MICRO-ANALYSIOS F ADENYLATCEY CLASEA ND CYCLICA MP-DEPENDENPTR OTEINK INASEA CTIVITIES IN HUMANM AMMARTYill 10RS by Terry Ann Woodford Dissertation submitted to the Graduate Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of DOCTORO F PHILOSOPHY in Zoology APPROVED: Dr. C. L. Rutherford Dr. E. R. Stout Dr. J, M. Conroy Dr. T. W. Keenan Dr. D. A. Stetler Dr. R. C. Krero.p August 1982 Blacksburg, Virginia This dissertation is dedicated to my mother, whose loss inspired my work, and to my father, each for their strength, encouragement, and continued faith in me. ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. Charles L. Rutherford for his guidance and support throughout the course of this study. I am also grateful to the members of my graduate committee, Dr. E. R. Stout, Dr. J.M. Conroy, Dr. D. A. Stetler, and Dr. R. C. Kramp, for their interest and valuable suggestions which contributed significantly to this work. Recognition is extended to my colleagues, Robert Taylor, Beth Gutmann, Jan Murphy, Ken Cooper, and also Michael Zink and Becky Grover, without whom various phases of the project would not be complete. I am very much indebted to Dr. Tom Keenan for joining my graduate committee during the last stages of my work, and also for his scientific insight, and generous friendship, and to Matthew White for his patient understanding and never-ending kindness. I am grateful to my brother, Wick, for his valuable advice, and to my dear friend Susan Anderson for making me laugh when life seemed all too serious. Also equally worthy of gratitude· are W.C. and Squeek for their companionship. My special thanks are most deservingly given to Linda Brown and Kerry Dylewski for their laborious time and effort in preparation of this manuscript, and to Dan Dylewski for photographic assistance. Finally, I gratefully acknowledge the financial assistance offered to me by the Department of Biology at Virginia Tech. The re- search was supported by Grant Number CA 24150 awarded by the National Cancer Institute. iii TABLE OF CONTENTS Chapter 1. INTRODUCTIO•N•• 1 1.1. Description of the Normal and Neoplastic Mammary Gland System • • • • • • 4 1.1.1. Normal Glandular Tissue 4 . . . . . 1.1.2. Neoplastic Glandular Tissue 5 . . . 1.1.2.1. Fibroadenoma. 5 . 1.1.2.2. Infiltrating Ductal Carcinoma 6 1.2. Cyclic 3': 5' Adenosine Monophosphate: Function and Implications in Carcinogenesis 7 1.3. Characterization of Adenylate Cyclase. 20 1.4. Characterization of Cyclic A."MP-Dependent Protein Kinase. 36 1.5. Statement of Objectives for the Biochemical Approach • • • 44 2. MATERIALSA NDM ETHODS 48 2 .1. Materials • • • . 48 2.1.1. Experimental Materials •• 48 2.1.2. Tissue Specimens. 49 2.2. Methods 49 2.2.1. Tissue Sections •• 49 2.2.2. Sample Preparation .• 50 2.2.2.1. Tissue Homogenates. so 2.2.2.2. Specimen Micro-Dissection . 51 2.2.3. Measurement of Dry Weight •.•.• 52 iv TABLEO F CONTENTS( CONTINUED) Chapter 2.2.4. Protein Determination. . • . • 54 2.2.5. Preparation of 125r-TME-ScAMP • 54 2.2.6. Preparation of Calmodulin. • 55 2.2.7. Solubilization of Adenylate Cyclase • 55 2.2.8. Preparation of Heat Stable Inhibitor of Protein Kinase from Tumor Extracts 56 2.2.9. Analytical Techniques. • • . • • • . 56 2.2.9.1. Radioinununoassay for Adenylate Cyclase ••••••••••• 56 2.2.9.2. Filter Binding Assay for Cyclic 3':5' AMP-Dependent Protein Kinase • • • . • • • • • . • 59 2.2.9.3. Spectrophotometric Assay for Adenosine Triphosphatase 60 2.2.9.4. Measurement of Endogenous Cyclic 3':5' AMPL evels. 61 2.2.9.5. Quantification of Cyclic 3':5' AMP Binding Protein. . • 61 2.2.9.6. Electrophoretic Analysis of Tumor Extracts . • • • • 62 2.2.9.7. Analysis of Endogenous Protein Phosphorylation. • • • • • • 62 2.2.9.8. DEAE-Sephacel Chromatography of the Protein Kinase Hol o enzyme 63 2.2.9.9. Separation of Regulatory and catalytic subunits by cAMP- Agarose Affinity Chroma- tography • • • • • • • • • • 64 V TABLEO F CONTENTS(C ONTINUED) Chapter 3. RESULTS •••• 66 3.1. Adenylate Cyclase Associated with Human Mammary Tumor Particulate Fractions. • 66 3.1.1. Evaluation of Adenylate Cyclase Assay . . . . Conditions 66 3.1.2. Assay Parameters in the Presence of . . . . . . MgATP. · 73 3.1.3. Comparison of Adenylate Cyclase Activity in Benign and Malignant Mammary Tumors • • • • • • • • • • • • • • • • 7 3 3.1.4. Calcium and Calmodulin Effects on Membrane-Bound Tumor Adenylate . . . . . . . . . . . . Cyclase. 79 . 3.1.5. Enzyme Activation by Guanyl Nucleotides 83 . . . . . . 3.1.6. Enzyme Activation by Fluoride 87 3.1.7. Effect of Additional Substances on Tumor Adenylate Cyclase ••••••••••• 92 3.1.8. Hormonal Responsiveness of Tumor Particle Adenylate Cyclase. • • • • • • • • 92 3.1.9. Thermal Stability of Tumor Adenylate Cyclase. • • • • • • • • • • • 102 3.1.10. Solubilization of Membrane-Bound Adenylate Cyclase ••••• 106 3.2. Adenylate Cyclase From Mammary Tumor Microsections. • • • • • 109 3.2.1. Determination of Enzyme Activity •• 109 3.2.2. Inter-and Intra-Tumor Comparisons • 111 3.2.3. Influence of Divalent Cations on Adenylate Cyclase ••••• 111 vi TABLE OF CONTENTS( CONTINUED) Chapter 3.2.4. Stimulation by Guanyl Nucleotides . . 115 3."2.5. Hormonal Responsiveness of Adenylate Cyclase. . . . . ..•...•. 120 3.2.6. Thermal Stability of Tissue-Associated Adenylate Cyclase. . . . . . • • . 128 3.2.7. Fractionation of Tissue-Associated Adenylate Cyclase. . . . . . . . . . 128 3.3. Protein Kinase Associated with Malignant and Benign Mammary Tumors. . . . . • . . . . 132 3.3.1. Evaluation of Protein Kinase Assay Conditions . . . . 132 3.3.2. Substrate Specificity, Substrate Affinity, and pH Optimum for Tumor- Derived Protein Kinase . . . . . . 133 3.3.3. Activation of Tumor-Derived Protein Kinase by Cyclic 3':5' AMP and Cyclic 3' :5' GMP ...•....... 135 3.3.4. Activation of Tumor-Derived Protein Kinase by Additional Cyclic Nucleotides .............. 141 3.3.5. Cyclic AMP-Dependent Protein Kinase Activity and Activity Ratios in a Series of Malignant and Benign Tumors . . • . . . . . . . . . . . . 141 3.3.6. Cyclic AMP-Dependent Protein Kinase Activity in Tumor Microsections. . . 147 3.3.7. Effect of Known Inhibitors of Protein Kinase . . 152 3.3.8. Thermal Stability of Tumor Protein Kinase . . . . . . . . . . . . . . . . 155 vii TABLE OF CONTENTS( CONTINUED) Chapter Page 3.3.9. Subcellular Localization of Tumor Protein Kinase Activity. 155 . 3.4. Mammary Tumor-Derived cAMP Binding Protein. 160 3.4.1. Evaluation of cAMP Binding Assay. . 160 3.4.2. Analysis of cAMP Binding Capacity in . . . . . Human Mammary Tumors . . 160 3.4.3. Estimation of Binding Affinity for cA.~P ; 163 3.5. Partial Purification of Protein Kinase Holoenzyme •... • . 163 3.5.1. DEAE-Sephacel Chromatography of Tumor Supernatant Fractions. . . • • • . 163 3.5.2. Separation of Catalytic and Regulatory Subunits by cAMP-Affinity Chromatography . • • . . . • . . 177 3.5.3. Reconstitution of the Protein Kinase Holoenzyme . . . . . . 180 3.6. Human Mammary Tumor-Derived Casein Kinase • 186 3.7. Endogenous Phosphorylation in Mammary Tumor Cytosolic Proteins . 190 4. DISCUSSION. . . 209 4.1. Statement of the Principle. . 209 4.2. Interpretation of the Data. 218 4.2.1. Adenylate Cyclase in ~eoplastic Mannnary Tissue • . . . • • 218 4.2.2. Protein Kinase Activity in Neoplastic Mammary Tissue . . . . . . • • 225 4.3. Implications for Future Study 238 viii TABLEO F CONTENTS(C ONTINUED) Chapter Page 5. SUMMAR•Y•• ••. ••• 241 6. LITERATUREC ITED• • • 245 VITA • • 271 ix LIST OF TABLES Table 1 EFFECTO F INCREASINGM n-ATP CONCENTRATIOONN TUMOR PARTICULATAE DENYLATCEY CLASE.• • • • • • • • • • 72 2 COMPARISOONF ADENYLATCEY CLASEA CTIVITYA SSOCIATED WITHP ARTICULATEFR ACTIONSF ROMB ENIGNA NDM ALIGNANT ~y TUMORS •••••••••••••••••• 77 3 TUMOR-ASSOCIATAEDD ENYLATCEY CLASEA CTIVITY IN THE PRESENCEO F Mn2 + , Mg2 + , AND Mn2 +- Ca 2+ ••••••. 80 4 EFFECT OF Ca2+ ANDI NCREASINGA TP CONCENTRATIOONN TUMOR-ASSOCIATAEDD ENYLATCEY CLASE• •....•. 81 5 EFFECT OF CALMODULIONN PARTICULATAE DENYLATCEY CLASE FROMM ALIGNANATN DB ENIGNT UMORS•• ••••••. 82 6 EFFECT OF CHOLERAT OXIN ON MAMMARTYU MOR-ASSOCIATE PARTICULATAE DENYLATCEY CLASEA CTIVITY ..•... 85 7 ACTIVATIONO F TUMOR-ASSOCIATAEDD ENYLATCEY CLASEB Y GUANYL-15-YL-IMIDODIPHOSPHATIEN THE PRESENCEO F Mn-ATP and Mg-ATP.• ••••..•••••••• 86 8 GUANYL-5'-YL-IMIDODIPHOSPHATE-ACTIVATOIFO N ADENYLATCEY CLASEA SSOCIATEDW ITHA PARTICULATE FRACTIONF ROMB ENIGNT UMORF A-3070 .•• 88 9 COMPARISOONF ADENYLATCEY CLASEA CTIVITY IN PARTICULATEFR ACTIONSF ROMB ENIGNA NDM ALIGNANT TUMORSIN THE PRESENCEO F GVANYL-5'-YL- IMIDODIPHOSPHAT..E.. ....•••••... 89 10 ' SODIUMF LUORIDES TIMULATIONO F TUMOR-ASSOCIATED ADENYLATCEY CLASEA CTIVITY .••.••.....• 90 11 SODIUMF LUORIDEA ND GUANYL-5'-YL-IMIDODIPHOSPHATE- INDUCEDS TIMULATIONO F ADENYLATCEY CLASEF ROM MALIGNANTTU MORID C-3669 •••••••• 93 12 INFLUENCEO F VARIOUSP OTENTIALE FFECTORSO N PARTICULATAE DENYLATCEY CLASEF ROMT WOH UMAN MAMMARTYU MORS• • • • • . • • • • • • • • • • • • • • 103 X
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