Christine A. Sikora

Manganese Superoxide Dismutase (SOD2) Inhibits Radiation-Induced Apoptosis by Stabilization of the Mitochondrial Membrane

Abstract Epperly, M. W., Sikora, C. A., DeFilippi, S. J., Gretton, J. A., Zhan, Q., Kufe, D. W. and Greenberger, J. S. Manganese Superoxide Dismutase (SOD2) Inhibits Radiation-Induced Apoptosis by Stabilization of the Mitochondrial Membrane. Radiat. Res. 157, 568–577 (2002). To define the molecular pathways involved in radiation-induced apoptosis and the role of the mitochondria, 32D cl 3 hematopoietic cells and subclones overexpressing either the human manganese superoxide dismutase (SOD2) transgene (1F2 and 2C6) or BCL2L1 (also known as Bcl-xl) transgene (32D-Bcl-xl) were compared for their response to radiation at the subcellular level, comparing nuclear to mitochondrial localized pathways. All cell lines showed complete detectable DNA repair by 30 min after irradiation, and clearly delayed migration of BAX and active stress-activated protein (SAP) kinases MAPK1 (also known as p38) and MAPK8 (also known as JNK1) to the mitochondria at 3 h. Radioresistant clonal lines 1F2, 2C6 and 32D-Bcl-xl showed significant decreases in mitochondrial membrane permeability, cytochrome C release, caspase 3 and poly(adenosine diphosphate-ribose) polymerase (PARP) activation at 6–12 h, and in apoptosis at 24 h. Since the nuclear-to-cytoplasm events preceding the release of cytochrome C were similar in all cell lines, and increased expression of either the SOD2 or the BCL2L1 transgene provided radiation protection, we conclude that events at the level of the mitochondria are critically involved in radiation-induced apoptosis.

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.

Modulation of Radiation-Induced Cytokine Elevation Associated with Esophagitis and Esophageal Stricture by Manganese Superoxide Dismutase-Plasmid/Liposome (SOD2-PL) Gene Therapy

Abstract Epperly, M. W., Gretton, J. A., DeFilippi, S. J., Sikora, C. A., Liggitt, D., Koe, G. and Greenberger, J. S. Modulation of Radiation-Induced Cytokine Elevation Associated with Esophagitis and Esophageal Stricture by Manganese Superoxide Dismutase-Plasmid/Liposome (SOD2-PL) Gene Therapy. Radiation of the esophagus of C3H/HeNsd mice with 35 or 37 Gy of 6 MV X rays induces significantly increased RNA transcription for interleukin 1 (Il1), tumor necrosis factor alpha (Tnf), interferon gamma inducing factor (Ifngr), and interferon gamma (Ifng). These elevations are associated with DNA damage that is detectable by a comet assay of explanted esophageal cells, apoptosis of the esophageal basal lining layer cells in situ, and micro-ulceration leading to dehydration and death. The histopathology and time sequence of events are comparable to the esophagitis in humans that is associated with chemoradiotherapy of non-small cell lung carcinoma (NSCLC). Intraesophageal injection of clinical-grade manganese superoxide dismutase-plasmid/liposome (SOD2-PL) 24 h prior to irradiation produced an increase in SOD2 biochemical activity in explanted esophagus. An equivalent therapeutic plasmid weight of 10 μg ALP plasmid in the same 500 μl of liposomes, correlated to around 52–60% of alkaline phosphatase-positive cells in the squamous layer of the esophagus at 24 h. Administration of SOD2-PL prior to irradiation mediated a significant decrease in induction of cytokine mRNA by radiation and decreased apoptosis of squamous lining cells, micro-ulceration, and esophagitis. Groups of mice receiving 35 or 37 Gy esophageal irradiation by a technique protecting the lungs and treating only the central mediastinal area were followed to assess the long-term effects of radiation. SOD2-PL-treated irradiated mice demonstrated a significant decrease in esophageal wall thickness at day 100 compared to irradiated controls. Mice with orthotopic thoracic tumors composed of 32D-v-abl cells that received intraesophageal SOD2-PL treatment showed transgenic mRNA in the esophagus at 24 h, but no detectable human SOD2 transgene mRNA in explanted tumors by nested RT-PCR. These data provide support for translation of this strategy of SOD2-PL gene therapy to studies leading to a clinical trial in fractionated irradiation to decrease the acute and chronic side effects of radiation-induced damage to the esophagus.

Manganese Superoxide Dismutase-Plasmid/ Liposome (MnSOD-PL) Administration Protects Mice from Esophagitis Associated with Fractionated Radiation

Intraesophageal administration of manganese superoxide dismutase‐plasmid/liposome (MnSOD‐PL) prior to single fraction radiation has been shown to protect mice from lethal esophagitis. In our study, C3H/HeNsd mice received fractionated radiation in two protocols: (i) 18 Gy daily for four days with MnSOD‐PL administration 24 hr prior to the first and third fraction, or (ii) 12 Gy daily for six days with MnSOD‐PL 24 hr prior to the first, third, and fifth fraction. Control radiated mice received either no liposomes only or LacZ (bacterial β‐galactosidase gene)‐plasmid/liposome (LacZ‐PL) by the same schedules. We measured thiol depletion and lipid peroxidation (LP) in whole esophagus and tested the effectiveness of a new plasmid, hemagglutinin (HA) epitope‐tagged MnSOD (HA‐MnSOD). In fractionation protocols, mice receiving MnSOD‐PL, but not LacZ‐PL (200 μl of plasmid/liposomes containing 200 μg of plasmid DNA), showed a significant reduction in morbidity, decreased weight loss, and improved survival. Four and seven days after 37 Gy single fraction radiation, the esophagus demonstrated a significant increase in peroxidized lipids and reduction in overall antioxidant levels, reduced thiols, and decreased glutathione (GSH). These reductions were modulated by MnSOD‐PL administration. The HA‐MnSOD plasmid product was detected in the basal layers of the esophageal epithelium 24 hr after administration and provided significant radiation protection compared to glutathione peroxidase‐plasmid/liposome (GPX‐PL), or liposomes containing MnSOD protein, vitamin E, co‐enzyme Q10, or 21‐aminosteroid. Thus, MnSOD‐PL administration significantly improved tolerance to fractionated radiation and modulated radiation effects on levels of GSH and lipid peroxidation (LP). These studies provide further support for translation of MnSOD‐PL treatment into human esophageal radiation protection.

Bone Marrow Origin of Cells with Capacity for Homing and Differentiation to Esophageal Squamous Epithelium

Abstract Epperly, M. W., Guo, H., Shen, H., Niu, Y., Zhang, X., Jefferson, M., Sikora, C. A. and Greenberger, J. S. Bone Marrow Origin of Cells with Capacity for Homing and Differentiation to Esophageal Squamous Epithelium. Radiat. Res. 162, 233–240 (2004). Our goal was to determine whether esophageal progenitor cells could be isolated from adult mouse esophagus or bone marrow and shown to home to and proliferate in the irradiated esophagus of recipient mice. Esophageal progenitor cells were isolated from adult male C3H/HeNsd or C57BL/6J green fluorescent protein (GFP+) mice by a serial in vitro preplate technique or the technique of side population cell sorting. When injected intravenously (i.v.), these cells homed to the 30-Gy-irradiated esophagus of GFP− female recipient mice and formed donor-origin esophageal foci. GFP+ whole murine bone marrow cells injected i.v. also formed donor-origin esophageal squamous cell foci and protected recipient GFP− mice from upper-body irradiation in a cell dose-dependent manner. Marrow chimeric GFP− mice reconstituted with GFP+ cells showed migration of GFP+ marrow cells to the esophagus after 30 Gy irradiation. Purified esophageal progenitor cells isolated from first-generation preplate cell recipients engrafted after i.v. injection to the esophagus of second-generation-irradiated recipient mice. These data establish that esophageal progenitor cells can home to the irradiated esophagus and show limited differentiation capacity to squamous epithelium.

Plasmid/Liposome Transfer of the Human Manganese Superoxide Dismutase Transgene Prevents Ionizing Irradiation-Induced Apoptosis in Human Esophagus Organ Explant Culture

Esophagitis is a major limiting factor in the treatment of lung cancer by radiation alone or in combination with chemotherapy. We have previously demonstrated that intraesophageal injection of manganese superoxide dismutase–plasmid/liposome (MnSOD–PL) complex into C3H/HeNsd mice blocks irradiation‐induced esophagitis. To determine whether the human esophagus can be similarly transfected, normal human esophageal sections obtained from the margins of esophagectomy specimens from esophageal cancer patients were transfected in vitro with alkaline phosphatase (AlkP)–PL complex and stained for AlkP activity, and the percent of cells expressing AlkP was calculated. At 24 hr after transfection with 20 or 200 μg of AlkP–PL complex, 55.0% and 85.8% of esophageal epithelial cells expressed detectable AlkP, respectively. Other sections transfected with MnSOD–PL complex showed transgene mRNA by nested reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay and increased MnSOD biochemical activity for at least 96 hr after transfection. Irradiated MnSOD–PL complex–transfected sections demonstrated a significantly decreased percentage of apoptotic cells when compared to irradiated control sections. Following 1,000 cGy, MnSOD–PL‐treated samples showed 7.5 ± 2.8% and 33.3 ± 7.3% apoptotic cells at 24 and 48 hr compared to 53.6 ± 6.9% and 59.0 ± 13.8% for nontransfected controls (P < 0.0001 and P < 0.1175). After 2,000 cGy, results at 24 and 48 hr were 25.0 ± 7.6% and 66.9 ± 4.9% for MnSOD‐transfected sections compared to 65.6 ± 4.3% and 90.0 ± 4.1% for control sections (P < 0.0001 and P = 0.0353), respectively. Thus, human esophageal sections can be transfected with MnSOD–PL complex in vitro and thereby protected against ionizing irradiation‐induced apoptosis. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 128–137 (2000).

Overexpression of the human manganese superoxide dismutase (Mnsod) Transgene prevents irradiation apoptosis of 32D cl 3 hematopoietic progenitor cells by stabilization of the mitochondria

Abstract Subclones of 32D cl 3, 32D-Bcl-xl, 1F2 and 2C6 which demonstrate overexpression of the human MnSOD transgene had increased radioresistance and decreased apoptosis compared to 32D cl 3. Comet assay demonstrated that DNA strand breaks were repaired in all cell lines by 30 minutes (mins) following 200, 400 or 600 cGy, and all had increased migration of Bax and SAP kinases p38 and Jnk1 to the mitochondria 3 hrs after irradiation. Only 32D cl 3 had increased cytochrome-C release, caspase-3 biochemical activity, and poly(adenosine diphosphate-ribose) polymerase (PARP) activation at 6 hrs. At 6 hrs, 10.6% of 32D cl 3 cells showed a decrease in mitochondrial membrane potential compared to 2.9, 1.7 and 1.8% for 1F2, 2C6 and 32D-Bcl-xl, respectively. 32D c1 3 showed a decrease in mitochondrial complex I and III activity, and decreased levels of adenosine triphosphate (ATP) compared to 1F2 and 2C6. 32D cl 3, 1F2, 2C6 or 32D-Bcl-xl were incubated with 5,5-dimethyl-1-pyroline N-oxide (DMPO) spin-trap chemical and irradiated. Electron paramagnetic (spin) resonance (EPR) spectroscopy of spin-trapped adducts demonstrated that 32D cl 3 or 32D-Bcl-xl cells had a higher concentration of free radicals following irradiation than 1F2 or 2C6. Peroxidation of mitochondrial-specific diphosphatidyl-glycerol (DPG) was increased in 32D cl 3 cells compared to 1F2. Thus, the irradiation protective role of MnSOD is due to stabilization of the mitochondria.

Intratracheal injection of manganese superoxide dismutase-plasmid/liposome (Mnsod-pl) Protects normal lung but not orthotopic thoracic tumors from irradiation

Abstract Major complications following total body irradiation (TBI) prior to bone marrow transplantation (BMT) include pneumonitis and lung fibrosis. Our laboratory previously demonstrated that intratracheal (IT) injection of C57BL/6J mice with MnSOD-PL complex 24 hours before irradiation reduces organizing alveolitis/fibrosis and increases survival. To demonstrate that overexpression of the human MnSOD transgene in the lung would not protect lung tumors, C57BL/6J mice were injected at the carina with 5×10 5 orthotopic 3LL Lewis lung carcinoma cells 10 days before MnSOD-PL injection, and sacrificed 24 hrs later. Lungs were removed, orthotopic tumor isolated, RNA extracted, and nested RT-PCR performed with primers specific for the human MnSOD transgene. Expression of the human MnSOD transgene was detected in lung cells but not in orthotopic tumors. Tumor bearing mice that received MnSOD-PL + 1800 cGy showed a significant increase in survival compared to LacZ-PL + 1800 cGy or 1800 cGy alone. Pulmonary MnSOD-PL treatment may compliment TBI by not only protecting the lungs from irradiation but also by increasing tumor control.