Arsenite Induces p53 Accumulation through an ATM-dependent Pathway in Human Fibroblasts¹

Arsenic compounds are ubiquitously distributed natural toxicants and are considered to be carcinogenic in humans (1). However, the molecular mechanisms of arsenic carcinogenicity remain elusive. Although arsenite by itself is thought to be inactive or typically too weak to induce gene point mutations (2, 3),increased frequencies of cytogenetic alterations, such as chromosome aberrations, sister chromatid exchanges, and micronucleus, have been found in peripheral lymphocytes of human populations with chronic exposure to arsenic through drinking water (4, 5, 6). In in vitro studies, sodium arsenite, a trivalent arsenic compound, induced similar cytogenetic alterations in a variety of cell systems (3, 7, 8, 9). At the same dose range,arsenite-induced cytogenetic alterations were closely associated with arsenite-induced morphological transformation in Syrian hamster embryo cells (10, 11). Our recent studies (12, 13)demonstrated that arsenite perturbs spindle dynamics and results in chromosome malsegregation during mitosis in cultured human cells. These investigations indicate that the genotoxicity and carcinogenicity of arsenite might be attributable to its activity on inducing cytogenetic alterations and/or genetic instability. Other mechanisms, such as modulation of DNA methylation (14) or DNA repair inhibition (15, 16, 17, 18), are also reportedly involved in arsenite genotoxicity and carcinogenicity.

Numerous reports have shown that arsenite treatment induces DNA strand breaks and DNA-protein cross-links in a variety of cell lines(19, 20, 21). DNA strand breaks usually trigger the accumulation of p53 protein, a short half-life protein denoted as the guardian of the genome. Only a few reports have described the relationship between arsenite exposure and p53 activation and/or accumulation (22, 23). The p53 protein plays a pivotal role in maintaining genome integrity. This task is achieved through the induction of growth arrest for repair of DNA damage or apoptosis for eliminating cells with irreparable damage (24, 25). In general, p53 is involved in diverse cellular processes, including DNA repair, cell cycle checkpoints, apoptosis, cell differentiation, and a variety of stress responses induced by endogenous and exogenous sources(26, 27). In response to diverse stresses, p53 activation is tightly regulated through a complicated signaling network, including protein stability, subcellular localization, and interaction with other proteins (28). NH₂-terminal phosphorylation of p53 is of importance to maintain p53 protein stability and its transactivation activities (29). Three members of the PI3-K³-related kinase family, DNA-dependent protein kinase (3),ATM, and ATR, are demonstrated to phosphorylate p53 at different sites in vitro, and two of them, ATM and ATR, possibly phosphorylate p53 on serine 15 in vivo(30, 31, 32). NH₂-terminal phosphorylation of p53 hampers the association of p53 with MDM-2, the negative regulator of p53, and hence prevents p53 from proteosome-mediated degradation (29, 33, 34, 35). p53 is therefore accumulated and transactivates its downstream genes that are also involved in cellular stress responses.

Because p53 reacts to different stress-promoting conditions and may serve as the center of a signal network responsible for appropriate responses to various cellular stresses, understanding how arsenite enhances p53 accumulation could help unveil cellular responses to arsenite-induced injury. HFW cells manifest normal and stable karyotypes and become senescent after several passages(12). We previously used HFW cells to investigate the effects of arsenite on cell cycle progression and cytogenetic alterations (7, 12). Our results showed that arsenite not only caused chromosome aberrations and micronuclei but also induced spindle defects and perturbed mitosis in HFW cells. Therefore, HFW cells were again used to investigate p53-dependent cellular responses to arsenite insults. In this study, we found that arsenite induces DNA strand breaks and results in p53 phosphorylation and accumulation in HFW cells through an ATM-dependent pathway.

HFW cells derived from newborn foreskin were kindly provided by Dr. W. N. Wen (National Taiwan University). GM3395 and GM3398 cells were obtained from Coriell Cell Repositories (Camden, NJ). Cells were routinely maintained in DMEM (Life Technologies, Inc., Grand Island,NY) supplemented with 10% fetal bovine serum (Hyclone Laboratories,Logan, UT), 0.37% sodium bicarbonate, 100 unit/ml penicillin, and 100μg/ml streptomycin. The cells were cultured at 37°C in an incubator with humidity-saturated air and 10% CO₂(36).

DNA strand breaks were measured by single-cell alkaline gel electrophoresis (comet assay) as described by Lynn et al.(20). Briefly, HFW cells after arsenite treatment were harvested, embedded in 1% agarose gel at a density of 1 × 10⁶/ml, and spread onto a fully frosted slide. The slides were immersed in ice-cold lysis buffer (10 mm Tris-HCl, 2.5 m NaCl,100 mm Na₂EDTA, 1% sodium N-lauryl sarcosinate, 1% Triton X-100, and 10% DMSO; pH 10) for 1 h at 4°C. Cellular DNA was denatured in electrophoresis buffer (300 mm NaOH and 1 mm Na₂EDTA) for 20 min at room temperature and then electrophoresed for 20 min at 25 V and 300 mA. Afterward, the slides were washed in distilled water, renatured in 0.4 m Tris-HCl (pH 7.5), stained with Sybr green(Molecular Probe, Eugene, OR), and examined under a fluorescence microscope (BX60; Olympus, Tokyo, Japan). Comets were classified into four groups based on the length and fluorescence intensity of the comet tail: type I, no tail (intact nuclei; Fig. 1,a, A–C); type II, comet with short tail (tail length less than the head diameter; Fig. 1,a, D–F); type III, comet with tail length longer than the head diameter and with low fluorescence intensity (Fig. 1,a, G and H); and type IV, comet with tail length longer than the head diameter and with high fluorescence intensity (Fig. 1 a, I; Ref. 37). Five hundred cells were examined for each treatment.

Immunoblotting was performed as described previously (12). In brief, after drug treatment HFW cells were scraped from culture dishes with the aid of a rubber policeman, lysed immediately in electrophoretic sample buffer, and heated at 95°C for 5 min(38). Protein concentrations were determined by Bradford analysis (39). An aliquot of 10 μg of cellular protein was loaded onto a 10% SDS-PAGE. After electrophoretic separation,polypeptides were transferred onto a nitrocellulose membrane by a semidry electrotransfer system (ATTO, Tokyo, Japan). After blocking in 5% skimmed milk in PBST (PBS containing 0.2% Tween 20)for 1 h, the membranes were incubated overnight at 4°C with primary antibodies that were appropriately diluted in blocking solution. p53, p21, and MDM-2 were reacted with anti-p53 monoclonal antibody (DO-1, sc-126; Santa Cruz Biotechnology, Santa Cruz, CA),anti-p21 monoclonal antibody (Transduction Laboratories, San Diego, CA), and anti-MDM-2 monoclonal antibody (Santa Cruz Biotechnology), respectively. The membranes were then extensively washed with PBST and incubated with secondary antibodies conjugated with horseradish peroxidase (Organon Teknika-Cappel, Turnhout,Belgium). Proteins were visualized using an enhanced chemiluminescence system according to the manufacturer’s instruction (Pierce,Rockford, IL). The levels of p53 were estimated using a densitometer(Densitometer 300S; Molecular Dynamics, Sunnyvale, CA).

To localize the p53 in cells, HFW cells were seeded onto glass coverslips. After treatment, cells on the coverslips were washed twice with PBS and then fixed in situ with methanol:acetone (1:1)at −20°C for 10 min. The coverslips were washed twice with PBS and incubated with anti-p53 monoclonal antibody (DO-1; Santa Cruz Biotechnology) at 4°C for 1 h. The unbound antibody was removed by extensively washing with PBST. The coverslips were further incubated with FITC-conjugated secondary antibody (Organon Teknika-Cappel) in the dark for 30 min. Chromosomes were counterstained with 0.1 μg/ml 4,6-diamino-2-phenylindole. After thoroughly rinsing with PBST, the coverslips were mounted with a 90% glycerol solution containing 1 mg/ml phenylenediamine (pH 8.0). For each treatment, 1000 cells were randomly selected and examined under a fluorescence microscope(Olympus, Tokyo, Japan). Cellular cyclin B1 was stained with rabbit anti-cyclin B1 antibodies (Santa Cruz Biotechnology) and visualized with rhodamine-conjugated secondary antibodies (Organon Teknika-Cappel).

After drug treatment, HFW cells were immediately lysed in lysis buffer(50 mm Tris-HCl, 5 mmNa₂EDTA, 150 mm NaCl, 0.5% NP40, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 0.5 mm sodium vanadate, and 1 mm sodium fluoride)for 20 min on ice. To immunoprecipitate cellular p53, an aliquot of 1 mg of cellular protein was incubated with 1 μg of anti-p53 monoclonal antibody (DO-1, sc-126; Santa Cruz Biotechnology) for 1 h at 4°C with agitation. Protein A-Sepharose beads (Sigma) were added to the mixture and incubated for another 1 h at 4°C. The immunocomplex was spun down by centrifugation, washed with lysis buffer five times,and boiled in SDS-PAGE sample buffer. The clear supernatants were subjected to immunoblot analysis for total p53 with another polyclonal p53 antibody (FL-393, sc-6243; Santa Cruz Biotechnology) and for phosphorylated p53 with an anti-p53 antibody specific to phosphorylated serine 15 (p53-S15, #3284S; New England BioLabs, Beverly, MA).

Logarithmically growing HFW cells were treated with 20 μmBrdUrd for 30 min. BrdUrd was then removed, and the cells were further incubated in medium with or without 5 μm arsenite. At various time points, cells were harvested for analysis of BrdUrd incorporation and DNA content. In brief, total cells were collected and fixed in cold 70% ethanol. After treatment with 2 n HCl for nicking DNA, 0.1 mNa₂B₄O₇was added to neutralize the reaction mixture. The incorporated BrdUrd was reacted with FITC-conjugated anti-BrdUrd antibodies (Boehringer Mannheim Biochemicals, Mannheim, Germany), and cellular DNA was stained with 4 μg/ml PI in PBS containing 1% Triton X-100 and 0.1 mg/ml RNase A. The fluorescent intensities of PI and FITC of individual cells were analyzed with a fluorescence-activated cell sorter (FACStar;Becton Dickinson Immunocytometry Systems) as described previously(36). The BrdUrd level in each cell cycle phase was determined using a computer program provided by the Becton Dickinson Immunocytometry System.

We showed previously that arsenite at a dose range of 1.25–5μ m significantly induces chromatid breaks and micronuclei in HFW cells (7, 12). In the present study, a recently developed and very sensitive method called “single-cell alkaline gel electrophoresis” or “comet assay” was adopted to determine whether arsenite causes DNA strand breaks in HFW cells. Treatment of HFW cells with 5 μm arsenite for 0–6 h time dependently increased the frequencies of type II comets from 10% to ∼60% and type III and IV comets from very low to 16 and 8%, respectively (Fig. 1 b). Types I–IV comets were assigned numerical scores of 0–3, respectively (40); the average scores for each time point of 5 μm arsenite treatment were 0.11 ± 0.03, 0.28 ± 0.06,0.54 ± 0.18, 0.77 ± 0.12,0.86 ± 0.21, 0.97 ± 0.18, and 1.15 ± 0.10. This result indicated that 5μ m arsenite significantly induced DNA strand breaks in HFW cells (P < 0.001, according to ANOVA analysis). According to dye exclusion assay, HFW cells remained viable and maintained intact membrane integrity at the time the comet assay was performed, and no apoptosis was detectable under the experimental conditions. Therefore, consistent with a previous report(20), the arsenite-induced DNA strand breaks identified through comet assay were not attributable to apoptotic or dead cells.

Because p53 is a well-documented DNA damage marker (41),the effects of arsenite on p53 protein levels were examined using immunoblotting. Treatment of HFW cells with arsenite at a dose range from 1.25 to 10 μm for 24 h dramatically increased p53 levels (Fig. 2,A). In a time-dependent experiment, p53 accumulation was observed at 1 h after arsenite treatment and increased to maximum accumulation at 4–6 h (Fig. 2,B). Afterward, p53 levels declined to one-third of the maximum and remained at a constant level for 24 h (Fig. 2,B). Using a densitometer, the intensities of p53 induced by 5 μm arsenite(0-, 1-, 2-, 4-, and 6-h time points in Fig. 2,B) were measured and plotted against the average comet scores (the same time points in Fig. 1,b). As shown in Fig. 2,D, the levels of p53 accumulation were linearly correlated to comet scores(r² = 0.929). These results indicated that during the first several hours, the levels of arsenite-induced p53 accumulation were parallel with the incidence of DNA strand breaks. In addition to p53 accumulation, the protein levels of p53 target genes, such as p21 and MDM-2, were concomitantly increased by arsenite treatment(Fig. 2 C).

Arsenite-induced p53 accumulation was further confirmed by immunofluorescence staining. Consistent with the results of immunoblotting (Fig. 2,B), p53 levels were very low in untreated HFW cells (Fig. 3, A and B). However, highly abundant p53 was frequently present in nuclei of arsenite-treated HFW (Fig. 3, C and D). The frequency of arsenite-treated cells showing bright fluorescence gradually increased from 5 to 30% during the 24-h treatment period (Fig. 3,E). For comparison, we examined the kinetics of p53 nuclear accumulation in nuclei of X-ray-irradiated HFW cells. After 1 Gy of X-ray irradiation, p53 accumulation in nuclei occurred immediately and transiently, i.e., 60% of nuclei showed bright fluorescence 1 h after irradiation and the frequency rapidly declined (Fig. 3,E), indicating that the kinetics of p53 accumulation to nuclei in arsenite-treated HFW cells are quite different from those in X-ray-irradiated cells. In another staining study, double staining for p53/cyclin B1 showed that arsenite-treated cells with p53 nuclear accumulation (Fig. 4,A, arrowheads) were completely separable from cyclin B1-positive cells (Fig. 4 B, arrows). Cyclin B1 is a marker for the G₂ phase cells; hence, this finding showed that cells with p53 nuclear accumulation were not at the G₂ phase.

Numerous reports have shown that p53 is phosphorylated by ATM kinase(30, 31), a member of the PI3-K-related protein kinase family, and hence is prevented from degradation and subsequently results in p53 accumulation (33, 42). To test whether ATM kinase is involved in the signaling of arsenite-induced p53 accumulation, HFW cells were cotreated with arsenite and wortmannin, an inhibitor of the PI3-K family (43). Wortmannin by itself did not affect the levels of p53 but effectively inhibited arsenite-induced p53 accumulation (Fig. 5,A). For comparison, wortmannin also inhibited X-ray-induced p53 accumulation (Fig. 5,B). However, wortmannin did not abrogate p53 accumulation in UV-irradiated or ALLN-treated cells (Fig. 5,B). ALLN is a calpain/proteosome inhibitor that induces p53 stabilization by inhibiting its degradation instead of altering its phosphorylation level (33). Similarly, wortmannin reduced MDM-2 levels in arsenite- or X-ray-treated HFW cells but had no effect in UV-irradiated and ALLN-treated cells (Fig. 5,B). Because ATM kinase phosphorylates p53 on serine 15 (30, 31), a p53-S15 antibody that specifically recognizes p53 with phosphorylated serine 15 was used to examine the phosphorylation status of arsenite-accumulated p53. As shown in Fig. 6, p53 that accumulated in arsenite-treated and X-ray-irradiated HFW cells strongly reacted to the p53-S15 antibody, whereas p53 in ALLN-treated and UV-irradiated HFW cells was much less reactive to the antibody. These results demonstrate that arsenite, similar to X-ray,induces phosphorylation of p53 on serine 15 but ALLN and UV do not. Accordingly, arsenite mimics X-ray in causing DNA strand breaks and activating ATM kinase, which phosphorylates p53 on serine 15 but follows different kinetics to produce these DNA strand breaks and p53 accumulation.

To further confirm the requirement of ATM for induction of p53 accumulation in arsenite-treated HFW cells, we examined the effects of arsenite on p53 accumulation in ATM^−/− cells. GM3395 cells are fibroblasts derived from an ataxia telangiectasia patient in which both alleles of the ataxia telangiectasia gene are mutated (atm^−/−) and extremely sensitive to X-ray-irradiation (44). GM3398^(atm+/?) cells, derived from the healthy brother of the same ataxia telangiectasia patient, were included for comparison. The results shown in Fig. 7, A and B, demonstrate that neither arsenite nor X-ray irradiation were able to induce p53 accumulation in GM3395^(atm−/−) cells, whereas the same treatment induced significant p53 accumulation in GM3398^(atm+/?) cells. Because a different pathway is involved in UV irradiation-induced p53 accumulation(45), p53 accumulation was also examined in UV-irradiated-GM3395 cells. UV irradiation expectedly and significantly enhanced p53 accumulation in GM3395^(atm−/−)cells (Fig. 7 C).

Induction of p53 accumulation often leads to cell cycle arrest and/or apoptosis. It is very difficult to induce apoptosis in HFW cells. By colony-forming assay, treatment of HFW cells with 5 μmarsenite for 24 h resulted in a 25% survival rate(12); however, no apoptosis was observed immediately after treatment. Even 24 h after arsenite was withdrawn from the medium,the incidence of nucleus-fragmented cells, a manifestation of apoptosis, was 1.2 ± 0.3% (average ± SD from four independent experiments). These results indicated that arsenite-treated HFW cells did not die through apoptosis.

To examine the effect of arsenite on cell cycle progression, HFW cells were first pulse-labeled with 20 μm BrdUrd for 30 min and then chased for 0–24 h in the presence or absence of 5μ m arsenite. The levels of BrdUrd were flow cytometrically detected after immunostaining with FITC-conjugated anti-BrdUrd antibodies, and cell cycle stages G₁,S, and G₂-M were concurrently analyzed by staining the cells with PI. Because the cell population with or without FITC could be easily distinguished and gated by the analysis software of FACStar, we could chase the effects of arsenite on the cell cycle progression of two cell populations with (S-phase cells) or without(non-S-phase cells) BrdUrd labeling. In untreated cultures, we observed a rapid decline of BrdUrd-labeled S-phase cells (Fig. 8,B), subsequently followed by an increase of BrdUrd-labeled G₂-M cells (Fig. 8,C) and G₁ cells (Fig. 8,A). A reappearance of BrdUrd-labeled S-phase cells occurred at 16 h after BrdUrd pulse labeling (Fig. 8,B), indicating that untreated cells normally and actively underwent their cell cycle. In arsenite-treated culture,BrdUrd-labeled S-phase cells declined as did untreated cells but at a slower rate (delay for at least 7 h; Fig. 8,B). However,the G₂-M cells continued to accumulate during arsenite treatment, indicating a G₂-M arrest. Furthermore, the appearance of BrdUrd-labeled G₁cells was first observed after a 16-h chase, at least 8 h later than in untreated cells (Fig. 8 A). These results support that arsenite treatment results in S-phase retardation and G₂-M arrest.

The kinetics of the increase in non-BrdUrd-labeled S phase in arsenite-treated HFW cells were similar to those of untreated cells(Fig. 8,E), indicating that arsenite did not affect the progression of non-BrdUrd-labeled G₁ cells to S-phase. However, the decline of non-BrdUrd-labeled G₂-M phase cells was apparently slower in arsenite-treated cells than in untreated cells (Fig. 8,F). These results indicate that arsenite treatment immediately affects the progression of G₂-M cells. The delay of G₂-M progression apparently results in a decline of non-BrdUrd-labeled G₁ phase cells because of a decrease in cell division (Fig. 8 D).

In the present study, using comet assay, BrdUrd labeling, and flow cytometry, we found that arsenite induces DNA strand breaks and G₂-M arrest. Concomitantly with DNA strand breaks, we observed p53 accumulation in HFW cells. Our results also showed that arsenite- and X-ray-induced p53 accumulation was inhibited by wortmannin, an inhibitor of the PI3-K family. In contrast, UV- or ALLN-induced p53 accumulation was insensitive to the inhibitory effects of wortmannin. We further confirmed that arsenite- and X-ray-induced p53 is phosphorylated at serine 15. Furthermore, both arsenite and X-ray irradiation, but not UV irradiation, failed to induce p53 accumulation in ATM^(−/−) fibroblasts, GM3395. These results support that arsenite causes DNA strand breaks in HFW cells and mimics X-ray irradiation to induce p53 phosphorylation and accumulation through an ATM-dependent pathway.

Nevertheless, arsenite and X-ray irradiation obviously follow different kinetics in terms of DNA strand break induction and p53 accumulation. In general, IR immediately causes DNA strand breaks and hence induces early and transient accumulation of p53 to nuclei (46). Alternatively, arsenite is gradually taken up by cells. The induction of DNA strand breaks and p53 accumulation by arsenite apparently follows a time-dependent course. The slow kinetics of DNA strand breaks and p53 accumulation are also manifested by a slow transport of p53 to nuclei in arsenite-treated cultures. Although arsenite showed different kinetics of DNA strand break induction and p53 accumulation from those of X-ray irradiation, other evidence indicated that arsenite and X-ray treatment of HFW cells share a similar signaling pathway for p53 accumulation, i.e., an ATM-dependent pathway. Our present results showing that p53 nuclear-accumulated cells are distinguishable from cyclin B-positive cells were similar to a previous report(47) that irradiation induces p53 nuclear accumulation in human fibroblasts, predominantly during the G₁phase and at the beginning of the S-phase of the cell cycle. Whether these cells lost their proliferation activity is an interesting question that remains to be answered.

Phosphorylation of p53 is a pivotal modification that regulates p53 stability and transactivation activities (48). Numerous studies have demonstrated that phosphorylation at serine 15 is required for p53 protein stabilization in response to DNA damage (32, 33). Furthermore, recent reports have also shown that phosphorylation of serine 15 of p53 is possibly involved in enhancing the p53s transactivation activities (49). At least two distinct signal transduction pathways can lead to p53 activation. One of these pathways involves ATM, the product of the gene mutated in patients with the cancer-predisposing disorder ataxia telangiectasia(50). ATM is a member of the PI3-K-related protein kinase family because its COOH terminus shows high similarities to the catalytic domain of PI3-K (51). The ATM pathway is activated upon exposure to IR and chemical compounds that induce DNA strand breaks (52). The other pathway, the ATM-independent pathway, is activated upon exposure to UV irradiation and cisplatin,which produce bulky DNA lesions and induce DNA strand breaks during repair processes (28, 53). Our present results demonstrate that the ATM-dependent pathway is involved in arsenite-induced p53 accumulation, supporting that arsenite exposure is indeed a genotoxic stress.

In addition to p53, a variety of targets have been identified for ATM catalytic activity, such as c-Abl, IκB, Chk2, and replication protein A (52), which are involved in ATM-mediated multiple responses to genotoxic stress including stress gene expression, DNA repair, and activation of cell cycle checkpoints (54, 55). ATM thus serves a surveillance role in maintaining genomic integrity. Elucidating the mode of transmission of ATM-mediated responses to arsenite-induced stress may help us better understand arsenite carcinogenesis and to develop chemopreventive strategies.

In the present study, arsenite treatment induced immediate G₂-M arrest. In response to DNA damages, p53 transcriptionally activates p21, the inhibitor of cyclin-dependent kinases, and leads to G₁ and G₂ arrest (56). Disruption of either the p53 or p21 gene abrogates the G₂ arrest induced by IR (57). Arsenite-induced G₂-M arrest might be a consequence mediated by p53-dependent p21 induction. However, the involvement of p21 in G₂-M arrest is controversial (58). A strong p53-dependent induction of the 14-3-3ς gene was observed in IR-induced G₂ arrest cells (59). The mammalian cell cycle checkpoint protein kinase Chk-2 is phosphorylated and activated in response to DNA damages in an ATM-dependent manner(55). Activated Chk-2 phosphorylates Cdc25C on serine 216. This phosphorylation promotes the binding of 14-3-3ς to Cdc25C, thereby negatively regulating Cdc25C phosphatase activities and hence preventing the onset of mitosis, which results in G₂ arrest (60, 61). Because both 14-3-3ς and Chk are downstream transducers of the ATM-dependent DNA damage checkpoint pathway, their involvement in arsenite-induced G₂-M arrest requires further investigation.

It has been reported that fibroblasts with wild-type p53 fail to enter into mitosis when DNA synthesis is blocked (62). The delayed progression of S-phase HFW cells probably resulted from retarded DNA polymerization in the presence of arsenite-induced DNA damages. Numerous reports have shown that ATM is also involved in the activation of S-phase checkpoints, such as the down-regulation of cyclinA/Cdk2 activity (63) and interference with replication protein A (64), and consequently reduces the rate of DNA synthesis.

Arsenite induces G₂-M arrest but does not trigger apoptosis in HFW cells. According to colony-forming assay, 75% of HFW cells lost their proliferation ability after treatment with 5μ m arsenite for 24 h (12), indicating that arsenite-induced injury eventually kills HFW cells. Nevertheless,we have demonstrated previously that arsenite at the same dose used in this study induces aneuploidy and other cytogenetic alterations in HFW colonies that survived (7, 12). Because the roles of p53 in triggering apoptosis are complex and not fully understood, the reason for the lack of apoptosis in arsenite-treated HFW cells is unclear. Arsenite is known to exert at least some of its toxic effects through interaction with sulfhydryl groups, and the nonprotein sulfhydryl glutathione appears to play an important role in the detoxification of arsenite (65). Disturbances in intracellular calcium homeostasis is also involved in arsenite-induced cytotoxicity (66). A cathepsin-like protease activity may also be involved in the killing effects of arsenite (67). These studies and speculation suggest that arsenite may induce other cellular injuries besides DNA damage to cause cell death.

Numerous studies have shown that p53 activation is signaled via distinct transduction pathways in response to different stresses and results in various cellular responses (28). Arsenite has been reported to induce DNA damage (19, 20, 21), oxidative stress (68, 69), oncogene expression(70, 71, 72), and gene amplification (73). In the present study, using a highly sensitive comet assay, we confirmed that arsenite treatment results in DNA strand breaks in HFW cells. At the same dose used in this study, we demonstrated previously that arsenite not only induces chromosome aberrations and micronuclei but also induces aneuploidy in HFW cells that survived (7, 12). DNA strand breaks are potential lesions that can be converted into such cytogenetic alterations. Although arsenite-induced oxidative stress is thought to play a crucial role in the induction of DNA strand breaks(20, 74), the exact mechanism of arsenite-induced DNA strand breaks and the roles of ATM pathway activation warrant our further concern.

We thank Douglas Platt for carefully reading the manuscript.