Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-dependent DNA Double-strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes*

Takahisa Furuta {ddagger}, Haruyuki Takemura {ddagger}, Zhi-Yong Liao {ddagger}, Gregory J. Aune {ddagger}, Christophe Redon {ddagger}, Olga A. Sedelnikova {ddagger}, Duane R. Pilch {ddagger}, Emmy P. Rogakou {ddagger}, Arkady Celeste §, Hua Tang Chen §, Andre Nussenzweig §, Mirit I. Aladjem {ddagger}, William M. Bonner {ddagger} and Yves Pommier {ddagger} 

From the {ddagger}Laboratory of Molecular Pharmacology and §Experimental Immunology Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255

Received for publication, January 8, 2003 , and in revised form, March 21, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX ({gamma}H2AX) foci. Here we show that {gamma}H2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting {gamma}H2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced {gamma}H2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This {gamma}H2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in {gamma}H2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form {gamma}H2AX. However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved {gamma}H2AX response for double-strand breaks induced by replication fork collision. {gamma}H2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Compact chromatin can be a structural barrier for DNA processing during replication, transcription, recombination, and DNA repair. Following DNA damage, chromatin must be modified to permit the access of repair proteins to the DNA lesions (1). Homologous recombination and non-homologous end joining are the main repair pathways for DNA double-strand breaks (2). Both processes are assumed to require chromatin alterations for DNA end processing, strand invasion, branch migration, DNA synthesis, and ligation as well as for recruiting checkpoint proteins (3).

The fundamental unit of chromatin is the nucleosome, which consists of an octamer of the four core histones, H2A, H2B, H3, and H4, around which the DNA is bound. Histone H2A includes three subfamilies whose members contain characteristic sequence elements that have been conserved independently throughout eukaryotic evolution: H2A1-H2A2, H2AZ, and H2AX (4, 5, 6). In mammals, H2AX represents 2–25% total H2A, whereas in yeast, the major H2A is the H2AX ortholog (7, 8).

H2AX phosphorylation (at its C terminus on serine 139) has been found at the sites of double-strand breaks in chromosomal DNA (9). This phosphorylated form of H2AX has been named {gamma}H2AX1 (8). {gamma}H2AX is rapidly formed in cells treated with ionizing radiation (IR) but also during V(D)J and class-switch recombination and apoptosis (8, 9, 10, 11, 12, 13). Because {gamma}H2AX appears within minutes after IR, {gamma}H2AX focus formation is considered to be a sensitive and selective signal for the existence of a DNA double-strand break (14). {gamma}H2AX foci co-localize with DNA repair and checkpoint proteins including Rad50, Rad51, and Brca1 (15). Recent studies (16, 17) carried out in H2AX knockout mouse embryonic stem cells and mice have demonstrated the involvement of {gamma}H2AX in the repair of DNA double-strand breaks induced by IR. These H2AX knock-out cells are hypersensitive to IR defective in DNA double-strand break rejoining and fail to form 53bp1, Brca1, and Nbs1 foci in response to IR (17).

An alternative source of DNA double-strand breaks is the conversion of single-strand breaks by advancing replication forks (18). DNA single-strand breaks can be produced by a variety of exogenous and endogenous DNA lesions and by the action of DNA topoisomerase I (top1) in DNA containing base damages (abasic sites, mismatches, oxidized bases, carcinogenic adducts, and UV lesions) and preexisting single-strand breaks (discover.nci.nih.gov/pommier/topo1.htm) (19, 20, 21, 22). Top1 is a ubiquitous enzyme that regulates the topological state of DNA during replication, transcription, recombination, and repair (23). The top1 catalytic cycle can be summarized as follows. 1) The enzyme binds to duplex DNA (Fig. 1A). 2) It cleaves one DNA strand by a trans-esterification reaction in which a top1 tyrosine-hydroxyl group becomes covalently linked to the 3'-phosphate of a DNA phosphodiester bond to generate a 5'-hydroxyl DNA terminus. This cleavage intermediate is commonly referred to as a cleavage (or cleavable) complex (Fig. 1B). 3) DNA supercoiling is relaxed by controlled rotation of the 5'-end of the broken DNA around the intact DNA strand (Fig. 1B). 4) After DNA relaxation is complete, the cleaved DNA is religated by nucleophilic attack from the 5'-hydroxyl DNA end and dissociation of the top1 tyrosyl residue from the 3'-end (Fig. 1A) (24). Top1 cleavage complexes are generally widely distributed throughout the genome and normally are transient.



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FIG. 1.
Generation of replication-mediated DNA double-strand breaks by top1 cleavage complexes. A, top1 binds non-covalently to duplex DNA (24). B, top1 produces DNA nicks in duplex DNA, which are referred to as cleavage complexes. Under normal conditions, cleavage complexes are readily reversible (from B to A). C, camptothecin traps top1 cleavage complexes by preventing top1-mediated DNA religation. Various DNA lesions such as abasic sites, mismatches, oxidized bases, UV lesions, single-strand breaks, and base alkylation also trap top1. D, after collision between replication fork and top1 cleavage complexes, the extension of the leading strand is terminated at the 5'-end of the template strand, which generates a DNA double-strand break (34). Replication on the leading strand is shown as a dashed arrow pointing to the left. Okazaki fragments are shown as dashed arrows pointing to the right.

 

Camptothecin and its derivatives (irinotecan and topotecan) are specific top1 inhibitors. Irinotecan has recently been approved by the FDA (Food & Drug Administration) for the treatment of colon cancers (25) and topotecan has been approved for the treatment of ovarian cancers (26, 27). Moreover, a recent clinical study revealed that irinotecan is superior to etoposide for the treatment of lung cancers (28). Therefore, top1 is an important cellular target for cancer chemotherapy (29, 30, 31). Camptothecin and its derivatives trap top1-DNA cleavage complexes by inhibiting the DNA religation step (Fig. 1C) (discover.nci.nih.gov/pommier/topo1.htm) (30, 31).

Despite the abundance of top1 throughout the cell cycle and in quiescent cells, the cytotoxicity of camptothecin is remarkably limited to replicating cells. For instance, when cells are pretreated with the DNA polymerase inhibitor, aphidicolin, they are protected from the cytotoxicity of camptothecins (32, 33). The replication dependence for the formation of lethal DNA lesions by camptothecins can be explained by the formation of replication-mediated DNA double-strand breaks (Fig. 1D). When a replication fork proceeds toward a top1 cleavage complex, the extension of the leading strand is blocked at the 5'-end of the cleaved DNA template with replication fork runoff, resulting in a DNA double-strand break (34, 35, 36).

In this study, we used camptothecin to investigate whether replication-mediated DNA double-strand breaks induce the formation of {gamma}H2AX and its concentration in nuclear foci. We also studied the upstream kinases implicated in the phosphorylation of H2AX and the relationship between {gamma}H2AX formation and the DNA double-strand break repair and checkpoint proteins, Mre11, Rad50, and Nbs1. Finally, the functional role of {gamma}H2AX was studied in H2AX knock-out mouse cells.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Human colon carcinoma HT29 and HCT116 cells were obtained from the Developmental Therapeutics Program, NCI. The SV40-transformed fibroblast cell lines from normal individual (GM00637) and from an ataxia telangiectasia (AT) homozygous patient (GM05849) were obtained from Dr. Michael Kastan (St. Jude Children's Research Hospital, Memphis, TN). The SV40-transformed fibroblast cell line from Nijmegen breakage syndrome (NBS) homozygous patient (GM15989) was purchased from Coriell Institute for Medical Research (Camden, NJ). They were grown at 37 °C in the presence of 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 µg/ml streptomycin. P388 and P388/CPT45 mouse leukemia cells were a kind gift from Drs. Michael R. Mattern and Randal K. Johnson (SmithKline Beecham). They were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C and 5% CO2. M059J/Fus1 and M059J/Fus9 cells were donated from Dr. Cordula U. Kirchgessner (Stanford University School of Medicine, Stanford, CA) and were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum containing 400 µg/ml G418 (Invitrogen). Ataxia telangiectasia and Rad3-related kinase dead (ATRkd) cells were donated from Dr. William A. Cliby (Mayo Clinic, Rochester, MN) and were incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum containing 400 µg/ml G418.

Drugs, Chemicals, and Antibodies—Camptothecin and UCN-01 were provided by the Drug Synthesis Chemistry Branch, Division of Cancer Treatment, NCI. Aphidicolin and wortmannin were purchased form Sigma. Aliquots were stored frozen at 10 mM in Me2SO and diluted further in medium immediately prior to each experiment. Anti-{gamma}H2AX mouse monoclonal antibody were purchased from Upstate Biotechnology (Altham, MA). Anti-{gamma}H2AX rabbit polyclonal antibody was purchased from Trevigen, (Gaithersburg, MD). Anti-Mre11 and Rad50 antibodies were purchased from Novus (Littleton, CO). Anti-Nbs1 antibody was donated from Dr. Nussenzweig (NCI, National Institutes of Health, Bethesda, MD). FITC-conjugated anti-mouse and anti-rabbit antibodies were purchased from Oncogene Research Products (Oncogene Science Inc., San Diego, CA). The second anti-mouse or anti-rabbit Ig antibodies conjugated with AlexaFluor 488 or 546 were purchased from Molecular Probes, Inc. (Eugene, OR). The second anti-mouse Ig antibody conjugated with horseradish peroxidase was purchased from Amersham Biosciences.

IR—Cells growing in T75 flask or on chamber slides were exposed to the indicated dose of IR from a 137Cs source in a Mark I irradiator (J. L. Shepherd and Associates). {gamma}H2AX analyses were performed 30 min after irradiation.

Laser Scanning Confocal Microscopy—Cells were grown on chamber slides. After treatment with indicated doses of camptothecin or IR with or without UCN-01, aphidicolin, or wortmannin at 37 °C, the cell preparations were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min, washed in PBS, permeabilized in 100% methanol at -20 °C for 20 min, washed, blocked with PBS containing 1% BSA and 5% goat serum (Jackson Immunolaboratories, West Grove, PA) for 1 h, incubated with one or two first antibodies at 800-fold dilution for 2 h, washed, incubated with a FITC, AlexaFluor 484, or AlexaFluor 546-conjugated goat anti-rabbit or anti-mouse Ig second antibody at 200-fold dilution for 1 h, and washed in PBS. Some slides were treated with 10 units/ml ribonuclease (Sigma) and stained with propidium iodide. The slides were mounted with mounting medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA) and viewed with a PCM2000 laser scanning confocal microscope (Nikon Co., Tokyo, Japan) using a x40 or x100 objectives. The projection was saved as a BMP file.

Laser Scanning Cytometry—Slides of cells stained with propidium iodide, the anti-{gamma}H2AX first antibody, and the FITC-conjugated second antibody prepared by the protocol as described above were subjected to analysis by laser scanning cytometry (LSC®, CompuCute, Cambridge, MA). The DNA content and intensity of FITC signal from each cell were determined.

Western Blot Analysis for {gamma}H2AX—Cells were grown to 50–80% confluence when treated with different agents. Cells were scraped and pelleted by centrifugation at 0 °C at 1,000 x g for 15 min. The pellets were washed twice in PBS, homogenized in 0.2 N H2SO4, and centrifuged at 13,000 x g. Histones were pelleted from the supernatant by adding 0.25 volume of 100% (w/v) trichloroacetic acid. The pellets were suspended in 100% ethanol overnight and centrifuged again at 13,000 x g. The pellets were dissolved in ultrapure water, and evaluated for protein concentration (Bio-Rad). Aliquot corresponding to 10 µg of protein was boiled in SDS sample buffer (Tris-glycine SDS sample buffer (twice), Invitrogen) and loaded onto the 4–20% Tris-Glycin precast gel (Novex, San Diego, CA). The separated proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA). The membrane was blocked with TBST (10 mM Tris-Cl, pH 7.4, 200 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk for 60 min prior to incubation with 50 ng/ml anti-{gamma}H2AX antibody for 2 h. The blots were washed in TBST and then incubated with horseradish peroxidase-conjugated anti-mouse antibody (1/1,000 dilution) and visualized by chemiluminescence using the Supersignal kit (SuperSignal® West Pico chemiluminescent substrate, Pierce). All of the presented data were confirmed in independent experiments.

Western Blotting Analysis for Nbs1—Cells were grown to 50–80% confluency when they were subjected to the treatment with different agents. Cells were harvested by scrape and washed twice with PBS, and then they were incubated on ice for 30 min in lysis buffer (0.3% Nonidet P-40, 25 mM NaF, 150 mM NaCl, 2 mM EGTA, 1 mM EDTA, 0.2% Triton X-100, 50 mM Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 100 µM leupeptin, 2 mM AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride)). Cell debris was removed by centrifugation at 12,000 x g for 15 min at 4 °C. The supernatant was evaluated for protein concentration and either used immediately for assays or stored at -70 °C.

Cell lysates containing 20 µg of total protein were electrophoresed in Tris-Glycin precast gels after being boiled with SDS sample buffer and then were electrophoretically transferred to Immobilon-P membranes for 1 h at 20 V. The membrane was blocked with 5% nonfat milk for 1 h prior to incubation with anti-Nbs1 antibody (1/10,000 dilution) for 2 h. After washing with TBST for 5 min three times, the membranes were incubated with secondary antibody (1/1,000 dilution) and visualized by chemiluminescence using the Supersignal kit. The presented result was confirmed in independent experiments.

MTT Assay for Cellular Response of H2AX+/+ and H2AX-/- Cells to Camptothecin—H2AX+/+ and H2AX-/- cells were seeded into 96-well microplates at 1,000 cells/90 µl in each well and incubated overnight. The cells were treated without or with the indicated concentrations of camptothecin for 72 h. After a 72-h incubation, 10 µl of MTT solution (5 mg/ml, Sigma) was added to the each well. 4 h later, the medium was aspirated and 100 µl of Me2SO was added to each well. The optical density of each well (wavelength 520 nm) was measured with a microplate reader (Emax, Molecular Devices Corp., Sunnyvale, CA). The survival of cells at each concentration of the drug was expressed as the percentage ratio of the optical density of cells treated with different concentrations of camptothecin to the mean optical density of the untreated cells. Measurements were performed in triplicate. Data were expressed as the mean ± S.D. The statistical difference in overall cellular responses between the two cell lines was assessed by the two-way ANOVA followed by the Scheffe's multiple comparison test. Difference in the survival ratios between the two cell lines at each concentration point was assessed by the Student's t test. When p value was <0.05, the difference was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Production of {gamma}H2AX by Top1 Cleavage Complex—Fig. 2A shows that when human colon carcinoma HCT116 cells were treated with camptothecin for 1 h, {gamma}H2AX foci were observed. {gamma}H2AX focus formation increased with camptothecin concentration (Fig. 2A, compare a, b, d, and f) and with increasing the time of exposure (Fig. 2A, compare b with c and d with e) (see panels g in Fig. 2A). Western blotting confirmed the time-dependent and concentration-dependent generation of {gamma}H2AX in camptothecin-treated cells (Fig. 2B, a and b). Fig. 2C also shows that {gamma}H2AX formation required top1, because camptothecin failed to induce {gamma}H2AX in top1-deficient mouse leukemia cells (37). By contrast, {gamma}H2AX was generated by IR in both top1-deficient and top1-proficient cells. Together, these results indicate for the first time that top1-mediated DNA lesions induce {gamma}H2AX in human and mouse cells in dose-dependent and time-dependent manners.



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FIG. 2.
Generation of {gamma}H2AX by camptothecin (CPT) in HCT116 cells. A, dose- and time-dependent {gamma}H2AX focus formation in camptothecin-treated cells. Cells were treated with the indicated concentration of camptothecin for the indicated time and stained with mouse anti-{gamma}H2AX antibody and goat anti-mouse antibody conjugated with FITC (green). Nuclei were stained with propidium iodide (PI) (red). Panel g shows the percentage of {gamma}H2AX-positive cells as a function of camptothecin concentration and time of camptothecin exposure. B, Western blotting analysis of dose- and time-dependent generation of {gamma}H2AX in HCT116 cells. {gamma}H2AX increased with the camptothecin concentration (a, 0.1, 1, and 10 µM for 1 h) and with the time of exposure (b, from 0 to 3 h with 1 µM camptothecin). C, Western blotting analysis of {gamma}H2AX induction by camptothecin (1 µM) and IR (10 Gy) in P388 leukemia cells expressing (+) or p388/CPT45 cells not expressing (-) top1.

 

Top1-mediated {gamma}H2AX Foci Are Attributed to Replication-mediated DNA Damage—The first suggestion of the replication-dependent induction of {gamma}H2AX by camptothecin was the observation that {gamma}H2AX focus formation was restricted to a fraction of the camptothecin-treated cells (Figs. 2A and 3A). By contrast, {gamma}H2AX focus formation was generally observed in all of the cells irradiated with 10 Gy (Figs. 3A and 4A) as reported previously (9). To determine which fraction of the cells formed {gamma}H2AX foci, we performed double staining for both {gamma}H2AX and PCNA, because PCNA levels are known to be highest in replicating cells (38). In the case of camptothecin-treated cells, {gamma}H2AX focus formation was restricted to PCNA-positive cells (Fig. 3A, panel a), whereas in the case of IR, {gamma}H2AX foci formed both in PCNA-positive and PCNA-negative cells (Fig. 3A, panel b). To quantify these results, slides corresponding to camptothecin (611 cells) or IR treatment (605 cells) were scored and cells were classified into four groups: PCNA-/{gamma}H2AX-, PCNA-/{gamma}H2AX+, PCNA+/{gamma}H2AX-, and PCNA+/{gamma}H2AX+ (Fig. 3B). In the case of camptothecin, {gamma}H2AX focus formation was observed in 51% of the cells. In these {gamma}H2AX-positive cells, 92% were also PCNA-positive. Conversely, 86% PCNA-negative cells were negative for {gamma}H2AX. By contrast, in cells treated with IR, the percentage of {gamma}H2AX-positive cells was similar in PCNA-positive and PCNA-negative cells (94%) (Fig. 3B).



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FIG. 3.
Camptothecin-induced {gamma}H2AX is restricted to S-phase. A, {gamma}H2AX focus formation in relation to cellular PCNA content after treatment with camptothecin (CPT) (a, 1 µM for 3 h) or IR (b, 30 min after 10 Gy). Cells were stained with anti-{gamma}H2AX antibody (green) or anti-PCNA antibody (red). Blue ellipses indicate the PCNA-negative cells. B, relationships between {gamma}H2AX and PCNA content in cells treated with CPT or IR. Statistical analysis was performed by the Chi-Square test. C, relationships between {gamma}H2AX cellular content and cell cycle in HCT116 cells. Untreated cells or cells treated with CPT (1 µM for 3 h) or IR (30 min after 10 Gy) were analyzed by laser scanning cytometry. x axis indicates DNA content (as determined by propidium iodide staining), and y axis indicates {gamma}H2AX content (in arbitrary units) (upper panels) or cell counts (lower panels). D, {gamma}H2AX foci (green) were not observed in a mitotic cell treated with CPT (1 µM) (a, white arrow) but were observed in a mitotic cell treated with 2 Gy of IR (b, white arrow).

 


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FIG. 4.
Effects of aphidicolin (APH), UCN-01 (7-hydroxystaurosporine), and wortmannin (Wort) on {gamma}H2AX formation by camptothecin (CPT) or IR in HCT116 cells. A, {gamma}H2AX focus formation (green) in HCT116 cells treated with CPT (1 µM, for 3 h) (e) or IR (10 Gy) (i) in the absence or presence of 1 µM aphidicolin (APH) (f and j). 0.1 µM UCN-01 (g and k), or 100 µM Wort (h and l). Cells were pretreated with APH, UCN-01, or Wort for 15 min prior to the treatment with CPT or IR. APH, UCN-01, or Wort was kept in the medium during the treatment with camptothecin (3 h) or 30 min after exposure to IR. B, Western blot analyses for the effects of APH, UCN-01, and Wort on {gamma}H2AX induction by camptothecin (b) or IR (c) in HCT116 cells. Treatment schedules were the same as described in A.

 

The relationship between {gamma}H2AX focus formation and DNA replication was further examined by comparing the cellular DNA and {gamma}H2AX contents using laser scanning cytometry (Fig. 3C). For camptothecin, cells in S-phase had the highest {gamma}H2AX contents, whereas {gamma}H2AX was lowest in the cells in the G1 and G2/M phases of the cell cycle. By contrast, for IR, {gamma}H2AX was equally distributed in all of the cell cycle phases. We also found that mitotic cells failed to form camptothecin-induced {gamma}H2AX foci, whereas {gamma}H2AX foci were observed after IR treatment (Fig. 3D, compare a with b) (9).

We next determined the effect of pretreatment with aphidicolin, a specific inhibitor of replication polymerases that blocks the formation of camptothecin-induced replication-mediated DNA double-strand breaks in camptothecin-treated cells (34). Fig. 4A shows that pretreatment with aphidicolin completely suppressed the formation of {gamma}H2AX foci by camptothecin (Fig. 4A, compare f with e). By contrast, IR-induced {gamma}H2AX foci remained clearly detectable in aphidicolin-treated cells (Fig. 4A, compare j with i). Western blotting analyses confirmed inhibition of {gamma}H2AX formation by aphidicolin in camptothecin-treated cells but not in IR-treated cells (Fig. 4B, b and c). These results indicate that camptothecin induces {gamma}H2AX focus formation in a DNA replication-dependent manner.

The Cell Cycle Checkpoint Modulators, UCN-01 and Wortmannin, Affect the Formation of {gamma}H2AX in Response to Top1-mediated and Replication-mediated DNA Damage—UCN-01, an anti-cancer drug in clinical trials, abrogates the S-phase checkpoint induced by camptothecin (39). Fig. 4A (panel c) shows that UCN-01 by itself did not induce {gamma}H2AX foci. However, pretreatment with UCN-01 markedly enhanced {gamma}H2AX focus formation in camptothecin-treated cells but not in cells exposed to IR (Fig. 4A, compare g with e and k with i). Western blotting analysis also demonstrated that UCN-01 enhanced {gamma}H2AX generation by camptothecin (Fig. 4B, b). These results are consistent with the findings that camptothecin-induced {gamma}H2AX formation by camptothecin is replication-dependent, because {gamma}H2AX formation increased in the absence of replication checkpoint (i.e. in UCN-01-treated cells) (see "Discussion").

We next determined the effect of wortmannin, a non-competitive and irreversible inhibitor of phosphatidylinositol 3-OH kinase-related kinases (PIKKs) (40, 41, 42) on the generation of {gamma}H2AX by camptothecin. Wortmannin inhibited the formation of both camptothecin-induced and IR-induced {gamma}H2AX foci (Fig. 4A, compare h with e and l with i). Western blotting analysis confirmed that wortmannin inhibited {gamma}H2AX generation by camptothecin and IR (Fig. 4B, b and c). The wortmannin results suggested that kinases from the PIKK family were implicated in the generation of {gamma}H2AX.

Generation of {gamma}H2AX by Replication-mediated DNA Double-strand Breaks Is Mediated by ATR and DNA-dependent Protein Kinase (DNA-PK)—To determine which of the three PIKKs, ATR, ATM, and DNA-PK, were implicated in the formation of {gamma}H2AX during replication-mediated DNA damage, we used cell lines with characterized deficiencies. For ATR, we used ATR dominant-negative cells expressing conditional ATRkd (43). To activate the ATRkd, cells were pretreated with 1 µg/ml doxycycline for 2 days. Fig. 5 shows that ATR inactivation blocked the formation of {gamma}H2AX by camptothecin (Fig. 5A, compare g with h, and B, b). By contrast, IR induced {gamma}H2AX foci both in ATR-proficient and ATR-deficient (ATRkd) cells (Fig. 5A, compare m with n) albeit with a reduced efficiency when quantitation was performed by Western blotting (Fig. 5B, c). {gamma}H2AX foci were induced either by camptothecin or IR in AT cells, which are deficient for the ATM gene (Fig. 5A, compare i with o). Western blotting indicated a reduced formation of {gamma}H2AX in the AT cells (Fig. 5B, e and f).



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FIG. 5.
ATR-dependent and DNA-PK-dependent formation of {gamma}H2AX foci. A, the indicated cell lines were treated with camptothecin (CPT) 1 µM for 3 h) or IR (10 Gy). The cells were stained with rabbit anti-{gamma}H2AX antibody and goat anti-rabbit Ig antibody conjugated with FITC and counterstained with propidium iodide (red). GM847/ATRkd cells were treated with 1 µg/ml doxycycline (Doxy +) to induce the expression of ATR kinase inactive, resulting in ATR kinase dominant-negative status. AT fibroblasts (deficient for ATM) (GM05849) were compared with normal fibroblasts (GM00637). M059J cells deficient from DNA-PKcs transfected with empty vector (M059J/Fus9) were compared with M059J/Fus1 cells transfected with the DNA-PKcs gene. B, Western blot analysis for {gamma}H2AX formation by CPT or IR. ATR- and ATR+ correspond to ATR dominant-negative cells (ATRkd Doxy +) and cells with functional ATR (ATRkd Doxy -), respectively. ATM- and ATM+ correspond to AT cells (GM05849) and ATM-proficient cells (normal fibroblasts, GM00637), respectively. DNA-PKcs- and + cells correspond to M059J/Fus9 cells transfected with control (empty) vector and M059J/Fus1 cells transfected with the DNA-PKcs gene, respectively.

 

To investigate the involvement of DNA-PK, we compared M059J/Fus9 and M059J/Fus1 cells (44). Human glioblastoma M059J cells are deficient for the DNA-PK catalytic subunit (DNA-PKcs) (45, 46, 47) and also have low ATM expression (48, 49). M059J/Fus1 cells were made by stable transfection of the DNA-PKcs gene into M059J cells. As control, M059J/Fus9 cells were made by transfection of the corresponding empty vector into M059J cells (44). Fig. 5 shows that {gamma}H2AX formation was reduced in camptothecin-treated M059J/Fus9 cells and was restored in the M059J/Fus1-complemented cells (Fig. 5A, compare k with l, and B, h). These results indicate that {gamma}H2AX is produced primarily by ATR, secondly by DNA-PK, and to a lesser degree by ATM in response to top1-induced replication-mediated DNA damage induced by camptothecin.

The Mre11-Rad50-Nbs1 (MRN) Complex Is Not Required for {gamma}H2AX Focus Formation—The MRN complex, which is conserved from yeasts to humans, binds to DNA double-strand breaks (50). Its activity is required for checkpoint kinase activation and RAD9 phosphorylation in yeast (51). While investigating MRN protein levels in human colon carcinoma cells from the NCI cell screen (Developmental Therapeutics Program, NCI) (dtp.nci.nih.gov/index.html), we found that HCT116 cells have almost undetectable levels of Mre11 and Rad50 and reduced levels of Nbs1. Fig. 6A shows a Western blotting analysis demonstrating low levels of Mre11, Rad50, and Nbs1 in HCT116 cells in comparison with those of HT29 cells, another colon carcinoma cell line from the NCI cell screen. Despite these differences in MRN levels, camptothecin-induced {gamma}H2AX foci were comparable in both cell lines (Fig. 6B). We also found that {gamma}H2AX foci were formed in NBS cells, which are deficient for Nbs1 (Fig. 6C, e and g). Moreover, neither Mre11 nor Rad50 formed foci in NBS cells (Fig. 6C, f and h). These results indicate that the formation of {gamma}H2AX foci by camptothecin does not require MRN, although the focus formation of Mre11 and Rad50 requires Nbs1.



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FIG. 6.
Mre11, Rad50, and Nbs1 are not required for the production of {gamma}H2AX foci in camptothecin (CPT)-treated cells. A, Western blotting analyses for Mre11, Rad50, and Nbs1 in HCT116 and HT29 cells. B, {gamma}H2AX foci formation in HT29 and HCT116 cells treated with CPT. C, focus formation for {gamma}H2AX, Mre11, and Rad50 in NBS cells treated with or without camptothecin. Cells were stained with anti-{gamma}H2AX, anti-Mre11, or Rad50 antibodies as indicated under each panel.

 

H2AX Is Required for MRN Focus Formation and Co-localization of the MRN and {gamma}H2AX Foci Induced by Camptothecin—We next determined the formation of MRN foci in H2AX+/+ or H2AX-/- MEFs (Fig. 7) (17). In H2AX+/+ MEFs, {gamma}H2AX foci formed in response to camptothecin and co-localized with MRN foci. By contrast, in H2AX-/- MEFs, MRN foci were not observed in response to camptothecin (Fig. 7, j, k, and i), although H2AX-/- MEFs had the same Mre11, Rad50, and Nbs1 protein levels as H2AX+/+ MEFs (data not shown). Upward mobility shift suggested that phosphorylation of Nbs1 was observed both in H2AX+/+ and H2AX-/- cells treated with camptothecin (Fig. 8B). Thus, the phosphorylation of MRN does not require H2AX. These results indicate that MRN focus formation requires H2AX and that MRN foci co-localize with the {gamma}H2AX foci in response to replication-mediated DNA damage induced by camptothecin.



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FIG. 7.
H2AX dependence for the formation of MRN foci and the co-localization of MRN and {gamma}H2AX foci in MEFs treated with camptothecin (CPT). A, MRN and {gamma}H2AX foci in H2AX+/+ MEFs at 3 h after treatment with camptothecin (1 µM for 3 h). B, similar experiments in H2AX knock-out (H2AX-/-) MEFs treated with CPT. Proteins targeted by antibodies are indicated under each panel. C, background levels for Mre11, Rad50, and Nbs1 in untreated (control, CL) H2AX-/- MEFs. Images were comparable in untreated H2AX+/+ cells (data not shown).

 


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FIG. 8.
Nbs1 (1 µM for 3 h) phosphorylation in H2AX-/- MEFs treated with camptothecin. A, Western blot analysis for {gamma}H2AX generation in H2AX+/+ and H2AX-/- MEFs. B, Western blot analysis for Nbs1 in H2AX+/+ and H2AX-/- MEFs.

 

H2AX-/- Cells Are Hypersensitive to Camptothecin—To investigate a potential functional role for {gamma}H2AX formation in response to replication-dependent DNA damage, we determined the survival responses of H2AX-proficient (+/+) or H2AX-deficient (-/-) MEFs to camptothecin by MTT assays. Fig. 9 shows that H2AX-/- MEFs were hypersensitive to camptothecin in comparison with H2AX+/+ MEFs (p < 0.001).



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FIG. 9.
Hypersensitivity of H2AX knock-out MEFs to camptothecin (CPT). Cytotoxic responses to camptothecin were analyzed by MTT assays. Curves were generated from three independent experiments. Statistical significance in cellular response to camptothecin between two cell lines was assessed by the two-way ANOVA with Sheffe's multiple comparison test and Student's t test. H2AX-/- cells were significantly hypersensitive from 0.03 to 1 µM of camptothecin (p < 0.001). *, p < 0.005 in comparison with H2AX-/- cells at the same concentration of camptothecin.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
This study demonstrates that {gamma}H2AX is induced and forms nuclear foci in response to replication-mediated DNA double-strand breaks induced by top1 cleavage complexes. This H2AX phosphorylation response appears to be mediated primarily both by ATR and DNA-PK and required for the recruitment of MRN foci at the sites of replication-mediated DNA double-strand breaks. Moreover, the hypersensitivity of H2AX knockout cells is consistent with a requirement for the {gamma}H2AX/MRN pathway for cellular processing of replication-mediated DNA double-strand breaks.

In top1-deficient cells (37), the generation of {gamma}H2AX was selectively suppressed for camptothecin but not for IR, findings that are consistent with the selectivity of camptothecin as a top1 inhibitor (52) and with the requirement of top1 cleavage complexes for the generation of {gamma}H2AX. However, cleavage complexes are not sufficient for {gamma}H2AX formation because aphidicolin, which has no effect on cleavage complex formation (32), blocked {gamma}H2AX formation. Aphidicolin is a DNA polymerase inhibitor (53), which prevents replication forks from proceeding to the site of top1 cleavage complexes, and thereby blocks the generation of replication-mediated DNA double-strand breaks in cells treated with camptothecin (Fig. 1) (34). Therefore, the suppression of camptothecin-induced {gamma}H2AX and {gamma}H2AX focus formation by aphidicolin demonstrates that {gamma}H2AX is produced by replication-mediated DNA double-strand breaks (Fig. 1).

Additional evidence for {gamma}H2AX formation by replication-mediated DNA damage include the laser scanning cytometry analysis shown in Fig. 3C, which reveals that {gamma}H2AX formation by camptothecin is selective for S-phase cells. {gamma}H2AX focus formation by camptothecin was also restricted to PCNA-positive cells and was not observed in mitotic cells. These results support the hypothesisthat{gamma}H2AXgenerationbycamptothecinisreplication-dependent. This cell-specific generation of {gamma}H2AX by camptothecin in replicating cells might be relevant for cancer chemotherapy, because one of the differences between normal and cancer cells is the enhanced PCNA content of cancer cells compared with normal cells (54).

Our finding that the cell cycle checkpoint abrogator, UCN-01 (7-hydroxystaurosporine), enhanced {gamma}H2AX formation by camptothecin is also consistent with the generation of {gamma}H2AX by replication-mediated DNA double-strand breaks. Under normal conditions, once double-strand breaks are generated, replication is stopped by the checkpoint mechanisms consisting of several checkpoint kinases, such as Chk1 and Chk2 (55, 56). UCN-01 abrogates the function and activation of these kinases (57, 58, 59, 60), and abrogates the S-phase checkpoint induced by camptothecin (39). A plausible explanation is that because of the checkpoint abrogation by UCN-01, replication forks cannot stop, and proceed until they collide with additional top1 cleavage complexes, resulting in the generation of new DNA double-strand breaks and in the further generation of {gamma}H2AX. This interpretation is consistent with the previously reported enhancement of cytotoxicity and the therapeutic synergism between S-phase-specific anticancer drugs (including camptothecins) and UCN-01 (39, 61, 62).

We next examined which kinases are implicated in {gamma}H2AX formation in response to the replication-mediated DNA double-strand breaks induced by camptothecin. First, we observed that wortmannin inhibited the generation of {gamma}H2AX. Because wortmannin is a broad specificity PIKK inhibitor with some selectivity for DNA-PK (40, 41, 42), it was likely that are involved in the phosphorylation of H2AX in response to replication-mediated double-strand breaks. ATM and DNA-PK have been previously implicated in the phosphorylation of H2AX in mammalian cells treated with IR (8, 63). Studies in Saccharomyces cerevisiae (10) demonstrated that Mec1 (the ATR ortholog) is required for H2AX phosphorylation after DNA damage. In this study, the phosphorylation of H2AX was observed in ATM-deficient cells and was markedly reduced in ATR dominant-negative cells (Fig. 4). In the M059J/Fus9 cells, which are deficient for DNA-PKcs and have low expression of ATM (45, 46, 47), low levels of {gamma}H2AX was observed. This low level of {gamma}H2AX in response to camptothecin was restored by transfection of the DNA-PKcs gene. Therefore, our experiments indicate that {gamma}H2AX generation in response to replication-mediated DNA double-strand breaks induced by camptothecin is mediated both by ATR and DNA-PK. The role of ATM in this replication-dependent phosphorylation appears not to be essential because AT cells were able to generate {gamma}H2AX in response to camptothecin. However, ATM may have a contributory role since we found a reduction in {gamma}H2AX formation in AT cells. We also observed phosphorylation of H2AX in AT cells in response to IR, albeit {gamma}H2AX formation was quantitatively reduced by Western blotting analysis in AT cells. A more essential role for ATM in the formation of {gamma}H2AX has been reported in mouse knock-out cell lines (63). However, in these mouse ATM-knockout cell lines, low levels of {gamma}H2AX were still detectable (63). Furthermore, a more recent study indicates that AT cells had delayed {gamma}H2AX formation in response to IR (64). Thus, it appears that ATM is important (possibly at the early times) for IR-induced {gamma}H2AX formation. In the case of replication-mediated DNA double-strand breaks, our data suggest that ATM is not essential but contributes to {gamma}H2AX formation. Recently, it has been proposed that ATR binds to the double-strand break sites in conjunction with ATRIP, independently of Rad9, Rad1, and Hus1 (9-1-1 complex), and phosphorylates neighboring H2AX (65, 66). Our data are consistent with the possibility that ATR is one of the sensors for replication-mediated DNA damage (65) and that phosphorylation of H2AX by ATR is an early event in the cellular response to top1 cleavage complexes in replicating cells. It is unlikely that {gamma}H2AX is induced by replication fork stalling rather than by the replication-mediated double-strand breaks because treatment with the DNA polymerase inhibitor aphidicolin failed to induce {gamma}H2AX within the same exposure time as camptothecin (Fig. 4A).

Recent reports using hydroxyurea or UV (66, 67) to induce replication stress showed {gamma}H2AX formation in an ATR-dependent manner. However, hydroxyurea and UV can produce a variety of other DNA modifications that activate diverse repair pathways (including nucleotide excision repair in the case of UV). On the other hand, the replication-mediated double-strand breaks induced by camptothecin are well defined (34, 36) and camptothecin is becoming more widely used as a specific inducer of such lesions in eukaryotic cells, including yeast (68, 69).

We also examined the association of {gamma}H2AX with the DNA double-strand break repair and checkpoint proteins, Mre11, Rad50, and Nbs1 (MRN complex). We found that Mre11, Rad50, and to a lesser extent, Nbs1 are much lower in HCT116 cells than in HT29 cells (Fig. 6). This finding is consistent with a recent report demonstrating that in HCT116 cells, the Mre11 gene contains a frameshift mutation because of the mismatch repair deficiency of HCT116 cells, resulting in a truncated and unstable Mre11 polypeptide (70). Despite their deficiency in Mre11, HCT116 cells produced comparable levels of {gamma}H2AX as HT29 cells, indicating that Mre11 is not required for the generation of {gamma}H2AX (Fig. 6B). Moreover, in Nbs1-deficient cells derived from a patient with NBS, {gamma}H2AX focus formation was observed (Fig. 6C). However, in such Nbs1-deficient cells, Mre11 and Rad50 focus formation was not observed. These observations indicate that MRN is not required for {gamma}H2AX formation in response to camptothecin treatment. Nevertheless, Nbs1 is required for Mre11 and Rad50 focus formation, which is consistent with the {gamma}H2AX/MRN pathway elicited by IR (8). In the case of IR, a recent study (64) demonstrates that the N-terminal domain (containing the BRCT and FHA motifs) of Nbs1 binds directly to {gamma}H2AX and that Nbs1 then recruits Mre11 to the double-strand break sites.

Using embryonic fibroblasts from the recently reported H2AX knock-out mice (17), we found that MRN foci failed to form in H2X-/- cells (Fig. 7B). Phosphorylation of Nbs1 remained detectable in the H2AX-/- MEFs, suggesting that focus formation is not required for phosphorylation of Nbs1 by ATR, ATM, or DNA-PK in response to replication-mediated DNA damage. Celeste et al. (17) report that Nbs1 did not form foci in response to IR in H2X-/- cells. A functional role for {gamma}H2AX in the recruitment of MRN complexes at the double-strand break sites appears common for both replication (this study) and IR (64). Also consistent with the IR response, we found that MRN foci generally co-localized with the {gamma}H2AX foci in camptothecin-treated cells (Fig. 7).

Our finding that H2AX knock-out fibroblasts were hypersensitive to camptothecin implies a functional role for {gamma}H2AX in cellular response to replication-mediated double-strand breaks. This finding is conserved in yeast because histone H2A serine 129 is necessary for yeast survival during top1-mediated DNA damage.2 Recently, Mre11-deficient yeast cells were also found to be hypersensitive to camptothecin (68, 69), which is consistent with the prior observation that cells from NBS patients are hypersensitive to camptothecin (71). Together, these observations demonstrate the functional importance of the {gamma}H2AX/MRN pathway for cellular response to replication-mediated double-strand breaks generated by top1.

In conclusion, we propose the following cellular responses to top1-induced replication-mediated DNA double-strand breaks (Figs. 1 and 10). When cells are exposed to camptothecin or to a variety of DNA lesions (19), top1 cleavage complexes are trapped throughout the genome. A fraction of these cleavage complexes is converted to replication-mediated DNA double-strand breaks when replication forks collide with these complexes. This step is inhibited by aphidicolin. Under physiological conditions, the replication-mediated DNA double-strand breaks elicit a checkpoint response, which blocks replication and prevents further collisions. By abrogating this S-phase checkpoint, UCN-01 increases the collision frequency and {gamma}H2AX formation. ATR and DNA-PK are activated by the replication-mediated double-strand breaks caused by top1 cleavage complexes and phosphorylate H2AX, which is converted to {gamma}H2AX. {gamma}H2AX is required to recruit repair proteins, possibly in conjunction with chromatin structure changes. Consequently, Mre11, Rad50, and Nbs1 complexes bind to the DNA double-strand breaks (Fig. 10). Deficiency in H2AX inactivates this pathway and increases the sensitivity of cells to replication-mediated DNA double-strand breaks induced by top1 cleavage complexes.



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FIG. 10.
Hypothetical model showing the {gamma}H2AX-dependent recruitment of MRN to a replication-mediated DNA double-strand break (DSB) site generated by a top1 cleavage complex. CPT, camptothecin.

 


    FOOTNOTES
 
* 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. Back

To whom correspondence should be addressed: NCI, National Institutes of Health, 37 Convent Dr., Bldg. 37, Rm. 5068A, Bethesda, MD 20892-4255. Tel.: 301-496-5941; Fax: 301-402-0752; E-mail: pommier{at}nih.gov.

1 The abbreviations used are: {gamma}H2AX, phosphorylated form of H2AX; IR, ionizing radiation; top1, topoisomerase I; AT, ataxia telangiectasia; NBS, Nijmegen breakage syndrome; ATR, ataxia telangiectasia and Rad3-related; ATRkd, ATR kinase dead; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA, analysis of variance; PCNA, proliferating cell nuclear antigen; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; PIKK, phosphatidylinositol 3-OH kinase-related kinase; ATM, ataxia telangiectasia mutated; MRN, Mre11-Rad50-Nbs1; MEF, mouse embryonic fibroblasts; Gy, gray; UCN-01, 7-hydroxystaurosporine. Back

2 C. Redon, D. R. Pilch, E. P. Rogakou, N. F. Lowndes, and W. M. Bonner, submitted for publication. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Michael Kastan for GM00637 and GM05849 cell lines, Dr. Cordula U. Kirchgessner for M059J/Fus1 and M059J/Fus9 cell lines, Dr. William A. Cliby, and Dr. Scott H. Kaufmann (Mayo Graduate School, Rochester, MN) for GM847/ATRkd cells. We appreciate Dr. Barbara J. Taylor (FACS core, NCI) for analysis by laser scanning cytometry. The help of Glenda Kohlhagen is greatly appreciated.



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