Overexpression of HSP25 reduces the level of TNF alpha-induced oxidative DNA damage biomarker, 8-hydroxy-2'-deoxyguanosine, in L929 cells.

Previously we and others have demonstrated that oxidative stress involving generation of reactive oxygen species (ROS) is responsible for the cytotoxic action of TNF alpha. Protective effect of small heat shock proteins (HSP) against diverse oxidative stress conditions has been suggested. Although overexpression of small HSP was shown to provide an enhanced survival of TNF alpha-sensitive cells when challenged with TNF alpha, neither the nature of TNF alpha-induced cytotoxicity nor the protective mechanism of small HSP has been completely understood. In this study, we have attempted to determine whether TNF alpha induces oxidative DNA damage in TNF alpha-sensitive L929 cells. We chose to measure the level of 8-hydroxy-2'-deoxyguanosine (8 ohdG), which has been increasingly recognized as one of the most sensitive markers of oxidative DNA damage. Our results clearly demonstrated that the level of 8 ohdG increased in L929 cells in a TNF alpha dose-dependent manner. Subsequently, we asked whether small HSP has a protective effect on TNF alpha-induced oxidative DNA damage. To accomplish this goal, we have stably transfected into L929 cells, which are devoid of endogenous small HSP, with the mouse small hsp cDNA (hsp25). We found that TNF alpha-induced 8 ohdG was decreased in cells overexpressing exogenous small HSP25. We also found that the cell-killing activity of TNF alpha was decreased in these cells as measured by clonogenic survival. Taken together, results from the current study show that a cytotoxic mechanism of TNF alpha involves oxidative damage of DNA, and that overexpression of the small HSP25 reduces this oxidative damage. We suggest that the reduction of oxidative DNA damage is an important protective mechanisms of small HSP against TNF alpha.