In situ visualization of DSBs to assess the extranuclear/extracellular effects induced by low-dose alpha-particle irradiation

Extranuclear/extracellular effects may have a significant effect on low-dose radiation risk assessment as well as on the shape of the dose-response relationship. Numerous studies using different end points such as sister chromatid exchanges, micronuclei and mutation have shown that this phenomenon exists in many cell types. However, these end points mostly reflect the late events after radiation damage, and little is known about the early response in this phenomenon. DNA double-strand breaks (DSBs) induced by ionizing radiation or carcinogenic chemicals can be visualized in situ using gamma-H2AX immunofluorescence staining, and there is evidence that the number of gamma-H2AX foci can be closely correlated with DSBs induced. Here we used gamma-H2AX as a biomarker to assess the extranuclear/extracellular effects induced by low-dose alpha particles in situ. The results show that a greater fraction of positive cells with DSBs (48.6%) was observed than the number of cells whose nuclei were actually traversed by the 1-cGy dose of alpha particles (9.2%). The fraction of DSB-positive cells was greatly reduced after treatment with either lindane or DMSO. These results suggest that in situ visualization of DSBs can be used to assess radiation-induced extranuclear/extracellular effects soon after irradiation. Moreover, the in situ DSB assay may provide a means to evaluate the spatial effect on unirradiated cells that are located in the neighboring region of cells irradiated by alpha particles.     

Detection of chromosomal instability in alpha-irradiated and bystander human fibroblasts

There is increasing evidence biological responses to ionizing radiation are not confined to those cells that are directly hit, but may be seen in the progeny at subsequent generations (genomic instability) and in non-irradiated neighbors of irradiated cells (bystander effects). These so called non-targeted phenomena would have significant contributions to radiation-induced carcinogenesis, especially at low doses where only a limited number of cells in a population are directed hit. Here we present data using a co-culturing protocol examining chromosomal instability in alpha-irradiated and bystander human fibroblasts BJ1-htert. At the first cell division following exposure to 0.1 and 1Gy alpha-particles, irradiated populations demonstrated a dose dependent increase in chromosome-type aberrations. At this time bystander BJ1-htert populations demonstrated elevated chromatid-type aberrations when compared to controls. Irradiated and bystander populations were also analyzed for chromosomal aberrations as a function of time post-irradiation. When considered over 25 doublings, all irradiated and bystander populations had significantly higher frequencies of chromatid aberrations when compared to controls (2-3-fold over controls) and were not dependent on dose. The results presented here support the link between the radiation-induced phenomena of genomic instability and the bystander effect.     

Role of homologous recombination in the alpha-particle-induced bystander effect for sister chromatid exchanges and chromosomal aberrations

The bystander effect for sister chromatid exchanges (SCEs) and chromosomal aberrations was examined in hamster cell lines deficient in either DNA-PKcs (V3 cells, deficient in nonhomologous end joining, NHEJ) or RAD51C (irs3 cells, deficient in homologous recombination, HR). Cells synchronized in G0/G1 phase were irradiated with very low fluences of alpha particles such that < 1% of the nuclei were traversed by an alpha particle. Wild-type cells showed a prominent bystander response for SCE induction; an even greater effect was observed in V3 cells. On the other hand, no significant induction of SCE was observed in the irs3 RAD51C-deficient bystander cells irradiated at various stages in the cell cycle. Whereas a marked bystander effect for chromosomal aberrations occurred in V3 cells, the induction of chromosomal aberrations in irs3 bystander cells was minimal and similar to that of wild-type cells. Based on these findings, we hypothesize that HR is essential for the induction of SCE in bystander cells; however, HR is unable to repair the DNA damage induced in NHEJ-deficient bystander cells that leads to either SCE or chromosomal aberrations.     

Nontargeted effects of ionizing radiation: implications for low-dose exposures

The traditional thinking has been that the biological effects of ionizing radiation occur in irradiated cells as a consequence of the DNA damage they incur. This implies that: 1) biological effects occur only in irratiated cells, 2) radiation traversal through the nucleus of the cell is a prerequisite to produce a biological response, and 3) DNA is the target molecule in the cell. Evidence has been emerging, however, for non-DNA targeted effects of radiation; that is, effects including mutations, chromosomal aberrations, and changes in gene expression which occur in cells that in themselves receive no radiation exposure. Two of these phenomena will be described in this paper. The first is radiation-induced genomic instability whereby biological effects, including elevated frequencies of mutations and chromosomal aberrations, arise in the distant descendants of irradiated cells. The second phenomenon has been termed the "bystander effect", whereby in a mixed population of irradiated and nonirradiated cells, biological effects arise in those cells that receive no radiation exposure. The damage signals are transmitted from cell to cell through gap junction channels, and the genetic effects observed in bystander cells appear to result from an upregulation of oxidative stress. The possible influence of these non-targeted effects of radiation of the respounse to low-dose exposures is discussed.     

H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues

A sequence variant of histone H2A called H2AX is one of the key components of chromatin involved in DNA damage response induced by different genotoxic stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress. γH2AX foci could be easily detected in cell nuclei using immunofluorescence microscopy that allows to use γH2AX as a quantitative marker of DSBs in various applications. H2AX is phosphorylated in situ by ATM, ATR, and DNA-PK kinases that have distinct roles in different pathways of DSB repair. The γH2AX serves as a docking site for the accumulation of DNA repair proteins, and after rejoining of DSBs, it is released from chromatin. The molecular mechanism of γH2AX dephosphorylation is not clear. It is complicated and requires the activity of different proteins including phosphatases and chromatin-remodeling complexes. In this review, we summarize recently published data concerning the mechanisms and kinetics of γH2AX loss in normal cells and tissues as well as in those deficient in ATM, DNA-PK, and DSB repair proteins activity. The results of the latest scientific research of the low-dose irradiation phenomenon are presented including the bystander effect and the adaptive response estimated by γH2AX detection in cells and tissues.     

γH2AX in Bystander Cells: Not Just a Radiation-Triggered Event,a Cellular Response to Stress Mediated by Intercellular Communication

The recent years have witnessed a rapid accumulation of experimental data showing that ionizing radiation elicits a plethora of biological effects in unirradiated cells receiving bystander signals from hit cells. This so-called radiation-induced bystander effect (RIBE) manifests in various ways including changes in gene expression, genetic and epigenetic alterations, as well as increases in cell transformation and cell death. Our group and others found that DNA double-stranded breaks (DSBs), directly measured by the γ-H2AX focus formation assay, accumulate in bystander cells in a number of experimental systems such as human cultured cells, human 3-dimensional tissue models and in mice. In addition, we recently found that various other sources of cell stress, including media from cancerous cells resulted in a DNA damage response (DDR) in normal human cells that is reminiscent of RIBE. These results suggest that the RIBE may be part of a more general stress response, however, the molecular mechanism underpinning the formation of DNA DSBs in bystander cells is still unclear. This extra view points to some possibilities that might explain why DDR in human cells can be observed under bystander conditions.     

DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.     

Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation

Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation, a process known as the bystander effect. Bystander effects can also lead to chromosomal/genomic instability within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The factors that mediate this cellular communication can be transferred between cells via gap junctions or released into the extracellular media following irradiation, but their nature has not been fully characterized. In this study we tested the hypothesis that the bystander effect mediator contains an RNA molecule that may be carried by exosomes. MCF7 cells were irradiated with 2 Gy of X rays and the extracellular media was harvested. RNase treatment abrogated the ability of the media to induce early and late chromosomal damage in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the bystander effect, and genomic instability, are at least in part mediated by exosomes and implicate a role for RNA.     

Heavy charged particles produce a bystander effect via cell-cell junctions.

Radiation-induced damage to living cells results from either a direct hit to cellular DNA, or from indirect action which leads to DNA damage from radiation produced radicals. However, in recent years there is evidence that biological effects such as cell killing, mutation induction, chromosomal damage and modification of gene expression can occur in a cell population exposed to low doses of alpha particles. In fact these doses are so low that not all cells in the population will be hit directly by the radiation. Using a precision alpha-particle microbeam, it has been recently demonstrated that irradiated target cells can induce a bystander mutagenic response in neighboring "bystander" cells which were not directly hit by alpha particles. Furthermore, these results suggest that gap-junction mediated cell-to-cell communication plays a critical role in this bystander phenomenon. The purpose of this section is to describe recent studies on bystander biological effects. The recent work described here utilized heavy charged particles for irradiation, and investigated the role of gap-junction mediated cell-cell communication in this phenomenon.     

DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects

Ojima, M., Ban, N. and Kai, M. DNA Double-Strand Breaks Induced by Very Low X-Ray Doses are Largely due to Bystander Effects. Radiat. Res. 170, 365-371 (2008).Phosphorylated ATM immunofluorescence staining was used to investigate the dose-response relationship for the number of DNA double-strand breaks (DSBs) induced in primary normal human fibroblasts irradiated with doses from 1.2 to 200 mGy. The induction of DSBs showed a supralinear dose-response relationship. Radiation-induced bystander effects may explain these findings. To test this hypothesis, the number of DSBs in cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; a supralinear dose-response relationship was not observed. Moreover, the number of DSBs obtained by subtracting the number of phosphorylated ATM foci in lindane-treated cells from the number of phosphorylated ATM foci in untreated cells was proportional to the dose at low doses (1.2-5 mGy) and was saturated at doses from 10-200 mGy. Thus the increase in the number of DSBs in the range of 1.2-5 mGy was largely due to radiation-induced bystander effects, while at doses >10 mGy, the DSBs may be induced mainly by dose-dependent direct radiation effects and partly by dose-independent radiation-induced bystander effects. The findings in our present study provide direct evidence of the dose-response relationship for radiation-induced bystander effects from broad-beam X rays.     

Bystander-mediated genomic instability after high LET radiation in murine primary haemopoietic stem cells

Communication between irradiated and unirradiated (bystander) cells can result in responses in unirradiated cells that are similar to responses in their irradiated counterparts. The purpose of the current experiment was to test the hypothesis that bystander responses will be similarly induced in primary murine stem cells under different cell culture conditions. The experimental systems used here, co-culture and media transfer, are similar in that they both restrict communication between irradiated and bystander cells to media borne factors, but are distinct in that with the media transfer technique, cells can only communicate after irradiation, and with co-culture, cells can communication before, during and after irradiation. In this set of parallel experiments, cell type, biological endpoint, and radiation quality and dose, were kept constant. In both experimental systems, clonogenic survival was significantly decreased in all groups, whether irradiated or bystander, suggesting a substantial contribution of bystander effects (BE) to cell killing. Genomic instability (GI) was induced under all radiation and bystander conditions in both experiments, including a situation where unirradiated cells were incubated with media that had been conditioned for 24h with irradiated cells. The appearance of delayed aberrations (genomic instability) 10-13 population doublings after irradiation was similar to the level of initial chromosomal damage, suggesting that the bystander factor is able to induce chromosomal alterations soon after irradiation. Whether these early alterations are related to those observed at later timepoints remains unknown. These results suggest that genomic instability may be significantly induced in a bystander cell population whether or not cells communicate during irradiation.     

The bystander effect-induced formation of micronucleated cells is inhibited by antioxidants, but the parallel induction of apoptosis and loss of viability are not affected

X-rays induce various DNA damages including strand breaks that lead to formation of micronuclei and chromosomal aberrations as well as increased number of apoptotic cells. Similar effects appear when non-irradiated cells are treated with medium collected from cultures of irradiated cells (irradiation conditioned medium - ICM). This phenomenon was termed "bystander effect". A number of studies suggest that bystander effect appears to be associated with up-regulation of oxidative metabolism. We thus compared the effects of antioxidant Vitamins C and E on the frequency of micronuclei and apoptotic cells in both directly irradiated cell cultures and in cultures exposed to ICM. Addition of Vitamins C or E (1-40 microg/ml) to culture medium after exposure to radiation or ICM reduced the frequency of micronuclei in a concentration-dependent manner. These vitamins had no effect on cell viability, clonogenic survival or the frequency of apoptotic cells under both conditions tested. These results show that the bystander effect causes micronucleation in addition to other known effects and suggest that the factors causing micronucleation by X-irradiation, oxidative DNA damage and incomplete repair, are regulated by apoptosis-independent pathways.     

Persistence of DNA double-strand breaks in normal human cells induced by radiation-induced bystander effect

Our previous study suggested that the DNA double-strand breaks (DSBs) induced by very low X-ray doses are largely due to bystander effects. The aim of this study was to verify whether DSBs created by radiation-induced bystander effects are likely to be repaired. We examined the generation of DSBs in cells by enumeration of phosphorylated ataxia telangiectasia mutated (ATM) foci, which are correlated with DSB repair, in normal human fibroblast cells (MRC-5) after X irradiation at doses ranging from 1 to 1000 mGy. At 24 h after irradiation, 100% (1.2 mGy), 58% (20 mGy), 12% (200 mGy) and 8.5% (1000 mGy) of the initial number of phosphorylated ATM foci were detected. The number of phosphorylated ATM foci in MRC-5 cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; phosphorylated ATM foci were not observed at 5 h (20 mGy) or 24 h (200 mGy) postirradiation. We also counted the number of phosphorylated ATM foci in MRC-5 cells cocultured with MRC-5 cells irradiated with 20 mGy. After 48 h of coculture, 81% of the initial numbers of phosphorylated ATM foci remained. These findings suggest that DSBs induced by the radiation-induced bystander effect persist for long periods, whereas DSBs induced by direct radiation effects are repaired relatively quickly.     

Dilution of irradiated cell conditioned medium and the bystander effect

While nontargeted and low-dose effects such as the bystander effect are now accepted, the mechanisms underlying the response have yet to be elucidated. It has been shown that the transfer of irradiated cell conditioned medium (ICCM) can kill cells that are not directly irradiated; however, to date the effect of ICCM concentration on cell killing has not been reported. The occurrence of a bystander effect was determined by measuring cell survival after exposure to various ICCM dilutions, using the colony-forming assay, in cells of six human cell lines with varied bystander responses and tumor/ p53 status. Autologous ICCM transfer for these cell lines induced a bystander effect as reported previously. ICCM from these cell lines was transferred to cells of a common reporter cell line (HPV-G) to investigate whether the lack of an induced bystander effect was due to their inability to generate or to respond to a bystander signal(s). ICCM from cells of four cell lines induced a bystander effect in HPV-G reporter cells, confirming that signal production is a critical factor. A saturation response was observed when ICCM was diluted. Survival was found to increase linearly until a plateau was reached and the bystander effect was abolished at 2x dilution. The effect of ICCM from the different cell lines reached a plateau at different dilutions, which were found to correlate with the cell line's radiosensitivity.     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

咖啡因抑制辐照区和旁区细胞的γ-H2AX foci的形成

辐射诱导的旁效应不仅在体外实验存在,也在动物体内存在,这对辐射剂量的计算和辐射风险的评估有重要影响。咖啡因是一个模式辐射敏感剂,显著增强辐射的细胞毒性。咖啡因也显著增强辐射诱导的旁效应,咖啡因处理使未受辐照的旁细胞对损伤信号更加敏感,但其机理并不清楚。在正常人原代细胞AG1522中,分析了咖啡因处理对组蛋白H2AX磷酸化的影响,咖啡因显著减少α粒子辐射区和旁区γ-H2AX foci阳性细胞的比率和每个细胞γ-H2AX foci点数,表明咖啡因处理抑制了H2AX磷酸化,后者启动细胞对DNA双链断裂的修复,从而提示咖啡因增强辐射旁效应可能的机理,对辐射治疗具有重要意义。     

The protective role of thiola and soybean seeds against the genotoxicity induced by potassium dichromate in mice

Available data suggesting the occurrence of "bystander effects" (i.e. damage induction in cells not traversed by radiation) were collected and critically evaluated, in view of the development of low-dose risk models. Although the underlying mechanisms are largely unknown, cellular communication seems to play a key role. In this context, the main features of cellular communication were summarised and a few representative studies on bystander effects were reported and discussed. Three main approaches were identified: (1) conventional irradiation of cell cultures with very low doses of light ions; (2) irradiation of single cells with microbeam probes; (3) treatment with irradiated conditioned medium (ICM), i.e. feeding of unexposed cells with medium taken from irradiated cultures. Indication of different types of bystander damage (e.g. cell killing, gene mutations and modifications in gene expression) has been found in each of the three cases. The interpretations proposed by the investigators were discussed and possible biases introduced by specific experimental conditions were outlined. New arguments and experiments were suggested, with the main purpose of obtaining quantitative information to be included in models of low-dose radiation action. Implications in interpreting low-dose data and modelling low-dose effects at cellular and supra-cellular level, including cancer induction, were analysed. Possible synergism with other low-dose specific phenomena such as adaptive response (AR) (i.e. low-dose induced resistance to subsequent irradiation) was discussed.     

Detection of chromosomal instability in bystander cells after Si490-ion irradiation

There is increasing evidence that two of the biological effects associated with low-dose ionizing radiation, genomic instability and bystander responses, may be linked. To verify and validate the link between the two phenomena, the ability of Si490 ions (high-energy particles associated with radiation risk in space) to induce bystander responses and chromosomal instability in human bronchial epithelial (HBEC-3kt) cells was investigated. These studies were conducted at both the population and single cell level in irradiated and nonirradiated bystander cells receiving medium from the irradiated cultures. At the general population level, transfer of medium from silicon-ion (Si490)-irradiated cultures (at doses of 0.073?Gy, 1.2?Gy and 2?Gy) to nonirradiated bystander cells resulted in small increases in the levels of chromosomal aberrations at the first division. Subsequently, single cell clones isolated from irradiated and bystander populations were analyzed for the appearance of de novo chromosome-type aberrations after ～50 population doublings using mFISH. Both irradiated and bystander clones demonstrated chromosomal instability (as seen by the de novo appearance of translocations and chromosomal fragments), albeit to different degrees, whereas sham-treated controls showed relatively stable chromosomal patterns. The results presented here highlight the importance of nontargeted effects of radiation on chromosomal instability in human epithelial cells and their potential relevance to human health.     

Protein tyrosine phosphatase (PTP) inhibition enhances chromosomal stability after genotoxic stress: Decreased chromosomal instability (CIN) at the expense of enhanced genomic instability (GIN)?

Inappropriate survival signaling after DNA damage may facilitate clonal expansion of genetically compromised cells, and it is known that protein tyrosine phosphatase (PTP) inhibitors activate key survival pathways. In this study we employed the genotoxicant, hexavalent chromium [Cr(VI)], which is a well-documented carcinogen of occupational and environmental concern. Cr(VI) induces a complex array of DNA damage, including DNA double strand breaks (DSBs). We recently reported that PTP inhibition bypassed cell cycle arrest and abrogated Cr(VI)-induced clonogenic lethality. Notably, PTP inhibition resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of DNA damage may lead to genomic instability (GIN), via cell cycle checkpoint bypass. The aim of the present study was to determine the effect of PTP inhibition on DNA DSB formation and chromosomal integrity after Cr(VI) exposure. Diploid human lung fibroblasts were treated with Cr(VI) in the presence or absence of the PTP inhibitor, sodium orthovanadate, for up to 24h, and cells were analyzed for DNA DSBs and chromosomal damage. Cr(VI) treatment induced a rapid increase in DNA DSBs, and a significant increase in total chromosomal damage (chromatid breaks and gaps) after 24h. In sharp contrast, PTP inhibition abrogated both DNA DSBs and chromosomal damage after Cr(VI) treatment. In summary, PTP inhibition in the face of Cr(VI) genotoxic stress decreases chromosomal instability (CIN) but increases mutagenesis, which we postulate to be a result of error-prone DNA repair.