Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward. Transport properties of a new variant, evidence for increased levels of a specific transport protein, and its partial characterization following affinity labeling.
Studies are reported on the characterization of a new isolate within a novel class of variants of the L1210 cell exhibiting markedly increased transport inward of folate analogues. This variant (L1210/R83), which was selected in the presence of the antifolate metoprine, exhibited a 40-fold increase in [3H]aminopterin influx compared to parental cells and a modest (4-5-fold) increase in [3H]aminopterin efflux. The increase in influx was associated with a comparable increase in influx Vmax for the one-carbon, reduced folate transport system and the same increase in the amount of specific binding of [3H]aminopterin on the cell surface. Values for influx Km for [3H]aminopterin and specificity for various folate structures were unchanged. The alteration in influx Vmax and more rapid efflux accounted for the different level of intracellular exchangeable level of drug at steady state in this variant compared with parental L1210 cells. Otherwise, membrane potential was unchanged. The N-hydroxysuccinimide ester of [3H]aminopterin was used to covalently label the specific binding protein for folate compounds in the plasma membrane of variant and parental L1210 cells. Incorporation of label into this protein was stable under a variety of conditions and accounted for 97 and 52% of total cellular labeling, respectively, for membrane derived from R83 and parental L1210 cells at a reagent concentration of 20 nM. Specific affinity labeling on the surface of parental and variant cells was decreased in the presence of aminopterin, methotrexate, or 5-formyltetrahydrofolate, but not in the presence of folic acid. Also, [3H]aminopterin influx in these cells was inhibited by the N-hydroxysuccinimide ester of aminopterin or methotrexate, but not the N-hydroxysuccinimide ester of folic acid. These findings, in addition to the increased affinity labeling of this variant, which corresponds to the increase in influx of [3H] aminopterin also seen, appears to identify the affinity labeled protein as a component of the "classical" one-carbon, reduced folate transport system in these cells. The affinity labeled protein from each cell type was solubilized in sodium dodecyl sulfate or extracted in detergent in the presence of proteinase inhibitors and was found to elute from Sephacryl S-300 and migrate during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single peak of Mr = 45,000-48,000. Recovery of labeled binding protein in these fractions from R83 variant cells was approximately 40 times greater than that from parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Yang
C H
CH
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Sirotnak
F M
FM
Mines
L S
LS
D000345:Affinity Labels; D000630:Aminopterin / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D002845:Chromatography; D004351:Drug Resistance / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D014644:Genetic Variation; D007700:Kinetics; D007939:Leukemia L1210 / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008727:Methotrexate / Q000378:metabolism; D008970:Molecular Weight; D011739:Pyrimethamine / Q000031:analogs & derivatives / Q000494:pharmacology; D011956:Receptors, Cell Surface; D013388:Succinimides / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D002793:Cholic Acids; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D013388:Succinimides; C006308:metoprine; D005492:Folic Acid; C056458:aminopterin N-hydroxysuccinimide ester; D000630:Aminopterin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D008727:Methotrexate; D011739:Pyrimethamine