Carcinogenesis vol.24 no.1 pp.53–61, 2003
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
1To
whom correspondence should be addressed
Email: [email protected]
Introduction
Lead is an ubiquitous toxic contaminant of our environment,
which was evaluated as a possible human carcinogen (group2B) 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
Abbreviations: 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.
© The Article Author(s). Published by Oxford University Press 2003
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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 UVL10 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.
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
Y.-W.Lin, S.-M.Chuang and J.-L.Yang
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 activatedERK1/2 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
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
54
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 (#sc571), AKT (#sc-8312), c-FOS (#sc-52) and α-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) β-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 [γ-32P]ATP
(6 000 Ci/mmol) to the substrate GST-cJUN(1–79) as described previously (33).
Transfection
Cells (4⫻105) were plated in a p60 dish one-day before transfection. Plasmid
(5–15 µg) containing a constitutively active form of MKK1 (∆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 [α-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 (ICPMS; 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
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
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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.
precipitates were discarded. The BCA protein assay kit (Pierce, Rockford, IL)
was adopted to determine protein concentrations using bovine serum albumin
as a standard.
ERK triggers NER and decreases Pb(II) mutagenesis
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 serumfree RPMI1640 medium for 24 h. To serve as positive control, the serumstarved 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.
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 GSTcJUN(1–79) as a substrate. As shown in Figure 1, 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 1 and data not shown). Additionally, the endogenous protein levels of the four MAPKs
were unaltered by Pb(II) (Figure 1).
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 2A 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 2B
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 2B). Pb(II) did not significantly influence
the endogenous ERK2 and AKT protein levels during the
time-course and recovery experiments (Figure 2). The above
results imply that Pb(II) activates ERK1/2 persistently and
AKT transiently in CL3 cells.
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 phosphoAKT levels. PD98059 (50 µM) and wortmannin (100 nM)
completely blocked phospho-ERK1/2 and phospho-AKT in
Pb(II)-treated cells, respectively (Figure 3). Wortmannin did
not influence the ERK1/2 activation and PD98059 did not
affect the AKT activation elicited by Pb(II) in CL3 cells
(Figure 3). The results suggest that Pb(II)-elicited ERK1/2 and
AKT signals do not communicate with each other.
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
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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 1. Western blot analyses of ERK2 and
AKT protein levels in the same WCE are shown in the lower panels.
Y.-W.Lin, S.-M.Chuang and J.-L.Yang
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 2.
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 1. Protein levels of α-tubulin in the
same WCE are shown in the lower panels as an internal control.
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 4A, 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 4B). The results indicate that
Pb(II)-elicited ERK1/2 participates in the induction of c-FOS
expression.
ERK1/2 activation by Pb(II) is positively correlated with anticytotoxicity 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
56
in serum-free medium and then subjected to the colonyforming 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, P ⬍ 0.01). In contrast, co-treatment with wortmannin did
not affect the Pb(II)-induced cytotoxicity (Figure 5A). Neither
PD98059 nor wortmannin alone exhibited a cytotoxic effect
in these experiments (Figure 5A). 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 5B, 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 5B, P ⬍ 0.01).
Conversely, wortmannin did not alter the low mutagenicity of
Pb(II) in CL3 cells (Figure 5B). 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 5D, 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.
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 6A). Again, PD98059 (50 µM) significantly
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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
1.01–4.95⫻106 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 1, and the western blot shown is one representative of
four independent experiments.
ERK triggers NER and decreases Pb(II) mutagenesis
elevated the cytotoxicity and mutagenicity in Pb(II)-treated
HFW cells (Figure 6B and 6C). 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.
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 7, 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 7C), despite
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 1.69–2.43⫻106 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 1.
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).
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 α-32P[dCTP] to examine the efficiency of NER
synthesis. As shown in Figure 8, 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 8).
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 8B). The results indicate that
Pb(II) exposure elevates cellular NER synthesis through the
ERK1/2 pathway.
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 9, 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
57
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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 1. 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 1.18–3.05⫻106 in each treatment for 6-TG assay.
Y.-W.Lin, S.-M.Chuang and J.-L.Yang
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 α-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%.
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.
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 10, 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.
58
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
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
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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 (4⫻105) 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 8. 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 phosphoERK1/2 and ERK2 in equal amounts of proteins derived from each WCE
are also shown in (B).
ERK triggers NER and decreases Pb(II) mutagenesis
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, PKCα 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
PKCα 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).
59
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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 NERproficient 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 NERdeficient 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 anticytotoxicity 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.
Y.-W.Lin, S.-M.Chuang and J.-L.Yang
Acknowledgements
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
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.
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.
4. Steenland,K. and Boffetta,P. (2000) Lead and cancer in humans: where
are we now? Am. J. Ind. Med., 38, 295–299.
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.
6. Gerber,G.B., Leonard,A. and Jacquet,P. (1980) Toxicity, mutagenicity and
teratogenicity of lead. Mutat. Res., 76, 115–141.
7. Goyer,R.A. (1993) Lead toxicity: current concerns. Environ. Health
Perspect., 100, 177–187.
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.
60
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.
10. Roy,N.K. and Rossman,T.G. (1992) Mutagenesis and comutagenesis by
lead compounds. Mutat. Res., 298, 97–103.
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.
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.
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.
14. Hartwig,A., Schlepegrell,R. and Beyersmann,D. (1990) Indirect mechanism
of lead-induced genotoxicity in cultured mammalian cells. Mutat. Res.,
241, 75–82.
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.
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.
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.
18. Kolch,W. (2000) Meaningful relationships: the regulation of the Ras/Raf/
MEK/ERK pathway by protein interactions. Biochem. J., 351, 289–305.
19. Whitmarsh,A.J. and Davis,R.J. (2000) A central control for cell growth.
Nature, 403, 255–256.
20. Davis,R.J. (2000) Signal transduction by the JNK group of MAP kinases.
Cell, 103, 239–252.
21. Chang,L. and Karin,M. (2001) Mammalian MAP kinase signaling cascades.
Nature, 410, 37–40.
22. Nebreda,A.R. and Porras,A. (2000) p38 MAP kinases: beyond the stress
response. Trends Biochem. Sci., 25, 257–260.
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.
24. Scheid,M.P. and Woodgett,J.R. (2001) PKB/AKT: functional insights from
genetic models. Nature Rev. Mol. Cell. Biol., 2, 760–768.
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.
26. Blume-Jensen,P. and Hunter,T. (2001) Oncogenic kinase signalling. Nature,
411, 355–365.
27. Ramesh,G.T., Manna,S.K., Aggarwal,B.B. and Jadhav,A.L. (1999) Lead
activates nuclear transcription factor-κB, activator protein-1, and aminoterminal c-Jun kinase in pheochromocytoma cells. Toxicol. Appl.
Pharmacol., 155, 280–286.
28. Lu,H., Guizzetti,M. and Costa,L.G. (2002) Inorganic lead activates the
mitogen-activated protein kinase kinase-mitogen-activated protein kinasep90RSK signaling pathway in human astrocytoma cells via a protein
kinase C-dependent mechanism. J. Pharmacol. Exp. Ther., 300, 818–823.
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.
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.
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.
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.
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.
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.
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.
Downloaded from http://carcin.oxfordjournals.org/ by guest on November 24, 2016
This phenomenon together with the finding that inhibition of
ERK1/2 could not affect Pb levels in CL3 cells (Figure 10)
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.
ERK triggers NER and decreases Pb(II) mutagenesis
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
64. Bressler,J., Kim,K.A., Chakraborti,T. and Goldstein,G. (1999) Molecular
mechanisms of lead neurotoxicity. Neurochem. Res., 24, 595–600.
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.
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.
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.
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.
Received July 10, 2002; revised August 25, 2002; accepted October 7, 2002
61
Downloaded from http://carcin.oxfordjournals.org/ by guest on November 24, 2016
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.
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.
38. Zimmermann,S. and Moelling,K. (1999) Phosphorylation and regulation
of Raf by Akt (protein kinase B). Science, 286, 1741–1744.
39. Karin,M., Liu,Z. and Zandi,E. (1997) AP-1 function and regulation. Curr.
Opin. Cell. Biol., 9, 240–246.
40. Tulchinsky,E. (2000) Fos family members: regulation, structure and role
in oncogenic transformation. Histol. Histopathol., 15, 921–928.
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.
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.
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.
44. Friedberg,E.C. (2001) How nucleotide excision repair protects against
cancer. Nature Rev. Cancer, 1, 22–33.
45. Hoeijmakers,J.H. (2001) Genome maintenance mechanisms for preventing
cancer. Nature, 411, 366–374.
46. Lindahl,T. and Wood,R.D. (1999) Quality control by DNA repair. Science,
286, 1897–1905.
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.
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.
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.
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.
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.
53. Chao,J.-I. and Yang,J.-L. (2001) Opposite roles of ERK and p38 mitogenactivated protein kinases in cadmium-induced genotoxicity and mitotic
arrest. Chem. Res. Toxicol., 14, 1193–1202.