The diffM did not show any time-dependent TNF�� or IFN�� release (Figure 2, A and B). In contrast, in actM (LPS 1 ng/ml, 5 ng/ml, and 10 ng/ml), concentrations of DAPT secretase buy TNF�� were significantly elevated (0 to 0.665 ng/ml), reaching a maximal release at 3 hours, whereas IFN�� secretion (0 to 0.002 ng/ml) was only slightly affected (Figure 2, A and B; P < 0.01). Because these cytokines were released by macrophages on their activation, they both were further investigated for their apoptosis-inducing effects in HCT116 tumor cells. Figure 2 TNF-�� and IFN-�� are significantly released by macrophage cell line U937 on activation. A and B: U937 cells were stimulated with PMA or stimulated and activated with PMA and LPS, and subsequently, the supernatants were analyzed by TNF-��-ELISA ...

TNF�� but Not IFN�� Exposure in HCT116 Tumor Cells Induced DAPK-Dependent Apoptosis We investigated whether TNF�� or IFN�� exposure alone as measured in the supernatant on LPS stimulation: 0.665 ng/ml for TNF�� and 0.002 ng/ml for IFN�� (Figure 2) were capable of inducing apoptosis in HCT116 tumor cells. IFN�� neither altered DAPK protein levels during the time frame of 6 hours to 72 hours, nor did it induce apoptosis (supplemental Figure S2 at http://ajp.amjpathol.org). In contrast, TNF�� treatment resulted in a significant increase in the apoptotic cell population in the Annexin-V assay (maximal induction at 48 hours: 3.94% to 19.2% apoptotic cells) accompanied by 2.8-fold up-regulation in caspase 3/7 activity (Figure 3, A and B).

Furthermore, TNF�� exposure led to an elevated DAPK protein level in HCT116 tumor cells (Figure 3C) paralleled by a decrease of the inactive DAPK protein phosphorylated at Ser308 (Figure 3C). The reduced phosphoDAPK-Ser308 amounts seemed to be controlled by a degradation-dependent pathway (supplemental Figure S3 at http://ajp.amjpathol.org). This gradual increase in DAPK level was accompanied by a pronounced induction of DAPK activity addressed by an in vitro kinase assay (Figure 3D) using mammalian ribosomal protein S6 as a substrate for DAPK.20 In further studies, we focused on signal transduction with 0.665 ng/ml TNF��, which best reflected the events that occurred in our experimental settings, although the same results (DAPK induction, apoptosis induction) were obtained when using a higher TNF�� concentration [30 ng/ml, supplemental Figure S4, A and B at http://ajp.

amjpathol.org]. Using identical conditioned macrophage Brefeldin_A supernatant either in the absence or presence of 10 ��g/ml of TNF-R2-Fc that is able to fully block TNF-R1-mediated cell death up to concentrations of 1 ��g/ml of TNF��21 we demonstrated a repression of the pro-apoptotic effect of macrophage supernatant by TNF-R2-Fc (supplemental Figure S4C at http://ajp.amjpathol.org). These data indicate that TNF�� is the major player in apoptosis induction and is capable of inducing apoptosis and simultaneously activates DAPK.