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.

A multicenter phase II study of cetuximab in combination with chest radiotherapy and consolidation chemotherapy in patients with stage III non-small cell lung cancer.

BACKGROUND: Cetuximab has demonstrated improved efficacy in combination with chemotherapy and radiotherapy. We evaluated the integration of cetuximab in the combined modality treatment of stage III non-small cell lung cancer (NSCLC). METHODS: Patients with surgically unresectable stage IIIA or IIIB NSCLC were treated with chest radiotherapy, 73.5 Gy (with lung and tissue heterogeneity corrections) in 35 fractions/7 weeks, once daily (63 Gy without heterogeneity corrections). Cetuximab was given weekly during radiotherapy and continued during consolidation therapy with carboplatin and paclitaxel up to a maximum of 26 weekly doses. The primary endpoint was overall survival. Baseline tumor tissue was analyzed for EGFR by fluorescence in situ hybridization (FISH). RESULTS: Forty patients were enrolled in this phase II study. The median overall survival was 19.4 months and the median progression-free survival 9.3 months. The best overall response rate in 31 evaluable patients was 67%. No grade 3 or 4 esophagitis was observed. Three patients experienced grade 3 rash; 16 patients (69%) developed grade 3/4 neutropenia during consolidation therapy. One patient died of pneumonitis, possibly related to cetuximab. EGFR gene copy number on baseline tumor tissues, analyzed by FISH, was not predictive of efficacy outcomes. CONCLUSIONS: The addition of cetuximab to chest radiotherapy and consolidation chemotherapy was tolerated well and had modest efficacy in stage III NSCLC. Taken together with the lower incidence of esophagitis, our results support evaluation of targeted agents instead of chemotherapy with concurrent radiotherapy in this setting.

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.

Methyltransferases mediate cell memory of a genotoxic insult.

Characterization of the direct effects of DNA-damaging agents shows how DNA lesions lead to specific mutations. Yet, serum from Hiroshima survivors, Chernobyl liquidators and radiotherapy patients can induce a clastogenic effect on naive cells, showing indirect induction of genomic instability that persists years after exposure. Such indirect effects are not restricted to ionizing radiation, as chemical genotoxins also induce heritable and transmissible genomic instability phenotypes. Although such indirect induction of genomic instability is well described, the underlying mechanism has remained enigmatic. Here, we show that mouse embryonic stem cells exposed to γ-radiation bear the effects of the insult for weeks. Specifically, conditioned media from the progeny of exposed cells can induce DNA damage and homologous recombination in naive cells. Notably, cells exposed to conditioned media also elicit a genome-destabilizing effect on their neighbouring cells, thus demonstrating transmission of genomic instability. Moreover, we show that the underlying basis for the memory of an insult is completely dependent on two of the major DNA cytosine methyltransferases, Dnmt1 and Dnmt3a. Targeted disruption of these genes in exposed cells completely eliminates transmission of genomic instability. Furthermore, transient inactivation of Dnmt1, using a tet-suppressible allele, clears the memory of the insult, thus protecting neighbouring cells from indirect induction of genomic instability. We have thus demonstrated that a single exposure can lead to long-term, genome-destabilizing effects that spread from cell to cell, and we provide a specific molecular mechanism for these persistent bystander effects. Collectively, our results impact the current understanding of risks from toxin exposures and suggest modes of intervention for suppressing genomic instability in people exposed to carcinogenic genotoxins.

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.

Gene therapy approaches for stem cell protection.

Cytotoxic exposure of bone marrow and other non-hematopoietic organs containing self-renewing stem cell populations is associated with damage to the supportive microenvironment. Recent evidence indicates that radical oxygen species resulting from the initial oxidative stress persist for months after ionizing irradiation exposure of tissues including oral cavity, esophagus, lung and bone marrow. Antioxidant gene therapy using manganese superoxide dismutase plasmid liposomes has provided organ-specific radiation protection associated with delay or prevention of acute and late toxicity. Recent evidence has suggested that manganese superoxide dismutase transgene expression in cells of the organ microenvironment contributes significantly to the mechanism of protection. Incorporating this knowledge into designs of novel approaches for stem cell protection is addressed in the present review.

Apoptotic interactions of cytochrome c: redox flirting with anionic phospholipids within and outside of mitochondria.

Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some - as yet unidentified - role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.

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.

Modeling stem cell population growth: incorporating terms for proliferative heterogeneity.

Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research. Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions. A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models. This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations. The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death. Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss. We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation. We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle-derived stem cells was observed. Finally, we demonstrate an improved estimation of the kinetic parameters. This study contributes to both the biological and mathematical understanding of stem cell dynamics. Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.

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.

Mechanisms of muscle stem cell expansion with cytokines.

Stem cell expansion and proliferation are important for cell transplantation and stem cell-mediated applications. While we have demonstrated that muscle stem cells can be obtained from adult skeletal muscle tissue, these cells represent only a small percentage of the muscle-derived cells and require in vitro expansion for successful stem cell-mediated therapies. In this study, we have examined the potential of several cytokines to stimulate stem cell growth by combining a non-exponential mathematical model with a unique cell culture system. The growth kinetics of two populations of muscle stem cells were characterized in culture medium supplemented with epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), FLT-3 ligand, hepatocyte growth factor, or stem cell factor (SCF). The division time (DT) and fraction of mitotically active cells were investigated as key parameters to further understand the mechanism of the expansion of the stem cell populations. Our results show that expansion of the freshly isolated, muscle-derived stem cells (MDSC) occurred by recruiting cells into the cell cycle in the presence of EGF, IGF-1, and SCF. However, expansion of the cultured stem cell clone, MC13, is attributed to a reduction of the length of the cell cycle in the presence of FGF-2, EGF, IGF-1, and SCF. Both MDSC and MC13 growth were inhibited in the presence of FLT-3 ligand by increasing the length of the cell cycle. Our results suggest that EGF, IGF-1, FGF-2, and SCF are important cytokines for stimulating the proliferation of MDSC. In addition, this study illustrates that expansion of stem cells occurs through different mechanisms, which consequently demonstrates the importance of monitoring several parameters of cell growth, such as DT and dividing fraction, following stimulation with growth factors.

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.