Overexpression of the human manganese superoxide dismutase (MnSOD) transgene in subclones of murine hematopoietic progenitor cell line 32D cl 3 decreases irradiation-induced apoptosis but does not alter G2/M or G1/S phase cell cycle arrest

Abstract

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 flow 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 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. © 1999 Wiley‐Liss, Inc.

Intratracheal injection of manganese superoxide dismutase (MnSOD) plasmid/liposomes protects normal lung but not orthotopic tumors from irradiation

“…The DNA was stained with propidium iodide and the length of the Comet tails measured using units established for Comet length, using a standard curve of Comet length. 31,32 At least 150 comets were measured for each mouse lung specimen. The data were analyzed using the Mann-Whitney confidence interval and test.…”

“…The data were analyzed using the Mann-Whitney confidence interval and test. 32 Quantification of cytokine mRNA in the lungs following irradiation and modulation by prior overexpression of MnSOD transgene C57BL/6J mice were IT-injected with 5 × 10 5 3LL-LacZ cells. Ten days later, the mice were injected with MnSOD-PL complex (500 g, plasmid DNA, as described above) and irradiated to 18 Gy.…”

Redox Regulation of T-Cell Function: From Molecular Mechanisms to Significance in Human Health and Disease

“…MnSOD is the cell's primary defense mechanism against free radical-mediated damage and is important in regulating oxidative and apoptotic signals (217). Another study has suggested that overexpression of the human MnSOD transgene in hematopoietic progenitor cell line 32D cl3 cells and other cell lines results in stabilization of the mitochondria and reduction in radiation-, TNFa-, or cytokine withdrawal-induced cell death (70). Thus, MnSOD stabilizes the mitochondrial membrane and reduces apoptosis.…”

Manganese superoxide dismutase gene therapy protects against irradiation-induced cystitis

“…Because O 2 Ϫ radicals are thought to mediate much of the radiation damage, strategies to decrease O 2 Ϫ have been tested as approaches to mitigating radiation damage. These strategies include overexpression of the MnSOD transgene in cells to protect them from irradiation-induced damage both in vitro and in vivo (12,14,15). In vitro, overexpression of MnSOD in 32D cl 3 murine hematopoietic progenitor cells prevents irradiation-induced apoptosis (12,15).…”

“…These strategies include overexpression of the MnSOD transgene in cells to protect them from irradiation-induced damage both in vitro and in vivo (12,14,15). In vitro, overexpression of MnSOD in 32D cl 3 murine hematopoietic progenitor cells prevents irradiation-induced apoptosis (12,15). This occurs at the level of the mitochondria, because increased MnSOD activity stabilizes the mitochondria, preventing mitochondrial membrane depolarization, release of cytochrome c, and activation of caspase 3, thereby preventing apoptosis (15).…”