Spatially Fractionated Radiation Induces Cytotoxicity and Changes in Gene Expression in Bystander and Radiation Adjacent Murine Carcinoma Cells

Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Radiation Research (Impact Factor: 2.91). 05/2012; 177(6):751-65. DOI: 10.2307/41545131
Source: PubMed

ABSTRACT Radiation-induced bystander effects have been extensively studied at low doses, since evidence of bystander induced cell killing and other effects on unirradiated cells were found to be predominant at doses up to 0.5 Gy. Therefore, few studies have examined bystander effects induced by exposure to higher doses of radiation, such as spatially fractionated radiation (GRID) treatment. In the present study, we evaluate the ability of GRID treatment to induce changes in GRID adjacent (bystander) regions, in two different murine carcinoma cell lines following exposure to a single irradiation dose of 10 Gy. Murine SCK mammary carcinoma cells and SCCVII squamous carcinoma cells were irradiated using a brass collimator to create a GRID pattern of nine circular fields 12 mm in diameter with a center-to-center distance of 18 mm. Similar to the typical clinical implementation of GRID, this is approximately a 50:50 ratio of direct and bystander exposure. We also performed experiments by irradiating separate cultures and transferring the medium to unirradiated bystander cultures. Clonogenic survival was evaluated in both cell lines to determine the occurrence of radiation-induced bystander effects. For the purpose of our study, we have defined bystander cells as GRID adjacent cells that received approximately 1 Gy scatter dose or unirradiated cells receiving conditioned medium from irradiated cells. We observed significant bystander killing of cells adjacent to the GRID irradiated regions compared to sham treated controls. We also observed bystander killing of SCK and SCCVII cells cultured in conditioned medium obtained from cells irradiated with 10 Gy. Therefore, our results confirm the occurrence of bystander effects following exposure to a high-dose of radiation and suggest that cell-to-cell contact is not required for these effects. In addition, the gene expression profile for DNA damage and cellular stress response signaling in SCCVII cells after GRID exposure was studied. The occurrence of GRID-induced bystander gene expression changes in significant numbers of DNA damage and cellular stress response signaling genes, providing molecular evidence for possible mechanisms of bystander cell killing.

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Available from: Eduardo G Moros, Sep 28, 2015
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    • "The current study examines the effects of exposure to copper (Cu), aluminum (Al) and external cobalt 60 gamma radiation on Atlantic salmon (Salmo salar L) to look at the impacts on a well-established low radiation dose effect—the bystander effect. This so-called non-targeted effect (NTE) of radiation is triggered at very low doses in the region of 2–4 mGy and activates stress signal pathways such as MAP kinase in irradiated cells (Lyng et al. 2006; Asur et al. 2010a, b; Ermakov et al. 2011; Asur et al. 2012; Mothersill and Seymour 2012a, b). It is known to involve depolarization of membranes followed by potassium, sodium and calcium ion-gated channel opening (Shao et al. 2006; Poon et al. 2007; Ubels et al. 2011). "
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    ABSTRACT: Very little is known about the combined effects of low doses of heavy metals and radiation. However, such "multiple stressor" exposure is the reality in the environment. In the work reported in this paper, fish were exposed to cobalt 60 gamma irradiation with or without copper or aluminum in the water. Doses of radiation ranged from 4 to 75 mGy delivered over 48 or 6 h. Copper doses ranged from 10 to 80 μg/L for the same time period. The aluminum dose was 250 μg/L. Gills and skin were removed from the fish after exposure and explanted in tissue culture flasks for investigation of bystander effects of the exposures using a stress signal reporter assay, which has been demonstrated to be a sensitive indicator of homeostatic perturbations in cells. The results show complex synergistic interactions of radiation and copper. Gills on the whole produce more toxic bystander signals than skin, but the additivity scores show highly variable results which depend on dose and time of exposure. The impacts of low doses of copper and low doses of radiation are greater than additive, medium levels of copper alone have a similar level of effect of bystander signal toxicity to the low dose. The addition of radiation stress, however, produces clear protective effects in the reporters treated with skin-derived medium. Gill-derived medium from the same fish did not show protective effects. Radiation exposure in the presence of 80 μg/L led to highly variable results, which due to animal variation were not significantly different from the effect of copper alone. The results are stressor type, stressor concentration and time dependent. Clearly co-exposure to radiation and heavy metals does not always lead to simple additive effects.
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    ABSTRACT: In this study, we sought to determine the therapeutic potential of variably sized (50 μm or 500 μm wide, 14 mm tall) parallel microbeam radiation therapy (MRT) alone and in combination with a novel anti-angiogenic peptide, anginex, in mouse mammary carcinomas (4T1)--a moderately hypoxic and radioresistant tumor with propensity to metastasize. The fraction of total tumor volume that was directly irradiated was approximately 25% in each case, but the distance between segments irradiated by the planar microbeams (width of valley dose region) varied by an order of magnitude from 150-1500 μm corresponding to 200 μm and 2000 μm center-to-center inter-microbeam distances, respectively. We found that MRT administered in 50 μm beams at 150 Gy was most effective in delaying tumor growth. Furthermore, tumor growth delay induced by 50 μm beams at 150 Gy was virtually indistinguishable from the 500 μm beams at 150 Gy. Fifty-micrometer beams at the lower peak dose of 75 Gy induced growth delay intermediate between 150 Gy and untreated tumors, while 500 μm beams at 75 Gy were unable to alter tumor growth compared to untreated tumors. However, the addition of anginex treatment increased the relative tumor growth delay after 500 μm beams at 75 Gy most substantially out of the conditions tested. Anginex treatment of animals whose tumors received the 50 μm beams at 150 Gy also led to an improvement in growth delay from that induced by the comparable MRT alone. Immunohistochemical staining for CD31 (endothelial cells) and αSMA (smooth muscle pericyte-associated blood vessels as a measure of vessel normalization) indicated that vessel density was significantly decreased in all irradiated groups and pericyte staining was significantly increased in the irradiated groups on day 14 after irradiation. The addition of anginex treatment further decreased the mean vascular density in all combination treatment groups and further increased the amount of pericyte staining in these tumors. Finally, evidence of tumor hypoxia was found to decrease in tumors analyzed at 1-14 days after MRT in the groups receiving 150 Gy peak dose, but not 75 Gy peak dose. Our results suggest that tumor vascular damage induced by MRT at these potentially clinically acceptable peak entrance doses may provoke vascular normalization and may be exploited to improve tumor control using agents targeting angiogenesis.
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    ABSTRACT: The application of a microchannel proton irradiation was compared to homogeneous irradiation in a three-dimensional human skin model. The goal is to minimize the risk of normal tissue damage by microchannel irradiation, while preserving local tumor control through a homogeneous irradiation of the tumor that is achieved because of beam widening with increasing track length. 20 MeV protons were administered to the skin models in 10- or 50-μm-wide irradiation channels on a quadratic raster with distances of 500 μm between each channel (center to center) applying an average dose of 2 Gy. For comparison, other samples were irradiated homogeneously at the same average dose. Normal tissue viability was significantly enhanced after microchannel proton irradiation compared to homogeneous irradiation. Levels of inflammatory parameters, such as Interleukin-6, TGF-Beta, and Pro-MMP1, were significantly lower in the supernatant of the human skin tissue after microchannel irradiation than after homogeneous irradiation. The genetic damage as determined by the measurement of micronuclei in keratinocytes also differed significantly. This difference was quantified via dose modification factors (DMF) describing the effect of each irradiation mode relative to homogeneous X-ray irradiation, so that the DMF of 1.21 ± 0.20 after homogeneous proton irradiation was reduced to 0.23 ± 0.11 and 0.40 ± 0.12 after microchannel irradiation using 10- and 50-μm-wide channels, respectively. Our data indicate that proton microchannel irradiation maintains cell viability while significantly reducing inflammatory responses and genetic damage compared to homogeneous irradiation, and thus might improve protection of normal tissue after irradiation.
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