(CANCER RESEARCH 32, 390-397, February 1972) Role of Catabolism in Pyrimidine Utilization for Nucleic Acid Synthesis in Vivol Geoffrey M. Cooper,2 W. F. Dunning, and Sheldon Greer Departments of Biochemistry (G. M. C., S. G./, Medicine [W. F. £>./,andMicrobiology ¡S.G./, University of Miami, Coral Gables, Florida 33146, and Papanicolaou Cancer Research Institute fW. F. D./, Miami, Florida 33136 SUMMARY is an irreversible inhibitor of dihydrouracil dehydrogenase, the first and rate-limiting enzyme in the catabolism of the The incorporation of pyrimidines into nucleic acids in vivo pyrimidine bases (4). The results of kinetic studies with a is increased by inhibition of pyrimidine catabolism with crude enzyme preparation from rat liver were consistent with diazouracil. The utilization of iodouracil or thymine for DNA the possibility that the mechanism of irreversible inhibition by synthesis can be increased approximately 20-fold by DU involves covalent bond formation at the enzymic active simultaneous administration of diazouracil and a purine site (10). This report deals with the effect of DU on the deoxyribonucleoside. The incorporation of iodouracil and utilization of pyrimidine bases and nucleosides for nucleic acid thymine, when administered at high doses, is elevated to synthesis in vivo. nearly that obtained with the corresponding pyrimidine In Ehrlich ascites cells (17) and human leukocytes (2), the deoxyribonucleosides, while at low doses thymidine is utilized incorporation of thymine or halogenated base analogs into preferentially over thymine. Diazouracil and purine DNA appears to be limited largely by the availability of deoxyribonucleosides do not appreciably affect the deoxyribose 1-phosphate for deoxyribonucleoside synthesis. incorporation of thymidine or iododeoxyuridine into DNA. The synthesis of ribonucleosides (5, 36) and ribonucleotides The utilization of fluorouracil and uracil is also elevated by (37) from uracil and FU is also limited by the availability of diazouracil but is not significantly affected by purine ribose 1-phosphate and phosphoribosyl pyrophosphate in ribonucleosides. Diazouracil has a similar effect on pyrimidine some, but not all, murine tumors which have been studied (17, incorporation in cells of the Dunning leukemia, rat liver, 24). In addition to limitation by the availability of reactants spleen, and small intestine, in spite of the differences in for anabolic conversion, the utilization of free pyrimidines, catabolic activity between these tissues, a finding that particularly in vivo, might also be limited by their catabolism. indicates the importance of systemic catabolism. The toxic The present study indicates that this is the case, since and antitumor activities of fluorouracil are potentiated equally inhibition of catabolism by administration of DU enhances the by diazouracil administration. incorporation of pyrimidine bases into nucleic acids. A preliminary report of some of this work has been presented (8). INTRODUCTION Pyrimidine catabolism may be relevant to the MATERIALS AND METHODS chemotherapeutic activity of the fluorinated pyrimidines (7, Chemicals. 125IU, 12SIUdR, and FU-6-3H were purchased 19, 20) and to the possible therapeutic use of the brominated and iodinated pyrimidines as tumor radiosensitizing agents (1, from Amersham/Searle Corporation, Des Plaines, 111. Uracil-6-3H, thymine-methyl-3H, and thymidine-methyl-3H 11, 18, 26, 43). One approach to elucidating the role of catabolism is the development of inhibitors of enzymes of the were purchased from New England Nuclear, Boston, Mass. DU catabolic pathway. Such inhibitors may also be of therapeutic was purchased from Sigma Chemical Company, St. Louis, Mo. interest in the potentiation of the antineoplastic activity of the Unlabeled pyrimidines, pyrimidine nucleosides, and purine halogenated pyrimidines. nucleosides were purchased from either Sigma Chemical Previous work in our laboratory (9, 10) indicated that DU3 Company or Calbiochem, Los Angeles, Calif. Actinomycin D is a product of Merck Sharp and Dohme, West Point, Pa. Tumor-bearing Animals. The Dunning leukemia IRC 741 'This investigation was supported by Grant DRG-1076 from the (13) was maintained by unilateral s.c. transplantation in the Damon Runyon Memorial Foundation, and by Grant ÇAI2522 and flank of adult male Fischer 344 rats. Animals were either bred Contract PH 43-64-80 from the National Cancer Institute, NIH. by W. F. Dunning or purchased from Microbiological "Predoctorat trainee supported by USPHS Training Grant HE-05463 Associates, Bethesda, Md., or from Charles River Breeding from the National Heart and Lung Institute and a Robert E. Maytag Laboratory, Wilmington, Mass. Purina laboratory chow and Fellowship from the University of Miami. 3The abbreviations used are: DU, 5-diazouracil; FU, 5-fluorouracil; water were provided ad libitum. IU, 5-iodouracil; lUdR, 5-iododeoxyuridine. Administration of Radioactive Pyrimidines. DNA precursors Received August 12. 1971; accepted November 3, 1971 (I2SIU, 125IUdR, 3H-labeled thymine, and 3H-labeled 390 CANCER RESEARCH VOL. 32 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1972 American Association for Cancer Research. In Vivo Inhibition of Pyrímidine CataboUsm thymidine) were administered daily for a 4-day period collected either by centrifugation or winding on a glass rod. beginning when the transplanted tumors were first palpable, The precipitate was washed 3 times with 95% ethanol and usually 9 or 10 days after implantation. 3H-Labeled FU and dissolved in 5 ml of 15 mM NaCl plus 1.5 mM sodium citrate. 3H-labeled uracil were administered in a single injection when 3H was determined by liquid scintillation counting. the tumors had grown to a diameter of approximately 3 cm, generally about 14 days after implantation. DU and «"I methotrexate were injected 2 hr prior to administration of the radioactive pyrimidines. Furine nucleosides were injected 1I 8 immediately after pyrimidine administration. All chemicals •¿[T were administered i.p. in 0.85% NaCl solution. The animals 7IPI were sacrificed 24 hr after injection, and tissues to be studied were removed and stored at —¿70°. Extraction of Nucleic Acids. For extraction of 3H-labeled 6•»MMif nucleic acids, approximately 3 g of tissue were homogenized in a Sorvall Omnimixer in 10 ml of distilled water at 0 . Fifteen ml of 10% perchloric acid were added, and the precipitate was collected by centrifugation, washed twice with 30 ml of 5% 4SSuS perchloric acid, and extracted 3 times with 30 ml of ethanol : ether (1:1). Nucleic acids were then hydrolyzed by heating at 90°for 45 min in 15 ml of 5% perchloric acid. 33 Insoluble material was removed by centrifugation. 125I-Labeled DNA was extracted by a modification of the 2!E method of Marmur (27). Approximately 2 g of tissue were - 1-f-rn- homogenized in 10 ml of 150 mM NaCl plus 15 mM sodium Contini 2 E 12 24 4l citrate at 0°in a Sorvall Omnimixer. Sodium dodecyl sulfate Tile after DUadministration{t[| was added to a final concentration of 4%, and the material was Chart 2. Recovery of dihydrouracil dehydrogenase activity after DU further homogenized in a glass tissue grinder, followed by administration. At varying times after DU administration (5 mg/kg), heating at 60°for 30 min. The preparation was then cooled to animals (Sprague-Dawley females) were sacrificed for determination of room temperature, NaCl was added to a final concentration of liver dihydrouracil dehydrogenase specific activity. Control animals did l M, and insoluble material was removed by centrifugation at not receive DU. Data from a representative experiment are presented as 0°for 30 min at 13,000 X g. DNA was precipitated from the in Chart 1. supernatant fluid by addition of 2 volumes of 95% ethanol and '= 12 Z 6 ÃŒ4 S 2 5 i - 2 24 2 24rrt 2 24 •¿ lime il Sacrilice tir f Nue ti DU DO»e AdministeredChemicals Chart 3. Inhibition by actinomycin D of recovery of dihydrouracil dehydrogenase activity following DU administration. DU was administered as a single injection of 5 mg/kg. Actinomycin D (AD), l mg/kg, was administered at the same time as DU and again 12 hr later. Animals (Sprague-Dawley females) receiving DU and/or actinomycin D 1 2 4 u usi were sacrificed at 2 and 24 hr after initial injection for determination of liver dihydrouracil dehydrogenase specific activity. Data are represented Chart 1. Inhibition of dihydrouracil dehydrogenase in vivo by DU. as mean ±S.D. of 2 to 8 animals. In animals receiving DU alone, the Two hr after i.p. injection of DU, animals were sacrificed and the activity of dihydrouracil dehydrogenase is significantly different specific activity of their liver dihydrouracil dehydrogenase was (p < 0.05) at 2 and 24 hr after injection, while in animals receiving DU determined. Sprague-Dawley females were used. Data are represented as and actinomycin D, enzyme activity is not significantly different mean ±S.D.of duplicate animals from a representative experiment. (p > 0.5) at the 2 times. FEBRUARY 1972 391 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1972 American Association for Cancer Research. Geoffrey M. Cooper, W. F. Dunning, and Sheldon Gréer Quenching was corrected by the method of channel ratios. In Vivo Metabolism of 12SIUdR. Swiss mice (Karwood 1251 was counted in a well-type y scintillation spectrometer. Farms, New City, N.Y.) received a single i.p. injection of DNA and deoxyribonucleotides were determined by the 12SIUdR in 0.85% NaCl solution. The mice were placed in diphenylamine reaction. Total nucleic acid content of metabolism cages, and urine, which contained 50 to 60% of hydrolysates was estimated by absorbance at 260 nm. the administered radioactivity, was collected 20 hr after Enzyme Assays. Dihydrouracil dehydrogenase was assayed injection. A 0.5-ml urine sample was placed on a 1-ml anión in the 105,000 X g supernatant fluid of all tissues by exchange column (Bio-Rad AG-1-X8-C1anion-exchange resin). measuring the TPNH-dependent dehalogenation of I2SIU as 12S1U and ' 2SIUdR were eluted as a single peak with 0.01 N HC1 and 12SI" was eluted with 5 M KI.