix human U133 genechip arrays upon treatment with LCA +/- SAHA. RT-4 and EJ-28 cells 2 express CDKN1A upon combined treatment with LCA + SAHA. LCA treatment leads to expression of the cytochrome P450 enzyme CYP3A4 mRNA in RT-4, RT-112 and HT1376 cells. However, the combined treatment with LCA + SAHA leads to CYP3A4 expression in EJ-28 cells. RT-4 cells stably transfected to over-express NCoR1, were treated with LCA and the target transcriptome investigated using Q-RT-PCRm. Three predominant groups of genes were induced; ABC transporter family of trans-membrane efflux pumps, detoxifying enzymes and cell cycle arrest proteins. EJ-28 cells were treated with LCA +/- SAHA and the target transcriptome investigated by hybridisation of cRNA to U133 Affymetrix genechip arrays. This yielded 3 predominant groups of targets; genes which drive cell proliferation and cell cycle progression, genes involved in transcription and post translational mRNA processing and genes involved in repair of damaged cellular components. Taken together, these two target transcriptomes suggest the presence of a xenobiotic protective response within the bladder urothelium which upon exposure to toxic compounds such as LCA inhibits cell division, expresses trans-membrane transporters and detoxifying enzymes to rid the cell of the toxin and finally initiates cellular repair. These findings may be harnessed for chemoprevention of bladder cancer by enhancing resistance to xenobiotics such as those derived from cigarette smoke. 3 Acknowledgements I would like to thank my supervisor, Dr Moray Campbell for appointing me to a research position in his group, for his constant support, encouragement and help without which this thesis would not have been completed. My deepest thanks and gratitude go to my wife, children, parents and in laws who have sacrificed enormously in supporting me through my years in research. I have been particularly fortunate in having a fantastic group of scientists around me who have been friends and key supporters of my research. In particular, I would like to thanks Serena Rakha, Sebastiano Battaglia and James Thorne who were all members of the Campbell Group. Amongst the other scientists who I would like to thank in particular are Chris Bruce and Ashraf Dallol from the Department of Medical Genetics and Katie Evans, Helen Pemberton, Dai Kim, Anna Stratford and Farhat Khanim from the Departments of Endocrinology and Biosciences. I have received vital help and support in the Affmetrix genechip experiments from Sim Sahota and Dr John Arrand from the Institute of Cancer Studies for which I am very grateful. I am indebted to the Department of Urology at the Queen Elizabeth Hospital, Birmingham for their vital support, in particular for appointing me to the research post jointly with Dr Moray Campbell and for organising research funding. I am particularly indebted to Messrs Mike Wallace, Andrew Arnold and Alan Doherty. This work was supported by the Department of Urology of the University Hospital Birmingham Foundation NHS Trust. 4 Abbreviations:- ABC ATP-binding cassette AIF apoptosis inducing factor AMV avian myeloblastosis virus AR Androgen receptor ATP Adenosine triphosphate ATRA All-trans retinoic acid 9 cRA 9 cis retinoic acid 22-HC 22-hydroxycholesterol cAMP cyclic adenosine monophosphate CAR Constitutive androsterone receptor CBP cAMP response element binding protein binding protein CDA chenodeoxycholic acid CDC25 cell division cycle 25 CDK cyclin dependent kinase CDKI cyclin dependent kinase inhibitor cDNA complimentary deoxyribonucleic acid ChIP chromatin immunoprecipitation CHO Chinese hamster ovary CIS Carcinoma in situ CoA co-activator 5 CoR co-repressor cRNA complimentary ribonucleic acid CYP cytochrome p450 enzyme CYP3A4 cytochrome p450 enzyme, family 3, subfamily A, polypeptide 4 CYP24 cytochrome p450 enzyme, 24-hydroxylase DBD DNA binding domain DEX dexamethasone DHA Cis 4,7,10,13,16,19 Docosahexaenoic acid DMEM Dulbecco‟s modified Eagle‟s medium DNA deoxyribonucleic acid DRIP vitamin D receptor interacting protein DTT Dithiothreitol ED25 Dose required to inhibit cell proliferation by 25% ED50 Dose required to inhibit cell proliferation by 50% EDTA ethylenediaminetetraacetic acid EGFR epidermal growth factor receptor ER estrogen receptor EPA Eicosapentaenoic acid ETYA 5,8,11,14-eicosatetraenoic acid FACS fluorescence activated cell sorter FXR Farnesoid X-activated receptor GAG Glycosaminoglycans GR glucocorticoid receptor 6 GSTM1 Glutathione-S-transferase M1 HAT histone acetyltransferase HDAC histone deacetylase HDACi histone deacetylase inhibitor HIV human immunodeficiency virus HNF4α hepatocyte nuclear factor 4α HRP horseradish peroxidase Kb kilo base KD kilo Dalton LCA Lithocholic acid LCOR Ligand-dependent nuclear receptor corepressor LBD ligand binding domain LXR Liver X receptor MMLV Moloney murine leukemia virus MNAR modulator of non-genomic action of estrogen receptor MR mineralocorticoid receptor mRNA messenger ribonucleic acid NAT2 N-acetyltransferase 2 Na Cl Sodium chloride NCOR1 Nuclear receptor co-repressor 1 NCOR2/SMRT Silencing mediator of retinoid and thyroid hormone receptors/Nuclear receptor co-repressor 2 NR nuclear receptor OD optical density 7 PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCN pregnenalone-16α-carbonitrile PCR polymerase chin reaction PI Propidium iodide PPAR Peroxisome proliferator activated receptor PR progesterone receptor PVDF polyvinylidene difluoride PXR pregnane X receptor Q-RT-PCR quantitative real time, reverse transcription polymerase chain reaction Q-RT-PCRM micro-fluidic quantitative real time, RT polymerase chain reaction RAR retinoic acid receptor Rb retinoblastoma protein RE response element RT reverse transcription RXR retinoid X receptor SAHA suberoylanilide hydroxamic acid or vorinostat SDS sodium dodecyl-sulphate SEM standard error of the mean SLIRP SRA stem loop-interacting rna-binding protein TCC Transitional cell carcinoma TRIP15/Alien Co-repressor Thyroid hormone receptor interactor 15/Alien TSA Trichostatin A UV ultra-violet 8 VDR vitamin D receptor 1 25(OH)2D3 1 25dihydroxyvitaminD3 XREM xenobiotic response enhancer module 9 Table of Contents Summary ..................................................................................................................................
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