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)

Enhanced DNA repair has been identified as a major mechanism of resistance to the anticancer drug cisplatin in murine leukemia L1210 cells. Studies of other cells have implicated the elevation of a variety of RNA transcripts in cisplatin resistance. This study investigated potential changes in transcription of these genes as well as genes involved in DNA repair. No elevation in any of the following transcripts was observed: thymidylate synthase, dihydrofolate reductase, DNA polymerase alpha, DNA polymerase beta, topoisomerase II, Ha-ras, beta-tubulin, metallothionein and the DNA repair genes ERCC1 and ERCC2. Thymidine kinase was increased no more than 2-fold. None of these RNA were induced by incubation with cisplatin. High levels of cisplatin produced selective decreases in certain RNA. These results demonstrate that the previous observations of elevated RNA can not be universally applied to all cisplatin-resistant cells.
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PMID:Analysis of various mRNA potentially involved in cisplatin resistance of murine leukemia L1210 cells. 197 66

Determinants of methotrexate (MTX) resistance in cell lines resistant to short, but not continuous, MTX exposure were investigated since such lines may have relevance to clinical resistance. CCRF-CEM R30dm (R30dm), cloned from CCRF-CEM R30/6 (a MTX-resistant subline of the CCRF-CEM human leukemia cell line), had growth characteristics similar to CCRF-CEM. R30dm was resistant to a 24-h exposure to levels as high as 300 microM MTX but was as sensitive as CCRF-CEM to continuous MTX exposure. MTX resistance of R30dm was stable for greater than 68 weeks in the absence of selective pressure. Initial velocities of MTX transport were comparable for R30dm and CCRF-CEM, as were dihydrofolate reductase specific activity and MTX binding. A 2-fold thymidylate synthase activity decrease for R30dm from that of CCRF-CEM was not a significant factor in R30dm MTX resistance. Decreased MTX poly(gamma-glutamate) synthesis resulted in lower levels of drug accumulation by R30dm. Decreased polyglutamylation was attributable to folylpolyglutamate synthetase (FPGS) activity in R30dm extracts which was 1, 2, and less than or equal to 10% of CCRF-CEM extracts with the substrates MTX, aminopterin, and naturally occurring folates, respectively. Comparison of cell lines with varying levels of resistance to short term MTX exposure indicated that the extent of MTX resistance was proportional to the reduction of FPGS activity. The evidence supported decreased FPGS activity as the mechanism of resistance to short MTX exposure in the cell lines investigated.
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PMID:Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines. 200 75

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.
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PMID:Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast. 201 65

In order to inhibit the in vitro translation of Plasmodium falciparum mRNA coding for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), oligodeoxynucleotides (ODNs) were directed against the translation initiation site or a site in the TS-coding region. In both cases considerable hybridization arrest, i.e. greater than 50% inhibition, was only achieved if the lengths of the ODNs to the two regions were 30 and 39 nucleotides, respectively, or longer. The ODN with the highest efficiency was a 49-mer directed against the TS-coding region (OTS49); 45 microM was sufficient to inhibit the expression of DHFR-TS by almost 90%. In this case the synthesis of DHFR-TS was interrupted at the binding site of OTS49 by a RNase H-independent mechanism. The resulting polypeptide was smaller (55 kDa) than one subunit of the native protein (71 kDa) and lacked TS activity.
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PMID:Hybridization arrest of cell-free translation of the malarial dihydrofolate reductase/thymidylate synthase mRNA by anti-sense oligodeoxyribonucleotides. 202 68

In order to determine the mechanism for the effects of homofolates on growth of Lactobacillus casei, polyglutamated derivatives of homofolate (HPteGlu), dihydrohomofolate and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of folate-requiring enzymes. The following L. casei enzymes were examined: thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase and dihydrofolate reductase. Polyglutamates of (6R,S)-H4HPteGlu are potent inhibitors of TS and GARFT. For example, the IC50 values of (6R,S)-H4HPteGlu6 are 0.7 microM for TS and 0.3 microM for GARFT. By contrast, the value for HPteGlu6 is greater than 10 microM for both TS and GARFT. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu derivatives increases with polyglutamate chain length. For TS, the Glu5 and Glu6 derivatives of (6R,S)-H4HPteGlu are 20 and 30 times more potent than the monoglutamate, respectively. For GARFT, the Glu2-6 derivatives are 2-3 times more potent than Glu1. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu polyglutamates is almost entirely due to the unnatural (6R) diastereomer at C-6. Homofolate derivatives are only weak inhibitors of aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase, and dihydrofolate reductase. We conclude that both TS and GARFT are potential targets of (6R)-H4HPteGlu polyglutamates.
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PMID:Tetrahydrohomofolate polyglutamates as inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase in Lactobacillus casei. 210 31

The pharmacologic responses of normal and malignant epidermal cells to chemotherapeutic agents were examined in a system which consists of a serum-free "defined" medium. The effects of methotrexate (MTX), fluorodeoxyuridine (FUDR), and hydroxyurea upon cell growth, DNA synthesis, thymidylate synthase activity, and dihydrofolate reductase (DHFR) activity were compared in normal human epidermal keratinocytes (NHEK), newborn epidermal cells (NEC), human squamous cell carcinoma (SCC-25), and a methotrexate-resistant human squamous cell carcinoma (SCC-15R). Normal keratinocytes were found to be 10(3) to 10(4) times less sensitive to the effects of MTX and FUDR than the malignant cells with respect to growth and DNA synthesis. The differential sensitivity to MTX and FUDR was not due to differences in growth media, increased target enzyme activity, e.g., DHFR and thymidylate synthase, or impaired transport of these drugs. The results indicate that the mechanism for the increased sensitivity of the squamous cell carcinoma to MTX and FUDR must involve a process which is distal to the de novo synthesis of dTMP.
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PMID:A comparison of the effects of antitumor agents upon normal human epidermal keratinocytes and human squamous cell carcinoma. 213 84

Pulsed field gel electrophoresis was used to identify the chromosome-size DNA of Pneumocystis carinii, a major pathogen of immunocompromised patients. Thirteen chromosomes of rodent Pneumocystis carinii, ranging in size from 300 to 700 kilobases (kb), were identified. The minimum genome size for P. carinii, estimated on the basis of the sizes of chromosomes, is 7,000 kb. Genetic heterogeneity among different P. carinii isolates was documented by demonstration of chromosomal size variability. By hybridization studies, the genes for topoisomerase I, dihydrofolate reductase, rRNA, actin, and thymidylate synthase were mapped to single chromosomes of approximately 650, 590, 550, 460, and 350 kb, respectively. Hybridization studies further confirmed the genetic heterogeneity of P. carinii.
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PMID:Identification of Pneumocystis carinii chromosomes and mapping of five genes. 216 Apr 29

The effects of the lipid-soluble dihydrofolate reductase inhibitor, trimetrexate, on the inhibition of thymidylate biosynthesis as a result of perturbation in cellular folate pools in H35 hepatoma cells in vitro has been investigated. Exposure of the cultures to increasing concentrations of trimetrexate between 2 and 20 nM causes a marked reduction in de novo thymidylate biosynthesis and a concomitant decrease in (6R)5,10-methylenetetrahydropteroylpolyglutamate (5,10-CH2H4PteGlun) from 2.0-0.2 microM, respectively. This is accompanied by an increase in H2PteGlun from 1.2 microM in control cultures to 4.7 microM in cultures exposed to 20 nM trimetrexate. The dependency of de novo thymidylate biosynthesis on intracellular 5,10-CH2H4PteGlun in trimetrexate-treated cells is compared with (a) the relationship of thymidylate biosynthesis on intracellular levels of 5,10-CH2H4PteGlun in folate-depleted cells supplemented with increments of folic acid and (b) the substrate (5,10-CH2H4PteGlun) dependence of purified thymidylate synthase from the same source. All three results are nearly identical demonstrating that trimetrexate-dependent inhibition of de novo thymidylate biosynthesis is primarily a result of substrate depletion. These results coupled with the weak inhibitory properties of H2PteGlun for thymidylate synthase Ki = 5.0 microM) suggest that H2PteGlun accumulation is not the major determinant in inhibiting thymidylate synthase following trimetrexate inhibition but under certain conditions has the potential to enhance the inhibition caused by substrate depletion.
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PMID:Role of substrate depletion in the inhibition of thymidylate biosynthesis by the dihydrofolate reductase inhibitor trimetrexate in cultured hepatoma cells. 216 50

The coding sequence of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from a moderately pyrimethamine-resistant strain (HB3) of Plasmodium falciparum was assembled in a pUC expression vector. The coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include all point mutations thus far linked to pyrimethamine resistance. Wild-type (3D7) and highly pyrimethamine-resistant (7G8) TS-DHFRs were made from this vector by cassette mutagenesis using Nco1-Xba1 fragments from the corresponding cloned TS-DHFR genes. Catalytically active recombinant TS-DHFRs were expressed in Escherichia coli, albeit at low levels. Both TS and DHFR coeluted upon gel filtration and copurified upon affinity and anion exchange chromatography. Gel filtration and SDS-PAGE indicated that the enzyme was a dimer with identical 67-kDa subunits, characteristic of protozoan TS-DHFRs. Amino-terminal sequencing gave 10 amino acids which perfectly matched the sequence predicted from the nucleotide sequence. The recombinant TS-DHFR was purified to homogeneity by 10-formylfolate affinity chromatography followed by Mono Q FPLC. The inhibition properties of pyrimethamine toward the purified recombinant enzymes show that the point mutations are the molecular basis of pyrimethamine resistance in P. falciparum.
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PMID:Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum. 217 83

In protozoa, thymidylate synthase (TS) and dihydrofolate reductase (DHFR) exist on the same polypeptide. The DHFR domain is on the amino terminus, TS is on the carboxy terminus, and the domains are separated by a junction peptide of varying size depending on the source. The native protein is a dimer of two such subunits and is 110-140 kDa. Most studies of bifunctional TS-DHFR have been performed with the protein from anti-folate resistant strains of Leishmania major, which show amplification of the TS-DHFR gene and overproduction of the bifunctional protein. The Leishmania TS-DHFR has also been highly expressed in heterologous systems. There is extensive communication between domains, and channeling of the H2folate product of TS to DHFR. Anti-folates commonly used to treat microbial infections are poor inhibitors of L. major DHFR. However, selective inhibitors of L. major vs human DHFR have been found. The TS-DHFR from Plasmodium falciparum has also been cloned and sequenced. Interestingly, pyrimethamine-resistant strains of P. falciparum have a common point mutation in the DHFR coding sequence which causes decreased binding of the folate analog. A detailed knowledge of the structure and function of protozoan TS-DHFRs will soon be available.
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PMID:Thymidylate synthase-dihydrofolate reductase in protozoa. 217 51


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