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

---

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

PG19T3 mouse melanoma cells were selected for resistance to methotrexate. Nine sub-lines that are resistant to concentrations of methotrexate ranging from 1.27 x 10(-7) M, to 1 x 10(-4) M methotrexate were selected and characterised in terms of their content of dihydrofolate reductase activity and their chromosomes. The intracellular level of dihydrofolate reductase activity increases with increasing resistance such that at the highest level of resistance PG19T3:MTXR10(-4)M cells contain approximately 1,000 fold more enzyme activity than the parental PG19T3 cells. It is shown that the enhanced activity is due to an increase in the amount of the enzyme rather than any structural change to the enzyme in resistant cells. Comparisons of pH activity profiles, profiles under different activating conditions and titrations with methotrexate suggest that the sensitive and resistant cells contain identical dihydrofolate reductases. Analysis of the chromosomes of resistant cells shows the presence of up to 5 large marker chromosomes which contain homogeneously staining regions after G-banding. These same regions stain intensely after C-banding and fluoresce brightly after staining with Hoechst 33258. The size of homogeneously staining regions increases throughout the process of selection. For one marker chromosome this increase may have been mediated via a ring chromosome.
...
PMID:The development of resistance to methotrexate in a mouse melanoma cell line. I. Characterisation of the dihydrofolate reductases and chromosomes in sensitive and resistant cells. 51 81

Bowes melanoma cells, which naturally produce tissue-type plasminogen activator (t-PA), were transfected with a plasmid containing a human t-PA cDNA under transcriptional control of the promoter/enhancer of the major immediate early gene of human cytomegalovirus (CMV) plus genes expressing geneticin (G418) resistance and dihydrofolate reductase (DHFR). In one of the initial geneticin-resistant transformants, t-PA mRNA transcribed from the chromosomally integrated plasmid had the same short half-life, 20-30 min, as did mRNA transcribed from the endogenous t-PA gene compared to 7-8 h for total poly(A)+ mRNA. After subsequent selection of such cells with methotrexate, a cell line was obtained in which the t-PA cDNA construct was co-amplified with the DHFR gene and which produced 10 times more t-PA protein than the original Bowes melanoma cells.
...
PMID:Endogenous gene and amplifiable cDNA construct both produce unstable t-PA mRNA in Bowes melanoma cells. 136 55

The rationale for melanoma-specific antitumor agents containing phenolic amines is based in part on the ability of the enzyme tyrosinase to oxidize these prodrugs to toxic intermediates. The phenolic amine compounds 4-S-cysteaminylphenol (4-S-CAP) and N-acetyl-4-S-cysteaminylphenol (N-Ac-4-S-CAP) inhibited in situ thymidylate synthase activity in pigmented melanoma cell lines but had little or no effect on nonpigmented and nonmelanoma cell lines. Theophylline, a cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor, increased tyrosinase activity and potentiated the inhibition of in situ thymidylate synthase by N-Ac-4-S-CAP. The inhibition of in situ thymidylate synthase by both drugs in pigmented melanoma cells correlated with the inhibition of DNA synthesis and cell growth and was not due to an indirect effect caused by inhibition of the enzyme dihydrofolate reductase. 4-S-CAP inhibition of thymidylate synthase activity in cell free extracts required oxidation of the drug. In the presence of tyrosinase, the concentration causing a 50% inhibition of thymidylate synthase activity (IC50) in cell-free extracts was less than 10 microM, but no inhibition was observed in its absence, even at a drug concentration of 500 microM. Two reducing agents, dithioerythritol and glutathione, effectively blocked the inhibition of thymidylate synthase by oxidized 4-S-CAP. In pigmented melanoma cells containing the enzyme tyrosinase, the quinone-mediated mechanism of inhibition of DNA synthesis via inhibition of thymidylate synthase may be uniquely important in the expression of phenolic amine cytotoxicity.
...
PMID:Thymidylate synthase as a target enzyme for the melanoma-specific toxicity of 4-S-cysteaminylphenol and N-acetyl-4-S-cysteaminylphenol. 150 78

Methotrexate (MTX), 6-thioguanine (6-TG) and cytosine arabinoside (ara-C) inhibited the replication of adenovirus (viral capacity) more in drug-sensitive than in resistant human melanoma cell lines. By comparison, inhibition of cellular DNA and RNA synthesis after short treatment periods (less than 48 hr) was not a good predictor of cellular sensitivity. MTX, an inhibitor of de novo nucleotide synthesis, was most effective when added to cells just before infection with virus and inhibited viral capacity at doses 10-1000-fold lower than those required to affect cell survival. The MTX-sensitive cell lines, members of a DNA repair deficient group sensitive also to killing by methylating agents (the Mer- phenotype), were not deficient in dihydrofolate reductase but exhibited DNA fragmentation after treatment with MTX for 48 hr. 6-TG and ara-C, inhibitors of purine and pyrimidine salvage, were most inhibitory to viral capacity when added greater than 36 hr before virus infection and were less effective than MTX (doses 5-7-fold and 4-24-fold higher than for cell survival respectively). No correlation was found between MTX sensitivity and sensitivity to 6-TG or ara-C. These results indicate that (i) inhibition of viral capacity is a more comprehensive test of antimetabolite cytotoxicity than inhibition of cellular DNA or RNA synthesis; (ii) the viral capacity assay correctly predicts cellular sensitivity to MTX, 6-TG and ara-C and therefore has potential for application to primary cultures of human tumours; and (iii) MTX-sensitive cell lines and adenovirus replication rely heavily on de novo nucleotide synthesis, which in Mer- cells appears to be linked to a DNA repair defect as yet undefined.
...
PMID:Effects of antimetabolites on adenovirus replication in sensitive and resistant human melanoma cell lines. 168 77

The gene that encodes the membrane-bound Mr 100,000 human melanoma-associated antigen (MAA) defined by mouse mAb 376.96, a leukocyte and fibroblast interferon-modulated glycoprotein having preferential distribution on melanoma and carcinoma cells, has been transfected into the mouse melanoma cell line B78H1 as a step toward molecular cloning and characterization of the MAA. Primary, secondary, and tertiary B78H1 transfectants expressing the Mr 100,000 MAA gene were generated by treatment with coprecipitated DNA from Mr 100,000 MAA+ human or transfectant mouse cells and they were detected by an indirect RBC rosetting assay. The Mr 100,000 MAA gene was also transferred into K-1735 mouse melanoma cells and into nonmalignant and malignant mouse fibroblast lines. The species immunoprecipitated by mAb 376.96 from human melanoma cells (Mr 100,000) and from mouse melanoma transfectant cells (Mr 97,000-100,000) were both converted to molecule(s) having an Mr of approximately 70,000 by enzymatic removal of asparagine-linked carbohydrate residues. Two independent secondary transformant clones of B78H1 cells express Mr 100,000 MAA antigenicity at levels significantly higher than those observed when one or two copies of the gene are present. Clone Mr 100,000 secondary-A spontaneously overexpresses Mr 100,000 MAA at least 5-fold and has greater than or equal to 10 times elevated levels of putatively Mr 100,000 MAA gene-associated human alu family repeat element (h-alu)-positive restriction fragments relative to "single" copy secondary transfectant cells. Clone Mr 100,000 secondary-B has increased copy number and expression of Mr 100,000 MAA as a consequence of a selective co-amplification procedure which is targeted to a mouse wild type dihydrofolate reductase (dhfr) gene expression vector. This vector was co-introduced into B78H1 cells in addition to the DNA of Mr 100,000 MAA+ primary transfectant cells and the initially selected aminoglycoside phosphotransferase (neo) gene vector. Stepwise selections of a secondary Mr 100,000 MAA+ transfectant clone with increasing concentrations of the dihydrofolate reductase-inhibitory antimetabolite methotrexate led to progressive increases in copy numbers of the introduced dhfr gene and to parallel increases in h-alu sequences, in cellular levels of dihydrofolate reductase protein, and in cellular mAb 376.96 reactivity. Levels of these entities ultimately reached 50-fold, relative to levels expressed prior to amplification. The array of h-alu+ restriction fragments amplified in Mr 100,000 secondary-B cell DNA is very similar to that observed in Mr 100,000 secondary-A cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Interspecific DNA-mediated transfer and amplification of a gene specifying a Mr 100,000 human melanoma-associated cell surface glycoprotein. 230 16

The new folate analog 10-ethyl-10-deaza-aminopterin (10EdAM) was equivalent to methotrexate (MTX) as an inhibitor of dihydrofolate reductase, but was more effectively transported and polyglutamylated in most tumor cells. Also, the transport and polyglutamylation of 10EdAM in tumor cells vis-a-vis normal proliferative tissue is substantially increased compared to MTX, favoring much greater accumulation of 10EdAM as cytotoxic polyglutamates in some of these tumor cells. 10EdAM was superior to MTX against 4 of 6 murine ascites tumors (L1210, S180, Ehrlich and Tapper) and far superior against 4 of 6 solid murine tumors (S180, Tapper, E0771 mammary AC, T241 fibrosarcoma). 10EdAM produced 10% to 30% complete regressions against S180, E0771 and T241 tumors. Both agents showed similar activity against P288 and 1498c leukemias and the Lewis lung tumor, but were inactive against B16 melanoma. Marked superiority of 10EdAM compared to MTX was also shown against the following human tumor xenografts: MX-1 (mammary carcinoma), LX-1 (small cell lung carcinoma) and CX-1 (colon carcinoma). 10EdAM produced 30% to 40% complete regressions against the MX-1 tumor.
...
PMID:10-Ethyl-10-deaza-aminopterin: structural design and biochemical, pharmacologic, and antitumor properties. 244 50

Trimetrexate (TMQ; NSC 352122) is a potent inhibitor of dihydrofolate reductase with good activity against murine i.p.-implanted B16 melanoma and colon 26 tumors. Preclinical antineoplastic activity, demonstrated schedule dependency, and data suggesting effectiveness against methotrexate-resistant cells prompted a Phase I clinical and pharmacokinetic study of trimetrexate using an i.v. daily x5 schedule. Forty-three good performance status patients were treated with 12 dose levels using daily doses varying from 0.5 to 15 mg/m2/d. Plasma and urine samples were obtained for pharmacokinetic analysis using a high-performance liquid chromatographic method. Myelosuppression was dose limiting and 15 mg/m2/d x5 was the maximum tolerated dose. White blood cell (WBC) and platelet toxicity were noted at doses of 1.6 mg/m2 and above. Median WBC and platelet nadirs occurred on approximately Days 11-12 with recovery by Days 15-18. Nonhematological toxicity included mucositis, nausea and vomiting, stomatitis, diarrhea, and rash. Evidence for antitumor activity was seen in seven patients. Trimetrexate elimination from plasma could be represented as either a bi- or triexponential process. Terminal elimination half-lives were in the range of 5-14 h in patients represented by a triexponential model. Approximately 10-20% of the dose administered was excreted in urine over a 24-h period. The recommended starting dose for patients in Phase II trials using the d x5 i.v. schedule is 8.0 mg/m2/d repeated every 21 days. Dose escalations may be possible depending on the extent of prior therapy and individual tolerance of the drug.
...
PMID:Phase I clinical and pharmacokinetic study of trimetrexate using a daily x5 schedule. 297 Feb 94

Chloroquinoxaline sulfonamide (CQS), a chlorinated derivative of sulfaquinoxaline (SQ), inhibited proliferation of murine B16 melanoma cells, but only when relatively high drug concentrations (1 mM) were used. The inhibition of cell growth by CQS was at least partially reversible by incubation in drug-free medium. Incubation of melanoma cells with CQS was associated with an arrest of the cell cycle in G0/G1 as measured by flow cytometry. The drug slightly decreased uptake of radiolabeled deoxyuridine and thymidine after 24- and 48-hr incubation periods but increased nucleoside incorporation at 72 hr. No evidence of intercalation with DNA was found. Because SQ previously was reported to inhibit an aspect of folate metabolism, we investigated the possibility that CQS limits tumor cell growth by altering folate homeostasis. This appears unlikely, however, in view of the following observations: (1) the cytotoxic effects of CQS could not be reversed by folinic acid; (2) deoxyuridine suppression of thymidine incorporation was not affected by CQS treatment; (3) CQS did not inhibit dihydrofolate reductase from mammalian or bacterial sources; and (4) CQS toxicity in mice was not reduced by folinic acid. Experiments performed with analogues modified in the quinoxaline and para-amino phenyl functions indicated that tumor cell inhibition did not require preservation of the conventional sulfonamide structure.
...
PMID:Cellular pharmacology of chloroquinoxaline sulfonamide and a related compound in murine B16 melanoma cells. 326 1

[3H] Methotrexate [( 3H]MTX) was covalently linked to monoclonal antibody (MoAb) 225.28S against human melanoma, to a rabbit anti-human melanoma IgG absorbed either with human red blood cells (AHMGR) or with red blood cells and a variety of normal human tissues (AHMGR + T), or to normal rabbit IgG (NRG). Human melanoma M21 cells were incubated at 0 degrees C or 37 degrees C with 10 microM free MTX or 10 microM MTX linked to one of the above carriers. The order of net uptake of MTX during 6 hours was MTX-MoAb 225.28S greater than MTX-AHMGR greater than MTX-AHMGR + T greater than MTX-NRG greater than or equal to MTX. This order of uptake by the three antibody conjugates corresponded to the amount of conjugate bound at equilibrium at 0 degrees C and to the immunofluorescence titers. Binding sites for MoAb 225.28S were more efficient for internalization of MTX than were those for the two polyclonal antibody preparations. When M21 cells preloaded with MTX by incubation at a drug concentration of 1.0 or 10 microM were incubated in drug-free medium, the amount of cell-associated MTX rapidly declined to 1.8 pmol/mg protein, i.e., the level of intracellular dihydrofolate reductase (DHFR). However, when cells preloaded to a drug content of 112 pmol/mg protein by incubation with 10 microM MTX linked to AHMGR were transferred to conjugate-free medium, 65 pmol MTX/mg remained cell associated after 12 hours. The efflux was inhibited by chloroquine. Both the efflux medium and M21 cells after a 9.5-hour incubation period had MTX-containing catabolic fragments that inhibited DHFR.
...
PMID:Uptake of methotrexate linked to polyclonal and monoclonal antimelanoma antibodies by a human melanoma cell line. 385 85

The additional segments of five large marker chromosomes of methotrexate-resistant mouse melanoma PG19T3 cells are shown to consist of C-banding material. In situ hybridization indicates that these additional segments also contain a high proportion of sequences that will cross hybridize with mouse satellite DNA. Analytical density gradient centrifugation suggests that up to 60% of the DNA in the additional segments may be in the form of satellite DNA. Incorporation of bromodeoxyuridine for one complete S phase and staining with the Hoechst 33258 fluorescence plus Giemsa technique reveals complex asymmetries within the additional segments. These asymmetries are interpreted as showing a large repeating unit, which is likely to be involved in the amplification of the dihydrofolate reductase gene.
...
PMID:Satellite DNA in large marker chromosomes of methotrexate-resistant mouse cells. 736 27


1 2 3 Next >>

---