Molecular cloning, subcellular localization, and rapid recruitment to DNA damage sites of chicken Ku70.

Ku70 is a multifunctional protein with pivotal roles in DNA repair via non-homologous end-joining, V(D)J recombination, telomere maintenance, and neuronal apoptosis control. Nonetheless, its regulatory mechanisms remain elusive. Chicken Ku70 (GdKu70) cDNA has been previously cloned, and DT40 cells expressing it have significantly contributed to critical biological discoveries. GdKu70 features an additional 18 amino acids at its N-terminus compared to mammalian Ku70, the biological significance of which remains uncertain. Here, we show that the 5' flanking sequence of GdKu70 cDNA is not nearly encoded in the chicken genome. Notably, these 18 amino acids result from fusion events involving the NFE2L1 gene on chromosome 27 and the Ku70 gene on chromosome 1. Through experiments using newly cloned chicken Ku70 cDNA and specific antibodies, we demonstrated that Ku70 localizes within the cell nucleus as a heterodimer with Ku80 and promptly accumulates at DNA damage sites following injury. This suggests that the functions and spatiotemporal regulatory mechanisms of Ku70 in chickens closely resemble those in mammals. The insights and resources acquired will contribute to elucidate the various mechanisms by which Ku functions. Meanwhile, caution is advised when interpreting the previous numerous key studies that relied on GdKu70 cDNA and its expressing cells.

Combined ataxia telangiectasia mutated and DNA-dependent protein kinase inhibition radiosensitizes Madin-Darby canine kidney cells.

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.

Feline XRCC4 undergoes rapid Ku-dependent recruitment to DNA damage sites.

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.

Inhibition of Crandell-Rees Feline Kidney cell proliferation by X-ray-induced senescence.

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 ß 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.

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

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.

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

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 SNPs. 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.

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

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.

Cloning, localization and focus formation at DNA damage sites of canine XRCC4.

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 changes 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.

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

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 ß-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.

Nuclear localization of mouse Ku70 in interphase cells and focus formation of mouse Ku70 at DNA damage sites immediately after irradiation.

To elucidate the mechanisms of DNA repair pathway is critical for developing next-generation radiotherapies and chemotherapeutic drugs for cancer. Ionizing radiation and many chemotherapeutic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). The classical nonhomologous DNA-end joining (NHEJ) (C-NHEJ) pathway repairs a predominant fraction of DSBs in mammalian cells. The C-NHEJ pathway appears to start with the binding of Ku (heterodimer of Ku70 and Ku80) to DNA break ends. Therefore, recruitment of Ku to DSB sites might play a critical role in regulating NHEJ activity. Indeed, human Ku70 and Ku80 localize in the nuclei and accumulate at microirradiated DSB sites. However, the localization and regulation mechanisms of Ku70 and Ku80 homologues in animal models, such as mice and other species, have not been elucidated in detail, particularly in cells immediately after microirradiation. Here, we show that EYFP-tagged mouse Ku70 localizes in the interphase nuclei of mouse fibroblasts and epithelial cells. Furthermore, our findings indicate that EYFP-mouse Ku70 accumulates with its heterodimeric partner Ku80 immediately at laser-microirradiated DSB sites. We also confirmed that the structure of Ku70 nuclear localization signal (NLS) is highly conserved among various rodent species, such as the mouse, rat, degu and ground squirrel, supporting the idea that NLS is important for the regulation of rodent Ku70 function. Collectively, these results suggest that the mechanisms of regulating the localization and accumulation of Ku70 at DSBs might be well conserved between the mouse and human species.

Dynamic changes in subcellular localization of cattle XLF during cell cycle, and focus formation of cattle XLF at DNA damage sites immediately after irradiation.

Clinically, many chemotherapeutics and ionizing radiation (IR) have been applied for the treatment of various types of human and animal malignancies. These treatments kill tumor cells by causing DNA double-strand breaks (DSBs). Core factors of classical nonhomologous DNA-end joining (C-NHEJ) play a vital role in DSB repair. Thus, it is indispensable to clarify the mechanisms of C-NHEJ in order to develop next-generation chemotherapeutics for cancer. The XRCC4-like factor (XLF; also called Cernunnos or NHEJ1) is the lastly identified core NHEJ factor. The localization of core NHEJ factors might play a critical role in regulating NHEJ activity. The localization and function of XLF have not been elucidated in animal species other than mice and humans. Domestic cattle (Bos taurus) are the most common and vital domestic animals in many countries. Here, we show that the localization of cattle XLF changes dynamically during the cell cycle. Furthermore, EYFP-cattle XLF accumulates quickly at microirradiated sites and colocalizes with the DSB marker γH2AX. Moreover, nuclear localization and accumulation of cattle XLF at DSB sites are dependent on 12 amino acids (288-299) of the C-terminal region of XLF (XLF CTR). Furthermore, basic amino acids on the XLF CTR are highly conserved among domestic animals including cattle, goat and horses, suggesting that the CTR is essential for the function of XLF in domestic animals. These findings might be useful to develop the molecular-targeting therapeutic drug taking XLF as a target molecule for human and domestic animals.

Impact of amino acid substitutions in two functional domains of Ku80: DNA-damage-sensing ability of Ku80 and survival after irradiation.

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.

Ku80 attentuates cytotoxicity induced by green fluorescent protein transduction independently of non-homologous end joining.

The green fluorescent protein (GFP) is the most commonly used reporter protein for monitoring gene expression and protein localization in a variety of living and fixed cells, including not only prokaryotes, but also eukaryotes, e.g., yeasts, mammals, plants and fish. In general, it is thought that GFP is nontoxic to cells, although there are some reports on the side effect of GFP. Further, details of the molecular mechanism concerning the side effect of GFP remain unclear. Here we show that Ku80, but not XRCC4, plays an important role in the mechanism of the resistance to cytotoxicity induced by enhanced GFP (EGFP). EGFP inhibited both cell proliferation and colony formation, and induced cell death in Ku80-deficient hamster cells, i.e., xrs-6 cells. In addition, Ku80 attenuated EGFP-induced cytotoxicity in the xrs-6 cells. No EGFP-induced cytotoxicity was observed in the NHEJ core protein XRCC4-deficient hamster cells, i.e., XR-1 cells. Furthermore, EGFP markedly enhanced X-ray-induced cytotoxicity in the xrs-6 cells. These results suggest that Ku80 plays a key role in the novel NHEJ-independent defense mechanism against EGFP-induced cytotoxicity. Caution should be taken in considering of the potential influence by the stress response mechanism, namely, the Ku80-dependent elimination mechanism of EGFP-induced cytotoxicity, being activated, even when using EGFP-expressing cells in which Ku80 functions normally.

The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation.

Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1-418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1-418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1-418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1-418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411-418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is important for the physiological function of Rad52 not only at a late stage following irradiation, but also at an early stage.

The defect of Ku70 affects sensitivity to x-ray and radiation-induced caspase-dependent apoptosis in lung cells.

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.

Establishment of hamster cell lines with EGFP-tagged human XRCC4 and protection from low-dose X-ray radiation.

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.

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

Ku70 and Ku80 play an essential role in the DNA double-strand break (DSB) repair pathway, i.e., nonhomologous DNA-end-joining (NHEJ). No accumulation mechanisms of Ku70 at DSBs have been clarified in detail, although the accumulation mechanism of Ku70 at DSBs plays key roles in regulating the NHEJ activity. Here, we show the essential domains for the accumulation and function of Ku70 at DSBs in living lung epithelial cells. Our results showed that EGFP-Ku70 accumulation at DSBs began immediately after irradiation. Our findings demonstrate that three domains of Ku70, i.e., the α/ß, DNA-binding, and Ku80-binding domains, but not the SAP domain, are necessary for the accumulation at or recognition of DSBs in the early stage after irradiation. Moreover, our findings demonstrate that the leucine at amino acid 385 of Ku70 in the Ku80-binding domain, but not the three target amino acids for acetylation in the DNA-binding domain, is involved in the localization and accumulation of Ku70 at DSBs. Furthermore, accumulations of XRCC4 and XLF, but not that of Artemis, at DSBs are dependent on the presence of Ku70. These findings suggest that Artemis can work in not only the Ku-dependent repair process, but also the Ku-independent process at DSBs in living epithelial cells.

Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation.

The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker γ-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful tool as a detection marker of DNA damaged sites.

KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair.

Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.

Establishment of ku70-deficient lung epithelial cell lines and their hypersensitivity to low-dose x-irradiation.

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.