There are two major pathways for repairing DNA double strand breaks in mammalian cells: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). The nonhomologous end joining repair is deficient in cells without Ku, whereas HRR is highly efficient in such cells compared with their wild-type counterparts. The mechanism remains unclear. We reported previously that Ku80−/− cells show a stronger ATM-dependent S-phase checkpoint response than Ku80+/+ cells after ionizing radiation (IR; X-Y. Zhou et al., Oncogene, 21:6377–6381, 2002). We report in this study that Ku80−/− cells also show a much stronger G2 accumulation than Ku80+/+ cells after IR. The stronger G2 checkpoint response in Ku80−/− cells is ATM independent but is accompanied with a higher activity of CHK1 kinase. Treatment with Chk1 antisense oligonucleotide abolishes the stronger G2 checkpoint response and sensitizes Ku80−/− cells to IR. These data indicate that the stronger G2 checkpoint response shown in Ku80−/− cells is CHK1 dependent and suggest that the CHK1-dependent checkpoint response contributes to the highly efficient HRR in such cells.

In response to IR,3 proliferating cells slow down the progress through the cell cycle by activating the DNA damage-induced checkpoints, G1, S, and G2 checkpoints, which are believed to promote DNA repair and to benefit genomic integrity (1, 2, 3, 4). Two major cDNA DSB repair pathways exist in mammalian cells, NHEJ and HRR. NHEJ repair is a fast process requiring Ku80, Ku70, DNA-dependent protein kinase catalytic subunit, ligase IV, and XRCC4 (5, 6). HRR repair is a slow process requiring Rad51, Rad52, and Rad54 as well as the Rad51 paralogues including XRCC2, XRCC3, Rad52B, Rad51C, and Rad51D (7). Deficiency in either NHEJ or HRR in animals leads to increased sensitivity to DNA damage inducers and to increased rates of neoplastic transformation (6), which suggests that both processes are important for genomic integrity. However, it is not known whether checkpoint activation benefits both NHEJ and HRR equally, or whether it is specifically adapted to assist just one of these processes.

Ku is an essential factor for NHEJ repair. Without Ku, the cells become very sensitive to IR (8, 9, 10, 11). On the other hand, without Ku, the cells have a higher HRR efficiency after DNA DSBs are induced (12, 13), indicating an overactivated HRR process in these cells and suggesting a complementary role of HRR in overcoming the NHEJ deficiency. The mechanism by which the highly efficient HRR occurs in Ku-deficient cells after DSBs is unclear. HRR mainly takes place in the S and G2 phases (14), suggesting that a longer S and G2 phase arrest will benefit such repair. Therefore, the stronger ATM-dependent S checkpoint response in Ku80−/− cells (15) might contribute to the highly efficient HRR in such cells.

In this study, we show that Ku80−/− cells have a much stronger G2 checkpoint response compared with their wild-type counterparts after IR. However, the stronger G2 checkpoint response is not dependent on the ATM pathway. Our data show that the stronger G2 checkpoint in irradiated Ku80−/− cells is accompanied by a stronger CHK1 activity, and Chk1 antisense oligonucleotide not only abolishes the stronger G2 checkpoint response but also efficiently sensitizes Ku80−/− cells to IR-induced killing. These results indicate that besides the overactivated ATM pathway, Ku80−/− cells also have an overactivated ATM-independent but CHK1-dependent pathway after IR, suggesting the contribution of the stronger checkpoint responses to the highly efficient HRR in Ku80−/− cells.

Cell Lines, Chemical Treatment, and Irradiation.

Immortalized Ku80+/+ and Ku80−/− mouse embryonic fibroblast cells were generated as described before (8, 16). Ku80–1 cells were generated by transfecting Ku80−/− cells with the plasmid pMV6 containing human Ku80 cDNA. All of these cells were adapted to growing in DMEM supplemented with 10% iron-supplemented calf serum (Sigma-Aldrich Co.). The incubations were at 37°C in an atmosphere of 5% CO2 and 95% air. Caffeine (Sigma-Aldrich Co.), wortmannin (Sigma-Aldrich Co.), or UCN-01 (National Cancer Institute) was added to the culture 30 min before the cells were exposed to X-rays (310 kV, 10 mA, 2-mm Al filter) and was kept in the culture until the cells were collected.

Flow Cytometry Assay.

As described (17), Ku cells were collected at required times and fixed in 70% ethanol. Cells were stained with the solution (62 μg/ml RNase A, 40 μg/ml propidium iodide, and 0.1% Triton X-100 in PBS) at room temperature for 1 h. The distribution of cells in the cell cycle was measured in a flow cytometer (Coulter Epics Elite).

Purification of GST-Cdc25C200–256.

BL21 cells were transformed with plasmid encoding GST-CDC25C200–256. The GST-CDC25C200–256 was purified by using the microspin GST purification module (Amersham Pharmacia Biotech, Inc.) according to the manufacturer’s instructions.

CHK1 Kinase Activity Assay.

For this purpose, cell extracts were prepared using the NE-PER kit (Pierce) according to the manufacturer’s instructions. The nuclear extracts (250 μg) were then mixed with 1 μg of CHK1 antibody (sc-7898; Santa Cruz) in the presence of 10 μl of a 50% (v/v) protein A-Sepharose slurry (RepliGen) as described previously (18).

CDK1 Phosphorylation and Kinase Activity Assay.

The CDK1 (also called CDC2) phosphorylation and CDK1 kinase assay are similar to the measurements of CDK2 phosphorylation and CDK2 kinase assay (18). For the kinase assay, cell extracts were prepared using the NE-PER kit (Pierce) according to the manufacturer’s instructions. The nuclear extracts (250 μg) were then mixed with 1 μg of CDK1 antibody (sc-54; Santa Cruz) in the presence of 10 μl of a 50% (v/v) protein A-Sepharose slurry (RepliGen) as the measurement of CDK2 activity described before (19).

Colony-forming Assay.

Cellular sensitivity to radiation was determined by the loss of colony-forming ability as described previously (17).

Treatment of Cells with Oligonucleotides.

The antisense oligonucleotide of Chk1 (5′-ggcactgccatgactcca-3′) was designed to specifically target the sequence of the start codon region of Chk1 mRNA (17). The oligonucleotide (5′-ACCATGAGTCTAGCACTC-3′) was designed as the control. The oligonucleotides were delivered to the cells by OligofectAMINE (Life Technologies, Inc.) according to the manufacturer’s instructions as described previously (18).

Stronger ATM-independent G2 Checkpoint Response Shown in Irradiated Ku80−/− Cells.

We showed previously that Ku80−/− cells had a stronger ATM-dependent S checkpoint response than Ku80+/+ cells after IR (15). Because ATM is also an important regulator of the G2 checkpoint (20, 21, 22), we further studied whether the overactivated ATM pathway also regulated a stronger G2 checkpoint response in Ku80−/− cells after IR. We analyzed the G2/M ratio with propidium iodide-stained Ku cells at different times after IR. Ku80−/− cells did show a stronger G2 checkpoint than Ku80+/+ cells after IR (Fig. 1). Although both Ku cells showed a clear G2-phase accumulation at 6 h after exposure to 2 Gy, the G2 accumulation became much less in Ku80+/+ cells and was even more in Ku80−/− cells at 10 h after IR (Fig. 1). Ku80-1 cells showed much less G2 accumulation than Ku80−/− cells at all time points after IR (Fig. 1), indicating that the IR-induced stronger G2 accumulation shown in Ku80−/− cells is because of Ku80 deficiency in such cells.

Then we examined the effect of wortmannin on the IR-induced G2 accumulation. Wortmannin, at 10 μm, which efficiently inhibits ATM and DNA-PK (15, 23), did not affect the IR-induced G2 accumulation in any of these Ku cells (Fig. 1), indicating that neither ATM nor DNA-PK is responsible for regulating the G2 checkpoint response. In line with this result, there is no difference in the G2/M checkpoint response between DNA-PK+/+ and DNA-PK−/− cells after IR (data not shown). To identify which kinase was involved in the ATM-independent process in Ku cells, we next tested the effects of caffeine and UCN-01 on the IR-induced G2 accumulation. Caffeine, at 4 mm, which inhibits ATM and ATR but not DNA-PK (24) and UCN-01, at 100 nm, which inhibits CHK1 (the downstream substrate of ATR) but not CHK2 (the downstream substrate of ATM; Refs. 25, 26), efficiently abolished the stronger G2 accumulation in irradiated Ku80−/− cells (Fig. 1), suggesting that the ATR/CHK1 pathway is responsible for this checkpoint response.

Stronger G2 Checkpoint Accompanies Highly Activated CHK1 Pathway in Irradiated Ku80−/− Cells.

To further study whether CHK1 was responsible for the stronger G2 accumulation in irradiated Ku80−/− cells, we examined the CHK1 pathway in these cells. Although we did not see the phosphorylation of CHK1, the reason might be that the signal of CHK1 phosphorylation is not sensitive enough to be detected with the regular one-dimensional gel in irradiated mammalian cells (27), we did observe higher CHK1 kinase activities in irradiated Ku cells than in nonirradiated controls (Fig. 2). At 10 h after IR, the CHK1 activity was reduced to the level of nonirradiated controls in Ku80+/+ cells but still kept the high level in Ku80−/− cells (Fig. 2) These results are consistent with the G2 arrest data (Fig. 1), suggesting that high CHK1 activity is necessary for maintaining the G2 arrest. The DNA damage checkpoint activated in G2 is believed to be mediated by an inhibition of the CDC25C phosphatase that activates the CDK1 kinase by removing inhibitory phosphates, thus allowing entry into mitosis (20, 28). CHK1 could phosphorylate CDC25C and therefore inactivate CDK1. To study whether the highly activated CHK1 kinase was related to inactivating CDK1 in irradiated Ku80−/− cells, we examined CDK1 activity and CDK1 phosphorylation. These results are consistent with the data of CHK1 activities (Fig. 2) and G2 arrest (Fig. 1). The CDK1 phosphorylations increased and the CDK1 activities decreased in Ku cells at 6 h after IR (Fig. 2). At 10 h after IR, the phosphorylation and activity of CDK1 were returned to the control levels in Ku80+/+ cells but remained at the similar levels with those at 6 h after IR in Ku80−/− cells (Fig. 2), indicating a correlation between CHK1 and CDK1 and suggesting the role of CHK1 in regulating the stronger G2 arrest in Ku80−/− cells.

Chk1 Antisense Oligonucleotide Abolished the Stronger G2 Checkpoint and Sensitized the Cells to IR.

To confirm that the CHK1 pathway is responsible for the stronger ATM-independent G2 accumulation in irradiated Ku80−/− cells, we examined the effects of Chk1 antisense oligonucleotide on this checkpoint response. The Chk1 antisense oligonucleotides specifically inhibited CHK1 expression in Ku80−/− cells (Fig. 3,A) and abolished the stronger G2 accumulation in irradiated Ku80−/− cells (Fig. 3,B). These results provide the direct evidence that CHK1 plays a key role in the stronger G2 arrest of irradiated Ku80−/− cells. To study the relationship between the G2 checkpoint response and radiosensitivity, we examined the radiosensitivity of Ku cells after abolishing their G2 checkpoint response by Chk1 antisense oligonucleotide. Chk1 antisense oligonucleotide sensitized both Ku80+/+ and Ku80−/− cells, but the sensitization in Ku80−/− cells was more efficient than in Ku80+/+ cells (Fig. 3 B), indicating that checkpoint responses are more important for protecting Ku80−/− cells from IR-induced killing.

In this study, our data indicate for the first time that the G2 checkpoint response in Ku80−/− cells is much stronger than in Ku80+/+ cells after IR and the stronger G2 checkpoint response in Ku80−/− cells is regulated by an ATM-independent but CHK1-dependent pathway. Besides CHK1, CHK2 is also involved in the G2 checkpoint response (29, 30, 31, 32), and CDC25C/CDK1 is also the downstream target of CHK2 (33, 34). However, after the cells are treated with wortmannin, the CHK1 activity is not affected (data not shown), and the G2 accumulation is not affected (Fig. 1), but the CHK2 activity is reduced (data not shown), excluding that CHK2 is mainly or directly involved in the stronger G2 checkpoint response in irradiated Ku80−/− cells.

It was reported recently that high concentrations of caffeine (5 mm) not only could abolish the IR-induced G2 checkpoint but also could by itself induce G1 arrest (35). The G2 results shown in this report, therefore, could also be explained because of caffeine or UCN-01-induced G1 arrest. To distinguish the effects of caffeine or UCN-01 on abolishing IR-induced G2 checkpoint from caffeine or UCN-01-induced G1 arrest, we observed the effects of caffeine (4 mm) combined with nocodazole (0.4 μg/ml) or UCN-01 (100 nm) combined with nocodazole (0.4 μg/ml) on the cells cycle progress. If caffeine or UCN-01 affects the checkpoint response, via abolishing IR-induced G2 arrest, as we explained the results shown in Fig. 1, either caffeine combined with nocodazole or UCN-01 combined with nocodazole-treated cells should arrest at the G2 phase because nocodazole prevents cells from going out of M phase; otherwise, if caffeine or UCN-01 via G1 arrest affects the IR-induced checkpoint response, either caffeine combined with nocodazole or UCN-01 combined with nocodazole-treated cells should arrest at the G1 phase. These results showed that most of the cells accumulated at the G2 phase after the cells were treated with either caffeine plus nocodazole or with UCN-01 plus nocodazole for 10 h (data not shown), confirming that caffeine or UCN-01 abolished IR-induced G2 arrest.

Abolishing the stronger G2 checkpoint response sensitizes Ku80−/− cells to IR-induced killing, indicating the importance of G2 checkpoint to cell survival. Ku80−/− cells are deficient in NHEJ, and the overactivated G2 checkpoint response is impossible to facilitate NHEJ repair; therefore, the logical explanation for the protective role of the G2 checkpoint in cell survival is that the G2 checkpoint facilitates HRR. The role of ATM in radiosensitivity is mainly dependent on HRR but not on NHEJ (36), the overactivated ATM pathway shown in irradiated Ku80−/− cells (15) partially explains why Ku-deficient cells have highly efficient HRR (12, 13). The results shown in this study provide additional explanation for the highly efficient HRR in Ku80−/− cells. NHEJ repair is not affected by the CHK1-regulated G2 checkpoint (17). Abolishing the CHK1-regulated G2 checkpoint sensitizes Ku80−/− cells to IR. These results suggest that the overactivated CHK1-regulated checkpoint also contributes to the highly efficient HRR in such cells.

Fig. 1.

Stronger ATM-independent G2 checkpoint response is shown in irradiated Ku80−/− cells. The Ku cells were collected at the indicated time points after 2 Gy of exposure. The preparation and measurement of flow cytometric profiles of cell cycle distribution are as described in “Materials and Methods.” The chemicals were added to the cell cultures 30 min before IR. Because wortmannin is easily degraded in water (37), the culture medium was changed with fresh wortmannin every 3 h.

Fig. 1.

Stronger ATM-independent G2 checkpoint response is shown in irradiated Ku80−/− cells. The Ku cells were collected at the indicated time points after 2 Gy of exposure. The preparation and measurement of flow cytometric profiles of cell cycle distribution are as described in “Materials and Methods.” The chemicals were added to the cell cultures 30 min before IR. Because wortmannin is easily degraded in water (37), the culture medium was changed with fresh wortmannin every 3 h.

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Fig. 2.

The overactivated CHK1 pathway is shown in irradiated Ku80−/− cells. The Ku cells were collected at the indicated time points after 2 Gy of exposure. The CHK1 protein and CHK1 kinase activity are measured with the CHK1-immunoprecipitated nuclear extracts from non- or irradiated Ku cells. The CDK1 protein and CDK1 activity were measured with the CDK1-immunoprecipitated nuclear extracts from non- or irradiated Ku cells. The detailed procedures were as described in “Materials and Methods.” The CDK1 phosphorylation was detected with Phospho-cdc2 (Tyr15) antibody (Cell Signaling).

Fig. 2.

The overactivated CHK1 pathway is shown in irradiated Ku80−/− cells. The Ku cells were collected at the indicated time points after 2 Gy of exposure. The CHK1 protein and CHK1 kinase activity are measured with the CHK1-immunoprecipitated nuclear extracts from non- or irradiated Ku cells. The CDK1 protein and CDK1 activity were measured with the CDK1-immunoprecipitated nuclear extracts from non- or irradiated Ku cells. The detailed procedures were as described in “Materials and Methods.” The CDK1 phosphorylation was detected with Phospho-cdc2 (Tyr15) antibody (Cell Signaling).

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Fig. 3.

Chk1 antisense oligonucleotide abolishes the G2 checkpoint response and sensitizes the Ku cells to IR. A, the levels of CHK1 expression were measured with the extracts from either Chk1 antisense or control oligonucleotide-treated Ku cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. B, the treatments of Chk1 antisense oligonucleotide are as described in “Materials and Methods.” The Ku80−/− cells were collected at the indicated time points after 2 Gy of exposure. The preparation and measurement of flow cytometric profiles of cell cycle distribution are as described in Fig. 1. C, as described in “Materials and Methods,” Ku cells were treated with oligonucleotides for 24 h and then were irradiated (2 Gy). After the cells were incubated for an additional 18 h, the cells were trypsinized and planted to new dishes for colony forming with the medium without oligonucleotide. Data shown are the average from three independent experiments. Oligo, oligonucleotide.

Fig. 3.

Chk1 antisense oligonucleotide abolishes the G2 checkpoint response and sensitizes the Ku cells to IR. A, the levels of CHK1 expression were measured with the extracts from either Chk1 antisense or control oligonucleotide-treated Ku cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. B, the treatments of Chk1 antisense oligonucleotide are as described in “Materials and Methods.” The Ku80−/− cells were collected at the indicated time points after 2 Gy of exposure. The preparation and measurement of flow cytometric profiles of cell cycle distribution are as described in Fig. 1. C, as described in “Materials and Methods,” Ku cells were treated with oligonucleotides for 24 h and then were irradiated (2 Gy). After the cells were incubated for an additional 18 h, the cells were trypsinized and planted to new dishes for colony forming with the medium without oligonucleotide. Data shown are the average from three independent experiments. Oligo, oligonucleotide.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by NIH Grants CA76203 (to Y. W.), CA56909 (to G. C. L.), and P30-CA56036.

3

The abbreviations used are: IR, ionizing radiation; DSB, double strand break; NHEJ, nonhomologous end joining; HRR, homologous recombination repair; ATM, ataxia telangiectasia mutated; DNA-PK, DNA-dependent protein kinase.

Special thanks go to Nancy Mott for help in the preparation of the manuscript and to Peggy Mammen for assistance with the laboratory work.

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