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Journal of Biochemistry Advance Access originally published online on August 19, 2009
Journal of Biochemistry 2009 146(5):693-703; doi:10.1093/jb/mvp130
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Differential Usage of the Transport Systems for Folic acid and Methotrexate in Normal Human T-Lymphocytes and Leukemic Cells

Bijesh Kumar Biswal and Rama Shanker Verma*

Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai-600036, India

*To whom Correspondence should be addressed. Tel: +91 44 2257 4109, Fax: +91 44 2257 4102, E-mail: vermars{at}iitm.ac.in

Received June 4, 2009; Accepted July 9, 2009


  Abstract

Methotrexate (MTX) has been used as an effective anti-cancer drug for a long time. Conceptually, it is accepted that MTX and folic acid are transported by folate receptors (FRs) in cancerous cells, but the exact mechanism of MTX uptake in human leukemia is unknown. The objective of this study was to investigate different transport systems for FA and MTX, and to delineate their uptake mechanism in MOLT4, K562, Hut78 leukemia cells and normal human T cells. In MOLT4, uptake of MTX was higher than FA, similar to that of K562, Hut78 and normal T cells. In MOLT4 cells, MTX uptake was maximum at pH 7.4 whereas FA uptake was maximum at pH 4.5. Uptake of FA and MTX was significantly inhibited by anions, suggesting anion-dependent transport system. FA uptake was found to be energy dependent whereas MTX uptake was energy independent. RT-PCR and immunofluorescence results demonstrated the presence of reduced folate carrier as well as proton coupled folate transporter and absence of FR in MOLT4 and normal T cells. These data suggest the existence of two separate and independent carrier-mediated transport systems for the uptake of FA and MTX in normal and leukemic human T cells.

Key Words: folate receptor, folic acid, methotrexate, MOLT4 cells, reduced folate carrier

Abbreviations: FA, folic acid; FR, folate receptor; MTX, methotrexate; PCFT, proton coupled folate transporter; RFC, reduced folate carrier; RT-PCR, reverse transcription polymerase chain reaction; PBS, phosphate buffered saline; HBS, hepes buffered saline; DEPC, diethylpyrocarbonate; FITC, fluorescein isothiocyanate


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