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Originally published In Press as doi:10.1074/jbc.M603747200 on August 10, 2006

J. Biol. Chem., Vol. 281, Issue 41, 30814-30823, October 13, 2006
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Defective Mre11-dependent Activation of Chk2 by Ataxia Telangiectasia Mutated in Colorectal Carcinoma Cells in Response to Replication-dependent DNA Double Strand Breaks*

Haruyuki Takemura1, V. Ashutosh Rao1, Olivier Sordet1, Takahisa Furuta, Ze-Hong Miao, LingHua Meng, Hongliang Zhang, and Yves Pommier2

From the Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-4255

Received for publication, April 19, 2006 , and in revised form, August 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Mre11·Rad50·Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA double strand breaks (DSBs)3 are among the most severe genomic lesions. Their repair requires cells to arrest cell cycle progression to avoid further damage as the broken DNA template is processed during replication or transcription (1-5). Cell cycle checkpoints may also allow the chromatin to switch from a metabolic (replicative) state to a repair state. DSBs can be produced directly by ionizing radiation and certain anticancer drugs that bind to DNA (bleomycin, neocarcinostatin, and topoisomerase II inhibitors such as etoposide or doxorubicin) (see Fig. 1a). DSBs can also result from the conversion of single strand breaks by DNA polymerase collisions or "replication fork collapse" at sites of damaged DNA sites (see Fig. 1b). These replication DSBs occur under routine growth conditions in cancer cells, but their frequency is markedly enhanced by topoisomerase I (Top1) inhibitors such as the camptothecin derivatives, topotecan and irinotecan (6-10). We refer to the latter as replication-mediated DSBs (or simply "Rep-DSBs") (see Fig. 1b).

Top1 is essential in metazoans (11, 12), because it removes DNA supercoiling (superhelical tension) generated during most DNA transactions (replication, transcription, chromatin remodeling, and repair). Top1 relaxes DNA by forming transient cleavage complexes (11, 12) (Top1-linked DNA single strand breaks) that are frequently distributed in the genome. Top1 nicking-closing activity must be coupled with replication to avoid DNA supercoiling buildup associated with the elongating replication forks. Slowing down the closing (re-ligation) reaction results in replication fork collisions and Rep-DSBs (6-8, 10). Top1 cleavage complexes can be stabilized under physiological conditions when a DNA base is damaged at a Top1 cleavage site (by oxidation, alkylation, mismatch, or base loss) or when the DNA contains carcinogenic adducts or is nicked within a few base pairs from the Top1 cleavage site (for review see Refs. 9 and 13). Top1 cleavage complexes can also be stabilized rapidly and reversibly by the alkaloid camptothecin (CPT) (10, 14). CPT traps Top1 cleavage complexes as it inter-calates between the base pairs flanking the DNA cleavage site within the Top1 cleavage complex (15). The CPT derivatives irinotecan and topotecan are clinically used for colon, ovarian, and lung carcinomas (16-18). However, despite detailed knowledge on the molecular mechanisms of Top1 trapping by camptothecins, predicting tumor responses remains empirical (9, 10, 19). In our studies we used the colon carcinoma cells of the NCI Anticancer Drug Screen that offers a diverse yet representative panel of cell lines for drug discovery (20, 21).

The importance of Chk2, a protein involved in cell cycle arrest due to DSBs, is supported by its conservation in all eukaryotes (22-25). The DNA damage-responsive elements, including the MRN complex (heterotrimer comprised of Mre11, Rad50, and Nbs1), Chk2, ATM, and histone H2AX, are activated by DSBs and serve both for DNA repair and checkpoint responses as well as for induction of apoptosis (1, 26-30) (reviewed as a molecular interaction map in Ref. 31 and at discover.nci.nih.gov/mim). Mre11 binds to and repairs DNA by its 3',5'-exonuclease and endonuclease activities (32). Mre11 also binds both Nbs1 and Rad50, which can bridge and keep together the broken DNA ends (33). Although Nbs1 regulates checkpoint response by binding to ATM (34) and to phosphorylated histone H2AX ({gamma}-H2AX) (35), the signal to sustain genomic stability generated by phosphorylation of H2AX and ATM is primarily maintained by a positive feedback loop via the adaptor protein MDC1 (36). ATM kinase is itself autophosphorylated on serine 1981 and activated within minutes following DSB (27). In turn, ATM activates Chk2 by phosphorylation on threonine 68 (37, 38). Hence, activated Chk2 can be viewed as an ATM "relay checkpoint kinase" for DSB response (31). Chk2 can activate both cell cycle arrest/checkpoint by phosphorylating Cdc25 phosphatases and apoptosis by phosphorylating p53, E2F1, and PML (24, 39). Germinal mutations inactivating ATM, Mre11, or Nbs1 give rise to ataxia telangiectasia (AT), ataxia telangiectasia-like disorder, and Nijmegen breakage syndrome (NBS), respectively (28). These three diseases are clinically related and predispose affected individuals to cancers. CHK2 mutations corresponding to Li-Fraumeni syndromes with normal p53 also increase cancer incidence (25, 40-42).

Defects in the DNA replication machinery itself can also predispose individuals to cancers (43). For instance, mismatch repair defects cause colon cancers because of the failure to remove nucleotides that are misincorporated in repeated sequences during replication (44). Bloom syndrome is associated with early onset of carcinomas because of a failure to repair abnormal replication forks (45) due to a lack of the BLM helicase. Replication fork abnormalities generated spontaneously or by agents such as hydroxyurea and UV activate the 911-ATRIP-ATR-Chk1 pathways; whereas MRN, ATM, and Chk2 have been implicated primarily in DSBs induced by ionizing radiation and radiomimetic drugs in the non-replicating genome (1, 39).

In the present study, we used CPT to examine Chk2 activation in response to Rep-DSBs and the role of MRN for this activation. We found rapid activation of Chk2 by ATM in response to Rep-DSBs. MRN appears essential for this activation, although in contrast to earlier reports for DSBs induced by ionizing radiation (34), in the case of Rep-DSBs, MRN is not required for ATM activation but is required for Chk2 activation downstream from ATM. We also show that MRN is defective in three of seven colon carcinoma cell lines from the NCI 60 Cell Anticancer Drug Screen, and that in all three cell lines, Chk2 activation is defective in response to Rep-DSBs. Moreover, we found that Chk2 expression is markedly reduced in three of four remaining cell lines with normal MRN. These findings demonstrate rapid Chk2 activation in response to Rep-DSBs indicating cross-talk between the replication and DSB checkpoint pathways. They also demonstrate defects in the MRN-Chk2 pathway in colorectal cancer cells because of either MRN or Chk2 defects.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Antibodies—The Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, NCI, National Institutes of Health, provided camptothecin. Aphidicolin, leupeptin, and aprotinin were obtained from Sigma. Anti-Mre11, -Rad50, and -Nbs1 polyclonal antibodies were purchased from Novus Biologicals (Littleton, CO). Anti-Mre11, -Rad50, and -Nbs1 monoclonal antibodies were obtained from GeneTex (San Antonio, TX). Phospho-anti-Nbs1, phospho-anti-Chk1, and phospho-T68-anti-Chk2 polyclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Chk1 and -Chk2 monoclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescent secondary Alexa Flour 488/568 anti-mouse serum IgG and Alexa Flour 488/568 anti-rabbit serum IgG were obtained from Molecular Probes (Eugene, OR). Cy3 anti-mouse and anti-rabbit IgG antibodies were obtained from Jackson Immuno-Research (West Grove, PA). Lab-Tek II chamber slide was obtained from Nalge Nunc (Rochester, NY). Hygromycin B was purchased from Invitrogen. G418 (Geneticin) was obtained from Mediatec (Herndon, VA).

Cell Culture—Human colon carcinoma HT29, HCT116, HCT15, HCC2998, KM12, COLO205, and SW620 cells were obtained from the Developmental Therapeutics Program (NCI, NIH). The SV40-transformed fibroblast cell line from a NBS homozygous patient was purchased from the Coriell Institute for Medical Research. Cells were grown at 37 °C in the presence of 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. YZ5 and pEBS7 fibroblast cell lines from an AT homozygous patient were a generous gift from Dr. Yosef Shiloh, Sackler School of Medicine, Tel Aviv University, Israel. 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, containing 100 µg/ml hygromycin B.

Cell Cycle Synchronization and Analyses, and CPT Treatments—Exponentially growing cells or synchronized cells were treated with 1 µM CPT as indicated. For cell synchronization, cells were first treated with 1 µM aphidicolin for 17 h, washed twice with phosphate-buffered saline, and incubated in fresh medium for 2 h. Then cells were treated with 1 µM CPT (or solvent for the untreated controls) for 75 min. Following two washes in phosphate-buffered saline, cells were incubated in culture medium for the designated times. For cell cycle analyses, cells were harvested and fixed in 70% ice-cold ethanol for 30 min at 4 °C. The fixed cells were washed twice with cold phosphate-buffered saline and stained with propidium iodide after treatment with 5 µg/ml RNase A. Cells were analyzed by FAC-Scan (BD Biosciences, San Jose, CA).


Figure 1
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  		FIGURE 1.
a, schematic representation of a DSB in duplex DNA. b, DSB induced by elongating DNA polymerase on the leading strand (Rep-DSBs). c, differential expression and co-regulation of Mre11, Rad50, and Nbs1 (MRN) proteins in the human colon carcinoma cell lines from the NCI Anticancer Screen. Three of the seven cell lines are deficient for all three molecular components of MRN. Molecular masses of the Mre11, Nbs1, and Rad50 polypeptides are indicated at the right.

 
Protein Extracts and Immunoblotting—Cells were grown to 70-80% confluence when treated with different agents. To prepare whole cell extracts, cells were harvested and washed twice with phosphate-buffered saline, then incubated on ice for 30 min in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.3% Non-idet P-40, 0.2% Triton X-100, 1 mM EDTA, 2 mM EGTA, 1 mM Na3VO4, 25 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, 5 µg/ml aprotinin, 100 µM/ml leupeptin). Lysates were clarified by centrifugation at 12,000 x g for 15 min at 4 °C. Protein concentration was quantified by Bradford method (46).

For immunoblotting, cells lysates were electrophoresed in SDS-PAGE precast gels (8% gels for Mre11 and Nbs1 and 6% gels for Rad50) and transferred onto Immobilon-P membranes (Millipore, Bedford, MA) using a semi-dry transfer apparatus. The immunoreactive bands were visualized by enhanced chemiluminescence (ECL Super Signal, Pierce) on autoradiographic films. All the presented data were confirmed in at least three different experiments. Confocal microscopy analyses were performed as described previously (47).

MRE11 Gene Transfection—The cDNA of Mre11 was obtained by PCR as a template with primers (P1, 5'-AGAGGCCTATGAGTACTGCAGATGCACTTGAT-3'; P2, 5'-GGCTCCGGAGTGCCAGTAAATATATTATCTTC-3'). cDNA was cloned by inserting into a pCR2.1 vector. Cloned cDNA was inserted into a pcDNA3.1 vector. Expression vectors containing of the genes of interest were confirmed by sequencing and transfected into HCT116 cells. For stable transfection Mre11 cDNA-containing cells were selected by culturing cells in the presence of 1.0 mg/ml G418.

Mre11 Silencing by RNA Interference—Mre11 expression was knocked down by transfection with an siRNA duplex (Qiagen) against the sequence GATGCCATTGAGGAATTAG of the Mre11 mRNA (48). Transfections were performed using LipofectamineTM 2000 (Invitrogen) according to the manufacturer's protocol. A negative control siRNA duplex from Qiagen (target DNA sequence: TTCTCCGAACGTGTCACGT) was used. The experiments were carried out 72 h after transfection.

DNA Synthesis Assays—DNA synthesis assay was performed by thymidine incorporation (49). Briefly, cells were prelabeled with 0.0025 µCi/ml of [14C]thymidine (53.6 mCi/mmol) for 48 h at 37 °C. The rate of DNA synthesis was measured by 10-min pulses with 1 µCi/ml of [methyl-3H]thymidine (80.9 Ci/mmol). 3H incorporation was stopped by washing cell cultures twice in ice-cold Hanks' balanced salt solution, and then scraping cells into 4 ml of ice-cold Hanks' balanced salt solution. Aliquots (1 ml) were then precipitated after addition of 100 µl of trichloroacetic acid in triplicate. Samples were vortexed, mixed, and centrifuged for 10 min at 12,000 x g at 4 °C. The precipitates were then dissolved overnight at 37 °C in 0.5 ml of 0.4 M NaOH. Samples were counted by dual label liquid scintillation and 3H values were normalized using 14C counts. Inhibition of DNA synthesis was calculated as the ratio of 3H:14C in the treated samples over the 3H:14C ratio in the untreated control samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MRN Deficiency in Three of Seven Colon Carcinoma Cell Lines from the NCI Anticancer Drug Screen Panel—Because MRN has emerged as an important component of DNA DSB repair and S-Phase checkpoint (29, 34), we tested the expression of Mre11, Rad50, and Nbs1 proteins in the colon carcinoma cell lines of the NCI Anticancer Drug Screen (50). Immunoblotting showed marked reduction of the three MRN proteins in HCT116, HCC2998, and KM12 cells compared with HT29, HCT15, COLO205, and SW620 cells (Fig. 1c). The Mre11 deficiency of HCT116 (30, 44) is due to a mismatch mutation in the MRE11 gene (44, 51). Thus, three of seven colon carcinoma cell lines from the NCI screen are globally deficient for MRN (i.e. all three molecular components are low or absent in HCT116, HCC2998, and KM12 cells), whereas the MRN complex is present in the four other cell lines (HT29, HCT15, COLO205, and SW620).

Defective Chk2 Phosphorylation/Activation in MRN-deficient Cells—Previous studies in HT29 and HCT116 cells (as well as the other colon carcinoma cell lines from the NCI Cell Screen) have shown that sensitivity to CPT is dependent on downstream pathways from Top1 cleavage complexes (21, 52). In particular, DNA repair and checkpoint activation favor cancer cell survival (9, 52). Accordingly, abrogation of the S-phase and G2 checkpoints markedly potentiates the antiproliferative activity of CPT (53). Because Chk2 and Chk1 are key effector kinases for the S and G2/M checkpoints, downstream from ATM and ATR, respectively (1), we compared the activation of Chk2 and Chk1 in HT29 and HCT116 treated with CPT. HT29 and HCT116 were chosen, because HT29 cells have the highest MRN levels, whereas HCT116 are well characterized Mre11-deficient cells (Fig. 1c) (30, 44).


Figure 2
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  		FIGURE 2.
Defective Chk2 phosphorylation in MRN-deficient cells treated with the Top1 inhibitor CPT. a, phosphorylations of Chk2 on Thr-68 (PT68-Chk2) and Chk1 on Ser-317 and Ser-345 (PS317-Chk1 and PS345-Chk1) were examined in exponentially growing HT29 and HCT116 cells (left and right panels, respectively) treated with 1 µM CPT for the indicated time periods. b, defective Chk2 phosphorylation in Nijmegen breakage syndrome (NBS) fibroblasts. Normal GM00637 fibroblasts are included for comparison. Chk2 and Chk1 correspond to 66- and 56-kDa polypeptides, respectively.

 


Figure 3
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  		FIGURE 3.
Phosphorylation of Chk2 (PT68-Chk2) persists after CPT removal and is associated with phosphorylations of Mre11 and Nbs1 in synchronized HT29 cells. a, S-phase synchronization protocol. Cells were treated with 1 µM aphidicolin (APH) overnight and with 1 µM CPT for 75 min. After CPT removal, cells were cultured in drug-free medium for the designated times. Arrowheads indicate sampling times. b, immunoblotting was performed using whole cell lysates at the indicated times. Nbs1 was immunoblotted using both total-Nbs1 antibody and phospho-Nbs1 antibody (PS343-Nbs1). Proliferating cell nuclear antigen (PCNA) was used as a loading control. Right panel, Nbs1 upper mobility shift is exemplified by a representative experiment showing electrophoretic mobility difference between untreated samples (0) and a CPT-treated sample (R2.5). Control (untreated) samples were loaded on each side of the CPT-treated (R2.5) sample to visualize the upper shift induced by CPT treatment.

 


Figure 4
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  		FIGURE 4.
Chk2 and Mre11 phosphorylations are induced by Rep-DSBs. a, schematic representation of Rep-DSBs induced by CPT. Left: reversible Top1 cleavage complex trapped by CPT (black rectangle). Right: the Top1 cleavage complex on the replicating leading strand leads to a Rep-DSBs by replication run-off (8). Aphidicolin (APH) prevents the formation of Rep-DSBs. b-d, HT29 cells were treated with 1 µM APH for 15 min prior to and during the CPT exposure (1 µM for the indicated times). Cells were examined for Chk2 phosphorylation (PT68-Chk2) by Western blotting (b) and immunofluorescence confocal microscopy (c) (1-h CPT exposure; nuclei are circled), and for Mre11 phosphorylation (upper-shifted band; panel d).

 
Fig. 2a shows Chk2 phosphorylation (on threonine 68 = PT68-Chk2) in HT29 but not in HCT116 in response to CPT. The HCT116 deficiency is not due to quantitative difference in Chk2 polypeptide, because total Chk2 protein levels are comparable in HT29 and HCT116 (Fig. 2a, second panels from top). Chk2 phosphorylation was confirmed in synchronized HT29 cells treated in S-phase with CPT (Fig. 3b). By contrast, Chk1 was phosphorylated both in HT29 and HCT116 on Ser-317 and Ser-345 (Fig. 2a); of which Ser-345 is regarded as a preferential target for phosphorylation/activation by ATR (54, 55). These experiments demonstrate dual activation of Chk2 and Chk1 in response to Top1-mediated DNA damage but defect in Chk2 activation in MRN-deficient HCT116 cells. To determine whether this lack of Chk2 activation was related to MRN deficiency, we tested Chk2 phosphorylation in response to CPT in Nbs1-deficient cells. Fig. 2b shows defective Chk2 phosphorylation in fibroblasts derived from an NBS patient. These results suggest that MRN is required for Chk2 activation but not for activation of Chk1 by Top1-mediated DNA damage.


Figure 5
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  		FIGURE 5.
ATM and Nbs1 are implicated in Chk2 phosphorylation in response to Rep-DSBs induced by Top1 cleavage complexes. a, to examine the role of ATM, Chk2 phosphorylation (PT68-Chk2) was examined at the indicated times after addition of 1 µM CPT in ATM-deficient (pEBS7) and ATM-complemented (YZ5) fibroblasts derived from an homozygous AT patient. b and c, immunofluorescence confocal microscopy of HT29 cells treated with 1 µM CPT for 1 h. Co-localization of PT68-Chk2 (red) and PS1981-ATM (green) is shown in b; co-localization of PT68-Chk2 (red) and Nbs1 (green) is shown in c. d, comparable ATM phosphorylation (PS1981-ATM) in HT29 (MRN-competent) and HCT116 (MRN-deficient) cells treated with 1 µM CPT. Nuclear contours are drawn as white circles. e, quantitative analysis showing comparable ATM activation/phosphorylation in HT29 and HCT116 cells treated with 1 µM CPT for the indicated times. At least 100 nuclei were counted for quantitation (bars, ± S.D.). f, schematic representation of the activation of the ATM-Chk2 axis in response to Rep-DSBs induced by Top1 cleavage complexes. Rep-DSBs activate (1) ATM autophosphorylation on serine 1981 (2), which is required (3) for full ATM activity. Phosphorylation of Chk2 by ATM (4) requires the MRN complex downstream from ATM (5). Symbol conventions are: green arrow, activation; green arrow with line, requirement; green circle and line, enzymatic activation; blue arrow with green dot, phosphorylation with the node representing the phosphorylated species (for further details see: discover.nci.nih.gov/mim see Chk2 molecular interaction map).

 
Mre11 and Nbs1 Phosphorylation in Response to Top1 Cleavage Complexes—Phosphorylation of Mre11 and Nbs1 was next examined in HT29 cells. Cells were synchronized in S-phase to focus on the Rep-DSBs response induced by CPT (Fig. 3a). CPT treatment caused the appearance of a second Mre11 band with reduced mobility compared with the bulk of Mre11 (Fig. 3b). This upper shift has been assigned to phosphorylated Mre11 (56). Mre11 phosphorylation was detectable later than PT68-Chk2 phosphorylation (Fig. 3b). Reduced mobility of Nbs1 was also observed 30 min after the addition of CPT and remained during CPT treatment as well as after drug removal. To better visualize the Nbs1 mobility shift, additional experiments were performed to compare the electrophoretic mobility of Nbs1 in extracts from untreated and CPT-treated cells (right panel in Fig. 3b). This reduced mobility has been assigned to hyperphosphorylated Nbs1 (57, 58). Nbs1 phosphorylation was further demonstrated with a phospho-specific Nbs1 antibody (PSer343) (Fig. 3b). Phosphorylation of Mre11, Nbs1, and Chk2 persisted for several hours after CPT removal (Fig. 3b).


Figure 6
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  		FIGURE 6.
a, MRE11 gene complementation in HCT116 cells restores Chk2 activation and protein levels for Nbs1 and Rad50. Mre11 cDNA-transfected HCT116 cells were selected with 1 mg/ml G418. Parental HCT116 cells and Mre11-complemented HCT116 cells (referred to as HCT116-Mre11 cells) were treated with 1 µM CPT for the indicated times. b, comparison of Chk2 activation between HCT116-Mre11 and HT29 cells treated with 1 µM CPT for the indicated times. c, knocking down Mre11 in HT29 cells decreases Chk2 activation and protein levels for Nbs1 and Rad50. HT29 cells were transfected with a duplex siRNA against Mre11 (siRNA-Mre11) or a negative control sequence (siRNA-Ctrl) and treated for 30 min with CPT at the indicated concentrations. d, Mre11 stabilizes Nbs1 and Rad50. HCT116 cells were treated with the proteasome inhibitor, MG132 for the indicated times and assayed for Rad50, Nbs1, and Mre11 by immunoblotting.

 
Phosphorylations of Chk2 and Mre11 Are Initiated by Rep-DSBs, and Chk2 Phosphorylation Requires MRN Downstream from ATM—CPT induces Rep-DSBs when advancing replication forks collide with the Top1 cleavage complexes stabilized by CPT on the leading replicating strand (6-8) (Fig. 4a). The DNA polymerase inhibitor, aphidicolin (APH) prevents CPT-induced cell death by avoiding the conversion of reversible cleavage complexes into Rep-DSBs (6-8, 30) (Fig. 4a). Fig. 4b shows reduced Chk2 phosphorylation in cells co-treated with CPT and APH. APH also inhibited the formation of Chk2 nuclear foci (Fig.4c) and Mre11 phosphorylation (Fig. 4d). These results indicate that Rep-DSBs induce Mre11 phosphorylation and the recruitment of phosphorylated PT68-Chk2 in nuclear foci.

To determine whether ATM is involved in Chk2 phosphorylation in response to the Rep-DSBs, we used the empty vector-transfected pEBS7 and the recombinant ATM-complemented YZ5 fibroblast cell lines from an AT homozygous patient. Fig. 5a shows lack of Chk2 phosphorylation in AT cells treated with CPT, whereas Chk2 phosphorylation is restored in ATM-complemented cells. These results implicateATMinChk2phosphorylation in response to Rep-DSBs.

Activated ATM (PS1981-ATM (27)) was found to co-localize in similar foci as activated Chk2 (PT68-Chk2) (Fig. 5b). Fig. 5c also shows co-localization of PT68-Chk2 and Nbs1 foci in CPT-treated cells. These results are consistent with the direct phosphorylation of Chk2 by ATM in response to Rep-DSBs.

To investigate whether MRN is required for ATM activation (34, 59) by Rep-DSBs, ATM activation was compared in HT29 and HCT116 cells treated with CPT. Similar activation of ATM was observed as PS1981-ATM foci in both cell lines (Fig. 5, d and e). ATM activation measured by Western blotting with PS1981-ATM antibodies was also similar in both cell lines and in the Mre11-complemented HCT116 cells (data not shown). Thus, we conclude that MRN is not required for ATM activation but that MRN acts downstream from ATM to activate Chk2 in response to Rep-DSBs (scheme in Fig. 5f).

Mre11 Complementation Restores Chk2 Phosphorylation, whereas Mre11 Down-regulation Reduces Chk2 Phosphorylation in Response to Rep-DSBs—Because HCT116 (deficient for the Mre11) are deficient for Chk2 phosphorylation in response to CPT, we examined whether Mre11 complementation could restore CPT-induced Chk2 phosphorylation. For this purpose, we established an Mre11-complemented HCT116 cell line, which we refer to as HCT116-Mre11. Fig. 6a shows restoration of Chk2 phosphorylation after Mre11 complementation. However, HCT116-Mre11 remained less effective than HT29 in inducing Chk2 phosphorylation (Fig. 6b). These results are consistent with a requirement of the MRN complex for Chk2 phosphorylation in response to Top1-induced Rep-DSBs in CPT-treated cells.

To further demonstrate the relationship between Mre11 and Chk2 activation, HT29 cells, which have the highest MRN levels, were transiently transfected with siRNA against Mre11 (48). Fig. 6c shows that Mre11 down-regulation reduces Chk2 phosphorylation. Together, these results demonstrate a close relationship between Mre11 and Chk2 activation.


Figure 7
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  		FIGURE 7.
a, differential Chk2 activation and protein levels in the colon carcinoma cell lines from the NCI 60 Developmental Therapeutics Program (DTP) Cell Screen. b, time-course analysis for differential Chk2 activation in HT29 and HCC2998 cells. Cells were treated with 1 µM CPT for the indicated times.

 
Mre11 Stabilizes Rad50 and Nbs1—Fig. 6a shows that HCT116 cells complemented for Mre11 also had increased levels for both Nbs1 and Rad50. Conversely, down-regulation of Mre11 in HT29 cells reduced both Nbs1 and Rad50. These observations, in addition to the fact that levels of MRN proteins tended to be expressed in parallel across the NCI 7 colon cell lines (see Fig. 1c) suggested stabilization of Rad50 and Nbs1 polypeptides by Mre11. To examine this possibility further, HTC116 cells were treated with the proteasome inhibitor, MG-132. Fig. 6d shows that MG-132 increased Rad50 and to a lesser extent Nbs1. These experiments suggest that Mre11 stabilizes Rad50 and Nbs1 (60) by preventing their degradation.

Defective Activation of Chk2 in at Least Five of Seven Human Colon Carcinoma Cell Lines from the NCI Anticancer Drug Screen—To further evaluate the importance of MRN for Chk2 activation by CPT, we examined Chk2 phosphorylation (on Thr-68) in each of the seven colon carcinoma cell lines from the NCI Anticancer Drug Screen following CPT treatment (Fig. 7a). Two of the three MRN-deficient cell lines (HCT116 and KM12) were globally deficient for Chk2 activation. The MRN-deficient HCC2998 cells phosphorylated Chk2 but to a lesser extent, as compared with the MRN-proficient HT29 cells (Fig. 7b).

Most noticeably, three of the four MRN-proficient cells (SW620, COLO205, and HCT15) were globally deficient for Chk2 activation (Fig. 7a). This observation suggests that lack of Chk2 activation is a common feature of colon cancer cells and that other defects besides MRN contribute to a lack of Chk2 activation. HCT15 cells express very low levels of Chk2 due to biallelic inactivation of the CHK2 gene (40, 61). Moreover, we found that the MRN-proficient SW620 and COLO205 cell lines, which are defective for Chk2 activation, also expressed very low levels of Chk2 protein. By contrast, all the MRN-deficient cells (HCC2998, HCT116, and KM12) express high levels of Chk2. Collectively, these experiments indicate that defective Chk2 activation can be due to either MRN deficiency or low expression of Chk2 protein.

Differential Cell Cycle Responses of Mre11/Chk2-proficient and -deficient Cells Treated with CPT—To examine the functional impact of MRN and Chk2 activation, experiments examining cell cycle progression after CPT treatment were performed. Fig. 8b shows that HT29, which have the highest MRN levels (Fig. 1c) and Chk2 activation (Fig. 7a) have a more pronounced DNA synthesis inhibition in response to CPT than HCT116, which are deficient for both MRN and Chk2. Moreover, following removal of CPT in S-phase-synchronized cells, S-phase arrest was more pronounced in HT29 than HCT116. The latter failed to arrest S-phase progression (compare the FACS profiles at 2.5 h following CPT removal in panels d and g in Fig. 8, see also panels e and h). At later times following CPT removal, HT29 arrested transiently in G2 (Fig. 8f), whereas HCT116 remained in G2/M for up to 10 h (Fig. 8i). These results suggest regulation of the S-phase checkpoint and cell cycle progression by the MRN-Chk2 pathway in response to Top1-mediated Rep-DSBs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Distinction between DSBs and Rep-DSBs—The present study shows that Rep-DSBs (Fig. 1b) activate ATM and Chk2 and induce Mre11 and Nbs1 phosphorylation, which is also the case of classic DSBs (see Fig. 1a). However, ATM activation by Rep-DSBs does not require MRN (Fig. 5, d and e), which contrasts with two recent reports concluding that MRN is required for ATM activation by DSBs (34, 59). Hence, in the case of Rep-DSBs induced by Top1 cleavage complexes, MRN activates Chk2 downstream from ATM (see scheme in Fig. 5f), whereas in the case of DSBs, MRN is upstream from ATM. The functional distinction between Rep-DSBs and DSBs is plausible, because the two lesions are anatomically different (see Fig. 1, a and b), and because Rep-DSBs activate ATR-Chk1 in addition to ATM-Chk2 (1, 39). We conclude that Rep-DSBs are likely to be recognized and repaired by distinct pathways compared with classic DSBs.

Rep-DSBs Activate the ATM-MRN-Chk2 Axis—CPT and its derivatives are potent anticancer drugs that produce well characterized replication fork damage (6-8, 10). Camptothecins are remarkably selective for Top1·DNA complexes, as demonstrated by the complete CPT resistance of yeast cells without Top1 (62). CPT binds reversibly at the interface of the Top1·DNA complex and prevents its dissociation (15, 63-65). Hence, CPT and its clinical derivatives represent a paradigm for interfacial inhibitors, which trap macromolecular complexes and turn out to be rather common among natural products (10, 66). Collisions of replication forks with the trapped Top1 cleavage complexes convert the single strand breaks into double strand breaks (see Figs. 1b and 4a) (8). It is likely that Rep-DSBs are produced under physiological conditions by a wide range of DNA alterations that trap Top1, including base mismatches, abasic sites, carcinogenic adducts, and nicks (reviewed in Refs. 9 and 13). CPT can therefore be used as a sharp tool to study Rep-DSBs both in mammalian and yeast. Although UV and hydroxyurea are traditionally used to induce "replication stress," the molecular lesions generated at replication forks by hydroxyurea and UV are not well defined.


Figure 8
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  		FIGURE 8.
Differential inhibition of DNA synthesis and cell cycle progression in response to CPT in MRN/Chk2-proficient (HT29) cells and in MRN/Chk2-deficient (HCT116) cells. a, protocol used to examine DNA synthesis inhibition by CPT (1 µM). Thymidine pulses (10 min) were performed at the indicated times (arrows). b, differential inhibition of DNA synthesis in HT29 and HCT116 cells. c, synchronization protocol used to examine alterations of cell cycle progression induced by short treatment of S-phase cells with CPT (1 µM for 75 min; the same protocol was used for Chk2 and MRN activation, see Fig. 3a). d and g, representative FACS profiles for untreated (CL) and CPT-treated HT29 and HCT116 cells at time 0 and 2.5 h after CPT removal. For CL cells, solvent without CPT was added at the same times. e-f and h-j, time course for cell cycle recovery following CPT treatment (protocol in panel c). Percentage of cells in S-phase (e and h) and in G2/M (f and i) were computed at the indicated times following removal of CPT or solvent without CPT (CL).

 
Replication stress is generally considered to activate the ATR-Chk1 pathway, whereas the ATM-Chk2 pathway is generally referred to as a cellular response to DSBs in non-replicating DNA (see Fig. 1a). Our studies demonstrate rapid activation of the ATM-Chk2 pathway in response to Rep-DSBs. Hence, both types of DSBs shown in Fig. 1 activate the ATM-Chk2 pathways. We previously reported that both types of DSBs also activate RPA2 phosphorylation (49), histone {gamma}-H2AX (30), and BLM phosphorylation (47). We now show that activated ATM (phosphorylated on Ser-1981) co-localizes with activated Chk2 (PT68-Chk2) (Fig. 5b). Therefore, both ATM and Chk2 are recruited together with MRN (Fig. 5, b and c) and {gamma}-H2AX (30). Moreover, both Mre11 and Nbs1 are phosphorylated (probably by ATM) (Figs. 3 and 4). Chk1 is also activated by Rep-DSBs (Fig. 2a), and CPT-induced activation of Chk1 has been attributed to ATR (67). Hence, both the ATM-MRN-Chk2 and ATR-Chk1 pathways are activated by Rep-DSBs induced by Top1 cleavage complexes.

MRN Is Required and Cooperates with ATM for Chk2 Activation in Response to Rep-DSBs—Despite normal Chk2 protein expression and normal ATM (Fig. 5, d and e), the colon carcinoma HCT116 cells are deficient for Chk2 activation in response to Rep-DSBs (Figs. 2a, 6b, and 7a). Complementation of the HCT116 cells with Mre11 restored Chk2 activation (Fig. 6a), and down-regulation of Mre11 in HT29 cells reduced Chk2 activation (Fig. 6c). NBS cells also fail to activate Chk2 in response to Rep-DSBs (Fig. 2b), and Nbs1 co-localizes with PT68-Chk2 foci (Fig. 5c). Two other colorectal cell lines from the NCI Cell Screen also fail to activate Chk2 and are MRN-deficient: HCC2998 and KM12 (see Fig. 7a and Table 1). We therefore conclude that MRN plays a critical role in Chk2 activation by ATM. Two recent reports indicate a direct requirement for MRN in activating ATM in response to classic DSBs (such as those shown in Fig. 1a). Thus, MRN appear to be "upstream" from ATM as MRN is required for ATM activation in response to DSBs. However, MRN must also function downstream from ATM because both Mre11 and Nbs1 are known to be phosphorylated by ATM in response to ionizing radiation and radiomimetic drugs (56-58, 68, 69). Both Mre11 and Nbs1 are phosphorylated in response to Rep-DSBs (Figs. 3 and 4). In the case of Rep-DSBs, MRN is not required to activate ATM (Fig. 5, d and e) and therefore acts primarily downstream from ATM possibly to facilitate Chk2 phosphorylation by ATM. Fig. 5f shows a model representing the ATM-Chk2 axis and the impact of MRN downstream from ATM.


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  		TABLE 1
Chk2 activation in response to Rep-DSB induced by camptothecin in the colon carcinoma cell lines from the NCI Anticancer DTP Screen

Relationship with Chk2 and MRN protein levels and with mismatch repair proficiency.

 
MRN and Chk2 Deficiencies in Colorectal Carcinomas—Our study shows that three of seven colon carcinomas cell lines in the NCI-DTP Anticancer Drug Screen are deficient for MRN (see Fig. 1c and Table 1). A recent study showed that 84% of colorectal cancers (41 out of a total of 49 tumors) had at least one mutation in MRE11 and that 39% of total tumors (19 tumors) had biallelic (fully inactivating) MRE11 mutations (51). Thus, Mre11 deficiency appears to be a common feature of colorectal tumors, and this may have been so far overlooked. The mechanism of MRE11 inactivation has been related to mismatch repair defects, which are frequent in colorectal tumors. The MRE11 gene contains a poly-T (11) repeat predisposing it to replication errors. Mismatch repair normally corrects for microsatellite instability. Mismatch repair deficiency, which was initially detected in colorectal tumors arising in patients with hereditary non-polyposis colorectal cancer, is also frequent in non-hereditary colorectal tumors. Mismatch repair defects lead to skipping of exon 5 and introduction of a premature stop codon (mutant MRE11 referred to as 484del88 transcript) (44). The four cell lines with normal MRN (HT29, SW620, COLO205, and HCT15) all have normal mismatch repair (44, 70), and the three cell lines with deficient MRN (HCT116, HCC2998, and KM12) are all mismatch repair-defective (44, 70, 71) (Table 1). It is therefore clear that mismatch repair deficiency is correlated with MRN deficiency. Furthermore, we observed high correlation between the relative levels of the three MRN proteins. Cells with low levels of Mre11 also have low Rad50 and Nbs1 levels (Fig. 1c). Because complementation of HCT116 cells with Mre11 restores Nbs1 and Rad50 protein levels, and down-regulation of Mre11 in HT29 reduces Nsb1 and Rad50 levels, we propose that Mre11 stabilizes Nbs1 and Rad50 polypeptides and may act as a chaperone for the MRN complex. In the absence of Mre11, Nbs1 and Rad50 are probably degraded by the proteasome as MG-132 elevates Nbs1 and Rad50 levels in HCT116 (Fig. 6d).

It is also striking that three of four colorectal cell lines with normal MRN (HCT15, SW620, and COLO205) are deficient for Chk2 activation as a result of low Chk2 protein expression (Fig. 7a and Table 1). Chk2 deficiency in the HCT15 cells is due to a biallelic inactivation of the CHK2 gene (40, 61). However, to our knowledge Chk2 deficiencies have not been reported previously for the COLO205 cells and SW620 cells.

Functional and Therapeutic Implications of MRN and Chk2 Deficiencies in Colorectal Carcinomas—HT29, which have the most robust MRN-Chk2 pathway among the seven colorectal cell lines of the NCI screen (Figs. 1c and 7a) arrest DNA synthesis and delay their S-phase progression in response to CPT (49, 53), whereas HCT116, which are the most Mre11- and Chk2-deficient (Figs. 1c and 7a) are defective in S-phase arrest and DNA synthesis inhibition (Fig. 8). Both the DNA synthesis and S-phase arrest in HT29 are due to checkpoint activation as the Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) (72, 73) can abrogate both DNA synthesis inhibition and S-phase arrest in CPT-treated cells (49, 53). Because HCT116 are more sensitive to CPT than HT29 cells (21), these experiments suggest a role of Chk2 for the repair of Top1 cleavage complexes and the regulation of cell cycle progression in S and G2.

Chk2 has a dual function as it activates both apoptosis and cell cycle checkpoints (31). Hence, in tumor tissues where apoptosis is defective, as in the case of tumors with mutated or inactivated p53, Chk2 inhibitors would be expected to act primarily by preventing DNA repair due to its main function as a checkpoint inhibitor that can induce cellular arrest in a p53-independent manner. On the other hand, in the normal tissues, a Chk2 inhibitor would be expected to limit apoptosis and therefore dose-limiting toxicity. The resulting net effect would then be an increased therapeutic index of the Top1 inhibitors (24, 31).

The critical functional importance of MRN for ATM-Chk2 activation and the known hypersensitivity of NBS-, AT-, and Chk2-deficient cells to CPT (9, 67, 74) suggest the potential therapeutic and prognosis value of systematically testing the MRN and Chk2 status of colorectal tumors treated with irinotecan and novel Top1 inhibitors in clinical development (10). Clinical investigations could also benefit from an understanding of the underlying genetic background that determines the activity of topotecan in ovarian cancers (75, 76).


   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

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Department of Health and Human Services, Convent Drive, Bldg. 37, Rm. 5068, Bethesda, MD 20892-425537. Tel.: 301-496-5944; Fax: 301-402-0752; E-mail: pommier{at}nih.gov.

3 The abbreviations used are: DSBs, DNA double strand breaks; MRN, Mre11·Rad50·Nbs1; Top1, topoisomerase I; Rep-DSBs, replication-mediated DSBs; CPT, camptothecin; {gamma}-H2AX, phosphorylated histone H2AX; AT, ataxia telangiectasia; NBS, Nijmegen breakage syndrome; APH, aphidicolin; MMR, Mismatch repair; ATM, ataxia telangiectasia mutated; ATR, AT and RAD-3-related; siRNA, small interference RNA. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Yosef Shiloh, Sackler School of Medicine, Tel-Aviv University, Israel for providing YZ5 and pEBS7 fibroblast cell lines. We thank Dr. Mirit Aladjem and Dr. Kurt W. Kohn for insightful discussions.



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