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Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter) were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associated β-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1Gy of X-rays. While the initial foci size was approximately 0.6microm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6microm or more in diameter at 24h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1, and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1Gy of X-rays and incubated for 24h, the grown large phosphorylated ATM foci (> or =1.6microm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6microm) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint. These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.

Formaldehyde is an aliphatic monoaldehyde and is a highly reactive environmental human carcinogen. Whereas humans are continuously exposed to exogenous formaldehyde, this reactive aldehyde is a naturally occurring biological compound that is present in human plasma at concentrations ranging from 13 to 97 micromol/L. It has been well documented that DNA-protein crosslinks (DPC) likely play an important role with regard to the genotoxicity and carcinogenicity of formaldehyde. However, little is known about which DNA damage response pathways are essential for cells to counteract formaldehyde. In the present study, we first assessed the DNA damage response to plasma levels of formaldehyde using chicken DT40 cells with targeted mutations in various DNA repair genes. Here, we show that the hypersensitivity to formaldehyde is detected in DT40 mutants deficient in the BRCA/FANC pathway, homologous recombination, or translesion DNA synthesis. In addition, FANCD2-deficient DT40 cells are hypersensitive to acetaldehyde, but not to acrolein, crotonaldehyde, glyoxal, and methylglyoxal. Human cells deficient in FANCC and FANCG are also hypersensitive to plasma levels of formaldehyde. These results indicate that the BRCA/FANC pathway is essential to counteract DPCs caused by aliphatic monoaldehydes. Based on the results obtained in the present study, we are currently proposing that endogenous formaldehyde might have an effect on highly proliferating cells, such as bone marrow cells, as well as an etiology of cancer in Fanconi anemia patients.

UV radiation causes cell death through the activation of various intracellular signaling molecules in both DNA damage-dependent and -independent manners. The ability of middle-wavelength UV (UVB) radiation to form DNA photoproducts is less than that of short-wavelength UV (UVC) radiation; however, the differences between UVB and UVC radiation in the extent of DNA damage-independent signaling and its contribution to cell death have not been well characterized. When cells were irradiated with UVB or UVC radiation at doses that generated equivalent amounts of DNA photoproducts, UVB radiation induced more clonogenic cell death, apoptotic cells, mitochondrial cytochrome C release, and intracellular oxidative stress. Among the signaling molecules examined, levels of p53 phosphorylated at Ser-392 and p38 were higher in UVB-irradiated cells than in UVC-irradiated cells. Both phosphorylations were reduced by treating cells with an antioxidant. Furthermore, an inhibitor of p38 also blocked the phosphorylation of p53 at Ser-392. These results suggest that UVB radiation activates the p38 pathway through the generation of oxidative stress, which merges with the DNA p53 pathway by phosphorylation of p53 at ser392. This greater contribution of the DNA damage-independent pathway in UVB-irradiated cells may explain the greater lethality of UVB radiation.

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 polymerase lambda (Pol lambda) is a DNA polymerase beta (Pol beta)-like enzyme with both DNA synthetic and 5'-deoxyribose-5'-phosphate lyase domains. Recent biochemical studies implicated Pol lambda as a backup enzyme to Pol beta in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol lambda and Pol beta in BER of DNA damage in living cells, we disrupted the genes for both enzymes either singly or in combination in the chicken DT40 cell line and then characterized BER phenotypes. Disruption of the genes for both polymerases caused hypersensitivity to H(2)O(2)-induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in cells after H(2)O(2) exposure was lower in Pol beta(-/-)/Pol lambda(-/-) cells than in Pol beta(-/-), wild-type, and Pol lambda(-/-) cells, which were equivalent. These results suggest that these polymerases can complement for one another in counteracting oxidative DNA damage. Similar results were obtained in assays for in vitro BER capacity using cell extracts. With MMS-induced cytotoxicity, there was no significant effect on either survival or BER capacity from Pol lambda gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol beta(-/-)/Pol lambda(-/-) and Pol beta(-/-) cells, suggesting that Pol beta had a dominant role in counteracting alkylation DNA damage in this cell system.

Werner syndrome (WS) is a premature aging syndrome caused by mutations of the WRN gene. Here, we demonstrate that a strain of WS fibroblast cells shows abnormal karyotypes characterized by several complex translocations and 50-fold more frequency of abnormal metaphases including dicentric chromosomes without fragments than normal cells when examined at a similar culture stage. Further, telomere fluorescence in situ hybridization indicates that the abnormal signals, extra telomere signal and loss of telomere signal, emerge two- to three-fold more frequently in WS cells than in normal cells. Taken together, these results indicate that chromosome instability including dysfunction of telomere maintenance is more prominent in WS cells than in normal cells. In addition, the accumulation of DNA double-strand breaks (DSBs) at the G(1) phase, including those at telomeres, detected by phosphorylated ATM (ataxia telangiectasia mutated) foci is accelerated in WS cells even at a low senescence level. The increased accumulation of DSBs in WS cells is reduced in the presence of anti-oxidative agents, suggesting that enhanced oxidative stress in WS cells is involved in accelerated accumulation of DSBs. These results indicate that WS cells are prone to accumulate DSBs spontaneously due to a defect of WRN, which leads to increased chromosome instability that could activate checkpoints, resulting in accelerated senescence.

Ionizing radiation induces genomic instability, transmitted over many generations through the progeny of surviving cells. It is manifested as the expression of delayed effects such as delayed cell death, delayed chromosomal instability and delayed mutagenesis. Induced genomic instability exerts its delayed effects for prolonged periods of time, suggesting the presence of a mechanism by which the initial DNA damage in the surviving cells is memorized. Our recent studies have shown that transmitted memory causes delayed DNA breakage, which in turn activates DNA damage checkpoint, and is involved in delayed manifestation of genomic instability. Although the mechanism(s) involved in DNA damage memory remain to be determined, we suggest that ionizing radiation-induced mega-base deletion destabilizes chromatin structure, which can be transmitted many generations through the progeny, and is involved in initiation and perpetuation of genomic instability. The possible involvement of delayed activation of a DNA damage checkpoint in the delayed induction of genomic instability in bystander cells is also discussed.

We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose-response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and 1 Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose.

Werner syndrome (WS) is an autosomal recessive disease characterized by multiple progeroid features. The gene responsible for WS, WRN, is a member of the human RecQ helicase family. WRN is unique among this family, associated with an exonuclease activity. In the present study, we established the human 293-derived cell lines, which expressed exogenously truncated WRN protein, lacking the N-terminal exonuclease domain but having normal helicase activity, and found that they were slightly, but nonetheless significantly, radiosensitive than control cell lines, into which the empty vector had been introduced. The truncated WRN-expressing cells also exhibited increased numbers of micronuclei, chromosome aberrations, and the foci of phosphorylated histone H2AX with X-rays. These results suggested a function of WRN exonuclease activity that is separable from helicase activity and is essential for the repair of radiation-induced DNA damages.

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.

Ionizing radiation induces genomic instability, which is transmitted over many generations after irradiation through the progeny of surviving cells. Induced genomic instability is manifested as the expression of the following delayed effects: delayed reproductive death or lethal mutation, chromosomal instability, and mutagenesis. Since induced genomic instability accumulates gene mutations (actually genomic instability is the process whereby gene mutation increases subtle difference) and gross chromosomal rearrangements, it has been thought to play a role in radiation-induced carcinogenesis. Radiation-induced genomic instability exerts its effects for prolonged periods of time, suggesting the presence of a mechanism by which the initial DNA damage in the surviving cells is memorized. Recent studies have shown that such memory transmission causes delayed DNA breakage, which in turn plays a role in the induction of delayed phenotypes. Although radiation-induced genomic instability has been studied for years, many questions remain to be answered. This review summarizes the current data on radiation-induced genomic instability. In particular, the mechanism(s) involved in the initiation and perpetuation of radiation-induced genomic instability, and a role of delayed activation of p53 protein are discussed.

To compare the genotoxic effects of high-LET ionizing radiation to those of low-LET radiation, we investigated the responses of human lymphoblastoid cells to DNA damage TK6 after treatment with either low-LET X rays or high-LET iron ions (1000 keV/microm). A highly localized distribution of gammaH2AX/RAD51 foci was observed in the nuclei of cells irradiated with iron ions, in sharp contrast to cells exposed to X rays, where the distribution of foci was much more uniform. This implied the occurrence of a relatively high frequency of closely spaced double-strand breaks, i.e. clustered DNA damage, after iron-ion exposure. Despite the well-established notion that clustered DNA damage is refractory to repair compared to isolated DNA lesions, there were no significant differences in the levels of clonogenic survival and apoptosis between cells treated with iron ions or X rays. Strikingly, however, cells accumulated in G(2)/M phase to a much lesser extent after iron-ion exposure than after X-ray exposure. This differential accumulation could be attributed to a much slower evacuation of the S-phase compartment in the case of cells irradiated with iron ions. Taken together, our results indicate that, relative to the situation for low-LET X rays, exposure to high-LET iron ions results in a substantially greater inhibition of S-phase progression as a result of a higher frequency of DNA replication-blocking clustered DNA damage.

Simple Summary: Canines have a high incidence of mammary tumors, which is a major problem in the veterinary field. Approximately half of canine mammary tumors are malignant and metastasize to nearby lymph nodes. In this study, we analyzed the expression levels of RAD51, a DNA homologous recombination repair-related molecule, in canine mammary tumor cell lines derived from the primary tumor CHMp and metastatic tumor CHMm, which were established from the same individual. The RAD51 expression level in CHMm was higher than that in CHMp. However, CHMp was more resistant than CHMm to irradiation and formed functional Rad51 foci, which form at DNA lesions for homologous recombination repair. The cell cycle regulator p21 was expressed upon radiation exposure in CHMp cells, but not in CHMm cells, indicating that CHMm did not induce cell cycle arrest after irradiation. These results suggest that the radioresistance of CHMm, with high RAD51 expression, is lower than that of CHMp. Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage. [ABSTRACT FROM AUTHOR]

RAD51 forms a complex with BRCA2 and plays a central role in the DNA damage response pathway that is associated with homologous recombination. The structures of RAD51 and its homologues are highly conserved from prokaryotes to higher eukaryotes. Although a large number of BRCA2 mutations have been reported, there are only a few reports on the mutations of RAD51, which have been shown in humans and dogs. However, several mutations of canine RAD51 were identified from mammary gland tumour tissues in a recent study. Some of these mutations seem to have an influence on the homo‐oligomerization or interaction with "Partner and localizer of BRCA2" (PALB2). In this study, we cloned the canine PALB2 homologue and investigated the effect on its interaction with the RAD51 mutants to evaluate the alteration in the function of RAD51 mutants. The A209S and T225S mutants of RAD51 show an attenuation of the interaction between RAD51 and PALB2. These results indicate that the canine RAD51 mutations can potentially alter the homologous recombination pathways in response to DNA damage in dogs. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROSs) are produced during normal cellular metabolism, particularly by respiration in mitochondria, and these ROSs are considered to cause oxidative damage to macromolecules, including DNA. In our previous paper, we found no indication that depletion of mitochondrial superoxide dismutase, SOD2, resulted in an increase in DNA damage. In this paper, we examined SOD1, which is distributed in the cytoplasm, nucleus, and mitochondrial intermembrane space. We generated conditional SOD1 knockout cells from chicken DT40 cells and analyzed their phenotypes. The results revealed that SOD1 was essential for viability and that depletion of SOD1, especially nuclear SOD1, increased sister chromatid exchange (SCE) frequency, suggesting that superoxide is generated in or near the nucleus and that nuclear SOD1 functions as a guardian of the genome. Furthermore, we found that ascorbic acid could offset the defects caused by SOD1 depletion, including cell lethality and increases in SCE frequency and apurinic/apyrimidinic sites. [ABSTRACT FROM AUTHOR]

Abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) is a base excision repair (BER) protein that binds to DNA single strand breaks (SSBs) and subsequently synthesizes and transfers poly(ADP-ribose) polymers to various nuclear proteins. Numerous biochemical studies have implicated PARP-1 as a modulator of BER; however, the role of PARP-1 in BER in living cells remains unclear partly due to lack of accurate quantitation of BER intermediates existing in cells. Since DT40 cells, chicken B lymphocytes, naturally lack PARP-2, DT40 cells allow for the investigation of the PARP-1 null phenotype without confounding by PARP-2. To test the hypothesis that PARP-1 is necessary for efficient BER during methylmethane sulfonate (MMS) exposure in vertebrate cells, intact DT40 cells and their isogenic PARP-1 null counterparts were challenged with different exposure scenarios for phenotypic characterization. With chronic exposure, PARP-1 null cells exhibited sensitivity to MMS but with an acute exposure did not accumulate base lesions or AP sites to a greater extent than wild-type cells. However, an increase in SSB content in PARP-1 null cell DNA, as indicated by glyoxal gel electrophoresis under neutral conditions, suggested the presence of BER intermediates. These data suggest that during exposure, PARP-1 impacts the stage of BER after excision of the deoxyribosephosphate moiety from the 5′ end of DNA strand breaks by polymerase β. [Copyright &y& Elsevier]

Abstract: Ionizing radiation can induce genomic instability in the progeny of irradiated cells, as was demonstrated in various experimental systems. Most in vitro studies have utilized replicating cells, but it is not clear whether radiation-induced genomic instability persists in quiescent cells. Here we show the induction of X-ray-induced genomic instability in normal human diploid cells irradiated and maintained in a quiescent state for up to 24 months while cells were subcultured approximately once every 2–3 months. Every 12 months, a fraction of the irradiated cell population was stimulated to divide by culturing at a low density, and we found that these cells showed increased frequencies of phosphorylated ATM foci, decreased colony-forming ability, and increased frequency of chromosomal aberrations. No significant increases in ROS levels were detected in long-term cultured cells. These results suggest that there are ROS-independent mechanism(s) induced by radiation, which can generate persistent delayed effects in quiescent cells, and could ultimately contribute to carcinogenesis. [Copyright &y& Elsevier]

Purpose: To determine whether glioma cells irradiated with a microplanar X-ray beam exert bystander effects. Methods and Materials: Microplanar beam irradiation of glioma cells in vitro was done using the SPring-8 synchrotron radiation facility. The amount of DNA double-strand breaks (dsbs) was measured by the fluorescence intensity of phosphorylated H2AX or the number of 53BP1 foci. The dose distribution in a cell population exposed to a single microplanar beam was determined by the amount of phosphorylated H2AX-positive cells. Bystander effects were determined by counting the number of 53BP1 foci in nonirradiated cells treated with conditioned medium from cultures of irradiated cells. Results: More DNA dsbs were detected in cells adjacent to an area irradiated by the single beam than in cells in distant, nonirradiated areas as a result of bystander effects caused by scattered X-rays and DNA dsbs. In support of this, more 53BP1 foci were observed in nonirradiated, conditioned medium–treated cells than in control cells (i.e., cells not treated with irradiation or conditioned medium). These results suggest that DNA dsbs were induced in nonirradiated cells by soluble factors in the culture medium. In addition, we observed cellular migration into areas irradiated with peak doses, suggesting that irradiated cells send signals that cause nonirradiated cells to migrate toward damaged cells. Conclusions: Bystander effects are produced by factors secreted as a result of slit-type microplanar X-ray beam irradiation. [Copyright &y& Elsevier]

Abstract: One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (γH2AX) at DNA double-strand breaks (DSBs). However, the role of γH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX −/S139A ) and examined their DNA repair capacity. H2AX −/S139A cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and γH2AX, we disrupted XRCC3 in H2AX −/S139A cells. XRCC3 −/− /H2AX −/S139A mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3 −/− /H2AX −/S139A cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly. [Copyright &y& Elsevier]

Abstract: It is well established that several checkpoint-, or repair factors, such as Ser1981-phosphorylated ATM protein, form discrete nuclear foci at the sites of DNA double-strand breaks (DSBs) caused by ionizing radiation (IR). The foci number decreases concurrently with DNA repair, but some fractions of the foci remain long after irradiation. While the mean size of the initial foci was approximately 0.6 μm in diameter 1 h after IR, the foci grew and the mean size reached ∼ 2.0 μm 24 h after irradiation. The percentage of cells with large foci, whose diameter is defined to be 1.6 μm or more, increased dose-dependently. All of the large foci of phosphorylated ataxia–telangiectasia mutated (ATM) colocalized with foci of Ser139-phosphorylated histone H2AX, mediator of DNA damage checkpoint (MDC)1, 53BP1 and Nijmegen breakage syndrome (NBS)1, which also grew similarly to phosphorylated ATM. When G0-synchronized cells were released soon after 1 Gy of X-rays, most of the cells with large phosphorylated ATM foci show no replication protein A (RPA) staining, which is the marker for the S phase cells, while RPA staining was frequently observed in cells with foci less than 1.6 μm in diameter. Furthermore, the percentage of cells with Ser15-phosphorylated p53 increased dependently on focus diameter of phosphorylated ATM, and more than 80% of the cells with large focus showed pan-nuclear staining of phosphorylated p53. These results indicate that the growth of IR-induced foci plays an essential role in amplifying DNA damage checkpoint signals in order to secure cells with a few residual DSBs or misrepaired DSBs against G1–S transition. [Copyright &y& Elsevier]