The Formation of Cytidine Nucleotides and RNA Cytosine from Orotic Acid by the Novikoff Tumor in Vitro*

Home , Cytidine, Cytidine triphosphate, Cytosine

HAROLD O. KAMMEN t AND ROBERT B. HURLBERT

(Biochemistry Department, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston f~5, Tezas)

The utilization of orotic acid for the formation the amino group of cytosine compounds in ma- of RNA ~uracil has been extensively studied in in- malian tissues may be derived from glutamine, intact animals, tissue slices, and cell suspensions (re- stead of ammonia. Eidinoff et al. (9) have shown viewed in Refs. 5, 31). The mechanism of the the glutamine antagonist, DON, to depress sig- utilization, via the initial conversion of orotic acid nificantly the incorporation of orotic acid into the to uridine nucleotides and the incorporation of uri- nucleic acid cytosine of rat tissues, both in vivo dine nucleotides into polynucleotides, has been and in tissue slices. Salzman et al. (33) have found partially described in intact animals (13, 17) and the amide group of glutamine to be the source of studied in detail in enzyme systems of microbial, the amino group of the polynucleotide cytosine avian, and mammalian origin (18, 36) (reviewed in HeLa cell cultures. In a preliminary report (31) in Refs. 33, 38, 39). The concomitant conversion we have described a soluble enzyme system, of orotic acid to RNA cytosine has usually been present in both the Novikoff rat tumor and rat observed in intact cell systems, in vivo and in liver, capable of the conversion of orotic acid to vitro, but is smaller, more variable, and apparent- uridine nucleotides and of the amination of the ly of secondary nature as compared with the con- uridine nucleotides to cytidine nucleotides. ATP version to RNA uracil (1, 7, 13, 14, 16, 18, 33, 34). and glutamine were required for the amination Lieberman (35) has demonstrated the direct step, guanosine nucleotides appeared to serve as amination of uridine triphosphate to form cytidine cofactors, and DON was inhibitory. triphosphate; the enzyme system, purified from In the work reported here, the objective was to Escherichia coli, required ammonia and ATP. The establish whether the enzymatic steps observed in existence of this reaction suggests strongly that the soluble system were part of the normal path- the uridine nucleotides and cytidine nucleotides way for the conversion of orotic acid to RNA cyto- are involved as intermediates in the conversion of sine in surviving Novikoff tumor tissue. The ef- orotic acid to RNA cytosine. fects of glutamine, other amino donors, glutamine Evidence has more recently been offered that antagonists, and uridine were examined in intact * Aided by grants from the American Cancer Society cell preparations of the tumor incubated with (P-146) and from the Anna Fuller Fund. With the assistance orotic acid-6-C TM. The labeling of the cytidine of Joyce Danielson Miller, Alice F. Wheeler, and Audrey F. nucleotides and RNA cytosine was compared with Posey. This work forms part of the dissertation submitted by the labeling of the uridine nucleotides and RNA Harold O. Kammen to the Department of Chemistry and uracil. To provide further perspective, the inter- Chemical Engineering, Stanford University, for the degree of Doctor of Philosophy, 1959. relationships among all the major "acid-soluble" pyrimidine metabolites of orotic acid were also I" Present address: Department of Pharmacology, Yale University, New Haven, Connecticut. examined. These metabolites (cytidine nucleo- ' The abbreviations used are: RNA (ribonucleieacid), DNA tides, cytidine, cytosine, uridine nucleotides, uri- (deoxyribonucleic acid), ATP (adenosine-5'-triphosphate), dine, and uracil) were separated from cold per- UMP (uridine-5'-phosphate), CMP (cytidine-5'-phosphate), chloric acid extracts of the tissue by ion-exchange DON (6-diazo-5-oxo-L-norleucine), and azaserine (0-diazo- and paper chromatographic methods. A brief re- acetyl-~serine). "Uridylic acid" and "cytidylic acid" represent the mixed $'- and 3'-monophosphates of uridine and cytidine, port has appeared (30). respectively, derived by alkaline hydrolysis of RNA. "Uridine nucleotides" and "cytidine nucleotides" represent the groups MATERIALS AND METHODS of nucleoside-5'-phosphate and -5'-pyrophosphate monomeric Materials.--The orotic acid-6-C 14 was pur- compounds extractable by cold percMoric acid and hydrolyz- chased from Tracerlab, Inc., the uniformly C 14- able to UMP and CMP, respectively. labeled cytidine from Schwarz Laboratories, Inc., Received for publication February 9, 1959. and the amino acids (L-isomers)from the Cali- 654

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1959 American Association for Cancer Research. KAMMEN AND HURLBERT--Formation of RNA Cytosine 655 fornia Corporation for Biochemical Research. The in a 13-ml. tube, and the precipitated tissue was re- azaserine bore the Parke, Davis and Company lot extracted with 0.4 N perchloric acid; the combined number 2342 X 35, and the DON s bore the Sloan- extracts (7-8 ml. total volume) constituted the Kettering Institute number SK 13,835 ab. The "cold perchloric acid extract." The precipitate was azaserine and DON materials were checked by further washed with two 8-ml. portions of cold published spectrophotometric criteria and found 0.4 ~r perchlorie acid, and 3 ml. of cold 12 per cent to be 59 per cent azaserine (3) and 87 per cent NaC1 was added. The suspension was neutralized DON (8), respectively. Other chemicals were com- carefully with a minimum volume of NaOH solu- mercial products of reagent quality. tion (phenol red indicator) and heated at 100~ C. The Novikoff transplantable hepatoma s (27) for 30 minutes to extract the sodium nucleate (12, was carried in 350-500-gm. female Holtzmann 16). The precipitate was centrifuged and re- Sprague-Dawley rats and was transplanted every extracted for a few minutes with 1 mi. of hot 10 5th day by the intraperitoneal injection of 100- per cent NaC1. The combined salt extracts were 300 mg. of fresh, minced tissue suspended in treated with 2.5 volumes of ethanol at - 10~ C. for 0.15 M NaC1. The tumor for the experimental work several hours to precipitate the sodium nucleate, was taken on the 5th day, when the rats were which was then washed twice with 5-ml. portions usually severely hemorrhagic. The large mass of of cold 80 per cent ethanol and dissolved in 2.0 ml. tumor in the greater omentum and a smaller mass of water. The sodium nucleate solution was brought adjacent to the spleen were removed, chilled, to 0.3 s NaOH and incubated overnight at 88 ~ C. cleaned of the larger blood clots, passed through The DNA was precipitated by the addition of 1 a stainless steel mesh in a large syringe, and sus- volume of 0.4 N perchloric acid (cold); the acid pended in the Krebs-Ringer phosphate-glucose solution and a 1-ml. wash with 0.1 N perchloric (KRPG) medium (see below). The free blood cells acid constituted the "RNA" fraction. The DNA were removed by suspending and sedimenting the was dissolved in 2 ml. of 0.1 s NaOH, the solution tumor tissue 2 or 3 times in the KRPG medium, was heated at 80 ~ C. for 5 minutes (I3), neutral- with centrifugation at about 100 X g for 1-2 ized, and treated with 2.5 volumes of cold ethanol. minutes at 2 ~ C. The packed tissue was diluted The precipitated sodium deoxyribonucleate was with the KRPG solution to a 30 per cent suspen, washed twice with 5-ml. portions of 80 per cent sion (v/v). The composition of the modified ethanol and dissolved in 3.0 pal. of water for KRPG solution was: 0.125 M NaCl, 0.015 M KCI, analysis. 0.003 ~ CaCb, 0.00067 M MgCl2, 0.006 M sodium Analysis and chromatography.--The RNA and DNA con- phosphate, pH 7.4, and 0.030 M D-glucose. In some tent of the sodium nucleate solution and DNA content of the cases the tissue was washed and suspended in the sodium deoxyribonucleate solution were determined. In addi- Krebs-Ringer salts solution, with separate addition, the RNA and DNA remaining in the tissue residue after extraction of the nucleates were estimated by analysis of an tion of the phosphate and glucose to the incuba- extract made with hot 0.4 N perchloric acid. All data have been tion flasks. calculated to include and correct for this residual RNA and Incubation and preparation of samples.--Ali- DNA (less than 10 per cent of the total). There was no de- quots of the tissue suspension were pipetted into tectable cross-contamination of the "RNA" and the sodium the incubation flasks; the relative amount of deoxyribonucleate fractious. The analyses for RNA were made by a modified orcinol procedure (19) and for DNA by Burton's tissue in each flask was estimated by analyses for modification of the Dische diphenylamine procedure (6). DNA at the end of the incubation. The incubation (Commercial yeast RNA and sperm DNA were used as mixtures were prepared and kept at 0 ~ in 50-ml. standards.) Erlenmeyer flasks, flushed with 95 per cent Oz-5 Each "RNA" fraction was brought to 1 N perchloric acid by the addition of 11.5 N perchloric acid, heated at 100~ C. for 80 per cent CO~ before !and after the addition of the minutes to destroy the purine nucleotides, and neutralized at tissue. During the incubation the stoppered flasks 0 ~ C. with the minimum volume of concentrated KOH. The were shaken about 100 cycles per minute at 38 ~ C. KC104 precipitate was washed twice by centrifugation with in a water bath. 'The pH of the medium decreased several ml. of cold water; the solution and washes were chro- to nearly 6.8 during the course of the incubations. matographed on a 9 cm. X I cm. column of Dowex-1 (formate) to separate the uridylic and cytidylic acids. The nucleotides To stop the incubations, the flasks were chilled in were eluted with formic acid by an improved procedure similar ice, and 0.1 volume of 4.4 rr perchloric acid was to one previously described (16), with modifications suitable added. The contents of each flask were centrifuged to the use of a longer column. To the "cold perchloric acid extract" was added 0.5/zmole 2 Generously supplied by Dr. H. C. Reilly of the Sloan- each of the carrier compounds UMP, CMP, uridine, cytidine, Kettering Institute for Cancer Research. Originally produced uracil, and cytosine. The solution was brought to 1 N perchloric by Parke, Davis & Company. acid and heated for 80 minutes at 100~ C. to hydrolyze the py- t We are grateful to Dr. Alex B. Novikoff for the shipment rimidine nucleotides to the monophosphates. The solution was of the original tumor-bearing rats of our series. chilled, adjusted with concentrated KOH to pH S-S, and

centrifuged. The supernatant solution and two cold water solvent of Magasanik et al. (93). The radioactivity of the uri- washes of the KC104 precipitate were chromatographed on a dine-uracil fraction was 70-100 per cent recovered in the Dowex-50 (H +) resin column to separate the CMP, cytidine, regions of known uridine and uracil, with no evidence of a third and cytosine. Fraction 1 from this chromatographic procedure component, by paper chromatography with the isobutyric was made to pH 10.5 with ammonia and chromatographed on acid-ammonia solvent or the butanol-water solvent of Mark- a Dowex-1 (formate) column to obtain fractions containing a ham and Smith (23). The cytidine and cytosine fractions were uridine-uracil mixture, UMP, and orotic acid. The elution contaminated with purines, although the cytosine appeared steps for the latter two chromatographic procedures are shown free of radioactive impurities when subjected to paper chro- in Table 1. matography with the latter solvents. The cytidine fraction The chromatographic fractions eluted by HC1 and by formic occasionally contained traces of a radioactive component with acid were chilled and evaporated to dryness in Erlenmeyer some of the chromatographic properties of ~-alanine. Large flasks over a 2:1 mixture of technical grade NaOH and CaC12 amounts of a salt-like material in the fraction made positive in evacuated desiccators warmed by infrared lamps. The dried identification of the latter component difficult. The best paper residues were each dissolved in 2.0 ml. of water for analysis. chromatographic resolution of the cytidine was obtained with The fractions eluted by ammonium formate were usually the isopropanol-HC1 solvent of Wyatt (93) after partial analyzed without prior drying. When necessary for paper separation from the salt by adsorption of the cytidine on chromatography, the uridine-uracil and UMP were dried as Dowex-50 (NH~-) columns and elution by dilute ammonia. The

TABLE 1 ION-EXCHANGE CtIROMATOGRAPHY OF "COLD PERCtILORIC ACID EXTRACTS"

DowEx-50 (H+) Dow~.x-1 (rxm~tATv.)

Ehant Eluant Fraction Fraction M1. Composition M1. Composition

,-- 10 0.001 M HC1 1 15 0.2 M NI~OH 10 0.001 M HC1 Uridine plus 20 0.1 M AmForm uracil 3 10 0.2 M HCI 3 * 5 0.1 M AmForm 4 CMP 20 0.2 M HCI 4 5 0.3 M AmForm 5 * 5 0.2 M HC1 5 UMP $0 0.3 M Am_Form 6 20 1.0 M HC1 6 * 5 0.3 1~I AmForm 7 Cytidine 25 1.0 M HC1 7 8 0.6 M AmForm 8 * 5 1.0 M HCI 80rotic acid 40 0.6 M AmForm 9 Cytosine 2O 2.0 M HCI 9 * 5 0.6 M AmForm 10 * 5 2.0 M HCI

The Dowex-1 (formate))< 8 resin, 200-400 mesh, was previously washed with 88 per cent formic acid and water (19) and the Dowex-50 (H +) )< 8 resin, 20(000 mesh, was washed with hot 1 N NaOH, water, 4 N HC1 and water. The resins were packed as columns of 9 cm. in height and 1 cm. in diameter. Fraction 1 also included the effluent from the sample placed on the column. The fractions marked (*) were frequently checked for light absorption at 260 m# and for content of radioactivity and were included with the main fraction if necessary. AmForm represents ammonium formate solutions of pH 5.

above, with stronger heating to remove the ammonium for- uridylic and cytidylic acids, as obtained by ion-exchange mate. chromatography, were adequately pure for measurement of Each fraction was prepared in duplicate for C ~4 analysis by specific activities; their purity was occasionally checked by use spreading and drying an aliquot within an inscribed ring on an of the isopropanol-HC1 solvent. A small radioactive peak, aluminum planchet. The samples were counted with a Nuclear- trailing the cytidylic acid peak, was separated by ion-exchange Chicago Corporation model D-47 gas flow counter and a micro- chromatography with the 9-cm. columns but was not resolved mil window for time periods sufficient to achieve a statistical when 4-cm. columns were tested. standard error within 5 per cent. Under these conditions of plating and counting, the self-absorption corrections for com- RESULTS AND DISCUSSION parable samples were small and were not applied. Aliquots of the uridylic and cytidylic acids were diluted in 0.1 N HCI for Presentation of data.mSince the amounts of spectrophotometric analysis. The spectrophotometric con- tissue in the suspension pipetted into the flasks stants used for calculation of the concentrations were ~260 = were not identical, analyses for the total DNA con- 9,900 for uridylic acid and ~s0 = 13,000 for cytidylic acid. The tent have been used to afford estimates of the rela- spectra of the chromatographed samples compared satisfac- torily with published spectra and known standards (4). tive amounts of tissue per flask. All incorporation The purity of each fraction was frequently checked by data have been calculated and compared "per mg. further chromatography. Although the CMP and UMP in the of tissue DNA" on the assumption that the total ion-exchange fractions were not completely spectrophoto- uptake of isotope was proportional to the amount metrically pure, no contaminant containing C ~4 could be de- tected by ion-exchange chromatography at a different pH or of tissue, at least over the short ranges of variation by paper chromatography in the isobutyric acid-ammonia within each experiment. The analyses for total

DNA content are included with each table and activity ratios, cytidylic acid/uridylic acid, pro- chart presented. vide a somewhat more vital and sensitive para- The radioactive compounds isolated in these meter, since they reflect the relative amounts of experiments represent all the quantitatively sig- the pyrimidines formed from orotic acid for the nificant pyrimidine metabolites of orotic acid-6- biosynthesis of RNA. A more rigorous interpreta- C TM. The ion-exchange fractions used to separate tion of the metabolic significance of these incor- these metabolites were frequently analyzed by poration data would require precise measurement paper chromatography under all the experimen- of specific activities and "pool sizes TM of all the tal conditions reported here, and no significant intermediates and products involved. amounts of other radioactive components were de- Discussion of data.--Chart 1 and Table ~ show tected (see "Materials and Methods"). The radio- the utilization of radioactivity from orotic acid- activity contents of the appropriate ion-exchange 6-C 14 by suspensions of the Novikoff tumor incu- fractions have accordingly been regarded as repre- bated in the presence of glutamine over a period sentative of the total radioactivity contents of the of ~ hours. To judge from the steady rates of in- pyrimidine compounds under study. Estimates of the total amounts of orotic acid utilized in each flask were made by a summation of UMP NU CLEOTIDES"~"~'-'~ the radioactivity recovered in the various pyrimi- 9 dine fractions. While the values so obtained are subject to summation errors, they provide a useful ~0 URIDYL.~ basis for comparison of the extent of the various ~5 ..,.. A C i L___.----.--~ metabolic pathways. At least 90--95 per cent of the UR ]~)'t NE..,,,~ i precursor radioactivity was recovered in the pyrim- SINE CPDS. idine and orotic acid fractions. The amounts of ~o radioactivity present in deoxyribo- derivatives and cf l intermediates of uracil catabolism were expected to be small and were not specifically measured. The data provide several comparisons of the .... : c~ T ,.o, ~.E ~ i relative amounts of uracil compounds and cytosine 0 30 60 120 compounds formed from orotic acid. The basic in- INCUBATION T/ME IN MINUTES formation is contained in the figures for the CH~.RT 1.--Formation of pyrimidine compounds from orotie amounts of radioactivity incorporated into the acid-6-C '4 by suspensions of Novikoff tumor tissue in the presence of glutamine. (Data expressed "per rag. of tissue uridine nucleotides (hydrolyzed and combined for DNA.") See Table 2. analysis as UMP), the cytidine nucleotides (combined as CMP), the RNA uridylic acid, and the corporation into DNA and RNA, the incubation RNA cytidylic acid. Although the incorporations conditions were adequate for metabolic survival of of C ~4 into uracil, uridine, cytosine, and cytidine the tissue, s Additional data (not shown) from the were also determined (see Chart 1), they are usual- same experiment indicated continuing survival ly not recorded individually here, because the and similar labeling patterns up to 4 hours. The values were relatively smaller and not greatly primary conversion of the orotic acid was to uracil variable under the different experimental condi- compounds; at zero time nearly all the detectable tions. Two additional columns of figures assist in radioactivity in the pyrimidine compounds was in interpretation of the experiments: the amounts of the uridine nucleotides, with a trace found in the radioactivity recovered as total cytosine com- cytidine nucleotides. (This incorporation occurred pounds (including the cytidine and cytosine) and while the tissue was present in the complete the calculated ratios of specific activities of the medium briefly at 0 ~ C. before the addition of RNA pyrimidines, cytidylic acid X 100/uridylic perchloric acid.) At 1 hour, 9.9 per cent of the acid. The values for the C 14 incorporated into radioactivity of the orotic acid had been utilized; "total cytosine compounds" (per mg. of tissue 4 Approximate "pool sizes," determined in the course of DNA) may be either compared directly as meas- other experiments, were (per rag. of tissue DNA): Uridine ures of the effects of the experimental conditions, nucleotides, 0.15 ~mole; cytidine nucleotides, 0.03 pmole; or used to calculate the percentage of the orotic RNA uridylic acid, 0.8 ~mole; RNA cytidylic acid, 1.1 ~mole; acid utilized which was converted to cytosine and RNA, 1.2 rag. compounds. The latter calculation affords a 6 The viability of the incubated tumor was tested several times. Rats given intraperitoneal injections of the tissue from measure of the relative total formations of uracil typical flasks, incubated from I to 3 hours, succumbed to the compounds and cytosine compounds. The specific tumor in 5-6 days.

of this amount, 33.5 per cent was present as RNA and glutamate plus ammonia on the metabolism of uridylie acid, 50 per cent as UMP nucleotides, 7.5 orotic acid have been compared. In the absence of per cent as uridine, 13.5 per cent as uracil, and an amino donor a small percentage of the orotic 6 per cent as total cytosine compounds. The RNA acid precursor utilized was converted to total cyto- cytidylic acid, cytidine nucleotides, cytidine, and sine compounds (figures calculated from Tables 3, cytosine represented 3.3 per cent, 1.9 per cent, 4, and 5 range from 0.9 to 3.7 per cent), and the 0.8 per cent, and 0.1 per cent, respectively, of the resulting ratios of specific activities "100 X cyti- radioactivity utilized. The formation of radio- dylic/uridylic" ranged from 1.7 to 3.6. Glutamine active cytidylic acid continued over the entire appeared to have two effects on the metabolism of time period, although the radioactivity in the orotic acid. One was a "general". effect, an im- cytidine nucleotides reached a maximum at 15 or provement in the total utilization of orotic acid 30 minutes. The rate of formation of cytosine and an increased labeling of RNA and DNA (see compounds from orotic acid clearly diminished also discussion of Table 6). The other was a specific effect on the formation of labeled cytosine com- TABLE 2 pounds, noted by increases of about threefold in FORMATION OF PYRIMIDINE COMPOUNDS FROM OROTIC the percentage of the "precursor utilized" con- ACID-6-C14 BY SUSPENSIONS OF NOVIKOFF TUMOR TIS- verted to total cytosine compounds (calculated SUE IN THE PRESENCE OF GLUTAMINE figures range from 4.3 to 8.8 per cent) and by (Supplementary data for Chart 1) three- to fourfold increases in the ratios of specific activities "100 • cytidylic/uridylic." The com-

RATIO OF bined "general" and specific effects resulted in in- SPECIFIC PRECURSOR creases of about fourfold in the absolute amounts ACTIVITIF~ INCUBATION DNA COSVENT UTIliZED of radioactivity found in the total cytosine com- TIME OF TISSUE (PER MG. OF pounds. In most of the experiments, glutamine TmSUSDNA) Cytidy!ie • 100 Uridylic was present at a concentration of 6.7 mM (30 gmoles/flask), although Table 3 shows one third of (rain.) (rag.) (counts/rain.) this concentration to be almost equally effective. 0 2.88 2,582 Glutamate (Table 3) and glutamate plus ammonia 15 2.66 13,133 10.8 30 2.90 16,516 9.5 (Table 4) did not exhibit the "general" stimula- 45 2.64 20,428 9.0 tion and, although effective in the specific stimu- 60 2.40 22,812 8.9 120 2.29 30,705 7.9 lation of formation of radioactive cytosine compounds, were not as effective as glutamine. As- Approximately 400 rag. of tissue was incubated at 38 ~ C. paragine (Table 3), while similar to glutamine in in 95 per cent Or-5 per cent C02 in 4.0 ml. of total volume con- the "general" improvement of the total utilization taining 2.8 ml. of Krebs-Ringer salts solution, 100 #moles of of orotic acid and RNA formation, was almost in- glucose, 40 gmoles of K-phosphate, pH 7.4, 50 gmoles of NaHC03, 30 #moles of glutamine, and 0.575 gmoles of orotic effective in specifically promoting cytosine forma- acid-6-C :4, 554,000 counts/min. In duplicates of the 45-minute tion from orotic acid. and 120-minute samples the tissue and the medium were sepa- The results with glutamine, asparagine, and rated by centrifugation at the end of the incubation and analyzed separately. The radioactive UMP, CMP, uridylic acid, glutamate are seen in Table 3 to be similar in the and cytidylic acid were found exclusively in the tissue portion, presence and absence of added nonradioactive while the radioactive pyrimidine nucleosides and bases were distributed in both tissue and medium. The medium contained uridine. Uridine has been previously observed to 82 and 74 per cent of the uridine, 90 and 78 per cent of the ura- stimulate the formation of cytosine compounds cil, 85 and 83 per cent of the cytidine, and 81 and 84 per cent from orotic acid several-fold (30). Additional data of the cytosine, in the 45-minute and 120-minute samples, respectively. presented in Table 3 show that uridine alone caused a 3.6-fold increase in the total conversion after 80-45 minutes, as shown by Chart 1 and by to cytosine compounds, without significant altera- the ratios of specific activities "cytidylic • 100/ tion in the amount of orotic acid utilized. Uridine uridylic" in Table 3, despite the continued increase did cause a 45 per cent reduction in the labeling in radioactivity of the uridine nucleotides. Unpub- of RNA uridylie acid, presumably by conversion lished data indicate that the initial rates of forma- to uridine nucleotides and dilution of the radio- tion of cytosine compounds from orotic acid are activity in the precursor of RNA uridylic acid. better maintained when nonradioactive uridine is The effects of uridine and glutamine are seen to be included in the medium. Table 3 also shows the independent; addition of the combination of extensive loss of labeled pyrimidine bases and glutamine and uridine to the basic system, as nudeosides into the medium. shown in Tables 3 and 4, increased the formation The effects of glutamine, asparagine, glutamate, of total cytosine compounds by factors of 8.8 and

COMPARISON OF AMINO DONORS IN THE FORMATION OF CYTOSINE COMPOUNDS FROM OROTIC ACID-6-C 14

C 14 CONTENT OF PYRIMIDINE COMPOUNDS (PER MG, OF TISSUE DNA) RATIO OF SPECIFIC ACTIVITIES CONDITIONS DNA Acid-soluble RNA CONTENT OF Total Precursor TISSUE DNA cytosine CytidylicX10~ utilized CMP UMP Uridylic Cytidylic compounds Uridylic Additions in #moles nucleo- nucleotides acid acid per flask tides

(mg.) (counts/ (counts/rain) (counts/ (counts/ (counts/ (counts/ (counts/ rain) rain) rain) rain) rain) rain) Basic system:* No amino donor 2.44 34 13,450 8,000 90 2,570 110 220 2.8 Glutamine, 10 2.75 133 15,680 9,090 330 2,97O 44O 835 9.4 Glutamine, 20 2.55 137 16,760 9,510 350 3,510 495 915 9.0 Glutamine, 30 2.55 135 16,550 9,670 360 3,330 470 910 9.2 Asparagine, 30 2.48 32 17,360 10,890 175 2,730 135 345 3.5 Basic system:t No amino donor 2 27 29 11,040 5,230 55 2,410 60 185 1.7 Glutamine, 30 2.62 127 15,I60 6,680 195 3,220 325 640 7.0 Glutamate, 30 2.40 53 11,110 4,990 85 2,050 110 315 3.7 Asparagine, 30 2.43 19 14,220 7,070 95 2,990 85 310 2.0 Basic systemt plus uridine, 5: No amino donor 2.88 29 11,290 5,950 180 1,310 240 490 12.8 Glutamine, 30 2.26 146 15,050 6,930 675 2,000 775 1,650 26.9 Glutamate, 30 2.31 54 11,120 5,280 235 1,200 305 680 18.3 Asparagine, 30 2.82 19 14,320 8,590 90 1,600 130 415 5.7

The "basic system" consisted of approximately 350 mg. of tumor tissue in 4.5 ml. of total volume containing 3.3 ml. of Krebs- Ringer phosphate-glucose solution plus 22 gmoles of extra K-PO4, 50 gmoles of KHCO3, and 0.575 gmoles of orotic acid-6-C 14, 380,000 counts/rain. Incubated for 60 minutes at 38 ~ C. in 95 per cent 02 (5 per cent C02). *t Separate experiments.

TABLE 4 EFFECT OF DON ON THE FORMATION OF CYTOSINE COMPOUNDS FROM OROTIC ACID-6-C t4

C 14 CONTENT OF p~amn~ COMPOUNDS (PER MG. OF TISSUE DNA) RATIO OF SPECIFIC CONmTIONS DNA ACTrVITIF~ CONTENT Acid-soluble RNA Total OF Precursor ., TISSUE cytosine CytidylicX 100 utilized Additions in #moles UMP CMP Uridylic Cytidylic compounds Uridylic per flask nucleotides nucleotides acid acid

(rag.) (counts/rain) (counts/rain) (counts/ (counts/ (counts/ (counts/ rain) min) rain) rain) Basic system: No inhibitor 1.98 14,390 8,750 155 3,060 155 395 3.6 DON, 0.4 1.85 14,860 9,480 30 2,670 10 110 0.3 DON, 4.0* 2.00 12,450 7,690 25 2,440 5 100 0.1 Basic system plus glutamine, 30: No inhibitor 1.87 17,720 9,780 575 4,380 815 1,560 13.3 DON, O.4 1.93 17,790 9,470 1,250 2,550 1,210 2,735 33.8 DON, 4.0* 1.87 15,6~0 10,040 100 2,370 180 390 5.2 Basic system plus glutamine, 30, and uridine, 5: No inhibitor 1.73 17,310 9,280 1,115 2,440 1,045 2,410 30.6 DON, 0,4 1.77 17,850 9,830 925 1,890 1,155 2,365 43.6 DON, 4.0 1.73 19,200 12,840 85 2,060 215 445 7.4 Basic system plus glutamate, 30, and NH4C1, 30: No inhibitor 1.72 12,860 7,700 365 1,910 350 825 13.2 DON, 0.4 1.57 12,760 8,190 95 1,800 100 290 4.0

The incubations were as described in Table 3 except that approximately 300 rag. of tumor tissue was used and no extra K-PO, was added. * From a separate experiment.

6.1, respectively (or by factors of 6.5 and 5.1, pounds, which are structural analogs of gluM- based on the percentage of the orotic acid pre- mine, appear to be specific antagonists of several cursor utilized). The ratios of the specific activities enzymatic reactions which utilize the amide group of cytidylic acid and uridylic acid were increased of glutamine with a requirement for ATP. A even more strikingly. This effect of uridine, similar specific competition between DON and glutamine to the substrate effect noted by Canellakis (7), has been observed in the formation of formyl- will be discussed more fully elsewhere. glycinamidine ribotide from formylglycinamide The experiments discussed above suggest that ribotide, glutamine, and ATP (24), and azaserine glutamine may be involved in the formation of has been shown to be a specific competitive in- cytosine compounds. More convincing evidence hibitor of glutamine as the donor of an -NH~ that glutamine is directly involved is offered in or = NH group in the formation of 5-phosphoribo-. Tables 4 and 5, where the inhibitory effects of sylamine from 5-phosphoribosylpyrophosphate DON and azaserine are recorded. These corn- (10), the formation of formylglycinamidine ribo-

TABLE 5 EFFECT OF AZASERINE ON THE FORMATION OF CYTOSINE COMPOUNDS FROM OROTIC ACID-6-C 14

C 14 CONTENT OF pTRIMIDINE COMPOUNDS (PER MG. OF TISSUE DNA) RATIO OF SPECIFIC ACTIVITIES CONDITIONS DNA Acid-soluble RNA CONTENT OF Total Precursor cytosine CytidylicX100 TISflUE CMP utilized UMP Uridylic Cytidylic compounds Additions in umoles Uridylic nucleotides nucho- acid acid per flask tides

(rag.) (counts/min) (counts/min) (counts/ (counts/min) (counts/ (counts/rain) rain) rain) Basic system: No inhibitor 1.81 12,610 7,550 95 2,880 95 270 2.1 Azaserine, 7 1.99 13,610 8,490 4O 2,850 15 125 0.3 Azaserine, 35 1.95 14,350 9,630 50 2,590 20 145 0.6 Plus glutamine, 30: No inhibitor 1.80 15,650 8,400 365 3,950 530 1,010 9.0 Azaserine, 7 1.82 16,110 8,400 755 3,430 880 1,745 16.2 Azaserine, 35 1.61 15,040 8,780 475 2,830 500 1,110 11.8

The incubations were as described in Table 4.

TABLE 6 EFFECT OF DON ON THE FORMATION OF PYRIMIDINE COMPOUNDS FROM U-C1LCYTIDINE

C 14 CONTENT OF PYRIMIDINE COMPOUND (pER MG. OF TISSUE DNA) RATIO OF SPECIFIC AC TI~'ITIF-~ CONDITIONS DNA Acid-sohbh RNA CONTENT OF Total Precursor uracil Uridylic X 100 TISSUE utilized UMP CMP Uridylic Cytidylic compounds Cytidylic Additions in umoles nucleo- nucleotides add acid per flask tides -. (rag.) (counts/rain) (counts/ (counts/Inin) (counts/ (counts/rain) (counts/rain) rain) min) Basic system: No inhibitor 1.70 8,590 435 2,210 95 1,430 3,960 10.3 DON, 0.40 1.56 8,570 460 2,120 95 1,460 3,960 9.5 Basic system plus glutamine, 80: No inhibitor 1.43 9,680 380 2,260 135 1,830 4,540 11.1 DON, 4.0 1.41 8,420 410 1,680 85 1,380 4,440 9.5

The flasks contained approximately 250 mg. of tumor tissue incubated as described in Table 4, except that the precursor was uniformly C14-hbeled cytidine, 1.0 gmole, 30,000 counts/min. The UMP was further purified by paper chromatography. Uridine and uracil contained approximately ~5 and 63 per cent, respectively, of the C 14 in the "Total Uracil Compounds" in all flasks.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1959 American Association for Cancer Research. KAMMEN AND HURLBERT--Formation of RNA Cytosine 661 tide (~4), and the formation of diphosphopyridine the labeled cytidine for the biosynthesis of RNA nucleotide from nicotinic acid-adenine dinucleo- is noteworthy, as well as significant conversions to tide (30). In our system (Table 4), DON at a con- UMP nucleotides and RNA uridylic acid (about centration of 0.089 mM inhibited the total forma- 6 per cent of the precursor utilized) and the formation of cytosine compounds from orotic acid al- tion of relatively large amounts of uridine and most completely in the absence of added glutamine uracil. and by 65 per cent in the presence of glutamate Interpretation of data.--The requirement for plus ammonia, but not at all in the presence of glutamine in the amination of uridine nucleotides 6.7 mM glutamine. DON at a concentration of to cytidine nucleotides and the inhibition of this 0.89 mM inhibited the formation by 75 per cent in amination step by DON have been previously the presence of 6.7 mM glutamine and by 81 per demonstrated in a soluble enzyme system from cent in the presence of glutamine plus uridine. the Novikoff tumor (~1). The data presented here Azaserine (Table 5), at a concentration of 1.55 mM provide evidence that in surviving tumor cells the in the absence of glutamine, partially inhibited the same enzymatic step is also of critical importance conversion to cytosine compounds, but in the in the formation of cytosine compounds from presence of glutamine was ineffective at a concen- orotic acid. The major evidence is as follows: tration of 7.8 mM. The effect of preincubation with 1. The initial conversion of orotic acid was ap- the inhibitors in the absence of glutamine was not parently to the uridine nucleotides, as found in tested. The inhibitors appeared to cause no altera- other tissues (13, 17, 18, ~6). No evidence that a tion in the conversion of orotic acid to uridine direct amination of orotic acid plays an important nucleotides but did cause significant reductions in role has yet been obtained in either the intact cell the labeling of RNA uridylic acid (10-15 per cent system or the soluble system, although the possi- in the absence of glutamine and 30-40 per cent in bility that such a reaction exists has not been the presence of glutamine). The latter inhibition directly excluded. appeared to be of a more general nature, reflected ~. The report of Eidinoff et al. (9) that the by decreased biosynthesis of RNA (see also Table glutamine antagonist, DON, inhibited the forma- 6) and related to the metabolic stimulation in- tion of RNA cytosine from orotic acid was con- duced by glutamine. firmed and extended. Glutamine was found to be An interesting effect was the increased forma- required by the Novikoff tumor for the optimal tion of C14-cytosine compounds observed with the formation of RNA cytosine from orotic acid. lower levels of DON and azaserine in the presence 3. The stimulatory effect of glutamine and the of glutamine (Tables 4 and 5). The conversion of inhibitory effect of glutamine antagonists were orotic acid to uracil compounds was unaffected, observed at the origin of the group of labeled although the subsequent incorporation of C 14 into cytosine compounds (cytidine nucleotides, RNA uridylic acid was decreased, and the incorporation cytosine, cytidine, and cytosine), rather than on into cytidylic acid was increased. This anomalous the formation of uridine nucleotides and RNA effect has not yet been further explored. Direct uracil or on the interconversion of the cytosine comparisons of the sensitivity of this system with compounds. the sensitivities of the other enzymatic reactions 4. The nucleosides and free bases appeared in of ghtamine to these irreversible inhibitors may these experiments as degradation products of the prove enlightening. The inhibitory levels of both uridine and cytidine nucleotides. Part of the sup- azaserine and DON in our system were far greater porting evidence was the release of uridine, cyti- than the levels of azaserine reported to inhibit dine, uracil, and cytosine from the tissue into the purine biosynthesis in Ehrlich ascites cells in vitro medium (Table ~); other evidence has been pro- (11). vided by the labeling patterns obtained in other In Table 6 the results of an experiment with experiments, with C14-uridine and C14-cytidine as uniformly C14-1abeled cytidine as precursor are precursors, similar to those reported in Table 6 and presented. Addition of glutamine improved the in a brief publication (~0). The nucleosides and general utilization of the precursor and the label- free bases also were clearly degradation products ing of RNA; this effect was reversed by the in broken cell preparations of the tumor (15). higher level of DON. There was no definite specific 5. The possibility of a pathway for the forma- effect, either on the formation of RNA cytidylic tion of cytosine compounds via amination of uri- acid or on the conversion to uracil compounds. dine to cytidine is apparently ruled out by the Similar results were obtained by Eidinoff et al. (9) anomalous effect of added unlabeled uridine, in an experiment on the utilization of C~-cytidylic which increased the formation of cytosine com- acid in rat tumor slices. The efficient utilization of pounds (Table 3), and by the effect of added un-

labeled cytidine, which inhibited the formation of This finding may be correlated with the report of all cytosine compounds from orotic acid rather Auerbach and Waisman that the Novikoff tumor than "trapping" the radioactivity as cytidine (30). is low in glutamine synthetase (3). A significant conversion of labeled cytidine to uracil compounds was observed (Table 6). Gluta- SUMMARY mine and DON did not affect this conversion, a The formation of cytidine nucleotides, RNA fact which may signify that the conversion did not cytidylic acid, and other pyrimidine compounds occur via a deamination of cytidine nucleotides to from orotic acid-6-C 14 was studied in suspensions uridine nucleotides. The deamination of cytidine of surviving tissue from the Novikoff rat tumor, to uridine did, however, appear to be a reaction of incubated under O2-CO2 in a supplemented Krebs- considerable metabolic significance in this tissue. Ringer bicarbonate medium. Most of the radio- We have previously proposed (30), on the basis of activity from the orotic acid utilized by the tissue labeling patterns obtained with radioactive orotic was recovered as uracil compounds, chiefly uridine acid, uridine, and cytidine as precursors, that nucleotides (hydrolyzed and combined for analysis cytidine is converted to RNA uracil via deamina- as uridine-5'-phosphate) and RNA uridylic acid, tion to uridine and phosphorylation of the uridine with lesser amounts as uridine and uracil. to uridine nucleotides. In the absence of other additives, approximately 3 per cent of the radioactivity from the orotic RNA acid utilized was recovered as cytosine compounds, URACIL CYTOSINE chiefly RNA cytidylic acid and cytidine nucleotides (hydrolyzed and combined as cytidine-5'- OROTIC _ URIDINE GLUTAMINE CYTIDINE ACID ---'~NUCLEOTIDES" ('BLOCKED'~)NUCLEOTIDES phosphate), with lesser amounts as cytidine and only small amounts as cytosine. Addition of glutamine to the incubation medium approxi- URIDINEII ~. CYTIDINEII mately tripled the formation of cytosine compounds, both on an absolute basis and relative to URACIL CYTOSINE the formation of uracil compounds; asparagine, glutamate, and glutamate plus ammonia were less CHART 2.--Metabolism of orotic acid in surviving Novikoff tumor tissue in vitro. Summary of discussion. effective in causing an increase. Addition of unlabeled uridine approximately doubled the forma- A diagrammatic summarization of the preceding tion of cytosine compounds. The effects of gluta- discussion of the metabolism of orotic acid in the mine and uridine were independent; in combina- Novikoff tumor is presented in Chart 2. tion they increased the formation by a factor of 6. The exact uridine and cytidine nucleotides The formation of cytosine compounds from participating in the amination step are unspeci- orotic acid was strongly suppressed by 0.09 mM fied; our besl: evidence to date with the soluble diazo-oxo-norleucine in the absence of glutamine enzyme system implicates the uridine nucleotides and by 0.9 mM diazo-oxo-norleucine in the at a high level of phosphorylation, consistent with presence of 6.7 mM glutamine. Azaserine was also the finding of Lieberman (35) that uridine triphos- inhibitory. The formation 'of uridine nucleotides phate is the compound aminated. In the interpre- and RNA uridylic acid from orotic acid was not tation of our data, the uridine nucleotide and the strongly affected by glutamine or diazo-oxo- cytidine nucleotide pools have also been assumed norleucine. The conversion of cytidine (as the to be (or to contain) direct precursors of the RNA added radioactive precursor) to RNA cytidylic uracil and RNA cytosine, respectively. Further in- acid or to uracil compounds was also not strongly vestigation of this assumption and of the exact affected by these agents. precursor-product relationships among the uridine These and other data are interpreted to indicate derivatives and cytidine derivatives will require that the predominant pathway for the formation measurement of the specific activities and degrees of :RNA cytosine from orotic acid in this mam- of equilibration of the individual compounds in- malian cell system proceeds via the initial con- volved. Experiments of this nature are under way. version of orotic acid to uridine nucleotides, fol- The extent of conversion of orotic acid to RNA lowed by amination of a member of the uridine cytosine, when the tissue was not supplemented nucleotide group to form the cytidine nucleotides. with glutamine, appeared to be much lower than Glutamine is specifically required in the amination the rates obtained with other tissues, both in vivo of uridine nucleotides to cytidine nucleotides, and and incubated as tissue slices (7, 9, 13, 14, 16, 84). the inhibition by diazo-oxo-norleucine is at this

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1959 American Association for Cancer Research. The Formation of Cytidine Nucleotides and RNA Cytosine from Orotic Acid by the Novikoff Tumor in Vitro

Harold O. Kammen and Robert B. Hurlbert

Cancer Res 1959;19:654-663.

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