To identify kinases that can be targeted to increase radiosensitivity things in HNSCC, it will be impor tant to explore multiple pathways. In this study, we used an antibody based array to quantify the expression Inhibitors,Modulators,Libraries levels of multiple phosphorylated kinases in a panel of HNSCC lines. The expression levels of these phospho kinases were correlated with radiosensitivity. Expression levels were measured in untreated and irradiated cells as both basal activity and activity induced by radiation of a ki nase could be important for cell survival after radiothe rapy. Inhibitors of the kinases that were associated with radiosensitivity were tested for their ability to enhance the radiotherapy effect in HNSCC. We identified several kinase inhibitors that have the potential to increase ra diosensitivity of tumors and thereby improve the out come of HNSCC patients.

Materials and methods Cell lines and chemicals Nine human head and neck squamous cell carcinoma cell lines were used in this study. The characteristics of the cell lines are shown in Table 1. Cell lines were not further authenticated or tested. Cells were cultured in T75 culture Inhibitors,Modulators,Libraries flasks, under humidified conditions, and passaged weekly or twice weekly in DMEM containing 2 mM L glutamine, 1% non essential amino acids, 20 mM Hepes, 10 units/ml penicillin, 10 units/ml streptomycin, and 10% fetal bovine serum. The following kinase inhibitors and concentrations were used Src Family Kinase inhibitor dasatinib, MEK1/2 inhibitor U0126. Human phospho kinase Inhibitors,Modulators,Libraries antibody array To determine levels of phospho kinases at baseline and after radiotherapy, cells were harvested after no treat ment or 1 h after a single dose of 4 Gy.

Cells were lysed using lysis buffer of the Human phospho kinase array kit and protein was quantitated using a standard Bradford absorbance assay. The Human phospho kinase Inhibitors,Modulators,Libraries array was performed ac cording the protocol of the manufacturer. In this array, 46 capture antibodies are Inhibitors,Modulators,Libraries spotted in duplicate on nitro cellulose membranes. The capture antibodies were di rected against the following antigen In short, cell lysates were incubated with the membrane overnight. Thereafter, the membranes were incubated with a cocktail of biotinylated detection antibodies and streptavidin HRP. Finally, proteins were detected using an ECL chemiluminescent system. To quantify expression levels, the integrated optical density of each spot was measured using ImageJ software.

IOD values were corrected for background signal and to compare different membranes sellectchem levels were normal ized to those of the positive controls on each membrane. Both the absolute expression levels after radiotherapy as well as the relative levels after radiotherapy were quantified. Radiosensitivity Clonogenic cell survival assays Cells were irradiated with graded doses at room temperature. After 1.