The relationship between folate transport activity at low pH and reduced folate carrier function in human Huh7 hepatoma cells

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Abstract

Transport of folates and antifolates in both hepatocytes and Huh7 human hepatoma cells is characterized by a low-pH optimum. Studies were undertaken to determine the extent to which this transport activity is mediated by the reduced folate carrier (RFC) in Huh7 human hepatoma cells. RFC expression was ablated by chemical mutagenesis and antifolate selective pressure with PT632 resulting in the PT632R subline in which RFC mRNA could not be detected. Methotrexate (MTX) influx in these cells at pH 7.4 was reduced by 70%, leaving substantial residual RFC-independent influx while influx of MTX and folic acid at pH 5.5 was not significantly decreased. The influx Kt for folic acid and MTX at pH 5.5 in PT632R cells was 0.36 and 1.5 μM, respectively. The affinity of the low pH transporter in PT632R cells was highest for pemetrexed (Ki = 140 nM), very low for PT632 (K i = 77 μM), and was stereospecific for the natural isomer (6S) of 5-formyltetrahydrofolate. In Huh7 cells transiently transfected with an RFC siRNA, RFC expression was reduced by 60% resulting in a 40% decrease in MTX influx at pH 7.4 but only a very small (5%) reduction in MTX or folic acid influx at pH 5.5. These data indicate that MTX transport in Huh7 cells at neutral pH is mediated largely by RFC while at pH 5.5 the predominant route of transport is independent of RFC.

Original languageEnglish (US)
Pages (from-to)57-64
Number of pages8
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1715
Issue number1
DOIs
StatePublished - Aug 30 2005

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Reduced Folate Carrier Protein
Folic Acid
Hepatocellular Carcinoma
Methotrexate
Folic Acid Antagonists
Pemetrexed
Mutagenesis
Leucovorin
Isomers
Small Interfering RNA
Hepatocytes

Keywords

  • Acidic pH
  • Folate transport
  • Folic acid
  • Methotrexate
  • Pemetrexed

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics

Cite this

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title = "The relationship between folate transport activity at low pH and reduced folate carrier function in human Huh7 hepatoma cells",
abstract = "Transport of folates and antifolates in both hepatocytes and Huh7 human hepatoma cells is characterized by a low-pH optimum. Studies were undertaken to determine the extent to which this transport activity is mediated by the reduced folate carrier (RFC) in Huh7 human hepatoma cells. RFC expression was ablated by chemical mutagenesis and antifolate selective pressure with PT632 resulting in the PT632R subline in which RFC mRNA could not be detected. Methotrexate (MTX) influx in these cells at pH 7.4 was reduced by 70{\%}, leaving substantial residual RFC-independent influx while influx of MTX and folic acid at pH 5.5 was not significantly decreased. The influx Kt for folic acid and MTX at pH 5.5 in PT632R cells was 0.36 and 1.5 μM, respectively. The affinity of the low pH transporter in PT632R cells was highest for pemetrexed (Ki = 140 nM), very low for PT632 (K i = 77 μM), and was stereospecific for the natural isomer (6S) of 5-formyltetrahydrofolate. In Huh7 cells transiently transfected with an RFC siRNA, RFC expression was reduced by 60{\%} resulting in a 40{\%} decrease in MTX influx at pH 7.4 but only a very small (5{\%}) reduction in MTX or folic acid influx at pH 5.5. These data indicate that MTX transport in Huh7 cells at neutral pH is mediated largely by RFC while at pH 5.5 the predominant route of transport is independent of RFC.",
keywords = "Acidic pH, Folate transport, Folic acid, Methotrexate, Pemetrexed",
author = "Rongbao Zhao and Marie Hanscom and Goldman, {I. David}",
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T1 - The relationship between folate transport activity at low pH and reduced folate carrier function in human Huh7 hepatoma cells

AU - Zhao, Rongbao

AU - Hanscom, Marie

AU - Goldman, I. David

PY - 2005/8/30

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N2 - Transport of folates and antifolates in both hepatocytes and Huh7 human hepatoma cells is characterized by a low-pH optimum. Studies were undertaken to determine the extent to which this transport activity is mediated by the reduced folate carrier (RFC) in Huh7 human hepatoma cells. RFC expression was ablated by chemical mutagenesis and antifolate selective pressure with PT632 resulting in the PT632R subline in which RFC mRNA could not be detected. Methotrexate (MTX) influx in these cells at pH 7.4 was reduced by 70%, leaving substantial residual RFC-independent influx while influx of MTX and folic acid at pH 5.5 was not significantly decreased. The influx Kt for folic acid and MTX at pH 5.5 in PT632R cells was 0.36 and 1.5 μM, respectively. The affinity of the low pH transporter in PT632R cells was highest for pemetrexed (Ki = 140 nM), very low for PT632 (K i = 77 μM), and was stereospecific for the natural isomer (6S) of 5-formyltetrahydrofolate. In Huh7 cells transiently transfected with an RFC siRNA, RFC expression was reduced by 60% resulting in a 40% decrease in MTX influx at pH 7.4 but only a very small (5%) reduction in MTX or folic acid influx at pH 5.5. These data indicate that MTX transport in Huh7 cells at neutral pH is mediated largely by RFC while at pH 5.5 the predominant route of transport is independent of RFC.

AB - Transport of folates and antifolates in both hepatocytes and Huh7 human hepatoma cells is characterized by a low-pH optimum. Studies were undertaken to determine the extent to which this transport activity is mediated by the reduced folate carrier (RFC) in Huh7 human hepatoma cells. RFC expression was ablated by chemical mutagenesis and antifolate selective pressure with PT632 resulting in the PT632R subline in which RFC mRNA could not be detected. Methotrexate (MTX) influx in these cells at pH 7.4 was reduced by 70%, leaving substantial residual RFC-independent influx while influx of MTX and folic acid at pH 5.5 was not significantly decreased. The influx Kt for folic acid and MTX at pH 5.5 in PT632R cells was 0.36 and 1.5 μM, respectively. The affinity of the low pH transporter in PT632R cells was highest for pemetrexed (Ki = 140 nM), very low for PT632 (K i = 77 μM), and was stereospecific for the natural isomer (6S) of 5-formyltetrahydrofolate. In Huh7 cells transiently transfected with an RFC siRNA, RFC expression was reduced by 60% resulting in a 40% decrease in MTX influx at pH 7.4 but only a very small (5%) reduction in MTX or folic acid influx at pH 5.5. These data indicate that MTX transport in Huh7 cells at neutral pH is mediated largely by RFC while at pH 5.5 the predominant route of transport is independent of RFC.

KW - Acidic pH

KW - Folate transport

KW - Folic acid

KW - Methotrexate

KW - Pemetrexed

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