The resistance to EGFRInhibitor is Aurora A required is irreversibly due to the substitution T790M acquired resistance to gefitinib and erlotinib h Frequently with a substitution mutation T790M kinase Dom ne of the EGFR associated, resulting in a protein that exhibits reduced binding to drugs, while maintaining the catalytic activity of t. Therefore, we decided that hosting the 272 HKI-resistant clones T790M mutation. Because endogenous EGFR-mutant alleles are RKT verst in the PC-9 cells, We used a sensitive assay, quantitative PCR and allele-specific amplification combined. In this assay, the primers are con Us so that only the mutation allelespecific verst Is strengthened and the amount of PCR product is then quantified relevant. The number of PCR cycles required to detect the mutation is a measure currently available for the target molecule in the reaction. The DCT defines the difference between the threshold cycle of the reaction and this mutant a positive control. Thus, the lower part of the DCT, the gr-Run representation of the mutation in the tested sample. Conversely, plus the DCT is the cutoff point, the less these samples contained mutations. We were able to confirm to that all our HKI 272-resistant clones of EGFR mutations found in Del15 get parental cell line, has best Firmed that the resistance is not acontaminating in the BX-795 702675-74-9 subpopulation of cells results in significant. In particular, all had 272 HKI-resistant clones and parental PC-9 cells has a value between 1 and DCT-2, the best Firmed that the mutation is present Del15 to a very high level in all cells. If the presence of EGFR T790M was tested, the resistant clones fell into two groups, the well defined on the two groups correspond to biochemically. Resistant clones class B, the EGFR phosphorylation in the presence of HKI retain 272 high T790M have in their genomic DNA, as determined by the lower DCT values. However, the class A clones in which EGFR phosphorylation was effectively suppressed by drugs, high values of DCT.
These values are in the N Height, or above the break point 11, indicating that these clones or not containing a mutation T790M, or that this mutation is present in low concentrations are below the sensitivity of the test. To ensure that T790M is in only one of two groups of resistant clones, we have a PCR reaction to specifically amplify exon 20, the codon 790 lt contains, And then sequenced individual clones. Securing previous results it was found that T790M is derived in a significant number of PCR subclones of clone 37th The parent-PC 9 cells and resistant clones of class A, but only wild-type sequences in the harbor 20th exon This closing S we find that the resistant clones, the EGFR phosphorylation in the presence of the drug to port T790M Rolipram mutation of EGFR hold. EGFR T790M mutation as a mechanism of resistance to HKI discovered 272 in cell culture model was unexpected since it has been reported that EGFR T790M resistance could HKI 272 to gefitinib and erlotinib overcome studies on cell cultures. In addition, a recent report showed a mouse model of NSCLC describes in connection with EGFR expression in cis with L858R T790M partial regression of adenocarcinomas.