To gain a better understanding of the role of reactive oxygen species (ROS) in aging, we determined the levels of various antioxidant enzymes in cultured human fibroblasts from normal individuals of different ages and also from patients with premature aging diseases. The fibroblast levels of catalase, glutathione reductase, glutathione peroxidase, glutathione-s-transferase, and glucose-6-phosphate dehydrogenase were all found to decrease with the age of the fibroblast donors. These fibroblasts also showed age-dependent increases of protein carbonyl and superoxide dismutase activity but lower levels of total glutathione (oxidized and reduced) and neutral protease activity. The cells obtained from young, premature aging patients exhibited enzyme activity patterns very similar to those obtained for old individuals. These results suggest that the antioxidant capacity declines with aging, and thus favors an increase in ROS-mediated damage. Using the amount of DNA as the indication of cell proliferation, we found that cells cultured under 3% oxygen grew faster than cells cultured under standard 20% oxygen. In addition, the basal activity of caspase 3-like proteases was slightly lower in cells cultured at 3% oxygen compared with cells cultured at 20% oxygen. When these cells were induced to activate caspase 3-like proteases by removal of FBS from the culture medium, cells from young individuals were more sensitive than cells from old or premature aging patients. These results suggest that lower oxygen concentrations might result in less oxidative damage to cells, leading to a decreased necessity for cells to commit apoptosis and thus facilitate the rate of cell proliferation. Taken together, these results indicate a causative role of ROS in aging. To further understand the mechanism by which ROS induce apoptosis, we treated HeLa cells with various concentrations of hydrogen peroxide. Treatment of HeLa cells with hydrogen peroxide resulted in activation of caspase 3-like proteases in a time- and concentration-dependent manner. This activation can be inhibited by a general caspase inhibitor (z-VAD-fmk) or a caspase- 3 specific inhibitor (DEVD-CHO). This activation appears to be caspase 9-independent and PI3K-dependent. Various proteins involved in the formation of the focal contact were also degraded, including FAK (focal adhesion kinase), Src, Shc, and paxillin, suggesting an effect of hydrogen peroxide treatment on the formation of the focal contact that is related to cell adhesion and actin polymerization. Finally, the effect of hydrogen peroxide on HeLa cells might be attributed to its effect on the release of arachidonic acid since the activation of caspase 3 and 7 was decreased by the addition of the specific PLA-2 (phospholipase A-2) inhibitor. Alternatively, membrane sphingomyelinase (divalent magnesium-dependent, -independent, and acidic) might be involved since it has been implicated to participate in ceramide release that appears to be an essential step for apoptosis. Current studies are aimed to resolve the pathways leading to activation of caspase 3-like proteases and the consequences of degradation of the focal adhesion complex.
