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Originally published In Press as doi:10.1074/jbc.M106287200 on November 7, 2001

J. Biol. Chem., Vol. 277, Issue 2, 1599-1606, January 11, 2002
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S Phase and G2 Arrests Induced by Topoisomerase I Poisons Are Dependent on ATR Kinase Function*

William A. ClibyDagger §, Kriste A. LewisDagger , Kia K. LillyDagger , and Scott H. Kaufmann

From the Dagger  Department of Obstetrics and Gynecology, Mayo Clinic and the  Department of Molecular Pharmacology, Mayo Graduate School, Rochester, Minnesota 55905

Received for publication, July 5, 2001, and in revised form, October 12, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G2 arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATR1 has been identified as one of the protein kinases that transduces signals to the cell cycle machinery during normal DNA replication (1) and after DNA damage (2-4). Like the structurally related kinases human ATM, Schizosaccharomyces pombe Rad3 (Rad3SP), and Saccharomyces cerevisiae Mec1 (Mec1Sc), ATR contains a conserved C-terminal kinase domain that phosphorylates downstream substrates (5). The nature of the DNA damage that activates ATR, the identity of its substrates, and the impact of ATR on cell cycle progression are currently the subject of extensive investigation.

Previous studies have suggested that ATR and ATM might have distinct but overlapping functions. In response to IR, ATR has been observed to phosphorylate and activate the checkpoint kinase Chk1 (4), which in turn phosphorylates Cdc25c, inactivating its phosphatase activity and contributing to the ensuing G2 arrest (6-8). In contrast, ATM, which appears to play the more critical role in response to IR, phosphorylates Chk2 (1, 9, 10). Despite these differences, Chen et al. (11) observed that Chk1 overexpression can complement the G2/M checkpoint defect in AT cells and restore IR resistance. These results suggest redundancy and overlap in the specific roles of ATM and ATR.

Several observations indicate that ATM and ATR are also important in the intra-S checkpoint, a series of biochemical reactions that inhibit DNA synthesis in the face of DNA damage or stalled replication forks (12-15). ATM has been shown recently to initiate signaling through Chk2 and Cdc25A to inhibit Cdk2 and prevent DNA synthesis after IR (1). The response of AT cells to other inhibitors of DNA replication, however, appears intact despite the absence of functional ATM (10). Moreover, the observation that several proteins known to be regulated by ATM can still be activated by IR in AT cells (10) indicates that at least one additional upstream regulatory protein can initiate the S phase checkpoint.

The possibility that ATR might play this role was initially suggested by the observation that ATR inhibition results in hypersensitivity to the replication inhibitors hydroxyurea and aphidicolin (2). Subsequent results indicated that ATR phosphorylates Chk1 in response to hydroxyurea (4). More recent experiments demonstrated that Xenopus ATR associates with chromatin in a replication-dependent manner, whereas ATM and DNA-PK do not (15). Interestingly, depletion of ATR in a cell-free Xenopus replication system blocked the Chk1 phosphorylation that ordinarily occurs after treatment with inhibitors of DNA replication (15). Collectively, these observations suggest that ATR is poised to respond to DNA damage occurring specifically during S phase.

In contrast to IR, which induces multiple types of DNA damage throughout the cell cycle (16), the topo I poison CPT and its derivatives produce a single type of DNA lesion that is largely replication-dependent (17-21). Early studies demonstrated that CPT is selectively toxic during S phase (22-25). Subsequent investigations demonstrated that this S phase selectivity reflects the formation of DNA ds breaks when advancing replication forks collide with drug-stabilized topo I-DNA complexes (26-29). Additional studies revealed that brief exposure of exponentially growing cells to high CPT concentrations produces a subsequent G2 arrest (30) as a consequence of impaired activation of cdc2-cyclin B complexes (31). In contrast, prolonged treatment with lower, therapeutically achievable CPT concentrations causes S phase slowing (32-34).

Although it has been observed that CPT-induced S phase arrest is abolished by 7-hydroxystaurosporine (33), an inhibitor of Chk1 and tac1 (35, 36), other events that lie upstream of the CPT-induced G2 and S phase arrests have remained unclear. The present study examines the role of ATR in the response to CPT derivatives. Because deletion of ATR is lethal at an early stage of development (37), studying the function of this kinase in mammalian cells has been difficult. Previous studies have demonstrated that ATR enzymatic activity and function can be abrogated by overexpression of a kinase-inactivated ATR allele (2). We have utilized cells expressing this kdATR allele in an inducible fashion to (i) evaluate the role of ATR in the G2 and S phase checkpoint activation after topo poisons, (ii) determine whether the abrogation of these checkpoints is related to altered phosphorylation of Chk1 and Chk2, and (iii) assess the effect altered ATR function on the cytotoxic affects of topo poisons. Results of these studies provide the first evidence that ATR plays a major role in checkpoint activation and cell survival in cells with replication-associated DNA ds breaks.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- TPT was obtained from the Pharmaceutical Resources Branch of the National Cancer Institute. SN-38 was provided by Pharmacia. Additional reagents were purchased from the following suppliers: etoposide from Biomol (Plymouth Meeting, PA), paclitaxel and doxorubicin from Sigma, nocodazole from Aldrich, and G418 from Invitrogen.

The following murine monoclonal antibodies were utilized: C-21 anti-topo I (Y.-C. Cheng, Yale University Medical School, New Haven, CT), Ki-S1 anti-topo IIalpha (13, 14) (Dr. Udo Kellner, Kiel, Germany), anti-Chk1 (Santa Cruz Biotechnology), anti-Chk2 (Dr. Junjie Chen, Mayo Foundation, Rochester, MN), and anti-BrdUrd (Becton Dickinson). In addition, affinity-purified epitope-specific rabbit antisera that recognize phospho-345Ser-Chk1 (Cell Signaling Technology, Beverly, MA) and phospho-Thr68-Chk2 (Junjie Chen), as well as goat anti-Chk1 (Upstate Biotechnology, Inc.), were used. Affinity-purified peroxidase-coupled secondary antibodies were purchased from Kierkegaard and Perry (Gaithersburg, MD).

Tissue Culture-- GM847 is an SV40-transformed fibroblast line. The GM847/kdATR line was previously constructed to contain a kinase-inactive allele of ATR under the positive control of a doxycycline-responsive promoter (2). GM847/kdATR cells were cultured in Dulbecco's minimal essential medium with 4.5 g/liter glucose, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine (medium A) supplemented with 400 µg/ml G418. At the start of each experiment, cells were grown in the absence or presence of 1 µg/ml doxycycline for 48 h in medium A. Varying concentrations of TPT or etoposide were then added for the indicated length of time. At the completion of this incubation, cells were then washed and plated in drug-free medium A in the continued presence or absence of doxycycline for the indicated length of time. Trypan blue exclusion, apoptotic morphological changes, and cell cycle distribution were then assayed as described below.

AT4BI, an SV40-transformed fibroblast line from a homozygous AT patient, was obtained from Patrick Concannon (Virginia Mason Research Center, Seattle, WA) and cultured in medium A. Treatments with TPT and etoposide were performed as described above.

For clonogenic assays, aliquots containing 5000 cells grown in the absence or presence of doxycycline were plated in triplicate 35-mm dishes, permitted to adhere overnight, treated with the indicated drug for 2 or 24 h, and incubated for 12-14 days to allow colonies to form. At the completion of this incubation, cells were stained with Coomassie Blue; and colonies containing >= 50 cells were manually counted. Control plates typically contained 140-180 colonies.

Assays for Cell Death-- To assess apoptotic morphological changes, adherent and floating cells were pooled, washed once with PBS, fixed with 3:1 (v/v) methanol:acetic acid, stained with 1 µg/ml Hoechst 33258, and examined by fluorescence microscopy (38). A duplicate unfixed sample was stained with 0.2% trypan blue to determine cell viability.

Flow Cytometry-- Control and treated cells were fixed in 50% ethanol, washed, digested with RNase A, stained with propidium iodide, and subjected to flow microfluorimetry on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) as described previously (38). For BrdUrd incorporation studies, cells were pretreated with 125 nM TPT or diluent for 8 h. BrdUrd was then added to a final concentration of 20 µM for 30 min. Cells were then washed and incubated for an additional 0-8 h in TPT or diluent. At the completion of the incubation, cells were trypsinized, centrifuged at 1000 × g, washed in ice-cold PBS, and fixed in 66% (v/v) ethanol at -20 °C. After rehydration with PBS, samples were incubated with 0.04% (w/v) pepsin in 0.1 N HCl for 30 min, washed in PBS containing 0.5% (w/v) Tween 20 (PBS-T), incubated in 2 N HCl for 30 min at 37 °C, neutralized with 0.1 M sodium borate, and washed again in PBS-T. All further steps were performed in the dark at 20-22 °C unless otherwise indicated. Samples were incubated with anti-BrdUrd antibody for >= 1 h, washed in PBS-T, treated with fluorescein-conjugated secondary antibody for 30 min, washed in PBS-T, incubated for 20 min at 37 °C in PBS-T supplemented with 20 µg/ml propidium iodide and 0.1 mg/ml RNase A, and subjected to flow cytometry. BrdUrd incorporation and cell cycle distribution were analyzed using Becton Dickinson CellQuest software.

Immunoblotting and Immunoprecipitation-- GM847/kdATR cells grown in the absence or presence of doxycycline for 48 h were solubilized in buffer consisting of 6 M guanidine hydrochloride, 250 mM Tris-HCl, 10 mM EDTA, 1 mM alpha -phenylmethylsulfonyl fluoride, and 150 mM beta -mercaptoethanol. After preparation for SDS-polyacrylamide gel electrophoresis as described previously (38), polypeptides (40 µg of protein) were separated on 5-15% (w/v) polyacrylamide gels and electrophoretically transferred to nitrocellulose. Blots were probed (38) using the reagents listed above.

The effects of TPT and etoposide on Chk1 and Chk2 phosphorylation were assessed by subjecting Chk1 and Chk2 immunoprecipitates to blotting with phosphoepitope-specific antibodies. This analysis was impaired by the presence of a heavy IgG band on immunoblots. To reduce the IgG signal, antibodies were covalently cross-linked to protein A-Sepharose beads using 20 mM dimethylpimelmidate in 0.2 M sodium borate, pH 9.0, as described in Ref. 39. The reaction was terminated by the addition of 0.2 M ethanolamine. After beads were thoroughly washed, cell lysates (prepared in buffer consisting of 20 mM Tris-HCl (pH 8.0), 100 mM NaC1, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml antipain, 10 nM microcystin, 2 µg/ml leupeptin, 1 mM Na3VO4, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerol phosphate, and 10 mM NaF) were reacted overnight with 15 µl of beads (packed volume) containing covalently bound Chk1 or Chk2 antibodies. After beads were washed twice with PBS, polypeptides were eluted in SDS sample buffer, separated on 12% acrylamide gels, and probed with antibodies as described in the individual figure legends.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments described below were designed to assess the potential role of ATR in modulating the response to DNA damage from topo poisons. For these studies, we employed GM847/kdATR, a recently described cell line that contains cDNA encoding kdATR under the positive control of a doxycycline-responsive promoter (2, 40). In the presence of doxycycline, the kdATR expressed by these cells diminishes the ability of endogenous ATR to phosphorylate itself (2) and downstream substrates such as p53 and human Chk1 in response to DNA damage (3, 4).

ATR Is Necessary for the G2/M Arrest after Brief Exposure to TPT-- To assess the effect of ATR on G2 arrest,2 cells grown in the absence or presence of doxycycline were exposed to 125 nM TPT for 2 h,3 washed, incubated in the continued absence or presence of doxycycline for 24 h, and harvested for flow cytometry. Cells grown in the absence of doxycycline underwent a TPT-induced G2 arrest, with the percentage of cells in G2/M increasing from 15% prior to TPT treatment to 42% 24 h later (Fig. 1A). In contrast, cells expressing the kdATR allele had a markedly diminished G2 arrest after TPT treatment, with the G2/M population increasing from 15% prior to treatment to only 24% after TPT exposure (Fig. 1B). Analysis at later time points demonstrated that this TPT-induced G2 arrest was transient in the presence of the kdATR allele, with return to essentially a pretreatment cell cycle distribution by 48 h (Fig. 1B). In contrast, the G2 arrest was more persistent in the presence of normal ATR function (Fig. 1A, 48 h).


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  		Fig. 1.   ATR is required for G2 checkpoint function after brief exposure to TPT. A and B, cell cycle profiles of GM847/kdATR cells grown in the absence (A, wild-type ATR function) or presence (B, kdATR allele overexpressed) of doxycycline (Doxy) were examined by flow cytometry. At t = 0, cells were harvested for analysis or exposed to 125 nM TPT for 2 h, incubated in fresh medium with or without doxycycline, and subsequently harvested for analysis at the indicated time points. Results are representative of three independent experiments.

ATR Is Also Required for Normal S Phase Arrest during Prolonged TPT Treatment-- Prolonged exposure to topo I poisons results in slowing of DNA replication (32-34), presumably because of activation of the S phase checkpoint by replication-induced conversion of the stabilized DNA-topo I complexes into frank DNA ds breaks (26-29). To evaluate the possibility that ATR might also function as part of this S phase checkpoint, we pretreated cells for 48 h with doxycycline (to inactivate ATR function) or diluent, then exposed them to 125 nM TPT for 8 h in the continued presence or absence of doxycycline. At this point cells were pulse-labeled with BrdUrd and returned to TPT-containing medium in the presence or absence of doxycycline for varying lengths of time. After staining with anti-BrdUrd and propidium iodide, the fate of the cells that synthesized DNA in the presence of TPT was then determined by multiparameter flow cytometry.

Control cells (no doxycycline or TPT) uniformly incorporated BrdUrd throughout S phase after a 30-min pulse (t = 0 indicates time after the BrdUrd pulse) (Fig. 2A, gated). By 8 h after completion of the pulse, the majority of the labeled cells had completed S phase in the absence of DNA damaging agents (Fig. 2A', gated). These results provide a base line for comparison.

When cells were exposed to TPT for 8 h (no doxycycline), an increased S phase population was observed (19% of total cells in Fig. 2B versus 11% in Fig. 2A), reflecting the previously reported S phase slowing. In addition, consistent with S phase checkpoint activity, these cells incorporated decreased amounts of BrdUrd into DNA, particularly when they were in late S phase (Fig. 2B, arrowhead). Moreover, with continued TPT incubation, fewer labeled cells progressed to G2 relative to untreated cells. By 8 h after the completion of the BrdUrd pulse, almost all of the labeled cells were still in late S phase (17% of total cell population, Fig. 2B', gated, versus 19% at start of incubation), again reflecting S phase arrest as a consequence of checkpoint activation.


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  		Fig. 2.   kdATR expression abrogates the S phase arrest after continuous TPT exposure. Cells were grown in the absence (panels A, A', B, and B') or presence (C and C') of doxycycline for 48 h. In the continued absence or presence of doxycycline, cells were treated with diluent (A and A') or 125 nM TPT (B, B', C, and C') for 8 h. Cells were then pulse-labeled with 20 µM BrdUrd for 30 min and immediately harvested (0 h, panels A, B, and C) or washed and treated with fresh medium with diluent (A'), 125 nM TPT (B'), or doxycycline + TPT (C') for 8 h. After treatment with anti-BrdUrd antibody and propidium iodide, cells were subjected to flow cytometry as described under "Materials and Methods." Scatter plots depict BrdUrd labeling (y axis, log scale) as a function of cell cycle distribution (x axis, propidium iodide content). Labeled cells were gated (box S) to exclude cells with G1 and G2 DNA contents and recorded as a percentage of all cells counted. Results are representative of three independent experiments.

Expression of the kdATR allele abrogated all of these features of the S phase checkpoint (Fig. 2, C and C'). In particular, cells grown in the presence of doxycycline appeared to incorporate label equally throughout S phase after TPT treatment (Fig. 2C, gated). Moreover, the majority of these cells had completed S phase 8 h after pulse labeling (Fig. 2C', gated), indicating that they progressed through S phase at a rate similar to that of untreated cells (Fig. 2A', gated). These findings demonstrate that ATR is required for TPT-induced inhibition of DNA replication.

TPT-induced Phosphorylation of Chk1 Is Dependent upon ATR Kinase Function, whereas Chk2 Phosphorylation Is Not-- The observation that the kdATR allele diminishes both G2 arrest (Fig. 1) and S phase slowing (Fig. 2) after TPT treatment suggested that ATR may be signaling for cell cycle arrest via Chk1 and/or Chk2, known upstream components of the G2 and S phase checkpoints. To assess these possibilities, GM847/kdATR cells grown in the absence or presence of doxycycline were exposed to 125 nM TPT for increasing lengths of time and then analyzed for the presence of phosphorylated Chk1 and Chk2. These experiments indicated that TPT treatment resulted in Chk1 phosphorylation (cf. Fig. 3A, lanes 3, 5, and 7). This phosphorylation was evident within 2 h (Fig. 3A, lane 3) and persisted for at least 24 h (Fig. 3A, lane 7). Expression of kdATR markedly diminished this Chk1 phosphorylation (Fig. 3A, lanes 4, 6, and 8). These results indicate that Chk1 phosphorylation correlates with the intra-S phase slowing (Fig. 2, B and B'), and abrogation of Chk1 phosphorylation correlates with loss of the S phase checkpoint (Fig. 2, C and C').


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  		Fig. 3.   Chk1 phosphorylation after TPT is regulated by ATR. GM847/kdATR cells grown in the absence (-) or presence (+) of doxycycline for 48 h were treated with diluent (0 h) or 125 nM TPT for 2-24 h, or IR. At the completion of the incubation, samples were immunoprecipitated with anti-Chk1 (A) or anti-Chk2 (B) antibody covalently linked to protein A-Sepharose. Immunoprecipitates were probed with anti-Chk1 or epitope-specific anti-phospho-Ser345-Chk1 (A) or anti-Chk2 or anti-phospho-Thr68-Chk2 (B) antisera.

In contrast, phosphorylation of Chk2 does not correlate with the effects of ATR. In particular, prolonged TPT treatment causes a detectable increase in Chk2 phosphorylation (cf. Fig. 3B, lanes 1 and 7) that occurs independent of ATR status (Fig. 3B, lane 8), suggesting that TPT-induced Chk2 phosphorylation occurs by an ATR-independent mechanism and is not responsible for the TPT-induced checkpoint activation.

Chk1 Activation and Cell Cycle Responses Observed after Etoposide Treatment Are Dependent upon ATR Kinase Activity-- Topo I poisons are almost exclusively dependent upon DNA replication for the formation of secondary DNA strand breaks, whereas topo II poisons such as etoposide depend somewhat more upon RNA transcription to generate these lesions (17, 18, 24, 41). Accordingly, topo II poisons are thought to generate DNA ds breaks throughout the cell cycle. In view of this dichotomy, we investigated whether functional inactivation of ATR affected the response of cells to the topo II poison etoposide.

As indicated in Fig. 4A, treatment of GM847/kdATR cells with 300 nM etoposide for 24 h resulted in a prominent G2 arrest (44% cells in G2 versus 16%, untreated). Induction of the kdATR allele for 24 h before addition of etoposide diminished the number of cells arrested in G2 (Fig. 4B, 35% versus 16% untreated), although the effect was smaller than that observed with TPT. In addition, there was a relative increase in the number of cells passing through M to G1 (42% G1 in the presence of doxycycline versus 26% G1 in its absence; Fig. 4, A and B).2 These results suggest that ATR also plays a role in the cell cycle response to etoposide-induced DNA damage. Consistent with this conclusion, etoposide-induced phosphorylation of Chk1 (Fig. 4C, lanes 3, 5, and 7), but not Chk2 (Fig. 4D, lanes 4 and 5), was abrogated by overexpression of the kdATR allele.


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  		Fig. 4.   Role of ATR in checkpoint after exposure to etoposide. A and B, cell cycle profiles of GM847/kdATR cells grown in the absence (A, wild-type ATR function) or presence (B, kdATR allele overexpressed) of doxycycline were examined by flow cytometry. At t = 0, cells were harvested for analysis or exposed to 300 nM etoposide for 24 h, and subsequently harvested for analysis. Results are representative of three independent experiments. C and D, effect of the kdATR allele on etoposide-induced phosphorylation of Chk1 and Chk2. GM847/kdATR cells grown in the absence (-) or presence (+) of doxycycline for 48 h were treated with diluent (0 h) or the indicated concentration of etoposide for 2, 8, or 24 h. At the completion of the incubation, samples were immunoprecipitated with anti-Chk1 (C) or anti-Chk2 (D) antibody covalently linked to protein A-Sepharose. Immunoprecipitates were probed with epitope-specific anti-phospho-Ser345-Chk1 (C) or anti-Chk2 (D). Cells were exposed to 20-Gray ionizing radiation as a positive control for Chk2 phosphorylation (D).

TPT- and Etoposide-induced Phosphorylation of Chk1 Occurs in an ATM-independent Manner-- To rule out the possibility that the effects of kdATR described above result from inhibition of the related kinase ATM, we analyzed the effects of topo poisons on the ATM-deficient SV40-transformed human fibroblast line AT4BI. Prolonged exposure of these cells to 125 nM TPT resulted in marked S phase accumulation (49% with TPT versus 25% untreated, Fig. 5A). In addition, prolonged exposure to 300 nM etoposide resulted in G2 arrest (41% with etoposide versus 18% untreated, Fig. 5B). These results indicate that ATM is not required for the TPT-induced S phase arrest or the etoposide-induced G2 arrest observed in Figs. 2A and 4A, respectively.


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  		Fig. 5.   TPT-induced checkpoint activation and Chk1 phosphorylation are independent of ATM function. A and B, cell cycle profiles of the AT-deficient AT4BI fibroblast cell line were examined by flow cytometry. At t = 0, cells were harvested for analysis or exposed to 125 nM TPT (A) or 300 nM etoposide (B) and subsequently harvested at 24 h. C, Chk1 phosphorylation in response to topo poisons in AT-deficient fibroblasts. AT4BI cells were treated with diluent (0 h) or the indicated concentration of TPT or etoposide for 8 h. After completion of incubations, samples were immunoprecipitated with anti-Chk1 antibody covalently linked to protein A-Sepharose. Immunoprecipitates were probed with anti-Chk1 or epitope-specific anti-phospho-Ser345-Chk1 antisera.

Our earlier results suggested that these cell cycle effects correlated with ATR-dependent Chk1 phosphorylation. If so, then the effects of these drugs on Chk1 phosphorylation would be predicted to occur normally in ATM-deficient cells. Consistent with this prediction, we observed that Chk1 phosphorylation at Ser345 was readily detectable when AT4BI cells were treated with TPT or etoposide (Fig. 5C, lanes 2 and 3). These results provide further support for the view that ATR is responsible for the activating phosphorylations of Chk1 after treatment with these drugs.

The kdATR Allele Enhances the Antiproliferative Effects of Topo I and II Poisons-- To determine whether abrogation of the ATR-induced signaling events altered the sensitivity of cells to the antiproliferative effects of topoisomerase poisons, GM847/kdATR cells grown in the absence or presence of doxycycline for 48 h were exposed to increasing concentrations of topo I or topo II poisons. As illustrated in Fig. 6A (open circles), a 24-h exposure to increasing concentrations of TPT in the absence of doxycycline decreased subsequent colony formation of GM847/kdATR cells with an IC50 of 75 nM and an IC90 of >200 nM. Treatment with doxycycline (Fig. 6A, closed circles) resulted in a 2-3-fold decrease in the IC50 (30 nM) and IC90 (100 nM), suggesting that ATR normally modulates the antiproliferative effects of TPT. Control experiments revealed that doxycycline had no effect on TPT sensitivity in parental GM847 cells (data not shown), indicating that the sensitization observed in GM847/kdATR cells was caused by overexpression of the kdATR allele.


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  		Fig. 6.   Expression of kdATR sensitizes cells to topo I poisons. A, C, and D, GM847/kdATR cells grown in the absence (open circles) or presence (closed circles) of doxycycline for 48 h were exposed for 24 h (A and D) or 2 h (C) to the indicated chemotherapeutic agent, washed, and allowed to form colonies. B, cells grown in the absence (open circles) or presence (closed circles) of doxycycline for 48 h were treated with 125 nM TPT for 24 h beginning at time 0 and collected at indicated time points. Apoptotic cells were quantitated as described under "Materials and Methods." E, lack of effect of the kdATR allele on topo I and topo II expression. After total cellular protein was harvested from cells growing in the absence (lanes 1-3) or presence (lanes 4-6) of doxycycline using previously described methods (38), aliquots containing 50 µg (lanes 1 and 4), 25 µg (lanes 2 and 5), or 12.5 µg (lanes 3 and 6) of total cellular protein were subjected to immunoblotting using anti-topo I (top panel) or anti-topo IIalpha (bottom panel) monoclonal antibodies. F, effects of topo poisons on polypeptide levels in cells expressing kdATR. GM847/kdATR cells grown in the absence or presence of doxycycline for 48 h were treated with 125 nM TPT or 300 nM etoposide for 12 or 24 h in the continued absence (-) or presence (+) of doxycycline. Whole cell lysates (50 µg of protein/well) were then subjected to SDS-PAGE followed by immunoblotting sequentially with anti-topo I (top panel), anti-topo IIalpha (middle panel), and poly(ADP-ribose) polymerase, which served as a loading control. Results are representative of three (panels A-E) or two (panel F) independent experiments.

To determine whether the effects of the clonogenic assays reflected altered survival rather than prolonged arrest, we examined survival and the development of apoptotic morphological changes. After a 24-h exposure to 125 nM TPT, the percentage of apoptotic cells as assessed by nuclear morphology was increased nearly 5-fold (9.7% versus 2.0%) when cells expressed the kdATR allele (Fig. 6B). After an additional 24-h incubation in fresh medium, the number of cells undergoing apoptosis increased further, but more cells expressing the kdATR allele still underwent apoptosis. When trypan blue exclusion was utilized to quantitate short term survival, complementary results were obtained, with survival after 24 and 48 h being 98 and 48%, respectively, for cells grown in the absence of doxycycline and 88 and 27% for cells grown in the presence of doxycycline.

Further experiments (Fig. 6C) demonstrated that expression of the kdATR allele also increased the sensitivity of cells to a 2-h TPT exposure, although higher concentrations of TPT were required to inhibit colony formation. A similar degree of sensitization was observed in cells treated for 24 h with another topo I poison, SN-38, the active metabolite of irinotecan (Fig. 6D), confirming that the effects of kinase-inactivated ATR were not unique to TPT.

Previous studies have suggested that pretreatment levels of topo I can affect sensitivity to topo I poisons (42, 43). More recently, CPT has also been shown to induce proteasome-mediated topo I degradation in resistant cell lines but not sensitive cell lines, raising the possibility that CPT-induced proteasome-mediated degradation might play a role in resistance to topo I poisons (44). To evaluate the possibility that ATR affected either pretreatment topo I levels or drug-induced topo I degradation, GM847/kdATR cells were grown in absence or presence of doxycycline for 48 h and harvested before or after treatment with TPT. These studies demonstrated that expression of the kdATR allele had no effect on basal topo I polypeptide levels (Fig. 6E). Moreover, expression of the kdATR allele did not affect the degradation of topo I in response to TPT. Whether the kdATR allele was expressed or not, topo I levels in these transformed fibroblasts remained constant during the course of TPT treatment (Fig. 6F). Likewise, expression of the kdATR allele did not affect basal levels of topo IIalpha (Fig. 6E). During the course of TPT or etoposide treatment, however, topo IIalpha levels increased (Fig. 6F), possibly reflecting the higher expression of topo II in S and G2 phase cells (45, 46). Once again, expression of the kdATR allele did not affect this change.

In a final series of experiments, the effect of the kdATR allele on the response to topo II poisons was examined. Previous reports have suggested that a brief exposure to high concentrations of etoposide kills cells in a manner that does not require ongoing DNA synthesis (41, 47). Consistent with this conclusion, expression of kdATR had no effect on the antiproliferative effects observed after exposure to high concentrations of etoposide for 2 h (data not shown). A different picture emerged after more prolonged etoposide treatment. Although the effects were somewhat smaller than those observed with TPT, presumably because only part of the toxic DNA strand breaks are being generated during S phase (24), expression of kdATR enhanced the antiproliferative effects during a 24-h exposure to lower etoposide concentrations (Fig. 7A). Similar effects were observed when etoposide was replaced doxorubicin (Fig. 7B), another agent that stabilizes topo II-DNA cleavage complexes (48). In contrast, the kdATR allele had little effect on the cytotoxicity of the microtubule stabilizing agent paclitaxel (Fig. 7C).


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  		Fig. 7.   Effect of kdATR on antiproliferative effects of etoposide, doxorubicin, and paclitaxel. GM847/kdATR cells grown in the absence (open circles) or presence (closed circles) of doxycycline for 48 h were exposed for 24 h to the indicated concentration of etoposide (A), doxorubicin (B), or paclitaxel (C), washed, and allowed to form colonies. Results in each panel are representative of at least three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Results of the present study have demonstrated that ATR kinase function is necessary for both the G2 and S phase arrests induced by topo I poisons. In particular, forced overexpression of the kdATR allele diminishes both the G2 arrest observed after a brief TPT exposure (Fig. 1) and the S phase arrest observed after prolonged treatment (Fig. 2). Abrogation of these TPT-induced checkpoints is accompanied by decreased Chk1 phosphorylation (Fig. 3A) but not by any change in Chk2 phosphorylation (Fig. 3B). Moreover, inhibition of ATR function, Chk1 phosphorylation and the ensuing arrests is associated with increased sensitivity to brief (Fig. 6C) or prolonged (Fig. 6, A, B, and D) exposures to the topo I poisons TPT and SN-38. Additional experiments have demonstrated that inhibition of ATR function also abrogates the Chk1 activation and cell cycle response observed after prolonged treatment with relatively low concentrations of the topo II poison etoposide (Fig. 4). These cellular responses to topo poisons appear to be independent of ATM function (Fig. 5). Collectively, these observations lead to the model outlined in Fig. 8. In addition, these observations have important implications for current understanding of the role of ATR in the response to DNA damage associated with replicating DNA, particularly damage initiated by topo I poisons.


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  		Fig. 8.   Proposed pathway for S and G2 arrests after treatment with topoisomerase poisons. The effects of the kdATR are described in the present report. The effects of aphidicolin (27,31,47) and UCN-01 (33) have been described previously. Likewise, the downstream effectors of Chk1 phosphorylation have been described recently (6-8).

The present study focused extensively on topo I poisons. These agents stabilize covalent intermediates between topo I and its DNA substrate, providing complexes that can be converted into frank DNA ds breaks through interactions with advancing replication forks (17, 20, 42, 49). In contrast to ionizing radiation, which produces a variety of DNA lesions, topo I poisons are currently thought to produce only a single type of cytotoxic DNA damage (17, 18). As a result, these agents have been recognized as unique tools for dissecting the biochemistry of checkpoint activation (31, 32).

The present results indicate that ATR plays a role in both of the cell cycle responses observed after exposure to topo I poisons. Brief exposure results in a subsequent G2 arrest, which is markedly attenuated by expression of the kdATR allele (Fig. 1B). The attenuation of this TPT-induced G2 arrest by kdATR is similar to the effect observed after ionizing radiation (9). The previously reported phosphorylation of Chk1 by ATR (12) provides a potential mechanism by which ATR may activate the G2 checkpoint after DNA damage. Consistent with this hypothesis, we observed that TPT treatment for as little as 2 h resulted in Chk1 phosphorylation that was inhibited by expression of the kdATR allele (Fig. 3A).

Prolonged exposure to topo I poisons leads to S phase slowing (32, 33). The observation that ATR is associated with DNA during replication (15) raised the possibility that ATR might also be involved in this response to topo I poisons. As reviewed by Naegeli (50), replication of damaged DNA is a primary mechanism of genetic instability because the rate of DNA synthesis determines the frequency of conversion to permanent changes in the DNA. The S phase checkpoint presumably functions to prevent the consequences of replication in the face of significant DNA damage (51). In budding yeast, the ATR homologue Mec1Sc is required for the S phase slowing seen after several forms of DNA damage; and interruption of Mec1Sc is associated with decreased survival after this damage (51). This Mec1Sc-dependent checkpoint involves slowing of DNA replication, thereby allowing adequate time for repair or for more tolerance to damage (52). Interestingly, yeast lacking Mec1Sc are able to progress through S phase despite significant DNA damage (52). This replication in the face of DNA damage correlates with late origin firing, suggesting that the unregulated DNA synthesis in Mec1Sc-deficient cells might result from a failure to prevent initiation at late origins (52). The parallels of the Mec1Sc-deficient yeast to cells expressing the kdATR allele are striking. In GM847 cells expressing wild-type ATR, the TPT-induced S phase slowing reflects two related changes: decreased synthesis of DNA, particularly during late S phase (arrowhead, Fig. 2B), and a decreased rate of S phase transit (Fig. 2B'). Both of these changes are abrogated by overexpression of the dominant negative kdATR protein (Fig. 2, C and C'). These results suggest that ATR, like Mec1Sc, slows S phase transit in response to DNA damage, possibly by inhibiting late firing DNA replicons.

This conclusion is consistent with other emerging information on the biological functions of ATR. Tibbetts et al. (53) recently demonstrated that ATR phosphorylates the tumor suppressor protein BRCA1 and colocalizes with this substrate at sites of stalled replication forks after treatment with aphidicolin or hydroxyurea. Coupled with the present results, these observations suggest that ATR is a key member of an intra-S phase checkpoint pathway.

Results of the present study also demonstrated quantitative and qualitative differences between the effects of the kdATR allele on sensitivities to topo I and topo II poisons. The kdATR allele affected sensitivity to topo I poisons more than topo II poisons (Fig. 6 (A and D) versus Fig. 7 (A and B)). Moreover, the kdATR allele sensitized cells to topo I poisons during exposures as brief as 2 h (Fig. 6C), whereas it had little effect on sensitivity to topo II poisons when they were applied for these brief time periods. These differences might be related to the fact that drug-stabilized topo I-DNA covalent complexes are particularly efficiently converted into cytotoxic lesions by advancing replication forks (24, 27, 47), whereas drug-stabilized topo II cleavage complexes can also be converted into cytotoxic lesions in a transcription-dependent manner, especially when cells are treated with high concentrations of these agents for brief periods (24, 41). The inability of kdATR to affect the cytotoxicity after brief exposure to these higher levels of topo II poisons raises the possibility that the resulting lesions are different from the DNA ds breaks that result from brief exposure to topo I poisons.

It is unclear at present how ATR and related kinases are activated by DNA damage. Current understanding suggests that DNA damage sensors, possibly Rad proteins, are recruited to sites of stalled replication forks and serve as scaffolds for the assembly and/or activation of these upstream signaling kinases (54, 55). Studies in yeast also suggest that Rad proteins, particularly Rad1, Rad9, Rad17, and Hus1, might be upstream of ATR and ATM homologs in DNA repair pathways (6). Consistent with this model, recent results have demonstrated that CPT and etoposide alter the association between the Rad9-Rad1-Hus1 complex and chromatin in K562 human leukemia cells (56). Further studies are required to assess whether these putative DNA damage sensors are upstream of ATR and to determine how topo I poisons and other types of DNA damage activate this pathway.

The present demonstration that ATR plays an important role in the cell cycle responses and survival after treatment of cells with topo I poisons does not rule out the possibility that other related kinases are also activated by these drugs. Recent experiments suggest that DNA-PK is activated after treatment with topo I poisons and, in turn, phosphorylates replication protein A (57). ATM also appears to play a role in the response to topo I poisons. A variety of studies (reviewed in Refs. 18 and 58) have indicated that fibroblasts from patients with AT are particularly sensitive to the antiproliferative effects of topo I-directed agents. More recently, Chk2 phosphorylation has been shown to occur in an ATM-dependent manner after exposure of mammalian cells to TPT (10).

Despite evidence that DNA-PK and ATM are activated in response to topo I poisons, the respective roles of these kinases in topo I poison-induced S phase checkpoint function has not been clear. On the one hand, the results of Chaturvedi et al. (10) have implicated ATM and Chk2 in the response to topo I poisons. On the other hand, Shao et al. reported that the S phase slowing induced by SN-38 can be abrogated by treatment with 7-hydroxystaurosporine (33). Because this agent does not alter the catalytic activities of either ATM (59)4 or Chk2, but instead inhibits Chk1 (35, 36), the observation of Shao et al. implicates kinases upstream of Chk1, which include ATR (4), in activation of the S phase checkpoint by topo I poisons. Our results (Figs. 2 and 3) likewise implicate ATR and Chk1 in the S phase checkpoint but do not rule out the possibility that other related kinases might also play a role.

In summary, the present results indicate that ATR plays an important role in the regulation of DNA replication after replication-associated DNA damage and, as such, may be a key component of a mammalian S phase DNA damage response pathway. As a result, cell survival after treatment with topoisomerase poisons, particularly topo I poisons, is enhanced by normal ATR function. These observations raise the possibility that alterations in ATR-dependent pathways might affect sensitivity to topo I poisons in the clinical setting. In addition, these observations suggest that ATR might be a good candidate for future development of biochemical modulating agents designed to enhance sensitivity of tumor cells to chemotherapeutic agents that target topoisomerases.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the kind gifts of antibodies from Junjie Chen, Y.-C. Cheng, and Udo Kellner, as well as, SN-38 from Pharmacia; helpful discussions with Junjie Chen, Larry Karnitz, and Jan Sarkaria; and secretarial assistance of Deb Strauss.

    FOOTNOTES

* This work was supported in part by National Institutes of Health R01 CA73709 and by grants from the Mayo Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Div. of Obstetrics and Gynecology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Tel.: 507-266-8685; Fax: 507-266-7953; E-mail: cliby.william@mayo.edu.

Published, JBC Papers in Press, November 7, 2001, DOI 10.1074/jbc.M106287200

2 Because GM847 cells lack p53 function, G1 arrest is not observed after DNA damage.

3 The concentration of 125 nM was chosen because it has minimal effect on cell survival after a 2-h exposure (Fig. 6C), yet alters cell cycle distribution.

4 J. Sarkaria, personal communication.

    ABBREVIATIONS

The abbreviations used are: ATR, mutated in ataxia telangiectasia-Rad-3-related kinase; AT, ataxia telangiectasia; ATM, mutated in ataxia telangiectasia; CPT, camptothecin; kd, kinase-dead; BrdUrd, 5-bromo-2'-deoxyuridine; ds, double-stranded; IR, ionizing radiation; PBS, calcium- and magnesium-free phosphate-buffered saline; PBS-T, phosphate-buffered saline plus Tween 20; SN-38, 7-ethyl-10-hydroxycamptothecin; topo, topoisomerase; TPT, topotecan; DNA-PK, DNA-dependent protein kinase.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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