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)

In order to increase the retention of drug activity, regiospecific coupling has been used to synthesize conjugates of methotrexate (MTX, 1) with normal rabbit IgG (NRG) and a mouse anti-human renal cancer monoclonal IgG (Dal K-20). MTX gamma-methyl ester (4) was produced either by selective esterification of MTX or by coupling of 4-amino-4-deoxy-N10-methylpteroic acid (2) with suitable glutamic acid derivatives. The MTX gamma-methyl ester (4) was then converted to the corresponding hydrazide 6. An amide-linked conjugate was formed when the MTX gamma-hydrazide (6) was converted to reactive acylating species 7 by using tert-butyl nitrite or trifluoroacetaldehyde, which were reacted with nucleophilic centers, presumably epsilon-amino groups, in native IgG. A hydrazone-linked conjugate was formed when MTX gamma-hydrazide (6) was reacted directly with IgG that had first been oxidized with periodate to form polyaldehyde IgG. The regiospecifically synthesized conjugates were somewhat more effective inhibitors in vitro of dihydrofolate reductase and of colony formation by human renal cancer (Caki-1) cells than were control nonregiospecific conjugates.
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PMID:Synthesis of methotrexate-antibody conjugates by regiospecific coupling and assessment of drug and antitumor activities. 281 Mar 30

During selection for methotrexate resistance, SV40-transformed human skin fibroblasts from patients with ataxia telangiectasia (A-T) underwent amplification of the dihydrofolate reductase (DHFR) gene, experienced nearly complete loss of the integrated SV40 sequences and showed a 3.6-fold increase in Ki-ras gene copy number. Over a period of months methotrexate-resistant (MTXr) A-T subclones were obtained, which were able to grow in progressively increasing MTX concentrations up to 100 microM. The ED50 values determined as the effective dose of MTX causing 50% growth inhibition in comparison to control cells increased from 3 x 10(-2) microM for MTXs AT5BI-VA cells to 250 microM MTX for the MTXr AX100 subclone. In contrast, human skin fibroblasts of healthy individuals did not show DHFR gene amplification and loss of SV40 sequences under comparable conditions and were unable to grow in MTX concentrations greater than 1 microM. Gene amplification and loss of DNA sequences are features underlying the genomic instability known to be a characteristic property of A-T cells and being probably responsible for the high cancer incidence in these patients.
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PMID:DHFR gene amplification in cultured skin fibroblasts of ataxia telangiectasia patients after methotrexate selection. 282 82

The biochemical rationale for the potentiation of the effects of 5-FU by MTX is based on an increased PRPP level or MTX polyglutamate produced by MTX. The cytotoxic action of MTX results not only from inhibition of DHFR but also depends upon thymidylate synthetase (TS), a key enzyme in DNA synthesis. We obtained a monoclonal antibody to TS using a hydrophilic peptide consisting of 20 amino acids in the TS amino acid sequence and demonstrated by PAP that TS was detectable in poorly differentiated adenocarcinoma cells but not in well differentiated adenocarcinoma cells. Upon clinical application of sequential doses of MTX and 5-FU, the median survival durations were 318 days and 156 days for scirrhous-type gastric cancer patients and non-scirrhous-type gastric cancer patients respectively. These results suggest that immunohistochemistry with TS antibody is available as an indicator of the effect of this drug regimen.
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PMID:[The role of thymidylate synthetase in sequential dose of MTX and 5-FU in the advanced scirrhous type gastric cancer]. 283 88

The mechanisms of acquired resistance to MTX were studied in P388 murine leukemia cell lines that were sensitive or resistant to ADR. The rate of MTX accumulation in ADR-sensitive cells that have acquired resistance to MTX was found to be lower than that measured in cells that were sensitive to both drugs. Furthermore, in contrast to drug-sensitive cells, in the ADR-sensitive MTX-resistant cells, most of the intracellular MTX (86.2%) was bound and MTX polyglutamation was not detected. The initial rate of MTX accumulation in cells that were resistant to both drugs was comparable to that measured in cells that were sensitive to both drugs or that were resistant only to ADR. However, in the cells that were resistant to both drugs, the rate of MTX accumulation was maintained at its initial level for a period that was considerably longer than that found in the other cell lines. After 3 h of exposure to MTX, the accumulation of MTX in cells that were resistant to both drugs was fourfold higher than that measured in cells that were sensitive to both drugs. Furthermore, while 65 to 70% of the intracellular MTX was free, in cells sensitive to both drugs, or resistant only to ADR, the corresponding value in cells that were resistant to both drugs was less than 1.5%, and a much lower proportion of the MTX was polyglutamated. The sensitivity to TMQ of ADR-sensitive, MTX-resistant cells was similar to that found in cells that were sensitive to ADR and MTX. However, ADR-resistant cells, sensitive or resistant to MTX, were markedly resistant to TMQ. The sensitivity of ADR-resistant MTX-sensitive cells to TMQ was restored by the presence of 10 microM verapamil. Such an effect was not observed in cells resistant to both drugs. It is suggested that P388 cells that have previously acquired resistance to ADR, when now selected by MTX, retain the MTX-transport system (in contrast to ADR-sensitive, MTX-resistant cells) and become resistant to MTX by increasing the activity of DHFR. The results obtained in ADR-resistant cells also suggested that resistance to TMQ was part of the multidrug resistance phenomenon.
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PMID:Mechanism of acquired resistance to methotrexate in P388 murine leukemia cells and in their doxorubicin-resistant subline. 297 48

The mechanisms of action of MTX and 5-FU have been further elucidated. Such studies will be important for the design of drug combinations and for the development of novel antifolate and fluoropyrimidine analogs. A greater understanding of MTX and ara-C transport and drug levels required to optimize transport may also aid in these endeavors. Pharmacokinetic parameters have been found to be predictors of relapse in children with acute leukemia, particularly with respect to MTX, 6-MP and ara-C. The intracellular terminal half-life of ara-C was correlated with remission duration in AML. Assay systems aimed at uncovering response predictors through biochemical analysis of patient tumor samples are being developed, including an interesting use of NMR spectroscopy to study the pharmacokinetics of fluorine-19-labeled 5-FU in vivo. Such an approach may yield valuable information on 5-FU anabolism in tumors in situ. A high frequency of resistance to MTX apparently may be generated within a single cell cycle by transient exposures to DNA synthesis inhibitors. The resistance may be based on either target enzyme amplification or altered membrane transport. These important studies provided bases for the rapid emergence of clinical resistance. Further, the multidrug-resistant phenotype appears to be a much broader based phenomenon as MTX resistance was found to be a frequent event in cells selected for multidrug resistance. A variety of novel approaches have been proposed to overcome antimetabolite resistance and to improve the selectivity of these agents, including the use of guanosine nucleotides, leucovorin and allopurines as biochemical modulators of 5-FU. Efficient techniques for the transfection of resistant DHFR into tissues using retroviruses have been reported. These studies serve as starting point for the ultimate development of more effective strategies for the treatment of human malignancies.
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PMID:Antimetabolites. 307 79

The response of hemopoietic and/or lymphosarcoma cells to methotrexate has been investigated. The proliferative capacity of hemopoietic tissue was measured by exogenous CFU assay. It was inhibited by a six times lower amount of MTX than the proliferative activity of lymphosarcoma cells as evaluated by liver-colony assay. The natural resistance of LS/BL lymphosarcoma cells to MTX might be attributed to the DHFR gene amplification resulting from DNA rearrangements appearing during malignant transformation.
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PMID:The cytotoxic effect of methotrexate as evaluated by colony-forming activity of hemopoietic and tumor cells. 321 32

To develop a highly efficient means for generating methotrexate resistant (MTXr) hematopoietic cells in vivo, a recombinant retroviral genome was constructed that encodes a MTXr dihydrofolate reductase (DHFRr). Cell lines producing high titers of virus capable of transmitting the DHFR gene were generated and used to infect mammalian cells in vitro. Analysis of infected fibroblasts indicated that the DHFRr gene was transmitted intact and conferred a high level of MTXr upon cells. Based on these findings, DHFRr-containing virus was used to infect murine bone marrow cells in vitro. Following infection, the transduced cells were introduced into lethally irradiated recipients via bone marrow transplantation techniques. The presence of the proviral sequences in cells of the spleen and bone marrow of engrafted recipients was associated with significantly increased survival of mice treated with otherwise lethal doses of MTX.
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PMID:Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate-resistant bone marrow. 329 24

A series of 5,8-dideaza analogues of folic acid, isofolic acid, aminopterin, and isoaminopterin were evaluated for inhibition of thymidylate synthase, TS, from mouse L1210 leukemia cells with 10-propargyl-5,8-dideazafolic acid, CB3717, 4a, as the reference inhibitor. These compounds were also tested as inhibitors of human dihydrofolate reductase, DHFR, obtained from WIL2 cells. None of the analogues studied were as potent as 4a toward TS; however, 9-methyl-5,8-dideazaisoaminopterin, 6d, was only 2.5-fold less effective. Compound 4a was prepared by direct alkylation of the di-tert-butyl ester of 5,8-dideazafolic acid followed by hydrolysis of the resulting diethyl ester, which resulted from concomitant transesterification. It was found to be identical with a sample of 4a prepared by earlier methodology by using a variety of spectroscopic techniques. Its isomer, 9-propargyl-5,8-dideazaisofolic acid, 4b, which was synthesized by an analogous approach, was found to be dramatically less inhibitory toward TS than 4a. Each of the 2,4-diamino derivatives, including those possessing an allyl or propargyl group at N9, was an excellent inhibitor of DHFR, having a level of potency similar to that of methotrexate, MTX. However, many of these 5,8-dideazaaminopterin analogues were far more inhibitory toward TS than MTX.
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PMID:Inhibition of murine thymidylate synthase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin. 333 15

Plasmid DNA containing EDF subunit cDNA and mouse dihydrofolate reductase (DHFR) cDNA was transfected into CHO DHFR- cells by the calcium-phosphate method. DHFR positive transformants secreted recombinant EDF (r-EDF) constitutively in an active form and accumulated it in the conditioned medium. Furthermore, cells which were resistant to methotrexate (MTX : 0.5 microM) secreted r-EDF up to 1 microgram/ml. r-EDF was identical to natural EDF (n-EDF) produced by human acute monocytic leukemia cell line, THP-1, as regards its dimeric structure and a biological activity.
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PMID:Expression of erythroid differentiation factor (EDF) in Chinese hamster ovary cells. 334 75

Lipophilic gamma-monoamide derivatives of aminopterin (AMT) were synthesized in high overall yield from 4-amino-4-deoxy-N10-formylpteroic acid and gamma-N-tert-alkyl-, gamma-N-aralkyl-, or gamma-N-arylamides of alpha-benzyl L-glutamate via a modification of the mixed carboxylic-carbonic anhydride coupling method. Coupling was also accomplished with p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate. Compounds obtained in this manner included the gamma-tert-butylamide, gamma-(1-adamantylamide), gamma-benzylamide, gamma-(3,4-dichlorobenzylamide), gamma-(2,6-dichlorobenzylamide), gamma-anilide, gamma-(3,4-methylenedioxyanilide), and gamma-(3,4-dihydroxanilide) derivatives of AMT. Also prepared, from 4-amino-4-deoxy-N10-methylpteroic acid via diethyl phosphorocyanidate coupling, was the gamma-(3,4-methylenedioxyanilide) of MTX. The methylenedioxyanilides were cleaved smoothly to dihydroxyanilides with boron tris(trifluoroacetate) in trifluoroacetic acid. All the gamma-monoamides were tested as inhibitors of purified dihydrofolate reductase (DHFR) from murine L1210 leukemia cells and as inhibitors of the growth of wild-type L1210 cells and a subline (L1210/R81) with high-level resistance to MTX and AMT based mainly on a defect in drug uptake via active transport. Several compounds were also tested against human leukemic lymphoblasts (CEM cells) and a resistant subline (CEM/MTX) whose resistance is likewise based on uptake. The IC50 of the gamma-monoamides against DHFR was 1.5- to 5-fold higher than that of the parent acids, but the IC50 against cultured cells varied over a much broader range, suggesting that uptake and/or metabolism rather than DHFR binding are principal determinants of in vitro growth inhibitory activity for these compounds. gamma-N-Aryl and gamma-N-aralkyl derivatives appeared to be more potent than gamma-N-tert-alkyl derivatives. Where comparison could be made, AMT gamma-monoamides were more potent than MTX gamma-monoamides. Several of the gamma-monoamides showed potency comparable to that of the parent acid against wild-type L1210 and CEM cells; all of them were more potent than MTX against the L1210/R81 subline; and some of the AMT gamma-monoamides were also more potent than the parent acid against resistant CEM/MTX cells. As a group, however, the gamma-monoamides were considerably more active against the murine cells than against the human cells, suggesting that the former may take up the amides better or may be able to metabolize them more efficiently than the parent acids. All the gamma-monoamides were tested in vivo against L1210 leukemia in mice.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Methotrexate analogues. 28. Synthesis and biological evaluation of new gamma-monoamides of aminopterin and methotrexate. 346 94


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