Dynamics of Ku80 in Living Hamster Cells with DNA Double-Strand Breaks Induced by Chemotherapeutic Drugs

Abstract

ABSTRACT. A variety of chemotherapeutic drugs, e.g., etoposide and bleomycin, are widely used in clinical practice to treat many types of animal malignancies. In the clinical situation, cellular resistance to chemotherapy is a significant component of tumor treatment failure. A variety of DNA repair factors, e.g., Ku80, might be a key contributor to chemoresistance to anticancer agents. In both cancer and normal cells, Ku80 plays a key role as a sensor of DNA double-strand break (DSB) induced by treatment with some chemotherapeutic drugs. Although the localization and mobility of Ku80 play a key role in regulating the physiological function of Ku80, it is not clear whether those of Ku80 are affected after treatment with chemotherapeutic drugs. We examined the localization and mobility of Ku80 in living hamster cells with or without DSBs, which were induced by treatment with chemotherapeutic drugs. Our data showed that Ku80, in contrast to H2AX, is highly mobile in the nuclei. We found that before and after the induction of DNA damage by treatment with etoposide or bleomycin, a major portion of Ku80 is exchanged by the same kinetics in the nuclei of interphase cells. These results suggest that the mobility of a major portion of Ku80 is not affected by DNA DSBs in order to find other DSBs. In addition, the information would be worthy to develop some new chemotherapeutic drugs to treat many types of animal malignancies.

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

“…In hamster cells, Ku70/Ku80 heterodimer accumulation at DSBs produced using a 405-nm laser starts immediately after irradiation [18], strongly supporting the idea that the heterodimer of Ku70 and Ku80 is a sensor of DSBs in the nuclei. Most recently, we have reported that Ku80, in contrast to H2AX, is highly mobile in the nuclei of hamster cells and that the mobility of a major portion of Ku80 is not affected by DNA DSBs produced by treatment with antitumor drugs [19]. Ku70 is mainly detected in the nuclei of normal human diploid lung fibroblasts, e.g., TIG-3 and MRC-5, and human cancer cells, e.g., HeLa and MCF-7 cells [11,13,15].…”

“…The cells were grown in a monolayer culture in DMEM (Nissui Seiyaku Co., Tokyo, Japan) supplemented with 10% fetal bovine serum and were maintained in a humidified incubator at 37°C under 5% CO 2 . Transient transfections were performed in Ku70-/-cells using FuGene6 (Roche Diagnostics K.K., Indianapolis, IN, U.S.A.) as described previously, and the cells were cultured for 2 days and then monitored using an FV300 confocal laser scanning microscope (Olympus, Tokyo, Japan) as previously described [18,19].…”

“…Immunofluorescence staining: Immunofluorescence staining was performed as previously described [19]. Briefly, cells were grown on culture slides, washed with PBS, and fixed at room temperature.…”

“…DNA was stained with DAPI fluorescent dye. The distribution of fluorescent signals was monitored using an FV300 confocal laser scanning microscope (Olympus) as previously described [18,19].…”

“…Unrepaired or improperly repaired DSBs can lead to chromosomal truncations and translocations, which can contribute to the development of cancer in higher eukaryotic organisms. DSBs are induced following exposure to ionizing radiation as well as treatment with etoposide or bleomycin [19,24,28]. Two major pathways exist in mammalian cells for repair of DNA DSBs: nonhomologous DNA-end-joining (NHEJ) repair and homologous recombination (HR) [9,25].…”