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)

BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 x 10(-5) and 1 x 10(-5), respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and surveys on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 micro M hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.
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PMID:Alteration of human breast tumor cell membrane functions by chromosome-mediated gene transfer. 23 36

New mutants of bacteriophage T4 that overproduce the enzyme dihydrofolate reductase were investigated. Unlike previously described overproducers of this enzyme (Johnson and Hall, 1974), these mutants did not overproduce deoxycytidylate deaminase. Overproduction of dihydrofolate reductase by the new mutants occurred because enzymatic activity continued to increase for a longer period of time in cells infected by the mutants than in cells infected by wild-type phage. This continued increase occurred even in the presence of rifampin, indicating that the overproduction is probably due to a post-transcriptional event. Both these new overproducers and the previously described overproducers were studied by using polyacrylamide gel electrophoresis. The two types of overproducers appeared to be very different. The previously described overproducers showed a delay and/or reduction in the synthesis of several proteins that normally started to be made 4 to 6 min after infection. Several proteins could be seen to be overproduced on the gels. The new overproducers did not show the delay in the synthesis of some proteins and only overproduced a few proteins. The new gene defined by the new overproducers is between the gene coding for thymidine kinase and the gene coding for lysozyme.
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PMID:Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants. 109 Jul 53

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

1. Methotrexate was administered immediately after partial (70%) hepatectomy, resulting in complete inhibition of dihydrofolate reductase in 24 h-regenerating liver. 2. At 48 h and 72 h after partial hepatectomy, thymidylate synthase activity was increased, whereas thymidine kinase was inhibited, by the injection of methotrexate. The DNA and RNA contents and the liver weight were also reduced in methotrexate-treated rats. 3. The immunoblotting assay showed that methotrexate stimulated the synthesis of thymidylate synthase protein in 48 h-regenerating liver. At the same time, thymidylate synthase activity was directly inhibited by methotrexate. The mechanisms of inhibition of these enzymes by methotrexate appeared to be different.
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PMID:Effects of methotrexate on rat liver regeneration after partial hepatectomy. 137 31

Resistance to methotrexate (MTX) is a well established clinical problem and strategies to circumvent this would obviously be beneficial. The human leukemia cell line, CCRF-CEM, was grown in folinic acid to study MTX resistance-reflecting more in vivo conditions. A 15.8-fold resistant subline, CEM/MTX, was evolved by stepwise increases in MTX exposure. The MTX resistant cell line exhibited both increased dihydrofolate reductase (DHFR) activity due to gene amplification as well as impaired MTX uptake. An additional mechanism of resistance to MTX was a 2-fold increase in thymidine kinase (TK). As a result of this increased TK activity, the CEM/MTX cells were collaterally sensitive to the nucleoside analogue, azidothymidine (AZT). CEM cells resistant to MTX possess properties that can be exploited by AZT and these studies may have clinical implications.
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PMID:Collateral sensitivity to azidothymidine in methotrexate resistant human leukemia cells. 138 29

We have shown that the vestigial (vg) mutant of D. melanogaster has a perturbed nucleotide metabolism compared to various wild-type strains. The mutant is particularly spontaneously resistant to aminopterin. The resistance seems to correlate with an increase in dihydrofolate reductase (DHFR) activity and quantity. The DHRF is a target enzyme of aminopterin. Our results suggest that the vg+ gene could be a regulatory gene acting on the DHFR gene. The wing mutant phenotype being due to a decrease in the thymidylate pool (dTMP) (Silber et al., 1989). In order to understand better the action of the mutant gene on nucleotide metabolism, we have induced suppressor genes of the mutant phenotype by mutagenesis with ethyl methanesulfonate (EMS) and bromouridine (BUR). The suppressor strains obtained display a phenotype intermediate between wild-type and vg phenotype. The action of three independent suppressor genes on eight parameters of nucleotide metabolism is reported here [three enzyme activities, resistance to aminopterin and to fluorodeoxyuridine (FUdR), auxotrophy test and the ability to use exogenous thymidine and uridine]. In comparison to the original vg strain, major changes for the parameters tested are observed. The most striking effects are obtained with the vgBUR27 strain, which is highly sensitive to aminopterin and to fluorodeoxyuridine and didplas the highest thymidine kinase (TK) and DHFR activities within the strains tested. The potential actions of suppressor genes on the vg mutant are discussed.
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PMID:vestigial suppressor genes and resistance to aminopterin in Drosophila melanogaster. 142 57

To determine the effect of different promoters on the expression of an altered dihydrofolate reductase (DHFR) gene conferring methotrexate (MTX) resistance in different cell types, double-copy retroviral vectors were constructed carrying a murine mutant DHFR under the control of five different promoters, i.e., human adenosine deaminase (ADA), simian virus 40 (SV40), thymidine kinase (TK), human beta-actin, and cytomegalovirus (CMV). Their expression was compared in NIH-3T3 cells, three human leukemia cell lines, and mouse bone marrow. The variant DHFR is readily expressed from these various promoters in retroviral vectors at a selectable level. In 3T3 cells, the DHFR constructs containing the SV40 promoter conferred the highest levels of resistance to MTX. In K562 and Raji cells, the construct with the TK promoter produced the highest level of resistance. However granulocyte-macrophage colony-forming unit (CFU-GM) colonies from mouse marrow were more resistant to MTX when infected with vectors containing the SV40 promoter and ADA promoter as compared to the other promoter constructs. These studies show that mouse fibroblast cell lines such as NIH-3T3 do not predict the effectiveness of retroviral-mediated gene transfer for marrow progenitor cells, and that the activity of retroviral vector-encoded promoters vary in an unpredictable manner from cell type to cell type. Possible implications for basic gene transfer studies and clinical applications are discussed.
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PMID:Comparison of the expression of a mutant dihydrofolate reductase under control of different internal promoters in retroviral vectors. 152 11

The murine B-cell hybridoma B9 requires interleukin-6 (IL-6) for its survival and proliferation in vitro. We show here that withdrawal of IL-6 from B9 cultures results in programmed death, concomitant with arrest of the cells in the G1 phase of the cell cycle. Unlike several other systems that undergo programmed cell death, no induction of transcripts corresponding to the testosterone-repressed message-2 or transglutaminase genes is observed during this process. Upon readdition of IL-6 to G1-arrested B9 cells, viability is maintained and entry into S phase occurs after a lag period of 10 to 12 hr. Northern blot analysis showed that the immediate-early mRNAs normally induced shortly after growth factor stimulation in quiescent fibroblasts (c-fos, c-jun, Egr-1, c-myc, JE, and KC), and other growth-related genes (2F1, c-Ha-ras, and p53), are either not induced or remain unchanged during G1 to S phase progression. A correlation was found, however, between the temporal pattern of expression of several G1/S phase genes (dihydrofolate reductase, thymidine kinase, transferrin receptor, and histone H3) and DNA synthesis. These results demonstrate that IL-6-induced viability and growth of hybridoma (and, presumably, plasmacytoma) cells is mediated via novel signal transduction pathways.
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PMID:Suppression of programmed death and G1 arrest in B-cell hybridomas by interleukin-6 is not accompanied by altered expression of immediate early response genes. 170 72

(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

A sensitive and versatile assay is described for the nuclear transport of 35S-labeled proteins obtained by the in vitro translation of SP6 plasmid-generated mRNAs. A specific nuclear accumulation of greater than 20-fold is observed for the transformation-related nuclear proteins, p53 and E1b, and the nuclear enzyme, thymidine kinase, whereas transport of the nonnuclear proteins, dihydrofolate reductase and simian virus 40 small t antigen, is negligible within 30 min.
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PMID:Nuclear transport of proteins translated in vitro from SP6 plasmid-generated mRNAs. 213 54


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