RESUMEN
PURPOSE: To determine whether increased expression of manganese superoxide dismutase (MnSOD) protects cells from irradiation by preventing the production of reactive oxygen species (ROS), a new approach to detecting free radical intermediates using ascorbate as an endogenous spin trap was used. MATERIALS AND METHODS: Cells from the 32D cl 3 hematopoietic cell line or a subclone overexpressing MnSOD (2C6) were incubated with dehydroascorbate for 30 min and irradiated to doses from 0 to 50 Gy. Radical intermediates reacting with spin traps or ascorbate were measured by electron spin resonance spectroscopy. Results were compared to irradiation-induced changes in thiol levels, irradiation survival curves, and accumulation of 8-OHdG as a measurement of DNA oxidative damage. RESULTS: Manganese superoxide dismutase-overexpressing 2C6 cells maintained higher levels of ascorbate (5.4 +/- 0.5 and 2.6 +/- 0.5 nmol/10(6) cells, respectively) and thiols (14.0 +/- 0.1 and 11.1 +/- 0.2 nmol/10(6) cells) compared to 32D cl 3 parent cells. Cells overexpressing MnSOD produced lower levels of ROS than did the parental 32D cl 3 cells, as evidenced by lower expenditure of ascorbate and GSH after irradiation. Increased ascorbate levels protected both 32D cl 3 and 2C6 cells from irradiation killing, as demonstrated by an increased shoulder on survival curves and decreased DNA 8-OHdG accumulation. CONCLUSIONS: Manganese superoxide dismutase overexpression protects 2C6 cells from irradiation damage by scavenging ROS that readily interact with major endogenous antioxidants--ascorbate and GSH--in nontransfected hematopoietic 32D cl 3 cells.
Asunto(s)
Ácido Ascórbico/metabolismo , Células Madre Hematopoyéticas/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Línea Celular , Supervivencia Celular , Daño del ADN , Espectroscopía de Resonancia por Spin del Electrón , Glutatión/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C3H , Oxidación-Reducción , Especies Reactivas de Oxígeno/análisisRESUMEN
We investigated the importance of mitochondrial localization of the SOD2 (MnSOD) transgene product for protection of 32D cl 3 hematopoietic cells from radiation-induced killing. Four plasmids containing (1) the native human copper/zinc superoxide dismutase (Cu/ZnSOD, SOD1) transgene, (2) the native SOD2 transgene, (3), the SOD2 transgene minus the mitochondrial localization leader sequence (MnSOD-ML), and (4) the SOD2 mitochondrial leader sequence attached to the active portion of the SOD1 transgene (ML-Cu/ZnSOD) were transfected into 32D cl 3 cells and subclonal lines selected by kanamycin resistance. Clonogenic in vitro radiation survival curves derived for each cell clone showed that Cu/ZnSOD- and MnSOD-ML-expressing clones had no increase in cellular radiation resistance (D0=0.89 +/- 0.01 and 1.08 +/- 0.02 Gy, respectively) compared to parent line 32D cl 3 (D0=1.15 +/- 0.11 Gy). In contrast, cell clones expressing either SOD2 or ML-Cu/ZnSOD were significantly radioresistant (D0=2.1 +/- 0.1 and 1.97 +/- 0.17 Gy, respectively). Mice injected intraesophageally with SOD2-plasmid/liposome (MnSOD-PL) complex demonstrated significantly less esophagitis after 35 Gy compared to control irradiated mice or mice injected intraesophageally with Cu/ZnSOD-PL or MnSOD-ML-PL. Mice injected with intraesophageal ML-Cu/ZnSOD-PL showed significant radioprotection in one experiment. The data demonstrate the importance of mitochondrial localization of SOD in the in vitro and in vivo protection of cells from radiation-induced cellular damage.
Asunto(s)
Esófago/efectos de la radiación , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/efectos de la radiación , Mitocondrias/enzimología , Mitocondrias/efectos de la radiación , Tolerancia a Radiación , Superóxido Dismutasa/metabolismo , Animales , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Línea Celular , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Clonación Molecular , Relación Dosis-Respuesta en la Radiación , Esófago/citología , Esófago/enzimología , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Ratones , Ratones Endogámicos C3H , Mitocondrias/ultraestructura , Dosis de Radiación , Superóxido Dismutasa/genética , Distribución Tisular , TransgenesRESUMEN
OBJECTIVE: Stabilization of the mitochondria in IL-3-dependent hematopoietic progenitor cell line 32D cl 3 by overexpression of the transgene for manganese superoxide dismutase (MnSOD) prior to ionizing radiation prevents apoptosis. We now demonstrate that overexpression of the MnSOD transgene also protects 32D cl 3 cells from apoptosis caused by exposure to tumor necrosis factor-alpha (TNF-alpha) or withdrawal of interleukin (IL)-3. MATERIALS AND METHODS: The hematopoietic progenitor cell line, 32D cl 3, and subclones overexpressing the human MnSOD transgene, 1F2 or 2C6, were radiated to 1000 cGy or were exposed to TNF-alpha (0 to 100 etag/mL) or were subjected to IL-3 withdrawal. The cells were then examined at several time points for DNA strand breaks using a comet assay, depolarization of the mitochondrial membrane, activation of caspase-3, PARP cleavage, and apoptosis, and also for changes in cell cycle distribution. RESULTS: Overexpression of the transgene for MnSOD resulted in increased survival following exposure to radiation, exposure to TNF-alpha, or IL-3 withdrawal. The cell lines overexpressing MnSOD (1F2 or 2C6) displayed decreased radiation-induced, TNF-alpha-induced, or IL-3 withdrawal-induced mitochondrial membrane permeability, caspase-3 and PARP activation, and apoptosis. CONCLUSIONS: Overexpression of the human MnSOD transgene in 32D cl 3 cells results in stabilization of the mitochondria and reduction in radiation-, TNF-alpha-, or IL-3 withdrawal-induced damage. Thus, MnSOD stabilization of the mitochondrial membrane is relevant to reduction of apoptosis by several classes of oxidative stress inducers.
Asunto(s)
Apoptosis , Superóxido Dismutasa/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Humanos , Interleucina-3/farmacología , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/efectos de la radiación , Ratones , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Transporte de Proteínas , Proteínas/metabolismo , Radiación Ionizante , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Transgenes , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Intratracheal injection of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) prior to single fraction or fractionated irradiation of C57BL/6J mouse lung has been demonstrated to protect the lung from irradiation-induced damage. MATERIALS AND METHODS: To determine whether irradiation-induced inflammatory cytokine levels influenced the recovery of tumors following single fraction irradiation, mice with orthotopic Lewis Lung Carcinoma (3LL) tumors received MnSOD-PL treatment 24 hours after tumor implantation and 24 hours prior to irradiation. Subgroups were implanted with Alzet pumps continuously replacing levels of inflammatory cytokines over 7 days. RESULTS: In cytokine-treated mice, there was no detectable significant alteration in radiotherapy-mediated improved survival (tumor regrowth delay) compared to irradiated control mice. Each group of mice that received MnSOD-PL had increased survival compared to irradiated controls. CONCLUSION: These results support the anticipated safety of intrapulmonary MnSOD-PL gene therapy in lung cancer patients for protection of normal lung tissue from irradiation while allowing effective irradiation-mediated tumor control.