Decreased Intracellular Superoxide Levels Activate Sindbis
Virus-induced Apoptosis*
Kuo-I
Lin
§,
Piera
Pasinelli
¶,
Robert H.
Brown
¶,
J. Marie
Hardwick§, and
Rajiv R.
Ratan
**
From the
Department of Neurology, Harvard Medical
School, and the
Beth Israel Deaconess Medical Center,
Boston, Massachusetts 02115, ¶ Massachusetts General Hospital,
Boston, Massachusetts 02129, and the § Department of
Molecular Microbiology and Immunology, Johns Hopkins University School
of Hygiene and Public Health, Baltimore, Maryland 21205
 |
ABSTRACT |
Infection of many cultured cell types with
Sindbis virus (SV), an alphavirus, triggers apoptosis through a
commonly utilized caspase activation pathway. However, the upstream
signals by which SV activates downstream apoptotic effectors, including
caspases, remain unclear. Here we report that in AT-3 prostate
carcinoma cells, SV infection decreases superoxide (O
2) levels
within minutes of infection as monitored by an aconitase activity
assay. This SV-induced decrease in O
2 levels appears to
activate or modulate cell death, as a recombinant SV expressing the
O
2 scavenging enzyme, copper/zinc superoxide dismutase (SOD),
potentiates SV-induced apoptosis. A recombinant SV expressing a mutant
form of SOD, which has reduced SOD activity, has no effect. The
potentiation of SV-induced apoptosis by wild type SOD is because of its
ability to scavenge intracellular O
2 rather than its ability
to promote the generation of hydrogen peroxide. Pyruvate, a peroxide
scavenger, does not affect the ability of wild type SOD to potentiate
cell death; and increasing the intracellular catalase activity via a
recombinant SV vector has no effect on SV-induced apoptosis. Moreover,
increasing intracellular O
2 by treatment of 3T3 cells with
paraquat protects them from SV-induced death. Altogether, our results
suggest that SV may activate apoptosis by reducing intracellular
superoxide levels and define a novel redox signaling pathway by which
viruses can trigger cell death.
 |
INTRODUCTION |
Apoptosis is a genetically controlled process, with distinct
morphological and biochemical features, by which cells commit suicide
during development and in disease states (1, 2). Sindbis virus
(SV),1 an alphavirus, belongs
to the growing list of viruses (3-10) that can trigger apoptosis in
infected host cells in vitro or in vivo. The
elimination of virus-infected cells via apoptosis is believed to be a
mechanism used by the host to limit the spread of progeny virus to
neighboring cells and thus to decrease the host viral burden (11).
However, if the primary targets in vivo are nonreplenishing
cells like neurons, virus-induced apoptosis can also have devastating
consequences for the host. For example, age-dependent
encephalitis and mortality induced by SV is correlated with the ability
of SV to induce apoptosis in neurons in the central nervous system of
mice (12, 13).
Mechanistic studies of SV-induced apoptosis indicate that SV engages an
apoptotic pathway used by many death stimuli (14). CrmA, a
caspase-inhibitory protein encoded by cowpox virus (15), inhibits
SV-induced apoptosis in cultured cells and reduces SV-induced mortality
in mice (14). SV-induced death can also be blocked by the pluripotent
anti-apoptotic protein, Bcl-2 (8, 16). These data suggest that SV
triggers a common death execution pathway, but the state changes
induced by SV which set this common pathway in motion remain unclear.
Maintenance of the cellular redox balance is believed to play an
important role in regulating survival (17), and multiple lines of
evidence indicate that disruption of the redox equilibrium in favor of
oxidants, a condition defined as "oxidative stress," may activate
apoptosis (18-20). In cultured cells, the addition of oxidants or
inhibition of antioxidant defenses leads to apoptosis (18-20).
Similarly, overexpression of antioxidant enzymes or treatment of cells
with antioxidants has been shown to prevent apoptotic death (21-24).
Apoptosis induced by a disruption in redox homeostasis appears to be a
mechanism of neuronal loss in a broad range of sporadic and inherited
neuropathological disorders, including Parkinson's disease (25),
Alzheimer's disease (26), amyotrophic lateral sclerosis (27), and
Huntington's disease (28). Moreover, genetic studies indicate that
inherited mutations in antioxidant defenses may also induce
neuropathology (29, 30). For example, a subset of patients with a
familial form of amyotrophic lateral sclerosis have been found to carry
mutations in the gene encoding the antioxidant enzyme, copper/zinc
superoxide dismutase (Cu,Zn-SOD) (29, 31, 32). Mutations in the
Cu,Zn-SOD gene appear to diminish the threshold for activation of
apoptosis in neurons, and antioxidants can restore the threshold for
activation of cell death to levels seen in cells carrying wild type
Cu,Zn-SOD (33). Taken together, these observations suggest that
oxidative stress is a mediator of apoptotic death, although cell death
induced by certain stimuli can be triggered independent of oxidative
stress (34, 35).
To examine whether apoptosis induced by SV is activated by oxidative
stress, we previously evaluated the effects of antioxidants on
SV-induced apoptosis in cultured cells (36). Our results showed that a
host of antioxidants, including metal chelators, inhibitors of lipid
peroxidation, or scavengers of peroxide failed to inhibit SV-induced
apoptosis, suggesting that oxidative stress is not a mediator of
SV-induced death (36). However, we did not monitor directly individual
reactive oxygen species (ROS), such as superoxide (O
2) or
peroxide (H2O2), so we could not definitively exclude a role for these ROS in regulating SV-induced death. In this
study, we examine the role of O
2 and
H2O2 in SV-induced apoptosis.
 |
MATERIALS AND METHODS |
Cell Culture and Viability Studies--
AT-3 rat prostate
carcinoma cells and 3T3 fibroblasts were cultured as described
previously (36, 37). For viability studies, 103 AT-3
cells/well or 104 3T3 fibroblasts/well were seeded in
96-well plates. 1 day after plating, recombinant SVs were added at a
multiplicity of infection (m.o.i.) of 10 plaque-forming units/cell.
Cell viability was determined by measuring the cell-associated lactate
dehydrogenase activity as described previously (37). For the
experiments involving pyruvate and paraquat
(1,1'-dimethyl-4,4'-bipyridinum dichloride) (Sigma), AT-3 cells or 3T3
fibroblasts were pretreated with pyruvate or paraquat for 1 h and
then infected with recombinant SVs. Viability assays were performed
24 h later as described above.
Generation of Recombinant Virus--
A cDNA fragment
containing Cu,Zn-SOD (SOD wt) or Cu,Zn-SOD with a glycine to serine
mutation at codon 41 (SOD M) was cut from the
PstI/EcoRI sites of PVL1392 and was then blunt
end ligated into the BstEII site of an SV vector (dsTE12Q)
in forward and reverse orientations. A polymerase chain
reaction-amplified DNA fragment containing catalase of Hemophilus
influenza from pWB5 plasmid (a gift from Dr. William Bishai) (38)
was also blunt end ligated into the BstEII site of a
recombinant SV vector in forward and reverse orientations. These
recombinant SV vectors were linearized with XhoI and then
transcribed in vitro with SP6 RNA polymerase (Life
Technologies, Inc.). The resultant RNA transcripts were transfected
into baby hamster kidney cells by the LipofectAMINE method as described
previously (37). Recombinant SVs were harvested from the supernatant of
the culture media of the transfected baby hamster kidney cells. Viral
titers were determined by standard plaque assays as described previously.
Immunoblotting--
106 AT-3 cells were infected
with recombinant SV-SODs (wt, M or reverse (R)) at an m.o.i. of 10. 16 h postinfection, cells were lysed as described previously (37).
20 µg of protein from cytoplasmic extracts was boiled in Laemmli
buffer and electrophoresed under reducing conditions on 15%
polyacrylamide gels. Proteins were then transferred to a polyvinylidene
difluoride membrane (Bio-Rad). Nonspecific binding was inhibited by
incubation in Tris-buffered saline and Tween (50 mM
Tris-HCl, pH 8.0, 0.9% NaCl, 0.1% Tween 20) containing nonfat dried
milk for 2 h. Human SOD antibody was diluted in 1% milk,
Tris-buffered saline and Tween at 1:5,000. After exposure of membranes
to horseradish peroxidase-conjugated secondary antibody for 1.5 h,
immunoreactive proteins were detected according to the enhanced
chemiluminescent protocol (Amersham Pharmacia Biotech).
Aconitase Assay--
106 AT-3 cells or 3T3
fibroblasts were either infected with SV (AR339) at an m.o.i. of 25 or
treated with paraquat. At various intervals after paraquat treatment or
SV infection, cells were lightly trypsinized, pelleted, and resuspended
in 100 µl of reaction mixture containing 50 mM Tris-HCl,
pH 7.4, 30 mM sodium citrate, 0.6 mM
MnCl2, 0.2 mM NADP+, and 1 unit/ml
isocitrate dehydrogenase (39-41). Cells were then homogenized by
Tekmar Tissumizer for 10 s in the reaction mixture, and aconitase
activity was then determined by the absorbance increase at 340 nm at
25 °C for 30 min in a Molecular Devices SpectraMax 250 thermostatted
microplate UV-visible spectrophotometer. The results were expressed as
(aconitase activity from each time point/aconitase activity from mock
infected or mock treated cells) × 100%.
SOD Activity Measurement--
SOD activity was determined by an
indirect inhibition assay developed by Oberley and Spitz (42). Briefly,
4 × 105 AT-3 cells were either mock infected or
infected with recombinant SV-SODs (wt, M, or R) at an m.o.i. of 10 for
16 h and were then trypsinized. Cell pellets were resuspended in
100 µl of potassium phosphate buffer (50 mM, pH 7.8) and
then were homogenized by Tekmar Tissumizer for 1 min. 10 µg of cell
homogenates was used for determining SOD activity in 200 µl of
reagent containing 50 mM potassium phosphate buffer, pH
7.8, 5.6 × 10
5 M nitro blue
tetrazolium, 0.1 mM xanthine, and 0.002 unit of xanthine
oxidase. The SOD activity of cell lysates was measured spectrophotometrically by monitoring the suppression of
superoxide-induced formation of formazan from nitro blue tetrazolium at
560 nm for 5 min.
Catalase Activity Measurement--
Catalase activity was
measured as described by Aebi (43). 4 × 105 AT-3
cells were mock infected or infected with recombinant SV-catalase (forward and reverse) for 20 h. Cell extracts were then prepared as described above. 10 µg of cell extracts was added to 100 µl of
10 mM H2O2 in 50 mM
potassium phosphate buffer, pH 7.8, and the rate of loss of absorbance
of H2O2 at 240 nm was monitored for 30 s
in a Beckman DU-50 spectrophotometer.
Sindbis Virus Production--
105 AT-3 cells or 3T3
fibroblasts were grown in 12-well plates and were infected with
recombinant SV-SOD viruses at an m.o.i. of 10. 2 h after
infection, cells were washed three times with phosphate-buffered
saline, and then the infectious media were harvested at 4, 8, and
24 h postinfection. Virus titers were determined by standard
plaque assay on baby hamster kidney cells. For paraquat experiments,
3T3 fibroblasts were treated with 250 µM paraquat 1 h prior to virus infection, and viabilities were assessed as described
above at the indicated intervals.
 |
RESULTS |
Infection with SV Reduces Intracellular Superoxide Levels in AT-3
Rat Prostate Carcinoma Cells--
Aconitase activity is a sensitive
and specific assay for measuring intracellular O
2 levels in
mammalian cells (44). To test whether O
2 levels change prior
to SV-induced apoptosis, we assayed aconitase activity after SV (AR339)
infection in AT-3 rat prostate carcinoma cells. Previous studies have
established that SV (AR399) triggers cell death with morphological and
biochemical features of apoptosis 18-24 h after infection in this cell
line (8, 36). SV (AR339) increased aconitase activity within minutes of binding to and/or infecting AT-3 cells, suggesting that SV decreases
O
2 prior to the onset of apoptosis (Fig.
1). To verify that aconitase activity is
a valid measure of O
2 levels in AT-3 cells, we exposed these
cells to paraquat, a redox cycling agent capable of generating
O
2 intracellularly. As expected, we found that paraquat
reduces aconitase activity beginning 5 min after drug exposure (Fig.
1).

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Fig. 1.
SV infection increases aconitase activity in
AT-3 cells. AT-3 cells were infected with SV (AR339) ( ) at an
m.o.i. of 25, and aconitase activity was determined at the indicated
time points as described under "Materials and Methods." In
parallel, AT-3 cells treated with paraquat ( ) were also harvested
for aconitase activity assays. The percentage of aconitase activity was
determined as (aconitase activity from each time point/aconitase
activity from mock infected/treated cells) × 100%. Results represent
the mean value from three to five independent experiments ± S.E.
|
|
Recombinant SV Expressing Wild Type SOD (SV-SOD1 wt) Potentiates
SV-induced Apoptosis--
To determine whether decreases in
O
2 levels regulate SV-induced apoptosis, we generated a
recombinant SV that carries the wild type human Cu,Zn-SOD gene (SV-SOD
wt). As controls, we inserted a mutated form of the human Cu,Zn-SOD
gene (SV-SOD M) and the wild type human Cu,Zn-SOD gene in the reverse
orientation (SV-SOD R), into the SV vector. SOD1 is a cytosolic enzyme
that catalyzes the conversion of O
2 into
H2O2. The mutated form of SOD1 used in this
study has a glycine to serine mutation at codon 41. This mutation,
which is predicted to disrupt the integrity of the
sheet initiated
at codon 41 and the adjoining active site loop containing the copper
ion, was identified previously in a pedigree of familial amyotrophic
lateral sclerosis patients and has been shown to cause a significant
reduction in SOD1 activity in cultured lymphoblasts of affected
patients compared with SOD1 activity in lymphoblasts from controls
(32).
Infection of AT-3 cells with SV-SOD wt significantly enhances cell
death compared with infection with recombinant SV-SOD M or recombinant
SV-SOD R (Fig. 2A). The
potentiating effect of wild type SOD1 on SV-induced apoptosis is also
observed in 3T3 fibroblasts where 47, 70, or 65% of cells survive
after infection with SV-SOD wt, SV-SOD M, or SV-SOD R, respectively
(not shown).

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Fig. 2.
SV-SOD1 wt, but not SV-SOD1 M or SV-SOD1 R,
potentiates SV-induced apoptosis. Panel A, cell
viability of AT-3 cells infected with recombinant SV-SOD wt
(dotted bar), SV-SOD M (gray bar), and SV-SOD R
(black bar) at 24 h postinfection. A small and
reproducible level of cell death (<3%) was observed in mock infected
cultures as monitored by phase-contrast microscopy, lactate
dehydrogenase, or 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide assay. Results are expressed as the mean ± S.E. from three independent experiments. Panel B, increased
SOD activity in recombinant SV-SOD wt-infected cells. AT-3 cells
infected with or without recombinant SV-SODs for 16 h were
harvested for SOD activity assay. Results represent the mean ± S.E. from six independent experiments. White bar, control;
other bars as in panel A. Panel C,
immunoblot verified that similar levels of heterologous SOD were
expressed in recombinant SV-SOD wt- and SV-SOD M-infected cells at
16 h postinfection. N.S. refers to a nonspecific
band.
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SOD enzymatic activity was determined in lysates of mock, SV-SOD wt-,
SV-SOD M-, or SV-SOD R-infected AT-3 cells. As expected, cells infected
with SV-SOD wt significantly increased SOD enzymatic activity
(1.7-fold) compared with mock infected cells. By contrast, SOD activity
in AT-3 cells infected with SV-SOD M or SV-SOD R is not significantly
different from mock infected cells (Fig. 2B). The
differences in SOD activity between SV-SOD wt- or SV-SOD M-infected
cells could not be attributed to different levels of heterologous SOD
protein expression as monitored by immunoblot analysis (Fig.
2C).
To evaluate whether wild type SOD potentiates cell death by
facilitating SV replication, viral titers were assessed in AT-3 cells
infected with each recombinant virus. SV-SOD wt, SV-SOD M, and SV-SOD R
replicate with equal efficiency at early time points after infection
and prior to the appearance of apoptotic morphology (Fig.
3). At 24 h postinfection, the virus
production was slightly lower in cells infected with SV-SOD wt compared
with virus production of cells infected with SV-SOD M and SV-SOD R. The
difference in viral titers detected at later time points likely relates
to the decreased viability of cells infected with SV-SOD wt (Fig. 3).
Taken together, our results suggest that decreased intracellular
O
2 levels enhance SV-induced apoptosis without affecting virus
replication.

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Fig. 3.
Recombinant SV-SOD wt ( ), SV-SOD M ( ),
and SV-SOD R ( ) replicate at similar rates in AT-3 cells.
Supernatants from AT-3 cells infected with recombinant SVs were
collected at 4, 8, and 24 h postinfection, and plaque assay was
performed in duplicate in baby hamster kidney cells. Results are
expressed as the mean ± S.E. from three independent experiments.
Error bars are hidden by the symbols.
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Inhibition of H2O2 Generation by Pyruvate
Does Not Prevent the Potentiating Effect of Wild Type SOD on SV-induced
Apoptosis--
Enforced expression of wild type SOD has been shown to
result in increased steady-state levels of hydrogen peroxide
(H2O2) (45). Hydrogen peroxide can induce
cytotoxicity by reacting with transition metals such as iron to form
highly reactive hydroxyl radicals. To examine whether enhanced
generation of H2O2 by wt SOD contributes to the
death-promoting effect of recombinant SV-SOD wt, we treated 3T3
fibroblasts with pyruvate, a nonenzymatic scavenger of peroxide (46),
1 h prior to infection with recombinant SV-SOD wt, SV-SOD M, or
SV-SOD R. Although 2 mM pyruvate completely inhibits cytotoxicity induced by 200 µM hydrogen peroxide in 3T3
(Fig. 4B) or AT-3 cells (not
shown), pyruvate (2 mM or 10 mM) has no effect
on the potentiation of SV-induced death by wild type SOD1 (Fig.
4A). These results suggest that potentiation of cell death by SV-SOD is not mediated through peroxide. However, we cannot exclude
the possibility that SV induces metabolic barriers that prevent
intracellular pyruvate from reaching a concentration required for
peroxide scavenging.

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Fig. 4.
Pyruvate, a peroxide scavenger, does not
reverse the potentiating effect of wild type SOD on SV-induced
apoptosis in 3T3 fibroblasts. Panel A, 3T3 fibroblasts
were pretreated with 2 mM or 10 mM pyruvate for
1 h and then infected with recombinant SV-SODs. Cell viability was
determined by lactate dehydrogenase assay at 24 h postinfection,
and the results are presented as the mean ± S.E. from three
independent experiments. A small and reproducible amount of cell death
(<3%) was observed in mock infected cultures. Dotted bars,
SV-SOD wt; gray bars, SV-SOD M; black bars,
SV-SOD R. Panel B, 3T3 fibroblasts were pretreated with 2 mM pyruvate for 1 h and then treated with 200 µM H2O2. Cell viability was
determined after 5 h of H2O2 treatment.
Results are expressed as the mean ± S.E. from three experiments.
Error bars are hidden by the symbols.
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To examine more definitively the role of H2O2
in SV-induced apoptosis, we generated a recombinant SV carrying a
catalase gene from H. influenza (SV-Cat.). Catalase is an
antioxidant enzyme that catalyzes H2O2 into
H2O and O2. As a control, we inserted catalase
in the reverse orientation into the SV vector. Catalase activity
measurements confirmed that infection of AT-3 cells with SV-Cat.
resulted in increased levels of catalase activity compared with mock or
SV-Cat. R-infected cells (Fig.
5A). To determine the effects
of enhanced catalase activity on SV-induced apoptosis, we measured the
viability of cells infected with various recombinant viruses at several
time points after infection. As shown in Fig. 5B, we found
that SV-Cat. and SV-Cat. R triggered cell death with similar kinetics
in AT-3 cells.

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Fig. 5.
Enforced expression of catalase using the
recombinant SV vector does not affect SV-induced death.
Panel A, AT-3 cells infected with recombinant SV-Cat.
(dotted bar) have elevated levels of catalase activity
compared with mock infected (white bar) or SV-Cat.
R-infected cells (black bar). AT-3 cells were either mock
infected or infected with recombinant SV-Cat. for 20 h and were
then harvested for catalase assay. Results represent the mean ± S.E. from four experiments. Panel B, cell viability of AT-3
cells infected with recombinant SV-Cat. ( ) and SV-Cat. R ( ) at 24 and 48 h postinfection. Results were expressed as the mean ± S.E. from three independent experiments.
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Taken together, these results are consistent with the notion that the
potentiating effects of wt SOD on SV-induced death relate to decreased
O
2 levels and not increased or decreased peroxide levels.
Furthermore and consistent with our previous observations (36, 47),
these data demonstrate that H2O2 is not a
regulator of SV-induced apoptosis.
Increasing Intracellular Superoxide by Paraquat Protects from
SV-induced Apoptosis--
If decreases in superoxide trigger
SV-induced death, then increases in intracellular O
2 should
abrogate SV cytotoxicity. To address this hypothesis, we treated 3T3
fibroblasts with 250 µM paraquat 1 h prior to
infection with SV-SOD wt, SV-SOD M, and SV-SOD R. At 24 h
postinfection, 250 µM paraquat significantly inhibits
cell death induced by SV-SOD wt, SV-SOD M, and SV-SOD R (Fig.
6A).

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Fig. 6.
Paraquat, a O 2 generator, inhibits
SV-induced apoptosis in 3T3 fibroblasts. Panel A, 3T3
cells were pretreated with 100 µM or 250 µM
paraquat and were then infected with recombinant SV-SODs 1 h later
at an m.o.i. of 10. Cell viability was determined at 24 h
postinfection, and the results are expressed as the mean ± S.E.
from three experiments. Dotted bars, control; gray
bars, 100 µM paraquat; black
bars, 250 µM paraquat. Panel B, paraquat
does not significantly influence recombinant SV-SOD wt replication. 3T3
fibroblasts were treated with 250 µM paraquat and then
infected with recombinant SV-SOD wt 1 h later at an m.o.i. of 10. Recombinant SV-SOD wt produced in 3T3 fibroblasts cultured supernatants
was collected at the indicated times postinfection, and plaque assay
was performed as described under "Materials and Methods." Results
represent the mean ± S.E. from three independent experiments.
, SV-SOD wt; , SV-SOD wt + paraquat.
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To determine whether paraquat impairs SV replication, virus production
from infected 3T3 fibroblasts with or without the treatment of paraquat
was measured. Concentrations of paraquat which impair apoptosis do not
interfere with virus replication at 4, 8, and 24 h postinfection
(Fig. 6B).
 |
DISCUSSION |
Stable overexpression of individual antioxidant enzymes in cell
lines has been shown to result in increased or decreased levels of
other antioxidant enzymes in these lines (48, 49). For example,
overexpression of SOD1 in NIH/3T3 cells has been shown to alter
endogenous glutathione peroxidase, manganase superoxide dismutase, and
glutathione transferase activities (48). Under these circumstances, the
primary biological effects of modulating the levels of a single
antioxidant enzyme may be difficult to evaluate. The recombinant SV
expression system overcomes this problem by allowing the expression of
heterologous proteins after infection, avoiding the need to generate
stable cell lines and limiting the amount of time in which secondary
changes can occur. Moreover, because recombinant SV can also induce
apoptosis, the role of the expressed heterologous proteins in
regulating apoptosis can be evaluated (15, 16, 37). Herein, we used the
recombinant SV system to overexpress SOD1 or catalase in AT-3 cells and
to demonstrate a role for O
2 but not peroxide in regulating
SV-induced death.
Several observations suggest that SV infection leads to reduced
intracellular O
2 levels, leading to activation of cell death. First, aconitase activity, an in situ measure whose levels
are inversely proportional to O
2 levels (50), increases almost immediately after SV infection. Second, expression of the O
2 scavenger, SOD, in a recombinant SV vector, accelerates SV-induced apoptosis, whereas a mutated form of SOD, with impaired enzymatic activity, does not (Fig. 2A). Third, the O
2
generator, paraquat, inhibits SV-induced apoptosis (Fig.
6A). These results suggest that intracellular O
2
may inhibit components of the death machinery required for SV-induced
apoptosis. Of note, intracellular O
2 has also been shown to be
an endogenous inhibitor of Fas-induced apoptosis (51), suggesting that
Fas- and SV-induced death may engage similar cell death pathways.
Indeed, previous evidence supports the notion that Fas- and SV-induced
apoptosis might activate a similar death pathway because both Fas- and
SV-induced apoptosis are sensitive to CrmA, a highly specific inhibitor
of caspases 1, 4, 5, or 8 (14).
Several lines of evidence indicate that Cu,Zn-SOD has a protective role
in regulating apoptosis. For example, microinjection of Cu,Zn-SOD
delays nerve growth factor deprivation-induced apoptosis in sympathetic
neurons (23). Also, overexpression of Cu,Zn-SOD inhibits apoptosis
induced by serum or growth factor withdrawal or calcium ionophore
treatment in nigral neural cells (52). Moreover, transgenic
Drosophila that carry a mutated Cu,Zn-SOD gene appeared to
have a shortened lifespan (53), whereas selective overexpression of the
wild type Cu,Zn-SOD gene in motor neurons of Drosophila with
defective Cu,Zn-SOD recover 60% of their lifespan (54). In mammals,
mice lacking Cu,Zn-SOD are more sensitive to axonal injury (55). These
converging lines of inquiry suggest that Cu,Zn-SOD can promote survival
in some circumstances. In contrast, transgenic mice overexpressing
Cu,Zn-SOD appeared to have significantly fewer thymocytes and are more
susceptible to lipopolysaccharide-mediated loss of thymocytes (56).
Moreover, neurons derived from transgenic mice overexpressing Cu,Zn-SOD also showed higher susceptibility to kainic acid-induced apoptosis (57). Therefore, constitutive expression of a O
2 scavenging enzyme may also sensitize cells to certain kinds of stimuli; these observations are consistent with our observations that overexpression of Cu,Zn-SOD exacerbates SV-induced apoptosis.
In contrast to established schemes whereby ROS are increased prior to
the activation of apoptosis (58), we find that intracellular ROS,
specifically O
2 levels, are decreased soon after SV infection (Fig. 1). The mechanism by which SV triggers the decrease of
intracellular O
2 is still unclear. However, this finding
combined with our previous observation that SV infection leads to
increased intracellular glutathione levels (36) suggests that SV may
activate the apoptotic pathway by perturbing the redox equilibrium
toward a reduced state rather than an oxidized state. Of note, a
decreased production of ROS has also been shown to be an early event in
dexamethasone-induced apoptosis in thymocytes (59), suggesting a more
general role for "reductive stress" in activating cell death.
What are the putative targets modified by decreased levels of
intracellular O
2 which regulate SV-induced death? Our results suggest that decreasing intracellular O
2 does not
facilitate SV replication because recombinant SV -SOD1 wt replicates
as well as the SV-SOD1 M and SV-SOD1 R (Fig. 3). Additionally, paraquat protects from SV-induced apoptosis and does not alter SV replication (Fig. 6B). Therefore, it seems likely that O
2
may play a role in changing the activity of as yet unidentified
cellular targets involved in regulating SV-induced apoptosis.
O
2 itself is capable of inactivating certain kinases, such as
mammalian creatine phosphokinase (60) and the NADH dehydrogenase
complex of the mitochondrial electron transport chain (61); however,
the roles of these proteins in apoptosis are still unknown. Another
potential target of O
2 are the
caspases-cysteine-dependent proteases whose activity has been shown to be altered by redox-active agents (62). It is unlikely
that the target modulated by superoxide levels which negatively
regulates SV-induced cell death is aconitase or its iron-deficient
analog IRP-1, as previous studies from our laboratory showed that the
iron chelators do not inhibit SV-induced death in AT3 cells (36). If
decreased IRP-1 activity or increased aconitase activity were the
mechanism by which the apoptotic signal is mediated, then iron
chelators, which enhance IRP-1 activity and decrease aconitase activity
(63), should attenuate SV-induced death.
In addition to O
2, nitric oxide has also been shown to inhibit
SV-induced apoptosis in cultured cells and diminish SV-mediated mortality in mice (64). Treatment of fetal mice with a nitric oxide
synthase inhibitor potentiates SV-induced encephalitis and mortality;
in addition, treatment of N18 neuroblastoma cells with nitric acid
donors enhances cell survival after SV infection. These results
combined with our observations suggest that O
2 and nitric
oxide are the free radical species that negatively regulate SV-induced apoptosis.
Previously, we have demonstrated that SV-induced apoptosis may not
result from the generation of ROS, as a panel of antioxidants failed to
protect from SV-induced apoptosis (36). Here, in an extension of our
previous study, we demonstrate that inhibition of intracellular
O
2 levels is required for SV-induced apoptosis. These results
not only provide insight into the mechanism by which SV transduces its
death signal but also broaden the concept of oxidative stress-induced
apoptosis; that is, reductive stress may also trigger cells to undergo apoptosis.
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ACKNOWLEDGEMENTS |
We thank Drs. Ron Morton and Carl W. White
for providing the aconitase activity assay protocol and Dr. Khalequz
Zaman for providing SOD activity assay and catalase activity assay
protocols. We thank Dr. William. R. Bishai for the pWB5 plasmid and Dr.
Bruce Demple for helpful suggestions.
 |
FOOTNOTES |
*
This work was supported by Grants NINDS R29, NS34943, and
KO8 NS01951 from the National Institutes of Health (to R. R. R.).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.
**
To whom correspondence should be addressed: Dept. of Neurology,
Harvard Medical School and the Beth Israel Deaconess Medical Center, 77 Avenue Louis Pasteur, Rm. 857, Boston, MA 02115. Tel.: 617-667-0802;
Fax: 617-667-0800; E-mail: rratan{at}caregroup.harvard.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
SV, Sindbis virus;
Cu, Zn-SOD, copper/zinc superoxide dismutase;
ROS, reactive oxygen
species;
O
2, superoxide;
m.o.i., multiplicity of infection;
SOD wt, wild type Cu,Zn-SOD;
SOD M, Cu,Zn-SOD with a glycine to serine
mutation at codon 41;
SOD R, reverse Cu,Zn-SOD;
SV-Cat., SV carrying a
catalase gene from H. influenza..
 |
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