Clonogenic assay to measure bystander cytotoxicity of targeted alpha-particle therapy.

Radiation therapy induces targeted effects in the cells that are irradiated and also non-targeted effects (i.e. bystander effects) in non-irradiated cells that are close to or at short distance (<∼1 mm) from irradiated cells. Bystander effects are mediated by intercellular communications and may result in cytotoxic and genotoxic modifications. Their occurrence and relative contribution to the irradiation outcome are influenced by several parameters among which the particle linear energy transfer seems to be prominent. Bystander effects were first observed after external radiation therapy, but have been described also following targeted radionuclide therapy. Therefore, we propose a method to investigate their occurrence in experimental conditions where cells are exposed to radiopharmaceuticals. In this approach, clonogenic cell death is the biological endpoint of the bystander effects caused by irradiation with alpha particles (a potent inducer of the bystander response).

The interaction of oxidative stress with MAPK, PI3/AKT, NF-κB, and DNA damage kinases influences the fate of γ-radiation-induced bystander cells.

Oxidative stress is associated with the induction of a plethora of effects on cellular macromolecules and signaling cascades. The onset of oxidative imbalance characterizes irradiated cells. The present study investigates the effects of ionizing radiation on oxidative stress induction in bystander cells and their interactions with critical cell signaling mediators. The effect of irradiated cell-conditioned medium (ICCM) from γ-irradiated hepatocellular carcinoma (HepG2) cells were studied in bystander HepG2 and normal liver (BRL-3A) cells at early (1 h, 2 h) and later (24 h) time points post-irradiation. Although ROS generation and lipid peroxidation showed the highest effects in both bystander cell groups at the early time points, antioxidant enzymes superoxide dismutase and catalase showed the lowest activity. Oxidative stress was persistent up to 24 h, but the highest level was seen in 1 h ICCM treated 8By cells. Although the levels of all pro-survival signaling factors (p-PI3K, p-Akt, p-p38MAPK, p-JNK, and p-NFκB) increased in bystander HepG2 cells, they showed a significant decrease in bystander BRL-3A cells. JAK2-STAT3 activation, however, was reduced only in BRL-3A cells, with no effect in HepG2 cells. However, in both bystander cell groups, activation of DNA damage sensors ATM, ATR, and cell cycle inhibitor p21 increased. Elevated ROS levels down-regulated the activation of PI3K, Akt, JNK, and NF-κB in BRL-3A cells but enhanced the activation of ATM and p21. In contrast, in HepG2 cells, increased ROS level elevated the activation of PI3K, JNK, p38MAPK, NF-κB with no effect on p-ATM or p21. ROS differentially influenced the interactions between the signaling mediators in the bystander cells. p-ATR levels, although increased in both bystander cell groups, showed no association with other factors. ICCM from the same HepG2 cells differently affected signaling factors in two groups of cells, highlighting the critical significance of the study in the field of radiation biology.

The cross-talk between Bax, Bcl2, caspases, and DNA damage in bystander HepG2 cells is regulated by γ-radiation dose and time of conditioned media transfer.

Although radiation-induced bystander effects have been broadly explored in various biological systems, the molecular mechanisms and the consequences of different regulatory factors (dose, time, cell type) on bystander responses are not clearly understood. This study investigates the effects of irradiated cell-conditioned media (ICCM) collected at different times post-irradiation on bystander cancer cells regarding DNA damage and apoptosis induction. Human hepatocellular carcinoma HepG2 cells were exposed to γ-ray doses of 2 Gy, 5 Gy, and 8 Gy. In the early and late stages (1 h, 2 h, and 24 h) after irradiation, the ICCM was collected and transferred to unirradiated cells. Compared to control, bystander cells showed an increased level of H2AX phosphorylation, mitochondrial membrane depolarization, and elevation of intrinsic apoptotic pathway mediators such as p53, Bax, cas9, cas-3, and PARP cleavage. These results were confirmed by phosphatidylserine (PS) externalization and scanning electron microscopic observations, suggesting a rise in bystander HepG2 cell apoptosis. Anti-apoptotic Bcl2-level and viability were lower in bystander cells compared to control. The highest effects were observed in 8 Gy γ radiation-induced bystander cells. Even though the bystander effect was persistent at all time points of the study, ICCM at the early time points (1 or 2 h) had the most significant impact on the apoptosis markers in bystander cells. Nevertheless, 24 h ICCM induced the highest increase in H2AX and p53 phosphorylation and Bax levels. The effects of ICCM of irradiated HepG2 cells were additionally studied in normal liver cells BRL-3A to simulate actual radiotherapy conditions. The outcomes suggest that the expression of the signaling mediators in bystander cells is highly dynamic. A cross-talk between those signaling mediators regulates bystander responses depending on the radiation dose and time of incubation post-irradiation.

A general theoretical framework to design base editors with reduced bystander effects.

Base editors (BEs) hold great potential for medical applications of gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 - 10 nucleotides). Here, to assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that explicitly evaluates the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model reproduces the experimental data of A3A-BE3 and its variants for targeting the "TC" motif and bystander editing. Analyzing this approach, we propose several general principles that can guide the design of BEs with a reduced bystander effect. These principles are then applied to design a series of point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verify experimentally that the new mutations provide different levels of stringency on reducing the bystander editing at different genomic loci, which is consistent with our theoretical model. Thus, our study provides a computational-aided platform to assist in the scientifically-based design of BEs with reduced bystander effects.

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.

Role of epigenetic mechanisms in propagating off-targeted effects following radiation based therapies - A review.

Despite being an important diagnostic and treatment modality, ionizing radiation (IR) is also known to cause genotoxicity and multiple side effects leading to secondary carcinogenesis. While modern cancer radiation therapy has improved patient recovery and enhanced survival rates, the risk of radiation-related adverse effects has become a growing challenge. It is now well-accepted that IR-induced side effects are not exclusively restricted to exposed cells but also spread to distant 'bystander' cells and even to the unexposed progeny of the irradiated cells. These 'off-targeted' effects involve a plethora of molecular events depending on the type of radiation and tumor tissue background. While the mechanisms by which off-targeted effects arise remain obscure, emerging evidence based on the non-mendelian inheritance of various manifestations of them as well as their persistence for longer periods supports a contribution of epigenetic factors. This review focuses on the major epigenetic phenomena including DNA methylation, histone modifications, and small RNA mediated silencing and their versatile role in the manifestation of IR induced off-targeted effects. As short- and long-range communication vehicles respectively, the role of gap junctions and exosomes in spreading these epigenetic-alteration driven off-targeted effects is also discussed. Furthermore, this review emphasizes the possible therapeutic potentials of these epigenetic mechanisms and how beneficial outcomes could potentially be achieved by targeting various signaling molecules involved in these mechanisms.

Not so sweet and simple: impacts of SARS-CoV-2 on the ß cell.

The link between COVID-19 infection and diabetes has been explored in several studies since the start of the pandemic, with associations between comorbid diabetes and poorer prognosis in patients infected with the virus and reports of diabetic ketoacidosis occurring with COVID-19 infection. As such, significant interest has been generated surrounding mechanisms by which the virus may exert effects on the pancreatic ß cells. In this review, we consider possible routes by which SARS-CoV-2 may impact ß cells. Specifically, we outline data that either support or argue against the idea of direct infection and injury of ß cells by SARS-CoV-2. We also discuss ß cell damage due to a "bystander" effect in which infection with the virus leads to damage to surrounding tissues that are essential for ß cell survival and function, such as the pancreatic microvasculature and exocrine tissue. Studies elucidating the provocation of a cytokine storm following COVID-19 infection and potential impacts of systemic inflammation and increases in insulin resistance on ß cells are also reviewed. Finally, we summarize the existing clinical data surrounding diabetes incidence since the start of the COVID-19 pandemic.

Virtual Reality or Augmented Reality as a Tool for Studying Bystander Behaviors in Interpersonal Violence: Scoping Review.

BACKGROUND: To provide participants with a more real and immersive intervening experience, virtual reality (VR) and/or augmented reality (AR) technologies have been integrated into some bystander intervention training programs and studies measuring bystander behaviors. OBJECTIVE: We focused on whether VR or AR can be used as a tool to enhance training bystanders. We reviewed the evidence from empirical studies that used VR and/or AR as a tool for examining bystander behaviors in the domain of interpersonal violence research. METHODS: Two librarians searched for articles in databases, including APA PsycInfo (Ovid), Criminal Justice Abstracts (EBSCO), Medline (Ovid), Applied Social Sciences Index & Abstracts (ProQuest), Sociological Abstracts (ProQuest), and Scopus till April 15, 2020. Studies focusing on bystander behaviors in conflict situations were included. All study types (except reviews) written in English in any discipline were included. RESULTS: The search resulted in 12,972 articles from six databases, and the articles were imported into Covidence. Eleven studies met the inclusion and exclusion criteria. All 11 articles examined the use of VR as a tool for studying bystander behaviors. Most of the studies were conducted in US young adults. The types of interpersonal violence were school bullying, dating violence, sexual violence/assault, and soccer-associated violence. VR technology was used as an observational measure and bystander intervention program. We evaluated the different uses of VR for bystander behaviors and noted a lack of empirical evidence for AR as a tool. We also discuss the empirical evidence regarding the design, effectiveness, and limitations of implementing VR as a tool in the reviewed studies. CONCLUSIONS: The reviewed results have implications and recommendations for future research in designing and implementing VR/AR technology in the area of interpersonal violence. Future studies in this area may further contribute to the use of VR as an observational measure and explore the potential use of AR to study bystander behaviors.

A Systems Pharmacology Model for Drug Delivery to Solid Tumors by Antibody-Drug Conjugates: Implications for Bystander Effects.

Antibody-drug conjugates (ADCs) are cancer drugs composed of a humanized antibody linked to a cytotoxic payload, allowing preferential release of payload in cancer cells expressing the antibody-targeted antigen. Here, a systems pharmacology model is used to simulate ADC transport from blood to tumor tissue and ADC uptake by tumor cells. The model includes effects of spatial gradients in drug concentration in a three-dimensional network of tumor blood vessels with realistic geometry and accounts for diffusion of ADC in the tumor extracellular space, binding to antigen, internalization, intracellular processing, and payload efflux from cells. Cells that process an internalized ADC-antigen complex may release payload that can be taken up by other "bystander" cells. Such bystander effects are included in the model. The model is used to simulate conditions in previous experiments, showing good agreement with experimental results. Simulations are used to analyze the relationship of bystander effects to payload properties and single-dose administrations. The model indicates that exposure of payload to cells distant from vessels is sensitive to the free payload diffusivity in the extracellular space. When antigen expression is heterogeneous, the model indicates that the amount of payload accumulating in non-antigen-expressing cells increases linearly with dose but depends only weakly on the percentage of antigen-expressing cells. The model provides an integrated mechanistic framework for understanding the effects of spatial gradients on drug distribution using ADCs and for designing ADCs to achieve more effective payload distribution in solid tumors, thereby increasing the therapeutic index of the ADC.

Complement-independent bystander injury in AQP4-IgG seropositive neuromyelitis optica produced by antibody-dependent cellular cytotoxicity.

Cellular injury in AQP4-IgG seropositive neuromyelitis spectrum disorder (herein called NMO) involves AQP4-IgG binding to astrocytes, resulting in astrocyte injury by complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) mechanisms. The rapid disease progression, severe tissue damage, and abundant leukocyte infiltration seen in some NMO patients suggest a more direct mechanism for demyelination and neurologic deficit than secondary injury from astrocyte loss. Here, we report evidence for an 'ADCC bystander mechanism' in NMO involving injury to nearby cells by leukocytes following their activation by AQP4-bound AQP4-IgG on astrocytes. In model cocultures containing AQP4-expressing and null CHO cells, AQP4-IgG and complement killed bystander null cells to ~ 100 µm away from AQP4-expressing cells; AQP4-IgG and NK cells produced bystander killing to ~ 300 µm, with perforin deposition seen on injured null cells. Bystander cytotoxicity was also seen with neutrophil-mediated ADCC and in astrocyte-neuron cocultures. Mechanistic studies, including real-time imaging, suggested that leukocytes activated by an AQP4-dependent ADCC mechanism injure bystander cells by direct targeted exocytosis on neighboring cells and not by diffusion of soluble granule contents. In support of this conclusion, ADCC bystander injury was preferentially reduced by an RGDS peptide that inhibits integrin adhesion. Evidence for ADCC bystander injury to oligodendrocytes and neurons was also found in mice following intracerebral injection of AQP4-IgG and NK cells, which was inhibited by RGDS peptide. These results establish a novel cellular pathogenesis mechanism in AQP4-IgG seropositive NMO and provide evidence that inflammatory mechanisms can cause widespread tissue damage in NMO independently of the secondary effects from astrocyte loss.

Common knowledge, coordination, and strategic mentalizing in human social life.

People often coordinate for mutual gain, such as keeping to opposite sides of a stairway, dubbing an object or place with a name, or assembling en masse to protest a regime. Because successful coordination requires complementary choices, these opportunities raise the puzzle of how people attain the common knowledge that facilitates coordination, in which a person knows X, knows that the other knows X, knows that the other knows that he knows, ad infinitum. We show that people are highly sensitive to the distinction between common knowledge and mere private or shared knowledge, and that they deploy this distinction strategically in diverse social situations that have the structure of coordination games, including market cooperation, innuendo, bystander intervention, attributions of charitability, self-conscious emotions, and moral condemnation.

Both irradiated and bystander effects link with DNA repair capacity and the linear energy transfer.

AIMS: In comparison with a low linear energy transfer (LET) radiation, a high-LET radiation induces more complex DNA damage. This study wonders whether radiation-induced bystander effect (RIBE) is dependent of LET. MATERIALS AND METHODS: Chinese hamster ovary CHO-9 cells and its subline EM-C11 cells (SSB repair deficient) and XR-C1 cells (DSB repair deficient) were irradiated by γ-rays, α-particles, or carbon ions with different LETs of 13, 30 and 70 keV/µm. Cell proliferation, cell death, DNA damage, cell cycle distribution and some protein expressions were measured with the cell counting kit-8 (CCK-8), colony formation, micronuclei (MN), flow cytometry and western blot, respectively. KEY FINDINGS: A series of cell responses were induced by these radiations in a LET-dependent manner, including proliferation inhibition, cell death, MN induction, G2/M phase arrest and the expression of γH2AX protein. These cell injuries were also depended on DNA repair capacity, and XR-C1 cells were the most sensitive to each radiation. Furthermore, when the cells were treated with the conditioned medium (CM) collected from irradiated CHO-9 cells, the MN induction and cell death response in the bystander cells of EM-C11 or XR-C1 increased along with LET of irradiation, and the bystander damage was easier to be induced in EM-C11 and XR-C1 cells than that in CHO-9 cells. SIGNIFICANCE: Both cellular DNA repair capacity and the LET value of radiation could deeply influence damage extents of not only the irradiated cells but also the bystander cells.

Beyond bystanders: Myeloid cells in chronic lymphocytic leukemia.

Tumor-promoting inflammation and escape from immune-mediated tumor destruction have been recognized as hallmarks of cancer, and myeloid cells are key players in these processes. By exploiting the tremendous plasticity of myeloid cells, tumors induce a variety of tumor-supportive and immunosuppressive cell phenotypes like tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). The relevance of these cell types in hematopoietic malignancies has only recently gained a stronger attention. Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that expand in secondary lymphoid organs and the bone marrow, and accumulate in the blood of patients. A large body of evidence suggests that the interactions between CLL cells and non-malignant cells in the tumor microenvironment play a key role in the pathology of this disease. CLL is associated with an inflammatory milieu and defective immune responses. A severe skewing of myeloid and T cells toward leukemia-supportive and immunosuppressive phenotypes have been observed in patient samples and the Eµ-TCL1 mouse model of CLL. Myeloid cells were thereby shown to enhance survival of CLL cells and contribute to apoptosis-resistance, to suppress anti-tumoral immunity, and to be involved in immune deficiency of leukemia patients. In addition, treatment regimens that are currently used for CLL target not only directly the malignant cells, but have also an impact on non-malignant bystander cells, including myeloid cells. This review summarizes current literature on these aspects and gives a perspective on how our current knowledge might be used to design novel immunotherapeutic approaches that can be combined with CLL-targeting drugs to achieve better therapeutic responses in CLL patients.

Amacrine cells coupled to ganglion cells via gap junctions are highly vulnerable in glaucomatous mouse retinas.

We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol. 527:159-173, 2019. © 2016 Wiley Periodicals, Inc.

Old Data-New Concepts: Integrating "Indirect Effects" Into Radiation Protection.

PURPOSE: To address the following key question, what are the consequences of nontargeted and delayed effects for linear nonthreshold models of radiation risk? This paper considers low-dose "indirect" or nontargeted effects and how they might impact radiation protection, particularly at the level of the environment. Nontargeted effects refer to effects in cells, tissues, or organisms that were not targeted by irradiation and that did not receive direct energy deposition. They include genomic instability and lethal mutations in progeny of irradiated cells and bystander effects in neighboring cells, tissues, or organisms. Low-dose hypersensitivity and adaptive responses are sometimes included under the nontargeted effects umbrella, but these are not considered in this paper. Some concepts emerging in the nontargeted effects field that could be important include historic dose. This suggests that the initial exposure to radiation initiates the instability phenotype which is passed to progeny leading to a transgenerational radiation-response phenotype, which suggests that the system response rather than the individual response is critical in determining outcome. CONCLUSION: Nontargeted effects need to be considered, and modeling, experimental, and epidemiological approaches could all be used to determine the impact of nontargeted effects on the currently used linear nonthreshold model in radiation protection.

Reactive oxygen species and nitric oxide signaling in bystander cells.

It is now well accepted that radiation induced bystander effects can occur in cells exposed to media from irradiated cells. The aim of this study was to follow the bystander cells in real time following addition of media from irradiated cells and to determine the effect of inhibiting these signals. A human keratinocyte cell line, HaCaT cells, was irradiated (0.005, 0.05 and 0.5 Gy) with γ irradiation, conditioned medium was harvested after one hour and added to recipient bystander cells. Reactive oxygen species, nitric oxide, Glutathione levels, caspase activation, cytotoxicity and cell viability was measured after the addition of irradiated cell conditioned media to bystander cells. Reactive oxygen species and nitric oxide levels in bystander cells treated with 0.5Gy ICCM were analysed in real time using time lapse fluorescence microscopy. The levels of reactive oxygen species were also measured in real time after the addition of extracellular signal-regulated kinase and c-Jun amino-terminal kinase pathway inhibitors. ROS and glutathione levels were observed to increase after the addition of irradiated cell conditioned media (0.005, 0.05 and 0.5 Gy ICCM). Caspase activation was found to increase 4 hours after irradiated cell conditioned media treatment (0.005, 0.05 and 0.5 Gy ICCM) and this increase was observed up to 8 hours and there after a reduction in caspase activation was observed. A decrease in cell viability was observed but no major change in cytotoxicity was found in HaCaT cells after treatment with irradiated cell conditioned media (0.005, 0.05 and 0.5 Gy ICCM). This study involved the identification of key signaling molecules such as reactive oxygen species, nitric oxide, glutathione and caspases generated in bystander cells. These results suggest a clear connection between reactive oxygen species and cell survival pathways with persistent production of reactive oxygen species and nitric oxide in bystander cells following exposure to irradiated cell conditioned media.

Impact of Co-Culturing with Fractionated Carbon-Ion-Irradiated Cancer Cells on Bystander Normal Cells and Their Progeny.

The purpose of this study was to compare the biological effects of fractionated doses versus a single dose of high-LET carbon ions in bystander normal cells, and determine the effect on their progeny using the layered tissue co-culture system. Briefly, confluent human glioblastoma (T98G) cells received a single dose of 6 Gy or three daily doses of 2 Gy carbon ions, which were then seeded on top of an insert with bystander normal skin fibroblasts (NB1RGB) growing underneath. Cells were co-cultured for 6 h or allowed to grow for 20 population doublings, then harvested and assayed for different end points. A single dose of carbon ions resulted in less damage in bystander normal NB1RGB cells than the fractionated doses. In contrast, the progeny of bystander NB1RGB cells co-cultured with T98G cells exposed to fractionated doses showed less damage than progeny from bystander cells co-cultured with single dose glioblastoma cells. Furthermore, inhibition of gap junction communication demonstrated its involvement in the stressful effects in bystander cells and their progeny. These results indicate that dose fractionation reduced the late effect of carbon-ion exposure in the progeny of bystander cells compared to the effect in the initial bystander cells.

Radiation exposure from depleted uranium: The radiation bystander effect.

Depleted uranium (DU) is a radioactive heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. In vivo studies have also demonstrated that DU is leukemogenic and genotoxic. DU possesses both a radiological (alpha particle) and chemical (metal) component but is generally considered a chemical biohazard. Studies have shown that alpha particle radiation does play a role in DU's toxic effects. Evidence has accumulated that non-irradiated cells in the vicinity of irradiated cells can have a response to ionization events. The purpose of this study was to determine if these "bystander effects" play a role in DU's toxic and neoplastic effects using HOS cells. We investigated the bystander responses between DU-exposed cells and non-exposed cells by co-culturing the two equal populations. Decreased cell survival and increased neoplastic transformation were observed in the non-DU exposed cells following 4 or 24h co-culture. In contrast Ni (II)- or Cr(VI)- exposed cells were unable to alter those biological effects in non-Ni(II) or non-Cr(VI) exposed co-cultured cells. Transfer experiments using medium from the DU-exposed and non-exposed co-cultured cells was able to cause adverse biological responses in cells; these results demonstrated that a factor (s) is secreted into the co-culture medium which is involved in this DU-associated bystander effect. This novel effect of DU exposure could have implications for radiation risk and for health risk assessment associated with DU exposure.

Overexpression of SKP2 Inhibits the Radiation-Induced Bystander Effects of Esophageal Carcinoma.

BACKGROUND: To investigate the effects of S-phase kinase protein 2 (SKP2) expression on the radiation induced bystander effect (RIBE) in esophageal cancer (EC) cells. MATERIALS AND METHODS: Western blot was used to detect the levels of SKP2, Rad51, and Ku70 in EC cells. Positive transfection, RNAi, micronucleus (MN), and γ-H2AX focus formation assay were used to investigate the effects of SKP2 on RIBE induced by irradiated cells. RESULTS: We found a significant negative correlation between SKP2 expression and MN frequency (p < 0.05) induced by RIBE. The results were further confirmed by positive transfection, RNAi, and rescue experiments.γ-H2AX focus formation assay results indicated that overexpression of SKP2 in the irradiated cells inhibited the DNA damage of RIBE cells. However, when SKP2 expression decreased in irradiated cells, the DNA damage of RIBE cells increased. Increased or decreased expression levels of SKP2 had effects on Rad51 expression under the conditions of RIBE. CONCLUSIONS: These results showed, for the first time, that SKP2 expression can inhibit RIBE of EC cells. The mechanism may function, at least partly, through the regulation of Rad51 in the ability to repair DNA damage.

Akt/mTOR mediated induction of bystander effect signaling in a nucleus independent manner in irradiated human lung adenocarcinoma epithelial cells.

Cytoplasm is an important target for the radiation-induced bystander effect (RIBE). In the present work, the critical role of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in the generation of RIBE signaling after X-ray irradiation and the rapid phosphorylation of Akt and mTOR was observed in the cytoplasm of irradiated human lung adenocarcinoma epithelial (A549) cells. Targeting A549 cytoplasts with individual protons from a microbeam showed that RIBE signal(s) mediated by the Akt/mTOR pathway were generated even in the absence of a cell nucleus. These results provide a new insight into the mechanisms driving the cytoplasmic response to irradiation and their impact on the production of RIBE signal(s).