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Studies on the biosynthesis of nucleic acids by Novikoff hepatoma tissue in vitro Scrimgeour, Kenneth Gray 1957

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STUDIES OM THE BIOSYNTHESIS OF NUCLEIC ACIDS  BY NOVIKOFF HEPATOMA. TISSUE IN VITRO by Kenneth Gray Scrimgeour A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biochemistry We accept this thesis as conforming to the standard required from candidates for the degree of MA.STER OF SCIENCE. Members of the Department of Biochemistry The University of British Columbia September, 1957. ABSTRACT Nucl e i c a c i d metabolism has been s t u d i e d i n v i t r o w i t h suspensions of Novikoff r a t hepatoma c e l l s . The formation of a c i d s o l u b l e and n u c l e i c a c i d purines from f o r m a t e - C ^ and a d e n i n e - 8 - C " ^ has been measured. In a l l cases, adenine had a higher s p e c i f i c a c t i v i t y than guanine. The a c i d s o l u b l e p u r i n e s were much more r a d i o a c t i v e than the n u c l e i c a c i d p u r i n e s . Adenine-8-Cp-4 g a V e a higher value f o r the r a t i o of the s p e c i f i c a c t i v i t i e s of adenine t o guanine n u c l e o t i d e s than t h a t obtainedfrom formate-C^. Both r i b o n u c l e i c a c i d and d e o x y r i b o n u c l e i c a c i d i n c o r p o r a t e d r a d i o a c t i v i t y i n t h i s system. A standardized set of c o n d i t i o n s s u i t a b l e f o r t e s t i n g the a c t i v i t y of p o s s i b l e chemotherapeutic and i n h i b i t o r y agents was e s t a b l i s h e d , and 4 such compounds were examined. Azaserine i n low doses was extremely e f f e c t i v e i n i n h i b i t i n g de novo purine b i o s y n t h e s i s . 6-Mereaptopurine a l s o blocked de novo synthesis of the p u r i n e s . A new p o s s i b l e a n t i m e t a b o l i c compoundj N-benzoylglycinamidine was t e s t e d . In low amounts, N-benzoylglycinamidine s t i m u l a t e d purine b i o s y n t h e s i s , but a l a r g e dose decreased both purine s y n t h e s i s and r e s p i r a t i o n . The No v i k o f f tumour c e l l suspension was used t o g a i n some knowledge of the mode of a c t i o n of actinomycin D. This a n t i b i o t i c i n h i b i t e d c e l l u l a r r e s p i r a t i o n , but not anaerobic g l y c o l y s i s . Actinomycin D decreased n u c l e i c a c i d b i o s y n t h e s i s and a c i d s o l u b l e guanine s y n t h e s i s , but d i d not a f f e c t the formation o f a c i d s o l u b l e adenine. Large amounts e i t h e r of calcium pantothenate or of coenzyme A were able t o reverse the i n h i b i t i o n of n u c l e i c a c i d metabolism, and t o reverse p a r t i a l l y the i n h i b i t i o n o f r e s p i r a t i o n . Coenzyme A was more e f f e c t i v e than pantothenate. These findings appear to support the suggestion that actinomycin D interferes with coenzyme A-dependent reactions. In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s t h e s i s for scholarly purposes may be granted by the Head of my Department or by his representative. It i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of / 5 ^ ^ ^ > u ^ L ^ / The University of B r i t i s h Colombia, Vancouver 8, Canada. ACKNOWLEDGMENTS The author gratefully acknowledges personal assistance from the National Research Council i n the form of a Bursary and two Summer Supplement allowances. Acknowledgment is made to Dr. S. H. Zbarsky for his direction, and to other members of the staff of the Biochemistry Department for their gracious assistance. Mrs. V. Creelman and Miss B. Findlay are sincerely thanked for preparation of the tumour-bearing rats. This research was supported by a grant from the National Cancer Institute of Canada. TABLE OF CONTENTS Page INTRODUCTION 1 EXPERIMENTAL 18 A. METHODS. I. Materials 18 II. Tissue preparation 19 III. Respiration . 20 IV. Glycolysis 20 V. Isolation of purines 21 VI. Radioactive counting procedures . ' 24 VII. Ultraviolet spectrophotometry 25 B. RESULTS. I. Characterization and standardization of the tissue suspension.; . (i) Respiration 26 ( i i ) Effect of fomate-C 1^ concentration . . . . 26 ( i i i ) Precision of specific a c t i v i t i e s . . . . . . 23 (iv) Time study of formate-C1^ incorporation . . . 29 (v) Time study of adenine-8-Cl4 incorporation . . 33 (vi) Incorporation of formate-C 1 4 into DNA and RNA . 36 (vii) Controls. (a) Heat inactivated tissue 37 (b) Blood cells 37 ( v i i i ) Amounts of purines isolated . 38 (ix:) Medium . 38 II. Inhibitors and antimetabolites. (i) Azaserine 40 ( i i ) 6-Mercaptopurine 40 ( i i i ) N-Benzoylglycinamidine 40 (iv) Actinomycin D (a) Effect on respiration 42 (b) Effect on glycolysis 43 (c) Effect on purine renewals . 44 (d) Reversals of inhibitions 44 DISCUSSION 49 SUMMARY 55 BIBLIOGRAPHY 57 TABLES Page I. Abbreviations used" . 5 II. Variability in specific activities . . . . . . . 30 III. Incorporation of formate-C 1 4 into DNA and RNA purines 36 IV. Amounts of purines isolated" 39 V. Effects of azaserine; 6-mercaptopurine, and N-benzoylglycinamidine 41 VI. Effect of actmnomycin D on respiration 42 VII. Effect of actinomycin D on glycolysis . . . . . . 43 VIII. Effect of actinomycin D on formate-C 1 4 incorporation . 45 IX. Effect of actinomycin D on DM and RNA . . . . . 46 X. Effect of actinomycin D on adenine-8-C 1 4 incorporation 46 XI. Pantothenate effect on actinomycin D inhibitions . . 47 XII. Coenzyme A effect on actinomycin D inhibitions . . . 47 FIGURES 1. Precursors of the purine ring 2 2. The pathway for de novo synthesis of purines . . . 4 5. Possible pathways for adenine ut i l i z a t i o n " . . . . 9 4. Effect of concentration on formate-C 1 4 incorporation . into acid soluble purines 27 5. Effect of. concentration on formate-C-"-4 incorporation into nucleic acid purines . " 28 6. Time study of formate-C 1 4 incorporation into acid soluble purines 31 7. Time study of formate-C 1 4 incorporation into nucleic acid purines 32 8. Time study of adenine-8-C 1 4 incorporation into acid soluble purines . . . . . . 34 9. Time study of adenine-8-C incorporation into nucleic acid purines 35 -1-INTRODUCTION Research i n the past few years has delineated the pathways of nucleic acid biosynthesis. The mechanisms for de novo synthesis of nucleotides and for u t i l i z a t i o n of preformed bases are well established. Enzymes that phosphorylate nucleotides and those that can synthesize the nucleic acids themselves have now been isolated. The nucleic acid metabolism of a tissue may be elucidated by the use of isotopically labeled compounds or of specific inhibitors or antimetabolites. The origin of each of the atoms of the purine skeleton: i s now known, primarily from the research of J. H. Buchanan and his associates. In 1948, studies on the u t i l i z a t i o n of a series of compounds labeled with 13 J-5 C and N for uric acid formation by the pigeon (1,2) showed that formate had given rise to carbons 2 and 8, carbon dioxide to carbon 6, and glycine to carbons 4 and 5 and nitrogen 7. Very recently, the origin of the other nitrogen atoms was determined (3,4). The amide nitrogen of glutamine supplies nitrogens 3 and 9, and the amino group of aspartate donates nitrogen 1. Figure 1 indicates the position of the precursors i n the purine structure. Because hypoxanthine i s the primary end-product of nitrogen metabolism i n pigeons, pigeon l i v e r has been used for much of the research to determine the steps i n the u t i l i z a t i o n of the precursors for purine formation. Studies on the mechanism of de novo purine biosynthesis from these precursors have shown that the ring formation occurs not with the purine bases themselves, but with the phosphorylated sugar derivatives, -2-COOH CH 2 C H - | \ | H 2 COOHJ © H ( ^ 0 0 H C o , © c»o CH-NH, COOH ' 9 © @ • .0 O H © H (_^,00H \ 9 / NH 2 C-0 9 H a ? H 2 CH-NH S COOH Figure 1. Precursors of the purine ring. -3-the nucleotides. The glycine and ribose-5-phosphate portions of the purine nucleotides are the largest precursor units. A pathway of purine synthesis, abbreviated to show only the steps referred to i n this thesis, i s shown i n Figure 2. Romberg, Lieberman, and Simms (5) have isolated 5-phosphoribosyl pyrophosphate (PHPP; see Table I for complete l i s t of abbreviations) and have shown that i t i s synthesized enzymatically from adenosine triphosphate (ATP) and ribose-5-phosphate. This sugar triphosphate is used for the formation of both purine and pyrimidine nucleotides. With the pyrimidines, the intact pyrimidine ring reacts with PRPP during de novo synthesis to form a nucleotide directly (6). With the purine pathway, however, the PRPP usually acts as a starting material for purine biosynthesis. Glycinamide ribotide (GAR) i s formed from glycine, PRPP, glutamine, and ATP (7 ,8) by extracts of pigeon l i v e r , probably through the intermediate 5-phosphoribosylamine (9). Next, a formate unit i s added to GAR through a f o l i c acid derivative (10) to form formylglycinamide ribotide (FGAR). Leveriberg and Buchanan have shown (11,12) that FGAR reacts with ATP and glutamine i n an enzyme system from pigeon l i v e r to produce 5-aminoimidazole ribotide through the intermediate formyl-glycinamidine ribotide (FGAM). Buchanan and his coworkers (13,14) have demonstrated that carbon dioxide i s added to the 4 position of 5-aminoimidazole ribotide to form 5-aminc—4~imidazolecarboxylic acid ribotide. This carboxylic acid then combines with aspartic acid to produce 5-amino-4-imidazole-N-succinocarboxamide ribotide, which i s then enzymatically cleaved by adenylosuccinase to fumaric acid and 5-amino-4-imidazolecarboxamide -4-C H 2 NH 2 o*c\ NH (HO> 2 -P -OH 2 C / ° \ H G L U T A M I N E G L Y C I N E ( H O ) - P - O H C / O 2 2 H \ H H 0 0 OH OH P R P P O-P-O-P-(OH) A T P 2 OH CH- NH 2 i ,C. CHO HN \ NH ( H O ) - P - O H C s 0 2 2 i OH OH C O , FGAM ASPARTATE 2 ATP H 2 N H 2 N / / / ^ S s N I H O I - P - O H . C 2 2 , H \ H H / H OH OH A T P H \ H H / H OH OH G A R FORMATE CH NH . 2 I .C. CHO 0' \ . NH GLUTAMINE I H 0 V P - ° H 2 C / ° H \ H H / H OH OH F G A R OH I N ' N' ( H O ) - P - O H C / O F O R M A T E j> • - ' 2 -> H \ H H / H OH OH A I C A R I M P Figure 2. The pathway for de novo synthesis of purines. -5-TABLE I Abbreviations used i n the text. Nucleic acids. DNA - -deoxyribonucleic acid RNA - • ribonucleic acid Nucleotides. These abbreviations are combinations of that" for the nucleoside and that indicating the phosphorylation le v e l . For example, AMP i s adenosine monophosphate. A- -- adenosine C- -- cytosine G- -- guanosine I- -- inosine T- -- thymidine ti- -- uridine X- -- xanthosine -MP -- monophosphate -DP; -- diphosphate -TP -- triphosphate DPN - diphosphopyridine nucleotide or coenzyme I. Purine precursors. PRPP - 5-phosphoribosyli pyrophosphate GAR - glycinamide ribotide FGAR - formylglycinamide ribotide FGAM - formylglycinamidine ribotide AICAR - 5-anu.no-4-imidazolecarboxamide ribotide -6-ribotide (AICAR). Greenberg has described (15) the requirements for the conversion of AICAR to inosinic acid (IMP). Formate, ATP, magnesium ions, and tetrahydrofolic acid are necessary for the addition of carbon 2 of the purine and for ring closure. The f i r s t complete purine ring to be formed probably is that of hypoxanthine, as IMP. Greenberg had evidence i n 1951 (16) that, with pigeon l i v e r homogenates, hypoxanthine was formed de novo from IMP. Abrams and Bentley (17) have shown that IMP can be converted by soluble enzymes from rabbit bone marrow to both adenylic acid (AMP) and guanylic acid (GMP). Furthermore, Edmonds and LePage (18) have demonstrated with mammalian tissue that IMP is synthesized from glycine-2-C-'-4 independently of AMP. Therefore, i t would appear that IMP is the main precursor of both AMP and GMP, although Carter and Cohen (19) had suggested that IMP might not be ah obligatory intermediate i n AMP synthesis. Lieberman (20) has isolated an enzyme, adenylosuccinate synthase, from Escherichia c o l i that catalyzes the GTP-dependent reaction between IMP and aspartate to produce adenylosuccinate. To complete the conversion of the 6-hydroxyl of IMP to an amino group, the adenylosuccinate can then be cleaved by cell-free extracts to yi e l d AMP. Previously, adenylosuccinase had been isolated from yeast (21), and i t had been shown that the formation of adenylosuccinic acid from AMP and fumaric acid was reversible. In the conversion of IMP to GMP, there i s f i r s t a DPN(coenzyme Independent oxidation (17,22,23) to xanthosine 5'-phosphate (XMP), and then an amination i n the 2 position to form GMP. The pigeon l i v e r (22) and the bone marrow (17) systems required glutamine for the amination, but a bacterial enzyme (24) u t i l i z e d ammonia. As Kbrnberg has pointed out i n a recent review (25), the biosynthesis of purine nucleotides from small molecules i s the main pathway. The incorporation of the free bases by the "preformed" or "salvage" pathways i s less common. Brown et a l had shown i n 1948 (26) that a d e n i n e - c o u l d be used by rats for both the adenine and guanine of nucleic acids. Guanine- similarly administered to rats was used very l i t t l e for nucleic acid formation. The formation of nucleotides from purines occurs primarily by reaction with PRPP under the influence of specific enzymes (27,28). However, a portion of the base may be incorporated by a two step mechanism, with the nucleosides as intermediates. Kalckar (29) was the f i r s t to completely characterize the nucleoside phosphorylase from rat l i v e r that catalyzed a reversible phosphorolysis of the nucleosides inosine and guanine to ribose-1-phosphate and the free purine. The equilibrium of this reaction favours the synthesis of the nucleosides, rather than the phosphorolytic s p l i t . Korn and Buchanan (30) have purified a similar enzyme from beef l i v e r for adenosine formation. The nucleosides are phosphorylated i n the 5' position by ATP through the agency of kinases, such as adenosine phosphokinase (31). Balis et a l (32) showed that nucleosides were not u t i l i z e d for nucleic acids as well as free purines were, but that breakdown to the bases and subsequent activation wqs not the sole pathway of their incorporation. Reichard and Skold(33) found that conversion of u r a c i l to IMP i n acetone powders of Ehrlich ascites cells occurs by the two pathways, but that most was formed via the nucleoside. Thus, in -8-base u t i l i z a t i o n , direct addition of PRPP i s the main scheme for purines, while nucleoside formation and i t s phosphorylation are probably more important for pyrimidines. Some of the ways by which adenine may reach the nucleotide level have been summarized i n Figure 3 . adenine can be converted to GMP through AMP and reversal of the adenylosuccinate reaction to IMP, or by catabolism'either of adenine to hypoxanthine by adenine deaminase or of adenosine to inosine by adenosine deaminase. As discussed above, enzymes have been shown to exist that produce nucleotides from hypoxanthine and ino-sine. Before'the nucleotides formed from the de novo and "salvage" pathways can be used for nucleic acid biosynthesis, i t i s possible that they must be phosphorylated to their 5 '-di- and triphosphates. Although the phosphates of adenosine have been known for quite a few years, i t has only been with the recent techniques of ion exchange chromatography that the polyphosphates of guanosine, uridine, and cytosine (34) have been isolated from the acid soluble extracts of animal tissue. Even more recently, LePage (35) has demonstrated the presence of deoxyadenosine triphosphate from tumour tissue, and Totter and his coworkers (36) have isolated mono-, d i - , and triphosphates of thymidine and deoxycytidine from thymus. Probably the remaining deoxynucleoside phosphates, similar to their ribonucleoside counterparts, w i l l soon be isolated, and the synthetic pathways of deoxynucleotides elaborated. In order to account for the complete spectrum of ribonucleoside phosphate found, enzymes Figure 3. Possible mechanisms for the u t i l i z a t i o n of adenine for nucleotide formation. were sought which could convert monophosphates to polyphosphates. Myokinase, or adenylate kinase, specific for adenylic acid, was the only known system. This enzyme catalyzes an equilibrium reaction between AMP and ATP to form two moles of AEP. An enzyme which could phosphorylate UMP to UDP or UTP, and GMP to GDP or GTP by transphosphorylation with ATP was purified from yeast (37) i n 1955. Rat l i v e r cytoplasm (38,39) can phosphorylate CMP and UMP to their corresponding d i - and triphosphates during oxidative phosphorylation. Investigations with P->2 indicated that ATP i s the intermediate phosphate acceptor i n these reactions, also. It can thus be seen that nucleotides can readily be phosphorylated by enzymes from a variety of sources. Enzymes for the synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) have been purified from bacteria. Ochoa and his associates have isolated a polynucleotide phosphorylase from Azotobacter vinelandii (40) which catalyzes the reversible synthesis of high molecular weight RNA-like polynucleotides from ribonucleoside diphosphates. The polynucleotide phosphorylase has also been demonstrated in other bacteria (4I-43) and i n extracts of spinach leaves (41). Enzymes have been found i n Escherichia c o l i by Korriberg and his coworkers (44) that form polymers of deoxyribonucleotides from the nucleoside triphosphates. A primer of DNA fragments, ATP, and two purified enzyme fractions are required for this DNA synthesis. Synthesis of complete nucleic acid molecules has not yet been demonstrated with cell-free animal tissue preparations. Belousova (45) has developed a non-isotopic system for the study of increase of amounts -11-of RNA and DNA i n homogenates and nuclei of normal arid neoplastic c e l l s . The nucleic acid synthesis, as judged by specific colour reactions, is a process of equilibrium that depends on the mass law and on the presence of nucleotides, ATP, and malic acid from the tricarboxylic acid cycle. Isotopic experiments, such as that of Goldwasser (46) on the u t i l i z a t i o n of labeled AMP for RNA in pigeon l i v e r homogenates or that of Herbert et a l (47) on orotic acid-6-C 1 4 u t i l i z a t i o n by rat l i v e r preparations, have shown incorporation of nucleotides into RNA linkages by renewal, but as yet no- de novo synthesis has been demonstrated. Paterson and LePage have reported that studies of RNA synthesis by homogenates of Flexner-Jobling carcinoma indicated that C^-labeled adenosine phosphates are added to the ends of existing RNA molecules, (48).. The purpose of this thesis was to study the biosynthesis of nucleic acids i n neoplastic tissue. The Novikoff hepatoma was used for the experimental work because i t was available. This hepatoma was very suitable because i t was a rapidly growing neoplasm - 5 days after i t s transplantation, i t supplied sufficient tissue for experiments - and because i t was easily reduced from solid form to a homogeneous single- ; c e l l suspension. The rate of nucleic acid biosynthesis i s very high i n tumour tissue, and therefore may be followed more easily than in normal tissues. This rapid metabolic rate made the hepatoma useful for the screening of possible metabolic.inhibitors. In v i t r o studies enable one to have controls with tissue identical to that being used for studies with the inhibitors. Metabolic studies with radioactive compounds require less isotopic material when done in vitro. The incorporation of sodium formate-C was used as a measure of the biosynthesis of nucleic acids i n these tumour cells because i t gives a good indication of the extent of the de novo purine pathway. Formate has other advantages in i t s a v a i l a b i l i t y and economy. The extent of formate-C"1"^ incorporation into the acid soluble and nucleic acid (acid insoluble) adenine and guanine was determined i n most experiments. There are several natural animal sources of the one carbon unit, formate. Siekevitz and Greenberg (49) showed that the methyl groups of methionine and choline, carbon 3 of serine, and carbon 2 of glycine a l l gave rise to formate i n rats. MacKenzie (50) reported that sarcosine was also a source of one carbon compounds, including formate. Formate enters positions 2 and 8 of the purine ring as a derivative of the vitamin f o l i c acid. Buchanan and Schulman (51) found, that citrovorum factor (N-5-formyl-5,6,7,8-tetrahydrofolic acid) stimulated IMP synthesis from glycine and formate. Greenberg (15) showed that the cofactor necessary for purine precursor formylation was derived from citrovorum factor. Jaenicke (52) and Greenberg et a l (53) provided evidence that the direct formate donor was W^-formyltetra-hydrofolic acid. A method was set up for preparing buffered suspensions of the hepatoma cells from the solid tumour. These preparations were f i r s t tested, for act i v i t y by measurement of the rate of respiration, and later by measurement of the incorporation of isotope (by isolation of the purines). It was found that for the amount of tissue used (about 60-80 milligrams dry weight per incubation flask) and for an incubation time of -13-14 140 minutes, 8 micromoles of formate-C was s u f f i c i e n t t o give maximum 14 i n c o r p o r a t i o n . Time s t u d i e s of the i n c o r p o r a t i o n o f formate-C and of adenine-8-C" 1^ i n t o a c i d s o l u b l e and n u c l e i c a c i d purines i n d i c a t e d t h a t the time f o r optimal i n c o r p o r a t i o n was at about 120 minutes. Subsequently, a l l experiments were c a r r i e d out a t 37° C. f o r 120 minutes, w i t h a standard amount o f t i s s u e and i s o t o p e . The rate of renewal of the a c i d s o l u b l e n u c l e o t i d e s , precursors of the n u c l e i c a c i d s , n a t u r a l l y was higher than t h a t of n u c l e i c a c i d p u r i n e s . RNA purines were renewed at a higher r a t e than DNA purines i n t h i s system. A f t e r these p r e l i m i n a r y experiments, 4 compounds were t e s t e d f o r p o s s i b l e i n h i b i t i o n , of n u c l e i c a c i d renewal under the standard set of c o n d i t i o n s . Two of the 4 compounds used were agents w e l l e s t a b l i s h e d as being i n h i b i t o r s of n u c l e i c a c i d b i o s y n t h e s i s - azaserine and 6-mercaptopurine. The other two compounds were N-benzoylglycinamidine and actinomycin D. Azaserine i s an a n t i b i o t i c f i r s t i s o l a t e d from Streptomyces i n 1954 (54), and i d e n t i f i e d as O-diazoacetyl-L-serine (N=N=CH'C0*0CH2-CH (NHg)•C0OH). Stock et a l (55) found t h a t i t i n h i b i t e d growth of Sarcoma 180. LePage, Greenlees, and Fernandes (56) observed t h a t i n c o r p o r a t i o n of glycine-2-C" 1"^ i n t o the purines of a s c i t e s c e l l s was i n h i b i t e d a t l e a s t 90 percent by ve r y s m a l l doses of az a s e r i n e . Glutamine a i d s i n overcoming the i n h i b i t i o n . Several i n v e s t i g a t o r s have found the accumulation o f the purine p r e c u r s o r s GAR and FGAR a f t e r treatment of t i s s u e s w i t h azaserine (8,57,58). Azaserine appears t o be bound q u i c k l y and i r r e v e r s i b l y t o an enzyme necessary f o r purine b i o s y n t h e s i s (57). -14-The enzyme being attacked i s that responsible for the conversion of FGAR to FGAM (59). With the Novikoff hepatoma suspensions, azaserine, at a dose level of 0.3 micrograms per flask, causes high inhibition of acid soluble adenine and nucleic acid purine renewal, with no effect on respiration. 6-Mercaptopurine is an antimetabolite that was synthesized by Hitchings and Elion (60). Clarke et a l (61) f i r s t showed that 6-mercaptopurine had an inhibitory effect on tumour growth. This unnatural purine is incorporated into both RNA and DNA (62). Skipper postulated (63) that 6-mercaptopurine blocks the u t i l i z a t i o n of MP for nucleic acid purines. Gots and Gollub have suggested (64) that 6-mercaptdpurine i s one of several purine analogues that may-act by preventing the use of exogenous purines and also by stopping de novo purine synthesis by a feed-back reaction. In studies with the hepatoma suspension, 1 milligram of 6-mercaptopurine per incubation flask inhibited respiration by 28 percent and blocked purine synthesis by 40 to 50 percent. 6-Mercaptopurine was fotind i n chromatographs of the acid soluble fraction, but none could be detected i n the nucleic acids. The third compound to be tested was N-benzoylglycinamidine It was thought that i t might be an antimetabolite of one of the purine precursors, possibly GAR or FGAM. This substance was added to the Novikoff hepatoma preparation at several dose levels. Doses as high as 'This was synthesized through the cooperation of Dr. W. R. Ashford by Merck and Company Limited, at the request of Dr. S. H. Zbarsky. -15-1 milligram per flask increased the specific activity of the purines without affecting respiration markedly. A dose of• 10 milligrams, however, blocked both acid soluble and nucleic acid purine labelling, and cut the respiratory rate by about one-half. The f i n a l compound, actinomycin D, was examined i n more detail. Actinomycin D i s a pigmented antibiotic isolated from Streptomyces parvullus broth cultures (65). It has a molecular weight of about 1200, and i t contains a.red chromophore and 5 amino acids - threonine, sarcosine, proline, valine, and N-methylvaline. Like the other actinomycins, actinomycin D is quite toxic. Roussos and Vining (66) have found by chromatography 7 components to be present i n each of the actinomycins, and have suggested that the difference i n the actinomycins from various species may be i n the relative amounts of these components. Actinomycin D, of a l l the compounds, i s closest to being homogeneous. Farber (67) has reported that actinomycin D has carcinolytic a c t i v i t y against.mouse tumours, and other workers have since tested i t on both experimental (68-70) and human (70,71) tumours. One of the few reports on the mode of action of actinomycin D has been made by Foley (72). Actinomycin D was examined i n several specific bacterial bioassay systems. Where inhibition of growth was observed, i t was found that the inhibition could be reversed by large amounts of pantothenate. This competitive inhibition suggested to Foley that perhaps actinomycin D interferes with coenzyme A and coenzyme A-dependent reactions. Methionine, adenine, orotic acid, and several dicarboxylic acids could also reverse inhibition, but were -16-non-competitive. Foley also proposed that actinomycin D blocked pantothenate-dependent reactions f o r the biosynthesis or u t i l i z a t i o n of amino acids by the bacteria used. Cobb and Walker (73) observed the effects of actinomycin D on the cytology of tissue cultures. The a n t i b i o t i c stopped migration and mitoses i n mouse f i b r o b l a s t s , and lowered the mitotic index of several human neoplasms. I t also reduced the amount of Feulgen-positive material (DNA) and altered the nuclear size of some neoplasms. 2 Actinomycin D had been tested by Creelman and Darrach f o r chemotherapeutic effects on the Novikoff hepatoma i n r a t s . Levels of 0.09 milligrams per kilogram increased the s u r v i v a l time of some r a t s , but double t h i s quantity was t o x i c . Actinomycin D showed a d e f i n i t e i n h i b i t i o n of r e s p i r a t i o n i n hepatoma c e l l s i n v i t r o . The rate of anaerobic g l y c o l y s i s was s l i g h t l y increased by larger amounts of the compound. Because res p i r a t i o n i s dependent on coenzyme A and gly c o l y s i s i s not, these r e s u l t s are i n accord with Foley's suggestion that actinomycin D interferes with reactions involving coenzyme A. When the incorporation of formate-C 1^ was followed, actinomycin D greatly lowered the s p e c i f i c a c t i v i t i e s of the purines, especially the guanine, of the mixed nucleic acids. The acid soluble adenine was not affected appreciably, but the acid soluble guanine formation had decreased. Adenine-8-C 1 4 incorporation was affected by Personal communication from V. Creelman and M. Darrach. the antibiotic i n the same way that formate-C uptake was. Another experiment demonstrated that both RNA and DNA purine renewal was blocked by actinomycin D-. Calcium pantothenate effected a complete reversal of the inhibition by actinomycin D of, purine formation and a partial reversal of respiration, but very large quantities of"; pantothenate (1 to 10 milligrams per 2.7 micrograms of actinomycin D) were required. Coenzyme A was more effective oh a molar basis for reversing the effects of actinomycin D, but large amounts s t i l l were required (1 to 5 milligrams per 2.7 micrograms of actinomycin D). The coenzyme A was p a r t i a l l y degraded to adenine nucleotide, and a two-fold increase i n the amount of acid, soluble adenine could be found when 5 milligrams of the coenzyme was added. This added source of adenine 14 caused a dilution i n the incorporation of formate-C into the purines. -18- -EXPERIMENTAL A. METHODS I. Materials. The Novikoff hepatoma was grown i n male Sprague-Dawley rats (180 grams, from the University of British-•Columbia colony). The tumours were transplanted by injection of 0.5 ml. of a 1 i n 5 dilution of tumour cells i n physiological saline into, each rat. In a period of 5 days after .the transplantations, the tumours had grown to a size of about 3.5 to 5 grams. Sodium 'formate- C 1 4 was obtained from Atomic Energy of Canada Limited and from Merck and Company Limited. The formate was administered as a solution (slightly alkaline) of either 14.1 mg. or 17 mg. per 5 -ml. A l l specific activities were corrected to a value of 2.6 X 10^ counts per minute (c.p.m.) per micromole (;uM) for the formate. Adenine-S-C1^ of specific activity 2.0 X 10^ c.v.m./pK was obtained from Merck and Company Limited as the sulphate hemihydrate, and was used as a solution made slightly acidic with hydrochloric acid. Azaserine, a g i f t of Dr. L. M. Long, Parke Davis and Company, was kept as an aqueous solution of 3 mg. per 100 ml. Actinomycin D, obtained through the courtesy of Dr. M. Darrach, was dissolved i n hydrochloric acid, then neutralized with sodium'hydroxide. Its f i n a l concentration was 0.135 mg. per 10 ml. / 6-Mercaptopurine from The Wellcome Research Laboratories was added i n solid form. N-Benzoylglycinamidine hydrochloride was used-as an aqueous solution of appropriate concentration, -19-or as a solid i f needed i n large quantities. Calcium pantothenate (dextrorotatory) from Merck and Company Limited was added either as a solution of 1 mg. per ml. or as the solid. Coenzyme A (80% pure) was purchased from Sigma Chemical Company. It was prepared as an aqueous solution of 10 mg. per ml. A l l solutions were kept frozen when not i n use. II. Tissue preparation. Each rat was k i l l e d by a blow on the head, and i t s tumour quickly excised. Tumour tissue from several animals was combined and passed through 2 syringes with No. 16 and No. 18 needles, i n that order. The tissue mass was passed from these syringes into a 50 ml. glass syringe with a No. 18 needle on i t . The tissue was diluted by drawing cold buffer solution (3 parts of buffer for respiration and aerobic experiments, and 15 parts for glycolysis studies) into the syringe. The syringe was inverted, a small amount of air was drawn in, and then the syringe was shaken and inverted several times to mix the tissue. Then the suspension was ejected onto a f i l t e r of 12 layers of grade 20 cheesecloth. The resulting f i l t r a t e was a suspension made up of single hepatoma cells and blood cells."'" Cells were obtained by centrifugation of aliquots of the f i l t e r e d suspensions, and dried Dr. H. E. Taylor of the Department of Pathology kindly examined a sample of the suspension microscopically. - 2 0 -i n vacuo over sodium hydroxide and sulphuric acid for measurement of dry weight. The suspensions usually contained 20 to 28 milligrams dry weight of tissue per m i l l i l i t r e . III. Respiration. A l l reactions were carried out i n Warburg flasks of 15 ml. volume. The Warburg apparatus was adjusted to the standard shaking rate (74) of 100 oscillations per minute at a stroke of 5 cm. at the manometer top, so that oxygen diffusion was not a limiting factor. A l l experiments with radioactive substrates were carried out under aerobic conditions at 37°C with 3 ml. of hepatoma c e l l suspension. Krebs-Ringer phosphate, pH 7 .4, was used for the buffer. The buffer solution was prepared as described i n Manometric Techniques (75) with the modification that only one-third the amount of calcium chloride was added, to prevent precipitation of calcium phosphate. The buffer kept the tissue medium within 0.2 pH units of 7.4 during the experiments. Respiration was followed by the direct method, and Qog's (microlitres of oxygen uptake per hour per milligram dry weight of tissue) were based on the i n i t i a l 30 minutes. Ten minutes of gassing with oxygen and equilibration preceeded the addition of radioactive substrates from the side-arms. Antimetabolites and inhibitors were added before the equilibration. The tissues were s t i l l respiring actively after 120 minutes, when most experiments were terminated. IV. Glycolysis. Several anaerobic glycolysis experiments were carried'out using ^21-evolution of carbon dioxide as a measure. Robinson's medium (76) as modified by LePage was used as the buffer. 5% Carbon dioxide and 35% nitrogen was used as the gas phase. The QQQ^'S (microlitres of carbon dioxide evolved per hour per milligram dry weight) were easily calculated because the glycolysis was linear. V. Isolation of purines. The purines were isolated by a method similar to that of LePage (77). The contents of each incubation flask were transferred! to a 15 ml. conical centrifuge tube. The flask was washed with 0.154 M sodium chloride and this rinsing was also added to the centrifuge tube. The medium was decanted after centrifuging, and the tissue rinsed with saline. The residue was extracted with 3 ml. of 2% perchloric acid at 0°C for 15 to 20 minutes to remove the acid soluble fraction. Two similar washings with 2 ml. of 2% perchloric acid each were added to the acid soluble supernatant solution. This solution was made to A% i n perchloric acid and hydrolyzed by heating at 90°C for 30 minutes. The mixed nucleic acid purines were extracted from the tissue with A% perchloric acid also by hydrolyzing at 90°C for 30 minutes. The residue was washed twice at room temperature with 2 ml. portions of 2% perchloric acid, and a l l the supernatants were combined. Adenine and guanine were purified from salts and pyrimidines by ion exchange chromatography on 6 X 15 mm. columns of Dowex-50 (200 to 400 mesh), H + form. Both the acid soluble and nucleic acid hydrolysates were treated i n the same manner. The purines were adsorbed Personal communication from G. A. LePage to A. R. P. Paterson. -22-by passing the hydrolyzed solution through the column and then 2 ml. of 0.1 N hydrochloric acid and 2 ml. of water were used to wash the column. The purines were recovered by elution with 4 ml. of 6 N hydrochloric acid. These eluates were taken to dryness i n desiccators i n vacuo. The adenine and guanine were separated by descending paper chromatography on Whatman No. 3 MM paper, using the isopropanol-hydrochloric acid solvent of Wyatt (78). The purines were located on the papers i n a beam of ultraviolet light. Adenine and guanine were eluted i n 4 ml. of 0.1 N hydrochloric acid and rechromatographed separately on Whatman No. 1 paper i n the same solvent. Two aliquots (of suitable size for radioactive counting) of each compound were rechromatographed to check the specific activity, except i n the case of the acid soluble guanines which were too small to divide. Some acid soluble guanine samples were contaminated with a fluorescent material which could be removed by further chromatography i n isobutyric acid-water-ammonia, 66:33:1 by volume (79). In the experiment using 6-mercaptopurine, adenine and 6-mercaptopurine ran with the same i n Wyatt's solvent. Therefore, the acid soluble adenine samples were further purified by paper chromatography i n 5% ammonium sulphate-5% isopropanol-water (62). When adenine-d-C^ was used as the isotopic precursor, any free adenine was removed from the acid soluble nucleotides by an ion exchange procedure devised by Dr. A. R. P. Paterson . The acid soluble Personal communication from A. R. P. Paterson. -23-extractd were prepared as usual, then neutralized in the cold with potassium hydroxide to bromcresol purple. The supernatant was decanted from the precipitate of potassium perchlorate onto a 5 X 30 mm. column of Dowex^ -1, CI form. The column was then washed with these solutions - 20 ml. of 0.01 N ammonium chloride, a water solution of 100 micrograms of non-isotopic adenine, 1.5 ml. of water, and 30 ml. 14 of 0.01 N ammonium chloride - to remove any unmetabolized adenine-8-C The nucleotides were eluted with 10 mi. of 1.5 N hydrochloric acid, diluted to be 1 N i n the acid, and hydrolyzed at 100°C for 60 minutes. The resulting purines were taken to dryness, and then purified on Dowex-50 and paper as above. The nucleic acid fraction was obtained by the normal procedure, but f i r s t the tissue was washed with 2 extra rinses of cold 2% perchloric acid. Where RNA and DNA purines were reported separately, the nucleic 4 acids were separated by a method based on hydrolysis of RNA by a l k a l i . The acid soluble fractions were removed as usual, but each sample of tissue again was washed twice more with the 2% perchlorate. The tissue residue from a typical flask was suspended i n 1 ml. of 10% sodium chloride, and neutralized with carbon dioxide-free sodium hydroxide i n 10% sodium chloride to the orange colour of phenol red. The mixed nucleic acids were extracted by heating to 100°C for 60 minutes. The supernatant was removed and the tissue re-extracted with 0.5 ml. of 10% sodium chloride for 30 minutes. To the combined supernatants were added 2 to 2 1/2 volumes of cold 35% ethanol. After the mixture had been chilled for 8 hours, i t was centrifuged and the supernatant discarded. The ^Personal communication from A. R. P. Paterson. -24-residue was washed with cold alcohol, and then dissolved i n 1 ml. of 0.1 N sodium hydroxide and incubated for 20 hours at 37°C. The solution of 2' ,3'-ribonucleotides and intact DNA was chilled and made to 0.1 N in hydrochloric acid. After the DNA had precipitated, the supernatant ribonucleotides were removed, made to 1 N with hydrochloric acid, and hydrolyzed for 60 minutes at 100°C. The purines were dried, purified on the Dowex-50 columns, and chromatographed on Whatman No. 1 i n Wyatts solvent. The DNA was purified by washing with cold 0.1 N hydrochloric acid, resuspending i n 0.5 ml. of 0.1 N sodium hydroxide, heating at 80°C for 20 minutes, and reprecipitating by making 0.1 N i n hydrochloric acid and c h i l l i n g . The DNA precipitate was hydrolyzed for 30 minutes at 100°C i n 1 N hydrochloric acid. The DNA hydrolysates were dried, and chromatographed directly i n the isopropanol-hydrochloric acid solvent. VI. Radioactive counting procedures. A l l radioactive counting was done i n a windowless gas flow counter. Suitable aliquots of the radioactive substrate^ were counted on aluminum planchets covered with lens paper. Purine samples were routinely counted by cutting discs of 26 mm. diameter from the paper chromatographs. The counts from the paper discs were multiplied by a factor of 2.7 to correct for the absorpt ion of the f i l t e r paper. This factor was determined by counting eluates of the paper discs on lens paper - aluminum planchets. Chromatographs were dried for at least 18 hours i n a fume hood before counting. Specific ac t i v i t i e s were expressed in a l l cases as c.p.m./uM. Some of the media were counted i n a -25-manner similar to that used by Oro and Rappoport (80) for their acid soluble fractions. Before counting, the medium was f i l t e r e d , and diluted to 10 ml. with dilute sodium carbonate. Duplicate lens paper planchets of 15 microlitres of this diluted medium were counted. Then the formate was volatilized from the planchets by the addition of 0.5 ml. of 50% formic acid and heating i n a hood with an infrared lam.pl Three such treatments removed a l l formate - volatile material. The planchets were then recounted. VII. Ultraviolet spectrophotometry. The adenine and guanine were eluted from the paper discs with 4 ml. of 0.1 N hydrochloric acid, or 2 ml., i f the quantity was small. The purines were l e f t overnight and then shaken i n a mechanical shaker for 30 minutes to ensure complete elution. A l l ultraviolet absorption spectra analyses were performed i n a Beckman D K H2 ratio recording spectrophotometer. Purine concentrations were calculated from these molar extintion coefficients : adenine - 13.5 X 10 5 at 261.5 millimicrons, guanine - 11.42 X 10^ at 248 millimicrons. 'Determined for this machine by A. Hori. B. RESULTS. I. Characterization and standardization of the tissue suspension, (i) Respiration. The respiration rate was used as a criterion of the general metabolic activity of the hepatoma c e l l s . Early experiments showed that the tumour cells were responsible for the oxygen uptake observed because the amount of uptake was always directly proportional to the weight of the tissue, and because the rate slowly decreased. If bacterial contamination were responsible for the respiration, then an increase i n the rate would be expected. Aureomycin (10 parts per million) and glucose (2 mg. per ml.) had no effect on the QQ^'S of the c e l l s . The QQ^'S observed varied from 6 to 11.5, but were quite constant i n any one experiment, unless altered by the addition of an inhibitor. The rates of respiration have been reported i n a l l experiments i n which possible inhibitory compounds were used. ( i i ) Effect of formate-C^ concentration. In order to determine what amount of formate to use i n routine experiments, both for maximal specific a c t i v i t y of the purines and for economy of the isotope, a preliminary study was made with different amounts of sodium formate-C"^. Hepatoma cells were incubated with 2,5, 10, and 15 micromoles of formate for 140 minutes. From the curves plotted (Figures 4 and 5), i t was concluded that 8 micromoles of formate per Warburg flask would be sufficient to saturate ©ither the enzyme systems , operating or the amounts of endogenous factors. This amount was used i n a l l further experiments. jJM OF FORMATE Figure 4. Effect of concentration on i n v i t r o incorporation of formate-C 4 into acid soluble purines by the Novikoff hepatoma. 140 minute incubation. -28-Figure 5. Effect of concentration on i n vitro incorporation of formate-Cl4 into nucleic acid purines by the Novikoff hepatoma. 140 minute incubation. -29-( i i i ) Precision of specific a c t i v i t i e s . Next, to determine the v a r i a b i l i t y i n formate incorporation i n samples from the same tissue preparation and hence the r e l i a b i l i t y and significance of any further results, 6 aliquot samples from a c e l l preparation were incubated under the same conditions. The results i n Table II showed that a difference greater than 12.5 % of control values i n studies with antimetabolites and inhibitors might be taken as being significant. In order to minimize the deviation, duplicate controls were run i n a l l • experiments, and their values averaged. (iv) Time study of formate-C incorporation. The incorporation of formate-C-''4 into the Novikoff hepatoma was followed by determining the specific a c t i v i t i e s of the tissue purines from flasks incubated for difference times. The values plotted i n Figures 6 and 7 were determined by averaging values of 2 (or i n some cases more) identical flasks. The acid soluble purines became- labeled very rapidly (Figure 6), adenine having the higher specific activity. Their specific a c t i v i t i e s were s t i l l increasing at 180 minutes. The nucleic acid compounds were not labeled as quickly. The guanine showed a definite lag period. Again, adenine had a higher specific activity. Once begun, the incorporation of isotope into both adenine and guanine remained linear, and their specific a c t i v i t i e s after 180 minutes were at least 4% of those of the corresponding acid soluble purines. A time of 120 minutes was chosen for a l l later experiments because purines of both fractions were s t i l l increasing i n specific -30-TABLE II Variability i n the specific a c t i v i t i e s of purines synthesized by samples from the same hepatoma tissue preparation. Incubated for 120 minutes with formate-C^-4. Flask Number \ Specific activity i n c.p.m./uM Acid soluble Nucleic acid Adenine Guanine Adenine Guanine 1 11.0 16 X 10 4 11.1 X 10 4 8.67 X 10 3 4.81 X 10 3 2 11.0 15.2 9.8 L0.1 4.89 3 10.4 14.9 8.5 7.76 4.46 4 11.2 19.8 11.9 9.6 5.80 5 11.2 19.2 10.7 . 9.99 4.67 6 11.5 - 20.3 10.2 • 9.76 5.79 Mean specific activity 17.6'\X 10 4 10.4 X 10 4 9.3 X 10 3 5.1 X 10 3 Relative mean deviation 12.5% 7.8# B.2% 3.5% -31-TIME IN MINUTES Figure 6 . Time study of i n vi t r o incorporation of formate-C into acid soluble purines by the Novikoff hepatoma. -32-Figure 7. Time study of i n vi t r o incorporation of formate-C into nucleic acid purines by the Novikoff hepatoma. -33-acti v i t i e s at this time and would thus give true values for synthetic reactions, and because the radioactivity of the nucleic acid purines would be sufficiently high to count accurately. (v) Time study of adenine-8-C^ incorporation. An experiment similar to that described above was carried out with adenine-8-C 1 4. A smaller amount of adenine (1 micromole) than formate was.used to minimize any toxic effects. The results from duplicate reaction vessels are plotted i n Figures 8 and 9. The pattern of incorporation was similar to that for formate. The a c t i v i t y of the acid soluble adenine was s t i l l increasing after 120 minutes, but the specific a c t i v i t y of the guanine remained constant throughout the period from 30 to 120 minutes. These findings indicated that even with the lower concentration of precursor, the system was s t i l l saturated at the end of the experiment. The nucleic acid adenine was labeled i n a linear fashion at f i r s t but declined sl i g h t l y i n rate of incorporation after 60 minutes. The guanine again demonstrated a lag i n incorporation, and i t s incorporation curve was almost identical to the nucleic acid guanine i n the formate-C"^ experiments. After 120 minutes, the nucleic acid adenine had a specific activity that was 3.7% of the acid soluble adenine activity, and the nucleic acid guanine specific a c t i v i t y was 1.7% of the acid soluble guanine. 14 The adenine-8-C was used more readily for the adenine compounds 14 than the guanine compounds, as compared to formate-C . The high specific activities of the purines isolated i n this experiment indicated that enzymes for u t i l i z a t i o n of purines by the "salvage" pathway were quite -34-9 0 0 , 0 0 0 CL O z 6 0 0 , 0 0 0 >-> (-< 3 0 0 , 0 0 0 o o UJ Q_ 00 ADENINE GUANINE 30 6 0 120 TIME IN M I N U T E S Figure 8 . Time study of i n vitro incorporation of adenine-8-C into acid soluble purines by the Novikoff hepatoma. 14 f -35-30 60 120 TIME IN MINUTES Figure 9. Time study of i n vitro incorporation of adenine-8-C 4 into nucleic acid purines by the Novikoff hepatoma. -36-active i n the Novikoff hepatoma. Despite the careful separation of adenine from the nucleotides of the acid soluble fraction, i t was possible that seme adenine-8-Cp-4 s t i l l was not removed. Dr. Paterson had found that his method might leave as much as 0.02% of the administered radioactivity as adenine with the nucleotide eluates. However, i t was f e l t , because much adenine would remain i n the medium and because the acid soluble adenine nucleotides contained so much radioactivity, that the values of the specific a c t i v i t i e s reported were accurate determinations for the acid soluble adenine samples. (vi) Incorporation of formate-C 1 4 into DNA and RNA. The rates of renewal of DM and RNA in vitro were compared i n an experiment reported i n Table III. The specific a c t i v i t i e s of the RNA purines were higher than those of the DNA bases. The RNA adenine was almost 5 times the guanine i n activity, but both purines of DNA had the same order of activity. TABLE III In vitro incorporation of formate- C 1 4 into DNA and RNA purxnes by the Novikoff hepatoma. Specific a c t i v i t y i n c.p.m./uM Adenine Guanine Acid soluble 71,500 21,600 DNA 295 265 RNA 4,520 945 -37-(vii) Controls. (a) Heat inactivated tissue. In order to make sure that there was no non-enzymatic incorporation of formate-C^ into purines, a sample of tumour suspension was inactivated by placing i t s Warburg flask into boiling water for 4 minutes. This tissue was incubated under normal conditions (with 8 micromoles of sodium formate-C 1 4 for 120 minutes at 37°C). Counts of samples from the acid soluble and nucleic acid purines were indistinguishable from the background count. (b) Blood c e l l s . Rough counts of hepatoma suspensions showed that there were about twice as many red blood c e l l s as tumour' c e l l s , although the blood cells were much smaller than the tumour c e l l s . Blood c e l l s have been regarded as relatively inert metabolically, as far as synthesis of new nucleic acid i s concerned. Goldwasser reported (81) that adenine-ft-C^ incorporation into RNA by avian erythrocytes was not appreciable. Nucleotides are present i n erythrocytes (82), but no report has been found of any anabolic enzymes other than nucleoside phosphorylase. Because mature red c e l l s have no nuclei, there probably is no DNA. The RNA concentration also drops on maturity (83). Attempts were made to separate the red cel l s from the tumour cel l s by differential centrifugation both i n Krebs-Ringer buffer and i n the sucrose solutions of Hogeboom et a l (84). No clear separations could be obtained. The red cells could be removed by preferential hemolysis and - 3 8 -then centrifugation, but not without damage to the respiration of the hepatoma ce l l s . A control experiment was performed, therefore, using a sample (0 .2 ml.) of blood drawn from the heart of a tumour-bearing rat into a syringe containing a small amount of heparin. This blood was diluted by addition to 3 ml. of buffer solution.in a Warburg flask, and was incubated with isotopic formate for 120 minutes. No appreciable respiration could be detected i n that time. After paper chromatography, the "nucleic acid" fraction exhibited only a non-radioactive fluorescent streak. The acid soluble portion also gave fluorescence and a small area of feint ultraviolet absorption. Only 50 c.p.m. of radioactivity could be accounted for i n the areas on the chromatograph where adenine and guanine are usually found. This amount was small enough so that any incorporation by erythrocytes may be neglected. ( v i i i ) Amounts of purines isolated. In order to estimate'the size of the acid soluble nucleotide pools and the approximate d i l u t i o n of isotope by nucleic acid purines, the chromatographs from 3 experiments were quantitatively analyzed (Table TV). It may be seen that the acid soluble purines were present i n concentrations much lower than their nucleic acid counterparts. This meant that the radioactivity from the acid soluble fraction was diluted considerably when i t was incorporated into the nucleic acids (by a factor of 10 for adenine, and 23 for guanine, in terms of specific a c t i v i t y ) . There was a 4 times higher concentration of adenine than guanine in the acid soluble extracts. (ix) Medium. When the media were counted, usually 70 to 80% of the formate-C^ that had been administered could be accounted for i n -39-the formate-volatile fraction. Considerable radioactivity remained after the vo l a t i l i z a t i o n procedure, but because this amount was not appreciably affected by any of the inhibitors tested,the media were not examined any further. TABLE IV Amounts of purine bases isolated by the technique used i n these experiments. The amounts are expressed as micromoles per 100 milligrams dry weight of tissue. Acid soluble. Nucleic acid Adenine Guanine • Adenine Guanine Value 0.38 .0.089 3.8 2.0 Precision + 0.04 + 0.013 '+ 0.4 + 0.4 Number of 27 9 2T. 20 Samples -40-II. Inhibitors and antimetabolites. (i) Azaserine. Azaserine was the f i r s t inhibitor to be tested. The results in Table V indicated that the purine biosynthesis of the Novikoff hepatoma was very readily blocked by this antibiotic. The renewal of both acid soluble and nucleic acid purines was blocked, without any decrease i n respiration. The results were expressed i n percent inhibition, or the difference from the control values i n percentage. A negative percent inhibition would indicate that there had been an increase i n specific activity over the controls. ( i i ) . 6-Hercaptopurine. Larger doses of 6-mercaptopurine than azaserine were used, i n line with those used by other investigators (57). The solid compound was added to the flasks because of the low water solubility of 6-mercaptopurine. Paper chromatographs i n the isopropanol-hydrochloric acid solvent revealed a straw coloured spot in the acid soluble hydrolysates with the same R^.as adenine. When this compound and the adenine were re chromatographed i n ammonium sulphate-isopropanol-water, the straw coloured compound separated from the adenine, and had the characteristic and fluorescence of 6-mercaptopurine. On this basis, i t was concluded that 6-mercaptopurine had been found i n the acid soluble fraction. Whether i t occurred as a free base or as a nucleotide was not determined. The 6-mercaptopurine decreased the acid soluble and nucleic acid purine act i v i t i e s and respiration. (Table 7). ( i i i ) N-Benzoylglycinamidine. Results from 2 experiments with N-benzoylglycinamidine are reported i n Table V. From these data, i t appeared that this compound i n low doses stimulated purine synthesis, possibly through hydrolysis to glycine. The large dose (10 mg.) inhibited -41-TABLE V Effects of azaserine, 6-mercaptbpurine^ and N-benzoyl-glycinamidine on i n vitro incorporation of formate-C 1 4 into purines by the Novikoff hepatoma. Compound Amount Q % Inhibition per °2 • Acid soluble Nucleic acid flask Adenine Guanine Adenine Guanine Azaserine 0 10.3 - - -0.37 10.3 . 89% 90% 81.5% 0.3 * 10.2 89 90.5 80 6-mercapto-purine 0 1 mg. 11.4 8.2 43.5% 51% 42% 1 mg. 8.2 45 . 49: 40 N-benzoyl-glycinamidine hydrochloride 0 1 mg. 7.9 7.0 5% -44% 15% -31% 0 6.3 - - - -1 ^  6.2 0% -7% 5% 8% 5 V 6.2 1.5 -15.5 -4 -3 100 X 6.3 -11.5 -14 -7 -14 1 mg. 5.7 -19.5 -26 -8.5 -16.5 10 mg. 3.4 51 57 72 85.5 -42-both purine renewal and respiration. The nucleic acid purines were inhibited more than the acid soluble purines. (iv) Actinomycin D. (a) Effect on respiration. Early experiments indicated that .actinomycin D i n low concentrations inhibited respiration. Therefore, an experiment was performed to measure the Q 's using increasing amounts of actinomycin D. °2 The gas phase used was a i r , not oxygen. The results are described i n Table 71. The slowing of the respiratory rate by small amounts of actinomycin D was quite definite. This was borne out by the Qo2*s ^ r o m the isotopic experiments with actinomycin D (Tables 7III to XII). TABLE 71 Effect of actinomycin D on respiration by the Novikoff hepatoma i n vitr o . 50.8 mg. dry weight of tissue per flask. Amount per flask 0 8.4 1 ( i n duplicate) ,0.031 y 7 . 7 ; 8.2 0.062 7.9 0.135 7.3; 7.5 0 .27 6.9 (in duplicate) 0.405 6.1 -43-(b) Effect on glycolysis. To determine whether the action of actinomycin D slowed oxygen uptake by limiting a reaction i n the glycolytic pathway, the studies described in Table VII were carried out. So that a l l the glucose would not be glycolyzed or that the buffer be completely overcome by acid production, smaller amounts of tissue were used. It was observed that there was no decrease i n carbon dioxide evolution (acid production from glucose) with increase i n actinomycin concentration, and with large amounts there was an increase. TABLE VII Effect of actinomycin D on anaerobic glycolysis by the Novikoff hepatoma i n vitr o . Experiment 1 had 10.2 mg. dry weight of' tissue per flask and experiment 2 had 10.5 mg. per flask. Amount per flask QN2 co 2 Experiment 1 Experiment 2 , 0 16.1; 16.5 12.8; 12.9 0.013 16.5; 18.0 -0.031 18.2; 19.8 13.4; 14.0 0.062 21.1; 23.5 13.7; 14.0 0.135 25.1; 26.1 14.3 0.405 - 1*.8 - 4 4 -(c) Effect on purine renewals. It was found that actinomycin D blocked formate-C^ incorporation into nucleic acid purines (Table VIII), the specific activities of guanine being most affected. The acid soluble adenine was relatively unaffected, but the acid soluble guanine formation was blocked. These effects were dependent on the concentration of actinomycin. There seemed to be no specific effect on either DNA or RNA (Table DC), as both were inhibited considerably. The incorporation of adenine-8-C"^ into guanine (both acid soluble and nucleic acid) was cut very markedly (Table X), while adenine nucleotide formation was barely unaffected. (d) Reversals of inhibitions. In separate experiments, pantothenate and coenzyme A were employed to reverse the effects of actinomycin D (Tables XI and XII). Percent inhibitions could only be reported for the pantothenate experiment, because the coenzyme A was pa r t i a l l y hydrolyzed to adenine nucleotides, during the experiment. Large quantities of calcium pantothenate were required to completely overcome the inhibition of purine synthesis by actinomycin D. The guanine compounds, whose formation was blocked more extensively by actinomycin, responded more to the pantothenate stimulation than did adenine. Even 10 mg. of pantothenate did not restore respiration completely. Coenzyme A was more effective than calcium pantothenate i n reversing the inhibitory effects of actinomycin D. The exact amount of reversal of purine renewal inhibition.could not be measured by the methods used because some of the coenzyme A was hydrolyzed, and diluted the acid soluble adenine pool. With 5 mg. of coenzyme A, there was twice as much acid soluble adenine at the end of the experiment as i n the controls. -45-TABLE VIII Effect of actinomycin D on i n vitro incorporation of formate-C 1 4 into purines by the Novikofl' hepatoma. Amount per flask Q °2 % Inhibition Acid Soluble Nucleic Acid Adenine Guanine Adenine Guanine 0 0.135 y 0.135 10.3 10.0 10.0 -7% -7 -21% -14 4% 12 0 1.35 y 1.35 1.1.4 7.8 9-1 . 29.5% 13.5 49% 43 0 7.9 - - - -i:.35 <r 6.4 -16% -3.5% 5.5% 27% 2.7 5.8 3.5 35 35 56 4.05 5.4 -1.5 26 46 49 -46-TABLE IX Effect of actinomycin D on i n vitro incorporation of formate-C 1 4 into the purines of DMA and RNA of Novikoff hepatoma. % Inhibition Amount Acid Soluble DNA RNA per flask °2 Adenine Guanine Adenine Guanine., Adenine Guanine 0 6.2 - - - - - -2.7 K 4.5 17% 54% 52% 56% • 55% 60% 2.7 4.5 18 58 45 51 77 77 TABLE X: 14 Effect oS- actinomycin D on i n vitro incorporation of adenine-8-C into purines by the Novikoff hepatoma. 1 uM of adenine-8-C 1 4 containing 2.0 X 10 6 c p •m. was added to each flask. % Inhibition Amount Respiration Acid Soluble Nucleic Acid.. per flask ' ' Adenine Guanine • Adenine Guanine 2.7 r 24% -5% 54% 24% 47% 2.7 21 1.5 47 -1 33 -47-TABLE XI Effect of calcium pantothenate and actinomycin D on in vitro incorporation of formate-C into purines by the Novikoff hepatoma. Flask contents Q % Inhibi tion Actinomycin Pantothenate Acid Soluble Nucleic Acid Adenine Guanine Adenine Guanine 0 0 8.5# - - - -5.4 * 0.1 mg. 4.2 21.5% 73% 51.5% 61% 2.7 0 e:2# 8 51 30 41 2,7 0.1 mg. 6.4 1.5 42 22 29 2.7 1.0 6.7# -23 25 7 7 2..7 10 7.8#. -14 -20 -11 -34 " - a l l values for these 4 conditions are averages of duplicate flasks. TABLE XII Effect of coenzyme A and actinomycin D on i n vitro incorporation of formate-C-1-4 into purines by the Novikoff hepatoma. Flask Contents Q V Specific activity i n c.p.m./uli Actinomycin Coenzyme A Acid Soluble Nucleic Acid Adenine Guanine Adenine Guanine 0 0 6.6$ 76,100 24,900 3230 865 2.7 Y 0 5.2# 63,600 11,900 1880 430 2.7 0.01 mg. 4.8 54,900 10,800 980 260 2.7 0.1 5.3 64,700 16,500 1460 346 2.7 1.0 5.8 64,400 12,200 2060 380 2.7 5.0.. 6-2 42,500 10,650 2380 350 # - a l l values for these 2 conditions are averages of duplicate flasks. -48-How the extra adenine affected the guanine and nucleic acid adenine values cannot be determined. The coenzyme A did not completely reverse the inhibition of respiration, but i t was more effective than pantothenate. -49-DISCUSSION Aerobic conditions were used for the study of nucleic acid metabolism in Novikoff hepatoma ce l l s i n the present study because of the simplicity of the suspension medium and so that the endogenous energy sources might be used f u l l y . LePage (77) found that glycine incorporation into purines occurred equally well under either aerobic or anaerobic conditions i n several tissues, whereas Harrington and Lavik (85), using P^2 as a measure of nucleic acid renewal i n Ehrlich ascites c e l l s , found that aerobic conditions were nesessary. Mannell and Rossiter (86) found that anaerobic conditions stopped formate-C^ incorporation into RNA by slices of guinea pig l i v e r . The time studies of the incorporation of both isotopic precursors showed that the acid soluble purines were rapidly labeled, and maintained this high level of specific activity. There was a lag period before the nucleic acids were labeled, then isotope incorporation occurred linearly with time u n t i l the experiments were terminated. This would indicate that the acid soluble nucleotides are the precursors of the nucleic acid purines. Similar results have been found by Paterson and Zbarsky (87) with intestinal mucosa suspensions. Bennett and Skipper (88) found that when formate-C^ was injected into tumour-bearing mice the acid soluble nucleotides reached maximal specific activity after 1 hour, but the RNA purines were not at their maximal activity until. 6 to 12 hours had elapsed. Haydar et a l (89) followed glycine-2-C 1 4 and formate-C 1 4 incorporation into the acid soluble and RNA purines of rat l i v e r i n vivo and observed that the RNA purines did not increase i n specific activity -50-after the acid soluble purines started to decrease i n activity. With glycine-2-C 1 4 as the precursor, the acid soluble guanine had a higher specific activity than the acid soluble adenine, as LePage (77) had found previously, but, when formate-C 1 4 was used as the precursor, the acid soluble adenine had the higher specific activity. Haydar and coworkers considered that formate incorporation only indicated the relative rates of synthesis of AMP and GMP from IMP (because of formate-C 1 4 exchange with the carbon 2 (51) of. IMP). Otherwise, a rate of adenine biosynthesis of more than 10 times that of guanine would be neeessary because the size of the acid soluble adenine pool was 8 times that of the guanine pool. Although the results reported i n this thesis d i f f e r from those of Haydar i n the labeling of the nucleic acids as a function of time, the higher concentration of acid soluble adenine (more than 4 times the guanine) and the higher specific activities of the adenine nucleotides derived from formate-C 1 4 are similar. However, the exchange of carbon 2 of existing nucleotide:•.molecules (51) with radioactive formate cannot be extensive i n the Novikoff hepatoma system because azaserine (which i n low concentrations i s a specific inhibitor of a reaction i n de novo purine biosynthesis) was extremely effective i n blocking formate incorporation into both the adenine and guanine nucleotides. The inhibitions were qualitatively similar to those found by LePage (56) using glycine-C 1 4 and by Tomisek et al (Figures 1 to 4 i n 58) with formate-C 1 4. Tomisek's results showed that FGAR and i t s riboside accumulated large amounts of radioactivity from formate-C 1 4, so that i t is l i k e l y that the labeling of the acid soluble adenine and guanine from formate i n our experiments must be due to -51-radioactivity i n both carbons 2 and 8, not just i n carbon 2. It would follow that incorporation of either glycine-C 1 4 _or formate-C 1 4 into the acid soluble purines, adenine and guanine, would be-a measure of the rate of conversion of IMP to the other nucleotides, i f both AMP and GMP originate solely from this precursor. These incorporations would also indicate the amounts of de novo purine biosynthesis. The discrepancy between the ratios of the specific activities of adenine and guanine when glycine-2-C 1 4 and formate-C^ were used i n the experiments by Haydar cannot be explained unless some reaction that allows formate incorporation into AMP but not IMP directly were existing. In any case, with the Novikoff hepatoma suspensions, the results with azaserine indicate strongly that formate-C^ incorporation into the purines i s a suitable measure of their de novo synthesis. The Novikoff hepatoma has been shown to have lower amounts of enzymes that catalyze purine breakdown than normal rat l i v e r (90). This fact could perhaps explain the low u t i l i z a t i o n of adenine-8-C 1 4 for guanine compounds. However, the preformed adenine was highly incorporated into adenine nucleotides. The direct conversion of adenine to AMP undoubtedly accounted for this u t i l i z a t i o n . The acid soluble nucleotides labeled from isotopic adenine were used to about the same extent as those 14 -labeled from formate-C for nucleic acid purine formation. There has been no distinction made i n these discussions between the terms "synthesis" and "renewal," as applied to nucleic acid formation. Examinations of P^2 incorporation into the nucleic acids of growing Escherichia c o l i and of tissue cultures of mammalian cells (91), and of formate-C^ incorporation i n vivo into tumour nucleic acid (88), have shown -52-that these tissues did not lose the radioactivity incorporated into their polynucleotides. However, other tissues, such as intestine and spleen (88) lost considerable amounts. The latter 2 tissues divide rapidly, normally not increasing i n size but replacing lost or damaged c e l l s . It would seem logic a l , then, that the incorporation of radioactivity into the nucleic acids of the hepatoma cells would be synthetic, not replacement or turnover. The Novikoff tumour has been shown to be a useful tissue for the examination of metabolic inhibitors. Azaserine and 6-mercaptopurine gave results similar to those observed with other tissues by other investigators. The value of N-benzoylglycinamidine as a chemotherapeutic agent deserves further study. Perhaps the large amount of this substance needed to show inhibitory effects would be toxic (indeed, i t greatly lowered respiration). The s t a b i l i t y of the compound also must be questioned. The observation that actinomycin D lowered respiration but not anaerobic glycolysis showed that the antibiotic had i t s effect on energy mobilization from endogenous sources,(probably carbohydrate) at a point past pyruvate. This, plus the effectiveness of pantothenate and especially of coenzyme A in reversing the inhibitions caused by actinomycin D, supports Foley's hypothesis (72) that actinomycin interfered with synthesis or biologic activity of coenzyme A. The degree to which the cells were permeable to coenzyme A was not determined. Because of the speed with which actinomycin D blocked respiration, i t would seem probable that i t i s not a stopping of coenzyme A synthesis, but an inhibition of a coenzyme A-dependent reaction that is effected by actinomycin D. -53-The mechanism by which actinomycin D inhibits nucleic acid purine and acid soluble guanine synthesis might be through the decrease in respiration. Mannell and Rossiter (86) suggested that respiratory poisons such as dinitrophenol might have effect on polynucleotide formation by lowering the rate of oxidative phosphorylation. If actinomycin D were active i n this way, then the synthesis of acid soluble adenine nucleotides (which also requires much energy) might also be blocked. The fact that both DNA and RNA synthsis were blocked, though, seems to support the inhibition of oxidative phosphorylation as the action of actinomycin D. Warburg (92) has commented that respiratory poisons may stop the growth of tumours by reducing respiration below a le t h a l minimum. Hepatoma cells have a lower number of mitochondria than l i v e r (93), and there is also a lack of a transhydrogenase,involving coenzyme II and DPN (94) i n the Novikoff hepatoma so that the hepatoma cells might be more susceptible than normal cells to any action involving respiration inhibition by actinomycin D. SUMMARY 1. Novikoff hepatoma cells have been employed for in vitro studies. The tumour preparations show a f a i r l y high respiratory rate and moderate anaerobic glycolysis. The cells were s t i l l actively metabolizing i n vitro after 3 hours. 2. Biosynthesis of acid soluble and nucleic acid purines from sodium formate-C 1 4 and adenine-8-C 1 4 by the tumour cells has been studied. 14 The hepatoma was able to incorporated formate-C into purines and to u t i l i z e preformed adenine for nucleotides. The specific activity of adenine (in both the acid soluble and mixed nucleic acid fractions) was higher than that of guanine when either isotopic precursor was used. Time studies of purine formation suggested that the .acid soluble purines were precursors of the nucleic acid purines. Ribonucleic acid purine renewal was higher than deoxyribonucleic acid renewal. 3. The erythrocytes that were present i n a l l hepatoma preparations #ere shown not to contribute significantly to the nucleic acid metabolism. 4. The in v i t r o hepatoma suspensions were used for the testing of 4 possible antimetabolites and chemotherapeutic agents. Azaserine and 6-mercaptopurine blocked synthesis of purines (as measured by i n vitro incorporation of formate-C1^) by the Novikoff tumour. These findings are similar to those found with other neoplastic tissues. 5. N-Benzoylglycinamidine was tested as a possible purine precursor antimetabolite. Low concentrations of this compounds stimulated purine renewal, but a high dose inhibited both respiration and purine biosynthesis. -55" 6. The action of actinomycin D on tumour c e l l metabolism was studied i n some detail. This antibiotic was found to slow down the rate of respiration of the cells but not the rate of anaerobic glycolysis. 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