Evidence that basal activity, but not transactivation, of the epidermal growth factor receptor tyrosine kinase is required for insulin-like growth factor I-induced activation of extracellular signal-regulated kinase in oral carcinoma cells

Evidence that basal activity, but not transactivation, of the epidermal growth factor receptor tyrosine kinase is required for insulin-like growth factor I-induced activation of extracellular signal-regulated kinase in oral carcinoma cells. RESULTS Gene expression data were obtained from the tumor collected at 4 different time points: (1) at initial diagnosis in 2001, (2) at recurrence after radiation therapy and before systemic chemotherapy or cetuximab treatment in 2004, (3) after cetuximab for 18 months and before AMG-479 treatment in 2007, and (4) after AMG-479 and before MTX treatment in 2007. Gene expression microarray data were also obtained from 2 areas of normal mucosal epithelium adjacent to the tumors: (1) at diagnosis in 2001 and (2) at recurrence after radiation therapy in 2004. Because the tumor rapidly grew during the AMG-479 treatment, effective target inhibition was confirmed by the decreased protein levels of total and phospho-IGF-1R and phospho-AKT after treatment with AMG-479 using Western blots (Physique 2). To examine the genes that were modulated by AMG-479 and the relatedness of the genes with numerous biological functions, differentially expressed genes with greater than 2-fold between pre-AMG-479 and post-AMG-479 treatment were determined by supervised analysis (Physique 3A and Supplemental Table, online only). Both the normal mucosa and tumor samples taken at the time of diagnosis before any treatment (samples obtained in 2001) differed in the MDV3100 expression signature when compared with samples at the time of recurrence (samples in 2004). This could be attributable to radiation effects because the recurrent tumor and normal mucosa samples were taken from the previously radiated field. Interestingly, after AMG-479 treatment, the expression signature reverted to the pattern observed in the pre-cetuximab-treated tumor which was sensitive to cetuximab. Open in a separate window Physique 2 Western blot MDV3100 comparison of protein levels in the tumors before and after AMG-479 treatment. IGF-1R, insulin-like growth factor-1 receptor; AKT, protein kinase B; DHFR, dihydrofolate reductase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Open in a separate window Physique 3 Hierarchical clustering of tumors and normal mucosal epithelia taken at 4 different time points. The gene expression experiments were performed in duplicate; At Dx NL 2001 FFPE – normal mucosal epithelium taken from a formalin-fixed paraffin-embedded (FFPE) tissue at original analysis in 2001, Pre-Cetux NL 2004 FFPE – regular mucosal epithelium extracted from a FFPE cells at recurrence after rays therapy and before systemic chemotherapy or cetuximab treatment in 2004, Pre-Cetux 2004 FFPE – tumor extracted from a FFPE cells at recurrence after rays therapy and before systemic chemotherapy or cetuximab treatment in 2004, Pre-AMG-479 2007 freezing – tumor extracted from a freezing cells used after cetuximab for 1 . 5 years and before AMG-479 treatment in 2007, and Pre-AMG-479 2007 freezing – tumor extracted from a freezing cells used after AMG-479 and before methotrexate treatment in 2007. (A) Rabbit Polyclonal to EGFR (phospho-Ser695) Gene manifestation data had been clustered using 2886 microarray probes which were differentially indicated between before and MDV3100 following the AMG-479 treatment. The genes indicated inside a blue box described the expression pattern following the AMG-479 treatment strongly. (B) Gene manifestation data had been clustered using genes in folate biosynthesis from Kyoto Encyclopedia of Genes and Genomes Pathway Data source. Dihydrofolate reductase was down-regulated following the AMG-479 treatment. The strength from the colours represents the number of gene manifestation levels: Reddish colored – higher manifestation, Green – smaller manifestation, and Dark – equal manifestation. The differentially expressed genes were interrogated using IPA further. The statistically significant systems of genes had been those involved with DNA replication, repair and recombination, cell cycle, cellular organization and assembly, cell signaling, and immune system response. Among the AMG-479-modulated genes with statistical significance was (Shape MDV3100 3B) that was downregulated MDV3100 by AMG-479. DHFR may be the binding focus on of MTX and its own energetic metabolite, which leads to S-phase cell routine inhibition.2 Eight additional genes in the folate biosynthesis pathway weren’t significantly altered by AMG-479. Reduction in the proteins degree of DHFR after AMG-479 treatment was verified by Traditional western blot (Shape 2). The tumor was adverse for human being papillomavirus disease or mutations in tyrosine kinase (TK) site or gene duplicate quantity by fluorescent in situ hybridization inside our previous.