Arsenite induces prominent mitotic arrest via inhibition of G2 checkpoint activation in CGL-2 cells
Ling-Huei Yih1,4,
Shun-Wen Hsueh1,
Wei-Shu Luu3,
Ted H. Chiu3 and
Te-Chang Lee2
1 Institute of Zoology and 2 Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China and 3 Institute of Pharmacology and Toxicology, Tzu Chi University, Hualien 970, Taiwan, Republic of China
4 To whom correspondence should be addressed Email: lhyih{at}gate.sinica.edu.tw
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Abstract
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Arsenic compounds, which are well-documented human carcinogens, are now used in cancer therapy. Knowledge of the mechanism by which arsenic exerts its toxicity may help in designing a more effective regimen for therapy. In this study, we showed that arsenite could induce prominent mitotic arrest in CGL-2 cells and demonstrated the presence of damaged DNA in arsenite-arrested mitotic cells. We then explored why these cells with arsenite-induced DNA damage were arrested at mitosis instead of G2 stage. When synchronized CGL-2 cells were treated with arsenite at stage G1, S or G2, all progressed into, and arrested at, the mitotic stage and contained damaged DNA, as demonstrated by the appearance of the DNA double-strand break marker, phosphorylated histone H2A.X (
-H2AX). Since X-irradiation induced G2 arrest in CGL-2 cells, these cells clearly have a functional G2 DNA damage checkpoint. However, treatment of X-irradiated CGL-2 cells with arsenite resulted in a decrease in G2 cells and an increase in mitotic cells, suggesting that arsenite may inhibit activation of the G2 DNA damage checkpoint and thus allow cells with damaged DNA to proceed from G2 into mitosis. Immunoblot analysis confirmed that arsenite treatment reduced the X-irradiation-induced phosphorylation of both ataxia-telangiectasia, mutated at serine 1981 and Cdc25C at serine 216, events which are crucial for G2 checkpoint activation and G2 arrest. Moreover, a higher frequency of apoptotic cells is observed in mitoticCGL-2 cells arrested by arsenite than those arrested by nocodazole or taxol. Our results show that the combined effects of arsenite in inducing DNA damages, inhibiting the activation of G2 checkpoint, and arresting cells with damaged DNA in the mitotic stage may subsequently enhance the induction of apoptosis in arsenite-arrested mitotic CGL-2 cells.
Abbreviations: APL, acute promyelocytic leukemia; ATM, ataxia-telangiectasia, mutated; Chk-1, checkpoint kinase 1; Chk-2, checkpoint kinase 2; FITC, fluorescein isothiocyanate;
-H2AX, phosphorylated histone H2A.X; PBS, phosphate-buffered saline; SDSPAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis
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Introduction
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Inorganic arsenite, arsenic trioxide, induces complete remission in a high proportion of patients with refractory acute promyelocytic leukemia (APL) (13) and has been approved for treatment of relapsed or refractory APL by the US Food and Drug Administration. The fact that arsenic trioxide is effective in patients refractory to conventional chemotherapy suggests that arsenic kills cancer cells via mechanisms different from those of conventional anticancer drugs. It is generally accepted that arsenic trioxide cures APL by induction of apoptosis (4,5). Recently, it was shown that inorganic arsenite could also induce apoptosis in a variety of solid tumor cells (69). However, it is not yet known how these cancer cells respond to arsenite and what determines their susceptibility.
The mechanism by which arsenic triggers apoptosis remains controversial. There is evidence that it down-regulates Bcl-2 protein and activates caspase-3-like caspase activity (4). Some reports have shown that arsenic-induced apoptosis is caused by a direct effect on the mitochondrial permeability transition pore, resulting in loss of the mitochondrial transmembrane potential (5). Other studies have demonstrated that arsenic compounds can disrupt mitosis and therefore induce apoptosis in a variety of cell systems (7,1013). The disruption of mitosis was shown to be due to interference with tubulin polymerization and disruption of mitotic spindles (7,10,14).
Mitotic abnormalities often arise directly from defects of centrosomes and/or mitotic spindles, which then induce prolonged mitotic arrest or delayed mitotic exit and trigger induction of apoptosis (15,16). Recent reports have demonstrated that entry into mitosis in the presence of damaged DNA leads to inactivation of centrosomes, formation of aberrant spindles and blockage of chromosome segregation, which consequently delays mitosis progression and induces mitotic abnormalities (17,18). In addition, chemical or pharmacological inhibition of the DNA damage checkpoint at the G2 stage induces premature entry into mitosis and subsequent initiation of apoptosis (19). However, the mechanism linking perturbation of the cell cycle to initiation of apoptosis is not fully understood.
In our previous study, we demonstrated that arsenite induces apoptosis in HeLa cells by arresting cells at the mitotic stage (11). In the present study, we demonstrated that arsenite induced more prominent mitotic arrest in CGL-2 cells than in other cell lines tested. Arsenite-arrested mitoticCGL-2 cells were also more susceptible to induction of apoptosis. CGL-2 cells, a fusion hybrid of the HeLa adenocarcinoma cell line and normal human fibroblasts, originally established for the analysis of transformation and tumorigenicity, are chromosomally stable and behave as transformed cells in culture, but their tumorigenic potential is suppressed (20). This high susceptibility of CGL-2 cells to arsenite-induced mitotic arrest and apoptosis provides an ideal model for studying the processes coupling arsenite induction of mitotic abnormalities to the onset of apoptosis. Elucidation of these processes might reveal sites of potential intervention for the use of arsenic in cancer chemotherapy.
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Materials and methods
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Cell culture
The CGL-2 cell line (21), derived from a hybrid (ESH5) of the HeLa variant, D98/AH2, and a normal human fibroblast strain, GM77, was kindly provided by Dr E.J.Stanbridge (University of California-Irvine). HeLa S3 cells were obtained from American Type Culture Collection (Manassas, VA). These cells were routinely maintained in Dulbecco's modified Eagle medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 0.37% sodium bicarbonate (GIBCO), 100 U/ml of penicillin (GIBCO), and 100 µg/ml of streptomycin (GIBCO) at 37°C in an humidified incubator in air and 10% CO2. The cells were routinely passaged twice per week.
Drug treatment
Logarithmically growing CGL-2 cells were treated for 24 h at 37°C with 110 µM arsenite, 0.010.1 µM nocodazole or 0.010.1 µM taxol, then harvested and analyzed. In most experiments, all the cells were used, but, in experiments in which floating and adherent cells were analyzed separately, the culture dishes were gently shaken and cells floating in the culture medium were collected by centrifugation at 800 g for 5 min. The remaining attached cells were collected by trypsinization.
X-irradiation
Logarithmically growing CGL-2 cells were irradiated at room temperature in complete medium using a Torrex 150D X-ray machine (EG&G, Astrophysics Research, Long Beach, CA) operating at 134 kV and 4 mA with a 1.2 mm beryllium filter at a dose rate of 6 Gy/min. A calibrated in-field ionizing monitor was used during each irradiation to ensure the accuracy of thedelivered dose.
Cytotoxicity assay
Logarithmically growing CGL-2 cells were treated for 24 h at 37°C with various concentrations of arsenite. The relative survival rate was then determined using a colony-forming assay as described previously (22). In brief, after arsenite treatment, the cells were collected, counted, serially diluted and seeded in triplicate at a density of 2002000 cells/dish in 60-mm Petri dishes and incubated for 10 days. Colonies were visualized and counted after fixing with 70% ethanol and staining with 3% Giemsa solution (Merck, Darmstadt, Germany). The plating efficiency of each treatment was calculated by dividing the number of colonies on each plate by the number of cells seeded. The relative survival rate was determined by normalization of the plating efficiency for each treatment with that for the untreated control.
Analysis of cell-cycle progression and mitotic index
Cell-cycle progression was monitored using DNA flow cytometry. In brief, the cells were trypsinized, washed once with phosphate-buffered saline (PBS), fixed with ice-cold 70% ethanol for 16 h, and stained with 4 µg/ml of propidium iodide in PBS containing 1% Triton X-100 and 0.1 mg/ml of RNase A. The DNA content of individual cells was analyzed using a fluorescence activated cell sorter (FACSTAR, Becton Dickinson Immunocytometry Systems), and the cell-cycle distribution of the cells determined using a computer program provided by Becton Dickinson Immunocytometry System as described previously (22). The cell suspension was then observed under a fluorescence microscope (Axioskop 2, Zeiss, Germany) to determine the mitotic index; at least 500 cells were scored in each sample and three independent measurements were performed to determine the mitotic index.
Synchronization of CGL-2 cells
The synchronization of G1-enriched cells was achieved by the protocol of double-thymidine block (23). Logarithmically growing CGL-2 cells at 50% confluence were treated with 2 mM thymidine for 12 h, switched to thymidine-free medium for 12 h, then again treated with 2 mM thymidine for 12 h. At this point, the majority of the cells were at the G1/S boundary. They were then allowed to progress forward by switching them to thymidine-free medium, and the cell-cycle progression was monitored at 24 h intervals using a DNA flow cytometer as described above.
Immunofluorescence staining of
-H2AX
CGL-2 cells seeded on glass coverslips were incubated for 24 h at 37°C with or without 2 µM arsenite, washed twice with PBS, and then fixed in situ with 90% methanol at 20°C for 10 min. The coverslips were again washed twice with PBS and incubated for 1 h at 37°C with anti-
-H2AX antibody (Upstate USA, Charlottesville, VA), then non-bound antibody was removed by extensive washing with PBS containing 0.2% Tween (PBST). The coverslips were then incubated for 30 min at 37°C in the dark with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Organon Teknika-Cappel, Belgium), and nuclei were simultaneously counterstained with 0.1 µg/ml of4,6-diamino-2-phenyl-indole (DAPI) (Sigma, St Louis, MO). After thorough rinsing with PBST, the coverslips were mounted with a 90% glycerol solution containing 1 mg/ml of phenylenediamine (pH 8.0, Merck, Gibbstown, NJ) and the
-H2AX foci in cells examined under a fluorescence microscope (Axioskop 2, Zeiss, Germany).
Immunoblots
Levels of cyclin B, Pds1, phospho-histone H3, phospho-Cdc25C (serine 216), total ataxia-telangiectasia, mutated (ATM) and phospho-ATM (serine 1981) proteins in total cell extracts were examined by western blotting as described previously (22). Briefly, CGL-2 cells after treatment were washed twice with ice-cold PBS, scraped off, collected in a 1.5 ml vial, and boiled in SDSPAGE sample buffer (24). Samples of cellular proteins (2050 µg) were resolved by 6 or 12% SDSPAGE, transferred onto polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ), and probed with the appropriate antibodies. ß-Actin was used as a loading control. The results shown are representative of at least two independent experiments. Rabbit polyclonal anti-cyclin B1 and goat polyclonal anti-ATM and anti-ß-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), mouse monoclonal anti-Pds1 antibody from NeoMarkers (Fremont, CA), rabbit polyclonal anti-phospho-H2AX (serine 139) antibodies from Upstate Biotechnology (Waltham, MA), mouse monoclonal anti-phospho-histone H3 (serine 10) antibody and rabbit polyclonal anti-phospho-Cdc25C (serine 216) antibodies from Cell Signaling Technology (Beverly, MA), and rabbit polyclonal anti-phospho-ATM (serine 1981) antibodies from Rockland (Gibertsville, PA). Protein concentrations were determined by Bradford analysis (25).
Apoptosis assay
The number of apoptotic cells was determined using an Annexin VFITC apoptosis detection kit (Oncogene, Boston, MA) as described previously (11). The cells were trypsinized, washed once with PBS, and resuspended in 100 µl of binding buffer containing 5 µl of 200 mg/ml FITC-conjugated Annexin V and 5 µl of 30 mg/ml propidium iodide. After a 10-min incubation at room temperature, the cells were examined under a fluorescence microscope (Axioskop 2, Zeiss, Germany). At least 500 cells were scored in each sample, and at least three independent measurements were performed to determine the percentage of apoptotic cells.
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Results
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Induction of prominent mitotic arrest by arsenite in CGL-2 cells
When logarithmically growing CGL-2 cells were treated for 24 h with 010 µM of arsenite, a dose-dependent increase in the G2/M cell population was seen by flow cytometric analysis over the concentration range of 04 µM arsenite, followed by a decrease at the concentration above 5 µM (Figure 1A). Microscopic examination confirmed that the increased G2/M population was predominantly mitotic cells (Figure 1A). As >50% of the cells were arrested in the mitotic stage by treatment with 25 µM arsenite for 24 h, the mitotic arrest induced by arsenite in CGL-2 cells was clearly more prominent than that reported previously in other cell types (1535% of cells arrested) (7,10,1214,22). Arsenite also induced cytotoxicity in CGL-2 cells in a dose-dependent manner as assessed by a colony-forming assay (Figure 1B). The level of mitotic arrest was inversely correlated with the relative survival rate in CGL-2 cells treated with 05 µM arsenite (Figure 1C), indicating that mitotic arrest was associated with arsenite-induced cell death. As mitotic cells are rounded-up, they can be separated from the attached interface cells by the shake-off technique. When the two cell populations were separated in this way from CGL-2 cells treated with 2 µM arsenite, the relative survival rates of the mitotic cells (filled square) and the remaining attached cells (empty square) were 4 and 96%, respectively, as compared with untreated cycling cells (Figure 1B). These results showed that arsenite induced mitosis-mediated cell death in CGL-2 cells.

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Fig. 1. Induction of prominent mitotic arrest and cytotoxicity in arsenite-treated CGL-2 cells. CGL-2 cells were treated with arsenite at the indicated concentration for 24 h, then total cells were analyzed for both cell-cycle distribution and cytotoxicity. (A) Concentration dependence of the cell-cycle distribution in arsenite-treated cells. (B) Concentration dependence of arsenite cytotoxicity. Clear and filled bars indicate the percentage of surviving cells in the attached and floating cells, respectively, in CGL-2 cultures treated with 2 µM arsenite. (C) Correlation between arsenite cytotoxicity and induction of mitotic arrest. The percentage survival of CGL-2 cells incubated with 05 µM arsenite for 24 h is plotted against the mitotic index of cultures treated with the corresponding arsenite concentration. The data presented are the mean ± SD of four independent experiments.
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Cell-cycle stage-independence of arsenite-induced mitotic arrest
To understand whether the arsenite-arrested mitotic cells originated from a specific cell-cycle stage, CGL-2 cells were synchronized at the G1 stage by double thymidine block. The S- and G2-enriched populations were then obtained by incubating the thymidine-blocked G1 cells in drug-free medium for 4 and 7 h, respectively (Figure 2A). As shown in Figure 2BD, addition of 2 µM arsenite to the G1-, S- or G2-enriched culture did not significantly retard cell-cycle progression until the cells went into mitotic stage. Once the arsenite-treated G1, S or G2 cells went into mitotic stage, they underwent cell-cycle arrest, as indicated by the limited increase in the G1 population at 1025 h after release of thymidine block. These results showed that, on treatment with arsenite, all the cells, whether treated at G1, S or G2 stage, progressed into, and arrested at, mitosis. However, it has been reported previously that, in other cell types, arsenite can induce cell-cycle arrest at the G1, S and G2 stage, and also mitotic arrest, but to a lesser degree (7,10,1214,22). Our present results showed that CGL-2 cells with arsenite-induced injuries entered mitosis without proper cell-cycle arrest, suggesting that, interference with the G2 checkpoint might be crucial in causing arsenite-induced mitotic arrest.

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Fig. 2. Cell-cycle independence of arsenite-induced mitotic arrest. CGL-2 cells were synchronized at G1 by double thymidine block and allowed to progress by releasing the thymidine block as described in the Materials and methods. The cells were then harvested at 24 h intervals and the cell-cycle distribution was analyzed. (A) Untreated CGL-2 cells. (BD) Arsenite (2 µM) was added at various times after the release of the thymidine block: (B) immediate after; (C) 4 h after; or (D) 7 h after. The data presented are the average of two independent experiments.
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Accumulation of
-H2AX in arsenite-arrested mitotic cells
Several reports have demonstrated that arsenite can induce DNA damage (2629). To determine whether arsenite-arrested mitotic CGL-2 cells contained damaged DNA, the level of
-H2AX, a robust marker for DNA double-strand breaks (30), was examined by both immunoblotting and immunofluorescence staining. In CGL-2 cells treated for 24 h with 2 µM arsenite,
-H2AX foci were clearly detectable in the interface nuclei (Figure 3B) and on the mitotic chromosomes (Figure 3C), whereas no
-H2AX signal was seen in untreated cells (Figure 3A). Immunoblot analysis showed that treatment of CGL-2 cells with 2 µM arsenite resulted in a time-dependent increase in the levels of
-H2AX (Figure 3D). Accumulation of
-H2AX showed that DNA damage occurred in arsenite-treated CGL-2 cells. To determine whether arsenite-induced DNA damage was involved in the induction of mitotic arrest in CGL-2 cells, we compared the cellular responses to the insults induced by arsenite, X-irradiation, nocodazole and taxol. Flow cytometric and mitotic index analysis of the total cell population showed that, at 14 h after irradiation with a dose of 10 Gy of X-rays, 72.4% of CGL-2 cells had a doubled DNA content, but the mitotic index was only 4.9% (Figure 4B), indicating that X-irradiation predominantly caused G2 arrest, whereas 24 h treatment with arsenite (2 and 4 µM), nocodazole (0.1 µM) or taxol (0.1 µM) induced substantial mitotic arrest (mitotic indices ranging from 54.3 to 73%, Figure 4CF). Immunoblot analysis (Figure 5) showed that the floating cells shaken off arsenite-, nocodazole- or taxol-treated cultures had high levels of cyclin B1, a marker of G2 and mitotic cells. These cells also had high levels of both phosphorylated histone H3 (at serine 10), a mitotic marker, and Pds1, an anaphase inhibitor. Together, these results indicate that these cells were arrested at the mitotic stage. Conversely, X-irradiated cells showed high levels of cyclin B1, but no accumulation of phosphorylated histone H3 or Pds1, confirming G2 arrest. In cells treated with nocodazole or taxol, spindle poisons but not DNA damaging agents, no
-H2AX signal was observed. However, in the arsenite-arrested mitotic cells, levels of
-H2AX were remarkably high, even higher than in 10 Gy X-irradiated cells, indicating that severe DNA damage had occurred in the arsenite-arrested mitotic CGL-2 cells.

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Fig. 4. Cell-cycle distribution of CGL-2 cells treated with arsenite, X-ray, nocodazole or taxol. Cells were either untreated (A) or incubated for 24 h with 2 or 4 µM arsenite (C and D), 0.1 µM nocodazole (E), or 0.1 µM taxol (F), or irradiated with a 10 Gy dose of X-rays and then allowed to recover for 14 h (B). The cells were then harvested for analysis of cell-cycle distribution and mitotic index. The data presented are representative of those obtained in three independent experiments.
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Override of X-irradiation-induced G2 arrest by arsenite in CGL-2 cells
The G2 DNA damage checkpoint is usually activated in response to DNA double-strand breaks and halts the cell cycle at the G2 stage for DNA repair. The cell-cycle distribution of CGL-2 cells in response to X-irradiation was analyzed to understand whether arsenite inhibits the activation of the G2 checkpoint and hence leads to the presence of damaged DNA in mitotic-arrested cells. When the mitotic index was examined at 30 min intervals starting immediately after irradiation with 5 Gy of X-rays, it fell from 6 to 0.7% within 2 h (Figure 6A, empty circles), indicating that the G2 checkpoint was immediately activated upon X-irradiation of CGL-2 cells. However, this effect was abolished in the presence of 5 mM caffeine (Figure 6A, filled circles), an agent known to abrogate the G2 checkpoint and override the G2 arrest induced by DNA damage (31), as indicated by the prevention of decrease in mitotic index. Thus, caffeine could override the immediate G2 arrest induced by X-rays in CGL-2 cells. Similar results were obtained using 2 and 5 µM arsenite, showing that arsenite mimicked caffeine in abrogating the G2 checkpoint and overriding the G2 arrest induced by X-irradiation in CGL-2 cells (Figure 6A, triangles). We also examined whether the G2 checkpoint in CGL-2 cells could accumulate the cells at the G2 stage when it was activated by X-irradiation. When G2 accumulation was examined 14 h after irradiation of CGL-2 cells with X-rays at doses ranging from 0 to 20 Gy (Figure 6B), there was a dose-dependent increase in the percentage of cells with a doubled DNA content (empty triangles) and a low mitotic index (empty bars), indicating accumulation of G2 cells. This effect was attenuated by 2 mM caffeine as indicated by the decrease in the percentage of cells with a doubled DNA content (Figure 6B, filled triangle) and the increase in mitotic index (Figure 6B, filled bar). However, when the cells were irradiated and incubated in the presence of 2 µM arsenite, the mitotic indices were significantly enhanced (Figure 6C, slashed bars). These results indicate that the dose-dependent G2 accumulation induced by X-irradiation was reduced and the G2-arrested cells were forced into mitosis by arsenite in the same manner as that of cells irradiated and incubated in the presence of caffeine. Since arsenite could also disrupt the function of mitotic spindles, these cells were therefore arrested at mitosis. These results indicated that arsenite could also mimic caffeine in attenuating the G2 accumulation induced by X-irradiation. Together, these results confirmed that the components of the G2 checkpoint pathway in CGL-2 cells responded normally to DNA damage-induced by X-irradiation and led to G2 arrest, suggested that arsenite could disrupt the function of G2 checkpoint and override X-ray-induced G2 arrest in CGL-2 cells. Similar results were also obtained in HeLa S3 cells showing that arsenite could force the X-ray-induced G2 arrested HeLa cells to mitotic stage and indicating that arsenite could also attenuate the G2 DNA damage checkpoint in HeLa S3 cells (data not shown).

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Fig. 6. Override of X-irradiation-induced G2 arrest by caffeine or arsenite. (A) Abolishment of X-irradiation-induced immediate G2 arrest by arsenite. CGL-2 cells were placed in medium containing 2 or 5 µM arsenite or 5 mM caffeine and immediately irradiated with a 5 Gy dose of X-rays. These cells were then incubated in the same medium for the indicated time prior to being harvested for analysis of the mitotic index. Abolishment of the immediate G2 arrest in X-irradiated cells by arsenite and caffeine was illustrated by prevention of the decrease in mitotic index. The data presented are the mean ± SD for five independent experiments. (B) Abrogation of X-irradiation-induced G2 accumulation by caffeine. CGL-2 cells were irradiated with 020 Gy of X-rays in drug-free medium (empty symbols and bars) or with 20 Gy of X-rays in medium containing 2 mM caffeine (filled symbols and bar), then incubated in the same medium for 14 h, prior to being harvested for analysis of cell-cycle distribution and mitotic index. (C) Attenuation of X-irradiation-induced G2 accumulation by arsenite. CGL-2 cells were irradiated with 020 Gy of X-rays in medium containing 2 µM arsenite, then incubated in the same medium for 14 h, prior to being harvested for analysis of cell-cycle distribution and mitotic index. The data presented are the average of two independent experiments.
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To further characterize the effects of arsenite on G2 accumulation, we followed the cell-cycle progression of CGL-2 cells X-irradiated in the presence or absence of 2 µM arsenite and then incubated the cells for 22 h in the same medium. After exposure to 10 Gy of X-rays, the percentage of G2/M cells increased at 8 h, peaked at 1114 h and then gradually declined to baseline by 22 h (Figure 7A, filled squares). Meanwhile the mitotic index decreased from 6 to 0.7% within 4 h, indicating the induction of immediate G2 arrest. The mitotic index remained low at 814 h, increased to 7.5% at 16 h and remained steady thereafter (Figure 7B, filled squares). These results showed that X-irradiation induced a transient G2 arrest in CGL-2 cells. In contrast, arsenite alone induced a time-dependent increase in the percentage of both G2/M and mitotic cells (Figure 7A and B, filled circles); similar results were seen in X-irradiated 2 µM arsenite-treated cells (Figure 7A and B, filled triangles), thereby confirming that arsenite treatment could override the G2 arrest induced by X-irradiation.

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Fig. 7. Induction of a time-dependent increase in mitotic index in X-irradiated cells by arsenite. (A) Percentage of cells in G2/M. (B) The mitotic index. CGL-2 cells were incubated for the indicated time with 2 µM arsenite or irradiated with a dose of 10 Gy of X-rays in the presence or absence of 2 µM arsenite, followed by incubation in the same medium for the indicated time. The data presented are the average of two independent experiments.
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Suppression of X-irradiation-activated phosphorylation of ATM and Cdc25C by arsenite
In response to DNA double-strand breaks, the ATM-dependent DNA damage checkpoint is activated; this initiates subsequent cellular responses, including cell-cycle arrest and DNA repair. Phosphorylation of serine residues 1981 of ATM kinase and 216 of Cdc25C phosphatase is crucial for the activation of the DNA damage checkpoint signaling pathway and G2 arrest (32,33). Therefore, we examined the effects of arsenite on these phosphorylation events after X-irradiation. As shown in Figure 8A, significant phosphorylation of the serine 1981 of ATM was seen at 1 h after 10 Gy X-irradiation, the level of phosphorylation then gradually decreased to 34 and 19% of the 1 h level at 7 and 14 h after X-irradiation, respectively (Figure 8A). When 2 µM arsenite was present during and after irradiation, the phosphorylation of serine 1981 of ATM at 1 h after 10 Gy X-irradiation was only 24% of that in cells irradiated with 10 Gy X-ray alone and decreased to 11 and 3% at 7 and 14 h, respectively (Figure 8A). Similar results were observed with 5 Gy X-irradiation. In addition, X-irradiation (416 Gy) induced significant serine 216 phosphorylation of Cdc25C, and 2 µM arsenite significantly suppressed this effect and increased the expression of the mitotic form of Cdc25C (Figure 8B). These results confirmed that arsenite could attenuate X-irradiation-induced activation of the G2 checkpoint.

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Fig. 8. Suppression of X-irradiation-induced phosphorylation of ATM (at serine 1981) and Cdc25C (at serine 216) by arsenite. (A) Arsenite inhibition of X-irradiation-induced ATM phosphorylation at serine 1981. CGL-2 cells were irradiated in drug-free or 2 µM arsenite-containing medium with 10 Gy of X-rays. The cells were then incubated in the same medium for 1, 7 or 14 h, prior to being harvested for immunoblot analysis of ATM phosphorylation using antibodies reactive with ATM phosphorylated at serine 1981 (upper panel) or ATM (lower panel). The basal level of ATM was used as the loading control. The number shown below the blot is the ratio of the intensity of the phosphorylated ATM band to the intensity of the ATM band expressed as a percentage of the ratio seen at 1 h after irradiation with 10 Gy. (B) Arsenite attenuation of X-irradiation-induced Cdc25C phosphorylation at serine 216 (Cdc25C-p-S216). CGL-2 cells were irradiated with 416 Gy of X-rays in drug-free or 2 µM arsenite-containing medium. The cells were then incubated in the same medium for 14 h prior to being harvested for immunoblot analysis of Cdc25C phosphorylation (top panel). The expression of total Cdc25C was also determined to verify the cell-cycle stage (center panel). ß-Actin was used as a loading control (bottom panel). The images presented are representative of those obtained in two independent experiments.
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Higher susceptibility of triggering apoptosis in arsenite-arrested mitotic CGL-2 cells than nocodazole- and taxol-arrested mitotic cells
Previous studies have shown that arsenite induces mitosis-mediated apoptosis (7,10,11,13). To examine the effect of the presence of DNA damage in arsenite-induced mitotic cells on the induction of apoptosis, we incubated arsenite-, nocodazole- and taxol-arrested mitotic CGL-2 cells for 24 h in drug-free medium and then examined them by Annexin V staining to assess apoptosis induction. When CGL-2 cells were treated for 24 h with arsenite, nocodazole or taxol at dose ranges that produced a similar frequency of mitotic cells, a higher frequency of apoptotic cells were seen in the arsenite-arrested mitotic cells than in those arrested by the other two spindle poisons (Table I). For example, treatment of CGL-2 cells with 4 µM arsenite, 100 nM nocodazole or 50 nM taxol resulted in 66, 65 and 71% of the cells arrested at mitotic stage, respectively. After an additional 24 h-incubation in drug-free medium, 61% of the arsenite-arrested mitotic cells underwent apoptosis, which is significantly higher than the 22 and 41% observed in that of nocodazole- or taxol-arrested mitotic cells. Since arsenite-arrested mitotic CGL-2 cells showed marked accumulation of
-H2AX, a marker of DNA damage, whereas nocodazole- or taxol-arrested mitotic cells did not (Figure 5), the presence of DNA damage in arsenite-arrested mitotic cells might play a role in enhancing the induction of apoptosis.
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Discussion
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Microtubule-targeted agents, such as paclitaxel and taxotere, are now often used in the treatment of a variety of human epithelial cancers. On binding to ß-tubulin, these agents decrease microtubule dynamic instability, interfere with G2/M transition, induce mitotic arrest, and subsequently trigger the molecular signaling for the mitochondrial pathway of apoptosis (15,16). Arsenite, which is effective in the treatment of relapsed or refractory APL, has also been demonstrated to attenuate spindle dynamics and thereby induce mitotic arrest and mitosis-mediated apoptosis (11,14). However, the mechanism involved in how the cell-cycle perturbation induced by these anti-microtubule agents triggers apoptosis remains to be elucidated. In this study, we demonstrated that arsenite induces a more prominent mitotic arrest in CGL-2 cells than in other cell lines studied (7,10,1214,22,34). In contrast to previous studies, arsenite did not alter the progression of each cell-cycle stage in CGL-2 cells. Furthermore, our results showed that arsenite could mimic caffeine in overriding X-irradiation-induced G2 arrest. Together, these results indicated that arsenite might disrupt the function of DNA damage checkpoint, and thereby allowing CGL-2 cells with damaged DNA to continue their cell-cycle progression. As arsenite also interferes with the function of mitotic spindles (7,10,14), the arsenite-treated CGL-2 cells are finally arrested in mitotic stage with the presence of DNA damage. As arsenite treatment causes DNA damage, inhibits G2 checkpoint activation and interferes with the function of spindle checkpoint (35), the arsenite-arrested mitotic CGL-2 cells consequently manifest higher susceptibility to induction of apoptosis than those treated with nocodazole or taxol.
In normal cells, DNA damage activates DNA damage checkpoint pathways that inhibit the progression of cells through the G1 and G2 phases and induces a transient delay in progression through S phase (36,37). Cell-cycle arrest before mitosis would allow cells time to repair DNA damage and, thereby preventing the transmission of genetic errors to daughter cells. Previous reports have demonstrated that arsenite could induce a significant cytostatic effect on human diploid fibroblasts, which contain wild-type p53 and normal cell-cycle checkpoint functions (12,38). On the other hand, arsenite induced G2 and mitotic arrest instead of G1 arrest in SV-40 transformed human diploid fibroblasts in which the p53 and Rb are inactivated by sequestration of SV-40 T antigens (12). Moreover, arsenite also induced a significant increase of cells at G1 stage in a myeloma cell line with wild-type p53 whereas arsenite induced G2 and mitotic arrest in myeloma cell lines with mutant p53 (34,39). These results indicated that arsenite might induce G1 arrest in a p53-dependent manner. We have demonstrated previously that arsenite could induce DNA damages and result in p53 accumulation and activation in normal diploid fibroblasts (38). Loss of p53 function might prevent the induction of G1 arrest and lead to G2 and mitotic arrest in arsenite-treated cells. Most cell lines that lack functional p53 protein are often arrested in the G2 stage due to DNA damage (40). CGL-2 cells are a fusion hybrid of HeLa cells and normal human fibroblasts and have no functional p53 protein. Therefore, our results showing an absence of G1 arrest in X-irradiated or arsenite-treated CGL-2 cells are expected. CGL-2 cells were shown to have a functional G2 DNA-damage checkpoint, as G2 arrest was observed in X-irradiated CGL-2 cells. However, arsenite-induced DNA damage did not stop the progression of S and G2 phase cells into mitosis and DNA damage was still present in arsenite-arrested mitotic cells. These results implied either that the arsenite-induced DNA damage could not properly activate the G2 DNA damage checkpoint or that arsenite might inactivate or abrogate the G2 DNA damage checkpoint in CGL-2 cells. In response to DNA double-strand breaks, the ATM-dependent DNA damage checkpoint is activated and initiates the subsequent cellular responses, including cell-cycle arrest and DNA repair (30). Ionizing irradiation induces rapid intermolecular autophosphorylation at serine 1981 of ATM and thus enhances the kinase activity of ATM (41). The enhanced kinase activity activates checkpoint kinase (Chk)-2 by phosphorylation at threonine 68. The activated Chk-2 subsequently phosphorylates Cdc25C at serine 216 and blocks its function (32,33). This inhibitory phosphorylation of serine 216 of Cdc25C increases its binding to 14-3-3, reduces its catalytic activity, and causes its sequestration in the cytoplasm (32,33). Without the phosphatase activity of Cdc25C, the mitotic cyclin-dependent kinase can no longer be activated; therefore, cells are arrested at the G2 stage. Based on our present results showing that arsenite treatment reduces phosphorylation of both the serine 1981 of ATM and the serine 216 of Cdc25C, we conclude that arsenite can attenuate or inactivate the G2 DNA-damage checkpoint. Arsenite-treated CGL-2 cells with damaged DNA therefore are not inhibited from entering mitosis, resulting in a prominent mitotic arrest. Our previous report has demonstrated that arsenite could significantly increase the histone H1 kinase activity of cyclin-dependent kinase 1 and also induce a significant mitotic arrest (35%) in HeLa S3 cells (14). In this study, similar effects of arsenite on G2 checkpoint abrogation were also observed in HeLa S3 cells. These results indicate that attenuation of G2 DNA damage checkpoint by arsenite might result in the prominent mitotic arrest in arsenite-treated CGL-2 and HeLa S3 cells and suggest that arsenite could inhibit the G2 checkpoint in other solid cancer cells. Further investigation of how arsenite attenuates the G2 DNA damage checkpoint is of great importance in elucidation of the action mechanism and the therapeutic implication of arsenite.
Pharmacological abrogation of the DNA damage checkpoint is frequently used in combination with DNA damaging agents in cancer chemotherapy. The targets are mainly kinases involved in the signaling pathway of DNA damage checkpoint, such as ATM, ATM-related, Chk-1 and Chk-2. The most efficacious checkpoint inhibitors found so far are caffeine, which inhibits DNA damage-proximal targets, such as ATM (31), UCN-01, which inhibits multiple targets, such as Chk-1 and Wee1 (42), and debromohymenialdisine, which inhibits Chk-1 and Chk-2 (43). These kinase inhibitors simply interfere with kinase activity without inducing DNA damage and mitotic arrest. A combination of checkpoint kinase inhibitors and DNA damaging agents is therefore used to increase the killing efficiency. On the other hand, over-expression of type I protein phosphatase releases the G2 checkpoint activated by DNA damage, induces aberrant mitoses, and sensitizes cells to DNA damage (44). In the present study, we demonstrated that the X-irradiation-induced G2 DNA damage checkpoint could be suppressed and DNA damage-induced G2 arrest overridden by arsenite in CGL-2 cells. We also demonstrated that arsenite deregulated the components involved in G2 DNA damage checkpoint pathways. Therefore, we suspect that abrogation of the G2 DNA damage checkpoint by arsenite might sensitize cancer cells to ionizing radiation. It has been reported that arsenic trioxide can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo, since co-treatment synergistically enhanced radiation-induced G2/M accumulation and apoptosis (6).
Arsenite-arrested mitotic CGL-2 cells were also more susceptible to apoptosis than those arrested by nocodazole or taxol, indicating that arsenite may act in a different mechanism to induce mitotic arrest. When mammalian cells with DNA replication defects or DNA damage (e.g. if the G2/M checkpoint is defective or is overruled) enter mitosis, their centrosomes break up during mitosis, multipolar spindles appear, and aneuploid cells are produced (17). Centrosome and spindle abnormalities may therefore be a general response of mammalian cells when DNA defects persist in mitosis. There is evidence for a strong link between the DNA damage-induced checkpoint signaling pathway and the induction of abnormal centrosomes and malfunctioned mitotic spindles, leading to the activation of spindle checkpoint and mitotic arrest (45,46). In addition to attenuating the spindle checkpoint and thus inducing the abnormal onset of anaphase (35), arsenite also causes DNA damage (2629). Thus, the combined effects of arsenite in inducing DNA damage, inhibiting the G2 checkpoint, and attenuating the spindle checkpoint may make arsenite-arrested mitotic CGL-2 cells more susceptible to induction of apoptosis than those treated with nocodazole or taxol.
Arsenic has pleiotropic effects on many biological systems and induces complex toxicopathological injuries (4750) including the generation of reactive oxygen species, induction of DNA damage, disruption of mitochondrial function, modification of gene and/or protein expression and intracellular signal transduction pathways, alteration of cell-cycle progression, and induction of cytogenetic aberrations and cellular transformation. These deleterious effects have been suggested to be involved in arsenite-induced apoptosis. These multiple actions of arsenite highlight the need for additional mechanistic studies to determine which actions mediate the diverse biological effects of this agent. Gaining a better insight into the mechanisms of action of arsenic compounds might allow us to increase their therapeutic effects while reducing toxic side-effects and to develop better regimens for cancer therapy. Although we do not yet fully understand how arsenite abrogates the DNA damage checkpoint, our results demonstrate that alteration of the DNA damage checkpoint plays a significant role in the induction of mitotic abnormalities and apoptosis by arsenite, which might be a great help in terms of cancer therapy, especially in tumors with p53 mutations or which have developed chemoresistance. This information will be critical in realizing the potential of the synergy between arsenite and other chemotherapeutic agents, thus providing enhanced benefit in cancer therapy.
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Acknowledgments
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The authors thank Drs K.Y.Jan and T.C.Wang (Institute of Zoology, Academia Sinica) for carefully reading the manuscript and assisting with the operation of the flow cytometer and fluorescence microscope. This work was supported by the Academia Sinica and grants from the National Science Council (NSC 91-2320-B001-050 and NSC 92-2320-B001-029), Republic of China.
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Received May 19, 2004;
revised August 27, 2004;
accepted September 27, 2004.