Abstract
ATM is autophosphorylated at Ser1981 and forms discrete foci in response to IR. After IR, the number of initially formed foci decreases time-dependently, but a few foci remain long after irradiation. We found that the remaining phosphorylated ATM foci grow, and G1 cells with foci of 1.6 μm or more in diameter were inhibited to progress into S phase. These lines of evidence suggests that the growth of phosphorylated ATM foci is critical for amplification of DNA-damage signals to the level sufficient for G1 arrest induction. Here, we examined a role of the foci growth in G1 arrest using ATM-specific inhibitor. First, G0-synchronized normal human diploid cells were irradiated with 1 Gy of X-rays, and then released from synchronization by replating into 35 mm dishes with BrdU-containing medium. Some of the cultures were treated with 10 μM KU55933, ATM-specific inhibitor. Cells were fixed and stained with anti-Ser1981-phosphorylated ATM and anti-BrdU antibodies 24 h after IR. The percentage of BrdU(+) cells was 32% and 38% in unirradiated cells and unirradiated-KU-treated cells, respectively. The percentage of BrdU(+) cells decreased to 8% with 1Gy of X-rays, whereas it remained at 25% in KU-treated-irradiated cells. In irradiated cells, approximately 20% of mock-treated cells had phosphorylated ATM foci of 1.6 μm or more in diameter, however, most of the KU-treated cells had foci 0.5 μm or less in diameter. These results indicate that the growth of the phosphorylated ATM foci amplify DNA-damage signals, which are necessary for the induction of G1 arrest.
