From the Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas, Austin, Texas 78712
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ABSTRACT |
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Acrolein is a highly electrophilic
Acrolein, an Extensive research has been done on the acute biochemical effects of
acrolein. However, the effects of subacute exposures have been little
studied, particularly at the molecular level. Recent work has shown
that acrolein can inhibit cell proliferation at doses that do not cause
lethality (3), and such information may have major significance in
terms of signal transduction pathways as well as, perhaps, in the
control of cell division and apoptosis. As a metabolite of
cyclophosphamide, acrolein may also play a role in the unique
antineoplastic efficacy of this drug through molecular effects
associated with low acrolein doses.
Myriad adverse cellular effects are seen following exposure to
acrolein, including growth inhibition, alterations in the levels of
glutathione (GSH), protein sulfhydryls, and thiol-containing enzymes,
and increased cell membrane permeability (4-8). The primary source of
acrolein's reactivity is its Some researchers have suggested that acrolein's antiproliferative
effects may be the result of its binding to RNA polymerase, thereby
serving as a transcriptional restraint (14). However, the fact that GSH
appears to play some role in cell division (15, 16) raises the
possibility that acrolein-mediated alterations in this tripeptide may
also be an important factor. Our previous data (3, 17) demonstrated
that inhibiting the proliferation of human lung adenocarcinoma A549
cells with acrolein correlated with acrolein-induced changes in GSH.
Although a cause-and-effect relationship between acrolein-induced
changes in GSH and proliferation has not been shown, it is apparent
that acrolein can alter redox-regulated cellular pathways. Nuclear
factor- NF- NF- Materials--
Dulbecco's modified Eagle's medium (DMEM),
acrolein (90%; water and dimers make up the other 10%), diethyl
maleate (DEM), and o-phthalaldehyde were obtained from
Sigma. Fetal bovine serum was purchased from Summit Biotechnology (Fort
Collins, CO). [3H]Thymidine (55 Ci/mmol) was obtained
from ICN (Costa Mesa, CA). All antibodies were secured from Santa Cruz
Biotechnology (Santa Cruz, CA) or New England Biolabs (Beverly, MA).
The double-stranded NF- Cell Culture--
A549 human lung adenocarcinoma cells, obtained
originally from the American Type Culture Collection (Manassas, VA),
were cultured in DMEM (pH 7.4) supplemented with 10% (v/v) fetal
bovine serum, 3.7 g/liter sodium bicarbonate, and 100 mg/liter
gentamicin. Cells were maintained at 37 °C with 5% CO2.
Cultures were passaged at confluency (approximately every 3 days) and
were removed from monolayer stock cultures with trypsin-EDTA. Cells
were counted with a T-890 Coulter counter (Miami, FL) and plated in
either Falcon 6-well dishes (9.6 cm2/well) or Corning 10-cm
tissue culture plates (55 cm2) with a medium volume of 2 ml/well and 10 ml/plate, respectively.
Cell Proliferation--
Changes in cell growth were
monitored by the uptake of [3H]thymidine. Cells were
seeded 48 h before treatment. DEM was dissolved in 100% ethanol
and added to culture dishes at an amount equivalent to 0.1% (v/v) of
the medium. For treatment with acrolein, cells were washed twice in one
volume/wash of Earl's balanced salt solution (EBSS). Cells were then
incubated for 30 min at 37 °C with 5% CO2 in sterile
EBSS containing the desired dose of acrolein. Incubation in EBSS was
essential because of the reactivity of acrolein with components of DMEM
(27). Following treatment, the cells were replenished with fresh DMEM + 10% fetal bovine serum. Additional washes to remove acrolein were not
incorporated because any residual acrolein would rapidly react with
nucleophiles present in the complete medium.
The uptake of exogenous 3H-labeled thymidine was measured
in cells treated with acrolein, DEM, or vehicle. Cells were pulsed for
2 h with 2.5 µCi/ml [3H]thymidine before isolating
the DNA (28). DNA was quantitated by fluorescence after treatment with
ethidium bromide (29).
Cell Counts--
Cell counts at the time of treatment (48 h
post-seeding) were obtained using the CyQuant cell proliferation assay.
This assay has a linear detection range of 50-50,000 cells/200 µl
and is dependent on a green dye (CyQuant-GR) that fluoresces when bound to cellular nucleic acids. Cell monolayers were washed twice with phosphate-buffered saline, trypsinized, suspended in phosphate-buffered saline, and pelleted at 200 × g. The supernatant was
carefully removed and the cells frozen at Total Glutathione Measurement--
Previous work in our
laboratory showed that acrolein treatment does not significantly alter
the level of glutathione disulfide (17). Therefore, only total
glutathione (GSH + glutathione disulfide) was measured by HPLC (30) or
enzymatically (31). Briefly, cells were seeded at 5000 cells/cm2 in six-well plates and treated with acrolein or
DEM. Cell monolayers were washed twice with PBS and lysed with 1 ml of
20 mM EDTA followed by sonication for 1 min. For HPLC
analyses, 250 µl of the lysate were combined with 83 µl of 25 mM NaH2PO4, pH 7.0. Samples were then processed, derivatized with o-phthalaldehyde, and
analyzed as described previously (17). For the enzymatic assay, 100 µl of cell lysate were combined with 600 µl of 0.2 M
KH2PO4 and 5 mM EDTA (pH 7.4) and
analyzed. Total protein in the lysates was determined (32) and compared
with a bovine serum albumin standard curve.
Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays were carried out after the method of Denison
et al. (33) as modified by Bowes et al. (34).
Briefly, cells were rinsed twice and lysed in ice-cold HEGD (25 mM HEPES, pH 7.6, 1.5 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.1 mg/ml phenylmethylsulfonyl fluoride,
0.75 mM spermidine, 0.15 mM spermine) by
homogenization. The homogenate was centrifuged at 12,000 × g for 10 min at 4 °C. Experiments examining the
activation of cytosolic latent NF-
To determine NF- NF-
Conditions for transfection of A549 cells were optimized using FuGENE
transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN).
2 µg of DNA were used for each transfection. 24 h
post-transfection, the cells were washed with EBSS and treated with
acrolein or DEM for 30 min as described previously. After treatment,
cells were washed with EBSS, and fresh DMEM medium with fetal bovine
serum containing 100 ng/ml TPA was added. 100 µl of media were
collected after 2 h for the alkaline phosphatase assay and stored
at Western Analyses--
Monolayer cells (106) were
lysed in 300 µl of lysis buffer (10 mM Tris-HCl (pH 7.4),
10 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 0.1% (v/v) Nonidet P-40, 100 µg/ml
phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin, and 1 mM sodium orthovanadate). The lysate was collected and
incubated on ice for 15 min. Samples were centrifuged at 16,000 × g for 10 min, and the supernatant was collected, assayed for
protein (36), and stored at Statistics--
Data are expressed as means ± S.E.
Comparisons between groups were done with a one-way analysis of
variance followed by the Student-Newman-Keul's test. A p
value of less than 0.05 was considered significant.
Acrolein and DEM Reduce Proliferation in A549 Cells--
Six-well
plates were seeded at 5000 cells/cm2 (48,000 cells/well)
and incubated 48 h before treatment for 30 min with 45 fmol of
acrolein/cell (6.7 µM) in EBSS or for 1 h with 6.7 pmol of DEM/cell (1 mM). DNA synthesis was reduced to 30 and 63% of vehicle-treated cells 2 h after acrolein or DEM
exposure, respectively (Table I). DNA
synthesis in acrolein-treated cells recovered to supranormal levels by
8 h post-treatment, whereas growth in DEM-treated cells remained
significantly suppressed, reaching only 54% of the level of growth in
vehicle-treated cells (64% of control cells) at 24 h.
Acrolein and DEM Decrease Cellular Glutathione--
Under slightly
different conditions, we have shown previously that acrolein rapidly
decreases total cellular GSH (3). In the current study, cells were
treated with 45 fmol of acrolein/cell or 6.7 pmol of DEM/cell. The
level of GSH in acrolein-treated cells declined to 13% of that in
vehicle-treated cells immediately following treatment and recovered to
normal or supranormal levels by 8 h post-treatment (Fig.
1). DEM-treated cells showed a smaller decline in GSH to 63% of the level in vehicle-treated cells with recovery again occurring by 8 h post-treatment (Fig. 1).
Acrolein and DEM Attenuate NF-
A time-response study of NF-
DEM also caused a decrease in NF-
The SEAP reporter assay confirmed that both acrolein and DEM diminished
the transcriptional activity of NF- Role of I
A more thorough analysis of the possibility of I Cytosolic versus Nuclear Effects--
To determine whether
acrolein blocked NF-
Analysis of the inhibition of activation of NF- The data presented here provide several avenues of support for the
hypothesis that both acrolein and DEM inhibit NF- Acrolein-mediated modifications of NF- The translocated NF- Most if not all agents activating NF- In the same way that acrolein affects NF- The key features of NF- In the current study, treating cells with 45 fmol of acrolein/cell for
30 min caused an 85% decline in DNA synthesis, which corresponded to a
55% decrease in NF- Results from DEM studies support the role of NF- In conclusion, acrolein causes a dramatic decline in NF-,
-unsaturated aldehyde to which humans are exposed in various
situations. In the present study, the effects of sublethal doses of
acrolein on nuclear factor
B (NF-
B) activation in A549 human lung
adenocarcinoma cells were investigated. Immediately following a 30-min
exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA
synthesis and NF-
B binding were reduced by more than 80%. All
parameters returned to normal or supranormal levels by 8 h
post-treatment. Pretreatment with acrolein completely blocked
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced
activation of NF-
B. Cells treated for 1 h with 1 mM
diethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA
synthesis, respectively. DEM also reduced NF-
B activation by 64% at
2 h post-treatment, with recovery to within 22% of control at
8 h. Both acrolein and DEM decreased NF-
B function ~50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h
after DEM treatment, but proliferation remained significantly depressed
for 24 h. Interestingly, DEM caused a profound decrease in NF-
B
binding, even at doses as low as 0.125 mM that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of
phosphorylated or nonphosphorylated inhibitor
B-
(I
B-
). Furthermore, acrolein decreased NF-
B activation in cells depleted of
I
B-
by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-
B activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-
B in
the cytosol prior to chemical dissociation from I
B with detergent. Together, these data support the conclusion that the inhibition of
NF-
B activation by acrolein and DEM is I
B-independent. The mechanism appears to be related to direct modification of thiol groups
in the NF-
B subunits.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated aldehyde, is a highly
electrophilic (1), volatile liquid with a pungent and irritating odor. It is produced by a wide variety of both natural and synthetic processes including incomplete combustion or pyrolysis of organic materials such as fuels, wood, synthetic polymers, food, and tobacco. In addition, patients treated with the cytostatic agent
cyclophosphamide are exposed to acrolein as a metabolite of the parent
drug (2).
,
-unsaturated carbon-carbon bond.
This molecule will react via a Michael addition in the presence of a
nucleophile to form an alkylated adduct. Acrolein's potential role as
a carcinogen is based on the observation that it binds GSH (9) and
nuclear chromatin (10) and can form a number of adducts with DNA
(11-13).
B (NF-
B)1 is one
of the most widely studied molecules affected by cellular redox status.
It was first identified as a factor that activated the Ig
-light
chain intron enhancer during B-lymphocyte development (18). High levels
of interest in this transcription factor are based on its broad role in
coordinately controlling a number of genes including those encoding
inflammatory cytokines, chemokines, interferons, proteins of the major
histocompatibility complex, growth factors, cell adhesion molecules,
and viruses (19).
B, which comprises a 50- and 65-kDa heterodimer complex, is the
prototype of a family of dimeric transcription factors consisting of
monomers that have approximately 300-amino acid Rel regions that bind
to DNA and interact with each other (20). These factors are normally
bound to a member of a family of inhibitory proteins known as inhibitor
B (I
B). The inhibitors all have 5-7 ankyrin repeat domains, each
with approximately 30 amino acids, that form a unit able to interact
with Rel regions. I
B-
, the best characterized member of this
family, binds the p50/p65 heterodimer of NF-
B and retains it in the
cytoplasm. The exposure of cells to NF-
B activators, including
oxidants, cytokines (such as tumor necrosis factor-
or
interleukin-1), or the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA), causes
phosphorylation of two serine residues of I
B-
(Ser32
and Ser36). This phosphorylation is the signal for
ubiquitination and degradation of I
B-
by the 26 S proteasome.
NF-
B is then released and translocated to the nucleus where it can
bind to
B sites on the DNA, thereby activating transcription of
target genes (21, 22).
B is thought to be under redox control at two distinct levels.
The activation and nuclear translocation of NF-
B involve reactive
oxygen intermediates and can be blocked by reducing agents such as
N-acetylcysteine and GSH (23, 24). In contrast, the DNA-binding activity of NF-
B is inhibited by oxidative agents and
potentiated by reducing thiols (25, 26). These are likely the results
of the requirement that cysteine residues present in the DNA-binding
domain of all members of the Rel protein family be reduced to bind DNA
(26). Acrolein's reactivity with nucleophiles suggests that it may
interfere with NF-
B binding either by altering the redox balance of
the nucleus or by forming adducts with NF-
B. In this study, we
describe acrolein's attenuation of NF-
B activation in A549 human
lung adenocarcinoma cells in a manner independent of I
B-
and
consistent with the formation of acrolein-NF-
B conjugates.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B consensus oligonucleotide was purchased
from Promega Corp. (Madison, WI). Phosphorylated and nonphosphorylated
I
B control cell extracts were obtained from New England Biolabs.
80 °C. At the time of
the assay, cells were thawed at room temperature and lysed in buffer
containing the CyQuant-GR dye prepared according to manufacturer's
instructions. Fluorescence was measured (excitation, 480 nm; emission,
520 nm) and compared with a standard curve for cell number determination.
B by detergents used the
12,000 × g supernatant fraction. Cytosol (4 µg of
protein) was pretreated with 0.8% (w/v) sodium deoxycholate and 1.1%
Nonidet P-40 for 10 min on ice before incubation with the labeled
oligonucleotide probe (35). NF-
B was assessed using the 12,000 × g pellet extracted with 20 µl of HEGDK (HEGD + 0.5 M KCl) for 1 h on ice. Extracted pellets were
centrifuged at 16,000 × g for 10 min at 4 °C, and
the supernatant containing the nuclear extracts was collected and
assayed for protein content (32). Extracts were frozen using liquid
nitrogen and stored at
80 °C until analyzed. 5-20 µg of
extracted protein were incubated in a reaction mixture consisting of
18.8 mM HEPES, 1.1 mM EDTA, 7.5% glycerol,
0.75 mM dithiothreitol, and 62.5 ng/ml poly(dI-dC) for 15 min at room temperature to reduce interference by nonspecific
DNA-binding proteins.
B binding activity, 0.1 ng of NF-
B labeled with
[
-32P]ATP (3000-5000 Ci/mmol; NEN Life Science
Products, Boston, MA) was added to the nuclear or cytosolic extracts
for 15 min. The specificity of the binding reaction was assessed using
unlabeled NF-
B, which competitively eliminated the induced band, or
with an excess of a non-NF-
B competitor oligonucleotide, which was without effect. Bound NF-
B was separated from the free probe on a
4% polyacrylamide nondenaturing gel for 2 h at 120 V. Gels were
dried under vacuum and exposed to Kodak XAR-5 film (Sigma) for 1-4 h
at
80 °C with intensifying screens. Gels were also evaluated with
a Packard Instant Imager and Packard imaging software (version 2.02, Packard Instrument Co.).
B Reporter Assay--
A549 cells were transfected with the
pNF-
B secreted alkaline phosphatase (SEAP) vector
(CLONTECH Laboratories, Palo Alto, CA). Induction
of the NF-
B pathway enables it to bind to the
enhancer element
located in the promoter region of the vector, thus activating
transcription of the reporter gene and leading to increases in alkaline
phosphatase activity in the culture medium. The alkaline phosphatase
assay was done using the Great EscAPe SEAP fluorescence detection kit
(CLONTECH) per the manufacturer's instructions.
20 °C.
20 °C. Thawed supernatants were mixed
1:3 with loading dye (4% (w/v) SDS, 20% (w/v) glycerol, 4% (w/v)
-mercaptoethanol, 0.2 M Tris-HCl (pH 6.8), and 0.02% (w/v) bromphenol blue) and separated on SDS-polyacrylamide gels (8-15%). Protein was transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and blocked from 1 h to
overnight in 5% (w/v) nonfat dry milk (Bio-Rad) in TBS-T (25 mM Tris-HCl (pH 7.6), 0.2 M NaCl, and 0.15%
Tween 20 (v/v)). Membranes were incubated with a polyclonal antibody
specific for the protein of interest (1:1500 dilution in TBS-T) for
1 h. After washing in TBS-T, the membranes were rinsed and
incubated with a horseradish peroxidase-conjugated secondary antibody
(1:3000 dilution in TBS-T; Amersham Pharmacia Biotech) for 1 h.
After the secondary antibody incubation, the membranes were rinsed with
TBS-T, and bound antibodies were detected using enhanced
chemiluminescence (ECL) with a kit from Amersham Pharmacia Biotech.
Developed film was scanned, and individual band densities were
integrated using NIH Image public domain software. Immunoblots
following the various treatments were run a minimum of two times.
Representative blots are shown in the figures.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
[3H]Thymidine incorporation
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Fig. 1.
Glutathione levels after treatment with
acrolein or DEM. Cells were seeded at 5000 cells/cm2
in six-well plates and incubated for 48 h at 37 °C in 5%
CO2 before treatment with 45 fmol of acrolein/cell (30 min)
or 1 mM DEM (1 h). Total glutathione was determined at the
indicated times as measured from the start of treatment. Data are
expressed as the mean percentage of total glutathione relative to
vehicle-treated cells ± S.E. (n = 5 for acrolein,
n = 2 for DEM).
B Activation--
Treating A549
cells with 35 fmol of acrolein/cell caused a significant decrease in
NF-
B activation relative to TPA-treated or serum-deprived controls
after as little as a 5-min exposure. This binding inhibition increased
with the time of exposure. However, NF-
B activation in
serum-deprived, vehicle-treated cells also began to decline at 2 h
post-treatment (Fig. 2). To minimize the effects of serum deprivation, 30-min acrolein treatments were selected
for further studies. With this length of treatment, both constitutive
and TPA-stimulated NF-
B activation were inhibited by acrolein (Fig.
2). Nonspecific binding was evident in this gel but did not correlate
with either time or acrolein treatment.
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Fig. 2.
NF- B binding after
serum deprivation or treatment with acrolein. Cells were seeded at
10,000 cells/cm2 in 10-cm plates and incubated for 48 h at 37 °C in 5% CO2 before treatment. Nuclear
fractions were harvested 4 h after beginning the treatment. 10 µg of total protein were loaded per lane. Lane 1, 30 min
in EBSS followed by 3.5 h with 100 ng/ml TPA in medium + serum.
Lane 2, 30 min in EBSS with 35 fmol of acrolein/cell
followed by 3.5 h with 100 ng/ml TPA in medium + serum. Lane
3, control untreated cells, 4 h in medium + serum. Lane
4, 5 min in EBSS followed by 4 h in medium + serum.
Lane 5, 5 min in EBSS with 35 fmol of acrolein/cell followed
by 4 h in medium + serum. Lane 6, 30 min in EBSS
followed by 3.5 h in medium + serum. Lane 7, 30 min in
EBSS with 35 fmol of acrolein/cell followed by 3.5 h in medium + serum. Lane 8, 2 h in EBSS followed by 2 h in
medium + serum. Lane 9, 2 h in EBSS with 35 fmol of
acrolein/cell followed by 2 h in medium + serum.
B activation in which cells were treated
with 45 fmol of acrolein/cell for 30 min (Fig.
3) showed inhibition and recovery
patterns very much like those seen when examining changes in DNA
synthesis (Table I) and total GSH (Fig. 1). Acrolein caused a dramatic
decline in NF-
B binding at 30 min and at 2 h post-treatment.
Some recovery of NF-
B activation was evident at 4 h, and the
inhibitory effect of 30 min of acrolein treatment was fully reversed by
8 h post-treatment (Fig. 3).
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Fig. 3.
Time course of NF- B
binding after treatment with acrolein. Cells were seeded at 10,000 cells/cm2 in 10-cm plates and incubated for 48 h at
37 °C in 5% CO2 before treatment. Nuclear fractions
were harvested at the indicated times as measured from the beginning of
treatment. 5 µg of total protein were loaded per lane. C,
control;
, vehicle (EBSS)-treated cells; +, acrolein-treated cells
(45 fmol/cell for 30 min).
B activation. There was a clear
dose-response relationship with NF-
B binding increasingly reduced
after 1-h exposures to DEM doses from 0.125 to 2 mM (Fig. 4). Interestingly, the inhibition seen in
NF-
B activation with 0.125 mM DEM was profound, yet
little or no change in total GSH was evident at this dose (data not
shown). Treating cells with 3.33 pmol of DEM/cell (1 mM)
for 1 h resulted in a NF-
B activation time response (Fig.
5) that was almost identical to that
obtained following acrolein exposure (Fig. 3), and again paralleled
changes in GSH (Fig. 1). NF-
B binding decreased dramatically at 1-2
h post-treatment and showed a recovery to near normal binding by 8-12
h. Nonspecific binding was more intense in this gel, but again did not
correlate with either time or DEM treatment.
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Fig. 4.
NF- B binding after
treatment with DEM. Cells were seeded at 10,000 cells/cm2 in 10-cm plates and incubated for 48 h at
37 °C in 5% CO2 before treatment. Cells were treated
with 2, 1, 0.5, 0.25, or 0.125 mM (left to
right) DEM (1 h). Nuclear fractions were harvested 4 h
after beginning the treatment. 10 µg of total protein were loaded per
lane. C, control; V, vehicle (0.1% (v/v)
ethanol-treated cells).
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Fig. 5.
Time course of NF- B
binding after treatment with DEM. Cells were seeded at 10,000 cells/cm2 in 10-cm plates and incubated for 48 h at
37 °C in 5% CO2 before treatment. Nuclear fractions
were harvested at the indicated times as measured from the beginning of
treatment. 10 µg of total protein were loaded per lane. C,
control;
, vehicle (0.1% (v/v) ethanol)-treated cells; +,
DEM-treated cells (1 mM for 1 h).
B. Two h after adding TPA to
cells pretreated for 30 min with either 45 fmol/cell acrolein or 1 mM DEM, SEAP activity was decreased by 51 and 45%, respectively.
B in Reduced NF-
B Binding--
Changes in NF-
B
activation are generally controlled by I
B. In a number of different
experiments, there were no consistent changes in the levels of
I
B-
up to 2 h after cells were treated for 30 min with 45 fmol of acrolein/cell or for 1 h with 3.33 pmol of DEM/cell (Fig.
6, A and B). To
further examine this phenomenon, changes in the level of phosphorylated
I
B-
were examined following treatment with acrolein. Once again,
no changes in the levels of this protein were observed (Fig.
6C), suggesting that acrolein blocks NF-
B activation by
an I
B-independent mechanism.
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Fig. 6.
I B-
levels are shown after treatment
with acrolein (A, B) or DEM (A), and
phosphorylated I
B-
is shown in panel C. Cells
were seeded at 10,000 cells/cm2 in 10-cm plates and
incubated for 48 h at 37 °C in 5% CO2 before
treatment. Western analyses were performed at the indicated times as
measured from the beginning of treatment (20 µg of protein/lane).
Panels A and B, C, control;
,
vehicle (EBSS or 0.1% ethanol)-treated cells; +A,
acrolein-treated cells (45 fmol/cell for 30 min); +D,
DEM-treated cells (1 mM for 1 h). Panel C, lane
1, untreated control; lane 2, 30 min in EBSS;
lane 3, 30 min in acrolein; lane 4, 1 h in
acrolein; lane 5, 2 h in acrolein; lane 6,
4 h in acrolein; lane 7, 4 h in EBSS; lane
8, 4 h untreated control; lane 9, positive
phosphorylated I
B control.
B-independent
changes in NF-
B activation involved examining the degradation of
I
B following stimulation with TPA, a tumor promoter that
up-regulates protein kinase C. Treatment with 100 ng/ml TPA caused a
temporary decrease in the levels of I
B (Fig.
7A). By stimulating cells with
TPA in the presence of the protein synthesis inhibitor cycloheximide (CHX), I
B levels were almost completely abrogated at 2 h
post-treatment (Fig. 7B). NF-
B activation was also
checked at this time to ensure that the treatment with CHX had not
affected NF-
B binding. Stimulating cells with TPA in the presence of
CHX resulted in maximum NF-
B activation at 2 h post-treatment
(Fig. 8), the same time that I
B levels
were at their nadir. Finally, cells that had been stimulated with TPA
for 1.5 h in the presence of CHX were treated with 45 fmol of
acrolein/cell for 30 min. Under these conditions, acrolein still caused
a 63% decline in NF-
B activation (Fig.
9), indicating that the effect of
acrolein was independent of I
B.
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Fig. 7.
I B-
levels
after treatment with TPA (A) and TPA + CHX
(B). Cells were seeded at 10,000 cells/cm2 in 10-cm plates and incubated for 48 h at
37 °C in 5% CO2 before treatment. Cells were treated
with 100 ng/ml TPA in the absence (A) or presence
(B) of 100 µg/ml CHX. Western analyses were performed at
the indicated times as measured from the beginning of treatment. 20 µg of protein/lane. C, control; V, vehicle
(0.1% (v/v) ethanol)-treated cells; S, phosphorylated I
B
standard.
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Fig. 8.
NF- B binding in the
presence of TPA and CHX. Cells were seeded at 10,000 cells/cm2 in 10-cm plates and incubated for 48 h at
37 °C in 5% CO2 before treatment. Cells were treated
with 100 ng/ml TPA in the presence of 100 µg/ml CHX (2 h). Nuclear
fractions were harvested at the indicated times as measured from the
beginning of treatment. 10 µg of total protein were loaded per lane.
C, control.
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Fig. 9.
NF- B binding in the
presence of TPA and CHX after treatment with acrolein. Cells were
seeded at 10,000 cells/cm2 in 10-cm plates and incubated
for 48 h at 37 °C in 5% CO2 before treatment.
Cells were treated with 100 ng/ml TPA in the presence of 100 µg/ml
CHX (1.5 h). Cells were then treated with 45 fmol of acrolein/cell or
vehicle (EBSS) for 30 min in the presence of CHX. Nuclear fractions
were harvested 2 h after the beginning of treatment. 10 µg of
total protein were loaded per lane.
B activation by acting in the cytoplasm or in
the nucleus, cytosolic extracts were obtained from cells pretreated for
30 min with 45 fmol/cell acrolein or vehicle. Treatment of vehicle
extracts with deoxycholate dissociated NF-
B from I
B, yielding
binding to the consensus sequence (Fig. 10, lane 4). In contrast,
extracts from acrolein-treated cells exhibited greatly diminished
binding (Fig. 10, lane 5), suggesting that cytosolic binding
occurs. Acrolein may also bind to NF-
B in the nucleus as shown by an
experiment in which cells were first treated with TPA to stimulate the
nuclear translocation of NF-
B followed by treatment with either
acrolein or DEM. This experiment revealed that both acrolein and DEM
decreased NF-
B activation under these conditions (data not shown),
suggesting that both can act within the nucleus.
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Fig. 10.
Binding of cytosolic
NF- B after latent activation with
detergent. Cells were seeded at 10,000 cells/cm2 in
10-cm plates and incubated for 48 h at 37 °C in 5%
CO2 before treatment. Cells were treated with 45 fmol of
acrolein/cell or vehicle (EBSS) for 30 min. Cytosol was then obtained
and treated with sodium deoxycholate and Nonidet P-40. 4 µg of total
protein were loaded per lane. Lane 1, empty lane; lane
2, vehicle; lane 3, acrolein; lane 4,
vehicle + detergent; lane 5, acrolein + detergent.
B was studied further
by treating the nuclear extracts of acrolein- and DEM-treated cells with either 100 µM GSH or 1%
-mercaptoethanol
(
-ME) in vitro to determine whether the
inhibitory effects were reversible. These data show that
neither GSH nor
-ME restores NF-
B binding in cells
treated with acrolein or DEM (Fig.
11). Pretreatment with TPA before
exposing the cells to acrolein or DEM had no effect on the
ability of GSH or
-ME to restore binding. Interestingly, the nuclear extracts from untreated control cells showed a
24% increase in NF-
B binding following the addition of
-ME but not GSH.
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Fig. 11.
Cells were seeded at 10,000 cells/cm2 in 10-cm plates and incubated for 48 h at
37 °C in 5% CO2 before treatment. Cells were
treated with 45 fmol of acrolein/cell (30 min) or 1 mM DEM
(1 h). Some cells were pretreated with 100 ng/ml TPA before incubation
with acrolein or DEM. Nuclear fractions were harvested 2 h after
the beginning of treatment. GSH or -mercaptoethanol was added to the
nuclear extracts in vitro to obtain 100 µM or
1% final concentration, respectively. 10 µg of total protein were
loaded per lane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation by an
I
B-independent mechanism. Most importantly, acrolein and DEM block
NF-
B binding without altering the cellular levels of either the
phosphorylated or nonphosphorylated forms of I
B-
. Acrolein and
DEM also block binding in the absence of I
B making it clear that
they must be interfering with the activation of NF-
B by some
mechanism other than modulation of I
B. Two possible mechanisms
exist: the direct inactivation of NF-
B; or the scavenging of nuclear
reducing equivalents required for NF-
B binding, such as GSH, thereby
lowering its binding affinity for DNA. This latter mechanism is
unlikely, however, because the effect of DEM occurred at doses that did
not alter GSH. Further, in cells treated with acrolein or DEM alone, or
when treatment followed TPA stimulation, NF-
B activation could not
be restored by the addition of either GSH or
-ME. An interesting
observation was that
-ME increased NF-
B binding in untreated
control cells. The nuclear redox status could be such that some
molecules of NF-
B are not in the reduced form necessary to
facilitate DNA binding. The addition of a strong reducing agent
in vitro may have forced the reduction of this pool of
NF-
B, resulting in the observed increase in binding.
B can occur in the cytosol of
intact cells as shown by the inability of detergents to release an
active form following acrolein treatment. This finding also indicates
that the site of modification is not blocked by I
B, matching
previous data from studies with other thiol reactive agents (37, 38).
NF-
B-like transcription factors belong to the Rel protein family and
have a highly conserved N-terminal region that is critical for DNA
binding. Within this region, the Cys62 residue on the p50
subunit and the Cys38 residue on the p65 subunit are
essential for DNA binding (26, 39, 40). Thiols present in these regions
may form conjugates with acrolein via a Michael addition. A mechanism
of this type has been described previously when various compounds
directly reacted with the p50 subunit to inhibit NF-
B binding (38). In a similar way, acrolein inhibits the DNA repair enzyme
O6-alkylguaninine-DNA alkyltransferase by acting
at an acrolein-sensitive thiol residue required for the catalytic
activity of the enzyme (41).
B must have a reduced environment if it is to
bind DNA (26, 37, 42). Therefore, in addition to forming conjugates
with NF-
B, acrolein may inhibit binding by depleting nuclear GSH. In
support of this mechanism, we observed a strong correlation between the
time necessary to recover intracellular GSH and the time it takes for
NF-
B binding to return to normal following an acrolein insult.
However, this seems unlikely to be the sole mechanism based on the
inability of deoxycholate to activate NF-
B after acrolein.
B tend to trigger the formation
of reactive oxygen species or are oxidants by themselves (43). Despite
the increased NF-
B activation seen in the presence of oxidants,
studies in two human T-cell lines have shown that a reduction in GSH by
buthionine sulfoximine inhibits the activation and translocation of
NF-
B (25, 44). It was also found that the addition of cysteine
caused a reversible oxidation of NF-
B (25), presumably by raising
intracellular glutathione disulfide levels. These data may point to the
existence of a critical intracellular redox balance. Increasing this
reactive oxygen species pool in the cytosol appears to activate
NF-
B, whereas increasing it in the nucleus blocks NF-
B binding,
which may enhance apoptosis (45, 46). In general, acrolein appears to
affect NF-
B binding directly by conjugating its protein subunits and
indirectly by alkylating GSH, thereby altering the nuclear redox balance.
B, our data suggest that
DEM decreases activation both directly and indirectly. The fact that
DEM blocks NF-
B binding in a manner not reversible with either GSH
or
-ME indicates that it has direct interactions with the NF-
B
subunits. Furthermore, because DEM is known to conjugate GSH through
the actions of glutathione S-transferase, it may act on
NF-
B indirectly by lowering the nuclear levels of GSH.
B transcriptional control are that it is
fast, versatile, and involved in many different gene systems (47). Of
particular interest to the current study is the involvement of NF-
B
with genes that regulate cell proliferation. The importance of NF-
B
subunits in cell proliferation is suggested by several studies. Snapper
et al. (48) showed that B cells from p50
/
knockout mice proliferated normally in response to some stimuli but
showed no response to other stimuli that were mitogenic in control
cells. Mice lacking the p50/p105 subunits developed normally but
exhibited defects in immune responses involving B cells (49). A more
critical role seems to belong to the p65 subunit. Vascular smooth
muscle cells treated with p65 antisense oligonucleotides showed a
concentration-dependent inhibition of both adherence and
proliferation (50). Even more dramatic is the report that p65 (Rel A)
null mice exhibited a dramatic phenotype-embryonic lethality,
apparently because of widespread apoptosis in the liver (51). This
function in the regulation of cell growth may be mediated through
c-myc, which is known to have two NF-
B sites in its
promoter/enhancer region (52). Furthermore, NF-
B is implicated in
the transcriptional regulation of the p53 gene (53).
B binding and an 87% loss of GSH. In all
instances, the measured parameters returned to normal levels or higher
by 8 h post-treatment. Although electrophoretic mobility shift
assay data are only semiquantitative, the correlation between
acrolein-mediated reductions in NF-
B activation and DNA synthesis
support a functional link. It may also be that acrolein inhibits growth
protein(s) in a manner similar to the proposed inhibition of NF-
B
activation, i.e. by conjugation with thiol residue(s)
critical to their function.
B in mediating
changes in DNA synthesis. Although DEM-treated cells posted a full
recovery of GSH by 8 h post-treatment, the 8-h levels of DNA
synthesis and NF-
B activation reached only 77 and 74% of the level
in vehicle-treated cells, respectively. However, NF-
B activation in
DEM-treated cells did return to normal by 12 h post-treatment, whereas DNA synthesis remained suppressed to 24 h.
B binding by
an I
B-independent mechanism, which most likely involves alkylation
of critical thiol sites within the DNA binding domain of the NF-
B
subunits. Identification of acrolein/DEM-p50 and/or acrolein/DEM-p65
conjugates will provide conclusive evidence of the mechanism by which
these chemicals alter NF-
B binding. It is apparent that GSH is
intimately involved with NF-
B activation and that both GSH and
NF-
B play a role in cell growth and apoptosis. The observed
attenuation of DNA synthesis following acrolein insult is probably the
downstream result of the effects of acrolein on GSH and NF-
B. In
addition, NF-
B is involved in regulating the expression of several
growth-related genes. As a result, the effects of acrolein on these
factors could have unrecognized toxic consequences, including a role in
the effectiveness of the anticancer drug cyclophosphamide.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grant HL48035 and by Center Grant ES07784.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. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ Supported by Grant 1 F32 ES05825 from NIEHS, National Institutes of Health. Current address: Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd., Ann Arbor, MI 48105.
¶ Gustavus and Louise Pfeiffer Professor of Toxicology. To whom correspondence should be addressed: Div. of Pharmacology and Toxicology, College of Pharmacy, University of Texas, Austin, TX 78712-1074. Tel.: 512-471-1107; Fax: 512-471-5002; E-mail: kehrerjim{at}mail.utexas.edu.
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ABBREVIATIONS |
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The abbreviations used are:
NF-B, nuclear
factor
B;
I
B, inhibitor
B;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
DMEM, Dulbecco's
modified Eagle's medium;
DEM, diethyl maleate;
EBSS, Earl's balanced
salt solution;
HPLC, high pressure liquid chromatography;
SEAP, secreted alkaline phosphatase;
CHX, cycloheximide;
-ME,
-mercaptoethanol.
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