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Mechanism of the cytolytic action of corticosteroids and its relationship to corticosteroid-resistance… Turnell, Roger William 1972

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M2G7 MECHANISM OF THE CYTOLYTIC ACTION OF CORTICOSTEROIDS AND ITS RELATIONSHIP TO CORTICOSTEROID-RESISTANCE IN MALIGNANT LYMPHOCYTES by ROGER WILLIAM TURNELL B.Sc, University of B r i t i s h Columbia, 1968. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1972 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 r e f e r e n c e and Study. I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f BIOCHEMISTRY The U n i v e r s i t y o f B r i t i s h C olumbia Vancouver 8, Canada Date J a n u a r y 13, 1972 i A B S T R A C T W h e n c e l l s o f t h e t h y m u s o r m o u s e l y m p h o s a r c o m a P1798S a r e t r e a t e d i n v i t r o w i t h 0.27 yM C o r t i s o l i n t i s s u e c u l t u r e m e d i u m w i t h 10% s e r u m o r 0.5% h u m a n a l b u m e n , n u c l e a r d a m a g e e n d i n g w i t h k a r y o r r h e x i s o c c u r s . T h e s t e r o i d - r e s i s t a n t s u b l i n e P1798R d o e s n o t s h o w t h e s e c h a n g e s . T h e c o r t i c o s t e r o i d - s e n s i t i v e l y m p h o c y t e s w h i c h u n d e r g o l y s i s d i f f e r i n s e v e r a l r e s p e c t s f r o m t h e r e s i s t a n t s u b l i n e . M o s t c r i t i c a l , a p p a r e n t l y , i s t h e c a p a c i t y f o r o x i d a t i o n o f f r e e f a t t y a c i d s ( F F A ) . T h i s w a s d e t e r m i n e d b y t h e e v o l u t i o n o f 1 ^ C G ^ f r o m l a b e l l e d s u b s t r a t e s , a n d i s o t o p e d i l u t i o n w i t h u n l a b e l l e d s u b s t r a t e . C o r t i c o s t e r o i d t r e a t m e n t i n v i v o f o r 2 h o u r s r a i s e d t h e F F A i n t h y m u s a n d P1798S c e l l s b y 77% a n d 58%, r e s p e c t i v e l y , w h i l e d e c r e a s i n g t h e r e s i s t a n t s u b l i n e P1798R b y 26%. I n v i t r o , C o r t i s o l h a d n o e f f e c t o n u p t a k e o f 1 ^ C - p a l m i t i c a c i d b u t d e c r e a s e d o x i d a t i o n b y 46% a n d 17% i n t h y m u s a n d P1798S, r e s p e c t i v e l y , w h i l e i n c r e a s i n g 9% i n P1798R. A f t e r i n c u b a t i o n o f s e n s i t i v e c e l l s i n m e d i u m c o n t a i n -i n g F F A c a l c u l a t e d t o . b e e q u i v a l e n t t o t h a t a c c u m u l a t e d a f t e r s t e r o i d t r e a t m e n t , e l e c t r o n m i c r o s c o p y r e v e a l e d t h a t c e r t a i n e f f e c t s o f c o r t i c o s t e r o i d s c o u l d b e r e p r o d u c e d b y f a t t y a c i d s o f c h a i n l e n g t h C - 9 a n d h i g h e r : n u c l e a r e d e m a , f o c a l d i s s o l u t i o n a n d d i s i n t e g r a t i o n o f t h e n u c l e a r m e m b r a n e and ultimately karyolysis. Steroid-resistant c e l l s show c y t o l o g i c a l changes only at tenfold higher concentrations of FFA. On the basis of these r e s u l t s the following scheme i s proposed as the mechanism by which c y t o l y s i s occurs i n c o r t i c o s t e r o i d - s e n s i t i v e lymphoid tissues: TG ^FFA ^Nuclear Damage Ac cumu1at ion C e l l l y s i s In an attempt to render r e s i s t a n t c e l l s sensitive to the c y t o l y t i c actions of c o r t i c o s t e r o i d s , studies were carried out where the oxidation of free f a t t y acids was i n h i b i t e d i n an attempt to induce an accumiilation of FFA. I n h i b i t i o n of oxidation by deoxycarnitine i n the s t e r o i d -r e s i s t a n t P1798R, L5178Y, and L1210 tumors results i n some l y s i s . Much more e f f e c t i v e , however, are compounds with branched structures, which cannot undergo 3-oxidation but can be oxidized to mono- or dicarboxylic acids which are l y t i c . In thymocytes, P1798S and P1798R, FFA did not a f f e c t the i n v i t r o incorporation of 1''C-leucine and 3H-uridine into a 5% TCA insoluble p r e c i p i t a t e . The incorporation of 3H-thymidine was only s l i g h t l y reduced i n the s t e r o i d -responsive c e l l s . This suggests that some st e r o i d e f f e c t s f o r example, those of immunosuppress ion, might be d i s s o c i a b l e f rom those i n v o l v i n g accumu la t i on o f FFA which l e a d to n u c l e a r damage and c y t o l y s i s of s e n s i t i v e c e l l s . i v TABLE OF CONTENTS Page ABSTRACT. i TABLE OF CONTENTS i v LIST OF TABLES . v i i LIST OF FIGURES . i x LIST OF APPENDICES x i ACKNOWLEDGEMENTS x i i ABBREVIATIONS USED x i i i INTRODUCTION . 1 The Question of Receptors 8 Glucocorticoid Effects on Carbohydrate Metabolism 13 Glucocorticoid E f f e c t s on Nucleic Acid and Protein Metabolism 14 The Present Problem 18 MATERIALS AND METHODS 20 Chemicals 20 Solvents 20 Radiochemicals 20 Materials 21 Animals 21 Methods-Biochemical 22 1) C e l l Suspensions 22 2) Incubations , . 2 3 3) Determination of Radioactivity 23 V TABLE OF CONTENTS (continued) Page 4) Assay for lkC02 Production 24 5) Preparation of Lipids 25 6) Separation of Neutral Lipids from Phospholipids 27 7) M i c r o t i t r a t i o n of Free Fatty Acids . 2 8 8) Spectrophotometric Assays 30 a) Total E s t e r i f i e d Fatty Acid . . . 30 b) L i p i d Phosphorous 3 0 9) Measurement of Incorporation of Radioactive Precursors of DNA, RNA and Protein into 5% TCA Insoluble Fraction 31 10) Uptake of 2-Deoxyglucose ( l - ^ C ) . . . 33 Methods-Electronmicroscopy 34 1) C e l l Suspensions and Incubations. . . 34 2) Fixation 34 a) Technique #1 34 b) Technique #2 35 3) Dehydration, Embedding, Sectioning, Staining, and Viewing 35 RESULTS 36 Preliminary Studies 36 Oxidation of Palmitic Acid-(1- 1^C) to 1 £ tC0 2 3 8 Oxidation of Short Chain Fatty Acids . . . 50 E f f e c t of C o r t i s o l and Palmitate on Carbohydrate Metabolism 54 v i TABLE OF CONTENTS (continued) Page E f f e c t of FFA on Mouse Lymphosarcoma C e l l Survival 54 Electronmicroscope Examination of the Effe c t s of FFA on Mouse Lymphoid C e l l s . . 56 Ef f e c t of Free Fatty Acids i n Inducing Lysis of Steroid-Resistant Tissues. . . 80 The Relationship of Cytolysis to Other Cor t i c o s t e r o i d Actions 82 DISCUSSION 98 BIBLIOGRAPHY 113 APPENDICES . 119 v i i LIST OF TABLES Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table I. Assay for 1 ''COproduction I I . Recovery of Lipids from S i l i c i c Acid Column II I . Comparison of Palmitic Acid-(1- 1 hC) Oxidation i n Different Media IV. E f f e c t of C o r t i s o l and/or Palmitic Acid on the Oxidation of Palmitic Acid- ( l - 1 *C) to lhC0z V. E f f e c t of Dexamethasone on Endogenous Free Fatty Acid Levels VI. E f f e c t of C o r t i s o l on Palmitic Acid Uptake VII. L i p i d Composition of Mouse Lympho-sarcoma P1798 and Thymus VIII. L i p i d D i s t r i b u t i o n of Mouse Lympho-sarcoma IX. Oxidation of Long and Short Chain Fatty Acids. Oxidation of Glucose by Mouse Lymphosarcoma C e l l s Uptake of 2-Deoxyglucose-(1- 1 kC) by Mouse Lymphoid C e l l s XII. E f f e c t of Free Fatty Acids on P1798 In V i t r o XIII. E f f e c t s of Increasing Azelaic Acid Concentration on Mouse Lymphosarcoma P1798 C e l l s In V i t r o XIV. E f f e c t of Deoxycarnitine on the Response of Tumor C e l l s to C o r t i s o l In V i t r o XV. E f f e c t of C i t r a l on L5178 In V i t r o X. XI. Page 26 32 45 47 49 51 52 53 55 57 58 59 60 81 83 v i i i LIST. OF TABLES, (continued) Table XVI. E f f e c t of C i t r a l on Tumors In Vivo Table XVII. Incorporation of Thymidine-(CH 2- 3H) into the Acid-Insoluble Fraction Table XVIII. Incorporation of Uridine-(5- 3H) into the Acid-Insoluble Fraction Table XIX. Incorporation of Leucine-( 1^C-U) into the Acid-Insoluble Fraction Page 84 94 96 97 i x LIST OF FIGURES Page F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e F i g u r e 8. 9. 10. 11. 12, 13. 14. 15. 16. S t r u c t u r e 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 w i t h L y m p h o c y t o l y t i c A c t i v i t y C o n c e n t r a t i o n of R a d i o a c t i v i t y i n Lymphat ic T i s s u e O u t l i n e o f the P r i n c i p a l Events t h a t have been I d e n t i f i e s i n the 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 s w i t h Thymus C e l l s In V i t r o A Model f o r the R e g u l a t i o n o f Lymphoid RNA and P r o t e i n B i o s y n t h e s i s by C o r t i s o l Counts Remaining i n the O r g a n i c Phase o f a CHCl 3 :MeOH (2:1 v/v) E x t r a c t i o n o f Mouse Lymphoid C e l l s Counts Remaining i n the Aqueous Phase of a CHC1 3:MeOH (2:1 v/v) E x t r a c t i o n o f Mouse Lymphoid C e l l s P r o d u c t i o n o f l l f C 0 2 b y Mouse Lymphoid T i s s u e s In V i t r o P r o d u c t i o n o f l l > C 0 2 f rom P a l m i t i c A c i d - ( 1 - 1 h C ) U n t r e a t e d C o n t r o l P1798S C e l l s U n t r e a t e d C o n t r o l P1798S C e l l s P a l m i t i c A c i d - T r e a t e d P1798S C e l l s A z e l a i c A c i d - T r e a t e d P1798S C e l l s P a l m i t i c A c i d - T r e a t e d P1798S C e l l s P a l m i t i c A c i d - T r e a t e d P1798S C e l l s A z e l a i c A c i d - T r e a t e d P1798S C e l l s A z e l a i c A c i d - T r e a t e d P1798S C e l l s 3 6 17 39 40 41 43 63 65 65 65 67 69 71 73 X LIST OF FIGURES (continued) Figure 17. Azelaic Acid-Treated P1798S C e l l s Figure 18. Palmitic Acid-Treated Thymocytes Figure 19. Palmitic Acid-Treated Thymocytes Figure 20. Proposed Scheme of Possible Factors Leading to Cytolysis Figure 21. Section of P1798R from Control Animal Figure 22. Section of P1798R from Control Animal Figure 23. Section of P1798R from Cit r a l - T r e a t e d Animal Figure 24. Section of P1798R from Citral-Treated Animal Figure 25. Mechanism of the C y t o l y t i c Action of Corticosteroids' Figure 26. Structure of Branched Chain Fatty Acids with Suggested Chemotherapeutic Value Figure 27. Outline of the Proposed Relation-ship of Events that occur Following Glucocorticoid Treatment P a g e 7 3 7 5 77 79 85 87 89 91 102 108 110 LIST OF APPENDICES Fischer's Medium for Leukemic C e l l s i n Mice (lx) Structures of Some Drugs and Chemicals Used Increased Mitochondrial Content of the Corticosteroid-Resistant Lymphosarcoma P179 8 Compared with Steroid-Sensitive Strain x i i ACKNOWLE DGEMENTS During the course of these studies, I have had the pleasure of working with a number of persons whose d i s -cussions have been most he l p f u l and to whom I am g r a t e f u l . To Judy, Ming, Denise, Hughe, Kash, Susan, Bruce and Brian, I express my sincere thanks. I wish to thank Dr. R.L. Noble, Director of the Cancer Research Centre, for the use of the f a c i l i t i e s at the Centre where t h i s research was conducted. The technical assistance of Mrs. R i l l a Hendry and Mrs. Hildegard Erber i n the preparation of the H&E sections of the P1798R tumor was g r a t e f u l l y appreciated. I am indebted to Mr. L. Hughe Clarke, a fellow graduate student from the Department of Pathology,.for his help i n the preparation and i n t e r -pretation of the electronmicrographs. The use of the micro-t i t r a t i o n apparatus belonging to Dr. G.I. Drummond of the Pharmacology Department i s g r a t e f u l l y acknowledged. F i n a l l y , most of a l l , I wish to thank Dr. A.F. Burton for his generous help and for the encouraging and enlight-ening discussions that were held throughout the preparation of t h i s thesis. I was a re c i p i e n t of a University of B r i t i s h Columbia Graduate Fellowship (1968-1969) and a Medical Research Council of Canada Studentship (1969-1972). X l l l ABBREVIATIONS USED ACTH a d r e n o c o r t i c o t r o p h i c hormone A6 20 nm a b s o r b a n c e a t 620 nm o r , any o t h e r w a v e l e n g t h s t a t e d cAMP c y c l i c a d e n o s i n e 3',5'-monophosphate cpm c o u n t s p e r m i n u t e DNA d e o x y r i b o n u c l e i c a c i d dpm d i s i n t e g r a t i o n s p e r m i n u t e EFA e s t e r i f i e d f a t t y a c i d ( s ) FA f a t t y a c i d ( s ) FFA f r e e f a t t y a c i d ( s ) KRP K r e b s - R i n g e r p h o s p h a t e b u f f e r mRNA m e s s e n g e r r i b o n u c l e i c a c i d NAD n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e NADH r e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e NADP n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e NADPH r e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o -t i d e p h o s p h a t e NEFA n o n - e s t e r i f i e d f a t t y a c i d ( s ) P1798S c o r t i c o s t e r o i d - s e n s i t i v e mouse l y m p h o -s a r c o m a P1798 P1798R c o r t i c o s t e r o i d - r e s i s t a n t mouse l y m p h o -s a r c o m a P1798 PPO 2 , 5 - d i p h e n y l o x a z o l e POPOP b i s ( 2 - ( 4 - m e t h y l - 5 - p h e n y l o x a z o l y i ) ) b e n z e n e RNA r i b o n u c l e i c a c i d TCA t r i c h l o r o a c e t i c a c i d XIV ABBREVIATIONS USED (cont inued) TEFA t o t a l e s t e r i f i e d f a t t y a c i d ( s ) U as i t r e f e r s " u n i v e r s a l l y t o r a d i o a c t i v i t y l a b e l l e d " means 1 INTRODUCTION The f i r s t observations on the e f f e c t of glucocorticoids on lymphocytes date from 1943 when Dougherty and White observed that i n various animal species a single i n j e c t i o n of ACTH would reduce the l e v e l of c i r c u l a t i n g lymphocytes (1). This lymphopenia was maximal i n three to nine hours and was dependent upon the amount of ACTH used. Within twenty-four hours the c i r c u l a t i n g lymphocyte levels had returned to normal. ACTH was.found to be inactive i n producing this- response i n adrenalectomized animals (2). Only adrenal c o r t i c a l steroids were able to produce a lympho-penic response i n adrenalectomized mice. Other hormones, such as i n s u l i n , epinephrine, and thyroxin, as well as cer t a i n s t e r o i d hormones were able to produce a lympho-penia provided they were administered to i n t a c t non-adrenal-ectomized animals (3). The investigation of the 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 -ships of steroids able to bring about lymphopenia indicated several features that were common to a l l : f i r s t of a l l , there must be an unsaturated A r i n g , a ketone at C-3; there must be a hydroxyl group or an oxygen function at po s i t i o n 11, and a side chain c h a r a c t e r i s t i c of the C-21 co r t i c o s t e r o i d s . The hydroxyl group at C-17 of C o r t i s o l i s not e s s e n t i a l to produce lymphopenia; however, the lack of i t causes a substantial loss i n lymphopenic a c t i v i t y . Early i n vivo studies showed that C o r t i s o l was most e f f e c t i v e 2 i n causing lymphocytolysis, followed by cortisone, cor-ticosterone, and 11-dehydrocorticosterone. More recent studies have shown that i h v i t r o only the 11-hydroxyl steroids have c y t o l y t i c a c t i v i t y (4,6,7,21,24). As shown i n Figure 1, the 11-keto steroids are active i n vivo- only, due to t h e i r p o t e n t i a l for being converted to t h e i r active 11-hydroxy forms by the enzyme 116-hydroxysteroid: NADP oxidoreductase, which occurs i n l i v e r (5,14). The kidney i s responsible f o r oxidation (14), the l i v e r for reduction. If glucocorticoids are administered to adrenalectomized-hepatectomized animals, they are without e f f e c t i n causing thymolysis (13). This may be due to the rapid conversion of 11-hydroxy steroids to 11-keto steroids by the kidney, and would indicate the necessity for the 11-hydroxy function i n maintaining thymolytic a c t i v i t y . There are three documented ways i n which steroids bring about involution of lymphatic tissue. They produce lymphocytokaryorrhexis. They i n h i b i t mitosis by destroy-ing c e l l s at metaphase and by i n h i b i t i n g the biosynthesis of DNA i n c e l l s that remain. The phenomenon of lympho-cytokaryorrhexis was the f i r s t to be described and i s assumed to be the p r i n c i p a l mechanism for the repression of lymphatic tissue ( 8 ) . By use of time-lapse dark-phase cinemiorography Dougherty has shown that aft e r administration of an "active" g l u c o c o r t i c o i d , lymphocytes shed t h e i r cytoplasm i n small 3 FIGURE 1: Structure of naturally occurring steroids with lymphocytolytic a c t i v i t y which can undergo interconversions. 4 b l e b s (40). L a t e r t h e n u c l e a r m e m b r a n e m a y d i s i n t e g r a t e w i t h t h e r e l e a s e o f n u c l e a r c o n t e n t s i n t o t h e c y t o p l a s m w h i c h m a y s t i l l b e i n t a c t . T h e c i n e m i c r o g r a p h i c s t u d i e s o f D o u g h e r t y h a v e b e e n c o n f i r m e d b y C o w a n a n d S o r e n s e n u s i n g t h e e l e c t r o n m i c r o -s c o p e , w h o l o o k e d a t m o u s e t h y m u s a f t e r i n j e c t i o n o f m i c e w i t h C o r t i s o l (11). M o r e r e c e n t l y , B u r t o n , S t o r r a n d D u n n h a v e d e v e l o p e d a n i n v i t r o s y s t e m f o r t r e a t i n g t h y m o c y t e s w i t h C o r t i s o l (24). T h e y o b s e r v e d t h a t t w o h o u r s a f t e r t r e a t m e n t o f t h y m o c y t e s w i t h a d o s e o f C o r t i s o l s u f f i c i e n t t o g i v e a m a x i m a l e f f e c t i n e i g h t h o u r s (0.27 y M ) , t h e n o r m a l c h r o m a t i n p a t t e r n h a d l a r g e l y d i s a p p e a r e d a n d t h e n u c l e u s a p p e a r e d s w o l l e n a n d r a t h e r h o m o g e n o u s . A f t e r f o u r h o u r s t h e r e w e r e c e l l s s h o w i n g d i s r u p t i o n o f n u c l e a r a n d c e l l u l a r m e m b r a n e s . P y k n o t i c c e l l s w e r e now r e c o g n i z e a b l e b y l i g h t m i c r o s c o p y . A t e i g h t h o u r s a m a x i m a l e f f e c t w a s o b s e r v e d , w i t h p r a c t i c a l l y e v e r y c e l l b e c o m i n g p y k n o t i c . T h e c o r t i c o s t e r o i d - s e n s i t i v e m o u s e l y m p h o s a r c o m a P1798 s h o w e d s i m i l a r h i s t o l o g i c e f f e c t s . D i s p e r s i o n o f c h r o m a t i n a n d r a t h e r h o m o g e n o u s a n d s w o l l e n n u c l e u s w e r e s e e n a f t e r f o u r h o u r s . T h e c o r t i c o s t e r o i d - r e s i s t a n t s u b l i n e o f t h i s l y m p h o s a r c o m a r e m a i n e d u n c h a n g e d o v e r t h i s p e r i o d . S i n c e t h e h a l f l i f e o f C o r t i s o l i s 40-50 m i n u t e s i n m a m m a l s a n d a l m o s t a l l o f a n i n j e c t e d d o s e i s e x c r e t e d a f t e r 100 m i n u t e s i t w a s o f i n t e r e s t t o d e t e r m i n e h o w l o n g a c y t o l y t i c d o s e o f g l u c o c o r t i c o i d w o u l d p e r s i s t i n t a r g e t 5 t i s s u e s . A f t e r i n j e c t i o n o f C o r t i s o l - (4- 1 ' 'C) t o m i c e t h e r e i s a p r o g r e s s i v e a c c u m u l a t i o n o f r a d i o a c t i v e h o r m o n e i n l y m p h a t i c t i s s u e w h i c h b e c o m e s m a x i m a l a f t e r t e n t o t w e l v e m i n u t e s a n d t h e n d r o p s t o i n s i g n i f i c a n t l e v e l s b y 100 m i n u t e s ( S e e F i g u r e 2). T h e d r a m a t i c e f f e c t s o f C o r t i s o l o n c y t o l y s i s a n d s u p p r e s s i v e e f f e c t s o n DNA s y n t h e s i s a n d m i t o s i s p e r s i s t f o r m a n y h o u r s . B u r t o n e t a l h a v e s h o w n t h a t i n v i t r o e x p o s u r e o f t h y m o c y t e s t o C o r t i s o l f o r a p e r i o d a s s h o r t a s o n e h o u r c a u s e d s u b -s t a n t i a l p y k n o s i s b y e i g h t h o u r s (24). S i n c e s t e r o i d s a r e o n l y m e t a b o l i z e d s l i g h t l y b y t h y m u s c e l l s u s p e n s i o n s (14,20) i t w o u l d a p p e a r t h a t g l u c o c o r t i c o i d s t r i g g e r a n e v e n t a t t h e c e l l u l a r l e v e l w h i c h p e r s i s t s l o n g a f t e r a l l o f t h e s t e r o i d h a s d i s a p p e a r e d . I n t h e i r s e a r c h f o r t h e p r i n c i p a l e v e n t s t h a t a r e i n v o l v e d i n t h e c y t o l y s i s o f l y m p h o c y t e s , i n v e s t i g a t o r s h a v e f o c u s s e d t h e i r a t t e n t i o n p r i m a r i l y o n g l u c o s e , p r o t e i n , a n d n u c l e i c a c i d m e t a b o l i s m . M o r e r e c e n t l y , t h e y h a v e t u r n e d t h e i r e f f o r t s t o t h e i d e n t i f i c a t i o n a n d i n t r a c e l l u l a r l o c a t i o n 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 a n d t o h o w i t w o u l d a f f e c t m e t a b o l i s m i n t h e s e a r e a s . F i g u r e 3 o u t l i n e s t h e p r i n c i p a l e v e n t s t h a t h a v e b e e n i d e n t i f i e d a f t e r g l u c o -c o r t i c o i d t r e a t m e n t , a n d t h e i r t e m p o r a l l o c a t i o n . E a c h o f t h e m a j o r a r e a s m e n t i o n e d a b o v e w i l l b e d i s c u s s e d i n s o m e d e t a i l . 6 W w o Q Q W EH U W *l O dP 1 0 -0.1 0.01 s p l e e n thymus lymph nodes I 10 -r-100 M I N U T E S A F T E R C 0 R T I S 0 L - 4 - 1 I N J E C T I O N FIGURE 2. C o n c e n t r a t i o n o f r a d i o a c t i v i t y i n l y m p h a t i c t i s s u e . Percentage o f r a d i o a c t i v i t y found i n the v a r i o u s l y m p h a t i c t i s s u e s a f t e r i n t r a v e n o u s i n j e c t i o n o f c o r t i s o l --4- C. (From Dougherty , B e r l i n e r , S c h n e e b e l i , a n d B e r l i n e r (12)). 7 Glucocorticoids thymus cells (ATP) Steroids 0 min « Specific binding< Irreversible step Act.O sensit. step cytoplasmic" complex f nuclear complex ) i (RNA) Sites 1 Nonspecific throughout f i nd ing cell J \ Nonspecific effects 15-.20 \ mm 'cyclohex. sensit. step..^ Protein I DECREASED GLUCOSE TRANSPORT \ Decreased glucose dependent ATP r 40 min Decreased protein synthesis Decreased RNA synthesis +4h Cell lysis F I G U R E 3 : O u t l i n e o f t h e p r i n c i p a l e v e n t s t h a t h a v e b e e n i d e n t i f i e d i n t h e 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 s w i t h t h y m u s c e l l s i n v i t r o a n d o f t h e p o s t u l a t e d r e l a t i o n s b e t w e e n t h e m . T h e t i m e s o n t h e l e f t g i v e a n i n d i c a t i o n o f t h e a p p r o x i m a t e t i m e a t w h i c h e a c h e v e n t b e g i n s d u r i n g a n i n c u b a t i o n w i t h C o r t i s o l a t 3 7 8 . B r a c k e t s a n d d a s h e d a r r o w s , r e s p e c t i v e l y , d e n o t e s u b s t a n c e s a n d s t e p s f o r t h e r o l e o f w h i c h a t p r e s e n t t h e r e i s o n l y i n d i r e c t e v i d e n c e . ( M o d i f i e d f r o m M u n c k ( 4 1 ) ) . 8 The Q u e s t i o n o f R e c e p t o r s : A i l s t e r o i d s y s t e m s s t u d i e d t o d a t e a p p e a r t o have a r e c e p t o r i n v o l v e d i n t h e i r mechanism o f a c t i o n . G l u c o -c o r t i c o i d s do n o t a p p e a r t o be an e x c e p t i o n t o t h i s r u l e . M u n c k 1 s g r o u p , u s i n g r a d i o a c t i v e l y l a b e l e d s t e r o i d s o f h i g h s p e c i f i c a c t i v i t y , and m e a s u r i n g t h e amount o f s t e r o i d bound t o c e l l s and c e l l f r a c t i o n s , has shown t h a t when r a t thymus c e l l s u s p e n s i o n s were t r e a t e d w i t h s t e r o i d s two d i f f e r e n t t y p e s o f p h y s i o l o g i c a l b i n d i n g o c c u r r e d : s p e c i f i c and n o n - s p e c i f i c b i n d i n g (20) . The m a g n i t u d e o f e q u i l i b r i u m b i n d i n g o f s t e r o i d showed no c o r r e l a t i o n w i t h s t e r o i d a c t i v i t y , b u t good c o r r e l a t i o n w i t h n o n - s p e c i f i c f u n c t i o n , s u c h as i n h i b i t i o n o f g l u c o s e u p t a k e a t h i g h c o n c e n t r a t i o n (10- 5M o r g r e a t e r ) ( 2 0 , 2 1 ) . F o r example, s t e r o i d s w h i c h have no g l u c o c o r t i c o i d a c t i v i t y , s u c h as d e o x y c o r t i c o s t e r o n e and p r o g e s t e r o n e , were bound 3 and 17 t i m e s more e f f e c t i v e l y t h a n was C o r t i s o l . T h i s b i n d i n g o f n o n - g l u c o c o r t i c o i d s t e r o i d s was i n t e r p r e t e d as a r e f l e c t i o n o f t h e n o n - s p e c i f i c b i n d i n g a c t i v i t y . I f c e l l s t h a t had b e e n t r e a t e d w i t h g l u c o c o r t i c o i d o r non-g l u c o c o r t i c o i d s t e r o i d s were d i l u t e d w i t h m e d i a i t was s e e n t h a t a f t e r 30 m i n u t e s a new e q u i l i b r i u m had b e e n a t t a i n e d w h i c h was e q u i v a l e n t t o t h a t c a l c u l a t e d f r o m t h e d i l u t i o n p r o d u c e d by t h e d i l u a n t . The m a i n f e a t u r e n o t e -w o r t h y i n s u c h d i s s o c i a t i o n c u r v e s was t h a t by one m i n u t e most o f t h e s t e r o i d had d i s s o c i a t e d . F o r example, f o r 9 d e o x y c o r t i c o s t e r o n e , t h e e q u i l i b r i u m c o n s t a n t ( ( S c ) / ( S ) e ) w e n t f r o m 8.0 a t 0 m i n u t e s t o 1.25 a t 1 m i n u t e . T h i s r a p i d l y d i s s o c i a t i n g f r a c t i o n i s c a l l e d t h e n o n - s p e c i f i c a c t i v i t y o f n o n - g l u c o c o r t i c o i d s (21,22). H o w e v e r , w h e n o n e l o o k s a t t h e d i s s o c i a t i o n c u r v e s f o r C o r t i s o l , t h e p a t t e r n o f d i s s o c i a t i o n i s d i f f e r e n t f r o m t h e n o n - g l u c o -c o r t i c o i d s . A s w i t h t h e n o n - g l u c o c o r t i c o i d s t h e r e w a s a n o n - s p e c i f i c f r a c t i o n t h a t d i s s o c i a t e d w i t h i n t h e f i r s t m i n u t e a n d t h e n a s e c o n d f r a c t i o n t h a t d i s s o c i a t e d s l o w l y o v e r t h e n e x t 15 m i n u t e s . T h i s s l o w l y d i s s o c i a t i n g f r a c t i o n i s a l s o c h a r a c t e r i s t i c o f o t h e r c o r t i c o s t e r o i d s s u c h a s d e x a m e t h a s o n e a n d c o r t i c o s t e r o n e t h a t e x e r t g l u c o c o r t i c o i d a c t i v i t y . T h e s p e c i f i c b i n d i n g o f C o r t i s o l a p p e a r s t o b e t e m p e r -o a t u r e d e p e n d e n t . I f t h e d i l u t i o n w a s c a r r i e d o u t a t 3 t h e n o n - s p e c i f i c f r a c t i o n d i s s o c i a t e d w h i l e t h e s p e c i f i c g l u c o c o r t i c o i d b i n d i n g f r a c t i o n d i d n o t d i s s o c i a t e u n t i l t h e t e m p e r a t u r e w a s r a i s e d t o 37° (20). T h e t w o d i f f e r e n t f o r m s o f b i n d i n g w e r e d i f f e r e n t w i t h r e s p e c t t o t h e i r s a t u r a t i o n k i n e t i c s . T h e n o n - s p e c i f i c f r a c t i o n d i d n o t a p p e a r t o b e c o m e s a t u r a t e d w i t h s t e r o i d s o v e r t h e r a n g e o f 1 0 - 1 0 t o 10-1* m o l a r , w h i l e t h e s e s p e c i f i c b i n d i n g s i t e s b e c a m e s a t u r a t e d a t 10 6 M , n e a r t h e u p p e r e n d o f t h e p h y s i o l o g i c a l r a n g e o f C o r t i s o l c o n c e n t r a t i o n s . F r o m t h e s a t u r a t i o n k i n e t i c s i t a p p e a r s t h a t t h e r e a r e a l i m i t e d n u m b e r o f b i n d i n g s i t e s ( a b o u t 5000) f o r s p e c i f i c i n t e r a c t i o n 10 w i t h g l u c o c o r t i c o i d s ( 2 0 ) e v e n t h o u g h s o m e 2.4 x 1 0 s m o l e c u l e s a r e b o u n d p e r c e l l ( 2 4 ) , t h e d i f f e r e n c e b e i n g t h o s e b o u n d n o n - s p e c i f i c a l l y . O t h e r s t e r o i d s w i l l c o m p e t e f o r t h e s e s p e c i f i c s i t e s m o r e o r l e s s i n p r o p o r t i o n t o t h e i r 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 v i t y . I f t h e s p e c i f i c b i n d i n g i n t e r a c t i o n s a r e t h e s i t e s o r s p e c i f i c a l l y t h e " r e c e p t o r s " t h r o u g h w h i c h g l u c o c o r -t i c o i d s e x e r t t h e i r m e t a b o l i c e f f e c t s o n t h e c e l l i t s h o u l d b e p o s s i b l e t o b l o c k t h e s e s i t e s a n d g l u c o c o r t i c o i d a c t i v i t y w i t h c o m p o u n d s w h i c h w i l l c o m p e t e f o r b i n d i n g t o t h e s e s i t e s . I t w a s f o u n d t h a t 1 0 " 5 M c o r t e x o l o n e w i l l c o m p e t e s p e c i f i c a l l y w i t h C o r t i s o l ( 1 0 6 M ) a n d v i r t u a l l y a b o l i s h t h e e f f e c t s o f C o r t i s o l o n g l u c o s e m e t a b o l i s m ( 2 0 , 2 5 ) . T h i s e f f e c t o f c o r t e x o l o n e i n p r e v e n t i n g C o r t i s o l f r o m p r o d u c i n g i t s p r o f o u n d e f f e c t s o n g l u c o s e m e t a b o l i s m h a s b e e n i n v e s t i g a t e d b y M e l y n k o v y c h a n d B i s h o p ( 2 3 ) u s i n g H e L a c e l l s . T h i s g r o u p w a s i n t e r e s t e d i n d e t e r m i n i n g w h e t h e r o r n o t c o r t e x o l o n e w o u l d h a v e a n y e f f e c t o n i n d u c t i o n b y p r e d n i s o l o n e o f a l k a l i n e p h o s p h a t a s e i n H e L a c e l l s . T h e y f o u n d t h a t a t t h e h i g h e s t l e v e l o f c o r t e x o l o n e u s e d ( 2 9 . 5 x 10 6 M ) , t h e i n d u c t i o n o f a l k a l i n e p h o s p h a t a s e w a s i n h i b i t e d b y 7 2 % , w h i l e n o e f f e c t w a s o b s e r v e d o n t h e b a s a l l e v e l o f e n z y m e i n c o n t r o l c u l t u r e s . I f m o r e p o t e n t s t e r o i d s s u c h a s t h e 9 a - f l u o r o p r e d n i s o l o n e s e r i e s o r l e s s p o t e n t s t e r o i d s s u c h a s C o r t i s o l w e r e u s e d i t w a s f o u n d t h a t c o r t e x o l o n e w a s m u c h l e s s e f f e c t i v e w i t h t h e p o t e n t 11 than with the weaker i n preventing a l k a l i n e phosphatase induction. These r e s u l t s , as do those of Munck and his co-workers, seem to support the assumption that the b i o l -o g i c a l a c t i v i t y of glucocorticoids i s r e f l e c t e d i n t h e i r a f f i n i t y for the binding s i t e s within the c e l l s . Making use of the previously mentioned fa c t that at 3° the s p e c i f i c binding f r a c t i o n does not dissociate, i t i s possible to assign a temporal.sequence to s p e c i f i c g l u c o c o r t i c o i d binding to receptors i n rat thymocytes. If c e l l s were incubated with 3 H - c o r t i s o l f o r 20 minutes at 3 7 ° , and d i l u t e d at 3 ° , the non-specific a c t i v i t y dissociated from the c e l l s while the s p e c i f i c receptor a c t i v i t y did not. After c e l l membranes and cytoplasmic materials were removed from the thymocytes by hypotonic shock i n 1.5 mM MgCl 2, the s p e c i f i c a l l y bound a c t i v i t y remained associated with the nuclear preparation ( 2 0 ) . However, when the c e l l s were incubated at 3° and disrupt-ed, the C o r t i s o l s p e c i f i c f r a c t i o n was i n the supernatant and not i n the nuclear f r a c t i o n ( 3 0 ) . I t has been suggested that the primary hormone in t e r a c t i o n i n some way d i r e c t s the receptor to i t s nuclear or "executive" place of action, where i t exerts i t s e f f e c t s on DNA,RNA and subsequent protein formation. I t i s the hormone receptor complex that i s active rather than the receptor or hormone alone. The g l u c o c o r t i c o i d receptor complex has been i s o l a t e d 12 from thymic nuclei preparations and separated from free C o r t i s o l on Sephadex G-25 where i t emerges with the void volume. The protein complex containing C o r t i s o l i s rapidly degraded by pronase. The rate at which the complex dissociates i s indistinguishable from the c h a r a c t e r i s t i c slow rate at which C o r t i s o l dissociates from the i s o l a t e d n u c l e i or from whole c e l l s (26). Under anaerobic conditions the e f f e c t s of C o r t i s o l on thymus c e l l s were abolished (29). Also under anaerobic conditions or conditions where no glucose i s present the formation of the nuclear receptor complex does not occur (26). The s p e c i f i c binding of C o r t i s o l i s intimately dependent upon the metabolic state of the c e l l and more prec i s e l y on the ATP l e v e l . Using the c o r t i c o s t e r o i d - s e n s i t i v e mouse lympho-sarcoma P1798, Rosen's group (28) has recently i s o l a t e d a s i m i l a r receptor complex using as a marker 3H-triamcin-ol i n e acetonide instead of 3 H - c o r t i s o l . They found, as did Munck, two binding fractions i n the c e l l s . One f r a c t i o n loosely binds 3H-triamcinoline acetonide which could be displaced by C o r t i s o l or i t s analogue 11-epi-c o r t i s o l , which i s devoid of g l u c o c o r t i c o i d a c t i v i t y . Only C o r t i s o l was e f f e c t i v e i n displacing the s p e c i f i c a l l y bound triamcinoline acetonide. The properties of t h e i r complex were s i m i l a r to those of Munck: i t separated with the void volume on Sephadex G-25, was sensitive to the actions of pronase and i n s e n s i t i v e to RNase and DNase. Similar glucocorticoid receptors have also been found 1 3 i n other stero i d responsive c e l l s ( 3 1 ) . Glucocorticoid Effects on Carbohydrate Metabolism: When glucocorticoids were injected into adrenal-ectomized fasted animals, l i v e r glycogen reserves were seen to increase. This e f f e c t took, about four hours. However, at least two hours before these e f f e c t s on l i v e r glycogen were evident, i t was observed by Munck and Koritz that blood glucose would r i s e 8 0 - 1 0 0 minutes following C o r t i s o l i n j e c t i o n ( 1 5 ) . This e f f e c t has been subsequent-ly confirmed by two other groups ( 1 6 , 1 7 ) . This rapid e f f e c t of glucocorticoids on blood glucose was thought to r e s u l t as a consequence of decreased glucose uptake by peripheral tissues. When sel e c t i v e tissues were investigated using 1^C-glucose i t was found that glucose uptake was increased i n gastrocnemius muscle, probably due to increased a v a i l a b i l i t y of glucose as a r e s u l t of elevated blood glucose ( 1 8 ) . In adipose tissue ( 1 9 , 3 3 ) and skin ( 3 4 ) there was decreased glucose u t i l i z a t i o n both i n vivo and i n v i t r o . Because of the catabolic actions of glucocorticoids on thymus, Munck and his group turned t h e i r attention to lymphoid tissue. Between 2 and 3 . 5 hours a f t e r i n j e c t i o n of C o r t i s o l the uptake of (U- 1^C)-glucose by whole tissue and incorporation into proteins and l i p i d s was decreased by 3 0 % ( 3 5 ) . Continuing work i n Munckrs laboratory con-1 4 f i r m e d t h a t d e c r e a s e d i n c o r p o r a t i o n o f l a b e l l e d g l u c o s e i n t o p r o t e i n a n d l i p i d w a s i n d e e d d u e t o d e c r e a s e d g l u c o s e u p t a k e ( 3 6 ) . I n v i t r o , C o r t i s o l a t c o n c e n t r a t i o n s a p p r o a c h -i n g p h y s i o l o g i c a l c o n c e n t r a t i o n s ( 1 0 7 - 1 0 6 M ) l e a d t o d e c r e a s e d g l u c o s e u p t a k e a n d u t i l i z a t i o n b y t h y m o c y t e s ( 2 1 , 2 2 , 3 6 ) . O n l y a c t i v e g l u c o c o r t i c o i d s w i l l e x h i b i t t h e s e e f f e c t s a t p h y s i o l o g i c a l c o n c e n t r a t i o n s . P o t e n t i a l l y a c t i v e c o r t i c o i d s s u c h a s c o r t i s o n e r e q u i r e c o n v e r s i o n to C o r t i s o l b e f o r e t h e y a r e active. R o s e n ' s g r o u p t e s t e d t w o s u b l i n e s o f t h e m o u s e l y m p h o -s a r c o m a P 1 7 9 8 : o n e t h a t i s s e n s i t i v e t o t h e c a t a b o l i c e f f e c t s o f C o r t i s o l a n d t h e o t h e r w h i c h i s r e s i s t a n t . G l u c o c o r t i c o i d s w e r e f o u n d t o h a v e p r o n o u n c e d i n h i b i t o r y e f f e c t s o n g l u c o s e u p t a k e a n d s u b s e q u e n t m e t a b o l i s m b y t h e s e n s i t i v e t u m o r b u t w e r e w i t h o u t e f f e c t i n t h e r e s i s t a n t t u m o r . A l t h o u g h g l u c o c o r t i c o i d s i n h i b i t e d u p t a k e o f g l u c o s e o r i t s a n a l o g u e 2 - d e o x y g l u c o s e b y 2 5 - 3 5 % a f t e r 2 h o u r s o f e x p o s u r e t h e r e w a s e s s e n t i a l l y n o c h a n g e i n h e x o k i n a s e a c t i v i t y ( 3 9 ) . T h e s e e f f e c t s w e r e s e e n t o p e r s i s t f o r a s l o n g a s e i g h t h o u r s f o l l o w i n g g l u c o c o r t i c o i d t r e a t m e n t . G l u c o c o r t i c o i d E f f e c t s o n N u c l e i c A c i d a n d P r o t e i n M e t a b o l i s m : W h e n o n e l o o k s a t t h e e f f e c t s o f g l u c o c o r t i c o i d s o n n u c l e i c a c i d a n d p r o t e i n m e t a b o l i s m t w o d i f f e r e n t a n d o p p o s i t e e f f e c t s a r e o b s e r v e d . T h e f i r s t i s a v e r y r a p i d s t i m u l a t o r y e f f e c t a n d t h e s e c o n d a n i n h i b i t o r y e f f e c t o f 1 5 t h e s t e r o i d h o r m o n e . T o d a t e , n o o n e h a s b e e n a b l e t o d e m o n s t r a t e t h a t g l u c o c o r t i c o i d s c a u s e i n c r e a s e d RNA s y n t h e s i s i n l y m p h o i d c e l l s s i m i l a r t o t h a t i n l i v e r , b u t t h e r e i s m u c h a c c u m -u l a t e d e v i d e n c e t o i n d i c a t e t h a t RNA s y n t h e s i s i s o b l i g a t o r y b e f o r e a n y o f t h e d e t r i m e n t a l e f f e c t s o f c o r t i c o i d s o n g l u c o s e m e t a b o l i s m a n d i o n t r a n s p o r t c a n b e o b s e r v e d . C o r t i s o l a l t e r e d t h e s y n t h e s i s o f r a p i d l y l a b e l l e d RNA i n l y m p h o i d c e l l s ( 4 2 ) . A s e a r l y a s 1 - 2 m i n u t e s a f t e r C o r t i s o l i n j e c t i o n t h e r a t e o f s y n t h e s i s o f RNA w a s d e c r e a s e d b y 16% a n d 23% i n l y m p h o i d c e l l s f r o m n o n -a d r e n a l e c t o m i z e d a n d a d r e n a l e c t o m i z e d r a t s r e s p e c t i v e l y . T h e s e e f f e c t s w e r e s e e n t o p e r s i s t f o r h o u r s f o l l o w i n g i n v i t r o g l u c o c o r t i c o i d t r e a t m e n t . T h e s e r e s u l t s t e n d t o i n d i c a t e t h a t i m m e d i a t e l y f o l l o w i n g g l u c o c o r t i c o i d t r e a t -m e n t RNA m e t a b o l i s m i s t u r n e d o f f . H o w e v e r , i n d i r e c t e v i d e n c e f r o m W h i t e ' s g r o u p i n d i c a t e d t h a t RNA s y n t h e s i s m u s t o c c u r i n o r d e r f o r t h e i n h i b i t o r y e f f e c t s o f C o r t i s o l o n r u b i d i u m i o n t r a n s p o r t , o n o r t h o p h o s p h a t e i n c o r p o r a t i o n , a n d o n g l u c o s e , 2 - d e o x y g l u c o s e , a n d 3 - 0 - m e t h y l g l u c o s e u p t a k e b y t h y m o c y t e s t o b e s e e n ( 4 3 ) . W h e n t h y m o c y t e s w e r e i n c u b a t e d w i t h C o r t i s o l a n d a c t i n o m y c i n D , a n i n h i b i t o r o f RNA b i o s y n t h e s i s , t h e u s u a l i n h i b i t o r y e f f e c t s o f C o r -t i s o l o n a b o v e p a r a m e t e r s w e r e n o t o b s e r v e d . W h e n a c t i n o -m y c i n D w a s a d d e d t o t h e t h y m o c y t e s y s t e m a f t e r t h e a d d i t i o n o f C o r t i s o l , t h e e f f e c t s o f C o r t i s o l w e r e n o l o n g e r p r e v e n t e d o r r e v e r s e d b y t h i s a n t i b i o t i c d u r i n g t h e 16 s u b s e q u e n t 1-2 h o u r s . T h i s w o u l d i n d i c a t e t h a t C o r t i s o l h a s a v e r y r a p i d e f f e c t o n t h e c e l l i n e n h a n c i n g RNA m e t a b o l i s m b e f o r e i t i s " t u r n e d o f f " . I n c o n t r a s t w i t h a c t i n o m y c i n D, i n h i b i t o r s o f p r o t e i n s y n t h e s i s s u c h a s c y c l o h e x i m i d e o r p u r o m y c i n r e v e r s e d t h e i n h i b i t o r y e f f e c t s o f C o r t i s o l w h e n g i v e n a t s o m e t i m e f o l l o w i n g C o r t i s o l a d m i n i s t r a t i o n . T h e i n h i b i t o r y e f f e c t s o f C o r t i s o l c a n b e r e v e r s e d b y t h e s e a n t i b i o t i c s a s l a t e a s 6 0 m i n u t e s f o l l o w i n g C o r t i s o l a d m i n i s t r a t i o n . A d i r e c t e f f e c t o f C o r t i s o l o n i n c r e a s e d p r o t e i n s y n t h e s i s i n l y m p h o i d c e l l s w a s o b s e r v e d b y K i d s o n ( 4 2 ) . T e n t o f i f t e e n m i n u t e s a f t e r t r e a t m e n t o f r a b b i t l y m p h n o d e c e l l s w i t h C o r t i s o l , i n c o r p o r a t i o n o f 3 H - l e u c i n e i n c r e a s e d t w o f o l d o v e r t h e l e v e l o b s e r v e d i n u n t r e a t e d c o n t r o l s . T w e n t y t o t h i r t y m i n u t e s a f t e r C o r t i s o l t r e a t m e n t t h e l e v e l f e l l a n d b y 60 m i n u t e s t h e l e v e l o f i n c o r p o r a t i o n h a d f a l l e n b e l o w t h e c o n t r o l l e v e l . T h e r e s u l t s i n d i c a t e t h a t t h e r e i s 5-10 m i n u t e s d e l a y , p r e s u m a b l y d u r i n g w h i c h RNA s y n t h e s i s o c c u r s , b e f o r e a " b u r s t " o f p r o t e i n s y n t h e s i s . T h i s " b u r s t -s y n t h e s i s " w o u l d p r o d u c e i n h i b i t o r y p r o t e i n w h i c h w o u l d h a v e i n h i b i t o r y e f f e c t s o n g l u c o s e m e t a b o l i s m , i o n t r a n s p o r t a n d p h o s p h o r y l a t i o n a n d a l s o " s h u t d o w n " c e l l m e t a b o l i s m a s i n d i c a t e d i n F i g u r e 4. T h e u l t i m a t e r e s u l t o f t h e s e i n h i b i t o r y e f f e c t s o f C o r t i s o l w o u l d b e c e l l l y s i s . M a k m a n , D v o r k i n a n d W h i t e h a v e a l s o r e c e n t l y p o s t u l a t e d t h e e x i s t e n c e o f a r e p r e s s o r p r o t e i n w h i c h w o u l d t u r n o f f t h e m e t a b o l i s m o f t h e c e l l ( 4 3 ) . 17 CORTISOL t Increased mRNA Synthesis Repression of mRNA Synthesis "Burst" Synthesis of Repressor Proteins Decreased Carbohydrate Metabolism, Ion Transport, Phosphorylation, etc. t Decreased Protein Biosynthesis I I I I T C e l l Death Figure 4: A model f o r the regulation of lymphoid RNA and protein biosynthesis by C o r t i s o l . 18 T h e l o n g t e r m r e p r e s s i v e e f f e c t s o f C o r t i s o l o n D N A , RNA a n d p r o t e i n b i o s y n t h e s i s h a v e b e e n s t u d i e d b y m a n y g r o u p s u s i n g t h y m o c y t e s a n d l y m p h o s a r c o m a c e l l s a s e x p e r i m e n t a l m o d e l s . A l l s t u d i e s h a v e t h e s a m e f e a t u r e s , t h a t b i o s y n t h e s i s i s s i g n i f i c a n t l y r e p r e s s e d o n e h o u r a f t e r C o r t i s o l t r e a t m e n t a n d t h e e f f e c t s i n v i t r o p e r s i s t f o r a s l o n g a s t h e s t u d i e s h a v e b e e n c a r r i e d o u t . T h e P r e s e n t P r o b l e m : A s m e n t i o n e d p r e v i o u s l y , a l l i n v e s t i g a t o r s , i n l o o k i n g f o r t h e m e c h a n i s m b y w h i c h g l u c o c o r t i c o i d s i n d u c e c y t o l y s i s o f l y m p h o c y t e s , h a v e f o c u s s e d t h e i r a t t e n t i o n s o n c a r b o -h y d r a t e , p r o t e i n a n d n u c l e i c a c i d m e t a b o l i s m . N o n e o f t h e s e a r e a s , I f e e l , explaini=j4dequately t h e d i s r u p t i o n o f l y m p h o c y t e s t h a t s e e m i n g l y b e g i n s w i t h t h e n u c l e u s a n d t h e n l e a d s t o t h e p r o g r e s s i v e d e g e n e r a t i o n o f t h e c e l l . T h i s s t u d y i s c o n c e r n e d w i t h t h e m e c h a n i s m o f t h e c y t o l y t i c a c t i o n o f c o r t i c o s t e r o i d s . T h e s t a r t i n g p o i n t f o r t h e s t u d y s t e m s f r o m s o m e e a r l i e r r e p o r t e d o b s e r v a t i o n s i n t h i s a n d o t h e r l a b o r a t o r i e s . T h e s e a r e : 1) t h e l o w c o n -c e n t r a t i o n o f f r e e f a t t y a c i d s t h a t d a m a g e c e l l s (24); 2) t h a t i n v i t r o f r e e f a t t y a c i d s i n d u c e p y k n o s i s o f t h y m o c y t e s a n d o f t h e c o r t i c o s t e r o i d - s e n s i t i v e m o u s e l y m p h o s a r c o m a P1798 w h i c h i s t e m p e r a t u r e - d e p e n d e n t 1 ; a n d 3) t h a t c o r t i c o s t e r o i d s a r e c a p a b l e o f i n d u c i n g t h e 1 B u r t o n , A . F . , U n p u b l i s h e d o b s e r v a t i o n s , 1967. 19 r e l e a s e o f FFA from t r i g l y c e r i d e s t o r e s (32) . T h i s s tudy w i l l be f o c u s s e d m a i n l y on the metabo l i sm o f f r e e f a t t y a c i d s by lymphocytes i n o r d e r t o determine whether or not a r e l a t i o n s h i p e x i s t s between f r e e f a t t y a c i d metabo l i sm and the s e n s i t i v i t y o r r e s i s t a n c e of m a l i g n a n t lymphocytes to c o r t i c o s t e r o i d s . 20 MATERIALS AND METHODS Chemicals: A l l Chemicals used i n thi s study, unless otherwise noted, were purchased from the Fischer Chemical outlet i n Vancouver, B.C.. Hyamine, PPO and POPOP were obtained from Packard Instrument Company, Downers Grove, I l l i n o i s . Tetrabutylammonium hydroxide 25% i n methanol was purchased from Matheson, Coleman and B e l l , Norwood, Ohio. Azelaic acid, p a l m i t i c acid, c a r n i t i n e and deoxycarnitine were obtained from Calbiochem, Los Angeles, C a l i f o r n i a . C o r t i s o l was from the Sigma Chemical Company, St. Louis, Missouri. Dexamethasone (decadron powder) was the g i f t of Merck and Company, Rahway, New Jersey, to Dr. M. Darrach i n t h i s department. C i t r a l was obtained from Eastman Kodak Co., Rochester, New York (for structure see Appendix I I ) . Solvents: A l l solvents used were of reagent grade and were p u r i f i e d by r e d i s t i l l a t i o n before use. Radiochemicals: The following radiochemicals were purchased from 21 Amersham S e a r l e C o r p o r a t i o n , w i t h s p e c i f i c a c t i v i t i e s as i n d i c a t e d : P a l m i t i c a c i d - ( l - 1 ^C) (35 . 2-56 . 9 m^/mM) , 1 - l e u c i n e -( 1 I f C-U) (331 mC^/mM) , sodium b i c a r b o n a t e ( - 1 "O (56 . 9mCi/mM) , a c e t i c a c i d (-1- 1 ' 'C) (52. 9mCi/mM) , sodium n-oc tanoate (-1- 1 ' 'C) (17.5 mCi/mM), D - G l u c o s e - ( 1 ^ C - U ) ( 3 0 9 mCj/mM), n-hexadecane-( l - 1 "O (120. 8 yCi/M) , thymid ine (methy l - 3 H) (26 C^/mM) and u r i d i n e (-5- 3 H) (30C-j/mM) . 2 -Deoxy-D-G lucose- ( 1 - 1 (53.3mCi/mM) was o b t a i n e d from New Eng land N u c l e a r . A l l r a d i o c h e m i c a l s were used upon r e c e i p t o r p u r i f i e d r o u t i n e l y by TLC o r paper chromatography. M a t e r i a l s : G l a s s f i b r e f i l t e r s used f o r the t r a p p i n g o f a c i d -i n s o l u b l e p r e c i p i t a t e s were o b t a i n e d from Reeve A n g e l , C l i f t o n , New J e r s e y . P l a s t i c c e n t r e w e l l s , o n e - h o l e d rubber serum s t o p p e r s , and 25 ml Er lenmeyer f l a s k s used i n the c o l l e c t i o n o f l l f C 0 2 were purchased from Kontes G l a s s Company, V i n e l a n d , New J e r s e y . S c i n t i l l a t i o n v i a l s were o b t a i n e d from a l o c a l supp ly house . A n i m a l s : The BALB/cJ and DBA/2J mice used i n these exper iments were o b t a i n e d from Jackson L a b o r a t o r i e s , Bar H a r b o r , Ma ine . The an ima ls were f e d a d i e t of P u r i n a Breeder Chow and water 22 ad libitum. The c o r t i c o s t e r o i d - s e n s i t i v e and - r e s i s t a n t mouse lymphosarcoma were maintained subcutaneously as s o l i d tumors i n the area of the f i r s t inguinal gland. Tumors were transplanted routinely every 15-17 days by grinding 100 mg tumor i n a tissue grinder with 5 ml 0.9% saline and i n j e c t i n g 0.1 ml of the c e l l suspension subcutaneously into BALB/cJ mice. The murine leukemias L517 8Y and L1210 were maintained i n DBA/2J mice as ascites tumors. Every 7-10 days ascites f l u i d was withdrawn from mice and 0.1 ml of t h i s f l u i d was injected into r e c i p i e n t s . Methods-Biochemical: 1) C e l l Suspensions : Thymus or tumor tissue was removed from BALB/cJ mice and ground i n a t e s t tube with a loose f i t t i n g glass pestle i n 0.9% NaCl- The suspension was f i l t e r e d through eight layers of s u r g i c a l cotton gauze and centrifuged at 1200xg for 2 minutes. It was washed once with 5 ml of 0.9% NaCl and p e l l e t e d . An aliquot of t h i s suspension was d i l u t e d with Fischer's Medium (Grand Island B i o l o g i c a l Company, Berkeley, C a l i f o r n i a ) , containing 10% horse serum (Hyland, Los Angeles, C a l i f o r n i a ) or 0.5% human albumin (Sigma, 23 S t . L o u i s , M i s s o u r i ) to g i v e a f i n a l c e l l c o n c e n t r a t i o n of 0 .5-4 m i l l i o n c e l l s / m l as determined by a hemacytometer. 2) I n c u b a t i o n s ; I n c u b a t i o n s were c a r r i e d out i n s tandard c o n i c a l g l a s s c e n t r i f u g e tubes s e a l e d w i t h s i l i c o n e rubber s t o p p e r s , e x c e p t f o r when 1 h C 0 2 was b e i n g a s s a y e d , when i n c u b a t i o n s were c a r r i e d out i n 25 ml Er lenmeyer f l a s k s . Samples were i n c u b a t e d i n an oven or water bath a t 3 8° f o r the r e q u i r e d t ime w i t h o u t a g i t a t i o n . C o r t i s o l was d i s s o l v e d i n e t h a n o l a t a c o n c e n t r a t i o n of 2 mg/ml and d i l u t e d w i t h s a l i n e b e f o r e a d d i t i o n t o samples . P a l m i t i c and a z e l a i c a c i d s as e t h a n o l s o l u t i o n s , were added t o the samples b e f o r e i n c u b a t i o n . A t no t ime d i d the e t h a n o l c o n c e n t r a t i o n i n i n c u b a t e d samples exceed 1%. 3) D e t e r m i n a t i o n of r a d i o a c t i v i t y : The sample t h a t was to be counted was p l a c e d i n a s c i n t i l l a t i o n v i a l and 10 ml o f l i q u i d s c i n t i l l a t i o n f l u i d was added. T h i s c o n s i s t e d o f 4 gm PPO and 100 mg POPOP per l i t e r o f t o l u e n e . For aqueous samples an a p p r o p r i a t e amount o f Hyandne s o l u b i l i z e r was added. Samples were counted u s i n g e i t h e r a Packard T r i - C a r b L i q u i d S c i n t i l l a t i o n Spect rometer (Model 3003) o r a N u c l e a r Ch icago U n i l u x I I I 24 Ambient Temperature Liquid S c i n t i l l a t i o n Counter. Sample quenching was monitored by the channels-Ratio Method ( 4 7 ) using a quench set prepared from n-hexadecane- ( 1 - 1 ''C) quenched with acetone or a commercially available set purchased from Nuclear Chicago. 4) Assay for 1 **C02 Production: Samples to be assayed for production of 1 1*C02 were incubated i n 2 5 ml Erlenmeyer f l a s k s . 0.9 ml of Fischer's Medium or KRP buffer were placed i n the flasks and to t h i s was added l a b e l l e d materials, C o r t i s o l , free f a t t y acids, and other chemicals as the experimental regimen dictated. 0.1 ml of the c e l l suspension was added and the flasks were closed with serum stoppers equipped with a centre well. 0.3 ml Hyamine was placed i n the centre well v i a a syringe and needle. The flasks were then placed i n an oven at 37° for the length of time required. At the end of the incubation period, flasks were removed from the oven and 0.3 ml 2N HaSCK was added to the incubation mixture v i a syringe through the serum stopper. Flasks were then placed i n a water bath at 37° and shaken at 1 2 0 cpm for at le a s t two hours. At the end of t h i s time the centre wells were removed, cut from the stems and placed i n s c i n t i l l a t i o n v i a l s . A further 0.5 ml of Hyamine was added and 10 ml pf s c i n t i l l a n t and r a d i o a c t i v i t y was determined. Samples 25 were corrected for quenching by the method of channel ra t i o s (47). As can be seen from Table I,the recovery of 1 "^ CC^  from NaH^COg i n KRP buffer was greater than 98%. Because Fischer's Medium contains added bicarbonate as one of i t s constituents (see Appendix I ) , t r i a l runs were made to determine whether or not the capacity of the Hyamine would be exceeded when this medium was used for incubating the c e l l suspensions. The r e s u l t s show that the capacity of the Hyamine i s great enough to trap a l l the C0 2 released from the medium. 5) Preparation of L i p i d s : A sample of minced tissue (0.5-2.0 gm) was homogenized i n 20 ml of chloroformimethanol (2:1 v/v) using a S o r v a l l -Omnimixer. The homogenate was f i l t e r e d under vacuum through a sintered glass funnel (fine porosity). T h e - s t a i n l e s s s t e e l tube and residue were re-extracted with an additional 20 ml of chloroform-methanol (2:1 v/v). The pooled l i p i d extracts were removed to a 50 ml extraction tube, 8 ml of 0.73% NaCl was added (48), and tubes were shaken vigor-ously for one minute. Upon separation of the emulsion, the upper aqueous phase was removed by suction and the lower chloroform-rich phase evaporated i n vacuo at 40°, using a rotary evaporator. The residue was extracted from 26 T A B L E I i A s s a y f o r C 0 2 P r o d u c t i o n S a m p l e R a d i o a c t i v i t y (dpm) M e d i a N a H 1 " * C 0 3 a d d e d 1 t , f C 0 2 -r e c o v e r e d ^ r e c o v e r e d 1. 2. 3. 4. 5. KRP 6 1 0 0 5 5 0 1 1 0 0 2 2 0 0 6 6 0 0 5 7 4 0 5 6 0 1 1 0 0 2 1 4 0 6 4 5 0 9 4 . 0 1 0 1 . 8 1 0 0 . 0 9 7 . 2 9 7 . 7 A v e r a g e r e c o v e r y : 9 8 . 1 ± 1 . 3 * 2 6 . 7. 8. 9. 1 0 . 1 1 . F I S C H E R 5 5 , 7 0 0 5 5 , 7 0 0 5 6 , 8 2 0 5 1 , 3 3 0 1 0 6 , 6 8 0 1 0 8 , 5 2 0 1 0 6 , 6 8 0 1 0 0 , 5 9 0 1 6 1 , 1 4 0 1 7 2 , 6 4 0 1 6 1 , 1 4 0 1 7 0 , 2 7 0 A v e r a g e r e c o v e r y 1 0 2 , 92. 1 0 1 . 94, 1 0 7 , 1 0 5 . 7 1 0 0 . 5 + 2 . 5 * ' * 1 1 hCOz w a s c o l l e c t e d a n d a c t i v i t y d e t e r m i n e d a s i n d i c a t e d i n t h e m e t h o d s s e c t i o n . F i g u r e s i n d i c a t e d a r e m e a n ± S E M . 27 t h e f l a s k u s i n g 10 m l o f c h l o r o f o r m - m e t h a n o l ( 2 : 1 v / v ) m i x t u r e , w a s t r a n s f e r r e d t o a t e s t - t u b e a n d e v a p o r a t e d t o d r y n e s s u n d e r a s t r e a m o f n i t r o g e n g a s . T h e l i p i d r e s i d u e w a s r e d i s s o l v e d i n E t O H : E t h e r ( 3 : 1 v / v ) a n d m a d e u p t o 5 o r 10 m l i n a v o l u m e t r i c f l a s k . 6) S e p a r a t i o n o f n e u t r a l l i p i d s f r o m p h o s p h o l i p i d s : A p p r o x i m a t e l y 6 gm o f s i l i c i c a c i d , w h i c h h a d b e e n a c t i v a t e d i n a n o v e n a t 1 1 0 ° f o r 24 h o u r s , w a s p l a c e d i n a c y l i n d r i c a l , s i n t e r e d - g l a s s f u n n e l ( f i n e p o r o s i t y ) a n d w a s h e d w i t h 40 m l o f l i g h t p e t r o l e u m e t h e r ( b p 6 0 ° - 8 0 ° ) . T h e l i p i d e x t r a c t w a s d i s s o l v e d i n 10 m l o f l i g h t p e t r o l e u m e t h e r a n d a d d e d t o t h e a b s o r b e n t . T h e a b s o r b e n t s o l v e n t m i x t u r e w a s s t i r r e d m e c h a n i c a l l y f o r 10 m i n u t e s . T h e p e t r o l e u m e t h e r e x t r a c t w a s i s o l a t e d f r o m t h e a b s o r b e n t b y v a c u u m f i l t r a t i o n . T h e s i l i c i c a c i d w a s t h e n w a s h e d 3 t i m e s w i t h 3 0 m l p o r t i o n s o f f r e s h l y d i s t i l l e d d i e t h y l e t h e r . T h e c o m b i n e d f i l t r a t e s , w h i c h c o n t a i n e d t h e n e u t r a l l i p i d f r a c t i o n , w e r e t a k e n t o d r y n e s s u s i n g a r o t a r y e v a p o r a t o r . T h e r e s i d u e w a s d i s s o l v e d i n 5 m l o f E t O H : E t h e r ( 3 : 1 v / v ) a n d s a m p l e s r e m o v e d f o r g r a v i m e t r i c a n d c o l o r i m e t r i c d e t e r m i n a t i o n o f t h e n e u t r a l l i p i d . T h e s i l i c i c a c i d w a s t h e n e x t r a c t e d 3 t i m e s w i t h 3 0 m l p o r t i o n s o f a n h y d r o u s m e t h a n o l . E x t r a c t s w e r e i s o l a t e d b y v a c u u m f i l t r a t i o n . T h e p o o l e d f i l t r a t e s c o n t a i n i n g 28 the p h o s p h o l i p i d s were taken to d ryness u s i n g a r o t a r y e v a p o r a t o r . The r e s i d u e was d i s s o l v e d i n 5 ml o f C H C I 3 : MeOH (3:1 v/v) and samples were removed f o r g r a v i m e t r i c and c o l o r i m e t r i c d e t e r m i n a t i o n . As shown i n T a b l e I I , the r e c o v e r y o f l i p i d s , on a we ight b a s i s , u s i n g t h i s method i s e s s e n t i a l l y comple te . In o r d e r to determine whether o r not the n e u t r a l l i p i d f r a c t i o n was contaminated w i t h any p h o s p h o l i p i d s t h a t may have been e l u t e d w i t h the d i e t h y l e t h e r s o l v e n t , phosphate a n a l y s e s were per formed on each f r a c t i o n . The r e s u l t s i n d i c a t e t h a t a l l the phosphorus was e l u t e d w i t h the p h o s p h o l i p i d f r a c t i o n . TLC o f the n e u t r a l l i p i d and p h o s p h o l i p i d f r a c t i o n s on S i l i c a Ge l p l a t e s r e v e a l e d t h a t each f r a c t i o n was f r e e from contaminates o f the o t h e r f r a c t i o n . 7) M i c r o t i t r a t i o n o f F r e e F a t t y A c i d s : A Rehberg m i c r o b u r e t o f 125 y l c a p a c i t y w i t h 0.2 y l d i v i s i o n s was used to t i t r a t e the o r g a n i c s o l u t i o n s con-t a i n i n g f r e e f a t t y a c i d s . The s o l u t i o n to be t i t r a t e d was s t i r r e d by b u b b l i n g n i t r o g e n gas through i t . The gas was f i r s t d r i e d by p a s s i n g through a column of C a C l 2 , then bubb led through .005 N NaOH (us ing bromthymol b l u e as an ac id/base i n d i c a t o r ) to remove any f r e e C O 2 and f i n a l l y d r i e d by p a s s i n g through a column of C a C l 2 and d r i e r i t e . 29 A phenol red indi c a t o r was prepared using a modified method of Kelley (49). A 0.01% (w/v) solution of phenol red i n absolute ethanol was prepared. This solution was dil u t e d with 200 ml of heptane, 1.0 ml of d i s t i l l e d water was added, and the r e s u l t i n g mixture s t i r r e d thoroughly. For t i t r a t i o n s of free f a t t y acid solutions t h i s stock solution was di l u t e d 2.5 f o l d with heptane and 1 ml portions used as indicators i n the t i t r a t i o n s . The indicator was t i t r a t e d from a lemon yellow colour to an orchid purple end point. A stock solution of t i t r a n t was prepared by d i l u t i n g 17.5 ml of tetrabutylammonium hydroxide which was 25% i n methanol to 100 ml with methanol. A fresh working solution of the t i t r a n t (approximately 0.01N) was prepared d a i l y by d i l u t i n g the stock 1:10 with methanol. A stock solution of free f a t t y acid was prepared by dis s o l v i n g 51.8 mg of palmitic acid i n 10 ml of MeOH. A working solution of free f a t t y acid containing 0.2 yEqv/ml was prepared by d i l u t i n g the stock 1:100 with heptane. Aliquots of organic l i p i d extracts or standard solutions of free f a t t y acids were made up to 3 ml i n heptane i n 15 x 85 mm t e s t tubes. One ml of the indicator was added, the solution was mixed using nitrogen gas and t i t r a t e d to a purple end point. The concentration of free f a t t y acid was determined from a standard curve c a l i b r a t e d from 0 - 0.6 yEqv of free f a t t y acid. 30 8) Spectrophotometric Assays: A l l spectrophotometric assays were performed using Cary 11 recording spectrophotometer to measure absorbance. a) Total E s t e r i f i e d Fatty Acid TEFA were analyzed according to the method of Stern and Shapero (51). Samples of l i p i d extracts were made up to 3 ml with Ethanol:Diethyl ether (3:1 v/v) i n 18 x 150 mm tes t tubes. To each tube was added 0.5 ml of 13.9% (w/v) hydroxylamine hydrochloride and 0.5 ml 3.5 N HaOH. The tubes were mixed thoroughly, capped with a marble and allowed to stand for 20 minutes at room temperature. At the end of 20 minutes 0.6 ml of 3.4 N HCI were added, tubes were shaken and then 0.5 ml of 0.37 M FeCl .6H 0 added, tubes 3 2 shaken and colour developed and measured at 525 nm. The concentration of TEFA was determined from a standard curve calibr a t e d using from 0-6 mEqv/1 of t r i a c e t i n . b) L i p i d Phosphorus L i p i d phosphorus was analyzed according to the method of Ames (50), which i s seven times more sensitive than the Fiske-Subbarow method. The l i p i d extracts to be analyzed were placed i n 13 x 100 mm tes t tubes and taken to dryness under a stream of nitrogen gas. To each tube 31 was added 30 y l of 10% (w/v) Mg(NO ),.6H,0 i n 95% EtOH. Solutions were taken to dryness and ashed by shaking the tubes over a strong flame u n t i l brown fumes appeared. The tubes were allowed to cool; then 0.3 ml of 0.5 N HCI was added. The tubes were capped with a marble and heated at 100° for 15 minutes. The tubes were cooled, and 0.7 ml of the phosphate assay mix was added. The assay mixture consisted of one part of 10% ascorbic acid and 6 parts of 4.2 gm ammonium molybdate tetrahydrate and 2 8.6 ml concen-trated s u l f u r i c acid made up to 1 l i t r e with d i s t i l l e d water. Then the samples were incubated at 45° for 20 minutes before determining the A660 nm. The amount of phosphate was calculated by comparing to a standard curve ca l i b r a t e d from 0-160 nM phosphate using NajHPOij. 7H20 as the standard. 9) Measurement of Incorporation of Radioactive Precursors of DNA, RNA and Protein into 5% TCA Insoluble Fraction: C e l l suspensions were incubated with radioactive precursors of DNA, RNA, or protein. At the end of the incubation period c e l l suspensions were layered on 5 ml of cold 0.9% NaCl i n a M i l l i p o r e F i l t r a t i o n Apparatus. Incubation tubes were washed with additional saline and washings added to the layered material. Gentle suction was applied and c e l l s were trapped on 2.4 cm diameter TABLE I I : Recovery of L i p i d s from S i l i c i c A c i d Column LIPID RECOVERED FROM COLUMN SAMPLE LIPID ADDED NEUTRAL LIPID PHOSPHOLIPID % RECOVERY TO COLUMN wt (mg) wt (mg) yM T?x wt (mg) yM P^  1. 34.95 11.17 - 18.05 - 83.6 2. 36.30 6.20 - 31.08 - 102.7 3. 14.25 7.52 0.00 5.30 3.7 90.0 4. 9.38 5.70 0.00 4.46 3.8 108.3 Average r e c o v e r y : 96.1+5.7% * 1 * 1 F i g u r e i n d i c a t e d i s the MEAN ± SEM. ro 3 3 glass f i b e r f i l t e r d i s c s . The f i l t e r s and funnels were washed with an additional 5 ml of saline to remove any remaining media and serum proteins. Then 5 ml of cold 5% TCA (w/v) were added to the funnel and allowed to f i l t e r through the f i l t e r s for at l e a s t 3 minutes. At the end of th i s period, suction was applied and the f i l t e r s were washed with an additional 5 ml of 5% TCA. When the funnel was removed the edges of the f i l t e r s were washed with cold 5% TCA to remove any acid-soluble r a d i o a c t i v i t y which may have been trapped under the funnel l i p . The f i l t e r s were removed to s c i n t i l l a t i o n v i a l s and dried i n vacuo at 8 0 ° . 1 0 ml of s c i n t i l l a t i o n f l u i d were added and r a d i o a c t i v i t y was determined. Results are ex-pressed i n cpm because the r a d i o a c t i v i t y was trapped on s o l i d supports. 1 0 ) Uptake of 2-Deoxyglucose- ( l - 1 ''C) : The uptake of 2-deoxyglucose was determined i n the same manner as the incorporation of l a b e l l e d precursors into DNA, RNA, and protein, with the following exceptions: a f t e r the samples and funnels were washed with cold 0 . 9 % NaCl, the funnels were removed and the edges of the f i l t e r s were washed with 0 . 9 % NaCl to remove any soluble a c t i v i t y that had been trapped under the funnel l i p . F i l t e r s were then removed to s c i n t i l l a t i o n v i a l s and treated as pre-3 4 viously described. Methods-Electronmicroscopy: 1 ) C e l l Suspensions and Incubations; The preparation of c e l l suspensions and incubations of the tumor P 1 7 9 8 was i d e n t i c a l to that described pre-viously for biochemical studies, except that the f i n a l concentration of c e l l s was adjusted to 1 - 2 m i l l i o n c e l l s / m l , as determined by a hemacytometer. 2 ) F i x a t i o n : Two techniques of i n i t i a l c e l l f i x a t i o n were used. The procedures gave i d e n t i c a l r e s u l t s . a) Technique # 1 : A f t e r incubation, c e l l suspensions were mixed with an equal volume of 0 . 1 % glutaraldehyde i n buffer before p e l l e t i n g , i n order to maintain c e l l shape. The c e l l s were then resuspended i n 2 . 5 % glutaraldehyde i n Millonig's Buffer f o r 3 0 minutes at 4 ° . A p e l l e t of these c e l l s was embedded i n Difco Agar ( 4 8 ° ) , drawn into a c a p i l l a r y tube, cooled, removed, and postfixed i n 1% OSCK i n Millonig's Buffer f o r 3 0 minutes at 4 ° . 35 b) Technique #2: After incubation, c e l l suspensions were pe l l e t e d , then resuspended i n 3.3% glutaraldehyde i n 0.1 N phosphate buffer for 30 minutes at room temperature. After a wash i n 0.1 N phosphate buffer the pelle t e d c e l l s were post-fix e d by the addition of 1% OsOi* i n 0.1 N b a r b i t o l buffer. Using 2 ml centrifuge tubes, i t was found that as few as 5 10 c e l l s could be routinely manipulated by t h i s technique without the necessity of more refined procedures. 3) Dehydration, Embedding, Sectioning, Staining and Viewing: After a buffer wash, samples fixed by each method were dehydrated i n an ascending ethanol s e r i e s , and embedded i n Epon 812 (44) or Maraglas (45). Sections were cut on an MT2 Sorval Ultramicrotome. Thick sections (0.5 microns) were stained with t o l u i d i n e blue (46). Thin sections were mounted on uncoated copper grids, stained with aqueous uranyl acetate and lead c i t r a t e and examined i n a Siemens Elmiskop I. 36 EXPERIMENTAL AND RESULTS Observations were made on c e l l s using a system that was developed i n t h i s laboratory for maintaining thymus c e l l suspensions i n culture for periods up to 24 hours i n good condition, a very important point i f early subtle changes are to be discerned. In addition to thymus, the c o r t i c o s t e r o i d - s e n s i t i v e mouse lymphosarcoma of BALB/cJ mice was also available. The c o r t i c o s t e r o i d - r e s i s t a n t subline of t h i s tumor, P1798R, as well as two other c o r t i -c osteroid-resistant lymphocytic leukemias of DBA mice, the L1210 and L5178Y, were available. These c e l l s a l l grow readily as ascites tumors and also as suspensions i n culture. The P1798S and R l i n e s also grow well as s o l i d subcutaneous growths. Treatment of the sensitive tumor with corticosteroids results i n rapid regression over a of period^several days while the r e s i s t a n t l i n e s continue to grow during treatment. This study of the c y t o l y t i c action of corticosteroids uses mainly the thymus, P1798S and P1798R c e l l s as exper-imental models with usefc-f the L5178Y and L1210.where resistance to corticosteroids i s being studied s p e c i f i c a l l y . Preliminary Studies: The incorporation of 1 "*C-acetate i n the l i p i d s of 37 P179 8 tumors were examined by incubating c e l l s f o r short periods of time, extracting with chloroform-methanol (2:1 v/v) and subjecting the extracts to paper chromato-graphy. There was a s i m i l a r l e v e l of incorporation of l a b e l l e d acetate into the l i p i d s of both the sensitive and r e s i s t a n t tumors. Autoradiograms revealed a q u a l i t a -t i v e l y s i m i l a r pattern of incorporation into the tumor li n e s of P179 8. The only apparent quantitative difference was a s i g n i f i c a n t increase of 1 4C-acetate incorporation into one phospholipid of the sensitive tumor (34.5+2.7% of t o t a l a c t i v i t y incorporated) when compared with the r e s i s t a n t tumor (27.7±0.6% t o t a l ) . The zone was ten-t a t i v e l y i d e n t i f i e d as l y s o l e c i t h i n . This was also true of the f i x a t i o n of 1^C-palmitic acid into the l i p i d s of these tumors. The metabolism of palmitic acid was looked at i n another way. Following the incubation of thymus or tumor c e l l s with palmitic acid-(1- 1 **C) , the d i s t r i b u t i o n of r a d i o a c t i v i t y i n the aqueous or organic phase of chloroform-methanol (2:1 v/v) extract was studied. The aqueous phase of such an extraction should contain the water-soluble acyl-coenzyme A and a c y l - c a r n i t i n e derivatives of the added free f a t t y a c id, while the'chloroform r i c h phase would contain the free and e s t e r i f i e d fatty acid. Oxidation of the f a t t y acid to C0 2 would be r e f l e c t e d by a disappearance of counts. C e l l s were incubated i n tissue culture medium for 3 0 minutes with l a b e l l e d substrate and 38 then extracted. The results of these extractions are i l l u s t r a t e d i n Figures 5 and 6. The results are expressed as a loss of the l a b e l when compared with controls taken as 1 0 0 % . Bars terminating above the dashed l i n e indicate loss of l a b e l while those terminating below indicate retention. A perusal of the figures indicates two d i f f e r e n t patterns of metabolism, loss of l a b e l i n corticosteroid^-r e s i s t a n t tumors and retention i n sensitive tumors. The r e s i s t a n t tumors, with one exception, showed increased loss of counts on the addition of C o r t i s o l or c a r n i t i n e . These re s u l t s were taken as suggestive evidence that the oxidation of FFA might be greater i n r e s i s t a n t c e l l s than i n s e n s i t i v e c e l l s and that carnitine was not apparently a l i m i t i n g factor i n the sensitive c e l l s . Oxidation of Palmitic Acid- ( 1 - 1 h C ) to 1 ""CO : 2— In order to determine i f there was a difference i n the oxidative capacity of c o r t i c o s t e r o i d - r e s i s t a n t and sensitive c e l l s , the oxidation of FFA to 1''CO was studied. Thymus, 2 P 1 7 9 8 S and P 1 7 9 8 R c e l l s were incubated i n K R P buffer ( 6 5 ) with palmitic acid- ( 1 - 1 4C) and 1''CO c o l l e c t e d f o r various 2 times up to a maximum of two hours. As can be seen from Figure 7, the production of 1 1 * C 0 2 increased over the f i r s t 30 minutes and was maximal between 30 and 6 0 minutes. Because a l l of the curves had the same common feature of a l i n e a r increase i n 1 "*C0 2 production over the f i r s t 30 39 140-THYMUS P 1 7 9 8 / S P 1 7 9 8 / R L 1 2 1 0 L 5 1 7 8 Y BJ3 WITH CORT ISOL ( l y / m l ) ^ WITH C A R N I T I N E ( l O O y / m l ) FIGURE 5: C e l l s were incubated i n Fischer's media and 10% horse serum at 37° for 30 minutes with 20,000 dpm of palmitic acid-(1- 1^C). At the end of the incubation period c e l l s and media were extracted with CHCl3:MeOH (2:1 v/v) and counts remaining i n the organic phase determined. ( --CONTROL VALUE) . 40 •FIGURE 6: C e l l s were incubated i n Fischer's media and 1 0 % horse serum at 37° for 30 minutes with 20,000 dpm of palmitic a c i d - ( 1 - 1 ^ C ) . At the end of the incubation period c e l l s and media were extracted with CHCl3:MeOH (2:lv/v) and counts remaining i n the aqueous phase determined. ( -CONTROL VALUE) . 41 time (minutes) FIGURE 7: P r o d u c t i o n of 1^002 by mouse lymphoid t i s s u e s i n v i t r o . C e l l s were i n c u b a t e d i n KRP b u f f e r w i t h p a l m i t i c a c i d - ( l - 1 "c) a n d 1 * C O 2 c o l l e c t e d , a) Thymus b) P1798S c) P1798R 42 minutes, a l l subsequent incubations looking at the e f f e c t s of the corticosteroids on FFA oxidation used a 30 minute incubation period. It was expected that any e a r l i e r subtle changes i n l i p i d metabolism would be detected using t h i s short incubation period. A number of experiments were performed to determine what were the oxidative capacities of these three c e l l l i n e s under various experimental conditions. The r e s u l t s are summarized i n Figure 8 . This data places the apparent oxidative capacity of three tissues i n r e l a t i v e order of t h e i r s e n s i t i v i t y to c o r t i c o s t e r o i d s : thymus greatest, followed by P 1 7 9 8 S and then P 1 7 9 8 R . This i s the reverse order to what would be expected i f the data were r e f l e c t i n g the maximum oxidative capacity of the c e l l s . The e f f e c t s of the various additives are d i f f i c u l t to i n t e r p r e t . In these experiments the l a b e l l e d substrate was the only added source of energy available to the c e l l s . Thymus c e l l s have been reported by Abramson and Blecher ( 5 8 ) to have a high l i p i d content of which 7 5 % i s neutral l i p i d consisting primarily of t r i g l y c e r i d e and a respiratory quotient that indicates that f a t i s being u t i l i z e d extensive-l y . I t has been calculated ( 5 9 ) that thymus c e l l s use 5% of t h e i r l i p i d stores per hour to account for t h e i r endogenous r e s p i r a t i o n . Spector ( 5 6 ) has found that tumor c e l l s u t i l i z e FFA r e a d i l y as an energy source. I t seemed possible, therefore, that the data obtained might be more an 43 •FIGURE 8: P r o d u c t i o n o f ^COz from p a l m i t i c a c i d - ( 1 - 1 ^ C ) . C e l l s were i n c u b a t e d i n KRP b u f f e r pH 7.4 a t 37° f o r 30 minutes w i t h 20,000 dpm p a l m i t i c a c i d - ( 1 - 1 ' ' C ) and 1 kCOz c o l l e c t e d . 44 i n d i c a t i o n of r e l a t i v e dependence upon fa t t y acid as an energy source than an i n d i c a t i o n of the capacity of the c e l l s f o r oxidation. Accordingly, experiments were carr i e d out i n a complete tissue culture medium (Fischer's, for composition see Appendix I ) , which, i t should be noted, i s the medium i n which the c y t o l y t i c action of c o r t i c o -steroids can be demonstrated. The u t i l i z a t i o n of l a b e l l e d palmitic acid i n t h i s medium was decreased i n a l l three c e l l l i n e s (Table I I I ) . The u t i l i z a t i o n was decreased much more i n the c o r t i c o s t e r o i d - s e n s i t i v e thymus and P1798S than i n the s t e r o i d r e s i s t a n t P1798R. Numerous experiments were undertaken to examine the e f f e c t s of C o r t i s o l and of c a r n i t i n e upon the oxidation of palmitic acid i n complete medium. While the r e p l i c a t i o n i n any one experiment was good, the e f f e c t of added constituents was variable from one experiment to the next, sometimes no e f f e c t being observed at a l l . One l i k e l y explanation i s that the "control" tissues might have i n f a c t been exposed to elevated c o r t i c o s t e r o i d levels p r i o r to t h e i r removal, and were thus i n e f f e c t , already treated. As the tumors grow larger, the animals become quite stressed, which would elevate c i r c u l a t i n g c o r t i c o s t e r o i d l e v e l s i n the mice (52,53). Also,the l a b e l l e d substrate was added only i n tracer amounts and the metabolism of a minute amount of fatty acid would be influenced by the s i z e of the endogenous pool at any given moment, a very variable 45 TABLE I I I : Comparison of Palmitic Acid-(1- 1^C) Oxidation i n D i f f erent Media 1.7C02 RECOVERED (dpm/M c e l l s ) CELL LINE KREB'S BUFFER FISCHER'S MEDIUM THYMUS 3 479 576 P1798S 5847 446 P1798R 551 111 Ce l l s were incubated i n Fischer's Medium or KRP for 30 minutes and 1 "*C02 produced from oxidation of palmitic acid- (1-11*C) c o l l e c t e d . Figures are the mean of 4-5 samples. 46 f a c t o r . Grea t v a r i a b i l i t y has been encountered i n t h i s l a b o r a t o r y and a l s o by o t h e r workers i n the uptake o f l a b e l l e d f a t t y a c i d s by tumor c e l l s i n v i v o ( 5 4 , 5 5 ) . When t h e i r l a b e l l e d FFA was d i l u t e d w i t h u n l a b e l l e d c a r r i e r the v a r i a b i l i t y was g r e a t l y reduced and r e p r o d u c i b l e r e s u l t s were o b t a i n e d . The e f f e c t o f u n l a b e l l e d s u b s t r a t e c o u l d a l s o g i v e i n f o r m a t i o n on the s i z e o f the endogenous p o o l and would more l i k e l y r e v e a l the t r u e o x i d a t i v e c a p a c i t y o f the c e l l s . R e s u l t s o f such e x p e r -iments a re shown i n Tab le IV. E x p r e s s e d as a percentage o f the c o n t r o l v a l u e s i n each c a s e , C o r t i s o l reduced the o x i d a t i o n o f p a l m i t i c a c i d i n s e n s i t i v e thymus and P1798S, w h i l e i n c r e a s i n g i t s l i g h t l y i n the r e s i s t a n t P1798R l i n e . The a d d i t i o n o f 78 yM depressed lkC02 p r o d u c t i o n 25% i n bo th s e n s i t i v e c e l l l i n e s , but o n l y s l i g h t l y dec reased t h a t o f the r e s i s t a n t c e l l l i n e . T h i s r e s u l t i n the r e s i s t a n t l i n e was i n t e r p r e t e d as i n d i c a t i n g a s m a l l e r endogenous p o o l and/or a g r e a t e r c a p a c i t y f o r o x i d a t i o n . C o r t i s o l produced a f u r t h e r d e c r e a s e i n the s e n s i t i v e c e l l s w i t h 78 yM p a l m i t i c a c i d , w h i l e a g a i n a s l i g h t s t i m u l a t i o n was observed i n the P1798R. The r e s u l t s i n s e n s i t i v e c e l l s c o u l d be i n t e r p r e t e d as an i n h i b i t i o n by C o r t i s o l o f f a t t y a c i d metabo l i sm r a t h e r than an i n c r e a s e i n the s i z e o f the endogenous p o o l o f f a t t y a c i d . T h e r e f o r e , the s i z e o f the endogenous pool was measured i n both c o n t r o l and C o r t i s o l t r e a t e d 47 TABLE I V : E f f e c t o f C o r t i s o l a n d / o r P a l m i t i c A c i d o n t h e O x i d a t i o n o f P a l m i t i c A c i d - ( 1 - 1 ^ C) t o ll*C02 TISSUE TREATMENT THYMUS P1798S P17 98R CONTROL 100% 100% 100% CORTISOL (2.7uM) 56% 8 3 % 1 0 9 % PALM I T I C ACID 7 5 % 7 3 % 9 0 % (78uM) CORTISOL (2.7uM) + PALMITIC ACID 5 0 % * 66% 9 5 % (78yM) The p e r c e n t a g e s r e f l e c t t h e amount o f lkCOz p r o d u c e d r e l a t i v e t o t h e c o n t r o l v a l u e . C e l l s w e r e i n c u b a t e d i n F i s c h e r ' s Medium a t 37° f o r 30 m i n u t e s w i t h p a l m i t i c a c i d -( 1 - 1 ' * C ) . F i g u r e s i n d i c a t e d a r e t h e mean o f 15-20 s a m p l e s e x c e p t f o r * w h i c h i s t h e mean o f 6 s a m p l e s . 48 t i s s u e s . S t e r o i d - t r e a t e d a n i m a l s w e r e i n j e c t e d w i t h 1 mg d e x a m e t h a s o n e i n 0.1 m l Sesame O i l i p ; c o n t r o l a n i m a l s w e r e i n j e c t e d w i t h t h e v e h i c l e a l o n e . Two h o u r s l a t e r P 1 7 9 8 S , P1798R a n d thymus t i s s u e w e r e r e m o v e d a n d FFA l e v e l s d e t e r m i n e d by m i c r o t i t r a t i o n . The r e s u l t s a r e r e p o r t e d i n T a b l e V. T h e s e r e s u l t s a r e i n a c c o r d w i t h t h e i n t e r p r e t a t i o n o f p r e v i o u s r e s u l t s ( c f T a b l e I V ) t h a t t h e e n d o g e n o u s l e v e l s o f FFA a r e i n c r e a s e d i n t h e s t e r o i d - s e n s i t i v e c e l l s a n d a c t u a l l y d e c r e a s e d i n t h e r e s i s t a n t P1798R. W h e t h e r o r n o t t h e i n c r e a s e d l e v e l s o f e n d o g e n o u s f r e e f a t t y a c i d w e r e a r e f l e c t i o n o f i n c r e a s e d r e l e a s e o f f r e e f a t t y a c i d s f r o m e n d o g e n o u s f a t s t o r e s o r i n c r e a s e d u p t a k e o f p l a s m a f r e e f a t t y a c i d s b y t h e s e s e n s i t i v e t i s s u e s i n v i v o c o u l d n o t be a s c e r t a i n e d f r o m t h e s e r e s u l t s . The e f f e c t o f C o r t i s o l on f a t t y a c i d u p t a k e i n v i t r o was t h e r e f o r e d e t e r m i n e d . C e l l s u s p e n s i o n s w e r e p r e p a r e d a n d i n c u b a t e d w i t h 7 8 yM p a l m i t i c a c i d w i t h o r w i t h o u t a d d e d C o r t i s o l (2.7 yM) f o r 2 h o u r s . I n o r d e r t o d e t e r m i n e w h e t h e r o r n o t C o r t i s o l h a d a f f e c t e d t h e u p t a k e o f p a l m i t i c a c i d , c e l l s w e r e p u l s e d w i t h p a l m i t i c a c i d - ( 1 - 1 ' ' C ) 30 m i n u t e s p r i o r t o t h e e n d o f t h e i n c u b a t i o n . A t t h e e n d o f t h e i n c u b a t i o n t h e c e l l s w e r e p e l l e t e d a t 1500 rpm i n a r e f r i g e r a t e d i n t e r n a t i o n a l c l i n i c a l c e n t r i f u g e . The medium was d e c a n t e d , t h e c e l l p e l l e t d i s s o l v e d i n 1.0 m l Hyamine a n d t h e u p t a k e o f l a b e l l e d p a l m i t i c a c i d d e t e r m i n e d . 49 TABLE V: E f f e c t of Dexamethasone on Endogenous Free Fatty Acid Levels LEVEL OF FFA (yEqv/gm) TISSUE EXPT # CONTROL LEVEL DEXAMETHASONE TREATED % INCREASE THYMUS I . * 1 9.554 16.885 +76% P1798S l . * 2 2. 8.140 3.434 12.870 3.904 +58% +14% P1798R 1, 2, 3.966 3.834 2.954 3.092 -26% -19% Animals were treated with 1 mg dexamethasone i n 0.1 ml Sesame o i l or with vehicle alone. Two hours l a t e r animals were s a c r i f i c e d , tissue removed and FFA level s determined by m i c r o t i t r a t i o n . * 1 Figures indicated are the mean value for a pooled sample of thymus from 5 female Balb/cJ mice weighing 17-20 gm. * 2 Figure indicated i s expressed i n nEqv FFA/M c e l l s . 50 The r e s u l t s are expressed i n Table VI. The uptake of palmitic acid was not affected s i g n i f i c a n t l y by C o r t i s o l i n any of the tissues studied. I t would appear that the increased endogenous levels of FFA must come from the l i p i d stores of the lymphoid c e l l s , these stores being t r i g l y c e r i d e . Abramson and Blecher (58)• have reported that i n rat thymocytes 2% of the t o t a l wet weight of tissue i s l i p i d , ' with over 60% of th i s l i p i d being neutral l i p i d . Further characterization showed that over 80% of the f r a c t i o n was t r i g l y c e r i d e . In the steroid sensitive c e l l s used i n th i s study i t was found that the t o t a l l i p i d content ranged from 4.2% f o r P1798S to 7.2% for thymus tissue with over 71% of the t o t a l f a t being neutral glyceride i n the case of P1798S. TLC of the l i p i d extracts of P1798S and P1798R showed that the only EFA detectable were phospholipid and t r i g l y c e r i d e , with traces of cholesterol esters observable under c e r t a i n conditions. The only NEFA detectable by chromatography was cholesterol. The Oxidation of Short Chain Fatty Acids: Spector (56) has reported that tumor c e l l s are limi t e d i n t h e i r capacity to oxidize short chain f a t t y acids as compared to long chain f a t t y acids. Accordingly, thymus and mouse lymphosarcoma c e l l s were incubated with equivalent concentrations of l a b e l l e d palmitic acid or sodium octanoate and 1 h t o z production monitored i n order 51 TABLE VI: E f f e c t of C o r t i s o l on Palmitic Acid Uptake PALMITIC ACID UPTAKE (dpm/M c e l l s ) TISSUE EXPT # CONTROL CORTISOL TREATED (2.7 yM) THYMUS 1. 1967±196 1753±371 2. 900±19 834±22 3. 978±36 1045115 P1798S 1. 923159 1039195 2. 609111 605121 P1798R l . * 1 1955159 1872+42 Ce l l s were incubated with 7 8 yM palmitic acid for 2 hours and 30 minutes p r i o r to the end of the incubation were pulsed with palmitic acid-(1- 1^C). Figures indicated are the MEAN 1 SEM for 4 samples. * 1 Figures indicated are the MEAN 1 SEM for 8 samples. 52 TABLE VII: L i p i d Composition of Mouse Lymphosarcoma P1798 and Thymus TISSUE TOTAL LIPID LIPID PHOSPHATE TEFA mg/gm yM/gm yM/gm P1798R * 1 23.2 5.4 74.5 P1798S *- 42.2 5.5 81.4 THYMUS * 2 71.6 Tissues were removed and t o t a l l i p i d , l i p i d phosphate and TEFA determined as indicated i n the methods section. ** Figures indicated are the mean of 3 separate deter-minations from at least 1 gm of tissue (wet weight). * 2% Figure indicated i s the mean of a 200 mg pooled sample from 5 female Balb/cJ mice (weight 17-20 gm). TABLE VIII: L i p i d D i s t r i b u t i o n of Mouse Lymphosarcoma LIPID COMPOSITION TISSUE' PHOSPHOLIPID NEUTRAL LIPID P1798S 28.9 71.0 P1798R 44.0 56.9 THYMUS(RAT)*1 38.5 61.5 (82.3%TG) L i p i d d i s t r i b u t i o n of mouse lymphosarcoma was determined by separating phospholipid from neutral l i p i d on activated s i l i c i c acid columns using technique described i n the methods section. * : From Abramson and Blecher (58),included for comparison purposes. 54 to see i f lymphoid c e l l s e x h i b i t e d c h a r a c t e r i s t i c s common to o t h e r tumor c e l l s . The r e s u l t s i n Table IX i n d i c a t e t h a t the r e v e r s e i s t r u e i n t h i s system. Lymphoid c e l l s have a g r e a t e r c a p a c i t y t o o x i d i z e s h o r t c h a i n f a t t y a c i d s , which i s s i m i l a r t o v a r i o u s normal t i s s u e s (57). E f f e c t of C o r t i s o l and P a l m i t a t e on Carbohydrate Metabolism: The long and s h o r t term e f f e c t s of C o r t i s o l and p a l m i t a t e on carbohydrate metabolism were s t u d i e d by examining the o x i d a t i o n of glucose to C0 2 and the uptake of 2 deoxyglucose- ( 1 - 1 ''C) . During the f i r s t 30 minutes f o l l o w i n g g l u c o c o r t i c o i d or p a l m i t a t e treatment no e f f e c t was observed on the metabolism of Glucose- ( 1 ^ C-U) t o ll*C02. Only 2 hours a f t e r treatment was there any e f f e c t on carbo-hydrate uptake (Table X I ) . C o r t i s o l depressed the uptake of 2-deoxyglucose, which competes w i t h g l u c o s e (60,61) onl y i n s t e r o i d r e s p o n s i v e t i s s u e s ; i t has no e f f e c t on P1798R. The e f f e c t s of p a l m i t a t e , except i n one case, were n e g l i g i b l e when compared w i t h the c o n t r o l v a l u e s . E f f e c t o f FFA on Mouse Lymphosarcoma C e l l S u r v i v a l : The r e s u l t s of i s o t o p e d i l u t i o n experiments (Table IV) i n d i c a t e d t h a t c e l l s s e n s i t i v e to c o r t i c o s t e r o i d would accumulate f r e e f a t t y a c i d . T h i s accumulation of f r e e f a t t y a c i d s c o u l d cause damage to c e l l s . Incubations were c a r r i e d out w i t h tumor c e l l s i n which the e f f e c t s 55 TABLE IX: Oxidation of Long and Short Chain Fatty Acids 1*COz PRODUCED FROM: TISSUE PALMITIC ACID-(1- 1 kC) SODIUM n-OCTANOATE-(1-1"c) THYMUS 799±64 11,1591418 P1798S 1539187 12,403+59 P1798R 701118 15,897162 C e l l s were incubated for 3 0 minutes with equivalent concentrations of palmitate or octanoate. 1 hCOz was assayed as previously described. Figures indicated are the MEAN 1 SEM for 4-5 samples. 56 of added f a t t y acids on c e l l v i a b i l i t y over a period of several hours were examined. As can be seen i n Table XII, both palmitic acid, a C-16, and a z e l a i c , a C-9 dicarbo-x y l i c acid (used also because of i t s greater s o l u b i l i t y i n aqueous so l u t i o n ) , increased the death of c e l l s i n culture. By contrast, the r e s i s t a n t subline P1798R was only p a r t i a l l y affected by concentrations of FFA ten times greater than that s u f f i c i e n t to k i l l a l l sensitive c e l l s i n 5.5 hours (Table XIII). A p a r a l l e l exists between s e n s i t i v i t y to FFA and s e n s i t i v i t y to c o r t i c o s t e r o i d s . Electronmicroscope Examination of the Effects of FFA on  Mouse Lymphoid C e l l s : The nature of the damage to sensitive c e l l s by FFA and i t s e f f e c t s on r e s i s t a n t c e l l s were examined i n greater d e t a i l using the electron microscope. Figure 9 shows the i n t e g r i t y of normal f i n e structure of untreated c o r t i c o s t e r o i d - s e n s i t i v e mouse lymphosarcoma P1798 c e l l s incubated i n Fischer's Medium f o r 4 hours. Maintenance of normal fi n e structure was also observed i n untreated thymocyte suspensions and i n c o r t i c o s t e r o i d -r e s i s t a n t mouse lymphosarcoma P1798 suspension, with or without added f a t t y acids. The presence of capsid-like structures both p a r t i a l l y formed, i n close association with the endoplasmic reticulum membrane, and more completely formed, found i n an i n t r a -57 TABLE X: Oxidation of Glucose by Mouse Lymphosarcoma C e l l s 1 ^  C0 2 PRODUCED (dpm/M c e l l s ) TISSUE EXPT # CONTROL CORTISOL TREATED (2.7 yM) PALMITATE TREATED (78 yM) P1798R 1. 2. 3, 54419 58517 690117 53413 587126 691+17 554118 586141 697110 P1798S 1. 2, 501 + 4 34214 51218 352 + 7 515119 347118 Ce l l s were incubated for 30 minutes with 370,000 dpm of Glucose- ( 1 "*C-U) and 1 kCC>2 c o l l e c t e d as previously de-scribed. Figures indicated are MEAN 1 SEM fbr 5 samples. 58 TABLE XI: Uptake of 2-Deoxyglucose-(1- 1^C) by Mouse Lymphoid C e l l s UPTAKE OF 2-DE0XYGLUC0SE (dpm/M c e l l s ) TREATMENT TISSUE EXPT # CONTROL CORTISOL PALMITATE (2.7 yM) (78 yM) THYMUS 1. 87±5 59±1 82±2 2. 91±4 68±5 149±11 P1798S 1. 135 + 8 118±13 120±12. 2. 154+12 88±3 145±11 P1798R 1. 36±2 35±2 40±3 2. 448±8 439±8 441±8 3. 59±3 62±13 57±4 C e l l s were incubated with 2.7 yM C o r t i s o l or 7 8 yM palmitic acid for 2 hours. Thirty minutes p r i o r to the termination of the incubation c e l l s were pulsed with 0.5yCi of 2-deoxyglucose-(1- 1^C). Figures indicated are MEAN ± SEM for 4-5 determinations. 59 TABLE XII: E f f e c t of Free Fatty Acids on P1798S In V i t r o VIABLE CELLS ±SEM DEAD CELLS!SEM (M CELLS/ML) (T CELLS/ML) CONTROL 1.725 ± 0.109 12.5 ± 12.5 PALMITATE TREATED 1.117 ± 0.217 * 250.0 ± 28.8 (20 yg/ml) AZELAIC ACID TREATED (10 yg/ml) 1.083 ± 0.174 * 283.3 ± 16.7 C e l l s were incubated i n Fischer's Medium with 10% Horse Serum at 37° for 4 hours. Viable c e l l s were determined by eosin exclusion. * p <0.05 compared with control value. 60 TABLE XIII: E f f e c t of Increasing Azelaic Acid Concentration on Mouse Lymphosarcoma P1798 C e l l s In V i t r o TUMOR AZELAIC ACID % LYSED CELLS A% CONCENTRATION P1798S 53 yM 100 (7) P1798R 0 yM 15.110.8 (7) 320 yM 18.110.9 (7) 3 <.05 420 yM 22.8+2.6 (7) 7.7 <.05 530 yM 25.0+2.6 (7) 9.9 <.01 Approximately 800T c e l l s were incubated i n Fischer's Medium with 10% Horse Serum for 5.5 hours at 37° C. The t o t a l and the number of lysed c e l l s were counted by a hemacytometer. 61 c i s t e r n a l p o s i t i o n (Figure 9; Inset, Figure 15) were noted i n P1798S c e l l s . These appeared i d e n t i c a l with so-c a l l e d A - p a r t i c l e s reported i n other murine leukemias and the normal tissue of neonatal mice (62) . Comparison of thick sections of control and treated P1798S suspensions af t e r 4 hours incubation when stained with t o l u i d i n e blue showed a marked increase i n c e l l degradation and necrosis i n treated sections (Figures 1 0 , 1 1 , 1 2 ) . Degenerative changes e s p e c i a l l y evident were the swelling and v e s i c u l a t i o n pf the cytoplasm. C e l l s undergoing necrosis were characterized by pyknosis and k a r y o l y s i s . The fine structure of P1798S c e l l s a f t e r 4 hours treatment confirmed the progressive nature of the degener-ative process. D i l a t a t i o n , swelling and subsequent v e s i c u l a t i o n of the endoplasmic reticulum (Figures 13 ,14 , 15,16) e s p e c i a l l y i n golgi regions (Figure 15) preceded c e l l death (Figure 1 7 ) . Noteworthy was the almost complete absence of membrane-bound ribosomes i n those c e l l s exhib-i t i n g minimal pathological changes at t h i s time. Marked d i l a t a t i o n of the perinuclear c i s t e r n a (Figure 14) usually progressed concomitantly with the v e s i c u l a t i o n of the endoplasmic reticulum. Mitochondrial a l t e r a t i o n s , except i n prenecrotic c e l l s , were not synchronous, but included only a portion of those within the c e l l (Figure 1 3 ) . Large dense granules (Figure 16) appeared i n the cytoplasm of treated but not control c e l l s at 4 hours. They were 62 e s p e c i a l l y conspicuous i n c e l l debris remaining a f t e r c y t o l y s i s . Detailed events i n the degenerative changes i n nuclear f i n e structure were d i f f i c u l t to assess because of the variable morphology of the nuclei of t h i s tumor and due to the asnychronous development of degeneration within c e l l populations. However, as a general r u l e , as cytoplasmic degeneration proceeded, the granularity of euchromatin became less pronounced and i t s usual sharp demarcation from heterochromatin less marked (compare Figure 9 with Figures 14 and 16). Figures 18 and 19 show the fine structure of thymo-cytes a f t e r 4 hours treatment with 78 yM palmitic acid. E s p e c i a l l y prominent at this time were those c e l l s under-going karyorrhexis. Areas of the nuclear membrane appeared to have become compromised with subsequent appearance of nuclear material, free and membrane bound, admixed with cytoplasm. A working hypothesis of the various factors which are involved i n bringing about corticosteroid-induced l y s i s i s i l l u s t r a t e d at Figure 20. The damage r e s u l t i n g from accumulation of FFA has been described; biochemical evidence that t h i s does i n fact occur has been obtained (Table V) . 63 FIGURE 9: Untreated control P1798S c e l l s after 4 hrs of incubation. The s t r u c t u r a l i n t e g r i t y of the incubated c e l l s i s well preserved. Maraglass X22,510. The inset shows membrane-associated and i n t r a c i s t e r n a l capsid-like structures that are commonly found i n the cytoplasm throughout the c e l l population. X59,190. 65 FIGURE 10: Untreated control P1798S c e l l s after 4 hrs of incubation. Good preservation of c e l l population i s evident. 0.5 micron thick section. Toluidine blue s t a i n . X1430. FIGURE 11: Palmitic acid-treated P179 8S c e l l s after 4 hrs of incubation. Cytoplasmic v e s i c u l a t i o n (arrows) i s very obvious. Extensive necrosis i s evidenced by pyknosis. Toluidine blue stain.X1430. FIGURE 12: Azelaic acid-treated P1798S c e l l s a f t e r 4 hrs of incubation. Extensive c e l l necrosis has occurred. Cytolysis i s a predominating event. Toluidine blue s t a i n . X1430. 6 6 7 \-67 FIGURE 13: P a l m i t i c a c i d - t r e a t e d P1798S c e l l s a f t e r 4 hours of i n c u b a t i o n . S w e l l i n g of the endop lasmic r e t i c u l u m , e s p e c i a l l y i n g o l g i r e g i o n s , i s prominent i n the e a r l y s tages of d e g e n e r a t i o n . Note the a lmost complete absence o f membrane-bound po lysomes . A l t e r a t i o n s i n m i t o c h o n d r i a and n u c l e a r mebrane are not obv ious a t t h i s t i m e . Marag las X30,870. 69 FIGURE 14: P a l m i t i c a c i d - t r e a t e d P179 8S c e l l s a f t e r 4 h o u r s o f i n c u b a t i o n . M a s s i v e v e s i c u l a t i o n o f t h e e n d o p l a s m i c r e t i c u l u m (ER) and s w e l l i n g o f t h e p e r i n u c l e a r c i s t e r n a a r e p r o m i n e n t e v e n t s i n t h e l a t e s t a g e o f d e g e n e r a t i o n . Note t h e f o c a l a l t e r a t i o n s i n t h e m i t o c h o n d r i a and t h e l o s s o f t h e s h a r p d i c h o t o m y between e u c h r o m a t i n and h e t e r o c h r o m a t i n . M a r a g l a s s X30,140. 70 71 FIGURE 15: Azelaic acid-treated P179 8S c e l l s a f t e r 4 hours incubation. Early swelling of the golgi zones and f o c a l al t e r a t i o n s i n the mitochondria are evident. Note the presence of capsid-like structures (arrow) within the ER. C e l l debris, a consequence of c y t o l y s i s , i s demonstrated i n the upper r i g h t area of the micrograph. Maraglas X26,370. 72 73 FIGURE 16: Azelaic acid-treated P1798S c e l l s a f t e r 4 hours of incubation. Progressive degeneration of the cytoplasm i s t y p i f i e d by swelling of both ER (arrow) and perinuclear c i s t e r n a . Isolated mitochondria show matrix a l t e r a t i o n s . Demarcation between euchromatin and heterochromatin i s reduced. The dense body at the l e f t of the micrograph i s found commonly i n treated c e l l s at 4 hours but not i n untreated c e l l s . Maraglas X20,420. FIGURE 17: Azelaic acid-treated P1798S c e l l s after 4 hours of incubation. Terminal s t r u c t u r a l a l t e r a t i o n i n prenecrotic c e l l s i s apparent by i t s progressive nature. The i n t e g r i t y of the nuclear membrane i s l o s t . Note the membrane bound p r o f i l e s within the nucleus (arrow). Degenerate mitochondria, vesiculated cisternae, and degranulation of ER are character-i s t i c of th i s terminal stage. Maraglas X31,640. 74 75 FIGURE 18: Palmitic acid-treated thymocytes a f t e r 4 hours of incubation. Note the process of karyorrhexis occurring i n these c e l l s . Portions of the nuclear material, sometimes membrane-bound (arrow), become separated from the nucleus and l i e within the cytoplasm. Note that the small degree of ER v e s i c u l a t i o n i s correlated with the paucity of ER within these c e l l s . Epon X22,760. 77 FIGURE 19: P a l m i t i c a c i d - t r e a t e d thymocytes a f t e r 4 hours of i n c u b a t i o n . P a r t i a l l y - b o u n d and unbound clumps (arrow) o f n u c l e a r m a t e r i a l l i e admixed w i t h i n the c y t o p l a s m . P e r i n u c l e a r c i s t e r n a l s w e l l i n g i s not obv ious i n these c e l l s undergo ing k a r y o r r h e x i s . Epon X27,710. FIGURE 20: Proposed scheme of possible factors leading to c y t o l y s i s . What the scheme proposes i s that FFA would be released from t r i g l y c e r i d e stores. An accumulation of FFA i n steroid-responsive tissues would lead to nuclear damage and ultimately c e l l l y s i s . S t e r o i d - r e s i s t a n t tissues would remove FFA by oxidation and possibly by r e - e s t e r i f i c a t i o n . 80 E f f e c t of Free Fatty Acids i n Inducing Lysis of Steroid- Resistant Tissues: In order to tes t further the c r i t i c a l importance of fa t t y acid i n the process of c y t o l y s i s , i t was reasoned that i n h i b i t i o n of f a t t y acid oxidation i n r e s i s t a n t c e l l s might then permit s u f f i c i e n t accumulation of thi s con-s t i t u e n t to cause damage to c e l l s . Deoxycarnitine, a s t r u c t u r a l analogue of ca r n i t i n e , lacks the oxygen function to which the fat t y acid i s e s t e r i f i e d i n ca r n i t i n e and i s thereby transported i n the mitochondria where i t under-goes 3-oxidation (for structure of carn i t i n e and deoxy-c a r n i t i n e , see Appendix I I ) . Deoxycarnitine functions as a competitive i n h i b i t o r i n the oxidation of FFA (95). Accordingly, c e l l s of the three s t e r o i d - r e s i s t a n t lympho-sarcomas were incubated with t h i s compound and the v i a b i l i t y of c e l l s was determined.(Table XIV). Approximately one t h i r d of the c e l l s of each tumor underwent l y s i s when treated with deoxycarnitine and C o r t i s o l together. The c e l l s remaining did not respond even to higher concentrations of i n h i b i t o r than.the 0.8 mM used i n these experiments. Injection of mice bearing these tumors i n the ascites form with deoxycarnitine produced s i m i l a r r e s u l t s . When the st e r o i d - r e s i s t a n t L517 8Y lymphosarcoma i s treated i n v i t r o with 10 ug of c i t r a l (3,7-dimethyl, 2,6-octadienal, for structure see Appendix I I ) , which, 81 TABLE XIV: E f f e c t of Deoxycarnitine on the Response of Tumor C e l l s to C o r t i s o l In V i t r o TUMOR VIABLE CELLS % OF CONTROLS P1798R 65+6 (6) L1210 63+14 (4) L5178Y 59±8 (4) Ce l l s were incubated i n tissue culture at 37° for 6 hours. Control samples were treated with 2.7 yM C o r t i s o l . Experimental samples had 0.8mM deoxycarnitine added as well. 82 i t was thought, because of i t s branched chain structure would i n h i b i t B-oxidation, i t was found that 30% of the c e l l s were rendered sensitive to the action of C o r t i s o l (Table XV). When the more soluble sodium b i s u l f i t e addition compounds of c i t r a l were tested, the number.? of c e l l s rendered sensitive was increased to 50% and 60% for concentrations of 20 and 100 yg of the compound respectively. In vivo treatment of the L517 8Y and L1210 with a c i t r a l suspension resulted i n 20% via b l e c e l l s remaining 6 hours aft e r treatment (Table XVI). The sodium b i s u l f i t e addition compound, although more e f f e c t i v e than c i t r a l i n v i t r o , was less e f f e c t i v e i n vivo (cf Table XV). When animals bearing the subcutaneous P1798R were treated with 5 mg c i t r a l i p , destructive effects of the compound were seen i n treated animals (compare Figure 21 and 22 with 23 and 24). Twenty-four hours following c i t r a l treatment large necrotic areas were observed i n treated tumors (compare Figure 21 with 23). High power observation of these areas indicated that the c e l l s had undergone karyolysis (compare Figure 22 with 24). The Relationship of Cytolysis to other C o r t i c o s t e r o i d Actions: It i s well documented that c o r t i c o s t e r o i d treatment of animals re s u l t s i n immunosuppression (66). I t was of in t e r e s t therefore to determine i f free f a t t y acids TABLE XV: E f f e c t of C i t r a l on L517 8 In V i t r o TREATMENT VIABLE CELLS % CONTROLS CITRAL 10 yg 69 (2) CITRAL SODIUM BISULFITE 20 yg 52 (2) CITRAL SODIUM BISULFITE 100 yg 42 (2) Cel l s were incubated i n Fischer's Medium with 10% Horse Serum at 37° for 6 hours. Controls were incubated with 2.7 yM C o r t i s o l only. TABLE XVI: E f f e c t of C i t r a l on Tumors In Vivo TUMOR TREATMENT VIABLE CELLS % CONTROLS L5178Y CITRAL SUSPENSION 20.0 (7) CITRAL SODIUM BISULFITE 60.0 (2) L1210 CITRAL SUSPENSION 16.6 (5) CITRAL SODIUM BISULFITE 90.0 (2) IP tumors 3 days after transplantation were treated i p with 5 mg CITRAL i n saline suspension. In some cases two in j e c t i o n s 2.5 hours apart were made. Ce l l s were ex-amined at 6 hours and the t o t a l lysed and unlysed c e l l s were counted. FIGURE 21: Section of P1798R from control animal. Hematoxylin and Eosin X52. 87 FIGURE 22: Section of P1798R from control animal. Hematoxylin and Eosin X206. 8 8 89 FIGURE 23: S e c t i o n o f P1798R taken from an imal 24 hours a f t e r t rea tment w i t h 5mg CITRAL i p . Note l a r g e areas of n e c r o s i s , (cf F i g u r e 2 1 ) . Hematoxy l in and E o s i n X52. 90 91 FIGURE 24: Section of P1798R taken from animal 24 hours afte r treatment with 5mg CITRAL i p . Note karyorrhexis has occurred, (cf Figure 22). Hematoxylin and Eosin X206. 92 93 reproduced a l l the known effects of c o r t i c o s t e r o i d i n lymphoid tissue. I t might be possible to separate the immunosuppressive and other a c t i v i t i e s of c o r t i c o s t e r o i d from i t s c y t o l y t i c actions. In order to explore t h i s p o s s i b i l i t y , thymus, Pl798S,and P1798R c e l l s were incubated o for 2 hours i n tissue culture medium at 37 with or without the addition of C o r t i s o l (2.7 yM) or palmitic acid (78 yM). T h i r t y minutes p r i o r to the end of the incubation, c e l l s were pulsed with l a b e l l e d precursors of DNA, RNA and protein and t h e i r incorporation into a 5% TCA insoluble f r a c t i o n determined. The r e s u l t s are presented i n Tables XVII, XVIII, and XIX. C o r t i s o l i n h i b i t s the uptake of thymidine into the acid-insoluble p r e c i p i t a t e of steroid-responsive tissues with one exception, but i s without e f f e c t i n the s t e r o i d -r e s i s t a n t P1798R (Table XVII). In the one experiment i n which the P1798S tumor was unresponsive to C o r t i s o l , the tumor was taken 19 days after transplant. At t h i s stage the tumor i s very large and may have already been treated with s t e r o i d due to a stress response (see previous comments, page 44). The effects p a r a l l e l those of C o r t i s o l to a lesser extent i n i n h i b i t i n g the incorporation of thymidine into DNA of steroid-responsive tissues and are without e f f e c t i n the r e s i s t a n t c e l l s . C o r t i s o l also i n h i b i t s the incorporation of uridine and leucine into the acid-insoluble f r a c t i o n of thymus and P1798S but i t i s without e f f e c t i n P1798R. (Tables XVIII 94 TABLE XVII: Incorporation of Thymidine-(CH 3- 3H) into the Acid-Insoluble Fraction TISSUE EXPT •.# INCORPORATION (dpm/M c e l l s ) CONTROL CORTISOL PALMITATE THYMUS 1. 2. 20731235 2219122 1584156 15991160 1823127 19531166 P1798S 1. 2.*1 1010131 30818 584120 30514 862+41 354112 P1798R 1. 2. 831127 854137 822114 772120 996134 860132 Samples were incubated for 2 hours i n tissue culture medium at 37° with or without added C o r t i s o l (2.7 yM) or palmitic acid (7 8 yM). 30 minutes pr i o r to the end of the incubation they were pulsed with 1 yC. of thymidine (CH3~ 3H), acid-insoluble f r a c t i o n was collected and r a d i o a c t i v i t y determined. Figures indicated are MEAN 1SEM for 4-5 samples. ** Figures indicated are for a large 19 day old tumor. 95 and X I X ) . P a l m i t i c acid h a s no s i g n i f i c a n t e f f e c t on the incorporation of leucine and uridine into TCA insoluble f r a c t i o n of thymus, P 1 7 9 8 S , and P1798R. The suppressive e f f e c t s of C o r t i s o l on the incorporation of thymidine, uridine and leucine into a 5% TCA insoluble f r a c t i o n and lack of e f f e c t on P1798R are i n agreement with the r e s u l t s of others (63,64) . TABLE XVIII: Incorporation of Uridine-(5- 3H) into the Acid-Insoluble Fraction INCORPORATION (dpm/M c e l l s ) TISSUE EXPT # CONTROL CORTISOL PALMITATE THYMUS 1. 619±33 4 8 5 ± 1 1 603±36 2. 6 5 8 + 1 1 415±7 698±16 P1798S 1. 1323120 890±9 1297±7 2. 1600±91 993+21 1511±43 P1798R 1. 1112168 985±22 1010+29 2. 1036±42 1065±20 1055128 Samples were incubated for 2 hours i n tissue culture medium at 37 with or without added C o r t i s o l (2.7 yM) or palmitic acid (7 8 yM). Thirty minutes p r i o r to the end of the incubation they were pulsed with 1 yC^ of Uridine-(5- 3H), acid-insoluble f r a c t i o n was coll e c t e d and radio-a c t i v i t y determined. Figures indicated are MEAN ± SEM for 4-5 samples. TABLE XIX: Incorporation of Leucine-i 1 kC-U) into the Acid-Insoluble Fraction INCORPORATION (dpm/M c e l l s ) TISSUE EXPT # CONTROL CORTISOL PALMITATE THYMUS 1. 172±2 12611' 167+3 2. 20914 164+4. 21616 P1798S 1. 501+11 385+6 51016 2. 436122 282111 451113 P1798R 1. 44818 43918 444118 2. 308114 29115 332118 Samples were incubated for 2 hours i n tissue culture medium at 37 with or without added C o r t i s o l (2.7 yM) or palmitic acid (7 8 yM). Thirty minutes p r i o r to the end of the incubation, they were pulsed with 0.5 yC^of leucine-( 1 4C-U), acid-insoluble f r a c t i o n was coll e c t e d and radio-a c t i v i t y determined. Figures indicated are MEAN 1 SEM for 4-5 samples. 98 DISCUSSION Most work on the action of corticosteroids has centered around the important metabolic events occurring i n l i v e r (68). Induction of s p e c i f i c RNA and enzymes by c o r t i c o i d s and the subsequent changes have been reviewed (68) . This report concerns a "peripheral" action of c o r t i c o s t e r o i d s : the c y t o l y t i c action on lymphoid tissue. The dramatic e f f e c t of corticosteroids upon ce r t a i n lymphoid c e l l s , e s p e c i a l l y those.of thymus, has been under investi g a t i o n for some time (69) . A s i m i l a r action occurs i n c e r t a i n malignant lymphocytes, notably the acute lymphocytic leukemias of childhood, and r e s u l t s i n l y s i s of c e l l s and remission of the disease for a variable period of time. Ultimately, as with other chemothera-peutic agents, the c e l l population becomes r e s i s t a n t to the action of the steroid (70). The work to be discussed here was undertaken to explore the mechanism whereby l y s i s occurs and the biochemical basis for the develop-ment of resistance. Destructive e f f e c t s of f a t t y acids on both malignant and non-malignant c e l l s have been observed i n v i t r o (24,71) and also i n vivo i n the case of tumor c e l l s (72). Ethyl palmitate has been reported to cause extensive destruction of the spleen i n several species (73). The actions of free f a t t y acids on mitochondria, i n causing 99 swelling are well known (74). Since previous studies i n t h i s laboratory had indicated that the l y t i c e f f e c t of free f a t t y acids was temperature-dependent, which p a r a l l e l s the l y t i c actions of steroids, studies were car r i e d out to determine i f C o r t i s o l would a f f e c t the metabolism of FFA i n corticosteroid-responsive and unresponsive tissues. Following C o r t i s o l treatment, the pattern of metabolism of FFA i n those two tissues was quite d i f f e r e n t . The steroid-responsive c e l l s retained FFA while the s t e r o i d - r e s i s t a n t c e l l s metabolized the FFA at a much higher rate than the unresponsive c e l l s , (see Figures 5 and 6). Results indicated that the oxidation of free f a t t y acids might be greater i n the r e s i s t a n t c e l l s . Electronmicrographic observations show that i f there i s an accumulation of FFA i n c o r t i c o s t e r o i d - s e n s i t i v e c e l l s , following c o r t i c o s t e r o i d treatment, th i s accum-ulation of FFA would have pronounced c y t o l y t i c e f f e c t s . The degenerative change set i n motion by FFA causes con-comitant nuclear and cytoplasmic a l t e r a t i o n s . The pro-gressive loss of normal dichotomy between heterochromatin and euchromatin 4 hours aft e r treatment i s s t r i k i n g l y s i m i l a r to that induced by c o r t i c o s t e r o i d (24). Bessis(82) has termed t h i s type of change nuclear edema. Indeed, due to d i l a t a t i o n of the endoplasmic reticulum and v e s i -c u l a t i o n i n FFA-treated P1798S c e l l s , the p o s s i b i l i t y of secondary a l t e r a t i o n i n ion and water balance within the 100 c e l l seems l i k e l y . The s t r u c t u r a l changes observed here are probably manifestations of the retrograde metabolic a l t e r a t i o n s which have been reported i n c o r t i c o s t e r o i d -induced degeneration: a decrease i n DNA synthesis (75,76) and i n DNA-dependent RNA polymerase a c t i v i t y (77,78), an increase i n DNase II a c t i v i t y (79,80), and changes i n the histone complement (81). It i s s i g n i f i c a n t that both c o r t i c o s t e r o i d (12) and FFA administration can cause karyorrhexis of the thymocyte nucleus at 4 hours. Great care was taken i n the i n t e r -pretation of electron micrographs to exclude those ex-amples which might even remotely be construed as due to tangential sectioning of the nuclear membrane. The f o c a l nature of the membrane degeneration i s noteworthy, for i t may imply s p a t i a l heterogeneity of function. The major cytoplasmic event induced by FFA i n P1798S c e l l s , progressive d i l a t a t i o n and eventual v e s i c u l a t i o n , was not noted at 4hours i n thymocyte suspensions, nor were these changes observed i n thymocytes treated with c o r t i c o -steroids a f t e r 4 hours (24). The c h a r a c t e r i s t i c paucity of endoplasmic reticulum i n c o r t i c a l lymphocytes (83) might account for t h i s . The swelling and d i l a t a t i o n of the perinuclear cisternae (Figure 14) might be predicted on the concept that t h i s space and that of the endoplasmic reticulum are co-existent i n mammalian c e l l s generally (84). A lack of d i l a t a t i o n of the perinuclear cisternae i n 101 thymocytes treated with FFA i s shown i n Figures 18 and 19. These c e l l s are undergoing karyorrhexis i n which swelling of the nuclear membrane does not seem to be an accompanying event. Other thymocytes treated with either FFA or c o r t i c o -steroids not undergoing karyorrhexis e x h i b i t nuclear swelling (24). The primary event i n FFA-induced c y t o l y s i s i s unknown. However, the s i m i l a r e f f e c t s of FFA and of corticosteroids on thymocytes and t h e i r c y t o l y t i c effects on c o r t i c o s t e r o i d -s e n s i t i v e lymphosarcoma c e l l s suggest a strong l i k e l i h o o d of a shared etiology. I t i s noteworthy that FFA admin-i s t r a t i o n does not seem to a f f e c t either the mitochondria nor the c e l l membrane primarily. A more complete version of the working hypothesis proposed i n Figure 20 to explain the mechanism of the c y t o l y t i c action i s now presented (Figure 25). As mentioned previously, an accumulation of FFA i n thymus and P179 8S tissues w i l l cause nuclear damage and ultimately c y t o l y s i s of these c e l l s . Concentrations of FA which cause c y t o l y s i s of the c o r t i c o s t e r o i d - s e n s i t i v e tissues are without e f f e c t on the P179 8R. The c r i t i c a l point i n whether or not a c e l l i s s e n s i t i v e to the c y t o l y t i c action of corticosteroids i s r e l a t e d to i t s capacity for oxidation of FA. Increased oxidative capacity of free f a t t y acids that are released from t r i g l y c e r i d e stores under the influence of c o r t i c o -s t e r o i d would render a c e l l r e s i s t a n t to the c y t o l y t i c TG Corticosteroid Sensitive Lipase cAMP RE-ESTERIFICATION OXIDATION FFA Accumulation NUCLEAR DAMAGE UPTAKE I CELL LYSIS FIGURE 25: Mechanism of the c y t o l y t i c action of corticosteroids, o 103 action of the s t e r o i d while decreased oxidative capacity would render i t sensitive to s t e r o i d action. This i s what was actually observed i n the lymphoid c e l l s studied: i n sensitive c e l l s following c o r t i c o s t e r o i d treatment the oxidation of palmitic acid to C0 2 was decreased (Table IV); indications of flooding of the FFA pool were observed (Table V). In r e s i s t a n t c e l l s just the opposite occurred: the oxidative capacity increased as the load of FFA increased, and the endogenous pool of FFA decreased (Table IV and V). Whether or not the decreased or increased rate of oxidation i n s e n s i t i v e or r e s i s t a n t tissues, respectively, represents a d i r e c t action of C o r t i s o l on the acyl c a r n i t i n e trans-ferase systems of mitochondria needs further i n v e s t i g a t i o n . Blecher and White (85) have reported that endogenous r e s p i r a t i o n i n suspensions of r a t thymic lymphocytes w i l l continue i n the absence of exogenous substrate and that the respiratory quotient i s i n d i c a t i v e of l i p i d oxidation. This oxidation i s , however, i n h i b i t e d by adrenal steroids. This data i s consistent with the view that corticosteroids might i n h i b i t the acyl c a r n i t i n e transferase i n the mitochondria of sensitive c e l l s and lead to an accumulation of FFA. Another important factor when considering oxidation i s the number of mitochondria that a c e l l contains. If r e s i s t a n t c e l l s contain more mitochondria than sensitive ones, i t would be expected that t h e i r capacity to handle free f a t t y acids would be increased (for a further consider-104 ation of t h i s point, see Appendix I I I ) . The a c t i v i t y of the acyl carnitine system i s dependent upon the presence of the c a r r i e r molecule, c a r n i t i n e . However, i n the sensitive tissues studied, c a r n i t i n e i s not a l i m i t i n g f a c t o r . In f a c t , the presence of added car n i t i n e i n sen s i t i v e c e l l s r e s u l t s i n decreased rate of oxidation (Figure 8), suggesting that exogenously added car n i t i n e i s competing for binding on the acyl c a r n i t i n e trans-ferase enzyme i n much the same manner as i t s analogue, deoxycarnitine, and thereby i n h i b i t i n g the transfer of acyl carnitine moeities from the cytosal into the mito-chondrion. Addition of c a r n i t i n e to P1798R r e s u l t s i n increased oxidation, i n d i c a t i v e of a greater oxidative capacity than sensitive c e l l s . Short chain f a t t y acids are transferred into the mitochondrion by a d i f f e r e n t transferase than C-16 or C-18 f a t t y acids (93,94). Spector (56), Weinhouse (91), and S c h o l e f i e l d (92) have reported.that tumor c e l l s appear to have l i t t l e capacity to oxidize short chain f a t t y acids. The mouse lymphosarcomas P1798S and P1798R do not follow the pattern observed by these workers. Like thymus (Table IX) and other peripheral tissues (57) , these c e l l s have a greater capacity f o r the oxidation of short chain than long chain fatty acids which are generally thought to be more e f f i c i e n t l y used i n l i p i d synthesis than energy production (57). 105 C o r t i c o s t e r o i d treatment of adrenalectomized diabetic rats resulted i n an increase i n the c i r c u l a t i n g l e v e l s of FFA (99). The increase l e v e l of FFA i n sensitive c e l l s , observed following c o r t i c o s t e r o i d treatment, may not be just a consequence of the release of FFA from endogenous stores, but may also be the r e s u l t of increased uptake because of these elevated c i r c u l a t i n g l e v e l s . The uptake of FFA i s probably not a major factor since s t e r o i d -induced c y t o l y s i s occurs i n v i t r o i n the absence of added FFA. The data of Table VI support t h i s point of view, and i n f a c t generally, a s l i g h t but not s i g n i f i c a n t decrease i n uptake was observed i n most cases following c o r t i c o s t e r o i d treatment. The mechanism of resistance of some malignant lympho-cytes to the c y t o l y t i c actions of corticosteroids might be dependent on not only increased oxidative capacity. From the mechanism outlined i n Figure 25, three other p o s s i b i l i t i e s are evident. F i r s t , c o r t i c o s t e r o i d - r e s i s t a n t lymphocytes might be able to r e - e s t e r i f y released f a t t y acids at a greater rate than sensitive tissues and thus prevent accumulation. In the P1798R tumor, th i s does not appear to be a major pathway. Other factors involved i n corticosteroid-resistance might be a decreased release of FFA or a decrease i n the t r i g l y c e r i d e stores from which the FFA are released. Both seem to be implicated i n the P1798R. Following dexamethasone treatment, the endogenous 106 l e v e l o f FFA i n the c e l l s dec reases (Table V ) . Whether t h i s d e c r e a s e i s a f a i l u r e o f the FFA to be r e l e a s e d o r a r e s u l t o f i n c r e a s e d o x i d a t i o n (Table IV) o r a comb ina t i on of bo th cannot be d i s c e r n e d . The endogenous l e v e l s o f f a t s t o r e s are lower i n the P1798R than the P1798S (Tables V I I and V I I I ) . As a consequence o f t h i s , the l e v e l s o f FFA r e l e a s e s are p r o b a b l y lower and the g r e a t e r o x i d a t i v e c a p a c i t y i n the p resence of C o r t i s o l would p r e v e n t an a c c u m u l a t i o n o f FFA and the r e s u l t i n g c e l l u l a r damage. By i n h i b i t i n g o x i d a t i o n or t r a n s f e r of FFA t o the m i t o c h o n d r i a , i t i s p o s s i b l e to render the r e s i s t a n t c e l l p o p u l a t i o n s e n s i t i v e to the a c t i o n s o f C o r t i s o l . Us ing d e o x y c a r n i t i n e , a s t r u c t u r a l analogue o f c a r n i t i n e , (see Appendix I I f o r s t r u c t u r e c o m p a r i s o n ) , which competes w i t h a c y l c a r n i t i n e f o r b i n d i n g on the a c y l c a r n i t i n e t r a n s f e r a s e (95) , a dec rease of one t h i r d i n the c e l l p o p u l a t i o n i s seen f o l l o w i n g concomi tant C o r t i s o l t rea tment (Table X I V ) . More e f f e c t i v e than d e o x y c a r n i t i n e , however, are b r a n c h e d - c h a i n compounds which because o f t h e i r branched s t r u c t u r e would be expected to i n h i b i t 3 - o x i d a t i o n ( 9 6 , 9 7 ) . A f t e r c i t r a l t r e a t m e n t , the v i a b l e c e l l p o p u l a t i o n i s dec reased up to 80% (Table X V I ) . T h i s a ldehyde i s r e a d i l y o x i d i z a b l e by bo th l i v e r and tumor t i s s u e to the c o r r e s p o n -d i n g a c i d , which has l y t i c a c t i v i t y . * 1 More e f f e c t i v e than * 1 K .H. Sim and A . F . B u r t o n , u n p u b l i s h e d o b s e r v a t i o n s , 1971. 1 0 7 c i t r a l alone, are emulsions of o l e i c acid and branched chain compounds l i k e c i t r a l . These emulsions when admin-is t e r e d i p to animals bearing the ascites form of the L 5 1 7 8 Y , L 1 2 1 0 , and P 1 7 9 8 R , w i l l cause 9 9 . 9 % destruction of the c e l l s within one hour.* 1 The tes t i n g of branched l i p i d s of chain length C - 1 6 or C - 1 8 with a general structure l i k e that of Figure 2 6 i s suggested for chemotherapeutic treatment of lymphocytic leukemias. These compounds would be expected to have a l l the properties of the described emulsions. They could enter $-oxidation, but once the branch point i s reached, 6-oxidation would be i n h i b i t e d . If the remaining C - 1 0 or C - 1 2 fragments were released, these would be l y t i c . Whether or not cAMP i s the modulator that activates the co r t i c o s t e r o i d - s e n s i t i v e lipase implicated i n thi s scheme i s a matter that awaits further i n v e s t i g a t i o n . However, some speculation can be advanced i n this area i f the p a r a l l e l that exists between adipocytes and lymphocytes holds ( 1 9 , 2 2 , 3 2 , 3 5 , 3 6 , 9 0 ) . The eff e c t s of corticosteroids i n i n h i b i t i n g uptake of glucose by lymphoid c e l l s ( 2 2 , 3 2 , 3 5 , 3 6 ) and adipocytes ( 1 9 , 9 0 ) and e f f e c t i n g the release of FFA and gl y c e r o l from adipocytes i s well documented ( 3 2 ) . The release of FFA under these conditions i s dependent upon the * 1 R.W. Tu r n e l l and A.F. Burton, observations not reported, 1 9 7 1 . I C H 2 C H 3 ^ C H 2 - C H ^ H ^ C H f C H 2 ^ C H 2 ^ y C O O H C H 3 ^ C H 2 ^ C H 2 ^ C H 2 ^ C H ^ C H ^ C H 2 ^ O O H CH I R FIGURE 26: Structure of branched chain f a t t y acids with suggested chemotherapeut value, a) fat t y acid with the branch point occurring on the odd numbered carbon, b) fat t y acid with the branch point on the even numbered carbon. The values of X and Y can be varied to produce a C-16 or C-18 fat t y acid with the branch point at varying distances from the terminal -COOH group. R = H, CH 3. 109 a c t i v a t i o n of a c o r t i c o s t e r o i d - s e n s i t i v e lipase by cAMP. This increased production of cAMP i s dependent upon the synthesis of RNA and protein (98). If synthesis i s blocked the production of cAMP and release of FFA by adipocytes under c o r t i c o s t e r o i d stimulation are also blocked. The ef f e c t s of corticosteroids on lymphocytes are also dependent upon RNA and protein synthesis (43). Yang and Vas (86) have reported that d i b u t y r y l cAMP w i l l i n h i b i t the growth of mouse leukemia L517 8Y-R C e l l s i n v i t r o . Masarocchia and Rosen (87) have noted recently that as P1798S and P 1 7 98R tumors become large the l e v e l of cAMP increases. It s t i l l remains to be determined whether changes i n cAMP are r e l a t e d to regression of lymphoid c e l l s following g l u c o c o r t i c o i d treatment. An important point noted i n t h i s investigation i s that the c y t o l y t i c a c t i v i t i e s of corticosteroids might be separated from other actions of corticosteroids which are believed to r e s u l t from a profound i n h i b i t o r y action on lymphoid tissue generally, for example, immunosuppression. The i n h i b i t o r y e f f e c t s of co r t i c o i d s on nucleic acid, protein, and glucose metabolism (Tables X,XVII-XIX) are not seen when c e l l s are treated with FFA which causes c y t o l y s i s . It seems quite possible that the two can be dissociated (see Figure 27), an important factor when the chemotherapeutic use of agents which promote c y t o l y s i s i s to be considered. 110 CORTISOL Cor t i c o i d - s e n s i t i v e lymphocytes "cytoplasmic complex" nuclear complex increased mRNA synthesis burst synthesis oJ proteins TG cAMP FFA nuclear damage c e l l l y s i s decreased carbohydrate metabolism, ion transport phosphorylation, etc. repression of mRNA synthesis decreased protein metabolism CYTOLYTIC EFFECTS METABOLIC EFFECTS IMMUNOSUPPRESSIVE EFFECTS FIGURE 27: Outline of the proposed rel a t i o n s h i p of events that occur following g l u c o c o r t i c o i d treatment. I l l The o b s e r v a t i o n s made i n t h i s t h e s i s t h a t f r e e f a t t y a c i d s are c y t o t o x i c a re suppor ted by r e c e n t o b s e r v a t i o n s from two o t h e r g roups . S a h l e r and G l i c k (88) observed t h a t mouse l i v e r ch romat in i s h i g h l y t o x i c t o L1210 mouse leukemia c e l l s . The c y t o t o x i c e f f e c t o f c h r o m a t i n - l i p i d i s caused by f r e e f a t t y a c i d s , e s p e c i a l l y o l e i c a c i d . A c o n c e n t r a t i o n Of 3 yg/ml o f f r e e f a t t y a c i d s would cause 100% c e l l l y s i s i n the L1210 l e u k e m i a . A c y t o t o x i c f a c t o r has a l s o been i d e n t i f i e d r e c e n t l y i n a lymph node e x t r a c t (89) . T h i s " c e l l u l a r l y t i c f a c t o r " was found to be c y t o t o x i c to tumor c e l l s such as MM2, F r i e n d , SR61, E h r l i c k , S180, and AH-66F. I t a l s o l y s e d the e r y t h r o c y t e s o f sheep , g u i n e a p i g , r a t , r a b b i t , and mouse. I n f r a r e d s p e c t r a , TLC , GLC, and i o n exchange chromatography e s t a b l i s h e d t h a t the " c e l l u l a r l y t i c f a c t o r " i s an u n s a t u r a t e d l ong c h a i n f a t t y a c i d . The r e s u l t s showed the l y t i c a c t i v i t y was p r o b a b l y due to the p resence o f o l e i c a c i d which i s a monoenoic a c i d ( 1 8 : 1 ) . The e f f e c t s o f t h i s " l y t i c f a c t o r " i n c a u s i n g c y t o l y s i s were temperature-dependent l i k e those p r e v i o u s l y observed i n t h i s l a b o r a t o r y . In c o n c l u s i o n , the o b s e r v a t i o n s made i n t h i s t h e s i s demonstrate t h a t t h e r e i s a s i m i l a r i t y i n the events i nduced by c o r t i c o s t e r o i d s and those by f r e e f a t t y a c i d s . The low c o n c e n t r a t i o n o f FFA a t which these events o c c u r r e d i s e s p e c i a l l y s i g n i f i c a n t . That d a t a s t r e n g t h e n the concept advanced , t h a t the c y t o l y t i c a c t i o n o f c o r t i c o s t e r o i d s on 112 normal and malignant lymphocytes i s mediated by FFA. The f a t t y acids would be released from endogenous t r i g l y -ceride.- stores by the action of a c o r t i c o s t e r o i d - s e n s i t i v e l i p a s e . In sensitive tissues an accumulation of FFA leads to nuclear damage and ultimately c e l l l y s i s . Resistance of malignant lymphocytes to the c y t o l y t i c action of cort i c o s t e r o i d s might be p a r t i a l l y attributed to the f a i l u r e of the FFA to be released from endogenous f a t stores, lower fat stores i n the s t e r o i d - r e s i s t a n t t i s s u e s , or to the greater capacity of r e s i s t a n t c e l l s to oxidize FFA. 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APPENDIX I FISCHER'S MEDIUM FOR LEUKEMIC CELLS IN MICE (IX) Component Mg/1 NaCl 8000.0 MgCl 2.6H 20 100.0 NaH'2POit'.H20 69.0 Na2HP0lt .7H20 113.0 KC1 400.0 CaCl 2.2H 20 91.0 Glucose 1000.0 L-Arginine 15.0 L-Histidine HCI 60.0 L-Isoleucine 75.0 L-Leucine 30.0 L-Lysine HCI 50.0 L-Methionine 100.0 L-Phenylalanine 60.0 L-Threonine 3 0.0 L-Valine 70.0 L-Tryptophan 10.0 L-Glutamine 204.0 L-Serine 15.0 L-Cystine 20.0 L-Tyrosine 60.0 L-Asparagine 10.0 Riboflavin 0.50 F o l i c Acid 10.0 Thiamine HCI 1.0 Nicotinamide 0.50 Ca pantothenate 0.50 Pyridoxal HCI ID. 50 Choline HCI 1.50 i - I n o s i t o l 1.50 B i o t i n 0.010 Phenol Red 5.0 NaHC03 1125.0 120. APPENDIX II STRUCTURES OF SOME DRUGS AND CHEMICALS USED ( C H 3 ) 3 I N + I C H 2 C H - R I C H 2 I C O O H C H O U S O H CARNITINE CITRAL cis = geranial (citral a) trans = neral (citral b) R S H DEOXYCARNITINE C H ^ H - - O H CORTISOL DEXAMETHASONE APPENDIX III I n c r e a s e d M i t o c h o n d r i a l Content o f the C o r t i c O s t e r o i d - R e s i s t a n t Lymphosarcoma P1798 Compared w i t h the S t e r o i d -Sens i t i v e S t r a i n . I t was the o p i n i o n of Mr. C l a r k e and m y s e l f t h a t when v i e w i n g s e c t i o n s o f the P1798R and P1798S under the e l e c t r o n mic roscope t h a t the number o f m i t o c h o n d r i a v i s i b l e pe r c r o s s - s e c t i o n a l f i e l d was g r e a t e r i n the r e s i s t a n t tumor. T h i s type of o b s e r v a t i o n , however, i s • ve ry d i f f i c u l t t o prove by e l e c t r o m i c r o s c o p i c means. Support o f t h i s o b s e r v a t i o n has been r e c e n t l y p r o v i d e d by a p u b l i c a t i o n of Schrek (67) . What he o b s e r v e d , u s i n g the e l e c t r o n m i c r o s c o p e , was t h a t normal non-mal ignant human lymphocytes had an average o f 5.9 m i t o c h o n d r i a pe r c r o s s - s e c t i o n w h i l e those from p a t i e n t s s u f f e r i n g from lymphosarcoma c e l l l eukemia had more m i t o c h o n d r i a and t h a t a g r e a t e r percentage o f these c e l l s had e l o n g a t e d m i t o -c h o n d r i a (26.5%). These r e s u l t s o f Schrek would suppor t the t h e s i s t h a t as a c e l l becomes more m a l i g n a n t ( i e i n t h i s c o n t e x t , t h a t i t s r e s i s t a n c e to c o r t i c o s t e r o i d - i n d u c e d l y s i s i n c r e a s e s ) i t s m i t o c h o n d r i a l c o n t e n t i n c r e a s e s and hence r e s i s t a n c e to c o r t i c o i d o r f a t t y a c i d a l s o i n c r e a s e s . 

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