Are MDCK cells transfected with the human MDR1 gene a good model of the human intestinal mucosa?
OBJECTIVE
To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa.


METHODS
P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDRI cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (K(M),Vmax) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (K(I)) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers.


RESULTS
MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [3H]-digoxin than did Caco-2 and MDCK-WT cells. K(M) values of vinblastine in Caco-2. MDCK-WT, and MDCK-MDR1 cells were 89.2+/-26.1, 24.5+/-1.1, and 252.8+/-134.7 microM, respectively, whereas Vmax values were 1.77+/-0.22, 0.42+/-0.01, and 2.43+/-0.86 pmolcm(-2)s(-1), respectively. Known P-gp substrates/inhibitors showed, in general, lower K(I) values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells.


CONCLUSIONS
These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.
Tang
Fuxing
F
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.
Horie
Kazutoshi
K
Borchardt
Ronald T
RT
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000818:Animals; D018938:Caco-2 Cells / Q000378:metabolism; D002460:Cell Line / Q000378:metabolism* / Q000502:physiology; D004077:Digoxin / Q000493:pharmacokinetics; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D019450:Genes, MDR / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004077:Digoxin