ERK Activation in Arsenite-Treated G1-Enriched CL3 Cells Contributes t
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《毒物学科学杂志》
Molecular Carcinogenesis Laboratory, Institute of Biotechnology and Department of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan
Department of Radiation Oncology, Taichung Veterans General Hospital, Taichung, Taiwan
ABSTRACT
Arsenite is known to induce chromosomal damage and extracellular signal-regulated kinases 1/2 (ERK) signaling transduction pathway. Arsenite also perturbs mitotic spindle and induces G2/M prolongation, leading to genomic instability. However, little is known concerning whether G1 phase is susceptible to arsenite in causing genomic instability and ERK activation. In this study, we investigate the roles of ERK activation in survival, micronucleus formation, and nucleotide excision repair (NER) synthesis in arsenite-treated G1-enriched CL3 human non-small-cell lung carcinoma cells. We found that G1 was the most insensitive phase to arsenite cytotoxicity, yet it was highly susceptible to arsenite in micronucleus induction. After arsenite exposure, the G1 cells exhibited a marked retard in the formation of binucleated cells when they were cultured in cytochalasin B, an inhibitor of cytokinesis, suggesting that arsenite delays the cell cycle progression. Arsenite activated sustained-ERK signal in G1 cells whose suppression further decreased cell proliferation and survival and could lower the micronucleus induction. The NER synthesis activity of G1 cells was inhibited by arsenite as a function of the extent of ERK activation. Intriguingly, blockage of ERK activation recovered NER synthesis activity in the arsenite-treated G1 cells. Together, these results suggest that ERK activation in arsenite-treated G1 cells counteracts cytotoxicity and contributes to genomic instability via NER synthesis inhibition and micronucleus induction.
Key Words: arsenite; MAPK; nucleotide excision repair; genomic instability; cell synchronization.
INTRODUCTION
Arsenic is a widely distributed natural toxicant that exists mainly in ground water (Nordstrom, 2002). Epidemiological evidence indicates a strong association between chronic arsenic exposure and increased incidences of lung, skin, bladder, kidney, and liver cancers in human (Bates et al., 1992; Chen et al., 1992, 1985; Chiou et al., 2001). Treatment of mammalian cells with arsenite causes several types of genomic injuries such as oxidative DNA adducts (Bau et al., 2002; Wang et al., 2001), DNA strand breaks (Bau et al., 2002; Dong and Luo, 1993; Wang et al., 2001; Yih and Lee, 2000), chromosomal aberrations (Jha et al., 1992; Lee et al., 1985b), micronuclei (Gurr et al., 1998; Liu and Huang, 1996, 1997; Wang and Huang, 1994; Wang et al., 1997; Yih and Lee, 1999), telomere shortening (Liu et al., 2003), and aneuploidy (Vega et al., 1995; Yih et al., 1997). Arsenite also acts as a tumor promoter via induction of cell transformation (Huang et al., 1999; Landolph, 1994; Lee et al., 1985b) and alteration of DNA methylation patterns (Mass and Wang, 1997; Zhao et al., 1997). Arsenite can enhance the genotoxicity of several mutagens including UV (Lee et al., 1985a; Yang et al., 1992), possibly due to interference with DNA repair processes (Bau et al., 2001; Chien et al., 2004; Hartwig et al., 1997; Lee-Chen et al., 1992; Okui and Fujiwara, 1986). Unlike typical carcinogens, arsenite is a weak mutagen in standard assay systems and causes mainly gene deletions (Hei et al., 1998; Wiencke et al., 1997). Nonetheless, exposure of human osteosarcoma TE85 cells to extremely low doses of arsenite for 20 or more generations markedly increases mutant frequency in the hprt gene (Mure et al., 2003). This delayed mutagenesis is postulated as a consequence of progressive epigenetic effects of arsenite such as alterations in signaling pathways (Mure et al., 2003).
The extracellular signal-regulated kinases 1/2 (ERK) are vital intracellular signaling components that become phosphorylated and activated in response to a wide diversity of extracellular stimuli including growth factors, cytokines, and environmental stresses (Kolch, 2000; Lewis et al., 1998). The best-characterized pathway leading to ERK activation is recruitment of the Raf/MEK/ERK three-kinase module to cell membrane via Ras to connect the upstream activated receptor tyrosine kinases (Kolch, 2000; Lewis et al., 1998). Activated ERK phosphorylates numerous substrates including transcription factors, other kinases, phosphatases, and cytoskeletal proteins for regulation of cell proliferation, differentiation, cell-cycle progression, transformation, migration, survival, and death. Previous studies have shown that arsenite can activate ERK dependent on Ras, Raf, and MEK (Liu et al., 1996; Ludwig et al., 1998). The arsenite-induced ERK activation is mediated via the EGF receptor and Shc adaptor (Chen et al., 1998) in a Src-dependent and ligand-independent mechanism for EGF receptor activation (Simeonova et al., 2002). Upon activation by arsenite, ERK increases the transcription of c-fos and c-jun and the DNA binding activity of AP-1 (Huang et al., 2001; Li et al., 2003), which is well correlated with a role of ERK activation in promoting anchorage-independent cell growth under arsenite exposure (Huang et al., 1999).
Several studies indicate that arsenite is a powerful inducer causing G2/M delay or arrest (Chen et al., 2002; Huang et al., 2000; Huang and Lee, 1998; McCabe et al., 2000; Yih et al., 1997; Yih and Lee, 2000). The arsenite-induced G2/M prolongation has been associated with perturbation of mitotic spindle (Huang and Lee, 1998; Yih et al., 1997), leading to cytogenetic alterations (Yih et al., 1997) or apoptosis (Huang et al., 2000; McCabe et al., 2000). Arsenite also induces G1 delay with a shorter duration than the G2/M delay (McCabe et al., 2000). However, the consequent fate of the G1 cells following arsenite exposure remains unknown. In this report, we investigate the effects of arsenite in (1) induction of cytotoxicity and micronucleus formation, (2) inhibition of nucleotide excision repair (NER) synthesis, and (3) activation of ERK in G1-enriched CL3 human non-small-cell lung carcinoma cells. By using specific inhibitors for ERK signaling, we further explored the roles of arsenite-induced ERK activation in cell proliferation, survival, micronucleus induction, and NER synthesis inhibition. Results obtained here suggest that G1 phase is sensitive to arsenite in ERK activation whose multi-functions can contribute to genomic instability.
MATERIALS AND METHODS
Cell culture.
The CL3 cell line established from a non-small-cell lung carcinoma was provided by Dr. P.-C. Yang (National Taiwan University Hospital, Taipei). Karyotype analysis revealed that 98% of the CL3 cells exhibit 46 chromosomes (Lee and Ho, 1994). Cells were cultured in RPMI1640 medium (Gibco/Life Technologies, Grand Island, NY) supplemented with 2.2% sodium bicarbonate, 0.03% L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum. Cells were maintained at 37°C in a humidified incubator containing 5% CO2 in air.
Centrifugal elutriation.
The G1-, S-, and G2/M-enriched cells were collected by the counterflow centrifugal elutriation using a Beckman J-6M centrifuge equipped with a JE-6B elutriation rotor (radius 8.6 cm) as previously described (Chao and Yang, 2001). Briefly, exponentially growing cells (1 x 108) were concentrated in 15 ml of RPMI1640. After delivering into the elutriation rotor, exponentially growing cells (1 x 108) were elutriated at a flow rate of 30 ml/min using RPMI1640 and fractionated at speed of 2000–1450 rpm (385–203 x g) at intervals of 50 rpm. A portion of the fractionated cells were subjected to flow cytometry analysis and showed that more than 90% those collected from centrifugal speed 2000–1850, 1750–1650, and 1550–1450 rpm were enriched respectively, at G1, S, and G2/M phases.
Flow cytometry.
Cells were trypsinized and fixed with 70% ethanol for at least 2 h at –20°C before centrifugation. The cell pellets were treated with propidium iodide (10 μg/ml) solution containing RNase A (100 μg/ml) and Triton X-100 (1%) for 30 min, followed by flow cytometry analysis using a FACScan and the CellQuest program (Becton-Dickinson, San Jose, CA).
Arsenite treatment.
Sodium arsenite (Merck, Darmstadt, Germany) was dissolved in MilliQ-purified water (Millipore, Bedford, MA). Cells (1 x 106) in exponentially growth or synchronous at the G1, S, or G2/M phases were exposed to arsenite (50–100 μM) in serum-free medium for 3 h. In experiments to determine the role of ERK signaling under arsenite exposure, G1 cells were treated with 5–50 μM arsenite for 3 h in serum-free medium in the presence or absence of 5–50 μM PD98059 (Calbiochem, San Diego, CA) or 1–5 μM U0126 (Calbiochem). After treatment, the cells were washed twice with phosphate-buffered saline (PBS), harvested immediately or allowed recovery for various times, and then subjected to the following analyses.
Cell proliferation.
After treatment, the cells were kept in a CO2 incubator for another 3 days. The cells were then trypsinized, and a portion of cells were mixed with 0.4% trypan blue for 15 min. The unstained cells were counted using an inverted microscope and a hemacytometer.
Colony-forming ability.
Cells were trypsinized and plated at a density of 200–1000 cells/60 mm-Petri dish in triplicate. Following incubation for 12–14 days the cell colonies were stained with 1% crystal violet solution (in 30% ethanol). The percent of survival was determined to be the number of colonies in the treated cells divided by those obtained in the untreated cells.
Cytokinesis-block micronucleus assay.
Micronuclei can be observed in binucleated cells that complete nuclear division and are blocked from cytokinesis using cytochalasin B, a microfilament-assembly inhibitor. This cytokinesis-block process (Fenech, 1993) was adopted to analyze arsenite-induced micronucleus formation. Briefly, cells were cultured in media containing 1 μg/ml of cytochalasin B for 24–48 h. Next, the cultures were washed with PBS, treated with 0.05% KCl for 3 min at room temperature, and then fixed in 3 ml of Carnoy's solution (20:1, methanol: acetic acid, v/v) for 15 min. The dishes were air-dried and stained for 15 min with freshly prepared Giemsa's solution (10% in 0.1 mM sodium phosphate buffer, pH 6.8). The dishes were blindly coded, and two thousand binucleated cells in each treatment were examined for scoring micronuclei per binucleated cells using an inverted microscope.
Preparation of whole-cell extract (WCE).
Cells were rinsed twice with cold PBS and lysed on ice in a WCE buffer (20 mM HEPES, pH 7.6, 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 0.1 mM Na3VO4, 50 mM NaF, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, and 100 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride). The cell lysate was collected by a rubber policeman, rotated at 4°C for 30 min, centrifuged at 10,000 rpm for 10 min, and the precipitates were discarded. The BCA protein assay kit (Pierce, Rockford, IL) was adopted to determine protein concentrations using bovine serum albumin as a standard.
Western blot analysis.
Equal amount of cellular proteins in WCE from each set of experiments were fractionated on 10% SDS–polyacrylamide gels. The protein bands were then transferred to PVDF membranes and probed with primary antibody followed with a horseradish peroxidase-conjugated second antibody. The antibodies against phospho-ERK(Thr202/Tyr204) (#9101) and ERK2 (sc-154) were obtained, respectively, from Cell Signaling (Beverly, MA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibody reaction was performed using the enhanced chemiluminescence detection procedure (Santa Cruz Biotechnology Inc.). To reprobe the membrane with another primary antibody, the blots were stripped from the membranes by a solution containing 2% SDS, 62.5 mM Tris–HCl (pH 6.8), and 0.7% -mercaptoethanol at 50°C for 15 min.
NER synthesis.
Supercoiled plasmid pUC19 (250 ng/μl in ddH2O) was irradiated with UV (254 nm, 400 J/m2) at a radiation intensity of 1–1.5 J/m2/sec. The UV-irradiated or unirradiated pUC19 (250 ng) were served as substrates to determine the NER synthesis efficiency of proteins (60 μg) derived from WCE in reaction mixtures (50 μl) containing 20 μM each of dGTP, dCTP, and dTTP, 8 μM dATP, 2 μCi -32P-dCTP (3000 Ci/mmol), 2 mM ATP, 45 mM HEPES-KOH (pH 7.5), 60 mM KCl, 7.5 mM MgCl2, 0.9 mM dithiothreitol, 0.4 mM EDTA, 3.4% glycerol, and 18 μg bovine serum albumin. Reactions were performed at 30°C for 1 h and terminated by adding EDTA to a final concentration of 20 mM. Following RNase A (80 μg/ml) and proteinase K (190 μg/ml)-SDS (0.5%) treatment, the plasmid DNA in the reaction mixtures was purified using phenol/chloroform extraction and ethanol precipitation, linearized with BamHI, and subjected to 0.8% agarose gel electrophoresis. The plasmid DNA in gel was stained with 0.5% ethidium bromide and visualized under near-UV transillumination. The gel was then dried and subjected to autoradiography.
RESULTS
G1 Phase is Less Sensitive than S and G2/M Phases to Arsenite Cytotoxicity
To explore whether cells at various cell cycle phases exhibit different sensitivities to arsenite, proliferating CL3 cells were separated into fractions of G1, S, and G2/M phases by counterflow centrifugal elutriation, and their duration in these phases were estimated to be 12, 5, and 7 h, respectively. Equal numbers of the G1-, S-, and G2/M-enriched and asynchronous cells were left untreated or treated with arsenite (50–100 μM) for 3 h, washed with PBS, and the percent of survival was determined by colony-forming ability assay. As shown in Figure 1, G1 cells were less sensitive than S, G2/M, and asynchronous cells to cytotoxicity caused by arsenite; e.g., 50 μM arsenite reduced the cell viability to 45, 35, 29, and 17% of the untreated levels in G1, asynchronous, S, and G2/M phases, respectively.
G1 Phase is Susceptible to Arsenite in Micronucleus Induction and Cell Cycle Delay
Previous reports have indicated that arsenite acts as both a clastogen and an aneugen to induce micronuclei in various mammalian cells (Gurr et al., 1998; Liu and Huang, 1996, 1997; Wang and Huang, 1994; Wang et al., 1997; Yih and Lee, 1999). To determine whether the induction of micronuclei by arsenite is dependent on cell cycle phases, after arsenite treatment the G1- and G2/M-enriched and asynchronous cells were kept cultured for 24 h in medium containing cytochalasin B (a microfilament-assembly inhibitor) to allow the accumulation of binucleated cells in those that underwent complete nuclear division. Microscopic examination showed that a high frequency of micronucleus was induced in arsenite (50 μM) exposed G1 cells; the average numbers per 1,000 binucleated cells in the untreated and arsenite-treated G1 cells were 21 ± 3 and 202 ± 8, respectively (Fig. 2A). The induced micronucleus level in arsenite-treated G1 cells was higher than those observed in asynchronous (151 ± 5) and G2/M cells (137 ± 8) (Fig. 2A). As shown in Fig. 2B, cytochalasin B incubation for 24 h could accumulate 50–70% of the untreated cells with binuclei; however, only 14% of the arsenite-treated G1 cells had binuclei, which was much lower than those observed in arsenite-treated G2/M (39%) and asynchronous (47%) cells.
To obtain more binucleated cells for evaluation of micronucleus induction, the untreated and arsenite-treated G1 cells were incubated with cytochalasin B for 36–48 h or recovery in medium for 12 h followed by incubation with cytochalasin B for another 24 h (36) before microscopic examination. These alternative recovery strategies indeed enhanced the populations of binucleated cells (Fig. 2B), suggesting that arsenite delays cell cycle progression of the G1 cells. As shown in Figure 2A, the numbers of micronuclei induced in the arsenite-treated G1 cells under a prolonged recovery period were still higher than those generated in the untreated cells, although they declined as the recovery time increased.
Sustained ERK Activation in Arsenite-Treated G1 Cells Counteracts Cytotoxicity
We next explored whether arsenite influences ERK activation in G1 cells. Figure 3A shows that arsenite (50–100 μM, 3 h) increased phospho-ERK in G1-enriched CL3 cells, and the induced level was continuing for at least 24 h after arsenite removal. The arsenite-activated ERK could be blocked by two structurally unrelated inhibitors for the ERK upstream kinases MEK1/2, PD98059 and U0126 (Fig. 3B). The effect of ERK inhibition in cell proliferation and survival of the arsenite-treated G1 cells were determined using trypan blue exclusion and colony-forming ability assays. As shown in Figure 4A, the cell proliferation was markedly inhibited in the arsenite-treated G1 cells, which was further suppressed by cotreatment with either PD98059 or U0126. Similarly, the two inhibitors for MEK1/2 significantly enhanced the decrease in survival of arsenite-exposed G1 cells (Fig. 4B). These results imply that ERK activation in G1 cells by arsenite counteracts cytotoxicity and thereby contributes to cell proliferation and survival.
ERK Activation in Arsenite-Treated G1 Cells Can Lead to Micronucleus Formation
The role of ERK activation in the micronucleus formation was examined by exposing G1 cells to arsenite (50 μM) for 3 h in the presence or absence of PD98059 or U0126, followed by recovery for 24 h or 40 h in medium containing cytochalasin B. PD98059 or U0126 significantly lowered the micronucleus levels in arsenite-treated G1 cells when they were recovered in cytochalasin B for 24 h but not 40 h (Fig. 5). The results suggest that ERK activation can lead to micronucleus formation in a population of arsenite-treated G1 cells, possibly those bypassing the delay of cell cycle progression. Yet, after a prolonged recovery period the micronucleus formation in arsenite-treated G1 cells appears to be independent of the ERK signal.
ERK Activation in Arsenite-Treated G1 Cells Is Involved in NER Synthesis Inhibition
The mechanism by which arsenite enhances the genotoxicity of UV has been implicated due to the inhibition of NER system (Bau et al., 2001; Chien et al., 2004; Hartwig et al., 1997; Lee-Chen et al., 1992; Okui and Fujiwara, 1986; Wang and Huang, 1994). To explore whether arsenite inhibits NER synthesis of cells at G1 phase and the involvement of ERK signal, the G1-enriched cells were exposed to arsenite for 3 h in the presence or absence of PD98059 or U0126 and then allowed recovery for 12 h before WCE preparation. The proteins in the WCE were incubated with UV-irradiated DNA, 4 dNTP, and -32P-dCTP for the determination of NER synthesis. As shown in Figure 6, protein extracts derived from G1-enriched cells could stimulate NER synthesis of UV-damaged pUC19 DNA but not that of the undamaged DNA. Conversely, the efficiency of NER synthesis was suppressed using protein extracts derived from arsenite-treated G1 cells in a dose-dependent manner, which was negatively correlated to an increased ERK activation by arsenite (Fig. 6). Intriguingly, PD98059 or U0126 cotreatment could rescue NER synthesis efficiency in the arsenite-treated G1 cells (Fig. 7). Quantitative analysis showed that 50 μM arsenite reduced the repair capability to 40% as compared with those derived from the untreated G1 cells; cotreatment with 50 μM PD98059 or 5 μM U0126 recovered the NER synthesis efficiency to 80% of the untreated levels (Fig. 7). The results suggest that ERK mediates the NER synthesis inhibition in arsenite-treated G1 cells.
DISCUSSION
Arsenite is known to perturb mitotic spindle dynamics, resulting in G2/M prolongation (Huang and Lee, 1998; Yih et al., 1997); however, less attention has been paid as to whether arsenite injuries G1 cells and what the consequent cell fate is. In this report, we show that the G1-enriched CL3 cells exposed to moderate cytotoxic concentrations of arsenite (50 μM for 3 h) exhibit a marked retard in the formation of binucleated cells under cytochalasin B, suggesting arsenite delays G1 cell cycle progression. Flow cytometry analysis confirmed that such an arsenite treatment delays the G1 progression for 12 h (Li, unpublished data). The finding is consistent with a report that continued exposure of G1-enriched U937 myelomonocytic leukemia cells to 5 μM arsenic trioxide (a cytostatic condition) delays the cell cycle progression (McCabe et al., 2000). Here, we further demonstrate that the G1-enriched CL3 cells are susceptible to arsenite in micronucleus induction; in particular, 14% of the arsenite-treated G1 cells that could timely form binuclei exhibit high levels of micronuclei, and increase in recovery time can reduce the micronucleus formation (Fig. 2). The results suggest that a small population of the arsenite-treated G1 cells may escape the G1 delay, leading to the formation of high levels of micronuclei; also, after arsenite exposure a prolonged recovery period may facilitate removal of the damaged cells. In fact, the levels of phospho-p53(Ser15), a hallmark of G1 checkpoint, were markedly elevated in the arsenite-treated G1-enriched CL3 cells during the recovery period (Li, unpublished data). On the other hand, arsenite induces sustained ERK activation in the G1-enriched CL3 cells, which mediates cell proliferation and survival and can trigger micronucleus formation, possibly in those bypassing the cell cycle delay. Thus, arsenite can trigger dual and opposing signals in the G1-enriched CL3 cells regarding induction or bypassing the temporary cell cycle arrest.
Arsenite is known to impair NER processes (Bau et al., 2001; Chien et al., 2004; Hartwig et al., 1997; Lee-Chen et al., 1992; Okui and Fujiwara, 1986; Wang and Huang, 1994). Consistently, we found that arsenite reduces the NER synthesis efficiency of the G1 cells. More intriguingly, results presented here indicate that the sustained ERK signal mediates NER synthesis inhibition in the arsenite-treated G1 cells. It is well-known that the NER machinery removes a variety of detrimental DNA lesions including oxidative DNA adducts and pyrimidine dimers (Friedberg, 2001; Hoeijmakers, 2001; Lindahl and Wood, 1999). Previous reports have shown that arsenite induces oxidative DNA adducts and DNA strand breaks in mammalian cells (Bau et al., 2002; Wang et al., 2001). Accordingly, it is hypothesized that the ERK-mediated NER synthesis inhibition caused by arsenite may facilitate the accumulation of DNA lesions in G1 cells, which can result in increased micronucleus formation when the damaged cells bypass the cell cycle delay and progress through the M phase.
The finding that arsenite-induced sustained ERK activation in G1-enriched CL3 cells could mediate in micronucleus formation is consistent with reports that this signal is involved in micronucleus formation induced by oncogenic Ras in NIH 3T3 (Saavedra et al., 1999) and in thyroid PCCL3 cells (Saavedra et al., 2000). The result also agrees with required ERK activation for cell transformation caused by arsenite in mouse Cl 41 cells (Huang et al., 1999). Furthermore, activation of calcium-dependent protein kinase C has been associated with arsenite-induced micronucleus formation in Chinese hamster ovary cells (Liu and Huang, 1997). Protein kinase C can mediate ERK activation at the Raf or MEK levels and also has a desensitization effect in Raf activation (Schonwasser et al., 1998). Activation of Raf-MEK-ERK signaling by protein kinase C can also be achieved via phosphorylation of the Raf kinase inhibitory protein that subsequently dissociates from Raf (Corbit et al., 2003). Moreover, the AP-1 transactivation function induced by arsenite requires both the ERK and protein kinase C activities in mouse epidermal JB6 cells (Huang et al., 2001). The foregoing suggests that the Ras-Raf-MEK-ERK pathway may couple with protein kinase C to trigger AP-1 transactivation and subsequent micronucleus formation under arsenite exposure. Also, it deserves further study on correlation of the responsible intracellular species elicited by arsenic, such as its methylated derivatives, calcium, and free radicals (Florea, 2005) for the signal transduction leading to genomic damage.
The capability of arsenite to inhibit DNA repair is possibly not the result of direct enzyme inhibition and has been suggested via interfering with the expression of DNA repair genes (Hu et al., 1998). Indeed, recent reports have showed that arsenite can decrease the expression of genes encoding NER enzymes, e.g., XPC in human epidermal keratinocytes (Hamadeh et al., 2002) and XPC, XPD, and DNA ligase-1 in human bronchial epithelial BEAS-2B cells (Andrew et al., 2003b). Moreover, the expression of ERCC1, XPF, and XPB genes of the NER complex are found to be inversely correlated with toenail arsenic levels in a case-control study (Andrew et al., 2003a). On the other hand, NER enzymes such as hHR23A, hHR23B, and replication protein A2 have been identified to be modified posttranslationally by the ERK pathway (Lewis et al., 2000), suggesting that this signal may regulate the repair efficiency by triggering phosphorylation of NER enzymes. Whether the ERK pathway mediates down-regulation of DNA repair genes under arsenite exposure and the underlying mechanisms warrant further investigation.
The finding that sustained ERK activation by arsenite contributes to genomic instability, however, is contradictory to the reports that ERK signal exhibits a protective role in maintaining genomic stability under a variety of carcinogens. Active ERK mediates the suppression of micronucleus induction by cadmium (Chao and Yang, 2001) and ionizing radiation (Yacoub et al., 2001) and the prevention of mutagenesis under lead exposure (Lin et al., 2003). ERK signal has been associated with increased expression of DNA repair genes. For instance, activation of the Ras-ERK pathway by insulin up-regulates ERCC-1 mRNA expression (Lee-Kwon et al., 1998), and ERK activation upon ionizing radiation increases the expression of ERCC-1 and XRCC1 at the transcription and translation levels (Yacoub et al., 2001). The foregoing suggests that ERK signaling may play dual roles in regulating genomic stability and that arsenite may induce negative effectors to counteract the protective role of ERK.
In summary, this report indicates that acute arsenite exposure (50 μM, 3 h) not only delays cell cycle progression of the G1 cells but also induces sustained ERK signal in these cells to support cell proliferation and survival. The arsenite-induced ERK signal in G1 cells also triggers NER synthesis inhibition and micronucleus formation. It is conceivable that under such an arsenite exposure ERK is a multifaceted signal to counteract the G1 delay by facilitating cells having unrepaired DNA lesions to progress through the next cell cycle phases and thereby contributes to genomic instability. Results obtained here also suggest that blockage of the ERK signal pathway may have therapeutic potential to decrease genotoxicity caused by acute arsenite exposure to human.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Kun-Yan Jan for critical discussion of the manuscript. This work was supported in part by Grant NSC93–3112-B-007–010 from the National Science Council, Taiwan, and by Grant VTG92-P4–23 from the Medical Research Advancement Foundation in Memory of Dr. Chi-Shuen Tsou, Taiwan.
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Department of Radiation Oncology, Taichung Veterans General Hospital, Taichung, Taiwan
ABSTRACT
Arsenite is known to induce chromosomal damage and extracellular signal-regulated kinases 1/2 (ERK) signaling transduction pathway. Arsenite also perturbs mitotic spindle and induces G2/M prolongation, leading to genomic instability. However, little is known concerning whether G1 phase is susceptible to arsenite in causing genomic instability and ERK activation. In this study, we investigate the roles of ERK activation in survival, micronucleus formation, and nucleotide excision repair (NER) synthesis in arsenite-treated G1-enriched CL3 human non-small-cell lung carcinoma cells. We found that G1 was the most insensitive phase to arsenite cytotoxicity, yet it was highly susceptible to arsenite in micronucleus induction. After arsenite exposure, the G1 cells exhibited a marked retard in the formation of binucleated cells when they were cultured in cytochalasin B, an inhibitor of cytokinesis, suggesting that arsenite delays the cell cycle progression. Arsenite activated sustained-ERK signal in G1 cells whose suppression further decreased cell proliferation and survival and could lower the micronucleus induction. The NER synthesis activity of G1 cells was inhibited by arsenite as a function of the extent of ERK activation. Intriguingly, blockage of ERK activation recovered NER synthesis activity in the arsenite-treated G1 cells. Together, these results suggest that ERK activation in arsenite-treated G1 cells counteracts cytotoxicity and contributes to genomic instability via NER synthesis inhibition and micronucleus induction.
Key Words: arsenite; MAPK; nucleotide excision repair; genomic instability; cell synchronization.
INTRODUCTION
Arsenic is a widely distributed natural toxicant that exists mainly in ground water (Nordstrom, 2002). Epidemiological evidence indicates a strong association between chronic arsenic exposure and increased incidences of lung, skin, bladder, kidney, and liver cancers in human (Bates et al., 1992; Chen et al., 1992, 1985; Chiou et al., 2001). Treatment of mammalian cells with arsenite causes several types of genomic injuries such as oxidative DNA adducts (Bau et al., 2002; Wang et al., 2001), DNA strand breaks (Bau et al., 2002; Dong and Luo, 1993; Wang et al., 2001; Yih and Lee, 2000), chromosomal aberrations (Jha et al., 1992; Lee et al., 1985b), micronuclei (Gurr et al., 1998; Liu and Huang, 1996, 1997; Wang and Huang, 1994; Wang et al., 1997; Yih and Lee, 1999), telomere shortening (Liu et al., 2003), and aneuploidy (Vega et al., 1995; Yih et al., 1997). Arsenite also acts as a tumor promoter via induction of cell transformation (Huang et al., 1999; Landolph, 1994; Lee et al., 1985b) and alteration of DNA methylation patterns (Mass and Wang, 1997; Zhao et al., 1997). Arsenite can enhance the genotoxicity of several mutagens including UV (Lee et al., 1985a; Yang et al., 1992), possibly due to interference with DNA repair processes (Bau et al., 2001; Chien et al., 2004; Hartwig et al., 1997; Lee-Chen et al., 1992; Okui and Fujiwara, 1986). Unlike typical carcinogens, arsenite is a weak mutagen in standard assay systems and causes mainly gene deletions (Hei et al., 1998; Wiencke et al., 1997). Nonetheless, exposure of human osteosarcoma TE85 cells to extremely low doses of arsenite for 20 or more generations markedly increases mutant frequency in the hprt gene (Mure et al., 2003). This delayed mutagenesis is postulated as a consequence of progressive epigenetic effects of arsenite such as alterations in signaling pathways (Mure et al., 2003).
The extracellular signal-regulated kinases 1/2 (ERK) are vital intracellular signaling components that become phosphorylated and activated in response to a wide diversity of extracellular stimuli including growth factors, cytokines, and environmental stresses (Kolch, 2000; Lewis et al., 1998). The best-characterized pathway leading to ERK activation is recruitment of the Raf/MEK/ERK three-kinase module to cell membrane via Ras to connect the upstream activated receptor tyrosine kinases (Kolch, 2000; Lewis et al., 1998). Activated ERK phosphorylates numerous substrates including transcription factors, other kinases, phosphatases, and cytoskeletal proteins for regulation of cell proliferation, differentiation, cell-cycle progression, transformation, migration, survival, and death. Previous studies have shown that arsenite can activate ERK dependent on Ras, Raf, and MEK (Liu et al., 1996; Ludwig et al., 1998). The arsenite-induced ERK activation is mediated via the EGF receptor and Shc adaptor (Chen et al., 1998) in a Src-dependent and ligand-independent mechanism for EGF receptor activation (Simeonova et al., 2002). Upon activation by arsenite, ERK increases the transcription of c-fos and c-jun and the DNA binding activity of AP-1 (Huang et al., 2001; Li et al., 2003), which is well correlated with a role of ERK activation in promoting anchorage-independent cell growth under arsenite exposure (Huang et al., 1999).
Several studies indicate that arsenite is a powerful inducer causing G2/M delay or arrest (Chen et al., 2002; Huang et al., 2000; Huang and Lee, 1998; McCabe et al., 2000; Yih et al., 1997; Yih and Lee, 2000). The arsenite-induced G2/M prolongation has been associated with perturbation of mitotic spindle (Huang and Lee, 1998; Yih et al., 1997), leading to cytogenetic alterations (Yih et al., 1997) or apoptosis (Huang et al., 2000; McCabe et al., 2000). Arsenite also induces G1 delay with a shorter duration than the G2/M delay (McCabe et al., 2000). However, the consequent fate of the G1 cells following arsenite exposure remains unknown. In this report, we investigate the effects of arsenite in (1) induction of cytotoxicity and micronucleus formation, (2) inhibition of nucleotide excision repair (NER) synthesis, and (3) activation of ERK in G1-enriched CL3 human non-small-cell lung carcinoma cells. By using specific inhibitors for ERK signaling, we further explored the roles of arsenite-induced ERK activation in cell proliferation, survival, micronucleus induction, and NER synthesis inhibition. Results obtained here suggest that G1 phase is sensitive to arsenite in ERK activation whose multi-functions can contribute to genomic instability.
MATERIALS AND METHODS
Cell culture.
The CL3 cell line established from a non-small-cell lung carcinoma was provided by Dr. P.-C. Yang (National Taiwan University Hospital, Taipei). Karyotype analysis revealed that 98% of the CL3 cells exhibit 46 chromosomes (Lee and Ho, 1994). Cells were cultured in RPMI1640 medium (Gibco/Life Technologies, Grand Island, NY) supplemented with 2.2% sodium bicarbonate, 0.03% L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum. Cells were maintained at 37°C in a humidified incubator containing 5% CO2 in air.
Centrifugal elutriation.
The G1-, S-, and G2/M-enriched cells were collected by the counterflow centrifugal elutriation using a Beckman J-6M centrifuge equipped with a JE-6B elutriation rotor (radius 8.6 cm) as previously described (Chao and Yang, 2001). Briefly, exponentially growing cells (1 x 108) were concentrated in 15 ml of RPMI1640. After delivering into the elutriation rotor, exponentially growing cells (1 x 108) were elutriated at a flow rate of 30 ml/min using RPMI1640 and fractionated at speed of 2000–1450 rpm (385–203 x g) at intervals of 50 rpm. A portion of the fractionated cells were subjected to flow cytometry analysis and showed that more than 90% those collected from centrifugal speed 2000–1850, 1750–1650, and 1550–1450 rpm were enriched respectively, at G1, S, and G2/M phases.
Flow cytometry.
Cells were trypsinized and fixed with 70% ethanol for at least 2 h at –20°C before centrifugation. The cell pellets were treated with propidium iodide (10 μg/ml) solution containing RNase A (100 μg/ml) and Triton X-100 (1%) for 30 min, followed by flow cytometry analysis using a FACScan and the CellQuest program (Becton-Dickinson, San Jose, CA).
Arsenite treatment.
Sodium arsenite (Merck, Darmstadt, Germany) was dissolved in MilliQ-purified water (Millipore, Bedford, MA). Cells (1 x 106) in exponentially growth or synchronous at the G1, S, or G2/M phases were exposed to arsenite (50–100 μM) in serum-free medium for 3 h. In experiments to determine the role of ERK signaling under arsenite exposure, G1 cells were treated with 5–50 μM arsenite for 3 h in serum-free medium in the presence or absence of 5–50 μM PD98059 (Calbiochem, San Diego, CA) or 1–5 μM U0126 (Calbiochem). After treatment, the cells were washed twice with phosphate-buffered saline (PBS), harvested immediately or allowed recovery for various times, and then subjected to the following analyses.
Cell proliferation.
After treatment, the cells were kept in a CO2 incubator for another 3 days. The cells were then trypsinized, and a portion of cells were mixed with 0.4% trypan blue for 15 min. The unstained cells were counted using an inverted microscope and a hemacytometer.
Colony-forming ability.
Cells were trypsinized and plated at a density of 200–1000 cells/60 mm-Petri dish in triplicate. Following incubation for 12–14 days the cell colonies were stained with 1% crystal violet solution (in 30% ethanol). The percent of survival was determined to be the number of colonies in the treated cells divided by those obtained in the untreated cells.
Cytokinesis-block micronucleus assay.
Micronuclei can be observed in binucleated cells that complete nuclear division and are blocked from cytokinesis using cytochalasin B, a microfilament-assembly inhibitor. This cytokinesis-block process (Fenech, 1993) was adopted to analyze arsenite-induced micronucleus formation. Briefly, cells were cultured in media containing 1 μg/ml of cytochalasin B for 24–48 h. Next, the cultures were washed with PBS, treated with 0.05% KCl for 3 min at room temperature, and then fixed in 3 ml of Carnoy's solution (20:1, methanol: acetic acid, v/v) for 15 min. The dishes were air-dried and stained for 15 min with freshly prepared Giemsa's solution (10% in 0.1 mM sodium phosphate buffer, pH 6.8). The dishes were blindly coded, and two thousand binucleated cells in each treatment were examined for scoring micronuclei per binucleated cells using an inverted microscope.
Preparation of whole-cell extract (WCE).
Cells were rinsed twice with cold PBS and lysed on ice in a WCE buffer (20 mM HEPES, pH 7.6, 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 0.1 mM Na3VO4, 50 mM NaF, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, and 100 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride). The cell lysate was collected by a rubber policeman, rotated at 4°C for 30 min, centrifuged at 10,000 rpm for 10 min, and the precipitates were discarded. The BCA protein assay kit (Pierce, Rockford, IL) was adopted to determine protein concentrations using bovine serum albumin as a standard.
Western blot analysis.
Equal amount of cellular proteins in WCE from each set of experiments were fractionated on 10% SDS–polyacrylamide gels. The protein bands were then transferred to PVDF membranes and probed with primary antibody followed with a horseradish peroxidase-conjugated second antibody. The antibodies against phospho-ERK(Thr202/Tyr204) (#9101) and ERK2 (sc-154) were obtained, respectively, from Cell Signaling (Beverly, MA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibody reaction was performed using the enhanced chemiluminescence detection procedure (Santa Cruz Biotechnology Inc.). To reprobe the membrane with another primary antibody, the blots were stripped from the membranes by a solution containing 2% SDS, 62.5 mM Tris–HCl (pH 6.8), and 0.7% -mercaptoethanol at 50°C for 15 min.
NER synthesis.
Supercoiled plasmid pUC19 (250 ng/μl in ddH2O) was irradiated with UV (254 nm, 400 J/m2) at a radiation intensity of 1–1.5 J/m2/sec. The UV-irradiated or unirradiated pUC19 (250 ng) were served as substrates to determine the NER synthesis efficiency of proteins (60 μg) derived from WCE in reaction mixtures (50 μl) containing 20 μM each of dGTP, dCTP, and dTTP, 8 μM dATP, 2 μCi -32P-dCTP (3000 Ci/mmol), 2 mM ATP, 45 mM HEPES-KOH (pH 7.5), 60 mM KCl, 7.5 mM MgCl2, 0.9 mM dithiothreitol, 0.4 mM EDTA, 3.4% glycerol, and 18 μg bovine serum albumin. Reactions were performed at 30°C for 1 h and terminated by adding EDTA to a final concentration of 20 mM. Following RNase A (80 μg/ml) and proteinase K (190 μg/ml)-SDS (0.5%) treatment, the plasmid DNA in the reaction mixtures was purified using phenol/chloroform extraction and ethanol precipitation, linearized with BamHI, and subjected to 0.8% agarose gel electrophoresis. The plasmid DNA in gel was stained with 0.5% ethidium bromide and visualized under near-UV transillumination. The gel was then dried and subjected to autoradiography.
RESULTS
G1 Phase is Less Sensitive than S and G2/M Phases to Arsenite Cytotoxicity
To explore whether cells at various cell cycle phases exhibit different sensitivities to arsenite, proliferating CL3 cells were separated into fractions of G1, S, and G2/M phases by counterflow centrifugal elutriation, and their duration in these phases were estimated to be 12, 5, and 7 h, respectively. Equal numbers of the G1-, S-, and G2/M-enriched and asynchronous cells were left untreated or treated with arsenite (50–100 μM) for 3 h, washed with PBS, and the percent of survival was determined by colony-forming ability assay. As shown in Figure 1, G1 cells were less sensitive than S, G2/M, and asynchronous cells to cytotoxicity caused by arsenite; e.g., 50 μM arsenite reduced the cell viability to 45, 35, 29, and 17% of the untreated levels in G1, asynchronous, S, and G2/M phases, respectively.
G1 Phase is Susceptible to Arsenite in Micronucleus Induction and Cell Cycle Delay
Previous reports have indicated that arsenite acts as both a clastogen and an aneugen to induce micronuclei in various mammalian cells (Gurr et al., 1998; Liu and Huang, 1996, 1997; Wang and Huang, 1994; Wang et al., 1997; Yih and Lee, 1999). To determine whether the induction of micronuclei by arsenite is dependent on cell cycle phases, after arsenite treatment the G1- and G2/M-enriched and asynchronous cells were kept cultured for 24 h in medium containing cytochalasin B (a microfilament-assembly inhibitor) to allow the accumulation of binucleated cells in those that underwent complete nuclear division. Microscopic examination showed that a high frequency of micronucleus was induced in arsenite (50 μM) exposed G1 cells; the average numbers per 1,000 binucleated cells in the untreated and arsenite-treated G1 cells were 21 ± 3 and 202 ± 8, respectively (Fig. 2A). The induced micronucleus level in arsenite-treated G1 cells was higher than those observed in asynchronous (151 ± 5) and G2/M cells (137 ± 8) (Fig. 2A). As shown in Fig. 2B, cytochalasin B incubation for 24 h could accumulate 50–70% of the untreated cells with binuclei; however, only 14% of the arsenite-treated G1 cells had binuclei, which was much lower than those observed in arsenite-treated G2/M (39%) and asynchronous (47%) cells.
To obtain more binucleated cells for evaluation of micronucleus induction, the untreated and arsenite-treated G1 cells were incubated with cytochalasin B for 36–48 h or recovery in medium for 12 h followed by incubation with cytochalasin B for another 24 h (36) before microscopic examination. These alternative recovery strategies indeed enhanced the populations of binucleated cells (Fig. 2B), suggesting that arsenite delays cell cycle progression of the G1 cells. As shown in Figure 2A, the numbers of micronuclei induced in the arsenite-treated G1 cells under a prolonged recovery period were still higher than those generated in the untreated cells, although they declined as the recovery time increased.
Sustained ERK Activation in Arsenite-Treated G1 Cells Counteracts Cytotoxicity
We next explored whether arsenite influences ERK activation in G1 cells. Figure 3A shows that arsenite (50–100 μM, 3 h) increased phospho-ERK in G1-enriched CL3 cells, and the induced level was continuing for at least 24 h after arsenite removal. The arsenite-activated ERK could be blocked by two structurally unrelated inhibitors for the ERK upstream kinases MEK1/2, PD98059 and U0126 (Fig. 3B). The effect of ERK inhibition in cell proliferation and survival of the arsenite-treated G1 cells were determined using trypan blue exclusion and colony-forming ability assays. As shown in Figure 4A, the cell proliferation was markedly inhibited in the arsenite-treated G1 cells, which was further suppressed by cotreatment with either PD98059 or U0126. Similarly, the two inhibitors for MEK1/2 significantly enhanced the decrease in survival of arsenite-exposed G1 cells (Fig. 4B). These results imply that ERK activation in G1 cells by arsenite counteracts cytotoxicity and thereby contributes to cell proliferation and survival.
ERK Activation in Arsenite-Treated G1 Cells Can Lead to Micronucleus Formation
The role of ERK activation in the micronucleus formation was examined by exposing G1 cells to arsenite (50 μM) for 3 h in the presence or absence of PD98059 or U0126, followed by recovery for 24 h or 40 h in medium containing cytochalasin B. PD98059 or U0126 significantly lowered the micronucleus levels in arsenite-treated G1 cells when they were recovered in cytochalasin B for 24 h but not 40 h (Fig. 5). The results suggest that ERK activation can lead to micronucleus formation in a population of arsenite-treated G1 cells, possibly those bypassing the delay of cell cycle progression. Yet, after a prolonged recovery period the micronucleus formation in arsenite-treated G1 cells appears to be independent of the ERK signal.
ERK Activation in Arsenite-Treated G1 Cells Is Involved in NER Synthesis Inhibition
The mechanism by which arsenite enhances the genotoxicity of UV has been implicated due to the inhibition of NER system (Bau et al., 2001; Chien et al., 2004; Hartwig et al., 1997; Lee-Chen et al., 1992; Okui and Fujiwara, 1986; Wang and Huang, 1994). To explore whether arsenite inhibits NER synthesis of cells at G1 phase and the involvement of ERK signal, the G1-enriched cells were exposed to arsenite for 3 h in the presence or absence of PD98059 or U0126 and then allowed recovery for 12 h before WCE preparation. The proteins in the WCE were incubated with UV-irradiated DNA, 4 dNTP, and -32P-dCTP for the determination of NER synthesis. As shown in Figure 6, protein extracts derived from G1-enriched cells could stimulate NER synthesis of UV-damaged pUC19 DNA but not that of the undamaged DNA. Conversely, the efficiency of NER synthesis was suppressed using protein extracts derived from arsenite-treated G1 cells in a dose-dependent manner, which was negatively correlated to an increased ERK activation by arsenite (Fig. 6). Intriguingly, PD98059 or U0126 cotreatment could rescue NER synthesis efficiency in the arsenite-treated G1 cells (Fig. 7). Quantitative analysis showed that 50 μM arsenite reduced the repair capability to 40% as compared with those derived from the untreated G1 cells; cotreatment with 50 μM PD98059 or 5 μM U0126 recovered the NER synthesis efficiency to 80% of the untreated levels (Fig. 7). The results suggest that ERK mediates the NER synthesis inhibition in arsenite-treated G1 cells.
DISCUSSION
Arsenite is known to perturb mitotic spindle dynamics, resulting in G2/M prolongation (Huang and Lee, 1998; Yih et al., 1997); however, less attention has been paid as to whether arsenite injuries G1 cells and what the consequent cell fate is. In this report, we show that the G1-enriched CL3 cells exposed to moderate cytotoxic concentrations of arsenite (50 μM for 3 h) exhibit a marked retard in the formation of binucleated cells under cytochalasin B, suggesting arsenite delays G1 cell cycle progression. Flow cytometry analysis confirmed that such an arsenite treatment delays the G1 progression for 12 h (Li, unpublished data). The finding is consistent with a report that continued exposure of G1-enriched U937 myelomonocytic leukemia cells to 5 μM arsenic trioxide (a cytostatic condition) delays the cell cycle progression (McCabe et al., 2000). Here, we further demonstrate that the G1-enriched CL3 cells are susceptible to arsenite in micronucleus induction; in particular, 14% of the arsenite-treated G1 cells that could timely form binuclei exhibit high levels of micronuclei, and increase in recovery time can reduce the micronucleus formation (Fig. 2). The results suggest that a small population of the arsenite-treated G1 cells may escape the G1 delay, leading to the formation of high levels of micronuclei; also, after arsenite exposure a prolonged recovery period may facilitate removal of the damaged cells. In fact, the levels of phospho-p53(Ser15), a hallmark of G1 checkpoint, were markedly elevated in the arsenite-treated G1-enriched CL3 cells during the recovery period (Li, unpublished data). On the other hand, arsenite induces sustained ERK activation in the G1-enriched CL3 cells, which mediates cell proliferation and survival and can trigger micronucleus formation, possibly in those bypassing the cell cycle delay. Thus, arsenite can trigger dual and opposing signals in the G1-enriched CL3 cells regarding induction or bypassing the temporary cell cycle arrest.
Arsenite is known to impair NER processes (Bau et al., 2001; Chien et al., 2004; Hartwig et al., 1997; Lee-Chen et al., 1992; Okui and Fujiwara, 1986; Wang and Huang, 1994). Consistently, we found that arsenite reduces the NER synthesis efficiency of the G1 cells. More intriguingly, results presented here indicate that the sustained ERK signal mediates NER synthesis inhibition in the arsenite-treated G1 cells. It is well-known that the NER machinery removes a variety of detrimental DNA lesions including oxidative DNA adducts and pyrimidine dimers (Friedberg, 2001; Hoeijmakers, 2001; Lindahl and Wood, 1999). Previous reports have shown that arsenite induces oxidative DNA adducts and DNA strand breaks in mammalian cells (Bau et al., 2002; Wang et al., 2001). Accordingly, it is hypothesized that the ERK-mediated NER synthesis inhibition caused by arsenite may facilitate the accumulation of DNA lesions in G1 cells, which can result in increased micronucleus formation when the damaged cells bypass the cell cycle delay and progress through the M phase.
The finding that arsenite-induced sustained ERK activation in G1-enriched CL3 cells could mediate in micronucleus formation is consistent with reports that this signal is involved in micronucleus formation induced by oncogenic Ras in NIH 3T3 (Saavedra et al., 1999) and in thyroid PCCL3 cells (Saavedra et al., 2000). The result also agrees with required ERK activation for cell transformation caused by arsenite in mouse Cl 41 cells (Huang et al., 1999). Furthermore, activation of calcium-dependent protein kinase C has been associated with arsenite-induced micronucleus formation in Chinese hamster ovary cells (Liu and Huang, 1997). Protein kinase C can mediate ERK activation at the Raf or MEK levels and also has a desensitization effect in Raf activation (Schonwasser et al., 1998). Activation of Raf-MEK-ERK signaling by protein kinase C can also be achieved via phosphorylation of the Raf kinase inhibitory protein that subsequently dissociates from Raf (Corbit et al., 2003). Moreover, the AP-1 transactivation function induced by arsenite requires both the ERK and protein kinase C activities in mouse epidermal JB6 cells (Huang et al., 2001). The foregoing suggests that the Ras-Raf-MEK-ERK pathway may couple with protein kinase C to trigger AP-1 transactivation and subsequent micronucleus formation under arsenite exposure. Also, it deserves further study on correlation of the responsible intracellular species elicited by arsenic, such as its methylated derivatives, calcium, and free radicals (Florea, 2005) for the signal transduction leading to genomic damage.
The capability of arsenite to inhibit DNA repair is possibly not the result of direct enzyme inhibition and has been suggested via interfering with the expression of DNA repair genes (Hu et al., 1998). Indeed, recent reports have showed that arsenite can decrease the expression of genes encoding NER enzymes, e.g., XPC in human epidermal keratinocytes (Hamadeh et al., 2002) and XPC, XPD, and DNA ligase-1 in human bronchial epithelial BEAS-2B cells (Andrew et al., 2003b). Moreover, the expression of ERCC1, XPF, and XPB genes of the NER complex are found to be inversely correlated with toenail arsenic levels in a case-control study (Andrew et al., 2003a). On the other hand, NER enzymes such as hHR23A, hHR23B, and replication protein A2 have been identified to be modified posttranslationally by the ERK pathway (Lewis et al., 2000), suggesting that this signal may regulate the repair efficiency by triggering phosphorylation of NER enzymes. Whether the ERK pathway mediates down-regulation of DNA repair genes under arsenite exposure and the underlying mechanisms warrant further investigation.
The finding that sustained ERK activation by arsenite contributes to genomic instability, however, is contradictory to the reports that ERK signal exhibits a protective role in maintaining genomic stability under a variety of carcinogens. Active ERK mediates the suppression of micronucleus induction by cadmium (Chao and Yang, 2001) and ionizing radiation (Yacoub et al., 2001) and the prevention of mutagenesis under lead exposure (Lin et al., 2003). ERK signal has been associated with increased expression of DNA repair genes. For instance, activation of the Ras-ERK pathway by insulin up-regulates ERCC-1 mRNA expression (Lee-Kwon et al., 1998), and ERK activation upon ionizing radiation increases the expression of ERCC-1 and XRCC1 at the transcription and translation levels (Yacoub et al., 2001). The foregoing suggests that ERK signaling may play dual roles in regulating genomic stability and that arsenite may induce negative effectors to counteract the protective role of ERK.
In summary, this report indicates that acute arsenite exposure (50 μM, 3 h) not only delays cell cycle progression of the G1 cells but also induces sustained ERK signal in these cells to support cell proliferation and survival. The arsenite-induced ERK signal in G1 cells also triggers NER synthesis inhibition and micronucleus formation. It is conceivable that under such an arsenite exposure ERK is a multifaceted signal to counteract the G1 delay by facilitating cells having unrepaired DNA lesions to progress through the next cell cycle phases and thereby contributes to genomic instability. Results obtained here also suggest that blockage of the ERK signal pathway may have therapeutic potential to decrease genotoxicity caused by acute arsenite exposure to human.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Kun-Yan Jan for critical discussion of the manuscript. This work was supported in part by Grant NSC93–3112-B-007–010 from the National Science Council, Taiwan, and by Grant VTG92-P4–23 from the Medical Research Advancement Foundation in Memory of Dr. Chi-Shuen Tsou, Taiwan.
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