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

[CANCER RESEARCH 34, 1619-1626, July 1974]

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 37Â°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 37Â°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

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 37Â°. 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 37Â°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 37Â°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 37Â°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 .-...... @.-. 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 37Â° for 45 mm. A 100% activity (7.6 x l0 10 mole of I @ dihydrofolate or thymidylate formed in 45 mm) represents the activity in 10_o io6 io@ the absenceof the folate compounds tested. Concentration, M

1620 CANCER RESEARCH VOL. 34

100 A, Methotrexate B, Dih@rofoIate >, ._-Q @ > 1OC ..fi.--.__'_ â€˜\ C (6 (5 6) @â€”,-z@ \@ 11 â€˜6 6) @ 50 C I5C>, E>,

0 0.1 0.2 0.4 Dihydrofolate reductase, units C) I I ( I @ 10_s 106 i@-@ 10@ Chart 2. Activity of dihydrofolate reductase on the activity of thymi Concentration.M dylate synthetase at pH 7.5 and 8.5. The activity of thymidylate synthetase Chart 3. The effect of methotrexate, dihydrofolate, and folate on the (10 Ml)was measuredafter incubation with dihydrofolate reductaseat pH activity of thymidylate synthetase in the presence of dihydrofolate 7.5 (0) and 8.5 (S) at 37Â°for 5 mm. The amount of dihydrofolate reductase. After thymidylate synthetase (10 zl) was preincubated with reductase shown is its activity in units, measured in the absenceof dihydrofolate reductase(40 Mi) at pH 7.5 and 37Â°for 5 mm, methotrexate, thymidylate synthetase at corresponding pH. The activity of thymidylate dihydrofolate, or folate, at the concentrations indicated, was added; then synthetase in the absence of dihydrofolate reductase at pH 7.5 is taken as (without further preincubation with methotrexate or other compounds) the 100% (0.017 unit). In this experiment, 2 controls were needed, I in which reaction was started by adding tritium-labeled dUMP to measure the dihydrofolate reductase was added with no thymidylate synthetasepresent activity of thymidylate synthetase. The activity measured in the absence of and 1 in which methylene-THF was added in the absence of thymidylate dihydrofolate and additional folate compounds was taken as 100%. synthetase. or with dihydrofolate and no longer binds to thymidylate the activity increased to the level observed in the absence of synthetase. If this is the case, the decrease of thyrnidylate dihydrofolate reductase at pH 7.5. This indicated a different synthetase activity in high concentrations of methotrexate interaction between these 2 enzymes at the different pH that occurs after the linear increase of activity may be values. Effect of Folate and Its Analogs on the Activity of explained by a direct inhibitory effect of methotrexate on Thymidylate Synthetase in the Presence of Dihydrofolate thyrnidylate synthetase. In contrast to the results obtained for methotrexate and dihydrofolate, treatment with folate Reductase. Since thymidylate synthetase was inhibited both did not give an increased activity of thymidylate synthetase, by folate compounds and by dihydrofolate reductase, it was but rather a slight decrease, perhaps because of the lower of interest to determine whether thymidylate synthetase was affinity of dihydrofolate reductase for folate under these more inhibited when treated with a folate compound (folate, conditions.4 A similar experiment, using extracts of LS2 methotrexate, or dihydrofolate) and dihydrofolate reduc and LM4 cells, is shown in Chart 7. In this case both tase. The experiments were carried out by preincubating enzymes were present in the soluble crude extracts and we thymidylate synthetase with dihydrofolate reductase at pH determined the effect of increasing methotrexate concentra 7.5 and 37Â°for 5 mm. Thenmethotrexate, dihydrofolate, or tion on the activity of thymidylate synthetase. We observed folate was added to the mixture of the 2 enzymes, and the increase in this activity, similar to that with the mixture of activity of thymidylate synthetase was compared to that of pure enzymes, but occurring at a lower concentration (l0@ thymidylate synthetase not treated with either folate com M). The difference may reflect the different ratios of pound or dihydrofolate reductase. Chart 3 shows the result enzymes in the extracts, a species difference or both. observed at different concentrations of the folate corn pound. Under these conditions thymidylate synthetase Change of Dihydrofolate Reductase Activity showed about 60% activity in the presence of dihydrofolate reductase, as previously (see Chart 2). On addition of Inhibition of Dihydrofolate Reductase by Thymidylate methotrexate or dihydrofolate, the activity of thymidylate Synthetase. Dihydrofolate reductase was inhibited when synthetase was not inhibited further but in fact increased preincubated with thyrnidylate synthetase. Chart 4 shows with increasing concentrations of rnethotrexate or dihy the change of activity of dihydrofolate reductase measured drofolate. There thus seems to be an interaction between at pH 8.5 with various concentrations of thyrnidylate thymidylate synthetase and dihydrofolate reductase such

that thymidylate synthetase is no longer inhibited by 4 Folate appears to bind to the same site of dihydrofolate reductase as dihydrofolate reductase when dihydrofolate or methotrex does dihydrofolate; and the affinity of folate for dihydrofolate reductase ate is present. Since these compounds have a strong affinity may be lower than that of dihydrofolate, since folate shows competitive inhibition of dihydrofolate reductase with respect to dihydrofolate and for dihydrofolate reductase, it might be that dihydrofolate gives an apparent K1 for folate at 1.8 x l0@ M compared with an apparent reductase dissociates from thymidylate synthetase when Km for dihydrofolate of 5.3 x 10@ M at pH 7.5 (M. Kawai, unpublished dihydrofolate reductase forms a complex with methotrexate observations).

JULY 1974 1621

reductase increased until, at 1 x l0@ MdUMP, the activity approximated the original activity of dihydrofolate reduc tase. Such an effect was time dependent, as shown in Chart 6 when various incubation times were used with a constant > concentration of dUMP. In this experiment, the concentra tion of dUMP was 1 x l0@ M. As seen in Chart 6 the recovery of dihydrofolate reductase activity increased with increasing incubation times. 6) Inhibition by 5, 10-Methylenetetrahydrofolate. It also seemed important to determine the effect of substrates and 2 products of 1 enzyme on the activity of the other enzyme. >â€˜ a Among those tested were 5 , lO-methylenetetrahydrofolate and dUMP. dUMP was found to have no effect on Thymidylate synthetase, units dihydrofolate reductase, but methylenetetrahydrofolate in Chart 4. Effect of thymidylate synthetase on the activity of dihydrofo hibited dihydrofolate reductase. As seen in Table 1, meth late reductase. After 10 @lof dihydrofolate reductase (0.03 unit) were ylenetetrahydrofolate, a substrate of thymidylate synthe preincubated with different amounts of thymidylate synthetase in the tase and a product of the serine transhydroxymethylase-cat presence of NADPH at 37Â° and pH 8.5 for S mm, the activity of dihydrofolate reductase in the mixture was measured. No change in the concentration of NADPH occurred until dihydrofolate was added.

synthetase. Similar results were obtained at pH 7.5 although 100 the decrease of the activity was less, about one-fourth of that at pH 8.5. The results again indicate an interaction @ between thymidylate synthetase and dihydrofolate reduc 80 6) tase as suggested by the change of thymidylate synthetase (5 0 activity in the presence ofdihydrofolate reductase (Chart 2). â€”4 It may be that the 2 enzymes interact with each other, so 60 C that access of substrates to the active sites is hindered or a 3 0â€”1/i@-@ â€˜ conformational change may occur to forms with decreased catalytic activity. [dUMP] , M Recovery of Dihydrofolate Reductase Activity Inhibited by Chart 5. The recovery of dihydrofolate reductase activity in the Thymidylate Synthetase when dUMP Is Present. In the presence ofthymidylate synthetase with increasing amounts ofdUMP. The experiment above, we have observed that methotrexate mixture of dihydrofolate reductase (10 al), thymidylate synthetase(20 gil), restores the activity of thyrnidylate synthetase when its and NADPH in Tris-HCI buffer, pH 8.5, was incubated at 37Â°for 5 mm. activity was inhibited by dihydrofolate reductase. Under On addition of dUMP, the mixture was further incubated for 5 mm; then these conditions, thymidylate synthetase may be dissociated the activity of dihydrofolate reductase was measured. The activity of from dihydrofolate reductase when a dihydrofolate reduc dihydrofolate reductase alone was taken as 100%. Control experiments consisted of reactions omitting thymidylate synthetase. tase-methotrexate complex is formed. In a similar way, when dU M P reacts with thymidylate synthetase, dihydrofo late reductase activity might no longer be inhibited by the thymidylate synthetase-dUMP complex. We therefore cx amined the effect of dUMP on the activity of dihydrofolate reductase in the presence of thymidylate synthetase. First we measured the increase ofdihydrofolate reductase activity after adding various concentrations ofdUMP to the 2 enzymes. Dihydrofolate reductase (10 jul) and thymidylate synthetase (20 jfl) were preincubated for S mm and dUMP was added. After a further 5 mm, the dihydrofolate reductase activity was measured by adding dihydrofolate. The interaction between thymidylate synthetase and dihy drofolate reductase was found to reach equilibrium within 5 mm because further decrease of the activity of dihydrofolate reductase was not observed when the incubation time for the 2 enzymes was prolonged more than 5 mm. The results are Time, mm shown in Chart 5. In the absence of dUMP, dihydrofolate Chart 6. Time dependence of the recovery of dihydrofolate reductase reductase mixed with thymidylate synthetase showed ca. activity in the presence of thymidylate synthetase and dUMP. The 60% of the dihydrofolate reductase activity measured in the procedure is the same as in Chart 5, except that a fixed concentration of absence of thymidylate synthetase at pH 8.5. With increas dUMP (1 x lfY5 M) was used and the incubation time with dUMP was ing concentrations of dUMP, the activity of dihydrofolate varied.

1622 CANCER RESEARCH VOL. 34

alyzed reaction, inhibited dihydrofolate reductase at both obtained a positive value when the 2 enzymes were mixed pH 7.5 and pH 8.5. The extent of the inhibition was higher (Table 2, Line 3). For comparison, we used the denatured at pH 7.5 than at pH 8.5. This suggests that the coenzyme, enzymes (heated at 100Â°for 3 mm and the precipitate methylenetetrahydrofolate, formed from tetrahydrofolate homogenized sonically). As indicated in Table 2, Lines 4, 5, by serine transhydroxyrnethylase, might regulate the activ and 6, mixing denatured enzymes did not cause a significant ity of dihydrofolate reductase. heat of the reaction compared to the heat of dilution of each Calorimetric Observation of the Interaction between the 2 denatured enzyme. Enzymes. The heat of reaction caused by the interaction If there is no reaction between thymidylate synthetase between thymidylate synthetase and dihydrofolate reduc and dihydrofolate reductase and Q0b5 is simply due to the tase at pH 7.5 and 37Â° was measured by using a mi dilution, Qob@obtained in mixing the 2 enzymes (Line 3) crocalorimeter. Upon mixing solutions of thymidylate syn would be equal to the sum of Q@b@measured in the dilution thetase and dihydrofolate reductase in the calorimeter, heat of each enzyme (Lines I and 2). However, the heat evolved was evolved that equalled the sum of the heats of dilution of by the combination of the 2 native enzymes, which is the each enzyme plus the heat of reaction between the 2 difference between the values obtained with the dilution of enzymes. The results obtained are listed in Table 2. Here, each enzyme (Lines 1 and 2) and the mixture of the 2 the experimental heat of reaction, [email protected] as positive enzymes (Line 3), is 11.6 x l0@ joule under the condition for the heat liberated. The heat of dilution of individual tested. This difference in the heat suggests that the state in enzymes was negative in sign (Lines 1 and 2), while we which the 2 enzymes interact with each other, e.g., in forming a â€œcomplex,â€•isthermodynamically more stable by Table 1 11.6 x l0@ joule than the state in which thymidylate InhibitoryreductaseControl efftct of methylenetetrahydrof'olate on dihydrofolate synthetase and dihydrofolate exist independently. Since the 100%activity(without 5, lO-methylenetetrahydrofolate) was taken as heat of reaction between the 2 enzymes is exothermic, the at each pH. The assay of dihydrofolate reductase is as described in â€œMaterialsand Methods,â€•with 5, lO-methylenetetrahydrofolateadded.Activity possibility of their association appears higher at a lower temperature. (%) In summary, the increase of Qobs on mixing the native Concentration of5, 10- enzymes is consistent with an interaction between them, methylenetetrahydrofolate8.50 pH 7.5 pH although it is not possible to describe the nature of the 1005x 100 interaction from these findings alone. The further calori 1.86lx104M105M 50Â± metric study of this interaction is in progress. 37Â±3.7 86Â±5.2 Action of Methotrexate in L1210 Cells aAverage Â±S.E.

Table 2 Effect of Metbotrexate on Thymidylate Synthetase Activ Heat of interaction between dihydrofolate reductase and thymidylate ity of the Cell Extracts. Since the effect of methotrexate on synthetase the activity of thymidylate synthetase appears to be related Solutions were of 0.05 M Tris-HCI, pH 7.5. Reactants I and 2 were to the concentration of dihydrofolate reductase, we exam mixed after temperature had equilibrated at 37Â°andthe heat formed, Q0@, med this effect in cell lines LS2 and LM4, which possess was measured. QÂ°@.istaken as positive in sign for heat liberated in the system. different activities of thymidylate synthetase and dihydro folate reductase (Table 3). In vitro studies on crude cx tracts of the cells were carried out as well as in vivo studies on intact cells. LineReactantsÂ°Q@.(l03joule)bI21Dihydrofolate Cell extracts were prepared after centrifuging the crude aloneâ€”4.52Buffer homogenate resulting from sonic disruption and assays reductaseBuffer performed as before with purified enzymes. The results are 1.53Dihydrofolate aloneThymidylate presented in Chart 7. In the extract of LS2 cells (which has synthetaseâ€” lower dihydrofolate reductase activity and higher thymidyl synthetase+4.64DenaturedreductaseThymidylate ate synthetase activity than did LM4 cells), the activity of thymidylate synthetase was lowered by increasing amounts

dihydrofolate Table 3 reductaseBufferaloneâ€”1.25Buffer Thymidylate synthetaseand dihydrofolate reductaseactivities ofLS2 and aloneDenatured LM4 cells thymidylate synthetase-@@06Denatured (units)Â°L52LM4Thymidylate

dihydrofolate thymidylate EnzymeActivity reductaseDenatured synthetaseâ€”0.8 synthetase6.91.4Dihydrofolate a Amounts of enzymes used are 40 @sgthymidylate synthetase and 2. 1 @g reductase13.31780 dihydrofolate reductase,each in I ml solution. Total volume of the reaction mixture is 2 ml. a Activity of the enzyme was determined at pH 7.5 in extracts of cells b Deviation of the measurement is Â±4%. and is expressedper 1O@cells.

JULY 1974 1623

M. Kawai and B. L. Hilicoat

drofolate reductase-methotrexate complex, thus increasing 08 the observed activity of thymidylate synthetase.

DISCUSSION @06 (6 I) The experimental results presented indicate that certain 4) interactions are possible between thymidylate synthetase, 5 0.4 C dihydrofolate reductase, their substrates, and 1 inhibitor, 4) methotrexate, as summarized in Chart 9. Thymidylate (5 synthetase is inhibited by a substrate of dihydrofolate V reductase and dihydrofolate reductase by methylenetetrahy E>( C drofolate which is a substrate of thymidylate synthetase or a product of the reaction catalyzed by serine transhydrox Câ€”.//@ â€˜ I I â€˜ 0 10@ 10@ 106 io@ i@-@ io@ ymethylase. There is an interaction between the thymidylate [Methotrexate]. M synthetase and dihydrofolate reductase enzymes themselves, which is modulated by methotrexate, dihydrofolate, and Chart 7. Effect of methotrexate on thymidylate synthetaseof LM4 and dUMP. The inhibition of thymidylate synthetase from the 152 cell extracts.For the assay,0.1ml of crudeextracts(l0@cells/mi) was other sources by folate compounds has also been reported. used.0, extract of LS2 cells; @,extractof LM4 cells.The enzymeactivity Borsa and Whitemore (5) investigated the inhibitory effect was measured at pH 7.5 and is expressedas units/0. I ml extract. of methotrexate on thymidylate synthetase from Ehrlich of methotrexate added, whereas the activity of thymidylate ascites carcinoma cells and from Escherichia coli and synthetase in the LM4 cell extract increased when the observed that high concentrations of the drug inhibited the concentration of methotrexate was increased up to ca. I x l0@ M, above which it began to decrease. These results are comparable with the results obtained for the effect of methotrexate on thymidylate synthetase activity in the purified enzymes systems as indicated in Charts 1 and 3, i.e., the activity of thymidylate synthetase was inhibited by a high concentration of dihydrofolate reductase, and such

inhibition was overcome by the presence of methotrexate. â€˜0

We also examined the effect of dUMP on the activity of x dihydrofolate reductase in LM4 and L52 extracts, but no E 0. effect was observed at either pH 7.5 or pH 8.5. 0 Effect of Methotrexate on the Incorporation of UdR in LM4 and LS2 Cells. To determine whether an increase of thymidylate synthetase activity occurred in vivo when methotrexate inhibited dihydrofolate reductase, we mea sured the rate of the incorporation of labeled UdR into 10.@ DNA via the thymidylate synthetase pathway. After the [Methotrexate],M cells were preincubated with methotrexate in Roswell Park Memorial Institute Medium 1640 for 60 mm at 37Â°, Chart 8. Effect of methotrexate on the incorporation of UdR-6-3H into DNA by LS2 and LM4 cells. Experimentswere formed as describedin UdR-6-3H was added to the cell suspension and the text. incorporation of the radioactivity at 45 mm was measured using the techniques described in â€œMaterialsand Methods.â€• D I HYDROFOLATE The rate of the incorporation was linear for at least 60 mm. @ @P 7 ;â€œ \ NADPH Chart 8 shows the rate of the UdR incorporation at \7 @----@ various concentrations ofmethotrexate in the LM4 and LS2 cells. The incorporation of UdR in LS2 cell was reduced 1@H@IDYLATEL'@..@J FOLATE with increasing concentrations of methotrexate, except for A@SYNTHETASEI7@1REDuCTAsE perhaps a slight increase at l0 â€˜Â°Mmethotrexate. Corn dUMP\----@ ;1 NADP pared with the LS2 cells, a significant increase in the incorporation was observed in LM4 cells when the concen METHYLENE- @--@â€œTETRAHYDOFOLATE T ETRAH@@AT@,,,,,@/# tration ofmethotrexate in the culture was b_b and l0@ M. At the higher concentrations of methotrexate, the rate of incorporation decreased. These observations, together with GLYCINE SERINE those presented earlier, suggested that thymidylate synthe Chart 9. Reactions (broken lines) that have been demonstrated to tase may be inhibited in vivo by high concentrations of affect interaction between thymidylate synthetase and dihydrofolate reduc dihydrofolate reductase. Appropriate concentrations of tase. Those of possible significance in vivo arc shown by double broken methotrexate form free thymidylate synthetase and a dihy lines. +, activation; â€”,inhibition; MTX, methotrexate.

1624 CANCER RESEARCH VOL. 34

enzyme directly. The inhibition of thymidylate synthetase ofdifferent pH on thymidylate synthetase and dihydrofolate from chicken embryo extracts by dihydrofolate is also reductase from pig thymus is unknown. In vivo, significant known. The inhibition was shown to be competitive against interaction may occur between thymidylate synthetase and

5 â€˜lO-methylenetetrahydrofolate (16). Tetrahydrofolate dihydrofolate reductase with inhibition of the activity of formed from dihydrofolate in the presence of dihydrofolate thymidylate by high levels of dihydrofolate reductase and reductase might also be expected to inhibit thymidylate the recovery of that activity at low concentrations of synthetase. The inhibitory effect of some tetrahydrofolate methotrexate. Chart 9 summarizes those interactions of analogs on thymidylate synthetase has been reported and possible significance in vivo, together with those that have the reduced forms have been found, in many cases, to act as been demonstrated in vitro. more potent inhibitors of thymidylate synthetase, as re In folate-requiring reactions, the interaction of enzyme viewed recently by Friedkin (7). Such an effect of tetrahy may not be restricted to dihydrofolate reductase and drofolate on thymidylate synthetase could be included in the thymidylate synthetase. Recently, MacKenzie (17) sug cyclic system in Chart 9, but demonstrating inhibition of gested the possibility of â€œcomplex-like formâ€•of 3 enzymes thymidylate synthetase by tetrahydrofolate was not possible from pig liver, 5 , lO-methylenetetrahydrofolate dehydro under our conditions since the formaldehyde present con genase, 5 , lO-methenyltetrahydrofolate cyclohydrolase, and verts tetrahydrofolate to 5 , lO-methylenetetrahydrofolate. lO-formyltetrahydrofolate synthetase, which copurified dur Also dihydrofolate reductase appears to be inhibited by ing the purification. Thymidylate synthetase has been shown other folate cofactors. Recently, dihydrofolate reductase to be inhibited also by a protein synthesized after phage 4e from bovine liver was shown to be inhibited, competitively infection of Bacillus subtilis ( I I). The protein inhibitor was with respect to dihydrofolate, by a number of 1-carbon probably not dihydrofolate reductase since it precipitated in derivatives of tetrahydrofolate including methylene-, 10- 40% ammonium sulfate solution. formyl-, and 5-formyltetrahydrofolate (18, 19). Although there is a difference in the ratios of activities of It is difficult, however, to be sure which interactions are thymidylate synthetase to dihydrofolate reductase in LS2 of importance in vivo. For example, the direct inhibition of and LM4 cell lines, the rate of incorporation of UdR was thymidylate synthesis by methotrexate may not be signifi almost the same in both cell lines. When there is a high cant in chemotherapy since these inhibitory concentrations production of dihydrofolate reductase in the methotrexate are too high to be achieved in patients. On the other hand, as resistant cell line, the activity of thymidylate could be we have shown, both in vivo and in vitro concentrations of depressed possibly through the interaction with dihydrofo methotrexate from 106 to lO_8 M can cause an increase in late reductase as we have suggested and the overall activity thymidylate synthetase activity, caused, we suggest, by the of the thymidylate synthetase cycle might be kept relatively reaction of this drug with dihydrofolate reductase, thereby constant by such an interaction. However, the action of decreasing the inhibitory effect of that enzyme on the methotrexate was quite different in these cells and suggests activity of the synthetase. Such an effect could be of that, in chemotherapy, low levels of methotrexate in cells importance in chemotherapy. with high dihydrofolate reductase activity might in fact Although we observe inhibition of dihydrofolate reduc increase the rate of thymidylate synthesis that the drug was tase by thymidylate synthetase and the relief of that intended to inhibit. inhibition in the presence of dUMP, this may not be significant in vivo, since relatively large amounts of thymi dylate synthetase are required to inhibit dihydrofolate reductase by 50% at pH 8.5. Such high levels of thymidylate REFERENCES synthetase relative to dihydrofolate reductase have not yet been found in any cell. Therefore, the inhibition of dihy 1. Aaronson, R. P., and Frieden, C. Rabbit Muscle Phosphofructoki drofolate reductase by thymidylate synthetase and the nase: Studies on the Polymerization. J. Biol. Chem., 247: 7502-7509, recovery of the activity by dUMP may occur with unphys 1972. iological amounts of thymidylate synthetase. Furthermore, 2. Albrecht, A. M., Pearce, F. K., and Hutchison, D. J. Folate Coenzymes in Amethopterin-sensitive and -resistant Strains of Strep the behavior of the enzyme at pH 8.5 may be different from tococcus faecalis: Enzymatic Formation and Metabolic Function. J. that at pH 7.5, because a different type of interaction is Biol.Chem.,241: 1036-1042,1966. apparent between the 2 enzymes at pH 8.5 (Chart 2). Such a 3. Blakley, R. L. Crystalline Dihydropteroylglutamic Acid. Nature, difference may be due to protonation of specific ionizable 188:231-232,1960. groups of these enzymes, which in turn might affect their 4. Blakley, R. L. The Biochemistryof Folic Acid and RelatedPteridines. interaction. Aaronson and Frieden (I), investigating the New York: John Wiley & Sons, Inc., 1969. change of the behavior of rabbit muscle phosphofructoki 5. Borsa, J., and Whitemore, G. F. Studies Relating to the Mode of nase between pH 6 and 8, reported that at pH 6 the enzyme Action of Methotrexate. III. Inhibition of Thymidylate Synthetase in exists in an inactive form and dissociated into its subunit. Tissue Culture Cells and in Cell-Free Systems. Mol. Pharmacol., 5: 318â€”332,1969. Thymidylate synthetase seems to be composed of 2 subunits 6. Dunlap, R. B., Harding, N. G. L., and Huennekens,F. M. Thymidyl in the case ofenzyme from a methotrexate-resistant clone of ate Synthetase from Amethopterin-Resistant Lactobacillus casei. Lactobacillus casei (15) and 4 subunits in the case of enzyme Biochemistry, JO: 88-97, 1971. from pig thymus (V. S. Gupta, private communication). 7. Friedkin, M. Thymidylate Synthetase. Advan. Enzymol., 38: 235-292, The subunits have identical primary structures but the effect 1973.

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8. Gauldie, J., and Hillcoat, B. L. Purification of Tetrahydrofolate from Deoxyuridylate-5-'H in Thymidylate Synthetase Assay. Anal. Dehydrogenase by Affinity Chromatography. Biochim. Biophys. Biochem..58:404-413, 1974. Acta, 268: 35-40, 1972. 15. Loeble, R. B., and Dunlap, R. B. Characterization of the Subunits of 9. Greewald,E. S. CancerChemotherapy.N. Y. J. Med., 72:2541-2556, Thymidylate Synthetase. Biochem. Biophys. Res. Commun., 49: 1972. 1671-1677,1972. 10. Gupta, V. S., and Meldrum, J. B. Purification and Properties of 16. Lorenson, M. Y., Maley, G. F., and Maley, F. The Purification and Thymidylate Synthetase from Pig Thymus. Can. J. Biochem., 50: Properties ofThymidylate Synthetase from Chick Embryo Extracts. J. 352â€”362,1972. Biol. Chem., 242: 3332-3344, 1967. 11. Haslam, E. A., Roscoe, D. H., and Tucker, R. G. Inhibition of 17. MacKenzie, R. E. Co-purification of Three Folate Enzymes from Thymidylate Synthetase in Bacteriophage-infected Bacillus subtilis. Porcine Liver. Biochem. Biophys. Res. Commun., 53: 1088-1095, Biochim. Biophys.Acta, 134:312â€”326,1967. 1973. 12. Hillcoat, B. L., Nixon, P. F.4and Blakley,R. L. Effectof Substrate 18. Rowe, P. B., and Lewis, G. P. Mammalian Fq)ate Metabolism. Decomposition on the Spectrophotometric Assay of Dihydrofolate Regulation of Folate Interconversion Enzymes. Biochemistry, 12: Reductase. Anal. Biochem., 21: 178-189, 1967. 1962-1969,1973. 13. Kammen, H. 0. A Rapid Assay for Thymidylate Synthetase. Anal. 19. Rowe, P. B., and Russel, P. J. Dihydrofolate Reductase. Studies on the Biochem., 17: 553-556, 1966. Activation of the Bovine Liver Enzyme. J. Biol. Chem., 248.' 984â€”991, 14. Kawai, M., and Hillcoat, B. L. Non-enzymatic Exchangeof Tritium 1973.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. Interaction of Thymidylate Synthetase and Dihydrofolate Reductase Enzymes in Vitro and in Vivo

Mutsufumi Kawai and Brian L. Hillcoat

Cancer Res 1974;34:1619-1626.

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