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. Author manuscript; available in PMC: 2014 Jul 2.
Published in final edited form as: J Cell Physiol. 2011 Feb;226(2):369–374. doi: 10.1002/jcp.22342

The Ku-Dependent Non-Homologous End-Joining Pathway Contributes to Low-Dose Radiation-Stimulated Cell Survival

XIAOYAN YU 1,2, HONGYAN WANG 1, PING WANG 1, BENJAMIN PC CHEN 3, YA WANG 1,*
PMCID: PMC4079012  NIHMSID: NIHMS592621  PMID: 20665702

Abstract

Low-dose (≤0.1 Gy) radiation-induced adaptive responses could protect cells from high-challenge dose radiation-induced killing. The protective role is believed to promote the repair of DNA double-strand breaks (DSBs) that are a severe threat to cell survival. However, it remains unclear which repair pathway, homologous recombination repair (HRR) or non-homologous end-joining (NHEJ), is promoted by low-dose radiation. To address this question, we examined the effects of low-dose (0.1 Gy) on high-challenge dose (2–4 Gy) induced killing in NHEJ- or HRR-deficient cell lines. We showed that 0.1 Gy reduced the high-dose radiation-induced killing for wild-type or HRR-deficient cells, but enhanced the killing for NHEJ-deficient cells. Interestingly, low-dose radiation also enhanced the killing for wild-type cells exposed to high-challenge dose radiation with high-linear energy transfer (LET). Because it is known that high-LET radiation induces an inefficient NHEJ, these results support that the low-dose radiation-stimulated protective role in reducing high-challenge dose (low-LET)-induced cell killing might depend on NHEJ. In addition, we showed that low-dose radiation activated the DNA-PK catalytic subunit (DNA-PKcs) and the inhibitor of DNA-PKcs destroyed the low-dose radiation-induced protective role. These results suggest that low-dose radiation might promote NHEJ through the stimulation of DNA-PKcs activity and; therefore, increase the resistance of cells to high-challenge dose radiation-induced killing.

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are a severe threat to cell killing. DNA DSBs are repaired by two major pathways: homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) in mammalian cells. Low doses at ≤0.1 Gy exposure could stimulate DNA repair (Ikushima et al., 1996) and induce cell resistance to high-challenge dose IR-induced killing (Raaphorst et al., 2006). However, it remains unclear which pathway, NHEJ, HRR or both, is involved in the low-dose IR stimulated repair.

Low-dose radiation-induced adaptive response was originally used to describe a phenomenon in which pre-exposure of cells to a low dose (adaptive dose) of radiation rendered cells less susceptible to damage induced by a subsequent high dose (challenge dose; Olivieri et al., 1984). Since then, many studies have shown that low-dose radiation-induced adaptive response could prevent genes from high-challenge dose IR-induced mutation (Sanderson and Morley, 1986; Laval, 1988; Schäppi-Büchi, 1994; Zhou et al., 1994; Rigaud et al., 1995; Sasaki, 1995; Azzam et al., 1996; Ueno et al., 1996; Broome et al., 2002; Lu et al., 2009), and prevent the mice from subsequent high-challenge dose IR-induced tumors (Redpath and Antoniono, 1998; Mitchel et al., 1999, 2004; Yu et al., 2009), although some studies still challenge the low-dose radiation-induced protective roles in preventing carcinogenesis (Brenner et al., 2003; Mullenders et al., 2009). The mechanism by which adaptive response reduces the high-challenge dose IR-induced damage in vitro and in vivo might be a complicated process that involves protein synthesis (Ikushima, 1989; Wolff et al., 1989), immune response (Liu, 1998), apoptosis (Cregan et al., 1999; Portess et al., 2007) and many other proteins including protein kinase C (Sasaki, 1995), poly-ADP ribose polymerase (Ueno et al., 1996) and nuclear factor (NF)-κB (Fan et al., 2007). The main purpose of this study is to address the question of which DNA DSB repair pathway, HRR, NHEJ or both, is stimulated by the low-dose IR, thus protecting cells from high-challenge dose IR-induced killing.

High linear energy transfer (LET) IR (induced by highly charged particles, high-energy ions or a special radiotherapy machine) can kill more cells than low-LET IR (such as X- or γ-rays) at the same doses. The higher relative biological effectiveness (RBE) on cell killing by high-LET IR is because of ineffective DNA repair (Goodhead et al., 1993; Rydberg et al., 1994; Stenerlow and Hoglund, 2002; Hill et al., 2004), which is mainly due to the inefficient Ku-dependent NHEJ pathway (Lind et al., 2003; Okayasu et al., 2006; Wang et al., 2008). Recently, we further demonstrated that high-LET IR when compared with low-LET IR only affects the Ku-dependent NHEJ but not HRR (Wang et al., 2008, 2010). The results related to high-LET IR affecting only NHEJ but not HRR (when compared with low-LET IR), help us identify which repair pathway (NHEJ or HRR) is stimulated by low-dose IR, by comparing the effects of the challenge dose with high- or low-LET IR on cell survival.

In this study, we examined the effects of low-dose IR (0.1 Gy of low-LET IR) on high-challenge dose (low- or high-LET) IR-induced killing among different cell lines: wild-type, HRR- or NHEJ-deficient. Our results suggest that the low-dose IR-induced adaptive response might be via promoting the Ku-dependent NHEJ that contributes primarily to protecting cells from high-challenge dose IR-induced killing.

Materials and Methods

Cell lines and drug treatment

CHO cells include AA8 (wild-type), irs1-SF (HRR-deficient; Liu et al., 1998), irs-20 [DNA-PK catalytic subunit (DNA-PKcs) mutant, abnormal NHEJ; Stackhouse and Bedford, 1993a,b, 1994; Priestley et al., 1998], V3 (DNA-PKcs and NHEJ-deficient) and V3 cells transfected with wild-type DNA-PKcs, A6 or A7 cluster mutant autophosphorylated DNA-PKcs (Chen et al., 2005, 2007). AA8 and irs1-SF cells were obtained from Dr. Thompson’s laboratory. Irs-20 cells were obtained from Dr. Bedford’s laboratory. V3 and V3 varies cell lines were as described previously (Chen et al., 2005, 2007). Transformed MEF cells that include Ku80+/+ cells and Ku80−/− cells were obtained from Gloria Li’s laboratory (Nussenzweig et al., 1996). MRC5SV (ATM+/+) and AT5BIVA (ATM−/−) cells are transformed human fibroblast cells that were as described previously (Wang and Wang, 2008). HeLa cells were obtained from American Type Culture Collection (ATCC). M059K (DNA-PKcs+/+) and M059J (DNA-PKcs−/−) cells were obtained from Dr. Allalunis-Turner’s Laboratory (Allalunis-Turner et al., 1993; Lees-Miller et al., 1995). These cells were adapted to grow in Dulbecco’s modified Eagle medium supplemented with 10% iron-supplemented calf serum (Sigma–Aldrich Co., St. Louis, MO) at 37°C in an atmosphere of 5% CO2 and 95% air. ATM inhibitor: Ku55933 and DNA-PKcs inhibitor: Nu7441 were a gift from Kudos. HeLa cells were treated with 10 μM of the inhibitor (Ku55933 or Nu7441) for 60 min and then exposed to radiation.

Irradiation

Low-LET radiation was carried out using an X-ray machine (X-RAD 320, N. Branford 320 kV, 10 mA, using a filtration with 2-mm aluminum) in our laboratory or using a cesium source (γ-ray) at the Brookhaven National Laboratory (BNL). High-LET IR was carried out using the alternating-gradient synchrotron (AGS, Fe ions, 1 GeV/amu, 150 keV/μm). All the irradiation was performed at room temperature. The dose rates for high doses were 2 Gy/min and for low-dose radiation were 0.1 Gy/min by reducing the electrical currency to 0.5 mA.

Cell survival assay

Cell sensitivity to radiation was determined by the loss of colony-forming ability. Briefly, after the cells were irradiated with low-dose IR (0.1 Gy), they were returned to the incubator for 6 h and then irradiated with low- or high-LET IR. After IR, the cells were collected and plated, aiming at a density of 20–100 colonies per flask. Two replicate flasks were prepared for each datum point, and cells were incubated for 1–2 weeks to allow colonies to develop. The control cells in the flasks were Sham-irradiated. Colonies were stained with crystal violet (100% methanol solution) before counting. All survival experiments were repeated by different laboratory personnel (X. Yu, H. Wang and P. Wang) in different experiments; therefore, survival data were collected from at least three independent experiments.

Synchronizing cells to G0/G1 phase

HeLa cells were grown in 10% calf serum to ~70% confluence. The cells were changed to medium without serum for 16 h. The cells were irradiated with 0.1 Gy and returned to the incubator for 6 h. The cells were irradiated with 4 Gy. After irradiation, the cells were either collected for colony forming (in the medium containing 10% calf serum) or continued culture for γ-H2AX foci assay.

Flow cytometry

The distribution of cells in the cell cycle was measured as described before (Yu et al., 2010). The cells were collected after growing in medium with (10% calf serum) or without serum for 12 h. Cells were stained with the solution [62 μg/ml RNase A, 40 μg/ml propidium iodide and 0.1% Triton X-100 in phosphate-buffered saline (PBS) buffer] at room temperature for 1 h in a flow cytometer (Beckman Cell Lab Quanta™ SC, Miami, FL).

γ-H2AX foci assay

The cells were growing on microscope cover glass in 35 mm dishes to about 70% confluence. After synchronized and irradiated as described above, the cell incubation continued in 37°C for different times. The cells were then fixed in 4% paraformaldehyde for 10 min, permeabilized for 10 min in 0.2% Triton X-100 and blocked in 10% normal goat serum for 1 h at room temperature. The cells on the cover glass were mixed with an anti-γ-H2AX antibody (Millipore, Billerica, MA) for 1 h, washed in PBS and mixed with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA). The cells were washed in PBS, stained and mounted by using a Vectashield solution containing 4,6 diamidino-2-phenylindole (Vector Laboratory, Burlingame, CA). The fluorescence γ-H2AX foci were observed by using a Zwiss Imager A1 epifluorescent microscope with Axiovision Rel.4.7 software and an AxioCam, MRm camera (Carl Zeiss MicroImaging, Inc, Thornwood, NY). For each sample, 100 cells were counted and the percentage of positive cells (foci number >10 in one cell) were calculated. Similar experiments were repeated two times.

Western blot

At 1 h after high-challenge dose IR, HeLa cells were collected and whole cell lyses were prepared in RIPA lysis buffer (50 mM Tris–HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EGTA; 1 mM PMSF; 1 μg/ml each of aprotinin, leupeptin, pepstatin; 1 mM Na3VO4; 1 mM NaF) and mixed with an equal volume of 2× protein loading buffer. Nucleic extracts were prepared by using a NE-PER™ kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. The polyclonal antibody against phosphorylated S2056 of DNA-PKcs was as described previously (Chen et al., 2007). Antibodies against DNA-PKcs (Ab-2; Neomarkers Inc., Fremont, GA), against ATM (D2E2; Cell Signaling Inc., Danvers, MA) and against phosphorylated ATM pS1981 (Rockland Inc., Gilbertsville, PA) were used in this study.

Results

Low-dose radiation protects wild-type and HRR-deficient cells but not NHEJ-deficient cells from high-challenge dose IR-induced killing

To address the question of which pathway, NHEJ or HRR or both, was promoted by low-dose IR and played the essential role in changing the sensitivity of the cells to high-challenge dose, we examined the effects of low-dose IR (0.1 Gy) on high-challenge dose (4 Gy) induced killing in wild-type, NHEJ or HRR-deficient CHO cells. The results showed that exposure to 0.1 Gy alone did not affect the surviving fraction (0.98–1.1) among these cell lines, indicating that 0.1 Gy by itself does not directly kill cells. However, low-dose (0.1 Gy) exposure could increase the resistance of the wild-type cells (AA8) and the HRR-deficient cells (irs1-SF) to the high-challenge dose (4 Gy; Fig. 1A). Interestingly, low-dose (0.1 Gy) exposure could not increase the resistance but sensitized the NHEJ-deficient cells (irs-20) to the high-challenge dose (4 Gy; Fig. 1A). These results suggest that low-dose IR increased the resistance of the cells to the high-challenge dose IR-induced killing might be via promoting NHEJ. To verify this hypothesis, we examined the effects of low-dose IR on the sensitivity of V3 cells to high-challenge doses because irs-20 cells are mutated in DNA-PKcs, still retain some residual protein function (Priestley et al., 1998) and V3 cells are completely deficient in DNA-PK dependent NHEJ (Whitmore et al., 1989). The results showed that 0.1 Gy pre-exposure made the DNA-PKcs-deficient cells become more sensitive to high-challenge dose (2 Gy) IR-induced killing (Fig. 1B). To further confirm the phenotypes, we did similar experiments with the mouse NHEJ-deficient cell line: Ku80−/− cells. The results showed that 0.1 Gy pre-exposure increased the resistance of wild-type (Ku80+/+) cells to high-challenge dose IR (Fig. 2), which is similar to that for AA8 (the wild-type CHO cells; Fig. 1A). However, 0.1 Gy pre-exposure sensitized the NHEJ-deficient (Ku80−/−) cells to high-challenge dose IR (Fig. 2), which is similar to that for V3 (the NHEJ-deficient CHO cells; Fig. 1B). These results provide additional evidence to support that low-dose IR promotes NHEJ, which results in increasing the resistance of the cells to high-challenge dose IR-induced killing.

Fig. 1.

Fig. 1

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Low-dose IR protects wild-type and HRR-deficient cells but not NHEJ-deficient CHO cells from high-challenge dose IR-induced killing. A: AA8 (wild-type), irs-20 (NHEJ-deficient cells) and irs1-SF (HRR-deficient cells) were first exposed to X-ray with 0.1 Gy. The cells were returned to a 37°C incubator and then exposed to 4 Gy at 6 h following low-dose exposure. After the high-dose exposure, the cells were collected and plated, aiming at a density of 20–100 colonies per flask. Data shown are the average ± standard error from five independent experiments. B: V3 cells were prepared using a clonogenic surviving assay as described above. Data shown are the average ± standard error from five independent experiments. *P < 0.05; **P < 0.01.

Fig. 2.

Fig. 2

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Low-dose IR sensitizes NHEJ-deficient MEF cells to high-challenge dose IR. Ku80+/+ (wild-type) and Ku80−/− (NHEJ-deficient) cells were exposed to 0.1 Gy first and then exposed to different doses of challenge dose radiation. The cells were collected to prepare a clonogenic assay as described in Figure 1. Data shown are the average ± standard error from three independent experiments. *P < 0.05; **P < 0.01.

The Low-dose-induced protective role could not be observed in the cells exposed to the high-challenge dose with high-LET IR

It has been reported that high-LET IR only induces inefficient Ku-dependent NHEJ but does not affect HRR (Wang et al., 2008, 2010). Therefore, if the low-dose IR-induced adaptive response is via promoting NHEJ but not HRR, a protective role should not be observed in high-LET irradiated cells. To confirm that a low-dose IR-induced protective role is via promoting NHEJ, we chose high-LET IR as the high-challenge dose and examined the effects of pre-exposed low-dose IR on high-challenge IR (with high-LET)-induced killing. The results showed that a low-dose (0.1 Gy) with low-LET IR could not protect wild-type Ku80+/+ cells from high-challenge dose (with high-LET) IR-induced killing as it did with high-challenge dose (with low-LET) IR. In contrast, low-dose IR sensitized the cells to the high-challenge dose (with high-LET) IR (Fig. 3). At the same time, low-dose IR sensitized the NHEJ-deficient Ku80−/− cells to high-challenge dose regardless of high-LET or low-LET IR (Fig. 3). The difference between the sensitivities of Ku80+/+ cells to low-LET IR and to high-LET IR at the 3 Gy point was 7.5-fold without 0.1 Gy pre-irradiation, and increased to 37.5-fold with 0.1 Gy pre-irradiation, although there is no difference between the sensitivities of Ku80−/− cells to low-LET IR and to high-LET IR at all dose points with or without 0.1 Gy pre-irradiation (Fig. S1). These data confirms our previous observation: the cells deficient in the Ku-dependent NHEJ showed similar sensitivities to low- or high-LET radiation (Wang et al., 2008, 2009). These data also support that the low-dose IR-stimulated the change in the sensitivity of the cells to the challenge dose depends on the Ku-related NHEJ pathway, suggesting that the low-dose IR increased the resistance of the cells to the high-challenge dose (with low-LET) is through stimulating the Ku-dependent NHEJ. To verify that HRR was not directly involved in the protective role of the cells resistant to the high-challenge dose, we examined the survival fraction of G0/G1 phase cells because NHEJ is independent of the cell cycle distribution (Metzger and Iliakis, 1991; Rothkamm et al., 2003), but HRR is only efficient in S and G2 phases (Takata et al., 1998). The results showed that when the cells were synchronized in the G1 phase (Fig. 4A), the low-dose IR still increased the resistance of G1 phase cells to the high-challenge dose (Fig. 4B). The γ-H2AX data indirectly reflect that low-dose IR stimulated the DNA repair (Fig. 4C). These results further support that low-dose IR increased the resistance of the cells to high-challenge dose is through promoting NHEJ.

Fig. 3.

Fig. 3

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Low-dose IR sensitizes cells to the high-challenge dose of high-LET IR. Ku80+/+ (wild-type) and Ku80−/− (NHEJ-deficient) cells were exposed to 0.1 Gy low-LET radiation first and then returned to the 37°C incubator. The cells were then exposed to different doses of low-LET or high-LET radiation as described in Materials and Methods Section. The cells were collected to prepare a clonogenic assay as described in Figure 1. Data shown are the average ± standard error from three independent experiments. *P < 0.05.

Fig. 4.

Fig. 4

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Low-dose IR increased the resistance of G1 phase cells to the high-challenge dose. A: Cell cycle distribution measured by flow cytometry. B: HeLa cells were synchronized in a G1 phase by starvation. The cells were exposed to 0.1 Gy and were returned to the incubator. The cells were irradiated with 4 Gy at 6 h later. The cells were then collected for the survival assay with 10% serum as described in the Materials and Methods Section. Data shown are the average ± standard error from three independent experiments. *P < 0.05. C: γ-H2AX foci indirectly reflected the DNA DSBs. For each sample, 100 cells were examined, and the percentage of the positive cells (foci >10 in one cell as the positive one) were calculated. Similar experiments were repeated three times. *P < 0.05.

Low-dose radiation-activated DNA-PKcs is responsible for the low-dose IR-protected cells from high-dose IR-induced killing

To further study how low-dose IR promotes the NHEJ pathway, we examined the activities of ATM and DNA-PKcs. Although both ATM and DNA-PKcs are involved in the cell radiosensitivity, DNA-PKcs is an essential factor for NHEJ and ATM mainly promotes HRR (Golding et al., 2004; Zhang et al., 2004; Beucher et al., 2009), although ATM is still involved in a NHEJ subset (~10%) to repair DNA DSBs (Jeggo and Löbrich, 2005). In addition, the change in the kinase activity was more sensitive than the change in the kinase protein level to low-dose IR (Bakkenist and Kastan, 2003). The results showed that the autophosphorylation signals of ATM or DNA-PKcs were induced by low-dose IR (Fig. 5A), indicating that low-dose IR could stimulate DNA-PKcs activity, and confirmed that low-dose IR could stimulate ATM (Bakkenist and Kastan, 2003). More importantly, the autophosphorylation signal of ATM or DNA-PKcs was stronger in the cells irradiated with 0.1 +4 Gy than in the cells irradiated with 4 Gy alone (Fig. 5A), indicating that low-dose IR plays a role in promoting the activation of these kinases. To further answer the question of which pathway, ATM, DNA-PKcs or both, played a key role in the low-dose IR-induced resistance of the cells to high-challenge dose IR, we examined the effects of the ATM inhibitor (Ku55933) or the DNA-PKcs inhibitor (Nu7441) on low-dose IR-induced resistance of HeLa cells to high-dose IR. The results showed that although the ATM inhibitor inhibited the ATM autophosphorylation activity (Fig. 5A), it did not affect the low-dose IR-increased resistance of HeLa to high-challenge dose IR (Fig. 5B). In contrast, when the DNA-PKcs inhibitor inhibited the DNA-PKcs autophosphorylation activity (Fig. 5A), the cells became sensitive to high-challenge dose IR (Fig. 5B). These results suggest that the DNA-PKcs pathway but not the ATM pathway contributes to the low-dose IR-increased resistance of the cells to high-challenge dose IR. To confirm this, we examined the effects of low-dose IR on the surviving response of ATM−/− or DNA-PKcs−/− human cells to high-dose IR. The results showed that low-dose IR still increased the resistance of ATM−/− cells to high-dose IR-induced killing (Fig. 5C), but sensitized DNA-PKcs−/− cells to high radiation-induced killing (Fig. 5D). These results support that the DNA-PKcs pathway but not the ATM pathway contributes to the low-dose IR-increased resistance of the cells to high-challenge dose IR.

Fig. 5.

Fig. 5

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Low-dose activated DNA-PKcs contributes to the protective role for increasing the resistance of the cells to high-dose IR-induced killing. A: The autophosphorylational signals of ATM or DNA-PKcs in HeLa cells following 0.1 or 4 Gy exposure were detected by Western blot. B: Comparison of the effects of the ATM or DNA-PKcs inhibitor on the sensitivity of HeLa cells to low- or high-dose IR-induced killing. Data shown are the average ± standard error from three independent experiments. C: Comparison of the sensitivity of ATM+/ + and ATM−/− cells to low- or high-dose IR-induced killing. Data shown are the average ± standard error from three independent experiments. D: Comparison of the sensitivity of DNA-PKcs+/+ and DNA-PKcs−/− cells to low- or high-dose IR-induced killing. Data shown are the average ± standard error from three independent experiments. **P < 0.05; **P < 0.01.

Discussion

In this study, we show for the first time that low-dose IR-induced the resistance of the cells to high-challenge dose IR-induced killing might be through promoting the NHEJ pathway, which is related to the low-dose IR-activated DNA-PKcs. Low-dose IR might promote NHEJ, which results in the cells being more dependent on the NHEJ pathway. When the cells have intact NHEJ, the cells could use the promoted NHEJ to become more resistant to the challenge dose IR. However, when the NHEJ pathway is not functional, more cells depend on the deficient pathway, which might result in more killing by radiation.

It was previously reported that the low-dose cisplatin-induced resistance of the cells to high-challenge dose might involve the HRR pathway (Raaphorst et al., 2006). This study showed a big difference in the low-dose of cisplatin-induced resistance between AA8 cells and irs1-SF cells to the high-challenge dose of cisplatin or IR. It is known that the cisplatin-induced DNA cross-link is mainly repaired by HRR (Wang et al., 2001; Aloyz et al., 2002), which is different from IR-induced DNA DSBs that is mainly repaired by NHEJ in mammalian cells; although HRR also contributes to the repair of DNA DSBs (occurs in S and G2 phases only). Low-dose IR did not affect cell cycle distribution (data not shown), but low-dose cisplatin might block cells to the S or G2 phase, which promotes HRR. Another group used an I-SceI induced DSB approach and reported that low-dose IR might promote HRR (Yatagai et al., 2008). We think that the different conclusions obtained by our two groups might be due to the approaches and the endpoints although we could not completely exclude the possibility that different cell cycle distribution might also contribute to the different results. We knew that transfection could induce non-target effects in the cells (Hu et al., 2001) and we were familiar with this approach (Wang et al., 2004; Hu et al., 2005). The I-SceI assay requires multi-transfections, which might change the cell functions in the different pathways. The report that DNA DSB repair detected by single-cell gel electrophoresis was stimulated by low-dose radiation at 2 h after IR (Ikushima et al., 1996) supports our thought because the repair data reflected in this study (Ikushima et al., 1996) was NHEJ but not HRR (HRR could not finish within 2 h). In addition, if low-dose radiation stimulates both NHEJ and HRR, we should see more resistance of the cells to high-challenge dose IR than we observed.

Although ATM and DNA-PKcs have some functional link (Riballo et al., 2004; Chen et al., 2007), it is generally thought that ATM primarily involves HRR (Golding et al., 2004; Zhang et al., 2004; Beucher et al., 2009) and DNA-PKcs primarily involves NHEJ. Our data clearly indicate that low-dose IR-induced resistance of the cells to high-challenge dose IR-induced killing depends on the NHEJ pathway but not the ATM pathway, which also supports that the ATM pathway mainly contributes to HRR. Autophosphorylation of DNA-PK plays an important role in maintaining DNA-PKcs dependent NHEJ and protects cells from IR-induced killing (Chan et al., 2002; Meek et al., 2007). Inhibition of DNA-PKcs activity including the autophosphorylation activity destroyed the low-dose IR-induced protective role, suggesting the importance of DNA-PKcs autophosphorylation for this role. Additional data showed that the V3 cell line transfected with a cluster mutation of DNA-PKcs autophosphorylation sites, 6A or 7A (Chen et al., 2007) had similar phenotypes to V3: low-dose IR could not induce any resistance of the cells to high-challenge dose IR. In fact, low-dose radiation sensitized these cells to high-challenge dose IR-induced killing (Fig. S2). These data further support that DNA-PKcs might be the key factor for the low-dose IR-induced protective role. Low-dose IR promotes ATM activation and the over-activated ATM may contribute to low-dose IR-induced long-term protective effects such as preventing carcinogenesis.

Taken together, our results suggest that the low-dose IR-induced adaptive response on protecting cell survival from high-dose IR-induced killing might be through promoting NHEJ. These discoveries could contribute useful information to improving radiation protection or radiation therapy.

Acknowledgments

Contract grant sponsor: Department of Energy (DOE) Grant;

Contract grant number: DE-FG02-09ER64755.

Contract grant sponsor: National Aeronautics and Space Administration NASA Grant;

Contract grant number: NNX09AF24G.

Contract grant sponsor: NASA Grant;

Contract grant number: NNX07AP84G.

This study was supported by Department of Energy (DOE) Grant (DE-FG02-09ER64755), National Aeronautics and Space Administration NASA Grant (NNX09AF24G) to Y.W. and NASA Grant (NNX07AP84G) to B.C. We thank Drs. Larry Thompson, Joel Bedford and Gloria Li for the cell lines, the support team in Brookhaven National Laboratory for help with the high-LET IR and Ms. Theune for help editing this manuscript.

Abbreviations

IR

ionizing radiation

DSBs

DNA double-strand breaks

HRR

homologous recombination repair

NHEJ

non-homologous end-joining

LET

linear energy transfer

DNA-PKcs

DNA-PK catalytic subunit

Footnotes

All authors declare that there is no conflict of interest.

Additional Supporting Information may be found in the online version of this article.

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