Figure 3

Curves of cell proliferation and EGFR modulation after addition of gefitinib at various concentrations. A, the graphs represent cell growth curves of the Ist-Mes-1, Ist-Mes-2 and MPP89 cell lines treated with 6.25, 12.5, 25 and 50 μM of drug (Gefitinib) compared to cells treated with DMSO at the same final concentration of that present in medium with drugs (CNTRL) as described in material and methods. The survival of cells was expressed as absorbance (A _{492 nm}-A_{690 nm}) and % of proliferation (on the right bottom). To evaluate the modulation of gefitinib on EGFR, Ist-Mes-1, Ist-Mes-2 and MPP89 cell lines were treated with 50 μM gefitinib for 48 h. The effect of the drug was evaluated by western blot (B) and quantitative PCR (C). The table (D) displays gefitinib IC₅₀ in the cell lines. E, effect of gefitinib on EGFR and AKT phosphorylation. Ist-Mes-1, Ist-Mes-2 and MPP89 cells were cultured in complete medium for 24 hours and then treated with gefitinib 50 μM as described in materials and methods. EGFR-phosphorylation (P-EGFR) and AKT-phosphorylation (P-AKT) were analyzed by western blot as described above. Cell treated with EGF were used to control for EGF-induced phosphorylation. DMSO was added to the medium of control to give a final concentration that matched DMSO present in medium containing drugs.