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)

This report examines the intracellular activity of dihydrofolate reductase using an in situ assay designed to measure enzymatic activity in intact cells. The rate of uptake of folic acid exceeded the rate of in situ dihydrofolate reductase activity suggesting that the reduction of folate to dihydrofolate, rather than transport, was the rate limiting step. In situ dihydrofolate reductase activity varied linearly with cell number. A comparison of the in situ activity revealed that a squamous cell carcinoma selected for methotrexate (MTX) resistant (SCC-15R) had 100 times greater dihydrofolate reductase (DHFR) activity than L1210 leukemia. In agreement with this finding, the in situ DHFR activity in SCC-15R cells was 50-fold less sensitive to the inhibitory effects of MTX than the L1210 in situ DHFR activity (IC50 = 1.1 x 10(-5) M and 2.4 x 10.7(-7) M respectively). The inhibition of in situ dihydrofolate reductase activity by MTX was found to correlate with the inhibition of growth, DNA synthesis (CdR incorporation) and in situ thymidylate synthase activity.
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PMID:A comparison of dihydrofolate reductase activity in intact leukemia cells and squamous cell carcinoma. 275 54

The novel tetrahydrofolate, 5,10-dideazatetrahydrofolic acid (DDATHF), was designed as an inhibitor of folate metabolism at a site other than dihydrofolate reductase. DDATHF has been shown to inhibit glycinamide ribonucleotide transformylase, a folate-requiring enzyme that catalyzes the first of two one-carbon transfer reactions in the de novo purine nucleotide biosynthetic pathway. Incubation of HL-60 promyelocytic leukemia cells with 5 x 10(-8) to 10(-5) M DDATHF resulted in a marked inhibition of growth after 48 h, with a complete cessation of cellular replication by day 4. Cell cycle analyses of DDATHF-treated HL-60 cells demonstrated an initial block in early S phase by day 3 followed by an accumulation of cells in the G1 and G2 + M phases of the cell cycle. Inhibition of growth was accompanied by a concentration-dependent increase in the percentage of mature myeloid cells that expressed nitroblue tetrazolium positivity, and a small increase in nonspecific esterase activity. Induction of differentiation and inhibition of growth by DDATHF were completely prevented by hypoxanthine and 5(4)-amino-4(5)-imidazole carboxamide, suggesting that depletion of intracellular purine nucleotide pools has an important role in the biological effects of this inhibitor. This possibility was confirmed by the finding that DDATHF caused a pronounced reduction in intracellular GTP and ATP levels within 2 h, with maximum decreases being observed by 24 h, a time interval which preceded the inhibition of cellular proliferation by this agent. Pyrimidine nucleoside triphosphate levels were markedly increased under these conditions. The findings indicate the importance of purine nucleotides to both the inhibition of growth and the induction of differentiation of HL-60 leukemia cells by DDATHF.
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PMID:Induction of HL-60 leukemia cell differentiation by the novel antifolate 5,10-dideazatetrahydrofolic acid. 275 15

A selected number of 1,3-diaminobenzo[f]quinazolines and 1,3-diamino-5,6-dihydrobenzo[f]quinazolines, which may be viewed as tricyclic analogues of the lipid-soluble antifolates pyrimethamine (PM), metoprine (DDMP), and etoprine (DDEP), were tested as inhibitors of purified dihydrofolate reductase (DHFR) from WI-L2 lymphoblasts, and as inhibitors of the growth of Streptococcus faecium ATCC 8043 and L1210 murine leukemia cells in culture. In addition, these tricyclic compounds were tested for antimalarial activity against Plasmodium berghei in mice, and for the ability to inhibit the growth of Pneumocystis carinii trophozoites in WI-38 human lung fibroblast cultures in the presence of leucovorin (LV). The most potent analogues were those with chlorine substitution in the ring distal to the 2,4-diaminopyrimidine moiety. Fully aromatic compounds tended to be more active than those in which the 5,6-bond was reduced, suggesting that planarity favors binding to the DHFR active site and may be favorable for cellular uptake. Several of the 2,4-diaminopyrimidine analogues showed greater potency than PM, DDMP or DDEP, and were more nearly comparable to the bicyclic 2,4-diaminopyrimidine antifolates trimetrexate (TMQ) or piritrexim (BW301U), which are known to be selectively toxic to P. carinii in the presence of LV. Two of the tricyclic compounds, 1,3-diamino-8-chlorobenzo[f]quinazoline and 1,3-diamino-9-chlorobenzo[f]quinazoline, proved to have activity similar to TMQ and BW301U in this system.
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PMID:Tricyclic 2,4-diaminopyrimidines with broad antifolate activity and the ability to inhibit Pneumocystis carinii growth in cultured human lung fibroblasts in the presence of leucovorin. 278 20

In order to explore the potential of retrovirus vectors for efficiently transferring foreign genes into mouse embryos, a replication-competent recombinant Moloney murine leukemia virus (Mo-MLV) vector carrying a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat was used to infect pre- and postimplantation embryos. When preimplantation mouse embryos were infected with the vector, as expected, the provirus integrated into the embryos and the germ line with the same efficiency as that observed with wild-type Mo-MLV, leading to inactivation of the recombinant virus. In contrast, when postimplantation mouse embryos were microinjected with virus-producing cells, between 90 to 100% of the surviving animals proved to be infected with the virus. The recombinant virus spread as efficiently as wild-type Mo-MLV in the infected embryos, resulting in up to three to five proviral copies per genome in heart, thymus, and brain tissues. Substantial expression of mutant DHFR*-coding viral message was found in all somatic tissues analyzed, the amounts correlating with the proviral copy number in the respective organ. These results suggest that replication-competent vectors are useful for efficient transfer and expression of foreign genes into tissues or whole animals when virus spread is needed.
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PMID:Transfer of a mutant dihydrofolate reductase gene into pre- and postimplantation mouse embryos by a replication-competent retrovirus vector. 279 20

A full-length human c-myb cDNA clone has been isolated from a CCRF-CEM leukemia cell cDNA library. The plasmid vector contains simian virus 40-derived promotor, splice, and polyadenylation sequences as well as a transcription unit for a dihydrofolate reductase cDNA. We have introduced this construct into Friend erythroleukemia (F-MEL) cells and have isolated a number of clones which contain intact and transcriptionally active human c-myb sequences. F-MEL clones expressing the highest levels of the human c-myb mRNA differentiate poorly in response to dimethyl sulfoxide. Two clones which initially expressed low levels of human c-myb transcripts and which differentiated normally were subsequently inhibited in their ability to differentiate when grown in successively higher concentrations of methotrexate, due to amplification and enhanced expression of plasmid sequences. The inhibitory effect on F-MEL differentiation appeared to be independent of the early decline in c-myc transcripts which were normally regulated in all cases examined. Our results indicate that constitutive expression of a nontruncated human c-myb cDNA can exert profound effects on erythroid differentiation and argue for a causal role of c-myb in the F-MEL differentiation process.
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PMID:Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation. 283 42

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.
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PMID:Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line. 283 83

Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.
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PMID:Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids. 289 31

A series of replication-competent Moloney murine leukemia virus vectors was constructed in which each vector contained a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat. Two of the resulting viruses, MLV (murine leukemia virus) DHFR*-5 and MLV DHFR*-7, were able to stably transfer methotrexate resistance to infected fibroblast cells upon multiple rounds of virus replication and in the absence of drug selection. Cell lines producing recombinant virus with high titers were established, which indicated that the insert did not grossly interfere with viral replication functions. These vectors should be useful for introducing and expressing foreign genes in vivo in tissues and whole animals in which virus spread is needed for efficient infection.
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PMID:Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance. 292 89

An assay system was developed for the detection and classification of methotrexate resistance in fresh human leukemic cells. Mechanisms of resistance to be identified were: overexpression of dihydrofolate reductase, decreased cellular uptake of methotrexate, decreased affinity of dihydrofolate reductase for methotrexate, decreased polyglutamylation of methotrexate, and low thymidylate synthase activity. The initial screening procedure utilizes 3H release after addition of [5-3H]-2'-deoxyuridine as a measure of intracellular activity of thymidylate synthase and of DNA synthesis; 3H release is assayed after 3-h incubations with methotrexate, trimetrexate, or gamma-fluoromethotrexate and after 4-h incubations with these agents followed by a 6-h incubation in drug free medium. The pattern of DNA synthesis inhibition and recovery under these two sets of conditions establishes the presence or absence of methotrexate resistance and allows a tentative classification of the resistance mechanism involved. In combination with determinations of dihydrofolate reductase activity, methotrexate titration studies, and the determination of intracellular drug accumulations in vitro, the system is readily able to classify CCRF-CEM human leukemia cell lines possessing well defined mechanisms of resistance. The findings in seven leukemia patients are also reported. Applying tentative reference values, four patients showed biochemical evidence of methotrexate resistance: two patients had only a transport defect, one patient had evidence of both a transport defect and low thymidylate synthase activity, and one patient appeared to have decreased methotrexate polyglutamylation. Application of the assay system in larger numbers of patients is feasible and is required to establish adequate reference values for the evaluation of biochemical-clinical correlates.
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PMID:Development of an assay system for the detection and classification of methotrexate resistance in fresh human leukemic cells. 294 4

Trimetrexate, a new nonclassical antifolate, was evaluated in a phase I trial in children with refractory cancer including nine with acute leukemia and 21 with solid tumors. The drug was administered as an i.v. bolus injection weekly for three doses, and courses were repeated every 28 days. The dose ranged from 35 to 145 mg/m2. Thirty patients who received a total of 33 courses were evaluable for toxicity, including 19 who were evaluable for hematological toxicity. The maximally tolerated dose for patients with a solid tumor and leukemia was 110 mg/m2. The dose-limiting toxicities were myelosuppression, mucositis and a pruritic, diffuse maculopapular rash. Other side effects observed included transient, mild elevations of serum transaminases, mild nausea and vomiting, and a local phlebitis at the site of injection at higher dose levels. A single patient with delayed drug clearance had evidence of renal toxicity with a transient increase in serum creatinine. The pharmacokinetics of trimetrexate were studied in 25 patients over the entire dose range. There was considerable interpatient variability in total drug clearance (range 9.2 to 215 ml/min/m2) and half-life (2.1 to 20 h). There was a suggestion of a correlation between plasma concentration at 24 h and the development of hematological toxicity at the highest dose level. Trimetrexate was cleared primarily by biotransformation with renal clearance accounting for only 10% of total clearance. Two metabolites of trimetrexate which inhibit the enzyme dihydrofolate reductase were identified in the urine. One of these appears to be a glucuronide conjugate.
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PMID:Pediatric phase I trial and pharmacokinetic study of trimetrexate. 295 48


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