Establishment of Ku70-Deficient Lung Epithelial Cell Lines and Their Hypersensitivity to Low-Dose X-Irradiation

Abstract

ABSTRACT. In clinical situations, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. The DNA repair protein Ku70 is a key contributor to chemoresistance to anticancer agents, e.g., etoposide and bleomycin, or radioresistance. Ku70 plays a key role as a sensor of DNA double-strand breaks (DSBs) induced following exposure to ionizing radiation as well as treatment with some chemotherapeutic drugs. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. However, it is not clear whether Ku70 plays a role in the low-dose radioresistance of lung epithelial cells. In this study, we established Ku70-deficient epithelial cell lines from murine lungs lacking Ku70. Ku70-/-lung epithelial cells exhibited reduced Ku80 expression. Moreover, Ku70-/-lung epithelial cells were more sensitive than controls (Ku70+/-lung epithelial cells) to low-dose X-irradiation (< 0.5 Gy). We also found that consistent with the Ku70 function as a sensor of DSBs, Ku70 mainly localized in the nuclei of murine lung epithelial cells. These findings clearly indicate that Ku70 plays a key role in regulation of the Ku80 expression level in and the radioresistance of lung epithelial cells. Our data also suggest that these cell lines might be useful not only for study of Ku70 functions and the DSB repair pathway, but also for study of the molecular mechanism underlying the sensitivity to chemotherapeutic drugs and radiation in lung epithelial cells.

The Defect of Ku70 Affects Sensitivity to X-Ray and Radiation-Induced Caspase-Dependent Apoptosis in Lung Cells

“…First, we confirmed the expression of Ku70. Consistent with our previous study [12], Western blot analysis showed that Ku70 was expressed in control (Ku70 +/−) MLE cells, but not in Ku70 −/− MLE cells (Fig. 1A).…”

“…Previously, our findings revealed that Ku70 −/− MLE cells are markedly sensitive to X-irradiation at low doses, from 0.125 to 2 Gy as compared with Ku70 +/− MEL cells (control cells) [12], suggesting that Ku70 plays a key role in radioresistance at doses lower than 2 Gy in lung epithelial cells. However, it remains unclarified whether Ku70 plays a role in radioresistance at doses higher than 2 Gy and works as an inhibitor of radiation-induced death of lung epithelial cells.…”

“…Ku70-deficient murine lung epithelial (MLE) cell lines established from Ku70 −/− mice and control mice (Ku70 +/−) were cultured as described in previous studies [11,12].…”

“…Western blot analysis: Western blot analysis was performed as previously described [9,12,13,16]. The supernatants were electrophoresed on 5-20% SDS-polyacrylamide gels (Wako, Osaka, Japan).…”

“…However, there are few reports about the epithelial cells from Ku70 −/− mice, although more than 90% of cancers are of epithelial cell origin. Recently, we have established and characterized Ku70-deficient murine lung epithelial (MLE) cell lines (Ku70 −/− MLE cells) [12]. These cells showed an increased sensitivity to X-irradiation compared with Ku70 +/− MLE cells.…”