A deficiency in the phosphorylation of DNA-PKcs in a radiosensitive human ESCC cell line

Abstract

DNA double strand breaks (DSBs) are the most deleterious and lethal form of DNA damage. DSBs are rejoined or repaired by two major pathways in mammalian cells: i.e. homologous recombination (HR) and non-homologous end-joining (NHEJ). DNA-PK is a nuclear, serine/threonine protein kinase consisting of three subunits of DNA-PKcs, Ku70 and Ku80, and involved in the NHEJ, V(D)J recombination and modulation of transcription [1]. Cells lacking DNA-PKcs activity are highly sensitive to DSBs-inducing agents such as ionizing radiations [2]. Douglas et al [3] identified 4 phosphorylation sites in the DNA-PKcs protein: i.e. Thr-2609, Ser-2612, Thr-2638 and Thr-2647. These sites were autophosphorylated with own DNA-PKcs. Chan et al [4] demonstrated that autophosphorylation of DNA-PKcs was required for the rejoining of DSBs. Radiation sensitivities of 31 human esophageal squamous cell carcinoma (ESCC) cell lines were investigated with a colony-formation assay. There was a large variation in radiosensitivity among 31 cell lines. In particular, the radiation sensitivity of one cell line (KYSE190) was distinct from a cluster of radiation sensitivities of other 30 cell lines. In order to understand the mechanism behind this hypersensitivity, we investigated the expression of ATM and DNA-PKcs proteins with a western-blotting method and the phosphorylation of DNA-PKcs with a immunohistochemical staining methd. The phosphorylation of DNA-PKcs was not observed in KYSE190 cells. No mutation was detected in the four phosphorylation sites, but one base change in FAT domain of DNA-PKcs gene was observed. These data seem to indicate that the high radiosensitivity of KYSE 190 cells results from the defect in the autophosphorylation of the DNA-PKcs protein