title: Establishment of optimized MDCK cell lines for reliable efflux transport studies.
abstract: Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results.
authors: Ruprecht-Karls-University, Institute of Pharmacy and Molecular Biotechnology, Heidelberg, 69120, Germany.
authors: Dominik
authors: D
authors: Gartzke
authors: Gert
authors: G
authors: Fricker
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D001693:Biological Transport, Active; D004077:Digoxin / Q000493:pharmacokinetics; D004285:Dogs; D005434:Flow Cytometry; D005452:Fluoresceins / Q000493:pharmacokinetics; D015870:Gene Expression; D006801:Humans; D061985:Madin Darby Canine Kidney Cells / Q000378:metabolism*; D011802:Quinidine / Q000493:pharmacokinetics; D014162:Transfection; D014747:Vinblastine / Q000493:pharmacokinetics
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005452:Fluoresceins; C085925:calcein AM; D014747:Vinblastine; D004077:Digoxin; D011802:Quinidine
keywords: ABC transporter; Cell lines; MDCK; P-glycoprotein; PCR; bidirectional transport assays; homogenous population