HUMANGGP:024500 - FACTA Search

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Query: HUMANGGP:024500 (thymidylate synthase)
2,970 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to gain insight into the mechanisms affecting combination treatment of tumor cells with interferon and halogenated pyrimidine antimetabolites, in vitro studies of murine colon adenocarcinoma 38 and HL-60 cell lines were undertaken. Interferons exhibited modest antiproliferative effects against these lines with DNA synthesis inhibited greater than RNA greater than protein synthesis, as studied by 3H-precursor incorporation. The adenocarcinoma cell line was considerably more sensitive to recombinant gamma-than to purified alpha/beta-interferon, while HL-60 was slightly more sensitive, in short term studies, to antiproliferative effects of recombinant alpha- than to gamma-interferon. Interferon treatment was further associated with suppression of a hyperdiploid component of the adenocarcinoma cell line, as detected by flow cytometry. Combination treatment of the adenocarcinoma cell line with interferon and halogenated pyrimidines, under 4-day continuous exposure conditions, revealed significant synergy for growth inhibition with gamma- much greater than alpha/beta-interferon and with 5-fluorodeoxyuridine greater than 5-fluorouracil much greater than 5-fluorouridine. Thus, synergy was much greater with the more antiproliferative interferon and with the antimetabolite derivative most likely to lead to thymidylate synthetase inhibition rather than RNA incorporation. Sequential 2-day + 2-day treatment revealed greater synergy when interferon preceded 5-fluorouracil or 5-fluorodeoxyuridine rather than the reverse protocol. The synergy of gamma-interferon and 5-fluorodeoxyuridine could be blocked by thymidine, which bypasses inhibition of thymidylate synthetase. HL-60 also exhibited thymidine antagonized synergistic growth-inhibitory effects of interferon and 5-fluorouracil. Analysis of this interaction by [3H]thymidine incorporation, which can reflect thymidylate synthase inhibition, revealed exaggerated responses of interferon-treated cells to either 5-fluorouracil or 5-fluorodeoxyuridine. These results indicate that interferon-halogenated pyrimidine antimetabolite synergistic interactions may be common to several cell types. Evidence is further presented for a mechanism of synergy entailing enhanced thymidylate synthetase inhibition by the antimetabolite of interferon-treated cells.
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PMID:Interferon effects upon the adenocarcinoma 38 and HL-60 cell lines: antiproliferative responses and synergistic interactions with halogenated pyrimidine antimetabolites. 245 31

The basis for the proliferation-dependent cytotoxicity of methotrexate has been investigated in mice bearing the L5178Y ascites leukemia. Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase tumor cells accumulated 8-fold higher levels of methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing tumor cells were observed in the cellular levels of unmetabolized methotrexate. Intestinal mucosa and bone marrow from non-tumor-bearing mice resembled slowly growing tumor cells and had markedly lower levels of methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of methotrexate polyglutamates in the logarithmically growing tumor cells was consistent with an increased synthesis of methotrexate polyglutamates in these cells. The enhanced methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular methotrexate transport, transmembrane efflux of methotrexate, or hydrolysis of methotrexate polyglutamates. Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells. Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased methotrexate polyglutamate accumulation and thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of methotrexate for tumors with a high growth fraction is a consequence of the rapid rates of both cellular methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by thymidylate synthase.
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PMID:Proliferation-dependent cytotoxicity of methotrexate in murine L5178Y leukemia. 245 28

A method is described herein for the isolation and quantitation of polyglutamates of the thymidylate synthase (TS) inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717) in tumor cells exposed to the drug in vitro. Cells were incubated with 50 microM 3H-CB3717 for 12 h and then disrupted by sonication. CB3717 and its polyglutamates were extracted by boiling in 0.01 M Tris-HCl pH 10. The extract was concentrated by lyophilization and analyzed by reverse phase HPLC (10 x 0.46-cm Polygosil 5-micron C18 column) using linear gradient elution (5-16% acetonitrile in 0.1 M sodium acetate, pH 5, over 15 min, 2 ml/min). Recovery of radioactivity at each stage of the method was greater than 70%. CB3717 and its polyglutamates were identified by co-chromatography with synthetic standards and by inhibition of partially purified TS. Quantitation was by means of radiochemical analysis. The 3H-CB3717 used in these studies was prepared by catalytic tritiation of diethyl-(2-chloro-4-nitrobenzoyl)-L-glutamate followed by consecutive alkylation with propargyl bromide and 2-amino-6-bromomethyl-3,4-dihydro-4-oxoquinazoline hydrobromide. The free diacid was prepared as required by hydrolysis in sodium hydroxide and purified by HPLC. Tritiation in only one position was confirmed by 3H NMR. Following the exposure of L1210 leukemia cells to 50 microM 3H-CB3717 for 12 h the total cellular radioactivity level was approximately 7 microM, of which 27% was present as polyglutamated metabolites with four and five glutamate residues.
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PMID:Development of an assay for the estimation of N10-propargyl-5,8-dideazafolic acid polyglutamates in tumor cells. 246 Nov 14

The authors examined the relationship between the level of thymidylate synthase (TS) and the sensitivity to 5-fluorouracil (5-FU) and UFT, a combined oral preparation of 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur) and uracil in a molar ratio of 1:4. For the studies we used the subrenal capsule (SRC) assay and 15 human gastric cancer tissues. The TS levels were assayed by the ligand-binding technique, using [6-3H]FdUMP. The relative variation of tumor size (delta TuS/TuS0) was calculated to be as follows: delta TuS/TuS0 = [(TuS6 - TuS0)/TuS0] x 100 (%), where TuS6 was the tumor size on day 6 and TuS0 on day 0. The chemosensitivity was considered to be positive when delta TuS/TuS0 in the treated group decreased to below -10%. Decrease in tumor size was marked in case of exposure to UFT (-19.8 +/- 13.0%) (mean +/- standard deviation), compared with that to 5-FU (-9.0 +/- 7.2%), with a statistically significant difference (P less than 0.001). The TS level varied from 1.7 to 30.8 pmol/g gastric cancer tissue and the mean was 7.1 +/- 7.2 pmol/g tissue. A correlation was noted between the TS level and decrease in size of the tumor exposed to 5-FU (r = -0.671) or UFT (r = -0.758): gastric cancer tissue with higher level of TS is more sensitive to 5-FU and UFT than is that with a lower TS level. These findings show that the sensitivity to 5-FU and UFT of gastric cancer tissue is related to the TS level and that UFT shows promise for the treatment of patients with gastric cancer.
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PMID:5-Fluorouracil and UFT-sensitive gastric carcinoma has a high level of thymidylate synthase. 249 66

Exposure of tumor cells to reduced folates before or with the fluoropyrimidines, 5-fluorouracil or 5-fluoro-2'deoxyuridine, results in a substantial increase in the activity of these drugs. Available evidence suggests that the mechanism of this synergism is a kinetic stabilization of complex formed between thymidylate synthase and fluorodeoxyuridylate that also involves a mole of the cofactor for the thymidylate synthase reaction, 5,10-methylenetetrahydrofolate. This effect results in an extended time of depletion of thymidine nucleotides with a resultant increased level of cell death. The biochemical aspects of this interaction are discussed and related to the therapeutics of this combination.
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PMID:Leucovorin enhancement of the effects of the fluoropyrimidines on thymidylate synthase. 252 10

Recent investigations have revealed a significant increase in cytotoxic response to (5-fluoropyrimidine, FP) agents in the presence of the folate folinic acid (CF). It has been suggested that CF provides a source of intracellular reduced folates which, in turn, enhances the inhibition of the cellular target thymidylate synthase (TS) by the FP metabolite 5-fluoro-2'-deoxyuridylate (FdUMP). The extent of variation in the response to FP-CF combinations is unknown but it is an important consideration in view of the utilization of these combinations for the therapy of colorectal carcinoma. In the present study, variation in the response to 5-fluoro-2'-deoxyuridine (FdUrd)-CF combinations was observed between two human colorectal tumor cell lines, RCA and C. The response of both cell lines to FdUrd increased with increasing CF, but the effect was more pronounced in cell line RCA. RCA was 4-fold less responsive than cell line C to FdUrd at low CF concentrations, whereas both cell lines exhibited similar sensitivity at high CF concentrations. RCA accumulated lower levels of TS folate cosubstrates after CF than did C; however, this was not the sole mechanism accounting for the differential response to FdUrd-CF. The two cell lines responded differently to equivalent intracellular levels of 5,10-methylenetetrahydrofolate (CH2H4PteGlu) derivatives, the folate ligands involved in tight-binding inhibition of TS by FdUMP. The differential response to CH2H4PteGlu was not due to lack of folate polyglutamation; the predominant CH2H4PteGlu derivative in both cells was the hexaglutamate form. The difference in response to CH2H4PteGlu was associated with a reduction in the affinity of the RCA TS for CH2H4PteGlu, relative to the C enzyme. Thus, a cell line has been identified that responds poorly to FdUrd at physiological levels of CF and that contains a variant TS enzyme.
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PMID:Modulation of 5-fluoro-2'-deoxyuridine response by folinic acid in human colonic tumor cell lines: the role of thymidylate synthase. 252 18

The antitumor effects of tegafur and UFT, fluorinated pyrimidines, were investigated in endometrial carcinoma bearing mice. Following administration of tegafur and UFT, each at a clinical dosage, the levels of 5-fluorouracil (5-Fu) and the inhibition of thymidylate synthase (TS) activity in the tumor tissue were measured. As a result, the mean levels of 5-Fu in the tumor tissue were approximately six times higher in the UFT group (29.2 +/- 10.7 ng/g) than in the tegafur group (4.9 +/- 4.8 ng/g) (p less than 0.01). The UFT group showed a mean inhibition of TS activity in the tumor tissue significantly higher (61.60 +/- 2.02%) than did the tegafur group (56.08 +/- 4.87%) (p less than 0.05). Hence, not only the higher tumor tissue levels of 5-Fu but also the higher inhibition of TS activity in the UFT group as compared with the tegafur group suggested that UFT had more potent antitumor effect.
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PMID:[Level of inhibition of thymidylate synthase activity and 5-fluorouracil in tumor tissues after administration of UFT or tegafur]. 253 Feb 93

Growth of human adenocarcinomas of the colon and rectum in immunoincompetent mice has allowed for a greater understanding of the interaction of 5-fluorouracil, its metabolism, and mechanism(s) of cytotoxicity under conditions of tumor growth in situ. Conversely, this agent has proven to be a useful tool in defining metabolic characteristics in human colon adenocarcinomas. Analysis of tumor sensitivity to 5-fluorouracil (FUra),5-fluorouridine (FUrd) and 5-fluoro-2'-deoxyuridine (FdUrd) suggests that growth inhibition in vivo is related to a DNA-directed event. Resistance, de novo appears to be a consequence of relatively transient inhibition of the target enzyme thymidylate synthase (dTMP-synthase), which may be a consequence of low concentrations of 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) or its polyglutamate forms within tumor cells in situ. In order to study the relationship between inhibition of dTMP-synthase and growth inhibition, mutant cells deficient in their ability to salvage dThd have been selected, and grown as xenografts. Data suggest that transient inhibition of dTMP-synthase and not dThd salvage is responsible for resistance de novo, and that prolonged inhibition of dTMP-synthase would be a lethal event in vivo. This would predict that a cell lacking dTMP-synthase activity would not be tumorigenic. This has been tested directly by selecting clones of GC3 colon adenocarcinoma cells deficient in dTMP-synthase (TS-) activity. Preliminary data indicate that each of 3 TS- clones is tumorigenic in athymic nude mice. The importance of dTMP-synthase as a target for drug development is discussed with respect to these findings.
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PMID:Biochemical mechanisms in colon xenografts: thymidylate synthase as a target for therapy. 266 84

The efficacy and toxicity of leucovorin 500 mg/m2 administered intravenously (IV) over 30 minutes daily for five days followed in one hour by fluorouracil (5-FU) 375 mg/m2 administered IV daily for five days, each given every 3 weeks, was assessed in 54 previously treated patients with metastatic breast cancer. An overall objective response rate of 24% was achieved (95% confidence interval, 13% to 38%), with an additional 56% of patients maintaining stable disease. Eleven of 12 patients who responded had received previous 5-FU therapy. Toxicity of this regimen included grade 3 diarrhea in 13%, grade 3 or 4 mucositis in 33%, grade 3 or 4 granulocytopenia in 65%, and grade 3 or 4 thrombocytopenia in 19%. Delay of treatment was required for hematologic toxicity in 44 patients. Thirty-eight patients required dose reductions due to toxicity. Biochemical evaluation of tumor biopsy specimens obtained from 17 patients used as their own controls with and without leucovorin was performed. These studies reveal an increased stabilization of the 5-fluorodeoxyuridylate (FdUMP)-thymidylate synthase (TS) folate ternary complex with the addition of leucovorin. There was a 71% +/- 14% occupancy or inhibition of the enzyme with the use of both 5-FU and leucovorin, v 30% +/- 13% for 5-FU alone (P2 less than .037). The percent TS bound in responding patients was substantially higher than in those patients with progressive disease. Finally, the mean total tumor TS pre-therapy in seven patients was 31 fmol/mg compared with a mean of 81 fmol/mg in these same seven patients 24 hours after therapy. This 2.6-fold increase suggests that there is an induction of the enzyme, TS, with 5-FU treatment.
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PMID:Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer. 266 35

Expansion of CH2THF pools in tissues of BALB/c mice bearing s.c.-implanted EMT6 mammary adenocarcinomas was measured after leucovorin administration. Twenty-four mice were treated with leucovorin at doses of 0, 45, 90, or 180 mg/kg/injection x 8 injections spaced over 48 h. Tumor and bone marrow cytosols were assayed for CH2THF by forming ternary complexes with thymidylate synthase and [3H]FdUMP. Tumor CH2THF pools were expanded significantly at the two higher doses. Marrow levels were not different from controls. Groups of tumor bearing mice were treated with saline, leucovorin, 5-fluorouracil or 5-fluourouracil plus leucovorin on an optimal dosage schedule. Measured plus leucovorin on an optimal dosage schedule. Measured from the last day of treatment, these tumors grew to 10 mm root-mean-square diameters in 3.5 +/- 1.4, 5.0 +/- 1.2, 6.5 +/- 1.5, and 9.3 +/- 1.2 days, respectively. Growth rates were significantly different from controls only in the latter two groups.
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PMID:Selective expansion of 5,10-methylenetetrahydrofolate pools and modulation of 5-fluorouracil antitumor activity by leucovorin in vivo. 278 34

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