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)

The purpose of this study was to characterize the transport properties of trimetrexate in WI-L2 human lymphoblastoid cells and determine the mode of resistance that had developed in a subline, WI-L2/TMQ, that was grown in increasing concentrations of trimetrexate. WI-L2/TMQ cells were 62-fold resistant to trimetrexate and 68- and 96-fold cross-resistant to the other lipophilic antifolates metoprine and piritrexim (BW 301U). No cross-resistance was observed with vincristine or doxorubicin, and sensitivity was not increased with 5 micrograms/ml of verapamil, indicating that it was not a typical multidrug resistance phenotype. WI-L2/TMQ exhibited a 2-fold increase in dihydrofolate reductase; however, this did not contribute significantly to the observed resistance, since these cells retained full sensitivity to methotrexate. Nor were there any kinetic alterations in dihydrofolate reductase toward trimetrexate or differences in the levels of thymidylate synthase. The major difference between the sensitive and resistant cell line was a 50% decrease in the influx rate of WI-L2/TMQ cells which produced a corresponding decrease in cellular trimetrexate at the steady state. No difference in efflux rates was detected nor were there any differences in intracellular water or metabolism of trimetrexate. Additional characterization of trimetrexate transport in WI-L2 showed that influx was nonsaturable up to 5 mM extracellular trimetrexate, relatively insensitive to sodium azide, and exhibited a Q10 of 2.7. Influx was, however, inhibited in a dose-dependent manner by concentrations of p-chloromercuribenzylsulfonate above 10 microM. Efflux studies revealed a large nonexchangeable fraction of trimetrexate that was well above the dihydrofolate reductase binding capacity and varied depending on the initial level of cell-associated drug. The intracellular exchangeable trimetrexate concentration at the steady state was always several-fold higher than the extracellular concentration. Retention of trimetrexate appeared to be coupled to some component of energy metabolism, since the presence of sodium azide stimulated this process by 2- to 3-fold. The data suggest that trimetrexate enters cells by passive diffusion but then is distributed and concentrated within the cell through more complex mechanisms which may involve energy coupling, compartmentation, or binding to macromolecules or organelles, although some type of carrier-mediated process cannot be ruled out.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of trimetrexate transport in human lymphoblastoid cells and development of impaired influx as a mechanism of resistance to lipophilic antifolates. 297 70

We describe studies of folate analogue transport in purified epithelial cell fractions isolated from mouse small intestine. Fractionation of these cells into immature proliferative and mature absorptive components and two components representative of intermediate stages of maturation was carried out by stepwise, nonenzymatic stripping of the everted organ. In contrast to the proliferative-specific enzyme markers, thymidine kinase and dihydrofolate reductase, folate analogue transport did not vary with the alteration in proliferative potential of these cells during maturation. The brush-border enzyme, alkaline phosphatase, was used as a positive marker for maturation. Initial influx of [3H]-aminopterin into both mature and immature cell fractions showed the same kinetics and did not exhibit pH dependence within the range of 6.0 to 7.8. A single saturable component (Km = 16 +/- 3 microM; V37 = 57 +/- 8 pmol/min/10(7) cells) was delineated, with the same temperature dependence (Q10 27-37 degrees = 3.2 +/- 0.4; Arrenhius constant = 11.1 +/- 3 kcal/mol) and same specificity for various folate compounds. Initial efflux of [3H]aminopterin was also similar in both cell types. Efflux was first-order (t1/2 37 degrees = 1.1 to 1.3 min; K37 = 0.53 +/- 0.04 min-1) and equal to the decay-time constant for approach to steady-state during accumulation of [3H]aminopterin, but showed higher-temperature dependence (Q10 27-37 degrees = 6.7 +/- 0.8; Arrenhius constant = 25.3 +/- 4 kcal/mol). Under the conditions used which do not allow polyglutamylation of [3H]aminopterin, steady-state levels of accumulation of exchangeable drug at 37 degrees in each cell fraction were accounted for by the various kinetic parameters for each flux. At all concentrations of [3H]aminopterin examined, both types of epithelial cells appeared to maintain a negative electrochemical gradient under physiological conditions. Overall, the data conform to a two-carrier model for folate analogue transport in which each flux is mediated by a separate system. However, specificity and saturability of influx for folate compounds, and inhibition of this flux by various agents was markedly different from that reported for various tumor cells.
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PMID:Similar characteristics of folate analogue transport in vitro in contrast to varying dihydrofolate reductase levels in epithelial cells at different stages of maturation in mouse small intestine. 648 81