Sensitivity of the cell lines carrying activating c-KIT mutations to dasatinib. (A) Western blot analysis of lysates prepared from HMC-1.1^{V560G+, D816V-} and HMC-1.2^{V560G+, D816V+} cell lines following incubation in the presence of imatinib or dasatinib. Cells were serum starved for 3 hours and exposed for 60 minutes to the inhibitors indicated at the concentrations listed (μM). p-kit indicates phospho-KIT (B) Cell-growth inhibition of HMC-1.1^{V560G+, D816V-} and HMC-1.2^{V560G+, D816V+} in the presence of varying concentrations of imatinib or dasatinib. Days in culture at the time of analysis are indicated. Each data point represents mean ± SEM from 3 separate experiments with duplicate counts. P < .05 at all concentrations of dasatinib or imatinib in HMC-1.1^{V560G+, D816V-} cells and at 0.5 and 1 mM dasatinib in HMC-1.2 ^{V560G+, D816V+} cells (2-tailed, paired t test on day-3 results). (C) Inhibition of cell growth of the P815 murine mastocytosis cell line harboring a c-KIT (D814V) mutation. Cells were grown in the presence of varying concentrations of dasatinib (i) or imatinib (ii). Days in culture at the time of analysis are indicated. (D) Annexin V binding of HMC-1.1^{V560G+, D816V-} and HMC-1.2^{V560G+, D816V+} cells as assessed by flow cytometry. Cells were cultured with various concentrations of inhibitors as shown and analyzed at 24 and 48 hours. Baseline values of annexin V-positive cells in untreated cultures were subtracted from each treatment. Each point represents mean ± SEM from duplicate measurements. Dasatinib, ▪; imatinib, ▨.