The impact of ionizing radiation on placental trophoblasts.

INTRODUCTION: Exposure to low-dose radiation is widespread and attributable to natural sources. However, occupational, medical, accidental, and terrorist-related exposures remain a significant threat. Information on radiation injury to the feto-placental unit is scant and largely observational. We hypothesized that radiation causes trophoblast injury, and alters the expression of injury-related transcripts in vitro or in vivo, thus affecting fetal growth. METHODS: Primary human trophoblasts (PHTs), BeWo or NCCIT cells were irradiated in vitro, and cell number and viability were determined. Pregnant C57Bl/6HNsd mice were externally irradiated on E13.5, and placentas examined on E17.5. RNA expression was analyzed using microarrays and RT-qPCR. The experiments were repeated in the presence of the gramicidin S (GS)-derived nitroxide JP4-039, used to mitigate radiation-induced cell injury. RESULTS: We found that survival of in vitro-irradiated PHT cell was better than that of irradiated BeWo trophoblast cell line or the radiosensitive NCCIT mixed germ cell tumor line. Radiation altered the expression of several trophoblast genes, with a most dramatic effect on CDKN1A (p21, CIP1). Mice exposed to radiation at E13.5 exhibited a 25% reduction in mean weight by E17.5, and a 9% reduction in placental weight, which was associated with relatively small changes in placental gene expression. JP4-039 had a minimal effect on feto-placental growth or on gene expression in irradiated PHT cells or mouse placenta. DISCUSSION AND CONCLUSION: While radiation affects placental trophoblasts, the established placenta is fairly resistant to radiation, and changes in this tissue may not fully account for fetal growth restriction induced by ionizing radiation.

Modulation of in utero total body irradiation induced newborn mouse growth retardation by maternal manganese superoxide dismutase-plasmid liposome (MnSOD-PL) gene therapy.

To determine the effects of manganese superoxide dismutase (MnSOD) plasmid liposome (PL) maternal radioprotection on fetal mice, timed pregnant female mice (E14 gestation) were irradiated to 3.0 Gy total body irradiation (TBI) dose, and the number, weight and growth and development over 6 months after birth of newborn mice was quantitated compared with irradiated controls. Maternal MnSOD-PL treatment at E13 improved pup survival at birth (5.4±0.9 per litter) compared with non-irradiated 3.0 Gy controls 4.9±1.1. There was no statistically significant difference in newborn abnormalities, male to female ratio in newborn litters, or other evidence of teratogenesis in surviving newborn mice from MnSOD-PL treated compared with irradiated controls. However, E14 3 Gy irradiated pups from gene therapy-treated mothers showed a significant increase in both growth and overall survival over 6 months after birth (P=0.0022). To determine if transgene product crossed the placenta pregnant E13 mice were injected intravenously with hemagglutinin-epitope-tagged MnSOD (100 µg plasmid in 100 µl liposomes), then after 24 h, fetal mice, placentas and maternal tissues were removed and tested by both immunohistochemistry and reverse transcriptase-PCR for transgene and product. There was no evidence of transgene or product in placenta or any fetal tissue while maternal liver was positive by both assays. The data provide evidence for fetal radioprotection by maternal MnSOD-PL gene therapy before irradiation, which is mediated by an indirect bystander effect and is associated with a significant improvement in both survival at birth and growth and development of newborn mice.

Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity.

Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600 rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900 rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle.

Intraesophageal MnSOD-plasmid liposome enhances engraftment and self-renewal of bone marrow derived progenitors of esophageal squamous epithelium.

We evaluated whether the improved esophageal radiation tolerance following Manganese Superoxide Dismutase (MnSOD)-Plasmid Liposomes was explained by improved engraftment of bone marrow-derived progenitors. C57BL/6NHsd female mice pretreated with intraesophageal MnSOD-PL were irradiated to 29 Gy to the esophagus and intravenously transplanted with marrow from male B6. 129S7-Gt (ROSA) 26S OR/J ROSA (Lac-Z+, G418-resistant) mice. After 14 days, esophagi were removed and side population and non-side population cells evaluated for donor multilineage (endothelin/vimentin/F480) positive esophageal cells. Serial intravenous transplantability was tested in second generation 29 Gy esophagus-irradiated mice. Esophagi from recipients receiving swallowed MnSOD-PL 24 h prior to irradiation demonstrated significantly increased esophageal repopulation with donor bone marrow-derived Lac-Z+, G418+, Y-probe+ multilineage cells (37.8+/-1.8>50 cell Lac-Z+ foci per esophagus) compared to irradiated controls (19.8+/-1.8) P<0.0001. Serial transfer to second-generation irradiated C57BL/6NHsd mice of intravenously injected SP or NSP first generation recipient esophagus cells was also significantly enhanced by MnSOD-PL intraesophageal pretreatment (74.4+/-3.6 SP-derived Lac-Z+ foci per esophagus, 48.6+/-5.4 NSP-derived) compared to irradiation controls (23.4+/-1.8 SP, 6.0+/-3.0 NSP), P<0.0001. Thus, intraesophageal MnSOD-PL administration enhances engraftment of marrow-derived progenitors.

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes.

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.

Inhalation delivery of manganese superoxide dismutase-plasmid/liposomes protects the murine lung from irradiation damage.

Intratracheal injection of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) complexes has been demonstrated to delay the onset and reduce the extent of ionizing irradiation-induced murine pulmonary organizing alveolitis/fibrosis. To facilitate translation of this modality to clinical fractionated radiotherapy, inhalation delivery of MnSOD-PL was developed using an ultrasonic nebulizer. Transgene product was quantitated by immunohistochemical quantitation and pulmonary tissue levels of MnSOD biochemical activity. C57BL/6NHsd female mice demonstrated a plasmid dose-dependent increased expression of MnSOD transgene product over the range of 250 microg-2.5 mg of MnSOD-PL administered over a constant 5 min interval. Delivery of a constant concentration of 500 microg of MnSOD-PL with varying times of administration ranging from 0.5 to 10 min demonstrated optimal MnSOD expression at 5 min. Mice pretreated by inhalation delivery of MnSOD-PL demonstrated significantly improved survival after 20 Gy single fraction irradiation to both lungs compared to LacZ-PL inhalation-treated or irradiated control mice. Mice receiving 10 fractions of 3.5 cGy demonstrated increased pulmonary MnSOD transgene product activity by a protocol of every Monday-Wednesday or daily inhalation of MnSOD-PL. Thus, inhalation radioprotective gene therapy using MnSOD-PL provides a practical and effective method for delivery of lung-specific radioprotection during fractionated radiotherapy protocols in a mouse model.

Radioprotective gene therapy.

Control of cancer by irradiation therapy alone or in conjunction with combination chemotherapy is often limited by organ specific toxicity. Ionizing irradiation toxicity is initiated by damage to normal tissue near the tumor target and within the transit volume of radiotherapy beams. Irradiation-induced cellular, tissue, and organ damage is mediated by acute effects, which can be dose limiting. A latent period follows recovery from the acute reaction, then chronic irradiation fibrosis (late effects) pose a second cause of organ failure. We have developed the technology for radioprotective gene therapy using the transgene for the antioxidant manganese superoxide dismutase, delivered to specific target organs (lung, esophagus, oral cavity, oropharynx, and bladder) using gene transfer vectors including plasmid/liposomes (PL) and adenovirus. Irradiation protection by MnSOD transgene overexpression at the cellular level has been demonstrated to be localized to the mitochondrial membrane. Using MnSOD transgene constructs lacking the mitochondrial localization leader sequence, and in other experiments attaching this localization signal to otherwise non-radioprotective cytoplasmic Cu/ZnSOD, mitochondrial localization has been demonstrated to be critical to protection. Organ specific injection of MnSOD-PL prior to irradiation demonstrates transgene expression for 48-72 hours, and an associated decrease in ionizing irradiation-induced expression of inflammatory cytokine mRNA and protein. Significant reduction of organ specific tissue injury has been demonstrated in several organ systems in rodent models. Application of MnSOD-PL gene therapy in the setting of fractionated chemo-radiotherapy is being tested in clinical trials for prevention of esophagitis during treatment of non-small cell carcinoma of the lung, and in prevention of mucositis during combination therapy of carcinomas of the head and neck. Encouraging results in pre-clinical models suggest that radioprotective gene therapy may facilitate dose escalation protocols to allow increases in the therapeutic ratio of cancer radiotherapy.

Cell phenotype specific kinetics of expression of intratracheally injected manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) during lung radioprotective gene therapy.

Intratracheal (IT) injection of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) complexes prior to whole lung irradiation of C57BL/6J mice provides significant protection from acute and chronic irradiation damage. We determined the duration of increased MnSOD biochemical activity and differential expression of a hemagglutinin (HA) epitope-tagged MnSOD transgene. HA-MnSOD-PL was IT injected at doses of 0-1000 microg, and mice were killed 1,2,3 or 4 days later. Other groups of mice were irradiated to 20 Gy to the pulmonary cavity 24 h after injection and killed at the same time points as non-irradiated mice. Both non-irradiated and irradiated groups of mice showed increased MnSOD biochemical activity with plasmid dose that plateaued at 100 microg of MnSOD plasmid DNA. In control mice, MnSOD biochemical activity decreased at 2, 3 or 4 days after injection. In irradiated mice, MnSOD biochemical activity decreased at day 2 but increased on days 3 and 4. HA-MnSOD expression decreased in broncheoalveolar macrophages and alveolar type-II cells 3 days after injection in non-irradiated and irradiated mice, but remained elevated in endothelial and epithelial cells past 4 days. The data provide a rationale for every second-day administration of intrapulmonary MnSOD-PL in clinical trials of radioprotective gene therapy. This should be sufficient to provide radioprotection during radiation treatments.

Modulation of redox signal transduction pathways in the treatment of cancer.

Reactive oxygen species (ROS)-mediated damage to DNA is associated with induction of stress-activated protein kinases leading to secondary and tertiary effects on the nuclear matrix, cytoplasmic transport mechanisms, and altered mitochondrial and cell membranes. The cellular defenses against ROS damage are associated with up-regulation of gene products that can significantly alter cell biology, including antiapoptotic Bax family proteins and inflammatory proteins. Altered cell integrity can occur either directly or by indirect paracrine and juxtacrine interactions within tissues. Previous approaches toward therapeutic intervention against ROS damage have included administration of radical scavenger compounds, use of novel drugs that increase cellular production of constitutive antioxidants, or pharmacologic agents that modify the intracellular transport of antioxidants. Strategies to modify the cellular effects of ROS in hyperbaric oxygen injury to the lung, reperfusion injury to transplanted organs, and cancer have led to novel approaches of gene therapy in which the transgenes for antioxidant proteins can be expressed in specific tissues. Reducing tissue-damaging effects of ROS may have relevance to cancer patients by ameliorating normal tissue damage from ionizing irradiation therapy, photodynamic therapy, and cancer chemotherapy.

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 beta-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 microl of plasmid/liposomes containing 200 microg 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.

Identification of respiratory complexes I and III as mitochondrial sites of damage following exposure to ionizing radiation and nitric oxide.

In 32D cl 3 hematopoietic progenitor cells, the overexpression of manganese superoxide dismutase (MnSOD, SOD2), the enzyme normally found in mitochondria, protects against the damaging effects of ionizing radiation. In the presence of a nitric oxide donor, which exacerbates the damage, inhibition of mitochondrial function can be demonstrated to be associated with respiratory complexes I (NADH dehydrogenase) and III (cytochrome c reductase), but not II (succinate dehydrogenase), IV (cytochrome c oxidase), or V (ATP synthase). The same pattern of inhibition is observed in the case of isolated bovine heart mitochondria exposed to ionizing radiation and the nitric oxide donor. The addition of authentic peroxynitrite (ONO2(-)) to isolated mitochondria also results in damage to complexes I and III (but not II, IV, and V), as shown by assays of electron-transfer activities and electron paramagnetic resonance (EPR) spectroscopic measurements, suggesting ONO2(-) to be responsible for most of the observed radiation damage in both the cultured cell lines and isolated mitochondria. It is argued that, in general, production of ONO2(-) is an important contributor to radiation damage in biological systems and the implications of these findings in relation to possible mechanisms of oxidant-linked apoptosis are briefly considered.

Overexpression of manganese superoxide dismutase (MnSOD) in whole lung or alveolar type II cells of MnSOD transgenic mice does not provide intrinsic lung irradiation protection.

Intratracheal (IT) injection of the transgene for human manganese superoxide dismutase in plasmid/liposome (SOD2-PL) complex prior to irradiation protects C57BL/6J mice from whole lung irradiation-induced organizing alveolitis/fibrosis. Transgene mRNA was detected in alveolar type II (AT-II) and tracheobronchial tree cells explanted to culture 48 hours after gene therapy. To determine whether constitutive overexpression of murine MnSOD (Sod2) in whole lung or surfactant promoter-restricted AT-II cells (SP1)-SOD2 mice would provide intrinsic radioresistance, transgenic mice of two strains were compared with age-matched controls. Other groups of surfactant promoter-restricted (SP1)-SOD2 transgenic mice or control FeVB/NHsd mice received IT SOD2-PL gene therapy prior to irradiation. There was no significant intrinsic lung protection in either strain of MnSOD transgenic mice. The SP1-SOD2 transgenic mice were protected from lung damage by IT injection of the human SOD2-PL complex 24 hours prior to irradiation. Thus, overexpression of either human SOD2 or murine Sod2 in the lungs of transgenic mice does not provide intrinsic lung irradiation protection. The overexpression of SOD2 in the SP1-SOD2 mice may have made the mice more sensitive to irradiation.

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 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.

Decreased pulmonary radiation resistance of manganese superoxide dismutase (MnSOD)-deficient mice is corrected by human manganese superoxide dismutase-Plasmid/Liposome (SOD2-PL) intratracheal gene therapy.

The pulmonary ionizing radiation sensitivity of C57BL/6 Sod2(+/-) mice heterozygous for MnSOD deficiency was compared to that Sod2(+/+) control littermates. Embryo fibroblast cell lines from Sod2(-/-) (neonatal lethal) or Sod2(+/-) mice produced less biochemically active MnSOD and demonstrated a significantly greater in vitro radiosensitivity. No G(2)/M-phase cell cycle arrest after 5 Gy was observed in Sod2(-/-) cells compared to the Sod2(+/-) or Sod2(+/+) lines. Subclonal Sod2(-/-) or Sod2(+/-) embryo fibroblast lines expressing the human SOD2 transgene showed increased biochemical activity of MnSOD and radioresistance. Sod2(+/-) mice receiving 18 Gy whole-lung irradiation died sooner and had an increased percentage of lung with organizing alveolitis between 100 and 160 days compared to Sod2(+/+) wild-type littermates. Both Sod2(+/-) and Sod2(+/+) littermates injected intratracheally with human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) complex 24 h prior to whole-lung irradiation showed decreased DNA strand breaks and improved survival with decreased organizing alveolitis. Thus underexpression of MnSOD in the lungs of heterozygous Sod2(+/-) knockout mice is associated with increased pulmonary radiation sensitivity and parallels increased radiation sensitivity of embryo fibroblast cell lines in vitro. The restoration of cellular radioresistance in vitro and in lungs in vivo by SOD2-PL transgene expression supports a potential role for SOD2-PL gene therapy in organ-specific radioprotection.

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 microgram 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).

Activation of the nitric oxide synthase 2 pathway in the response of bone marrow stromal cells to high doses of ionizing radiation.

Reverse transcription-polymerase chain reaction and immunofluorescence analysis of D2XRII murine bone marrow stromal cells showed that gamma irradiation with doses of 2-50 Gy from (137)Cs stimulated expression of nitric oxide synthase 2 (Nos2, also known as iNos). The activation of Nos2 was accompanied by an increase in the fluorescence of 4,5-diaminofluorescein diacetate, a nitric oxide trap, and accumulation of 3-nitrotyrosine within cellular proteins in a dose-dependent manner. These effects were inhibited by actinomycin D and by N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride, a specific inhibitor of Nos2. The induction of Nos2 expression and Nos2-dependent release of nitric oxide in D2XRII cells was observed within 24 h after irradiation and was similar in magnitude to that observed in cultures incubated with Il1b and Tnf. We conducted (1) confocal fluorescence imaging of 3-nitrotyrosine in bone marrow cells of irradiated C57BL/6J mice and (2) 3-nitrotyrosine fluorescence imaging of FDC-P1JL26 hematopoietic cells that were cocultured with previously irradiated D2XRII bone marrow stromal cells. Exposure to ionizing radiation increased the production of 3-nitrotyrosine in irradiated bone marrow cells in vivo and in nonirradiated FDC-P1JL26 cells cocultured with irradiated D2XRII cells for 1 or 4 h. We suggest that nitrative/oxidative stress to the transplanted multilineage hematopoietic cells due to exposure to nitric oxide released by host bone marrow stromal cells may contribute to the genotoxic events associated with malignant alterations in bone marrow tissue of transplant recipients who are prepared for engraftment by total-body irradiation.

Intratracheal injection of manganese superoxide dismutase (MnSOD) plasmid/liposomes protects normal lung but not orthotopic tumors from irradiation.

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 beta-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 (D0 2.022 and 2.153, respectively), or when irradiation was delivered 24 h after coculture (D0 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, and a 20% and 30% survival at day 50, respectively. Mice receiving MnSOD-PL complex followed by 18 Gy showed prolonged survival of 45% at 50 days after irradiation (P < 0.001). Nested RT-PCR assay for the human MnSOD transgene demonstrated expression at 24 h in normal lung, but not in orthotopic tumors. Decreased irradiation induction of TGF-beta1, TGF-beta2, TGF-beta3, MIF, TNF-alpha, and IL-1 at 24 h was detected in lungs, but not orthotopic tumors from MnSOD-PL-injected mice (P < 0.001). Thus, pulmonary radioprotective MnSOD-PL therapy does not provide detectable 'bystander' protection to thoracic tumors.

The use of adeno-associated virus to circumvent the maturation-dependent viral transduction of muscle fibers.

Muscle-based gene therapy using adenovirus, retrovirus, and herpes simplex virus has been hindered by viral cytotoxicity, host immune response, and the maturation-dependent viral transduction of muscle fibers. The development of new mutant vectors has greatly reduced the toxicity and the immune rejection problems, but the inability of viral vectors to penetrate and transduce mature myofibers remains an important issue. Research has been focused on the characterization of barriers to viral transduction in mature myofibers to develop strategies to circumvent the maturation-dependent viral transduction of myofibers. Here, we report that adeno-associated virus (AAV) can be used to overcome the maturation-dependent viral transduction of myofibers. We have investigated by which mechanism AAV can penetrate and efficiently transduce mature muscle fibers, and have shown that this viral vector is not blocked by the basal lamina and that AAV transduction of myofibers is independent of myoblast mediation. Although AAV can efficiently transduce mature myofibers, a differential transduction is still observed among the different types of myofibers that correlates with the expression of the heparan sulfate proteoglycan receptors, the muscle maturity, the number of viral particles used, and the time postinjection. The identification of the mechanisms by which AAV transduces mature myofibers will help in the development of strategies to achieve an efficient muscle-based gene therapy for inherited and acquired diseases.

Prevention of irradiation-induced esophagitis by plasmid/liposome delivery of the human manganese superoxide dismutase transgene.

Esophagitis is a major toxicity of radiation therapy for nonsmall-cell lung cancer. Intraesophageal injection of manganese superoxide dismutase (MnSOD) plasmid/liposome complexes (1 mg of the pRK5-MnSOD plasmid containing the human MnSOD transgene in a 0.15 ml volume of lipofectin) before irradiation was carried out to attempt to prevent irradiation esophagitis. In control noninjected male C3H/HeNsd mice, esophagitis was induced by single fraction 3,500 cGy irradiation. Histopathology at 4 days revealed vacuole formation in squamous lining cells, separation of the squamous layer from the underlying muscle layer, ulceration at 7 days, and dehydration and death by 30 days. MnSOD plasmid/liposome complex-injected mice showed transcription of the human MnSOD transgene message in esophageal squamous lining cells by nested reverse transcriptase-polymerase chain reaction (RT-PCR) increased MnSOD biochemical activity 24 h after injection, decreased vacuole formation at day 4 (P < 0.001) after 3,500 cGy thoracic irradiation, and improved survival (P = 0.0009). In contrast, groups of mice receiving LacZ (bacterial beta-galactosidase gene) plasmid/liposome complexes or liposomes containing no DNA before 3,500 cGy irradiation showed an unaltered irradiation histopathology and decreased survival. Mice receiving intraesophageal MnSOD plasmid/liposomes followed 8 h later by human equivalent doses of Taxol (1.4 mg/kg) and carboplatin (2.5 mg/kg), then 15 h later 3,300 cGy irradiation, showed increased survival, compared with irradiated control or LacZ plasmid/liposome groups. Thus, overexpression of the human MnSOD transgene in the esophagus can prevent irradiation-induced esophagitis in the mouse model.