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Impact of Amino Acid Substitutions in Two Functional Domains of Ku80: DNA-Damage-Sensing Ability of Ku80 and Survival after Irradiation

ABSTRACT 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.

Chemotherapy and radiotherapy are well-established treatments for cancer. Various chemotherapeutic drugs, such as etoposide and bleomycin, and ionizing radiation (IR) have been clinically applied for the treatment of many types of animal and human malignancies. On the other hand, in clinical settings, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. 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.
DNA double-strand breaks (DSBs) are considered the most critical DNA damage [3]. Unrepaired or incorrectly repaired DSBs can lead to chromosomal truncations and translocations, which can contribute to the development of cancer in higher eukaryotic organisms. In addition, one DSB is sufficient to kill a cell, when it is not repaired. DSBs are induced following exposure to ionizing radiation as well as treatment with etoposide or bleomycin [15,22,27]. At nascent DSB sites, histone H2AX molecules near these sites are rapidly phosphorylated at serine 139 (γH2AX) [23]. The detection of γH2AX using a specific antibody is the most sensitive method currently available for identifying DSBs.
The heterodimer formation between Ku70 (also named XRCC6) and Ku80 (also named XRCC5) and the DSBsensing ability of Ku might play a key role in the regulation of Ku-dependent DNA repair activity [1,6,17]. The nonhomologous DNA-end-joining (NHEJ) repair process is responsible for repairing a major fraction of DSBs in mammalian cells [2,3,17]. The NHEJ repair pathway starts with the binding of Ku70 and Ku80 to a DNA end. Ku70 and Ku80 accumulate at laser-induced DSB sites immediately following irradiation, and these are essential for the accumulation of core-NHEJ factors, such as DNA-PKcs, XLF and XRCC4, but not other core-NHEJ factor, i.e., Artemis, at DSB sites [6,9,10,17,18].
Previously, we generated and analyzed cell lines stably expressing chimeric constructs of EGFP and the wild-type Ku80 protein or its mutant protein to which mutations were introduced by the site-directed mutagenesis technique [5,7]. Xrs-6 cells are a DSB repair mutant of the CHO-K1 line and are defective in a core-NHEJ factor Ku80 [3,21]. Our data revealed that the EGFP-Ku80 transformants showed higher etoposide resistance than xrs-6 cells. Moreover, our data showed that the Ku70-binding-site mutations (A453H/ V454H) of Ku80 and nuclear localization signal (NLS)dysfunctional mutations (K565A/K566A/K568A) affected the ability to complement etoposide sensitivity. However, it remains unclear whether these Ku80 mutations modify the radiosensitivity of cells and the DSB-sensing ability of Ku80.
In this study, to clarify whether the Ku70-binding-site mutations (A453H/V454H) of Ku80 and NLS-dysfunctional mutations (K565A/K566A/K568A) affect the ability to complement radiosensitivity, we first analyzed the radiosensitivity of CHO-K1, xrs-6 and three transformants. Next, we examined whether these mutations in the two domains of Ku80 affect Ku80 accumulation at DSBs.
X-irradiation: Cells were exposed to X-rays at room temperature, as described previously [5]. X-rays were generated at 200 kVp/20 mA and filtered through 0.5 mm Cu and Al filters.
Western blot analysis: Western blot analysis was performed as described previously [10,12]. The following antibodies were used: a rabbit anti-GFP polyclonal antibody ( Fluorescence microscopy: The fluorescence in cells was visualized as previously described [6,16]. Briefly, the cells were fixed in PBS containing 4% paraformaldehyde. The fixed cells were then blocked using a blocking solution and incubated for 30 min at room temperature with a mouse anti-γ-H2AX monoclonal antibody (JBW301) (Upstate Biotechnology Inc., Charlottesville, VA, U.S.A.). After washing with PBS, antibody binding was detected using Alexa fluor 568-conjugated secondary antibodies (Molecular Probes, Eugene, OR, U.S.A.). DNA was stained with 0.025 µg/ml 4,6-diamino-2-phenylindole (DAPI) fluorescent dye (Boehringer Mannheim, Mannheim, Germany). The cells were examined under an Olympus IX70 fluorescence microscope (Olympus, Tokyo, Japan). Images were acquired using an FV300 confocal laser scanning microscope (Olympus).
Localized DNA damage induction using laser and cell imaging: Localized DNA damage induction using a laser and confocal imaging were performed as described previously [6,9,10]. Briefly, confocal images of living cells expressing EGFP-tagged proteins were obtained using an FV300 confocal scanning laser microscopy system (Olympus). A 1% power scan (for 1 sec) from a 405 nm laser was used to induce localized DSBs.

RESULTS
Ku80 is a key contributor to chemoresistance to anticancer agents, such as etoposide, or radioresistance in CHO-K1 cells [3,8,25]. First, we confirmed the expression of Ku80 in CHO-K1 and Ku80-deficient xrs-6 cells. Consistent with previous studies [8,24], Western blot analysis showed that Ku80 was expressed in CHO-K1 cells, but not in xrs-6 cells (Fig. 1). Expectedly, the Ku70 expression level was markedly reduced in the xrs-6 cells. A core-NHEJ factor XRCC4 as well as β-actin was expressed in both cell lines.
DSBs are considered the most critical DNA damage [3]. One DNA DSB is sufficient to kill a cell, when it is not repaired. γH2AX is a sensitive indicator of DSBs induced by X-irradiation [23]. To confirm the roles of Ku80 in γH2AX elimination after X-irradiation in CHO-K1 cells, we examined X-irradiation-induced H2AX phosphorylation and γH2AX elimination in xrs-6 cells and CHO-K1 cells by confocal laser scanning microscopy using the anti-γH2AX antibody (Fig. 2). Following X-irradiation, γH2AX foci were detected in both cell lines at 1 hr postirradiation. Although γH2AX elimination from 1 to 25 hr after X-irradiation was detected in both cell lines, the elimination from xrs-6 cells was slower than that from the CHO-K1 cells. These findings confirm the persistence of unrepaired DSBs in Ku80deficient xrs-6 cells.
Previously, we established and characterized stable cell lines expressing EGFP-Ku80 or EGFP in xrs-6 cells [5]. In addition, we established and characterized cell lines stably expressing EGFP-Ku80 mutants with point mutations in either or in both the NLS and heterodimerization domains [EGFP-Ku80 (K565A/K566A/K568A), EGFP-Ku80 (A453H/V454H) or EGFP-Ku80 (A453H/V454H/K565A/ K566A/K568A)] using Ku80-deficient xrs-6 cells [7]. In this study, we reconfirmed that the wild-type Ku80 fusion protein or its mutant Ku80 fusion proteins were expressed in these transformants by Western blot analysis (Fig. 3). We also reconfirmed that hamster Ku70 was detected in extracts prepared from the xrs-6 cells transformed with EGFP-Ku80 or EGFP-Ku80 (K565A/K566A/K568A) and from the CHO-K1 cells, but not in those from the other transformants, confirming that mutations of Ku80 at amino acids 453 and 454 affect the interaction of Ku80 with hamster Ku70 (Fig.  3). To determine whether the Ku80 point mutations in the 2 domains in Ku80 affect the sensitivity of xrs-6 cells to X-rays, we examined the radiosensitivity of EGFP-Ku80/ xrs-6 cells, xrs-6 cells and the three transformants by colony formation assay. As shown in Fig. 4, the EGFP-Ku80/xrs-6 cells exhibited higher radioresistance than xrs-6 cells, which is consistent with our previous report [5]. The double mutants with mutations in 2 functional domains were unable to complement the radiosensitivity of xrs-6 cells (Fig. 4), suggesting that these domains of Ku80 are important for complementing the radiosensitivity of Ku80-deficient cells. The NLS-dysfunctional Ku80 transformant EGFP-Ku80 (K565A/K566A/K568A)/xrs-6 cells exhibited a slightly higher radiosensitivity than the EGFP-Ku80/xrs-6 cells, suggesting that the NLS of Ku80 is important for Ku-dependent radioresistanse. Similarly, another domain transformant EGFP-Ku80 (A453H/V454H)/xrs-6 cells exhibited a slightly higher radiosensitivity than the EGFP-Ku80 transformants. These findings suggest that both or either of the Ku80 point mutations in the 2 domains of Ku80 affects the sensitivity of xrs-6 cells to X-rays.
Previously, we showed that EGFP-Ku80 accumulation at DSBs began immediately after laser microirradiation in EGFP-Ku80/xrs-6 cells [6]. We examined whether the mutations in both the NLS and heterodimerization domains of Ku80 affect the accumulation of Ku80 at DSBs (Fig. 5A-D). Expectedly, EGFP-Ku80 was localized in the nucleus and accumulated at microirradiated sites in the living cells (Fig.  5B). On the other hand, EGFP-Ku80 (A453H/V454H/ K565A/K566A/K568A) was observed to be localized in the cytoplasm, and the mutant did not accumulate at DSBs (Fig.  5C). As shown in Fig. 5D, EGFP alone was localized in the nucleus and cytoplasm and not accumulated at microirradiated sites. These findings demonstrate that the Ku80 point mutations in the 2 domains of Ku80 affect the accumulation of Ku80 at DSBs.

DISCUSSION
It is important to elucidate the molecular mechanisms underlying the radioresistance, as well as the chemoresistance, of human and animal cells in order to develop next-generation medical treatments for cancer. DNA repair genes, particularly core-NHEJ genes, are key players in radioresistance and chemoresistance. We previously identified and characterized the Ku70-binding-site of Ku80 and the NLS of Ku80 [4,14]. In this study, our findings showed that either or both of the amino acid substitutions in the 2 functional domains of Ku80, i.e., the Ku70-binding-site mutations (A453H/V454H) and NLS-dysfunctional mutations (K565A/K566A/K568A), affect the ability to complement X-ray sensitivity. Moreover, our findings showed that these mutations affect the ability to repair DNA damage of Ku80 at microirradiation-induced DSBs.
Radiation and etoposide induce DSBs via different mechanisms and with different efficiencies. The rates of rejoining DSBs differ between etoposide and radiation [19]. In previous studies, we demonstrated that EGFP-Ku80 used in this study complemented not only X-ray sensitivity but also etoposide sensitivity in EGFP-Ku80/xrs-6 cells [5]. Others and we demonstrated that the two mutations of Ku80 at amino acids 453 and 454 in the Ku70 binding domain affect the interaction of Ku70 with Ku80 [8,14,20]. In addition, we showed that the Ku70-binding-site mutations (A453H/ V454H) of Ku80 affected slightly the ability to complement etoposide sensitivity. In this study, Ku70-binding-site mutations (A453H/V454H) of Ku80 only affected slightly the ability to complement radiosensitivity. Another minor DSB repair pathway, such as homologous recombination, may be activated complementarily in EGFP-Ku80 (A453H/V454H)/ xrs-6 cells, although further studies are needed to clarify this.  Alternatively, we cannot completely exclude the possibility that the Ku70 binding activity and DNA binding activity may remain slightly below the detection sensitivity in vivo.
Previously, we showed that in addition to Ku80 NLS, the heterodimerization between Ku70 and Ku80 is also important for nuclear entry of Ku80 [4,8,14]. In this study, NLS-dysfunctional mutations (K565A/K566A/K568A) also only affected slightly the ability to complement radiosensitivity. On the other hand, the double mutants EGFP-Ku80 (A453H/V454H/K565A/K566A/K568A) with mutations in the 2 functional domains were unable to complement the radiosensitivity of xrs-6 cells. In addition, the mutant was not able to accumulate at DSBs, when the DSBs were induced using our simple live-cell imaging technique with a laser, suggesting that the mutations affect the ability to repair DNA damage of Ku80. Altogether, we consider that the double mutants were unable to repair DSBs of xrs-6 cells, since the double mutants were not able to localize and accumulate at DSBs. These findings support the idea that the localization and accumulation mechanism of Ku80 at DSBs might play a key role in regulating NHEJ activity [6,17]. The DNA repair system protects the genome in humans and animals. It has been reported that genetic variations in NHEJ genes can affect DNA repair capacity and confer predisposition to several types of clinical human cancer [26]. This study and further studies will lead to a better understanding of not only the molecular mechanism underlying the cellular response via the NHEJ pathway to radiation and chemotherapeutic drugs, but also carcinogenesis induced by defects of NHEJ functions in mammalian cells.