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INTRODUCTION |
Pulmonary epithelial cell injury is an unfortunate consequence of
therapy with supraphysiological concentrations of oxygen (hyperoxia)
and a prominent feature of acute inflammatory lung injury (1, 2).
Oxidant-induced cell injury and death are generally thought to occur
via reactive oxygen species. Although exogenous hydrogen peroxide
(H2O2) and paraquat induce apoptosis in
cultured alveolar epithelial (A549) cells, hyperoxia kills these cells
via a mode of cell death that is distinct from apoptosis both
morphologically and biochemically (3-5). These observations raise the
possibility that hyperoxia actually inhibits apoptosis in pulmonary
epithelial cells.
There are several possible ways that hyperoxia might inhibit apoptosis
in pulmonary epithelial cells. First, one or more enzymes in the
apoptotic pathway might be sensitive to direct oxidation by high levels
of molecular O2, resulting in an irreversible abrogation of
the apoptotic pathway in these cells. Alternatively, hyperoxia may
activate pathways that inhibit apoptosis. Several laboratories have
shown that activation of the transcription factor NF-
B can prevent
apoptosis induced by chemotherapeutic agents and ionizing radiation in
cultured cells (6-9). In addition, activation of NF-
B may be
responsible for the protective effect of low-dose amyloid
in
neuronal cell death (10) and in the survival and function of
hematopoietic stem and progenitor cells (11). We have previously
demonstrated that hyperoxia activates NF-
B in A549 cells (5). In
this report, we examined whether preexposure of cultured epithelial
cells to hyperoxia prevents subsequent oxidant-induced apoptosis and
whether NF-
B activation or up-regulation of antioxidant enzymes
(AOEs)1 mediate this
inhibitory process.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human lung adenocarcinoma A549 cells (ATCC,
Manassas, VA) were grown in F12K medium supplemented with
10% fetal bovine serum, 1% glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (Life Technologies, Inc.) and maintained
at 37 °C in 5% CO2/95% room air. HeLa-20 and
HeLa-80 cells were grown in Ham's F-10 medium (Life Technologies,
Inc.), supplemented with 15% fetal bovine serum, 1% glutamine, 100 units/ml penicillin, 100 units/ml streptomycin and maintained in 2%
CO2/98% room air at 37 °C. In all experiments, cells
were seeded at 20-40% confluence on plastic culture dishes and were
allowed to adhere overnight prior to exposure. Cell cultures exposed
directly to H2O2 were seeded slightly lower
than those preexposed to hyperoxia, to reach cell densities similar to
those of the preexposed cultures at the time of
H2O2 exposure, because cell density affects the
sensitivity to the cytotoxic effects of H2O2
(data not shown). Hyperoxic conditions were achieved by growing cells
in 40, 60, or 95% O2/5% CO2 at 37 °C in
sealed, humidified chambers for up to 48 h. Some cell cultures
were treated with 500 units/ml (10 ng/ml) TNF-
(Pharmingen, San
Diego, CA) for 30 min, with 2-5 mM
H2O2 or 10 mM paraquat for up to
24 h. Media and oxidants were refreshed each day when cells were
cultured for more than 1 day. For this reason, and because the cultures were subconfluent, neither ATP nor glucose levels were depleted by hyperoxia.
Assays for Antioxidant Enzyme Activities--
AOE activities
were measured in A549, HeLa-20, and HeLa-80 cells as described
previously (12-14). Briefly, a competitive inhibition assay was used
to measure total superoxide dismutase activity spectrophotometrically through the inhibition of cytochrome
c/xanthine-xanthine oxidase oxidation. Inhibition of 50%
cytochrome c oxidation is defined as 1 unit of superoxide
dismutase activity at 550 nm (12). Catalase activity was assayed
spectrophotometrically by following the disappearance of
H2O2 at 240 nm (13). Glutathione peroxidase activity was determined through the glutathione disulfide formation spectrophotometrically following the absorbance decrease of
NADPH to NADP at 340 nm (14).
Western Blots--
Cell lysates were prepared according to the
procedures as described (2, 5). Proteins from each sample were loaded
onto 10% SDS-polyacrylamide gels (Bio-Rad). After electrophoresis, proteins were transferred to Immobilon-P membranes (Millipore, Bedford,
MA). For I
B
detection, blots were incubated with rabbit anti-I
B
antibodies as recommended by the supplier (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Antibody binding was detected by
the enhanced chemiluminescent reagent CDP-Star (Roche Molecular Biochemicals) and by exposing the filter to x-ray film.
Immunofluorescence--
Detection of the p65 subunit of NF-
B
was performed as described with minor modification (5). In brief,
formalin-fixed cells grown on coverslips were first incubated with
ice-cold 100% methanol at
20 °C and blocked with 1% bovine serum
albumin (Panvera, Madison, WI). Cells were then incubated with 2.5 µg/ml (in 1% w/v bovine serum albumin) anti-NF-
B p65 antibodies
(Santa Cruz Biotechnology Inc.) for 60 min and washed with three
changes of 1× Tris-buffered saline (0.1 M Tris-Cl, pH 7.4, 0.15 M NaCl). Sheep anti-rabbit IgG-fluorescein
isothiocyanate (Roche Molecular Biochemicals) was used as the secondary
antibody. Cells were counterstained with 2 µg/ml of
4',6-diamidine-2-phenylindole dihydrochloride (DAPI) (Roche Molecular
Biochemicals) to visualize the nuclei, and the results were examined by
immunofluorescence microscopy using the B-2 filter for fluorescein
isothiocyanate and the UV-2A filter for DAPI (Nikon Inc., Melville, NY).
Assays for Apoptosis--
The terminal transferase dUTP nick end
labeling (TUNEL) assay was performed with cells seeded on coverslips
and fixed in formalin as described (2, 3). The protocol utilized for
TUNEL staining was as described previously except that proteinase K
treatment was omitted. TUNEL reagents, including rhodamine-conjugated
anti-digoxigenin Fab fragment, were obtained from Roche Molecular
Biochemicals. Cells were counterstained with 2 µg/ml DAPI for 8 min
at room temperature. The TUNEL assay results were examined by
immunofluorescence microscopy using the G-2A filter for rhodamine and
the UV-2A filter for DAPI.
The Annexin V incorporation assay was performed with cells grown
in six-well dishes. After exposure to oxidants, cells were rinsed with
phosphate-buffered saline, trypsinized and combined with cells detached
during the oxidative exposure. Cells were pelleted (200 × g for 5 min), rinsed with ice-cold phosphate-buffered saline, and then incubated with Annexin V-fluorescein isothiocyanate according to the vendor's instructions (R&D systems, Minneapolis, MN).
The relative fluorescence intensity was measured with excitation at 488 nm and emission at 520 nm, and the cell size was determined using the
forward scatter method with a Becton Dickson flow cytometer. The
extent of apoptosis was assessed by fluorescence intensity or condensed
cell size using Cell Quest software (Becton Dickson, Franklin Lakes,
NJ). Cells with fluorescence intensity higher than 210 relative
fluorescence units or cell size smaller than 345 units were scored as
apoptotic. The extent of apoptosis between groups were analyzed using
Kolmogorov-Smirnov analysis using Cell Quest software, and
p < 0.05 is considered statistically significant (15).
Image Analysis--
To quantify the extent of apoptosis by TUNEL
assay, samples were illuminated with UV light to visualize either
TUNEL-positive nuclei (590 nm) or total, DAPI-stained nuclei (420 nm)
and analyzed by computer-aided analysis as described previously (2). To quantify the extent of nuclear condensation, nuclei were stained with
DAPI. The DAPI fluorescence was examined and images were captured with
a charge-coupling device video camera. Uniform camera control
settings were used for image capture, and image threshholding was
identical for all images. The captured images were analyzed using the
Metamorph system (Universal Imaging, West Chester, PA), running on a
personal computer. At least 25 fields were analyzed on each coverslip
from a minimum of two independent experiments for each group. The
percentage of apoptotic cells was calculated as the percentage of
TUNEL-positive apoptotic nuclei divided by the total number of nuclei
(detected by DAPI staining). The numbers of the nuclear sizes were
analyzed by Maple distribution (Maple math software). The data were
analyzed for statistical significance using the Student's t
test and analysis of variance, with p < 0.05 considered significant.
Transfection and Stable Cell Lines--
A549 cells were
transfected with 5 µg of I
Bdn-pUSEamp, a plasmid that carries a
dominant-negative construct of I
B with mutations at Ser-32 and
Ser-36 (Upstate Biotechnology, Lake Placid, NY), followed by selection
with 0.8 mg/ml G418 (Life Technologies, Inc.). At least three stable
cell lines that express less than 50% of NF-
B activity of those in
vector clones were established. At the same time, more than three
clones expressing the pUSEamp vector alone were also established. To
determine the levels of NF-
B activity in these cells, a reporter
gene expression assay was performed. Cells were transiently
cotransfected with 50 ng of pCMV-SPORT-
-galactosidase, 1 µg of
pBlue, and 50 ng of pNF-
B-luc, a reporter plasmid containing 5 NF-
B binding sites inserted upstream of the luciferase reporter
(Stratagene, La Jolla, CA). Twenty-four hours after transfection, cells
were assayed for luciferase activity, which was normalized on the basis
of
-galactosidase expression according to the vendor's
instructions, and data were analyzed for statistical significance using
Student's t tests and analysis of variance.
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RESULTS |
Preexposure of Alveolar Epithelial Cells to Hyperoxia Inhibits
Oxidant-induced Apoptosis--
To determine whether hyperoxia can
inhibit apoptosis, alveolar epithelial A549 cells were exposed either
to 5 mM H2O2 to directly induce
apoptosis (3) or to 95% O2 for 24 h and then exposed to H2O2. Fig. 1,
A-D, shows the nuclear morphology of the various treatment
groups visualized with DAPI staining. The nuclei of A549 cells
condensed and showed intense fluorescence with DAPI staining when
undergoing apoptosis (Fig. 1C), as is typical of most
apoptotic cells (16). Preexposure to hyperoxia reduced the fluorescent
intensity and nuclear condensation (Fig. 1D) when compared
with H2O2 alone (Fig. 1C). We
further examined the change in nuclear size induced by exposure to
H2O2 by computer-aided image analysis.
Approximately 78% of the nuclei were condensed (nuclear size <500
pixel) and showed bright fluorescent staining when cells were exposed
to H2O2 alone (Fig. 1, C and
E). In contrast, when cells were preexposed to hyperoxia,
only 11% of cells demonstrated this pattern of nuclear condensation
(Fig. 1, D and E). Fig. 1E reveals a
clear shift in the nuclear size distribution, from 424 ± 19 to
790 ± 24 pixels of the mean of nuclear area, when cells were
preexposed to hyperoxia (p < 0.05). These results
suggest that the nuclear condensation induced by exposure to
H2O2 is inhibited by preexposure to
hyperoxia.

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Fig. 1.
Hyperoxia inhibits
H2O2-induced nuclear condensation in A549
cells. A-D, A549 cells treated with 5 mM H2O2 for 4 h with or
without preexposure to 95% O2 for 24 h and stained
with DAPI to visualize all nuclei. Control, cells were
cultured in medium alone (A and B);
H2O2, cells were cultured in 5 mM H2O2 (C and
D); hyperoxia, cells were exposed to 95%
O2 for 24 h (B and D). Apoptotic
nuclei appear condensed and brighter (C and D).
E, Maple distribution of the nuclear size (area of nucleus)
of A549 cells exposed to H2O2 with
(O2/H2O2) or
without (RA/H2O2) preexposure to
hyperoxia (p < 0.001). A shift to the left
indicates the nuclear condensation of these cells.
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To determine whether hyperoxia interferes with internucleosomal
cleavage, we assayed for apoptosis using the TUNEL assay. Fig.
2A shows that exposing cells
to hyperoxia prior to H2O2 exposure reduced the
number of TUNEL-positive nuclei in comparison to control cells
preexposed to room air. Computer-aided image analysis revealed that the
percentage of TUNEL-positive nuclei/DAPI-positive nuclei (total nuclei)
decreased from 70 ± 5 to 27 ± 5% after hyperoxic preexposure (p < 0.05). Another indicator of apoptosis
is the externalization of phosphatidylserine from the cytosolic surface to the extracellular surface (17). Using fluorescein
isothiocyanate-conjugated Annexin V that detects phosphatidylserine
translocation, we analyzed the extent of apoptosis in cells exposed to
H2O2 with or without preexposure to hyperoxia.
Fig. 2B shows that 42% of cells treated directly with
H2O2 had high intensity for Annexin V binding,
compared with 12% of cells preexposed to hyperoxia followed by
H2O2 exposure. In addition, shrinkage of the
cell size induced by exposure to H2O2 was
reduced with hyperoxic preexposure (data not shown).

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Fig. 2.
Hyperoxia inhibits
H2O2-induced apoptosis in A549 cells.
A, A549 cells were treated with 2 mM
H2O2 for 8 h with or without preexposure
to 95% O2 for 24 h and assayed for apoptosis by the
TUNEL assay. Control, cells were cultured in medium alone;
H2O2, cells were cultured in 2 mM H2O2; hyperoxia,
cells were preexposed to 95% O2 for 24 h. The
bright red staining indicates the TUNEL-positive nucleus.
Insets correspond to the respective fields with all nuclei
visualized with DAPI. B, A549 cells were treated with 3 mM H2O2 for 4 h with or
without preexposure to 95% O2 for 24 h. The Annexin V
incorporation (measured by flow cytometry) assay was used to assess
apoptosis. RA/medium, cells were cultured in medium alone;
RA/H2O2, cells were
cultured in 3 mM H2O2 without
preexposure to 95% O2.
O2/H2O2, cells
were cultured in 3 mM H2O2 with
preexposure to 95% O2 for 24 h. Relative
fluorescence units represents the intensity of Annexin V
incorporation. A shift to the right indicates
phosphatidylserine translocation, an indicator for cells undergoing
apoptosis. Both TUNEL and Annexin V incorporation assays revealed
statistically significant reductions in apoptosis in response to
preexposure to hyperoxia.
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To examine whether preexposure to hyperoxia can inhibit apoptosis
induced by other oxidants, we exposed A549 cells to 10 mM paraquat (an intracellular superoxide generator) for 24 h, with or
without preexposure to hyperoxia. Studies from our and other laboratories have demonstrated that cytotoxicity induced by paraquat occurs via apoptosis (3, 18, 19). Fig. 3
shows that 24 h of preexposure to hyperoxia significantly
inhibited paraquat-induced apoptosis, assessed by both the Annexin V
incorporation assay (Fig. 3A) and an analysis of cell
shrinkage (Fig. 3B).

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Fig. 3.
Hyperoxia inhibits paraquat-induced apoptosis
in A549 cells. A549 cells were exposed to 95% O2 for
24 h or to room air prior to the treatment with 10 mM
paraquat for 24 h. Apoptosis was determined by Annexin V
incorporation (A) and cell shrinkage (B) assays
and analyzed by flow cytometry as described under "Experimental
Procedures." RA/Para, cells were exposed to paraquat
directly; O2/Para, cells were exposed to
hyperoxia prior to the paraquat exposure. Apoptosis is represented by
an increase in the intensity of Annexin V incorporation (a shift to the
right in A) and by the cell shrinkage (a shift to
the left in B). A significant reduction
(p < 0.05) in apoptosis was noted in cells preexposed
to hyperoxia.
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The inhibition of H2O2-induced apoptosis was
observed in A549 cells preexposed to hyperoxia (95%) for a time period
as short as 30 min and as long as 48 h. However, there was no
direct association between the reduction in apoptosis and the oxygen
concentration or the duration of preexposure. For example, preexposure
to 40, 60, or 95% O2 resulted in 17, 20, or 12%,
respectively, of cells undergoing apoptosis in comparison to 42% of
control cells. In addition, increasing the duration of preexposure to
hyperoxia did not provide additional benefits. A 40% reduction in
apoptosis was observed with preexposure to 95% O2 for 30 min compared to a 52% reduction in cells preexposed for 48 h.
One possible explanation for this inhibition is that hyperoxia might
interfere with the activation of proapoptotic pathways as a consequence
of overall oxygen toxicity suffered by the cells. One prediction of
this notion is that mutant cells that are not sensitive to oxygen
toxicity would not be protected by hyperoxic preexposure, as the cells
would not be poisoned by hyperoxia prior to secondary oxidant insults.
To test this, we repeated the above experiments in hyperoxia-resistant
HeLa-80 cells, which proliferate normally in 80% O2, a
lethal dose to many other cell types (20, 21). The parental HeLa cells
(referred to as HeLa-20) were used as controls. Both HeLa-20 and
HeLa-80 cells were propagated in room air and then either preexposed to
hyperoxia for 24 h prior to H2O2 or
treated with H2O2 directly. Fig.
4 shows that preexposure to hyperoxia
blunted subsequent H2O2-induced apoptosis in
both cell lines. Because HeLa-80 cells are not metabolically poisoned when grown in 80% O2, these results suggest that the
inhibition of apoptosis by hyperoxia is derived from the activation of
survival pathways rather than a general poisoning of these epithelial
cells.

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Fig. 4.
Inhibition of oxidant-induced apoptosis by
hyperoxia is not a result of oxygen toxicity. HeLa-80
(hyperoxia-resistant cells) and HeLa-20 (control HeLa cells) were
preexposed to 80%O2/5%CO2 or room air for
24 h and then exposed to 4 mM
H2O2 for 4 h.
RA/H2O2, cells were
cultured in 4 mM H2O2 without
preexposure to 95% O2.
O2/H2O2,
cells were cultured in 4 mM H2O2
with preexposed to 80% O2 for 24 h. Relative
fluorescence units represents the intensity of Annexin V
incorporation. Apoptosis is represented by an increase in Annexin V
incorporation (a shift to the right in both panels). A
significant reduction (p < 0.05) in apoptosis was
noted in cells preexposed to hyperoxia in both HeLa-80 and HeLa-20
cells.
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Antioxidant Enzymes Are Not Responsible for This
Inhibition--
The ability of hyperoxia to confer resistance to
further oxidative injury has raised the question of how endogenous
antioxidant systems respond to hyperoxic exposure. We have focused on
the classic antioxidant enzymes, including superoxide dismutase,
catalase, and glutathione peroxidase, all of which may play crucial
roles in responding to oxidative stress by scavenging excess reactive oxygen species. To determine whether this inhibitory effect of hyperoxia is due to increased AOE activities, we assessed the enzymatic
activities of total superoxide dismutase, catalase, and glutathione
peroxidase in cells with and without preexposure to hyperoxia. Fig.
5 shows that not only was there no
increase in AOE activities observed during and after hyperoxic
exposure, but a slight decrease was associated with the preexposure.
However, the differences between the activities in room air controls
and those in hyperoxic cells were not statistically significant. These data demonstrate that this inhibition of apoptosis is not related to an
increase in endogenous AOE activities.

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Fig. 5.
AOE activities are not up-regulated following
hyperoxic exposure. A549 cells were grown in either room air
(RA) or in 95%O2/5%CO2
(O2) for 24 h. Total cell lysates were prepared
from the trypsinized cells, and enzymatic activities of total
superoxide dismutase, catalase, and glutathione peroxidase were assayed
as described under "Experimental Procedures." The y axis
represents the relative enzymatic activities normalized to the total
amounts of protein. U/mg protein, units of AOE activity/mg
of total protein in the lysates.
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Preexposure to TNF-
Inhibits the Extent of Oxidative
Apoptosis--
Previously, we have shown that hyperoxia induces
NF-
B activation in A549 cells (5). To determine whether activation
of NF-
B plays a role in the protection of oxidative apoptosis, we tested whether activating NF-
B by means other than hyperoxia can
also provide protection against oxidative apoptosis. A549 cells were
treated with TNF-
prior to exposure to
H2O2. Treatment of A549 cells with 500 units/ml
TNF-
induced nuclear translocation of NF-
B (Fig.
6C), indicating that NF-
B
was activated in TNF-
-exposed cells. In contrast, NF-
B is
sequestered in the cytoplasm in control cells that are not exposed to
TNF-
(Fig. 6B). A549 cells are fairly resistant to the
cytotoxic effects of TNF-
(24, 25), and no morphological evidence
for apoptosis was apparent after exposure to TNF-
alone for 30 min,
or even for up to 24 h (data not shown). Although
H2O2-treated cells still underwent apoptosis, the extent of cell shrinkage was reduced from 64 to 42%
(p < 0.05) in cells pretreated with TNF-
(Fig.
6A). In addition, increases in the activities of superoxide
dismutase, catalase, and glutathione peroxidase were not observed in
TNF-
-treated cells (data not shown). These results suggest that
NF-
B activation plays an important role in the extent of
oxidant-induced apoptosis.

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Fig. 6.
Preexposure to TNF-
activates NF- B and inhibits
oxidant-induced apoptosis in A549 cells. A, TNF-
pretreatment inhibits oxidant-induced apoptosis. A549 cells were
exposed to 5 mM H2O2 for 4 h
with or without preexposure to 500 units/ml of TNF- for 30 min.
RA/medium, cells were cultured in medium alone;
RA/H2O2, cells were
cultured in 5 mM H2O2 without
preexposure to TNF- .
TNF- /H2O2,
cells were cultured in 5 mM H2O2
with preexposure to TNF- for 30 min. Apoptosis was assessed by cell
shrinkage (a shift to the left). Preexposure to TNF-
resulted in a significant reduction in cell shrinkage when cells were
subsequently exposed to H2O2 (p < 0.05). B and C, localization of NF- B by an
immunofluorescent assay. Activation of NF- B was assessed by nuclear
translocation of p65 subunit of NF- B from the cytoplasm in control
medium (B) or in TNF- -treated cells (C).
Insets (B and C) show the
corresponding nuclei visualized with DAPI staining.
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Activation of NF-
B via I
B Degradation Is Involved in the
Inhibition of Apoptosis by Hyperoxic Preexposure--
To determine
whether NF-
B activation induced by hyperoxia is via the I
B kinase
(IKK) signal transduction pathway, we examined the steady-state levels
of I
B (inhibitor of NF-
B). NF-
B normally remains sequestered
in the cytoplasm by I
B. Upon exposure to stimuli, I
B is
phosphorylated by IKK, ubiquitinated, and degraded (23). NF-
B is
then released and translocates to the nucleus, regulating gene
expression. Fig. 7A shows that
the levels of I
B
decreased shortly after exposure to hyperoxia
(i.e. 30 min) and remained lower than room air control cells
for up to 48 h. Previously, we demonstrated that the p65 subunit
of NF-
B translocates from the cytoplasm to the nucleus in cells
exposed to hyperoxia for periods as short as 30 min and as long as
48 h (5). This time frame closely correlates with the reduction in
the extent of apoptosis by hyperoxic preexposure, suggesting that
NF-
B activation via activation of the IKK signal transduction
pathway is involved in this inhibition. Similarly, to determine whether
inhibition of apoptosis by TNF-
preexposure is also via the IKK
pathway, we examined the steady-state levels of I
B
in TNF-
exposed cells. Fig. 7B shows that TNF-
induced I
B
degradation. These results suggest that either hyperoxia or TNF-
induces NF-
B activation via activation of the IKK signal
transduction pathway. This pathway may play an important role in
inhibiting oxidant-induced apoptosis.

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Fig. 7.
Hyperoxia- and
TNF- -induced NF- B
activation is via the IKK signal transduction pathway in A549
cells. A, A549 cells were exposed to
95%O2/5% CO2 for the time indicated (from 0 to 48 h), and steady-state levels of I B were determined by
Western blotting as described under "Experimental Procedures."
B, the steady-state levels of I B in control medium or
in TNF- -treated cells were assessed by Western blotting. Both
hyperoxia and TNF- induced the degradation of I B , an indicator
for the activation of the IKK pathway.
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To test whether suppression of NF-
B activity via the IKK pathway
alters the extent of apoptosis induced by exposure to
H2O2, we transfected A549 cells with a
construct that encodes I
B with dominant-negative mutations that
prevent its degradation. Stable clones that express less than 50%
(46-49%) of NF-
B activity compared with those in vector clones
were established as the dominant-negative mutant (DNM) clones and
exposed to H2O2. The extent of apoptosis induced by exposure to H2O2 was determined in
untransfected A549 cells, in three A549 clones transfected with
vector alone, and in three different DNM clones. Fig.
8 shows that the extent of apoptosis,
assessed by cell shrinkage and Annexin V incorporation, was increased
significantly in the DNM cell line (I
B DNM, 67%) compared to the
vector cell line (25%) and the untransfected A549 cells (31%). These
results suggest that NF-
B activation via the IKK signal transduction
pathway is involved in regulating the extent of oxidative
apoptosis.

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Fig. 8.
Augmentation of oxidant-induced apoptosis by
suppressing NF- B activity via interfering with
the IKK pathway. A549 cells were transfected with either a plasmid
containing a DNM construct of I B or a plasmid with the vector
only, and at least three stable cell lines each were established.
Untransfected A549 cells (WT A549), three stable I B DNM
cell lines expressing less than 50% NF- B activity compared to the
vector clones (I B DNM), and three
stable cell lines expressing vector alone (vector) were
exposed to 4 mM H2O2 for 4 h.
Apoptosis was assessed by cell shrinkage and Annexin V incorporation
assays and analyzed by flow cytometry. One representative each from the
I B DNM and vector cell lines is shown here. Apoptosis is represented
by cell shrinkage (a shift to the left in A) and
by an increase in the intensity of Annexin V incorporation (a shift to
the right in B). A significant increase
(p < 0.05) in apoptosis was noted in the DNM cells
compared with either the wild-type cells or vector cells, suggesting
that inhibiting NF- B activity by inhibiting phosphorylation of
I B enhances oxidative apoptosis.
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DISCUSSION |
We have shown that preexposure of epithelial cells to hyperoxia
inhibits the onset of apoptosis caused by exposure to
H2O2 or paraquat. Because this protection
occurs in cells that do not suffer from oxygen toxicity, this effect is
not merely a consequence of overall cell poisoning of an apoptotic
pathway but appears to result from the activation of NF-
B-mediated
survival pathways. That these pathways might be activated by NF-
B
was demonstrated not only by the activation of NF-
B by both
hyperoxia and TNF-
, which afford similar protection from oxidative
apoptosis, but also by the augmentation of oxidative apoptosis in cells
with suppressed NF-
B activity.
Resistance to Oxidative Injury Is Mediated by Specific Survival
Pathways Instead of Oxygen Toxicity--
Exposure to sublethal levels
of hyperoxia has been shown to increase resistance to further hyperoxic
injury and improve survival rates in animal models (26, 27).
Preexposing cultured mammalian cells to oxidants can also prime or
"desensitize" the cells to further oxidative injury (22, 28).
However, mechanisms for this acquired resistance have not been well
delineated and, based on correlative data, may in some cases be
a consequence of preinduction of antioxidant enzyme defenses (29, 30).
Our observations in A549 cells (Fig. 5) indicate that resistance to
oxidant-induced apoptosis is not mediated through increased activities
of endogenous AOEs. This notion is further supported by results from
HeLa-80 cells preexposed to hyperoxia and TNF-
-treated A549 cells
that do not up-regulate AOE activities but show resistance to
oxidant-induced apoptosis.
Although prolonged exposure to hyperoxia is lethal to most cells, it
does not kill pulmonary epithelial cells via apoptosis. On the other
hand, oxidants such as H2O2 and paraquat cause
these cells to undergo apoptosis (3, 4, 18, 19). The reasons for this
remain unclear. It is likely that at least some of the organellar
and/or macromolecular sites of O2 damage are different from
sites affected by other oxidants, because molecular O2
diffuses throughout the cell and can target virtually all organelles
and cytosolic molecules. One possible explanation for the lack of apoptosis associated with hyperoxia in these cells is that one or more
steps in the oxidative pathway to apoptosis might be sensitive to
direct oxidation by high levels of molecular O2. For
example, caspases may become oxidized and lose their ability to
initiate and execute apoptosis (31). However, our results demonstrate that cells preexposed to hyperoxia still undergo apoptosis upon subsequent exposure to H2O2, suggesting that
apoptotic pathways are either incompletely inhibited by molecular
O2 or circumvented by subsequent oxidant injury. Our
studies further suggest that hyperoxia inhibits apoptosis by activation
of prosurvival pathways, rather than via inactivating the apoptotic
pathway through oxidization of the macromolecules.
Activation of NF-
B via the IKK Pathway in Hyperoxic Preexposed
Cells May Be Responsible for the Inhibition of Oxidant-induced
Apoptosis--
NF-
B is an oxidative stress-responsive transcription
factor that can be activated by a variety of agents, including
cytokines and oxidants (32, 33), and has been shown to protect cells from apoptosis by regulating Bcl-2 family members and caspases (34,
35). Hyperoxia activates NF-
B in both animal models and epithelial
cells (5, 36, 37). Our results demonstrate that preexposure to
hyperoxia inhibits subsequent oxidant-induced apoptosis. This
inhibition was effective even with limited (30 min) preexposure to
hyperoxia. This time frame of inhibition, from 30 min to 48 h,
associates closely with the activation of NF-
B (5) and I
B
degradation (Fig. 7A), an indicator for the activation of
the IKK signal transduction pathway. These data suggest that activation
of NF-
B via the IKK pathway is involved in this inhibitory process.
This hypothesis is supported by the observations that the extent of
oxidative apoptosis is concomitantly affected by the levels of NF-
B
activity altered either by preexposure to TNF-
(Fig. 6) or by
transfection with a DNM construct of I
B in A549 cells (Fig. 8).
Furthermore, hyperoxia-induced inhibition corresponds to an increase of
nuclear translocation of NF-
B in oxygen-resistant HeLa-80 cells
exposed to hyperoxia (data not shown). Because hyperoxia activates
NF-
B in both rat and mouse lungs (36, 37), activation of NF-
B may
also be important in conferring resistance to further oxidative injury
by reducing oxidant-induced apoptosis in these animal models.
The mechanism by which NF-
B may inhibit oxidant-induced apoptosis is
unclear. One possibility is that activation of NF-
B may change the
oxidative equilibrium in the cellular milieu to a less oxidative state.
This notion is supported by the studies reported by Haddad and
colleagues (38). An increase in NF-
B activation can be achieved by
treating cells with an antioxidant, N-Acetyl-L-Cysteine,
suggesting a correlation between NF-
B activation and increased
antioxidant capacity. On the other hand, inhibiting NF-
B activation
by pyrrolidine dithiocarbamate favors an oxidative equilibrium of
glutathione (GSH) by lowering the ratio of GSH to its oxidized form,
glutathione disulfide (38). NF-
B may also participate in the
protective process by up-regulating anti-apoptotic Bcl-2 family members
and/or survival and stress-response factors such as heme oxygenase-1
(HO-1). HO-1 can be markedly up-regulated by hyperoxia (30, 39) and
over-expression of HO-1 protects A549 cells from further hyperoxic
injury (40). Interestingly, the 5'-upstream region of HO-1 contains
some potential NF-
B binding consensus sequences (41) and
over-expression of NF-
B can specifically enhance the expression of
HO-1 (42). Further work on characterizing these signal transduction
pathways will lead to greater understanding of how these pathways can
be regulated.