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.
ABSTRACT. 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.
The skin is an external organ that is most frequently exposed to radiation. High-dose radiation initiates and promotes acute radiation injury. Thus, it is important to investigate the influence of high-dose radiation exposure on the skin at the molecular level. The post-translational modification of p53 plays a central role in radiation responses, including apoptosis and cell growth arrest. Although it is well known that ataxia telangiectasia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK) can phosphorylate Ser15/Ser18 of p53 in vitro, the post-translational modification pattern and the modifier of p53 in the skin after exposure to high-dose X-rays are not yet well understood. Here we show that the phosphorylation of p53 on Ser15/Ser18, as well as the phosphorylation of histone H2AX on Ser139, was detected in the keratinocytes of the mouse skin and human skin models after high-dose X-ray irradiation. Following high-dose X-ray irradiation, both proteins were also phosphorylated in the skin keratinocytes of both ATM gene knockout mice and DNA-PK-deficient SCID mice.
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