Microinjection of the restriction endonuclease HaeIII, which causes DNA double-strand breaks with blunt ends, induces nuclear accumulation of p53 protein in normal and xeroderma pigmentosum (XP) primary fibroblasts. In contrast, this induction of p53 accumulation is not observed in ataxia telangiectasia (AT) fibroblasts. HaeIII-induced p53 protein in normal fibroblasts is phosphorylated at serine 15, as determined by immunostaining with an antibody specific for phosphorylated serine 15 of p53. This phosphorylation correlates well with p53 accumulation. Treatment with lactacystin (an inhibitor of the proteasome) or heat shock leads to similar levels of p53 accumulation in normal and AT fibroblasts, but the p53 protein lacks a phosphorylated serine 15. Following microinjection of HaeIII into lactacystin-treated normal fibroblasts, lactacystin-induced p53 protein is phosphorylated at serine 15 and stabilized even in the presence of cycloheximide. However, neither stabilization nor phosphorylation at serine 15 is observed in AT fibroblasts under the same conditions. These results indicate the significance of serine 15 phosphorylation for p53 stabilization after DNA doublestrand breaks and an absolute requirement for ATM in this phosphorylation process.Upon various types of cellular stresses, protein levels of the tumor suppressor p53 increase rapidly, mainly by posttranslational mechanisms. The elevation of p53 in turn induces inhibition of cell cycle progression and/or apoptosis (17). This system is important for the maintenance of the integrity of genetic information and for elimination of abnormal cells. Defects in this system result in a high incidence of tumor progression (6) or in abnormal development (25). The stresses which induce the accumulation of p53 include genotoxic radiation (X ray, ␥ ray, UV, etc.), genotoxic drugs (8), inhibitors of DNA replication and transcription (32), and cellular stresses (9, 30) (heat shock, osmotic shock, hypoxia, etc.). How these stresses are sensed by cells and how the signals are transduced to effector molecules in cells are subjects of great interest.Ataxia telangiectasia (AT) is an autosomal recessive disorder which is characterized by radiosensitivity of the affected individual. Patients suffering from AT often develop neoplasia, and thus a link between radiosensitivity and predisposition to develop cancer is suspected. AT cells are sensitive to ionizing radiation (IR) and show radioresistant DNA synthesis after IR. The gene responsible for AT has recently been identified (ATM), but its role and function in radiosensitivity and predisposition to develop cancer are not known. The AT gene encodes a protein whose homology suggest it to be a member of the phosphatidylinositol 3-kinase family (27). In AT cells, or cells derived from ATM gene knockout mice, p53 accumulation after IR is blunted or delayed compared with that of normal cells (15). However, the p53 responses of AT cells against other genotoxic agents such as UV are normal (4). For this reason, ATM has been sugges...