Pyrimidine Metabolism in Human Leukocytes II

[CANCER RESEARCH 26 Part 1, 2276-2281,November 1966]

Pyrimidine Metabolism in Human Leukocytes II. Metabolism of the Thymine Nucleotide Pools in Normal and Leukemic Leukocytes

RICHARD A. COOPER,1 SEYMOUR PERRY, AND T. R. BREITMAN2

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

Summary newly synthesized DNA-thymine which was derived from exoge The incorporation of thymidine (TclR)-3H into the thymidine nous TdR was linearly related to the log of the TdR concentra tion, increasing from 13% to 87% over a range of TdR concentra diphosphate (TDP) and thymidine triphosphate (TTP) pools tions from 0.03 Â¿IMto300 Â¿IM.Thepurpose of this report is to of intact leukocytes from normal donors and patients with describe both the synthesis of thymine nucleotides from TdR and chronic myelogenous leukemia (CML) reached a maximum within 5 min at 37Â°C.Asteady state was maintained during the the dynamics of the thymine nucleotide pools in intact human leukocytes. subsequent 20 min. The time to reach maximal incorporation of TdR-3H into the thymidine monophosphate (TMP) pool was slower than into the TDP and TTP pools. In leukemic cells the Materials and Methods rate of equilibration of TMP with TdR-3H was related directly Leukocytes were isolated as described (8) from 4 patients with to the TdR concentration and inversely to the temperature. CML, who were in relapse and were receiving no therapy, and With increasing concentrations of TdR, expansion of the TMP from pooled normal buffy coat, obtained by the differential cen- pool derived from TdR was greater than expansion of the TDP trifugation of dextran-sedimented whole blood from 25 normal and TTP pools. In leukemic leukocytes the TDP and TTP pools donors. Incubations were carried out as described (8), com were approximately equal in size and accounted for 65% of the mencing within 3 and 6 hr after blood was obtained from CML thymine nucleotides in the presence of 1 Â¿IMTdRbut only 10% and normal donors, respectively. CML leukocytes were sus in the presence of 300 Â¿IMTdR.In normal leukocytes at these pended at a final concentration of 5 X 107/ml and normal leuko TdR concentrations the TDP and TTP pools accounted for 35% cytes at 2 X 10s/ml. TdR-3H was obtained from Schwarz Bio- and 5% of the total thymine nucleotides. Research, Orangeburg, New York. Analysis of intracellular thy Over the range of TdR concentrations studied, incorporation mine nucleotide pools and TdR-3H incorporation into DNA was of TdR-3H into the TMP pool was 2- to 5-fold greater and in carried out as described (8). corporation into the TDP and TTP pool was 6- to 15-fold greater in CML than in normal leukocytes. Incorporation of 3 JUM TdR-3H into DNA was 30-fold greater in CML than in normal Results and Discussion leukocytes. KINETICS OF TdR INCORPORATION INTO THYMINE NUCLEOTIDES. In the presence of TdR at a concentration of 14 Â¿IMtheturn When CML leukocytes were incubated at 37Â°Cwith TdR-3H at over rate of the thymine nucleotide pools was 3 min. Approxi a concentration of 4 Â¿IM,labelingof TDP and TTP pools reached mately 35% of the TTP turnover was due to its incorporation a maximum within 5 min and remained constant for the subse into DNA. quent 20 min (Chart IA). Similar results were obtained at all TdR concentrations examined between 0.17 Â¿IMand 300 Â¿ixi. Introduction Equilibration of the TMP pool with 4 Â¿IMTdR-3H,however, was During on investigation of TdR3 incorporation into the DNA not attained within the 25-min experimental period. In normal leukocytes incubated with 4 Â¿IMTdR-3Hat 37Â°C,maximal label of human leukemic leukocytes (8), it was observed that the % of ing of the TDP and TTP pools also occurred at approximately 5 1Present address: II and IV (Harvard) Medical Service, Boston min and equilibration of the TMP pool was reached by 18 min City Hospital, Boston, Mass. (Chart 1J3). Incorporation of TdR into DNA of both normal and 1To whom requests for reprints should be addressed at the Lab CML leukocytes was linear during the time interval between 5 oratory of Physiology, National Cancer Institute, NIH, Bethesda, and 30 min. Md. 3The abbreviations used are: TdR, thymidine; TMP, TDP, and FACTORSAFFECTINGEQUILIBRATION.Equilibrationof the TMP TTP, thymidine-5'-mono-, di-, and tri-phosphate; dUMP, deoxy- pool of CML leukocytes occurred more rapidly with increasing uridine-5'-monophosphate; and CML, chronic myelogenous leuke TdR concentrations (Chart 2). Lower temperatures had a varying mia. effect on the rate of equilibration of the 3 thymine nucleotide Received January 28, 1966; accepted May 24, 1966. pools (Chart 3). The TDP and TTP pools equilibrated progres-

2276 CANCER RESEARCH VOL. 26

100

10 20 25 MINUTES CHART2.Equilibration of the thymidine monophosphate (TMP) pool as a function of thymidine (TdR)-3H concentration. V, 100 AIMTdR; O, 4 MMTdR; â€¢0.3 pMTdR. Cells from 1 patient with chronic myelogenous leukemia were incubated at 37Â°Cand ana lyzed as in Methods.

which intact cells were used, there are at least 2 factors to be 10 20 30 10 20 30 considered: (a) in cell-free extracts the time course of thymine MINUTES nucleotide formation is influenced by differing stabilities of the CHART1. A, Incorporation of thymidine (TdR)-3H into the enzyme activities (3, 4); and (6) disruption of the intracellular thymine nucleotide pools of intact leukemic leukocytes. For com localization of enzymes may be an important determinant in the parison each point was calculated as the % of incorporation into equilibrium achieved. Gentry et al. (10), using intact Novikoff the thymidine monophosphate (TMP) pool at 25 min. Results are tissue culture cells, have also found that the thymine nucleotide from 3 separate experiments using leukocytes obtained from 3 pa pool equilibrated with exogenous TdR within 10 min and that tients with chronic myelogenous leukemia. Incorporation into TdR was incorporated into the thymine nucleotide pool at 4Â°C. TMP at 25 min was 7 m/nmoles/1010cells. TdR-3H concentration was 4pM.Incubation at 37Â°Candanalysis as described in Methods. EXPANSION OF THE THYMINE NUCLEOTIDE POOL AS A FUNCTION OFEXOGENOUSTdRCONCENTRATION.Theeffect of concentration TDP and TTP are thymidine di- and tri-phosphate. B, Incorpora on the incorporation of TdR-3H into the thymine nucleotide tion of thymidine (TdR)-'H into the thymine nucleotide pools of intact normal leukocytes. For comparison each point was calcu pools is shown in Chart 4. In CML leukocytes, as the TdR con lated as the % of incorporation into the thymidine monophosphate centration was raised from 1 fiu to 300 Â¿IMtheTMP pool derived (TMP) pool at 25 min. Results are from pooled leukocytes from from exogenous TdR increased 150-fold and the TDP and TTP 25 normal donors. Incorporation into TMP at 25 min was 1.4 pools increased 10-fold. In normal leukocytes, over the same con intimÃ³les/1010cells.TdR-'H concentration was 4/iM. Incubation at centrations, incorporation of TdR into the TMP pool increased 37Â°Cand analysis as described in Methods. 300-fold and incorporation into the TDP and TTP pools in creased 25-fold. sively more slowly at 23Â°Cand 5Â°C,whereas equilibration of the While the relative increase in incorporation of TdR into the TMP pool occurred more rapidly at lower temperatures. Sus thymine nucleotide pools of CDL and normal leukocytes was tained activity of thymidine kinase and the thymine nucleotide similar over the range of TdR concentrations examined, the ab kinases at 5Â°Cwasin contrast to the apparent loss of DNA poly- solute incorporation of TdR into these pools was always greater merase activity, as indicated by the lack of TdR-3H incorporation in CML than in normal cells. The greatest difference was in the into DXA at this temperature. TDP and TTP pools, which were 15-fold larger in CML than in The enzymes involved in TdR phosphorylation and incorpora normal cells at a TdR concentration of l Â¿IMand6-fold larger at tion into DNA have been extensively studied in cell-free extracts a TdR concentration of 300 Â¿IM.Therewas less disparity between of mammalian tissues (1, 3, 5, 11, 18). Equilibration of the thy the size of the TMP pool in normal and in CML leukocytes, this mine nucleotide pools with radioactive TdR, as reported here, has pool being 5-fold greater in CML than in normal cells at a TdR not, in general, been observed. In comparing results of experi concentration of l Â¿IMandonly 2-fold greater at a TdR concentra ments utilizing cell-free extracts with results in this study in tion of 300 nil.

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10 15 20 25 30 0 5 10 15 20 25 30 35 MINUTES CHARTS. Effect of temperature on thymidine (TdR)-3H incorporation into the thymine nucleotide pools. â€¢TMP; A, TDP; O, TTP Cells from 1 patient with chronic myelogenous leukemia were incubated and analyzed as in Methods. Incorporation into TMP at 30 min was 8.5 imtmoles/10 cells at 23Â°Cand 9 m/umoles/10 cells at 5Â°C.TMP, TOP, and TTP are thymidine mono-, di-, and tri-phosphate.

The size of the TDP pool was approximately equal to that of the accompanying study (8) were morphologically mature and the TTP pool at all TdR concentrations in both CML and normal were not undergoing DNA synthesis (J. Moxley and S. Perry, un leukocytes. Together they accounted for 65% of the thymine published observations). If these, like normal leukocytes, ac nucleotides in CML cells incubated with 1 /UMTdR. This de cumulated TMP in excess of TDP and TTP, 2 otherwise incon creased to 10% in the presence of 300 Â¿UMTdR.In normal leu sistent findings can be explained: kocytes TDP and TTP accounted for 35% and 5% of the total a) Evidence has been presented that TDP results from phos- thymine nucleotides at TdR concentrations of l Â¿UMand300 /UM, phorylation of TMP and that TTP is derived from TDP rather respectively. The composition of the thymine nucleotide pool of than from pyrophosphorylation of TMP (11, 12). The rate at leukocytes is in contrast to that of intact Novikoff tissue culture which maximal radioactivity of a product is reached when its cells, where TTP accounted for 80-90% of the thymine nucleotide precursor is derived from both a radioactive and a nonradioactive pool and TDP for the remaining 10-20% over the range of TdR source depends on the rate at which maximal specific activity of concentrations from l /UMto 1 HIM(10). this precursor is attained. The finding that the radioactivity of The substantial incorporation of TdR into the TMP pool of TDP and TTP reached a maximum more rapidly than that of normal leukocytes is consistent with reports that normal leuko TMP is not consistent with the sequence cytes, although lacking measurable thymidylate synthetase ac tivity (16), have thymidine kinase activity comparable to that in dUMP TdR-3H TMP -Â»TDP -* TTP CML leukocytes (7). Competitive inhibition of thymidine kinase by TTP in cell-free extracts of a variety of mammalian tissue (6, 7, 13), including normal and leukemic human leukocytes, is com This finding is compatible, however, with the existence of 2 pools patible with the linear relationship between TdR concentration of TMP, one which equilibrates rapidly and serves as precursor and TMP accumulation observed in Chart 4. The lesser incor for TDP and the other which labels more slowly and does not poration of TdR into the TDP and TTP pools of normal as com contribute to the TDP pool. Although it is known that some me pared with CML leukocytes correlates with the lesser amount of tabolites exist in more than 1 intracellular pool, the mixture of DNA synthesized by normal leukocytes. Incorporation of 3 /UM mature leukocytes with immature leukocytes, the latter actively TdR into DNA was 30-fold greater in CML than in normal leu synthesizing DNA, may in itself constitute 2 TMP pools. A kocytes. problem of interpretation similar to our own was discussed by Approximately 75% of the CML leukocytes used in this and Potter and Nygaard (15), who found that the maximum specific

2278 CANCER RESEARCH VOL. 26

TT| '

THYMINE NUCLEOTJDE POOLS

C ML NORMAL TMP â€¢ O TOP TTP â€¢ o

IOZ -

3.0 10 30 TdR CONCENTRATION CHART 4. Incorporation of thymidine (TdR)-'H into the thymine nucleotide pools and DNA of normal and leukemic leukocytes Data using cells from 3 patients with chronic myelogenous leukemia (CML) are plotted with solid lines and solid bar, and data obtained from pooled normal leukocytes with dashed lines and hatched bar. TDP and TTP are plotted on a single line. The TdR concentration for the DNA synthesis part of this experiment was 3 /IM. Incubations were performed under standard conditions at 37Â°Cfor 30 min. An alysis was as described in Methods. TMP, TDP, and TTP are thymidine mono-, di-, and tri-phosphate. activity of TDP in the thymus of rats injected with TdR-14C was leukemic cells were mature and similar to normal cells and that 25% of its precursor, TMP. in only the remaining 25% of these cells did the composition of 6) In a previous study 36% of the newly synthesized DNA- TMP reflect the composition of newly synthesized DXA-thy- thymine was found to be derived from TdR at a concentration of mine.4 0.3 MMand 87 % at a TdR concentration of 300 AIM(8).From these TURNOVER OF THE THYMINE NUCLEOTIDE POOLS. It has been data and the data in Chart 4 it was calculated that the contribu shown that the size of the thymine polyphosphate pool is a func tion to the TDP and TTP pools from synthesis de novo was con tion of the exogenous TdR concentration (Chart 7 in Ref. 8). stant at all TdR concentrations (8). A similar calculation applied The rate at which each pool reaches equilibrium with its precur to the TMP pool derived from TdR yielded results which indi sor derived from TdR is dependent on both the rate of expansion cated that the TMP pool from synthesis de novo increased in the presence of increasing TdR concentrations. However, the TMP 4 Because of the small size of the TDP and TTP pools of normal pool from synthesis de novo was found to be constant when these cells compared with CML cells, a similar calculation did not sig data were recalculated with the consideration that 75% of the nificantly alter the data obtained in CML cells.

NOVEMBER 1966 2279

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0 15 20 IO 15 20 IO 15 20 t t t MINUTES CHART5. Turnover of the thymine nucleotide pools in leukemic leukocytes. Data from 2 separate experiments using cells from dif ferent donors are plotted together. â€¢Control cells incubated from 0 time with 14JIMthymidine (TdR) containing 200/ic TdR-3H/mmole TdR; A, value for control cells at 15 min; all other points are expressed as a % of this; d, cells incubated with 14 /ÃŒMTdRcontaining 800/ic TdR-3H/mmole TdR beginning at 0 time. At 10 min nonradioactive TdR was added, maintaining the TdR concentration at 14 itti but lowering the specific activity of the TdR-3H to 200//c/mmole TdR. O, Cells incubated with unlabeled 14/Â»MTdRfrom 0 time. At 10 min TdR-3H was added, maintaining the TdR concentration at 14/IM,but raising the specific activity of TdR-3H to 200/ic/mmole. Analysis performed as in Methods. Incubations were at 37Â°C.TMP, TDP, and TTP are thymidine mono-, di-, and tri-phosphate. of the total pool and its rate of turnover. Chart 5 demonstrates to TMP was suggested by IvÃ©s,whodescribed phosphomonester- the turnover rates of these pools in CML leukocytes. In this ex ase and nucleoside triphosphatase activity in extracts of Novikoff periment control cells were incubated with TdR-3H at a concen hepatoma but only marginal nucleoside diphosphatase activity tration of 14 /UM.Experimental cells were incubated with an equal (12). Deoxynucleotide monophosphatase activity is greatest in concentration of TdR in the absence of TdR-3H or with TdR-3H cells not actively synthesizing DNA (2, 14). at a specific activity 4 times that of the control. After 10 min, Previous studies (8) demonstrated that the rate of TdR in when TdR incorporation into the TDP and TTP pools was maxi corporation into DNA was constant at TdR concentrations be mal, the specific activity of TdR in each of the 2 experimental tween 0.5 and 300 pM. The data in Chart 4 can be used to deter cultures was adjusted to equal that of the control. The size of the mine that, in CML leukocytes, the amount of TdR-3H thymine nucleotide pools remained constant. Rapid turnover of incorporated into DNA in 30 min is 3 times the size of the TTP the thymine nucleotide pools is indicated by the rate at which pool derived from TdR at a concentration of 14 JUM.Sinceat this the radioactivity of these pools reached a new equilibrium as a concentration the TTP pool turns over every 3 min, approxi consequence of the change in the specific activity of extracellular mately 35% of the total turnover results from the incorporation TdR-3H. From these data the half life of thymine nucleotides in of TTP into DNA. the presence of 14 /UMTdR was 60-90 sec. Equilibration of the TDP and TTP pools with TdR-3H introduced at 10 min was References slightly slower than in the control. This probably resulted from 1. Behki, R. M., and Morgan, W. S. Studies on the Phosphoryla- expansion of the TMP pool during the 1st 10 min of incubation. tion of Thymidine in Regenerating Rat Liver. Arch. Biochem. Turnover of the TTP pool could result from both incorporation Biophys., 107: 427-34, 1964. of TTP into DNA and dephosphorylation of TTP to TDP. Turn 2. Beltz, R. E. Comparison of the Content of Thymidylate Syn- over of the TMP and TDP pools could result from both phos- thetase, Deoxycytidylate Deaminase, and Deoxyriboncleoside phorylation and dephosphorylation. Degradation of thymine nu Kinases in Normal and Regenerating Rat Liver. Ibid., 99: cleotides by phosphatases has been described in mammalian 304-12, 1962. tissues (9, 12, 17). That TDP once formed is not readily degraded 3. Bianchi, P. A., Butler, J. A. V., Crathorn, A. R., and Shooter,

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K. V. The Thymidine Phosphorylating Kinases. Biochim. 11. Grav, H. J., and Smellie, R. M. S. The Mechanism of Forma Biophys. Acta, SS: 123-31, 1961. tion of Thymidine 5'-Triphosphate by Enzymes from 4. Bianchi, P. A., Farina, M. V., and Polli, E. Phosphorylation Landschutz Ascites-Tumor Cells. Biochem. J., 89: 486-91,1963. of Thymidine Diphosphate in Resting and Proliferating Mam 12. IvÃ©s,D. H. Evidence for Thymidine Diphosphate as the malian Cells. Ibid., 91: 323-25, 1964. Precursor of Thymidine Triphosphate in Tumor-Transfer of 5. Bollum, F. J., and Potter, V. R. Nucleic Acid Metabolism in the Terminal Phosphate of Adenosine Triphosphate to Thy- Regenerating Rat Liver. VI. Soluble Enzymes which Convert midyate. J. Biol. Chem., 240: 819-24, 1965. Thymidine to Thymidine Phosphates and DNA. Cancer Res., 13. IvÃ©s,D. H., Morse, P. A., Jr., and Potter, V. R. Feedback 19: 561-65, 1959. Inhibition of Thymidine Kinase by Thymidine Triphosphate. (i. Breitman, T. R. The Feedback Inhibition of Thymidine Ki- Ibid., 238: 1467-74, 1963. nase. Biochim. Biophys. Acta, 67: 153-55, 1963. 14. Maley, F., and Maley, G. F. Nucleotide Interconversions. IV. 7. Bresnick, E., and Karjala, R. J. End Product Inhibition of Activities of Deoxycytidylate Deaminase and Thymidylate Thymidine Kinase Activity in Normal and Leukemic Human Synthetase in Normal Rat Liver and Hepatomas. Cancer Res., Leukocytes. Cancer Res., W: 841-46, 1964. il- 1421-27, 1961. 8. Cooper, R. A., Perry, S., and Breitman, T. R. Pyrimidine 15. Potter, R. L., and Nygaard, O. F. The Conversion of Thymi Metabolism in Human Leukocytes. I. Contribution of Exog dine to Thymine Nucleotides and Deoxyribonucleic Acid in enous Thymidine to DNA-Thymine and Its Effect on Thy- Vivo. J. Biol. Chem., 2S8: 2150-55, 1963. mine Nucleotide Synthesis in Leukemic Leukocytes. Ibid., 16. Silber, R., Gabrio, B. W., and Hnennekens, F. M. Studies on 26: 2265-73, I960. Normal and Leukemic Leukocytes. VI. Thymidylate Synthe 9. Eker, P. Properties and Assay of Thymine Deoxyribonucleo- tase and Deoxycytidylate Deaminase. J. Clin. Invest., 42: 1913-21, 1963. tide Phosphatase of Mammalian Cells in Tissue Culture. J. Biol. Chem., 240: 419-22, 1965. 17. Weissman, S. M., Paul, J., Thomson, R. Y., Smellie, R. M. S., and Davidson, J. N. Thymidine Kinases in Mammalian Tis 10. Gentry, G. A., Morse, P. A., Jr., IvÃ©s,D.H., Gebert, R., and sues. Biochem. J., 76: 1P-2P, 1960. Potter, V. R. Pyrimidine Metabolism in Tissue Culture Cells 18. Weissman, S. M., Smellie, R. M. S., and Paul, J. Studies on the Derived from Rat Hepatomas. II. Thymidine Uptake in Sus Biosynthesis of Deoxyribonucleic Acid by Extracts of Mam pension Cultures Derived from the Novikoff Hepatoma. Can malian Cells. IV. The Phosphorylation of Thymidine. Bio cer Res., 26: 509-16, 1965. chim. Biophys. Acta, 45: 101-10, 1960.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1966 American Association for Cancer Research. Pyrimidine Metabolism in Human Leukocytes: II. Metabolism of the Thymine Nucleotide Pools in Normal and Leukemic Leukocytes

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

Cancer Res 1966;26:2276-2281.

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