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Purine nucleotide biosynthesis in ehrlich ascites carcinoma cells in vitro effects of actinomycin d and… Nair, M.S. Parameswaran 1968

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PURINE NUCLEOTIDE BIOSYNTHESIS IN EHRLICH ASCITES CARCINOMA CELLS in EFFECTS OF ACTINOMYCIN  vitro  D AND GLUCOSE  by M.S. PARAMESWARAN NAIR B.Sc., U n i v e r s i t y o f Madras, 1951 M . S c , A l i g a r h M u s l i m U n i v e r s i t y , 1953  THESIS SUBMITTED IN PARTIAL FULFILMENT OF . THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n t h e Department of Biochemistry  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d f o r t h e degree o f DOCTOR OF PHILOSOPHY  THE UNIVERSITY OF BRITISH COLUMBIA November, 196 8  In p r e s e n t i n g an  this  thesis  advanced degree at  the  Library  I further for  shall  the  his  of  this  agree that  written  of  be  for financial  -  g r a n t e d by  gain  Biochemistry  12  -  1968.  for  for extensive the  It i s understood  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, Canada  23  British  available  permission.  Department of  Date  University  permission  representatives. thesjs  f u l f i l m e n t of  make i t f r e e l y  s c h o l a r l y p u r p o s e s may  by  in p a r t i a l  Columbia  shall  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and copying of  that  not  the  that  Study.  this  thesis  Department  c o p y i n g or  for  or  publication  allowed without  my  . .  • 1 -• •  ABSTRACT The b i o s y n t h e s i s o f p u r i n e n u c l e o t i d e s i n E h r l i c h a s c i t e s c a r c i n o m a c e l l s was i n v e s t i g a t e d under d i f f e r e n t c o n d i t i o n s . I n i n i t i a l studies the e f f e c t of actinomycin  D was examined.  The  n u c l e o t i d e s from t h e a c i d s o l u b l e f r a c t i o n o f E h r l i c h a s c i t e s carcinoma c e l l s incubated w i t h actinomycin  D and ^ C - f o r m a t e were  adsorbed on c h a r c o a l and e l u t e d w i t h a m i x t u r e ethanol.  The e l u t e d n u c l e o t i d e s were s e p a r a t e d  o f p y r i d i n e and by two d i m e n s i o n a l  paper chromatography u s i n g i s o b u t y r i c acid-ammonia-water i n t h e f i r s t d i r e c t i o n - a n d aqueaus ammonium a c e t a t e - e t h a n o l n u c l e o t i d e s were e s t i m a t e d  i n t h e second. .The  by u l t r a v i o l e t s p e c t r o p h o t o m e t r y and  the r a d i o a c t i v i t y i n c o r p o r a t e d was d e t e r m i n e d by l i q u i d t i o n counting.  As t h e r e s u l t s o f t h e s e s t u d i e s u s i n g  scintilla-  small  amounts o f c e l l s were i n c o n c l u s i v e due t o v a r i a t i o n s from e x p e r i m e n t t o e x p e r i m e n t , s i m i l a r s t u d i e s were c a r r i e d o u t u s i n g l a r g e r amounts o f c e l l s u s p e n s i o n s . from t h e s e e x p e r i m e n t s were s e p a r a t e d graphy on D E A E - c e l l u l o s e  The a c i d s o l u b l e n u c l e o t i d e s by ion-exchange chromato-  and f i n a l l y by paper chromatography.  I t was o b s e r v e d t h a t t h e r e was an a c c u m u l a t i o n o f a c i d s o l u b l e n u c l e o t i d e s i n E h r l i c h a s c i t e s carcinoma c e l l s incubated 1k  C - f o r m a t e and a c t i n o m y c i n  D.  with  The s p e c i f i c a c t i v i t i e s ' o f  these  n u c l e o t i d e s were n o t s i g n i f i c a n t l y d i f f e r e n t from t h o s e o f t h e c o n t r o l experiments.  The i n c o r p o r a t i o n o f  n u c l e i c a c i d s was i n h i b i t e d by a c t i n o m y c i n From t h e s e o b s e r v a t i o n s  111  C-formate i n t o  D i n these c e l l s .  i t was c o n c l u d e d t h a t a c t i n o m y c i n  D did  not i n h i b i t t h e b i o s y n t h e s i s o f p u r i n e n u c l e o t i d e s i n E h r l i c h  11  a s c i t e s c a r c i n o m a c e l l s in vitro. e f f e c t o f actinomycin  I t i s suggested t h a t the  D on n u c l e i c a c i d m e t a b o l i s m i s t h e r e f o r e ,  a t a s t a g e beyond t h e s y n t h e s i s o f n u c l e o t i d e s . F u r t h e r s t u d i e s on t h e e f f e c t o f a c t i n o m y c i n  D revealed  that the a n t i b i o t i c i n h i b i t e d the r e s p i r a t i o n o f E h r l i c h a s c i t e s carcinoma.cells  in vitro  slightly.  The g l y c o l y s i s i n E h r l i c h  a s c i t e s carcinoma c e l l s was unaffected.-by  a c t i n o m y c i n D.  I n e x p e r i m e n t s where t h e e f f e c t o'f g l u c o s e poration of radioactive precursors  on t h e i n c o r -  i n t o n u c l e o t i d e s and n u c l e i c  a c i d s o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s was examined, c o n t r a r y t o t h e r e s u l t s o f many, a d e c r e a s e i n i n c o r p o r a t i o n was o b s e r v e d . T h i s d e c r e a s e i n i n c o r p o r a t i o n was independent o f t h e p r e s e n c e ++ of Ca  ions  in  t h e i n c u b a t i o n medium, t h e b u f f e r and o f t h e r a d i o -  a c t i v e p r e c u r s o r s used i n t h e s e i n c u b a t i o n s .  I t was observed  t h a t t h e r e were two f a c t o r s c o n t r o l l i n g t h e i n c o r p o r a t i o n o f radioactive precursors  in. vitro  i n t o t h e n u c l e o t i d e s and n u c l e i c  acids o f E h r l i c h a s c i t e s carcinoma c e l l s i n presence o f glucose; the c o n c e n t r a t i o n o f g l u c o s e of c e l l s i n s u s p e n s i o n .  I n d i l u t e c e l l s u s p e n s i o n s (packed c e l l  volume l e s s than 5%) g l u c o s e creased  i n t h e medium and t h e c o n c e n t r a t i o n  a t a c o n c e n t r a t i o n o f 5.5 mM de-  whereas i n dense c e l l s u s p e n s i o n s  above 8%) t h e same c o n c e n t r a t i o n o f g l u c o s e incorporation of l a b e l l e d precursors n u c l e o t i d e s and n u c l e i c , a c i d s . glucose  (packed c e l l volume increased the  i n t o both a c i d s o l u b l e  2 - D e o x y g l u c o s e , an analogue o f  a t a s i m i l a r c o n c e n t r a t i o n decreased the i n c o r p o r a t i o n  1 "4  of  C-formate i n d i l u t e as w e l l as i n dense c e l l  suspension.  D i n i t r o p h e n o l , an u n c o u p l e r o f o x i d a t i v e phosphory-  l a t i o n , a l s o d e c r e a s e d t h e i n c o r p o r a t i o n o f '*C-formate i n t h e s e 1  - c e l l s w h i c h was more marked i n d i l u t e c e l l s u s p e n s i o n s i n p r e s e n c e of g l u c o s e .  I t was c o n c l u d e d from t h e s e o b s e r v a t i o n s  main f a c t o r c o n t r o l l i n g t h e i n c o r p o r a t i o n in vitro precursors  into nucleotides  that the  of radioactive  and n u c l e i c a c i d s o f E h r l i c h a s c i t e s  c a r c i n o m a c e l l s was t h e t r a n s i e n t d e p l e t i o n and r e g e n e r a t i o n o f ATP i n t h e s e c e l l s i n p r e s e n c e o f g l u c o s e . G l u c o s e i n c r e a s e d t h e i n c o r p o r a t i o n o f '*C-formate i n t o 1  the s e r i n e o f t h e a c i d s o l u b l e f r a c t i o n o f E h r l i c h a s c i t e s c a r c i n oma c e l l s .  A marked i n c r e a s e i n t h e i n c o r p o r a t i o n o f  1If  i n t o s e r i n e was o b s e r v e d i n p r e s e n c e o f 2-deoxyglucose. e f f e c t s were i n d e p e n d e n t o f t h e c o n c e n t r a t i o n pension.  C-formate These  o f c e l l s i n sus-  I t was suggested t h a t when t h e a v a i l a b i l i t y o f t h e  common p r e c u r s o r , N - N 5  1 0  methylenetetrahydrofolic  a c i d was i n -  c r e a s e d p a r t i c u l a r l y due t o a d e c r e a s e i n i n c o r p o r a t i o n i n t o n u c l e i c a c i d s , more o f t h e l a b e l l e d p r e c u r s o r into serine.  may be i n c o r p o r a t e d  Iv TABLE OF CONTENTS Page INTRODUCTION  1  Actinomycins"arid Their B i o l o g i c a l A c t i v i t y  2  Biosynthesis of Purine Nucleotides  12  Biosynthesis of Nucleic Acids  15  . Present  Investigation  18  MATERIALS AND METHODS.  .• 25  1.  E h r l i c h A s c i t e s . Carcinoma C e l l s  25  2.  Determination  26  3.  Radioactive Materials..  4.  Actinomycin  5.  Determination  of Glucose  27  6.  Determination  o f Phosphate  27  7.  Determination  of Nucleic Acids....  8.  Radioautog.raphy  28;  9.  L i q u i d S c i n t i l l a t i o n Counting  28  10.  o f Packed C e l l Volume  D. ...... ,  General Procedure f o r Incubations E h r l i c h A s c i t e s Carcinoma C e l l s .  26 . .  26  ...  in vitro  27  of 29  11.  C e l l R e s p i r a t i o n Studies  30  12.  I s o l a t i o n o f N u c l e i c A c i d Components from E h r l i c h A s c i t e s Carcinoma C e l l s -  30  a) I s o l a t i o n o f a c i d s o l u b l e n u c l e o t i d e s  30  b) N u c l e i c a c i d s  31  13.  I s o l a t i o n and D e t e r m i n a t i o n o f P u r i n e and P y r i m i d i n e Bases f rom N u c l e o t i d e s and N u c l e i c A c i d s  32  14.  E l u t i o n and E s t i m a t i o n o f N u c l e i c A c i d from Chromatograms.....  33  Components  V  Page 15..  Separation  of A c i d S o l u b l e N u c l e o t i d e s  a) I s o l a t i o n o f n u c l e o t i d e s by adsorption  34  charcoal  34  b) Paper chromatography o f n u c l e o t i d e s  35  c) Ion-exchange chromatography o f n u c l e o t i d e s . . .  36  SECTION I  39  EFFECT OF ACTINOMYCIN D ON THE BIOSYNTHESIS OF PURINE NUCLEOTIDES EXPERIMENTAL. . 1.  2.  3.  39  Time Course o f I n c o r p o r a t i o n o f ^ C - F o r m a t e i n t o N u c l e i c A c i d Components o f E h r l i c h A s c i t e s Carcinoma Cells. E f f e c t o f A c t i n o m y c i n D on t h e I n c o r p o r a t i o n o f Formate i n t o the Bases o f N u c l e o t i d e s and N u c l e i c A c i d s o f E h r l i c h A s c i t e s Carcinoma C e l l s Chromatography of A c i d S o l u b l e N u c l e o t i d e s Chromatography - Development o f a Method  lk  39  C-  by Paper  41 48  a) Paper chromatography o f r i b o n u c l e o t i d e s  48  b) Paper chromatography o f a c i d s o l u b l e n u c l e o t i d e s from E h r l i c h a s c i t e s c a r c i n o m a c e l l s . . .  52  c) Removal o f s u b s t a n c e s i n t e r f e r i n g i n paper chromatography o f a c i d s o l u b l e n u c l e o t i d e s . . .  53  i ) D e s a l t i n g n u c l e o t i d e s u s i n g DEAE-  iii)  cellulose  53  D e s a l t i n g by g e l f i l t r a t i o n  54  i i i ) A d s o r p t i o n o f n u c l e o t i d e s on c h a r c o a l . . d) C h a r a c t e t i o n o f s u b s t a n c e s e l u t e d from chromatograms 4.  E f f e c t o f A c t i n o m y c i n D on t h e I n c o r p o r a t i o n o f *CFormate i n t o t h e A c i d S o l u b l e N u c l e o t i d e s o f E h r l i c h A s c i t e s Carcinoma C e l l s . . . .  55 59  ll  63  vi Page 5.  E f f e c t o f A c t i n o m y c i n D on t h e I n c o r p o r a t i o n o f C - F o r m a t e in vitro by t h e I n t e s t i n a l Mucosa o f t h e Rat 14  6.  E f f e c t o f Varying Concentrations the I n c o r p o r a t i o n o f ^ C - F o r m a t e A s c i t e s Carcinoma C e l l s  7.  Separation of Acid Soluble Nucleotides of E h r l i c h A s c i t e s Carcinoma C e l l s by Chromatography on DEAEC e l l u l o s e Column.  8.  67  o f A c t i n o m y c i n D on. i n t o RNA o f E h r l i c h 70  70  E f f e c t o f A c t i n o m y c i n D on t h e B i o s y n t h e s i s o f P u r i n e N u c l e o t i d e s i n E h r l i c h A s c i t e s Carcinoma C e l l s in vitro  9. 10.  74  E f f e c t o f A c t i n o m y c i n D on t h e R e s p i r a t i o n o f E h r l i c h A s c i t e s Carcinoma C e l l s . . . . . 83 E f f e c t o f A c t i n o m y c i n D on t h e G l y c o l y s i s A s c i t e s Carcinoma C e l l s  DISCUSSION  by E h r l i c h  .  87 91  SECTION I I  101  EFFECT OF GLUCOSE ON THE INCORPORATION OF ^C-FORMATE INTO NUCLEIC ACID COMPONENTS OF EHRLICH ASCITES CARCINOMA CELLS in  vitro.  EXPERIMENTAL..... 1.  2.  .101  Time Course o f I n c o r p o r a t i o n o f ^ C - F o r m a t e . i n t o V a r i o u s N u c l e i c A c i d Components i n P r e s e n c e o f Glucose..  102  Effect of C a on t h e I n c o r p o r a t i o n o f ^C-Formate i n t o N u c l e i c A c i d Components o f E h r l i c h A s c i t e s Carcinoma C e l l s  109  1  + +  1  3.  E f f e c t o f B u f f e r on t h e I n c o r p o r a t i o n o f ^C-Formate i n t o E h r l i c h A s c i t e s Carcinoma C e l l s i n P r e s e n c e o f Glucose. 114  4.  E f f e c t o f G l u c o s e on t h e I n c o r p o r a t i o n o f 2- *CG l y c i n e by E h r l i c h A s c i t e s Carcinoma C e l l s in vitro.  1  1!  123  vii Page .  5.  The I n c o r p o r a t i o n o f ^C-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s Suspended i n D i f f e r e n t Buffers  127  6.  I n c r e a s e d I n c o r p o r a t i o n o f C-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s i n P r e s e n c e o f G l u c o s e  131  7.  E f f e c t o f V a r y i n g C o n c e n t r a t i o n o f G l u c o s e on t h e I n c o r p o r a t i o n o f C - F o r m a t e by E h r l i c h A s c i t e s Carcinoma C e l l s in vitro  135  8.  I n c o r p o r a t i o n o f C-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s as a F u n c t i o n o f C e l l C o n c e n t r a t i o n .  137  9.  E f f e c t o f 2-Deoxyglucose on t h e I n c o r p o r a t i o n o f '*C-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s in  1  llf  14  ll+  1  vitro  10.  11.  ..  141  I n c o r p o r a t i o n o f C-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s Under V a r i o u s C o n d i t i o n s A f f e c t i n g ATP F o r m a t i o n .  146  E f f e c t o f G l u c o s e and 2-Deoxyglucose on t h e I n c o r p o r a t i o n o f C-Formate i n t o S e r i n e o f E h r l i c h A s c i t e s Carcinoma C e l l s  151  1If  llf  DISCUSSION SUMMARY BIBLIOGRAPHY.  157 ..... . / ..  :  171 176  viii TABLES Page I.  E f f e c t o f a c t i n o m y c i n D on t h e i n c o r p o r a t i o n o f C - f o r m a t e i n t o the bases o f n u c l e i c a c i d compone n t s o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in vitro 1 4  II. III. IV. V.  VI. VII.  VIII.  IX.  X.  XI. XII. XIII. XIV.  45  R„ v a l u e s o f r i b o n u c l e o t i d e s . F Recovery o f n u c l e o t i d e s adsorbed on c h a r c o a l by e l u t i o n w i t h t h r e e d i f f e r e n t s o l v e n t systems Phosphorus d e t e r m i n a t i o n o f n u c l e o t i d e s from chromatograms  50 58  obtained 62  E f f e c t o f a c t i n o m y c i n D on the i n c o r p o r a t i o n o f Cformate i n t o p u r i n e n u c l e o t i d e s o f i E h r l i c h a s c i t e s carcinoma c e l l s  65  E f f e c t o f a c t i n o m y c i n D on the i n c o r p o r a t i o n formate by the i n t e s t i n a l mucosa o f the r a t  69  XIf  of  1If  C-  E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f a c t i n o m y c i n D on the i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o RNA o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in vitro  71  C o n c e n t r a t i o n o f p u r i n e n u c l e o t i d e s i s o l a t e d from ' E h r l i c h a s c i t e s carcinoma c e l l s incubated w i t h a c t i n o - . mycin D in vitro 79 E f f e c t o f a c t i n o m y c i n D on the i n c o r p o r a t i o n o f Cformate i n t o p u r i n e n u c l e o t i d e s o f E h r l i c h a s c i t e s carcinoma c e l l s . .  80  E f f e c t o f a c t i n o m y c i n D on the c o n c e n t r a t i o n o f p y r i m i d i n e n u c l e o t i d e s o f E h r l i c h a s c i t e s carcinoma c e l l s in vitro  82  E f f e c t o f a c t i n o m y c i n D on the r e s p i r a t i o n o f E h r l i c h a s c i t e s carcinoma c e l l s  86  lh  Effect of C a on the i n c o r p o r a t i o n o f C - f o r m a t e i n p r e s e n c e o f g l u c o s e by E h r l i c h a s c i t e s c a r c i n o m a c e l l s 115 + +  14  Change i n pH'of E h r l i c h a s c i t e s c a r c i n o m a c e l l s pension incubated with glucose  sus-  E f f e c t o f g l u c o s e on the i n c o r p o r a t i o n o f ^ C - f o r mate i n t o RNA n u c l e o t i d e s and thymine o f E h r l i c h a s c i t e s carcinoma c e l l s . . . .  117  1  122  ix Page XV. XVI. XVII.  XVIII.  E f f e c t o f g l u c o s e on t h e i n c o r p o r a t i o n o f 2- '*Cg l y c i n e by E h r l i c h a s c i t e s c a r c i n o m a c e l l s in vitro...  128  I n c o r p o r a t i o n o f '*C-formate by E h r l i c h a s c i t e s oma c e l l s suspended i n two d i f f e r e n t b u f f e r s  130  1  1  carcin-  E f f e c t o f g l u c o s e on t h e i n c o r p o r a t i o n o f '*C-formate i n t o n u c l e i c a c i d components o f E h r l i c h a s c i t e s carcinoma:..cells  132  E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f g l u c o s e on t h e i n c o r p o r a t i o n o f '*C-formate i n t o n u c l e i c a c i d components of E h r l i c h a s c i t e s carcinoma c e l l s  136  I n c o r p o r a t i o n o f ''C-formate i n p r e s e n c e of. g l u c o s e by d i f f e r e n t c o n c e n t r a t i o n s o f E h r l i c h a s c i t e s c a r c i n oma c e l l s u s p e n s i o n s  139  I n c o r p o r a t i o n of C-formate i n presence of glucose i n t o bases o f DNA by d i f f e r e n t c o n c e n t r a t i o n s o f E h r l i c h a s c i t e s carcinoma c e l l s u s p e n s i o n s  140  E f f e c t o f 2-deoxyglucose on t h e i n c o r p o r a t i o n formate by E h r l i c h a s c i t e s carcinoma c e l l s  143  1  1  XIX.  XX.  XXI. XXII.  XXIII.  XXIV.  XXV.  XXVI.  1  llf  of  llf  CC-  E f f e c t o f 2-deoxyglucose i n t h e i n c o r p o r a t i o n . o f formate i n t o t h e a c i d s o l u b l e p u r i n e s o f E h r l i c h a s c i t e s carcinoma c e l l s  lk  144  E f f e c t o f 2-deoxyglucose on t h e i n c o r p o r a t i o n o f '*Cformate i n t o t h e bases o f DNA o f E h r l i c h a s c i t e s carcinoma c e l l s  145  I n c o r p o r a t i o n o f ^ C - f o r m a t e by- E h r l i c h a s c i t e s c a r cinoma c e l l s under d i f f e r e n t c o n d i t i o n s a f f e c t i n g t h e ATP i n t h e c e l l  149  I n c o r p o r a t i o n o f '*C-formate i n t o t h e a c i d s o l u b l e p u r i n e s o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s under d i f f e r e n t conditions a f f e c t i n g ATP concentration i n the cell.. . ...  150  R a d i o a c t i v i t y i n the a c i d s o l u b l e f r a c t i o n of E h r l i c h a s c i t e s carcinoma c e l l s i n c u b a t e d w i t h C-formate a f t e r t h e removal o f n u c l e o t i d e s  153  1  1  1If  X FIGURES Page 1.  Structure  o f A c t i n o m y c i n D..  2.  Proposed model f o r b i n d i n g deoxyguanosine o f DNA  3.  Biosynthesis  4.  I n c o r p o r a t i o n Of '*C-formate i n t o a c i d s o l u b l e f r a c t i o n o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in vitro  40  5.  I n c o r p o r a t i o n o f ^ C - f o r m a t e into.RNA o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in vitro  42  6.  I n c o r p o r a t i o n o f *C-formate i n t o DNA o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in vitro .  43  7.  Schematic r e p r e s e n t a t i o n o f two d i m e n s i o n a l paper chromatogram o f r i b o n u c l e o t i d e s  51  8.  S e p a r a t i o n o f a c i d s o l u b l e n u c l e o t i d e s from E h r l i c h a s c i t e s c a r c i n o m a c e l l s on D E A E - c e l l u l o s e column  73  9.  Chromatography on D E A E - c e l l u l o s e o f a c i d s o l u b l e n u c l e o t i d e s from E h r l i c h a s c i t e s c a r c i n o m a c e l l s w i t h ^C-formate. -  76  Chromatography on D E A E - c e l l u l o s e o f a c i d s o l u b l e n u c l e o t i d e s from E h r l i c h a s c i t e s c a r c i n o m a c e l l s w i t h *C-formate and a c t i n o m y c i n D  77  11.  E f f e c t o f a c t i n o m y c i n D on oxygen consumption o f E h r l i c h a s c i t e s carcinoma c e l l s . . .  85  12.  E f f e c t o f a c t i n o m y c i n D on t h e g l y c o l y s i s o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in vitro ...  89  13.  E f f e c t o f g l u c o s e on the i n c o r p o r a t i o n o f C-formate i n t o a c i d s o l u b l e f r a c t i o n by E h r l i c h a s c i t e s c a r c i n oma c e l l s suspended i n Krebs R i n g e r phosphate b u f f e r . . 105  14.  E f f e c t o f g l u c o s e on the i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o RNA o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s suspended i n Krebs R i n g e r phosphate b u f f e r 106  15.  E f f e c t o f g l u c o s e on the i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o DNA o f / E h r l i c h a s c i t e s c a r c i n o m a c e l l s suspended i n Krebs R i n g e r phosphate b u f f e r 107  actinomycin D t o the  of Inosinic acid  9 14  1  1  11  1  10.  4  lI  xi  Page 16.  17.  E f f e c t o f g l u c o s e on the i n c o r p o r a t i o n o f ^Cformate i n t o a c i d s o l u b l e f r a c t i o n o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s suspended i n c a l c i u m f r e e Krebs R i n g e r phosphate b u f f e r . . . . 1  I l l  E f f e c t o f g l u c o s e on the i n c o r p o r a t i o n o f Cformate i n t o RNA o f E h r l i c h a s c i t e s carcinoma c e l l s suspended i n c a l c i u m f r e e Krebs R i n g e r phosphate buffer.  112  E f f e c t o f g l u c o s e on the i n c o r p o r a t i o n o f Cformate i n t o DNA o f E h r l i c h a s c i t e s carcinoma c e l l s suspended i n c a l c i u m f r e e Krebs R i n g e r phosphate buffer  113  E f f e c t o f g l u c o s e oh the i n c o r p o r a t i o n o f ^Cformate i n t o t h e a c i d s o l u b l e f r a c t i o n o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s suspended i n Krebs R i n g e r bicarbonate buffer  119  E f f e c t o f g l u c o s e on t h e i n c o r p o r a t i o n o f Cformate i n t o the RNA o f E h r l i c h a s c i t e s carcinoma c e l l s suspended i n Krebs R i n g e r b i c a r b o n a t e b u f f e r . .  120  21.  E f f e c t o f g l u c o s e on the i n c o r p o r a t i o n of Ref o r m a t s i n t o the DNA of E h r l i c h a s c i t e s carcinoma c e l l s suspended i n Krebs R i n g e r b i c a r b o n a t e b u f f e r . .  121  22.  E f f e c t o f g l y c i n e on the i n c o r p o r a t i o n o f Cg l y c i n e by E h r l i c h a s c i t e s c a r c i n o m a c e l l s . .  125  23.  E f f e c t o f g l u c o s e and 2-deoxyglucose on the i n c o r p o r a t i o n o f ' C - f o r m a t e i n t o the s e r i n e of t h e acid.s o l u b l e f r a c t i o n o f E h r l i c h a s c i t e s carcinoma c e l l s . 155  18.  19.  20.  1If  llf  1  lk  1  lk  14  I /  xii LIST OF ABBREVIATIONS AMP) ADP) ATP) -  5'-mono,; d i , and t r i p h o s p h a t e s o f Adenosine  CMP) CDP) CTP)  5'-mono, d i , and t r i p h o s p h a t e s o f c y t o s i n e  Dansyl  1-Dimethyl aminonaphthalene 5-sulphonyl c h l o r i d e  DEAE  Diethylamino  DNA  Desoxyribonucleic  DNP  2, 4 - D i n i t r o p h e n o l  EDTA  Ethylenediaminetetraacetic acid  FGAR  F o r m y l g l y c i n e amide r i b o t i d e  GAR. GMP) GDP) GTP) IMP NAD, NADHNADP, NADPH PP PRPP RNA TMP UMP) UDP) UTP)  Glycineamide  ethyl acid  ribotide  5'-mono, d i , and t r i p h o s p h a t e s o f guanosine 5'-phosphate o f i n o s i n e N i c o t i n a m i d e adenine d i n u c l e o t i d e and i t s reduced form N i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate and I t s reduced form Pyrophosphate Phosphoribosy1  pyrophosphate  Ribonucleic acid 5'-phosphate o f t h y m i d i n e 5'-mono-, d i , and t r i phosphates o f u r i d i n e  dUMP  5'-phosphate o f d e o x y u r i d i n e  XMP  5'-phosphate o f x a n t h o s i n e  x m  ACKNOWLEDGEMENT The  a u t h o r w i s h e s t o e x p r e s s h i s g r a t i t u d e t o Dr. S.H.  Z b a r s k y f o r h i s guidence and c o u n s e l d u r i n g t h e c o u r s e o f these i n v e s t i g a t i o n s . The  author  i s i n d e b t e d t o Dr. A.R.P. P a t e r s o n o f McEachern  Laboratories, U n i v e r s i t y of A l b e r t a , f o r providing the E h r l i c h a s c i t e s c a r c i n o m a c e l l s . ' The many h e l p f u l d i s c u s s i o n s w i t h Dr. M. Smith on t h e s e p a r a t i o n o f n u c l e o t i d e s and withl.Dr. G.H. D i x o n oh t h e s e p a r a t i o n o f amino a c i d s from a c i d s o l u b l e f r a c t i o n s were g r e a t l y a p p r e c i a t e d .  M r s . D. Hawkins  cooperated  w i t h some a s p e c t s o f t h e study and M i s s M. H a r r i s o n o f U.B.C. H e a l t h S e r v i c e s was h e l p f u l i n d e v e l o p i n g autograms.  t h e exposed r a d i o -  - 1 • ... 1  INTRODUCTION Substances w h i c h b l o c k s p e c i f i c b i o s y n t h e t i c r e a c t i o n s have been o f g r e a t v a l u e i n c l a r i f y i n g t h e normal mechanisms by w h i c h macromolecules a r e s y n t h e s i z e d by l i v i n g The a c t i n o m y c i n s a r e o f s p e c i a l importance  cells.  i n t h i s regard,  as they a r e shown t o be s p e c i f i c i n h i b i t o r s o f RNA s y n t h e s i s , and hence a r e used a t an ever i n c r e a s i n g r a t e , i n t h e e l u c i d a t i o n o f mechanisms o f g e n e t i c t r a n s m i s s i o n , c e l l u l a r  dif-  ferentiation, protein synthesis, virus multiplication etc. Because o f t h e s i m p l i c i t y o f t h e m i c r o b i a l organisms and t h e ease w i t h w h i c h enzymes and o t h e r substances from t h e s e organisms,  c o u l d be i s o l a t e d  many o f t h e i n v e s t i g a t i o n s i n these  d i r e c t i o n s a r e c a r r i e d o u t i n b a c t e r i a l systems.  The complex  n a t u r e o f h i g h l y d i f f e r e n t i a t e d c e l l s make s i m i l a r s t u d i e s a l l t h e more d i f f i c u l t i n mammalian systems. Much o f t h e i n v e s t i g a t i o n c a r r i e d o u t u s i n g a c t i n o m y c i n as a b l o c k i n g agent p e r t a i n s t o i t s e f f e c t on m a c r o m o l e c u l a r synthesis.  Only l i m i t e d i n f o r m a t i o n i s a v a i l a b l e on t h e  e f f e c t o f a c t i n o m y c i n D on t h e s y n t h e s i s o f s m a l l m o l e c u l e s w h i c h a r e p r e c u r s o r s o f macromolecules e s p e c i a l l y o f n u c l e i c acids. Bacillus  P r e v i o u s s t u d i e s showed t h a t when t h e growth o f subtilis  was i n h i b i t e d by t h e a d d i t i o n o f a c t i n o m y c i n  D t o t h e medium, t h e r e was an a c c u m u l a t i o n t i d e s i n the c e l l s  o f guanine n u c l e o -  ( 1 ) . The i n v e s t i g a t i o n s r e p o r t e d i n t h i s  t h e s i s were o r i g i n a l l y u n d e r t a k e n  t o e l u c i d a t e the e f f e c t of  a c t i n o m y c i n D on t h e a c i d s o l u b l e n u c l e o t i d e p o o l o f mammalian  systems.  E h r l i c h a s c i t e s carcinoma c e l l s - a c e l l  line  h a v i n g m i c r o b i a l s i m p l i c i t y I n c u l t i v a t i o n and mammalian c o m p l e x i t y i n m e t a b o l i c r e a c t i o n s were chosen f o r t h e s e studies.  As an i n t r o d u c t i o n t o t h e s e i n v e s t i g a t i o n s a  d i s c u s s i o n Of t h e n a t u r e and e f f e c t o f a c t i n o m y c i n D p a r t i c u l a r l y on n u c l e i c a c i d m e t a b o l i s m  seems t o be most  appropriate. As  1  .'  "*C-formate was used as a p r e c u r s o r o f n u c l e o t i d e s  i n t h e experiments r e p o r t e d i n t h i s t h e s i s , t h e b a s i c s t e p s by which formate i s i n c o r p o r a t e d i n t o n u c l e o t i d e s and n u c l e i c acids are also o u t l i n e d . A c t i n o m y c i n s and T h e i r B i o l o g i c a l A c t i v i t y  .  A c t i n o m y c i n s a r e a group o f chromopeptide produced  by m i c r o - o r g a n i s m s  •St-reptomyc.es. ( 2 ) .  antibiotics  b e l o n g i n g l a r g e l y t o t h e genus  The f i r s t c r y s t a l l i n e a n t i b i o t i c o f t h e  s e r i e s , a c t i n o m y c i n A, was i s o l a t e d by Waksman and Woodruff i n 1940  (.3). S i n c e then more than f i f t y c h e m i c a l forms o f these  compounds have been i s o l a t e d from d i f f e r e n t c u l t u r e s of^ s t r e p t myces by d i f f e r e n t groups o f i n v e s t i g a t o r s .  These have been  named from l e t t e r s o f t h e a l p h a b e t r a n g i n g from A t o Z, by num b e r s , and a l s o by Greek sub l e t t e r s . d i f f e r e n t t r a d e names.  They a r e a l s o known unde  Though Waksman s u g g e s t e d  (4) a system-  a t i c nomenclature  f o r t h e s e compounds, t h i s .has n o t been  followed s t r i c t l y  i n l i t e r a t u r e , p r o b a b l y due t o t h e l i m i t e d  use o f t h e l e s s e r known compounds. The a c t i n o m y c i n s a r e c h a r a c t e r i z e d by t h e i r b r i g h t orange red  c o l o u r and complex b i o l o g i c a l p r o p e r t i e s .  Brockmann e t  - 3 al.  (5,6) have shown by d e g r a d a t i o n experiments  c o n t a i n t h e same chromophoric moiety a c t i n o c i n  t h a t a l l o f them (2-amino 4,6-  d i m e t h y i 3-oxophenoxazine 1 , 9 - d i c a r b o x y l i c a c i d ) and t h a t they d i f f e r o n l y i n t h e s u b s t i t u t i o n among t h e p o l y p e p t i d e c h a i n s j o i n e d t o t h e chromophoric  moiety.  Among t h e a c t i n o m y c i n s , t h e b e s t known compound i s a c t i n o mycin D, d i s c o v e r e d by V i n i g and Waksman i n (7) 1954. ( F i g u r e 1).  Of t h e many complex a c t i n o m y c i n s i s o l a t e d , a c t i n o m y c i n D  i s shown t o be c h r o m a t o g r a p h i c a l l y t h e most homogeneous substance (8).  Actinomycin D i s a l s o h i g h l y t o x i c , although i t i s  l e s s t o x i c than t h e o t h e r s .  However,the a b i l i t y , o f a c t i n o m y c i n  D t o e x e r t a marked e f f e c t on n u c l e i c a c i d s y n t h e s i s and v i r u s m u l t i p l i c a t i o n has been p r o f i t a b l y e x p l o i t e d by b i o l o g i s t s and b i o c h e m i s t s p a r t i c u l a r l y i n t h e e l u c i d a t i o n o f mechanisms o f transmission of genetic information. The a b i l i t y o f a c t i n o m y c i n D t o i n h i b i t b o t h p r o t e i n -and RNA s y n t h e s i s was r e p o r t e d by S l o t n i c k i n 1959 ( 9 ) . Soon a f t e r that, many i n v e s t i g a t o r s  (10, 11) showed t h a t a c t i n o m y c i n D  can s e l e c t i v e l y i n h i b i t RNA s y n t h e s i s both i n micro-organisms and i n mammalian c e l l s . mycin D suppressed i n host c e l l s  Further., i t was a l s o shown t h a t a c t i n o -  t h e growth o f DNA virus,, but n o t RNA v i r u s  ( 1 2 ) . S i m i l a r l y t h e a n t i b i o t i c was shown t o  i n h i b i t RNA s y n t h e s i s by c e l l f r e e systems from b o t h mammalian (13) and m i c r o b i a l s o u r c e s can a l s o be suppressed  (14) .  DNA s y n t h e s i s i n t h e c e l l s '  by h i g h e r c o n c e n t r a t i o n o f a c t i n o m y c i n  D than r e q u i r e d f o r t h e i n h i b i t i o n o f RNA s y n t h e s i s ( 1 1 ) . H u r w i t z e t a l . have shown (14) t h a t t h e a c t i v i t y o f DNA p o l y merase in vitro  i s l e s s s e n s i t i v e t o a c t i n o m y c i n D than t h a t  of RNA p o l y m e r a s e .  F i g u r e 1.  A c t i n o m y c i n D. The sequence of amino a c i d s i s L - t h r e o n i n e , D - v a l i n e ( D - V a l ) , L - p r o l i n e (LP r o ) , s a r c o s i n e (Sar) and N-methyl-valine.  -  To e l u c i d a t e  5 -  t h e c o r r e l a t i o n between b i o l o g i c a l a c t i v i t y  and m o l e c u l a r s t r u c t u r e , many c h e m i c a l l y  a l t e r e d actinomycins  have been produced and many n a t u r a l l y o c c u r r i n g have been i s o l a t e d (15,16).  actinomycins  I n a study w i t h a s e r i e s o f  d i f f e r e n t a c t i n o m y c i n s R e i c h e t a l . (17) have shown t h a t t h e i n h i b i t o r y a c t i o n o f t h e a n t i b i o t i c on t h e growth o f HeLa c e l l s and on t h e i s o l a t e d RNA polymerase r e a c t i o n c o u l d be c o r r e l a t e d t o i t s a b i l i t y t o form complexes w i t h DNA. tions at d i f f e r e n t portions  Altera-  of the a n t i b i o t i c molecule  could  a f f e c t t h e i n h i b i t o r y e f f e c t s t o a g r e a t e r o r l e s s e r degree. Thus a c t i n o m y c i n s p r o v i d e a unique example o f c o r r e l a t i n g b i o l o g i c a l a c t i v i t y with molecular structure.  Several  character-  i s t i c f u n c t i o n a l groups p r e s e n t i n t h e a c t i n o mycin m o l e c u l e (see F i g u r e 1) appear t o be e s s e n t i a l f o r t h e b i o l o g i c a l a c t i v i t y of the a n t i b i o t i c . These f u n c t i o n a l groups  are:-  (1) t h e f r e e amino group a t p o s i t i o n 3 o f t h e chromophore. (2) t h e unreduced q u i n o i d a l oxygen a t p o s i t i o n 4 and (3) t h e lactone  rings of the peptide side  chains.  Removal o f t h e amino group on p o s i t i o n 3 o r s u b s t i t u t i o n o f t h e hydrogens o f t h e amino group w i t h a l k y l groups the a c t i v i t y c o m p l e t e l y . system reduces t h e b i n d i n g i n low a c t i v i t y the  lactone  (18).  Reduction of the q u i n o i d a l  abolishes ring  o f a c t i n o m y c i n w i t h DNA, r e s u l t i n g  Similarly, hydrolysis  o f one o r b o t h o f  r i n g s o r removal o f t h e p e p t i d e s i d e c h a i n s reduces  the a c t i v i t y o f t h e a n t i b i o t i c (19) markedly. The  s t r u c t u r a l features  o f t h e DNA m o l e c u l e n e c e s s a r y f o r  t h e e f f e c t i v e b i n d i n g o f a c t i n o m y c i n D are the guanine base and the h e l i c a l s t r u c t u r e o f  DNA.  The maximal amount o f a c t i n o m y c i n bound t o DNA the guanine c o n t e n t o f the DNA; proportional to i t . thymidylate  though i t i s not  A p u r i n i c DNA.,  synthetic  parallels  directly  deoxyadenylate  (dA:T) and s y n t h e t i c d e o x y i n o s i n i c d e o x y c y t i d y l i c  copolymer ( d l r d C ) do not i n t e r a c t w i t h a c t i n o m y c i n whereas a p y r i m i d i n i c a c i d DNA,  s y n t h e t i c deoxyguanylic d e o x y c y t i d y l i c  copolymer (dG:dC) and n a t u r a l l y o c c u r r i n g DNA The r e q u i r e m e n t  bind actinomycin.  o f h e l i c a l c o n f i g u r a t i o n o f DNA  b i n d i n g i s suggested  by the o b s e r v a t i o n t h a t  f o r maximal  heat-denatured  DNA  b i n d s a c t i n o m y c i n t o a l e s s e r e x t e n t than h e l i c a l DNA  DNA  o f t h e same base c o m p o s i t i o n  (20).  The b i n d i n g o f a c t i n o m y c i n D t o DNA enzymic a c t i v i t y o f DNA  dependent RNA  i n t e r f e r e s w i t h the  polymerase.  t i o n s o f Hartmann_ e t ' a l . (21) and by G o l d b e r g the i n h i b i t i o n o f RNA  or  The  observa-  e t a l . (13) t h a t  s y n t h e s i s by a c t i n o m y c i n D cannot be  i n f l u e n c e d by changing  c o n c e n t r a t i o n s o f the enzyme, c o f a c t o r s  o r n u c l e o t i d e p r e c u r s o r s , but can be v a r i e d by c o n c e n t r a t i o n s o f DNA,  s u p p o r t s the above p r o p o s i t i o n . They have f u r t h e r  shown t h a t the i n h i b i t o r y e f f e c t of d i f f e r e n t a c t i n o m y c i n v a t i v e s p a r a l l e l s t h e i r a b i l i t y t o form complexes w i t h Substances such as dA:T, bind i n RNA  s y n t h e s i s by RNA  DNA.  p o l y r i b o n u c l e o t i d e s e t c . which do  the a n t i b i o t i c can e f f e c t i v e l y f u n c t i o n as polymerase i n presence  deri-  not  templates  of actinomycin  D. The b i n d i n g o f a c t i n o m y c i n a t r e l a t i v e l y h i g h c o n c e n t r a t i o n s  t o DNA was shown t o a l t e r t h e p h y s i c a l p r o p e r t i e s o f DNA t o some e x t e n t .  K r e s t e n e t a i . (22) and R e i c h  (23) have shown  t h a t b i n d i n g o f a c t i n o m y c i n i n c r e a s e d t h e temperature a t w h i c h t h e secondary  s t r u c t u r e o f DNA m e l t s o u t .  (Tm) This  e f f e c t l i k e t h e o t h e r s i s a l s o dependent on t h e e x t e n t o f binding.  The s t a b i l i t y o f complexes t o v a r i a t i o n i n pH,  p a r t i c u l a r l y i n a c i d i c range i s g r e a t e r than t h e a c i d o f DNA a l o n e .  stability  I n b o t h cases t h e d i s s o c i a t i o n o f t h e complex i s  accompanied by d e n a t u r a t i o n and s t r a n d s e p a r a t i o n o f t h e DNA. The v i s c o s i t y o f DNA i s a l s o i n c r e a s e d when i t i s bound t o actinomycin D (12). Reich  (23) has c o n c l u d e d  from h i s e x p e r i -  ments, t h a t t h e f o r c e s b i n d i n g a c t i n o m y c i n D t o DNA a r e s t r o n g e r than those m a i n t a i n i n g t h e DNA s t r u c t u r e i t s e l f .  The  c o n c e n t r a t i o n s o f a c t i n o m y c i n w h i c h produce t h e change i n p h y s i c a l p r o p e r t i e s was a l s o found t o i n h i b i t t h e r e p l i c a t i o n o f DNA by DNA p o l y m e r a s e , a p r o c e s s i n which s e p a r a t i o n o f template s t r a n d occurs.  -Lower c o n c e n t r a t i o n s o f a c t i n o m y c i n  D w h i c h i n h i b i t s RNA s y n t h e s i s do n o t a f f e c t t h e DNA r e p l i c a tion. I n a d d i t i o n t o . t h e i n h i b i t i o n o f b o t h RNA and DNA p o l y merase r e a c t i o n s (both r e q u i r e DNA t e m p l a t e s ) r e p o r t e d t o i n h i b i t m e t h y l a t i o n o f DNA. reported t o i n h i b i t nucleases  actinomycin i s  Actinomycin i s also  ( 1 8 ) . Based on a l l t h e s e  t i o n s R e i c h has proposed t h e f o l l o w i n g h y p o t h e s i s .  observa-  "The  s u r f a c e o f t h e DNA h e l i x i s d i f f e r e n t i a t e d i n t o two s p i r a l /  grooves o f u n e q u a l s i z e , and t h e a n t i p a r a l l e l p o l a r i t y o f t h e complementary s t r a n d s o f DNA causes each f u n c t i o n a l group o f  t h e base p a i r s t o be always i n f i x e d p o s i t i o n s r e l a t i v e t h e two g r o o v e s .  to  I t appears p o s s i b l e t h a t enzymes which  c a t a l y s e r e a c t i o n s i n v o l v i n g DNA  (such as p o l y m e r a s e s , methy-  l a t i n g and g l u o o s y l a t i n g enzymes, and n u c l e a s e s ) may  act  on  o r a t t a c h t o v a r i o u s c o m b i n a t i o n s of f u n c t i o n a l groups on s u r f a c e of t h e h e l i x .  I f t h i s were so, the  the  differential  s e n s i t i v i t y of the p r o c e s s t o a n t i b i o t i c a c t i o n c o u l d depend on the .'.steric r e l a t i o n s h i p between the s i t e s w h i c h b i n d a c t i n o mycin and enzyme r e s p e c t i v e l y " ( 1 8 ) . suggested t h a t actinomycins  A c c o r d i n g l y he  i n h i b i t RNA  has  polymerase by a s t e r i c  b l o c k on the s u r f a c e on w h i c h the enzyme a c t s , whereas the s y n t h e s i s i s i n h i b i t e d by an i n d i r e c t e f f e c t due t i o n of s t r a n d s e p a r a t i o n by the  t o the  DNA  inhibi-  antibiotic.  Based on X-ray and model b u i l d i n g s t u d i e s H a m i l t o n e t a l . (24) have f o r m u l a t e d . a  model f o r the DNA:actinomycin D complex.  I n t h i s model the a c t i n o m y c i n DNA  occupies  h e l i x where i t i s bound t o the DNA  the minor groove of by seven hydrogen bonds,  f o u r of w h i c h are c o n t r i b u t e d by the p e p t i d e mycin and the r e s t by a c t i n o m y c i n  c h a i n s of a c t i n o -  chromophore.  The  geometric  d i s t r i b u t i o n of the l a t t e r t h r e e hydrogen bonds were s t u d i e d by H a m i l t o n e t a_l_. i n d e t a i l and were found t o be  steriochemi-  c a l l y s a t i s f a c t o r y . These i n t e r a c t i o n s are. shown i n F i g u r e Most of the known f a c t s r e g a r d i n g w i t h DNA  the r e a c t i o n of  and the a s s o c i a t e d i n h i b i t i o n o f DNA |  model.  the model a c c o u n t s f o r the r o l e o f the s t r u c t u r e of known t o be r e q u i r e d f o r b i o l o g i c a l a c t i v i t y .  actinomycin  dependent  polymerase c o u l d be deduced from t h i s m o l e c u l a r  The  2.  RNA Thus  actinomycin reduction  - .9 -  F i g u r e 2.  Proposed model f o r the b i n d i n g o f a c t i n o m y c i n D t o deoxyguanosine o f DNA. Hydrogen bonds between a c t i n o m y c i n and DNA i n d i c a t e d by and between guanine and c y t o s i n e i n DNA by  - 10 -  '  ;  '~ •'  o f t h e q u i r n i d a l oxygen, w i l l d e s t r o y t h e a b i l i t y o f oxygen t o f u n c t i o n as a hydrogen a c c e p t o r .  Changes i n the amino group  o f t h e chromophore would s i m i l a r l y i n t e r f e r e w i t h i t s a b i l i t y t o form hydrogen bonds w i t h t h e DNA  c o n s t i t u e n t s . . The p e p t i d e  s i d e c h a i n s can be v i s u a l i z e d as b e i n g s t a b i l i z e d by t h e l a c t o n e r i n g system i n a c o n f o r m a t i o n p e r m i t t i n g t h e f o r m a t i o n o f . f o u r a d d i t i o n a l H-bonds-'between  t h e p e p t i d e N.H-groups,  oxygen o f t h e p h o s p h o d i e s t e r  group o f DNA  and t h e  strand, opposite to  t h a t c o n t a i n i n g t h e guanine i n t e r a c t i n g w i t h t h e chromophore. I t c o u l d be seen t h a t t h e r e i s no i n v o l v e m e n t o f p o s i t i o n 7 i n the complex f o r m a t i o n as i t p r o j e c t s away from t h e DNA i n t h e model.  helix  G o l d b e r g e t a l . (25) . have r e p o r t e d t h a t t h e  p r e s e n c e o f b u l k y s u b s t i t u e n t groups a t t h i s p o s i t i o n does not i n t e r f e r e w i t h t h e b i n d i n g o f a c t i n o m y c i n t o DNA w h i c h i s i n agreement w i t h t h e model.  That t h e s u b s t i t u t i o n a t p o s i t i o n 7  d i d n o t a f f e c t t h e b i o l o g i c a l a c t i v i t y o f a c t i n o m y c i n has been shown e a r l i e r by M u l l e r ( 2 6 ) . The complex f o r m a t i o n a l s o depends on t h e s t r u c t u r e p r e s e n t i n DNA and a l s o i t s c o n f i g u r a t i o n . ' T h i s f a c t i s a l s o amply accounted f o r by t h e proposed model.  Thus o n l y guanine can  p r o v i d e t h e hydrogen i n t h e minor groove o f the DNA the quino>idal oxygencan s e r v e as an a c c e p t o r .  The  f o r which disposition  o f t h e v a r i o u s p a r t i c i p a t i n g groups i n t h e B - c o n f o r r a a t i c n o f the DNA h e l i x seems t o be v e r y i m p o r t a n t f o r t h e b i n d i n g , as a c t i n o m y c i n b i n d s o n l y p o o r l y w i t h s i n g l e s t r a n d e d DNA,  sRNA,  and Reo v i r u s RNA, w h i c h are supposed t o e x i s t i n the A-conformation.  T h a t . t h e complex f o r m a t i o n was r e s t r i c t e d t o t h e minor  - 11 groove o f t h e DNA has been f u r t h e r v e r i f i e d by R e i c h and co-workers (27).  The f i n d i n g t h a t t h e s u b s t i t u t i o n p f groups i n the minor  groove and n o t i n t h e major groove a b o l i s h e d t h e complex  formation  f u r t h e r c o n f i r m e d t h e p o s i t i o n o f a c t i n o m y c i n D i n t h e minor groove o f DNA i n t h e complex. R e c e n t l y t h e v a r i o u s r e a c t i o n s by w h i c h RNA r e p l i c a t i o n .brought about by RNA polymerase have been worked o u t be Anthony e t a l . (28) and a l s o by Ishihama and Kameyama ( 2 9 ) .  The e f f e c t  of a c t i n o m y c i n D on t h e e n z y m a t i c r e a c t i o n c a t a l y s e d . b y DNA dependent RNA polymerase has been s t u d i e d by R i c h a r d s o n ( 3 0 ) . R e i c h e t a l . (12) had p o s t u l a t e d e a r l i e r t h a t some o f t h e b i n d i n g s i t e s o f t h e enzyme may be b l o c k e d by a c t i n o m y c i n , when i t b i n d s t o t h e t e m p l a t e DNA.  R i c h a r d s o n has conducted  s t u d i e s on t h e b i n d i n g o f t h e enzyme i n t h e p r e s e n c e o f a c t i n o mycin and has c o n c l u d e d  t h a t actinomycin a t concentrations which -  i n h i b i t RNA polymerase d i d n o t r e d u c e t h e number o f b i n d i n g s i t e s f o r t h e enzyme on t h e DNA.  I t a l s o d i d not a f f e c t the a f f i n i t y  constant f o r the b i n d i n g e i t h e r .  He c o n c l u d e d  from h i s s t u d i e s  that actinomycin D i n h i b i t s the polymerisation of nucleotide more t h a n t h e i n i t i a t i o n o f t h e c h a i n .  That a c t i n o m y c i n D i n h i b i t s  c h a i n e l o n g a t i o n by RNA polymerase when n a t i v e DNA was used as t e m p l a t e v/as a l s o r e p o r t e d r e c e n t l y by Sen.tenac e t a l . (31) . Though t h e e f f e c t o f a c t i n o m y c i n D i s p r i m a r i l y on t h e i n h i b i t i o n o f DNA dependent RNA polymerase and t h e consequent i n h i b i t i o n o f p r o t e i n s y n t h e s i s and o t h e r r e l a t e d c e l l u l a r  activi-  t i e s , many o t h e r m e t a b o l i c p r o c e s s e s have been shown t o be a f f e c t e d . b y a c t i n o m y c i n D.  L a s z l o e t a l . (32) observed t h a t  a c t i n o m y c i n D i n h i b i t e d r e s p i r a t i o n and g l y c o l y s i s i n l e u c e m i c leucocytes.  The i n h i b i t i o n o f b i o l u m i n e s c e n c e  by a c t i n o m y c i n  D i n c e r t a i n marine f l a g e l l a t e s - n o t a b l y t h e f o r m a t i o n o f Euglena  c h l o r o p l a s t s by l i g h t s e n s i t i v e r e a c t i o n has been  r e p o r t e d by M c C a l l a and A l l e n  (33) .  The i n h i b i t o r y e f f e c t o f  a c t i n o m y c i n D on p r o t e i n s s y n t h e s i s u n r e l a t e d t o t h e s y n t h e s i s o f mRNA has been observed by H o r n i n g and R a b i n o w i t z  (34).  A  d i f f e r e n t i a l e f f e c t o f a c t i n o m y c i n D on t h e f o r m a t i o n o f enzymes i n Bacillus  licheniformis  o b s e r v e d by P o l l a c k (35) .  and Bacillus  cereus was  Actinomycin D supressed the induc-  t i o n o f g - g l u c o s i d a s e and p e n i c i l l i n a s e i n Bacillus  cereus  where as i t i n h i b i t e d t h e i n d u c t i o n o f o n l y B - g l u c o s i d a s e and n o t p e n i c i l l i n a s e i n Bacillus  licheniformis.  Similar  inhibi-  t i o n o f a c t i n o m y c i n D on t h e f o r m a t i o n o f a-amylase, p r o t e a s e s and a l k a l i n e phosphatase, subtilis  but not ribonuclease i n  i s r e p o r t e d by Kadowaki and coworkers  e t a l . (37) o b s e r v e d t h e organism  Bacillus  (36).  Yoshida  an i n h i b i t o r y a c t i o n o f a c t i n o m y c i n on  producing the a n t i b i o t i c .  t i n g e f f e c t o f a c t i n o m y c i n D on Bacillus  That t h e growth subtilis  inhibi-  c a n be  a b o l i s h e d by i n t e r a c t i o n o f t h e drug w i t h t h y r o x i n has been observed by Kim e t a l . (38). Biosynthesis of Purine Nucleotides. Previous observations revealed that the incorporation of 1  Re-formate  into nucleic acids i n E h r l i c h ascites  c e l l s was i n h i b i t e d by a c t i n o m y c i n D (39).  carcinoma  The complex and  v a r i e d s t e p s i n t h e b i o s y n t h e s i s o f p u r i n e n u c l e o t i d e s and t h e involvement  o f formate i n t h i s p r o c e s s were e l u c i d a t e d by t h e  - 13 i n v e s t i g a t i o n s o f Buchanan and by Greenberg and a s s o c i a t e s i n t h e l a t e r 1950's (40).  They have d e f i n e d the b i o s y n t h e t i c  sequence i n a s t e p w i s e manner so t h a t the o r i g i n o f each atom o f t h e p u r i n e r i n g i s now c l e a r l y known.  The pathway thus  e l u c i d a t e d has i t s f i r s t s t e p i n the f o r m a t i o n o f p h o s p h o r i bosylamine  from 5 p h o s p h o r i b o s y l p y r o p h o s p h a t e  (PRPP) and  g l u t a m i n e and c u l m i n a t e s i n the f o r m a t i o n o f i n o s i n i c a c i d as shown i n the F i g u r e 3. The  c o n v e r s i o n o f i n o s i n i c a c i d t o a d e n y l i c and g u a n y l i c  a c i d have been shown t o be t h r o u g h the p a r t i c i p a t i o n o f a s p a r t i c a c i d , and g l u t a m i c a c i d r e s p e c t i v e l y  (41).  The f o r m a t i o n  o f AMP from IMP u t i l i z e s t h e . h i g h energy bond o f GTP,  t o form  a d e n y l o s u c c i n i c a c i d w h i c h i s s u b s e q u e n t l y b r o k e n down t o a d e n y l i c a c i d and f u m a r i c a c i d .  The c o n v e r s i o n o f IMP t o GMP  i n v o l v e s the o x i d a t i o n o f i n o s i n i c a c i d f i r s t t o XMP f o l l o w e d by a m i n a t i o n u s i n g the amide group o f g l u t a m i n e  (42).  This  r e a c t i o n r e q u i r e s ATP. In  a d d i t i o n t o the de novo s y n t h e s i s o u t l i n e d , o t h e r  mechanisms o f n u c l e o t i d e f o r m a t i o n have been demonstrated. Thus the preformed p u r i n e s can be c o n v e r t e d t o n u c l e o t i d e s u t i l i z i n g t h e v e r s a t i l e substance t i d e pyrophosphorylase  PRPP and a p p r o p r i a t e n u c l e o -  (43).  Base + PRPP ===== n u c l e o t i d e + P-P A n o t h e r minor r o u t e may i n v o l v e the enzyme n u c l e o s i d e phosphory l a s e d i s c o v e r e d by K a l c k a r (44). Adenine + Ribose-l-PCH  • •• • Adenosine + P i  "  - 14 -  CO;  ^  J Cs C i l l •c i  Aspartate—N I «"->Ci "Format*"  r T  X C|»—"Formate" •  (  Glycine  \  GlutAmine amide N .  l  •  m..i  Ribose-S'-P  A T P ~ ^ ,  AMP«^  >rRPP  (5'-phoaphoribosyl-r-pyrophosphate) Glycine + A T P  Qlutamine ~Y  NH, £  A D P+ P  GluUmate + P?<r-^X_»H N—R-5'-P (Pbosphoriboeylamine)  ° *  :  ^IH—R-5'-P  Glycinamide ribotide  V FH, NH /  \  CH,  Glutamate  CHO  +  A D P +  *•  P  NH  Glutamine + A T P  CH,  ^CHO  o=c  •'HN=i  ^IH—R-5'-P  •R-o'-P  Formylfflycinamide ribotide  Formyljdycinamidine ribotide ATP ^ A D P  +  P  COOH CO, H -i ^ '  I!  CH •  NH,—6  ^N—R-5'-P  'N—R-5'-P  O COOH i 1 Aspartate A D P CH, CH, C N. + A T P + P I / X / X V V > HC NH C X j . —Z—\ I n CH COOH NH,—C •  N*—R-o'-P  Aminoimidaiolecarboxylic acid ribotide  Aminoimidajole ribotide  Aminoimidaiole succinocarboxarrude ribotide Fumarate  O  O  C NH  V  CH  C  N^.  \CH  Inoainic a r i d (Incline monopha*p h a t * . IMP, hyi>oTar,t h i n e nucleotide)  F i g u r e 3.  H,0  ^xv  / X / NH, C l| '  0 n  J  VX .  CH  F"<  v  H FHcN»-C==0  NH,  N-R-o'-P  HC  V F o r i n a m idoimidaznlf?c a r bHo x a m i d u  ribotide  Biosynthesis of I n o s i n i c Acid.  Aminoimidtizolecarboxamidc ribotide  - 15 -  Adenosine .kinase c a t a l y s e s t h i s r e a c t i o n A d e n o s i n e + ATP v Besides  .  ^ AMP + ADP e t c .  t h e i n v o l v e m e n t o f formate i n t h e b i o s y n t h e s i s  o f p u r i n e n u c l e o t i d e 5 , t h i s one carbon fragment a l s o c o n t r i b u t e s t h e m e t h y l group o f TMP., The f o r m a t i o n o f thymine n u c l e o t i d e s de novo by t h e d i r e c t m e t h y l a t i o n f r e e e x t r a c t s o f E. ooli Kornberg (45) .  o f dUMP by c e l l  were f i r s t shown by F r i e d k i n and  I t i s now w e l l e s t a b l i s h e d t h a t t h e s y n t h e s i s  o f thymine n u c l e o t i d e de novo o c c u r s p r i n c i p a l l y through t h e methylation  o f dUMP and t h a t t e t r a h y d r o f o l a t e and formate a r e  " s u b s t r a t e s . The mechanism o f t h e r e a c t i o n has been e l u c i d a t e d r e c e n t l y i n g r e a t e r d e t a i l by Reyes and H e i d e l b e r g e r (46) u s i n g h i g h l y p u r i f i e d enzymes and s o p h i s t i c a t e d Biosynthesis of Nucleic  techniques.  Acids  The mechanism by which DNA i s s y n t h e s i z e d  i n t h e c e l l s was  f i r s t e l u c i d a t e d by Kornberg and coworkers u s i n g an enzyme i s o l a t e d from E. ooli has  (47). The enzyme, known as DNA polymerase,  s i n c e been i s o l a t e d from d i f f e r e n t s o u r c e s i n c l u d i n g mamma-  lian cells  ( 4 8 ) . The r e a c t i o n b r o u g h t about by t h e enzyme i s  a polymerization of the four deoxyribonucleoside  triphosphates  i n p r e s e n c e o f a p r i m e r DNA m o l e c u l e t o form a new p o l y m e r i z e d product.  The p r i m e r DNA can be from a n i m a l , b a c t e r i a l ,  viral  or p l a n t s o u r c e s and can be e i t h e r n a t i v e o r d e n a t u r e d . A l l the f o u r d e o x y r i b o n u c l e o s i d e r e a c t i o n , omission  triphosphates  are required f o r the  o f one o r more would reduce t h e a c t i v i t y o f  . t h e enzyme c o n s i d e r a b l y .  The p r i m e r DNA'was shown merely t o a c t  - 16 as a t e m p l a t e and t h e polymerase was shown t o copy t h e n u c l e o s i d e sequence o f t h e p r i m e r t o form t h e p r o d u c t  (49).  Recent i n v e s t i g a t i o n on t h e mechanism o f r e p l i c a t i o n o f DNA by Oish'i (50) suggest t h a t t h e i n i t i a l p r o d u c t o f b a c t e r i a l DNA s y n t h e s i s i s a s i n g l e s t r a n d e d  fragment o f a DNA m o l e c u l e .  He has a l s o i s o l a t e d a second i n t e r m e d i a t e subtilis having  w h i c h i s a double s t r a n d e d single.stranded structure.  s t a g e o f DNA r e p l i c a t i o n .  Bacillus  from  DNA w i t h a p a r t o f i t T h i s he c l a i m s as t h e second  S i m i l a r discontinuous  c h a i n growth  f o r DNA r e p l i c a t i o n has been s u g g e s t e d by O k a z a k i e t a l . The  (51).  f i n a l p r o d u c t may be o b t a i n e d by t h e a c t i o n o f t h e p o l y -  n u c l e o t i d e j o i n i n g enzyme r e c e n t l y demonstrated by G e l l e r t (52) . S t u d i e s on t h e b i o s y n t h e s i s o f RNA b o t h in vivo vitro  and in  have shown t h a t RNA I s s y n t h e s i z e d from' the f o u r r i b o -  n u c l e o s i d e d i p h o s p h a t e s by a DNA-dependent enzyme system d i s c o v e r e d by Weiss and G l a s t o n e  (53).  These enzymes, t h e DNA  dependent RNA polymerases have s i n c e been o b t a i n e d bacteria  (54) p l a n t s  valent Mg  + +  or Mn  + +  from  (55) and mammalian s o u r c e s (56).  A di-  i o n and a l l t h e f o u r r i b o n u c l e o s i d e  phosphates a r e r e q u i r e d f o r t h e r e a c t i o n .  tri-  The a c t i v i t y o f  the enzyme i s dependent on t h e p r e s e n c e o f DNA which a c t s as the t e m p l a t e f o r RNA s y n t h e s i s . nearest neighbour frequencies  The base c o m p o s i t i o n  and t h e  o f t h e n u c l e o t i d e s o f t h e RNA  formed were shown t o be complementary t o t h e DNA p r i m e r  (57).  Though H u r w i t z et. a l . (58) have shown t h a t b o t h s t r a n d s o f the p r i m e r DNA a r e c o p i e d by t h e enzyme in vitro,  t h i s does  ' -17—;' not seem t o be the case in vivo.  H a y a s h i and Spiegelman (59)'  have shown t h a t o n l y one o f t h e s t r a n d s o f the p r i m i n g DNA i s c o p i e d in vivo, E.  ooli  by the RNA p o l y m e r a s e .  Using h i g h l y p u r i f i e d  polymerase and n a t i v e <j> x.\174 RF-DNA, they have f u r t h e r  shown t h a t o n l y one  s t r a n d i s c o p i e d in vitro  a l s o the. same s t r a n d u t i l i z e d in  (60) and t h i s i s  vivo.  R e c e n t l y Anthony e t a l . (28) and Ishihama and Kameyama (29) have r e p o r t e d e v i d e n c e t o show t h a t RNA s y n t h e s i s by DNA-dependent RNA  polymerase occurs  i n three d i f f e r e n t steps.  a) A s s o c i a t i o n :  DNA + Enzyme  b) I n i t i a t i o n :  DNA Enzyme + p u r i n e  They a r e :  . ' DNA-Enzyme nucleotide  ======= [BNA-Enzyme p u r i n e  nucleotide]  c) P o l y m e r i s a t i o n ! [DNA-Enzyme p u r i n e n u c l e o t i d e ] + NTP. •  DNA-Enzyme o l i g o r i b o nucleotides.  They showed t h a t the f i r s t s t e p o f the p r o c e s s  can be i n h i b i t e d  i n s o l u t i o n by h i g h i o n i c s t r e n g t h , w h i l e the second s t e p , v i z . f o r m a t i o n o f the DNA-enzyme-purine n u c l e o t i d e complex, i s u n a f f e c t e d . They f u r t h e r showed t h a t the i n i t i a t i o n i s r a t e - l i m i t i n g a t low concentrations of nucleoside triphosphate.  T h i s l i m i t a t i o n can be o v e r -  come by r e l a t i v e l y h i g h c o n c e n t r a t i o n o f p u r i n e n u c l e o t i d e s . B e s i d e s DNA-dependent RNA polymerase t h e r e are o t h e r enzyme systems i s o l a t e d which are c a p a b l e The  of polymerizing ribonucleotides.  enzyme, p o l y n u c l e o t i d e p h o s p h o r y l a s e ,  d i s c o v e r e d by Ochoa and  c o w o r k e r s , u t i l i z e s r i b o n u c l e o s i d e d i p h o s p h a t e s as s u b s t r a t e s f o r p o l y m e r i z a t i o n r e a c t i o n (61). which u t i l i z e s RNA as a t e m p l a t e  The e x i s t e n c e o f an enzyme - RNA dependent RNA  - 18 polymerase  (replicase)-has  (62).  enzyme  The  polymerase, served  by  In merase,  i s very  except  RNA  that  instead  contrast  to  of  D  polymerase  reactions of  no  (14).  complex  is  the  primary  Because  of  the  specificity  polymerase,  for  demonstrating  in  bio-logical  to  be  the  Present  of  actinomycin  biotic.  specific  and it  The by  protein  other is well  synthesis, biotic  this  case  DNA-dependent  D  the  lends and  action  support  is  the of  has  to  be  concerned  RNA  with  of  that  the  DNA-dependent a  DNA-directed  DNA-dependent  view  antibiotic. the  proved  poly-  RNA  the  formation the  poly-  either  to  of  of  RNA  RNA-dependent  inhibition  valuable RNA  cellular  tool  synthesis  polymerase  appears  synthesis  on  very  about  the  mechanism  by  which  i n h i b i t e d by  of  D  an  important  has for  been the  development  hormone at  action, a  actinomycin  little  synthesis  i s known and  of  molecular D  about  the  utilization  modern  of  small  theories  transport, Though  nucleic  action  poly-  of  level.  inhibits  of  anti-  RNA  part  active  mode the  this  DNA-dependent  processes  that  is  the  of  synthesis,  known  and  available  inhibition  basis  cellular  the  D,  is  macromolecules  actinomycin  experimental  about  of  information  of  the  in  described  Investigations  biosynthesis  merase  workers  previously  function  the  on  DNA  existence  mostly  Considerable action  to  mode o f  systems.  enzyme  or This  actinomycin the  many  i n h i b i t o r y e f f e c t on  (10)  actinomycin  RNA  by  DNA.  has  phosphorylase  the  template  i t s e f f e c t s on  actinomycin  binding  demonstrated  s i m i l a r to  the  nucleotide  the  been  of  acid the  anti-  precursor  RNA.  molecules o f n u c l e i c a c i d s .  I f t h e prime e f f e c t . o f a c t i n o -  mycin D i s o n l y t o i n h i b i t t h e p o l y m e r i z a t i o n o f r i b o n u c l e o t i d e s , and n o t t o a f f e c t t h e de novo s y n t h e s i s o f t h e s e  sub-  s t a n c e s , then an i n c r e a s e i n t h e c e l l u l a r c o n c e n t r a t i o n o f n u c l e o t i d e s c o u l d be e x p e c t e d when c e l l s a r e t r e a t e d w i t h actinomycin  D.  case o f Bacillus mycin A3 (63).  Such an e f f e c t has been demonstrated i n t h e subtilis  with actinomycin  D (1) and chromo-  Wheeler and B e n n e t t (64) have r e p o r t e d  that  D i n t e r f e r e d w i t h the de novo s y n t h e s i s o f p u r i n e  actinomycin  r i b o n u c l e o t i d e s i n L. leichmannii  and H.Ep.2 c e l l s .  t r a s t ^ stimulatory e f f e c t o f actinomycin  I n con-  on t h e f o r m a t i o n  o f GDP and GTP from guanine i n E h r l i c h a s c i t e s tumour c e l l s was  r e p o r t e d by Harbers and M u l l e r  (66) o b s e r v e d t h a t t h e a c t i n o m y c i n  (65).  Lowy and W i l l i a m s  does n o t i n h i b i t e i t h e r t h e  de novo s y n t h e s i s o f p u r i n e n u c l e o t i d e s o r t h e i n t e r c o n v e r s i o n of p u r i n e n u c l e o t i d e s i n r a b b i t r e t i c u l o c y t e s . . Recently, J a c o l i and  Zbarsky  (1) have r e p o r t e d t h a t t h e r e i s an a c c u m u l a t i o n o f  guanine n u c l e o t i d e s i n t h e a c i d s o l u b l e f r a c t i o n o f subtilis,  t h e growth o f w h i c h i s i n h i b i t e d by a c t i n o m y c i n D.  In a d d i t i o n , o t h e r metabolic actinomycin  D.  that actinomycin  e f f e c t s have been r e p o r t e d f o r  Thus,Horning and R a b i n o w i t z  (34) have shown  D i n h i b i t s p r o t e i n synthesis unrelated t o the  s y n t h e s i s o f mRNA and, glucose.  Bacillus  t h i s i n h i b i t i o n c o u l d be r e v e r s e d by  I t has a l s o been r e p o r t e d t h a t a c t i n o m y c i n  i n h i b i t i o n o f r e s p i r a t i o n and g l y c o l y s i s i n l e u k e m i c  D causes leucocytes  (32) .  The  present  i n v e s t i g a t i o n s were u n d e r t a k e n t o c l a r i f y some  - 20 of the above mentioned p o i n t s , p a r t i c u l a r l y t o see whether i s any e f f e c t f o r a c t i n o m y c i n  there  D on the de novo s y n t h e s i s o f  n u c l e o t i d e s i n mammalian systems.  I t was a l s o o f i n t e r e s t t o  see whether t h e r e was any a c c u m u l a t i o n o f n u c l e o t i d e s i n mamm a l i a n c e l l s by a c t i n o m y c i n  D as i n Bacillus  subtilis.  Ehrlich  a s c i t e s c a r c i n o m a c e l l s were used f o r the s t u d y r e p o r t e d i n the t h e s i s because o f t h e i r advantages (67).  These mammalian c e l l  l i n e s are h i g h l y d e d i f f e r e n t i a t e d and have a h i g h m i t o t i c r a t e . The  a s c i t e s tumour c e l l s can be r e p r o d u c e d w i t h homogeneity and  are e a s i l y p r o p a g a t e d .  Moreover u n i f o r m  s u s p e n s i o n s o f these  mammalian c e l l s c o u l d be e a s i l y p r e p a r e d . As the problem was  p a r t i c u l a r l y p e r t a i n i n g t o the study o f  the e f f e c t o f a c t i n o m y c i n  D on the de novo s y n t h e s i s o f n u c l e o -  t i d e s o f the a c i d s o l u b l e p o o l , i t was n e c e s s a r y t o s e p a r a t e and e s t i m a t e  these substances.  Most o f the s a t i s f a c t o r y  methods f o r the s e p a r a t i o n o f n u c l e o t i d e s depend on column chromatography i n w h i c h the s u b s t a n c e s become g r e a t l y d i l u t e d i n the e l u t i o n s o l v e n t and l a r g e q u a n t i t i e s o f s t a r t i n g m a t e r i a l are r e q u i r e d f o r i s o l a t i o n o f even s m a l l q u a n t i t i e s o f p r o ducts.  I n the f i n a l s t a g e s  t o paper chromatography.  o f s e p a r a t i o n , one has  to resort  As the i n i t i a l e x p e r i m e n t s were  c a r r i e d out w i t h s m a l l q u a n t i t i e s o f c e l l s u s p e n s i o n s ,  separa-  t i o n o f the a c i d s o l u b l e n u c l e o t i d e s from c e l l e x t r a c t s by column'chromatography was not p r a c t i c a l .  Hence paper chroma-  t o g r a p h i c methods were d e v e l o p e d f o r the s e p a r a t i o n o f s m a l l q u a n t i t i e s o f n u c l e o t i d e s from the a c i d s o l u b l e e x t r a c t s . t o chromatography the n u c l e o t i d e s were adsorbed on c h a r c o a l  Prior  - 21 and e l u t e d w i t h p y r i d i n e - a l c o h o l m i x t u r e .  This  treatment  gave b e t t e r r e s o l u t i o n o f the m i x t u r e on paper chromatograms. E h r l i c h a s c i t e s carcinoma c e l l suspensions in vitro  with  1If  C - f o r m a t e and a c t i n o m y c i n  were D.  incubated  The a c i d s o l u b l e  n u c l e o t i d e s were i s o l a t e d , and s e p a r a t e d by paper chromatography.  The c o n c e n t r a t i o n o f p u r i n e n u c l e o t i d e s and the  amount o f r a d i o a c t i v i t y i n c o r p o r a t e d i n t o these  nucleotides  were d e t e r m i n e d . The  r e s u l t s o f t h e s e e x p e r i m e n t s were i n c o n c l u s i v e due  t o v a r i a t i o n s , though t h e r e was a t r e n d towards an i n c r e a s e of n u c l e o t i d e s i n incubations c o n t a i n i n g higher of a c t i n o m y c i n  D.  Therefore  volumes o f c e l l s u s p e n s i o n actinomycin  concentrations  s i m i l a r experiments u s i n g  larger  were c a r r i e d out i n the presence o f  D and the a c i d s o l u b l e n u c l e o t i d e s from  these  e x p e r i m e n t s were s e p a r a t e d by chromatography on DEAE c e l l u l o s e columns. was  The r e s u l t s from t h e s e e x p e r i m e n t s proved t h a t t h e r e  an a c c u m u l a t i o n  of acid soluble nucleotides i n Ehrlich  carcinoma c e l l s incubated w i t h The  llf  C - f o r m a t e and a c t i n o m y c i n D.  i n c r e a s e was the h i g h e s t i n the case o f adenine n u c l e o t i d e s .  I n the case o f Bacillus i n guanine n u c l e o t i d e s  subtilis (1).  the major i n c r e a s e was seen  F u r t h e r , i t was seen t h a t the  c o n c e n t r a t i o n s o f the n u c l e o s i d e t r i p h o s p h a t e s are  increased  many times o v e r the c o n t r o l s though a c o r r e s p o n d i n g  increase i n  the monophosphates was not seen.  discussed  i n the l i g h t o f o t h e r The  These r e s u l t s are  observations.  e f f e c t of actinomycin  D on the i n c o r p o r a t i o n o f C-  - formate i n t o b o t h RNA and DNA was a l s o examined.  lh  I n agreement  • -22- • • with other reported r e s u l t s  (39) i t was observed  that the i n -  c o r p o r a t i o n o f t h i s p r e c u r s o r i n t o b o t h t h e n u c l e i c a c i d s was i n h i b i t e d by a c t i n o m y c i n .  However, t h e i n c o r p o r a t i o n o f R e 1  format e i n t o RNA was i n h i b i t e d t o a g r e a t e r e x t e n t than i n t o DNA. S i m i l a r e f f e c t s were observed  i n studies with r a t i n t e s t i n a l  mucosa. F u r t h e r , t h e e f f e c t o f a c t i n o m y c i n D on o t h e r  cellular'  p r o c e s s e s , v i z . , r e s p i r a t i o n and g l y c o l y s i s , was i n v e s t i g a t e d . C e l l r e s p i r a t i o n was measured by t h e c l a s s i c a l procedure o f Warburg manometry.  G l y c o l y s i s was f o l l o w e d by t h e d i s a p p e a r a n c e  o f added g l u c o s e i n t h e presence' and absence o f a c t i n o m y c i n D i n suspensions  o f E h r l i c h a s c i t e s carcinoma  cells.  From these e x p e r i -  ments i t was seen t h a t t h e r e was a s l i g h t i n h i b i t i o n • o f i n presence  o f a c t i n o m y c i n D.  respiration  However, t h e r e was no c o r r e l a t i o n  between t h e e x t e n t o f i n h i b i t i o n o f r e s p i r a t i o n and i n h i b i t i o n of n u c l e i c a c i d s y n t h e s i s .  I n t h e case o f g l y c o l y s i s , t h e r e  was no e f f e c t e x e r t e d by t h e a n t i b i o t i c on t h e g l u c o s e t i o n of the c e l l s . observations  utiliza-  T h i s i s i n c o n t r a s t t o some o f t h e  (32) and i n agreement w i t h some o t h e r s ( 6 8 ) .  I t has been r e p r o t e d by many i n v e s t i g a t o r s t h a t i n E h r l i c h carcinoma  (69, 70, 71, 72)  c e l l s t h e i n c o r p o r a t i o n in vitro  of  r a d i o a c t i v e p r e c u r s o r s i n t o n u c l e o t i d e and n u c l e i c a c i d p u r i n e s was markedly s t i m u l a t e d by t h e a d d i t i o n o f g l u c o s e t o t h e i n c u b a t i o n medium.  Harrington  (69) suggested  that this stimula-  t o r y e f f e c t was due t o t h e c o n v e r s i o n o f g l u c o s e t o phosphoribosylpyrophosphate  (PRPP)  w h i c h i s one o f t h e s t a r t i n g m a t e r i a l s  of p u r i n e n u c l e o t i d e b i o s y n t h e s i s .  T h i s v i e w has been q u e s t i o n e d  - 23  -  by Henderson and LePage (70) , who  suggested t h a t the o b s e r v e d  s t i m u l a t i o n r e s u l t e d from the energy s u p p l i e d by g l y c o l y s i s . Thomson e t a l . (71) are of the view t h a t g l u c o s e was t o the r i b o s e - 5 - p h o s p h a t e r e q u i r e d f o r p u r i n e Herscovics  converted  biosynthesis.  and J o h n s t o n (72) o b s e r v e d an i n c r e a s e i n g l u t a m i n e  i n c e l l s incubated  w i t h g l u c o s e and hence s u g g e s t e d t h a t  s t i m u l a t o r y e f f e c t o f g l u c o s e was  due  t o the i n c r e a s e d  avail-  a b i l i t y of g l u t a m i n e f o r the b i o s y n t h e s i s of p u r i n e s . attempt t o i n c r e a s e the i n c o r p o r a t i o n of nucleotides  purines  In  "^C-formate and  an  into  and n u c l e i c a c i d s of E h r l i c h a s c i t e s c a r c i n o m a  c e l l s , incubated The  1  the  in  vitro,  of v a r i o u s  g l u c o s e was  f r a c t i o n s were i s o l a t e d and  of ^ C - f o r m a t e i n c o r p o r a t e d was 1  were o b t a i n e d  determined.  i n these experiments.  t r a r y t o the r e p o r t e d  added t o the medium.  Varying r e s u l t s  I n many i n s t a n c e s , con-  f i n d i n g s , a d e c r e a s e i n the  o f '*C-formate i n t o the n u c l e i c a c i d p u r i n e s was 1  T h i s problem was  examined i n g r e a t e r d e t a i l and  e s t i n g r e s u l t s were o b t a i n e d .  I t was  the amount  incorporation observed.  several  inter-  seen i n s e v e r a l e x p e r i -  ments t h a t the i n c o r p o r a t i o n of '*C-formate i n t o the a c i d 1  soluble f r a c t i o n increased while incorporation i n t o nucleic a c i d s was  decreased.  However, i n cases where t h e r e was  a  d e c r e a s e of i n c o r p o r a t i o n i n the n u c l e i c a c i d s , a s i m i l a r d e c r e a s e i n the i n c o r p o r a t i o n of s o l u b l e p u r i n e was  also noticed.  much of the r a d i o a c t i v i t y was  Ilf  C - f o r m a t e i n the a c i d On f u r t h e r i n v e s t i g a t i o n ,  found t o be c o n c e n t r a t e d  i n serine  of the a c i d s o l u b l e f r a c t i o n , making the over a l l a c t i v i t y the f r a c t i o n h i g h e r  of  than the c o n t r o l s e x p e r i m e n t s , though the  -incorporation of r a d i o a c t i v i t y i n t o the acid soluble was l o w e r .  purines  The e f f e c t o f g l u c o s e e i t h e r t o i n c r e a s e o r t o  -decrease t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o t h e n u c l e i c 1  a c i d purines  was found t o depend on t h e c o n c e n t r a t i o n  c e l l suspension.  I n d i l u t e c e l l s u s p e n s i o n s t h e e f f e c t was  always i n h i b i t o r y .  S i m i l a r r e s u l t s were o b s e r v e d when  g l y c i n e was used as t h e n u c l e i c a c i d p r e c u r s o r . for  of the  1  "*C-  The r e a s o n  t h i s d e c r e a s e d 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 i t y was suspec-  t e d t o be due t o t h e d e l a y e d r e s y n t h e s i s  o f t h e much needed  energy s o u r c e ATP, w h i c h becomes t e m p o r a r i l y  depleted  a d d i t i o n of glucose through the hexokinase r e a c t i o n . i n f e r e n c e was s t r e n g t h e n e d by t h e o b s e r v a t i o n glucose,  on t h e This  t h a t 2-deoxy-  an agent c a p a b l e o f d e p l e t i n g c e l l u l a r ATP l e v e l s ,  caused an i n h i b i t i o n o f i n c o r p o r a t i o n o f *C-formate even i n 1!  dense s u s p e n s i o n s o f E h r l i c h a s c i t e s carcinoma c e l l s i n which case g l u c o s e always gave a s t i m u l a t i o n r a t h e r t h a n an i n h i b i t i o n of incorporation.  Uncoupling of o x i d a t i v e phosphoryla-  t i o n by d i n i t r o p h e n o l a l s o caused an i n h i b i t i o n o f i n c o r p o r a t i o n of l a b e l . for  These r e s u l t s and t h e o t h e r p o s s i b l e reasons  t h e d e c r e a s e d i n c o r p o r a t i o n o f *C-formate i n p r e s e n c e o f  glucose are discussed.  ll  -  25 -  MATERIALS AND METHODS 1) E h r l i c h A s c i t e s Carcinoma C e l l s The  a s c i t e s c a r c i n o m a c e l l s used i n t h e s t u d y r e p o r t e d I n  t h i s t h e s i s were grown i n t h e p e r i t o n e a l  c a v i t y o f Swiss mice  o f t h e Ha/ICR s t r a i n (A.R. Schmidt & Co.).  The tumour c e i l  l i n e s were m a i n t a i n e d by Dr. A.R.P. E a t e r s o n . o f the, U n i v e r s i t y of A l b e r t a Cancer Research U n i t , Edmonton.  The t r a n s p l a n t a t i o n  o f tumour c e l l s was k i n d l y c a r r i e d o u t a t t f i e above i n s t i t u t e and  t h e a n i m a l s were s h i p p e d i m m e d i a t e l y t o t h e Department of'  B i o c h e m i s t r y , U.B.C.  The a n i m a l s were m a i n t a i n e d on P u r i n a  Chow and w a t e r ad libitum,  u n t i l t h e y were s a c r i f i c e d .  Seven t o e i g h t days a f t e r t r a n s p l a n t a t i o n  o f t h e tumour  c e l l s , t h e a n i m a l s were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n and the tumour c e l l s c o l l e c t e d .  F o r t h e c o l l e c t i o n o f c e l l s , 1 ml  o f c o l d p h y s i o l o g i c a l s a l i n e was i n j e c t e d i n t o t h e p e r i t o n e a l c a v i t y , t h e s k i n o f t h e abdominal r e g i o n was ^removed, t h e abdomen c u t open by s c i s s o r s and t h e p e r i t o n e a l  fluid  containing  tumour c e l l s was d r a i n e d i n t o a 40 ml g r a d u a t e d c e n t r i f u g e -kept c o o l e d i n i c e .  The c e n t r i f u g e  30 I . U n i t s o f h e p a r i n coagulation  tube,  tubes als-o c o n t a i n e d about  ( N u t r i t i o n a l B i o c h e m i c a l Co.) t o p r e v e n t  of the ascites f l u i d .  I f t h e a s c i t i c f l u i d was  b a d l y c o n t a m i n a t e d w i t h r e d c e l l s , t h e tumour; c e l l s were n o t used f o r t h e i n v e s t i g a t i o n . The  tumour c e l l s were s e p a r a t e d from t h e a s c i t i c plasma  by c e n t r i f u g a t i o n i n a r e f r i g e r a t e d c e n t r i f ugse. ( I n t e r n a t i o n a l  - 26 model PR-2)  a t 2500 rpm f o r 5 m i n u t e s .  The s e p a r a t e d c e l l s  were washed w i t h 3 volumes o f c o l d p h y s i o l o g i c a l s a l i n e and centrifuged again.  The c e l l volume was n o t e d and t h e super-  n a t a n t f l u i d removed.  The washed c e l l s were then suspended  i n 9 volumes o f b u f f e r s o l u t i o n t o g i v e a 1:10 s u s p e n s i o n . 2) D e t e r m i n a t i o n o f Packed C e l l Volume. Theppacked c e l l volume o f t h e s u s p e n s i o n was determined when r e q u i r e d by c e n t r i f u g i n g t h e E h r l i c h carcinoma c e l l  sus-  p e n s i o n i n Wintrobe h e m a t o c r i t e t u b e s , i n an I n t e r n a t i o n a l C l i n i c a l C e n t r i f u g e w i t h head No. 211 a t maximum p e r m i s s a b l e speed f o r 30 minutes  (73).  3) R a d i o a c t i v e M a t e r i a l s . Radioactive  llf  C-sodium formate was purchased  R a d i o c h e m i c a l C e n t r e , Amersham, England. ved i n water made a l k a l i n e carbonate.  from t h e  The s a l t was d i s s o l -  (pH 8) by t h e a d d i t i o n o f sodium  The s o l u t i o n thus p r e p a r e d c o n t a i n e d 200 micro--  curies per ml. 2 - ^ C - g l y c i n e was a p r o d u c t o f Merck Sharp and Dohme o f 1  Canada L t d . to  T h i s was d i s s o l v e d i n s u f f i c i e n t volume o f w a t e r  g i v e a s o l u t i o n c o n t a i n i n g 200 m i c r o c u r i e s p e r m l .  4) A c t i n o m y c i n D. T h i s was a g i f t t o P r o f e s s o r S.H. Zbarsky from Merck Sharp Dohme c f Canada L t d .  The p r o d u c t was l a b e l l e d 'Lyo' m e r a c t i n o -  mycin and was p r o v i d e d i n v i a l s each c o n t a i n i n g 0.5 mg o f a c t i n o m y c i n D.  T h i s was d i s s o l v e d i n 20% e t h a n o l t o g i v e t h e  d e s i r e d c o n c e n t r a t i o n o f e i t h e r 200 ug/ml c r 100 ug/ml (74). These s o l u t i o n s were s t o r e d a t 5) D e t e r m i n a t i o n o f G l u c o s e was  -20°.  Glucose^  e s t i m a t e d u s i n g t h e G l u c o s t a t method.  G l u c o s t a t k i t was  purchased  from W o r t h i n g t o n  The  B i o c h e m i c a l Cor-  p o r a t i o n and e s t i m a t i o n s c a r r i e d out a c c o r d i n g t o t h e d i r e c t i o n s provided;by  the manufacturers  w i t h minor m o d i f i c a t i o n s . A l i -  quots o f t h e c e l l s u s p e n s i o n s were s u i t a b l y d i l u t e d and  de-  p r o t e i n i z e d by a d d i n g e q u a l volumes o f ZnSO>> (2%) and Ba(OH)2 (1.8%) s o l u t i o n s  (75) .  The y e l l o w c o l o u r d e v e l o p e d  i n c u b a t i o n f o r 30 minutes a t 37° was ford  r e a d a t 400 mu  after in a Gil-  spectrophotometer.  6) D e t e r m i n a t i o n o f Phosphate. The phosphorus c o n t e n t o f v a r i o u s n u c l e o t i d e s e l u t e d from chromatograms was  determined  a c c o r d i n g t o t h e method o f F i s k e  and SubbaRow (76) as m o d i f i e d by B a r t l e t t produced was  r e a d a t 800 mu  (77).  The b l u e c o l o u r  i n a Cary s p e c t r o p h o t o m e t e r  c o n t e n t o f phosphorus o b t a i n e d from a s t a n d a r d curve using s i m i l a r  and  the  prepared  procedure.  7) D e t e r m i n a t i o n of N u c l e i c A c i d s . The DNA  i s o l a t e d from a s c i t e s c e l l s was  amine r e a c t i o n of D i s c h e DNA  (Mann Research  (78).  e s t i m a t e d by  dipheny  H i g h l y p o l y m e r i z e d c a l f thymus  L a b o r a t o r i e s ) was  used as a s t a n d a r d i n  these e s t i m a t i o n s . RNA  was  e s t i m a t e d by t h e o r c i n o l method o f Mejbaum  m o d i f i e d by Munro e t a l . (80).  (79)  T h e . f i n a l c o n c e n t r a t i o n s of  - 28 o r c i n o l and f e r r i c c h l o r i d e i n t h e m o d i f i e d method were 0.3% and 0.01% r e s p e c t i v e l y .  The f i n a l c o n c e n t r a t i o n o f H C l was  6N. Adenosine monophosphate was used as a s t a n d a r d and c o n c e n t r a t i o n s were e x p r e s s e d i n terms o f umoles o f a d e n y l i c acid 8)  (81) . Radioautography. R a d i o a c t i v e areas on chromatograms and e l e c t r o p h o r o g r a m s ,  were d e t e c t e d by r a d i o a u t o g r a p h y . between two X-ray f i l m s p e r i o d o f 2 t o 3 weeks.  Chromatograms were p l a c e d  (Kodak M e d i c a l , No Screen) The exposed  for a  f i l m s were then  developed,  f i x e d and washed a t t h e dark room f a c i l i t i e s a v a i l a b l e a t t h e U n i v e r s i t y H e a l t h S e r v i c e o f U.B.C. 9) L i q u i d S c i n t i l l a t i o n  Counting.  R a d i o a c t i v i t y o f t h e v a r i o u s ^ - l a b e l l e d compounds was 1  determined u s i n g P a c k a r d T r i r - C a r b l i q u i d s p e c t r o p h o t o m e t e r model 314-X.  scintillation  I n some, l a t e r  experiments,  d e t e r m i n a t i o n s were made u s i n g N u c l e a r Chicago l i q u i d  scinti-  l l a t i o n c o u n t e r Mark I I . Each c o u n t i n g v i a l c o n t a i n e d 0.5 ml of  a s o l u t i o n o f t h e r a d i o a c t i v e m a t e r i a l , 0.5 ml o f IM hya.-  mine h y d r o x i d e i n methanol and 5 ml o f s c i n t i l l a t i o n f l u i d o f the f o l l o w i n g c o m p o s i t i o n (82). 1, 4-diox'ane-6 p a r t s by volume 1, 2-diiaethoxyethane-1 p a r t by volume a n i s o l e - 1 p a r t by volume PPO  (Kent C h e m i c a l s , Vancouver) was d i s s o l v e d i n the' above  s o l v e n t system t o a c o n c e n t r a t i o n o f 1.46% and POPOP- (Packard  - 29 I n s t r u m e n t Co.) t o a c o n c e n t r a t i o n o f 0.06%.  The hyamine hydro-  x i d e was p r e p a r e d from hyamine 10-X, a p r o d u c t o f Rohm and Haas.  The m a t e r i a l was p u r i f i e d  (83) and c o n v e r t e d t o t h e  h y d r o x i d e by t h e p r o c e d u r e d e s c r i b e d by E i s e n b e r g ( 8 4 ) . 10) G e n e r a l P r o c e d u r e f o r I n c u b a t i o n s in vitro A s c i t e s Carcinoma  of E h r l i c h  Cells.  The r a d i o a c t i v e i n c o r p o r a t i o n s t u d i e s were c a r r i e d o u t i n 250 ml Erlenmeyer f l a s k s i n a manner s i m i l a r t o t h a t d e s c r i b e d by S t e w a r t and Zbarsky  (85) .  Each f l a s k c o n t a i n e d 25 u l o f a  s o l u t i o n o f r a d i o a c t i v e formate a c t i v e formate as a c a r r i e r .  (5 uC), 20 ymole o f non r a d i o -  T h i s c o n c e n t r a t i o n o f non r a d i o -  a c t i v e formate was optimum f o r t h e maximum c o r p o r a t i o n o f ^C1  formate by a s c i t e s carcinoma c e l l s e i t h e r i n Krebs R i n g e r phosphate  (86).  Enough c e l l s u s p e n s i o n  (87) b u f f e r o r Krebs R i n g e r  b i c a r b o n a t e b u f f e r pH 7.8 was added t o make t h e t o t a l volume 10 m l .  The f l a s k s were k e p t c o o l e d i n i c e d u r i n g a d d i t i o n s .  The gas phase was e i t h e r 0  2  o r a m i x t u r e o f 95% 0  2  depending on t h e b u f f e r used t o suspend t h e c e l l s .  and 5% C 0  The f l a s k s  were s t o p p e r e d a f t e r t h e r m a l e q u i l i b r i u m f o r 3 m i n u t e s . b a t i o n was c a r r i e d o u t i n s h a k i n g w a t e r b a t h t i o n s p e r minute) a t 37° f o r 2 h o u r s .  2  Incu-  (100-110 o s c i l a -  F o r e v e r y experiment  i n c u b a t i o n s i n d u p l i c a t e were c a r r i e d o u t . When t h e time c o u r s e o f 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 formate under v a r i o u s c o n d i t i o n s by E h r l i c h a s c i t e s  carcinoma  c e l l s was s t u d i e d , i n c u b a t i o n was c a r r i e d o u t e i t h e r i n 500 ml o r 1000 ml f l a s k s .  C o n c e n t r a t i o n o f t h e p r e c u r s o r added t o t h e  - 30 i n c u b a t i o n medium was  such t h a t e v e r y 10 ml o f s u s p e n s i o n con-  t a i n e d 25 u l (5 m i c r o c u r i e s ) c o n t e n t of non per ml.  The  of r a d i o a c t i v e f o r m a t e .  r a d i o a c t i v e formate i n the medium was  f l a s k was  c l o s e d w i t h a one  through w h i c h a narrow p o l y e t h y l e n e or a m i x t u r e o f 95% Oxygen and used, was  The 2 umole  holed rubber stopper  tube was  inserted.  Oxygen  5% C O 2 depending cn the b u f f e r ,  passed i n t o the f l a s k t h r o u g h the p o l y e t h y l e n e  T h i s p r e v e n t e d any  tube.  change i n gas phase when samples were w i t h -  drawn a t i n t e r v a l s , by s u c t i o n . w a t e r b a t h a t 37°C and was  The  f l a s k was  incubated  in a  shaken d u r i n g the p e r i o d of  incuba-  t i o n a t 100-110 o s c i l l a t i o n s per minute 11)  Cell Respiration  •.  Studies.  S t u d i e s of c e l l r e s p i r a t i o n were c a r r i e d out by convent i o n a l Warburg manometric t e c h n i q u e s 20 mg  approximately  of c e l l s i n a t o t a l volume of 3 ml o f Krebs R i n g e r  phosphate b u f f e r pK 12)  (87) u s i n g  7.8.  I s o l a t i o n of N u c l e i c A c i d Components from E h r l i c h A s c i t e s Carcinoma C e l l s . a) I s o l a t i o n c f a c i d - s o l u b l e n u c l e o t i d e s : - The  p e n s i o n a f t e r i n c u b a t i o n was  cell  sus-  transferred into a centrifuge  tube c h i l l e d i n i c e , and c e n t r i f u g e d a t 15,000 x g f o r 20 minut e s i n a S o r v a l l RC  II B centrifuge.  O f f , the t i s s u e p e l l e t was  15 m i n u t e s , and  supernatant was  washed w i t h c o l d b u f f e r and  suspension c e n t r i f u g e d again. w i t h 3 ml c f 0.2  The  The  drained  the  washed p e l l e t w a s mixed w e l l  M p e r c h l o r i c a c i d (cold) and k e p t i n i c e f o r t h e n c e n t r i f u g e d f o r 10 m i n u t e s .  The  supernatant  - 31 -  s o l u t i o n was c o l l e c t e d and t h e p r e c i p i t a t e e x t r a c t e d more w i t h  2 ml o f c o l d p e r c h l o r i c a c i d .  once  The e x t r a c t s were  combined. The  < p o o l e d e x t r a c t was a d j u s t e d t o pH 6.5-7 w i t h 10. N  c o l d p o t a s s i u m h y d r o x i d e , s o l u t i o n and t h e r e s u l t i n g m i x t u r e cooled w e l l i n i c e t o p r e c i p i t a t e the potassium  perchlorate.  A f t e r c e n t r i f u g a t i o n , the supernatant s o l u t i o n containing the nucleotides  was s e p a r a t e d and s t o r e d a t -20° u n t i l  analysed  further. b) N u c l e i c  a c i d s : - The n u c l e i c a c i d f r a c t i o n s from a s c i t e s  c a r c i n o m a c e l l s were p r e p a r e d by t h e method o f Hecht and P o t t e r (88)  with  some m o d i f i c a t i o n s .  the e x t r a c t i o n o f n u c l e o t i d e s  The p r e c i p i t a t e o b t a i n e d a f t e r was washed t w i c e w i t h  2 ml  p o r t i o n s . o f 0. 2M p e r c h l o r i c a c i d and once witih 95% e t h a n o l , and  the: washings d i s c a r d e d .  The r e s i d u e was suspended i n 1 ml  of 10% NaCl and n e u t r a l i z e d by t h e d r o p w i s e acddition o f IN NaOH i n 10% NaCl u s i n g p h e n o l r e d as an i n t e r n a l i n d i c a t o r . The  s u s p e n s i o n was heated i n a b o i l i n g water )bath f o r 1 hour,  centrifuged  and t h e s u p e r n a t a n t f l u i d was s e p a r a t e d .  r e s i d u e was e x t r a c t e d  w i t h a n o t h e r 0.5 m l o f .10% NaCl f o r 30  minutes a t 100° and t h e e x t r a c t s  combined.  Ifhe sodium n u c l e a t e  was  p r e c i p i t a t e d by t h e a d d i t i o n o f 3 volumes; o f c o l d  The  m i x t u r e was s t o r e d  ethanol.  i n the c o l d overnight and the p r e c i p i -  t a t e c o l l e c t e d by c e n t r i f u g a t i o n . n u c l e a t e s was d i s s o l v e d a t 37° f o r 2 2 h o u r s .  The  The p r e c i p i t a t e o f sodium  i n 1 ml c f 0.1 N NaOH and i n c u b a t e d  T h i s p r o c e d u r e h y d r o l y s e d t h e RNA.  the s o l u t i o n , t h e u n r e a c t e d DNA was p r e c i p i t a t e d by t h e  From  32  -  a d d i t i o n of N HCl t o a f i n a l c o n c e n t r a t i o n c f 0.1 N. p e n s i o n was  c h i l l e d and c e n t r i f u g e d i n the c o l d and the  n a t a n t s o l u t i o n of RNA  from N u c l e i c A c i d s and  DNA  o f P u r i n e and P y r i m i d i n e Bases  Nucleotides.  a c i d s o l u b l e f r a c t i o n s and the r i b o n u c l e o t i d e p r e p a r a -  t i o n s obtained.as  d e s c r i b e d above, were e v a p o r a t e d t o d r y n e s s  i n a vacuum d e s i c c a t o r over cone. H2SCH and sodium Each r e s i d u e t h u s o b t a i n e d was  hydroxide.  h y d r c l y s e d w i t h 72% p e r c h l o r i c  a c i d a c c o r d i n g t o the p r o c e d u r e of Marshak and V o g e l Each h y d r o l y s a t e was w i t h ION  super-  (89).  I s o l a t i o n and D e t e r m i n a t i o n  The  sus-  n u c l e o t i d e s and the p r e c i p i t a t e of  were c o l l e c t e d s e p a r a t e l y 13)  The  (90).  d i l u t e d w i t h 1 ml of w a t e r , n e u t r a l i z e d  c o l d potassium hydroxide  p o t a s s i u m p e r c h l o r a t e was  and the p r e c i p i t a t e of  removed a f t e r c o o l i n g , by  centrifu-  gation. The  supernatant  s o l u t i o n c o n t a i n i n g the bases was  i n t o a 10 ml beaker and e v a p o r a t e d t o d r y n e s s in r e s i d u e was  placed  vacuo.  The  t a k e n up i n a s m a l l volume of w a t e r , and the s o l u t i o n  a p p l i e d t o f i l t e r paper f o r d e s c e n d i n g chromatography.  The  chromatograms were d e v e l o p e d i n Wyatt's s o l v e n t system  (91)  containing iscpropancl-water-HCl  (170:39:41 v / v ) .  were much f a s t e r and b e t t e r i n K i r b y ' s c o n t a i n i n g methanol)" water^ and HCl used i n l a t e r  Separations  s o l v e n t system  (92)  (70: ice 2C v/v) , which  was  experiments.  I n o r d e r t o remove the f l u o r e s c e n t m a t e r i a l which i n t e r f e r e d w i t h the s e p a r a t i o n of bases i n chromatography, the h y d r c l y s a t e s from the a c i d s o l u b l e f r a c t i o n were adsorbed on  •  - 33  Dowex-50-X8 ( H  +  -  form) p r i o r t o chromatography  and e l u t e d w i t h  h y d r o c h l o r i c a c i d a c c o r d i n g t o the p r o c e d u r e o f LePage (93). When o n l y t h e p u r i n e bases were t o be o b t a i n e d from t h e n u c l e o t i d e s , h y d r o l y s i s was c a r r i e d o u t by t h e method o f V i s c h e r and C h a r g a f f (9 4) u s i n g 1 N HCl ( a t 100°  f o r i hour).  The s o l u t i o n was e v a p o r a t e d t o d r y n e s s and bases s e p a r a t e d as described  above.  Adenine and h y p o x a n t h i n e form a s i n g l e s p o t cn  chromato-  graphy e i t h e r i n Wyatt's s o l v e n t system c r i n K i r b y ' s s o l v e n t system.  To s e p a r a t e them t h e m a t e r i a l from t h i s s p o t was  e l u t e d and rechromatographed by Hershey et_ a l . (28%) and water  i n the s o l v e n t system d e s c r i b e d  (95) w h i c h c o n t a i n e d i s c p r o p a n o l - a m m c n i a -  (85:1.3:15  v/v).  Rechromatcgraphy  of "guanine  s p o t s " i n t h i s s o l v e n t system f a c i l i t a t e d t h e e l i m i n a t i o n c f the  i n t e r f e r i n g f l u o r e s c e n t m a t e r i a l from guanine.  14) E l u t i o n and E s t i m a t i o n o f N u c l e i c A c i d Components from Chromatograms. The n u c l e i c a c i d components on chromatograms were l o c a t e d by u l t r a v i o l e t l i g h t . cut  Each U.V.  a b s o r b i n g a r e a cn the paper  was  i n t o s e v e r a l s m a l l p i e c e s and" the m a t e r i a l e x t r a c t e d by the  method of M e z e i and Zbarsky (96) w i t h 0.1 N H C l .  The  absorption  spectrum o f each c f t h e e l u t e d s u b s t a n c e s was d e t e r m i n e d i n a Cary model 15 s p e c t r o p h o t o m e t e r . a r e a s of the paper w i t h no U.V. blanks i n the e s t i m a t i o n s .  E l u a t e s from c o r r e s p o n d i n g a b s o r b i n g m a t e r i a l s e r v e d as  The c o n c e n t r a t i o n was  mined from, the molar e x t i n c t i o n c o e f f i c i e n t s  (97) .  then deter-  .- 34 15)  S e p a r a t i o n of A c i d S o l u b l e N u c l e o t i d e s . a) I s o l a t i o n of n u c l e o t i d e s by c h a r c o a l a d s o r p t i o n : -  For  t h e s e p a r a t i o n c f the v a r i o u s n u c l e o s i d e phosphates o f the a c i d s o l u b l e f r a c t i o n , paper chromatography was method was  employed.  A  new  d e v e l o p e d f o r the s e p a r a t i o n of t h e a c i d s o l u b l e  n u c l e o t i d e s as d e s c r i b e d under r e s u l t s .  Before  chromatography  the a c i d s o l u b l e n u c l e o t i d e s were i s o l a t e d from the a c i d s o l u b l e f r a c t i o n by a d s o r b i n g e l u t i n g from i t . stances  Zhivkov  and  Thus the n u c l e o t i d e s were f r e e d of the sub-  i n t e r f e r i n g i n chromatography.  and e l u t i o n was  The  charcoal, a d s o r p t i o n  c a r r i e d out a c c o r d i n g t o the p r o c e d u r e of  (98) w i t h minor m o d i f i c a t i o n s .  The  a c i d s o l u b l e f r a c t i o n was  . S o r v a l l c e n t r i f u g e tube. t o 1-2  them on a c t i v a t e d c h a r c o a l  w i t h 5 N HC1.  c o l d s o l u t i o n , 0.2 c o a l i n w a t e r was  The  The  t r a n s f e r r e d i n t o a 12  pH of the s o l u t i o n was  s o l u t i o n was  The  tube was  shaken w e l l and At  the  intervals  shaken t o d i s p e r s e the c h a r c o a l i n the s o l u t i o n  which otherwise s i o n was  to-the  of a c t i v a t e d c h a r -  a d s o r p t i o n a l l o w e d t o p r o c e e d f o r 30 m i n u t e s . the tube was  adjusted  c o o l e d i n i c e and  ml of a 10% s u s p e n s i o n added.  ml  s e t t l e s t o the bottom.  The  c h a r c o a l suspen-  t h e n c e n t r i f u g e d a t 15000 x g f o r 10 minutes i n a  S o r v a l l RC  I I - 3 c e n t r i f u g e , and  moved q u i c k l y .  The  supernatant  unadsorbed m a t e r i a l by the U.V. sedimented c h a r c o a l was w a t e r , the s u s p e n s i o n washings d i s c a r d e d .  the supernatant s o l u t i o n was  solution re-  checked f o r any  a b s o r p t i o n a t 260 my.  washed w i t h 3 ml of c o l d d i s t i l l e d  c e n t r i f u g e d a g a i n as b e f o r e and The  The  the  l a s t drops of water were removed by.  w i p i n g the s i d e s w i t h t i s s u e paper.  The n u c l e o t i d e s adsorbed on t h e c h a r c o a l were e l u t e d w i t h •8 ml o f a m i x t u r e o f p y r i d i n e and 60% e t h a n o l (5:95 v / v ) .  The  s o l v e n t was added t o the c h a r c o a l and mixed w e l l and t h e s u s p e n s i o n was k e p t i n a water b a t h a t 37°C f o r 3 h o u r s . was  The tube  c l o s e d w i t h p a r a f i l m t o p r e v e n t any l o s s o f s o l v e n t .  At  i n t e r v a l s t h e tubes were shaken t o d i s p e r s e the c h a r c o a l i n the s o l v e n t f o r complete e l u t i o n .  F i n a l l y , t h e c h a r c o a l was r e -  moved by c e n t r i f u g a t i o n and t h e s u p e r n a t a n t s o l u t i o n e v a p o r a t e d to  dryness i n a f l a s h e v a p o r a t o r , keeping the bath temperature  at  20°C.  The r e s i d u e was e x t r a c t e d from t h e f l a s k s w i t h 3 t o  4 ml of w a t e r and t h e e x t r a c t t r a n s f e r r e d t o a s m a l l t u b e . The water from the e x t r a c t was removed by l y o p h i l i z a t i o n .  The  r e s i d u e t h u s o b t a i n e d was d i s s o l v e d i n a s m a l l amount o f water and a p p l i e d on paper f o r chromatography. b) Paper chromatography  o f n u c l e o t i d e s : - Two d i m e n s i o n a l  paper chromatography was used f o r t h e s e p a r a t i o n o f t h e a c i d • s o l u b l e nucleotides..  R e c t a n g u l a r s h e e t o f Whatman No. 40  p a p e r , 56 x.46 cms, was used f o r chromatography.  The  filter  sample  was a p p l i e d t o one c o r n e r o f t h e paper 8 cms away from e i t h e r edge o f t h e paper. pinking shears.  The bottom edge of the paper was  Chromatography  was c a r r i e d out i n l a r g e  r e c t a n g u l a r g l a s s t a n k s (Shandon & Co.) technique.  employing t h e d e s c e n d i n g  The s o l v e n t system used i n the f i r s t  had t h e f o l l o w i n g c o m p o s i t i o n : Isobutyric acid Ammonia  cut with  586 ml 35 ml  separation  - 36 water  369 ml  0.000 1 M EDTA  10 ml  pH o f s o l u t i o n 4.3 a d j u s t e d w i t h i s o b u t y r i c a c i d The s o l v e n t was a l l o w e d t o f l o w f o r 28-30 hours by w h i c h t i m e t h e s o l v e n t f r o n t r e a c h e d t h e bottom and f l o w e d o v e r . The paper was then removed and d r i e d i n a i r . were l o c a t e d and marked under U.V. l i g h t .  The n u c l e o t i d e s  The paper was  s u i t a b l y trimmed t o reduce i t s s i z e and t o remove t h e U.V. a b s o r b i n g band g e n e r a l l y seen a t t h e bottom o f t h e paper a f t e r the  f i r s t run.  The. s h e e t o f paper was t u r n e d t h r o u g h 90° so  t h a t t h e n u c l e o t i d e s were now near t h e t o p edge o f t h e paper. The bottom edge was c u t w i t h a p i n k i n g s h e a r s and d e s c e n d i n g chromatography was c a r r i e d o u t i n t h e second d i r e c t i o n .  The  s o l v e n t system employed i n t h e second d i r e c t i o n was o f t h e following composition: Ammonium A c e t a t e  1 M  E t h a n o l , 9.5% A d j u s t e d t o pH 7 w i t h a c e t i c The paper chromatogram  -  3 vol  -  7.5 v o l  acid.  was r u n f o r 36 hours i n t h i s  a f t e r w h i c h t h e paper was removed and d r i e d i n a i r .  solvent The_:nucleo-  t i d e s on t h e paper were d e t e c t e d and marked o u t under U.V. c) Ion-exchange chromatography:- When c o m p a r i t i v e l y  larger  q u a n t i t i e s o f n u c l e o t i d e s were t o be s e p a r a t e d , as was n e c e s s a r y i n some l a t e r e x p e r i m e n t s , i o n exchange chromatography was employed  f o r the separation of a c i d s o l u b l e nucleotides of  a s c i t e s carcinoma c e l l s .  The p r o c e d u r e f o l l o w e d f o r t h e s e p a r a -  t i o n was t h a t d e s c r i b e d by Oikawa and Smith ( 9 9 ) .  - 37 The cells  acid soluble nucleotides  were e x t r a c t e d  (about 10 gm wet w e i g h t ) w i t h  (50 m l ) .  from t h e  1 M cold perchloric  acid  A f t e r t h e e x t r a c t i o n t h e s o l u t i o n was b r o u g h t t o  pH 7 by slow a d d i t i o n o f c o l d 10 N sodium h y d r o x i d e .  The  n e u t r a l i z e d s o l u t i o n was e v a p o r a t e d i n a f l a s h e v a p o r a t o r , k e e p i n g t h e b a t h t e m p e r a t u r e a t 25°C. extracted  f i r s t with  The r e s i d u e was  50 ml and t h e n w i t h 30 m l o f c o l d 95%  e t h a n o l t o remove t h e s o d i u i t o p e r c h l o r a t e .  The r e m a i n i n g un-  d i s s o l v e d m a t e r i a l was c o l l e c t e d by c e n t r i f u g a t i o n . material  containing  the nucleotides  was d i s s o l v e d  This  i n 4-5 ml  w a t e r , and t h e s o l u t i o n was a p p l i e d t o a column o f D E A E - c e l l u l o s e , 25 x 1 cm i n t h e c a r b o n a t e form p r e p a r e d and packed a c c o r d i n g t o t h e p r o c e d u r e o f T o m l i n s o n and T e n e r ( l O O ) . A f t e r t h e s o l u t i o n had been r u n i n t o t h e column, the n u c l e o t i d e s creasing  were e l u t e d  concentration  from t h e column by a l i n e a r l y i n -  o f ammonium b i c a r b o n a t e pH 8.6.  The  g r a d i e n t was a c h i e v e d by g r a d u a l and c o n t i n u e d a d d i t i o n o f ammonium b i c a r b o n a t e ( 0 . 2 M) t o w a t e r i n a m i x i n g chamber (101) from which s o l u t i o n i s withdrawn t o t h e column. was r e g u l a t e d collected.  The f r a c t i o n s were examined a t 260 mu f o r t h e  a s i n g l e peak were p o o l e d .  and t h e f r a c t i o n s f o r m i n g  The e l u t i o n o f t h e n u c l e o t i d e s v/as  complete as t h e s a l t c o n c e n t r a t i o n  r e a c h e d 0.11 M.  The  o f t h e s o l u t i o n i n t h e tubes was o b t a i n e d by t h e  r e f r a c t i v e i n d e x method. The  rate  a t 1 ml/minute and 5 ml o r 10 ml f r a c t i o n s were  p r e s e n c e o f U.V. a b s o r b i n g m a t e r i a l  molarity  The f l o w  w a t e r and ammonium b i c a r b o n a t e from t h e combined  -  38 -  f r a c t i o n f o r m i n g t h e "peaks" were removed i n a f l a s h and f i n a l l y i n a l y o p h i l i z e r .  evaporator  The n u c l e o t i d e s i n t h e r e s i d u e  were f u r t h e r s e p a r a t e d and c h a r a c t e r i z e d by paper chromato;  graphy e i t h e r i n i s o b u t y r i c - a m m o n i a - w a t e r  s o l v e n t system o r i n  ammonium a c e t a t e - e t h a n o l s o l v e n t system.  A u t h e n t i c samples o f  n u c l e o s i d e phosphates (Pabst B i o c h e m i c a l s , C a l i f o r n i a ) were used as s t a n d a r d s .  I n some cases two d i m e n s i o n a l paper chroma-  tography was c a r r i e d o u t u s i n g b o t h t h e s o l v e n t systems as d e s c r i b e d above.  The substances  were e l u t e d from paper and  were f u r t h e r i d e n t i f i e d by t h e i r a b s o r p t i o n s p e c t r a .  - 39 SECTION I EFFECT OF ACTINOMYCIN D O N  THE BIOSYNTHESIS OF  PURINE NUCLEOTIDES IN EHRLICH ASCITES CARCINOMA CELLS in  Vitro.  EXPERIMENTAL 1) Time Course o f I n c o r p o r a t i o n o f ^P-Formate' i n t o N u c l e i c 1  A c i d Components o f E h r l i c h A s c i t e s Carcinoma C e l l s . As a p r e l i m i n a r y s t e p i n t h e study o f t h e e f f e c t o f a c t i n o mycin D on t h e a c i d s o l u b l e p o o l o f a s c i t e s c a r c i n o m a  cells,  the time c o u r s e 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  :  into  the a c i d s o l u b l e m a t e r i a l s as w e l l as i n t o RNA and DNA was determined.  Formate which i s w e l l known as a p r e c u r s o r  purine r i n g of nucleotides studies.  of the  and n u c l e i c a c i d s was used i n t h e s e  The E h r l i c h a s c i t e s c a r c i n o m a c e l l s u s p e n s i o n was  p r e p a r e d i n Krebs R i n g e r phosphate b u f f e r pH 7.8 and i n c u b a t i o n s were c a r r i e d o u t as d e s c r i b e d  under methods.  A t i n t e r v a l s ' a 10  ml sample o f t h e s u s p e n s i o n was withdrawn from t h e i n c u b a t i o n m i x t u r e and t h e a c i d s o l u b l e f r a c t i o n , RNA h y d r o l y s a t e were  and DNA  obtained. The a c i d s o l u b l e f r a c t i o n was n e u t r a l i z e d w i t h 10 N c o l d KOH.  The r a d i o a c t i v i t y i n t h e n e u t r a l s o l u t i o n was d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n counting perchlorate. determined.  a f t e r t h e removal o f p o t a s s i u m  The o p t i c a l d e n s i t y o f t h e s o l u t i o n a t 260 mu was The r a d i o a c t i v i t y i n c o r p o r a t e d  i n t o the a c i d s o l u b l e  f r a c t i o n a g a i n s t time o f i n c u b a t i o n i s g i v e n i n F i g u r e The r i b o n u c l e o t i d e c o n c e n t r a t i o n  i n t h e RNA  4.  hydrolysate  - 40 -  - 41 was e s t i m a t e d by t h e m o d i f i e d o r c i n o l method as a s t a n d a r d . was e x p r e s s e d  (80) u s i n g  AMP  The 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 i t y i n t o  RNA  i n terms o f s p e c i f i c a c t i v i t y d e f i n e d as  p e r minute p e r umole o f AMP.  counts  The time course o f i n c o r p o r a t i o n  i s g i v e n i n F i g u r e 5. The c o n c e n t r a t i o n o f DNA was e s t i m a t e d by t h e  diphenylamine  r e a c t i o n and t h e 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 i t y d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n counting.  The r a d i o a c t i v i t y i n c o r p o r a t e d  i n t o DNA p l o t t e d a g a i n s t time i s g i v e n i n F i g u r e 6. Examination 1  o f t h e f i g u r e s show t h a t t h e i n c o r p o r a t i o n o f  '*C-formate i n t o RNA and DNA o f a s c i t e s carcinoma c e l l s  l i n e a r f o r the p e r i o d of i n c u b a t i o n .  was  The 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 i t y i n t o t h e a c i d s o l u b l e m a t e r i a l s was maximum a f t e r about 30 m i n u t e s a f t e r w h i c h t h e l e v e l remained c o n s t a n t . T h i s may be due t o a s t e a d y s t a t e o f s y n t h e s i s o f RNA and DNA b e i n g reached  i n w h i c h the l a b e l l e d n u c l e o t i d e s may be removed  at a constant r a t e . have observed  F o r , a s i m i l a r study R i c h a r d s e t a l . (102)  t h a t , i n case o f thymus c e l l s u s p e n s i o n t h e  i n c o r p o r a t i o n o f '*C-formate i n t o DNA was l i n e a r f o r a p e r i o d 1  of 6 hours. 2) E f f e c t o f A c t i n o m y c i n  D on t h e I n c o r p o r a t i o n o f ' C-Formate 1  t  i n t o Bases o f N u c l e o t i d e s and N u c l e i c A c i d s o f E h r l i c h A s c i t e s Carcinoma C e i l s . As t h e r e were c o n f l i c t i n g r e p o r t s as t o t h e e f f e c t o f a c t i n o m y c i n D ori t h e a c i d s o l u b l e p o o l  ( 1 , 64, 65) and o n l y  l i t t l e i s known about t h e e f f e c t oh s y n t h e s i s o f a c i d s o l u b l e n u c l e o t i d e s i n mammalian t i s s u e s , t h e p r e s e n t i n v e s t i g a t i o n s  - 42  I •  20  40  60  80  100  TIME (minutes) F i g u r e 5.  I n c o r p o r a t i o n o f C - f o r m a t e i n t o RNA o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in vitro. llt  120  - 43 -  - 44 were u n d e r t a k e n , As a s t e p towards the study,, the i n c o r p o r a t i o n o f  lk  C-  formate i n t o the v a r i o u s n u c l e i c a c i d components of E h r l i c h a s c i t e s carcinoma c e l l s at d i f f e r e n t concentrations mycin D was  determined.  Tumour c e l l s u s p e n s i o n s were made i n  Krebs R i n g e r phosphate b u f f e r pH 7.8, c a r r i e d out as d e s c r i b e d  of a c t i n o -  and  incubation  was  i n 2 50 ml s t o p p e r e d Erlenmeyer f l a s k s . •  A c t i n o m y c i n D d i s s o l v e d i n 20% e t h a n o l was t o g i v e the d e s i r e d c o n c e n t r a t i o n . t a i n e d e q u a l volumes o f 20%  The  added t o the f l a s k s  c o n t r o l f l a s k s con-  ethanol.  A f t e r i n c u b a t i o n s , the a c i d s o l u b l e f r a c t i o n , RNA and DNA  were o b t a i n e d  under methods.  from c e l l s by the p r o c e d u r e s  The  d r y DNA,  RNA  n u c l e o t i d e s were h y d r o l y s e d  s u l p h u r i c a c i d and  n u c l e o t i d e s , and  The  were s e p a r a t e d by paper chromatography. the c o n c e n t r a t i o n  a c t i v i t y incorporated  sodium  acid soluble  t o t h e i r c o n s t i t u e n t bases' by  method of Marshak and V o g e l (90).  from paper and  described  These f r a c t i o n s were e v a p o r a t e d t o . d r y n e s s i n  vacuum d e s i c c a t o r s over c o n c e n t r a t e d hydroxide.  hydrolysate,  purines The  and  the  pyrimidine  bases were e x t r a c t e d  of each base and  i n t o each were d e t e r m i n e d .  the r a d i o The  results  are g i v e n i n T a b l e I . I t i s seen from T a b l e I t h a t the i n c o r p o r a t i o n o f  l>i  C-  formate i n t o the bases of the a c i d s o l u b l e n u c l e o t i d e s was I n h i b i t e d . b y the p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s actinomycin  D i n the incubation.medium.  There was  not  of  a slight  i n c r e a s e i n the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o b o t h adenine and guanine.  The  v e r y h i g h count o b t a i n e d  i n the case  Table I E f f e c t o f a c t i n o m y c i n D on t h e i n c o r p o r a t i o n o f '*C-formate i n t o t h e bases o f t h e 1  n u c l e i c a c i d components o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in v i t r o . S p e c i f i c a c t i v i t y - counts p e r minute p e r umole. Acid-soluble nucleotides A c tc xi nn o -D /ml  RNA Adenine  Guanine  5420  437  113  676  5484  447  1879  908  5502  5804  1448  979  5716  1802  731  Adenine  Gua- • C y t - Ura- . nine osine c i l  0  5250  1559  775  0.05  5806  1718  0.5  5845  1.0 1.5  DNA C y t - Ura-. osine c i l  Ade. nine  Guanine  C y t - Thyos i n e mine  28  79  118  122  100  1745  83 '  32  94  117  99  120  1793  >fe Ul  428  88  37  67  101  78  86  1717  1  6079  347  22  7  42  125  85  92  1605  5492  3327  41  50  100  120  70  85  1680  1  - 46  o f u r a c i l may thymine.  -  be due t o the c o n t a m i n a t i o n o f t h e samples  by  I n Wyatt'.s s o l v e n t system, t h e s e two bases r u n c l o s e  t o each o t h e r .  Moreover  the c o n c e n t r a t i o n o f thymine i n t h e  a c i d s o l u b l e f r a c t i o n i s t o o low i n t h e sample t o d e t e c t i t on paper under U.V.  light.  The r a d i o a c t i v i t y i n c o r p o r a t e d  into  thymine i s always h i g h w h i c h i s a l s o r e f l e c t e d i n t h e thymine o b t a i n e d from  DNA.  An e x a m i n a t i o n o f the i n c o r p o r a t i o n o f ^ C - f ormate i n t o the RNA  and DNA  agrees w i t h many o f t h e o b s e r v a t i o n s r e p o r t e d by  other i n v e s t i g a t o r s .  I n the case o f RNA  t i o n of i n c o r p o r a t i o n of guanine.  1  t h e r e i s an  inhibi-  "*C-formate i n t o b o t h adenine and  However, t h e i n h i b i t i o n o f i n c o r p o r a t i o n i s g r e a t e r  i n t h e case o f guanine than adenine o f RNA.  Zbarsky has  (39)  r e p o r t e d p r e v i o u s l y a s i m i l a r e f f e c t o f a c t i n o m y c i n D on the i n c o r p o r a t i o n of  1!  *C-f ormate by N o v i k o f f hepatoma c e l l s  E h r l i c h a s c i t e s carcinoma c e l l s .  I n t h e case o f DNA,  h i g h e s t s p e c i f i c a c t i v i t y o b t a i n e d i s f o r t h e base  the  thymine.  I t i s known t h a t the m e t h y l group o f thymine i s formed one-carbon s o u r c e s (45).  and  from  S m e H i e e t a l . (103) have observed  a h i g h e r 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 i t y i n thymine, t h a n i n t o p u r i n e s when  1h  C ~ f o r m a t e . was used as a p r e c u r s o r .  suggested t h a t o f DNA may  They have  the h i g h e r l a b e l l i n g o f thymine compared t o p u r i n e s  be due t o the s m a l l p o o l s i z e o f thymine  In t h e case o f t h e p u r i n e s t h e i n c o r p o r a t i o n o f  14  precursors.  C-formate i s  v e r y much l e s s because of the r e l a t i v e l y l a r g e p o o l s i z e of nucleic acid precursors. i n the i n c o r p o r a t i o n o f  Here a l s o t h e r e was 1!  a s l i g h t decrease  *C-f ormate i n t o guanine as observed  - 47 -  by Zbarsky i n h i s e x p e r i m e n t s ( 3 9 ) . As t h e s u b j e c t o f g r e a t e r i n t e r e s t was t o study t h e e f f e c t o f a c t i n o m y c i n D on t h e f o r m a t i o n o f a c i d s o l u b l e n u c l e o t i d e s , f u r t h e r i n v e s t i g a t i o n s were c a r r i e d o u t .  I t was o b s e r v e d  p r e v i o u s l y t h a t t h e r e was no i n h i b i t i o n 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 i n t o t h e bases o f a c i d s o l u b l e n u c l e o tides;. llf  However, t h e r e was a d e c r e a s e i n t h e i n c o r p o r a t i o n o f  C - f o r m a t e i n t o t h e bases o f RNA. I t i s known t h a t t h e  corresponding  nucleoside triphosphates are required f o r the  s y n t h e s i s o f b o t h R N A and DNA.  I n t h e de now  pathway o f  s y n t h e s i s o f p u r i n e r i b o n u c l e o t i d e s , "*(:-formate 1  i s known t o be i n -  c o r p o r a t e d b e f o r e t h e f o r m a t i o n o f t h e A M P and GMP. F a i l u r e o f t h e s e m a t e r i a l s t o form t h e h i g h e r phosphate may thus r e duce t h e 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 i t y i n t o t h e n u c l e i c a c i d s though t h e r e may be i n c o r p o r a t i o n o f C - f o r m a t e i n t o t h e mono1 4  phosphates.  Estimation of the 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 i t y  i n t h e bases o f t h e a c i d s o l u b l e s thus may n o t g i v e any i n f o r m a t i o n r e g a r d i n g t h e f o r m a t i o n o f t h e t r i p h o s p h a t e s which a r e e s s e n t i a l f o r p o l y m e r i z a t i o n t o form b o t h  DNSL  and  RNA.  Further.,  i f t h e r e i s o n l y an i n h i b i t i o n o f p o l y m e r i z a t i o n o f t h e n u c l e o s i d e t r i p h o s p h a t e and no i n h i b i t i o n o f t h e i r f o r m a t i o n ,  then  an i n c r e a s e i n c o n c e n t r a t i o n o f t h e t r i p h o s p h a t e s c o u l d be expected i n the a c i d s o l u b l e p o o l .  So i t was n e c e s s a r y  to iso-  l a t e t h e v a r i o u s n u c l e o s i d e phosphates o f t h e a c i d s o l u b l e p o o l and t o e s t i m a t e them t o d e t e r m i n e whether t h e observed t i o n of i n c o r p o r a t i o n of C - f o r m a t e 1 k  of a c t i n o m y c i n  inhibi-  i n t o R N A was due t o t h e e f f e c t  D o n l y on t h e p o l y m e r i z a t i o n r e a c t i o n , o r on t h e  -  48  -  de novo s y n t h e s i s o f r i b o n u c l e o t i d e s . 3) S e p a r a t i o n o f A c i d S o l u b l e N u c l e o t i d e s by Paper Chromat o g r a p h y - Development o f a Method. a) Paper chromatography o f r i b o n u c l e o t i d e s : - S e p a r a t i o n o f r i b o n u c l e o t i d e s by paper chromatography i s b e s e t w i t h s p e c i a l problems as these compounds c o n t a i n one o r more h i g h l y p o l a r phosphoric  a c i d groups, the i o n i z a t i o n o f which c o n s i d e r a b l y  a f f e c t the r e s o l u t i o n s .  To d e v e l o p a method o f s e p a r a t i o n ,  a l a r g e v a r i e t y o f s o l v e n t systems were t r i e d u s i n g  authentic  s t a n d a r d n u c l e o t i d e m i x t u r e s , b u t t h e s e p a r a t i o n s were nevertheless not satisfactory. (104)  S e v e r a l y e a r s ago Krebs and Hems  employed two d i m e n s i o n a l  paper chromatography f o r t h e  s e p a r a t i o n o n l y o f adenosine a n d ; i n o s i n e mixtures.  phosphates from r e a c t i o n  S i m i l a r l y B e r g k v i s t and Deutsch (105) r e p o r t e d t h e  s e p a r a t i o n o f mono, d i , and t r i phosphates o f g u a n o s i n e , adenos i n e , i n o s i n e and u r i d i n e by two d i m e n s i o n a l graphy.  paper chromato-  However t h i s method f a i l e d t o s e p a r a t e u r i d i n e and  c y t i d i n e phosphates which always m i g r a t e d systems employed.  together i n the solvent  The h i g h c o n c e n t r a t i o n o f ammonium s u l p h a t e  i n t h e second s o l v e n t system l i m i t s t h e use o f t h i s method f o r the q u a n t i t a t i v e e s t i m a t i o n o f n u c l e o t i d e s and t h e measurement of  radioactivity. From r e s u l t s o f chromatography employing d i f f e r e n t s o l v e n t  systems i n one d i r e c t i o n , i n f o r m a t i o n was o b t a i n e d , w h i c h p e r m i t t e d the combination  o f two d i f f e r e n t s o l v e n t systems which  were found s a t i s f a c t o r y f o r t h e s e p a r a t i o n p f r i b o n u c l e o t i d e s  - 49 by two d i m e n s i o n a l The  paper chromatography a r e g i v e n on page 35.  s o l v e n t system used f o r chromatographyv.in- the  first  d i r e c t i o n was a m o d i f i c a t i o n o f t h e s o l v e n t system recommended for  t h e r e s o l u t i o n o f guanine n u c l e o t i d e s  (97).  The s o l v e n t  system used i n t h e second d i r e c t i o n was o r i g i n a l l y developed by P a l a d i n i and L e l o i r  (106).  The r e l a t i v e m o b i l i t i e s o f t h e  v a r i o u s 5 ' — r i b o n u c l e o t i d e s i n each o f these s o l v e n t systems are g i v e n i n T a b l e I T , These were d e t e r m i n e d by c a r r y i n g o u t d e s c e n d i n g chromatography o f a u t h e n t i c n u c l e o s i d e phosphates (Pabst B i o c h e m i c a l s , Milwaukee)^ No.  40 f i l t e r p a p e r s .  on l a r g e s h e e t s o f Whatman  The t r u e R^ v a l u e s - t h e r a t i o o f d i s -  tance o f s o l u t e m i g r a t i o n t o t h a t o f s o l v e n t - was n o t e s t i m a t e d because t h e s o l v e n t was a l l o w e d t o f l o w o f f t h e paper f o r h o u r s . Instead, the r a t e of migration r e l a t i v e t o t h a t of a standard n u c l e o t i d e , 7AMP, i s g i v e n i n T a b l e I I .  I t c a n be seen from t h e  t a b l e t h a t a w e l l marked s e p a r a t i o n o f t h e common r i b o n u c l e o t i d e s can be o b t a i n e d by c a r r y i n g o u t two d i m e n s i o n a l graphy u s i n g t h e two s o l v e n t systems d e s c r i b e d . which are not separated  chromato-  Substances  i n t h e f i r s t d i r e c t i o n f o r example,  GMP, UMP, e t c . a r e s e p a r a t e d w e l l on chromatography i n t h e second s o l v e n t system. Two d i m e n s i o n a l a mixture  chromatography was c a r r i e d o u t a f t e r a p p l y i n g  o f t h e r i b o n u c l e o t i d e s on t o t h e paper and d e v e l o p i n g  the chromatogram i n t h e f i r s t d i r e c t i o n i n  isobutyric-ammonia-  water system and then, i n t h e second d i r e c t i o n i n ammonium acetate-ethanol  system.  are shown i n F i g u r e 7.  The r e l a t i v e p o s i t i o n s o f t h e n u c l e o t i d e s I t can be seen from t h e diagram t h a t  - 50 Table I I 'Rg, v a l u e s o f r i b o n u c l e o t i d e s i n two s o l v e n t Solvent I.  I s o b u t y r i c - ammonia - w a t e r pH  S o l v e n t I I . Ammonium a c e t a t e - e t h a n o l pH  F (AMP) Nucleotide .Solvent I  Solvent I I  AMP  1.00  1.00  ADP  0.72  0.44  ATP  0.49  0.20  GMP  0.43  0.72  GDP  0.28  0.33  GTP  0.21  0.16  UMP  0.43  1.20  UDP  0.29  0.54  UTP  0.22  0.31  CMP  0.85  0.94  CDP  0.54  0.42  CTP  0.40  0.19  IMP  0.43  0.85  TMP  0.79  1.80  NAD  0.84  0.54  7.  systems. 4.3.  Solvent I origin  ^—' UTpO  (Q  CfcP  A T P  ^,  D  QjDP  D p  Q N A D  O  G M P  OiMP  ^CMP ^  Q  AMP  QjMP  F i g u r e 7.  A Schematic r e p r e s e n t a t i o n o f two d i m e n s i o n a l paper chromatogram p f r i b o nucleotides. S o l v e n t I . I s o b u t y r i c - a m m o n i a - w a t e r (30 h o u r s ) . S o l v e n t I I . Ammonia a c e t a t e - e t h a n o l ' ( 3 6 h o u r s ) .  the mixture  o f t h e 12 r i b o n u c l e o t i d e s a r e w e l l s e p a r a t e d i n  chromatography i n t h e s o l v e n t systems d e s c r i b e d . t i e s of the deoxynucleotides  The m o b i l i -  were n o t d e t e r m i n e d .  t h a t t h e y a l s o move w i t h t h e c o r r e s p o n d i n g  I t i s likely  ribonucleotides.  However, t h e c o n c e n t r a t i o n o f d e o x y n u c l e o t i d e s  a r e low compared  t o t h e r i b o n u c l e o t i d e s , t o i n t e r f e r e w i t h q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s (107) i n t h e p r e s e n t i n v e s t i g a t i o n s . b) Paper chromatography o f a c i d s o l u b l e n u c l e o t i d e s  from  E h r l i c h a s c i t e s c a r c i n o m a c e l l s : - To t e s t t h e e f f i c a c y o f t h e method i n s e p a r a t i n g n u c l e o t i d e s from b i o l o g i c a l m a t e r i a l , an a c i d s o l u b l e e x t r a c t was made from 5-6 ml o f E h r l i c h a s c i t e s carcinoma c e l l s . was  The p e r c h l o r i c a c i d e x t r a c t o f n u c l e o t i d e s  n e u t r a l i z e d i n t h e c o l d w i t h 10 N KOH and t h e p o t a s s i u m p e r -  c h l o r a t e was removed by c e n t r i f u g a t i o n a f t e r c o o l i n g t h e mixt u r e f o r s e v e r a l hours.  The s u p e r n a t a n t f l u i d c o n t a i n i n g t h e  n u c l e o t i d e s was l y o p h i l i z e d and t h e r e s i d u e e x t r a c t e d w i t h a known volume o f water.-. A volume o f t h i s n u c l e o t i d e s o l u t i o n corresponding  t o 1 ml o f packed c e l l s was a p p l i e d on t o t h e  c o r n e r o f a l a r g e s h e e t o f Whatman No. 1 paper.  The chromato-  gram was d e v e l o p e d i n i s o b u t y r i c acid-ammonia-water i n t h e f i r s t d i r e c t i o n f o r 32 h o u r s , and t h e U.V. a b s o r b i n g  spots were  marked a f t e r d r y i n g t h e paper i n t h e a i r .  There was no s a t i s -  f a c t o r y s e p a r a t i o n of n u c l e o t i d e s achieved  by t h i s  Instead of c l e a r separate m a t e r i a l was o b s e r v e d .  process.  s p o t s , a l a r g e s t r e a k o f U.V.  absorbin  No f u r t h e r r e s o l u t i o n was o b t a i n e d by  .chromatography i n t h e second d i r e c t i o n .  I t was f e l t t h a t t h e  f a i l u r e to obtain s a t i s f a c t o r y separation of nucleotides  from  E h r l i c h a s c i t e s tumour c e l l e x t r a c t s was. due t o t h e p r e s e n c e of s a l t s i n t h e n u c l e o t i d e m i x t u r e a p p l i e d on paper.  The presence  of s a l t s i n t h e m i x t u r e has been r e p o r t e d t o a l t e r t h e m o b i l i t i e s of n u c l e i c a c i d components on chromatograms (108).  Further,  a preliminary run of a mixture pf authentic nucleotides  with  sodium c h l o r i d e added t o i t gave chromatograms s i m i l a r t o t h a t o b s e r v e d w i t h t h e a c i d s o l u b l e e x t r a c t from E h r l i c h a s c i t e s carcinoma c e l l s .  I t was d e s i r a b l e t o remove t h e s a l t from t h e  • n u c l e o t i d e e x t r a c t e d from E h r l i c h a s c i t e s c e l l s p r i o r t o chromatography . The d i f f e r e n t methods t e s t e d f o r t h e i s o l a t i o n o f n u c l e o t i d e s from a s c i t e s s o l u b l e f r a c t i o n f r e e o f i n t e r f e r i n g m a t e r i a l s i n chromatographs  are described  below.  c) Removal o f s u b s t a n c e s i n t e r f e r i n g i n chromatography o f acid soluble nucleotides:i ) .:. d e s a l t i n g o f n u c l e o t i d e s u s i n g D E A E - c e l l u l o s e : R u s h i z k y and Sober  have used D E A E - c e l l u l o s e  o l i g o n u c l e o t i d e s from s a l t s o l u t i o n s (109).  f o r adsorption of From t h e c e l l u l o s e  n u c l e o t i d e s can be e l u t e d by ammonium' c a r b o n a t e w h i c h can be e a s i l y removed by l y o p h i l i z a t i o n . DEAE-cellulose  T h i s method o f a d s o r p t i o n on  and e l u t i o n from i t was t r i e d f o r s e p a r a t i n g  n u c l e o t i d e s from s a l t s o f t h e a c i d s o l u b l e f r a c t i o n .  DEAE-  c e l l u l o s e p r e p a r e d i n t h e c a r b o n a t e form a c c o r d i n g t o t h e p r o c e d u r e o f Tomlinson and Tener(lOO) was packed i n t o a column 25 x 1 cm under p r e s s u r e  (5 l b s / s q . i n . ) .  Ther-neutral s o l u t i o n  of a c i d s o l u b l e n u c l e o t i d e s e x t r a c t e d from E h r l i c h a s c i t e s  - 54 c a r c i n o m a c e l l s was s o l u t i o n was t i o n was  d i l u t e d w i t h 4 volumes o f water and  added t o the top of the column.  A f t e r the s o l u -  passed i n t o the column, the column was  w a t e r , and the e f f l u e n t was  m o n i t o r e d f o r U.V.  washed w i t h absorbing  m a t e r i a l using a G i l s o n Medical E l e c t r o n i c s monitor. e f f l u e n t from the column was  the  The  found t o c o n t a i n a c o n s i d e r a b l e  amount of u l t r a v i o l e t - - a b s o r b i n g m a t e r i a l e l u t e d by the wash. T h i s may  p a r t l y be the e l u t i o n of n u c l e o t i d e s due  t o the  p r e s e n c e of s a l t s because n u c l e o t i d e s can be r e l e a s e d from DEAE-cellulose  column by s a l t s o l u t i o n s .  So, the method  not s a t i s f a c t o r y f o r the p r e s e n t s e p a r a t i o n o f s a l t s and tides.  The  was nucleo-  n a t u r e of s u b s t a n c e s which were e l u t e d from the  column on washing was  not examined f u r t h e r .  i i ) d e s a l t i n g by g e l f i l t r a t i o n : - G e l e x c l u s i o n c h r o matography i s w i d e l y used t o . s e p a r a t e molecular  size.  The  u s i n g B i o - G e l P-2 was  substances d i f f e r i n g i n  s e p a r a t i o n of n u c l e o t i d e s by t h i s  (Bio-Rad  technique  L a b o r a t o r i e s , Richmond, C a l i f o r n i a )  r e p o r t e d by U z i e l and Cohn (110).  T h i s method was  tried  f o r the s e p a r a t i o n of n u c l e o t i d e s o f the a c i d s o l u b l e e x t r a c t from the s u b s t a n c e s i n t e r f e r i n g i n chromatography. B i o - G e l P-2 night.  The  (50-100 mesh) was  e q u i l i b r a t e d w i t h water o v e r -  s w o l l e n beads were a l l o w e d t o s e t t l e i n t o a column  of s i z e 50 x 1 cm.  The  a c i d s o l u b l e e x t r a c t prepared  from 5.6  ml.  of E h r l i c h a s c i t e s carcinoma c e l l s , a f t e r n e u t r a l i z a t i o n and removal of p o t a s s i u m p e r c h l o r a t e , was  lyophilized.  The  residue  e x t r a c t e d w i t h a s m a l l q u a n t i t y of water and the s o l u t i o n added t o the top of the column.  E l u t i o n was  s t a r t e d w i t h water a t a  was  - 55 f l o w r a t e of 2 ml per sq. cm a r e a of c r o s s s e c t i o n o f the column per m i n u t e .  5 ml f r a c t i o n s were c o l l e c t e d i n a f r a c t i o n c o l -  lector.  f r a c t i o n s were examined a t 260 my  The  of any U.V.  absorbing  peak combined. was  observed.  Only one The  e v a p o r a t o r and  m a t e r i a l and  f o r the p r e s e n c e  the samples f o r m i n g a s i n g l e  s i n g l e l a r g e peak w i t h U.V.  p o o l e d sample was  absorbance  evaporated i n a f l a s h  t h e n e x t r a c t e d w i t h a known volume o f w a t e r .  a l i q u o t sample of t h i s s o l u t i o n c o r r e s p o n d i n g t o 1 ml o f volume was paper.  a p p l i e d on t o a l a r g e s h e e t of Whatman No.  Two  described  dimensional  before.  chromatography was  cell  1 filter  t h e n c a r r i e d out  as  E x a m i n a t i o n of the chromatograms under  l i g h t showed s t r e a k i n g as was  seen p r e v i o u s l y , w i t h o u t  c l e a r s e p a r a t i o n of n u c l e o t i d e s .  An  U z i e l has  U.V.  any  l a t e r pointed  that gel f i l t r a t i o n  of n u c l e o t i d e s  i s i n f l u e n c e d by  f a c t o r s such as pH,  t e m p e r a t u r e , bead s i z e e t c .  out  various  (111).  More-  o v e r , i m m e d i a t e l y a f t e r the e l u t i o n of the m o n o n u c l e o t i d e s , the s a l t s are e l u t e d from the column and of overlaps.  t h e r e i s the  possibility  I t i s l i k e l y t h a t any. p r o t e i n i n the a c i d s o l u b l e  e x t r a c t w i l l be e l u t e d w i t h the n u c l e o t i d e s and  could i n t e r f e r e  w i t h the s e p a r a t i o n of n u c l e o t i d e s on chromatograms. iii)  adsorption  of: n u c l e o t i d e s  t i o n of p u r i n e s , p y r i d i n e s and c h a r c o a l and  on c h a r c o a l : - Adsorp-  t h e i r d e r i v a t i v e s on a c t i v a t e d  e l u t i o n w i t h s a l t f r e e s o l u t i o n i s w i d e l y used f o r  s e p a r a t i o n of t h e s e compounds from s a l t s o l u t i o n s . the c h a r c o a l has  t o be p u r i f i e d and  the experiments described  Before  s p e c i a l l y prepared.  h e r e i n , the c h a r c o a l was  use,  For  p r e p a r e d by  a combination  o f methods d e s c r i b e d by Thomson (112)  and  by  P l a i s t e d and Riggs (113) . One i n 1000 The  hundred grams of Darco G.  60 c h a r c o a l was  ml of 2 N HCl and the m i x t u r e was  c h a r c o a l was  suspended  r e f l u x e d f o r 3 hours  t h e n c o l l e c t e d by f i l t r a t i o n on a Buchner  f u n n e l and washed w i t h d i s t i l l e d water u n t i l the f i l t r a t e neutral.  t h e n suspended In  The washed c h a r c o a l was  2 M a c e t i c a c i d and the m i x t u r e was suspension  was  1000  ml  r e f l u x e d f o r 2 hours.  of The  f i l t e r e d on a Buchner f u n n e l a n d ^ w h i l e s t i l l  the f u n n e l ^ t h e c h a r c o a l was u n t i l the f i l t r a t e was o f ammoniacal e t h a n o l  (Amm:18 v o l , H 0 2  amounts  43 v o l , ' e t h a n o l 50 v o l ) .  a g a i n washed w i t h d i s t i l l e d water  u n t i l n e u t r a l and the e f f l u e n t . h a d an a b s o r p t i o n of 0.05 The  on  washed w i t h g l a s s - d i s t i l l e d water  n e u t r a l and then w i t h copious  F i n a l l y , t h e c h a r c o a l was  a t 260 my.  was,  c h a r c o a l was  or l e s s  d r i e d and weighed and t h e n sus-  pended i n d i s t i l l e d water t o g i v e a c o n c e n t r a t i o n of 10 gms 100  ml. For a d s o r p t i o n e x p e r i m e n t s ,  was  per  an a l i q u o t of t h i s  suspension  used. The  ^ e f f i c a c y of the method was  nucleotides.  Two  t a i n i n g a mixture  standard  f i r s t tested with authentic  s o l u t i o n s of n u c l e o t i d e s one  con-  of adenine n u c l e o t i d e s and the o t h e r c o n t a i n i n g  guanine n u c l e o t i d e s were p r e p a r e d .  A known c o n c e n t r a t i o n of  these n u c l e o t i d e s i n a t o t a l volume o f 4 ml was ml of. a 10% c h a r c o a l s u s p e n s i o n . t i d e s on c h a r c o a l was described previously.  The  mixed w i t h  a d s o r p t i o n of the  0.2  nucleo-  c a r r i e d out a c c o r d i n g t o the p r o c e d u r e For the purpose of e l u t i o n t h r e e  different  - .57'- ''  s o l v e n t systems were employed and t h e r e c o v e r y o f m a t e r i a l i n each s o l v e n t m i x t u r e  determined.  S o l v e n t s employed, were:  1) cone, ammonia-ethanol-water (5:50:45 v/v) 2) t r i e t h y l a m i n e - e t h a n o l - w a t e r 60%  (112)  (5 v o l o f t r i e t h y l a m i n e i n 95 v o l  ethanol).  3) p y r i d i n e - e t h a n o l - w a t e r  (5 v o l o f p y r i d i n e i n 95 v o l o f  60% e t h a n o l ) . ( 9 8 ) . The  s u b s t a n c e s were e l u t e d by m i x i n g t h e c h a r c o a l w i t h 8  ml o f s o l v e n t under e x a m i n a t i o n b a t h a t 37°C f o r 3 h o u r s .  and k e e p i n g  t h e tubes i n a water  The tubes were c l o s e d t o p r e v e n t  any e v a p o r a t i o n and shaken o c c a s i o n a l l y t o d i s p e r s e t h e c h a r coal.  I n many cases e i t h e r t h e s o l v e n t •exhibited', h i g h U.V.  a b s o r p t i o n o r i t d i s p e r s e d f i n e carbon p a r t i c l e ' s t o g i v e a b l a c k s o l u t i o n on e x t r a c t i o n and hence t h e e s t i m a t i o n o f c o n c e n t r a t i o n o f n u c l e o t i d e s i n t h e e i u a t e by d i r e c t s p e c t r o p h o t o m e t r y was  not p o s s i b l e .  A f t e r e l u t i o n t h e s o l v e n t was e v a p o r a t e d i n  a f l a s h evaporator,  the residue d i s s o l v e d i n a small quantity  of water and t h e s o l u t i o n a p p l i e d on t o Whatman No. 4 0 f i l t e r paper.  The chromatogram was d e v e l o p e d i n i s o b u t y r i c . a c i d -  ammonia-water system, t h e U.V. a b s o r b i n g  s p o t s on t h e chromato-  gram were marked, e l u t e d and t h e e l u a t e s made up t o a known volume..  The c o n c e n t r a t i o n o f t h e n u c l e o t i d e s i n s o l u t i o n was  d e t e r m i n e d by s p e c t r o p h o t o m e t r y and t h e r e c o v e r y was c a l c u l a t e d . The  d a t a "are p r e s e n t e d  i n Table I I I .  From t h e t a b l e i t i s seen t h a t p ' y r i d i n e - e t h a n o l m i x t u r e was  t h e b e s t s u i t e d s o l v e n t system f o r t h e e l u t i o n o f n u c l e o -  t i d e s from c h a r c o a l .  Thomson (112) has r e p o r t e d t h a t t h e  -  58 -  Table I I I Recovery o f n u c l e o t i d e s adsorbed on c h a r c o a l by e l u t i o n w i t h t h r e e d i f f e r e n t s o l v e n t systems. D e t a i l s are given i n text. P e r c e n t a g e Recovery Nucleotide mixture  Concentration as t h e monophosphate (ymoles)  Solvent I  Solvent I I  Solvent I I I  Adenine Nucleotides (AMP, ADP & ATP)  0.299  95.5  96  81.8  0.430  91.2  81.3  78.7  Guanine Nucleotides (GMP, GDP & GTP)  0.417  88.8  72  67.0  0.625  92.7  67.5  68.4  Solvent I .  Pyridine-ethanol-water.  Solvent I I .  Triethylamine -ethanol-water.  Solvent I I I  Ammonia-ethanol-water.  - 59 -  e t h a n o l i c ammonia system g i v e s l e s s complete e l u t i o n t h a n t h e p y r i d i n e w a t e r s o l v e n t system.  The t r i e t h a n o l a m i r i e - e t h a n o l -  w a t e r system was found t o d i s p e r s e much o f t h e f i n e c h a r c o a l i n the s o l v e n t forming a black suspension.  Thomson has p o i n t e d o u t  t h a t though the, p u r i f i c a t i o n o f c h a r c o a l " i s messy and l a b o r i o u s , i t cannot s a f e l y be a b r i d g e d "  (112).  For e f f i c i e n t  adsorption,  of t h e n u c l e o t i d e s t h e pH o f t h e n u c l e o t i d e s o l u t i o n t o be maintained  between 1 and 2.. I t was s u g g e s t e d by Thomson and by  ^Grav t h a t a r e c o v e r y o f more t h a n 60% o f t h e absorbed m a t e r i a l i s c o n s i d e r e d t o be s a t i s f a c t o r y  (112, 1 0 7 ) . I n t h e p r e s e n t  e x p e r i m e n t s t h e r e c o v e r y r a t e s a r e h i g h e r and hence t h e method was used t o s e p a r a t e n u c l e o t i d e s from s a l t s o f t h e a c i d s o l u b l e fraction. d) C h a r a c t e r i z a t i o n o f t h e s u b s t a n c e s e l u t e d from chromatograms:- A f t e r t h e methods f o r t h e r e c o v e r y o f n u c l e o t i d e s from s o l u t i o n s by a d s o r p t i o n on and e l u t i o n from c h a r c o a l were s t a n d a r d i z e d , these methods were a p p l i e d t o t h e a c i d s o l u b l e f r a c t i o n s from E h r l i c h a s c i t e s c a r c i n o m a c e l l s .  The a c i d s o l u b l e  n u c l e o t i d e s were i s o l a t e d from E h r l i c h a s c i t e s c e l l e x t r a c t s by t h e t e c h n i q u e  o f c h a r c o a l a d s o r p t i o n and e l u t i o n .  The r e -  s u l t i n g s o l u t i o n o f n u c l e o t i d e s f r e e d from o t h e r s u b s t a n c e s i n t e r f e r i n g i n chromatography was a p p l i e d on t o a s h e e t o f Whatman No.. 1 f i l t e r paper and two d i m e n s i o n a l formed as d e s c r i b e d .  On e x a m i n a t i o n  chromatography was p e r -  o f chromatograms under U.V.  l i g h t c l e a r s e p a r a t i o n s o f many m a t e r i a l s i n t o d i s t i n c t were o b s e r v e d .  spots  These s p o t s were marked o u t , e l u t e d and i d e n t i f i e d  - 60  -  by the f o l l o w i n g p r o c e d u r e s . From the p a t t e r n o f d i s t r i b u t i o n o f a u t h e n t i c m i x t u r e  of  n u c l e o t i d e s on chromatograms, the p o s i t i o n s of many o f the. n u c l e o t i d e s were known.  The  a b s o r p t i o n s p e c t r a of s u b s t a n c e s  a f t e r e l u t i o n from chromatogram were o b t a i n e d u s i n g a Cary r e c o r d i n g s p e c t r o p h o t o m e t e r , model 15.  I t was  o b s e r v e d from  the chromatogram t h a t the m o b i l i t y o f a p a r t i c u l a r d e c r e a s e d w i t h i n c r e a s e i n the degree of  nucleotide  phosphorylation.  Thus the monophosphate move f a s t e r than the d i p h o s p h a t e s the t r i p h o s p h a t e s  are l e a s t m i g r a t o r y  the same spectrum.  The  and  of the compounds g i v i n g  c o n c e n t r a t i o n of some of the  nucleo-  t i d e s i n the a c i d s o l u b l e f r a c t i o n from 1 ml of E h r l i c h a s c i t e s c e l l s are too low t o be c l e a r l y d e t e c t e d  on paper.  To know  t h e . p o s i t i o n s of t h e s e s u b s t a n c e s on chromatogram, a m o d i f i e d technique  o f ' f i n g e r p r i n t i n g ' was  employed.  The  s o l u t i o n of  a c i d s o l u b l e n u c l e o t i d e s from 2 ml of c e l l s u s p e n s i o n was i d e d i n t o two were added. was  e q u a l p a r t s and t o one p a r t a u t h e n t i c The  div-  nucleotides  a u t h e n t i c n u c l e o t i d e s added were whose  low i n the a c i d s o l u b l e e x t r a c t .  The  two  a p p l i e d on t o s h e e t s of f i l t e r paper and two chromatography was  content  s o l u t i o n s were dimensional  c a r r i e d out under i d e n t i c a l c o n d i t i o n s .  two  chromatograms were viewed under U.V.  The  d i f f e r e n t i n t e n s i t i e s of t e s t m a t e r i a l on the chromatograms  can be e a s i l y r e c o g n i z e d  l i g h t and  The  compared.  thus v e r i f y i n g the p o s i t i o n of  p a r t i c u l a r s u b s t a n c e which was  the  d i f f i c u l t t o l o c a t e i n the  matogram of a c i d s o l u b l e e x t r a c t s .  As a s t e p f u r t h e r , co-  chromatography of some of the s u b s t a n c e s i s o l a t e d from the  chro-  - 61 chrpmatograms w i t h a u t h e n t i c samples was a l s o  tried.  For f u r t h e r c o n f i r m a t i o n o f t h e substances i s o l a t e d , the amount o f phosphorus  p r e s e n t i n t h e s e compounds was d e t e r m i n e d .  The e l u t e d samples o f t h e same n u c l e o t i d e s were p o o l e d and t h e volume o f the. s o l u t i o n reduced by l y o p h i l i z a t i o n . .  The d i f f e r e n t  s o l u t i o n s o f t h e n u c l e o t i d e s were d i l u t e d so t h a t t h e c o n c e n t r a t i o n o f n u c l e o t i d e s e x p r e s s e d as O.D./ml was n e a r l y ^ e q u a l i n a l l cases.  As t h e s t u d y p e r t a i n e d m a i n l y t o p u r i n e n u c l e o t i d e s ,  the a n a l y s e s were c a r r i e d o u t o n l y f o r t h e s e compounds.  The  r e s u l t s a r e g i v e n i n Table IV. The v a l u e f o r phosphorus side' phosphates  indicated the d i f f e r e n t nucleo-  from t h e chromatograms.  This f u r t h e r confirms  the paper c h r o m a t o g r a p h i c s e p a r a t i o n o f t h e d i f f e r e n t n u c l e o s i d e phosphates  by t h e method employed.  I n t h e chromatograms, o b t a i n e d by t h e method employed i n a d d i t i o n t o t h e t h r e e adenine n u c l e o t i d e s , another  substance  h a v i n g t h e same s p e c t r a l c h a r a c t e r i s t i c o f adenine n u c l e o t i d e appeared.  The p o s i t i o n o f t h e substance on chromatogram was  c l o s e t o ADP and t h i s s u b s t a n c e can e a s i l y be m i s t a k e n f o r ADP. However, t h e m a t e r i a l was i d e n t i f i e d as NAD.  For i d e n t i f i c a t i o n ,  the change i n t h e s p e c t r a o f t h e c y a n i d e complex o f t h e subs t a n c e , as w e l l as t h e reduced NADH, was o b t a i n e d i n Cary spectrophotometer.  The NAD was reduced by t h e a l c o h o l  dehydro-  genase (Worthington B i o c h e m i c a l Corp.) a c c o r d i n g t o t h e p r o cedure g i v e n i n P a b s t C i r c u l a r - 1 0 p l e x and t h e NADH  +  (97).  Both t h e c y a n i d e com-  show a new a b s o r p t i o n peak a t 340 mu.  S i n c e t h e s e methods were d e v e l o p e d , s i m i l a r  procedures  - 62 T a b l e IV Phosphorus d e t e r m i n a t i o n s  on p u r i n e  nucleotides  -obtained from t w o - d i m e n s i o n a l chromatograms. T h e o r e t i c a l v a l u e s were c a l c u l a t e d u s i n g E ^ max  Nucleotide  Concentration (O.D./ml)  Phosphorus c o n c e n t r a t i o n (yg/ml) Found  Theoretical  AMP  1.78  4.0  3.75  ADP  1.88  7.2  7.56  ATP  1.83  11.0  11.27  GMP  1.85  3.6  4.18  GDP  1.70  7.3  7.6  GTP  1.2  7.8  8.1  - 63  -  were r e p o r t e d by o t h e r s f o r t h e s e p a r a t i o n of a c i d s o l u b l e n u c l e o t i d e s from p l a n t  (114)  4) E f f e c t of A c t i n o m y c i n  and  animal t i s s u e s  (115).  D on the I n c o r p o r a t i o n of  i n t o the A c i d S o l u b l e N u c l e o t i d e s  1,lf  C-For mate  of E h r l i c h A s c i t e s Carcinoma  Cells. Once the method o f paper chromatography was  developed f o r  the s e p a r a t i o n o f v a r i o u s r i b o n u c l e o t i d e s , the e f f e c t o f a c t i n o mycin D on the f o r m a t i o n of a c i d s o l u b l e n u c l e o t i d e s i n E h r l i c h a s c i t e s c a r c i n o m a c e l l s was  examined.  Incubation  experiments  s i m i l a r t o the one  d e s c r i b e d p r e v i o u s l y were c a r r i e d o u t .  incubation mixture  contained  u c u r i e of  1  "^-formate and  20 umoles of sodium f o r m a t e , 5  10 ml o f E h r l i c h a s c i t e s carcinoma  c e l l s u s p e n s i o n i n Krebs R i n g e r phosphate b u f f e r pH Actinomycin  D d i s s o l v e d i n 20% e t h a n o l was  amounts t o the a p p r o p r i a t e e t h a n o l was  Each  f l a s k s and  7.8.  added i n a p p r o p r i a t e  t o the c o n t r o l s o n l y  20%  added.  A f t e r i n c u b a t i o n the a c i d s o l u b l e n u c l e o t i d e s were e x t r a c t e d w i t h 0.2  M c o l d p e r c h l o r i c a c i d , n e u t r a l i z e d w i t h c o l d KOH  s t o r e d a t -20°  and  i f not used i m m e d i a t e l y a f t e r removal of p o t a s s i u m  perchlorate. The  a c i d s o l u b l e f r a c t i o n was  t i o n of 2 N'HCl. 0.2  ml o f 10%  c h a r c o a l was  The  b r o u g h t t o pH 1-2  n u c l e o t i d e s were adsorbed by  by the adding  s u s p e n s i o n of a c t i v a t e d c h a r c o a l i n w a t e r . c o l l e c t e d and the n u c l e o t i d e s e l u t e d w i t h  pyridine-ethanol solvent mixture. the r e s i d u e was  The  e l u a t e was  Two- d i m e n s i o n a l  chromatography was  The  the  evaporated,  d i s s o l v e d i n a s m a l l q u a n t i t y of water  a p p l i e d on t o a l a r g e s h e e t of Whatman No.  addi-  and  40 f i l t e r paper.  c a r r i e d out f o r the  separation  of the n u c l e o t i d e s . marked under U.V.  A f t e r chromatography t h e n u c l e o t i d e s were  and the s p o t s were e l u t e d w i t h water  c o l l e c t e d i n 10 ml b e a k e r s .  The w a t e r was  and the r e s i d u e i n the beaker was volume of 0.1 s o l u t i o n was  N HCl.  The  e v a p o r a t e d in  vacuo  diluted with a definite  c o n c e n t r a t i o n of t h e . n u c l e o t i d e s i n  d e t e r m i n e d by u l t r a v i o l e t s p e c t r o p h o t o m e t r y .  samples o f n u c l e o t i d e s were counted i n a l i q u i d counter  and  and t h e s p e c i f i c a c t i v i t y c a l c u l a t e d .  H a l f ml  scintillation The  results  are  g i v e n i n T a b l e V. Though these e x p e r i m e n t s were l a b o r i o u s and time consuming, the r e s u l t s o b t a i n e d were r a t h e r d i s a p p o i n t i n g . the d a t a p r e s e n t e d i n c o r p o r a t i o n of  14  E x a m i n a t i o n of  i n t a b l e s s h o w s v a r y i n g r e s u l t s b o t h on :  C - f o r m a t e i n t o the n u c l e o t i d e s and the  c o n c e n t r a t i o n o f the n u c l e o t i d e s i s o l a t e d .  I n these  the total  experiments  the r a d i o a c t i v e p r e c u r s o r i n c o r p o r a t e d i n t o t h e n u c l e o t i d e s i s expressed  i n terms of s p e c i f i c a c t i v i t y w h i c h i s dependent on  the c o n c e n t r a t i o n of the s u b s t a n c e s i n . s o l u t i o n . to  the de novo s y n t h e s i s , wherein . -  n u c l e o t i d e s may The  llf  In a d d i t i o n  C-formate i s incorporated,  a l s o be formed i n the c e l l by o t h e r mechanisms.  i n c r e a s e i n c o n c e n t r a t i o n o f n u c l e o t i d e s thus may  the s p e c i f i c a c t i v i t y t o some e x t e n t .  There i s no  decrease  correlation,  between the i n c r e a s e i n c o n c e n t r a t i o n of n u c l e o t i d e s observed and the- d e c r e a s e i n s p e c i f i c a c t i v i t y .  I t may  be p o i n t e d  out  t h a t i n the event of a normal s y n t h e s i s of n u c l e o t i d e s i n the c e l l i n p r e s e n c e of a c t i n o m y c i n RNA, The  an a c c u m u l a t i o n  D and i n h i b i t i o n of s y n t h e s i s . o f  of n u c l e o t i d e s can be e x p e c t e d i n the  cell.  i n c r e a s e i n c o n c e n t r a t i o n of n u c l e o t i d e s o b s e r v e d i n c e l l s  Table V Experiment  I  E f f e c t o f a c t i n o m y c i n D on t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o p u r i n e 1  n u c l e o t i d e s o f E h r l i c h a s c i t e s carcinoma c e l l s . by t w o - d i m e n s i o n a l  paper chromatography.  Specific activity ( (counts per minute p e r ymole) Act. D yg/ml  0  N u c l e o t i d e s were s e p a r a t e d  0.05  0.5  1.5  Concentration mymoles/10 ml s u s p e n s i o n 0  0.05  0.5  1.5  AMP  1821  2345  4280  5338  270.2  323.0  368.2  320.0  • ADP  1724  2310  4144  5273  420.0  298.0  321.2  640.1  ATP  1283  1877  3420  4445  225.5  201.4  200*7  624.5  GMP  1499  1368  1423  1327  50.4  50.4  59  76.8  GDP  1537  1230  1300  1121  49.0  42.6  42.6  96.0  GTP  723  587  1127  1240  14.5  23.0  13.0  23.0  Table  V(continued)  Concentration mymole/10 m l s u s p e n s i o n  Specific activity ( c o u n t s p e r m i n u t e p e r umole) Act. D yg/ml Expt.  0  1  2  'A  4  O  :.8  3202 3193 2944  3785 3607 3598  4654 3207 3458  3012 3010 2959  4801 4661 4368 Total  3050 2433 2457  GMP GDP GTP  2900 2790 2635  2628 2803 2629  2091 1856 1842  3526 2789 2645 Total  AMP ADP ATP  258. 0 421. 0 424. 0  2  217. 0 432. 0 563. 0  1103. 0. 1212 36. 8 52. 2 71. 9 70. 7 . . 2.9.0 . . 4.9 .6  4  8  196. 0 338. 0 427. 0 961  224. 0 421. 0 522. 0 1167  544. 0 368. 0 313. 0 1225. 0  36. 27 121. 7 60. 2 63. 4 121. 7 61. 5 21. 7 . .'.2.9.. 07 14. 5 128. 67 213. 6  151. 9  158. 3  143. 8  201 .3 394 .7 522 .0  193. 3 322. 6 337. 0  254. 3 508.6 177. 5 322.6 320. 0 400. 0 .3.59..0 . .5.1.4. . 2.90. .  3 4174 3693 3404  4917 4222 3855  2877 2717 3467  3613 3441 3056  3642 3707 3372  T o t a l . .11.18 .0 GMP GDP GTP  l  2  AMP ADP ATP  Expt.  i  3531 3504 3203  3863. 3211 2721  1370 2168 2135  2729 2436 2480  2625 2092 2067 Total.  39 .3 56 .0 . . . .4.4 .7 , 140 .0  852. ,0. .856. 5 .11.6.8..3. 1121.2 43. 0 111.8 32. 5 39. 9 68.3 52. 4 69. 0 50. 0 3 . . .18.1 2 , . .4.2. 29. 0 . . .3.0. 111. 5  122. 5  154. 8  19 8.2  -  can be due  67  -  t o such a mechanism o p e r a t i n g , though no  explana-  t i o n i s a v a i l a b l e f o r the d e c r e a s e i n c o n c e n t r a t i o n o b s e r v e d . 5) E f f e c t of A c t i n o m y c i n in vitro  by the  D on the I n c o r p o r a t i o n o f ^C-Formate 1  I n t e s t i n a l Mucosa of  Rat.  E h r l i c h c a r c i n o m a c e l l s , l i k e any o t h e r tumour c e l l s , m e t a b d l i c a l l y abnormal.  The d i f f e r e n t s u s c e p t i b i l i t y of  a n t i b i o t i c to d i f f e r e n t c e l l generations  may  are  the  be the cause of  the v a r i a t i o n s i n the r e s u l t s o b t a i n e d i n s t u d i e s u s i n g a s c i t e s tumour c e l l s .  To see the e f f e c t of the a c t i n o m y c i n  D on  b i o s y n t h e s i s of p u r i n e n u c l e o t i d e s i n a normal t i s s u e , s t u d i e s t o those r e p o r t e d were c a r r i e d out u s i n g the mucosal c e l l s of the r a t . llf  T h i s t i s s u e was  much the.same way  T h i s t i s s u e was  similar  intestinal  shown t o i n c o r p o r a t e  C - f o r m a t e i n t o a c i d s o l u b l e n u c l e o t i d e s , the RNA  DNA  the  and t o the  as i n E h r l i c h a s c i t e s c a r c i n o m a c e l l s  (85).  a l s o under i n v e s t i g a t i o n i n the l a b o r a t o r y f o r  the p r e s e n c e o f v a r i o u s enzymes. Rat i n t e s t i n a l mucosa was  scraped  o f f a f t e r f l u s h i n g the  i n t e s t i n e s w i t h c o l d Krebs R i n g e r phosphate and i n c u b a t i o n c a r r i e d out a c c o r d i n g t o p r o c e d u r e of S t e w a r t and The  s c r a p i n g s were p o o l e d  placed into f l a s k s .  Zbarsky  and weighed amounts (0.8-1 gm)  of mucosa.  a c t i v e formate (0.5 m i c r o c u r i e s per ml of s u s p e n s i o n ) and formate (2 umole/ml of s u s p e n s i o n ) were added.  was  (85). were  Enough c o l d Krebs R i n g e r phosphate b u f f e r  added t o each f l a s k t o g i v e a 10% s u s p e n s i o n  i n 20% e t h a n o l was  was  Radiosodium  Actinomycin  added t o the a p p r o p r i a t e f l a s k s .  D  Incubation  c a r r i e d out as f o r the E h r l i c h a s c i t e s carcinoma, c e l l s . The  a c i d s o l u b l e n u c l e o t i d e s were e x t r a c t e d as i n the case  of a s c i t e s c a r c i n o m a c e l l s .  was  They were chromatographed on paper  - 68  -  a f t e r a d s o r p t i o n on c h a r c o a l and e l u t i o n from i t .  The  nucleo-  t i d e s were i s o l a t e d and r a d i o a c t i v i t y i n each e s t i m a t e d . content  of the d i and t r i phosphates were low f o r e s t i m a t i o n  and hence were e x t r a c t e d and counted t o g e t h e r . fore given  f o r combined counts o f d i - a n d RNA  was  The  the n u c l e o t i d e s s e p a r a t e d  by two d i m e n s i o n a l  by the o r c i n o l method and  RNA -per se was  also obtained.  The  v a l u e s are  there-  of  i s o l a t e d , hydrolyzed  i n the case o f a c i d s o l u b l e n u c l e o t i d e s . estimated  The  ..triphosphates  adenosine and guanosine.  was  The  and  chromatography as  The  content  of  RNA  the i n c o r p o r a t i o n i n t o  r e s u l t s of these  experiments  are g i v e n i n T a b l e V I . The  i n c o r p o r a t i o n of ^ C - f o r m a t e i n t o the a c i d s o l u b l e  n u c l e o t i d e s v a r i e d i n p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n actinomycin  D.  Here a l s o , as i n the case of E h r l i c h a s c i t e s  carcinoma c e l l s , no d e f i n i t e c o n c l u s i o n can be drawn as t o e f f e c t , of actinomycin  nucleotide obtained  of  1If  the  D on n u c l e o t i d e b i o s y n t h e s i s from the  p a t t e r n of i n c o r p o r a t i o n .  The  of  The  c o n c e n t r a t i o n of the a c i d s o l u b l e  from i n t e s t i n a l mucosa was  a l s o very  small.  r e s u l t s showed a 60-66% i n h i b i t i o n i n the i n c o r p o r a t i o n  C - f o r m a t e i n t o RNA  a t the h i g h e s t c o n c e n t r a t i o n of a c t i n o -  mycin used.  The  i n h i b i t i o n of i n c o r p o r a t i o n o f  i n t o the RNA  n u c l e o t i d e s a l s o corresponded t o t h i s value.  the case of the RNA  n u c l e o t i d e s , t h e r e was  radioactivity  a greater  In  inhibition  o b s e r v e d i n the case of guanine n u c l e o t i d e s t h a n i n adenine nucleotides.  This observation  i s i n agreement w i t h r e s u l t s ob-  t a i n e d i n the case of a s c i t e s carcinoma c e l l s . The (85).  DNA  o b t a i n e d was  too low f o r any q u a n t i t a t i v e a n a l y s i s  E f f e c t o f a c t i n o m y c i n D on t h e i n c o r p o r a t i o n o f "*C-formate by t h e I n t e s t i n a l 1  mucosa o f t h e r a t . S p e c i f i c a c t i v i t y - c o u n t s p e r minute p e r umo.le Acid-soluble nucleotides Actinomycin D  AMP  cpm/O.D. ADP+ATP  GMP  cpm/O.D. GDP+GTP  RNA  % Inhibition i n T o t a l RNA . .  AMP  GMP  CMP  UMP  Expt. I  Expt.:  0  5913  218  5576  500  177  163  34  19  (Taken as - 0)  2.5  6848  247  5934  481  175  150  6  16  2  2  5.0  6471  204  5311  -  131  113  7  10  23  20  10.0  5436  157  7038  _  55  40  6  8  60  66  - 70 6) E f f e c t o f V a r y i n g C o n c e n t r a t i o n o f A c t i n o m y c i n D on t h e I n c o r p o r a t i o n o f ^ C - F o r m a t e in vitro  i n t o RNA -of E h r l i c h  A s c i t e s Carcinoma C e l l s . As t h e r e was v a r i a t i o n i n r e s u l t s o b t a i n e d i n these experiments  w i t h i n c r e a s i n g amount  o f a c t i n o m y c i n D, i t . w a s  d e s i r a b l e t o f i n d Out t h e e f f e c t a t a f i n a l c o n c e n t r a t i o n o f a c t i n o m y c i n D w h i c h would m a x i m a l l y E h r l i c h a s c i t e s carcinoma was observed  cells.  i n h i b i t RNA s y n t h e s i s i n  In the previous studies' i t  t h a t a c t i n o m y c i n D a t a c o n c e n t r a t i o n o f 10 ug/ml  w i l l i n h i b i t t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o . RNA o f r a t i n t e s t i n a l mucosa by about 66%. a s c i t e s carcinoma  Similar studies using E h r l i c h  c e l l s t o f i n d out a concentration of a c t i n o -  mycin D r e q u i r e d f o r maximum i n h i b i t i o n o f I n c o r p o r a t i o n o f llf  C - f o r m a t e i n t o RNA v/ere u n d e r t a k e n .  experiment  i s given i n Table V I I .  The r e s u l t s o f one such  I t i s seen from t h e t a b l e  t h a t t h e maximum i n h i b i t i o n o f RNA s y n t h e s i s i n E h r l i c h  ascites  c e l l s I s a t c o n c e n t r a t i o n o f 5 ug/ml o f a c t i n o m y c i n D.  However,  many i n v e s t i g a t o r s have used a c o n c e n t r a t i o n o f 10 ug/ml o r h i g h e r f o r s t u d y i n g t h e e f f e c t o f a c t i n o m y c i n D on many c e l l u l a r processes.  I n t h e l a t e r experiments  d e s c r i b e d i n t h i s thesis.,  a c o n c e n t r a t i o n o f 10 ug/ml o f a c t i n o m y c i n was used. 7) S e p a r a t i o n o f A c i d S o l u b l e N u c l e o t i d e s o f E h r l i c h A s c i t e s Carcinoma C e l l s by Chromatography on D E A E - c e l l u l o s e Co1umn-. I t was r e a l i z e d from p r e v i o u s experiments  t h a t where s m a l l  q u a n t i t i e s o f m a t e r i a l was used f o r a n a l y s i s , a s m a l l change i n c o n c e n t r a t i o n g e t s m a g n i f i e d and becomes s i g n i f i c a n t l y low o r high.  S i m i l a r l y as t h e c o n c e n t r a t i o n o f m a t e r i a l under examina-  - 71 Table V I I E f f e c t of varying concentrations of actinomycin D on t h e i n c o r p o r a t i o n o f Ehrlich ascites  1 k  C - f o r m a t e i n t o RNA by  c a r c i n o m a c e l l s in  vitro.  S p e c i f i c a c t i v i t y - counts p e r minute per umole Actinomycin D ug/ml  AMP  0 0.5  H  GMP  CMP  UMP  117  70  143  9  I  37  58  0 5  1.0  79  16  35  2.0  59  4  38  00  3  20  0  2  38  0  5.0 10.0  11  t i b n was l o w , a s m a l l change i n r a d i o a c t i v i t y i s l i a b l e t o change an i n h i b i t i o n t o a s t i m u l a t i o n o f i n c o r p o r a t i o n .  Because  o f t h e v a r i a t i o n i n r e s u l t s o b t a i n e d i n many e x p e r i m e n t s i n w h i c h s m a l l q u a n t i t i e s o f n u c l e o t i d e s were s e p a r a t e d by paper chromatography,  I t was d e s i r a b l e t o use l a r g e r q u a n t i t i e s o f  m a t e r i a l s f o r study..  For the separation of l a r g e r concentrations  o f n u c l e o t i d e s from E h r l i c h a s c i t e s c a r c i n o m a c e l l s ,  chromato-  graphy on D E A E - c e l l u l o s e - c o l u m n , was c a r r i e d , p u t . For t h e s t a n d a r d i z a t i o n o f t h e t e c h n i q u e  of separation of  n u c l e o t i d e s , a p r e l i m i n a r y experiment using a c i d s o l u b l e nucleot i d e s from E h r l i c h a s c i t e s carcinoma c e l l s was performed.  The •  p r o c e d u r e f o l l o w e d f o r t h e i s o l a t i o n and s e p a r a t i o n o f n u c l e o t i d e s was a c c o r d i n g t o t h e method o f Oikawa and Smith ( 9 9 ) . About 4 gm wet w e i g h t o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s were e x t r a c t e d w i t h 20-25 ml o f 1 M c o l d p e r c h l o r i c a c i d .  The  s o l u t i o n was brought t o a pH o f 6.5-7 u s i n g 10 N sodium hydrox i d e and t h e water e v a p o r a t e d i n a f l a s h e v a p o r a t o r .  The  r e s i d u e was e x t r a c t e d w i t h 50 m l o f 95% e t h a n o l t o remove t h e sodium p e r c h l o r a t e .  The n u c l e o t i d e s which r e m a i n as t h e r e s i d u e  were e x t r a c t e d w i t h a s m a l l volume o f water and t h e s o l u t i o n a p p l i e d on t o a column o f D E A E - c e l l u l o s e  25 x 1 cm i n t h e c a r -  bonate form, p r e p a r e d and packed a c c o r d i n g t o t h e p r o c e d u r e of T o m l i n s o n and Tener(lOO).  E l u t i o n o f t h e n u c l e o t i d e s was  c a r r i e d o u t w i t h a l i n e a r g r a d i e n t o f ammonium b i c a r b o n a t e (0.2 M) a t pH 8.6, and 5 ml f r a c t i o n s were c o l l e c t e d . s t a n c e s e l u t e d were i s o l a t e d and f u r t h e r s e p a r a t e d i z e d by paper chromatography  The sub-  and c h a r a c t e r -  and a b s o r p t i o n s p e c t r a .  The  e l u t i o n p r o f i l e i s g i v e n i n F i g u r e 8. • I t i s seen from t h e  ABSORBANCE AT 260 my  H"  n 00  H 3 H HM P  to o  1 £ 0 OJ t5! «+1— H S CD g CU 3 rt 1  » H-  \ 4r-  Ul  3  O  H rt o a < CD Hi 1 H 0 c < D Hi D PJ M > H "« tn tt H-  o  o Ul  to Ul  A  CD  o  «  o  •  H  o  tr o  D ts - tr H O  o  CD C H< H CD i O O cn rt c CD H1  Cb  •a  o o o  — CD to cn  Hi  •3 3  - o H y cn CD O D a w rt • 3  1  < 0  H Hi ' ui >l rt  o3 W  co o  >-3 H O  .a' o  cn  o o  tr M HO tr  •3 tr 3 cu cn  o H-  >  n  < CD 1-i rt H CU CU CD H o cn ! cn rt O H- o i-3 5: 0 cu •d CD 3 1"! • • Ci-iD cn o  s:  H CD o 1 h 3 a CD CU >.  to O  O  M  CTl  o  D O CoD 0  -  M H H M H CD cn 1 O G rt .—, S CD •X) Cu *»  rt 3 cn Ul — •  0 3  O  o to  M o l a r i t y of Ammonium bicarbonate' - ZL -  - 74 -  e l u t i o n p r o f i l e that a s a t i s f a c t o r y r e s o l u t i o n of the nucleot i d e s can be a c h i e v e d by a c o m b i n a t i o n o f column chromatography and paper  chromatography.  8) E f f e c t o f A c t i n o m y c i n D on t h e B i o s y n t h e s i s o f P u r i n e N u c l e o t i d e s i n E h r l i c h A s c i t e s Carcinoma C e l l s in  vitro.  To s t u d y t h e e f f e c t o f a c t i n o m y c i n D on t h e i n c o r p o r a t i o n of  ll  *C-formate  i n t o t h e a c i d s o l u b l e n u c l e o t i d e s , tumour c e l l s  from 12 mice were c o l l e c t e d and p o o l e d . cells).  ( T o t a l volume 20 ml  The p o o l e d c e l l s were suspended i n Krebs  Ringer  phosphate b u f f e r pH 7.8 t o g i v e a 1 i n 10 s u s p e n s i o n . formate (2. mole/ml s u s p e n s i o n ) / a n d u  1  Sodium  ^'C-formate. (0.5 uc/ml  suspension) were added t o t h e s o l u t i o n and mixed w e l l .  The  s o l u t i o n was then d i v i d e d i n t o two e q u a l p o r t i o n s i n 500 ml Erlenmeyer  flasks.  A c t i n o m y c i n D d i s s o l v e d i n 20% e t h a n o l t o  a c o n c e n t r a t i o n o f 10 mg/ml was added t o one and an e q u a l volume of 20% e t h a n o l added t o t h e o t h e r .  A l l t h e s e o p e r a t i o n s were  done i n t h e c o l d room. These f l a s k s were g a s s e d w i t h oxygen, c l o s e d w i t h rubber s t o p p e r s and i n c u b a t e d i n a water b a t h a t 37°C f o r 2 hours. < D u r i n g t h e p e r i o d o f i n c u b a t i o n t h e • f l a s k s were shaken a t 100-110 o s c i l l a t i o n s p e r minute.  A f t e r the i n c u b a t i o n the c e l l s  separ-  a t e d by c e n t r i f u g a t i o n a t 15,000 x g f o r 20 m i n u t e s , washed once w i t h 5 volumes o f c o l d Krebs R i n g e r phosphate and c e n t r i fuged a g a i n .  A f t e r removing  t h e s u p e r n a t a n t f l u i d , t h e wet  w e i g h t o f t h e c e l l s was d e t e r m i n e d .  The c e l l s were e x t r a c t e d  w i t h 1 M c o l d p e r c h l o r i c a c i d , and t h e e x t r a c t was n e u t r a l i z e d w i t h c o l d 10 N sodium h y d r o x i d e and s t o r e d a t -20°C u n t i l  used.  The  s o l u t i o n of a c i d s o l u b l e . n u c l e o t i d e s was  on a f l a s h e v a p o r a t o r  evaporated  (bath t e m p e r a t u r e 25°C) and the  residue  r e p e a t e d l y e x t r a c t e d w i t h e t h a n o l t o remove t h e sodium p e r chlorate.  The  s o l u t i o n of s o d i u m - p e r c h l o r a t e  and the' r e s i d u e was T h i s s o l u t i o n was chromatography was  was  centrifuged off  e x t r a c t e d w i t h a s m a l l volume of w a t e r .  a p p l i e d on t o a column of D E A E - c e l l u l o s e  and  c a r r i e d out a c c o r d i n g t o t h e method d e s c r i b e d .  The p r o f i l e s of e l u t i o n from DEAE-cellulose-  column of  nucleo-  t i d e s of the a c i d s o l u b l e f r a c t i o n s , from t i s s u e s i n c u b a t e d i n the absence-and p r e s e n c e of a c t i n o m y c i n  D are g i v e n i n F i g u r e 9 and  F i g u r e 10 r e s p e c t i v e l y . E x a m i n a t i o n of the e l u t i o n p r o f i l e s shows t h a t the p a t t e r n of e l u t i o n of n u c l e o t i d e s i s more or l e s s the same i n b o t h the cases and s i m i l a r t o the p a t t e r n o b t a i n e d p r e v i o u s l y .  Compari-  son of the e l u t i o n p r o f i l e s c l e a r l y shows the i n c r e a s e i n conc e n t r a t i o n of the n u c l e o t i d e s from E h r l i c h a s c i t e s carcinoma c e l l s incubated in.presence  of a c t i n o m y c i n  D.  Though t h e r e  was  a g e n e r a l i n c r e a s e of a l l n u c l e o t i d e s , i t can be seen t h a t t h i s i n c r e a s e was  maximum i n the case of the n u c l e o s i d e  p a r t i c u l a r l y of ATP. e x e r t any  T h i s may  triphoshates  mean t h a t a c t i n o m y c i n  i n h i b i t o r y e f f e c t on the f o r m a t i o n o f the  D does not nucleoside  triphosphates. I t i s known from the r e s u l t s r e p o r t e d i n t h i s t h e s i s , w e l l as from those r e p o r t e d by o t h e r s t h a t a c t i n o m y c i n b i t e d the i n c o r p o r a t i o n of s y n t h e s i s of RNA  (39).  llf  C - f o r m a t e i n t o RNA  I f actinomycin  D inhi-  p u r i n e s and  D does not i n h i b i t  de novo s y n t h e s i s of n u c l e o s i d e t r i p h o s p h a t e s and o n l y f e r e s v/ith the p o l y m e r i z a t i o n of the n u c l e o s i d e  as  the the  inter-  triphosphates  +J  1.0  o  0.8-  Xi u u ra  n  •H  c o  J  0.6T  0.2  cn  4-1  o  0.4  >1  +J  -  •H  u  0.1  rH  o s  0.  40  120  80 FRACTION  F i g u r e 9.  160  NOS,  Chromatography o f a c i d s o l u b l e n u c l e o t i d e s from E h r l i c h a s c i t e s c a r c i n o m a c e l l s (10.9 gm) i n c u b a t e d w i t h **C-formate. F r a c t i o n s (10 mlr ) were c o l l e c t e d a t 10 minute i n t e r v a l s . Peaks were s e p a r a t e d and i d e n t i f i e d as d e s c r i b e d i n t e x t . D E A E - c e l l u l o s e column (25 x 1 cm. ) was used f o r chromatography. 1  g  o  KO CM  E-»  0.2  <  W  U CQ Cd  O  to CQ  40  80  120  160  FRACTION NOS. F i g u r e 10,  Chromatography o f a c i d s o l u b l e n u c l e o t i d e s from E h r l i c h a s c i t e s c a r c i n o m a c e l l s (10.7 gm) i n c u b a t e d w i t h ^ C - f ormate and a c t i n o m y c i n D (10 yg/ml o f s u s p e n s i o n ) . F r a c t i o n s (10 ml.--) were c o l l e c t e d a t 10 m i n u t e i n t e r v a l s . Peaks were s e p a r a t e d and i d e n t i f i e d as d e s c r i b e d i n t e x t . D E A E - c e l l u l o s e column (25 x 1 cm::) was used f o r chromatography.  - 78  t o form RNA,  -  t h e n an i n c r e a s e i n the c o n c e n t r a t i o n of  substances i n c e l l s t r e a t e d w i t h actinomycin The  s o l u t i o n of each  c e l l u l o s e : c o l u m n was due was  U.V.  can be  these expected.  a b s o r b i n g peak from the  BEAE  e v a p o r a t e d and the s u b s t a n c e s i n the  f u r t h e r s e p a r a t e d by paper chromatography u s i n g  resi-  the  i s o b u t y r i c - a m m o n i a - w a t e r s o l v e n t m i x t u r e o r i n the ammonium a c e t a t e - e t h a n o l system.  The a b s o r p t i o n s p e c t r a of the m a t e r i a l s  e l u t e d from the paper and t h e i r c o n c e n t r a t i o n s were d e t e r m i n e d by u l t r a v i o l e t s p e c t r o p h o t o m e t r y .  H a l f ml samples of s o l u -  t i o n s of the p u r i n e n u c l e o t i d e s were counted and the r a d i o a c t i v i t y i n c o r p o r a t e d i n t o each d e t e r m i n e d .  The  concentration  o f the p u r i n e n u c l e o t i d e s i s o l a t e d from c o n t r o l e x p e r i m e n t s and"cells incubated with actinomycin The  D i s g i v e n i n Table  r a d i o a c t i v i t y i n c o r p o r a t e d . i n t o the v a r i o u s p u r i n e  V I I I .  nucleo-  t i d e s Is g i v e n i n T a b l e I X . I t can be seen from t h e s e t a b l e s t h a t i n b o t h  experiments,  the c o n c e n t r a t i o n of the n u c l e o t i d e s i n c r e a s e d i n c e l l s with actinomycin  D.  incubated  T h i s shows t h a t the main a c t i o n of a c t i n o -  mycin D i s t o i n h i b i t the p o l y m e r i z a t i o n of n u c l e o s i d e  triphos-  phates t o form RNA  nucleo-  tides.  and not t o i n h i b i t the s y n t h e s i s of  As the i n c r e a s e i n c o n c e n t r a t i o n was  n u c l e o s i d e t r i p h o s p h a t e s , i t may  seen m a i n l y  be s u g g e s t e d t h a t  i n the  actinomycin  does not i n h i b i t the f o r m a t i o n of the t r i p h o s p h a t e s .  However,  the i n c o r p o r a t i o n of '*C-formate i n t o the p u r i n e n u c l e o t i d e s i s 1  s l i g h t l y less i n c e l l s incubated with actinomycin d e c r e a s e i s not v e r y s i g n i f i c a n t . of  111  The  D, though the  decrease i n i n c o r p o r a t i o n  C - f o r m a t e i n t o n u c l e o t i d e s becomes l e s s a p p a r e n t when the  - 79 -  Table V I I I Concentrations of purine nucleotides i s o l a t e d from E h r l i c h a s c i t e s c a r c i n o m a c e l l s i n c u b a t e d w i t h a c t i n o m y c i n D (10 yg/ml) in vitro. centrations  Con-  a r e e x p r e s s e d as my moles p e r gram  wet w e i g h t o f c e l l s .  Experiment I Acid-soluble Nucleotide AMP  control 23.44  control + Act. D 25.68  . Experiment I I . . . control 33.41  control + Act. D 31.35  ADP  133.6  175.26  150.5  239.5  ATP  288.8  662.4  698.8  1077.9  GMP  7.86  8.09  17.83  14.38  GDP  -  3.3  46.6  65.22  GTP  21.61  85.4  104.82  51.75 .  - 80 T a b l e IX E f f e c t of actinomycin llf  D on t h e i n c o r p o r a t i o n o f  C-formate i n t o the purine nucleotides of a c i d  s o l u b l e f r a c t i o n of E h r l i c h a s c i t e s carcinoma c e l l s in vitro.  Data r e c o r d e d  as s p e c i f i c  activity  (counts p e r minute p e r umole).  Experiment I Acid soluble nucleotides  Control  Control + Act. D  Experiment I I Control  Control + Act. D  AMP  4163  3841  3982  3497  ADP  4725  4986  4238  3805  ATP  4663  4285  3998  3600  GMP  3575  3123  4842  3989  1882  1742  1732  1688  1721  1103  GDP GTP  2740  l a r g e i n c r e a s e i n c o n c e n t r a t i o n of t h e n u c l e o t i d e s i n i n c u b a t i o n s c o n t a i n i n g actinomycin D i s taken i n t o c o n s i d e r a t i o n . I t may  be p o i n t e d out t h a t i n the c e l l s t h e r e are mechanisms  o t h e r t h a n the de novo s y n t h e s i s by which n u c l e o t i d e s formed.  are  As the p o l y m e r i z a t i o n of the n u c l e o t i d e s i s - i n h i b i t e d  i n i n c u b a t i o n s c o n t a i n i n g a c t i n o m y c i n D, t h e s i z e o f the  nucleo-  t i d e p o o l i n c r e a s e s , w h i c h would r e s u l t i n t h e d i l u t i o n o f the r a d i o a c t i v e n u c l e o t i d e s formed. t i d e s i n the c e l l may  The  a c c u m u l a t i o n of  nucleo-  p r e v e n t f u r t h e r de.novo. s y n t h e s i s of n u c l e o  t i d e s by feedback i n h i b i t i o n  (116) which w i l l a l s o reduce the  i n c o r p o r a t i o n of ^ C - f o r m a t e i n t o n u c l e o t i d e s , r e s u l t i n g i n low 1  specific  activity.  Since.these  i n v e s t i g a t i o n s were u n d e r t a k e n Lowy e t a l . (66)  have shown i n s t u d i e s w i t h r a b b i t r e t i c u l o c y t e s and that actinomycin  D does not i n h i b i t the de novo  erythrocytes  s y n t h e s i s of  n u c l e o t i d e s or the f o r m a t i o n of n u c l e o t i d e s from p u r i n e s .  The  p r e s e n t i n v e s t i g a t i o n shows s i m i l a r r e s u l t s i n E h r l i c h a s c i t e s carcinoma c e l l s . A general increase i n concentration of other was  nucleotides  a l s o observed i n c e l l s incubated w i t h actinomycin  D.  c o n c e n t r a t i o n of the p y r i m i d i n e n u c l e o t i d e s i s o l a t e d by methods d e s c r i b e d i s p r e s e n t e d  i n T a b l e X.  actinomycin  p.  the  l i t can be seen from .  the r e s u l t s t h a t t h e r e v/as a l s o an i n c r e a s e o:f the n u c l e o t i d e s i n c e l l s when the RNA  The  pyrimidine  synthesis i s inhibited  by  K i d a e t a l . (63) have i n v e s t i g a t e d the e f f e c t of  chromomycin A 3 , an a n t i b i o t i c s i m i l a r i n a c t i t o n t o D, on the n u c l e i c a c i d m e t a b o l i s m of Bacillus  subtilis  actinomycin SB-15.  - 82 -  Table X E f f e c t o f a c t i n o m y c i n D.on t h e c o n c e n t r a t i o n o f p y r i m i d i n e n u c l e o t i d e s and NAD o f E h r l i c h carcinoma  c e l l s in vitro.  ascites  A c t i n o m y c i n D concen-  t r a t i o n 10 ug/mL.Concentration  e x p r e s s e d as mumoles  - p e r gram wet w e i g h t o f t i s s u e .  Nucleotide  Control  Control + Actinomycin D  CMP  6.18  CDP  50.3 •.  67.13  CTP  76.4  97.12  UMP  16.83  28.78  UDP  35.35  37.88  UTP  63.4  NAD  29.16  6.606  127.5 35.12  - 83 They a l s o o b s e r v e d a q u a n t i t a t i v e change i n the  nucleotide  components of the c e l l s , w h i c h i s i n agreement w i t h the p r e s e n t findings. 9) E f f e c t o f A c t i n o m y c i n D on the R e s p i r a t i o n of E h r l i c h A s c i t e s Carcinoma C e l l s . I t has been r e p o r t e d i n h i b i t s r e s p i r a t i o n and H o r n i n g and  recently  g l y c o l y s i s i n leucemic  D  leucocytes.  R a b i n o w i t z have shown t h a t the r a t e o f oxygen  t a k e of Sarcoma 3 7 . a s c i t e s absence of a c t i n o m y c i n y e a r s ago  (32) t h a t a c t i n o m y c i n  c e l l s incubated  D was  up-  i n the p r e s e n c e or  not s i g n i f i c a n t l y d i f f e r e n t .  Scrimgeour o b s e r v e d  (68) t h a t a c t i n o m y c i n  the r e s p i r a t i o n i n N o v i k o f f hepatoma p r e p a r a t i o n s . i n t e r e s t t o study the e f f e c t of a c t i n o m y c i n  Some  D inhibited I t was  of  D on the r e s p i r a t i o n  of E h r l i c h a s c i t e s c a r c i n o m a c e l l s used f o r s t u d i e s  described  in this thesis. S t u d i e s on r e s p i r a t i o n of E h r l i c h a s c i t e s carcinoma c e i l s were c a r r i e d out by the u s u a l t e c h n i q u e of Warburg manometry u s i n g f l a s k s w i t h s i d e arms. c o l l e c t e d as d e s c r i b e d 1500  rpm  E h r l i c h a s c i t e s c e l l s were  previously.  They were c e n t r i f u g e d  i n an I n t e r n a t i o n a l r e f r i g e r a t e d c e n t r i f u g e  P.R-11) f o r 3 m i n u t e s , t h e s u p e r n a t a n t was  at  (model  removed, and  the  cells  were washed w i t h t h r e e volumes o f c o l d Krebs R i n g e r phosphate buffer.  The  3 minutes and  s u s p e n s i o n was  c e n t r i f u g e d a g a i n a t 2000 rpm  the s u p e r n a t a n t was  removed.  The  for  c e l l s were  d i l u t e d w i t h 9 volumes of c o l d Krebs R i n g e r phosphate b u f f e r ..pH  7.8.  Two  ml o f t h e c e l l s u s p e n s i o n was  i n a t o t a l volume o f 3.ml. t a i n e d 0.2  ml o f 10% KOH.  The  p r e s e n t i n each f l a s k  c e n t r e w e l l o f the- f l a s k s  con-  S u b s t r a t e s whenever used were t i p p e d  from t h e s i d e arm o f t h e f l a s k s t o the main compartment a f t e r t h e r m a l e q u i l i b r a t i o n f o r 15 m i n u t e s .  The  gas phase was a i r  and the i n c u b a t i o n s were c a r r i e d out a t 37°C f o r 1 hour. The u p t a k e o f oxygen w i t h time by t h e c e l l s u s p e n s i o n i n t h e presence  and absence o f a c t i n o m y c i n D i s g i v e n i n F i g u r e  It can be seen from t h e f i g u r e t h a t the r a t e o f r e s p i r a t i o n c e l l s i n t h e absence o f a c t i n o m y c i n D was T h i s was  observed  2 hours.  After  respiration".  The presence  c a r r i e d out f o r  a s m a l l f a l l i n the  of a c t i n o m y c i n D does not change the  r a t e o f r e s p i r a t i o n up t o 30 m i n u t e s , decrease  though t h e r e was  i n t h e oxygen uptake i n about 60 m i n u t e s .  v a l u e s are g i v e n i n T a b l e X I . i n the Q02  v a l u e s i n presence  a small  The  The d a t a show a s m a l l  Qo  decrease  I n a s i m i l a r study L a s z l o e t  al.  (32) have shown t h a t t h e oxygen uptake by leukemic  was  118  156  u l of the c o n t r o l s f o r a 5 hour p e r i o d .  leucocytes  o f 50 ug/ml o f a c t i n o m y c i n D compared t o They have con-  c l u d e d from t h i s t h a t a c t i n o m y c i n D i n h i b i t s r e s p i r a t i o n leucemic  leucocytes.  2  of a c t i n o m y c i n D s u g g e s t i n g a  small i n h i b i t i o n i n respiration.  u l i n presence  of  l i n e a r w i t h time.  even when t h e i n c u b a t i o n was  t h i s p e r i o d t h e r e was  11.  Scrimgeour observed  of  an i n h i b i t i o n of  r e s p i r a t i o n o f N o v i k o f f hepatoma i n presence  of a c t i n o m y c i n  D.  The p r e s e n t r e s u l t s are comparable t o t h e s e o b s e r v a t i o n s . Glucose  i n h i b i t e d t h e oxygen uptake o f E h r l i c h a s c i t e s  c e l l s by almost by o t h e r s  (117).  45%.  carcinoma  T h i s i s i n agreement w i t h the o b s e r v a t i o n s  T h i s i n h i b i t i o n o f r e s p i r a t i o n by  glucose  -  85  -  20  40 TIME  F i g u r e 11.  60  (minutes)  E f f e c t of a c t i n o m y c i n D on the oxygen consumption of E h r l i c h a s c i t e s carcinoma c e l l s . Each f l a s k c o n t a i n e d 19.7 mg o f d r y w e i g h t of t i s s u e i n a t o t a l volume o f 3.0 ml o f Krebs R i n g e r phosphate b u f f e r . Gas phase was a i r and i n c u b a t i o n ' w a s a t 37°C Control ( oe ) + Actinomycin D ( A )  - 86 -  Table XI E f f e c t o f a c t i n o m y c i n D on t h e r e s p i r a t i o n o f E h r l i c h a s c i t e s carcinoma c e l l s .  Each mano-  m e t r i c f l a s k c o n t a i n e d 19.5 mg d r y w e i g h t o f t i s s u e i n a t o t a l volume o f 3 ml. A c t i n o m y c i n D when p r e s e n t was 10 yg/ml and g l u c o s e was . 5.5 mM.  Additions  Control  0_  % Inhibition by A c t i n o m y c i n  11.02, 10.97  + Act. D (10 yg/ml)  9.58,  9.56  + glucose  6.13/  6.20  + glucose + A c t . D.  5.4  5.47  13.14, 12.85  11.90, 11.90  i s p r o b a b l y due t o t h e o p e r a t i o n o f the C r a b t r e e e f f e c t  (118).  A c t i n o m y c i n D d e c r e a s e s t h e oxygen uptake i n t h e presence o f g l u c o s e by another 12%. However, t h e i n h i b i t i o n o f r e s p i r a t i o n produced  by a c t i n o m y c i n i s o f t h e same magnitude i n t h e  presence o f added s u b s t r a t e as w e l l as i n t h e absence, o f any exogenous s u b s t r a t e .  This i n h i b i t i o n of r e s p i r a t i o n i s  much l e s s compared t o t h e i n h i b i t i o n o f RNA: s y n t h e s i s caused by the- same- amount o f a c t i n o m y c i n added t o t h e medium and' hence t h e r e may n o t be any c o r r e l a t i o n between these, two processes. 10) E f f e c t o f A c t i n o m y c i n D on G l y c o l y s i s  by E h r l i c h A s c i t e s  Carcinoma C e l l s . There a r e c o n f l i c t i n g r e p o r t s i n t h e l i t e r a t u r e about t h e e f f e c t o f a c t i n o m y c i n on t h e g l y c o l y s i s by d i f f e r e n t Scrimgeour  tissues.  observed t h a t a c t i n o m y c i n D d i d n o t i n h i b i t g l y c o l y s i s  i n N o v i k o f f hepatoma (68).  Prave and K r o n i n g  (119) r e p o r t e d  t h a t a c t i n o m y c i n I reduced b o t h a e r o b i c and a n a e r o b i c i n E h r l i c h a s c i t e s carcinoma  cells.  glycolysis  R e c e n t l y i t was r e p o r t e d  by L a s z l o e t a l . (32) t h a t a c t i n o m y c i n D i n h i b i t e d t h e g l y c o l y s i s i n leukemic l e u c o c y t e s .  I t was o f i n t e r e s t t o study t h e e f f e c t  of a c t i n o m y c i n D on t h e u t i l i z a t i o n o f g l u c o s e by E h r l i c h c a r c i noma c e l l s in  vitro.  A t e n percent suspension of E h r l i c h a s c i t e s  carcinoma  was made i n Krebs R i n g e r phosphate b u f f e r pH 7.8. g l u c o s e , as a 5% s o l u t i o n centration  cells  Sufficient  i n b u f f e r , was added t o g i v e a con-  o f 1 mg/ml o f s u s p e n s i o n and t h e s u s p e n s i o n mixed w e l l .  - 88 I t was  d i v i d e d i n t o two p o r t i o n s and a c t i n o m y c i n D d i s s o l v e d  i n 20% e t h a n o l was  added t o one p o r t i o n t o g i v e a c o n c e n t r a t i o n  o f 10 mg/ml o f s u s p e n s i o n .  To the o t h e r p o r t i o n w h i c h s e r v e d  as the c o n t r o l , an e q u a l volume of 20% e t h a n o l was  added.  The  S o l u t i o n s were i n c u b a t e d a t 37°C i n a w a t e r bath and were k e p t shaken d u r i n g the p e r i o d o f i n c u b a t i o n . o f the s u s p e n s i o n was  A t i n t e r v a l s one  t r a n s f e r r e d from t h e f f l a s k s , and depro-  t e i n i z e d w i t h 2 ml o f each o f z i n c s u l p h a t e hydroxide solution, The  g l u c o s e was  ml  (2%) and b a r i u m  a c c o r d i n g t o the method o f Somyogi (75).  e s t i m a t e d u s i n g 1 ml o f the p r o t e i n f r e e  fil-  t r a t e by the g l u c o s e o x i d a s e method u s i n g a g l u c o s t a t k i t (Worthington Biochemical  Corp).  A s t a n d a r d c u r v e showing the c o n c e n t r a t i o n o f g l u c o s e i n s o l u t i o n a g a i n s t the o p t i c a l d e n s i t y was  p r e p a r e d by u s i n g a  s e r i e s of standard glucose s o l u t i o n s t r e a t e d s i m i l a r l y . c o n c e n t r a t i o n o f g l u c o s e i n the t e s t samples was u s i n g the s t a n d a r d . c u r v e .  The  The  determined  c o n c e n t r a t i o n of glucose a t  d i f f e r e n t i n t e r v a l s of i n c u b a t i o n i s given i n F i g u r e  12.  I t can be seen from the F i g u r e 12 t h a t the f a l l i n conc e n t r a t i o n o f g l u c o s e w i t h time i s not a f f e c t e d by the  presence  o f a c t i n o m y c i n D i n the incubation.medium.  suggest  T h i s would  i n t u r n t h a t a c t i n o m y c i n D e x e r t e d no i n h i b i t o r y e f f e c t on g l y c o l y s i s o f E h r l i c h a s c i t e s carcinoma  cells.  the  This r e s u l t i s  i n c o n t r a s t w i t h the o b s e r v a t i o n r e p o r t e d f o r a c t i n o m y c i n I by Prave and Koning  i n E h r l i c h a s c i t e s carcinoma  c e l l s and w i t h  the f i n d i n g s o f L a z l o e t a l . (32) f o r a c t i n o m y c i n D i n l e u c e m i c •leucocytes.  Scrimgeour s t u d i e d the a f f e c t o f a c t i n o m y c i n D on  - 89 -  800  600 3  fi O  •H  -P -  "ta u. +>  fi 400 cu  D fi O U CO  in o  o rH  U  200  30  15  TIME Figure  12.  45  60  (minutes)  E f f e c t o f a c t i n o m y c i n D on g l y c o l y s i s by a s c i t e s carcinoma c e l l s in vitro. Control  (  ©—  )  + Actinomycin D (10 yg/ml)  (-  Ehrlich )  - 90 t h e g l y c o l y s i s o f N o v i k o f f hepatoma u s i n g t h e Warburg manometric techniques  (68).  He observed no i n h i b i t i o n o f g l y c o l y s i s i n  t h i s t i s s u e i n presence o f a c t i n o m y c i n D.  In the p r e s e n t i n v e s -  t i g a t i o n s , g l y c o l y s i s was measured by the d i s a p p e a r a n c e g l u c o s e from t h e c e l l s u s p e n s i o n which agrees w i t h the o b t a i n e d by manometric t e c h n i q u e s , used by  Scrimgeour.  of results  -  91  -  DISCUSSION I n the p a s t few y e a r s d i f f e r e n t a n t i b i o t i c s w h i c h can i n h i b i t n u c l e i c a c i d and p r o t e i n s y n t h e s i s i n c e l l u l a r systems, i n c e l l f r e e e x t r a c t s , and p u r i f i e d enzyme p r e p a r a t i o n s , have been i s o l a t e d .  The mechanism o f a c t i o n o f these a n t i b i o t i c s  has  been i n v e s t i g a t e d i n d e t a i l w h i c h c l a r i f i e d the modern conc e p t s about the i n t e r r e l a t i o n s h i p o f p r o t e i n s y n t h e s i s and n u c l e i c a c i d s y n t h e s i s , the mechanisms o f a c t i o n o f hormones, o f a c t i v e t r a n s p o r t , i n d u c t i o n of enzymes, the growth o f v i r u s and many o t h e r c e l l u l a r One  processes.  o f the w i d e l y used a n t i b i o t i c s t o study t h e s e  a t a m o l e c u l a r l e v e l i s a c t i n o m y c i n D.  processes  The m o l e c u l a r mechanisms  by w h i c h a c t i n o m y c i n D can i n h i b i t l i v i n g p r o c e s s e s have been amply documented (12).  Because of the s p e c i f i c i t y o f a c t i o n of  a c t i n o m y c i n D i n i n h i b i t i n g the DNA  dependent RNA  polymerase,  t h i s a n t i b i o t i c has been used t o i n v e s t i g a t e the i n v o l v e m e n t RNA  i n b i o l o g i c a l processes.  The  s p e c i f i c i t y of a c t i o n of  a c t i n o m y c i n has been a t t r i b u t e d t o the a b i l i t y o f the t o form m o l e c u l a r complexes w i t h DNA, p l a t e f o r b o t h the s y n t h e s i s o f RNA  substance  w h i c h f u n c t i o n s as a temand  The p r o c e s s of r e p l i c a t i o n c f RNA  DNA. by DNA  dependent  RNA  polymerase has been r e p o r t e d t o t a k e p l a c e i n t h r e e d i s t i n c t separate processes  (28, 29):  t o s p e c i f i c s i t e s on the DNA  of  1) the b i n d i n g o f RNA  and  polymerase  w h i c h f u n c t i o n as a t e m p l a t e i n t h e  r e a c t i o n , 2) the i n i t i a t i o n o f RNA  s y n t h e s i s by the enzyme-DNA-  complex formed f i r s t r e a c t i n g w i t h p u r i n e n u c l e o t i d e s , t o form a second i n t e r m e d i a t e , and 3) the p o l y m e r i z a t i o n o f f o u r k i n d s  -92-  o f r i b o n u c l e o t i d e s t o form a polymer under the d i r e c t i o n of t h e DNA RNA  template.  The  polymerase t o DNA  sucrose  e f f e c t of a c t i n o m y c i n was  i n v e s t i g a t e d by R i c h a r d s o n (30)  d e n s i t y g r a d i e n t c e n t r i f u g a t i o n and a l s o by  s t u d i e s of the complex. which c o u l d i n h i b i t RNA  I t was  F u r t h e r , the k i n e t i c s of the RNA  He has o b s e r v e d t h a t t h e RNA  dependent RNA  D were s t u d i e d by  s y n t h e s i s by RNA  From t h e s e o b s e r v a t i o n s  and has  actinomycin  he has  stops i n  concluded t h a t polymerase  Similar observation  D i n h i b i t s the c h a i n e l o n g a t i o n i n RNA  when n a t i v e DNA  Richardson.  l e s s e f f e c t on the i n i t i a t i o n of  t h e s i s t h a n on the p o l y m e r i z a t i o n .  poly-  polymerase s t a r t s  D does not a f f e c t t h e b i n d i n g of RNA  templates,  D in  f o r the b i n d i n g r e a c t i o n .  r a p i d l y and t h e n i m m e d i a t e l y b e g i n s t o slow down and  t o DNA  at l e v e l s  and t h a t a c t i n o m y c i n  s y n t h e s i s by DNA  merase i n p r e s e n c e of a c t i n o m y c i n  actinomycin  by  s y n t h e s i s d i d not reduce the number of  a f f e c t e d the a f f i n i t y c o n s t a n t  about 5 m i n u t e s .  of  sedimentation  observed t h a t actinomycin  b i n d i n g s i t e s f o r RNA-polymerase on DNA, no way  D on the b i n d i n g  syn-  that  synthesis  used as a t e m p l a t e has been r e p o r t e d r e c e n t l y  by Sentenac e t a l . (31). • I n c o n t r a s t t o t h i s , R i c h a r d s o n has o b s e r v e d t h a t p r o f l a v i n , a n o t h e r i n h i b i t o r of DNA w i t h the b i n d i n g o f RNA  dependent RNA  polymerase, i n t e r f e r e s  polymerase t o DNA  t e m p l a t e s and  also  i n h i b i t s t h e ' i n i t i a t i o n of the s y n t h e s i s more than the  elongation  of the c h a i n by the p o l y m e r i z a t i o n r e a c t i o n .  i t has been  r e p r o t e d by Mizuno e t a l . (120) and B44  Recently  t h a t the a n t i b i o t i c s , r i f a m y c i n  ( s t r e p t o v a r a c i n ) i n h i b i t the RNA  polymerase of m i c r o b i a l  - 93 systems by i n h i b i t i n g t h e i n i t i a t i o n o f t h e p o l y m e r i z a t i o n reaction.  However,these a n t i b i o t i c s d i d n o t i n h i b i t t h e RNA  polymerase i s o l a t e d from E h r l i c h a s c i t e s c a r c i n o m a c e l l s . Many o f t h e s t u d i e s on t h e e f f e c t s o f a c t i n o m y c i n  D are  concerned w i t h b i o s y n t h e s i s o f macromolecules and l i t t l e i s known about t h e e f f e c t o f t h e a n t i b i o t i c on t h e s y n t h e s i s o f precursors  of nucleic acids.  Wheeler and B e n n e t t (121)  r e p o r t e d i n 1960 and l a t e r i n 1962 (64) t h a t a c t i n o m y c i n i n h i b i t e d t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e and 2- " ^ - g l y c i n e i n t o 1  a c i d s o l u b l e n u c l e o t i d e s o f L. arabinosus. ll+  Incorporation of  C - f o r m a t e i n t o s o l u b l e n u c l e o t i d e s o f H.Ep.#2 c e l l s grown i n  t i s s u e c u l t u r e was a l s o r e p o r t e d t o be i n h i b i t e d by a c t i n o m y c i n D.  From, a comparison o f d a t a o b t a i n e d  i n studies with  C-  lh  formate and 8 - ^ C - h y p o x a n t h i n e they have c o n c l u d e d t h a t a c t i n o 1  mycin D d i d n o t i n t e r f e r e w i t h t h e b i o s y n t h e s i s o f t h e p u r i n e m o i e t y de novo,  i n E h r l i c h a s c i t e s carcinoma c e l l s in  I t was a l s o o b s e r v e d t h a t a c t i n o m y c i n interconversion  vivo.  D did not i n h i b i t the  of purine r i b o n u c l e o t i d e s .  differences of the e f f e c t of actinomycin  S i m i l a r i t i e s and  D on t h e p u r i n e and  n u c l e i c a c i d s y n t h e s i s l e d them t o suggest "two m e t a b o l i c b l o c k s , one on t h e s y n t h e s i s o f p u r i n e n u c l e o t i d e s de novo, and one on t h e u t i l i z a t i o n o f guanine n u c l e o t i d e s f o r s y n t h e s i s o f RNA"..  The second s i t e o f i n h i b i t i o n seems t o be common t o a l l  the systems s t u d i e d and they f u r t h e r s u g g e s t e d t h a t t h e b l o c k . of de novo s y n t h e s i s o b s e r v e d may be a secondary e f f e c t . c o u l d v e r y w e l l be t h e case i n t h e l i g h t o f t h e p r e s e n t  This inves-  t i g a t i o n and t h e l a t e r known f a c t s about t h e e f f e c t s o f a c t i n o mycin D .  - 94 Harbers and M i i l l e r  (65) s t u d i e d t h e e f f e c t o f  actinomycin  D on t h e i n c o r p o r a t i o n o f 8- ^C-guanine i n t o RNA p u r i n e s i n 1  E h r l i c h a s c i t e s carcinoma c e l l s .  They have o b s e r v e d t h a t t h e  i n c o r p o r a t i o n o f guanine t o RNA i s i n h i b i t e d by a c t i n o m y c i n They have f u r t h e r o b s e r v e d t h a t a c t i n o m y c i n  D.  d i d not prevent the  f o r m a t i o n o f l a b e l l e d n u c l e o t i d e s from 8- "*C-guanine i n these 1  systems.  W h i l e p r e s e n t i n v e s t i g a t i o n s were i n p r o g r e s s , t h e  e f f e c t of actinomycin  D on the f o r m a t i o n  and,. i n t e r c o n v e r s i o n  of p u r i n e n u c l e o t i d e s was s t u d i e d by Lowy and W i l l i a m s a system w h i c h i s i n c a p a b l e o f any RNA s y n t h e s i s . reported  (66) i n  They p r e v i o u s l y  (122) t h a t r a b b i t r e t i c u l o c y t e s were u n a b l e t o s y n t h e s i z e  RNA a l t h o u g h ,  these c e l l s are capable  n u c l e o t i d e s de novo.  of s y n t h e s i z i n g purine  The e f f e c t s o f a c t i n o m y c i n  D on t h e b i o -  s y n t h e s i s o f p u r i n e n u c l e o t i d e s was s t u d i e d i n t h i s system u s i n g llf  C - f o r m a t e as a r a d i o a c t i v e p r e c u r s o r .  I t was o b s e r v e d t h a t  a c t i n o m y c i n D a t a c o n c e n t r a t i o n as h i g h as 50 mg/ml had no i n h i b i t o r y e f f e c t on., t h e f o r m a t i o n o f p u r i n e n u c l e o t i d e s o r on t h e f o r m a t i o n o f n u c l e o t i d e s from preformed p u r i n e s .  Actinomycin  a l s o had no e f f e c t on t h e i n t e r c o n v e r s i o n o f p u r i n e i n t h i s system. actinomycin  J a c o l i and Zbarsky  nucleotides  (1) s t u d i e d t h e e f f e c t o f  on t h e s y n t h e s i s o f n u c l e o t i d e s i n Bacillus  and o b s e r v e d an a c c u m u l a t i o n  D  subtilis  o f guanine n u c l e o t i d e s i n the a c i d  s o l u b l e f r a c t i o n o f c e l l s t h e growth o f which was i n h i b i t e d by actinomycin  D.  The p r e s e n t s t u d i e s were u n d e r t a k e n t o f i n d o u t t h e e f f e c t of a c t i n o m y c i n  D i n a mammalian system v i z . E h r l i c h a s c i t e s  -carcinoma c e l l s .  E h r l i c h a s c i t e s carcinoma c e l l suspensions  in  Krebs R i n g e r phosphate b u f f e r was i n c u b a t e d i n p r e s e n c e o f a c t i n o m y c i n D.  1  ^C-Formate was used as a p r e c u r s o r o f n u c l e o -  t i d e s and n u c l e i c a c i d , i n these s t u d i e s .  The v a r i o u s components  o f t h e a c i d s o l u b l e n u c l e o t i d e s from c e l l s i n c u b a t e d  with  a c t i n o m y c i n D as w e l l as from c o n t r o l e x p e r i m e n t s were by two d i f f e r e n t t e c h n i q u e s .  separated  A method o f s e p a r a t i o n by paper  chromatography developed d u r i n g t h e course o f t h e s e i n v e s t i g a t i o n s was used t o s e p a r a t e  small q u a n t i t i e s of a c i d s o l u b l e  n u c l e o t i d e s from E h r l i c h a s c i t e s tumour c e l l s u s p e n s i o n s . l a r g e r q u a n t i t i e s o f m a t e r i a l was t o be s e p a r a t e d , on D E A E - c e l l u l o s e  When  chromatography  was c a r r i e d o u t .  The r e s u l t s o f s t u d i e s i n s e p a r a t i o n and e s t i m a t i o n o f a c i d s o l u b l e m a t e r i a l from s m a l l q u a n t i t i e s o f c e l l s were i n c o n c l u s i v e due t o v a r i a t i o n s i n r e s u l t s from e x p e r i m e n t t o e x p e r i m e n t . s t u d i e s employing l a r g e r q u a n t i t i e s o f c e l l s u s p e n s i o n ,  From  i t was  found t h a t t h e amounts o f a c i d s o l u b l e n u c l e o t i d e s i n c r e a s e d i n c e l l s i n c u b a t e d w i t h a c t i n o m y c i n D.  A systematic a n a l y s i s of  the components o f t h e a c i d s o l u b l e n u c l e o t i d e s r e v e a l e d t h e marked i n c r e a s e i n c o n c e n t r a t i o n o f t h e p u r i n e n u c l e o t i d e s . i n c r e a s e was p a r t i c u l a r l y n o t i c e a b l e i n b o t h ATP and GTP.  This An  u n u s u a l f i n d i n g was t h a t • t h e i n c r e a s e i n c o n c e n t r a t i o n o f ATP was g r e a t e r t h a n a c o r r e s p o n d i n g  i n c r e a s e o f GTP.  t i o n s o f t h e d i p h o s p h a t e s were a l s o i n c r e a s e d .  The  concentra-  F u r t h e r , an i n -  crease i n concentrations of a l l the other pyrimidine nucleotides were a l s o o b s e r v e d i n c e l l s i n c u b a t e d  i n presence of actinomycin  D, p a r t i c u l a r l y o f UTP. The i n c o r p o r a t i o n o f ^ C - f ormate i n t o t h e v a r i o u s  purine  n u c l e o t i d e s was e s t i m a t e d .  The s p e c i f i c  a c t i v i t i e s of the  v a r i o u s substances i s o l a t e d  i n presence of actinomycin  D were  l o w e r t h a n t h e c o n t r o l s , though t h e d e c r e a s e i s n o t v e r y significant.  This decrease i n s p e c i f i c  c e l l s incubated with actinomycin causes.  a c t i v i t y observed i n  D.'.may be due t o d i f f e r e n t  I t may be n o t e d t h a t t h e c o n c e n t r a t i o n o f t h e n u c l e o -  t i d e s i n t h e s e cases has i n c r e a s e d c o n s i d e r a b l y and as t h e specific tration  activity  i s a f u n c t i o n o f c o n c e n t r a t i o n , when concen-  o f t h e n u c l e o t i d e p o o l i s i n c r e a s e d t h e r e may be a  decrease i n the s p e c i f i c specific tration  activity  activity.  r  However, t h e d e c r e a s e i n  i s s m a l l compared t o t h e i n c r e a s e i n concen-  of the nucleotides.  From t h e p r e s e n t  i n v e s t i g a t i o n s as w e l l as from t h e o b s e r -  v a t i o n s o f o t h e r s i t i s known t h a t a c t i n o m y c i n normal s y n t h e s i s o f RNA i n t h e c e l l s .  D inhibited  the  I n t h e normal m e t a b o l i s m  o f RNA, t h i s compound may be b r o k e n down t o n u c l e o t i d e s which may a g a i n be r e u t i l i z e d .  In presence of actinomycin  D, t h e  r e u t i l i z a t i o n o f t h e n u c l e o t i d e s formed by RNA breakdown i s n o t u t i l i z e d f u r t h e r and t h i s can d i l u t e  the r a d i o a c t i v i t y  a c i d s o l u b l e p o o l w h i c h may a g a i n lower t h e . s p e c i f i c the n u c l e o t i d e s . decreasing  the s p e c i f i c  i n h i b i t the conversion  a c t i v i t y of the acid s o l u b l e nucleotide.  activity.  D d i d not  o f preformed p u r i n e s t o n u c l e o t i d e s o r  the i n t e r c o n v e r s i o n o f n u c l e o t i d e s . converted  a c t i v i t y of  There i s a l s o y e t a n o t h e r p o s s i b i l i t y f o r  I t was o b s e r v e d by many (65, 66) t h a t a c t i n o m y c i n  purines  of the  Non-radioactive,  preformed  i n t o n u c l e o t i d e s can a l s o reduce t h e s p e c i f i c  Finally,  the accumulation of nucleotides i n the c e l l s  may produce a feedback i n h i b i t i o n on t h e de novo s y n t h e s i s o f purine nucleotides  (116) whereby t h e f o r m a t i o n o f r a d i o a c t i v e  n u c l e o t i d e s may be d e c r e a s e d .  However, as t h e d e c r e a s e i n  s p e c i f i c a c t i v i t y i s c o n s i d e r a b l y l e s s compared t o marked i n c r e a s e i n c o n c e n t r a t i o n o f n u c l e o t i d e s , i t may be assumed t h a t t h e p u r i n e n u c l e o t i d e s formed by a l l t h e s e a l t e r n a t e pathways a l s o i s s m a l l . that actinomycin  I t i s c o n c l u d e d from t h e s e s t u d i e s  D does n o t i n h i b i t t h e de novo s y n t h e s i s o f  p u r i n e n u c l e o t i d e s i n E h r l i c h a s c i t e s carcinoma c e l l s in  vitro.  R e c e n t l y K i d a , e t a l . (63) have r e p o r t e d t h e e f f e c t o f chromomycin A 3 on n u c l e i c a c i d m e t a b o l i s m o f Bacillus Chromomycin A 3  l i k e actinomycin  sublitis.  D inhibits selectively  cellular  and enzymic s y n t h e s i s o f RNA by DNA dependent RNA polymerase. T h i s s p e c i f i c i n h i b i t i o n has been c o n f i r m e d (123).  by Hartmann e t a l .  K i d a e t a l . have s t u d i e d t h e i n c o r p o r a t i o n o f  the a c i d s o l u b l e n u c l e o t i d e s o f Bacillus  subtilis  P  into  i n h i b i t e d by  chromomycin A 3 . They have a l s o i s o l a t e d and e s t i m a t e d s o l u b l e n u c l e o t i d e s from t h e m i c r o - o r g a n i s m s t e s t e d . s o l u b l e n u c l e o t i d e s were s e p a r a t e d  3 2  on Dowex-l-column.  the a c i d The a c i d They have  o b s e r v e d a marked i n c r e a s e o f s e v e r a l n u c l e o t i d e components, particularly  t h o s e o f guanine and c y t o s i n e .  Though t h e r e  was  an i n c r e a s e i n u r i d i n e n u c l e o t i d e s and adenine n u c l e o t i d e s , i t was n o t so marked as t h e o t h e r two.  However, t h e y have observed  a decrease i n the r a d i o a c t i v i t y of the a c i d s o l u b l e n u c l e o t i d e s by i n c r e a s i n g t h e i n c u b a t i o n t i m e . p o s s i b l e degradation longed incubation.  T h i s , they a t t r i b u t e t o t h e  o f the r a d i o a c t i v e n u c l e o t i d e s d u r i n g  pro-  These r e s u l t s a r e v e r y much i n agreement  - 98 with the results reported herein. have observed  Horning and R a b i n o w i t z (124)  e l e v a t e d l e v e l s o f ATP,  ADP and AMP, when Sarcoma  37 a s c i t e s were i n c u b a t e d i n t h e presence  o f a c t i n o m y c i n D.  C a s h e l and G a l l e n t (125) r e c e n t l y r e p o r t e d t h a t a c t i n o m y c i n D does n o t i n h i b i t t h e c o n v e r s i o n o f u r i d i n e t o DTP and CTP i n Ei  coli  cells.  They have observed  an a c c u m u l a t i o n o f b o t h UTP  and CTP i n c e l l s t r e a t e d w i t h e i t h e r a c t i n o m y c i n D o r p r o f l a v i n . The agreement o f r e s u l t s i n m i c r o b i a l and mammalian systems, s u g g e s t ' t h a t t h e r e i s no i n h i b i t i o n o f t h e de novo s y n t h e s i s o f p u r i n e n u c l e o t i d e s by t h e s e a n t i b i o t i c s i n h i b i t i n g RNA polymerase. secondary  The a c c u m u l a t i o n o f t h e n u c l e o t i d e s seen a r e t h e  e f f e c t produced, by t h e i n h i b i t i o n o f n u c l e i c a c i d  synthesis.  The a c c u m u l a t i o n o f t h e n u c l e o t i d e may f u r t h e r i n -  h i b i t t h e de novo s y n t h e s i s t h r o u g h t h e known mechanisms o f regulation of purine nucleotide synthesis.  A p a r t from t h e s e ,  t h e r e seems t o be no i n h i b i t i o n caused by a c t i n o m y c i n D on n u c l e o t i d e b i o s y n t h e s i s in  vitro.  It. has been r e p o r t e d by Acs e t a l . (126) t h a t i n subtilisj  Bacillus  t h e newly formed RNA-of h i g h m o l e c u l a r w e i g h t i s  r a p i d l y degraded i n presence  o f a c t i n o m y c i n D.. As these com-  ponents c o u l d n o t be r e u t i l i z e d because o f t h e i n h i b i t i o n o f RNA  polymerase by a c t i n o m y c i n , one c o u l d n a t u r a l l y expect an  increase i n the acid soluble nucleotides i n incubations containi n g a c t i n o m y c i n D.  However, t h e r e a r e r e p o r t s i n t h e l i t e r a t u r e  c o n t r a r y t o t h e o b s e r v a t i o n s o f Acs e t a h . Cbantrenne (127) has s t u d i e d t h e t u r n o v e r o f RNA i n presence Bacillus  cereus.  He has c o n c l u d e d  of actinomycin i n  " t h a t t h e r e i s no r e a s o n t o  b e l i e v e t h a t a c t i n o m y c i n caused t h e d e s t r u c t i o n o f RNA by any  - 99 d i r e c t a c t i o n " and t h a t a c t i n o m y c i n o n l y i n h i b i t e d the  RNA  s y n t h e s i s "making o b v i o u s the d e g r a d a t i v e phase of the t u r n over" .  Leivi  E.  c e l l s i n h i b i t e d by a c t i n o m y c i n D.  ooli  (128) has s t u d i e d t h e d e g r a d a t i o n o f RNA  " t h e r e i s t h e r e f o r e no e v i d e n c e  He has  in  observed:  t h a t actinomycin induces  breakdown i n the b a c t e r i a i n w h i c h i t has been t e s t e d " . a l s o has suggested  t h a t the breakdown o f any RNA  He  observed  experiments  r e f l e c t s , o n l y the "normal breakdown o f l a b i l e  t i o n o f RNA  and n o t an a r t i f a c t o f a c t i n o m y c i n a c t i o n " .  l i g h t o f t h e s e e v i d e n c e s as w e l l as from the s p e c i f i c of substance concluded  observed  RNA  in fracIn the  activities  i n the p r e s e n t i n v e s t i g a t i o n s , i t can  t h a t the observed  be  i n c r e a s e i n n u c l e o t i d e i n the a c i d  s o l u b l e p o o l i s n o t due t o breakdown o f RNA  i n d u c e d by a c t i n o -  mycin D. Because o f the c o n f l i c t i n g r e p o r t s i n the l i t e r a t u r e about t h e e f f e c t s o f a c t i n o m y c i n s i n g e n e r a l and a c t i n o m y c i n D i n p a r t i c u l a r , on r e s p i r a t i o n and g l y c o l y s i s by d i f f e r e n t t h e s e were examined i n E h r l i c h a s c i t e s carcinoma  cells.  r e s u l t s i n d i c a t e d t h a t a c t i n o m y c i n D i n h i b i t e d the i n - E h r l i c h a s c i t e s carcinoma nificant.  c e l l s though i t was  tissues,  respiration  not v e r y  S i m i l a r e f f e c t s o f a c t i n o m y c i n are r e p o r t e d by  investigators  (68).  The  sigother  I t i s not known whether t h i s i n h i b i t i o n o f  r e s p i r a t i o n by a c t i n o m y c i n D a f f e c t s n u c l e o t i d e s y n t h e s i s i n cells.  The p r e s e n t o b s e r v a t i o n s as w e l l as those r e p o r t e d by  o t h e r s t h a t the n u c l e o t i d e s are i n c r e a s e d i n c e l l s w i t h a c t i n o m y c i n D suggest  incubated  t h a t t h i s i n h i b i t i o n of r e s p i r a t i o n  does n o t a f f e c t n u c l e o t i d e s y n t h e s i s .  Glycolysis i n Ehrlich  - 100 a s c i t e s carcinoma added g l u c o s e .  c e l l s was f o l l o w e d by t h e d i s a p p e a r a n c e o f  I t i s known t h a t about 80% o f t h e d i s a p p e a r a n c e  of g l u c o s e i n E h r l i c h a s c i t e s carcinoma f o r by t h e p r o d u c t i o n o f l a c t i c a c i d  c e l l s can be  (117).  accounted  From t h e p r e s e n t  i n v e s t i g a t i o n s , i t was c o n c l u d e d t h a t a c t i n o m y c i n D has no e f f e c t on t h e g l u c o s e uptake o f E h r l i c h a s c i t e s carcinoma  cells.  T h i s would mean t h a t t h e g l y c o l y t i c enzymes a r e n o t i n h i b i t e d by a c t i n o m y c i n D.  - 101  -  SECTION I I EFFECTS OF GLUCOSE ON THE  INCORPORATION OF  1  "* C—FORMATE  .INTO NUCLEIC ACID COMPONENTS OF EHRLICH ASCITES CARCINOMA CELLS in  vitro.  EXPERIMENTAL I t was  o b s e r v e d from p r e v i o u s  experiments t h a t  u t i l i z a t i o n i n E h r l i c h a s c i t e s c a r c i n o m a c e l l s was h i b i t e d by a c t i n o m y c i n  D.  mycin D i n h i b i t e d the RNA and R a b i n o w i t z  I t was  glucose not i n -  a l s o observed t h a t a c t i n o -  synthesis i n these c e l l s .  (81) r e p o r t e d t h a t a c t i n o m y c i n  o f o t h e r a n t i b i o t i c s i n h i b i t e d the RNA  Horning  D and a v a r i e t y  s y n t h e s i s i n Sarcoma 37  a s c i t e s c e l l s i n the p r e s e n c e of g l u c o s e , but the p r o t e i n s y n t h e s i s was  o n l y i n h i b i t e d i n the absence of g l u c o s e .  e x p l a i n these observations f o r ATP cell.  and The  t h e y suggested a compartmentation  a r e g u l a t o r y r o l e f o r l a b i l e RNA a b i l i t y of g l u c o s e  of a c t i n o m y c i n  D was  To  s p e c i e s i n the  t o p r e v e n t the i n h i b i t o r y e f f e c t  r e l a t e d t o the h i g h e r a c c e s s i b i l i t y of  d e r i v e d from g l y c o l y s i s a t the b i o s y n t h e t i c s i t e s i n the I n e x p l a i n i n g the e f f e c t of g l u c o s e  ATP  cells.  on the i n c o r p o r a t i o n of  l a b e l l e d n u c l e i c a c i d p r e c u r s o r s by E h r l i c h a s c i t e s c a r c i n o m a c e l l s , Henderson and LePage (70) have suggested t h a t the g l y c o l y t i c energy was acids.  used f o r b i o s y n t h e s i s of n u c l e o t i d e s and n u c l e i c  I n the s t u d i e s of i n c o r p o r a t i o n of '*C-formate by 1  E h r l i c h a s c i t e s c a r c i n o m a c e l l s i n p r e s e n c e of a c t i n o m y c i n was  o b s e r v e d t h a t a low i n c o r p o r a t i o n o f the •I  D, i t  radioactivity  •  r e s u l t e d i n a wide v a r i a t i o n i n the s p e c i f i c a c t i v i t i e s of substances i s o l a t e d f o r d i f f e r e n t experiments.  To m i n i m i z e  the the  - 102 v a r i a t i o n i t was 1 lf  1  C-formate.  -  desirable to increase  the i n c o r p o r a t i o n  I n an attempt t o i n c r e a s e  *C-formate. i n t o n u c l e o t i d e  c a r c i n o m a c e l l s , g l u c o s e was  and  the i n c o r p o r a t i o n  added t o the medium, and  components o f E h r l i c h a s c i t e s c e l l s e s t i m a t e d . t o the o b s e r v a t i o n lk  blem  was The  of others  was  investigated i n greater  for incubation.  On  1If  C - f o r m a t e was  irres-  t o g l y c o l y s i s , of  i o n s i n the medium o r o f the b u f f e r used f u r t h e r e x a m i n a t i o n i t was  e f f e c t of g l u c o s e depended on the c e l l d e n s i t y tions.  Hence t h i s p r o -  detail.  p e c t i v e of. the f a l l i n pH of the medium due + +  I n many e x p e r i -  (69-71) a d e c r e a s e  observed.  d e c r e a s e i n i n c o r p o r a t i o n of  the p r e s e n c e of C a  the  nucleic acid  1  i n i n c o r p o r a t i o n of C-formate  of  n u c l e i c a c i d s , of E h r l i c h a s c i t e s  i n c o r p o r a t i o n of ^'C-formate i n t o the v a r i o u s  ments c o n t r a r y  of  observed i n the  In a c o n c e n t r a t e d c e l l s u s p e n s i o n , g l u c o s e  that incuba-  increased  the i n c o r p o r a t i o n whereas i n a d i l u t e c e l l s u s p e n s i o n same concentration  of g l u c o s e d e c r e a s e d the i n c o r p o r a t i o n .  These  e x p e r i m e n t s s u g g e s t e d t h a t the main f a c t o r c o n t r o l l i n g the i n c o r p o r a t i o n of p r e c u r s o r s t h e s e c e l l s was ATP  i n t o n u c l e i c a c i d components of  the t r a n s i e n t d e p l e t i o n and  i n the c e l l s i n p r e s e n c e of g l u c o s e .  always i n c r e a s e d  the i n c o r p o r a t i o n of  11  regeneration  of  However, g l u c o s e  *C-formate i n t o s e r i n e  of the a c i d s o l u b l e f r a c t i o n of E h r l i c h a s c i t e s c a r c i n o m a c e l l s , i n v e s t i g a t i o n s are p r e s e n t e d below. 1) Time Course' of i n c o r p o r a t i o n of ''C-Porraate i n t o V a r i o u s 1  N u c l e i c A c i d Components i n P r e s e n c e o f G l u c o s e . E h r l i c h a s c i t e s c a r c i n o m a c e l l s were c o l l e c t e d from  8-10  - 103  -  a n i m a l s a week a f t e r tumour i m p l a n t a t i o n  as d e s c r i b e d b e f o r e . '  The  c e l l s were c e n t r i f u g e d  rpm  ( I n t e r n a t i o n a l PR-IT, model) f o r 3 minutes and  n a t a n t f l u i d was  removed.  i n a refrigerated centrifuge  The  c e l l s were washed w i t h  t i m e s the c e l l volume of c o l d 0.9% fuged a g a i n a t 2000 rpm was  removed, and  the  at  1500  superthree  sodium c h l o r i d e and c e n t r i -  f o r 4 minutes.  The  supernatant f l u i d  the c e l l s were t h e n suspended i n 9 volumes o f  c o l d Krebs R i n g e r phosphate b u f f e r pH  7.8.  yc/ml s u s p e n s i o n ) and  formate (2 ymoles/ml)  non  radioactive  ll  *C-Formate  were added t o t h e . c e l l s u s p e n s i o n and mixed w e l l . s i o n was  d i v i d e d i n t o two  equal portions  The  (0.5  suspen-  i n s e p a r a t e 1000  ml  Erlenmeyer f l a s k s . The  f l a s k s were c l o s e d w i t h a one  holed rubber stopper  through which a narrow p o l y e t h y l e n e tube was was  inserted.  passed c o n t i n u o u s l y i n t o the f l a s k s p l a c e d i n a w a t e r b a t h  a t 37°C and  shaken a t 100-110 o s c i l l a t i o n s per minute.  thermal e q u i l i b r a t i o n f o r 3 minutes, glucose s o l u t i o n added t o one  The  c o n t e n t s were mixed w e l l and  continued.•  was  incubation  withdrawn from each f l a s k  from the c e l l s , the a c i d s o l u b l e  f r a c t i o n , RNA  as d e s c r i b e d under methods.  The  and  perchloric  e x t r a c t of a c i d s o l u b l e n u c l e o t i d e s  was  p o t a s s i u m h y d r o x i d e t o pH  the p r e c i p i t a t e of  c h l p r a t e was  (5%)  ,  A t i n t e r v a l s a 10 ml sample was  extracted  After  of the f l a s k s t o g i v e a c o n c e n t r a t i o n of 1 mg/ml  of s u s p e n s i o n .  and  Oxygen  removed by  t i o n f o r s e v e r a l hours.  6.5-7  and  DNA acid  neutralized with  c e n t r i f u g a t i o n a f t e r c o o l i n g the The  were  10  N  persolu-  supernatant s o l u t i o n containing  the  - 104 acid soluble nucleotides a s collected separately.  H a l f ml samples  w  of t h i s s o l u t i o n were counted i n a l i q u i d s c i n t i l l a t i o n The O.D.  counter.  o f t h i s s o l u t i o n a t 260 mu was o b t a i n e d a f t e r d i l u t i n g  5 times w i t h w a t e r .  The r a d i o a c t i v i t y i n c o r p o r a t e d i n t o t h e  a c i d s o l u b l e f r a c t i o n p e r O.D. i n F i g u r e 13.  u n i t p l o t t e d a g a i n s t time i s g i v e n  i n some o f t h e l a t e r e x p e r i m e n t s samples o f t h e  a c i d s o l u b l e f r a c t i o n were mixed w i t h hyamine w i t h o u t  previous  n e u t r a l i z a t i o n and counted.  A s o l u t i o n o f 0.2 M p e r c h l o r i c a c i d  was- used as a b l a n k i n these  estimations.  A . p o r t i o n o f t h e RNA n u c l e o t i d e s o b t a i n e d by h y d r o l y s i s o f RNA d u r i n g t h e s e p a r a t i o n from DNA was d i l u t e d 8 t o 10 times w i t h water.  The r i b o s e c o n t e n t o f t h e n u c l e o t i d e m i x t u r e was .  e s t i m a t e d by t h e m o d i f i e d o i r c i n o l r e a c t i o n ( 8 0 ) . AMP ( C a l biochemicals)  was used as a s t a n d a r d  i n these e s t i m a t i o n s ( 8 1 ) .  The c o n c e n t r a t i o n s o f n u c l e o t i d e s were c a l c u l a t e d i n terms o f umoles o f adenine n u c l e o t i d e .  The r a d i o a c t i v i t y  i n t o t h e s e n u c l e o t i d e s was d e t e r m i n e d by l i q u i d counting.  incorporated scintillation  The r e s u l t s o f t h e time course o f i n c o r p o r a t i o n i s  g i v e n i n F i g u r e 14. The DNA was d i s s o l v e d i n 0.5 ml o f 0.1 N NaOH and d i l u t e d 10 t i m e s .  The c o n c e n t r a t i o n o f t h e DNA i n s o l u t i o n was  by t h e d i p h e n y l a m i n e r e a c t i o n .  Highly polymerized  DNA  was used as a s t a n d a r d .  (Mann Research C o r p o r a t i o n )  ml samples o f t h e s o l u t i o n were counted i n a l i q u i d  estimated  c a l f thymus Half  scintillation  spectrophotometer t o o b t a i n the r a d i o a c t i v i t y incorporated. r e s u l t s a r e g i v e n i n F i g u r e 15. T h i s was t h e p r o c e d u r e used f o r a l l t h e time course o f  The  - 105 -  30  60  90  120  TIME (minutes) F i g u r e 13.  E f f e c t of g l u c o s e (5.5 mM) on the i n c o r p o r a t i o n o f C - f o r m a t e i n t o , the a c i d s o l u b l e f r a c t i o n o f E h r l i c h a s c i t e s carcinoma c e l l s suspended i n Krebs R i n g e r phosphate b u f f e r pH 7.3. llf  Control  (  o  ©  )  + g l u c o s e (——A  )  - 1-05 -  600  ft r-i O  e \  a, o  400  4J  •H > •H  -)->  O  D •H  m  •H  o  Q) Cu  Ui  200 "  60  90  120  180  TIME (minutes) F i g u r e 14.  E f f e c t o f g l u c o s e (5.5 mM) on the i n c o r p o r a t i o n o f "*C-formate i n t o RNA by E h r l i c h a s c i t e s carcinoma c e l ' l s suspended i n Krebs R i n g e r phosphate b u f f e r pH 7.8. 1  Control  (  -)  + glucose G  -k  )  - 107 -  30  60  90  TIME Figure  15.  120  (minutes)  E f f e c t o f g l u c o s e (5.5 mil) o n t h e i n c o r p o r a t i o n o f C-£ormate i n t o DNA o f E h r l i c h a s c i t e s c a r c i n o m a cells s u s p e n d e d i n K r e b s R i n g e r p h o s p h a t e b u f f e r p H 7.8. lh  Control  (  O  ©  )  + -glucose  (  &  )  - 108 incorporation studies described  i n the t h e s i s .  t h e r e a r e changes o f b u f f e r s o r s u b s t r a t e s The g e n e r a l p r o c e d u r e s o f i n c u b a t i o n s t h e same as d e s c r i b e d  Where e v e r  t h e s e a r e mentioned.  and e s t i m a t i o n s  were  here.  Examination of the Figure  13 shows t h a t t h e i n c o r p o r a t i o n  of r a d i o a c t i v i t y i n t o a c i d s o l u b l e f r a c t i o n was m a r k e d l y w i t h time i n presence of glucose. the observations  of other  increased  T h i s was i n agreement w i t h  investigators.  However, from f i g u r e s  14 and 15 i t c a n be seen t h a t t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e 1  i n t o b o t h RNA and DNA was d e c r e a s e d w i t h time i n t h e i n c u b a t i o n s c o n t a i n i n g g l u c o s e w h i c h was i n c o n t r a s t w i t h r e s u l t s by  obtained  others. Because o f t h e d i s a g r e e m e n t i n t h e r e s u l t from t h a t o b s e r v e d  by o t h e r s , t h e e x p e r i m e n t was r e p e a t e d s e v e r a l t i m e s under s i m i lar conditions. of  lh  C-formate  I n a l l cases an i n c r e a s e i n t h e i n c o r p o r a t i o n  i n t o t h e a c i d s o l u b l e f r a c t i o n was n o t i c e d .  ever l a t e r experiments revealed  t h a t t h i s i n c r e a s e cannot be a c -  counted f o r e n t i r e l y by t h e i n c o r p o r a t i o n o f nucleotides  alone.  How-  1If  C-formate  into  I n s p i t e o f an a p p a r e n t i n c r e a s e , when t h e  incorporation of ^C-formate  i n t o n u c l e o t i d e p u r i n e s was d e t e r -  mined, a d e c r e a s e was n o t i c e d i n many cases i n p r e s e n c e o f g l u c o s e . L a t e r i t was a l s o found t h a t much o f t h e ^ C - f o r m a t e was i n c o r 1  p o r a t e d i n t o s e r i n e i n p r e s e n c e o f g l u c o s e w h i c h appeared i n t h e a c i d s o l u b l e f r a c t i o n c o n t r i b u t i n g t o the apparent i n c r e a s e i n specific  acitivity.  I n t h e case o f RNA, t h e i n c o r p o r a t i o n o f C-formate lh  was  l o w e r t h a n c o n t r o l s i n many e x p e r i m e n t s i n p r e s e n c e o f g l u c o s e ,  - 109 though the d e c r e a s e was  -  not always s i g n i f i c a n t .  Similar re-  s u l t s were a l s o o b t a i n e d when the i n c o r p o r a t i o n of '*C-formate 1  i n t o DNA  was  s t u d i e d i n E h r l i c h a s c i t e s carcinoma c e l l s .  Because of the d i s a g r e e m e n t i n r e s u l t s from those o b t a i n e d o t h e r s , w i t h r e s p e c t t o the e f f e c t of g l u c o s e  by  on i n c o r p o r a t i o n  o f '*C-formate by E h r l i c h carcinoma c e l l s , v a r i o u s  other  1  f a c t o r s a f f e c t i n g the i n c o r p o r a t i o n s were s t u d i e d . 2) E f f e c t of C a  + +  on the I n c o r p o r a t i o n of  14  C-Formate i n t o  N u c l e i c A c i d Components of E h r l i c h A s c i t e s Carcinoma C e l l s . Previous  i n v e s t i g a t o r s have o m i t t e d c a l c i u m from the b u f f e r  s o l u t i o n i n w h i c h the t i s s u e was  suspended.  They have a l s o  o b s e r v e d an i n c r e a s e i n the i n c o r p o r a t i o n of p r e c u r s o r of c l e i c a c i d s i n the p r e s e n c e o f g l u c o s e .  nu-  I n the e x p e r i m e n t s  d e s c r i b e d h e r e i n , E h r l i c h a s c i t e s c e l l s were suspended i n Krebs R i n g e r phosphate b u f f e r c o n t a i n i n g 0.86- umoles/ml of chloride.  calcium  C a l c i u m i s w e l l known as an a c t i v a t o r of ATPase  an i n h i b i t o r of o x i d a t i v e p h o s p h o r y l a t i o n  (129).  and  Recently  c a l c i u m has been r e p o r t e d t o p r e v e n t the o x i d a t i o n of  citrate,  thereby  a f f e c t i n g t h e . o x i d a t i o n of s u b s t r a t e i n many t i s s u e s  (130).  To a v o i d the p o s s i b i l i t y of c a l c i u m a f f e c t i n g the i n -  c o r p o r a t i o n of  xl>  C - f o r m a t e I n t o n u c l e i c a c i d components i n  p r e s e n c e of g l u c o s e , the f o l l o w i n g e x p e r i m e n t was I n c u b a t i o n was  c a r r i e d out.  c a r r i e d out as d e s c r i b e d b e f o r e , but i n  t h i s c a s e , the c e l l s were d i l u t e d w i t h Krebs R i n g e r The  phosphate  b u f f e r pH 7.8,  c o n t a i n i n g no c a l c i u m .  v i d e d i n t o two  a f t e r the a d d i t i o n of the ^ C - f o r m a t e and  r a d i o a c t i v e formate as d e s c r i b e d b e f o r e .  s u s p e n s i o n was d i -  1  G l u c o s e was  non  added t o  -  no  -  one o f t h e f l a s k s t o have a c o n c e n t r a t i o n o f 1 mg/ml o f s u s p e n s i o n , and 0  2  was f l u s h e d t h r o u g h t h e f l a s k s .  Samples were  w i t h d r a w n a t i n t e r v a l s from b o t h t h e i n c u b a t i o n m i x t u r e s .  The  a c i d s o l u b l e f r a c t i o n and t h e n u c l e i c a c i d s were o b t a i n e d from, the c e l l s .  The c o n c e n t r a t i o n o f each o f t h e components and t h e  r a d i o a c t i v i t y i n c o r p o r a t e d were d e t e r m i n e d .  The r e s u l t s a r e  g i v e n i n F i g u r e s '. . 16, 17 and 18. I t can be seen from F i g u r e 16 t h a t t h e i n c o r p o r a t i o n o f •^-C-formate i n c r e a s e d markedly i n p r e s e n c e o f g l u c o s e p e r i o d o f 1 hour.  f o r -a  On l o n g e r i n c u b a t i o n s t o 2 hours t h e r e was  a d e c r e a s e i n i n c o r p o r a t i o n o f '*C-formate i n b o t h t h e c o n t r o l 1  and i n i n c u b a t i o n s c o n t a i n i n g  glucose.  I n t h e case o f b o t h RNA and DNA, t h e i n c o r p o r a t i o n o f  1,lf  C  d e c r e a s e d i n p r e s e n c e o f g l u c o s e , s i m i l a r t o t h a t o b s e r v e d when c e l l s were suspended i n Krebs R i n g e r phosphate s o l u t i o n c o n t a i n ing calcium.  So t h e e f f e c t e x e r t e d by g l u c o s e i n d e c r e a s i n g t h e  i n c o r p o r a t i o n o f '*C-formate i n t o t h e n u c l e i c a c i d may n o t be 1  due  t o t h e p r e s e n c e o f c a l c i u m i n t h e i n c u b a t i o n medium. ++  : • c .In..order t o see whether Ca poration of  1If  C - f o r m a t e i n p r e s e n c e o f g l u c o s e , the ' f o l l o w i n g  e x p e r i m e n t was c a r r i e d o u t . suspension  E h r l i c h a s c i t e s carcinoma c e l l  was made i n Krebs R i n g e r phosphate b u f f e r pH 7.8  c o n t a i n i n g - no c a l c i u m .  1  '*C-Formate (0.5 uC / m l ) , sodium f o r -  mate (2 umole/ml) and g l u c o s e pension  had any e f f e c t on t h e i n c o r -  and mixed w e l l .  (1 mg/ml) were added t o t h e sus-  The s o l u t i o n was d i v i d e d i n t o two  h a l v e s and enough c a l c i u m c h l o r i d e s o l u t i o n was added t o one of t h e i n c u b a t i o n m i x t u r e s  t o g i v e a c o n c e n t r a t i o n o f 2.58  Ill  30  60  90  120  TIME (minutes) F i g u r e 16.  E f f e c t o f g l u c o s e (5.5 mM) on the i n c o r p o r a t i o n o f C - f o r m a t e i n t o a c i d s o l u b l e f r a c t i o n of E h r l i c h a s c i t e s c a r c i n o m a suspended i n Ca f r e e Krebs R i n g e r phosphate b u f f e r pH 7.8. llf  ~  Control  (  •©•  )  + glucose (  A-  A  )  - 112  30  -  60  90  120  TIME (minutes) F i g u r e 17.  E f f e c t o f g l u c o s e (5.5 mM) on the i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o t h e 3jlN_A of E h r l i c h a s c i t e s c a r c i n o m a c e l l s suspended i n Ca f r e e Krebs R i n g e r phosphate b u f f e r pH 7 . 8 . Control  (  o  ©  -)  + glucose (  -A  A  )  - 113 -  900  "  30  F i g u r e 18.  60 90 120 TIME (minutes) E f f e c t :of'.glucose (5.5 mM) on the i n c o r p o r a t i o n o f C-formate i n t o the D_NA of E h r l i c h a s c i t e s c a r c i n o m a c e l l s suspended i n Ca f r e e Krebs R i n g e r phosphate b u f f e r pH'7.8. lh  T  Control  (  e  ©  )  + glucose (  A-—A  )  - 114 umoles/ml.  The s u s p e n s i o n s were i n c u b a t e d  as b e f o r e  and samples  were withdrawn a t i n t e r v a l s from t h e i n c u b a t i o n m i x t u r e s .  The  c e l l s were c o l l e c t e d by c e n t r i f u g a t i o n and t h e a c i d s o l u b l e nucleotides  and t h e n u c l e i c a c i d s were e x t r a c t e d from t h e c e l l s .  The c o n c e n t r a t i o n incorporated  o f t h e v a r i o u s components and t h e r a d i o a c t i v i t y  i n t o t h e s e components were d e t e r m i n e d .  These a r e  given i n Table X I I . From t h e t a b l e i t can be seen t h a t c a l c i u m i o n s e x e r t no i n h i b i t o r y e f f e c t on t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o t h e nucleic acid.  Contrary  to inhibition a slight  e f f e c t on t h e i n c o r p o r a t i o n o f  llf  stimulatory  C-formate i n t o n u c l e i c a c i d i s  seen i n p r e s e n c e o f c a l c i u m . However t h e r e was a s l i g h t d e c r e a s e i n t h e i n c o r p o r a t i o n o f llf  C-formate i n t o the a c i d s o l u b l e f r a c t i o n i n incubations  t a i n i n g calcium.  I t i s n o t known whether t h i s d e c r e a s e i n  i n c o r p o r a t i o n i s because o f h i g h e r nucleotides  con-  i n c o r p o r a t i o n of the l a b e l l e d  i n t o the n u c l e i c acids.  However, from t h e s e e x p e r i m e n t s i t may be c o n c l u d e d t h a t t h e decrease i n i n c o r p o r a t i o n of C - f o r m a t e i n t o the various 14  a c i d components o f E h r l i c h a s c i t e s c e l l s i n c u b a t e d  nucleic  with glucose  was n o t due t o t h e p r e s e n c e o f c a l c i u m i n t h e i n c u b a t i o n medium. So i n subsequent e x p e r i m e n t s , c a l c i u m was n o t o m i t t e d  from t h e  J i u f f e r s o l u t i o n used t o suspend t h e c e l l s . 3) E f f e c t o f B u f f e r on t h e I n c o r p o r a t i o n o f ^ C - F o r m a t e by E h r l i c h A s c i t e s Carcinoma i n P r e s e n c e o f G l u c o s e . E h r l i c h a s c i t e s carcinoma c e l l s are remarkable i n t h e i r a b i l i t y t o g l y c o l y z e sugars.  As t h e s e c e l l s e x h i b i t b o t h C r a b t r e e  - 115 Table X I I E f f e c t of C a  + +  on t h e i n c o r p o r a t i o n o f  lh  C-formate  by E h r l i c h a s c i t e s c a r c i n o m a c e l l s i n p r e s e n c e o f glucose  (5.5 mM).  E x p e r i m e n t a l i n c u b a t i o n s con-  t a i n e d 2.5 umoles/ml o f c a l c i u m c h l o r i d e and c a l c i u m was a b s e n t from-the c o n t r o l s .  Acid-soluble Fraction (cpm/O.D. )  RNA (cpm/umole) AMP  DNA (cpm/mg)  Control  Exptl..  Control  Exptl ^  Control  7.5  3429  2664  58  88  127  142  15  4439  3109  135  102  300  272  30  5415  3753  129  169  352  350  45  4983  4366  227  190  527  471  60  6331  4665  205  266  548  700  90  5538  5576  298  403  1015  988  120  4318  5722  554  643  847  1107  Time  Exptl,  - 116 effect  (118) and t h e P a s t e u r e f f e c t , the l a c t i c a c i d produced i s  not maximally o x i d i z e d  (131).  The a c c u m u l a t i o n o f l a c t i c  reduces t h e pH o f the s u s p e n s i o n c o n s i d e r a b l y . of  pH may  The l o w e r i n g  a f f e c t the a c t i v i t y of t h e n u c l e i c a c i d  polymerases  as the pH optima f o r t h e s e enzymes a r e w e l l above pH 7. example t h e pH optimum f o r RNA.polymerase from E. r e p o r t e d t o be pK 7.9 and below pH 6.5 (132).  The pH optimum  of: DNA  acid  ooli  For is  the enzyme i s i n a c t i v a t e d  polymerase f o r i n t e s t i n a l  mucosa, o f r a t i s found t o be 7.8  (133).  The d e c r e a s e in. pH  w i t h time of E h r l i c h a s c i t e s carcinoma c e l l s u s p e n s i o n i n Krebs R i n g e r phosphate b u f f e r pH 7.8 (5.5 mM)  incubated with glucose  i s g i v e n i n T a b l e X I I I . I t i s seen from the t a b l e  t h a t the pH o f the s u s p e n s i o n dropped from 7.35 6.7  a t t h e end o f 2 hours o f i n c u b a t i o n .  a t 0 time t o  I t i s a l s o seen t h a t  t h e r e was no d e c r e a s e i n pH i n i n c u b a t i o n s which d i d n o t cont a i n glucose.  To overcome the change i n pH due t o g l y c o l y s i s ,  Krebs R i n g e r b i c a r b o n a t e b u f f e r was p r e f e r r e d t o phosphate buffer. R e c e n t l y P o o l e (134) has r e p o r t e d t h a t the f a l l i n pH o f g l y c o l y z i n g E h r l i c h a s c i t e s carcinoma c e l l s c o r r e s p o n d e d w i t h the  p r o d u c t i o n of l a c t i c a c i d though the l a c t a t e p r o d u c t i o n was  about the same i n both Krebs R i n g e r phosphate buffers.-  and b i c a r b o n a t e  She a l s o observed t h a t t h e f a l l o f pH was g r e a t e r i n  Krebs R i n g e r phosphate b u f f e r t h a n i n b i c a r b o n a t e b u f f e r to  due  the b e t t e r b u f f e r i n g e f f i c i e n c y of the b i c a r b o n a t e b u f f e r . F u r t h e r s t u d i e s of the' e f f e c t o f g l u c o s e on the i n c o r p o r a -  t i o n of '*C-formate was c a r r i e d out i n Krebs R i n g e r b i c a r b o n a t e 1  - 117 Table X I I I Decrease i n pH w i t h time o f E h r l i c h  ascites  carcinoma c e l l s u s p e n s i o n s i n Krebs R i n g e r phosphate b u f f e r pH 7.8, i n c u b a t e d w i t h g l u c o s e in  vitro.  5.5  A - C o n t r o l , B - Control + glucose  mM.  pH o f Suspension Time  A  0  .  7.35-  7.5 15 30  B 7.35  7.3  7.00  7.3  6.80  . . 7.3  6.85  45  7.3  6.8  60  7.3  6.75  90 120  '-<V.  I  /  V |  7.3  6.7  '••  7.3  6.7  - 118 -  buffer.  A l l o t h e r c o n d i t i o n s o f i n c u b a t i o n were t h e same e x c e p t  t h a t t h e gas phase was a m i x t u r e o f 95% O2 and 5% CO2•  The pH  o f t h e s u s p e n s i o n was checked a t i n t e r v a l s and found t o be s t a t i o n a r y , a t 7.4 b o t h i n c o n t r o l and i n p r e s e n c e o f g l u c o s e .  The  t i m e c o u r s e o f i n c o r p o r a t i o n o f '*C-formate i n p r e s e n c e o f 1  g l u c o s e . i n Krebs R i n g e r b i c a r b o n a t e  buffer into acid soluble  f r a c t i o n , RNA and DNA a r e g i v e n i n F i g u r e s  19, 20 and 21 r e s p e c -  tively. The 111  r e s u l t s from t h e f i g u r e s show t h a t t h e i n c o r p o r a t i o n o f  C-formate was i n c r e a s e d  i n the acid soluble f r a c t i o n i n  p r e s e n c e o f g l u c o s e w h i l e t h e same was d e c r e a s e d i n t h e RNA and the DNA.  T h i s was s i m i l a r t o t h e f i n d i n g o b t a i n e d  i n the experi  ments i n w h i c h Krebs R i n g e r phosphate was used t o suspend t h e cells.  Though t h e r e was no d i f f e r e n c e i n t h e p a t t e r n o f i n -  c o r p o r a t i o n , t h e pH o f t h e i n c u b a t i o n m i x t u r e was s t a b i l i z e d by b i c a r b o n a t e  b u f f e r as o b s e r v e d from a d i r e c t  determination,  because o f t h e b e t t e r b u f f e r i n g e f f i c i e n c y o f t h e Krebs R i n g e r bicarbonate  b u f f e r compared, t o t h e Krebs R i n g e r phosphate b u f f e r  As t h e r e was d e c r e a s e d i n c o r p o r a t i o n o f C - f o r m a t e i n t o 1 4  b o t h RNA and DNA i n p r e s e n c e o f g l u c o s e i t was o f i n t e r e s t t o see t h e e x t e n t o f i n h i b i t i o n i n t h e n u c l e o t i d e s bases o f DNA.  The n u c l e o t i d e s  obtained  o f RNA and  by h y d r o l y s i s o f t h e  RNA f r o m - t h e s e e x p e r i m e n t s were adsorbed on c h a r c o a l ,  eluted  w i t h p y r i d i n e - a l c o h o l m i x t u r e and were s e p a r a t e d by two dimens i o n a l paper chromatography.  The DNA was h y d r o l y s e d  bases s e p a r a t e d a l s o by paper chromatography. incorporated  and t h e  The r a d i o a c t i v i t y  i n t o t h e s e s u b s t a n c e s were e s t i m a t e d  and a r e g i v e n  - 119 -  ,  i  i  30 Figure  19.  «  60 90 TIME (minutes)  •  ^  120  E f f e c t o f g l u c o s e (5.5 mM) on the i n c o r p o r a t i o n o f '*C-formate i n t o the a c i d s o l u b l e f r a c t i o n o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s suspended i n Krebs R i n g e r b i c a r b o n a t e b u f f e r pH 7.8. 1  Control  (  ©  ©  )  + glucose (  A  A-——)  120  30  -  60  90  TIME F i g u r e 20.  120  (minutes)  E f f e c t o f g l u c o s e (5.5 mM) on the i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o the RNA o f E h r l i c h a s c i t e s carcinoma c e l l s suspended i n Krebs R i n g e r b i c a r b o n a t e b u f f e r pH 7.8. 1  Control  (  ©  «>  )  + glucose  (  A  -A  )  - 121 -  30  60  90  TIKE F i g u r e 21.  120  (minutes)  E f f e c t of g l u c o s e (5.5 mM) on the i n c o r p o r a t i o n o f C - f o r m a t e i n t o the DNA o f E h r l i c h a s c i t e s carcinoma c e l l s suspended i n Krebs R i n g e r b i c a r b o n a t e b u f f e r pH 7.8. 1H  Control  (—:—©  o  )  + glucose  (  A  A  )  T a b l e XIV I n c o r p o r a t i o n o f ^C-formate i n t o RNA n u c l e o t i d e s and DNA thymine o f E h r l i c h 1  ascites  c a r c i n o m a c e l l s in  vitro,  incubated with glucose  were suspended i n Krebs R i n g e r b i c a r b o n a t e b u f f e r pH 7.8.  (5.5 mM).  The c e l l s  A - Control, B -  Control + glucose. Specific activity  counts p e r minute p e r umole  RNA AMP  Time min- • utes  A  7.5  DNA CMP  GMP B  A  B;  A  46  21  0  0  30  72  44  50  60  165  52  87 .  120  661  441  301  ...  UMP  Thymine B  B  A  B  A  12  21  2  0  196  119  8  35  11  2  0  552  323  42  84  31  72  0  743  449  208  208  71  126  16  1780  626  . - 123 -  i n Table  XIV..  I n DNA, thymine.  the i n c o r p o r a t i o n o f ^ C - f o r m a t e was m a i n l y i n the  Though t h e r e was a s l i g h t i n c o r p o r a t i o n of r a d i o -  a c t i v i t y i n the p u r i n e bases o f DNA, they were too low t o have any  significance.  E x a m i n a t i o n o f the r e s u l t s show t h a t g l u c o s e  d e c r e a s e d the i n c o r p o r a t i o n o f '*C-formate i n t o the 1  nucleotides  o f RNA and  t o the thymine o f DNA.  4) E f f e c t o f G l u c o s e on t h e I n c o r p o r a t i o n E h r l i c h A s c i t e s Carcinoma C e l l s in In studying  purine  o f 2T.* ^ C - G l y c i n e by  vitro.  the i n c o r p o r a t i o n o f C - f o r m a t e i n t o the llf  n u c l e i c acids o f various  t i s s u e s , S m e l l i e e t a l . (103)  observed  t h a t the 1 c a r b o n u n i t formed from formate can exchange w i t h c a r b o n 2 (C )  o f the p u r i n e  o f the p u r i n e  r i n g , '*C-formate i s a l s o i n c o r p o r a t e d  2  ring.  I n a d d i t i o n t o the  1  :  m e t h y l group o f thymine.  Henderson and  formation  i n t o the  LePage (70) p o i n t e d  out  t h a t because o f thesev-non s p e c i f i c r e a c t i o n s , formate i s i n f e r i o r t o g l y c i n e f o r . t h e s t u d y o f de novo s y n t h e s i s o f p u r i n e s . They have recommended r a d i o a c t i v e g l y c i n e i n p r e f e r e n c e t o f o r mate as a p r e c u r s o r glucose exerted  of purines.  I n o r d e r t o see whether,  an e f f e c t on the i n c o r p o r a t i o n o f 2 - ^ C - g l y c i n e 1  s i m i l a r t o t h a t o b s e r v e d f o r ^ C - f o r m a t e , an experiment was p e r 1  formed u s i n g  2 - ^ C - g l y c i n e as the r a d i o a c t i v e p r e c u r s o r o f 1  nucleic acids. B e f o r e i n v e s t i g a t i n g the e f f e c t o f g l u c o s e ,  the  requirement  of non r a d i o a c t i v e g l y c i n e f o r the optimum i n c o r p o r a t i o n o f 2 - ^ C - g l y c i n e by E h r l i c h a s c i t e s c a r c i n o m a was i n v e s t i g a t e d . 1  E h r l i c h a s c i t e s c e l l s u s p e n s i o n s were p r e p a r e d i n Krebs  R i n g e r b i c a r b o n a t e b u f f e r pH  7.8  as d e s c r i b e d  before.  In-  c u b a t i o n s were c a r r i e d out i n 250 ml E r l e n m e y e r f l a s k s i n a w a t e r b a t h a t 37°C.  T w e n t y - f i v e m i c r o l i t r e s of a s o l u t i o n  of g l y c i n e c o n t a i n i n g glycine/ml)  was  0.2  uC of 2 - ^ C - g l y c i n e / m l (14.5  added t o each f l a s k . . Non  of  radioactive glycine  d i s s o l v e d i n Krebs R i n g e r b i c a r b o n a t e b u f f e r was appropriate  mg  1  added t o  f l a s k s t o y i e l d the d e s i r e d c o n c e n t r a t i o n s .  The  f l a s k s were i n c u b a t e d f o r 2 hours a t 37°C and were k e p t shaken d u r i n g the p e r i o d o f i n c u b a t i o n . t i s s u e was and  of the p e r i o d ,  the  r e c o v e r e d by c e n t r i f u g a t i o n , washed w i t h c o l d b u f f e r  the washings were d i s c a r d e d .  were e x t r a c t e d  The  acid soluble  nucleotides  from the washed t i s s u e p e l l e t w i t h 0.2  perchloric acid.  The  soluble nucleotides The  A t the end  r a d i o a c t i v i t y incorporated  M cold  i n t o the  d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n  i n c o r p o r a t i o n of 2 - ^ C - g l y c i n e a t d i f f e r e n t 1  of added g l y c i n e i s g i v e n i n F i g u r e  22.  acid  counting.  concentrations  E x a m i n a t i o n of  the  f i g u r e shows t h a t the i n c o r p o r a t i o n of the r a d i o a c t i v e  glycine  i n t o the a c i d s o l u b l e f r a c t i o n d e c r e a s e d v/ith i n c r e a s e  i n concentra-  t i o n , of the non The  r a d i o a c t i v e g l y c i n e i n the i n c u b a t i o n medium.  d e c r e a s e d i n c o r p o r a t i o n of 2 - C - g l y c i n e i n presence 1 4  of added g l y c i n e may and  be due  t o the enlargement of the p o o l s i z e  the d i l u t i o n of the r a d i o a c t i v e compound.  o u t t h a t t h i s sample of g l y c i n e had (.1 mC/72.5 mg  I t may  be  a low s p e c i f i c a c t i v i t y  of g l y c i n e ) . . No o t h e r sample o f g l y c i n e v/as  a v a i l a b l e and under these c i r c u m s t a n c e s t h i s sample was t h e r e was  pointed  no r e q u i r e m e n t of any  t i o n of 2 - ^ C - g l y c i n e . 1  recently reported  added g l y c i n e f o r the  used  incorpora-  Johnstone and S c h o l e f i e l d (135)  t h a t amino a c i d s are t r a n s p o r t e d  and  into  have and  - 125  10 F i g u r e 22.  -  20 Added g l y c i n e  30 (ymole/10 ml  40 suspension)  E f f e c t o f g l y c i n e on t h e i n c o r p o r a t i o n of 2 - ^ C - g l y c i n e . by E h r l i c h a s c i t e s carcinoma c e l l s in vitro. 1  - 126 - '. concentrated process. ly.  i n E h r l i c h a s c i t e s c a r c i n o m a . c e l l s by an a c t i v e  T h i s was a l s o s u g g e s t e d by C h r i s t e n e e n  I t i s p o s s i b l e t h a t t h e s m a l l amount o f r a d i o a c t i v e g l y -  c i n e added i s c o n c e n t r a t e d cells non  (136) p r e v i o u s -  by t h e E h r l i c h a s c i t e s carcinoma  t o g i v e maximum i n c o r p o r a t i o n .  No f u r t h e r a d d i 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 was n e c e s s a r y  f o r t h e maximum i n c o r -  poration of•radioactive glycine. T o c t e s t t h e e f f e c t o f g l u c o s e on t h e i n c o r p o r a t i o n o f 21  R c - g l y c i n e , i n c u b a t i o n s were c a r r i e d o u t w i t h E h r l i c h a s c i t e s  cells  i n Krebs R i n g e r b i c a r b o n a t e  buffer.  t a i n e d 10 ml o f 10% v/v c e l l s u s p e n s i o n s t i o n of 2- ^C-glycine 1  (0.2 pC/ml).  Each f l a s k con-  and 25 u l o f a s o l u -  G l u c o s e was added t o t h e  a p p r o p r i a t e f l a s k t o g i v e a c o n c e n t r a t i o n o f 5.5 mM. t i o n was c a r r i e d o u t i n 250 ml f l a s k s as d e s c r i b e d .  IncubaAfter  i n c u b a t i o n t h e a c i d s o l u b l e f r a c t i o n and t h e n u c l e i c a c i d s were e x t r a c t e d from t h e c e l l s .  The r a d i o a c t i v e g l y c i n e i n c o r p o r a t e d  i n t o each o f t h e f r a c t i o n s was d e t e r m i n e d by l i q u i d counting.  F u r t h e r , t h e a c i d s o l u b l e n u c l e o t i d e s were adsorbed  on c h a r c o a l and e l u t e d w i t h a m i x t u r e The  scintillation  o f p y r i d i n e and a l c o h o l .  p u r i n e n u c l e o t i d e s were h y d r o l y z e d  o f V i s c h e r and C h a r g a f f  t o t h e bases by t h e method  (94) and s e p a r a t e d  by chromatography i n  K i r b y ' s s o l v e n t system and i n s o l v e n t system, o f Hershey e t a l . (95). The  s u b s t a n c e s were e l u t e d from t h e chromatograms and t h e  s p e c i f i c . . a c t i v i t y of. t h e p u r i n e bases d e t e r m i n e d .  The n u c l e o -  t i d e s o b t a i n e d by h y d r o l y s i s o f RNA v/ere i s o l a t e d by c h a r c o a l a d s o r p t i o n and e l u t i o n and s e p a r a t e d The  by paper chromatography.  DNA was h y d r o l y z e d w i t h 72% p e r c h l o r i c a c i d and t h e bases  - 127 s e p a r a t e d by chromatography. purines Table  The  s p e c i f i c a c t i v i t i e s of  of a c i d s o l u b l e n u c l e o t i d e s  RNA  and  DNA  the  are given  in  XV. I t can be seen from the t a b l e t h a t the s p e c i f i c a c t i v i t y  of a c i d s o l u b l e p u r i n e s  d e c r e a s e d i n p r e s e n c e of g l u c o s e .  The  r e s u l t s a l s o show t h a t the i n c o r p o r a t i o n of 2- "*C-glycine  into  1  b o t h p u r i n e of RNA t a i n i n g glucose. even g r e a t e r  and The  than i n  DNA  was  decreased i n i n c u b a t i o n  d e c r e a s e i n i n c o r p o r a t i o n i n t o RNA  conis  DNA.  I n t h e s e e x p e r i m e n t s , the e f f e c t of g l u c o s e on the 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  incor-  i n t o the n u c l e i c a c i d com-  ponents o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s i n c u b a t e d in  vitro  was  obtained  i n v e s t i g a t e d under v a r y i n g c o n d i t i o n s .  The  results  .so f a r showed t h a t the e f f e c t of g l u c o s e does not depend on radioactive precursor were seen w i t h  l!  the  employed as the same or s i m i l a r e f f e c t s  *C-formate and w i t h 2- ^ C - g l y c i n e . 1  The  effect  a l s o does not seem t o depend on the b u f f e r s o l u t i o n used t o suspend the c e l l s , though Krebs R i n g e r b i c a r b o n a t e b u f f e r maint a i n e d the pH i n p r e s e n c e of g l u c o s e .  Study u s i n g the d i f f e r e n t  b u f f e r s a l s o showed t h a t the e f f e c t o b s e r v e d f o r g l u c o s e was due  t o the l o w e r i n g  5) The  Incorporation  o f pH  not  caused by g l y c o l y s i s .  of ^C-Formate by E h r l i c h A s c i t e s Carcinoma 1  C e l l s Suspended i n D i f f e r e n t B u f f e r s . I n the e x p e r i m e n t s c a r r i e d out t o s t u d y the e f f e c t of g l u c o s e on i n c o r p o r a t i o n of n u c l e i c a c i d p r e c u r s o r s  by E h r l i c h  a s c i t e s carcinoma, c e l l s , c e l l s u s p e n s i o n s were made e i t h e r i n Krebs R i n g e r phosphate b u f f e r or Krebs R i n g e r b i c a r b o n a t e b u f f e r .  T a b l e XV E f f e c t o f g l u c o s e on t h e i n c o r p o r a t i o n o f 2- "*C-glycine i n t o t h e v a r i o u s 1  n u c l e i c a c i d f r a c t i o n s and p u r i n e s o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in  vitro.  S p e c i f i c a c t i v i t y - c o u n t s p e r minute p e r umole. Additions  A. s o l u b l e  RNA  —  x 10 cpm  1  DNA .  -  cpm/ cpm/ ymole mg AMP  Acid soluble fraction •  •  RNA  DNA  :  Adenine  Guanine  Hypoxt-n-Adean thine, n i n e  Guanine  Adenine  Guanine  134  821  660  2167  9741  4371  250  181  118  107  140  955  517  2432  10015  6185  269  225  104  118  1  126  181  871  1703  5130  3655  91  38  85  81  2  150  825  669  1957  5997  5089  128  96  85 ]  cell suspension 2  cell + glucose 111  -129 As t h e r e was v a r i a t i o n , i n t h e amount o f i n c o r p o r a t i o n o f t h e l a b e l l e d compounds i n t h e s e b u f f e r s , i t was o f i n t e r e s t t o study the i n c o r p o r a t i o n , of  llf  C - f o r m a t e v/ith r e s p e c t t o t h e  b u f f e r s used. E h r l i c h a s c i t e s c a r c i n o m a c e l l s were c o l l e c t e d and washed w i t h c o l d normal s a l i n e .  The c e l l s thus p r e p a r e d were d i v i d e d  i n t o two and one p o r t i o n was suspended i n Krebs R i n g e r phosphate b u f f e r and t h e o t h e r i n Krebs R i n g e r b i c a r b o n a t e t o g i v e a 1 i n 10 s u s p e n s i o n i n each case. formate (0.5 uC/ml) and sodium formate t o t h e s u s p e n s i o n s and mixed w e l l .  buffer  Enough o f ' C1Jf  (2 umole/ml) were added  Ten ml f r a c t i o n s o f t h e s e  s u s p e n s i o n s i n 250 ml Erlenmeyer f l a s k s were used f o r t h e i n c o r p o r a t i o n s t u d i e s i n presence of glucose.  Appropriate  con-  t r o l e x p e r i m e n t s w i t h c e l l s suspended i n each' o f t h e b u f f e r s were a l s o c a r r i e d o u t .  After incubations  t h e c e l l s were sep-  a r a t e d , and t h e a c i d s o l u b l e f r a c t i o n and t h e n u c l e i c a c i d s were e x t r a c t e d .  The '*C-formate i n c o r p o r a t e d 1  f r a c t i o n s was d e t e r m i n e d .  i n t o each o f t h e  The r e s u l t s a r e g i v e n i n T a b l e XVI.  From t h e d a t a g i v e n i n t h e t a b l e i t appears t h a t t h e amount o f i n c o r p o r a t i o n o f '*C-formate i s s l i g h t l y h i g h e r i n 1  c e l l s suspended i n phosphate b u f f e r .  I t i s conceivable  that  much o f t h e i n o r g a n i c phosphate i s used i n t h e b i o s y n t h e s i s o f nucleotides  and n u c l e i c a c i d s .  poration i n bicarbonate  b u f f e r may be r e l a t e d t o t h e d e c r e a s e d  a v a i l a b i l i t y of Inorganic recently reported  The g e n e r a l l y d e c r e a s e d i n c o r -  phosphate.  Hershko e t . a l .  (137) have  t h a t p r e s e n c e o f i n o r g a n i c phosphate i n t h e  i n c u b a t i o n medium markedly s t i m u l a t e d t h e i n c o r p o r a t i o n o f  - 130 -  T a b l e XVI I n c o r p o r a t i o n o f "^C-formate i n t o t h e n u c l e i c 1  a c i d components of, E h r l i c h a s c i t e s  carcinoma  c e l l s suspended i n two d i f f e r e n t b u f f e r s . C o n d i t i o n s o f i n c u b a t i o n s were same i n b o t h experiments.  Krebs R i n g e r phosphate buffer Additions  c  Acid soluble P /O.D. m  Krebs R i n g e r B i c a r b o n a t e ... b u f f e r  RNA DNA A c i d s o l u b l e RNA. DNA cpm/u cpm/ cpm/O.D. cpm/y cpm/ mole. AMP. mg mole. AMP. mg  2066  204  683  1450  350  616  Cell Suspension  1878  229  548  1460  250  '773  Cell suspension . + glucose (5.5 mM)  3776  13 8  324  2809  226  781  3511  111  374  3134  238  700  - 131 purines  into ribonucleotides i n rabbit erythrocytes.  s t i m u l a t i o n was Wu  (138)  has  This  a t t r i b u t e d t o an i n c r e a s e i n PRPP i n the  reported  cinoma c e l l s tions .  -  that glycolysis, i n E h r l i c h ascites car-  i s r e g u l a t e d by the i n o r g a n i c phosphate  Henderson and  cells.  Khoo (139)  concentra-  o b s e r v e d t h a t g l u c o s e can  PRPP i n E h r l i c h a s c i t e s c a r c i n o m a c e l l s . ments Krebs R i n g e r phosphate was  form  So i n f u r t h e r e x p e r i -  used t o suspend the E h r l i c h  a s c i t e s carcinoma c e l l s . 6) I n c r e a s e d  I n c o r p o r a t i o n of  xtt  C-Formate by E h r l i c h A s c i t e s  Carcinoma C e l l s i n P r e s e n c e of G l u c o s e . I t has been g e n e r a l l y a c c e p t e d by o t h e r s increased  that glucose  the 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  i n t o nuc-  l e i c a c i d components of E h r l i c h a s c i t e s tumour c e l l s .  Contrary  to t h i s ,  i n the p r e s e n t i n v e s t i g a t i o n s v a r y i n g r e s u l t s were  obtained.  Under t h e c o n d i t i o n s of t h e s e i n v e s t i g a t i o n s the  g e n e r a l e f f e c t of g l u c o s e was 1!  t o d e c r e a s e the i n c o r p o r a t i o n of  *C-formate i n t o n u c l e i c a c i d components of E h r l i c h a s c i t e s  carcinoma c e l l s .  However, i n some e x p e r i m e n t s i n agreement w i t h  the r e s u l t s of o t h e r s , an i n c r e a s e i n i n c o r p o r a t i o n of formate was  o b s e r v e d when E h r l i c h c a r c i n o m a c e l l s were  with glucose.  The  r e s u l t s of one  lif  C-  incubated  such experiment i s g i v e n i n  Table. X V I I . I t can be seen from the t a b l e t h a t the i n c o r p o r a t i o n of lf  *C-formate d o u b l e d i n some cases i n p r e s e n c e of g l u c o s e .  This  i  i s i n agreement /with r e s u l t s r e p o r t e d by  others.  Glucose metabolism i n E h r l i c h a s c i t e s carcinoma c e l l s s t u d i e d by many i n v e s t i g a t o r s .  was  Glucose i s phosphorylated q u i c k l y  - 132 -  Table  XVII E f f e c t o f g l u c o s e on t h e i n c o r p o r a t i o n o f 1  ^ C - f o r m a t e in  vitro  i n t o the various n u c l e i c ,  a c i d components o f E h r l i c h a s c i t e s  carcinoma  cells.  S p e c i f i c A c t i v i t y - cpm/umole Acid-soluble fraction A d d i t i o n s Adenine Guan- Hypoxine anthine 1  A RNA  DNA  AMP  GMP  Thymine  2738  3374  2970  163  287  1568  2  2476  3158  2797  173  242  1746  1  4428  5581  5162  289  482  1743  5620  7604  7376  385  605  1675  Cell suspension  Cell suspension + glucose 2  -133and g l y c o l y z e d t o g i v e l a c t i c a c i d . lactic acid i s limited  (131).  The  o x i d a t i o n of t h i s  Thomson e t a l . (71) have  o b s e r v e d t h a t the n e t amount of ATP  decreased i n E h r l i c h a s c i t e s  carcinoma c e l l s allowed to u t i l i z e glucose. b o l i s m and t h e ATP  The  l e v e l s of E h r l i c h a s c i t e s carcinoma c e l l s  were s t u d i e d by Overgaard-Hansen r e c e n t l y (73) . t h a t when g l u c o s e was  r e g e n e r a t i o n o f ATP  t o normal.  under t h e s e c o n d i t i o n s was  d e p l e t i o n of ATP  of E h r l i c h  a  The  comparatively  l e v e l d i d not r e t u r n  i s b r o u g h t about by a s e r i e s  of r e a c t i o n s i n w h i c h h e x o k i n a s e , The  observed  a sudden d e p l e t i o n of ATP.  and even a f t e r 2 hours the ATP The  She  added t o a . d i l u t e s u s p e n s i o n  a s c i t e s c a r c i n o m a c e l l s t h e r e was  slow p r o c e s s  g l u c o s e meta-  myokinase, and  adenylate  deaminase t a k e p a r t  (73).  n u c l e o t i d e s were h y d r o l y z e d  the n u c l e o s i d e s and  t h e s e s u b s t a n c e s escaped i n t o the medium  i n which the c e l l s were suspended. the process  of g l y c o l y s i s was  The ATP  regenerated  through  not a b l e t o b r i n g back the  t i d e pool to i t s o r i g i n a l l e v e l .  Overgaard-Hansen has  to  nucleo-  also  o b s e r v e d t h a t the d e p l e t i o n of adenine n u c l e o t i d e s i s l e s s , regeneration i s q u i c k e r i n presence of glucose  in cell  and  suspensions  o f h i g h e r c o n c e n t r a t i o n as d e t e r m i n e d by packed c e l l volume. R e c e n t l y Laws and S t r i c k l a n d (140) c o n c e n t r a t i o n o f ATP The  de  have a l s o r e p o r t e d a  i n ascites c e l l s treated with  novo s y n t h e s i s of n u c l e o t i d e s and the  lower,  glucose. phosphoryla-  t i o n s t o g i v e n u c l e o s i d e t r i p h o s p h a t e s are a l l r e a c t i o n s r e q u i r i n g ATP.  P u r i n e n u c l e o t i d e s a r e r e p o r t e d t o be r e q u i r e d f o r the  i n i t i a t i o n of the p o l y m e r i z a t i o n r e a c t i o n by which RNA (28).  A d e c r e a s e i n ATP  i s formed  l e v e l s i n the c e l l s t h u s can d e c r e a s e  the i n c o r p o r a t i o n o f "*G-formate b o t h i n t o n u c l e o t i d e s and 1  nucleic acids. I n s e e k i n g an e x p l a n a t i o n f o r t h e d e c r e a s e d i n c o r p o r a t i o n of l a b e l l e d p r e c u r s o r s i n t o n u c l e i c a c i d components i n E h r l i c h a s c i t e s tumour c e l l s .  i n p r e s e n c e o f g l u c o s e , i t may be sug-  g e s t e d t h a t g l u c o s e r a p i d l y d e p l e t e d t h e ATP o f t h e c e l l s , t h e r e g e n e r a t i o n o f w h i c h was d e l a y e d The  i n dilute cell  suspensions.  i n c r e a s e i n i n c o r p o r a t i o n o f ^ C - f o r m a t e . s e e n i n some' e x p e r i 1  ments may be due t o t h e r a p i d r e s t o r a t i o n Of the.ATP l e v e l and i t s continued  s y n t h e s i s i n denser c e l l s u s p e n s i o n s ,  by Overgaard-Hansen.  as o b s e r v e d  From t h e r e s u l t s o b t a i n e d i n t h e p r e s e n t  i n v e s t i g a t i o n s , as w e l l as those r e p o r t e d by Overgaard-Hansen, i t was f e l t t h a t t h e r e a r e two f a c t o r s c o n t r o l l i n g t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e by E h r l i c h a s c i t e s c a r c i n o m a c e l l s i n p r e s 1  ence o f g l u c o s e .  The f i r s t i s t h e c e l l c o n c e n t r a t i o n i n suspen-  s i o n and t h e second t h e sugar c o n c e n t r a t i o n i n t h e s u s p e n s i o n . By v a r y i n g t h e sugar c o n c e n t r a t i o n a t a p a r t i c u l a r c e l l t i o n i t may be p o s s i b l e t o v a r y t h e d e p l e t i o n o f adenine t i d e s and r e g e n e r a t i o n o f ATP.  nucleo-  The same e f f e c t may be produced  by v a r y i n g t h e c e l l c o n c e n t r a t i o n i n s u s p e n s i o n sugar c o n c e n t r a t i o n .  concentra-  at a particular  These may be r e f l e c t e d i n t h e s t i m u l a t i o n  or i n h i b i t i o n o f i n c o r p o r a t i o n o f '*C-formate by E h r l i c h a s c i t e s 1  carcinoma c e l l s . . To v e r i f y t h i s h y p o t h e s i s performed.  two s e r i e s o f e x p e r i m e n t s were  In the f i r s t s e r i e s , using a p a r t i c u l a r c e l l  concen-  t r a t i o n t h e sugar c o n c e n t r a t i o n was v a r i e d and t h e i n c o r p o r a t i o n of  llf  C - f o r m a t e a t d i f f e r e n t sugar c o n c e n t r a t i o n s i n t o v a r i o u s  n u c l e i c a c i d f r a c t i o n s was d e t e r m i n e d .  I n t h e second s e r i e s t h e  - 135 sugar c o n c e n t r a t i o n t h e s u s p e n s i o n was k e p t c o n s t a n t , and t h e c e l l c o n c e n t r a t i o n v a r i e d and t h e i n c o r p o r a t i o n s t u d i e s c a r r i e d out. 7) E f f e c t o f V a r y i n g C o n c e n t r a t i o n o f G l u c o s e on t h e I n c o r p o r a t i o n o f ''C-Formate by E h r l i c h A s c i t e s Carcinoma C e i l s 1  in  vitro.  E h r l i c h a s c i t e s c a r c i n o m a c e l l s were c o l l e c t e d as d e s c r i b e d before.  The c e l l s were d i l u t e d w i t h 8 volumes o f Krebs R i n g e r  phosphate b u f f e r pH 7.8.  The packed c e l l volume o f t h i s suspen-  s i o n was d e t e r m i n e d by c e n t r i f u g i n g i n a hematocriti.. tube i n a c l i n i c a l c e n t r i f u g e as d e s c r i b e d under methods. (The packed  cell  volume o f c e l l s u s p e n s i o n p r e p a r e d by t h e u s u a l p r o c e d u r e o f d i l u t i o n w i t h 9 volumes o f b u f f e r , v a r i e d from 5-7%.)  Incuba-  t i o n s were c a r r i e d o u t i n 250 ml Erlenmeyer f l a s k s as d e s c r i b e d under methods.  Each f l a s k c o n t a i n e d 10 ml o f c e l l s u s p e n s i o n  5 uC.L o f "*C-formate, 20 umoles o f sodium formate and v a r y i n g 1  concentrations of glucose. A f t e r i n c u b a t i o n t h e a c i d s o l u b l e f r a c t i o n , RNA and DNA were i s o l a t e d from t h e c e l l s .  They were h y d r o l y z e d and t h e bases  s e p a r a t e d by paper chromatography. chromatograms photometry.  The s u b s t a n c e s from t h e  were e l u t e d and e s t i m a t e d by u l t r a v i o l e t s p e c t r o The r a d i o a c t i v i t y i n t h e bases was d e t e r m i n e d by  l i q u i d s c i n t i l l a t i o n counting.  The r e s u l t s a r e g i v e n i n T a b l e  XVIII. E x a m i n a t i o n o f t h e d a t a i n t h e t a b l e shows t h a t , i n some c a s e s , t h e i n c o r p o r a t i o n o f C-formate lh  i n t o the various n u c l e i c  a c i d components was i n c r e a s e d i n p r e s e n c e o f g l u c o s e .  On f u r t h e r  Table  XVIII Effects of varying concentration 1  o f g l u c o s e on t h e i n c o r p o r a t i o n o f  ''C-formate i n t o t h e v a r i o u s n u c l e i c a c i d components o f E h r l i c h a s c i t e s  c a r c i n o m a c e l l s in  vitro.  Packed c e l l volume 8.5%. S p e c i f i c a c t i v i t y - c o u n t s p e r minute p e r umole. . . .  Acid soluble f r a c t i o n Concentrat i o n of cpm/O.D. glucose mg %  RNA  DNA  Ade  Gua  Hypx.  Ade  Gua  Ade  Gua  Cyt  2496  4904  4562  6350  581  418  83  96  42  1970  50.0  2665  7778  5252  8798  582  465  114  112  71  2141  100.0  3297  7966  5502  9289  849  607  131  126  60  2202  500.0  9182  4171  4071  5442  570  366  .189  100  61  1943  0  Ade - A d e n i n e , Gua - Guanine, Hypx. - H y p o x a n t h i n e , C y t -  Cytosine.  Thymine  - 137  -  i n c r e a s e of the c o n c e n t r a t i o n of g l u c o s e t o 500 mg%, c o r p o r a t i o n of  "*C-formate was  1  S t e w a r t and  Zbarsky  the i n -  decreased.  (141)  have  reported s i m i l a r results  i n s t u d y i n g the i n c o r p o r a t i o n of ^ C - f o r m a t e by  intestinal  mucosa and by E h r l i c h a s c i t e s carcinoma c e l l s .  They have  t a i n e d an i n c r e a s e i n the i n c o r p o r a t i o n of c o n c e n t r a t i o n of 50 mg%  llt  ob-  C-formate w i t h a  of g l u c o s e i n the case of E h r l i c h a s c i t e s  carcinoma c e l l suspensions.  The  increase i n incorporation  r e p o r t e d by them i s comparable t o the p r e s e n t  observations.  8) I n c o r p o r a t i o n of C - F o r m a t e by E h r l i c h A s c i t e s Carcinoma u  C e l l s as a F u n c t i o n of C e l l  Concentration.  I n the second s e r i e s of e x p e r i m e n t s the c e l l was  v a r i e d and sugar c o n c e n t r a t i o n was  concentration  k e p t c o n s t a n t a t 100  A s c i t e s c a r c i n o m a c e l l s were c o l l e c t e d and washed as  mg%.  described  previously. The  c e l l s were suspended in'::twice the volume of Krebs  R i n g e r phosphate b u f f e r pH 7.8.  This suspension  s e r i a l l y to give three d i f f e r e n t concentrations. c e l l volume of each s u s p e n s i o n u s i n g hematocrit;. t u b e s .  was  was The  diluted packed  d e t e r m i n e d by c e n t r i f u g a t i o n  I n c u b a t i o n was  c a r r i e d out i n E r l e n -  meyer f l a s k s i n a water b a t h a t 37°C f o r 2 hours and the was  k e p t shaken d u r i n g the p e r i o d of i n c u b a t i o n .  c o n t a i n e d , 1 mg  of g l u c o s e , 0.5  uC of  l4  was  Each f l a s k  C - f o r m a t e and 2 umoles  of sodium formate per ml of c e l l s u s p e n s i o n . suspension  flask  Enough  cell  measured i n t o each f l a s k so as t o g i v e about 1  .ml of c e l l volume. A f t e r i n c u b a t i o n , the a c i d s o l u b l e f r a c t i o n , RNA  and  DNA  - 138 were o b t a i n e d  from each f l a s k , t h e y were h y d r o l y s e d  t o g i v e the bases and graphy.  The  U.V.  and e s t i m a t e d  suitably,  the bases s e p a r a t e d by paper" chromato-  absorbing  m a t e r i a l from t h e paper was  by u l t r a v i o l e t s p e c t r o p h o t o m e t r y . . The  a c t i v i t y incorporated  i n t o the v a r i o u s f r a c t i o n s was  by l i q u i d s c i n t i l l a t i o n c o u n t i n g g i v e n i n T a b l e s XIX  and  procedures.  The  eluted radio-  determined  r e s u l t s are  XX.  E x a m i n a t i o n of the t a b l e s shows t h a t t h e i n c o r p o r a t i o n of ll  *C-formate i n c r e a s e d w i t h the i n c r e a s e i n c o n c e n t r a t i o n  c e l l s i n suspension.  Thus the i n c o r p o r a t i o n o f  of  radioactivity  i n t o each of the f r a c t i o n s i s the h i g h e s t i n c e l l s h a v i n g a packed c e l l volume; under the c o n d i t i o n s of t h e T h i s v a l u e d e c r e a s e s as the c e l l c o n c e n t r a t i o n In t h e s e i n c u b a t i o n s constant  and  the volume of c e l l s was  25%  experiment. i s decreased.  more or l e s s k e p t  the volume of b u f f e r s o l u t i o n v a r i e d so t h a t  the  t o t a l volume o f c e l l s u s p e n s i o n v a r i e d from e x p e r i m e n t t o e x p e r i ment.  The  d e c r e a s e i n the i n c o r p o r a t i o n of r a d i o a c t i v i t y  served  i n decreasing  be due  t o ' t h e d i l u t i o n of the r a d i o a c t i v i t y caused by  the d e n s i t y of c e l l s u s p e n s i o n s may  obpartly  the  i n c r e a s e i n volume of the i n c u b a t i o n medium. The  e f f e c t of g l u c o s e v a r i e d from a s t i m u l a t i o n of i n c o r -  p o r a t i o n i n dense s u s p e n s i o n t o i n h i b i t i o n i n d i l u t e s o l u t i o n . Thus the i n c o r p o r a t i o n of ^ C - f o r m a t e i n t o the. a c i d s o l u b l e 1  f r a c t i o n , RNA and DNA bases  -yas h i g h e r  i n incubations containing  g l u c o s e e x c e p t f o r the l a s t e x p e r i m e n t , where t h e r e was decrease n o t i c e d . volume was  I n the i n c u b a t i o n where the packed  the l o w e s t ,  t h e r e may  a cell  be a d e p l e t i o n o f ATP and  the  T a b l e XIX Incorporation of ^ - f o r m a t e i n t o the purines of a c i d soluble 1  nucleotides  and o f RNA o f E h r l i c h a s c i t e s tumour c e l l s i n p r e s e n c e o f g l u c o s e ent c o n c e n t r a t i o n s o f c e l l s u s p e n s i o n . 5.5  A - Control, B - Control +  .  S p e c i f i c a c t i v i t y - c o u n t s p e r minute p e r . ;;; ymo.le. ...  ./„.•.'.. ..  A c i d s o l u b l e f r a c t i o n ......  25% 15%  Adenine A  glucose  mM.  ;  Packed cell volume  at d i f f e r -  :  Guanine B  A  Hypoxanthine B  14019 53491 11776 50661  A  B  28390  65880  RNA  I  Adenine  Guanine  : A .... B 1208 4811  M  : A 312  B 907  9466 33406  7405 24412  13875  31590  845  2611  178  403  7%  5633  8529  5094  5960  12162  15097  583  646  120  173  3.5%  872  685  1842  1040  282  166  57  48  co  ^  T a b l e XX I n c o r p o r a t i o n o f **C-formate i n t o t h e bases o f DNA o f E h r l i c h a s c i t e s 1  c e l l s i n p r e s e n c e o f g l u c o s e (5.5 mM) suspension.  tumour  at d i f f e r e n t concentrationstof c e l l s i n  A - C o n t r o l , B - C o n t r o l + g l u c o s e (5.5 mM). S p e c i f i c a c t i v i t y - c o u n t s p e r minute, p e r umole  Packed Cell volume  Adenine  Cytosine  Thymine  • A  .  Guanine B  A  B  A  B  .  A  B  :  M  25%  191.  522  312  907  133  272  5461  5410  15%  155  240  178  403  84  175  3558  2811  7%  69  68  120  173  50  64  1776  933  28  21  57  49  34  27  822  387  3.5%  1  - 141 r e g e n e r a t i o n o f t h i s i s n o t as f a s t as i n case o f dense sion.  suspen-  I n a d d i t i o n t o t h e d e c r e a s e i n ATP, t h e r e may be t h e non-  a v a i l a b i l i t y o f o t h e r c o f a c t o r s w h i c h c o n t r i b u t e s "to t h e decreased i n c o r p o r a t i o n of ^ C - f o r m a t e i n t o the n u c l e i c  acid  components i n d i l u t e s o l u t i o n s i n p r e s e n c e o f g l u c o s e . 9)  E f f e c t o f 2-deoxyglucose on t h e I n c o r p o r a t i o n o f  1  Re-Formate  vitro.  by E h r l i c h A s c i t e s Carcinoma C e l l s i n  To f u r t h e r v e r i f y t h e r e l a t i o n between t h e d e p l e t i o n o f ATP and t h e d e c r e a s e i n i n c o r p o r a t i o n o f C-formate lk  i n Ehrlich  a s c i t e s c a r c i n o m a c e l l s a n o t h e r e x p e r i m e n t was c a r r i e d o u t u s i n g 2-deoxyglucose i n s t e a d o f g l u c o s e .  2-Deoxyglucose  i s t a k e n up  by E h r l i c h a s c i t e s c a r c i n o m a c e l l s , l i k e g l u c o s e and i s phosp h o r y l a t e d t o g i v e t h e 2-deoxyglucose 6-phosphate u s i n g ATP as t h e phosphate donor  (117).  by h e x o k i n a s e  As t h e p h o s p h o r y l a t e d  2-deoxyglucose i s n o t f u r t h e r m e t a b o l i z e d , t h e r e i s no p o s s i b i l i t y of  r e g e n e r a t i o n o f ATP t h r o u g h g l y c o l y s i s .  can be used t o d e p l e t e t h e ATP i n t h e c e l l circumstances, the i n c o r p o r a t i o n of  1If  So 2-deoxyglucose (142) and under such  C - f o r m a t e s h o u l d be  i n h i b i t e d i n dense as w e l l as i n d i l u t e d c e l l s u s p e n s i o n s .  This  was t e s t e d i n t h e f o l l o w i n g e x p e r i m e n t . E h r l i c h a s c i t e s c e l l s were c o l l e c t e d and washed a c c o r d i n g to  the procedure described before.  The c e l l volume a f t e r  c e n t r i f u g a t i o n v/as n o t e d and c e l l s were suspended i n f o u r of  Krebs R i n g e r phosphate b u f f e r pH 7.8.  volumes  A p o r t i o n of the  s u s p e n s i o n was d i l u t e d w i t h e q u a l volume o f t h e same b u f f e r . C e l l volume o f t h e s e s u s p e n s i o n s w e r e d e t e r m i n e d .  I n c u b a t i o n s were  c a r r i e d o u t i n 250 ml E r l e n m e y e r f l a s k s a t 37°C f o r 120 m i n u t e s .  - 142 The  gas phase was  formate and icals  -  oxygen.. Each f l a s k c o n t a i n e d  20 umole o f sodium f o r m a t e .  A grade - g l u c o s e f r e e ) was  f l a s k to give a f i n a l concentration  5 uc of  "*C-  l  2-Deoxyglucose (£!albiochem-  added t o the of 5.5  mM.  appropriate An amount of  c e l l s u s p e n s i o n c o r r e s p o n d i n g t o 1 ml o f c e l l volume was t o each f l a s k . RNA  and DNA  i n t o RNA  A f t e r the i n c u b a t i o n the a c i d s o l u b l e  were i s o l a t e d .  The  were f u r t h e r h y d r o l y z e d  scribed:  radioactivity  a c i d s o l u b l e f r a c t i o n and DNA  d e s c r i b e d : u n d e r methods. DNA  The  The  fraction,  incorporated  were d e t e r m i n e d as  a c i d s o l u b l e n u c l e o t i d e s , RNA  and  t o g i v e the bases by methods de-  bases were s e p a r a t e d by paper chromatography.  r a d i o a c i t i v i t y incorporated f r a c t i o n s was  added  i n t o the p u r i n e b a s e s of the  The  various  d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g .  r e s u l t s are g i v e n i n T a b l e s XXI, X X I I and  The  XXIII.  E x a m i n a t i o n of the r e s u l t s show a marked d e c r e a s e i n the i n c o r p o r a t i o n of  llf  C - f o r m a t e i n t o a l l the f r a c t i o n s of n u c l e i c  acids i n incubations  c o n t a i n i n g 2-deoxyglucose.  Though the  t o t a l a c t i v i t y i n the a c i d s o l u b l e f r a c t i o n i n presence of d e o x y g l u c o s e was  2-  i n c r e a s e d m a r k e d l y , on s e p a r a t i o n and  estima-  t i o n of r a d i o a c t i v i t y i n the bases of the a c i d s o l u b l e  nucleo-  t i d e s , a d e c r e a s e i n the i n c o r p o r a t i o n was r e s u l t s were o b t a i n e d incubation mixture.  noticed.  p r e v i o u s l y when g l u c o s e was  Similar  p r e s e n t i n the  I n the case of i n c o r p o r a t i o n s of r a d i o -  a c t i v i t y t o the n u c l e i c a c i d b a s e s , the d e c r e a s e was  greater  compared t o the d e c r e a s e caused by g l u c o s e under s i m i l a r c o n d i tions.  I t i s seen from the t a b l e t h a t 2-deoxyglucose d e c r e a s e d  the i n c o r p o r a t i o n of  1!t  C-formate both i n concentrated  and  in  - 143 -  T a b l e XXI E f f e c t o f 2-deoxyglucose on t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o t h e n u c l e i c a c i d components 1  o f E h r l i c h a s c i t e s carcinoma c e l l s in  vitro.  A - C o n t r o l , B - C o n t r o l + 2-deoxyglucose (5.5 mM).  Acid soluble Fraction . cpm/O.D. Packed c e l l volume  RNA  DNA  cpm/umole AMP  epm/mg  A  B  A  B  A  B  1  1571  7064  464  113  1377  202  2  1693 .  7053  476  98  1472  204  1  1944  .7028  417  100  1351  162  2  2033  402  70  1297  138  14%  6.5% 7113  Table XXII E f f e c t o f 2-deoxyglucose on t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o t h e 1  p u r i n e s o f a c i d s o l u b l e n u c l e o t i d e s and RNA o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in  vitro.  A - C o n t r o l , B - C o n t r o l + 2-deoxyglucose (5.5 niM) . S p e c i f i c a c t i v i t y - c o u n t s p e r minute p e r umole  Acid-Soluble nucleotides Packed cell volume  Adenine A  Guanine B  A  3666  230  3347  3572  131  3665 3581  .  RNA  . . . Hypoxanthine B  Adenine  Guanine  A  B  A  B  A  B  1596  3660  648  376  26  255  91  2650  1303  2899  591  363  38  258  81  115  3010  1312  3540  551  317  14  188  #7  114  2394  1283  3487  228  310  23  185  83  14%  6.5%  Table XXIII E f f e c t o f 2-deoxy g l u c o s e on t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o t h e 1  bases o f DNA o f E h r l i c h a s c i t e s c a r c i n o m a c e l l s in A - C o n t r o l , B - C o n t r o l + 2-deoxyglucose  vitro.  (5.5 mM)  S p e c i f i c a c t i v i t y - c o u n t s p e r minute p e r umole Packed cell volume  Adenine  Guanine B  255  A  Cytosine B  Thymine B  B  319  13  36  1806  258  325  32  36  1819  297  47  1679  177  1668  168  14% 294  16  38  48  54  52  6.5% 23  72  21  -  dilute-cell  suspensions.  146  -  This i s i n contrast with the r e s u l t  o b t a i n e d w i t h g l u c o s e , where i n h i b i t i o n was o b t a i n e d o n l y i n d i l u t e c e l l suspensions.  T h i s may be due t o t h e d i f f e r e n c e i n  r e s t o r a t i o n o f adenine n u c l e o t i d e p o o l when 2-deoxyglucose i s p r e s e n t i n the—medium.  Overgaard-Hansen (73) has observed t h a t  i n t h e p r e s e n c e o f 2-deoxyglucose., t h e adenine n u c l e o t i d e p o o l i n E h r l i c h a s c i t e s c a r c i n o m a c e l l was d e p l e t e d extent than i n the presence of glucose..  to.greater  The d e p l e t i o n i n t h e  former case i s n o t r e v e r s e d because t h e 2-deoxyglucose i s n o t metabolized  f u r t h e r than the i n i t i a l p h o s p h o r y l a t i o n .  Hansen has a l s o o b s e r v e d an i n c r e a s e i n t h e t o t a l  Overgaard-  concentration  of i n o s i n e , adenosine and h y p o x a n t h i n e as t h e n u c l e o t i d e p o o l i s being depleted.  The l a c k o f r e g e n e r a t i o n o f ATP, even i n dense  c e l l suspension  i n p r e s e n c e o f 2-deoxyglucose may be t h e r e a s o n  for  t h e i n h i b i t i o n o f i n c o r p o r a t i o n o f ^ C - f o r m a t e observed i n  dense s u s p e n s i o n  o f E h r l i c h a s c i t e s carcinoma c e l l s .  The g r e a t e r  i n h i b i t i o n Of i n c o r p o r a t i o n i n p r e s e n c e o f 2-deoxyglucose, t h a n i n p r e s e n c e o f g l u c o s e under s i m i l a r c o n d i t i o n s may a l s o be due to t h e g r e a t e r d e p l e t i o n o f ATP i n t h e c e l l s . 10)  I n c o r p o r a t i o n o f '*C-Formate by E h r l i c h A s c i t e s Carcinoma 1  C e l l s Under V a r i o u s C o n d i t i o n s A f f e c t i n g ATP  Formation.  I f t h e c o n c e n t r a t i o n o f adenine n u c l e o t i d e s , p a r t i c u l a r l y ATP, i s i n c r e a s e d i n t h e c e l l by some mechanism, t h e n t h e r e i s a p o s s i b i l i t y o f i n c o r p o r a t i n g more ^ C - f o r m a t e i n t o t h e v a r i o u s 1  n u c l e i c a c i d components.  As t h e c e l l membrane i s impermeable  t o p o l y p h o s p h a t e s , ATP may n o t pass through i t and t h e c o n c e n t r a t i o n o f t h i s m a t e r i a l cannot be r a i s e d i n t h e c e l l by t h e d i r e c t  - 147 a d d i t i o n o f ATP t o c e l l s u s p e n s i o n s . and LePage (143) o b s e r v e d t h a t carcinoma c e l l suspension  C-AMP added t o E h r l i c h a s c i t e s  was found t o be i n c o r p o r a t e d . i n t o t h e  n u c l e i c acids of the c e l l . Racker  XIt  Some y e a r s ago Edmonds  Based on t h i s o b s e r v a t i o n Wu and  (144) c a r r i e d o u t i n c u b a t i o n s o f E h r l i c h a s c i t e s c a r c i n o m a  c e l l s i n p r e s e n c e o f g l u c o s e and AMP and o b s e r v e d an i n c r e a s e of ATP c o n c e n t r a t i o n i n t h e c e l l .  T h i s p r o c e d u r e was u n d e r t a k e n  t o i n c r e a s e ATP i n c e l l s and t h e r e b y t i o n o f C-formate lk  carcinoma c e l l s .  t o increase the incorpora-  into the nucleic acids of E h r l i c h ascites These e x p e r i m e n t s a r e d e s c r i b e d below.  E h r l i c h a s c i t e s carcinoma c e l l suspensions  were made i n  Krebs R i n g e r phosphate b u f f e r pH 7.8 t o g i v e a c o n c e n t r a t i o n o f about 10% ( v / v ) .  I n c u b a t i o n s were c a r r i e d o u t i n a t o t a l volume  of 10 ml o f t h i s s u s p e n s i o n  i n 250 ml E r l e n m e y e r f l a s k s under  c o n d i t i o n s as d e s c r i b e d p r e v i o u s l y . ml s u s p e n s i o n , formate.  Each f l a s k c o n t a i n e d  9.5  5 y.'C o f ^C-formate and 20 umoles o f sodium1  The f l a s k s were k e p t c o o l e d i n i c e d u r i n g t h e a d d i t i o n  of s u b s t r a t e s .  The c o n c e n t r a t i o n o f g l u c o s e where ever  was 5.5 mM and AMP was 1 mM.  present  D i n i t r o p h e n o l , a w e l l known un-  c o u p l i n g agent o f o x i d a t i v e p h o s p h o r y l a t i o n  (129) was added t o  some i n c u b a t i o n s when r e q u i r e d t o s t o p t h e f o r m a t i o n o f ATP. The  f i n a l c o n c e n t r a t i o n o f t h e d i n i t r o p h e n o l used was 0.1 mM.  The  t o t a l volume was made t o 10 ml w i t h b u f f e r , whenever  necessary.  A f t e r i n c u b a t i o n , t h e a c i d s o l u b l e f r a c t i o n , RNA,  and DNA were s e p a r a t e d . hydrolyzed The  The a c i d s o l u b l e n u c l e o t i d e s were  and t h e bases s e p a r a t e d  by paper chromatography.  c o n c e n t r a t i o n o f DNA and RNA were d e t e r m i n e d as d e s c r i b e d  - 148 previously.  The r a d i o a c t i v i t y i n t h e samples was  by l i q u i d s c i n t i l l a t i o n c o u n t i n g .  estimated  The r e s u l t s a r e g i v e n i n  T a b l e s XXIV and XXV. The  r e s u l t s o f these experiments f u r t h e r confirm the r e -  l a t i o n between ATP g e n e r a t i o n  and '*C-formate i n c o r p o r a t i o n by  E h r l i c h a s c i t e s carcinoma c e l l s . i n t r a c e l l u l a r concentration  1  The a t t e m p t t o r a i s e t h e  o f ATP by t h e a d d i t i o n o f AMP and  g l u c o s e t o t h e medium f a i l e d t o g i v e t h e d e s i r e d e f f e c t .  The  p r e s e n c e o f AMP i n t h e i n c u b a t i o n s l i g h t l y d e c r e a s e d t h e i n c o r p o r a t i o n o f C-formate  i n t o t h e p u r i n e bases o f t h e a c i d  lh  soluble nucleotides.  T h i s may p a r t l y be due t o t h e d i l u t i o n  e f f e c t caused by non r a d i o a c t i v e AMP added t o t h e c e l l u l a r  pool,  and p a r t l y due t o t h e feedback i n h i b i t i o n o f t h e de novo s y n t h e s i s by AMP  (116).  However, AMP had no a p p r e c i a b l e  effect  on t h e i n c o r p o r a t i o n o f C - f o r m a t e i n t o t h e n u c l e i c a c i d s . 1 4  The  absence o f any e f f e c t o f AMP on t h e i n c o r p o r a t i o n o f  1h  C-  formate may s u g g e s t t h e p o s s i b i l i t y t h a t AMP may n o t e n t e r t h e cell.  W a l l a c h and U l l r e y (145) r e p o r t e d  phorylated cell.  t h a t AMP i s dephos-  a t t h e c e l l membrane p r i o r t o e n t r y i n t o t h e a s c i t e s  I n such a c a s e , r e g e n e r a t i o n  of the nucleotide  a s e r i e s o f r e a c t i o n i n w h i c h ATP i s i n v o l v e d . incorporation of  11+  requires  A d e c r e ase i n  C-formate i s noticed i n a l l n u c l e i c a c i d  components i n i n c u b a t i o n s may be due t o v a r i o u s  c o n t a i n i n g b o t h g l u c o s e and AMP.  This  f a c t o r s , among w h i c h , i s o t o p e d i l u t i o n by  added n u c l e o t i d e , end p r o d u c t i n h i b i t i o n o f s y n t h e s i s , a l t e r a t i o n of t h e e q u i l i b r i u m o f t h e n u c l e o t i d e p o o l , and d e p l e t i o n o f adenine n u c l e o t i d e by g l u c o s e a r e more  important.  - 149  T a b l e XXIV I n c o r p o r a t i o n o f *C-formate  by E h r l i c h  l>  c a r c i n o m a c e l l s in vitro  ascites  under d i f f e r e n t  condi-  t i o n s a f f e c t i n g ATP c o n c e n t r a t i o n i n t h e c e i l . Concentration DNP 0.1 mM.  o f glucose  5.5 mM.  AMP 1 mM.  Packed c e l l volume E x p t . I 6.5,  E x p t . I I 7%.  Acid soluble fraction cpm/O.D. Expt.I  Expt.II  2945  1512  .glucosel0135  Additions  RNA  DNA  cpm/umole Expt.I  cpm/mg  Expt.II  Expt.I  Expt.II  993  1017  1626  1828  9447  677  ' 774  835  790  4804  2423  925  954  1682  1814  + glucose + AMP 7380  7367  543  683  842  1096  + glucose + AMP +DNP 13424  4280  339  201  226  292  DNP*  1625  Nil +  + AMP  * I n Expt. I I only.  440  1418  - 150 -  T a b l e XXV Incorporation of  llf  C-formate i n t o purines of the  a c i d s o l u b l e n u c l e o t i d e s of. E h r l i c h  ascites  c a r c i n o m a c e l l s under d i f f e r e n t c o n d i t i o n s  affect-  i n g ATP c o n c e n t r a t i o n i n t h e c e l l .  S p e c i f i c a c t i v i t y - c o u n t s p e r minute per umole Adenine Additions  Guanine  Hypoxanthine  Expt.I  Expt.II  Expt.I  Expt.II  Expt.I  6025  5529  7611  3954  7047  6228  6744  .6017  4048  3411  8462  - .  + AMP  4352  4931  4200  3360  5084  4480  + glucose + AMP  1383  ,-2015  1319  1258  1297  2764  + glucose + AMP + DNP  524  2801  778  580  2003  Nil +  glucose  + DNP*  -  '• 3395  (-) samples l o s t , * I n E x p t . I I o n l y .  1621  Expt.II  4430  - 151 Dinitrophenol phosphorylation  i s w e l l known as an u n c o u p l e r o f o x i d a t i v e  (129).  I t i s seen t h a t d i n i t r o p h e n o l i n con-  c e n t r a t i o n enough t o u n c o u p l e o x i d a t i v e p h o s p h o r y l a t i o n the i n c o r p o r a t i o n o f ^ C - f o r m a t e i n t o t h e a c i d s o l u b l e  nucleo-  1  t i d e s and t o n u c l e i c a c i d s . that  :.DNP,  Gross e t a i . (146) have  i n h i b i t e d the synthesis  t ;  t r a t i o n o f DNP used was much h i g h e r  reported  o f RNA, b u t t h e concen-  than t h e c o n c e n t r a t i o n r e -  quired for uncoupling of oxidative phosphorylation. observation  The p r e s e n t  t h a t DNP can i n h i b i t t h e i n c o r p o r a t i o n o f  formats a t concentration  decreased  1  Re-  enough t o uncouple o x i d a t i v e phosphory-  -4 lation  (10  M) may be due t o t h e u n a v a i l a b i l i t y o f ATP f o r  continued synthesis incubations  of the various  n u c l e i c a c i d components.  In  c o n t a i n i n g AMP, g l u c o s e and DNP, t h e i n h i b i t i o n o f  i n c o r p o r a t i o n i s maximum. of o x i d a t i v e phosphorylation  T h i s may be due t o t h e u n c o u p l i n g i n addition t o the various  reasons  s t a t e d above. 11)  E f f e c t o f G l u c o s e and 2-Deoxyglucose on t h e I n c o r p o r a t i o n of ^Re-Formate i n t o S e r i n e  o f E h r l i c h A s c i t e s Carcinoma C e l l s .  I t was o b s e r v e d i n many e x p e r i m e n t s w i t h E h r l i c h a s c i t e s c a r c i n o m a c e l l s t h a t t h e i n c o r p o r a t i o n o f C - f o r m a t e .into t h e 1 k  a c i d s o l u b l e f r a c t i o n was g r e a t l y i n c r e a s e d glucose.  i n presence of  When t h e s p e c i f i c a c t i v i t y o f t h e a c i d s o l u b l e n u c l e o -  t i d e bases were e s t i m a t e d , i t was found t h a t g l u c o s e a c t u a l l y decreased the i n c o r p o r a t i o n of C - f o r m a t e 1 k  i n t o the nucleotide  b a s e s , though t h e t o t a l a c t i v i t y i n t h e a c i d s o l u b l e f r a c t i o n was higher.  T h i s d i f f e r e n c e was a l s o o b s e r v e d when t h e c e l l s were  i n c u b a t e d w i t h 2-deoxyglucose.  2-Deoxyglucose d e c r e a s e d t h e  • - 152 - . • i n c o r p o r a t i o n of ^C-formate i n t o a l l the n u c l e i c a c i d f r a c t i o n s , 1  irrespective of concentration  of c e l l s i n suspension.  But.the  t o t a l , r a d i o a c t i v i t y i n t h e a c i d s o l u b l e f r a c t i o n was h i g h e r i n presence of 2-deoxyglucose, s i m i l a r t o the observations  with  glucose.  The. n u c l e o t i d e s  from t h e a c i d ' s o l u b l e f r a c t i o n o f c e l l s  incubated  w i t h 2-deoxyglucose and o f c o n t r o l e x p e r i m e n t s were  removed by c h a r c o a l a d s o r p t i o n .  The r a d i o a c t i v i t y p r e s e n t i n t h e  solution after adsorption of nucleotides  i s g i v e n i n T a b l e XXVI.  I t can be seen from t h e t a b l e t h a t t h e s o l u t i o n no U.V. a b s o r b i n g  materials a f t e r adsorption  ever, the s o l u t i o n contained  bn c h a r c o a l .  some r a d i o a c t i v e s u b s t a n c e s .  a c t i v i t y o f t h e s o l u t i o n s were h i g h e r 2-deoxyglucose.  contained  i n incubations  HowThe  containing  These f i n d i n g s l e d t o t h e s u s p i c i o n t h a t t h e  i n c r e a s e i n r a d i o a c t i v i t y seen i n t h e a c i d s o l u b l e f r a c t i o n i n p r e s e n c e o f g l u c o s e and 2-deoxyglucose may be due t o something o t h e r than t h e n u c l e o t i d e s . Herscovics  and J o h n s t o n e ( 7 2 ) r e p o r t e d  t h a t the presence of  glucose^ i n t h e i n c u b a t i o n medium i n c r e a s e d t h e i n c o r p o r a t i o n o f 1  ^C-.formate i n t o s e r i n e and p r o t e i n s o f E h r l i c h a s c i t e s c a r -  cinoma c e l l s .  I t was t h e r e f o r e s u s p e c t e d t h a t t h e r a d i o a c t i v i t y  may be c o n c e n t r a t e d  i n serine of the a c i d s o l u b l e f r a c t i o n of  E h r l i c h a s c i t e s carcinoma c e l l s which c o n t r i b u t e d f o r the apparent high i n c o r p o r a t i o n i n t o a c i d s o l u b l e . i t was n e c e s s a r y t o s e p a r a t e and e s t i m a t e  To v e r i f y  this  the r a d i o a c t i v i t y i n  s e r i n e from t h e a c i d s o l u b l e f r a c t i o n s o f E h r l i c h a s c i t e s c a r cinoma c e l l s from d i f f e r e n t i n c u b a t i o n s . The i s o l a t i o n and s e p a r a t i o n o f s e r i n e from t h e a c i d  - 153 T a b l e XXVI Radioactivity i n the acid soluble  fraction of  E h r l i c h a s c i t e s carcinoma c e l l s incubated llf  C-formate  nucleotides  Addition  Nil  + 2-deoxy glucose (5.5 mM)  Nil  + 2-deoxy glucose (5.5 mM)  Packed c e l l volume  1 4 %  14%  6.5%  6.5%  in  vitro  with  a f t e r t h e removal o f  by a d s o r p t i o n on c h a r c o a l .  O.D./260  O.D./280  cpm/ml  0.072  0.052  5174  0.067  0.051  5518  0.041  0.018  17758  0.027  0.012  15998  0.060  0.048  5994  0.053  0.041  6386  0.004  0.002  12304  0.007  0.006  10640  - 154 s o l u b l e f r a c t i o n was c a r r i e d o u t i n t h e f o l l o w i n g way.  The  a c i d s o l u b l e n u c l e o t i d e s were adsorbed on c h a r c o a l and removed from t h e s o l u t i o n .  The amino a c i d s w h i c h remain i n s o l u t i o n  were adsorbed on a column o f 5 cm x 1 cm o f Doxex-8 r e s i n , i n the H+ form a d j u s t e d  t o pH 2.5.  A f t e r the adsorption  o f t h e amino a c i d s , t h e column was  washed w i t h 0.01 M a c e t i c a c i d t o remove t h e s a l t s and o t h e r impurities.  F i n a l l y , t h e amino a c i d s adsorbed on t h e column  were e l u t e d w i t h 5 ml o f 1 M ammonia. and  The ammonia was removed  t h e r e s i d u e was e x t r a c t e d w i t h 1 ml o f w a t e r .  One hundred  m i c r o l i t r e s o f t h e r e s u l t i n g s o l u t i o n was a p p l i e d on t o a s t r i p o f Whatman No. 1 paper 6 5 cm x 20 cm.  The m i x t u r e o f  amino a c i d s was s e p a r a t e d by h i g h v o l t a g e e l e c t r o p h o r e s i s a t 4000 v o l t s f o r 45 minutes i n formate b u f f e r pH 1.9.  In e l e c t r o -  p h o r e t i c s e p a r a t i o n , samples from i n c u b a t i o n c o n t a i n i n g and  from c o n t r o l s e x p e r i m e n t s were s p o t t e d a l o n g s i d e  same paper.  phoresis.  i n the  A m i x t u r e o f n e u t r a l amino a c i d s c o n t a i n i n g  a r g i n i n e as a v i s u a l marker s e r v e d  as a s t a n d a r d  F r e e s e r i n e was a l s o r u n as a s t a n d a r d  This lane c o n t a i n i n g s e r i n e standard  glucose  "Dansyl"  i n electroon t h e paper.  was c u t o u t a f t e r t h e  e l e c t r o p h o r e s i s and s p r a y e d w i t h n i n h y d r i n .  The p o s i t i o n o f  s e r i n e on t h e e l e c t r o p h o r o g r a m i n t h e m a t e r i a l from t h e i n c u b a t i o n s was d e t e r m i n e d from t h e p o s i t i o n o f t h e s e r i n e and  standard  t h a t o f " D a n s y l " a r g i n i n e w h i c h can be e a s i l y v i s u a l i z e d . The e l e c t r o p h o r o g r a m c o n t a i n i n g t h e r a d i o a c t i v e s p o t s  exposed t o X-ray f i l m s f o r 2 t o 3 weeks i n t h e dark. 'were t h e n d e v e l o p e d and t h e s p o t s v i s u a l i z e d .  was  The f i l m s  The r a d i o a u t o g r a m s  - 155 -  Co 3  s;  • VI4/S  "Figure  23.  E f f e c t o f g l u c o s e ( 5 . 5 mM) a n d 2 - d e o x y g l u c o s e (5.5 mM) on the i n c o r p o r a t i o n of C-formate into the-serine of a c i d soluble fraction of Ehrlich ascites carcinoma c e l l s at different concentrations c f c e l l suspensions. 1 1 +  - 156 are shewn i n F i g u r e 23.  I t was i m m e d i a t e l y seen from the  r a d i o a u t o g r a m -that t h e i n c o r p o r a t i o n o f ^ C - f o r m a t e 1  s e r i n e was i n c r e a s e d  into  i n p r e s e n c e o f g l u c o s e of 2-deoxyglucose.  T h i s i n c r e a s e i n 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 i t y was n o t depend e n t on t h e c o n c e n t r a t i o n  of c e l l s i n s u s p e n s i o n .  of 2-deoxyglucose, where i n c o r p o r a t i o n o f '*C- i n t o 1  and n u c l e i c a c i d s was  i n h i b i t e d markedly, a high  i n t o s e r i n e i s observed.  I n t h e case nucleotides incorporation  - 157 DISCUSSION The  e f f e c t o f g l u c o s e on the i n c o r p o r a t i o n o f l a b e l l e d  precursors  i n t o the p u r i n e s  o f a c i d s o l u b l e n u c l e o t i d e s and  n u c l e i c a c i d s has' been i n v e s t i g a t e d by v a r i o u s groups o f workers (69-72). 1  I t i s g e n e r a l l y o b s e r v e d t h a t the i n c o r p o r a t i o n o f  '*C-forraate in vitro  i s s t i m u l a t e d by the p r e s e n c e o f g l u c o s e  i n the medium i n a l m o s t a l l the systems s t u d i e d . was  o b s e r v e d by Thomson e t a l . (14 7 ) , t h a t g l u c o s e  the i n c o r p o r a t i o n o f C-formate r a b b i t bone marrow in vitro, thymine was enhanced.  diminished  diminished  i n t o n u c l e i c a c i d purines  lh  DNA  However i t  in  whereas the i n c o r p o r a t i o n i n t o I n r a b b i t thymus t i s s u e g l u c o s e  the i n c o r p o r a t i o n o f '*C-formate i n t o DNA thymine 1  t o about h a l f the c o n t r o l v a l u e .  I n a l a t e r r e p o r t , Thomson  e t a l . (71) have shown t h a t i n r a b b i t bone marrow the n u c l e i c a c i d b i o s y n t h e s i s i s not a f f e c t e d by the p r e s e n c e o f g l u c o s e . Various  explanations  have been o f f e r e d f o r the  e f f e c t o f g l u c o s e on ^ C - f o r m a t e i n c o r p o r a t i o n . 1  Thus,  (69) has suggested t h a t g l u c o s e a c t e d as a p r e c u r s o r of r i b o s e  5-POi,  o r PRPP.  stimulatory Harrington  possibly  The view t h a t g l u c o s e p r o v i d e d  the  much needed energy s o u r c e f o r the s y n t h e t i c r e a c t i o n i s e x p r e s s e d by Henderson-and LePage (70).  They have a l s o observed  t h a t the e f f e c t o f g l u c o s e i s a b o l i s h e d by i o d o a c e t a t e a c e t a t e and al.  d i n i t r o p h e n o l i n support o f t h e i r view.  o r iodo-  Thomson e t  (71) are o f the o p i n i o n t h a t g l u c o s e c o n t r i b u t e d the  5-POit  required for purine biosynthesis.  (148)  o b s e r v e d an i n c r e a s e d p r o d u c t i o n  ribose  E l l i s and S c h o l e f i e l d of acid soluble  nucleo-  t i d e s from adenine i n p r e s e n c e o f g l u c o s e and suggested  the  - 158 p o s s i b l e r o l e o f g l u c o s e as an energy s o u r c e . Johnstone  H e r s c o v i c s and  (72) a r e o f t h e view t h a t g l u c o s e l e d t o - a n i n c r e a s e d  a v a i l a b i l i t y o f g l u t a m i n e which i s n e c e s s a r y f o r t h e de novo synthesis of the purine r i n g . I t i s p o s s i b l e t h a t a l l t h e s e views a r e v a l i d as t h e r e a r e e v i d e n c e s t o s u p p o r t them.  I n E h r l i c h a s c i t e s carcinoma  the c o m b i n a t i o n o f a l l t h e s e p o s s i b i l i t i e s e x i s t .  cells  Tumour c e l l s  a r e c h a r a c t e r i z e d by a h i g h r a t e o f g l y c o l y s i s and so g l u c o s e may be a b l e t o p r o v i d e energy by t h i s p r o c e s s . has shown t h a t b o t h glucose-6-phosphate  Ashman (149)  and 6-phosphogluconate  dehydrogenases a r e p r e s e n t i n a s c i t e s c e l l s .  K i t (150) has  shown t h a t hexosephosphate o x i d a t i v e pathway i s a c t i v e i n E h r l i c h a s c i t e s carcinoma c e l l s showing t h e a v a i l a b i l i t y and p r o d u c t i o n of r i b o s e - 5 - p h o s p h a t e  from g l u c o s e .  F u r t h e r , Henderson and Khoo  (139) have observed t h a t i n E h r l i c h a s c i t e s carcinoma g l u c o s e i n c r e a s e d t h e p r o d u c t i o n o f PRPP.  cells  I t i s w e l l known t h a t  PRPP p l a y s a key r o l e i n n u c l e o t i d e b i o s y n t h e s i s .  I t i srequired  f o r t h e f i r s t r e a c t i o n o f t h e de novo s y n t h e s i s o f p u r i n e n u c l e o t i d e s as w e l l as f o r t h e f o r m a t i i o n o f n u c l e o t i d e s from p r e formed bases.  Kvamme and Svenneby (151) r e p o r t e d t h a t g l u c o s e  prevented the deamination o f glutamine present i n a s c i t e s carcinoma c e l l s by t h e g l u t a m i n a s e p r e s e n t i n t h e c e l l . of H e r s c o v i c s and Johnstone(72)  The c l a i m  t h a t g l u c o s e i n f l u e n c e d t h e Clk  formate i n c o r p o r a t i o n by i n c r e a s i n g t h e g l u t a m i n e c o n c e n t r a t i o n is also  valid.  In a d d i t i o n t o a l l t h e s e f a c t o r s t h e p r e s e n t s e r i e s o f experiments  show t h a t t h e e f f e c t o f g l u c o s e a l s o depends on t h e  -  159  ~  c o n c e n t r a t i o n of c e l l s i n s u s p e n s i o n .  By v a r y i n g the c o n c e n t r a -  t i o n o f c e l l s i n s u s p e n s i o n from a h i g h e r v a l u e to" a lower v a l u e t h e e f f e c t o f g l u c o s e can a l s o be v a r i e d . h i g h c e l l c o n c e n t r a t i o n as determined  Thus a t a  by packed c e l l volume,  g l u c o s e s t i m u l a t e s , i n c o r p o r a t i o n whereas a t lower c o n c e n t r a t i o n s g l u c o s e i n h i b i t s t h e i n c o r p o r a t i o n o f '*C-formate t o a l l 1  f r a c t i o n s of n u c l e i c a c i d s .  B i k i s and Q u a s t e l  p o i n t e d out t h a t t h e c e l l d e n s i t y i n d i f f e r e n t  (152) have experiments  s h o u l d be k e p t c o n s t a n t i f comparable r e s u l t s are t o be o b t a i n e d . I t i s also.observed t h a t at a p a r t i c u l a r concentration of c e l l suspension, s t i m u l a t o r y or I n h i b i t o r y e f f e c t of glucose depends on the amount of g l u c o s e p r e s e n t i n s o l u t i o n .  There  i s an optimum c o n c e n t r a t i o n of g l u c o s e which.may i n c r e a s e i n c o r p o r a t i o n w h i l e higher c o n c e n t r a t i o n of glucose  decreases  t h e i n c o r p o r a t i o n . The optimum c o n c e n t r a t i o n o f g l u c o s e t o have a maximum s t i m u l a t o r y e f f e c t a g a i n may  depend on the con-  c e n t r a t i o n o f c e l l s u s p e n s i o n ; t h i s optimum c o n c e n t r a t i o n i n c r e a s e s v/ith i n c r e a s e i n c e l l d e n s i t y . The g l y c o l y t i c a b i l i t y of E h r l i c h A s c i t e s carcinoma i s well.known. of  These c e l l s are p e c u l i a r i n t h e i r  utilization  g l u c o s e , i n t h a t they e x h i b i t b o t h C r a b t r e e e f f e c t  (the  d e c r e a s e d r e s p i r a t i o n due t o g l u c o s e ) and P a s t e u r e f f e c t decreased g l y c o l y s i s due t o i n c r e a s e d o x i d a t i o n ) . g l y c o l y s i s and o x i d a t i o n o f g l u c o s e r e g u l a t e t h e of ATP  i n the  The of  cells  Both  (the the  availability  cell.  importance  o f ATP  i n the c e l l f o r de novo s y n t h e s i s  p u r i n e n u c l e o t i d e s i s seen from the e q u a t i o n s -  • - 160 G l y c i n e + R i b o s e 5-P0 — 9ATP + 9H 0  4  + 3NH* + HCO^ + 2HC00  -  > IMP. + SH 0 2  -> 3ADP + 8HP07 + AMP + HP 07 + 9 H  2  +  2  The s t i m u l a t o r y o r t h e i n h i b i t o r y e f f e c t o f g l u c o s e on t h e i n c o r p o r a t i o n o f C-formate lk  c o u l d be a t t r i b u t e d i n p a r t t o t h e  c o n d i t i o n s o f a v a i l a b i l i t y o f ATP. i n t h e c e l l s i n p r e s e n c e o f glucose Thomson e t a l . (71) have shown t h a t E h r l i c h a s c i t e s c a r cinoma c e l l s i n c u b a t e d  a e r o b i c a l l y i n y i t r o w i t h o u t any added  s u b s t r a t e : h a v e a h i g h c o n t e n t o f ATP.  A d d i t i o n of glucose  under a e r o b i c c o n d i t i o n s d e c r e a s e d t h e amount o f ATP i n t h e c e l l . Overgaard-Hansen  (73) has i n v e s t i g a t e d t h e adenine  t i d e p o o l i n a s c i t e s c a r c i n o m a c e l l s under v a r i o u s Her  observations  nucleo-  conditions.  a r e i n l i n e w i t h t h e p r e s e n t f i n d i n g s as -re-  f l e c t e d on n u c l e o t i d e and n u c l e i c a c i d b i o s y n t h e s i s .  It is  w e l l known t h a t E h r l i c h a s c i t e s c a r c i n o m a c e l l s c o n t a i n a powerf u l hexokinase.  G l u c o s e - under a e r o b i c c o n d i t i o n s i s shown t o  produce a t r a n s i e n t d e p l e t i o n o f t h e a d e n i n e n u c l e o t i d e v i a the hexokinase r e a c t i o n .  pool  C l o s e l y associated w i t h the i n i t i a l  b u r s t o f g l u c o s e consumption t h e r e a r e o t h e r r e a c t i o n s  taking  p l a c e i n t h e c e l l s w h i c h markedly d e c r e a s e t h e ATP c o n t e n t o f the c e l l s .  These a r e b r o u g h t about by t h e enzymes myokinase  a d e n y l a t e deaminase, 5 ' - n u c l e o t i d a s e , also pointed  nucleosidase  e t c . She has  o u t t h a t t h e e f f e c t o f g l u c o s e on adenine  t i d e p o o l i n d e n s e s u s p e n s i o n i s much l e s s pronounced. :  nucleoThe  d e p l e t i o n o f adenine n u c l e o t i d e p o o l i s a l s o l e s s i f o n l y q u a n t i t i e s o f sugar a r e added w h i c h may n o t produce an ATP  small  - 161 shortage. t h a t was  -  A c o n c e n t r a t i o n of g l u c o s e w h i c h i s h i g h e r  than  u t i l i z e d d u r i n g the i n i t i a l b u r s t of consumption  produces a d e p l e t i o n of the n u c l e o t i d e p o o l w h i c h i s i n i t i a t e d by the d e a m i n a t i o n of AMP  t o IMP,  which i n t u r n i s f u r t h e r  decomposed t o i n o s i n e and h y p o x a n t h i n e .  These m e t a b o l i t e s were  shown t o i n c r e a s e i n the medium as g l u c o s e was (73).  b e i n g used  up  However, a f t e r the sudden b u r s t , when g l y c o l y s i s i s  c o n t r o l l e d , the h y p o x a n t h i n e i s t a k e n up by the c e l l and  reutil-  i z e d f o r the r e s t o r a t i o n of the d e p l e t e d adenine n u c l e o t i d e pool.  The  p e r i o d of r e s t o r a t i o n of the adenine n u c l e o t i d e p o o l  is significantly  prolonged  i n d i l u t e c e l l suspensions.  Overgaeird-Hansen has a l s o shown t h a t i n d i l u t e c e l l  Further,  suspensions  the d e p l e t i o n of the adenine n u c l e o t i d e p o o l i s more s e v e r e , whereas i n dense s u s p e n s i o n s ,  the s m a l l d e c r e a s e i n adenine  n u c l e o t i d e p o o l seen i s q u i c k l y r e s t o r e d by the ATP mechanisms. of AMP  However, under a n a e r o b i c  t a k e s p l a c e though AMP  regeneration  c o n d i t i o n s no d e a m i n a t i o n  c o n c e n t r a t i o n i n c r e a s e s through  h e x o k i n a s e and myokinase a c t i v i t i e s , and no d e p l e t i o n of adenine n u c l e o t i d e p o o l i s seen.  I t was  a l s o shown t h a t under a e r o b i c  c o n d i t i o n s , 2-deoxyglucose induces  an i r r e v e r s i b l e d e p l e t i o n of  adenine n u c l e o t i d e i n the a s c i t e s c e l l s .  T h i s i s because 2-  d e o x y g l u c o s e m e t a b o l i s m does not go f a r t h e r t h a n i t s i n i t i a l phosphorylation A cellular  f o r w h i c h ATP  is utilized  (117).  d e c r e a s e i n the t o t a l a v a i l a b i l i t y  a e r o b i c c o n d i t i o n s i n the p r e s e n c e of g l u c o s e  of ATP  under  i s also reported  r e c e n t l y by many o t h e r i n v e s t i g a t o r s . Thus a f t e r 4 5 minutes of i n c u b a t i o n of E h r l i c h a s c i t e s c a r c i n o m a c e l l s w i t h  glucose  - 162 a t o t a l l o s s o f 20% o f t h e c e l l u l a r a c i d s o l u b l e n u c l e o t i d e p o o l has been r e p o r t e d by Hotham e t . a l .  (153).  Similarly  Laws and S t r i c k l a n d (140) have shown a reduced c o n c e n t r a t i o n of ATP i n E h r l i c h a s c i t e s carcinoma c e l l s i n c u b a t e d w i t h in. p r e s e n c e o f oxygen.  glucose  M e i k l e e t a l . (154) have shown t h e  e x c r e t i o n o f U.V. a b s o r b i n g m a t e r i a l • :(at. 260 my) into:' t h e medium o f a s c i t e s c e l l s u s p e n s i o n s  i n c u b a t e d w i t h glucose.'..  T h i s e x c r e t i o n o f m e t a b o l i t e s i n c r e a s e s w i t h time t o about 20 m i n u t e s , and t h e r e b e g i n s  to f a l l .  But when c e l l s a r e t r e a t e d  w i t h g l u c o s e and i o d o a c e t a t e o r w i t h 2-deoxyglucose, t h e conc e n t r a t i o n o f U.V. a b s o r b i n g m a t e r i a l i n c r e a s e s w i t h time and does n o t d e c r e a s e a t a l l . product  I t i s l o g i c a l to think that the  o f ATP breakdown i n d u c e d by g l u c o s e d i f f u s e s i n t o t h e  medium and i s t a k e n up by t h e c e l l s under c o n d i t i o n s i n which ATP and o t h e r n u c l e o t i d e b i o s y n t h e s i s i s i n i t i a t e d .  In the  p r e s e n c e o f i o d o a c e t a t e and 2-deoxyglucose r e s y n t h e s i s may n o t occur. for  These a u t h o r s have f u r t h e r o b s e r v e d t h a t ATP i s r e q u i r e d  the reconversion of the degradation  products mainly  inosine  and hypoxanthine,back t o t h e n u c l e o t i d e s and t h a t r e s y n t h e s i s of n u c l e o t i d e s does n o t t a k e p l a c e i n p r e s e n c e o f 2-deoxyglucose or i o d o a c e t a t e due t o t h e u n a v a i l a b i l i t y o f t h i s energy I t i s l o g i c a l t o assume from a l l these o b s e r v a t i o n s  source. that  g l u c o s e produces a t r a n s i e n t d e p l e t i o n o f adenine n u c l e o t i d e s , i n p a r t i c u l a r ATP i n E h r l i c h a s c i t e s c a r c i n o m a c e l l s when i n cubated a e r o b i c a l l y . T h i s d e p l e t i o n may depend on c o n c e n t r a t i o n of c e l l suspensions  when t h e g l u c o s e  c o n c e n t r a t i o n i s c o n s t a n t and  depend on c o n c e n t r a t i o n o f g l u c o s e when c o n c e n t r a t i o n o f c e l l  -  suspension  i s constant.  163 -  The i n h i b i t o r y and s t i m u l a t o r y e f f e c t  of g l u c o s e on "^C-formate i n c o r p o r a t i o n i n t o p u r i n e s o f n u c l e o 1  t i d e s " and n u c l e i c a c i d s can p a r t l y be e x p l a i n e d on t h e b a s i s o f these  observations. As. p o i n t e d o u t p r e v i o u s l y an i n c r e a s e i n c o n c e n t r a t i o n o f  ATP can f a c i l i t a t e r e a c t i o n s w h i c h r e q u i r e t h e u t i l i z a t i o n o f ATP and de novo s y n t h e s i s o f p u r i n e n u c l e o t i d e i s dependent • bn ATP.  F u r t h e r t h e r o l e of. ATP and p u r i n e : n u c l e o t i d e s i n  g e n e r a l i n t h e b i o s y n t h e s i s o f RNA has been e l u c i d a t e d by Anthony e t a l . (28) .  They have shown t h a t t h e c h a i n  initiation  r e a c t i o n i n t h e mechanism o f RNA polymerase r e a c t i o n i s dependent on p u r i n e n u c l e o t i d e s and has a s p e c i f i c r e q u i r e m e n t f o r ATP o r GTP.  As ATP p l a y s a c e n t r a l r o l e i n t h e r e g u l a t i o n o f p u r i n e  n u c l e o t i d e m e t a b o l i s m , i t c o u l d be c o n c e i v a b l e t h a t .a d e c r e a s e i n ATP w i l l a l s o reduce the i n i t i a t i o n r e a c t i o n s .  Recently,  C a s h e l and G a l l e n t (125) have i n v e s t i g a t e d t h e r e g u l a t i o n o f RNA s y n t h e s i s i n E. coli,  by amino a c i d s .  They o b s e r v e d t h a t  the f o r m a t i o n o f n u c l e o s i d e t r i p h o s p h a t e s a r e amino a c i d dependent and a l s o t h a t ATP f o r m a t i o n showed a severe amino a c i d dependence. gest-decrease  I t was a l s o o b s e r v e d t h a t the ATP p o o l s u f f e r s the l a r on amino a c i d s t a r v a t i o n .  From t h e s e and o t h e r  o b s e r v a t i o n s , they suggest t h a t t h e r e may be a d e c r e a s e d r a t e o f c h a i n i n i t i a t i o n due t o t h e d e c r e a s e d s u p p l y o f ATP. observations  The p r e s e n t  a r e a l s o i n agreement o f t h i s view.  Further p o s s i b i l i t i e s  o f i n h i b i t i o n o f t h e de novo s y n t h e s i s  by g l u c o s e may be due t o the secondary e f f e c t s produced subsequent t o the a c c u m u l a t i o n  of degradation  products  o f adenine n u c l e o t i d e s .  -  Henderson  (155,156)  inhibition sibility  PRPP  these  of  glucose  in  cells  accumulation the  purine  that  condition for  of  of  formation  substance that  of  tion  PRPP  such  as  markedly there  i s also  required pathway  ATP of  for and  These  the  markedly  the  are  i n c o r p o r a t i o n of  both  in dilute  cose  i s known  as to  well be  hexokinase. s i m i l a r go  beyond  Yushok  further  the  (157)  by  initial have  the  as  the  of  from  (139)  form of  of  substance  Ehrlich  the  that  forma-  under  inhibitors  synthesis  there  this  observed  in  for  metabolic  cell,  since  have  PRPP  ATP  the  requires  observation  then  of  PRPP  that  is also  the  by  de  effect  of  both  was  when  v i z . PRPP w h i c h  nucleotides  x l  explained  by  the  incorporation. *C-formate  i n dense up  by  glucose.  by  cells But  that  in  I t was  Ehrlich This  cell  is  novo  ascites  inhibition  is  is  (117).  respiring  Krebs  seen  2-Deoxyglu-  phosphorylated  metabolic  step  2-  observed  suspensions.  and  the  phosphorylation  reported  bases  Khoo can  2-deoxyglucose.  taken to  i s evident  purines.  ^ C - f ormate  inhibited  demand  i s conceivable  important  preformed  views  increased  d i n i t r o p h e n o l , the  biosynthesis  from  on  most  The  requirement by  pos-  under  and  glucose  It  i s the  operative  preformed  i n presence  (139).  There  feedback  c o n t r o l experiments,  Henderson  deficiency in  the  deoxyglucose  or  exert  be  glucose  from  The  bases  novo.  also  to  suggested  or  diminished  deficiency  that  cells.  conditions  i s an  with  conditions,  iodoacetate  de  may  compared  However,  carcinoma  purine  phosphorylation.  incubated  aerobic  that  mechanisms  nucleotides  ascites  anaerobic  shown  -  biosynthesis  bases  (43).  under  of  has  164  steps  by do  McComb Ringer  not and ascites  - 165  -  c a r c i n o m a c e l l s , p h o s p h o r y l a t i o n o f 2-deoxyglucose i n d u c e s a d r a s t i c d e p l e t i o n o f ATP.  They a l s o o b s e r v e d t h e dephosphory-  l a t i o n and d e a m i n a t i o n o f ATP t o IMP t h r o u g h ADP  and  AMP.  F u r t h e r i n o s i n e accumulated i n t h e r e a c t i o n m i x t u r e under t h e s e conditions.  They have suggested the Involvement o f h e x o k i n a s e ,  a d e n y l a t e k i n a s e , a d e n y l a t e deaminase, and 5 ' - n u c l e o t i d a s e , f o r the  o p e r a t i o n o f t h e s e pathways.  The a c t i v i t i e s o f t h e s e en-  zymes i n c a r c i n o m a c e l l s were found t o be adequate t o b r i n g about such a s e r i e s o f ATP d e g r a d a t i o n r e a c t i o n s . s i m i l a r t o t h i s a l s o have been r e p o r t e d by (73). the  She  Observations  Overgaard-Hansen  has a l s o o b s e r v e d t h a t u n l i k e i n t h e case o f g l u c o s e ,  d e p l e t i o n of the adenine n u c l e o t i d e p o o l caused by 2-deoxy-  g l u c o s e under a e r o b i c c o n d i t i o n s .is  irreversible.  The i n h i b i t i o n o f i n c o r p o r a t i o n of • ^C-formate by E h r l i c h 1  a s c i t e s c a r c i n o m a c e l l s i n p r e s e n c e o f 2-deoxyglucose can be e x p l a i n e d from the above o b s e r v a t i o n s .  I t i s seen t h a t u n l i k e  g l u c o s e , 2-deoxyglucose i n h i b i t s i n c o r p o r a t i o n o f . C-formate 1  lf  i n t o n u c l e i c a c i d components i n dense c e l l s u s p e n s i o n s .  This  i n h i b i t i o n i n dense c e l l s u s p e n s i o n by 2-deoxyglucose c o u l d be e x p l a i n e d by the o b s e r v a t i o n o f Overgaard-Hansen  t h a t the d e p l e -  t i o n o f adenine n u c l e o t i d e pool, by 2-deoxyglucose i s i r r e v e r s i b l In d i l u t e c e l l s u s p e n s i o n the i n h i b i t o r y e f f e c t o f 2-deoxyglucos can a l s o be e x p l a i n e d on the b a s i s o f d e p l e t i o n of ATP. d e c r e a s e i n i n c o r p o r a t i o n of of  14  However  C - f o r m a t e o b s e r v e d i n presence  g l u c o s e under s i m i l a r c o n d i t i o n s i s l e s s marked.  I t may  be  r e a l i z e d t h a t c o n t r a r y t o the o b s e r v a t i o n s w i t h 2-deoxyglucose, g l u c o s e i n c r e a s e d the i n c o r p o r a t i o n o f  14  C-formate into nucleic  - 166 acid  components i n d e n s e c e l l  of packed c e l l The effect  last  volume above  on  the  carcinoma c e l l s .  As  i.e. in  to prevent the  cell  depletion  of  1 4  by  i n E h r l i c h a s c i t e s carcinoma c e l l s  was  increased.  ATP  content  conditions,  the  Hence t h i s method was  i n the  cell  and  also  Wu  production  ferent  was  ATP  o f ATP.  conditions  Incorporation  o f ATP  production  E x p e r i m e n t s i n w h i c h AMP the  ATP  concentration  i n the  results,  a d e c r e a s e i n the  bases of  acid soluble  observed. of  The  added AMP  decrease i n incorporation i n the d i d not  enter  observed  t h a t AMP,  ADP  'stages t o i n o r g a n i c  little  s e e n was  the and  and  ATP  1 4  ATP  of  and cells  increase  C~formate  incubations, phosphorylation  to the  to the  the  lk  d e p l e t i o n was  1!  difstudied.  increase  expected  *C-formate  into  nucleic acids  e f f e c t on This  the may  p r i m a r i l y due  W a l l a c h and  be  (144),  C - f o r m a t e under  were h y d r o l y z e d  p h o s p h a t e and  to  I n some  T h e s e f i n d i n g s may cell.  not  w i t h AMP  e f f e c t on  contrary  nucleic acids.  medium.  added AMP  and  incorporation  had  Racker  oxidative of  could  g l u c o s e were added t o  cell,  nucleotides  r a d i o a c t i v i t y i n t o the  of glucose  and  and  attempted  the  and medium  content of the  study the  added t o p r e v e n t  cells  incubated  i n t o n u c l e i c a c i d under these c o n d i t i o n s . dinitrophenol  ascites  these t o the  l e v e l s i n the  reported  the  i m p e r m e a b l e t o ATP  d i r e c t a d d i t i o n of o f ATP  t o show  C - f o r m a t e by  membranes a r e  However, i t was  g l u c o s e under a e r o b i c  and  suspension  7-8%.  incorporation  a l s o sugar phosphate, the  that  suspension,  s e r i e s o f e x p e r i m e n t s were d e s i g n e d  o f ATP  undertaken.  -  was  incorporation mean t h a t to the  presence  i n d i c a t e that, Ullrey through  the  the  (145) successive  adenosine p r i o r to t h e i r  entry  - 167 i n t o the c e l l .  In support of t h i s increased  concentration  o f adenine and h y p o x a n t h i n e were o b s e r v e d when t h e a c i d s o l u b l e p u r i n e s were e s t i m a t e d  i n the present i n v e s t i g a t i o n s .  Another o b s e r v a t i o n  i n these experiments i s t h a t  phenol a t l e v e l s s u f f i c i e n t t o i n h i b i t o x i d a t i v e s e v e r e l y i n h i b i t e d C-formate  phosphorylation  The i n h i b i t i o n i s p a r t i c u l a r l y marked  i n the presence of glucose. that concentrations  Gross e t a l . (146) have  much h i g h e r  have r e c e n t l y r e p o r t e d  reported  t h a n t h a t r e q u i r e d for. u n c o u p l i n g  o x i d a t i v e p h o s p h o r y l a t o n i n h i b i t e d RNA polymerase. (158)  dinitro-  i n c o r p o r a t i o n i n t o RNA and t o  lh  a. l e s s e r degree, t o DNA.  :  t h a t i n . 2?. coli  Simon e t a l .  2,4—dinitrophenol  caused a marked i n h i b i t i o n o f RNA s y n t h e s i s w h i l e l i t t l e e f f e c t was seen on t h e s y n t h e s i s c o f DNA and p r o t e i n . Richardson  (30) s t u d i e d t h e mechanism o f a c t i o n o f DNP on  DNA dependent RNA p o l y m e r a s e , t h e enzyme r e s p o n s i b l e of RNA i n t h e c e l l .  f o r synthesi  He has shown t h a t a t a c o n c e n t r a t i o n  of 3  -2 x 10  M d i n i t r o p h e n o l t h e attachment o f t h e enzyme t o DNA,  w h i c h i s t h e f i r s t s t e p i n t h e p r o c e s s o f polymerase r e a c t i o n c o u l d be c o m p l e t e l y  prevented.  He has a l s o shown t h a t t h e  a f f i n i t y c o n s t a n t o f t h e enzyme f o r DNA was a l s o reduced by dinitrophenol. C r e a s e r and S c h o l e f i e l d (159) have i n v e s t i g a t e d t h e i n f l u e n c of DNP on t h e  3 2  P metabolism of E h r l i c h a s c i t e s carcinoma c e l l s .  They have o b s e r v e d t h a t i n t h e p r e s e n c e o f DNP, t h e r e was a decrease i n the formation and  aerobic conditions.  o f l a b e l l e d ATP and ADP under a n a e r o b i c Further,  they have o b s e r v e d a r a p i d l o s s  of b o t h ATP and ADP w i t h i n 5 m i n u t e s o f t h e a d d i t i o n o f DNP.  - 168 Thus^DNP n o t o n l y p r e v e n t e d t h e f o r m a t i o n  o f ATP.but a l s o i n -  c r e a s e d i t s r a t e o f removal i n a s c i t e s carcinoma c e l l s . investigators and  Many  (160,161) have shown t h a t b o t h amino a c i d uptake  i n c o r p o r a t i o n w i t h p r o t e i n s by E h r l i c h a s c i t e s carcinoma  c e l l s a r e i n h i b i t e d by DNP.  These r e a c t i o n s a r e energy r e q u i r -  i n g p r o c e s s e s and t h e e f f e c t o f DNP was shown t o be  reversed  by s u p p l y i n g energy by g l y c o l y s i s . On t h e b a s i s o f theseobservations  i t i s tempting t o speculate  that the i n h i b i t o r y  e f f e c t seen on t h e i n c o r p o r a t i o n o f ^'C-formate i n t o RNA i n 1  p r e s e n c e o f DNP i s a l s o due t o u n a v a i l a b i l i t y o f ATP. becomes more e v i d e n t  This  from t h e f i n d i n g t h a t t h e i n h i b i t i o n s a r e  more marked i n samples c o n t a i n i n g DNP and g l u c o s e .  It is  p o s s i b l e t h a t i n t h e s e samples t h e r e i s more d e p l e t i o n o f ATP t h a n i n t h e samples c o n t a i n i n g DNP o n l y .  Other f a c t o r s such as  a d i r e c t i n h i b i t i o n o f RNA polymerase as shown by Gross e t a l . (146)  and by R i c h a r d s o n (30) may a l s o c o n t r i b u t e f o r t h e i n h i b i -  t i o n o f "*C-f ormate . i n c o r p o r a t i o n i n t o RNA by E h r l i c h a s c i t e s 1  c e l l s i n p r e s e n c e o f DNP. I t has been o b s e r v e d by H e r s c o v i c s  and Johnstone (72)  that  g l u c o s e s t i m u l a t e s t h e i n c o r p o r a t i o n o f added l a b e l l e d formate i n t o f r e e s e r i n e of E h r l i c h a s c i t e s carcinoma c e l l s .  This i n -  c o r p o r a t i o n i n t o s e r i n e i s shown t o be g r e a t e r under a n a e r o b i c c o n d i t i o n s than under a e r o b i c c o n d i t i o n s . of  1!  But t h e i n c o r p o r a t i o n  *C-formate i n t o n u c l e i c a c i d s by t h e same t i s s u e was 25-50%  lower under a n a e r o b i c t h a n a e r o b i c c o n d i t i o n s . s e r i e s of experiments a l s o the i n c o r p o r a t i o n of i n t o s e r i n e was seen t o be i n c r e a s e d  In the present 14  C-formate  i n the presence of glucose.  - 169 Further> t h e i n c r e a s e was a l s o o b s e r v e d under c o n d i t i o n s where the i n c o r p o r a t i o n i n t o n u c l e i c a c i d i s markedly i n h i b i t e d as i n t h e case o f i n c u b a t i o n w i t h Herscovics  2-deoxyglucose..  and Johnstone(162) o b s e r v e d t h a t NADPH and  g l u t a m i n e a r e t h e f a c t o r s l i m i t i n g t h e I n c o r p o r a t i o n o f ^C1  formate e i t h e r t o s e r i n e o r t o n u c l e i c a c i d v i a f o r m y l g l y c i n e mide r i b o t i d e . N A D P H added t o c e l l f r e e systems s y n t h e s i z i n g b o t h FGAR and s e r i n e v/ere shown t o i n h i b i t t h e former p r o c e s s by 50-%, w h i l e s e r i n e s y n t h e s i s was d o u b l e d . which g r e a t l y s t i m u l a t e s always i n h i b i t e d  llf  S i m i l a r l y , glutamine  ^ C - f ormate i n c o r p o r a t i o n i n t o GAR  C - f o r m a t e i n c o r p o r a t i o n i n t o s e r i n e by 30%.  Thus t h e r e i s a c o m p e t i t i o n  f o r t h e common one carbon fragment  between s e r i n e and FGAR s y n t h e s i s . out t h a t the c o n c e n t r a t i o n  However i t may be p o i n t e d  o f b o t h NADPH and g l u t a m i n e a r e low  i n E h r l i c h a s c i t e s carcinoma c e l l s  ( 1 1 7 , 163).  glucose could increase the concentration  Addition of  of both, e s p e c i a l l y  NADPH v i a t h e HMP pathway which is. a c t i v e i n E h r l i c h a s c i t e s carcinoma c e l l s  (150).  However, t h i s e x p l a n a t i o n  account f o r t h e i n c r e a s e d  fails to  i n c o r p o r a t i o n o f '*C-formate i n t o 1  s e r i n e i n p r e s e n c e o f 2-deoxyglucose, which i s n o t m e t a b o l i z e d farther; than the i n i t i a l phosphorylation.  I t i spossible to  t h i n k t h a t when t h e a v a i l a b i l i t y o f a c t i v e one carbon fragments ( " a c t i v e formaldehyde" o r -* N - N 5  1 0  methylenetetrahydrofolic  a c i d ) i s i n c r e a s e d by t h e d e c r e a s e d s y n t h e s i s o f n u c l e o t i d e s , i t may be u t i l i z e d f o r t h e s y n t h e s i s o f s e r i n e .  Formation of s e r i n e  by t h e i n c o r p o r a t i o n o f t h e " a c t i v e formaldehyde" i s an e q u i l i b r i u m r e a c t i o n and i t i s n o t known whether t h i s r e q u i r e s energy  (164).  - 170 The f o r m a t i o n o f p u r i n e from g l y c i n e and " a c t i v e formaldehyde" r e q u i r e s c o n s i d e r a b l e amounts o f energy.  I t i s conceivable  t h a t under c o n d i t i o n s where t h e energy r e q u i r e m e n t s a r e n o t adequate t o meet t h e demand f o r s y n t h e s i s o f n u c l e o t i d e s , more o f t h e C - f o r m a t e may be used t o form s e r i n e . 1 4  The mechanism  o f t h e c o m p e t i t i o n between g l y c i n e and GAR f o r t h e " a c t i v e formaldehyde" i s n o t c l e a r .  - 171 SUMMARY  (1)  The v a r i o u s  purine  nucleotides  carcinoma  cells  of  i n a mammalian system v i z . E h r l i c h a s c i t e s  were  g l y c i n e were used  factors a f f e c t i n g the biosynthesis  investigated.  1  ^ C - F o r m a t e a n d 2- Clk  as t h e r a d i o a c t i v e p r e c u r s o r s  f o r these  studies.  (2) the  A method acid soluble  developed. tography the  the  and  by  small  involved  amounts  f o r the separation  to interfering these  a method  interfering  for their  were i s o l a t e d  adsorption  mixture  .90-95% r e c o v e r i e s  purine  The  amounts results.  prior  soluble  of the  was  i n  When  soluble  nucleo-  the separations i t was  to  necessary  The  fraction  This  were  chromatography nucleo-  of the  and e l u t i o n w i t h  p y r i d i n e and e t h a n o l .  method  a  cells  solvent gave  nucleotides.  e f f e c t of actinomycin  nucleotides  chromatography  from the a c i d  of acid  standardized.  on a c t i v a t e d c h a r c o a l  containing  (3)  r e m o v a l was  (pH 4.3)  acetate-ethanol  Therefore  materials  chroma-  to the f i r s t .  cells,  substances.  from  was,:  paper  system  i n t h e ammonium  i n a direction perpendicular tried  of c e l l s  two d i m e n s i o n a l  from E h r l i c h a s c i t e s carcinoma  remove  tides  from  d i r e c t i o n and then  (pH 7)  p o o r due to  The method  m e t h o d was  tides  fraction  of ribonucleotides  i n an i s o b u t y r i c acid-ammonia-water  first  system  f o r the separation  examined.  f o r the separation  D on t h e b i o s y n t h e s i s Initial  using  of free nucleotides  of E h r l i c h a s c i t e s carcinoma I n experiments where  studies  cells  gave  of  paper  from  small  inconclusive  l a r g e r volume o f c e l l s  were  used  - 172 the a c i d s o l u b l e n u c l e o t i d e s were s e p a r a t e d by chromatography on D E A E - c e l l u l o s e  columns.  The c o n c e n t r a t i o n s o f n u c l e o t i d e s  were d e t e r m i n e d by u l t r a v i o l e t s p e c t r o p h o t o m e t r y and the r a d i o a c t i v i t y i n c o r p o r a t e d was measured by l i q u i d counting-.  scintillation  From these s t u d i e s i t was o b s e r v e d t h a t t h e concen-  t r a t i o n of. t h e n u c l e o t i d e s i n t h e c e l l s i n c u b a t e d w i t h a c t i n o mycin D was i n c r e a s e d .  T h i s i n c r e a s e was more marked i n t h e  c o n c e n t r a t i o n s o f the n u c l e o s i d e t r i p h o s p h a t e s , p a r t i c u l a r l y o f ATP and DTP.  The r a d i o a c t i v i t y i n c o r p o r a t e d i n t o . the. p u r i n e  n u c l e o t i d e s was s l i g h t l y d e c r e a s e d i n p r e s e n c e o f D.  actinomycin  B u t the d e c r e a s e i n i n c o r p o r a t i o n was v e r y much l e s s i n  degree compared t o the i n c r e a s e i n c o n c e n t r a t i o n o f the n u c l e o tides.  The 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 i n t o t h e .  - n u c l e i c a c i d s was c o n s i d e r a b l y d e c r e a s e d by a c t i n o m y c i n D b o t h i n E h r l i c h a s c i t e s c a r c i n o m a c e l l s and i n t h e i n t e s t i n a l of t h e r a t , i n w h i c h i t was t e s t e d .  mucosa  T h i s d e c r e a s e was more  marked i n the case o f guanine o f RNA than o f a d e n i n e , which was i n agreement w i t h the o b s e r v a t i o n s v a t i o n s i t was concluded the  de  novo  of others.  that actinomycin  From these  obser-  D d i d not i n h i b i t  synthesis of purine n u c l e o t i d e s i n E h r l i c h a s c i t e s  c a r c i n o m a c e l l s and t h a t the s i t e o f a c t i o n o f a c t i n o m y c i n a stage beyond the f o r m a t i o n o f the n u c l e o s i d e (4)  Actinomycin  triphosphates.  D i n h i b i t e d the r e s p i r a t i o n o f E h r l i c h  a s c i t e s carcinoma c e l l s only s l i g h t l y .  The g l y c o l y s i s i n  E h r l i c h a s c i t e s c a r c i n o m a c e l l s was u n a f f e c t e d by D.  I t was c o n c l u d e d  D is  from these o b s e r v a t i o n s  actinomycin  t h a t the i n h i b i t i o n  of RNA s y n t h e s i s o b s e r v e d i n t h e p r e s e n c e o f a c t i n o m y c i n  D was  - 173 not due  to the i n h i b i t i o n of energy u t i l i z a t i o n or non  a b i l i t y of (5)  avail-  ATP. The  e f f o r t s to i n c r e a s e the i n c o r p o r a t i o n o o f r a d i o -  active precursors  i n t o the n u c l e i c a c i d components by  a d d i t i o n of glucose  the  to the medium gave v a r y i n g r e s u l t s , mostly  a decrease i n the i n c o r p o r a t i o n .  Hence the v a r i o u s f a c t o r s  i n f l u e n c i n g the 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  into  the n u c l e i c a c i d components of E h r l i c h ..ascites carcinoma i n presence of glucose were examined. the e f f e c t of C a  + +  of c e l l suspensions, The  f a c t o r s examined i n c l u d e d  i o n s i n the medium, the e f f e c t of d i f f e r e n t  b u f f e r s , the v a r i a t i o n s . o f pH  medium.  The  i n the medium, of the  concentration  and of the c o n c e n t r a t i o n of glucose  decrease i n i n c o r p o r a t i o n of p r e c u r s o r s  i n the  into nucleic  a c i d components caused by glucose d i d not depend on the presence or absence of Ca  ions i n the medium.  t i o n of p r e c u r s o r s  i n t o n u c l e i c a c i d s components i n presence  of glucose was Ringer  Decrease i n i n c o r p o r a -  observed both i n Krebs Ringer  bicarbonate  phosphate and  Krebs  b u f f e r s , though the l a t t e r b u f f e r s t a b i l i z e d  the pH of the medium d u r i n g g l y c o l y s i s . t h a t the e f f e c t of glucose  This f i n d i n g i n d i c a t e d  c o u l d not be e x p l a i n e d on the b a s i s  of a change i n pH of the medium because of g l y c o l y s i s .  The  i n c o r p o r a t i o n of ^ C - f ormate was  phosphate  b u f f e r than i n b i c a r b o n a t e  higher  buffer.  i n Krebs Ringer  T h i s has been a t t r i b u t e d to  the i n c r e a s e d a v a i l a b i l i t y of phosphate i n the former. r e s u l t s were o b t a i n e d when 2- ''C-glycine was 1  a c t i v e p r e c u r s o r of n u c l e o t i d e s and 1  ^C-formate.  Similar  used as a r a d i o -  n u c l e i c a c i d s i n s t e a d of  - 174 -  (6)  /Among t h e f a c t o r s c o n t r o l l i n g t h e i n c o r p o r a t i o n o f  p r e c u r s o r s i n t o n u c l e i c a c i d components, t h e f o l l o w i n g . s e e m t o be more i m p o r t a n t ;  (1) the c o n c e n t r a t i o n o f g l u c o s e i n t h e  medium and (2) t h e c o n c e n t r a t i o n o f c e l l s u s p e n s i o n . p o r a t i o n pf p r e c u r s o r s was Increased  i n dilute cell  The i n c o r suspensions  w i t h low c o n c e n t r a t i o n s o f g l u c o s e , w h i l e h i g h e r c o n c e n t r a t i o n s decreased of g l u c o s e  the incorporation.  At a physiological concentration  (5.5 mM) t h e i n c o r p o r a t i o n was i n c r e a s e d i n dense  c e l l suspensions  (packed c e l l volume above 8%) and d e c r e a s e d i n  d i l u t e c e l l suspension  (PCV below 5 % ) . i n between t h e s e two  c o n c e n t r a t i o n s o f c e l l s u s p e n s i o n , a 5.5 mM c o n c e n t r a t i o n o f g l u c o s e gave wide v a r i a t i o n s i n i n c o r p o r a t i o n s . (7) cells,  2-Deoxyglucose, a s u b s t a n c e which d e p l e t e d ATP i n t h e a t a concentration s i m i l a r t o that of glucose  decreased  t h e i n c o r p o r a t i o n o f C-formate  into nucleic  lh  components o f E h r l i c h a s c i t e s carcinoma 14%) as w e l l as i n a d i l u t e  (5.5 mM)  cells  (PCV 6.5%) c e l l  acid  i n a dense (PCV suspension.  (8)  D i n i t r o p h e n o l , an u n c o u p l e r o f o x i d a t i v e p h o s p h o r y l a t i o n  decreased  t h e i n c o r p o r a t i o n o f C-formate  components o f E h r l i c h a s c i t e s carcinoma more marked i n d i l u t e c e l l s u s p e n s i o n s (9)  into nucleic  lk  I t was c o n c l u d e d  cells.  acid  T h i s d e c r e a s e was  i n presence  of glucose.  from t h e above o b s e r v a t i o n s t h a t t h e  main f a c t o r c o n t r o l l i n g t h e i n c o r p o r a t i o n o f p r e c u r s o r s t o n u c l e i c a c i d components In t h e s e c e l l s  was t h e t r a n s i e n t d e p l e -  t i o n and r e g e n e r a t i o n o f ATP i n t h e c e l l s  i n presence  of glucose.  - 175 (10)  Glucose increased the i n c o r p o r a t i o n of  14  C-formate  i n t o serine of the a c i d soluble f r a c t i o n of E h r l i c h a s c i t e s carcinoma c e l l s .  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