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 correlat es with a late increase in reduction of the superoxide scavenger (4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), suggestin g an increase in MTT reduction per viable cell. This effect was studie d in two TNF-sensitive and in five different TNF-resistant clones. Wit hin 36 hrs TNF promoted a 7-fold increase in the reduction of MTT in T NF-sensitive cells. Exogenous ceramide induced a similar effect prior to cell death. Four of the five TNF-resistant clones were also resista nt to ceramide and displayed no increase in MTT reduction with either TNF or ceramide. The remaining TNF-resistant clone was sensitive to ce ramide, 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 tha t ceramide can serve as a second messenger for TNF in cell death. (C) 1996 Academic Press, Inc.