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

British Journal of Cancer (1997) 76(6), 734-738 © 1997 Cancer Research Campaign Confocal microscopy visualization of antifolate uptake by the reduced folate carrier in human leukaemic cells J Jolivet1, M-P Faure2, SC Wong3, JW Taub4 and LH Matherly3 1Centre de Recherche, Cenre hospitalier de l'Universit6 de Montr6al, Pavillon; 2H1tel-Dieu, 3840 rue Saint-Urbain, Montreal, Canada H2W 1T8; Advanced Bioconcept Inc., 1801 McGill College, Suite 720, Montr4al, Canada H3A 2N4; 3Developmental Therapeutics Program, Karmanos Cancer Institute and the Department of Pharmacology, Wayne State University School of Medicine, Detroit, Ml, USA; 4Department of Pediatrics, Childrens Hospital of Michigan, Detroit, Ml, USA Summary Confocal microscopy was used to visualize the intracellular uptake of the fluorescent methotrexate analogue, fluorescein-MTX (FMTX), 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 transport The 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 (Kaufman et al, 1978; Rosowsky et al, 1982; Received 29 October 1996 Revised 24 February 1997 Accepted 6 March 1997 Correspondence to: J Jolivet 734 Assaraf and Schimke, 1987; Trippett et al, 1992; Matherly et al, 1995). The most extensively studied of these compounds, fluorescein MTX (F-MTX), has been reported to penetrate cells slowly over several hours and to accumulate to high levels in both MTXsensitive and transport-impaired cells (Assaraf and Schimke, 1987), presumably by a non-RFC uptake process. As F-MTX binds avidly to DHFR, it has frequently been used to detect elevated levels of this enzyme target in MTX-resistant cells by flow cytometry (Kaufman et al, 1978). Whereas capacities for MTX membrane transport can also be assayed with F-MTX and flow cytometry, by following the loss of cellular fluorescence due to the competitive displacement of DHFR-bound F-MTX with exogenous MTX (Assaraf and Schimke, 1987), the sensitivity of this indirect assay of RFC function is limited by the range of displacing MTX concentrations used. On this basis, we sought to develop a more direct and sensitive approach for assaying RFC function in intact cells that might be amenable to the study of clinical specimens. In this report, we describe the use of confocal microscopy to visualize directly a RFCmediated uptake of F-MTX that is potently inhibited by RFC-transport substrates and is completely abolished in cultured cells with impaired MTX transport. Initial experiments are also described that extend the use of this approach to the detection of RFC transport competent and impaired leukaemic blasts from children with ALL. Our data show a high level of sensitivity for confocal analysis, reflecting its enhanced image resolution over standard fluorescence microscopy and an ability to establish the intracellular (as opposed to surface) localization of the fluorescent drug. Confocal microscopy and antifolate transport 735 A B E F G H J K L D Figure 1 Confocal detection of F-MTX uptake by wild-type, transport-impaired and RFC-transfected K562 cells. A-D are cross-sections of wild-type K562 cells after 15-min incubations with 1 gM F-MTX alone (A), 15 min with added ZD1 694 (B), 24 h alone (C) or 24 h with added ZD1 694 (D). E-H are cross-sections of K500E cells after 15-min incubations with F-MTX alone (E), 15 min with added ZD1 694 (F); 24 h alone (G) or 24 h with added ZD1694 (H). l-L are crosssections of K43-6 cells after 15-min incubations with F-MTX alone (I), 15 min with added ZD1 694 (J), 24 h alone (K) or 24 h with added ZD1 694 (L). The final ZD1 694 concentration was 100 gM. The darkened area totally devoid of fluorescence staining in most sections is the cell nucleus. All images were printed with a Focus camera system using false colours to simulate fluorescence intensity, with lowest to highest intensities represented sequentially by the colours blue, green, yellow, red and white. The scale bar represents 5 gm MATERIALS AND METHODS Chemicals F-MTX was purchased from Molecular Probes (Eugene, OR, USA) and MTX was obtained from Sigma Chemical (St. Louis, MO, USA). ZD1694 (N-(5-(N-(3,4-dihydro-2-methyl-4-oxyquinazolin6-ylmethyl)-N-methylamino)-2-then (...truncated)