1 Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown 26505; and 2 Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia 26506
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ABSTRACT |
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The present study investigates whether reactive oxygen species (ROS)
are involved in p53 activation, and if they are, which species is
responsible for the activation. Our hypothesis is that hydroxyl radical
(·OH) functions as a messenger for the activation of this tumor
suppressor protein. Human lung epithelial cells (A549) were used to
test this hypothesis. Cr(VI) was employed as the source of ROS due to
its ability to generate a whole spectrum of ROS inside the cell. Cr(VI)
is able to activate p53 by increasing the protein levels and enhancing
both the DNA binding activity and transactivation ability of the
protein. Increased cellular levels of superoxide radicals
(O2·), hydrogen peroxide
(H2O2), and ·OH radicals were detected on the
addition of Cr(VI) to the cells. Superoxide dismutase, by enhancing the
production of H2O2 from O2
·
radicals, increased p53 activity. Catalase, an
H2O2 scavenger, eliminated ·OH radical
generation and inhibited p53 activation. Sodium formate and aspirin,
·OH radical scavengers, also suppressed p53 activation. Deferoxamine,
a metal chelator, inhibited p53 activation by chelating Cr(V) to make
it incapable of generating radicals from H2O2.
NADPH, which accelerated the one-electron reduction of Cr(VI) to Cr(V)
and increased ·OH radical generation, dramatically enhanced p53
activation. Thus ·OH radical generated from Cr(VI) reduction in A549
cells is responsible for Cr(VI)-induced p53 activation.
Cr(VI) carcinogenesis; reactive oxygen species
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INTRODUCTION |
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THE TUMOR
SUPPRESSOR PROTEIN p53 plays an important role in protecting
cells from tumorgenetic alteration (19, 22).
Mutational inactivation of p53 has been found to be involved in various
human cancers, which indicates the importance of p53 in human
carcinogenesis (42). It has been reported that >50% of
human cancers contain mutations in the p53 gene (15,
22). The p53 is activated in response to a variety of
stimuli, such as UV, radiation, hypoxia, nucleotide deprivation,
etc. (7, 10, 18,
23). The activation of p53 may cause either cell division
cycle arrest or apoptosis (1, 9,
19, 40).
The p53 can be considered as one of the oxidative stress response transcription factors (42). There are several cysteine residues in the central domain of the p53 protein. These residues are crucial for the p53 protein binding to the specific DNA sequence. Redox modulation at a posttranslational level often occurs by reduction or oxidation of the disulfide bond. A reduced state is required for these cysteine residues to ensure that p53 protein would bind to specific consensus DNA and transactivate target genes (12, 27, 29). Among these genes, more than one-half of them are reported to be involved in the metabolism of reactive oxygen species (ROS) (28). ROS are also believed to be involved in the activation of p53 protein induced by UV and ion irradiation (11, 44). Questions remain to be answered concerning which species among ROS plays a key role in the process of p53 activation.
Cr(VI)-containing compounds are considered to be well-established
carcinogens (4). They are potential inducers of tumors in
experimental animals and active agents in the induction of DNA damage,
such as DNA strand breakage (4, 13,
20, 21, 41, 43,
46). Industrial exposure to these compounds is reported to
be associated with a higher incidence of human lung cancer (13, 21). Environmental exposure to Cr(VI)
could induce lung toxicity in the short term and carcinogenecity over
the long term (6). Cr(VI) and Cr(III) are the two stable
chromium oxidation states found in nature. Unlike Cr(III), Cr(VI) can
enter the cell through the anion transport system (3,
5). After it enters the cell, Cr(VI) is reduced by
cellular reductants to its lower oxidation states, Cr(V) and Cr(IV)
(31). These reactive chromium intermediates are capable of
generating a whole spectrum of ROS (33, 35,
36, 39). ROS generated by these reactions can cause DNA strand breaks, base modification, lipid peroxidation, and
nuclear transcription factor NF-B activation (30,
33, 35, 36, 39,
48). In the present studies, Cr(VI) was used as the source
of ROS to investigate the role of ROS in p53 activation, and the
following questions will be answered: 1) whether Cr(VI) is
able to activate p53 protein, and 2) if so, whether ROS play a role in this process and which species is responsible for p53 activation.
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MATERIALS AND METHODS |
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Materials. Sodium dichromate (Na2Cr2O7) and chromium fluoride [Cr(III)] were purchased from Aldrich (Milwaukee, WI). Deferoxamine, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), superoxide dismutase (SOD), catalase, sodium formate, aspirin, NADPH, and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO). Dihydroethidium (DE) was purchased from Molecular Probes (Eugene, OR). The spin trap, DMPO, was purified by charcoal decolorization and vacuum distillation and was free of electron spin resonance (ESR) detectable impurities.
Cell culture. A human lung epithelial cell line (A549 cells) was obtained from American Type Culture Collection (Rockville, MD). The cells were maintained in F-12K (Kaighn's modification) nutrient mixture medium (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 1,000 U/ml penicillin-streptomycin.
Nuclear extraction.
Nuclear extracts were prepared by a modified method of Ye et al.
(47). A549 cells suspended in F-12K plus 10% FBS were
cultured in 35-mm cell culture plates at 5 × 106
cells/plate for 24 h. The cells were treated with chromium and other agents for 3 h. After washing the cells with 5 ml PBS twice, we added fresh F-12K medium with 10% FBS. The cells were then incubated at 37°C for various times. At the end of the culture period, cells were harvested and treated with 500 µl of lysis buffer
[50 mM KCl, 0.5% Nonidet P-40 (NP-40), 25 mM HEPES (pH 7.8), 1 mM
phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 20 µg/ml
aprotinin, and 100 µM dithiothreitol (DTT)] on ice for 4 min. After
centrifugation at 14,000 rpm for 1 min, we saved the supernatant as a
cytoplasmic extract. The nuclei were washed once with the same
buffer without NP-40. The washed nuclei were suspended in a 100-µl
volume of extraction buffer (500 mM KCl, 10% glycerol with the same
concentrations of HEPES, PMSF, leupeptin, aprotinin, and DTT as the
lysis buffer) and pipetted three times for proper mixing. After
centrifugation at 14,000 rpm for 5 min, we harvested the supernatant
and stored this nuclear protein extract at 70°C. The
concentration was determined using a bicinchoninic acid protein assay
reagent (Pierce, Rockford, IL).
Western blot. The nuclear extraction proteins were used for Western blot analysis to determine the p53 protein level. This analysis was carried out as described before (8). Briefly, samples (20 µg protein) denatured with SDS were electrophoretically separated on 10% Tris-glycine gels (Novex, San Diego, CA) and were transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH). The membrane was preblocked in milk buffer [Tris-buffered saline Tween (TBST) containing 5% nonfat milk] for 20 min and exposed for 1 h to 0.8 µg/ml affinity-purified mouse antibody to p53 protein in fresh milk buffer. The membrane was rinsed and incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG for 1 h. The membrane was then washed with TBST, and antibody binding sites were visualized by enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). All the antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
Oligonucleotides.
Oligonucleotides were synthesized by the phosphoramidite method on a
DNA/RNA synthesizer (model 392; Applied Biosystems, Foster City, CA). A p53 binding sequence (5'-AGACATGCCTAGACATTGT-3') was used
to synthesize a p53 binding oligonucleotide. The synthesized single-stranded oligonucleotides were deprotected at 50°C overnight, dried in a speed vacuum, and then dissolved in the Tris-EDTA buffer. Complementary strands were denatured at 80°C for 5 min and annealed at room temperature. The double-stranded probe was labeled with [-32P]ATP (Amersham, Arlington Heights, IL) using a T4
kinase (Bethesda Research Laboratories, Gaithersburg, MD).
Electrophoretic mobility shift assay.
The DNA-protein binding reaction was conducted in a 24-µl reaction
mixture including 1 µg poly[dI-dC] (Sigma), 3 µg nuclear protein
extract, 3 µg BSA, 4 × 104 cpm of
32P-labeled oligonucleotide probe (1 µg), 0.1 µg p53
antibody Ab-1 (Oncogene, Cambridge, MA), 3 µl distilled water, and 12 µl of 2× reaction buffer (24% glycerol, 24 mM HEPES, pH 7.9, 8 mM
Tris · HCl, pH 7.9, 2 mM EDTA, and 2 mM DTT). This mixture was
incubated on ice for 10 min in the absence of the radiolabeled probe,
then incubated for 20 min at room temperature in the presence of the radiolabeled probe. After the incubation, the DNA-protein complexes were resolved on a 3.5% acrylamide gel that had been prerun at 210 V
for 30 min with 0.5× Tris-boric acid-EDTA buffer. The loaded gel was
run at 210 V for 60 min, then dried and placed on Kodak X-OMAT film
(Eastman Kodak, Rochester, NY) for autoradiography. The film was
developed after overnight exposure at 70°C.
Assay for p53 transcriptional activation.
Human lung epithelial A549 cells (3 × 105) suspended
in 1 ml of 10% FBS F-12K medium were seeded into each well of a
six-well plate. After being incubated at 37°C for 24 h, the
cells were transit transfected by 1 µg p53-luciferase reporter
plasmid and 1 µg -gal plasmid in reduced-serum medium. The cells
were incubated overnight and then washed. Fresh 10% FBS F-12K medium
was added. Twenty-four hours later, the cells were exposed to various
concentrations of Cr(VI) for 3 h. After being washed, the
transfected cells were incubated overnight in fresh F-12K medium. The
cells were then extracted with 400 µl reporter lysis buffer in 4°C
for 2 h. The luciferase activity was measured with 80 µl of cell
lysate using a Monolight luminometer (model 3010; Analytical
Luminescence Laboratory, Sparks, MD).
-gal activity was determined
by using a method described before (47). The results are
expressed as relative p53 activity compared with controls after
normalizing by
-gal activity.
ESR measurements. ESR spin trapping was used to examine free radical generation. The use of this method is necessary because of the reactive nature of the free radicals to be studied. This technique involves an addition-type reaction of a short-lived radical with a diamagnetic compound (spin trap) to form a relatively long-lived free radical product, the so-called spin adduct, which can be studied by conventional ESR. The intensity of the spin adduct signal corresponds to the amount of short-lived radicals trapped, and the hyperfine splittings of the spin adduct are generally characteristic of the original, short-lived trapped radical.
All measurements were conducted with a Varian E9 ESR spectrometer and a flat cell assembly. Hyperfine couplings were measured (to 0.1 Gauss) directly from magnetic field separation with the use of potassium tetraperoxochromate (K3CrO8) and 1,1-diphenyl-2-picrylhydrazyl as reference standards. An EPRDAP 2.0 program (US EPR, Clarksville, MD) was employed for data acquisition and analysis. Reactants were mixed in test tubes to a total final volume of 0.5 ml. The reaction mixture was then transferred to a flat cell for ESR measurement.Cellular O2· and H2O2
assay.
DE is a superoxide radical (O2
·)-specific dye
(2, 26). DE was dissolved in DMSO at 2 mM and
kept at
20°C. The cells were plated onto a glass slip in the
24-well plate at 1 × 104/well 16 h before
treatment. DE was added to the cell culture together with the various
treatments, and the cells were incubated at 37°C for 30 min. The
final DMSO concentration in the cell culture was 0.1%. After the cells
were stained, they were washed with PBS and fixed with 10% buffered
formalin. The slip was mounted on a glass slide and observed with the
use of a Saratro 2000 (Molecular Dynamics, Sunnyvale, CA)
laser scanning confocal microscope (Optiphot-2; Nikon, Melville, PA)
fitted with an argon-ion laser. DCFH-DA at 2 µM was used to monitor
the H2O2 level inside the cells according to
the methods reported earlier (14, 17,
49).
Oxygen consumption measurements. Oxygen consumption measurements were carried out with a Gilson oxygraph equipped with a Clark electrode (Gilson Medical Electronics, Middleton, WI). These measurements were made from mixtures containing 1.0 × 106/ml cells and various treatments in a total volume of 1.5 ml. The oxygraph was calibrated with media equilibrated with oxygen of known concentrations.
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RESULTS |
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The p53 activation by Cr(VI) is dose and time dependent.
Human lung epithelial A549 cells were used to study induction of p53
activation by Cr(VI). The cells were incubated with different concentrations of Cr(VI) for 3 h. After washing the cells, fresh F-12K medium supplemented with 10% FBS was added. The cells were then
incubated at 37°C overnight. The p53 protein level and DNA binding
activity were analyzed in the nuclear extracts. As shown in Fig.
1A, the p53 protein
level increased after exposure to increasing concentrations of Cr(VI).
The DNA binding activity of the same protein was also investigated by
electrophoretic mobility shift assay. The results are shown in Fig.
1B. From this figure, a similar concentration-dependent
pattern was found, i.e., the higher the concentration of Cr(VI), the
higher the binding activity of p53 protein.
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Detection of free radicals.
ESR spin trapping was used to detect free radical generation from
Cr(VI)-stimulated A549 cells. Fig.
4A shows the spectrum recorded
from a mixture containing Cr(VI), A549 cells, and DMPO (a spin-trapping
reagent). This spectrum consists of a 1:2:2:1 quartet with splittings
of aH = aN = 14.9 G, where
aH and aN denote hyperfine splittings of the
-hydrogen and the nitroxyl nitrogen, respectively. On the basis of
these splitting constants, the 1:2:2:1 quartet was assigned to
DMPO/·OH adduct. The arrow at the right side shows a small peak that
was assigned to a Cr(V)-NADPH complex as reported earlier
(38). Addition of SOD, a O2
· scavenger
whose function is to convert O2
· to
H2O2, dramatically increased the DMPO/·OH
adduct signal (Fig. 4B). Catalase, an
H2O2 scavenger, and sodium formate, a scavenger of ·OH radical, decreased the generation of ·OH radical (Fig. 4,
C and D). A metal chelator, deferoxamine,
suppressed the DMPO/·OH signal (Fig. 4E). NADPH, a
cofactor of certain flavoenzymes, such as GSSG-R (glutathione
reductase), which catalyzes the conversion of Cr(VI) to Cr(V), enhanced
the generation of both Cr(V) and ·OH radical (Fig. 4F).
H2O2 dramatically increased the DMPO/·OH signal with reduction of the Cr(V) peak (Fig. 4G).
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Oxygen consumption.
As shown before, ·OH radicals were produced via a Cr(V)-mediated
Fenton-like reaction [H2O2 + Cr(V) Cr(VI) + ·OH + OH
]. The
H2O2 was generated by the dismutation of
O2
· radicals. O2
· radicals were
generated by one-electron reduction of molecular oxygen during the
reduction of Cr(VI) by flavoenzymes in the presence of NADPH. A Gilson
oxygraph equipped with a Clark electrode was used to measure oxygen
consumption in Cr(VI)-stimulated A549 cells. As shown in Fig.
6, addition of Cr(VI) enhanced the oxygen
consumption, and NADPH slightly accelerated it. Cr(III) did not have
any significant effect.
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·OH radical is responsible for Cr(VI)-induced p53 activation.
A549 cells were incubated with Cr(VI) in the presence of various
reagents for 3 h (Fig. 7). After the
cells were washed, the fresh F-12K medium with 10% FBS was added. The
cells were then incubated overnight and were harvested for extraction
of nuclear proteins. The protein level was analyzed by Western blot.
The results are shown in Fig. 7. As shown in this figure, the p53 protein level in the untreated A549 cells (lane 1) was below
the detection limits. Cells treated with Cr(VI) showed an elevated p53
protein level (lane 2). Cr(III), because of its inability to
enter the cell, had no effect (lane 3). SOD increased the
protein level through the generation of H2O2
and ·OH radical (lane 4), whereas catalase, sodium
formate, and aspirin scavenged ·OH radical and diminished the protein
level (lanes 5-7). Deferoxamine chelated Cr(V) and
decreased p53 protein induction (lane 8). NADPH, on the
other hand, enhanced the generation of ·OH radical and increased p53
protein induction in A549 cells (lane 9). These results
suggest that ·OH radical is responsible for Cr(VI)-induced p53
protein activation.
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DISCUSSION |
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The present studies show that Cr(VI) is able to induce the activation of p53 protein in the epithelial cell line A549. ·OH radicals play a key role in this Cr(VI)-induced p53 activation.
With the use of ESR with a loop-gap resonator, previous studies have
demonstrated that reduction of Cr(VI) in live animals generates Cr(V)
(24). Flavoenzymes, such as GSSG-R, are likely reductants
for Cr(V) generation. With the use of a noncellular chemical system,
our earlier studies have shown that reduction of Cr(VI) by GSSG-R in
the presence of NADPH generates Cr(V) (31). The results
obtained from the present studies show that Cr(VI) can be reduced by
the whole cells to Cr(V). During the reduction process, molecular
oxygen is reduced to O2· radical, which generates
H2O2 via dismutation. Cr(V) reacts with
H2O2 to generate ·OH radicals via a
Fenton-like reaction [Cr(V) + H2O2
Cr(VI) + ·OH+ OH
]
(Fig. 8).
The ·OH radical is the key species among ROS responsible for p53
activation. The following experimental observations support the above
conclusion. 1) Sodium formate, an ·OH radical scavenger, inhibited p53 activation. 2) Deferoxamine, which chelated
Cr(V) to make it incapable of generating ·OH radical from
H2O2, diminished p53 activation. 3)
SOD enhanced the p53 activation instead of attenuating it. This is due
to the fact that SOD increased the H2O2
generation by catalyzing O2· dismutation, as
demonstrated by fluorescent staining, and increased the ·OH radical
generation through a Fenton-like reaction, as demonstrated by ESR spin
trapping. 4) A mixture of Cr(VI) and A549 cells increased
molecular oxygen consumption. 5) Catalase, which depleted
H2O2 and blocked ·OH radical generation,
inhibited the p53 activation.
H2O2 is an endogenous oxidant that is commonly used to investigate the role of ROS in p53 activation. However, inconsistent observations were reported from different laboratories (16, 27). For example, it has been observed that H2O2 can activate p53 protein and its target gene Bax (16). It has also been reported that H2O2 decreased p53 transactivation by weakening its binding to the specific DNA sequence (27). The results obtained from present study show that whereas H2O2 itself is unable to cause p53 activation, it can indirectly activate p53 through its ability to generate ·OH radical on reaction with metal ions. The following experimental observations support this conclusion. 1) Sodium formate, an ·OH radical scavenger, inhibited p53 activation. This ·OH radical scavenger did not react with H2O2. Similar results were obtained with the use of a different ·OH radical scavenger, aspirin. 2) Deferoxamine, which inhibited p53 activation due to chelation of Cr(V) to make this metal ion unable to generate ·OH radical from H2O2, did not react with H2O2. Although H2O2 is not a direct p53 stimulating agent, it functions as a precursor for ·OH radical generation. Thus elimination of H2O2 inhibited p53 activation. This may explain the reported controversy concerning the role of H2O2 in p53 activation.
As for O2· radical, it is not a direct p53
stimulating agent either. As shown in the present studies, a specific
O2
· radical scavenger, SOD, enhanced p53 activation
instead of causing inhibition. The enhancement of p53 activation is due
to the generation of H2O2 from
O2
· radical catalyzed by SOD.
O2
· radical is generated via reduction of molecular
oxygen as demonstrated by the oxygen consumption assay. Thus the
following scheme can best accommodate p53 activation induced by ROS
reaction
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It may be noted that many other metal ions, mineral particles, and chemical carcinogens, such as cobalt, nickel, vanadium, asbestos, and silica are reported to be capable of generating ·OH radical (25, 32, 34, 37, 45). It is possible that those agents may have the same function as Cr(VI); i.e., they may cause p53 activation through ·OH radical as a common messenger. Because p53 is involved in various biological processes such as regulation of genes involved in the cell cycle, cell growth arrest after DNA damage, and apoptosis, ·OH radical could be an important messenger in signal transduction pathways involved in carcinogenesis processes.
In conclusion, 1) Cr(VI) is able to induce the p53 activation, 2) ROS is involved in its mechanism of activation, and 3) ·OH radical functions as a messenger in the p53 activation processes.
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FOOTNOTES |
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Address for reprint requests and other correspondence: X. Shi, Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV 26505 (E-mail: xas0{at}cdc.gov).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 27 December 1999; accepted in final form 27 March 2000.
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