FIGURE 4  EGFR mutations and differential effects on the sensitivity of the receptor towards inhibition by erlotinib and gefitinib

Figure 4.  EGFR mutations and differential effects on the sensitivity of the receptor towards inhibition by erlotinib and gefitinib

COS-7 cells transiently transfected with EGFR plasmid constructs (wild-type, L858R, E884K, and L858R + E884K [created by site-directed mutagenesis as previously described¹⁶]) were treated with increasing concentrations of either (A) erlotinib or (B) gefitinib, in the presence of EGF (100 ng/ml, 30 min). Whole cell lysates were collected after EGF stimulation, separated on 7.5% SDS-PAGE and electrotransferred onto nitrocellulose membrane for immunoblotting. The membrane was probed with an antibody against phospho-EGFR [pY1068] (upper panel) and -actin (lower panel) as a loading control. Equivalent receptor expression was also confirmed with an anti-EGFR immunoblot (see Supplementary Figure 1 online). Two separate immunoblotting experiments were used for quantitative analysis of the phospho-EGFR signal intensity. U, cells untreated with TKI; WT, wild-type. (C) Percentage (%) phospho-EGFR signal level compared with the untreated control (100%) plotted for EGFR^L858R-COS-7and EGFR^{L858R + E884K}-COS-7 cell lines treated with either of the two EGFR TKIs at 0.1 M. The EGF-stimulated phospho-EGFR activation of L858R mutation is modulated by the E884K mutation differentially with erlotinib (resistant) and gefitinib (sensitizing) as shown here at the in vitro concentration of 0.1 M. The percentage phospho-EGFR signal level (%) relative to untreated control (100%) was shown for both the L858R and L858R + E884K mutations to illustrate how E884K differentially modulates the sensitizing effect of L858R on the receptor towards the two different inhibitors. (D) Multiple sequence alignment performed by the CLUSTAL W alignment program for exon 22 of ErbB1, 2, and 3 of the ErbB receptor family. The black arrow shows the conserved nucleotide G that was mutated in our patient's tumor EGFR gene and the arrow outline and bold text shows the conserved amino acid residue glutamic acid, E884. The methods are further described in the Supplementary methods section; please go to the article online for details. ^*Conserved nucleotides among the three sequences; IB, immunoblot.

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