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Telomeric-associated long non-coding RNAs TERRA and TERC can be quantified in culture cell lines and human PBMCs using peptide nucleic acid probes with flow cytometry (RNA-Flow).

Mesenchymal stem cells (MSCs) are multilineage adult stem cells with considerable potential for cell‐based regenerative therapies. In vitro expansion changes their epigenetic and cellular properties, with a poorly understood impact on DNA damage response (DDR) and genome stability. We report here results of a transcriptome‐based pathway analysis of in vitro‐expanded human bone marrow‐derived mesenchymal stem cell (hBM‐MSCs), supplemented with cellular assays focusing on DNA double‐strand break (DSB) repair. Gene pathways affected by in vitro aging were mapped using gene ontology, KEGG, and GSEA, and were found to involve DNA repair, homologous recombination (HR), cell cycle control, and chromosomal replication. Assays for the recognition (γ‐H2AX + 53BP1 foci) and repair (pBRCA1 + γ‐H2AX foci) of X‐ray‐induced DNA DSBs in hBM‐MSCs show that over a period of 8 weeks of in vitro aging (i.e., about 10 doubling times), cells exhibit a reduced DDR and a higher fraction of residual DNA damage. Furthermore, a distinct subpopulation of cells with impaired DNA DSB recognition was observed. Several genes that participate in DNA repair by HR (e.g., Rad51, Rad54, BRCA1) show a 2.3‐ to fourfold reduction of their mRNA expression by qRT‐PCR. We conclude that the in vitro expansion of hMSCs can lead to aging‐related impairment of the recognition and repair of DNA breaks.

Background and PurposeIncreased levels of the chaperone protein GRP78 have been implicated in poorer outcomes of cancer therapy. We have therefore explored the functional connection between the expression of GRP78 and the development of radioresistance and metastatic behavior in HNSCC.Material and MethodsThe association between gene expression of GRP78 and survival in HNSCC patients was examined using the TCGA database. The influence of ionizing radiation on the GRP78 levels in HNSCC cell lines, their secreted extracellular vesicles (EV) and non-irradiated EV-recipient cells was investigated by Western Blot and FACS. The consequences of chemical inhibition or experimental overexpression of GRP78 on radioresistance and migration of HNSCC cells were analyzed by clonogenic survival and gap closure assays.ResultsElevated levels of GRP78 RNA in HNSCC correlated with poorer overall survival. Radiation increased GRP78 protein expression on the surface of HNSCC cell lines. Experimental overexpression of GRP78 increased both radioresistance and migratory potential. Chemical inhibition of GRP78 impaired cell migration. EVs were identified as a potential source of increased GRP78 content as elevated levels of surface GRP78 were found in EVs released by irradiated cells. These vesicles transferred GRP78 to non-irradiated recipient cells during co-cultivation.ConclusionsWe have identified the chaperone protein GRP78 as a potential driver of increased radioresistance and motility in HNSCC. The uptake of GRP78-rich EVs originating from irradiated cells may contribute to a poorer prognosis through bystander effects mediated by the transfer of GRP78 to non-irradiated cells. Therefore, we consider the chaperone protein GRP78 to be an attractive target for improving radiotherapy strategies.

Abstract Background Mesenchymal stem cells (MSCs) are attracting increasing interest for cell-based therapies, making use of both their immuno-modulating and regenerative potential. For such therapeutic applications, a massive in vitro expansion of donor cells is usually necessary to furnish sufficient material for transplantation. It is not established to what extent the long-term genomic stability and potency of MSCs can be compromised as a result of this rapid ex vivo expansion. In this study, we investigated the DNA damage response and chromosomal stability (indicated by micronuclei induction) after sub-lethal doses of gamma irradiation in murine MSCs at different stages of their in vitro expansion. Methods Bone-marrow-derived tri-potent MSCs were explanted from 3-month-old female FVB/N mice and expanded in vitro for up to 12 weeks. DNA damage response and repair kinetics after gamma irradiation were quantified by the induction of γH2AX/53BP1 DSB repair foci. Micronuclei were counted in post-mitotic, binucleated cells using an automated image analyzer Metafer4. Involvement of DNA damage response pathways was tested using chemical ATM and DNA-PK inhibitors. Results Murine bone-marrow-derived MSCs in long-term expansion culture gradually lose their ability to recognize endogenous and radiation-induced DNA double-strand breaks. This impaired DNA damage response, indicated by a decrease in the number of γH2AX/53BP1 DSB repair foci, was associated with reduced ATM dependency of foci formation, a slower DNA repair kinetics, and an increased number of residual DNA double-strand breaks 7 h post irradiation. In parallel with this impaired efficiency of DNA break recognition and repair in older MSCs, chromosomal instability after mitosis increased significantly as shown by a higher number of micronuclei, both spontaneously and induced by γ-irradiation. Multifactorial regression analysis demonstrates that in vitro aging reduced DNA damage recognition in MSCs after irradiation by a multiplicative interaction with dose (p

Background and Purpose: Cardiotoxicity is a well-known adverse effect of radiation therapy. Measurable abnormalities in the heart function indicate advanced and often irreversible heart damage. Therefore, early detection of cardiac toxicity is necessary to delay and alleviate the development of the disease. The present study investigated long-term serum proteome alterations following local heart irradiation using a mouse model with the aim to detect biomarkers of radiation-induced cardiac toxicity.Materials and Methods: Serum samples from C57BL/6J mice were collected 20 weeks after local heart irradiation with 8 or 16 Gy X-ray; the controls were sham-irradiated. The samples were analyzed by quantitative proteomics based on data-independent acquisition mass spectrometry. The proteomics data were further investigated using bioinformatics and ELISA.Results: The analysis showed radiation-induced changes in the level of several serum proteins involved in the acute phase response, inflammation, and cholesterol metabolism. We found significantly enhanced expression of proinflammatory cytokines (TNF-α, TGF-β, IL-1, and IL-6) in the serum of the irradiated mice. The level of free fatty acids, total cholesterol, low-density lipoprotein (LDL), and oxidized LDL was increased, whereas that of high-density lipoprotein was decreased by irradiation.Conclusions: This study provides information on systemic effects of heart irradiation. It elucidates a radiation fingerprint in the serum that may be used to elucidate adverse cardiac effects after radiation therapy.

Glioblastoma stem-like cells (GSLCs) in glioblastoma limit effective treatment and promote therapeutic resistance and tumor recurrence. Using a combined radiation and drug-screening platform, we tested the combination of a histone deacetylase inhibitor (HDACi) and MAPK/ERK kinase inhibitor (MEKi) with radiation to predict the efficacy against GSLCs. To mimic a stem-like phenotype, glioblastoma-derived spheres were used and treated with a combination of HDACi (MS-275) and MEKi (TAK-733 or trametinib) with 4 Gy irradiation. The sphere-forming ability after the combined radiochemotherapy was investigated using a sphere formation assay, while the expression levels of the GSLC markers (CD44, Nestin and SOX2) after treatment were analyzed using Western blotting and flow cytometry. The combined radiochemotherapy treatment inhibited the sphere formation in both glioblastoma-derived spheres, decreased the expression of the GSLC markers in a cell-line dependent manner and increased the dead cell population. Finally, we showed that the combined treatment with radiation was more effective at reducing the GSLC markers compared to the standard treatment of temozolomide and radiation. These results suggest that combining HDAC and MEK inhibition with radiation may offer a new strategy to improve the treatment of glioblastoma.

Radiation-induced cardiovascular disease is associated with metabolic remodeling in the heart, mainly due to the inactivation of the transcription factor peroxisome proliferator-activated receptor alpha (PPARα), thereby inhibiting lipid metabolic enzymes. The objective of the present study was to investigate the potential protective effect of fenofibrate, a known agonist of PPARα on radiation-induced cardiac toxicity. To this end, we compared, for the first time, the cardiac proteome of fenofibrate- and placebo-treated mice 20 weeks after local heart irradiation (16 Gy) using label-free proteomics. The observations were further validated using immunoblotting, enzyme activity assays, and ELISA. The analysis showed that fenofibrate restored signalling pathways that were negatively affected by irradiation, including lipid metabolism, mitochondrial respiratory chain, redox response, tissue homeostasis, endothelial NO signalling and the inflammatory status. The results presented here indicate that PPARα activation by fenofibrate attenuates the cardiac proteome alterations induced by irradiation. These findings suggest a potential benefit of fenofibrate administration in the prevention of cardiovascular diseases, following radiation exposure.

Radiation-induced inflammation leading to the permeability of the endothelial barrier may increase the risk of cardiovascular disease. The aim of this study was to investigate potential mechanisms in vitro at the level of the proteome in human coronary artery endothelial cells (HCECest2) that were exposed to radiation doses of 0, 0.25, 0.5, 2.0 and 10 Gy (60Co-γ). Proteomics analysis was performed using mass spectrometry in a label-free data-independent acquisition mode. The data were validated using bioinformatics and immunoblotting. The low- and moderate-dose-irradiated samples (0.25 Gy, 0.5 Gy) showed only scarce proteome changes. In contrast, an activation of DNA-damage repair, inflammation, and oxidative stress pathways was seen after the high-dose treatments (2 and 10 Gy). The level of the DNA damage response protein DDB2 was enhanced early at the 10 Gy dose. The expression of proteins belonging to the inflammatory response or cGAS-STING pathway (STING, STAT1, ICAM1, ISG15) increased in a dose-dependent manner, showing the strongest effects at 10 Gy after one week. This study suggests a connection between the radiation-induced DNA damage and the induction of inflammation which supports the inhibition of the cGAS-STING pathway in the prevention of radiation-induced cardiovascular disease.

Radiation biology is a highly interdisciplinary field at the interface of biology, physics, and medicine. It is characterized by rapid advances in biological and technical knowledge. The potential for using these advances to optimize medical care, radiation protection, and related fields can be exploited only with complementary activities to support the education of young academics. A small number of academic institutions have committed resources into radiation-related courses and curricula; however, few offer a comprehensive interdepartmental research and training program. At the Technical University of Munich (TUM), a full Master of Science (MSc) course in radiation biology has been established. This article describes the TUM MSc radiation biology program, discusses the scope of the field, the teaching goals, and the interdisciplinary curriculum. Detailed information on the full MSc program can be found continuously updated at www.radonc.med.tum.de/masterradiationbiology.

Prenatal exposure to stress such as increased level of reactive oxygen species or antiviral therapy are known factors leading to adult heart defects. The risks following a radiation exposure during fetal period are unknown, as are the mechanisms of any potential cardiac damage. The aim of this study was to gather evidence for possible damage by investigating long-term changes in the mouse heart proteome after prenatal exposure to low and moderate radiation doses. Pregnant C57Bl/6J mice received on embryonic day 11 (E11) a single total body dose of ionizing radiation that ranged from 0.02 Gy to 1.0 Gy. The offspring were sacrificed at the age of 6 months or 2 years. Quantitative proteomic analysis of heart tissue was performed using Isotope Coded Protein Label technology and tandem mass spectrometry. The proteomics data were analyzed by bioinformatics and key changes were validated by immunoblotting. Persistent changes were observed in the expression of proteins representing mitochondrial respiratory complexes, redox and heat shock response, and the cytoskeleton, even at the low dose of 0.1 Gy. The level of total and active form of the kinase MAP4K4 that is essential for the embryonic development of mouse heart was persistently decreased at the radiation dose of 1.0 Gy. This study provides the first insight into the molecular mechanisms of cardiac impairment induced by ionizing radiation exposure during the prenatal period.

Mesenchymal stem cells (MSCs) are a source of adult multipotent cells important in tissue regeneration. Murine MSCs are known to proliferate poorly in vitro under normoxia. The aim of this study is to analyze the interaction of nonphysiological high oxygen and low-dose γ-irradiation onto growth, senescence, and DNA damage. Tri-potent bone marrow-derived MSCs from p53 wildtype and p53−/− mice were cultured under either 21% or 2% O2. Long-term observations revealed a decreasing ability of wildtype mMSCs to proliferate and form colonies under extended culture in normoxia. This was accompanied by increased senescence under normoxia but not associated with telomere shortening. After low-dose γ-irradiation, the normoxic wildtype cells further increased the level of senescence. The number of radiation-induced γH2AX DNA repair foci was higher in mMSCs kept under normoxia but not in p53−/− cells. P53-deficient MSCs additionally showed higher clonogeneity, lower senescence levels, and fewer γH2AX repair foci per cell as compared to their p53 wildtype counterparts irrespective of oxygen levels. These results reveal that oxygen levels together with γ-irradiation and p53 status are interconnected factors modulating growth capacity of BM MSCs in long-term culture. These efforts help to better understand and optimize handling of MSCs prior to their therapeutic use.

Patients suffering from brain malignancies are treated with high-dose ionising radiation. However, this may lead to severe learning and memory impairment. Preventive treatments to minimise these side effects have not been possible due to the lack of knowledge of the involved signalling pathways and molecular targets. Mouse hippocampal neuronal HT22 cells were irradiated with acute gamma doses of 0.5 Gy, 1.0 Gy and 4.0 Gy. Changes in the cellular proteome were investigated by isotope-coded protein label technology and tandem mass spectrometry after 4 and 24 hours. To compare the findings with the in vivo response, male NMRI mice were irradiated on postnatal day 10 with a gamma dose of 1.0 Gy, followed by evaluation of the cellular proteome of hippocampus and cortex 24 hours post-irradiation. Analysis of the in vitro proteome showed that signalling pathways related to synaptic actin-remodelling were significantly affected at 1.0 Gy and 4.0 Gy but not at 0.5 Gy after 4 and 24 hours. We observed radiation-induced reduction of the miR-132 and Rac1 levels; miR-132 is known to regulate Rac1 activity by blocking the GTPase-activating protein p250GAP. In the irradiated hippocampus and cortex we observed alterations in the signalling pathways similar to those in vitro. The decreased expression of miR-132 and Rac1 was associated with an increase in hippocampal cofilin and phospho-cofilin. The Rac1-Cofilin pathway is involved in the modulation of synaptic actin filament formation that is necessary for correct spine and synapse morphology to enable processes of learning and memory. We suggest that acute radiation exposure leads to rapid dendritic spine and synapse morphology alterations via aberrant cytoskeletal signalling and processing and that this is associated with the immediate neurocognitive side effects observed in patients treated with ionising radiation.

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.

MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis.

BACKGROUND:microRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer. PRINCIPAL FINDINGS:Expression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity. CONCLUSION:The transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.

BackgroundRadiation therapy treatment of breast cancer, Hodgkin's disease or childhood cancers expose the heart to high local radiation doses, causing an increased risk of cardiovascular disease in the survivors decades after the treatment. The mechanisms that underlie the radiation damage remain poorly understood so far. Previous data show that impairment of mitochondrial oxidative metabolism is directly linked to the development of cardiovascular disease.Methodology/principal findingsIn this study, the radiation-induced in vivo effects on cardiac mitochondrial proteome and function were investigated. C57BL/6N mice were exposed to local irradiation of the heart with doses of 0.2 Gy or 2 Gy (X-ray, 200 kV) at the age of eight weeks, the control mice were sham-irradiated. After four weeks the cardiac mitochondria were isolated and tested for proteomic and functional alterations. Two complementary proteomics approaches using both peptide and protein quantification strategies showed radiation-induced deregulation of 25 proteins in total. Three main biological categories were affected: the oxidative phophorylation, the pyruvate metabolism, and the cytoskeletal structure. The mitochondria exposed to high-dose irradiation showed functional impairment reflected as partial deactivation of Complex I (32%) and Complex III (11%), decreased succinate-driven respiratory capacity (13%), increased level of reactive oxygen species and enhanced oxidation of mitochondrial proteins. The changes in the pyruvate metabolism and structural proteins were seen with both low and high radiation doses.Conclusion/significanceThis is the first study showing the biological alterations in the murine heart mitochondria several weeks after the exposure to low- and high-dose of ionizing radiation. Our results show that doses, equivalent to a single dose in radiotherapy, cause long-lasting changes in mitochondrial oxidative metabolism and mitochondria-associated cytoskeleton. This prompts us to propose that these first pathological changes lead to an increased risk of cardiovascular disease after radiation exposure.