Mammalian Tissue Response to Low Dose Ionizing Radiation: The Role of Oxidative Metabolism and Intercellular Communication

The objective of the project was to elucidate the mechanisms underlying the biological effects of low dose/low dose rate ionizing radiation in organs/tissues of irradiated mice that differ in their susceptibility to ionizing radiation, and in human cells grown under conditions that mimic the natural in vivo environment. The focus was on the effects of sparsely ionizing cesium-137 gamma rays and the role of oxidative metabolism and intercellular communication in these effects. Four Specific Aims were proposed. The integrated outcome of the experiments performed to investigate these aims has been significant towards developing a scientific basis to more accurately estimate human health risks from exposures to low doses ionizing radiation. By understanding the biochemical and molecular changes induced by low dose radiation, several novel markers associated with mitochondrial functions were identified, which has opened new avenues to investigate metabolic processes that may be affected by such exposure. In particular, a sensitive biomarker that is differentially modulated by low and high dose gamma rays was discovered

Kinetics of propagation of bystander effects in human cells cultures exposed to low fluences of high LET radiations

International audienceWe and others have previously shown, in confluent cell cultures exposed to low fluences of a particles, that the proportion of cells that upregulate stress-inducible proteins is much higher that the number of cells irradiated. This phenomenon, called bystander effect, is now well accepted and is thought to impact the health risks of exposure to ionizing radiation. Here, we investigate the kinetics of propagation of signaling events that lead to induction of DNA damage in bystander cells in confluent normal human AG1522 fibroblasts exposed to a mean dose of 0.2 cGy from 3.2 MeV a particles (LET ; 124 keV/lm) or 1 GeV/n iron ions (LET ; 151 keV/lm). We evaluated the formation of 53BP1 foci (p53 binding protein 1), which localizes at sites of DNA double strand breaks, as a function of time after irradiation. The fraction of cells whose nuclei were traversed by an irradiating particle was derived from Poisson statistics and estimates of cell geometry, particle fluence and energy loss. At a mean dose of 0.2 cGy, only 1.4% and 1.2% of the cells are traversed through the nucleus by a particle or iron ion tracks, respectively. The number of 53BP1 foci in control cells was ; 0.61 foci per cell. In a particle-irradiated cell cultures, the mean number of foci per cell was 0.73 (p,0.001) at 15 min; it reached 0.90 by 3h (p,0.001) after irradiation, following which a decrease was observed. The same trend was detected when the fraction of cells with foci was considered: it reached 61% at 3h which is higher than the expected 47.4% of the cells (46% in control + 1.4% traversed). The increase in foci formation over the expected value was eliminated when the cells were incubated with a specific inhibitor of ATM (Ataxia Telangiectasia Mutated protein). Analyses of bystander effects in iron ion-irradiated cell cultures and the effect of partial oxygen tension on the kinetic of 53BP1 foci formation in low fluences alpha particle-irradiated cell populations are in progress

Non-targeted Stressful Effects in Normal Human Fibroblast Cultures Exposed to Low Fluences of High Charge, High-Energy (HZE) Particles: Kinetics of Biologic Responses and Significance of Secondary Radiations.

International audienceThe induction of nontargeted stressful effects in cell populations exposed to low fluences of high-charge (Z) and high-energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ∼151 keV/μm] or 600 MeV/u silicon ions (LET ∼50 keV/μm) at mean absorbed doses as low as 0.2 cGy, wherein 1-3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21(Waf1) (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ∼109 keV/μm) that targets ∼1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ∼13 keV/μm) by which, on average, ∼13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 μm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles

Radiation Type- and Dose-Specific Transcriptional Responses across Healthy and Diseased Mammalian Tissues

Ionizing radiation (IR) is a genuine genotoxic agent and a major modality in cancer treatment. IR disrupts DNA sequences and exerts mutagenic and/or cytotoxic properties that not only alter critical cellular functions but also impact tissues proximal and distal to the irradiated site. Unveiling the molecular events governing the diverse effects of IR at the cellular and organismal levels is relevant for both radiotherapy and radiation protection. Herein, we address changes in the expression of mammalian genes induced after the exposure of a wide range of tissues to various radiation types with distinct biophysical characteristics. First, we constructed a publicly available database, termed RadBioBase, which will be updated at regular intervals. RadBioBase includes comprehensive transcriptomes of mammalian cells across healthy and diseased tissues that respond to a range of radiation types and doses. Pertinent information was derived from a hybrid analysis based on stringent literature mining and transcriptomic studies. An integrative bioinformatics methodology, including functional enrichment analysis and machine learning techniques, was employed to unveil the characteristic biological pathways related to specific radiation types and their association with various diseases. We found that the effects of high linear energy transfer (LET) radiation on cell transcriptomes significantly differ from those caused by low LET and are consistent with immunomodulation, inflammation, oxidative stress responses and cell death. The transcriptome changes also depend on the dose since low doses up to 0.5 Gy are related with cytokine cascades, while higher doses with ROS metabolism. We additionally identified distinct gene signatures for different types of radiation. Overall, our data suggest that different radiation types and doses can trigger distinct trajectories of cell-intrinsic and cell-extrinsic pathways that hold promise to be manipulated toward improving radiotherapy efficiency and reducing systemic radiotoxicities

Considerations for application of benchmark dose modeling in radiation research: workshop highlights

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Expression of NES-hTERT in Cancer Cells Delays Cell Cycle Progression and Increases Sensitivity to Genotoxic Stress

Telomerase is a reverse transcriptase associated with cellular immortality through telomere maintenance. This enzyme is activated in 90% of human cancers, and inhibitors of telomerase are currently in clinical trials to counteract tumor growth. Many aspects of telomerase biology have been investigated for therapy, particularly inhibition of the enzyme, but little was done regarding its subcellular shuttling. We have recently shown that mutations in the nuclear export signal of hTERT, the catalytic component of telomerase, led to a mutant (NES-hTERT) that failed to immortalize cells despite nuclear localization and catalytic activity. Expression of NES-hTERT in primary fibroblast resulted in telomere-based premature senescence and mitochondrial dysfunction. Here we show that expression of NES-hTERT in LNCaP, SQ20B and HeLa cells rapidly and significantly decreases their proliferation rate and ability to form colonies in soft agar while not interfering with endogenous telomerase activity. The cancer cells showed increased DNA damage at telomeric and extra-telomeric sites, and became sensitive to ionizing radiation and hydrogen peroxide exposures. Our data show that expression of NES-hTERT efficiently counteracts cancer cell growth in vitro in at least two different ways, and suggest manipulation with the NES of hTERT or its subcellular shuttling as a new strategy for cancer treatment

Mammalian Tissue Response to Low Dose Ionizing Radiation: The Role of Oxidative Metabolism and Intercellular Communication

The objective of the project was to elucidate the mechanisms underlying the biological effects of low dose/low dose rate ionizing radiation in organs/tissues of irradiated mice that differ in their susceptibility to ionizing radiation, and in human cells grown under conditions that mimic the natural in vivo environment. The focus was on the effects of sparsely ionizing cesium-137 gamma rays and the role of oxidative metabolism and intercellular communication in these effects. Four Specific Aims were proposed. The integrated outcome of the experiments performed to investigate these aims has been significant towards developing a scientific basis to more accurately estimate human health risks from exposures to low doses ionizing radiation. By understanding the biochemical and molecular changes induced by low dose radiation, several novel markers associated with mitochondrial functions were identified, which has opened new avenues to investigate metabolic processes that may be affected by such exposure. In particular, a sensitive biomarker that is differentially modulated by low and high dose gamma rays was discovered

Increased Frequency of Spontaneous Neoplastic Transformation in Progeny of Bystander Cells from Cultures Exposed to Densely Ionizing Radiation

An increased risk of carcinogenesis caused by exposure to space radiation during prolonged space travel is a limiting factor for human space exploration. Typically, astronauts are exposed to low fluences of ionizing particles that target only a few cells in a tissue at any one time. The propagation of stressful effects from irradiated to neighboring bystander cells and their transmission to progeny cells would be of importance in estimates of the health risks of exposure to space radiation. With relevance to the risk of carcinogenesis, we investigated, in model C3H 10TK mouse embryo fibroblasts (MEFs), modulation of the spontaneous frequency of neoplastic transformation in the progeny of bystander MEFs that had been in co-culture 10 population doublings earlier with MEFs exposed to moderate doses of densely ionizing iron ions (1 GeV/nucleon) or sparsely ionizing protons (1 GeV). An increase (P,0.05) in neoplastic transformation frequency, likely mediated by intercellular communication through gap junctions, was observed in the progeny of bystander cells that had been in cocultur