Cloning, localization and focus formation at DNA damage sites of canine Ku70

Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

doi:10.1292/jvms.16-0649

Cited by 16 publications

(38 citation statements)

References 33 publications

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“…Expectedly, EYFP, used as a control, was distributed throughout the cell (with the exclusion of the nucleoli) in pEYFP‐transfected cells (Fig. D), consistent with our previous reports . Meanwhile, during mitosis, EYFP‐canine Ku80 was detected throughout the cytoplasm of the transfected cells, but was not localized to mitotic chromosomes (Fig.…”

Section: Resultsmentioning

confidence: 95%

“…Local DNA damage induction using laser and subsequent cell imaging was carried out as described previously . Immunocytochemistry was carried out using a mouse anti‐γH2AX monoclonal antibody (JBW301; Upstate Biotechnology Inc., Charlottesville, VA, USA) and an Alexa Fluor 568‐conjugated secondary antibody (Molecular Probes, Eugene, OR, USA), as previously described .…”

Section: Methodsmentioning

confidence: 99%

“…pEYFP-canine Ku80 or pEYFP-C1 was transiently transfected into cells using Lipofectamine 3000 (Invitrogen). Post-transfection, cells were cultured for 2 days and then observed under an FV300 confocal laser-scanning microscope (Olympus, Tokyo, Japan), as previously described [8,[13][14][15][16][17][18]. DNA in the fixed cells was stained with 4,6-diamino-2-phenylindole (DAPI) fluorescent dye, as previously described [18].…”

Section: Cell Lines Cultures and Transfectionsmentioning

confidence: 99%

“…Local DNA damage induction using laser and subsequent cell imaging was carried out as described previously [9,15,17]. Immunocytochemistry was carried out using a mouse anti-cH2AX monoclonal antibody (JBW301;…”

Section: Local Dna Damage Induction Using Laser and Cell Imagingmentioning

confidence: 99%

“…Upstate Biotechnology Inc., Charlottesville, VA, USA) and an Alexa Fluor 568-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA), as previously described [14,15,18].…”

Section: Local Dna Damage Induction Using Laser and Cell Imagingmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

Section: Cell Lines Cultures and Transfectionsmentioning

confidence: 99%

Section: Local Dna Damage Induction Using Laser and Cell Imagingmentioning

confidence: 99%

“…Upstate Biotechnology Inc., Charlottesville, VA, USA) and an Alexa Fluor 568-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA), as previously described [14,15,18].…”

Section: Local Dna Damage Induction Using Laser and Cell Imagingmentioning

confidence: 99%

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

2017

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Feline XLF accumulates at DNA damage sites in a Ku‐dependent manner

2019

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Resistance to radiotherapy and chemotherapy is a common problem in the treatment of cancer in humans and companion animals, including cats. There is thus an urgent need to develop new treatments. Molecularly targeted therapies hold the promise of high specificity and significant cancer‐killing effects. Accumulating evidence shows that DNA double‐strand break ( DSB ) repair proteins, which function in Ku‐dependent non‐homologous DNA ‐end joining ( NHEJ ), are potential target molecules for next‐generation cancer therapies. Although cancer radioresistance in cats has been previously described, there are no reports on feline Ku‐dependent NHEJ . Here, we cloned and sequenced feline XLF cDNA and characterized X‐ray repair cross‐complementing protein 4‐like factor ( XLF ), which is one of the core NHEJ proteins. We demonstrated that feline XLF localizes to the nuclei of feline cells and that feline XLF immediately accumulates at laser‐induced DSB sites in a Ku‐dependent manner. Amino acid sequence alignment analysis showed that feline XLF has only 80.9% identity with human XLF protein, while the predicted nuclear localization signal and putative 14‐3‐3‐binding motif are perfectly conserved among human, cat, dog, chimpanzee, and mouse. These findings are consistent with the hypothesis that regulation of subcellular localization is important for the function of XLF . Furthermore, these findings may be useful in clarifying the mechanisms underlying feline Ku‐dependent DSB repair and feline cell radioresistance, and possibly facilitate the development of new molecularly targeted therapies that target common proteins in human and feline cancers.

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Feline XRCC4 undergoes rapid Ku‐dependent recruitment to DNA damage sites

2022

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Radiation and chemotherapy resistance remain some of the greatest challenges in human and veterinary cancer therapies. XRCC4, an essential molecule for nonhomologous end joining repair, is a promising target for radiosensitizers. Genetic variants and mutations of XRCC4 contribute to cancer susceptibility, and XRCC4 is also the causative gene of microcephalic primordial dwarfism (MPD) in humans. The development of clinically effective molecular‐targeted drugs requires accurate understanding of the functions and regulatory mechanisms of XRCC4. In this study, we cloned and sequenced the cDNA of feline XRCC4. Comparative analysis indicated that sequences and post‐translational modification sites that are predicted to be involved in regulating the localization of human XRCC4, including the nuclear localization signal, are mostly conserved in feline XRCC4. All examined target amino acids responsible for human MPD are completely conserved in feline XRCC4. Furthermore, we found that the localization of feline XRCC4 dynamically changes during the cell cycle. Soon after irradiation, feline XRCC4 accumulated at laser‐induced DNA double‐strand break (DSB) sites in both the interphase and mitotic phase, and this accumulation was dependent on the presence of Ku. Additionally, XRCC4 superfamily proteins XLF and PAXX accumulated at the DSB sites. Collectively, these findings suggest that mechanisms regulating the spatiotemporal localization of XRCC4 are crucial for XRCC4 function in humans and cats. Our findings contribute to elucidating the functions of XRCC4 and the role of abnormal XRCC4 in diseases, including cancers and MPD, and may help in developing XRCC4‐targeted drugs, such as radiosensitizers, for humans and cats.

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Cloning, localization and focus formation at DNA damage sites of canine Ku70

Cited by 16 publications

References 33 publications

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

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

Feline XLF accumulates at DNA damage sites in a Ku‐dependent manner

Feline XRCC4 undergoes rapid Ku‐dependent recruitment to DNA damage sites

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