Abstract
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.
Highlights
The folate analogue, methotrexate (MTX) is an important component in the chemotherapy of childhood acute lymphocytic leukaemia (ALL)
We describe the use of confocal microscopy to visualize directly a RFCmediated uptake of F-MTX that is potently inhibited by reduced folate carrier (RFC)-transport substrates and is completely abolished in cultured cells with impaired MTX transport
Confocal microscopy images for wild-type K562 cells (A-D), their transport deficient subline (K500E; E-H) and KSOOE cells transfected with the human RFC cDNA are shown in Figure 1, following incubations with 1,UM F-MTX
Summary
ChemicalsF-MTX was purchased from Molecular Probes (Eugene, OR, USA) and MTX was obtained from Sigma Chemical Tissue culture reagents and supplies were purchased from assorted vendors except iron-supplemented calf serum, which was obtained from Hyclone Laboratories (Logan, UT, USA). The wild-type K-562 human erythroleukaemia cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). The transport-impaired K500E subline was selected from wild-type K562 cells by cloning in soft agar in the presence of 500 nm MTX (Matherly et al, 1992). K500E cells were approximately 90% transport impaired by direct assay with [3H]MTX (Wong et al, 1997). The K500E cell line was transfected with the human RFC cDNA-pcDNA3 construct (Wong et al, 1995) using lipofectin (Buonocore and Rose, 1991). A G418-resistant clone (designated K43-6) was selected by cloning in soft agar and characterized for increased MTX sensitivity and completely restored
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