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)

Efforts to use fresh human sarcoma cells for evaluating antifolate resistance with an in situ thymidylate synthesis assay using 5-[3H] deoxyuridine were unsuccessful because of low thymidylate synthesis activity in enzymatically disaggregated tumors. By incubating tumor cell suspensions in supplemented RPMI-1640 medium with 10% fetal bovine serum for 3 days, activity of the in situ thymidylate synthesis assay markedly increased (1.42 versus 0.03 pmol/h/10(7) cells), thus allowing 75% of samples to be evaluated for antifolate sensitivity. By criteria developed with a methotrexate-resistant and -sensitive cell line, this assay indicated that most sarcomas are naturally resistant to methotrexate (12 of 15). Natural resistance to 10-ethyl-10-deazaaminopterin and trimetrexate was also observed in 60% of the samples (nine of 15, respectively). The results from the 3-day in situ assay were confirmed by specific tests for resistance mechanisms in most sarcoma samples. The resistance mechanisms detected were impaired polyglutamylation, an increased level of dihydrofolate reductase, and amplification of this gene. These results encourage further exploration of this assay to predict response to antifolates in individual patients and to evaluate efficacy of new antifolates as candidates for clinical trial.
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PMID:Mechanisms of natural resistance to antifolates in human soft tissue sarcomas. 137 15

A human fibrosarcoma cell line, HT-1080, and four new cell lines (HS-16, HS-28, HS-30, and HS-42) were established from untreated patients with mesenchymal chondrosarcoma, peripheral nerve sheath sarcoma, malignant hemangiopericytoma, and mixed mesodermal tumor, respectively, and were used for analysis of mechanisms of intrinsic resistance to methotrexate. All four new cell lines were resistant to methotrexate as determined by inhibition of thymidylate synthase in whole cells and by growth inhibition, as compared with HT-1080, a methotrexate sensitive cell line. Methotrexate uptake, level of dihydrofolate reductase, and inhibition of this enzyme by methotrexate in the four cell lines were comparable to HT-1080 cells. However, levels of long chain polyglutamates (glu3-5) of methotrexate achieved after a 24-h incubation with this drug were much lower in the four new cell lines as compared to the HT-1080 cell line (5- to 20-fold lower). The low levels of methotrexate polyglutamates formed is likely the major cause of intrinsic methotrexate resistance in these new sarcoma cell lines.
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PMID:Intrinsic resistance to methotrexate in human soft tissue sarcoma cell lines. 137 1

A methylcholanthrene-induced rat sarcoma that can be propagated in vitro or in vivo was evaluated for resistance to antifolates and was found to be relatively resistant to methotrexate and 10-ethyl-10-deazaaminopterin but sensitive to trimetrexate. Rat sarcoma cell extracts contained low levels of dihydrofolate reductase activity, the target enzyme of methotrexate, and inhibition of this enzyme by these three antifolates was similar. Transport studies showed poor uptake of both methotrexate and 10-ethyl-10-deazaaminopterin. In contrast, trimetrexate achieved high intracellular levels. The poor uptake of methotrexate was not due to lack of polyglutamylation. Thus, the basis for natural resistance to methotrexate and 10-ethyl-10-deazaaminopterin, compared with trimetrexate, in this rat sarcoma cell line was due to decreased transport of these drugs.
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PMID:Mechanisms of sensitivity and natural resistance to antifolates in a methylcholanthrene-induced rat sarcoma. 171 70

The methotrexate concentrations in the lungs or cutaneous metastases of patients with osteogenic or soft-tissue sarcoma were determined at different times after a high-dose methotrexate therapy. The levels in the metastases were 0.964 to 2.96 X 10(-7) molar six to nine days after the end of MTX infusion. They were thus 7.8 to 28 times higher than the corresponding serum levels. At the same time, an appreciable rise of dihydrofolate reductase activity was observed in the metastases. After chromatographic separation over Sephadex G15, MTX polyglutamates could be demonstrated in all tumor samples investigated so far; these amounted up to 68.3% of the total MTX. Taking into account the slower efflux of MTX polyglutamates compared to unchanged MTX, a new hypothesis for the principle of action of high-dose methotrexate therapy is discussed: the very high MTX doses lead to such high intracellular MTX concentrations even in transport-resistant tumor cells that at least part of the MTX is converted into MTX polyglutamates. Unchanged MTX flows relatively rapidly out of the cells, whereas the MTX polyglutamates only break down very slowly and thus can be cytostatically effective over a long period of time.
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PMID:Methotrexate and methotrexate polyglutamates in human sarcoma metastases after high-dose methotrexate therapy. 619 16

The transcriptional promoter of the Harvey sarcoma virus long terminal repeat has been used to construct a biologically active dihydrofolate reductase chimera. The construction placed the long terminal repeat at the 5' end of a dihydrofolate reductase cDNA. This chimera mediated methotrexate resistance when introduced into wild-type NIH3T3 mouse cells by transfection. The chimeric sequences were expressed in the form of polyadenylated RNA and dihydrofolate reductase protein and were amplified when the methotrexate-resistant transfectants were selected to grow in increasing methotrexate concentrations. This chimera was dominant acting and able to confer a methotrexate-resistant phenotype on wild-type NIH3T3 cells. It has been used in cotransfection experiments with DNA from human tumor cells to obtain foci of methotrexate-resistant transformed NIH3T3 cells resulting from uptake of exogenous DNA. The transfected methotrexate-resistant cells carried double minute chromosomes that appeared to contain DNA acquired during transfection.
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PMID:Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera. 629 6

A series of increasingly drug-resistant cell populations were selected and cloned from C-46 murine neuroblastoma with the chemotherapeutic drugs maytansine, vincristine, adriamycin, or Baker's antifol. All clones demonstrated reciprocal cross-resistance to these structurally and functionally diverse drugs and failed to accumulate radiolabeled vincristine, colchicine, or Baker's antifol despite normal drug binding to cell homogenates. Initial isolates of drug-resistant populations were genetically unstable, rapidly reverting to a drug-sensitive phenotype when grown without drug, at 0.05 reversion per cell division. After prolonged growth in drug, this drug-resistant genotype stabilized. Mean chromosome number increased 300% in an initially isolated 20-fold maytansine-resistant clone, which also displayed numerous double-minute chromosomes. Descendants 240-fold more resistant than the parent, also unstable, possessed the wild-type complement of 80 chromosomes, but 45% of these cells possessed 24 double-minute chromosomes per cell; such chromosomes were absent from the drug-sensitive parental clone. Only 1.0 and 1.2 double-minute chromosomes per cell were seen in a 7-fold stably resistant revertant or 1200-fold stably resistant descendants, respectively. Double-minute chromosomes containing amplified genes for the drug target dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been reported in an unstable methotrexate-resistant R1-A sarcoma. These extrachromosomal gene copies were absent in stably resistant progeny. The presence of similar particles in unstably drug-resistant uptake mutants of neuroblastoma and their diminution in stably resistant descendants supports and extends their possible role in the rapid onset and instability of epigenetic drug resistance in cancer chemotherapy.
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PMID:Correlation of double-minute chromosomes with unstable multidrug cross-resistance in uptake mutants of neuroblastoma cells. 694 68

Expression systems were developed for evaluating recombinant human thrombomodulin (TM) production in different host cell lines by investigating the performance of five mammalian expression vectors. The expression vectors were constructed so that they contain multiple monocistronic gene cassettes which include a gene encoding a dominant selectable marker, HyR (hygromycin B phosphotransferase), under the regulation of the thymidine kinase promoter, the target gene which encodes a truncated human re-TM under the regulation of various promoters, an amplifiable gene (Dhfr) encoding murine dihydrofolate reductase under the regulation of either the SV40 early or late promoter along with the SV40 enhancer and the SV40 ori. We tested the performance of the five expression vectors in human embryonic kidney cells (HEK293), baby hamster kidney cells (BHK), human melanoma cells (CHL-1) and Dhfr- Chinese hamster ovary cells (CHO/Dhfr-). We found that the efficiency of DNA uptake, transient expression and stable expression of the different expression vectors were all cell-line dependent. However, the myeloproliferative sarcoma virus (MPSV) LTR promoter consistently showed higher expression levels in all cell lines, particularly in HEK293 cells. These results were confirmed by the distribution curves of the level of expression of individual clones. Furthermore, by amplifying Dhfr in transfected CHO/Dhfr- cells with 100 nM methotrexate, we achieved a 20-fold increase in re-TM production using the SV40 late promoter to control murine Dhfr expression. Our data from DNA and mRNA analysis reveal that pMPSV-TM has a high transcription efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression efficiency of the human thrombomodulin-encoding gene in various vector and host systems. 752 46

To determine the effect of in vivo pharmacological selective pressure on the insertion and expression of new gene sequences, retroviral-mediated transfer of a methotrexate-resistant dihydrofolate reductase (Mtxr-DHFR) gene in hematopoietic tissue was investigated using a murine syngeneic bone marrow transplant system. A series of recombinant retroviral vectors were constructed to contain long terminal repeat (LTR) regions from different murine retroviruses associated with various proliferative disorders of the lymphohematopoietic system, including Moloney leukemia virus, spleen focus-forming virus (anemia strain) and myeloproliferative sarcoma virus. High-titer DHFR virus (10(7) colony-forming units/ml) was generated by gene amplification adapting virus-producer cell lines to grow in medium containing increasing concentrations of Mtx. This high-titer DHFR virus was used to introduce the Mtxr-DHFR gene into murine hematopoietic tissue by injecting DHFR virus-exposed marrow into lethally irradiated syngeneic recipient mice that subsequently were administered Mtx. Southern blot analysis of spleen DNA demonstrated insertion of the DHFR provirus in all surviving mice transplanted with DHFR virus-exposed marrow. However, enzymatic assay of crude spleen extracts demonstrated the presence of Mtxr-DHFR activity only in mice that were administered Mtx; nonadministered animals or animals transplanted with control (neo) virus-infected marrow contained undetectable drug-resistant enzyme activity. These results suggest the selective outgrowth of hematopoietic tissue harboring and expressing a DHFR provirus in animals administered Mtx and have implications for the application of drug-resistance gene insertion in somatic tissues of animals and humans.
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PMID:Selective expression of methotrexate-resistant dihydrofolate reductase (DHFR) activity in mice transduced with DHFR retrovirus and administered methotrexate. 824 74

The mechanisms of intrinsic and acquired resistance to methotrexate (MTX) in human tumors are reviewed herein. In blasts from patients with acute lymphocytic leukemia, resistance mechanisms found are decreased uptake and increased dihydrofolate reductase (DHFR) activity. A major cause of intrinsic resistance to MTX in soft tissue sarcoma cells and in acute myelocytic leukemia appears to be a lack of drug retention, due mainly to low levels of polyglutamylation. A novel association between lack of the retinoblastoma protein and intrinsic MTX resistance has been found. This has been attributed to an increase in DHFR activity, due to an increased rate of transcription of this gene, stimulated by an increase in levels of free E2F, not sequestered by hypophosphorylated retinoblastoma protein.
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PMID:Resistance mechanisms to methotrexate in tumors. 882 Sep 44

We examined the antitumor effects of two antifolate inhibitors of thymidylate synthesis, N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theno yl-L-glutamic acid (D1694; Tomudex) and 1843U89 as well as a folate-based inhibitor of purine synthesis, 5, 10-dideazatetrahydrofolic acid (DDATHF) on human soft tissue sarcoma cell lines having intrinsic resistance to methotrexate (MTX) due to impaired accumulation of polyglutamates of MTX (HS-16 and HS-42 cells) and to increased levels of dihydrofolate reductase and thymidylate synthase activity (HS-18 cells). Growth inhibition studies showed that ED50 values for D1694 and 1843U89 after a 24-h exposure were 11-19-fold and 22-222-fold lower, respectively, than those for MTX in HT-1080, a MTX-sensitive cell line, and the three MTX-resistant cell lines. In contrast, DDATHF was less cytotoxic than MTX in both the MTX-sensitive and the three resistant sarcoma cell lines. Uptake of D1694, 1843U89, or DDATHF was 2.5-4.5-fold higher than MTX in these sarcoma cell lines. However, D1694 and 1843U89, unlike MTX, accumulate in HS-16 and HS-42 cells as polyglutamate forms, reaching 70% of the total intracellular drug level after 24 h. DDATHF polyglutamates (9.4-24%) were less in the same cell lines. Much lower Km values for D1694 and 1843U89 as compared to MTX for folylpolyglutamate synthase were measured in the sarcoma cell lines, with Vmax values equal to or slightly higher than those obtained with MTX. D1694 and 1843U89 are significantly more cytotoxic than MTX in intrinsically MTX-resistant sarcoma cell lines as a result of extensive formation of polyglutamates. These two thymidylate synthase inhibitors should be evaluated in patients with soft tissue sarcomas.
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PMID:Antitumor activity of antifolate inhibitors of thymidylate and purine synthesis in human soft tissue sarcoma cell lines with intrinsic resistance to methotrexate. 981 25


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