H2AX phosphorylation in response to DNA double-strand break formation during bystander signalling: effect of microRNA knockdown

Upon DNA double-strand break (DSB) formation, hundreds of H2AX molecules in the chromatin flanking the break site are phosphorylated on serine residue 139, termed gamma-H2AX, so that virtually every DSB site in a nucleus can be visualised within 10 min of its formation using an antibody to gamma-H2AX. One application of this sensitive assay is to examine the induction of DNA double-strand damage in subtle non-targeted cellular effects such as the bystander effect. Here whether microRNA (miRNA) serve as a primary signalling mechanism for bystander effect propagation by comparing matched human colon carcinoma cell lines with wild-type or depleted levels of mature miRNAs was investigated. No major differences were found in the levels of induced gamma-H2AX foci in the tested cell lines, indicating that though miRNAs play a role in bystander effect manifestation, they appear not to be the primary bystander signalling molecules in the formation of bystander effect-induced DSBs.     

Experimental techniques for studying bystander effects in vitro by high and low-LET ionising radiation

Ionising radiation can induce responses within non-exposed neighbouring (bystander) cells which potentially have important implications on the estimates of risk from low dose or low dose rate exposures of ionising radiations. A range of strategies have been developed for investigating bystander effects in vitro for both high-LET alpha particles or low-LET ultrasoft X rays using either partial shielding (grids, half-shields and slits) or by using a co-culture system where two physically separated populations of cells can be cultured together, allowing one population of cells to be irradiated while the second population remains unirradiated. The techniques described provide a useful tool to study bystander effects and complement microbeam studies. Studies using these systems show significant increases in the unirradiated bystander cells for various end points including the induction of chromosomal instability in haemopoetic stem cells and transformation in CGL1 cells.     

Non-targeted effects of radiation: bystander responses in cell and tissue models

The standard paradigm for radiation effects in cellular systems has involved direct damage to DNA and in particular, DNA double strand breaks as the triggering lesions leading to mutation, cell death and transformation. Recently, however, a growing body of evidence has reported non-targeted effects, which are not a direct consequence of the initial lesions produced in cellular DNA. These have included bystander responses, genomic instability, gene induction, adaptive responses and low dose hypersensitivity. A common observation of these responses is that they dominate at low doses and saturate with increasing dose. Non-targeted effects may therefore have consequences for extrapolation of risk estimates to low doses if these are important in vivo. A range of experimental techniques is being used to study non-targeted responses, including microbeam approaches. Microbeams have considerable advantages in that they allow individual cells and subcellular targets to be selected within populations with precise low doses and, if required, exact dose rates. Recent advances also allow targeting of 3-D cell systems. The mechanisms underlying non-targeted responses appear to involve production of reactive oxygen species and direct cell-to-cell signalling via gap junctional intercellular communication although significant differences exist in different cell types. The triggering lesions for these responses remain unclear however. Some non-targeted responses may be inter-related, for example in the case of bystander responses and instability and may be part of a general stress response system in irradiated populations. Some non-targeted effects may also act as protective mechanisms; if they lead to the removal of potentially damaged cells from the population.     

Novel approaches with track segment alpha particles and cell co-cultures in studies of bystander effects

There is now a significant body of data that indicate that the effects of ionising radiation may extend to more than those cells that directly suffer damage to DNA in the cell nucleus. Cells neighbouring those cells that are irradiated, or even well separated from those that are irradiated demonstrate several responses that are recorded in hit cells as a function of absorbed dose. That is, the responding non-hit cells are bystanders of hit cells. A protocol has been devised which allows for examination of one means of eliciting bystander responses, specifically, effects on non-contacting cells. Cell culture chambers are set up such that a population of cells is physically separate from the energy depositions of track segment charged particles. Absorption of energy in sub-millimetre distances in the cell culture medium ensures that one population of cells can only respond to factors generated in the irradiated medium or in another population of irradiated co-cultured cells, which may be of similar or dissimilar origin. For irradiation of medium alone, enhanced levels of micronuclei, and of delays in cell cycle progression occur in normal human fibroblasts, but not epithelial cells. This procedure allows for a defining of the factors responsible for initiating bystander effects and for determining their quantitative relevance.     

Bystander responses in three-dimensional cultures containing radiolabelled and unlabelled human cells

Research on the radiation-induced bystander effect has been carried out mainly in 2-D tissue culture systems. This study uses a 3-D model, wherein apparently normal human diploid fibroblasts (AG1522) are grown in a carbon scaffold, to investigate the induction of a G(1) checkpoint in bystander cells present alongside radiolabelled cells. Cultures were simultaneously pulse-labelled with (3)H-deoxycytidine ((3)HdC) to selectively irradiate a minor fraction of cells, and bromodeoxyuridine (BrdU) to identify the radiolabelled cells. After thorough washing of cultures, iododeoxyuridine (IdU) was administered to detect proliferating bystander cells. The cultures were harvested at various times thereafter, and cells were reacted with two monoclonal antibodies specific to IdU/BrdU or BrdU, respectively, stained with propidium iodide, and subjected to multi-parameter flow cytometry. Cell-cycle progression was followed in radiolabelled cells (BrdU(+)) that were chronically irradiated by low energy beta particles emitted by DNA-incorporated (3)H, and in unlabelled bystander cells (BrdU(-)) by a flow cytometry based cumulative labelling index assay. As expected, radiolabelled cells were delayed, in a dose-dependent manner, in G(2) and subsequently G(1). No delay occurred in progression of bystander cells through G(1), when the labelled cells were irradiated at dose rates up to 0.32 Gy h(-1).     

Genomic instability and the role of radiation quality

Genomic instability (GI) is a hallmark of tumorigenic progression and is observed as delayed genetic damage in the progeny of irradiated and unirradiated bystander cells. The expression of GI can be influenced by genotype, cell type and radiation quality. While several studies have demonstrated the induction of GI by high and low-linear energy transfer (LET) radiation, our work on human and mouse primary cell systems has shown LET-dependent differences in the induction and expression of GI. These differences might be attributed to differences in radiation track structure, dose rate, contribution of bystander cells and radiation dose. This paper reviews the role of radiation quality in the induction of GI and describe the possible mechanisms underlining the observed differences between radiation types on its induction. The experimental results presented suggest that dose might be the most significant factor in determining induction of GI after low-LET radiation.     

Prediction of dose response for radiation induced exchange aberrations taking cell cycle delays into account

Chromosomal aberrations (CAs) are regarded as one of the most sensitive biological indicators of genetic alterations. The aberration frequency is routinely determined in the first metaphase. Yet, the data interpretation can be complicated due to radiation induced mitotic delays. To investigate the effect of delays on CA frequency in the first mitosis, human lymphocytes were irradiated with X rays and Giemsa detectable CAs were measured at different sampling times. Besides, a computer simulation was performed reproducing the main effects under investigation, that is, CA induction and cell progression through the mitotic cycle. The CA formation model takes into account the structural organisation of interphase chromosomes in a lymphocyte nucleus, DNA double-strand break (DSB) induction and their rejoining/misrejoining. Lymphocyte transition through the cell cycle was simulated by a Monte Carlo technique. The delay was proposed to result from DNA DSBs. The predicted ratios of first/second/third cycle metaphases agree with the experimental data for control and irradiated samples. Both experimental and calculated CA frequencies in the first mitosis were nearly time-independent. This was proposed to result from de-synchronisation of the lymphocyte population.     

A model for the induction of chromosome aberrations through direct and bystander mechanisms

A state vector model (SVM) for chromosome aberrations and neoplastic transformation has been adapted to describe detrimental bystander effects. The model describes initiation (formation of translocations) and promotion (clonal expansion and loss of contact inhibition of initiated cells). Additional terms either in the initiation model or in the rate of clonal expansion of initiated cells, describe detrimental bystander effects for chromosome aberrations as reported in the scientific literature. In the present study, the SVM with bystander effects is tested on a suitable dataset. In addition to the simulation of non-linear effects, a classical dataset for neoplastic transformation in C3H 10T1/2 cells after alpha particle irradiation is used to show that the model without bystander features can also describe LNT-like dose responses. A published model for bystander induced neoplastic transformation was adapted for chromosome aberration induction and used to compare the results obtained with the different models.     

Radiation-induced bystander effects induce radioadaptive response by low-dose radiation

When normal human fibroblast cells (MRC-5) received a priming irradiation of 3-20 mGy 4 h prior to irradiation with 1000 mGy, the number of DNA double-stranded breaks (DSBs) decreased significantly to 18.2-18.7 per cell compared with 21 per cell when there was no priming irradiation. This result indicates that a priming irradiation of 3-20 mGy induces a radioadaptive response in MRC-5. The authors' previous study had indicated that DSBs induced by ≤ 20 mGy are due to a radiation-induced bystander effect. These findings suggest that radiation-induced bystander effects might contribute to induction of the radioadaptive response. To test this hypothesis, MRC-5 were suspended in lindane, an inhibitor of radiation-induced bystander effects, which was added to the medium for the priming irradiation of 3-20 mGy. Lindane inhibited the protective effect of priming irradiation on DSBs caused by subsequent irradiation with 1000 mGy. Thus, radiation-induced bystander effects may play a role in radioadaptive responses.     

First attempts at prediction of DNA strand-break yields using nanodosimetric data

We present the first results of our attempts to correlate yields of ionisation clusters in a gas model of DNA and corresponding double-strand break (DSB) yields in irradiated plasmids, using a simple statistical model of DNA lesion formation. Based on the same statistical model, we also provide a comparison of simulated nanodosimetric data for electrons and published DSB yields obtained with the PARTRAC code.     

Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.     

Coupling of cell fate selection model enhances DNA damage response and may underlie BE phenomenon

Double‐strand break‐induced (DSB) cells send signal that induces DSBs in neighbour cells, resulting in the interaction among cells sharing the same medium. Since p53 network gives oscillatory response to DSBs, such interaction among cells could be modelled as an excitatory coupling of p53 network oscillators. This study proposes a plausible coupling model of three‐mode two‐dimensional oscillators, which models the p53‐mediated cell fate selection in globally coupled DSB‐induced cells. The coupled model consists of ATM and Wip1 proteins as variables. The coupling mechanism is realised through ATM variable via a mean‐field modelling the bystander signal in the intercellular medium. Investigation of the model reveals that the coupling generates more sensitive DNA damage response by affecting cell fate selection. Additionally, the authors search for the cause‐effect relationship between coupled p53 network oscillators and bystander effect (BE) endpoints. For this, they search for the possible values of uncertain parameters that may replicate BE experiments’ results. At certain parametric regions, there is a correlation between the outcomes of cell fate and endpoints of BE, suggesting that the intercellular coupling of p53 network may manifest itself as the form of observed BEs.Inspec keywords: biological effects of ionising particles, molecular biophysics, biochemistry, DNA, cellular biophysics, physiological models, biomolecular effects of radiation, cellular effects of radiation, biological effects of X‐rays, oscillations, proteinsOther keywords: three‐mode two‐dimensional oscillators, p53‐mediated cell fate selection, globally coupled DSB‐induced cells, coupled model consists, coupling mechanism, ATM variable, bystander signal, intercellular medium, sensitive DNA damage response, coupled p53 network oscillators, intercellular coupling, cell fate selection model, double‐strand break‐induced cells, DSBs, neighbour cells, oscillatory response, excitatory coupling, plausible coupling model     

Feasibility of a neutron detector-dosemeter based on single-event upsets in dynamic random-access memories

In an attempt to investigate the effect of radiation quality, dose and specific repair pathways on correct and erroneous rejoining of DNA double strand breaks (DSBs), an assay was applied that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionising radiation. While substantial misrejoining occurs in mammalian cells after high acute irradiation doses, decreasing misrejoining frequencies were observed in dose fractionation experiments with X rays. In line with this finding, continuous irradiation with gamma rays at low dose rate leads to no detectable misrejoining. This indicates that the probability for a DSB to be misrejoined decreases drastically when DSBs are separated in time and space. The same dose fractionation approach was applied to determine DSB misrejoining after alpha particle exposure. In contrast to the results with X rays, there was no significant decrease in DSB misrejoining with increasing fractionation. This suggests that DSB misrejoining after alpha irradiation is not significantly affected by a separation of particle tracks. To identify the enzymatic pathways that are involved in DSB misrejoining, cell lines deficient in non-homologous end-joining (NHEJ) were examined. After high X ray doses, DSB misrejoining is considerably reduced in NHEJ mutants. Low dose rate experiments show elevated DSB misrejoining in NHEJ mutants compared with wild-type cells. The authors propose that NHEJ serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.     

Luminescence of CsGd2F7 crystals

The induction of mutations at the Hprt locus and minisatellite sequences was studied in V79 cells, peripheral blood lymphocytes (PBL) and lymphoblastoid cells (CCRF-CEM) exposed to gamma rays. In V79 cells the Hprt mutant frequency increased with dose at least up to 6.0 Gy, whereas the number of HPRT mutant lymphocytes increased up to 3 Gy. Clones derived from single irradiated cells were screened for mutations at minisatellite sequences by DNA fingerprint analysis. In V79 cells, a dose-response curve for minisatellite alterations was obtained up to 4.5 Gy. In contrast, very few mutations at minisatellite sequences (2/137) were detected among clones isolated from PBL of two donors irradiated with 1-4 Gy. Similar results were observed in lymphoblastoid CCRF-CEM cells irradiated with 2-3 Gy (4 mutants/180 clones), suggesting that in human lymphoid cells minisatellite DNA is more stable than in other mammalian and human cell lines.     

A review of the bystander effect and its implications for low-dose exposure

Current models for the interaction between ionising radiation and living cells or tissues are based on direct genetic damage produced by energy deposition in cellular DNA. An important observation which has questioned this basic assumption is the radiation-induced bystander response, in which cells which have not been directly targeted respond if their neighbours have been exposed. This response predominates at low doses of relevance to radiation risk analysis (<0.2 Gy) and therefore needs to be fully characterised. The development of microbeams, which allow individual cells within populations to be targeted with precise doses of radiation, has provided a useful tool for quantifying this response. The authors' studies have targeted individual human and mouse cells with counted protons and helium ions and monitored neighbouring cells for the production of bystander responses. Bystander responses have been measured after exposures as low as a single proton or helium ion delivered to an individual cell. An important aspect is that these responses saturate with increasing dose to the single target cell, thus the relative roles of direct and indirect (non-targeted) responses change with dose. Studies with multicellular, tissue-based models are providing evidence that bystander responses may have a complex phenotype involving multiple pathways and the overall response may be a balance between multiple signalling processes and responses to radiation exposure. Current models for radiation risk assume a linear non-threshold response and have generally been extrapolated from high-dose exposures. The involvement of competing processes at low doses may have important consequences for understanding the effects of low-dose exposure.     

Characterisation of a bystander effect induced in human tissue explant cultures by low let radiation

The existence of a bystander effect following both alpha and gamma irradiation of many cell lines is not now in dispute. The significance of this effect for cancer risk assessment and radiotherapy treatment planning requires demonstration of its relevance in vivo. The problem in demonstrating the existence of the effect in vivo is that other systemic effects may mask or confound the effect being investigated and it is practically impossible to attribute an effect in a particular cell to a signal produced in another irradiated cell. To approach this problem, an assay has been developed where fragments of human tissue can be irradiated ex vivo and the media harvested and added to unirradiated, clonogenic cells which have a well characterised and stable response to the bystander signal. The variation in the production of a signal from patient to patient can thus be assessed. The results of a study using tissue from over 100 patients attending Beaumont and St Vincent's Hospitals in Dublin for investigation of urological disorders including follow-up after treatment for transitional cell carcinoma (TCC) and resection of suspect prostatic lesions, are now available. Blood samples from the prostate group were also obtained. The results show that there is variation in the effect of the signal produced by irradiated tissue from different patients. This holds for bladder, prostate and blood. Gender, smoking status and the existence of a malignancy influence the expression of the signal by normal tissue. Male gender, smoking and a pre-existing malignancy all reduce the amount or effect of the signal produced into medium when the tissue is exposed. The effects of exposure to medium containing the signal are transmitted to distant progeny of the exposed cell population. The results may be important not only for understanding radiation risk mechanisms for protection but also for radiotherapy treatment planning where they may open new avenues for development of drugs for combined therapy.     

Genotoxic damage in non-irradiated cells: contribution from the bystander effect

It has always been accepted dogma that the deleterious effects of ionising radiation such as mutagenesis and carcinogenesis are due mainly to direct damage to DNA. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, it is shown here that non-irradiated cells acquire the mutagenic phenotype through direct contact with cells whose nuclei are traversed with 2 alpha particles each. Pre-treatment of cells with lindane, a gap junction inhibitor, significantly decreased the mutant yield. Furthermore, when irradiated cells were mixed with control cells in a similar ratio as the in situ studies, no enhancement in bystander mutagenesis was detected. Our studies provide clear evidence that genotoxic damage can be induced in non-irradiated cells, and that gap junction mediated cell-cell communication plays a critical role in the bystander phenomenon.     

The production of single strand and double strand breaks in DNA in aqueous solution by vacuum UV photons below 10 eV

The mechanisms of break formation in fully hydrated DNA have been investigated using monochromatic photons below 10 eV. This has been achieved by developing a novel 'wet cell' for irradiating DNA in aqueous solution. Our preliminary data show that 7-10 eV photons readily induce strand breaks even though almost all of the energy is absorbed in the water. Therefore, the mechanism for the induction of single and double strand breaks (SSBs and DSBs) most likely involves indirect damage by OH radicals and is substantiated by data from studies in the presence of the OH radical scavenger Tris, which showed a substantial protective effect. The dose-effect curve for DSB induction is seen to be linear, or near-linear, indicating the involvement of 1-hit mediated induction of DSBs. These data point to single-event induction of DSBs being a significant pathway with all radiation types.     

Track structure calculations on hypothetical subcellular targets for the release of cell-killing signals in bystander experiments with medium transfer

Track structure studies using PARTRAC have been performed with the aim to investigate the possibility of revealing information on initiating targets and mechanisms of bystander effects mediated by signals released into the culture medium. Dependences on radiation dose have been assessed for alternative signal emission scenarios, defined by required energy deposits in a number of subcellular targets, mimicking e.g. mitochondria as hypothetical targets for the release of signals. The simulation results agree with target theory, and elucidate the characteristic dose for signal emission as a function of target topology, size and activation energy. The observed dose dependence of bystander cell kill in medium transfer experiments is not as steep as predicted by the considered simple signal emission scenarios with a single or even multiple hits to the hypothetical targets. This has been resolved by accounting for variations in cellular characteristics among the irradiated cells.