Effect of ABCG2 overexpression on gefitinib accumulation, efflux, and uptake.
Characterization of HEK293/R2 overexpressing cells: (A) Cells were lysed and 50μg of proteins for H292, H460 and HEK293 or 10μg for HEK293/R2 cells loaded to assess ABCG2 protein expression by Western blot analysis. Different amounts of proteins were loaded to avoid the signal saturation in the sample from ABCG2 overexpressing cells. Data are from a representative experiment. Each experiment, repeated twice, yielded similar results. (B) ABCG2 protein levels on cell surface were quantified by flow-cytometry and expressed as molecular equivalent of fluorochrome (MEF) as described in the Methods Section. (C) Cells were loaded with 1 μM Hoechst 33342 in the presence or in the absence of 10 μM Fumitremorgin C. After 4 hours of incubation, Hoechst 33342 was removed and its fluorescence was determined by luminometer. Relative ABCG2 activity was defined as the ratio of Hoechst 33342 accumulation per μg of protein between Fumetrimorgin C treated cells and untreated cells and was expressed as fold increase. The mean values of three independent measurements (± SD) are shown. The cellular accumulation (D), efflux (E) and uptake (F) of 0.1 μM [3H]gefitinib was determined in overexpressing ABCG2 transporter HEK293/R2 cells and in the parental cells (HEK293), after 4 hours of gefitinib treatment. The percentage of efflux was calculated after 5 min of incubation with fresh medium, while initial velocity (5 min) of [3H] gefitinib uptake was measured after 4 hours of 0.1 μM gefitinib treatment (***P < 0.001).