An investigation of clustered radiation-induced lesions in DNA

Recent track structure modelling studies indicate that radiation induced damage to DNA consists of a spectrum of different lesions of varying complexity. There is considerable evidence to suggest that, in repair-proficient systems, it is only the small proportion of more complex forms that is responsible for most of the biological effect. The complex lesions induced consist initially of clustered radical sites and a knowledge of their special chemistry is important in modelling how they react to form the more stable products that are processed by the repair systems. However, much of the current understanding of the chemical stage of radiation has developed from single-radical systems and there is a need to translate this to the more complex reactions that are likely to occur at the important multiple radical sites. With low LET radiation, DNA dsb may derive either from single-radical attack that damages both strands by a transfer mechanism, or from pairs of radical sites induced in close proximity, with one or more radical on each strand. With high LET radiation, modelling studies indicate that there is an increased probability of dsb arising from sites with more than two radical centres, leading to a greater frequency of more complex types of break. The spectrum of these lesions depends on the overall outcome of consecutive physical and chemical processes. The initial pattern of radical damage is determined by the energy depositions on and around the DNA, according to the type of radiation. This pattern is then modified by scavengers that inhibit the formation of radicals on the DNA, and by agents that either chemically repair (e.g. thiols) or fix (e.g. oxygen) a large fraction of these radicals. The reaction kinetics associated with clustered radical sites will differ from those of single sites: (1) because of the opportunities for interactions between the radicals themselves; and (2) because certain endpoints, e.g. a dsb, may require a combination of the products of two or more radicals. Fast response techniques using pulsed low and high LET irradiation have been established to measure the reactions of radical sites on pBR322 plasmid DNA with oxygen and thiols with a view to obtaining information about cluster size. This paper describes experimental approaches to explore the role of the chemical stage of the radiation effect in relation to lesion complexity.

Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9 +/- 0.5 h and a slow component with a half-life of 16 +/- 9 h. For a particles, a fast component with a half-life of 0.7 +/- 0.4 h and a slow component with a half-life of 12 5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37 +/- 0.12) relative to low-LET radiation (0.22 +/- 0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites. (c) 2005 by Radiation Research Society.

ATR-dependent radiation-induced gammaH2AX foci in bystander primary human astrocytes and glioma cells.

Radiotherapy is an important treatment for patients suffering from high-grade malignant gliomas. Non-targeted (bystander) effects may influence these cells' response to radiation and the investigation of these effects may therefore provide new insights into mechanisms of radiosensitivity and responses to radiotherapy as well as define new targets for therapeutic approaches. Normal primary human astrocytes (NHA) and T98G glioma cells were irradiated with helium ions using the Gray Cancer Institute microbeam facility targeting individual cells. Irradiated NHA and T98G glioma cells generated signals that induced gammaH2AX foci in neighbouring non-targeted bystander cells up to 48 h after irradiation. gammaH2AX bystander foci were also observed in co-cultures targeting either NHA or T98G cells and in medium transfer experiments. Dimethyl sulphoxide, Filipin and anti-transforming growth factor (TGF)-beta 1 could suppress gammaH2AX foci in bystander cells, confirming that reactive oxygen species (ROS) and membrane-mediated signals are involved in the bystander signalling pathways. Also, TGF-beta 1 induced gammaH2AX in an ROS-dependent manner similar to bystander foci. ROS and membrane signalling-dependent differences in bystander foci induction between T98G glioma cells and normal human astrocytes have been observed. Inhibition of ataxia telangiectasia mutated (ATM) protein and DNA-PK could not suppress the induction of bystander gammaH2AX foci whereas the mutation of ATM- and rad3-related (ATR) abrogated bystander foci induction. Furthermore, ATR-dependent bystander foci induction was restricted to S-phase cells. These observations may provide additional therapeutic targets for the exploitation of the 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.

Radiation-induced bystander and adaptive responses in cell and tissue models.

The use of microbeam approaches has been a major advance in probing the relevance of bystander and adaptive responses in cell and tissue models. Our own studies at the Gray Cancer Institute have used both a charged particle microbeam, producing protons and helium ions and a soft X-ray microprobe, delivering focused carbon-K, aluminium-K and titanium-K soft X-rays. Using these techniques we have been able to build up a comprehensive picture of the underlying differences between bystander responses and direct effects in cell and tissue-like models. What is now clear is that bystander dose-response relationships, the underlying mechanisms of action and the targets involved are not the same as those observed for direct irradiation of DNA in the nucleus. Our recent studies have shown bystander responses even when radiation is deposited away from the nucleus in cytoplasmic targets. Also the interaction between bystander and adaptive responses may be a complex one related to dose, number of cells targeted and time interval.