Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

ATF7 mediates TNF-α-induced telomere shortening.

Telomeres maintain the integrity of chromosome ends and telomere length is an important marker of aging. The epidemiological studies suggested that many types of stress including psychosocial stress decrease telomere length. However, it remains unknown how various stresses induce telomere shortening. Here, we report that the stress-responsive transcription factor ATF7 mediates TNF-α-induced telomere shortening. ATF7 and telomerase, an enzyme that elongates telomeres, are localized on telomeres via interactions with the Ku complex. In response to TNF-α, which is induced by various stresses including psychological stress, ATF7 was phosphorylated by p38, leading to the release of ATF7 and telomerase from telomeres. Thus, a decrease of ATF7 and telomerase on telomeres in response to stress causes telomere shortening, as observed in ATF7-deficient mice. These findings give credence to the idea that various types of stress might shorten telomere.

Molecular cloning, subcellular localization, and rapid recruitment to DNA damage sites of chicken Ku70.

Ku70 is a multifunctional protein with pivotal roles in DNA repair via non-homologous end-joining, V(D)J recombination, telomere maintenance, and neuronal apoptosis control. Nonetheless, its regulatory mechanisms remain elusive. Chicken Ku70 (GdKu70) cDNA has been previously cloned, and DT40 cells expressing it have significantly contributed to critical biological discoveries. GdKu70 features an additional 18 amino acids at its N-terminus compared to mammalian Ku70, the biological significance of which remains uncertain. Here, we show that the 5' flanking sequence of GdKu70 cDNA is not nearly encoded in the chicken genome. Notably, these 18 amino acids result from fusion events involving the NFE2L1 gene on chromosome 27 and the Ku70 gene on chromosome 1. Through experiments using newly cloned chicken Ku70 cDNA and specific antibodies, we demonstrated that Ku70 localizes within the cell nucleus as a heterodimer with Ku80 and promptly accumulates at DNA damage sites following injury. This suggests that the functions and spatiotemporal regulatory mechanisms of Ku70 in chickens closely resemble those in mammals. The insights and resources acquired will contribute to elucidate the various mechanisms by which Ku functions. Meanwhile, caution is advised when interpreting the previous numerous key studies that relied on GdKu70 cDNA and its expressing cells.

The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation.

Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1-418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1-418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1-418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1-418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411-418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is important for the physiological function of Rad52 not only at a late stage following irradiation, but also at an early stage.

Acetylation of the nuclear localization signal in Ku70 diminishes the interaction with importin-α.

Proteins are functionally regulated by various types of posttranslational modifications (PTMs). Ku, a heterodimer complex of Ku70 and Ku80 subunits, participates in DNA repair processes. Ku is distributed not only in the nucleus but also in the cytoplasm, suggesting that the function of Ku is regulated by its subcellular localization. Although Ku70 undergoes PTMs including phosphorylation or acetylation, it remains unknown whether the PTMs of Ku70 affect the subcellular localization of Ku. Using a cell-free pull-down assay technique, we show that Nε-acetylation of lysine residues in the synthetic peptide matched to Ku70's nuclear localization signal (NLS) reduces the peptide's interaction with the nuclear transport factor importin-α. The reduced interaction by acetylation was supported by molecular simulation analysis. In addition, when expressed in the endogenous Ku80-defective Chinese hamster ovary xrs-6 cells, some full-size human Ku70 mutants with substitutions of glutamine, a possible structural mimetic of Nε-acetyl-lysine, for lysine at the specific NLS positions exhibited no nuclear distribution. These findings imply that acetylation of particular lysine residues in the Ku70 NLS regulates nuclear localization of Ku.

A possible overestimation of the effect of acetylation on lysine residues in KQ mutant analysis.

Acetylation of lysine residues, one of the most common protein post-transcriptional modifications, is thought to regulate protein affinity with other proteins or nucleotides. Experimentally, the effects of acetylation have been studied using recombinant mutants in which lysine residues (K) are substituted with glutamine (Q) as a mimic of acetyl lysine (KQ mutant), or with arginine (R) as a mimic of nonacetylated lysine (KR mutant). These substitutions, however, have not been properly validated. The effects lysine acetylation on Ku, a multifunctional protein that has been primarily implicated in DNA repair and cell survival, are characterized herein using a series of computer simulations. The binding free energy was reduced in the KQ mutant, while the KR mutant had no effect, which is consistent with previous experimental results. Unexpectedly, the binding energy between Ku and DNA was maintained at almost the same level as in the wild type protein despite full acetylation of the lysine residues. These results suggest that the effects of acetylation may be overestimated when the KQ mutant is used as a mimic of the acetylated protein.

Accumulation of Ku70 at DNA double-strand breaks in living epithelial cells.

Ku70 and Ku80 play an essential role in the DNA double-strand break (DSB) repair pathway, i.e., nonhomologous DNA-end-joining (NHEJ). No accumulation mechanisms of Ku70 at DSBs have been clarified in detail, although the accumulation mechanism of Ku70 at DSBs plays key roles in regulating the NHEJ activity. Here, we show the essential domains for the accumulation and function of Ku70 at DSBs in living lung epithelial cells. Our results showed that EGFP-Ku70 accumulation at DSBs began immediately after irradiation. Our findings demonstrate that three domains of Ku70, i.e., the α/ß, DNA-binding, and Ku80-binding domains, but not the SAP domain, are necessary for the accumulation at or recognition of DSBs in the early stage after irradiation. Moreover, our findings demonstrate that the leucine at amino acid 385 of Ku70 in the Ku80-binding domain, but not the three target amino acids for acetylation in the DNA-binding domain, is involved in the localization and accumulation of Ku70 at DSBs. Furthermore, accumulations of XRCC4 and XLF, but not that of Artemis, at DSBs are dependent on the presence of Ku70. These findings suggest that Artemis can work in not only the Ku-dependent repair process, but also the Ku-independent process at DSBs in living epithelial cells.

KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair.

Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.

Combined ataxia telangiectasia mutated and DNA-dependent protein kinase inhibition radiosensitizes Madin-Darby canine kidney cells.

Uncovering radiation toxicity is critical for the adaptation and expansion of advanced radiation therapies and for the development of novel cancer radiotherapy. In the near future, advanced radiotherapies, including heavy ion beam treatment, are expected to be applied in the treatment of dogs, but further basic research on the effects of radiation using canine normal and cancer cells is necessary to actually apply these techniques and achieve high therapeutic efficacy. The radiation sensitivity is varied by the activities of DNA damage response (DDR) and DNA repair. The development of radiosensitizers that target DDR- and DNA repair-kinases, like ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), is progressing and is expected to be introduced into canine radiotherapy. However, there are no cytotoxicity reports on using the combination of radiation and these sensitizers as treatment in canine cells. In this study, we examined the cytotoxic effects of X-rays and/or radiosensitizers on the Madin-Darby canine kidney (MDCK) cell line. Our results show that X-rays suppress MDCK cell colony formation and proliferation in a dose-dependent manner. Additionally, our observations imply that the combination treatment with ATM inhibitor KU-55933 and DNA-PK inhibitor NU7441 significantly increased X-ray cytotoxicity in MDCK cells compared with the drugs alone. Furthermore, our findings further suggest that MDCK cells might be useful in clarifying the cytotoxicity in canine epithelial cells due to radiation and/or radiosensitizers, such as molecule-targeted drugs.

Feline XRCC4 undergoes rapid Ku-dependent recruitment to DNA damage sites.

Radiation and chemotherapy resistance remain some of the greatest challenges in human and veterinary cancer therapies. XRCC4, an essential molecule for nonhomologous end joining repair, is a promising target for radiosensitizers. Genetic variants and mutations of XRCC4 contribute to cancer susceptibility, and XRCC4 is also the causative gene of microcephalic primordial dwarfism (MPD) in humans. The development of clinically effective molecular-targeted drugs requires accurate understanding of the functions and regulatory mechanisms of XRCC4. In this study, we cloned and sequenced the cDNA of feline XRCC4. Comparative analysis indicated that sequences and post-translational modification sites that are predicted to be involved in regulating the localization of human XRCC4, including the nuclear localization signal, are mostly conserved in feline XRCC4. All examined target amino acids responsible for human MPD are completely conserved in feline XRCC4. Furthermore, we found that the localization of feline XRCC4 dynamically changes during the cell cycle. Soon after irradiation, feline XRCC4 accumulated at laser-induced DNA double-strand break (DSB) sites in both the interphase and mitotic phase, and this accumulation was dependent on the presence of Ku. Additionally, XRCC4 superfamily proteins XLF and PAXX accumulated at the DSB sites. Collectively, these findings suggest that mechanisms regulating the spatiotemporal localization of XRCC4 are crucial for XRCC4 function in humans and cats. Our findings contribute to elucidating the functions of XRCC4 and the role of abnormal XRCC4 in diseases, including cancers and MPD, and may help in developing XRCC4-targeted drugs, such as radiosensitizers, for humans and cats.

Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation.

The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker γ-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful tool as a detection marker of DNA damaged sites.

Establishment of ku70-deficient lung epithelial cell lines and their hypersensitivity to low-dose x-irradiation.

In clinical situations, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. The DNA repair protein Ku70 is a key contributor to chemoresistance to anticancer agents, e.g., etoposide and bleomycin, or radioresistance. Ku70 plays a key role as a sensor of DNA double-strand breaks (DSBs) induced following exposure to ionizing radiation as well as treatment with some chemotherapeutic drugs. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. However, it is not clear whether Ku70 plays a role in the low-dose radioresistance of lung epithelial cells. In this study, we established Ku70-deficient epithelial cell lines from murine lungs lacking Ku70. Ku70-/- lung epithelial cells exhibited reduced Ku80 expression. Moreover, Ku70-/- lung epithelial cells were more sensitive than controls (Ku70+/- lung epithelial cells) to low-dose X-irradiation (< 0.5 Gy). We also found that consistent with the Ku70 function as a sensor of DSBs, Ku70 mainly localized in the nuclei of murine lung epithelial cells. These findings clearly indicate that Ku70 plays a key role in regulation of the Ku80 expression level in and the radioresistance of lung epithelial cells. Our data also suggest that these cell lines might be useful not only for study of Ku70 functions and the DSB repair pathway, but also for study of the molecular mechanism underlying the sensitivity to chemotherapeutic drugs and radiation in lung epithelial cells.

Detection of double-stranded DNA breaks and apoptosis induced by bleomycin in mouse intestine.

The gastrointestinal tract is exposed to a myriad of mutagens, making the DNA damage response (DDR) essential to maintain intestinal homeostasis. In vivo models to study DDRs are necessary to understand the mechanisms of disease development caused by genetic disorders such as colorectal cancer. A double-stranded break (DSB) in DNA is the most toxic type of DNA damage; it can be induced by either X-rays or chemicals, including anticancer agents. If DSBs in DNA cannot be repaired, cells can die by apoptosis to be removed from tissues. Here, we show that the DDRs observed as the phosphorylation of H2AX (γH2AX) and caspase-3-dependent apoptosis-induction are under critical control in the intestine of C57BL mice that were injected intraperitoneally with bleomycin, a natural glycopeptide used clinically as an antitumor agent. We found a significant increase in γH2AX expression 2-6 hr post-treatment in mouse ileum, cecum, and colon tissues by Western blotting and immunostaining. Apoptotic cells were observed after 6-24 hr by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunofluorescence of active caspase-3. We observed that γH2AX expression and apoptotic cells were distributed in the lower part of the crypt. The experimental protocol described here is a simple procedure that can be used generally as an in vivo intestinal toxicity assay. Our experimental approach provides a useful method for examining the effects of various bioactive compounds on the DDR, which is essential for understanding intestinal homeostasis.

Inhibition of Crandell-Rees Feline Kidney cell proliferation by X-ray-induced senescence.

Radioresistance and radiotoxicity have been reported following cancer treatments in felines. Optimizing radiation doses to induce cytotoxic effects to only cancer cells and not normal cells is critical in achieving effective radiation therapy; however, the mechanisms of radiation resistance, radiotoxicity, and DNA damage response (DDR) in feline cells have not yet been elucidated. A DNA double-strand break (DSB) is the most toxic type of DNA damage induced by X-rays and heavy ion beams used in treating cancers. Crandell-Rees Feline Kidney (CRFK) cells is one of the most widely used cat cells in life science research. Here, we report that DSB-triggered senescence induced by X-rays is important in inhibiting the proliferation of CRFK cells. We demonstrated through cell proliferation assay that X-rays at doses 2 Gy and 10 Gy are toxic to CRFK cells that irradiating CRFK cells inhibits their proliferation. In X-irradiated CRFK cells, a dose-dependent increase in DSB-triggered senescence was detected according to morphological changes and using senescence-associated ß galactosidase staining assay. Moreover, our data indicated that in CRFK cells, the major DDR pathway, which involves the phosphorylation of H2AX at Ser139, was normally activated by ATM kinases. Our findings are useful in the understanding of X-rays-induced cellular senescence and in elucidating biological effects of radiation, e.g., toxicity, in feline cells. Furthermore, our findings suggest that the CRFK cell line is an excellent matrix for elucidating radioresistance and radiotoxicity in cat cells.

Dynamics of Ku80 in living hamster cells with DNA double-strand breaks induced by chemotherapeutic drugs.

A variety of chemotherapeutic drugs, e.g., etoposide and bleomycin, are widely used in clinical practice to treat many types of animal malignancies. In the clinical situation, cellular resistance to chemotherapy is a significant component of tumor treatment failure. A variety of DNA repair factors, e.g., Ku80, might be a key contributor to chemoresistance to anticancer agents. In both cancer and normal cells, Ku80 plays a key role as a sensor of DNA double-strand break (DSB) induced by treatment with some chemotherapeutic drugs. Although the localization and mobility of Ku80 play a key role in regulating the physiological function of Ku80, it is not clear whether those of Ku80 are affected after treatment with chemotherapeutic drugs. We examined the localization and mobility of Ku80 in living hamster cells with or without DSBs, which were induced by treatment with chemotherapeutic drugs. Our data showed that Ku80, in contrast to H2AX, is highly mobile in the nuclei. We found that before and after the induction of DNA damage by treatment with etoposide or bleomycin, a major portion of Ku80 is exchanged by the same kinetics in the nuclei of interphase cells. These results suggest that the mobility of a major portion of Ku80 is not affected by DNA DSBs in order to find other DSBs. In addition, the information would be worthy to develop some new chemotherapeutic drugs to treat many types of animal malignancies.

Combined deletions of IHH and NHEJ1 cause chondrodystrophy and embryonic lethality in the Creeper chicken.

The Creeper (Cp) chicken is characterized by chondrodystrophy in Cp/+ heterozygotes and embryonic lethality in Cp/Cp homozygotes. However, the genes underlying the phenotypes have not been fully known. Here, we show that a 25 kb deletion on chromosome 7, which contains the Indian hedgehog (IHH) and non-homologous end-joining factor 1 (NHEJ1) genes, is responsible for the Cp trait in Japanese bantam chickens. IHH is essential for chondrocyte maturation and is downregulated in the Cp/+ embryos and completely lost in the Cp/Cp embryos. This indicates that chondrodystrophy is caused by the loss of IHH and that chondrocyte maturation is delayed in Cp/+ heterozygotes. The Cp/Cp homozygotes exhibit impaired DNA double-strand break (DSB) repair due to the loss of NHEJ1, resulting in DSB accumulation in the vascular and nervous systems, which leads to apoptosis and early embryonic death.

Feline XLF accumulates at DNA damage sites in a Ku-dependent manner.

Resistance to radiotherapy and chemotherapy is a common problem in the treatment of cancer in humans and companion animals, including cats. There is thus an urgent need to develop new treatments. Molecularly targeted therapies hold the promise of high specificity and significant cancer-killing effects. Accumulating evidence shows that DNA double-strand break (DSB) repair proteins, which function in Ku-dependent non-homologous DNA-end joining (NHEJ), are potential target molecules for next-generation cancer therapies. Although cancer radioresistance in cats has been previously described, there are no reports on feline Ku-dependent NHEJ. Here, we cloned and sequenced feline XLF cDNA and characterized X-ray repair cross-complementing protein 4-like factor (XLF), which is one of the core NHEJ proteins. We demonstrated that feline XLF localizes to the nuclei of feline cells and that feline XLF immediately accumulates at laser-induced DSB sites in a Ku-dependent manner. Amino acid sequence alignment analysis showed that feline XLF has only 80.9% identity with human XLF protein, while the predicted nuclear localization signal and putative 14-3-3-binding motif are perfectly conserved among human, cat, dog, chimpanzee, and mouse. These findings are consistent with the hypothesis that regulation of subcellular localization is important for the function of XLF. Furthermore, these findings may be useful in clarifying the mechanisms underlying feline Ku-dependent DSB repair and feline cell radioresistance, and possibly facilitate the development of new molecularly targeted therapies that target common proteins in human and feline cancers.

Tissue-specific DNA-PK-dependent H2AX phosphorylation and gamma-H2AX elimination after X-irradiation in vivo.

Histone H2AX rapidly undergoes phosphorylation at Ser139 (gamma-H2AX) in response to DNA double-strand breaks. Although ATM kinase and DNA-PK phosphorylate Ser139 of H2AX in culture cells, the regulatory mechanism of gamma-H2AX level remains unclear in vivo. Here, we detected the phosphorylation of H2AX and the elimination of gamma-H2AX in the mouse skin after X-irradiation. Furthermore, following X-irradiation, the level of gamma-H2AX also increased in mice lacking either ATM or DNA-PK. Although the elimination after X-irradiation was detected in the skin of these mutant mice, the elimination in DNA-PK-deficient mice was slower than that in C3H and ATM knockout mice, suggesting that a fraction of gamma-H2AX in the skin is eliminated in a DNA-PK-dependent manner. Although the DNA-PK-dependent elimination of gamma-H2AX was also detected in the liver, kidney, and spleen, the DNA-PK-dependent phosphorylation of H2AX was detected in the spleen only. These results suggest that the regulatory mechanism of gamma-H2AX level is tissue-specific.

Histone H2AX phosphorylation independent of ATM after X-irradiation in mouse liver and kidney in situ.

Histone H2AX undergoes phosphorylation at Ser-139 (gamma-H2AX) rapidly in response to DNA double-strand breaks (DSBs) induced by ionizing radiation. The post-translational modification of H2AX plays a central role in responses to radiation, including the repair of DSBs. Although ataxia telangiectasia mutated (ATM) kinase phosphorylates Ser-139 of H2AX in vitro, the post-translational modification pattern and the modifier of H2AX in organs in vivo are not yet well understood. In this study, we detected phosphorylation of H2AX at Ser-139 in cells of the mouse ear, liver, and kidney after X-irradiation. Moreover, the phosphorylation of H2AX was regulated depending on not only the cell type, but also the organ type and the localization of a cell type in an organ. Following X-irradiation, H2AX was phosphorylated in the liver and kidney of ATM gene knockout mice, suggesting that ATM kinase is not essential for phosphorylation of H2AX in these organs after X-irradiation in vivo.

Characterization of Ninjurin and TSC22 induction after X-irradiation of normal human skin cells.

The skin is an external organ that is most frequently exposed to radiation. It is important to elucidate the influence of radiation exposure on the skin at the molecular level. To identify radiation-responsive genes in human skin cells, we used microarray technology to examine the effects of irradiation on 641 genes in normal human epidermal keratinocytes at 4 h and 8 h postirradiation with a cytotoxic dose of X-ray (10 Gy). We found that 18 genes were upregulated and 35 genes were downregulated in keratinocytes at 4 h and/or 8 h postirradiation. Ninjurin, whose function remains unknown in keratinocytes, was induced most strongly by X-irradiation. Several known apoptosis-related genes, such as TSC22, were also upregulated. We characterized Ninjurin and TSC22 induction after X-irradiation of normal human skin cells. The induction of the expression of Ninjurin and TSC22 mRNA in keratinocytes following high-dose X-irradiation was confirmed by northern blot analysis. In dermal fibroblasts, Ninjurin, but not TSC22, was induced after X-ray irradiation. The dependence of both gene expression on the status of an apoptosis regulator, p53, was found. In addition, the expression of both mRNA was induced upon treatment with an apoptosis inducer, etoposide. On the other hand, TSC22, but not Ninjurin, was induced and accumulated in keratinocytes upon treatment with an apoptosis inducer, anisomycin. However, in transient expression assay, EYFP-TSC22, as well as EYFP-Ninjurin or EYFP alone, did not induce apoptosis in keratinocytes in contrast to EYFP-GADD45. Taken together, these findings have important implications on the understanding of the mechanism underlying the complex response of skin cells following X-irradiation.