Pyrimidine Metabolism in Human Leukocytes I. Contribution of Exogenous Thymidine to DNA-Thymine and Its Effect on Thymine Nucleotide Synthesis in Leukemic Leukocytes1

[CANCER RESEARCH 26 Part 1, 2267-2275, November 1966]

RICHARD A. COOPER,2 SEYMOUR PERRY, AND T. R. BREITMAN3

Medicine Branch and Laboratory of Physiology, National Cancer Institute, NIH, Bethesda, Maryland

Summary phosphorylation of TMP in cells actively synthesizing DNA (17, The pathways for the formation of DNA-thymine from syn 40). Induction of thymidine kinase during periods of DNA syn thesis (3, 7, 8, 30, 43), and feedback inhibition of this enzyme by thesis de novo and from exogenous thymidine (TdR) have been studied in intact leukocytes from patients with chronic myelog- TTP (6, 8, 43) suggest that the salvage pathway may be an im portant source of DNA-thymine. Moreover, results with cell- enous leukemia. The most significant observations were as free extracts of regenerating rat liver (3) and with tissue culture follows: (a) DNA-thymine derived from exogenous TdR in cells incubated in the presence of amethopterin or 5-fluoro-2'- creases from 13% to 87% over a range of TdR concentrations deoxyuridine (21, 33) demonstrate that the DNA-thymine re from 0.03 /Â¿Mto300 /XM.(b) TdR causes expansion of the total quirement can be met entirely from the salvage pathway. thymidine diphosphate and thymidine triphosphate (TTP) Two questions regarding the salvage pathway that had not pool but does not influence the contribution of the pathway been answered adequately and that prompted this investigation de novo to this pool. Thus, the increasing contribution of exogenous TdR to the formation of DNA-thymine occurs be are: (a) To what extent does incorporation of exogenous TdR into DNA decrease incorporation of thymine synthesized de novo? (b) cause of a progressive dilution of TTP synthesized de novo Does the availability of an exogenous source for thymine nucleo- with TTP derived from exogenous TdR. (c) In concentrations tide synthesis affect thymine nucleotide synthesis de novo? greater than 0.3 /Â¿M,TdRinhibits DNA synthesis but not RNA synthesis. This inhibition is dependent on the continued presence of TdR in the medium and is reversed by the addition of deoxy- Materials and Methods cytidine. LABELEDCOMPOUNDS.TdR-3H,1.9 and 10 c/mmole, GdR-3H, 500 ^e/inmole, and UR-2-14C,26 mc/mmole, were obtained from Introduction Schwarz BioResearch, Inc., Orangeburg, New York. OA-6-14C, In human leukocytes 2 pathways are known for the synthesis 32 mc/mmole, was obtained from Nuclear-Chicago Corp., Des of TMP4: methylation of dUMP by thymidylate synthetase (41), Plaines, Illinois. 3H-labeled compounds were labeled in the base. the last step in the pathway de novo, and phosphorylation of TdR LEUKOCYTEDONORS.Leukocytes were obtained from 10 pa by thymidine kinase (2), the salvage pathway. TdR is available tients with CML in relapse. All had the Philadelphia chromosome from both the DNA of catabolized cells (11, 39) and the DNA and WBC greater than 100,000/cu mm. None were in blastic contained in dietary sources. In addition, TdR results from de- crisis. Seven had received no antileukemic therapy. One patient received radiation to the spleen 8 months prior to this study, and 1Part of this work has been published in abstract form; see 2 had received busulfan 4 and 18 months, respectively, before this study. One of the busulfan-treated patients received cytosine Cooper and Perry (13). 2Present address: II and IV (Harvard) Medical Service, Boston arabinoside until 2 weeks prior to study. City Hospital, Boston, Mass. LEUKOCYTEISOLATIONANDINCUBATION.Leukocytes were iso *To whom requests for reprints should be addressed at the Lab lated from freshly drawn blood by a dextran sedimentation, oratory of Physiology, National Cancer Institute, NIH, Bethesda, hypotonie shock method, omitting the use of streptokinase and Md. streptodornase (18). After washing once with 0.85% NaCl the 4The following abbreviations are used: TdR, thymidine; TMP, cells were suspended at a final concentration of 2-6 X 107/ml in TDP, and TTP, thymidine-5'-mono-, di-, and tri-phosphate; UdR, deoxyuridine; dUMP, deoxyuridine-5'-monophosphate; UR, uri- a medium containing 1 HIMMgCU; 1-5 HIMCaCl2; 3 IHMKC1; dine; UMP, uridine-5'-monophosphate; CdR, deoxycytidine; 109 HIMNaCl; 30 mM sodium phosphate buffer, pH 7.4; heparin, dCDP, deoxycytidine-5'-diphosphate; CR, cytidine; CDP, cyti- 10 units/ml; and glucose, 1 mg/ml. Incubations were carried out at 37Â°Cin siliconized glass vials open to air in a Dubnoff meta dine-5'-diphosphate; GdR, deoxyguanosine; GR, guanosine; AR, adenosine; dATP, deoxyadenosine-5'-triphosphate; AdR, deoxy- bolic shaker (100 oscillations/min) and were completed within adenosine; OA, orotic acid; CML, chronic myelogenous leukemia/- 5 hr after obtaining the cells. Cells were counted in an electronic r, correlation coefficient. counting machine (Coulter Electronics, Inc., Chicago, Illinois). Received January 28, 1966; accepted May 24, 1966. RADIOACTIVITYOFNUCLEICACIDS.For measurement of the ra-

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5 Â«"100 JHâ€”-L ro (9) (4) (2) J 80 - (3) l60^ o: o o 5 40 - I 5/rf error of Mean ( ) Number of Determinations 20 -

0.03 0.3 3 30 300 TdR CONCENTRATION CHART 1. Incorporation of thymidine (TdR)-'H into DNA. Incubations were carried out under standard conditions for 60 min. The MCof TdR-3H added/ml of incubation mixture varied with the final TdR concentration as follows: 0.5 MMTdR, 5 MC;4 Â¡anTdR, 10 MC; 16.5 MMTdR, 15 MC;31.5 MMTdR, 15 MC;62 MMTdR, 20 MC;152.5 MMTdR, 25 MC;302.5 MMTdR, 25 MC.Values were normalized to 100% at 3.5 MMTdR. Mean incorporation of TdR at a concentration of 3.5 MMwas 13.4 niMiiioles/lO10 cells. dioactivity of DNA, the reaction was stopped by addition of an The difference between the total radioactivity and the radio equal volume of 1.0 MKOH. After 18 hr at 37Â°Cthemixture was activity of DNA (which accounted for 6-20% of the total) was cooled in an ice bath and neutralized with trichloroacetic acid. taken as the radioactivity of RNA. DNA was precipitated by addition of trichloroacetic acid to a ANALYSIS OF INTHACELLULAR THYMINE NÃœCLEOTIDE POOLS. final concentration of 5% (free acid), collected and washed on The reaction was stopped by pipetting 2-ml aliquots of the in Whatman GF/C glass fiber filters. The precipitate was dried cubation mixture into tubes containing 1 /Â¿moleof unlabeled and was then dissolved in 1 ml of hydroxide of Hyamine (l M in TMP, TOP, and TTP immersed in a Dry Ice-alcohol bath. After methanol; Packard Instrument Co., La Grange, Illinois). After partially thawing and refreezing the samples 5 times to disrupt addition of 10 ml of toluene phosphor solution containing 3 gm the cells, the tubes were placed in a boiling water bath for 2 min, of 2, 5-diphenyloxazole and 0.05 gm of 1, 4-bis-2-(5-phenyloxa- cooled, and centrifuged. Similar results were obtained when the zolyl)-benzene/liter, the mixture was counted in a liquid scintil samples were boiled prior to disrupting the cells. The super lation spectrometer. Count ing efficiencies were 13% for 3H and natant fluids were added to 1- x 2-cm AG-X8 (200-400 mesh) 50% for MC. Quenching was monitored by the channels ratio chloride columns. The columns were washed with 20 bed volumes method. Of the radioactivity recorded as DNA, 2-4% was from of water to elute TdR and its catabolites. TMP was eluted with contaminating RNA as measured by the amount of radioactive 12 bed volumes of 0.0085 M HC1-0.085 M LiCl; TDP with uracil from OA-14Cand UR-14C in the DNA fraction after per 12 bed volumes of 0.05 M HC1-0.15 M LiCl; and TTP with chloric acid hydrolysis and Chromatographie separation (see be 20 bed volumes of 0.2 M HC1-0.2 M LiCl. The eluates were low). collected in vials and dried. The residue was dissolved in 1.5 ml For determination of the radioactivity of DNA-thymine, the of water and 20 ml of Bray's solution (5) and counted in a liquid trichloroacetic acid precipitate was collected by centrifugation, scintillation spectrometer. The degree of quenching was deter lyophilized, and hydrolyzed with 0.1 ml of 60% perchloric acid mined by means of an external standard. for 1 hr in a boiling water bath. Each sample was passed through DEGRADATIONOFTdR. TdR degradation was determined by a 1- x 2-cm AG-1-X8 (200-400 mesh, Bio-Rad, Los Angeles, Cal chromatography of the trichloroacetic acid supernatant fraction ifornia) formate column to remove perchlorate ions. The column of the incubation mixture on Whatman No. 3MM filter paper was washed with 4 bed volumes of water, and the combined ef using Fink's solvent 8 (19). The rate of degradation was constant fluent was lyophilized. The dry material was taken up in 50 jul for at least 1 hr, and in the presence of 2 X IO7cells/ml, it ranged of water and chromatographed (descending) with known stand from 0.18 m/Â¿mole/hrat an initial TdR concentration of 0.3 /Â¿M ards on Whatman No. 3MM filter paper using 86% butanol- to 33 m/xmoles/hr at 300 JUMTdR. water. After drying, the paper was cut into strips and counted S. E., S. D., and correlation coefficient were calculated accord hi the scintillation spectrometer with toluene phosphor solution. ing to Snedecor (42). For measurement of the radioactivity of RNA, aliquots of the incubation mixture were pipetted into cold trichloroacetic acid Results (final concentration 5%). The precipitate, containing the total INCORPORATIONOFEXOGENOUSTdR. Chart 1 shows the in acid-insoluble radioactivity, was filtered and counted as for DNA. corporation of varying concentrations of TdR-3H into DNA. At

226S CANCER RESEARCH VOL. 26

2000 â€”i 1 r

100 1800 -

2 50

I Sta. error of Mean 25 (1 Number oÃDeterminations

0.03 0.3 3 30 300 10,000 TdR CONCENTRATION CHART3. Effect of thymidine (TdR) on incorporation of deoxy guanosine (GdR)-3H into DNA. Incorporation in the absence of TdR was set to 100%. Incubations were carried out under standard conditions for 60 min with the addition of 5 pMGdR-'H (2.5 juc/ml) and various concentrations of unlabeled TdR. Incorporation of IO 20 30 40 50 60 GdR-3H into DNA in the absence of TdR was 4.9 to 17 maniÃ³les/ IO10cells.

CHART2. Time course of the inhibition by thymidine (TdR) of deoxyguanosine (GdR)-3H incorporation into DNA. Incubations that the amount of DNA-thymine synthesized via the salvage were carried out under standard conditions with the addition of pathway was constant at concentrations of exogenous TdR above 6 MMGdR-3H (2.5 ^c/ml) and either no TdR or 1 HIMTdR. In the 0.5 MM,they do not provide a measure of the total amount of absence of TdR, incorporation of GdR-3H into DNA in 60 min was DNA-thymine synthesized or the % of the total which was de 17 m/imoles/1010 cells. rived from the salvage pathway. To examine the effect of TdR on total DNA-thymine synthesis, GdR-3H incorporation into DNA least 4 concentrations were tested with each of 9 leukocyte sam was used to measure DNA synthesis. At the concentration used ples. For comparison, the results have been calculated as % of (o Â¿IM),GdRdid not influence DNA synthesis as measured by incorporation of 3.5 /Â¿MTdRsince incorporation at this concen incorporation of low concentrations of TdR-3H into DNA (see tration was measured in all samples. As the concentration of TdR below). The addition of 1 HIMTdR resulted in 46% inhibition of was increased from 0.03 JUMto 0.5 IJ.M,the amount of TdR in GdR incorporation into DNA (Chart 2). This % inhibition was corporated progressively increased. However, increasing the TdR observed at 3 min and remained constant over a period of 1 hr. concentration from 0.5 /UMto 300 pM.resulted in little additional The effect of various concentrations of TdR on incorporation of incorporation. The incorporation of TdR at a concentration of GdR into DNA and RNA of leukocytes from 7 donors was meas 3.5 JUMranged from 7.5 to 28.9 m/tmoles/lO10 cells hi 60 min ured. At TdR concentrations greater than 0.3 Â¿IMtherewas a (mean = 13.4 mamÃ³les Â±S.D. 2.2). This correlated best with progressive decrease of GdR incorporation into DNA (Chart 3). the % myelocytes (r = 0.97; P < 0.001), less with the % myelo- Incorporation of the guanine of GdR into RNA was not affected blasts + promyelocytes -f myelocytes (r = 0.79; 0.01 < P < by TdR concentrations up to 300 /IM. 0.05), and least with the % myeloblasts (r = 0.56; 0.05 < P < There was no inhibition of GdR incorporation into DNA when 0.1). Although the absolute incorporation of TdR, and all other cells were incubated with 1 IBMTdR at 37Â°Cfor 10 min, washed, nucleic acid precursors used, varied among the leukocyte samples and resuspended in TdR-free medium. Studies in the accompany studied, comparison of the data on the basis of an internal con ing paper (14) have shown that, following removal of TdR, the stant (% of control, % of maximal incorporation) resulted in half life of TMP, TDP and TTP derived from TdR at a concen little variation from the mean. tration of 14 Â¿mis60-90 sec at 37Â°C.Thus, the continued pres EFFECT OF EXOGENOUS TdR AND OTHER NUCLEOSIDES ON DNA ence of TdR or its phosphorylated derivatives is necessary for SYNTHESIS.While the data presented in Chart 1 demonstrate inhibition to occur.

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30 -

15 -

I ! 10 100 1000 10,000 I 10 100 1000 CONCENTRATION(jiM) CHART4. A, Effect of pyrimidine nucleosides on incorporation of 5 MMdeoxyguanosine (GdR)-3H (2.5/iC/ml) into DNA. The abbrevi ations used are: UdR, deoxyuridine; UR, uridine; CR, cytidine; TdR, thymidine; CdR, deoxycytidine. Incubations for 60 min under standard conditions. B, Effect of purine nucleosides on 0.5 to 1.3 MMthymidine (TdR)-3H (1.9-5 MC/ml) incorporation into DNA. The abbreviations used are: AdR, deoxyadenosinejGdR, deoxyguanosine; AR, adenosine. Incubations for 60 min under standard conditions.

The pyrimidine nucleosides UR, CR, UdR, and CdR did not 100 T inhibit DNA synthesis (Chart 4A). RNA formation was also unaffected by concentrations of these nucleosides up to 300 Â¿in. The inhibition of DNA synthesis caused by 300 JUMTdR was completely reversed by 3 MMCdR (Chart 5), while UR, CR, and 80 UdR, at concentrations up to 300 JUM,did not affect this inhibi tion. The effect of purine nucleosides on DNA synthesis was evalu ated by their influence on incorporation of TdR-3H into DNA 60 ribonucleotides from OA and UR. Two factors are responsible for the decreased OA and UR incorporation into DNA-thymine in the presence of exogenous thesis from the salvage pathway; and (6) at concentrations of TdR: (a) incorporation of TTP synthesized via the salvage path TdR greater than 0.3 /IM, a reduction in the total amount of way rather than TTP synthesized de novo resulting from inhibi DNA-thymine synthesized. In the presence of exogenous TdR tion of TTP synthesis de novo and/or dilution with TTP syn- at a concentration of 3 MMthe total decrease of OA and UR in-

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100 TABLE 1 r SYNTHESIS OF DNA-THYMiNE IN THE PRESENCE OF EXOGENOUSTdRÂ» 90 UR i)\.\-thymine Total DXA- DXA tby- TdR-'H in (A) mine synthe TdR con thymine mine synthe sized from corporation fromTdRD 80 centration synthesized sized de novo TdR (B - C) into DNA + E X100B012.735.655.167.187.4 (MM)00.030.33.030.0300.0(B)(mamÃ³les/ (mamÃ³les/ (rajuÃ±Ã³les/ (m/itnoles/ 10'Â»cells/hr)25.325.325.324.320.215.9(C)D\A-thy-10"cells/hr)25.321.516.710.97.31.5(D)IOÂ«cells/hr)03.88.613.412.914.4(E)10>Â°celis/hr)02.69.413.414.213.4(K)'â€¢;

70 i o Â£ 60 o o fe 50 Iâ€” Â«TdR, thymidine. SE OA UJ Â£540 Q_ obtained are close to values obtained by a direct measure of TdR 30 incorporation into DNA (Chart 1), shown in Table 1, Column E. The % contribution of the salvage pathway to the synthesis of DNA-thymine in the presence of various TdR concentrations was 20 calculated (Table 1, Column F). It is linearly related to the log of the TdR concentration accounting for 13% of the DNA- thymine at 0.03 AIMand 87% at 300 /Ã•MTdR. IO - EFFECTOF EXOGENOUSTdR ON THYMINENUCLEOTIDESYN THESISDE NOVO.Although there was a contribution to DNA- thymine by TTP synthesis de novo at all concentrations of TdR investigated, the data presented in Table 1 do not indicate 0.03 0.3 3 30 300 whether the amount of TTP synthesized de novo was affected by TdR CONCENTRATION the availability of exogenous TdR. The values presented in Table CHART6. Effect of thymidine (TdR) on orotic ocid (OA)-14C and 1, Column F, reflect the composition of the TTP pool from syn uridine (UR)-"C incorporation into DNA-thymine. Incorporation thesis de novo and from exogenous TdR and are equal to the % in the absence of TdR was set to 100%. Incubations were carried of the TTP pool which was derived from TdR-3H.6 Thus, the out under standard conditions for 60 min. Incorporation was linear size of the total TTP pool can be calculated from a measurement with time. OA concentration, 40 Â¡OIL,1.4Â¿ic/ml;UR concentration, of the TTP pool derived from TdR-3H and the values in Table 2.5 MM,0.1 ^c/ml. Each OA point represents the average of 3 inde pendent experiments with cells obtained from separate donors. 1, Column F. UR incorporation was determined in cells from a 4th donor. Incor Results in the accompanying paper (14) demonstrated that, for poration of OA into DNA-thymine in the absence of TdR was 1.8- each concentration of TdR studied here, the radioactive TDP 6.4 rmimoles/1010 cells; UR incorporation was 1.8 m/jmoles/lO10 and TTP pools reached a maximum within 5 min and remained cells. constant for at least the subsequent 20 min. The combined value of TDP and TTP was treated as a sum because of the rapid corporation into DNA-thymine was 57% (Chart 6, average). Of equilibration between these pools. this, 4% was due to inhibition of DNA synthesis (Chart 3). Chart 7 illustrates the size of the TdR polyphosphate (TDP Thus, incorporation of TTP synthesized via the salvage pathway and TTP) pool derived from TdR-3H, as measured (14), and resulted in 53% inhibition of incorporation of TTP synthesized the size of the pool from synthesis de novo, as calculated from de novo. Since the mean incorporation of TdR-3H at a concentra tion of 3 /IM was 13.4 mjumoles/1010 cells/hr (Chart 1), total this measurement and the data in Table 1, Column F. Over the DNA-thymine synthesis in the absence of exogenous TdR was range of TdR concentrations from 0.17 /IMto 300 /Ã•Mthecontribu 25.3 m/imoles/1010 cellsAr (13.4 x 0.53-1)- tion to the size of the TdR polyphosphate pool from synthesis Total DNA-thymine synthesis at various TdR concentrations de novo was constant, while there was a progressive expansion of this pool attributable to the salvage pathway." was calculated from this latter value and the data on TdR inhibi tion of DNA synthesis in Charts (Table 1, Column B). Similarly, the contribution from the pathway de novo was calculated from 5 Implicit in this discussion is the assumption that there is no the data on OA-14Cand UR-14C incorporation in Chart 6 (Table preferential utilization of TTP synthesized de novo or from exoge 1, Column C). The difference between total DNA-thymine syn nous TdR. thesized and DNA-thymine synthesized de novo is the contribu 6There was insufficient incorporation of OA and UR into the tion of the salvage pathway (Table 1, Column D). Values so TdR polyphosphate pool to make this measurement directly.

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14 100 0.17 24 280 03 27 280 4 30 300 2 86 3 95 THYMIDINECONCENTRATION(/iMI CHART7. Thymidine diphosphate (TOP) + thymidine triphosphate (TTP) pools in the presence of various thymidine (TdR) con centrations. Data using cells from 3 donors are shown. Hatched bar represents TOP + TTP pools de rived from exogenous TdR-3H. Solid bar represents TDP +TTP from synthesis de novo calculated from the relationship: TDP + TTP from synthesis de novo =

(TOP + TTP from TdR) X (10090 - % DNA-thymine from TdR) (yc DNA-thymine from TdR

Incubations were carried out under standard conditions for 30 min. TdR-3H incorporation into TDP and TTP was determined as de scribed in Methods.

Discussion to dCDP (31). This was substantiated in intact murine lympho- blast cells with the demonstration that TdR inhibited UR con This study demonstrates that in CML leukocytes concentra version to CdR nucleotides but not its conversion to CR tions of exogenous TdR as low as 0.03 UMcontribute a significant nucleotides (32). The conversion of CDP to dCDP in cell-free % of the DNA-thymine synthesized. It has been reported that TdR concentrations greater than this occur in the spleen (5.4- systems is inhibited by TTP (37). 7.8 m/imoles/gm of tissue) and thymus (14.4-21.4 m/umoles/gm Exogenous TdR at concentrations to 300 /Ã•Mdid not inhibit incorporation of labeled GdR, OA, or UR into the RNA of CML of tissue) of rats and that approximately 25% of the TDP and leukocytes. In contrast, 400 Â¿UMTdRwas found to inhibit UR TTP pools in the spleen are derived from TdR (35). incorporation into the RNA of HeLa (34) and murine lympho- This study also demonstrates that, although the % contribu tion of TdR to the synthesis of DNA-thymine increased pro blast (31) cells. A variable effect of TdR on DNA synthesis in different cell lines also exists, as shown by the lack of inhibition gressively at increasing TdR concentrations, TTP synthesis de of DNA synthesis in chick embryo mince in the presence of 250 novo occurred at all concentrations of TdR investigated. Expan Ã•JMTdR(28) and in Ehrlich ascites cells in the presence of 2 HIM sion of the thymine nucleotide pool due to the presence of exoge TdR (26). nous TdR did not lead to a decreased net thymine nucleotide Inhibition of cell proliferation or nucleic acid synthesis has synthesis de novo. been observed in the presence of AdR (12, 26, 29, 36, 44) and INHIBITIONOFNUCLEICACIDSYNTHESIS.Several investigators GdR (24, 29, 44). dATP, a potent inhibitor of ribonucleotide have observed inhibition of cell growth hi tissue cultures con reducÃase (37), accumulates in cells incubated with AdR (12, taining TdR (4, 16, 28, 33, 34, 44). The lowest concentration of 26). AR, which prevents this accumulation of dATP, reverses TdR which inhibited DNA synthesis in our studies (3 /*M)was similar to the lowest growth-inhibiting concentrations noted by the AdR inhibition of DNA synthesis (26). AdR inhibition has also been reversed by the addition of CdR plus GdR (26), and others (31, 34). Removal of TdR from the medium or addition of GdR inhibition has been reversed by the addition of CdR plus CdR to the medium resulted in resumption of the normal rate AdR (24). Thus, as with the inhibition of DNA synthesis caused of DNA synthesis in our studies and, in previous reports, resump tion of the normal rate of cell growth (4, 31, 33, 44). by TdR, the purine deoxynucleosides appear to inhibit DNA The observation that cells lacking TdR kinase activity are not synthesis by interfering with ribonucleotide reducÃase. inhibited by TdR implicated a phosphorylated derivative of TdR CONTROL OF THYMINE NUCLEOTIDE SYNTHESIS. It Would appear as the inhibitor substance (16, 31). Reversal of inhibition by efficient for the cell to cease TTP synthesis de novo and rely CdR but not CR, or any other pyrimidine nucleoside, indi entirely on the salvage pathway when the amount of TTP syn cated that the enzymatic step blocked was the reduction of CDP thesized by that pathway was adequate to supply the needs of

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TdR TdR IMP TMP CARBAMYL PHOSPHATE + - -CARBAMYL ASPARTATE- -DIHYDROOROTATE ASPARTATE

URIDINE UMP- â€¢OROTIDINEMONOPHOSPHATE -*â€¢ â€¢OROTATE

dUDP- -dUMP- -UdR dCMP-

dCDP" TdR :TMP URACIL

TTP TOP. TMP- -TdR

CHART8. Pyrimidine nucleotide synthesis. Direction of arrows represents flow. Hatched arrows represent reactions inhibited in cell- free systems by compounds shown. The abbreviations used are: Udii, deoxyuridine; dUMP and dUDP, deoxyuridine-5'-mono- and diphosphate; dCMP, dCDP, and dCTP, deoxycytidine-5'-mono-, di-, and tri-phosphate; TdR, thymidine; TMP, TDP, and TTP, thymidine-5'-mono-, di-, and tri-phosphate; UMP, UDP, and UTP, uridine-S'-mono-, di-, and tri-phosphate; CÃ¼P, cytidine-5'-di-phosphate; and CTP, cytidine-5'-triphosphate. The following enzymatic reactions are designated by numbers: 1, aspartate transcarbamylase; 2, dihydroorotase ; 3, ribonucleotide reducÃase; 4, thymidylate synthetase ; 5, pyrimidine trans-W-deoxyribosylase ; and 6, thymidine kin ase. the cell. Evidence has been obtained in mammalian cell-free DNA synthesis, as has been suggested (20, 31, 40), then the in systems for the feedback control of thymine nucleotide biosyn creased size of the thymine nucleotide pool brought about by thesis de novo at 4 enzymatic sites (Chart 8). However, this study addition of TdR should have resulted in an increase in the total has indicated that the yield of thymine nucleotides synthesized amount of DNA synthesized. This did not occur, indicating that, de novo was not altered over the range of TdR concentrations in cells with a complement of enzymes capable of synthesizing tested despite a 4- to 5-fold increase in the size of the TDP and DNA, TTP is not limiting. The elevation hi the activities of TTP pools. thymidylate synthetase (22, 27) and TdR kinase (3, 43) in re Aspartic transcarbamylase (Enzyme 1, Chart 8) and dihydro generating rat liver suggests that the size of the thymine nucleo orotase (Enzyme 2, Chart 8) are inhibited by TdR and TMP tide pool is controlled through the mechanism of enzyme dere- (8, 9). If these reactions are rate limiting in the synthesis of UMP, pression. inhibition might be reflected both as a decreased TDP and TTP Synthesis of dUMP from UMP requires reduction of either synthesis de novo and, because of less dilution with UMP syn UDP or CDP to the corresponding deoxynucleotide diphosphate thesized de novo, an increased incorporation of OA-14Cand UR-14C (Enzymes 3A and 3B, Chart 8). Although both are reduced by into RNA. Similarly, noncompetitive inhibition of thymidylate the same enzyme fraction, it is not known whether there is a synthetase (Enzyme 4, Chart 8) by TMP, as has been observed single ribonucleotide reducÃaseor whether each pyrimidine nucle in a crude enzyme system from Ehrlich ascites cells (38), would otide diphosphate is a substrate for a separate enzyme (1). dUMP be reflected as a decreased synthesis de novo of TTP and TDP can also be synthesized by phosphorylation of UdR. The elevated in the presence of the increased concentrations of TMP7 derived TTP level, which results from the presence of exogenous TdR, from TdR. Such consequences of these feedback controls were inhibits reduction of CDP (37) (and therefore possibly reduction not observed. of UDP). Although transfer of deoxyribose from exogenous TdR Although our data indicate the absence of feedback control to uracil (Enzyme 5, Chart 8) could offer an alternate source of over thymine nucleotide synthesis de novo in intact leukemic cells, dUMP for the pathway de novo of TMP synthesis (15, 45), com it cannot be ruled out that thymine nucleotide synthesis de novo petition between the UdR so formed and the high concentrations was under the influence of maximal feedback inhibition at the of TdR for the same phosphorylating enzyme may limit this lowest concentration of TdR used in this study. In either case, pathway. Since, in mammalian cells, CdR probably does not if production of thymine nucleotides is the controlling factor in participate in the deoxyribose exchange (15), a similar circum vention of the ribonucleotide reductase reaction cannot be ac 7Expansion of the TMP pool is discussed in the accompanying complished by cytosine plus TdR. paper (14). Expansion of the TTP pool with subsequent inhibition of

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1966 American Association for Cancer Research. Richard A. Cooper, Seymour Perry, and T. R. Breitman ribonucleotide reductase is the probable cause of the observed Thymine and Uracil Metabolism in the Liver and Hepatoma. decrease in DNA synthesis in the presence of exogenous TdR. J. Nati. Cancer Inst., 24: 13-29, 1960. Feedback control of TdR kinase (Enzyme 6, Chart 8) by TTP 16. Dubbs, D. R., and Kit, S. Effect of Halogenated Pyrimidines and Thymidine on Growth of L-Cells and a Subline Lacking would limit the amount of TTP synthesized from TdR. If this Thymidine Kinase. Exptl. Cell Res., 33: 19-28, 1964. control mechanism is operative in the intact cell, it appears to be 17. Eker, P. Properties and Assay of Thymine Deoxyribonucleo- insufficient to protect the cell from accumulating growth-inhibit tide Phosphatase of Mammalian Cells in Tissue Culture. J. ing concentrations of TTP, a result consistent with the com Biol. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1966 American Association for Cancer Research. Pyrimidine Metabolism in Human Leukocytes: I. Contribution of Exogenous Thymidine to DNA-Thymine and Its Effect on Thymine Nucleotide Synthesis in Leukemic Leukocytes

Richard A. Cooper, Seymour Perry and T. R. Breitman

Cancer Res 1966;26:2267-2275.

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