Stress signaling between human lymphocytes after induction of bystander effect by exposure to ionizing radiation in adaptive doses

We previously reported that the consequence of human lymphocytes irradiation by the adaptive doses (X-rays, 10 cGy) was a transposition of the homologous chromosome loci in the cell nucleus (FISH method); this phenomenon was mediated by the increase of nucleolus activity. They both are transmited to non-irradiated cells by the bystander effect (BE). We shown that the reaction of stress signaling is induced by the DNA fragments of irradiated lymphocytes. The study shows that after the inhibition of caspase 3 activity in irradiating lymphocytes or the blockade TLR9 in bystander cells the transposition was not observed. A signaling way of BE from irradiated lymphocytes apoptosis to bystander cells receptors is discussing.     

Transposition of chromosome loci in bystander cells upon exposure to an adaptive dose of ionizing radiation

During the process of the realization of the bystander effect the trans of the Signal from irradiated cells to the intact cell (bystander cells) happens. So both type of cells (irradiated and intact cells) have the same damages and reactions. There are new data about bystander effect as the transduction mechanism of the adaptive response and we have investigated this phenomenon. There are an incubation of the intact (bystander cells) and the exposed (X-radiation of 10 cGy) human lymphocytes and we analyze the location of the centromeric loci of the first chromosome. We observed hat for the first time that after X-ray exposition of the adaptive doses the transposition of the chromosome loci from the peripheral to the central parts of the nucleus in intact (bystander cell) G0-lymphocytes which were incubated in the growth environment cells with irradiated cells removal. We support that the starting states of the adaptive response is the loci extrication of the matrix, the transposition and the approach homologous chromosomes. This process is necessary for the DNA double strand breaks reparation (in the case of injured dose X-radiation) with the participation of the homologous recombination.     

Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. This study investigated the role of changes in calcium levels in bystander responses leading to chromosomal damage in nonirradiated T98G glioma cells and AG01522 fibroblasts that had been either exposed to conditioned medium from irradiated cells or co-cultured with a population where a fraction of cells were individually targeted through the nucleus or cytoplasm with a precise number of microbeam helium-3 particles. After the recipient cells were treated with conditioned medium from T98G or AG01522 cells that had been irradiated through either nucleus or cytoplasm, rapid calcium fluxes were monitored in the nonirradiated recipient cells. Their characteristics were dependent on the source of the conditioned medium but had no dependence on radiation dose. When recipient cells were co-cultured with an irradiated population of either T98G or AG01522 cells, micronuclei were induced in the nonirradiated cells, but this response was eliminated by treating the cells with calcicludine (CaC), a potent blocker of Ca(2+) channels. Moreover, both the calcium fluxes and the bystander effect were inhibited when the irradiated T98G cells were treated with aminoguanidine, an inhibitor of nitric oxide synthase (NOS), and when the irradiated AG01522 cells were treated with DMSO, a scavenger of reactive oxygen species (ROS), which indicates that NO and ROS were involved in the bystander responses generated from irradiated T98G and AG01522 cells, respectively. Our findings indicate that calcium signaling may be an early response in radiation-induced bystander effects leading to chromosome damage.     

Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in CHO cells

This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells.     

Spatially fractionated radiation induces cytotoxicity and changes in gene expression in bystander and radiation adjacent murine carcinoma cells

Radiation-induced bystander effects have been extensively studied at low doses, since evidence of bystander induced cell killing and other effects on unirradiated cells were found to be predominant at doses up to 0.5 Gy. Therefore, few studies have examined bystander effects induced by exposure to higher doses of radiation, such as spatially fractionated radiation (GRID) treatment. In the present study, we evaluate the ability of GRID treatment to induce changes in GRID adjacent (bystander) regions, in two different murine carcinoma cell lines following exposure to a single irradiation dose of 10 Gy. Murine SCK mammary carcinoma cells and SCCVII squamous carcinoma cells were irradiated using a brass collimator to create a GRID pattern of nine circular fields 12 mm in diameter with a center-to-center distance of 18 mm. Similar to the typical clinical implementation of GRID, this is approximately a 50:50 ratio of direct and bystander exposure. We also performed experiments by irradiating separate cultures and transferring the medium to unirradiated bystander cultures. Clonogenic survival was evaluated in both cell lines to determine the occurrence of radiation-induced bystander effects. For the purpose of our study, we have defined bystander cells as GRID adjacent cells that received approximately 1 Gy scatter dose or unirradiated cells receiving conditioned medium from irradiated cells. We observed significant bystander killing of cells adjacent to the GRID irradiated regions compared to sham treated controls. We also observed bystander killing of SCK and SCCVII cells cultured in conditioned medium obtained from cells irradiated with 10 Gy. Therefore, our results confirm the occurrence of bystander effects following exposure to a high-dose of radiation and suggest that cell-to-cell contact is not required for these effects. In addition, the gene expression profile for DNA damage and cellular stress response signaling in SCCVII cells after GRID exposure was studied. The occurrence of GRID-induced bystander gene expression changes in significant numbers of DNA damage and cellular stress response signaling genes, providing molecular evidence for possible mechanisms of bystander cell killing.     

Influence of the bystander phenomenon on the chromosome aberration pattern in human lymphocytes induced by in vitro α-particle exposure

A recent publication on both chromosome-type and chromatid-type aberrations in lymphocytes of patients during treatment with radium-224 for ankylosing spondilitis has revived the question of whether the chromatid-type aberrations may be the consequence of factors released by irradiated cells. Therefore, the aim of the present study was to investigate the influence of such a bystander phenomenon on the chromosome aberration pattern of lymphocytes. Monolayers of human lymphocytes were irradiated with 1 Gy of α-particles from an americium-241 source in the absence or presence of whole blood, autologous plasma or culture medium. In the presence of any liquid covering the monolayer during irradiation, the chromatid-type aberrations were, contrary to expectation, elevated. Whereas the intercellular distribution of dicentrics was significantly overdispersed, the chromatid-type aberrations showed a regular dispersion. It can be concluded that the enhanced frequency of chromatid aberrations is the result of a damage signal or a bystander phenomenon released by irradiated cells.     

DNA damaging bystander signalling from stem cells,cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these ‘nearest neighbour’ cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.     

Radiation-induced bystander effect in healthy G(o) human lymphocytes: biological and clinical significance

To study the bystander effects, G(0) human peripheral blood lymphocytes were X-irradiated with 0.1, 0.5 and 3 Gy. After 24h, cell-free conditioned media from irradiated cultures were transferred to unexposed lymphocytes. Following 48 h of medium transfer, viability, induction of apoptosis, telomere shortening, reactive oxygen species (ROS) levels and micronuclei (after stimulation) were analyzed. A statistically significant decrement in cell viability, concomitant with the loss of mitochondrial membrane potential, telomere shortening, increases in hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(-)) with depletion of intracellular glutathione (GSH) level, and higher frequencies of micronuclei, were observed in bystander lymphocytes incubated with medium from 0.5 and 3 Gy irradiated samples, compared to lymphocytes unexposed. Furthermore, no statistically significant difference between the response to 0.5 and 3 Gy of irradiation in bystander lymphocytes, was found. However, when lymphocytes were irradiated with 0.1 Gy, no bystander effect with regard to viability, apoptosis, telomere length, and micronuclei was observed, although a high production of ROS level persisted. Radiation in the presence of the radical scavenger dimethyl sulfoxide (DMSO) suppressed oxidative stress induced by 3 Gy of X-rays with the effective elimination of bystander effects, suggesting a correlation between ROS and bystander signal formation in irradiated cells. The data propose that bystander effect might be mostly due to the reactions of radiation induced free radicals on DNA, with the existence of a threshold at which the bystander signal is not operative (0.1 Gy dose of X-rays). Our results may have clinical implications for health risk associated with radiation exposure.     

植物转基因座位形成的分子机制

转基因座位是指染色体上插入的转基因及相邻的特定DNA序列。大多数转基因座位是以转基因片段、基因组片段和填充DNA相间而存在,仅少数含有完整的单拷贝转基因,这是由于在转基因整合过程中,转基因及基因组DNA发生缺失、重复和染色体的重排。转基因整合主要通过双链DNA断裂修复中的异常重组所产生,而同源重组也发挥了一定的作用。异常重组主要由单链复性、合成依赖链复性和依赖Ku蛋白的非同源末端连接途径调节。     

Bystander effect: biological endpoints and microarray analysis

In cell populations exposed to ionizing radiation, the biological effects occur in a much larger proportion of cells than are estimated to be traversed by radiation. It has been suggested that irradiated cells are capable of providing signals to the neighboring unirradiated cells resulting in damage to these cells. This phenomenon is termed the bystander effect. The bystander effect induces persistent, long-term, transmissible changes that result in delayed death and neoplastic transformation. Because the bystander effect is relevant to carcinogenesis, it could have significant implications for risk estimation for radiation exposure. The nature of the bystander effect signal and how it impacts the unirradiated cells remains to be elucidated. Examination of the changes in gene expression could provide clues to understanding the bystander effect and could define the signaling pathways involved in sustaining damage to these cells. The microarray technology serves as a tool to gain insight into the molecular pathways leading to bystander effect. Using medium from irradiated normal human diploid lung fibroblasts as a model system we examined gene expression alterations in bystander cells. The microarray data revealed that the radiation-induced gene expression profile in irradiated cells is different from unirradiated bystander cells suggesting that the pathways leading to biological effects in the bystander cells are different from the directly irradiated cells. The genes known to be responsive to ionizing radiation were observed in irradiated cells. Several genes were upregulated in cells receiving media from irradiated cells. Surprisingly no genes were found to be downregulated in these cells. A number of genes belonging to extracellular signaling, growth factors and several receptors were identified in bystander cells. Interestingly 15 genes involved in the cell communication processes were found to be upregulated. The induction of receptors and the cell communication processes in bystander cells receiving media from irradiated cells supports the active involvement of these processes in inducing bystander effect.     

Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells

Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal.     

DNA-signaling pathway mediating development of a radiation-induced bystander effect in human cells

Low doses of ionizing radiation induce the adaptive effect (AE) development in human cells which is followed by a number of cell responses. These responses can be transmitted from irradiated cells to non-irradiated ones (bystander effect, BE). The major role in radiation-induced BE is played by an oxidative stress (OS) and a DNA-signaling pathway, in which extracellular DNA fragments (ecDNA) are the factors of stress-signalization. We propose the following sequence of events in this signaling system: irradiation-OS-DNA modification-apoptosis of irradiated cells-ecDNA-signal acceptance by non-irradiated cells-OS-DNA modification, etc. We observed a radiation-induced BE which is accompanied by DNA-signaling pathway in differentiated and undifferentiated human cells forming monolayer or suspension cultures. Here we discuss several aspects of the radiation-induced BE mechanism and its persistence possibilities.     

Role of extracellular DNA oxidative modification in radiation induced bystander effects in human endotheliocytes

The development of the bystander effect induced by low doses of irradiation in human umbilical vein endothelial cells (HUVECs) depends on extracellular DNA (ecDNA) signaling pathway. We found that the changes in the levels of ROS and NO production by human endothelial cells are components of the radiation induced bystander effect that can be registered at a low dose. We exposed HUVECs to X-ray radiation and studied effects of ecDNA(R) isolated from the culture media conditioned by the short-term incubation of irradiated cells on intact HUVECs. Effects of ecDNA(R) produced by irradiated cells on ROS and NO production in non-irradiated HUVECs are similar to bystander effect. These effects at least partially depend on TLR9 signaling. We compared the production of the nitric oxide and the ROS in human endothelial cells that were (1) irradiated at a low dose; (2) exposed to the ecDNA(R) extracted from the media conditioned by irradiated cells; and (3) exposed to human DNA oxidized in vitro. We found that the cellular responses to all three stimuli described above are essentially similar. We conclude that irradiation-related oxidation of the ecDNA is an important component of the ecDNA-mediated bystander effect.     

Particularities of blood lymphocyte response to irradiation in vitro in breast cancer patients

DNA breaks and their repair efficiency were analyzed in irradiated in vitro lymphocytes (at doses 1 Gy, gamma-radiation of 60Co, dose rate 1 Gy/min) isolated from peripheral blood of 41 untreated patients with breast cancer and 25 healthy donors using the DNA comet assay under non-denaturing conditions (mainly double-strand DNA breaks (DSB), as well as apoptotic cell death using the DNA halo assay. To estimate the expression of bystander effect, the cells were incubated in a culture medium obtained from lymphocytes irradiated in vitro at doses 1 Gy. The average DSB level in blood lymphocytes of breast cancer patients was shown to be significantly higher (p < 0.05) compared with that in control donors. In general, the following effects were observed in irradiated in vitro lymphocytes of cancer patients: (1) increased sensitivity to y-radiation-induced DNA DSBs compared with lymphocytes from healthy donors, (2) reduced repair efficiency of these damages. Incubation of irradiated blood lymphocytes in a medium from irradiated cells led to an increased relative number of DNA DSBs and an elevated fraction of cells dying through apoptotic pathway both in blood lymphocytes from cancer patients and control donors. However, these non-targeted effects were more expressed for the blood lymphocytes of breast cancer patients.     

The role of mitochondria in the radiation-induced bystander effect in human lymphoblastoid cells

Cells without intact mitochondrial DNA have been shown to lack the bystander effect, which is an energy-dependent process. We hypothesized that cells harboring mutations in mitochondrial genes responsible for ATP synthesis would show a decreased bystander effect compared to normal cells. Radiation-induced bystander effects were analyzed in two normal and four mitochondrial mutant human lymphoblastoid cells. Medium from previously irradiated cells (conditioned medium) was transferred to unirradiated cells from the respective cell lines and evaluated for the bystander effect using the cytokinesis-block micronucleus assay. Unlike normal cells that were used as a control, mitochondrial mutant cells neither generated nor responded to the bystander signals. The bystander effect was inhibited in normal cells by adding the mitochondrial inhibitors rotenone and oligomycin to the culture medium. Time-controlled blocking of the bystander effect by inhibitors was found to occur either for prolonged exposure to the inhibitor prior to irradiation with an immediate and subsequent removal of the inhibitors or immediate post-application of the inhibitor. Adding the inhibitors just prior to irradiation and removing them immediately after irradiation was uneventful. Fully functional mitochondrial metabolic capability may therefore be essential for the bystander effect.     

Induction of replication protein A in bystander cells

The bystander effect is a biological phenomenon whereby cells not directly targeted by DNA-damaging agents elicit a response similar to that of targeted cells. Understanding the mechanisms underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for radiotherapy of tumors. Identification of DNA repair and signal transduction proteins that are induced specifically in bystander cells may help in deducing the molecular mechanism(s) responsible for this complex phenomenon. With this objective, we have studied the expression of replication protein A (RPA), which is involved in various DNA metabolic activities such as replication, repair and recombination. We analyzed RPA expression by immunofluorescence and Western blot techniques in both gamma-irradiated primary human fibroblast cells and bystander cells that were recipients of conditioned growth medium harvested from gamma-irradiated cell cultures. A two- to threefold induction of RPA was observed in bystander MRC5 cells treated with conditioned medium collected from gamma-irradiated WI38 or MRC5 cells. Lack of induction of RPA in sham-manipulated MRC5 cells treated with irradiated medium alone (without cells) indicates that the signal elicited from the irradiated cells is responsible for induction of RPA in bystander cells. RPA was induced more effectively in bystander cells than in irradiated cells at the earliest time analyzed (30 min), and the RPA level declined to that of sham-treated control cells by 24 h after treatment. In addition to RPA, apurinic/apyrimidinic endonuclease (APE, a key enzyme of the base excision repair pathway) also showed enhanced expression in bystander cells. Our findings suggest that the induction of RPA and APE is due to a combination of DNA strand breaks and oxidized base lesions in the genomic DNA of bystander cells.     

The presence of conjugative transposon Tn916 in the recipient strain does not impede transfer of a second copy of the element.

Transfer of the conjugative transposon Tn916 from the chromosome of Bacillus subtilis to a transposon-free Streptococcus pyogenes strain occurs at the same frequency as transfer to a Tn916-containing recipient. This rules out a model for conjugal transfer of Tn916 in which a copy of the element in the recipient represses transposition of a copy introduced by conjugation. Homology-directed integration of the incoming transposon into the resident one is less frequent than insertion elsewhere in the chromosome. This shows that after conjugation, transposition occurs more frequently than homologous recombination. However, because transconjugants arising from homologous recombination can be selected, it is possible to use Tn916 as a shuttle for gram-positive organisms for which there is no easy means of introducing DNA.     

The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.     

Human neonatal lymphocytes immortalized after microinjection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV) is a highly efficient acute transforming agent in human cells, provided that the intact virus is used. To investigate the ability of viral DNA alone to transform cells, we introduced the EBV genome into human lymphocytes. After microinjection of EBV DNA into neonatal B lymphocytes, we established a cell line that in early passages contained multiple viral fragments. This cell line retained sequences from the short, unique (Us) region of the EBV genome and sequences from EcoRI-E. The viral sequences were not expressed; however, the cells expressed a 2.3-kilobase polyadenylated message homologous to the c-fgr oncogene, a cellular locus believed to be activated by EBV infection [M. S. C. Cheah, T. J. Ley, S. R. Tronick, and K. C. Robbins, Nature (London) 319:238-240.]. The cell line was monoclonal with rearrangement at the immunoglobulin locus and had a reciprocal translocation t(1;7)(p34;q34) and a deletion of sequences within the locus for the beta chain of the T-cell receptor. The close proximity of the translocation to the chromosomal loci for c-fgr on chromosome 1 and the T-cell receptor beta chain on chromosome 7 suggests that structural alteration of these genes was critical to this transformation event.     

Investigation of radiation-induced "bystaner effect" using model of adaptive response in mixed lymphocyte culture from humans of different gender

The novel method for the investigation of radiation-induced "bystander effect" has been tested on the model of mixed lymphocyte culture from humans of different gender. The "bystander effect" was estimated by the ability of nonirradiated female/male cells to develop an adaptive response in mixed culture with irradiated at the dose 0.05 Gy of X-rays G0 lymphocytes of opposite gender. The preliminary results indicate that both irradiated lymphocytes and non-irradiated but neighbouring with pre-exposed cells are less susceptible to the genetic damages manifested as chromosome aberrations induced in G1 lymphocytes by a subsequent high dose of X-ray (1.0 Gy). Direct adaptive response as well as indirect one were expressed more obvious in female lymphocytes.