Pyrimidine Metabolism in Human Leukocytes III. the Utilization of Thymine for DNA-Thymine Synthesis by Leukemic Leukocytes

[CANCER RESEARCH 26 Part 1, 2282-2285, November 19661

Pyrimidine Metabolism in Human Leukocytes III. The Utilization of Thymine for DNA-Thymine Synthesis by Leukemic Leukocytes

T. R. BREITMAN,1 SEYMOUR PERRY, AND RICHARD A. COOPER2

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

For measurement of isotope incorporation into TdR, thymine, Summary DHT, and BUIB, 0.5-ml aliquots of the incubation mixture were There was only slight incorporation of thymine-3H into DNA pipetted into tubes containing 0.1 ml of ice-cold 30% trichloro- of intact human leukemic leukocytes in vitro in the absence of an acetic acid, held in an ice bath for approximately 10 min, and exogenous source of deoxyribose. Thymine-3H incorporation was then centrifuged. Then 100-^1 aliquots of the supernatant fluid promoted by both purine and pyrimidine deoxynucleosides. The were spotted on Whatman No. 3MM filter paper and chromato- net conversion of thymine-3H to thymidine (TdR)-3H was greatest graphed (descending) using Fink's solvent 8 (4). The paper was in the presence of TdR or deoxyuridine. Addition of deoxycytidine cut into strips and counted in a liquid scintillation spectrometer resulted in less net conversion, and the purine deoxynucleosides with toluene phosphor solution. Thymine-3H (6.7 c/mmole) and produced little netTdR-3H. Addition of pyrimidine deoxynucleo TdR-14C (30 mc/mmole) were obtained from the New England sides but not purine deoxynucleosides resulted in decreased thy- Nuclear Corporation, Boston, Massachusetts. mine-3H catabolism. The results indicate that human leukocytes have the enzymatic Results and Discussion capability to convert thymine to TdR. This enzymatic capability is not expressed in the absence of added deoxynucleosides because EFFECT OF DEOXYNUCLEOSIDES ON THYMINE INCORPORATION of rapid catabolism of TdR and thymine and a limited supply of INTODNA. The incorporation of thymine-3H (at a concentration an available deoxyribose source. of 1 MM)into DNA was slight in the absence of UdR but increased with increasing UdR concentrations and was maximum at a UdR

Introduction EFFECT OF UdR ON THYMINE INCORPORATION INTO DNA Studies with mammalian tissues in vivo and in vitro have indi T cated that thymine is incorporated poorly or not at all into DNA (1, 5, 7). Two factors could be responsible for this: (a) rapid catabolism of thymine to DHT3 (2), and (b) insufficient net con version of thymine to TdR due to lack of enzymatic capability, lack of a source of deoxyribose, or rapid catabolism of TdR to thymine. The purpose of the present report is to describe thy mine utilization and the influence of deoxynucleosides on its me tabolism in human leukocytes.

Materials and Methods Leukocytes were obtained from 2 patients with CML in re lapse. The methods for leukocyte isolation and incubation and for determination of isotope incorporation into DNA were as de scribed (3).

"To whom requests for reprints should be addressed at the Lab oratory of Physiology, National Cancer Institute, NIH, Bethesda, I I I 1 I I I 1 Md. O.I 0.2 0.3 0.4 05 0.6 07 0.8 0.9 1.0 2Present address: II and IV (Harvard) Medical Service, Boston City Hospital, Boston, Mass. THYMINE (fiMl 3The abbreviations used are: DHT, dihydrothymine; TdR, thy UdR(mM) midine; BUIB,o-ureidoisobutyric acid; UdR, deoxyuridine; CdR, CHART1.Effect of deoxyuridine (UdR) on thymine-3H incorpo deoxycytidine; AdR, deoxyadenosine; GdR, deoxyguanosine; ration into DNA. Leukocytes at a concentration of 2 X IO7cells/ml CML, chronic myelogenous leukemia. were incubated for 100min. DNA was isolated and its radioactiv Received January 28, 1966; accepted May 24, 1966. ity determined as described in Methods.

2282 CANCER RESEARCH VOL. 26

DHT+ BUIB THYMINE -TdR

90 0 30 60 90 MINUTES CHART2. Effect of deoxynucleosides on metabolism of thymine-'H. Leukocytes at a concentration of 2 X IO7cells/ml. Thymine con centration was l /IM. The abbreviations used are: TdR, thymidine; DHT, dihydrothymine; BUIB, j3-ureidoisobutyric acid; UdR, deoxyuridine; CdR, deoxycytidine; AdR, deoxyadenosine; and GdR, deoxyguanosine; TdR and UdR concentrations: â€¢1 MM;O, 10 /IM; A 0.1 mM; O, 1 mM; â€¢ 10 mM. Other deoxynucleosides at a concentration of 1 HIM:O, CdR; A, AdR; T, GdR. No deoxynucleoside addition (control),^. Chromatographie separation of metabolites and determination of their radioactivity are as described in Methods. Approximately 99% of the initial total radioactivity was accounted for in thymine, TdR, DHT, and BUIB. concentration of 300 /IM (Chart 1). At this UdR concentration equal concentrations (1 mM), CdR was not as effective as UdR the rate of thymine-8H incorporation was directly related to thy or TdR in promoting thymine conversion to TdR. This probably mine concentration from 0.1 to 1.0 /tM. reflects a rate limiting initial deamination to UdR. Addition of other deoxynucleosides also markedly enhanced It was previously reported that the rate of TdR degradation thymine incorporation into DNA (Table 1). For comparison, in the presence of 2 X 10' cells/ml ranged from 0.18 m/nmole/ml/ rates of TdR incorporation into DNA in the presence and absence hr at an initial TdR concentration of 0.3 Â¿XMto33 m/imoles/hr of deoxynucleosides are also presented. It is seen that, while thy at 300 /Â¿MTdR(3). As seen in Chart 3, UdR and CdR share this mine incorporation into DNA is enhanced, TdR incorporation degradative pathway with TdR. Thus high concentrations of into DNA is inhibited by addition of deoxynucleosides. Thus, the CdR, UdR, and TdR can spare from degradation the small presence of deoxynucleosides markedly decreases the difference amounts of TdR-3H formed in their presence. between the rates of thymine and TdR incorporation. An additional factor promoting TdR-3H accumulation in the KFFECT OF DEOXYNUCLEOSIDES ON THYMINE ANABOLISM AND presence of pyrimidine deoxynucleosides was decreased catabo CATABOLISM.Toexamine further the effects of deoxynucleosides lism of thymine-3H to DHT-3H and BUIB-'H (Chart 2). How on thymine metabolism, the anabolism of thymine to TdR and ever, the increased anabolism of thymine-3H to TdR-3H exceeded its catabolism to DHT and BUIB [the end catabolite of thymine the decreased catabolism of thymine, the net result being a de in human leukocytes (6)] were measured. Pyrimidine deoxynu crease in mean thymine concentration. Raising the TdR concen cleosides increased the rate of TdR-3H formation (Chart 2). At tration from 10 Â¿IMto1 mM resulted in a 65% decrease in the

NOVEMBER 1966 2283

TABLE 1 EFFECT OF DEOXYNUCLEOSIDESONTHYMINEAND THYMIDINE INCORPORATIONINTODNA"

DEOXYXUCLEO- SIDEADDITION (MM)NoneUdR:25250300300CdR:300AdR:300300GdR:300300THYMINE1Â«0.41.00.41.00.40.40.4Incorporation6<0.010.183.88.42.51.32.4THYMIDINE0M0.41.02.52.52.50.41.00.41.0Incorporationh100Â«130Â«134Â«803450Â«65Â«00Â«78Â«

CdR

dR-1-P04 CHART3.Pyrimidine deoxynucleoside metabolism. The abbrevi ations used are: T, thymine; TdR, thymidine; A, adenine; G, gua nine; AdR, deoxyadenosine; GdR, deoxyguanosine; U, uracil; UdR, deoxyuridine; CdR, deoxycytidine; dR-l-PO4, 2-deoxyribose- " Leukocytes at a concentration of 2 X IO7 cells/ml were in l-PO-4, DHT, dihydrothymine; and DHU, dihydrouracil. The cubated for 60 min. DNA was isolated and its radioactivity de following enzymatic reactions are designated by small letters: a, termined as described in Methods. UdR, CdR, GdR, and AdR purine deoxynucleoside phosphorylase; 6 and 6', thymidine phos are the deoxynucleosides of uracil, cytosine, guanine, and adenine. phorylase; c, pyrimidine /rans-A^deoxyribosylase; and d, dihy- 6 The AmÃ³les incorporated into DNA/108 cells/hr. dropyrimidine dehydrogenase. Â«Calculated from data in Table 1 and Chart 4 of Cooper et al. (3). no significant change in thymine-3H catabolism (Chart 2C and C'). This indicates that purine deoxynucleosides are capable of formation of DHT-3H and BUIB-3H but a 470% increase in the formation of TdR-3H. The net effect of this on mean thymine-3H enhancing thymine conversion to TdR at a rate approximately concentration after the 90-min incubation was a decrease from equal to the sum of the rates at which TdR is converted to TMP by thymidine kinase and degraded to thymine by thymidine 0.70 MMto 0.55 Â¿tM.Similar results were obtained with UdR and phosphorylase. CdR. Although the mode of deoxyribose transfer cannot be ascer The data indicate that enhanced incorporation of thymine into DNA and accumulation of TdR-3H derived from thymine-3H in tained from these experiments, the results indicate that it is not lack of enzymatic capability to convert thymine to TdR which the presence of pyrimidine deoxynucleosides resulted from 3 prevents leukocytes and possibly other mammalian tissues from mechanisms: (a) a decrease in the rate of TdR degradation to effectively incorporating thymine into DNA. Rather, as the re thymine; (b) a decrease in the rate of thymine degradation to sults with AdR and GdR indicate, this enzymatic capability is DHT; and (c) an increase in the rate of thymine conversion to TdR either directly by Reaction c, shown in Chart 3 (transdeoxy- not expressed in the absence of added deoxynucleosides because ribosylation), or indirectly via Reactions 6 and 6' (coupled thy- of a limited supply of endogenously produced deoxyribose sub strate to support conversion of thymine to TdR at a rate great midine phosphorylation). The uracil produced by either of these enough to overcome the rapid catabolism of TdR and thymine. pathways competes with thymine for reduction to DHT. Evi dence has been presented that the thymidine phosphorylase and pyrimidine Â¿rans-W-deoxyribosylase activities are associated with References the same protein (8, 9). AdR and GdR probably enhance TdR formation as a result of 1. Brown, G. B., Roll, P. M., and Weinfeld, H. Biosynthesis of a coupling of Reaction a in Chart 3 (purine deoxynucleoside Nucleic Acids. In: W. D. McElroy and H. B. Glass (eds.), Phos phorus Metabolism, Vol. 2, pp. 385-406.Baltimore: Johns Hop phosphorylase) with Reaction b in Chart 3 (thymidine phos phorylase), though it cannot be ruled out that the 2-deoxyribose- kins Press, 1951. 2. Canellakis, E. S., Jaffe, J. J., Mantsavinos, R., and Krakow, 1-PÃ›4 produced inhibits the action of thymidine phosphorylase J. S. Pyrimidine Metabolism. IV. A Comparison of Normal and on TdR synthesized from thymine and an endogenous source of Regenerating Rat Liver. J. Biol. Chem., 234: 2096-99, 1959. deoxyribose. In contrast to the increased TdR-3H concentration 3. Cooper, R. A., Perry, S., and Breitman, T. R. Pyrimidine Me observed in the presence of TdR, UdR, and CdR, addition of tabolism in Human Leukocytes. I. Contribution of Exogenous AdR and GdR resulted in a slight accumulation of TdR-3H and Thymidine to DNA-Thymine and Its Effect on Thymine Nu-

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cleotide Synthesis in Leukemic Leukocytes. Cancer Res., 26: Plentl, A. A., and Schoenheimer, R. Studies in the Metabolism 2265-73, 1966. of Purines and Pyrimidines by Means of Isotopie Nitrogen. J. 4. Fink, K., Cline, R. E., Henderson, R. B., and Fink, R. M. Me Biol. Chem., 15S: 203-17, 1944. tabolism of Thymine (Methyl-C" or -2-C") by Rat Liver in Zimmerman, M. Deoxyribosyl Transfer. II. Nucleoside:Pyrim Vitro. 3. Biol. Chem., 231: 425-33, 1956. idine Deoxyribosyltransferase Activity of Three Partially Puri 5. Hakala, M. T., and Taylor, E. The Ability of Furine and Thy fied Thymidine Phosphorylases. Ibid., 239: 2622-27, 19(>4. mine Derivatives and of Glycine to Support the Growth Zimmerman, M., and Seidenberg, J. Deoxyribosyl Transfer. I. of Mammalian Cells in Culture. Ibid., 234: 126-28, 1959. Thymidine Phosphorylase and Nucleoside Deoxyribosyl 6. Marsh, J. C., and Perry, S. The Reduction of Thymine by Hu Transferase in Normal and Malignant Tissues. Ibid., %S9: man Leukocytes. Arch. Biochem. Biophys., 104: 146-49, 1964. 2618-21, 1964.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1966 American Association for Cancer Research. Pyrimidine Metabolism in Human Leukocytes: III. The Utilization of Thymine for DNA-Thymine Synthesis by Leukemic Leukocytes

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

Cancer Res 1966;26:2282-2285.

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