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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,  U n i v e r s i t y 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 t h e s i s as conforming required  t o the  standard  THE UNIVERSITY OF BRITISH COLUMBIA January,  1972  In p r e s e n t i n g an  this  thesis  in partial  advanced degree a t the U n i v e r s i t y  the  Library  I further for  shall  agree  make that  i t freely  permission  his representatives.  of  this  written  thesis  BIOCHEMISTRY  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  Date  gain  January  13, 1972  Columbia  shall  I agree  copying of this  b y t h e Head o f my  It i s understood  for financial  Columbia,  that  f o r r e f e r e n c e and S t u d y .  f o r extensive  permission.  Department o f  of B r i t i s h  available  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  f u l f i l m e n t of the requirements f o r  that  thesis  Department o r  copying or p u b l i c a t i o n  n o t be a l l o w e d w i t h o u t  my  i  ABSTRACT  When  c e l l s  of  the  thymus  or  mouse  with  0.27  lymphosarcoma  P1798S  are  tissue  culture  medium  with  10% s e r u m  damage  ending  with  karyorrhexis  nuclear  treated  s t e r o i d - r e s i s t a n t  i n  v i t r o  subline  P1798R  yM  0.5%  or  does  C o r t i s o l  i n  human  occurs.  not  show  albumen,  The these  changes. The undergo  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 s i s  d i f f e r  subline.  Most  oxidation  of  by  the  and  free  i n  P1798S  C o r t i s o l  vivo  the  had  decreased  FFA  after  by  while  calculated  of  fatty  acids  focal  dissolution  uptake  46% a n d  of  to.be  treatment,  effects of  on  subline  of  and  the  1  17% i 9%  in  to  in  that  could  and  disintegration  thymus while  and  In  v i t r o ,  acid  but  P1798S,  P1798R.  electronmicroscopy  C-9  in  26%.  thymus  c e l l s  equivalent  and  C o r t i c o s t e r o i d  FFA  by  for  determined  ^C-palmitic  n  resistant  substrates,  P1798R  sensitive  length  was  respectively,  corticosteroids  chain  the  capacity  substrate.  58%,  increasing  incubation  steroid  certain  e f f e c t  This  raised  which  from the  labelled  unlabelled 2 hours  is  (FFA).  from  77% a n d  oxidation  After  acids  ^CG^  for by  respects  apparently,  resistant  no  respectively,  ing  1  with  c e l l s  decreasing  fatty of  d i l u t i o n  treatment  several  c r i t i c a l ,  evolution  isotope  i n  lymphocytes  be  higher: of  the  medium  contain-  accumulated revealed  that  reproduced  by  nuclear  edema,  nuclear  membrane  and  ultimately  karyolysis.  Steroid-resistant  c e l l s show  c y t o l o g i c a l changes only a t t e n f o l d h i g h e r c o n c e n t r a t i o n s of  FFA. On  the  b a s i s 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 in corticosteroid-sensitive  TG  lymphoid  ^FFA  occurs  tissues:  ^Nuclear Damage Ac cumu1at i o n  Cell In an t o the  attempt to render r e s i s t a n t c e l l s  c y t o l y t i c a c t i o n s of c o r t i c o s t e r o i d s ,  c a r r i e d out where the i n h i b i t e d i n an  o x i d a t i o n of  can  be  are  lytic.  accumiilation  L1210  oxidized  In thymocytes, P1798S and i n v i t r o incorporation  i n t o a 5% TCA  insoluble  of  P1798R, FFA 1  precipitate.  The  in  3-oxidation  a c i d s which  3  affect  H-uridine  incorporation  of H-thymidine was  only s l i g h t l y reduced i n the  responsive c e l l s .  T h i s suggests t h a t  3  steroid-  d i d not  ''C-leucine and  FFA.  compounds  which cannot undergo  to mono- or d i c a r b o x y l i c  was  of  tumors r e s u l t s  Much more e f f e c t i v e , however, are  w i t h branched s t r u c t u r e s , but  s t u d i e s were  d e o x y c a r n i t i n e i n the  r e s i s t a n t P1798R, L5178Y, and some l y s i s .  sensitive  free f a t t y acids  attempt to induce an  I n h i b i t i o n of o x i d a t i o n by  the  lysis  steroid-  some s t e r o i d  effects  for  example,  from those nuclear  those of  involving  i m m u n o s u p p r e s s i o n , m i g h t be accumulation  damage and c y t o l y s i s  of  of  FFA w h i c h  sensitive  dissociable  lead  cells.  to  iv  TABLE OF CONTENTS Page ABSTRACT.  i  TABLE OF CONTENTS  iv  LIST OF TABLES  .  LIST OF FIGURES  .  v i i i  LIST OF APPENDICES  xi  ACKNOWLEDGEMENTS  x  ABBREVIATIONS USED INTRODUCTION  i  i  xiii  .  1  The Question o f Receptors Glucocorticoid Metabolism  x  8  E f f e c t s on Carbohydrate 13  G l u c o c o r t i c o i d E f f e c t s on N u c l e i c A c i d and P r o t e i n 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 2) Incubations  22 ,  3) Determination o f R a d i o a c t i v i t y  .23 23  V  TABLE OF CONTENTS  (continued) Page  4) Assay f o r C0 lk  2  Production  24  5) P r e p a r a t i o n of L i p i d s  25  6) S e p a r a t i o n of N e u t r a l L i p i d s from P h o s p h o l i p i d s  27  7) M i c r o t i t r a t i o n of Free F a t t y A c i d s 8) Spectrophotometric  .  28  Assays  30  a) T o t a l E s t e r i f i e d F a t t y A c i d  . . .  30  b) L i p i d Phosphorous 9) Measurement of I n c o r p o r a t i o n of R a d i o a c t i v e P r e c u r s o r s of DNA, RNA and P r o t e i n i n t o 5% TCA Insoluble Fraction 10)  Uptake of 2-Deoxyglucose  30  31  (l-^C).  .  .  Methods-Electronmicroscopy  33 34  1) C e l l Suspensions and Incubations.  .  2) F i x a t i o n  .  34 34  a) Technique #1  34  b) Technique #2  35  3) Dehydration, Embedding, S e c t i o n i n g , S t a i n i n g , and Viewing RESULTS  35 36  Preliminary Studies  36  O x i d a t i o n of P a l m i t i c A c i d - ( 1 - ^ C ) 1  to  1£t  C0  38  2  O x i d a t i o n of Short Chain F a t t y A c i d s 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 Carbohydrate Metabolism  . . .  50  on 54  vi  TABLE OF CONTENTS  (continued) Page  E f f e c t o f FFA on Mouse Lymphosarcoma Cell Survival  54  E l e c t r o n m i c r o s c o p e Examination of the E f f e c t s o f FFA on Mouse Lymphoid C e l l s .  .  56  E f f e c t o f Free F a t t y A c i d s i n Inducing Lysis of Steroid-Resistant Tissues. .  .  80  The R e l a t i o n s h i p of C y t o l y s i s t o Other Corticosteroid Actions  82  DISCUSSION  98  BIBLIOGRAPHY APPENDICES  113 .  119  vii  LIST OF TABLES  Page Table  I.  Table  II.  Table  III.  Table  IV.  Assay  for  1  26  ''COproduction  Recovery o f L i p i d s A c i d Column  from  Comparison o f P a l m i t i c Oxidation i n Different  Silicic  32  Acid-(1- C) Media 1 h  E f f e c t of C o r t i s o l and/or P a l m i t i c A c i d on the O x i d a t i o n of P a l m i t i c A c i d - ( l - *C) t o C0  47  E f f e c t o f Dexamethasone on Endogenous Free F a t t y A c i d L e v e l s  49  E f f e c t o f C o r t i s o l on P a l m i t i c Uptake  51  1  lh  z  Table  V.  Table  VI.  Table  VII.  Table  VIII.  Table  IX.  Table  X.  Table  XI.  Table  XII.  Table  XIII.  Table  Table  XIV.  XV.  45  Acid  L i p i d Composition o f Mouse Lymphosarcoma P1798 and Thymus  52  Lipid Distribution sarcoma  53  o f Mouse Lympho-  O x i d a t i o n of Long and Short Fatty Acids.  Chain 55  O x i d a t i o n o f Glucose by Mouse Lymphosarcoma C e l l s  57  Uptake of 2-Deoxyglucose-(1- C) by Mouse Lymphoid C e l l s  58  E f f e c t of Free F a t t y A c i d s on P1798 In V i t r o  59  E f f e c t s of Increasing A z e l a i c Acid C o n c e n t r a t i o n on Mouse Lymphosarcoma P1798 C e l l s In V i t r o  60  E f f e c t o f D e o x y c a r n i t i n e on the Response o f Tumor C e l l s t o C o r t i s o l In V i t r o  81  E f f e c t o f C i t r a l on L5178 In V i t r o  83  1k  viii  LIST. OF TABLES,  (continued)  Page Table  XVI.  Table  XVII.  Table Table  XVIII. XIX.  E f f e c t of C i t r a l on Tumors In V i v o  84  I n c o r p o r a t i o n of Thymidine-(CH - H) i n t o the A c i d - I n s o l u b l e F r a c t i o n  94  I n c o r p o r a t i o n of U r i d i n e - ( 5 - H ) i n t o the A c i d - I n s o l u b l e F r a c t i o n  96  I n c o r p o r a t i o n of L e u c i n e - ( ^ C - U ) i n t o the A c i d - I n s o l u b l e F r a c t i o n  97  3  2  3  1  ix  L I S T OF FIGURES  Page Figure  Figure  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 Steroids with Lymphocytolytic Activity  3  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 Lymphatic T i s s u e  6  Figure  O u t l i n e of the P r i n c i p a l Events t h a t have b e e n I d e n t i f i e s i n t h e Interactions of Glucocorticoids w i t h Thymus C e l l s I n V i t r o  Figure  A Model f o r the R e g u l a t i o n L y m p h o i d 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  Figure  of  17  Counts Remaining i n the O r g a n i c P h a s e o f a C H C l : M e O H ( 2 : 1 v/v) E x t r a c t i o n o f Mouse L y m p h o i d C e l l s  39  C o u n t s R e m a i n i n g i n t h e Aqueous P h a s e o f a CHC1 :MeOH ( 2 : 1 v/v) E x t r a c t i o n o f Mouse L y m p h o i d C e l l s  40  Production of C 0 b y Mouse Lymphoid T i s s u e s In V i t r o  41  Production o f C02 from Palmitic Acid-(1- C)  43  3  Figure  3  Figure Figure  l l f  2  8.  l l >  1 h  Figure  9.  Untreated Control  P1798S  Cells  63  Figure  10.  Untreated Control  P1798S  Cells  65  Figure  11.  Palmitic  Figure  12,  A z e l a i c A c i d - T r e a t e d P1798S  Figure  13.  Palmitic  A c i d - T r e a t e d P1798S  Cells  67  Figure  14.  Palmitic  A c i d - T r e a t e d P1798S  Cells  69  Figure  15.  A z e l a i c A c i d - T r e a t e d P1798S  Cells  71  Figure  16.  A z e l a i c A c i d - T r e a t e d P1798S  Cells  73  A c i d - T r e a t e d P1798S  Cells Cells  65 65  X  LIST OF FIGURES  (continued) P a  Figure  17.  A z e l a i c Acid-Treated  Figure  18.  Figure Figure Figure Figure  g  e  P1798S C e l l s  73  Palmitic Acid-Treated  Thymocytes  75  19.  P a l m i t i c Acid-Treated  Thymocytes  77  20.  Proposed Scheme o f P o s s i b l e F a c t o r s Leading t o C y t o l y s i s  79  S e c t i o n o f P1798R from C o n t r o l Animal  85  S e c t i o n o f P1798R from C o n t r o l Animal  87  21. 22.  Figure  23.  S e c t i o n of P1798R from C i t r a l - T r e a t e d Animal  89  Figure  24.  S e c t i o n o f P1798R from C i t r a l - T r e a t e d Animal  91  Figure  25.  Mechanism of the C y t o l y t i c Action of C o r t i c o s t e r o i d s '  102  Figure  26.  S t r u c t u r e o f Branched Chain F a t t y A c i d s w i t h Suggested Chemotherapeutic Value  108  O u t l i n e of the Proposed R e l a t i o n s h i p of Events t h a t occur F o l l o w i n g G l u c o c o r t i c o i d Treatment  110  Figure  27.  LIST OF APPENDICES  F i s c h e r ' s Medium f o r Leukemic C e l l s i n Mice (lx) S t r u c t u r e s of Some Drugs and Chemicals Used Increased M i t o c h o n d r i a l Content of 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 P179 8 Compared w i t h Steroid-Sensitive Strain  xii  ACKNOWLE DGEMENTS  During  the course o f these s t u d i e s , I have had the  p l e a s u r e o f working w i t h a number o f persons whose d i s c u s s i o n s have been most h e l p f u l and t o whom I am g r a t e f u l . To Judy, Ming, Denise,  Hughe, Kash, Susan, Bruce and  B r i a n , I express my s i n c e r e  thanks.  I wish t o thank Dr. R.L. Noble, D i r e c t o r o f the Cancer Research Centre,  f o r the use o f the f a c i l i t i e s  at the Centre where t h i s r e s e a r c h was conducted. t e c h n i c a l a s s i s t a n c e o f Mrs.  The  R i l l a Hendry and Mrs.  Hildegard  E r b e r i n the p r e p a r a t i o n of the H&E s e c t i o n s o f the P1798R tumor was g r a t e f u l l y a p p r e c i a t e d .  I am indebted t o Mr. L.  Hughe C l a r k e , a f e l l o w graduate student from the Department of P a t h o l o g y , . f o r h i s h e l p i n the p r e p a r a t i o n and i n t e r p r e t a t i o n of the e l e c t r o n m i c r o g r a p h s . t i t r a t i o n apparatus belonging  t o Dr. G.I. Drummond o f the  Pharmacology Department i s g r a t e f u l l y F i n a l l y , most of a l l ,  The use o f the micro-  acknowledged.  I wish t o thank Dr. A.F. Burton  f o r h i s generous h e l p and f o r the encouraging  and e n l i g h t -  ening d i s c u s s i o n s t h a t were h e l d throughout the p r e p a r a t i o n of t h i s  thesis.  I was a r e c i p i e n t o f a U n i v e r s i t y o f B r i t i s h Graduate F e l l o w s h i p  (1968-1969) and a M e d i c a l  C o u n c i l o f Canada Studentship  (1969-1972).  Columbia  Research  X l l l  ABBREVIATIONS  USED  ACTH  adrenocorticotrophic  A 6 20 nm  absorbance wavelength  hormone  a t 6 2 0 nm o r , a n y stated  cAMP  cyclic  adenosine  cpm  counts  per minute  DNA  deoxyribonucleic  dpm  d i s i n t e g r a t i o n s p e r minute  EFA  esterified  FA  fatty  FFA  free  KRP  Krebs-Ringer phosphate  mRNA  messenger r i b o n u c l e i c  NAD  nicotinamide  NADH  reduced nicotinamide  NADP  nicotinamide adenine phosphate reduced nicotinamide t i d e phosphate  NADPH  other  3',5'-monophosphate  acid  fatty  acid(s)  acid(s) fatty  acid(s)  adenine  acid dinucleotide adenine  dinucleotide  dinucleotide adenine  dinucleo-  NEFA  non-esterified  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 sarcoma P1798  lympho-  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 sarcoma P1798  lympho-  PPO  2,5-diphenyloxazole  POPOP  fatty  buffer  acid(s)  bis(2-(4-methyl-5-phenyl benzene  RNA  ribonucleic  acid  TCA  trichloroacetic acid  oxazolyi))  XIV  ABBREVIATIONS  USED  (continued)  TEFA  total  esterified  fatty  acid(s)  U  as i t r e f e r s t o r a d i o a c t i v i t y means "universally labelled"  1  INTRODUCTION The f i r s t o b s e r v a t i o n s on the e f f e c t of g l u c o c o r t i c o i d s on lymphocytes date from 1943 when Dougherty and White observed t h a t i n v a r i o u s animal s p e c i e s a s i n g l e of  injection  ACTH would reduce the l e v e l of c i r c u l a t i n g lymphocytes ( 1 ) .  T h i s lymphopenia was maximal i n three t o nine hours and was dependent upon the amount o f ACTH used.  W i t h i n twenty-  four hours the c i r c u l a t i n g lymphocyte l e v e l s had r e t u r n e d to  normal.  ACTH was.found t o be i n a c t i v e i n producing  this- response i n adrenalectomized animals (2). Only a d r e n a l c o r t i c a l s t e r o i d s were able to produce a lymphop e n i c response i n adrenalectomized mice.  Other hormones,  such as i n s u l i n , e p i n e p h r i n e , and t h y r o x i n , as w e l l as c e r t a i n s t e r o i d hormones were able t o produce a lymphop e n i a p r o v i d e d they were administered t o i n t a c t non-adrenalectomized animals (3). The i n v e s t i g a t i o n o f the s t r u c t u r e - a c t i v i t y s h i p s o f s t e r o i d s able t o b r i n g about lymphopenia  relationindicated  s e v e r a l f e a t u r e s t h a t were common t o a l l : f i r s t o f a l l , there must be an u n s a t u r a t e d A r i n g , a ketone a t C-3; t h e r e must be a h y d r o x y l group or an oxygen f u n c t i o n a t p o s i t i o n 11, and a s i d e c h a i n c h a r a c t e r i s t i c of the C-21 corticosteroids.  The h y d r o x y l group a t C-17 o f C o r t i s o l  i s not e s s e n t i a l t o produce lymphopenia; however, the l a c k o f i t causes a s u b s t a n t i a l l o s s i n lymphopenic  activity.  E a r l y i n v i v o s t u d i e s showed t h a t C o r t i s o l was most e f f e c t i v e  2  i n causing l y m p h o c y t o l y s i s , f o l l o w e d by c o r t i s o n e , c o r t i c o s t e r o n e , and 1 1 - d e h y d r o c o r t i c o s t e r o n e . s t u d i e s have shown t h a t i h v i t r o o n l y the s t e r o i d s have c y t o l y t i c a c t i v i t y  More r e c e n t 11-hydroxyl  (4,6,7,21,24).  As shown  i n F i g u r e 1, the 11-keto s t e r o i d s are a c t i v e i n vivodue  t o t h e i r p o t e n t i a l f o r being converted t o t h e i r  only,  active  11-hydroxy forms by the enzyme 116-hydroxysteroid: NADP o x i d o r e d u c t a s e , which occurs i n l i v e r  (5,14).  The  kidney  i s r e s p o n s i b l e f o r o x i d a t i o n (14), the l i v e r f o r r e d u c t i o n . I f g l u c o c o r t i c o i d s are a d m i n i s t e r e d t o hepatectomized thymolysis  adrenalectomized-  animals, they are without e f f e c t i n c a u s i n g  (13).  T h i s may  be due  to the r a p i d c o n v e r s i o n  of 11-hydroxy s t e r o i d s t o 11-keto s t e r o i d s by the  kidney,  and would i n d i c a t e the n e c e s s i t y f o r the 11-hydroxy f u n c t i o n i n maintaining thymolytic a c t i v i t y . There are t h r e e documented ways i n which s t e r o i d s b r i n g about i n v o l u t i o n of lymphatic t i s s u e . lymphocytokaryorrhexis.  They produce  They i n h i b i t m i t o s i s by d e s t r o y -  i n g c e l l s a t metaphase and by i n h i b i t i n g the b i o s y n t h e s i s of DNA  i n c e l l s t h a t remain.  c y t o k a r y o r r h e x i s was  The phenomenon of lympho-  the f i r s t to be d e s c r i b e d and i s  assumed t o be the p r i n c i p a l mechanism f o r the r e p r e s s i o n of lymphatic t i s s u e  (8).  By use of t i m e - l a p s e dark-phase  cinemiorography  Dougherty has shown t h a t a f t e r a d m i n i s t r a t i o n 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 s m a l l  3  FIGURE 1: 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 which can undergo i n t e r c o n v e r s i o n s .  4  blebs with  Later  (40). the  which  release  may The  s t i l l  of be  by  who  looked  C o r t i s o l  have  developed  with  C o r t i s o l  maximal  system observed  with in  eight  observed,  with  showing  Pyknotic At  eight  p r a c t i c a l l y  c o r t i c o s t e r o i d - s e n s i t i v e similar  rather  homogenous  hours.  The  lymphosarcoma Since  after  the  and  100  histologic  half  almost  dose  a  c e l l  mouse  l i f e a l l  i t of  unchanged  of  was  of an of  C o r t i s o l injected interest  glucocorticoid  is  were  to  would  was  pyknotic.  P1798  of  chromatin  seen  subline  a f t e r  of  this  period.  40-50  dose  four  and  e f f e c t  becoming  this  the  recognizeable  Dispersion  over  and  nuclear  maximal  nucleus  the  After  lymphosarcoma  effects. swollen  yM),  now  c o r t i c o s t e r o i d - r e s i s t a n t  remained  minutes  c y t o l y t i c  and  were  hours  every  s u f f i c i e n t  homogenous.  c e l l s  Dunn  after  disappeared  of  mice  and  hours  (0.27  disruption  micro-  thymocytes  C o r t i s o l  hours  rather  c e l l s  two  been  of  Storr  treating  of  and  microscopy.  mammals  dose  swollen  light  four  a  have  i n j e c t i o n  that  appeared  by  showed  for  largely  were  cytoplasm  electron  Burton,  had  there  the  the  after  pattern  membranes.  a  thymus  v i t r o  e f f e c t  disintegrate  Dougherty  using  recently,  They  c e l l u l a r  and  Sorensen  thymocytes  may  into  of  chromatin  nucleus  The  contents  studies  More  in  membrane  .  mouse  (24).  to  hours  at  an  of  a  and  (11).  treatment  normal  nuclear i n t a c t  Cowan  with  give  nuclear  cinemicrographic  confirmed scope,  the  is  minutes  in  excreted  determine persist  how in  long target  5  tissues. there i n  A f t e r  is  a  progressive  lymphatic  tissue  twelve  minutes  by  minutes  100  C o r t i s o l  on  synthesis al  have  i n j e c t i o n  and  shown a  have and  t h e i r in  nucleic t h e i r  location affect  of  trigger  search  for  acid  glucocorticoid  metabolism  in  events  c o r t i c o i d  treatment,  the  some  major  d e t a i l .  the  that  areas  these have and  hour  et  to sub-  s l i g h t l y  the  c e l l u l a r  the  steroid  on  areas.  and  i d e n t i f i e d  above  w i l l  level disappeared.  that  are  glucose, they  and  to  Figure  temporal  that  investigators  recently,  receptors  by  has  events  i d e n t i f i c a t i o n  mentioned  DNA  caused  appear  at  More  t h e i r  of  Burton  would  primarily  been  on  thymocytes  lymphocytes,  metabolism. to  i t  of  attention  efforts  one  to  levels  hours.  p r i n c i p a l  of  ten  effects  effects  of  hormone  (24).  event  the  cytolysis  t h e i r  as  mice  after  dramatic  metabolized  a l l  to  radioactive  many  hours  an  after  the  for  (14,20)  ''C)  i n s i g n i f i c a n t  exposure  only  1  maximal  The  short  long  of  suppressive  eight are  p r i n c i p a l  of  as  suspensions  focussed  turned  by  steroids  persists  involved  in  period  glucocorticoids  In  and  to  2).  v i t r o  pyknosis  which  Figure  that  s t a n t i a l  c e l l  drops  p e r s i s t  for  thymus  becomes  mitosis  C o r t i s o l  Since  then  (See  C o r t i s o l - (4-  accumulation  which  cytolysis  and  of  3  how  have  i n t r a c e l l u l a r i t  would  outlines a f t e r  location. be  p r o t e i n ,  the  glucoEach  discussed  in  6  spleen  10-  W  w o  thymus  Q  Q W EH U W  0.1  *l  O  lymph nodes  dP  0.01  -r-  I  100  10 MINUTES  AFTER  C0RTIS0L-4-  1  INJECTION  FIGURE 2. Concentration of r a d i o a c t i v i t y i n lymphatic tissue. 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 lymphatic tissues a f t e r intravenous i n j e c t i o n of c o r t i s o l -4C. (From D o u g h e r t y , 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  thymus cells  Glucocorticoids  Steroids  (ATP)  Specific binding< 0 min «  Irreversible step Act.O sensit. step  cytoplasmic" complex  f  nuclear complex  ) i  Sites 1 Nonspecific throughout f i n d i n g cell J  \  Nonspecific effects  (RNA)  'cyclohex. sensit. step..^ 15-.20 \ mm  Protein  I \  DECREASED GLUCOSE TRANSPORT  r  Decreased glucose dependent ATP  4 0 min Decreased protein synthesis  +4h  Decreased RNA synthesis  Cell lysis  FIGURE 3: Outline of the p r i n c i p a l events that have been i d e n t i f i e d i n the interactions of glucocorticoids w i t h thymus c e l l s i n v i t r o and of the postulated relations between them. The times on the l e f t g i v e an i n d i c a t i o n of the approximate time at which each event begins d u r i n g an i n c u b a t i o n w i t h C o r t i s o l at 37 . Brackets and dashed arrows, respectively , denote substances and steps for the r o l e of which at present there is only i n d i r e c t evidence. ( M o d i f i e d f r o m Munck (41)). 8  8  The  Question  Ail a  of  steroid  receptor  do  Munck s  steroid when two  thymus  and  cell  high  example,  and  of  17  times of  that  had  glucocorticoid seen  that  after  of  dilution  produced  of  in  such  the  the  as  have  treated were  minutes  a  equivalent by  the  steroid  good  of  greater)  was  (20,21).  Cortisol.  This  was  interpreted activity.  media  equilibrium  had  calculated  non-  i t  was  been from  main  feature  was  that  by  For  If  or  The  dissociated.  activity, bound  diluted with  diluant.  glucose  were  binding  that  showed  correlation  glucocorticoid  d i s s o c i a t i o n curves  s t e r o i d had  that  steroids  glucocorticoid  steroids  to  of  shown with  progesterone,  new  steroids  occurred:  of  but  or  no  with  rule.  amount  inhibition  non-specific  steroids 30  binding  5  and  the  Gluco-  (20) .  (10- M  which  have  this  labeled  binding  e f f e c t i v e l y than  been  w h i c h was  most  such  concentration  attained  worthy  function,  to  treated  activity,  non-glucocorticoid  reflection  cells  binding  steroid  more  were  to  action.  f r a c t i o n s , has  equilibrium  steroids  of  exception  physiological  deoxycorticosterone  binding a  and  appear  measuring  suspensions  of  date  radioactively  non-specific  magnitude  at  as  an  cell  non-specific  such  be  cells  correlation with  For  as  using  d i f f e r e n t types  uptake  to  bound  The  3  appear  and  to  to  t h e i r mechanism  activity,  rat  with  studied  specific  specific  no  not  in  group,  1  high  systems  involved  corticoids  of  Receptors:  one  example,  the  noteminute for  9  deoxycorticosterone, went  from  rapidly  of  looks  pattern  the  dissociation  dissociation As  non-specific  f r a c t i o n  over is  and  the  also  is  with  then  next  a  15  the  and  1 minute.  c a l l e d  the  (21,22).  curves  non-specific  second  f r a c t i o n  the  minutes.  This  of  non-gluco-  the  that  exert  a  f i r s t slowly  dissociating  corticosteroids  corticosterone  was  dissociated  slowly  other  the  there  within  that  when  C o r t i s o l ,  from  dissociated  This  However,  f o r  d i f f e r e n t  ((Sc)/(S)e)  non-glucocorticoids  that  c h a r a c t e r i s t i c  dexamethasone  is  constant  at  1.25  f r a c t i o n  c o r t i c o i d s .  minute  to  non-glucocorticoids  at  of  equilibrium  0 minutes  dissociating  a c t i v i t y one  at  8.0  the  f r a c t i o n  such  as  glucocorticoid  a c t i v i t y . The  s p e c i f i c  binding  of  C o r t i s o l  appears  to  be  temper-  o ature the  dependent.  non-specific  glucocorticoid the  of  appear 10  to  became  become 10 * -1  saturated  saturation of  f r a c t i o n  raised  were  to  saturated molar,  with  6  range  of  C o r t i s o l  kinetics  i t  appears  sites  while  not  with  near  (about  the  The  respect  over  s p e c i f i c upper  to  5000)  there f o r  u n t i l  t h e i r d i d  the  not  range  binding  end of  are  3  d i f f e r e n t  a  sites  the  concentrations. that  at  s p e c i f i c  two  f r a c t i o n  steroids  the  out  dissociate  (20).  these  10  binding  carried  non-specific  while M,  did  37°  d i f f e r e n t The  was  dissociated  at  physiological  number  was  k i n e t i c s .  to  - 1 0  d i l u t i o n  f r a c t i o n  binding  saturation  the  binding  temperature  forms  of  If  From  the  limited  s p e c i f i c  i n t e r a c t i o n  10  with  glucocorticoids  molecules those for  bound  these  t h e i r  s p e c i f i c  exert  binding  metabolic  effects  found  that  10" M  with of  C o r t i s o l  e f f e c t  producing  using  whether  its  c e l l s .  or  not  found x  10  i n h i b i t e d basal  steroids potent  on  by  This  M),  the  induction  enzyme as  steroids  cortexolone  the such was  no  in 9 as  was  the  of  e f f e c t  less  c e l l  i t  to  should a c t i v i t y  these  w i l l  compete  v i r t u a l l y  abolish  (20,25).  preventing  C o r t i s o l  on  glucose  and  interested have  any  of  in  in  on  induction  c e l l s .  cortexolone  used  phosphatase  observed  used  e f f e c t i v e  determining  HeLa  cultures.  were  (23)  e f f e c t  alkaline was  metabolism  Bishop  a-fluoroprednisolone C o r t i s o l  sites  metabolism  l e v e l  control  much  to  glucocor-  binding  and  in  the  which  phosphatase  highest  72%, while  such  M)  would  the  of  6  effects  group  at  compete  glucocorticoid  Melynkovych  that  being  w i l l  are  cortexolone  cortexolone profound  s  proportion  on  for  glucose  alkaline  by  l e v e l  (10  of  6  and  5  cortexolone  prednisolone  (29.5  of  investigated  HeLa  sites  C o r t i s o l  in  through  compete  was  difference  interactions  w i l l  It  10  a c t i v i t y .  "receptors"  their  x  2.4  steroids  less  which  been  that  or  compounds  effects  They  more  these  This  by  Other  block  s p e c i f i c a l l y  has  the  some  ( 2 4 ) , the  to  sites.  from  sites  s p e c i f i c  possible  the  c e l l  glucocorticoid  s p e c i f i c a l l y  with  per  though  n o n - s p e c i f i c a l l y .  the  t i c o i d s be  bound  s p e c i f i c If  or  are  (20) e v e n  If  on  the  more  series  was  potent or  less  i t  was  found  with  the  potent  11  than with the weaker i n p r e v e n t i n g a l k a l i n e phosphatase induction.  These r e s u l t s , as do those of Munck and h i s  co-workers, seem to support  the assumption t h a t the  biol-  o g i c a l a c t i v i t y of g l u c o c o r t i c o i d s 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 f o r the b i n d i n g s i t e s w i t h i n the  cells.  Making use of the p r e v i o u s l y mentioned f a c t t h a t a t 3 ° the s p e c i f i c b i n d i n g f r a c t i o n does not d i s s o c i a t e , i t i s p o s s i b l e to a s s i g n 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 b i n d i n g to r e c e p t o r s i n r a t thymocytes. I f c e l l s were incubated w i t h  3  H - c o r t i s o l f o r 2 0 minutes  at 3 7 ° , and d i l u t e d at 3 ° , the n o n - s p e c i f i c a c t i v i t y d i s s o c i a t e d from the c e l l s w h i l e the s p e c i f i c a c t i v i t y d i d not.  A f t e r c e l l membranes and  receptor  cytoplasmic  m a t e r i a l s were removed from the thymocytes by  hypotonic  shock i n 1.5  activity  mM  M g C l , the s p e c i f i c a l l y bound 2  remained a s s o c i a t e d w i t h the n u c l e a r p r e p a r a t i o n  (20).  However, when the c e l l s were incubated at 3 ° and d i s r u p t 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  and not i n the n u c l e a r f r a c t i o n  I t has been  t h a t the primary  (30).  supernatant  hormone i n t e r a c t i o n i n some way  suggested  directs  the r e c e p t o r to i t s n u c l e a r or " e x e c u t i v e " p l a c e of a c t i o n , where i t e x e r t s i t s e f f e c t s on DNA,RNA and p r o t e i n formation.  subsequent  I t i s the hormone r e c e p t o r complex  t h a t i s a c t i v e r a t h e r than the r e c e p t o r or hormone a l o n e . The  g l u c o c o r t i c o i d r e c e p t o r complex has been i s o l a t e d  12  from thymic n u c l e i p r e p a r a t i o n s and C o r t i s o l on Sephadex G-25 volume.  The  protein  free  where i t emerges w i t h the  complex c o n t a i n i n g  degraded by pronase. dissociates  separated from  The  Cortisol i s rapidly  r a t e at which the  i s indistinguishable  from the  slow r a t e at which C o r t i s o l d i s s o c i a t e s  void  complex  characteristic  from the  isolated  n u c l e i or from whole c e l l s  (26).  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 a b o l i s h e d  A l s o under anaerobic c o n d i t i o n s  Under anaerobic  or c o n d i t i o n s  glucose i s p r e s e n t the  formation of the  complex does not  (26).  occur  Cortisol i s intimately of the  c e l l and  Using the  The  where  (29).  no  nuclear receptor  s p e c i f i c binding  dependent upon the m e t a b o l i c  more p r e c i s e l y on the ATP  of state  level.  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  s i m i l a r r e c e p t o r complex u s i n g as a marker o l i n e acetonide i n s t e a d d i d Munck, two  conditions  of  binding  3  H-cortisol.  f r a c t i o n s i n the  f r a c t i o n l o o s e l y binds H - t r i a m c i n o l i n e 3  c o u l d be d i s p l a c e d  by  H-triamcin-  They found, as cells.  C o r t i s o l or i t s analogue 11-epiactivity.  e f f e c t i v e i n d i s p l a c i n g the  bound t r i a m c i n o l i n e  One  acetonide which  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 Only C o r t i s o l was  3  isolated a  acetonide.  The  specifically  properties  of t h e i r  complex were s i m i l a r to those of Munck: i t separated w i t h the v o i d volume on Sephadex G-25, actions  of pronase and  Similar  was  s e n s i t i v e to  i n s e n s i t i v e to RNase and  the  DNase.  g l u c o c o r t i c o i d r e c e p t o r s have a l s o been found  13  i n other s t e r o i d responsive c e l l s ( 3 1 ) .  G l u c o c o r t i c o i d E f f e c t s on Carbohydrate  Metabolism:  When g l u c o c o r t i c o i d s were i n j e c t e d i n t o a d r e n a l ectomized f a s t e d animals, l i v e r glycogen r e s e r v e s were seen t o i n c r e a s e .  T h i s e f f e c t took, about four hours.  However, a t l e a s t two hours b e f o r e these e f f e c t s on l i v e r glycogen were e v i d e n t , i t was observed by Munck and K o r i t z t h a t b l o o d g l u c o s e would r i s e 8 0 - 1 0 0 minutes (15).  Cortisol injection  following  T h i s e f f e c t has been  l y confirmed by two o t h e r groups  (16,17).  subsequent-  This rapid  e f f e c t o f g l u c o c o r t i c o i d s on blood g l u c o s e was thought to r e s u l t as a consequence of decreased g l u c o s e uptake by p e r i p h e r a l t i s s u e s .  When s e l e c t i v e t i s s u e s were  i n v e s t i g a t e d u s i n g ^ C - g l u c o s e i t was found t h a t g l u c o s e 1  uptake was i n c r e a s e d i n gastrocnemius muscle,  probably  due t o i n c r e a s e d a v a i l a b i l i t y of glucose as a r e s u l t of e l e v a t e d b l o o d glucose and s k i n  In adipose t i s s u e  (18).  (19,33)  ( 3 4 ) there was decreased glucose u t i l i z a t i o n both  i n v i v o and i n v i t r o . Because o f the c a t a b o l i c a c t i o n s o f g l u c o c o r t i c o i d s on thymus, Munck and h i s group lymphoid  tissue.  turned t h e i r a t t e n t i o n t o  Between 2 and 3 . 5 hours a f t e r  injection  of C o r t i s o l the uptake o f (U- ^C)-glucose by whole t i s s u e 1  and i n c o r p o r a t i o n i n t o p r o t e i n s and l i p i d s was decreased by 3 0 % ( 3 5 ) .  C o n t i n u i n g work i n Munck s l a b o r a t o r y conr  1 4  firmed into  that  protein  In  active  w i l l  physiological  concentrations.  before  of  or  i t s  of  exposure  ( 3 9 ) .  opposite  one  acid  was  to  have  at  are  e f f e c t  by  the  were  i n  after  to  of  i n  an  by resistant  of  glucose  hours  2  hexokinase  p e r s i s t  f o r  Metabolism:  glucocorticoids  two d i f f e r e n t i s  as  treatment.  and P r o t e i n  The f i r s t  second  the  uptake  glucocorticoid  Acid  i n h i b i t o r y  metabolism  change  seen  metabolism  and the  resistant.  e f f e c t  no  lympho-  catabolic  i s  2 5 - 3 5 %  the effects  observed.  t h e mouse  i n h i b i t e d  on N u c l e i c  and p r o t e i n  these  conversion  pronounced  without  following  looks  to  and subsequent  effects  Effects  exhibit  Potentially  of  which  essentially  These  effects  stimulatory  other  b u t were  hours  Glucocorticoid  sublines  glucocorticoids  there  to  thymocytes  require  sensitive  2-deoxyglucose  analogue  eight  found  uptake  tumor  Although  two  i s  approach-  active.  and the  were  glucose  sensitive  nucleic  tested  C o r t i s o l  on  cortisone  are  one that  Glucocorticoids  When  they  group  e f f e c t s  as  glucose  lead  6  glucocorticoids  P 1 7 9 8 :  as  - 1 0 M)  active  sarcoma  long  7  Only  Rosen's  a c t i v i t y  decreased  by  such  glucose  concentrations  ( 1 0  concentrations  c o r t i c o i d s  tumor.  at  to  and u t i l i z a t i o n  to C o r t i s o l  the  C o r t i s o l  due  l a b e l l e d  uptake  at  e f f e c t s  indeed  of  glucose  ( 2 1 , 2 2 , 3 6 ) .  effects  was  v i t r o ,  physiological  decreased  incorporation  and l i p i d  ( 3 6 ).  uptake ing  decreased  a  i n h i b i t o r y  on  and  very  rapid  e f f e c t  of  15  the  steroid To  hormone.  date,  no  glucocorticoids c e l l s  similar  evidence  before  any  that to  the  altered  lymphoid  C o r t i s o l  the  adrenalectomized These i n  effects  v i t r o  were  metabolism  RNA  evidence  of  by  on  mycin  D  of  above was  C o r t i s o l ,  prevented  or  As  of  to  the  can  1-2  as  synthesis  lymphoid  persist  turned group  for  to  be  Cortisol the  to  the  effects  reversed  by  usual were  on  RNA  after  RNA  was  from  non-  respectively.  hours  following  results  tend  inhibitory  of  system were  a n t i b i o t i c  glucose  thymocytes D,  an  i n h i b i t o r of  When  after no  Cortisol  incorporation,  effects  observed.  t r e a t -  synthesis  effects  When  to  i n d i r e c t  RNA  actinomycin  C o r t i s o l  this  of  that  (43).  thymocyte of  minutes  3-0-methyl  and  not  obligatory  labelled  orthophosphate  and  is  However,  inhibitory  seen  accum-  glucocorticoid  off.  on  much  observed.  rats  indicated  the  be  These  following  lymphoid  corticoids  cells  for  treatment.  parameters  added  of  rapidly  early  rate  is  biosynthesis,  t i s o l  transport of  is  that  in  synthesis  effects  2-deoxyglucose,  with  RNA  adrenalectomized  thymocytes  incubated  RNA  ion  transport,  glucose,  uptake were  order  ion  that  23% i n  White's  i n  rubidium  l i v e r ,  immediately  from  occur  on  there  glucocorticoid  ment  and  but  seen  that  on  in  the  indicate  must  synthesis  (42).  and  demonstrate  RNA  synthesis  16% a n d  to  increased  and  c e l l s  by  able  detrimental  i n j e c t i o n  decreased  been  indicate  metabolism  C o r t i s o l in  of  has  cause  to  ulated  glucose  one  the  longer  during  the  Coractinoaddition  16  subsequent  1-2  has  rapid  a  very  metabolism In  before  such  inhibitory following  as  60 minutes  synthesis  c e l l s two  with  Twenty  5-10  to  have and as  Cortisol  reversed  of  or  after  produce  inhibitory  in  Figure  effects  Dvorkin  the  existence  the  metabolism  of  and  of of  a  4.  glucose  Cortisol  White  have  repressor the  "shut  The  c e l l  the  time effects  as  late  by  3  Kidson  rabbit  increased  the  l e v e l  incorporation  had  f a l l e n  indicate which  that  RNA  protein  down"  c e l l result  would  c e l l  be  recently which  there  is  synthesis This  which  metabolism,  protein  node  controls.  synthesis.  also  (42).  lymph  H-leucine  ultimate  (43) .  protein  treatment  during  protein  also  some  untreated  of  would  and  at  of  Cortisol  of  on  of  in  "burst"  effects  reversed  increased  results  presumably  protein  inhibitory  observed  l e v e l The  of  antibiotics  treatment  after  l e v e l .  delay,  on  a  indicated  given  RNA  administration.  observed  the  enhancing  The  incorporation  minutes  Cortisol  inhibitors  these  was  that  puromycin  when  by  c e l l s  l e v e l  phosphorylation  Makman,  D,  minutes  inhibitory  inhibitory  actinomycin  minutes  before  synthesis"  off".  Cortisol  control  in  "turned  e f f e c t  60  minutes  c e l l  Cortisol  the  indicate  administration.  be  t h i r t y  the  occurs,  of  C o r t i s o l ,  by  the  cycloheximide  lymphoid  over  and  below  in  would  following  f i f t e e n  f o l d  f e l l  can  d i r e c t  to  as  on  is  with  C o r t i s o l  C o r t i s o l  Ten  i t  effects  of  This  e f f e c t  contrast  synthesis  A  hours.  "burst-  would  ion  transport  metabolism of  these  lysis. postulated  would  turn  off  17  CORTISOL  t Increased mRNA S y n t h e s i s  Repression o f mRNA Synthesis  "Burst"  Synthesis  of Repressor P r o t e i n s  Decreased Carbohydrate Metabolism, Ion T r a n s p o r t , Phosphorylation, e t c .  t  Decreased P r o t e i n Biosynthesis I I I I  T  C e l l Death  F i g u r e 4:  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 .  18  The  long  term  repressive  DNA,  RNA a n d p r o t e i n  many  groups  using  experimental that after as  The  Present  long  As for of  models.  C o r t i s o l  for  as  the  previously,  I  by which  have  protein  areas,  f e e l ,  that  seemingly  study  is  concerned  this  that  2)  from  and other  centration  of  i n  thymocytes  1  3)  Burton,  with  some  free  and of  that  acid  one  carried  as  features, hour  v i t r o  p e r s i s t  out.  free the  the the  of  the  reported These  acids  fatty  that  acids  None  nucleus  the mechanism  e a r l i e r  is  corticosteroids  A . F . , Unpublished  and  point  1)  damage  induce  capable  f o r  the  the  observations,  c e l l s  con-  (24); of  mouse  inducing  1967.  i n  low  pyknosis  of  then  c y t o l y t i c  observations  are:  of  This  temperature-dependent are  of  c e l l .  c o r t i c o s t e r o i d - s e n s i t i v e  which  carbo-  disruption  the  The s t a r t i n g  looking  cytolysis  on  metabolism.  of  fatty  P1798  attentions  with  i n  induce  degeneration  laboratories.  v i t r o  lymphosarcoma and  same  i n  by  c e l l s  investigators,  their  begins  corticosteroids.  stems  been  on  studied  repressed  explaini=j4dequately  the progressive  study  the  glucocorticoids  and n u c l e i c  to  of  have  effects  a l l  focussed  leads  action  been  lymphosarcoma  and the have  C o r t i s o l  Problem:  lymphocytes,  lymphocytes  and  studies  studies  of  have  s i g n i f i c a n t l y  treatment  the mechanism  these  A l l  i s  mentioned  hydrate,  biosynthesis  thymocytes  biosynthesis  effects  1  ; the  19  release will acids  of  FFA f r o m t r i g l y c e r i d e  (32).  be f o c u s s e d m a i n l y on t h e m e t a b o l i s m o f by l y m p h o c y t e s  a relationship and t h e to  stores  i n order  exists  sensitivity  corticosteroids.  This free  t o determine whether  between f r e e  or resistance  fatty  acid  of malignant  study  fatty or  not  metabolism lymphocytes  20  MATERIALS AND METHODS  Chemicals:  A l l Chemicals used i n t h i s study, u n l e s s otherwise noted, were purchased from the F i s c h e r Chemical o u t l e t i n Vancouver, B.C..  Hyamine, PPO and POPOP were o b t a i n e d from  Packard Instrument Company, Downers Grove,  Illinois.  Tetrabutylammonium hydroxide 25% i n methanol was purchased from Matheson, Coleman and B e l l , Norwood, Ohio.  Azelaic  a c i d , p a l m i t i c a c i d , c a r n i t i n e and d e o x y c a r n i t i n e were o b t a i n e d from Calbiochem, Los Angeles, C a l i f o r n i a .  Cortisol  was from the Sigma Chemical Company, S t . L o u i s , M i s s o u r i . Dexamethasone (decadron powder) was the g i f t o f Merck and Company, Rahway, New J e r s e y , t o Dr. M. Darrach i n t h i s department.  C i t r a l was o b t a i n e d from Eastman Kodak Co.,  Rochester, New York  ( f o r s t r u c t u r e see Appendix I I ) .  Solvents:  A l l s o l v e n t s 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 b e f o r e use.  Radiochemicals:  The f o l l o w i n g r a d i o c h e m i c a l s were purchased from  21  Amersham S e a r l e indicated: (  1 I f  Corporation, with s p e c i f i c  Palmitic  a c i d - ( l - ^C) (35 . 2-56 . 9 m^/mM) , 1  C - U ) (331 mC^/mM) , s o d i u m b i c a r b o n a t e ( - " O  a c i d ( - 1 - ' ' C ) (52. 9mCi/mM) , s o d i u m n - o c t a n o a t e 1  ( 1 7 . 5 mCi/mM), D - G l u c o s e - ( ^ C - U ) ( 3 0 9 1  1  "O  (120. 8 yCi/M) , t h y m i d i n e  u r i d i n e ( - 5 - H ) (30C-j/mM) . 3  was o b t a i n e d  mCj/mM),  1-leucine-  (-1- ''C) 1  n-hexadecane-  ( m e t h y l - H ) (26 C^/mM)  and  3  2-Deoxy-D-Glucose- ( 1 -  f r o m New E n g l a n d N u c l e a r .  were u s e d upon r e c e i p t  as  (56 . 9mCi/mM) ,  1  acetic  (l-  activities  All  (53.3mCi/mM)  1  radiochemicals  or p u r i f i e d r o u t i n e l y  by TLC o r  paper  chromatography.  Materials:  Glass insoluble Clifton,  fibre  filters  precipitates New J e r s e y .  serum s t o p p e r s , collection  of  l l f  used f o r  were o b t a i n e d Plastic  and 25 ml E r l e n m e y e r C02  of  f r o m Reeve  centre wells, flasks  from a l o c a l  supply  acid-  Angel,  one-holed used i n  were p u r c h a s e d f r o m K o n t e s  Company, V i n e l a n d , New J e r s e y . obtained  the t r a p p i n g  Scintillation  rubber  the  Glass vials  were  house.  Animals:  The  B A L B / c J and DBA/2J m i c e u s e d i n t h e s e  were o b t a i n e d  from J a c k s o n L a b o r a t o r i e s ,  The a n i m a l s were f e d a d i e t  of  experiments  Bar H a r b o r ,  Maine.  P u r i n a B r e e d e r Chow and w a t e r  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 i n g u i n a l g l a n d . transplanted routinely  every 15-17  Tumors were  days by g r i n d i n g  tumor i n a t i s s u e g r i n d e r w i t h 5 ml 0.9%  100  s a l i n e and  mg  injecting  0.1 ml of the c e l l suspension subcutaneously i n t o BALB/cJ mice. The murine leukemias L517 8Y and L1210 i n DBA/2J mice as a s c i t e s tumors. f l u i d was injected  were maintained  Every 7-10  days  ascites  withdrawn from mice and 0.1 ml of t h i s f l u i d into  was  recipients.  Methods-Biochemical:  1) C e l l Suspensions  :  Thymus or tumor t i s s u e was and ground i n 0.9% layers for  removed from BALB/cJ mice  i n a t e s t tube w i t h a l o o s e f i t t i n g g l a s s  NaCl-  The suspension was  f i l t e r e d through  of s u r g i c a l c o t t o n gauze and c e n t r i f u g e d  2 minutes.  I t was  eight  a t 1200xg  washed once w i t h 5 ml of 0.9%  and p e l l e t e d .  An a l i q u o t of t h i s suspension was  with Fischer's  Medium  (Grand I s l a n d  NaCl  diluted  B i o l o g i c a l Company,  B e r k e l e y , C a l i f o r n i a ) , c o n t a i n i n g 10% horse serum Los Angeles, C a l i f o r n i a ) or 0.5%  pestle  human albumin  (Hyland,  (Sigma,  23  St.  Louis,  0.5-4  2)  Missouri)  million  to give  cells/ml  a final  concentration  Incubations;  centrifuge except  tubes  f o r when  incubated  sealed with 1 h  were c a r r i e d o u t  C02  silicone  rubber  agitation.  to  solutions,  of  samples.  flasks.  Samples  3 8° f o r  2 mg/ml and d i l u t e d w i t h  were added t o t h e  A t no t i m e d i d  the e t h a n o l  samples exceed  1%.  3) D e t e r m i n a t i o n o f  incubations  the  and a z e l a i c  concentration  in  ethanol  saline  acids  samples b e f o r e  as  was a d d e d . liter  amount o f  vial  This of  incubation. incubated  radioactivity:  and 10 ml o f  consisted of  toluene.  counted using e i t h e r  liquid  samples  was a d d e d .  a Packard T r i - C a r b  (Model 3003)  scintillation  a fluid  4 gm PPO and 100 mg POPOP  F o r aqueous  Hyandne s o l u b i l i z e r  Spectrometer  before  ethanol  The s a m p l e t h a t was t o be c o u n t e d was p l a c e d i n scintillation  were  required  C o r t i s o l was d i s s o l v e d i n  Palmitic  glass  stoppers,  was b e i n g a s s a y e d , when  i n 25 ml E r l e n m e y e r  a concentration  addition  i n standard c o n i c a l  i n an o v e n o r w a t e r b a t h a t  time without  per  of  as d e t e r m i n e d by a h e m a c y t o m e t e r .  I n c u b a t i o n s were c a r r i e d o u t  at  cell  an  appropriate  Samples were Liquid  Scintillation  or a Nuclear Chicago Unilux  III  24  Ambient Temperature  Liquid Scintillation  Counter.  Sample  quenching was monitored by the c h a n n e l s - R a t i o Method ( 4 7 ) u s i n g a quench s e t prepared from n-hexadecane- ( 1 - ''C) 1  quenched w i t h acetone o r a commercially a v a i l a b l e s e t purchased from Nuclear Chicago.  4 ) Assay f o r **C02 P r o d u c t i o n : 1  Samples t o be assayed f o r p r o d u c t i o n o f *C0 were 11  2  incubated i n 2 5 ml Erlenmeyer f l a s k s .  0 . 9 ml o f F i s c h e r ' s  Medium o r KRP b u f f e r were p l a c e d i n the f l a s k s and to t h i s was added l a b e l l e d m a t e r i a l s , C o r t i s o l , f r e e f a t t y and other chemicals as the experimental regimen  acids,  dictated.  0 . 1 ml o f the c e l l suspension was added and the f l a s k s were c l o s e d w i t h serum stoppers equipped w i t h a c e n t r e well.  0 . 3 ml Hyamine was p l a c e d i n the c e n t r e w e l l v i a a  s y r i n g e and needle. at  The f l a s k s were then p l a c e d i n an oven  3 7 ° f o r the l e n g t h o f time r e q u i r e d .  A t the end o f the  i n c u b a t i o n p e r i o d , f l a s k s were removed from the oven and 0.3  ml 2 N HaSCK was added t o the i n c u b a t i o n mixture v i a  s y r i n g e through the serum stopper.  F l a s k s were then p l a c e d  i n a water bath a t 3 7 ° and shaken a t 1 2 0 cpm f o r a t l e a s t two hours.  A t the end o f t h i s time the c e n t r e w e l l s were  removed, c u t from the stems and p l a c e d i n s c i n t i l l a t i o n vials. pf  A f u r t h e r 0 . 5 ml o f Hyamine was added and 1 0 ml  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 c o r r e c t e d f o r quenching by the method of channel ratios  (47).  As  can be  seen from Table I,the recovery  NaH^COg i n KRP  b u f f e r was  F i s c h e r ' s Medium contains constituents  greater  than 98%.  added bicarbonate  (see Appendix I ) , t r i a l  of  1  "^CC^  from  Because as one  of i t s  runs were made to  determine whether or not the c a p a c i t y of the Hyamine would be exceeded when t h i s medium was cell  suspensions.  The  used f o r i n c u b a t i n g  the  r e s u l t s show t h a t the c a p a c i t y  the Hyamine i s g r e a t enough to t r a p a l l the C0  of  released  2  from the medium.  5) P r e p a r a t i o n  of  Lipids:  A sample of minced t i s s u e  (0.5-2.0 gm)  i n 20 ml of chloroformimethanol Omnimixer.  The  homogenate was  a sintered glass funnel s t e e l tube and  (2:1 v/v)  o u s l y f o r one  using  a Sorvall-  The- stainless  r e s i d u e were r e - e x t r a c t e d w i t h an a d d i t i o n a l (2:1 v / v ) .  e x t r a c t s were removed t o a 50 ml NaCl was  homogenized  f i l t e r e d under vacuum through  (fine p o r o s i t y ) .  20 ml o f chloroform-methanol  0.73%  was  added minute.  (48), and  The  pooled  e x t r a c t i o n tube, 8 ml  removed  emulsion,  by s u c t i o n and  lower c h l o r o f o r m - r i c h phase evaporated i n vacuo at u s i n g a r o t a r y evaporator.  The  of  tubes were shaken v i g o r -  Upon s e p a r a t i o n of the  the upper aqueous phase was  lipid  r e s i d u e was  the 40°,  extracted  from  26  TABLE  Ii  Assay  for  C0  Production  2  Radioactivity NaH "*C0 added  1t, C0 recovered  1  Sample  Media  1. 2. 3. 4. 5.  KRP  f  3  2  6100 550 1100 2200 6600  FISCHER  55,700  recovery:  Average  *  1  1  h  COz  was  collected  indicated Figures  in  the  indicated  and  methods are  94.0 101.8 100.0 97.2 97.7  a c t i v i t y  recovery  determined  section.  mean  98.1  ±  SEM.  ±  1.3*  2  102,  56,820 51,330 108,520 100,590 172,640 170,270  55,700 106,680 106,680 161,140 161,140  8. 9. 10. 11.  ^recovered  5740 560 1100 2140 6450 Average  6 . 7.  (dpm)  92. 101. 94, 107, 105.7 100.5  as  +  2.5*'  27  the  flask  using  mixture,was dryness was 5  under  10  ml  ml  a  in  stream in  a  Separation  in  c y l i n d r i c a l , washed The  with  l i p i d  ether  and  mixture  an  at  110°  added  to  ether. l i p i d  The  ml  f r a c t i o n ,  (3:1  The v/v)  colorimetric The  portions  by  vacuum  The  of  to  samples  acid  was  anhydrous  f i l t r a t i o n .  10  up  to  The for  freshly  the  was  in  for  pooled  a  5  petroleum  washed d i e t h y l the  neutral  rotary  ml  of  EtOH:  gravimetric  neutral  80°).  The  then  using  -  absorbent  contained  extracted  methanol.  the  a  solvent  d i s t i l l e d  dissolved  of  light  in  and  60°  minutes.  acid  removed  (bp  of  from  which  placed  absorbent  10  been  porosity)  ml  dryness  then  The  residue  had  was  ether  isolated  was  determination  of  in  s i l i c i c  taken  residue and  s i l i c i c  ml  was  f i l t r a t e s ,  were  made  which  hours,  petroleum  portions  combined  evaporator. Ether  30  l i p i d  to  phospholipids:  (fine  absorbent.  extract  and  acid,  24  mechanically  f i l t r a t i o n .  with  for  dissolved  the  s t i r r e d  from  funnel  l i g h t  was  v/v)  evaporated  The  v/v)  s i l i c i c  sintered-glass  extract  gas.  (3:1  l i p i d s  oven  of  and  (2:1  flask.  of  ml  test-tube  nitrogen  gm  by  times  a  6  ether  3  of  neutral  petroleum vacuum  to  volumetric  of  40  was  chloroform-methanol  EtOH:Ether  Approximately activated  of  transferred  redissolved  or  6)  10  and  l i p i d . 3  times  Extracts f i l t r a t e s  were  with  3 0  i s o l a t e d  containing  28  the p h o s p h o l i p i d s evaporator.  were t a k e n t o d r y n e s s u s i n g a  The r e s i d u e was d i s s o l v e d i n  MeOH ( 3 : 1 v/v)  basis,  In o r d e r  II,  using this  the recovery of  method i s  f r a c t i o n was c o n t a m i n a t e d w i t h  lipids,  the n e u t r a l  ether  all  t h a t may  The  phosphate  results  t h e p h o s p h o r u s was e l u t e d w i t h  phospholipid fraction. phospholipid  lipid  solvent,  a n a l y s e s were p e r f o r m e d on e a c h f r a c t i o n .  fractions  e a c h f r a c t i o n was f r e e  TLC o f  the n e u t r a l  on S i l i c a  from contaminates  the  lipid  Gel plates  on a  complete.  any p h o s p h o l i p i d s  have been e l u t e d w i t h t h e d i e t h y l  that  gravimetric  essentially  to determine whether or not  indicate  CHCI3:  determination.  As shown i n T a b l e weight  5 ml o f  and s a m p l e s were removed f o r  and c o l o r i m e t r i c  rotary  and  revealed  of  the  that  other  fraction.  7) M i c r o t i t r a t i o n  of  Free Fatty  A Rehberg m i c r o b u r e t divisions taining  fatty  acids.  was s t i r r e d by b u b b l i n g  capacity  the organic  The s o l u t i o n  with  t o be  indicator)  d r i e d by p a s s i n g t h r o u g h a c o l u m n o f  C O 2 and  CaCl  2  and  con-  The  CaCl , 2  (using bromthymol b l u e  t o remove any f r e e  yl  titrated  n i t r o g e n gas t h r o u g h i t .  .005 N NaOH  0.2  solutions  d r i e d by p a s s i n g t h r o u g h a column o f  bubbled through acid/base  125 y l  was u s e d t o t i t r a t e  free  was f i r s t  of  Acids:  as  gas then an  finally drierite.  29  A phenol r e d i n d i c a t o r was prepared u s i n g a m o d i f i e d method o f K e l l e y  (49).  A 0.01% (w/v) s o l u t i o n o f phenol  red i n a b s o l u t e e t h a n o l was prepared.  This  s o l u t i o n was  d i l u t e d w i t h 200 ml o f heptane, 1.0 ml o f 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  For t i t r a t i o n s o f f r e e f a t t y a c i d s o l u t i o n s  thoroughly. t h i s stock  s o l u t i o n was d i l u t e d 2.5 f o l d w i t h heptane and 1 ml p o r t i o n s used as i n d i c a t o r s i n the t i t r a t i o n s . t i t r a t e d from a lemon y e l l o w c o l o u r end  The i n d i c a t o r was  t o an o r c h i d  purple  point. A stock s o l u t i o n o f t i t r a n t was prepared by d i l u t i n g  17.5  ml o f tetrabutylammonium hydroxide which was 25% i n  methanol t o 100 ml w i t h methanol. of the t i t r a n t  A f r e s h working  solution  (approximately 0.01N) was prepared d a i l y  by d i l u t i n g the stock 1:10 w i t h methanol. A stock s o l u t i o n o f f r e e f a t t y a c i d was prepared by d i s s o l v i n g 51.8 mg of p a l m i t i c a c i d i n 10 ml o f MeOH. A working s o l u t i o n o f f r e e f a t t y a c i d c o n t a i n i n g was  0.2 yEqv/ml  prepared by d i l u t i n g the stock 1:100 w i t h heptane. Aliquots  of organic l i p i d extracts  o r standard  solutions  of f r e e f a t t y a c i d s were made up t o 3 ml i n heptane i n 15 x 85 mm t e s t tubes. the  s o l u t i o n was mixed u s i n g n i t r o g e n  a p u r p l e end p o i n t . was  One ml o f the i n d i c a t o r was added, gas and t i t r a t e d t o  The 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  determined from a standard curve c a l i b r a t e d from  0 - 0.6 yEqv o f f r e e f a t t y a c i d .  30  8) Spectrophotometric Assays: All  s p e c t r o p h o t o m e t r i c assays were performed u s i n g  Cary 11 r e c o r d i n g spectrophotometer t o measure absorbance.  a) T o t a l E s t e r i f i e d F a t t y A c i d  TEFA were analyzed a c c o r d i n g t o the method o f S t e r n and Shapero to  (51).  Samples o f l i p i d e x t r a c t s were made up  3 ml w i t h E t h a n o l : D i e t h y l e t h e r (3:1 v/v) i n 18 x 150  mm t e s t tubes. (w/v)  To each tube was added 0.5 ml o f 13.9%  hydroxylamine h y d r o c h l o r i d e and 0.5 ml 3.5 N HaOH.  The tubes were mixed t h o r o u g h l y , capped w i t h a marble and allowed t o stand f o r 20 minutes a t room temperature. the  end o f 20 minutes  At  0.6 ml of 3.4 N HCI were added, tubes  were shaken and then 0.5 ml o f 0.37 M F e C l .6H 0 added, tubes 3  2  shaken and c o l o u r developed and measured a t 525 nm.  The  c o n c e n t r a t i o n o f TEFA was determined from a standard curve c a l i b r a t e d u s i n g 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 of the  Ames  was analyzed a c c o r d i n g t o the method  (50), which i s seven times more s e n s i t i v e than  Fiske-Subbarow  method.  The l i p i d e x t r a c t s t o be  analyzed were p l a c e d i n 13 x 100 mm t e s t tubes and taken to  dryness under a stream o f n i t r o g e n gas.  To each  tube  31  was added 30 y l of 10% (w/v) Mg(NO ),.6H,0 i n 95% EtOH. S o l u t i o n s were taken to dryness and ashed by shaking the tubes over a s t r o n g flame u n t i l brown fumes appeared. The tubes were allowed t o c o o l ; then 0.3 ml o f 0.5 N HCI was added.  The tubes were capped w i t h a marble and heated  at 100° f o r 15 minutes. of  the phosphate  The tubes were c o o l e d , and 0.7 ml  assay mix was added.  The assay mixture  c o n s i s t e d o f one p a r t o f 10% a s c o r b i c a c i d and 6 p a r t s o f 4.2 gm ammonium molybdate  t e t r a h y d r a t e and 2 8.6 ml concen-  t r a t e d s u l f u r i c a c i d made up t o 1 l i t r e w i t h d i s t i l l e d water.  Then the samples were incubated a t 45° f o r 20  minutes b e f o r e determining the A660 nm. phosphate was c a l c u l a t e d by comparing c a l i b r a t e d from 0-160 nM phosphate the  The amount o f  t o a standard curve  u s i n g NajHPOij. 7H 0 as 2  standard.  9) Measurement o f I n c o r p o r a t i o n o f R a d i o a c t i v e P r e c u r s o r s o f DNA, RNA and P r o t e i n i n t o 5% TCA I n s o l u b l e F r a c t i o n :  C e l l suspensions were i n c u b a t e d w i t h r a d i o a c t i v e p r e c u r s o r s o f DNA, RNA, o r p r o t e i n . incubation period c e l l of  A t the end o f the  suspensions were l a y e r e d on 5 ml  c o l d 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.  I n c u b a t i o n tubes were washed w i t h a d d i t i o n a l s a l i n e and washings added t o the l a y e r e d m a t e r i a l .  Gentle suction  was a p p l i e d and c e l l s were trapped on 2.4 cm diameter  TABLE I I :  Recovery of  Lipids  from S i l i c i c  Acid  Column  L I P I D RECOVERED FROM COLUMN SAMPLE  L I P I D ADDED TO COLUMN wt (mg)  NEUTRAL L I P I D wt  (mg)  yM  PHOSPHOLIPID  T?  x  wt  (mg)  % RECOVERY  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 :  *  1  Figure  indicated  is  96.1+5.7%  *  1  t h e MEAN ± SEM.  ro  33  glass f i b e r f i l t e r discs.  The f i l t e r s  and funnels were  washed w i t h an a d d i t i o n a l 5 ml of s a l i n e t o remove any remaining media and serum p r o t e i n s . 5 % TCA  Then 5 ml o f c o l d  (w/v) were added t o the f u n n e l and allowed t o f i l t e r  through the f i l t e r s  f o r a t l e a s t 3 minutes.  At the end o f  t h i s p e r i o d , s u c t i o n was a p p l i e d and the f i l t e r s were washed w i t h an a d d i t i o n a l 5 ml of 5 % TCA.  When the f u n n e l  was removed the edges o f the f i l t e r s were washed w i t h c o l d 5 % TCA t o remove any a c i d - s o l u b l e r a d i o a c t i v i t y which may have been trapped under the f u n n e l l i p . The  f i l t e r s were removed t o s c i n t i l l a t i o n v i a l s and  d r i e d i n vacuo a t 8 0 ° .  1 0 ml o f 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.  R e s u l t s are ex-  pressed i n cpm because the r a d i o a c t i v i t y was trapped on solid  10)  supports.  Uptake o f 2-Deoxyglucose- ( l - ''C) : 1  The  uptake o f 2-deoxyglucose was determined i n the  same manner as the i n c o r p o r a t i o n o f l a b e l l e d i n t o DNA,  RNA,  precursors  and p r o t e i n , w i t h the f o l l o w i n g e x c e p t i o n s :  a f t e r the samples and f u n n e l s were washed w i t h c o l d 0 . 9 % NaCl, the f u n n e l s were removed and the edges o f the f i l t e r s were washed w i t h 0 . 9 %  NaCl t o remove any s o l u b l e  t h a t had been trapped under the f u n n e l l i p .  activity  F i l t e r s were  then removed t o s c i n t i l l a t i o n v i a l s and t r e a t e d as p r e -  34  viously described.  Methods-Electronmicroscopy:  1)  C e l l Suspensions  and I n c u b a t i o n s ;  The p r e p a r a t i o n of c e l l suspensions and i n c u b a t i o n s of  the tumor P 1 7 9 8  was  i d e n t i c a l to that described pre-  v i o u s l y f o r b i o c h e m i c a l s t u d i e s , except t h a t the c o n c e n t r a t i o n of c e l l s was  final  adjusted to 1 - 2 m i l l i o n  cells/ml,  as determined by a hemacytometer.  2)  Fixation:  Two  techniques of i n i t i a l  The procedures gave i d e n t i c a l  a) Technique  c e l l f i x a t i o n were used. results.  #1:  A f t e r i n c u b a t i o n , c e l l suspensions were mixed w i t h an equal volume of 0 . 1 % g l u t a r a l d e h y d e i n b u f f e r b e f o r e p e l l e t i n g , i n order t o m a i n t a i n c e l l shape.  The  cells  were then resuspended  i n 2 . 5 % glutaraldehyde i n M i l l o n i g ' s  B u f f e r f o r 3 0 minutes  at 4 ° .  embedded i n D i f c o Agar  A p e l l e t of these c e l l s  ( 4 8 ° ) , drawn i n t o a c a p i l l a r y  c o o l e d , removed, and p o s t f i x e d i n 1% B u f f e r f o r 3 0 minutes  at 4 ° .  OSCK  was tube,  i n Millonig's  35  b) Technique  #2:  A f t e r i n c u b a t i o n , c e l l suspensions were p e l l e t e d , then resuspended  i n 3.3% g l u t a r a l d e h y d e i n 0.1 N  b u f f e r f o r 30 minutes i n 0.1 N phosphate  a t room temperature.  phosphate  A f t e r a wash  b u f f e r the p e l l e t e d c e l l s were p o s t -  f i x e d by the a d d i t i o n o f 1% OsOi* i n 0.1 N b a r b i t o l b u f f e r . Using 2 ml c e n t r i f u g e tubes, i t was found t h a t as few as 5  10  c e l l s c o u l d be r o u t i n e l y manipulated by t h i s technique  w i t h o u t the n e c e s s i t y of more r e f i n e d procedures. 3) Dehydration, Embedding, S e c t i o n i n g , S t a i n i n g and Viewing:  A f t e r a b u f f e r wash, samples f i x e d by each method were dehydrated i n an ascending e t h a n o l s e r i e s , and embedded i n Epon 812  (44) or Maraglas  MT2 S o r v a l Ultramicrotome.  (45). S e c t i o n s were c u t on an Thick sections  were s t a i n e d w i t h t o l u i d i n e blue  (0.5 microns)  (46). T h i n s e c t i o n s were  mounted on uncoated copper g r i d s , s t a i n e d w i t h aqueous u r a n y l a c e t a t e and l e a d c i t r a t e and examined i n a Siemens Elmiskop I .  36  EXPERIMENTAL AND  Observations were made on was  RESULTS  c e l l s u s i n g a system  developed i n t h i s l a b o r a t o r y  f o r m a i n t a i n i n g thymus  c e l l suspensions i n c u l t u r e f o r p e r i o d s up good c o n d i t i o n ,  a very important p o i n t  changes are to be d i s c e r n e d .  that  to 24 hours i n  i f early  In a d d i t i o n  subtle  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 subline  also available.  corticosteroid-resistant  of t h i s tumor, P1798R, as w e l l as two  costeroid-resistant the  The  L1210  and  lymphocytic leukemias of DBA  L5178Y, were a v a i l a b l e .  grow r e a d i l y as a s c i t e s tumors and culture.  The  other  P1798S and  subcutaneous growths.  corti-  mice,  These c e l l s a l l  a l s o as suspensions i n  R l i n e s a l s o grow w e l l as  Treatment of the  solid  s e n s i t i v e tumor  with c o r t i c o s t e r o i d s r e s u l t s i n rapid regression  over a  of  period^several  days w h i l e the r e s i s t a n t l i n e s continue  to  grow d u r i n g treatment. This  study of the  uses mainly the  c y t o l y t i c a c t i o n of  thymus, P1798S and  P1798R c e l l s as  i m e n t a l models w i t h usefc-f the L5178Y and resistance Preliminary  The  corticosteroids exper-  L1210.where  t o c o r t i c o s t e r o i d s i s being s t u d i e d  specifically.  Studies:  incorporation  of  1  "*C-acetate i n the  lipids  of  37  P179 8 tumors were examined by i n c u b a t i n g c e l l s f o r s h o r t p e r i o d s of time, e x t r a c t i n g w i t h (2:1 v/v) graphy.  chloroform-methanol  and s u b j e c t i n g the e x t r a c t s to paper chromatoThere was  a s i m i l a r l e v e l of i n c o r p o r a t i o n of  l a b e l l e d a c e t a t e i n t o the l i p i d s o f both the s e n s i t i v e and r e s i s t a n t tumors.  Autoradiograms r e v e a l e d a q u a l i t a -  t i v e l y s i m i l a r p a t t e r n of i n c o r p o r a t i o n l i n e s of P179 8. was  The  i n t o the tumor  only apparent q u a n t i t a t i v e d i f f e r e n c e  a s i g n i f i c a n t i n c r e a s e of  14  C-acetate  incorporation  i n t o one p h o s p h o l i p i d of the s e n s i t i v e tumor of t o t a l a c t i v i t y r e s i s t a n t tumor  incorporated)  (34.5+2.7%  when compared with  (27.7±0.6% t o t a l ) .  The  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 .  zone was  T h i s was  the  ten-  also true  of the f i x a t i o n of ^ C - p a l m i t i c a c i d i n t o the l i p i d s  of  1  these  tumors. The metabolism of p a l m i t i c a c i d was  another way.  looked at i n  F o l l o w i n g the i n c u b a t i o n of thymus or tumor  c e l l s with p a l m i t i c a c i d - ( 1 - **C) , the d i s t r i b u t i o n 1  r a d i o a c t i v i t y i n the aqueous or o r g a n i c phase methanol  (2:1 v/v)  e x t r a c t was  studied.  The  of  of chloroform-  aqueous  phase of such an e x t r a c t i o n should c o n t a i n the waters o l u b l e acyl-coenzyme A and a c y l - c a r n i t i n e d e r i v a t i v e s of the added f r e e f a t t y a c i d , w h i l e  the'chloroform  rich  phase would c o n t a i n the f r e e and e s t e r i f i e d f a t t y a c i d . O x i d a t i o n of the f a t t y a c i d t o C0 a disappearance of counts.  2  would be r e f l e c t e d  C e l l s were incubated  c u l t u r e medium f o r 3 0 minutes with  by  i n tissue  l a b e l l e d substrate  and  38  then e x t r a c t e d .  The  r e s u l t s of these e x t r a c t i o n s  i l l u s t r a t e d i n F i g u r e s 5 and as a l o s s of the as 1 0 0 % .  6.  The  r e s u l t s are  are expressed  l a b e l when compared w i t h c o n t r o l s  Bars t e r m i n a t i n g above the dashed l i n e  l o s s of l a b e l w h i l e those t e r m i n a t i n g below retention.  A p e r u s a l of the  figures  taken indicate  indicate  indicates  two  different  p a t t e r n s of metabolism, l o s s of l a b e l 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 tumors and  retention  r e s i s t a n t tumors, w i t h one l o s s of counts on  the  i n s e n s i t i v e tumors.  The  e x c e p t i o n , showed i n c r e a s e d  addition  of C o r t i s o l or  carnitine.  These r e s u l t s were taken as s u g g e s t i v e evidence t h a t  the  oxidation  than  in  of FFA  might be  s e n s i t i v e c e l l s and  greater i n r e s i s t a n t c e l l s  that  a l i m i t i n g f a c t o r i n the  c a r n i t i n e was  sensitive  apparently  cells.  A c i d - ( 1 - C ) to  O x i d a t i o n of P a l m i t i c  not  1h  1  ""CO  :  —  2  In order to determine i f there was oxidative  capacity  c e l l s , the  a difference  of c o r t i c o s t e r o i d - r e s i s t a n t  oxidation  of FFA  to ''CO  was  1  and  studied.  in  the  sensitive Thymus,  2 P1798S  and  P1798R  with p a l m i t i c  c e l l s were incubated i n K R P  a c i d - ( 1 - C)  and  14  ''CO  buffer  (65)  c o l l e c t e d f o r various  1  2  times up  to a maximum of two  F i g u r e 7 , the p r o d u c t i o n of  hours. 1 1  *C0  2  As  can  maximal between 3 0 and  Because a l l of the  curves had  a l i n e a r increase i n  1  "*C0  2  seen from  i n c r e a s e d over the  3 0 minutes and was  the  be  first  6 0 minutes.  same common f e a t u r e  p r o d u c t i o n over the  first  of 30  39  140-  THYMUS  BJ3  P1798/S  WITH C O R T I S O L  P1798/R  (ly/ml)  L1210  ^  L5178Y  WITH C A R N I T I N E  (lOOy/ml)  FIGURE 5: C e l l s were incubated i n F i s c h e r ' s media and 10% horse serum a t 37° f o r 30 minutes w i t h 20,000 dpm o f palmitic acid-(1- ^C). At the end of the i n c u b a t i o n p e r i o d c e l l s and media were e x t r a c t e d w i t h CHCl3:MeOH (2:1 v/v) and counts remaining i n the o r g a n i c phase determined. ( --CONTROL VALUE) . 1  40  •FIGURE 6: C e l l s were i n c u b a t e d i n F i s c h e r ' s media and 1 0 % horse serum a t 37° f o r 30 minutes w i t h 2 0 , 0 0 0 dpm o f p a l m i t i c a c i d - ( 1 - ^ C ) . At the end o f the i n c u b a t i o n p e r i o d c e l l s and media were e x t r a c t e d w i t h CHCl3:MeOH ( 2 : l v / v ) and counts remaining i n the aqueous phase determined. ( -CONTROL VALUE) . 1  41  time  (minutes)  FIGURE 7 : P r o d u c t i o n o f ^002 by mouse l y m p h o i d t i s s u e s in vitro. 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 - "c) a n d * C O c o l l e c t e d , a) Thymus b) P1798S c) P1798R 1  1  1  2  42  minutes, a l l subsequent i n c u b a t i o n s effects  l o o k i n g at  of the c o r t i c o s t e r o i d s on FFA  a 30 minute i n c u b a t i o n p e r i o d . I t was  o x i d a t i o n used expected t h a t  e a r l i e r s u b t l e changes i n l i p i d metabolism detected  using t h i s short incubation  the  would  any  be  period.  A number of experiments were performed to determine what were the o x i d a t i v e c a p a c i t i e s of these three l i n e s under v a r i o u s experimental c o n d i t i o n s . are summarized i n F i g u r e  8.  o x i d a t i v e c a p a c i t y of three  This data places  order  P1798S  and  to what would be  then  results  the  apparent  P1798R.  of  greatest,  T h i s i s the  reverse  expected i f the data were r e f l e c t i n g  the maximum o x i d a t i v e c a p a c i t y of the c e l l s . of the v a r i o u s  The  t i s s u e s i n r e l a t i v e order  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 f o l l o w e d by  cell  The  effects  a d d i t i v e s 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 s u b s t r a t e was only added source o f energy a v a i l a b l e to the  the  cells.  Thymus c e l l s have been r e p o r t e d by Abramson and B l e c h e r to have a h i g h  l i p i d content of which 7 5 % i s n e u t r a l  c o n s i s t i n g p r i m a r i l y of t r i g l y c e r i d e and quotient ly.  been c a l c u l a t e d  lipid  a respiratory  t h a t i n d i c a t e s t h a t f a t i s being u t i l i z e d  I t has  (5 8)  ( 5 9 ) t h a t thymus c e l l s  extensiveuse  5% of t h e i r l i p i d s t o r e s per hour to account f o r t h e i r endogenous r e s p i r a t i o n . c e l l s u t i l i z e FFA  Spector  ( 5 6 ) has  found t h a t tumor  r e a d i l y as an energy source.  p o s s i b l e , t h e r e f o r e , that the data obtained  I t seemed  might be more an  43  •FIGURE 8: P r o d u c t i o n o f ^COz f r o m p a l m i t i c a c i d - ( 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 m i n u t e s w i t h 2 0 , 0 0 0 dpm p a l m i t i c a c i d - ( 1 - ' ' C ) and CO collected. 1  1  1  k  z  44  i n d i c a t i o n of r e l a t i v e dependence upon f a t t y a c i d as energy source than an i n d i c a t i o n of the c e l l s for oxidation. out  capacity  of  (Fischer's,  composition see Appendix I ) , which, i t should be i s the medium i n which the  c y t o l y t i c a c t i o n of  steroids  can  palmitic  a c i d i n t h i s medium was  cell  lines  demonstrated.  (Table I I I ) .  much more i n the  The  The  for  noted,  cortico-  u t i l i z a t i o n of  labelled  decreased i n a l l t h r e e  u t i l i z a t i o n was  corticosteroid-sensitive  P1798S than i n the  the  A c c o r d i n g l y , experiments were c a r r i e d  i n a complete t i s s u e c u l t u r e medium  be  an  decreased  thymus  and  s t e r o i d r e s i s t a n t P1798R.  Numerous experiments were undertaken to examine e f f e c t s of C o r t i s o l and of p a l m i t i c i n any  one  of c a r n i t i n e upon the  a c i d i n complete medium. experiment was  constituents  was  variable  from one  the  replication  of added  experiment to the  sometimes no e f f e c t b e i n g observed at a l l . e x p l a n a t i o n i s that  oxidation  While the  good, the e f f e c t  the  One  next,  likely  " c o n t r o l " t i s s u e s might have i n  f a c t been exposed to e l e v a t e d c o r t i c o s t e r o i d l e v e l s p r i o r to t h e i r removal, and As  the  were thus i n e f f e c t , a l r e a d y  tumors grow l a r g e r , the  treated.  animals become q u i t e  stressed,  which would e l e v a t e 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).  A l s o , t h e l a b e l l e d s u b s t r a t e was  o n l y i n t r a c e r amounts and  the metabolism of a minute  amount of f a t t y a c i d would be the  endogenous p o o l at any  added  influenced  by  the  given moment, a very  size  of  variable  45  TABLE I I I : Comparison of P a l m i t i c A c i d - ( 1 - ^ C ) O x i d a t i o n i n D i f f e r e n t Media 1  1  CELL LINE  .7C0  2  RECOVERED (dpm/M c e l l s )  KREB'S BUFFER  FISCHER'S MEDIUM  THYMUS  3 479  576  P1798S  5847  446  P1798R  551  111  C e l l s were incubated i n F i s c h e r ' s Medium o r KRP f o r 30 minutes and "*C0 produced from o x i d a t i o n o f p a l m i t i c a c i d - (1- *C) c o l l e c t e d . F i g u r e s are the mean of 4-5 samples. 1  2  11  46  factor. Great v a r i a b i l i t y laboratory labelled  the  and a l s o by o t h e r w o r k e r s  fatty  When t h e i r  has b e e n e n c o u n t e r e d  acids  labelled  variability  by tumor c e l l s  also  was g r e a t l y  give  oxidative  of  iments  are  of  control values  the  oxidation while  of  of  both s e n s i t i v e of  it  slightly  78 yM cell  cells  lines, cell  C0  line.  of  78 yM  palmitic  as an i n h i b i t i o n  in  fatty  acid.  i n the  Therefore,  25% i n  decreased in a  for  the  line.  the  smaller oxidation.  sensitive  again a  slight  P1798R.  cells  by C o r t i s o l o f  t h a n an i n c r e a s e  in  the  and P 1 7 9 8 S , P1798R  result  capacity  acid, while  sensitive  exper-  percentage  thymus  slightly  decrease  the  true  such  production  2  This  a greater  produced a f u r t h e r  The r e s u l t s  pool  the  the r e s i s t a n t lk  substrate  C o r t i s o l reduced  sensitive in  carrier  the  reveal  Results  but only  s t i m u l a t i o n was o b s e r v e d i n  pool of  unlabelled  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  with  rather  (54,55).  E x p r e s s e d as a  depressed  endogenous p o o l a n d / o r Cortisol  IV.  acid in  the r e s i s t a n t  resistant  of  i n each c a s e ,  palmitic  increasing  of  reproducible  on t h e s i z e o f  the c e l l s .  shown i n T a b l e  The a d d i t i o n  that  The e f f e c t  and w o u l d more l i k e l y  capacity  the uptake  in vivo  r e d u c e d and  information  endogenous p o o l  this  FFA was d i l u t e d w i t h u n l a b e l l e d  r e s u l t s were o b t a i n e d . could  in  in  c o u l d be  fatty size of the  was m e a s u r e d i n b o t h c o n t r o l  interpreted  acid  metabolism  the  endogenous  s i z e of  the  and C o r t i s o l  endogenous treated  47  TABLE I V : E f f e c t o f C o r t i s o l and/or 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 - ^C) t o *C02 1  ll  TISSUE TREATMENT  THYMUS  CONTROL  100%  100%  100%  56%  83%  109%  75%  73%  90%  66%  95%  CORTISOL  (2.7uM)  PALMITIC ACID (78uM)  CORTISOL (2.7uM) + PALMITIC ACID 50%* (78yM)  P1798S  P17 98R  T h e p e r c e n t a g e s r e f l e c t t h e a m o u n t o f COz produced relative t o the control value. C e l l s were i n c u b a t e d i n F i s c h e r ' s M e d i u m 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- '*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 1 5 - 2 0 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 . lk  1  48  tissues.  Steroid-treated  animals were i n j e c t e d  d e x a m e t h a s o n e i n 0.1 m l S e s a m e O i l i p ; c o n t r o l were  injected  with  the vehicle  P1798S, P1798R and thymus levels  determined  reported the  i n T a b l e V.  resistant  levels  cells  and a c t u a l l y  of  uptake in  acid  of plasma  effect  could  therefore  free  fatty  levels  acids  n o t be a s c e r t a i n e d  determined.  incubated with  with IV)  i n the i n the  with  or  increased tissues  results.  palmitic  the incubation  the c e l l s  in  a refrigerated  international  to determine  the c e l l  palmitic  acid-(1- ''C)  clinical  and  added  the uptake of  were p e l l e t e d  The  was  or without  30  1  t o t h e end o f t h e i n c u b a t i o n .  decanted,  release  uptake i n v i t r o  In order  of  medium was  endogenous  sensitive  from these  acid  f o r 2 hours.  were p u l s e d w i t h  minutes p r i o r  Hyamine  are  C e l l suspensions were prepared  7 8 yM p a l m i t i c  ( 2 . 7 yM)  cells  of  by t h e s e  whether o r n o t C o r t i s o l had a f f e c t e d acid,  decreased  from endogenous f a t s t o r e s  o f C o r t i s o l on f a t t y a c i d  Cortisol  (cf Table  were a r e f l e c t i o n of i n c r e a s e d  fatty acids  vivo  FFA  P1798R.  fatty  free  mg  later  are i n accord  o f FFA a r e i n c r e a s e d  Whether o r not the i n c r e a s e d free  hours  The r e s u l t s  results  1  animals  were removed and  These r e s u l t s  of previous  t h e endogenous  steroid-sensitive  tissue  Two  by m i c r o t i t r a t i o n .  interpretation  that  alone.  with  A t t h e end a t 1500  rpm  centrifuge.  pellet dissolved  and t h e uptake o f l a b e l l e d p a l m i t i c  i n 1.0  acid  The ml  determined.  49  TABLE V: E f f e c t of Dexamethasone on Endogenous Free F a t t y A c i d L e v e l s  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 t r e a t e d w i t h 1 mg dexamethasone i n 0.1 ml Sesame o i l o r w i t h v e h i c l e a l o n e . Two hours l a t e r animals were s a c r i f i c e d , t i s s u e removed and FFA l e v e l s determined by m i c r o t i t r a t i o n . * F i g u r e s i n d i c a t e d are the mean value f o r a pooled sample o f thymus from 5 female B a l b / c J mice weighing 1720 gm. 1  *  2  F i g u r e i n d i c a t e d i s expressed i n nEqv FFA/M  cells.  50  The  r e s u l t s are expressed i n Table VI.  The uptake o f  p a l m i t i c a c i d was not a f f e c t e d s i g n i f i c a n t l y by C o r t i s o l in  any o f the t i s s u e s s t u d i e d .  I t would appear t h a t the  i n c r e a s e d endogenous l e v e l s of FFA must come from the lipid  s t o r e s of the lymphoid c e l l s , these s t o r e s  being  triglyceride. Abramson and B l e c h e r  (58)• have r e p o r t e d  that i n r a t  thymocytes 2% o f the t o t a l wet weight o f t i s s u e i s l i p i d , ' w i t h over 60% o f t h i s l i p i d being  neutral l i p i d .  Further  c h a r a c t e r i z a t i o n showed t h a t over 80% o f the f r a c t i o n was triglyceride.  In the s t e r o i d s e n s i t i v e c e l l s used i n t h i s  study i t was found t h a t the t o t a l l i p i d  content  ranged  from 4.2% f o r P1798S t o 7.2% f o r thymus t i s s u e w i t h over 71%  o f the t o t a l f a t being  o f P1798S.  n e u t r a l g l y c e r i d e i n the case  TLC o f the l i p i d e x t r a c t s o f P1798S and P1798R  showed t h a t the only EFA d e t e c t a b l e were p h o s p h o l i p i d and t r i g l y c e r i d e , w i t h t r a c e s of c h o l e s t e r o l e s t e r s o b s e r v a b l e under c e r t a i n c o n d i t i o n s .  The only NEFA d e t e c t a b l e by  chromatography was c h o l e s t e r o l . The  Oxidation Spector  o f Short  Chain F a t t y  (56) has r e p o r t e d  Acids:  t h a t tumor c e l l s are  l i m i t e d i n t h e i r capacity to o x i d i z e short chain a c i d s as compared t o long c h a i n f a t t y a c i d s .  fatty  Accordingly,  thymus and mouse lymphosarcoma c e l l s were incubated equivalent  concentrations  sodium octanoate and  1 h  to  with  of l a b e l l e d p a l m i t i c a c i d o r z  production  monitored i n order  51  TABLE V I :  E f f e c t o f C o r t i s o l on P a l m i t i c A c i d  PALMITIC ACID UPTAKE  Uptake  (dpm/M c e l l s )  TISSUE  EXPT #  CONTROL  CORTISOL TREATED (2.7 yM)  THYMUS  1. 2. 3.  1967±196 900±19 978±36  1753±371 834±22 1045115  P1798S  1. 2.  923159 609111  1039195 605121  P1798R  l . *  1955159  1872+42  1  C e l l s were incubated w i t h 7 8 yM p a l m i t i c a c i d f o r 2 hours and 30 minutes p r i o r t o the end of the i n c u b a t i o n were p u l s e d w i t h p a l m i t i c a c i d - ( 1 - ^ C ) . Figures i n d i c a t e d are the MEAN 1 SEM f o r 4 samples. 1  *  1  Figures  i n d i c a t e d are the MEAN 1 SEM f o r 8 samples.  52  TABLE V I I :  TISSUE  P1798R *  TOTAL LIPID mg/gm  1  P1798S *THYMUS *  and  L i p i d Composition o f Mouse Lymphosarcoma P1798 and Thymus  2  LIPID PHOSPHATE yM/gm  TEFA yM/gm  23.2  5.4  74.5  42.2  5.5  81.4  71.6  T i s s u e s were removed and t o t a l l i p i d , l i p i d phosphate TEFA determined as i n d i c a t e d i n the methods s e c t i o n .  ** F i g u r e s i n d i c a t e d are the mean o f 3 separate d e t e r minations from a t l e a s t 1 gm o f t i s s u e (wet w e i g h t ) . * % F i g u r e i n d i c a t e d i s the mean o f a 200 mg p o o l e d sample from 5 female B a l b / c J mice (weight 17-20 gm). 2  TABLE V I I I :  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  38.5  61.5  THYMUS(RAT)*  1  (82.3%TG)  L i p i d d i s t r i b u t i o n o f mouse lymphosarcoma was determined by s e p a r a t i n g p h o s p h o l i p i d from n e u t r a l l i p i d on a c t i v a t e d s i l i c i c a c i d columns u s i n g technique d e s c r i b e d i n the methods section. * From Abramson and B l e c h e r purposes. :  (58),included f o r comparison  54  to  see i f lymphoid  to  o t h e r tumor c e l l s .  that  cells  The  the r e v e r s e i s true  have a g r e a t e r c a p a c i t y acids,  which  Effect  o f C o r t i s o l and  The  l o n g and  examining  the o x i d a t i o n  glucocorticoid  after  hydrate uptake  i n steroid The  which  tissues  The indicated  were s t u d i e d  by  2  and  the 30  accumulate fatty  free  acids  were c a r r i e d  minutes  t h e r e any  e f f e c t on  C o r t i s o l depressed the  competes w i t h g l u c o s e i t has  sensitive  could  cause  o u t w i t h tumor c e l l s  i n which  2  carbo-  on  case,  values.  Survival:  accumulation of  damage t o c e l l s .  *C0 .  uptake  no e f f e c t  to c o r t i c o s t e r o i d  This  ll  (60,61)  o f p a l m i t a t e , e x c e p t i n one  fatty acid.  effect to  1  of i s o t o p e d i l u t i o n experiments  cells  uptake  o f G l u c o s e - ( ^C-U)  on Mouse Lymphosarcoma C e l l  that  Metabolism:  o r p a l m i t a t e t r e a t m e n t no  responsive tissues;  results  (57).  and  were n e g l i g i b l e when compared w i t h t h e c o n t r o l  E f f e c t o f FFA  cells  chain fatty  During the f i r s t  t r e a t m e n t was  effects  indicate  of C o r t i s o l  of glucose to C0  (Table X I ) .  2-deoxyglucose,  common  Lymphoid  short  term e f f e c t s  on t h e m e t a b o l i s m  2 hours  P1798R.  system.  carbohydrate metabolism  observed  only  i n T a b l e IX  P a l m i t a t e on C a r b o h y d r a t e  1  Only  of  i n this  2 d e o x y g l u c o s e - ( 1 - ''C) .  was  results  to oxidize  short  on  following  characteristics  i s s i m i l a r t o v a r i o u s normal  palmitate  of  exhibited  ( T a b l e IV) would free  Incubations the  effects  55  TABLE IX: O x i d a t i o n o f Long and Short Chain F a t t y A c i d s  1  TISSUE  *COz  PRODUCED FROM:  PALMITIC A C I D - ( 1 - C ) 1k  SODIUM n-OCTANOATE-(1- "c) 1  THYMUS  799±64  11,1591418  P1798S  1539187  12,403+59  P1798R  701118  15,897162  C e l l s were incubated f o r 3 0 minutes w i t h e q u i v a l e n t c o n c e n t r a t i o n s o f p a l m i t a t e or octanoate. C O z was assayed as p r e v i o u s l y d e s c r i b e d . F i g u r e s i n d i c a t e d are the MEAN 1 SEM f o r 4-5 samples. 1 h  56  of added f a t t y a c i d s on c e l l v i a b i l i t y over a p e r i o d o f s e v e r a l hours were examined.  As can be seen i n Table X I I ,  both p a l m i t i c a c i d , a C-16, and a z e l a i c , a C-9 xylic acid  (used a l s o because o f i t s g r e a t e r  i n aqueous s o l u t i o n ) , i n c r e a s e d culture.  By c o n t r a s t ,  dicarbo-  solubility  the death o f c e l l s i n  the r e s i s t a n t s u b l i n e  P1798R  was o n l y p a r t i a l l y a f f e c t e d by c o n c e n t r a t i o n s o f FFA ten times g r e a t e r than t h a t s u f f i c i e n t t o k i l l c e l l s i n 5.5 hours  (Table X I I I ) .  a l l sensitive  A p a r a l l e l e x i s t s between  s e n s i t i v i t y t o FFA and s e n s i t i v i t y t o c o r t i c o s t e r o i d s .  E l e c t r o n m i c r o s c o p e Examination o f the E f f e c t s o f FFA on Mouse Lymphoid C e l l s : The and  nature of the damage t o s e n s i t i v e c e l l s by FFA  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  g r e a t e r d e t a i l u s i n g the e l e c t r o n  microscope.  F i g u r e 9 shows the i n t e g r i t y of normal f i n e  structure  of u n t r e a t e d 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 F i s c h e r ' s  Medium f o r 4 hours.  Maintenance o f normal f i n e s t r u c t u r e was a l s o observed i n u n t r e a t e d 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, w i t h o r without added f a t t y The  acids.  presence o f c a p s i d - l i k e s t r u c t u r e s  formed, i n c l o s e  both p a r t i a l l y  a s s o c i a t i o n w i t h the endoplasmic  reticulum  membrane, and more completely formed, found i n an i n t r a -  57  TABLE X:  O x i d a t i o n of Glucose by Mouse Lymphosarcoma C e l l s  1 ^C 0  2  PRODUCED (dpm/M c e l l s )  CONTROL  CORTISOL TREATED (2.7 yM)  PALMITATE TREATED (78 yM)  1. 2. 3,  54419 58517 690117  53413 587126 691+17  554118 586141 697110  1. 2,  501 + 4 34214  51218 352 + 7  515119 347118  TISSUE  EXPT #  P1798R  P1798S  C e l l s were incubated f o r 30 minutes w i t h 370,000 dpm of Glucose- ( "*C-U) and CC>2 c o l l e c t e d as p r e v i o u s l y described. F i g u r e s i n d i c a t e d are MEAN 1 SEM f b r 5 samples. 1  1k  58  TABLE X I : Uptake o f 2-Deoxyglucose-(1- ^C) by Mouse Lymphoid C e l l s 1  UPTAKE OF 2-DE0XYGLUC0SE (dpm/M c e l l s ) TREATMENT TISSUE  EXPT #  CONTROL  CORTISOL ( 2 . 7 yM)  PALMITATE (78 yM)  THYMUS  1. 2.  87±5 91±4  59±1 68±5  82±2 149±11  P1798S  1. 2.  135 + 8 154+12  118±13 88±3  120±12. 145±11  P1798R  1. 2. 3.  36±2 448±8 59±3  35±2 439±8 62±13  40±3 441±8 57±4  C e l l s were incubated w i t h 2.7 yM C o r t i s o l o r 7 8 yM p a l m i t i c a c i d f o r 2 hours. T h i r t y minutes p r i o r t o the t e r m i n a t i o n o f the i n c u b a t i o n c e l l s were pulsed w i t h 0.5yCi of 2-deoxyglucose-(1- ^C). F i g u r e s i n d i c a t e d are MEAN ± SEM f o r 4-5 d e t e r m i n a t i o n s . 1  59  TABLE X I I : E f f e c t o f Free F a t t y on P1798S In V i t r o  Acids  VIABLE CELLS ±SEM (M CELLS/ML)  DEAD CELLS!SEM (T CELLS/ML)  CONTROL  1.725 ± 0.109  12.5 ± 12.5  PALMITATE TREATED (20 yg/ml)  1.117 ± 0.217 *  250.0 ± 28.8  AZELAIC ACID TREATED (10 yg/ml) 1.083 ± 0.174 *  283.3 ± 16.7  C e l l s were incubated i n F i s c h e r ' s Medium w i t h 10% Horse Serum a t 37° f o r 4 hours. V i a b l e c e l l s were determined by e o s i n e x c l u s i o n . *  p <0.05 compared w i t h c o n t r o l v a l u e .  60  TABLE X I I I : E f f e c t of Increasing A z e l a i c Acid Concentration on Mouse Lymphosarcoma P1798 C e l l s In V i t r o  TUMOR  AZELAIC ACID CONCENTRATION  % LYSED CELLS  100  A%  P1798S  53 yM  (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 F i s c h e r ' s Medium w i t h 10% Horse Serum f o r 5.5 hours a t 37° C. The t o t a l and the number o f l y s e d 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; I n s e t , F i g u r e noted i n P1798S c e l l s .  15) were  These appeared i d e n t i c a l w i t h  c a l l e d A - p a r t i c l e s reported  i n other murine leukemias  soand  the normal t i s s u e of neonatal mice ( 6 2 ) . Comparison of t h i c k s e c t i o n s of c o n t r o l and  treated  P1798S suspensions a f t e r 4 hours i n c u b a t i o n when s t a i n e d w i t h t o l u i d i n e blue d e g r a d a t i o n and  showed a marked i n c r e a s e i n c e l l  necrosis i n treated sections  (Figures  10,11,12).  Degenerative changes e s p e c i a l l y e v i d e n t were the s w e l l i n g and  v e s i c u l a t i o n pf the cytoplasm.  C e l l s undergoing  n e c r o s i s were c h a r a c t e r i z e d by pyknosis and k a r y o l y s i s . The  f i n e s t r u c t u r e of P1798S c e l l s a f t e r  treatment confirmed the p r o g r e s s i v e a t i v e process.  nature of the  D i l a t a t i o n , s w e l l i n g and  e s p e c i a l l y i n g o l g i regions  c e l l death  (Figure 1 7 ) .  degener-  subsequent  v e s i c u l a t i o n of the endoplasmic r e t i c u l u m 15,16)  4 hours  (Figures 1 3 , 1 4 ,  (Figure 15) preceded  Noteworthy was  the almost complete  absence of membrane-bound ribosomes i n those c e l l s i t i n g minimal p a t h o l o g i c a l changes a t t h i s time.  exhibMarked  d i l a t a t i o n of the p e r i n u c l e a r c i s t e r n a (Figure 14) u s u a l l y p r o g r e s s e d concomitantly endoplasmic r e t i c u l u m .  w i t h the v e s i c u l a t i o n of Mitochondrial  a l t e r a t i o n s , except  i n p r e n e c r o t i c c e l l s , were not synchronous, but only a p o r t i o n of those w i t h i n the c e l l Large dense g r a n u l e s  the  included  (Figure 1 3 ) .  (Figure 16) appeared i n the  of t r e a t e d but not c o n t r o l c e l l s at 4 hours.  cytoplasm  They were  62  e s p e c i a l l y conspicuous  i n c e l l d e b r i s remaining  after  cytolysis. D e t a i l e d events i n the degenerative changes i n n u c l e a r f i n e s t r u c t u r e were d i f f i c u l t of  the v a r i a b l e morphology of the n u c l e i o f t h i s tumor  and due  t o the asnychronous development of  within c e l l populations.  euchromatin  demarcation  degeneration  However, as a g e n e r a l r u l e ,  as c y t o p l a s m i c degeneration proceeded, of  t o assess because  the g r a n u l a r i t y  became l e s s pronounced and i t s u s u a l sharp  from heterochromatin  F i g u r e 9 w i t h F i g u r e s 14 and  l e s s marked  (compare  16).  F i g u r e s 18 and 19 show the f i n e s t r u c t u r e of thymocytes a f t e r 4 hours treatment with 78 yM p a l m i t i c  acid.  E s p e c i a l l y prominent at t h i s time were those c e l l s undergoing k a r y o r r h e x i s . to  Areas of the n u c l e a r membrane  appeared  have become compromised w i t h subsequent appearance of  n u c l e a r m a t e r i a l , f r e e and membrane bound, admixed w i t h cytoplasm. A working h y p o t h e s i s of the v a r i o u s f a c t o r s which are i n v o l v e d i n b r i n g i n g about 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 s i l l u s t r a t e d a t F i g u r e 20. from accumulation  The damage r e s u l t i n g  of FFA has been d e s c r i b e d ; b i o c h e m i c a l  evidence t h a t t h i s does i n f a c t occur has been o b t a i n e d (Table V) .  63  FIGURE 9: incubation.  Untreated c o n t r o l P1798S c e l l s a f t e r 4 h r s o f The s t r u c t u r a l i n t e g r i t y of the i n c u b a t e d c e l l s  i s well preserved.  Maraglass  X22,510.  The i n s e t shows  membrane-associated and i n t r a c i s t e r n a l c a p s i d - l i k e t h a t are commonly found i n the cytoplasm throughout p o p u l a t i o n . X59,190.  structures the c e l l  65  FIGURE 10: incubation.  Untreated c o n t r o l P1798S c e l l s Good p r e s e r v a t i o n  0.5 micron t h i c k s e c t i o n .  FIGURE 11: incubation. Extensive  a f t e r 4 hrs of  of c e l l p o p u l a t i o n  i s evident.  T o l u i d i n e blue s t a i n . X1430.  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 r s of  Cytoplasmic v e s i c u l a t i o n (arrows) i s v e r y obvious.  necrosis  i s evidenced by p y k n o s i s . T o l u i d i n e blue  stain.X1430.  FIGURE 12: incubation.  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 f t e r 4 h r s o f Extensive  c e l l n e c r o s i s has occurred.  i s a predominating event.  Cytolysis  T o l u i d i n e blue s t a i n . X1430.  6  7  \-  6  67  FIGURE of  13:  incubation.  especially of  Palmitic  S w e l l i n g of  in golgi  degeneration.  acid-treated  is  prominent  the almost complete  membrane-bound p o l y s o m e s . n u c l e a r mebrane  the endoplasmic  regions,  Note  P1798S c e l l s  Alterations  are not obvious  at t h i s  after  4 hours  reticulum,  i n the e a r l y absence  of  i n mitochondria time.  stages  Maraglas  and X30,870.  69  FIGURE of  14:  Palmitic  incubation.  reticulum  (ER)  acid-treated  Massive and  events  the  focal  alterations  the  sharp  dichotomy  Maraglass  i n the  X30,140.  vesiculation  swelling  prominent  P 1 7 9 8S  late  i n the  between  of  the  stage  of  cells the  cisterna  degeneration.  mitochondria euchromatin  and  and  4  hours  endoplasmic  perinuclear of  after  the  are  Note  loss  of  heterochromatin.  70  71  FIGURE 15: incubation.  A z e l a 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 hours E a r l y s w e l l i n g o f the g o l g i  zones and f o c a l  a l t e r a t i o n s i n the m i t o c h o n d r i a are e v i d e n t . presence of c a p s i d - l i k e s t r u c t u r e s  Note the  (arrow) w i t h i n the ER.  C e l l d e b r i s , 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 o f the micrograph. Maraglas X26,370.  72  73  FIGURE 16:  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 f t e r 4 hours  of i n c u b a t i o n .  P r o g r e s s i v e degeneration  o f the cytoplasm  i s t y p i f i e d by s w e l l i n g o f both ER (arrow) and p e r i n u c l e a r cisterna.  I s o l a t e d 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 a t the l e f t o f the micrograph i s  found commonly i n t r e a t e d c e l l s a t 4 hours but not i n untreated  cells.  FIGURE 17:  Maraglas X20,420.  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 f t e r 4 hours  of i n c u b a t i o n .  Terminal  structural alteration i n prenecrotic  c e l l s i s apparent by i t s p r o g r e s s i v e nature. of the n u c l e a r membrane i s l o s t . p r o f i l e s w i t h i n the nucleus  Note the membrane bound  (arrow).  Degenerate  v e s i c u l a t e d c i s t e r n a e , and d e g r a n u l a t i o n i s t i c o f t h i s t e r m i n a l stage.  The i n t e g r i t y  mitochondria,  of ER are c h a r a c t e r -  Maraglas X31,640.  74  75  FIGURE 18: incubation.  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 o f Note the process o f k a r y o r r h e x i s  these c e l l s . membrane-bound and  occurring i n  P o r t i o n s o f the n u c l e a r m a t e r i a l , sometimes (arrow),  become separated  l i e w i t h i n the cytoplasm.  from the nucleus  Note t h a t the small degree  of ER v e s i c u l a t i o n i s c o r r e l a t e d w i t h the p a u c i t y o f ER w i t h i n these c e l l s .  Epon X22,760.  77  FIGURE  19:  Palmitic  of  incubation.  of  nuclear material  Perinuclear  acid-treated  Partially-bound lie  cisternal  after  4 hours  and unbound clumps  (arrow)  admixed w i t h i n  swelling  undergoing k a r y o r r h e x i s .  thymocytes  is  the  cytoplasm.  not obvious  Epon X 2 7 , 7 1 0 .  i n these  cells  FIGURE 20: Proposed scheme o f p o s s i b l e f a c t o r s l e a d i n g t o c y t o l y s i s . What the scheme proposes i s t h a t FFA would be r e l e a s e d from t r i g l y c e r i d e stores. An accumulation o f FFA 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 would l e a d to n u c l e a r damage and u l t i m a t e l y c e l l l y s i s . Steroid-resistant tissues would remove FFA by o x i d a t i o n and p o s s i b l y by r e - e s t e r i f i c a t i o n .  80  E f f e c t o f Free F a t t y A c i d s i n Inducing L y s i s o f S t e r o i d Resistant Tissues: In  o r d e r t o t e s t f u r t h e r the c r i t i c a l  importance o f  f a t t y a c i d i n the process o f 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 might then p e r m i t s u f f i c i e n t accumulation o f t h i s s t i t u e n t t o cause damage t o c e l l s . s t r u c t u r a l analogue  cells  con-  Deoxycarnitine, a  o f c a r n i t i n e , l a c k s the oxygen f u n c t i o n  to which t h e f a t t y a c i d i s e s t e r i f i e d i n c a r n i t i n e and i s thereby t r a n s p o r t e d i n the m i t o c h o n d r i a where i t undergoes 3 - o x i d a t i o n  ( f o r s t r u c t u r e o f c a r n i t i n e and deoxy-  c a r n i t i n e , see Appendix I I ) .  D e o x y c a r n i t i n e f u n c t i o n s as  a c o m p e t i t i v e i n h i b i t o r i n the o x i d a t i o n o f FFA (95). Accordingly, c e l l s o f the three s t e r o i d - r e s i s t a n t  lympho-  sarcomas were incubated w i t h 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 X I V ) . Approximately  one t h i r d o f the c e l l s o f each tumor  underwent l y s i s when t r e a t e d w i t h d e o x y c a r n i t i n e and C o r t i s o l together.  The c e l l s remaining d i d not respond  even t o h i g h e r c o n c e n t r a t i o n s of i n h i b i t o r than.the used i n these experiments.  0.8 mM  I n j e c t i o n o f mice b e a r i n g  these tumors i n the a s c i t e s form w i t h d e o x y c a r n i t i n e produced  similar  results.  When the s t e r o i d - r e s i s t a n t L517 8Y lymphosarcoma i s t r e a t e d i n v i t r o w i t h 10 ug o f c i t r a l  (3,7-dimethyl,  2 , 6 - o c t a d i e n a l , f o r s t r u c t u r e see Appendix I I ) , which,  81  TABLE XIV:  TUMOR  E f f e c t o f D e o x y c a r n i t i n e on the Response o f Tumor C e l l s t o C o r t i s o l In V i t r o  VIABLE CELLS % OF CONTROLS  P1798R  65+6  (6)  L1210  63+14  (4)  L5178Y  59±8  (4)  C e l l s were incubated i n t i s s u e c u l t u r e a t 37° f o r 6 hours. C o n t r o l samples were t r e a t e d w i t h 2.7 yM C o r t i s o l . Experimental samples had 0.8mM d e o x y c a r n i t i n e added as w e l l .  82  i t was thought, because would i n h i b i t  o f i t s branched  chain structure  B - o x i d a t i o n , i t was found t h a t 30% o f the  c e l l s were rendered s e n s i t i v e t o t h e a c t i o n o f C o r t i s o l (Table XV).  When the more s o l u b l e sodium  bisulfite  a d d i t i o n compounds of c i t r a l were t e s t e d , the number.? o f c e l l s rendered s e n s i t i v e was i n c r e a s e d t o 50% and 60% for  c o n c e n t r a t i o n s o f 20 and 100 yg o f the compound  respectively. In v i v o treatment of the L517 8Y and L1210 w i t h a citral  suspension r e s u l t e d i n 20% v i a b l e c e l l s  6 hours a f t e r treatment  (Table XVI).  remaining  The sodium  a d d i t i o n compound, although more e f f e c t i v e than in vitro  , was l e s s e f f e c t i v e i n v i v o  citral  P1798R were  i p , d e s t r u c t i v e e f f e c t s o f the  compound were seen i n t r e a t e d animals and 22 w i t h 23 and 24).  citral  (cf Table XV).  When animals b e a r i n g the subcutaneous t r e a t e d w i t h 5 mg c i t r a l  bisulfite  Twenty-four  (compare F i g u r e 21 hours  following  treatment l a r g e n e c r o t i c areas were observed i n  t r e a t e d tumors  (compare F i g u r e 21 w i t h 23).  High power  o b s e r v a t i o n o f these areas i n d i c a t e d t h a t the c e l l s had undergone k a r y o l y s i s  (compare F i g u r e 22 with 24).  The R e l a t i o n s h i p o f C y t o l y s i s t o o t h e r C o r t i c o s t e r o i d A c t i o n s : I t i s w e l l documented t h a t c o r t i c o s t e r o i d of  animals r e s u l t s i n immunosuppression  i n t e r e s t t h e r e f o r e t o determine  treatment  (66). I t was o f  i f free fatty  acids  TABLE XV:  E f f e c t o f 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)  C e l l s were incubated i n F i s c h e r ' s Medium w i t h 10% Horse Serum a t 37° f o r 6 hours. C o n t r o l s were incubated w i t h 2.7 yM C o r t i s o l o n l y .  TABLE XVI:  E f f e c t of C i t r a l on Tumors In V i v o  TUMOR  TREATMENT  L5178Y  CITRAL SUSPENSION  20.0 (7)  CITRAL SODIUM BISULFITE  60.0 (2)  CITRAL SUSPENSION  16.6 (5)  CITRAL SODIUM BISULFITE  90.0 (2)  L1210  VIABLE CELLS % CONTROLS  IP tumors 3 days a f t e r t r a n s p l a n t a t i o n were t r e a t e d i p w i t h 5 mg CITRAL i n s a l i n e suspension. In some cases two i n j e c t i o n s 2.5 hours a p a r t were made. C e l l s were examined a t 6 hours and the t o t a l l y s e d and u n l y s e d c e l l s were counted.  FIGURE 21:  S e c t i o n of P1798R from c o n t r o l animal.  Hematoxylin and E o s i n X52.  87  FIGURE 22:  S e c t i o n o f P1798R from c o n t r o l animal.  Hematoxylin and E o s i n X206.  8 8  89  FIGURE 2 3 : after  Section of  treatment with  necrosis,  (cf F i g u r e  P1798R t a k e n f r o m a n i m a l 24 h o u r s  5mg CITRAL i p .  Note l a r g e a r e a s  2 1 ) . H e m a t o x y l i n and E o s i n X 5 2 .  of  90  91  FIGURE 24:  S e c t i o n o f P1798R taken from animal 24 hours  a f t e r treatment w i t h 5mg CITRAL i p . has  occurred,  Note k a r y o r r h e x i s  (cf F i g u r e 22). Hematoxylin and E o s i n X206.  92  93  reproduced a l l the known e f f e c t s of c o r t i c o s t e r o i d i n lymphoid t i s s u e .  I t might be p o s s i b l e  immunosuppressive and  to separate  o t h e r a c t i v i t i e s of  from i t s c y t o l y t i c a c t i o n s .  the  corticosteroid  In order to e x p l o r e 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 f o r 2 hours i n t i s s u e c u l t u r e medium at 37 the  addition  of C o r t i s o l  (2.7  T h i r t y minutes p r i o r t o the  yM)  end  w i t h or without  or p a l m i t i c  of the  and  their incorporation  f r a c t i o n determined. XVII, XVIII, and  The  (78  incubation,  were p u l s e d w i t h l a b e l l e d p r e c u r s o r s of DNA, protein  acid  RNA  i n t o a 5% TCA  cells  and  insoluble  r e s u l t s are p r e s e n t e d i n Tables  XIX.  C o r t i s o l i n h i b i t s the uptake of thymidine i n t o acid-insoluble w i t h one  p r e c i p i t a t e of s t e r o i d - r e s p o n s i v e  e x c e p t i o n , but  i s without e f f e c t i n the  r e s i s t a n t P1798R (Table XVII).  In the one  which the  P1798S tumor was  tumor was  taken 19 days a f t e r t r a n s p l a n t .  the  tumor i s very l a r g e  w i t h s t e r o i d due page 44).  The  and  may  incorporation  and  P1798S but  At t h i s stage treated  (see p r e v i o u s comments,  and  of thymidine  lesser  into  are without e f f e c t  cells.  C o r t i s o l a l s o i n h i b i t s the leucine  the  e f f e c t s p a r a l l e l those of C o r t i s o l to a  resistant  and  steroid-  experiment i n  have a l r e a d y been  of 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 n the  the  tissues  unresponsive to C o r t i s o l ,  to a s t r e s s response  e x t e n t i n i n h i b i t i n g the DNA  yM).  i n t o the  incorporation  acid-insoluble  of  uridine  f r a c t i o n of thymus  i t i s without e f f e c t i n P1798R. (Tables XVIII  94  TABLE XVII: I n c o r p o r a t i o n o f Thymidine-(CH - H) i n t o the Acid-Insoluble Fraction 3  3  INCORPORATION  (dpm/M c e l l s )  TISSUE  EXPT •..#  CONTROL  CORTISOL  PALMITATE  THYMUS  1. 2.  20731235 2219122  1584156 15991160  1823127 19531166  P1798S  1. 2.*  1010131 30818  584120 30514  862+41 354112  831127 854137  822114 772120  996134 860132  P1798R  1  1. 2.  Samples were incubated f o r 2 hours i n t i s s u e c u l t u r e medium a t 37° w i t h o r without added C o r t i s o l (2.7 yM) o r p a l m i t i c a c i d (7 8 yM). 30 minutes p r i o r t o the end o f the i n c u b a t i o n they were p u l s e d w i t h 1 yC. o f thymidine (CH3~ H), a c i d - i n s o l u b l e f r a c t i o n was c o l l e c t e d and r a d i o a c t i v i t y determined. F i g u r e s i n d i c a t e d are MEAN 1SEM f o r 4-5 samples. 3  **  Figures indicated  are f o r a l a r g e  19 day o l d tumor.  95  and  XIX).  P a l m i t i c a c i d h a s no s i g n i f i c a n t e f f e c t on the  incorporation  o f l e u c i n e and u r i d i n e i n t o T C A i n s o l u b l e  f r a c t i o n of thymus, P 1 7 9 8 S , and P 1 7 9 8 R .  T h e suppressive  e f f e c t s o f C o r t i s o l on the i n c o r p o r a t i o n  o f thymidine,  u r i d i n e and l e u c i n e i n t o a 5% T C A i n s o l u b l e f r a c t i o n and l a c k o f e f f e c t on P 1 7 9 8 R are i n agreement w i t h the r e s u l t s of o t h e r s  (63,64) .  TABLE XVIII: I n c o r p o r a t i o n o f U r i d i n e - ( 5 - H ) i n t o the Acid-Insoluble Fraction 3  INCORPORATION TISSUE  EXPT #  THYMUS  (dpm/M c e l l s )  CONTROL  CORTISOL  PALMITATE  1. 2.  619±33 658+11  485±11 415±7  603±36 698±16  P1798S  1. 2.  1323120 1600±91  890±9 993+21  1297±7 1511±43  P1798R  1. 2.  1112168 1036±42  985±22 1065±20  1010+29 1055128  Samples were i n c u b a t e d f o r 2 hours i n t i s s u e c u l t u r e medium a t 37 w i t h or w i t h o u t added C o r t i s o l (2.7 yM) o r p a l m i t i c a c i d (7 8 yM). T h i r t y minutes p r i o r t o the end of the i n c u b a t i o n they were p u l s e d w i t h 1 yC^ of U r i d i n e (5- H), a c i d - i n s o l u b l e f r a c t i o n was c o l l e c t e d and r a d i o a c t i v i t y determined. F i g u r e s i n d i c a t e d are MEAN ± SEM f o r 4-5 samples. 3  TABLE XIX:  I n c o r p o r a t i o n o f L e u c i n e - i C - U ) i n t o the Acid-Insoluble Fraction 1 k  INCORPORATION TISSUE  EXPT #  (dpm/M c e l l s )  CONTROL  CORTISOL  PALMITATE  THYMUS  1. 2.  172±2 20914  12611' 164+4.  167+3 21616  P1798S  1. 2.  501+11 436122  385+6 282111  51016 451113  P1798R  1. 2.  44818 308114  43918 29115  444118 332118  Samples were incubated f o r 2 hours i n t i s s u e c u l t u r e medium a t 37 w i t h o r without added C o r t i s o l (2.7 yM) o r p a l m i t i c a c i d (7 8 yM). T h i r t y minutes p r i o r t o the end of the i n c u b a t i o n , they were p u l s e d w i t h 0.5 yC^of l e u c i n e ( C - U ) , a c i d - i n s o l u b l e f r a c t i o n was c o l l e c t e d and r a d i o a c t i v i t y determined. F i g u r e s i n d i c a t e d are MEAN 1 SEM f o r 4-5 samples. 14  98  DISCUSSION  Most work on the a c t i o n of c o r t i c o s t e r o i d s centered around the important m e t a b o l i c events in liver  (68).  c o r t i c o i d s and This report the  I n d u c t i o n of s p e c i f i c RNA the  occurring enzymes by  subsequent changes have been reviewed  lymphoid t i s s u e .  dramatic e f f e c t of c o r t i c o s t e r o i d s upon c e 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 i n v e s t i g a t i o n f o r some time  (69) .  been under  A similar action  occurs i n c e r t a i n malignant lymphocytes, notably acute lymphocytic leukemias of c h i l d h o o d , and l y s i s of c e l l s and  remission  of time.  peutic  agents, the c e l l p o p u l a t i o n  a c t i o n of the  here was  ment of  steroid  results in  as w i t h other chemothera-  (70).  becomes r e s i s t a n t to  The  work to be  discussed  undertaken to e x p l o r e the mechanism whereby  l y s i s occurs and  the b i o c h e m i c a l b a s i s  f o r the  develop-  resistance.  Destructive and  Ultimately,  the  of the d i s e a s e f o r a v a r i a b l e  period  the  e f f e c t s of f a t t y a c i d s on both malignant  non-malignant c e l l s have been observed i n v i t r o  (24,71) and  a l s o i n v i v o i n the case of tumor c e l l s  Ethyl palmitate destruction actions  (68) .  concerns a " p e r i p h e r a l " a c t i o n of c o r t i c o s t e r o i d s :  c y t o l y t i c a c t i o n on The  and  has  has  of the  been reported  (72).  to cause e x t e n s i v e  spleen i n several species  (73).  The  of f r e e f a t t y a c i d s on m i t o c h o n d r i a , i n causing  99  s w e l l i n g are w e l l known Since previous  (74).  s t u d i e s i n t h i s l a b o r a t o r y had  t h a t the l y t i c e f f e c t of f r e e f a t t y a c i d s was  indicated  temperature-  dependent, which p a r a l l e l s the l y t i c a c t i o n s of s t e r o i d s , s t u d i e s were c a r 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 and  unresponsive t i s s u e s .  i n corticosteroid-responsive  Following  the p a t t e r n of metabolism of FFA quite d i f f e r e n t .  The  Cortisol  treatment,  i n those two  steroid-responsive  tissues  cells  was  retained  FFA w h i l e the s t e r o i d - r e s i s t a n t c e l l s m e t a b o l i z e d the a t a much h i g h e r r a t e than the unresponsive c e l l s , Figures  5 and  6).  Results  FFA  (see  i n d i c a t e d t h a t the o x i d a t i o n  of  f r e e f a t t y a c i d s might be g r e a t e r i n the r e s i s t a n t c e l l s . Electronmicrographic  observations  i s an accumulation o f FFA cells,  show t h a t i f there  in corticosteroid-sensitive  f o l l o w i n g c o r t i c o s t e r o i d treatment, t h i s accum-  u l a t i o n of FFA would have pronounced c y t o l y t i c e f f e c t s . The  d e g e n e r a t i v e change s e t i n motion by FFA  comitant n u c l e a r  and  cytoplasmic  causes con-  alterations.  The  pro-  g r e s s i v e l o s s o f normal dichotomy between heterochromatin and  euchromatin 4 hours a f t e r treatment i s s t r i k i n g l y  s i m i l a r t o t h a t induced by c o r t i c o s t e r o i d (24). has  termed t h i s type o f change n u c l e a r  due  to d i l a t a t i o n of the endoplasmic r e t i c u l u m and  c u l a t i o n i n FFA-treated  edema.  Bessis(82) Indeed, vesi-  P1798S c e l l s , the p o s s i b i l i t y  secondary a l t e r a t i o n i n i o n and water balance w i t h i n  of 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 m a n i f e s t a t i o n s of the r e t r o g r a d e m e t a b o l i c a l t e r a t i o n s which have been r e p o r t e d i n c o r t i c o s t e r o i d induced d e g e n e r a t i o n : a decrease i n DNA and i n DNA-dependent RNA  polymerase  an i n c r e a s e i n DNase I I a c t i v i t y the h i s t o n e complement  s y n t h e s i s (75,76)  activity  (77,78),  (79,80), and changes i n  (81).  I t i s s i g n i f i c a n t t h a t both c o r t i c o s t e r o i d  (12)  and  FFA a d m i n i s t r a t i o n can cause k a r y o r r h e x i s o f the thymocyte nucleus a t 4 hours.  Great care was  p r e t a t i o n of e l e c t r o n micrographs  taken i n the i n t e r -  t o exclude those  ex-  amples which might even remotely be construed as due t a n g e n t i a l s e c t i o n i n g of the n u c l e a r membrane.  The  nature of the membrane degeneration i s noteworthy, may  to focal  for i t  imply s p a t i a l h e t e r o g e n e i t y of f u n c t i o n . The major c y t o p l a s m i c event induced by FFA i n P1798S  c e l l s , p r o g r e s s i v e d i l a t a t i o n and e v e n t u a l v e s i c u l a t i o n , was  not noted a t 4hours i n thymocyte suspensions, nor were  these changes observed i n thymocytes t r e a t e d w i t h s t e r o i d s a f t e r 4 hours o f endoplasmic  (24).  The c h a r a c t e r i s t i c p a u c i t y  r e t i c u l u m i n c o r t i c a l lymphocytes  might account f o r t h i s . perinuclear cisternae  The  cortico-  (83)  s w e l l i n g and d i l a t a t i o n of the  (Figure 14) might be p r e d i c t e d on  the concept t h a t t h i s space and t h a t of the  endoplasmic  r e t i c u l u m are c o - e x i s t e n t i n mammalian c e l l s g e n e r a l l y (84). A l a c k of d i l a t a t i o n of the p e r i n u c l e a r c i s t e r n a e i n  101  thymocytes t r e a t e d w i t h FFA  i s shown i n F i g u r e s 18 and  These c e l l s are undergoing k a r y o r r h e x i s i n which of the  n u c l e a r membrane does not  event.  an  swelling  accompanying  Other thymocytes t r e a t e d w i t h e i t h e r FFA  steroids  not  swelling  or c o r t i c o -  undergoing k a r y o r r h e x i s e x h i b i t n u c l e a r  (24).  The  primary event i n FFA-induced c y t o l y s i s i s unknown.  However, the on  seem to be  19.  s i m i l a r e f f e c t s of FFA  thymocytes and  and  of  corticosteroids  t h e i r c y t o l y t i c e f f e c t s on  corticosteroid-  s e n s i t i v e lymphosarcoma c e l l s suggest a s t r o n g l i k e l i h o o d of a shared e t i o l o g y . i s t r a t i o n does not nor  the  I t i s noteworthy t h a t FFA  seem to a f f e c t e i t h e r the  of the working h y p o t h e s i s  proposed i n F i g u r e 20 to e x p l a i n c y t o l y t i c a c t i o n i s now previously,  the mechanism of  presented  an accumulation of FFA  (Figure  of these c e l l s .  25).  i n thymus and  t i s s u e s w i l l cause n u c l e a r damage and  on  mitochondria  c e l l membrane p r i m a r i l y .  A more complete v e r s i o n  of the  admin-  ultimately  the  As  mentioned  P179 8S cytolysis  C o n c e n t r a t i o n s of FA which cause c y t o l y s i s  c o r t i c o s t e r o i d - s e n s i t i v e t i s s u e s are without e f f e c t  the P179 8R.  The  c r i t i c a l point  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 a c t i o n of  i s r e l a t e d to i t s c a p a c i t y oxidative  capacity  i n whether or not  for oxidation  of f r e e f a t t y a c i d s  from t r i g l y c e r i d e s t o r e s  under the  corticosteroids  of FA.  t h a t are  influence  a  Increased released  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  RE-ESTERIFICATION  OXIDATION  TG  Corticosteroid Sensitive Lipase cAMP  NUCLEAR  FFA  Accumulation  I  UPTAKE  FIGURE 25:  Mechanism of the c y t o l y t i c a c t i o n  DAMAGE  CELL LYSIS of c o r t i c o s t e r o i d s ,  o  103  a c t i o n of the s t e r o i d w h i l e decreased o x i d a t i v e would render i t s e n s i t i v e t o s t e r o i d a c t i o n . what was  a c t u a l l y observed i n the  capacity  This i s  lymphoid c e l l s  studied:  i n s e n s i t i v e c e l l s f o l l o w i n g c o r t i c o s t e r o i d treatment the o x i d a t i o n of p a l m i t i c a c i d to C0 i n d i c a t i o n s of f l o o d i n g of the FFA (Table V ) .  was  decreased  the endogenous p o o l of FFA  (Table  IV);  p o o l were observed  In r e s i s t a n t c e l l s j u s t the o p p o s i t e  the o x i d a t i v e c a p a c i t y i n c r e a s e d and  2  occurred:  as the l o a d of FFA  decreased  Whether or not the decreased or i n c r e a s e d  increased,  (Table IV and r a t e of  V).  oxidation  i n s e n s i t i v e or r e s i s t a n t t i s s u e s , r e s p e c t i v e l y ,  represents  a d i r e c t a c t i o n of C o r t i s o l 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 systems of m i t o c h o n d r i a needs f u r t h e r i n v e s t i g a t i o n . Blecher  and White  (85) have r e p o r t e d  t h a t endogenous  r e s p i r a t i o n i n suspensions o f r a t thymic lymphocytes w i l l continue i n the absence of exogenous s u b s t r a t e the r e s p i r a t o r y q u o t i e n t  and  i s i n d i c a t i v e of l i p i d  that  oxidation.  T h i s o x i d a t i o n i s , however, i n h i b i t e d by adrenal  steroids.  This data i s c o n s i s t e n t w i t h the view that c o r t i c o s t e r o i d s might i n h i b i t 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 i n the m i t o c h o n d r i a of s e n s i t i v e c e l l s and of FFA.  l e a d to an  accumulation  Another important f a c t o r when c o n s i d e r i n g  i s the number of m i t o c h o n d r i a t h a t a c e l l c o n t a i n s .  oxidation If  r e s i s t a n t c e l l s c o n t a i n more m i t o c h o n d r i a than s e n s i t i v e ones, i t would be expected t h a t t h e i r c a p a c i t y to handle f r e e f a t t y a c i d s would be i n c r e a s e d  (for a f u r t h e r  consider-  104  a t i o n of t h i s p o i n t , see Appendix I I I ) .  The  activity  of  the a c y l c a r n i t i n e 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  s e n s i t i v e t i s s u e s s t u d i e d , c a r n i t i n e i s not a l i m i t i n g factor.  In f a c t , the presence of added c a r n i t i n e i n  s e n s i t i v e c e l l s r e s u l t s i n decreased r a t e of (Figure 8), suggesting  oxidation  t h a t exogenously added c a r n i t i n e  i s competing 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 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 t r a n s f e r of  acyl c a r n i t i n e moeities chondrion. increased  from the c y t o s a l i n t o the mito-  A d d i t i o n of c a r n i t i n e to P1798R r e s u l t s i n o x i d a t i o n , i n d i c a t i v e of a g r e a t e r  oxidative  c a p a c i t y than s e n s i t i v e c e l l s . Short  c h a i n f a t t y a c i d s are t r a n s f e r r e d i n t o the  mitochondrion by a d i f f e r e n t t r a n s f e r a s e than C-16 f a t t y acids  (93,94).  Scholefield  (92) have r e p o r t e d . t h a t  to have l i t t l e acids.  The  Spector  (56), Weinhouse  (91),  tumor c e l l s  c a p a c i t y to o x i d i z e s h o r t c h a i n  mouse lymphosarcomas P1798S and  and  have a g r e a t e r  other p e r i p h e r a l t i s s u e s  (57).  fatty not  L i k e thymus  c a p a c i t y f o r the o x i d a t i o n o f s h o r t  to be more e f f i c i e n t l y used i n l i p i d s y n t h e s i s production  appear  (57) , these  than long c h a i n f a t t y a c i d s which are g e n e r a l l y  C-18  and  P1798R do  f o l l o w the p a t t e r n observed by these workers. (Table IX)  or  cells chain  thought than energy  105  C o r t i c o s t e r o i d treatment of adrenalectomized d i a b e t i c r a t s r e s u l t e d i n an i n c r e a s e 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  in sensitive cells,  observed f o l l o w i n g c o r t i c o s t e r o i d treatment, may j u s t a consequence o f the r e l e a s e of FFA s t o r e s , but may  of FFA  be  from endogenous  a l s o be the r e s u l t of i n c r e a s e d  because of these e l e v a t e d  not  circulating levels.  uptake The  uptake  i s probably not a major f a c t o r s i n c e 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 added FFA. view, and  The  data of Table VI support t h i s p o i n t  i n f a c t g e n e r a l l y , a s l i g h t but not  decrease i n uptake was corticosteroid The  i n the absence o f of  significant  observed i n most cases f o l l o w i n g  treatment.  mechanism of r e s i s t a n c e of some malignant lympho-  cytes to the c y t o l y t i c a c t i o n s of c o r t i c o s t e r o i d s might be dependent on not only  increased  oxidative  From the mechanism o u t l i n e d i n F i g u r e p o s s i b i l i t i e s are e v i d e n t .  25,  capacity.  three  other  First, corticosteroid-resistant  lymphocytes might be able to r e - e s t e r i f y r e l e a s e d  fatty  a c i d s at a g r e a t e r r a t e than s e n s i t i v e t i s s u e s and  thus  prevent accumulation.  In the P1798R tumor, t h i s does not  appear to be a major pathway.  Other f a c t o r s i n v o l v e d i n  c o r t i c o s t e r o i d - r e s i s t a n c e might be a decreased r e l e a s e FFA  of  or a decrease i n the t r i g l y c e r i d e s t o r e s from which  the FFA P1798R.  are r e l e a s e d . Following  Both seem to be i m p l i c a t e d i n the  dexamethasone treatment, the endogenous  106  level this  of  decrease  a result of  FFA i n  of  is  the  cells  decreases  a failure  of  stores  are  lower i n  and V I I I ) .  are probably  capacity  in  population  it  oxidation  is  to  the  structure  acyl carnitine  population  for  (Table X I V ) .  *  1  of  FFA t o  of  carnitine,  one t h i r d  the  cell  by b o t h  liver  Sim and A . F .  activity.*  Burton,  1  however,  their  inhibit  and tumor t i s s u e  lytic  treatment  Cortisol  This  (see  carnitine  in  the v i a b l e  cell  competes  compounds w h i c h b e c a u s e o f  80% ( T a b l e X V I ) .  the  Using  Cortisol.  on t h e a c y l  treatment,  an  damage.  than d e o x y c a r n i t i n e ,  citral  FFA  the r e s i s t a n t  of  analogue  binding  effective  a c i d , w h i c h has  K.H.  of  More  d e c r e a s e d up t o  ding  VII  concomitant  After  oxidizable  (Tables  oxidative  cellular  b r a n c h e d s t r u c t u r e w o u l d be e x p e c t e d t o  is  fat  seen f o l l o w i n g  are branched-chain  (96,97).  of  levels  of  or  combination  levels  comparison), which  (95), a decrease is  the  to render actions  Appendix  transferase  this,  or t r a n s f e r  possible  a structural  with  a  C o r t i s o l would p r e v e n t  deoxycarnitine, for  of  FFA and t h e r e s u l t i n g  sensitive  II  or  l o w e r and t h e g r e a t e r  the presence of  By i n h i b i t i n g  IV)  t h e P1798R t h a n t h e P1798S  releases  mitochondria,  (Table  The endogenous  As a c o n s e q u e n c e  accumulation of  Whether  t h e FFA t o be r e l e a s e d  increased oxidation  b o t h c a n n o t be d i s c e r n e d .  (Table V ) .  3 oxidation -  cell  population  aldehyde to the  is  readily  correspon-  More e f f e c t i v e  unpublished observations,  than  1971.  107  c i t r a l a l o n e , are emulsions of o l e i c a c i d and branched c h a i n compounds l i k e c i t r a l .  These emulsions when admin-  i s t e r e d i p t o animals b e a r i n g the a s c i t e s form of the L5178Y,  of  L1210,  and P 1 7 9 8 R ,  w i l l cause 9 9 . 9 % d e s t r u c t i o n  the c e l l s w i t h i n one h o u r . *  1  The t e s t i n g o f 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 t h a t o f F i g u r e 2 6 i s suggested f o r chemotherapeutic treatment o f lymphocytic leukemias.  These compounds would  be expected t o have a l l the p r o p e r t i e s o f the d e s c r i b e d emulsions.  They c o u l d enter $ - o x i d a t i o n , but once the  branch p o i n t i s reached, 6 - o x i d a t i o n would be i n h i b i t e d . If  the remaining C - 1 0 o r C - 1 2 fragments were r e l e a s e d ,  these would be l y t i c . Whether o r not cAMP i s the modulator t h a t a c t i v a t e s the 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 i m p l i c a t e d i n t h i s scheme i s a matter t h a t awaits f u r t h e r i n v e s t i g a t i o n .  However,  some s p e c u l a t i o n can be advanced i n t h i s area i f the p a r a l l e l t h a t e x i s t s between a d i p o c y t e s and lymphocytes holds 32,35,36,90).  The e f f e c t s of c o r t i c o s t e r o i d s i n i n h i b i t i n g  uptake o f g l u c o s e by lymphoid c e l l s adipocytes  (19,22,  (19,90)  (22,32,35,36)  and  and e f f e c t i n g the r e l e a s e of FFA and  g l y c e r o l from a d i p o c y t e s i s w e l l documented  ( 3 2 ) . The  r e l e a s e of FFA under these c o n d i t i o n s i s dependent upon the  *  1  1971.  R.W. T u r n e l l and A.F. Burton, o b s e r v a t i o n s not r e p o r t e d ,  I  CH CH3^  CH ^ 3  C H  C H  2  2-CH^ ^CHf H  2^CH ^ 2  C H  C H  2^CH ^  2^CH^  2  C H  C O O H y  ^CH ^  O O H  2  CH I R FIGURE 26: S t r u c t u r e o f branched c h a i n f a t t y a c i d s w i t h suggested chemotherapeut value, a) f a t t y a c i d w i t h the branch p o i n t o c c u r r i n g on the odd numbered carbon, b) f a t t y a c i d w i t h the branch p o i n t on the even numbered carbon. The v a l u e s o f X and Y can be v a r i e d t o produce a C-16 or C-18 f a t t y a c i d w i t h the branch p o i n t at v a r y i n g d i s t a n c e s from the t e r m i n a l -COOH group. R = H, CH . 3  109  a c t i v a t i o n o f 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 by cAMP. This increased synthesis  production  of cAMP i s dependent upon the  o f RNA and p r o t e i n  the p r o d u c t i o n  (98).  If synthesis  i s blocked  o f cAMP and r e l e a s e o f FFA by a d i p o c y t e s  under c o r t i c o s t e r o i d s t i m u l a t i o n are a l s o b l o c k e d .  The  e f f e c t s o f c o r t i c o s t e r o i d s on lymphocytes are a l s o dependent upon RNA and p r o t e i n s y n t h e s i s have r e p o r t e d  (43).  Yang and Vas (86)  t h a t 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 . and  Rosen  (87) have noted r e c e n t l y t h a t as P1798S and  P 1 7 98R tumors become l a r g e the l e v e l o f cAMP It s t i l l  increases.  remains t o be determined whether changes i n cAMP  are r e l a t e d t o r e g r e s s i o n glucocorticoid An  Masarocchia  o f lymphoid c e l l s  following  treatment.  important p o i n t noted i n t h i s i n v e s t i g a t i o n i s  t h a t the c y t o l y t i c a c t i v i t i e s o f c o r t i c o s t e r o i d s might be  separated from o t h e r a c t i o n s o f c o r t i c o s t e r o i d s which  are b e l i e v e d t o r e s u l t from a profound i n h i b i t o r y a c t i o n on The  lymphoid t i s s u e g e n e r a l l y ,  f o r example, immunosuppression.  i n h i b i t o r y e f f e c t s of c o r t i c o i d s on n u c l e i c a c i d ,  p r o t e i n , and g l u c o s e metabolism not  (Tables X,XVII-XIX) are  seen when c e l l s are t r e a t e d w i t h FFA which causes  cytolysis.  I t seems q u i t e p o s s i b l e t h a t the two can be  dissociated  (see F i g u r e  27), an important f a c t o r when the  chemotherapeutic use o f agents which promote c y t o l y s i s i s t o be  considered.  110  CORTISOL Corticoid-sensitive lymphocytes "cytoplasmic complex" nuclear complex i n c r e a s e d mRNA synthesis b u r s t s y n t h e s i s oJ proteins  TG cAMP FFA  decreased carbohydrate metabolism, i o n t r a n s p o r t phosphorylation, e t c .  repression of mRNA s y n t h e s i s  decreased protein metabolism  nuclear damage  cell lysis CYTOLYTIC EFFECTS  METABOLIC EFFECTS  IMMUNOSUPPRESSIVE EFFECTS  FIGURE 27: O u t l i n e o f the proposed r e l a t i o n s h i p o f events t h a t occur f o l l o w i n g g l u c o c o r t i c o i d treatment.  Ill  The o b s e r v a t i o n s acids  are  cytotoxic  f r o m two o t h e r  made i n  are  this  groups.  Sahler  chromatin i s  leukemia c e l l s .  The c y t o t o x i c  c a u s e d by f r e e  concentration 100% c e l l has  in  "cellular  tumor c e l l s  acids,  guinea p i g ,  rat,  TLC,  GLC,  fatty  acid.  probably  also  l y s e d the  rabbit,  lytic  (18:1).  causing c y t o l y s i s  previously  demonstrate  that  that  there  is  of  A  cause factor  of  Infrared  lytic  oleic  spectra,  established  "lytic  that  chain  activity  this  to  sheep,  was  a c i d which i s  of  (89).  S180,  an u n s a t u r a t e d l o n g  The e f f e c t s  a factor"  like  those  laboratory.  a similarity  in  the  and t h o s e by f r e e  this  cytolytic  thesis  events fatty  FFA a t w h i c h t h e s e e v e n t s  significant. the  erythrocytes  t h e o b s e r v a t i o n s made i n  low c o n c e n t r a t i o n especially  SR61, E h r l i c k ,  showed t h e  i n d u c e d by c o r t i c o s t e r o i d s  advanced,  is  this  acid.  A cytotoxic  were t e m p e r a t u r e - d e p e n d e n t  observed i n  In c o n c l u s i o n ,  oleic  a c i d s would  chromatography  factor"  t o L1210 mouse  a lymph node e x t r a c t  and mouse.  The r e s u l t s  acid  in  observed  was f o u n d t o be c y t o t o x i c  due t o t h e p r e s e n c e o f  monoenoic  is  fatty  fatty  chromatin-lipid  especially  free  factor"  and i o n e x c h a n g e  "cellular  of  free  observations  (88)  toxic  effect  s u c h as MM2, F r i e n d , It  The  highly  t h e L1210 l e u k e m i a .  lytic  and A H - 6 6 F .  in  and G l i c k  a l s o been i d e n t i f i e d r e c e n t l y  This  the  fatty  Of 3 yg/ml o f  lysis  that  s u p p o r t e d by r e c e n t  t h a t mouse l i v e r  is  thesis  acids. occurred  That d a t a strengthen the action of  concept  corticosteroids  on  112  normal and malignant lymphocytes i s mediated by The f a t t y a c i d s would be r e l e a s e d ceride.- s t o r e s lipase.  by the a c t i o n o f a  FFA.  from endogenous  trigly-  corticosteroid-sensitive  In s e n s i t i v e t i s s u e s an accumulation o f FFA  to n u c l e a r damage and u l t i m a t e l y  cell lysis.  leads  Resistance  of malignant lymphocytes to 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 might be p a r t i a l l y a t t r i b u t e d f a i l u r e o f the FFA t o be r e l e a s e d stores,  lower f a t s t o r e s  from endogenous f a t  i n the s t e r o i d - r e s i s t a n t  or t o the g r e a t e r c a p a c i t y FFA.  to the  of r e s i s t a n t c e l l s t o  tissues, oxidize  The f a c t t h a t they are r e s i s t a n t t o the c y t o l y t i c  e f f e c t of exogenously added f a t t y a c i d strengthens the concept t h a t o x i d a t i o n  i s the most important f a c t o r i n  r e n d e r i n g c e l l s r e s i s t a n t t o the a c t i o n o f c o r t i c o s t e r o i d s .  113  BIBLIOGRAPHY  1.  Dougherty, T.F., and White, A., S c i e n c e , 98_: 3 6 7 , ( 1 9 4 3 ) .  2.  Dougherty, T.F., and White, A., E n d o c r i n o l . ,  3.  Dougherty, T.F., and White, A., J . Lab. C l i n . Med.,32: 584, (1947).  4.  Makman, M.H., Dvorkin, B., and White, A., J . B i o l . Chem., 241: 1646, (1966).  5.  Bush, I.E., and Mahesh, V.B., Biochem. J . , 7_1: 7 0 5 , ( 1 9 5 9 ) .  6.  G a b o u r e l , J.D., and Aranow, A., J . Pharmacol. E x p t l . Therap., 1 3 6 : 2 1 3 , ( 1 9 6 6 ) .  7.  B e r l i n e r , D.L., and Ruhmann, A.G., E n d o c r i n o l . , 7_8: 37 3, (1966).  8.  Dougherty, T.F., and White, A., Am. J . A n a t . , 7 7 :  9.  Frank, F.A., and Dougherty, T.F., Proc. Soc. E x p t l . Med. , 8_2: 1 7 , (1953) .  3 5 : 1,  (1944).  8 1 , (1945). Biol.  10.  S c h n e e b e l i , G., and Dougherty, T.F., C i n e . Res., 2: 3 42, (1948).  11.  Cowan, W.K., (1964) .  12.  Dougherty, T.F., B e r l i n e r , M.L., S c h n e e b e l i , G.L., and B e r l i n e r , D.L., Ann. N.Y. Acad. S c i . 1 1 3 : 8 2 5 , ( 1 9 6 4 ) .  13.  H o f e r t , J . F . , and White, A., E n d o c r i n o l . , 77_: 5 7 4 , ( 1 9 6 5 ) .  14.  Burton, A.F. , E n d o c r i n o l . , 7_7: 3 2 5 , ( 1 9 6 5 ) .  15.  Munck, A., and K o r i t z , S.B., A c t a E n d o c r i n o l . , 35 (Suppl. 5 1 ) : 8 2 1 , (1960).  16.  Glenn, E.M., M i l l e r , W.L., and S c h l a g e l C.A., Rec. Prog. Horm. Res., 1 9 : 1 0 7 , ( 1 9 6 3 ) .  17.  Long, C.N.H., F r y , E.G., and B o n n y c a s t l e , M., E n d o c r i n o l . , 3_5 (Suppl. 5 1 ) : 8 1 9 , ( 1 9 6 0 ) .  18.  Munck, A., and K o r i t z , S.B., Biochim. Biophys. A c t a , 57: 3 1 0 , (1962).  and Sorenson, G.D.,  Lab. I n v e s t . , 13_: 353  Acta  114  19.  Munck, A., Biochim. Biophys. A c t a , 5_7: 3 1 8 , ( 1 9 6 2 ) .  20.  Munck, A., and Brinck-Johnsen, T., J . B i o l . Chem., 2 4 3 : 5556,  (1968).  21.  K a t t w i n k e l , J . , and Munck, A., E n d o c r i n o l . , 7 9 : 3 8 7 , ( 1 9 6 6 ) .  22.  Munck, A., E n d o c r i n o l . , 7 7 : 3 5 6 , ( 1 9 6 5 ) .  23.  Melynykovych, G., and Bishop, C.F., Biochem. Biophys. Res. Comm. , 3_2: 2 3 3 , ( 1 9 6 8 ) .  24.  Burton, A.F., S t o r r , J.M., and Dunn, W.L. , Can. J . Biochem., 45:  25.  289, (1967).  Mosher, K.M., Young, D.A., and Munck, A., J . B i o l . Chem., 246:  654, (1971).  26.  Wira, C , and Munck, A., J . B i o l . Chem., 245: 3436,  (1970).  27.  Jensen, E.V., Suzuki, T., Kawashima, T., Stumpf, W.E., J u n g b l u t t , P.W., and DeSombre, E.R., Proc. Nat. Acad. S c i . 5_9: 632, (1968) .  28.  K i r k p a t r i c k , A.F., M i l h o l l a n d , R.J., and Rosen, F., Nature, ( i n p r e s s ) .  29.  Munck, A., J . B i o l . Chem., 243: 1039, (1968).  30.  Wira, C.R., R o c h e f o r t , H., and B a u l i e u , E.E., A c t a E n d o c r i n o l . , 67: (Suppl. 153): 223, (1971).  .31.  Schaumberg, B.P., and BjzSjensen, E. , Biochim. Biophys. A c t a , 170: 172, (1968).  32.  Mahler, R.F., and S t a f f o r d , W.L., I n : J.K. Grant (ed.), The C o n t r o l of L i p i d Metabolism, B i o c h e m i c a l S o c i e t y Symposium No. 24, p. 155, New York: Academic P r e s s , 1963.  33.  F a i n , J.N., Scow, R.O., and Chernick, S.S., J . B i o l . Chem., 238: 54, (1963).  34.  O v e r e l l , B.G., Condon, S.E., and Petrow, V., J . Pharm. Pharmacol., 12/. 150, (1960).  35.  B a r t l e t t , D., M o r i t a , Y., and Munck, A., Nature, 196: 897, (1962).  36.  M o r i t a , Y., and Munck, A., Biochjm. Biophys. A c t a , 93: 150, (1964).  37.  Hedeskov, C.J., and Esmann, V., Biochim, Biophys. A c t a , 148: 372, (1967).  115  38.  Rosen, J.M., F i n a , J . J . , M i l h o l l a n d , R.F., and Rosen F., J . B i o l . Chem., 2 4 5 : 2 0 7 4 , ( 1 9 7 0 ) .  39.  Rosen, J.M., M i l h o l l a n d , R.F., and Rosen, F., Biochim. Biophys. A c t a , 2 1 9 : 4 4 7 , ( 1 9 7 0 )  40.  Dougherty,  41> •  Munck, A., P e r s p e c t . B i o l . Med., 1 4 : 2 6 5 , ( 1 9 7 1 ) .  42.  Kidson, C., Nature, 2 1 3 : 7 7 9 , ( 1 9 6 7 ) .  43.  Makman,M.H., Dvorkin, B., and White, A., Proc. Nat. Acad. S c i . , 68: 1269, (1971).  44.  L u f t , J.H. , Biophys. Biochem. C y t o l . , 9_: 409, (1961).  45.  S p u r l o c k , B.O., K a t t i n e , V.E., and Freeman, J.H., J . C e l l B i o l . , 17: 203, (1963).  46.  Trump, B., Smuckler, E., and B e n d i t t , E., J . U l t r a s t r . R e s . , 5 : 4 3 , (1961).  47.  Bruno, G.A., and C h r i s t i a n , J.E., A n a l . Chem. 33: 650, (1961).  48.  F o l c h , J.H., Lees, M., and S l o a n e - S t a n l e y , G.H., J . B i o l . Chem., 226: 497, (1957).  49.  K e l l e y , T.F., A n a l . Chem., 37: 1079, (1965).  50.  Ames, B.N., I n : S.P. Colowick and N.O Kaplan (eds.), Methods i n Enzymology, Volume 8, p.115, New York: Academic P r e s s , 1966.  51.  S t e r n , I . , and Shapero, B., J . C l i n . Path. 6_: 158, (1953).  52.  M i l k o v i c , S., and Bates, R.W. , E n d o c r i n o l . ,7_4: 617, (1964) .  53.  L e v i n e , S., and Treiman, D.M., E n d o c r i n o l . , 7_5: 142, (1964).  54.  J o h a l , K.S., S t u d i e s on the Uptake and U t i l i z a t i o n of P a l m i t i c A c i d and O c t a n o i c A c i d i n Rat Kidney Carcinoma Astrocytoma, and Other T i s s u e s . , B.Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, 1971.  55.  Eaton, P., and S t e i n b e r g , D. , J . L i p i d (1961).  T.F., P h y s i o l . Revs., 3 2 : 3 7 9 , ( 1 9 5 2 ) .  Res., 2_: 376  116  56.  S p e c t o r , A., Cancer R e s 2 7 :  1587, (1967).  57.  S c h e i g , R., I n : J.R. Senior (ed.), Medium Chain T r i g l y c e r i d e s , p.39, P h i l a d e l p h i a : U n i v e r s i t y of P e n n s y l v a n i a P r e s s , 1968.  58.  Abramason, D., and B l e c h e r , M., Biochim. Biophys. A c t a , _98: 117, (1965) .  59.  Konings, A.W.T., and Loomeijer, F . J . , Biochim. Biophys. A c t a , 202: 216, (1970).  60.  K i p n i s , D.M., and C o r i , C.F., J . B i o l . Chem., 235: 3070, (1960).  61.  Rosen, J.M., F i n a , J . J . , M i l h o l l a n d , R.J. and Rosen, F., J . B i o l . Chem., 245: 2074, (1970).  62.  A o k i , T., Stuck, B., Hammerling, U., Old, L . J . , and de Harven, E. , Cancer Res., 3_0: 244, (1970).  63.  Makman, M.H., Nakagawa, S., and White, A., Rec. Prog. Hormone Res., 2_3: 195, (1967).  64.  Rosen, F., M i l h o l l a n d , R.J., and N i c h o l , C.A., A b s t r a c t s of the V l l t h I n t e r n a t i o n a l Congress of B i o c h e m i s t r y , p.867, Tokyo, 1967.  65.  Krebs, H.A., and E g g l e s t o n , L.V., Biochem.,J., 442, (1960).  66.  P e t r a n y i , J r . , G., Benczur, M., and A l f o l d y , P., Immunol., 21: 151, (1971).  67.  Schrek, R. , Proc. Am. Assoc. Cancer Res., 12_: 18, (1971).  68.  Rosen, F., and N i c h o l , C.A., Vitamins and Hormones, 21: 135, (1965).  69.  Dougherty, T.F., I n : J.W. Rebuck (ed.), The Lymphocyte and Lymphatic T i s s u e , p. 112, New York: Paul B. Hoeber Inc., 1960.  70.  Damashek, W., Ann. N.Y. Acad. S c i . , 82: 924, (1959).  71.  Bennett, L.R., and Cannon, F.E., J . Nat. Cancer 19: 999, (1957).  72.  Townsend, G.F., Morgan J.F., and H a z l e t t , B., Nature, 183: 1270, (1959).  73.  P r o s n i t z , L., Kawasaki,S., Cohen, G.S., Dineen, J . L . ,  34:  Inst.,  117  P e r i l l e , P.E., and F i n c h , S.C., Soc., 6:487, (1969).  J. Reticuloendothelial  74.  Lehninger, A.L., I n : The Mitochondrion,p.185, York: W.A. Benjamin Inc., 1964.  75.  Stevens, W., C o l e s s i d e s , C., and Dougherty, E n d o c r i n o l . , 76:1100, (1965).  T.F. ,  76.  Stevens, W., C o l e s s i d e s , C., and Dougherty, E n d o c r i n o l . , 7_8: 600, (1966).  T.F.,  77.  Nakagawa, S., and White, A., Proc. Nat. Acad. S c i . , 5_5:900, (1966).  78.  Nakagawa, S.,  79.  Raina, P.N., and Rosen, F., Arch. I n t . Pharmacodyn., 182:14, (1969).  80.  W e i r n i k , P l H . , and MacLeod, R.H., 49:138, (1965).  81.  W h i t f i e l d , J.F., P e r r i s , A.D., and Youdale, E x p t l . C e l l Res., 52:349, (1968).  82.  B e s s i s , M., In: A.V.S. de Rueck and J . K n i g h t . ( e d s . ) , p.287, London: J&A C h u r c h i l l L t d . , 1964.  83.  Abe,  84.  Watson, M.L.,  85.  B l e c h e r , M., and White, A., 15:391, (1959).  86.  Yang, T . J . , and Vas, S.I., Proc. Can. Fed. B i o l . 13:132, (1970).  87.  Masaracchia, R.A., and Rosen, F., I n : P h y s i o l o g y and Pharmacology o f C y c l i c AMP an I n t e r n a t i o n a l Conference, p. 114, Book o f A b s t r a c t s : M i l a n , I t a l y , 1971.  88.  S a h l e r , C., and G l i c k , J.L., Proc. Am. Res., 12:11, (1971).  89.  Okudaira, H., Kataoka, T., Okada, H., F u r u s e - I r i e , R . , Kawachi, S., Nojima, S., and N i s h i o k a , K., J . Biochem., 68:379, (1970).  90.  Munck, A., Biochim. Biophys. A c t a , 57:310,  K.,  New  and White, A., E n d o c r i n o l . , 83^:861, (1967).  Acta Endocrinol., T.,  and I t o , T . , Z. Z e l l f o r s c h , 110:321, J . Biophys.Biochem.  (1970).  C y t o l . , 1:257,(1955).  Recent Progr. Hormone Res.,  Assoc.  Soc.,  Cancer  (1961).  118.  91.  W e i n h o u s e , S . , A l l e n , A . , and M i l l i n g t o n , Cancer R e s . , 13:367, (1953).  R.H.,  92.  Scholefield, Cancer R e s . ,  93.  Fritz, Chem.,  I.B., Schultz, S.K., 238: 2509, (1963).  94.  Norum,  K.R. , B i o c h i m .  95.  F r i t z , I . B . , and S c h u l t z , 2188, (1965).  96.  S t e i n b e r g , D., Herndon, J r . , J . H . , U h l e n d o r f , B.W., Mize, C . E . , A v i g a n , M i l n e , G.W.A., Science 156:1740, (1967).  97.  S p e c t o r , A . I n : R. P a o l e t t i ( e d . ) , P r o g r e s s i n B i o c h e m i c a l P h a r m a c o l o g y , Volume V I , p . 13 0, B a s e l : S. K a r g e r , 1 9 7 1 .  98.  Fain, J.N., (1971).  99.  McCraw, E . F . , W a g l e , S . R . , L i f e S c i . 8:505, (1969).  P . G . , S a t o , S . , and W e i n h o u s e , 20_:661, ( 1 9 6 0 ) .  Dodd, A . ,  and S r e r e ,  Biophys.  Acta,  S.K.,  and N o v a k ,  J.  P.A.,  Patterson,  J.  89;: 9 5 ,  Biol.  L.,  S.,  C ,  (1964).  Chem.,  Metabol.,  Biol.  240:  20:  109,  and A s h m o r e , J . ,  119.  APPENDIX I  FISCHER'S MEDIUM FOR LEUKEMIC CELLS IN MICE  (IX)  Component  Mg/1  NaCl MgCl .6H 0 NaH' POit'.H 0 Na HP0 .7H 0 KC1 CaCl .2H 0 Glucose L-Arginine L - H i s t i d i n e HCI L-Isoleucine L-Leucine L - L y s i n e HCI L-Methionine L-Phenylalanine L-Threonine L-Valine L-Tryptophan L-Glutamine L-Serine L-Cystine L-Tyrosine L-Asparagine Riboflavin F o l i c Acid Thiamine HCI Nicotinamide Ca pantothenate P y r i d o x a l HCI C h o l i n e HCI i-Inositol Biotin Phenol Red NaHC0  8000.0 100.0 69.0 113.0 400.0 91.0 1000.0 15.0 60.0 75.0 30.0 50.0 100.0 60.0 3 0.0 70.0 10.0 204.0 15.0 20.0 60.0 10.0 0.50 10.0 1.0 0.50 0.50 ID. 50 1.50 1.50 0.010 5.0 1125.0  2  2  2  2  2  lt  2  2  2  3  120.  APPENDIX I I  STRUCTURES OF SOME DRUGS AND CHEMICALS USED  (CH ) I N + I CH 3  3  2  CH-R I CH I COOH 2  US O H  CHO  CARNITINE  CITRAL c i s = geranial trans = neral  RS H  ( c i t r a l a) ( c i t r a l b)  DEOXYCARNITINE  CH^H  --OH  CORTISOL  DEXAMETHASONE  APPENDIX I I I  I n c r e a s e d M i t o c h o n d r i a l Content of 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 t h e S t e r o i d Sens i t i v e S t r a i n .  I t was t h e o p i n i o n o f M r . viewing sections microscope  that  cross-sectional This  type  of  of  t h e number o f  this of  Schrek  lymphosarcoma c e l l  the  (26.5%).  thesis  this lysis  that  context,  hence r e s i s t a n c e  was t h a t  of  these  as a c e l l its  its  normal  non-malignant  suffering  had e l o n g a t e d  of  Schrek would  becomes more m a l i g n a n t  resistance  to  mitochondrial  using  5.9 m i t o c h o n d r i a  cells  to c o r t i c o i d or  provided  What he o b s e r v e d ,  l e u k e m i a had more m i t o c h o n d r i a  These r e s u l t s  that  increases)  (67).  those from p a t i e n t s  a greater percentage chondria  difficult  means.  had an a v e r a g e o f  cross-section while  • very  electron  tumor.  o b s e r v a t i o n has b e e n r e c e n t l y  electronmicroscope,  human l y m p h o c y t e s  is  when  per  i n the r e s i s t a n t  however,  t o p r o v e by e l e c t r o m i c r o s c o p i c  the  that  mitochondria v i s i b l e  f i e l d was g r e a t e r  by a p u b l i c a t i o n  and m y s e l f  t h e P1798R and P1798S u n d e r t h e  observation,  Support of  Clarke  per  from and  that  mitosupport (ie  in  corticosteroid-induced  content  fatty  increases  acid also  and  increases.  

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