Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Koike, Manabu; Ikuta, Togo; Miyasaka, Tsutomu; Shiomi, Tadahiro

doi:10.1038/sj.onc.1203247

Cited by 68 publications

(104 citation statements)

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“…The nuclear translocation of Ku70 is, at least in part, controlled at a nuclear localization signal (NLS)-recognition step by NLS receptors and regulated by the heterodimerization with Ku80 [12][13][14][15]17]. 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.…”

Section: Discussionmentioning

confidence: 57%

“…It was also reported that Ku70 is translocated from the cytoplasm to the nucleus in rat and mouse fibroblasts after X-irradiation [2,29]. On the other hand, we have demonstrated that the nuclear translocation of Ku70 is, at least in part, controlled at a nuclear localization signal (NLS)-recognition step by NLS receptors and regulated by heterodimerization with Ku80 [12][13][14][15]17].…”

mentioning

confidence: 81%

“…Briefly, the SV40 T antigen expression vector (pLNCLT) was transfected using Effectene (Qiagen Inc., Chatsworth, CA, U.S.A.), and stable cell lines were established using G418 (0.3 mg/mL), as described previously [16]. A cell line of a human tumor, HeLa, was cultured as described in previous studies [10,12]. 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 .…”

Section: Mice Cell Lines Cultures and Transfectionmentioning

confidence: 99%

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

Koike¹,

Yutoku

Koike

2011

The Journal of Veterinary Medical Science

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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.

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Section: Discussionmentioning

confidence: 57%

mentioning

confidence: 81%

Section: Mice Cell Lines Cultures and Transfectionmentioning

confidence: 99%

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

Koike¹,

Yutoku

Koike

2011

The Journal of Veterinary Medical Science

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“…The subcellular localization of Ku has been controversial, as discussed in Koike et al (Koike et al, 1999). Reports of purely nuclear (Koike et al, 1999;Bakalkin et al, 1998), membrane (Dalziel et al, 1992), cytoplasmic (Bakalkin et al, 1998) and both nuclear and cytoplasmic (Fewell and Kuff, 1996) localization of Ku have been published (reviewed in Koike et al, 1999). The discrepancy is probably due to differences in the detection methods used or to a change in Ku's subcellular localization during the cell cycle.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, by indirect immunofluorescence, Ku70 and Ku86 were found distributed over both the cytoplasm and nuclei of CHO K1 and xrs-5 cells, with enhanced perinuclear localization of Ku86 in the xrs-5 cells (Yasui et al, 1999). The subcellular localization of Ku has been controversial, as discussed in Koike et al (Koike et al, 1999). Reports of purely nuclear (Koike et al, 1999;Bakalkin et al, 1998), membrane (Dalziel et al, 1992), cytoplasmic (Bakalkin et al, 1998) and both nuclear and cytoplasmic (Fewell and Kuff, 1996) localization of Ku have been published (reviewed in Koike et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Analysis of the DNA replication competence of thexrs-5 mutant cells defective in Ku86

Matheos

Novac

Price

et al. 2003

Journal of Cell Science

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The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein,previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2,PCNA, DNA polymerase ϵ and δ, Primase and Topoisomerase IIα)were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oriβ) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oriβ origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 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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Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

Cited by 68 publications

References 54 publications

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

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

Analysis of the DNA replication competence of thexrs-5 mutant cells defective in Ku86

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

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