MICRO-ANALYSIS OF ADENYLATE CYCLASE AND CYCLIC AMP-DEPENDENT PROTEIN ...

293 Pages·2017·12.61 MB·English

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