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Version actuelle en date du 28 décembre 2015 à 06:14

The supportive data for this hypothesis includes the in vitro observations that these are genes induced when an EGFR Discussion The epidermal growth factor receptor family is of tremen dous biological and clinical importance for many solid epithelial tumors dependent cell line is freed from growth inhibition via EGFR inhibitors and the in vivo associations between the high expression of these signa tures and genes including HRAS, KRAS and EGFR itself. Regardless of the classical markers of activation of the Discussion The epidermal growth factor receptor family is of tremen dous biological and clinical importance for many solid epithelial tumors EGFR RAS MEK pathway, the strong associations between these expression profiles and patient outcomes in two dif ferent data sets suggest that they are important profiles. Currently, we have chosen only to validate our profiles using additional microarray Discussion The epidermal growth factor receptor family is of tremen dous biological and clinical importance for many solid epithelial tumors data sets, as opposed to using western blots or quantitative PCR of the training set, since each of these signatures represents a large number of genesproteins. A better understanding of each subtypes EGFR signaling pathway will have an impact on identifying and determining treatment as the gene expression signature may more readily be associated with activation of the pathway than EGFR status alone. Methods Cell culture SUM102 and SUM149 cells were a gift from Steve Ethier of Wayne State University and represent cell lines derived from ER and HER2 basal like breast tumors.

The SUM cell lines were maintained in an Epithelial Growth Medium developed by the Tissue Culture Facility at the University of North Carolina at Chapel Hill, and the SUM149 line was further supplemented with 5% FBS. The MCF 7, ZR 75 1, HME CC and ME16C cell lines were obtained and maintained as previously described. Cytotoxicity assay Cell line sensitivities to drugs were assessed using a mito chondrial dye conversion assay as described previously with the following modifications. Cells were seeded into trip licate 96 well plates and allowed to adhere over night. Cells were treated for 72 h with a range of doses of individual drugs. Carboplatin, doxorubicin, 5 fluorour acil, paclitaxel, and LY294002 were purchased from Sigma. Gefitinib was a gift from Astra Zeneca and cetuximab was purchased from the UNC Hos pitals Pharmacy Storeroom. U0126 was purchased from Cell Signaling. The inhib itory concentration that caused a 50% reduction in MTT dye conversion dose was determined as previously described. Drug combination interactions were analyzed using methods developed by Chou and Talalay. Using cell lines plated as described above, seven treatment combina tions consisting of constant ratios of IC50 doses were applied to cells and growth compared to untreated controls using the MTT assay. Four treatment schedules were tested 72 h concurrent, 72 h inhibitor followed by 72 h chemotherapeutic, 72 h chemotherapeutic followed by 72 h inhibitor, and a 144 h concurrent dose with a media change at 72 h. CalcuSyn was used to determine the combination index, which is a measure ment of the type of drug interactions. A combination index of one indicates an additive response, less than one indicates a synergistic response, and greater than one indicates an antagonistic response. Collection of mRNA for cell line experiments For each treatment, the SUM102 cells were grown in 15 cm dishes until 5060% confluence.