Confocal microscopy visualization of antifolate uptake by the reduced folate carrier in human leukaemic cells

Abstract

Summary 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 giM) 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 reestablished 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.Keywords: folate; fluorescein methotrexate; methotrexate; reduced folate carrier; membrane transportThe folate analogue, methotrexate (MTX) is an important component in the chemotherapy of childhood acute lymphocytic leukaemia (ALL). Although long-term disease-free survival for children with ALL has continued to increase and now approaches 70% (Pizzo and Poplack, 1993), further improvements in ALL treatment will have better results if patients who may benefit from more intensive therapies can be identified.Critical determinants of MTX sensitivity and resistance have been previously described in cultured cells (Jolivet et al, 1983;Goldman and Matherly, 1985). MTX is transported into cells by the reduced folate carrier (RFC) where it binds to dihydrofolate reductase (DHFR) and is metabolized to MTX polyglutamates by folylpolyglutamate synthetase (FPGS). Lymphoblasts from children with ALL also synthesize long-chain MTX polyglutamates (Whitehead et al, 1990), and correlations have been established between accumulation of these metabolites and characteristic patient prognostic features (i.e. lineage, hyperdiploidy, etc.) or MTX dose (Whitehead et al, 1992;Barredo et al, 1994;Synold et al, 1994).In recent years, fluorescent analogues of MTX have fostered studies of MTX resistance in cultured cells and leukaemic blasts by flow cytometry...

Methods to Evaluate the Effect of Ethanol on the Folate Analogue: Fluorescein Methotrexate Uptake in Human Proximal Tubular Cells

“…Masereeuw et al [ 19 ] suggested that cellular accumulation of FL-MTX is a result of diffuse entry and it is energy-independent. FL-MTX transport by RFC in human leukemia cell lines and leukemia blasts was demonstrated by confocal laser scanning microscopy [ 20 ]. Li et al [ 21 ] observed that the transport of FL-MTX is mediated by the reduced folate transporter (RFC) in brush border membrane vesicles of rat small intestine.…”

Implications of the Dominant Role of Transporters in Drug Uptake by Cells (Supplementary Material)

“…However, it is often difficult to source radiolabelled compounds. Another way of labelling is to conjugate the drug to a fluorescent moiety so uptake can be followed by confocal microscopy, as demonstrated in a study on the reduced folate carrier of fluoresceinmethotrexate [233][234][235]. However, the attachment of fluorescent moieties can potentially alter the nature of the drug-transporter interaction.…”

Transport of fluorescein methotrexate by multidrug resistance-associated protein 3 in IEC-6 cells

“…Fluorescence labeling of the transporter in situ could be used for detecting and isolating cells by flow cytometry and further monitoring localization and conformation of the transporter within the membrane (7). Recently, F-MTX transport by RFC in human leukemia cell lines and leukemiablasts has also been demonstrated by confocal laser scanning microscopy (19). We (30) have also suggested that the transport of F-MTX in the small intestine is mediated partly by RFC by using the in vitro everted intestine segments of rats.…”