From the Department of Anatomy and Cell Biology, North Texas Eye
Research Institute at University of North Texas Health Science Center,
Fort Worth, Texas 76107, the § Department of Pediatrics,
Louisiana State University Medical Center, Shreveport, Louisiana 71130, the Department of Molecular Oncology,
Cytokine Research Laboratory, University of Texas M. D. Anderson
Cancer Center, Houston, Texas 77030, and the ¶ Department of
Ophthalmology and Visual Sciences, University of Illinois at Chicago,
Chicago, Illinois 60612
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ABSTRACT |
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The mechanisms of photoreceptor cell death via
apoptosis, in retinal dystrophies, are largely not understood. In the
present report we show that visible light exposure of mouse cultured
661W photoreceptor cells at 4.5 milliwatt/cm2 caused
a significant increase in oxidative damage of 661W cells, leading to
apoptosis of these cells. These cells show constitutive expression of
nuclear factor- Nuclear factor- Most of the earlier studies on NF- In several experimental models of retinal dystrophies, including
certain forms of retinitis pigmentosa, photoreceptor cells of the
retina have been shown to undergo apoptosis (20-23). A number of
studies have identified the primary mutations involved in the etiology
of these diseases (24). These studies, however, do not completely
explain the ultimate phenotypic manifestation of the disease namely
apoptosis of photoreceptor cells. The difficulty in studying the
disease process of retinal dystrophies is further compounded by non
availability of a homogenous permanent photoreceptor cell line, since
retina is a complex tissue of multiple cell types. In the current
studies we have used a transformed mouse photoreceptor cell line
661W.2
In vivo studies have shown that exposure of rats to constant
light results in apoptosis of photoreceptor cells (25-29). Even moderate intensities of light exposure have been shown to damage the
retinas of rats (30). Since then, light has been extensively studied as
an initiator of retinal cell death in a number of in vivo
(31-33) and in vitro experimental conditions.
In the current study, we assessed the contribution of an oxidative
stress paradigm to the propensity of photoreceptor cells to proceed to
cell death via apoptosis, using cultured photoreceptor cells. We
provide evidence in this paper that visible light exposure to
photoreceptor cells results in oxidative damage leading to apoptosis
via down-modulation of NF- Materials--
The following materials were purchased from the
indicated sources: fetal bovine serum from JRH Biosciences, Lenexa, KS;
paraformaldehyde and H2O2 from EM Sciences,
Gibbstown, NJ; HEPES, phenylmethylsulfonyl fluoride, ALLN, and
dithiothreitol from Sigma; poly(dI-dC)·poly(dI-dC) from Amersham
Pharmacia Biotech; and polynucleotide kinase from New England Biolabs,
Beverly, MA.
Antibodies--
p50 subunit of NF- Cell Culture--
The 661W cells were originally isolated from a
transgenic mouse line expressing the construct HIT1 comprising of SV40
T-antigen driven by the human interphotoreceptor retinol binding
protein promoter (35). The construct HIT1 resulted in SV40 T-antigen expression and retinal and brain tumors. 661W cells are routinely grown
in complete medium consisting of Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum and 1% penicillin/streptomycin, at
37 °C in a humidified atmosphere of 5% CO2 and 95% air.
Light Exposure of the Cells--
The 661W cells were seeded
either in 35/60/100-mm tissue culture dishes or on round coverslips
kept in 35-mm dishes and exposed to fluorescent visible light at 4.5 milliwatt/cm2 for varying durations up to 4 h at
37 °C in tissue culture. The accompanying control cells were
shielded from light for similar intervals and left in similar
conditions as the cells in light-exposed paradigm.
3' End Labeling of Fragmented DNA by Terminal Deoxynucleotidyl
Transferase-mediated Fluoresceinated dUTP Nick End Labeling
(TUNEL)--
The TUNEL procedure as described by Gavrieli et
al. (36) was employed to study apoptosis, using a commercially
available apoptosis kit (in situ cell death detection
kit, Boehringer Mannheim) as per the supplier's instructions.
Measurements of Membrane Lipid Peroxidation and Reduced
Glutathione (GSH) Levels--
The membrane lipid peroxidation of
light-exposed cultured cells was studied by measuring the
malonyldialdehyde levels by a colorimetric method involving
thiobarbituric acid adduct formation (37). The GSH levels in
light-exposed cells was studied by using the
5,5'-dithiobis(2-nitrobenzoic acid) reagent (38).
Immunoblot Analysis--
Protein extracts from 661W cultured
cells exposed to light were subjected to immunoblot analysis (39, 40)
using specific antibodies for I Immunolocalization Studies--
The 661W cells were exposed to
light and fixed in 4% paraformaldehyde. The immunofluorescence for p65
subunit of NF- Preparation of Cytoplasmic and Nuclear Extracts--
The 661W
cells were exposed to light for the desired amount of time, and the
nuclear and cytoplasmic extracts were prepared (41). Briefly, the cells
were suspended in 100 µl of buffer C (10 mM HEPES, pH
7.9, 1.5 mM MgCl2, 10 mM KCl, 10%
glycerol, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride) and incubated on ice for 15 min. 3 µl
of 10% Nonidet P-40 was added to the suspension and briefly vortexed.
Following this, the nuclei were pelleted by centrifugation at low
speed. The supernatant (cytoplasmic extract) was collected and stored
at Electrophoretic Mobility Shift Assays (EMSA)--
A
double-stranded oligonucleotide containing the NF- Transfection of 661W with the Dominant Negative
I Membrane Lipid Peroxidation and Depletion of Glutathione Are
Observed in Cultured Photoreceptor Cells Exposed to Light--
The
661W cells were exposed to light for up to 4 h, and membrane lipid
peroxide formation and GSH levels were measured. There was almost a
2-fold increase in malonyldialdehyde formation following light
exposure, as compared with controls (Table
I) and inclusion of NAC in the medium
before light exposure of cultured cells, prevented the increase in MDA
levels (Table I). These results indicate that photic injury to
photoreceptor cells occurs due to a possible involvement of an
oxidative pathway. To explore this possibility further, we used other
anti-oxidants such as thiourea and mannitol in our studies. As shown,
photo-oxidative stress resulted in significant lowering of GSH levels
as compared with control cells maintained in dark. The presence of
thiourea (7 mM) in the medium of 661W cells was protective
against photo-oxidative damage, as seen by the maintenance of GSH
levels close to control values (Table I).
NF-
The specificity of the binding of NF- Pretreatment of Photoreceptor Cells with Antioxidants Protects
against Down-modulation of NF- Treatment with H2O2 Does Not Significantly
Alter the NF- Effect of Photo-oxidative Stress on NF- Immunoblot Analysis of I Cultured Photoreceptor Cells Undergo Cell Death via Apoptosis
upon Light Exposure--
To establish that oxidative damage along with
a down-modulation of NF- Photo-oxidative Stress and Immunocytochemical Localization of
NF- Pretreatment with the Proteasome Inhibitor ALLN Does Not Protect
661W Cells against Photo-oxidative Stress-induced
Apoptosis--
Since our results suggested that preservation of
NF-
The TUNEL assay revealed that ALLN pretreatment also caused some of the
cells maintained in the dark, to undergo apoptosis (Fig. 6b,
panel B). Furthermore, ALLN treatment did not protect against photo-oxidative stress induced apoptosis (Fig. 6b,
panel D) compared with cells exposed to light without any
pretreatment of ALLN (Fig. 6b, panel C). ALLN, by
virtue of being a proteasome inhibitor, could block I Transient Transfection of 661W Cells with a Dominant Negative
I
The NF-
The TUNEL assay revealed that mere transfection of the I
Thus, transfection with the super-repressor hastens the kinetics of
down-modulation of NF- Pretreatment with the Caspase-1 Inhibitor (YVAD-CMK) Protects
against Down-modulation of NF-
To further study if the caspase-1 inhibitor could protect against
light-induced apoptosis, we performed TUNEL assay of these cells
exposed to light in presence of the inhibitor. As seen in Fig.
8b, pretreatment with the caspase-1 inhibitor could protect against photo-oxidative stress induced apoptosis (panel C)
as compared with 661W cells exposed to light in absence of caspase-1 inhibitor (panel B), and dark-exposed controls (panel
A). Similar studies with caspase-3 inhibitor showed that it did
not render protection of 661W cells against photo-oxidative
stress-induced apoptosis (data not shown). These studies indicate that
down-modulation of NF- Apoptosis of photoreceptor cells is a common phenotype in retinal
dystrophies, shared by patients with age-related macular degeneration
and autosomal dominant retinitis pigmentosa, a family of disorders
characterized by photoreceptor cell degeneration and a corresponding
loss of vision. Point mutations of several genes involved in visual
transduction have been identified in retinitis pigmentosa and other
forms of retinal dystrophies (24). These studies, while pointing to the
primary cause of the disease, do not provide clues to the downstream
effectors that play a crucial role in the progression of the disease,
ultimately leading to photoreceptor cell death by apoptosis.
Light has been extensively used as a initiator of photoreceptor cell
death in a number of in vivo and in vitro
experimental conditions (31-33). In vivo studies have also
shown that exposure of rats to constant light results in apoptosis of
photoreceptor cells (25-30). Production of lipid hydroperoxides has
been observed in light-exposed retinas (44). Retina has been shown to
be susceptible to lipid peroxidation (45, 46) despite having high
levels of antioxidants (47-49).
In the current study, we have assessed the involvement of oxidative
damage in the apoptotic cell death of 661W photoreceptor cells in an
in vitro model. The cell line expresses several markers of
differentiated photoreceptors, including opsin, arrestin,
rds/peripherin, phosducin, and interphotoreceptor retinol binding
protein.2 The 661W cell line, therefore, is a valuable cell
line to study photoreceptor cell death. When the cells exposed to light
for 4 h were subjected to an apoptotic TUNEL assay, it was seen
that 80% of the cells were labeled with fluoresceinated dUTP,
suggestive of apoptosis. In a time course study, the extent of
apoptosis of 661W cells had a direct correlation to duration of light
exposure, and inclusion of NAC was protective of this effect. This
experimental system thus, afforded a convenient model to analyze, the
molecular events leading to apoptosis of photoreceptors by an oxidative pathway.
The important role of oxidative events has been further emphasized for
a variety of biological processes, such as signal transduction and gene
expression (19). A few studies have demonstrated that in particular,
activation of NF- By electrophoretic mobility shift assay, we found constitutive NF- In the cell, NF- The role of NF- In the 661W cells the NF-B (NF-
B), and light exposure of photoreceptor
cells results in lowering of NF-
B levels in both the nuclear and
cytosolic fractions in a time-dependent manner. Immunoblot
analysis of I
B
and p50, and p65 (RelA) subunits of NF-
B,
suggested that photo-oxidative stress results in their depletion.
Immunocytochemical studies using antibody to RelA subunit of NF-
B
further revealed the presence of this subunit constitutively both in
the nucleus and cytoplasm of the 661W cells. Upon exposure to
photo-oxidative stress, a depletion of the cytoplasmic and nuclear RelA
subunit was observed. The depletion of NF-
B appears to be mediated
through involvement of caspase-1. Furthermore, transfection of these
cells with a dominant negative mutant I
B
greatly enhanced the
kinetics of down modulation of NF-
B, resulting in a faster
photo-oxidative stress-induced apoptosis. Taken together, these studies
show that the presence of NF-
B RelA subunit in the nucleus is
essential for protection of photoreceptor cells against apoptosis
mediated by an oxidative pathway.
INTRODUCTION
Top
Abstract
Introduction
References
B
(NF-
B)1 is a widely
distributed transcription factor that plays a role in the regulation of
a number of cellular and viral genes involved in early defense
reactions in higher organisms (1). NF-
B exists in an inactive form
bound to the inhibitory protein I
B
or I
B
(2-4). Treatment
of cells with inducers such as lipopolysaccharide, interleukin-1, and
tumor necrosis factor-
(TNF-
), generally result in degradation of I
B proteins. This releases NF-
B of its inhibitory constraint, facilitating its translocation to the nucleus, resulting in regulation of expression of genes encoding cytokines, hematopoietic growth factors, and cellular adhesion molecules. NF-
B exhibits its DNA binding activity in its dimeric form, and the most commonly occurring dimer is that of the p50 and the p65 (RelA) subunits. NF-
B has been
shown to be constitutively active in several cell types, including B
cells (5), thymocytes (6), and neurons (7).
B focused on its role in
immunological and inflammation responses (1, 8, 9). Recent reports
suggest that NF-
B is also activated by oxidative signaling (10-13).
It has been suggested in many of these studies that reactive oxygen
intermediates (ROI) may be involved in the activation of NF-
B.
Another area of research, where NF-
B involvement is gaining momentum, is the regulation of apoptosis. One of the earliest significant observations in this direction was made by Beg et al. (14), who demonstrated extensive apoptosis of liver cells leading to embryonic death of mice lacking the RelA subunit. Subsequent work by Beg and Baltimore (15) demonstrated that treatment of RelA-deficient (RelA
/
) mouse fibroblasts and
macrophages, with TNF-
, resulted in a significant reduction in cell
viability. Along similar lines, Wang et al. (16), Van
Antwerp et al. (17), and Liu et al. (18) showed a
role of NF-
B in suppression of TNF-
-induced apoptosis. There is
also evidence of pro-apoptotic aspects of RelA activity. For instance,
it was shown that serum starvation of 293 cells causes cell death
accompanied by the activation of RelA containing NF-
B (19).
B. NF-
B, which was constitutively expressed in the 661W cells, was found to be progressively
down-regulated upon exposure of the cells to light. By
immunocytochemistry using NF-
B RelA antibody, the NF-
B activity
appeared to be localized both in the nucleus and cytoplasm of
dark-exposed 661W cells. Upon exposure to light the nuclear and
cytoplasmic NF-
B RelA immunolabeling was largely diminished in these
cells. Pretreatment of the cells with various antioxidants prevented to
a great extent the down-modulation of NF-
B and also protected the
cells from apoptosis. Furthermore, transient transfection of the 661W
cells with a dominant negative I
B
N (super-repressor) caused a
rapid decline in NF-
B binding activity in the cells, leading to a
faster kinetics of photo-oxidative stress-induced apoptosis.
Down-modulation of NF-
B in these cells appears to be mediated by
caspase-1. Our results suggest that NF-
B, which is constitutively
expressed in 661W photoreceptor cells, undergoes degradation when
subjected to oxidative stress leading to apoptosis of the photoreceptor cells. Thus, the presence of NF-
B in the nucleus is essential for
photoreceptor cell survival and protection against oxidative stress
induced apoptosis.
EXPERIMENTAL PROCEDURES
B, a goat polyclonal IgG;
p65, of NF-
B, a rabbit polyclonal IgG; and I
B
rabbit
polyclonal IgG were from Santa Cruz Biotechnology, Santa Cruz, CA.
GAPDH (chicken anti-rabbit GAPDH immunoaffinity-purified monospecific
antibody) was kindly supplied by Drs. Glaser and Cross (34).
-Tubulin, a mouse monoclonal antibody, was from Sigma.
Peroxidase-labeled secondary antibodies either anti-rabbit IgG or
anti-mouse IgG were from Kirkegaard and Perry Laboratories Inc.,
Gaithersburg, MD. Anti-cyclin D1 antibody was against amino acids
1-295, which represents full-length cyclin D1 of human origin, and was
obtained from Santa Cruz Biotechnology. Fluorescein
isothiocyanate-labeled anti-rabbit IgG from Vector Laboratories,
Burlingame, CA.
B
, p50, and RelA subunit of
NF-
B at 1:500 dilution. Cytoplasmic extracts were used for I
B
analysis, whereas nuclear extracts were used to study RelA and p50
subunits of NF-
B. Control blots were run using total cellular
extracts and an antibody to GAPDH at 1:1000 dilution. The binding of
primary antibodies was detected by using peroxidase labeled appropriate
secondary antibodies, which were detected by using diaminobenzidine as substrate.
B was done by using a specific antibody against p65
and a fluorescein isothiocyanate-labeled goat anti-rabbit secondary
antibody (20). The immunofluorescent cells were photographed using a
Nikon Microphot-FXA photomicroscope.
80 °C. The nuclear pellet was resuspended in 70 µl of buffer
D (20 mM HEPES, pH 7.9, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20%
glycerol, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride). The suspension was incubated for 20 min
at 4 °C followed by a centrifugation at 8000 g for 5 min. The
supernatant containing the nuclear protein extract was transferred to a
fresh microcentrifuge tube and stored at
80 °C. Protein
concentrations of the cytoplasmic and the nuclear extracts were
measured with a detergent-compatible Protein Assay Kit (Bio-Rad), using
bovine serum albumin as a standard.
B DNA-binding
consensus sequence, 5'-AGT TGA GGG GAC TTT CCC AGG C-3', and
a double-stranded mutant oligonucleotide, 5'-AGT TGA GGC GAC TTT CCC AGG C-3' (Santa Cruz
Biotechnology) were used to study the DNA binding activity of NF-
B
by EMSA as described (42). For supershift assay, 4 µg of nuclear
extract was incubated with 1 µg of antibodies for 30 min at room
temperature and analyzed by EMSA.
B
--
The dominant negative I
B
(super-repressor)
construct I
B
N was obtained from Dr. Dean Ballard, Vanderbilt
University, Nashville, TN (43). I
B
N is a deletion mutant in
which the N-terminal 36 amino acids are deleted from the I
B
protein. The 661W cells were transiently transfected with the construct
using the LipofectAMINE reagent (Life Technologies, Inc.), as per the
manufacturer's instructions using 8 µl of LipofectAMINE and 5 µg
of the I
B
super-repressor plasmid DNA for 1 ml of transfection
mix. The untransfected control cells were treated in a similar manner
except for the exclusion of the plasmid DNA. The transfected cells and
their controls were used 48 h post-transfection for making either
total cellular extract for immunoblot analysis or cytoplasmic and
nuclear extracts for EMSAs and for TUNEL assay as described above.
RESULTS
Effect of photo-oxidative stress on malonyldialdehyde formation and
glutathione levels in cultured photoreceptor cells
B Activity Is Down-modulated upon Exposure of Photoreceptor
Cells to Light--
The photoreceptor cells were exposed to light for
various time intervals for 15, 30, and 60 min. There was no change in
NF-
B activity up to 30 min of light exposure in both the nucleus
(Fig. 1a, lanes 2 and 3 for nucleus) and cytoplasm (Fig. 1a,
lanes 6 and 7) compared with dark-exposed control
cells (Fig. 1a, lanes 1 and 5, for
nucleus and cytoplasm, respectively). Upon 60 min of light exposure,
there was approximately 80% loss of NF-
B binding activity in both
the nucleus and cytoplasm (lanes 4 and 8,
respectively). These results indicate that the cultured photoreceptor
cells express NF-
B constitutively and that the activity of NF-
B
decreases on exposure to photo-oxidative stress.
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Fig. 1.
Effect of a time course of light exposure on
NF- B levels in 661W cells. a,
661W cells express NF-
B constitutively (lanes 1 and
5, for nuclear and cytoplasmic fractions, respectively).
Lanes 2 and 6, 3 and 7, and 4 and 8 represent NF-
B binding activity in nucleus and cytoplasm,
respectively, after 15, 30, and 60 min of light exposure to the cells.
For quantitation of the bands of the autoradiogram, a value of 100%
was taken for dark-exposed controls and the values for other samples
were calculated as percent of control and are shown below each lane.
"D" and "L" represent dark-exposed
controls and light-exposed cells respectively. b, the
identity of the shifted band seen in the EMSA was confirmed to be that
of NF-
B by competition EMSA using molar excesses of cold consensus
and mutant NF-
B oligonucleotides. Lanes 1 and
5 show NF-
B levels in dark-exposed 661W cells in the
absence of any competitor oligonucleotide. Competition EMSA using 50-, 100-, and 200-fold molar excess of cold consensus NF-
B
oligonucleotide resulted in reduction of the NF-
B binding to the
consensus sequence (Fig. 1b, lanes 2-4). On the
other hand competition with cold mutant NF-
B oligonucleotide using
50-, 100-, and 200-fold molar excess did not result in a reduction of
the NF-
B binding to consensus sequence (Fig. 1b,
lanes 6-8). c, furthermore, the specificity of
NF-
B binding was established by super shift assays using p50 and p65
antibodies. Anticyclin D1 was used as an unrelated antibody serving as
a negative control. The results show that both p65 and p50 antibodies
resulted in decrease in NF-
B band intensity, whereas anticyclin D1
did not have any effect on the binding reaction (c).
B was shown by competition with
cold NF-
B consensus oligonucleotide (Fig. 1b, lanes 2-4). As expected, a lack of competition was observed with cold NF-
B mutant oligonucleotide (Fig. 1b, lanes
6-8). The DNA protein complex seen in the EMSA appears to be a
heterodimer of p50 and p65 subunits of NF-
B, as revealed by a
decrease in the binding upon additional incubation with the antibodies
to the p65 and p50 (Fig. 1c) subunits, in a supershift
assay. An unrelated antibody, anti-cyclin D1 used as a negative
control, did not inhibit the DNA protein complex formation (Fig.
1c). These results confirm the identity of the NF-
B
DNA-protein complex seen in the EMSAs.
B upon Exposure to Light--
In
order to establish the involvement of oxidative damage in the lowering
of NF-
B activity during conditions of photo-oxidative stress, we
studied the effects of antioxidants, namely NAC, mannitol, and thiourea
under these conditions (Fig.
2a). Lanes 1,
5, and 9 represent NF-
B binding activity in
dark-exposed cells. Lanes 2, 6, and 10 represent NF-
B binding activity in dark-exposed cells in the
presence of NAC, mannitol, and thiourea, respectively. The presence of
these anti-oxidants did not appreciably alter the NF-
B binding
activity in dark-exposed control cells. As expected, the activity of
NF-
B decreased on exposure to photo-oxidative stress (lanes
3, 7, and 11). The inclusion of NAC,
mannitol, and thiourea in the growth medium prior to light exposure
partially protected against the down-modulation of NF-
B (Fig.
2a, lanes 4, 8, and 12,
respectively) in light-exposed 661W cells. The differences in the
extent of protection of NF-
B levels in these groups may be
attributed to differences in efficacy of these anti-oxidants in
affording protection against photo-oxidative damage. These results
indicate that oxidative damage plays a major role in decreasing the NF-
B activity in cultured photoreceptor cells exposed to light.
It remains to be seen if these antioxidants offer an additive protection of NF-
B levels, if used simultaneously.
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Fig. 2.
Pretreatment of 661W cells with antioxidants
protects against the down-modulation of nuclear
NF- B levels upon light exposure.
a, pretreatment of 661W cells with NAC, mannitol, and
thiourea protects against the down-modulation of NF-
B levels
(lanes 4, 8, and 12, respectively)
compared with light-exposed 661W cells (lanes 3,
7, and 11, respectively). NF-
B binding
activity in dark-exposed controls are shown in lanes 1,
5, and 9 and dark-exposed control cells treated
with NAC (2 mM), mannitol (50 mM), and thiourea
(7 mM) are shown in lanes 2, 6, and
10, respectively. "D" and "L"
represent dark-exposed controls and light-exposed cells respectively.
"NAC," "Man," and "Thio"
indicate N-acetylcysteine, mannitol, and thiourea,
respectively. b, 661W cells were also treated with 300 µM H2O2 for 30, 60, and 120 min,
and NF-
B binding activity was studied by EMSA. There was little
change in NF-
B binding activity upon treatment with
H2O2 for 30 and 60 min in both the nucleus
(Fig. 2b, lanes 2 and 3) and cytoplasm
(Fig. 2b, lanes 6 and 7), compared
with the activity in the untreated cells (Fig. 2b,
lanes 1 and 5 corresponding to nucleus and
cytoplasm, respectively). A modest increase in the NF-
B binding
activity was seen after 120 min of treatment with
H2O2 in both nucleus (lane 4) and
cytoplasm (lane 8).
B Binding Activity and Apoptosis of the 661W
Cells--
Our results so far suggest that light may down-modulate
NF-
B through generation of ROIs. To further confirm the role of ROIs in this process we treated the cells with H2O2
for various times up to 120 min and measured NF-
B activation and
apoptosis. The EMSA revealed no significant change in the NF-
B
binding activity in these cells treated with 300 µM
H2O2 for 30 and 60 min (Fig. 2b,
lanes 2 and 3 and lanes 6 and
7 for nuclear and cytoplasmic extracts, respectively),
compared with untreated control cells (lanes 1 and
5 for nucleus and cytoplasm, respectively). However, treatment of H2O2 for 120 min resulted in a
modest increase in the NF-
B binding activity both in the nucleus
(lane 4) and the cytoplasm (lane 8). The TUNEL
assay revealed no significant increase in the number of apoptotic cells
on treatment with H2O2 compared with untreated
controls for all the durations of H2O2
treatment (data not shown). Therefore, this data indicate that ROIs
alone are not sufficient for light induced down-regulation of NF-
B and activation of apoptosis in these cells.
B Levels in Madin-Darby
Canine Kidney (MDCK) Cells--
To assess the specificity of response
of 661W cells to photo-oxidative stress, we studied the effect of light
exposure on MDCK cells, using them as an unrelated control. The light
exposure of MDCK cells did not cause a decrease in NF-
B binding
activity, in both the nucleus and cytoplasm (data not shown). These
results indicate that the cell-specific response of 661W cells to light is different from that of MDCK cells.
B
, p50, and RelA Subunit of NF-
B
in 661W Cells Exposed to Light--
To further confirm the
down-modulation of NF-
B, the protein levels of I
B
, p50, and
RelA subunits of NF-
B were studied in 661W cells exposed to light,
with or without pretreatment with NAC and thiourea, by immunoblot
analysis using specific antibodies. The light-exposed cells showed
lowering of I
B
, p50, and RelA subunit of NF-
B, as compared
with dark controls. Pretreatment of the cells with both anti-oxidants
protected the levels of NF-
B p50, RelA, and I
B
subunits,
albeit partially, upon exposure to light (Fig.
3). To ensure that light exposure does
not result in a generalized protein degradation, a control protein
GAPDH was included, which was not greatly altered in all samples under these experimental conditions. On quantitation, there was a
50% decrease of I
B
protein levels on 2-h light exposure. On the other
hand, there was
90% decrease in p50 and RelA subunit with no change
in GAPDH levels under similar conditions. Inclusion of anti-oxidants
protected to a large extent against degradation of these proteins.
I
B
was protected 100%, whereas p50 and p65 were protected to
40-45% of control values. Based on the results of GAPDH protein
levels, these data suggest that down-modulation of I
B
and
p50 and p65 subunit of NF-
B is not due to random protein
degradation.
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Fig. 3.
Immunoblot analysis of
NF- B p50, RelA,
I
B
subunit in 661W
cells exposed to photo-oxidative stress. The cells were exposed to
light for 2 h in presence or absence of NAC (2 mM) and
thiourea (7 mM), and the levels of NF-
B subunits p50,
RelA, and I
B
subunit were studied by immunoblot analysis. There
was a decrease in the levels of I
B
, p50, and RelA subunit of
NF-
B upon light exposure (lane 2), and these were
protected to varying extents by pretreatment of the cells with NAC and
thiourea (lanes 3 and 4), respectively. The GAPDH
levels were not altered between the experimental groups studied.
"D" and "L" represent dark- and
light-exposed 661W cells, respectively. "Nac" and
"Thio" indicate N-acetylcysteine and
thiourea, respectively.
B results in apoptosis of these cells, we
studied the effect of photo-oxidative stress on apoptosis of these
cells in presence or absence of the anti-oxidant, NAC. We found that
exposure of 661W cells to light up to 1 h did not result in any
significant apoptosis of cultured photoreceptor cells (Fig.
4A). However, after 2 and
4 h of light exposure, many cells underwent apoptosis (Fig. 4,
C and E, respectively), compared with
dark-exposed control cells as seen by incorporation of fluoresceinated
dUTP in the nuclei of apoptotic cells containing fragmented DNA.
Approximately, 80% of the cells were seen to undergo apoptosis in 661W
cells exposed to light for 4 h (Fig. 4E). Inclusion of
NAC protected the cells from apoptosis at the 1-, 2-, and 4-h intervals
of light exposure, respectively (Fig. 4, B, D,
and F). As expected, dark-exposed control cells did not show
any TUNEL-positive apoptotic cells (data not shown).
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Fig. 4.
Fluorescent TUNEL labeling of 661W cells
after exposure to light for various time durations in presence or
absence of NAC. 661W cells with or without pretreatment with NAC
were exposed to light for various durations, fixed with 4%
paraformaldehyde, and processed for TUNEL labeling. There was a
time-dependent increase in the number of the cells labeled
with the fluoresceinated dUTP suggestive of apoptosis (white
arrows) in 661W cells, with increasing duration of light exposure
for 1 h (A), 2 h (C), and 4 h
(E). Not all the cells were undergoing apoptosis in 2-h
light-exposed group (C, arrowheads). Inclusion of
NAC in the culture medium before light exposure, protected the cells
from apoptosis (B, D, and F) for 1, 2, and 4 h of light exposure, respectively. There were also a few
apoptotic cells in the NAC pretreated cells exposed to light for 4 h (F, arrow). Non-apoptotic cells were devoid of
fluorescence.
B RelA Subunit in 661W Cells--
To study the effect of
photo-oxidative stress on the localization of RelA subunit of NF-
B,
immunocytochemistry was performed in these cells in presence and
absence of NAC, using RelA-specific antibody. It was seen that RelA was
present in the nucleus and also in the cytoplasm of dark-exposed
control cells (Fig. 5A). Upon
exposure to light, the nuclear and cytoplasmic labeling of NF-
B
diminished to a large extent (Fig. 5B). But, in the presence of NAC, a number of cells exposed to light retained positive
immunoreactivity of RelA, in the nucleus as well as cytoplasm (Fig.
5C). There were still some cells, which had diminished RelA
immunoreactivity in their nuclei and cytoplasm (Fig. 5C).
These data indicate that light exposure caused the degradation of RelA
subunit from the nucleus as well as cytoplasm by an oxidative
pathway.
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Fig. 5.
Photo-oxidative stress and immunocytochemical
localization of NF- B RelA subunit in 661W cells. The RelA
subunit of NF-
B was predominantly present in the nucleus and in the
cytoplasm of dark-exposed control cells (A,
arrows). Upon exposure to light, the nuclear and cytoplasmic
labeling diminished considerably (B, arrows). In
the presence of NAC, a number of cells showed positive immunoreactivity
in the nucleus as well as cytoplasm (C, arrows).
Some nuclei devoid of RelA were also seen in NAC pretreated cells
exposed to light (C, arrowheads). These data
indicate that light exposure brought about lowering of NF-
B levels
in the nucleus as well as cytoplasm by an oxidative pathway.
B levels in the cells protects against apoptosis, we investigated
the down-regulation of constitutive NF-
B activity and its effect on
apoptosis, by a proteasome inhibitor, ALLN. ALLN pretreatment of the
661W cells did not protect the NF-
B binding activity, upon exposure
to light (Fig. 6a, lanes
4 and 8 for nucleus and cytoplasm, respectively) compared with
NF-
B binding activity in both the nucleus and the cytoplasm of 661W
cells exposed to light without any pretreatment (Fig. 6a,
lanes 3 and 7 for nucleus and cytoplasm,
respectively). ALLN treatment to cells maintained in the dark also did
not alter their NF-
B activity (Fig. 6a, lanes
2 and 6 for nucleus and cytoplasm, respectively).
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Fig. 6.
NF- B binding
activity and apoptosis of ALLN-pretreated, dark- and light-exposed 661W
cells. a, 661W cells were pretreated with ALLN (100 µM) and exposed to light for 1 h. Pretreatment with
ALLN did not protect against the down-regulation of NF-
B binding
activity upon light exposure (lanes 4 and 8 for
nucleus and cytoplasm, respectively). Lanes 1 and
5 represent NF-
B binding activity in nucleus and
cytoplasm of dark-exposed control cells. Lanes 2 and
6 show the NF-
B binding activity in ALLN-pretreated,
dark-exposed control cells. The NF-
B binding activity was decreased
upon exposure to light (lanes 3 and 7 for nucleus and cytoplasm, respectively). b, TUNEL assay of
ALLN-pretreated, dark- and light-exposed 661W cells. Pretreatment with
ALLN caused a few cells to undergo apoptosis even in cells maintained
in dark (B, arrows), compared with the untreated
controls (A). Upon light exposure, a number of cells were
TUNEL-positive (C, arrows). Pretreatment with
ALLN did not protect against photo-oxidative stress-induced apoptosis
(D, arrows).
B
degradation, thereby, NF-
B activation could be inhibited thus
leading to apoptosis. This provides further confirmation of our
hypothesis that NF-
B is involved in blocking apoptosis.
B
Accelerates Photo-oxidative Stress-induced Apoptosis by
Down-modulating NF-
B Binding Activity--
To further delineate the
role of NF-
B in apoptosis we also transfected the cells with
I
B
super-repressor and examined its effects on NF-
B binding
activity and apoptosis upon exposure to photo-oxidative stress. To
determine this, 661W cells were transiently transfected with I
B
super-repressor and evaluated for their I
B
and I
B
N
levels, NF-
B binding activity, and DNA fragmentation by TUNEL.
Immunoblot analysis of the transfected cells revealed higher protein
levels of I
B
in these cells, compared with the mock-transfected
controls (Fig. 7a, compare
lane 2 with lane 1). A faster moving band
corresponding to the super-repressor was also detected in the
transfected cells representing a truncated form of I
B
, the
I
B
N, but not in the mock-transfected cells (Fig.
7a, compare lane 2 with lane 1). A
control protein,
-tubulin, did not appear to change between the two
samples (Fig. 7a, bottom panel).
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Fig. 7.
Effect of
I B
super-repressor
expression on NF-
B levels and apoptosis of
661W cells. a, 661W cells transfected with the
super-repressor I
B
(+) and their corresponding control cells (
)
were used for an immunoblot analysis of I
B
and I
B
N
expression. There was a higher amount of I
B
expression in the
transfected cells (lane 2) compared with the
mock-transfected control cells (lane 1). A faster moving
band corresponding to the super-repressor I
B
N was also
detected in the transfected cells, but not in the mock-transfected
cells. The lower panel shows a duplicate immunoblot of
-tubulin levels, used as a control to show equal loading of protein
in each lane. b, EMSA of NF-
B binding activity in 661W
cells transfected with the I
B
super-repressor, exposed to light
for different time durations. The untransfected cells showed higher
NF-
B binding activity, in comparison with the transfected cells both
in the nucleus (compare lane 1 with lane 2) and
the cytoplasm (compare lane 9 with lane 10). A
short time course of light exposure caused a rapid loss of NF-
B
binding activity in the transfected cells in nucleus (lanes
4, 6, and 8 for 15, 30, and 60 min of light
exposure) and cytoplasm (lanes 12, 14, and
16 for 15, 30, and 60 min of light exposure). The
untransfected cells showed much slower kinetics of NF-
B
down-regulation, showing practically no change in the activity up to 30 min of light exposure in both nucleus (lanes 3 and
5) and cytoplasm (lanes 11 and 13). A
significant decrease in the NF-
B binding activity in the
untransfected cells was seen only upon 60 min of light exposure in both
nucleus (lane 7) and cytoplasm (lane 15). For
quantitation of the bands of the autoradiogram, a value of 100% was
taken for dark treated controls for both the transfected and
untransfected cells and the values for other samples were calculated as
percent of control for each group and are shown below each lane.
"D" and "L" represents dark and light,
respectively. c, TUNEL assay of super-repressor I
B
N
transfected 661W cells exposed to light for various time durations.
There were no TUNEL-positive cells in the untransfected cells
maintained in the dark (A) and in those exposed to light for
15 (B) and 30 (C) min. There was induction of
apoptosis only after 60 min of light exposure (D,
arrows), in the untransfected cells. In contrast,
transfection with the I
B
super-repressor caused a few cells to
undergo apoptosis even in the cells maintained in the dark
(E, arrows). There was a rapid increase in the
number of TUNEL positive cells, in the transfected cells exposed to
light for 15 (F), 30 (G), and 60 min
(H) of light exposure (arrows).
B binding activity in the untransfected cells was higher than
that of the super-repressor transfected cells (Fig. 7b,
lanes 1 and 2). Upon exposure to light, there was
a rapid decline in NF-
B activity in the transfected cells compared
with that in the untransfected cells, both in the nucleus and cytoplasm (Fig. 7b, lanes 4, 6, and 8
compared with lanes 3, 5, and 7 for nucleus; lanes 12, 14, and 16 compared
with lanes 11, 13, and 15 for
cytoplasm, for 15, 30, and 60 min of light exposure, respectively). Quantitation of a typical EMSA autoradiogram showed practically no
change in NF-
B binding activity of control cells exposed to light up
to 30 min, with a 75-85% decrease in binding activity upon 60 min of
light exposure in nucleus as well as cytoplasm. In contrast, in
I
B
super-repressor transfected cells, there was a rapid loss of
NF-
B binding activity, which decreased by 40 and 95% at 30 and 60 min in the nucleus and by 50, 80, and 90% at 15, 30, and 60 min of
light exposure in the cytoplasm. There was no decrease in the NF-
B
binding activity of nucleus at 15 min of light exposure in the
transfected cells.
B
N
super-repressor caused a number of cells to undergo apoptosis, even
without being exposed to light (Fig. 7c, panel
E). There was a rapid increase in the number of TUNEL positive
cells in the transfected cells by as early as 15 min of exposure to
light (Fig. 7c, panel F), when compared with
untransfected 661W cells (Fig. 7c, panel B). It
reached a maximum by 30 min of light exposure in the I
B
super-repressor transfected group (Fig. 7c, panel G), in contrast with just a few apoptotic cells in the
untransfected 661W cells exposed to light for 60 min (Fig.
7c, panel D).
B binding activity as well as apoptotic cell
death of 661W cells upon exposure to light.
B and Apoptosis of 661W Cells upon
Exposure to Light--
In order to identify proteases involved in the
down-regulation of NF-
B in the 661W cells, we studied the effect of
caspase inhibitors on NF-
B binding activity and apoptosis of 661W
cells exposed to photo-oxidative stress. Pretreatment of 661W cells with the caspase-1 inhibitor, YVAD-CMK (100 µM) could
protect against down-regulation of NF-
B upon exposure of the cells
to light in both nucleus (Fig.
8a, compare lane 4 to lane 3) and cytoplasm (Fig. 8a, compare
lane 8 to lane 7). On the other hand, inclusion
of caspase-3 inhibitor, DEVD-CHO did not protect the levels of NF-
B
in the light-exposed cells (data not shown).
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Fig. 8.
Effect of pretreatment with caspase-1
inhibitor on NF- B levels and apoptosis of 661W
cells, upon exposure to light. a, 661W cells were
pretreated with caspase-1 inhibitor (YVAD-CMK) (100 µM)
and NF-
B levels were studied in nuclear and cytoplasmic fractions,
after exposure of the cells to light for 2 h. The inclusion of
YVAD-CMK resulted in protection of NF-
B levels in light-exposed 661W
cells in both the nuclear (compare lane 4 with lane
3) and cytoplasmic fractions (compare lane 8 with
lane 7). Inclusion of the caspase-1 inhibitor in
dark-exposed control cells did not alter NF-
B levels in both nuclear
(lane 2 compared with lane 1) and cytoplasmic
fractions (lane 6 compared with lane 5),
respectively. "D" and "L" represent dark-
and light-exposed 661W cells. b, fluorescent TUNEL labeling
of caspase-1 inhibitor pretreated 661W cells exposed to light. The
cells maintained in the dark were devoid of apoptotic cells
(A, arrowheads). A number of TUNEL-positive cells
were seen in the light-exposed group (B, arrows).
Pretreatment with the caspase-1 inhibitor was protective against
apoptosis as seen by decreased TUNEL labeling in these cells
(C, arrowheads). A few cells undergoing apoptosis
were also seen in this group (C, arrows).
B may be due to an activation of a specific
caspase, namely, caspase-1.
DISCUSSION
B requires oxidative signaling, i.e. its
expression is dependent on the redox state of the cell (1, 10).
Treatment of several cell lines with H2O2
resulted in activation of NF-
B (50, 51), and this can be blocked by inclusion of antioxidants (52-54).
B
activity, both in the nucleus and the cytoplasm of 661W photoreceptor
cells. Visible light exposure to photoreceptor cells creates conditions
of photoxidative stress leading to oxidative damage. This causes the
cells to proceed to cell death via apoptosis. The NF-
B activity in
661W cells was found be progressively down-regulated upon exposure of
the cells to light. Pretreatment of the cells with antioxidants namely
NAC, thiourea, and mannitol partially prevented the down-modulation of
NF-
B and NAC also protected the cells from apoptosis, indicating
involvement of hydroxyl radicals and superoxide anions in the pathway
leading to cell death via apoptosis. The NF-
B activity profile in
the photoreceptor cells exposed to light appears to be radically
different from that seen in the unrelated MDCK cells. While the light
exposure of 661W cells leads to decrease in NF-
B binding activity
and apoptosis, the same stimulus does not greatly alter the nuclear
NF-
B activity and does not lead to cell death in the MDCK cells.
This points to a cell type specificity of 661W cells' response to light.
B is stored in the cytoplasm in its inactive state
by interaction with I
B
. On activation, I
B
undergoes degradation through an ubiquitin-dependent pathway (55,
56), allowing translocation of NF-
B to nucleus (57, 58) and
subsequently binding to DNA regulatory elements within NF-
B target
genes. Under our experimental conditions we find degradation not only of I
B
, but also the NF-
B p50 and RelA subunits. How the p50 and p65 subunits of NF-
B are degraded in this oxidative stress paradigm is not fully understood. It is plausible that exposure of
photoreceptor cells to light causes activation of one or more proteases
which leads to degradation of not only I
B
, but also the p50 and
p65 subunits. One of the protease responsible for degradation of
NF-
B proteins in the 661W cells appears to be the interleukin
1
-converting enzyme also called caspase-1 according to the new
nomenclature. Recently, it was shown that caspase-3, which activates
apoptosis, can also induce proteolytic cleavage of I
B
(59). In
addition, the ubiquitin-conjugating enzymes that control I
B
degradation are known to be induced by ROI (60). Thus, in our system,
it appears that exposure of photoreceptor cells to light generates ROI,
which activates caspase-1, resulting in proteolytic cleavage of NF-
B
proteins leading to apoptosis. This is consistent with studies which
show that NF-
B activation is essential for cell survival (61).
B in apoptosis is not very clear with reports of both
pro- and anti-apoptotic aspects appearing in literature. It was
demonstrated recently that NF-
B is needed for TNF-
mediated induction of IAP-2, a protein belonging to the inhibitors of apoptosis (IAP) protein family. When overexpressed in mammalian cells, cIAP-2 activates NF-
B and suppresses TNF cytotoxicity (62). Exposure of
human alveolar epithelial (A 549) cells to hyperoxia resulted in
activation of NF-
B leading to necrotic cell death (63). These
authors speculated that apoptosis occurs in the absence of NF-
B
activation but protection from cell death by NF-
B is limited to
apoptosis. It was further shown that TNF-
induces cell death in
RelA
/
mouse fibroblast cells, whereas
RelA+/+ cells were unaffected, demonstrating the role of
RelA in protection of the cells from TNF-
induced apoptosis (15). A
similar antiapoptotic role of the RelA subunit was also demonstrated by
Wang et al. (16) and Van Antwerp et al. (17).
These studies indicate that either the inhibition of NF-
B RelA
subunit or the prevention of its translocation to nucleus is essential
to induce apoptosis (15). However, several groups have suggested that
NF-
B may function pro-apoptotically under some conditions and in
certain cell lines (19, 64, 65).
B protein does not seem to follow the
prototype pathway of its activation, upon exposure to an oxidative stimuli. For instance we do not observe an increase in NF-
B binding activity in these cells treated with an oxidizing agent like
H2O2. Similarly, exposure of 661W cells to
photo-oxidative stress, which causes a degradation of I
B
, does
not activate NF-
B, but in fact degrades the p50 and p65 subunits. It
therefore appears that in these cells, oxidative damage activates other
cellular mechanisms that trigger selective protein degradation,
culminating in apoptosis. It needs to be emphasized that NF-
B
signaling is indeed essential for survival of these cells, as seen by
the induction of apoptosis in the 661W cells upon transfection with the
I
B super-repressor. But the overwhelming stimulus for cell death
appears to result from the oxidative degradation of NF-
B, as seen by
the greatly accelerated kinetics of apoptosis in the
super-repressor-transfected 661W cells. A similar response was observed
when the cells were pretreated with the proteasome inhibitor, ALLN,
which also inhibits calpain I and II, cathepsin B, cathepsin L, and
neutral cysteine proteases. Treatment with ALLN would slow down the
degradation of I
B
, thereby perturbing the NF-
B signaling in
the cell and exacerbate cell death via apoptosis. The results of
ALLN treatment further suggest that the down-modulation of RelA subunit
is not due to generalized protein degradation, but may indeed require an activation of a specific caspase involved in apoptosis (66). Evidence is accumulating to attest to the fact that a constitutive expression/activation of NF-
B is essential for a cell to survive an
apoptotic insult (61, 67, 68). All these observations underscore
the importance of the protective role of NF-
B against photo-oxidative stress induced apoptosis in the 661W cells. A schematic
representation of the molecular events occurring in the course of
photo-oxidative stress-induced apoptosis of photoreceptor cells are
shown in Fig. 9.
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Fig. 9.
Summary of the proposed mechanism of
photo-oxidative stress induced apoptosis of photoreceptor
cells.
Taken together, these results suggest that the RelA subunit of NF-B
constitutively expressed in 661W photoreceptor cells may be important
for photoreceptor cell survival. Exposure of the cells to visible light
creates conditions of photo-oxidative stress, causing the production of
reactive oxygen intermediates leading to oxidative damage as evidenced
by depletion of glutathione and increase in malonyldialdehyde
formation. These oxidative events further result in the down-modulation
of NF-
B (predominantly the RelA subunit), thereby exacerbating the
oxidative damage and channeling the cells along a pathway of cell
death, via apoptosis.
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ACKNOWLEDGEMENTS |
---|
We appreciate Drs. Robert W Gracy and James E. Turner for their support and Dr. Victoria Rudick for providing MDCK cells. We also thank Dr. Larry Oakford, Terry Opera, and Anne-Marie Brun for the photographic work reported in this paper and Drs. Ashok Kumar and Sunil Manna for their initial involvement with the immunoblot analysis.
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FOOTNOTES |
---|
* 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.
Recipient of a grant from Foundation Fighting Blindness and
Knights Templer Eye Foundation, Inc.
** To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. Tel.: 817-735-2094; E-mail: nagarwal{at}hsc.unt.edu.
The abbreviations used are:
NF-B, nuclear
factor-
B; EMSA, electrophoretic mobility shift assay; TUNEL, terminal deoxynucleotidyl transferase mediated fluoresceinated dUTP
nick end labeling; GAPDH, glyceraldehyde phosphate dehydrogenase; GSH, glutathione-reduced; NAC, N-acetylcysteine; ALLN, N-acetylleucylleucylnorleucinal; I
B
, inhibitory
subunit of NF-
B; I
B
N, super-repressor of I
B
; FITC, fluorescein isothiocyanate; TNF-
, tumor necrosis factor-
; ROI, reactive oxygen intermediates; MDCK, Madin-Darby canine kidney.
2 M. J. Crawford, R. R. Krishnamoorthy, H. J. Sheedlo, D. T. Organisciak, R. S. Roque, N. Agarwal, and M. R. Al-Ubaidi, submitted for publication.
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REFERENCES |
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