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Nuclear retention of topical glucocorticoids and absence of cross-linking with DNA in cultured dermal… Au, Diana Shu-Lian 1981

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cl NUCLEAR RETENTION OF TOPICAL GLUCOCORTICOIDS AND ABSENCE OF CROSS-LINKING WITH DNA IN CULTURED DERMAL FIBROBLASTS """""^  DIANA SHU-LIAN JkU B.Sc. (Pharm), N a t i o n a l Taiwan U n i v e r s i t y , 1973 M.S. (Pharm), The Ohio S t a t e U n i v e r s i t y , 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES (The D i v i s i o n of Pharmaceutics i n the F a c u l t y o f Pharmaceutical Sciences) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA by DOCTOR OF PHILOSOPHY i n January, 1981 ( c T ) Diana Shu-Lian Au, 1981 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of Pharmaceutical Sciences The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date February 25, 1981 i . ABSTRACT The p r i n c i p a l mechanism by which g l u c o c o r t i c o i d s exert t h e i r phy-s i o l o g i c a l e f f e c t s i s by a l t e r a t i o n s of DNA metabolism. Studies have been done to c l a r i f y the nuclear s i t e s of human and mouse L-929 f i b r o b l a s t s , w i t h which the t o p i c a l g l u c o c o r t i c o i d s , hydrocortisone (HC) and triam c i n o l o n e ace-tonide (TA), are l i k e l y to i n t e r a c t f o r i n i t i a t i n g the a l t e r a t i o n s i n DNA r e a c t i o n s , e s p e c i a l l y DNA i t s e l f and chromatin p r o t e i n s . F u r t h e r , data have been added on the uptake and r e t e n t i o n of TA i n the n u c l e i of L-929 f i b r o b l a s t s . A l l s t u d i e s have been done using a c t i v e l y m e t a b o l i z i n g c e l l s i n c u l t u r e over extended periods of time of exposure to the s t e r o i d s (0-96 h r ) . The p o s s i b l e d i r e c t glucocorticoid-DNA i n t e r a c t i o n - by c r o s s - l i n k i n g was i n v e s t i g a t e d by hydroxyapatite chromatography and thermal scanning analy-s i s , using DNA i s o l a t e d from 8-methoxypsoralen t r e a t e d and UV-A i r r a d i a t e d mouse L-929 f i b r o b l a s t s as p o s i t i v e c o n t r o l s . C r o s s - l i n k i n g was not d e t e c t a -b l e under wi d e l y v a r y i n g experimental c o n d i t i o n s . The f a t e of the g l u c o c o r t i c o i d i n n u c l e i was st u d i e d by measuring 3 the amount of s p e c i f i c a l l y r e t a i n e d TA. The s p e c i f i c r e t e n t i o n of H-TA at a —8 conce n t r a t i o n of 10 M i n c u l t u r e s of L-929 f i b r o b l a s t s was evaluated i n i n -t a c t n u c l e i , chromatin and d e p r o t e i n i z e d DNA. The d i s t r i b u t i o n of s p e c i f i c a l l y r e t a i n e d TA between cytoplasm and n u c l e i , and i t s i n t r a n u c l e a r d i s t r i b u t i o n between the nucleoplasm and chromatin a l s o were determined as a f u n c t i o n of time. Approximately, 10% of c y t o s o l r e t a i n e d TA was t r a n s l o c a t e d i n t o the nu-cl e u s . The highest l e v e l , 0.4 fmole/ug nuclear p r o t e i n or 2.8 fmole/ug DNA, was reached a f t e r 6 hours. R e l a t i v e l y constant l e v e l s , approximately h a l f of , the highest l e v e l , were observed ther e a f t e r up to 96 hr. The m a j o r i t y of TA i n the nucleus, 56-75%, was as s o c i a t e d w i t h chromatin. The l e v e l of TA r e -tained i n chromatin reached the highest l e v e l , 0.6 fmole/ug chromosomal pro-t e i n or 1.1 fmole/ug DNA, a f t e r 6 hours of i n c u b a t i o n . R e l a t i v e l y constant i i . l e v e l s , approximately 60% of the highest l e v e l , were observed t h e r e a f t e r up to 96 hr. 3 A l t e r n a t i v e s i t e s of subchromatin l o c a l i z a t i o n of the H-TA, i n terms of v a r i o u s c l a s s e s of histone and nonhistone p r o t e i n s , were s t u d i e d i n a p r e l i m i n a r y i n v e s t i g a t i o n by the s e l e c t i v e d i s s o c i a t i o n method of immo-b i l i z e d chromatin on hydroxyapatite. The TA was l o c a t e d i n unbound chromoso-mal p r o t e i n s (75%), histone p r o t e i n s (13%) and nonhistone p r o t e i n s (12%). None of the g l u c o c o r t i c o i d was found i n f r a c t i o n s of n u c l e i c a c i d s , thus con-f i r m i n g the unlild-iness of d i r e c t glucocorticoid-DNA i n t e r a c t i o n s , but i n d i c a -t i n g that the primary s i t e s of TA-nuclear i n t e r a c t i o n s r e s i d e i n chromatin p r o t e i n s . The suppressive e f f e c t of triamcinolone acetonide, at concentrations —8 —6 of 10 and 5.0 x 10 M i n c u l t u r e media, on nuclear p r o t e i n s and DNA of mouse L-929 f i b r o b l a s t s was determined f o r v a r i o u s i n c u b a t i o n i n t e r v a l s . The suppression was demonstrated to be a l a t e , 24 hours or longer, consequence of the treatment of TA. The chemical s t a b i l i t y of the g l u c o c o r t i c o i d s used was examined i n storage s o l u t i o n s and i n media removed from t e s t c u l t u r e s by a g a s - l i q u i d chro-matographic assay developed f o r the purpose. The assay involved double d e r i -v a t i z a t i o n of the g l u c o c o r t i c o i d s with methoxyamine and N - t r i m e t h y l s i l y l i m i -dazole, and i s capable of d e t e c t i n g nanogram q u a n t i t i e s of h y d r o c o r t i s o n e , triamcinolone acetonide and desonide. i i i . TABLE OF CONTENTS PAGE LIST OF TABLES v i l l LIST OF FIGURES x LIST OF ABBREVIATIONS x i v INTRODUCTION . i LITERATURE SURVEY 3 I . C l i n i c a l uses and adverse e f f e c t s of t o p i c a l g l u c o c o r t i -coids 3 I I . S t r u c t u r e - a c t i v i t y - r e l a t i o n s h i p of g l u c o c o r t i c o i d s 6 I I I . Biochemical events accompanying the c l i n i c a l and adverse e f f e c t s i n the s k i n 10 IV. Mechanism of g l u c o c o r t i c o i d a c t i o n s - an overview 12 V. Receptor models of the mechanism of g l u c o c o r t i c o i d a c t i o n 15 A. N o n - s p e c i f i c binding before uptake by target c e l l s 17 B. G l u c o c o r t i c o i d passage across the c e l l membrane 18 C. Cytoplasmic receptor binding 18 D. A c t i v a t i o n and t r a n s l o c a t i o n of g l u c o c o r t i c o i d - r e c e p -t o r complex 21 E. Nuclear acceptor binding and gene expression 22 VI. Physicochemical c h a r a c t e r i s t i c s of g l u c o c o r t i c o i d receptor and receptor binding 23 V I I . Nuclear binding of g l u c o c o r t i c o i d s 27 V I I I . Nuclear components associated w i t h g l u c o c o r t i c o i d binding 30 IX. Drug i n t e r a c t i o n s with DNA 36 X. G l u c o c o r t i c o i d s as p o t e n t i a l i n t e r c a l a t o r s 41 XI. Nuclear r e s i d u a l forms of g l u c o c o r t i c o i d - r e c e p t o r complexes 45 i v . PAGE X I I . Techniques f o r de t e c t i n g c r o s s - l i n k s 48 A. Hydroxyapatite chromatography 50 B. Thermal scanning a n a l y s i s 52 X I I I . Regulation of gene expression 58 XIV. Dichotomy and time p r o f i l e s of g l u c o c o r t i c o i d e f f e c t s 62 XV. S u b c e l l u l a r l o c a l i z a t i o n of g l u c o c o r t i c o i d s 63 EXPERIMENTAL 66 SECTION I . G a s - l i q u i d chromatographic a n a l y s i s of hydrocortisone,  triamcinolone acetonide and desonide i n c u l t u r e media of mouse and human dermal f i b r o b l a s t s 68 A. I n t r o d u c t i o n 68 B. Experimental 69 1. M a t e r i a l s 69 2. Preparation of d e r i v a t i v e s 71 3. D i f f e r e n t i a l scanning c a l o r i m e t r y 72 *4. G a s - l i q u i d chromatography 72 5. Gas chromatography-mass spectrometry 72 6. E x t r a c t i o n procedure 73 7 . Recovery studies 73 8. Assays on b i o l o g i c a l samples 74 C. Results and d i s c u s s i o n 74 1. Confirmation of p u r i t i e s of t e s t g l u c o c o r t i c o i d s 74 2. S e l e c t i o n of e x t r a c t i n g solvent 75 3. S e l e c t i o n of d e r i v a t i z i n g agents 75 H. Column s e l e c t i o n 76 5. Optimum r e a c t i o n time f o r s i l y l a t i o n and s t a b i l i t y of d e r i v a t i v e s 76 6. Confirmation of d e r i v a t i v e formation using GC-MS 76 PAGE 7. Resolution of MO-TMS d e r i v a t i v e s on OV-7 and OV-17 columns 87 8 . C a l i b r a t i o n curves 90 9. B i o l o g i c a l data 95 10. Me t a b o l i t e s 99 11. A p p l i c a b i l i t y of MO-TMS r e a c t i o n to other g l u c o c o r t i -coids 100 SECTION I I . I n v e s t i g a t i o n of c r o s s - l i n k i n g of DNA induced by g l u -c o c o r t i c o i d s and nuclear r e t e n t i o n o f TA 101 MATERIALS AND METHODS 1 0 1 A. C e l l c u l t u r e s and g l u c o c o r t i c o i d s 101 1. C e l l c u l t u r e s 101 a. C h a r a c t e r i s t i c s of c e l l l i n e s 101 b. Maintenance o f c u l t u r e d c e l l s 101 2. Treatment with g l u c o c o r t i c o i d s 102 a. Preparation of g l u c o c o r t i c o i d stock s o l u t i o n s 102 b. Incubation of c e l l s with g l u c o c o r t i c o i d s 103 B. I s o l a t i o n and P u r i f i c a t i o n of DNA 103 1. Homogenization 103 2. D e p r o t e i n i z a t i o n 103 3. Chromatography on hydroxyapatite column 104 4. Concentration of the pooled DNA by Amicon f i l t r a t i o n 105 C. Burton's diphenylamine assay f o r DNA 105 1. Preparation of s o l u t i o n s 105 2. Procedure 106 D. Bio-Rad p r o t e i n assay 106 1. S e l e c t i o n of p r o t e i n standard 107 2. Procedure 107 v i . PAGE E. Thermal scanning a n a l y s i s , Tm and hyperchromicity 107 F. UV-A i r r a d i a t i o n of c u l t u r e d f i b r o b l a s t s 108 G. I s o l a t i o n and s o l u b i l i z a t i o n of n u c l e i 110 H. I s o l a t i o n of chromatin from i s o l a t e d n u c l e i 111 I . L i q u i d s c i n t i l l a t i o n counting 112 EXPERIMENTS 113 A. Test f o r p o s s i b l e c r o s s - l i n k i n g of DNA induced by gluco-c o r t i c o i d s 113 3 B. Retention of H-TA during the i s o l a t i o n of DNA 114 3 C. S p e c i f i c r e t e n t i o n of H-TA i n whole n u c l e i of c u l t u r e d mouse L-929 f i b r o b l a s t s 114 D. S p e c i f i c r e t e n t i o n of H-TA i n chromatin of c u l t u r e d mouse L-929 f i b r o b l a s t s 115 E. Subchromatin l o c a l i z a t i o n of TA 116 i RESULTS AND DISCUSSION 117 A. Morphology of g l u c o c o r t i c o i d - t r e a t e d and g l u c o c o r t i c o i d -f r e e mouse L-929 dermal f i b r o b l a s t s 117 B. E v a l u a t i o n of c r o s s - l i n k i n g of DNA with g l u c o c o r t i c o i d s 117 1. Quantity and q u a l i t y of i s o l a t e d DNA 117 2. Examination of c r o s s - l i n k i n g of DNA 120 3 C. Retention of H-TA during the i s o l a t i o n of DNA 133 D. S p e c i f i c r e t e n t i o n o f TA i n whole n u c l e i of mouse L-929 f i b r o b l a s t s 133 1. D e s c r i p t i o n of the preparation of n u c l e i 133 2. Time p r o f i l e of s p e c i f i c r e t e n t i o n i n whole n u c l e i 136 a. Gross uptake per pooled c e l l s of 5 p l a t e s 136 3 b. S p e c i f i c nuclear r e t e n t i o n of H-TA normalized f o r equal amount of DNA or nuclear p r o t e i n 139 v i i . PAGE 3. E f f e c t s of TA on t o t a l nuclear p r o t e i n and DNA content i n n u c l e i 145 a. S e l e c t i o n and r e l i a b i l i t y of p r o t e i n and DNA assays 145 b. V a r i a t i o n i n the nuclear p r o t e i n l e v e l of TA-tr e a t e d and TA-free mouse L-929 f i b r o b l a s t s as a f u n c t i o n of time 146 c. V a r i a t i o n i n the DNA content i n n u c l e i of TA-treated and TA-free mouse L-929 f i b r o b l a s t s as a fu n c t i o n of time 148 H. I n t r a c e l l u l a r d i s t r i b u t i o n of H-TA as a f u n c t i o n of time 155 E. S p e c i f i c r e t e n t i o n of H-TA i n chromatin 160 I. C h a r a c t e r i z a t i o n o f i s o l a t e d chromatin 160 2. Time p r o f i l e of s p e c i f i c r e t e n t i o n i n chromatin 160 V 3 3. I n t r a n u c l e a r d i s t r i b u t i o n of H-TA 161 3 F. Subchromatin l o c a l i z a t i o n of H-TA 165 SUMMARY 172 APPENDIX 175 I . M a t e r i a l s 175 I I . Apparatus 180 REFERENCES i «*> v i i i . LIST OF TABLES P A G E I . Diseases with which the a p p l i c a t i o n of t o p i c a l g l u c o c o r t i -coids i s accepted therapy 4 I I . R e l a t i v e c l i n i c a l potencies of t o p i c a l g l u c o c o r t i c o i d s 9 I I I . Binding parameters of n a t u r a l l y o c c u r r i n g and s y n t h e t i c g l u -c o c o r t i c o i d s to receptors 26 IV. DNA l e s i o n s produced i n mammalian c e l l s by va r i o u s agents 49 V. D i s s o c i a t i o n p a t t e r n of histone and nonhistone chromosomal p r o t e i n s by NaCl and urea from hydroxyapaptite 53 VI. Retention time of parent and d e r i v a t i z e d g l u c o c o r t i c o i d s 78 VII . E s timation of HC a f t e r e x t r a c t i o n from spiked medium samples 91 V I I I . E s t i m a t i o n of TA a f t e r e x t r a c t i o n from spiked medium samples 92 IX. Estimation of DSN a f t e r e x t r a c t i o n from spiked medium samples 94 X. Levels o f HC i n media as a f u n c t i o n of time a f t e r i n c u b a t i o n with c u l t u r e d human dermal f i b r o b l a s t s 96 XI. Levels o f TA i n media as a f u n c t i o n of time a f t e r i n c u b a t i o n w i t h c u l t u r e d mouse L-929 dermal f i b r o b l a s t s 97 X I I . Levels of DSN i n media as a f u n c t i o n of time a f t e r incuba-t i o n with c u l t u r e d mouse L-929 dermal f i b r o b l a s t s 98 X I I I . Conditions examined to i n v e s t i g a t e the formation of c r o s s -l i n k e d DNA by g l u c o c o r t i c o i d s 126 3 XIV. HAP chromatography of deproteinized DNA from H-TA t r e a t e d mouse L-929 f i b r o b l a s t s 1 3 4 XV. Q u a n t i t a t i v e removal of r a d i o a c t i v i t y by d e p r o t e i n i z a t i o n w i t h CHC^/BuOH mixture 1 3 5 3 XVI. Gross uptake of H-TA i n t o the n u c l e i of pooled c e l l s from 5 p l a t e s 1 3 7 i x . PAGE XVII. S p e c i f i c r e t e n t i o n of TA i n whole n u c l e i 142 XV I I I . T o t a l nuclear p r o t e i n l e v e l s i n TA-free and TA-treated c u l -tured mouse L-929 f i b r o b l a s t s at va r i o u s incubation i n t e r v a l s 149 XIX. DNA l e v e l s i n n u c l e i o f TA-free and TA-treated c u l t u r e d mouse L-929 f i b r o b l a s t s at various incubation i n t e r v a l s 153 3 XX. I n t r a c e l l u l a r d i s t r i b u t i o n Of H-TA between cytoplasm and nu-cleus i n the absence or the presence of 500-fold unlabeled TA 157 XXI. S p e c i f i c r e t e n t i o n o f TA i n chromatin 162 3 XXII. I n t r a n u c l e a r d i s t r i b u t i o n of H-TA between nucleoplasm and chromatin i n the absence or the presence of 500-fold un-label e d TA 166 X. LIST OF FIGURES PAGE 1. Some s t r u c t u r a l m o d i f i c a t i o n s of s y n t h e t i c g l u c o c o r t i c o i d s 7 2. Arachidonic a c i d - p r o s t a g l a n d i n transformation cascade 14 3. The molecular mechanisms of s t e r o i d hormone a c t i o n (receptor model) 16 4. Proposed c y c l e of events c o n t r o l l i n g the binding s t a t e and c e l l u l a r l o c a t i o n of g l u c o c o r t i c o i d receptor 25 5. The k i n e t i c s and r e v e r s i b l e e f f e c t s of nuclear binding of dexa-methasone-receptor complexes i n i n t a c t HTC c e l l s 29 6. Independence of nuclear binding of concentration of receptor-dexamethasone complex i n the cytoplasm of i n t a c t HTC c e l l s a t 37°C 31 7. I d e a l i z e d s e c t i o n of a nucleus, showing a l l the main components 33 8. G l u c o c o r t i c o i d receptors bind to DNA but not to RNA 35 9. Drug-induced macromolecular damage of nuclear DNA 38 10. DNA undergoing wave-like d i s t o r t i o n i n i t s s t r u c t u r e 39 11. Schematic i l l u s t r a t i o n of dynamic concepts of DNA s t r u c t u r e i n v o l v e d i n DNA breathing and i n drug i n t e r c a l a t i o n 40 4 12. Conformations of A -3-one A r i n g 42 13- Hydrogen bonding i n the DOC-adenine complex viewed perpendicu-l a r to the adenine plane 43 14. Stacking of adenine and the A -3-one region of DOC 44 3 15. Release of receptor- H-dexamethasone complexes (RD) from HTC c e l l n u c l e i by NaCl 47 16. Basic a c t i o n of DNA with i n c r e a s i n g temperature 55 17. Hyperchromia e f f e c t of DNA upon heating 57 x i . PAGE 18. Models f o r two types of p o s s i b l e i n t r a n u c l e a r c o n t r o l over s t e r o i d - r e c e p t o r complex i n t e r a c t i o n s w i t h DNA 60 19. Chemical s t r u c t u r e s of t e s t g l u c o c o r t i c o i d s 70 20. T y p i c a l chromatograms of g l u c o c o r t i c o i d s and e x t r a c t s (a) Authentic g l u c o c o r t i c o i d s (b) E x t r a c t of blank medium with 10% serum (c) G l u c o c o r t i c o i d s e x t r a c t e d from serum-containing medium 77 21. Reaction k i n e t i c s of HC, T and TA w i t h s i l y l a t i n g agent at 100°C 79 22. (a) Electron-impact mass spectrum of (M0) 2-(TMS)^-HC 80 (b) Electron-impact mass spectrum of (M0) 2~(TMS) 1 J-T 81 (c) Electron-impact mass spectrum of (MO) 1-(TMS) 2~TA 82 (d) Electron-impact mass spectrum of (MO) 1-(TMS) 2-DSN 83 (e) Electron-impact mass spectrum of (M0) 2~PRG 84 23. (a) Electron-impact mass spectrum of (MO)^-(TMS)^-DSN 88 (e) Electron-impact mass spectrum of (MO) 1 -(TMS^-TA 89 2k. T y p i c a l d e n a t u r a t i o n - r e n a t u r a t i o n p r o f i l e of DNA i s o l a t e d from c u l t u r e d f i b r o b l a s t s 109 25. Morphology of g l u c o c o r t i c o i d - t r e a t e d and g l u c o c o r t i c o i d - f r e e mouse L-929 dermal f i b r o b l a s t s 118 26. Representative chromatogram of d e p r o t e i n i z e d c e l l homogenate a p p l i e d to HAP column 122 27. Representative chromatogram of nati v e and denatured DNA a p p l i e d to HAP column 123 28. HAP chromatography of DNA i s o l a t e d from (8-M0P)-treated and UV-A i r r a d i a t e d mouse L-929 f i b r o b l a s t s 125 29. HAP chromatography of DNA i s o l a t e d from HC-, TA- or TA-UV treated mouse L-929 f i b r o b l a s t s 127 XIX. PAGE 30 . Thermal scanning a n a l y s i s of DNA i s o l a t e d from (8-MOP)-treated and UV-A i r r a d i a t e d mouse L-929 f i b r o b l a s t s 129 3 1 . Thermal scanning a n a l y s i s of DNA i s o l a t e d from UV-A i r r a d i a t e d mouse L-929 f i b r o b l a s t s 130 32 . Thermal scanning a n a l y s i s of DNA i s o l a t e d from HC-, TA- or (TA-UV)-treated mouse L-929 f i b r o b l a s t s 131 33. Thermal scanning a n a l y s i s of DNA i s o l a t e d from untreated mouse L-929 f i b r o b l a s t s 132 •3 34. Gross uptake of H-TA i n t o whole n u c l e i of pooled c e l l s of 5 p l a t e s 138 35. S p e c i f i c r e t e n t i o n of TA i n whole n u c l e i , normalized on DNA b a s i s 140 36. S p e c i f i c r e t e n t i o n of TA i n whole n u c l e i , normalized on nuclear p r o t e i n b a s i s 141 3 7 . T o t a l nuclear p r o t e i n l e v e l s i n TA-free and TA-treated c u l t u r e s as a f u n c t i o n of time 147 38 . Suppression of t o t a l nuclear p r o t e i n s induced by TA at l e v e l s of 1 0 " 8 M and 5.01 x 1 0 - 6 M 150 39. DNA l e v e l s i n n u c l e i of TA-free and TA-treated mouse L-929 f i b r o b l a s t s as a f u n c t i o n of time 151 40. Suppression of DNA l e v e l i n n u c l e i induced by TA at concentra-p c t i o n s of 10" M and 5.01 x 10" M 154 •3 41. I n t r a c e l l u l a r d i s t r i b u t i o n of H-TA between cytoplasm and nucleus i n the absence or presence of 500-fold unlabeled TA 156 •3 42. Percent d i s t r i b u t i o n of t o t a l c e l l u l a r H-TA between c y t o -plasm and nucleus 159 4 3 . S p e c i f i c r e t e n t i o n of TA i n chromatin, normalized on DNA b a s i s 163 x i i i . PAGE 44. S p e c i f i c r e t e n t i o n o f TA i n chromatin, normalized on chromo-somal p r o t e i n basis 164 45. I n t r a n u c l e a r d i s t r i b u t i o n of H-TA between nucleoplasm and chromatin i n the absence or the presence of 500-fold unlabeled TA 167 46. Percent d i s t r i b u t i o n of t o t a l nuclear H-TA between nucleo-plasm and chromatin 168 47. D i s s o c i a t i o n pattern of t o t a l chromosomal p r o t e i n s from immobi-l i z e d chromatin on HAP column as e l u t e d w i t h NaCl followed by NaCl and urea 169 48. D i s s o c i a t i o n of H-TA from chromatin as eluted w i t h NaCl f o l -lowed by NaCl and urea 171 x i v . LIST OF ABBREVIATIONS cAMP e y e l i e adenosine 3',5'-monophosphate C.V. c o e f f i c i e n t of v a r i a t i o n DMEM Dulbecco's modified Eagle medium DNA deoxyribonucleic a c i d DSC d i l u t e s a l i n e c i t r a t e DSN desonide EDTA eth y l e n e d i a m i n e t e t r a a c e t i c a c i d FCS f e t a l c a l f serum FID f l a m e - i o n i z a t i o n d e t e c t i o n GLC g a s - l i q u i d chromatography GC-MS gas chromatography - mass spectrometry HAP hydroxyapatite chromatography HC hydrocortisone HTC hepatoma t i s s u e c u l t u r e i . d. i n t e r n a l diameter LSC l i q u i d s c i n t i l l a t i o n counting MO methoxime d e r i v a t i v e MOP 8-methoxypsoralen PBS phosphate buffered s a l i n e PRG progesterone S.D. standard d e v i a t i o n S.D.S. sodium dodecyl s u l f a t e S.E. standard e r r o r SSC standard s a l i n e c i t r a t e T triamcinolone XV. TA triamcinolone acetonide TMS t r i m e t h y l s i l y l d e r i v a t i v e UV-A u l t r a v i o l e t r a d i a t i o n with long wavelength, 320-400 nm xv i . ACKNOWLEDGEMENTS I am g r a t e f u l to Dr. J.O. R u n i k i s f o r h i s encouragement and guidance throughout t h i s p r o j e c t , as w e l l as h i s f r i e n d s h i p . I wish to thank my research committee members, Drs. F.S. Abbott, J.E. Axelson, J.B. Hudson, J.H. M c N e i l l , R.H. Pearce and Wm.D. Stewart, f o r t h e i r help and v a l u a b l e advice during t h i s study. I e s p e c i a l l y a p p r eciate the time and e f f o r t given by Dr. R.H. Pearce f o r reading the f i n a l t h e s i s . I owe g r a t i t u d e to Mr. A. Bandenieks f o r a s s i s t a n c e w i t h c e l l c u l t u r e s , and to Mr. R. Burton f o r a s s i s t a n c e w i t h the use of the gas chromatograph-mass spectrometer. I am t h a n k f u l f o r f i n a n c i a l support from the U n i v e r s i t y of B r i t i s h Columbia, the Medical Research C o u n c i l of Canada, the B.C. Health Care Research Foundation and the Canadian Dermatology Foundation. DEDICATION To Mom, Dad and Shiu-Leung f o r t h e i r confidence i n and, above a l l , f o r t h e i r love and encouragement INTRODUCTION Advancing knowledge i n pharmaceutics i n d i c a t e s a p r o g r e s s i v e l y greater need f o r the a p p l i c a t i o n of biochemical techniques to the s o l u t i o n of research problems i n pharmaceutics. A case i n point i s the problem of t o p i c a l g l u c o c o r t i c o i d s . Formulation of a t o p i c a l g l u c o c o r t i c o i d cream, f o r i n s t a n c e , i n -volves not only a s e l e c t i o n of the g l u c o c o r t i c o i d , the v e h i c l e i n g r e d i e n t s and t h e i r c o n c e n t r a t i o n s , but a l s o an e v a l u a t i o n of the potency of the f i -nished pr e p a r a t i o n and i t s p o t e n t i a l to produce adverse e f f e c t s . At present there are only e m p i r i c a l potency bioassays a v a i l a b l e which are based,on such gross p h y s i o l o g i c endpoints as s k i n b l a n c h i n g , erythema, edema or t h i n n -ing of the s k i n ( H a l e b l i a n , 1976; Stoughton, 1977; Maibach and Stoughton, 1975). These bioassays r e l a t e at best i n d i r e c t l y to the b a s i c mechanism of g l u c o c o r t i c o i d a c t i o n . To e s t a b l i s h a more r a t i o n a l r e l a t i o n s h i p between the p h y s i c a l chemical p r o p e r t i e s of the dosage form of a t o p i c a l g l u c o c o r t i c o i d and i t s c l i n i c a l p r o p e r t i e s , i t i s necessary to know the mechanism on which the g l u c o c o r t i c o i d a c t i o n depends. However, at present, even the s i t e w i t h i n the nucleus where the g l u c o c o r t i c o i d acts i s u n c e r t a i n . G l u c o c o r t i c o i d s act on DNA metabolism, e s p e c i a l l y t r a n s c r i p t i o n , to exert t h e i r p h y s i o l o g i c a l e f f e c t s (Baxter and Rousseau, 1979). However, i t i s not c l e a r whether the g l u c o c o r t i c o i d acts by d i r e c t b i n d i n g to DNA or by p e r t u r b a t i o n of some other nuclear components (Baxter and Rousseau, 1979; Higgins et a l . , 1979).It seemed a t t r a c t i v e t h e r e f o r e to i n v e s t i g a t e whether a d i r e c t e f f e c t on DNA could be detected by studying i t s p o s s i b l e c r o s s - l i n k - * ing by g l u c o c o r t i c o i d (Duax, 1976). The p r i n c i p a l o b j e c t i v e of t h i s research has been the search f o r a d i r e c t e f f e c t of g l u c o c o r t i c o i d s on DNA i n i n t a c t m e t a b o l i z i n g c e l l s , and to gather p r e l i m i n a r y i n f o r m a t i o n on a l t e r n a t i v e nuclear components which have the " s p e c i f i c " (high a f f i n i t y ) b i n d i n g a b i l i t y presumably nece-ssary i n i n i t i a l r e a c t i o n s i t e s f o r the g l u c o c o r t i c o i d s . A l s o , part of the research p r o j e c t has been the development of a q u a n t i t a t i v e g a s - l i q u i d chromatographic assay f o r monitoring the chemical s t a b i l i t y of g l u c o c o r t i c o i d s during prolonged exposure to c u l t u r e s of a c t i v e -l y m e t a b o l i z i n g c e l l s . 3. LITERATURE SURVEY I. C l i n i c a l uses and adverse e f f e c t s of t o p i c a l g l u c o c o r t i c o i d s T o p i c a l g l u c o c o r t i c o i d s are by f a r the most commonly pr e s c r i b e d t h e r a p e u t i c agents i n dermatology today. More than 50% of dermatological p r e s c r i p t i o n s are w r i t t e n for g l u c o c o r t i c o i d s s i n g l y or i n combination with other i n g r e d i e n t s (Stoughton, 1977) . This does not mean that t h e i r use i s l i m i t e d to dermatologists. Rather, v i r t u a l l y a l l s p e c i a l t i e s as w e l l as general p r a c t i c e , make use of these t o p i c a l agents i n one form or another. Now that 0.5% hydrocortisone t o p i c a l preparations have been released f o r dispensing under pharmacists' s u p e r v i s i o n without p r e s c r i p -t i o n , the use and p o s s i b l e misuse of t o p i c a l g l u c o c o r t i c o i d s are of cur-rent i n t e r e s t to v i r t u a l l y a l l health p r o f e s s i o n s . T o p i c a l g l u c o c o r t i c o i d s are remarkably e f f e c t i v e f o r a wide v a r i e t y of s k i n d i s o r d e r s . At l e a s t 56 diseases l i s t e d i n Table I have been reported to be responsive to the treatment with t o p i c a l g l u c o c o r t i -c o i d s . Their c h i e f e f f e c t s are a profound l o c a l anti-inflammatory a c t i o n , i n h i b i t i o n of the growth rat e of epidermal and some other p e r i -pheral c e l l s , immunosuppression and an a n t i p r u r i t i c a c t i o n . T o p i c a l l y a p p l i e d g l u c o c o r t i c o i d s have become more and more potent with the con-tinued development of new analogs. Refinements by s u b s t i t u t i o n of f l u o r i n e , c h l o r i n e , methyl- and other groups on the s t e r o i d nucleus or by a d d i t i o n of various side chains have made more potent compounds e i t h e r by i n c r e a s i n g t h e i r i n t r i n s i c a c t i v i t y at the s i t e of a c t i o n , by promoting t h e i r speed of p e n e t r a t i o n through the s k i n b a r r i e r , or by delaying t h e i r metabolism to i n a c t i v e d e r i v a t i v e s . In a d d i t i o n , more s o p h i s t i c a t e d v e h i c l e s have increased absorption of g l u c o c o r t i c o i d s by the s k i n . However, the prolonged use of t o p i c a l g l u c o c o r t i c o i d s causes a number of adverse e f f e c t s both s y s t e m i c a l l y and l o c a l l y . S y s t e m i c a l l y , 4. Table I Diseases with which the a p p l i c a t i o n of t o p i c a l g l u c o c o r t i c o i d s i s accepted therapy (Maddin et a l . , 1975) Inflamroat ions Acne v u l g a r i s Acute r a d i o d e r m a t i t i s A n g i i t i s Atopic d e r m a t i t i s Diaper d e r m a t i t i s Eczema Lichen planus Lichen simplex chronicus O t i t i s externa P a n n i c u l i t i s Plasma c e l l b a l a n i t i s P o l y c h o n d r i t i s Primary i r r i t a n t contact d e r m a t i t i s Seborrheic d e r m a t i t i s V a s c u l i t i s Immunologic Disorders A l l e r g i c contact d e r m a t i t i s A l l e r g i c v a s c u l i t i s Berlocque d e r m a t i t i s Lupus erythematosus Pemphigus, pemphigoid Pyoderma gangrenosum U r t i c a r i a V i t i l i g o Wegener's granulomatosis Disorders of  C e l l u l a r Function F o l l i c u l a r keratoses K e l o i d Lichen myxedematosus Mastocytosis Myxoid cy s t I n f e c t i o n s Herpes simplex, recurrent Impetigo Leprosy R i c k e t t s i a l diseases Zoster Malignancies Lymphoma Mycosis fungoides Diseases of C e l l u l a r P r o l i f e r a t i o n E x f o l i a t i v e d e r m a t i t i s Hypertrophic scar and k e l o i d . H i s t i o c y t o s i s X Keratoacanthoma P s o r i a s i s I d i o p a t h i c Disorders A l o p e c i a areata Aphthous s t o m a t i t i s P o l y a r t e r i t i s S a r c o i d o s i s Scleroderma Diseases of Ne u t r o p h i l Accumulation Subcorneal p u s t u l a r Sweet's syndrome Development defects Epidermolysis bullosum I c h t h y o s i s Metabolic Disorders Gaucher's disease N e c r o b i o s i s l i p o i d i c a Myxedema Fu n c t i o n a l Disorders D y s h i d r o s i s Livedo r e t i c u l a r i s P r u r i t u s suppression of adrenal c o r t i c a l f u n c t i o n occurs from absorption through the s k i n to a f f e c t i n t e r n a l organs, e s p e c i a l l y when the s t e r o i d i s used under o c c l u s i o n and i n c h i l d r e n . Most c u r r e n t l y a v a i l a b l e t o p i c a l c o r t i -c o s t e r o i d s e a s i l y cause adrenal suppression i n the m a j o r i t y of p a t i e n t s . Some, such as c l o b e t a s o l propionate cause profound suppression, even without o c c l u s i o n . With acute usage, the e f f e c t i s t r a n s i e n t and rev e r -s i b l e but Carruthers et a l . (1975) present evidence r e l a t e d to i n s u l i n s t r e s s that i n d i c a t e s decreased reserve occurs i n p a t i e n t s on prolonged therapy, even though depressed C o r t i s o l l e v e l s r e t u r n to normal values. There i s a good deal of p a t i e n t v a r i a t i o n , unmeasurable by any c u r r e n t l y known p r o p e r t i e s of the c o r t i c o s t e r o i d s . Some i n d i v i d u a l s demonstrate e f f e c t s considerably at variance with other s u b j e c t s . A l s o , variance occurs between compounds - some s t e r o i d s show potency by granu-loma i n h i b i t i o n and other anti-inflammation t e s t s , but are impotent by t o p i c a l a p p l i c a t i o n , f o r reasons inapparent to us at present (Stoughton, 1975). P e n e t r a t i o n s t u d i e s , as Stoughton (1975) p o i n t s out, are s i m i l a r between betamethasone a l c o h o l and betamethasone-17-valerate, but th e r a -p e u t i c e f f e c t i s r a d i c a l l y d i f f e r e n t . The cutaneous adverse e f f e c t s are common and c o n s t i t u t e a more vexing problem than systemic e f f e c t s . Untoward cutaneous side e f f e c t s i n c l u d e s t r i a e , erythema, t e l a n g i e c t a s i a , purpura, r o s a c e a - l i k e dermati-t i s , rebound d e r m a t i t i s , epidermal atrophy and dermal atrophy. S k i n (epidermal and dermal) atrophy i s perhaps the most undesirable s i d e e f f e c t of g l u c o c o r t i c o i d treatment. The major e f f e c t m i c r o s c o p i c a l l y occurs i n the r e t i c u l a r dermis with degeneration of e l a s t i c f i b e r s , c o l l a g e n f i b r i l bundle d i s o r g a n i z a t i o n , aggregation of m i c r o f i b r i l s and decreased numbers of f i b r o b l a s t s (Jablonska et a l . , 1979). In severe atrophy, t i s s u e bulk w i l l improve only about 50$ a f t e r c e s s a t i o n o f therapy (Voorhees, 1977b). Other side e f f e c t s from t o p i c a l use are s u p e r i n f e c t i o n , a d d i c -t i o n (Kligman and Frosh, 1979), and a l l e r g i c contact d e r m a t i t i s (Coskey, 1978) to the s t e r o i d compounds. Each of these poses i t s own problems f o r s o l u t i o n , and must be considered c l i n i c a l l y . P h y s i c i a n s must s t i l l use g l u c o c o r t i c o i d s on a t r i a l - a n d - e r r o r b a s i s ( E p s t e i n , 1979). Therefore, i t would be h i g h l y d e s i r a b l e to e s t a -b l i s h an o b j e c t i v e c r i t e r i o n for s e l e c t i n g g l u c o c o r t i c o i d s on a r a t i o n a l -l y determined, t h e o r e t i c a l l y v a l i d b e n e f i t / r i s k r a t i o b a s i s . Only one assay i s a v a i l a b l e f o r the e v a l u a t i o n of t o p i c a l g l u c o c o r t i c o i d potency i n man, namely the v a s o c o n s t r i c t i o n assay, o r i g i n a l l y developed by McKen-z i e and Stoughton(1962). This assay p r e d i c t s reasonably w e l l the r e l a -t i v e c l i n i c a l potencies of the g l u c o c o r t i c o i d s , but' the r e l a t i o n s h i p of i t s endpoint - s k i n blanching - to c l i n i c a l potencies cannot be explained b i o c h e m i c a l l y or p h y s i o l o g i c a l l y . I t has been demonstrated however th a t v a s o c o n s t r i c t i o n can be c o r r e l a t e d with concentration of s t e r o i d , and that a c r i t i c a l l e v e l must be present to produce i t (Wallace et a l . , 1979) . There are many v a s o c o n s t r i c t i v e drugs which have no a n t i - i n f l a m -matory a c t i o n . The comparative assay of g l u c o c o r t i c o i d i n h i b i t o r y e f f e c t on mouse L-929 f i b r o b l a s t p r o l i f e r a t i o n has been used s i m i l a r l y to rank the g l u c o c o r t i c o i d r e l a t i v e potency ( B e r l i n e r , 1967a), but there are l i m i t a t i o n s i n e x t r a p o l a t i n g the data to human study. Some compounds have more or l e s s c l i n i c a l anti-inflammatory a c t i v i t y than p r e d i c t e d by these assays, e.g. betamethasone v a l e r a t e . I I . S t r u c t u r e - a c t i v i t y - r e l a t i o n s h i p of g l u c o c o r t i c o i d s G l u c o c o r t i c o i d s have a 21-carbon skeleton as shown i n Figure 1. Group M o d i f i c a t i o n s and R£ Double bond R„ or R. F or C l or CH_ 3 4 3 R 5 OH or CH 3 R, or R-, E s t e r i f i c a t i o n R r and R, Acetonide formation Some s t r u c t u r a l m o d i f i c a t i o n s of s y n t h e t i c g l u c o c o r t i c o i d s : I n d i c a t e s f u n c t i o n a l groups e s s e n t i a l f o r g l u c o c o r t i c o i d a c t i v i t y Of major importance i n s t e r o i d chemistry i s the c r u c i a l r e l a t i o n s h i p between s t r u c t u r e and f u n c t i o n a l a c t i v i t y . C e r t a i n f e a t u r e s are abso-l u t e l y e s s e n t i a l f o r the p r e s e r v a t i o n of b i o l o g i c a l a c t i o n of a l l g l u c o -c o r t i c o i d hormones. The e s s e n t i a l f u n c t i o n a l groups are A -3-ketone, 11B-0H and 17B-ketol side chain (Foye, 197*0. A number of s u b s t i t u t i o n s at other s i t e s have produced s y n t h e t i c analogues with enhanced glucocor-t i c o i d a c t i v i t i e s . These m o d i f i c a t i o n s i c l u d e C^  = double bond, f l u o r i n a t i o n , c h l o r i n a t i o n or methylation at Cg or Cg, h y d r o x y l a t i o n or methylation at C^, e s t e r i f i c a t i o n of hydroxyl group at C ^ or C 2 1, and acetonide formation with hydroxyl groups o f C ^ and C ^ (Stewart et a l . , 1973) . The r e l a t i v e potencies of these continuously developed s y n t h e t i c g l u c o c o r t i c o i d s have been reported p r i m a r i l y based on v a s o c o n s t r i c t i o n bioassay i n man and, to a l e s s e r degree, on c l i n i c a l s t u d i e s . However, there are too many s i g n i f i c a n t gaps i n experimental and c l i n i c a l data to support any f i r m e r ranking. Moreover, the rankings reviewed i n d i f f e r e n t a r t i c l e s demonstrate considerable discrepancy (Table I I ) . For example, those i n the cases of desonide, t r i a m c i n o l o n e acetonide, betamethasone-s - v a l e r a t e and f l u o c i n o l o n e acetonide. T h i s , again, s t r e s s e s the impor-tance of searching f o r more r e l i a b l e , o b j e c t i v e parameters from biochemi-c a l events which c o n s t i t u t e the receptor model of g l u c o c o r t i c o i d a c t i o n f o r e s t a b l i s h i n g a v a l i d and r e p r o d u c i b l e ranking of a l l g l u c o c o r t i c o i d s u s e f u l i n t o p i c a l therapy. Ponec et a l . (1980) report t h e i r attempts to r e l a t e c l i n i c a l g l u c o c o r t i c o i d e f f i c a c y with e f f e c t s on human f i b r o b l a s t growth i n h i b i -t i o n i n c u l t u r e . They show s i g n i f i c a n t d i f f e r e n c e s i n potency i n some compounds, that are confirmed c l i n i c a l l y , but p a r a d o x i c a l responses i n Table I I R e l a t i v e c l i n i c a l potencies of t o p i c a l g l u c o c o r t i c o i d s G l u c o c o r t i c o i d Stewart et a l . 1978 (a) R e l a t i v e Ranking Maibach and Stoughton, 1975 (b) Haynes and Lamer, 1975 (c) Hydrocortisone Prednisolone F l u d r o c o r t i s o n e Methylprednisolone * Triamcinolone acetonide Beclomethasone dipropionate * Betamethasone-17-v a l e r a t e Betamethasone-17-benzoate * Desonide Betamethasone Dexamethasone Betamethasone dipropionate * Fluocinolone acetonide Fluocinolone acetonide acetate IV IV I I I I I I I I I (0.1%) I (0.5%) I I I I I I VI VI V (cream, foam, l o t i o n ) I (ointment) V (cream) I (ointment) I V V ( s o l u t i o n ) I (cream, ointment) 1 A 10 5 25 25 (a) I:strongest IV:weakest (b) I : strongest VI: weakest (c) r e l a t i v e potencies i n numerical values (Hydrocortisone: weakest) * demonstrates discrepancy other c l i n i c a l l y a c t i v e agents. They caution against e x t r a p o l a t i n g these c u l t u r e r e s u l t s to the c l i n i c a l s i t u a t i o n . I l l . Biochemical events accompanying the c l i n i c a l and adverse e f f e c t s i n  the s k i n G l u c o c o r t i c o i d s have the c a p a c i t y to prevent or suppress the development of the l o c a l heat, redness, s w e l l i n g and tenderness by which inflammation i s recognized at the gross l e v e l of observation (Haynes and Larner, 1975). At the c e l l u l a r or metabolic l e v e l s , g l u c o c o r t i c o i d s have suppressive e f f e c t s on the e n t i r e inflammatory process i n c l u d i n g c a p i l -l a r y p e r m e a b i l i t y , antibody formation and the r e p a i r process (Spain, 1975). This i s i n contrast to n o n - s t e r o i d a l anti-inflammatory agents, such as a s p i r i n and s a l i c y l a t e s , phenylbutazone, indomethacin and mefena-mic a c i d . The n o n - s t e r o i d a l anti-inflammatory agents i n h i b i t the exuda-t i v e rather than the p r o l i f e r a t i v e phases of inflammation (Foye, 1974), t h e i r a c t i o n s being l i m i t e d to the i n h i b i t i o n of p r o s t a g l a n d i n synthe-s i s . Therefore, they suppress histamine- or bradykinin-induced inflamma-t i o n but not that dependent on n e u t r o p h i l s , the l a t t e r not being i n -fluenced by p r o s t a g l a n d i n s (Wintroub, 1980). In inflamed s k i n , g l u c o c o r t i c o i d s a f f e c t both the epidermis and the dermis. In the inflamed epidermis, the stratum corneum i s thickened and l o o s e l y organized. C e l l maturation or d i f f e r e n t i a t i o n i s incomplete, and c e l l p r o l i f e r a t i o n i s increased. In the dermis, blood v e s s e l s are markedly d i l a t e d during inflammation, and inflammatory polymorphonuclear leucocytes and mononuclear c e l l s are found. When a g l u c o c o r t i c o i d pre-p a r a t i o n i s applied t o p i c a l l y , the g l u c o c o r t i c o i d a f f e c t s c e l l s i n each l a y e r of the s k i n as i t penetrates. As a r e s u l t , the rate of c e l l matur-a t i o n i s normalized, and complete c e l l u l a r d i f f e r e n t i a t i o n occurs as they migrate to the stratum corneum. D i l a t e d blood v e s s e l s are returned to t h e i r normal diameter. F i b r o b l a s t p r o l i f e r a t i o n i s suppressed and the amount of dermal co l l a g e n i s reduced. Inflammatory c e l l s then d i s -appear. The stratum corneum becomes more compact and b e t t e r organized (Voorhees, 1977b). The cutaneous adverse e f f e c t s induced by prolonged t o p i c a l ap-p l i c a t i o n of g l u c o c o r t i c o i d s are thought to be mediated, i n l a r g e p a r t , by the d i r e c t a c t i o n of the s t e r o i d on the f i b r o b l a s t s . One of the ex-planations proposed f o r these connective t i s s u e m a n i f e s t a t i o n s , i n c l u d i n g t h i n n i n g of the s k i n , s t r i a e and poor wound h e a l i n g , i s a reduction i n the collagen content of the involved t i s s u e (David et a l . , 1970; Sim et a l . , 1976; Jablonska et a l . , 1979). The l o s s of c o l l a g e n content may be due to i n h i b i t i o n of or a l t e r a t i o n i n c o l l a g e n s y n t h e s i s , or the exces-si v e degradation of p r e v i o u s l y deposited c o l l a g e n as shown by the r e s u l t s of g l u c o c o r t i c o i d treatment of k e l o i d i n man ( G r i f f i t h , 1966; Ketchum et a l . , 1966; Wilson, 1965). E l a s t i c t i s s u e and glvcosaminoglycans (Jab l o n -ska et a l . , 1979) are a l s o a l t e r e d i n t h i s process. Studies i n animals, on the other hand, have i n d i c a t e d an i n h i b i t i o n of c o l l a g e n s y n t h e s i s , rather than accelerated degradation, to be r e s p o n s i b l e f o r these e f f e c t s (Smith, 1967; K i v i r i k k o et a l . , 1965). The i n h i b i t i o n of c o l l a g e n s y n t h e s i s has been proposed due to e i t h e r (a) a generalized a n t i a n a b o l i c e f f e c t of g l u c o c o r t i c o i d s on f i b r o -b l a s t s (Thompson and Lippman, 1974), p a r t i c u l a r l y apparent f o r c o l l a g e n because the p r i n c i p a l s y n t h e t i c a c t i v i t y of the c e l l i s devoted to t h i s p r o t e i n which normally accumulates e x t r a c e l l u l a r l y , or (b) an e f f e c t on the enzyme, p r o l y l hydroxylase, which acts as a key enzyme i n the biosyn-t h e t i c sequence and i s s a i d to be s p e c i f i c a l l y i n h i b i t e d by g l u c o c o r t i -coids (Cutroneo and Counts, 1975; Cutroneo et a l . , 1971). Ponec et a l . take issue with the l a t t e r . They d i d demonstrate reduced c o l l a g e n formation (Ponec et a l . , 1979). IV. Mechanism o f g l u c o c o r t i c o i d a c t i o n s - an overview The mechanisms of anti-inflammatory a c t i o n and cutaneous adverse e f f e c t s are s t i l l not e n t i r e l y understood, although many attempts have been made. A number of theo r i e s have been proposed to e x p l a i n g l u c o c o r -t i c o i d s a c t i o n s since 1949 when the f i r s t g l u c o c o r t i c o i d ( c o r t i s o n e ) was t r i e d f o r the treatment of rheumatoid a r t h r i t i s (Hensch et a l . , 1949) and 1952 when g l u c o c o r t i c o i d was f i r s t t o p i c a l l y a p p l i e d (Sulzberger and Witten, 1952). These mechanisms have included (1) the theory of l y s o s o -mal membrane s t a b i l i z a t i o n , (2) models i n v o l v i n g reduction of arac h i d o n i c a c i d and i n h i b i t i o n of pr o s t a g l a n d i n s y n t h e s i s , (3) the hypothesis of p a r a l l e l a c t i o n of cAMP and g l u c o c o r t i c o i d s , and (4) the receptor model theory. The model of lysosomal membrane s t a b i l i z a t i o n suggested that a s i g n i f i c a n t p o r t i o n of the therapeutic a c t i o n of g l u c o c o r t i c o i d s r e s u l t s from a d i r e c t e f f e c t of g l u c o c o r t i c o i d s on membranes. According to t h i s theory, g l u c o c o r t i c o i d s are incorporated i n t o biomembranes s t a b i l i z i n g them by a l t e r i n g the movement or shape of membrane phospholipids. This s t a b i l i z a t i o n has been considered r e s p o n s i b l e f o r the a b i l i t y of glu c o -c o r t i c o i d s to i n h i b i t the release of lysosomal enzymes and to modify the a c t i v i t y o f other membrane-associated enzymes ( E p s t e i n , 1977; Weissman and G o l d s t e i n , 1976). Vertebrate c e l l s that maintain c e l l - c e l l r e l a t i o n -s h i p s , and i n f l u e n c e t h e i r environment may do so by r e l e a s i n g , i n s o l u b l e or p a r t i c u l a t e form, s e l e c t e d c e l l surface molecules. This process, known as shedding, i s to be d i s t i n g u i s h e d from s e c r e t i o n . Shedding i s an important aspect of normal p r o t e i n turnover i n the c e l l membrane, and p r o t e i n s , l i p o p r o t e i n s , g l y c o p r o t e i n s , g l y c o l i p i d s , glycosaminoglycans or c e l l surface receptors may be shed, e i t h e r alone or i n combinations. This process occurs i n growing c e l l s and i n c e l l s a c t i v a t e d by v a r i o u s s t i m u l a n t s . Shedding may be a f f e c t e d i n malignant c e l l s by c o r t i c o s -t e r o i d s , and i t i s h i g h l y l i k e l y that these compounds a f f e c t shedding of normal c e l l s (Black, 1980; Schimke, 1975). This a f f e c t s both c e l l beha-v i o u r and i t s environment. The second hypothesis was postulated based on the observation that t o p i c a l g l u c o c o r t i c o i d s i n p s o r i a s i s apparently blocked the a c t i o n of the enzyme phospholipase A^, which releases free arachidonic a c i d from c e l l membranes (Hammarstrom et a l . , 1977), and thereby i n h i b i t e d the formation of prostaglandins, as w e l l as the p r o s t a g l a n d i n - r e l a t e d sub-stances, the thromboxanes and 12 - L-hydroxy-5,8,10,14-eicosatetraenoic a c i d (HETE). The sequence of events involved i n t h i s "arachidonic a c i d -p r o s t a g l a n d i n transformation cascade" i s shown i n Figure 2. The substan-ces synthesized i n t h i s cascade are among the most powerful b i o a c t i v e compounds known and are thought to mediate inflammation i n c l u d i n g p o l y -morphonuclear leucocyte chemotaxis. The concentration of free arachido-n i c a cid i t s e l f and HETE were markedly increased i n l e s i o n s of p s o r i a s i s (Hammarstrom et a l . , 1975). I t was assumed that the reduction of the t i s s u e content of these substances could be a s i g n i f i c a n t aspect of the a c t i o n of t o p i c a l g l u c o c o r t i c o i d s i n p s o r i a s i s . The t h i r d hypothesis, now g e n e r a l l y disregarded, s t a t e s t h a t , as the second messenger of many hormones, cAMP a l s o mediated g l u c o c o r t i c o i d a c t i o n . I t i s s t i l l not s e t t l e d what r o l e cAMP plays i n g l u c o c o r t i c o i d c o n t r o l of c e l l metabolism, but i t seems u n l i k e l y that cAMP mediation i s necessary f o r g l u c o c o r t i c o i d s to act (Voorhees et a l . , 1975; Thompson and Lippman, 1974). Instead, i t now appears that metabolic s i g n a l s induced 14. Membrane phospholipid Phopholipase A2 (Blocked by glucocorticoids Free arachidonic acid lipoxygenase (HPETE) 1 -cycloxygenase (PGG 2 ) HETE (PGI2) (TXA2) PGE2 PGF2(x 1 T X B 2 * the compounds within brackets (PGG , TXA PGI HPETE) are unstable intermediates which have not yet been d i r e c t l y de-monstrated i n epidermis. HPETE HETE PGG P G H TXiC TXB^ PGE: PGF 2a 12L-hydroperoxy-5,8,10,14-eicosatetraenoic a c i d 12L-hydroxy-5,8,10,14-eicosatetraenoic acid prostaglandin G^, an endoperoxide intermediate an unstable derivative of arachidonic acid thromboxane A2 thromboxane prostaglandin E 0 prostaglandin F 2a Figure 2 : Arachidonic acid - prostaglandin transformation cascade (Hammarstrom et a l . , 1977) by e i t h e r cAMP or g l u c o c o r t i c o i d s , a f f e c t the c e l l metabolism i n the same d i r e c t i o n s , but by d i f f e r e n t mechanisms of a c t i o n (Thompson and Lippman, 1974). As an example, g l u c o c o r t i c o i d s seem to enhance the p r o l i f e r a t i o n o f thymocytes induced by cAMP (Munck and Leung, 1977), and i n t u i t i v e l y , the pharmacologic i n h i b i t i o n of inflammatory s k i n disease might be en-hanced by cAMP (Voorhees, 1977a). Therefore, the t h e r a p e u t i c range of s t e r o i d s might have been extended by cAMP. These models of the mechanism of g l u c o c o r t i c o i d a c t i o n a l l have the weakness that they e x p l a i n some g l u c o c o r t i c o i d a c t i o n s but not others, f o r instance, the a n t i p r u r i t i c and a n t i a n a b o l i c a c t i o n s are un-s a t i s f a c t o r i l y accounted f o r . The only theory that has the p o t e n t i a l a b i l i t y to e x p l a i n the broader spectrum of g l u c o c o r t i c o i d a c t i v i t y i s the receptor model theory. V. Receptor models of the mechanism of g l u c o c o r t i c o i d a c t i o n G l u c o c o r t i c o i d s d i f f e r from other hormonal s t e r o i d s by a f f e c t i n g almost a l l t i s s u e s and c e l l types. The nature of the p h y s i o l o g i c a l r e -sponse v a r i e s according to the c e l l or system being examined. The g l u c o -c o r t i c o i d mediates a l a r g e number of p h y s i o l o g i c a l and biochemical e f f e c t s , as reviewed by Leung and Munck (1975). The work on the mechanism of g l u c o c o r t i c o i d a c t i o n s has not advanced as f a r as that on sex hormones or progesterone. Enough observa-t i o n s t e s t i f y however that the general model of s t e r o i d a c t i o n , derived from studies on progesterone as depicted i n Figure 3, a l s o a p p l i e s to g l u c o c o r t i c o i d s . The "receptor" theory has been reviewed i n d e t a i l i n G l u c o c o r t i -c o i d Hormone A c t i o n (Baxter and Rousseau, 1979) which contains the most recent, comprehensive reviews i n chapters by Rousseau and Baxter (Chap. cell membrane nuclear membrane *• RNA polymerase DNA structural gene j transcription —^—• primary transcript J, modification M e ^ r £ > mRNA it acceptor site irk effector site 3 : The molecular mechanism of s t e r o i d hormone a c t i o n (receptor model) (O'Malley and B u l l e r , 1977) 3 ) , Higgins et a l . (Chap. 8 ) , Johnson et a l . (Chap. 17) and Aronow (Chap. 18). P e r t i n e n t a l s o i s information i n the older volumes of Receptors and  Mechanisms o f A c t i o n of S t e r o i d Hormones ( P a s q u a l i n i , 1976 and 1977), e s p e c i a l l y chapters 1 and 8 by Munck and Leung, and i n Receptors and Hor-mone Ac t i o n (O'Malley and Birnbaumer, 1977 and 1978) with chapters by Clar k and Peck (Chap. 11) i n Volume I and those by Stormshak et a l . (Chap. 3 ) , F e i g e l s o n et a l . (Chap. 9 ) , Baxter and I v a r i e (Chap 10), Yama-moto and Ringold (Chap. 11) i n Volume I I , as w e l l as the chapter by O'Malley and a s s o c i a t e s i n Volume I I (Chap. 8 ) . C r i t i q u e of the theory has been w r i t t e n i n the Annual Review of Physiology by Gorski and Gannon (1976). P r a t t (1978) has summarized the mechanism o f g l u c o c o r t i c o i d e f f e c t s i n f i b r o b l a s t s . The receptor model of the mechanism of the a c t i o n of g l u c o c o r t i -coids can be summarized as f o l l o w s : (A) N o n - s p e c i f i c binding before uptake by ta r g e t c e l l s N a t u r a l l y o c c u r r i n g g l u c o c o r t i c o i d s (<(s^ c i r c u l a t e throughout the body predominantly complexed with plasma p r o t e i n s , mainly with e o r t i -c o s t e r o i d - b i n d i n g g l o b u l i n , CBG or t r a n s c o r t i n , (approximately 77$), and s l i g h t l y with albumin (about 15$). Over the normal p h y s i o l o g i c range of 5 to 25 ug/mL f o r t o t a l hydrocortisone c o n c e n t r a t i o n i n the plasma, the amount of the s t e r o i d bound to CBG increases i n a nearly p r o p o r t i o n a l manner to the t o t a l hydrocortisone l e v e l . The unbound hydrocortisone remains at about 8$ of the t o t a l l e v e l ( T a i t and B u r s t e i n , 1964). Human CBG binds s y n t h e t i c g l u c o c o r t i c o i d s only s l i g h t l y except f o r prednisolone ( B a l l a r d , 1979). The d i s t r i b u t i o n of g l u c o c o r t i c o i d s between unbound and protein-bound forms i s dependent on both the type of g l u c o c o r t i c o i d s and the l e v e l s of plasma p r o t e i n s , e s p e c i a l l y of CBG, and profoundly a f f e c t s the a v a i l a b i l i t y of g l u c o c o r t i c o i d s to target c e l l s . 18. (B) G l u c o c o r t i c o i d passage across the c e l l membrane The mechanism of g l u c o c o r t i c o i d entry i n t o t arget c e l l s has r e -ceived r e l a t i v e l y l i t t l e a t t e n t i o n . I t has been g e n e r a l l y assumed that g l u c o c o r t i c o i d s r e a d i l y pass across the c e l l membrane v i a passive d i f f u -s i o n by v i r t u e of t h e i r small s i z e and l i p o p h i l i c nature. Uptake s t u d i e s i n hepatoma c e l l s (Plageman and Erbe, 1976) and thymocytes (Mayer et a l . , 1976) have been c o n s i s t e n t with t h i s view; however, s e v e r a l systems, such as mouse L-929 and rat l i v e r c e l l s , accumulates g l u c o c o r t i c o i d s against a concentration gradient (Gross and Aronow, 1970; Rao et a l . , 1976). I n pa r t , such an accumulation against a concentration g r a d i e n t may be ex-pla i n e d by binding to i n t r a c e l l u l a r g l u c o c o r t i c o i d receptors, but there seems to be an a d d i t i o n a l uptake which may represent e i t h e r low a f f i n i t y membrane-binding s i t e s , or a f a c i l i t a t e d s t e r o i d t r a n s p o r t system. At present, the evidence f o r a general existence of e i t h e r inward or outward f a c i l i t a t e d t r a n s p o r t remains i n c o n c l u s i v e . (C) Cytoplasmic receptor binding A c e n t r a l concept i n t h i s model of s t e r o i d a c t i o n i s that the g l u c o c o r t i c o i d must bind to a s p e c i f i c receptor p r o t e i n i n order to exert i t s e f f e c t i n the c e l l . W i thin t a r g e t c e l l s , which can be d i s t i n g u i s h e d from non-target c e l l s by autoradiographic technique, the entering g l u c o -c o r t i c o i d i n t e r a c t s with cytoplasmic receptor (designated as R -R. i n 3 D F i g . 3) i n a non-covalent manner with high a f f i n i t y , s t e r e o - s p e c i f i c i t y and f i n i t e c a p a c i t y (Feigelson et a l . , 1978) to y i e l d a g l u c o c o r t i c o i d -receptor complex. The idea of receptor dimers was o r i g i n a l l y formulated f o r the progesterone receptor by O'Malley's l a b o r a t o r y . However, g l u c o c o r t i c o i d ( B u l l e r et a l . , 1976), estrogen and androgen ( N a r r i s and Kohler, 1976) binding a c t i v i t y could be e l u t e d from various ion-exchange r e s i n s as p a i r s of peaks reminiscent of the oviduct progesterone system (O'Malley and B u l l e r , 1976). The two subunits of cytoplasmic receptor have pre-v i o u s l y been c l a s s i f i e d as the R and R. p r o t e i n s on the b a s i s of a b t h e i r d i f f e r e n t a f f i n i t i e s for nuclear c o n s t i t u e n t s . Subunit R binds a to DNA, but not to chromatin, whereas subunit R^ binds to chromatin, and l e s s r e a d i l y to DNA. Both p r o t e i n s have i d e n t i c a l hormone-binding s p e c i f i c i t i e s and k i n e t i c s (O'Malley and B u l l e r , 1976). O'Malley pro-posed that subunit R i s t r u l y a r e g u l a t o r y N p r o t e i n which might be ex-pected to a l t e r t r a n s c r i p t i o n . Conversely, subunit R^ i s merely a s p e c i f i e r p r o t e i n that c a r r i e s the R p r o t e i n to the neighborhood of responsive genes and i s i n e f f e c t i v e i n s t i m u l a t i n g chromatin t r a n s c r i p -t i o n . In the e a r l y 1960's, i n t r a c e l l u l a r g l u c o c o r t i c o i d - b i n d i n g com-ponents were i d e n t i f i e d i n r a t l i v e r using r a d i o a c t i v e s t e r o i d , but t h e i r involvement i n hormone a c t i o n was debatable (King and Mainwaring, 1974). A major breakthrough occurred when binding studies were conducted on i s o -l a t e d c e l l s f o r which s t e r o i d concentrations were known with p r e c i s i o n and competition and k i n e t i c experiments could be performed, and recourse made to s y n t h e t i c g l u c o c o r t i c o i d s that d i d not i n t e r a c t s i g n i f i c a n t l y w i t h nonreceptor components of the c e l l . C r i t i c a l i n v e s t i g a t i o n s by Munck (Munck and Brinck-Johnsen, 1968) and Schaumburg (Schaumburg and Bojesen, 1968) on r a t thymocytes provided the f i r s t c l e a r demonstration o f g l u c o c o r t i c o i d r e c e p t o r s . I n these c e l l s , a l i m i t e d number of s i t e s could be detected that bound s t e r o i d s with an a f f i n i t y c o n s i s t e n t w i t h t h e i r g l u c o c o r t i c o i d a c t i v i t y . These f i n d i n g s were extended to g l u c o c o r -t i c o i d - s e n s i t i v e c e l l l i n e s i n c u l t u r e : human HeLa c e l l s (Melnykovych and Bishop, 1 9 6 9 ) , mouse L-929 f i b r o b l a s t s (Hackney et a l . , 1 9 7 0 ) , and P1798 lymphosarcoma ( K i r k p a t r i c k et a l . , 1 9 7 1 ) , r a t hepatoma t i s s u e c u l t u r e (HTC) (Baxter and Tomkins, 1970), and S*»9 lymphoma c e l l s (Baxter et a l . , 1971). Mammalian s k i n apparently contains receptor p r o t e i n s i n both epidermis and dermis (Voorhees, 1977b). P r e l i m i n a r y evidence demonstrat-ed the presence of a s i n g l e c l a s s o f h i g h - a f f i n i t y b i n d i n g p r o t e i n s i n mouse s k i n (Slaga et a l . , 1977) and i n primary c u l t u r e s of epidermal c e l l s ( E p s t e i n , 1977; Ponec et a l . , 1980). When c e l l s c o n t a i n i n g bound s t e r o i d were f r a c t i o n a t e d , the bound s t e r o i d was found p r i m a r i l y i n the^ nucleus (Baxter and Tomkins, 1970; Baxter et a l . , 1971). In co n t r a s t , when binding was examined i n e x t r a c t s of c e l l s that had not been exposed to the s t e r o i d , a c t i v i t y was r e s t r i c t -ed to the c y t o s o l (Baxter and Tomkins, 1971; P r a t t and I s h i i , 1972). These observations provided the i n i t i a l i n d i c a t i o n t h a t the g l u c o c o r t i -c o i d receptor system i s s i m i l a r to that of other s t e r o i d - r e s p o n s i v e sys-tems, i n that i t possesses c y t o s o l receptors which, upon bindi n g an a c t i v e s t e r o i d , a s s o c i a t e with nucleus. The nature of the nuclear b i n d -in g r e a c t i o n i s discussed elsewhere. The model systems studied i n the e a r l y seventies included r a t HTC c e l l s (Baxter and Tomkins, 1970; Baxter and Tomkins, 1971; Rousseau et a l . , 1972a), l i v e r (Rousseau et a l . , 1972a; Beato and F e i g e l s o n , 1972; Litwack et a l . , 1973) and thymocytes (Schaumburg, 1972; B e l l and Munck, 1973), mouse L-929 f i b r o b l a s t s ( P r a t t and I s h i i , 1972), lymphosarcoma P1798 ( K i r k p a t r i c k et a l . , 1972) and p i t u i t a r y tumors (Watanabe et a l . , 1973), chick embryo r e t i n a (Chader et a l . , 1972) and c u l t u r e d mammary c e l l s from r a t (Gardner and W i t t l i f f , 1973) and mouse (Shyamala, 1974). These i n v e s t i g a t i o n s gave some i n s i g h t i n t o the binding k i n e t i c s and physicochemical p r o p e r t i e s of the receptor s i t e s , and e s t a b l i s h e d the involvement of the receptor i n g l u c o c o r t i c o i d hormone a c t i o n on firme r ground. For a d e t a i l e d account of the l i t e r a -ture on the subje c t , comprehensive reviews are a v a i l a b l e ( P a s q u a l i n i , 1976 and 1977; O'Malley and Birnbaumer, 1977 and 1978; Baxter and Rous-seau, 1979). Ponec et a l . (1980) have attempted to r e l a t e c y t o s o l receptor binding i n human c u l t u r e d f i b r o b l a s t s , to growth i n f l u e n c e d by g l u c o c o r -t i c o i d s . Under c e r t a i n circumstances growth i n h i b i t o r y e f f e c t s were l o s t without binding being a f f e c t e d . I t would seem another mechanism, perhaps s t e r o i d i n d u c t i o n of other c o n t r o l l i n g f a c t o r s , may be i n v o l v e d . The c y t o s o l binding of g l u c o c o r t i c o i d s c o n s t i t u t e s both s p e c i -f i c and n o n - s p e c i f i c binding. Munck and Leung (1977) defined the s p e c i -f i c i t y of binding as the a b i l i t y to form complexes with molecules of s t r u c t u r e s that l i e w i t h i n the range of stereochemical c o n f i g u r a t i o n s a s s o c i a t e d with a p a r t i c u l a r hormonal a c t i v i t y . N o n s p e c i f i c i t y r e f e r r e d to a r e l a t i v e lack of such s e l e c t i v i t y . Therefore, not a l l g l u c o c o r t i -coid binding i s receptor binding, nor a l l bindings r e s u l t i n g l u c o c o r t i -c o i d a c t i v i t i e s . This p r o j e c t i s mainly concerned with the determination of " s p e c i f i c b i n d i n g " . (D) A c t i v a t i o n and t r a n s l o c a t i o n of g l u c o c o r t i c o i d - r e c e p t o r complex The g l u c o c o r t i c o i d - r e c e p t o r complex undergoes an " a c t i v a t i o n " needed for t r a n s l o c a t i o n i n t o the nucleus. This a c t i v a t i o n i s b e l i e v e d to i n v o l v e conformational change of the receptor by mechanisms e i t h e r of d r i v i n g the e q u i l i b r i u m between two forms of receptors towards the form of higher a f f i n i t y to g l u c o c o r t i c o i d (Monod et a l . , 1965), or of "induced f i t " model (Koshland and Neet, 1968; Atkinson, 1966). The rate of the a c t i v a t i o n i s h i g h l y dependent on temperature (Milgrom et a l . , 1973; Jensen et a l . , 1972) and i o n i c strength (Milgrom et a l . , 1973). I t i s now thought that only 40-60$ of g l u c o c o r t i c o i d - r e c e p t o r complexes become a c t i v a t e d (Higgins et a l . , 1979). The t r a n s l o c a t i o n of a c t i v a t e d g l u c o c o r t i c o i d - r e c e p t o r complex from cytoplasm i n t o nucleus i s not deniable, but very l i t t l e i s known about how i t a c t u a l l y occurs. The g l u c o c o r t i c o i d remains bound to the same receptor p r o t e i n i n both cytoplasmic and nuclear compartments during the t r a n s l o c a t i o n process. The passage of the complex across the nuclear membrane i s not a r a t e - l i m i t i n g step i n the nuclear accumulation of the complex ( B u l l e r et a l . , 1975). (E) Nuclear acceptor binding and gene expression The t r a n s l o c a t e d g l u c o c o r t i c o i d - r e c e p t o r complex a s s o c i a t e s w i t h acceptor s i t e s i n the chromatin, i n an as yet undefined manner to form a ternary c h r o m a t i n - g l u c o c o r t i c o i d - r e c e p t o r complex. These events lead to su b t l e a l t e r a t i o n s i n the patter n of gene t r a n s c r i p t i o n , and f i n a l l y , the a l t e r e d or new mRNA, enzyme or other p r o t e i n l e v e l s a f f e c t the hormonally evoked c e l l u l a r and p h y s i o l o g i c a l responses. However, the process le a d i n g to the t r a n s c r i p t i o n a l a l t e r a t i o n s and, f u r t h e r , to changes i n c e l l u l a r physiology i s the l e a s t understood aspect of the receptor model theory. Some of the problems r e q u i r i n g f u r -ther e x p l o r a t i o n and a more p r e c i s e d e f i n i t i o n are: (a) the p r e c i s e chemical nature o f the i n t e r a c t i o n between g l u -c o c o r t i c o i d - r e c e p t o r complex and nuclear components, as f o r example, whether the binding i s i o n i c , hydrophobic or covalent i n nature, (b) the component of g l u c o c o r t i c o i d - r e c e p t o r complex i n v o l v e d i n the nuclear b i n d i n g , i . e . whether the g l u c o c o r t i c o i d or the receptor p r o t e i n , or both are in v o l v e d i n the i n t e r a c t i o n , (c) the nuclear component r e s p o n s i b l e f o r the s p e c i f i c acceptor binding, among which DNA, his t o n e and non-histone p r o t e i n s have been pro-posed ( F e i g e l s o n et a l . , 1978; Wong and Aronow, 1976: O'Malley and B u l l e r , 1977), (d) the d i s s o c i a t i o n of g l u c o c o r t i c o i d from receptor s i t e s and of g l u c o c o r t i c o i d - r e c e p t o r complex from acceptor s i t e s , as w e l l as the g l u c o c o r t i c o i d e l i m i n a t i o n from the c e l l , (e) The mechanism of r e g u l a t i o n of gene expression induced by binding of g l u c o c o r t i c o i d - r e c e p t o r complex, e s p e c i a l l y why only a small percentage of genes are a f f e c t e d , ( f ) the c o r r e l a t i o n of nuclear binding with b i o l o g i c a l r e -sponse, and (g) the unexplained dichotomy of g l u c o c o r t i c o i d e f f e c t s which may be due to d i f f e r e n c e s i n c e l l types, c e l l metabolic s t a t e s or t h e i r s t a t e of d i f f e r e n t i a t i o n as w e l l as on d i f f e r e n c e s i n g l u c o c o r t i c o i d con-c e n t r a t i o n s and on the lengths of time of exposure of c e l l s to g l u c o c o r -t i c o i d . Reviews on these c o n t r o v e r s i a l or i n s u f f i c i e n t s t u d i e s are pre-sented f u r t h e r i n t h i s survey. VI. Physicochemical c h a r a c t e r i s t i c s o f g l u c o c o r t i c o i d receptor and recep-t o r b i n d i n g The receptors are proteinaceous i n nature, s i n c e t h e i r b i n d i n g a c t i v i t y to g l u c o c o r t i c o i d s i s destroyed by pronase, t r y p s i n , papain and chymotrypsin, but not by DNAse, RNAse, collagenase, hyaluronidase, neur-aminidase and lysosomal enzymes. Phospholipases A and C devoid of pro-t e o l y t i c a c t i v i t y i n a c t i v a t e receptor binding i n mouse L-929 f i b r o b l a s t s and r a t thymocytes, suggesting that phospholipids are important f o r receptor s t a b i l i t y or that the products of the phospholipase r e a c t i o n denature the receptor (Hackney and P r a t t , 1971; Schulte et a l . , 1976). The amphoteric character of receptor p r o t e i n i s revealed by i t s a b i l i t y to bind to both an i o n i c (phosphocellulose, DNA) and c a t i o n i c ( d i e t h y l -a m i n o e t h y l c e l l u l o s e ) polymers. G l u c o c o r t i c o i d receptors are u b i q u i t o u s l y d i s t r i b u t e d as physio-l o g i c r e g u l a t o r s i n mammalian t i s s u e s (Rousseau and Baxter, 1979). The d i s t r i b u t i o n of g l u c o c o r t i c o i d receptors i n mammalian t i s s u e s has been compiled by Munck and Leung (1977). I t has been proposed that the receptor i s cycled through d i f -f e r e n t binding s t a t e s and l o c a t i o n s i n the c e l l (Middlebrook et a l . , 1975; Munck et a l . , 1972; I s h i i et a l . , 1972; N i e l s e n et a l . , 1977). A s i n g l e receptor p r o t e i n may be a c t u a l l y p a r t i c i p a t i n g s e v e r a l times i n the process of s t e r o i d binding and gene a c t i v a t i o n . A model of the receptor c y c l e proposed by N i e l s e n et a l . (1977) i s presented i n Figure ^. The c y c l i n g of the receptor i n i t s bound form from cytoplasm to nucleus i s regarded as followed by regeneration of unbound receptor i n t o the cytoplasm. The receptor c y c l e may have an important r o l e i n deter-mining the a b i l i t y of the c e l l to change r a p i d l y the i n t e n s i t y of i t s p h y s i o l o g i c a l response i n response to changing plasma g l u c o c o r t i c o i d l e v e l s ( P r a t t , 1978). The binding parameters, the e q u i l i b r i u m d i s s o c i a t i o n constant, Kp, and the b i n d i n g c a p a c i t y , ^ m a x> °f g l u c o c o r t i c o i d s have been determined us i n g Scatchard a n a l y s i s by a number of i n v e s t i g a t o r s . Most of the st u d i e s have been c a r r i e d out with dexamethasone and to a l e s s e r extent w i t h hydrocortisone and triamcinolone acetonide i n v a r i o u s r a t or mouse t i s s u e s . Only a few st u d i e s have involved other g l u c o c o r t i c o i d s or human c e l l s . The data summarized i n Table I I I represent the s t u d i e s r e p o r t i n g the apparent e q u i l i b r i u m d i s s o c i a t i o n constants o f g l u c o c o r t i -c o i d receptor binding done i n the past decade. V a r i a t i o n s among the values obtained are l a r g e , but most f a l l w i t h i n one order of magnitude of 25. NUCLEUS CYTOPLASM dephosphorylated, i n a c t i v e receptor phosphorylated receptor s t e r o i d - r e c e p t o r complex a c t i v a t e d s t e r o i d - r e c e p t o r complex s t e r o i d processes that appear to r e q u i r e energy i n L - c e l l s Figure 4 : Proposed c y c l e of events c o n t r o l l i n g the b i n d i n g s t a t e and c e l l u l a r l o c a t i o n of g l u c o c o r t i c o i d receptor (Nielsen et a l . , 1977) 1 R a RS RS S n Table I I I Binding parameters of n a t u r a l l y o c c u r r i n g and s y n t h e t i c g l u c o c o r t i c o i d s to receptors G l u c o c o r t i c o i d T i s s u e / c e l l l i n e K^nM) B max Reference Hydrocortisone Triamcinolone acetonide Dexamethasone Mouse L-929 fb Mouse L-929 fb (a) 2.8 A.3 (b) P r a t t et al.,1975 Middlebrook and Aronow, 1977 Rat HTC 11 0 . 6 3 ( c ) Rousseau et a l . , 1972a Mouse L-929 f b . 1.6 - P r a t t et a l . ,1975 Mouse L-929 f b . Human s k i n f b . 0.06 O.A-8.6 ^ ( b ) 1.5-11.A Middlebrook and Aronow, 1977 (d) ..Bruning et a l . , 1979 Mouse L-929 f b . 7.6 - P r a t t et al.,1975 Human s k i n f b . A.5 0 . 1 2 ( C ) Bauknecht, 1977 Human amniotic f l u i d c e l l s 19 0.375 ( c ) Bauknecht,1977 Rat HTC 3.1 0.66 ( C ) Rousseau et al.,1972a Rat thymocytes 1.9 - Schaumburg,1972 Rat l i v e r 3.7 - K o b l i n s k i et al.,1972 Rat kidney 8.A 0 . 3 1 ( C ) R a f e s t i n - O b l i n et a l . , 1977 Rat adipose t i s s u e Rat t e s t i s 6 3 ± 2 (c) 0.2 Feldman and Loose,1977 (c) 0.2 ±0.05 Evain et al.,1976 Mouse thymocytes A i o ( e > Duval et al.,1976 Mouse lymphoma 20 - Rousseau et al.,1972b Rabbit lung c e l l s f r e s h l y i s o l a t e d 6.9 7500 c u l t u r e d 1-2 weeks 7.9 26500 c u l t u r e d , f e t a l 10.2 13150 ^ Ballard,1977 ( f ) ( f ) (a) f i b r o b l a s t s (b) nM, (c) fmol/pg p r o t e i n , (d) fmol/u g DNA, (e) fmol/10 c e l l s , ( f ) s i t e s / c e l l each other. I n t e r p r e t a t i o n of the binding c a p a c i t y , ^ m a x ^ * s roade unce r t a i n by i n c o n s i s t e n c i e s i n the manner of data p r e s e n t a t i o n which by va r i o u s authors are expressed v a r i o u s l y as fmol per ug p r o t e i n , fmol per yg DNA, fmol per 10^ c e l l s or the number of bindi n g s i t e s per c e l l . S tudies of binding k i n e t i c s have confirmed that the g l u c o c o r t i -c o i d - r e c e p t o r a s s o c i a t i o n i s second order and d i s s o c i a t i o n i s f i r s t order (Baxter and Tomkins, 1971). The rate of a s s o c i a t i o n to the receptor has been found to be slower for g l u c o c o r t i c o i d s with high a f f i n i t y than f o r those with low a f f i n i t y (Rousseau et a l . , 1972a; P r a t t et a l . , 1975; B a l l a r d et a l . , 1975; Kaine et a l . , 1975). G l u c o c o r t i c o i d s that bind with high a f f i n i t y have been found to have a slow rate of d i s s o c i a t i o n . For instance, the potent hydrocortisone analog, tri a m c i n o l o n e acetonide, binds exceedingly slowly and has extremely high a f f i n i t y due to the f a c t th a t , once bound, i t shows v i r t u a l l y no d i s s o c i a t i o n during the time period of observation ( P r a t t and I s h i i , 1972; Watanabe et a l . , 1973; Goral and W i t t l i f f , 1975). V I I . Nuclear binding of g l u c o c o r t i c o i d s I t i s now g e n e r a l l y recognized that although c y t o s o l g l u c o c o r t i -coid receptor binding i s a necessary step, the nuclear i n t e r a c t i o n s o f the complex play the c e n t r a l r o l e of many aspects of s t e r o i d hormone a c t i o n . C e l l f r a c t i o n a t i o n (Rousseau et a l . , 1973; Jensen et a l . , 1971; Shyamala and G o r s k i , 1969; Wira and Munck, 1974; O'Malley et a l . , 1971) and whole c e l l autoradiographic experiments (Bogoroch, 1969) have shown that r a d i o l a b e l l e d s t e r o i d hormones are r e t a i n e d and concentrated i n the n u c l e i of target c e l l s of a l l t i s s u e s of the mammalian body. Removal of the c e l l nucleus, e.g. with c y t o c h a l a s i n B, prevents g l u c o c o r t i c o i d s from inducing t y r o s i n e aminotransferase i n hepatoma (HTC) c e l l s ( I v a r i e et a l . , 1975). The removal of g l u c o c o r t i c o i d from c e l l n u c l e i by washing stops i n i t i a t i o n of t r a n s c r i p t i o n i n r a t p i t u i t a r y tumor c e l l s (Johnson et a l . , 1979b). G l u c o c o r t i c o i d s become s p e c i f i c a l l y bound to the nucleus only a f t e r binding of g l u c o c o r t i c o i d to c y t o s o l receptors. N u c l e i i s o l a t e d from untreated c e l l s do not bind g l u c o c o r t i c o i d s (Higgins et a l . , 1973a). The k i n e t i c s of nuclear bind i n g have been studied i n HTC c e l l s , r a t thymocytes and mouse L-929 c e l l s . In HTC c e l l s exposed to dexametha-sone at 37°, nuclear binding of s t e r o i d reaches a maximum w i t h i n 30 min and l e v e l s o f f (Figure 5 ) . The plateau p e r s i s t s as long as the s t e r o i d i s present (Rousseau et a l . , 1973). With r a t thymocytes, there i s a l a g period of l e s s than one minute before dexamethasone-receptor complex s t a r t s to appear i n the nucleus. The nuclear binding then l e v e l s o f f a f t e r 5-10 min (Munck et a l . , 1979). In other c e l l l i n e s , the steady s t a t e of l e v e l s of bin d i n g are a t t a i n e d more sl o w l y , but are g e n e r a l l y reached w i t h i n approximately 2 hr (Middlebrook et a l . , 1975). The m a j o r i t y of i n v e s t i g a t o r s whose work dominates the current views on g l u c o c o r t i c o i d mechanism of a c t i o n s , such as Baxter and Rousseau (1979), P r a t t (1978), Aronow (1979), Munck and Leung (1977), and O'Malley and Birnbaumer (1977, 1978) emphasize i n t h e i r experimental design the 0-2 hr, sometimes 0-6 hr time period f o r s t u d i e s . They have assumed that a f t e r nuclear binding has reached the steady s t a t e the g l u c o c o r t i c o i d -receptor complex has no f u r t h e r i n f l u e n c e on the c e l l u l a r metabolism. Their assumption should be tested by f o l l o w i n g g l u c o c o r t i c o i d i n f l u e n c e through at l e a s t two c e l l c y c l e periods (about 96 hr f o r human f i b r o -b l a s t s , 48 hr f o r L-929 f i b r o b l a s t s ) . The nuclear binding of g l u c o c o r t i c o i d s i n HTC c e l l s has been found r a p i d l y r e v e r s i b l e a f t e r removal of the s t e r o i d from the i n c u b a t i o n 29. 5 10 30 60 90 120 TIME , min. Figure 5 : The k i n e t i c s and r e v e r s i b l e e f f e c t s of nuclear b i n d i n g of dexamethasone-receptor complexes i n i n t a c t HTC c e l l s . Cytosol ( c i r c l e s ) and nuclear ( t r i a n g l e s ) content of recep-t o r i s presented as a f u n c t i o n of time f o l l o w i n g a d d i t i o n of dexamethasone ( • , • ) or of the v e h i c l e ( A ) to i n -t a c t HTC c e l l s . Dashed l i n e corresponds to data obtained f o l l o w i n g removal of s t e r o i d from the c u l t u r e at 30 min. (Rousseau, 1975) medium (Figure 5). The disappearance of the s t e r o i d from the nucleus has been observed to be complete w i t h i n 1 hr at 37° (Rousseau, 1975). The nuclear binding was b e l i e v e d i n i t i a l l y to be a s a t u r a b l e process with high a f f i n i t y . However, Chamness et a l . (1974) and Milgrom et a l . (1975, 1976) and Higgins et a l . (1979) have demonstrated that apparent s a t u r a t i o n of nuclear s i t e s i s due to competition of excess non-receptor components w i t h s p e c i f i c b i n d i n g s i t e s . In the presence of a constant concentration of nonreceptor p r o t e i n , nuclear binding i s found to be a nonsaturable l i n e a r f u n c t i o n of the concentration of the com-plex. Higgins et a l . (1973c) have a l s o suggested that saturable binding i n v i t r o i s a r t i f a c t u a l by demonstrating that n u c l e i from c e l l s p r e t r e a t -ed with s t e r o i d i n v i v o are s t i l l capable of subsequent binding of the complex i n v i t r o . Hence, most l a b o r a t o r i e s now appear to agree t h a t nuclear b i n d i n g of complexes i s not r e a d i l y s a t u r a b l e . Lacking s a t u r a -t i o n , the Scatchard p l o t f o r nuclear binding has a slope of zero (Figure 6), and binding a f f i n i t y and c a p a c i t y can no longer be estimated by t h i s a n a l y s i s as i t can be done f o r c y t o s o l receptor binding (Yamamoto and A l b e r t , 1976; Higgins et a l . , 1979). Lack of s a t u r a t i o n can be i l l u s -t r a t e d by F i g u r e 6 which represents the data of Higgins et a l . on dexa-methasone-receptor accumulation i n HTC c e l l n u c l e i . V I I I . Nuclear components as s o c i a t e d with g l u c o c o r t i c o i d b i n d i n g The nucleus p l a y s an important r o l e i n the mechanism of g l u c o -c o r t i c o i d a c t i o n . Both the accumulation of g l u c o c o r t i c o i d - r e c e p t o r com-plex and the m o d i f i c a t i o n of gene expression occur i n n u c l e i of t a r g e t c e l l s . In order to d i s c u s s the p o s s i b l e mechanism of these two events, a b r i e f review of the nuclear s t r u c t u r e i s i n order. 31. CO CP 9 t1 O o "D| C D O - Q I k_ 03 Q) O z co 0 X & 2 o o o co o • - - • • 1 2 3 4 N uclear-bound receptor-dexamethasone complexes, pmol/mgDNA Figure 6 : Independence of nuclear b i n d i n g of conc e n t r a t i o n of re c e p t o r -dexamethasone complex i n the cytoplasm of i n t a c t HTC c e l l s at 37°. Shown i s a Scatchard p l o t of the data where concentration of , the t o t a l c y t o s o l receptor-dexamethasone complexes and nuclear-bound complexes were measured. (Higgins et a l . , 1979) The nucleus (Fi g u r e 7) i s surrounded by the outer (ONM) and inner nuclear (INM) semipermeable membranes that enclose the p e r i n u c l e a r space (PNS) which i s a part of the rough endoplasmic r e t i c u l u m and has r i b o -somes (Rb) attached. Between the chromatin and the inner membrane l i e s the lamina densa (LD) which i s t h i n n e r i n f r o n t of the nuclear pores (NP). The chromatin i s found as heterochromatin (HC), nucleolus-asso-c i a t e d chromatin (NC), and euchromatin (EC). The nucleolus shows the granular (G) components and f i b r i l l a r centers (FC). In the b o r d e r l i n e of the chromatin, many perichromatin granules (PG) and a l a y e r of p e r i c h r o -matin f i b r i l s (PF) are found. F i n a l l y , i n the interchromatin space, a c l u s t e r of interchromatin granules (IG), granular nuclear body (GNB), a simple nuclear body (SNB), a c o i l e d body (CB), and an i n t r a n u c l e a r r o d l e t (INR) are seen. Busch (1979) tabulated the d e t a i l e d nuclear s t r u c t u r e s i n terms of nuclear envelope, chromatin, nucleolus, nuclear RNP network and nuclear p a r t i c l e s . The nucleus i s part of an i n t e g r a t e d system by which c e l l s of various organs respond to e x t r a c e l l u l a r and i n t r a c e l l u l a r stimu-l i . These s t i m u l i i n t e r a c t with nuclear i n f o r m a t i o n a l system^ to produce s p e c i f i c products that permit response of the cytoplasm to the environ-ments and i t s f u n c t i o n a l demands. The nucleus contains the major r e p o s i t o r y of genetic informa-t i o n , chromatin, which comprises DNA and i t s a s s o c i a t e d p r o t e i n s . When i n t a c t c e l l s are exposed to g l u c o c o r t i c o i d s and the n u c l e i are then f r a c -t i o n a t e d , g l u c o c o r t i c o i d - r e c e p t o r complexes are found i n chromatin but not i n the nucleolus, nucleoplasm, or nuclear membranes (Higgins et a l . , 1979). A s i m i l a r d i s t r i b u t i o n i s seen i n l i v e r n u c l e i a f t e r b i n d i n g of g l u c o c o r t i c o i d - r e c e p t o r s i n v i t r o (Beato et a l . , 1973). In chromatin, nuclear-bound r e c e p t o r - g l u c o c o r t i c o i d complexes are found e x t e n s i v e l y 33. Nuclear envelope Nuclear p a r t i c l e s ONM outer nuclear membrane PG perichromatin granules INM inner nuclear membrane PF perichromatin f i b r i l s PNS p e r i n u c l e a r space IG i n t e r c h r o m a t i n granules NP nuclear pores GNB granular nuclear body Rb ribosomes SNB simple nuclear body CB c o i l e d body Chromat in-chromosomes INR i n t r a n u c l e a r r o d l e t HC heterochromatin LD lamina densa EC euchromatin mRNP precursor p a r t i c l e s NC nu c l e o l u s - a s s o c i a t e d chromatin The nu c l e o l u s n u c l e o l i i n v a r i o u s stages of c e l l f u n c t i o n G granular components FC f i b r i l l a r centers Pre-rRNA I n t e r l o c k s of rRNA and ribosomal p r o t e i n s y n t h e s i s The n u c l e o l a r channel system Figure 7: I d e a l i z e d s e c t i o n of a nucleus, showing a l l the main components ( B o u t e i l l e et a l . , 1974) d i s t r i b u t e d i n both the t r a n s c r i p t i o n a l l y a c t i v e and i n a c t i v e f r a c t i o n s (Levy and Baxter, 1976). G l u c o c o r t i c o i d - r e c e p t o r complexes do not bind to RNA (Rousseau et a l . , 1975). The evidence f o r t h i s comes from c e l l - f r e e systems. In •3 these systems, c y t o s o l receptor i s bound with H-dexamethasone, then a c t i v a t e d by heating to 20° f o r 30 minutes and incubated with e i t h e r n a t i v e HTC DNA or E. c o l i rRNA. F r a c t i o n a t i o n through a Sepharose 4B column f i n d s the g l u c o c o r t i c o i d - r e c e p t o r complexes bound with DNA but not with RNA (Figure 8 ) . The p r e c i s e nature of g l u c o c o r t i c o i d - r e c e p t o r binding to nuclear acceptor s i t e s i n chromatin i s s t i l l a matter of dis p u t e . D i f f e r e n t groups of i n v e s t i g a t o r s have va r i o u s views. Fe i g e l s o n et a l . (1978) suggested DNA i s the major binding com-ponent of the g l u c o c o r t i c o i d - r e c e p t o r complex at acceptor s i t e s . He has demonstrated that b r i e f treatment of r a t l i v e r n u c l e i w i t h DNAse I caused a l o s s of about "\0% of nuclear DNA, but r e s u l t e d i n more than B>0% o f l o s s i n nuclear binding c a p a c i t y f o r g l u c o c o r t i c o i d s . Thus, i t appears t h a t g l u c o c o r t i c o i d - r e c e p t o r complex i s bound to a small p o r t i o n of the genome, which i s r e a d i l y s e n s i t i v e to DNAse. Baxter et a l . (1972) and Higgins et a l . (1973b) reported s i m i l a r observations to confirm the i n -volvement of DNA i n acceptor b i n d i n g s i t e s . O'Malley and B u l l e r (1977) proposed that non-histone p r o t e i n s play an e s s e n t i a l r o l e i n nuclear b i n d i n g o f s t e r o i d s and would e x p l a i n the t i s s u e s p e c i f i c i t y of target c e l l s f o r g l u c o c o r t i c o i d s . Non-histone p r o t e i n s , u n l i k e histone p r o t e i n s , are q u i t e d i v e r s e from c e l l to c e l l w i t h i n a species or among species. I t has been demonstrated that s p e c i -f i c b inding of s t e r o i d to chromatin can be t r a n s f e r r e d from a t a r g e t t i s s u e to a non-target t i s s u e merely by switching s u b f r a c t i o n s of the 35. E c O CD CM -•—• co CD O c 03 V-O CO < Fraction number Figure 8 : G l u c o c o r t i c o i d receptors bind to DNA but not to RNA R: fr e e r e c e p t o r - s t e r o i d complexes S: fr e e s t e r o i d (Rousseau et a l . , 1975) non-histone p r o t e i n s (Spelsberg, 1975). A major c l a s s of non-histone, a c i d i c p r o t e i n s from oviduct, AP^, has been shown to co n t a i n such a t a r g e t - t i s s u e s p e c i f i c a c t i v i t y (O'Malley et a l . , 1972). Puca et a l . (1974) and Mainwaring et a l . (1976) on the other hand proposed that b a s i c rather than a c i d i c non-histone p r o t e i n s were re s p o n s i b l e f o r the t i s s u e s p e c i f i c i t y of nuclear b i n d i n g . Wong and Aronow (1976) detected an a l t e r a t i o n i n the p a t t e r n of histone p r o t e i n s i n L-929 f i b r o b l a s t s f o l l o w i n g exposure to gluco-c o r t i c o i d s . They thought that g l u c o c o r t i c o i d - r e c e p t o r complexes bind to chromatin by e i t h e r d i s p l a c i n g or modifying histone p r o t e i n s . D i r e c t binding of the g l u c o c o r t i c o i d - r e c e p t o r complexes to DNA occurs i n c e l l - f r e e systems and has been al l u d e d to before. I t s occur-rence i s a d e f i n i t e although inadequately i n v e s t i g a t e d p o s s i b i l i t y . IX. Drug i n t e r a c t i o n s with DNA During the past few years, important advances have taken place i n understanding the molecular b a s i s of drug-DNA i n t e r a c t i o n s . These advances include the use of the technique of X-ray c r y s t a l l o g r a p h y i n determining the three-dimensional s t r u c t u r e s of a l a r g e number of drug-n u c l e i c a c i d c r y s t a l l i n e complexes. Types of drug-DNA i n t e r a c t i o n s i n c l u d e i n t e r c a l a t i o n , hydrogen bonding, van der Waals f o r c e s , e l e c t r o -s t a t i c and hydrophobic i n t e r a c t i o n s . Among these i n t e r a c t i o n s , i n t e r -c a l a t i o n has received the most a t t e n t i o n , because i t i s b e l i e v e d to be involved i n the mechanisms of point mutation, carcinogenesis, c y t o t o x i -c i t y and anticancer a c t i v i t y of drugs. The mechanism i s b e l i e v e d to i n -volve damage to DNA by v i r t u e of s i n g l e strand breaks, double strand breaks, i n t e r s t r a n d c r o s s - l i n k s and DNA-protein c r o s s - l i n k s . The charac-t e r i s t i c s of such drug-induced macromolecular damage of nuclear DNA are depicted i n Figure 9. I n t e r c a l a t i o n of drugs i n t o DNA i s defined as the i n s e r t i o n of drug molecules between two successive bases i n DNA, and i t i s a l s o termed base-stacking i n t e r a c t i o n (Weeks et a l . , 1975). I n t e r c a l a t i o n i s a type of strong i n t e r a c t i o n between drug molecule and DNA. Known i n t e r c a l a t -ing agents are the b i f u n c t i o n a l echinomycin, b i s a c r i d i n e , b i s e t h i d i u m , mixed e t h i d i u m - a c r i d i n e ( W a k i l i n and Waring, 1976; Ughetto and Waring, 1977; LePacq et a l . , 1975; Butour et a l . , 1978; Dervan and Becker, 1978), actinomycin ( S o b e l l and J a i n , 1972; S o b e l l et a l . , 1977b), ethidium ( J a i n et a l . , 1977), aminoacridine (Sakore et a l . , 1977 and 1979; Reddy et a l . , 1979), e l l i p t i c i n e , tetramethyl-N-methylphenanthrolinium ( J a i n et a l . , 1979) irehdiamine (Mahler et a l . , 1968; S o b e l l et a l . , 1977a), psoralen (Cole, 1971; Lown and Sim, 1978; Ley et a l . , 1977; Wiesehahn and Hearst, 1978) and bleomycin (Umezawa et a l . , 1973; M u l l e r et a l . , 1972; Bearden et a l . , 1977) Besides the s t e r o i d a l diamine, irehdiamine, these i n t e r -c a l a t i n g agents are a l l planar h e t e r o c y c l i c i n s t r u c t u r e . A current concept of S o b e l l (1979) and others consider i n t e r -c a l a t i o n to r e s u l t from the dynamic nature of DNA conformational changes. According to t h i s view, the DNA molecule undergoes wave-like propagation i n i t s polymeric s t r u c t u r e , that i s e x c i t e d through impulses generated at random by the continuous bombardment of DNA by solvent mole-cules along i t s length. The wave propagation may i n v o l v e the motions of bending, s t r e t c h i n g , shearing and unwinding, and r e s u l t s i n s t r u c t u r a l d i s t o r t i o n of DNA. At some c r i t i c a l o s c i l l a t i o n amplitude, a l t e r n a t e sugars "snap i n t o " a C^' endo sugar conformation with a concomitant p a r t i a l unstacking of base p a i r s . Such s t r u c t u r e i s denoted B-kinked DNA (Figure 10) and i s favorable f o r drug i n t e r c a l a t i o n (Figure 11). A s i m i -38. Single-strand breaks Denaturing conditions Alkali-labile sites Alkali Double-strand, breaks Spontaneously Interstrand cross-link Denaturing conditions DNA-protein %ZB> cross-1 ink , . Figure 9 : Drug-induced macromolecular damage of nuclear DNA (Kohn, 1979) (A) B DNA drawn by computer graphics (B) (C) 3-kinked DNA, drawn by computer (D) graphics B DNA, Corey-Pauling-Koltun (CPK) space f i l l i n g model B-kinked DNA, CPK space f i l l i n g model Figure 10 : DNA undergoing wave-like d i s t o r t i o n i n i t s s t r u c t u r e ( S o b e l l , 1979) 40. ure 11 : Schematic i l l u s t r a t i o n of dynamic concepts of DNA s t r u c t u r e i n v o l v e d i n DNA breathing and i n drug i n t e r c a l a t i o n ( S o b e l l , 1979) l a r B-kinked conformation i s proposed as a key intermediate i n DNA un-winding f o r RNA s y n t h e s i s i n v i v o . X. G l u c o c o r t i c o i d s as p o t e n t i a l i n t e r c a l a t o r s G l u c o c o r t i c o i d s and other A -3-one A - r i n g c o n t a i n i n g s t e r o i d s , such as progesterone and deoxycorticosterone, have been proposed by Duax et a l . , (1976) as p o t e n t i a l n u c l e i c a c i d i n t e r c a l a t o r s , based on t h e i r s t u d i e s on the s t r u c t u r a l X-ray a n a l y s i s of deoxycorticosterone (DOC)-adenine c r y s t a l l i n e complex. The conformation of A -3-one A- r i n g was found between the 1a-sofa and 1a, 2B-half c h a i r forms (Fi g u r e 12) from c r y s t a l l o g r a p h i c a n a l y s i s (Duax and Norton, 1975). In the optimized molecules, the r i n g was c o n s i s t e n t l y c l o s e r to the 1a, 2B-half c h a i r conformation (Schmit and Rousseau, 1978; Duax et a l . , 1976), and was very planar (Duax et a l . , 1976). In the c r y s t a l s t r u c t u r e of DOC-adenine complex, two kinds o f i n t e r a c t i o n s , hydrogen bonds (Figure 13) and steroid-base s t a c k i n g (Figure 14), have been observed. Weeks et a l . (1975) suggested t h a t although the nature of the i n t e r a c t i o n s between the hormone-receptor com-plex and the DNA molecule i s unclear, the mere f a c t that a s t a b l e DOC-adenine c r y s t a l l i n e complex can be formed i s i n t e r e s t i n g and p o s s i b l y s i g n i f i c a n t . They a l s o considered the hydrogen bond p a i r i n g between the c o r t i c o i d side chain and the adenine u n l i k e l y to be a b i o l o g i c a l l y s i g n i -f i c a n t i n i t i a l r e a c t i o n between a c o r t i c o s t e r o i d and DNA since the ade-nine atoms involved i n the hydrogen bonds normally p a r t i c i p a t e i n base p a i r i n g i n t e r a c t i o n s . However, t h i s c o r t i c o i d - b a s e p a i r i n g could s t a b i -l i z e a DNA conformation i n which a break had already been made. On the other hand, the f a c t that adenine stacks over the unsaturated DOC A - r i n g , i n preference to forming stacks of adenine molecules alone, i n d i c a t e s 42. Figure 12 : Conformations of A - 3 - one A r i n g (Schmit and Rousseau, 1979) A3. i i • • (Weeks et a l . , 1975) (Duax et a l . , 1976) Figure 13 : Hydrogen bonding i n the DOC-adenine complex viewed perpendicular to the adenine plane 44. Figure 14 : Stacking of adenine and the A - 3 - one region of DOC that i t may not be unreasonable to regard the A H-3-one A-rings of pro-gesterone and the c o r t i c o i d s , i n c l u d i n g g l u c o c o r t i c o i d s , as p o t e n t i a l i n t e r c a l a t o r s . There i s no evidence that d i r e c t i n t e r a c t i o n of DNA with gluco-c o r t i c o i d s precedes the appearance of any g l u c o c o r t i c o i d - i n d u c e d e f f e c t . The presence of a g l u c o c o r t i c o i d - r e c e p t o r complex i n n u c l e i i s needed to e l i c i t the response. Many i n v e s t i g a t o r s have reported i n t e r a c t i o n s o f g l u c o c o r t i c o i d - r e c e p t o r complexes with DNA i n c e l l - f r e e systems: S t e r o i d s have been shown to bind to p u r i f i e d n a t i v e and denatured DNA (Cohen and Kidson, 1970; Arya and Yang, 1975) and to pr o t e c t the DNA secondary s t r u c t u r e from thermal denaturation (Arya and Yang, 1975). S i m i l a r ob-se r v a t i o n s were reported by Milgrom et a l . (1976), Atger and Milgrom (1978) and Kidson et a l . (1970). G l u c o c o r t i c o i d - r e c e p t o r complexes were found to i n t e r a c t with chromatin (O'Malley et a l . , 1977; O'Malley and B u l l e r , 1976; King and Gordon, 1972; Baxter et a l . , 1972; Cake et a l . , 1978; Duncan and Duncan, 1973; K a l i m i et a l . , 1975). Duax et a l . (1976) proposed t h a t , at the time of i n t e r a c t i o n o f the s t e r o i d - r e c e p t o r complex with the DNA, the s t e r o i d may be at the i n t e r f a c e between the p r o t e i n and the DNA. The p r o t e i n may i n i t i a t e un-coupling of DNA and the s t e r o i d i n t e r a c t i o n s t a b i l i z e i t , or v i c e versa (Duax et a l . , 1976). Moreover, O'Malley has r e c e n t l y demonstrated t h a t i n the case of progesterone, the receptor i s a dimer binding two s t e r -o i d s , one of which i n t e r a c t s with non-histone chromosomal p r o t e i n and the other with n u c l e o t i d e residue of DNA (Schrader et a l . , 1975). XI. Nuclear r e s i d u a l forms of g l u c o c o r t i c o i d - r e c e p t o r complexes Studies on s u b c e l l u l a r d i s t r i b u t i o n of H-TA i n mouse L-929 f i b r o b l a s t s have revealed a f r a c t i o n o f bound g l u c o c o r t i c o i d i n the nuclear p e l l e t that i s r e s i s t a n t to e x t r a c t i o n with 0.3 M KC1 (Middle-brook et a l . , 1975). This f r a c t i o n , designated as "nuclear r e s i d u a l form" of g l u c o c o r t i c o i d - r e c e p t o r complex, was b e l i e v e d to be t i g h t l y bound to chromatin. A s i m i l a r r e s i d u a l nuclear binding has a l s o been described i n dexamethasone-treated HTC c e l l s (Higgins et a l . , 1973c). As shown i n Figure 15, not more than 80$ of dexamethasone-receptor complexes bound to HTC n u c l e i could be e x t r a c t e d by up to 0.5 M NaCl i n i n t a c t c e l l s or c e l l - f r e e experiments. The existence of such "nuclear r e s i d u a l forms" would imply strong g l u c o c o r t i c o i d b i n d i n g to the chromatin, per-haps to the DNA, which might be preserved during i s o l a t i o n procedures of DNA. Therefore, i t was considered worthwhile to t e s t the hypothesis that , a f t e r the nuclear binding of g l u c o c o r t i c o i d - r e c e p t o r complex to chromatin, the g l u c o c o r t i c o i d molecule might become- i n t e r c a l a t e d i n t o the DNA h e l i x at some poi n t of time during the metabolic c y c l e of the c e l l as proposed but not f u r t h e r studied by Duax et a l . (1976) f o r DOC-adenine complexes. Such i n t e r c a l a t e d g l u c o c o r t i c o i d s would be t i g h t l y bound to chromatin, and might be r e s p o n s i b l e f o r the nuclear r e s i d u a l b i n d i n g of g l u c o c o r t i c o i d s . When the c u l t u r e d c e l l s have been incubated w i t h g l u c o -c o r t i c o i d f o r a prolonged period of time, i t i s p o s s i b l e , at c e r t a i n stages of the c e l l c y c l e , to have the required c r i t i c a l B-kinked confor-mation of DNA s t r u c t u r e , f a v o r a b l e f o r i n t e r c a l a t i o n to occur. The psoralens, l i k e a l a r g e number of other drugs that i n t e r a c t s t r o n g l y with n u c l e i c a c i d s , are b e l i e v e d to i n t e r c a l a t e between base p a i r s (Wiesehahn and Hearst, 1978; Cole, 1971). Subsequent exposure to long wave-length u l t r a v i o l e t r a d i a t i o n (320-400 nm) leads to the c r o s s -l i n k i n g between psoralen double bonds and pyrimidines on opposite strands of a DNA double h e l i x (Cole, 1971; Dall'Acqua et a l . , 1972). This s t a b i -NaCl, M Release of receptor- JH-dexamethasone complexes (RD) from HTC c e l l s n u c l e i by NaCl. (Higgins et a l . , 1973c) 3 Washed HTC c e l l n u c l e i c o n t a i n i n g H-RD complexes bound i n i n t a c t c e l l ( • ) or i n a c e l l - f r e e system ( O ) were exposed to i n c r e a s i n g concentrations of NaCl. A f t e r 30 min. at 0°C, the n u c l e i were washed and assayed f o r r e t a i n e d complexes l i z e s the DNA h e l i x and can be detected by HAP chromatography and de-n a t u r a t i o n - r e n a t u r a t i o n a n a l y s i s . The c r o s s - l i n k i n g of DNA induced by psoralens w i t h UV i r r a d i a t i o n has been e s t a b l i s h e d w i t h p u r i f i e d DNA (Pathak et a l . , 1974; Pathak et a l . , 1977), i n human and mouse t i s s u e c u l t u r e c e l l s (Cech and Pardue, 1976; Wiesehahn et a l . , 1977) and i n v i v o i n mammalian s k i n (Pathak and Kramer, 1969; Dall'Acqua et a l . , 1972). Psoralen plus long-wavelength UV i r r a d i a t i o n was t h e r e f o r e s e l e c t e d as a model of i n t e r c a l a t i o n f o r t e s t i n g whether the presence of i n t e r c a l a t i o n of g l u c o c o r t i c o i d s i n t o DNA of c u l t u r e d mouse L-929 and human dermal f i b r o b l a s t s can be demonstrated. Drugs b e l i e v e d to i n t e r c a -l a t e DNA in c l u d e the trypanocide (ethidium bromide), the antitumor a n t i -b i o t i c s (daunomycin and adriamycin), the a n t i m a l a r i a l drug ( c h l o r o q u i n e ) , the carcinogen (3,4-benzpyrene) and the r a d i c a l i o n of the t r a n q u i l l i z e r (chlorpromazine) (Waring, 1968). Each of these drugs binds to DNA by i n t e r c a l a t i o n , but r e s u l t i n d i v e r s e b i o l o g i c a l m a n i f e s t a t i o n s . I n t e r c a -l a t i o n i s , t h e r e f o r e , a process which provides a s t r i k i n g l y v e r s a t i l e molecular b a s i s f o r drug a c t i o n (Waring, 1970). V a r i a t i o n s i n DNA l e s i o n s induced by i n t e r c a l a t o r s are demonstrated i n Table IV. Among these agents, psoralen e l i c i t s e f f e c t s on DNA s i m i l a r to those produced by g l u c o c o r t i c o i d s . Both are a n t i - p s o r i a t i c agents, and both have a n t i -p r o l i f e r a t i v e e f f e c t on dermal c e l l s . The major DNA l e s i o n induced by psoralen plus UV i s i n t e r s t r a n d c r o s s - l i n k i n g thought to prevent the strand separation which must occur f o r normal DNA r e p l i c a t i o n and t r a n s -c r i p t i o n . X I I . Techniques f o r d e t e c t i n g c r o s s - l i n k s A number of physicochemical p r o p e r t i e s of, c h a r a c t e r i s t i c o f i n t e r c a l a t i o n , DNA have been reported. They i n c l u d e : Table IV DNA lesions produced i n mammalian c e l l s by various agents Agent SSB Direct Enzymic Base DSB ISC DPC damage X-rays UV l i g h t (254 nm) Bleomycin Nitrogen mustards and mitomycin Chloroethylnitroso-ureas (BCNU, CCNU, chlorozotocin) Cis-Pt (II) Trans-Pt (II) Adriamycin and other i n t e r c a l a t o r s Psoralen plus l i g h t + + + + SSB, single-strand breaks; DSB, double-strand breaks; ISC, interstrand c r o s s - l i n k s ; DPC, DNA-protein c r o s s - l i n k s . (Kohn, 1979) (a) increased thermal s t a b i l i t y as demonstrated i n aminoacri-dine-DNA complex (Gersch and Jordan, 1965) and i n a n t h r a c y c l i n e a n t i b i o -t i c s , daunomycin and nogalamycin, i n t e r c a l a t e d DNA (Kersten and Kersten, 1968). (b) increased r a t e of e l u t i o n o f DNA by a l k a l i n e f i l t e r method (Kohn, 1978 and 1979) with i n t e r c a l a t i n g agents causing strand breaks and decreased e l u t i o n r a t e with those e x e r t i n g c r o s s - l i n k s . (c) increased v i s c o s i t y and decreased sedimentation c o e f f i c i e n t as observed with a c r i d i n e or with ethidium i n t e r c a l a t e d i n closed c i r c u -l a r I x 174 RF DNA (Kersten et a l . , 1966; O'Brien et a l . , 1966; M u l l e r and Crothers, 1968; Waring, 1970), and (d) s h i f t of v i s i b l e spectrum as reported i n systems of amino-a c r i d i n e s with DNA (Bradley and Wolf, 1959; Peacocke and S k e r r e t t , 1956). As i l l u s t r a t e d i n Figure 9, c r o s s - l i n k e d DNA behaves d i f f e r e n t l y upon thermal denaturation from re g u l a r DNA. Regular DNA responds to thermal denaturation with complete strand separation which r e s u l t s i n a kinky s u p e r c o i l e d s i n g l e - s t r a n d e d form, when the denatured sample i s cooled. On the other hand, the c r o s s - l i n k e d DNA demonstrates p a r t i a l strand separation, but the separated strands are r e a d i l y reannealed when co n d i t i o n s of h e l i x s t a b i l i t y are re s t o r e d on c o o l i n g . Renaturation occurs because the p a r t i a l l y separated strands are s t i l l held i n the v i c i n i t y to each by the i n t e r c a l a t i n g molecule. I t has been reported by Geiduschek (1961) that a s i n g l e c r o s s - l i n k i n an unnicked, double-strand-ed DNA molecule i s s u f f i c i e n t to hold the complementary strands i n r e g i s -t e r during denaturation and thus le a d i n g to r a p i d r e n a t u r a t i o n . (A) Hydroxyapatlte chromatography Hydroxyapatite (HAP), C a 1 Q ( P 0 l ( ) 6 ( 0 H ) 2 , i s a complex o f Ca(0H) 2 3 C a 3 ( P 0 M ) 2 , I t o r i g i n a l l y developed by T i s e -l i u s , Hjerten and Levin (1956) f o r p r o t e i n chromatography, and was f i r s t used with n u c l e i c acids by Semenza (1957) working i n T i s e l i u s ' l a b o r a t o r y and by Main and Cole (1957). Hydroxyapatite chromatography has been widely used i n p r e p a r a t i v e biochemistry, and has proven i t s e l f a v e r s a -t i l e technique f o r the f r a c t i o n a t i o n and p u r i f i c a t i o n of p r o t e i n s ( H j e r -ten, 1959; T i s e l i u s et a l . , 1956), enzymes ( L i n d s t a d t et a l . , 1970), n u c l e i c a c i d s (Meinke et a l . , 1974; Coleman et a l . , 1978), v i r u s e s (Smith and Lee, 1978) and other macromolecules (Garola and McGuire, 1977; T r a f -ford et a l . , 1975; P a v l i k and Coulson, 1976; Peck and C l a r k , 1977). The b a s i s of r a p i d i s o l a t i o n o f p u r i f i e d DNA from c e l l l y s a t e by hydroxyapatite chromatography l i e s i n the d i f f e r e n t i a l a f f i n i t i e s of DNA, RNA, p r o t e i n s , carbohydrates and various low-molecular-weight substances (Bernardi, 1971a). In p r i n c i p l e , the high a f f i n i t y of double-stranded DNA to hydroxyapatite permits the i s o l a t i o n of DNA, f r e e of contaminants which are l e s s t e n a c i o u s l y bound, simply by l o a d i n g the t i s s u e l y s a t e onto hydroxyapatite column and then e l u t i n g with phosphate b u f f e r s of appropriate concentrations (Markov and Ivanov, 1974). The main f a c t o r i n v o l v e d i n the adsorption of n u c l e i c acids on hydroxyapatite column seems to be the i n t e r a c t i o n between the phosphate groups of n u c l e i c a c i d s and calcium ions on the surface of hydroxyapatite c r y s t a l s ( B e r n a r d i , 1971a). No s i g n i f i c a n t changes i n the p h y s i c a l , chemical and b i o l o g i c a l p r o p e r t i e s of n a t i v e DNA take place upon the a d s o r p t i o n - e l u t i o n process ( B e r n a r d i , 1969a). Native DNA i s eluted at 0.20-0.25 M potassium phos-phate (KP). The denatured DNA and RNA are e l u t e d at potassium phosphate m o l a r i t i e s which are d i s t i n c t l y lower (0.10-0.15 M) ( P a r i s h , 1972). O l i -gonucleotides are e l u t e d at even lower m o l a r i t i e s (0.001-0.015 M), and most p r o t e i n s are adsorbed much l e s s s t r o n g l y than n a t i v e DNA. Polysac-charides and small organic molecules are adsorbed very weakly, i f at a l l ( B e r n a r d i , 1969a) . Thus, n a t i v e and denatured DNA can be e a s i l y separat-ed by e i t h e r stepwise or gradient e l u t i o n . The reduction i n the e l u t i o n m o l a r i t y required which occurs when r i g i d double-stranded molecules are denatured i s a s p e c i a l case i n the ge n e r a l l y observed tendency of more f l e x i b l e molecules to have lower e l u -t i o n m o l a r i t i e s than more r i g i d ones (B e r n a r d i , 1971a; Martinson, 1973a) . I t has been proposed that decreased a f f i n i t y of the more f l e x i -b l e denatured DNA molecule r e s u l t s from an unfavorable c o n f i g u r a t i o n a l entropy change imposed on f l e x i b l e molecules (Martinson, 1973a) . The X-ray d i f f r a c t i o n a n a l y s i s of secondary s t r u c t u r e s of both n a t i v e and denatured DNA i n d i c a t e d that the a f f i n i t y of a r i g i d , h e l i c a l n u c l e i c acid f o r hydroxyapatite i s governed by the s t e r i c a v a i l a b i l i t y of i t s backbone phosphates f o r adsorptive i n t e r a c t i o n s (Martinson, 1973b) . The method of the f r a c t i o n a t i o n of chromosomal p r o t e i n s by the hydroxyapatite d i s s o c i a t i o n technique was developed by Bloom and Anderson (1978) , based on the a b i l i t y of hydroxyapatite to bind n a t i v e chromatin i n s o l u t i o n s which do not d i s s o c i a t e chromosomal p r o t e i n s from the DNA. The p r o t e i n s were then s e l e c t i v e l y d i s s o c i a t e d from the immobilized chro-matin by treatment with NaCl, or with NaCl p l u s urea. The hydroxyapatite d i s s o c i a t i o n method represented a ra p i d one-step f r a c t i o n a t i o n procedure which r e s u l t e d i n the q u a n t i t a t i v e recovery of chromosomal p r o t e i n s de-void of n u c l e i c acids (Bloom and Anderson, 1978 ) . The patterns of d i s s o c i a t i o n of both histone and non-histone chromosomal p r o t e i n s by NaCl and urea, on the b a s i s of the d i f f e r e n t i a l a f f i n i t i e s of chromosomal p r o t e i n s to DNA, were reported by Bloom and Anderson (1978) as presented i n Table V. (B) Thermal scanning a n a l y s i s Denaturation of DNA i s defined as the t r a n s i t i o n of d o u b l e - h e l i -Table V Diss o c i a t i o n pattern of histone and nonhistone chromosomal proteins by NaCl and urea from hydroxyapatite column Fract ion Eluent* Components of eluate NaP(mM) NaCl(M) Urea(M) A 10 - - Unbound chromosomal proteins B 80 - - Unbound chromosomal proteins C 80 0.25 - Loosely bound nonhistone proteins(NHP) D 80 0.5 - Very l y s i n e - r i c h histone protein, E 80 0.75 - Very l y s i n e - r i c h histone protein, H^ F 80 1.25 - Moderately l y s i n e - r i c h histone proteins, H 2A & H 2B G 80 2 - A r g i n i n e - r i c h histone proteins, H„ & H. 3 A H 80 2 2 Tight l y bound nonhistone proteins(NHP) I 80 2 A Tigh t l y bound nonhistone proteins(NHP) J 80 2 8 Tig h t l y bound nonhistone proteins(NHP) K 500 2 5 Nucleic acid (NA) A l l eluents contained 5 mM NaHSO^ to reduce p r o t e o l y t i c a c t i v i t y (Panyim et a l . , 1968). c a l form of DNA i n t o disordered random c o i l s by d i s r u p t i o n of hydrogen bonds between base p a i r s and of hydrophobic i n t e r a c t i o n s between neigh-bouring stacked bases (Lehninger, 1975) . Denaturation of DNA can occur at extreme pH or at elevated temperature. In the l a t t e r case, i t i s c a l l e d thermal denaturation. The b a s i c mechanism of denaturation i s de-p i c t e d i n Figure 16 . Based upon both theory and experiment, h e l i x - c o i l t r a n s i t i o n i n DNA c o n s i s t s o f the f o l l o w i n g key elements: (a) A n u c l e a t i o n step, which i s the denaturation of the f i r s t base p a i r i n a h e l i c a l segment. In t h i s step, both hydrogen bonds i n the base p a i r s on e i t h e r side of the denatured base p a i r are destroyed.' (b) A propagation step, which i s the breaking of the hydrogen bonds i n a base p a i r next to a denatured ( c o i l e d ) segment. Only one s t a c k i n g i n t e r a c t i o n with the adjacent base p a i r i n the h e l i c a l segment has to be disrupted i n t h i s step; because of t h i s d i f f e r e n c e i n s t a c k i n g i n t e r a c t i o n s , there i s a tendency f o r a c o i l e d segment to grow in s t e a d of generating more short c o i l e d segment which i s the basis f o r cooperative t r a n s i t i o n when a DNA i s denatured. (c) A strand separation step, which i n v o l v e s a p h y s i c a l separa-t i o n o f both complementary strands of DNA. (d) Since a G C p a i r i s thermodynamically more s t a b l e than an A T p a i r , more f r e e energy i s needed to denature a G C p a i r than an A T p a i r (Marmur and Doty, 1962; Crothers et a l . , 1965; Crothers, 1968; Tino-co et a l . , 1973). (e) E l e c t r o s t a t i c r e p u l s i o n between phosphates on the negative-l y charged phosphate l a t t i c e o f DNA tends to d e s t a b i l i z e the h e l i c a l s t r u c t u r e ; charge n e u t r a l i z a t o n on phosphates by c a t i o n s s t a b i l i z e s g r e a t l y the double h e l i c a l DNA (Marmur and Doty, 1962; Dove and Davidson, Double-helical DNA heat Partially unwound DNA Separated strands of DNA in random coi Is Figure 16 : Basic a c t i o n of DNA wi t h i n c r e a s i n g temperature. The complimentary strands of DNA unwind or denature upon heating. The denaturation or strand separation i s followed s p e c t r o p h o t o m e t r i c a l l y by an increase i n absorbance at 260 nm 1962; S c h i l d k r a u t and L i f s o n , 1965; Record, 1967; Gruenwedel and Hsu, 1969; Manning, 1972). Such h e l i x - c o i l t r a n s i t i o n i n DNA has been discussed i n many a r t i c l e s and t r e a t e d very e x t e n s i v e l y i n the books of Poland and Sheraga (1970) and of B l o o m f i e l d , Crothers and Tinoco (1974). Denaturation of a d o u b l e - h e l i c a l DNA i s accompanied by an i n -crease i n absorbance. This phenomenon i s c a l l e d hyperchromism and i s a t t r i b u t e d to the d e s t r u c t i o n of s t a c k i n g i n t e r a c t i o n among n u c l e o t i d e s i n DNA (Tinoco, 1960; Tinoco, 1961; DeVoe, 1965; F e l s e n f e l d and H i r s c h -man, 1965). The s p e c i f i c e f f e c t s of elevated temperatures on the DNA absorption spectra are shown i n Figure 17. The absorption spectrum of a DNA sample was taken at various temperatures and superimposed f o r com-parison. As the temperature changes g r a d u a l l y from 60° to 80°, there i s a sharp increase i n absorption centered around 260 nm. Further e l e v a -t i o n of temperature shows a subsequent increase to a maximum. I t was observed that denaturation of DNA occurred i n a narrow temperature range (Marmur and Doty, 1962). The temperature at which 50% of n a t i v e DNA i s denatured i s defined as the melting temperature, T , of the DNA. Both T and hyperchromicity are parameters d e f i n i n g the m thermal denaturation of a given DNA sample. Thermal scanning technique i n v o l v e s the e l e v a t i o n of temperature up to beyond the melting range with small increments, not greater than 1° per min, and the monitoring of absorbance at 260 nm, ^260' ^ e denaturation p r o f i l e i s constructed by p l o t t i n g the r e l a t i v e absorbance against temperature. The r e n a t u r a t i o n p r o f i l e i s s i m i l a r l y obtained by r e v e r s i n g the temperature. This technique i s of use i n c h a r a c t e r i z i n g a DNA d e n a t u r a t i o n - r e n a t u r a t i o n p r o f i l e which i s s e n s i t i v e to i n t e r a c t i o n s w i t h i n DNA h e l i x and i n t e r a c t i o n between p r o t e i n and DNA. Therefore, 57. -220 240 260 280 300 320 Wavelength, nm Figure 17 : Hyperchromia e f f e c t of DNA upon he a t i n g . Note that absorption increases are centered around 260 nm as the temperature increases from 60° to 80^. This i n c r e a s e (hyperchromicity) stems from thermal denaturation of the double strands of the DNA molecule. thermal scanning a n a l y s i s i s of p o t e n t i a l a p p l i c a t i o n i n r e v e a l i n g the p o s s i b l e i n t e r a c t i o n s between complex and DNA or between g l u c o c o r t i c o i d and DNA. Such a p p l i c a t i o n s have been demonstrated by Cech et a l . (1979) and L i (1977). X I I I . Regulation of gene expression The molecular mechanism by which the receptor-genome i n t e r a c t i o n regulates genetic expression i n target c e l l s has been e s p e c i a l l y s c r u t i -nized over the l a s t decade. However, at the present time, even the best data s t i l l do not reve a l how the s t e r o i d - r e c e p t o r complex i s i n v o l v e d i n t h i s r e g u l a t i o n ( G o r s k i and Gannon, 1976). In the past few years, almost every p o s s i b l e c o n t r o l mechanism inv o l v e d i n gene expression has been advocated, i n c l u d i n g t r a n s c r i p t i o n a l c o n t r o l p o s t t r a n s c r i p t i o n a l c o n t r o l , t r a n s l a t i o n a l c o n t r o l and changes i n enzyme and mRNA s t a b i l i t i e s ( F e i g e l -son et a l . , 1978), but Gorski and Gannon (1976) commented that many of the proposed c o r r e l a t i o n s between s t e r o i d - r e c e p t o r nuclear binding and gene expression appeared i n v a l i d when c l o s e l y examined, and confirmed c o r r e l a t i o n s f r e q u e n t l y lack general a p p l i c a b i l i t y . Moreover, as pointed out by Stormshak et a l . (1978), the ma j o r i t y of research e f f o r t s have neglected to study i n d e t a i l the s t e r o i d - r e c e p t o r r e g u l a t i o n of DNA r e -p l i c a t i o n . I t i s recognized that nuclear binding of g l u c o c o r t i c o i d s can be only one of the t r i g g e r i n g steps i n the complex sequence of the r e a c t i o n , l e a d i n g through t r a n s c r i p t i o n a l , and perhaps other DNA-dependent func-t i o n s , to the great d i v e r s i t y of g l u c o c o r t i c o i d t i s s u e - s p e c i f i c respon-ses. Such t i s s u e - s p e c i f i c g l u c o c o r t i c o i d a c t i o n s range, f o r example, from i n d u c t i o n of enzymes, as t y r o s i n e aminotransferase i n hepatoma t i s -sue c u l t u r e (Rousseau et a l . , 1972a) and proteinaceous hormones, as growth hormone i n c u l t u r e d p i t u i t a r y c e l l s (Kohler et a l . , 1968), i n h i b i -t i o n of DNA s y n t h e s i s by unknown mechanisms, as i n human epidermal c e l l s (Voorhees, 1977b) and i n mouse L-929 f i b r o b l a s t s ( P r a t t and Aronow, 1966) to s t i m u l a t i o n of DNA synthesis i n various f i b r o b l a s t c u l t u r e s ( R u n i k i s et a l . , 1978; K i r k and Mittwoch, 1977; Thrash et a l . , 1974), and c y t o l y -s i s of s t e r o i d - s e n s i t i v e lymphocytes (Baxter et a l . , 1971). However, these s p e c i f i c but divergent c e l l u l a r responses o f v a r i o u s target c e l l s are thought to o r i g i n a t e at the l e v e l of the nucleus through m o d i f i c a t i o n of gene expression. What remains unclear i s what molecules are involved besides the g l u c o c o r t i c o i d - r e c e p t o r complex and whether the r e a c t i o n s i n v o l v e DNA d i r e c t l y or i n d i r e c t l y (Higgins et a l . , 1979). A number of models f o r r e a c t i o n s i n v o l v i n g gene expression have been suggested. Thompson et a l . (1977) described a model of negative or p o s i t i v e c o n t r o l s as depicted i n Figure 18. The repressor model of nega-t i v e c o n t r o l supposes that the s t e r o i d - r e c e p t o r complex i n h e r e n t l y pos-sesses a high a f f i n i t y for appropriate gene s i t e s . An example of such negative c o n t r o l might be a h i s t o n e or other p r o t e i n , which block genes that would otherwise have high a f f i n i t y f o r the s t e r o i d - r e c e p t o r complex. The adapter model of p o s i t i v e c o n t r o l supposes that some mole-c u l e intervenes between the hormone-receptor complex and the genes i t a f f e c t s . T h i s molecule would bind to the complex and thereby confer upon i t c i s t r o n s p e c i f i c i t y . An array of such c o n t r o l molecules would be r e -quired to provide s p e c i f i c l i nkage to the v a r i e t y of s p e c i f i c genes s t e r o i d s a f f e c t . The question of how d i f f e r e n t i a t e d c e l l s determine which c o n t r o l molecules to produce i s not addressed by these models. A number of experiments have been c a r r i e d out t r y i n g to i d e n t i f y such mole-cul e s , and candidates have been found i n both the h i s t o n e and non-histone Gene 2 SR Gene 1 t SR SR Repressor model B SR fa •i Gene 1 Adapter model * Gene 2 Figure 18 : Models f o r two types of p o s s i b l e i n t r a n u c l e a r c o n t r o l over s t e r o i d - r e c e p t o r complex i n t e r a c t i o n s w i t h DNA (A) A pure n e g a t i v e - c o n t r o l system, w i t h s p e c i f i c a l l y designed p r o t e i n s b l o c k i n g access of a c t i v a t e d complex (SR*) by binding to one gene, w h i l e l e a v i n g open another f o r i n t e r -a c t i o n w i t h SR*. (B) A pure p o s i t i v e - c o n t r o l model, i n which a c t i v a t e d s t e r o i d receptor (SR*) i n the nucleus binds to a s p e c i f i c s i t e on an intermediary acceptor molecule, which i n turn s p e c i f i e s , by v i r t u e of i t s second, g e n e - s p e c i f i c s i t e , the gene to which the ternary complex w i l l b ind. f r a c t i o n s of the nuclear p r o t e i n s (O'Malley et a l . , 1972; Spelsburg et a l . , 1972; Puca et a l . , 1974). Of course, small molecules could be p a r t of the c o n t r o l s operative i n e i t h e r model (Cake et a l . , 1976). This p o s i t i v e c o n t r o l model p r e d i c t s that c e l l s d i f f e r e n t i a t e by l o s i n g or f a i l i n g to make the intermediary adapter molecules. In a c e l l w ith f u n c t i o n a l s t e r o i d receptors, the choice of s p e c i f i c a l l y induced products, whether few or many, would depend e n t i r e l y on the presence of the p o s i t i v e adapters. Overlapping models are p o s s i b l e . Johnson et a l . (1979a) proposed " s t o i c h i o m e t r i c " or " c a t a l y t i c " models. The s t o i c h i o m e t r i c model assumes that r e c e p t o r - s t e r o i d complexes bind s p e c i f i c a l l y at c e r t a i n s i t e s on chromatin to regulate t r a n s c r i p t i o n of s p e c i f i c mRNAs. The s i t e - s p e c i f i c l o c a l i z a t i o n of the g l u c o c o r t i c o i d -receptor complex would be due e i t h e r to s p e c i f i c sequences on DNA that bind the complexes, or to chromatin p r o t e i n s l o c a t e d at s p e c i f i c s i t e s that bind or i n f l u e n c e the binding of complexes. The c a t a l y t i c model assumes that the complex e i t h e r a c t i v a t e s an enzyme, or i n some other ways modify molecules important f o r gene expression. Yamamoto and A l b e r t s (1976) hypothesized that s p e c i f i c hormone e f f e c t s required c l u s -tered receptor complex bi n d i n g , s i n c e only t h i s l e d , i n t h e i r view, to formation o f a l a r g e enough patch to in c l u d e both a s p e c i f i c promotor and i t s s t r u c t u r a l gene. The s p e c i f i c i t y of the g l u c o c o r t i c o i d responses may e x i s t as c o n t r o l s on the a c c e s s i b i l i t y of s p e c i f i c genes f o r binding, by v i r t u e o f c e l l d i f f e r e n t i a t i o n , as proposed i n repressor and adaptor models. Or, i t may.be at l e v e l s d i s t a l to the receptor-acceptor i n t e r a c t i o n that only the t r i g g e r o f modified gene expression i s modulated as suggested i n s t o i c h i o m e t r i c and c a t a l y t i c models. None o f the models as yet, have been c o n v i n c i n g l y defined i n biochemical terms. However, i t i s g e n e r a l l y agreed by Thompson et a l . (1977) and Johnson et a l . (1979a) that b i n d i n g of r e c e p t o r - s t e r o i d complex to most of acceptor s i t e s does not e l i c i t b i o l o g i c a l l y r e levant responses. Only a few of these acceptors, w i t h a f f i n i t y same as or higher than most of the detectable acceptors, are lo c a t e d at s i t e s where the expression o f s p e c i f i c genes can be modified. XIV. Dichotomy and time p r o f i l e s of g l u c o c o r t i c o i d e f f e c t s The b i o l o g i c a l responses to g l u c o c o r t i c o i d s may d i f f e r between or w i t h i n animal species. For example, lymphocytes from rodents are lysed by hydrocortisone, while hydrocortisone reduces the number of human lymphocytes by inducing t h e i r migration from c i r c u l a t i o n . Human and mouse c e l l s respond d i f f e r e n t l y to g l u c o c o r t i c o i d s i n c u l t u r e ( R u n i k i s et a l . , 1978). P r o l i f e r a t i o n of L-929 c e l l s during l o g growth i s i n h i b i t e d by g l u c o c o r t i c o i d s ( P r a t t and Aronow, 1966; B e r l i n e r and Ruhmann, 1967) but human s k i n f i b r o b l a s t s have increased growth rate under the same con-d i t i o n s ( R u n i k i s et a l . , 1978; K i r k and Mittwoch, 1977). Moreover, the e f f e c t of g l u c o c o r t i c o i d on col l a g e n s y n t h e s i s i s often reported as i n h i -b i t o r y , yet the s t i m u l a t i o n e f f e c t has a l s o been claimed (Harvey et a l . , 1974; Doherty and S a a r n i , 1976). These discrepant observations have no reasonable explanation, but underline the importance of r e p o r t i n g c e l l types used f o r experiments. In a d d i t i o n , even w i t h i n the same c e l l l i n e , dichotomic e f f e c t i s observed. Stormshak reported that chronic i n j e c t i o n s of estrogens i n t o ovariectomized mice i n i t i a l l y s t i m u l a t e d , then depressed u t e r i n e c e l l d i v i s i o n , DNA s y n t h e s i s , and DNA polymerase a c t i v i t y . I t c l e a r l y demonstrated a dose- and time-dependent r e f r a c t o r i n e s s (Stormshak et a l . , 1978). A time-dependence of s t e r o i d a c t i o n a l s o i s demonstrated i n another s i m i l a r study (Glasser et a l . , 1972). I n j e c t i o n of e s t r a d i o l i n t o the immature r a t stimulated an increase i n u t e r i n e DNA s y n t h e s i s by 18 hr a f t e r treatment. Maximal s y n t h e s i s of DNA occurred by 24 hr then subsided to c o n t r o l l e v e l by 36 hr. The e f f e c t of e s t r a d i o l on DNA p o l y -merase a c t i v i t i e s followed a s i m i l a r course. Therefore, i t i s of p a r t i c u l a r i n t e r e s t to e l u c i d a t e the time p r o f i l e of g l u c o c o r t i c o i d - i n d u c e d biochemical events which occur during prolonged periods of exposure to g l u c o c o r t i c o i d . The time-dependence of s t e r o i d a c t i o n may be another important f a c t o r other than c e l l - t y p e and concentration-dependences i n r a t i o n a l i z i n g the dichotomy of g l u c o c o r t i -coid e f f e c t s . A recent study of the e f f e c t of time and concentration on the ac t i o n of hydrocortisone on f i b r o b l a s t s i n v i t r o measuring DNA, hyaluro-n i c a c i d and p r o t e i n synthesis (Saarni and Tammi, 1978) demonstrated s e v e r a l impressive e f f e c t s i n DNA s y n t h e s i s . They observed a lower H-thymidine uptake by hydrocortisone-exposed c e l l s than by c o n t r o l c e l l s , demonstrating that the hydrocortisone i n h i b i t s scheduled DNA sy n t h e s i s _5 (at the l e v e l of 10 M). S i m i l a r l y , they found that both g l u c o c o r t i -coid s t i m u l a t i o n and i n h i b i t i o n of the formation of s e v e r a l metabolites on the pathway of the connective t i s s u e synthesis depended on the gluco-c o r t i c o i d i n c u b a t i o n time. This discovery, together with the u n c e r t a i n mechanism of s t e r o i d a c t i o n , leads to the conc l u s i o n that s t u d i e s of the secondary e f f e c t s o f g l u c o c o r t i c o i d , such as collagen metabolism, RNA s y n t h e s i s , c e l l p r o l i -f e r a t i o n , e i t h e r i n c u l t u r e d c e l l s or i n the s k i n , have only a l i m i t e d value u n t i l the more fundamental questions of g l u c o c o r t i c o i d primary e f f e c t ( s ) on nuclear acceptor s i t e s have been c l a r i f i e d . XV. S u b c e l l u l a r l o c a l i z a t i o n of g l u c o c o r t i c o i d s Studies of s u b c e l l u l a r l o c a l i z a t i o n of g l u c o c o r t i c o i d s have been r e l a t i v e l y l i m i t e d . None have been done with s k i n c e l l s other than mouse L-929 f i b r o b l a s t s . Most of the work done has d e a l t only with the d i s t r i -bution between cytoplasm and nucleus. The techniques employed i n these studies g e n e r a l l y have involved the l y s i s of whole c e l l s followed by u l t r a c e n t r i f u g a t i o n of the crude l y s a t e . This approach provides l e s s p r e c i s e i n f o r m a t i o n concerning the g l u c o c o r t i c o i d l o c a l i z a t i o n between cytoplasm and n u c l e i than i s o l a t i o n of i n t a c t n u c l e i p r i o r to the mea-surements of the re t a i n e d r a d i o a c t i v e g l u c o c o r t i c o i d s . In respect to studies of the d i s t r i b u t i o n of g l u c o c o r t i c o i d s w i t h i n the nuclear compartment, there . have been even fewer studies r e -ported. Middlebrook et a l . (1975) d i s t i n g u i s h e d two types of nuclear r e t e n t i o n of g l u c o c o r t i c o i d s , designated as nuclear e x t r a c t a b l e and nuclear r e s i d u a l p o r t i o n s , based on d i f f e r e n t i a l e x t r a c t a b i l i t y of these two f r a c t i o n s by 0.3 M KC1. These i n v e s t i g a t i o n s d i d not c o r r e l a t e c l e a r l y with any s p e c i f i c component of the nucleus. Higgins et a l . (1979) reported i n t h e i r recent review a r t i c l e that g l u c o c o r t i c o i d - r e c e p -t o r complexes have been found i n chromatin but not i n the nu c l e o l u s , nucleoplasma or nuclear membrane. Very l i t t l e i s known about the g l u c o -c o r t i c o i d d i s t r i b u t i o n w i t h i n a nucleus. The subchromatin l o c a l i z a t i o n of g l u c o c o r t i c o i d s w i t h i n the chromatin i s equally poorly understood. Retention of g l u c o c o r t i c o i d i n chromatin has been as s o c i a t e d w i t h the internucleosomal regions or w i t h the DNAse-sensitive p o r t i o n of the nucleosome, si n c e DNAse I , which pre-f e r e n t i a l l y a t t a c k s the internucleosomal regions of chromatin, a b o l i s h e s the r e t e n t i o n o f g l u c o c o r t i c o i d i n chromatin (Baxter et a l . , 1972; Hig-g i n s et a l . 1973b; N o l l , 1976). Bloom and Anderson (1978) have used the hydroxyapatite d i s s o c i a t i o n method to f r a c t i o n a t e the chromatin i n t o un-bound chromosomal p r o t e i n s , loosely-bound non-histone p r o t e i n , h i s t o n e p r o t e i n s (H^, E^k & H 2B, and & H l j), and t i g h t l y bound non-his t o n e p r o t e i n . They found the estrogen-receptor complexes approximate-l y 70$ e l u t e d with l o o s e l y bound non-histone p r o t e i n , l e s s than 5$ w i t h h i s t o n e p r o t e i n s , and 25$ with t i g h t l y bound non-histone p r o t e i n s . No such a study was known f o r g l u c o c o r t i c o i d s , and no p r e c i s e subchromatin l o c a l i z a t i o n of g l u c o c o r t i c o i d has been confirmed. The r e t e n t i o n of g l u c o c o r t i c o i d s i n n u c l e i or chromatin as a f u n c t i o n o f time has been g e n e r a l l y studied only up to 6 hr. I t i s of p a r t i c u l a r i n t e r e s t to examine t h e i r behaviour f o r longer periods, s i n c e chromatin i s not a s t a t i c e n t i t y , but undergoes many chemical and s t r u c -t u r a l changes (Pederson, 1972). Such changes might w e l l a f f e c t the amount of g l u c o c o r t i c o i d r e t a i n e d as w e l l as the type of i n t e r a c t i o n be-tween chromatin and complex. Anderson et a l . (1973) s t u d i e d the c o r r e l a -t i o n of estrogen-induced u t e r o t r o p i c and growth responses with nuclear s t e r o i d - r e c e p t o r complex content. They found that, i n general, growth responses were p r o p o r t i o n a l to the q u a n t i t y of estrogen-receptor complex that remained bound to the nucleus f o r 6 hours, not the q u a n t i t y d r i v e n i n t o the nucleus immediately a f t e r a d m i n i s t r a t i o n of l a r g e dose of e s t r a -d i o l i n v i v o . Thus, nuclear r e t e n t i o n , r a t h e r than i n i t i a l nuclear up-take, of receptors, determined such complex responses as c e l l u l a r growth and r e p l i c a t i o n (O'Malley and B u l l e r , 1976). The time p r o f i l e s of g l u c o c o r t i c o i d r e t e n t i o n i n various sub-c e l l u l a r f r a c t i o n s , are reported i n t h i s t h e s i s to i n v e s t i g a t e the time dependence of g l u c o c o r t i c o i d s u b c e l l u l a r l o c a l i z a t i o n . EXPERIMENTAL The "Outline of Research" shows the i n t e r - r e l a t i o n s h i p of the various experiments and methods comprising t h i s thesis as rel a t e d to the three types of experiments: (1) Gas-liquid chromatographic assay of hydrocortisone, triam-cinolone acetonide and desonide. (2) I s o l a t i o n of DNA from i n t a c t c e l l s and examination of these DNA for c r o s s - l i n k i n g with non-radioactive glucocorticoids over incubation periods of varying lengths of time up to 96 hours. (3) Examination of s u b c e l l u l a r d i s t r i b u t i o n of t r i t i a t e d TA under in t a c t c e l l conditions for determining: 3 (a) retention of H-TA i n DNA, 3 (b) retention of H-TA i n n u c l e i and i n t r a c e l l u l a r d i s t r i b u -3 t i o n of H-TA, 3 (c) retention of H-TA i n chromatin and intranuclear d i s t r i -3 bution of H-TA, 3 (d) subchromatin d i s t r i b u t i o n of H-TA. Outline of the Research P r o l i f e r a t i n g c e l l s with 90% confluency (about 4 x 10 c e l l s per plate) Administration of gl u c o c o r t i c o i d s with change of medium Af t e r desired period of incubation GLC-FID assay of stock s o l u t i o n Non-radioactive g l u c o c o r t i c o i d Media C e l l s J 1 H-glucocorticoid with and without 500-fold excess of unlabeled glucocorticoid GLC assay I s o l a t i o n of DNA J Media 1 Characterization of DNA (Quantity, purity) 1. Burton's assay for DNA Assay by l i q u i d s c i n t i l l a t i o n counting (LSC) C e l l s - J 2. 3. 4. 5. A260/ A280 r a t i ° Hyperchromicity T measurement m Bio-Rad protein assay Is o l a t i o n of DNA J HAP chromo-tography A260 a n d LSC Examination of c r o s s - l i n k i n g of DNA induced by gl u c o c o r t i c o i d s 1. HAP chromatography 2. Thermal denaturation-renaturation k i n e t i c s I s o l a t i o n of nu c l e i J S o l u b i l i z a t i o n of n u c l e i I LSC, DNA and protein assays Determination of the s p e c i f i c nuclear retention and i n t r a -c e l l u l a r d i s t r i b u t i o n I s olation of chromatin I S o l u b i l i z a t i o n of chromatin LSC, DNA and protein assays Determination of s p e c i f i c retention i n chromatin and intranuclear d i s t r i b u t i o n 1 HAP chromo-tography J 230 ,LSC 4 subchromatin d i s t r i b u t i o n 68. S e c t i o n I . G a s - l i q u i d chromatographic a n a l y s i s of hydrocortisone, t r i a m -c i n o l o n e acetonide and desonide i n c u l t u r e media of mouse and  human dermal f i b r o b l a s t s A q u a n t i t a t i v e g a s - l i q u i d chromatographic (GLC) assay with f l a m e - i o n i z a t i o n d e t e c t i o n (FID) was developed. The development and the a p p l i c a b i l i t y of t h i s assay are described i n t h i s s e c t i o n . (A) I n t r o d u c t i o n S y n t h e t i c g l u c o c o r t i c o i d s vary i n t h e i r c l i n i c a l potencies as w e l l as i n t h e i r p o t e n t i a l f o r causing dermal atrophy, the major adverse e f f e c t of long-term t o p i c a l g l u c o c o r t i c o i d therapy (Stewart et a l . , 1978). The m a j o r i t y of the studies on the pathogenesis of t h i s adverse e f f e c t have used c u l t u r e d dermal c e l l s as models, and the incubation of g l u c o c o r t i c o i d s with c u l t u r e d c e l l s has l a s t e d from hours to days. How-ever, no assays of g l u c o c o r t i c o i d s i n c u l t u r e media appear to have been published. I t has been assumed that the amounts of g l u c o c o r t i c o i d s added to c e l l c u l t u r e media represent t h e i r b i o l o g i c a l l y a c t i v e concentrations and the metabolism of g l u c o c o r t i c o i d s during the period of i n c u b a t i o n has been n e g l i g i b l e . The development of an assay, t h e r e f o r e i s an important t e s t of the v a l i d i t y of such assumptions. The requirements f o r q u a n t i t a t i v e assay i n b i o l o g i c a l systems are that s e n s i t i v i t y should be i n the low nanogram range because gluco-c o r t i c o i d s are p h y s i o l o g i c a l l y a c t i v e at these low concentrations, and the technique should be able to i d e n t i f y p o s s i b l e metabolites or decompo-s i t i o n products. Due to ready instrument a v a i l a b i l i t y , GLC-FID assay was i n v e s t i g a t e d f o r the determination of g l u c o c o r t i c o i d s i n c e l l c u l t u r e systems. 69. Few q u a n t i t a t i v e HPLC ( R y r f e l d t et a l . , 1979; Loo et a l . , 1977; Loo and Jordan, 1977; Wortman et a l . , 1973; Roth et a l . , 1980) and GLC (Martin and Amos, 1978; Mason and F r a s e r , 1975; Simpson, 1973) assays have been reported f o r the a n a l y s i s of s y n t h e t i c g l u c o c o r t i c o i d s i n b i o -l o g i c a l f l u i d s . Among HPLC assays, the work of R y r f e l d t et a l . ( 1979) measuring plasma l e v e l s of budesonide i n dogs with the a i d of r a d i o a c t i v e d e t e c t i o n achieved s e n s i t i v i t y as low as 0.05 ng/mL plasma. Among GLC techniques, Simpson (1973) estimated triamcinolone acetonide, t r i a m c i n o -lone and prednisolone i n r a t muscle, using e l e c t r o n capture d e t e c t i o n and t r i m e t h y l s i l y l d e r i v a t i v e , w i t h s e n s i t i v i t s s between 2 to 4 ng of the' g l u c o c o r t i c o i d : M a r t i n and Amos (1978) reported a GC-MS assay f o r prednisone and prednisolone as m e t h o x i m e - t r i m e t h y l s i l y l d e r i v a t i v e to ng/mL plasma l e v e l s . Methoxime- t r i m e t h y l s i l y l d e r i v a t i v e had not been applied to the assay of other s y n t h e t i c g l u c o c o r t i c o i d s (Martin and Amos, 1 9 7 8 ) , although i t had been used suc-c e s s f u l l y f o r the assays of n a t u r a l l y - o c c u r r i n g s t e r o i d s (Prost and Maume, 1 9 7 4 ) . No s p e c i f i c assay techniques have been reported so f a r f o r desonide. (B) Experimental (1) M a t e r i a l s The d e s c r i p t i o n s of chemicals, reagents and apparatus used are compiled i n Appendices I and II. The t e s t g l u c o c o r t i c o i d s included the s y n t h e t i c f l u o r i n a t e d g l u -c o c o r t i c o i d s , triamcinolone acetonide (TA) and triamcinolone (T), t h e i r n o n - f l u o r i n a t e d analog, desonide (DSN) and the n a t u r a l l y o c c u r r i n g hydro-c o r t i s o n e (HC). Their chemical s t r u c t u r e s are shown i n Fig u r e s 19. Methanol and e t h y l acetate were d i s t i l l e d i n g l a s s , and p y r i d i n e was the s i l y l a t i n g grade. GLUCOCORTICOID Hydrocortisone H H H H OH Triamcinolone 1 A F OH OH Triamcinolone acetonide 1 A - F X Desonide 1 A H Figure 19 : Chemical s t r u c t u r e s of t e s t g l u c o c o r t i c o i d s Methoxyamine hydrochloride, d i s s o l v e d i n p y r i d i n e , 100 mg/mL (Pfaffenberger and Horning, 1975), was stored i n a c o n i c a l R e a c t i - v i a ^ - ^ f i t t e d with a T e f l o n - l i n e d M i n i n e r t v a l v e ^ a t 4°C i n a d e s i c c a t o r . N - t r i m e t h y ] s i l y l imidazole (TMSI), was t r a n s f e r r e d to a c o n i c a l R e a c t i - v i a F ^ f i t t e d with a Mjn i n e r t v a l v i ^ immediately a f t e r each ampoule was opened, and the v i a l was kept at 4°C i n a d e s i c c a t o r . (2) P r e p a r a t i o n of d e r i v a t i v e s M e t h o x i m e - t r i m e t h y l s i l y l (MO-TMS) d e r i v a t i v e s : The procedure of P f a f f enberger and Horning (1975) was adapted as f o l l o w s : Test g l u c o c o r t i c o i d (not more than 800 yg) was placed i n 1 mL R e a c t i - v i a i ^ - ^ f i t t e d with a T e f l o n - l i n e d screw cap. One hundred yL o f methoxyamine h y d r o c h l o r i d e / p y r i d i n e stock s o l u t i o n was added to d i s s o l v e the compound, and the r e s u l t i n g s o l u t i o n was heated at 70°C f o r 15 min-utes. One hundred yL of TMSI was added and s i l y l a t i o n was conducted a t 100°C for 10 to 30 minutes depending on the compound te s t e d . One to 2 yL of the f i n a l r e a c t i o n mixture (volume o f 200 yL) was i n j e c t e d d i r e c t l y i n t o the GLC or GC-MS. t - B u t y l d i m e t h y l s l l y l (BDMS) d e r i v a t i v e s : The procedure was modified from those reported i n the l i t e r a t u r e ( G a s k e l l and Brooks, 1976; K e l l y and Ta y l o r , 1976; P h i l l i p o u et a l . , 1975). Test g l u c o c o r t i c o i d (not more than 800 yg) was placed i n a 1 mL R e a c t i - v i a T ^ - ^ f i t t e d with T e f l o n - l i n e d screw cap. One hundred yL of t - b u t y l d i m e t h y l s i l y l c h l o r i d e mixture (1.0 mmole t - b u t y l d i m e t h y l s i l y l c h l o r i d e , 2.5 mmole i m i d a z o l e per mL of anhydrous N,N-dimethylfonnamide) was added to d i s s o l v e the com-pound, and the r e s u l t i n g s o l u t i o n was incubated at 100°C f o r 1 hour. A f t e r the r e a c t i o n , 100 yL of d i s t i l l e d water was added and the d e r i v a -t i v e s were ext r a c t e d i n t o chloroform p r i o r to GLC or GC-MS a n a l y s i s . 72. (?) D i f f e r e n t i a ] scanning c a l o r i m e t r y A d i f f e r e n t i a l scanning c a l o r i m e t e r was employed. A l l samples were crimped. The rate of temperature increase was 10°C/min f o r each run. (4) G a s - l i q u i d chromatography A Hewlett-Packard g a s - l i q u i d chromatograph (Model 5830A) equip-ped with a terminal and a f l a m e - i o n i z a t i o n detector was used. The c o i l e d g l a s s column, 1.8 m x 2 mm i . d . , contained 3% OV-7. coated onto 80-100 mesh Chromosorb W (HP). A l l columns were conditioned at 100°C f o r 15 hours, and then the oven temperature was increased by 0.1°C/min up to 285°C, at which i t was allowed to remain f o r approximately 15 hours. The flow ra t e o f c a r r i e r gas during the c o n d i t i o n i n g period was 10 mL/min. The operating temperatures f o r ro u t i n e a n a l y s i s were: i n j e c t i o n p ort, 265°C; column, 285°C and detector, 300°C. The gas flow r a t e s were: c a r r i e r gas (helium), 60 mL/min; hydrogen, 40 mL/min; and a i r , 300 mL/min. (5) Gas chromatography-mass spectrometry A computerized Varian MATT-111 gas chromatograph-electron-impact mass spectrometer was used to study the fragmentation patt e r n of the M0-TMS d e r i v a t i v e s f o r confirming the chemical s t r u c t u r e s of the d e r i v a -t i v e s . The f o l l o w i n g c o n d i t i o n s were used f o r the GC: i n j e c t i o n port temperature, 230°C; column temperature was programmed from 200°C to 260°C at a rate of 10°C/min; and c a r r i e r gas (helium) flow r a t e , 20 mL/min. The 2 m x 2 mm i . d . g l a s s column was packed w i t h 3% OV-17 coated onto 80-100 mesh Chromosorb W (HP). For the MS, the i o n i z a t i o n beam energy was 80 eV; the source analyzer temperature was 250°C: and the separator was at 280°C. The modes of detection included t o t a l ion current and s e l e c t e d ion monitoring. (6) E x t r a c t i o n procedure The g l u c o c o r t i c o i d s were double extracted from the c e l l c u l t u r e medium c o n t a i n i n g 10$ f e t a l c a l f serum (FCS) i n Dulbecco Modified Eagle Medium (DMEM) as f o l l o w s : S o l i d sodium c h l o r i d e was added to 5 to 10 mL a l i q u o t s of the medium, decanted from c e l l c u l t u r e s , to make i t 1 N. The aqueous phase was extracted twice with an equal volume of g l a s s - d i s t i l l e d e t h y l ace-t a t e . A f t e r c e n t r i f u g a t i o n to separate the l a y e r s , the organic e x t r a c t s were combined and evaporated to dryness under a g e n t l e stream of n i t r o g e n at ambient temperature. The residue was t r a n s f e r r e d to a 1 mL R e a c t i -v i a r - " ^ c o n t a i n i n g 100 uL of i n t e r n a l standard (0.50 pg/mL i n methanol) by r i n s i n g three times with 0.2 mL of f r e s h e t h y l acetate. The r e s u l t i n g mixture was evaporated to dryness and subjected to d e r i v a t i z a t i o n . A white i n t e r f a c i a l p r e c i p i t a t e occurred i n the e x t r a c t i o n o f g l u c o c o r t i c o i d s from serum-containing medium. The a d d i t i o n of NaCl to make a 1 N s o l u t i o n avoided t h i s d i f f i c u l t y . (7) Recovery st u d i e s A s e r i e s of known amounts of t e s t g l u c o c o r t i c o i d s o l u t i o n i n methanol (HC, TA or DSN) were added to i n d i v i d u a l c e n t r i f u g e tubes and evaporated to dryness under a stream of n i t r o g e n . F i v e mL o f 10$ FCS-c o n t a i n i n g medium, e i t h e r f r e s h l y prepared or decanted from c o n t r o l c e l l s were added to each g l u c o c o r t i c o i d residue. The spiked samples were ex-t r a c t e d by the procedure described above.. Percentage r e c o v e r i e s were c a l c u l a t e d from standard curves obtained from g l u c o c o r t i c o i d samples which were d e r i v a t i z e d without e x t r a c t i o n . (8) Assays on b i o l o g i c a l samples The g l u c o c o r t i c o i d stock s o l u t i o n as w e l l as the g l u c o c o r t i c o i d -c o n t a i n i n g media were assayed before a d d i t i o n to the c e l l s to a s c e r t a i n the extent of agreement between c a l c u l a t e d and observed g l u c o c o r t i c o i d concentrations. Deviations of more than 15$ of expected values were ob-served on occasion because of d i l u t i o n e r r o r s . Only experimentally determined concentrations have been reported i n the f o l l o w i n g t a b l e s . The g l u c o c o r t i c o i d - c o n t a i n i n g medium was incubated with e i t h e r mouse L-929 or human dermal f i b r o b l a s t s a f t e r the c e l l s had reached con-fluence i n d u p l i c a t e c u l t u r e p l a t e s f o r time periods up to 96 hours. The decanted media from the 2 p l a t e s c o n t a i n i n g 6 to 8 x 10^ c e l l s were pooled and stored at -4°C u n t i l a n a l y s i s . The c e l l s were simultaneous-l y harvested f o r biochemical s t u d i e s of g l u c o c o r t i c o i d e f f e c t s on c e l l u -l a r DNA reported l a t e r i n t h i s t h e s i s . (C) R e sults and d i s c u s s i o n (1) Confirmation of p u r i t i e s of t e s t g l u c o c o r t i c o i d s A l l s t e r o i d s were drie d at 40°C f o r 1 hour, and at ambient temperature overnight i n a vacuum oven before use. The weight l o s t due to the drying was l e s s than 1$ i n a l l cases. The m e l t i n g p o i n t s of these compounds were determined by both c a p i l l a r y m. p. determination and d i f f e r e n t i a l scanning c a l o r i m e t r y . The p u r i t i e s of HC, DSN and PRG as received were s a t i s f a c t o r y . The samples of T and TA had to be r e c r y s t a l l i z e d by d i s s o l v i n g the compound i n metha-nol with the a i d of steam, and a l l o w i n g the f i l t e r e d r e s u l t i n g s o l u t i o n to stand i n a hood f o r three days before c o l l e c t i n g the c r y s t a l s by f i l -t r a t i o n . The p u r i t i e s of a l l compounds were confirmed by HPLC. (2) S e l e c t i o n of e x t r a c t i n g solvent Solvents of e t h y l acetate, d i e t h y l ether, chloroform and methy-lene c h l o r i d e were evaluated f o r t h e i r e x t r a c t i o n e f f i c i e n c y of g l u c o c o r -t i c o i d from serum-free and serum-containing media. E t h y l acetate was chosen, because i t had higher e x t r a c t i o n e f f i c i e n c y , and permitted e a s i e r withdrawal of e x t r a c t s . The presence of serum i n the medium decreases the e x t r a c t a b i l i t y of g l u c o c o r t i c o i d by s i n g l e e x t r a c t i o n and, t h e r e f o r e , double e x t r a c t i o n was used as a r o u t i n e procedure. (3) S e l e c t i o n of d e r i v a t i z i n g agents For i n i t i a l experiments, t - b u t y l d i m e t h y l s i l y l c h l o r i d e was s e l e c t e d as a p o t e n t i a l d e r i v a t i z i n g reagent based on the advantages des-c r i b e d i n the l i t e r a t u r e ( G a s k e l l and Brooks, 1976; K e l l y and T a y l o r , 1976; P h i l l i p o u , 1975). These were ( i ) high s e l e c t i v i t y of the r e a c t i o n by which s i l y l a t i o n occurs only at the unhindered C^-OE group of the g l u c o c o r t i c o i d , ( i i ) increased s t a b i l i t y o f t - b u t y l d i m e t h y l s i l y l ethers towards h y d r o l y s i s , which i s 10 times higher than that of t r i m e t h y l -s i l y l ethers, and ( i i i ) the s i m p l i c i t y of the mass spectra of t - b u t y l d i -m e t h y l s i l y l ethers, because these compounds give intense (M-57) + ions. By monitoring t h i s c h a r a c t e r i s t i c i o n , MS has the p o t e n t i a l to be a sen-s i t i v e and s e l e c t i v e technique f o r q u a n t i t a t i v e a n a l y s i s of g l u c o c o r t i -c o i d s . The r e s u l t s obtained with t h i s d e r i v a t i v e , however, were g e n e r a l -l y d i s a p p o i n t i n g . The t-BDMS d e r i v a t i v e s had u n s a t i s f a c t o r y GLC proper-t i e s , such as r e t e n t i o n times longer than those of t h e i r parent compounds and adsorption to the column occurred due to the remaining f r e e func-t i o n a l groups. Therefore, the f u r t h e r use of t - b u t y l d i m e t h y l s i l y l c h l o r -ide was discontinued. The MO-TMS d e r i v a t i v e s of t e s t g l u c o c o r t i c o i d s proved to be sup e r i o r to t h e i r corresponding t-BDMS d e r i v a t i v e s based on t h e i r short r e t e n t i o n times and sharp symmetrical peaks with higher molar response than t h e i r parent compounds. (4) Column s e l e c t i o n Columns packed with 3% OV-7, OV-17 and 0V-25 were examined f o r t h e i r s u i t a b i l i t y to resolve peaks of the i n t e r n a l standard, g l u c o c o r t i -c o i d s and any endogenous component i n serum. The OV-7 and OV-17 columns gave s u p e r i o r e l u t i o n p r o f i l e s to the OV-25 column. However, with OV-17, the serum associated peaks prevented s a t i s f a c t o r y r e s o l u t i o n of the i n -t e r n a l standard, PRG, a d i f f i c u l t y not present w i t h the OV-7 column (Figure 20-b and 20-c). Therefore, OV-7 packing was se l e c t e d f o r the assay notwithstanding the longer r e t e n t i o n times observed (Table V I ) . (5) Optimum r e a c t i o n time for s i l y l a t i o n and s t a b i l i t y of d e r i v a t i v e s The optimum time f o r s i l y l a t i o n was evaluated by r e a c t i n g sam-pl e s c o n t a i n i n g equivalent amounts of HC, T or TA f o r various times at 100°C. Known amounts of separately d e r i v a t i z e d methoxime i n t e r n a l standards were then added. The y i e l d of the d e r i v a t i v e , as observed by i t s peak area r a t i o to that of the i n t e r n a l standard, was monitored. Figure 21 showed that the optimum y i e l d was obtained when r e a c t i o n times were 10, 15 and 30 minutes f o r HC, T and TA r e s p e c t i v e l y . The MO-TMS d e r i v a t i v e s were s t a b l e i n excess o f 24 hours at 4°C when stored i n t i g h t l y capped r e a c t i o n v i a l s . (6) Confirmation of d e r i v a t i v e formation u s i n g GC-MS The fragmentation patterns of d e r i v a t i z e d HC, T, TA and DSN when analyzed by e l e c t r o n impact GLC-mass spectrometry confirmed the formation of MO-TMS d e r i v a t i v e s and PRG as the MO d e r i v a t i v e (Figure 22 a-e). There i s l i t t l e i n f o r m a t i o n a v a i l a b l e on the fragmentation of the MO-TMS BLANK: Medium + 10% Serum Figure 20 : T y p i c a l chromatograms of g l u c o c o r t i c o i d s and e x t r a c t s (a) Authentic g l u c o c o r t i c o i d s (b) Extrac t of blank medium w i t h 10% serum (c) G l u c o c o r t i c o i d s extracted from serum-containing medium Table VI Retention time of parent and derivatized glucocorticoids Retention time, min Glucocorticoid Underivatized compound(k) 3% OV-17 ' MO-TMS deri v a t i v e 3% OV-17 3% OV-7 Progesterone 1.31 1.38 2.57 Prednisolone - 2.20 -Hydrocortisone A. 09 2.10 A.50 Triamcinolone 5. OA 3.53 -Desonide 7.25 3.69 6.36 Triamcinolone acetonide 8.59 A. 38 7.37 (a) Three or more values are averaged. (b) A l l compounds except progesterone gave more than one peak due to thermal decomposition. The value given i s that of the major peak of each sample. Figure 21 : Reaction k i n e t i c s of HC, T and TA w i t h s i l y l a t i n g agent at 100°C P l o t of area r a t i o of d e r i v a t i z e d compounds to d e r i v a t i z e d PRG as a f u n c t i o n of time Figure 22-c : Electron-impact mass spectrum of (MO).-(TMS)„ - TA 85. d e r i v a t i v e s o f the s y n t h e t i c g l u c o c o r t i c o i d s . Those o f s t e r o i d s present i n adrenal gland e x t r a c t s of r a t s , p r i m a r i l y c o r t i c o s t e r o n e d e r i v a t i v e s , have been reported by Prost and Maume (1974). T h e i r scheme of charac-t e r i s t i c cleavage form the b a s i s f o r the i n t e r p r e t a t i o n of the observed spectra (Figure 22 a-e). The w e l l known l o s s e s of O-methoxy (m/e 3 D from O-methoxime groups and of s i l a n o l (m/e 89) from t r i m e t h y l s i l y l groups (Prost and Maume, 1974; Harvey and Vouros, 1979) were observed i n these reported experiments. (a) Fragmentation patt e r n of HC d e r i v a t i v e , (MOU-(TMS)^-HC The peak at m/e 636 (1.8$) corresponds to the molecular i o n peak; other c h a r a c t e r i s t i c peaks were observed at m/e 605 (10.5$) ((M-0CH 3) +), 361 (3-5$), 246 (6.1$), 147 (7.0$) ( ( C H ^ S i = O S i ( C H 3 ) 3 ) (Engel and Orr, 1972), 103 (11.4$) ( CH, + 0 -S i ( C H 3 ) 3 ) , ( s i ( C H 3 ) 3 ) . 89 (12.3$) (•* S i ( C H 3 ) 3 ) , ( C K ^ j O y - y CK3O1 m/e 361 and 73 (100$) C H 2 -OSKCKjlj C « f\IH2 C -OSiOt f 3 C H 2 m/e 246 (b) Fragmentation p a t t e r n o f T d e r i v a t i v e , (M0K - (TMS)^ - T The peak at m/e 739 (0.7$) corresponds to one l e s s thn the mole-c u l a r ion ( ( M - 1 ) + ) . The c h a r a c t e r i s t i c peaks are 709 (3.0$) ( ( M - 0 C H 3 ) + ) , 689 (1.4$) ((M-0CH 3-HF) +), 618 (2.6$) (M-F-(< O S i ( C H 3 ) 3 ) ) + , 147 (8.8$), 103 (12.3$) and 73 (100$). [CH, (c) Fragmentation patt e r n of TA d e r i v a t i v e , (M0) 1 - (TMS)^ - TA The peak at m/e 607 (0.4$) corresponds to the molecular i o n . 86. The c h a r a c t e r i s t i c peaks are 587 (1.1$) ((M-HF)t), 576 (1.6$) ( ( M - O C H 3 ) + ) , 556 (10.7$) ((M-OCH 3-HF)t), 121 ( 9 . 0 $ ) , 103 (18.1$) and 73 (100$). m/e 121 (d) Fragmentation patt e r n of DSN d e r i v a t i v e , (M0) 1 - (TMS)^ - DSN The peak at m/e 589 (0 . 8 $ ) corresponds to the molecular i o n . The c h a r a c t e r i s t i c peaks are 558 (4.9$) ( ( M - 0 C H 3 ) + ) , 468 (6 . 8$ ) ((M-121)?), 121 (10.1$), 103 (13.7$) and 73 (100$). (e) Fragmentation pattern of PRG d e r i v a t i v e , (MO)^ - PRG The peak at m/e 372 (100$) corresponds to the molecular i o n . The c h a r a c t e r i s t i c peaks are 3^1 (72.8$) ((M-OCH^*), 286 (29.5$), 273 (46.2$), 220 (11.5$), 153 (57.4$), 137 (40.2$), 125 (65.6$), 100 (75.5$) and 87 (39.1$). m/e 286 m/e 153 m/e 273 CK£> m/e 137 m/e 125 m/e 220 CHL 1 C «N - 0 C H 3 1 H 3 CH «= CHo v *• * C H 2 m/e 1 0 0 m/e 87 (7) Resolution of MO-TMS d e r i v a t i v e s on OV-7 and OV-17 columns The GLC chromatograms o f a l l four g l u c o c o r t i c o i d d e r i v a t i v e s appeared as s i n g l e peaks when eluted from OV-7 columns (Figure 20-a and 20-c). Compounds TA and DSN, however, resolved i n t o two peaks, a minor peak A w i t h shorter r e t e n t i o n time and occupying l e s s than 10$ o f peak area, and a major one B, when OV-17 packings were used due to a higher degree o f r e s o l u t i o n obtainable with OV-17 columns. A GC-MS examination showed that the chemical s t r u c t u r e of the d e r i v a -t i v e e l u t i n g as the minor peak A corresponded to (MO)^ - (TMS)^ -DSN, m/e = 660 (M-1) + whereas that of the major peak B corresponded to (MO) - (TMS) 2 - DSN-, m/e = 589 (mt) as i l l u s t r a t e d i n Fi g u r e 23-a and 22-d, r e s p e c t i v e l y . We a s c r i b e the formation of peak A as being due to p a r t i a l eno-l i z a t i o n of the keto group at C^Q. The keto group at C^Q i n DSN as w e l l as i n TA, i n contrast to HC and T, i s hindered by the C^g, C ^ acetonide group and remains l a r g e l y r e s i s t a n t to formation of the MO de-r i v a t i v e . Under favourable c o n d i t i o n s such as exposure of the r e a c t i o n mixtures to s u n l i g h t r a d i a t i o n , and excess s i l y l a t i n g reagent, some eno-l i z a t i o n , however, can occur which then would lead to the formation of the minor (TMS)^-derivative observed. I n d i r e c t evidence f o r the existence of the e n o l i z a t i o n r e a c t i o n was obtained upon exposure of the r e a c t i o n mixture to s u n l i g h t . S u n l i g h t has been long recognized as a c a t a l y s t f o r the e n o l i z a t i o n of a wide v a r i e t y o f ketones (Hart, 1979). Exposure to s u n l i g h t increased the r e -l a t i v e magnitude of peak A to peak B as expected of a s u n l i g h t - c a t a l y z e d r e a c t i o n . Removal o f the r e a c t i o n mixture from s u n l i g h t reversed the magnitude o f peak A back to i t s o r i g i n a l l e v e l s , i n d i c a t i n g the r e v e r s i -b i l i t y of the sunl i g h t - i n d u c e d e n o l i z a t i o n r e a c t i o n . Prolonging d e r i v a -t i z a t i o n r e a c t i o n time led to an increase i n the peak A at expense of the magnitude o f peak B, suggesting the formation o f A from B. The f a c t that the chromatographic peaks from OV-7 columns repre-sent two d e r i v a t i z a t i o n products o f TA and DSN, however, d i d not i n t e r -f e r e with the assay, because the c o n t r i b u t i o n s by the (TMS^-deriva-t i v e s were l e s s than 10$ of the area of the peaks and, moreover, were i n ne a r l y constant r a t i o to those of the major ( T M S ^ - d e r i v a t i v e s . I t i s important that the d e r i v a t i z a t i o n r e a c t i o n time i s kept a t 30 minutes and l i g h t be excluded. TA shared a l l the p r e v i o u s l y mentioned c h a r a c t e r i s t i c s of d e r i -v a t i z a t i o n and gave (M0) 1-(TMS) 2-TA, m/e = 607 (m*) as the. major product and (MO^-CTMS^-TA, m/e = 679 (m*) as the minor product (Fig u r e 22-c and 23-b). The presence of a n t i b i o t i c s i n the medium showed no i n t e r f e r e n c e on the chromatograms. (8) C a l i b r a t i o n curves The c a l i b r a t i o n curves f o r HC, TA and DSN e x t r a c t e d from medium co n t a i n i n g 10$ FCS were constructed. The curves were l i n e a r w i t h i n the ranges s t u d i e d , namely, 40-1600 ng/yL d e r i v a t i z e d s o l u t i o n f o r HC ( e q u i -v alent to 1.6 - 65.8 yg/mL medium), 60-1600 ng/yL d e r i v a t i z e d s o l u t i o n f o r TA (equivalent to 2.4-63.7 yg/mL medium) and 20-300 ng/yL d e r i v a t i z e d s o l u t i o n f o r DSN (equivalent to 0.8-12.0 yg/mL medium) (Table V I I , V I I I and I X ) . Two c a l i b r a t i o n curves of HC were determined with a time lapse of s i x months between them. The r e l a t i v e standard d e v i a t i o n i n slope values (0.905 and 0.946) was 3»1$- The combined data gave a s t r a i g h t 2 l i n e of y = 0.947 x - 0.005 with r = 0.998 where y = peak area r a t i o , Table VII Estimation of HC a f t e r e x t r a c t i o n from spiked medium samples Amount added Amount to 5 mL FCS- Weight Mean peak area recovered, Mean recovery DMEM, U g r a t i o ( a ) , x r a t i o ( b ) , y Vig(c) % March/78 16.45 0. 135 0.105 + 24.67 0. 203 0.144 + 41.42 0. 337 0.280 61.68 0. 506 0.439 + 164.48 1. 348 1. 193 + g./78 8.22 0. 067 0.057 + 16.45 0. 134 0.141 + 24.67 0. 200 0.180 + 41.12 0. 334 0.355 + 61.68 0. 501 0.492 + 82.24 0. 668 0.649 + 164.48 1. 335 1.312 4-328.96 2. 670 2.533 + 0.010 ( d ) 13.95 84. 82 + 6.86 0.002 18.69 75. 75 + 1.00 0.020 34.86 84. 77 + 5.84 0.028 53.75 87. 15 + 5.36 0.052 143.38 87. 17 + •3.71 0.001 7.92 97. 51 + 5.52 0.008 18.39 111. 82 + 5.54 0.001 21.65 90. 16 + 4.39 0.022 44.11 107. 28 + 6.48 0.024 60.55 98. 17 + 4.63 0.021 79.34 96. 47 + 3.08 0.020 158.93 96. 63 + 1.42 0.047 305.40 92. ,84 + 1.69 (d) (a) Weight of HC/ weight of PRG (b) Area of the peak of d e r i v a t i z e d HC/area of the peak of d e r i v a t i z e d PRG. (c) C a l c u l a t e d from the standard curve of HC without e x t r a c t i o n , y=mx + c, 2 2 where m= 1.026 and c =-0.009, r = 0.999, where r = c o e f f i c i e n t of 2 determination, y = mx + c, where m = 0.947 and c =-0.005, r = 0.998 f o r extracted samples, as p l o t t e d mean area r a t i o vs_ weight r a t i o . The average mean recovery was 93.12 ± 9.74%. (d) Mean ± S.D., c a l c u l a t e d from nine measurements of three s e r i e s of samples Table VIII Estimation of TA aft e r extraction from spiked medium samples Amount added Amount to 5 mL FCS- Weight Mean peak area recovered, Mean recovery DMEM, yg r a t i o ( a ) , x r a t i o ( b ) f y Ug( c) % A p r i l / 7 8 25.92 0. 142 0.073 ± 39.00 0.214 0.123 ± 64.80 0.354 0.214 ± 86.40 0.531 0.292 ± 129.13 0.708 0.408 ± 257.70 1.412 0.805 ± Oct./78 11.94 0.097 0.051 ± 15.92 0.129 0.070 ± 23.88 0. 194 0.103 ± 39.80 0.323 0.178 ± 59.70 0.485 0.278 ± 79.60 0.646 0.363 ± 159.20 1 .292 0.750 ± 318.40 2.584 1.551 ± Feb./79 11.94 0.119 0.066 ± 15.92 0.159 0.093 ± 23.88 0.234 0.144 ± 39.80 0.396 0.233 ± 0. ,0 1 3 ( d ) 22. 75 87.75 + 11.09 0. ,022 34. 95 89.62 + • 12.36 0. ,009 57. 13 88.17 + 3.33 0. ,022 75. 99 87.96 + 6.15 0. ,031 104. 23 80.65 + 4.37 0. .035 220. 53 77.82 + 2.49 0. .003 11. 79 98.76 + 4.25 0. ,001 14. 84 93.21 + 1.24 0. ,011 20. 35 85.20 + 7.82 0. ,001 32. ,60 81.92 + 0.20 0. ,008 48. ,98 82.04 4 2.08 0. ,005 62. ,92 79.05 + 1.09 0. ,028 126. ,29 79.33 + 2.90 0. ,036 257. ,58 80.90 + 1.83 0. ,004 11 . ,63 97.37 + 4.90 0. ,006 15. ,27 95.93 + 4.95 0. .011 21. ,99 92.08 + 6.21 0. .003 33, ,94 85.28 + 0.91 (d) (continued...) Table V I I I (continued) 59.70 79.60 159.20 318.40 0.595 0.356 ± 0.025 50.32 0.793 0.508 ± 0.035 70.69 1.586 0.913 ± 0.096 124.72 3.171 1.640 ± 0.004 221.88 84.28 ± 5.66 88.81 ± 5.86 73.34 ± 8.07 69.69 ± 0.17 (a) Weight of TA/Weight of PRG (b) Area of the peak of d e r i v a t i z e d TA/Area of the peak of d e r i v a t i z e d PRG (c) C a l c u l a t e d from the standard curve of TA without e x t r a c t i o n , y = mx + c, 2 2 where m = 0.750 and c = -0.021, r = 0.990, where r = c o e f f i c i e n t of 2 determination, y = mx + c, where m = 0.533 and c = 0.012, r = 0.996 f o r extracted samples, as p l o t t e d mean area r a t i o vs weight r a t i o . The average mean recovery was 85.64 ± 7.19%. (d) Mean ± S.D., c a l c u l a t e d from nine measurements of three s e r i e s of samples. Table IX Estimation of DSN after extraction from spiked medium samples Amount added Amount to 5 mL FCS- Weight Mean peak area recovered, Mean recovery DMEM, pg r a t i o ( a \ x r a t i o ( b ) , y ug<c) % 4 .0 0.079 0.040 + 0.003 3.95 98.77 + 4.72 8, .0 0.163 0.106 + 0.006 8.15 101.97 + 4.70 12, .0 0.242 0.169 4 0.006 12.12 101.03 + 3.01 16, .0 0.325 0.236 + 0.005 16.32 102.03 + 1.81 24. .0 0.468 0.350 + 0.008 23.49 97.91 + 2.05 40, ,0 0.726 0.555 + 0.026 36.42 91.04 + 4.03 60. ,0 1 .092 0.847 + 0.091 54.81 91.36 + 9.53 (a) Weight of DSN/Weight of PRG (b) Area of the peak of der i v a t i z e d DSN/Area of the peak of der i v a t i z e d PRG, (c) Calculated from the standard curve of DSN without extraction, y = mx + c, 2 2 where m = 0.790 and c = -0.023, r = 0.999, where r = c o e f f i c i e n t of 2 determination. y = mx + c, where m = 0.713 and c = -0.004, r = 0.999 for extracted samples as plotted mean area r a t i o vs weight r a t i o . The average mean recovery was 97.73 ± 4.72%. (d) Mean i S.D., calculated from nine measurements of three series of samples. x = weight r a t i o and r " i s the c o e f f i c i e n t of determination (Winer, 1971 i n d i c a t i n g a s a t i s f a c t o r y r e p r o d u c i b i l i t y of the assay. Three c a l i b r a t i o n curves of TA were determined w i t h i n a year. The r e l a t i v e standard d e v i a t i o n of slope values (0.515, 0.599 and 0.579) was 7.6$. The combined data gave a s t r a i g h t l i n e of y = 0.553 x + 0.012 2 2 with r = 0.996, where y = peak area r a t i o , x = weight r a t i o and r i s the c o e f f i c i e n t of determination. The s a t i s f a c t o r y r e p r o d u c i b i l i t y o f the assay confirmed the v a l i d i t y of using the sum of double-peak areas, (M0) 1-(TMS) 2-TA and (MO) 1 -(TMS^-TA f o r assay based on the as-sumption that the y i e l d r a t i o of these two peaks was a constant. The r e c o v e r i e s of three g l u c o c o r t i c o i d s added i n v a r i o u s amounts were a l s o tabulated (Tables V I I - I X ) . The average mean r e c o v e r i e s ± S.D. were 93-12 ± 9.74$, 85.64 ± 7.19$ and 97.73 ± 4.72$ f o r HC, TA and DSN r e s p e c t i v e l y . The amounts of g l u c o c o r t i c o i d s e xtracted f o r the preparation of the c a l i b r a t i o n curves are l a r g e compared to what i s needed f o r the a c t i -v i t y of potent g l u c o c o r t i c o i d s , e.g., TA i n c e l l c u l t u r e s . Therefore, the e n t i r e volume of 10 mL of the c u l t u r e medium or the pooled media of s e v e r a l p l a t e s had to be extracted i n order to o b t a i n enough TA f o r assay. On the other hand, the amounts used f o r c a l i b r a t i o n curves are r e a l i s t i c f o r assays of stock s o l u t i o n concentrates and s i m i l a r s o l u t i o n s of pharmaceutical i n t e r e s t . (9) B i o l o g i c a l data The a p p l i c a b i l i t y of the reported assay was demonstrated by determining the HC, TA and DSN l e v e l s i n FCS-containing c e l l c u l t u r e medium a f t e r v a r i o u s time periods of incubation with c u l t u r e d human or mouse dermal f i b r o b l a s t s . The r e s u l t s were summarized i n Table X, XI and X I I . Table X Levels of HC i n media as a fu n c t i o n of time a f t e r i n c u b a t i o n with c u l t u r e d human dermal f i b r o b l a s t s Concentration assayed, yg/mL medium Incubation time, hr. n A-2 (a) (% remaining i n t a c t ) A- A ( b ) 0 2 0. ,731 ± 0. 134 (100.0 ± 18 . 3 ) ( C ) -0.25 1 0. 462 2 2 0. 392 ± 0. 005 4 2 0. ,389 ± 0. 035 (53.3 ± 4 .7) 0. ,519 ± o. 009 6 2 0. ,474 ± o. 009 8 2 0. ,434 ± o. 016 12 2 0. ,436 ± o. 013 24 2 0. ,386 ± 0. 044 (52.9 ± 6 • 0) 0. ,346 (n = - 1) 48 2 0. ,455 ± 0. ,050 (a) Human dermal f i b r o b l a s t s , F.H. passage 6. (b) Human dermal f i b r o b l a s t s , W.P. passage 4. (c) The concentration of HC recovered from medium co n t a i n i n g no c e l l s was defined as 100% of i n t a c t drug. * data given as mean ± S.D. Table XI Levels of TA in media as a function of time a f t e r incubation with cultured mouse L-929 dermal f i b r o b l a s t Incubation n Concentration assayed, Intact TA remaining time, hr. yg/mL medium in media, % 0 5 1.265 + 0.082 100.0 + 6.5 ( a ) 2 2 1.032 + 0.003 81.6 + 0.2 4 2 1.025 + 0.001 81.0 + 0.1 8 2 1.030 + 0.010 81.5 + 0.8 11 2 1.011 + 0.005 79.9 + 0.4 24 2 1.060 + 0.005 83.8 + 0.4 48 1 1.011 79.9 72 2 1.028 + 0.002 81.3 + 0.1 83.5 2 1.079 + 0.030 85.3 + 2.4 94 2 0.919 + 0.022 72.7 + 1.7 (a) The concentration of TA recovered from medium containing no c e l l s was defined as 100% of in t a c t drug. * data given as mean - S.D. Table XII Levels of DSN i n media as a f u n c t i o n of time a f t e r i n c u b a t i o n with c u l t u r e d mouse L-929 dermal f i b r o b l a s t s Incubation Concentration assayed, I n t a c t DSN remaining time, hr. n ug/mL medium i n media, % 0 4 0.607 ± 0.012 100.0 ± 2 . 0 ( a ) 4 2 0.514 ± 0.101 84.6 ±11.7 24 2 0.496 ± 0.033 81.7 ± 5.4 (a) The concentration of DSN recovered from medium c o n t a i n i n g no c e l l s was defined as 100% of i n t a c t drug. * data given as mean ± S.D. Prolonged incubation of TA w i t h c u l t u r e d mouse L-929 dermal f i b r o b l a s t s up to 8 3 . 5 hours d i d not cause appreciable decrease of i n t a c t TA i n media. About 20$ of TA was removed from the c u l t u r e media by the mechanisms of absorption and/or adsorption by c e l l s two hours a f t e r the TA a d m i n i s t r a t i o n . The l e v e l s of TA at va r i o u s incubation periods be-tween 2 hours and 8 3 . 5 hours d i d not vary a p p r e c i a b l y . The s i g n i f i c a n t decrease of TA l e v e l a f t e r 94 hours was observed, but unexplained. To e l i m i n a t e the p o s s i b i l i t y of decomposition, the s t a b i l i t y o f TA i n medium upon incubation was studied at 37°C i n the absence of c e l l s f o r 72 and 96 hours. There was no appreciable decomposition due to the incubation at 37°C. The l e v e l s of DSN a f t e r 4 and 24 hours of incubation with mouse L-929 f i b r o b l a s t s a l s o i n d i c a t e d about 20$ l o s s of DSN from the medium. Therefore, f o r any b i o l o g i c a l e f f e c t of s y n t h e t i c g l u c o c o r t i -coids observed at the dose l e v e l o f 1 ug/mL medium, the true b i o a v a i l a b l e dose was probably not more than 0.2 yg/mL medium. The l e v e l s of HC as a f u n c t i o n of time had been measured twice a t va r i o u s i n t e r v a l s . The l o s s of HC from the media due to c e l l s was l a r g e r than that of s y n t h e t i c g l u c o c o r t i c o i d s . The HC a l s o had rat h e r constant l e v e l s as incubated with human dermal f i b r o b l a s t s between 0.25 and 48 hours. (10) M e t a b o l i t e s No ex t r a peaks were observed i n samples incubated with c e l l s f o r 0-108 hours and no appreciable change i n the magnitudes of endogenous peaks i n the mixture due to medium and 10$ serum. I t was proposed that no metabolite was apparently present unless i t s MO-TMS d e r i v a t i v e had the same r e t e n t i o n time of the d e r i v a t i z e d parent compound, or that the amounts of metabolites were too small to be detected. 1 0 0 . (11) A p p l i c a b i l i t y of MO-TMS r e a c t i o n to other g l u c o c o r t i c o i d s Another g l u c o c o r t i c o i d , d i f l o r a s o n e d i a c e t a t e , was d e r i v a t i z e d under the same r e a c t i o n c o n d i t i o n s ; however, the r e a c t i o n product gave four peaks i n GLC chromatogram i n d i c a t i n g m u l t i p l e d e r i v a t i z i n g products presumably due to the h y d r o l y s i s of acetates by hydrochloride generated i n the r e a c t i o n w i t h methoxyamine hydrochloride and subsequent s i l y l a -t i o n s at C ^ and/or f r e e hydroxyl groups. Therefore, d i f l o r a s o n e d i a c e t a t e and probably other ester-group c o n t a i n i n g g l u c o c o r t i c o i d s , are not amenable to the GLC a n a l y s i s using the subsequent d e r i v a t i z a t i o n w i t h methoxyamine hydrochloride and N - t r i -m e t h y l s i l y l imidazole. Prednisolone could be d e r i v a t i z e d to y i e l d the (M0) 2 (TMS)^ - prednisolone d e r i v a t i v e with the same r e a c t i o n procedure. 101. Section I I . I n v e s t i g a t i o n of c r o s s - l i n k i n g of DNA induced by glucocor -t i c o i d s and nuclear r e t e n t i o n of TA  M a t e r i a l s and Methods The d e s c r i p t i o n s of chemicals, reagents and apparatus used are compiled i n Appendices- I and IT, (A) C e l l c u l t u r e s and g l u c o c o r t i c o i d s (1) C e l l c u l t u r e s (a) C h a r a c t e r i s t i c s of c e l l l i n e s Human dermal f i b r o b l a s t s are i s o l a t e d by us from explants o f b i o p s i e s from 25 -35 year o l d males, stored at -193°C i n l i q u i d n i t r o -gen. The c e l l s were grown between 4 t h and 8 t h s u b c u l t u r e i n monolayer c u l t u r e s to confluence i n p l a s t i c c u l t u r e dishes. Mouse L-929 s k i n f i b r o b l a s t s were derived from a c h e m i c a l l y transformed clone i n 1943 ( E a r l e , 1943) and have since been maintained by Flow L a b o r a t o r i e s . This c e l l l i n e was recommended f o r assays of t o p i c a l g l u c o c o r t i c o i d s ( B e r l i n e r , 1967a) , but d i f f e r e d from d i p l o i d human s k i n f i b r o b l a s t s i n important ways. L -929 c e l l s are aneuploid, having an increased but v a r i a b l e chromosone count, 63 to 69 and they are not c e l l -density i n h i b i t e d . A c t u a l l y , they have been long considered mistakenly to be equivalent to human f i b r o b l a s t s i n response to t o p i c a l g l u c o c o r t i -c o i d s . Their growth rates are i n h i b i t e d by g l u c o c o r t i c o i d s , while growth rates of human s k i n f i b r o b l a s t s under i d e n t i c a l experimental c o n d i t i o n s are stimulated ( R u n i k i s et a l . , 1978; Thrash et a l . , 1974 ) . (b) Maintenance of c u l t u r e d c e l l s Both c u l t u r e d mouse L -929 and human dermal f i b r o b l a s t s were main-ta i n e d i n Dulbecco's Modified Eagle Medium, pH 6 . 8 (DMEM) supplemented with 10$ f e t a l c a l f serum (FCS), sodium bicarbonate 3-7 g / l i t r e medium, and the a n t i b i o t i c s , p e n i c i l l i n 100 units/mL, streptomycin 100 pg/mL and amphotericin B (Fungizone) 0.25 yg/mL. The c e l l s were incubated a t 37°C i n 5% (v/v) C0 2 - 95% (v/v) a i r . The medium was changed twice weekly. The c e l l u l a r morphology was examined r e g u l a r l y . Any p l a t e w i t h signs of m i c r o b i a l contamination, c e l l u l a r degeneration or poor growth was discarded. P l a t e s of about 4 x 10^ c e l l s per p l a t e were used f o r st u d i e s . U s u a l l y , 5 p l a t e s were pooled f o r a s i n g l e sample unless other-wise s p e c i f i e d . (2) Treatment with g l u c o c o r t i c o i d s (a) Preparation o f g l u c o c o r t i c o i d stock s o l u t i o n s G l u c o c o r t i c o i d s i n v e s t i g a t e d were HC and TA. 3 Stock s o l u t i o n s of the g l u c o c o r t i c o i d s , 10 yg/mL i n propylene g l y c o l v e h i c l e , were prepared using the method of Brotherton (1971). C o n t r o l s o l u t i o n s of the propylene g l y c o l v e h i c l e were prepared i n a s i m i l a r f a s h i o n . The procedure of s o l u t i o n p r e p a r a t i o n was as f o l l o w s : S o l u t i o n A contained 50% of propylene g l y c o l i n methanol. S o l u -3 t i o n B contained 10 yg/mL g l u c o c o r t i c o i d i n methanol. The stock g l u -3 c o c o r t i c o i d s o l u t i o n , 10 yg/mL i n propylene g l y c o l , was obtained by mixing two volumes of s o l u t i o n A with one volume of s o l u t i o n B i n a 500 mL vacuum f l a s k , followed by the removal o f methanol under vacuum. The c o n t r o l s o l u t i o n was prepared by mixing two volumes of s o l u t i o n A with one volume of pure methanol. A f t e r the removal of the methanol, the r e s u l t a n t c o n t r o l and g l u c o c o r t i c o i d s o l u t i o n s were t r a n s f e r r e d to s t e r i l e amber b o t t l e s of 60 mL ca p a c i t y and stored at -4°C i n a c o l d room. The stock s o l u t i o n s thus prepared were assayed by GLC-FID analy-s i s before use to confirm the concentration and s t a b i l i t y of the s o l u t i o n . Immediately p r i o r to i n c u b a t i o n , the g l u c o c o r t i c o i d stock s o l u -t i o n was d i l u t e d to the desired c o n c e n t r a t i o n with the c u l t u r e medium. 103. C o n t r o l c u l t u r e s received equivalent volumes of stock v e h i c l e s . The pro-pylene g l y c o l content of the media thus prepared d i d not i n t e r f e r e w i t h c e l l growth. (b) Incubation of c e l l s with g l u c o c o r t i c o i d s The 90% confluent c u l t u r e s , approximately four days a f t e r seed-5 ing with 5 x 10 c e l l s per p l a t e , were incubated i n g l u c o c o r t i c o i d - c o n -t a i n i n g media at 37°C. The concentrations i n v e s t i g a t e d ranged from 10" 8 M (4.4 ng/mL DMEM) to 5.01 x 10' 6 M (2.2 yg/mL DMEM) f o r TA, from 2.76 x 1 0 - 6 M (1.0 u g/mL DMEM) to 2.76 x 10~ 5 M (10 yg/mL DMEM) fo r HC, and 2.40 x 10~ 5 M (1.0 yg/mL DMEM) f o r DSN. The incubation was maintained f o r varying periods up to 108 hr. At the end of the e x p e r i -ment, the g l u c o c o r t i c o i d - c o n t a i n i n g media were removed f o r f u r t h e r t e s t -i n g and the c e l l s were harvested with the a i d of rubber policemen f o r var i o u s s t u d i e s . (B) I s o l a t i o n and p u r i f i c a t i o n of DNA (1) Homogenization of c e l l s The homogenization s o l u t i o n was composed of 8 M urea, 1 mM EDTA and 1$ (w/v) S.D.S. i n 0.24 M sodium phosphate b u f f e r . The pH of the s o l u t i o n was adjusted to pH 6.8. The s o l u t i o n was stored at 4°C f o r no longer than three months (Meinke et a l . , 1974; DuVivier et a l . , 1978). Ten mL of homogenization s o l u t i o n were d i s t r i b u t e d among p l a t e s , and the s o l u t i o n was l e f t i n contact with c e l l s f o r 5 min. at 4°C. The c e l l suspension was t r a n s f e r r e d with the a i d of a rubber policeman i n t o a gl a s s - T e f l o n homogenizer. There i t was manually homogenized with 10 to 20 strokes u n t i l an uniformly viscous homogenate was obtained. (2) D e p r o t e i n i z a t i o n This homogenate was t r a n s f e r r e d i n t o an Erlenmeyer f l a s k where 104. it was brought up to 1 N NaCl by adding 585 mg of NaCl(s) to f a c i l i t a t e the d i s s o c i a t i o n of chromosomal p r o t e i n s from the chromatin (Meneghini, 1976). The d e p r o t e i n i z a t i o n was done by gentle shaking with s i x volumes of chloroform/butanol (4:1, v/v), and the aqueous phase was separated by c e n t r i f u g a t i o n . The d e p r o t e i n i z a t i o n of the aqueous phase was repeated two times with f r e s h chloroform/butanol mixture. ( 3) Chromatography on hydroxyapatite column (a) Preparation of the column Fours grams of hydroxyapatite powder (HAP) were hydrated i n 50 mL of 0.01 M phosphate b u f f e r , pH 6.8, w i t h g e n t l e s w i r l i n g (when hydrat-ed, B i o - g e l HAP swelled to 2 -3 mL/g dry wt.) The suspension was b o i l e d i n a water bath f o r 30 min. and allowed to s e t t l e f o r 15 min. The f i n e s i n the cloudy upper l e v e l and at the top of the s e t t l e d g e l were decant-ed. The paste was resuspended i n 20 mL of the same b u f f e r f o r column pouring. The column was packed i n a 2 cm ( i . d . ) x 10 cm g l a s s column by wet pouring technique. The set-up of the HAP chromatography c o n s i s t e d of a gradient mixer, a p e r i s t a l t i c pump and a f r a c t i o n c o l l e c t o r i n a d d i t i o n to the packed column. (b) I s o l a t i o n of DNA by chromatograrjry The combined deproteinized aqueous phase was a p p l i e d on the top of the packed hydroxyapatite column and chromatographed with a l i n e a r gradient of 0.01 M to 0.5 M of phosphate b u f f e r . The flow rate of the 2 eluent was c o n t r o l l e d to about 30 mL/hr/cm by the p e r i s t a l t i c pump. 2 Bernardi (1971a) recommended flow rates of 5 - 50 mL/hr/cm to avoid the d i s t o r t i o n of chromatographic peaks. The f r a c t i o n s of 3.2 mL each were monitored by measuring the absorbance at 260 nm ( A 5 f i n ) , and f r a c t i o n s c o n t a i n i n g DNA were pooled. 105. (4) Concentration of the pooled DNA by Amicon f i l t r a t i o n The s t i r r i n g f i l t r a t i o n u n i t (Model 12, Amicon) was used with prehydrated D i a f l o XM 100A membranes (Amicon), which r e t a i n molecules of s i z e l a r g e r than 100,000. The s o l u t i o n was introduced i n t o the transparent sleeve with the a i d of a 5 mL syringe before applying p o s i t i v e pressure with h i g h - p u r i t y n i t r o g e n . The solvent and any molecule having M.W. smaller than the pore s i z e passed through the membrane, l e a v i n g the DNA i n r e t e n t a t e . Two a l i q u o t s of 5 mL of d i l u t e s a l i n e - c i t r a t e (DSC, 0.015 M NaCl and 0.0015 M N a 2 - c i t r a t e pH 6.85) were added to wash the re t e n t a t e f o r removal of any phosphate ions and other c a t i o n s . A f t e r the wash, the membrane was soaked i n a t e s t tube with 5 mL of DSC overnight at 4°C i n order to ensure the maximum recovery of DNA from the re t e n t a t e and the membrane (Bohnert, 1978). (C) Burton's diphenylamine assay f o r DNA A modified v e r s i o n of Burton's assay (1955) was employed, i n which the s e n s i t i v i t y of the assay was improved by adding acetaldehyde to the reagents and by a l l o w i n g the s o l u t i o n to stand f o r 16-20 hr. at 30°C instead of heating i t at 100°C (Burton, 1956). Diphenylamine reagent reacts with the deoxyribose moieties which are bound to purines i n DNA and forms blue-colored complex. DNA can there f o r e be qu a n t i t a t e d c o l o r i m e t r i c a l l y by measuring the absorbance at 600 nm. C a l i b r a t i o n curves were constructed w i t h i n the concentration range of 2 to 50 yg/mL. (1) Preparation of s o l u t i o n s : DNA stock standard s o l u t i o n of 1 mg/mL ;Calf thymus DNA was d i s -solved i n 5 mM NaOH (Chandra and Appel, 1973). The s o l u t i o n could be stored i n r e f r i g e r a t o r f o r about 6 months. The DNA working standard s o l u t i o n was prepared by mixing equal volumes of stock standard s o l u t i o n and 1 N HClO^, and heating at 70°C f o r 15min. The s o l u t i o n was then cooled slowly at room temperature and d i l u t e d to the concentration of 100 ug/mL with 0.5 N HCIO^ for c o n s t r u c t i n g the c a l i b r a t i o n curve. The s o l u t i o n could be stored i n r e f r i g e r a t o r f o r 3 weeks. Diphenylamine reagent, 1.5$ (w/v), was prepared by d i s s o l v i n g 1.5 gm of diphenylamine i n 100 mL a c e t i c a c i d and 1.5 mL concentrated H^SOjj. Immediately p r i o r to use, 0.1 mL of aqueous acetaldehyde, 16 mg/mL, was added f o r each 20 mL of reagent. (2) Procedure a. the working standard or t e s t s o l u t i o n s , 0.2 to 1.0 mL, were taken, and the volumes were made up to 1.0 mL w i t h 0.5 N HC10^. b. the r e s u l t i n g DNA s o l u t i o n was mixed with 2 volumes o f diphenylamine s o l u t i o n c o n t a i n i n g acetaldehyde, and allowed to stand f o r 16-20 hr. at 30°C. c. The absorbance at 600 nm was measured. C a l i b r a t i o n was repeated f o r each assay. (D) Bio-Rad p r o t e i n assay The Bio-Rad p r o t e i n assay i s a dye-binding assay based on the d i f f e r e n t i a l c o l o r change of the Coomassie B r i l l i a n t Blue G-250, i n r e -sponse to i t s binding to various concentrations of p r o t e i n (Bradford, 1 9 7 6 ) . When the binding occurs, the absorbance maximum f o r the a c i d i c dye s o l u t i o n s h i f t s from 465 nm to 595 nm (Reisner et a l . , 1975; Sedmak and Grossberg, 1 9 7 7 ) . By monitoring the absorbance at 595 nm, the pro-t e i n concentrations can be determined. 107. (1) S e l e c t i o n of p r o t e i n standard The best p r o t e i n standard i s a p u r i f i e d preparation of the pro-t e i n being assayed; however, the i d e n t i t y of the p r o t e i n was not known i n t h i s research, and therefore was not a v a i l a b l e . Of the two commercially a v a i l a b l e standards, bovine serum albumin was used instead o f bovine gamma g l o b u l i n , because bovine serum albumin gave a c o l o r y i e l d c l o s e r to non-histone and histone p r o t e i n s - c o n t a i n i n g samples than the g l o b u l i n standard does. An example of the s u i t a b i l i t y of albumin as a standard f o r assaying a complex p r o t e i n mixture (adrenal gland s u b c e l l u l a r f r a c -t i o n s ) has been given by Polland et a l . (1978). (2) Procedure The f o l l o w i n g microassay procedure, s u i t a b l e f o r amounts of pro-t e i n ranging from 1 to 20 yg, was employed (Bio-Rad b u l l e t i n , 1977). A standard curve was prepared each time the assay was perform-ed. A s e r i e s of d i l u t i o n s o f p r o t e i n standards c o n t a i n i n g from 1 to 25 yg/mL i n 0.8 mL of blank solvent, i d e n t i c a l to that of the t e s t sample, were placed i n clean, dry t e s t tubes. Same volume of sample solvent served as blank. Dye reagent concentrate, 0.2 mL, was added, followed by a gentle vortex avoiding excess foaming. ^595 were measured against reagent blank 5 minutes a f t e r the a d d i t i o n of the dye reagent and A,-^ ,-was p l o t t e d against the amount of p r o t e i n . (E) Thermal scanning a n a l y s i s , T^ and hyperchromicity (1) Denaturation p r o f i l e Four cuvettes containing 0.25 mL of the solvent, d i l u t e s a l i n e -c i t r a t e (DSC), and three t e s t DNA samples r e s p e c t i v e l y , were heated from 30 °C to 90 °C with an increment of 1°C/min. The cuvettes were scanned 20 seconds per c y c l e . The A ? f i n of each sample was recorded as 108. a f u n c t i o n o f temperature to generate the denaturation p r o f i l e (Figure 24). (2) Renaturation p r o f i l e The temperature of the cuvettes were brought down r a p i d l y from 90 °C to 50°C w i t h i n 2 minutes c o n t r o l l e d with the thermoprogrammer a f t e r the completion of thermal denaturation. The temperature was a l l o w -ed to stay at 50°C f o r 10 minutes, then a f u r t h e r c o o l i n g to 10°C was conducted i n an i d e n t i c a l f a s h i o n . The A^^Q'S were s i m i l a r l y p l o t t e d as a f u n c t i o n o f temperature to generate the r e n a t u r a t i o n p r o f i l e ( F i g u r e 24). (3) Data a n a l y s i s Denaturation p r o f i l e : The melting p o i n t s of the DNA samples were c a l c u l a t e d from the denaturation p r o f i l e as shown i n Figure 24, based on the d e f i n i t i o n of T as that temperature at which h a l f of the m absorbance increase i s achieved. Besides T , the hyperchromicity was a l s o measured as the r a t i o of t o t a l absorbance change to the i n i t i a l absorbance before denaturation, ^ A 2 g 0 ) 50° • 90° ^ A 2 6 0 ^ 50° ' e x ~ pressed i n percentage, to c h a r a c t e r i z e the denaturation p r o f i l e . Renaturation p r o f i l e : The hypochromic e f f e c t of r e n a t u r a t i o n due to c o o l i n g was measured. The $ r e n a t u r a t i o n was c a l c u l a t e d as ( ( A A 2 6 0 ) 90° • S O 0 7 ^ A 2 6 0 ) 50° • 90° ) x 1 0 ° * f o r c o o l i n g t o 5 0 ° C and ( ( A A 2 6 0 ) 9(f__+ 1 0 ° / ( A A 2 6 o ) 5 0 ° • 9 o ° ) x 1 0 ° * f o r c o o l i n g to 10°C. (F) UV-A i r r a d i a t i o n of c u l t u r e d f i b r o b l a s t s Human and L-929 f i b r o b l a s t were i r r a d i a t e d i n the p l a t e s where they were grown to 90$ confluency. Immediately before i r r a d i a t i o n , the c u l t u r e media i n the p l a t e s were replaced with PBS or PBS pl u s t e s t com-Denaturation plateau : Renaturation 8/ rv • 7& Lag f • 4 • • • Tm Buffer, DSC 50 60 70 80 Temperature, ° C 90*60 60—10 10 ;ure 24 : T y p i c a l denaturation - r e n a t u r a t i o n p r o f i l e of DNA i s o l a t e d from cu l t u r e d f i b r o b l a s t s 1 1 0 . pound ( g l u c o c o r t i c o i d s or 8-methoxypsoralen) to about 0.15 cm i n depth (8 ml / p l a t e ) . The p l a t e s were kept from l i g h t by wrapping them with a l u m i -num f o i l u n t i l placed i n the i r r a d i a t i o n incubator, where they were shaken slowly (100 strokes/hr) with t h e i r l i d s on, at 30°C. The incubator was equipped with black l i g h t blue fluorescence lamps e m i t t i n g l i g h t w i t h i n the range of 310 nm to 360 nm, with most of the energy f l u x at 330 nm which reached the c e l l s at an i n t e n s i t y of 0.6 2 mW/cm . Varying i r r a d i a t i o n periods of time were used. The c e l l s were examined f o r the i n f l u e n c e of the i r r a d i a t i o n on t h e i r v i a b i l i t y . They were examined under microscope f o r p e r s i s t e n c e of t h e i r attachment to the p l a t e s . Then, 0.H% trypan blue was allowed to contact the c e l l s for 1 minute. A f t e r r i n s i n g 5 times with PBS, the c e l l s were re-examined f o r s t a i n i n g . Since only dead c e l l s s t a i n , the proportion of non-stained c e l l s to stained c e l l s provided an i n d i c a t i o n of c e l l v i a b i l i t y . (G) I s o l a t i o n and s o l u b i l i z a t i o n of n u c l e i A l l procedures were c a r r i e d out at 4°C unless s p e c i f i e d other-wise. 7 - 8 Two to f i v e x 10 c e l l s were incubated i n DMEM with 10 M t r i t i a t e d TA at 37°C i n the absence or presence of 5 x 10"^ M un-la b e l e d TA (Johnson et a l . , 1979a; I s h i i et a l . , 1972; Garola and McGuire, 1977). At varous times, the r e a c t i o n s were stopped by r e p l a c i n g the DMEM i n each p l a t e with 1 mL of i c e - c o l d C a + + - and Mg + + - f r e e PBS co n t a i n i n g 5 x 10 ^  M unlabeled TA. The samples then were washed three times with PBS by resuspension and c e n t r i f u g a t i o n a t 1,000 x g. C e l l s were lysed by 5 mL of 0.5% T r i t o n X-100 i n EDTA/saline c o n t a i n i n g 5 x 10~^ M unlabeled TA, to release cytoplasmic m a t e r i a l s while l e a v i n g the n u c l e i i n t a c t (Meneghini, 1976"). To ensure the complete rupture of cyt o -plasmic membranes, the c e l l suspension was vortexed v i g o r o u s l y three times f o r 2 minutes each. The n u c l e i were c o l l e c t e d by c e n t r i f u g a t i o n a t 1,000 x g for 10 minutes. The p e l l e t s were resuspended with PBS, and examined under the phase-contrast microscope at 150 X m a g n i f i c a t i o n (Hudson and Dimmock, 1977). A f t e r the removal of PBS, the p e l l e t s were s o l u b i l i z e d i n 2.5 mL of hypotonic s o l u b i l i z i n g b u f f e r , pH 7.4, c o n t a i n i n g C a C l 2 , MgCl 2, d i t h i o t h r e i t o l and g l y c e r o l . The suspension was sheared manually 10 times with a syringe with 25 gauge 5/8 inch hypodermic needle whenever the homogeneity of the s o l u b i l i z e d n u c l e i p r e p a r a t i o n was not s a t i s f a c -t o r y . (H) I s o l a t i o n o f chromatin from i s o l a t e d n u c l e i A l l procedures were c a r r i e d out at 4°C unless s p e c i f i e d other-wise. The chromatin was prepared from i n t a c t i s o l a t e d n u c l e i . The procedure o f Johnson and Baxter (1978) was used w i t h t h e i r m o d i f i c a t i o n s of c o n d i t i o n s of washing and storage (Johnson et a l . , 1979b). The chromatin was i s o l a t e d by a stepwise lowering o f the i o n i c s t r e n g t h . F i r s t , the nuclear p e l l e t was resuspended i n 10 mL of 80 mM NaCl, 20 mM EDTA, pH 6.3, and the ex t r a c t e d nuclear p e l l e t was c e n t r i f u -ged at 5,000 x g f o r 10 minutes and the supernatant removed. The p e l l e t was then resuspended i n 10 mL of 1.5 mM NaCl, 0.15 mM Na,>-c i t r a t e (0.01 SSC) pH 7.0 and r e c e n t r i f u g e d again. The p u r i f i e d chroma-t i n , as a c l e a r hydrated g e l , was s o l u b i l i z e d i n a b u f f e r c o n t a i n i n g 0.01 SSC and 25% (v/v) g l y c e r o l , and stored at -20°C. 112. ( I ) L i q u i d s c i n t i l l a t i o n counting The counting e f f i c i e n c y , background counts and quench e f f e c t s of the various solvents and c e l l components present i n experiments of s c i n -t i l l a t i o n counting were as f o l l o w s . The counting e f f i c i e n c y of t r i t i a t e d samples i n 10 mL of Aqua-•3 s o l - 2 using 50 mg of H-toluene standard was 45.5 ± 0.5$ (n=26). The background counts of various s o l v e n t s , i n c l u d i n g DMEM (20 y L ) , PBS (100 yL), T r i t o n X-100 with cytoplasm ( 100 yL), s o l u b i l i z i n g b u f f e r with n u c l e i or chromatin (200 yL) and 0 . 0 1 - 0.5 M Nap b u f f e r (3.2ml each), i n 10 mL o f Aquasol - 2 ranged from 2 1 - 2 9 cpm. Aquasol - 2 a l s o gave a background of 20 cpm. Thus, these solvents c o n t r i b u t e d no s i g n i f i c a n t counts. The quench e f f e c t of these v a r i o u s solvents with or without ad-d i t i v e s (cytoplasm, n u c l e i or chromatin) was n e g l i g i b l e . The r e c o v e r i e s 3 of r a d i o a c t i v i t y i n these a l i q u o t s , measured with known amounts of H-toluene standard ranged from 91 to 9 8 $ . Aquasol - 2 formed s i n g l e phase systems with these reagents. No quench c o r r e c t i o n therefore was a p p l i e d because experimental v a r i a t i o n s due to other sources were considered to be at l e a s t 5$. EXPERIMENTS (A) Test f o r p o s s i b l e c r o s s - l i n k i n g of DNA induced by g l u c o c o r t i c o i d s Tests f o r c r o s s - l i n k i n g of DNA were done with a e l l s incubated w i t h HC- or TA-containing DMEM. The concentrations were 1 yg/mL DMEM or 10 yg/mL DMEM f o r both g l u c o c o r t i c o i d s . The incu b a t i o n time v a r i e d between 0 and 96 hr. In stu d i e s with UV-A i r r a d i a t i o n , the c e l l s were i r r a d i a t e d by long wavelength UV-A f o r 20 min, 1 hr or 2 hr p r i o r to the harvest of c e l l s . The DNA was i s o l a t e d from harvested c e l l s using the procedure described i n M a t e r i a l s and Methods subsection B, and i t s p u r i t y was exa-mined by determining the *260/'A280 r a t i o > ^ n e amount of p r o t e i n r e -maining with the DNA as w e l l as the T and hyperchromicity. The amount m of the i s o l a t e d DNA was measured by Burton's diphenylamine assay or by estim a t i o n from A^gQ. Two techniques were u t i l i z e d to detect the presence of c r o s s -l i n k e d DNA by g l u c o c o r t i c o i d s : ( i ) hydroxyapatite chromatography of DNA samples, which were thermally denatured at 95° f o r 5 min, followed by rap i d c o o l i n g i n ice-water bath f o r another 5 min, and ( i i ) thermal scan-ning a n a l y s i s of n a t i v e DNA samples f o r e s t a b l i s h i n g the denaturation-r e n a t u r a t i o n p r o f i l e s . The DNA i s o l a t e d from mouse L-929 f i b r o b l a s t s , i r r a d i a t e d with UV-A i n the presence o f 8-methoxypsoralen (8-M0P), 5 pg/mL PBS, served as the p o s i t i v e c o n t r o l of c r o s s - l i n k e d DNA to confirm that ( i ) the i s o l a -t i o n procedures of DNA d i d not conceal the presence of c r o s s - l i n k i n g and ( i i ) the two techniques selected to detect the c r o s s - l i n k i n g , HAP chroma-tography and den a t u r a t i o n - r e n a t u r a t i o n k i n e t i c s , are s u f f i c i e n t to detect c r o s s - l i n k i n g of DNA (Cech et a l . , 1979; Dall'Acqua et a l . , 1972; Cole, 1971). (B) Retention of H-TA during the i s o l a t i o n o f DNA This s e r i e s o f experiments i d e n t i f i e d steps i n i s o l a t i o n proce-dures, i f any, which removed JH-TA from nuclear DNA. I t i s s u f f i c i e n t to examine the steps of d e p r o t e i n i z a t i o n and HAP chromato-graphy . The n u c l e i i s o l a t e d from mouse L-929 f i b r o b l a s t s , t r e a t e d with 3 H-TA f o r 6 or 96 hours, were homogenized manually and deproteinized three times as described i n M a t e r i a l s and Methods B-1 and B - 2 . The three CHCl^/BuOH e x t r a c t s were c o l l e c t e d separately and evaporated to dryness under a ni t r o g e n stream. The residues were d i s s o l v e d i n 1 mL of 0.1 N NaOH (Kobayashi, 1978) and 10 mL of Aquasol - 2 was added f o r counting. The d e p r o t e i n i z e d DNA i n aqueous phase was chromatographed as described i n M a t e r i a l s and Methods B - 3 . F r a c t i o n s c o l l e c t e d were monitored f o r DNA with A^gQ measurement and for r a d i o a c t i v i t y with l i q u i d s c i n t i l l a t i o n counting by adding 3.2 mL of each f r a c t i o n to 10 mL of Aquasol - 2 . 3 (C) S p e c i f i c r e t e n t i o n of H-TA i n whole n u c l e i of c u l t u r e d mouse  L-929 f i b r o b l a s t s 3 The nuclear r e t e n t i o n of H-TA was determined by l i q u i d s c i n -t i l l a t i o n counting. T r i p l i c a t e samples o f 200 p i a l i q u o t s of s o l u b i l i z e d n u c l e i , obtained as described i n M a t e r i a l s and Methods s e c t i o n G, were counted i n v i a l s o f 10 mL of Aquasol - 2 . The r a d i o a c t i v i t y was normalized f o r nuclear p r o t e i n determined by Bio-Rad p r o t e i n assay, and f o r DNA de-termined by Burton's assay f o l l o w i n g the p r a c t i c e s of Johnson and Baxter (1978). The s p e c i f i c a l l y bound TA was determined by the commonly accept-ed procedure of s u b t r a c t i n g the n o n s p e c i f i c binding, determined i n p a r a l -l e l incubations c o n t a i n i n g 5 x 10~^ M unlabeled TA, from the t o t a l ( s p e c i f i c and n o n - s p e c i f i c ) binding measured i n the absence of unlabeled TA (Johnson et a l . , 1979a; I s h i i et a l . , 1972; Garola and McQuire, 1977). The data were converted from cpm/ug nuclear p r o t e i n and cpm/ug DNA to fmole/ug nuclear p r o t e i n and fmole/ug DNA, r e s p e c t i v e l y , by m u l t i -p l y i n g with the f a c t o r of 3.296 x 10 fmole/cpm. This conversion made the data i n u n i t s comparable with the l i t e r a t u r e data of cytoplasmic r e -ceptor b i n d i n g . (D) S p e c i f i c r e t e n t i o n of H-TA i n chromatin of c u l t u r e d mouse L-929  f i b r o b l a s t s 3 The s p e c i f i c r e t e n t i o n of H-TA was determined by l i q u i d s c i n -t i l l a t i o n counting. T r i p l i c a t e samples of 200 \L a l i q u o t s of s o l u b i l i z e d chromatin, prepared as described i n M a t e r i a l s and Methods subsection H, were counted i n v i a l s of 10 mL of Aquasol-2. The r a d i o a c t i v i t y was nor-malized f o r chromosomal p r o t e i n determined by Bio-Rad p r o t e i n assay, and f o r DNA determined by Burton's assay. The s p e c i f i c a l l y r e t a i n e d TA was determined by s u b t r a c t i n g the n o n s p e c i f i c a l l y r e tained TA, determined simultaneously i n i n c u b a t i o n s c o n t a i n i n g 5 x 10~^ M unlabeled TA, from the t o t a l ( s p e c i f i c and non-s p e c i f i c ) b i nding, measured i n the absence of unlabeled TA. The data were expressed as fmole/ug chromosomal p r o t e i n and fmoleAug DNA. The s p e c i f i c r e t e n t i o n of TA i n chromatin was p l o t t e d against the duration of experiments. 116. (E) Subchromatin l o c a l i z a t i o n of TA The HAP d i s s o c i a t i o n method, described by Bloom and Anderson (1978), f r a c t i o n a t e s chromosomal p r o t e i n s i n r e l a t i o n to t h e i r b i n d i n g p r o p e r t i e s to DNA. Hydroxyapatite powder, 2 gm, were prehydrated i n 10 mM NaP buf-f e r , pH 7.0. Columns as described p r e v i o u s l y ( M a t e r i a l s and Methods B-3-a) were used. Chromatin samples were then eluted s e q u e n t i a l l y with 20 mL p o r t i o n s of the 11 solvents l i s t e d i n Table V. The flow r a t e s were kept at 30 mL/hr/cm^ at H°C. F r a c t i o n s of 10 mL were c o l l e c t e d . The c o l l e c t e d f r a c t i o n s were monitored f o r H-TA by l i q u i d s c i n t i l l a t i o n counting of 1 mL of each f r a c t i o n i n 10 mL of Aquasol - 2 , and f o r d i s s o c i a t e d p r o t e i n s by measurements of absorbance at 230 nm (Bluthmann, 1977). The r a d i o a c t i v i t y and w e r e p l o t t e d against f r a c t i o n numbers. The subchromatin l o c a l i z a t i o n o f TA was i n d i c a t e d by these two p l o t s . V RESULTS AND DISCUSSION (A) Morphology of g l u c o c o r t i c o i d - t r e a t e d and g l u c o c o r t i c o i d - f r e e mouse  L-929 dermal f i b r o b l a s t s G l u c o c o r t i c o i d s induce d e f i n i t e morphological changes i n f i b r o -b l a s t s (Figure 25). G l u c o c o r t i c o i d - t r e a t e d L-929 f i b r o b l a s t s were l a r g e r , f l a t t e r , l e s s densely packed, and often more polygonal and e p i -t h e l i a l i n appearance than g l u c o c o r t i c o i d - f r e e c o n t r o l s . Such morpholo-g i c a l changes have been discussed i n d e t a i l by P r a t t (1978), Rasche and Ulmer (1968) and B e r l i n e r (1967b), and were considered to represent a phenotypic r e v e r s i o n from a "transformed, or tumor c e l l - l i k e s t a t e " to a more t i g h t l y regulated growth s t a t e (Wigler et a l . , 1975). (B) E v a l u a t i o n of c r o s s - l i n k i n g of DNA with g l u c o c o r t i c o i d s (1) Quantity and q u a l i t y of i s o l a t e d DNA y The q u a n t i t i e s of DNA i s o l a t e d by techniques described i n M a t e r i a l s and Methods subsection B from mouse L-929 and human dermal f i b r o b l a s t s were measured using e i t h e r Burton's assay or estimation from *260" ^ n e a b s o r D a n c e u n i t at 260 nm was assumed to be equivalent to 50 yg of DNA/mL (DuVivier et a l . , 1976). P a r i s h (1972) has suggested t h a t one u n i t of A^gQ represents 66.7 Ug of DNA/mL. The value of 50 yg/mL was used, however, since the l a t t e r value provided b e t t e r agreement be-tween the two techniques f o r DNA q u a n t i t a t i o n . With human c e l l s , the DNA ranged from 6.8 to 9.2 yg per 10^ c e l l s , which were comparable to the l i t e r a t u r e value, 8.30 ± 0.45 yg per 10^ c e l l s (Fujimoto et a l . , 1977). The amounts of DNA recovered from mouse L-929 f i b r o b l a s t s were higher, from 8.4 to 17.5 y g per 10^ c e l l s . The higher DNA content of mouse L-929 f i b r o b l a s t s can be e x p l a i n -Incubation time (hr) G l u c o c o r t i c o i d -t r e a t e d G l u c o c o r t i c o i d -f ree 0,2,4,6,24 48,72,96 Figure 25 : Morphology of g l u c o c o r t i c o i d - t r e a t e d and g l u c o c o r t i c o i d -f r e e mouse L-929 f i b r o b l a s t s ed by the aneuploidy of mouse L-929 c e l l s , which have upwards of 65 chro-mosomes per c e l l compared to human f i b r o b l a s t s with 46 chromosomes. The p u r i t y was accepted as adequate f o r t h i s research i f the i s o l a t e d DNA was as pure as commercial DNA (Sigma, type I ) . The l a t t e r i s claimed by the manufacturer to be e s s e n t i a l l y f r e e of RNA and p r o t e i n , and i s a widely used reference standard. I t i s not p o s s i b l e to i s o l a t e a b s o l u t e l y pure DNA from c e l l s . The f o l l o w i n g three c r i t e r i a were used. (a) The r a t i o of absorbance, ^GO^ZSO The reference standard of commercial DNA (Sigma, type I) gave a mean value ± S.D. of the r a t i o of A 2 6 o / A 2 8 0 ' 1 , 6 9 1 0 , ° ^ ^ n = 8 ^ ' t h e r e f o r e , the DNA i s o l a t e d from c u l t u r e d f i b r o b l a s t s were acceptable i f the r a t i o A 2 6 c / A 2 8 0 w a s n^Sher than 1.65. The l i t e r a t u r e c r i t e r i o n r e q u i r i n g a r a t i o higher than 1.80 (DuVivier et a l . , 1978) was not adopt-ed f o r the above mentioned p r a c t i c a l reason. (b) P r o t e i n content: v DNA preparations c o n t a i n i n g l e s s than 5.4$ (w/w) of p r o t e i n , measured by the Bio-Rad assay, were admissible f o r f u r t h e r s t u d i e s . The usual c r i t e r i o n f o r " s u b s t a n t i a l l y " p r o t e i n - f r e e DNA i s l e s s than 1$ (Meneghini, 1976; P a r i s h , 1972). Since Sigma DNA showed a l s o higher pro-t e i n content ( 5 . 4 $ ) , we accept the r e s u l t as a r t i f a c t u a l l y high due to high blank values i n r e l a t i o n to the small amounts of the p r o t e i n found i n DNA. (c) T_ and hyperchromicity m *-The melting p o i n t , T^, and the hyperchromicity value of the i s o l a t e d DNA from c u l t u r e d f i b r o b l a s t s were examined. The i s o l a t e d DNA i n DSC had a melting point of 71.2 ± 0.7°C (n = 9) and hyperchromicity o f 30.0 ± 4.1$ (n = 9 ) . These values were com-parable to those of the standard of c a l f thymus DNA, 70.3 ± 0.3°C (n = 5 ) , and f e l l w i t h i n the range of l i t e r a t u r e values (Marmur and Doty, 1962; Spodheim-Maurizot et a l . , 1979). The m e l t i n g curves of i s o l a t e d DNA corresponded to those r e p o r t -ed i n l i t e r a t u r e f or s u b s t a n t i a l l y pure DNA. The data of thermal scan-ning a n a l y s i s was ther e f o r e accepted as evidence of adequate p u r i t y o f the DNA, i s o l a t e d by the techniques reported here, f o r the purpose of t h i s p r o j e c t . The discrepancies observed between the A^gg/^gQ r a^^° as w e l l as the p r o t e i n values observed and the r e s p e c t i v e l i t e r a t u r e values were a t t r i b u t a b l e to methodological l i m i t a t i o n s . (2) Examination of c r o s s - l i n k i n g of DNA (a) Hydroxyapatite chromatography Hydroxyapatite chromatography was employed f o r three d i f f e r e n t purposes. F i r s t l y , i t was applied f o r i s o l a t i n g DNA from homogenate of c e l l l y s a t e . Secondly, hydroxyapatite chromatography was adapted to d i s -t i n g u i s h denature^ single-stranded DNA from n a t i v e double-stranded or from renatured n a t i v e - l i k e double-stranded DNA. T h i r d l y , i t was a p p l i e d f o r f r a c t i o n a t i n g chromosomal p r o t e i n s s e l e c t i v e l y from chromatin, pre-sented i n Results and Di s c u s s i o n subsection F. Examination of c r o s s -l i n k i n g of DNA u t i l i z e d the f i r s t two p r o p e r t i e s of hydroxyapatite to separate DNA from other components o f c e l l l y s a t e , i n i t s n a t i v e form, and to d i s t i n g u i s h between i t s denatured s i n g l e - s t r a n d e d and renatured double-stranded forms when subjected to heat denaturation. A l i n e a r concentration gradient i n s t e a d of stepwise e l u t i o n technique was employed, to avoid the a r t i f a c t u a l " f a l s e peak", which has been reported to be associated with the stepwise e l u t i o n technique ( T i s e -l i u s et a l . , 1956; Hjerten, 1959). Sodium phosphate (NaP), which i s s l i g h t l y l e s s e f f e c t i v e than potassium phosphate (KP), was used as the e l u t i n g agent. The DNA of deproteinized c e l l homogenates was w e l l resolved from other c e l l u l a r components, presumably RNA and p r o t e i n , and e l u t e d between 0 .28 and 0 . 33 M sodium phosphate b u f f e r , pH 6 . 8 (Figure 2 6 ) . The e a r l y peak of the chromatogram, el u t e d at 0.04 - 0 .08 M sodium phosphate b u f f e r , consisted of some u n i d e n t i f i e d A2gQ-absorbing species and homogenization b u f f e r . Meinke et a l . (1974) found these low s a l t f r a c t i o n s contained p r o t e i n and sheared n u c l e i c a c i d s (RNA and/or DNA). Traces of chloroform/butanol l e f t a f t e r the d e p r o t e i n i z a t i o n step made no c o n t r i b u t i o n to the magnitude of t h i s peak. Two peaks e l u t i n g between 0 .12-0.25 M were u n i d e n t i f i e d . How-ever, they were believed to be r i b o n u c l e i c a c i d (RNA) on the basis of the reported chromatographic behaviour of RNA (Bernardi, 1971a) . The a b i l i t y of hydroxyapatite to separate n a t i v e double-stranded and denatured sin g l e - s t r a n d e d DNA was demonstrated i n Figure 27. The thermally denatured single-stranded DNA was eluted from hydroxyapatite columns at a sodium phosphate m o l a r i t y of 0 .18 - 0 .23 M, d i s t i n c t l y lower than that of e l u t i n g n a t i v e DNA, 0 .27 - 0.31 M. The renatured as w e l l as denatured but c r o s s - l i n k e d DNA behaved i d e n t i c a l l y to the n a t i v e DNA and were seen to be eluted at the same m o l a r i t y as n a t i v e DNA, as expected from l i t e r a t u r e reports (Bernardi, 1969b) . The recovery of n a t i v e DNA was q u a n t i t a t i v e , whereas that of denatured DNA was only 34-60$. This was expected due to the aggregation of the denatured DNA molecules caused by r e s i d u a l p r o t e i n and/or i n t e r -molecular base p a i r i n g (Bernardi, 1971b) . The DNA i s o l a t e d from (8-MOP)-treated and ( U V - A ) - i r r a d i a t e d mouse L -929 f i b r o b l a s t s were s p l i t i n t o two a l i q u o t s . One a l i q u o t was thermally denatured at 95°C f o r 5 minutes followed by r a p i d c o o l i n g i n an ice-water bath f o r another 5 minutes. The denatured a l i q u o t and the 0 10 20 30 40 50 60 Fraction No. Figure 26 : Representative chromatogram of deproteinized c e l l homogenate applied to HAP column i—» Fraction No. Figure 27 : Representative chromatogram of n a t i v e and denatured DNA applied to HAP column S.S. : s i n g l e - s t r a n d e d DNA D.S. : double-stranded DNA to n a t i v e counterpart were eluted through the i n d i v i d u a l hydroxyapatite columns simultaneously. The nativ e DNA preparation contained only double-stranded DNA, while the denatured DNA con s i s t e d of both denatured single-stranded and renatured double-stranded DNA. The renatured double-stranded peak i n d i -cated the presence of c r o s s - l i n k e d DNA. The magnitudes of the peak o f the c r o s s - l i n k e d DNA were s i m i l a r i n cases o f UV-A i r r a d i a t i o n s from 20 minutes to two hours i n the presence of 8-M0P. The hydroxyapatite chro-matograms of native and denatured DNA i s o l a t e d from (8-M0P)-UVA t r e a t e d mouse L-929 f i b r o b l a s t s (Figure 28), t h e r e f o r e , served as a p o s i t i v e con-t r o l f o r d e t e c t i n g the presence of c r o s s - l i n k e d DNA induced by gluco c o r -t i c o i d and glucocorticoid-UVA treatments. The DNA i s o l a t e d from hydrocortisone- or triamcinolone aceto-n i d e - t r e a t e d mouse or human dermal f i b r o b l a s t s , w i t h or without UV i r r a -d i a t i o n fhad no detectable double-stranded DNA remaining a f t e r the b o i l -c o o l denaturation. The g l u c o c o r t i c o i d - t r e a t e d c e l l s were, t h e r e f o r e , u n l i k e l y to possess c r o s s - l i n k e d DNA molecules at a detectable l e v e l . Twenty-three studies with various combinations o f (1) type of g l u c o c o r t i -c o i d , hydrocortisone or triamcinolone acetonide, (2) conc e n t r a t i o n of g l u c o c o r t i c o i d , 1 yg/mL or 10 pg/mL media, (3) c e l l l i n e , human or mouse L-929 dermal f i b r o b l a s t , (4) incu b a t i o n p e r i o d , 0,2,4,8,12,24,48,72,84,96 or 108 hours, and (5) with or without UV-A i r r a d i a t i o n , as w e l l as va r i o u s periods of i r r a d i a t i o n , 20 minutes, 1 hour or 2 hours, were con-ducted as summarized i n Table X I I I . However, the denatured DNA from a l l these s t u d i e s showed only a s i n g l e peak i n d i c a t i n g that only s i n g l e -stranded molecules were present. Therefore, hydroxyapatite chromato-graphy showed no evidence f o r c r o s s - l i n k i n g induced by g l u c o c o r t i c o i d or glucocorticoid-UVA treatments ( F i g u r e 29). Fraction No. Figure 28 : HAP chromatography of DNA i s o l a t e d f rom (8—MOP)—treated and UV—A i r r a d i a t e d mouse L—929 f i b r o b l a s t s ho Table X I I I Conditions examined to i n v e s t i g a t e the formation of c r o s s - l i n k e d DNA by g l u c o c o r t i c o i d s G l u c o c o r t i c o i d Concentration C e l l l i n e yg/mL media Incubation time, hr. UV-A i r r a d i a t i o n i n t e r v a l , hr. L-929 HC TA 10 1 1 10 10 10 0,2,4,8,12,24,48,72, 96 4, 12,24, 96 0,2,4,8,12,24,48, 96 96 0 24, 96 1/3, 1, 2 2 Human HC TA 4, 12,24,48,72,84,96 0,2,4,8,12,24,48,72,84,96, 108 (*) : Twenty-three independent experiments were c a r r i e d out with v a r i o u s combinations of conditions l i s t e d i n the t a b l e . r > 0 - 0 Before thermal denaturation Fraction No. ;ure 29 : HAP chromatography of DNA i s o l a t e d from HC-, TA- or TA-UV treated mouse L-929 f i b r o b l a s t s (b) Denaturetion-renaturation k i n e t i c s Thermal scanning a n a l y s i s o f DNA has been used c o n v i n c i n g l y by Pathak et a l . (1977) to demonstrate c r o s s - l i n k i n g of s k i n DNA by 8-MOP a f t e r exposure to UV-A i r r a d i a t i o n . With t h i s technique, the c r o s s - l i n k -ed DNA i s detectable by v i r t u e of i t s c h a r a c t e r i s t i c d e naturation-r e n a t u r a t i o n p r o f i l e (Figure 3 0 ) . I n v e s t i g a t i o n f o r c r o s s - l i n k i n g of DNA from g l u c o c o r t i c o i d t r e a t e d and g l u c o c o r t i c o i d p l u s UV-A tr e a t e d f i b r o -b l a s t s were made using t h i s technique with a p o s i t i v e c o n t r o l of DNA i s o -l a t e d from 8-MOP treated and UV-A i r r a d i a t e d mouse L-929 f i b r o b l a s t s . The r e s u l t s of t h i s study are summarized with the a i d of the o p t i c a l melting curves and re n a t u r a t i o n p r o f i l e s shown i n F i g u r e s 31-33. The denaturation p r o f i l e s o f Figure s 30-33 demonstrated no ap-p r e c i a b l e d i f f e r e n c e i n e i t h e r T m or hyperchromicity between DNA sam-ples of untreated f i b r o b l a s t s (Figure 33) and those of f i b r o b l a s t s w i t h various treatments (Figures 30-32). However, the DNA i s o l a t e d from 8-MOP and UV-A tr e a t e d f i b r o b l a s t s , when cooled to 50 °C a f t e r thermal de-na t u r a t i o n up to 90°C, showed c l e a r evidence of r e n a t u r a t i o n r e f l e c t e d i n the r e n a t u r a t i o n p r o f i l e ( F i g u r e 3 0 ) . The percent r e n a t u r a t i o n of c r o s s - l i n k e d DNA was 77.7 ± 6.0$ (mean ± S.E. n = 5). On the other hand, DNA i s o l a t e d from ( i ) untreated L-929 and ( i i ) U V - i r r a d i a t e d L-929, as w e l l as the c a l f thymus DNA standard gave only 9.5 ± 1.8$ (mean ± S.E., n = 4 each) of re n a t u r a t i o n under the same c o o l i n g c o n d i t i o n s ( F i g u r e s 33 and 31 r e s p e c t i v e l y ) . The DNA samples i s o l a t e d from hydrocortisone- or triamcinolone acetonide-treated, or triamcinolone acetonide - UV trea t e d human or mouse L-929 f i b r o b l a s t s c o n s i s t e n t l y had l e s s than 10$ (8.1 ± 1.1$, n = 6) of re n a t u r a t i o n as the denatured samples were cooled to 50°C, demonstrat-i n g no detectable c r o s s - l i n k i n g due to the treatment o f g l u c o c o r t i c o i d or 30 Thermal scanning a n a l y s i s of DNA i s o l a t e d from (8-MOP)-treated and UV-A i r r a d i a t e d mouse L-929 f i b r o b l a s t s Temperature, ° C Figure 31 : Thermal scanning a n a l y s i s of DNA i s o l a t e d from UV-A i r r a d i a t e d mouse L-929 f i b r o b l a s t s U3 o Denaturation p r o f i l e Renaturation p r o f i l e F i g ure 32 : Thermal scanning a n a l y s i s of DNA i s o l a t e d from HC-, TA- or (TA-UV)-treated mouse L-929 f i b r o b l a s t s ure 33 : Thermal scanning a n a l y s i s of DNA i s o l a t e d from untreated mouse L-929 f i b r o b l a s t s 133. to the combination o f triamcinolone acetonide treatment and UV-A i r r a d i a -t i o n ( F i g u r e 3 2 ) . The approximate l i m i t of the d e t e c t a b i l i t y of c r o s s - l i n k s by the o p t i c a l r e n a t u r a t i o n method was k% (Cech et a l . , 1 9 7 9 ) . Based on the data of both HAP chromatography and denaturation-r e n a t u r a t i o n k i n e t i c s , n e i t h e r the treatments w i t h hydrocortisone or triamc i n o l o n e acetonide nor the combination of triamcinolone acetonide and UV-A i r r a d i a t i o n caused detectable c r o s s - l i n k i n g of DNA i n c u l t u r e d human and mouse L - 9 2 9 dermal f i b r o b l a s t s under the experimental c o n d i -t i o n s . (C) Retention o f H-triamcinolone acetonide during the i s o l a t i o n o f DNA When the deproteinized c e l l homogenates were chromatographed through HAP columns, only background l e v e l s of r a d i o a c t i v i t y could be recovered i n the el u a t e s (Table XIV). I t was concluded, t h e r e f o r e , t h a t 3 d e p r o t e i n i z a t i o n of H-TA c o n t a i n i n g n u c l e i by t r i p l e e x t r a c t i o n w i t h CHCl^/BuOH removed q u a n t i t a t i v e l y a l l t r a c e s of r a d i o a c t i v i t y (Table XV). This observation r u l e d out the p o s s i b i l i t y of recovering DNA bound-g l u c o c o r t i c o i d from whole c e l l s by the use of t h i s r a t h e r d r a s t i c i s o l a -t i o n procedure. I t does not exclude the p o s s i b i l i t y of a weak hydrogen bonding between the complex and the DNA as suggested by Duax et a l . (1976) f o r deoxycorticosterone and adenine. (D) S p e c i f i c r e t e n t i o n o f TA i n whole n u c l e i of mouse L - 9 2 9 f i b r o b l a s t s ( 1) D e s c r i p t i o n of the pr e p a r a t i o n of n u c l e i The clean preparations of the n u c l e i were r e p r o d u c i b l y obtained as judged by examination under the phase-contrast microscope. The n u c l e i were grey, o v a l p a r t i c l e s with black n u c l e o l i and re g u l a r membrane sur-1 134. Table X i v HAP chromatography of de p r o t e i n i z e d DNA from H-TA tre a t e d mouse L-929 f i b r o b l a s t s Appearance B K G Recovered counts F r a c t i o n NaP (3.2 mL i n Count Recovery due to ^ H - T A , cpm No. (M) 10 mL Aquasol-2) (cpm) % (b) Gross Net 1 0.01 Gel 21.5 66.9 32.3 10.7 3 0.35 24.5 67.0 30.4 5.9 5 0.05 25.8 70.8 29.0 3.2 7 0.065 25.7 69.2 28.9 3.2 9 0.085 25.6 67.0 33.2 7.6 11 0. 1 25.5 67.3 27.5 2.0 13 0.115 Gel wi t h l i q u i d 26.7 67.0 32.7 6.0 15 0.13 26.3 67.6 29.3 3.0 17 0.15 26.9 68.2 28.4 1.5 19 0.165 24.9 66.3 27.3 2.4 21 0.18 27.4 65.1 31.1 3.7 23 0.2 Clear l i q u i d 22.9 68.9 29.4 6.5 25 0.215 26.2 68.3 28.9 2.7 27 0.23 29.8 70.2 30.2 0.4 29 0.245 28.4 68.4 26.5 -1.9 31 0.26 28.1 75.6 30.3 2.2 33 0.28 24.6 77.0 27.5 2.9 35 0.295 25.6 79.1 24.7 -0.9 37 0.31 25.9 80.7 26.5 0.6 39 0.325 L i q u i d w i t h ppt. 27.4 80.6 27.5 0.1 41 0.345 27.3 82.7 25.6 -1.7 43 0.36 25.0 81.9 27.1 2.1 45 0.375 27.5 83.4 25.7 -1.8 47 0.39 25.1 85.3 28.8 3.7 49 0.41 25.9 81.6 28.7 2.8 51 0.425 25.1 89.2 26.8 1.7 53 0.44 27.8 87.0 24.9 -2.9 55 0.46 26.1 84.9 27.0 0.9 57 0.475 27.4 88.6 28.1 0.7 59 0.49 23.9 86.0 24.7 0.8 (a) Background counts of i n d i v i d u a l eluents 3 (b) Recoveries obtained w i t h H-toluene standards T n h l e XV Q u a n t i t n t t v n rcmnv.nl o f r a d f o n c t 1 v i tv bv d o p r o t p i n i za11 on w i t h CHC1. / RtiOH m i x t u r e Appearance (0.5 mL, Recovered counts 0.1 N NnOH . . due to 3H-TA, cpm CHCl^BuOH In 10 mL BKG Recovery Cross' e x t r a c t Aquasol-2) Count, cpm % (b) (n = 4) Net (c) F i r s t C l e a r l i q u i d 1807.8 Second Turbid l i q u i d A3.8 Third Turbid l i q u i d 119.6 T o t a l 88.2 2555.8 + 75.9 7A8.0 848.1 88.7 355.8 ± 11.0 312.0 351.8 85.6 555.0 ± 28.9 435.A 508.6 1708.5 ±93.3 (a) Background counts of i n d i v i d u a l e x t r a c t s 3 (b) Recoveries obtained w i t h H-toluene standards (c) Adjusted f o r % recovery T o t a l r a d i o a c t i v i t y i n n u c l e i before d e p r o t e i n i z a t i o n was 1705 cpm; th e r e f o r e , the t o t a l recovery of r a d i o a c t i v i t y due to d e p r o t e i n i z a t i o n was 100.2 ± 5.5% faces. Only a f a i n t " s k e l e t o n " of cytoplasm was n o t i c e d . Most of the granular cytoplasmic contents had been s o l u b i l i z e d . The u n i f o r m i t y o f the pr e p a r a t i o n was s a t i s f a c t o r y as r e f l e c t e d by c o e f f i c i e n t s of v a r i a t i o n of l e s s than 10$, among t r i p l i c a t e samples of measurements o f r a d i o a c t i v i t y , DNA and p r o t e i n . (2) Time p r o f i l e of s p e c i f i c r e t e n t i o n on whole n u c l e i (a) Gross uptake per pooled c e l l s o f 5 p l a t e s 3 The H-TA taken up i n t o the n u c l e i was measured by s o l u b i l i z -ing the i s o l a t e d n u c l e i from the pooled c e l l s of 5 p l a t e s , c o n t a i n i n g approximately 4 - 6 x 10^ per p l a t e , followed by the l i q u i d s c i n t i l l a -3 t i o n counting o f a l i q u o t s of the H-TA i n s o l u b i l i z e d n u c l e i . The time p r o f i l e of t h i s process of uptake was examined by ha r v e s t i n g c e l l s 0.5, 3 6, 24, 48, 72 and 96 hours a f t e r a d d i t i o n o f H-TA to the c e l l s . 3 The amounts of H-TA r e t a i n e d by n u c l e i were tabulated as cpm/5 p l a t e s and fmole/5 p l a t e s i n Table XVI. The nuclear r e t e n t i o n o f 3 H-TA increased with time from 12 to 361 fmole/5 p l a t e s reaching a maximum at 72 hr. The data were uncorrected f o r v a r i a t i o n s i n nuclear DNA and p r o t e i n content which occurred during the period of 96 hr incuba-t i o n examined. 3 In Figure 34, the amount of H-TA t r a n s l o c a t e d i n t o the n u c l e i increased markedly from 0.5 to 6 hours of i n c u b a t i o n , approximately t e n -3 f o l d . The amount of H-TA i n n u c l e i was then maintained r e l a t i v e l y 3 unchanged u n t i l 48 hours, at which time, the H-TA was f u r t h e r taken up to about twice the l e v e l at 24 hours. The amount o f nuclear r e t e n t i o n then l e v e l e d o f f again over the remaining time period examined o f 48 to 96 hours. 3 The increase i n nuclear r e t e n t i o n of H-TA during the f i r s t 6 Table XVI Gross uptake of H-TA Into the n u c l e i of pooled c e l l s from 5 p l a t e s Gross nuclear r e t e n t i o n Incubation cpm/5 p l a t e s fmole/5 p l a t e s time, hr. A< C ) B (d) A ( C ) B ( d> 0.5 366 + 79 ( 2 0 % ) ( e ) 48 + 49 ( 1 0 0 % ) ( e ) 12.1 ± 2.6 1.6 ± 1.6 6 4035 + 1054 (26%) 182 + 95 (52%) 131.5 ± 34.7 6.0 ± 3.1 24 3483 + 441 (13%) 235 + 49 (21%) 114.8 ± 14.5 7.7 ± 1.6 48 7861 + 1824 (23%) 341 + 174 (51%) 259.1 ± 60.1 11.2 ± 5.7 72 10962 + 2451 (22%) 907 + 310 (34%) 361.3 ± 80.8 29.9 ± 10.2 96 7519 + 1819 (24%) 707 + 564 (80%) 247.8 ± 60.0 23.3 f 18.6 (a) n = 6, average of s i x measurements from two independent s e r i e s of experiments (b) TA : 3.296 x 10~ 2 fmole/cpm (c) 10~ 8 M 3H-TA without unlabeled TA (d) 10~ 8 M 3H-TA w i t h unlabeled TA (e) C.V. ( c o e f f i c i e n t of v a r i a t i o n ) =(S.D./mean)x 100% 12 ^ 10 8 a 6 E 3 n = 6 Bar : 10" 8 M 3H-TA without unlabeled TA 10 8 M 3H-TA w i t h 500 X unlabeled TA of two independent experiments y ± 1 S.E. R • A T A \ • T A • • • • A T • • A T A T A • _ A T A T A • A • A • A T A T A T A • A • A • A • A • A T A • A • A T A T A T A T A T A T A T A T A T A T A T A T A T A T A T A T A T A T A T A T AXT A T A T A T A T A T A T A T A T A T A T A T A T 24 48 72 Incubation time, hr. 96 V) CD a 3 1 0 a> "5 o o E x 1 o Figure 34 : Gross uptake of H-TA i n t o whole n u c l e i of pooled c e l l s of 5 p l a t e s . 139. hours of i n c u b a t i o n was gradual since the DNA content and the number of c e l l s d i d not change appreciably during t h i s p eriod. (b) S p e c i f i c nuclear r e t e n t i o n of H-TA normalized f o r equal  amount of DNA or nuclear p r o t e i n The data presented above were normalized on the basis of DNA or nuclear p r o t e i n s since both new DNA and p r o t e i n s were synthesized during the incubation periods up to 96 hr. The normalized data are presented i n Figures 35 and 36 . 3 The H-TA s p e c i f i c a l l y r e t a i n e d i n n u c l e i , expressed as fmole/yg DNA, ranged from 0.762 ± 0.180 (mean ± S.E., n = 9 of three i n -dependent experiments) to 2.775 ± 0.645 (n = 6) fmole/yg DNA v a r y i n g w i t h the periods of i n c u b a t i o n time (Table X V I I ) . These data were comparable to l i t e r a t u r e values f o r other l i n e s of c e l l s : f o r dexamethasone, i t was 2 .3 fmole/yg DNA i n GC c e l l s and 1.5 fmole/yg DNA i n S49 c e l l s a f t e r 4 hours of incubation (Johnson et a l . , 1979), and 1.29 ± 0.2 (mean ± S.E., n = 17) pmole/mg DNA i n r a t hepatoma c e l l s a f t e r at l e a s t 30 minute incubation (Rousseau et a l . , 1973). 3 The time p r o f i l e of t h i s s p e c i f i c nuclear r e t e n t i o n of H-TA i s i l l u s t r a t e d i n Figure 35. As can be seen, a marked increase i n nuclear r e t e n t i o n occurred between 0.5 and 6 hours, then a s i g n i f i c a n t drop was observed at 24 hours. The s p e c i f i c a l l y r e t a i n e d TA l e v e l s i n n u c l e i remained constant afterwards up to 96 hours (Figure 35). A s i m i l a r p r o f i l e of s p e c i f i c nuclear r e t e n t i o n was observed when data were normalized f o r equal amounts of nuclear p r o t e i n s ( F i g u r e 3 6 ) . The maximal r e t e n t i o n was achieved by 6 hours with a s i g n i f i c a n t drop to a plateau l e v e l afterwards which remained at constant l e v e l up to the 96 hours measured. The k i n e t i c s of nuclear binding of g l u c o c o r t i c o i d s during the 3 < z Q O) Q) O E 1 h 9 • • • • .5 • • 9 9 • • • • TT7 m m • • • • 9 • • • • 15 • • • • • • • • • • 24 48 72 96 I ncubation time , hr, Figure 35 : S p e c i f i c r e t e n t i o n of TA i n whole n u c l e i , normalized on DNA b a s i s o • J.B • • 9 • • • • • • 9 • • 15 • • • • • • 6 24 48 72 Incubation time , hr. 96 Figure 36 : S p e c i f i c r e t e n t i o n of TA i n whole n u c l e i , normalized on nuclear p r o t e i n basl Table XVII S p e c i f i c r e t e n t i o n of TA i n whole n u c l e i Incubation No. of S p e c i f i c r e t e n t i o n ^ )  time,hr. samples( a) fmole/yg DNA fmole/yg nuclear p r o t e i n s 0.5 9 . 0.762 + 0.180 0.073 + 0.011 6 6 2.775 + 0.645 0.396 + 0.042 24 9 1.076 + 0.293 0.264 + 0.053 48 9 1.125 + 0.248 0.287 + 0.041 72 9 0.998 + 0.115 0.283 + 0.011 96 15 1.115 + 0.140 0.206 + 0.042 (a) Number of samples of 2 - 5 independent experiments (b) Mean ± S.E. 143. f i r s t few hours have been widely studied by various procedures (e.g. Rousseau et a l . , 1973; Rousseau, 1975; I s h i i et a l . , 1972; Middlebrook et a l . , 1 9 7 5 ) . However, no data have been reported on the e f f e c t o f pro-longed incubation on the nuclear r e t e n t i o n of TA. Rousseau et a l . (1973) reported that i n hepatoma c e l l s exposed to dexamethasone at 37°C, the nuclear binding of s t e r o i d reached a maximum w i t h i n 30 minutes and then l e v e l e d o f f . Rousseau ( 1975) a l s o claimed that the plateau p e r s i s t e d as long as the s t e r o i d was present. However, h i s study of the nuclear binding of dexamethasone l a s t e d only 90 minutes, with only the 90 minute data point beyond 30 minutes to demon-s t r a t e the plateau (Figure 5 ) . I s h i i et a l . ( 1972) and Middlebrook et a l . ( 1975) studied the 3 s u b c e l l u l a r d i s t r i b u t i o n of H-triamcinolone acetonide i n mouse L-929 f i b r o b l a s t s , and reported that the time p r o f i l e of the TA-binding i n the s o l u b l e c y t o s o l f r a c t i o n reached a maximum by 20 minutes and remained constant f o r 6 hours ( I s h i i et a l . , 1 9 7 2 ) . However, no comparable time p r o f i l e f o r nuclear binding was reported i n t h e i r s t u d i e s . There are good reasons to i n v e s t i g a t e nuclear r e t e n t i o n beyond the 6 hours commonly studied, e s p e c i a l l y when the c e l l s are exposed to l a r g e concentration g r a d i e n t s over long periods i n dermatological uses of g l u c o c o r t i c o i d s . The generation time of the L - 9 2 9 f i b r o b l a s t s i s about 23 hours (Baserga, 1 9 7 6 ) . During t h i s time, the s u s c e p t i b i l i t y of the c e l l s to drug a c t i o n v a r i e s . New p r o t e i n s and n u c l e i c a c i d s are synthesized. The c e l l d i v i d e s a f t e r 23 hours. A DNA c y c l e with p r o g r e s s i v e l y i n c r e a s i n g * number of strand breaks has been suggested ( C o l l i n s , 1977), posing the question whether DNA i s more a c c e s s i b l e to g l u c o c o r t i c o i d s at one time than another. 144. The g l u c o c o r t i c o i d s , according to some (e.g. Voorhees, 1977b) , act during the phase of the c e l l c y c l e as i n h i b i t o r s of m i t o s i s . According to others, g l u c o c o r t i c o i d s act at a l l stages of the c e l l c y c l e (Baserga, 1976) . The issue i s u n s e t t l e d . More information on glucocor-t i c o i d a c t i o n i n n u c l e i i s needed. C l i n i c a l l y , the prolonged exposure o f g l u c o c o r t i c o i d s i s of i n t e r e s t , since both the development of r e s i s t a n c e to g l u c o c o r t i c o i d a c t i o n (DuVivier, 1976; DuVivier and Stoughton, 1977) and adverse e f f e c t s require a long time to develop. Another aspect of the previous s t u d i e s of nuclear r e t e n t i o n o f g l u c o c o r t i c o i d s , which may introduce u n c e r t a i n t y , i s that the published data have been obtained by means of measurement o f crude homogenates. Rousseau et a l . ( 1973) , f o r instance, separated the c e l l homogenate of 3 H-dexamethasone-incubated hepatoma c e l l s by c e n t r i f u g a t i o n at 1,200 x g f o r 5 minutes i n t o c y t o s o l - c o n t a i n i n g supernatant and a p e l l e t . A f t e r washing, the r a d i o a c t i v i t y of the p e l l e t was measured. S i m i l a r l y , I s h i i et a l . (1972) and Middlebrook et a l . (1975) f r a c t i o n a t e d the ruptured c e l l s by c e n t r i f u g a t i o n at 7 ,000 x g f o r 3 minutes i n t o c y t o s o l - c o n t a i n -i n g supernatant and the nuclear p e l l e t . In c o n t r a s t , the measurements reported here involved the i s o l a t i o n of clean i n t a c t n u c l e i , r a t h e r a crude f r a c t i o n of a nuclear p e l l e t , p r i o r to the measurement of r a d i o -a c t i v i t y . The data obtained here had much l e s s p o s s i b i l i t y of b e i n g d i s -t o r t e d by contamination with c y t o s o l r a d i o a c t i v i t y . 3 This study c l e a r l y demonstrated the presence of H-TA i n the n u c l e i of c u l t u r e d f i b r o b l a s t s throughout the time p e r i o d o f 96 hr examined. Therefore, the absence of c r o s s - l i n k i n g of DNA with glucocor-t i c o i d s reported i n Re s u l t s and D i s c u s s i o n s e c t i o n B could not be a s c r i b -ed to an absence of g l u c o c o r t i c o i d s i n the n u c l e i of the f i b r o b l a s t s examined f o r c r o s s - l i n k i n g . 145 . (?) E f f e c t s of TA on t o t a l nuclear p r o t e i n and DNA content In n u c l e i (a) S e l e c t i o n and r e l i a b i l i t y of p r o t e i n and DNA assays Bio-Rad p r o t e i n assay k i t was employed to q u a n t i t a t e the con-taminating p r o t e i n of p u r i f i e d DNA, and the p r o t e i n contents o f prepara-t i o n s of n u c l e i or chromatin. Commercially obtained c a l f thymus DNA which i s widely accepted as s u b s t a n t i a l l y p r o t e i n - f r e e p r e p a r a t i o n served as a c o n t r o l . This assay has advantages over other p r o t e i n assays, e s p e c i a l l y the widely used Lowry method (Lowry et a l . , 1951), because ( i ) i t i s much eas i e r to use, r e q u i r i n g one reagent and f i v e minutes to perform as com-pared to three reagents and 30-10 minutes t y p i c a l f o r the Lowry assay, ( i i ) the absorbance of dye-protein complex i s r e l a t i v e l y s t a b l e , not r e q u i r i n g the c r i t i c a l timing necessary f o r the Lowry assay and ( i i i ) i t i s f r e e from most of the i n t e r f e r e n c e s which l i m i t the a p p l i c a t i o n of the Lowry assay. The s e n s i t i v i t y l i m i t of the assay i s 1 yg per sample, com-parable to that of Lowry method. The r e p r o d u c i b i l i t y of the c a l i b r a t i o n curve was s a t i s f a c t o r y . The C.V. of the slopes of the curves was l e s s than 5% among runs. The reagent blank c o n t a i n i n g the sample b u f f e r and the dye r e -agent r e s u l t e d i n a reddish-brown s o l u t i o n with an absorbance charac-t e r i s t i c a l l y about 0.425. The r e l a t i v e l y high c o l o r y i e l d of the blank was normal (Bio-Rad b u l l e t i n , 1979), and d i d not a f f e c t the l i n e a r i t y , r e p r o d u c i b i l i t y or s e n s i t i v i t y o f the assay. DNA i n t e r f e r e s with the assay, however, when present i n concen-t r a t i o n s of more than 1 mg/mL (Bio-Rad b u l l e t i n , 1979). The highest DNA * content i n the samples of t h i s p r o j e c t was kept to l e s s than 0.2 mg/mL; the r e f o r e , no i n t e r f e r e n c e by DNA was noted. The s o l u b i l i z i n g b u f f e r used does not i n t e r f e r e w i t h the Bio-Rad 146. assay, although a l l f i v e of i t s components, T r i s - H C l , CaCl,,, MgCl 2, d i t h i o t h r e i t o l and g l y c e r o l are known to i n t e r f e r e with the Lowry assay. The widely used Burton's technique f o r DNA determinations gave s a t i s f a c t o r y r e p r o d u c i b i l i t y . Maximum d i f f e r e n c e i n slopes of c a l i b r a -t i o n curves was 3% (C.V.). I t was found however that our use of d i t h i o -t h r e i t o l - c o n t a i n i n g b u f f e r f o r s o l u b i l i z i n g nucleus required p r i o r d i a l y -s i s against 0.5 N HC10^, otherwise, no c o l o r was developed i n the pre-sence of the s o l u b i l i z i n g b u f f e r . Apparently, the reducing agent, d i t h i o t h r e i t o l , prevented the formation of S c h i f f ' s base between the amine and deoxyribose. The d i a l y s i s removed the i n t e r f e r e n c e completely. The technique i s s p e c i f i c f o r nuclear DNA. RNA, i n amounts of 2000 ug g i v e s a detectable c o l o r e q u i v a l e n t to 1.4 yg of DNA (Burton, 1956). Considering the minute q u a n t i t i e s of RNA present i n n u c l e i , the extent of t h i s i n t e r f e r e n c e was considered n e g l i g i b l e . There was no i n t e r f e r e n c e due to the presence of p r o t e i n i n t e s t samples. (b) V a r i a t i o n s i n the nuclear p r o t e i n l e v e l o f TA-treated and  TA-free mouse L-929 f i b r o b l a s t s as a f u n c t i o n of time The t o t a l nuclear p r o t e i n l e v e l i n pooled c e l l s of 5 p l a t e s were determined u s i n g t r i p l i c a t e a l i q u o t s of s o l u b i l i z e d n u c l e i by Bio-Rad p r o t e i n assay. In Figure 37, the t o t a l nuclear p r o t e i n contents, i n mg, were p l o t t e d against the i n c u b a t i o n time, 0.5 - 96 hours. C o n t r o l c u l t u r e s , without TA treatment, possessed r e l a t i v e l y constant l e v e l s up to 6 hours, which corresponds to the time required f o r the g l u c o c o r t i c o i d to reach i t s maximal nuclear r e t e n t i o n . The nuclear p r o t e i n l e v e l s approximately doubled, f i r s t , by 24 hours, and then again by 48 hours. Beyond 48 hours, the nuclear p r o t e i n contents no longer increased and l e v e l e d o f f up to 96 hours (Fi g u r e 37). 2.0 Without TA -8 o°l With 10 M TA G 3 With 5.01 x 10 6 M TA 1.5 § 1.0 .5 .5 24 48 72 Incubation time. hr. • • a • • • a l a • a • a • • • • 96 Figure 37 T o t a l nuclear p r o t e i n l e v e l s i n TA-free and TA-treated c u l t u r e s as a f u n c t i o n of time. Maximum C.V. i s 5%, n = 3. 148. TA-treated c u l t u r e s at two concentrations of 10~ M and 5.01 x 10"^ M showed profoundly lower nuclear p r o t e i n contents (Table X V I I I ) . P l o t t e d as percent suppression r e l a t i v e to c o n t r o l c u l t u r e p r o t e i n l e v e l s against time (Figure 38), i t i s seen that the i n h i b i t i o n of p r o t e i n f o r -mation becomes more pronounced at the l a t e r stages of i n c u b a t i o n , 24 to 96 hr. The nuclear p r o t e i n l e v e l s were nearly 50% lower than i n u n t r e a t -ed c u l t u r e s a f t e r 24 to 96 hr of TA i n c u b a t i o n . P r a t t i n h i s review (1978) sta t e d that general c e l l u l a r p r o t e i n s y n t h e s i s i n L c e l l s was not i n h i b i t e d by g l u c o c o r t i c o i d s , but t h a t s e l e c t i v e i n h i b i t i o n to the synthesis of some i n d i v i d u a l p r o t e i n s might occur. Wong and Aronow (1976) reported s e l e c t i v e i n h i b i t i o n of i n c o r -p o r a t i o n of r a d i o a c t i v e amino acid s i n t o a l y s i n e - r i c h h istone component i n L c e l l n u c l e i . No f u r t h e r studies of TA e f f e c t on nuclear p r o t e i n l e v e l s f o r various incubation times seem to have been reported. The higher dose of TA, 5.01 x 10~^ M exerted appreciably greater e f f e c t —8 than that of 10" M only a f t e r 48 hours of i n c u b a t i o n . (c) V a r i a t i o n s i n the DNA content i n n u c l e i of TA-treated and TA-f r e e mouse L-929 f i b r o b l a s t s as a f u n c t i o n of time The DNA content i n pooled c e l l s of 5 p l a t e s were assayed w i t h t r i p l i c a t e a l i q u o t s of s o l u b i l i z e d n u c l e i by Burton's diphenylamine tech-nique. In Figure 39, the t o t a l DNA contents, i n mg, were p l o t t e d against i n c u b a t i o n time. C o n t r o l c u l t u r e s without the treatment of TA maintained r e l a t i v e l y constant l e v e l s of DNA up to 6 hours. The amount of DNA increased more than two-fold by 24 hours, and about f o u r - f o l d by 48 hours. The DNA l e v e l s remained r e l a t i v e l y constant beyond 48 hours up to 96 hours ( F i g u r e 39). The DNA content of TA-treated c u l t u r e s showed, w i t h respect to Table XVIII Total nuclear protein l e v e l s i n TA-free and TA-treated cultured mouse L-929 f i b r o b l a s t s at various incubation i n t e r v a l s Incubation . time, hr. Nuclear protein content (a) TA-free co n t r o l 1.0 x 10 M TA yg % suppression —6 5.0 x 10 M TA (b) % suppression (b) 0.5 505.8 378.5 25.2 481.0 5.1 6 508.3 477.7 '6.0 500.1 1.6 24 942.2 801.7 14.9 821.5 12.8 48 1804.4 1308.6 27.5 1060.7 41.2 72 2008.4 1037.3 48.4 719.1 64.2 96 1702.5 917.4 46.1 731.5 57.0 (a) average of t r i p l i c a t e measurements. Maximum C.V. i s 5% (b) calculated as (nuclear protein) -LA.— .. - (nuclear •free protein),,,. v TA-treated x 100% (nuclear p r o t e i n ) ^ -free VO 60 |DO| 10 8 M TA 1=3 5.01 x 10 6 M TA § 40 c "8 20 CD O • • • • • • • • • • • • • • • .5 • • • • • • • • n r • • • o • • • • • • • • • • • • 24 48 Incubation time, hr 72 96 Figure 38 : Suppression of t o t a l nuclear p r o t e i n s induced by TA at l e v e l s of 10 and 5.01 x 10 M Maximum C.V. i s 5%, n = 3 .8 .6 O) E "5 c .E .4 < z Q (0 2 o • E 3 Without TA With 10" 8 M TA With 5.01 x I 0 " 6 M TA a a a a a a a a a a a a a D • a • o • o • a a a a • • a • • a • a • • D .5 6 24 48 Incubation time, hr. 72 96 F i g u r e 39 : DNA l e v e l s i n n u c l e i of TA-free and TA-treated mouse L-929 f i b r o b l a s t s as a f u n c t i o n of time. Maximum C.V. i s 5%, n = 3 152. the above c o n t r o l s , suppression of DNA. The suppression was s i m i l a r to the TA e f f e c t on nuclear p r o t e i n s , but occurred e a r l i e r , a f t e r 24 hours rather than 48 hours. The percent of suppression of DNA content v a r i e d —8 with the concentration of the g l u c o c o r t i c o i d , and was 40-65$ f o r 10 M and 50-73$ f o r 5.01 x 10~ D M TA under the same incu b a t i o n c o n d i t i o n s (Table XIX, Figure 40). At 24 hours, both concentrations o f TA exerted s i m i l a r degrees of suppression of DNA l e v e l s . At 48-96 hours, c u l t u r e s r e c e i v i n g 5.01 x 10~° M TA had a greater e f f e c t . These i n h i b i t o r y e f f e c t s were expected from known g l u c o c o r t i c o i d a c t i o n s . The suppressive e f f e c t on DNA synthesis has been evaluated by 3 measuring decreased i n c o r p o r a t i o n of H-thymidine i n t o DNA ( P r a t t , 1978; P r a t t and Aronow, 1966; Armelin and Armelin, 1978; A d o l f and Swetly, 1979). P r a t t and Aronow studied the i n h i b i t i o n of DNA synth e s i s _7 i n L-929 f i b r o b l a s t s by f l u o c i n o l o n e acetonide, 5 x 10 M, and by hydrocortisone, 5 x 10~^ M at 3, 6, 12 and 24 hours. They found that the i n h i b i t i o n was i n i t i a l l y evident w i t h i n 6 hours. A f t e r 24 hours o f exposure, the i n c o r p o r a t i o n of thymidine was i n h i b i t e d by about 50$. S i m i l a r s tudies on L-929 f i b r o b l a s t s over s e v e r a l days of exposure to hydrocortisone demonstrated that the maximum i n h i b i t i o n of thymidine i n -c o r p o r a t i o n was about 70$ and was a t t a i n e d at 24 hours ( S e i f e r t and H i l z , 1966). Armelin and Armelin (1978) reported that t h i s i n h i b i t o r y a c t i o n of hydrocortisone, at the p h y s i o l o g i c a l l e v e l o f 0.3 Pg/mL i n ST 1 c e l l s , increased p r o g r e s s i v e l y with time of in c u b a t i o n , reaching a maximum a f t e r 20 hours. In Adolf and Swetly's s t u d i e s (1979), i t was observed t h a t a f t e r 48 hours of incubation of human lymphoid c e l l s with 10 uM t r i a m -c i n o l o n e , the i n h i b i t i o n of DNA s y n t h e s i s was 86 ± 10$, and t h i s e f f e c t was dose-dependent. The data from t h i s study show the same magnitude of suppression Table XIX DNA l e v e l s i n n u c l e i of TA-free and TA-tre.-i red cultured mouse L-929 f i b r o b l a s t s at various incubation i n t e r v a l s Incubation time, hr. DNA c o n t e n t ^ TA-free control 1.0 x 10~ 8 M TA 5.0 x 10 6 M TA Kg i'g „ . (b) A suppression i'g - . (b) h suppression 0.5 137.7 - - 131.2 4.7 6 153.6 - - 176. 1 -24 512.4 223.4 56.4 225.3 56.0 48 774.3 462.8 40.2 317.4 59.0 72 852.8 298.8 65 234.3 72.5 96 710. 1 371.6 47.7 280.4 60.5 (a) Average of t r i p l i c a t e measurements. Maximum C.V. i s 5%. Incubation time, hr. Suppression of DNA l e v e l i n n u c l e i induced by TA at concentrations and 5.01 x 10~ 6 M. Maximum C.V. i s 5%, n = 3 155. of DNA l e v e l by TA by d i r e c t measurement of the amount of DNA i n n u c l e i as those reported i n l i t e r a t u r e by i n d i r e c t methods and demonstrated that although no d i r e c t e f f e c t o f TA on the DNA s t r u c t u r e ( c r o s s - l i n k i n g ) could be e s t a b l i s h e d , b i o l o g i c a l e f f e c t s c l e a r l y i n v o l v i n g DNA, and depending on the presence o f TA, such as the i n h i b i t i o n of nuclear pro-t e i n and DNA formation, do indeed occur. The data reported here confirm-ed that the c e l l l i n e used f o r i n v e s t i g a t i n g the g l u c o c o r t i c o i d - i n d u c e d c r o s s - l i n k i n g i s responsive to the treatment of g l u c o c o r t i c o i d . 3 (4) I n t r a c e l l u l a r d i s t r i b u t i o n of JH-TA as a f u n c t i o n of time 3 The i n t r a c e l l u l a r d i s t r i b u t i o n s of H-TA upon the incubation w i t h L-929 f i b r o b l a s t s at 37°C for varying periods of time were deter-mined. The harvested c e l l s were washed three times w i t h 5 mL of i c e - c o l d phosphate buffered s a l i n e (PBS) to remove the adhering e x t r a c e l l u l a r TA. In order to prevent the l o s s of i n t r a c e l l u l a r TA i n the washing process, unlabeled TA, 5 x 10 ° M, was added i n PBS to maintain the same concen-t r a t i o n gradient between the i n s i d e and the outside of the cytoplasmic membrane. The washed c e l l s were lysed with 0.5% of T r i t o n X-100 i n EDTA/saline followed by one wash with i c e - c o l d PBS. The r a d i o a c t i v i t i e s present i n the T r i t o n X-100 l y s a t e and i n the subsequent PBS wash were defined as the cytoplasmic TA. The nuclear TA was measured as the r a d i o -a c t i v i t y i n the preparation of the lysed n u c l e i . The data on i n t r a c e l l u l a r d i s t r i b u t i o n were obtained from two to seven independent s t u d i e s , each c o n s i s t i n g of an average of three mea-—8 3 surements. In the cases of incubation with 10" M H-TA alone, the t o t a l amount o f TA taken up ' i n t o the c e l l s increased p r o p o r t i o n a l l y between 0.5 and 1 hours, and then was constant up to 96 hours (Figure 11; Table XX). The d i s t r i b u t i o n of i n t r a c e l l u l a r TA between cytoplasm" and nucleus was r e l a t i v e l y constant with v a r i e d incubation times. The per-.5 4 6 24 Incubation time, hr. 48 72 96 Figure A l : I n t r a c e l l u l a r d i s t r i b u t i o n of H-TA between cytoplasm and nucleus i n the absence or the presence of 500-fold unlabeled TA Table XX I n t r a c e l l u l a r d i s t r i b u t i o n of H-TA between cytoplasm and nucleus i n the absence or the presence of 500-fold unlabeled TA Without unlabeled TA With 500-fold unlabeled TA  Incubation No. of T o t a l i n t r a c e l l u l a r % d i s t r i b u t i o n T o t a l i n t r a c e l l u l a r % d i s t r i b u t i o n time, hr. samples a c t i v i t y , cpm Cytoplasm N u c l e i a c t i v i t y , cpm Cytoplasm N u c l e i 0.5 15 12493 + 1821 95.3 + 0.9 4.7 + 0.9 4120 + 899 97.1 + 0.8 2.9 + 0.8 2 6 71088 + 3174 88.5 + 3.5 11.5 + 3.5 7678 + 1037 95.5 + 1.8 4.5 + 1.8 4 6 111694 + 6761 91.0 + 0.2 9.0 + 0.2 9376 + 1429 97.1 + 0.4 3.0 + 0.4 6 18 116396 + 21077 94.0 + 0.9 6.1 + 0.9 16295 + 4386 97.7 + 0.5 2.4 + 0.5 24 12 100451 + 15906 95.0 + 0.8 5.0 + 0.8 17369 + 3752 98.4 + 0.3 1.6 + 0.3 48 9 112491 + 14957 90.8 + 1.4 9.2 + 1.4 27344 + 5036 98.9 + 0.5 1.1 + 0.5 72 9 114417 + 14563 89.4 + 0.4 10.6 + 0.4 39567 + 9804 97.8 + 0.8 2.2 + 1.4 96 12 114835 + 24335 93.5 + 1.0 6.5 + 1.0 45711 + 6893 98.6 + 0.2 1.4 + 0.2 data were presented as mean ± S.E. centage of c e l l u l a r TA i n n u c l e i were i n the range o f 4.7 to 11.5$ (Figure 42). On the other hand, when 500-fold of unlabeled TA was i n c u -3 bated together with H-TA, the c e l l u l a r TA was mainly l o c a t e d i n c y t o -plasm. Cytoplasmic TA c o n s t i t u t e d 95.5 to 98.9$ of c e l l u l a r TA. The 3 H-TA taken up i n t o n u c l e i under t h i s incubation c o n d i t i o n (with un-l a b e l e d competitors) was at a n e g l i g i b l e l e v e l . The t o t a l amount of 3 H-TA t r a n s f e r r e d i n t o c e l l s increased g r a d u a l l y upon prolonged incuba-t i o n s between 6 and 96 hours; however, no appreciable change was observed i n t h e i r nuclear l e v e l s (Figure 41). Middlebrook et a l . (1975) determined the s u b c e l l u l a r d i s t r i b u -t i o n of g l u c o c o r t i c o i d receptors i n mouse L-929 f i b r o b l a s t s by i n c u b a t i n g c e l l s at 37 °C f o r 1 hour with 10' 8 M 3H-TA, with and without the a d d i t i o n of 1000-fold unlabeled g l u c o c o r t i c o i d as competitors. They found i n t r a c e l l u l a r TA-receptor complexes located i n cytoplasmic, nuclear e x t r a c t a b l e and nuclear r e s i d u a l f r a c t i o n s with percentages of 80 ± 8, 11 ± 7 and 9 ± 4$, r e s p e c t i v e l y . The nuclear r e t e n t i o n of H-TA was approximately double the value reported here. However, the design of the experiment was d i f f e r e n t i n s e v e r a l important aspects. In t h e i r s t u d i e s , the c e l l s were ruptured with a hypotonic b u f f e r a f t e r 1 hour of incuba-t i o n with TA, followed by a b r i n g i n g up the c e l l homogenate to an i s o -t o n i c c o n d i t i o n p r i o r to the c e n t r i f u g a t i o n at 7,000 x g. They defined the supernatant as the cytoplasmic f r a c t i o n . Nuclear e x t r a c t a b l e f r a c -t i o n s were those e x t r a c t e d from nuclear p e l l e t s w i t h 0.3 M KC1-0.01 M T r i s b u f f e r , l e a v i n g the non-extractable p o r t i o n as the "nuclear r e s i d u a l f r a c t i o n " . In the work of Middlebrook et a l . , the nuclear p e l l e t s were composed of both n u c l e i and cytoplasmic d e b r i s ; t h e r e f o r e , i t i s not sur-p r i s i n g that t h e i r r e s u l t s gave the higher r a d i o a c t i v i t y i n nuclear f r a c -t i o n s than that reported here, because of contamination due to c y t o p l a s -ion •*—> c CD 8> v_ cellu 100 • 80 • total 60 • o 40 ' 6^ 20 • Cytoplasm F l M l l Nucleus 15 , 6 18 tlL_ 12 to 4 6 24 48 Incubation time, hr 72 ft 12 i S L . 96 Figure 42 : Percent d i s t r i b u t i o n of t o t a l c e l l u l a r H-TA between cytoplasm and nucleus mic d e b r i s . In t h i s t h e s i s , the c e l l l y s i s was done more s e l e c t i v e l y by r u p t u r i n g only the cytoplasmic membrane. Moreover, the nuclear r e t e n t i o n 3 of H-TA was measured a f t e r i s o l a t i n g i n t a c t n u c l e i to ensure minimal contamination due to cytoplasmic p a r t i c u l a t e . The leakage of nuclear components and TA through the nuclear membrane i n the process of i s o l a t i n g n u c l e i was a p o s s i b i l i t y . As an' a d d i t i o n a l precaution, unlabeled TA was included i n both l y s i n g agent and washing b u f f e r f o r maintaining constant concentration gradient o f TA between the i n s i d e and the outside of the nuclear membrane, which i n 3 t u r n , would a i d i n preventing net l o s s of H-TA from n u c l e i regardless of the p o s s i b l e leakage of other nuclear components. (E) S p e c i f i c r e t e n t i o n of^H-TA i n chromatin (1) C h a r a c t e r i z a t i o n o f i s o l a t e d chromatin The c r i t e r i o n employed to c h a r a c t e r i z e the i s o l a t e d chromatin was the r a t i o o f protein/DNA i n chromatin. The range of t h i s r a t i o f o r the samples reported here was from 1.33 to 1.82. The composition of chromatin from p u r i f i e d n u c l e i o f P 815 c e l l s had protein/DNA r a t i o o f 1.50 ( P r e s c o t t , 1977). The chromatin i s o l a t e d from r a t l i v e r , which has been used as a model f o r chromatins i n general, possessed a histone/DNA r a t i o of 1.0 and non-histone chromosomal p r o t e i n to DNA i n r a t i o of 0.6, or a combined r a t i o of t o t a l chomosomal p r o t e i n to DNA of 1.6 (Bonner, 1979). A r a t i o s u b s t a n t i a l l y greater than 1.6 suggests contamination by nonchromosomal p r o t e i n s adherent to the chromatin (Bonner, 1979). (2) Time p r o f i l e o f s p e c i f i c r e t e n t i o n i n chromatin The s p e c i f i c r e t e n t i o n of TA i n chromatin was i n v e s t i g a t e d by i s o l a t i n g chromatin from i n t a c t n u c l e i , and the r a d i o a c t i v i t y was measur-ed a f t e r the s o l u b i l i z a t i o n of chromatin with 0.01 S S C - g l y c e r o l . The TA s p e c i f i c a l l y r e t a i n e d i n chromatin was normalized f o r equal amounts of DNA or chromosomal p r o t e i n (Table XXI). The maximum r e t e n t i o n o f TA i n chromatin, 1.1 fmole/yg DNA was seen to occur between 4 and 6 hours a f t e r i n c u b a t i o n , while a decrease i n the r e t e n t i o n was observed a t 24 hours, a f t e r which time the l e v e l was maintained up to 96 hours (Figure 43). Normalization of the r a d i o a c t i v e data by equal amounts o f chromosomal p r o t e i n gave a s i m i l a r p r o f i l e of the s p e c i f i c r e t e n t i o n o f TA i n chroma-t i n (Figure 44) with the maximum r e t e n t i o n of 0.6 fmole/yg chromosomal p r o t e i n occuring between 4 and 6 hours a f t e r i n c u b a t i o n . 3 The s p e c i f i c r e t e n t i o n of H-TA i n chromatin over extended periods of incu b a t i o n time as reported i n t h i s t h e s i s has not been r e -ported before. Middlebrook et a l . (1975) and Aronow (1979) d i d f i n d a 3 t i g h t l y bound f r a c t i o n of H-TA i n the n u c l e i of L-929 c e l l s which could not be d i s s o c i a t e d from the n u c l e i with 0.3 M KC1. They however worked with crude nuclear p e l l e t s r a t h e r than chromatin and thus only had a t e n t a t i v e b a s i s f o r t h e i r suggestion that TA i s re t a i n e d w i t h i n the chromatin. The time p r o f i l e of the s p e c i f i c r e t e n t i o n of the TA here 3 described confirms that the uptake of H-TA i n t o the n u c l e i indeed r e -s u l t s i n a t i g h t a s s o c i a t i o n with the chromatin, i n the v i c i n i t y of DNA molecules, over the 96 hours examined. 3 (3) I n t r a n u c l e a r d i s t r i b u t i o n of H-TA The d i s t r i b u t i o n of nuclear TA between nucleoplasm and chromatin was a l s o i n v e s t i g a t e d . The TA r e t a i n e d i n nucleoplasm was defined opera-t i o n a l l y as the f r a c t i o n s of r a d i o a c t i v i t y which could be removed by l y s i s of nuclear membrane with NaCl-EDTA (80 mM NaCl, 20 mM EDTA, pH 6.3) and subsequent wash with 0.01 SSC (1.5 mM NaCl, 0.15 mM N a 2 - c i t r a t e pH 7.0). The r a d i o a c t i v i t y remaining i n the g e l - l i k e chromatin p e l l e t r e -presented the TA re t a i n e d i n chromatin. The percent of nuclear TA i n Table XXI S p e c i f i c r e t e n t i o n of TA i n chromatin Incubation No. of S p e c i f i c r e t e n t i o n  time, hr. samples( a) fmole/ug DNA fmole/ug chromosomal p r o t e i n s 0.5 9 0.052 + 0.019 0.032 + 0.007 2 6 0.436 + 0.046 0.339 + 0.025 4 6 1.065 + 0.182 0.530 + 0.026 6 9 1.025 + 0.297 0.565 + 0.026 24 6 0.747 + 0.038 0.297 + 0.083 48 3 0.712 + 0.040 0.423 + 0.033 72 3 0.728 + 0.031 0.315 + 0.021 96 6 0.726 + 0.053 0.266 + 0.012 (a) Number of samples of 1 - 3 independent experiments. (b) Mean ± S.E. 1.2 < z Q O) D _CD o E .8 EJsD .5 2 4 6 24 Incubation time, hr. f * l • • • • • • • • • • • • • • 3 3 • • • • • • • • • • • • • • • • • • • • 48 72 96 Figure 43 : S p e c i f i c r e t e n t i o n of TA i n chromatin, normalized on DNA b a s i s ON u> c '53 •*—> 2 CL "Hi .6 .4 o E 9 o ZJ O E 6 24 48 72 96 Incubation time, hr. Figure 44 : S p e c i f i c r e t e n t i o n of TA i n chromatin, normalized on chromosomal p r o t e i n b a s i s 165. chromatin was always more than 50$, ranging from 56.5 ± 6.4$ to 73-9 ± 5.6$ of t o t a l TA r e t a i n e d i n n u c l e i , v a r y i n g w i t h the inc u b a t i o n times. The remaining TA was i n nucleoplasm, ranging from 26.1 ± 5.6$ to 43.5 ± 6.4$ (Table XXII; Figures 45 and 46). The incubation with unlabeled competitor caused the H-TA up-take i n t o n u c l e i to become n e g l i g i b l e . Under these c o n d i t i o n s , most nuclear TA was located i n the nucleoplasm (71.2 ± 7.0$) f o r f i r s t 2 hours of i n c u b a t i o n , and remained between 43.1 ± 6.4$ and 47.9 ± 7.4$ f o r the r e s t of incubation i n t e r v a l s . Higgins et a l . reported that when i n t a c t c e l l s were exposed to s t e r o i d s and c e l l n u c l e i were then f r a c t i o n a t e d , g l u c o c o r t i c o i d - r e c e p t o r complexes were found only i n chromatin but not i n the nucleolus, nucleo-plasm and nuclear membrane, quoting t h e i r unpublished observations ( H i g -gin s et a l . , 1979). However, i n our s t u d i e s , chromatin only r e t a i n e d the ma j o r i t y o f nuclear TA, 57-74$. With the remaining TA l o c a t e d i n e x t r a -chomosomal compartments of the nucleus. Higgins et a l . used d i f f e r e n t c e l l l i n e s from ours f o r the study, which might account f o r t h i s discrepancy. (F) Subchromatin l o c a l i z a t i o n of^H-TA A p r e l i m i n a r y attempt was made using the technique of Bloom and 3 Anderson (1978) to f r a c t i o n a t e the H-TA c o n t a i n i n g chromatin i n t o v a r i o us c l a s s e s of histone and non-histone p r o t e i n s by hydroxyapatite chromatography. The f r a c t i o n s o f HAP el u a t e s were monitored by both A^^Q measurement and l i q u i d s c i n t i l l a t i o n counting. S p e c i f i c d i s s o c i a t i o n p a t t e r n s of t o t a l chromosome p r o t e i n s are i l l u s t r a t e d i n Figure 47. I t was obtained from chromatin immobilized on HAP as e l u t e d with NaCl and NaCl plus urea s o l u t i o n s . To examine the d i s s o c i a t i o n p a t t e r n of TA r e t a i n e d i n chromatin, Table XXII I n t r a n u c l e a r d i s t r i b u t i o n of H-TA between nucleoplasm and chromatin i n the absence or the presence of 500-fold unlabeled TA Without unlabeled TA With 500-fold unlabeled TA  Incubation No. of T o t a l i n t r a n u c l e a r % d i s t r i b u t i o n T o t a l i n t r a n u c l e a r % d i s t r i b u t i o n  time, cpm samples a c t i v i t y , cpm Nucleoplasm chromatin a c t i v i t y cpm Nucleoplasm chromatin 0.5 9 704 + 177 43.5 + 6.4 56.5 + 6.4 191 + 66 71.2 + 11.1 28.8 + 11.1 2 6 7097 + 1114 31.0 + 0.8 69.1 + 0.8 365 + 185 71.2 + 7.0 28.8 + 7.0 4 6 9980 + 470 26.1 + 5.6 73.9 + 5.6 280 + 74 45.5 + 3.0 54.5 + 3.0 6 12 7944 + 1268 27.0 + 4.5 73.0 + 4.5 397 + 146 43.1 + 6.4 56.9 + 6.4 24 6 6525 + 1846 33.2 + 3.0 66.8 + 3.0 291 + 115 36.6 + 3.3 63.5 + 3.3 48 3 16564 + 833 44.5 + 2.3 55.5 + 2.3 165 + 43 46.1 + 5.2 53.9 + 5.2 72 3 14515 + 680 24.7 + 1.6 75.3 + 1.6 406 + 22 53.0 + 2.9 47.0 + 2.9 96 6 9315 + 3569 32.6 + 12.0 67.4 + 12.0 593 + 13 47.9 + 7.4 52.1 + 7.4 data were presented as mean ± S.E. E 16 Q. O T— >N 12 > o 03 O 8 "D E "D 0 tain 4 CD DC H Nucleoplasm Chromatin Nucleoplasm Chromatin without unlabeled TA wit h unlabeled TA 4 6 24 48 Incubation time, hr. 72 96 Figure 45 : I n t r a n u c l e a r d i s t r i b u t i o n of H-TA between nucleoplasm and chromatin i n the absence the presence of 500-fold unlabeled TA .5 2 4 6 24 48 72 96 Incubation time, hr. 3 Figure 46 : Percent d i s t r i b u t i o n of t o t a l nuclear H-TA between nucleoplasm and chromatin E c o CO CM co CD o c CO n i _ o CO n < 1 . 0 r .8 L .6 .4 t .2 A B C D E F G NHP H1 H 2 A H 3 H 2 B H 4 _ n H i NHP K NA 9 11 13 15 Fraction No. 17 19 21 23 Figure 47 : D i s s o c i a t i o n pattern of t o t a l chromosomal proteins from immobilized chromatin on HAP column as eluted with NaCl followed by NaCl and urea Letters,A-K, NHP, NA, H , H^A, H^B, II 3 and H 4,are referred to Table V. ON 170. JH-TA-containing and TA-free chromatin were chromatographed i n a p a r a l -l e l f a s h i o n . Approximately 75$ of the s p e c i f i c a l l y bound TA was d i s s o -c i a t e d from the HAP-immobilized chromatin along with p r o t e i n s designated by Bloom and Anderson (1978) as those of unbound chromosomal p r o t e i n s ( F r a c t i o n A, B and C, eluted with 0 to 0.25 M NaCl). Less than 13$ of TA w as d i s s o c i a t e d along with histone p r o t e i n s by i n c r e a s i n g the NaCl con-c e n t r a t i o n to 2 M ( F r a c t i o n s D, E, F and G). The remaining 12$ was eluted along with the t i g h t l y bound non-histones by treatment w i t h 2 M NaCl i n the presence of urea ( F r a c t i o n s H, I and J) (Figure 48). The t i g h t l y bound TA, reminiscent of the t i g h t l y bound estrogen receptor complexes reported by Bloom and Anderson were thought to be the nuclear r e s i d u a l forms of g l u c o c o r t i c o i d . Bloom and Anderson (1978) pro-posed the i n t e r a c t i o n of the t i g h t l y bound estrogen as a d i r e c t a s s o c i a -t i o n w i t h DNA or p o s s i b l y the a s s o c i a t i o n w i t h acceptor p r o t e i n s which i n turn were bound t i g h t l y to the DNA (Spelsberg, 1974; Puca et a l . , 1975; Mainwaring et a l . , 1976). G l u c o c o r t i c o i d s are be l i e v e d to share these p o s s i b i l i t i e s with estrogens except that the d i r e c t a s s o c i a t i o n of c r o s s - l i n k i n g i n case of g l u c o c o r t i c o i d has been found u n l i k e l y to be present. Bugany and Beato (1977) proposed the p o s s i b i l i t y that the TA-receptor complexes recognize other components of the chromatin, such as the chromosomal p r o t e i n s . Reports from other s t e r o i d target t i s s u e s have shown an i n t e r a c t i o n of the corresponding receptors with b a s i c and a c i d i c p r o t e i n s of the chroma-t i n (O'Malley et a l . , 1972; Puca et a l . , 1975). The data of t h i s study supported the view of Bugany and Beato that the H-TA which was s p e c i -f i c a l l y r e t a i n e d i n chromatin, was l a r g e l y a s s o c i a t e d with chromosomal pr o t e i n s r a t h e r than with DNA. Open histograms: without unlabeled TA Shaded histograms: w i t h 500-fold unlabeled TA L e t t e r s of A-K are r e f e r r e d to Table V. ^ 172 . SUMMARY A g a s - l i q u i d chromatographic a n a l y t i c a l technique was developed to quan-t i t a t e hydrocortisone, triamcinolone acetonide and desonide l e v e l s i n t i s s u e c u l t u r e media. The assay detects as l i t t l e as 20 ng of the gluco-c o r t i c o i d s . The a n a l y s i s of the three g l u c o c o r t i c o i d s i n propylene g l y c o l stock s o l u -t i o n s has demonstrated that the g l u c o c o r t i c o i d s are s t a b l e upon storage. The s t u d i e s of the g l u c o c o r t i c o i d s i n c u l t u r e media i n the presence of serum and f i b r o b l a s t s have shown that the g l u c o c o r t i c o i d s are chemically s t a b l e with no detectable formation of metabolites under the c o n d i t i o n s of i n c u b a t i o n . The concentrations of triamcinolone acetonide and desonide i n medium of c e l l c u l t u r e s f e l l 20% w i t h i n 2 hr. There a f t e r , the concentrations remained constant up to 84 hr. Therefore, f o r any b i o l o g i c a l e f f e c t of s y n t h e t i c g l u c o c o r t i c o i d s observed at the dose of 1 ug/mL medium, the g l u c o c o r t i -c o i d r e s p o n s i b l e f o r the observed e f f e c t was not more than 20% of the dose. The l o s s of HC from the media due to c e l l s was more than 40%, l a r g e r than that of s y n t h e t i c g l u c o c o r t i c o i d s . The HC a l s o had r e l a t i v e l y constant l e v e l s during the prolonged periods of in c u b a t i o n . The c r o s s - l i n k i n g of DNA wi t h g l u c o c o r t i c o i d s was i n v e s t i g a t e d by hydroxy-a p a t i t e chromatography and thermal scanning a n a l y s i s using (a) two c e l l l i n e s , mouse L-929 and human dermal f i b r o b l a s t s , (b) two g l u c o c o r t i c o i d s , HC and TA, (c) two g l u c o c o r t i c o i d c o n c e n t r a t i o n s , 1 ug/mL medium and 10 ug/mL medium, (d) various times of in c u b a t i o n ranging up to 96 hours, (e) wit h or without UV-A i r r a d i a t i o n . No c r o s s - l i n k i n g was detectable under these experimental c o n d i t i o n s . 173. (3) The uptake and r e t e n t i o n of JH-TA i n t o the nucleus and the e f f e c t of TA 3 on the amount of nuclear p r o t e i n s and DNA were determined. H-TA was pre-sent i n n u c l e i of c u l t u r e d f i b r o b l a s t s and suppressed the amount of nuclear p r o t e i n s and DNA. Thus, the absence of c r o s s - l i n k i n g of DNA w i t h glucocor-t i c o i d s was not due to the f a i l u r e of the t r a n s l o c a t i o n of TA i n t o the n u c l e i , and was not due to the unresponsiveness of the c e l l s to the gluco-c o r t i c o i d s . (4) The s p e c i f i c r e t e n t i o n of triamcinolone acetonide, at a c o n c e n t r a t i o n of —8 10 M i n c u l t u r e media, reached the highest l e v e l , 0.4 fmole/ug nuclear p r o t e i n or 2.8 fmole/ug DNA, i n i n t a c t n u c l e i of mouse L-929 f i b r o b l a s t s a f t e r 6 hours of i n c u b a t i o n . R e l a t i v e l y constant,lower l e v e l s , 0.2 -0.3 fmole/ug nuclear p r o t e i n or 1.0-1.1 fmole/ug DNA, were observed between 24 and 96 hours. (5) Approximately 10% of c y t o s o l - r e t a i n e d TA was t r a n s l o c a t e d i n t o the nucleus. The f r a c t i o n t r a n s l o c a t e d i n the nucleus remained r e l a t i v e l y unchanged up to 96 hours. (6) The s p e c i f i c r e t e n t i o n of t r i a m c i n o l o n e acetonide, at a c o n c e n t r a t i o n of —8 10 M i n c u l t u r e media, reached the highest l e v e l , 0.6 fmole/ug chromo-somal p r o t e i n or 1.1 fmole/ug DNA, i n chromatin of mouse L-929 f i b r o -b l a s t s a f t e r 6 hours of i n c u b a t i o n . R e l a t i v e l y constant,lower l e v e l s , 0.3-0.4 fmole/ug chromosomal p r o t e i n or 0.7 fmole/ug DNA, were observed between 24 and 96 hours. 3 —8 (7) The i n t r a n u c l e a r d i s t r i b u t i o n of H-TA, at the l e v e l of 10 M i n c u l t u r e medium, between nucleoplasm and chromatin was examined. The m a j o r i t y of * TA i n the nucleus, 56-75%, was a s s o c i a t e d w i t h chromatin, and the remain-i n g nuclear TA was l o c a t e d i n nucleoplasm. (8) A p r e l i m i n a r y i n v e s t i g a t i o n of subchromatin l o c a l i z a t i o n of "'H - T A was performed. Approximately 75% of the s p e c i f i c a l l y r e t a i n e d T A was d i s s o -c i a t e d from the HAP-immobilized chromatin along w i t h p r o t e i n s designated by Bloom and Anderson as those of unbound chromosomal p r o t e i n s . Less than 13% of TA was d i s s o c i a t e d along w i t h h i s t o n e p r o t e i n s . The remain-i n g 12% was e l u t e d along w i t h the t i g h t l y bound nonhistone p r o t e i n s . 3 V i r t u a l l y , no H-TA was e l u t e d w i t h n u c l e i c a c i d f r a c t i o n s ; t h e r e f o r e , t h i s study strengthens the view that g l u c o c o r t i c o i d s do not reach DNA i n i n t a c t c e l l s but, i n the form of a g l u c o c o r t i c o i d - r e c e p t o r complex, exert t h e i r e f f e c t s on DNA i n d i r e c t l y through some as yet to be charac-t e r i z e d p e r t u r b a t i o n of the s t r u c t u r e of nuclear p r o t e i n s . 175. APPENDIX I MATERIALS 1. 2. 3. 4. 5. 6. ACETALDEHYDE, Cat. No. 468, Eastman Kodak Co., Rochester, New York. ANTIBIOTIC-ANTIMYCOTIC MIXTURE (100 X), P e n i c i l l i n 10,000 units/mL, Streptomycin 10,000 yg/mL, Fungizone 25 yg/mL, Cat. No. 600-5240, Gibco, Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d , New York. AQUASOL-2, U n i v e r s a l L.S.C. C o c k t a i l , Lot. No. 229 AT9, New England Nuclear, Boston, Massachusetts. BIO-RAD P r o t e i n ASSAY KIT, c o n s i s t i n g of dye reagent ( c o n t a i n i n g phosphoric a c i d and methanol) concentrate ( c o n t r o l no. 18291) and p r o t e i n standard ( c o n t r o l no. 18329), Bio-Rad L a b o r a t o r i e s , 32nd & G r i f f i n , Richmond, C a l i f o r n i a . CALF THYMUS DNA, Type I , h i g h l y polymerized, Lot 48C-9580, Sigma Chemical Co., St. Lo u i s , M i s s o u r i . Ca , Mg - c o n t a i n i n g PBS ( f o r photoreaction) 0.002 M C a C l 2 C a ^ and M g ^ - f r e e PBS, (phosphate buffered s a l i n e ) , pH = 7.2. 0.05 M sodium phosphate b u f f e r (pH 6.9) 0.10 M NaCl 0.001 M MgCl 2 KCl 0.2 gm/L 0.2 gm/L 1.15 gm/L NaCl 8.0 gm/L P r e p a r a t i o n : Soln A KCl 4 gm/100 mL (Lot BBR, M a l l i n c k r o d t ) 4 gm/100 mL ( F i s h e r , Lot 742804) 176. Soln B Na 2HP0 l j 23 gm/500 mL (Lot BNP, M a l l i n c k r o d t ) For making 1 L: 8 gm of NaCl + 5 ml of s o l n A + 25 ml of s o l n B ? a d j u s t pH^H20 q.s. 8. DESONIDE, Lot No. CS-1-27, Dome l a b o r a t o r i e s , D i v i s i o n M i l e s Lab. Inc., West Haven, Connecticut. 9. DIALYSIS TUBING, S p e c t r a p o r ^ tubing, M.W. c u t o f f 12,000-14,000, c y l i n d . d i a . 6.4 mm., Spectrum Medical I n d u s t r i e s Inc., Terminal Annex, Los Angeles, C a l i f o r n i a . 10. DILUTE SALINE-CITRATE, DSC, 0.015 M NaCl and 0.0015 M sodium c i t r a t e pH 6.85. 11. DIPHENYLAMINE, Reagent ACS, Lost C8A, Eastman Kodak Co., Rochester, New York. 12. DIS0DIUM ETHYLENEDIAMINETETRAACETATE (Na 2~EDTA), Lot 770619, Cer-t i f i e d A.C.S., F i s h e r S c i e n t i f i c Co., F a i r Lawn, New Jersey. 13. DULBECCO'S MODIFIED EAGLE MEDIUM (DMEM), Powder, Cat. No. H-16, Gibco, Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d , New York. 14. ETHYL ACETATE, d i s t i l l e d i n g l a s s , Caledon, Georgetown, Ontario. 15. FETAL CALF SERUM, mycoplasm tested and v i r u s screened, Cat. No. 200-6140, Gibco, Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d , New York. 16. GLACIAL ACETIC ACID, Reagent A.C.S., Code 001019-005-59-0, A l l i e d Chemical Canada L t d . , Pointe C l a i r e , Quebec. 17. HYDROCORTISONE, Lot No. 402-9511, P f i z e r Co. L t d . , Montreal. 18. HYDROXYAPATITE, DNA grade, B i o - G e l ^ H T P , C o n t r o l No. 19350, BioRad L a b o r a t o r i e s , Richmond, C a l i f o r n i a . 19. HYPOTONIC SOLUBILIZING BUFFER, pH 7.4 c o n s i s t i n g o f 20 mM T r i s - H C l ( T r i s (hydroxymethyl) aminomethane, 177. J.T. Baker Chem. Co., N.J.) 2 mM C a C l 2 ( F i s h e r ) 2 mM MgCl 2 (J.T. Baker Chem. Co. N.J., Lot 601-6809) 3 mM d i t h i o t h r e i t o l (Lot 117C-0287, Sigma, stored at 0-5°C i n d e s s i c a t o r ) 5% g l y c e r o l 20. METHANOL, d i s t i l l e d i n g l a s s , Caledon, Georgetown, Ontario. 21. METHOXYAMINE HYDROCHLORIDE, Lot # 04239.19, P i e r c e Chemical Co., Rockford, I l l i n o i s . 22. 8-METH0XYPS0RALEN, 8-MOP, Lot 34C-1660, s o l u t i o n i n 50% EtOH, 100 ug/mL, Sigma, St. L o u i s , M i s s o u r i . 23. MOUSE L-929 DERMAL FIBROBLASTS, Passage No. of 578, 584 and 564, Flow L a b o r a t o r i e s , McLean, V i r g i n i a . 24. N-TRIMETHYLSILYL IMIDAZOLE, Lot # 0211774, 10 x 1 gm ampoules, P i e r c e Chemical Co., Rockford, I l l i n o i s . 25. PERCHLORIC ACID, HCIO^, 60$, a n a l y t i c a l reagent, Lot ENV, M a l l i n c -k r o d t Inc., St. Lou i s , M i s s o u r i . 26. PROGESTERONE, Lot No. 113C-0190, Sigma Chemical Co., St. Loui s , Mis-s o u r i . 27. PROPYLENE GLYCOL, Laboratory grade, F i s h e r S c i e n t i f i c Co., F a i r Lawn, New Jersey. 28. PYRIDINE, S i l y l a t i n g grade, P i e r c e Chemical Co., Rockford, I l l i n o i s . 29. REACTI-VIAL, P i e r c e Chemical Co., Rockford, I l l i n o i s . 30. SALINE/EDTA, pH = 7.4 c o n s i s t i n g of 100 mM NaCl (American S c i e n t i f i c & Chemical, Lot 63719) 10 mM Na2_EDTA ( C e r t i f i e d ACS. F i s h e r S c i e n t i f i c Co., Lot 770619) 178. 31. SODIUM BICARBONATE, a n a l y t i c a l reagent grade, M a l l i n c k r o d t Inc., S t . Louis , M i s s o u r i . 32. SODIUM CHLORIDE, c e r t i f i e d A.C.S., Lot 766294, F i s h e r S c i e n t i f i c Co., F a i r Lawn, New Jersey. 33. SODIUM CITRATE, t r i b a s i c , Lot 80029, a n a l y t i c a l reagent, BDH Chemi-c a l s , Toronto. 34. SODIUM DODECYL SULFATE (S.D.S.), Lot 772002, Laboratory grade, F i s h e r S c i e n t i f i c Co., F a i r Lawn, New Jersey. 35. SODIUM HYDROXIDE, Lot 764282, C e r t i f i e d A.C.S., F i s h e r S c i e n t i f i c Co., F a i r Lawn, New Jersey. 36. SODIUM PHOSPHATE BUFFER, pH 6.8, 0.24 M ( d i l u t e 480 mL of 0.5 M b u f f e r to 1 L) 0.01 M ( d i l u t e 20 mL of 0.5 M b u f f e r to 1 L) SODIUM PHOSPHATE, MONOBASIC, monohydrate, granular, Code 2312, SODIUM PHOSPHATE, DIBASIC, anhydrous, Lot 5074, B & A, A l l i e d Chemi-c a l , General Chemical Div., Pointe C l a i r e , Quebec. 37. STANDARD SALINE-CITRATE, SSC, 0.15 M NaCl and 0.015 M sodium c i t r a t e pH 6.85. 38. SULFURIC ACID, CONCENTRATED 93-98$, F i s h e r S c i e n t i f i c Co., F a i r Lawn, New Jersey. 39. t-BUTYLDIMETHYLSILYL CHLORIDE MIXTURE, c o n t i n i n g 1.0 mmole t - b u t y l -d i m e t h y l s i l y l c h l o r i d e , 2.5 mmole imidazole per mL of anhydrous N,N-dimethylformamide, Lot # 18028, Applied Sciences L a b o r a t o r i e s Inc., Pennsylvania. 40. TISSUE CULTURE DISH, 3003 O p t i l u x ^ / 100 x 20 mm S t y l e Dish, F a l -con, Div. Becton, Dickinson an Co., C o c k e y s v i l l e , Maryland. 0.5 M (0.293 M Na^PO^ and 0.207 M NaHgPCy HgO) 179, 41. TRIAMCINOLONE, USP, Code #2014, Batch # Z5040, Cyanamid of Canada L t d . , Montreal. 42. TRIAMCINOLONE ACETONIDE, USP, Code #45645, Batch #431, Cyanamid of Canada L t d . , Montreal. 43. TRITIATED-HYDROCORTISONE 3H-HC, Batch 38, s p e c i f i c a c t i v i t y U2 Ci/mmole i n benzene/EtOH (9:1), Amersham/Searle, A r l i n g t o n Heights, I l l i n o i s . 44. TRITIATED-TOLUENE STANDARD, Code No. 188280, Lot H177, a c t i v i t y 1.297 x 10 6 dpm/g ± 0.2$, Date October 1/77, Amersham, A r l i n g t o n Heights, I l l i n o i s . 45. TRITIATED-TRIAMCINOLONE ACETONIDE 3H-TA, (a) NET-470, Lot 853-017, s p e c i f i c a c t i v i t y 2.16 Ci/mmol i n EtOH, (b) NET-470, Lot 998-277, s p e c i f i c a c t i v i t y 31.3 Ci/mmol i n ben-zene/EtOH (9:1), New England Nuclear Co., Boston, Massachusetts. 46. TRITON X-100, Lot 87C-0075, polyethylene g l y c o l p - i s o o c t y l p h e n y l -25 ether ( a l k y l p olyether a l c o h o l ) d^ = 1.0595, non-ionic detergent, Sigma, St. Lou i s , M i s s o u r i . (CH3)3 C CH2- C — 0 " (CH2CH20)x H (CH3)2 47. TRYPSIN, s t e r i l e , 10X, 2 .5$ i n modified Hanks' balanced s a l i n e s o l u -t i o n , without Mg & Ca, Lot 16893026, Flow L a b o r a t o r i e s , McLean, V i r -g i n i a . 48. UREA, Lot 12G08, p r i l l e d , reagent A.C.S., Matheson Coleman & B e l l Manufacturing Chemists, Norwood, Ohio. 180. APPENDIX I I APPARATUS 1. AMICON FILTRATION UNIT, S t i r r i n g f i l t r a t i o n u n i t , Model 12, and D i a -f l o XM 100 A membrane, Amicon Co., Lexington, Massachusetts. 2. BECKMAN DB-GT SPECTROPHOTOMETER and 10" l i n e a r recorder equipped with 1P28A p h o t o m u l t i p l i e r , deuterium and tungsten sources, Beckman Instruments, Inc., F u l l e r t o n , C a l i f o r n i a . 3. BENCH-TOP CLINICAL CENTRIFUGE, Cat. No. 809, AC or DC, with b u i l d - i n f i x e d angle r o t o r , I n t e r n a t i o n a l Equipment Co., Boston, Massachu-s e t t s . 4. BLACK LIGHT BLUE FLUORESCENT LAMP, UV-A 330 (310-360 nm), 0.6 2 mW/cm i n i n t e n s i t y , S y l v a n i a F20T12-BLB, Toronto, Ontario. 5. CARBON DIOXIDE-CELL INCUBATOR, Model 3221-14, N a t i o n a l Appliance Co., (a Heinicke Co.), P o r t l a n d , Oregon. 6. CALCULATOR, Model HP-97, Program SD-03A, Hewlett-Packard, Avondale, Pennsylvania. 7 . CONSTANT TEMPERATURE BATH, HAAKE, Model E-51, Gehruder Haake K.G., B e r l i n , Germany. 8. DIFFERENTIAL SCANNING CALORIMETER, Model 1B, Perkin-Elmer, Norwalk, Connecticut. 9. GAS-CHROMATOGRAPH-ELECTRON-IMPACT MASS SPECTROMETER, V a r i a n MAT-111, Varia n , Pala A l t o , C a l i f o r n i a . 10. GAS-LIQUID CHR0MAT0GRAPH, Model 583 OA, equipped with a t e r m i n a l , Model 18850A, Hewlett-Packard, Avondale, Pennsylvania. 11. GILS0N MICRO FRATI0NAT0R, equipped with sensors f o r time or drops, Terochem. L a b o r a t o r i e s L t d . , Edmonton, A l b e r t a . 181. 12. GRADIENT MIXER, Model GM-1, c o n s i s t i n g of two r e s e r v o i r c y l i n d e r s of 300 mL capacity and low speed, 250 rpm synchronous motor, Pharmacia Fine Chemicals, Sweden. 13. HEMACYTOMETER, Neubauer improved, A l b e r t Sass. 14. INVERTED MICROSCOPE, with attached camera (100 X, 200 X, 400X), Nikon ^4012, Japan, Nikon M-35S camera, Nikon 38527 l i g h t source, Nikon 4FM camera adapter. 15. ISOCAP/300 PROGRAMMABLE LIQUID SCINTILLATION SYSTEM, Model 6868, ambient temperature, Nuclear Chicago, Des P l a i n e s , I l l i n o i s . 16. MELTING POINT DETERMINATION APPARATUS, Thomas-Hoover, Arthur H. Thomas Co., P h i l a d e l p h i a , Pennsylvania. 17. PERISTALTIC PUMP, Desaga Model 131900, STA multipurpose, Brinkman, Heidelberg. 18. PHASE-CONTRAST MICROSCOPE, 150X, M40-58916, Wild Heerbrugg, S w i t z e r -land. 19. RADIOMETER pH METER, Type PHM 26 equipped with standard g l a s s and calomel e l e c t r o d e s and b u i l t - i n p r e c i s i o n s c a l e expander, Radiometer A/S, Copenhagen, Denmark. 20. S0RVALL SUPERSPEED AUTOMATIC REFRIGERATED CENTRIFUGE, RC2-B, equip-ped with fixed-angle r o t o r SM-24 of r ( r a d i u s ) = 4.34 inches, Sor-v a l l , Norwalk, Connecticut. 21. TEFLON-FITTED GLASS HOMOGENIZER, A.H.T. Co., P h i l a d e l p h i a , Pennsyl-vania. 22. UV-VIS SPECTROPHOTOMETER, G i l f o r d , Model 250, equipped with automa-t i c cuvette thermoprogrammer, Model 2527, Automatic reference com-pensator and cuvette s e l e c t o r , Model 2535, Analog m u l t i p l e x e r , Model 6046, Recorder, Model 6050, O b e r l i n , Ohio. 23. VORTEX-GENIE, Model 3,061,280, 10 speed c o n t r o l s , F i s h e r S c i e n t i f i c Co., F a i r Lawn, New Jersey, 182 . REFERENCES Adolf, G.R. and Swetly, P. (1979). 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