Opposite Roles of ERK and p38 Mitogen-ActivatedProtein 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 proteinkinase (MAPK) in guarding genome stability and regulating cell cycle progression were exploredin 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-G1populations; however, this did not alter the levels of Cdc2 and cyclin B1. These results suggestthat Cd elicits mitotic arrest without affecting the progression of G2 to mitosis. Usingcounterflow 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 cellcycle phase to Cd for the induction of ERK and p38 MAPK activities, cytotoxicity, apoptosis,micronucleus, and intracellular peroxide; despite that similar Cd accumulation was observedin G1-, S-, and G2/M-cells. Co-treatment early-G2 cells with Cd and SB202190, an inhibitor ofp38 MAPK, significantly decreased the induction of micronucleus, mitotic arrest, and apoptosis.Conversely, PD98059, an inhibitor of the ERK upstream activators MKK1/2, enhancedmicronucleus and apoptosis in Cd-treated early-G2 cells. Together, the results suggest thatintracellular 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, whereasthe activated-ERK may help to maintain genome integrity and survival.

Introduction

The ERK1 and p38 MAPK cascades are critical in thecontrol of cell growth, differentiation, and apoptosis (1-5). In general, ERK signaling cascade is activated bygrowth factors and required for cell proliferation. Con-versely, p38 MAPK pathway is involved in growth arrestand apoptosis in response to genotoxic agents. However,cumulative reports have indicated that these signalingpathways exhibit more complex roles in the regulationof distinct cellular effects. While transient ERK activationpromotes proliferation in fibroblasts, persistent activationmediates growth arrest or differentiation in neuronalcells, T cells, and muscle cells (1). On the other hand,activation of p38 MAPK has been implicated to berequired for cell survival, proliferation, and differentia-tion (5). The particular function regulated by ERK or p38MAPK 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 multipleeffects on cell cycle progression. ERK is essential for

meiotic maturation in mouse (6) and Xenopus oocytes (7)and mitotic processes in Xenopus egg extracts (8-10).However, inappropriate activation of ERK induces re-pression of DNA synthesis in starfish eggs (11) andarrests cells at G2 (9, 12) and mitotic phases (9, 13) inXenopus egg extracts. In mammalian cell lines, ERKactivation and localization in nucleus is required for theprogression of G0/G1 to S phase (14-17). Phosphorylationof ERK is also essential for the progression from G2 tomitosis (18). Moreover, normal mitotic progression ishighly associated with the activation/inactivation andlocalization of ERK (19, 20). In general, ERK activationis essential for entry into a specific cell cycle transitionand its inactivation, re-localization, and reactivation maybe required for exit from that stage and entry into thesubsequent phase. Thus, the activity of ERK oscillatesthrough different cell cycle phases to control cell prolif-eration. Phosphorylation of nuclear transcription factorsby ERK is a crucial step in the regulation of geneexpression (21). ERK may also regulate the associationbetween chromosomes and microtubules through itsinteraction with the kinetochore motor protein CENP-E(20). On the other hand, p38 MAPK activation is involvedin the inhibition of serum-stimulated cell cycle progres-sion at G1/S by cdc42Hs (22). Opposing to the function ofERK pathway, p38 MAPK signal cascade inhibits themitogen-induced cyclin D1 expression (23). Activation ofp38 MAPK is shown to be associated with mitotic arrestin NIH 3T3 cells induced by nocodazole, a microtubule

* To whom correspondence should be addressed. Phone: 886-3-5742756. Fax: 886-3-5645782. E-mail: jlyang@life.nthu.edu.tw.

† Molecular Carcinogenesis Laboratory.‡ Medical Technology Laboratory.1 Abbreviations: ERK, extracellular signal-regulated kinase; MAPK,

mitogen-activation protein kinase; Cd, cadmium; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetra-zolium bromide; FITC, fluorescein isothiocyanate; DCF, dichlorofluo-rescein; ICP-MS, inductively coupled plasma-mass spectrometer; ROS,reactive oxygen species.

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

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

depolymerization agent (24). Furthermore, the MKK6-p38γ signal is required for the G2 arrest upon γ-radiation(25).

Cd is a ubiquitous environmental toxicant that hasbeen evaluated as a human carcinogen (26). Cd caninduce DNA damage, morphological transformations,micronuclei, chromosomal aberrations, and gene muta-tions in cultured mammalian cells (27-33) and tumorsin animal models (34). Cd compounds have also beencharacterized as a spindle poison (35, 36). Cd has beenreported to activate p38 MAPK and ERK in CL3 humanlung adenocarcinoma cells (37), U937 human promono-cytic leukemia cells (38), and 9L rat brain tumor cells(39). Persistent activation of p38 MAPK by Cd is medi-ated in apoptosis, whereas ERK activation by Cd playsan opposite role in CL3 cells (37). Although ERK activityis critical to control cell cycle progression and p38 MAPKmay cause G1/S, G2, or mitotic arrest, the roles of thesesignals elicited by Cd in affecting genome integrity andcell cycle progression remain unknown. Here we showthat CdCl2 induced irreversible mitotic arrest withoutsignificant influence the levels of Cdc2 and cyclin B1 inCL3 cells. G2/M was the most sensitive cell cycle phaseto Cd in the induction of ERK and p38 MAPK activities,cytotoxicity, apoptosis, micronucleus, and intracellularperoxide, although Cd accumulation was independent ofcell cycle phase. The effects of ERK and p38 MAPK onCd-induced micronucleus and mitotic arrest were studiedusing PD98059, a specific inhibitor of the ERK upstreamactivators MKK1/2 (40), and SB202190, a specific inhibi-tor of p38 MAPK (41, 42), respectively. Together, thefindings suggest that intracellular peroxide elicited byCd is highly associated with the activation of ERK andp38 MAPK that play opposite roles in Cd-induced geno-toxicity and mitotic arrest.

Experimental Procedures

Cell Culture. The CL3 cell line established from a non-small-cell lung carcinoma tumor of a 60-year-old male patient inTaiwan (43) was provided by Dr. P. C. Yang at the Departmentof Internal Medicine and Clinical Pathology, National TaiwanUniversity Hospital, Taipei. Cells were cultured in RPMI1640medium (Gibco, Life Technologies, Grand Island, NY) supple-mented 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 weregrown to near confluence and fed with serum-free medium for2 days. The cells were plated at a density of 1.5 × 106 cells pera p60-dish in medium containing 10% fetal calf serum and 1µg/mL aphidicolin (Sigma, St. Louis, MO). One day later, thecells synchronized at the G1/S-border were washed withRPMI1640 medium, re-fed with culture medium, and kept in aCO2 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 treat-ment, the cells were washed twice with PBS and kept in theincubator for various times before they were analyzed by flowcytometry.

Alternatively, G1-, S-, and G2/M-enriched cells were collectedby the counterflow centrifugal elutriation using a Beckman J-6Mcentrifuge equipped with a JE-6B elutriation rotor (44, 45).Briefly, exponentially growing cells (1 × 108) were concentratedin 15 mL of RPMI1640 containing 1% fetal calf serum andelutriated at a flow rate of 30 mL/min. After the cells settledinto the chamber, fractions were collected at a speed of 2100-1450 rpm at intervals of 50 rpm. Aliquots of the samples were

subjected to cell cycle analysis by flow cytometry to determinethe cell cycle phase of each fraction. Following elutriation, cellswere treated with Cd for 2 h and assayed for the activation ofERK and p38 MAPK, cytotoxicity, apoptosis, micronucleus, Cduptake, and intracellular peroxide levels.

In experiments to determine the roles of ERK and p38 MAPKin Cd-induced micronuclei and cell cycle inhibition, cells wereco-treated with Cd and specific kinase inhibitors PD98059(Calbiochem, San Diego, CA) and SB202190 (Calbiochem),respectively.

Flow Cytometry. Cell cycle phases were analyzed using afluorescence-activated cell sorter (FACScan, Becton-Dickinson,San Jose, CA) with CellQuest and Modfit LT softwares. Aliquotsof 1 × 106 cells were fixed with 70% ethanol for at least 2 h at-20 °C before centrifugation. The cell pellets were treated withpropidium iodine (4 µg/mL) solution containing RNase (100 µg/mL) and Triton X-100 (1%) for 30 min. The stained cells wereanalysis using a FACScan with a pulse processing protocolaccording to the manufacturer’s instructions.

Mitotic Index Analysis. Exponentially growing cells wereplated at a density of 5 × 104 cells/cm2 in 60-mm dishes 1 daybefore treatment. Cells were left untreated or treated with 0.5µM nocodazole (Sigma) for 4 h, washed with PBS, and thentreated with Cd for 2 h in RPMI1640 medium. After removingCd, the cells were washed twice with PBS, and kept culturedfor 0, 3, 6, 12, and 24 h. Next, the cells were trypsinized andtreated with 0.05% KCl for 5 min at room temperature. Aftercentrifugation, the cells were fixed with methanol/acetic acidsolution (3:1, v/v). An aliquot of the cells in suspension wasdropped onto a clean slide, air-dried and stained with a 10%Giemsa solution (Merck). At least 200 cells were scored for thedetermination of mitotic index in each culture.

Cytotoxicity. Cells were seeded in 96-well plates at a densityof 1 × 104 cells/well and treated with Cd for 2 h. After treatment,the cells were washed twice with PBS and kept cultured inRPMI1640 containing 10% fetal calf serum for 48 h. The cellswere then treated with 500 µg/mL MTT (Sigma) and kept in aCO2 incubator for 4 h. Viable cells can convert MTT to formazanthat generates blue color when dissolved in dimethyl sulfoxide(46). The intensity was measured using a reader for enzyme-linked immunosorbent assay and an absorption wavelength of565 nm.

Apoptosis. Annexin V-FITC binding assay was adopted forthe determination of apoptosis. Immediately after Cd treatment,2 × 105 cells were washed twice with PBS and suspended inbinding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, and 2.5mM CaCl2). The cells were stained with annexin V-FITC (1 ng/µL; Medical & Biological Lab., LTD, Japan) and propidiumiodide (5 ng/µL) for 15 min in the dark. The stained cells wereanalyzed using flow cytometry and the CellQuest program.Apoptotic cells were detected by those stained with annexinV-FITC but not with propidium iodide.

Micronucleus Assay. The cells 1 × 105 in 60-mm Petri dishwere cultured overnight and exposed to Cd (0-80 µM) for 2 hin RPMI1640 medium. Next, the cultures were washed threetimes with PBS, treated with 1 µg/mL cytochalasin B (Sigma)in RPMI1640 medium containing 10% fetal calf serum andincubated for another 24 h. At the end of incubation, the cultureswere washed with PBS once, and incubated in 0.05% KCl for 5min at room temperature and then fixed in 3 mL of Carnoy’ssolution (20:1, methanol: acetic acid, v/v) for 15 min. The disheswere air-dried and stained with freshly Giemsa’s solution for15 min. The numbers of micronucleus per binucleated cells werescored under a microscope and five hundred binucleated cellsper dish were examined.

Measurement of Intracellular Peroxide Level. The in-tracellular peroxide level was estimated by the activation andoxidation of 2′,7′-dichlorofluorescin diacetate to DCF (47). AfterCd treatment, 1 × 106 cells were suspended in PBS and incu-bated with 80 µM of 2′,7′-dichlorofluorescin diacetate (EstmanKodak, Rochester, NY) for 30 min in the dark. The cells werethen centrifuged and the cell pellets were kept in ice and

1194 Chem. Res. Toxicol., Vol. 14, No. 9, 2001 Chao and Yang

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 intensitywas detected using a fluorescence spectrophotometer withexcitation and emission wavelength at 502 and 523 nm,respectively.

Determination of Cellular Cd Level. Cells were exposedto various Cd concentrations (0-80 µM) in serum-free mediumfor 2 h. Following treatment, the cells were washed three timeswith PBS and the numbers of cells were determined. One millionof cells were centrifuged and the cell pellet was sonicated inMilliQ-purified water. Total cellular Cd concentrations wereanalyzed by an ICP-MS (SCIEX ELAN 5000, Perkin-Elmer,Norwalk, CT). The ICP-MS conditions were as followed: powerof 1000 W, plasma flow rate of 15 L/min, auxiliary flow rate of0.8 L/min, and sample flow rate of 1 mL/min.

Western Blot Analysis. Cells were lysed in whole cellextract buffer containing 20 mM HEPES at pH 7.6, 75 mMNaCl, 2.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5mM DTT, 0.1 mM Na3VO4, 50 mM NaF, 0.5 µg/mL leupeptin, 1µg/mL aprotinin, and 100 µg/mL 4-(2-aminoethyl)benzene-sulfonyl fluoride. The cell lysate was rotated at 4 °C for 30 min,centrifuged at 10 000 rpm for 10 min, and the precipitates werediscarded. Protein concentrations were determined by the BCAprotein assay kit (Pierce, Rockford, IL) using bovine serumalbumin as a standard.

Equal amounts of proteins (20-60 µg) extracted from eachsets of experiments were loaded onto 10% SDS-polyacrylamidegels. The protein bands were then transferred electrophoreti-cally to PVDF membranes (NEN, Boston, MA). Membranes wereprobed with primary antibody and followed with a horseradishperoxidase-conjugated second antibody (BioRad Co., Hercules,CA). Phospho-specific antibodies for p38 MAPK (no. 9211), andERK (no. 9101) were purchased from New England BioLabs Inc.(Beverly, MA). Anti-ERK2 (C-14), anti-p38 MAPK (C-20), andanti-Cdc2 (H-297) antibodies were purchased from Santa CruzBiotechnology, Inc. (Santa Cruz, CA). Anti-cyclin B1 (Ab-2) waspurchased from Oncogene Sciences (Cambridge, MA). Antibodyreaction was detected using the enhanced chemiluminescencedetection procedure according to the manufacturer’s recom-mendations (NEN, Boston, MA). In some experiments, antibod-ies were stripped from membranes using solution containing2% SDS, 62.5 mM Tris-HCl, pH 6.8, and 0.7% (w/w) â-mercap-toethanol at 50 °C for 15 min before reprobing with anotherprimary antibody. The relative protein intensities on blots werequantitated using a computing densitometer equipped with theImagQuant analysis program (Molecular Dynamics, Sunnyvale,CA).

Results

Cd Causes Mitotic Arrest. CL3 cells at exponentialgrowth were left untreated or treated with CdCl2 (40 µM)in serum-free media for 2 h, washed with PBS, keptcultured for 3-24 h, and then subjected to flow cytometryanalysis to examine how does Cd interfere cell cycleprogression. The G1 fraction significantly decreased(Figure 1A) while the G2/M fraction increased (Figure 1B)6-24 h after Cd treatment. Conversely, Cd did notsignificantly affect the S phase fractions (Figure 1C). Tofurther characterize whether Cd affects M-phase, themitotic index of Cd-treated asynchronous growing cellswas examined by chromosome spreading. As shown inFigure 1D, a 2-h Cd exposure markedly increased themitotic index 6-24 h later. The results of chromosomespreading and flow cytometry analysis indicate that Cdinduces mitotic arrest and does not seem to affect the G2

phase.Moreover, cells were enriched in M-phase using no-

codazole (0.5 µM, 4 h), left untreated or treated with Cd

for 2 h, washed with PBS, kept cultured for 3-24 h andthen subjected to mitotic index analysis. The nocodazole-enriched mitotic cells declined to basal levels within 24h, while the fraction of mitotic cells accumulated at highlevels in those treated with Cd plus nocodazole (Figure2). This result suggests that Cd-induced mitotic arrestis irreversible which is different from the mechanism ofnocodazole.

Next, we examined the effect of Cd on the progressionof G2 phase to the next cell cycle phases. CL3 cells weresynchronized at G1/S border using serum-starvation (2days) and aphidicolin (1 µg/mL, 24 h). Flow cytometry

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

Figure 2. Different mechanisms of mitotic arrest induced byCd and nocodazole. Exponential growing CL3 cells were exposedto 0.5 µM of nocodazole for 4 h, left untreated or treated with40 µM of CdCl2 for 2 h, washed with PBS, kept cultured for3-24 h and then subjected to mitotic index analysis. Barsrepresent the SEM of three independent experiments.

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analyses showed that these synchronous cells enteredinto S-phase rapidly after aphidicolin removal and mostof the cells progressed to early-G2 phase 4 h later (Figure3). The early-G2 cells were left untreated or treated with40 µM of Cd for 2 h, washed with PBS, kept cultured for0-22 h (6-28 h after aphidicolin removal), and analyzedby flow cytometry. As shown in Figure 3, the untreatedcells progressed to the next G1-phase while Cd-treatedcells remained at G2/M phase 12 h after aphidicolinremoval. Chromosome spreading examination showedthat most of the Cd-treated cells exhibited mitotic figuresat 9-12 h after aphidicolin removal, suggesting the Cd-treated early-G2 cells accumulate at M-phase (data notshown). In addition, a high frequency of sub-G1 popula-tion 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

analysis to understand how Cd causes mitotic arrest. CL3cells were synchronized at early-G2 phase, left untreatedor treated with Cd for 2 h as described in Figure 3 andthe whole cell extracts were harvested immediately orat various times after Cd withdrawal. As shown in Figure4 the amounts of cyclin B1 and Cdc2 in the control cellspeaked at 8 h after aphidicolin removal (R2) and de-creased gradually when the cells progressed to the nextcell cycle phase. Exposing the early-G2 cells to Cd did notaffect the levels of cyclin B1 and Cdc2 in cells obtainedat various recovery times (Figure 4). Also, a slower-migrating 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 Cd-treated cells (Figure 4). The lack of affecting Cdc2/cyclinB1 cell cycle engine in Cd-treated cells suggests that Cddoes not elicit G2 arrest.

Persistent Activation of ERK and p38 MAPK inCd-Treated Early-G2 Cells. Cd activates ERK and p38MAPK in CL3 cells (37). Activation of ERK is essentialfor the progression from G2 to mitosis (18) and mitoticprogression (19, 20); also, inappropriate ERK activationmay result in G2 or mitotic arrest (9, 13). Activation ofp38 MAPK by nocodazole and γ-radiation has beenassociated with mitotic arrest and G2 arrest, respectively(24, 25). We therefore examined the ERK and p38 MAPK

Figure 3. Cd delays the early-G2 cells entering into the nextG1 phase and subsequently induces the sub-G1 populations. CL3cells were grown to near confluence, fed with serum-freemedium for 2 days, and plated in culture medium containing 1µg/mL of aphidicolin for 1 day to allow cells synchronized atthe G1/S-border. The cells were then washed with RPMI1640medium, re-fed with culture medium, and kept in an incubatorfor 4 h to progress to early-G2 phase. The early-G2 cells wereleft untreated or treated with 40 µM of Cd for 2 h in serum-freemedium, washed twice with PBS, and kept cultured for varioustimes before they were analyzed by flow cytometry (R0-R22represent recovery 0-22 h, number in parentheses is time afteraphidicolin removal). The fractions of each cell cycle phases wereaveraged from three to six experiments.

Figure 4. Cd does not alter the levels of cyclin B1 or Cdc2.Cells were synchronized and treated with Cd as described inFigure 3. Whole cell extracts were harvested from the controlG1/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 recovery0-22 h). Western blot analysis was performed using antibodiesagainst cyclin B1 or Cdc2. The relative intensity was quanti-tated using a computing densitometer. Results were obtainedfrom three to four experiments and the bar represents SEM.

1196 Chem. Res. Toxicol., Vol. 14, No. 9, 2001 Chao and Yang

activities in the early-G2 cells treated with Cd as de-scribed in Figure 3, using specific antibodies for the dualphosphorylation sites of the catalytic domain. Althoughan increased level of phosphorylated-p38 MAPK wasobserved in the control G1/S-cells obtained immediatelyafter aphidicolin removal, it decreased later (Figure 5),which indicates aphidicolin transiently elicits p38 MAPKactivity. Figure 5 shows that Cd markedly induced theamounts of dual phosphorylated-p38 MAPK in early-G2

cells (R0), which remained at high levels 2-22 h afterCd withdrawal. On the other hand, the amounts ofphosphorylated-ERK in the control cells oscillated withthe progression of cell cycle (Figure 5). After Cd exposurefor 2 h, the amounts of phosphorylated-ERK steadilyincreased to 2-fold of the untreated levels (Figure 5).Additionally, the protein amounts of p38 or ERK ex-tracted in each recovery times of Cd-treated cells wereabout the same as in the untreated cells (Figure 5). Theresults suggest that persistent activation of p38 MAPKor ERK by Cd may be correlated with the ability of thismetal to irreversibly induce mitotic arrest.

To determine the effects of p38 MAPK and ERKactivation on cell cycle progression and apoptosis, cellsenriched at the early G2-phase as described in Figure 3were treated with Cd (40 µM) for 2 h in the presence ofSB202190, 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 hafter Cd exposure showed that 50 µM of PD98059reduced the number of cells reentry into G1 phase andincreased the numbers of sub-G1 cells induced by Cd(Figure 6). Conversely, 10 µM of SB202190 reduced thenumbers of sub-G1 cells and enhanced the number of cellsreentry into G1 phase (Figure 6). PD98059 or SB202190alone did not affect the cell cycle progression (data notshown). These findings suggest that the p38 MAPKactivated by Cd may transmit signals to induce mitoticarrest and subsequently lead to apoptosis, while the Cd-activated ERK may enhance mitotic exit and protect fromapoptosis.

G2/M Is the Most Sensitive Phase to Cd in theActivation of p38 MAPK and ERK. To further char-acterize the ability of Cd to activate p38 MAPK and ERKat different cell cycle phases, CL3 cells were separatedby counterflow centrifugal elutriation into fractions of G1,S, and G2/M phases. Examples of typical flow cytometricprofiles of DNA from asynchronous cells and thosecollected from centrifugal speed 1900 rpm (G1-phase),1700 rpm (S-phase), and 1500 rpm (G2/M-phase) areshown in Figure 7A. These G1-, S-, or G2/M-cells wereexposed to Cd for 2 h and the whole cell extracts wereisolated for the determination of the levels of dualphosphorylated-ERK and p38 MAPK. As shown in Figure7B, the highest level of the p38 MAPK activated by Cdwas observed in G2/M-cells. Cd also activated p38 MAPKin S-cells. However, Cd did not induce the p38 MAPKactivity in G1-cells. G2/M was also the most sensitivephase for the activation of ERK by Cd (Figure 7B). Inthe untreated populations, a higher ERK activity wasobserved in S-phase (Figure 7B).

G2/M Is the Most Sensitive Phase to Cd in theInduction of Cytotoxicity, Apoptosis, and Micro-

Figure 5. Persistent activation of p38 MAPK and ERK in theCd-treated early G2-cells. Cells were synchronized and treatedwith Cd as described in Figure 3. Whole cell extracts wereharvested from the control G1/S cells and the early-G2 cells thatwere left untreated (G2) or treated with 40 µM of Cd for 2 h(R0-R22 represent recovery 0-22 h). Activation of p38 MAPKand ERK in whole cell extracts were examined using phospho-specific antibodies. Western blot analyses of the p38 MAPK andERK2 protein levels are showed in the lower panels. The relativeintensity was quantitated using a computing densitometer.Results were obtained from three to five experiments and thebar represents SEM. Student t-test was adopted to compare thesignificance between Cd-treated and untreated cells. (*) P < 0.05and (**) P < 0.01, respectively.

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

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nucleus. To investigate the relative sensitivities ofdifferent cell cycle phases to Cd-induced toxicity, equalnumbers of G1-, S-, and G2/M-cells derived from counter-flow centrifugal elutriation were exposed to 0-80 µM ofCd for 2 h. Cytotoxicity was determined using MTT assay48 h later. As shown in Figure 8A, G2/M was the mostsensitive phase to the cytotoxicity induced by Cd. Cellsat the S-phase exhibited similar Cd sensitivity to theasynchronous cells, while the G1-cells were less sensitiveto Cd (Figure 8A). The fractions of apoptotic cells wereexamined using annexin V-FITC assay and flow cytom-etry. As shown Figure 8B, Cd induced significantly moreapoptotic cells in the G2/M-phase than the other cell cyclephases. The apoptotic cells observed in untreated cells

were 3-4% of those that were increased to 8, 10, 15, and23% in the G1-, asynchronous, S-, and G2/M-cells exposedto 80 µM of Cd, respectively (Figure 8B). The formationof micronuclei is a reflection of DNA damage, defectivemitosis, and loss of genetic materials. This assay wasthen adopted to explore the genotoxicity induced by Cdat various cell cycle phases. As shown in Figure 8C, Cdinduced significantly higher numbers of micronuclei inthe G2/M- and S-cells than the asynchronous- and G1-

Figure 7. G2/M was the most sensitive cell cycle phase to theactivation of p38 MAPK and ERK by Cd. (A) G1-, S-, and G2/M-cells were fractionated from proliferating CL3 cells usingcounterflow centrifugal elutriation and analyzed by flow cytom-etry as described in the Experimental Procedures. The fractionsof each cell cycle phases were averaged from five experiments.(B) G1-, S-, and G2/M-cells obtained from counterflow centrifugalelutriation were exposed to Cd for 2 h, and their whole cellextracts were subjected to the determination of p38 MAPK andERK activation using phospho-specific antibodies. After detec-tion of phosphorylated proteins, antibodies were stripped frommembranes and then reprobed with primary antibodies againstp38 MAPK and ERK2. Western blots of the p38 MAPK andERK2 protein levels are showed in the lower panels. The relativeintensity was quantitated using a computing densitometer. Thenumbers between western blots of phosphorylated and totalproteins were the relative activities of kinases obtained fromaveraging five experiments.

Figure 8. G2/M was the most sensitive cell cycle phase to theinduction of cytotoxicity, apoptosis and micronucleus by Cd. (A)Following counterflow centrifugal elutriation, cells synchronizedat G1-, S-, and G2/M were left untreated or treated with Cd for2 h, washed twice with PBS, kept cultured for 2 days, and thensubjected 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 determinationof apoptosis using annexin V-FITC binding assay and analyzedby flow cytometry. (C) After Cd treatment, the cells were washedwith PBS and incubated for another 24 h in culture mediumcontaining 1 µg/mL of cytochalasin B. The cells were thenprepared for micronucleus assay and the numbers of micronucleiper binucleated cells were scored using a microscope. Resultswere obtained from three to four experiments and the barrepresents SEM. Student t-test was adopted to compare thesignificance between Cd-treated synchronous and asynchronouscells; (*) P < 0.05 and (**) P < 0.01, respectively.

1198 Chem. Res. Toxicol., Vol. 14, No. 9, 2001 Chao and Yang

cells. The spontaneous numbers of micronuclei wereabout the same (21.3-23.3/1000 binucleus cells) in theasynchronous, G2/M-, S-, and G1-cells (Figure 8C).

p38 MAPK and ERK May Involve Oppositely inthe Induction of Micronucleus by Cd. The roles ofp38 MAPK and ERK activated by Cd in genotoxicitywere explored using kinase-specific inhibitors. G1-, S-, orG2/M-cells derived from counterflow centrifugal elutria-tion were exposed to 80 µM of Cd in the presence ofSB202190 or PD98059 for 2 h. As shown in Figure 9,SB202190 (10 µM) significantly decreased the numbersof micronuclei induced by Cd in G2/M- and S-phases.Conversely, PD98059 (50 µM) enhanced the numbers ofmicronuclei induced by Cd in G2/M- and S-phases (Figure9).

Cd Accumulation Is Independent of Cell CyclePhases. The levels of Cd accumulated in cells mayinfluence toxicity. Therefore, the amounts of intracellularCd in the asynchronous, G1-, S-, or G2/M-cells weremeasured using ICP-MS. Cells were exposed to Cd for 2h, washed three times with PBS, and subjected to theanalysis of the intracellular Cd amounts. As shown inTable 1, similar amounts of Cd were accumulated in theasynchronous, G1-, S-, and G2/M-cells. This result indi-cates that the cell cycle phase-dependent induction ofcytotoxicity, apoptosis, micronucleus, and the activities

of p38 MAPK and ERK by Cd do not correlate with theintracellular Cd levels.

G2/M Is the Most Sensitive Phase to Cd in theInduction of Intracellular Peroxide. Oxidative stresshas been shown to be highly associated with Cd geno-toxicity (32, 48-51). The intracellular peroxide levelsinduced by Cd in different cell cycle phases were thenmeasured to elucidate the correlation between oxidativestress and genotoxicity. After Cd treatment, the asyn-chronous, G1-, S-, or G2/M-cells (1 × 106 cells/each) weresuspended in PBS and incubated with 80 µM of 2′,7′-dichlorofluorescin diacetate for 30 min in the dark. Thereaction of intracellular peroxides with activated 2′,7′-dichlorofluorescein diacetate would result in DCF thatintensity was detected by a fluorescence spectrophotom-eter. As shown in Figure 10, the DCF fluorescenceintensity induced by Cd was dependent on the cell cyclephases; G2/M was the most sensitive phase to Cd in theinduction of DCF fluorescence, followed was S-phase.Whereas, Cd-treated G1-cells produced lower levels ofDCF fluorescence than those induced in asynchronouscells (Figure 10). This result suggests that the intra-cellular peroxide induced by Cd may be the causative ofthe cell cycle phase-dependent induction of cytotoxicity,apoptosis, micronucleus, and the p38 MAPK and ERKactivities.

DiscussionIn this manuscript, we have demonstrated that Cd

induces irreversible mitotic arrest and G2/M is the most

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 0.54 ( 0.29(3) 2.17 ( 1.67 (3) 1.77 ( 1.59 (3) 1.08 ( 0.83 (3)40 84.12 ( 13.65 (3) 78.64 ( 5.33 (4) 71.63 ( 7.98 (4) 69.42 ( 7.75 (4)80 160.97 ( 17.09 (4) 152.87 ( 27.98 (4) 160.81 ( 20.95 (5) 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 numberof experiments.

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

Figure 10. G2/M was the most sensitive cell cycle phase tothe induction of intracellular peroxide levels by Cd. Synchroni-zation of cells was as described in Figure 7. Immediately aftertreatment, the cells were washed with PBS, trypsinized andincubated with 80 µM of 2′,7′-dichlorofluorescin diacetate for30 min at 37 °C in the dark. The DCF intensity was detectedusing a fluorescence spectrophotometer. Results were obtainedfrom three to five experiments and the bar represents SEM.Student t-test was adopted to compare the significance betweenCd-treated synchronous and asynchronous cells; (*) P < 0.05and (**) P < 0.01, respectively.

Roles of MAPKs in Cd Genotoxicity and Mitotic Arrest Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1199

sensitive phase to Cd causing dose-dependent inductionof cytotoxicity, apoptosis, micronucleus, and p38 MAPKand ERK activities in CL3 human lung adenocarcinomacells. The ability of Cd to arrest cells at mitotic phase isconsistent with previous findings that it depolymerizesmicrotubules and actins in cultured cells (35, 36). Inter-estingly, Cd does not affect the levels of phosphorylated-Cdc2 critical for triggering the G2/M transition or thedegradation of cyclin B1 that occurs in anaphase. There-fore, Cd may not elicit the checkpoint control of G2/Mtransition; rather, Cd may interfere with the progressionof anaphase and cytokinesis, thereby failing in mitoticexit. The mechanism of mitotic arrest caused by Cd isdifferent from that induced by nocodazole (a microtubuledepolymerizer) (24) or arsenite (a microtubule stabilizer)(52), which are associated with a delayed inactivation ofCdc2/cyclin B1.

Recently, we have shown that Cd persistently elicitsp38 MAPK and ERK signals (37). Here, we furtherdemonstrated that activation of p38 MAPK and ERK byCd is cell cycle dependent. Cd elicits markedly strongerp38 MAPK activity in G2/M-cells than in S-cells followedby G1-cells. Similarly, Cd significantly activates ERK inG2/M-cells. The pyridinylimidazole inhibitors of p38R andp38â MAPK including SB202190 and SB203580 have noeffect on the activity of many other protein kinases,including other MAPK family members (41, 42). Also, Cd-induced apoptosis can be enhanced by transient trans-fection of a wild-type p38 vector (37). The present studyshows that co-administrating SB202190 significantlydecreases Cd-elicited mitotic arrest, apoptosis, and thefrequency of micronucleus, suggesting the Cd-activatedp38 MAPK is involved in these events. However, wecannot exclude the possibility that SB202190 may inhibitother unknown targets participating in Cd-induced ge-nome instability, apoptosis, and mitotic arrest. Previ-ously, p38 MAPK activation is reported to be associatedwith nocodazole-induced mitotic arrest (24). Neverthe-less, the present study has shown for the first time thatp38 MAPK activation by genotoxic agents can potentiallytransmit signals to disturb genome integrity.

On the other hand, we have demonstrated thatPD98059 at a dose that completely blocks ERK phos-phorylation (data not shown) enhances the Cd-inducedsub-G1 fraction and micronucleus, suggesting that theactivated-ERK potentially contributes to survival andgenome stability. The involvement of ERK in preventinggenotoxicity is consistent with the reports that activationof MEK2, an ERK upstream activator, by ionizing radia-tion enhances the ability of cells to survival and to recoverfrom the G2/M cell cycle checkpoint arrest (53). ERKphosphorylation protects DNA strand breakage andapoptosis induced by hyperoxia (54). Similarly, c-Fos, adownstream target of ERK, protects survival and chro-mosomal integrity upon many types of genotoxic damage(55). However, ERK has been shown to mediate themicronucleus induction by v-Ras in NIH 3T3 cells thatlack functional tumor suppressor p53 protein (56). In theabsence of p53 abnormal centrosome amplification occurs(57), however, the multiple copies of centrosomes in mostof the p53-/- cells sequester to the spindle poles andsuccessfully participate in the formation of bipolar spindlesand proceed through cytokinesis with a normal karyotype(57, 58). Overexpression of the Mos/ERK pathway greatlyenhanced chromosome instability in p53-/- cells, possiblydue to Mos/ERK interfering with the formation of astral

microtubules (58). Conversely, constitutive activation ofthe Mos/ERK pathway in cells having functional p53allows G2-cells progress to M phase but fails to undergocytokinesis (59). CL3 cells have wild-type p53 sequence(J. Y. Shew, personal communication), and Cd can inducephosphorylation at Ser15 of p53 in CL3 cells (J.-I Chao,unpublished data). The contradictory effects of ERKsignal cascade on maintaining genome integrity in p53deficient and proficient cells deserve further investiga-tion.

Although G2/M is the most sensitive phase to Cd inthe induction of p38 MAPK and ERK activities, apoptosis,and micronucleus, similar levels of Cd accumulation areobserved in asynchronous, G1-, S-, and G2/M-cells. Thisimplies that Cd itself may not directly elicit theseresponses. Recent reports have shown that ROS acts asa signal transduction molecule. For example, platelet-derived growth factor elicits intracellular peroxide thatassociates with tyrosine phosphorylation, ERK stimula-tion, and DNA synthesis in rat vascular smooth musclecells (60). Additionally, H2O2 activates ERK and p38MAPK in cultured mammalian cells including CL3 (61,62). Moreover, several studies have implicated that ROSis involved in Cd-induced genotoxicity in cultured cells,including the induction of DNA strand breaks, 8-hydroxy-2′-deoxyguanosine adducts, chromosomal aberrations,and mutations (32, 48-51). We have demonstrated herethat Cd-treated G2/M-cells generate markedly higherlevels of intracellular peroxide than the other cell cyclephases, suggesting that ROS is the causative of theinduction of MAPK signals, genotoxicity, and apoptosisin Cd-treated cells.

Cd induces higher levels of intracellular peroxide andgenotoxicity in G2/M-cells. However, Cd does not signifi-cantly cause G2 arrest; rather, it leads to mitotic arrest.Moreover, exposing G1-cells to Cd does not affect cell cycleprogression to S-phase (data not shown). It is a well-established hypothesis that in response to DNA damagesuch as γ-radiation, cell cycle progression can be arrestedat the G1, S, and G2 phases that provides the cell moretime to repair damage in DNA before progressing to thenext phase of the cycle (63-65). The marked mitoticarrest and insignificant G1 and G2 arrests elicited by Cdsuggest that spindle damage may be more critical thanDNA damage in inducing signals to affect genomeintegrity upon Cd exposure. These results also suggestthat the mitotic spindle apparatus may be the Cd targetto generate ROS. Spindle damage provokes two signals,one leading to prevent anaphase onset and the otherpreventing cytokinesis and mitotic exit (66-68). Inresponse to unattached kinetochores, checkpoint kinasessuch as Bub1 promote the formation of Mad2-Cdc20-APCcomplexes, thereby inhibiting anaphase onset by pre-venting the degradation of Pds mediated by APC-Cdc20(66-68). In response to distinct events, possibly occurringat the spindle poles, the Bub2-dependent pathway pre-vents activation of the mitotic exit network including theTem1 GTPase and Cdc14 phosphatase (66-68). It isinteresting to explore whether Cd affects these spindlecheckpoint signals.

It should be noticed that Cd cytotoxicity is also affectedby cell culture conditions, e.g., cells that have beencultured in media containing serum (10%) before Cd-treatment (the procedure used in this study) are moresensitive to Cd than those cultured in serum-free mediaprior to Cd exposure (37). However, the differential

1200 Chem. Res. Toxicol., Vol. 14, No. 9, 2001 Chao and Yang

cytotoxicity observed in the two culture conditions iscorrelated with the levels of ROS and Cd uptake (datanot shown). Also, these different cell culture conditionsdo not affect the patterns of ERK or p38 MAPK activatedby Cd.

In summary, we have demonstrated that Cd per-sistence activates ERK and p38 MAPK particularly inthe G2/M phase and elicits mitotic arrest and subse-quently apoptosis. The activation of ERK and p38 MAPKby Cd is associated with the levels of intracellularperoxide but not Cd accumulation. The generation of ROSby Cd may subsequently lead to the activation of ERKand p38 MAPK, and the induction of micronuclei. WhileERK potentially plays a role in guarding genome integ-rity, p38 MAPK may trigger genome instability andapoptosis in Cd-treated cells. The present study providesevidence that genome integrity can be modulated byepigenetic activation of MAPK pathways in response togenotoxic agents.

Acknowledgment. The authors are grateful to DrP.-C. Yang for providing the CL3 cells and Dr S.-M.Chuang for critical discussion of the manuscript. Thiswork was supported in part by Grant NSC89-2311-B-007-027 from the National Science Council, Taiwan, and byGrant VTG89-G4-04 from the Medical Research Advance-ment Foundation in Memory of Dr. Chi-Shuen Tsou,Taiwan.

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