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Research Articles: Therapeutics

Effect of combined DNA repair inhibition and G₂ checkpoint inhibition on cell cycle progression after DNA damage

¹ Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada and ² Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California

Requests for reprints: Michel Roberge, Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. Phone: 604-822-2304; Fax: 604-822-5227. E-mail: michelr{at}interchange.ubc.ca

Abstract

In response to DNA damage, cell survival can be enhanced by activation of DNA repair mechanisms and of checkpoints that delay cell cycle progression to allow more time for DNA repair. Inhibiting both responses with drugs might cause cancer cells to undergo cell division in the presence of lethal amounts of unrepaired DNA. However, we show that interfering with DNA repair via inhibition of DNA-dependent protein kinase (DNA-PK) reduces the ability of checkpoint inhibitors to abrogate G₂ arrest and their radiosensitizing activity. Cells exposed to the DNA-PK inhibitor AMA37, DNA-PK-deficient cells, and nonhomologous end joining–deficient cells all enter prolonged G₂ arrest after exposure to ionizing radiation doses as low as 2 Gy. The checkpoint kinase Chk2 becomes rapidly and transiently overactivated, whereas Chk1 shows sustained overactivation that parallels the prolonged accumulation of cells in G₂. Therefore, in irradiated cells, DNA repair inhibition elicits abnormally strong checkpoint signaling that causes essentially irreversible G₂ arrest and strongly reduces the ability of checkpoint kinase inhibitors to overcome G₂ arrest and radiosensitize cells. Variable levels of proteins controlling DNA repair have been documented in cancer cells. Therefore, these results have relevance to the development of DNA-PK inhibitors and G₂ checkpoint inhibitors as experimental therapeutic approaches to enhance the selective killing of tumor cells by radiotherapy or DNA-damaging chemotherapeutic agents. [Mol Cancer Ther 2006;5(4):885–92]

Introduction

DNA damage can elicit a variety of cellular responses (1). Some, such as apoptosis, mitotic catastrophe, and premature senescence, remove damaged cells from proliferating populations. Others, such as DNA repair and cell cycle checkpoints, which delay cell cycle progression and allow more time for DNA repair, promote cell survival and subsequent proliferation. Considerable efforts are being invested into identifying chemicals that modulate these responses and investigating whether they have potential as cancer therapy agents. In particular, it has been proposed that drugs that inhibit DNA repair or cell cycle checkpoints might enhance the efficacy of current DNA-damaging cancer treatments, such as ionizing radiation and many chemotherapeutic drugs.

According to current models, DNA damage activates the phosphoinositide 3-kinase–like kinase family members DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), and ataxia telangiectasia and rad3 related (ATR). Repair of double-strand breaks, the most lethal DNA lesions caused by ionizing radiation, is accomplished through two major pathways: nonhomologous end joining (NHEJ) and homologous recombination repair (2). NHEJ is a G₁-S-predominant process controlled by DNA-PK (a complex of the catalytic subunit DNA-PK_CS, Ku70/86), DNA Ligase IV, Artemis, and XRCC4 (3). Homologous recombination repair is a late S-G₂-predominant process that requires Rad52, Rad54, and various Rad51 paralogues (4, 5). ATM and ATR phosphorylate and activate the checkpoint kinases Chk1 and Chk2, which phosphorylate a variety of downstream targets resulting in cell cycle delay at the G₁ and G₂ phases of the cell cycle and slow down of S-phase traversal.

More than 50% of human cancers have a mutated form of p53, resulting in an inoperative G₁ checkpoint (6) but a functional, albeit weakened G₂ checkpoint. Some inhibitors of the G₂ checkpoint can selectively sensitize p53-defective cells to DNA-damaging agents, likely as a result of undergoing mitosis in the presence of damaged DNA (reviewed in ref. 7). DNA-PK-defective cells show increased sensitivity to ionizing radiation, and it was recently shown that DNA-PK inhibitors can radiosensitize tumors in vitro and in vivo (8, 9).

We reasoned that cells treated with ionizing radiation and a DNA-PK inhibitor would arrest in G₂ phase with more unrepaired DNA than cells exposed to irradiation alone. Exposure of these G₂-arrested cells to checkpoint inhibitors would then force them to divide in the presence of more heavily damaged DNA, which might translate into increased killing. However, in the absence of NHEJ repair, DNA damage caused a stronger activation of checkpoint kinases and a more pronounced and prolonged cell cycle arrest, which G₂ checkpoint inhibitors were less able to overcome and increase cell killing. These observations are relevant to efforts to develop checkpoint inhibitors and DNA-PK inhibitors as tumor-selective radiosensitizers for cancer therapy.

Materials and Methods

Cell Lines, Irradiation, and Checkpoint Inhibitor Treatment
MO59K, MO59J, and MO59J/Fus1 cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (Life Technologies, Gaithersburg, MD). MO59J/Fus1 cells were additionally supplemented with 250 µg/mL G418 (Invitrogen, San Diego, CA). All Chinese hamster ovary (CHO) cells were cultured in DMEM with 10% fetal bovine serum. CHO K1 and XRCC4 cells were obtained from the Coriell Cell Repository (Camden, NJ). CHO V3 cells were transfected with either empty vector or hDNA-PK_CS cDNA (10) and were a gift from Susan Lees-Miller. CHO 51D1 and 51D1.3 cells were a gift from Larry H. Thompson. MCF-7 mp53 cells were cultured as previously described (11, 12). Cells were irradiated at a rate of 0.75 Gy/min using a ⁶⁰Co source (Gammacell 220, Atomic Energy Commission of Canada, Chalk River, Ontario, Canada). Caffeine (Sigma-Aldrich Co., St. Louis, MO), UCN-01 (National Cancer Institute), isogranulatimide (13), and debromohymenialdesine (14) were used to abrogate G₂ arrest, together with 300 ng/mL nocodazole (Sigma-Aldrich) to arrest cells in mitosis. AMA37 (Calbiochem, La Jolla, CA) was added to cells 30 minutes before treatment and refreshed every 8 hours.

Flow Cytometry and Checkpoint Inhibitor Assay
Cells were prepared for flow cytometry analysis exactly as described in (15). Checkpoint inhibition was assessed as described in ref. 15, except that CHO cells were irradiated with 6.5 Gy and incubated for 8 hours followed by checkpoint inhibitor plus nocodazole addition for an additional 4 hours.

Cell Clonogenicity Assays
To assess radiosensitization by checkpoint inhibitors and AMA37, MCF-7 cells were plated in serial dilutions into six-well plates. They were allowed to attach for 24 hours and then irradiated in the presence or absence of drug. After 24 hours, drugs were washed off, and cells were grown in fresh medium for 10 days. Colonies were stained with Malachite Green and counted. Clonogenicity was expressed as % colonies relative to untreated cells.

Kinase Assays
Following mock irradiation or exposure to 6.5 Gy, cells were further cultured as indicated. Cells were then harvested via trypsinization, washed twice in PBS, assayed for CDK1 (16), Chk1/2 (17), or ATM/ATR (18) as described with the following modifications. Protein concentration was determined using the Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, IL), with 125, 250, or 500 µg total protein lysate used for immunoprecipitation of CDK1, Chk1/2, or ATM/ATR, respectively. Antibodies used were 2 µg anti-ATR antibody (Santa Cruz Biotechnology, Santa Cruz, CA), 1 µg anti-CDK1 antibody (KAP-CC001E, Stressgen), 1 µg anti-Chk2 antibody (Santa Cruz Biotechnology), 1 µg anti-Chk1 antibody (Santa Cruz Biotechnology), or 2 µg anti-ATM antibody (Ab-3 PC116, Oncogene, Uniondale, NY). Substrates used were 1 µg histone H1 (Sigma, St. Louis, MO), 1 µg GST-CDC25C_200-256 (19), or 2 µg PHAS-1 (AG Scientific, San Diego, CA) for CDK1, Chk1/2, or ATM/ATR assays, respectively. Samples were separated via SDS-PAGE, exposed to Kodak X-OMAT AR film, and developed according to the manufacturer's instructions.

Results and Discussion

DNA-PK Inhibition and NHEJ Deficiency Decrease the Ability of Cancer Cells to Respond to G₂ Checkpoint Inhibitors
Caffeine, an inhibitor of ATM and ATR (20, 21), and UCN-01, isogranulatimide, and debromohymenialdesine, inhibitors of Chk1 or Chk1/Chk2, can abrogate the G₂ checkpoint and potentiate the lethality of ionizing radiation (13, 14, 21–23). DNA-PK inhibitors can radiosensitize tumor cells by impairing DNA repair via the NHEJ system (8, 24). The arylmorpholine AMA37 is a potent in vitro inhibitor of DNA-PK (IC₅₀ = 0.27 µmol/L) that does not inhibit ATM or ATR and inhibits phosphoinositide 3-kinases poorly (25). We wished to determine whether checkpoint inhibitors are able to overcome G₂ arrest in the presence of a DNA-PK inhibitor.

The interplay between DNA-PK inhibition and G₂ checkpoint inhibition was first examined in human breast carcinoma MCF-7 mp53 cells. These cells are NHEJ-proficient, have an inactive G₁ checkpoint (11, 26), and have been used extensively to study the G₂ checkpoint (11, 13–15). To measure G₂ checkpoint inhibition, MCF-7 mp53 cells were exposed to AMA37 or the drug carrier DMSO for 30 minutes and then irradiated with 6.5 Gy (Fig. 1A ). At 16 hours, when G₂ arrest was maximal, cells were treated for 8 hours with 2 mmol/L caffeine, 100 nmol/L UCN-01, 100 µmol/L isogranulatimide, or 40 µmol/L debromohymenialdesine, together with nocodazole to trap cells that escaped G₂ arrest in mitosis. The cells were then analyzed by two-dimensional flow cytometry using the mitosis-specific antibody GF-7 to distinguish M cells from G₂ cells (ref. 15; see Supplementary Fig. S1³ for examples). Quantitation of G₂ checkpoint inhibition after various treatments is presented in Fig. 1A. Caffeine, UCN-01, isogranulatimide, and debromohymenialdesine caused clear G₂ checkpoint inhibition. AMA37 reduced the ability of UCN-01, isogranulatimide, and debromohymenialdesine, but not caffeine, to overcome G₂ arrest ( < 0.05). By contrast, 200 nmol/L wortmannin, which inhibits phosphoinositide 3-kinases but not ATM or DNA-PK (27), did not reduce the activity of the checkpoint inhibitors (Fig. 1A). The effect of AMA37 on -H2AX phosphorylation was consistent with selective inhibition of DNA-PK in vivo (see Supplementary Fig. S2)³, indicating that the effects of AMA37 were likely due to DNA-PK inhibition and not to modulation of other phosphoinositide 3-kinase–like kinase involved in the checkpoint response.

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of G2 checkpoint inhibitors on cells lacking DNA-PK or NHEJ activity. A, MCF-7 mp53 cells were exposed to DMSO, 20 µmol/L AMA37, or 200 nmol/L wortmannin 30 min before irradiation with 6.5 Gy. Sixteen hours later, when arrested in G2, cells were exposed to checkpoint inhibitors plus nocodazole for 8 h and harvested as described in Materials and Methods. GF-7-positive cells represent those trapped in mitosis. B, MO59 cells were irradiated and treated as in A. C, CHO cells were irradiated with 6.5 Gy. Eight hours after ionizing radiation, checkpoint inhibitors and nocodazole were added for 4 h. Columns, average between different experiments (n 2); bars, SD.

These data indicated that DNA-PK activity is required for cells to resume cell cycle progression after G₂ arrest, or that the reduced activity of checkpoint inhibitors is a consequence of reduced DNA repair via NHEJ in cells lacking DNA-PK activity. To address this issue, we monitored checkpoint inhibition in an additional six related CHO cell lines. CHO K1 cells are considered wild type. CHO XR-1 cells do not express XRCC4, making them defective for NHEJ repair (36). CHO V3 cells are deficient in DNA-PK_CS (37). CHO V3 cells stably transfected with either empty vector (CHO V3) or human DNA-PK_CS cDNA (CHO V3 hDNA-PK_CS) differ by the presence or absence of DNA-PK_CS (10). CHO 51D1 lack both alleles expressing Rad51D, whereas 51D1.3 are a Rad51D-reconstituted isogenic match. DNA-PK_CS-deficient V3 cells were less responsive to checkpoint inhibitors than their DNA-PK-proficient counterparts (Fig. 1C). Interestingly, XRCC4-deficient XR-1 cells were not responsive at all to checkpoint inhibitors, whereas K1 cells with functional NHEJ were. CHO 51D1 cells, deficient in Rad51D, were less responsive to UCN-01 and isogranulatimide, but not caffeine, than their Rad51D-expressing counterparts, 51D1.3. These data provide strong evidence that it is a lack of DNA repair that reduces the capacity of checkpoint inhibitors to abrogate G₂ arrest.

DNA-PK Inhibition Leads to Reduced Radiosensitization by Checkpoint Inhibitors
To determine whether the decreased ability of checkpoint inhibitors to abrogate G₂ arrest translates into reduced radiosensitization, we assessed the clonogenicity of MCF-7 mp53 cells following combined treatment with AMA37 and checkpoint inhibitors. Table 1 lists the effect of different drug combinations on clonogenicity. Exposure of cells to 6.5 Gy ionizing radiation alone lead to a decrease in survival to 15.4 ± 2.0% of unirradiated controls. Irradiation in the presence of caffeine, UCN-01, or isogranulatimide caused a further decrease in clonogenicity, indicating radiosensitization. Inhibition of DNA-PK with AMA37 also lead to radiosensitization. The checkpoint kinase inhibitors UCN-01 and isogranulatimide were unable to radiosensitize cells in the presence of AMA37, consistent with their inability to overcome G₂ arrest. Caffeine, the only compound that retained checkpoint inhibitory activity in the presence of AMA37 (Fig. 1A), was also able to radiosensitize in the presence of AMA37. Caffeine inhibits ATM and ATR and acts upstream of the Chk kinase inhibitors UCN-01 and isogranulatimide. Caffeine is also a less selective checkpoint inhibitor than UCN-01 and isogranulatimide. It inhibits several other cellular activities, including DNA repair itself (reviewed in ref. 7). We speculate that caffeine's radiosensitizing effect in the presence of AMA37 may due to its inhibition of DNA repair and/or its ability to down-regulate more branches of the checkpoint response than UCN-01 and isogranulatimide.

View larger version (12K): [in this window] [in a new window]   Figure 2. Effect of ionizing radiation on G2 arrest in cells lacking DNA-PK or NHEJ activity. Cells were irradiated at time 0 and sampled at the indicated times, and the G2-M populations were quantitated using flow cytometry. A, MCF-7 mp53 cells were exposed to DMSO, 20 µmol/L AMA37, 200 nmol/L wortmannin, or 50 µmol/L LY294002 30 min before irradiation with 6.5 Gy. Another cell population was irradiated with 6.5 Gy, and AMA37 was added 16 h later. Cells were harvested at the indicated times and analyzed by flow cytometry. B, MO59K, MO59J, or MO59J/Fus1 cells were exposed to 6.5 Gy. C, CHO cells were exposed to 2 Gy. Points, average between different experiments (n 3); bars, SD.

Quantitation of the G₂-M peaks as a function of time after irradiation of MO59 cells is shown in Fig. 2B. Exposure to 6.5 Gy caused a large increase in the G₂-M peak that was maximal at 16 hours for all three cell lines. Most of the MO59K cells escaped G₂ arrest between 16 and 24 hours and cycled thereafter as a loosely synchronous population. Similar to AMA37 treatment, MO59J cells remained arrested in G₂ for at least 48 hours after ionizing radiation. Cell populations with sub-G₁ DNA content became increasingly evident at time points longer than 48 hours, indicating the onset of cell death (data not shown). Cyclin B1 also accumulated in G₂-arrested MO59J cells (Supplementary Fig. S3C).³ MO59J/Fus1 cells began to exit G₂ arrest 16 hours after exposure 6.5 Gy, much like MO59K cells (Fig. 2B). Additional ionizing radiation doses were tested in these cell lines: a lower dose of 2 Gy caused a significant increase in the G₂-M peak only in MO59J cells and remained for upwards of 48 hours. A higher dose of 10 Gy caused a large increase in G₂-M that peaked at 16 to 24 hours in all cell lines, with MO59K and MO59J/Fus1 cells recovering from this arrest. However, MO59J/Fus1 cells remained arrested longer and fewer cells finally exited G₂ arrest at this dose (P < 0.0001; see Supplementary Fig. S3A).³ Interestingly, MO59J/Fus1 cells have about 60% of the DNA-PK activity of MO59K (29), and they exited G₂ arrest significantly later and to a lesser extent than M059K after exposure to 10 Gy but not lower doses, suggesting a relationship between the level of cellular DNA-PK activity and the ability of cells to escape G₂ arrest. At 10 Gy, MO59J/Fus1 cells were equally unresponsive to checkpoint inhibitors as MO59J (data not shown), whereas MO59K cells remained equally responsive.

The different CHO cell lines were subjected to irradiation with 2 Gy, and G₂ arrest was monitored over time. Ionizing radiation elicited a transient G₂ arrest in K1 cells and V3 hDNA-PK_CS cells, both of which have DNA-PK_CS and NHEJ activity (Fig. 2C). However, DNAPK_CS-defective V3 cells and NHEJ-defective XR-1 cells showed strong sustained G₂ arrest (Fig. 2C). Exit from G₂ arrest started after 8 hours in K1 and V3 hDNA-PK_CS cells, whereas V3 and XR-1 cells remained arrested in G₂ for at least 48 hours. For unclear reasons, G₂ arrest was also accompanied by an increase in 8n population in XR-1 and V3 cells, and a large increase in apoptosis was observed at the higher dose of 6.5 Gy (see Supplementary Fig. S3D).³ The G₂ arrest profile of 51D1 and 51D1.3 cells irradiated with 2 Gy was similar to that of wild-type K1 cells (Fig. 2C), but 6.5 Gy caused a slightly prolonged G₂ arrest in 51D1 cells (see Supplementary Fig. S3D).³ Together, these results show that lack of repair at the time of DNA damage causes prolonged G₂ arrest that cannot be abrogated by checkpoint inhibitors, likely caused by the persistence of DNA lesions.

Ionizing Radiation Causes Overactivation of Chk1 and Chk2 in Cells Lacking DNA-PK Activity
We next wished to examine the mechanism underlying prolonged G₂ arrest in cells lacking DNA repair. The G₂ checkpoint is generally believed to comprise two convergent pathways: ATM activation followed by Chk2 activation and ATR activation followed by Chk1 activation, both leading to CDC25 and CDK1 inhibition (39, 40). We therefore monitored the activation of the canonical checkpoint pathways using in vitro kinase assays.

We first measured the activity of the kinases ATR, Chk1, and CDK1 in M059 cells. Cells were exposed or not to 6.5 Gy and incubated for 16 hours to achieve maximal G₂ arrest, and the activity of the immunoprecipitated kinases was measured in vitro using substrate proteins or peptides (see Materials and Methods). The protein levels of ATR, Chk1, and CDK1 (p34^cdc2) were similar in the three cell lines and did not change after irradiation (Fig. 3A ). Ionizing radiation caused an increase in ATR activity in MO59K and MO59J/Fus1 cells but not in MO59J (Fig. 3A). No increase in Chk1 kinase activity was observed in MO59K or MO59J/Fus1 cells following ionizing radiation. However, a clear increase in Chk1 kinase activity was observed in irradiated MO59J cells (Fig. 3A), which required several hours after ionizing radiation to become apparent. This result was unexpected because Chk1 is typically activated poorly by ionizing radiation (40), and the upstream activator of Chk1 is ATR (41). As expected, CDK1 kinase activity decreased following DNA damage in all cell lines, but the decrease was slightly stronger in MO59J cells (Fig. 3A), likely due to increased Chk1 activity. Interestingly, an ionizing radiation dose of 50 Gy was required to elicit a similarly high level of Chk1 activation in DNA-PK-complemented MO59J/Fus1 cells (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 3. Overactivation of Chk1 and Chk2 in DNA-PK-deficient cells. A, ATR, Chk1, and CDK1 activity in irradiated cells. Cells were harvested 16 h after mock irradiation (–) or exposure to 6.5 Gy (+). Cellular levels of ATR, Chk1, and CDK1 protein were measured by Western blotting, and their kinase activity was measured in immunoprecipitates as described in Materials and Methods. B, MCF-7 mp53 cells were treated without or with AMA37 30 min before irradiation, and cellular levels and kinase activity of ATR and Chk1 were measured as above 16 h after irradiation. C, ATM and Chk2 activity in irradiated MCF-7 mp53 cells harvested 1 or 16 h after ionizing radiation (IR). D, Chk2 kinase activity of MO59 cells measured at 1 or 16 h after ionizing radiation.

We next examined the activity of the kinases ATM and Chk2. MCF-7 mp53 cells were preincubated with AMA37 and irradiated. One hour after exposure to 6.5 Gy, ATM activity in both DMSO- and AMA37-treated cells increased slightly and then decreased by 16 hours (Fig. 3C). In the absence of AMA37, Chk2 activity was increased at both 1 and 16 hours after irradiation. However, very strong Chk2 activity was observed in AMA37-treated cells 1 hour after ionizing radiation (Fig. 3C). To confirm that this strong transient increase was indeed due to lack of DNA-PK activity, M059K, M059J, and M059J/Fus1cells were irradiated, and Chk2 activity was measured 1 and 16 hours later. Chk2 was clearly overactivated in DNA-PK-deficient M059J cells compared with DNA-PK-complemented MO59J/Fus1 cells and M059K cells (Fig. 3D). We were unable to examine checkpoint kinase activity in CHO cells because available antibodies showed insufficient selectivity towards rodent proteins.

Overall, the results show that irradiation of cells lacking DNA-PK activity or cells exposed to the DNA-PK inhibitor AMA37 causes overactivation of Chk1 and Chk2. This abnormally high checkpoint signaling is likely the mechanism underlying the prolonged G₂ arrest. Prolonged G₂ arrest dependent on Chk1 overactivation has also been observed in human AT cells after DNA damage, and this requires S-phase traversal (42), as well as in mouse Ku86-deficient cells (19). The replication of damaged DNA in S phase could generate secondary lesions that overactivate the checkpoint in G₂ (42). This explanation likely also applies to cells lacking DNA-PK activity. In addition, we observed that cells lacking DNA-PK activity showed abnormally high Chk2 activity within 1 hour of irradiation. In normal cells, Chk2 activation is probably limited because rapid DNA repair restricts the magnitude of the damage signal.

The overactivation of Chk1 and Chk2 in cells lacking DNA-PK activity also adequately explains why cells show a reduced response to G₂ checkpoint inhibitors that target Chk1 and/or Chk2. At concentrations that can be used on cells, these inhibitors may not counteract the overactivated pathway sufficiently to override G₂ arrest. There may also be additional differences between cells undergoing transient G₂ arrest and cells undergoing prolonged G₂ arrest that render checkpoint inhibitors less effective. At time points longer than 48 hours, most G₂-arrested MO59J cells showed sub-G₁ DNA peaks, indicating apoptosis. However, many surviving cells were observed, and they showed signs of senescence, including a large size, flattened shape, and increased acidic ß-galactosidase activity (data not shown). Recently, it has been shown that senescent human fibroblasts exhibit similar markers to DNA damage checkpoint-arrested cells, such as colocalization of phosphorylated H₂AX with 53BP1, MDC1, and NBS1, along with overactivation of the checkpoint kinases Chk1/Chk2 (43). Below a threshold level of G₂ checkpoint signaling, cells may undergo transient G₂ arrest to increase time for DNA repair and then resume cell cycle progression, whereas above this threshold, cells may arrest permanently and enter a senescent state that is unresponsive to G₂ checkpoint inhibitors. Drugs targeting DNA-PK might be useful to drive cancer cells into a senescent state.

Variable levels of DNA-PK activity and DNA-PK_CS, Ku70, and Ku86 protein levels have been documented in a variety of cancers (44–48). The observation that the level of DNA-PK activity influences the ability of cells to resume cell cycle progression after exposure to a clinical dose of 2 Gy may, therefore, have relevance to the fate of tumor cells after radiotherapy or treatment with DNA-damaging chemotherapeutic agents, and ultimately to the clinical outcome of these therapeutic approaches. In addition, combination therapy with DNA-damaging agents and G₂ checkpoint inhibitors or DNA-PK inhibitors has been proposed as a way to increase the selective killing of tumor cells (7–9, 24). The observation that DNA repair inhibition strongly affects the response of cells to checkpoint inhibitors should be taken into consideration when further developing these experimental therapeutic approaches.

Acknowledgments

We thank Susan Lees-Miller (University of Calgary, Calgary, Alberta, Canada); Cordula Kirchgessner (Stanford University, Stanford, CA), Katheryn Meek (Michigan State University, East Lansing, MI), and Larry H. Thompson (Lawrence Livermore National Laboratory, Livermore, CA), for their generous gifts of MO59K, MO59J, MO59J/Fus1, CHO V3 vector- and hDNA-PK_CS–transfected cells, and 51D1/51D1.3 cells, respectively; Peter Davies (Albert Einstein College of Medicine, Bronx, NY), for GF-7 antibody; Ya Wang (Thomas Jefferson University, Philadelphia, PA), for GST-CDC25C_200-256 expression plasmid; and Hilary Anderson for critical reading of the article.

Footnotes

Grant support: National Cancer Institute of Canada (M. Roberge). C.M. Sturgeon is a recipient of a National Sciences and Engineering Research Council postgraduate scholarship.

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.

³ Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Received 9/ 7/05; revised 1/19/06; accepted 2/15/06.

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