Abstract
It is generally accepted that p53 phosphorylation by ATM is important for radiation-induced G1 arrest, however, relationship between spatiotemporal dynamics of activated ATM and p53 phosphorylation in individual cells remains unknown. Previously, we demonstrated that residual foci of activated ATM (Ser1981-phosphorylated ATM) grow in size time-dependently. Therefore, in the present study, we examined the relationship between the growth of phosphorylated ATM foci and p53 phosphorylation. G0-synchronized normal human diploid cells were treated with Nutlin-3, which stabilizes p53 by inhibition of p53-MDM2 interaction, to make p53 levels constant in all cells. Then, cells were irradiated with 1 Gy of X-rays, and were fixed at 2, 4, 8, 24 h after irradiation, followed by immunofluorescence staining for Ser1981-phosphorylated ATM and Ser15-phosphorylated p53. We measured fluorescence intensity of the both proteins in the nucleus, and found that intensity of both proteins decreased time-dependently on the whole. Interestingly, some of the cells persisted strong fluorescence intensity of the both proteins until 24 h after 1 Gy, like cells at 2 h after 1 Gy, though most of the initial foci disappeared by this time. All of such cells had the large foci (≥1.6 μm), therefore, we investigated p53-phosphorylating ability of a single phosphorylated ATM focus at 24 h after irradiation. We observed clear correlation between diameter of a single phosphorylated ATM focus and fluorescence intensity of phosphorylated p53. These results indicate that p53 phosphorylation is maintained by growth of residual phosphorylated ATM foci long after irradiation.
