Affiliations of authors: M. O. Ripple, S. R. Schwarze, Institute on Aging, University of Wisconsin, Madison, and Geriatric Research, Education and Clinical Center, William S. Middleton Memorial Veterans Hospital, Madison; W. F. Henry, G. Wilding, Department of Medicine, University of Wisconsin Comprehensive Cancer Center, Madison; R. Weindruch, Institute on Aging, University of Wisconsin, Geriatric Research, Education and Clinical Center, William S. Middleton Memorial Veterans Hospital, and Department of Medicine, University of Wisconsin Comprehensive Cancer Center, Madison.
Correspondence to: George Wilding, M.D., Department of Medicine, University of Wisconsin, K6/550 CSC, 600 Highland Ave., Madison, WI 53792.
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ABSTRACT |
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INTRODUCTION |
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Earlier, we (12) linked androgen exposure to production of reactive oxygen species (ROS) in cultured prostate cancer cells. ROS (e.g., superoxide radical, hydrogen peroxide, and hydroxyl radical) cause macromolecular damage and may play important roles in tumor development and aging (11,13,14). ROS are also important regulatory molecules acting as intracellular second messengers and regulators of protein function (15-18).
Studies of dietary antioxidants provide encouraging data regarding prostate cancer
prevention. Data from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (19) show that long-term -tocopherol supplementation reduces
prostate cancer incidence by 32% and prostate cancer mortality by 41% in cigarette
smokers. Similar evidence links intake of lycopene, a potent carotenoid, and selenium
supplementation with reduced prostate cancer risk (20,21).
Understanding how antioxidants act to reduce cancer risk will aid in developing prevention
strategies.
This study examined the effect of androgens and antioxidants on transcription factor binding
at AP-1 and NF-B DNA-binding sites. AP-1 and NF-
B are ubiquitous protein
complexes that mediate cellular response to various external signals by binding to distinct DNA
sites (22-24). Knowing that androgen treatment influences the
intracellular oxidation state in cultured cells and that AP-1 and NF-
B are redox regulated,
we sought to determine if androgens stimulate the binding activity of these transcription factors
and if antioxidants can influence the effect.
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MATERIALS AND METHODS |
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Androgen-responsive, androgen receptor-positive LNCaP and androgen-independent DU145 human prostate carcinoma cells were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained at 37 °C in an atmosphere of 5% carbon dioxide and 95% air in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD) and 1x antibiotic-antimycotic (Sigma Chemical Co., St. Louis, MO, from which all chemicals, unless otherwise noted, were obtained). Cells were passaged weekly, and medium was replaced every 3 or 4 days. LNCaP cells of passages 50-90 and DU145 cells of passages 60-70 were used for all experiments.
Experiments were conducted in medium containing 1% FBS and 4%
charcoal-stripped FBS (CSS) to limit the adverse growth effects noted with 5% CSS,
which are unrelated to hormone depletion (12). For 96-well assays, 4000
cells were plated per well in 100 µL of DMEM plus 1% FBS and 4% CSS.
Then 4 days later, cells were treated with 100 µL of DMEM plus 1% FBS and
4% CSS containing a 2x concentration of the specified treatment. The final
concentration of treatment was 0, 25, 50, 100, or 500 µM vitamin C (ascorbic acid)
and vitamin E (-tocopherol succinate) in 1 nM R1881, a synthetic androgen (Du
Pont NEN, Boston, MA), or vehicle control medium (containing 0.01% ethanol).
Cells harvested for electrophoretic mobility-shift assay were plated at 106cells per 100-mm tissue culture plate in medium containing 5% FBS. The following day, medium was replaced with fresh medium containing 1% FBS and 4% CSS. Cells were treated 3 or 4 days later with fresh medium containing R1881 or the vehicle control. After the appropriate treatment period, cells were washed, scraped from the plate, resuspended in cold Kreb's Ringer buffer, and centrifuged for 5 minutes at 4 °C at 1000 rpm. The cell pellet was frozen at -80 °C.
Measurement of ROS and DNA Content
We harvested 96-well plates for estimation of cellular peroxides and hydroxyl radical levels using 2',7'-dichlorofluorescein diacetate (DCF) (Molecular Probes, Inc., Eugene, OR) after 24, 72, and 96 hours of treatment as described elsewhere (25). Fluorescent units were measured in each well after a 45-minute incubation with DCF (1 µg/0.1 mL) at 37 °C by use of an LS 50B Luminescence Spectrometer (The Perkin-Elmer Corp., Norwalk, CT). The same plates were frozen at -80 °C, and the DNA content per well was determined after thawing by a fluorometric assay using the dye Hoechst 33258 (26). The DNA fluorescence units were used as a measure of cell growth, and the DCF fluorescence units normalized to DNA (DCF/DNA) were used as a measure of ROS levels.
Electrophoretic Mobility-Shift Assay
Frozen cell pellets were processed to obtain cytoplasmic and nuclear extracts in the presence of aprotinin (2 µg/mL), leupeptin (2-5 µg/mL), and pepstatin A (5 µg/mL) (27). Each cell pellet was resuspended in a 5x volume of Buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, and 1 mM polymethylsulfonyl fluoride) and was incubated on ice for 15 minutes. Nuclear pellets were obtained after centrifugation (14 000 rpm at room temperature for 10 seconds) in a microcentrifuge; supernatant was saved as cytoplasmic extract after a second centrifugation (13 000 rpm at 4 °C for 10 minutes). The nuclear pellet was resuspended in 2x original pellet volume of Buffer C (20 mM HEPES [pH 7.9], 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM ethylene diamine tetraacetate [EDTA], 1 mM dithiothreitol, and 1 mM polymethylsulfonyl fluoride). Cells were incubated on ice for 30 minutes and centrifuged (14 000 rpm at 4 °C for 30 minutes) in a microcentrifuge, and the supernatant was saved as nuclear extract. All extracts were stored at -80 °C. The protein content was measured with the use of the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL).
Nuclear extracts, 2-6 µg, were brought to 4 µL with water and combined with 5 µL of mixtwo parts H2O, one part poly(dI-dC), and two parts 5x gel shift buffer (375 mM NaCl, 75 mM Tris-HCl [pH 7.5], 37.5% glycerol, 7.5 mM EDTA, 7.5 mM dithiothreitol, 50 mM 1.5% Nonidet P-40, and 100 µg/mL bovine serum albumin). They were then incubated for 20 minutes on ice. Oligonucleotides (Promega Corp., Madison, WI) end labeled with 32P were added and incubated with the samples for 20 minutes at room temperature. Protein-DNA complexes were run through a 1.5-mm 4.5% acrylamide gel in 0.25x Tris-borate-EDTA buffer. Gels were run at 175 V for 1.5 hours, dried, and imaged with the use of PhosphorImager and ImageQuant analysis software (Molecular Dynamics, Sunnyvale, CA).
Immunoblots
Nuclear extracts, 25-30 µg of protein per lane, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the use of 4%-12% Bis-Tris gels (NOVEX, San Diego, CA) and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) with the use of a semidry system. Immunoblots were blocked overnight at 4 °C in 5% dried milk (wt/vol) in phosphate-buffered saline (PBS) (pH 7.4) plus 0.05% Tween 20. Blots were incubated with polyclonal primary antibodies to c-Fos, c-Jun, p50, and p65 (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or androgen receptor (gift of Dr. C. Chang, University of Rochester, Rochester, NY) in PBS plus 0.05% Tween 20 for 1-2 hours at room temperature. The blots were washed and exposed to a horseradish peroxidase-labeled secondary antibody (Amersham Pharmacia Biotech, Arlington Heights, IL) for 45 minutes at room temperature. Washes and luminescent detection were carried out by use of the ECLplus detection system according to the instructions of the manufacturer (Amersham Pharmacia Biotech). Densitometric analyses of scanned x-ray films were performed with the use of Adobe Photoshop and National Institutes of Health image software.
Statistical Analyses
Experiments were conducted in 96-well plates (n = 6). Each treatment was compared
with either the vehicle control treatment or the R1881 treatment by use of the unpaired,
two-tailed Student's t test. Sample sizes were adequate to detect statistically
significant differences. Western and immunoblot analyses were done at least twice (n2).
The mean density of specific bands from scanned x-ray films was compared by use of a paired,
two-tailed Student's t test. All data were tested for statistical significance at the
nominal .05 significance level. The reported P values are two-sided and reflect the data
shown. All experiments were repeated at least two times.
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RESULTS |
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Growth (DNA per well) and ROS levels as measured by DCF/DNA of LNCaP
cells were unaffected by treatment with 1 nM R1881 for 24
hours (Fig. 1, A and B). At 96 hours, growth was 40%
lower (Fig. 1,
D; P<.0001) and ROS levels were 86%
higher in R1881-treated cells than in untreated control cells (Fig. 1,
C;
P<.001).
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AP-1 DNA-Binding Activity in Cells Treated With R1881
Treatment of LNCaP cells with 0.001-10 nM R1881 for 96
hours resulted in increased DNA-binding activity to the AP-1 consensus
sequence with 0.25, 1, and 10 nM R1881 (Fig.
2, A; 0.25-nM data not shown). Increased
binding activity was also noted with 1 nM5
-dihydrotestosterone (DHT). All of the doses that increased
binding activity were previously shown to be associated with elevated
levels of DCF-determined ROS content (12). Increased
DNA-binding activity to the AP-1 site was first noted 5 hours after
treatment with 1 nM R1881 (Fig. 2,
B). Binding activity
increased with time, peaking at 96 hours, and remained elevated for at
least 120 hours (Fig. 2,
C). Androgen-independent DU145 cells are
unresponsive to R1881 based on DCF or DNA measures and to R1881-induced
alterations in DNA-binding activity to AP-1 sites (data not shown).
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Treatment of LNCaP cells with equimolar concentrations of vitamins C
plus E abrogated R1881-induced binding to the AP-1 site (Fig. 2, D).
Treatment with 50, 100, or 500 µM vitamins C plus E for 24
hours diminished binding at the AP-1 site in both vehicle
control-treated and R1881-treated LNCaP cells. The 500-µM
dose produced the largest decrease in binding activity at 24 hours and
the only clear decrease in AP-1 DNA-binding activity at 96 hours
(96-hour data not shown).
NF-B DNA-Binding Activity in Cells Treated With R1881
DNA binding of nuclear extracts of LNCaP cells to the NF-B
consensus sequence was elevated by 1 and 10 nM R1881 after 96
hours of treatment (Fig. 3,
A). Doses of 0.001-0.1
nM R1881 had no stimulatory effect on DNA binding. NF-
B
DNA-binding activity was induced by 1 nM R1881 at 36 hours
(Fig. 3,
B). At 72 and 96 hours, very strong binding activity of the
slower migrating complex was observed (upper arrow, Fig. 3,
B).
Binding activity remained elevated for at least 120 hours.
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Equimolar concentrations of vitamins C plus E lowered the
androgen-induced DNA binding at the NF-B site after 96 hours of
treatment (Fig. 3,
C). Only the 500-µM dose influenced
DNA-binding activity to the NF-
B site. This dose increased the
binding activity of the faster migrating NF-
B DNA-protein complex
in cells not treated with R1881. A 24-hour treatment with vitamins C
plus E did not influence DNA-binding activity at the NF-
B site
(data not shown).
Protein Levels of c-Jun, c-Fos, p50, p65, and Androgen Receptor in LNCaP Cells Treated With R1881
Nuclear extracts from LNCaP cells treated with 0-1 nM
R1881 for 24 and 96 hours showed no increase in AP-1 (c-Jun and
c-Fos)-associated proteins, NF-B (p50 and p65)-associated
proteins, or androgen receptor (Table 1)
. After 24
and 96 hours, 1 nM R1881 caused a marked decrease in the
protein level of c-Fos (P = .047, 24 hours; P =
.0005, 96 hours).
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DISCUSSION |
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We observed that androgens administered at doses previously found to induce oxidative
stress (12) can increase the DNA-binding activity of LNCaP nuclear
proteins to both AP-1 and NF-B sites. In contrast, R1881 and DHT had no effect on AP-1
and NF-
B DNA-binding activities or oxidative state in androgen-independent DU145 cells.
AP-1 and NF-
B DNA-binding activities are known to be sensitive to ROS (34-36). DNA binding of AP-1 and NF-
B and of several other ROS-sensitive
transcription factors is activated by reducing agents and inhibited by oxidizing agents in
vitro, but the effects are often reversed in intact cells, which is suggestive of a more
complex form of cellular regulation (36-38). DNA-binding activity
induced in LNCaP cells by hydrogen peroxide at AP-1 sites and by tumor necrosis factor-
at NF-
B sites was similar in magnitude to that caused by androgen treatment (data not
shown).
AP-1, a ubiquitous transcriptional activator, mediates responses to external signals by regulating the expression of genes involved in growth, differentiation, and stress responses (22,38). It is composed of members of the Jun and Fos families that form homodimers (c-Jun:c-Jun) and heterodimers (c-Jun:c-Fos) that bind to a specific DNA response element. Neoplastic transformation may be induced simply by altering the expression or structure of c-Jun or c-Fos (22). Regulation of AP-1 activity is complex and involves mechanisms that increase the levels of Jun and Fos proteins as well as those that stimulate the activity of pre-existing Jun and Fos (22,39). Redox regulation of AP-1-binding activity occurs through a conserved cysteine residue in the DNA-binding domain of c-Fos and c-Jun proteins (34). Western blot analyses of protein levels in nuclear extracts of LNCaP cells showed no androgen-induced increase in the level of c-Fos or c-Jun. In fact, c-Fos levels were statistically significantly lower in LNCaP cells treated with 1 nM R1881, suggesting that the increased DNA binding is due to a change in activity and not protein level.
There is evidence that transcriptional activation by nuclear receptors, like the androgen receptor, is influenced by interactions with other transcription factors (40-42). Androgen receptor-induced transcriptional activity can be inhibited by members of the Jun and Fos families (43,44) or can be enhanced by c-Jun (45), depending on the conditions. Conversely, androgen receptor inhibits c-Jun-induced AP-1 transcriptional activity in several cell lines transfected with androgen receptor, c-Jun:c-Fos, and an AP-1-responsive reporter vector (43,44,46). These varying results may derive from inherent differences in the cell lines used, the presence of other co-activators or co-repressors, the level of androgen, and, perhaps most importantly, in the ratio of androgen receptor to c-Jun/c-Fos.
c-Jun can interact with the DNA- and ligand-binding domains of the androgen receptor (44). Whether this interaction occurs in vivo and whether it regulates endogenous AP-1 or androgen receptor-induced transactivation is unknown. Our attempts to immunoprecipitate LNCaP androgen receptor with a c-Jun antibody were unsuccessful (data not shown).
Like AP-1, NF-B is a ubiquitous transcription factor involved in growth,
differentiation, and stress response (23,24). It is a dimer of members from
the Rel/NF-
B family (e.g., p65:p50). NF-
B exists in an inactive form bound to
I
B proteins in the cytoplasm and thus does not require de novo protein synthesis
for activation (23). Upon activation, I
B is degraded and the active
NF-
B complex is translocated to the nucleus (38). Redox regulation
of NF-
B is thought to be modulated in part through a conserved cysteine residue in the p50
subunit and through I
B release (37,47). Western blot analyses
indicated no substantial change in p50 or p65 protein levels in the nuclear extracts of LNCaP
cells treated with R1881. Other Rel/NF-
B family members may be involved in the
DNA-binding response. Another explanation for androgen-induced increases in NF-
B
DNA-binding activity is that the proteins themselves are altered (e.g., by oxidative modulation of
sulfhydryl groups, phosphorylation status, etc.).
The ability of treatment with vitamins C plus E to diminish androgen-induced AP-1 DNA-binding activity suggests that oxidative stress contributes to the regulation of this activity. Vitamins C plus E blocked the rise in DCF fluorescence induced in LNCaP cells by R1881. DCF fluorescence decreased after 24 hours, but this decrease occurred only in cells treated with the highest doses of antioxidants (100 or 500 µM). The antioxidant effect on DCF was greater at 96 hours. However, vitamins C plus E decreased DNA binding to the AP-1 site in both the vehicle control-treated and the R1881-treated cells most effectively after 24 hours. Only the 500-µM antioxidant treatment substantially decreased androgen-induced DNA binding to the AP-1 site at 96 hours after treatment. The differential regulation of AP-1 by specific types of ROS may explain these results. Hydrogen peroxide and superoxide radical are known to elevate AP-1 (36,38). DCF fluorescence, which does not detect superoxide radicals, increases after 36-48 hours of treatment with 1 nM R1881 (12), while AP-1 DNA-binding activity is elevated after 5 hours of treatment. Hydrogen peroxide, the product of superoxide radical dismutation by superoxide dismutase enzymes, may rise in response to an earlier elevation in superoxide radical levels.
Androgen-induced NF-B activity is elevated much later than AP-1 DNA-binding
activity in LNCaP cells. Unlike AP-1, NF-
B is not sensitive to superoxide radical and is
most potently induced by hydrogen peroxide (18,35,38). In accordance
with our DCF data, NF-
B DNA-binding activity is elevated after 36 hours of androgen
treatment. NF
B DNA-binding activity is decreased only in cells treated for 96 hours with
the highest dose of antioxidants, suggesting that the ROS involved in androgen-induced AP-1
and NF-
B regulation are not identical.
Androgen-induced NF-B and AP-1 DNA-binding activity and DCF fluorescence
remain elevated for prolonged periods. Other antioxidants (N-acetyl cysteine, sodium
selenite, and
-tocopherol acetate) were less effective at reducing DCF fluorescence or
altering DNA-binding activity (data not shown). The DCF assay may be a useful tool for
optimizing antioxidant combinations that regulate androgen-induced redox-sensitive
transcription factor activity. Some of the downstream events following androgen exposure are
likely related to changes in the activities of redox-sensitive transcription factors. Defining the
pathways involving androgens, oxidative stress, and prostate carcinogenesis and determining the
capacity for antioxidants to favorably intervene appear to be topics worthy of further pursuit.
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NOTES |
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Supported by the CaPCURE Foundation (i.e., Foundation for the Cure of Prostate Cancer) and Department of Defense grant DAMD17-98-1-8505 (G. Wilding) and by Public Health Service grant T32AG00213 (R.Weindruch) from the National Institute on Aging, National Institutes of Health, Department of Health and Human Services.
We thank Dawn Church and Chuma Odenigwe for their assistance with immunoanalyses and the electrophoretic mobility-shift assay. We also thank Dr. Terry Oberley, Dr. John Ramsey, and Dr. Shigeki Miyamoto for their helpful comments regarding this manuscript.
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Manuscript received September 22, 1998; revised May 20, 1999; accepted May 26, 1999.
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