Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:024500 (thymidylate synthase)
 2,970 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The formation and stability of the covalent ternary complex formed between thymidylate synthase (E.C. 2.1.1.45), 5-fluoro 2'-deoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) has been examined in cytosols derived from xenografts of human colon adenocarcinomas. The rate of association (ka) for FdUMP was low being between 3.4 +/- 0.9 and 10.2 +/- 2.6 X 10(6) M-1 min-1, with the lowest ka value being determined in cytosols from a tumor (HxELC2) which has demonstrated some sensitivity to 5-fluoropyrimidines. Relative to reported ka values for human leukemic cells, the rate of association of FdUMP was 20- to 59-fold lower. This difference is not a consequence of FdUMP catabolism, or metabolism of CH2-H4PteGlu. In cytosols the apparent Km values for dUMP (3.6-4.2 microM) and and [6RS]- CH2-H4PteGlu (25-26.7 microM) were similar to reported values for human enzyme. Data derived from cytosols were similar to those derived using affinity purified enzyme from HxVRC5 colon adenocarcinoma xenografts. The net dissociation of [6-3H] FdUMP from the covalent ternary complex was 31-33 min in the absence of added CH2-H4PteGlu, and the rate of dissociation was dependent upon the concentration of cofactor. The concentration of [6RS]-CH2-H4PteGlu required to stabilize ternary complex derived from HxELC2 cytosols was slightly lower than that required for the same degree of stabilization of complex formed in cytosols from resistant tumors (HxGC3,HxVRC5). Addition of 5-CHO-H4PteGlu, 5-CH3-H4PteGlu, H2PteGlu, and PteGlu did not stabilize the covalent complex, but H4PteGlu substituted for CH2-H4PteGlu.
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PMID:Binding of 5-fluorodeoxyuridylate to thymidylate synthase in human colon adenocarcinoma xenografts. 373 54

We have identified amplification of the thymidylate synthase gene in a colonic tumor that had developed resistance to 5-fluorouracil/leucovorin combination chemotherapy. The tumor had previously undergone a partial response to this combination but began to progress following a prolonged period of continuous therapy and relative disease stabilization. Since thymidylate synthase is a target enzyme for 5-fluorouracil, it is likely that the observed gene amplification is responsible for the resistance. Thus, gene amplification may be a relevant mechanism of acquired resistance to fluoropyrimidine chemotherapy in the clinic.
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PMID:Thymidylate synthase gene amplification in a colon tumor resistant to fluoropyrimidine chemotherapy. 381 93

A human T-lymphoblast cell line, CCRF-CEM/R1, resistant to methotrexate by virtue of increased dihydrofolate reductase activity, was grown in stepwise increasing concentrations of methotrexate. This additional selection pressure resulted in a cell line, CCRF-CEM/R2, resistant to methotrexate by virtue of both an elevation of dihydrofolate reductase activity and a marked decrease in methotrexate transport. The R1 and R2 cells were approximately 70- and 350-fold more resistant to methotrexate than were the parent cells. The effects of three folate antagonists were studied on these cell lines and also on CCRF-CEM/R3 cells, characterized by impaired methotrexate transport but normal levels of dihydrofolate reductase. The elevated reductase subline CCRF-CEM/R1 was cross-resistant to triazinate [Baker's antifol, NSC 139105; ethanesulfonic acid compounded with alpha-(2-chloro-4-[4,6-diamino-2,2-dimethyl-S-triazine-1-(2H)-yl] phenoxyl)-N,N-dimethyl-m-toluamide (1:1)] and trimetrexate (NSC 249008, JB-11, TMQ; 2,4-diamino-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline), two nonclassical folate antagonists. In contrast, the transport defective subline, CCRF-CEM/R3 was not cross-resistant to these two compounds. In cells resistant to MTX by virtue of both mechanisms, CCRF-CEM/R2, triazinate, and trimetrexate were partially cross-resistant. All three methotrexate-resistant sublines showed minor cross-resistance to isoaminohydroxyquinazoline (IAHQ, NSC 289517; 5,8-dideazaisopteroylglutamate), a folate antagonist inhibitor of thymidylate synthase. These data demonstrate that methotrexate-resistant tumor cells may be effectively inhibited by antifolates with different route of entry into cells or with different enzyme targets.
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PMID:Cytotoxic effects of folate antagonists against methotrexate-resistant human leukemic lymphoblast CCRF-CEM cell lines. 385 84

Dihydrofluorouracil (FUH2), the initial catabolite of 5-fluorouracil (FUra), was examined to determine whether this derivative had antitumor activity or host cell (bone marrow) toxicity. Studies were undertaken with Ehrlich ascites tumor and bone marrow cells isolated from CF-1 mice. Cells were exposed for 1 h either to no drug (control) or to varying concentrations, ranging from 1 to 250 microM, of either FUra, FUH2, or alpha-fluoro-beta-alanine. Cells were then cultured and colony formation was assessed after 10 to 14 days. Ehrlich ascites tumor cells were more sensitive to FUra [50% lethal dose (LD50) = 18 microM] than to FUH2 [LD50 = 50 microM], with no sensitivity to alpha-fluoro-beta-alanine even at 250 microM. Bone marrow cells had a toxicity profile similar to that of FUra (LD50 = 10 microM) but were relatively insensitive to FUH2 (LD50 greater than 250 microM), with no sensitivity to alpha-fluoro-beta-alanine. Subsequent studies examined colony formation of the human breast carcinoma cell line MCF-7 following 1 h exposure to varying concentrations of FUra and FUH2. These cells were less sensitive to both FUra (LD50 approximately 80 microM) and FUH2 (LD50 approximately 350 microM). Initial studies on the mechanism of toxicity of FUH2 demonstrated that this FUra catabolite could produce inhibition of thymidylate synthase activity in Ehrlich ascites tumor cells with a pattern similar to that resulting from exposure to FUra. This is the first study to demonstrate that FUH2 (a quantitatively important catabolite of FUra) is cytotoxic, and it suggests that FUH2 may contribute to the toxicity of FUra in vivo, possibly by being anabolized to FUra.
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PMID:Dihydrofluorouracil, a fluorouracil catabolite with antitumor activity in murine and human cells. 392 43

Changes in reduced folates upon exposure of Krebs ascites cells and L1210 murine leukemia cells to methotrexate (MTX) have been measured by stoichiometric entrapment of tissue methylenetetrahydrofolate into a stable ternary complex with thymidylate synthase and tritiated 5-fluoro-2'-deoxy-uridine-5'-monophosphate. Tetrahydrofolate and 5-methyltetrahydrofolate were determined after conversion to methylenetetrahydrofolate. In both tumor cell lines, treatment with methotrexate at levels which had little effect on methylenetetrahydrofolate and tetrahydrofolate concentrations resulted in nearly complete elimination of the methyltetrahydrofolate pool. Thus, an initial effect of methotrexate on folate metabolism appears to be on methyltetrahydrofolate.
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PMID:Effects of methotrexate on folates in Krebs ascites and L1210 murine leukemia cells. 394 80

Synergy of sequential MTX and 5-FU has been shown in several in vitro and in vivo systems. In the present study the influence of time interval between MTX and 5-FU and MTX dose on 5-FU accumulation in tumor cells has been examined in Sarcoma 180 in vivo. There was a clear relationship between MTX dose applied and amount of 5-FU detected in the acid-soluble fraction, the RNA fraction and the thymidylate synthase complex fraction. Also, the MTX-5-FU time interval affected clearly the amount of 5-FU detected in all three fractions, the optimum time interval being 8-12 hr. The results indicate that for sequential application of MTX and 5-FU selection of an adequate MTX dose and a sufficient time interval is crucial to achieve synergistic action.
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PMID:The effect of methotrexate pretreatment on 5-fluorouracil kinetics in sarcoma 180 in vivo. 401 17

Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolated:dUMP C-methyltransferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP), and poly(gamma-glutamyl) conjugates of pteroate and methotrexate (MTX) has been examined as a basis for the sequence-dependent synergism of the 5-fluorouracil-MTX combination in inhibiting viability of L1210 murine tumor cells. A 1.4-log (25-fold) increase in the inhibition of soft agar colony formation was observed when MTX preceded 5-fluorouracil as compared to the reverse sequence. L1210 cells converted 39% of the total intracellular MTX into MTX poly(gamma-glutamate)s within 4 hr of exposure to 1 microM MTX. MTX and MTX(gamma-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and I-5,10-methylenetetrahydropteroylpentaglutamate stoichiometric binding of FdUMP to two sites on thymidylate synthase was observed. The dissociation constants for FdUMP in the ternary complexes formed in the presence of MTX, MTX(gamma-glutamate), 7,8-dihydropteroylpentaglutamate, and I-5-10-methylenetetrahydropteroylpentaglutamate were estimated to be 370, 27, < 10, and < 10 nM, respectively, by equilibrium dialysis. We propose that the sequence-dependent effect of MTX plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.
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PMID:5-fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyridylate binding to thymidylate synthase by dihydropteroylpolyglutamates. 616 May 78

5-Formyltetrahydrofolate was found to reverse the binding of methotrexate to dihydrofolate reductase in the Ehrlich ascites tumor in vitro. When cells pretreated with methotrexate were resuspended in methotrexate-free buffer containing 5-formyltetrahydrofolate (or 5-methyltetrahydrofolate), net dissociation of the antifolate from the enzyme was observed. Methotrexate associated with the enzyme under these conditions was below the enzyme binding capacity. However, glucose or azide increased the fraction of dihydrofolate reductase associated with methotrexate and abolished the effect of tetrahydrofolates on this intracellular component. Addition of 5-fluoro-2'-deoxyuridine had no effect on this response to the reduced folate, thereby precluding a direct role for the thymidylate synthase-dependent generation of dihydrofolate in this dissociation of methotrexate from dihydrofolate reductase. Enzyme-bound methotrexate could also be reduced by exposure to 5-formyltetrahydrofolate prior to uptake and efflux of free methotrexate. When cells were incubated under conditions which favored formation of methotrexate polyglutamate derivatives, subsequent treatment with 5-formyltetrahydrofolate had no effect on the binding of the conjugated antifolate to dihydrofolate reductase. These findings support a role for dihydrofolate reductase as a locus for competitive binding interactions between reduced folates and methotrexate that may be a basis for the ability of 5-formyltetrahydrofolate to prevent the biochemical effects of this antifolate. These data suggest that the presence of methotrexate polyglutamate derivatives and cellular energy metabolism may be critical determinants of the responsiveness of methotrexate-treated cells to reduced folates and may play important roles in the selectivity of 5-formyltetrahydrofolate rescue.
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PMID:Role of methotrexate polyglutamylation and cellular energy metabolism in inhibition of methotrexate binding to dihydrofolate reductase by 5-formyltetrahydrofolate in Ehrlich ascites tumor cells in vitro. 618 86

Computer modeling has been a valuable tool for clarifying the mechanism of action of antifolates. Some consequences of folyl and antifolyl polyglutamate synthesis can be addressed by adaptation of a network thermodynamic computer model of methotrexate action. Reversal or prevention of methotrexate cytotoxicity by 5-formyltetrahydrofolate has widely been assumed to occur through the delivery of reduced folate in substrate amounts for thymidylate synthesis, by-passing the effects of methotrexate at dihydrofolate reductase. This mechanism is inconsistent with experimental data which shows that "rescue" is a competitive phenomenon and that the transport process is incapable of delivering reduced folate at an adequate rate. Computer modeling studies are presented which predict that expansion of the total folate pool as folylpolyglutamates with "rescue" would reduce the inhibitory effect of MTX on thymidylate synthesis. Dihydrofolate polyglutamates could then accumulate to the high level needed to displace methotrexate from the small fraction of sites on dihydrofolate reductase that are sufficient to sustain tetrahydrofolate synthesis. Experimental studies with Ehrlich ascites tumor cells support this prediction. It is likely that a critical step in the protection of normal host tissues in high dose-rescue treatment regimens is the conversion of exogenously supplied 5-formyltetrahydrofolate to polyglutamyl derivatives and accumulation of total intracellular folate to higher than normal levels. Other computer simulations are presented which examine the potential significance of direct inhibition of thymidylate synthase by polyglutamyl forms of methotrexate. The model predicts that in cells with biochemical properties similar to methotrexate sensitive L1210 cells, inhibition of dihydrofolate reductase would still be the predominant site of action unless the thymidylate synthase Ki for a methotrexate polyglutamate is below about 0.1 microM. However, in methotrexate-resistant cells with elevated dihydrofolate reductase but normal membrane transport and polyglutamylation, thymidylate synthase may be the more important target enzyme.
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PMID:Predictions of a network thermodynamics computer model relating to the mechanism of methotrexate rescue by 5-formyltetrahydrofolate and to the importance of inhibition of thymidylate synthase by methotrexate-polyglutamates. 619 92

A close analogue of the antileukemic agent 5,8-dideaza-N10 propargylfolic acid (2) was synthesized by replacing the propargyl moiety of 2 with a cyanomethyl group. This compound, N10-(cyanomethyl)-5,8-dideazafolic acid (3), was evaluated for its antifolate and antitumor activities in several biological test systems. Alkylation of diethyl N-(4-aminobenzoyl)-L-glutamate with bromoacetonitrile gave diethyl N-[4-[(cyanomethyl)amino]benzoyl]-L-glutamate (7). Reaction of 7 with 2 amino-6-(bromomethyl)-4-hydroxyquinazoline (9) in dimethylacetamide gave the corresponding diethyl ester 11, which was hydrolyzed to the target compound 3. The known antileukemic agent 2 was also synthesized for comparative studies by employing a modified procedure, which resulted in a better yield of this product. Both compounds 2 and 3 were evaluated for their antifolate activities by using two folate-requiring microorganisms, Streptococcus faecium and Lactobacillus casei. They were further evaluated as inhibitors of thymidylate synthase and dihydrofolate reductase derived from the above organisms, as well as for their antitumor activity by using selected tumor cells in culture. Compound 2 was found to be as equally potent as methotrexate (MTX) against S. faecium, and it was an excellent inhibitor of L. casei thymidylate synthase. The cyanomethyl analogue 3 was less active than 2 in all the test systems, except the inhibition of dihydrofolate reductase.
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PMID:Folate analogues. 21. Synthesis and antifolate and antitumor activities of N10-(cyanomethyl)-5,8-dideazafolic acid. 640 10


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