title: MDR1 haplotypes significantly minimize intracellular uptake and transcellular P-gp substrate transport in recombinant LLC-PK1 cells.
abstract: 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 MDR1wt 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 microM) were evaluated in control and recombinant cells. Additionally, the transepithelial transport of R123 (1 microM) and Vinca alkaloids (5 microM) was evaluated. Except for MDR1(2677T) and MDR1(1236T/2677T/3435T), cells expressing MDR1 variants displayed intermediate R123 intracellular accumulation (1.5-2-fold higher) and lower effluxed R123 (10-20% vs. 52%) compared to those expressing MDR1wt. Efflux ratios across MDR1wt expressing cells were significantly larger for R123 (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 (approximately 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.
authors.affiliation: Department of Pharmaceutics, Box 357610, University of Washington, Seattle, Washington 98195-7610, USA. nsalama@u.washington.edu
authors.forename: Noha N
authors.initials: NN
authors.lastname: Salama
authors.forename: Ziping
authors.initials: Z
authors.lastname: Yang
authors.forename: Tot
authors.initials: T
authors.lastname: Bui
authors.forename: Rodney J Y
authors.initials: RJ
authors.lastname: Ho
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression; D019450:Genes, MDR / Q000235:genetics*; D006239:Haplotypes; D018374:LLC-PK1 Cells; D020641:Polymorphism, Single Nucleotide; D020112:Rhodamine 123 / Q000378:metabolism; D013552:Swine; D014162:Transfection; D014747:Vinblastine / Q000378:metabolism; D014750:Vincristine / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D020112:Rhodamine 123; D014750:Vincristine; D014747:Vinblastine