Figure 6

Gefitinib treatment reduced RWGT2 tumour EGFR and MAPK phosphorylation at 78 h but not tumour volume. (A) Comparison of EGFR and MAPK phosphorylation as assessed in untreated and gefitinib-treated RWGT2 tumours by Western blots. Tumours from untreated RWGT2 mice had greater phosphorylation of both EGFR phosphotyrosine residue 1068 and ERK1/2 when compared to tumour lysates from RWGT2 gefitinib-treated mice. Furthermore, all lysates had equivalent EGFR and ERK1/2 expression. Equal amounts of total protein (10 μg) were separated by electrophoresis using 4–20% tris-glycine sodium dodecyl sulphate-polyacrylamide gels, transferred onto nitrocellulose membranes, and analysed by immunoblotting using rabbit polyclonal antibodies to phosphotyrosine 1068 and phospho ERK1/2. Blots were stripped and reprobed with rabbit polyclonal antibodies against EGFR and ERK1/2. The blots were stripped a final time and reprobed with a polyclonal antibody to β-actin. Experiments were repeated three times, and the data from a representative blot are shown. (B) Tumour volumes were measured serially as described in ‘Materials and methods.’ No reduction in tumour volume was present (pretreatment, 48 and 78 h) between gefitinib-treated and untreated mice at any time point. Each bar represents the mean tumour volume of eight or nine mice; bars, s.e.m. ^*P=0.048 vs treated at the pretreatment time point even though the animals were randomised. (C) Gefitinib treatment of mice with RWGT2 xenografts did not cause an increase in apoptosis. Representative samples at 78 h from gefitinib-treated and untreated tumours. H&E staining revealed similar numbers of apoptotic cells represented as shrunken cells with eosinophilic cytoplasm and karyorrhectic or pyknotic nuclei in both the treated and untreated mice (black, arrows); bar 100 μm. Fluorescent TUNEL staining for apoptosis (black, arrows) revealed similar numbers of apoptotic cells throughout the tumour parenchyma. × 400. (D) Comparison of numbers of apoptotic cells in gefitinib-treated and untreated RWGT2 tumours at 78 h. Ten × 100 fields from a fluorescent TUNEL slide for each mouse were counted to assess the number of apoptotic cells per field. The mean apoptotic cell number per × 100 field was represented as the black bar for gefitinib-treated and white bar for the untreated. No significant differences in mean apoptotic cells in tumours were present between the gefitinib-treated vs untreated mice.