P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope.
Madin Darby canine kidney (MDCK) cells transfected with the multidrug resistance mdr1 gene, MDR1-MDCK (Pastan et al., 1988, Proc. Natl. Acad. Sci. USA 85 4486-4470), were used in a combined approach to study expression, localisation and functionality of the P-glycoprotein (P-gp) membrane transporter in the same cell culture preparations. Cells were characterised with regard to their growth curve, transepithelial electrical resistance (TEER), and cytoarchitecture. Efflux of the P-gp substrate rhodamine123 (rho123) was monitored with confocal laser scanning microscopy (CLSM). The transfected cells grew in multilayers. After reaching confluence they exhibited a complete tight junction (TJ) network. P-gp was strongly expressed at the uppermost apical surface of the multilayer already after 4 days in culture. The lower cell layers were not clearly polarised. P-gp-mediated transport could be followed by efflux of the fluorescent rho123 from the cells into the apical extracellular space. Verapamil, a P-gp inhibitor, significantly decreased efflux. For MDCK parent cells the rho123 assay was negative up to about day 20, and only at later times (day 25) low P-gp activity was detected. These results clearly show that despite the fact that the transfected cells form irregular layers, they provide a good model for screening of P-gp substrates and inhibitors.
Hämmerle
S P
SP
Biopharmacy, Department of Applied BioSciences, ETH Zürich, CH-8057, Zürich, Switzerland.
Rothen-Rutishauser
B
B
Krämer
S D
SD
Günthert
M
M
Wunderli-Allenspach
H
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D002455:Cell Division; D002460:Cell Line; D003599:Cytoskeleton / Q000648:ultrastructure; D004285:Dogs; D018432:Drug Resistance, Multiple; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D007668:Kidney; D018613:Microscopy, Confocal / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D019108:Tight Junctions / Q000648:ultrastructure; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011994:Recombinant Proteins