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

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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] Pyrimidine Metabolism in Human Leukocytes I. Contribution of Exogenous Thymidine to DNA-Thymine and Its Effect on Thymine Nucleotide Synthesis in Leukemic Leukocytes1 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 /¿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 /¿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 37°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- NOVEMBER 1966 2267 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1966 American Association for Cancer Research. Richard A. Cooper, Seymour Perry, and T. R. Breitman 120 5 «"100 JH—-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 ¡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 37°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 /¿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/¿mole/hrat an initial TdR concentration of 0.3 /¿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).
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