CONFOCAL MICROSCOPY VISUALIZATION OF ANTIFOLATE UPTAKE BY THE REDUCEDFOLATE CARRIER IN HUMAN LEUKEMIC-CELLS

Confocal microscopy was used to visualize the intracellular uptake of the fluorescent methotrexate analogue, fluorescein-MTX (F-MTX), in hum an leukaemic cell lines and leukaemic blasts. Cytosolic labelling of w ild-type K562 human erythroleukaemia cells was detected during 3-60 mi n incubations with F-MTX (1 mu M) and was completely inhibited by co-e xposure 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 (appro ximately 10% of wild type) in membrane transport by the reduced folate carrier (RFC). F-MTX uptake was reestablished in K500E cells transfec ted with a cDNA to human RFC, establishing a role for RFC in the cellu lar uptake of this compound. High levels of intracellular labelling we re 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 childr en with acute lymphoblastic leukaemia and could be blocked with ZD1694 . In leukaemic blasts with a documented defect in MTX uptake, F-MTX ac cumulation was abolished in almost all the cells. These results displa y the power of confocal microscopy for directly visualizing RFC-mediat ed anti-folate uptake. Over short intervals, F-MTX uptake is mediated by RFC, however, RFC-independent processes predominate during long dru g exposures. Direct assay by confocal microscopy may be better suited than other indirect methods (i.e. flow cytometry) for detecting low le vels of RFC transport in leukaemic blasts from patients undergoing che motherapy with methotrexate.