Figure 3. Expression of EGFR with a L858R/T790M or Del/T790M mutation more effectively promotes gefitinib resistance in HCC827 and H3255 cells.

(A) The single-mutant WT/T790M (WT/T), the double-mutant L858R/T790M (L/T) and Del/T790M (Del/T), and GFP (as a control) were introduced into HCC827 cells. The resulting cell lines were subjected to an MTS survival assay in increasing concentrations of gefitinib. (B) The same retroviruses as in A were used to infect H3255 cells, and resulting cell lines were subjected to MTS survival assays in increasing doses of gefitinib. (C) Lysates from HCC827 cells expressing EGFR mutants or GFP control and exposed to increasing concentrations of gefitinib for 12 hours were probed with the indicated antibodies. (D) Xenografts in nu/nu mice were generated from either HCC827 Del/T790M or HCC827 GFP cells. Gefitinib was administered by oral gavage, and tumors were measured 3 times weekly. Mean tumor volumes are shown. (E) The H3255 cell lines were subjected to an MTS survival assay in the presence of increasing concentrations of CL-387,785 and gefitinib. (F) H3255 L858R/T790M and H3255 GFP cells were seeded together at different ratios to determine whether a minor percent of L858R/T790M cells could confer resistance to cells with no T790M mutation. Cells were seeded in the indicated ratios (in parentheses) of H3255 GFP cells to H3255 L858R/T790M cells and subjected to an MTS survival assay.