Epperly Michael W, Gretton Joan E, Sikora Christine A, Jefferson Mia, Bernarding Michael, Nie Suhua, Greenberger Joel S
Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213, USA.
Radiat Res. 2003 Nov;160(5):568-78. doi: 10.1667/rr3081.
We investigated the importance of mitochondrial localization of the SOD2 (MnSOD) transgene product for protection of 32D cl 3 hematopoietic cells from radiation-induced killing. Four plasmids containing (1) the native human copper/zinc superoxide dismutase (Cu/ZnSOD, SOD1) transgene, (2) the native SOD2 transgene, (3), the SOD2 transgene minus the mitochondrial localization leader sequence (MnSOD-ML), and (4) the SOD2 mitochondrial leader sequence attached to the active portion of the SOD1 transgene (ML-Cu/ZnSOD) were transfected into 32D cl 3 cells and subclonal lines selected by kanamycin resistance. Clonogenic in vitro radiation survival curves derived for each cell clone showed that Cu/ZnSOD- and MnSOD-ML-expressing clones had no increase in cellular radiation resistance (D0=0.89 +/- 0.01 and 1.08 +/- 0.02 Gy, respectively) compared to parent line 32D cl 3 (D0=1.15 +/- 0.11 Gy). In contrast, cell clones expressing either SOD2 or ML-Cu/ZnSOD were significantly radioresistant (D0=2.1 +/- 0.1 and 1.97 +/- 0.17 Gy, respectively). Mice injected intraesophageally with SOD2-plasmid/liposome (MnSOD-PL) complex demonstrated significantly less esophagitis after 35 Gy compared to control irradiated mice or mice injected intraesophageally with Cu/ZnSOD-PL or MnSOD-ML-PL. Mice injected with intraesophageal ML-Cu/ZnSOD-PL showed significant radioprotection in one experiment. The data demonstrate the importance of mitochondrial localization of SOD in the in vitro and in vivo protection of cells from radiation-induced cellular damage.
我们研究了超氧化物歧化酶2（MnSOD）转基因产物的线粒体定位对于保护32D cl 3造血细胞免受辐射诱导杀伤的重要性。将四个质粒转染到32D cl 3细胞中，这四个质粒分别含有：（1）天然人铜/锌超氧化物歧化酶（Cu/ZnSOD，SOD1）转基因；（2）天然SOD2转基因；（3）去除线粒体定位前导序列的SOD2转基因（MnSOD-ML）；（4）连接到SOD1转基因活性部分的SOD2线粒体前导序列（ML-Cu/ZnSOD），并通过卡那霉素抗性选择亚克隆系。为每个细胞克隆绘制的体外克隆形成辐射存活曲线显示，与亲本系32D cl 3（D0 = 1.15±0.11 Gy）相比，表达Cu/ZnSOD和MnSOD-ML的克隆细胞辐射抗性没有增加（D0分别为0.89±0.01和1.08±0.02 Gy）。相反，表达SOD2或ML-Cu/ZnSOD的细胞克隆具有显著的抗辐射性（D0分别为2.1±0.1和1.97±0.17 Gy）。与对照照射小鼠或经食管注射Cu/ZnSOD-PL或MnSOD-ML-PL的小鼠相比，经食管注射SOD2-质粒/脂质体（MnSOD-PL）复合物的小鼠在接受35 Gy辐射后食管炎明显减轻。在一项实验中，经食管注射ML-Cu/ZnSOD-PL的小鼠显示出显著的辐射防护作用。数据表明，超氧化物歧化酶的线粒体定位在体外和体内保护细胞免受辐射诱导的细胞损伤中具有重要作用。
