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Ann Arbor, MI 48106 PROTEIN INDUCTION AND ESTROGEN METABOLISM IN SUBCELLULAR PREPARATIONS AND TISSUE SLICES OF THE GOLDEN SYRIAN HAMSTER DISSERTATION Presented in Partial Fulfillment of the Requirements for the degree of Philosophy in the Graduate School of The Ohio State University By Mustapha Ahmed Beleh, B.S. **** The Ohio State University 1992 Dissertation Committee Approved by: Robert W. Brueggemeier, Ph.D. Young C. Lin, D.V.M., Ph.D. Duane D. Miller, Ph.D. Adviser Dennis R. Feller, Ph.D. College of Pharmacy Dedication To the sweetest sister, Dalia ii ACKNOWLEDGMENTS I would like to express my gratitude and thanks to everyone who aided in making the past five years successful: My family, especially my late mother, my father, stepmother, sister and brother for all the support and care they offered over the years. My adviser Dr. Robert Brueggemeier for his guidence, patience and teachings over the course of my graduate career. The members of my graduate committee, especially Dr. Young Lin for his help and advise. Denise Donley and Patricia Goetz, both for their assisting in some of the studies performed within this dissertation work and for their friendship, Nancy Katlic for her help with different aspects in the laboratory, George Chang for aiding in cell culture related techniques and Jack Fowble and John Miller for their various technical help. Soheila Ebrahimian, my friend and laboratory partner for five years, and my friend and roommate Nader Moawad for all the help and support they supplied. My fellow graduate students, especially Kimberly Markovich, Allen Hopper, Meri Slavica and Anne "cool" Quinn. Yasser AbdelGhany, a friend who is always there, and all my friends over the years, Vimon, Yushi, Becky and Nagy. Finally, I would like to thank my friends who stood by me during the final phase of my graduate studies: Helen, Saroj, Yasmine and Patricia White for their help in preparation of this dissertation, Farida, Michelle and Shamim for their friendship and support. VITA February 24, 1963 ........................ Bom - Egypt June 1985 ...................................... B.S. pharmacy, University of Alexandria School of Pharmacy, Egypt 1987 - 1988 ................................... Graduate Teaching Associate The Ohio State University Columbus, Ohio 1988 - 1991 ................................... Graduate Research Associate The Ohio State University Columbus, Ohio 1991 - present ............................... Proctor and Gamble fellowship The Ohio State University Columbus, Ohio PUBLICATIONS AND ABSTRACTS 1.Brueggemeier, R. W.; Tseng, K.; Katlic, N. E.; Beleh, M. A.; Lin, Y. C. Estrogen metabolism in primary kidney cell cultures from Syrian hamsters. J. Steroid Biochem. 1990, 36, 325-331. 2.Brueggemeier, R, Tseng, K., Katlic, N., Beleh, M., Lin, Y. Estrogen metabolism in primary kidney cell cultures from Syrian hamsters, American Association of Cancer Research, Washington D.C., May 1990, Abst. No. 1330. v 3. Beleh, M., Donley, D., Lin, Y., Brueggemeier, Estrogen metabolism in subcellular preparations and tissue cultures of Syrian hamster liver and kidney, Internationa Symposium on Hormonal Carcinogenesis, Cancun, Mexico, March 1991. 4.Beleh, M., Donley, D., Lin, Y., Brueggemeier, Estrogen metabolism in subcellular preparations and tissue cultures of Syrian hamster liver and kidney, The Endocrine Society meeting, Washington D.C., June 1991, Abst. No. 532. FIELD OF STUDY Major field : Pharmacy TABLE OF CONTENTS PAGE DEDICATION......................................................................................................... ii ACKNOWLEDGMENTS...................................................................................... iii VITA................ v LIST OF FIGURES................................................................................................. x LIST OF TABLES................................................................................................xiv LIST OF PLATES.................................................................................................xvi CHAPTER I. INTRODUCTION............................................................................1 1.1 Cancer .................................................................................................................1 1.1.1 Historical perspective .......................................................................... 1 1.1.2 The cancer epidemic ........................................................................... 3 1.1.3 Cancer terminologies ........................................................................... 4 1.1.4 Treatment of cancer ............................................................................ 5 1.1.5 Risk factors and cancer ....................................................................... 9 1.2 Hormone-dependent cancer ..............................................................................10 1.2.1 Endocrinology and hormones ........................................................10 1.2.2 Steroid hormones and their receptors ................................................12 1.2.3 Estrogens and their physiological role .............................................. 17 1.2.4 Estrogen biosynthesis and metabolism ...................... 21 1.2.5 The role of estrogens in carcinogenesis ...........................................23 1.3 The golden Syrian hamster : an animal model for estrogen-dependent cancer .............................................................. 26 1.3.1 Induction of renal adenocarcinomas .................................................26 1.3.2 Morphology and histology of the tumors ........................................ 29 1.3.3 The role of hormonal and carcinogenic activity of estrogens in tumorigenesis .............................................................33 1.4 Growth factors, oncogenes and cell transformation .......................................36 1.4.1 Growth factors : general aspects ................................................ 36 1.4.2 Growth factors : a closer look .................................................... 41 1.4.3 Oncogenes and tumor suppressor genes ............................................44 1.4.4 The role of growth factors, oncogenes and tumor supressor genes in breast cancer .........................................................47 1.5 Estrogen metabolism........................................................................................50 1.5.1 Oxidative metabolism of estrogens .....................................................50 1.5.2 Estrogen metabolism in Syrian hamsters ...........................................53 1.5.3 The role of DNA and protein adducts and cell death in carcinogenesis ................................................................58 CHAPTER II. OBJECTIVES AND EXPERIMENTAL......................................62 2.1 Statement of objectives .....................................................................................62 2.2 Experimental ..................................................................................................... 64 2.2.1 Materials and methods ........................................................................ 64 2.2.2 Preparation of primary kidney cell cultures ..................................... 66 2.2.3 Preparation of kidney and liver microsomes ..................................... 67 2.2.4 Preparation of kidney and liver slices ............................................... 68 2.2.5 Protein biosynthesis in primary kidney cell cultures........................68 2.2.6 Separation of proteins using one and two dimensional electrophoresis ................................................................69 2.2.7 Western blotting .................................................................................. 71 2.2.8 Immunoprecipitation