Impact of overexpression of the reduced folate carrier (RFC1), an anion exchanger, on concentrative transport in murine L1210 leukemia cells

Rongbao Zhao, Richard Seither, Kevin E. Brigle, Iraida G. Sharina, Pi J. Wang, I. David Goldman

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Abstract

Transport of reduced folates in murine leukemia cells is mediated by the bidirectional reduced folate carrier (RFC1) and independent unidirectional exit pumps. RFC1 has been proposed to be intrinsically equilibrating, generating transmembrane gradients by exchange with inorganic and organic anions. This paper defines the role of high level carrier expression, through transfection with RFC1 cDNA, on concentrative transport of the folate analog, methotrexate (MTX) in murine L1210 leukemia cells. RFC1 was expressed in the MTX(r)A line, which lacks a functional endogenous carrier to obtain the MTX(r)A-R16 clonal derivative. Influx was increased ~9-fold in MTX(r)A-R16 cells without a change in K(m). The efflux rate constant was increased by a factor of 5.1 relative to L1210 cells, and this resulted in only a 2.1-fold increase in the steady-state level of free intracellular MTX, [MTX](i), when [MTX](c) was 1 μM. The concentrative advantage for RFC1 (the ratio of [MTX](i) in MTX(r)A-R16 to L1210 cells) increased from 1.8 at 0.1 μM MTX to 3.8 at an [MTX](c) level of 30 μM. Augmented transport in MTX(r)A-R16 cells was accompanied by a 2-fold increase in accumulation of MTX polyglutamate derivatives and a ~50% decrease in the EC50 for 5-formyltetrahydrofolate and folic acid and the MTX IC50 relative to L1210 cells. These alterations paralleled changes in [MTX](i) and not the much larger change in influx at low [MTX](e) levels, consistent with the critical role that free intracellular folates and drug play in meeting cellular needs for folates and as a determinant of antifolate activity, respectively. The data indicate that RFC1 produces a large and near symmetrical increase in the bidirectional fluxes of MTX resulting in only a small increase in the transmembrane chemical gradient at low extracellular folate levels. Hence, increased expression of RFC1, alone, may not be an efficient adaptive response to folate deprivation, and other factors may come into play to account for the marked increases in concentrative folate transport which occur when cells are subjected to low folate-selective pressure.

Original languageEnglish (US)
Pages (from-to)21207-21212
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number34
DOIs
StatePublished - Aug 22 1997

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Reduced Folate Carrier Protein
Leukemia L1210
Methotrexate
Anions
Folic Acid
Derivatives
Folic Acid Antagonists

ASJC Scopus subject areas

  • Biochemistry

Cite this

Impact of overexpression of the reduced folate carrier (RFC1), an anion exchanger, on concentrative transport in murine L1210 leukemia cells. / Zhao, Rongbao; Seither, Richard; Brigle, Kevin E.; Sharina, Iraida G.; Wang, Pi J.; Goldman, I. David.

In: Journal of Biological Chemistry, Vol. 272, No. 34, 22.08.1997, p. 21207-21212.

Research output: Contribution to journalArticle

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abstract = "Transport of reduced folates in murine leukemia cells is mediated by the bidirectional reduced folate carrier (RFC1) and independent unidirectional exit pumps. RFC1 has been proposed to be intrinsically equilibrating, generating transmembrane gradients by exchange with inorganic and organic anions. This paper defines the role of high level carrier expression, through transfection with RFC1 cDNA, on concentrative transport of the folate analog, methotrexate (MTX) in murine L1210 leukemia cells. RFC1 was expressed in the MTX(r)A line, which lacks a functional endogenous carrier to obtain the MTX(r)A-R16 clonal derivative. Influx was increased ~9-fold in MTX(r)A-R16 cells without a change in K(m). The efflux rate constant was increased by a factor of 5.1 relative to L1210 cells, and this resulted in only a 2.1-fold increase in the steady-state level of free intracellular MTX, [MTX](i), when [MTX](c) was 1 μM. The concentrative advantage for RFC1 (the ratio of [MTX](i) in MTX(r)A-R16 to L1210 cells) increased from 1.8 at 0.1 μM MTX to 3.8 at an [MTX](c) level of 30 μM. Augmented transport in MTX(r)A-R16 cells was accompanied by a 2-fold increase in accumulation of MTX polyglutamate derivatives and a ~50{\%} decrease in the EC50 for 5-formyltetrahydrofolate and folic acid and the MTX IC50 relative to L1210 cells. These alterations paralleled changes in [MTX](i) and not the much larger change in influx at low [MTX](e) levels, consistent with the critical role that free intracellular folates and drug play in meeting cellular needs for folates and as a determinant of antifolate activity, respectively. The data indicate that RFC1 produces a large and near symmetrical increase in the bidirectional fluxes of MTX resulting in only a small increase in the transmembrane chemical gradient at low extracellular folate levels. Hence, increased expression of RFC1, alone, may not be an efficient adaptive response to folate deprivation, and other factors may come into play to account for the marked increases in concentrative folate transport which occur when cells are subjected to low folate-selective pressure.",
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N2 - Transport of reduced folates in murine leukemia cells is mediated by the bidirectional reduced folate carrier (RFC1) and independent unidirectional exit pumps. RFC1 has been proposed to be intrinsically equilibrating, generating transmembrane gradients by exchange with inorganic and organic anions. This paper defines the role of high level carrier expression, through transfection with RFC1 cDNA, on concentrative transport of the folate analog, methotrexate (MTX) in murine L1210 leukemia cells. RFC1 was expressed in the MTX(r)A line, which lacks a functional endogenous carrier to obtain the MTX(r)A-R16 clonal derivative. Influx was increased ~9-fold in MTX(r)A-R16 cells without a change in K(m). The efflux rate constant was increased by a factor of 5.1 relative to L1210 cells, and this resulted in only a 2.1-fold increase in the steady-state level of free intracellular MTX, [MTX](i), when [MTX](c) was 1 μM. The concentrative advantage for RFC1 (the ratio of [MTX](i) in MTX(r)A-R16 to L1210 cells) increased from 1.8 at 0.1 μM MTX to 3.8 at an [MTX](c) level of 30 μM. Augmented transport in MTX(r)A-R16 cells was accompanied by a 2-fold increase in accumulation of MTX polyglutamate derivatives and a ~50% decrease in the EC50 for 5-formyltetrahydrofolate and folic acid and the MTX IC50 relative to L1210 cells. These alterations paralleled changes in [MTX](i) and not the much larger change in influx at low [MTX](e) levels, consistent with the critical role that free intracellular folates and drug play in meeting cellular needs for folates and as a determinant of antifolate activity, respectively. The data indicate that RFC1 produces a large and near symmetrical increase in the bidirectional fluxes of MTX resulting in only a small increase in the transmembrane chemical gradient at low extracellular folate levels. Hence, increased expression of RFC1, alone, may not be an efficient adaptive response to folate deprivation, and other factors may come into play to account for the marked increases in concentrative folate transport which occur when cells are subjected to low folate-selective pressure.

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