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Development and Characterization of Carboplatin, Docetaxel, and Combined Carboplatin/Docetaxel Resistant Ovarian Cancer Cell Lines

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Development and Characterization of Carboplatin, Docetaxel, and Combined Carboplatin/Docetaxel Resistant Ovarian Cancer Cell Lines DEVELOPMENT AND CHARACTERIZATION OF CARBOPLATIN, DOCETAXEL, AND COMBINED CARBOPLATIN/DOCETAXEL RESISTANT OVARIAN CANCER CELL LINES by STEPHEN ROBERT ARMSTRONG Thesis submitted as a partial requirement in the Master of Science (M.Sc.) in Biology School of Graduate Studies Laurentian University Sudbury, Ontario © Stephen R. Armstrong, 2010 Library and Archives Bibliothgque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'6dition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votra r6f6rence ISBN: 978-0-494-70639-8 Our file Notre r6f6rence ISBN: 978-0-494-70639-8 NOTICE: AVIS: The author has granted a non- L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduce, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, queiques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. I+I Canada Thesis Abstract Platinating agents (cisplatin or carboplatin) and taxanes (paclitaxel or docetaxel) are two standard classes of chemotherapy drugs used to treat ovarian cancer, typically after surgery to remove the primary tumour. Patients often exhibit drug resistance to single agent therapy, and dual agent therapy has proven to provide a better clinical response than administration of single agents. However, resistance occurs commonly in dual agent therapy. It is known that specific changes in gene expression occur in single agent resistance, some of which likely play a role in the acquisition of drug resistance, but it is not known whether resistance to combined chemotherapy is accompanied by changes not seen with single agent resistant cells. Using the chemotherapy-naive A2780 ovarian cancer cell line, we have generated three drug resistant subpopulations of these cell lines by exposing A2780 cells to increasing concentrations of drug until a maximally tolerated dose was reached. Cell lines exhibiting resistance to carboplatin (A2780CBN), docetaxel (A2780DXL), or a combination of carboplatin and docetaxel (A2780CBNDXL) were obtained. The greatest resistance was obtained for docetaxel (4000 fold) compared to carboplatin (12 fold), and the combination of carboplatin and docetaxel (13 fold). Full genome oligo microarray analysis was then used to identify differences in gene expression between the drug resistant cell lines and their co-cultured control cell lines. Over 70% of all gene expression changes occurring in A2780CBNDXL were unique to that cell line suggesting that novel gene expression changes may be involved in dual drug resistance. Gene expression changes associated with dual drug resistance may be used to develop clinically relevant biomarkers for carboplatin/docetaxel resistance in ovarian cancer. iii Acknowledgements I would like to acknowledge the following individuals and organizations that gave key contributions to this project, and without which the project would not have been possible: • Dr. Carita Lanner, the project leader and principal investigator, who designed, funded, supervised, and guided the research project, also teaching the author critical laboratory practices necessary to perform cell culture, cell line selection, clonogenic assays, and RNA preparation • Dr. Amadeo Parissenti, who also participated in the design and funding of the project, contributing a large portion of materials necessary for the project including microarray chips, fetal bovine serum, the cytotoxic drugs carboplatin and docetaxel, and important laboratory resources at the Sudbury Regional Cancer Center including the apparatuses necessary to perform microarray analysis and RIN value determination, and reviewed the thesis document for publication • Dr. Baoqing Guo, who conducted the RIN value assays of prepared RNA, participated in and taught the author cRNA preparation and array hybridization in the microarray analysis protocol, and who scanned the microarray chips, extracted the raw microarray data and processed the data in Partek Genomics Suite • Dr. Eric Gauthier and Dr. Mazen Saleh, members of the supervisory committee who gave insights and critique to the design of project during its development and progression, and who reviewed the thesis document for publication • Dr. Matthew Hall who agreed to externally review the thesis, and who gave his insight to improve the thesis document iv • Dr. Stacey Santi, who handled the cytotoxic drugs carboplatin and docetaxel, and distributed them to the author for the project under Dr. Amadeo Parissenti • The Northern Ontario School of Medicine, for the use of their research laboratory for tissue culture work including cell line selection and Clonogenic assays, and RNA preparation for microarray analysis • The Sudbury Regional Cancer Center, for the use of their research laboratory for RIN value determination, cRNA preparation, and array hybridization for microarray analysis. • St. Joseph's Hospital, for allowing the use of their Gene Pix Scanner for scanning the microarray chips • Julianna McConnell, and Jamie & Robert Armstrong for their love, encouragement and support v Table of Contents Thesis abstract iii Acknowledgements iv Table of contents vi List of figures xii List of tables xiv List of abbreviations xv 1.0 Introduction 1 1.1 Chemotherapy and ovarian cancer 1 1.1.1 Platinating agents - Mechanisms of action 1 1.1.2 Taxanes - Mechanisms of action 4 1.2 Drug resistance in ovarian cancer 5 1.2.1 Mechanisms of resistance to platinating agents 5 1.2.2 Mechanisms of resistance to taxanes 11 1.2.3 Combination chemotherapy 14 1.2.4 Mechanisms of resistance to combination chemotherapy 16 1.3 Genetic profiling of drug resistance 18 1.3.1 Development and application of biomarkers 20 1.4 Study design and hypothesis .21 vi 2.0 Materials and Methods 23 2.1 Cell lines and culture 23 2.2 Cell line selection 24 2.3 Cell viability assay 25 2.3.1 Preparation of methylcellulose stock solution 25 2.3.2 Clonogenic assay for drug sensitivity 26 2.3.3 IC50 determination 27 2.3.4 Statistical determination of IC50 significance 27 2.4 RNA isolation and quality analysis 28 2.4.1 RNA preparation - Qiagen kit 28 2.4.2 Quantification of RNA by absorbance spectroscopy 29 2.4.3 RNA agarose gels 29 2.4.4 RIN determination 30 2.5 Gene expression analysis 31 2.5.1 Preparation of cRNA 31 2.5.2 Array hybridization 32 2.5.3 Array scanning and feature extraction 33 2.5.4 Statistical analysis 34 2.5.5 Identifying unique and shared changes in gene expression 34 3.0 Results 36 3.1 Cell line selection 36 3.1.1 Generation of carboplatin, docetaxel and carboplatin/docetaxel resistant cell 36 lines 3.1.2 Characterization of the A2780CBN cell line 38 3.1.3 Characterization of the A2780DXL cell line 43 3.1.4 Characterization of the A2780CBNDXL cell line 48 3.2 Gene expression analysis 55 3.2.1 Comparison of gene expression between the A2780CBN cell line and the 55 A2780CC cell line 3.2.2 Comparison of gene expression between the A2780DXL cell line and the 55 A2780CC cell line 3.2.3 Comparison of gene expression between the A2780CBNDXL cell line and 57 the A2780CC cell line 3.2.4 Comparison of gene expression amongst the A2780CBN, A2780DXL, and 58 A2780CBNDXL cell lines 4.0 Discussion 62 4.1 Generation of resistant cell lines 62 4.1.1 Generation of the A2780CBN cell line 65 4.1.2 Generation of the A2780DXL cell line 66 4.1.3 Generation of the A2780CBNDXL cell line 67 viii 4.1.4 Dual resistance vs. cross resistance 68 4.2 Gene expression analysis 69 4.2.1 Unique gene expression changes in the A2780CBN cell line 71 4.2.2 Unique gene expression changes in the A2780DXL cell line 77 4.2.3 Unique gene expression changes in the A2780CBNDXL cell line 80 4.2.4 Gene expression changes shared between the A2780CBN and A2780DXL 84 cell lines 4.2.5 Gene expression changes shared between the A2780CBN and 86 A2780CBNDXL cell lines 4.2.6 Gene expression changes shared between the A2780DXL and 89 A2780CBNDXL cell lines 4.2.7 Gene expression changes shared between all three resistant cell lines 91 4.3 Conclusions and Future Directions 99 References 106 Appendices 136 Appendix I - Up-regulated gene expression changes in the A2780CBN cell line 137 Appendix II - Down-regulated gene expression
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