Responses of hematopoietic cells after ionizing-irradiation in anemic adult medaka (Oryzias latipes).

PURPOSE: Hematopoietic tissues of vertebrates are highly radiation sensitive and the effects of ionizing radiation on the hematopoiesis have been studied in mammals and teleosts for decades. In this study, radiation responses in the kidney, the main hematopoietic organ in teleosts, were investigated in Japanese medaka (Oryzias latipes), which has been a model animal and a large body of knowledge has been accumulated in radiation biology. METHODS: Kidney, the main hematopoietic tissue of adult medaka fish, was locally irradiated using proton and carbon ion beams irradiation system of Takasaki Ion Accelerator for Advanced Radiation Application (TIARA), QST, and the effects on peripheral blood cells and histology of the kidney were investigated. RESULTS: When only kidneys were locally irradiated with proton or carbon ion beam (15 Gy), the hematopoietic cells in the irradiated kidney and cell density in the peripheral blood decreased 7 days after the irradiation in the same manner as after the whole-body irradiation with γ-rays (15 Gy). These results demonstrate that direct irradiation of the hematopoietic cells in the kidney induced cell death and/or cell cycle arrest and stopped the supply of erythroid cells. Then, the cell density in the peripheral blood recovered to the control level within 4 days and 7 days after the γ-ray and proton beam irradiation (15 Gy), respectively, while the cell density in the peripheral blood did not recover after the carbon ion beam irradiation (15 Gy). The hematopoietic cells in the irradiated kidneys temporarily decreased and recovered to the control level within 21 days after the γ-ray or proton beam irradiation (15 Gy), while it did not recover after the carbon ion beam irradiation (15 Gy). In contrast, the recovery of the cell density in the peripheral blood delayed when anemic medaka were irradiated 1 day after the administration of phenylhydrazine. With and without γ-ray irradiation, a large number of hematopoietic cells was still proliferating in the kidney 7 days after the anemia induction. CONCLUSIONS: The results obtained strongly suggest that the hematopoietic stem cells in medaka kidney prioritize to proliferate and increase peripheral blood cells to eliminate anemia, even when they are damaged by high-dose irradiation.

Strain difference in transgene-induced tumorigenesis and suppressive effect of ionizing radiation.

Transgenic expression in medaka of the Xiphophorus oncogene xmrk, under a pigment cell specific mitf promoter, induces hyperpigmentation and pigment cell tumors. In this study, we crossed the Hd-rR and HNI inbred strains because complete genome information is readily available for molecular and genetic analysis. We prepared an Hd-rR (p53+/-, p53-/-) and Hd-rR HNI hybrid (p53+/-) fish-based xmrk model system to study the progression of pigment cells from hyperpigmentation to malignant tumors on different genetic backgrounds. In all strains examined, most of the initial hyperpigmentation occurred in the posterior region. On the Hd-rR background, mitf:xmrk-induced tumorigenesis was less frequent in p53+/- fish than in p53-/- fish. The incidence of hyperpigmentation was more frequent in Hd-rR/HNI hybrids than in Hd-rR homozygotes; however, the frequency of malignant tumors was low, which suggested the presence of a tumor suppressor in HNI genetic background fish. The effects on tumorigenesis in xmrk-transgenic immature medaka of a single 1.3 Gy irradiation was assessed by quantifying tumor progression over 4 consecutive months. The results demonstrate that irradiation has a different level of suppressive effect on the frequency of hyperpigmentation in purebred Hd-rR compared with hybrids.

Comparative histology of wild-type and p53-deficient medaka (Oryzias latipes): nephrotoxic effect of ultraviolet A radiation.

Ultraviolet radiation is an ecological factor that directly affects terrestrial organisms through suppression of immunity or damage to internal organs. The present study assessed the effects of ultraviolet A (UVA) radiation on the kidneys of both wild-type (WT) and p53-deficient medaka (Oryzias latipes) and evaluated which strain was more resistant to the effects of UVA. Fish were divided into four groups: control group 1 (Cwt and Cp53), kept for 3 days without UVA exposure; group 2 (1wt and 1p53), fish exposed daily to UVA for 1 h day-1 for 3 days; group 3 (2wt and 2p53), fish exposed daily to UVA for 2 h day-1 for 3 days; and group 4 (3wt and 3p53), fish exposed daily to UVA for 3 h day-1 for 3 days. Samples of tissues were obtained 24 h after UVA exposure. The most obvious histopathological changes induced by UVA radiation in kidney tissues of both strains of medaka (WT and p53-deficient) were high levels of vacuolation of tubular cells followed by necrosis. The tubular segments lost their normal shape which appeared like a network structure and their cells with clear cytoplasm. Necrosis of lymphoid tissues and spots of brown pigmentation (possibly melanomacrophages) were sporadically seen in interstitial lymphoid tissues, while shrinkage of glomeruli, diminution of periodic acid-Schiff staining, and increased amount of collagenous fibers were observed. Our results confirmed the harmful effects of UVA radiation on kidney tissues of both WT and p53-deficient medaka. However, WT medaka was affected more than p53-deficient medaka.

Protective effect of p53 knockout on 4-nonylphenol-induced nephrotoxicity in medaka (Oryzias latipes).

In the past few decades, environmental pollutants have become common because of misused nonionic surfactants and detergents. Nonylphenol ethoxylates (NPs) are one of the most important contaminants of water. Therefore, the present study aimed to investigate the protective blocking effect of apoptosis (deficient P53 gene) on 4-nonylphenol (4-NP)-induced nephrotoxicity of medaka (Oryzias latipes). We divided 36 fish into six groups: two different control groups of wild type (Wt; Hd-rR) control and p53 (-/-) control, and four different treated with 4-nonylphenol (50 µg/L and 100 µg/L) for 15 days. Histology, immunochemistry, and TUNEL assays confirmed that 4-NP causes nephrotoxicity. Our results showed that 4-NP administration significantly disturbed the kidney structure and function and 4-NP-treated fish showed dilated glomerular vessels, had less glomerular cellular content, decreased expression of glomerular proteins, and an increased level of apoptosis compared with a Wt control group (P < 0.05). As p53 is an apoptotic inducer, some protection in p53-deficient medaka was found as nephrotoxic effects of 4-NP were minimized significantly. Our study demonstrated for the first time to our knowledge that 4-NP induces apoptosis, causing nephrotoxicity in medaka. We found that blocking apoptosis blocking was able to protect the kidney from the toxic effects of 4-NP.

Oocyte atresia in WT (HdrR) and P53 (-/-) medaka (Oryzias latipes) exposed to UVA.

The negative effects of ambient ultraviolet (UVA) on the water environment have been recently highlighted; UVA can create deleterious effects by stimulating stress on pelagic organisms. Little is known about UVA effects on oocyte characteristics of female fish. In the present study we explored the effects of exposure to ecologically relevant levels of simulated UVA radiation on ovaries of two major strains WT (HdrR) and P53 (-/-) of medaka (Oryzias latipes) mature female. Fish were assigned to control and three UVA-exposed groups as (15 min, 30 min, and 60 min/day) for three days and sample selection was 24 h and 14 days after exposure. Histological alterations and oocyte atresia percentage were analyzed in the UVA-exposed fish compared to control. Alteration comprised hyperthrophied follicular cells with increased thickness, breakdown of egg chorion (zona radiata), damage of cortical alveoli, and distorted nucleus and cytoplasm. The atresia percentages significantly increased with higher UVA exposure dose and time for both the wild type and the p53 deficient fish. The wild type displayed significantly higher oocyte atresia percentage than the p53 mutant. These results suggested that UVA exposure provoked histological alterations in both p53 and WT medaka oocytes leading to follicular atresia, which reduce female reproductive ability and larval production. UVA oocyte response showed p53 dependent and independent histological alteration, however, the p53 mutant was less sensitive to UVA than the wild type in medaka fish.

Prognosis of Hippocampal Function after Sub-lethal Irradiation Brain Injury in Patients with Nasopharyngeal Carcinoma.

This work emphasizes the value of assessing hippocampal function by making a timely MRI-based prognosis following a minor dose of hippocampal irradiation after nasopharyngeal carcinomas (NPC) radiotherapy. A quasi-experiment with case-control design and functional assessments (e.g., neuroimaging analysis with fMRI) was conducted to assess hippocampal function after radiotherapy. We delivered 70 Gy of irradiation to nasopharyngeal carcinomas by 6MV helical radiotherapy and collected data from twenty NPC patients and 24 healthy age-matched subjects. Inevitably, hippocampi also received an average dose of 6.89 Gy (range, 2.0-14 Gy). Seed-based functional connectivity of the hippocampus was applied to estimate the cognitive alteration by time before, one month, and four months after irradiation. Afterward, longitudinal-and-cross-sessional statistical inference was determined with time-dependent measurement analysis of variance (ANOVA) with controlled covariance. Over time, there were longitudinal changes in the functional connectivity of hippocampal-related cortices, including the right middle frontal lobe, left superior temporal lobe, and left postcentral gyrus. The findings indicate the presence of functional plasticity, demonstrating how minor irradiation affects functional performance during the early delayed phase of irradiation-induced brain injury.

Immunostaining of UVA-induced DNA damage in erythrocytes of medaka (Oryzias latipes).

Some authors have recently reported that UVA induces double-strand breaks (DSBs) in DNA. Only a few researchers have reported on the induction of DSBs upon UVA exposure, as measured using the Comet assay and γ-H2AX as markers of DSB formation. In the present study, we have investigated for the first time the dose-dependent induction of DSBs by UVA in medaka (Oryzias latipes) erythrocytes. Adult female medaka fish were exposed to UVA for 15, 30, and 60min/day for three continuous days; an unirradiated control group was kept in the same laboratory conditions. At 0h and 24h after UVA exposure, blood was collected to detect DNA damage and repair. The number of γ-H2AX foci was higher than the control value at 0h after UVA exposure and decreased within a 24h. the comet assay showed that DNA repair began during the recovery period. These findings confirm our pervious findings of genotoxic effects after UVA exposure in medaka erythrocytes and suggest that the replication-independent formation of UVA-induced DSBs is mediated through the generation of reactive oxygen species. In conclusion, these results suggest that DNA damage and repair occur after UVA exposure in medaka fish. UVA is the main component of solar UV radiation and is used for artificial UV exposure. Our results may have implications for skin cancer research.

An Approach to Elucidate NBS1 Function in DNA Repair Using Frequent Nonsynonymous Polymorphism in Wild Medaka (Oryzias latipes) Populations.

Nbs1 is one of the genes responsible for Nijmegen breakage syndrome, which is marked with high radiosensitivity. In human NBS1 (hNBS1), Q185E polymorphism is known as the factor to cancer risks, although its DSB repair defect has not been addressed. Here we investigated the genetic variations in medaka (Oryzias latipes) wild populations, and found 40 nonsynonymous single nucleotide polymorphisms (SNPs) in medaka nbs1 (olnbs1) gene within 5 inbred strains. A mutation to histidine in Q170 residue in olNbs1, which corresponds to Q185 residue of hNBS1, was widely distributed in the closed colonies derived from the eastern Korean population of medaka. Overexpression of H170 type olNbs1 in medaka cultured cell lines resulted in the increased accumulation of olNbs1 at laser-induced DSB sites. Autophosphorylation of DNA-dependent protein kinase at T2609 was suppressed after the γ-ray irradiation, which was followed by prolonged formation of γ-H2AX foci and delayed DSB repair. These findings suggested that the nonsynonymous SNP (Q170H) in olnbs1, which induced DSB repair defects, is specifically distributed in the eastern Korean population of medaka. Furthermore, examination using the variation within wild populations might provide a novel method to characterize a driving force to spread the disease risk alleles.

Apoptotic cell death in erythrocytes of p53-deficient medaka (Oryzias latipes) after γ-irradiation.

PURPOSE: Previous studies have examined the effects of γ-irradiation (γ-IR) on wild-type and p53 mutant Medaka (Oryzias latipes) 24 hours after irradiation and in the present work, apoptosis and alterations in erythrocytes of 4, 8 and 24 h and 14 days after gamma-ray irradiation were reported as genotoxic biomarkers of γ-irradiation. MATERIALS AND METHODS: Sexually mature wild-type, WT (Hd-rR) and p53(-/-) adult female medaka (O. latipes) were exposed to 4 Gy dose of γ-IR and sampling were collected after 4, 8 and 24 h and 14 days. RESULTS: Apoptosis and morphological alterations were observed from 4 h after irradiation and remarkably increased 8 h after irradiation in the wild-type. Apoptotic cell death has been observed 8 h after irradiation most prominently but subtle in p53 mutant medaka. All these phenotypes were recovered 14 days after irradiation in both strains. Although no micronuclei were seen in any group, nuclear abnormalities were observed in red blood cells. Both apoptosis and morphological alterations in erythrocytes were decreased after 24 and 14 days after γ-irradiation. CONCLUSIONS: We conclude that apoptosis and malformations caused by 4 Gy γ-irradiation in the erythrocytes of medaka fish occurs from 4-24 h and the initial response until 8 h was p53-dependent.

In vivo 3D analysis of systemic effects after local heavy-ion beam irradiation in an animal model.

Radiotherapy is widely used in cancer treatment. In addition to inducing effects in the irradiated area, irradiation may induce effects on tissues close to and distant from the irradiated area. Japanese medaka, Oryzias latipes, is a small teleost fish and a model organism for evaluating the environmental effects of radiation. In this study, we applied low-energy carbon-ion (26.7 MeV/u) irradiation to adult medaka to a depth of approximately 2.2 mm from the body surface using an irradiation system at the National Institutes for Quantum and Radiological Science and Technology. We histologically evaluated the systemic alterations induced by irradiation using serial sections of the whole body, and conducted a heart rate analysis. Tissues from the irradiated side showed signs of serious injury that corresponded with the radiation dose. A 3D reconstruction analysis of the kidney sections showed reductions in the kidney volume and blood cell mass along the irradiated area, reflecting the precise localization of the injuries caused by carbon-beam irradiation. Capillary aneurysms were observed in the gill in both ventrally and dorsally irradiated fish, suggesting systemic irradiation effects. The present study provides an in vivo model for further investigation of the effects of irradiation beyond the locally irradiated area.

Apoptosis and morphological alterations after UVA irradiation in red blood cells of p53 deficient Japanese medaka (Oryzias latipes).

Morphological alterations in red blood cells were described as hematological bioindicators of UVA exposure to investigate the sensitivity to UVA in wild type Japanese medaka (Oryzias latipes) and a p53 deficient mutant. The fewer abnormal red blood cells were observed in the p53 mutant fish under the control conditions. After exposure to different doses of UVA radiation (15min, 30min and 60min/day for 3days), cellular and nuclear alterations in red blood cells were analyzed in the UVA exposed fish compared with non-exposed controls and those alterations included acanthocytes, cell membrane lysis, swollen cells, teardrop-like cell, hemolyzed cells and sickle cells. Those alterations were increased after the UVA exposure both in wild type and the p53 deficient fish. Moreover, apoptosis analyzed by acridine orange assay showed increased number of apoptosis in red blood cells at the higher UVA exposure dose. No micronuclei but nuclear abnormalities as eccentric nucleus, nuclear budding, deformed nucleus, and bilobed nucleus were observed in each group. These results suggested that UVA exposure induced both p53 dependent and independent apoptosis and morphological alterations in red blood cells but less sensitive to UVA than Wild type in medaka fish.

Acute Toxicity of Gamma Radiation to the Monogonont Rotifer Brachionus koreanus.

We examined the tolerance of the monogonont rotifer Brachionus koreanus in response to gamma radiation. In order to determine the median lethal dose (LD50) of rotifers against gamma radiation, we irradiated B. koreanus with gamma rays from 0 to 7000 grays (Gy). The LD50s were 2900 and 2300 Gy at 24 h (LD50-24 h) and 96 h (LD50-96 h) after irradiation, respectively. In addition, the no observed effect levels (NOEL) were 1500 and 1000 Gy at 24 and 96 h, respectively. This is the first determination of lethal doses of gamma radiation for B. koreanus, which could be useful in ecological assessment of gamma radiation toward aquatic life and could be useful for understanding toxic mechanisms over sublethal doses.

Irradiation-injured brain tissues can self-renew in the absence of the pivotal tumor suppressor p53 in the medaka (Oryzias latipes) embryo.

The tumor suppressor protein, p53, plays pivotal roles in regulating apoptosis and proliferation in the embryonic and adult central nervous system (CNS) following neuronal injuries such as those induced by ionizing radiation. There is increasing evidence that p53 negatively regulates the self-renewal of neural stem cells in the adult murine brain; however, it is still unknown whether p53 is essential for self-renewal in the injured developing CNS. Previously, we demonstrated that the numbers of apoptotic cells in medaka (Oryzias latipes) embryos decreased in the absence of p53 at 12-24 h after irradiation with 10-Gy gamma rays. Here, we used histology to examine the later morphological development of the irradiated medaka brain. In p53-deficient larvae, the embryonic brain possessed similar vacuoles in the brain and retina, although the vacuoles were much smaller and fewer than those found in wild-type embryos. At the time of hatching (6 days after irradiation), no brain abnormality was observed. In contrast, severe disorganized neuronal arrangements were still present in the brain of irradiated wild-type embryos. Our present results demonstrated that self-renewal of the brain tissue completed faster in the absence of p53 than wild type at the time of hatching because p53 reduces the acute severe neural apoptosis induced by irradiation, suggesting that p53 is not essential for tissue self-renewal in developing brain.

Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells.

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3' portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.

Embryonic Medaka Model of Microglia in the Developing CNS Allowing In Vivo Analysis of Their Spatiotemporal Recruitment in Response to Irradiation.

Radiation therapy (RT) is pivotal in the treatment of many central nervous system (CNS) pathologies; however, exposure to RT in children is associated with a higher risk of secondary CNS tumors. Although recent research interest has focused on the reparative and therapeutic role of microglia, their recruitment following RT has not been elucidated, especially in the developing CNS. Here, we investigated the spatiotemporal dynamics of microglia during tissue repair in the irradiated embryonic medaka brain by whole-mount in situ hybridization using a probe for Apolipoprotein E (ApoE), a marker for activated microglia in teleosts. Three-dimensional imaging of the distribution of ApoE-expressing microglia in the irradiated embryonic brain clearly showed that ApoE-expressing microglia were abundant only in the late phase of phagocytosis during tissue repair induced by irradiation, while few microglia expressed ApoE in the initial phase of phagocytosis. This strongly suggests that ApoE has a significant function in the late phase of phagocytosis by microglia in the medaka brain. In addition, the distribution of microglia in p53-deficient embryos at the late phase of phagocytosis was almost the same as in wild-type embryos, despite the low numbers of irradiation-induced apoptotic neurons, suggesting that constant numbers of activated microglia were recruited at the late phase of phagocytosis irrespective of the extent of neuronal injury. This medaka model of microglia demonstrated specific recruitment after irradiation in the developing CNS and could provide a useful potential therapeutic strategy to counteract the detrimental effects of RT.

Progerin, the protein responsible for the Hutchinson-Gilford progeria syndrome, increases the unrepaired DNA damages following exposure to ionizing radiation.

INTRODUCTION: Progerin, the protein responsible for the Hutchinson-Gilford Progeria Syndrome (HGPS), is a partially deleted form of nuclear lamin A, and its expression has been suggested as a cause for dysfunctional nuclear membrane and premature senescence. To examine the role of nuclear envelop architecture in regulating cellular aging and DNA repair, we used ionizing radiation to increase the number of DNA double strand breaks (DSBs) in normal and HGPS cells, and analyzed possible relationship between unrepaired DSBs and cellular aging. RESULTS: We found that HGPS cells are normal in repairing a major fraction of radiation-induced double strand breaks (M-DSBs)but abnormal to show increased amount of residual unrepaired DSBs (R-DSBs). Such unrepaired DSBs were 2.6 times (CI 95 %: 2.2-3.2) higher than that in normal cells one week after the irradiation, and 1.6 times (CI 95 %: 1.3-1.9) higher even one month after the irradiation. These damages tend to increase as the nuclear envelope become abnormal, a characteristic of both HGPS and normal human cells which undergo replicative senescence. The artificial, enforced over-expression of progerin further impaired the repair of M-DSBs, implying lamin A-associated nuclear membrane has an important role for DNA DSB repair. Introduction of telomerase gene function in HGPS cells reversed such aging phenotypes along with upregulation of lamin B1 and downregulation of progerin, which is a hallmark of young cells. CONCLUSION: We suggest that lamin A- or progerin-associated nuclear envelope is involved in cellular aging associated with DNA damage repair.

Nuclear and cytoplasmic changes in erythrocytes of p53-deficient medaka fish (Oryzias latipes) after exposure to gamma-radiation.

Previous studies have examined the effects of gamma-radiation on Japanese fish, in particular medaka (Oryzias latipes). In the present work, alterations in erythrocytes were recorded as haematological bio-indicators of exposure to gamma-radiation. After exposure of medaka fish to two different doses of radiation (2 Gy and 10 Gy), many malformations in red blood cells were observed in the irradiated fish compared with control fish. These malformations included acanthocytes, crenated cells, amoeboid cells, and sickle cells. More malformations were seen at the higher radiation dose. No micronuclei were seen in any group, but nuclear abnormalities were observed. We conclude that gamma-radiation causes morphological malformations of erythrocytes and is harmful to medaka fish.

p53-Dependent suppression of genome instability in germ cells.

Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2(-/-) males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2(-/-) and wild-type fish. By contrast, irradiated p53(-/-) fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2(-/-) fish, but negligible levels in p53(-/-) fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells.

Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after γ-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of γH2AX foci after γ-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of γH2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after γ-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the number of 53BP1 foci compared with wild-type cells. These results suggest that low level of DNA-PK activity causes aberrant DNA-PKcs autophosphorylation in RIC1 cells. It is known that 53BP1 is involved in both DNA-PK dependent and independent NHEJ. Therefore we suggest that DNA-PK independent NHEJ repair DSBs under the condition of decreased DNA-PK activity, which causes reduction of HR efficiency.

Unrepairable DNA double-strand breaks that are generated by ionising radiation determine the fate of normal human cells.

After an exposure to ionising radiation, cells can quickly repair damage to their genomes; however, a few unrepairable DNA double-strand breaks (DSBs) emerge in the nucleus in a prolonged culture and perpetuate as long as the culture continues. These DSBs may be retained forever in cells such as non-dividing ageing tissues, which are resistant to apoptosis. We show that such unrepairable DSBs, which had been advocated by the classical target theory as the 'radiation hit', could account for permanent growth arrest and premature senescence. The unrepairable DSBs build up with repeated irradiation, which accounts for an accumulated dose. Because these DSBs tend to be paired, we propose that the untethered and 'torn-off' molecular structures at the broken ends of the DNA result in an alteration of chromatin structure, which protects the ends of the DNA from genomic catastrophe. Such biochemical responses are important for cell survival but may cause gradual tissue malfunction, which could lead to the late effects of radiation exposure. Thus, understanding the biology of unrepairable damage will provide new insights into the long-term effects of radiation.