MDR1 haplotypes significantly minimize intracellular uptake and transcellular P-gp substrate transport in recombinant LLC-PK1 cells

To date, research on the effect of single nucleotide polymorphisms (SNPs) on P-glycoprotein (P-gp) expression and functionality has rendered inconsistent results. This study systematically evaluates the impact of MDR1 haplotypes (1236/2677, 1236/3435, 2677/3435, 1236/2677/3435) on P-gp functionality compared to individual SNPs (1236, 2677, and 3435) in validated stable recombinant epithelial cells. Recombinant LLC-PK1 cells expressing MDR1(wt) or its variants were developed and validated for this purpose. Intracellular accumulation and time-dependant efflux of a P-gp substrate, Rhodamine 123 (R123, 5 mu M) were evaluated in control and recombinant cells. Additionally, the transepithelial transport of 8123 (1 mu M) and Vinca alkaloids (5 mu M) was evaluated. Except for MDR1(2677T) and MDR1(1236T/2677T/3435T), cells expressing MDR1 variants displayed intermediate 8123 intracellular accumulation (1.5-2-fold higher) and lower effluxed 8123 (10-20% vs. 52%) compared to those expressing MDR1(wt). Efflux ratios across MDR1(wt) expressing cells were significantly larger for 8123 (3.95 +/- 1.1), Vinblastine (3.75 +/- 0.26), and Vincristine (2.8 +/- 0.29). Recombinant cells expressing MDR1 variants displayed 0%-22.7% P-gp activity (similar to 80%-100% efflux loss). Results suggest that MDR1 polymorphisms at the 1236, 2677, and/or 3435 positions significantly minimize P-gp functionality in vitro, the extent of which appears to be substrate dependant. (c) 2006 Wiley-Liss, Inc.