Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60601
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ABSTRACT |
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The intracellular signaling
pathways that control O2 deprivation (anoxia)-induced
apoptosis have not been fully defined in lung epithelial cells.
We show here that the lung epithelial cell line A549 releases
cytochrome c and activates caspase-9 followed by DNA
fragmentation and plasma membrane breakage in response to anoxia. The
antiapoptotic protein Bcl-XL prevented the
anoxia-induced cell death by inhibiting the release of cytochrome
c and caspase-9 activation. A549 cells devoid of
mitochondrial DNA (°-cells) and lacking a functional electron
transport chain were resistant to anoxia-induced apoptosis.
A549 cells preconditioned with either hypoxia (1.5% O2) or
tumor necrosis factor-
, which activated the transcription
factors hypoxia-inducible factor-1 or nuclear factor-
B,
respectively, did not provide protection from anoxia-induced cell
death. These results indicate that A549 cells require a functional electron transport chain and the release of cytochrome c for
anoxia-induced apoptosis.
Bcl-XL; hypoxia; hypoxia inducible factor-1; tumor
necrosis factor-; nuclear factor-
B
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INTRODUCTION |
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APOPTOSIS IS A GENETICALLY ENCODED, regulated multistep process of cell death (32). Presently, there are two discrete mechanisms that can initiate apoptosis (6, 29). The first mechanism, commonly associated with growth factor withdrawal and radiation, involves the activation of proapoptotic Bcl-2 family members such as Bax or Bak (18, 34). These proteins initiate the loss of outer mitochondrial membrane integrity, resulting in the release of cytochrome c and other proapoptotic proteins that normally reside in the mitochondrial intermembrane space (8, 12). Cytochrome c released into the cytoplasm initiates the formation of a complex known as the "apoptosome" by binding to the adaptor molecule apoptotic protease-activating factor (Apaf)-1 (17, 19). This triggers the oligomerization of Apaf-1 followed by the recruitment and activation of procaspase-9. Activated caspase-9 can activate downstream effector caspases-3 and -7, resulting in morphological features of apoptosis. Cytochrome c release and subsequent caspase-9 activation are considered early events in apoptosis (6). Bcl-XL or Bcl-2 inhibits apoptosis in response to a variety of death stimuli by preserving mitochondrial integrity and preventing cytochrome c release (11, 31, 35). The second apoptotic mechanism involves the ligation of death receptors such as Fas/CD95, which recruit and activate caspase-8 (22, 23). In a variety of cells, caspase-8 is sufficient to activate downstream caspases, such as caspase-3 or -7, that execute the morphological features associated with apoptosis. By contrast, some cells display a weak caspase-8 activation and thereby require caspase-8 to cleave Bid, a proapoptotic factor. Cleavage of Bid induces the loss of mitochondrial integrity leading to cytochrome c release and caspase-9 activation (16, 20).
Clinically, O2 deprivation-induced cell death is an important phenomenon (31). The mechanisms responsible for O2 deprivation-induced cell death in lung epithelial cells have relevance to clinical lung injury after severe shock or lung transplantation. Cells that become exposed to PO2 <1 Torr (anoxia) for prolonged periods of time begin to undergo cell death. Previous studies have suggested that a variety of nonlung cells die through apoptosis in response to anoxia (9, 10, 25, 28). These studies have also shown that the antiapoptotic Bcl-2 family members such as Bcl-XL or caspase inhibitors prevent anoxia-induced cell death. However, to date studies have not examined the mechanisms underlying anoxia-induced apoptosis in lung epithelial cells. In the present study, we examined whether mitochondrial electron transport and the release of mitochondrial protein cytochrome c are involved in initiating anoxia-induced apoptosis in the human lung epithelial A549 cells.
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MATERIALS AND METHODS |
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Cell culture.
A549 cells were cultured at 30-50% confluence in Dulbecco's
modified Eagle's medium (DMEM) supplemented with HEPES (10 mM), pyruvate (1 mM), penicillin (100 units/ml), streptomycin (100 µg/ml)
and 10% heat-inactivated fetal bovine serum (GIBCO). The Bcl-XL and the control neomycin vector (Neo) were gifts of
Dr. Craig Thompson (30). A549 cells transfected with
Bcl-XL or Neo were cultured in DMEM with 1 mg/ml of G418,
and stable clones were isolated. Wild-type A549 cells were incubated in
DMEM containing ethidium bromide (100 ng/ml), sodium pyruvate (1 mM),
and uridine (100 µg/ml) to generate °-A549 cells. Anoxic
conditions (0% O2, 85% N2, 10%
H2, and 5% CO2) were achieved in an anaerobic
workstation (BugBox, Ruskinn Technologies). Hypoxic conditions (1.5%
O2, 93.5% N2, and 5% CO2) were
achieved in a variable anaerobic workstation (INVIVO O2;
Ruskinn Technologies).
Measurement of cell death. We assayed cell death by measuring lactate dehydrogenase (LDH) activity in culture supernatants using the cytotoxicity detection kit (Roche Molecular Biochemicals) according to the manufacturer's protocol. We detected apoptosis either as a percentage of cells that had condensed/fragmented nuclei by staining with Hoechst no. 33258 (1 µg/ml; Sigma) as previously described (4) or by measuring cytoplasmic histone-associated DNA fragments after cell death using the Cell Death Detection ElizaPlus(Roche Molecular Biochemicals) according to the manufacturer's protocol.
Cytochrome c immunostaining. A549 cells were plated on 60-mm culture dishes at 20-30% confluence and exposed to experimental conditions. Both adherent and nonadherent cells were collected and centrifuged for 5 min at 200 g. The resulting pellet was then washed with PBS and centrifuged again for 5 min at 200 g. The cells were subsequently resuspended in 100 µl of PBS and centrifuged to microscope glass plates at 200 g for 5 min (Cytospin 3 Cytocentrifuge; Shandon). The cells were then incubated at 20°C for 5 min in methanol-acetone (1:1) and blocked for 45 min in PBS containing 0.1% bovine serum albumin (BSA; Sigma). After being blocked, the cells were removed, air-dried, and incubated for 2 h with a 1:500 dilution of the 7H8.2C12 anti-cytochrome c monoclonal antibody (PharMingen) at 37°C in a humidified environment. The cells were then washed for 4 × 15 min in PBS containing 0.1% BSA, air-dried, and incubated for 1 h with a 1:50 dilution of a rhodamine-conjugated secondary antibody (Chemicon International). Subsequently, the cells were washed as before, air-dried, and stained with 4,6-diamidino-2-phenylindole and 1,4-diazabicyclo-(2.2.2)octane. The cells were then visualized via microscopy.
Measurement of caspase activity. Caspase-9 enzymatic activity was measured with a fluorometric assay kit specific to the caspase (R&D Systems). Cell were plated onto 100-mm culture dishes at 40-60% confluence, and caspase activity was measured according to the manufacturer's protocol with a fluorescent microplate reader. Data were normalized using total protein concentration as determined by the Bio-Rad protein assay (Bio-Rad). The caspase inhibitor z-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk; Enzyme Systems Products) was administered at a concentration of 100 µM 1 h before incubation of the cells under O2 deprivation.
Immunoblotting analysis of hypoxia-inducible factor-1.
A549 cells were plated on 100-mm plates at 60-80% confluence and
assayed for hypoxia-inducible factor (HIF)-1
protein levels from
nuclear extracts by Western analysis as previously described (5).
Transfection and reporter gene assays.
A549 cells were plated on 35-mm culture dishes at 40-60%
confluence and transfected with 0.5 µg plasmid using LipofectAMINE reagent (Life Technologies) according to the manufacturer's protocol. Transfections were performed using either a HIF-1-responsive luciferase reporter plasmid containing a trimer of hypoxia response elements (HRE)
from the murine Epo 3' enhancer or a nuclear factor
(NF)-B-responsive luciferase reporter plasmid containing two
canonical
B sites (1, 5). Cells were exposed to various
conditions 24 h after transfection. Cells were lysed and assayed
for luciferase reporter activity using the Luciferase Assay System (Promega).
Measurement of mitochondrial membrane potential.
To assess the change in mitochondrial membrane potential () in
cells exposed to anoxia, cells were plated onto 60-mm culture dishes at
40-60% confluence and incubated 1 h before the time point in
the presence of two fluorescent probes, tetramethylrhodamine ethyl
ester (TMRE, excitation = 550 nm and emission = 580 nm; 200 nM) and Mitotracker green (MITO, excitation = 490 nm and
emission = 515 nm; 1 µM) (Molecular Probes). Cells were lysed
with 1% (vol/vol) Triton X-100, and fluorescence was measured on a
SpectraMax Gemini microplate reader (Molecular Devices). TMRE localizes
within mitochondria, and its fluorescence increases in proportion to
the
. MITO fluorescence localizes to mitochondria independently of
and reflects the number of mitochondria within a given cell. The
ratio between TMRE fluorescence and MITO fluorescence reflects
normalized to the number of mitochondria. As a control for each
condition, cells were incubated with both TMRE and MITO in the presence
of the protonophore carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP, 20 µM; Sigma), which dissipates the
. For each condition, the ratio of TMRE and MITO was subtracted from the TMRE-to-MITO ratio
in the presence of FCCP [(TMRE/MITO)
(TMRE/MITO)FCCP].
Statistical analysis. Data are presented as mean values ± SE. The experimental n values reported reflect the number of independent experiments. Data were analyzed using one-way analysis of variance (ANOVA). When the ANOVA indicated that a significant difference was present, we explored individual differences with the Student's t-test, using the Bonferroni correction for multiple comparisons. Statistical significance was determined at the 0.05 level. Experimental samples were compared with control cells exposed to 21% O2.
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RESULTS |
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Bcl-XL protects A549 cells from anoxia-induced
apoptosis.
A549 cells stably transfected with Neo or Bcl-XL were
exposed to 21 or 0% O2 for 24 and 48 h. Cellular
death was detected by assaying the samples for LDH release. Neo control
cells and Bcl-XL cells did not display a significant
increase in cell death after 24-h exposure to 0% O2. After
48 h of anoxia, there was a significant increase in cell death in
the Neo A549 cells compared with cells transfected with
Bcl-XL (Fig. 1A).
To determine if the cellular death due to anoxia was apoptotic, Neo
and Bcl-XL A549 cells were examined for DNA fragmentation
after 48 h of 0% O2. Neo A549 cells revealed a
4.3 ± 0.23-fold increase in DNA fragmentation over basal levels,
whereas Bcl-XL A549 cells did not show a significant
increase in fragmentation (Fig. 1B). These observations were
further confirmed by examining condensed/fragmented nuclei with Hoechst
staining in fixed Neo and Bcl-XL A549 cells exposed to 0%
O2 for 48 h. Neo control cells under 0%
O2 alone displayed apoptotic nuclei, whereas
Bcl-XL cells under 0% O2 did not display
apoptotic nuclei (Fig. 1C). These results illustrate that Bcl-XL protects cells from anoxia-induced
apoptosis.
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Bcl-XL prevents anoxia-induced release of cytochrome c
from mitochondria.
The release of mitochondrial cytochrome c is an early event
in a variety of cells undergoing apoptosis (11, 31,
35). To examine whether cells release cytochrome c
during anoxia, we exposed Neo and Bcl-XL A549 cells to 24 or 36 h of 21 or 0% O2. Neo and Bcl-XL
A549 cells exposed to 21% O2 with an anticytochrome c antibody gave a punctate immunofluorescent staining
pattern characteristic of mitochondrial localization (Fig.
2, A and C). Neo
and Bcl-XL A549 cells exposed to 24 h of 0%
O2 also displayed a punctate staining indicative of
cytochrome c localization of mitochondria (data not shown).
By contrast, Neo A549 cells after 36 h of 0% O2
displayed a diffuse staining pattern characteristic of a loss of
mitochondrial cytochrome c staining (Fig. 2B).
Bcl-XL A549 cells after 36 h of 0% O2
continued to exhibit a punctate staining pattern indicative that
cytochrome c was localized to mitochondria (Fig.
2D). The irreversible broad-range caspase inhibitor zVAD-fmk
(100 µM) did not prevent the release of cytochrome c (data
not shown), indicating that anoxia-induced release of cytochrome c was not dependent on caspase activation. Collectively,
these results suggest that Bcl-XL inhibits anoxia-induced
apoptosis by preventing the release of cytochrome c.
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Bcl-XL prevents anoxia-induced caspase-9 activation.
Cytochrome c released from mitochondria has been shown to
bind to Apaf-1, which then undergoes a conformational change that allows the cleavage and activation of caspase-9 (17, 19). The activated form of caspase-9 subsequently triggers a caspase cascade
that results in the death of the cell. To test whether the release of
cytochrome c during anoxia led to the activation of
caspase-9, Neo- and Bcl-XL-transfected A549 cells were
exposed to 0% O2 for 24 and 36 h, and caspase-9
activity was measured (Fig.
3A). Neo control cells exhibit
a significant increase in caspase-9 activity after 36 h of anoxia
compared with cells transfected with Bcl-XL. To further
understand the role of caspases during anoxia induced cell death, we
treated A549 cells with zVAD-fmk (100 µM) before exposure to 0%
O2 conditions for 48 h. Cells exposed to 0%
O2 displayed a significant increase in cell death compared with cells exposed to 21% O2 or cells exposed to 0%
O2 in the presence of zVAD-fmk (Fig.
3B). These results suggest that caspases are
important executioners of the cell death pathway in A549 cells after
exposure to anoxia.
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Bcl-XL maintains a partial during anoxia.
Previous studies have shown that loss of cytochrome c is
accompanied by a loss of
(11, 31, 35). We examined
changes in
in Neo control and Bcl-XL transfected A549
cells exposed to 0% O2 for 12, 24, 36, and 48 h.
was measured using the ratio of TMRE and MITO. TMRE fluorescence is
dependent on
, whereas MITO fluorescence is independent of
and reflects the number of mitochondria within a given cell. Neo A549
cells had a significant drop in potential at the point of cytochrome
c release as demonstrated by a 78.3 ± 4.8% and
84.8 ± 3.2% decrease in potential after 36 and 48 h of 0%
O2, respectively (Fig.
4A). Bcl-XL
maintained a potential of 49.7 ± 5.8% and 54.5 ± 3.0%
after 36 and 48 h of 0% O2 after 48 h. Thus
Bcl-XL prevents the loss of mitochondrial integrity during
anoxia as indicated by the maintenance of a
and the prevention of
cytochrome c release in A549 cells overexpressing Bcl-XL.
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Anoxia-induced cell death requires a functional mitochondrial
electron transport chain.
To determine whether electron transport chain was required for
anoxia-induced death in A549 cells, wild-type and °-A549 cells were exposed to 21 or 0% O2 for 24 or 48 h and
assayed for cell death.
°-cells have been depleted of
mitochondrial DNA, which encodes for 13 genes, including critical
subunits of the electron transport chain (3). Previous
studies have demonstrated
°-cells were able to die in response to
growth factor withdrawal (7). However, our current results
indicate that
°-A549 cells did not undergo death after exposure to
anoxia. Wild-type A549 cells exposed to 0% O2 exhibited
40.2 ± 4.8% cell death compared with 13.4 ± 1.9% cell
death in
°-A549 cells (Fig. 5). Thus
these results indicate that a functional electron transport is required
for anoxia-induced cell death.
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Hypoxia activates HIF-1 but does not prevent anoxia-induced cell
death in A549 cells.
HIF-1 is a basic helix-loop-helix/PAS protein consisting of 120-kDa
HIF-1 and 91- to 94-kDa HIF-1
subunits. HIF-1
protein is
present only in hypoxic cells, whereas HIF-1
protein levels are
constitutively expressed and not significantly affected by O2. HIF-1-targeted genes include the glycolytic enzymes and
glucose transporters (27). We tested whether the
activation of HIF-1 would prevent anoxia-induced cell death by exposing
wild-type A549 cells to 1.5% O2 for 24 h to activate
HIF-1, followed by a 48-h exposure to anoxia. Figure
6A demonstrates that an
HIF-1-dependent luciferase reporter construct increased 130 ± 21-fold after 24 h of 1.5% O2 compared with 21%
O2. Furthermore, a Western blot analysis to detect HIF1-
showed a drastic increase at 1.5% O2 (Fig. 6A).
However, wild-type A549 cells exposed to 1.5% O2 for 24 h to activate HIF-1 and subsequently exposed to 0%
O2 for 48 h displayed similar level of cell death as
cells that were exposed to 48 h of anoxia without the hypoxic
preconditioning (Fig. 6B). A549 cells exposed to 1.5%
O2 for 48 h did not display an increase cell death
compared with cells under 21% O2. Collectively, these results indicate that hypoxic preconditioning activates HIF-1 but does
not affect anoxia-induced cell death.
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Tumor necrosis factor- activates NF-
B but does not prevent
anoxia-induced cell death in A549 cells.
Tumor necrosis factor (TNF)-
can activate either the intracellular
death machinery or a signaling pathway, resulting in stimulation of the
transcription factor NF-
B (2). Hypoxia can stimulate NF-
B activation, which itself can then activate a variety of antiapoptotic genes. To examine whether activation of NF-
B by TNF-
would prevent anoxia-induced cell death, wild-type A549 cells
were exposed to TNF-
for 24 h followed by exposure to 0% O2 for 48 h. TNF-
activation of NF-
B was
measured with a luciferase reporter construct containing two NF-
B
sites. A 9.0 ± 2.3-fold increase was seen in NF-
B activity
after 24 h of exposure to TNF-
, whereas a smaller increase of
4.6 ± 1.6-fold was found after exposure to 1.5% O2
for 24 h (Fig. 7A).
Exposure of A549 cells to TNF-
(10 ng/ml) for 48 h at 21%
O2 did not trigger cell death. A549 cells exposed to
TNF-
(10 ng/ml) for 24 h to activate NF-
B and subsequently
exposed to 0% O2 for 48 h displayed a similar level
of cell death as cells that were exposed to 48 h of anoxia without
the TNF-
preconditioning (Fig. 7B). Taken together, these results indicate that TNF-
preconditioning resulting in NF-
B activation does not prevent anoxia-induced cell death.
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DISCUSSION |
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An early feature of apoptosis in response to a variety of
death stimuli is the disruption of outer mitochondrial membrane integrity resulting in the release of the cytochrome c and
other proapoptotic molecules from the mitochondria to the cytosol
(6). In the present study we examined whether a loss of
mitochondrial integrity was an early event in apoptosis induced
by anoxia in A549 cells. Cytochrome c release, followed by
caspase-9 activation, was observed before plasma membrane breakage and
DNA fragmentation in A549 cells exposed to anoxia. The loss of
cytochrome c was preceded by a depolarization in ,
whereas an almost complete dissipation of
was observed after
cytochrome c release. The broad-based caspase inhibitor
zVAD-fmk did not prevent the release of cytochrome c during
anoxia, which suggests that caspases are not involved in the initiation
of cytochrome c release. However, zVAD-fmk was able to
significantly prevent anoxia-induced cell death, indicating that
caspases play an important role in cell death after cytochrome
c release. By contrast, Bcl-XL, an
antiapoptotic protein that localizes to the outer mitochondrial
membrane, was able to prevent the dissipation of
, release of
cytochrome c, caspase-9 activation, and cell death during
anoxia. These results indicate that the loss of mitochondrial integrity
is an important early regulatory step in the apoptotic pathway
induced by anoxia.
A possible mechanism proposed, in a variety of cells, for cytochrome c release and mitochondrial depolarization is the opening of the PTP (13). This pore is a large-conductance channel that spans both mitochondrial membranes. Opening of the pore is characterized by an abrupt increase in the permeability of the inner mitochondrial membrane to molecules as large as 1,500 Da. Primary evidence for a PTP model comes from experiments showing that cyclosporin A, an inhibitor of PTP formation, prevents cell death. However, our present data indicate that cyclosporin A does not prevent cell death in response to anoxia in A549 cells. These data suggest that PTP may not be a major regulator of death in these cells. Other possible mechanisms for cytochrome c release could be specific cytochrome c-conducting channels in the outer membrane or the nonspecific disruption of the outer membrane as a result of proapoptotic Bcl-2 family members such as Bax or Bak (21). In this case, antiapoptotic proteins such as Bcl-XL would bind to Bax or Bak to prevent cytochrome c release.
A fundamental question in understanding anoxia-induced
apoptosis is determining the initiating mechanism for cell
death. Under normal physiological conditions, the oxidation of NADH is
coupled to the reduction of O2 through the respiratory
chain (24). O2 is reduced to water by
cytochrome c oxidase. Electron transfer through the
respiratory chain is coupled to the directional movement of protons
across the inner mitochondrial membrane. This movement across the
membrane establishes an electrochemical potential that provides the
thermodynamic driving force for the F1F0-ATP
synthase to generate ATP in the matrix. Thus a potential mechanism to
initiate anoxia-induced apoptosis is that a lack of
O2 inhibits cytochrome c oxidase within the
mitochondrial electron transport chain, resulting in the activation of
programmed cell death. Experimental evidence in the present study
indicates that °-A549 cells that lack mitochondrial DNA do not
undergo anoxia-induced cell death. Mitochondrial DNA encodes the three
catalytic subunits of cytochrome c oxidase, whereas nuclear
DNA encodes the proapoptotic protein cytochrome c.
Previous studies have shown that
°-cells are able to undergo cell
death in response to a variety of apoptotic stimuli, including growth factor withdrawal, ligation of death receptors, and
staurosporine (7). Therefore our current results indicate
that the initiating mechanism of apoptosis during anoxia is
different from other classic apoptotic stimuli.
A potential therapeutic mechanism to prevent anoxia-induced
apoptosis could be to activate genes encoded by the
transcription factors HIF-1 and NF-B. HIF-1 is able to induce the
expression of glycolytic genes and the angiogenic factor vascular
endothelial growth factor (VEGF) (26). HIF-1 is
necessary for the normal embryonic and cardiovascular system
development and is also implicated in cancer progression and
apoptosis. Activation of HIF-1 in vivo could prevent cells from
being exposed to anoxic conditions by stimulating VEGF production,
thereby promoting angiogenesis. By contrast, the activation of HIF-1 in
vitro could activate glycolytic enzymes that could provide ATP that is
normally generated by electron transport during anoxia to prevent cell
death. Our current data suggest that activation of HIF-1 is not
sufficient to prevent cell death during anoxia in vitro. A549 cells
preconditioned with 24 h of 1.5% O2 to activate HIF-1
target genes did not prevent subsequent anoxia-induced cell death.
Hence, glycolysis upregulation via HIF-1 is not likely to be an
important regulator of anoxia-induced cell death.
The NF-B pathway is also a key mediator of genes involved in the
control of apoptosis (14). Antiapoptotic genes
that are directly activated by NF-
B include the cellular inhibitors
of apoptosis (c-IAP1, c-IAP2, and IXAP), the TNF-
receptor-associated factors (TRAF1 and TRAF2), and IEX-1L.
TNF-
is one of the classical inducers of NF-
B and can induce
cells to undergo apoptosis (33). The sensitivity
of different cell types to TNF-
-induced apoptosis can vary
dramatically, but most cells become very sensitive upon simultaneous
treatment with inhibitors of protein synthesis. It has been suggested
therefore that a gene, or set of genes, is induced upon TNF-
receptor activation that downregulates the apoptosis signal.
Recent results have shown that NF-
B activated by TNF-
is at least
partly responsible for this effect. Indeed, our current results show
that A549 cells do not undergo cell death with TNF-
treatment alone.
TNF-
is sufficient to activate NF-
B in these cells. However, A549
cells that were preconditioned for 24 h with TNF-
to activate
NF-
B could not protect against subsequent anoxia-induced cell death.
These results suggest that NF-
B target genes are not likely to
regulate anoxia-induced apoptosis.
In summary, our findings provide evidence that anoxia-induced
apoptosis requires the inhibition of a functional electron
transport chain and the release of cytochrome c in lung
epithelial cells. The antiapoptotic Bcl-XL protein can
prevent the release of cytochrome c and subsequent cell
death during anoxia, whereas preconditioning with hypoxia or TNF-
does not prevent cell death during anoxia. Collectively, these results
indicate that the main regulatory step of anoxia-induced
apoptosis is at the level of the mitochondria.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of General Medicine Sciences Grant GM-60472-02 (to N. S. Chandel) and the Crane Asthma Center.
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. S. Chandel, Div. of Pulmonary & Critical Care, Tarry Bldg. 14-707, 300 E. Superior St., Chicago, IL 60611-3010 (E-mail: nav{at}northwestern.edu).
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.
10.1152/ajplung.00281.2001
Received 24 July 2001; accepted in final form 6 November 2001.
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