Thymidylate synthase inhibition in cells with arrested DNA synthesis is not due to an allosteric interaction in the replitase complex

Activity of thymidylate synthase was measured in situ in leukemia cells by tritium release from [5-3H]dUrd. Aphidicolin, an inhibitor of DNA polymerase alpha, but not thymidylate synthase, caused a time dependent inhibition of the enzyme when added to the cells after [5-3H]dUrd. Cells treated with hydroxyurea and aphidicolin in sequence before addition of [5-3H]dUrd had a high initial thymidylate synthase activity that decreased with time. This pattern indicates that thymidylate synthase activity is linked to DNA synthesis; however, its inhibition by drugs that inhibit DNA synthesis may be due to accumulation of thymidine nucleotide(s), rather than to an allosteric interaction in the replitase complex.     

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.     

Simple separation of tritiated water and [3H]deoxyuridine from [5-3H]deoxyuridine 5'-monophosphate in the thymidylate synthase assay

A simple micromethod was developed for the accurate measurement of the activity of dTMP synthase in rat liver crude extracts. The reaction product of dTMP synthase activity assay, i.e., tritiated water, generated by the release of tritium from carbon-5 of [5-3H]deoxyuridine 5'-monophosphate (dUMP), was separated simply by 100% KOH absorption from [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]dUMP during the enzyme reaction. Tritiated water was trapped in three droplets of 100% KOH deposited on the underside of the vessels' lids, while [3H]dUrd remained in the bottom of vessels after absorption of the substrate, [5-3H]dUMP, from the reaction mixture by charcoal treatment. Under standard assay conditions in the crude extract of rat liver, the specific activities of dTMP synthase and dUMP phosphatase were 0.092 +/- 0.002 and 0.351 +/- 0.013 nmol/h/mg protein, respectively. This method was also adapted for dTMP synthase assay in crude extracts of rat hepatoma 3924A. The major advantages of this procedure are the elimination of the phosphatase activity which interferes with the estimation of dTMP synthase activity in crude extracts, one-step separation of 3H2O, high sensitivity (with a limit of detection of 10 pmol of 3H2O production), high reproducibility (less than +/- 4.3%), and capability to measure activity in small amounts of sample (30-45 micrograms protein).     

Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.     

Differential affinities of pyrimidine nucleoside analogues for deoxythymidine and deoxycytidine kinase determine their incorporation into murine leukemia L1210 cells

A number of 5-substituted pyrimidine deoxyribonucleoside (dThd or dCyd) derivatives have been evaluated for their effects on the incorporation of dThd and dCyd into the nucleotide pool and nucleic acids of murine leukemia L1210 cells. Several observations indicate that the dThd kinase and dCyd kinase activity of the cells and the differential affinities of these enzymes for the pyrimidine deoxyribonucleosides determine the incorporation of dThd and dCyd into the cells: (i) dThd and dCyd were not incorporated into mutant L1210 cells deficient in either dThd kinase or dCyd kinase activity; (ii) for a series of 5-substituted dThd and dCyd analogues a strong correlation was found between their inhibitory effects on the incorporation of dThd or dCyd into cell material and their Ki/Km for dThd kinase and dCyd kinase (r = 0.92 and 0.97, respectively); (iii) inhibitors of DNA synthesis (i.e. araC) and RNA synthesis (i.e. actinomycin D) suppressed the incorporation of dThd, most likely due to an inhibitory activity at the dThd kinase level (through the allosteric action of dTTP or slow regeneration of dThd kinase).     

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A

The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea pig lymphocytes incorporate [3H]dThd, [3H]dCyd, [3H]dUrd, [3H]Cyd and [3H]Urd into DNA-thymines and DNA-cytosines in a highly conserved distribution pattern. DNA-thymines were labeled only by dThd and dUrd, while DNA-cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA-thymines and DNA-cytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.     

Methionine synthase assay based on coupling with thymidylate synthase reaction

Methionine synthase reaction may be coupled with thymidylate synthase-catalysed tritium release from [5-3H]dUMP via non-enzymatic reaction of formaldehyde with tetrahydrofolate. A convenient and sensitive assay of methionine synthase activity, based on this principle, is described.     

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after cessation of dihydrofolate reductase activity in stationary versus log phase L1210 leukemia cells.

Previous studies from this laboratory established that the rapid but partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure of L1210 leukemia cells to antifolates cannot be due to direct feedback inhibition of thymidylate synthase by dihydrofolate or any other endogenous folylpolyglutamates when dihydrofolate reductase activity is abolished by antifolates. Rather, the data suggested this preservation of tetrahydrofolate cofactor pools is likely due to a fraction of cellular folates unavailable for oxidation to dihydrofolate. This paper explores the role of cell cycle phase in L1210 leukemia cells in logarithmic versus stationary phase growth as a factor in the rate and extent of tetrahydrofolate cofactor interconversion to dihydrofolate after exposure of cells to the dihydrofolate reductase inhibitor trimetrexate. The S phase fraction was reduced by inoculating L1210 leukemia cells at high density to achieve a stationary state. Flow cytometric analysis of DNA content indicated that log phase cultures were 53.0% S phase; this decreased to 42.1% at 24 h and 24.1% at 48 h in stationary phase cultures. 5-Bromo-2'-deoxyuridine incorporation into DNA decreased 80 and 96%, while [3H]dUrd incorporation into DNA declined 70 and 95% for stationary cultures at 24 and 48 h, respectively, as compared with the log phase rates. Log phase cells interconverted 28.0% of the total pool of radiolabeled folates to dihydrofolate with a half-time of approximately 30 s. Stationary cells at 24 h interconverted 20.4% of the total folate pool with a t1/2 of approximately 3 min, and at 48 h, net interconversion to dihydrofolate decreased further to 12.1% with a t1/2 of approximately 6 min. The decrease in the extent of tetrahydrofolate cofactor interconversion to dihydrofolate in stationary phase cells was directly proportional to the decrease in the S phase fraction determined by total DNA content. This suggests that tetrahydrofolate cofactor depletion occurs only in S phase cells. The much larger drop in [3H]dUrd and 5-bromo-2'-deoxyuridine incorporation into DNA in comparison with the decline in the S phase fraction measured by DNA content along with the reduced rate of tetrahydrofolate cofactor interconversion to dihydrofolate indicates that the rate of DNA synthesis is decreased in S phase cells in stationary cultures. Network thermodynamic simulations suggest that a reduction in the number of S phase cells and their thymidylate synthase catalytic activity would account for the observed decrease in the rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after trimetrexate in stationary phase cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides

The two thymidine (dThd) kinases in human cells, the cytosolic, S-phase-specific TK1 and the mitochondrial, constitutively expressed TK2 were purified to homogeneity as judged from sodium dodecyl sulfate-gel electrophoresis. The substrate specificity of TK1 and TK2 toward natural substrates and important nucleoside analogues was compared. With TK1, the Km values for 5-fluorodeoxyuridine (FdUrd), 3'-azido-2',3'-dideoxythymidine (AZT), and 3'-fluoro-2',3'-dideoxythymidine (FLT) were 2.2, 0.6, and 2.1 microM as compared to 0.5 microM for dThd and 9 microM for deoxyuridine (dUrd). With TK2, dUrd, deoxycytidine (dCyd), and 5-fluorodeoxyuridine (FdUrd) were efficiently phosphorylated, but with distinctly different kinetics: Michaelis-Menten kinetics with dCyd, dUrd, and FdUrd; negative cooperativity with dThd. Negative cooperativity was also observed with AZT, although this drug was a very poor substrate for TK2 with a Vmax of 5-6% of that with dThd. FLT, 2',3'-dideoxycytidine (ddCyd), and arabinofuranosylcytosine (araC) were not substrates for TK2, and 2',3'-didehydrodideoxy-thymidine (D4T) was not a substrate for TK1 or TK2. On the other hand, AZT, FLT, and D4T were competitive inhibitors with Ki values of 0.6, 6, and 2073 microM for TK1, and 2, 10, and 78 microM for TK2, respectively. The much lower tolerance for modifications of the deoxyribose moiety of TK2 as compared to TK1 is important for the design of new antiviral nucleoside analogues intended for use in cells with different expression of TK1 and TK2.     

Thymidylate synthase activity in the development of Hymenolepis diminuta

Extracts of the tapeworm, Hymenolepis diminuta, catalyse N5,10-methylene-tetrahydrofolate-dependent release of tritium from [5-3H]dUMP, indicating the presence of thymidylate synthase. The enzyme activity was found in immature, mature and gravid proglottids, as well as in immature and mature oncospheres. The reaction showed pH optimum at 7.5. Its Michaelis constants were approximately 2 and 15 microM for dUMP and (+/-), L-N5,10-methylenetetrahydrofolate, respectively. Incubation of the tapeworm extracts with 5-F-[3H]dUMP and N5,10-methylenetetrahydrofolate resulted in formation of a labelled complex, separable under conditions of SDS polyacrylamide electrophoresis (mol. wt. of approx. 34,000), corresponding to thymidylate synthase subunit. Results of gel filtration of the above complex, under nondenaturing conditions, pointed to a dimeric structure of the enzyme.     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

The effects on 4-aminoantifolates on 5-formyltetrahydrofolate metabolism in L1210 cells. A biochemical basis of the selectivity of leucovorin rescue

This report describes studies designed to evaluate possible inhibitory effects of diaminoantifolates on folate-dependent biosynthetic enzymes in intact L1210 leukemia cells. A novel approach is described which involves an assessment of the metabolism of and biosynthetic flux of the one-carbon moiety from (6S)5-formyltetrahydrofolate in folate-depleted cells. Pretreatment with methotrexate (10 microM), resulting in the formation of methotrexate polyglutamates, or continuous incubation with trimetrexate (1 microM) inhibited growth of folate-depleted L1210 cells in the presence of folic acid or 5-formyltetrahydrolate. In both control and drug-treated cells, double-labeled (6S)-5-[14C]formyl[3H]tetrahydrofolate was rapidly metabolized with the loss of the [14C]formyl group. Under all conditions, the predominant metabolite was 10-formyl[3H]tetrahydrofolate, detectable both intracellularly and extracellularly. In drug-treated cells, there was a remarkably small decrease in the level of 10-formyl[3H]tetrahydrofolate (approximately 30%) and a 10-fold rise in the level of [3H]dihydrofolate to less than 20% of the total folate pool. The incorporation of [14C]formyl group from 5-[14C]formyltetrahydrofolate into thymidylate, serine, and methionine was unaffected by the presence of 1 microM trimetrexate, consistent with the generation of sufficient 5,10-[14C]methylenetetrahydrofolate to drive these reactions. Similarly, the presence of methotrexate polyglutamates had no effect at the level of amino acid synthesis; however, carbon transfer into thymidylate was markedly inhibited. Even though 10-formyltetrahydrofolate was readily formed from 5-formyltetrahydrofolate in this model, the net incorporation of 14C from 5-[14C]formyltetrahydrofolate into purine nucleotides was inhibited by both methotrexate and trimetrexate treatments. Similar findings were obtained when [14C]glycine incorporation into purine nucleotides was monitored in cells incubated with unlabeled 5-formyltetrahydrofolate. Finally, in antifolate-treated cells incubated with unlabeled 5-formyl-tetrahydrofolate, transfer of 14C from [14C]formate or [14C]serine into biosynthetic products or incorporation of [3H]deoxyuridine into nucleic acids was potently inhibited. These results suggest that insufficient levels of tetrahydrofolate and 5, 10-methylenetetrahydrofolate were formed to drive these reactions despite the presence of high levels of 10-formyltetrahydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)     

A study of the influence of newly synthesized acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline derivatives on deoxythymidine and deoxycytidine kinase activities in human neurofibrosarcoma and ovarian cancer

The influence of nine newly synthesized uracil acyclonucleosides, and 36 derivatives of 1,2,3,4-tetrahydroisoquinoline on the activity of enzymes catalysing dTMP and dGMP synthesis, on the content of dTTP and dGTP in acid soluble fraction and on the incorporation of [14C]dThd and [14C ]dGuo into DNA in tumour homogenates was studied. The influence of the compounds was studied in the cytosol from intraoperatively excised human tumours - neurofibrosarcoma and ovarian cancer. It was shown that dTMP and dGMP synthesis is inhibited competitively by 34.1+/-4.0% in both types of tumours by 0.2 mM 1-N-(3'-hydroxypropyl)-6-methyluracil (1) and 0.2 mM 1-N-(3'-hydroxypropyl)- 5,6- tetramethyleneuracil (2). The mentioned acyclonucleosides reduced the content of dTTP and dGTP in the acid soluble fraction of tumours (59.7+/-3.1% of control). 1-(4-chlorophenyl)-6,7-dihydroxy- 1,2,3,4-tetrahydroisoquinoline (3), 1-(2,3-dichlorophenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (4) and 1-(3-methoxyphenyl)-6,7-dihydroxy 1,2,3,4-tetrahydroisoquinoline (5) at 0.2 mM concentration caused a mixed type inhibition of the synthesis of dTMP and dGMP by, on average, 33.2+/-4.4%, and reduced the content of dTTP and dGTP in the acid soluble fraction (52.6+/-3.7% of control) but were active only in the cytosol of neurofibrosarcoma. While acyclonucleosides undergo phosphorylation in the cytosol by cellular kinases, with their triphosphates being active acyclonucleoside metabolites, active 1,3,4,5-tetrahydroisoquinoline derivatives (compounds not containing a deoxyribose moiety), cannot be phosphorylated. ACN and THI derivatives which inhibit dThd and dCyd kinase activities, inhibit also the incorporation of [14C]dThd and [14C]dGuo (ACN - 50.2+/-2.7%, THI - 53.4+/-3.9% of incorporation inhibition) into tumour DNA. The obtained results point to the mechanism of uracil acyclonucleosides and 1,2,3,4-tetrahydroisoquinoline biological activity consisting in inhibiting the synthesis of DNA components.     

Folate-pool interconversions and inhibition of biosynthetic processes after exposure of L1210 leukemia cells to antifolates. Experimental and network thermodynamic analyses of the role of dihydrofolate polyglutamylates in antifolate action in cells

Folate analogs that inhibit dihydrofolate reductase result in only partial interconversion of tetrahydrofolate cofactors to dihydrofolate with preservation of the major portion of reduced cellular folate cofactors in L1210 leukemia cells. One possible explanation for this phenomenon is that low levels of dihydrofolate polyglutamates that accumulate in the presence of antifolates block thymidylate synthase to prevent depletion of reduced folate pools. This paper correlates biochemical analyses of rapid interconversions of radiolabeled folates and changes in purine and pyrimidine biosynthesis in L1210 murine leukemia cells exposed to antifolates with network thermodynamic computer modeling to assess this hypothesis. When cells are exposed to 1 microM trimetrexate there is an almost instantaneous inhibition of [3H] deoxyuridine or [14C]formate incorporation into nucleotides which is maximal within 5 min. This is associated with a rapid rise in cellular dihydrofolate (t1/2 approximately 1.5 min), which reaches a steady state that represents only 27.9% of the total folate pool. Pretreatment of cells with fluorodeoxyuridine, to inhibit thymidylate synthase by about 95% followed by trimetrexate only slows the rate of folate interconversion (t1/2 approximately 25 min) but not the final dihydrofolate level achieved. This is consistent with computer simulations which predict that direct inhibition of thymidylate synthase by 97, 98, and 99% should increase the half-time of dihydrofolate rise after trimetrexate to 40, 60, and 124 min, respectively, but the final level achieved is always the same as in cells with normal thymidylate synthase activity. The data reflect the high degree of catalytic activity of thymidylate synthase relative to tetrahydrofolate cofactor pools in the cells and the enormous extent of inhibition of this enzyme that is necessary to slow the rate of folate interconversions after addition of antifolates. The model predicts, and the data demonstrate, that virtually any residual thymidylate synthase activity will permit the interconversion of all tetrahydrofolate cofactors available for oxidation to dihydrofolate when dihydrofolate reductase activity is abolished, but the rate of interconversion will be slowed. Additional simulations indicate that the time course of cessation of tetrahydrofolate-dependent purine and pyrimidine biosynthesis after antifolates in these cells can be accounted for solely on the basis of tetrahydrofolate cofactor depletion alone. These data exclude the possibility that direct inhibition of thymidylate synthase by dihydrofolate polyglutamates, or any other intracellular folates that accumulate in cells after antifolates, can account for the rapid but partial interconversion of reduced folate cofactors to dihydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical evidence for the existence of thymidylate synthase in the obligate intracellular parasite Chlamydia trachomatis.

Since eucaryotic cell-derived thymidine or thymidine nucleotides are not incorporated into Chlamydia trachomatis DNA, we hypothesized that C. trachomatis must obtain dTTP for DNA synthesis by converting dUMP to dTMP. In most cells, this reaction is catalyzed by thymidylate synthase (TS) and requires 5,10-methylenetetrahydrofolate as a cofactor. We used C. trachomatis serovar L2 and a mutant CHO K1 cell line with a genetic deficiency in folate metabolism as a host for chlamydial growth. This cell line lacks a functional dihydrofolate reductase (DHFR) gene and, as a result, is unable to carry out de novo synthesis of dTTP. C. trachomatis inclusions form normally when DHFR- cells are starved for thymidine 24 h prior to and during the course of infection. When [6-3H]uridine is used as a precursor to label C. trachomatis-infected CHO DHFR- cells, radiolabel is readily incorporated into chlamydia-specific DNA. When DNA from [6-3H]uridine-labelled infected cultures is acid hydrolyzed and subjected to high-performance liquid chromatography analysis, radiolabel is detected in thymine and cytosine nucleobases. By using the DHFR- cell line as a host and [5-3H]uridine as a precursor, we could monitor intracellular C. trachomatis TS activity simply by following the formation of tritiated water. There is a good correlation between in situ TS activity and DNA synthesis activity during the chlamydial growth cycle. In addition, both C. trachomatis-specific DNA synthesis and 3H2O release are inhibited by exogenously added 5-fluorouridine but not by 5-fluorodeoxyuridine. Finally, we demonstrated in vitro TS activity in crude extracts prepared from highly purified C. trachomatis reticulate bodies. The activity is dependent on the presence of methylenetetrahydrofolic acid and can be inhibited with 5-fluoro-dUMP. Taken together, these results indicate that C. trachomatis contains a TS for the synthesis of dTMP.     

Improved measurement of thymidylate synthetase activity by a modified tritium-release assay

Accurate quantitation of thymidylate synthetase activity using a tritium-release assay is dependent upon measurement of only that tritium released from deoxy[5-³H]uridine monophosphate ([³H]dUMP) during the biosynthesis of thymidylate. Removal of remaining [³H]dUMP on completion of the assay by charcoal adsorption and correction for the nonenzymatic release of tritium are necessary. Although over 99% of [³H]dUMP is removed immediately following addition of charcoal, these studies demonstrate that sufficient [³H]dUMP can remain to prevent accurate measurement of low levels of thymidylate synthetase activity. By delaying measurement of radioactivity for at least 24 h following addition of charcoal, this problem is minimized. To account for nonenzymatic release of tritium, a blank containing enzyme extract with omission of $\text{±,}\text{l}\text{-5,10-}\text{methylenetetrahydrofolate}$ is demonstrated to be more effective than the commonly used blank in which water is substituted for enzyme extract. In samples containing 5-fluoro-2′-deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthetase activity, an alternative blank containing a high concentration of FdUMP (approximately 1mM) is useful in demonstrating a theoretical maximal or complete inhibition of thymidylate synthetase activity.     

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthase

The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thymidylate synthetase-positive and -negative murine mammary FM3A carcinoma cells as a useful system for detecting thymidylate synthetase inhibitors

The murine mammary FM3A/O and the thymidylate (dTMP) synthetase-deficient FM3A/TS^? carcinoma cell lines can be considered as a novel and useful test system for the detection of nucleoside analogues which are directly aimed at the thymidylate synthetase. These compounds should be inhibitory for FM3A/O but not for FM3A/TS^? cells, and their inhibitory effects on FM3A/O cell growth should be readily reversed by exogenous dThd within the concentration range of 5–20 μM.