Murley Jeffrey S, Kataoka Yasushi, Baker Kenneth L, Diamond Alan M, Morgan William F, Grdina David J
Department of Radiation and Cellular Oncology, University of Chicago, Illinois 60637, USA.
Radiat Res. 2007 Apr;167(4):465-74. doi: 10.1667/RR0758.1.
RKO36 cells, a subclone of RKO colorectal carcinoma cells that have been stably transfected with the pCMV-EGFP2Xho vector, were grown to confluence and then exposed to either the radioprotector WR-1065, i.e. the active thiol form of amifostine, for 30 min at doses of 40 microM and 4 mM or the cytokine tumor necrosis factor alpha (TNFalpha, TNFA) for 30 min at a concentration of 10 ng/ml and then washed. Total protein was isolated as a function of time up to 32 h after these treatments. Both doses of WR-1065 as well as the concentration of TNFalpha used were effective in elevating intracellular levels of the antioxidant protein SOD2 (also known as MnSOD) at least 15-fold over background levels as determined by Western blot analysis, while measured SOD2 activity was elevated between 5.5- and 6.9-fold. SOD2 reached a maximal level 24 h and 20 h after WR-1065 and TNFalpha treatments, respectively. The antioxidant proteins catalase and glutathione peroxidase (GPX) were also monitored over the 32-h period. In contrast to the robust changes observed in intracellular levels of SOD2 as a function of time after exposure of cells to WR-1065, catalase levels were elevated only 2.6-fold over background as determined by Western blot analysis, while GPX activity was unaffected by WR-1065 exposure. GPX protein levels were extremely low in cells, and analysis of GPX activity using a spectrophotometric method based on the consumption of reduced NADPH also revealed no measurable change as a function of WR-1065 or TNFalpha exposure. RKO36 cells either were irradiated with X rays in the presence of either 40 microM or 4 mM WR-1065 or 10 ng/ml TNFalpha or were irradiated 24 or 20 h later, respectively, when SOD2 protein levels were most elevated. The concentrations and exposure conditions used for WR-1065 and TNFalpha were not cytotoxic and had no effect on plating efficiencies or cell survival compared to untreated controls. No protection or sensitization was observed for cells irradiated in the presence of 40 microM WR-1065 or TNFalpha. Survival was elevated 1.90-fold for cells irradiated in the presence of 4 mM WR-1065. When RKO36 cells were irradiated with 2 Gy 24 h after 40 microM or 4 mM WR-1065 and 20 h after TNFalpha treatments when SOD2 levels were the most increased, survival was elevated 1.42-, 1.48- and 1.36-fold, respectively. This increased survival represents a SOD2-mediated delayed radioprotective effect. SOD2 appears to be an important antioxidant gene whose inducible expression is an important element in adaptive cellular responses in general, and the delayed radioprotective effect in particular. It can be induced by a range of agents including cytoprotective nonprotein thiols such as WR-1065 and pleiotropic cytokines such as TNFalpha.
RKO36细胞是已用pCMV-EGFP2Xho载体稳定转染的RKO结肠癌细胞的一个亚克隆，将其培养至汇合状态，然后分别用辐射防护剂WR-1065（即氨磷汀的活性硫醇形式）以40微摩尔/升和4毫摩尔/升的剂量处理30分钟，或用细胞因子肿瘤坏死因子α（TNFα）以10纳克/毫升的浓度处理30分钟，然后洗涤。在这些处理后长达32小时的时间内，根据时间分离总蛋白。通过蛋白质印迹分析确定，WR-1065的两种剂量以及所使用的TNFα浓度均能有效提高抗氧化蛋白SOD2（也称为锰超氧化物歧化酶）的细胞内水平，使其比背景水平至少高15倍，而测得的SOD2活性提高了5.5至6.9倍。WR-1065和TNFα处理后，SOD2分别在24小时和20小时达到最高水平。在32小时期间还监测了抗氧化蛋白过氧化氢酶和谷胱甘肽过氧化物酶（GPX）。与细胞暴露于WR-1065后SOD2细胞内水平随时间的显著变化相反，通过蛋白质印迹分析确定，过氧化氢酶水平仅比背景高2.6倍，而GPX活性不受WR-1065暴露的影响。细胞中GPX蛋白水平极低，使用基于还原型NADPH消耗的分光光度法分析GPX活性也显示，其活性不会因WR-1065或TNFα暴露而发生可测量的变化。RKO36细胞在存在40微摩尔/升或4毫摩尔/升WR-1065或10纳克/毫升TNFα的情况下接受X射线照射，或者分别在SOD2蛋白水平最高的24小时或20小时后接受照射。与未处理的对照相比，WR-1065和TNFα所使用的浓度和暴露条件没有细胞毒性，对平板接种效率或细胞存活没有影响。在存在40微摩尔/升WR-1065或TNFα的情况下照射的细胞未观察到保护或增敏作用。在存在4毫摩尔/升WR-1065的情况下照射的细胞存活率提高了1.90倍。当RKO36细胞在40微摩尔/升或4毫摩尔/升WR-1065处理后24小时以及TNFα处理后20小时（此时SOD2水平增加最多）接受2戈瑞照射时，存活率分别提高了1.42倍、1.48倍和1.36倍。这种存活率的提高代表了一种SOD2介导的延迟辐射防护作用。SOD2似乎是一个重要的抗氧化基因，其诱导表达通常是适应性细胞反应中的一个重要因素，尤其是在延迟辐射防护作用中。它可以被一系列试剂诱导产生，包括细胞保护非蛋白硫醇如WR-1065和多效细胞因子如TNFα。
