title: Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry.
abstract: Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies.
authors.affiliation: Department of Medical Oncology, Free University Hospital, Amsterdam, The Netherlands.
authors.forename: N
authors.initials: N
authors.lastname: Feller
authors.forename: C M
authors.initials: CM
authors.lastname: Kuiper
authors.forename: J
authors.initials: J
authors.lastname: Lankelma
authors.forename: J K
authors.initials: JK
authors.lastname: Ruhdal
authors.forename: R J
authors.initials: RJ
authors.lastname: Scheper
authors.forename: H M
authors.initials: HM
authors.lastname: Pinedo
authors.forename: H J
authors.initials: HJ
authors.lastname: Broxterman
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000493:pharmacokinetics; D018528:ATP-Binding Cassette Transporters / Q000032:analysis* / Q000493:pharmacokinetics; D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics; D003630:Daunorubicin / Q000493:pharmacokinetics; D018432:Drug Resistance, Multiple / Q000502:physiology*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes; D006801:Humans; D007624:KB Cells; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020112:Rhodamine 123; D012235:Rhodamines / Q000493:pharmacokinetics; D012680:Sensitivity and Specificity
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000964:Antimetabolites, Antineoplastic; D003630:Daunorubicin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D020112:Rhodamine 123; D012235:Rhodamines; C085925:calcein AM