Wortmannin, a yeast metabolite, is really a specific inhibitor from the phosphatidylinositol 3-kinase (PI3K) family, including double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the results of wortmannin on DNA damage in DNA-PK-deficient cells acquired from severe combined immunodeficient rodents (SCID cells). Survival of wortmannin-treated cells decreased inside a concentration-dependent manner. After treatment with 50 |¨¬M wortmannin, survival decreased to 60% of this of untreated cells. We observed that treatment with 20 and 50 |¨¬M wortmannin caused DNA damage equal to that by .37 and .69 Gy, correspondingly, of |?-ray radiation. The buildup of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Furthermore, the existence of DSBs was confirmed by ale nuclear extracts from |?-ray-irradiated SCID cells to create in vitro phosphorylation of histone H2AX. These results claim that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.