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Analysis of Messenger Rnas Detectable in Medium Conditioned by Tumour Cells and in Serum from Breast Cancer Patients

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Analysis of Messenger Rnas Detectable in Medium Conditioned by Tumour Cells and in Serum from Breast Cancer Patients Analysis of Messenger RNAs Detectable in Medium Conditioned by Tumour Cells and in Serum from Breast Cancer Patients A thesis submitted for the degree of Ph.D. Dublin City University By Elaine Kenny B.Sc. (Biotechnology) The research work described in thesis was performed under the supervision of Prof. Martin Clynes and Dr Lorraine O’Driscoll National Institute for Cellular Biotechnology Dublin City University June 2006 I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D. 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.: 1~C)0/S~ Date: sh Acknowledgements I would like to sincerely thank Professor Martin Clynes and Dr. Lorraine O’Driscoll for taking a chancc on me and letting me do some research work before I finished my degree. It helped me to realise that research work is were I want to be (and encouraged me to study harder for my exams so that I could do a PhD here!). Throughout my PhD the constant encouragement (and crossed fingers!) got me through many dark days and confusing results. Thanks Paddy (“the human encyclopaedia*’), for all the help and training when 1 first started. Joe, I'm convinced the place would come to a standstill without you, thanks for all the “special runs” I know we’re a demanding lot! To all who have advised me on techniques/ software and listened patiently to my (sometimes insane) ideas; Jai, Niall, Paudie, Ann Marie and Norma; thanks a mill, I couldn’t have done it without you. A special thanks also to Irene, you’re the best coffee/ McDonalds mate a girl could ever ask for. You and Bella made it so easy for me at the end. To my lunch buddies Olga (and Yegor for 8 'A months!), Jason, Ann Marie and Helena, thanks. To Yvonne, Carol, Mairead, Eadaoin, Sweta, Mick, Finbar, Paula, Will, Laura and everyone else who’s friendship made working in the NCTCC/ NICB such a happy experience for me thank-you. Thanks especially to my family. Mam, Dad, Ed, Paul, Trev, my twinny Darren, sisters(-ish) Hilary, Lorraine and Meabh for all your encouragement and support and yes, I’m finally going to get a real job! And above all, to Paul, without your encouragement I may never have started (or finished!) this work. This achievement is as much yours as it is mine, thanks for putting up with all the crankiness and tears and for always being there for me. Bring on Hawaii!! ABBREVIATIONS A Absent AFP Alpha feto protein ATCC American Tissue Culture Collection cDNA Complementary DNA CDS Coding Sequence CEA Carcinoembryonic antigen CK Cytokeratin CM Conditioned Media COV Coefficient of Variation cRNA Complementary RNA DEPC Diethyl Pyrocarbonate DMEM Dublecco’s Minimum Essential Medium DMSO Dimethyl Sulfoxide EDTA Ethylenediaminetctraacetic Acid EST Expressed sequence tag FCS Foetal Calf Semm H NCI HI299 HCC Hepatocellular carcinoma hCG Human chorionic gonadotropin-p HNSCC Head and neck squamous cell carcinoma HT NCI HI299 TAX IHC Immunohistochcmistry LOH Loss of Heterozygosity M MDA-F MA MDA-F-ADR MMLV-RT - Moloney Murine Leukaemia Virus- Revc mRNA Messenger RNA MT MDA-F-TAX NPC Nasopharyngeal carcinoma NSCLC Non-small cell lung cancer Ill PorM - Present or Marginal PBS - Phosphate Buffered Saline Post - Post-operation Pre - Pre-operation R - RPMI 2650 ref - relative centrifugal field (also= xg) RM - RPMI 2650 ML Rpm - Revolutions per minute RT - RPMI 2650 TAX RT-PCR - Reverse Transcription Polymerase Chain Reaction SD - Standard Deviation Tris - Tris (hydroxymethyl) aminomethane TV - 0.25% Trypsin/ 0.01% EDTA solution in PBS UTR - Untranslated Region IV ABSTRACT 1 1.1 T um our Markers and B iom arkers in C a n c er ............................................................4 1.2 Serum/ T issue T umour Ma r k e r s ...................................................................................... 6 1.2.1 a-Fetoprotein.................................................................................................................6 1.2.2 Human chorionic gonadotropin- fi (hCG)..............................................................7 1.2.3 CA 125............................................................................................................................8 1.2.4 C EA................................................................................................................................ 8 1.2.5 Cytokeratins..................................................................................................................9 1.2.6 EG F receptor family..................................................................................................11 1.2.6.1 Epidermal growth factor receptor......................................................................... 12 1.2.6.2 HER2/NEU ................................................................................................................... 13 1.2.7 Cancer cell- secreted proteins.................................................................................14 1.2.8 Exploitation of the immune system for cancer biomarkers................................15 1.3 D etection of circulating tum ou r cells in cancer patients............................. 15 1.4 C ell-free N ucleic Acids in S erum / P lasm a...............................................................26 1.4.1 Serum / Plasma DNA.................................................................................................27 1.4.1.1 Cancer Testing.............................................................................................................27 1.4.1.1.1 Quantification of total DNA ............................................................................ 27 1.4.1.1.2 Quantification of gene expression..................................................................29 1.4.1.1.3 Detection of microsatellite alterations including LOH ............................29 1.4.1.1.4 Detection of mutations in specific genes......................................................30 1.4.1.1.5 Detection of aberrantly methylated DNA ................................................... 31 1.4.1.1.6 Detection of viral sequences........................................................................... 32 1.4.1.2 Prenatal Diagnosis......................................................................................................34 1.4.2 Serum/ Plasma RNA..................................................................................................35 1.4.2.1 Cancer testing.............................................................................................................. 35 1.5 AIMS OF THESIS ....................................................................................................................39 2 MATERIALS & METHODS .....................................................................................................4 0 2.1 W A TER........................................................................................................................................41 2.1.1 DEPC treated water..................................................................................................41 2.2 GLASSWARE...........................................................................................................................41 2.3 STERILISATION .....................................................................................................................42 2.4 MEDIA PREPARATION .......................................................................................................42 2.5 CELL LINES.............................................................................................................................. 43 2.5.1 Subculture of A dherent Lines..................................................................................45 2.5.2 Cell Counting..............................................................................................................45 2.5.3 Cell Freezing...............................................................................................................46 2.5.4 Cell Thawing............................ 46 2.5.5 Sterility Checks......................................................................................................... 4 7 2.6 MYCOPLASMA ANALYSIS.............................................................................................. 47 2.6.1 Indirect Staining Procedure.....................................................................................47 2.6.2 Direct Staining...........................................................................................................48 2.7 C ell grow th and Conditioned M edia (C M )/ serum collectio n ...................... 49 V 2.7.1 Identifying a suitable time point for CM collection........................................... 49 2.7.2 Study to find a reproducible minimum detection level.......................................50 2.7.3 Analysis of transcripts in RNA isolated from cell lines and CM......................51 2.7.4 Concentration protocol 1 .........................................................................................51 2.7.5 Concentration protocol 2 .........................................................................................53 2.7.6 Concentration protocol 3 .........................................................................................54 2.7.7 Pilot DLKP microarray study.................................................................................55
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