Phenotypic and Proteomic Analysis of 5-Fluorouracil T reated Normal and Carcinoma Cells A thesis submitted for the degree of Ph.D. Dublin City University By William Bryan, B.Sc. The research work described in this thesis was performed under the supervision of Prof. Martin Clynes National Institute for Cellular Biotechnology Dublin City University 2006 I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph-.O.-... (insert title of degree for which registered) is entirely my own work and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed (Candidate) ID No.: ffg5~72o3X Date:? )-/< - O b _________________ Acknowledgements Firstly, I’d like to thank Prof. Martin Clynes and Dr. Paula Meleady for the opportunity to pursue this Ph.D. in the National Institute for Cellular Biotechnology at Dublin City University and for their mentoring over the years - it’s very much appreciated. Also I’d like to thank the proteomics crew, Andrew, Paul, Jon and Mick for all the technical discussions on casting the perfect 2D gels and mastering of the mass spectrum. Also I’d like to thank the proteomics/toxicology crew, Lisa and Joanne, for the useful discussions over the years. Also I have to thank Lisa for the friendly ‘slagging’ towards the end of our Ph.D.’s, I guess you won the Ph.D. race, barely, by just 30 minutes. Cheers to Bella and Eadaoin for making those otherwise painful weekends pleasant. Also I have to thank the old Differentiation lab, Finbar, Brendan, and Jason for showing me the ropes when I started and for the many crazy surreal moments. I can’t forget the diabetes lab, cheers, particularly Elaine and Irene - always up for a bit of craic. Thanks to Helena and Annemarie for the useful invasion assay discussions. Cheers Toxicology folk particularly Vanessa for helpful discussions on drug metabolism and Molecular folk particularly Paudie, Mohan and Jai for all the Bioinfomatics tips. Thanks to Bella for all the help at the end of my Ph.D. especially during my crazy scientist stage. Thanks to Paul for the games of pool and the use of the office while writing up the thesis - it helped maintain my sanity. Thanks to Joe, Ultan, Mairead, Carol and Yvonne, the crucial cogs in the machine that is the NICB, especially to Carol and Yvonne for the help at the end, cheers. -ABBREVIATIONS- 5-FU - 5-Fluorouracil 52FdU - 5 -Fluoro-2 ’ deoxyuri dine 55FdU - 5-Fluoro-5 ’deoxyuridine Adr - Adriamycin ATCC - American Type tissue Culture Collection ATR - ataxia-telangiectasia-and-RAD3-related ATM - ataxia telangiectasia mutated BrdU - Bromodeoxyuridine DMEM - Dulbeccos modified Eagle Medium DMSO - Dimethyl sulphoxide ECM - Extracellular Matrix elF - eukaryotic Initiation Factor EEF - Eukayotic Elongation Factor ER - Endoplasmic Reticulum FAK - Focal Adherence Kinase FCS - Fetal Calf Serum HSPB1 - Heat shock protein 27 IC - Inhibitory concentration ID - Inhibitor of DNA binding kDa - KiloDaltons PBS-A - Phosphate Buffered Saline - Autoclaved NSCLC - Non-Small Cell Lung Carcinoma MMP - Matrix Metalloproteinase MALDI-ToF - Matrix Assisted Laser Desorption/Ionisation - Time of Flight MS - Mass Spectrometer RNA - ribonucleic acid SDS - Sodium Dodecyl Sulphate PAGE - Polyacryamide Gel Electrophoresis mRNA - messenger RNA rRNA - Ribosomal RNA tRNA - transcriptional RNA RT - Room temperature TP - Thymidine Phosphorylase (Platelet Derived Growth Factor) TS - Thymidylate Synthetase UHP - Ultra high pure water UPR - Unfolded protein response v/v - volume/volume w/v - weight/volume XBP1 - X-Box binding protein 1 rpm - revolutions per minute -Abstract- The anti-metabolite - 5-Fluorouracil (5-FU) - is the most widely used chemotherapeutic drug. It exerts its anti cancer effect through incorporation into DNA and RNA. Characterisation of this drugs mode of action is crucial in the development of future therapies. There have been many DNA microarray experiments performed in order to gain such information. However, only two proteomic experiments have been performed to date that look at the effect of 5-FU treatment. Here the proteomic alterations induced by IC8o 5-FU treatment of normal and cancer cells of epithelial origin of the lung and breast are investigated. These cell lines include a lung adenocarcinoma (A549), a non small cell lung carcinoma cell line (DLKP), normal bronchial epithelial cell line (NHBE), a breast adenocarcinoma (MCF-7) and human mammary epithelial cells (HMEC). Phenotypes were characterised and 5-FU was found to induce and reduce invasion in various cell lines. Adherence was altered in one of the three cancer cell lines to the extracellular matrix proteins collagen type IV and fibronectin. Differential regulated proteins were quantified using 2 dimensional difference gel electrophoresis (2D-DIGE) and differentially regulated proteins were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF MS). Data shows the NHBE showed a dose dependent response to 5-FU treatment. Proteins that were found regulated are discussed in terms of microtubule dynamics and future drug combinations, translation control and stress, cytosketetal dynamics and invasion, and inhibition of apoptosis. 5-Fluorouracil is being increasing replaced in clinical trials with 3rd generation 5-FU drugs such as capecitabine. Capecitabine is converted in the liver to 5-fluoro-5’- deoxyuridine (55FdU) and this is converted to 5-FU by thymidine phosphorylase whose over expression is induced by radiotherapy. 5-FU is converted to 5-fluoro-2’- deoxyuridine in the cytoplasm. A comparison using 2D-DIGE between DLKP treated 5- FU and the fluoropyrimidines - 52FdU and 55FdU at ICgo cocentrations to determine similar proteomic alterations. Results demonstrated that the down regulation of Stathmin as a common fluoropyrimidine response. Furthermore this data may indicate a role for the use of vinca alkoloids or taxanes in combination with 5-FU. Resistance to 5-FU is a major clinical problem as mediated predominantly by over expression of Thymidylate Synthetase. A variant of DLKP was generated by pulse selection with 55FdU and showed a ~4 fold resistance to 5-FU. Proteomic analysis using 2D-DIGE identified several proteins involved in uracil metabolism and oxidative metabolism differentially regulated between DLKP and DLKP-55. DLKP is a heterogeneous cell line composed of at least three subpopulations. These subpopulations were isolated and were assigned the names DLKP-SQ, DLKP-I and DLKP-M. In this thesis data is presented demonstrating highly significant differences in motility and invasion between the clonal subpopulation. Analysis of proteomic alteration was carried out using 2D-DIGE on both the total cell lysate and the hydrophobic proteomes. Proteins were identified in the total cell lysates that suggest that DLKP-M is mesenchymal-like in nature as originally described and that the interconversion process observed between the clones is regulated at least by key proteins involved in protein metabolism. Analysis of the hydrophobic proteome found at least 300 proteins that correlated with motility/invasion and collagen synthesis. Identification of these proteins demonstrated increased association of microfilaments to the cellular membranes a process important in cellular motility. Furthermore 3 poorly described proteins were identified that correlated with motility/invasion and collagen synthesis. In addition data generated by these experiments indicates that fluoropyrimidine treatment of DLKP does not result in the selection of one of the subpopulations of DLKP and that DLKP-55 is not a subpopulation of DLKP. Data also shows the presence of protein in the heterogeneous population that are not present or are over expressed in the clonal populations indicating cell-cell communication. Table of contents 1.0 Introduction 5 1.1.1 Development of the fluoropyrimidines and 5-Fluorouracil 6 1.1.2 Clinical usage of 5-FU 6 1.1.3 5-Fluorouracil metabolism and method of action 7 1.1.4 DNA damage and repair 10 1.1.5 RNA damage and RNA Decay 11 1.1.6 Catabolism 11 1.1.7 5 -Fluorouracil clinical treatment 13 1.1.8 Global mRNA analysis 5-Fluorouracil treatments 13 1.1.9 Global Protein expression analysis of 5-Fluororuacil treatment 15 1.1.10 Environmental stress mechanisms 16 1.1.11 Genotoxic stress 16 1.1.12 ER stress/Unfolded protein response 17 1.1.13 Genotoxic stress and ER stress 18 1.1.14 HSPs and apoptosis 19 1.2 Fluoropyrimidines 25 1.3 Fluorouracil resistance 26 1.4 The cell line DLKP and its subpopulations 30 1.4.1 Epithelial-Mesenchymal transition 30 1.4.2 Cell Matrix interactions 33 1.4.3 Integrins 34 1.4.4 Rho family members and actin dynamics 37 1.4.5 Integrin and proteases 37 1.4.6 Syndecans 37 1.4.7 Cytoskeletal alteration during migration 39 1.4.7.1 Assembly of Actin Filaments 39 1.4.7.2 The Arp2/3 Complex 39 1.4.7.3 Elongation and Annealing of F-actin 39 1.4.7.4 ADF/Cofilin 40 1.4.7.5 Profilin 43 1.4.7.6 Gelsolin Superfamily 43 1.4.7.7 CapZ, a barbed-end capping protein 43 1.5 Aims of thesis 45 2.0 Materials and Methods 49 2.1 Ultrapure water 50 2.2 Glassware 50 2.3 Sterilisation Procedures 50 2.4 Preparation of cell culture media 51 2.5 Cells and Cell Culture 52 2.5.1 Subculturing of cell lines 53 2.5.2 Cell counting 55 2.5.3 Cryopreservation of cells 55 2.5.4 Thawing of cryopreserved cells 55 1 2.5.5 Monitoring of sterility of cell culture solutions 56 2.6 Mycoplasma analysis