Overexpression of the human manganese superoxide dismutase (Mnsod) Transgene prevents irradiation apoptosis of 32D cl 3 hematopoietic progenitor cells by stabilization of the mitochondria

Subclones of 32D cl 3, 32D-Bcl-xl, 1F2 and 2C6 which demonstrate overexpression of the human MnSOD transgene had increased radioresistance and decreased apoptosis compared to 32D cl 3. Comet assay demonstrated that DNA strand breaks were repaired in all cell lines by 30 minutes (mins) following 200, 400 or 600 cGy, and all had increased migration of Bax and SAP kinases p38 and Jnk1 to the mitochondria 3 hrs after irradiation. Only 32D cl 3 had increased cytochrome-C release, caspase-3 biochemical activity, and poly(adenosine diphosphate-ribose) polymerase (PARP) activation at 6 hrs. At 6 hrs, 10.6% of 32D cl 3 cells showed a decrease in mitochondrial membrane potential compared to 2.9, 1.7 and 1.8% for 1F2, 2C6 and 32D-Bcl-xl, respectively. 32D c1 3 showed a decrease in mitochondrial complex I and III activity, and decreased levels of adenosine triphosphate (ATP) compared to 1F2 and 2C6. 32D cl 3, 1F2, 2C6 or 32D-Bcl-xl were incubated with 5,5-dimethyl-1-pyroline N-oxide (DMPO) spin-trap chemical and irradiated. Electron paramagnetic (spin) resonance (EPR) spectroscopy of spin-trapped adducts demonstrated that 32D cl 3 or 32D-Bcl-xl cells had a higher concentration of free radicals following irradiation than 1F2 or 2C6. Peroxidation of mitochondrial-specific diphosphatidyl-glycerol (DPG) was increased in 32D cl 3 cells compared to 1F2. Thus, the irradiation protective role of MnSOD is due to stabilization of the mitochondria.