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.

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 microgram ALP plasmid in the same 500 microliter 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.

Radiation pneumonitis remains a critical dose-limiting toxicity of total body irradiation (TBI) for use in bone marrow transplantation. The acute and chronic phases of radiation damage in the mouse lung have been shown to correlate with mouse strain genotype and are dependent on fraction size, total dose, and total lung volume. Our prior studies demonstrated effective prevention of irradiation-induced lung damage and improved survival in C57BL/6J mice by MnSOD plasmid/liposome gene therapy. In the present studies, we investigated the kinetics of irradiation-induced upregulation of mRNA for acute phase cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha, and fibrosis-associated transforming growth factor (TGF)-beta and isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) in 2000 cGy whole-lung irradiated C57BL/6J mice, a strain known to develop dose and volume-dependent organizing alveolitis/fibrosis. The results demonstrate increase in mRNA for IL-1 between days 1 and 14 after irradiation with return to baseline levels out to 120 days. TNF-alpha mRNA levels were not initially elevated but increased between 80 and 100 days and then decreased by 120 days. The mRNA levels for TGF-beta1 demonstrated an initial increase within the first 14 days after total lung irradiation with a decrease to baseline levels out to 100 days. Then, in striking contrast to the other two cytokines, an increase in TGF-beta2 mRNA occurred at around 120 days and correlated with the detection of organizing alveolitis/radiation fibrosis and mortality. These results are consistent with a two-phase mechanism in the molecular pathology of irradiation lung injury, in which IL-1 cytokine mRNA levels correlated with the acute pneumonitis phase and delayed elevation of TNF-alpha (80-100 days), TGF-beta1 (100 days), and TGF-beta2 (120 days) were associated with the fibrosis phase. Insight into the cell-specific and tissue-specific molecular mechanisms of ionizing irradiation induction of mRNA for pulmonary cytokines may provide new strategies for treatment of radiation pneumonitis in TBI patients.

Pulmonary toxicity is a major complication of total body irradiation used in preparation of patients for bone marrow transplantation. The mechanism of the late pulmonary damage manifested by fibrosis is unknown. In C57BL/6NHsd mice, manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) intratracheal injection 24 hours prior to 20 Gy single-fraction irradiation to both lungs significantly reduced late irradiation damage. Single intratracheal injections of MnSOD-PL, at concentrations as low as 250 microg of plasmid DNA, in a constant volume of 78 microL of liposomes, reduced late damage. To determine whether a slowly proliferating population of cells in the lung was responsible for initiation of fibrosis and was altered by MnSOD-PL therapy, 20 Gy total lung-irradiated mice were examined at serial time points for bromodeoxyuridine (BrdU) uptake in sites of cell division. There was low-level, but nonsignificant, increased cell proliferation detected at 80 days, with a significant increase at 100 days, 120 days, and at the time of death. Immunohistochemical assay for up-regulation of adhesion molecules associated with recruitment, transendothelial migration, and proliferation of bronchoalveolar macrophages revealed significant up-regulation of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) at 100 days with further increases up to the time of death. Increases were first detected in endothelin-positive endothelial cells. MnSOD-PL administration prior to irradiation decreased both BrdU incorporation and delayed expression of VCAM-1 and ICAM-1. The data indicate that the appearance of late irradiation-induced pulmonary fibrosis is associated with the up-regulation of adhesion molecules and suggest that potential targets for intervention may focus on the pulmonary vascular endothelium.

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.

To determine whether intratracheal (IT) lung protective manganese superoxide-plasmid/liposomes (MnSOD-PL) complex provided 'bystander' protection of thoracic tumors, mice with orthotopic Lewis lung carcinoma-bacterial ␤-galactosidase gene (3LL-LacZ) were studied. There was no significant difference in irradiation survival of 3LL-LacZ cells irradiated, then cocultured with MnSOD-PL-treated compared with control lung cells (D 0 2.022 and 2.153, respectively), or when irradiation was delivered 24 h after coculture (D 0 0.934 and 0.907, respectively). Tumor-bearing control mice showed 50% survival at 18 days and 10% survival at 21 days. Mice receiving liposomes with no insert or LacZ-PL complex plus 18 Gy had 50% survival at 22 days,

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.

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.