Accumulation of Ku70 at DNA double-strand breaks in living epithelial cells

Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

doi:10.1016/j.yexcr.2011.07.018

Cited by 27 publications

(69 citation statements)

References 44 publications

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“…The control mechanisms underlying the subcellular local- ization of Ku70 and Ku80 play a key role in regulating the physiological function of not only Ku itself, but also other NHEJ core factors [6,11,17,20]. Recently, using Ku70 −/− MLE cells, we have shown that Ku70 is essential for the accumulations of XRCC4 and XLF, but not those of Artemis and p21, at DSBs in the early stage following irradiation of lung epithelial cells [10,11]. Thus, the accumulation of the NHEJ core factors XRCC4 and XLF is dependent on Ku; however, that of another NHEJ factor, Artemis, which plays a role in both the NHEJ and HR pathways, is independent of Ku in MLE cells.…”

Section: Discussionmentioning

confidence: 99%

“…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].…”

Section: Cell Lines and Culturesmentioning

confidence: 99%

“…Fluorescence microscopy: The fluorescence in cells was visualized as previously described [8,11]. Briefly, the cells were fixed in PBS containing 4% paraformaldehyde.…”

Section: Cell Lines and Culturesmentioning

confidence: 99%

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

Koike¹,

Yutoku

Koike³

2013

The Journal of Veterinary Medical Science

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ABSTRACT. The DNA repair protein Ku70 is a key player in chemoresistance to anticancer agents (e.g., etoposide) or radioresistance. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. Previously, we established and characterized Ku70-deficient murine lung epithelial (Ku70 −/− MLE) cells and found that these cells are more sensitive than Ku70 +/− MLE cells (control cells) to X-irradiation, as determined by clonogenic survival assay; however, the mechanism underlying this sensitivity remains unclear. In this study, we examined the mechanism by which X-irradiation triggers the death of Ku70 −/− MLE cells. Our results showed that Ku70 −/− MLE cells were more sensitive to radiation-induced apoptosis than control cells, although X-irradiation activated caspase-3 and caspase-7, and cleaved PARP in both cell lines. We also examined the expression level of phosphorylated H2AX (γH2AX), which is a marker of DSB, and observed the phosphorylation of H2AX and the elimination of γH2AX in both cell lines after X-irradiation. The elimination in Ku70 −/− MLE cells was slower than that in control cells, suggesting that DSB repair activity in the Ku70 −/− MLE cells is lower than that in control cells. These findings suggest that Ku70 might play a key role in the inhibition of apoptosis through the DSB repair pathway in lung epithelial cells. Our findings also suggest that these cell lines might be useful for the study of Ku70 functions and the Ku70-dependent DSB repair pathway in lung epithelial cells. KEY WORDS: anticancer treatment, epithelial cell, γH2AX, Ku70, Ku80.

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Section: Discussionmentioning

confidence: 99%

“…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].…”

Section: Cell Lines and Culturesmentioning

confidence: 99%

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

Koike¹,

Yutoku

Koike³

2013

The Journal of Veterinary Medical Science

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“…DNA was stained with 0.025 µg/ml 4,6-diamino-2-phenylindole (DAPI) fluorescent dye (boehringer Mannheim, Mannheim, Germany), and the cells were examined under an Olympus IX 70 fluorescence microscope (Olympus) to determine the localization of proteins. Images were acquired using an FV300 confocal laser scanning microscope (Olympus) as previously described [17,19,21].…”

Section: Cell Lines and Culturesmentioning

confidence: 99%

“…Recently, we have shown that in living lung epithelial cells, but not Ku70-deficient cells, human XRCC4 localizes and accumulates at DSBs after irradiation [21]. Therefore, the mechanisms underlying the control of subcellular localization of XRCC4 are dependent on Ku70 and Ku80 and might play a key role in the regulation of the physiological function of XRCC4 in both hamster and mouse lung cells.…”

mentioning

confidence: 99%

Establishment of Hamster Cell Lines with EGFP-Tagged Human XRCC4 and Protection from Low-Dose X-Ray Radiation

Koike

Yutoku

Koike

2012

The Journal of Veterinary Medical Science

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AbSTRAcT. In clinical settings, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. The mechanisms underlying the control of localization of DNA repair proteins play a key role in the regulation of DNA repair activity. The DNA repair protein XRCC4, which is a regulator of DNA ligase IV activity, might be a key contributor to not only chemoresistance to anticancer agents, e.g., etoposide, but also radioresistance. However, it remains unclear whether XRCC4, which is a key player in nonhomologous DNA-end-joining (NHEJ), plays a role in low-dose radioresistance. In this study, we confirmed that human XRCC4 tagged with the enhanced green fluorescent protein (EGFP-XRCC4), as well as the DNA damage sensor Ku80 tagged with EGFP, mainly localized in the nuclei and its accumulation at DNA damaged sites began immediately after microirradiation. Moreover, we generated and characterized cell lines expressing EGFP-XRCC4 in XRCC4-deficient cells, i.e., XR-1 cells derived from the Chinese hamster ovary. Our findings showed that XR-1 cells were more sensitive than controls (CHO-K1) to low-dose X-irradiation (<0.5 Gy), whereas the radiosensitive phenotype of XR-1 cells was rescued by the expression of EGFP-XRCC4. We also confirmed that EGFP-XRCC4 expressed stably in XR-1 cells stabilizes DNA ligase IV. Altogether, these cell lines might be useful for the study of not only the dynamics and function of XRCC4, but also the molecular mechanism underlying the cellular resistance via the NHEJ pathway to low-dose radiation in mammalian cells.

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Cloning of canine Ku80 and its localization and accumulation at DNA damage sites

2017

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Molecularly targeted therapies have high specificity and significant cancer‐killing effect. However, their antitumor effect might be greatly diminished by variation in even a single amino acid in the target site, as it occurs, for example, as a consequence of SNP s. Increasing evidence suggests that the DNA repair protein Ku80 is an attractive target molecule for the development of next‐generation radiosensitizers for human cancers. However, the localization, post‐translational modifications (PTMs), and complex formation of Ku80 have not been elucidated in canines. In this study, for the first time, we cloned, sequenced, and characterized canine Ku80 cDNA . Our data show that canine Ku80 localizes in the nuclei of interphase cells and is quickly recruited at laser‐induced double‐strand break sites. Comparative analysis shows that canine Ku80 had only 82.3% amino acid identity with the homologous human protein, while the nuclear localization signal ( NLS ) in human and canine Ku80 is evolutionarily conserved. Notably, some predicted PTM sites, including one acetylation site and one sumoylation site within the NLS , are conserved in the two species. These findings suggest that the spatial and temporal regulation of Ku80 might be conserved in humans and canines. However, our data indicate that the expression of Ku80 is considerably lower in the canine cell lines examined than in human cell lines. These important findings might be useful to better understand the mechanism of the Ku80‐dependent DNA repair and for the development of potential next‐generation radiosensitizers targeting common targets in human and canine cancers.

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Accumulation of Ku70 at DNA double-strand breaks in living epithelial cells

Cited by 27 publications

References 44 publications

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

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

Establishment of Hamster Cell Lines with EGFP-Tagged Human XRCC4 and Protection from Low-Dose X-Ray Radiation

Cloning of canine Ku80 and its localization and accumulation at DNA damage sites

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