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DNA double strand breaks (DSBs) are the most deleterious and lethal form of DNA damage. DSBs are rejoined or repaired by two major pathways in mammalian cells: i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ). DNA-PK is a nuclear, serine/threonine protein kinase consisting of three subunits of DNA-PKcs, Ku70 and Ku80, and involved in the NHEJ, V(D)J recombination and modulation of transcription [1]. Cells lacking DNA-PKcs activity are highly sensitive to DSBs-inducing agents such as ionizing radiations [2]. Douglas et al [3] identified 4 phosphorylation sites in the DNA-PKcs protein: i.e. Thr-2609, Ser-2612, Thr-2638 and Thr-2647. These sites were autophosphorylated with own DNA-PKcs. Chan et al [4] demonstrated that autophosphorylation of DNA-PKcs was required for the rejoining of DSBs. Radiation sensitivities of 31 human esophageal squamous cell carcinoma (ESCC) cell lines were investigated with a colony-formation assay. There was a large variation in radiosensitivity among 31 cell lines. In particular, the radiation sensitivity of one cell line (KYSE190) was distinct from a cluster of radiation sensitivities of other 30 cell lines. In order to understand the mechanism behind this hypersensitivity, we investigated the expression of ATM and DNA-PKcs proteins with a western-blotting method and the phosphorylation of DNA-PKcs with a immunohistochemical staining methd. The phosphorylation of DNA-PKcs was not observed in KYSE190 cells. No mutation was detected in the four phosphorylation sites, but one base change in FAT domain of DNA-PKcs gene was observed. These data seem to indicate that the high radiosensitivity of KYSE 190 cells results from the defect in the autophosphorylation of the DNA-PKcs protein.

Normal human and ataxia telangiectasia (AT) homozygote fibroblasts were irradiated with 2 Gy X-rays, 70 keV/um carbon (290MeV/n) and 200 keV/um iron ions (500MeV/n) at plateau phase cultures, and the kinetics of phosphorylation in various proteins associated with DNA double strand break (DSB) repair were compared. In normal cells, peaks of GammaH2AX foci formation were observed 30 to 60 min post-irradiation for three kinds of radiations, and the disappearance of gamma-H2AX foci was much faster for X-irradiated samples than that for samples irradiated with heavy ions. In AT cells, on the other hand, the peaks of gamma-H2AX phosphorylation for carbon and iron samples were delayed for one to two hours when compared to that for X-rays. The phosphorylation of DNA-PKcs (at threonine 2609) in normal cells was significantly delayed in carbon and iron irradiated cells when compared to X-irradiated cells. Disappearance of DNA-PKcs sites in normal cells was much faster in X-irradiated samples than carbon and iron samples as in the case of gamma-H2AX. However, we were not able to detect DNA-PKcs phosphorylation signals in AT cells irradiated with high LET heavy ions, although DNA-PKcs foci were observed with AT cells irradiated with X-rays. Moreover, in the case of ATM protein phosphorylation (serine 1981) in normal cells, iron irradiation alone caused a significant initial delay, but the kinetics of disappearance is similar for iron and carbon samples with much higher number of remaining foci in iron samples than those for X-rays and carbon ions. \nThese results indicate that 1) high LET irradiation induces complex and/or severe DNA DSB damage which affects the functions of crucial DSB repair proteins, 2) Both ATM and DNA-PKcs may recognize the complexity of DSBs, and 3) ATM may be critical in repairing the complex form of DSB damage such as the one induced by high LET radiation., Radiation Research Society 53rd Annual Meeting

Comparison of phosphorylation kinetics in DNA repair proteins after exposure to high and low LET radiations \nR. Okayasu (1), A. Okabe (1,2) and K. Takakura (2) (1) National Institute of Radiological Sciences, Chiba, Japan, (2) International Christian University, Tokyo, Japan (rokayasu@nirs.go.jp /Fax: +81 43-251-4531 / Phone: +81 43-206-3230) \nWe irradiated plateau phase normal human fibroblasts with 2 Gy X-rays, 70 keV/um carbon (290MeV/n) and 200 keV/um iron ions (500 MeV/n) and observed the kinetics of phosphorylation in various proteins associated with DNA double strand break (DSB) repair. GammaH2AX foci, a marker for DSBs, were detected immediately after irradiation, and the peak of phosphorylation was seen 30 to 60 min post-irradiation for three kinds of radiations. Disappearance of gamma-H2AX foci was much faster for X-irradiated samples than that for heavy ion irradiated samples; the phosphorylation kinetics for carbon and iron ions are similar for gamma-H2AX foci. In contrast, phosphorylation of an NHEJ protein, DNA-PKcs (threonine 2609) was significantly delayed in carbon and iron irradiated cells when compared to X-irradiated cells. Disappearance of DNA-PKcs sites was much faster in X-irradiated samples than carbon and iron samples which showed a similar pattern as in the case of gamma-H2AX. Furthermore, in the case of ATM protein phosphorylation (serine 1981), iron irradiation alone caused a significant initial delay, but the kinetics of disappearance is similar for iron and carbon samples with much higher remaining number of foci in iron samples than those for X-rays and carbon ions. These results suggest that 1) high LET irradiation induces complex and/or severe DNA DSB damage which affects the function of DSB repair proteins, 2) Both ATM and DNA-PKcs may recognize the complexity of DSBs, but ATM may be more sensitive to detecting the complexity of DSB damage, 3) gamma-H2AX may just recognize DSBs, but not the complexity of them. Our studies also indicate that high LET radiation would be a useful tool to elucidate the complexity of initial DSB damage and the mechanism of repair on these damages., The 36th COSPAR Scientifi Assembly

We used an antibody to DNA-PKcs phosphorylated at threonine 2609(T2609) to study the status of autophosphorylation of DNA-PKcs, which is one of the key proteins for Non Homologous End Joining(NHEJ). The number of foci of T2609 induced by X rays in normal human fibroblast cells (HFLIII) and Ligase IV deficient cells (180BR) were counted under fluorescent microscope. DNA-PKcs was autophosphorylated 10 minutes after irradiation and the number of foci reached the peak at 1hour after irradiation in both cell lines. Although the number of foci were gradually reduced and returned to almost the background levels after 24 hours, the remaining number of foci in 180BR cells was much higher than that in normal cells. To investigate the interaction with other proteins, the colocalization of T2609 was studied with phosphorylated ATM at serine 1981(S1981) and gamma H2AX. The focus of T2609 was formed over nucleus and did not correspond with the sites of S1981 and gamma H2AX foci. However the foci of T2609 were accumulated and almost all of them were colocalized with S1981 and gamma H2AX 4 hours after irradiation. In unirradiated cells, gamma H2AX and S1981 foci were not formed at the sites of T2609 foci, although there were some foci of phosphorylated DNA-PKcs. After high LET carbon ion(70keV/um) irradiation, the peak for the number of T2609 foci appearance was significantly delayed in HFLIII and 180BR when compared with X-irradiated cells. This delay was more distinct in irradiated 180BR cells. These results indicate both DNA-PKcs and ATM may function at DSB sites simultaneously. Besides, DNA-PKcs should be phosphorylated not at the sites of DSB. Therefore, the phosphorylation of DNA-PKcs should have not only repair function but also other functions. The results from experiments using high LET heavy ion irradiation are also presented., 第５２回 Radiation Research Society annual meeting

DNA double strand breaks (DSBs) are the most deleterious and lethal form of DNA damage. DSBs are rejoined or repaired by two major pathways in mammalian cells: i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ). DNA-PK is a nuclear, serine/threonine protein kinase consisting of three subunits of DNA-PKcs, Ku70 and Ku80, and involved in the NHEJ, V(D)J recombination and modulation of transcription [1]. Cells lacking DNA-PKcs activity are highly sensitive to DSBs-inducing agents such as ionizing radiations [2]. Douglas et al [3] identified 4 phosphorylation sites in the DNA-PKcs protein: i.e. Thr-2609, Ser-2612, Thr-2638 and Thr-2647. These sites were autophosphorylated with own DNA-PKcs. Chan et al [4] demonstrated that autophosphorylation of DNA-PKcs was required for the rejoining of DSBs. Radiation sensitivities of 31 human esophageal squamous cell carcinoma (ESCC) cell lines were investigated with a colony-formation assay. There was a large variation in radiosensitivity among 31 cell lines. In particular, the radiation sensitivity of one cell line (KYSE190) was distinct from a cluster of radiation sensitivities of other 30 cell lines. In order to understand the mechanism behind this hypersensitivity, we investigated the expression of ATM and DNA-PKcs proteins with a western-blotting method and the phosphorylation of DNA-PKcs with a immunohistochemical staining methd. The phosphorylation of DNA-PKcs was not observed in KYSE190 cells. No mutation was detected in the four phosphorylation sites, but one base change in FAT domain of DNA-PKcs gene was observed. These data seem to indicate that the high radiosensitivity of KYSE 190 cells results from the defect in the autophosphorylation of the DNA-PKcs protein., The 5th Japan-France Workshop on Radiobiology and Medical Imaging