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Carcinogenesis, Vol. 24, No. 1, 53-61, January 2003
© 2003 Oxford University Press

CANCER BIOLOGY

Persistent activation of ERK1/2 by lead acetate increases nucleotide excision repair synthesis and confers anti-cytotoxicity and anti-mutagenicity

Yun-Wei Lin, Show-Mei Chuang and Jia-Ling Yang1

Molecular Carcinogenesis Laboratory, Department of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan, Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lead, a possible human carcinogen, affects signal transduction pathways in many aspects, yet exhibits low mutagenicity in human cells. In this study, we explore whether signaling pathways including the four MAPKs and AKT affect DNA repair and mutagenicity in the exposure of mammalian cells to lead acetate [Pb(II)]. Pb(II) increased the phosphorylated ERK1/2 and phosphorylated AKT but not the phosphorylated ERK5, phosphorylated p38 and JNK activity in human non-small cell lung adenocarcinoma CL3 cells. The duration of ERK1/2 activation was much longer than AKT activation and these two signals were independently activated by Pb(II) in CL3 cells. Intriguingly, a MKK1/2 inhibitor PD98059 (25–50 µM) markedly suppressed ERK1/2 activation and greatly promoted the hprt mutation frequency and cytotoxicity in Pb(II)-treated CL3 cells. Conversely, inhibition of the AKT signal by wortmannin did not exhibit such effects. Inhibition of the persistently activated ERK1/2 in Pb(II)-treated diploid human fibroblasts by PD98059 also markedly increased the mutagenicity and cytotoxicity. The Pb(II)-induced mutagenicity and cytotoxicity were significantly higher in nucleotide excision repair (NER)-deficient UVL-10 rodent cells than their counterpart AT3-2 cells; also, ERK1/2 activation by Pb(II) was observed in AT3-2 but not UVL-10 cells. Furthermore, cellular NER synthesis was enhanced by Pb(II) exposure, which was markedly suppressed by PD98059. Activation of ERK1/2 by expressing a constitutively active form of MKK1 in CL3 cells also elevated cellular NER synthesis. Together, these results indicate that persistent activation of ERK1/2 signaling by Pb(II) enhances cellular NER synthesis, thereby conferring anti-cytotoxicity and anti-mutagenicity.

Key Words: ERK, extracellular signal-regulated kinase • ICP-MS, inductively coupled plasma-mass spectrometer • JNK, c-JUN N-terminal kinase • MAPK, mitogen-activation protein kinase • MKK, MAPK kinase • MKKK, MKK kinase • NER, nucleotide excision repair • Pb(II), lead acetate • PBS, phosphate-buffered saline • PI3K, phosphatidylinositol 3-kinase • PKC, protein kinase C • 6-TG, 6-thioguanine • WCE, whole cell extract


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Lead is an ubiquitous toxic contaminant of our environment, which was evaluated as a possible human carcinogen (group-2B) in 1987 by the International Agency for Research on Cancer (1) (based on sufficient animal data and insufficient human data). More recently, epidemiologic studies of lead smelter or battery workers provided some evidence of an increased risk of lung, stomach and bladder cancers; however, the results may be biased by confounders such as tobacco smoking and arsenic (2–5). Lead acetate [Pb(II)] causes kidney, brain and lung cancers in experimental rodents and acts synergistically with other carcinogens (1,6,7). Pb(II) increases gene mutations in cultured rodent cells (8–12), yet, the induction levels are much weaker than typical carcinogens. Pb(II) does not enhance gene mutations in diploid human fibroblasts, although it induces anchorage-independent phenotypes in the same cells (13). Pb(II) does not cause DNA strand breaks in HeLa cells (14), however, in vitro studies have demonstrated that Pb(II) is able to interact with the phosphate backbone of nucleic acids (15) and induce DNA strand breaks and 8-hydroxydeoxyguanosine adducts (16). Due to its weak genotoxicity and co-genotoxicity with UV and alkylating carcinogens, the Pb(II) genotoxicity has been attributed to indirect mechanisms such as interference with DNA repair processes (10,14).

The four subfamilies of mammalian mitogen-activation protein kinase (MAPKs), i.e. the extracellular signal-regulated kinases (ERK1/2), the c-JUN N-terminal kinases (JNKs), the p38 kinases and ERK5 (also termed big MAP kinase 1), as well as AKT (also termed as protein kinase B) are vital signaling transducers differentially activated in response to a wide diversity of extracellular stimuli including growth factors, cytokines and environmental stresses (17–26). Activation of MAPKs is regulated through a three-kinase module composed of a MAPK, a MAPK kinase (MKK) and a MKK kinase (MKKK) (17–23). These MAPK modules are connected to the cell surface receptor and activated through interaction with a family of small GTPases and MKKK kinases. Activation of MAPKs requires a dual-phosphorylation of the Thr and Tyr residues within the motif Thr–Glu–Tyr (ERK1/2 and ERK5), Thr–Pro–Tyr (JNK) and Thr–Gly–Tyr (p38) in the subdomain VIII of the catalytic domain. In general, the activated ERKs control cell proliferation and differentiation (17–19), whereas, the stimulated JNK and p38 pathways regulate growth arrest, apoptosis, cell survival, transformation, proliferation and invasion (17,20–23). The particular function regulated by MAPKs is likely to depend on the cell type, the stimulus and the duration and strength of kinase activities. On the other hand, AKT is recruited to the plasma membrane by phosphatidylinositol 3-kinase (PI3K)-dependent phospholipid binding and full activation of AKT required phosphorylation at Thr308 and Ser473 residues (24–26). Activation of AKT results in increasing cellular proliferation and protection from apoptosis through phosphorylation and inactivation of several effectors including Bad, caspase-9, the forkhead family of transcription factors, GSK-3, p27 and p21 (24–26).

Pb(II) has been recently reported to activate ERK1/2 and JNK in a rat pheochromocytoma cell line PC-12 (27), and ERK1/2 but not AKT signaling in a human astrocytoma cell line 1232N1 (28). Conversely, Pb(II) does not activate ERK1/2 and JNK, while it stimulates p38 phosphorylation and subsequently Hsp27 phosphorylation in bovine adrenal chromaffin cells (29). Whether Pb(II) can activate these signals in cell types other than neural-derived cells and what are the physiological roles of their activation remains unknown. Here we show that Pb(II) activates ERK1/2 persistently and AKT transiently, but does not stimulate ERK5, JNK and p38 in a human non-small cell lung adenocarcinoma cell line, CL3. We further demonstrate that inhibition of the activated-ERK1/2 but not AKT signaling greatly increases Pb(II) cytotoxicity and mutagenicity. Moreover, we provide evidence to reveal that the activated-ERK1/2 signal is essential for the enhanced cellular nucleotide excision repair (NER) synthesis in Pb(II)-treated cells, which may account for the low mutagenicity of this metal in mammalian cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
The CL3 cell line established from a non-small-cell lung carcinoma tumor was provided by Dr P.-C.Yang at the Department of Internal Medicine and Clinical Pathology, National Taiwan University Hospital, Taipei. The human diploid fibroblast line HFW was given by Dr W.-N.Wen at the Institute of Biochemistry, National Taiwan University, Taipei. The AT3-2 (NER proficiency) and UVL–10 (ERCC1 deficiency) lines derived from Chinese hamster ovary (CHO) cells were provided by Dr G.M.Adair at MD Anderson Cancer Center, Smithville, Texas (30–32). CL3, HFW and CHO cells were cultured in RPMI-1640, DMEM and McCoy’s 5A media (Gibco, Life Technologies, Grand Island, NY), respectively, 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%). CL3 and CHO cells were maintained at 37°C in a humidified incubator containing 5% CO2 in air, while HFW cells were cultured in a 10% CO2 incubator.

Treatment
Cells in exponential growth were plated before serum starvation for 16–18 h. Lead acetate (Merck, Darmstadt, Germany) was dissolved in MilliQ-purified water (Millipore, Bedford, MA). Serum-starved cells were then exposed to lead acetate for 15 min–24 h in serum-free media. In experiments to determine the effects of protein kinase inhibitors, serum-deprived cells were pre-treated with PD98059 (Calbiochem, San Diego, CA), a MKK1/2 inhibitor, for 1 h or wortmannin (Sigma Chemical Co., St Louis, MO), a PI3K inhibitor, for 30 min before the addition of Pb(II). The serum-starved cells were also irradiated with UV (254 nm, 15 J/m2) or exposed to H2O2 (300 µM, 5 min) to serve as positive controls. The radiation intensity of UV was 1 J/m2/s when measured with an UVX radiometer (UVP Inc., CA).

Cytotoxicity assay
Immediately after treatment, cells were washed with phosphate-buffered saline (PBS) and trypsinized for the determination of cell numbers using a hemocytometer. The cells were plated at a density of 100–200 cells per 60 mm Petri dish in triplicate for each treatment. The cells were then cultured for 7–14 days and cell colonies were stained with 1% crystal violet solution (in 30% ethanol). Cytotoxicity was determined to be the number of colonies in the treated cells divided by the number of colonies in the untreated control (12).

Mutagenicity assay
The Pb(II)-treated or untreated cells were maintained in exponential growth for 7 days to allow for the expression of resistance to 6-thioguanine (6-TG). One million cells from each treatment were plated onto ten 100 mm Petri dishes in a selective medium containing 40 µM (CL3 and HFW) or 66 µM (AT3-2 and UVL–10) of 6-TG, followed by incubation for 7–14 days. Plating efficiency of cells at the time of selection was also assayed in a non-selective medium to correct the observed hprt mutant frequency. The hprt mutant frequency was calculated to be the total number of 6-TG resistant colonies divided by the total number of clonable cells at selection time (12).

Preparation of whole cell extract (WCE)
The Pb(II)-treated or untreated cells were rinsed twice with cold PBS and lysed in the WCE buffer containing 20 mM HEPES, pH 7.6, 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 0.1 mM Na3VO4, 50 mM NaF, 0.5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin and 100 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride. The cell lysate was rotated at 4°C for 30 min, centrifuged at 10 000 r.p.m. for 15 min and the precipitates were discarded. The BCA protein assay kit (Pierce, Rockford, IL) was adopted to determine protein concentrations using bovine serum albumin as a standard.

Western blot analysis
Equal amounts of proteins in WCE from each set of experiments were subjected to western blot analyses as described previously (33). The polyclonal antibodies specific against phospho-ERK1/2(Thr202/Tyr204) (#9101), phospho-p38(Thr180/Tyr182) (#9211) and phospho-AKT(Ser473) (#9271) were purchased from Cell Signaling (Beverly, MA). The polyclonal antibody against phospho-ERK5(Thr218/Tyr220) (#44–612) was purchased from BIOSOURCE International (Camarillo, CA). The polyclonal antibody against ERK2 (#sc-154), ERK5 (#sc-5626), p38 MAP kinase (#sc-535), JNK1 (#sc-571), AKT (#sc-8312), c-FOS (#sc-52) and {alpha}-tubulin (#sc-8035) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody reaction was detected using the enhanced chemiluminescence detection procedure according to the manufacturer’s recommendations (NEN, Boston, MA). To re-probe the membrane with another primary antibody, antibodies in the blot were stripped from membranes by a solution containing 2% SDS, 62.5 mM Tris–HCl, pH 6.8, and 0.7% (w/w) ß-mercaptoethanol at 50°C for 15 min. The relative protein intensities on blots were quantitated using a computing densitometer equipped with the ImagQuant analysis program (Molecular Dynamics, Sunnyvale, CA).

JNK kinase assay
JNK in WCE (50 µg proteins) was reacted with GST-cJUN(1–79) (5 µg) and glutathione–Sepharose 4B beads (Amersham Pharmacia Biotech, Arlington Heights, IL) and the kinase activity was performed by transferring [{gamma}-32P]ATP (6 000 Ci/mmol) to the substrate GST-cJUN(1–79) as described previously (33).

Transfection
Cells (4x105) were plated in a p60 dish one-day before transfection. Plasmid (5–15 µg) containing a constitutively active form of MKK1 ({Delta}N3/S218E/S222D; MKK1-CA) (34), kindly provided by Dr N.G.Ahn at the Department of Chemistry and Biochemistry, University of Colorado, Boulder, was transfected into CL3 cells by calcium phosphate co-precipitation. After incubation for 6 h, the cells were washed with PBS, kept cultured in complete media for 2 days and then subjected to the preparation of WCE and repair synthesis assay.

NER synthesis
The efficiency of cellular NER synthesis was measured according to the procedure of Dr R.D.Wood (35,36) with modification. Briefly, the pUC19 plasmid substrates were prepared by alkaline lysis method and stored at -20°C. The purified plasmid substrates (250 ng/µl in ddH2O) were irradiated with UV (254 nm, 400 J/m2) at a radiation intensity of 1–1.5 J/m2/s. NER synthesis reaction mixtures (50 µl) contained 60 µg of proteins derived from human WCE, 250 ng of UV-irradiated or un-irradiated plasmid substrates, 20 µM each of dGTP, dCTP and dTTP, 8 µM dATP, 2 µCi [{alpha}-32P]dCTP (3000 Ci/mmol), 2 mM ATP, 45 mM HEPES-KOH, pH 7.5, 60 mM KCl, 7.5 mM MgCl2, 0.9 mM dithiothreitol, 0.4 mM EDTA, 3.4% glycerol and 18 µg bovine serum albumin. Reactions were performed at 30°C for 1 h and terminated by adding EDTA to a final concentration of 20 mM. The samples were then treated with 80 µg/ml RNaseA for 10 min and 190 µg/ml proteinase K and 0.5% SDS for 30 min at 37°C. The plasmid DNA in the reaction mixtures was purified by phenol/chloroform extraction and ethanol precipitation, linearized with BamHI and subjected to agarose gel (0.8%) electrophoresis. The plasmid DNA in gel was stained with ethidium bromide (0.5%) and visualized under near-UV transillumination. The gel was then dried and subjected to autoradiography. The band intensities were measured with a computing densitometer equipped with the ImageQuant analysis program.

Determination of intracellular lead level
Cells were exposed to various Pb(II) concentrations in serum-free medium for 24 h. Following treatment, the cells were washed three times with PBS and the numbers of cells were determined. One million cells were centrifuged and the cell pellet was sonicated in MilliQ-purified water. Total cellular Pb level was analyzed by an inductively coupled plasma-mass spectrometer (ICP-MS; SCIEX ELAN 5000, Perkin Elmer, Norwalk, CT). The ICP-MS conditions were as follows: power of 1000 W, plasma flow rate of 15 l/min, auxiliary flow rate of 0.8 l/min, carrier gas flow rate of 0.8 l/min and sample flow rate of 1 ml/min.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Pb(II) elicits ERK1/2 and AKT phosphorylation in CL3 cells
The ability of Pb(II) to activate the four MAPKs was investigated by exposing CL3 cells to various concentrations of lead acetate in serum free medium for 15 min–24 h. The activation of ERK1/2, ERK5 and p38 kinase was determined by western blots using antibodies specific to recognize phospho-ERK1/2, phospho-ERK5 and phospho-p38, respectively. The JNK kinase activity was performed by in vitro kinase assay using GST-cJUN(1–79) as a substrate. As shown in Figure 1Go, exposure of cells to Pb(II) for 24 h increased ERK1/2 phosphorylation in a dose-dependent manner, e.g. 10–100 µM Pb(II) enhanced the phosphorylated ERK1/2 levels 3.1–4.5 fold of the untreated control. Pb(II) also induced the phospho-ERK1/2 levels in a time-dependent manner (15 min–24 h; data not shown). In contrast, exposure of CL3 cells to Pb(II) (10–500 µM) for 15 min, 1 or 24 h did not activate ERK5, p38 and JNK, although these MAPKs could be activated in the same cell line by positive control stimuli (Figure 1Go and data not shown). Additionally, the endogenous protein levels of the four MAPKs were unaltered by Pb(II) (Figure 1Go).



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  		Fig. 1. Pb(II) elicits ERK1/2 but not ERK5, p38 and JNK signals in CL3 cells. Cells were left untreated or treated with 10–100 µM Pb(II) in serum-free RPMI1640 medium for 24 h. To serve as positive control, the serum-starved cells were irradiated with UV (254 nm, 15 J/m2) for ERK1/2, p38 and JNK activation or exposed to H2O2 (300 µM, 5 min) for the induction of ERK5 (PS; positive stimuli). The WCE were prepared immediately after Pb(II) and H2O2 treatments, and 30 min after UV irradiation. The activation of ERK1/2, ERK5 and p38 in WCE were examined using phospho-specific antibodies. JNK activity in equal amounts of proteins was examined using GST-cJun(1–79) as a substrate. The relative activities shown under each blot were determined by densitometric analysis, which were calculated from the average of seven (phospho-ERK1/2) or three [phospho-ERK5, phospho-p38 and GST-cJun(1–79)] independent experiments and were normalized by arbitrarily setting the densitometry of control cells to 1. Western blot analyses of ERK2, ERK5, p38 and JNK protein levels in the same WCE are shown in the lower panels.

 
We also investigated the ability of Pb(II) to activate AKT, a potent survival signal, by western blots using antibody specific to recognize phospho-AKT(Ser473). Figure 2AGo shows that Pb(II) could activate the phospho-AKT in CL3 cells, however, the activated levels were variable as the exposure time increased. To determine the duration of ERK and AKT signals activated by Pb(II), after a 24-h exposure to 30 µM Pb(II), CL3 cells were washed with PBS and allowed recovery in serum-free media for 0–8 h before WCE isolation. Figure 2BGo shows that the phospho-ERK1/2 activated by Pb(II) remained at high levels during the recovery times. Conversely, the Pb(II)-activated AKT diminished rapidly after removing the metal from media (Figure 2BGo). Pb(II) did not significantly influence the endogenous ERK2 and AKT protein levels during the time-course and recovery experiments (Figure 2Go). The above results imply that Pb(II) activates ERK1/2 persistently and AKT transiently in CL3 cells.



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  		Fig. 2. Duration of the activation of ERK1/2 and AKT by Pb(II) in CL3 cells. In (A), cells were left untreated or treated with 30 µM Pb(II) in serum-free media for the indicated time period before preparation of WCE. In (B), after exposure of cells to 30 µM Pb(II) for 24 h, the cells were washed twice with PBS, kept cultured in serum-free medium for 0–8 h and subjected to WCE preparation. The activation of ERK1/2 and AKT were determined using phospho-specific antibodies. The relative activities shown under each blot were determined from the average of four independent experiments as described in Figure 1Go. Western blot analyses of ERK2 and AKT protein levels in the same WCE are shown in the lower panels.

 
Pb(II) independently elicited ERK1/2 and AKT phosphorylation
It has been reported that AKT can phosphorylate and inactivate RAF, which is an upstream ERK1/2 activator (37,38). To inspect the relationship between the ERK1/2 and AKT signaling pathways elicited by Pb(II), CL3 cells were exposed to Pb(II) in the presence of PD98059, an inhibitor of the ERK1/2 upstream kinases MKK1/2, or wortmannin, an inhibitor of the AKT upstream activator PI3K, and then subjected to examination of the phospho-ERK1/2 and phospho-AKT levels. PD98059 (50 µM) and wortmannin (100 nM) completely blocked phospho-ERK1/2 and phospho-AKT in Pb(II)-treated cells, respectively (Figure 3Go). Wortmannin did not influence the ERK1/2 activation and PD98059 did not affect the AKT activation elicited by Pb(II) in CL3 cells (Figure 3Go). The results suggest that Pb(II)-elicited ERK1/2 and AKT signals do not communicate with each other.



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  		Fig. 3. Independent activation of ERK1/2 and AKT by Pb(II). Cells were pretreated with PD98059 (50 µM) for 1 h or wortmannin (100 nM) for 30 min before the addition of various concentrations of Pb(II) for 24 h. The levels of ERK1/2 and AKT activation were determined from three independent experiments as described in Figure 2Go.

 
Inhibition of ERK suppresses the c-fos levels induced by Pb(II)
In response to growth factors and mitogens, the ERK1/2 activity is required for the expression of the immediate early gene c-fos through phosphorylation and activation of transcriptional activators TCF and SRF (39,40). Also, sustained ERK activity can phosphorylate and stabilize c-FOS protein (41). We therefore examined the ability of Pb(II) to induce c-FOS and the involvement of ERK1/2 signal. CL3 cells were exposed to 30 µM Pb(II) for various times and the protein levels of c-FOS were determined by western blot analysis. As shown in Figure 4AGo, Pb(II) increased c-FOS protein levels 2- to 3-fold of the untreated control. In the presence of PD98059 (25 µM), Pb(II) could not elevate the c-FOS protein levels in CL3 cells (Figure 4BGo). The results indicate that Pb(II)-elicited ERK1/2 participates in the induction of c-FOS expression.



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  		Fig. 4. ERK1/2-dependent induction of c-FOS expression by Pb(II) in CL3 cells. In (A), cells were treated with 30 µM Pb(II) in serum-free media for the indicated time period. In (B), cells were left untreated or pretreated with PD98059 (25 µM) for 1 h before exposure to 30 µM Pb(II) for 24 h. The WCE was subjected to western analysis using antibody against c-fos. The relative intensities shown are calculated from the average of four experiments as described in Figure 1Go. Protein levels of {alpha}-tubulin in the same WCE are shown in the lower panels as an internal control.

 
ERK1/2 activation by Pb(II) is positively correlated with anti-cytotoxicity and anti-mutagenicity
To investigate the roles of ERK1/2 or AKT activation in cytotoxicity and mutagenicity, CL3 cells were left untreated or pre-treated with 50 µM PD98059 for 1 h or 100 nM wortmannin for 30 min before co-exposure to Pb(II) for 24 h in serum-free medium and then subjected to the colony-forming ability and 6-TG assays. Approximately 40% of the cells survived when they were exposed to 500 µM Pb(II), which was reduced to 20% by co-exposure to PD98059 (Figure 5A Go, P< 0.01). In contrast, co-treatment with wortmannin did not affect the Pb(II)-induced cytotoxicity (Figure 5AGo). Neither PD98059 nor wortmannin alone exhibited a cytotoxic effect in these experiments (Figure 5AGo). After treatment with Pb(II) and/or inhibitors, CL3 cells were cultured for 7 days at exponential growth before selection for the hprt mutations. As shown in Figure 5BGo, 300–500 µM Pb(II) did not induce the hprt mutation frequency in CL3 cells. Intriguingly, 50 µM PD98059 co-treatment dramatically increased the hprt mutation frequency in CL3 cells exposed to Pb(II) (Figure 5BGo, P< 0.01). Conversely, wortmannin did not alter the low mutagenicity of Pb(II) in CL3 cells (Figure 5BGo). To further illustrate the correlation between ERK1/2 inactivation and Pb(II) mutagenesis, CL3 cells were exposed to various concentrations of PD98059 for 1 h before treatment of 300 µM Pb(II) for 24 h. As shown in Figure 5C and 5DGo, PD98059 at 25–50 µM markedly enhanced the hprt mutation frequency in Pb(II)-treated cells, in which the ERK1/2 activity was completely suppressed. Conversely, 10 µM PD98059 that did not completely suppress the ERK1/2 activity showed no effect on the low mutagenicity in Pb(II)-treated cells.



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  		Fig. 5. PD98059 enhances Pb(II)-induced cytotoxicity and mutagenicity in CL3 cells, while wortmannin does not. Cells were pretreated with PD98059 (50 µM) for 1 h or wortmannin (100 nM) for 30 min before exposure to various concentrations of Pb(II) for 24 h. Alternatively, cells were pretreated with various concentrations of PD98059 for 1 h before exposure to 300 µM Pb(II) for 24 h. The cytotoxicity and mutagenicity were performed by colony-forming ability (A) and 6-TG resistant (B and C) assays, respectively. Results were obtained from four to fourteen experiments and bars represent SEM. Total numbers of viable cells examined were 1.01–4.95x106 in each treatment for 6-TG assay. **P < 0.01 using Student’s t-test for the comparison between cells exposed to Pb(II) and Pb(II) plus PD98059. The ERK1/2 activation (D) was examined as described in Figure 1Go, and the western blot shown is one representative of four independent experiments.

 
The phenomenon that persistent activation of ERK1/2 participates in anti-cytotoxicity and anti-mutagenicity in Pb(II)-treated cells was further studied in a diploid human fibroblast line HFW. Western blot analysis showed that Pb(II) (10–500 µM) could dose-dependently activate ERK1/2 in HFW cells (Figure 6AGo). Again, PD98059 (50 µM) significantly elevated the cytotoxicity and mutagenicity in Pb(II)-treated HFW cells (Figure 6B and 6CGo). Recently, PD98059 has been demonstrated to inhibit the activation of MKK5->ERK5 as well as MKK1/2->ERK1/2 pathways (42,43). However, Pb(II) (10–500 µM) did not activate ERK5 in HFW cells (data not shown). The above results indicate that activation of the ERK1/2 signaling pathway protects human cells from cytotoxicity and mutagenicity upon Pb(II) exposure.



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  		Fig. 6. Sustained ERK1/2 activation by Pb(II) confers anti-cytotoxicity and anti-mutagenesis in HFW diploid human fibroblasts. In (A), cells were treated with Pb(II) for 24 h in low-serum medium (0.1%), washed twice with PBS and kept cultured in low-serum medium for 0, 1 or 8 h. The relative activities of ERK1/2 shown are determined from the average of three experiments as described in Figure 1Go. In (B) and (C), cells were pretreated with PD98059 (50 µM) for 1 h before exposure to Pb(II) for 24 h. The Pb(II)-induced cytotoxicity and mutagenicity were determined by colony-forming ability (B) and 6-TG resistant assays (C), respectively. Results were obtained from four to nine experiments and bars represent SEM. **P < 0.01 using Student’s t-test for the comparison between cells exposed to Pb(II) and Pb(II) plus PD98059. Total numbers of viable cells examined were 1.18–3.05x106 in each treatment for 6-TG assay.

 
NER participates in preventing Pb(II)-induced cytotoxicity and mutagenicity
NER is a major error-free DNA repair process to remove a broad variety of DNA lesions caused by environmental carcinogens (44–46). The genotoxicity of Pb(II) has been attributed to indirect mechanisms such as interference with NER because it enhances the genotoxicity of strong mutagens such as UV (10,14). However, evidence that Pb(II) alone affects the NER pathway has never been demonstrated. We therefore examined the cytotoxicity and the hprt mutation frequency in Pb(II)-exposed cells using a rodent UVL-10 cell line that is defective in NER and its counterpart AT3-2 cells (30,31) to reveal the involvement of NER in preventing Pb(II) genotoxicity. UVL-10 cells are unable to produce ERCC1 protein due to a point mutation located at exon 5 of its encoded gene (32), thereby failing in incision 5' to the site of base damage (44). As shown in Figure 7Go, UVL-10 cells were significantly more sensitive than AT3-2 cells to Pb(II)-induced cytotoxicity and mutagenicity. The results suggest that Pb(II) may induce DNA lesions that can be repaired through the NER pathway. Intriguingly, ERK1/2 activation by Pb(II) was observed in AT3-2 but not UVL-10 cells (Figure 7CGo), despite the endogenous ERK1/2 activity being 2.4-fold higher in the latter. The results suggest that ERK1/2 activation may be correlated to NER capability in cells exposed to Pb(II).



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  		Fig. 7. Comparison of cytotoxicity, mutagenicity and ERK1/2 activation induced by Pb(II) in NER-proficient and NER-deficient rodent cells. AT3-2 and UVL-10 cells were treated with Pb(II) for 24 h and the cytotoxicity and mutagenicity were determined by colony-forming ability (A) and 6-TG resistant assays (B), respectively. Results were obtained from four experiments and bars represent SEM. **P < 0.01 using Student’s t-test for the comparison between AT3-2 cells and UVL-10 exposed to Pb(II). Total numbers of viable cells examined were 1.69–2.43x106 in each treatment for 6-TG assay. The high background hprt mutation frequencies of these cells have been reported previously (31). The relative activities of ERK1/2 in AT3-2 cells and UVL-10 exposed to Pb(II) are determined from the average of six experiments as described in Figure 1Go.

 
Persistent activation of ERK1/2 enhances NER synthesis
We next adopted a NER synthesis assay (35,36) to explore whether Pb(II)-activated ERK1/2 signal is involved in regulating DNA repair processes. CL3 cells were left untreated or treated with 10–100 µM Pb(II) for 24 h in the presence or absence of PD98059 and then allowed recovery in serum-free media for 8 h before extraction of the proteins. The proteins in the WCE were incubated with UV-irradiated plasmid DNA, 4 dNTP, and {alpha}-32P[dCTP] to examine the efficiency of NER synthesis. As shown in Figure 8Go, the capability of WCE derived from the Pb(II)-treated cells to incorporate nucleotides into UV-damaged DNA was higher than that derived from the untreated control cells. Furthermore, PD98059 markedly decreased the Pb(II)-stimulated NER synthesis (Figure 8Go). Quantitative analysis showed that cells treated with 30 µM Pb(II) increased the NER synthesis by 166% of the untreated cells; in the presence of PD98059 this level was 22% lower than the untreated cells (Figure 8BGo). The results indicate that Pb(II) exposure elevates cellular NER synthesis through the ERK1/2 pathway.



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  		Fig. 8. Pb(II) increases the efficiency of NER synthesis in CL3 cells in an ERK1/2-dependent manner. Cells were left untreated or pretreated with PD98059 (50 µM) for 1 h before exposure to 10–100 µM of Pb(II) for 24 h in serum-free medium. The cells were then washed twice with PBS and kept cultured in serum-free medium for 8 h before WCE preparation. The NER synthesis efficiency of equal amounts of proteins (60 µg) in WCE was determined by reaction with pUC19 plasmid (250 ng) that had been irradiated with UV (400 J/m2) as described in Materials and methods. Untreated pUC19 plasmid (-UV) was incubated with WCE derived from untreated cells to serve as a negative control. In (A), upper panel: autoradiograph of gels, showing the incorporation of {alpha}-32P[dCTP]. Lower panel: photograph of the same gels stained with ethidium bromide showing equal amounts of DNA used in each reaction. In (B), the relative activities were determined by densitometric analysis, which were calculated from the average of four to eight independent experiments and were normalized by arbitrarily setting the densitometry of control cells to 100%.

 
To further examine the role of the ERK1/2 signal in NER synthesis, we manipulated the ERK1/2 activity by transfection of MKK1-CA, a constitutive active mutant of MKK1 vector, into CL3 cells and allowed expression for 2 days before preparation of WCE for the NER synthesis assay. As shown in Figure 9Go, the WCE derived from cells transfected with 5–15 µg of MKK1-CA had elevated phospho-ERK1/2 and exhibited markedly higher NER synthesis than that derived from cells expressing a control vector. Taken together, the above results indicate that activation of ERK1/2 enhances cellular NER synthesis and that the low mutagenicity of Pb(II) in NER-proficient mammalian cells may be due mainly to the sustained-ERK1/2 signal to enhance the error-free NER synthesis.



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  		Fig. 9. Expression of MKK1-CA, a constitutively active form of MKK1, enhances the efficiency of NER synthesis in CL3 cells. Cells (4x105) were plated in a 60 mm dish 1 day before transfection. Vectors pcDNA3 or MKK1-CA (5–15 µg) were transfected into CL3 cells and allowed expression for 2 days. The WCE was harvested to determine the efficiency of NER synthesis as described in Figure 8Go. An example of gel pattern is shown in (A) and the quantitative results averaged from four independent experiments are shown below the autoradiograph. The levels of phospho-ERK1/2 and ERK2 in equal amounts of proteins derived from each WCE are also shown in (B).

 
PD98059 does not affect the intracellular Pb levels
The levels of Pb accumulated in cells may influence toxicity. We therefore measured the amounts of Pb accumulation in the presence or absence of PD98059 using ICP-MS. CL3 cells were exposed to 300–500 µM of Pb(II) for 24 h, washed three times with PBS and subjected to the analysis of the intracellular Pb amounts. As shown in Figure 10Go, similar levels of Pb were accumulated in the presence or absence of PD98059. This result indicates that the ability of PD98059 to enhance the cytotoxicity and mutagenicity and decrease NER synthesis in Pb(II)-treated cells is not due to differential intercellular Pb levels.



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  		Fig. 10. ERK1/2 signaling does not affect Pb accumulation. CL3 cells were left untreated or pretreated with PD98059 (50 µM) for 1 h before exposure to 300–500 µM of Pb(II) for 24 h in serum-free medium. The cells were then washed three times with PBS, centrifuged and the cell pellet was sonicated. Total intracellular Pb amounts were analyzed by an ICP-MS. Results were obtained from three experiments and bars represent SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Previous reports have indicated that Pb(II) exhibits weak genotoxicity in cultured rodent cells and does not cause mutations in human cells (8–14,47,48). However, in vitro evidence has demonstrated that this non-essential toxic metal can destabilize DNA helical structure and induce DNA strand breaks and oxidative DNA adducts (15,16). These contradictory results suggest that the etiology of Pb(II) genotoxicity is rather complex. Because Pb(II) enhances the genotoxicity of strong mutagens such as UV and alkylating carcinogens, it has been proposed that Pb(II) impedes NER machinery (10,14). Nonetheless, evidence of cellular NER affected by Pb(II) alone has never been demonstrated. Here we show for the first time that NER synthesis is indeed elevated in Pb(II)-exposed human cells and that Pb(II) induces significantly higher cytotoxicity and mutagenicity in the NER-deficient than in the NER-proficient rodent cells. These results clearly suggest that exposure of mammalian cells to Pb(II) stimulates NER and confers anti-mutagenicity. In the presence of H2O2, Pb(II) dose-dependently induces 8-hydroxydeoxyguanosine adducts in calf thymus DNA, which can be prevented by singlet oxygen scavengers (16). Cellular NER machinery removes many kinds of base damage including oxidative DNA adducts (44–46). Whether Pb(II) can induce oxidative DNA adducts in NER-deficient cells deserved further investigation.

Recent studies on neural-derived cells have shown that Pb(II) affects signal transduction pathways including ERK1/2 (27,28), however, the physiological role remains largely unknown. In this study, we found that Pb(II) could activate ERK1/2 among the four MAPKs and AKT survival signals in human CL3 cells. We further demonstrated that inhibition of the persistent activated-ERK1/2 by PD98059 markedly potentiated the cytotoxicity and mutagenicity of Pb(II) in human CL3 and HFW cells. Conversely, blockage of the transient activated-AKT in CL3 cells did not affect the low mutagenicity of Pb(II). Intriguingly, Pb(II) could stimulate ERK1/2 in the NER-proficient but not the NER-deficient rodent cells. Moreover, the Pb(II)-induced NER synthesis was diminished by inhibiting the persistent activated-ERK1/2 in CL3 cells; also, constitutive expression of MKK1 -> ERK1/2 signal could significantly increase NER synthesis. Taken together, these observations indicate that Pb(II) persistently activates ERK1/2 which triggers NER, thereby conferring anti-cytotoxicity and anti-mutagenicity in mammalian cells.

ERK1/2 exemplify one class of MAPKs that undergoes activation by a range of stimuli including growth factors, cytokines, cell adhesion, tumor-promoting phorbol esters and oncogenes (19). It is well known that ERK1/2 activation is necessary for cell growth because it phosphorylates and activates numerous substrates involved in nucleotide synthesis, gene transcription, protein synthesis and cell cycle progression (19). Recently, three global NER enzymes involved in the recognition of and binding to damaged DNA, i.e. hHR23A, hHR23B and replication protein A2, have been identified as novel targets which are posttranslationally modified by the ERK1/2 pathway using functional proteomics and mass spectrometry techniques (49). The hHR23B that tightly complexes with XPC to stimulate the initiation step of NER (44–46) is rapidly proteolytically processed and phosphorylated in response to ERK1/2 signaling induced by UV (49). Accordingly, ERK1/2 may regulate the repair efficiency by posttranslational modification of enzymes involved in NER. Alternatively, ERK1/2 may phosphorylate transcription factors to upregulate the expression of genes involved in NER and thereby enhance the repair efficiency. In human cells, NER is composed of at least 30 proteins involved in DNA damage recognition, dual incision of the DNA strand containing a lesion, DNA synthesis and ligation to replace error-free an excised 25–30 oligonucleotide (44–46). The role of ERK1/2 signaling in regulating the expression of NER genes and posttranslational control of NER proteins warrants further investigation.

Both the ERK1/2 and p38 pathways contribute to TCF activation and c-fos transcription in response to UV (50). Here, we also observed that c-FOS induced by Pb(II) required ERK1/2 and was possibly independent of p38. This is consistent with the finding that sustained phospho-ERK docking to the DEF domain of c-FOS protein resulted in phosphorylation and prolongation of its biological effect (41). Our present finding that ERK1/2 could be involved in preventing genotoxicity is also consistent with previous reports showing that c-FOS play a role in cellular defense systems, in which mouse fibroblasts lacking c-fos are hypersensitive to a wide variety of genotoxic agents in the induction of cytotoxicity, apoptosis and chromosomal breakages (51,52). Recently, ERK1/2 signaling has been implicated in protecting apoptosis and micronucleus formation induced by Cd (33,53), enhancing cellular viability and recovery from the G2/M cell cycle checkpoint arrest upon ionizing radiation (54) and reducing DNA strand breakage and apoptosis induced by hyperoxia (55). Moreover, a recent report has shown that ERK1/2 activated upon ionizing radiation is associated with increased expression of ERCC1 and XRCC1, repairing of apurinic sites and decreased micronucleus formation (56). All the above data suggest that ERK1/2 signal activated by certain DNA damage agents can function in providing protection from genomic instability. On the other hand, activation of the ERK1/2 through constitutive expression of oncoproteins such as Mos and Ras greatly enhances chromosome instability (57–59). The finding that ERK1/2 participated in chromosome instability caused by Mos has been associated with the loss of p53 function to induce cell cycle arrest and apoptosis in mouse embryo fibroblasts (57). However, ERK1/2-mediated chromosome instability induced by Ras is also observed in thyroid PCCL3 cells containing wild-type p53 (59), yet these cells are resistant to apoptosis upon ionizing radiation (60). The contradictory role of ERK1/2 signaling induced by DNA damage agents and oncoproteins in maintaining genome integrity is obviously an interesting issue for further exploration.

Previous reports have indicated that Pb(II) can substitute for Ca(II) and bind with Ca(II) binding proteins (61,62). In CL3 cells, Pb(II) could increase intracellular Ca(II) levels, however, PD98059 did not alter the Ca(II) levels induced by Pb(II) (data not shown). Moreover, pretreatment CL3 cells with a membrane-permeable form of Ca(II) chelator, (acetoxymethyl)-1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (20 µM) did not affect Pb(II)-induced ERK1/2 activation (data not shown). ERK1/2 activation by Pb(II) in CL3 cells may thus be independent of intracellular Ca(II) levels. Pb(II) can activate protein kinase C (PKC) in many neural-derived cells (63,64). The PKC activity is implicated in the induction of AP-1 DNA binding activity and c-fos expression by Pb(II) in PC-12 cells (65,66). More recently, PKC{alpha} is identified as an upstream signal for the activation of ERK1/2 in human astrocytoma cells exposed to Pb(II) for 15–30 min; however, longer exposure (24 h) results in down-regulation of both PKC{alpha} and ERK1/2 (28). In contrast to the finding in human astrocytoma cells (28), we observed that longterm Pb(II) exposure persistently activated ERK1/2 in human CL3 cells. Whether PKC plays a role in ERK1/2 activation in our system is currently under investigation.

Noticeably, contradictory to the finding of WCE derived from Pb(II)-exposed cells, our unpublished data showed that the efficiency of NER synthesis was inhibited by adding Pb(II) in vitro, which is consistent with a previous observation (67). This phenomenon together with the finding that inhibition of ERK1/2 could not affect Pb levels in CL3 cells (Figure 10Go) suggest that once entering the cells the metal itself may not directly interfere with NER machinery, while in a cell-free system Pb(II) may interact with NER proteins and decrease their enzymatic activities. However, an in vitro study has shown that Pb(II) does not affect the DNA binding activity of mouse XPA protein (68), a NER metalloprotein that preferentially binds to damaged DNA (44–46). Pb(II) may block other NER proteins in a cell-free system, nevertheless, this does not reflect the physiological role of Pb(II).

Exposure to Pb(II) could induce cellular NER synthesis, however, our unpublished data showed that co-exposure of CL3 cells to benzo[a]pyrene diol epoxide and Pb(II) synergistically enhanced mutagenicity and cytotoxicity in CL3 cells. Intriguingly, no activation of the ERK1/2 signal was observed in this co-treatment (Lin et al., manuscript in preparation). These results suggest that the co-genotoxicity of Pb(II) is due partly to down-regulation of the ERK1/2 activity. It is therefore important to notice that although Pb(II) itself can activate ERK1/2 to prevent genotoxicity, complex signaling transduction pathways would be generated when Pb(II) was combined with other environmental carcinogens that may result in a different cell fate.

In conclusion, we have demonstrated for the first time that persistent activation of ERK1/2 by Pb(II) is essential for the stimulation of NER synthesis conferring anti-cytotoxicity and anti-mutagenicity in mammalian cells. Conversely, the AKT signal transiently induced by Pb(II) does not influence the genotoxicity of Pb(II). Although Pb(II) itself is capable of inducing an error-free repair mechanism, multifaceted DNA damage checkpoint signals can be stimulated when there is a combination of exposures to other environmental mutagens, which may decrease the protecting effect generated by Pb(II). Moreover, we identify a novel role of ERK1/2 in regulating DNA repair machinery and guarding genome integrity.


   Notes
 
1 To whom correspondence should be addressed Email: jlyang{at}life.nthu.edu.tw Back


   Acknowledgments
 
This work was supported by Grants from the National Science Council (NSC90-2311-B-007-007), and Program for Promoting Academic Excellence of Universities (89-B-FA04-1–4), the Ministry of Education, Republic of China.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. IARC (1987) Overall evaluation of carcinogenicity: an updating of IARC monographs. In IARC Monographs on the Evaluation of Carcinogenic Risks to Human, Volumes 1–42, Supplement 7. International Agency for Research on Cancer, Lyon, pp. 230–232.
  2. Fu,H. and Boffetta,P. (1995) Cancer and occupational exposure to inorganic lead compounds: a meta-analysis of published data. Occup. Environ. Med., 52, 73–81.[Abstract]
  3. Anttila,A., Heikkila,P., Pukkala,E., Nykyri,E., Kauppinen,T., Hernberg,S. and Hemminki,K. (1995) Excess lung cancer among workers exposed to lead. Scand. J. Work. Environ. Health, 21, 460–469.[ISI][Medline]
  4. Steenland,K. and Boffetta,P. (2000) Lead and cancer in humans: where are we now? Am. J. Ind. Med., 38, 295–299.[CrossRef][ISI][Medline]
  5. Englyst,V., Lundstrom,N.G., Gerhardsson,L., Rylander,L. and Nordberg,G. (2001) Lung cancer risks among lead smelter workers also exposed to arsenic. Sci. Total Environ., 273, 77–82.[CrossRef][Medline]
  6. Gerber,G.B., Leonard,A. and Jacquet,P. (1980) Toxicity, mutagenicity and teratogenicity of lead. Mutat. Res., 76, 115–141.[CrossRef][ISI][Medline]
  7. Goyer,R.A. (1993) Lead toxicity: current concerns. Environ. Health Perspect., 100, 177–187.[ISI][Medline]
  8. Oberly,T.J., Piper,C.E. and McDonald,D.S. (1982) Mutagenicity of metal salts in the L5178Y mouse lymphoma assay. J. Toxicol. Environ. Health, 9, 367–376.[ISI][Medline]
  9. Zelikoff,J.T., Li,J.H., Hartwig,A., Wang,X.W., Costa,M. and Rossman,T.G. (1988) Genetic toxicology of lead compounds. Carcinogenesis, 9, 1727–1732.[Abstract/Free Full Text]
  10. Roy,N.K. and Rossman,T.G. (1992) Mutagenesis and comutagenesis by lead compounds. Mutat. Res., 298, 97–103.[CrossRef][ISI][Medline]
  11. Ariza,M.E. and Williams,M.V. (1996) Mutagenesis of AS52 cells by low concentrations of lead(II) and mercury(II). Environ. Mol. Mutagen., 27, 30–33.[CrossRef][ISI][Medline]
  12. Yang,J.-L., Yeh,S.-C. and Chang,C.-Y. (1996) Lead acetate mutagenicity and mutational spectrum in the hypoxanthine guanine phosphoribosyltransferase gene of Chinese hamster ovary K1 cells. Mol. Carcinogen., 17, 181–191.[ISI][Medline]
  13. Hwua,Y.-S. and Yang,J.-L. (1998) Effect of 3-aminotriazole on anchorage independence and mutagenicity in cadmium- and lead-treated diploid human fibroblasts. Carcinogenesis, 19, 881–888.[Abstract/Free Full Text]
  14. Hartwig,A., Schlepegrell,R. and Beyersmann,D. (1990) Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells. Mutat. Res., 241, 75–82.[CrossRef][ISI][Medline]
  15. Tajmir-Riahi,H.A., Naoui,M. and Ahmad,R. (1993) The effects of Cu2+ and Pb2+ on the solution structure of calf thymus DNA: DNA condensation and denaturation studied by Fourier transform IR difference spectroscopy. Biopolymers, 33, 1819–1827.[CrossRef][ISI][Medline]
  16. Yang,J.-L., Wang,L.-C., Chang,C.-Y. and Liu,T.-Y. (1999) Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8-hydroxydeoxyguanosine adduct by lead acetate. Environ. Mol. Mutagen., 33, 194–201.[CrossRef][ISI][Medline]
  17. 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.[CrossRef][ISI][Medline]
  18. Kolch,W. (2000) Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem. J., 351, 289–305.[CrossRef][ISI][Medline]
  19. Whitmarsh,A.J. and Davis,R.J. (2000) A central control for cell growth. Nature, 403, 255–256.[CrossRef][Medline]
  20. Davis,R.J. (2000) Signal transduction by the JNK group of MAP kinases.Cell, 103, 239–252.[CrossRef][ISI][Medline]
  21. Chang,L. and Karin,M. (2001) Mammalian MAP kinase signaling cascades.Nature, 410, 37–40.[CrossRef][Medline]
  22. Nebreda,A.R. and Porras,A. (2000) p38 MAP kinases: beyond the stress response. Trends Biochem. Sci., 25, 257–260.[CrossRef][ISI][Medline]
  23. Kyriakis,J.M. and Avruch,J. (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev., 81, 807–869.[Abstract/Free Full Text]
  24. Scheid,M.P. and Woodgett,J.R. (2001) PKB/AKT: functional insights from genetic models. Nature Rev. Mol. Cell. Biol., 2, 760–768.[CrossRef][ISI][Medline]
  25. Brazil,D.P. and Hemmings,B.A. (2001) Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem. Sci., 26, 657–664.[CrossRef][ISI][Medline]
  26. Blume-Jensen,P. and Hunter,T. (2001) Oncogenic kinase signalling.Nature, 411, 355–365.[CrossRef][Medline]
  27. Ramesh,G.T., Manna,S.K., Aggarwal,B.B. and Jadhav,A.L. (1999) Lead activates nuclear transcription factor-{kappa}B, activator protein-1, and amino-terminal c-Jun kinase in pheochromocytoma cells. Toxicol. Appl. Pharmacol., 155, 280–286.[CrossRef][ISI][Medline]
  28. Lu,H., Guizzetti,M. and Costa,L.G. (2002) Inorganic lead activates the mitogen-activated protein kinase kinase-mitogen-activated protein kinase-p90RSK signaling pathway in human astrocytoma cells via a protein kinase C-dependent mechanism. J. Pharmacol. Exp. Ther., 300, 818–823.[Abstract/Free Full Text]
  29. Leal,R.B., Cordova,F.M., Herd,L., Bobrovskaya,L. and Dunkley,P.R. (2002) Lead-stimulated p38MAPK-dependent Hsp27 phosphorylation. Toxicol. Appl. Pharmacol., 178, 44–51.[CrossRef][ISI][Medline]
  30. Clarkson,J.M., Mitchell,D.L. and Adair,G.M. (1983) The use of an immunological probe to measure the kinetics of DNA repair in normal and UV-sensitive mammalian cell lines. Mutat. Res., 112, 287–299.[CrossRef][ISI][Medline]
  31. MacLeod,M.C., Adair,G. and Humphrey,R.M. (1988) Differential efficiency of mutagenesis at three genetic loci in CHO cells by a benzo[a]pyrene diol epoxide. Mutat. Res., 199, 243–254.[ISI][Medline]
  32. Rolig,R.L., Lowery,M.P., Adair,G.M. and Nairn,R.S. (1998) Characterization and analysis of Chinese hamster ovary cell ERCC1 mutant alleles. Mutagenesis, 13, 357–365.[Abstract/Free Full Text]
  33. 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.[Abstract/Free Full Text]
  34. Whalen,A.M., Galasinski,S.C., Shapiro,P.S., Nahreini,T.S. and Ahn,N.G. (1997) Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase. Mol. Cell. Biol., 17, 1947–1958.[Abstract]
  35. Wood,R.D., Robins,P. and Lindahl,T. (1988) Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts.Cell, 53, 97–106.[CrossRef][ISI][Medline]
  36. Aboussekhra,A., Biggerstaff,M., Shivji,M.K., Vilpo,J.A., Moncollin,V., Podust,V.N., Protic, M., Hubscher,U., Egly,J.M. and Wood,R.D. (1995) Mammalian DNA nucleotide excision repair reconstituted with purified protein components.Cell, 80, 859–868.[CrossRef][ISI][Medline]
  37. Rommel,C., Clarke,B.A., Zimmermann,S., Nunez,L., Rossman,R., Reid,K., Moelling,K., Yancopoulos,G.D. and Glass,D.J. (1999) Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt.Science, 286, 1738–1741.[Abstract/Free Full Text]
  38. Zimmermann,S. and Moelling,K. (1999) Phosphorylation and regulation of Raf by Akt (protein kinase B).Science, 286, 1741–1744.[Abstract/Free Full Text]
  39. Karin,M., Liu,Z. and Zandi,E. (1997) AP-1 function and regulation. Curr. Opin. Cell. Biol., 9, 240–246.[CrossRef][ISI][Medline]
  40. Tulchinsky,E. (2000) Fos family members: regulation, structure and role in oncogenic transformation. Histol. Histopathol., 15, 921–928.[ISI][Medline]
  41. Murphy,L.O., Smith,S., Chen,R.H., Fingar,D.C. and Blenis,J. (2002) Molecular interpretation of ERK signal duration by immediate early gene products. Nature Cell Biol., 4, 556–564.[ISI][Medline]
  42. Mody,N., Leitch,J., Armstrong,C., Dixon,J. and Cohen,P. (2001) Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway. FEBS Lett., 502, 21–24.[CrossRef][ISI][Medline]
  43. Suzaki,Y., Yoshizumi,M., Kagami,S., Koyama,A.H., Taketani,Y., Houchi,H., Tsuchiya,K., Takeda,E. and Tamaki,T. (2002) Hydrogen peroxide stimulates c-Src-mediated big mitogen-activated protein kinase 1 (BMK1) and the MEF2C signaling pathway in PC12 cells: potential role in cell survival following oxidative insults. J. Biol. Chem., 277, 9614–9621.[Abstract/Free Full Text]
  44. Friedberg,E.C. (2001) How nucleotide excision repair protects against cancer. Nature Rev. Cancer, 1, 22–33.[CrossRef][Medline]
  45. Hoeijmakers,J.H. (2001) Genome maintenance mechanisms for preventing cancer.Nature, 411, 366–374.[CrossRef][Medline]
  46. Lindahl,T. and Wood,R.D. (1999) Quality control by DNA repair.Science, 286, 1897–1905.[Abstract/Free Full Text]
  47. Tai,E.C.H. and Lee,T.-C. (1990) Induction of sister chromatid exchanges by lead compounds in Chinese hamster ovary cells. Bull. Inst. Zool. Acad. Sin., 29, 121–125.
  48. Lin,R.H., Lee,C.H., Chen,W.K. and Lin-Shiau,S.Y. (1994) Studies on cytotoxic and genotoxic effects of cadmium nitrate and lead nitrate in Chinese hamster ovary cells. Environ. Mol. Mutagen., 23, 143–149.[ISI][Medline]
  49. Lewis,T.S., Hunt,J.B., Aveline,L.D., Jonscher,K.R., Louie,D.F., Yeh,J.M., Nahreini,T.S., Resing,K.A. and Ahn,N.G. (2000) Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry. Mol. Cell, 6, 1343–1354.[CrossRef][ISI][Medline]
  50. Price,M.A., Cruzalegui,F.H. and Treisman,R. (1996) The p38 and ERK MAP kinase pathways cooperate to activate Ternary Complex Factors and c-fos transcription in response to UV light. EMBO J., 15, 6552–6563.[ISI][Medline]
  51. Haas,S. and Kaina,B. (1995) c-Fos is involved in the cellular defence against the genotoxic effect of UV radiation. Carcinogenesis, 16, 985–991.[Abstract/Free Full Text]
  52. 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.[Abstract/Free Full Text]
  53. Chao,J.-I. and Yang,J.-L. (2001) Opposite roles of ERK and p38 mitogen-activated protein kinases in cadmium-induced genotoxicity and mitotic arrest. Chem. Res. Toxicol., 14, 1193–1202.[CrossRef][ISI][Medline]
  54. 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.[Abstract/Free Full Text]
  55. 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.[Medline]
  56. Yacoub,A., Park,J.S., Qiao,L., Dent,P. and Hagan,M.P. (2001) MAPK dependence of DNA damage repair: ionizing radiation and the induction of expression of the DNA repair genes XRCC1 and ERCC1 in DU145 human prostate carcinoma cells in a MEK1/2 dependent fashion. Int. J. Radiat. Biol., 77, 1067–1078.[CrossRef][ISI][Medline]
  57. 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.[Abstract]
  58. 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.[Abstract/Free Full Text]
  59. Saavedra,H.I., Knauf,J.A., Shirokawa,J.M., Wang,J., Ouyang,B., Elisei,R., Stambrook,P.J. and Fagin,J.A. (2000) The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway. Oncogene, 19, 3948–3954.[CrossRef][ISI][Medline]
  60. Yang,T., Namba,H., Hara,T., Takmura,N., Nagayama,Y., Fukata,S., Ishikawa,N., Kuma,K., Ito,K. and Yamashita,S. (1997) p53 induced by ionizing radiation mediates DNA end-jointing activity, but not apoptosis of thyroid cells. Oncogene, 14, 1511–1519.[CrossRef][ISI][Medline]
  61. Belloni-Olivi,L., Annadata,M., Goldstein,G.W. and Bressler,J.P. (1996) Phosphorylation of membrane proteins in erythrocytes treated with lead. Biochem. J., 315, 401–406.[Medline]
  62. He,L., Poblenz,A.T., Medrano,C.J. and Fox,D.A. (2000) Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore. J. Biol. Chem., 275, 12175–12184.[Abstract/Free Full Text]
  63. Costa,L.G., Guizzetti,M., Lu,H., Bordi,F., Vitalone,A., Tita,B., Palmery,M., Valeri,P. and Silvestrini,B. (2001) Intracellular signal transduction pathways as targets for neurotoxicants. Toxicology, 160, 19–26.[CrossRef][ISI][Medline]
  64. Bressler,J., Kim,K.A., Chakraborti,T. and Goldstein,G. (1999) Molecular mechanisms of lead neurotoxicity. Neurochem. Res., 24, 595–600.[CrossRef][ISI][Medline]
  65. Chakraborti,T., Kim,K.A., Goldstein,G.G. and Bressler,J.P. (1999) Increased AP-1 DNA binding activity in PC12 cells treated with lead. J. Neurochem., 73, 187–194.[CrossRef][ISI][Medline]
  66. Kim,K.A., Chakraborti,T., Goldstein,G.W. and Bressler,J.P. (2000) Immediate early gene expression in PC12 cells exposed to lead: requirement for protein kinase C. J. Neurochem., 74, 1140–1146.[CrossRef][ISI][Medline]
  67. Calsou,P., Frit,P., Bozzato,C. and Salles,B. (1996) Negative interference of metal(II) ions with nucleotide excision repair in human cell-free extracts. Carcinogenesis, 17, 2779–2782.[Abstract/Free Full Text]
  68. Asmuss,M., Mullenders,L.H., Eker,A. and Hartwig,A. (2000) Differential effects of toxic metal compounds on the activities of Fpg and XPA, two zinc finger proteins involved in DNA repair. Carcinogenesis, 21, 2097–2104.