Thus we chose to focus on the results of the caspase 3 7 screen to initially identify regulators of TRAIL and use the caspase 8 and cell viability screening data to corrob orate our findings. We defined putative negative regula tors of TRAIL as those genes for which at least three of the four siRNAs tested caused an increase in TRAIL induced caspase 3 7 activation Sorafenib Tosylate two standard deviations or more from the TRAIL induced caspase 3 7 activation, seen with the control siNeg siRNA. This corresponded to a 10. 28 fold change for the kinase and additional gene set screens and a 7. 96 fold change for the phosphatase gene set. These fold changes were comparable with that seen after silencing of the negative regulator FLIP.

These criteria identified 83 kinases or kinase related genes, four phosphatases, and 63 genes from Inhibitors,Modulators,Libraries the add itional gene set, whose silencing augmented TRAIL induced caspase 3 7 activity. The screen identified several known negative regulators Inhibitors,Modulators,Libraries of apoptosis as negative regulators of TRAIL induced caspase 3 7 activation, in cluding BCL2L1, BCL2L2, BIRC2, and BIRC3. Also we assessed whether any genes act as positive regulators of TRAIL activity. We defined positive regula tors of TRAIL induced caspase activation as those genes in which at least three of four siRNAs resulted in TRAIL Inhibitors,Modulators,Libraries induced caspase 3 7 activation that was two or more standard deviations less than that seen in cells treated with the siNeg control. Interestingly, with these cri teria, no positive regulators of TRAIL induced caspase 3 7 activation were identified.

Silencing of CASP8 clearly inhibited TRAIL induced activation of caspase 3 7 by more than 2 standard deviations, indicating that the screen was capable of identifying such Inhibitors,Modulators,Libraries genes. Relaxing the criteria to siRNAs that resulted in more than a 1 standard deviation reduction in TRAIL induced caspase 3 7activation compared with the siNeg control, identified eight genes as putative positive regulators of TRAIL induced caspase 3 7 activation. Gene network analysis and experimental corroboration of negative regulators of TRAIL We focused our subsequent analysis on putative negative regulators of TRAIL induced caspase 3 7 activation ra ther than on positive regulators because of the number of genes identified and because they may be potential targets that, when inhibited, will enhance TRAIL induced apoptosis.

Given the relatively large number of putative negative regulators of TRAIL induced apoptosis, we sub jected the 150 genes to network gene analysis to aid in identification of common regulatory networks in which these genes function. Of the 147 genes with curated inter action data, the largest network identified connected 79 genes. Of these 79 genes, Inhibitors,Modulators,Libraries 42 were connected principally via four genes with seven or more interactions. The genes (-)-Nutlin-3 situated at these nodes are BCL2L1, IKBKB, PDPK1, and SRC.