The relationship between folate transport activity at low pH and reduced folate carrier function in human Huh7 hepatoma cells.
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 PT632(R) 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 K(t) for folic acid and MTX at pH 5.5 in PT632(R) cells was 0.36 and 1.5 microM, respectively. The affinity of the low pH transporter in PT632(R) cells was highest for pemetrexed (K(i)=140 nM), very low for PT632 (K(i)=77 microM), 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.
Zhao
Rongbao
R
Department of Medicine and the Albert Einstein Cancer Center, Albert Einstein College of Medicine, Chanin 628, Bronx, NY 10461, USA. rzhao@aecom.yu.edu
Hanscom
Marie
M
Goldman
I David
ID
D001692:Biological Transport; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D005492:Folic Acid / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008113:Liver Neoplasms / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism; D034741:RNA, Small Interfering; D058978:Reduced Folate Carrier Protein; D014162:Transfection
D026901:Membrane Transport Proteins; D034741:RNA, Small Interfering; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D005492:Folic Acid; D008727:Methotrexate