Confocal microscopy visualization of antifolate uptake by the reduced folate carrier in human leukaemic cells.
Confocal microscopy was used to visualize the intracellular uptake of the fluorescent methotrexate analogue, fluorescein-MTX (F-MTX), in human leukaemic cell lines and leukaemic blasts. Cytosolic labelling of wild-type K562 human erythroleukaemia cells was detected during 3-60 min incubations with F-MTX (1 microM) and was completely inhibited by co-exposure to either methotrexate or the thymidylate synthase inhibitor, ZD1694. There was no significant intracellular F-MTX accumulation over this period in a K562 subline (K500E) with a documented defect (approximately 10% of wild type) in membrane transport by the reduced folate carrier (RFC). F-MTX uptake was re-established in K500E cells transfected with a cDNA to human RFC, establishing a role for RFC in the cellular uptake of this compound. High levels of intracellular labelling were detected in all cell lines after prolonged (24 h) F-MTX incubations, however F-MTX accumulation at this time was not inhibited by ZD1694. F-MTX uptake by RFC was also detected in leukaemic blasts from children with acute lymphoblastic leukaemia and could be blocked with ZD1694. In leukaemic blasts with a documented defect in MTX uptake, F-MTX accumulation was abolished in almost all the cells. These results display the power of confocal microscopy for directly visualizing RFC-mediated anti-folate uptake. Over short intervals, F-MTX uptake is mediated by RFC, however, RFC-independent processes predominate during long drug exposures. Direct assay by confocal microscopy may be better suited than other indirect methods (i.e. flow cytometry) for detecting low levels of RFC transport in leukaemic blasts from patients undergoing chemotherapy with methotrexate.
Jolivet
J
J
Centre de Recherche, Centre hospitalier de l'Université de Montréal, Pavillon, Canada.
Faure
M P
MP
Wong
S C
SC
Taub
J W
JW
Matherly
L H
LH
D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal / Q000379:methods*; D011799:Quinazolines / Q000494:pharmacology*; D058978:Reduced Folate Carrier Protein; D013876:Thiophenes / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011799:Quinazolines; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; D013876:Thiophenes; C068874:raltitrexed