ATM-dependent Pathway in Human Fibroblasts Arsenite Induces p53 Accumulation through an

Arsenic compounds are potent human carcinogens. Accumulated evidence has shown that arsenite-induced cytogenetic alterations are associated with the carcinogenicity of arsenic. Because p53 plays a guarding role in maintaining genome integrity and accuracy of chromosome segregation, the mechanistic effects of arsenite on p53 activation were analyzed. In the present study, arsenite-induced DNA strand breaks were confirmed by alkaline single-cell gel electrophoresis (comet assay) in human fibroblast (HFW) cells. Accompanying the appearance of DNA strand breaks was a significant accumulation of p53 in arsenite-treated HFW cells, as demonstrated by immunoblotting and immunofluorescence techniques. p53 downstream proteins, such as p21 and the human homologue of murine double minute-2, were also significantly induced by arsenite treatment. Cell cycle retardation and G2-M arrest were observed in 5-bromo2*-deoxyuridine pulse-labeled HFW cells by flow cytometry. Wortmannin, an inhibitor of phosphatidylinositol 3-kinases, inhibited arseniteor X-ray irradiation-induced p53 accumulation but did not alter UV irradiationor N-acetyl-Leu-Leu-norleucinal-induced p53 accumulation. p53 phosphorylation on serine 15 was also confirmed by immunoblotting technique in arseniteand X-ray-treated HFW cells but was not observed in UVor N-acetyl-Leu-Leu-norleucinal-treated HFW cells. These results suggest the involvement of a phosphatidylinositol 3-kinase-related protein kinase in arsenite-induced p53 accumulation. For confirmation, we demonstrated that arsenite treatment, similar to X-ray irradiation, did not induce p53 accumulation in GM3395 fibroblasts derived from a patient with ataxia telangiectasia. In contrast, UV irradiation did cause p53 accumulation in these cells. Together, these findings infer that arsenite-induced DNA strand breaks may lead to p53 phosphorylation and accumulation through an ataxia telangiectasia mutated-dependent pathway in HFW cells.