Park Y M, Han M Y, Blackburn R V, Lee Y J
Department of Biology, Inchon University College of Natural Science, Korea.
J Cell Physiol. 1998 Jan;174(1):27-34. doi: 10.1002/(SICI)1097-4652(199801)174:1<27::AID-JCP4>3.0.CO;2-I.
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.
此前我们和其他人已经证明，涉及活性氧（ROS）生成的氧化应激是TNFα细胞毒性作用的原因。有人提出小热休克蛋白（HSP）对多种氧化应激条件具有保护作用。尽管当用TNFα刺激时，小HSP的过表达显示能提高TNFα敏感细胞的存活率，但TNFα诱导的细胞毒性的本质以及小HSP的保护机制都尚未完全了解。在本研究中，我们试图确定TNFα是否会在TNFα敏感的L929细胞中诱导氧化性DNA损伤。我们选择测量8-羟基-2'-脱氧鸟苷（8-OHdG）的水平，它已越来越被认为是氧化性DNA损伤最敏感的标志物之一。我们的结果清楚地表明，L929细胞中8-OHdG的水平以TNFα剂量依赖性方式增加。随后，我们询问小HSP是否对TNFα诱导的氧化性DNA损伤具有保护作用。为了实现这一目标，我们将小鼠小hsp cDNA（hsp25）稳定转染到缺乏内源性小HSP的L929细胞中。我们发现，在过表达外源性小HSP25的细胞中，TNFα诱导的8-OHdG减少。我们还发现，通过克隆形成存活率测量，这些细胞中TNFα的细胞杀伤活性降低。综上所述，本研究结果表明，TNFα的细胞毒性机制涉及DNA的氧化损伤，并且小HSP25的过表达减少了这种氧化损伤。我们认为，氧化性DNA损伤的减少是小HSP对TNFα的重要保护机制。
