The tumor samples were collected from client owned animals that have developed the disease naturally. All procedures per formed on these animals with regards to tumor collection were strictly for treatment purposes selleck chem and nothing was done different because of the drug perturbation study. All pro cedures were performed according to standard of care regardless of whether an animal had its tumor sampled. For the generation of the experimental data, the canine osteosarcoma primary cell cultures were plated in 384 well plates at a seeding density of 2000 cells per well over graded concentrations of 60 small molecule kinase inhibitors. Each inhibitor was plated individually at four concentrations predicted to bracket the IC50 for that drug.
Cells were cultured in RPMI 1640 supplemented with 2mM glutamine, 2mM sodium pyruvate, 2mM HEPES, 1% penicillin streptomycin, and 10% fetal bovine serum for 72 hours. At the end of the 72 hour incubation, cell viability Inhibitors,Modulators,Libraries was assessed using the MTS assay. All values were normal ized to the mean of seven wells on each plate containing no drug. The IC50 for Inhibitors,Modulators,Libraries each drug was then determined by identification of the two concentrations bracketing 50% cell viability and application of the following formula DA where cell viabil ity value above 50% A and cell viability value below 50% B. The experimentally generated IC50 values are included as Additional file 2. The experimentally gener ated sensitivities of the 60 drugs are then scaled to values between 0 and 1. Among the 60 drugs on the drug screen, 46 drugs have known target inhibition profiles.
of these 46 drugs, 2 pro vide information only on the target mTOR and analysis of these drugs Inhibitors,Modulators,Libraries are triv ial. Thus, the remaining 44 drugs are used to generate the TIMs. These target profiles were extracted from several literature sources Inhibitors,Modulators,Libraries based on experimental quan titative dissociation Inhibitors,Modulators,Libraries constants which are treated as EC50 values for each drug across kinase target assays with more than 300 targets. The target profiles of the drugs are shown in Additional file 3. Figures 2 and 3 represent the equivalent TIM cir cuits generated from experimental data for Bailey and Sy respectively. The TIM circuits for Charley and Cora are included in Additional file 1. To emphasize the biological relevance provided by the TIM framework employed in the analysis of the biologi cal data, we present a more in depth analysis of the TIM circuit devised for the canine patient Bailey.
The vast majority of human osteosarcomas con tain genetic or post translational abnormalities in one or both of the tumor suppressors p53 and pRb. The first target identified in this circuit is PKC alpha. PKC alpha modifies CDKN1A, selleck Lenalidomide which is the primary mediator of p53 tumor suppressor activity. PSMB5 represents the proteasome.