title: Loss of folic acid exporter function with markedly augmented folate accumulation in lipophilic antifolate-resistant mammalian cells.
abstract: We previously described a 1,000-fold pyrimethamine-resistant Chinese hamster ovary cell line (PyrR100) which retains parental dihydrofolate reductase activity and methotrexate (MTX) sensitivity. This study characterizes the basis for the 14-fold decrease in folic acid and leucovorin concentrations required for clonogenic growth of PyrR100 cells relative to parental AA8 cells. Under conditions in which folic acid reduction was blocked by trimetrexate, PyrR100 cells displayed relative to parental AA8 cells a: 1) 17- and 5-fold increase in the net transport of folic acid and MTX, respectively; 2) 23- and 5-fold decrease in the efflux rate constant for folic acid and MTX, respectively; and 3) 2-fold increase in folic acid influx with no significant change in MTX influx. The markedly increased net folic acid transport in PyrR100 cells could not be explained by cellular folic acid binding, mitochondrial sequestration, polyglutamylation, nor by a decreased membrane potential. The effect of energy deprivation on folic acid and MTX transport in both cell lines was quite different. Glucose and pyruvate deprivation nearly abolished the increase in net folic acid transport in PyrR100 cells. In contrast, energy deprivation increased net MTX transport in AA8 cells, whereas no change was seen with PyrR100 cells. Furthermore, while folic acid influx in PyrR100 and AA8 cells was markedly reduced with energy deprivation, MTX influx was not affected. Provision of glucose and pyruvate to energy-deprived cells resulted in a rapid onset of MTX efflux from parental AA8 cells but not from PyrR100 cells. Taken together these results indicate that the markedly enhanced net transport of folic acid and MTX in PyrR100 cells is largely due to the complete loss of exit pump activity. Furthermore, the energy source that sustains the augmented levels of folic acid appears linked to the influx process and is distinct from the energy source that sustains MTX gradients under these conditions. We conclude that the loss of folic acid efflux is an efficient means of augmenting cellular uptake of folate cofactors and subsequent survival on picomolar folate concentrations. This constitutes the first demonstration of the loss of folic acid exporter activity in mammalian cells as a response to lipophilic antifolate selective pressure.
authors.affiliation: Department of Biology, The Technion, Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Y G
authors.initials: YG
authors.lastname: Assaraf
authors.forename: I D
authors.initials: ID
authors.lastname: Goldman
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D002955:Leucovorin / Q000378:metabolism; D050356:Lipid Metabolism; D008727:Methotrexate / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists; D011956:Receptors, Cell Surface; D019289:Pyruvic Acid; D013762:Tetrahydrofolate Dehydrogenase; D005947:Glucose; D002955:Leucovorin; D008727:Methotrexate