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Acquisition and Loss of Amplified Genes 300 N. Zeeb Rd. Ann Arbor Order Number 0201776 Acquisition and loss of amplified genes Wani, Maqsood Ahmad, Ph.D. The Ohio State University, 1991 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 ACQUISITION AND LOSS OF AMPLIFIED GENES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University by Maqsood Ahmad Wani, B.S., B.V.S., M.V.S. The Ohio State University 1991 Dissertation Committee: Approved by Robert M. Snapka, Ph.D. s i a I s ' Marshall Williams, Ph.D. Deborah S. Parris, Ph.D. Advisor Steven D. Ambrosio, Ph,D. Department of Medical Microbiology & Immunology Copyright by Maqsood Ahmad Wani 1991 To my Family, Friends and Teachers ii ACKNOWLEDGEMENTS It gives me great pleasure to express my sincere gratitude to my principal advisor, Robert M. Snapka, Ph.D., who provided me an opportunity to undertake my graduate education under his supervision. His countless efforts, valuable scientific planning, critical evaluation, constructive criticism are greatly acknowledged. I highly appreciate his support and understanding attitude. Sincere gratitude is also due to Steven M. D’Ambrosio, Ph.D. for his friendliness and consistent help; Deborah Parris, Ph.D. and Marshall Williams, Ph.D. for their valuable instruction, encouragement and enthusiam. I appreciate all the whole hearted help rendered from by John M. Strayer, Cha- gyun Shin, Paskasari Perm ana, Grant Marquit, Steve Santangelo, Bassem Hassan, Susan Baird, Paul Kelner and Marie Powelson. Over my five years at this university I was fortunate enough to live with my family, Altaf A. Wani, Gulzar Wani, Baraq Wani and Najum Wani. I want to place on record my deep appreciation for their profound affection. Expression in formal words is no substitute for all that they have done for me during my entire academic career. It is always difficult to single out a few from the many to whom gratitude is due but I owe many thanks to all the members of The Department of Radiology with whom I was in close association during the course of my stay at The Ohio State University. The author owes special thanks to Altaf A. Wani, Ph.D., Edie Yamasaki, Gulzar Wani, Ruth D’Ambrosio, Jasna Arczina, Shahabuddin, Jennifer Williams, Robin Shanfeld, Rebecca Shepard, Jonna Gill, Ahsia Khan, Kim Chrifield and Sundaresan Venkatachalam for their technical and moral support. Thanks are also due to Phyllis Byard and Debbie Marcus, the non-academic staff of Radiobiology Division. I also want to thank all the academic and non-academic staff of The Department of Medical Microbiology and Immunology. Special thanks are to my parents, brothers, sisters, friends and all other well wishers. VITA October 1, 1958 Date of Birth Srinagar Kashmir, India Place of Birth November, 1978 .... Bachelor of Science in Biology, Srinagar, India October, 1982 ......... Degree in Veterinary Medicine, C.S.A. Univ., Kanpur, India October, 1985 ......... Master’s in Veterinary Medicine, C.S.A. Univ., Kanpur, India Octber, 1985-1986 Clinical Pathologist, J&K, India October 1986-87 Research Assistant, Radiobiology, OSU Coumbus, Imm., The Ohio State v PUBLICATIONS Wani, M.A. (1985) Histamine and 5-Hydroxytryptamine concentrations in peripheral blood and certain reproductive tissues during estrous cycle and pregnancy in Buffalo. M.V.Sc. Thesis. Wani, M.A., and Snapka, R.M. (1989) Methotrexate resistance in NIH3T3 cells expressing polyoma virus oncogenes. Teratogen. Carcinogen. Mutagen. 9:369-382 Wani, M.A. and Snapka, R.M. (1990) Drug-induced loss of unstably amplified genes. Cancer Invest. 8:586-91 Wani, M.A., Strayer, J.M. and Snapka, R.M. (1990) Hypersensitivity to low-level cytotoxicity stress in mouse cells with high level DHFR gene amplification. Anticancer Drugs 1:67-75 Shin, C.G., Strayer, J., Wani, M.A. and Snapka, R.M. (1990) Rapid evaluation of Topoisomerase Inhibitors: Caffeine Inhibition of Topoisomerase in vivo. Teratogen. Carcinogen. Mutagen. 10:41-52 FIELDS OF STUDY Major Field: Medical Microbiology & Immunology 1982 Veterinary Medicine at College of Veterinary Sciences Mathura (U.P.), India 1985 Pharmacology: Biogenic amines and their role in reproduction in buffaloes. M. Sabir, Ph.D., Departmenty of Pharmacology, College of Veterinary Sciences, Mathura (U.P), India vi 1986-1991 Cancer Chemotherapy. Role of polyoma oncogenes in methotrexate resistance. Role of chemotherapy in reversion of methotrexate resistance. Dr. Robert M. Snapka, Ph.D., Departments of Radiology, Medical Microbiolgy and Immunology, The Ohio State University, Columbus Ohio, USA. TABLE OF CONTENTS ACKNOWLEDGEMENT............................................................................................. iii VITA ...................................................................................................................................v LIST OF TABLES .........................................................................................................xii LIST OF FIGURES.................................................................................................... xiii CHAPTER PAGE I. INTRODUCTION .............................................................................................1 PURPOSE........................................................................................................... 1 GENERAL INTRODUCTION ................................................................ 5 Thymidylate Synthesis .............................................................................. 5 Methotrexate, an Inhibitor of Dihydrolate Reductase .............................9 Mechanism of Cell Death by Methotrexate ............................................11 Transport of Methotrexate ..................................................................... 11 Metabolism of Methotrexate ................................................................... 12 Resistance to Methotrexate ................................................................... 14 AMPLIFICATION OF DIHYDROFOLATE REDUCTASE AND METHOTREXATE RESISTANCE ...............................................................15 LOSS OF UNSTABLY AMPLIFIED GENES..............................................22 II. MATERIALS AND METHODS ...................................................................24 MATERIALS ..................................................................................................24 Reagents ....................................................................................................24 Cell Lines and Cell Culture Supplies .....................................................24 Plasmids and Cloned P robes ...................................................................25 Bacteriological Supplies ..........................................................................26 GROWTH AND SELECTION FOR METHOTREXATE RESISTANCE..................................................................................................26 Cell Culture .............................................................................................26 Selection of Cells for Methotrexate Resistance .................................... 26 Plating Efficiency ................................................................................... 28 Growth Curves ........................................................................................ 28 Measurement of Cell Viability .................................................................29 viii CELL SORTING AND FLOW CYTOMETRY 29 QUANTITATION OF GENE DOSAGE......................................................30 Isolation of Genomic DNA fromi C ells ................................................. 31 Quantitation of DNA by Hoechst A ssay ............................................... 32 Preparation of Cytoplasm for Cytodot ..................................................32 DNA-DNA Hybridization ...................................................................... 33 Prehybridization and Hybridization ......................................................34 Radiolabelling of DNA with 32P ...........................................................35 PREPARATION OF DNA FOR RADIOLABELLING...............................37 Growth of Bacteria ..................................................................................37 Preparation of Competent C e lls ............................................................. 37 Transformation of Competent Cells ...................................................... 38 Isolation of Plasmid D N A ...................................................................... 38 Isolation and Elution of DNA Fragments from Agarose G e l 40 RESULTS.......................................................................................................42 EFFECT OF POLYOMA T ANTIGENS ON METHOTREXATE RESISTANCE IN N1H 3T3 CELLS.............................................................42 Clonal Variation in Frequencies of Methotrexate Resistance in CC1 C ells ........................................................................................... 42 Frequencies of Methotrexate Resistance in Cells Expressing Polyoma Oncogences ............................................................................. 44 Plating Efficiencies of Polyoma Oncogene Expressing Cell Lines . 47 Evaluation of Dihydrofolate Reductase and ras Gene Copy Number in Methotrexate Resistant C e lls .............................................................53 Quantitation of Dihydrofolate Reductase Gene Expression in Methotrexate Resistant C e lls ..................................................................63
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