A new method for a quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells.
A rapid, functional and quantitative diagnostic method for the estimation of the P-glycoprotein (P-gp)-dependent multidrug resistance is required in the clinical treatment of human tumours, as chemotherapy protocols and resistance-reversing agents could be applied accordingly. In the present work, by using a calcein accumulation method in combination with immunorecognition and drug-resistance studies, a new method is described for the quantitative estimation of the expression and function of the multidrug transporter. MDR1-transfected and drug-selected tumour cell lines with various levels of drug resistance were examined. The expression of P-gp and its cell-surface appearance were assessed by quantitative immunoblotting and by immunofluorescence cytometry. The transport function of the P-gp was assessed by measuring the extrusion of calcein acetoxymethyl ester (AM) with fluorometry and flow cytometry, while in parallel experiments drug resistance was directly examined in cell survival assays. The MDR1 activity factor (MAF), calculated from the calcein AM extrusion assay, is demonstrated to provide a reliable quantitative measure for MDR1 specific activity, reflecting cellular drug resistance. This relatively simple and rapid new functional P-gp assay surpasses the formerly used techniques in both sensitivity and reproducibility.
Homolya
L
L
National Institute of Haematology, Blood Transfusion and Immunology, Budapest, Hungary.
Holló
M
M
Müller
M
M
Mechetner
E B
EB
Sarkadi
B
B
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism / Q000502:physiology; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry; D005452:Fluoresceins / Q000032:analysis / Q000494:pharmacology; D005470:Fluorometry; D006801:Humans; D007624:KB Cells / Q000187:drug effects / Q000378:metabolism; D007941:Leukemia P388 / Q000188:drug therapy / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000188:drug therapy / Q000378:metabolism; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009374:Neoplasms, Experimental / Q000188:drug therapy* / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D005452:Fluoresceins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014700:Verapamil; C007740:fluorexon