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

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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. The specific activity of DNA thymine and the nucleic acid pynimidines of tumor, liver, and intestine following administration of orotic acid RNA uracil was not significantly altered at this C'4 to rats beaning H.Ep. @{3tumors are presented concentration, while the specific activity of the in Table 1. The figures inside the parentheses refer corresponding cytosine was depressed by factors of to the relative activities (treated/control) and in @9and 14, respectively. At a concentration of dicate the extent to which the specific activity in 10—sM, the thymine specific activity was de the treated animals has been depressed relative to pressed by a factor of 7, while that for the DNA the controls. In relation to the other pynimidines, cytosine was depressed 100-fold. At this concen

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tration, the uracil specific activity was not appre Experiments rekiling to site of metabolic inter ciably changed, while the RNA cytosine activity ference.—When uniformly labeled cytidylic acid was reduced 40-fold. C'4 (@‘@‘mixedisomers) was used as a precursor of All the host animals used in the experiments re nucleic acid pyrimidines in 11.5. #1 tumor slices, ported in Tables 1—3had been exposed to x-radia there was no appreciable effect of DON at the tion and cortisone administration. In Table 4 liver same concentration used in the two preceding ex slices were obtained from rats that were tumor periments, 10@ M (Table 5). However, when free and had not been exposed to x-radiation and uridylic acid-C'4 was used as a precursor, the addi cortisone treatment. Although the specific activi tion of DON resulted in a significant depression of ties of thymine and uracil were lowered very the RNA cytosine specific activity (Table 6). The slightly, more than tenfold depressions in specific RNA uracil specific activity was not affected. The activity were observed for nucleic acid cytosine. extent of incorporation into DNA cytosine was too These results demonstrate that this effect did not small to allow a measurement of the specific activ arise as the result of the pretreatment given to the ity. host animal. The extent to which glutamine in tissue slice

TABLE 2 EFFECT OF DON ON INCORPORATION OF URE1D0SuCcINIC ACID-C'4 INTO NUCLEIC ACID PYRIMIDINES OF RATs BEARING H.S. #1 TUMORS (Single dose: 3 mg/kg)

SPECIFIC ACTIVITY (CousTs/ais/MMo@@) DNA RNA Gaoui. T@sau@ Thymine Cytosine Uracil Cytosine Control Tumor 245 478 2,780 1,090 Liver 72 1,180 16,300 7,900 Intestine 162 260 763 536 Spleen 77 146 320 460 DON Tumor 261 37 1,940 108 treated (1.1)@ (0.078) (0.70) (0.10) Liver 39 310 11,700 2,510 (0.54) (0.26) (0.72) (0.32) Intestine 63 30 840 106 (0.39) (0.12) (1.1) (0 . 20) Spleen t <20 185 78 (<0.14) (0.58) (0.17)

C Numbers in parentheses refer to relative activities: treated/control. t Sample lost during analysis. TABLE S EFFECT OF DON ON INCORPORATION OF ORoTIc ACID-C'4 INTO NUCLEIC ACID PYRIMIDINES OF HS. #1 TUMOR SLICES

SraCIFIC ACTIVITY RELATIVE ACTIVITY MOLAR CONC. DON: 0 1O@ 10—' 10—i 10—s (counta/minh&mole) (treated/control) DNA Thymine 1,450 1,450 197 1.0 0.14 Cytosine 540 19 6 0.035 0.01 RNA Uracil 17,400 13,400 15,100 0.77 0.87 Cytosine 1,970 141 47 0.072 0.024

TABLE 4 TABLE 5

EFFECTOF DON ON INCORPORATiON OF OROTIC OF DON ON INCORPORATION OF CYTIDYLIC ACID iNTO NUCLEIC ACID PYRIMIDLNES C'4 INTO THE NucLEic ACID PYRIMIDINES OF H.S. #1 TuMoR SLICES OF RAT LIVER SLICES DON concentration, 10@ M. cytidylic acid-C'4 (DON concentration, 10@ M) mixed isomers, uniformly labeled. RELATIVE RELATIVE ACTIVITYControlSPECIFIC ACTIVITYACm-C'4 ACTIVITYEFFECT SPECI@'@CACTIVITY Treated Treated (trested/ control)DNAThymine (counts/min/@smole)(treated/ control)Control (counts/min/ismole) 6 5 .8 Thymine 57 60 1.05 1.27RNAUracilCytosine 128 90 0.07DNA Cytosine 157 200 8,430 6,350 Uracil 131 106 0.81 Cytosine 2,480 2210.75 0.089RNA Cytosine 926 822 0.89

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incubation mixtures might reverse the effect of The results are in accord with a tentative hy DON on the incorporation of orotic acid into nu pothesis that DON interferes with one of the reac cleic acid cytosine was studied (Table 7). The ad tions leading from a metabolite containing the dition of glutamine at a concentration of 0.01 M uracil moiety to one containing the cytosine slightly depressed the specific activity of the nu moiety. These metabolites in the case of E. coli B cleic acid cytosines (column 8). The extensive de were found to be uridine triphosphate and cytidine pression of these activities by DON alone (10@ M) triphosphate (11). The absence of any appreciable is shown in column 4. When glutamine and DON effects when labeled cytidylic acid was used (Table (addedat the sametime)werepresentat concen 5) indicates strongly that the site of action of DON is not between a metabolite at the cytidine (or TABLE 6 cytidylic) level and nucleic acid cytosine. The de EFFECT OF DON ON INCORPORATION OF URIDYL1C AcID pressed RNA cytosine specific activity in the case C'4 INTO THE NUCLEiC ACID PYRIMIDINES of precursor uridylic acid (Table 6) is in accord OF H.Ep. #s TUMOR SLICES with the tentative hypothesis suggested above. DON concentration, I0@ M. Uridylic acid-C'4 = The absence of an effect of DON on the specific 2',S' mixed isomers, uniformly labeled. activity of RNA uracil (Table 6) is in accord with RELATIVE the results obtained with orotic acid and ureido SPECIFIC ACTIVITY ACTIVITY Control Treated (treated/ succinic acid precursors (Tables 1—5). (counts/min/@tmole) control) The uridylic and cytidylic acids (Tables 6 and DNA Thymine 32 26 0.8 7) were added as @‘,8'mixed isomers. Leibman and Cytosine 4 4 Heidelberger (9) have presented evidence for the RNA Uracil @ 8@ :@ dephosphorylation of ribonucleoside @‘and 8' Cytosine phosphates incubated with tissue slices of rat liver * Radiocarbon content too small for specific activity to be calculated. and Flexner-Jobling carcinoma. In rats, cytidylic

TABLE 7 EFFECT OF DON AND GLUTAMINE ON THE INCORPORAT1ON OF OR0TIc ACID-C'4 INTO NucLEIc AcID PYRIMIDINES OF H.Ep. #3 TUMOR SLICES

Control K A B D DON concentration: 104M 104u 10's Glutamine concentration: 10'M 103M 10'M Specific activity (counts/znin/@imole) DNA Thymine 110 83 39 25 32 Cytosine 45 19 0 0 6 RNA Uracil 3,220 3,400 2,660 1,750 1,420 Cytosine 218 187 4 10 45

Relative activity (treated/control) DNA Thymine 0.75 0.35 0.23 0.29 Cytosine 0.42 0.0 0.0 0.13 RNA Uracil 1.05 0.83 0.54 0.44 Cytosine 0.86 0.02 0.05 0.21

trations of 10_2 M and 10@ M, respectively (column acid (@‘,3')and cytidine are equally effective as 6), there was a partial reversal of the effect of precursors of nucleic acid pyrimidines (8). DON. The relative activity (treated/control) rose Inhibition by DON of the incorporation of from 0.0 and 0.0@ to 0.13 and 0.@1 for DNA and formate into nucleic acid purines has been reported RNA cytosine, respectively. At a glutamine con by Barclay et al. and Maxwell and Nickel (@,1@). centration only 10 X that of DON (column 5), Levenberg et al. demonstrated that DON was a this effect was very small. competitive inhibitor of glutamine in a specificre action concerned with inosinic acid biosynthesis DISCUSSION (10). The results in Table 7 indicate that the addi The results of the in vivo as well as in vitro stud tion of glutamine can reverse to some extent the ies show that DON significantly depressed incor depression of the specific activity of nucleic acid poration of orotic and ureidosuccinic acids into cytosine brought about by DON. However, cell nucleic acid cytosine. Incorporation into nucleic permeability in this experiment may be an impor acid uracil and thymine was affected to a small tant factor. The preparation of soluble systems extent relative to cytosine. containing enzymes related to pyrimidine inter

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conversions will permit a more precise definition of Acids. In: E. CHARGAFFandJ. N. DAVIDSON(eds.), The the site of action of DON along metabolic path Nucleic Acids, 2:350. New York: Academic Press, 1955. 4. Ci..utan, D. A.; REILLY, H. C.; and S@rocx,C. C. 6-Diazo ways leading to nucleic acid cytosine. 5-oxo-i@-norleucine, a New Tumor Inhibitory Substance. Effects on Mouse Sarcoma 180. Abstr., 12M, 129th Meet SUMMARY ing, American Chemical Society, April, 1956. The incorporation of ureidosuccinic and orotic 5. @.A Comparative Study of 6-Diazo-5-oxo-t-norleu cine and 0-Diazoacetyl-L-serine on Sarcoma 180. Proc. Am. acid into the cytosine moiety of the nucleic acids of Assoc. Cancer Research, 2: 100, 1956. some mammalian tissues was significantly de 6. DI0N, H. W.; Funs.rn, S. A.; J@xunowsni, Z. L.; Zoit&, pressed by DON (6-diazo-5-oxo.-z@-norleucine) rela J. G.; and BARTZ, Q. R. 6-Diazo-5-oxo-t-norleucine, a tive to the effect on nucleic acid uracil and thy New Tumor Inhibitory Substance. II. Isolation and Char acterization. J. Am. Chem. Soc., 78:3075—77, 1956. mine. This “cytosineeffect―was found in the case 7. EHRLIcR, J.; Coyrny, G. L.; FISHER, M. W.; HILLEGAS, of liver, intestine, and tumor of rats bearing hu A. B.; KORBERGER,D.L.; MAcaas&sut,H. E.; RIOHTSEL, man tumor transplants H.S. #1 and H.Ep. #8 fol W. A.; and ROEGNER, F. R. 6-Diazo-5-oxo-i@-norleucine, lowing single-dose intraperitoneal administration a New Tumor Inhibitory Substance. I. Biological Studies. Antibiotics and Chemotherapy, 6:487—97, 1956. at a level of 3 mg/kg. This effect was also found in 8. HURLBERT, R. B., and Porran, V. R. A Survey of the in vitro incorporation studies with rat liver as well Metabolism of Orotic Acid in the Rat. J. Biol. Chem., as with tumor slices. 195:257—70,1952. 9. Lnmae@, K. C., and HEIDELBERGER, C. Metabolism of These results suggest that DON (or a compound P'LLabeled Nucleotides. J. Biol. Chem., 216:823—30, derived biosynthetically from the latter) is interfer 1955. ing along a metabolic pathway leading from a 10. LEVENBERG, B. ; MELNIcK, I.; and BUCHANAN, J. M. Bio uracil-containing metabolite to one containing the synthesis of the Purines. XV. The Effect of Aza-z@-serine and 6-diazo-5-oxo-@-norleucine on Inosinic Acid Bio cytosine moiety. When uniformly labeled cytidylic synthesis de novo. J. Biol. Chem., 225: 163—76,1957. acid-C'4 was used as a precursor in vitro with tu 11. LIEBERMAN, I. Enzymatic A.mination of Uridine Tn mor slices, DON had no appreciable effect on the phosphate to Cytidine Triphosphate. J. Biol. Chem., 222: 765—75, 1956. incorporation of radiocarbon into the nucleic acid 12. MAXWELL, R. E., and Nicxx@, V. S. 6-Diazo-5-oxo-i- pyrimidines. When uniformly labeled uridylic norleucine, a New Tumor Inhibitory Substance. V. Micro acid-C'4 was used, the incorporation of radiocar biological Studies of Modes of Action. Antibiotics & Chem hon into RNA cytosine was appreciably reduced, otherapy, 7:81—89, 1957. @ 13. MEEKER, P. C.; TELLER, M. N.; J. E.; and Wooi, relative to uracil. These results are in accord with LEY, G. W. Effect of Actinomycin D on Two Human Tn the mechanism suggested above. The addition of mors Growing in Conditioned Rats. Antibiotics & Chemo relatively large concentrations of glutamine in an therapy,7:247—50,1957. 14. Nyc, J. F., and MITcHELL, H. K. Synthesis of Orotic Acid in vitro system partially reversed this effect of from Aspartic Acid. J. Am. Chem. Soc., 69: 1882—84,1947. DON. 15. ROLL, P. M., and WELIKY, I. The Utilization of Nucleo tides by the Mammal. II. N'5-labeled Purine Nucleotides. ACKNOWLEDGMENTS J. Biol. Chem., 213:509—22, 1955. The Human Tumor Laboratory, DivisionofSteroidBiology 16. SCHMIDT,G., and TRANNHAUSER,S. J. A Method for the of this Institute, kindly supplied the animals bearing the Determination of Desoxynibonucleic Acid and Phospho H.S. #1 and H.Ep. #3 transplants. The authors are indebted to proteins in Animal Tissues. J. Biol. Chem., 161:83-89, C. P. Rhoads, G. B. Brown, and C. C. Stock of this Institute 1945. and to H. E. Skipper of the Southern Research Institute for 17. Tooi.AN, H. W. Transplantable Human Neoplasms Main helpful discussions and suggestions. tamed in Cortisone-treated Laboratory Animals: H.S. #1, H.Ep. #1, H.Ep. #2, H.Ep. #3, and H. Emb. Rh #1. Cancer REFERENCES Research, 14:660—66, 1954. I . ANDERSON, E. P.; YEN, C. Y.; M@mEL, H. G.; and 18. Uaenssrr, W. W.; Bunais, R. H.; and STAUFFER, J. F. SMITH, P. K. Ureidosuccinic Acid as a Precursor of Nucleic Manometric Techniques and Related Methods for the Acid Pyrimidines in Normal and Tumor-Bearing Mice. Study of Tissue Metabolism, p. 119. Minneapolis: Burgess J. Biol. Chem., 213:625—33, 1955. Pub. Co.,1951. 2. BARcLAY, R. K.; G@utrnxni., E.; and Prnzms, M. Effects 19. WYATT, G. R. Separation of Nucleic Acid Components by of DON on Incorporation of Precursors into Nucleic Acids. Chromatography on Filter Paper. In: E. CHARGAFFand Proc. Am. Assoc. Cancer Research, 2:98, 1956. J. N. DAVIDSON (eds.), The Nucleic Acids, Vol. 1, Chap. 7. 3. BROWN, G. B., and Rozi, P. M. Biosynthesis of Nucleic New York: Academic Press, 1955.

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Maxwell L. Eidinoff, Joseph E. Knoll, Benjamin Marano, et al.

Cancer Res 1958;18:105-109.

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