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It has long been known that the level of radiosensitivity between individuals covers a considerable range. This range is reflected in analysis of patient cell lines with some cell lines showing significantly reduced sensitivity to in vitro radiation exposure. Our increased exposure to radiation from diagnostic medical procedures and other life style changes has raised concerns that there may be individuals who are at an elevated risk from the harmful impact of acute or chronic low dose radiation exposure. Additionally, a subset of patients show an enhanced normal tissue response following radiotherapy, which can cause significant discomfort and, at the extreme, be life threatening. It has long been realised that the ability to identify sensitive individuals and to understand the mechanistic basis underlying the range of sensitivity within the population is important. A reduced ability to efficiently repair DNA double strand breaks (DSB) and/or activate the DSB damage response underlies some, although not necessarily all, of this sensitivity. In this article, we consider the utility of the recently developed γH2AX foci analysis to provide insight into radiation sensitivity within the population. We consider the nature of sensitivity including the impact of radiation on cell survival, tissue responses and carcinogenesis and the range of responses within the population. We overview the current utility of the γH2AX assay for assessing the efficacy of the DNA damage response to low and high dose radiation and its potential future exploitation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The genetic diversity of a range of ionising radiation sensitive mutants of cultured mammalian cell lines has been examined. Hybrids were constructed from suitably marked diploid cells by cell fusion and selected using resistance to HAT and ouabain. Hybrids were examined for ploidy and gamma-ray sensitivity. The data suggest that at least 8 and possibly 9 complementation groups exist which confer sensitivity to ionising radiation. Mutants in at least 3 distinct complementation groups have a reduced ability to rejoin DNA double-strand breaks.

Mutants with defects in the rejoining of DNA double-strand breaks (dsbs) have been identified and characterised from E. coli and the yeast, Saccharomyces cerevisiae. More recently, 3 mammalian cell mutants with defective dsb rejoining have also been described. These mutants are xrs, XR-1 and L5178Y/S, and they are derived from at least two distinct complementation groups. The aim of this article is to review the current status of the studies with these mammalian cell mutants which are defective in dsb rejoining and, in particular, to compare their properties with those mutants identified from lower organisms. Possible mechanistic differences in the process of dsb rejoining between prokaryotes and lower and higher eukaryotes are discussed. All the mammalian mutants defective in dsb rejoining, are sensitive primarily to ionising radiation with little cross-sensitivity to UV-radiation. This is similar to the rad52 mutants of S. cerevisiae but contrasts to the majority of the E. coli mutants with defective dsb rejoining. Where studied, the mammalian cell mutants show enhanced resistance to ionizing radiation in late S/G2 phase, which, in one case, correlates with an enhanced ability to rejoin dsbs. This, together with other evidence, suggests that two mechanisms of dsb rejoining may exist in higher eukaryotes, one which operates uniquely in S/G2 phase and a second mechanism operating throughout the cell cycle and dependent upon the xrs and XR-1 gene products (although whether the xrs and XR-1 dependent pathways are distinct cannot at present be ascertained). Since duplicate homologues will be present in late S/G2 phase cells, this pathway may involve a recombinational mechanism. The xrs-dependent pathway might involve illegitimate recombination, but the xrs mutants do not appear to have a major defect in homologous recombination (involving plasmid DNA) and in this respect are distinct from rad52 mutants.

A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D10 values 5-10-fold of wild-type D10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D10 values less than 2-fold of wild-type D10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks.

Six CHO mutants have previously been described as being sensitive to ionizing radiation and bleomycin treatment, with little or no cross sensitivity to UV-radiation (Jeggo and Kemp, 1983). Their ability to rejoin single- and double-strand breaks has been examined here. Using two techniques, gradient sedimentation and alkaline elution, no difference could be observed between wild-type and mutant strains in the initial number of single-strand breaks induced, the rate of rejoining, or the final level of single-strand breaks rejoined. Thus, a major inability to rejoin single-strand breaks is not the basis for sensitivity in these mutants. In contrast, all 6 mutants showed a decreased ability to rejoin the double-strand breaks induced by gamma-irradiation as measured by neutral elution. Rejoining of half of the breaks occurred in 37 min in wild-type cells and reached a maximum level of 72% after 2 h. All the mutants showed a decreased rate of rejoining, and the final level was 17% of that observed in the wild-type in the most defective mutant, and ranged from 35 to 69% in the other 5 mutants. These are the first mammalian cell mutants to be described with a defect in double-strand break rejoining.

Six X-ray-sensitive strains (xrs) of the Chinese hamster ovary (CHO) cell line, all of which have a defect in double-strand break (dsb) rejoining, have been investigated for their proficiency in DNA transfection assays. All 6 strains and clonal isolates derived from them, show a decreased stable transfection frequency using the plasmids pSV2neo and pSV2gpt after transfection by either the CaPh method or the polybrene method. The magnitude of this effect is DNA concentration dependent and is more marked after transfection with higher DNA concentrations (5-20 micrograms DNA). A spontaneous X-ray-resistant reactivant (or revertant) of one xrs strain also acquired the elevated transfection frequency of the wild-type strain providing evidence for a causal relationship between the decreased transfection frequency and the xrs phenotype. In contrast, the strains show no defect when transfection is assayed using a transient transfection system. Since the transient transfection assay only depends on the uptake and transcriptional activity of foreign DNA, and does not necessitate DNA integration, this suggests that the xrs strains do not have a defect in the uptake of foreign DNA, but might have a defect in integration or the processing of DNA molecules prior to integration.

The genetic diversity of 6 X-ray-sensitive (xrs) mutants of the CHO cell line has been investigated. Hybrids were constructed by fusing ouabain- and 6-thioguanine-resistant cells to ouabain- and 6-thioguanine-sensitive cells and selecting in HAT and ouabain medium. Hybrids were examined for ploidy and X-ray sensitivity. Crosses between xrs mutants and wild-type showed that each mutant was recessive. Crosses between different xrs mutants showed that all were in the same complementation group. Although all the mutants are primarily sensitive to ionizing radiation and bleomycin, and all have a defect in double-strand break rejoining, their cross-sensitivity to other DNA-damaging agents differed to some degree. One explanation is that this repair gene is involved in a pleiotropic response to DNA damage.

6 mutants of the CHO cell line hypersensitive to ionizing radiation have been previously described (Jeggo et al., 1982; Jeggo and Kemp, 1983). In this report, the degree of radiosensitivity of DNA synthesis in these mutant strains has been investigated. 5 of the 6 mutant strains showed a greater degree of inhibition of DNA synthesis following gamma-irradiation compared to the wild-type strains, one mutant showed a similar response to the parent strain. An analysis of the size of the DNA made after irradiation in mutant and wild-type strains indicated that initiation events were more inhibited than chain elongations. This enhanced radiosensitivity is in contrast to results observed using ataxia telangiectasia fibroblasts, in which DNA synthesis is radioresistant in comparison to normal skin fibroblasts. These results are interpreted on the basis of sensitivity resulting from a defect in DNA repair in these mutant strains.

The frequency of both spontaneous and X-ray- (95 rad) induced cytogenetical aberrations has been determined for 2 X-ray-sensitive strains (xrs-6 and xrs-7) of the Chinese hamster ovary cell line, and their wild-type parent (CHO-K1). Increased levels of spontaneous aberrations were not a general feature of the xrs strains, although xrs-7 did show a 2-fold increase in chromatid gaps. Unsynchronied populations of xrs cells, estimated to have been irradiated in late S and G2, showed a 3-5-fold increase in chromatid gaps, breaks and exchanges compared to CHO-K1. The irradiation of synchronised populations of xrs-7 and CHO-K1 in G1 demonstrated a 3-5-fold increase in chromosome breaks, gaps and exchanges in xrs-7. In addition xrs-7 displayed a large increase in chromatid-type aberrations, particularly triradials. These X-ray-sensitive strains have previously been shown to have a defect in double-strand break rejoining (Kemp et al., 1984), and an increased number of double-strand breaks (DBSs) remain in their DNA after irradiation compared to wild-type cells. The increased number of DSBs remaining in these strains 20 min after irradiation, correlates well with the increase in chromosome breaks.