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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 vitro EFFECTS OF ACTINOMYCIN D AND GLUCOSE by M.S. PARAMESWARAN NAIR B.Sc., U n i v e r s i t y of Madras, 1951 M.Sc, A l i g a r h Muslim 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 the Department of Biochemistry We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard f o r the degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF BRITISH COLUMBIA November, 196 8 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h C olumbia, I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s j s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Biochemistry The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 2 3 - 1 2 - 1 9 6 8 . . . • 1 -• • ABSTRACT The b i o s y n t h e s i s 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 carcinoma 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 . In i n i t i a l s t u d i e s the e f f e c t of actinomycin D was examined. The nu c l e o t i d e s from 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 incubated w i t h actinomycin D and ^C-formate were adsorbed on ch a r c o a l and e l u t e d w i t h a mixture of p y r i d i n e and ethanol. The e l u t e d n u c l e o t i d e s were separated by two dimensional paper chromatography using i s o b u t y r i c acid-ammonia-water i n the f i r s t d i r e c t i o n - a n d aqueaus ammonium acetate-ethanol i n the second. .The nu c l e o t i d e s were estimated by u l t r a v i o l e t spectrophotometry 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 determined by l i q u i d s c i n t i l l a -t i o n counting. As the r e s u l t s of these s t u d i e s using small amounts of c e l l s were i n c o n c l u s i v e due to v a r i a t i o n s from experiment t o experiment, s i m i l a r s t u d i e s were c a r r i e d out using l a r g e r amounts of c e l l suspensions. 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 experiments were separated by ion-exchange chromato-graphy on DEAE-cellulose and f i n a l l y by paper chromatography. I t was observed t h a t there was an accumulation 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 carcinoma c e l l s incubated w i t h 1 kC-formate and actinomycin D. 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 not s i g n i f i c a n t l y d i f f e r e n t from those of the c o n t r o l experiments. The i n c o r p o r a t i o n of 1 1 1C-formate i n t o n u c l e i c acids was i n h i b i t e d by actinomycin D i n these c e l l s . From these observations i t was concluded t h a t actinomycin D d i d not i n h i b i t the b i o s y n t h e s i s of purine n u c l e o t i d e s i n E h r l i c h 1 1 a s c i t e s carcinoma c e l l s in vitro. I t i s suggested t h a t the e f f e c t of actinomycin D on n u c l e i c a c i d metabolism i s therefore, at a stage beyond the synt h e s i s of n u c l e o t i d e s . Further s t u d i e s on the e f f e c t of actinomycin D revealed t h a t 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 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 in vitro 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 carcinoma c e l l s was unaffected.-by actinomycin D. In experiments where the e f f e c t o'f glucose on 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 precursors 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 carcinoma c e l l s was examined, con t r a r y to the r e s u l t s of many, a decrease i n i n c o r p o r a t i o n was observed. This decrease i n i n c o r p o r a t i o n was independent of the presence ++ of Ca ions i n the i n c u b a t i o n medium, the b u f f e r and of the r a d i o -a c t i v e precursors used i n these i n c u b a t i o n s . I t was observed t h a t there were two f a c t o r s 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 r a d i o a c t i v e precursors in. vitro i n t o the n u c l e o t i d e s and n u c l e i c acids of E h r l i c h a s c i t e s carcinoma c e l l s i n presence of glucose; the c o n c e n t r a t i o n of glucose i n the medium and the con c e n t r a t i o n of c e l l s i n suspension. In d i l u t e c e l l suspensions (packed c e l l volume l e s s than 5%) glucose at a co n c e n t r a t i o n of 5.5 mM de-cr e a s e d whereas i n dense c e l l suspensions (packed c e l l volume above 8%) the same c o n c e n t r a t i o n of glucose increased the i n c o r p o r a t i o n of l a b e l l e d precursors i n t o both 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 . 2-Deoxyglucose, an analogue of glucose at 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 uncoupler of o x i d a t i v e phosphory-l a t i o n , a l s o decreased the i n c o r p o r a t i o n of 1'*C-formate i n these - c e l l s which was more marked i n d i l u t e c e l l suspensions i n presence of glucose. I t was concluded from these observations 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 in vitro of r a d i o a c t i v e precursors i n t o n u c l e o t i d e s and n u c l e i c acids of E h r l i c h a s c i t e s carcinoma c e l l s was the t r a n s i e n t d e p l e t i o n and regeneration of ATP i n these c e l l s i n presence of glucose. Glucose increased the i n c o r p o r a t i o n of 1'*C-formate i n t o the 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 -oma c e l l s . A marked increase i n the i n c o r p o r a t i o n of 1 I fC-formate i n t o s e r i n e was observed i n presence of 2-deoxyglucose. These e f f e c t s were independent of the co n c e n t r a t i o n of c e l l s i n sus-pension. I t was suggested t h a t when the a v a i l a b i l i t y of the common pr e c u r s o r , N 5-N 1 0 m e t h y l e n e t e t r a h y d r o f o l i c a c i d was i n -creased p a r t i c u l a r l y due to a decrease 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 of the l a b e l l e d precursor may be incorporated i n t o s e r i n e . I v 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 B i o s y n t h e s i s of Purine Nucleotides 12 Bi o s y n t h e s i s of N u c l e i c Acids 15 . Present I n v e s t i g a t i o n 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 of Packed C e l l Volume 26 3. Ra d i o a c t i v e M a t e r i a l s . . 26 4. Actinomycin D. ...... , . . 26 5. Determination of Glucose 27 6. Determination of Phosphate 27 7. Determination of N u c l e i c A c i d s . . . . ... 27 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. General Procedure f o r Incubations in vitro of E h r l i c h A s c i t e s Carcinoma C e l l s . 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 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 - 30 a) I s o l 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 30 b) N u c l e i c acids 31 13. I s o l a t i o n and Determination of Purine and Pyri 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 Acids 32 14. E l u t i o n and E s t i m a t i o n of N u c l e i c A c i d Components from Chromatograms..... 33 V Page 15.. Separation of A c i d Soluble Nucleotides 34 a) I s o l a t i o n of n u c l e o t i d e s by cha r c o a l a d s o r p t i o n 34 b) Paper chromatography of n u c l e o t i d e s 35 c) Ion-exchange chromatography of 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. . 39 1. Time Course of 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 . 39 2. E f f e c t of Actinomycin D on the I n c o r p o r a t i o n of lkC-Formate i n t o the Bases of Nucleotides and N u c l e i c Acids of E h r l i c h A s c i t e s Carcinoma C e l l s 41 3. Chromatography of A c i d Soluble Nucleotides by Paper Chromatography - Development of a Method 48 a) Paper chromatography of r i b o n u c l e o t i d e s 48 b) Paper chromatography of a c i d s o l u b l e nucleo-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 . . . 52 c) Removal of substances i n t e r f e r i n g i n paper chromatography of 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 using DEAE-c e l l u l o s e 53 i 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 54 i i i ) A d sorption of n u c l e o t i d e s on c h a r c o a l . . 55 d) Characte t i o n of substances e l u t e d from chromatograms 59 4. E f f e c t of Actinomycin D on the I n c o r p o r a t i o n of ll*C-Formate i n t o the A c i d Soluble Nucleotides of E h r l i c h A s c i t e s Carcinoma C e l l s . . . . 63 v i Page 5. E f f e c t of Actinomycin D on the I n c o r p o r a t i o n of 1 4C-Formate in vitro by the I n t e s t i n a l Mucosa of the Rat 67 6. E f f e c t of Varying Concentrations of Actinomycin D on. the I n c o r p o r a t i o n of ^ C-Formate 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 70 7. Separation of A c i d 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 DEAE-C e l l u l o s e Column. 70 8. E f f e c t of Actinomycin D on the B i o s y n t h e s i s of Purine Nucleotides i n E h r l i c h A s c i t e s Carcinoma C e l l s in vitro 74 9. E f f e c t of Actinomycin 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 . . . . . 83 10. E f f e c t of Actinomycin D on the 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 87 DISCUSSION . 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 0 1 1. Time Course of In c o r p o r a t i o n of 1^C-Formate.into Various N u c l e i c A c i d Components i n Presence of Glucose.. 102 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 1^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 109 3. E f f e c t of B u f f e r on the I n c o r p o r a t i o n of 1^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 Presence of Glucose. 114 4. E f f e c t of Glucose on the I n c o r p o r a t i o n of 2-1!*C-Gl 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. 123 v i i Page . 5. The I n c o r p o r a t i o n of 1 ^ 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 B u f f e r s 127 6. Increased I n c o r p o r a t i o n of l l fC-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s i n Presence of Glucose 131 7. E f f e c t of Varying Concentration of Glucose on the In c o r p o r a t i o n of 1 4C-Formate 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 of l l +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 Function of C e l l Concentration. 137 9. E f f e c t of 2-Deoxyglucose on the In c o r p o r a t i o n of 1'*C-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s in vitro . . 141 10. I n c o r p o r a t i o n of 1 I fC-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s Under Various Conditions A f f e c t i n g ATP Formation. 146 11. E f f e c t of Glucose and 2-Deoxyglucose on the Incor-p o r a t i o n of l l fC-Formate i n t o Serine of E h r l i c h A s c i t e s Carcinoma C e l l s 151 DISCUSSION 157 SUMMARY ..... . / 171 BIBLIOGRAPHY. ..: 176 v i i i TABLES Page I . E f f e c t of actinomycin D on t h e i n c o r p o r a t i o n of 1 4C-formate i n t o the bases of n u c l e i c a c i d compon-ents of E h r l i c h a s c i t e s carcinoma c e l l s in vitro 45 I I . R„ values of r i b o n u c l e o t i d e s . 50 F I I I . Recovery of nu c l e o t i d e s adsorbed on charc o a l by e l u t i o n w i t h three d i f f e r e n t s o l v e n t systems 58 IV. Phosphorus determination of nu c l e o t i d e s obtained from chromatograms 62 V. E f f e c t of actinomycin D on the i n c o r p o r a t i o n of X I fC-formate i n t o purine 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 VI. E f f e c t of actinomycin D on the i n c o r p o r a t i o n of 1 I fC-formate by the i n t e s t i n a l mucosa of the r a t 69 V I I . E f f e c t of va r y i n g concentrations of actinomycin D on the i n c o r p o r a t i o n of ^ C-formate 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 in vitro 71 V I I I . Concentration of purine 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 IX. E f f e c t of actinomycin D on the i n c o r p o r a t i o n of lhC-formate i n t o purine n u c l e o t i d e s of E h r l i c h a s c i t e s carcinoma c e l l s . . 80 X. E f f e c t of actinomycin D on the c o n c e n t r a t i o n of pyr i m i d i n e n u c l e o t i d e s of E h r l i c h a s c i t e s carcinoma c e l l s in vitro 82 XI. E f f e c t of actinomycin 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 86 X I I . 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 1 4C-formate i n presence of glucose by E h r l i c h a s c i t e s carcinoma c e l l s 115 X I I I . Change i n pH'of E h r l i c h a s c i t e s carcinoma c e l l s sus-pension incubated w i t h glucose 117 XIV. E f f e c t of glucose on the i n c o r p o r a t i o n of 1 ^ C - f o r -mate i n t o RNA nu c l e o t i d e s and thymine of E h r l i c h a s c i t e s carcinoma c e l l s . . . . 122 i x Page XV. E f f e c t of glucose on the i n c o r p o r a t i o n of 2-1'*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... 128 XVI. I n c o r p o r a t i o n of 1'*C-formate 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 two d i f f e r e n t b u f f e r s 130 XVII. E f f e c t of glucose on the i n c o r p o r a t i o n of 1'*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:..cells 132 XV I I I . E f f e c t of v a r y i n g concentrations of glucose on the i n c o r p o r a t i o n of 1'*C-formate i n t o n u c l e i c a c i d compon-ents of E h r l i c h a s c i t e s carcinoma c e l l s 136 XIX. I n c o r p o r a t i o n of 1''C-formate i n presence of. glucose by d i f f e r e n t concentrations of 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 suspensions 139 XX. I n c o r p o r a t i o n of l l fC-formate i n presence of glucose i n t o bases of DNA by d i f f e r e n t concentrations of E h r l i c h a s c i t e s carcinoma c e l l suspensions 140 XXI. E f f e c t of 2-deoxyglucose on the i n c o r p o r a t i o n of l l fC-formate by E h r l i c h a s c i t e s carcinoma c e l l s 143 XXII. E f f e c t of 2-deoxyglucose i n the i n c o r p o r a t i o n . o f lkC-formate i n t o the a c i d s o l u b l e purines of E h r l i c h a s c i t e s carcinoma c e l l s 144 X X I I I . E f f e c t of 2-deoxyglucose on the i n c o r p o r a t i o n of 1'*C-formate i n t o the bases of DNA of E h r l i c h a s c i t e s carcinoma c e l l s 145 XXIV. I n c o r p o r a t i o n of ^C-formate 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 the ATP i n the c e l l 149 XXV. I n c o r p o r a t i o n of 1'*C-formate i n t o the a c i d s o l u b l e purines of E h r l i c h a s c i t e s carcinoma 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 ATP c o n c e n t r a t i o n i n the c e l l . . . ... 150 XXVI. 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 incubated w i t h 1 I fC-formate a f t e r the removal of n u c l e o t i d e s 153 X FIGURES Page 1. S t r u c t u r e of Actinomycin D.. 4 2. Proposed model f o r b i n d i n g actinomycin D to the deoxyguanosine of DNA 9 3. B i o s y n t h e s i s of I n o s i n i c a c i d 14 4. I n c o r p o r a t i o n Of 1'*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 of E h r l i c h a s c i t e s carcinoma c e l l s in vitro 40 5. I n c o r p o r a t i o n of 1^C-formate into.RNA of E h r l i c h a s c i t e s carcinoma c e l l s in vitro 42 6. I n c o r p o r a t i o n of 1 1*C-formate i n t o DNA of E h r l i c h a s c i t e s carcinoma c e l l s in vitro . 43 7. Schematic r e p r e s e n t a t i o n of two dimensional paper chromatogram of r i b o n u c l e o t i d e s 51 8. Separation 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 carcinoma c e l l s on DEAE-cellulose column 73 9. Chromatography on DEAE-cellulose 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 carcinoma c e l l s w i t h 1 ^C-formate. - 76 10. Chromatography on DEAE-cellulose 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 carcinoma c e l l s w i t h l I*C-formate and actinomycin D 77 11. E f f e c t of actinomycin D on oxygen consumption of 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 of actinomycin D on the g l y c o l y s i s of E h r l i c h a s c i t e s carcinoma c e l l s in vitro ... 89 13. E f f e c t of glucose on the 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 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 Ringer phosphate b u f f e r . . 105 14. E f f e c t of glucose on the i n c o r p o r a t i o n of ^C-formate 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 suspended i n Krebs Ringer phosphate b u f f e r 106 15. E f f e c t of glucose on the i n c o r p o r a t i o n of ^C-formate 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 Krebs Ringer phosphate b u f f e r 107 x i Page 16. E f f e c t of glucose on the i n c o r p o r a t i o n of 1^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 of E h r l i c h a s c i t e s carcinoma c e l l s suspended i n calcium f r e e Krebs Ringer phosphate b u f f e r . . . . I l l 17. E f f e c t of glucose on the i n c o r p o r a t i o n of 1 I fC-formate 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 suspended i n calcium f r e e Krebs Ringer phosphate b u f f e r . 112 18. E f f e c t of glucose on the i n c o r p o r a t i o n of l l fC-formate i n t o 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 calcium f r e e Krebs Ringer phosphate b u f f e r 113 19. E f f e c t of glucose oh the i n c o r p o r a t i o n of 1^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 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 Ringer bicarbonate b u f f e r 119 20. E f f e c t of glucose on the i n c o r p o r a t i o n of lkC-formate i n t o the RNA 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 Ringer bicarbonate b u f f e r . . 120 E f f e c t of glucose on the i n c o r p o r a t i o n of 1 Re-formats 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 Ringer bicarbonate b u f f e r . . 121 E f f e c t of g l y c i n e on the i n c o r p o r a t i o n of lkC-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 . . 125 23. E f f e c t of glucose and 2-deoxyglucose on the i n c o r -p o r a t i o n of ' 1 4C-formate i n t o the s e r i n e of the acid.-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 . 155 21. 22. I / x i i LIST OF ABBREVIATIONS AMP) ADP) ATP) -CMP) CDP) CTP) Dansyl DEAE DNA DNP EDTA FGAR GAR. GMP) GDP) GTP) IMP NAD, NADH-NADP, NADPH PP PRPP RNA TMP UMP) UDP) UTP) dUMP XMP 5'-mono,; d i , and triphosphates of Adenosine 5'-mono, d i , and triphosphates of c y t o s i n e 1-Dimethyl aminonaphthalene 5-sulphonyl c h l o r i d e Diethylamino e t h y l D esoxyribonucleic a c i d 2, 4-Dinitrophenol E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d Formylglycine amide r i b o t i d e Glycineamide r i b o t i d e 5'-mono, d i , and triphosphates of guanosine 5'-phosphate of i n o s i n e Nicotinamide adenine d i n u c l e o t i d e and i t s reduced form Nicotinamide adenine d i n u c l e o t i d e phosphate and Its reduced form Pyrophosphate Phosphoribosy1 pyrophosphate R i b o n u c l e i c a c i d 5'-phosphate of thymidine 5'-mono-, d i , and t r i phosphates of u r i d i n e 5'-phosphate of deoxyuridine 5'-phosphate of xanthosine x m ACKNOWLEDGEMENT The author wishes to express h i s g r a t i t u d e t o Dr. S.H. Zbarsky f o r h i s guidence and counsel during the course of these i n v e s t i g a t i o n s . The author i s indebted to Dr. A.R.P. Paterson of McEachern L a b o r a t o r i e s , U n i v e r s i t y of A l b e r t a , f o r p r o v i d i n g the E h r l i c h a s c i t e s carcinoma 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 the sep a r a t i o n of n u c l e o t i d e s and withl.Dr. G.H. Dixon oh the sep a r a t i o n of amino acids 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 appreciated. Mrs. D. Hawkins cooperated w i t h some aspects of the study and Miss M. Ha r r i s o n of U.B.C. Health S e r v i c e s was h e l p f u l i n developing the exposed r a d i o -autograms. - 1 -• ... 1 INTRODUCTION Substances which block 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 of great value i n c l a r i f y i n g the normal mechanisms by which macromolecules are synthesized by l i v i n g c e l l s . The actinomycins are of s p e c i a l importance i n t h i s regard, as they are shown to be s p e c i f i c i n h i b i t o r s of RNA s y n t h e s i s , and hence are used at an ever i n c r e a s i n g r a t e , i n the e l u c i -d a t i o n of mechanisms of genetic t r a n s m i s s i o n , c e l l u l a r d i f -f e r e n t i a t i o n , p r o t e i n s y n t h e s i s , v i r u s m u l t i p l i c a t i o n e t c . Because of the s i m p l i c i t y of the m i c r o b i a l organisms and the ease w i t h which enzymes and other substances could be i s o l a t e d from these organisms, many of the 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 are c a r r i e d out i n b a c t e r i a l systems. The complex nature of 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 the more d i f f i c u l t i n mammalian systems. Much of the i n v e s t i g a t i o n c a r r i e d out using actinomycin as a b l o c k i n g agent p e r t a i n s to i t s e f f e c t on macromolecular s y n t h e s i s . 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 the e f f e c t of actinomycin D on the s y n t h e s i s of small molecules which are precursors of macromolecules e s p e c i a l l y of n u c l e i c a c i d s . Previous s t u d i e s showed t h a t when the growth of Bacillus subtilis was i n h i b i t e d by the a d d i t i o n of actinomycin D to the medium, there was an accumulation of guanine nucleo-t i d e s i n the c e l l s (1). The i n v e s t i g a t i o n s reported i n t h i s t h e s i s were o r i g i n a l l y undertaken to e l u c i d a t e the e f f e c t of actinomycin D on the a c i d s o l u b l e n u c l e o t i d e pool of 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 l i n e having m i c r o b i a l s i m p l i c i t y In c u l t i v a t i o n and mammalian complexity i n metabolic r e a c t i o n s were chosen f o r these s t u d i e s . As an i n t r o d u c t i o n to these 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 the nature and e f f e c t of actinomycin 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 metabolism seems to be most appro p r i a t e . .' As 1 "*C-formate was used as a precursor of nu c l e o t i d e s i n the experiments reported i n t h i s t h e s i s , the b a s i c steps by which formate i s incorporated 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 a l s o o u t l i n e d . Actinomycins and Their B i o l o g i c a l A c t i v i t y . Actinomycins are a group of chromopeptide a n t i b i o t i c s produced by micro-organisms belonging l a r g e l y to the genus •St-reptomyc.es. (2). 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 of the s e r i e s , actinomycin A, was i s o l a t e d by Waksman and Woodruff i n 1940 (.3). Since then more than f i f t y chemical forms of 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 of 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 of the alphabet ranging from A to Z, by num bers, and al s o by Greek sub l e t t e r s . They are a l s o known unde d i f f e r e n t trade names. Though Waksman suggested (4) a system-a t i c nomenclature f o r these compounds, t h i s .has not been followed s t r i c t l y i n l i t e r a t u r e , probably due to the l i m i t e d use of the l e s s e r known compounds. The actinomycins are 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 lour and complex b i o l o g i c a l p r o p e r t i e s . Brockmann et - 3 -a l . (5,6) have shown by degradation experiments t h a t a l l of them co n t a i n the same chromophoric moiety a c t i n o c i n (2-amino 4,6-dimethyi 3-oxophenoxazine 1 , 9 - d i c a r b o x y l i c acid) and t h a t they d i f f e r only i n the s u b s t i t u t i o n among the polypeptide chains j o i n e d t o the chromophoric moiety. Among the actinomycins, the best known compound i s a c t i n o -mycin D, discovered by V i n i g and Waksman i n (7) 1954. (Figure 1). Of the many complex actinomycins i s o l a t e d , actinomycin D i s shown to be chromatographically the most homogeneous sub-stance (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 the others. However,the a b i l i t y , of actinomycin D to exe 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 biochemists p a r t i c u l a r l y i n the e l u c i d a t i o n of mechanisms of tra n s m i s s i o n of gene t i c i n f o r m a t i o n . The a b i l i t y of actinomycin D to i n h i b i t both p r o t e i n -and RNA synt h e s i s was reported 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 actinomycin D can s e l e c t i v e l y i n h i b i t RNA sy n t h e s i s both i n micro-organisms and i n mammalian c e l l s . Further., i t was a l s o shown th a t a c t i n o -mycin D suppressed the growth of DNA virus,, but not RNA v i r u s i n host c e l l s (12). S i m i l a r l y the a n t i b i o t i c was shown to i n h i b i t RNA sy n t h e s i s by c e l l f r e e systems from both mammalian (13) and m i c r o b i a l sources (14) . DNA sy n t h e s i s i n the c e l l s ' can a l s o be suppressed by higher c o n c e n t r a t i o n of actinomycin D than r e q u i r e d f o r the i n h i b i t i o n of RNA synt h e s i s (11). Hurwitz et a l . have shown (14) t h a t the a c t i v i t y of DNA poly-merase in vitro i s l e s s s e n s i t i v e to actinomycin D than t h a t of RNA polymerase. F i g u r e 1. Actinomycin D. The sequence of amino acids i s L-threonine, D-valine (D-Val), L - p r o l i n e (L-P r o ) , sarcosine (Sar) and N-methyl-valine. - 5 -To e l u c i d a t e the 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 molecular s t r u c t u r e , many chem 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 actinomycins have been i s o l a t e d (15,16). In a study w i t h a s e r i e s of d i f f e r e n t actinomycins Reich et a l . (17) have shown t h a t the i n h i b i t o r y a c t i o n of the a n t i b i o t i c on the growth of HeLa c e l l s and on the i s o l a t e d RNA polymerase r e a c t i o n could 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. A l t e r a -t i o n s at d i f f e r e n t p o r t i o n s of the a n t i b i o t i c molecule could a f f e c t the i n h i b i t o r y e f f e c t s to a greater or l e s s e r degree. Thus actinomycins provide a unique example of 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 w i t h molecular s t r u c t u r e . Several c h a r a c t e r -i s t i c f u n c t i o n a l groups present i n the a c t i n o mycin molecule (see Figure 1) appear to be e s s e n t i a l f o r the 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) the f r e e amino group at p o s i t i o n 3 of the chromophore. (2) the unreduced q u i n o i d a l oxygen at p o s i t i o n 4 and (3) the lactone r i n g s of the peptide s i d e chains. Removal of the amino group on p o s i t i o n 3 or s u b s t i t u t i o n of the hydrogens of the amino group w i t h a l k y l groups abo l i s h e s the a c t i v i t y completely. Reduction of the q u i n o i d a l r i n g system reduces the bi n d i n g of actinomycin w i t h DNA, r e s u l t i n g i n low a c t i v i t y (18). S i m i l a r l y , h y d r o l y s i s of one or both of the lactone r i n g s or removal of the peptide side chains reduces the a c t i v i t y of the a n t i b i o t i c (19) markedly. The s t r u c t u r a l features of the DNA molecule necessary f o r the e f f e c t i v e b i n d i n g of actinomycin D are the guanine base and the h e l i c a l s t r u c t u r e of DNA. The maximal amount of actinomycin bound to DNA p a r a l l e l s the guanine content of the DNA; though i t i s not d i r e c t l y p r o p o r t i o n a l t o i t . A p u r i n i c DNA., s y n t h e t i c deoxyadenylate thymidylate (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 (dlrdC) do not i n t e r a c t w i t h actinomycin 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 b i n d actinomycin. The requirement of h e l i c a l c o n f i g u r a t i o n of DNA f o r maximal b i n d i n g i s suggested by the observation t h a t heat-denatured DNA binds actinomycin t o a l e s s e r extent than h e l i c a l DNA or DNA of the same base composition (20) . The b i n d i n g of actinomycin D to DNA i n t e r f e r e s w i t h the enzymic a c t i v i t y of DNA dependent RNA polymerase. The observa-t i o n s of Hartmann_ e t ' a l . (21) and by Goldberg et a l . (13) 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 by actinomycin D cannot be i n f l u e n c e d by changing concentrations of the enzyme, c o f a c t o r s or 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 concentrations of DNA, supports 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 actinomycin d e r i -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 DNA. Substances such as dA:T, p o l y r i b o n u c l e o t i d e s e t c . which do not bin d 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 templates i n RNA s y n t h e s i s by RNA polymerase i n presence of actinomycin D. The b i n d i n g of actinomycin at r e l a t i v e l y high concentrations t o DNA was shown to a l t e r the p h y s i c a l p r o p e r t i e s of DNA t o some extent. Kresten e t a i . (22) and Reich (23) have shown th a t b i n d i n g of actinomycin increased the temperature (Tm) at which the secondary s t r u c t u r e of DNA melts out. This e f f e c t l i k e the others i s a l s o dependent on the extent of b i n d i n g . The s t a b i l i t y of complexes to 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 greater than the a c i d s t a b i l i t y of DNA alone. In both cases the d i s s o c i a t i o n of the complex i s accompanied by denat u r a t i o n and strand s e p a r a t i o n of the DNA. The v i s c o s i t y of DNA i s a l s o increased when i t i s bound to actinomycin D (12). Reich (23) has concluded from h i s e x p e r i -ments, t h a t the forces b i n d i n g actinomycin D to DNA are stronger than those m a i n t a i n i n g the DNA s t r u c t u r e i t s e l f . The concentrations of actinomycin which produce the 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 to i n h i b i t the r e p l i c a t i o n of DNA by DNA polymerase, a process i n which s e p a r a t i o n of template strand occurs. -Lower concentrations of actinomycin D which i n h i b i t s RNA synt h e s i s do not a f f e c t the DNA r e p l i c a -t i o n . In a d d i t i o n to.the i n h i b i t i o n of both RNA and DNA pol y -merase r e a c t i o n s (both r e q u i r e DNA templates) actinomycin i s reported to i n h i b i t m e t h y l a t i o n of DNA. Actinomycin i s a l s o reported to i n h i b i t nucleases (18). Based on a l l these observa-t i o n s Reich has proposed t he f o l l o w i n g hypothesis. "The surface of the 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 of unequal s i z e , and the a n t i p a r a l l e l p o l a r i t y of the complementary strands of DNA causes each f u n c t i o n a l group of the base p a i r s to 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 to the two grooves. 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 polymerases, methy-l a t i n g and g l u o o s y l a t i n g enzymes, and nucleases) may act on or a t t a c h to v a r i o u s combinations of f u n c t i o n a l groups on the surface of the h e l i x . I f t h i s were so, the d i f f e r e n t i a l s e n s i t i v i t y of the process to a n t i b i o t i c a c t i o n could depend on the .'.steric r e l a t i o n s h i p between the s i t e s which bind a c t i n o -mycin and enzyme r e s p e c t i v e l y " ( 1 8 ) . A c c o r d i n g l y he has suggested t h a t actinomycins i n h i b i t RNA polymerase by a s t e r i c b l o ck on the surface on which the enzyme a c t s , whereas the DNA 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 to the i n h i b i -t i o n of strand s e p a r a t i o n by the a n t i b i o t i c . Based on X-ray and model b u i l d i n g s t u d i e s Hamilton e t a l . (24) have formulated.a model f o r the DNA:actinomycin D complex. In t h i s model the actinomycin occupies the minor groove of DNA h e l i x where i t i s bound to the DNA by seven hydrogen bonds, four of which are c o n t r i b u t e d by the peptide chains of a c t i n o -mycin and the r e s t by actinomycin chromophore. The geometric d i s t r i b u t i o n of the l a t t e r three hydrogen bonds were st u d i e d by Hamilton e t a_l_. i n d e t a i l and were found to be s t e r i o c h e m i -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 Figure 2. Most of the known f a c t s regarding the r e a c t i o n of actinomycin w i t h DNA and the a s s o c i a t e d i n h i b i t i o n of DNA dependent RNA polymerase could | be deduced from t h i s molecular model. Thus the model accounts f o r the r o l e of the s t r u c t u r e of actinomycin known to 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 . The r e d u c t i o n - .9 -F i g u r e 2. Proposed model f o r the bi n d i n g of actinomycin D to deoxyguanosine of DNA. Hydrogen bonds between actinomycin 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 - ' ; '~ •' of the quirnidal oxygen, w i l l destroy the a b i l i t y of oxygen t o f u n c t i o n as a hydrogen acceptor. Changes i n the amino group of the 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 to form hydrogen bonds w i t h the DNA c o n s t i t u e n t s . . The peptide s i d e chains can be v i s u a l i z e d as being s t a b i l i z e d by the lactone r i n g system i n a conformation p e r m i t t i n g the formation o f . f o u r a d d i t i o n a l H-bonds-'between the peptide N.H-groups, and the oxygen of the phosphodiester group of DNA s t r a n d , opposite to t h a t c o n t a i n i n g the guanine i n t e r a c t i n g w i t h the chromophore. I t could be seen t h a t there i s no involvement of p o s i t i o n 7 i n the complex formation as i t p r o j e c t s away from the DNA h e l i x i n the model. Goldberg et a l . (25) . have reported t h a t the presence of bulky s u b s t i t u e n t groups at 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 the b i n d i n g of actinomycin to DNA which i s i n agreement w i t h the model. That the s u b s t i t u t i o n at p o s i t i o n 7 d i d not a f f e c t the b i o l o g i c a l a c t i v i t y of actinomycin has been shown e a r l i e r by M u l l e r (26). The complex formation a l s o depends on the s t r u c t u r e present i n DNA and a l s o i t s c o n f i g u r a t i o n . ' This f a c t i s a l s o amply accounted f o r by the proposed model. Thus only guanine can provide the hydrogen i n the minor groove of the DNA f o r which the quino>idal oxygencan serve as an acceptor. The d i s p o s i t i o n of the v a r i o u s p a r t i c i p a t i n g groups i n the B-conforraaticn of the DNA h e l i x seems to be very important f o r the b i n d i n g , as actinomycin binds only p o o r l y w i t h s i n g l e stranded DNA, sRNA, and Reo v i r u s RNA, which are supposed to e x i s t i n the A-conforma-t i o n . That.the complex formation was r e s t r i c t e d to the minor - 11 -groove of the DNA has been f u r t h e r v e r i f i e d by Reich and co-workers (27). The f i n d i n g t h a t the s u b s t i t u t i o n pf groups i n the minor groove and not i n the major groove a b o l i s h e d the complex formation f u r t h e r confirmed the p o s i t i o n of actinomycin D i n the minor groove of DNA i n the complex. Recently the va r i o u s r e a c t i o n s by which RNA r e p l i c a t i o n .brought about by RNA polymerase have been worked out be Anthony et a l . (28) and a l s o by Ishihama and Kameyama (29). The e f f e c t of actinomycin D on the enzymatic r e a c t i o n catalysed.by DNA dependent RNA polymerase has been s t u d i e d by Richardson (30). Reich 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 of the b i n d i n g s i t e s of the enzyme may be blocked by actinomycin, when i t binds to the template DNA. Richardson has conducted s t u d i e s on the b i n d i n g of the enzyme i n the presence of a c t i n o -mycin and has concluded t h a t actinomycin at concentrations which -i n h i b i t RNA polymerase d i d not reduce the number of b i n d i n g s i t e s f o r the enzyme on the 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 concluded from h i s st u d i e s t h a t actinomycin D i n h i b i t s the p o l y m e r i s a t i o n of n u c l e o t i d e more than the i n i t i a t i o n of the c h a i n . That actinomycin 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 template v/as a l s o reported r e c e n t l y by Sen.tenac e t a l . (31) . Though the e f f e c t of actinomycin D i s p r i m a r i l y on the i n -h i b i t i o n of DNA dependent RNA polymerase and the consequent i n -h i b i t i o n of p r o t e i n s y n t h e s i s and other r e l a t e d c e l l u l a r a c t i v i -t i e s , many other metabolic processes have been shown to be affected.by actinomycin D. L a s z l o et a l . (32) observed that actinomycin 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 leucemic l e u c o c y t e s . The i n h i b i t i o n of bioluminescence by actinomycin D i n c e r t a i n marine f l a g e l l a t e s - notably the formation of 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 reported by McCalla and A l l e n (33) . The i n h i b i t o r y e f f e c t of actinomycin 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 to the synt h e s i s of mRNA has been observed by Horning and Rabinowitz (34). A d i f f e r e n t i a l e f f e c t of actinomycin D on the formation of enzymes i n Bacillus licheniformis and Bacillus cereus was observed by P o l l a c k (35) . Actinomycin D supressed the induc-t i o n of g-glucosidase 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 the i n d u c t i o n of only B-glucosidase and not p e n i c i l l i n a s e i n Bacillus licheniformis. S i m i l a r i n h i b i -t i o n of actinomycin D on the formation of a-amylase, proteases and a l k a l i n e phosphatase, but not ribon u c l e a s e i n Bacillus subtilis i s reported by Kadowaki and coworkers (36). Yoshida e t a l . (37) observed an i n h i b i t o r y a c t i o n of actinomycin on the organism producing the a n t i b i o t i c . That the growth i n h i b i -t i n g e f f e c t of actinomycin D on Bacillus subtilis can be abol i s h e d by i n t e r a c t i o n of the drug w i t h t h y r o x i n has been observed by Kim e t a l . (38). B i o s y n t h e s i s of Purine N u c l e o t i d e s . Previous observations revealed t h a t the i n c o r p o r a t i o n of 1 Re-formate i n t o n u c l e i c acids i n E h r l i c h a s c i t e s carcinoma c e l l s was i n h i b i t e d by actinomycin D (39). The complex and v a r i e d steps i n the b i o s y n t h e s i s of purine n u c l e o t i d e s and the involvement of formate i n t h i s process were e l u c i d a t e d by the - 13 -i n v e s t i g a t i o n s of Buchanan and by Greenberg and a s s o c i a t e s i n the l a t e r 1950's (40). They have defined the b i o s y n t h e t i c sequence i n a stepwise manner so t h a t the o r i g i n of each atom of the purine 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 step i n the formation of phosphori-bosylamine from 5 phosphoribosylpyrophosphate (PRPP) and glutamine and culminates i n the formation of 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 conversion of i n o s i n i c a c i d to ad e n y l i c and g u a n y l i c a c i d have been shown to be through the p a r t i c i p a t i o n of aspar-t i c a c i d , and glutamic a c i d r e s p e c t i v e l y (41). The formation of AMP from IMP u t i l i z e s the.high energy bond of GTP, to form a d e n y l o s u c c i n i c a c i d which i s subsequently broken down to ad e n y l i c a c i d and fumaric a c i d . The conversion of IMP to GMP in v o l v e s the o x i d a t i o n of i n o s i n i c a c i d f i r s t to XMP foll o w e d by amination u s i n g the amide group of glutamine (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 to the de novo s y n t h e s i s o u t l i n e d , other mechanisms of n u c l e o t i d e formation have been demonstrated. Thus the preformed purines can be converted to nu c l e o t i d e s u t i l i z i n g the v e r s a t i l e substance PRPP and appropriate nucleo-t i d e pyrophosphorylase (43). Base + PRPP ===== n u c l e o t i d e + P-P Another minor route may i n v o l v e the enzyme nucleoside phosphory l a s e discovered by Kalc k a r (44). " Adenine + Ribose-l-PCH • •• • Adenosine + P i - 14 -CO; ^ Aspartate—NT J Cs C X I i l l C|»—"Formate" «"->Ci • c( i • Glycine \ lGlutAmine amide N . "Format*  r • m..i A T P ~ ^ , Ribose-S'-P A M P « ^ > r R P P (5'-phoaphoribosyl-r-pyrophosphate) Qlutamine ~Y Glycine + A T P A D P + P £ GluUmate + P?<r -^X_»H :N—R -5 ' -P ° * NH, (Pbosphori-boeylamine) ^IH—R-5 ' -P Glycinamide ribotide V NH Glutamate / \ + A D P + P CH, CHO *• •'HN=i Glutamine + A T P •R-o ' -P Formyljdycinamidine ribotide FH, NH CH, ^CHO o=c ^ I H — R - 5 ' - P Formylfflycinamide ribotide A T P ^ A D P + P COOH CO, H -i ^  ' I! CH COOH CH, ' N — R - 5 ' - P Aminoimidajole ribotide NH,—6 • ^ N — R - 5 ' - P Aminoimidaiole-carboxylic acid ribotide O i 1 Aspartate A D P , 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 Aminoimidaiole succino-carboxarrude ribotide Fumarate O C N^ . NH V \ CH C Inoainic ar id (Incl ine monopha*-phat*. IMP, hyi>oTar,-thine nucleotide) H,0 CH ^ x v 0 n HC V H O / X / V F " < NH, C X. v l| ' CH J N-R-o'-P H FHcN»-C==0 NH, Aminoimidt izolecar-boxamidc ribotide F o r i nam idoimidaznlf?-carboxamidu ribotide Figure 3. B i o s y n t h e s i s of I n o s i n i c A c i d . - 15 -Adenosine .kinase c a t a l y s e s t h i s r e a c t i o n . Adenosine + ATP v ^ AMP + ADP e t c . Besides the involvement of formate i n the b i o s y n t h e s i s of purine 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 -butes the methyl group of TMP., The formation of thymine n u c l e o t i d e s de novo by the d i r e c t methylation of dUMP by c e l l f r e e e x t r a c t s of E. ooli were f i r s t shown by F r i e d k i n and Kornberg (45) . I t i s now w e l l e s t a b l i s h e d t h a t the synt h e s i s of thymine n u c l e o t i d e de novo occurs p r i n c i p a l l y through the methylation of dUMP and that t e t r a h y d r o f o l a t e and formate are "substrates. The mechanism of the 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 greater d e t a i l by Reyes and Heidelberger (46) using 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 techniques. B i o s y n t h e s i s of N u c l e i c Acids The mechanism by which DNA i s synthesized i n the 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 using an enzyme i s o l a t e d from E. ooli (47). The enzyme, known as DNA polymerase, has s i n c e been i s o l a t e d from d i f f e r e n t sources i n c l u d i n g mamma-l i a n c e l l s (48). The r e a c t i o n brought about by the enzyme i s a p o l y m e r i z a t i o n of the four deoxyribonucleoside triphosphates i n presence of a primer DNA molecule to form a new polymerized product. The primer DNA can be from animal, b a c t e r i a l , v i r a l or p l a n t sources and can be e i t h e r n a t i v e or denatured. A l l the four deoxyribonucleoside triphosphates are req u i r e d f o r the r e a c t i o n , omission of one or more would reduce the a c t i v i t y of . the enzyme considerably. The primer DNA'was shown merely to act - 16 -as a template and the polymerase was shown to copy the nucleo-s i d e sequence of the primer to form the product (49). Recent i n v e s t i g a t i o n on the mechanism of r e p l i c a t i o n of DNA by Oish'i (50) suggest t h a t the i n i t i a l product of b a c t e r i a l DNA sy n t h e s i s i s a s i n g l e stranded fragment of a DNA molecule. He has a l s o i s o l a t e d a second intermediate from Bacillus subtilis which i s a double stranded DNA wi t h a p a r t of i t having s i n g l e . s t r a n d e d s t r u c t u r e . This he claims as the second stage of DNA r e p l i c a t i o n . S i m i l a r discontinuous chain growth f o r DNA r e p l i c a t i o n has been suggested by Okazaki e t a l . (51). The f i n a l product may be obtained by the a c t i o n of the poly-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) . Studies on the b i o s y n t h e s i s of RNA both in vivo and in vitro have shown tha t RNA Is synthesized from' the four r i b o -nucleoside diphosphates by a DNA-dependent enzyme system d i s -covered by Weiss and Glastone (53). These enzymes, the DNA dependent RNA polymerases have s i n c e been obtained from b a c t e r i a (54) p l a n t s (55) and mammalian sources (56). A d i -v a l e n t Mg + + or Mn + + i o n and a l l the four r i b o n u c l e o s i d e t r i -phosphates are r e q u i r e d f o r the r e a c t i o n . The a c t i v i t y of the enzyme i s dependent on the presence of DNA which acts as the template f o r RNA sy n t h e s i s . The base composition and the nearest neighbour frequencies of the nuc l e o t i d e s of the RNA formed were shown to be complementary to the DNA primer (57). Though Hurwitz et. a l . (58) have shown t h a t both strands of the primer DNA are copied by the enzyme in vitro, t h i s does ' -17—;' not seem to be the case in vivo. Hayashi and Spiegelman (59)' have shown t h a t only one of the strands of the priming DNA i s copied in vivo, by the RNA polymerase. Using h i g h l y p u r i f i e d E. ooli 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 only one strand i s copied in vitro (60) and t h i s i s a l s o the. same strand u t i l i z e d in vivo. Recently Anthony et a l . (28) and Ishihama and Kameyama (29) have reported evidence to show t h a t RNA sy 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. They are: a) A s s o c i a t i o n : DNA + Enzyme . ' DNA-Enzyme b) I n i t i a t i o n : DNA Enzyme + purine n u c l e o t i d e ======= [BNA-Enzyme purine n u c l e o t i d e ] c) P o l y m e r i s a t i o n ! [DNA-Enzyme purine n u c l e o t i d e ] + NTP. • DNA-Enzyme o l i g o r i b o -n u c l e o t i d e s . They showed t h a t the f i r s t step of the process can be i n h i b i t e d i n s o l u t i o n by high i o n i c s t r e n g t h , w h i l e the second step, v i z . formation of the DNA-enzyme-purine n u c l e o t i d e complex, i s unaffected. 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 at low con-c e n t r a t i o n s of nucleoside t r i p h o s p h a t e . This l i m i t a t i o n can be over-come by r e l a t i v e l y high c o n c e n t r a t i o n of purine n u c l e o t i d e s . Besides DNA-dependent RNA polymerase there are other enzyme systems i s o l a t e d which are capable of pol y m e r i z i n g r i b o n u c l e o t i d e s . The enzyme, p o l y n u c l e o t i d e phosphorylase, discovered by Ochoa and coworkers, u t i l i z e s r i b o n u c l e o s i d e diphosphates as substrates 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). The existe n c e of an enzyme which u t i l i z e s RNA as a template - RNA dependent RNA - 18 -p o l y m e r a s e ( r e p l i c a s e ) - h a s b e e n d e m o n s t r a t e d by many w o r k e r s ( 6 2 ) . The enzyme i s v e r y s i m i l a r t o t h e p r e v i o u s l y d e s c r i b e d p o l y m e r a s e , e x c e p t t h a t t h e t e m p l a t e f u n c t i o n i n t h i s c a s e i s s e r v e d by RNA i n s t e a d o f DNA. I n c o n t r a s t t o i t s e f f e c t s on t h e DNA-dependent RNA p o l y -m e r a s e , a c t i n o m y c i n D has no i n h i b i t o r y e f f e c t on e i t h e r 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 (10) o r on t h e RNA-dependent RNA p o l y m e r a s e r e a c t i o n s (14). T h i s l e n d s s u p p o r t t o t h e v i e w t h a t 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 a n d t h e f o r m a t i o n o f t h e c omplex i s t h e p r i m a r y mode o f a c t i o n o f t h e a n t i b i o t i c . B e c a u s e o f t h e s p e c i f i c i t y o f i n h i b i t i o n o f t h e DNA-dependent RNA p o l y m e r a s e , a c t i n o m y c i n D h a s p r o v e d t o be a v a l u a b l e t o o l f o r d e m o n s t r a t i n g t h e e x i s t e n c e o f D N A - d i r e c t e d RNA s y n t h e s i s i n b i o - l o g i c a l s y s t e m s . DNA-dependent RNA p o l y m e r a s e a p p e a r s t o be t h e enzyme m o s t l y c o n c e r n e d w i t h c e l l u l a r s y n t h e s i s o f RNA. P r e s e n t I n v e s t i g a t i o n s C o n s i d e r a b l e i n f o r m a t i o n i s a v a i l a b l e a b o u t t h e mode o f a c t i o n o f a c t i n o m y c i n D, and t h e mechanism by w h i c h t h e b i o s y n t h e s i s o f m a c r o m o l e c u l e s i s i n h i b i t e d b y t h i s a n t i -b i o t i c . The s p e c i f i c i n h i b i t i o n o f DNA-dependent RNA p o l y -m e r ase by a c t i n o m y c i n D has b e e n a n i m p o r t a n t p a r t o f t h e e x p e r i m e n t a l b a s i s f o r t h e d e v e l o p m e n t o f modern t h e o r i e s a b o u t p r o t e i n s y n t h e s i s , hormone a c t i o n , a c t i v e t r a n s p o r t , and o t h e r c e l l u l a r p r o c e s s e s a t a m o l e c u l a r l e v e l . Though i t i s w e l l known t h a t a c t i n o m y c i n D i n h i b i t s n u c l e i c a c i d s y n t h e s i s , v e r y l i t t l e i s known a b o u t t h e 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 s y n t h e s i s and u t i l i z a t i o n o f s m a l l p r e c u r s o r molecules of n u c l e i c a c i d s . I f the prime e f f e c t . o f a c t i n o -mycin D i s only 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 ri b o n u c l e o -t i d e s , and not to a f f e c t the de novo s y n t h e s i s of these sub-stances, then an in c r e a s e i n the c e l l u l a r c o n c e n t r a t i o n of nuc l e o t i d e s could be expected when c e l l s are t r e a t e d w i t h actinomycin D. Such an e f f e c t has been demonstrated i n the case of Bacillus subtilis w i t h actinomycin D (1) and chromo-mycin A3 (63). Wheeler and Bennett (64) have reported t h a t actinomycin 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 of purine 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 . In con-t r a s t ^ s t i m u l a t o r y e f f e c t of actinomycin on the formation of 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 reported by Harbers and M u l l e r (65). Lowy and Willia m s (66) observed t h a t the actinomycin does not i n h i b i t e i t h e r the de novo s y n t h e s i s of purine n u c l e o t i d e s or the i n t e r c o n v e r s i o n of purine 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 reported t h a t there i s an accumulation of 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 of Bacillus subtilis, the growth of which i s i n h i b i t e d by actinomycin D. In a d d i t i o n , o t h e r metabolic e f f e c t s have been reported f o r actinomycin D. Thus,Horning and Rabinowitz (34) have shown that actinomycin D i n h i b i t s p r o t e i n s y n t h e s i s u n r e l a t e d to the synt h e s i s of mRNA and, t h i s i n h i b i t i o n could be reversed by glucose. I t has a l s o been reported t h a t actinomycin D causes i n h i b i t i o n of r e s p i r a t i o n and g l y c o l y s i s i n leukemic leucocytes (32) . The present i n v e s t i g a t i o n s were undertaken to 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 to see whether there i s any e f f e c t f o r actinomycin D on the de novo s y n t h e s i s of nuc l e o t i d e s i n mammalian systems. I t was a l s o of i n t e r e s t t o see whether there was any accumulation of nu c l e o t i d e s i n mam-malian c e l l s by actinomycin D as i n Bacillus subtilis. E h r l i c h a s c i t e s carcinoma c e l l s were used f o r the study reported i n the t h e s i s because of 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 high 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 reproduced w i t h homogeneity and are e a s i l y propagated. Moreover uniform suspensions of these mammalian c e l l s c o uld be e a s i l y prepared. 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 to the study of the e f f e c t of actinomycin D on the de novo s y n t h e s i s of nucleo-t i d e s of the a c i d s o l u b l e p o o l , i t was necessary to separate and estimate these substances. Most of 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 of nu c l e o t i d e s depend on column chromatography i n which the substances 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 of 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 of even s m a l l q u a n t i t i e s of pro-ducts. In the f i n a l stages of s e p a r a t i o n , one has to r e s o r t to paper chromatography. As the i n i t i a l experiments were c a r r i e d out w i t h small q u a n t i t i e s of c e l l suspensions, separa-t i o n of 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-tographic methods were developed f o r the s e p a r a t i o n of small q u a n t i t i e s of nu 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 . P r i o r to chromatography the n u c l e o t i d e s were adsorbed on charcoal - 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 mixture. This treatment gave b e t t e r r e s o l u t i o n of the mixture on paper chromatograms. E h r l i c h a s c i t e s carcinoma c e l l suspensions were incubated in vitro w i t h 1 I fC-formate and actinomycin D. 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 separated by paper chromato-graphy. The co n c e n t r a t i o n of purine n u c l e o t i d e s and the amount of 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 n u c l e o t i d e s were determined. The r e s u l t s of these experiments were i n c o n c l u s i v e due to v a r i a t i o n s , though there was a trend towards an increase of 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 higher concentrations of actinomycin D. Therefore s i m i l a r experiments using l a r g e r volumes of c e l l suspension were c a r r i e d out i n the presence of actinomycin 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 experiments were separated by chromatography on DEAE c e l l u l o s e columns. The r e s u l t s from these experiments proved t h a t there was an accumulation 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 carcinoma c e l l s incubated w i t h l l fC-formate and actinomycin D. The increase was the highest i n the case of adenine n u c l e o t i d e s . In the case of Bacillus subtilis the major increase was seen i n guanine n u c l e o t i d e s (1). F u r t h e r , i t was seen th a t the concentrations of the nucleoside triphosphates are increased many times over the c o n t r o l s though a corresponding increase i n the monophosphates was not seen. These r e s u l t s are discussed i n the l i g h t of other observations. The e f f e c t of actinomycin D on the i n c o r p o r a t i o n of lhC-- formate i n t o both RNA and DNA was a l s o examined. In agreement • -22- • • w i t h other reported r e s u l t s (39) i t was observed t h a t the i n -c o r p o r a t i o n of t h i s precursor i n t o both the n u c l e i c a c i d s was i n h i b i t e d by actinomycin. However, the i n c o r p o r a t i o n of 1 Re-format e i n t o RNA was i n h i b i t e d t o a greater extent than i n t o DNA. S i m i l a r e f f e c t s were observed i n s t u d i e s w i t h r a t i n t e s t i n a l mucosa. F u r t h e r , the e f f e c t of actinomycin D on other c e l l u l a r ' processes, 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 the c l a s s i c a l procedure of Warburg manometry. G l y c o l y s i s was followed by the disappearance of added glucose i n the presence' and absence of actinomycin D i n suspensions of E h r l i c h a s c i t e s carcinoma c e l l s . From these e x p e r i -ments i t was seen t h a t there was a s l i g h t i n h i b i t i o n • o f r e s p i r a t i o n i n presence of actinomycin D. However, there was no c o r r e l a t i o n between the extent of i n h i b i t i o n of 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 . In the case of g l y c o l y s i s , there was no e f f e c t exerted by the a n t i b i o t i c on the glucose u t i l i z a -t i o n of the c e l l s . This i s i n c o n t r a s t to some of the observations (32) and i n agreement w i t h some others (68). I t has been reproted by many i n v e s t i g a t o r s (69, 70, 71, 72) th a t i n E h r l i c h carcinoma c e l l s the 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 precursors 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 purines was markedly s t i m u l a t e d by the a d d i t i o n of glucose to the i n -cubation medium. Harrington (69) suggested t h a t t h i s s t i m u l a -t o r y e f f e c t was due to the conversion of glucose to phospho-ribosylpyrophosphate (PRPP) which i s one of the s t a r t i n g m a t e r i a l s of purine n u c l e o t i d e b i o s y n t h e s i s . This view has been questioned - 23 -by Henderson and LePage (70) , who suggested t h a t the observed 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 et a l . (71) are of the view t h a t glucose was converted to the ribose-5-phosphate r e q u i r e d f o r purine b i o s y n t h e s i s . Herscovics and Johnston (72) observed an increase i n glutamine i n c e l l s incubated w i t h glucose and hence suggested t h a t the s t i m u l a t o r y e f f e c t of glucose was due to the increased a v a i l -a b i l i t y of glutamine f o r the b i o s y n t h e s i s of p u r i n e s . In an attempt to i n c r e a s e the i n c o r p o r a t i o n of 1 "^C-formate and 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 of E h r l i c h a s c i t e s carcinoma c e l l s , incubated in vitro, glucose was added to the medium. The purines of v a r i o u s f r a c t i o n s were i s o l a t e d and the amount of 1^C-formate i n c o r p o r a t e d was determined. Varying r e s u l t s were obtained i n these experiments. In many i n s t a n c e s , con-t r a r y to the reported f i n d i n g s , a decrease i n the i n c o r p o r a t i o n of 1'*C-formate i n t o the n u c l e i c a c i d purines was observed. This problem was examined i n greater d e t a i l and s e v e r a l i n t e r -e s t i n g r e s u l t s were obtained. I t was 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 1'*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 increased w h i l e i n c o r p o r a t i o n i n t o n u c l e i c acids was decreased. However, i n cases where there was a decrease 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 decrease i n the i n c o r p o r a t i o n of I l fC-formate i n the a c i d s o l u b l e purine was a l s o n o t i c e d . On f u r t h e r i n v e s t i g a t i o n , much of the r a d i o a c t i v i t y was found to be concentrated i n 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 , making the over a l l a c t i v i t y of the f r a c t i o n higher than the c o n t r o l s experiments, though the -incorporation of r a d i o a c t i v i t y i n t o the a c i d s o l u b l e purines was lower. The e f f e c t of glucose e i t h e r to increase or to -decrease the i n c o r p o r a t i o n of 1^C-formate i n t o the n u c l e i c a c i d purines was found t o depend on the con c e n t r a t i o n of the c e l l suspension. In d i l u t e c e l l suspensions the 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 observed when 1 "*C-g l y c i n e was used as the n u c l e i c a c i d precursor. The reason f o r t h i s decreased 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 was suspec-ted to be due t o the delayed r e s y n t h e s i s of the much needed energy source ATP, which becomes te m p o r a r i l y depleted on the a d d i t i o n of glucose through the hexokinase r e a c t i o n . This i n f e r e n c e was strengthened by the observation t h a t 2-deoxy-glucose, an agent capable of 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 of i n c o r p o r a t i o n of 1 !*C-formate even i n dense suspensions of E h r l i c h a s c i t e s carcinoma c e l l s i n which case glucose always gave a s t i m u l a t i o n r a t h e r than an i n h i b i -t i o n of i n c o r p o r a t i o n . 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 of in c o r p o r a -t i o n of l a b e l . These r e s u l t s and the other p o s s i b l e reasons f o r the decreased i n c o r p o r a t i o n of l l*C-formate i n presence of glucose are discussed. - 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 carcinoma c e l l s used i n the study reported In t h i s t h e s i s were grown i n the p e r i t o n e a l c a v i t y of Swiss mice of the Ha/ICR s t r a i n (A.R. Schmidt & Co.). The tumour c e i l l i n e s were maintained by Dr. A.R.P. Eaterson. of 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 of tumour c e l l s was k i n d l y c a r r i e d out at tfie above i n s t i t u t e and the animals were shipped immediately t o the Department of' Biochemistry, U.B.C. The animals were maintained on Pur i n a Chow and water ad libitum, u n t i l they were s a c r i f i c e d . Seven to 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 of the tumour c e l l s , the animals 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 . For the c o l l e c t i o n of c e l l s , 1 ml of 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 the p e r i t o n e a l c a v i t y , the s k i n of the abdominal region was ^ removed, the abdomen cut open by s c i s s o r s and the p e r i t o n e a l f l u i d c o n t a i n i n g tumour c e l l s was drained i n t o a 40 ml graduated c e n t r i f u g e tube, -kept cooled i n i c e . The c e n t r i f u g e tubes als-o contained about 30 I . Units of heparin ( N u t r i t i o n a l B i o c h e m i c a l Co.) to prevent c o a g u l a t i o n of the a s c i t e s f l u i d . I f the a s c i t i c f l u i d was badly contaminated wi t h red c e l l s , the tumour; c e l l s were not used f o r the i n v e s t i g a t i o n . The tumour c e l l s were separated from the 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) at 2500 rpm f o r 5 minutes. The separated c e l l s were washed w i t h 3 volumes of c o l d p h y s i o l o g i c a l s a l i n e and c e n t r i f u g e d again. The c e l l volume was noted and the super-natant f l u i d removed. The washed c e l l s were then suspended i n 9 volumes of b u f f e r s o l u t i o n to give a 1:10 suspension. 2) Determination of Packed C e l l Volume. Theppacked c e l l volume of the suspension was determined when r e q u i r e d by c e n t r i f u g i n g the E h r l i c h carcinoma c e l l sus-pension i n Wintrobe hematocrite tubes, 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 at maximum permissable speed f o r 30 minutes (73). 3) Radioactive M a t e r i a l s . R a d i o a c t i v e l l fC-sodium formate was purchased from the Radiochemical Centre, Amersham, England. The s a l t was d i s s o l -ved i n water made a l k a l i n e (pH 8) by the a d d i t i o n of sodium carbonate. The s o l u t i o n thus prepared contained 200 micro--c u r i e s per ml. 2- 1^C-glycine was a product of Merck Sharp and Dohme of Canada L t d . This was d i s s o l v e d i n s u f f i c i e n t volume of water to give a s o l u t i o n c o n t a i n i n g 200 micro c u r i e s per ml. 4) Actinomycin D. This was a g i f t to Professor S.H. Zbarsky from Merck Sharp Dohme c f Canada L t d . The product was l a b e l l e d 'Lyo' meractino-mycin and was provided i n v i a l s each c o n t a i n i n g 0.5 mg of actinomycin D. This was d i s s o l v e d i n 20% ethanol to give the d e s i r e d c o n c e n t r a t i o n of 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 at -20°. 5) Determination of Glucose^ Glucose was estimated using the G l u c o s t a t method. The G l u c o s t a t k i t was purchased from Worthington Biochemical Cor-p o r a t i o n and estimations c a r r i e d out according to the 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 of the c e l l suspensions 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 adding equal volumes of ZnSO>> (2%) and Ba(OH)2 (1.8%) s o l u t i o n s (75) . The yellow colour developed a f t e r i n c u b a t i o n f o r 30 minutes at 37° was read at 400 mu i n a G i l -f o r d spectrophotometer. 6) Determination of Phosphate. The phosphorus content of 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 to the method of F i s k e and SubbaRow (76) as modified by B a r t l e t t (77). The blue colour produced was read at 800 mu i n a Cary spectrophotometer and the content of phosphorus obtained from a standard curve prepared using s i m i l a r procedure. 7) Determination 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 estimated by dipheny amine r e a c t i o n of Dische (78). Highly polymerized c a l f thymus DNA (Mann Research Laboratories) was used as a standard i n these e s t i m a t i o n s . RNA was estimated by the o r c i n o l method of Mejbaum (79) modified by Munro et a l . (80). T h e . f i n a l concentrations 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 the modified 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 of HCl was 6N. Adenosine monophosphate was used as a standard and concentrations were expressed i n terms of umoles of a d e n y l i c a c i d (81) . 8) Radioautography. Radioactive areas on chromatograms and electrophorograms, were detected by radioautography. Chromatograms were placed between two X-ray f i l m s (Kodak M e d i c a l , No Screen) f o r a p e r i o d of 2 to 3 weeks. The exposed f i l m s were then developed, f i x e d and washed at the dark room f a c i l i t i e s a v a i l a b l e at the U n i v e r s i t y Health S e r v i c e of 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 of the var i o u s 1 ^ - l a b e l l e d compounds was determined using Packard Trir-Carb l i q u i d s c i n t i l l a t i o n spectrophotometer model 314-X. In some, l a t e r experiments, determinations were made using Nuclear Chicago l i q u i d s c i n t i -l l a t i o n counter Mark I I . Each counting v i a l contained 0.5 ml of a s o l u t i o n of the r a d i o a c t i v e m a t e r i a l , 0.5 ml of IM hya.-mine hydroxide i n methanol and 5 ml of s c i n t i l l a t i o n f l u i d of the f o l l o w i n g composition (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 Chemicals, Vancouver) was d i s s o l v e d i n the' above so l v e n t system to a con c e n t r a t i o n of 1.46% and POPOP- (Packard - 29 -Instrument Co.) t o a c o n c e n t r a t i o n of 0.06%. The hyamine hydro-x i d e was prepared from hyamine 10-X, a product of Rohm and Haas. The m a t e r i a l was p u r i f i e d (83) and converted to the hydroxide by the procedure described by Eisenberg (84). 10) General Procedure f o r Incubations in vitro of E h r l i c h  A s c i t e s Carcinoma C e l l s . 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 out 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 described by Stewart and Zbarsky (85) . Each f l a s k contained 25 u l of a s o l u t i o n of r a d i o a c t i v e formate (5 uC), 20 ymole of non r a d i o -a c t i v e formate as a c a r r i e r . This c o n c e n t r a t i o n of non r a d i o -a c t i v e formate was optimum f o r the maximum c o r p o r a t i o n of 1^C-formate by a s c i t e s carcinoma c e l l s (86). Enough c e l l suspension e i t h e r i n Krebs Ringer phosphate (87) b u f f e r or Krebs Ringer bicarbonate b u f f e r pH 7.8 was added to make the t o t a l volume 10 ml. The f l a s k s were kept cooled i n i c e during a d d i t i o n s . The gas phase was e i t h e r 0 2 or a mixture of 95% 0 2 and 5% C0 2 depending on the b u f f e r used to suspend the c e l l s . The f l a s k s were stoppered a f t e r thermal e q u i l i b r i u m f o r 3 minutes. Incu-b a t i o n was c a r r i e d out i n shaking water bath (100-110 o s c i l a -t i o n s per minute) a t 37° f o r 2 hours. For every experiment incubations i n d u p l i c a t e were c a r r i e d out. When the time course of i n c o r p o r a t i o n : .of r a d i o a c t i v e 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 out e i t h e r i n 500 ml or 1000 ml f l a s k s . Concentration of the precursor added to the - 30 -i n c u b a t i o n medium was such t h a t every 10 ml of suspension con-t a i n e d 25 u l (5 microcuries) of r a d i o a c t i v e formate. The content of non r a d i o a c t i v e formate i n the medium was 2 umole per ml. The f l a s k was c l o s e d w i t h a one holed rubber stopper through which a narrow polyethylene tube was i n s e r t e d . Oxygen or a mixture of 95% Oxygen and 5% C O 2 depending cn the buffer, used, was passed i n t o the f l a s k through the polyethylene tube. This prevented any change i n gas phase when samples were w i t h -drawn at i n t e r v a l s , by s u c t i o n . The f l a s k was incubated i n a water bath at 37°C and was shaken during the p e r i o d of incuba-t i o n at 100-110 o s c i l l a t i o n s per minute •. 11) C e l l R e s p i r a t i o n S tudies. Studies 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 conven-t i o n a l Warburg manometric techniques (87) using approximately 20 mg of c e l l s i n a t o t a l volume of 3 ml of Krebs Ringer phosphate b u f f e r pK 7.8. 12) 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 c e l l sus-pension a f t e r i n c u b a t i o n was t r a n s f e r r e d i n t o a c e n t r i f u g e 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 at 15,000 x g f o r 20 minu-tes i n a S o r v a l l RC I I B c e n t r i f u g e . The supernatant w a s drained Off, the t i s s u e p e l l e t was washed w i t h c o l d b u f f e r and the suspension c e n t r i f u g e d again. The washed p e l l e t w a s mixed w e l l w i t h 3 ml c f 0.2 M p e r c h l o r i c a c i d (cold) and kept i n i c e f o r 15 minutes, and then c e n t r i f u g e d f o r 10 minutes. The supernatant - 31 -s o l u t i o n was c o l l e c t e d and the p r e c i p i t a t e e x t r a c t e d once more w i t h 2 ml of c o l d p e r c h l o r i c a c i d . The e x t r a c t s were combined. < The pooled e x t r a c t was adjusted to pH 6.5-7 wi t h 10. N c o l d potassium hydroxide, s o l u t i o n and the r e s u l t i n g mixture cooled w e l l i n i c e to p r e c i p i t a t e the potassium p e r c h l o r a t e . 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 c o n t a i n i n g the nu c l e o t i d e s was separated and sto r e d a t -20° u n t i l analysed f u r t h e r . 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 carcinoma c e l l s were prepared by the method of Hecht and P o t t e r (88) w i t h some m o d i f i c a t i o n s . The p r e c i p i t a t e obtained a f t e r the e x t r a c t i o n of nu c l e o t i d e s was washed t w i c e w i t h 2 ml po r t i o n s . of 0. 2M p e r c h l o r i c a c i d and once witih 95% etha n o l , and the: washings discarded. The residue was suspended i n 1 ml of 10% NaCl and n e u t r a l i z e d by the dropwise acddition of IN NaOH i n 10% NaCl using phenol red as an i n t e r n a l i n d i c a t o r . The suspension was heated i n a b o i l i n g water )bath f o r 1 hour, c e n t r i f u g e d and the supernatant f l u i d was separated. The residue was e x t r a c t e d w i t h another 0.5 ml of .10% NaCl f o r 30 minutes at 100° and the e x t r a c t s combined. Ifhe sodium nucleate was p r e c i p i t a t e d by the a d d i t i o n of 3 volumes; of c o l d ethanol. The mixture was stored 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 . The p r e c i p i t a t e of sodium nucleates was d i s s o l v e d i n 1 ml c f 0.1 N NaOH and incubated at 37° f o r 2 2 hours. This procedure hydrolysed the RNA. From the s o l u t i o n , the unreacted DNA was p r e c i p i t a t e d by the 3 2 -a d d i t i o n of N HCl to a f i n a l c o n c e n t r a t i o n c f 0.1 N. The sus-pension 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 super-natant s o l u t i o n of RNA 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 DNA were c o l l e c t e d s e p a r a t e l y (89). 13) I s o l a t i o n and Determination of Purine and P y r i m i d i n e Bases  from N u c l e i c Acids and N u c l e o t i d e s . The 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 prepara-t i o n s obtained.as described above, were evaporated to dryness i n a vacuum d e s i c c a t o r over cone. H2SCH and sodium hydroxide. Each residue thus obtained was hydrclysed w i t h 72% p e r c h l o r i c a c i d according to the procedure of Marshak and Vogel (90). Each hydr o l y s a t e was d i l u t e d w i t h 1 ml of water, n e u t r a l i z e d w i t h ION c o l d potassium hydroxide and the p r e c i p i t a t e of potassium p e r c h l o r a t e was removed a f t e r c o o l i n g , by c e n t r i f u -g a t i o n . The supernatant s o l u t i o n c o n t a i n i n g the bases was placed i n t o a 10 ml beaker and evaporated to dryness in vacuo. The residue was taken up i n a small volume of water, and the s o l u t i o n a p p l i e d to f i l t e r paper f o r descending chromatography. The chromatograms were developed i n Wyatt's s o l v e n t system (91) c o n t a i n i n g iscpropancl-water-HCl (170:39:41 v / v ) . Separations were much f a s t e r and b e t t e r i n K i r b y ' s s o l v e n t system (92) c o n t a i n i n g methanol)" water^ and HCl (70: ice 2C v/v) , which was used i n l a t e r experiments. In order to 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 red 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 to 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 according to the procedure of LePage (93). When only the purine bases were to be obtained from the 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 out by the method of V i s c h e r and Chargaff (9 4) using 1 N HCl (at 100° f o r i hour). The s o l u t i o n was evaporated to dryness and bases separated as de s c r i b e d above. Adenine and hypoxanthine form a s i n g l e spot 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 separate them the m a t e r i a l from t h i s spot was e l u t e d and rechromatographed i n the s o l v e n t system described by Hershey et_ a l . (95) which contained iscpropanol-ammcnia-(28%) and water (85:1.3:15 v / v ) . Rechromatcgraphy of "guanine spots" 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 the 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 of 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 . Each U.V. absorbing area cn the paper was cut i n t o s e v e r a l s m a l l pieces and" the m a t e r i a l e x t r a c t e d by the method of Mezei and Zbarsky (96) w i t h 0.1 N HCl. The absorption spectrum of each c f the e l u t e d substances was determined i n a Cary model 15 spectrophotometer. Eluates from corresponding areas of the paper w i t h no U.V. absorbing m a t e r i a l served as blanks i n the e s t i m a t i o n s . The c o n c e n t r a t i o n was then deter-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) . .- 34 -15) Separation of A c i d Soluble 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 the s e p a r a t i o n c f the v a r i o u s nucleoside phosphates of the a c i d s o l u b l e f r a c t i o n , paper chromatography was employed. A new method was developed f o r the s e p a r a t i o n of the a c i d s o l u b l e n u c l e o t i d e s as described 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 adsorbing them on a c t i v a t e d c h a r c o a l and e l u t i n g from i t . Thus the n u c l e o t i d e s were fr e e d of the sub-stances i n t e r f e r i n g i n chromatography. The charcoal, adsorption and e l u t i o n was c a r r i e d out according to the procedure of Zhivkov (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 t r a n s f e r r e d i n t o a 12 ml . S o r v a l l c e n t r i f u g e tube. The pH of the s o l u t i o n was adjusted to 1-2 w i t h 5 N HC1. The s o l u t i o n was cooled i n i c e and to-the c o l d s o l u t i o n , 0.2 ml of a 10% suspension of a c t i v a t e d char-c o a l i n water was added. The tube was shaken w e l l and the adsorption allowed to proceed f o r 30 minutes. At i n t e r v a l s the tube was shaken to 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 e t t l e s to the bottom. The c h a r c o a l suspen-s i o n was then c e n t r i f u g e d at 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 the supernatant s o l u t i o n r e -moved q u i c k l y . The supernatant s o l u t i o n was checked f o r any unadsorbed m a t e r i a l by the U.V. absorption at 260 my. The sedimented cha r c o a l was washed wi t h 3 ml of c o l d d i s t i l l e d water, the suspension c e n t r i f u g e d again as before and the washings discarded. The l a s t drops of water were removed by. wiping 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 the ch a r c o a l were e l u t e d w i t h •8 ml of a mixture of p y r i d i n e and 60% ethanol (5:95 v / v ) . The s o l v e n t was added to the c h a r c o a l and mixed w e l l and the sus-pension was kept i n a water bath at 37°C f o r 3 hours. The tube was c l o s e d w i t h p a r a f i l m to prevent any l o s s of s o l v e n t . At i n t e r v a l s the tubes were shaken to 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 , the ch 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 the supernatant s o l u t i o n evaporated to dryness i n a f l a s h evaporator, keeping the bath temperature at 20°C. The residue was e x t r a c t e d from the f l a s k s w i t h 3 to 4 ml of water and the e x t r a c t t r a n s f e r r e d to a small tube. 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 residue thus obtained was d i s s o l v e d i n a small amount of water and a p p l i e d on paper f o r chromatography. b) Paper chromatography of n u c l e o t i d e s : - Two dimensional paper chromatography was used f o r the se p a r a t i o n of the a c i d • s o l u b l e nucleotides.. Rectangular sheet of Whatman No. 40 f i l t e r paper, 56 x.46 cms, was used f o r chromatography. The sample was a p p l i e d to one corner of the paper 8 cms away from e i t h e r edge of the paper. The bottom edge of the paper was cut w i t h p i n k i n g shears. 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 tanks (Shandon & Co.) employing the descending technique. The s o l v e n t system used i n the f i r s t s e p aration had the f o l l o w i n g composition: I s o b u t y r i c a c i d 586 ml Ammonia 35 ml - 36 -water 369 ml 0.000 1 M EDTA 10 ml pH of s o l u t i o n 4.3 adjusted 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 allowed to flow f o r 28-30 hours by which time the so l v e n t f r o n t reached the bottom and flowed over. The paper was then removed and d r i e d i n a i r . The n u c l e o t i d e s were l o c a t e d and marked under U.V. l i g h t . The paper was s u i t a b l y trimmed to reduce i t s s i z e and to remove the U.V. absorbing band g e n e r a l l y seen at the bottom of the paper a f t e r the f i r s t run. The. sheet of paper was turned through 90° so t h a t the n u c l e o t i d e s were now near the top edge of the paper. The bottom edge was cut w i t h a p i n k i n g shears and descending chromatography was c a r r i e d out i n the second d i r e c t i o n . The so l v e n t system employed i n the second d i r e c t i o n was of the f o l l o w i n g composition: Ammonium Acetate 1 M - 3 v o l Ethanol, 9.5% - 7.5 v o l Adjusted t o pH 7 w i t h a c e t i c a c i d . The paper chromatogram was run f o r 36 hours i n t h i s s o l v e n t a f t e r which the paper was removed and d r i e d i n a i r . The_:nucleo-t i d e s on the paper were detected and marked out under U.V. c) Ion-exchange chromatography:- When c o m p a r i t i v e l y l a r g e r q u a n t i t i e s of n u c l e o t i d e s were to be separated, as was necessary i n some l a t e r experiments, i o n exchange chromatography was employed f o r the sepa 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 of a s c i t e s carcinoma c e l l s . The procedure fo l l o w e d f o r the separa-t i o n was t h a t described by Oikawa and Smith (99). - 37 -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 from the c e l l s (about 10 gm wet weight) w i t h 1 M c o l d p e r c h l o r i c a c i d (50 ml). A f t e r the e x t r a c t i o n the s o l u t i o n was brought to pH 7 by slow a d d i t i o n of c o l d 10 N sodium hydroxide. The n e u t r a l i z e d s o l u t i o n was evaporated i n a f l a s h evaporator, keeping the bath temperature at 25°C. The residue was ex t r a c t e d f i r s t w i t h 50 ml and then w i t h 30 ml of c o l d 95% ethanol to remove the sodiuitoperchlorate. The remaining 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 . This m a t e r i a l c o n t a i n i n g the nu c l e o t i d e s was d i s s o l v e d i n 4-5 ml water, and the s o l u t i o n was a p p l i e d to a column of DEAE-cellulose, 25 x 1 cm i n the carbonate form prepared and packed according to the procedure of Tomlinson and Tener(lOO). A f t e r the s o l u t i o n had been run i n t o the column, the n u c l e o t i d e s were e l u t e d from the column by a l i n e a r l y i n -c r e a s i n g c o n c e n t r a t i o n of ammonium bicarbonate pH 8.6. The gradient was achieved by gradual and continued a d d i t i o n of ammonium bicarbonate(0.2 M) to water i n a mixing chamber (101) from which s o l u t i o n i s withdrawn to the column. The flow r a t e was regu l a t e d at 1 ml/minute and 5 ml or 10 ml f r a c t i o n s were c o l l e c t e d . The f r a c t i o n s were examined at 260 mu f o r the presence of U.V. absorbing m a t e r i a l and the f r a c t i o n s forming a s i n g l e peak were pooled. The e l u t i o n of the nu c l e o t i d e s v/as complete as the s a l t c o n c e n t r a t i o n reached 0.11 M. The m o l a r i t y of the s o l u t i o n i n the tubes was obtained by the r e f r a c t i v e index method. The water and ammonium bicarbonate from the combined - 38 -f r a c t i o n forming the "peaks" were removed i n a f l a s h evaporator and f i n a l l y i n a l y o p h i l i z e r . The n u c l e o t i d e s i n the residue were ; f u r t h e r separated 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 isobutyric-ammonia-water s o l v e n t system or i n ammonium acetate-ethanol s o l v e n t system. Authentic samples of nucleoside phosphates (Pabst Biochemicals, C a l i f o r n i a ) were used as standards. In some cases two dimensional paper chroma-tography was c a r r i e d out using both the s o l v e n t systems as desc 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 sorption s p e c t r a . - 39 -SECTION I EFFECT OF ACTINOMYCIN DON THE BIOSYNTHESIS OF PURINE NUCLEOTIDES IN EHRLICH ASCITES CARCINOMA CELLS in Vitro. EXPERIMENTAL 1) Time Course of I n c o r p o r a t i o n of 1 ^ P-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 . As a p r e l i m i n a r y step i n the study of the e f f e c t of a c t i n o -mycin D on the a c i d s o l u b l e pool of a s c i t e s carcinoma c e l l s , : the time course of i n c o r p o r a t i o n of r a d i o a c t i v e precursor i n t o 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 precursor of the purine r i n g of n u c l e o t i d e s and n u c l e i c a c i d s was used i n these s t u d i e s . The E h r l i c h a s c i t e s carcinoma c e l l suspension was prepared i n Krebs Ringer phosphate b u f f e r pH 7.8 and incu b a t i o n s were c a r r i e d out as described under methods. At i n t e r v a l s ' a 10 ml sample of the suspension was withdrawn from the i n c u b a t i o n mixture and the a c i d s o l u b l e f r a c t i o n , RNA hydro l y s a t e and DNA were 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 the n e u t r a l s o l u t i o n was determined by l i q u i d s c i n t i l l a t i o n counting a f t e r the removal of potassium p e r c h l o r a t e . The o p t i c a l d e n s i t y of the s o l u t i o n at 260 mu was determined. 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 of i n c u b a t i o n i s given i n Figure 4. 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 the RNA hydrolysate - 40 -- 41 -was estimated by the modified o r c i n o l method (80) using AMP as a standard. 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 RNA was expressed i n terms of s p e c i f i c a c t i v i t y defined as counts per minute per umole of AMP. The time course of i n c o r p o r a t i o n i s given i n Figure 5. The c o n c e n t r a t i o n of DNA was estimated by the diphenylamine r e a c t i o n and 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 determined 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 corporated i n t o DNA p l o t t e d a g a i n s t time i s given i n Figure 6. Examination of the f i g u r e s show t h a t the i n c o r p o r a t i o n of 1'*C-formate i n t o RNA and DNA of a s c i t e s carcinoma c e l l s was 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 . 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 the 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 minutes a f t e r which the l e v e l remained constant. This may be due to a steady s t a t e of syn t h e s i s of RNA and DNA being reached i n which 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 . For,a s i m i l a r study Richards e t a l . (102) have observed that, i n case of thymus c e l l suspension the i n c o r p o r a t i o n of 1'*C-formate i n t o DNA was l i n e a r f o r a p e r i o d of 6 hours. 2) E f f e c t of Actinomycin D on the I n c o r p o r a t i o n of 1' tC-Formate  i n t o Bases of Nucleotides and N u c l e i c Acids of E h r l i c h  A s c i t e s Carcinoma C e i l s . As there were c o n f l i c t i n g r e p o r t s as to the e f f e c t of actinomycin D ori the a c i d s o l u b l e pool (1, 64, 65) and only l i t t l e i s known about the e f f e c t oh syn t h e s i s 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 mammalian t i s s u e s , the present i n v e s t i g a t i o n s - 42 I • 20 40 60 80 100 120 TIME (minutes) Figure 5. I n c o r p o r a t i o n of l l tC-formate 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 in vitro. - 43 -- 44 -were undertaken, As a step towards the study,, the i n c o r p o r a t i o n of lkC-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 of a c t i n o -mycin D was determined. Tumour c e l l suspensions were made i n Krebs Ringer phosphate b u f f e r pH 7.8, and i n c u b a t i o n was c a r r i e d out as d e s c r i b e d i n 2 50 ml stoppered Erlenmeyer f l a s k s . • Actinomycin D d i s s o l v e d i n 20% ethanol was added to the f l a s k s t o give the d e s i r e d c o n c e n t r a t i o n . The c o n t r o l f l a s k s con-t a i n e d equal volumes of 20% e t h a n o l . A f t e r incubations, the 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 , and DNA were obtained from c e l l s by the procedures described under methods. These f r a c t i o n s were evaporated to.dryness i n vacuum d e s i c c a t o r s over concentrated s u l p h u r i c a c i d and sodium hydroxide. The dry DNA, RNA n u c l e o t i d e s , and a c i d s o l u b l e n u c l e o t i d e s were hydrolysed to t h e i r c o n s t i t u e n t bases' by the method of Marshak and Vogel (90). The purines and p y r i m i d i n e were separated by paper chromatography. The bases were e x t r a c t e d from paper and the c o n c e n t r a t i o n of each base and the r a d i o -a c t i v i t y i n c orporated i n t o each were determined. The r e s u l t s are given i n Table I. I t i s seen from Table I t h a t the i n c o r p o r a t i o n of l>iC-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 not I n h i b i t e d . b y the presence of i n c r e a s i n g concentrations of actinomycin D i n the incubation.medium. There was a s l i g h t i ncrease 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 both adenine and guanine. The very high count obtained i n the case Table I E f f e c t of actinomycin D on the i n c o r p o r a t i o n of 1'*C-formate i n t o the bases of the 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 in v i t r o . S p e c i f i c a c t i v i t y - counts per minute per umole. A c i d - s o l u b l e n u c l e o t i d e s RNA DNA Actxno-c i n D /ml Ade-nine Gua- • nine Cyt-osine Ura- . c i l Ade-nine Gua-nine Cyt-osine Ura-. c i l Ade-. nine Gua-nine Cyt-os ine Thy-mine 0 5250 1559 775 5420 437 113 28 79 118 122 100 1745 1 0.05 5806 1718 676 5484 447 83 ' 32 94 117 99 120 1793 >fe Ul 0.5 5845 1879 908 5502 428 88 37 67 101 78 86 1717 1 1.0 5804 1448 979 6079 347 22 7 42 125 85 92 1605 1.5 5716 1802 731 5492 3327 41 50 100 120 70 85 1680 - 46 -of u r a c i l may be due to the contamination of the samples by thymine. In Wyatt'.s s o l v e n t system, these two bases run c l o s e t o each other. Moreover the c o n c e n t r a t i o n of thymine i n the a c i d s o l u b l e f r a c t i o n i s too low i n the sample t o detect i t on paper under U.V. l i g h t . 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 thymine i s always high which i s a l s o r e f l e c t e d i n the thymine obtained from DNA. An examination of the i n c o r p o r a t i o n of ^ C - f ormate i n t o the RNA and DNA agrees w i t h many of the observations reported by other i n v e s t i g a t o r s . In the case of RNA there i s an i n h i b i -t i o n of i n c o r p o r a t i o n of 1 "*C-formate i n t o both adenine and guanine. However, 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 i s greater i n the case of guanine than adenine of RNA. Zbarsky has (39) reported p r e v i o u s l y a s i m i l a r e f f e c t of actinomycin 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 and E h r l i c h a s c i t e s carcinoma c e l l s . In the case of DNA, the highest s p e c i f i c a c t i v i t y obtained i s f o r the base thymine. I t i s known t h a t the methyl group of thymine i s formed from one-carbon sources (45). SmeHie et a l . (103) have observed a higher 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 thymine, than i n t o purines when 1 hC~formate. was used as a prec u r s o r . They have suggested t h a t the higher l a b e l l i n g of thymine compared to purines of DNA may be due to the small pool s i z e of thymine precursors. In the case of the purines the i n c o r p o r a t i o n of 1 4C-formate i s very much l e s s because of the r e l a t i v e l y l a r g e pool s i z e of n u c l e i c a c i d p r e c u r s o r s . Here a l s o there was a s l i g h t decrease i n the i n c o r p o r a t i o n of 1 !*C-f ormate i n t o guanine as observed - 47 -by Zbarsky i n h i s experiments (39). As the sub j e c t of greater i n t e r e s t was t o study the e f f e c t of actinomycin D on the formation of a c i d s o l u b l e nucleo-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 out. I t was observed p r e v i o u s l y t h a t there was no i n h i b i t i o n of i n c o r p o r a t i o n of r a d i o a c t i v e precursor i n t o the bases of a c i d s o l u b l e nucleo-tides;. However, there was a decrease i n the i n c o r p o r a t i o n of l l fC-formate i n t o the bases of RNA. I t i s known t h a t the corresponding nucleoside triphosphates are r e q u i r e d f o r the sy n t h e s i s of both RNA and DNA. In the de now pathway of sy n t h e s i s of purine r i b o n u c l e o t i d e s , 1 "*(:-formate i s known to be i n -corporated before t h e formation of the AMP and GMP. F a i l u r e of these m a t e r i a l s to form the higher phosphate may thus r e -duce 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 the n u c l e i c acids though there may be i n c o r p o r a t i o n of 1 4C-formate i n t o the mono-phosphates. E s t i m a t i o n 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 the bases of the a c i d s o l u b l e s thus may not give any i n f o r -mation regarding the formation of the tri p h o s p h a t e s which are 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 to form both DNSL and RNA. Further., i f there i s only an i n h i b i t i o n of p o l y m e r i z a t i o n of the nucleo-s i d e triphosphate and no i n h i b i t i o n of 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 co n c e n t r a t i o n of the tri p h o s p h a t e s could be expected i n the a c i d s o l u b l e p o o l . So i t was necessary to i s o -l a t e the var i o u s nucleoside phosphates of the a c i d s o l u b l e pool and t o estimate them to determine whether the observed i n h i b i -t i o n of i n c o r p o r a t i o n of 1 kC-formate i n t o RNA was due to the e f f e c t of actinomycin D only on the p o l y m e r i z a t i o n r e a c t i o n , or on the - 48 -de novo s y n t h e s i s of r i b o n u c l e o t i d e s . 3) Separation of A c i d Soluble Nucleotides by Paper Chroma- tography - Development of a Method. a) Paper chromatography of r i b o n u c l e o t i d e s : - Separation of r i b o n u c l e o t i d e s by paper chromatography i s beset w i t h s p e c i a l problems as these compounds c o n t a i n one or 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 of 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 develop a method of s e p a r a t i o n , a l a r g e v a r i e t y of so l v e n t systems were t r i e d using a u t h e n tic standard n u c l e o t i d e mixtures, but the separations were never-t h e l e s s not s a t i s f a c t o r y . Several years ago Krebs and Hems (104) employed two dimensional paper chromatography f o r the sep a r a t i o n only of adenosine and;inosine phosphates from r e a c t i o n mixtures. S i m i l a r l y B e r g k v i s t and Deutsch (105) reported the sep a r a t i o n of mono, d i , and t r i phosphates of guanosine, adeno-s i n e , i n o s i n e and u r i d i n e by two dimensional paper chromato-graphy. However t h i s method f a i l e d to separate u r i d i n e and c y t i d i n e phosphates which always migrated together i n the so l v e n t systems employed. The high c o n c e n t r a t i o n of ammonium sulphate i n the second s o l v e n t system l i m i t s the use of 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 of n u c l e o t i d e s and the measurement of r a d i o a c t i v i t y . From r e s u l t s of 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 obtained, which per-m i t t e d the combination of 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 the sepa r a t i o n pf r i b o n u c l e o t i d e s - 49 -by two dimensional paper chromatography are given on page 35. The s o l v e n t system used f o r chromatographyv.in- the f i r s t d i r e c t i o n was a m o d i f i c a t i o n of the so l v e n t system recommended f o r the r e s o l u t i o n of guanine n u c l e o t i d e s (97). The sol v e n t system used i n the 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 of the vari o u s 5 ' — r i b o n u c l e o t i d e s i n each of these s o l v e n t systems are given i n Table IT, These were determined by c a r r y i n g out descending chromatography of aut h e n t i c nucleoside phosphates (Pabst Biochemicals, Milwaukee)^ on l a r g e sheets of Whatman No. 40 f i l t e r papers. The true R^ values - the r a t i o of d i s -tance of s o l u t e m i g r a t i o n to t h a t of so l v e n t - was not estimated because the so l v e n t was allowed to flow o f f the paper f o r hours. Instead, the r a t e of m i g r a t i o n r e l a t i v e to t h a t of a standard n u c l e o t i d e , 7AMP, i s given i n Table I I . I t can be seen from the t a b l e t h a t a w e l l marked se p a r a t i o n of the common r i b o n u c l e o -t i d e s can be obtained by c a r r y i n g out two dimensional chromato-graphy using the two so l v e n t systems described. Substances which are not separated i n the f i r s t d i r e c t i o n f o r example, GMP, UMP, e t c . are separated w e l l on chromatography i n the second s o l v e n t system. Two dimensional chromatography was c a r r i e d out a f t e r applying a mixture of the r i b o n u c l e o t i d e s on to the paper and developing the chromatogram i n the f i r s t d i r e c t i o n i n isobutyric-ammonia-water system and then, i n the second d i r e c t i o n i n ammonium acetate-ethanol system. The r e l a t i v e p o s i t i o n s of the nucl e o t i d e s are shown i n Figure 7. I t can be seen from the diagram t h a t - 50 -Table I I 'Rg, values of 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 systems. Solvent I . I s o b u t y r i c - ammonia - water pH 4.3. Solvent I I . Ammonium acetate - ethanol pH 7. F (AMP) N u c l e o t i d e .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 Solvent I o r i g i n —^' (Q A T P UTpO CfcDP ^ , D p QjDP Q N A D O G M P O i M P ^CMP ^ Q AMP QjMP Figur e 7. A Schematic r e p r e s e n t a t i o n of two dimensional paper chromatogram pf r i b o -n u c l e o t i d e s . Solvent I. Isobutyric-ammonia-water (30 hours). Solvent I I . Ammonia acetate-ethanol'(36 hours). the mixture of the 12 r i b o n u c l e o t i d e s are w e l l separated i n chromatography i n the so l v e n t systems des c r i b e d . The m o b i l i -t i e s of the deoxynucleotides were not determined. I t i s l i k e l y t h a t they a l s o move w i t h the corresponding r i b o n u c l e o t i d e s . However, the co n c e n t r a t i o n of deoxynucleotides are low compared to the r i b o n u c l e o t i d e s , to 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 the present i n v e s t i g a t i o n s . b) Paper chromatography 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 carcinoma c e l l s : - To t e s t the e f f i c a c y of the method i n se 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 of E h r l i c h a s c i t e s carcinoma c e l l s . The p e r c h l o r i c a c i d e x t r a c t of n u c l e o t i d e s was n e u t r a l i z e d i n the c o l d w i t h 10 N KOH and the potassium per-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 the mix-ture f o r s e v e r a l hours. The supernatant f l u i d c o n t a i n i n g the nu c l e o t i d e s was l y o p h i l i z e d and the residue e x t r a c t e d w i t h a known volume of water.-. A volume of t h i s n u c l e o t i d e s o l u t i o n corresponding to 1 ml of packed c e l l s was a p p l i e d on to the corner of a l a r g e sheet of Whatman No. 1 paper. The chromato-gram was developed i n i s o b u t y r i c acid-ammonia-water i n the f i r s t d i r e c t i o n f o r 32 hours, and the U.V. absorbing spots were marked a f t e r d r y i n g the paper i n the 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 process. Instead of c l e a r separate spots, a l a r g e streak of U.V. absorbin m a t e r i a l was observed. No f u r t h e r r e s o l u t i o n was obtained by .chromatography i n the second d i r e c t i o n . I t was f e l t t h a t the f a i l u r e to o b t a i n 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 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 to the presence of s a l t s i n the n u c l e o t i d e mixture a p p l i e d on paper. The presence of s a l t s i n the mixture has been reported to a l t e r the 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). F u r t h e r , a p r e l i m i n a r y run of a mixture pf authentic n u c l e o t i d e s w i t h sodium c h l o r i d e added to i t gave chromatograms s i m i l a r to t h a t observed w i t h the 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 to remove the s a l t from the •nucleotide 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 chroma-tography . The d i f f e r e n t methods t e s t e d f o r the i s o l a t i o n of nucleo-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 of 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 of substances i n t e r f e r i n g i n chromatography of a c i d s o l u b l e n u c l e o t i d e s : -i ) .:. d e s a l t i n g of n u c l e o t i d e s using DEAE-cellulose: -Rushizky and Sober have used DEAE-cellulose f o r adsorption of 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). From the 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' carbonate which can be e a s i l y removed by l y o p h i l i z a t i o n . This method of adsorption on DEAE-cellulose and e l u t i o n from i t was t r i e d f o r separating n u c l e o t i d e s from s a l t s of the 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 prepared i n the carbonate form according to the procedure of Tomlinson and Tener(lOO) was packed i n t o a column 25 x 1 cm under pressure (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 -carcinoma c e l l s was d i l u t e d w i t h 4 volumes of water and the s o l u t i o n was added to the top of the column. A f t e r the s o l u -t i o n was passed i n t o the column, the column was washed w i t h water, and the e f f l u e n t was monitored f o r U.V. 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. The e f f l u e n t from the column was found to c o n t a i n a considerable 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. This 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 to the presence 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 was not s a t i s f a c t o r y f o r the present separation of s a l t s and nucleo-t i d e s . The nature of substances 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 : - Gel e x c l u s i o n chro-matography i s widely used to.separate substances d i f f e r i n g i n molecular s i z e . The separation of n u c l e o t i d e s by t h i s technique using Bio-Gel P-2 (Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a ) was reported by U z i e l and Cohn (110). This method was t r i e d 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 of the a c i d s o l u b l e e x t r a c t from the substances i n t e r f e r i n g i n chromatography. Bio-Gel P-2 (50-100 mesh) was e q u i l i b r a t e d w i t h water over-n i g h t . The swollen beads were allowed to 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 potassium p e r c h l o r a t e , was l y o p h i l i z e d . The residue was e x t r a c t e d w i t h a small q u a n t i t y of water and the s o l u t i o n added to 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 at a - 55 -flow r a t e of 2 ml per sq. cm area of cross s e c t i o n of the column per minute. 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 -l e c t o r . The f r a c t i o n s were examined at 260 my f o r the presence of any U.V. absorbing m a t e r i a l and the samples forming a s i n g l e peak combined. Only one s i n g l e l a r g e peak w i t h U.V. absorbance was observed. The pooled sample was evaporated i n a f l a s h evaporator and then e x t r a c t e d w i t h a known volume of water. An a l i q u o t sample of t h i s s o l u t i o n corresponding to 1 ml of c e l l volume was a p p l i e d on to a l a r g e sheet of Whatman No. 1 f i l t e r paper. Two dimensional chromatography was then c a r r i e d out as described before. Examination of the chromatograms under U.V. 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 , without any 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 . U z i e l has l a t e r pointed out t h a t g e l 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 v a r i o u s f a c t o r s such as pH, temperature, bead s i z e e t c . (111). More-over, immediately a f t e r the e l u t i o n of the mononucleotides, the s a l t s are e l u t e d from the column and there i s the p o s s i b i l i t y of o v e r l a p s . 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. i i i ) a dsorption of: n u c l e o t i d e s on c h a r c o a l : - Adsorp-t i o n of p u r i n e s , p y r i d i n e s and 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 c h a r c o a l and 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 widely used f o r s e p a r a t i o n of these compounds from s a l t s o l u t i o n s . Before use, the c h a r c o a l has to be p u r i f i e d and s p e c i a l l y prepared. For the experiments described h e r e i n , the charcoal was prepared by a combination of methods described by Thomson (112) and by P l a i s t e d and Riggs (113) . One hundred grams of Darco G. 60 c h a r c o a l was suspended i n 1000 ml of 2 N HCl and the mixture was r e f l u x e d f o r 3 hours The c h a r c o a l was then c o l l e c t e d by f i l t r a t i o n on a Buchner funnel 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 was, n e u t r a l . The washed char c o a l was then suspended In 1000 ml of 2 M a c e t i c a c i d and the mixture was r e f l u x e d f o r 2 hours. The suspension was f i l t e r e d on a Buchner funnel and^while s t i l l on the funnel^the c h a r c o a l was washed w i t h g l a s s - d i s t i l l e d water u n t i l the f i l t r a t e was n e u t r a l and then w i t h copious amounts of ammoniacal ethanol (Amm:18 v o l , H 20 43 vol,' ethanol 50 vol) . F i n a l l y , t h e c h a r c o a l was again 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 absorption of 0.05 or l e s s at 260 my. The c h a r c o a l was d r i e d and weighed and then sus-pended i n d i s t i l l e d water to give a c o n c e n t r a t i o n of 10 gms per 100 ml. For adsorption experiments, an a l i q u o t of t h i s suspension was used. ^ The e f f i c a c y of the method was f i r s t t e s t e d w i t h authentic n u c l e o t i d e s . Two standard s o l u t i o n s of n u c l e o t i d e s one con-t a i n i n g a mixture of adenine n u c l e o t i d e s and the other c o n t a i n i n g guanine n u c l e o t i d e s were prepared. A known con 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 of 4 ml was mixed wi t h 0.2 ml of. a 10% c h a r c o a l suspension. The adsorption of the nucleo-t i d e s on c h a r c o a l was c a r r i e d out according t o the procedure described p r e v i o u s l y . For the purpose of e l u t i o n three d i f f e r e n t - .57'- '' s o l v e n t systems were employed and the recovery of m a t e r i a l i n each s o l v e n t mixture determined. Solvents employed, were: 1) cone, ammonia-ethanol-water (5:50:45 v/v) (112) 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 (5 v o l of t r i e t h y l a m i n e i n 95 v o l 60% e t h a n o l ) . 3) pyridine-ethanol-water (5 v o l of p y r i d i n e i n 95 v o l of 60% e t h a n o l ) . (98). The substances were e l u t e d by mixing the char c o a l w i t h 8 ml of so l v e n t under examination and keeping the tubes i n a water bath at 37°C f o r 3 hours. The tubes were c l o s e d to prevent any evaporation and shaken o c c a s i o n a l l y to d i s p e r s e the char-c o a l . In many cases e i t h e r the sol v e n t •exhibited', high U.V. absorption or i t dispersed f i n e carbon p a r t i c l e ' s to give a black s o l u t i o n on e x t r a c t i o n and hence the e s t i m a t i o n of concentra-t i o n of n u c l e o t i d e s i n the eiu a t e by d i r e c t spectrophotometry was not p o s s i b l e . A f t e r e l u t i o n the s o l v e n t was evaporated i n a f l a s h evaporator, the residue d i s s o l v e d i n a small q u a n t i t y of water and the s o l u t i o n a p p l i e d on to Whatman No. 4 0 f i l t e r paper. The chromatogram was developed i n i s o b u t y r i c . a c i d -ammonia-water system, the U.V. absorbing spots on the chromato-gram were marked, e l u t e d and the eluates made up to a known volume.. The con 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 s o l u t i o n was determined by spectrophotometry and the recovery was c a l c u l a t e d . The data "are presented i n Table I I I . From the t a b l e i t i s seen t h a t p'yridine-ethanol mixture was the best s u i t e d s o l v e n t system f o r the e l u t i o n of nucleo-t i d e s from c h a r c o a l . Thomson (112) has reported that the - 58 -Table I I I Recovery of n u c l e o t i d e s adsorbed on ch a r c o a l by e l u t i o n w i t h three 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 t e x t . Concentration as the monophosphate (ymoles) Percentage Recovery Nu c l e o t i d e mixture Solvent I Solvent I I Solvent I I I Adenine Nucleotides (AMP, ADP & ATP) 0.299 0.430 95.5 96 91.2 81.3 81.8 78.7 Guanine Nucleotides (GMP, GDP & GTP) 0.417 0.625 88.8 72 92.7 67.5 67.0 68.4 Solvent I . Pyri d i n e - e t h a n o l - w a t e r . Solvent I I . Tri e t h y l a m i n e -ethanol-water. Solvent I I I Ammonia-ethanol-water. - 59 -e t h a n o l i c ammonia system gives l e s s complete e l u t i o n than the p y r i d i n e water 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 -water system was found to di s p e r s e much of the 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 pointed out th a t though the, p u r i f i c a t i o n of ch 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 abridged" (112). For e f f i c i e n t a d s o r p t i o n , of the n u c l e o t i d e s the pH of the n u c l e o t i d e s o l u t i o n to be maintained between 1 and 2.. I t was suggested by Thomson and by ^Grav t h a t a recovery of more than 60% of the absorbed m a t e r i a l i s considered to be s a t i s f a c t o r y (112, 107). In the present experiments the recovery r a t e s are higher and hence the method was used to separate n u c l e o t i d e s from s a l t s of the a c i d s o l u b l e f r a c t i o n . d) C h a r a c t e r i z a t i o n of the substances e l u t e d from chromato-grams:- A f t e r the methods f o r the recovery of nu c l e o t i d e s from s o l u t i o n s by adsorption on and e l u t i o n from ch a r c o a l were stan-d a r d i z e d , these methods were a p p l i e d t o the 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 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 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 the technique of c h a r c o a l adsorption 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 of n u c l e o t i d e s f r e e d from other substances i n -t e r f e r i n g i n chromatography was a p p l i e d on to a sheet of Whatman No.. 1 f i l t e r paper and two dimensional chromatography was per-formed as des c r i b e d . On examination of chromatograms under U.V. l i g h t c l e a r separations of many m a t e r i a l s i n t o d i s t i n c t spots were observed. These spots were marked out, 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 procedures. From the p a t t e r n of d i s t r i b u t i o n of a u t h e n t i c mixture 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 of the. n u c l e o t i d e s were known. The absorption s p e c t r a of substances a f t e r e l u t i o n from chromatogram were obtained using a Cary re c o r d i n g spectrophotometer, model 15. I t was observed from the chromatogram t h a t the m o b i l i t y of a p a r t i c u l a r n u c l e o t i d e decreased w i t h increase i n the degree of phosphorylation. Thus the monophosphate move f a s t e r than the diphosphates and the triphosphates are l e a s t migratory of the compounds g i v i n g the same spectrum. The 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 to be c l e a r l y detected on paper. To know t h e . p o s i t i o n s of these substances on chromatogram, a modified technique of ' 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 suspension was d i v -ided i n t o two equal p a r t s and to one p a r t a u t h e n t i c n u c l e o t i d e s were added. The authentic n u c l e o t i d e s added were whose content was low i n the a c i d s o l u b l e e x t r a c t . The two s o l u t i o n s were a p p l i e d on to sheets of f i l t e r paper and two dimensional chromatography was 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 . The two chromatograms were viewed under U.V. l i g h t and compared. 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 recognized thus v e r i f y i n g the p o s i t i o n of the p a r t i c u l a r substance which was d i f f i c u l t to l o c a t e i n the chro-matogram of a c i d s o l u b l e e x t r a c t s . As a step f u r t h e r , co-chromatography of some of the substances i s o l a t e d from the - 61 -chrpmatograms w i t h a u t h e n t i c samples was a l s o t r i e d . For f u r t h e r c o n f i r m a t i o n of the substances i s o l a t e d , the amount of phosphorus present i n these compounds was determined. The e l u t e d samples of the same n u c l e o t i d e s were pooled and the volume of 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 of the n u c l e o t i d e s were d i l u t e d so t h a t the concentra-t i o n of n u c l e o t i d e s expressed as O.D./ml was nearly^equal i n a l l cases. As the study p e r t a i n e d mainly to purine n u c l e o t i d e s , the analyses were c a r r i e d out only f o r these compounds. The r e s u l t s are given i n Table IV. The value f o r phosphorus i n d i c a t e d the d i f f e r e n t nucleo-side' phosphates from the chromatograms. This f u r t h e r confirms the paper chromatographic s e p a r a t i o n of the d i f f e r e n t nucleoside phosphates by the method employed. In the chromatograms, obtained by the method employed i n a d d i t i o n to the three adenine n u c l e o t i d e s , another substance having the same s p e c t r a l c h a r a c t e r i s t i c of adenine n u c l e o t i d e appeared. The p o s i t i o n of the substance on chromatogram was clo s e to ADP and t h i s substance can e a s i l y be mistaken f o r ADP. However, the 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 the sp e c t r a of the cyanide complex of the sub-stance, as w e l l as the reduced NADH, was obtained i n Cary spectrophotometer. The NAD was reduced by the a l c o h o l dehydro-genase (Worthington Biochemical Corp.) according to the pro-cedure given i n Pabst C i r c u l a r - 1 0 (97). Both the cyanide com-plex and the NADH+ show a new absorption peak at 340 mu. Since these methods were developed, s i m i l a r procedures - 62 -Table IV Phosphorus determinations on purine n u c l e o t i d e s -obtained from two-dimensional chromatograms. T h e o r e t i c a l values were c a l c u l a t e d using E ^ max Nucleotide Concentration Phosphorus c o n c e n t r a t i o n (yg/ml) (O.D./ml) Found T h e o r e t i c a l 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 reported by others f o r the 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) and animal t i s s u e s (115). 4) E f f e c t of Actinomycin D on the I n c o r p o r a t i o n of 1 , l fC-For mate i n t o the A c i d Soluble Nucleotides of E h r l i c h A s c i t e s Carcinoma  C e l l s . Once the method of paper chromatography was developed f o r the s e p a r a t i o n of 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 of a c t i n o -mycin D on the formation 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 carcinoma c e l l s was examined. Incubation experiments s i m i l a r to the one described p r e v i o u s l y were c a r r i e d out. Each i n c u b a t i o n mixture contained 20 umoles of sodium formate, 5 u c u r i e of 1 "^-formate and 10 ml of E h r l i c h a s c i t e s carcinoma c e l l suspension i n Krebs Ringer phosphate b u f f e r pH 7.8. Actinomycin D d i s s o l v e d i n 20% ethanol was added i n appropriate amounts to the appropriate f l a s k s and to the c o n t r o l s only 20% ethanol was 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 and s t o r e d at -20° i f not used immediately a f t e r removal of potassium p e r c h l o r a t e . The a c i d s o l u b l e f r a c t i o n was brought to pH 1-2 by the addi-t i o n of 2 N'HCl. The n u c l e o t i d e s were adsorbed by adding 0.2 ml of 10% suspension of a c t i v a t e d c h a r c o a l i n water. The c h a r c o a l was 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 the p y r i d i n e - e t h a n o l s o l v e n t mixture. The e l u a t e was evaporated, the residue was d i s s o l v e d i n a small q u a n t i t y of water and a p p l i e d on to a l a r g e sheet of Whatman No. 40 f i l t e r paper. Two- dimensional chromatography was c a r r i e d out f o r the s e p a r a t i o n of the n u c l e o t i d e s . A f t e r chromatography the n u c l e o t i d e s were marked under U.V. and the spots were e l u t e d w i t h water and c o l l e c t e d i n 10 ml beakers. The water was evaporated in vacuo and the residue i n the beaker was d i l u t e d w i t h a d e f i n i t e volume of 0.1 N HCl. The 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 s o l u t i o n was determined by u l t r a v i o l e t spectrophotometry. Half ml samples of n u c l e o t i d e s were counted i n a l i q u i d s c i n t i l l a t i o n counter and the 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 . The r e s u l t s are given i n Table V. Though these experiments were l a b o r i o u s and time consuming, the r e s u l t s obtained were r a t h e r d i s a p p o i n t i n g . Examination of the data presented i n tables :shows v a r y i n g r e s u l t s both on the i n c o r p o r a t i o n of 1 4C-formate i n to the n u c l e o t i d e s and the t o t a l 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 s o l a t e d . In these experiments the r a d i o a c t i v e precursor i n c o r p o r a t e d i n t o the 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 which i s dependent on the c o n c e n t r a t i o n of the substances i n .solution. In a d d i t i o n to the de novo s y n t h e s i s , wherein -. l l fC-formate i s i n c o r p o r a t e d , n u c l e o t i d e s may a l s o be formed i n the c e l l by other mechanisms. The increase 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 thus may decrease the s p e c i f i c a c t i v i t y to some extent. There i s no c o r r e l a t i o n , between the increase 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- decrease i n s p e c i f i c a c t i v i t y . I t may be pointed out t h a t i n the event of a normal syn 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 presence of actinomycin D and i n h i b i t i o n of s y n t h e s i s . o f RNA, an accumulation of n u c l e o t i d e s can be expected i n the c e l l . The increase 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 i n c e l l s Table V Experiment I E f f e c t of actinomycin D on the i n c o r p o r a t i o n of 1^C-formate i n t o purine n u c l e o t i d e s of E h r l i c h a s c i t e s carcinoma c e l l s . N u c l e o t i d e s were separated by two-dimensional paper chromatography. S p e c i f i c (counts a c t i v i t y ( per minute per ymole) Concentration mymoles/10 ml suspension Act. D yg/ml 0 0.05 0.5 1.5 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 T a b l e V(continued) S p e c i f i c a c t i v i t y (counts per minute per umole) C o n c e n t r a t i o n mymole/10 ml suspension A c t . D 0 1 2 'A 4 :.8 yg/ml O i l 2 4 8 Expt. 2 AMP 3202 3785 4654 3012 4801 258. 0 217. 0 196. 0 224. 0 544. 0 ADP 3193 3607 3207 3010 4661 421. 0 432. 0 338. 0 421. 0 368. 0 ATP 2944 3598 3458 2959 4368 424. 0 563. 0 427. 0 522. 0 313. 0 T o t a l 1103. 0. 1212 961 1167 1225. 0 GMP 3050 2900 2628 2091 3526 52. 2 36. 8 60. 2 36. 27 121. 7 GDP 2433 2790 2803 1856 2789 70. 7 71. 9 61. 5 63. 4 121. 7 GTP 2457 2635 2629 1842 2645 . . 2.9. 0 . . 4.9 . 6 21. 7 . .'.2.9.. 07 14. 5 T o t a l 151. 9 158. 3 143. 8 128. 67 213. 6 Expt. 3 AMP 4174 4917 2877 3613 3642 201 .3 193. 3 177. 5 254. 3 508.6 ADP 3693 4222 2717 3441 3707 394 .7 322. 6 320. 0 400. 0 322.6 ATP 3404 3855 3467 3056 3372 522 .0 337. 0 .3.59.. 0 . .5.1.4. . 2.90. . T o t a l . .11.18 .0 852. ,0. .856. 5 .11.6.8.. 3. 1121.2 GMP 3531 3863. 1370 2729 2625 39 .3 32. 5 39. 9 43. 0 111.8 GDP 3504 3211 2168 2436 2092 56 .0 50. 0 52. 4 69. 0 68.3 GTP 3203 2721 2135 2480 2067 . . . .4.4, .7 29. 0 . . .3.0. 2 , . .4.2. 3 . . .18.1 T o t a l . 140 .0 111. 5 122. 5 154. 8 19 8.2 - 67 -can be due to 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 decrease i n c o n c e n t r a t i o n observed. 5) E f f e c t of Actinomycin D on the I n c o r p o r a t i o n of 1^C-Formate  in vitro by the I n t e s t i n a l Mucosa of Rat. E h r l i c h carcinoma c e l l s , l i k e any other tumour c e l l s , are 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 the 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 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 obtained i n s t u d i e s using a s c i t e s tumour c e l l s . To see the e f f e c t of the actinomycin D on the b i o s y n t h e s i s of purine n u c l e o t i d e s i n a normal t i s s u e , s i m i l a r s t u d i e s to those reported were c a r r i e d out u s i n g the i n t e s t i n a l mucosal c e l l s of the r a t . This t i s s u e was shown to i n c o r p o r a t e l l fC-formate 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 and to the DNA much the.same way as i n E h r l i c h a s c i t e s carcinoma c e l l s (85). This t i s s u e was 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 presence of 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 Ringer phosphate and i n c u b a t i o n was c a r r i e d out according to procedure of Stewart and Zbarsky (85). The scrapings were pooled and weighed amounts (0.8-1 gm) were placed i n t o f l a s k s . Enough c o l d Krebs Ringer phosphate b u f f e r was added to each f l a s k to give a 10% suspension of mucosa. Radio-a c t i v e formate (0.5 m i c r o c u r i e s per ml of suspension) and sodium formate (2 umole/ml of suspension) were added. Actinomycin D i n 20% ethanol was added to the appropriate f l a s k s . Incubation was 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 carcinoma c e l l s . They were chromatographed on paper - 68 -a f t e r adsorption 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 estimated. The 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 together. The values are there-f o r e given f o r combined counts of di-and ..triphosphates of adenosine and guanosine. The RNA was i s o l a t e d , hydrolyzed and the n u c l e o t i d e s separated by two dimensional chromatography as i n the case of a c i d s o l u b l e n u c l e o t i d e s . The content of RNA was estimated by the o r c i n o l method and the i n c o r p o r a t i o n i n t o RNA -per se was a l s o obtained. The r e s u l t s of these experiments are given i n Table VI. 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 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 presence of i n c r e a s i n g c o n c e n t r a t i o n of 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 to the e f f e c t , of actinomycin 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 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 n u c l e o t i d e obtained from i n t e s t i n a l mucosa was a l s o very s m a l l . The 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 of 1 I fC-formate i n t o RNA at the highest 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 of r a d i o a c t i v i t y i n t o the RNA n u c l e o t i d e s a l s o corresponded to t h i s value. In the case of the RNA n u c l e o t i d e s , there was a greater i n h i b i t i o n observed i n the case of guanine n u c l e o t i d e s than i n adenine n u c l e o t i d e s . 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 DNA obtained 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 (85). E f f e c t of actinomycin D on the i n c o r p o r a t i o n of 1 "*C-formate by the I n t e s t i n a l mucosa of the r a t . S p e c i f i c a c t i v i t y -counts per minute per umo.le A c i d - s o l u b l e n u c l e o t i d e s RNA % I n h i b i t i o n i n T o t a l RNA . . A c t i n o -mycin D AMP cpm/O.D. ADP+ATP GMP cpm/O.D. GDP+GTP 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 of Varying Concentration of Actinomycin D on the In c o r p o r a t i o n of ^C-Formate 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 there was v a r i a t i o n i n r e s u l t s obtained i n these experiments w i t h i n c r e a s i n g amount of actinomycin D, it.was d e s i r a b l e to f i n d Out the e f f e c t at a f i n a l c o n c e n t r a t i o n of actinomycin D which would maximally i n h i b i t RNA synt h e 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 . In the previous studies' i t was observed t h a t actinomycin D at a con c e n t r a t i o n of 10 ug/ml w i l l i n h i b i t the i n c o r p o r a t i o n of ^C-formate i n t o . RNA of r a t i n t e s -t i n a l mucosa by about 66%. S i m i l a r s t u d i e s using E h r l i c h a s c i t e s carcinoma c e l l s to f i n d out a con c e n t r a t i o n 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 of I n c o r p o r a t i o n of l l fC-formate i n t o RNA v/ere undertaken. The r e s u l t s of one such experiment i s given i n Table V I I . I t i s seen from the t a b l e t h a t the maximum i n h i b i t i o n of RNA synt h e s i s i n E h r l i c h a s c i t e s c e l l s Is at co n c e n t r a t i o n of 5 ug/ml of actinomycin D. However, many i n v e s t i g a t o r s have used a co n c e n t r a t i o n of 10 ug/ml or higher f o r studying the e f f e c t of actinomycin D on many c e l l u l a r processes. In the l a t e r experiments described i n t h i s thesis., a c o n c e n t r a t i o n of 10 ug/ml of actinomycin was used. 7) Separation of A c i d 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 DEAE-cellulose Co1umn-. I t was r e a l i z e d from previous experiments t h a t where small q u a n t i t i e s of m a t e r i a l was used f o r a n a l y s i s , a small change i n con c e n t r a t i o n gets magnified and becomes s i g n i f i c a n t l y low or high. S i m i l a r l y as the co n c e n t r a t i o n of m a t e r i a l under examina-- 71 -Table V I I E f f e c t of v a r y i n g concentrations of actinomycin D on the i n c o r p o r a t i o n of 1 k C - f o r m a t e 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 in vitro. S p e c i f i c a c t i v i t y - counts per minute per umole Actinomycin D ug/ml AMP GMP CMP UMP 0 117 70 143 9 0.5 H I 37 58 0 1.0 79 16 35 5 2.0 59 4 38 00 5.0 3 20 0 10.0 11 2 38 0 t i b n was low, a small change i n r a d i o a c t i v i t y i s l i a b l e to change an i n h i b i t i o n to 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 . Because of the v a r i a t i o n i n r e s u l t s obtained i n many experiments i n which small q u a n t i t i e s of n u c l e o t i d e s were separated by paper chromatography, I t was d e s i r a b l e to use l a r g e r q u a n t i t i e s of m a t e r i a l s f o r study.. For the sepa r a t i o n of l a r g e r concentrations of 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 , chromato-graphy on DEAE-cellulose-column, was c a r r i e d , put. For the s t a n d a r d i z a t i o n of the technique 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 nucleo-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 was performed. The • procedure followed f o r the i s o l a t i o n and s e p a r a t i o n of nucleo-t i d e s was according to the method of Oikawa and Smith (99). About 4 gm wet weight of E h r l i c h a s c i t e s carcinoma c e l l s were e x t r a c t e d w i t h 20-25 ml of 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 to a pH of 6.5-7 using 10 N sodium hydro-xide and the water evaporated i n a f l a s h evaporator. The residue was e x t r a c t e d w i t h 50 ml of 95% ethanol to remove the 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 remain as the residue were e x t r a c t e d w i t h a small volume of water and the s o l u t i o n a p p l i e d on to a column of DEAE-cellulose 25 x 1 cm i n the car-bonate form, prepared and packed according t o the procedure of Tomlinson and Tener(lOO). E l u t i o n of the n u c l e o t i d e s was c a r r i e d out wit h a l i n e a r g r a d i e n t of ammonium bicarbonate (0.2 M) at 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 . The sub-stances e l u t e d were i s o l a t e d and f u r t h e r separated and character-i z e d by paper chromatography and absorption s p e c t r a . The e l u t i o n p r o f i l e i s given i n Figure 8. • I t i s seen from the H" n 00 • H 3 H H- CD M P o 1 £ 0 OJ t5! «+ 1—1 H S CD g CU 3 rt » H- O H rt o a < CD Hi 1 H 0 c < D Hi D PJ M > H "« tn tt H-ABSORBANCE AT 260 my D - tr CD tr o ts < i c •a o o o •3 3 - o y cn CD D a w rt H O C H-1 H CD O O cn rt CD H-Cb — CD to cn Hi o-H1 3 O W 3 tr n >-3 H O .a' o cn to o o o co o o o A \ • M < 0 H-H Hi O l ' ui tr > rt •3 tr 3 cu to cn O > o H-< CD 1-i rt H CU CU CD H o cn ! cn rt O H- o i-3 5: 0 cu O •d CD 3 1"! • • i-i cn o CD s: H CD o M 1 h 3 CTl a CD CU o >. D O o - 0 CD M H H M H CD cn 1 O G rt .—, S CD •X) Cu *» rt 3 cn Ul —• o to Ul « o Ul o Ul 4r-3 H O o to 0 3 Molar i ty of Ammonium bicarbonate' - ZL -- 74 -e l u t i o n p r o f i l e t h a t 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 nucleo-t i d e s can be achieved by a combination of column chromatography and paper chromatography. 8) E f f e c t of Actinomycin D on the B i o s y n t h e s i s of Purine Nucleotides 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 study the e f f e c t of actinomycin D on the i n c o r p o r a t i o n of l l*C-formate 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 , tumour c e l l s from 12 mice were c o l l e c t e d and pooled. (Total volume 20 ml c e l l s ) . The pooled c e l l s were suspended i n Krebs Ringer phosphate b u f f e r pH 7.8 to g i v e a 1 i n 10 suspension. Sodium formate (2. umole/ml suspension)/and 1 ^ 'C-formate. (0.5 uc/ml suspension) were added to the 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 equal p o r t i o n s i n 500 ml Erlenmeyer f l a s k s . Actinomycin D d i s s o l v e d i n 20% ethanol to a c o n c e n t r a t i o n of 10 mg/ml was added to one and an equal volume of 20% ethanol added t o the other. A l l these operations were done i n the c o l d room. These f l a s k s were gassed w i t h oxygen, c l o s e d w i t h rubber stoppers and incubated i n a water bath at 37°C f o r 2 hours. < During the p e r i o d of i n c u b a t i o n t h e • f l a s k s were shaken at 100-110 o s c i l l a t i o n s per minute. A f t e r the i n c u b a t i o n the c e l l s separ-ated by c e n t r i f u g a t i o n at 15,000 x g f o r 20 minutes, washed once w i t h 5 volumes of c o l d Krebs Ringer phosphate and c e n t r i -fuged again. A f t e r removing the supernatant f l u i d , the wet weight of the c e l l s was determined. 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 the 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 hydroxide and stored at -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 evaporated on a f l a s h evaporator (bath temperature 25°C) and the residue repeatedly e x t r a c t e d w i t h ethanol to remove the sodium per-c h l o r a t e . The s o l u t i o n of sodium-perchlorate was c e n t r i f u g e d o f f and the' residue was e x t r a c t e d w i t h a small volume of water. This s o l u t i o n was a p p l i e d on to a column of DEAE-cellulose and chromatography was c a r r i e d out according to the method described. 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 incubated i n the absence-and presence of actinomycin D are given i n Figure 9 and Figure 10 r e s p e c t i v e l y . Examination of the e l u t i o n p r o f i l e s shows th 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 both the cases and s i m i l a r to the p a t t e r n obtained 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 increase i n con-c 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 actinomycin D. Though there was a general increase 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 ncrease was maximum i n the case of the nucleoside t r i p h o s h a t e s p a r t i c u l a r l y of ATP. This may mean t h a t actinomycin D does not e x e r t any i n h i b i t o r y e f f e c t on the formation of the nucleoside t r i p h o s p h a t e s . I t i s known from the r e s u l t s reported i n t h i s t h e s i s , as w e l l as from those reported by others t h a t actinomycin D i n h i -b i t e d the i n c o r p o r a t i o n of l l fC-formate i n t o RNA purines and the s y n t h e s i s of RNA (39). I f actinomycin D does not i n h i b i t the de novo s y n t h e s i s of nucleoside triphosphates and only i n t e r -f e r e s v/ith the p o l y m e r i z a t i o n of the nucleoside triphosphates 1.0 0.8-0.6T 0 .4 -0. n J 0.2 0.1 40 80 FRACTION NOS, 120 160 +J o Xi u u ra •H c o 4-1 o >1 +J •H u rH o s cn F i g u r e 9. Chromatography 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 carcinoma c e l l s (10.9 gm) incubated w i t h 1 **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 separated 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 . DEAE-cellulose column (25 x 1 cm. ) was used f o r chromatography. g o KO CM E-» < W U C Q C d O to CQ 0.2 40 80 FRACTION NOS. 120 160 Fi g u r e 10, Chromatography 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 carcinoma c e l l s (10.7 gm) incubated w i t h ^ C - f ormate and actinomycin D (10 yg/ml of suspension). F r a c t i o n s (10 ml.--) were c o l l e c t e d at 10 minute i n t e r v a l s . Peaks were separated and i d e n t i f i e d as de s c r i b e d i n t e x t . DEAE-cellulose column (25 x 1 cm::) was used f o r chromatography. - 78 -to form RNA, then an increase i n the c o n c e n t r a t i o n of these substances i n c e l l s t r e a t e d w i t h actinomycin can be expected. The s o l u t i o n of each U . V . absorbing peak from the B E A E c e l l u l o s e : c o l u m n was evaporated and the substances i n the r e s i -due was f u r t h e r separated by paper chromatography using the isobutyric-ammonia-water s o l v e n t mixture or i n the ammonium acetate-ethanol system. The absorption 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 centrations were determined by u l t r a v i o l e t spectrophotometry. Half ml samples of s o l u -t i o n s of the purine 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 determined. The c o n c e n t r a t i o n of the purine 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 experiments a n d " c e l l s incubated w i t h actinomycin D i s g i v e n i n Table V I I I . 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 v a r i o u s purine nucleo-t i d e s Is given i n Table I X . I t can be seen from these t a b l e s t h a t i n both 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 incubated w i t h actinomycin D. This shows t h a t the main a c t i o n of a c t i n o -mycin D i s to i n h i b i t the p o l y m e r i z a t i o n of nucleoside t r i p h o s -phates to form RNA and not to i n h i b i t the s y n t h e s i s of nucleo-t i d e s . As the increase i n c o n c e n t r a t i o n was seen mainly i n the nucleoside t r i p h o s p h a t e s , i t may be suggested t h a t actinomycin does not i n h i b i t the formation 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 1'*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 s l i g h t l y l e s s i n c e l l s incubated w i t h actinomycin D, though the decrease i s not very s i g n i f i c a n t . The decrease i n i n c o r p o r a t i o n of 1 1 1C-formate i n t o n u c l e o t i d e s becomes l e s s apparent when the - 79 -Table V I I I Concentrations of purine 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 actinomycin D (10 yg/ml) in vitro. Con-c e n t r a t i o n s are expressed as my moles per gram wet weight of c e l l s . Experiment I . Experiment I I . . . A c i d - s o l u b l e c o n t r o l c o n t r o l + c o n t r o l c o n t r o l + Nuc l e o t i d e Act. D Act. D AMP 23.44 25.68 33.41 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 51.75 . 85.4 104.82 - 80 -Table IX E f f e c t of actinomycin D on the i n c o r p o r a t i o n of l l fC-formate i n t o the purine n u c l e o t i d e s 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 recorded as s p e c i f i c a c t i v i t y (counts per minute per umole). Experiment I Experiment I I A c i d s o l u b l e n u c l e o t i d e s C o n t r o l C o n t r o l + C o n t r o l C o n t r o l + Act. D Act. D AMP ADP ATP 4163 4725 4663 3841 3982 4986 4238 4285 3998 3497 3805 3600 GMP 3575 GDP -GTP 2740 3123 4842 1882 1742 1688 1721 3989 1732 1103 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 the n u c l e o t i d e s i n incuba-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 pointed out t h a t i n the c e l l s t h e r e are mechanisms other than 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 are formed. 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 cubations c o n t a i n i n g actinomycin D, the s i z e of the nucleo-t i d e pool i n c r e a s e s , which would r e s u l t i n the d i l u t i o n of the r a d i o a c t i v e n u c l e o t i d e s formed. The accumulation of nucleo-t i d e s i n the c e l l may prevent f u r t h e r de.novo. s y n t h e s i s of nucleo 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 1^C-formate 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 s p e c i f i c a c t i v i t y . Since.these i n v e s t i g a t i o n s were undertaken Lowy et 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 e r y t h r o c y t e s t h a t actinomycin D does not i n h i b i t the de novo synthesis of n u c l e o t i d e s or the formation of n u c l e o t i d e s from purines. The present 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 c o n c e n t r a t i o n of o t h e r n u c l e o t i d e s was a l s o observed i n c e l l s incubated w i t h actinomycin D. The 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 the methods described i s presented i n Table X. l i t can be seen from . the r e s u l t s t h a t there v/as a l s o an increase o:f the pyrimidine n u c l e o t i d e s i n c e l l s when the RNA synthesis i s i n h i b i t e d by actinomycin p. Kida et 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 actiton to actinomycin D, on the n u c l e i c a c i d metabolism of Bacillus subtilis SB-15. - 82 -Table X E f f e c t of actinomycin D.on the co n c e n t r a t i o n of pyr i m i d i n e n u c l e o t i d e s and NAD of E h r l i c h a s c i t e s carcinoma c e l l s in vitro. Actinomycin D concen-t r a t i o n 10 ug/mL.Concentration expressed as mumoles - per gram wet weight of t i s s u e . N ucleotide C o n t r o l C o n t r o l + Actinomycin D CMP 6.18 6.606 CDP 50.3 •. 67.13 CTP 76.4 97.12 UMP 16.83 28.78 UDP 35.35 37.88 UTP 63.4 127.5 NAD 29.16 35.12 - 83 -They a l s o observed a q u a n t i t a t i v e change i n the n u c l e o t i d e components of the c e l l s , which i s i n agreement w i t h the present f i n d i n g s . 9) E f f e c t of Actinomycin 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 reported r e c e n t l y (32) t h a t actinomycin D i n h i b i t s r e s p i r a t i o n and g l y c o l y s i s i n leucemic leucocytes. Horning and Rabinowitz have shown tha t the r a t e of oxygen up-take of Sarcoma 37.ascites c e l l s incubated i n the presence or absence of actinomycin D was not s i g n i f i c a n t l y d i f f e r e n t . Some years ago Scrimgeour observed (68) t h a t actinomycin D i n h i b i t e d 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 t was of i n t e r e s t to study the e f f e c t of actinomycin 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 used f o r s t u d i e s described i n t h i s t h e s i s . Studies 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 usual technique of Warburg manometry using f l a s k s w i t h side arms. 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 as described p r e v i o u s l y . They were c e n t r i f u g e d at 1500 rpm 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 (model P.R-11) f o r 3 minutes, the supernatant was removed, and the c e l l s were washed w i t h three volumes of c o l d Krebs Ringer phosphate b u f f e r . The suspension was c e n t r i f u g e d again at 2000 rpm f o r 3 minutes and the supernatant was removed. The 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 Ringer phosphate b u f f e r ..pH 7.8. Two ml of the c e l l suspension was present i n each f l a s k i n a t o t a l volume of 3.ml. The centre w e l l of the- f l a s k s con-t a i n e d 0.2 ml of 10% KOH. Substrates whenever used were ti p p e d from the s i d e arm of the f l a s k s to the main compartment a f t e r thermal e q u i l i b r a t i o n f o r 15 minutes. The gas phase was a i r and the incubations were c a r r i e d out at 37°C f o r 1 hour. The uptake of oxygen w i t h time by the c e l l suspension i n the presence and absence of actinomycin D i s given i n Figure 11. It can be seen from the f i g u r e t h a t the r a t e of r e s p i r a t i o n of c e l l s i n the absence of actinomycin D was l i n e a r w i t h time. This was observed even when the i n c u b a t i o n was c a r r i e d out f o r 2 hours. A f t e r t h i s p e r i o d there was a s m a l l f a l l i n the r e s p i r a t i o n " . The presence of actinomycin D does not change the r a t e of r e s p i r a t i o n up to 30 minutes, though there was a small decrease i n the oxygen uptake i n about 60 minutes. The Qo 2 values are given i n Table XI. The data show a small decrease i n the Q02 values i n presence of actinomycin D suggesting a small i n h i b i t i o n i n r e s p i r a t i o n . In a s i m i l a r study L a s z l o et a l . (32) have shown th a t the oxygen uptake by leukemic leucocytes was 118 u l i n presence of 50 ug/ml of actinomycin D compared to 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 . They have con-cluded from t h i s t h a t actinomycin D i n h i b i t s r e s p i r a t i o n of leucemic leucocytes. Scrimgeour observed an i n h i b i t i o n of r e s p i r a t i o n of N o v i k o f f hepatoma i n presence of actinomycin D. The present r e s u l t s are comparable to these observations. Glucose i n h i b i t e d the oxygen uptake of E h r l i c h a s c i t e s carcinoma c e l l s by almost 45%. This i s i n agreement w i t h the observations by others (117). This i n h i b i t i o n of r e s p i r a t i o n by glucose - 8 5 -20 40 60 TIME (minutes) Fi g u r e 11. E f f e c t o f actinomycin 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 contained 19.7 mg of dry weight of t i s s u e i n a t o t a l volume of 3.0 ml of Krebs Ringer phosphate b u f f e r . Gas phase was a i r and incubation'was at 37°C C o n t r o l ( o- e ) + Actinomycin D ( A ) - 86 -Table XI E f f e c t of actinomycin 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 . Each mano-me t r i c f l a s k contained 19.5 mg dry weight of t i s s u e i n a t o t a l volume of 3 ml. Actinomycin D when present was 10 yg/ml and glucose was . 5.5 mM. A d d i t i o n s 0_ % I n h i b i t i o n by Actinomycin C o n t r o l 11.02, 10.97 + Act. D (10 yg/ml) 9.58, 9.56 13.14, 12.85 + glucose 6.13/ 6.20 + glucose + Act. D. 5.4 5.47 11.90, 11.90 i s probably due to the op e r a t i o n of the Crabtree e f f e c t (118). Actinomycin D decreases the oxygen uptake i n the presence of glucose by another 12%. However, the i n h i b i t i o n of r e s p i r a -t i o n produced by actinomycin i s of the same magnitude i n the presence of added su b s t r a t e as w e l l as i n the absence, of 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 to the i n h i b i t i o n of RNA: synthesis caused by the- same- amount of actinomycin added to the medium and' hence there may not be any c o r r e l a t i o n between these, two processes. 10) E f f e c t of Actinomycin 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 are 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 the e f f e c t of actinomycin on the g l y c o l y s i s by d i f f e r e n t t i s s u e s . Scrimgeour observed t h a t actinomycin D d i d not i n h i b i t g l y c o l y s i s i n N ovikoff hepatoma (68). Prave and Kroning (119) reported t h a t actinomycin I reduced both aerobic and anaerobic 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 . Recently i t was reported by L a s z l o et a l . (32) t h a t actinomycin D i n h i b i t e d the 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 of i n t e r e s t to study the e f f e c t of actinomycin D on the u t i l i z a t i o n of glucose by E h r l i c h c a r c i -noma c e l l s in vitro. A ten percent suspension of E h r l i c h a s c i t e s carcinoma c e l l s was made i n Krebs Ringer phosphate b u f f e r pH 7.8. S u f f i c i e n t glucose, as a 5% s o l u t i o n i n b u f f e r , was added to give a con-c e n t r a t i o n of 1 mg/ml of suspension and the suspension 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 actinomycin D d i s s o l v e d i n 20% ethanol was added to one p o r t i o n to give a c o n c e n t r a t i o n of 10 mg/ml of suspension. To the other p o r t i o n which served as the c o n t r o l , an equal volume of 20% ethanol was added. The S o l u t i o n s were incubated at 37°C i n a water bath and were kept shaken during the p e r i o d of i n c u b a t i o n . At i n t e r v a l s one ml of the suspension was 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 of each of z i n c sulphate (2%) and barium hydroxide s o l u t i o n , according to the method of Somyogi (75). The glucose was estimated using 1 ml of the p r o t e i n f r e e f i l -t r a t e by the glucose oxidase method using a g l u c o s t a t k i t (Wor-t h i n g t o n Biochemical Corp). A standard curve showing the c o n c e n t r a t i o n of glucose 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 prepared by using 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 . The c o n c e n t r a t i o n of glucose i n the t e s t samples was determined using the standard.curve. The c o n c e n t r a t i o n of glucose at 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 Figure 12. I t can be seen from the Figure 12 t h a t the f a l l i n con-c e n t r a t i o n of glucose w i t h time i s not a f f e c t e d by the presence of actinomycin D i n the incubation.medium. This would suggest i n t u r n t h a t actinomycin D exerted no i n h i b i t o r y e f f e c t on the g l y c o l y s i s of E h r l i c h a s c i t e s carcinoma c e l l s . 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 reported f o r actinomycin 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 of L a z l o e t a l . (32) f o r actinomycin D i n leucemic •leucocytes. Scrimgeour s t u d i e d the a f f e c t of actinomycin D on - 89 -800 3 fi O •H -P -"ta u. +> fi cu D fi O U CO in o o rH U 600 400 200 15 30 45 TIME (minutes) 60 Figure 12. E f f e c t of actinomycin 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 in vitro. C o n t r o l ( © — ) + Actinomycin (-D (10 yg/ml) ) - 90 -the g l y c o l y s i s of N o v i k o f f hepatoma using the Warburg manometric techniques (68). He observed no i n h i b i t i o n of 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 of actinomycin D. In the present 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 disappearance of glucose from the c e l l suspension which agrees w i t h the r e s u l t s obtained by manometric techniques, used by Scrimgeour. - 91 -DISCUSSION In the past few years d i f f e r e n t a n t i b i o t i c s which 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 of a c t i o n of 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 which c l a r i f i e d the modern con-cepts about the i n t e r r e l a t i o n s h i p of 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 of a c t i o n of hormones, of 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 of v i r u s and many other c e l l u l a r processes. One of the widely used a n t i b i o t i c s to study these processes at a molecular l e v e l i s actinomycin D. The molecular mechanisms by which actinomycin D can i n h i b i t l i v i n g processes have been amply documented (12). Because of the s p e c i f i c i t y of a c t i o n of actinomycin 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 to i n v e s t i g a t e the involvement of 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 actinomycin has been a t t r i b u t e d to the a b i l i t y of the substance to form molecular complexes w i t h DNA, which f u n c t i o n s as a tem-p l a t e f o r both the s y n t h e s i s of RNA and DNA. The process of r e p l i c a t i o n c f RNA by DNA dependent RNA polymerase has been reported to take place i n three d i s t i n c t and separate processes (28, 29): 1) the b i n d i n g of RNA polymerase to s p e c i f i c s i t e s on the DNA which f u n c t i o n as a template i n the r e a c t i o n , 2) the i n i t i a t i o n of 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 purine n u c l e o t i d e s , to 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 of four kinds -92-of r i b o n u c l e o t i d e s to form a polymer under the d i r e c t i o n of the DNA template. The e f f e c t of actinomycin D on the b i n d i n g of RNA polymerase to DNA was i n v e s t i g a t e d by Richardson (30) by sucrose 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 sedimentation s t u d i e s of the complex. I t was observed t h a t actinomycin at l e v e l s which could i n h i b i t RNA s y n t h e s i s d i d not reduce the number of b i n d i n g s i t e s f o r RNA-polymerase on DNA, and t h a t actinomycin D i n no way a f f e c t e d the a f f i n i t y constant f o r the b i n d i n g r e a c t i o n . F u r t h e r , the k i n e t i c s of the RNA s y n t h e s i s by DNA dependent RNA p o l y -merase i n presence of actinomycin D were st u d i e d by Richardson. He has observed t h a t the RNA s y n t h e s i s by RNA polymerase s t a r t s r a p i d l y and then immediately begins to slow down and stops i n about 5 minutes. From these observations he has concluded t h a t actinomycin D does not a f f e c t the b i n d i n g of RNA polymerase to DNA templates, and has l e s s e f f e c t on the i n i t i a t i o n of syn-t h e s i s than on the p o l y m e r i z a t i o n . S i m i l a r observation t h a t actinomycin D i n h i b i t s the chain e l o n g a t i o n i n RNA s y n t h e s i s when n a t i v e DNA used as a template has been reported r e c e n t l y by Sentenac et a l . (31). • In c o n t r a s t to t h i s , Richardson has observed t h a t p r o f l a v i n , another i n h i b i t o r of DNA dependent RNA polymerase, i n t e r f e r e s w i t h the b i n d i n g of RNA polymerase to DNA templates and a l s o 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 e l o n g a t i o n of the chain by the p o l y m e r i z a t i o n r e a c t i o n . Recently i t has been reproted by Mizuno et a l . (120) 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 and B44 ( 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 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 . However,these a n t i b i o t i c s d i d not i n h i b i t the 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 carcinoma c e l l s . Many of the stu d i e s on the e f f e c t s of actinomycin D are concerned w i t h b i o s y n t h e s i s of macromolecules and l i t t l e i s known about the e f f e c t of the a n t i b i o t i c on the synt h e s i s of precursors of n u c l e i c a c i d s . Wheeler and Bennett (121) reported i n 1960 and l a t e r i n 1962 (64) t h a t actinomycin i n -h i b i t e d the i n c o r p o r a t i o n of ^C-formate and 2- 1 " ^ - g l y c i n 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 of L. arabinosus. I n c o r p o r a t i o n of l l +C-formate i n t o s o l u b l e n u c l e o t i d e s of 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 reported to be i n h i b i t e d by actinomycin D. From, a comparison of data obtained i n s t u d i e s w i t h lhC-formate and 8- 1^C-hypoxanthine they have concluded t h a t a c t i n o -mycin D d i d not i n t e r f e r e w i t h the b i o s y n t h e s i s of the purine moiety 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 vivo. I t was a l s o observed t h a t actinomycin D d i d not i n h i b i t the i n t e r c o n v e r s i o n of purine r i b o n u c l e o t i d e s . S i m i l a r i t i e s and d i f f e r e n c e s of the e f f e c t of actinomycin D on the purine 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 to suggest "two metabolic b l o c k s , one on the synthesis of purine n u c l e o t i d e s de novo, and one on the u t i l i z a t i o n of guanine n u c l e o t i d e s f o r synthesis of RNA".. The second s i t e of i n h i b i t i o n seems t o be common to a l l the systems s t u d i e d and they f u r t h e r suggested t h a t the block . of de novo s y n t h e s i s observed may be a secondary e f f e c t . This could very w e l l be the case i n the l i g h t of the present inves-t i g a t i o n and the l a t e r known f a c t s about the e f f e c t s of 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 the e f f e c t of actinomycin D on the i n c o r p o r a t i o n of 8- 1^C-guanine i n t o RNA purines i n E h r l i c h a s c i t e s carcinoma c e l l s . They have observed t h a t the i n c o r p o r a t i o n of guanine to RNA i s i n h i b i t e d by actinomycin D. They have f u r t h e r observed t h a t actinomycin d i d not prevent the formation of l a b e l l e d n u c l e o t i d e s from 8-1 "*C-guanine i n these systems. While present i n v e s t i g a t i o n s were i n progress, the e f f e c t of actinomycin D on the formation and,. i n t e r c o n v e r s i o n of purine n u c l e o t i d e s was s t u d i e d by Lowy and Wi l l i a m s (66) i n a system which i s incapable of any RNA s y n t h e s i s . They p r e v i o u s l y reported (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 unable t o synthesize RNA although, these c e l l s are capable of s y n t h e s i z i n g purine n u c l e o t i d e s de novo. The e f f e c t s of actinomycin D on the b i o -s y n t h e s i s of purine 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 using l l fC-formate 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 observed t h a t actinomycin D at a c o n c e n t r a t i o n as high as 50 mg/ml had no i n -h i b i t o r y e f f e c t on., the formation of purine n u c l e o t i d e s or on the formation of n u c l e o t i d e s from preformed purin e s . Actinomycin D a l s o had no e f f e c t on the i n t e r c o n v e r s i o n of purine n u c l e o t i d e s i n t h i s system. J a c o l i and Zbarsky (1) s t u d i e d the e f f e c t of actinomycin on the syn t h e s i s of n u c l e o t i d e s i n Bacillus subtilis and observed an accumulation of 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 of c e l l s the growth of which was i n h i b i t e d by actinomycin D. The present s t u d i e s were undertaken to f i n d out the e f f e c t of actinomycin 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 i n Krebs Ringer phosphate b u f f e r was incubated i n presence of actinomycin D. 1^C-Formate was used as a precursor of nucleo-t i d e s and n u c l e i c acid, i n these s t u d i e s . The va r i o u s components of 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 s incubated w i t h actinomycin D as w e l l as from c o n t r o l experiments were separated by two d i f f e r e n t techniques. A method of se p a r a t i o n by paper chromatography developed during the course of these i n v e s t i g a -t i o n s was used to separate 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 suspensions. When l a r g e r q u a n t i t i e s of m a t e r i a l was to be separated, chromatography on DEAE-cellulose was c a r r i e d out. The r e s u l t s of 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 of a c i d s o l u b l e m a t e r i a l from small q u a n t i t i e s of c e l l s were i n c o n c l u s i v e due to v a r i a t i o n s i n r e s u l t s from experiment to experiment. From s t u d i e s employing l a r g e r q u a n t i t i e s of c e l l suspension, i t was found t h a t the amounts of a c i d s o l u b l e n u c l e o t i d e s increased i n c e l l s incubated w i t h actinomycin D. A systematic a n a l y s i s of the components of the a c i d s o l u b l e n u c l e o t i d e s revealed the marked in c r e a s e i n c o n c e n t r a t i o n of the purine n u c l e o t i d e s . This 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 both ATP and GTP. An unusual f i n d i n g was th a t • t h e increase i n co n c e n t r a t i o n of ATP was greater than a corresponding increase of GTP. The concentra-t i o n s of the diphosphates were a l s o increased. F u r t h e r , an i n -crease i n concentrations of a l l the other p y r i m i d i n e n u c l e o t i d e s were a l s o observed i n c e l l s incubated i n presence of actinomycin D, p a r t i c u l a r l y of UTP. The i n c o r p o r a t i o n of ^ C - f ormate i n t o the various purine n u c l e o t i d e s was estimated. The s p e c i f i c a c t i v i t i e s of the va r i o u s substances i s o l a t e d i n presence of actinomycin D were lower than the c o n t r o l s , though the decrease i s not very s i g n i f i c a n t . This decrease i n s p e c i f i c a c t i v i t y observed i n c e l l s incubated w i t h actinomycin D.'.may be due to d i f f e r e n t causes. I t may be noted t h a t the con c e n t r a t i o n of the nucleo-t i d e s i n these cases has increased c o n s i d e r a b l y and as the s p e c i f i c a c t i v i t y i s a f u n c t i o n of c o n c e n t r a t i o n , when concen-t r a t i o n of the n u c l e o t i d e pool i s increased there may be a r decrease i n the s p e c i f i c a c t i v i t y . However, the decrease i n s p e c i f i c a c t i v i t y i s small compared to the increase i n concen-t r a t i o n of the n u c l e o t i d e s . From the present i n v e s t i g a t i o n s as w e l l as from the obser-v a t i o n s of others i t i s known t h a t actinomycin D i n h i b i t e d the normal s y n t h e s i s of RNA i n the c e l l s . In the normal metabolism of RNA, t h i s compound may be broken down to n u c l e o t i d e s which may again be r e u t i l i z e d . In presence of actinomycin D, the r e u t i l i z a t i o n of the n u c l e o t i d e s formed by RNA breakdown i s not 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 of the a c i d s o l u b l e pool which may again lower the. s p e c i f i c a c t i v i t y of the n u c l e o t i d e s . There i s a l s o yet another p o s s i b i l i t y f o r decreasing the s p e c i f i c 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 . I t was observed by many (65, 66) t h a t actinomycin D d i d not i n h i b i t the conversion of preformed purines to n u c l e o t i d e s or the i n t e r c o n v e r s i o n of n u c l e o t i d e s . Non-radioactive, preformed purines converted i n t o n u c l e o t i d e s can a l s o reduce the s p e c i f i c a c t i v i t y . F i n a l l y , the accumulation of n u c l e o t i d e s i n the c e l l s may produce a feedback i n h i b i t i o n on the de novo s y n t h e s i s of purine n u c l e o t i d e s (116) whereby the formation of r a d i o a c t i v e n u c l e o t i d e s may be decreased. However, as the decrease 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 to marked inc 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 , i t may be assumed t h a t the purine n u c l e o t i d e s formed by a l l these a l t e r n a t e pathways a l s o i s s m a l l . I t i s concluded from these s t u d i e s t h a t actinomycin D does not i n h i b i t the de novo s y n t h e s i s 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 carcinoma c e l l s in vitro. Recently K i d a , et a l . (63) have reported the e f f e c t of chromomycin A 3 on n u c l e i c a c i d metabolism of Bacillus sublitis. Chromomycin A 3 l i k e actinomycin D i n h i b i t s s e l e c t i v e l y c e l l u l a r and enzymic sy n t h e s i s of RNA by DNA dependent RNA polymerase. This s p e c i f i c i n h i b i t i o n has been confirmed by Hartmann e t a l . (123). Kida et a l . have s t u d i e d the i n c o r p o r a t i o n of 3 2 P 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 of Bacillus subtilis 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 estimated the a c i d s o l u b l e n u c l e o t i d e s from the micro-organisms t e s t e d . The a c i d s o l u b l e n u c l e o t i d e s were separated on Dowex-l-column. They have observed a marked increase of s e v e r a l n u c l e o t i d e components, p a r t i c u l a r l y those of guanine and c y t o s i n e . Though there was an increase 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 not so marked as the other two. However, they 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 the i n c u b a t i o n time. T h i s , they a t t r i b u t e to the p o s s i b l e degradation of the r a d i o a c t i v e n u c l e o t i d e s during pro-l o n g e d i n c u b a t i o n . These r e s u l t s are very much i n agreement - 98 -wi t h the r e s u l t s reported h e r e i n . Horning and Rabinowitz (124) have observed elevated l e v e l s of ATP, ADP and AMP, when Sarcoma 37 a s c i t e s were incubated i n the presence of actinomycin D. Cashel and G a l l e n t (125) r e c e n t l y reported t h a t actinomycin D does not i n h i b i t the conversion of u r i d i n e to DTP and CTP i n Ei coli c e l l s . They have observed an accumulation of both 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 actinomycin D or p r o f l a v i n . The agreement of r e s u l t s i n m i c r o b i a l and mammalian systems, suggest'that there i s no i n h i b i t i o n of the de novo synthesis of purine n u c l e o t i d e s by these a n t i b i o t i c s i n h i b i t i n g RNA polymerase. The accumulation of the n u c l e o t i d e s seen are the secondary e f f e c t produced, by the 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 . The accumulation of the 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 the de novo s y n t h e s i s through the known mechanisms of r e g u l a t i o n of purine n u c l e o t i d e s y n t h e s i s . Apart from these, there seems to be no i n h i b i t i o n caused by actinomycin D on nuc l e o t i d e b i o s y n t h e s i s in vitro. It. has been reported by Acs et a l . (126) t h a t i n Bacillus subtilisj the newly formed RNA-of high molecular weight i s r a p i d l y degraded i n presence of actinomycin D.. As these com-ponents could not be r e u t i l i z e d because of the i n h i b i t i o n of RNA polymerase by actinomycin, one could n a t u r a l l y expect an increase i n the a c i d s o l u b l e n u c l e o t i d e s i n incubations c o n t a i n -i n g actinomycin D. However, there are re p o r t s i n the l i t e r a t u r e c ontrary to the observations of Acs e t a h . Cbantrenne (127) has st u d i e d the turnover of RNA i n presence of actinomycin i n Bacillus cereus. He has concluded "that there i s no reason to b e l i e v e that actinomycin caused the d e s t r u c t i o n of RNA by any - 99 -d i r e c t a c t i o n " and t h a t actinomycin only i n h i b i t e d the RNA s y n t h e s i s "making obvious the degradative phase of the t u r n -over" . L e i v i (128) has s t u d i e d the degradation of RNA i n E. ooli c e l l s i n h i b i t e d by actinomycin D. He has observed: "there i s t h e r e f o r e no evidence t h a t actinomycin induces RNA breakdown i n the b a c t e r i a i n which i t has been t e s t e d " . He a l s o has suggested t h a t the breakdown of any RNA observed i n experiments r e f l e c t s , only the "normal breakdown of l a b i l e f r a c -t i o n of RNA and not an a r t i f a c t of actinomycin a c t i o n " . In the l i g h t of these evidences as w e l l as from the s p e c i f i c a c t i v i t i e s of substance observed i n the present i n v e s t i g a t i o n s , i t can be concluded t h a t the observed increase 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 pool i s not due to breakdown of RNA induced by a c t i n o -mycin D. Because of 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 the e f f e c t s of actinomycins i n general and actinomycin 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 i s s u e s , these were examined i n E h r l i c h a s c i t e s carcinoma c e l l s . The r e s u l t s i n d i c a t e d t h a t actinomycin D i n h i b i t e d the r e s p i r a t i o n i n - E h r l i c h a s c i t e s carcinoma c e l l s though i t was not very s i g -n i f i c a n t . S i m i l a r e f f e c t s of actinomycin are reported by other i n v e s t i g a t o r s (68). I t i s not known whether 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 by actinomycin 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 c e l l s . The present observations as w e l l as those reported by others t h a t the n u c l e o t i d e s are increased i n c e l l s incubated w i t h actinomycin D suggest 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 not 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 . G l y c o l y s i s i n E h r l i c h - 100 -a s c i t e s carcinoma c e l l s was followed by the disappearance of added glucose. I t i s known t h a t about 80% of the disappearance of glucose i n E h r l i c h a s c i t e s carcinoma c e l l s can be accounted f o r by the production of l a c t i c a c i d (117). From the present i n v e s t i g a t i o n s , i t was concluded t h a t actinomycin D has no e f f e c t on the glucose uptake of E h r l i c h a s c i t e s carcinoma c e l l s . This would mean t h a t the g l y c o l y t i c enzymes are not i n h i b i t e d by actinomycin 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 observed from previous experiments t h a t glucose 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 carcinoma c e l l s was not i n -h i b i t e d by actinomycin D. I t was a l s o observed t h a t a c t i n o -mycin D i n h i b i t e d the RNA s y n t h e s i s i n these c e l l s . Horning and Rabinowitz (81) reported t h a t actinomycin D and a v a r i e t y of other a n t i b i o t i c s i n h i b i t e d the RNA synthesis i n Sarcoma 37 a s c i t e s c e l l s i n the presence of glucose, but the p r o t e i n s y n t h e s i s was only i n h i b i t e d i n the absence of glucose. To e x p l a i n these observations they suggested a compartmentation f o r ATP and 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 species i n the c e l l . The a b i l i t y of glucose to prevent the i n h i b i t o r y e f f e c t of actinomycin D was r e l a t e d t o the higher a c c e s s i b i l i t y of ATP d e r i v e d from g l y c o l y s i s at the b i o s y n t h e t i c s i t e s i n the c e l l s . In e x p l a i n i n g the e f f e c t of glucose 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 precursors by E h r l i c h a s c i t e s carcinoma 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 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 a c i d s . In the s t u d i e s of i n c o r p o r a t i o n of 1'*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 presence of actinomycin D, i t was observed t h a t a low 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 i t y •I • 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 the substances i s o l a t e d f o r d i f f e r e n t experiments. To minimize the - 102 -v a r i a t i o n i t was d e s i r a b l e 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 1 l fC-formate. In an attempt to i n c r e a s e the i n c o r p o r a t i o n of 1*C-formate. i n t o n u c l e o t i d e and n u c l e i c acids, of E h r l i c h a s c i t e s carcinoma c e l l s , glucose was added to the medium, and the i n c o r p o r a t i o n of 1^'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 c e l l s estimated. In many e x p e r i -ments contrary to the observation of others (69-71) a decrease i n i n c o r p o r a t i o n of lkC-formate was observed. Hence t h i s pro-blem was i n v e s t i g a t e d i n greater d e t a i l . The decrease i n i n c o r p o r a t i o n of 1 I fC-formate was i r r e s -p e c t i v e of. the f a l l i n pH of the medium due t o g l y c o l y s i s , of the presence of C a + + ions i n the medium or o f the b u f f e r used f o r i n c u b a t i o n . On f u r t h e r examination i t was observed t h a t e f f e c t of glucose depended on the c e l l d e n s i t y i n the incuba-t i o n s . In a concentrated c e l l suspension, glucose 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 suspension same conc e n t r a t i o n of glucose decreased the i n c o r p o r a t i o n . These experiments suggested 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 precursors i n t o n u c l e i c a c i d components of these c e l l s was the t r a n s i e n t d e p l e t i o n and regeneration of ATP i n the c e l l s i n presence of glucose. However, glucose always increased the i n c o r p o r a t i o n of 1 1*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 carcinoma c e l l s , i n v e s t i g a t i o n s are presented below. 1) Time Course' of i n c o r p o r a t i o n of 1''C-Porraate i n t o Various  N u c l e i c A c i d Components i n Presence of Glucose. E h r l i c h a s c i t e s carcinoma c e l l s were c o l l e c t e d from 8-10 - 103 -animals 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 before. ' The c e l l s were c e n t r i f u g e d i n a r e f r i g e r a t e d c e n t r i f u g e at 1500 rpm ( I n t e r n a t i o n a l PR-IT, model) f o r 3 minutes and the super-natant f l u i d was removed. The c e l l s were washed w i t h three times the c e l l volume of c o l d 0.9% sodium c h l o r i d e and c e n t r i -fuged again at 2000 rpm f o r 4 minutes. The supernatant f l u i d was removed, and the c e l l s were then suspended i n 9 volumes of c o l d Krebs Ringer phosphate b u f f e r pH 7.8. l l*C-Formate (0.5 yc/ml suspension) and non r a d i o a c t i v e formate (2 ymoles/ml) were added to t h e . c e l l suspension and mixed w e l l . The suspen-s i o n was d i v i d e d i n t o two equal p o r t i o n s i n separate 1000 ml Erlenmeyer f l a s k s . The f l a s k s were cl o s e d w i t h a one holed rubber stopper through which a narrow polyethylene tube was i n s e r t e d . Oxygen was passed continuously i n t o the f l a s k s placed i n a water bath at 37°C and shaken at 100-110 o s c i l l a t i o n s per minute. A f t e r 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 (5%) was added to one of the f l a s k s to give a c o n c e n t r a t i o n of 1 mg/ml of suspension. The contents were mixed w e l l and i n c u b a t i o n continued.• , At i n t e r v a l s a 10 ml sample was withdrawn from each f l a s k and 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 and DNA were ex t r a c t e d as described under methods. The p e r c h l o r i c a c i d 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 n e u t r a l i z e d w i t h 10 N potassium hydroxide to pH 6.5-7 and the p r e c i p i t a t e of per-c h l p 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 the s o l u -t i o n f o r s e v e r a l hours. The supernatant s o l u t i o n c o n t a i n i n g the - 104 -a c i d s o l u b l e n u c l e o t i d e s w a s c o l l e c t e d s e p a r a t e l y . Half ml samples 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 counter. The O.D. of t h i s s o l u t i o n at 260 mu was obtained a f t e r d i l u t i n g 5 times w i t h water. 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 per O.D. u n i t p l o t t e d a g a i n s t time i s given i n Figure 13. i n some of the l a t e r experiments samples of the 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 without 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 of 0.2 M p e r c h l o r i c a c i d was- used as a blank i n these e s t i m a t i o n s . A . p o r t i o n of the RNA n u c l e o t i d e s obtained by h y d r o l y s i s of RNA during the sep a r a t i o n from DNA was d i l u t e d 8 to 10 times w i t h water. The r i b o s e content of the n u c l e o t i d e mixture was . estimated by the modified o i r c i n o l r e a c t i o n (80). AMP ( C a l -biochemicals) was used as a standard i n these estimations (81). The concentrations of 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 of umoles of 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 incorporated i n t o these n u c l e o t i d e s was determined 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 of the time course of i n c o r p o r a t i o n i s given i n Figure 14. The DNA was d i s s o l v e d i n 0.5 ml of 0.1 N NaOH and d i l u t e d 10 times. The concen t r a t i o n of the DNA i n s o l u t i o n was estimated by the diphenylamine r e a c t i o n . Highly polymerized c a l f thymus DNA (Mann Research Corporation) was used as a standard. Half ml samples of the 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 spectrophotometer to o b t a i n the r a d i o a c t i v i t y incorporated. The r e s u l t s are given i n Figure 15. This was the procedure used f o r a l l the time course of - 105 -30 60 90 120 TIME (minutes) Figure 13. 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 of l l fC-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 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 Ringer phosphate b u f f e r pH 7.3. C o n t r o l ( o © ) + glucose (——A ) - 1-05 -600 ft r-i O e \ a, o 4J •H > •H -)-> O D •H m •H o Q) Cu Ui 400 200 " 60 90 TIME (minutes) 120 180 Figure 14. 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 of 1 "*C-formate i n t o RNA by E h r l i c h a s c i t e s carcinoma cel'ls suspended i n Krebs Ringer phosphate b u f f e r pH 7.8. C o n t r o l ( -) + glucose G -k ) - 107 -30 60 90 120 TIME ( m i n u t e s ) F i g u r e 15. E f f e c t o f g l u c o s e (5.5 mil) on t h e i n c o r p o r a t i o n o f lhC-£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 c e l l s 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 pH 7.8. C o n t r o l ( O © ) + - g l u c o s e ( & ) - 108 -i n c o r p o r a t i o n s t u d i e s described i n the t h e s i s . Where ever there are changes of b u f f e r s or substrates these are mentioned. The general procedures of incubations and estimations were the same as described here. Examination of the Figure 13 shows tha t 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 a c i d s o l u b l e f r a c t i o n was markedly increased w i t h time i n presence of glucose. This was i n agreement w i t h the observations of other i n v e s t i g a t o r s . However, from f i g u r e s 14 and 15 i t can be seen t h a t the i n c o r p o r a t i o n of 1^C-formate i n t o both RNA and DNA was decreased w i t h time i n the incubations c o n t a i n i n g glucose which was i n c o n t r a s t w i t h r e s u l t s obtained by others. Because of the disagreement i n the r e s u l t from t h a t observed by o t h e r s , the experiment was repeated s e v e r a l times under s i m i -l a r c o n d i t i o n s . In a l l cases an increase i n the i n c o r p o r a t i o n of lhC-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 was n o t i c e d . How-ever l a t e r experiments revealed t h a t t h i s increase cannot be ac-counted f o r e n t i r e l y by the i n c o r p o r a t i o n of 1 I fC-formate i n t o n u c l e o t i d e s alone. In s p i t e of an apparent i n c r e a s e , when 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 o t i d e purines was deter-mined, a decrease was n o t i c e d i n many cases i n presence of glucose. L a t e r i t was a l s o found t h a t much of the 1^C-formate was i n c o r -porated i n t o s e r i n e i n presence of glucose which appeared i n the 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 to the apparent increase i n s p e c i f i c a c i t i v i t y . In the case of RNA, the i n c o r p o r a t i o n of lhC-formate was lower than c o n t r o l s i n many experiments i n presence of glucose, - 109 -though the decrease was not always s i g n i f i c a n t . S i m i l a r r e -s u l t s were a l s o obtained when the i n c o r p o r a t i o n of 1'*C-formate 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 disagreement i n r e s u l t s from those obtained by o t h e r s , w i t h respect to the e f f e c t of glucose on i n c o r p o r a t i o n of 1'*C-formate by E h r l i c h carcinoma c e l l s , v a r i o u s other 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 1 4C-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 omitted calcium from the b u f f e r s o l u t i o n i n which the t i s s u e was suspended. They have a l s o observed an increase i n the i n c o r p o r a t i o n of precursor of nu-c l e i c a c i d s i n the presence of glucose. In the experiments described 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 Ringer phosphate b u f f e r c o n t a i n i n g 0.86- umoles/ml of calcium c h l o r i d e . Calcium i s w e l l known as an a c t i v a t o r of ATPase and an i n h i b i t o r of o x i d a t i v e phosphorylation (129). Recently calcium has been reported to prevent the o x i d a t i o n of c i t r a t e , 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 avoid the p o s s i b i l i t y of calcium a f f e c t i n g the i n -c o r p o r a t i o n of x l >C-formate Into n u c l e i c a c i d components i n presence of glucose, the f o l l o w i n g experiment was c a r r i e d out. Incubation was c a r r i e d out as described before, but i n t h i s case, the c e l l s were d i l u t e d w i t h Krebs Ringer phosphate b u f f e r pH 7.8, c o n t a i n i n g no calcium. The suspension was d i -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 1^C-formate and non r a d i o a c t i v e formate as described before. Glucose was added to - n o -one of the f l a s k s to have a co n c e n t r a t i o n of 1 mg/ml of sus-pension, and 0 2 was f l u s h e d through the f l a s k s . Samples were withdrawn at i n t e r v a l s from both the i n c u b a t i o n mixtures. The a c i d s o l u b l e f r a c t i o n and the n u c l e i c acids were obtained from, the c e l l s . The co n c e n t r a t i o n of each of the components 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 were determined. The r e s u l t s are given i n Figures '. . 16, 17 and 18. I t can be seen from Figure 16 t h a t the i n c o r p o r a t i o n of •^-C-formate increased markedly i n presence of glucose f o r -a p e r i o d of 1 hour. On longer i n c u b a t i o n s to 2 hours there was a decrease i n i n c o r p o r a t i o n of 1'*C-formate i n both the c o n t r o l and i n incubations c o n t a i n i n g glucose. In the case of both RNA and DNA, the i n c o r p o r a t i o n of 1,lfC decreased i n presence of glucose, s i m i l a r to t h a t observed when c e l l s were suspended i n Krebs Ringer phosphate s o l u t i o n c o n t a i n -i n g calcium. So the e f f e c t exerted by glucose i n decreasing the i n c o r p o r a t i o n of 1'*C-formate i n t o the n u c l e i c a c i d may not be due to the presence of calc i u m i n the i n c u b a t i o n medium. ++ : • c .In..order to see whether Ca had any e f f e c t on the i n c o r -p o r a t i o n of 1 I fC-formate i n presence of glucose, the 'following experiment was c a r r i e d out. E h r l i c h a s c i t e s carcinoma c e l l suspension was made i n Krebs Ringer phosphate b u f f e r pH 7.8 containing- no calcium. 1'*C-Formate (0.5 uC /ml), sodium f o r -mate (2 umole/ml) and glucose (1 mg/ml) were added to the sus-pension and mixed w e l l . The s o l u t i o n was d i v i d e d i n t o two halves and enough calcium c h l o r i d e s o l u t i o n was added to one of the i n c u b a t i o n mixtures to give a co n c e n t r a t i o n of 2.58 I l l 30 60 90 120 TIME (minutes) Fig u r e 16. 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 of l l fC-formate 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 carcinoma suspended i n Ca f r e e Krebs Ringer phosphate b u f f e r pH 7.8. C o n t r o l ( •©• ) + glucose ( A- A ) - 112 -30 60 90 120 TIME (minutes) Figure 17. 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 of ^C-formate i n t o the 3jlN_A of E h r l i c h a s c i t e s carcinoma c e l l s suspended i n Ca f r e e Krebs Ringer phosphate b u f f e r pH 7 . 8 . C o n t r o l ( o © -) + glucose ( -A A ) - 113 -900 " 30 60 90 120 TIME (minutes) Fig u r e 18. 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 of lhC-formate i n t o the D_NA of E h r l i c h a s c i t e s carcinoma c e l l s suspended i n Ca T f r e e Krebs Ringer phosphate b u f f e r pH'7.8. C o n t r o l ( e © ) + glucose ( A - — A ) - 114 -umoles/ml. The suspensions were incubated as before and samples were withdrawn a t i n t e r v a l s from the i n c u b a t i o n mixtures. 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 the a c i d s o l u b l e n u c l e o t i d e s and the n u c l e i c a c i d s were e x t r a c t e d from the c e l l s . The c o n c e n t r a t i o n of the var i o u s components 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 these components were determined. These are given i n Table X I I . From the t a b l e i t can be seen th a t calcium ions exert no i n h i b i t o r y e f f e c t on the i n c o r p o r a t i o n of ^C-formate i n t o the n u c l e i c a c i d . Contrary to i n h i b i t i o n a s l i g h t s t i m u l a t o r y e f f e c t on the i n c o r p o r a t i o n of l l fC-formate i n t o n u c l e i c a c i d i s seen i n presence of calcium. However there was a s l i g h t decrease i n the i n c o r p o r a t i o n of l l fC-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 con-t a i n i n g calcium. I t i s not known whether t h i s decrease i n i n c o r p o r a t i o n i s because of higher i n c o r p o r a t i o n of the l a b e l l e d n u c l e o t i d e s i n t o the n u c l e i c a c i d s . However, from these experiments i t may be concluded t h a t the decrease i n i n c o r p o r a t i o n of 1 4C-formate i n t o the various 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 c e l l s incubated w i t h glucose was not due to the presence of calcium i n the i n c u b a t i o n medium. So i n subsequent experiments, calcium was not omitted from the Ji u f f e r s o l u t i o n used to suspend the c e l l s . 3) E f f e c t of B u f f e r on the I n c o r p o r a t i o n of ^C-Formate by E h r l i c h  A s c i t e s Carcinoma i n Presence of Glucose. 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 to g l y c o l y z e sugars. As these c e l l s e x h i b i t both Crabtree - 115 -Table X I I 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 lhC-formate by E h r l i c h a s c i t e s carcinoma c e l l s i n presence of glucose (5.5 mM). Experimental incubations con-t a i n e d 2.5 umoles/ml of calcium c h l o r i d e and calcium was absent from-the c o n t r o l s . A c i d - s o l u b l e RNA (cpm/umole) DNA (cpm/mg) F r a c t i o n AMP (cpm/O.D. ) Time C o n t r o l E x p t l . . C o n t r o l E x p t l ^  C o n t r o l Exptl, 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 - 116 -e f f e c t (118) and the Pasteur 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 accumulation of l a c t i c a c i d reduces the pH of the suspension c o n s i d e r a b l y . The lowering of pH may a f f e c t the a c t i v i t y of the n u c l e i c a c i d polymerases as the pH optima f o r these enzymes are w e l l above pH 7. For example the pH optimum f o r RNA.polymerase from E. ooli i s reported to be pK 7.9 and below pH 6.5 the enzyme i s i n a c t i v a t e d (132). The pH optimum of: DNA polymerase f o r i n t e s t i n a l mucosa, of r a t i s found to be 7.8 (133). The decrease 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 suspension i n Krebs Ringer phosphate b u f f e r pH 7.8 incubated w i t h glucose (5.5 mM) i s given i n Table X I I I . I t i s seen from the t a b l e t h a t the pH of the suspension dropped from 7.35 at 0 time to 6.7 at the end of 2 hours of i n c u b a t i o n . I t i s a l s o seen t h a t there was no decrease i n pH i n incubations which d i d not con-t a i n glucose. To overcome the change i n pH due to g l y c o l y s i s , Krebs Ringer bicarbonate b u f f e r was p r e f e r r e d to phosphate b u f f e r . Recently Poole (134) has reported t h a t the f a l l i n pH of 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 corresponded w i t h the p roduction of l a c t i c a c i d though the l a c t a t e production was about the same i n both Krebs Ringer phosphate and bicarbonate buffers.- She a l s o observed t h a t the f a l l of pH was greater i n Krebs Ringer phosphate b u f f e r than i n bicarbonate b u f f e r due to 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 bicarbonate b u f f e r . Further s t u d i e s of the' e f f e c t of glucose on the i n c o r p o r a -t i o n of 1'*C-formate was c a r r i e d out i n Krebs Ringer bicarbonate - 117 -Table X I I I Decrease i n 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 suspensions i n Krebs Ringer phos-phate b u f f e r pH 7.8, incubated w i t h glucose in vitro. A - C o n t r o l , B - C o n t r o l + glucose 5.5 mM. pH of Suspension Time A B 0 . 7.35- 7.35 7.5 7.3 7.00 15 7.3 6.80 30 . . 7.3 6.85 45 7.3 6.8 60 7.3 6.75 90 V | 7.3 6.7 120 '-<V. '•• 7.3 6.7 I / - 118 -b u f f e r . A l l other c o n d i t i o n s of i n c u b a t i o n were the same except t h a t the gas phase was a mixture of 95% O2 and 5% CO2• The pH of the suspension was checked at i n t e r v a l s and found to be s t a t -i o n a r y , a t 7.4 both i n c o n t r o l and i n presence of glucose. The time course of i n c o r p o r a t i o n of 1'*C-formate i n presence of g l u c o s e . i n Krebs Ringer bicarbonate b u f f e r i n t o a c i d s o l u b l e f r a c t i o n , RNA and DNA are given i n Figures 19, 20 and 21 respec-t i v e l y . The r e s u l t s from the f i g u r e s show t h a t the i n c o r p o r a t i o n of 1 1 1 C-formate was increa s e d 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 glucose w h i l e the same was decreased i n the RNA and the DNA. This was s i m i l a r to the f i n d i n g obtained i n the e x p e r i ments i n which Krebs Ringer phosphate was used to suspend the c e l l s . Though there was no d i f f e r e n c e i n the p a t t e r n of i n -c o r p o r a t i o n , the pH of the i n c u b a t i o n mixture was s t a b i l i z e d by bicarbonate b u f f e r as observed from a d i r e c t determination, because of 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 Krebs Ringer bicarbonate b u f f e r compared, t o the Krebs Ringer phosphate b u f f e r As there was decreased i n c o r p o r a t i o n of 1 4C-formate i n t o both RNA and DNA i n presence of glucose i t was of i n t e r e s t to see the extent of i n h i b i t i o n i n the nu c l e o t i d e s of RNA and bases of DNA. The n u c l e o t i d e s obtained by h y d r o l y s i s of the RNA from-these experiments were adsorbed on c h a r c o a l , 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 mixture and were separated by two dimen-s i o n a l paper chromatography. The DNA was hydrolysed and the bases separated a l s o by paper chromatography. 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 these substances were estimated and are given - 119 -, i i « • ^ 30 60 90 120 TIME (minutes) Figure 19. 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 of 1'*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 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 Ringer bicarbonate b u f f e r pH 7.8. C o n t r o l ( © © ) + glucose ( A A-——) 120 -30 60 90 120 TIME (minutes) Figure 20. 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 of 1^C-formate i n t o the RNA 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 Ringer bicarbonate b u f f e r pH 7.8. C o n t r o l ( © «> ) + glucose ( A -A ) - 121 -30 60 90 120 TIKE (minutes) F i g u r e 21. 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 of 1 HC-formate 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 Ringer bicarbonate b u f f e r pH 7.8. C o n t r o l (—:—© o ) + glucose ( A A ) Table XIV I n c o r p o r a t i o n of 1 ^ C-formate i n t o RNA n u c l e o t i d e s and DNA thymine of E h r l i c h a s c i t e s carcinoma c e l l s in vitro, incubated w i t h glucose (5.5 mM). The c e l l s were suspended i n Krebs Ringer bicarbonate b u f f e r pH 7.8. A - C o n t r o l , B -C o n t r o l + glucose. Time min- • utes S p e c i f i c a c t i v i t y counts p e r minute per umole ... RNA DNA AMP GMP CMP UMP Thymine A B A B ; A B A B A B 7.5 46 21 0 0 12 21 2 0 196 119 30 72 44 50 8 35 11 2 0 552 323 60 165 52 87 . 42 84 31 72 0 743 449 120 661 441 301 208 208 71 126 16 1780 626 . - 123 -i n Table XIV.. In DNA, the i n c o r p o r a t i o n of ^ C-formate was mainly i n the thymine. Though there 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 purine bases of DNA, they were too low to have any s i g n i f i c a n c e . Examination of the r e s u l t s show t h a t glucose decreased the i n c o r p o r a t i o n of 1'*C-formate i n t o the purine n u c l e o t i d e s of RNA and t o the thymine of DNA. 4) E f f e c t of Glucose on the I n c o r p o r a t i o n of 2T.* ^ C-Glycine by  E h r l i c h A s c i t e s Carcinoma C e l l s in vitro. In studying the i n c o r p o r a t i o n of l l fC-formate i n t o the n u c l e i c acids of va r i o u s 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 carbon u n i t formed from formate can exchange w i t h carbon 2 (C 2) of the purine r i n g . In a d d i t i o n to the formation of the purine r i n g , : 1'*C-formate i s a l s o incorporated i n t o the methyl group of thymine. Henderson and LePage (70) pointed out t h a t because of 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 to g l y c i n e for. the study of de novo synthesis of purines. They have recommended r a d i o a c t i v e g l y c i n e i n preference to f o r -mate as a precursor of pu r i n e s . In order to see whether, glucose exerted an e f f e c t on the i n c o r p o r a t i o n of 2- 1^C-glycine s i m i l a r to that observed f o r 1^C-formate, an experiment was per-formed using 2 - 1 ^ C - g l y c i n e as the r a d i o a c t i v e precursor of n u c l e i c a c i d s . Before i n v e s t i g a t i n g the e f f e c t of glucose, 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 of 2- 1^C-glycine by E h r l i c h a s c i t e s carcinoma was i n v e s t i g a t e d . E h r l i c h a s c i t e s c e l l suspensions were prepared i n Krebs Ringer bicarbonate b u f f e r pH 7.8 as d e s c r i b e d before. I n -cubations were c a r r i e d out i n 250 ml Erlenmeyer f l a s k s i n a water bath at 37°C. Twenty-five micro 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 0.2 uC of 2- 1^C-glycine/ml (14.5 mg of glycine/ml) was added to each f l a s k . . Non r a d i o a c t i v e g l y c i n e d i s s o l v e d i n Krebs Ringer bicarbonate b u f f e r was added to appropriate f l a s k s to 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 incubated f o r 2 hours at 37°C and were kept shaken during the p e r i o d of i n c u b a t i o n . At the end of the p e r i o d , the t i s s u e was recovered 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 and the washings were discarded. 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 from the washed t i s s u e p e l l e t 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 . 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 n u c l e o t i d e s determined by l i q u i d s c i n t i l l a t i o n counting. The i n c o r p o r a t i o n of 2- 1^C-glycine at d i f f e r e n t concentrations of added g l y c i n e i s given i n Figure 22. Examination 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 g l y c i n 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 decreased v/ith increase i n concentra-tion, of the non 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. The decreased i n c o r p o r a t i o n of 2 - 1 4 C - g l y c i n e i n presence of added g l y c i n e may be due to the enlargement of the pool s i z e and the d i l u t i o n of the r a d i o a c t i v e compound. I t may be pointed out t h a t t h i s sample of g l y c i n e had a low s p e c i f i c a c t i v i t y (.1 mC/72.5 mg of g l y c i n e ) . . No other sample of g l y c i n e v/as a v a i l a b l e and under these circumstances t h i s sample was used and there was no requirement of any added g l y c i n e f o r the i n c o r p o r a -t i o n of 2 - 1 ^ C - g l y c i n e . Johnstone and S c h o l e f i e l d (135) have r e c e n t l y reported t h a t amino acids are t r a n s p o r t e d i n t o and - 125 -10 20 30 40 Added g l y c i n e (ymole/10 ml suspension) Fig u r e 22. E f f e c t of g l y c i n e on the i n c o r p o r a t i o n of 2- 1^C-glycine . by E h r l i c h a s c i t e s carcinoma c e l l s in vitro. - 126 - '. concentrated i n E h r l i c h a s c i t e s carcinoma.cells by an a c t i v e process. This was a l s o suggested by Christeneen (136) previous-l y . I t i s p o s s i b l e t h a t the sm a l l amount of r a d i o a c t i v e g l y -c i n e added i s concentrated by the E h r l i c h a s c i t e s carcinoma c e l l s to give 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 of non r a d i o a c t i v e precursor was necessary f o r the maximum 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 g l y c i n e . Toctest the e f f e c t of glucose on the i n c o r p o r a t i o n of 2-1 R c - g l y c i n e , incubations were c a r r i e d out w i t h E h r l i c h a s c i t e s c e l l s i n Krebs Ringer bicarbonate b u f f e r . Each f l a s k con-t a i n e d 10 ml of 10% v/v c e l l suspensions and 25 u l of a s o l u -t i o n of 2- 1^C-glycine (0.2 pC/ml). Glucose was added to the appropriate f l a s k to give a con c e n t r a t i o n of 5.5 mM. Incuba-t i o n was c a r r i e d out i n 250 ml f l a s k s as described. 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 and the n u c l e i c acids were e x t r a c t e d from the c e l l s . The r a d i o a c t i v e g l y c i n e incorporated i n t o each of the f r a c t i o n s was determined by l i q u i d s c i n t i l l a t i o n counting. F u r t h e r , 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 e l u t e d w i t h a mixture of p y r i d i n e and a l c o h o l . The purine n u c l e o t i d e s were hydrolyzed t o the bases by the method of V i s c h e r and Chargaff (94) and separated by chromatography i n Kirb y ' s s o l v e n t system and i n s o l v e n t system, of Hershey et a l . (95). The substances were e l u t e d from the chromatograms and the s p e c i f i c . . a c t i v i t y of. the purine bases determined. The nucleo-t i d e s obtained by h y d r o l y s i s of RNA v/ere i s o l a t e d by charcoal adsorption and e l u t i o n and separated by paper chromatography. The DNA was hydrolyzed w i t h 72% p e r c h l o r i c a c i d and the bases - 127 -separated by chromatography. The s p e c i f i c a c t i v i t i e s of the purines 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 are given i n Table 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 purines decreased i n presence of glucose. 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- 1 "*C-glycine i n t o both purine of RNA and DNA was decreased i n i n c u b a t i o n con-t a i n i n g glucose. The decrease i n i n c o r p o r a t i o n i n t o RNA i s even greater than i n DNA. In these experiments, the e f f e c t of glucose on 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 precursors i n t o the n u c l e i c a c i d com-ponents of E h r l i c h a s c i t e s carcinoma c e l l s incubated in vitro was 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 r e s u l t s obtained .so f a r showed tha t the e f f e c t of glucose does not depend on the r a d i o a c t i v e precursor employed as the same or s i m i l a r e f f e c t s were seen w i t h l !*C-formate and w i t h 2- 1 ^C-glycine. The e f f e c t a l s o does not seem to depend on the b u f f e r s o l u t i o n used to suspend the c e l l s , though Krebs Ringer bicarbonate b u f f e r main-t a i n e d the pH i n presence of glucose. Study using the d i f f e r e n t b u f f e r s a l s o showed tha t the e f f e c t observed f o r glucose was not due t o the lowering of pH caused by g l y c o l y s i s . 5) The I n c o r p o r a t i o n of 1^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 B u f f e r s . In the experiments c a r r i e d out to study the e f f e c t of glucose 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 precursors 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 suspensions were made e i t h e r i n Krebs Ringer phosphate b u f f e r or Krebs Ringer bicarbonate b u f f e r . Table XV E f f e c t of glucose on the i n c o r p o r a t i o n of 2- 1 "*C-glycine i n t o the 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 and purines of E h r l i c h a s c i t e s carcinoma c e l l s in vitro. S p e c i f i c a c t i v i t y -counts per minute per umole. A d d i t i o n s A. s o l u b l e RNA DNA A c i d s o l u b l e f r a c t i o n RNA DNA — . - • • : x 10 cpm/ cpm/ cpm ymole mg Ade- Guanine Hypoxt-n-Ade- Gua- Ade- Gua-AMP nine an thine, nine nine nine nine 1 134 821 660 2167 9741 4371 250 181 118 107 c e l l suspension 2 140 955 517 2432 10015 6185 269 225 104 118 1 126 181 871 1703 5130 3655 91 38 85 81 c e l l + glucose 2 150 825 669 1957 5997 5089 128 96 85 ] 111 - 1 2 9 -As there was v a r i a t i o n , i n the amount of i n c o r p o r a t i o n of the l a b e l l e d compounds i n these b u f f e r s , i t was of i n t e r e s t to study the i n c o r p o r a t i o n , of l l fC-formate v/ith respect to the b u f f e r s used. E h r l i c h a s c i t e s carcinoma 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 prepared 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 Ringer phos-phate b u f f e r and the other i n Krebs Ringer bicarbonate b u f f e r t o give a 1 i n 10 suspension i n each case. Enough of ' 1 J fC-formate (0.5 uC/ml) and sodium formate (2 umole/ml) were added to the suspensions and mixed w e l l . Ten ml f r a c t i o n s of these suspensions i n 250 ml Erlenmeyer f l a s k s were used f o r the 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 experiments w i t h c e l l s suspended i n each' of the b u f f e r s were a l s o c a r r i e d out. A f t e r i n c u b a t i o n s the c e l l s were sep-ara t e d , and the a c i d s o l u b l e f r a c t i o n and the n u c l e i c acids were e x t r a c t e d . The 1'*C-formate inc o r p o r a t e d i n t o each of the f r a c t i o n s was determined. The r e s u l t s are given i n Table XVI. From the data given i n the t a b l e i t appears t h a t the amount of i n c o r p o r a t i o n of 1'*C-formate i s s l i g h t l y higher i n c e l l s suspended i n phosphate b u f f e r . I t i s conceivable t h a t much of the i n o r g a n i c phosphate i s used i n the 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 a c i d s . The g e n e r a l l y decreased i n c o r -p o r a t i o n i n bicarbonate b u f f e r may be r e l a t e d to the decreased a v a i l a b i l i t y of I n o r g a n i c phosphate. Hershko e t . a l . (137) have r e c e n t l y reported t h a t presence of i n o r g a n i c phosphate i n the i n c u b a t i o n medium markedly s t i m u l a t e d the i n c o r p o r a t i o n of - 130 -Table XVI Inc o r p o r a t i o n of 1 "^C-formate i n t o the 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 suspended i n two d i f f e r e n t b u f f e r s . Conditions of incubations were same i n both experiments. Krebs Ringer phosphate Krebs Ringer Bicarbonate b u f f e r ... b u f f e r A c i d s o l u b l e RNA DNA A c i d s o l u b l e RNA. DNA Ad d i t i o n s cP m/O.D. cpm/u cpm/ cpm/O.D. cpm/y cpm/ mole. AMP. mg mole. AMP. mg 2066 204 683 1450 350 616 C e l l Suspension 1878 229 548 1460 250 '773 C e l l suspension . + 3776 glucose (5.5 mM) 3511 13 8 111 324 374 2809 3134 226 781 238 700 - 131 -purines i n t o r i b o n u c l e o t i d e s i n r a b b i t e r y t h r o c y t e s . This s t i m u l a t i o n was a t t r i b u t e d to an increase i n PRPP i n the c e l l s . Wu (138) has reported t h a t 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 -cinoma c e l l s i s r e g u l a t e d by the i n o r g a n i c phosphate concentra-t i o n s . Henderson and Khoo (139) observed t h a t glucose can form PRPP i n E h r l i c h a s c i t e s carcinoma c e l l s . So i n f u r t h e r e x p e r i -ments Krebs Ringer phosphate was used to suspend the E h r l i c h a s c i t e s carcinoma c e l l s . 6) Increased I n c o r p o r a t i o n of x t tC-Formate by E h r l i c h A s c i t e s  Carcinoma C e l l s i n Presence of Glucose. I t has been g e n e r a l l y accepted by others t h a t glucose increased 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 precursors 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 present 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 the c o n d i t i o n s of these i n v e s t i g a t i o n s the general e f f e c t of glucose was to decrease the i n c o r p o r a t i o n of 1 !*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 experiments i n agreement w i t h the r e s u l t s of o t h e r s , an increase i n i n c o r p o r a t i o n of l i fC-formate was observed when E h r l i c h carcinoma c e l l s were incubated w i t h glucose. The r e s u l t s of one such experiment i s given i n Table. XVII. 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 l f*C-formate doubled i n some cases i n presence of glucose. This i i s i n agreement /with r e s u l t s reported 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 was s t u d i e d by many i n v e s t i g a t o r s . Glucose i s phosphorylated q u i c k l y - 132 -Table XVII E f f e c t of glucose on the i n c o r p o r a t i o n of 1^C-formate in vitro i n t o the various 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 . S p e c i f i c A c t i v i t y - cpm/umole A c i d - s o l u b l e A RNA DNA f r a c t i o n A d d i t i o n s Adenine Guan- Hypox- AMP GMP Thymine ine anthine 1 2738 3374 2970 163 287 1568 C e l l suspension 2 2476 3158 2797 173 242 1746 1 4428 5581 5162 289 482 1743 C e l l suspension + glucose 2 5620 7604 7376 385 605 1675 -133-and g l y c o l y z e d to g i v e l a c t i c a c i d . The o x i d a t i o n of t h i s l a c t i c a c i d i s l i m i t e d (131). Thomson et a l . (71) have observed t h a t the net 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. The glucose meta-bolism and the ATP 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 stud i e d by Overgaard-Hansen r e c e n t l y (73) . She observed th a t when glucose was added to a . d i l u t e suspension of E h r l i c h a s c i t e s carcinoma c e l l s there was a sudden d e p l e t i o n of ATP. The regeneration of ATP under these c o n d i t i o n s was a comparatively slow process and even a f t e r 2 hours the ATP l e v e l d i d not r e t u r n to normal. The d e p l e t i o n of ATP i s brought about by a s e r i e s of r e a c t i o n s i n which hexokinase, myokinase, and adenylate deaminase take p a r t (73). The n u c l e o t i d e s were hydrolyzed to the nucleosides and these substances escaped i n t o the medium i n which the c e l l s were suspended. The ATP regenerated through the process of g l y c o l y s i s was not able to b r i n g back the nucleo-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 a l s o observed 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 , and regeneration i s q u i c k e r i n presence of glucose i n c e l l suspensions of higher c o n c e n t r a t i o n as determined by packed c e l l volume. Recently Laws and S t r i c k l a n d (140) have a l s o reported a lower, c o n c e n t r a t i o n of ATP i n a s c i t e s c e l l s t r e a t e d w i t h glucose. The de novo s y n t h e s i s of n u c l e o t i d e s and the phosphoryla-t i o n s to give nucleoside triphosphates are a l l r e a c t i o n s r e q u i r i n g ATP. Purine n u c l e o t i d e s are reported to 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 i s formed (28). A decrease i n ATP l e v e l s i n the c e l l s thus can decrease the i n c o r p o r a t i o n of 1 "*G-formate both 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 . In seeking an exp l a n a t i o n f o r the decreased i n c o r p o r a t i o n of l a b e l l e d precursors 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 presence of glucose, i t may be sug-gested t h a t glucose r a p i d l y depleted the ATP of the c e l l s , the regeneration of which was delayed i n d i l u t e c e l l suspensions. The increase i n i n c o r p o r a t i o n of 1^C-formate.seen i n some' e x p e r i -ments may be due to the 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 synthesis i n denser c e l l suspensions, as observed by Overgaard-Hansen. From the r e s u l t s obtained i n the present i n v e s t i g a t i o n s , as w e l l as those reported by Overgaard-Hansen, i t was f e l t t h a t there are two f a c t o r s c o n t r o l l i n g the in c o r p o r a -t i o n of 1^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 pres-ence of glucose. The f i r s t i s the 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 the second the sugar conce n t r a t i o n i n the suspension. By v a r y i n g the sugar c o n c e n t r a t i o n at a p a r t i c u l a r c e l l concentra-t i o n i t may be p o s s i b l e to vary the d e p l e t i o n of adenine nucleo-t i d e s and regeneration of ATP. The same e f f e c t may be produced by v a r y i n g the c e l l c o n c e n t r a t i o n i n suspension at a p a r t i c u l a r sugar c o n c e n t r a t i o n . These may be r e f l e c t e d i n the s t i m u l a t i o n or i n h i b i t i o n of i n c o r p o r a t i o n of 1'*C-formate by E h r l i c h a s c i t e s carcinoma c e l l s . . To v e r i f y t h i s hypothesis two s e r i e s of experiments were performed. 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 the sugar c o n c e n t r a t i o n was v a r i e d and the i n c o r p o r a t i o n of l l fC-formate at d i f f e r e n t sugar concentrations i n t o v arious n u c l e i c a c i d f r a c t i o n s was determined. In the second s e r i e s the - 135 -sugar c o n c e n t r a t i o n the suspension was kept constant, and the c e l l c o n c e n t r a t i o n v a r i e d and the 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 of Varying Concentration of Glucose on the Incorpora- t i o n of 1 ''C-Formate by E h r l i c h A s c i t e s Carcinoma C e i l s in  vitro. E h r l i c h a s c i t e s carcinoma c e l l s were c o l l e c t e d as described before. The c e l l s were d i l u t e d w i t h 8 volumes of Krebs Ringer phosphate b u f f e r pH 7.8. The packed c e l l volume of t h i s suspen-s i o n was determined 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 described under methods. (The packed c e l l volume of c e l l suspension prepared by the usual procedure of d i l u t i o n w i t h 9 volumes of 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 out i n 250 ml Erlenmeyer f l a s k s as described under methods. Each f l a s k contained 10 ml of c e l l suspension 5 uC.L of 1 "*C-formate, 20 umoles of sodium formate and v a r y i n g concentrations of glucose. 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 were i s o l a t e d from the c e l l s . They were hydrolyzed and the bases separated by paper chromatography. The substances from the chromatograms were e l u t e d and estimated by u l t r a v i o l e t spectro-photometry. The r a d i o a c t i v i t y i n the bases was determined 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 are given i n Table X V I I I . Examination of the data i n the t a b l e shows t h a t , i n some cases, the i n c o r p o r a t i o n of lhC-formate i n t o the various n u c l e i c a c i d components was increased i n presence of glucose. On f u r t h e r Table XVIII E f f e c t s of v a r y i n g c o n c e n t r a t i o n of glucose on the i n c o r p o r a t i o n of 1 ''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 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 -counts per minute per umole. . . . A c i d s o l u b l e f r a c t i o n RNA DNA Concentra-t i o n of cpm/O.D. Ade Gua Hypx. Ade Gua Ade Gua Cyt Thymine glucose mg % 0 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 Ade - Adenine, Gua - Guanine, Hypx. - Hypoxanthine, Cyt - Cy t o s i n e . - 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 glucose to 500 mg%, the i n -c o r p o r a t i o n of 1 "*C-formate was decreased. Stewart and Zbarsky (141) have reported s i m i l a r r e s u l t s i n studying the i n c o r p o r a t i o n of ^C-formate by i n t e s t i n a l 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 ob-t a i n e d an increase i n the i n c o r p o r a t i o n of l l tC-formate w i t h a c o n c e n t r a t i o n of 50 mg% of glucose 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 i n c o r p o r a t i o n reported by them i s comparable to the present observations. 8) I n c o r p o r a t i o n of uC-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s as a Function of C e l l Concentration. In the second s e r i e s of experiments the c e l l c o n c e n t r a t i o n was v a r i e d and sugar c o n c e n t r a t i o n was kept constant at 100 mg%. A s c i t e s carcinoma c e l l s were c o l l e c t e d and washed as described p r e v i o u s l y . The c e l l s were suspended in'::twice the volume of Krebs Ringer phosphate b u f f e r pH 7.8. This suspension was d i l u t e d s e r i a l l y to give three d i f f e r e n t c o ncentrations. The packed c e l l volume of each suspension was determined by c e n t r i f u g a t i o n using hematocrit;. tubes. Incubation 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 bath at 37°C f o r 2 hours and the f l a s k was kept shaken during the p e r i o d of i n c u b a t i o n . Each f l a s k contained, 1 mg of glucose, 0.5 uC of l 4C-formate and 2 umoles of sodium formate per ml of c e l l suspension. Enough c e l l suspension was measured i n t o each f l a s k so as to give 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 obtained from each f l a s k , they were hydrolysed s u i t a b l y , to give the bases and the bases separated by paper" chromato-graphy. The U.V. absorbing m a t e r i a l from the paper was e l u t e d and estimated by u l t r a v i o l e t spectrophotometry. . 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 v a r i o u s f r a c t i o n s was determined by l i q u i d s c i n t i l l a t i o n counting procedures. The r e s u l t s are given i n Tables XIX and XX. Examination of the t a b l e s shows t h a t the i n c o r p o r a t i o n of l l*C-formate increased w i t h the increase i n c o n c e n t r a t i o n of c e l l s i n suspension. Thus 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 each of the f r a c t i o n s i s the highest i n c e l l s having a 25% packed c e l l volume; under the c o n d i t i o n s of the experiment. This value decreases as the c e l l c o n c e n t r a t i o n i s decreased. In these incubations the volume of c e l l s was more or l e s s kept constant and 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 of c e l l suspension v a r i e d from experiment to e x p e r i -ment. The decrease 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 ob-served i n decreasing the d e n s i t y of c e l l suspensions may p a r t l y be due to'the 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 increase i n volume of the i n c u b a t i o n medium. The e f f e c t of glucose 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 suspension to 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 1^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 , RNA and DNA bases -yas higher i n incubations c o n t a i n i n g glucose except f o r the l a s t experiment, where there was a decrease n o t i c e d . In the i n c u b a t i o n where the packed c e l l volume was the lowest, there may be a d e p l e t i o n of ATP and the Table XIX I n c o r p o r a t i o n of 1 ^ - f o r m a t e i n t o the purines of a c i d s o l u b l e n u c l e o t i d e s and of RNA of E h r l i c h a s c i t e s tumour c e l l s i n presence of glucose at d i f f e r -ent c o n c e n t r a t i o n s o f c e l l suspension. A - C o n t r o l , B - C o n t r o l + glucose 5.5 mM. S p e c i f i c a c t i v i t y -counts per minute per ; . ./„.•.'.. .. . ;;; ymo.le. ... A c i d s o l u b l e f r a c t i o n ...... Packed Adenine Guanine Hypoxanthine c e l l :  volume A B A B A B 25% 14019 53491 11776 50661 28390 15% 9466 33406 7405 24412 13875 7% 5633 8529 5094 5960 12162 3.5% 872 685 1842 1040 RNA I Adenine Guanine M co : : ^ A .... B A B 65880 1208 4811 312 907 31590 845 2611 178 403 15097 583 646 120 173 282 166 57 48 Table XX I n c o r p o r a t i o n of 1 **C-formate i n t o the bases of DNA of E h r l i c h a s c i t e s tumour c e l l s i n presence of glucose (5.5 mM) at d i f f e r e n t concentrationstof c e l l s i n suspension. A - C o n t r o l , B - C o n t r o l + glucose (5.5 mM). S p e c i f i c a c t i v i t y - counts per minute, per umole Packed Adenine Guanine Cytosine Thymine C e l l volume • A B A B A B A B 1 . . : 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 3.5% 2 8 21 57 49 34 27 822 387 - 141 -regeneration of t h i s i s not as f a s t as i n case of dense suspen-s i o n . In a d d i t i o n to the decrease i n ATP, there may be the non-a v a i l a b i l i t y of other c o f a c t o r s which c o n t r i b u t e s "to the 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 a c i d components i n d i l u t e s o l u t i o n s i n presence of glucose. 9) E f f e c t of 2-deoxyglucose on the I n c o r p o r a t i o n of 1 Re-Formate by E h r l i c h A s c i t e s Carcinoma C e l l s i n vitro. To f u r t h e r v e r i f y the r e l a t i o n between the d e p l e t i o n of ATP and the decrease i n i n c o r p o r a t i o n of lkC-formate i n E h r l i c h a s c i t e s carcinoma c e l l s another experiment was c a r r i e d out using 2-deoxyglucose i n s t e a d of glucose. 2-Deoxyglucose i s taken up by E h r l i c h a s c i t e s carcinoma c e l l s , l i k e glucose and i s phos-phorylated to give the 2-deoxyglucose 6-phosphate by hexokinase using ATP as the phosphate donor (117). As the phosphorylated 2-deoxyglucose i s not f u r t h e r metabolized, there i s no p o s s i b i l i t y of regeneration of ATP through g l y c o l y s i s . So 2-deoxyglucose can be used to deplete the ATP i n the c e l l (142) and under such circumstances, the i n c o r p o r a t i o n of 1 I fC-formate should 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 suspensions. This was t e s t e d i n the f o l l o w i n g experiment. 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 according 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 noted and c e l l s were suspended i n four volumes of Krebs Ringer phosphate b u f f e r pH 7.8. A p o r t i o n of the suspension was d i l u t e d w i t h equal volume of the same b u f f e r . C e l l volume of these suspensions w e r e determined. Incubations were c a r r i e d out i n 250 ml Erlenmeyer f l a s k s at 37°C f o r 120 minutes. - 142 -The gas phase was oxygen.. Each f l a s k contained 5 uc of l"*C-formate and 20 umole of sodium formate. 2-Deoxyglucose (£!albiochem-i c a l s A grade - glucose free) was added t o the appropriate f l a s k to give a f i n a l c o n c e n t r a t i o n of 5.5 mM. An amount of c e l l suspension corresponding t o 1 ml of c e l l volume was added to each f l a s k . 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 f r a c t i o n , RNA and DNA were i s o l a t e d . 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 RNA a c i d s o l u b l e f r a c t i o n and DNA were determined as described:under methods. The 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 were f u r t h e r hydrolyzed t o give the bases by methods de-s c r i b e d : The bases were separated by paper chromatography. The r a d i o a c i t i v i t y i n corporated i n t o the purine bases of the various f r a c t i o n s was determined 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 are given i n Tables XXI, XXII and X X I I I . Examination of the r e s u l t s show a marked decrease i n the i n c o r p o r a t i o n of l l fC-formate 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 2-deoxyglucose was increased markedly, 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 decrease i n the i n c o r p o r a t i o n was n o t i c e d . S i m i l a r r e s u l t s were obtained p r e v i o u s l y when glucose was present i n the i n c u b a t i o n mixture. In 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 to the n u c l e i c a c i d bases, the decrease was greater compared to the decrease caused by glucose under s i m i l a r condi-t i o n s . I t i s seen from the t a b l e t h a t 2-deoxyglucose decreased the i n c o r p o r a t i o n of 1 ! tC-formate both i n concentrated and i n - 143 -Table XXI E f f e c t of 2-deoxyglucose on the i n c o r p o r a t i o n of 1^C-formate i n t o the 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 in vitro. A - C o n t r o l , B - C o n t r o l + 2-deoxyglucose (5.5 mM). A c i d s o l u b l e RNA DNA F r a c t i o n . cpm/O.D. cpm/umole AMP epm/mg Packed c e l l volume A B A B A B 1 1571 7064 464 113 1377 202 14% 2 1693 . 7053 476 98 1472 204 1 1944 .7028 417 100 1351 162 6.5% 2 2033 7113 402 70 1297 138 Table XXII E f f e c t of 2-deoxyglucose on the i n c o r p o r a t i o n of 1^C-formate i n t o the purines of a c i d s o l u b l e n u c l e o t i d e s and RNA of 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 niM) . S p e c i f i c a c t i v i t y - counts per minute per umole A c i d - S o l u b l e n u c l e o t i d e s . RNA Packed c e l l Adenine Guanine . . . Hypoxanthine Adenine Guanine volume A B A B A B A B A B 3666 230 3347 1596 3660 648 376 26 255 91 14% 3572 131 2650 1303 2899 591 363 38 258 81 3665 115 3010 1312 3540 551 317 14 188 #7 6.5% 3581 114 2394 1283 3487 228 310 23 185 83 Table X X I I I E f f e c t of 2-deoxy glucose on the i n c o r p o r a t i o n of 1^C-formate i n t o the bases of DNA of 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) S p e c i f i c a c t i v i t y -counts per minute per umole Packed c e l l volume Adenine B Guanine Cytosine Thymine A B B B 14% 6.5% 255 294 38 54 16 319 325 48 52 13 32 23 36 36 47 72 21 1806 1819 1679 1668 258 297 177 168 - 146 -d i l u t e - c e l l suspensions. This i s i n c o n t r a s t w i t h the r e s u l t obtained w i t h glucose, where i n h i b i t i o n was obtained only i n d i l u t e c e l l suspensions. This may be due to the d i f f e r e n c e i n r e s t o r a t i o n of adenine n u c l e o t i d e pool when 2-deoxyglucose i s present i n the—medium. Overgaard-Hansen (73) has observed t h a t i n the presence of 2-deoxyglucose., the adenine n u c l e o t i d e pool i n E h r l i c h a s c i t e s carcinoma c e l l was depleted t o . g r e a t e r extent than i n the presence of glucose.. The d e p l e t i o n i n the former case i s not reversed because the 2-deoxyglucose i s not metabolized f u r t h e r than the i n i t i a l p hosphorylation. Overgaard-Hansen has a l s o observed an increase i n the t o t a l c o n c e n t r a t i o n of i n o s i n e , adenosine and hypoxanthine as the nu c l e o t i d e pool i s being depleted. The l a c k of regeneration o f ATP, even i n dense c e l l suspension i n presence of 2-deoxyglucose may be the reason f o r 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 of ^C-formate observed i n dense suspension of E h r l i c h a s c i t e s carcinoma c e l l s . The greater 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 presence of 2-deoxyglucose, than i n presence of glucose 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 the greater d e p l e t i o n of ATP i n the c e l l s . 10) I n c o r p o r a t i o n of 1'*C-Formate by E h r l i c h A s c i t e s Carcinoma  C e l l s Under Various Conditions A f f e c t i n g ATP Formation. I f the co n c e n t r a t i o n of 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 increased i n the c e l l by some mechanism, then there i s a p o s s i b i l i t y of i n c o r p o r a t i n g more 1^C-formate i n t o the various n u c l e i c a c i d components. As the c e l l membrane i s impermeable to polyphosphates, ATP may not pass through i t and the concentra-t i o n of t h i s m a t e r i a l cannot be r a i s e d i n the c e l l by the d i r e c t - 147 -a d d i t i o n of ATP to c e l l suspensions. Some years ago Edmonds and LePage (143) observed t h a t XItC-AMP added t o E h r l i c h a s c i t e s carcinoma c e l l suspension was found to be i n c o r p o r a t e d . i n t o the n u c l e i c acids of the c e l l . Based on t h i s observation Wu and Racker (144) c a r r i e d out incubations of E h r l i c h a s c i t e s carcinoma c e l l s i n presence of glucose and AMP and observed an inc r e a s e of ATP co n c e n t r a t i o n i n the c e l l . This procedure was undertaken to increase ATP i n c e l l s and thereby to increase the i n c o r p o r a -t i o n of lkC-formate i n t o the 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 carcinoma c e l l s . These experiments are described 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 Ringer phosphate b u f f e r pH 7.8 to give a co n c e n t r a t i o n of about 10% (v/v). Incubations were c a r r i e d out i n a t o t a l volume of 10 ml of t h i s suspension i n 250 ml Erlenmeyer f l a s k s under c o n d i t i o n s as described p r e v i o u s l y . Each f l a s k contained 9.5 ml suspension, 5 y.'C of 1 ^ C-formate and 20 umoles of sodium-formate. The f l a s k s were kept cooled i n i c e during the a d d i t i o n of s u b s t r a t e s . The co n c e n t r a t i o n of glucose where ever present was 5.5 mM and AMP was 1 mM. D i n i t r o p h e n o l , a w e l l known un-cou p l i n g agent of o x i d a t i v e phosphorylation (129) was added to some incubations when r e q u i r e d to stop the formation of ATP. The f i n a l c o n c e n t r a t i o n of the 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 to 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 , the a c i d s o l u b l e f r a c t i o n , RNA, and DNA were separated. The a c i d s o l u b l e n u c l e o t i d e s were hydrolyzed and the bases separated by paper chromatography. The c o n c e n t r a t i o n of DNA and RNA were determined as described - 148 -p r e v i o u s l y . The r a d i o a c t i v i t y i n the samples was estimated 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 are given i n Tables XXIV and XXV. The r e s u l t s of these experiments f u r t h e r confirm the r e -l a t i o n between ATP generation and 1'*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 . The attempt to r a i s e the i n t r a c e l l u l a r c o n c e n t r a t i o n of ATP by the a d d i t i o n of AMP and glucose to the medium f a i l e d to give the d e s i r e d e f f e c t . The presence of AMP i n the i n c u b a t i o n s l i g h t l y decreased the i n -c o r p o r a t i o n of lhC-formate i n t o the purine 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 . This may p a r t l y be due to the 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 to the c e l l u l a r p o o l , and p a r t l y due to the feedback i n h i b i t i o n of the de novo syn-t h e s i s by AMP (116). However, AMP had no appreciable e f f e c t on the i n c o r p o r a t i o n of 1 4C-formate i n t o the n u c l e i c a c i d s . The absence of any e f f e c t of AMP on the i n c o r p o r a t i o n of 1 h C-formate may suggest the p o s s i b i l i t y t h a t AMP may not enter the c e l l . Wallach and U l l r e y (145) reported t h a t AMP i s dephos-phorylated at the c e l l membrane p r i o r to entry i n t o the a s c i t e s c e l l . In such a case, regeneration of the n u c l e o t i d e r e q u i r e s a s e r i e s of r e a c t i o n i n which ATP i s i n v o l v e d . A decre ase i n i n c o r p o r a t i o n of 1 1 +C-formate i s n o t i c e d i n a l l n u c l e i c a c i d components i n incubations c o n t a i n i n g both glucose and AMP. This may be due to var i o u s f a c t o r s , among which, isotope d i l u t i o n by added n u c l e o t i d e , end product i n h i b i t i o n of s y n t h e s i s , a l t e r a t i o n of the e q u i l i b r i u m of the n u c l e o t i d e p o o l , and d e p l e t i o n of adenine n u c l e o t i d e by glucose are more important. - 149 Table XXIV I n c o r p o r a t i o n of l>*C-formate by E h r l i c h a s c i t e s carcinoma c e l l s in vitro 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 con c e n t r a t i o n i n the c e i l . C oncentration of glucose 5.5 mM. AMP 1 mM. DNP 0.1 mM. Packed c e l l volume Expt. I 6.5, Expt. I I 7%. A c i d s o l u b l e f r a c t i o n cpm/O.D. RNA cpm/umole DNA cpm/mg Ad d i t i o n s Expt.I E x p t . I I Expt.I E x p t . I I Expt.I E x p t . I I N i l 2945 1512 993 1017 1626 1828 + .glucosel0135 9447 677 ' 774 835 790 + AMP 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 440 1418 * In Expt. I I only. - 150 -Table XXV I n c o r p o r a t i o n of l l fC-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 a s c i t e s carcinoma 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 ATP con c e n t r a t i o n i n the c e l l . S p e c i f i c a c t i v i t y -counts per minute per umole Adenine Guanine Hypoxanthine A d d i t i o n s Expt.I E x p t . I I Expt.I E x p t . I I Expt.I E x p t . I I N i l 6025 5529 7611 3954 7047 6228 + glucose 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 + DNP* '• 3395 1621 4430 (-) samples l o s t , * In Expt. I I onl y . - 151 -D i n i t r o p h e n o l i s w e l l known as an uncoupler of o x i d a t i v e phosphorylation (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 to uncouple o x i d a t i v e phosphorylation decreased the i n c o r p o r a t i o n of 1^C-formate i n t o the a c i d s o l u b l e nucleo-t i d e s and to n u c l e i c a c i d s . Gross e t a i . (146) have reported t h a t :.DNP, t ; i n h i b i t e d the sy n t h e s i s of RNA, but the concen-t r a t i o n of DNP used was much higher than the co n c e n t r a t i o n r e -qu i r e d f o r uncoupling of o x i d a t i v e phosphorylation. The present ob s e r v a t i o n t h a t DNP can i n h i b i t the i n c o r p o r a t i o n of 1 Re-formats at co n c e n t r a t i o n enough to uncouple o x i d a t i v e phosphory--4 l a t i o n (10 M) may be due t o the u n a v a i l a b i l i t y of ATP f o r continued s y n t h e s i s of the var i o u s n u c l e i c a c i d components. In incub a t i o n s c o n t a i n i n g AMP, glucose and DNP, 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 i s maximum. This may be due to the uncoupling of o x i d a t i v e phosphorylation i n a d d i t i o n to the var i o u s reasons s t a t e d above. 11) E f f e c t of Glucose and 2-Deoxyglucose on the I n c o r p o r a t i o n of ^Re-Formate i n t o Serine of E h r l i c h A s c i t e s Carcinoma C e l l s . I t was observed i n many experiments w i t h E h r l i c h a s c i t e s carcinoma c e l l s t h a t the i n c o r p o r a t i o n of 1 k C - f o r m a t e .into the 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 increased i n presence of glucose. When the s p e c i f i c a c t i v i t y of the a c i d s o l u b l e nucleo-t i d e bases were estimated, i t was found t h a t glucose a c t u a l l y decreased the i n c o r p o r a t i o n of 1 k C - f o r m a t e i n t o the n u c l e o t i d e bases, 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 was higher. This d i f f e r e n c e was a l s o observed when the c e l l s were incubated w i t h 2-deoxyglucose. 2-Deoxyglucose decreased the • - 152 - . • i n c o r p o r a t i o n of 1^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 , i r r e s p e c t i v e of c o n c e n t r a t i o n 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 the a c i d s o l u b l e f r a c t i o n was higher i n presence of 2-deoxyglucose, s i m i l a r t o the observations w i t h glucose. The. n u c l e o t i d e s from the acid' s o l u b l e f r a c t i o n of c e l l s incubated w i t h 2-deoxyglucose and of c o n t r o l experiments were removed by ch 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 present i n the s o l u t i o n a f t e r a d s o r p t i o n of n u c l e o t i d e s i s given i n Table XXVI. I t can be seen from the t a b l e t h a t the s o l u t i o n contained no U.V. absorbing m a t e r i a l s a f t e r adsorption bn c h a r c o a l . How-ever, the s o l u t i o n contained some r a d i o a c t i v e substances. The a c t i v i t y of the s o l u t i o n s were higher i n incubations c o n t a i n i n g 2-deoxyglucose. These f i n d i n g s l e d t o the s u s p i c i o n t h a t the incr e a s e i n r a d i o a c t i v i t y seen 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 glucose and 2-deoxyglucose may be due to something other than the n u c l e o t i d e s . Herscovics and Johnstone(72) reported t h a t the presence of glucose^ i n the i n c u b a t i o n medium increased the i n c o r p o r a t i o n of 1 ^ C-.formate i n t o s e r i n e and p r o t e i n s of E h r l i c h a s c i t e s car-cinoma c e l l s . I t was t h e r e f o r e suspected t h a t the r a d i o a c t i v i t y may be concentrated i n 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 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 . To v e r i f y t h i s i t was necessary to separate and estimate the r a d i o a c t i v i t y i n s e r i n e from the a c i d s o l u b l e f r a c t i o n s of 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 of s e r i n e from the a c i d - 153 -Table XXVI 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 incubated w i t h l l fC-formate in vitro a f t e r the removal of nu c l e o t i d e s by adsorption on c h a r c o a l . Packed c e l l A d d i t i o n volume O.D./260 O.D./280 cpm/ml N i l 1 4 % 0.072 0.052 5174 0.067 0.051 5518 + 2-deoxy glucose (5.5 mM) 14% 0.041 0.018 17758 0.027 0.012 15998 N i l 6.5% 0.060 0.048 5994 0.053 0.041 6386 + 2-deoxy glucose (5.5 mM) 6.5% 0.004 0.007 0.002 0.006 12304 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 out i n the 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 char c o a l and removed from the s o l u t i o n . The amino acid s which remain i n s o l u t i o n were adsorbed on a column of 5 cm x 1 cm of Doxex-8 r e s i n , i n the H+ form adjusted t o pH 2.5. A f t e r the adsorption of the amino a c i d s , the column was washed w i t h 0.01 M a c e t i c a c i d to remove the s a l t s and other i m p u r i t i e s . F i n a l l y , the amino acid s adsorbed on the column were e l u t e d w i t h 5 ml of 1 M ammonia. The ammonia was removed and the residue was e x t r a c t e d w i t h 1 ml of water. One hundred micro l i t r e s of the 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 to a s t r i p of Whatman No. 1 paper 6 5 cm x 20 cm. The mixture of amino acid s was separated by high v o l t a g e e l e c t r o p h o r e s i s at 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 glucose and from c o n t r o l s experiments were spotted alongside i n the same paper. A mixture of n e u t r a l amino acids c o n t a i n i n g "Dansyl" a r g i n i n e as a v i s u a l marker served as a standard i n e l e c t r o -p h o r e s i s . Free s e r i n e was a l s o run as a standard on the paper. This lane c o n t a i n i n g s e r i n e standard was cut out a f t e r the e l e c t r o p h o r e s i s and sprayed w i t h n i n h y d r i n . The p o s i t i o n of s e r i n e on the electrophorogram i n the m a t e r i a l from the incuba-t i o n s was determined from the p o s i t i o n of the s e r i n e standard and t h a t of "Dansyl" a r g i n i n e which can be e a s i l y v i s u a l i z e d . The electrophorogram c o n t a i n i n g the r a d i o a c t i v e spots was exposed to X-ray f i l m s f o r 2 to 3 weeks i n the dark. The f i l m s 'were then developed and the spots v i s u a l i z e d . The radioautograms - 155 -C o 3 s; • VI4/S " F i g u r e 23. E f f e c t o f g l u c o s e (5.5 mM) and 2 - d e o x y g l u c o s e (5.5 mM) on t h e i n c o r p o r a t i o n o f 1 1 + C - f o r m a t e i n t o t h e - s e r i n e o f 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 a t d i f f e r e n t c o n c e n t r a t i o n s c f c e l l s u s p e n s i o n s . - 156 -are shewn i n Fig u r e 23. I t was immediately seen from the radioautogram -that the i n c o r p o r a t i o n of 1^C-formate i n t o s e r i n e was increased i n presence of glucose of 2-deoxyglucose. This i n c r e a s e i n 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 was not depen-dent on the co n c e n t r a t i o n of c e l l s i n suspension. In the case of 2-deoxyglucose, where i n c o r p o r a t i o n of 1'*C- 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 was i n h i b i t e d markedly, a high 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 observed. - 157 -DISCUSSION The e f f e c t of glucose on the i n c o r p o r a t i o n of l a b e l l e d precursors i n t o the purines of 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 acids has' been i n v e s t i g a t e d by vari o u s groups of workers (69-72). I t i s g e n e r a l l y observed t h a t the i n c o r p o r a t i o n of 1'*C-forraate in vitro i s s t i m u l a t e d by the presence of glucose i n the medium i n almost a l l the systems s t u d i e d . However i t was observed by Thomson e t a l . (14 7), t h a t glucose diminished the i n c o r p o r a t i o n of lhC-formate i n t o n u c l e i c a c i d purines i n r a b b i t bone marrow in vitro, whereas the i n c o r p o r a t i o n i n t o DNA thymine was enhanced. In r a b b i t thymus t i s s u e glucose diminished the i n c o r p o r a t i o n of 1'*C-formate i n t o DNA thymine to about h a l f the c o n t r o l value. In a l a t e r r e p o r t , Thomson et a l . (71) have shown tha 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 presence of glucose. Various explanations have been o f f e r e d f o r the s t i m u l a t o r y e f f e c t of glucose on 1^C-formate i n c o r p o r a t i o n . Thus, Harrington (69) has suggested t h a t glucose acted as a precursor p o s s i b l y of r i b o s e 5 - P O i , or PRPP. The view t h a t glucose provided the much needed energy source f o r the s y n t h e t i c r e a c t i o n i s expressed by Henderson-and LePage (70). They have a l s o observed t h a t the e f f e c t of glucose i s a b o l i s h e d by iodoacetate or iodo-acetate and d i n i t r o p h e n o l i n support of t h e i r view. Thomson et a l . (71) are of the o p i n i o n that glucose c o n t r i b u t e d the r i b o s e 5 - P O i t r e q u i r e d f o r purine b i o s y n t h e s i s . E l l i s and S c h o l e f i e l d (148) observed an increased production of a c i d s o l u b l e nucleo-t i d e s from adenine i n presence of glucose and suggested the - 158 -p o s s i b l e r o l e of glucose as an energy source. Herscovics and Johnstone (72) are of the view t h a t glucose l e d to-an increased a v a i l a b i l i t y of glutamine which i s necessary f o r the de novo s y n t h e s i s 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 these views are v a l i d as there are evidences to support them. In E h r l i c h a s c i t e s carcinoma c e l l s the combination of a l l these p o s s i b i l i t i e s e x i s t . Tumour c e l l s are c h a r a c t e r i z e d by a high r a t e of g l y c o l y s i s and so glucose may be able to provide energy by t h i s process. Ashman (149) has shown t h a t both glucose-6-phosphate and 6-phosphogluconate dehydrogenases are present 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 the a v a i l a b i l i t y and production of ribose-5-phosphate from glucose. 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 c e l l s glucose increased the production of PRPP. I t i s w e l l known th a t PRPP plays 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 s r e q u i r e d f o r the f i r s t r e a c t i o n of the de novo s y n t h e s i s of purine nucleo-t i d e s as w e l l as f o r the f o r m a t i i o n of nu c l e o t i d e s from pre-formed bases. Kvamme and Svenneby (151) reported t h a t glucose prevented the deamination of glutamine present i n a s c i t e s carcinoma c e l l s by the glutaminase present i n the c e l l . The c l a i m of Herscovics and Johnstone(72) t h a t glucose i n f l u e n c e d the lkC-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 the glutamine c o n c e n t r a t i o n i s a l s o v a l i d . In a d d i t i o n to a l l these f a c t o r s the present s e r i e s of experiments show t h a t the e f f e c t of glucose a l s o depends on the - 159 ~ c o n c e n t r a t i o n of c e l l s i n suspension. By v a r y i n g the concentra-t i o n of c e l l s i n suspension from a higher value to" a lower value the e f f e c t of glucose can a l s o be v a r i e d . Thus at a high c e l l c o n c e n t r a t i o n as determined by packed c e l l volume, glucose stimulates, i n c o r p o r a t i o n whereas at lower concentra-t i o n s glucose i n h i b i t s the i n c o r p o r a t i o n of 1'*C-formate to a l l 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 Quastel (152) have pointed out t h a t the c e l l d e n s i t y i n d i f f e r e n t experiments should be kept constant i f comparable r e s u l t s are to be obtained. I t i s also.observed t h a t a t a p a r t i c u l a r c o n c e n t r a t i o n 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 glucose present 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 glucose which.may increase 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 the 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 of glucose to have a maximum s t i m u l a t o r y e f f e c t again may depend on the con-c e n t r a t i o n of c e l l suspension; t h i s optimum co n c e n t r a t i o n i n -creases v/ith i n c rease 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 c e l l s i s well.known. These c e l l s are p e c u l i a r i n t h e i r u t i l i z a t i o n of glucose, i n t h a t they e x h i b i t both Crabtree e f f e c t (the decreased r e s p i r a t i o n due to glucose) and Pasteur e f f e c t (the decreased g l y c o l y s i s due to increased o x i d a t i o n ) . Both the g l y c o l y s i s and o x i d a t i o n of glucose r e g u l a t e the a v a i l a b i l i t y of ATP i n the c e l l . The importance of ATP i n the c e l l f o r de novo s y n t h e s i s of purine n u c l e o t i d e s i s seen from the equations -• - 160 Glyc i n e + Ribose 5-P0 4 + 3NH* + HCO^ + 2HC00 -— > IMP. + SH 20 9ATP + 9H 20 -> 3ADP + 8HP07 + AMP + HP 207 + 9H + The s t i m u l a t o r y or the i n h i b i t o r y e f f e c t of glucose on the i n c o r p o r a t i o n of lkC-formate could be a t t r i b u t e d i n p a r t t o the co n d i t i o n s of a v a i l a b i l i t y of ATP. i n the c e l l s i n presence of 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 incubated 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 substrate:have a high content of ATP. A d d i t i o n of glucose under aerobic c o n d i t i o n s decreased the amount of ATP i n the c e l l . Overgaard-Hansen (73) has i n v e s t i g a t e d the adenine nucleo-t i d e pool i n 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 . Her observations are i n l i n e w i t h the present 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 . I t i s w e l l known t h a t E h r l i c h a s c i t e s carcinoma c e l l s c o n t a i n a power-f u l hexokinase. Glucose - under aerobic c o n d i t i o n s i s shown to produce a t r a n s i e n t d e p l e t i o n of the adenine n u c l e o t i d e pool v i a the hexokinase r e a c t i o n . C l o s e l y a s s o c i a t e d w i t h the i n i t i a l b u r s t of glucose consumption there are other r e a c t i o n s t a k i n g place i n the c e l l s which markedly decrease the ATP content of the c e l l s . These are brought about by the enzymes myokinase adenylate deaminase, 5'-nucleotidase, nucleosidase e t c . She has al s o pointed out tha t the e f f e c t of glucose on adenine nucleo-t i d e pool i n dense :suspension i s much l e s s pronounced. The d e p l e t i o n of adenine n u c l e o t i d e pool i s a l s o l e s s i f only small q u a n t i t i e s of sugar are added which may not produce an ATP - 161 -shortage. A c o n c e n t r a t i o n of glucose which i s higher than t h a t was u t i l i z e d d uring 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 pool which i s i n i t i a t e d by the deamination of AMP to IMP, which i n t u r n i s f u r t h e r decomposed to i n o s i n e and hypoxanthine. These metabolites were shown to i n c r e a s e i n the medium as glucose was being used up (73). 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 hypoxanthine i s taken up by the c e l l and r e u t i l -i z e d f o r the r e s t o r a t i o n of the depleted adenine n u c l e o t i d e p o o l . 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 pool i s s i g n i f i c a n t l y prolonged i n d i l u t e c e l l suspensions. F u r t h e r , Overgaeird-Hansen has a l s o shown th a t i n d i l u t e c e l l suspensions the d e p l e t i o n of the adenine n u c l e o t i d e pool i s more severe, whereas i n dense suspensions, the s m a l l decrease i n adenine n u c l e o t i d e pool seen i s q u i c k l y r e s t o r e d by the ATP regeneration mechanisms. However, under anaerobic c o n d i t i o n s no deamination of AMP takes place though AMP c o n c e n t r a t i o n increases through hexokinase 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 pool i s seen. I t was a l s o shown th a t under aerobic 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 . This i s because 2-deoxyglucose metabolism does not go f a r t h e r than i t s i n i t i a l p h o sphorylation f o r which ATP i s u t i l i z e d (117). A c e l l u l a r decrease i n the t o t a l a v a i l a b i l i t y of ATP under aerobic c o n d i t i o n s i n the presence of glucose i s a l s o reported r e c e n t l y by many other 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 carcinoma c e l l s w i t h glucose - 162 -a t o t a l l o s s of 20% of the 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 pool has been reported by Hotham e t .a l . (153). S i m i l a r l y 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 incubated w i t h glucose in. presence of oxygen. Meik l e e t a l . (154) have shown the e x c r e t i o n of U.V. absorbing m a t e r i a l • :(at. 260 my) into:' the medium of a s c i t e s c e l l suspensions incubated w i t h glucose.'.. This e x c r e t i o n of metabolites i n c r e a s e s w i t h time to about 20 minutes, and there begins to f a l l . But when c e l l s are t r e a t e d w i t h glucose and iodoacetate or w i t h 2-deoxyglucose, the con-c e n t r a t i o n of U.V. absorbing m a t e r i a l increases w i t h time and does not decrease at a l l . I t i s l o g i c a l to t h i n k t h a t the product of ATP breakdown induced by glucose d i f f u s e s i n t o the medium and i s taken up by the c e l l s under c o n d i t i o n s i n which ATP and other 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 presence of iodoacetate and 2-deoxyglucose r e s y n t h e s i s may not occur. These authors have f u r t h e r observed t h a t ATP i s required f o r the reconversion of the degradation products mainly i n o s i n e and hypoxanthine,back to the 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 not take place i n presence of 2-deoxyglucose or iodoacetate due to the u n a v a i l a b i l i t y of t h i s energy source. I t i s l o g i c a l to assume from a l l these observations t h a t glucose produces a t r a n s i e n t d e p l e t i o n of 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 carcinoma c e l l s when i n -cubated a e r o b i c a l l y . This d e p l e t i o n may depend on concentration of c e l l suspensions when the glucose c o n c e n t r a t i o n i s constant and depend on co n c e n t r a t i o n of glucose when co n c e n t r a t i o n of c e l l - 163 -suspension i s constant. 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 glucose on 1 "^C-formate i n c o r p o r a t i o n i n t o purines of nucleo-tides" and n u c l e i c acids can p a r t l y be explained on the b a s i s of these observations. As. pointed out p r e v i o u s l y an increase i n co n c e n t r a t i o n of ATP can f a c i l i t a t e r e a c t i o n s which r e q u i r e the u t i l i z a t i o n of ATP and de novo s y n t h e s i s of purine n u c l e o t i d e i s dependent • bn ATP. Further the 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 general i n the b i o s y n t h e s i s of RNA has been e l u c i d a t e d by An-thony e t a l . (28) . They have shown t h a t the chain i n i t i a t i o n r e a c t i o n i n the mechanism of RNA polymerase r e a c t i o n i s dependent on purine n u c l e o t i d e s and has a s p e c i f i c requirement f o r ATP or GTP. As ATP plays a c e n t r a l r o l e i n the r e g u l a t i o n of purine n u c l e o t i d e metabolism, i t could be conceivable t h a t .a decrease 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, Cashel and G a l l e n t (125) have i n v e s t i g a t e d the r e g u l a t i o n of RNA synt h e s i s i n E. coli, by amino a c i d s . They observed t h a t the formation of nucleoside triphosphates are amino a c i d depen-dent and a l s o t h a t ATP formation showed a severe amino a c i d dependence. I t was a l s o observed t h a t the ATP pool s u f f e r s the l a r -gest-decrease on amino a c i d s t a r v a t i o n . From these and other o b s e r v a t i o n s , they suggest t h a t there may be a decreased r a t e of chain i n i t i a t i o n due to the decreased supply of ATP. The present observations are a l s o i n agreement of t h i s view. Further p o s s i b i l i t i e s of i n h i b i t i o n of the de novo synthesis by glucose may be due to the secondary e f f e c t s produced subsequent to the accumulation of degradation products of adenine n u c l e o t i d e s . - 164 -H e n d e r s o n (155,156) has shown t h a t p u r i n e b a s e s e x e r t f e e d b a c k i n h i b i t i o n o f p u r i n e b i o s y n t h e s i s de novo. T h e r e i s t h e p o s -s i b i l i t y t h a t t h e s e mechanisms may a l s o be o p e r a t i v e u n d e r c o n d i t i o n o f g l u c o s e p h o s p h o r y l a t i o n . The i n c r e a s e d demand f o r PRPP i n c e l l s i n c u b a t e d w i t h g l u c o s e i s e v i d e n t f r o m t h e a c c u m u l a t i o n o f b a s e s compared t o c o n t r o l e x p e r i m e n t s , s i n c e 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 f r o m p r e f o r m e d b a s e s r e q u i r e s t h i s s u b s t a n c e ( 4 3 ) . However, H e n d e r s o n and Khoo (139) h a v e o b s e r v e d t h a t u n d e r a e r o b i c c o n d i t i o n s , g l u c o s e c a n f o r m 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 . The r e q u i r e m e n t o f ATP f o r t h e f o r m a -t i o n o f PRPP i s a l s o s u g g e s t e d by t h e o b s e r v a t i o n t h a t u n d e r a n a e r o b i c c o n d i t i o n s o r i n p r e s e n c e o f m e t a b o l i c i n h i b i t o r s s u c h as i o d o a c e t a t e o r d i n i t r o p h e n o l , t h e s y n t h e s i s o f PRPP was m a r k e d l y d i m i n i s h e d ( 1 3 9 ) . I t i s c o n c e i v a b l e t h e n t h a t when t h e r e i s an ATP d e f i c i e n c y i n t h e c e l l , t h e r e i s a l s o t h e d e f i c i e n c y o f t h e most i m p o r t a n t s u b s t a n c e v i z . PRPP w h i c h i s r e q u i r e d f o r t h e b i o s y n t h e s i s o f n u c l e o t i d e s b o t h by de novo pathway and f r o m p r e f o r m e d p u r i n e s . T h e s e v i e w s a r e f u r t h e r e x p l a i n e d by t h e e f f e c t o f 2-d e o x y 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 . I t was 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 x l * C - f o r m a t e by E h r l i c h a s c i t e s i s m a r k e d l y i n h i b i t e d by 2 - d e o x y g l u c o s e . T h i s i n h i b i t i o n i s s e e n b o t h i n d i l u t e as w e l l as i n d e n s e c e l l s u s p e n s i o n s . 2 - D e o x y g l u -c o s e i s known t o be t a k e n up by c e l l s and p h o s p h o r y l a t e d by h e x o k i n a s e . s i m i l a r t o g l u c o s e . B u t t h e m e t a b o l i c s t e p s do n o t go b e y o n d t h e i n i t i a l p h o s p h o r y l a t i o n s t e p ( 1 1 7 ) . McComb and Yushok (157) h a v e r e p o r t e d t h a t i n r e s p i r i n g K r e b s R i n g e r a s c i t e s - 165 -carcinoma c e l l s , phosphorylation of 2-deoxyglucose induces a d r a s t i c d e p l e t i o n of ATP. They a l s o observed the dephosphory-l a t i o n and deamination of ATP to IMP through ADP and AMP. Further i n o s i n e accumulated i n the r e a c t i o n mixture under these c o n d i t i o n s . They have suggested the Involvement of hexokinase, adenylate k i n a s e , adenylate deaminase, and 5'-nucleotidase, f o r the o p e r a t i o n of these pathways. The a c t i v i t i e s o f these en-zymes i n carcinoma c e l l s were found to be adequate to b r i n g about such a s e r i e s of ATP degradation r e a c t i o n s . Observations s i m i l a r to t h i s a l s o have been reported by Overgaard-Hansen (73). She has a l s o observed t h a t u n l i k e i n the case of glucose, the d e p l e t i o n of the adenine n u c l e o t i d e p ool caused by 2-deoxy-glucose under aerobic c o n d i t i o n s .is i r r e v e r s i b l e . 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 of • 1^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 presence of 2-deoxyglucose can be explained from the above observations. I t i s seen t h a t u n l i k e glucose, 2-deoxyglucose i n h i b i t s i n c o r p o r a t i o n of 1. l fC-formate i n t o n u c l e i c a c i d components i n dense c e l l suspensions. This i n h i b i t i o n i n dense c e l l suspension by 2-deoxyglucose could be explained by the observation of Overgaard-Hansen t h a t the deple-t i o n of 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 suspension the i n h i b i t o r y e f f e c t of 2-deoxyglucos can a l s o be explained on the b a s i s of d e p l e t i o n of ATP. However decrease i n i n c o r p o r a t i o n of 1 4C-formate observed i n presence of glucose 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 to the observations w i t h 2-deoxyglucose, glucose increased the i n c o r p o r a t i o n of 1 4C-formate i n t o n u c l e i c - 166 -a c i d components i n dense c e l l suspension, i . e . i n suspension of packed c e l l volume above 7-8%. The l a s t s e r i e s of experiments were designed to show the e f f e c t of ATP on the i n c o r p o r a t i o n of 1 4 C - f o r m a t e by a s c i t e s carcinoma c e l l s . As c e l l membranes are impermeable to ATP and a l s o sugar phosphate, the d i r e c t a d d i t i o n of these t o the medium to prevent the d e p l e t i o n of ATP l e v e l s i n the c e l l s c o u l d not be undertaken. However, i t was r e p o r t e d by Wu and Racker (144), t h a t 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 AMP and 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 , the ATP content of the c e l l s was i n c r e a s e d . Hence t h i s method was attempted to i n c r e a s e the ATP content i n the c e l l and a l s o study the e f f e c t on lkC~formate 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 . In some i n c u b a t i o n s , d i n i t r o p h e n o l was added to prevent the o x i d a t i v e p h o s p h o r y l a t i o n and p r o d u c t i o n of ATP. I n c o r p o r a t i o n of 1 4 C - f o r m a t e under d i f -f e r e n t c o n d i t i o n s of ATP p r o d u c t i o n and ATP d e p l e t i o n was s t u d i e d . Experiments i n which AMP and glucose were added to i n c r e a s e the ATP c o n c e n t r a t i o n i n the c e l l , c o n t r a r y to the expected r e s u l t s , a decrease i n the i n c o r p o r a t i o n of 1 !*C-formate i n t o bases of a c i d s o l u b l e n u c l e o t i d e s and to the n u c l e i c a c i d s was observed. The added AMP had l i t t l e e f f e c t on 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 the n u c l e i c a c i d s . T h i s may mean t h a t the decrease i n i n c o r p o r a t i o n seen was p r i m a r i l y due to the presence of glucose i n the medium. These f i n d i n g s may i n d i c a t e that, the added AMP d i d not e n t e r the c e l l . Wallach and U l l r e y (145) observed t h a t AMP, ADP and ATP were h y d r o l y z e d through s u c c e s s i v e 'stages to i n o r g a n i c phosphate and adenosine p r i o r to t h e i r e n t r y - 167 -i n t o the c e l l . In support of t h i s increased c o n c e n t r a t i o n of adenine and hypoxanthine were observed when the a c i d s o l u b l e purines were estimated i n the present i n v e s t i g a t i o n s . : Another ob s e r v a t i o n i n these experiments i s t h a t d i n i t r o -phenol a t l e v e l s s u f f i c i e n t to i n h i b i t o x i d a t i v e phosphorylation s e v e r e l y i n h i b i t e d lhC-formate i n c o r p o r a t i o n i n to RNA and to a. l e s s e r degree, to DNA. 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. Gross e t a l . (146) have reported t h a t concentrations much higher than t h a t r e q u i r e d for. uncoupling o x i d a t i v e phosphorylaton i n h i b i t e d RNA polymerase. Simon e t a l . (158) have r e c e n t l y reported t h a t i n . 2?. coli 2,4—dinitrophenol caused a marked i n h i b i t i o n of RNA synthesis w h i l e l i t t l e e f f e c t was seen on the 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 the mechanism of a c t i o n of DNP on DNA dependent RNA polymerase, the enzyme r e s p o n s i b l e f o r s y n t h e s i of RNA i n the c e l l . He has shown t h a t at a co 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 the attachment of the enzyme to DNA, which i s the f i r s t step i n the process of polymerase r e a c t i o n could be completely prevented. He has a l s o shown th a t the a f f i n i t y constant of the enzyme f o r DNA was a l s o reduced by d i n i t r o p h e n o l . Creaser 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 the i n f l u e n c of DNP on the 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 observed t h a t i n the presence of DNP, there was a decrease i n the formation of l a b e l l e d ATP and ADP under anaerobic and aerobic c o n d i t i o n s . F u r t h e r , they have observed a r a p i d l o s s of both ATP and ADP w i t h i n 5 minutes of the a d d i t i o n of DNP. - 168 -Thus^DNP not only prevented the formation of ATP.but a l s o i n -creased i t s r a t e of removal i n a s c i t e s carcinoma c e l l s . Many i n v e s t i g a t o r s (160,161) have shown t h a t both amino a c i d uptake and 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 are i n h i b i t e d by DNP. These r e a c t i o n s are energy r e q u i r -i n g processes and the e f f e c t of DNP was shown to 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 the b a s i s of these-observations i t i s tempting to speculate t h a t the i n h i b i t o r y e f f e c t seen on the i n c o r p o r a t i o n of 1 ^ 'C-formate i n t o RNA i n presence of DNP i s a l s o due to u n a v a i l a b i l i t y of ATP. This becomes more evident from the f i n d i n g t h a t the i n h i b i t i o n s are more marked i n samples c o n t a i n i n g DNP and glucose. I t i s p o s s i b l e t h a t i n these samples there i s more d e p l e t i o n of ATP than i n the samples c o n t a i n i n g DNP only. 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 of RNA polymerase as shown by Gross et a l . (146) and by Richardson (30) may a l s o c o n t r i b u t e f o r the i n h i b i -t i o n of 1 "*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 c e l l s i n presence of DNP. I t has been observed by Herscovics and Johnstone (72) t h a t glucose s t i m u l a t e s the i n c o r p o r a t i o n of 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 to be greater under anaerobic c o n d i t i o n s than under aerobic c o n d i t i o n s . But the i n c o r p o r a t i o n of 1 !*C-formate i n t o n u c l e i c a c i d s by the same t i s s u e was 25-50% lower under anaerobic than aerobic c o n d i t i o n s . In the present 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 1 4C-formate i n t o s e r i n e was seen to be increased i n the presence of glucose. - 169 -Further> the inc r e a s e was a l s o observed 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 the case of i n c u b a t i o n w i t h 2-deoxyglucose.. Herscovics and Johnstone(162) observed t h a t NADPH and glutamine are the f a c t o r s l i m i t i n g the I n c o r p o r a t i o n of 1^C-formate e i t h e r to s e r i n e or to n u c l e i c a c i d v i a formyl g l y c i n e -mide ribotide.NADPH added to c e l l f r e e systems s y n t h e s i z i n g both FGAR and s e r i n e v/ere shown to i n h i b i t the former process by 50-%, w h i l e s e r i n e s y n t h e s i s was doubled. S i m i l a r l y , glutamine which g r e a t l y s t i m u l a t e s ^ C - f ormate i n c o r p o r a t i o n i n t o GAR always i n h i b i t e d l l fC-formate 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 there i s a competition f o r the common one carbon fragment between s e r i n e and FGAR s y n t h e s i s . However i t may be pointed out t h a t the con c e n t r a t i o n of both NADPH and glutamine are low i n E h r l i c h a s c i t e s carcinoma c e l l s (117, 163). A d d i t i o n of glucose could increase the con c e n t r a t i o n of both, e s p e c i a l l y NADPH v i a the 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 f a i l s to account f o r the increased i n c o r p o r a t i o n of 1'*C-formate i n t o s e r i n e i n presence of 2-deoxyglucose, which i s not metabolized fa r t h e r ; than the i n i t i a l p hosphorylation. I t i s p o s s i b l e to thin k t h a t when the a v a i l a b i l i t y of a c t i v e one carbon fragments ("active formaldehyde" or -* N 5-N 1 0 m e t h y l e n e t e t r a h y d r o f o l i c acid) i s increased by the decreased syn t h e s i s of 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 the synt h e s i s of s e r i n e . Formation of s e r i n e by the i n c o r p o r a t i o n of the " 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 not known whether t h i s r e q u i r e s energy (164). - 170 -The formation of purine 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 of energy. I t i s conceivable t h a t under c o n d i t i o n s where the energy requirements are not adequate to meet the demand f o r sy n t h e s i s of n u c l e o t i d e s , more of the 1 4C-formate may be used to form s e r i n e . The mechanism of the competition between g l y c i n e and GAR f o r the " a c t i v e formaldehyde" i s not c l e a r . - 171 -SUMMARY (1) The v a r i o u s f a c t o r s a f f e c t i n g 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 a mammalian s y s t e m v i z . 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 e r e i n v e s t i g a t e d . 1 ^ C - F o r m a t e and 2-lkC-g l y c i n e w e r e u s e d a s 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 t h e s e s t u d i e s . (2) A m e t h o d f o r t h e 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 f r o m t h e a c i d s o l u b l e f r a c t i o n f r o m s m a l l amounts o f c e l l s was,: d e v e l o p e d . The m e t h o d i n v o l v e d two d i m e n s i o n a l p a p e r c h r o m a -t o g r a p h y i n an i s o b u t y r i c a c i d - a m m o n i a - w a t e r s y s t e m (pH 4.3) i n t h e f i r s t d i r e c t i o n a n d t h e n i n t h e ammonium a c e t a t e - e t h a n o l s y s t e m (pH 7) i n a d i r e c t i o n p e r p e n d i c u l a r t o t h e f i r s t . When t h e m e t h o d was t r i e d f o r t h e 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 f r o m 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 e s e p a r a t i o n s w e r e p o o r due t o i n t e r f e r i n g s u b s t a n c e s . T h e r e f o r e i t was n e c e s s a r y t o remove t h e s e i n t e r f e r i n g m a t e r i a l s p r i o r t o c h r o m a t o g r a p h y and a m e t h o d f o r t h e i r r e m o v a l was s t a n d a r d i z e d . The n u c l e o -t i d e s w e r e i s o l a t e d f r o m t h e a c i d s o l u b l e f r a c t i o n o f t h e c e l l s b y a d s o r p t i o n o n a c t i v a t e d c h a r c o a l a n d e l u t i o n w i t h a s o l v e n t m i x t u r e c o n t a i n i n g p y r i d i n e a nd e t h a n o l . T h i s m e t h o d g a v e .90-95% r e c o v e r i e s o f t h e n u c l e o t i d e s . (3) The 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 was e x a m i n e d . I n i t i a l s t u d i e s u s i n g p a p e r c h r o m a t o g r a p h y f o r t h e s e p a r a t i o n o f f r e e n u c l e o t i d e s f r o m s m a l l amounts 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 g a v e i n c o n c l u s i v e r e s u l t s . I n e x p e r i m e n t s w h e r e l a r g e r v o l u m e o f c e l l s w e r e u s e d - 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 separated by chromatography on DEAE-cellulose columns. The concentrations of n u c l e o t i d e s were determined by u l t r a v i o l e t spectrophotometry 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 s c i n t i l l a t i o n counting-. From these s t u d i e s i t was observed t h a t the concen-t r a t i o n of. the nu c l e o t i d e s i n the c e l l s incubated w i t h a c t i n o -mycin D was incre a s e d . This i n c r e a s e was more marked i n the concentrations of the nucleoside 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 of 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 into. the. purine n u c l e o t i d e s was s l i g h t l y decreased i n presence of actinomycin D. But the decrease i n i n c o r p o r a t i o n was very much l e s s i n degree compared to the increase i n c o n c e n t r a t i o n of the nucleo-t i d e s . 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 precursor i n t o t h e . - n u c l e i c a c i d s was considerably decreased by actinomycin D both i n E h r l i c h a s c i t e s carcinoma c e l l s and i n the i n t e s t i n a l mucosa of the r a t , i n which i t was t e s t e d . This decrease was more marked i n the case of guanine of RNA than of adenine, which was i n agreement w i t h the observations of others. From these obser-v a t i o n s i t was concluded that actinomycin D d i d not i n h i b i t the de novo 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 carcinoma c e l l s and th a t the s i t e of a c t i o n of actinomycin D i s a stage beyond the formation of the nucleoside t r i p h o s p h a t e s . (4) Actinomycin D i n h i b i t e d 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 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 carcinoma c e l l s was unaffected by actinomycin D. I t was concluded from these observations t h a t the i n h i b i t i o n of RNA syn t h e s i s observed i n the presence of actinomycin 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 v a i l -a b i l i t y of ATP. (5) The e f f o r t s to increase the incorporationoof radio-active precursors into the nucleic acid components by the addition of glucose to the medium gave varying r e s u l t s , mostly a decrease i n the incorporation. Hence the various factors influencing the incorporation of radioactive precursors into the nucleic acid components of E h r l i c h ..ascites carcinoma i n presence of glucose were examined. The factors examined included the e f f e c t of C a + + ions i n the medium, the e f f e c t of d i f f e r e n t buffers, the variations.of pH i n the medium, of the concentration of c e l l suspensions, and of the concentration of glucose i n the medium. The decrease i n incorporation of precursors into nucleic acid components caused by glucose did not depend on the presence or absence of Ca ions i n the medium. Decrease i n incorpora-t i o n of precursors into nucleic acids components i n presence of glucose was observed both i n Krebs Ringer phosphate and Krebs Ringer bicarbonate buffers, though the l a t t e r buffer s t a b i l i z e d the pH of the medium during g l y c o l y s i s . This finding indicated that the e f f e c t of glucose could not be explained on the basis of a change i n pH of the medium because of g l y c o l y s i s . The incorporation of ^ C - f ormate was higher i n Krebs Ringer phosphate buffer than i n bicarbonate buffer. This has been attributed to the increased a v a i l a b i l i t y of phosphate i n the former. Similar r e s u l t s were obtained when 2- 1 ''C-glycine was used as a radio-active precursor of nucleotides and nucleic acids instead of 1^C-formate. - 174 -(6) /Among the f a c t o r s 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 precursors i n t o n u c l e i c a c i d components, the following.seem to be more important; (1) the c o n c e n t r a t i o n of glucose i n the medium and (2) the c o n c e n t r a t i o n of c e l l suspension. The i n c o r -p o r a t i o n pf precursors was Increased i n d i l u t e c e l l suspensions w i t h low concentrations of glucose, w h i l e higher concentrations decreased the i n c o r p o r a t i o n . At a p h y s i o l o g i c a l c o n c e n t r a t i o n of glucose (5.5 mM) the i n c o r p o r a t i o n was increased i n dense c e l l suspensions (packed c e l l volume above 8%) and decreased i n d i l u t e c e l l suspension (PCV below 5%). i n between these two concentrations of c e l l suspension, a 5.5 mM co n c e n t r a t i o n of glucose 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) 2-Deoxyglucose, a substance which depleted ATP i n the c e l l s , a t a co n c e n t r a t i o n s i m i l a r to th a t of glucose (5.5 mM) decreased the i n c o r p o r a t i o n of lhC-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 i n a dense (PCV 14%) as w e l l as i n a d i l u t e (PCV 6.5%) c e l l suspension. (8) D i n i t r o p h e n o l , an uncoupler of o x i d a t i v e phosphorylation decreased the i n c o r p o r a t i o n of lkC-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 . This decrease was more marked i n d i l u t e c e l l suspensions i n presence of glucose. (9) I t was concluded from the above observations 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 precursors to n u c l e i c a c i d components In these c e l l s was the t r a n s i e n t deple-t i o n and regeneration of ATP i n the 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 1 4C-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 carcinoma c e l l s . This increase was more marked when there was a decrease i n i n c o r p o r a t i o n of 1^C-formate i n t o n u c l e i c a c i d components. 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