Opposite Roles of ERK and p38 Mitogen-Activated Protein Kinases in

Chem. Res. Toxicol. 2001, 14, 1193-1202

1193

Opposite Roles of ERK and p38 Mitogen-Activated Protein Kinases in Cadmium-Induced Genotoxicity and Mitotic Arrest Jui-I Chao†,‡ and Jia-Ling Yang*,† Molecular Carcinogenesis Laboratory, Department of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan, Republic of China, and Medical Technology Laboratory, Hsinchu Hospital, Department of Health, The Executive Yuan, Taiwan, Republic of China Received February 22, 2001

The roles of extracellular signal-regulated kinase (ERK) and p38 mitogen-activation protein kinase (MAPK) in guarding genome stability and regulating cell cycle progression were explored in CL3 human lung adenocarcinoma cells treated with cadmium (Cd), a human carcinogen. Exposing asynchronous cells to CdCl2 for 2 h (45% viability) caused irreversible mitotic arrest. Exposing early-G2 cells to Cd markedly delayed mitotic exit and subsequently induced sub-G1 populations; however, this did not alter the levels of Cdc2 and cyclin B1. These results suggest that Cd elicits mitotic arrest without affecting the progression of G2 to mitosis. Using counterflow centrifugal elutriation and flow cytometry analysis, CL3 cells synchronized at G1-, S-, and G2/M-phases were collected and treated with CdCl2. G2/M was the most sensitive cell cycle phase to Cd for the induction of ERK and p38 MAPK activities, cytotoxicity, apoptosis, micronucleus, and intracellular peroxide; despite that similar Cd accumulation was observed in G1-, S-, and G2/M-cells. Co-treatment early-G2 cells with Cd and SB202190, an inhibitor of p38 MAPK, significantly decreased the induction of micronucleus, mitotic arrest, and apoptosis. Conversely, PD98059, an inhibitor of the ERK upstream activators MKK1/2, enhanced micronucleus and apoptosis in Cd-treated early-G2 cells. Together, the results suggest that intracellular peroxide may participate in the activation of ERK and p38 MAPK by Cd; also, the activated-p38 MAPK may contribute to mitotic arrest and genome instability, whereas the activated-ERK may help to maintain genome integrity and survival.

Introduction The ERK1 and p38 MAPK cascades are critical in the control of cell growth, differentiation, and apoptosis (15). In general, ERK signaling cascade is activated by growth factors and required for cell proliferation. Conversely, p38 MAPK pathway is involved in growth arrest and apoptosis in response to genotoxic agents. However, cumulative reports have indicated that these signaling pathways exhibit more complex roles in the regulation of distinct cellular effects. While transient ERK activation promotes proliferation in fibroblasts, persistent activation mediates growth arrest or differentiation in neuronal cells, T cells, and muscle cells (1). On the other hand, activation of p38 MAPK has been implicated to be required for cell survival, proliferation, and differentiation (5). The particular function regulated by ERK or p38 MAPK is likely to depend on the cell type, the stimulus, and the duration and strength of kinase activities (1, 5). The ERK signal transduction pathway has multiple effects on cell cycle progression. ERK is essential for * To whom correspondence should be addressed. Phone: 886-35742756. Fax: 886-3-5645782. E-mail: [email protected]. † Molecular Carcinogenesis Laboratory. ‡ Medical Technology Laboratory. 1 Abbreviations: ERK, extracellular signal-regulated kinase; MAPK, mitogen-activation protein kinase; Cd, cadmium; PBS, phosphatebuffered saline; MTT, 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide; FITC, fluorescein isothiocyanate; DCF, dichlorofluorescein; ICP-MS, inductively coupled plasma-mass spectrometer; ROS, reactive oxygen species.

meiotic maturation in mouse (6) and Xenopus oocytes (7) and mitotic processes in Xenopus egg extracts (8-10). However, inappropriate activation of ERK induces repression of DNA synthesis in starfish eggs (11) and arrests cells at G2 (9, 12) and mitotic phases (9, 13) in Xenopus egg extracts. In mammalian cell lines, ERK activation and localization in nucleus is required for the progression of G0/G1 to S phase (14-17). Phosphorylation of ERK is also essential for the progression from G2 to mitosis (18). Moreover, normal mitotic progression is highly associated with the activation/inactivation and localization of ERK (19, 20). In general, ERK activation is essential for entry into a specific cell cycle transition and its inactivation, re-localization, and reactivation may be required for exit from that stage and entry into the subsequent phase. Thus, the activity of ERK oscillates through different cell cycle phases to control cell proliferation. Phosphorylation of nuclear transcription factors by ERK is a crucial step in the regulation of gene expression (21). ERK may also regulate the association between chromosomes and microtubules through its interaction with the kinetochore motor protein CENP-E (20). On the other hand, p38 MAPK activation is involved in the inhibition of serum-stimulated cell cycle progression at G1/S by cdc42Hs (22). Opposing to the function of ERK pathway, p38 MAPK signal cascade inhibits the mitogen-induced cyclin D1 expression (23). Activation of p38 MAPK is shown to be associated with mitotic arrest in NIH 3T3 cells induced by nocodazole, a microtubule

10.1021/tx010041o CCC: $20.00 © 2001 American Chemical Society Published on Web 08/10/2001

1194

Chem. Res. Toxicol., Vol. 14, No. 9, 2001

depolymerization agent (24). Furthermore, the MKK6p38γ signal is required for the G2 arrest upon γ-radiation (25). Cd is a ubiquitous environmental toxicant that has been evaluated as a human carcinogen (26). Cd can induce DNA damage, morphological transformations, micronuclei, chromosomal aberrations, and gene mutations in cultured mammalian cells (27-33) and tumors in animal models (34). Cd compounds have also been characterized as a spindle poison (35, 36). Cd has been reported to activate p38 MAPK and ERK in CL3 human lung adenocarcinoma cells (37), U937 human promonocytic leukemia cells (38), and 9L rat brain tumor cells (39). Persistent activation of p38 MAPK by Cd is mediated in apoptosis, whereas ERK activation by Cd plays an opposite role in CL3 cells (37). Although ERK activity is critical to control cell cycle progression and p38 MAPK may cause G1/S, G2, or mitotic arrest, the roles of these signals elicited by Cd in affecting genome integrity and cell cycle progression remain unknown. Here we show that CdCl2 induced irreversible mitotic arrest without significant influence the levels of Cdc2 and cyclin B1 in CL3 cells. G2/M was the most sensitive cell cycle phase to Cd in the induction of ERK and p38 MAPK activities, cytotoxicity, apoptosis, micronucleus, and intracellular peroxide, although Cd accumulation was independent of cell cycle phase. The effects of ERK and p38 MAPK on Cd-induced micronucleus and mitotic arrest were studied using PD98059, a specific inhibitor of the ERK upstream activators MKK1/2 (40), and SB202190, a specific inhibitor of p38 MAPK (41, 42), respectively. Together, the findings suggest that intracellular peroxide elicited by Cd is highly associated with the activation of ERK and p38 MAPK that play opposite roles in Cd-induced genotoxicity and mitotic arrest.

Experimental Procedures Cell Culture. The CL3 cell line established from a non-smallcell lung carcinoma tumor of a 60-year-old male patient in Taiwan (43) was provided by Dr. P. C. Yang at the Department of Internal Medicine and Clinical Pathology, National Taiwan University Hospital, Taipei. Cells were cultured in RPMI1640 medium (Gibco, Life Technologies, Grand Island, NY) supplemented with sodium bicarbonate (2.2%, w/v), L-glutamine (0.03%, w/v), penicillin (100 units/mL), streptomycin (100 µg/ mL), and fetal calf serum (10%). Cells were maintained at 37 °C in a humidified incubator containing 5% CO2 in air. Cell Synchronization and Cd Treatment. Cells were grown to near confluence and fed with serum-free medium for 2 days. The cells were plated at a density of 1.5 × 106 cells per a p60-dish in medium containing 10% fetal calf serum and 1 µg/mL aphidicolin (Sigma, St. Louis, MO). One day later, the cells synchronized at the G1/S-border were washed with RPMI1640 medium, re-fed with culture medium, and kept in a CO2 incubator for 4 h to allow cells to progress to early-G2 phase. The early-G2 cells were exposed to CdCl2 (Merck, Darmstadt, Germany) for 2 h in serum-free medium. At the end of treatment, the cells were washed twice with PBS and kept in the incubator for various times before they were analyzed by flow cytometry. Alternatively, 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 (44, 45). Briefly, exponentially growing cells (1 × 108) were concentrated in 15 mL of RPMI1640 containing 1% fetal calf serum and elutriated at a flow rate of 30 mL/min. After the cells settled into the chamber, fractions were collected at a speed of 21001450 rpm at intervals of 50 rpm. Aliquots of the samples were

Chao and Yang subjected to cell cycle analysis by flow cytometry to determine the cell cycle phase of each fraction. Following elutriation, cells were treated with Cd for 2 h and assayed for the activation of ERK and p38 MAPK, cytotoxicity, apoptosis, micronucleus, Cd uptake, and intracellular peroxide levels. In experiments to determine the roles of ERK and p38 MAPK in Cd-induced micronuclei and cell cycle inhibition, cells were co-treated with Cd and specific kinase inhibitors PD98059 (Calbiochem, San Diego, CA) and SB202190 (Calbiochem), respectively. Flow Cytometry. Cell cycle phases were analyzed using a fluorescence-activated cell sorter (FACScan, Becton-Dickinson, San Jose, CA) with CellQuest and Modfit LT softwares. Aliquots of 1 × 106 cells were fixed with 70% ethanol for at least 2 h at -20 °C before centrifugation. The cell pellets were treated with propidium iodine (4 µg/mL) solution containing RNase (100 µg/ mL) and Triton X-100 (1%) for 30 min. The stained cells were analysis using a FACScan with a pulse processing protocol according to the manufacturer’s instructions. Mitotic Index Analysis. Exponentially growing cells were plated at a density of 5 × 104 cells/cm2 in 60-mm dishes 1 day before treatment. Cells were left untreated or treated with 0.5 µM nocodazole (Sigma) for 4 h, washed with PBS, and then treated with Cd for 2 h in RPMI1640 medium. After removing Cd, the cells were washed twice with PBS, and kept cultured for 0, 3, 6, 12, and 24 h. Next, the cells were trypsinized and treated with 0.05% KCl for 5 min at room temperature. After centrifugation, the cells were fixed with methanol/acetic acid solution (3:1, v/v). An aliquot of the cells in suspension was dropped onto a clean slide, air-dried and stained with a 10% Giemsa solution (Merck). At least 200 cells were scored for the determination of mitotic index in each culture. Cytotoxicity. Cells were seeded in 96-well plates at a density of 1 × 104 cells/well and treated with Cd for 2 h. After treatment, the cells were washed twice with PBS and kept cultured in RPMI1640 containing 10% fetal calf serum for 48 h. The cells were then treated with 500 µg/mL MTT (Sigma) and kept in a CO2 incubator for 4 h. Viable cells can convert MTT to formazan that generates blue color when dissolved in dimethyl sulfoxide (46). The intensity was measured using a reader for enzymelinked immunosorbent assay and an absorption wavelength of 565 nm. Apoptosis. Annexin V-FITC binding assay was adopted for the determination of apoptosis. Immediately after Cd treatment, 2 × 105 cells were washed twice with PBS and suspended in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). The cells were stained with annexin V-FITC (1 ng/ µL; Medical & Biological Lab., LTD, Japan) and propidium iodide (5 ng/µL) for 15 min in the dark. The stained cells were analyzed using flow cytometry and the CellQuest program. Apoptotic cells were detected by those stained with annexin V-FITC but not with propidium iodide. Micronucleus Assay. The cells 1 × 105 in 60-mm Petri dish were cultured overnight and exposed to Cd (0-80 µM) for 2 h in RPMI1640 medium. Next, the cultures were washed three times with PBS, treated with 1 µg/mL cytochalasin B (Sigma) in RPMI1640 medium containing 10% fetal calf serum and incubated for another 24 h. At the end of incubation, the cultures were washed with PBS once, and incubated in 0.05% KCl for 5 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 with freshly Giemsa’s solution for 15 min. The numbers of micronucleus per binucleated cells were scored under a microscope and five hundred binucleated cells per dish were examined. Measurement of Intracellular Peroxide Level. The intracellular peroxide level was estimated by the activation and oxidation of 2′,7′-dichlorofluorescin diacetate to DCF (47). After Cd treatment, 1 × 106 cells were suspended in PBS and incubated with 80 µM of 2′,7′-dichlorofluorescin diacetate (Estman Kodak, Rochester, NY) for 30 min in the dark. The cells were then centrifuged and the cell pellets were kept in ice and

Roles of MAPKs in Cd Genotoxicity and Mitotic Arrest resuspended in 2 mL of cold PBS before fluorescence detection. The oxidation of intracellular peroxide with activated 2′,7′dichlorofluorescein diacetate would result in DCF that intensity was detected using a fluorescence spectrophotometer with excitation and emission wavelength at 502 and 523 nm, respectively. Determination of Cellular Cd Level. Cells were exposed to various Cd concentrations (0-80 µM) in serum-free medium for 2 h. Following treatment, the cells were washed three times with PBS and the numbers of cells were determined. One million of cells were centrifuged and the cell pellet was sonicated in MilliQ-purified water. Total cellular Cd concentrations were analyzed by an ICP-MS (SCIEX ELAN 5000, Perkin-Elmer, Norwalk, CT). The ICP-MS conditions were as followed: power of 1000 W, plasma flow rate of 15 L/min, auxiliary flow rate of 0.8 L/min, and sample flow rate of 1 mL/min. Western Blot Analysis. Cells were lysed in whole cell extract buffer containing 20 mM HEPES at pH 7.6, 75 mM NaCl, 2.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 0.1 mM Na3VO4, 50 mM NaF, 0.5 µg/mL leupeptin, 1 µg/mL aprotinin, and 100 µg/mL 4-(2-aminoethyl)benzenesulfonyl fluoride. The cell lysate was rotated at 4 °C for 30 min, centrifuged at 10 000 rpm for 10 min, and the precipitates were discarded. Protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. Equal amounts of proteins (20-60 µg) extracted from each sets of experiments were loaded onto 10% SDS-polyacrylamide gels. The protein bands were then transferred electrophoretically to PVDF membranes (NEN, Boston, MA). Membranes were probed with primary antibody and followed with a horseradish peroxidase-conjugated second antibody (BioRad Co., Hercules, CA). Phospho-specific antibodies for p38 MAPK (no. 9211), and ERK (no. 9101) were purchased from New England BioLabs Inc. (Beverly, MA). Anti-ERK2 (C-14), anti-p38 MAPK (C-20), and anti-Cdc2 (H-297) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-cyclin B1 (Ab-2) was purchased from Oncogene Sciences (Cambridge, MA). Antibody reaction was detected using the enhanced chemiluminescence detection procedure according to the manufacturer’s recommendations (NEN, Boston, MA). In some experiments, antibodies were stripped from membranes using solution containing 2% SDS, 62.5 mM Tris-HCl, pH 6.8, and 0.7% (w/w) β-mercaptoethanol at 50 °C for 15 min before reprobing with another primary antibody. The relative protein intensities on blots were quantitated using a computing densitometer equipped with the ImagQuant analysis program (Molecular Dynamics, Sunnyvale, CA).

Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1195

Figure 1. Cd induces mitotic index and decreases G1 fractions of asynchronous cells. Exponential growing CL3 cells were left untreated or treated with 40 µM of CdCl2 for 2 h, washed twice with PBS, kept cultured for 3-24 h, and then subjected to flow cytometry (A-C) or mitotic index analyses (D). Bars represent the SEM of three to four independent experiments. Student t-test was adopted to compare the significance between Cdtreated and untreated cells at a particular recovery time. (**) P < 0.01.

Results Cd Causes Mitotic Arrest. CL3 cells at exponential growth were left untreated or treated with CdCl2 (40 µM) in serum-free media for 2 h, washed with PBS, kept cultured for 3-24 h, and then subjected to flow cytometry analysis to examine how does Cd interfere cell cycle progression. The G1 fraction significantly decreased (Figure 1A) while the G2/M fraction increased (Figure 1B) 6-24 h after Cd treatment. Conversely, Cd did not significantly affect the S phase fractions (Figure 1C). To further characterize whether Cd affects M-phase, the mitotic index of Cd-treated asynchronous growing cells was examined by chromosome spreading. As shown in Figure 1D, a 2-h Cd exposure markedly increased the mitotic index 6-24 h later. The results of chromosome spreading and flow cytometry analysis indicate that Cd induces mitotic arrest and does not seem to affect the G2 phase. Moreover, cells were enriched in M-phase using nocodazole (0.5 µM, 4 h), left untreated or treated with Cd

Figure 2. Different mechanisms of mitotic arrest induced by Cd and nocodazole. Exponential growing CL3 cells were exposed to 0.5 µM of nocodazole for 4 h, left untreated or treated with 40 µM of CdCl2 for 2 h, washed with PBS, kept cultured for 3-24 h and then subjected to mitotic index analysis. Bars represent the SEM of three independent experiments.

for 2 h, washed with PBS, kept cultured for 3-24 h and then subjected to mitotic index analysis. The nocodazoleenriched mitotic cells declined to basal levels within 24 h, while the fraction of mitotic cells accumulated at high levels in those treated with Cd plus nocodazole (Figure 2). This result suggests that Cd-induced mitotic arrest is irreversible which is different from the mechanism of nocodazole. Next, we examined the effect of Cd on the progression of G2 phase to the next cell cycle phases. CL3 cells were synchronized at G1/S border using serum-starvation (2 days) and aphidicolin (1 µg/mL, 24 h). Flow cytometry

1196


Figure 3. Cd delays the early-G2 cells entering into the next G1 phase and subsequently induces the sub-G1 populations. CL3 cells were grown to near confluence, fed with serum-free medium for 2 days, and plated in culture medium containing 1 µg/mL of aphidicolin for 1 day to allow cells synchronized at the G1/S-border. The cells were then washed with RPMI1640 medium, re-fed with culture medium, and kept in an incubator for 4 h to progress to early-G2 phase. The early-G2 cells were left untreated or treated with 40 µM of Cd for 2 h in serum-free medium, washed twice with PBS, and kept cultured for various times before they were analyzed by flow cytometry (R0-R22 represent recovery 0-22 h, number in parentheses is time after aphidicolin removal). The fractions of each cell cycle phases were averaged from three to six experiments.

analyses showed that these synchronous cells entered into S-phase rapidly after aphidicolin removal and most of the cells progressed to early-G2 phase 4 h later (Figure 3). The early-G2 cells were left untreated or treated with 40 µM of Cd for 2 h, washed with PBS, kept cultured for 0-22 h (6-28 h after aphidicolin removal), and analyzed by flow cytometry. As shown in Figure 3, the untreated cells progressed to the next G1-phase while Cd-treated cells remained at G2/M phase 12 h after aphidicolin removal. Chromosome spreading examination showed that most of the Cd-treated cells exhibited mitotic figures at 9-12 h after aphidicolin removal, suggesting the Cdtreated early-G2 cells accumulate at M-phase (data not shown). In addition, a high frequency of sub-G1 population was observed 22 h after Cd exposure (Figure 3), suggesting that Cd triggers these cells to apoptosis. Cd Does Not Affect Cdc2/cyclin B1 Machinery. The cellular amounts of two mitotic regulatory proteins, cyclin B1 and Cdc2, were examined using western blot

Chao and Yang

Figure 4. Cd does not alter the levels of cyclin B1 or Cdc2. Cells were synchronized and treated with Cd as described in Figure 3. Whole cell extracts were harvested from the control G1/S cells and the early-G2 cells that were left untreated (G2) or treated with 40 µM of Cd for 2 h (R0-R22 represent recovery 0-22 h). Western blot analysis was performed using antibodies against cyclin B1 or Cdc2. The relative intensity was quantitated using a computing densitometer. Results were obtained from three to four experiments and the bar represents SEM.

analysis to understand how Cd causes mitotic arrest. CL3 cells were synchronized at early-G2 phase, left untreated or treated with Cd for 2 h as described in Figure 3 and the whole cell extracts were harvested immediately or at various times after Cd withdrawal. As shown in Figure 4 the amounts of cyclin B1 and Cdc2 in the control cells peaked at 8 h after aphidicolin removal (R2) and decreased gradually when the cells progressed to the next cell cycle phase. Exposing the early-G2 cells to Cd did not affect the levels of cyclin B1 and Cdc2 in cells obtained at various recovery times (Figure 4). Also, a slowermigrating form of Cdc2 (possibly hyper-phosphorylated) increased at 8-10 h after aphidicolin removal (R2-R4) and then decreased later in both the control and Cdtreated cells (Figure 4). The lack of affecting Cdc2/cyclin B1 cell cycle engine in Cd-treated cells suggests that Cd does not elicit G2 arrest. Persistent Activation of ERK and p38 MAPK in Cd-Treated Early-G2 Cells. Cd activates ERK and p38 MAPK in CL3 cells (37). Activation of ERK is essential for the progression from G2 to mitosis (18) and mitotic progression (19, 20); also, inappropriate ERK activation may result in G2 or mitotic arrest (9, 13). Activation of p38 MAPK by nocodazole and γ-radiation has been associated with mitotic arrest and G2 arrest, respectively (24, 25). We therefore examined the ERK and p38 MAPK

Roles of MAPKs in Cd Genotoxicity and Mitotic Arrest


Figure 6. Effects of SB202190 and PD98059 on cell cycle progression of the Cd-treated early-G2 cells. The early-G2 cells prepared as described in Figure 3 were exposed to Cd for 2 h in the presence of SB202190 or PD98059, washed twice with PBS, and kept incubation for 22 h before they were analyzed by flow cytometry. The fractions of each cell cycle phases were averaged from 4 experiments.

Figure 5. Persistent activation of p38 MAPK and ERK in the Cd-treated early G2-cells. Cells were synchronized and treated with Cd as described in Figure 3. Whole cell extracts were harvested from the control G1/S cells and the early-G2 cells that were left untreated (G2) or treated with 40 µM of Cd for 2 h (R0-R22 represent recovery 0-22 h). Activation of p38 MAPK and ERK in whole cell extracts were examined using phosphospecific antibodies. Western blot analyses of the p38 MAPK and ERK2 protein levels are showed in the lower panels. The relative intensity was quantitated using a computing densitometer. Results were obtained from three to five experiments and the bar represents SEM. Student t-test was adopted to compare the significance between Cd-treated and untreated cells. (*) P < 0.05 and (**) P < 0.01, respectively.

activities in the early-G2 cells treated with Cd as described in Figure 3, using specific antibodies for the dual phosphorylation sites of the catalytic domain. Although an increased level of phosphorylated-p38 MAPK was observed in the control G1/S-cells obtained immediately after aphidicolin removal, it decreased later (Figure 5), which indicates aphidicolin transiently elicits p38 MAPK activity. Figure 5 shows that Cd markedly induced the amounts of dual phosphorylated-p38 MAPK in early-G2 cells (R0), which remained at high levels 2-22 h after Cd withdrawal. On the other hand, the amounts of phosphorylated-ERK in the control cells oscillated with the progression of cell cycle (Figure 5). After Cd exposure for 2 h, the amounts of phosphorylated-ERK steadily increased to 2-fold of the untreated levels (Figure 5). Additionally, the protein amounts of p38 or ERK extracted in each recovery times of Cd-treated cells were about the same as in the untreated cells (Figure 5). The results suggest that persistent activation of p38 MAPK or ERK by Cd may be correlated with the ability of this metal to irreversibly induce mitotic arrest.

To determine the effects of p38 MAPK and ERK activation on cell cycle progression and apoptosis, cells enriched at the early G2-phase as described in Figure 3 were treated with Cd (40 µM) for 2 h in the presence of SB202190, a p38 MAPK inhibitor (41, 42), or PD98059, an inhibitor of the ERK upstream activators MKK1/2 (40). Flow cytometry analysis of the cells harvested 22 h after Cd exposure showed that 50 µM of PD98059 reduced the number of cells reentry into G1 phase and increased the numbers of sub-G1 cells induced by Cd (Figure 6). Conversely, 10 µM of SB202190 reduced the numbers of sub-G1 cells and enhanced the number of cells reentry into G1 phase (Figure 6). PD98059 or SB202190 alone did not affect the cell cycle progression (data not shown). These findings suggest that the p38 MAPK activated by Cd may transmit signals to induce mitotic arrest and subsequently lead to apoptosis, while the Cdactivated ERK may enhance mitotic exit and protect from apoptosis. G2/M Is the Most Sensitive Phase to Cd in the Activation of p38 MAPK and ERK. To further characterize the ability of Cd to activate p38 MAPK and ERK at different cell cycle phases, CL3 cells were separated by counterflow centrifugal elutriation into fractions of G1, S, and G2/M phases. Examples of typical flow cytometric profiles of DNA from asynchronous cells and those collected from centrifugal speed 1900 rpm (G1-phase), 1700 rpm (S-phase), and 1500 rpm (G2/M-phase) are shown in Figure 7A. These G1-, S-, or G2/M-cells were exposed to Cd for 2 h and the whole cell extracts were isolated for the determination of the levels of dual phosphorylated-ERK and p38 MAPK. As shown in Figure 7B, the highest level of the p38 MAPK activated by Cd was observed in G2/M-cells. Cd also activated p38 MAPK in S-cells. However, Cd did not induce the p38 MAPK activity in G1-cells. G2/M was also the most sensitive phase for the activation of ERK by Cd (Figure 7B). In the untreated populations, a higher ERK activity was observed in S-phase (Figure 7B). G2/M Is the Most Sensitive Phase to Cd in the Induction of Cytotoxicity, Apoptosis, and Micro-

1198


Figure 7. G2/M was the most sensitive cell cycle phase to the activation of p38 MAPK and ERK by Cd. (A) G1-, S-, and G2/ M-cells were fractionated from proliferating CL3 cells using counterflow centrifugal elutriation and analyzed by flow cytometry as described in the Experimental Procedures. The fractions of each cell cycle phases were averaged from five experiments. (B) G1-, S-, and G2/M-cells obtained from counterflow centrifugal elutriation were exposed to Cd for 2 h, and their whole cell extracts were subjected to the determination of p38 MAPK and ERK activation using phospho-specific antibodies. After detection of phosphorylated proteins, antibodies were stripped from membranes and then reprobed with primary antibodies against p38 MAPK and ERK2. Western blots of the p38 MAPK and ERK2 protein levels are showed in the lower panels. The relative intensity was quantitated using a computing densitometer. The numbers between western blots of phosphorylated and total proteins were the relative activities of kinases obtained from averaging five experiments.

nucleus. To investigate the relative sensitivities of different cell cycle phases to Cd-induced toxicity, equal numbers of G1-, S-, and G2/M-cells derived from counterflow centrifugal elutriation were exposed to 0-80 µM of Cd for 2 h. Cytotoxicity was determined using MTT assay 48 h later. As shown in Figure 8A, G2/M was the most sensitive phase to the cytotoxicity induced by Cd. Cells at the S-phase exhibited similar Cd sensitivity to the asynchronous cells, while the G1-cells were less sensitive to Cd (Figure 8A). The fractions of apoptotic cells were examined using annexin V-FITC assay and flow cytometry. As shown Figure 8B, Cd induced significantly more apoptotic cells in the G2/M-phase than the other cell cycle phases. The apoptotic cells observed in untreated cells

Chao and Yang

Figure 8. G2/M was the most sensitive cell cycle phase to the induction of cytotoxicity, apoptosis and micronucleus by Cd. (A) Following counterflow centrifugal elutriation, cells synchronized at G1-, S-, and G2/M were left untreated or treated with Cd for 2 h, washed twice with PBS, kept cultured for 2 days, and then subjected for the determination of cytotoxicity using MTT assay. (B) The untreated or Cd-treated cells were washed with PBS, kept cultured for 16 h, and then subjected to the determination of apoptosis using annexin V-FITC binding assay and analyzed by flow cytometry. (C) After Cd treatment, the cells were washed with PBS and incubated for another 24 h in culture medium containing 1 µg/mL of cytochalasin B. The cells were then prepared for micronucleus assay and the numbers of micronuclei per binucleated cells were scored using a microscope. Results were obtained from three to four experiments and the bar represents SEM. Student t-test was adopted to compare the significance between Cd-treated synchronous and asynchronous cells; (*) P < 0.05 and (**) P < 0.01, respectively.

were 3-4% of those that were increased to 8, 10, 15, and 23% in the G1-, asynchronous, S-, and G2/M-cells exposed to 80 µM of Cd, respectively (Figure 8B). The formation of micronuclei is a reflection of DNA damage, defective mitosis, and loss of genetic materials. This assay was then adopted to explore the genotoxicity induced by Cd at various cell cycle phases. As shown in Figure 8C, Cd induced significantly higher numbers of micronuclei in the G2/M- and S-cells than the asynchronous- and G1-



Table 1. Accumulation of Cd in Various Cell Cycle Phases of CL3 Cellsa Cd (ng/106 cells)b CdCl2 (µM, 2 h)

asynchronous

G1

S

G2/M

0 40 80

0.54 ( 0.29(3) 84.12 ( 13.65 (3) 160.97 ( 17.09 (4)

2.17 ( 1.67 (3) 78.64 ( 5.33 (4) 152.87 ( 27.98 (4)

1.77 ( 1.59 (3) 71.63 ( 7.98 (4) 160.81 ( 20.95 (5)

1.08 ( 0.83 (3) 69.42 ( 7.75 (4) 165.84 ( 20.74 (5)

a Cells were synchronized and treated with Cd as described in Figure 7. b Mean ( SEM. Numbers in parentheses indicate the number of experiments.

Figure 9. SB202190 attenuates, whereas PD98059 potentiates the frequency of micronucleus induced by Cd. G1-, S-, and G2/ M-cells obtained from counterflow centrifugal elutriation were exposed to Cd (80 µM) plus SB202190 (10 µM) or PD98059 (50 µM) for 2 h then subjected to micronucleus examination as described in Figure 8. Results were obtained from three experiments and the bar represents SEM. Student t-test was adopted to compare the significance between synchronous cells treated with Cd alone and those co-treated with kinase inhibitors; (*) P < 0.05 and (**) P < 0.01, respectively.

cells. The spontaneous numbers of micronuclei were about the same (21.3-23.3/1000 binucleus cells) in the asynchronous, G2/M-, S-, and G1-cells (Figure 8C). p38 MAPK and ERK May Involve Oppositely in the Induction of Micronucleus by Cd. The roles of p38 MAPK and ERK activated by Cd in genotoxicity were explored using kinase-specific inhibitors. G1-, S-, or G2/M-cells derived from counterflow centrifugal elutriation were exposed to 80 µM of Cd in the presence of SB202190 or PD98059 for 2 h. As shown in Figure 9, SB202190 (10 µM) significantly decreased the numbers of micronuclei induced by Cd in G2/M- and S-phases. Conversely, PD98059 (50 µM) enhanced the numbers of micronuclei induced by Cd in G2/M- and S-phases (Figure 9). Cd Accumulation Is Independent of Cell Cycle Phases. The levels of Cd accumulated in cells may influence toxicity. Therefore, the amounts of intracellular Cd in the asynchronous, G1-, S-, or G2/M-cells were measured using ICP-MS. Cells were exposed to Cd for 2 h, washed three times with PBS, and subjected to the analysis of the intracellular Cd amounts. As shown in Table 1, similar amounts of Cd were accumulated in the asynchronous, G1-, S-, and G2/M-cells. This result indicates that the cell cycle phase-dependent induction of cytotoxicity, apoptosis, micronucleus, and the activities

Figure 10. G2/M was the most sensitive cell cycle phase to the induction of intracellular peroxide levels by Cd. Synchronization of cells was as described in Figure 7. Immediately after treatment, the cells were washed with PBS, trypsinized and incubated with 80 µM of 2′,7′-dichlorofluorescin diacetate for 30 min at 37 °C in the dark. The DCF intensity was detected using a fluorescence spectrophotometer. Results were obtained from three to five experiments and the bar represents SEM. Student t-test was adopted to compare the significance between Cd-treated synchronous and asynchronous cells; (*) P < 0.05 and (**) P < 0.01, respectively.

of p38 MAPK and ERK by Cd do not correlate with the intracellular Cd levels. G2/M Is the Most Sensitive Phase to Cd in the Induction of Intracellular Peroxide. Oxidative stress has been shown to be highly associated with Cd genotoxicity (32, 48-51). The intracellular peroxide levels induced by Cd in different cell cycle phases were then measured to elucidate the correlation between oxidative stress and genotoxicity. After Cd treatment, the asynchronous, G1-, S-, or G2/M-cells (1 × 106 cells/each) were suspended in PBS and incubated with 80 µM of 2′,7′dichlorofluorescin diacetate for 30 min in the dark. The reaction of intracellular peroxides with activated 2′,7′dichlorofluorescein diacetate would result in DCF that intensity was detected by a fluorescence spectrophotometer. As shown in Figure 10, the DCF fluorescence intensity induced by Cd was dependent on the cell cycle phases; G2/M was the most sensitive phase to Cd in the induction of DCF fluorescence, followed was S-phase. Whereas, Cd-treated G1-cells produced lower levels of DCF fluorescence than those induced in asynchronous cells (Figure 10). This result suggests that the intracellular peroxide induced by Cd may be the causative of the cell cycle phase-dependent induction of cytotoxicity, apoptosis, micronucleus, and the p38 MAPK and ERK activities.

Discussion In this manuscript, we have demonstrated that Cd induces irreversible mitotic arrest and G2/M is the most

1200


sensitive phase to Cd causing dose-dependent induction of cytotoxicity, apoptosis, micronucleus, and p38 MAPK and ERK activities in CL3 human lung adenocarcinoma cells. The ability of Cd to arrest cells at mitotic phase is consistent with previous findings that it depolymerizes microtubules and actins in cultured cells (35, 36). Interestingly, Cd does not affect the levels of phosphorylatedCdc2 critical for triggering the G2/M transition or the degradation of cyclin B1 that occurs in anaphase. Therefore, Cd may not elicit the checkpoint control of G2/M transition; rather, Cd may interfere with the progression of anaphase and cytokinesis, thereby failing in mitotic exit. The mechanism of mitotic arrest caused by Cd is different from that induced by nocodazole (a microtubule depolymerizer) (24) or arsenite (a microtubule stabilizer) (52), which are associated with a delayed inactivation of Cdc2/cyclin B1. Recently, we have shown that Cd persistently elicits p38 MAPK and ERK signals (37). Here, we further demonstrated that activation of p38 MAPK and ERK by Cd is cell cycle dependent. Cd elicits markedly stronger p38 MAPK activity in G2/M-cells than in S-cells followed by G1-cells. Similarly, Cd significantly activates ERK in G2/M-cells. The pyridinylimidazole inhibitors of p38R and p38β MAPK including SB202190 and SB203580 have no effect on the activity of many other protein kinases, including other MAPK family members (41, 42). Also, Cdinduced apoptosis can be enhanced by transient transfection of a wild-type p38 vector (37). The present study shows that co-administrating SB202190 significantly decreases Cd-elicited mitotic arrest, apoptosis, and the frequency of micronucleus, suggesting the Cd-activated p38 MAPK is involved in these events. However, we cannot exclude the possibility that SB202190 may inhibit other unknown targets participating in Cd-induced genome instability, apoptosis, and mitotic arrest. Previously, p38 MAPK activation is reported to be associated with nocodazole-induced mitotic arrest (24). Nevertheless, the present study has shown for the first time that p38 MAPK activation by genotoxic agents can potentially transmit signals to disturb genome integrity. On the other hand, we have demonstrated that PD98059 at a dose that completely blocks ERK phosphorylation (data not shown) enhances the Cd-induced sub-G1 fraction and micronucleus, suggesting that the activated-ERK potentially contributes to survival and genome stability. The involvement of ERK in preventing genotoxicity is consistent with the reports that activation of MEK2, an ERK upstream activator, by ionizing radiation enhances the ability of cells to survival and to recover from the G2/M cell cycle checkpoint arrest (53). ERK phosphorylation protects DNA strand breakage and apoptosis induced by hyperoxia (54). Similarly, c-Fos, a downstream target of ERK, protects survival and chromosomal integrity upon many types of genotoxic damage (55). However, ERK has been shown to mediate the micronucleus induction by v-Ras in NIH 3T3 cells that lack functional tumor suppressor p53 protein (56). In the absence of p53 abnormal centrosome amplification occurs (57), however, the multiple copies of centrosomes in most of the p53-/- cells sequester to the spindle poles and successfully participate in the formation of bipolar spindles and proceed through cytokinesis with a normal karyotype (57, 58). Overexpression of the Mos/ERK pathway greatly enhanced chromosome instability in p53-/- cells, possibly due to Mos/ERK interfering with the formation of astral

Chao and Yang

microtubules (58). Conversely, constitutive activation of the Mos/ERK pathway in cells having functional p53 allows G2-cells progress to M phase but fails to undergo cytokinesis (59). CL3 cells have wild-type p53 sequence (J. Y. Shew, personal communication), and Cd can induce phosphorylation at Ser15 of p53 in CL3 cells (J.-I Chao, unpublished data). The contradictory effects of ERK signal cascade on maintaining genome integrity in p53 deficient and proficient cells deserve further investigation. Although G2/M is the most sensitive phase to Cd in the induction of p38 MAPK and ERK activities, apoptosis, and micronucleus, similar levels of Cd accumulation are observed in asynchronous, G1-, S-, and G2/M-cells. This implies that Cd itself may not directly elicit these responses. Recent reports have shown that ROS acts as a signal transduction molecule. For example, plateletderived growth factor elicits intracellular peroxide that associates with tyrosine phosphorylation, ERK stimulation, and DNA synthesis in rat vascular smooth muscle cells (60). Additionally, H2O2 activates ERK and p38 MAPK in cultured mammalian cells including CL3 (61, 62). Moreover, several studies have implicated that ROS is involved in Cd-induced genotoxicity in cultured cells, including the induction of DNA strand breaks, 8-hydroxy2′-deoxyguanosine adducts, chromosomal aberrations, and mutations (32, 48-51). We have demonstrated here that Cd-treated G2/M-cells generate markedly higher levels of intracellular peroxide than the other cell cycle phases, suggesting that ROS is the causative of the induction of MAPK signals, genotoxicity, and apoptosis in Cd-treated cells. Cd induces higher levels of intracellular peroxide and genotoxicity in G2/M-cells. However, Cd does not significantly cause G2 arrest; rather, it leads to mitotic arrest. Moreover, exposing G1-cells to Cd does not affect cell cycle progression to S-phase (data not shown). It is a wellestablished hypothesis that in response to DNA damage such as γ-radiation, cell cycle progression can be arrested at the G1, S, and G2 phases that provides the cell more time to repair damage in DNA before progressing to the next phase of the cycle (63-65). The marked mitotic arrest and insignificant G1 and G2 arrests elicited by Cd suggest that spindle damage may be more critical than DNA damage in inducing signals to affect genome integrity upon Cd exposure. These results also suggest that the mitotic spindle apparatus may be the Cd target to generate ROS. Spindle damage provokes two signals, one leading to prevent anaphase onset and the other preventing cytokinesis and mitotic exit (66-68). In response to unattached kinetochores, checkpoint kinases such as Bub1 promote the formation of Mad2-Cdc20-APC complexes, thereby inhibiting anaphase onset by preventing the degradation of Pds mediated by APC-Cdc20 (66-68). In response to distinct events, possibly occurring at the spindle poles, the Bub2-dependent pathway prevents activation of the mitotic exit network including the Tem1 GTPase and Cdc14 phosphatase (66-68). It is interesting to explore whether Cd affects these spindle checkpoint signals. It should be noticed that Cd cytotoxicity is also affected by cell culture conditions, e.g., cells that have been cultured in media containing serum (10%) before Cdtreatment (the procedure used in this study) are more sensitive to Cd than those cultured in serum-free media prior to Cd exposure (37). However, the differential


cytotoxicity observed in the two culture conditions is correlated with the levels of ROS and Cd uptake (data not shown). Also, these different cell culture conditions do not affect the patterns of ERK or p38 MAPK activated by Cd. In summary, we have demonstrated that Cd persistence activates ERK and p38 MAPK particularly in the G2/M phase and elicits mitotic arrest and subsequently apoptosis. The activation of ERK and p38 MAPK by Cd is associated with the levels of intracellular peroxide but not Cd accumulation. The generation of ROS by Cd may subsequently lead to the activation of ERK and p38 MAPK, and the induction of micronuclei. While ERK potentially plays a role in guarding genome integrity, p38 MAPK may trigger genome instability and apoptosis in Cd-treated cells. The present study provides evidence that genome integrity can be modulated by epigenetic activation of MAPK pathways in response to genotoxic agents.

Acknowledgment. The authors are grateful to Dr P.-C. Yang for providing the CL3 cells and Dr S.-M. Chuang for critical discussion of the manuscript. This work was supported in part by Grant NSC89-2311-B-007027 from the National Science Council, Taiwan, and by Grant VTG89-G4-04 from the Medical Research Advancement Foundation in Memory of Dr. Chi-Shuen Tsou, Taiwan.

References (1) Marshall, C. J. (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signalregulated kinase activation. Cell 80, 179-185. (2) Whitmarsh, A. J., and Davis, R. J. (1996) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med. 74, 589-607. (3) Garrington, T. P., and Johnson, G. L. (1999) Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11, 211-218. (4) Keyse, S. M. (2000) Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr. Opin. Cell Biol. 12, 186-192. (5) Nebreda, A. R., and Porras, A. (2000) p38 MAP kinases: beyond the stress response. Trends Biochem. Sci. 25, 257-260. (6) Verlhac, M. H., de Pennart, H., Maro, B., Cobb, M. H., and Clarke, H. J. (1993) MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. Dev. Biol. 158, 330-340. (7) Haccard, O., Lewellyn, A., Hartley, R. S., Erikson, E., and Maller, J. L. (1995) Induction of Xenopus oocyte meiotic maturation by MAP kinase. Dev. Biol. 168, 677-682. (8) Abrieu, A., Fisher, D., Simon, M. N., Doree, M., and Picard, A. (1997) MAPK inactivation is required for the G2 to M-phase transition of the first mitotic cell cycle. EMBO J. 16, 6407-6413. (9) Bitangcol, J. C., Chau, A. S., Stadnick, E., Lohka, M. J., Dicken, B., and Shibuya, E. K. (1998) Activation of the p42 mitogenactivated protein kinase pathway inhibits Cdc2 activation and entry into M-phase in cycling Xenopus egg extracts. Mol. Biol. Cell 9, 451-467. (10) Guadagno, T. M., and Ferrell, J. E., Jr. (1998) Requirement for MAPK activation for normal mitotic progression in Xenopus egg extracts. Science 282, 1312-1315. (11) Tachibana, K., Machida, T., Nomura, Y., and Kishimoto, T. (1997) MAP kinase links the fertilization signal transduction pathway to the G1/S-phase transition in starfish eggs. Embo J. 16, 43334339. (12) Walter, S. A., Guadagno, T. M., and Ferrell, J. E., Jr. (1997) Induction of a G2-phase arrest in Xenopus egg extracts by activation of p42 mitogen-activated protein kinase. Mol. Biol. Cell 8, 2157-2169. (13) Takenaka, K., Gotoh, Y., and Nishida, E. (1997) MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts. J. Cell Biol. 136, 1091-1097.

Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1201 (14) Pages, G., Lenormand, P., L’Allemain, G., Chambard, J. C., Meloche, S., and Pouyssegur, J. (1993) Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc. Natl. Acad. Sci. U.S.A. 90, 8319-8323. (15) Brunet, A., Roux, D., Lenormand, P., Dowd, S., Keyse, S., and Pouyssegur, J. (1999) Nuclear translocation of p42/p44 mitogenactivated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J. 18, 664-674. (16) Talarmin, H., Rescan, C., Cariou, S., Glaise, D., Zanninelli, G., Bilodeau, M., Loyer, P., Guguen-Guillouzo, C., and Baffet, G. (1999) The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G1 phase progression in proliferating hepatocytes. Mol. Cell. Biol. 19, 6003-6011. (17) Hulleman, E., Bijvelt, J. J., Verkleij, A. J., Verrips, C. T., and Boonstra, J. (1999) Nuclear translocation of mitogen-activated protein kinase p42MAPK during the ongoing cell cycle. J. Cell. Physiol. 180, 325-333. (18) Wright, J. H., Munar, E., Jameson, D. R., Andreassen, P. R., Margolis, R. L., Seger, R., and Krebs, E. G. (1999) Mitogenactivated protein kinase kinase activity is required for the G2/M transition of the cell cycle in mammalian fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 96, 11335-11340. (19) Shapiro, P. S., Vaisberg, E., Hunt, A. J., Tolwinski, N. S., Whalen, A. M., McIntosh, J. R., and Ahn, N. G. (1998) Activation of the MKK/ERK pathway during somatic cell mitosis: direct interactions of active ERK with kinetochores and regulation of the mitotic 3F3/2 phosphoantigen. J. Cell Biol. 142, 1533-1545. (20) Zecevic, M., Catling, A. D., Eblen, S. T., Renzi, L., Hittle, J. C., Yen, T. J., Gorbsky, G. J., and Weber, M. J. (1998) Active MAP kinase in mitosis: localization at kinetochores and association with the motor protein CENP-E. J. Cell Biol. 142, 1547-1558. (21) Hill, C. S., and Treisman, R. (1995) Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80, 199-211. (22) Molnar, A., Theodoras, A. M., Zon, L. I., and Kyriakis, J. M. (1997) Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK. J. Biol. Chem. 272, 13229-13235. (23) Lavoie, J. N., L’Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996) Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J. Biol. Chem. 271, 20608-20616. (24) Takenaka, K., Moriguchi, T., and Nishida, E. (1998) Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest. Science 280, 599-602. (25) Wang, X., McGowan, C. H., Zhao, M., He, L., Downey, J. S., Fearns, C., Wang, Y., Huang, S., and Han, J. (2000) Involvement of the MKK6-p38γ cascade in γ-radiation-induced cell cycle arrest. Mol. Cell. Biol. 20, 4543-4552. (26) International Agency for Research on Cancer (1993) Cadmium and cadmium compounds. IARC Monographs on the evaluation of carcinogenic risks to humans: cadmium, mercury, beryllium and exposures in the glass industry, Vol. 58, pp 119-237, International Agency for Research on Cancer, Lyon, France. (27) DiPaolo, J. A., and Casto, B. C. (1979) Quantitative studies of in vitro morphological transformation of Syrias hamster cells by inorganic metal salts. Cancer Res. 39, 1008-1013. (28) Ochi, T., and Ohsawa, M. (1983) Induction of 6-thioguanineresistant mutants and single-strand scission of DNA by cadmium chloride in cultured Chinese hamster cells. Mutat. Res. 111, 6978. (29) Ochi, T., Mogi, M., Watanabe, M., and Ohsawa, M. (1984) Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride. Mutat. Res. 137, 103-109. (30) Berces, J., Otos, M., Szirmai, S., Crane-Uruena, C., and Koteles, G. J. (1993) Using the micronucleus assay to detect genotoxic effects of metal ions. Environ. Health Perspect. 101 (Suppl. 3), 11-13. (31) Seelbach, A., Fissler, B., and Madle, S. (1993) Further evaluation of a modified micronucleus assay with V79 cells for detection of aneugenic effects. Mutat. Res. 303, 163-169. (32) Yang, J.-L., Chao, J.-I, and Lin J.-G (1996) Reactive oxygen species may participate in the mutagenicity and mutational spectrum of cadmium in Chinese hamster ovary-K1 cells. Chem. Res. Toxicol. 9, 1360-1367. (33) Hwua, Y.-S., and Yang, J.-L. (1998) Effect of 3-aminotriazole on anchorage independence and mutagenicity in cadmium- and leadtreated diploid human fibroblasts. Carcinogenesis 19, 881-888. (34) Waalkes, M. P., and Rehm, S. (1994) Chronic toxic and carcinogenic effects of cadmium chloride in male DBA/2NCr and NFS/ NCr mice: strain-dependent association with tumors of the

1202

(35) (36) (37)

(38)

(39)

(40)

(41)

(42)

(43) (44)

(45) (46)

(47)

(48)

(49)


hematopoietic system, injection site, liver, and lung. Fundam. Appl. Toxicol. 23, 21-31. Li, W., Zhao, Y., and Chou, I. N. (1993) Alterations in cytoskeletal protein sulfhydryls and cellular glutathione in cultured cells exposed to cadmium and nickel ions. Toxicology 77, 65-79. Wang, Z., and Templeton, D. M. (1996) Cellular factors mediate cadmium-dependent actin depolymerization. Toxicol. Appl. Pharmacol. 139, 115-121. Chuang, S.-M., Wang, I.-C., and Yang, J.-L. (2000) Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis 21, 1423-1432. Galan, A., Garcia-Bermejo, M. L., Troyano, A., Vilaboa, N. E., de Blas, E., Kazanietz, M. G., and Aller, P. (2000) Stimulation of p38 mitogen-activated protein kinase is an early regulatory event for the cadmium-induced apoptosis in human promonocytic cells. J. Biol. Chem. 275, 11418-11424. Hung, J. J., Cheng, T. J., Lai, Y. K., and Chang, M. D. (1998) Differential activation of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases confers cadmiuminduced HSP70 expression in 9L rat brain tumor cells. J. Biol. Chem. 273, 31924-31931. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489-27494. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler. J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., and Young (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372, 739-746. Lee, J. C., Kassis, S., Kumar, S., Badger, A., and Adams, J. L. (1999) p38 mitogen-activated protein kinase inhibitorss mechanisms and therapeutic potentials. Pharmacol. Ther. 82, 389-397. Yang, P.-C., Luh, K.-T., Wu, R., and Wu, C.-W. (1992) Characterization of the mucin differentiation in human lung adenocarcinoma cell lines. Am. J. Respir. Cell Mol. Biol. 7, 161-171. Liesveld, J. L., Keng, P. C., Rowe, J. M., DiPersio, J. F., and Abboud, C. N. (1994) Effects of GM-CSF on Ki67 expression and cell cycle traverse in acute myelogenous leukemia specimens and cell lines. Leuk. Res. 18, 609-616. Terui, Y., Furukawa, Y., Kikuchi, J., and Saito, M. (1995) Apoptosis during HL-60 cell differentiation is closely related to a G0/G1 cell cycle arrest. J. Cell Physiol. 164, 74-84. Plumb, J. A., Milroy, R., and Kaye, S. B. (1989) Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res. 49, 44354440. Sanchez-Ferrer, A., Santema, J. S., Hilhorst, R., and Visser, A. J. (1990) Fluorescence detection of enzymatically formed hydrogen peroxide in aqueous solution and in reversed micelles. Anal. Biochem. 187, 129-132. Ochi, T., Ishiguro, T., and Ohsawa, M. (1983) Participation of active oxygen species in the induction of DNA single-strand scissions by cadmium chloride in cultured Chinese hamster cells. Mutat. Res. 122, 169-175. Ochi, T., and Ohsawa, M. (1985) Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells. Mutat. Res. 143, 137142.

Chao and Yang (50) Hassoun, E. A., and Stohs, S. J. (1996) Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures. Toxicology 112, 219-226. (51) Mikhailova, M. V., Littlefield, N. A., Hass, B. S., Poirier, L. A., and Chou, M. W. (1997) Cadmium-induced 8-hydroxydeoxyguanosine formation, DNA strand breaks and antioxidant enzyme activities in lymphoblastoid cells. Cancer Lett. 115, 141-148. (52) Yih, L. H., Ho, I. C., and Lee, T. C. (1997) Sodium arsenite disturbs mitosis and induces chromosome loss in human fibroblasts. Cancer Res. 57, 5051-5059. (53) Abbott, D. W., and Holt, J. T. (1999) Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation. J. Biol. Chem. 274, 2732-2742. (54) Buckley, S., Driscoll, B., Barsky, L., Weinberg, K., Anderson, K., and Warburton, D. (1999) ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2. Am. J. Physiol. 277, L159-166. (55) Kaina, B., Haas, S., and Kappes, H. (1997) A general role for c-Fos in cellular protection against DNA-damaging carcinogens and cytostatic drugs. Cancer Res. 57, 2721-2731. (56) Saavedra, H. I., Fukasawa, K., Conn, C. W., and Stambrook, P. J. (1999) MAPK mediates RAS-induced chromosome instability. J. Biol. Chem. 274, 38083-38090. (57) Fukasawa, K., Choi, T., Kuriyama, R., Rulong, S., and Vande Woude, G. F. (1996) Abnormal centrosome amplification in the absence of p53. Science 271, 1744-1747. (58) Fukasawa, K., and Vande Woude, G. F. (1997) Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability. Mol. Cell Biol. 17, 506-518. (59) Fukasawa, K., and Vande Woude, G. F. (1995) Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle. Proc. Natl. Acad. Sci. U.S.A. 92, 3430-3434. (60) Sundaresan, M., Yu, Z. X., Ferrans, V. J., Irani, K., and Finkel, T. (1995) Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270, 296-299. (61) Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q., and Holbrook, N. J. (1996) Activation of mitogen-activated protein kinase by H2O2: role in cell survival following oxidant injury. J. Biol. Chem. 271, 4138-4142. (62) Chuang, S.-M., Liou, G.-Y., and Yang, J.-L. (2000) Activation of JNK, p38 and ERK mitogen-activated protein kinases by chromium(VI) is mediated through oxidative stress but does not affect cytotoxicity. Carcinogenesis 21, 1491-1500. (63) Shackelford, R. E., Kaufmann, W. K., and Paules, R. S. (1999) Cell cycle control, checkpoint mechanisms, and genotoxic stress. Environ. Health Perspect. 107 (Suppl. 1), 5-24. (64) Dasika, G. K., Lin, S. C., Zhao, S., Sung, P., Tomkinson, A., and Lee, E. Y. (1999) DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene 18, 7883-7899. (65) Lowndes, N. F., and Murguia, J. R. (2000) Sensing and responding to DNA damage. Curr. Opin. Genet. Dev. 10, 17-25. (66) Taylor, S. S. (1999) Chromosome segregation: dual control ensures fidelity. Curr. Biol. 9, R562-R564. (67) Burke, D. J. (2000) Complexity in the spindle checkpoint. Curr. Opin. Genet. Dev. 10, 26-31. (68) Cerutti, L. and Simanis, V. (2000) Controlling the end of the cell cycle. Curr. Opin. Genet. Dev. 10, 65-69.

TX010041O

Opposite Roles of ERK and p38 Mitogen-Activated Protein Kinases in

Recommend Documents