著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
公益社団法人 日本獣医学会
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
pp.16-0381, (Released:2016-09-18)
被引用文献数
7

Various chemotherapies and radiation therapies are useful for killing cancer cells mainly by inducing DNA double-strand breaks (DSBs). Uncovering the molecular mechanisms of DSB repair processes is crucial for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. XRCC4 plays a critical role in Ku-dependent nonhomologous DNA-end joining (NHEJ) in human cells and is one of the core NHEJ factors. The localization of core NHEJ factors, such as human Ku70 and Ku80, might play a crucial role in regulating NHEJ activity. Recently, companion animals, such as canines, have been proposed to be a good model in many aspects of cancer research. However, the localization and regulation mechanisms of core NHEJ factors in canine cells have not been elucidated. Here, we show that the expression and subcellular localization of canine XRCC4 change dynamically during the cell cycle. Furthermore, EYFP-canine XRCC4 accumulates quickly at laser-microirradiated DSB sites. The structure of a putative human XRCC4 nuclear localization signal (NLS) is highly conserved in canine, chimpanzee and mouse XRCC4. However, the amino acid residue corresponding to the human XRCC4 K210, thought to be important for nuclear localization, is not conserved in canine XRCC4. Our findings might be useful for the study of the molecular mechanisms of Ku-dependent NHEJ in canine cells and the development of new radiosensitizers that target XRCC4.
著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
JAPANESE SOCIETY OF VETERINARY SCIENCE
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
pp.22-0061, (Released:2022-09-15)

Uncovering radiation toxicity is critical for the adaptation and expansion of advanced radiation therapies and for the development of novel cancer radiotherapy. In the near future, advanced radiotherapies, including heavy ion beam treatment, are expected to be applied in the treatment of dogs, but further basic research on the effects of radiation using canine normal and cancer cells is necessary to actually apply these techniques and achieve high therapeutic efficacy. The radiation sensitivity is varied by the activities of DNA damage response (DDR) and DNA repair. The development of radiosensitizers that target DDR- and DNA repair-kinases, like ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), is progressing and is expected to be introduced into canine radiotherapy. However, there are no cytotoxicity reports on using the combination of radiation and these sensitizers as treatment in canine cells. In this study, we examined the cytotoxic effects of X-rays and/or radiosensitizers on the Madin–Darby Canine Kidney (MDCK) cell line. Our results show that X-rays suppress MDCK cell colony formation and proliferation in a dose-dependent manner. Additionally, our observations imply that the combination treatment with ATM inhibitor KU-55933 and DNA-PK inhibitor NU7441 significantly increased X-ray cytotoxicity in MDCK cells compared with the drugs alone. Furthermore, our findings further suggest that MDCK cells might be useful in clarifying the cytotoxicity in canine epithelial cells due to radiation and/or radiosensitizers, such as molecule-targeted drugs.
著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
JAPANESE SOCIETY OF VETERINARY SCIENCE
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
pp.20-0679, (Released:2021-03-17)
被引用文献数
5

Radioresistance and radiotoxicity have been reported following cancer treatments in felines. Optimizing radiation doses to induce cytotoxic effects to only cancer cells and not normal cells is critical in achieving effective radiation therapy; however, the mechanisms of radiation resistance, radiotoxicity, and DNA damage response (DDR) in feline cells have not yet been elucidated. A DNA double-strand break (DSB) is the most toxic type of DNA damage induced by X-rays and heavy ion beams used in treating cancers. Crandell-Rees Feline Kidney (CRFK) cells is one of the most widely used cat cells in life science research. Here, we report that DSB-triggered senescence induced by X-rays is important in inhibiting the proliferation of CRFK cells. We demonstrated through cell proliferation assay that X-rays at doses 2 Gy and 10 Gy are toxic to CRFK cells that irradiating CRFK cells inhibits their proliferation. In X-irradiated CRFK cells, a dose-dependent increase in DSB-triggered senescence was detected according to morphological changes and using senescence-associated β galactosidase staining assay. Moreover, our data indicated that in CRFK cells, the major DDR pathway, which involves the phosphorylation of H2AX at Ser139, was normally activated by ATM kinases. Our findings are useful in the understanding of X-rays-induced cellular senescence and in elucidating biological effects of radiation, e.g., toxicity, in feline cells. Furthermore, our findings suggest that the CRFK cell line is an excellent matrix for elucidating radioresistance and radiotoxicity in cat cells.
著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
公益社団法人 日本獣医学会
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
vol.79, no.3, pp.554-561, 2017 (Released:2017-03-23)
参考文献数
32
被引用文献数
11

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair machinery, specifically non-homologous DNA-end joining (NHEJ), is crucial for developing next-generation radiotherapies and common chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, might play vital roles for regulation of NHEJ activity. The human Ku heterodimer (Ku70/Ku80) is a core NHEJ factor in the NHEJ pathway and is involved in sensing of DSBs. Companion animals, such as canines, have been proposed to be an excellent model for cancer research, including development of chemotherapeutics. However, the post-translational modifications, localization and complex formation of canine Ku70 have not been clarified. Here, we show that canine Ku70 localizes in the nuclei of interphase cells and that it is recruited quickly at laser-microirradiated DSB sites. Structurally, two DNA-PK phosphorylation sites (S6 and S51), an ubiquitination site (K114), two canonical sumoylation consensus motifs, a CDK phosphorylation motif, and a nuclear localization signal (NLS) in the human Ku70 are evolutionarily conserved in canine and mouse species, while the acetylation sites in human Ku70 are partially conserved. Intriguingly, the primary candidate nucleophile (K31) required for 5’dRP/AP lyase activity of human and mouse Ku70 is not conserved in canines, suggesting that canine Ku does not possess this activity. Our findings provide insights into the molecular mechanisms of Ku-dependent NHEJ in a canine model and form a platform for the development of next-generation common chemotherapeutics for human and animal cancers.
著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
公益社団法人 日本獣医学会
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
pp.16-0649, (Released:2017-02-06)
被引用文献数
11

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair machinery, specifically non-homologous DNA-end joining (NHEJ), is crucial for developing next-generation radiotherapies and common chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, might play vital roles for regulation of NHEJ activity. The human Ku heterodimer (Ku70/Ku80) is a core NHEJ factor in the NHEJ pathway and is involved in sensing of DSBs. Companion animals, such as canines, have been proposed to be an excellent model for cancer research, including development of chemotherapeutics. However, the post-translational modifications, localization and complex formation of canine Ku70 have not been clarified. Here, we show that canine Ku70 localizes in the nuclei of interphase cells and that it is recruited quickly at laser-microirradiated DSB sites. Structurally, two DNA-PK phosphorylation sites (S6 and S51), an ubiquitination site (K114), two canonical sumoylation consensus motifs, a CDK phosphorylation motif, and a nuclear localization signal (NLS) in the human Ku70 are evolutionarily conserved in canine and mouse species, while the acetylation sites in human Ku70 are partially conserved. Intriguingly, the primary candidate nucleophile (K31) required for 5’dRP/AP lyase activity of human and mouse Ku70 is not conserved in canines, suggesting that canine Ku does not possess this activity. Our findings provide insights into the molecular mechanisms of Ku-dependent NHEJ in a canine model and form a platform for the development of next-generation common chemotherapeutics for human and animal cancers.
著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
公益社団法人 日本獣医学会
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
pp.16-0440, (Released:2016-10-14)
被引用文献数
7

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair processes, especially nonhomologous DNA-end joining (NHEJ), is critical for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, such as human Ku70 and Ku80, might play critical roles in controlling NHEJ activity. XRCC4-like factor (XLF) is a core NHEJ factor and plays a key role in the Ku-dependent NHEJ repair process in human cells. Recently, companion animals, such as canines, have been proposed to be a good model for many aspects of cancer research, including the development of chemotherapeutics. However, the localization and regulation of core NHEJ factors in canine cells have not been elucidated. Here, we show that the localization of canine XLF changes dynamically during the cell cycle. EYFP-canine XLF localizes in the nuclei of interphase cells and accumulates immediately at microirradiated DSB sites. The structure of a putative human XLF nuclear localization signal (NLS) and a putative 14-3-3 binding motif are evolutionarily conserved in canine, chimpanzee and mouse XLF. However, the putative β-TRCP-recognizable degron of human XLF is not conserved in canine and mouse. Additionally, some vital human XLF phosphorylation sites, including the ATM major phosphorylation site (S251), are not conserved in canine XLF. Our findings might be useful for the study of the molecular mechanisms of NHEJ in canine cells and for the development of new radiosensitizers that target XLF.
著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
公益社団法人 日本獣医学会
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
pp.13-0283, (Released:2013-09-10)
被引用文献数
4

Various chemotherapeutic drugs, such as etoposide, and ionizing radiation (IR) have been clinically applied for the treatment of many types of animal and human malignancies. IR and chemotheraputic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). On the other hand, unrepaired or incorrectly repaired DSBs can lead to chromosomal truncations and translocations, which can contribute to the development of cancer in humans and animals. Thus, it is important to clarify the molecular mechanisms underlying the chemosensitivity or radiosensitivity of mammalian cells in order to develop medical treatments and next-generation chemotherapeutic drugs for cancer. Previously, we established and analyzed cell lines stably expressing chimeric constructs of EGFP and the wild-type Ku80 (XRCC5) protein or its mutant protein to which mutations were introduced by the site-directed mutagenesis. We found that the Ku70 (XRCC6)-binding-site mutations (A453H/V454H) of Ku80 and nuclear localization signal (NLS)-dysfunctional mutations (K565A/K566A/K568A) affected the ability to complement etoposide sensitivity. In this study, we examined the radiosensitivity of these cell lines. We found that either or both amino acid substitutions in two functional domains of Ku80, i.e., Ku70-binding-site mutations (A453H/V454H) and NLS-dysfunctional mutations (K565A/K566A/K568A), affect the ability to complement radiosensitivity. Moreover, these mutations in the two domains of Ku80 affect the DSB-sensing ability of Ku80. These information and Ku80 mutant cell lines used might be useful for the study of not only the dynamics and function of Ku80, but also the molecular mechanism underlying the cellular response to IR and chemotherapeutic drugs in mammalian cells.
著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
公益社団法人 日本獣医学会
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
pp.12-0333, (Released:2012-11-14)
被引用文献数
4 11

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.
著者
Manabu KOIKE Yasutomo YUTOKU Aki KOIKE
出版者
公益社団法人 日本獣医学会
雑誌
Journal of Veterinary Medical Science (ISSN:09167250)
巻号頁・発行日
pp.12-0112, (Released:2012-05-23)
被引用文献数
3 7

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 chemoresistance to not only anticancer agents, e.g., etoposide, but also radioresistance. However, it is 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.