Loss of folic acid exporter function with markedly augmented folate accumulation in lipophilic antifolate-resistant mammalian cells

Yehuda G. Assaraf, I. David Goldman

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

We previously described a 1,000-fold pyrimethamine-resistant Chinese hamster ovary cell line (Pyr(R100)) 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 Pyr(R100) cells relative to parental AA8 cells. Under conditions in which folic acid reduction was blocked by trimetrexate, Pyr(R100) 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 Pyr(R100) 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 Pyr(R100) cells. In contrast, energy deprivation increased net MTX transport in AA8 cells, whereas no change was seen with Pyr(R100) cells. Furthermore, while folic acid influx in Pyr(R100) 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 Pyr(R100) cells. Taken together these results indicate that the markedly enhanced net transport of folic acid and MTX in Pyr(R100) 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.

Original languageEnglish (US)
Pages (from-to)17460-17466
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number28
DOIs
StatePublished - 1997

Fingerprint

Folic Acid Antagonists
Folic Acid
Cells
Methotrexate
Pyruvic Acid
Trimetrexate
Glucose
Cell Line
Pyrimethamine
Tetrahydrofolate Dehydrogenase
Leucovorin

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{76bb9353d4c748f6ab7f61251997ce04,
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 (Pyr(R100)) 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 Pyr(R100) cells relative to parental AA8 cells. Under conditions in which folic acid reduction was blocked by trimetrexate, Pyr(R100) 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 Pyr(R100) 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 Pyr(R100) cells. In contrast, energy deprivation increased net MTX transport in AA8 cells, whereas no change was seen with Pyr(R100) cells. Furthermore, while folic acid influx in Pyr(R100) 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 Pyr(R100) cells. Taken together these results indicate that the markedly enhanced net transport of folic acid and MTX in Pyr(R100) 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.",
author = "Assaraf, {Yehuda G.} and Goldman, {I. David}",
year = "1997",
doi = "10.1074/jbc.272.28.17460",
language = "English (US)",
volume = "272",
pages = "17460--17466",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "28",

}

TY - JOUR

T1 - Loss of folic acid exporter function with markedly augmented folate accumulation in lipophilic antifolate-resistant mammalian cells

AU - Assaraf, Yehuda G.

AU - Goldman, I. David

PY - 1997

Y1 - 1997

N2 - We previously described a 1,000-fold pyrimethamine-resistant Chinese hamster ovary cell line (Pyr(R100)) 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 Pyr(R100) cells relative to parental AA8 cells. Under conditions in which folic acid reduction was blocked by trimetrexate, Pyr(R100) 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 Pyr(R100) 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 Pyr(R100) cells. In contrast, energy deprivation increased net MTX transport in AA8 cells, whereas no change was seen with Pyr(R100) cells. Furthermore, while folic acid influx in Pyr(R100) 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 Pyr(R100) cells. Taken together these results indicate that the markedly enhanced net transport of folic acid and MTX in Pyr(R100) 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.

AB - We previously described a 1,000-fold pyrimethamine-resistant Chinese hamster ovary cell line (Pyr(R100)) 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 Pyr(R100) cells relative to parental AA8 cells. Under conditions in which folic acid reduction was blocked by trimetrexate, Pyr(R100) 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 Pyr(R100) 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 Pyr(R100) cells. In contrast, energy deprivation increased net MTX transport in AA8 cells, whereas no change was seen with Pyr(R100) cells. Furthermore, while folic acid influx in Pyr(R100) 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 Pyr(R100) cells. Taken together these results indicate that the markedly enhanced net transport of folic acid and MTX in Pyr(R100) 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.

UR - http://www.scopus.com/inward/record.url?scp=0030839959&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030839959&partnerID=8YFLogxK

U2 - 10.1074/jbc.272.28.17460

DO - 10.1074/jbc.272.28.17460

M3 - Article

VL - 272

SP - 17460

EP - 17466

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 28

ER -