Cytokine-induced apoptosis in epithelial HT-29 cells is independent of nitric oxide formation - Evidence for an interleukin-13-driven phosphatidylinositol 3-kinase-dependent survival mechanism

A combination of the pro-inflammatory cytokines interleukin (IL)-1 alpha, i nterferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the hum an colon carcinoma cell line HT-29, This can be inhibited by pretreatment w ith IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wri ght, K., Ward, S. G., Kolios, G., and Westwick, J. (1997) J. Biol. Chem. 27 2, 12626-12633), Since NO has been implicated in regulating mechanisms lead ing to cell death, while activation of PI 3-kinase-dependent signaling casc ades are thought to be involved with promoting cell survival events, we hav e investigated the outcome of these cytokine treatments on apoptosis and ce ll survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-gamma/TNF-alpha, IFN-gamma/CD95, IL-1 alpha/IFN-gamma, and IL-1 alpha/IFN-gamma/TNF-alpha to varying extents. Induction of apoptot ic markers by HT-29 cells in response to cytokine treatment is not dependen t on NO production. Pretreatment with IL-13 protects against IL-1 alpha/IFN -gamma/TNF-alpha- and IFN-gamma/TNF-alpha- as well as IFN-gamma/CD95-induce d (but not IL-1 alpha/IFN-gamma-induced) cell death. In addition, IFN-gamma /TNF-alpha and IL-1 alpha/IFN-gamma/TNF-alpha stimulate activation of caspa se-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effect or, protein kinase B, The PI 3-kinase inhibitors wortmannin and LY294002 in hibit IL-13 stimulation of protein kinase B as well as the cell survival ef fects of IL-13, These data demonstrate that cytokine-induced apoptosis of H T-29 cells is NO-independent and that the activation of a PI 8-kinase-depen dent signaling cascade by IL-13 is a key signal responsible for the inhibit ion of apoptosis.