title: Activation of the nitric oxide synthase 2 pathway in the response of bone marrow stromal cells to high doses of ionizing radiation.
abstract: Reverse transcription-polymerase chain reaction and immunofluorescence analysis of D2XRII murine bone marrow stromal cells showed that gamma irradiation with doses of 2-50 Gy from (137)Cs stimulated expression of nitric oxide synthase 2 (Nos2, also known as iNos). The activation of Nos2 was accompanied by an increase in the fluorescence of 4,5-diaminofluorescein diacetate, a nitric oxide trap, and accumulation of 3-nitrotyrosine within cellular proteins in a dose-dependent manner. These effects were inhibited by actinomycin D and by N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride, a specific inhibitor of Nos2. The induction of Nos2 expression and Nos2-dependent release of nitric oxide in D2XRII cells was observed within 24 h after irradiation and was similar in magnitude to that observed in cultures incubated with Il1b and Tnf. We conducted (1) confocal fluorescence imaging of 3-nitrotyrosine in bone marrow cells of irradiated C57BL/6J mice and (2) 3-nitrotyrosine fluorescence imaging of FDC-P1JL26 hematopoietic cells that were cocultured with previously irradiated D2XRII bone marrow stromal cells. Exposure to ionizing radiation increased the production of 3-nitrotyrosine in irradiated bone marrow cells in vivo and in nonirradiated FDC-P1JL26 cells cocultured with irradiated D2XRII cells for 1 or 4 h. We suggest that nitrative/oxidative stress to the transplanted multilineage hematopoietic cells due to exposure to nitric oxide released by host bone marrow stromal cells may contribute to the genotoxic events associated with malignant alterations in bone marrow tissue of transplant recipients who are prepared for engraftment by total-body irradiation.
authors.affiliation: Department of Radiation Oncology, Environmental and Occupational Health, University of Pittsburgh, PA 15261, USA.
authors.forename: N V
authors.initials: NV
authors.lastname: Gorbunov
authors.forename: K L
authors.initials: KL
authors.lastname: Pogue-Geile
authors.forename: M W
authors.initials: MW
authors.lastname: Epperly
authors.forename: W L
authors.initials: WL
authors.lastname: Bigbee
authors.forename: R
authors.initials: R
authors.lastname: Draviam
authors.forename: B W
authors.initials: BW
authors.lastname: Day
authors.forename: N
authors.initials: N
authors.lastname: Wald
authors.forename: S C
authors.initials: SC
authors.lastname: Watkins
authors.forename: J S
authors.initials: JS
authors.lastname: Greenberger
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000201:enzymology / Q000528:radiation effects*; D002450:Cell Communication; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006412:Hematopoietic Stem Cells / Q000502:physiology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011829:Radiation Dosage; D011839:Radiation, Ionizing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000201:enzymology / Q000528:radiation effects; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse