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[Cancer Research 65, 6874-6881, August 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Molecular Basis for G2 Arrest Induced by 2'-C-Cyano-2'-Deoxy-1-ß-D-Arabino-Pentofuranosylcytosine and Consequences of Checkpoint Abrogation

Xiaojun Liu1, Ying Guo1, Yexiong Li1, Yingjun Jiang1,2, Sherri Chubb1, Atsushi Azuma1, Peng Huang1,2, Akira Matsuda3, Walter Hittelman1,2 and William Plunkett1,2

1 Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center and 2 The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas and 3 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Requests for reprints: William Plunkett, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Box 71, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3335; Fax: 713-794-4316; E-mail: wplunket{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosylcytosine (CNDAC) is a nucleoside analogue with a novel mechanism of action that is currently being evaluated in clinical trials. Incorporation of CNDAC triphosphate into DNA and extension during replication leads to single-strand breaks directly caused by ß-elimination. These breaks, or the lesions that arise from further processing, cause cells to arrest in G2. The purpose of this investigation was to define the molecular basis for G2 checkpoint activation and to delineate the sequelae of its abrogation. Cell lines derived from diverse human tissues underwent G2 arrest after CNDAC treatment, suggesting a common mechanism of response to the damage created. CNDAC-induced G2 arrest was instituted by activation of the Chk1-Cdc25C-Cdk1/cyclin B checkpoint pathway. Neither Chk2, p38, nor p53 was required for checkpoint activation. Inhibition of Chk1 kinase with 7-hydroxystaurosporine (UCN-01) abrogated the checkpoint pathway as indicated by dephosphorylation of checkpoint proteins and progression of cells through mitosis and into G1. Cell death was first evident in hematologic cell lines after G1 entry. As indicated by histone H2AX phosphorylation, DNA damage initiated by CNDAC incorporation was transformed into double-strand breaks when ML-1 cells arrested in G2. Some breaks were manifested as chromosomal aberrations when the G2 checkpoint of CNDAC-arrested cells was abrogated by UCN-01 but also in a minor population of cells that escaped to mitosis during treatment with CNDAC alone. These findings provide a mechanistic rationale for the design of new strategies, combining CNDAC with inhibitors of cell cycle checkpoint regulation in the therapy of hematologic malignancies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The genetic stability of mammalian cells is continuously threatened by multiple causes of genotoxic stress. The most prevalent resulting damage is in the form of DNA single-strand breaks (SSB; refs. 1–3). For instance, DNA damage in sugar residues, termed "direct SSBs," can arise endogenously from chemical attack by reactive oxygen species, other electrophilic molecules, and the intrinsic instability of DNA (4–6). In addition, physical agents, such as ionizing radiation or UV light, can directly induce SSBs (7–9). Abortive topoisomerase I cleavage, which nicks and reseals one strand of DNA, is another direct cause of SSBs. Uncoupling of such breaks from topoisomerase I activity potentiates the cytotoxicity of a class of anticancer agents (e.g., camptothecins; ref. 10). In contrast, SSBs arise indirectly as a result of cellular responses to nucleobase damage. Damaged bases generated by chemical adducts, oxidation processes, cytosine deamination, and depurination are removed by the glycolytic enzymes of the base excision repair process. The resulting apurinic/apyrimidinic site attracts AP endonuclease (APE1/HAP1; refs. 11, 12), the diesterase activity of which cleaves the sugar phosphate backbone of DNA generating a SSB. If left unrepaired, SSBs can cause potentially lethal chromosomal double-strand breaks (DSB) during DNA replication and by chromosome segregation at mitosis. Mutations in certain SSB repair genes have been shown to be associated with human genetic diseases, particularly neurodegenerative disorders (2, 13).

2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosylcytosine (CNDAC) is a deoxycytidine analogue conceptualized as a mechanism-based DNA self-strand–breaking nucleoside (14–16). Chemical models and evidence from in vitro systems showed that ligation of the 3' hydroxyl of CNDAC, either chemically (14–17) or on extension with a deoxynucleotide (18), initiates a ß-elimination process. This results in rearrangement of the CNDAC molecule to form 2'-C-cyano-2',3'didehydro-2',3'-dideoxycytidine (CNddC), a de facto chain terminator that is the signature of this process. Recent studies have shown the formation of CNddC in the DNA of tumor cell lines exposed to CNDAC (19, 20) consistent with the direct formation of SSBs. Interestingly, this unique action of CNDAC was associated with an arrest of cell cycle progression in the G2 phase (20).

It remains a challenge to understand the mechanistic basis by which CNDAC-induced SSBs activate the G2 checkpoint. Our approach to this problem was to characterize the molecular pharmacology of G2 checkpoint activation by CNDAC in several hematologic malignancy lines. Because the G2 checkpoint is viewed as a resistance mechanism that could permit cells to repair potentially lethal damage, such as SSBs, we dysregulated the checkpoint pathway to allow cell cycle progression through mitosis, only after which apoptosis was activated. Subsequently, we identified signaling events involved in these processes and revealed chromosomal abnormalities typical of DSBs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. ML-1, a human acute myelogenous leukemia cell line containing wild-type p53, was a gift from Dr. Michael B. Kastan (St. Jude Children's Research Hospital, Memphis, TN). Cells were maintained in exponential growth phase in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. The population doubling time of ML-1 was ~22 to 24 hours. Other hematologic cell lines (CCRF-CEM, K562, Raji, and U937) were cultured under the same condition as ML-1. All cells were free of Mycoplasma as determined by an ELISA kit (Life Technologies MycoTest kit; Life Technologies, Grand Island, NY).

Chemicals and antibodies. The nucleoside analogue CNDAC was synthesized as described (14). A stock solution (15-25 mmol/L) was prepared in PBS (pH 6.5), sterilized by filtration, stored at –20°C, and diluted in sterile PBS just before use. UCN-01 (NSC 638850) was provided by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). Aliquots of UCN-01 were stored as 10 mmol/L in DMSO at –20°C and diluted in serum-free medium immediately before each experiment. All other chemicals were reagent grade.

Sources of antibodies are as follows: rabbit polyclonal antibodies against phospho-Ser317 and Ser345 of Chk1 (2344 and 2341), phospho-Thr68 of Chk2 (2661), phospho-Ser15 of p53 (9284), phospho-Thr180/Tyr182 of p38 MAP kinase (MAPK, 9211), p38 MAPK (9212), phospho-Ser216 of Cdc25C (9528), and phospho-Tyr15 of Cdk1 (9111; Cell Signaling Technology, Beverly, MA); mouse monoclonal antibody against p53 (OP43; Oncogene Research Products, Boston, MA); rabbit anti-Chk1 (sc-7898), mouse anti-Cdk1 (sc-54), mouse anti-p21 (6246) and mouse anti–cyclin B1 (sc-245; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal antibody to Chk2 (07-057), mouse monoclonal antibodies to Cdc25C (05-507) and phospho-Ser139 of H2AX (05-636; Upstate Biotechnology, Charlottesville, VA); mouse anti–poly(ADP-ribose) polymerase (PARP; 66391A) and rabbit anti-caspase-3 (557035; BD PharMingen International, San Diego, CA); mouse monoclonal antibodies to ß-actin (A1978) and phospho-Ser10 of histone H3 (H-6409; Sigma-Aldrich (St. Louis, MO); and anti-mouse or anti-rabbit IgG horseradish peroxidase (HRP)–conjugated antibody (Amersham Biosciences, Piscataway, NJ). MPM-2 antibody was a kind gift from Dr. Jian Kuang (University of Texas M.D. Anderson Cancer Center, Houston, TX).

Cell cycle analysis. Cells were washed with ice-cold PBS (pH 7.4) and fixed in 70% ethanol. Fixed cells were washed with PBS before incubation with 50 µg/mL propidium iodide (PI; Sigma-Aldrich) and 2.5 µg/mL DNase-free RNase A (Roche, Indianapolis, IN). Fluorescence was measured on a Becton Dickinson FACSCalibur flow cytometer (San Jose, CA). At least 20,000 cells were measured for each sample.

Terminal deoxynucleotidyl transferase–mediated nick-end labeling assay. To identify apoptotic cells, a flow cytometric terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) assay was done using a DeadEnd fluorometric TUNEL kit (Promega, Madison, WI) according to the manufacturer's protocol.

Immunofluorescent detection of phosphorylated histone H3. Phosphorylated histone H3 was immunostained according to the method of Xu et al. (21) with some modifications. Briefly, 70% ethanol-fixed cells were permeabilized with 0.5% Triton X-100 in PBS. After centrifugation, the cell pellet was incubated in 100 µL PBS containing 1% bovine serum albumin (BSA) and 7.5 µg of a monoclonal antibody specifically recognizing phospho-Ser10 on histone H3 at room temperature. After being washed with PBS containing 1% BSA, cells were incubated with FITC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted at a ratio of 1:60 in the dark. Washed again, the cells were incubated with PBS containing PI and RNase A before the fluorescence was measured by flow cytometry.

Quantification of mitotic index. After centrifugation of cells to slides by Cytospin (Thermo Electron Corp., Waltham, MA), cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) and mounted in Vectashield mounting medium with 1.5 µg/mL 4',6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA). Mitotic morphology was identified by the appearance of nuclear DNA condensation by epifluorescence microscopy (Nikon, Tokyo, Japan). At least 200 cells per field in a minimum of three randomly selected fields were counted on three slides for each sample.

Immunoblotting and immunoprecipitation. Cells pellets were washed with ice-cold PBS and lysed at 4°C by sonication in radioimmunoprecipitation assay buffer [RIPA; PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA (pH 8), 1 mmol/L sodium orthovanadate (pH 10), 50 mmol/L NaF, 10 mmol/L ß-glycerophosphate, 20 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL pepstatin, 2 mmol/L phenylmethylsulfonyl fluoride]. Lysates were centrifuged at 14,000 rpm for 10 minutes, and the supernatants were subjected to protein content determination using a detergent-compatible protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. An equal volume of 2x SDS sample loading buffer containing 100 mmol/L Tris-HCl (pH 6.8), 10% glycerol, 4% SDS, 0.05% bromophenol blue, and 5% ß-mercaptoethanol was added to cell lysates before heating at 95°C to 100°C for 5 minutes. Aliquots of 40 to 50 µg total cell protein were loaded onto 8% to 10% SDS-polyacrylamide gels (percentages depended on sizes of target proteins). Proteins were separated by electrophoresis at constant voltage (80-200 V) and electrotransferred to nitrocellulose membranes at 150 mA overnight at 4°C. Membranes were blocked at room temperature for 1 hour in TBS/0.05% Tween 20 (TBS-T buffer, pH 7.4) containing 5% nonfat dried milk and incubated with primary antibodies overnight at 4°C. Membranes were washed with TBS-T buffer thrice and incubated with secondary antibodies conjugated to HRP for 1 hour. After washing thrice, the blots were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Pierce, Rockford, IL).

Cdk1 immunocomplexes were immunoprecipitated with Cdk1 antibody from cell lysates prepared in TS buffer [50 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl] containing protease and phosphatase inhibitors at the same concentrations as in RIPA. Immunoprecipitates were washed with TS buffer containing 0.1% Tween 20 thrice and boiled in 2x SDS sample loading buffer. Supernatants were subjected to gel separation and immunoblotting as described above.

Cytogenetic analysis. Colcemid (100 ng/mL) was added to cell cultures 2 hours before harvesting. Cells were resuspended and swollen in 0.075 mol/L KCl at 37°C for 10 minutes before being fixed in fresh methanol/acetic acid (3:1, v/v) at room temperature for 20 minutes. Following three washes with ice-cold methanol/acetic acid (3:1, v/v), fixed cells were spread onto Superfrost microscopic slides (Erie Scientific, Portsmouth, NH) and air-dried overnight. Spreads were stained with 4% (v/v) Giemsa (Karyomax Giemsa stock solution, Invitrogen Corp., Carlsbad, CA) freshly prepared in Gurr buffer (Invitrogen) at room temperature for 20 minutes. Following rinsing with Millipore water, stained slides were air-dried overnight before mounting using Eukitt Histomount (Zymed Laboratories, Inc., South San Francisco, CA). All slides were blinded before quantitation of chromosomal aberrations (including gaps and breaks) by microscopic examination. At least 60 metaphase spreads were analyzed on three separate slides for each sample. For each treatment, variations between slides were not remarkable as determined by statistical assays (data not shown).

Confocal microscopy of immunofluorescent {gamma}-H2AX. Drug-treated ML-1 cells were centrifuged onto slides, fixed in 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. After blocking with PBS containing 6% goat serum (ICN Biochemicals, Inc., Irvine, CA), cells were incubated with {gamma}-H2AX (phospho-Ser139) antibody at 500-fold dilution in 3% goat serum in PBS for 2 hours followed by three washes with PBS containing 0.1% NP40. Then, cells were incubated for 1 hour with a FITC-conjugated goat anti-mouse IgG antibody diluted at a ratio of 1:100 followed by three washes. Cell nuclei were stained in PBS with PI and RNase. The slides were mounted with mounting medium Vectashield (Vector Laboratories) and sealed with nail polish. {gamma}-H2AX and nuclear staining were viewed with a LSM 510 laser scanning confocal microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) using a 63x objective. The projections of multiple slices were saved as TIF files.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
G2 arrest induced by 2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosylcytosine. Earlier investigations developed a model for CNDAC-induced G2 arrest in exponentially growing ML-1 cells (20). To evaluate the prevalence of this action, we tested the response of nine additional cell lines from diverse sources to a range of CNDAC concentrations for at least a population doubling time (Fig. 1A). Although the percentages of cells with a G2-M DNA content in exponentially growing populations varied 2-fold (14-29%) among the cell lines, CNDAC more than doubled the G2-M phase population in most lines. The cell line–dependent differences in the G2-arresting concentrations, which were minimally toxic as judged by lack of cells with a sub-G1 DNA content, may reflect differing capacities to metabolize the analogue or unequal thresholds for activating the G2 checkpoint.



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  		Figure 1. Actions of CNDAC in human cell lines. G2 arrest caused by CNDAC in diverse human cell lines. A, effect of CNDAC on cell cycle distribution is concentration and time dependent. ML-1 cells were treated with (B) various concentrations of CNDAC for 24 hours or (C) 2 µmol/L CNDAC for 3 days. Cell cycle distribution (x, sub-G1; {blacktriangleup}, G1; {circ}, S; {blacksquare}, G2-M) was determined by flow cytometry.

 
To characterize the CNDAC-induced G2 arrest in greater detail, we focused our investigations on the concentration-time response in exponentially growing ML-1 cells. The maximum G2 arrest after a 24-hour incubation occurred with 2 µmol/L CNDAC (Fig. 1B). Concentrations of <1 µmol/L failed to exert a profound G2 arrest, whereas concentrations exceeding 4 µmol/L were toxic as judged by >10% cells with a sub-G1 DNA content. To determine the time dependency of CNDAC-induced G2-M arrest, ML-1 cells were exposed to various concentrations of CNDAC for 3 days (Fig. 1C). Following a transient S-phase delay 12 hours after treatment with 2 µmol/L CNDAC, the population with a G2-M DNA content peaked at 24 hours. In fact, the low incidence of mitotic figures, minor level of histone H3 phosphorylation, and absence of MPM-2 epitope phosphorylation strongly indicated that these cells were arrested in G2 phase (see below). By 48 hours, the duration of two normal population doublings, there was a decrease in G2-M cells, which was mirrored by an increase in the population with a sub-G1 DNA content. Thus, the G2 arrest was considerably maintained (~3-fold relative to nontreated cells) for as long as 72 hours by 2 µmol/L CNDAC. Similar concentration and time dependencies in CNDAC-induced G2 arrest were also observed in CCRF-CEM T-cell lymphoblasts, Raji B-cell lymphoma, and U937 histiocytic lymphoma (Supplementary Fig. S1).

2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosylcytosine activates the Chk1-Cdc25C-Cdk1/cyclin B1 checkpoint signaling pathway. To determine the mechanism of CNDAC-induced G2 arrest, we first investigated the checkpoint pathway (22–24) activated by other cell cycle active agents, such as topoisomerase I poisons (25–29), cisplatin, and ionizing radiation. On incubation of ML-1 cells with 2 µmol/L CNDAC, the DNA damage kinase Chk1 became phosphorylated on both Ser317 and Ser345, indicating its activation by upstream sensors (Fig. 2). This was associated with phosphorylation of Chk1 target protein Cdc25C on Ser216, consistent with the inhibitory regulation of its phosphatase activity (23, 30). Thus, Cdk1 accumulated in a Tyr15-phosphorylated state. This was accompanied by an increase in the cyclin B1 level as cells accumulated in G2. These changes are consistent with G2 checkpoint activation. In addition, phosphorylation of Ser15 on p53 was evident 12 hours after CNDAC treatment. This was associated with an increase in total cellular p21 by 24 hours, an event that could contribute to the G2 arrest (see below). In contrast to Chk1, the phosphorylation of another DNA damage checkpoint kinase, Chk2 on Thr68, was not significant until 48 hours after CNDAC incubation. This indicated that Chk2 activation is not a major participant in transducing DNA damage signals generated by CNDAC. The mitogen-activated protein kinase p38, which has been shown to participate in activation of the G2 checkpoint in response to UV (31), is constitutively activated in ML-1 cells. However, there were no apparent changes in the level of this protein or its phosphorylation status during a 24-hour incubation with CNDAC (Supplementary Fig. S2, 12 and 24 hours), suggesting that it is not involved in the G2 checkpoint activation.



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  		Figure 2. Chk1 but not Chk2 is the DNA damage checkpoint kinase responsible for activation of the G2 checkpoint in response to CNDAC. Exponentially growing ML-1 cells were incubated with 2 µmol/L CNDAC, and the G2 arrest was maintained for as long as 3 days. Equal amounts of cell lysate proteins were subjected to immunoblot analysis with indicated antibodies. ß-Actin was used for a loading control.

 
7-Hydroxystaurosporine abrogates 2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosylcytosine–induced G2 checkpoint and triggers apoptosis. Arrest in G2 might be a protective mechanism that may allow cells to repair damage to their DNA (26). To overcome this potential defense mechanism, we sought to dysregulate this G2 checkpoint with the Chk1 kinase inhibitor, 7-hydroxystaurosporine (UCN-01; refs. 32–34). The population of cells in G2 increased ~4-fold during a 24-hour incubation with 2 µmol/L CNDAC, a distribution that remained stable through 36 hours (Supplementary Fig. S3). When 100 nmol/L UCN-01 was added, the G2 population (53%) declined rapidly within 3 hours to the level of untreated cells (15%) and was <5% by 6 hours (Fig. 3). This was accompanied by a transient increase in cells with a G1 DNA content, which peaked at 6 hours after UCN-01 addition, followed by a precipitous decline. This decrease in the G1 population was mirrored by the initiation of apoptosis as indicated by the increase in the sub-G1 population 6 hours after UCN-01 addition. The kinetics of the apoptotic process measured by the TUNEL assay positivity also reflected the decline of the G1 population (Fig. 3). Similar abrogation of CNDAC-induced G2 arrest by UCN-01 was observed in several other cell lines (Supplementary Table S1; Supplementary Fig. S3). In contrast, treatment of ML-1 cells with 100 nmol/L UCN-01 alone had no discernible effect on population doubling time or cell cycle distribution (data not shown).



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  		Figure 3. UCN-01 abrogates CNDAC-induced G2 arrest and triggers apoptosis. ML-1 cells were incubated with 2 µmol/L CNDAC continuously for 36 hours ({circ}). At 24 hours, the culture was split and 100 nmol/L UCN-01 was added (arrow) to one portion ({bullet}). Samples were collected at the indicated times and analyzed by flow cytometry alone or combined with TUNEL assay.

 
7-Hydroxystaurosporine dysregulated the G2 checkpoint pathway. Molecular pharmacology studies of G2 checkpoint proteins indicated that addition of UCN-01 to CNDAC-arrested cells resulted in the rapid loss of phospho-Ser216 Cdc25C, the direct substrate of Chk1, suggesting that the activity of Chk1 kinase had been inhibited (Fig. 4A). Concomitant with this was the loss of the Tyr15-phosphorylated form of Cdk1 (Fig. 4B), indicating a biochemical basis for checkpoint abrogation characterized by the observed progression of G2-arrested cells. That Chk1 became even more intensely phosphorylated on Ser317 and Ser345 at 6 and 9 hours after UCN-01 addition (Fig. 4A) was likely caused by DNA damage as cells began the apoptotic process. This may reflect the activation of a sensor that responds to a form of DNA damage other than that eliciting G2 arrest. In a separate study, exposure of ML-1 cells to 100 nmol/L UCN-01 alone for 6 hours led to a mild increase in the phosphorylated forms of Chk1 (data not shown), suggesting a possible role of upstream sensor(s) activated in response to UCN-01 directly or indirectly. Nevertheless, because UCN-01 occupies the ATP-binding pocket of Chk1 (35), the kinase activity is suppressed although the Chk1 protein is in a superphosphorylated status. Evaluation of the status of Chk2 (Fig. 4C) indicated that it was not activated by phosphorylation on Thr68 during G2 arrest induced by a 24-hour treatment with CNDAC, but this site was phosphorylated 3 hours after UCN-01 addition to CNDAC-arrested cells. That this became more intense at 6 to 12 hours is another indication of the activation of upstream sensors of DNA damage as cells initiated apoptosis.



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  		Figure 4. Changes in DNA damage response proteins caused by CNDAC alone and in combination with UCN-01. Exponentially growing ML-1 cells were incubated with 2 µmol/L CNDAC for 24 hours before the cells were split and 100 nmol/L UCN-01 was added to one portion. Cell lysates were prepared at indicated times. A, response of Chk1 kinase and Cdc25C phosphatase. Equal amounts of protein lysates were fractionated by SDS-PAGE and subjected to immunoblot analysis with indicated antibodies. ß-Actin was used for a loading control. B, response of Cdk1 kinase. Immunoprecipitates of Cdk1 were subjected to immunoblotting with antibody against phospho-Tyr15 on Cdk1. The relative level of phosphorylated Cdk1 was normalized to that of total Cdk1. Arrows, selective diminution of Tyr15-phosphorylated Cdk1. Responses: Chk2 kinase (C); p53, p21, and cell death pathway (D). C and D, cell lysates were processed as in (A), and blots were probed with antibodies as indicated.

 
The biochemistry of apoptosis was seen by the presence of cleaved (activated) caspase-3 and cleavage of PARP 6 hours after UCN-01 addition (Fig. 4D). Concomitant with this, p53 phosphorylation on Ser15 became more intense after UCN-01 addition, coincident with the initiation of apoptosis (Fig. 4D). In addition, p21 was increasingly expressed in association with p53 phosphorylation and transactivation during the CNDAC-alone phase of G2 arrest (Fig. 4D). This was not evident in cells lacking p53 (data not shown). Following UCN-01 addition and the activation of apoptosis at 6 hours, cellular p21 levels decreased rapidly, which is likely due to cleavage by caspase-3 (36–38).

G2 arrest–abrogated cells progress through mitosis before the onset of apoptosis. Within 3 hours after UCN-01 addition, approximately two thirds of the CNDAC-arrested ML-1 cells progressed out of G2 (Fig. 3). To better characterize this process, we investigated changes in cellular and molecular levels of several well-defined mitotic markers. That cells were arrested in G2 rather than M phase before the addition of UCN-01 was indicated by the lack of mitoses as determined by scoring of mitotic figures (Fig. 5A) and the absence of histone H3 phosphorylation (Fig. 5B) in cells before UCN-01 addition. However, shortly after the addition of UCN-01 to the CNDAC-treated cells, there was a coincident wave of cells entering mitosis and that with histone H3 phosphorylation on Ser10 as well as that with MPM-2 epitope phosphorylation (Fig. 5C; refs. 39, 40), indicating that cells were arrested in late G2 phase and underwent a level of synchronous cell division without activating a mitotic checkpoint. These mitotic markers peaked 1 to 1.5 hours after UCN-01 addition, which explains the 1.5-hour lag before the sharp decrease in cells with a G2-M DNA content (Fig. 3).



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  		Figure 5. CNDAC-arrested ML-1 cells progressed through mitosis after UCN-01 addition. Exponentially growing ML-1 cells were incubated with 2 µmol/L CNDAC for 24 hours before the cells were split and 100 nmol/L UCN-01 was added to one portion. Cells were fixed at indicated times and subjected to scoring of mitotic figures (A) and immunostaining of Ser10-phosphorylated histone H3 coupled with flow cytometric analysis (B), respectively. C, cell lysates were collected at counterpart time points and immunoblotted for the MPM-2 epitopes. Nocodazole (400 ng/mL) and colcemid (100 ng/mL) were used as positive controls.

 
2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosylcytosine causes double-strand breaks in ML-1 cells. After incorporation into DNA by ML-1 cells, CNDAC causes SSBs through ß-elimination (20). However, we found by fluorescent immunostaining that phospho-Ser139 H2AX ({gamma}-H2AX) formed discrete nuclear foci in ML-1 cells after a 24-hour exposure to CNDAC, suggesting the formation of DSBs (Fig. 6A). There was little change in the intensity of {gamma}-H2AX foci 1 hour after UCN-01 addition into CNDAC-arrested cells (data not shown); the foci became significantly more intense after 9 hours. Those cells with intense, large nuclear bodies were undergoing apoptosis as judged by their nuclear morphology (Fig. 6A). This time course is consistent with cell cycle distribution and biochemistry of DNA damage response proteins (Figs. 3 and 4). Immunoblotting of ML-1 lysates showed a steady increase in {gamma}-H2AX level during 36 hours of CNDAC treatment (Fig. 6B). However, addition of UCN-01 to CNDAC-arrested cells resulted in greater elevation in the {gamma}-H2AX level compared with CNDAC alone. Thus, it seems that the change in {gamma}-H2AX protein level is consistent with that of the immunocytochemistry. {gamma}-H2AX has been regarded as a sensitive marker for DNA DSBs (41), which responds rapidly in cells treated with genotoxic stress, such as ionizing radiation. Based on our observation of nuclear foci formation, together with the increased protein level of {gamma}-H2AX in response to CNDAC, we hypothesized that CNDAC treatment may in fact lead to formation of DSBs.



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  		Figure 6. Histone H2AX phosphorylation and chromosomal aberrations caused by CNDAC or addition of UCN-01 to CNDAC-arrested cells. Exponentially growing ML-1 cells were incubated with 2 µmol/L CNDAC for 24 hours before the cells were split and 100 nmol/L UCN-01 was added to one portion. A, cells were fixed at indicated times and subjected to fluorescent staining of Ser139-phosphorylated H2AX ({gamma}-H2AX). {gamma}-H2AX was colored in green and nuclear staining with PI in red. Representative images taken by confocal microscopy are shown for the green channel alone and merged with the red channel. B, cell lysates were collected at indicated time points and analyzed by immunoblotting with antibodies. C, exponentially growing ML-1 cells (control, I) were treated with 100 nmol/L UCN-01 for 2 hours either alone (II) or following 24-hour incubation with 2 µmol/L CNDAC (III). Colcemid (100 ng/mL) was added 2 hours (I-III) before preparation of metaphase chromosome spreads. In cells treated with CNDAC alone (IV), colcemid was introduced 12 hours after addition of CNDAC and coincubated for additional 12 hours. Representative images of I-IV. Long arrows and short arrows, breaks and gaps in chromosomes, respectively. D, quantitation of chromosomal aberrations in ML-1 cells incubated with 2 µmol/L CNDAC for 24 hours ± 100 nmol/L UCN-01 for 2 hours.

 
To test this hypothesis, we did cytogenetic analysis on ML-1 cells collected in metaphase with colcemid. Representative photographs of mitotic spreads are shown in Fig. 6C. These cells have ~90 chromosomes per cell (Fig. 6D), indicating that ML-1 cells are pseudotetraploid. As we showed above, CNDAC-arrested cells progressed through mitosis within 1.5 hours after addition of UCN-01. This provides an opportune time for visualizing existing abnormalities in chromosomes. We treated CNDAC-arrested cells with UCN-01 and colcemid for 2 hours to enrich mitotic cells. By scoring 104 well-resolved metaphases, we detected an average of 5.82 chromatid type aberrations per cell, including gaps and breaks (Fig. 6D). In contrast, there were very few aberrations found in 167 metaphases of control (mock-treated) cells (average, 0.06 per cell). No aberrations were detected in 61 metaphases of cells treated with UCN-01 alone for 2 hours. Among these treatments, there was no significant difference in chromosome numbers per cell. Because of the slow rate of escape from G2 arrest, reflecting the stringent nature of the checkpoint, the actions of CNDAC alone on chromosomal integrity were evaluated by incubating cells with 2 µmol/L CNDAC for 12 hours, when colcemid was added for an additional 12 hours to accumulate mitotic cells (Fig. 6D). Chromosomal aberrations were evident (1.42 per cell) although significantly less than in cells treated with the combination. Thus, CNDAC alone and in combination with UCN-01 was associated with a significantly larger number of chromosomal abnormalities (i.e., DNA DSBs) relative to controls or cells treated with UCN-01 alone. More than 97% of cells treated with CNDAC and UCN-01 had aberrations (Supplementary Fig. S4), with ~80% having more than two (Fig. 6D). More than 70% of cells incubated with CNDAC alone had aberrations but <20% of those exhibited more than two.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies have provided a more comprehensive understanding of cellular responses to the actions of CNDAC. CNDAC-induced activation of the G2 checkpoint pathway is an action that is common among cells lines from diverse tissue origins (Fig. 1A). This blockade of cell cycle transit is relatively sustained, as it was observed to hold over several population doubling times without inducing cell death (Fig. 1; Supplementary Fig. S1). Molecular pharmacology studies in ML-1 cells indicated an activation of the Chk1-Cdc25C-Cdk1/cyclin B1 pathway. Although p53 became phosphorylated and expression of p21 increased during the onset of the checkpoint, cells lacking p53 function (CCRF-CEM, Raji, and U937) also arrested in response to CNDAC (Fig. 1A; Supplementary Table S1; Supplementary Fig. S3), suggesting that these proteins are not required in the cellular response. Finally, there was no change in the phosphorylation status of either Chk2 kinase (Fig. 4C) or p38 (Supplementary Fig. S2) under conditions that caused G2 arrest. The phosphorylation of H2AX by one or more of the phosphatidylinositol 3-kinases, ATM, ATR, or DNA-PK, is well documented as a response to DSBs (42–49), although recent evidence suggests that it may also be activated by unusual chromosomal configurations as well (50). It may be assumed that one or more of the phosphatidylinositol 3-kinases activate the elements of the G2 checkpoint pathway.

The generation of CNDAC-induced DNA damage and its nature is likely to depend on several factors. First, the rate of ß-elimination that generates SSBs following CNDAC nucleotide incorporation is likely a stochastic process. This is predicted by the simultaneous presence of CNDAC in 5'-3' phosphodiester linkage in DNA as well as the ß-elimination product CNddC at the 3' termini (20). Second, the amount of analogue incorporated into DNA is related to the duration of exposure to each cell during its transit through the S phase. This may account for why only a portion of the population became positive for {gamma}-H2AX (Fig. 6A). Third, studies in model systems showed that CNDAC incorporation blocks primer extension (51), and some transient accumulation of CNDAC-treated cells in the S phase was evident (Supplementary Fig. S3). Thus, CNDAC incorporation may in part cause replication fork stalling, giving rise to structures that activate phosphorylation of H2AX. Finally, attempts to repair the SSBs generated by this de facto chain terminator may result in processing errors that generate DSBs. Clearly, additional investigations will be required to determine the nature of the CNDAC-induced DNA damage, the proteins that respond to repair damage, and those that activate the G2 checkpoint pathway.

Inhibition of Chk1 with UCN-01 rapidly reversed the inhibitory phosphorylations on Ser216 on Cdc25C and Tyr15 Cdk1, consistent with abrogation of the checkpoint pathway and cell cycle progression in several hematologic cell lines. Although UCN-01 is not entirely specific for Chk1, new compounds with greater Chk1 specificity gave similar results (52). Although p21 levels became depleted, this was only evident 6 hours following UCN-01 addition when most cells had moved through mitosis, suggesting it may be secondary to the activation of caspases (Fig. 4D; refs. 36–38). The onset of cell death several hours after checkpoint abrogation was evident in several cell lines, which was associated with such markers of apoptosis as sub-G1 DNA content, TUNEL positivity, caspase-3 activation, and PARP cleavage. The precise signaling mechanisms that generate these responses remain to be elucidated.

Considering that UCN-01 alone did not contribute to the generation of chromosome aberrations and that {gamma}-H2AX was positively stained and increased with time in ML-1 cells treated with CNDAC alone, we conclude that the DNA DSBs are solely induced by cellular responses to CNDAC incorporation into DNA. On the other hand, as reflected by the fact that cells treated with CNDAC alone presented far fewer aberrations than those treated with the combination, the G2 checkpoint activated by CNDAC does seem to serve as a defense mechanism against such potentially lethal DNA damage. Abrogation of the G2 checkpoint ablated the survival opportunity of these cells and therefore enhanced cell death. However, because only 37 of 127 cells scored lacked aberrations, excluding damage not visualized by this assay, cells entering mitosis with chromosomal aberrations will likely be captured by the G1 checkpoint and destined to apoptosis.

In summary, incorporation of CNDAC triphosphate into DNA and extension during replication leads to SSBs directly caused by ß-elimination. These breaks, or the lesions that arise from further processing, initiate signals that activate the G2 checkpoint pathway. This occurs in cell lines derived from diverse human tissues and therefore is likely to be a common response mechanism to the damage created by CNDAC. Abrogation of the checkpoint pathway by inhibiting Chk1 kinase permits cells to progress through mitosis and into G1. DNA damage initiated by ß-elimination-induced SSBs is transformed into DSBs as cells are arrested in G2. Some of these breaks manifest as chromosomal aberrations when the G2 checkpoint of CNDAC-arrested cells is abrogated by UCN-01 but also in a minor population of cells that escapes to mitosis during treatment with CNDAC alone. This damage most likely generates signals for cell death in hematologic malignancies, the consequences of which are first evident only after cells progress through mitosis to G1. Thus, these findings illustrate a portion of the mechanism by which increased tumor cell killing may be achieved by a therapeutic strategy of checkpoint dysregulation.


   Acknowledgments
 
Grant support: National Cancer Institute, Department of Health and Human Services grants CA28596 and CA55164 and Cancer Center support grant P30 CA16672 and Sankyo Co. Ltd. (Tokyo, Japan).

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Y. Li is currently at the Department of Radiation Oncology, Cancer Hospital, Chinese Academy of Medical Sciences, Beijing 100021, People's Republic of China.

Received 1/28/05. Revised 4/ 8/05. Accepted 5/17/05.


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 Introduction
 Materials and Methods
 Results
 Discussion
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