Suhua Nie

Mitochondrial Localization of Superoxide Dismutase is Required for Decreasing Radiation-Induced Cellular Damage

Abstract Epperly, M. W., Gretton, J. E., Sikora, C. A., Jefferson, M., Bernarding, M., Nie, S. and Greenberger, J. S. Mitochondrial Localization of Superoxide Dismutase is Required for Decreasing Radiation-Induced Cellular Damage. Radiat. Res. 160, 568–578 (2003). 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.

Overexpression of the transgene for manganese superoxide dismutase (MnSOD) in 32D cl 3 cells prevents apoptosis induction by TNF-α, IL-3 withdrawal, and ionizing radiation

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.

Mitochondrial Targeting of a Catalase Transgene Product by Plasmid Liposomes Increases Radioresistance In Vitro and In Vivo

Abstract Epperly, M. W., Melendez, J. A., Zhang, X., Nie, S, Pearce, L., Peterson, J., Franicola, D., Dixon, T., Greenberger, B. A., Komanduri, P., Wang, H. and Greenberger, J. S. Mitochondrial Targeting of a Catalase Transgene Product by Plasmid Liposomes Increases Radioresistance In Vitro and In Vivo. Radiat. Res. 171, 588-595 (2009). To determine whether increased mitochondrially localized catalase was radioprotective, a human catalase transgene was cloned into a small pSVZeo plasmid and localized to the mitochondria of 32D cl 3 cells by adding the mitochondrial localization sequence of MnSOD (mt-catalase). The cell lines 32D-Cat and 32D-mt-Cat had increased catalase biochemical activity as confirmed by Western blot analysis compared to the 32D cl 3 parent cells. The MnSOD-overexpressing 32D cl 3 cell line, 2C6, had decreased baseline catalase activity that was increased in 2C6-Cat and 2C6-mt-Cat subclonal cell lines. 32D-mt-Cat cells were more radioresistant than 32D-Cat cells, but both were radioresistant relative to 32D cl 3 cells. 2C6-mt-Cat cells but not 2C6-Cat cells were radioresistant compared to 2C6 cells. Intratracheal injection of the mt-catalase-plasmid liposome complex (mt-Cat-PL) but not the catalase-plasmid liposome complex (Cat-PL) increased the resistance of C57BL/6NHsd female mice to 20 Gy thoracic irradiation compared to MnSOD-plasmid liposomes. Thus mitochondrially targeted overexpression of the catalase transgene is radioprotective in vitro and in vivo.

Ethyl Pyruvate, a Potentially Effective Mitigator of Damage after Total-Body Irradiation

Abstract Epperly, M., Jin, S., Nie, S., Cao, S., Zhang, X., Franicola, D., Wang, H., Fink, M. P. and Greenberger, J. S. Ethyl Pyruvate, a Potentially Effective Mitigator of Damage after Total-Body Irradiation. Radiat. Res. 168, 552–559 (2007). Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to improve survival and ameliorate organ damage in animal models of sepsis, ischemia/reperfusion injury and hemorrhagic shock. Incubating IL3-dependent mouse hematopoietic progenitor cell 32Dcl3 cells before or after irradiation with 10 mM EP increased resistance to radiation as assessed by clonogenic radiation survival curves, decreased release of mitochondrial cytochrome C into the cytoplasm, and decreased apoptosis. EP inhibited radiation-induced caspase 3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in 32Dcl3 cells in a concentration-dependent fashion. EP was given i.p. to C57BL/6NHsd mice irradiated with 9.75 Gy total-body irradiation (TBI). This treatment significantly improved survival. The survival benefit was apparent irrespective of whether treatment with EP was started 1 h before TBI and continued for 5 consecutive days after TBI or the compound was injected only 1 h before or only for 5 days after TBI. In all of the in vitro and in vivo experiments, ethyl lactate, an inactive analogue of EP, had no detectable radioprotective or mitigating effects. EP may be an effective radioprotector and mitigator of the hematopoietic syndrome induced by TBI.

Effects of MnSOD-Plasmid Liposome Gene Therapy on Antioxidant Levels in Irradiated Murine Oral Cavity Orthotopic Tumors

Abstract Epperly, M. W., Wegner, R., Kanai, A. J., Kagan, V., Greenberger, E. E., Nie, S. and Greenberger, J. S. Effects of MnSOD-Plasmid Liposome Gene Therapy on Antioxidant Levels in Irradiated Murine Oral Cavity Orthotopic Tumors. Radiat. Res. 167, 289–297 (2007). 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.

Synthetic Protection Short Interfering RNA Screen Reveals Glyburide as a Novel Radioprotector

Abstract To assist in screening existing drugs for use as potential radioprotectors, we used a human unbiased 16,560 short interfering RNA (siRNA) library targeting the druggable genome. We performed a synthetic protection screen that was designed to identify genes that, when silenced, protected human glioblastoma T98G cells from γ-radiation-induced cell death. We identified 116 candidate protective genes, then identified 10 small molecule inhibitors of 13 of these candidate gene products and tested their radioprotective effects. Glyburide, a clinically used second-generation hypoglycemic drug, effectively decreased radiation-induced cell death in several cell lines including T98G, glioblastoma U-87 MG, and normal lung epithelial BEAS-2B and in primary cultures of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective in vivo (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, P  =  0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors.

Increased Radioresistance, G2/M Checkpoint Inhibition, and Impaired Migration of Bone Marrow Stromal Cell Lines Derived from Smad3−/− Mice

Abstract Epperly, M. W., Goff, J. P., Zhang, X., Niu, Y., Shields, D. S., Wang, H., Shen, H., Franicola, D., Bahnson, A. B., Nie, S., Greenberger, E. E. and Greenberger, J. S. Increased Radioresistance, G2/M Checkpoint Inhibition, and Impaired Migration of Bone Marrow Stromal Cell Lines Derived from Smad3−/− Mice. Radiat. Res. 165, 671–677 (2006). 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 D0 = 2.25 ± 0.14 Gy compared to Smad3+/+ cells with a D0 = 1.75 ± 0.03 (P = 0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D0 = 1.49 0.10 (P = 0.028) for Smad3−/−(3) cells. More Smad3−/− cells than Smad3+/+ cells were in the G2/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 G2/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.