Absence of nNOS Increases Longevity of Long Term Bone Marrow Cultures and Radiation Resistance.

Neuronal nitric oxide synthase (nNOS) has been shown to be localized to the mitochondrial membrane. The mitochondria play an important role in irradiation-induced apoptosis. Following irradiation, there is increased production of superoxide as well as an induction of nitric oxide in mitochondria. The combination of superoxide and nitric oxide results in production of peroxynitrite, a very strong oxidant that produces lipid peroxidation. Previous data have demonstrated that lack of nNOS protects the bladder from ionizing irradiation damage. To determine the role of nNOS in hematopoiesis, we established long term bone marrow cultures (LTBMCs) from nNOS deletion recombinant negative (knockout) mice as well as nNOS+/+ littermate mice. LTBMCs from nNOS knockout mice compared to +/+ control demonstrated increased total cumulative cell production (32.2 x 10⁶ and 15.9 x 10⁶ non-adherent cells, respectively), cobblestone island formation (5073 and 1106, respectively), and increased cumulative generation of day 14 CFU-GEMM colony forming cells per 10⁵ plated (325 ± 30.4 and 9 ± 2.5 colonies, respectively) over 20 weeks in culture. IL-3 dependent non-adherent cell lines derived from the nNOS-/- and the nNOS+/+ cultures were tested for radiosensitivity. Cells from the nNOS−/− cell line demonstrated decreased radiation apoptosis 24 hours following irradiation, 5.89 ± 0.71% apoptotic cells compared to 10.42 ± 1.19% for the control littermates (p=0.041). Cell cycle analysis of littermate cells at 24 hours after 10 Gy demonstrated a G₀/G₁ and a G₂/M block while there was no change in the cell cycle distribution of the nNOS−/− cells. The data demonstrate that absence of nNOS increases the longevity of hematopoiesis in LTBMCs and increases the radiation resistance of hematopoietic cell lines derived from such cultures.