To determine whether overexpression of the human MnSOD transgene protected 32D cl 3 hematopoietic progenitor cells from ionizing irradiation, 32D cl 3 cells were co-electroporated with the pRK5 plasmid containing the human MnSOD transgene and SV2-neo plasmid with G418-resistant colonies selected. Two clones (1F2 and 2C6) were identified to overexpress the human MnSOD transgene by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and increased biochemical activity. Measurement of irradiation-induced damage was determined in cells removed from G418 for 1 week before irradiation. Irradiation survival curves, apoptosis tunnel assay, and Comet assay was performed. Cell cycle distribution was determined for each line at 0, 1, 3, 6, 24, and 48 hr after 500 cGy by fixing the cells in 70% ethanol, staining with propidium iodide, and analysis by now cytometer. Biochemical MnSOD activity in U/mg protein was 2.6 for 32D cl 3 and significantly elevated to 8.4 and 6.6 (P < 0.001) U/mg protein for subclones 1F2 and 2C6, respectively. Irradiation survival curves demonstrated an increased shoulder on the irradiation survival curve for 1F2 and 2C6 cells with an (n) over bar of 4.95 +/- 0.48 (P = 0.042) and 4.95 +/- 0.13 (P = 0.011), compared with 2.77 +/- 0.20 for 32D cl 3. A higher percent of 32D cl 3 cells demonstrated apoptosis at 24 and 48 hr after 1,000 cGy irradiation, compared with 1F2 and 2C6 cells (at 24 hr, 29.37% +/- 2.01% of 32D cl 3 cells were apoptotic compared with 5.21 +/- 2.61 (P = 0.018) and 5.27 +/- 2.58 (P = 0.004) for 1F2 and 2C6, respectively). Significantly more DNA strand breaks were detected by Comet assay in 32D cl 3 cells (Comet length at 600 cGy of 103.4 +/- 50.3 units, compared with 69.7 +/- 36.3 (P < 0.001) and 48.9 +/- 27.5 (P < 0.001) for 1F2 and 2C6, respectively). In contrast, irradiation-induced cell cycle arrest was similar between the cell lines with a G2/M phase arrest at 6 hr and a G1/S phase arrest at 24 and 48 hr after irradiation. While overexpression of MnSOD increases the shoulder on the irradiation survival curve of 32D cl 3 cells, decreases irradiation-induced apoptosis, and DNA strand breaks by Comet assay, irradiation-induced alterations in cell cycle distribution were not significantly altered. These 32D cl 3 subclonal lines overexpressing MnSOD provide a potentially valuable system with which to study the mechanism of irradiation-induced cell cycle arrest separate from irradiation-induced apoptosis. Radiat. Oncol. Invest. 7:331-342, 1999. (C) 1999 Wiley-Liss, Inc.
