Neuronal/Mitochondrial Nitric Oxide Synthase Homologous Deletion Recombinant Negative Mice (NOS1 −/−) Long-Term Bone Marrow Cultures (LTBMCs) Demonstrate Increased Longevity and Radioresistance of Derived Cell Lines.

Neuronal NOS (NOS1) is localized to mitochondria. Ionizing irradiation results in influx of calcium into mitochondria stimulating production of nitric oxide (NO), and also increases production of superoxide which reacts with NO to produce peroxynitrite. Peroxidation of mitochondrial lipids, release of cytochrome C and apoptosis is directly related to mitochondrial peroxynitrite. We hypothesized that reduction of mitochondrial NO production should provide radioresistance. In addition, since ROS production is associated with aging NOS1−/− mouse LTBMCs should demonstrate greater hematopoietic longevity. LTBMCs established from NOS1 −/− mice demonstrated increased cumulative adherent cobblestone islands (adherent stem cell containing islands), production of total nonadherent cells, and cumulative day 7 and day 14 CFU-GEMM hematopoietic multi-lineage colony forming cells (over 65 weeks) compared to NOS1 +/+ controls (22 weeks) (p < 0.0001). Seven and 14 day CFU-GEMM production in nonadherent cell harvests from NOS1 −/− LTBMCs continued for 65 weeks compared to 15 weeks for NOS1 +/+ LTBMCs (p < 0.0001). Marrow stromal cell lines derived from NOS1 −/− and NOS1 +/+ culture were irradiated to doses from 0 to 800 cGy, plated in 4 well tissue culture plates, stained with crystal violet 7 days later and colonies of greater than 50 cells were counted. NOS1 −/− stromal cell lines had an increased shoulder on the survival curve compared to the NOS1 +/+ cells (n = 32.15 ± 1.21 and 10.47 ± 3.20, respectively, p=0.0026). Cell cycle analysis of NOS1 −/− and NOS1 +/+ cell lines following 10 Gy irradiation demonstrated a G1 arrest 6 hr after irradiation, in both; however, by 24 hr, NOS1 +/+ but not NOS1−/− cells resumed normal cycling. To determine whether the radioresistance of NOS1−/− cells was attributable to expected higher levels of antioxidants, cells were analyzed for glutathione (GSH) and glutathione peroxidase (GPX). NOS1 −/− cells demonstrated increased GSH compared to NOS1 +/+ cells at 0, 30 min and 24 hr following irradiation (p < 0.0001) with no significant difference in GPX before or after irradiation. NOS1 −/− compared to NOS1+/+. IL-3 dependent hematopoietic cells from NOS1 −/− LTBMCs had significantly decreased apoptosis, 24 hrs following 10 Gy irradiation (5.3 ± 2.4 vs. 14.8 ± 3.3 %, respectively, p = 0.049). Therefore, reduction of NOS1 in bone marrow increases hematopoietic longevity in LTBMCs and radioresistance of derived cell lines.