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Alkylation Damage in Mammalian Cells: an Analysis of the Mutagenic Potential of 04-Methylthymine Adducts

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Alkylation Damage in Mammalian Cells: an Analysis of the Mutagenic Potential of 04-Methylthymine Adducts ALKYLATION DAMAGE IN MAMMALIAN CELLS: AN ANALYSIS OF THE MUTAGENIC POTENTIAL OF 04-METHYLTHYMINE ADDUCTS by KARA DENISE BEST ALTSHULER B.S., Texas A&M University, 1987 SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY MARCH 1994 o Massachusetts Institute of Technology, 1994 () Signature of Author ,Y Division of Toxicology March 15, 1994 Certified byr John M. Essigmann Thesis Supervisor Accepted by Steven R. Tannenbaum MASSACHLUSETS 4YUSTITUT ........- ,,"1h Y Chairman, Commitee on Graduate Students APR 2 0 1994 This doctoral thesis has been examined by a Committee of the Division of Toxicology as follows: Professor William G. Thilly Chairman Professor John M. Essigmanr. , . - Thesis Supervisor -~ I Professor Helmut Zarbl Professor Edward Loechler ( ,... ~-' ,Y~~.. .. ~. _,-,, ALKYLATION DAMAGE IN MAMMALIAN CELLS: AN ANALYSIS OF THE MUTAGENIC POTENTIAL OF 0 4-METHYLTHYMINE ADDUCTS by Kara Denise Best Altshuler Submitted to the Division of Toxicology on March 15, 1994 in partial fulfillment of the requirements for the Degree of Doctor of Philosophy ABSTRACT The mutagenic effects of two alkyl-DNA adducts were investigated in mammalian cells with differing repair phenotypes. A shuttle vector system was used that consisted of a plasmid, pCNheI, which contains a pML2 bacterial origin of replication, an SV40 mammalian origin of replication, the neomycin resistance gene and SV40 polyadenylation sequences. This plasmid can integrate into and replicate within the genome of mammalian cells. Three different oligonucleotides were positioned within a single NheI site in the pCNheI shuttle vector: the control oligonucleotide, 5'-GCTAGC-3', an oligonucleotide with the same sequence containing 04MeThy in place of the single thymine, and an oligonucleotide with 06MeGua replacing the second guanine. These modified shuttle vectors were then transfected into two types of Chinese hamster ovary (CHO) cells: one CHO type possessed an endogenous 06 -methylguanine DNA methyltransferase (MGMT) enzyme (mex') and the other was deficient in this repair activity (mex-). The progeny shuttle vectors were amplified from the cellular genomic DNA by using polymerase chain reaction (PCR) technology. The mutation frequencies arising from the replication of the adducts were analyzed by NheI digestion. The mutation frequencies arising from both adducts were high in the repair deficient cells (28-50% for 0 4MeThy and 7-8.5% for O6MeGua). The mutation frequencies for O4MeThy were significantly higher than those for O6MeGua in the same cell line. In methyltransferase proficient cells (mex+), the mutation frequency for O4MeThy remained high (22-42%), whereas the mutation frequency for O6MeGua dropped to background levels (<1%) in these cells. These results indicated that the 04MeThy adduct was not a substrate for repair by the mammalian methyltransferase. In addition, the high mutation frequency values arising due to replication of the adducted thymine in both mex- and mex + cells indicated that this adduct may not be a substrate for repair by any repair system in the CHO cells. Sequence analysis of the PCR products showed the mutations to be exclusively T--C transitions for 04MeThy and G---A transitions for O'MeGua. Individual G418' clones were selected from CHO cells that had been transfected with O4MeThy and O6MeGua modified shuttle vectors. Mutation frequency values for the mex- clones arising from both vector types were determined in order to obtain more information into how each cell processed the alkylated bases. Thesis Supervisor: Dr. John M. Essigmann Title: Professor of Chemistry and Toxicology Acknowledgements A great number of very special people deserve credit for helping me complete my thesis work and it is a pleasure for me to mention them here. First, I thank my parents, Jim and Carol, for being the terrific people they are, for supporting me throughout my every endeavor and for giving me such a great foundation which allowed me to reach for my highest goals. I also want to thank my sister, Kristi, for being such a good friend and confidant and for showing me that sisters are treasures to have. I owe many thanks to my advisor, John Essigmann, for his support, his understanding in times of trouble and for letting me go on my extended vacations. Thanks also to the members of my committee, Bill Thilly, Helmut Zarbl and Ed Loechler for their helpful suggestions and their never-ending search for the scientific truth. I am grateful to Colette Hodes and Manjit Dosanjh not only for the shuttle vectors and adducted oligos that I used in my thesis, but also for their scientific insight and their friendship. Both of them have shown me that you can get what you want out of life; it just may take traveling down a different road. I owe a big debt of gratitude to Kim, Denise and Laurie for keeping it all together in the lab and for suffering through my endless baby stories. Denise, you'll always be my Garth Brooks informant! I also want to thank my UROP, Cindy Parrish, for her able assistance and cheery personality. I would like to give a special thanks to Roberta Pastorelli. Roberta has been a dear friend, showing me how to be excited about science even when nothing is working and teaching me how to look for the beautiful things in life. In addition to teaching me Italian (Ciao, bonazza!), she expanded my cultural horizons and shows me still that if you look at it the right way, an ocean's span is really just right next door. I owe a debt of gratitude to a few members of the Stubbe lab for maintaining my mental health while I was completing my thesis. These people are responsible for filling my days with laughter and helping me enjoy many fun nights at the Border. Thanks go to Squire Booker (it work!) for being my "Texas Connection". Special thanks go to Michael Absalon for being such a good friend, helping me with my project, listening to my ideas, and showing me the characteristics of a true scientist. I would like to thank my friend and benefactor, Lisa Naser Bradley, for the countless things she has done for me during and after her time in the lab. I would also like to thank Susan Verghis for her scientific advice, generosity and discussions about graduate school and family. I also thank Elizabeth Trimmer for her friendship, her constant willingness to listen, and for being such a nice person and Debbie Kreutzer for lifting my spirits with her bright personality, encouraging me with her supportiveness and for being a great friend. Extra special thanks go to Dipti Mathur for innumerable reasons. She has proven that a true friend loves you unconditionally. We've laughed together, cried together and with our husbands, solved the world's problems together. I thank my in-laws, Tom and Suzanne, for being so supportive and giving me a great place to live and to finish my thesis. I thank Chanter, Jesse and Cody for their love, devotion and their understanding when I tried to pack more members into our family! I want to thank my precious son, Joshua, for being so accomodating when I dragged him into work with me for months on end. In addition to being the world's happiest baby and biggest flirt, Josh has given my life balance and has shown me the true meaning of sacrifice and love. Finally, I want to thank my husband, Tom, for being my best friend and fan. I dedicate this thesis to him, for without his belief in me, I would never have been able to accomplish this work. His constant love, encouragement and understanding have enabled me to reach for the stars. This is for you, babe. ........ ....... TABLE OF CONTENTS Title Page 1 Committee Page 2 Abstract 3 Acknowledgements 5 Table of Contents 7 List of Figures 9 List of Tables 11 List of Abbreviations 12 I. Introduction 13 II. Literature Survey 16 A. Introduction 17 B. The role of 04AlkThy adducts in carcinogenesis 18 C. Mutagenesis by 04AlkThy adducts 26 D. Repair of 04AlkThy and O6AlkGua adducts 30 E. The role of O6AlkGua in carcinogenesis 48 F. Shuttle vector systems for investigating mutagenesis in mammalian cells 52 III. Materials and Methods 61 IV. Results 76 A. Use of the shuttle vector system to investigate 0 4MeThy mutagenesis 77 B. Introduction of a single 0 4MeThy or O6MeGua into the NheI site of the shuttle vector pCNheI 82 C. Mutagenesis by 0 4MeThy and 06MeGua in CHO cells 101 D. Mutational specificity of 0 4MeThy and O6MeGua in CHO cells 119 E. Analysis of individual clones that have arisen during G418 selection 125 V. Discussion 130 A. Advantages and limitations of the shuttle vector system used to determine mutagenesis in mammalian cells 133 B. Types of mutations induced by 04MeThy in O6MeGua-DNA methyltransferase deficient mammalian cells 136 C. Comparison of the mutation frequencies of 04MeThy and 06MeGua in mammalian cells of differing repair capacities 139 D. Repair of 04MeThy and O6MeGua in CHO cells possessing O6MeGua-DNA methyltransferase repair protein 143 E. Mutation frequencies of individual clones arising from mex- and mex ÷ cells transfected with 04MeThy and 06MeGua shuttle vectors 145 F. Probability distribution of mutations in CHO mex- and mex+ cells using shuttle vectors transfected by the calcium phosphate precipitate technique 150 G. Experimental error in the transfection protocol 175 VI. Concluding remarks and suggestions for future research 178 References 189 Appendix A. Mutation Frequency Analysis by Oligonucleotide Hybridization 207 Appendix B. Mutation Frequency Analysis by Mismatch Amplification Mutation Assay (MAMA) 220 Biographical Sketch 252 LIST OF FIGURES Figure No. Title Page 1. Sites of in vivo and in vitro alkylation in nucleic acids 19 2.
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