Determining the Role of p53 Mutation in Human Breast Cancer Progression Using Recombinant Mutant/Wild-Type p53 Heterozygous Human Mammary Epithelial Cell Culture Models Item Type text; Electronic Dissertation Authors Junk, Damian Jerome Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 28/09/2021 18:36:12 Link to Item http://hdl.handle.net/10150/193600 DETERMINING THE ROLE OF P53 MUTATION IN HUMAN BREAST CANCER PROGRESSION USING RECOMBINANT MUTANT/WILD-TYPE P53 HETEROZYGOUS HUMAN MAMMARY EPITHELIAL CELL CULTURE MODELS by Damian Jerome Junk _________________________ A Dissertation Submitted to the Faculty of the GRADUATE INTERDISCIPLINARY PROGRAM IN CANCER BIOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2008 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Damian Jerome Junk entitled Determining the Role of p53 Mutation in Human Breast Cancer Progression Using Recombinant Mutant/Wild-Type p53 Heterozygous Human Mammary Epithelial Cell Culture Models and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy _______________________________________________________________________ Date: 4/18/08 Bernard W. Futscher, Ph.D. _______________________________________________________________________ Date: 4/18/08 Anne E. Cress, Ph.D. _______________________________________________________________________ Date: 4/18/08 Jesse D. Martinez, Ph.D. _______________________________________________________________________ Date: 4/18/08 David W. Mount, Ph.D. _______________________________________________________________________ Date: 4/18/08 Mark A. Nelson, Ph.D. Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: 4/18/08 Dissertation Director: Bernard W. Futscher, Ph.D. 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: Damian Jerome Junk 4 ACKNOWLEDGEMENTS Fist and foremost I want to thank God for all of the blessings in my life. Most importantly, I am thankful for all of the teachers throughout my life that took the extra time to encourage, instruct, and challenge me. I would like to thank Dr. Bernard Futscher for the amazing opportunity to work in his lab. His guidance and instruction, not only about science but also life in general demonstrate his unique ability to mentor. I would like to thank the members of my committee for their thoughtful insights that have improved the research and my ability to conduct it. I thank Dr. Jesse Martinez for his p53 expertise, the E6 plasmid constructs, and assistance with the sub-cellular localization assay. I want to thank the members of the Futscher lab for all of their help and making the lab a place you look forward to going each and every day. I especially want to thank Dr. Marc Oshiro for his day-to-day interest in my scientific development. He was always there to challenge me and demonstrate proper scientific technique, and I have greatly valued his friendship over the years. I want to thank my family who encouraged me to continue my education. I would like to thank the new additions to my family, the Watterson’s, for their love and support. I thank my brothers Danial and Dennis for always being there. I thank my parents Daniel and Mary Jane Thomas, William Junk, and Janet Blair for being my first teachers and continuing to be role models and involved in my education. Finally I thank my wife Summer and daughter Ella. Their love and support make all things possible. They have sacrificed more for this work than I. 5 DEDICATION This work is dedicated to my little girl Ella McKenzie. I hope she is blessed with the creativity and imagination to dream big. And I hope she is gifted with the determination, strength, courage, perseverance, and faith to make those dreams a reality. 6 TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................... 9 LIST OF TABLES ......................................................................................................... 11 LIST OF ABBREVIATIONS ....................................................................................... 12 ABSTRACT .................................................................................................................... 19 INTRODUCTION .......................................................................................................... 21 Breast Cancer ............................................................................................................. 21 Breast cancer incidence and mortality .................................................................... 21 Breast cancer risk factors .........................................................................................22 Types of breast cancer ............................................................................................. 22 Breast cancer screening and treatment ................................................................... 24 Human mammary epithelial cell culture .................................................................. 26 The p53 Tumor Suppressor ...................................................................................... 29 The p53 gene and protein structure ......................................................................... 29 Protein levels of p53 in normal cells ....................................................................... 31 Activation of p53 in normal cells ............................................................................. 32 Function of p53 in normal cells ............................................................................... 37 Inactivation of p53 in cancer cells ........................................................................... 43 The role of p53 mutation in breast cancer ............................................................... 47 Epigenetic Mechanisms of Transcriptional Control ............................................... 49 Epigenetic modifications in normal cells ................................................................. 49 Epigenetic modifications in cancer cells ................................................................. 52 Epigenetic silencing of MASPIN and DESMOCOLLIN3 ........................................ 54 Cooperation of Genetic and Epigenetic Dysfunction .............................................. 56 Dissertation Aims ....................................................................................................... 59 MATERIALS AND METHODS .................................................................................. 60 Materials ..................................................................................................................... 60 Adenoviruses ............................................................................................................ 60 E6 constructs ............................................................................................................ 60 Methods ....................................................................................................................... 60 Cell culture ............................................................................................................... 60 Adenoviral infections ............................................................................................... 61 Nucleic acid isolation .............................................................................................. 62 MCF10A E6 cell line production ............................................................................. 62 Mutant p53 cloning .................................................................................................. 64 pENTR cloning ........................................................................................................ 65 7 TABLE OF CONTENTS - Continued LR clonase generation of UBC lentivirus ................................................................ 65 Topo cloning generation of CMV lentivirus ...........................................................