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On Incorpora Tion of Precursors Into Nucleic Acid Pyrimidines *

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On Incorpora Tion of Precursors Into Nucleic Acid Pyrimidines * Pyrimidine Studies I. Effect of DON (6-Diazo-5-oxo-L-norleucine) on Incorpora tion of Precursors into Nucleic Acid Pyrimidines * MAXWELL L. EIDIN0FF, JOSEPH E. KNOLL, BENJAMIN MARANO, AND LORETTA CHEONG (Dieision of Biophysics, Sloan-Kettering Institute, Memorial Center, New York, N.Y.) As a part of a study related to the effect of Se formly labeled,' was a mixture of @‘,3'-phosphate lected compounds on the formation of nucleic acid isomers and had a specific activity of 0.45 jzc/ pynimidines, the tumor-inhibitory antibiotic, 6- j@mole. diazo-5-oxo-i,.norleucine, has been tested. The In vivo experiments (Tables 1, 2).—The preparation and im chemical and tumor-inhibitory properties of this plantation of the tumor tissue, as well as the care of the am mats during the pro- and postimplantation petiod, was carried compound, referred to below as DON, have been out by the Human Tumor Laboratory, Division of Steroid reported (4—7).Following the administration of Biology, at Sloan-Kettering Institute. Young female rats DON, the incorporation of ureidosuccinicacid and (Carworth Farms Wistar and Charles River Breeding Labs. orotic acid (labeled with radiocarbon) into nucleic Wistar), weighing approximately 60 gui. were used. The tu acid cytosine was selectively depressed relative to mor-bearing rats had been treated with x-radiation and cor tisone (13, 17). Incorporation studies were performed 9 days uracil and thymine. This effect was observed in the after tumor implantatioi@. The radiocarbon-labeled ureidosuc case of tumor, liver, and intestine of rats bearing cinic acid and orotic acid, in the potassium salt forms, were ad two types of human tumor transplants as well as ministered intraperitoneally at the level of 20 pc/O0-gm in slice experiments with tumor tissue or rat liver. weight. The control group of five to six animals was sacrificed under ether anesthesia 7 hours after administration of the la The incorporation data suggest that the site of beled precursors. The time interval selected was based on the interference by DON in these studies is along a experiments of Hurlbert and Potter (8) and Anderson et a!. pathway leading from a metabolite containing the (1) and the necessity of obtaining a measurable specific activity unacil moiety to one containing the cytosine in the DNA pyrimidines. The animals were given food (Purina Chow) and water ad libitum during this period. In the treated moiety. group the labeled precursor was administered 30 minutes after MATERIALS AND METHODS the injection of DON (in saline solution), and the animals were sacrificed 7 hours later. To minimize the effect of biological Ureidosuccinic acid, labeled in the ureido car variation, the tissue from each group of animals was pooled and bon, was synthesized by an adaptation of the analyzed as described below. method of Nyc and Mitchell (14). Urea-C'4 was Tissue slice experiments (Tables 8-7).--Tumors were excised reacted with potassium carbonate to give labeled from the host rats 11 days after implantation, the necrotic por tions removed, and the remaining tissue pooled and minced potassium cyanate. The latter was then treated finely with scalpel blades. The resulting slices (in the controls) with DL-aspartic acid to yield ureidosuccinic acid, were incubated for 2 hours at 37°C. in a Krebs-Ringer-bicar which was then purified by recrystallization. The bonate medium containing 0.1 per cent glucose and the labeled specific activity was approximately 3 sc/mg. precursor (18). When DON was present, the labeled precursor was added 15 minutes after the incubation had started. The Onoti& acid-6-C'4 had a specific activity of 1.09 tissue was constantly shaken in 500-mi. Erlenmeyer flasks @ic/@zmole.Its purity was checked by paper chro under an oxygen: carbon dioxide (95:5) atmosphere. Incuba matographic methods and ultraviolet absorption. lions were stopped by the addition of an equal volume of ethyl Uridylic acid-C'4, uniformly labeled,' was a mix alcohol. Approximately 10 gm. of wet tissue was contained in ture of @‘,3'-phosphateisomers and had a specific 60 ml. of incubation medium. Twenty pc. of orotic acid-C'4 were present in each incubation flask (Tables 3, 4, 7). In Tables @ activity of 0.17 @ic/@mole.Cytidylic acid-C'4, uni 5 and 6, 5 and 10 of labeled cytidylic and uridylic acids, 4 Aided by research grants from the U.S. Atomic Energy respectively, were used. Commission AT (30-1)910. Analy8is for nucleic acid pyrtmzdines.—The combined so diuxn nucleates were obtained from the homogenized, dried, 1 Orotic acid-6-C'4 was obtained from Bio-Rad Co., uridyl ic acid-C'4 and cytidylic acid-C'4 from Schwarz Labs.; gluts defatted tissue by extraction with 10 per cent sodium chloride mine (reagent) from the California Found. for Biochemical solution at 100°C. for 6 hours and precipitation with ethanol Research; and DON was supplied by Parke-Davis & Co. (15). The barium ribonucleotides and DNA were obtained by a modification of the Schmidt-Thannhauser procedure (15, 16) Received for publication August 1, 1957. and hydrolyzed in 70 per cent perchloric acid at 100°C.for 45 105 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1958 American Association for Cancer Research. 106 Cancer Research VOL. 18, J@uu@@ 1958 minutes (5 mg. to 0.1 ml. HC1O4). The mixture was then di the specific activity of the nucleic acid cytosine ‘ luted by a factor of five and the char centrifuged off. The was most depressed by the administration of hydrolysate containing the purine and pyrimidine bases was placed in a band of Whatman No. 1 paper and was developed DON. This was observed in each of the six cytosine with the use of a descending propanol-HC1 solution. This con assays of Table 1. In the case of liver and intestine, sisted of a solution containing 65 ml. of peroxide-free isopropa the RNA uracil specific activities were not sig nol and 17 ml. 12 N HC1 made up to 100 ml. with water (19). nificantly affected, while the cytosine activities Approximate R, values in this system for guanine, adenine, were depressed by a factor of 5. cytosine, uradil, and thymine are 0.25, 0.36, 0.47, 0.68, and 0.77, respectively. The more slowly moving purine bands thus In rats beaning H.S. #1 tumors, the nucleic acid did not interfere with the pyrimidine analysis. The pyrimidine cytosine activity was also depressed most by the bands were cut out and eluted with distilled water descending administration of DON (Table @).This was ob the paper strip and dripping off the other pointed end. The served in each of the eight cytosine assays in this eluant was again applied to filter paper, and the pyrimidines developed in an ascending manner with the use of n-butanol table. In this experiment, the DNA thymine spe saturated with water (19). cific activity of the tumor was not appreciably The rechromatographed pyrimidines, together with appro changed, while that for the cytosine was depressed priate filter paper blanks, were eluted as described above and by a factor of approximately [email protected] factor for the brought up to a volume of S ml. One ml. of this solution was intestine RNA cytosine was 5, while the come diluted to S ml. with 0.01 N HC1, and the optical densities were determined by a Beckmann Model DU spectrophotometer. sponding uracil activity was not affected. The Optical density ratios (250/260 and 280/260) were Checked latter results are similar to those in Table 1. TABLE 1 EFFECT OF DON ON INCORPORATiON OF OROTIC AcID-C'4 INTO NUCLEIC ACID PYRIMI DiNES OF RA@NBEARING H.Ep. @3TUMORS (Single dose: 3 mg/kg) SPacInc @tCTiViT@(couars/ws/@ioi@a) DNA RNA Gaour Tisava Thymine Cytoaine Uracil Cyto.ine Control Tumor 370 543 748 916 Liver 935 1,700 21,400 10,900 Intestine 710 1,100 1,980 1,940 DON Tumor 16@ 29 460 52 treated (044)@ (0.053) (0.62) (0.057) Liver 386 80 22,400 1,950 (0.41) (0.047) (1.05) (0.18) Intestine @06 167 2,460 430 (0. 29) (0.15) (1.24) (0.22) a Numbers in parentheses refer to relative activities: treated/control. with literature values, and measurements of standard solutions The incorporation of these labeled precursors after filter paper blanks were subtracted. The purity of the was greater for the RNA pyrimidines. The largest isolated pyrimidine bases was checked by the ultraviolet ab sorption measurements. The absence of appreciable radioactive specific activities in these and other in t,ivo experi impurity was demonstrated by preliminary experiments in ments (after 7 hours) were uniformly found in the which two solvent systems were compared in the ascending liver, relative to the other organs listed in Tables 1 chromatography step: butanol saturated with water and and @. butanol-aniinonia (85 ml. conc. NH4OH in 250 ml. n-butanol) Tumor siwe experiment,,.—The specific activities (19). The specific activities Checked within the experimental error (about 3 per cent). Radioactivity measurements were of the nucleic acid pynimidines in a tissue slice ex made under conditions of infinite thinness with aluminum pemiment containing DON at a concentration of planchets of 20 sq. cm. area and a gas flow counter. Very little 10—sand 10@ M in the incubation medium are pre activity could be detected in the purine fractions. sented in Table 3. The specific effect of DON on RESULTS the incorporation of precursor into nucleic acid cytosine is demonstrated by the results in columns In vivo experimenta.—The specific activity of .3 and 5.
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