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)

Plasma pharmacokinetics of high-dose (500 mg/m2) leucovorin calcium (dl-5-formyltetrahydrofolic acid [dl-CF]) and fluorouracil (FUra) have been evaluated in patients with advanced colorectal cancer treated with the combination of FUra and dl-CF by two different intravenous (IV) schedules: (A) In patients with no prior chemotherapy, dl-CF was administered by a two-hour IV infusion and FUra by rapid IV injection one hour after the start of the dl-CF infusion and (B) in previously treated patients, dl-CF and FUra were administered by five-day continuous IV infusion (CI). Following the two-hour infusion of dl-CF, mean peak plasma concentration and elimination half-life of I-5-formyltetrahydrofolic acid (I-CF) were 24 +/- 6 mumol/L and 0.8 +/- 0.1 hour, respectively. CI of dl-CF over five days yielded a mean steady-state plasma level of I-CF of only 1.2 +/- 0.5 mumol/L. Peak and steady-state plasma concentrations of the metabolite 5-methyl tetrahydrofolic acid were comparable in the two schedules (17 +/- 8 mumol/L for the two-hour infusion and 12 +/- 5 mumol/L for the CI). Areas under the concentration v time curve (AUC) of total reduced folates were significantly greater under conditions of CI: 89.0 v 16.7 mmol/L/min for the two-hour infusion. In tumor tissue, 5,10-methylenetetrahydrofolate increased eight-fold two to four hours following the two-hour infusion and two-fold during the CI of dl-CF and FUra. Inhibition of thymidylate synthase (dTMP-S) by the two-hour and CI infusion schedules were 66% v 39%, respectively. The observed differences in the intracellular dTMP-S folate cofactor pools and the degree of inhibition of dTMP-S achieved in patients treated by two different schedules may be due to differences in the biochemical properties and/or to differences in the modulation of FUra metabolism by folate of tumor tissues obtained from newly diagnosed and previously treated patients.
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PMID:Plasma and tumor tissue pharmacology of high-dose intravenous leucovorin calcium in combination with fluorouracil in patients with advanced colorectal carcinoma. 326 Jun 21

About 30 kinds of biochemical modulators of 5-fluorouracil (5-FU) are described. They modify the metabolism and cytotoxic action of 5-FU through an alteration in (1) intracellular concentration of 5-FU or its metabolites, (2) intracellular pool of co-substrates essential for 5-FU metabolism, and (3) intracellular pool of normal substrates which compete with 5-FU metabolism. As a result, they affect either its inhibitory effect on thymidylate synthase or incorporation into RNA, or both. Thus, some of these modulators can improve antitumor activity of 5-FU by potentiating its tumor-selective toxicity or reducing its host toxicity selectively. From such various points of view, a variety of biochemical modulators of 5-FU are reviewed.
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PMID:[Potentiation of the antitumor activity of 5-fluorouracil by biochemical modulators]. 327 10

Asynchronous populations of Ehrlich ascites tumor cells grown in vivo were separated by centrifugal elutriation into fractions of G1-, S-, and G2/M-phase cells with less than 10% cross-contamination. Cytoplasmic mRNA from phase-synchronous cells was used to prepare cDNA which was ligated with bacteriophage lambda gt10 arms and amplified in Escherichia coli C600 hfl-. EcoRI digests of DNA isolated from the sublibraries (G1, S, G2/M) were submitted to Southern hybridizations with radiolabeled probes either (a) for genes whose phase-specific expression is clearly documented, thymidine kinase, dihydrofolate reductase, and thymidylate synthase, or (b) for genes whose change of expression during the cell cycle is likely, lamin C, beta-actin, alpha- and beta-tubulin, c-myc, c-fos, p53. The cDNA sequences for genes of group (a) were found to be significantly enriched in DNA of the S-phase library indicating that the cell cycle phase-specific patterns of the respective mRNA levels are conserved in the sublibraries. Sequences belonging to group (b) were also found to be enriched in DNA isolated from the sublibraries: c-fos in G1 phase, lamin C, beta-actin, tubulins, c-myc in S phase, and p53 in G1/S phase. The unexpected prevalence of c-myc and alpha-tubulin in the S-phase library is supported by Northern analysis of RNA from phase-synchronous cells. Non-phase-specific, randomly chosen sequences hybridized equally strong with DNA isolated from the different sublibraries. No significant changes of the patterns of hybridization signals were observed with DNA from different amplifications of the sublibraries when analyzed with the same DNA probe indicating that the cDNA complexities are well conserved during amplifications. Consequently, the sublibraries are useful to obtain information about the cell cycle phase-specific expression of mRNAs for other genes of interest. Since the sublibraries reflect mRNA levels of the cells growing in vivo they supply data on the physiological in vivo pattern of gene expression undisturbed by potentially unphysiological in vitro conditions.
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PMID:Cell cycle phase-specific cDNA libraries reflecting phase-specific gene expression of Ehrlich ascites cells growing in vivo. 333 23

The method for measuring polyglutamate forms of CH2-H4PteGlu and H4PteGlu, by entrapment in ternary complexes with [6-3H]5-fluoro-2'-deoxyuridylate and Lactobacillus casei thymidylate synthase has been characterized. Results demonstrated that (a) the relationship between concentration of CH2-H4PteGlu and complex isolated on nondenaturing gels was dependent upon the number of glutamyl residues, and an alternative method for data analysis has been presented, (b) the relationship was linear over a 100-fold change in concentration, (c) formation of isolatable complex was time dependent, (d) noncovalent complexes formed with PteGlu2-5 could be isolated only at concentrations considerably higher than those required for CH2-H4PteGlu1-6, and (e) endogenous deoxyuridylate would be unlikely to interfere significantly with the assay. The distribution of polyglutamates of CH2-H4PteGlu and the combined pools of CH2-H4PteGlu plus H4PteGlu were subsequently examined in three human colon adenocarcinoma xenografts. In each tumor, the pentaglutamate of CH2-H4PteGlu and H4PteGlu was the most prominent species, followed by the hexaglutamate, constituting 68 to 92% of the CH2-H4PteGlu pool, and greater than 93% of the combined pools. A small percentage of di-, tri-, and tetraglutamates were also detected. Using a catalytic assay, the combined pool of CH2-H4PteGlu and H4PteGlu was estimated in the range of 0.5 to 2.7 microM in cell water, and for CH2-H4PteGlu, from 185 nM to 1.7 microM. Using thymidylate synthase purified from colon adenocarcinoma HxVRC5, CH2-H4PteGlu5 (where the subscript digit attached to the glutamate portion equals the number of glutamate residues) stabilized the covalent ternary complex at greater than 200-fold lower concentration in comparison to CH2-H4PteGlu1. Data indicated that in each colon tumor, the concentrations of CH2-H4PteGlun or CH2-H4PteGlun plus H4PteGlun were suboptimal for the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase, and would predict for relatively transient inhibition of thymidylate synthase after treatment with 5-fluorouracil. These data support therapeutic modulation to increase the concentration of CH2-H4PteGlun in the treatment of colon adenocarcinomas with 5-fluorouracil.
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PMID:Characterization of the pools of 5,10-methylenetetrahydrofolates and tetrahydrofolates in xenografts of human colon adenocarcinoma. 336 96

The putative thymidylate synthase (TS) gene of herpesvirus ateles, a T-lymphotropic tumor virus of New World primates, has a single large open reading frame encoding a polypeptide of 32.9 kilodaltons. The gene is transcribed into an unspliced 2.4-kilobase mRNA that is abundantly expressed late in virus replication. The AT-rich 5' untranslated leader sequence of TS mRNA in herpesvirus ateles-infected cells is remarkable in length (1,184 nucleotides), containing 29 minicistrons; this may indicate a role in translation regulation.
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PMID:Thymidylate synthase gene of herpesvirus ateles. 340 83

The goals of new antifolate development are: 1) improved selectivity, 2) improved penetration into pharmacologic sanctuaries, and 3) effectiveness vs. tumors either with intrinsic or acquired resistance to methotrexate (MTX). The major target for antifolate development has been dihydrofolate reductase (DHFR), but other critical folate-dependent enzymes, i.e., thymidylate synthase, methionine synthetase, and folylpolyglutamate synthetase are also important targets for new antifolate development. The possibility that DHFR from tumor tissue differs significantly from normal tissue DHFR now seems improbable, and the ideas of the late Bill Baker to design specific inhibitors of the tumor enzyme vs. the normal tissue DHFR are unlikely to succeed. However, the experience with triazinate (Baker's antifol; TZT) indicates that transport of antifols could be exploited to provide selective toxicity, as well as to provide agents effective vs. MTX-resistant cells. This work led to a second generation of "nonclassical" folate antagonists, of which trimetrexate (JB-11; TMQ) is now in clinical trial. Uptake of TMQ is via an MTX-independent membrane system, and extremely high intracellular levels of this drug are achieved in human leukemia cells.
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PMID:Design and rationale for novel antifolates. 343 93

The active metabolite of FUra, 5-fluorodeoxyuridine monophosphate (5-FdUMP), requires the presence of reduced folates to form a covalent ternary complex with the target enzyme thymidylate synthase (TS). In vitro and in vivo studies have demonstrated a potentiation of the cytotoxic effects of FUra when combined with the reduced folate, leucovorin. We have applied this concept to the treatment of metastatic breast cancer in a phase II trial, as recent clinical studies on patients with colorectal carcinoma have suggested an enhanced efficacy for the combination of FUra plus leucovorin. Patients entered on the present study are undergoing treatment with a 5-day daily regimen of leucovorin (500 mg/m2, iv) followed by FUra (375 mg/m2, iv). Toxicity and response data are currently being collected on patients who have failed "standard" combination regimens that included FUra. In patients with accessible tumor, serial biopsies are being obtained during treatment with the combination of FUra and leucovorin and during therapy with FUra alone to assess the degree of 5-FdUMP binding to the target enzyme, TS, in the presence and absence of exogenously administered leucovorin. Preliminary results from the biochemical studies suggest an enhanced saturation of TS by the fluorinated pyrimidine when administered with leucovorin.
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PMID:Preliminary results of a phase II trial for the treatment of metastatic breast cancer with 5-fluorouracil and leucovorin. 350 43

Novel antitumor agents related to levodopa and dopamine exhibit a selective and rapid inhibition of DNA synthesis as measured by thymidine incorporation. Our investigations have attempted to determine the biochemical basis of the selective inhibition of tumor cells and in this present study we examined the effects of these agents on thymidylate synthase. The dihydroxybenzene derivatives were found to inhibit thymidylate synthase in situ at concentrations ranging between 100 and 800 microM. The quinols did not inhibit partially purified thymidylate synthase, although the oxidized quinones did cause inhibition at concentrations between 10 and 100 microM. Time course experiments suggested that the inhibition of thymidylate synthase in situ by the dihydroxybenzene derivatives occurs after the inhibition of thymidine incorporation, indicating that an earlier event was critical to the inhibition of DNA synthesis. With the use of a novel in situ assay which measured the release of [3H]water from [5-3H] uridine in intact cells, we were able to show that one of the earliest biochemical events is the inhibition of ribonucleotide reductase and that the inhibition of thymidylate synthase, which is delayed by approximately 30 min, was indirectly mediated possibly through effects on ribonucleotide reductase.
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PMID:Sequential inhibitory effects of antitumor agents related to levodopa and dopamine upon DNA synthetic enzymes. 351 Jun 22

The formation and isolation of [6-3H]FdUMP-thymidylate synthase-5,10-methylenetetrahydrofolate covalent complex have been examined in tumor cytosols incubated with albumin-dextran coated charcoal used to remove endogenous nucleotide. Charcoal suspension (10% charcoal, 0.5% albumin, 0.05% dextran) absorbed greater than 98% of dUMP added to cytosols, but it reduced by 42-87% covalent complex isolated from subsequent incubation with [6-3H]FdUMP and cofactor using cytosols from different tumors. Initial treatment of ternary complex with charcoal suspension did not cause a decrease in stability of covalent complex during subsequent incubation (37 degrees), but complex separated from free ligand by 10% charcoal suspension was not stable to further treatment with 4% charcoal suspension. Treatment of tumor cytosols with 10% charcoal suspension, to remove nucleotide, did not decrease the rate at which enzyme catalyzed the release of 3H2O from [5-3H]dUMP, or release active enzyme from the ternary complex. Based on these observations, a sensitive procedure for determining thymidylate synthase activity has been developed in which unbound nucleotides (dUMP, FdUMP) are removed prior to assay of enzyme activity. The procedure is suitable for assay of small samples of tissue or of tissues with a low (or inhibited) level of thymidylate synthase activity.
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PMID:Determination of thymidylate synthase activity in colon tumor tissues after treatment with 5-fluorouracil. 359 15

The clonal cytotoxic effects and mechanism of action of a new series of 2-amino-4-hydroxyquinazoline folate analogues (5,8-dideazafolates) have been assessed using the human colon tumor cell line HCT-8. Of these compounds only 5-methyl-5,8-dideazafolate was potentially more effective than a compound previously identified, 5,8-dideazaisofolate (H-338, NSC 289517). HCT-8 sublines resistant to methotrexate, 5-fluorodeoxyuridine, and H-338 were either minimally or not cross-resistant to the other agents. The cytotoxicity of H-338 was strongly dependent on the time of exposure; at exposure times shorter than 8 h it was essentially nontoxic. Thymidine alone, as well as leucovorin or folic acid, protected against the cytotoxic effects of H-338. This is consistent with thymidylate synthase (TS) as its only locus of action. Studies with dihydrofolate reductase and TS isolated from HCT-8 cells indicated that these quinazolines were weaker inhibitors of dihydrofolate reductase than was methotrexate, but they were not particularly potent TS inhibitors. However, synthetic poly-gamma-glutamate derivatives of quinazolines showed dramatically increased TS, but not dihydrofolate reductase, inhibition. TS inhibition increased as the polyglutamate chain length increased. Using isolated HCT-8 folylpolyglutamate synthetase, all the parent quinazolines containing L-glutamate were found to be substrates. With H-338, the results indicated that tetraglutamate or longer derivatives could be synthesized intracellularly. These results are consistent with our hypothesis that cytotoxicity by such quinazolines necessarily involves "lethal synthesis" from a prodrug; i.e., the nontoxic parent drug must be converted to polyglutamates before TS inhibition and subsequent cytotoxicity can occur.
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PMID:Mechanism of action of 5,8-dideazaisofolic acid and other quinazoline antifols in human colon carcinoma cells. 366 1


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