RESUMEN
Intraoral manganese superoxide dismutase (SOD2)-plasmid liposome (PL) radioprotective gene therapy prolongs the survival of mice with orthotopic oral cavity tumors within the irradiated field. To determine whether the mechanism involved effects in antioxidant pool, C57BL/6J mice bearing orthotopic oral cavity squamous cell carcinoma SCC-VII tumors received intraoral or intravenous MnSOD-PL gene therapy 24 h prior to 18 Gy irradiation to the head and neck region. Glutathione (GSH) levels and levels of radiation-generated nitric oxide and peroxynitrite were measured in orthotopic tumors and in adjacent oral mucosa. MnSOD-PL transfection of the SCC-VII tumor cells, but not normal embryo fibroblasts, produced acute radiosensitization. Furthermore, SCC-VII tumor cells demonstrated increased relative hydrogen peroxide (the product of MnSOD superoxide dismutation)-induced apoptosis in vitro. Radiation decreased levels of GSH and increased GPX in both tumor and normal cells in vitro, effects that were blunted by MnSOD-PL treatment. In vivo irradiation decreased GSH and GPX more effectively in tumors, and the decrease was not reversed by MnSOD-PL therapy. Intravenous but not intraoral administration of epitope-tagged hemagglutinin MnSOD-PL resulted in significant uptake in orthotopic tumors and decreased the levels of radiation-induced nitric oxide and peroxynitrite. Thus normal tissue radioprotective MnSOD-PL gene therapy radiosensitizes tumor cell lines in vitro and has a therapeutic effect on orthotopic tumors in part through its effects on tumor antioxidants.
Asunto(s)
Antioxidantes/metabolismo , Terapia Genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/terapia , Plásmidos/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Modelos Animales de Enfermedad , Peróxido de Hidrógeno/toxicidad , Liposomas , Ratones , Boca/metabolismo , Boca/efectos de la radiación , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Nitratos/metabolismo , Superóxido Dismutasa/administración & dosificación , Factores de TiempoRESUMEN
OBJECTIVE: Neuronal nitric oxide synthase (NOS1, mitochondrial NOS, neuronal NOS) homozygous deletion recombinant negative mice demonstrate ionizing irradiation resistance in vivo, attributable to the decrease in mitochondrial-localized production of peroxynitrite, a potent lipid toxic free radical species resulting from the combination of nitric oxide and superoxide. The present studies were designed to determine whether reduced mitochondrial generation of toxic radical oxygen species in NOS1-/- mice also increased the longevity of hematopoiesis in continuous bone marrow cultures and conferred radioresistance to cells in vitro. MATERIALS AND METHODS: Long-term bone marrow cultures (LTBMCs) were established from NOS1-/- and NOS1+/+ littermate mice. Radiation resistance of hematopoietic and marrow stromal cells was measured. Cell cycle analysis and measurement of glutathione and glutathione peroxidase were carried out on irradiated clonal bone marrow stromal cell lines. RESULTS: A significant increase in longevity of hematopoiesis was detected in NOS1-/- mouse LTBMCs for over 64 weeks in culture compared to 20 weeks for NOS1+/+ mouse LTBMCs (p < 0.001). Permanent bone marrow stromal cell lines derived from NOS1-/- mouse LTBMCs demonstrated increased radioresistance in vitro reflected by an increased shoulder on the survival curve with n = 32.15 +/- 1.21 compared to NOS1+/+ cells n = 10.47 +/- 3.2 (p = 0.0026), interleukin-3-dependent NOS1-/- hematopoietic progenitor cell lines also demonstrated decreased apoptosis after 10 Gy irradiation. Both pre- and postirradiation stabilization of the cellular antioxidant pool was detected in NOS1-/- cells. NOS1-/- cells showed a prolonged G1 cell cycle arrest after 10 Gy. CONCLUSIONS: Prolonged hematopoiesis in LTBMCs correlates with intrinsic radioresistance of hematopoietic and marrow stromal cells from NOS1-/- mice. The data confirm the importance to hematopoiesis of mitochondrial localized nitric oxide in both radioresistance and longevity of hematopoiesis in continuous bone marrow cultures.
Asunto(s)
Células de la Médula Ósea/citología , Hematopoyesis , Células Madre Hematopoyéticas/efectos de la radiación , Óxido Nítrico Sintasa de Tipo I/genética , Células del Estroma/efectos de la radiación , Animales , Células de la Médula Ósea/enzimología , Técnicas de Cultivo de Célula , Células Cultivadas , Homocigoto , Ratones , Ratones Mutantes , Mitocondrias/metabolismo , Mutación , Óxido Nítrico/metabolismo , Óxido Nítrico/fisiología , Efectos de la Radiación , Factores de TiempoRESUMEN
Smad3 protein is a prominent member of the Tgfb receptor signaling pathway. Smad3(-/-) mice display decreased radiation-induced skin fibrosis, suggesting a defect in both Tgfb-mediated fibroblast proliferation and migration. We established bone marrow stromal cell lines from Smad3(-/-) mice and homozygous littermate(+/+) mice. Smad3(-/-) cells displayed a significant increase in radiation resistance with a D(0)=2.25+/- 0.14 Gy compared to Smad3(+/+) cells with a D(0)=1.75+/- 0.03 (P=0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D(0)=1.49 0.10 (P=0.028) for Smad3(-/-)(3) cells. More Smad3(-/-) cells than Smad3(+/+) cells were in the G(2)/M phase; Smad3(-/-)(3) cells were similar to Smad3(+/+) cells. Smad3(+/+) cells exhibited increased apoptosis 24 h after 5 Gy (15%) or 8 Gy (43%) compared to less than 1% in Smad3(-/-) cells exposed to either dose. The movement of Smad3(-/-) cells, measured in an automated cell tracking system, was slower than that of Smad3(+/+) cells. Smad3(-/-)(3) cells resembled Smad3(+/+) cells. These studies establish concordance of a defective Tgfb signal transduction pathway, an increased proportion of G(2)/M cells, and radioresistance. The decreased migratory capacity of Smad3(-/-) cells in vitro correlates with decreased radiation fibrosis in vivo in mice deficient in Tgfb signaling.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína smad3/metabolismo , Animales , Apoptosis/efectos de la radiación , Células de la Médula Ósea/efectos de la radiación , Línea Celular , Movimiento Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Genes cdc/fisiología , Genes cdc/efectos de la radiación , Ratones , Tolerancia a Radiación/fisiología , Células del Estroma/citología , Células del Estroma/fisiología , Células del Estroma/efectos de la radiaciónRESUMEN
BACKGROUND: Thalidomide (TL), due to its antiangiogenic effects, has been postulated to be a potential radiosensitizer of multiple myeloma and squamous tumors in vivo. MATERIALS AND METHODS: To determine whether TL was a radiosensitizer, 32D cl 3 cells (hematopoietic progenitor) as well as SCC-VII (squamous cell carcinoma), OPM1 or OPM2 (multiple myeloma) tumor cells were irradiated to doses ranging from 0 to 8 Gy and then plated in 0, 50 or 150 microM TL in each of three protocols: i) 1 hour before irradiation; ii) 1 hour before irradiation and also in medium following irradiation; or iii) placed in TL containing medium following irradiation. RESULTS: Using 150 microM TL (which did not stimulate cell growth) the 32D cl 3 cells had increased radiation sensitivity compared to the control irradiated cells. In contrast, the SCC-VII, OPMI or OPM2 cells showed no detectable radiosensitization when incubated in TL before, during or after irradiation compared to the control irradiated cells. CONCLUSION: These results demonstrated that TL may be a selective radiosensitizer.