Effects of arsenic on telomerase and telomeres in relation to cell proliferation and apoptosis in human keratinocytes and leukemia cells in vitro
Tong-Cun Zhang1,
Michael T. Schmitt2 and
Judy L. Mumford2,3
1 National Research Council, 2001 Wisconsin Avenue, NW, Washington DC, 20007 and 2 Human Studies Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA
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Abstract
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Telomeres are critical in maintaining chromosome and genomic stability. Arsenic, a human carcinogen as well as an anticancer agent, is known for its clastogenicity. To better understand molecular mechanisms of arsenic actions, we investigated arsenite effects on telomere and telomerase and determined cell growth and apoptosis in HL-60 and HaCaT cells in vitro. Low concentrations (0.11 µM in HaCaT and 0.10.5 µM in HL-60) of arsenite increased telomerase activity, maintained or elongated telomere length, and promoted cellular proliferation. High concentrations (>140 µM) of arsenite decreased telomerase activity, telomere length and induced apoptosis. Results from the studies comparing cell lines with and without telomerase activity suggested that telomerase was involved in arsenic-induced apoptosis. The spin trap agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was effective in protecting the arsenite-induced telomere attrition and apoptosis, suggesting that reactive oxygen species may play an important role in the shortening of telomeres and apoptosis induced by arsenic. These findings suggest the carcinogenic effects of arsenic may be partly attributed to increase in telomerase activity leading to promotion of cell proliferation and its anticancer effects by exerting oxidative stress and leading to telomeric DNA attrition and apoptosis.
Abbreviations: DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; hTERT, human telomerase reverse transcriptase; ROS, reactive oxygen species; TRAP, telomere repeat amplification protocol; TRF, terminal restriction fragment
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Introduction
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Arsenic is ubiquitous in the environment. Chronic exposure to arsenic has been associated with increased incidence of cancers in skin, lung, bladder, liver and kidney in humans (1,2). In vitro and in vivo studies have shown arsenicals cause chromosome abnormalities, including elevation of the frequency of micronuclei, sister chromatid exchanges, chromosome aberrations and gene amplification (35). Arsenicals also have been reported to induce oxyradicals, DNA and chromosome damage, inhibit DNA repair and modulate DNA methylation in mammalian cells (46). Animal studies have shown that arsenic functions as a co-mutagen and is a tumor promoter (4). Arsenic has been reported as a human carcinogen and paradoxically it also has been used therapeutically for treatment of acute promyelocytic leukemia (7,8).
Although progress has been made in identifying the possible mechanisms of arsenic carcinogenesis, the precise mechanisms are not yet clear due to the lack of appropriate animal models. The most putative modes of action for arsenic carcinogenesis are chromosomal abnormalities, oxidative stress and promotion of cell proliferation (4). In this study, we investigated the effects of arsenic on telomeres, which play a crucial role in maintaining the integrity of chromosome, cell proliferation and apoptosis.
Telomeres, the nucleoprotein structures at chromosome termini, are composed of simple repetitive G/C-rich DNA complexed with specific telomere binding proteins as well as histones (9,10). The human telomeric DNA sequence is (TTAGGG)n and the length of the terminal restriction fragment (TRF) containing the (TTAGGG)n tract varies in different cell types (10,11). Telomeres play a critical role in maintaining chromosome stability by protecting chromosome ends from degradation and fusion (1214). Because of the inability of DNA synthesis to copy the 3' termini of chromosomes during cellular replication, there is a gradual loss of telomeric sequence under normal conditions. For this reason, the chromosomes shorten with progressive cell division, eventually triggering either senescence or apoptosis when telomere length becomes critically short. Activation of telomerase, a special ribonucleoprotein reverse transcriptase, is important in maintaining telomere length. The expression of human telomerase reverse transcriptase (hTERT) is a rate-limiting step for telomerase activity. Telomerase is necessary for the sustained growth of most human tumors and plays an important role in tumorigenesis (9,11,14). Telomerease activity has been detected in different types of tumor cells (11,12). Since regulation of telomerase activity and telomere length are critical in maintaining stability of chromosome and arsenic is known for its clastogenicity in human cells, alterations in telomerase activity and telomere may be associated with the chromosomal damage exerted by arsenic. Chou et al. show that arsenic inhibits telomerase transcription leading to chromosomal end lesions in human cells in vitro (15). The objective of this study was to examine the effects of arsenite on telomerase activity, telomere length and cell viability in two types of human cell lines, including HaCaT (human epidermal keratinocytes), and HL-60 cells (promyelocytic leukemia cells). Human epidermal keratinocytes are primary targets of arsenic because skin cancer has been associated with chronic exposure to arsenic. Promyelocytic leukemia cells are also used to investigate the effects of arsenic because arsenic trioxide has been used as an anticancer agent for acute promyelocytic leukemia cases.
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Materials and methods
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Cells
HaCaT and HL-60 are the two main cell lines under investigation for the effects of arsenic in this study. SW13 (human fibroblasts) and HT1080 (human fibrosarcoma) were used as the telomerase negative and positive controls, respectively, for immunohistochemical detection of telomerase hTERT protein. The telomerase negative cell lines, including SW13 and SW480 (human colon adenocarcinoma), and the telomerase positive cell line, HT1080 were also used to investigate the effect of telomerase on arsenic-induced apoptosis. HaCaT cells were a gift from Dr Norbert E.Fusenig, German Cancer Institute, Heidelberg, Germany. All other cell lines were from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
Cell treatment and viability
A stock solution of 1 mM sodium arsenite (Sigma, St Louis, MO) in RPMI 1640 medium was prepared. After 24 h of cell seeding with cells showing 5060% confluence, sodium arsenite was added to the cultures to reach the final concentrations of 0.140 µM. After 1, 3 and 5 days of arsenite treatment, cell growth, including cell viability and total cell number, was determined by MTT assay, a cell proliferation kit (Roche Molecular Biochemicals, Indianapolis, IN) and trypan blue exclusive method. In MTT assays, the cells were plated in the 96-well plates (triplicate wells for each treatment) at a density of 1 x 104 cells in 0.2 ml of medium/well and cell growth was measured at 560 nm absorbance and expressed as the percentage of controls. In each experiment, three replicates were included for each treatment and control. Three experiments were conducted for each treatment and control in both cell lines.
Cell apoptosis
The treated and control cells were harvested, centrifuged, washed in PBS buffer and resuspended in PBS buffer at 105/ml. The cells were stained for 15 min with 10 µM Hoechst 33342 (Ho342, Molecular Probes, Eugene, OR) and then with 10 µM propidium iodide (PI, Molecular Probes, Eugene, OR) for 15 min. The percentage of apoptotic cells were determined by Ho342 staining, while damage to the plasma membrane indicating cell necrosis was detected by PI staining. The defining characteristic of apoptosis is the change in cell morphology, including cell shrinkage, chromatin margination, nuclear condensation and then segmentation and division into apoptotic bodies (16). From each treatment, at least 300 cells were counted, and the percentage of apoptotic and necrotic cells was calculated.
Reactive oxygen species and arsenic effects
In order to assess the role of reactive oxygen species (ROS) on cell growth, apoptosis and telomere length, a spin trap agent (5,5-dimethyl-1-pyrroline-N-oxide or DMPO from Sigma) was added at the concentration of 2.5 mM to the cell cultures treated with arsenite and controls for 3 days (for telomere assay) and 5 days (for cell growth and apoptosis assays). Cell viability, apoptosis and telomere length (see below) were assessed to determine the effects of DMPO on arsenic-treated cells.
Immunostaining of telomerase protein
Protein expression of hTERT was detected by avidinbiotinperoxidase immunocytochemistry staining methods as described previously with minor modifications (17). Briefly, a monolayer of the cells on glass microscope slides was prepared using a cytospin and fixed in 10% formalin. The primary antibody, polyclonal rabbit antibody against hTERT (EST21A, Alpha Diagnostic International, San Antonio, TX) diluted to 10 µg/ml (18) and secondary antibody from the ABC ELITE rabbit IgG detection Kit (Vector Laboratories, Burlingame, CA) was used. The peroxidase activity of the sample was detected using hydrogen peroxide as substrate and diaminobenzidine (Stable DAB, Research Genetics, Huntsville, AL) as a chromogen. HT1080 and SW13 were used as positive and negative controls, respectively.
Telomerase activity
Telomerase activity in cell extracts was measured by the PCR-based telomere repeat amplification protocol (TRAP) using TRAPeze kit (Intergen, Gaithersburg, MD). Briefly, the cells were washed in phosphate-buffered saline and homogenized in the CHAPS lysing buffer for 30 min on ice. Then, protein (50100 ng) from each cell extract was analyzed in the TRAP reaction. The cell extracts were added directly to the TRAP reaction mixture containing dNTPs, TS primer (6 x 105 c.p.m.), reverse primer mixture and Taq DNA polymerase. Then, the extended telomerase products were amplified by two-step PCR (94°C, 30 s and 60°C, 30 s) for 27 cycles. The bands indicating telomerase activity were analyzed using the Lumi-Imager (Boehringer Mannheim Corporation, Indianapolis, IN) with LumiAnalyst Software. Telomerase activity was quantified by measuring the signal of telomerase ladder bands and was calculated as the ratio to the internal standard and expressed as percent of the controls (19).
Telomerase and apoptosis
In order to assess the role of telomerase on arsenic-induced apoptosis, telomerase negative (SW13, SW480) and positive cells (HT1080), HL-60 and HaCaT were treated with arsenic ranging 0 to 40 µM for 5 days and percent of apoptotic cells was determined.
Analysis of telomere length by Southern blotting
DNA of the cell cultures was isolated using QIAamp Blood Kit (Qiagen, Valencia, CA). Determination of TRF was used to assess telomere length using a Telomere Length Assay Kit (Roche Molecular Biochemicals, Indianapolis, IN). TRF length corresponds qualitatively to telomere length. Purified genomic DNA (2 µg) was digested with Hinf I and Rsa I and separated by agarose gel electrophoresis. The DNA fragments were transferred to a positively charged nylon membrane by capillary transfer and fixed by UV- crosslinking (120 mJ). The membrane was hybridized with telomere-specific probe (TTAGGG)4, labeled with digoxigenin (DIG) and incubated with anti-DIG-alkaline phosphatase according to the manufacturer's protocol. The chemiluminescence signals and TRF length were detected using the Lumi-Imager.
Statistical analysis
The values of mean and standard deviation from the replicates of independent experiments were calculated. Student's t-test was used to test whether the treated cultures were significantly different from the controls.
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Results
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Arsenic effects on cell growth
The effects of arsenite on cell growth of HL-60 and HaCaT by MTT assays are shown in Figure 1. When cells were exposed to arsenite ranging from 0.1 to 40 µM for 1, 3 and 5 days, cellular growth was increased at low doses showing maximal effect at 0.5 µM, whereas cell growth was significantly decreased at concentrations
1 µM in both cell lines. The increase in cell growth was significantly different from the controls (P < 0.05) for 5 days treatment for both cell lines. These results indicated that arsenite induced cell proliferation at low concentrations and inhibited cell growth at higher concentrations. Similar results were also found using trypan blue method (data not shown).

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Fig. 1. Effects of arsenite on cell growth by MTT assay and apoptosis by Hoechst/PI staining. HL-60 and HaCaT were treated with arsenite at 040 µM for 15 days. In MTT assays, data shown are cell viability measured by optical density (OD) at 560 nm and expressed as percent of controls. Apoptosis was expressed as the percent of apoptotic cells. All assays were conducted in three replicates for each treatment. Data shown are mean ± SD from three independent experiments.
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Arsenic effects on apoptosis
In all the cell cultures treated with various concentrations of arsenite, the necrotic cells (showing PI- and trypan blue- positive staining cells) were all below 5%. Dose- and time-related apoptotic effects from arsenite exposure after 1, 3 and 5 days were observed in both cell lines (Figure 1). When the concentrations of arsenite were <1 µM, induction of cell apoptosis by arsenite was not significantly increased. However, the percentage of apoptotic cells was significantly increased (P < 0.01 and P < 0.05 for HL-60 and HaCaT, respectively for 5 days treatment) when arsenite concentrations were higher than 1 µM. In comparison between the two cell lines, HL-60 cells were more susceptible to apoptosis from arsenite treatment than HaCaT cells. These results showed that arsenic induced cell proliferation and apoptosis in both cell lines, depending on the dose of arsenite.
Effects of DMPO on arsenic-induced cell growth and apoptosis
The results of adding DMPO, a scavenger of ROS, to the arsenic-treated cultures significantly decreased the arsenic-induced apoptosis in both cell lines (P < 0.01 and P < 0.05 for HL-60 and HaCaT, respectively) as shown in Figure 2. The cultures treated with 40 µM arsenic showed a decrease of 34 and 40%, respectively, in HL-60 and HaCaT in apoptosis by DMPO. However, DMPO showed no effects on the cell proliferation. No significant effects were observed on cell viability from DMPO treated alone (data not shown). These results suggest that ROS may be involved in the arsenic-induced apoptosis but not involved in the arsenic-induced cell growth.

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Fig. 2. Effects of DMPO on arsenite-induced cell growth and apoptosis in HL-60 and HaCaT cells. All cells were treated for 5 days with arsenite at 040 µM or arsenite at the same concentrations combined with 2.5 mM DMPO. Cell viability and apoptotic cells were determined by MTT assay and Hoechst/PI staining assay, respectively. All assays were conducted in three replicates for each treatment. Data shown are mean ± SD from three independent experiments.
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Arsenic effects on telomerase activity
The effects of arsenite on telomerase activity or hTERT protein expression were shown in Figure 3. The control cells of HL-60 (a tumor cell line) and HaCaT (an immortal cell line) were positive in protein expression as expected (see Figure 3C and F). Protein expression of hTERT in both cell lines showed an increase at 0.5 µM arsenite treatment and a decrease at 20 µM as shown in Figure 3D, E, G and H.

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Fig. 3. hTERT protein expression by immunocytochemistry staining. (A) SW13 cells as the negative controls. (B) HT1080 cells as the positive controls. (CE) HL-60 cells treated with arsenite at 0, 0.5 and 20 µM, respectively, for 3 days. (FH) HaCaT cells treated with arsenite at 0, 0.5 and 20 µM, respectively, for 3 days. Arsenite significantly elevated hTERT protein expression at low dose (0.5 µM) as shown in (D) and (G), whereas arsenite significantly inhibited hTERT protein expression at high dose (20 µM) as shown in (E) and (H) in both cell lines.
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The effects of arsenite on telomerase activity by TRAP assays were shown in Figure 4A and B (for quantification). At a low dose (0.5 µM) telomerase activity was elevated 2-fold for HL-60 and 3-fold for HaCaT whereas at high doses (10 and 20 µM) the activity was inhibited. There is a dose-dependent decrease in telomerase activity following arsenite treatment at 10 and 20 µM concentrations in HL-60. Since the results showed that telomerase activity had a 23-fold increase resulting from arsenite treatment at low doses, we further investigated the effects of low dose arsenite treatment (0.1, 0.5 and 1.0 µM) in HL-60. The results showed a dose-dependent increase in telomerase activity up to 0.5 µM and then decreased at 1 µM as shown in Figure 4C. In addition, a time-dependent course of telomerase activity, both up- and down-regulation, were shown in both cell lines exposed to arsenite (data not shown). The up-regulation (at 0.5 µM) and down-regulation (at 10 and 20 µM) of telomerase activity were also in good agreement with the results of hTERT protein expression as described above.

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Fig. 4. Effects of arsenite on telomerase activity. (A) Telomerase activity by TRAPeze assay in HL-60 and HaCaT cells. Cells were treated with 0, 0.5, 10 and 20 µM of arsenite for 3 days and telomerase activity was determined. The experiment was repeated and the results were in agreement. (B) Quantification of telomerase activity from TRAPeze assay in (A). Telomerase activity was quantified by measuring the signal of telomerase ladder bands, and dividing by the intensity of the bands from the internal controls. The values were expressed as percent of the controls. (C) Telomerase activity at low doses by TRAPeze assay in HL-60. Cells were treated with 0, 0.1, 0.5 and 1 µM of arsenite for 3 days and telomerase activity was determined.
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Telomerase and arsenic-induced apoptosis
The results comparing the telomerase negative and positive cell lines and HL-60 and HaCaT showed that the telomerase negative cells, SW480 and SW13 were more resistant to arsenite-induced apoptosis, whereas the telomerase positive cells (HT1080) were more sensitive to arsenic-induced apoptosis as shown in Figure 5. The apoptotic rates of HL-60 and HaCaT were also shown in Figure 5 to compare with these telomerase negative and positive cells. These results suggest that telomerase activity may be related to arsenic-induced apoptosis.

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Fig. 5. Comparing telomerase-negative and -positive cells to investigate the effects of telomerase on arsenic-induced apoptosis. All cell lines were treated with arsenite 040 µM for 5 days. Cell apoptosis was determined by Hoechst/PI staining assays. All assays were conducted in three replicates for each treatment and data shown are mean ± SD from three independent experiments.
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Arsenic effects on telomere length and co-treatment with DMPO
The results of determining telomere length by TRF analysis are shown in Figure 6. The TRF length of the cells treated with 0.5 µM maintained original length in HL60 cells and showed some elongation in HaCaT cells. Conversely, when the cells were exposed to higher concentrations of arsenite (10 and 20 µM), TRF length was shortened significantly. When DMPO was added to the arsenite treated cultures (10 µM) in HL-60, telomere length of the DMPO-treated cells was elongated when compared with the arsenite treated cells as shown in Figure 7. This result showed that DMPO provided some protection against telomere shortening induced by arsenite and ROS may play some role in the telomere attrition.

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Fig. 6. Effects of arsenite on telomere length by Southern blot. Cells were exposed to 020 µM of arsenite for 3 days and terminal restriction fragments of DNA from the treated or control cells were determined.
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Fig. 7. Effects of DMPO on telomere length. HL-60 cells were treated for 3 days with 10 µM arsenite or with 10 µM arsenite combined with 2.5 mM DMPO. Telomere length was determined by Southern blot.
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Discussion
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In this study, we investigated the effects of arsenite on telomeres, telomerase and cell viability in human cells in vitro to better understand the mechanisms of arsenic carcinogenesis and anticancer effects. We found that arsenite exerted effects on telomerase activity and telomere length and correspondingly also showed induction of cell proliferation or apoptosis depending on the dose. Previous studies have implicated that telomere shortening may serve as an important checkpoint to limit the potential of human cells to proliferate (11,13). When the telomeres are shortened to a critical length, the signal for senescence or cell apoptosis is activated. Tumor cells are able to bypass this growth control checkpoint by activation of telomerase. Telomerase activity or maintenance of telomere stability is necessary for the continued proliferation of cells and is critical in immortalization and tumor progression of human cells (1113). Inhibition of telomerase activity suppresses the growth of human cancer cells and leads to chromosomal damage and apoptosis (20). Here, we showed that arsenite altered telomerase activity and telomere length in HL-60 and HaCaT cells. At low concentrations (<1.0 µM) arsenite promoted telomerase activity and increased or at least maintained telomere length. These observations corresponded with induction of cell proliferation. Treatment with high concentrations of arsenite (>1.0 µM) decreased telomerase activity and showed a rapid and dramatic loss of telomeric DNA, leading to cell apoptosis. Chou et al. reported that arsenic trioxide at 0.751.0 µM inhibited telomerase transcription and resulted in chromosomal end lesions, which promote either genomic instability toward carcinogenesis or cancer cell death in NB4 cells (15). Telomere and telomerase play a dual role in tumorigenesis and replicate senescence. In immortal cells, the maintenance of telomerase and telomere can stabilize genomic instability and chromosomal abnormalities and thus permit unlimited cell proliferation, whereas in non-immortal cells, inhibition of telomerase activity and telomere shortening causes either cell apoptosis or promotion of chromosomal end-to-end fusion which may lead to initiation of carcinogenesis (21). The observed changes here in telomerase activity and telomere length by arsenic may partly explain paradoxically carcinogenic and anticancer effects of arsenic.
Our studies comparing the telomerase negative and positive cells showed that telomerase-active cell lines are sensitive to arsenic-apoptosis and the telomerase-negative cell lines are more resistant. These observations suggested that telomerase plays some role in the arsenic-induced apoptosis. In this study, arsenic at high dose induced dramatic loss of telomeric DNA and showed a rapid increase in apoptosis in HL-60 and HaCaT cells. This may be in part due to inhibition of telomerase and also due to the massive telomere loss during the early stage of DNA damage induced by arsenic. Proficient telomeric end-capping function is crucial in chromosomal stability and cell viability. It has been reported that massive telomere loss is an early event of DNA damage-induced apoptosis (22). Ramirez et al. showed that the cells undergoing apoptosis upon DNA damage exhibit a rapid and dramatic loss of telomeric sequences. This telomere loss occurs at early stages of apoptosis, because it does not require caspase-3 activation and is induced by loss of mitochondrial membrane potential and ROS production. These observations suggest a direct effect of mitochondrial dysfunction on telomeres. It is possible that high dose of arsenic may act like other DNA damaging agents that can trigger apoptosis initially by other mechanisms and cause the degradation of telomere repeats (22,23).
The mechanisms for arsenite-induced the alterations of telomerase activity and telomere length in this study are still unclear. In this study, we showed increased telomerase and cell proliferation by arsenite at low dose. Some possible mechanisms are the up-regulation of heat shock proteins (such as Hsp90) and some cell growth factors (such as EGF, TGF-
), which can be induced by arsenic and also are positive regulators of telomerase and cell proliferation (2427). In addition, other signaling pathways mediated by arsenic can also alter gene expression systems, such as c-myc, p53, c-Jun N-terminal kinases and ataxia-telangiectasia mutated, which are also known to play an important role in regulating telomerase (2733). In this study we showed that high concentrations of arsenite decreased telomerase activity and telomere length and induced apoptosis. Oxidative radicals have been shown to cause damage to telomeric-DNA. Arsenic is known for generation of oxyradicals that consequently deranges cellular signaling pathways and causes DNA strand breaks and chromosome damage (4,23). Liu et al. reported that arsenic-induced oxidative stress promotes telomere attrition and apoptotic cell death in mouse embryos (34). In this study, co-treatment of DMPO, an oxygen free radical scavenger, with arsenite protected cells against arsenic-induced apoptosis at high dose but did not protect cells in cell proliferation with low dose of arsenic treatments. DMPO also provided some protection against shortening of telomere length induced by arsenic. These observations suggest that oxidative radicals may at least in part be attributed to the arsenic-induced telomere shortening and apoptosis at high dose.
Some differences in sensitivity to arsenic were observed in the two different human cell types investigated in this study. HL-60 cells, which are promyelocytic leukemia cells, were more sensitive than the skin keratinocytes, HaCaT cells, in decreasing telomerase activity (and shortening of telomeres) and induction of apoptotic effects by arsenite. This finding may explain why arsenic has been shown to be effective as a therapeutic agent for treatment of acute promyelocytic leukemia patients. In this study we also showed that telomerase negative cells, SW13 and SW480, were resistant to arsenic-induced apoptosis. This may be due to the fact that these telomerase negative cells are capable of maintaining long heterogeneous telomeres by the ALT (alternative lengthening of telomeres) mechanisms and are resistant to anti-telomerase drug therapies (35) and arsenic in this case. Whereas, telomerase positive cells were more sensitive to arsenic-induced apoptosis. These findings substantiated that arsenic is targeting on the cells with telomerase activity in inducing apoptosis.
In summary, this study demonstrated that arsenite altered telomere length and telomerase activity, and showed effects on cell proliferation and apoptosis in human cells. This is the first report showing that low concentrations of arsenic increased telomerase activity, maintained or elongated telomere length, and correspondingly promoted cell proliferation, which may lead to tumor progression or tumorigenesis. High concentrations of arsenic decreased telomerase activity and telomere length and induced apoptosis. These results may, in part, explain the paradoxical phenomena of arsenic actions in carcinogenic and anticancer effects and provide some insight into possible mechanisms of arsenic effects in human cells. The findings showing that mode of action in human cells varies depending on the arsenic dose may have potential implications on the shape of the doseresponse curves for low and high levels of arsenic exposure in arsenic risk assessment in humans.
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Notes
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3 To whom correspondence should be addressed Email: mumford.judy{at}epa.gov 
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Acknowledgments
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This work was performed while T.-C.Zhang was a NRC Research Associate at Human Studies Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, North Carolina. This manuscript has been reviewed by National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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Received March 20, 2003;
revised June 25, 2003;
accepted August 3, 2003.