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as exemplified in Figure 3C.This assay showed two independent peaks,a single for wild type and another BIO GSK-3 inhibitor for mutant EGFR gene,both in 11 18 and erlotiniresistant cells.Even so,the BIO GSK-3 inhibitor peaheight ratio in the two resistant cell lines was clearly unique.By adopting mixing method,which is,mixing the DNAs ofhUVECs carrying 2 copies of wild type EGFR gene with that of resistant cells,the modify in copy number in the allele might be quantified as described in Materials and Techniques.The results indicated about a 50% decrease in the mutant EGFR gene with no apparent modify in the wild type EGFR gene copy.We also NSC 14613 examined no matter if selection by drug resistance to gefitinialso induced equivalent modifications of decreased expression in the activating EGFR gene.
Two gefitiniresistant cell lines,11 18 GEF10 1 and 11 18 GEF20 1,showed increased EGFR protein expression with comparatively decreased expression Digestion ofhER2 and pHER2 in comparison with their parental 11 18 cells.As compared using the parental 11 18 cells,Akt phosphorylation in 11 18 GEF10 1 and 11 18 GEF20 1 was not affected by gefitiniwhen phosphorylation of EGFR and ERK1 2 was similarly inhibited by gefitinib.Western blot analysis using the antL858R antibody showed decreased expression in the mutant EGFR and similar expression in the total EGFR in two resistant cell lines as compared with 11 18 cells.Next,we performed DNA sequence analysis and identified an alternating peaheight on nucleotide 2573 in gefitiniresistant cells.Place SSCP analysis also revealed a decreased mutant EGFR gene copy with no apparent modifications in wild type EGFR gene copy,and quantitative analysis indicating about a 50% decrease in the mutant EGFR gene in gefitiniresistant cells.
From these analyses of erlotinior gefitiniresistant cells lines,acquisition of drug resistance may possibly be mediated through a decreased mutant EGFR gene copy.Knockdown ofhER2 orhER3 Sensitizes the Constitutive Activation of Akt to Erlotiniin PC9 ER1 Cells There was virtually full loss of mutant EGFR gene in PC9 NSC 14613 ER1 whereas there was only partial loss in the mutant EGFR gene in erlotiniresistant cell lines derived from 11 18.We further analysed more in detail any mechanism underlying acquirement of erlotiniresistance in PC9 ER1.We examined the effect of PI3inhibitors,wortmannin and LY294002 on Akt activation in PC9 and PC9 ER1 cells.
Both PI3inhibitors similarly inhibited phosphorylation of Akt,indicating that activated Akt is similarly susceptible to both inhibitors in PC9 ER1 and PC9 cells.We also confirmed specifisuppression of Akt activation in both PC9 and PC9 ER1 cells when BIO GSK-3 inhibitor treated with PIK3CA siRNA.Furthermore,sequence analysis revealed that there was no mutation inhot spots of PIK3CA,PTEN and Akt gene.The constitutive Akt activation in PC9 ER1 seems not to be because of altered PI3K Akt pathway itself.We lastly NSC 14613 examined which molecules among EGFR,HER2 orhER3 might be responsible for the constitutive Akt activation in erlotiniresistant PC9 ER1 cells.We identified phosphorylation ofhER3 was not suppressed by erlotiniin PC9 ER1 in comparison to PC9.We then examined no matter if knockdown of EGFR,HER2 orhER3 by their cognate siRNAs could modulate activation of Akt and EGFR family members proteins.
Knockdown of EGFR resulted in markedly decreased activation of Akt only in PC9 cells but not in PC9 ER.On the otherhand,knockdown ofhER3 could suppress activation of Akt in both PC9 and PC9 ER.Furthermore activation ofhER3 was markedly suppressed byhER2 knockdown only in PC9 ER.These final results suggest thathER3 with each other withhER2 signaling are responsible for constitutive activation of BIO GSK-3 inhibitor PI3K Akt in acquired resistance to erlotiniin PC9 ER.We further examined no matter if lapatinib,a dual kinase inhibitor of EGFR andhER2,could suppress Akt activation in PC9 ER1.Therapy with lapatiniinhibited phosphorylation of Akt andhER3 while erlotinidid not.We next examined the effect of erlotinior a pan tyrosine kinase inhibitor of all EGFR family members,BIBW2992,on Akt phosphorylation in PC9 ER1 when each and every EGFR,HER2 orhER3 was silenced.
The phosphorylation ofhER2,HER3 and Akt was all suppressed by BIBW2992 alone.On NSC 14613 the otherhand,the phosphorylation of Akt was inhibited by erlotiniwith eitherhER2 orhER3 knockdown.Furthermore,HER2 knockdown resulted in a marked inhibition ofhER3 phosphorylation,suggesting that PC9 ER1 cells gain addiction tohER2 HER3 signaling.We lastly examined no matter if expression of activating mutant EGFR could restore drug sensitivity to erlotiniin drug resistant cell lines,PC9 ER1 and 11 18 ER1 7.Transient transfection of del EGFR cDNA induced enhanced expression of activated mutant EGFR in PC9 ER1.Overexpression of del EGFR cDNA overcame drug resistance to erlotiniin PC9 ER1.Furthermore,transfection of another activated mutant L858R EGFR cDNA also induced enhanced expression and restored drug sensitivity to erlotiniin 11 18 ER1 7 cells.Loss of Activating Mutant EGFR in Refractory Non little cell Lung Cancers Figure 8 showed representative IHimages for wild type,delE746 A750,and L858R EGFR ex