Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Roswell Park Memorial Institute 4265 human lymphoblasts were grown with three dihydrofolate reductase inhibitors: a 2,4-diaminopteridine, methotrexate; a 2,4-diaminoquinazoline, chlorasquin; and, a 2,4-diaminotriazine, triazinate. In the absence of inhibitor, dihydrofolate reductase activity increased to a peak at mid-log growth and then declined during the later growth stages. When cells were grown with 10(-8) M antifolate, cell growth was not affected, but dihydrofolate reductase activity (assayed at pH 7.0) remained at approximately initial levels throughout the growth cycle. This represented 60 to 70% less activity at the mid-log stage of growth, as compared to control cells. Dihydrofolate reductase activity in cells grown with 10(-8) M methotrexate, when assayed at pH 8.5, reached levels twice those in control cells. Enzyme activity in cells grown with 10(-8) M chlorasquin, when assayed at pH 8.5, was also higher than at pH 7.0, but it was not as high as that observed in methotrexate-treated cells. Activity in cells grown with 10(-8) M triazinate was approximately the same when assayed at either pH 7.0 or 8.5. At 10(-8) M, the three antifolates had no effect on the activities of thymidylate synthetase, thymidine kinase, serine trans-hydroxymethylase, 5,10-methylenetetrahydrofolate dehydrogenase, 10-formyltetrahydrofolate synthetase, and thymidylate kinase. However, when concentrations were used which completely inhibited growth (10(-7) to 10(-5) M methotrexate or chlorasuin; 10(-6) to 10(-5) M triazinate), dihydrofolate reductase was progressively inhibited, and there was a two- and a threefold elevation of thymidylate synthetase and thymidine kinase activity, respectively. Quantitatively, the elevation of either enzyme was similar over the range of growth-inhibitory concentrations studied. The activities of the other enzymes were unaffected. Methotrexate and chlorasquin inhibited thymidylate synthetase in a noncompetitive manner (with respect to 5,10-methylenetetrahydrofolate) with approximate Ki values of 4.5 X 10(-5) M and 4.9 X 10(-6) M, respectively. Triazinate, at 10(-3) M, had no significant effect on thymidylate synthetase activity. At 10(-3) M, the antifolates produced a negligible inhibition of thymidine kinase. Deoxyuridine 5'-monophosphate (10(-5) M) effectively protected thymidylate synthetase from heat inactivation in vitro. Dihydrofolate or 5,10-methylenetetrahydrofolate, at 10(-3) M, only partially protected thymidylate synthetase. Concentrations of methotrexate (10(-7) to 10(-6) M), chlorasquin (10(-7) M), and triazinate (10(-6) to 10(-5) M), which produced thymidylate synthetase elevation in vivo, did not protect the enzyme from heat inactivation in vitro. Methotrexate at 10(-5) M and chlorasquin at 10(-6) M gave slight protection. Thymidine kinase was stabilized only by thymidine.
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PMID:Elevation of dihydrofolate reductase, thymidylate synthetase, and thymidine kinase in cultured mammalian cells after exposure to folate antagonists. 127 51

The cDNA (DHFR) encoding the wild-type (wt) dihydrofolate reductase (DHFR) was used as a dominant selectable marker in the transfection of murine hybridoma Sp2/0-Ag14 cells by protoplast fusion. The initial clones contained 100-400 copies of integrated plasmid DNA, and the high level of wt DHFR protein produced enabled the cells to survive the drug selection at 100 nM methotrexate (MTX). The expression of the gene of interest was several fold higher than when the mutant DHFR with decreased MTX binding was used as the selection marker, presumably because the clones were more sensitive to the stress induced by MTX. When the clones were propagated at higher concentrations of MTX, expression of both DHFR and the gene of interest increased. This induction is freely reversible, and we have shown that it is controlled at the transcriptional level, by nuclear run-off transcription assays.
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PMID:The use of a wild-type dihydrofolate reductase-encoding cDNA as a dominant selectable marker and induction of expression by methotrexate. 144 34

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an enzyme involved in the metabolism of nucleic acids; it is also an important target for folate antagonists such as methotrexate (MTX). The distribution and expression of DHFR both in human HeLa BU-25 cell line and in methotrexate-resistant (MTX-R) variant, deriving from the human VA2-B cell line (having the DHFR gene amplified) was studied by tetrazolium salt method and by flow cytometric analysis. The immunohistochemical labelling of DHFR was achieved by using the streptavidinbiotinilated complex technique. DHFR activity was low in the human HeLa BU-25 cell line, while it was very high in the MTX-R cell line; the activity level increased with the increasing concentration of the MTX. The results obtained with cytochemical and immunohistochemical technique were compared. These findings showed that the hyperproduction of DHFR is strictly related with the cells having the DHFR gene amplified. Since MTX resistance is a common finding in the cells of patients with acute leukaemia, these studies may be extended to tumour-bearing patients at onset and following chemotherapy with methotrexate.
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PMID:Immunohistochemistry of dihydrofolate reductase in methotrexate-sensitive and -resistant human cell lines by flow cytometry: a comparison with the cytochemical tetrazolium salt method. 152 71

Dihydrofolate reductase reduces folic acid to tetrahydrofolate as a prerequisite to one-carbon metabolism, which is required for normal embryonic de novo DNA synthesis. The developmental toxicity of methotrexate (MTX) has been attributed to MTX's ability to inhibit the activity of dihydrofolate reductase and thereby indirectly suppress one-carbon metabolism. The compound 1-(p-tosyl)-3,4,4-trimethylimidazolidine (TTI), which is structurally unrelated to folate, reestablishes one-carbon metabolism by the biomimetic transfer of single carbon units. Whether the developmental toxicity of MTX is indeed caused via suppressed one-carbon metabolism was tested in New Zealand white rabbits following concurrent maternal treatment with MTX and TTI. TTI reduced MTX developmental toxicity judged by increased mean fetal body weights, decreased percentage of malformed fetuses, and reduced incidences of major malformations. Two doses of TTI (90 mg/kg, each) at 1 hr prior to and 1 hr after MTX also reduced the developmental toxicity, but was no more effective than the single-injection regimen. Treatment with TTI alone caused no developmental toxicity. Histologically, MTX caused enlarged intercellular spaces in limb bud mesenchyme that began at 6-8 hr and increased in size until 16 hr. Mesenchymal nuclei appeared basophilic, with angular contours. Pretreatment with TTI delayed MTX-induced histological changes until 20-24 hr after MTX in 36-50% of embryos and completely protected the remainder. The sequence of MTX-induced changes was not altered among affected embryos, although the severity of the lesions did not appear as great. Saline-only or TTI-only treatments caused no alterations in limb buds. These data are consistent with the concept that impaired one-carbon metabolism is indeed the fundamental process underlying MTX developmental toxicity.
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PMID:Methotrexate-induced developmental toxicity in rabbits is ameliorated by 1-(p-tosyl)-3,4,4-trimethylimidazolidine, a functional analog for tetrahydrofolate-mediated one-carbon transfer. 163 81

A stepwise selection procedure was used to obtain from Mtx-5011 Aedes albopictus cells, variants with increased resistance to methotrexate (mtx). On the basis of growth, the Mtx-5011 derivatives were 270- to 3,000-fold more resistant to mtx than wild-type mosquito cells. Properties associated with mtx resistance in these cells were consistent with amplification of the dihydrofolate reductase (DHFR) gene. The cells overproduced DHFR protein, were enriched with DHFR mRNA, and DNA from resistant cells was enriched for a band that likely contained the DHFR coding sequence. Karyotype analysis indicated that high levels of resistance were accompanied by a conversion to tetraploidy, chromosome rearrangements, and an apparent duplication of one of the mosquito chromosomes.
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PMID:Genetic changes in methotrexate-resistant mosquito cells. 172 12

Dihydrofolate reductase is an intracellular target enzyme for folate antagonists, including the anticancer drug methotrexate. In order to design novel drugs with altered binding properties, a detailed description of protein-drug interactions in solution is desirable to understand the specificity of drug binding. As a first step in this process, heteronuclear three-dimensional NMR spectroscopy has been used to make sequential resonance assignments for more than 90% of the residues in human dihydrofolate reductase complexed with methotrexate. Uniform enrichment of the 21.5-kDa protein with 15N was required to obtain the resonance assignments via heteronuclear 3D NMR spectroscopy since homonuclear 2D spectra did not provide sufficient 1H resonance dispersion. Medium- and long-range NOE's have been used to characterize the secondary structure of the binary ligand-enzyme complex in solution.
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PMID:Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution. 173 71

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an important enzyme involved in DNA metabolism. In this connection the cell cycle modulation of DHFR levels in HeLa S3 and HL 60 cell lines was investigated by flow cytometric analysis. A concentration of 4 micrograms/ml of aphidicolin was employed to synchronize the cell lines. DHFR was cytochemically detected by using tetrazolium salt and immunofluorescence techniques; DNA content was evaluated by means of propidium iodide staining. At 0, 2, 4, 6, 8, 10, 12 hrs. after the removal of the drug we observed a low DHFR level in G0-G1 phase, followed by an increase during late S and G2/M phases. The variations of this enzyme may represent, under well defined conditions, a marker of cycling cells.
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PMID:Modulation of cytochemically detected dihydrofolate reductase during cell cycle. 180 92

Dihydrofolate reductase (DHFR; EC 1.5.1.3) is required in folate metabolism for the synthesis of purines, thymidine, and glycine. Although there have been several reports of induction of DHFR enzyme by methotrexate (MTX), a drug that competitively inhibits DHFR, there are no studies reported that examine the effect of MTX on DHFR gene transcription. We have examined the effect of MTX and other inhibitors of DNA synthesis on DHFR transcription using a transient expression assay. MTX stimulates transient expression in a concentration-dependent manner from a hamster DHFR promoter construct containing 150 base pairs 5' to the start of transcription. Addition of either tetrahydrofolate or hypoxanthine plus thymidine prevents the promoter induction in response to MTX, suggesting that stimulation by MTX results from inhibition of these metabolites. Furthermore, two other antimetabolic drugs--fluorodeoxyuridine and hydroxyurea--also stimulate the DHFR promoter in a concentration-dependent manner. In contrast, aphidicolin, which blocks cell growth through inhibition of DNA polymerase alpha, has no effect on the DHFR promoter. The potential relevance of these results to cross-resistance to chemotherapeutic agents and to the process of gene amplification is discussed.
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PMID:Stimulation of dihydrofolate reductase promoter activity by antimetabolic drugs. 183 62

Dihydrofolate reductase was obtained from Pneumocystis carinii isolated from heavily infected lungs of female Sprague-Dawley rats infected by transtracheal inoculation. The enzyme differed significantly from other forms of dihydrofolate reductase in response to KCl and to antifolate drugs. Dihydrofolate reductase from P. carinii was used to assess activity of analogs of pyrimethamine, methotrexate, and trimetrexate. One pyrimethamine analog was selective for P. carinii dihydrofolate reductase; potency was in the micromolar range. In contrast, 21 methotrexate analogs and 2 trimetrexate analogs were selective for P. carinii dihydrofolate reductase; potencies for these were in the nanomolar range.
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PMID:Inhibition of Pneumocystis dihydrofolate reductase by analogs of pyrimethamine, methotrexate and trimetrexate. 184 Jan 46

Dihydrofolate reductase (DHFR) cDNA sequences were isolated from a methotrexate-resistant mouse L5178Y cell line previously shown to contain methotrexate-resistant dihydrofolate reductase enzyme activity. Specifically-primed reverse transcription products were amplified using the polymerase chain reaction and then cloned into a mammalian expression plasmid. Candidate clones were identified by restriction analysis and then functionally tested by transfection into mouse 3T3 fibroblasts, selecting for methotrexate-resistant colonies. Sequence analysis of the cDNA clones demonstrated the substitution of tryptophan (TGG) in place of the wild-type phenylalanine (TTC) at codon 31. Sequencing of PCR-amplified genomic DNA extracted from the drug-resistant L5178Y cells confirmed the tryptophan codon at position 31. Transfection of mammalian tissue culture cells with expression plasmids containing the trp31 DHFR sequence resulted in substantial methotrexate-resistant colony formation. Recombinant trp31 DHFR enzyme activity expressed in stably-transfected Chinese hamster ovary cells was approximately 20-fold less sensitive to methotrexate inhibition than wild-type mouse DHFR enzyme activity. We conclude that the cloned Trp31 DHFR sequence encodes an enzyme substantially resistant to methotrexate which confers a drug-resistance phenotype to cells in which it is expressed.
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PMID:Isolation and characterization of a variant dihydrofolate reductase cDNA from methotrexate-resistant murine L5178Y cells. 226 62


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