Irreversible TKI, we modeled the Lenvatinib E7080 resistance to the HKI-kinase inhibitor irreversibly dual EGFR/HER2 272 in a NSCLC cell culture model. Materials and methods to produce cell culture study resistant subclones of PC 9 cells, they were treated with 400 Ag / ml ethyl methanesulfonate overnight. They were allowed to recover for several passages, then at a density of 6104 cells / dish 10 cm 2 in 0.2 amol / l seeded HKI 272 t. The relative resistance of these cells to HKI 272, compared with the reversible inhibitors gefitinib and erlotinib was obtained by inoculating 3000 cells / well in 96-well plate in the presence of various concentrations of the drugs, follow-up 72 h by fixation of cells in 4% formaldehyde, F staining with Syto 60 and quantification of cell mass with the Odyssey infrared imaging system. NIH3T3 cells were grown in DMEM, erg complements With 10% f Fetal bovine serum, glutamine and penicillin / streptomycin. Epidermal growth factor at a final concentration of 100 ng / ml. Gefitinib and erlotinib were obtained from the pharmacy at the Massachusetts General Hospital. HKI 272 was provided by Wyeth Pharmaceuticals, is available. Wild-type EGFR, EGFR C797A, EGFR L858R and EGFR C797A/L858R were subcloned into pUSEamp for transient transfection LY2109761 700874-71-1 of 293T cells. EGFR wild-type EGFR T790M and PRV HygR constructions have already been described. EGFR L858R, EGFR Del746 750, C797A/L858R EGFR and EGFR-C797A / Del746 750 were subcloned into pBABE puro to infect NIH3T3 cells. For retroviral infection studies were EGFR and pBabe puro pRV HygR constructs in transfected 293T derivatives BOSC 23 packaging cell lines. NIH3T3 cells were transfected with retroviral supernatant twice infected in the presence of polybrene. The infected cells were selected using hygromycin or puromycin. Immunoblot analysis the cells were lysed with ice-cold buffer containing 1 mmol / L sodium orthovanadate and a protease inhibitor cocktail. Debris was removed by centrifugation. Clarified Rten lysates were boiled in loading buffer and by gel 4% to 15% gradient SDS-PAGE.
The proteins Were electroblotted onto nitrocellulose and specific antique Rpern demonstrated. The phosphorylated EGFR Y992 Antique Body was obtained from Biosource, total EGFR antibody was Body from Santa Cruz Biotechnology, phosphorylated tyrosine residues on antique From BD Transduction Laboratories body was, and tubulin Antique Body was from Abcam, it was h a secondary horseradish peroxidase-conjugated Ren Antique body followed. The bands were visualized using verst Rkter chemiluminescence followed by autoradiography. EGFRMutations analysis of several clones of exon 20 were sequenced in Temsirolimus order to investigate codon 790th Scorpion ARMS primers for the detection of E746A750del and T790M was done with an EGFR Scorpion kit according to Kimura et al. Briefly, all reactions in 25 volumes using 5 AL AL Of the target DNA, carried out 16 AL mixture of reaction buffer, 3.2 AL water, and 0.8 U Taq polymerase. All reagents were included in this kit. Real-time PCR was performed using Applied Biosystems 7500 under the following conditions: denaturation at anf ngliche 95jC for 10 min, 50 cycles of 30 s 95jC and 61jC for 60 s at fluorescence measurement value at the end of each cycle. The data analysis was performed using the ABI Prism 7500 software. The threshold cycle was as in the h Chsten plane defined.