Modulation of immune responses using adjuvants to facilitate therapeutic vaccination.

Therapeutic vaccination offers great promise as an intervention for a diversity of infectious and non-infectious conditions. Given that most chronic health conditions are thought to have an immune component, vaccination can at least in principle be proposed as a therapeutic strategy. Understanding the nature of protective immunity is of vital importance, and the progress made in recent years in defining the nature of pathological and protective immunity for a range of diseases has provided an impetus to devise strategies to promote such responses in a targeted manner. However, in many cases, limited progress has been made in clinical adoption of such approaches. This in part results from a lack of safe and effective vaccine adjuvants that can be used to promote protective immunity and/or reduce deleterious immune responses. Although somewhat simplistic, it is possible to divide therapeutic vaccine approaches into those targeting conditions where antibody responses can mediate protection and those where the principal focus is the promotion of effector and memory cellular immunity or the reduction of damaging cellular immune responses as in the case of autoimmune diseases. Clearly, in all cases of antigen-specific immunotherapy, the identification of protective antigens is a vital first step. There are many challenges to developing therapeutic vaccines beyond those associated with prophylactic diseases including the ongoing immune responses in patients, patient heterogeneity, and diversity in the type and stage of disease. If reproducible biomarkers can be defined, these could allow earlier diagnosis and intervention and likely increase therapeutic vaccine efficacy. Current immunomodulatory approaches related to adoptive cell transfers or passive antibody therapy are showing great promise, but these are outside the scope of this review which will focus on the potential for adjuvanted therapeutic active vaccination strategies.

Hyperacetylation of Cardiac Mitochondrial Proteins Is Associated with Metabolic Impairment and Sirtuin Downregulation after Chronic Total Body Irradiation of ApoE -/- Mice.

Chronic exposure to low-dose ionizing radiation is associated with an increased risk of cardiovascular disease. Alteration in energy metabolism has been suggested to contribute to radiation-induced heart pathology, mitochondrial dysfunction being a hallmark of this disease. The goal of this study was to investigate the regulatory role of acetylation in heart mitochondria in the long-term response to chronic radiation. ApoE-deficient C57Bl/6J mice were exposed to low-dose-rate (20 mGy/day) gamma radiation for 300 days, resulting in a cumulative total body dose of 6.0 Gy. Heart mitochondria were isolated and analyzed using quantitative proteomics. Radiation-induced proteome and acetylome alterations were further validated using immunoblotting, enzyme activity assays, and ELISA. In total, 71 proteins showed peptides with a changed acetylation status following irradiation. The great majority (94%) of the hyperacetylated proteins were involved in the TCA cycle, fatty acid oxidation, oxidative stress response and sirtuin pathway. The elevated acetylation patterns coincided with reduced activity of mitochondrial sirtuins, increased the level of Acetyl-CoA, and were accompanied by inactivation of major cardiac metabolic regulators PGC-1 alpha and PPAR alpha. These observations suggest that the changes in mitochondrial acetylation after irradiation is associated with impairment of heart metabolism. We propose a novel mechanism involved in the development of late cardiac damage following chronic irradiation.

Total body exposure to low-dose ionizing radiation induces long-term alterations to the liver proteome of neonatally exposed mice.

Tens of thousands of people are being exposed daily to environmental low-dose gamma radiation. Epidemiological data indicate that such low radiation doses may negatively affect liver function and result in the development of liver disease. However, the biological mechanisms behind these adverse effects are unknown. The aim of this study was to investigate radiation-induced damage in the liver after low radiation doses. Neonatal male NMRI mice were exposed to total body irradiation on postnatal day 10 using acute single doses ranging from 0.02 to 1.0 Gy. Early (1 day) and late (7 months) changes in the liver proteome were tracked using isotope-coded protein label technology and quantitative mass spectrometry. Our data indicate that low and moderate radiation doses induce an immediate inhibition of the glycolysis pathway and pyruvate dehydrogenase availability in the liver. Furthermore, they lead to significant long-term alterations in lipid metabolism and increased liver inflammation accompanying inactivation of the transcription factor peroxisome proliferator-activated receptor alpha. This study contributes to the understanding of the potential risk of liver damage in populations environmentally exposed to ionizing radiation.

Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose rate gamma radiation.

Chronic low-dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low-dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation-induced senescence. Cellular proteins were quantified using isotope-coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence-related biological pathways were influenced by radiation, including cytoskeletal organization, cell-cell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation-induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low-dose irradiation.

PPAR alpha: a novel radiation target in locally exposed Mus musculus heart revealed by quantitative proteomics.

Radiation exposure of the thorax is associated with a markedly increased risk of cardiac morbidity and mortality with a latency period of decades. Although many studies have confirmed the damaging effect of ionizing radiation on the myocardium and cardiac endothelial structure and function, the molecular mechanism behind this damage is not yet elucidated. Peroxisome proliferator-activated receptor alpha (PPAR alpha), a transcriptional regulator of lipid metabolism in heart tissue, has recently received great attention in the development of cardiovascular disease. The goal of this study was to investigate radiation-induced cardiac damage in general and the role of PPAR alpha in this process in particular. C57BL/6 mice received local heart irradiation with X-ray doses of 8 and 16 gray (Gy) at the age of 8 weeks. The mice were sacrificed 16 weeks later. Radiation-induced changes in the cardiac proteome were quantified using the Isotope Coded Protein Label (ICPL) method followed by mass spectrometry and software analysis. Significant alterations were observed in proteins involved in lipid metabolism and oxidative phosphorylation. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPAR alpha. This was reflected as decreased expression of its target genes involved in energy metabolism and mitochondrial respiratory chain confirming the proteomics data. This study suggests that persistent alteration of cardiac metabolism due to impaired PPAR alpha activity contributes to the heart pathology after radiation.

Integrative proteomic and microRNA analysis of primary human coronary artery endothelial cells exposed to low-dose gamma radiation.

High doses of ionising radiation significantly increase the risk of cardiovascular disease (CVD), the vascular endothelium representing one of the main targets. Whether radiation doses lower than 500 mGy induce cardiovascular damage is controversial. The aim of this study was to investigate radiation-induced expression changes on protein and microRNA (miRNA) level in primary human coronary artery endothelial cells after a single 200 mGy radiation dose (Co-60). Using a multiplex gel-based proteomics technology (2D-DIGE), we identified 28 deregulated proteins showing more than ±1.5-fold expression change in comparison with non-exposed cells. A great majority of the proteins showed up-regulation. Bioinformatics analysis indicated "cellular assembly and organisation, cellular function and maintenance and molecular transport" as the most significant radiation-responsive network. Caspase-3, a central regulator of this network, was confirmed to be up-regulated using immunoblotting. We also analysed radiation-induced alterations in the level of six miRNAs known to play a role either in CVD or in radiation response. The expression of miR-21 and miR-146b showed significant radiation-induced deregulation. Using miRNA target prediction, three proteins found differentially expressed in this study were identified as putative candidates for miR-21 regulation. A negative correlation was observed between miR-21 levels and the predicted target proteins, desmoglein 1, phosphoglucomutase and target of Myb protein. This study shows for the first time that a low-dose exposure has a significant impact on miRNA expression that is directly related to protein expression alterations. The data presented here may facilitate the discovery of low-dose biomarkers of radiation-induced cardiovascular damage.

Long-term effects of acute low-dose ionizing radiation on the neonatal mouse heart: a proteomic study.

Epidemiological studies establish that children and young adults are especially susceptible to radiation-induced cardiovascular disease (CVD). The biological mechanisms behind the elevated CVD risk following exposure at young age remain unknown. The present study aims to elucidate the long-term effects of ionizing radiation by studying the murine cardiac proteome after exposure to low and moderate radiation doses. NMRI mice received single doses of total body (60)Co gamma-irradiation on postnatal day 10 and were sacrificed 7 months later. Changes in cardiac protein expression were quantified using isotope-coded protein label and tandem mass spectrometry. We identified 32, 31, 66, and 34 significantly deregulated proteins after doses of 0.02, 0.1, 0.5, and 1.0 Gy, respectively. The four doses shared 9 deregulated proteins. Bioinformatics analysis showed that most of the deregulated proteins belonged to a limited set of biological categories, including metabolic processes, inflammatory response, and cytoskeletal structure. The transcription factor peroxisome proliferator-activated receptor alpha was predicted as a common upstream regulator of several deregulated proteins. This study indicates that both adaptive and maladaptive responses to the initial radiation damage persist well into adulthood. It will contribute to the understanding of the long-term consequences of radiation-induced injury and developmental alterations in the neonatal heart.

Rapid proteomic remodeling of cardiac tissue caused by total body ionizing radiation.

Accidental nuclear scenarios lead to environmental contamination of unknown level. Immediate radiation-induced biological responses that trigger processes leading to adverse health effects decades later are not well understood. A comprehensive proteomic analysis provides a promising means to identify and quantify the initial damage after radiation exposure. Early changes in the cardiac tissue of C57BL/6 mice exposed to total body irradiation were studied, using a dose relevant to both intentional and accidental exposure (3 Gy gamma ray). Heart tissue protein lysates were analyzed 5 and 24 h after the exposure using isotope-coded protein labeling (ICPL) and 2-dimensional difference-in-gel-electrophoresis (2-D DIGE) proteomics approaches. The differentially expressed proteins were identified by LC-ESI-MS-MS. Both techniques showed similar functional groups of proteins to be involved in the initial injury. Pathway analyses indicated that total body irradiation immediately induced biological responses such as inflammation, antioxidative defense, and reorganization of structural proteins. Mitochondrial proteins represented the protein class most sensitive to ionizing radiation. The proteins involved in the initial damage processes map to several functional categories involving cardiotoxicity. This prompts us to propose that these early changes are indicative of the processes that lead to an increased risk of cardiovascular disease after radiation exposure.

Low-dose irradiation causes rapid alterations to the proteome of the human endothelial cell line EA.hy926.

High doses of ionising radiation damage the heart by an as yet unknown mechanism. A concern for radiological protection is the recent epidemiological data indicating that doses as low as 100-500 mGy may induce cardiac damage. The aim of this study was to identify potential molecular targets and/or mechanisms involved in the pathogenesis of low-dose radiation-induced cardiovascular disease. The vascular endothelium plays a pivotal role in the regulation of cardiac function and is therefore a potential target tissue. We report here that low-dose radiation induced rapid and time-dependent changes in the cytoplasmic proteome of the human endothelial cell line EA.hy926. The proteomes were investigated at 4 and 24 h after irradiation at two different dose rates (Co-60 gamma ray total dose 200 mGy; 20 mGy/min and 190 mGy/min) using 2D-DIGE technology. Differentially expressed proteins were identified, after in-gel trypsin digestion, by MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses. We identified 15 significantly differentially expressed proteins, of which 10 were up-regulated and 5 down-regulated, with more than ±1.5-fold difference compared with unexposed cells. Pathways influenced by the low-dose exposures included the Ran and RhoA pathways, fatty acid metabolism and stress response.

Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics.

Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the fixation process, proteins undergo degradation and cross-linking, making conventional protein analysis technologies problematic. In this study, we have compared several extraction and separation methods for the analysis of proteins in FFPE tissues. Incubation of tissue sections at high temperature with a novel extraction buffer (20 mM Tris-HCl, pH 8.8, 2% SDS, 1% beta-octylglucoside, 200 mM DTT, 200 mM glycine, and a mixture of protease inhibitors) resulted in improved protein recovery. Protein separation by 1-DE followed by LC-ESI MS/MS analysis was the most effective approach to identify proteins, based on the number of peptides reliably identified. Interestingly, a number of peptides were identified in regions of the 1DE not corresponding to their native molecular weights. This is an indication of the formation of protein-protein complexes by cross-linking, and of protein fragmentation due to prolonged sample storage. This study will facilitate the development of future proteomic analysis of FFPE tissue and provide a tool for the validation in archival samples of biomarkers of exposure, prognosis and disease.

Changes in the effective gravitational field strength affect the state of phosphorylation of stress-related proteins in callus cultures of Arabidopsis thaliana.

In a recent study it was shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly in amount (P <0.05) after 2 h of hypergravity (18 up-regulated, 10 down-regulated) could be identified. The corresponding proteins were largely involved in stress responses, including the detoxification of reactive oxygen species (ROS). In the present study, these investigations are extended to phosphorylated proteins. For this purpose, callus cell cultures of Arabidopsis thaliana were exposed to hypergravity (8 g) and simulated weightlessness (random positioning; RP) for up to 30 min, a period of time which yielded the most reliable data. The first changes, however, were visible as early as 10 min after the start of treatment. In comparison to 1 g controls, exposure to hypergravity resulted in 18 protein spots, and random positioning in 25, respectively, with increased/decreased signal intensity by at least 2-fold (P <0.05). Only one spot (alanine aminotransferase) responded the same way under both treatments. After 30 min of RP, four spots appeared, which could not be detected in control samples. Among the protein spots altered in phosphorylation, it was possible to identify 24 from those responding to random positioning and 12 which responded to 8 g. These 12 proteins (8 g) are partly (5 out of 12) the same as those changed in expression after exposure to 2 h of hypergravity. The respective proteins are involved in scavenging and detoxification of ROS (32%), primary metabolism (20.5%), general signalling (14.7%), protein translation and proteolysis (14.7%), and ion homeostasis (8.8%). Together with our recent data on protein expression, it is assumed that changes in gravitational fields induce the production of ROS. Our data further indicate that responses toward RP are more by post-translational protein modulation (most changes in the degree of phosphorylation occur under RP-treatment) than by protein expression (hypergravity).

Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures.

In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS-PAGE. Image analysis of Sypro Ruby-stained gels showed that approximately 28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning.

Low-dose radiation differentially regulates protein acetylation and histone deacetylase expression in human coronary artery endothelial cells.

PURPOSE: Ionizing radiation induces cardiovascular disease, the endothelium being the main target. The exact mechanism of the damage is unclear but the involvement of multiple signaling pathways is probable. Reversible lysine acetylation is a posttranslational protein modification that regulates activity across a broad range of signaling pathways. The aim of this study was to determine if a low radiation dose results in acetylome alteration in endothelial cells. MATERIALS AND METHODS: Human coronary artery endothelial cell line was irradiated with Cs-137 gamma-rays (0.5 Gy) and proteomics analysis was performed using enriched acetylated peptides and all peptides. Data were validated using immunoblotting, deacetylase activity assay, and RhoA activity assay. RESULTS: Nearly a hundred proteins were found to have an altered acetylation status 24 h after irradiation, primarily due to an overall decrease in acetylation. The expression of specific deacetylases was significantly increased, coinciding with an enhancement in global deacetylase activity. Proteins changed in their acetylation status belonged to several pathways including protein synthesis, cytoskeleton-related processes, protein folding and calcium signaling. The predicted changes in the RhoA/actin cytoskeleton pathway were validated by immunoassay. CONCLUSIONS: This study shows that protein acetylation is an important mediator of radiation responses in human cardiac coronary endothelial cells. Increased knowledge of the endothelial response to radiation is crucial for the development of normal tissue-sparing modalities during radiation therapy.

Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways.

PURPOSE: Epidemiological studies indicate that radiation doses as low as 0.5 Gy increase the risk of cardiovascular disease decades after the exposure. The aim of the present study was to investigate whether this radiation dose causes late molecular alterations in endothelial cells that could support the population-based data. MATERIALS AND METHODS: Human coronary artery endothelial cells were irradiated at 0.5 Gy (X-ray) and radiation-induced changes in the proteome were investigated after different time intervals (1, 7 and 14 d) using ICPL technology. Key changes identified by proteomics and bioinformatics were validated by immunoblotting and ELISA. RESULTS: The radiation-induced alteration of the endothelial proteome was characterized by sustained perturbation of Rho GDP-dissociation inhibitor (RhoGDI) and nitric oxide (NO) signalling pathways. At later time-points, this was accompanied by reduced proteasome activity, enhanced protein carbonylation indicating augmented oxidative stress, and senescence. CONCLUSIONS: These molecular changes are indicative of long-term premature endothelial dysfunction and provide a mechanistic framework to the epidemiological data showing increased risk of cardiovascular disease at 0.5 Gy.

A mechanistic model for atherosclerosis and its application to the cohort of Mayak workers.

We propose a stochastic model for use in epidemiological analysis, describing the age-dependent development of atherosclerosis with adequate simplification. The model features the uptake of monocytes into the arterial wall, their proliferation and transition into foam cells. The number of foam cells is assumed to determine the health risk for clinically relevant events such as stroke. In a simulation study, the model was checked against the age-dependent prevalence of atherosclerotic lesions. Next, the model was applied to incidence of atherosclerotic stroke in the cohort of male workers from the Mayak nuclear facility in the Southern Urals. It describes the data as well as standard epidemiological models. Based on goodness-of-fit criteria the risk factors smoking, hypertension and radiation exposure were tested for their effect on disease development. Hypertension was identified to affect disease progression mainly in the late stage of atherosclerosis. Fitting mechanistic models to incidence data allows to integrate biological evidence on disease progression into epidemiological studies. The mechanistic approach adds to an understanding of pathogenic processes, whereas standard epidemiological methods mainly explore the statistical association between risk factors and disease outcome. Due to a more comprehensive scientific foundation, risk estimates from mechanistic models can be deemed more reliable. To the best of our knowledge, such models are applied to epidemiological data on cardiovascular diseases for the first time.

Transcriptomic and proteomic analysis of mouse radiation-induced acute myeloid leukaemia (AML).

A combined transcriptome and proteome analysis of mouse radiation-induced AMLs using two primary AMLs, cell lines from these primaries, another cell line and its in vivo passage is reported. Compared to haematopoietic progenitor and stem cells (HPSC), over 5000 transcriptome alterations were identified, 2600 present in all materials. 55 and 3 alterations were detected in the proteomes of the cell lines and primary/in vivo passage material respectively, with one common to all materials. In cell lines, approximately 50% of the transcriptome changes are related to adaptation to cell culture, and in the proteome this proportion was higher. An AML 'signature' of 17 genes/proteins commonly deregulated in primary AMLs and cell lines compared to HPSCs was identified and validated using human AML transcriptome data. This also distinguishes primary AMLs from cell lines and includes proteins such as Coronin 1, pontin/RUVBL1 and Myeloperoxidase commonly implicated in human AML. C-Myc was identified as having a key role in radiation leukaemogenesis. These data identify novel candidates relevant to mouse radiation AML pathogenesis, and confirm that pathways of leukaemogenesis in the mouse and human share substantial commonality.

Chronic low-dose-rate ionising radiation affects the hippocampal phosphoproteome in the ApoE-/- Alzheimer's mouse model.

Accruing data indicate that radiation-induced consequences resemble pathologies of neurodegenerative diseases such as Alzheimer´s. The aim of this study was to elucidate the effect on hippocampus of chronic low-dose-rate radiation exposure (1 mGy/day or 20 mGy/day) given over 300 days with cumulative doses of 0.3 Gy and 6.0 Gy, respectively. ApoE deficient mutant C57Bl/6 mouse was used as an Alzheimer´s model. Using mass spectrometry, a marked alteration in the phosphoproteome was found at both dose rates. The radiation-induced changes in the phosphoproteome were associated with the control of synaptic plasticity, calcium-dependent signalling and brain metabolism. An inhibition of CREB signalling was found at both dose rates whereas Rac1-Cofilin signalling was found activated only at the lower dose rate. Similarly, the reduction in the number of activated microglia in the molecular layer of hippocampus that paralleled with reduced levels of TNFα expression and lipid peroxidation was significant only at the lower dose rate. Adult neurogenesis, investigated by Ki67, GFAP and NeuN staining, and cell death (activated caspase-3) were not influenced at any dose or dose rate. This study shows that several molecular targets induced by chronic low-dose-rate radiation overlap with those of Alzheimer´s pathology. It may suggest that ionising radiation functions as a contributing risk factor to this neurodegenerative disease.

Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells.

Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium.

Ionising radiation induces persistent alterations in the cardiac mitochondrial function of C57BL/6 mice 40 weeks after local heart exposure.

BACKGROUND AND PURPOSE: Radiotherapy of thoracic and chest-wall tumours increases the long-term risk of radiation-induced heart disease. The aim of this study was to investigate the long-term effect of local heart irradiation on cardiac mitochondria. METHODS: C57BL/6 and atherosclerosis-prone ApoE(-/-) mice received local heart irradiation with a single X-ray dose of 2 Gy. To investigate the low-dose effect, C57BL/6 mice also received a single heart dose of 0.2 Gy. Functional and proteomic alterations of cardiac mitochondria were evaluated after 40 weeks, compared to age-matched controls. RESULTS: The respiratory capacity of irradiated C57BL/6 cardiac mitochondria was significantly reduced at 40 weeks. In parallel, protein carbonylation was increased, suggesting enhanced oxidative stress. Considerable alterations were found in the levels of proteins of mitochondria-associated cytoskeleton, respiratory chain, ion transport and lipid metabolism. Radiation induced similar but less pronounced effects in the mitochondrial proteome of ApoE(-/-) mice. In ApoE(-/-), no significant change was observed in mitochondrial respiration or protein carbonylation. The dose of 0.2 Gy had no significant effects on cardiac mitochondria. CONCLUSION: This study suggests that ionising radiation causes non-transient alterations in cardiac mitochondria, resulting in oxidative stress that may ultimately lead to malfunctioning of the heart muscle.

Cell survival following radiation exposure requires miR-525-3p mediated suppression of ARRB1 and TXN1.

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.