Therapeutic Fractional Doses of Ionizing Radiation Promote Epithelial-Mesenchymal Transition, Enhanced Invasiveness, and Altered Glycosylation in MCF-7 Breast Cancer Cells.

The clinical outcome of radiation therapy is restricted due to the acquired radio-resistance of a subpopulation of tumour cells that may cause tumour relapse and distant metastasis. While the effects of ionizing radiation (IR) such as DNA damage and cell stress are well-documented, the potential role of IR in inducing invasive potential in cancer cells has not been broadly studied, therefore we aimed to investigate it in this study. MCF-7 cells irradiated with 0 Gy (control) or 2 Gy X-ray therapeutic doses of IR were assessed for cell viability, percentage of apoptotic cells, and reactive oxygen species (ROS) levels, DNA fragmentation, Matrigel invasion, assessment of epithelial-mesenchymal transition (EMT) markers and Helix pomatia agglutinin (HPA) binding at 30 min, 4- or 24-h post-IR. Reduction in cell viability, increase in apoptotic cells, ROS positive cells, and DNA fragmentation were observed, while functional invasiveness and EMT were exacerbated together with altered glycosylation in MCF-7 cells irradiated with 2 Gy X-ray compared to control cells. These findings indicate that despite the detrimental effects of 2 Gy X-ray IR on MCF-7 cells, a subpopulation of cells may have gained increased invasive potential. The exacerbated invasive potential may be attributed to enhanced EMT and altered glycosylation. Moreover, deregulation of transforming growth factor-beta (TGF-ß) following IR may be one of the elements responsible for these changes, as it lies in the intersection of these invasion-promoting cell processes.

MiRNA-Mediated Fibrosis in the Out-of-Target Heart following Partial-Body Irradiation.

Recent reports have shown a link between radiation exposure and non-cancer diseases such as radiation-induced heart disease (RIHD). Radiation exposures are often inhomogeneous, and out-of-target effects have been studied in terms of cancer risk, but very few studies have been carried out for non-cancer diseases. Here, the role of miRNAs in the pathogenesis of RIHD was investigated. C57Bl/6J female mice were whole- (WBI) or partial-body-irradiated (PBI) with 2 Gy of X-rays or sham-irradiated (SI). In PBI exposure, the lower third of the mouse body was irradiated, while the upper two-thirds were shielded. From all groups, hearts were collected 15 days or 6 months post-irradiation. The MiRNome analysis at 15 days post-irradiation showed that miRNAs, belonging to the myomiR family, were highly differentially expressed in WBI and PBI mouse hearts compared with SI hearts. Raman spectral data collected 15 days and 6 months post-irradiation showed biochemical differences among SI, WBI and PBI mouse hearts. Fibrosis in WBI and PBI mouse hearts, indicated by the increased deposition of collagen and the overexpression of genes involved in myofibroblast activation, was found 6 months post-irradiation. Using an in vitro co-culture system, involving directly irradiated skeletal muscle and unirradiated ventricular cardiac human cells, we propose the role of miR-1/133a as mediators of the abscopal response, suggesting that miRNA-based strategies could be relevant for limiting tissue-dependent reactions in non-directly irradiated tissues.

Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum.

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.

Early Responses to Low-Dose Ionizing Radiation in Cellular Lens Epithelial Models.

Cataract is the leading cause of visual impairment which can result in blindness. Cataract formation has been associated with radiation exposure; however, the mechanistic understanding of this phenomenon is still lacking. The goal of this study was to investigate mechanisms of cataract induction in isolated lens epithelial cells (LEC) exposed to ionizing radiation. Human LECs from different genetic backgrounds (SV40 immortalized HLE-B3 and primary HLEC cells) were exposed to varying doses of 137Cs gamma rays (0, 0.1, 0.25 and 0.5 Gy), at low (0.065 Gy/min) and higher (0.3 Gy/min) dose rates. Different assays were used to measure LEC response for, e.g., viability, oxidative stress, DNA damage studies, senescence and changes to telomere length/telomerase activity at two time points (1 h and 24 h, or 24 h and 15 days, depending on the type of assay and expected response time). The viability of cells decreased in a dose-dependent manner within 24 h of irradiation. Measurement of reactive oxygen species showed an increase at 1 h postirradiation, which was alleviated within 24 h. This was consistent with DNA damage results showing high DNA damage after 1 h postirradiation which reduced significantly (but not completely) within 24 h. Induction of senescence was also observed 15 days postirradiation, but this was not attributed to telomere erosion or telomerase activity reduction. Overall, these findings provide a mechanistic understanding of low-dose radiation-induced cataractogenesis which will ultimately help to inform judgements on the magnitude of risk and improve existing radiation protection procedures.

Non-targeted effects of radiation: a personal perspective on the role of exosomes in an evolving paradigm.

PURPOSE: Radiation-induced non-targeted effects (NTE) have implications in a variety of areas relevant to radiation biology. Here we evaluate the various cargo associated with exosomal signaling and how they work synergistically to initiate and propagate the non-targeted effects including genomic instability and bystander effects. CONCLUSIONS: Extra cellular vesicles, in particular exosomes, have been shown to carry bystander signals. Exosome cargo may contain nucleic acids, both DNA and RNA, as well as proteins, lipids, and metabolites. These cargo molecules have all been considered as potential mediators of NTE. A review of current literature shows mounting evidence of a role for ionizing radiation in modulating both the numbers of exosomes released from affected cells as well as the content of their cargo, and that these exosomes can instigate functional changes in recipient cells. However, there are significant gaps in our understanding, particularly regarding modified exosome cargo after radiation exposure and the functional changes induced in recipient cells.

Introduction to the Special LDLensRad Focus Issue.

Recent epidemiological and experimental animal data, as well as reanalyses of data previously accumulated, indicate that the lens of the eye is more radiosensitive than was previously thought. This has resulted in a reduction of the occupational lens dose limit within the European Union countries, Japan and elsewhere. This Commentary introduces the work done by the LDLensRad Consortium contained within this Focus Issue, towards advancement of understanding of the mechanisms of low dose radiation cataract.

Ionising Radiation Promotes Invasive Potential of Breast Cancer Cells: The Role of Exosomes in the Process.

Along with the cells that are exposed to radiation, non-irradiated cells can unveil radiation effects as a result of intercellular communication, which are collectively defined as radiation induced bystander effects (RIBE). Exosome-mediated signalling is one of the core mechanisms responsible for multidirectional communication of tumor cells and their associated microenvironment, which may result in enhancement of malignant tumor phenotypes. Recent studies show that exosomes and exosome-mediated signalling also play a dynamic role in RIBE in cancer cell lines, many of which focused on altered exosome cargo or their effects on DNA damage. However, there is a lack of knowledge regarding how these changes in exosome cargo are reflected in other functional characteristics of cancer cells from the aspects of invasiveness and metastasis. Therefore, in the current study, we aimed to investigate exosome-mediated bystander effects of 2 Gy X-ray therapeutic dose of ionizing radiation on the invasive potential of MCF-7 breast cancer cells in vitro via assessing Matrigel invasion potential, epithelial mesenchymal transition (EMT) characteristics and the extent of glycosylation, as well as underlying plausible molecular mechanisms. The findings show that exosomes derived from irradiated MCF-7 cells enhance invasiveness of bystander MCF-7 cells, possibly through altered miRNA and protein content carried in exosomes.

Out-of-Field Hippocampus from Partial-Body Irradiated Mice Displays Changes in Multi-Omics Profile and Defects in Neurogenesis.

The brain undergoes ionizing radiation exposure in many clinical situations, particularly during radiotherapy for brain tumors. The critical role of the hippocampus in the pathogenesis of radiation-induced neurocognitive dysfunction is well recognized. The goal of this study is to test the potential contribution of non-targeted effects in the detrimental response of the hippocampus to irradiation and to elucidate the mechanisms involved. C57Bl/6 mice were whole body (WBI) or partial body (PBI) irradiated with 0.1 or 2.0 Gy of X-rays or sham irradiated. PBI consisted of the exposure of the lower third of the mouse body, whilst the upper two thirds were shielded. Hippocampi were collected 15 days or 6 months post-irradiation and a multi-omics approach was adopted to assess the molecular changes in non-coding RNAs, proteins and metabolic levels, as well as histological changes in the rate of hippocampal neurogenesis. Notably, at 2.0 Gy the pattern of early molecular and histopathological changes induced in the hippocampus at 15 days following PBI were similar in quality and quantity to the effects induced by WBI, thus providing a proof of principle of the existence of out-of-target radiation response in the hippocampus of conventional mice. We detected major alterations in DAG/IP3 and TGF-ß signaling pathways as well as in the expression of proteins involved in the regulation of long-term neuronal synaptic plasticity and synapse organization, coupled with defects in neural stem cells self-renewal in the hippocampal dentate gyrus. However, compared to the persistence of the WBI effects, most of the PBI effects were only transient and tended to decrease at 6 months post-irradiation, indicating important mechanistic difference. On the contrary, at low dose we identified a progressive accumulation of molecular defects that tended to manifest at later post-irradiation times. These data, indicating that both targeted and non-targeted radiation effects might contribute to the pathogenesis of hippocampal radiation-damage, have general implications for human health.

Induction of Genomic Instability in a Primary Human Fibroblast Cell Line Following Low-Dose Alpha-Particle Exposure and the Potential Role of Exosomes.

PURPOSE: To study the induction of genomic instability (GI) in the progeny of cell populations irradiated with low doses of alpha-particles and the potential role of exosome-encapsulated bystander signalling. METHODS: The induction of GI in HF19 normal fibroblast cells was assessed by determining the formation of micronuclei (MN) in binucleate cells along with using the alkaline comet assay to assess DNA damage. RESULTS: Low dose alpha-particle exposure (0.0001-1 Gy) was observed to produce a significant induction of micronuclei and DNA damage shortly after irradiation (assays performed at 5 and 1 h post exposure, respectively). This damage was not only still evident and statistically significant in all irradiated groups after 10 population doublings, but similar trends were observed after 20 population doublings. Exosomes from irradiated cells were also observed to enhance the level of DNA damage in non-irradiated bystander cells at early times. CONCLUSION: very low doses of alpha-particles are capable of inducing GI in the progeny of irradiated cells even at doses where <1% of the cells are traversed, where the level of response was similar to that observed at doses where 100% of the cells were traversed. This may have important implications with respect to the evaluation of cancer risk associated with very low-dose alpha-particle exposure and deviation from a linear dose response.

Phenotypic and Functional Characteristics of Exosomes Derived from Irradiated Mouse Organs and Their Role in the Mechanisms Driving Non-Targeted Effects.

Molecular communication between irradiated and unirradiated neighbouring cells initiates radiation-induced bystander effects (RIBE) and out-of-field (abscopal) effects which are both an example of the non-targeted effects (NTE) of ionising radiation (IR). Exosomes are small membrane vesicles of endosomal origin and newly identified mediators of NTE. Although exosome-mediated changes are well documented in radiation therapy and oncology, there is a lack of knowledge regarding the role of exosomes derived from inside and outside the radiation field in the early and delayed induction of NTE following IR. Therefore, here we investigated the changes in exosome profile and the role of exosomes as possible molecular signalling mediators of radiation damage. Exosomes derived from organs of whole body irradiated (WBI) or partial body irradiated (PBI) mice after 24 h and 15 days post-irradiation were transferred to recipient mouse embryonic fibroblast (MEF) cells and changes in cellular viability, DNA damage and calcium, reactive oxygen species and nitric oxide signalling were evaluated compared to that of MEF cells treated with exosomes derived from unirradiated mice. Taken together, our results show that whole and partial-body irradiation increases the number of exosomes, instigating changes in exosome-treated MEF cells, depending on the source organ and time after exposure.

Radiation-Induced Senescence Bystander Effect: The Role of Exosomes.

Ionizing Radiation (IR), especially at high doses, induces cellular senescence in exposed cultures. IR also induces "bystander effects" through signals released from irradiated cells, and these effects include many of the same outcomes observed following direct exposure. Here, we investigate if radiation can cause senescence through a bystander mechanism. Control cultures were exposed directly to 0, 0.1, 2, and 10 Gy. Unirradiated cells were treated with medium from irradiated cultures or with exosomes extracted from irradiated medium. The level of senescence was determined post-treatment (24 h, 15 days, 30 days, and 45 days) by ß-galactosidase staining. Media from cultures exposed to all four doses, and exosomes from these cultures, induced significant senescence in recipient cultures. Senescence levels were initially low at the earliest timepoint, and peaked at 15 days, and then decreased with further passaging. These results demonstrate that senescence is inducible through a bystander mechanism. As with other bystander effects, bystander senescence was induced by a low radiation dose. However, unlike other bystander effects, cultures recovered from bystander senescence after repeated passaging. Bystander senescence may be a potentially significant effect of exposure to IR, and may have both beneficial and harmful effects in the context of radiotherapy.

Does Direct and Indirect Exposure to Ionising Radiation Influence the Metastatic Potential of Breast Cancer Cells.

Ionising radiation (IR) is commonly used for cancer therapy; however, its potential influence on the metastatic ability of surviving cancer cells exposed directly or indirectly to IR remains controversial. Metastasis is a multistep process by which the cancer cells dissociate from the initial site, invade, travel through the blood stream or lymphatic system, and colonise distant sites. This complex process has been reported to require cancer cells to undergo epithelial-mesenchymal transition (EMT) by which the cancer cells convert from an adhesive, epithelial to motile, mesenchymal form and is also associated with changes in glycosylation of cell surface proteins, which may be functionally involved in metastasis. In this paper, we give an overview of metastatic mechanisms and of the fundamentals of cancer-associated glycosylation changes. While not attempting a comprehensive review of this wide and fast moving field, we highlight some of the accumulating evidence from in vitro and in vivo models for increased metastatic potential in cancer cells that survive IR, focusing on angiogenesis, cancer cell motility, invasion, and EMT and glycosylation. We also explore the indirect effects in cells exposed to exosomes released from irradiated cells. The results of such studies need to be interpreted with caution and there remains limited evidence that radiotherapy enhances the metastatic capacity of cancers in a clinical setting and undoubtedly has a very positive clinical benefit. However, there is potential that this therapeutic benefit may ultimately be enhanced through a better understanding of the direct and indirect effects of IR on cancer cell behaviour.

Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.

Radiation-induced Chromosome Instability: The Role of Dose and Dose Rate.

Nontargeted effects include radiation-induced genomic instability (RIGI) which is observed in the progeny of cells exposed to ionizing radiation and can be manifested in different ways, including chromosomal instability and micronucleus (MN) formation. Since genomic instability is commonly observed in tumors and has a role in tumor progression, RIGI has the potential of being an important mechanism for radiation-induced cancer. The work presented explores the role of dose and dose rate on RIGI, determined using a MN assay, in normal primary human fibroblast (HF19) cells exposed to either 0.1 Gy or 1 Gy of X-rays delivered either as an acute (0.42 Gy/min) or protracted (0.0031 Gy/min) exposure. While the expected increase in MN was observed following the first mitosis of the irradiated cells compared to unirradiated controls, the results also demonstrate a significant increase in MN yields in the progeny of these cells at 10 and 20 population doublings following irradiation. Minimal difference was observed between the two doses used (0.1 and 1 Gy) and the dose rates (acute and protracted). Therefore, these nontargeted effects have the potential to be important for the low-dose and dose-rate exposure. The results also show an enhancement of the cellular levels of reactive oxygen species after 20 population doublings, which suggests that ionising radiation (IR) could potentially perturb the homeostasis of oxidative stress and so modify the background rate of endogenous DNA damage induction. In conclusion, the investigations have demonstrated that normal primary human fibroblast (HF19) cells are susceptible to the induction of early DNA damage and RIGI, not only after a high dose and high dose rate exposure to low linear energy transfer, but also following low dose, low dose rate exposures. The results suggest that the mechanism of radiation induced RIGI in HF19 cells can be correlated with the induction of reactive oxygen species levels following exposure to 0.1 and 1 Gy low-dose rate and high-dose rate x-ray irradiation.

Effects of ionizing radiation on telomere length and telomerase activity in cultured human lens epithelium cells.

PURPOSE: To investigate the effects of ionizing radiation on telomere length and telomerase activity in human lens epithelial cells. There are studies suggesting evidence of telomere length in association with opacity of the lens; however, these studies have been conducted on Canine Lens cells. Our study was designed to understand further the effects of different doses of ionizing radiation on telomere length and telomerase activity in cultured human lens epithelium cells from three Donors. MATERIALS AND METHODS: For this study, embryonic human lens epithelial (HLE) cells from three donors, obtained commercially were cultured. Telomere length and telomerase activity were measured after each passage until cells stopped growing in culture. This was repeated on irradiated (0.001 Gy, 0.01 Gy, 0.02 Gy, 0.1 Gy, 1 Gy and 2 Gy) cells. DNA damage response using the H2AX and telomere dysfunction foci assays were also examined at 30 mins, 24 hours, 48 hours and 72 hours postirradiation. RESULTS AND CONCLUSION: We have demonstrated genetic changes in telomere length and oxidative stress, which may be relevant to cataractogenesis. Our study shows that in control cells telomere length increases as passage increases. We have also demonstrated that telomere length increases at higher doses of 1.0 Gy and 2.0 Gy. However, telomerase activity decreases dose dependently and as passages increase. These results are not conclusive and further studies ex vivo measuring lens opacity and telomere length in the model would be beneficial in a bigger cohort, hence confirming a link between telomere length, cataractogenesis and genetic factors.

The future impacts of non-targeted effects.

Ionizing radiation was traditionally thought to exert its detrimental effects through interaction with sensitive cellular targets, nuclear DNA being of most importance. This theory has since merged with a more recently described radiation response called non-targeted effects (NTE). This review will briefly look at the various types of NTE and the potential implications they may have for radiobiology research and its applications. The most well-known NTE are genomic instability (GI) and bystander effects (BE). Other NTE include abscopal effects, which are similar to bystander effects but are generally based in a clinical environment with immune involvement as the defining feature. Currently, our understanding of NTE is limited to certain signaling pathways/molecules, and as yet there is no theory that describes or can accurately predict the occurrence or outcome of these NTE. There are numerous groups investigating these processes in vitro and in vivo, and thus steady progress is being made. Developing a deeper understanding of NTE has potential impacts for therapy and diagnosis, safer occupational exposures, space flight and our general understanding of radiation biology.

When a duck is not a duck; a new interdisciplinary synthesis for environmental radiation protection.

This consensus paper presents the results of a workshop held in Essen, Germany in September 2017, called to examine critically the current approach to radiological environmental protection. The meeting brought together participants from the field of low dose radiobiology and those working in radioecology. Both groups have a common aim of identifying radiation exposures and protecting populations and individuals from harmful effects of ionising radiation exposure, but rarely work closely together. A key question in radiobiology is to understand mechanisms triggered by low doses or dose rates, leading to adverse outcomes of individuals while in radioecology a key objective is to recognise when harm is occurring at the level of the ecosystem. The discussion provided a total of six strategic recommendations which would help to address these questions.

Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

Extracellular Vesicles Mediate Radiation-Induced Systemic Bystander Signals in the Bone Marrow and Spleen.

Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EVs) in mediating radiation-induced bystander signals, but in vivo investigations are still lacking. Here, we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), and 24 h later, EVs were isolated from the bone marrow (BM) and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similar to direct radiation, EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of eight differentially expressed microRNAs (miRNA) was identified in the EVs originating from both low- and high-dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic, and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects, and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms.

Exosome-Mediated Telomere Instability in Human Breast Epithelial Cancer Cells after X Irradiation.

In directly irradiating cells, telomere metabolism is altered and similar effects have been observed in nontargeted cells. Exosomes and their cargo play dominant roles in communicating radiation-induced bystander effects with end points related to DNA damage. Here we report novel evidence that exosomes are also responsible for inducing telomere-related bystander effects. Breast epithelial cancer cells were exposed to either 2 Gy X rays, or exposed to irradiated cell conditioned media (ICCM), or exosomes purified from ICCM. Compared to control cells, telomerase activity decreased in the 2 Gy irradiated cells and both bystander samples after one population doubling. At the first population doubling, telomere length was shorter in the 2 Gy irradiated sample but not in the bystander samples. By 24 population doublings telomerase activity recovered to control levels in all samples; however, the 2 Gy irradiated sample continued to demonstrate short telomeres and both bystander samples acquired shorter telomeres. RNase treatment of exosomes prevented the bystander effects on telomerase and telomere length that were observed at 1 population doubling and 24 population doublings, respectively. Thermal denaturation by boiling eliminated the reduction of telomere length in bystander samples, suggesting that the protein fraction of exosomes also contributes to the telomeric effect. RNase treatment plus boiling abrogated all telomere-related effects in directly irradiated and bystander cell populations. These findings suggest that both proteins and RNAs of exosomes can induce alterations in telomeric metabolism, which can instigate genomic instability in epithelial cancer cells after X-ray irradiation.