Ceramide reproduces late appearance of oxidative stress during TNF-mediated cell death in L929 cells

Tumor necrosis factor alpha mediated cell death in L929 cells correlates with a late increase in reduction of the superoxide scavenger 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), suggesting an increase in MTT reduction per viable cell. This effect was studied in two TNF-sensitive and in five different TNF-resistant clones. Within 36 hrs TNF promoted a 7-fold increase in the reduction of MTT in TNF-sensitive cells. Exogenous ceramide induced a similar effect prior to cell death. Four of the five TNF-resistant clones were also resistant to ceramide and displayed no increase in MTT reduction with either TNF or ceramide. The remaining TNF-resistant clone was sensitive to ceramide, displaying an increase in MTT reduction. Our results suggest a late increase in superoxide production prior to cellular destruction during TNF and ceramide mediated cell death and support the notion that ceramide can serve as a second messenger for TNF in cell death. (C) 1996 Academic Press, Inc.