CHANGES IN GENE EXPRESSION IN CYCLOSPORINE A TREATED GINGIVAL FIBROBLASTS by Jeffrey S. Wallis A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences Summer 2005 Copyright 2005 Jeffrey S. Wallis All rights reserved UMI Number: 1428256 Copyright 2005 by Wallis, Jeffrey S. All rights reserved. UMI Microform 1428256 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 Changes in Gene Expression In Cyclosporine A Treated Gingival Fibroblasts by Jeffrey S. Wallis Approved: ______________________________________________________ Mary C. Farach-Carson, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: ______________________________________________________ Daniel D. Carson, Ph.D. Chair of the Department of Biological Sciences Approved: ______________________________________________________ Thomas Apple, Ph.D. Dean of the College of Arts and Sciences Approved: ______________________________________________________ Conrado M. Gempesaw II, Ph.D. Vice Provost for Academic and International Programs DEDICATION I would like to give very special thanks to my advisor Dr. Cindy Farach-Carson for being an incredible mentor and teacher. Over the past 3 years I have been fortunate to have been given the opportunity to explore a subject of great personal interest in an environment that has allowed me to grow both as a scientist and a person. Her knowledge and patience is an inspiration to all those around her. I would not be the individual I am today nor would my life be headed in its current direction if it were not for her and I will be forever grateful for everything she has taught me. I would like to thank Dr. Daniel Carson for his guidance and valuable opinions. His advice on where the direction of my research should go has been integral to its success. I would like to thank Dr. David Usher for all his contributions towards my project as well as keeping me employed as a teaching assistant during my time at the University of Delaware. I thank you and perhaps most importantly, my parents thank you. I would also like to thank Dr. Jonathan Korostoff, whose expert knowledge in the field of dentistry, specifically periodontics, has been both a valuable asset to my scientific endeavors and an inspiration for me as I pursued my acceptance to dental school. I must also thank all the members of the Carson and Farach-Carson labs. I could not have made it through the rigors of graduate school without their help and friendship. I will always have fond memories of my time spent with you all. Finally, I would like to thank my parents for their love and support as I move on to my next challenge. I hope that I can continue to make you both proud and become the doctor I so admire in the both of you. I love you both very much. iii ACKNOWLEDGEMENTS • National Institutes of Health, R03 Grant • I would like to thank the following people for their personal contributions towards my research: Ben Rohe and Dr. Chu Zhang for their assistance in RT-PCR, Q- PCR and cell staining work; Joanne Julian for her advice and expertise involving cell culture and staining; Anissa Brown, Melissa Brayman, and Caroline Muir for their help with western blot techniques and analysis; Lynn Schwarting and John O’Connor for their help and assistance in microarray analysis. Doreen Anderson for her help in formatting and submission of my thesis. iv TABLE OF CONTENTS TABLE OF CONTENTS……………………………………………………………….v LIST OF TABLES……………………………………………………………………..vii LIST OF FIGURES……………………………………………………………………viii LIST OF ABBREVIATIONS………………………………………………………......ix ABSTRACT………………………………………………………………………….......xi HYPOTHESIS………………………………………………………………………….20 Chapter 1 Introduction……..……………….…………………………………………..1 1.1 Gingival Overgrowth.…………………………………………………………….1 1.2 Gene Changes Associated with CsA…..………………………………………….8 1.3 Microarray Analysis ………………………………………………………..….. 16 Chapter 2 Materials and Methods………………..……………………………………21 2.1 Cell Line and Culture……………………………………………………………21 2.2 RNA Extraction and Northern Blot……………………………………………..22 2.3 Microarray Analysis……………………………………………………………..22 2.4 Microarray Statistics…………………………………………………………….23 2.5 Reverse-transcript PCR (RT-PCR) and Real-Time PCR……………………….23 2.6 Antibodies……………………………………………………………………….25 2.7 Western Blots……………………………………………………………………26 2.8 Cell Staining……………………………………………………………………..28 Chapter 3 Results……………………………………………………………….………30 v 3.1 RNA Gel Electrophoresis and Northern Blot…………………………………...30 3.2 Microarray Results………………………………………………………………32 3.3 Real-Time PCR………………………………………………………………….36 3.4 Western Blots……………………………………………………………………40 3.5 Cell Staining……………………………………………………………………..42 Chapter 4 Discussion and Conclusion…………………………………………………46 4.1 Discussion……………………………………………………………………….46 4.2 Conclusions……………………………………………………………………...53 Chapter 6 Future Work………………………………………………………………...55 References……………………………………………………………………………….57 Appendix………………………………………………………………………………...64 vi TABLES Table 1. Relative Intensities of Array Genes Chosen for Validation………..………….35 Table 2. List of Extracellular Matrix Genes…………………………………………….64 Table 3. List of MAP Kinase Genes…………………………………………………….67 vii FIGURES Figure 1. Cross-Section of Tooth Microenvironment…...………………………………..3 Figure 2. Molecular Structure of Cyclosporine A…………..…………………………..14 Figure 3. Proposed Mechanism of Action of Cyclosporine A ..………………………...15 Figure 4. Sample protocol of GEArray™ Q Series Microarray……….………...………18 Figure 5. Sample of Microarray………………………………………………………....19 Figure 6. RNA Gel and Northern Blot Analysis………………………………………...31 Figure 7. Extracellular Matrix Microarray Images of 6 Day Vehicle vs. CsA………….33 Figure 8. MAP Kinase Microarray Images of 6 Day Vehicle vs. CsA………………….34 Figure 9. Q-PCR of Collagen 1 alpha 1…………………………………………………38 Figure 10. Q-PCR of Fibronectin…………………………..……………………………39 Figure 11. Western Blot Analysis………………………………………………………..41 Figure 12. Trypan Blue Staining………………………………………………………...43 Figure 13. Crystal Violet Staining………………………………………………………44 Figure 14. Syto13 and Phalloidin Staining………….…………………………………..45 Figure 15. Fold Difference of Collagen 1 alpha 1 Q-PCR cycle threshold using ∆∆ct method……………………………………………………………………..…70 viii ABBREVIATIONS Bp base pairs CaN calcineurin CD1 cyclin D1 cDNA complementary DNA CEACAM5 CarcinoEmbryonic Antigen Cell Adhesion Molecule 5 Col1α1 collagen type 1 alpha 1 chain CpN cyclophilin CsA cyclosporine A dNTP deoxynucleoside 5'-triphosphate ECM extracellular matrix EDTA ethylenediaminetetraacetic EGR-1 Early Growth Response 1 FACS fluorescence activated cell sorter FBS fetal bovine serum FDA Food and Drug Administration FK506 tacrolimus FN-1 fibronectin GAGs glycosaminoglycans GAPDH glyceraldehyde-3-phosphate dehydrogenase HGFs human gingival fibroblasts IFN-γ interferon gamma ix IL-2 interleukin-2 MEM minimal essential media mRNA messenger RNA NF-AT Nuclear Factor of Activated T Cell PBS phosphate buffered saline PBS-T PBS + Tween 20 solution PIC Protease Inhibitor Cocktail PMSF phenylmethylsulphonylfluoride Q-PCR quantitative RT-PCR or real time RT-PCR RT-PCR reverse transcript-DNA polymerase chain reaction SEB Sample Extraction Buffer SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SPARC Secreted Protein, Acidic, Cysteine-Rich Ta annealing temperature TCA trichloroacetic acid TGFβ transforming growth factor β Tm melt temperature x ABSTRACT Cyclosporine A (CsA) is a potent immunosuppressant drug, produced by the fungus Tolypocladium inflatum, used to prevent allograft rejection after organ transplantation. It is distributed under the name Sandimmune® and has increased the success rate of transplants to approximately 95% over a one year period. The immunosuppressant effect is due to the effect of CsA on the serine-threonine Ca2+-calmodulin protein phosphatase calcineurin. Calcineurin is responsible for activation of NF-AT (nuclear factor of activated T cell) which translocates to the nucleus of the T cell to activate interleukin-2 for T cell maturation and proliferation. CsA complexes with cyclophilin and binds to calcineurin to inhibit its phosphatase activity thereby preventing T cell proliferation. A common side effect of CsA is gingival overgrowth. The exact cause of gingival overgrowth remains unsolved and controversy remains over whether the overgrowth is a result of hypertrophic growth or hyperplastic growth. In this study, gingival fibroblasts were used to examine whether gene changes after treatment with CsA cause hypertrophic growth, hyperplastic growth, or a combination of both. Gene arrays representing pathway specific genes associated with each form of growth, as well as cellular stains to examine nuclear and cytoskeletal changes, were employed to address the problem. Five genes expressed by gingival fibroblasts were identified from the arrays and selected for validation. The results indicate that Collagen 1α1 mRNA expression decreases after six days of treatment with CsA, but that overall there were modest changes in gene expression in gingival fibroblasts treated with CsA over a six day period. It was xi concluded that gingival fibroblasts are not solely responsible for the overgrowth seen in patients treated with CsA. Overgrowth is more likely due to