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The purpose of this study was to isolate Caco-2 subclones that express high levels of multidrug resistance protein (MDR1) and to characterize their kinetics and affinity parameters for MDR1 substrate/inhibitors. The subclones were selected by a dilution cloning technique. The polarized efflux of [3H]-vinblastine across subclone cell monolayers was quantified by measuring the apparent permeability coefficients (Papp) of [3H]-vinblastine in the basolateral (BL)-to-apical (AP) direction and in the AP-to-BL direction (Papp BL-to-AP/Papp AP-to-BL) across the cell monolayers. The expression of MDR1 in the Caco-2 subclones compared with the parental Caco-2 cells was confirmed by Western blotting analysis. The kinetics parameters (Km, Vmax) of [3H]-vinblastine and the inhibitory constants (KI) of several known MDR1 substrates/inhibitors on the transport of [3H]-digoxin determined in the parental Caco-2 cells and Caco-2 subclones were also compared. Three subclones (#1, #20, #21) were selected based on their polarized efflux of [3H]-vinblastine. The Papp BL-to-AP/Papp AP-to-BL ratios for #1, #20, and #21 were 110, 140, and 112, respectively, and were about 6-fold higher than the ratio observed for the parental Caco-2 cells. In the presence of GF-120918 (2 microM), a known MDR1-specific inhibitor, the Papp BL-to-AP/Papp AP-to-BL ratios were significantly decreased, suggesting that these cells were overexpressing MDRI. The Km values observed for vinblastine in the Caco-2 subclones were nearly identical to the value observed in the parental Caco-2 cells. In contrast, the Vmax values observed in the subclones were approximate 26-69% higher. The KI values observed for various known MDR1 substrates/inhibitors on [3H]-digoxin transport were nearly identical to those in the parental Caco-2 cells and Caco-2 subclones. The high functional efflux activities of these subclones were stable up to 6 months. Subclones #1, #20, #21 express high levels of MDR1. These Caco-2 subclones may be useful models for profiling drugs for their MDR1 substrate activity and for establishing structure-transport relationships for this efflux transporter.


Kazutoshi Horie, Fuxing Tang, Ronald T Borchardt. Isolation and characterization of Caco-2 subclones expressing high levels of multidrug resistance protein efflux transporter. Pharmaceutical research. 2003 Feb;20(2):161-8

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PMID: 12636153

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