Lam E W, Zwacka R, Engelhardt J F, Davidson B L, Domann F E, Yan T, Oberley L W
Radiation Research Laboratory, Department of Radiology, The University of Iowa, Iowa City 52242-1101, USA.
Cancer Res. 1997 Dec 15;57(24):5550-6.
As a first step in evaluating the tumor suppressor activity of the manganese superoxide dismutase (MnSOD) gene on established tumors in vivo, we used adenovirus-mediated gene transfer as a means of delivering the MnSOD cDNA to hamster cheek pouch carcinoma (HCPC-1) cells in vitro. HCPC-1 cells were transduced with the adenovirus-MnSOD construct (AdMnSOD) at multiplicities of infectivity (MOI) of 0, 10, 25, 50, 100, 150, and 200 MOI or with the adenovirus-LacZ reporter gene construct (AdLacZ) at 100 MOI. Dose-dependent increases in MnSOD immunoreactivity were seen on Western blotting and indirect immunofluorescence microscopy with increasing AdMnSOD titers. Maximal immunoreactivity was observed at 100 MOI AdMnSOD with both techniques. Moreover, we observed a concomitant 6-7-fold increase in MnSOD activity compared with parental cell levels that also peaked at 100 MOI AdMnSOD. To determine the effect of transgene-expressed MnSOD on tumor cell behavior, we examined cell growth, plating efficiency, and anchorage-dependent growth in soft agar. Cell number measured on day 13 decreased approximately 50% with 100 MOI AdMnSOD (P < 0.05) compared with parental cells. Moreover, cell doubling time increased from 38 to 44 h with 100 MOI AdMnSOD. Plating efficiency and cell growth in soft agar decreased approximately two-thirds with 100 MOI AdMnSOD (P < 0.001). These assays of the transformed phenotype in vitro all appeared to show maximal effect with 100 MOI AdMnSOD. As tumor growth in vivo is most predictable by a combination of these in vitro data, our results suggest that if MnSOD can be effectively delivered to a tumor in vivo using the adenovirus paradigm, effective tumor growth suppression can be observed.
作为评估锰超氧化物歧化酶（MnSOD）基因对体内已形成肿瘤的抑癌活性的第一步，我们采用腺病毒介导的基因转移方法，将MnSOD cDNA体外导入仓鼠颊囊癌（HCPC-1）细胞。用腺病毒-MnSOD构建体（AdMnSOD）以感染复数（MOI）为0、10、25、50、100、150和200 MOI转导HCPC-1细胞，或以腺病毒-LacZ报告基因构建体（AdLacZ）以100 MOI转导。随着AdMnSOD滴度增加，蛋白质免疫印迹法和间接免疫荧光显微镜检查可见MnSOD免疫反应性呈剂量依赖性增加。两种技术在AdMnSOD为100 MOI时均观察到最大免疫反应性。此外，与亲代细胞水平相比，我们观察到MnSOD活性同时增加了6至7倍，在AdMnSOD为100 MOI时也达到峰值。为了确定转基因表达的MnSOD对肿瘤细胞行为的影响，我们检测了细胞生长、接种效率和软琼脂中的锚定依赖性生长。与亲代细胞相比，在第13天测量的细胞数量在AdMnSOD为100 MOI时减少了约50%（P<0.05）。此外，AdMnSOD为100 MOI时细胞倍增时间从38小时增加到44小时。AdMnSOD为100 MOI时接种效率和软琼脂中的细胞生长减少了约三分之二（P<0.001）。这些体外转化表型分析似乎在AdMnSOD为100 MOI时均显示出最大效应。由于体内肿瘤生长最能通过这些体外数据的组合来预测，我们的结果表明，如果使用腺病毒模式将MnSOD有效递送至体内肿瘤，则可观察到有效的肿瘤生长抑制。
