Delayed cytoprotection after enhancement of Sod2 (MnSOD) gene expression in SA-NH mouse sarcoma cells exposed to WR-1065, the active metabolite of amifostine
Delayed cytoprotection after enhancement of Sod2 (MnSOD) gene expression in SA-NH mouse sarcoma cells exposed to WR-1065, the active metabolite of amifostine
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DOI：
10.1667/0033-7587(2002)158
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发表时间：
2002-07-01
影响因子：
3.4
通讯作者：
Grdina, DJ
中科院分区：
文献类型：
--
作者：
Murley, JS;Kataoka, Y;Grdina, DJ
将SA-NH小鼠肉瘤细胞培养成融合细胞，然后将其暴露于40um或4 mM的WR-1065(即活性硫醇形式的氨磷汀)中30分钟，然后洗涤。染毒后不同时间提取总RNA和总蛋白，最高可达24111。两种浓度的WR-1065在影响Sod2(也称为MnSOD)基因表达和蛋白水平方面同样有效。用小鼠cDNA探针进行Northern印迹分析，发现有3个Sod2转录本，大小分别为1、4和6kb。4kb和6kb转录本的表达分别增加了20%和60%，并在4-20h内保持高水平。蛋白质印迹分析表明，Sod2蛋白水平在同一时间段内比背景对照水平增加了15倍。用活性凝胶对Sod2蛋白进行鉴定，发现其具有活性。SA-NH细胞在40微米或4 mM WR-1065存在下或在去除Sod2蛋白水平最高的24小时后接受X射线照射。在40微米的WR-1065照射下，细胞未观察到保护作用。而4 mM WR-1065照射的SA-NH细胞在照射2Gy后存活率分别提高1.27倍、1.14倍和1.20倍，照射后24 h细胞存活率分别提高1.27倍、1.14倍和1.20倍。在暴露于WR-1065后24小时观察到的存活水平的增加代表了WR-1065的延迟辐射防护作用，并与Sod2蛋白水平最高的时间相对应。这些数据显示了WR-1065的一种新的辐射防护机制，并提出了关于肿瘤保护问题的新的潜在关注。(C)2002年，由辐射研究学会提供。
SA-NH mouse sarcoma cells were grown to confluence and then exposed to either 40 muM or 4 mM of WR-1065, i.e. the active thiol form of amifostine, for 30 min and then washed. Total RNA and protein were isolated at various times up to 24 111 after exposure. Both concentrations of WR-1065 were equally effective in affecting Sod2 (also known as MnSOD) gene expression and protein levels. Northern blot analysis using a mouse cDNA probe revealed three Sod2 transcripts of 1, 4 and 6 kb. Expression of both the 4- and 6-kb transcripts increased by 20 and 60%, respectively, and remained elevated over a period of 4 to 20 h. Sod2 protein levels, as determined by Western blot analysis, increased 15-fold over background control levels over the same interval. Sod2 protein was evaluated using activity gels and was found to be active. SA-NH cells were irradiated with X rays either in the presence of 40 muM or 4 mM WR-1065 or 24 h later after its removal, when Sod2 protein levels were most elevated. No protection was observed for cells irradiated in the presence of 40 muM WR-1065. In contrast, survival after a dose of 2 Gy was elevated 1.27-, 1.14- and 1.20-fold in SA-NH cells irradiated in the presence of 4 mM WR-1065 or 24 h after exposure of the cells to 40 KM and 4 mM WR-1065, respectively. The increased survival levels observed 24 h after exposure to WR-1065 represents a delayed radioprotective effect of WR-1065 and corresponds to the time at which Sod2 protein levels are most elevated. These data demonstrate a novel mechanism for radioprotection by WR-1065 and suggest a new potential concern regarding the issue of tumor protection. (C) 2002 by Radiation Research Society.