Abstract

The significance of the human multidrug resistance gene (MDR1) G1199A polymorphism, resulting in a Ser400Asn modification in P-glycoprotein (P-gp), remains unclear. We have developed stable recombinant LLC-PK1 epithelial cells expressing either MDR1_wt or MDR1₁₁₉₉ to evaluate functional consequences of G1199A [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide]. P-gp activity observed in MDR1_wt and MDR1₁₁₉₉ cells was completely inhibited in the presence of the specific P-gp inhibitor GF120918. Comparable expression of mRNA and protein in the MDR1-expressed cells and correct localization of P-gp in the apical membrane of recombinant cells was verified. Mean intracellular rhodamine-123 (R123) accumulation, measured by flow cytometry, was approximately 4.75-fold higher in MDR1₁₁₉₉ recombinant cells than MDR1_wt cells. Cytotoxicity studies have shown that MDR1_wt and MDR1₁₁₉₉ cells exhibited similar resistance, as measured by EC₅₀ values, to doxorubicin (155 ± 68 versus 120 ± 32 nM); however, MDR1₁₁₉₉ cells were more resistant to vinblastine (1.41 ± 0.51 versus 15.7 ± 4.0 nM; p < 0.001) and vincristine (1.18 ± 0.56 versus 3.41 ± 1.47 nM; p < 0.05). The apparent transepithelial permeability ratios of R123 in MDR1_wt and MDR1₁₁₉₉ cells were 3.54 ± 0.94 and 2.02 ± 0.51 (p < 0.05), respectively. Therefore, the G1199A polymorphism alters the efflux and transepithelial permeability of a fluorescent substrate and sensitivity to select cytotoxic agents, which may influence drug disposition and therapeutic efficacy of some P-gp substrates.