Role of Catabolism in Pyrimidine Utilization for Nucleic Acid Synthesis in Vivol

(CANCER RESEARCH 32, 390-397, February 1972)

Geoffrey M. Cooper,2 W. F. Dunning, and Sheldon Greer

Departments of Biochemistry (G. M. C., S. G./, Medicine [W. F. Â£>./,andMicrobiology Â¡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 Ã‡AI2522 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

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 Â«"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 â€¢¿[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 â€”¿70Â°. Extraction of Nucleic Acids. For extraction of 3H-labeled 6â€¢Â»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 90Â°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 0Â°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 60Â°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 0Â°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

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S 2 5 i - 2 24 2 24rrt 2 24 â€¢¿ lime il Sacrilice tir f Nue ti DU DOÂ»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 Â±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 Â±S.D.of duplicate animals from a representative experiment. (p > 0.5) at the 2 times.

FEBRUARY 1972 391

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 aniÃ³n 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. All urinary previously described (10). The reaction mixture contained 0.2 mM 12SIU (0.1 juCi), 2 mM TPNH, and 10 mM phosphate radioactivity chromatographed as III and lUdR or as I". buffer, pH 7.4. The conditions for all assays were such that product (12SI~) formation was linear with incubation time, and enzyme activity was proportional to the amount of RESULTS enzyme extract added to the incubation mixture. Protein was estimated by the method of Warburg and Christian (44). In Vivo Inhibition of Pyrimidine Catabolism by DU. The effect of in vivo administration of DU was studied by determination of dihydrouracil dehydrogenase activity in the Table 1 Incorporation of '3 5W and '2 sWdR into DNA 105,000 X g supernatant fraction of livers excised from animals that had previously been given injections of DU. of Leukemia IRC 741 Chemicals were administered daily for a 4-day period starting 10 Maximal inhibition, which varied from 80 to 95% (Charts 1 to days after IRC 741 implantation: methotrexate, 0.1 mg/kg; DU, 5.0 3), was attained with a DU dose between 2.5 and 5.0 mg/kg mg/kg; guanosine and deoxyguanosine, 100 mg/kg; '2 5IU, 25 mg (104 (Chart 1). Similar results were obtained with both Fischer and Mmoles)/kg, 0.1 /uCi/Mmole; 125IUdR, 25 mg (71 Mmoles)/kg, 0.1 Sprague-Dawley rats. /iCi/jimole. Twenty-four hr after the final injection the animals were Inhibition of catabolism in vivo was also determined in sacrificed, and the specific activity of the isolated tumor DNA was studies of the metabolism of '2 5lUdR after its administration determined. Data are presented as nmoles of administered precursor incorporated per mg of DNA; mean Â±S.D. of the number of animals to Swiss mice. Radioactive metabolites excreted in the urine given in parentheses. Statistical analysis of selected illustrative data was were analyzed 20 hr after administration of 25 mg/kg by means of the Student t test. 125IUdR (1 /jCi/mouse). In duplicate mice that received 5 mg/kg of DU 2 hr before '2 5lUdR injection, 15% of the total urinary radioactivity was 125I", whereas 12SI" constituted Chemicals administered in Radioactive addition to methotrexate DNA precursor sIU(nmoles/mgDNA) 53% of the radioactivity excreted by control mice not receiving DU. Since DU does not inhibit the phosphorolysis of NoneDeoxyguanosineGuanosineDUDU 0.03(8)0.520.150.621.80.752.10.19 (4)a0.05 lUdR by either thymidine phosphorylase or uridine phosphorylase (G. M. Cooper and S. GrÃ©er,unpublished (2)0.10(4)Â°0.6 observations), the 72% inhibition of 12SJ~ formation in vivo anddeoxyguanosineDU (9)a0.05 resulting from DU administration represents inhibition of andguanosineNoneSIU5IU5IU5IUS1U5IUMUdRDU (2)0.3 dihydrouracil dehydrogenase. (7)1.7 The duration of inhibition was studied by assaying 12SIU and deoxyguanosine 25lUdR0.11 0.7(10) catabolism in extracts of livers removed from animals ' Significantly different from control values (p < 0.05). sacrificed at varying times after administration of a single dose

Table 2 Incorporation ofl2SIU and '2 5WdR into DNA of liver, spleen, and small intestine The experimental details were as described in Table 1. DNA was extracted from the indicated normal tissues of tumor-bearing rats. Data aie represented as nmoles of administered precursor incorporated per mg DNA; mean Â±S.D.of the number of animals indicated in parentheses. Statistical analysis of selected data was by means of the Student t test. 125IU (nmoles/mg DNA)

Chemicals administered in Radioactive additionprecursorNoneDeoxyguanosineGuanosineDUDU to methotrexate DNA intestine0.047

(4)0.0505 Â±0.005 (4)0.043Â±0.002 (4)0.35Â±0.005 (2)0.01Â±0.02 (2)0.022Â±0.010 (2)0.065Â±0.05 (2)0.0450Â±0.001 (2)0.050Â±0.002 (2)0.20Â±0.005 (2)Â°0.23Â±0.005 (2)"0.21Â±0.010 Â±0.02(2)"0.88 anddeoxyguanosineDU Â±0.04(4)6N.D.C0.20 (4)60.085Â±0.04 Â±0.18(4)b0.20 andguanosineNone5IU5IU5IU5IUSIU5IUMUdRSpleen0.01 (2)0.20Â±0.015 (2)0.75Â±0.02 Â±0.02(2)Liver0.022 Â±0.05(2)Small Â±0.05(2) 0 Significantly different from control values (p < 0.05). 6 Significantly different from control values (p < 0.01). c N.D., not determined.

392 CANCER RESEARCH VOL. 32

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1972 American Association for Cancer Research. In Vivo Inhibition of Pyrimidine Catabolism of DU (Chart 2). Dihydrouracil dehydrogenase activity doses of methotrexate inhibit tumor growth and reduce the increased slowly and did not reach the control level until 24 to incorporation of *2 s lUdR into DNA, perhaps as a result of 48 hr after DU administration. reutilization of thymidine released from killed cells (3, 14, 28, The recovery of dihydrouracil dehydrogenase activity is 38). '2 s IU is incorporated to only 5% the extent of '2 s lUdR. prevented by administration of actinomycin D (Chart 3). This Administration of deoxyguanosine, which presumably acts as a is consistent with the possibility that enzyme synthesis is source of deoxyribose 1-phosphate, increases 125IU required for recovery, as would be expected from the incorporation 5-fold. Guanosine has no effect, a fact indicating irreversibility of DU inhibition. The effect of actinomycin D specificity for the deoxyribose moiety. Deoxyadenosine can alone probably results from inhibition of normal enzyme substitute for deoxyguanosine. Inhibition of pyrimidine turnover. catabolism by DU results in a 6-fold increase in the Utilization of Pyrimidines for Nucleic Acid Synthesis. The incorporation of '2 5IU. Combined administration of DU and effect of DU and purine nucleosides on the incorporation of deoxyguanosine increases 125IU incorporation 18-fold to 125IU and 12SIUdR into DNA of the IRC 741 leukemia is approximately the level attained by the administration of illustrated in Table 1. In this experiment, methotrexate, 0.1 125IUdR (note that the molar quantity of 12SIUdR mg/kg, was administered to all animals. This is an optimal dose administered is 30% less than that of 125IU). The resulting in a 50 to 100% increase in 125IUdR incorporation incorporation of the deoxyribonucleoside, 125IUdR, is not over that in control animals receiving I25IUdR alone. Larger significantly affected by DU and deoxyguanosine.

Table 3 Incorporation of 3H-labeled thymine and thymidine into DNA ofleukemic tissue Chemicals were administered daily for a 4-day period starting 9 days after IRC 741 implantation: DU, 5 mg/kg; 3H-labeled thymine and thymidine, either 10 Mmoles/kg (20 iiCi/Aimole) or 100 Mrnoles/kg (2 Â¿iCi/Mmole);deoxyguanosine, 100 mg/kg. Methotrexate was not administered. Data are represented as nmoles of administered precursor incorporated per mg DNA; mean Â±S.D. of the number of animals given in parentheses. Statistical analysis of selected data was by means of the Student t test. 3H-Labeled thymine (nmoles/mg DNA) Dose of labeled precursor

AdministeredchemicalsNone DNAprecursorThymine jumoles/kg0.093

(3) Â±0.5(2) DU Thymine 0.3 0.1 (2) 7.5 Â±0.1(2) Deoxyguanosine Thymine 0.2 0.1 (2) 5.1 Â±0.1(2) DU + deoxyguanosine Thymine 0.7 0.1 (2)Â° 15 Â±1 (2)Â° None Thymidine 8.5 1.5 (2)lOO/jmoles/kg3.827 Â±3(2) DU + deoxyguanosine3H-LabeledThymidine10 9.0 (1)0.009 38 Â±6(2)

" Significantly different from control values (p < 0.01).

Table 4 Utilization of 3H-labeled FU for RNA synthesis in various tissues Single injections of chemicals were administered 24 hr before the animals were sacrificed: 3H-labeled FU, 20 mg (155 Mmoles)/kg (1.9 pCi//umole); DU, 5 mg/kg; deoxyguanosine and guanosine, 100 mg/kg. Total nucleic acids were extracted and incorporation of 3H-labeled FU is expressed as dpm/A26 â€ž¿unitof the nucleic acid hydrolysate. Data are represented as mean Â±S.D. of the number of animals given in parentheses. Statistical analysis of selected data was by means of the Student / test. 3H-Labeled FU (dpm/A^,, unit)

Administered chemicalsNoneGuanosineDeoxyguanosineDUDU74188 intestine38

Â±20(2)110Â± Â±1(2)21 Â±5(2)34 Â±6(2)46 14(2)72(1)340 Â±5(2)20(1)62 Â±5(2)42 Â±6(2)48 Â±14(2)93 Â±4(2)90 Â±15(2)Â°420 Â±2(2)Â°94 Â±9(2)Â°138 Â±2(2)Â°110 andguanosineDU Â±45(2)420 Â±12(2)118+2 Â±18(2)158 Â±10(2)130Â± anddeoxyguanosinePooled Â±100(2)93 (2)22 Â±34(2)35 30(2)44 data:Without DUWith Â±21(5)390 Â±3(5)92 Â±11(6)130Â± Â±7(6)110 DUIRC Â±120(6)Â°Spleen23Â±29(6)Â°Liver28 35 (6)"Small Â±25(6)Â° 1Significantly different from animals not receiving DU (p < 0.05).

FEBRUARY 1972 393

Table 5 Comparison between levels of pyrimidine catabolism and stimulation of pyrimidine utilization by DU in different tissues Incorporation of 'J 5IU into DNA and 3H-labeled FU into RNA in the presence and absence of DU in different tissues was determined as described in Tables 1 to 4. The effect of DU on FU incorporation is represented by the ratio of 3H-labeled FU incorporated following DU administration to 3H-labeled FU incorporated without DU administration (3H dpm/A260 unit with DU: 3H dpm/A,60 unit without DU). A similar ratio is used to represent the effect of DU on 125IU incorporation (nmoles ' *5 lU/mg DNA with DU: nmoles '2 5lU/mg DNA without DU). The specific activity of dihydrouracil dehydrogenase was determined in the 105,000 X g supernatant fluid of all tissues. Data are expressed as mean Â±S.D. of the number of determinations given in parentheses.

Ratio of pyrimidine incorporation with and without administered DUÂ°

Dihydrouracil dehydrogenase 3H-Labeled FU incorporation 12s IU incorporation (nmoles '2 51"/hr/mg protein) Tissue (with DU:without DU) (withDU:withoutDU)

IRC741SpleenSmall 0.05(2)< Â±0.9(5)4.4* Â±0.9(10)3.8 0.2(4)2.2 1.3(5)2.5 Â±0.8(6)3.3 intestineIntestinal Â±0.2(5)3.1 Â±0.4(6)N.D.b3.7 Â±0.6(6)N.D.3.7 mucosaLiver< Â±0.2(3)8.8 Â±0.4(8)4.5 Â±0.3(6)4.7 Â±1.1(6) 0 This ratio, the increase in pyrimidine incorporation obtained by inhibition of catabolism with DU, was not altered by administration of purine nucleosides. 6 N.D., not determined.

Stimulation of '2 s IU incorporation by deoxyguanosine and 16Â» DU was observed in the spleen, liver, and small intestine, as well as in cells of the IRC 741 leukemia (Table 2). In these i 150 normal tissues, as in leukemic tissue, combined administration of DU and deoxyguanosine increased the incorporation of 12SIU to that found with ' 2sIUdR. Â£140i.s2 Inhibition of catabolism by DU administration also increases the incorporation of thymine into DNA (Table 3). At doses of both 10 and 100 Â¿tmoles/kg, the incorporation of 120^ thymine is stimulated by DU and deoxyguanosine. At the high â€”¿r*~J-i dose, the incorporation of the base is elevated by DU and Â«o.E5loeâ€”-PT: deoxyguanosine to approximately 50% of that obtained with thymidine. However, at a dose of 10 Â¿/moles/kg,preferential utilization of thymidine is observed. Administration of DU and deoxyguanosine with a high dose of thymidine appears to l 15 30 45 60i'â€žâ€” 0 2.5 5.0"10 15 result in a small increase, approximately 40%, in thymidine Fllint ut/Hi incorporation. 1/11l/S 1/5 1/115/5 0/10 1/5 2/105/109/10 The increase in thymine incorporation resulting from Tuie Deaths administration of DU is less than the increase obtained in IU Withoutn Miiiistutiii Â«itkDuÂ«dmiimtiilioii incorporation (Table 1). This may be related to differences in Chart 4. Effect of DU on therapy of leukemia IRC 741 with FU. metabolism of the 2 pyrimidines or to Â«utilization of labeled Injections of DU (5 mg/kg) and varying doses of FU were administered catabolic products of 3H-labeled thymine which may not daily for 4 days beginning 1 day after unilateral s.c. tumor readily occur with 12SIU (40). Administration of implantation. Control animals not receiving FU received injections of methotrexate with 12SIU but not with 3H-labeled thymine 0.85% NaCl solution. Data are represented as the mean survival time of may also be a factor affecting the degree of stimulation animals that died of leukemia; percentage of controls Â±S.D.The mean obtained with DU. survival time of control animals was 16 days and was not affected by DU administration. The fraction of animals which died of chemical The incorporation of FU into RNA (Table 4) is also toxicity represents animals which died between 7 and 14 days after stimulated by inhibition of catabolism with DU in the tumor implantation and showed no signs of leukemia on autopsy. In leukemic and normal tissues studied. Both guanosine and the case of rats receiving DU and 15 mg/kg FU, the mean survival time deoxyguanosine produce only a minor stimulation of refers to the survival of the single rat not dying from chemical toxicity. incorporation. Since there is no specific stimulation by the ribonucleoside, it appears that pyrimidine utilization in these tissues is not limited by the availability of ribose 1-phosphate Systemic and Tissue-specific Catabolism. Table 5 illustrates or phosphoribosyl pyrophosphate for ribonucleotide synthesis. the lack of relationship between the specific activity of The utilization of3 H-labeled uracil for nucleic acid synthesis is dihydrouracil dehydrogenase in the 4 tissues investigated and similarly affected by DU and purine nucleosides in leukemic the stimulation of pyrimidine incorporation obtained with DU tissue, small intestine, and liver. in these tissues. In spite of the variation in catabolic activities,

394 CANCER RESEARCH VOL. 32

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1972 American Association for Cancer Research. In Vivo Inhibition offyrimidine Catabolism and particularly the absence of catabolism in leukemic tissue base and the limited availability of deoxyribose 1-phosphate. and spleen, pyrimidine incorporation in all tissues is similarly However, when the incorporation of thymine and thymidine is increased by DU administration. That there is no relationship compared at low doses (10 jumoles/kg) the incorporation of between tissue catabolic activity and increase in pyrimidine thymidine is 10-fold higher than that of thymine even when utilization resulting from inhibition of catabolism suggests that DU and deoxyguanosine are administered simultaneously. This systemic catabolism, rather than catabolism in the target additional preference for deoxyribonucleoside utilization at tissue, is the primary factor that limits pyrimidine utilization low doses may be related to differences between nucleosides in vivo. and free bases regarding their mode of entry into cells. Tumor Therapy with FU. Since DU stimulates the Pyrimidine bases permeate cells by free diffusion (22), whereas incorporation of FU into RNA, it is expected that inhibition nucleosides are transported by a carrier-mediated mechanism of catabolism will also potentiate the cytotoxicity of FU. The (23, 25, 29-31) and, once inside cells, may be trapped by experiments illustrated in Chart 4 demonstrate that both the rapid phosphorylation (31). This would lead to a preferential antitumor effects and the toxicity of FU are potentiated by uptake of the nucleoside at low concentrations, at which DU. The FU dose required to produce both toxicity and carrier-mediated transport is most active (23, 30), and tumor inhibition is decreased approximately 5-fold by DU accounts for preferential incorporation of thymidine administration. DU itself has no toxic or antitumor effect at compared to thymine into DNA in vivo when low doses are the doses administered. At a given level of toxicity, similar administered. degrees of tumor inhibition by FU are observed whether or It might be anticipated that the incorporation of thymidine not DU is administered, a fact indicating that inhibition of or lUdR would be increased by administration of DU and catabolism with DU does not affect the chemotherapeutic deoxyguanosine, since these compounds would facilitate selectivity of FU. reutilization of thymine or IU formed from phosphorolysis of the administered deoxyribonucleosides. However, due to the low efficiency of utilization of the free base compared to the DISCUSSION deoxyribonucleoside, administration of DU and deoxygua We have previously described irreversible inhibition of nosine would not be expected to increase deoxyribonucleoside dihydrouracil dehydrogenase by DU in vivo and in a crude incorporation by more than a maximum of 50 to 75%. Such enzyme preparation from rat liver (10). Inhibition of rat liver an effect, which is of only marginal significance, is observed in dihydrouracil dehydrogenase by 5-cyanouracil has been the case of thymidine incorporation. Studies on inhibition of reported by Dorsett et al. (12), and this compound is also nucleoside phosphorolysis, in progress in our laboratory, may active in inhibiting the catabolism of low doses (1.5 to 2.0 elucidate the role of catabolism in pyrimidine nucleoside mg/kg) of uracil and FU in vivo (16). In contrast to DU, utilization. 5-cyanouracil is a reversible inhibitor (9, 10), and may Although cells of the IRC 741 leukemia and the spleen have therefore be less adaptable to in vivo applications than DU, no detectable catabolic activity, DU administration increases particularly with regard to inhibition of the catabolism of large pyrimidine incorporation in these tissues to the same extent as doses of pyrimidines similar to doses administered in in liver. This result implies that pyrimidine utilization in the chemotherapy and in this study. Administration of DU, 5.0 tumor and spleen is affected by circulating levels of precursors mg/kg, daily for a 4-day period is tolerated without significant which are limited by systemic catabolism occurring largely in weight loss or gross signs of toxicity. The toxic effects of the liver. If only catabolism of pyrimidines in the tissue under larger doses of DU (chronic 50% lethal dose in Fischer rats is study limited their incorporation into nucleic acids, then DU 10 mg/kg/day for 4 days) may be related to cytotoxic effects should affect pyrimidine utilization to a greater extent in of DU other than inhibition of pyrimidine catabolism (39,41, tissues with active catabolic pathways, such as the liver, than 42). in tissues lacking catabolic activity. DU administration results in a marked increase in the Pyrimidine catabolism is reduced or lacking in a number of utilization of pyrimidine bases for nucleic acid synthesis. This tumors studied (6, 7, 15, 21, 34-36) and shows a marked effect of inhibiting catabolism is analogous to increasing negative correlation with growth rate in the Morris hepatoma xanthine and hypoxanthine utilization by administration of series (15, 35). Chaudhuri et al. (7) and Heidelberger (20) have allopurinol, an inhibitor of xanthine oxidase (32, 33). On the suggested that the lack of catabolic activity in tumors is basis of our studies (8, 10), Ferdinandus and Weber (J.A. responsible for the chemotherapeutic selectivity of FU. Ferdinandus and G. Weber, personal communication) have However, the present results are not compatible with this utilized DU and confirmed that inhibition of catabolism hypothesis, since the utilization of pyrimidines in tissues increases thymine incorporation into DNA of developing rat lacking catabolic activity is controlled by systemic catabolism liver. in the liver. In order for a correlation to exist between the Combined administration of DU and deoxyguanosine tumor selectivity of FU and the lack of catabolism in tumors, increases the incorporation of high doses (100/mioles/kg of the role of catabolism in limiting FU utilization for RNA 125IU and 3H-labeled thymine into DNA to nearly the same synthesis and anabolism to fluorodeoxyuridylate would have level of incorporation attained by administration of ' 2SIUdR to be tissue specific rather than systemic. Futhermore, if or 3H-labeled thymidine. This suggests that under these catabolism of FU in normal tissues but not in tumors was conditions the low incorporation of the free base relative to significant to the chemotherapeutic activity of FU, then the deoxyribonucleoside results from catabolism of the free simultaneous administration of DU should increase the

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Geoffrey M. Cooper, W. F. Dunning and Sheldon Greer

Cancer Res 1972;32:390-397.

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