Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-alpha-mediated apoptosis

Tumour necrosis factor-alpha (TNF-alpha) was found to be a cell cycle-indep endent apoptogenic cytokine in cultured fibroblast L929 cells. This asserti on is based on the observations (1) TNF-alpha increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytom etry, and (2) TNF-alpha induced DNA fragmentation as resolved by agarose ge l electrophoresis. When cells were exposed to TNF-alpha (50 ng/ml), a slow rise in intracellular free Ca2+ level and a delayed increase in the product ion of reactive oxygen species (ROS) (both observed 3 h after the addition of TNF-alpha) were observed in fluo-3 and fura-red or dichlorofluorescein l oaded cells, respectively. Interestingly, challenge of cells with TNF-alpha in the presence of BAPTA/AM, an intracellular Ca2+ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidin ooxy (4OH-TEMPO) blocked the TNF-alpha-mediated Ca2+ rise. Moreover, when c ells were exposed to TNF-alpha. with both COH-TEMPO and BAPTA/AM, more viab le cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca2+ are two cross-talk messeng ers important in TNF-alpha-mediated apoptosis.