Intracellular content of gefitinib in NSCLC cell lines and its effect on EGFR autophosphorylation. A) Time course of 0.1 μM [3H]gefitinib accumulation in H322 (●) and H1299 (○) cell lines. Each point represents the mean ± SD of four independent determinations. B) H322 cells were incubated for 0.5 h and 24 h with the indicated extracellular concentrations of gefitinib before stimulation with 0.1 μg/ml EGF for 5 min. Western blot analysis was performed by using monoclonal antibodies directed to p-EGFR(p-Tyr1068) and to EGFR. The immunoreactive spots were quantified by densitometric analysis, ratios of phosphotyrosine/total EGFR were calculated and values are expressed as percentage of inhibition versus control. The experiment, repeated three times, yielded similar results. C) H322 cells were incubated with 0.1 μM gefitinib for 0.5 h or 24 h and then the initial rate (5 min) of 0.1 μM [3H]gefitinib uptake was measured. Each bar represents the mean ± SD of four independent determinations. D) H322 cells were exposed to 0.1 μM [3H]gefitinib for 0.5 h and 24 h, in the absence or in the presence of 10 μM Fumetrimorgin C (F) or PSC833 (P) and then intracellular gefitinib content was determined. Values given are the means (± SD) of three independent determinations (***P < 0.001).