A novel MDR1 GT1292-3TG (Cys431Leu) genetic variation and its effect on P-glycoprotein biologic functions

Abstract

P-glycoprotein (P-gp) is a membrane-bound transporter protein that is encoded by the human multidrug resistance gene MDR1 (ABCB1). P-gp recognizes a wide range of xenobiotics, is pivotal in mediating cancer drug resistance, and plays an important role in limiting drug penetration across the blood-brain barrier. MDR1 genetic variation can lead to changes in P-gp function and may have implications on drug pharmacokinetics. We have identified a novel MDR1 (GT1292-3TG) (Cys431Leu) genetic variation through systematic profiling of subjects with leukemia. The cellular and transport function of this variation was investigated with recombinant human embryonic kidney cells expressing MDR1. Compared with the wild type, MDR1 (GT1292-3TG) recombinant cells exhibited a lower drug resistance phenotype for a panel of chemotherapeutic agents. When compared with wild type, MDR1 (GT1292-3TG) recombinant cells exposed exhibited a 75% decrease in IC₅₀ for doxorubicin (162.6 ± 17.4 to 37.9 ± 2.6 nM) and a 50% decrease in IC(50) for paclitaxel (155.7 ± 27.5 to 87.7 ± 9.2 nM), vinblastine (128.0 ± 15.9 to 65.9 ± 5.1 nM), and vincristine (593.7 ± 61.8 to 307.3 ± 17.0 nM). The effects of the Cys431Leu variation, due to MDR1 (GT1292-3TG) nucleotide transition, on P-gp-dependent intracellular substrate accumulation appeared to be substrate dependent where doxorubicin, vinblastine, and paclitaxel exhibit an increased accumulation (p < 0.05), while verapamil and Hoechst33342 exhibit a decreased intracellular concentration compared with wild type (p < 0.05). Collectively, these data suggest MDR1 (GT1292-3TG) variation of P-gp may reduce drug resistance and that subjects with this genotype undergoing chemotherapy with drugs that are transported by P-gp could potentially be more responsive to therapy than those with MDR1 wild-type genotype.

Fig. 1

Comparison of P-gp protein expression in recombinant MDR1 and host HEK cells. Total cell lysates from recombinant cell expressing MDR1 _WT (lane 2), MDR1 _GT1292-3TG (lane 3), or MDR1 negative HEK host (lane 1) were separated by gel electrophoresis, then blotted onto a PVFD membrane. The membrane was probed with the F4 anti-P-gp monoclonal antibody, developed with a ECL reagent, and exposed to X-ray film to detect anti-P-gp reactive protein bands at ~150 kDa

Fig. 2

Effects of P-gp inhibitor GF120918 on rhodamine123 efflux in preloaded P-gp recombinant cells. Recombinant cells expressing control and MDR1 variants were seeded into 12-well tissue culture plates in quadruplicate and grown overnight at 37°C. Medium was removed and preincubated with serum-free medium or medium containing 1 μM GF120918 for 5 min before replacing the medium with 1 μM R123 or 1 μM R123 plus 1 μM GF120918 and incubated for 30 min at 37°C. Medium was removed, and the plates were washed with warm PBS. Serum-free medium or medium containing 1 μM GF120918 was added and incubated at 37°C for 30 min. After extensive washing of the cells, cells were lysed with 0.5% DOC. The intracellular R123 fluorescence (λ _ex = 485 nm; λ _em = 535 nm) was measured on a Victor III multiplate reader. White bars rhodamine123, black bars rhodamine123 + GF120918

Fig. 3

Comparison of intracellular substrate concentration in cells expressing MDR1 _WT and MDR1 _GT1292-3TG. Cells were seeded out into 96-well plates and grown overnight. The growth media was then removed and replaced with 1 μM rhodamine123 (R123), 5 μM doxorubicin (Dox), 1 μM Paclitaxel-Oregon Green® 488 (Pac), 1 μM Bodipy FL-Vinblastine (VB), 0.3 μM Bodipy FL-Verapamil (Ver), or 0.4 μM Hoechst33342 (H) in DMEM for 30 min at 37°C. Cells were washed three times with cold PBS and lysed in 0.5% DOC. The respective drug concentrations were determined based on fluorescent intensity measurement and expressed as a percentage of control host HEK cells without MDR1. Results were normalized for protein content. Data expressed are mean and standard deviation of quadruplicate samples. Black columns HEK (−) control, hatched columns MDR1 _WT, dotted columns MDR _GT1292-3TG. *p < 0.05; **p < 0.01