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Interaction of Thymidylate Synthetase and Dihydrofolate Reductase Enzymes in Vitro and in Vivo'

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Interaction of Thymidylate Synthetase and Dihydrofolate Reductase Enzymes in Vitro and in Vivo' [CANCER RESEARCH 34, 1619-1626, July 1974] Interaction of Thymidylate Synthetase and Dihydrofolate Reductase Enzymes in Vitro and in Vivo' Mutsufumi Kawai2 and Brian L. Hilicoat Department of Biochemistry, McMaster University, Hamilton, Ontario, L8S 4)9 Canada SUMMARY reactions plays an important role in the chemotherapy of cancer (4, 9). Besides this functional connection between Thymidylate synthetase and dihydrofolate reductase these 2 enzymes, further relationships are suggested by the modify each other's activities in vitro. Folate, dihydrofolate, association of increased thymidylate synthetase activity in and metliotrexate inhibit thymidylate synthetase in the bacterial mutants resistant to antifolate drugs because of absence of dihydrofolate reductase, but dihydrofolate and increased levels of dihydrofolate reductase (2, 6). methotrexate appear to prevent the interaction between the In the present investigation, possible functional and two enzymes, thus increasing the activity of thymidylate physical interaction between the 2 enzymes has been studied synthetase. Dihydrofolate reductase, inhibited by thymidyl using the enzymes purified from the same source and ate synthetase, is also reactivated by deoxyuridine S'-mono measuring the activity of each enzyme in the presence of the phosphate, a substrate of thymidylate synthetase. The other, as well as by microcalorimetric investigations on the former behavior seems significant in vivo, but not the latter. protein-protein interaction. Crude extracts of methotrexate-resistant L 1210 cells, which have high dihydrofolate reductase activity and low thymi dylate synthetase activity, show increased activity of thymi MATERIALS AND METHODS dylate synthetase in the presence of methotrexate. With intact cells, the rate of incorporation of deoxyuridine into Chemicals. Folic acid, tetrahydrofolic acid, NADPH, and DNA is enhanced in the presence of low concentrations of UdR3 were obtained from Sigma Chemical Co., St. Louis, methotrexate. Wild-type L I2 10 cells show neither effect. Mo., while UdR-6-3H and dUMP-5-3H were purchased Methotrexate may thus increase thymidylate synthetase from Amersham-Searle, Arlington Heights, Ill. In some activity in cells possessing relativel@' large amounts of earlier experiments, dUMP-5-3H from Calatomic, Los dihydrofolate reductase and small amounts of thymidylate Angeles, Calif., was used. We prepared dihydrofolate by the synthetase. reduction of folic acid with sodium dithionite (3) and 5 , lO-methylenetetrahydrofolate from tetrahydrofolate by the method of Dunlap et a!. (6). INTRODUCTION Assays. Thymidylate synthetase was assayed by an iso tope method that measured the release of tritium from The de novo synthesis of thymidylate from deoxyuridy dUMP-5-3H into the solvent (13). The final concentration in late and 5 , lO-methylenetetrahydrofolate is catalyzed by the reaction mixture was 0.05 M Tris-HC1, pH 7.5; 0.12 M thymidylate synthetase and results in the formation of 2-mercaptoethanol; 0.05 M MgCl2; 1.67 x l0@ M 5, 10- @ dihydrofolate (Equation A), which in turn is reduced by methylenetetrahydrofolate; 6 x l0 M dUMP-5-3H, 0.56 NADPH under the action ofdihydrofolate reductase (Equa Ci/mmole; and enzyme solution in a total volume of 0.3 ml. tion B). The reaction mixture was incubated at 37°for 45 mm. In each assay, the corresponding mixture without the enzyme dUMP + 5 , lO-methylenetetrahydrofolate. was used as a blank, since tritium exchange from dUMP-5- TMP + dihydrofolate (A) 3H can occur in the absence of the enzyme (14). In this paper 1 unit of activity is taken as l0@ mole of TMP Dihydrofolate + NADPH + H@ . formed per mm. tetrahydrofolate + NADP@ (B) The activity of dihydrofolate reductase was measured by the change in absorbance of NADPH with dihydrofolate at Such reduction is necessary to maintain the pool of 340 nm and 37°in a reaction mixture containing 0. 1 M reduced folate cofactors in the cell. Inhibition of these Tris-HCI, 0.15 M KC1, 1.0 x l0@ M 2-mercaptoethanol, 1.44 x l0@ M NADPH, I x l0@ M dihydrofolate, and the enzyme in a total volume of 1.0 ml (12). One unit of activity 1 This work is supported by Medical Research Council of Canada Grant MT-3380. ofthis enzyme is defined as l0@ mole ofNADPH caused to 2 Fellow of the Medical Research Council of Canada, to whom reprint requests should be addressed. Received December 3, 1973; accepted March 22@1974. 3 The abbreviation used: UdR, deox@yuridine. JULY 1974 1619 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. M. Kawai and B. L. Hillcoat react per mm. The reaction mixture contained additions as each containing 0. 1 ml enzyme solution in 0.05 MTris-HC1, indicated subsequently. In all experiments appropriate pH 7.5. In the reference cells, 2 samples, each 1 ml of the controls were carried out. same buffer solution, were used. The measurement was Purification of Enzymes. Thymidylate synthetase was made at 37°. purified from pig thymus by column chromatography after ammonium sulfate fractionation as described by Gupta and RESULTS Meldrum (10), except that Sephadex G-200 and hydrox yapatite steps were applied after Sephadex G- 100 chroma Change of the Activity of Thymidylate Synthetase tography. Carboxymethyl-Sephadex was not used. The activity of the thymidylate synthetase from the final purifi cation was I .7 units/mI at pH 7.5 and 0.9 unit/ml at pH 8.5. Effect of Folate and Related Compounds. First we exam The specific activity was 4.5 units/mg protein at pH 7.5. med the change in thymidylate synthetase activity in the We purified dihydrofolate reductase from pig thymus by presence of folate, methotrexate, and dihydrofolate. The affinity chromatography using agarose to which methotrex reaction mixture was incubated at pH 7.5 and 37°for 45 ate had been coupled. The ammonium sulfate fraction, 60 to mm, and TMP formed from dUMP was measured. The 85%, was applied to the column as described previously (8). results indicated that thymidylate synthetase was inhibited The activity of dihydrofolate reductase obtained was 4.9 by those compounds tested, as shown in Chart 1. As seen units/mi at pH 7.5 and 3.3 units/mi at pH 8.5. The specific from Chart 1, methotrexate significantly inhibited thymi activity was 66 units/mg protein at pH 7.5. dylate synthetase in the high concentration (ca. 50% inhibi Each enzyme preparation was shown to be free of activity tion by l0@ M methotrexate). Dihydrofolate also inhibited of the other, and each gave a single protein band on the enzyme, although the effect was not as marked as in the polyacrylamide gel electrophoresis. case of methotrexate. Folate also showed some inhibitory Experiments with Intact Cells. The rate of incorporation effect on thymidylate synthetase activity, but the inhibition of UdR into DNA was used as a measure of DNA synthesis was quite weak compared to that of the other 2 compounds via the conversion ofdUMP to TMP, i.e., via the thymidyl tested. These results indicated that an inhibitor of dihy ate synthetase-dihydrofolate reductase pathway. Cells were drofolate reductase, methotrexate, inhibits thymidylate syn chosen that contained different amounts of these enzymes thetase and that dihydrofolate which is a substrate of relative to each other. One cell line was the wild-type clone dihydrofolate reductase inhibits the synthetase also. of the Ll210 line, LS2, and the other was a methotrexate Effect of Dihydrofolate Reductase. Chart 2 shows the resistant clone of the same line, LM4. The activities of the 2 change of thymidylate synthetase activity measured in the enzymes in each cell line are shown in Table 3. Methotrex presence of different concentrations of dihydrofolate reduc ate was used to inhibit dihydrofolate reductase over a range tase at pH 7.5 and 8.5. At pH 7.5, the activity of of concentrations to determine the effect on UdR incorpora thymidylate synthetase decrea;ed with increasing concen tion into DNA. tration of dihydrofolate reduc@ase. In contrast, an opposite UdR Incorporation. The incorporation of UdR-6-3H into effect of dihydrofolate reductase on thymidylate synthetase DNA in LS2 and LM4 cells was determined by precipitat activity occurred when the reaction was carried out at pH ing DNA with acid on a glass filter. Methotrexate was 8.5. The pH for optimumactivityofthymidylatesynthetase added at varying concentrations to 0.98 ml of cell suspension from pig thymus in the absence ofdihydrofolate reductase is (2 x l0@ cells/mi medium) and incubated at 37°for 1 hr. about 7.0 (10). As seen in Chart 2, the activity of Then UdR-6-3H was added to a final concentration of 1 thymidylate synthetase in the absence of dihydrofolate zCi/ml (l0@ M UdR) and the culture incubated at 37°for reductase at pH 8.5 is about 45% less than at 7.5. However, a further 45 mm. The incubation was terminated by adding the activity of the synthetase at pH 8.5 was increased by increasing the concentration ofdihydrofolate reductase, and 5 ml ice-cold 10% trichloroacetic acid, and the mixture was poured through a Whatman GF/A glass filter. The incuba tion flask was washed out with 3 portions of S ml cold 10% trichloroacetic acid, and the precipitate was further washed with 10 ml 70% ethanol and dried. The filter was placed in scintillation fluid, and the radioactivity was counted in a >; 100 Nuclear-Chicago liquid scintillation counter. > Calorimetric Measurement. Calorimetric studies were (6 carried out using an LKB 10700 batch microcalorimeter by 11 .-...... [email protected]. C @ measuring the heat evolved on mixing 2 solutions of I ml, Ir' @,50 A, Methotrexate Chart 1. Activity of thymidylate synthetase in the presenceof various B, concentrations of methotrexate, dihydrofolate, and folate. Thymidylate C, Folate synthetase (10 @il)was incubated in 0. 1 M Tris-HCI, pH 7.5, with the various concentration of folate compounds in a total volume of 0.3 ml as indicated at 37° for 45 mm.
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