From the Departments of Molecular and Cellular Physiology and
Medicine, Louisiana State University Health Sciences
Center, Shreveport, Louisiana 71130-3932
Received for publication, August 23, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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The current study examines the contribution of
mitochondria-derived reactive oxygen species (ROS) in
tert-butyl-hydroperoxide (TBH)-induced apoptotic signaling
using clones of undifferentiated pheochromocytoma (PC-12) cells that
stably overexpress the human mitochondrial or cytoplasmic forms of
superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD,
respectively). Exposure of wild type cells to TBH caused an early
generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine
pretreatment; however, N-acetylcysteine was ineffective in
cytoprotection when added after TBH-induced ROS formation. Stable
overexpression of SOD isoforms caused a 2- and 3.5-fold elevation in
CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria,
respectively, and 3-fold increases in cellular GSH content.
Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced
mitochondrial ROS generation and cell apoptosis. Whereas transient
Mn-SOD expression similarly prevented PC-12 apoptosis, this was
associated with increases in SOD activity but not GSH, indicating that
cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS
elimination and not due to increases in cellular GSH content per
se. Stable or transient CuZn-SOD overexpression exacerbated cell
apoptosis in conjunction with accelerated caspase-3 activation,
regardless of cell GSH levels. Collectively, our results support a role
for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated
by Mn-SOD overexpression and is independent of cellular GSH levels
per se.
The involvement of the mitochondria in apoptotic signaling is a
generally accepted paradigm for the activation of cellular apoptosis
mediated by oxidants (1). However, the contribution of mitochondrial
generated ROS to the initiation of apoptotic signaling and the roles
that the mitochondrial antioxidant enzyme, manganese superoxide
dismutase (Mn-SOD),1 and the
cytoplasmic copper zinc superoxide dismutase (CuZn-SOD) play in
modulating oxidant-induced cell apoptosis are not completely resolved.
Previous studies in animal and cell models of SOD overexpression have
demonstrated that overexpression of CuZn-SOD and Mn-SOD can both be
beneficial and detrimental. For instance, different studies have shown
that CuZn-SOD overexpression rendered mice more susceptible to
infection (2), induced muscle aberrations (3), and destruction of axons
in neuromuscular junctions (4) and death of trisomy 16 neuronal cells
(5), yet other studies showed that enzyme overexpression decreased
myocardial ischemia/reperfusion injury (6). In comparison,
overexpression of Mn-SOD has generally exerted protective effects, such
as protection of murine fibrosarcoma cells from
5-azacytadine-dependent apoptosis (7), attenuation of
hyperglycemic-induced bovine endothelial cells oxidative stress (8),
and protection against adriamycin-induced acute cardiac toxicity (9).
Moreover, increased oxidative damage has been found to be associated
with altered mitochondrial function in heterozygous Mn-SOD knockout
mice (10). However, increased invasiveness of tumor metastasis has been
associated with Mn-SOD overexpression (11).
The quantitative significance of SOD overexpression in cell apoptosis
is poorly defined. Moreover, a direct link between SOD overexpression
and the intracellular antioxidant status, such as GSH, has not been
vigorously explored. Previous studies have shown that as compared with
control animals, transgenic mice overexpressing the heterozygous form
of Mn-SOD (Mn-SOD The objective of the current study was designed to address these
questions using tert-butyl-hydroperoxide (TBH) as a model hydroperoxide and undifferentiated pheochromocytoma (PC-12) cells, a
cell model that has been previously characterized by Greene and
Tischler (14), to study the cellular and molecular aspects of neuronal
apoptosis following induction of cell differentiation in culture with
nerve growth factor. To address our hypothesis that mitochondrial
derived ROS are an important functional mediator of TBH-induced
apoptosis in PC-12 cells, we have generated PC-12 clones that
overexpress the human mitochondrial Mn-SOD or the human cytoplasmic
CuZn-SOD. Given the central role that the mitochondria play in the
initiation of the apoptotic cascade mediated by oxidants and redox
imbalance (1, 15), we determined the contribution of mitochondrial ROS
and cellular redox shifts to cell apoptosis in PC-12 cells that stably
or transiently overexpress Mn-SOD or CuZn-SOD.
Materials--
The following chemicals were obtained from Sigma:
agarose, TBH, 4',6-diamidino-2-phenylindole (DAPI), and DNA markers
( Cell Culture and Incubations--
Pheochromocytoma cells (PC-12)
were a generous gift from Dr. Nikki Holbrook (Yale University, New
Haven, CT). Wild type PC-12 cells as well as PC-12 clones that were
stably transfected with the pRetro-Off vector or pRetro-Off vector
containing human Mn-SOD or CuZn-SOD were cultured in Dulbecco's
modified Eagle's medium containing 25 mM glucose with 10%
heat-inactivated fetal bovine serum, 5% heat-inactivated horse serum,
and 2 mM glutamine at 37 °C in a 95% air, 5%
CO2 humidified environment. PC-12 clones were selected in
puromycin (1.5 nmol/ml), and the culture medium was changed every 2 days. For all experiments, wild type PC12 cells or PC-12 clones were
plated at a specified density the day before the experiment was
performed. On the day of the experiment, the culture media were
replaced with fresh Dulbecco's modified Eagle's medium. TBH was added
to cell cultures at a final concentration of 100 µM. In
designated experiments, cells were treated with the following agents at
the specified final concentrations: NAC, 5 mM; rotenone, 50 µM; antimycin A, 1 µM.
Detection of Apoptosis by DAPI Staining--
DAPI staining was
performed according to the method of Wang et al. (16).
Briefly, 1 × 105 wild type cells or PC12 clones were
grown on 12-mm circular glass cover slips in 24-well plates. Cells were
treated with 100 µM TBH for 24 h, washed with PBS,
and fixed with cold 4% paraformaldehyde for 15 min. Cells were then
washed with PBS, fixed with cold 70% ethanol at Measurements of GSH and GSSG--
Cellular glutathione (GSH and
GSSG) was determined by the high performance liquid chromatography
method of Reed et al. (17). Cells (2 × 106) were cultured in 100-mm culture plates and exposed to
TBH in 10 ml of Dulbecco's modified Eagle's medium. At time points
ranging from 0 to 6 h, cells were washed with PBS and then
harvested by scraping in ice-cold 5% trichloroacetic acid. The cell
suspensions were centrifuged to remove the trichloroacetic
acid-insoluble proteins. The acid supernatant was derivatized with 6 mM iodoacetic acid and 1% 2,4-dinitrophenyl fluorobenzene
to yield the S-carboxymethyl and 2,4-dinitrophenyl
derivatives of GSH and GSSG, respectively. Separation of GSH and GSSG
derivatives was performed on a 250 × 4.6-mm Alltech Lichrosorb
NH2 10-µm column. Cellular GSH and GSSG contents were
quantified by comparison with standards derivatized in the same manner.
Preparation of Cell Lysates and Western Analyses of
CPP32--
PC12 clones (2 × 106) were plated in a
T-25 flask. After treatment with 100 µM TBH for specified
times from 0-6 h, cells were ruptured with 300-600 µl of lysis
buffer containing 300 mM NaCl, 50 mM Tris-HCl,
0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1.8 mg/ml
iodoacetamide for 30 min at 4 °C. Adherent cells were scraped, and
whole cell extracts were prepared by homogenization. Cell extracts were
stored at Western Analyses of PARP Cleavage--
PC12 clones were plated
at a density of 2 × 106 cells and were harvested at
the indicated times (0-6 h) after TBH treatment according to the
method of Stefanis et al. (18). Cells were scraped and
collected into buffer (25 mM HEPES, pH 7.5, 5 mM EDTA, 2 mM EDTA, 1% Triton X-100, 10 µg/ml each pepstatin and leupeptin, and 1 mM
phenylmethylsulfonyl fluoride) and sonicated. The nuclear pellets were
separated from supernatants by centrifugation (1,500 rpm, 5 min). The
pellets were stored at Determination of Total Cellular and Mitochondrial Oxidant
Production--
Total cellular oxidant formation was measured using
the oxidant-sensitive nonfluorescent probe, dihydrorhodamine 123 (DHR). Previous studies have utilized oxidation of DHR for the detection of
general cellular oxidant production given that DHR oxidation is
mediated by biological oxidants like peroxynitrite and a variety of
secondary hydrogen peroxide-dependent intracellular
reactions such as H2O2-cytochrome c
and H2O2-Fe2+ (19, 20).
Intracellular two-electron oxidation of DHR results in the formation of
the fluorescent product, rhodamine 123, which preferentially
accumulates in mitochondria according to the trans-mitochondrial potential although DHR can be oxidized in different compartments throughout the cell. Measurement of rhodamine fluorescence at the
excitation/emission wavelengths of 500 nm/536 nm provides reasonable
quantification of the overall cellular oxidant production. DHR was
prepared as a 25 mM stock solution in nitrogen-purged dimethyl formamide and kept stored in the dark at
Mitochondrial oxidant production was assessed using the
mitochondria-specific ROS-sensitive fluorescence probe, Mito Tracker Red. PC-12 cells stably transfected with pRetro-Off vector control or
Mn-SOD were grown on coverslips and treated with 100 µM
TBH for 0-45 min. Cells were washed twice with PBS and then incubated for 15 min with 50 nM Mito Tracker Red
(CM-H2XROS; Molecular Probes, Inc., Eugene, OR). To verify
that mitochondria were the site of ROS formation, co-incubations were
performed with 50 nM Mito Tracker Green FM (Molecular
Probes), a mitochondria-specific fluorescence probe. Cells loaded with
the fluorescence probes were imaged using a Nikon E600 fluorescence
microscope, with ×40/1.0 numerical aperture oil or ×60/1.4 numerical
aperture oil objectives and excitation/emission wavelength pairs of 490 nm/516 nm and 581 nm/644 nm, for Mito Tracker Green and Mito Tracker
Red, respectively. Illumination was provided by a 50-watt X-Cite
metal-halide source (EFOS, Inc., Mississauga, Canada), which gave
constant illumination intensity across the microscope field. All images
were captured using a SynSys digital camera (Photometrics, Tuscon, AZ)
at equal exposure time (500 ms) as determined from the fluorescent
emission from untreated pRetro-Off control cells. For the overlaid
images, the exposures were made at the same plane of focus for both
excitation wavelengths at equal times. These images were computer
graphically overlaid and analyzed using MetaMorph Software (Universal
Imaging, Downingtown, PA).
Production of Mammalian Expression Vectors Containing Mn-SOD or
CuZn-SOD--
The human Mn-SOD cDNA was isolated from pBluescript
following NotI and SalI digestion and ligated
into the NotI and SalI sites of pBI. This
resulting vector was digested with NotI and XmnI to remove a fragment of DNA containing the Mn-SOD cDNA ligated to
the SV40 polyadenylation signal. pRetro-Off was digested with BamHI and treated with Klenow to produce blunt end termini.
After further digestion of pRetro-Off with NotI, the Mn-SOD
NotI-XmnI fragment was ligated into pRetro-Off.
The human CuZn-SOD was isolated from pBluescript following
BamHI digestion and ligated into the BamHI site
of pRetro-Off. The orientation of the cDNAs of Mn-SOD and CuZn-SOD
in pRetro-Off was confirmed by restriction enzyme analysis and sequencing.
Transfection of PC-12 Cells--
Transfection of expression
vectors was performed by electroporation. The transfection efficiency
of PC-12 cells was initially tested by electroporating pGreenlantern
(Invitrogen), a plasmid that expresses the green fluorescent protein,
into cells using voltages of 150-350 V. After 24-48 h, transfection
efficiency was assessed by the percentage of cells expressing green
fluorescent protein by flow cytometry. At 200 V, the transfection
efficiency was typically 35-40%. Test (Mn-SOD-pRetro-Off or
CuZn-SOD-pRetro-Off) or control (pRetro-Off) vectors (10 µg) were
mixed in 200 µl of sterile PBS and used to suspend 5 × 106 cells. Cell and DNA mixtures were subjected to 200 V at
a capacitance of 960 microfarads. The cells were resuspended in growth
media and allowed to grow for 2-3 days. After replating at a
population density of 5 × 103/cm2, cells
were grown in puromycin (1.5 nmol/ml) to select for the cells that have
integrated the plasmid DNA into their genome. For generation of stable
Mn-SOD- or CuZn-SOD-overexpressing cell lines, cells were medium
changed every 2 days until single colonies formed. The colonies were
isolated and individually expanded to form the respective cell lines.
Genomic DNA was prepared from cells using the Qiamp DNA blood minikit
(Qiagen) and tested for the presence of vector DNA incorporation in the
genome using PCR.
Determination of SOD Activities--
As previously described,
total SOD activity (cytoplasmic CuZn-SOD and mitochondrial Mn-SOD) was
determined by the ability of cell-free extracts to inhibit the
oxidation of cytochrome c by xanthine oxidase in the absence
of cyanide (21). The oxidation of cytochrome c was measured
by a change in the absorbance at 550 nm. Mn-SOD activity was determined
in the presence of cyanide to inhibit CuZn-SOD activity. CuZn-SOD
activity was calculated as the difference between the total SOD
activity and that of Mn-SOD. Results are expressed as units/mg protein.
Protein Assay--
Protein was measured using the Bio-Rad
Protein Assay kit according to the manufacturer's protocol.
Statistical Analysis--
Results are expressed as mean ± S.E. Data were analyzed using a one-way analysis of variance with
Bonferroni corrections for multiple comparisons or using Student's
t test. p values of <0.05 were considered as
statistically significant.
TBH-induced PC-12 Apoptosis Is Associated with ROS Generation and
the Abrogation by NAC--
For our studies, we have generated three
clones of pRetro-Off as well as three clones of Mn-SOD-overexpressing
and two clones of CuZn-SOD-overexpressing cells. The results of a
complete set of studies using one clone from each of pRetro-Off,
Mn-SOD, and CuZn-SOD are presented. Initial experiments show that the
other respective clones behaved similar to one another and gave
reproducible results in response to TBH.
Fig. 1 summarizes the results on the
effect of TBH on cellular production of ROS and cell apoptosis in
undifferentiated wild type PC-12 cells. Fig. 1A shows that
treatment of cells with 100 µM TBH caused a 3-fold
increase in rhodamine fluorescence at 30 min, consistent with an early
generation of ROS. This TBH-induced DHR oxidation was eliminated by
pretreatment of cells with the thiol antioxidant, NAC. The production
of ROS caused by TBH elicited ~37% cell apoptosis at 24 h,
which was completely prevented by NAC pretreatment (Fig.
1B). Notably, the addition of NAC at 1 h after TBH
exposure and at a time after ROS increase did not confer cytoprotection
against TBH challenge (Fig. 1B). Collectively, these results
are consistent with the suggestions that ROS are a mediator of
TBH-induced PC-12 apoptosis and that the initiating signaling occurred
within the first 30 min after cells were treated with TBH.
Mn-SOD and CuZn-SOD Differentially Affect Apoptosis in PC12
Cells--
To determine the intracellular source of ROS
production, we generated PC-12 clones that overexpress the human
mitochondrial Mn-SOD or the human cytoplasmic CuZn-SOD cDNAs. The
insertion of Mn-SOD and CuZn-SOD pRetro-Off mammalian expression
vectors into the genome of PC-12 cells was confirmed by PCR (data not shown). The extent of Mn-SOD and CuZn-SOD overexpression was quantified by the respective enzyme activities of the SOD isoforms. The data in
Fig. 2A show that transfection
with Mn-SOD resulted in a 3.5-fold increase in cellular Mn-SOD enzyme
activity, whereas transfection with CuZn-SOD resulted in a 2-fold
elevation in CuZn-SOD activity as compared with the respective SOD
activities in wild type PC-12 cells and pRetro-Off vector controls.
Differential fractionation of cell extracts confirmed that the
transfected Mn-SOD and CuZn-SOD proteins were localized to the
respective mitochondrial and cytoplasmic compartments (Fig.
2B). In each instance, the endogenous enzyme activity of the
other SOD isoform was unaffected by the transfections, indicating that
the overexpression of either Mn-SOD or CuZn-SOD did not cause a
compensatory up- or down-regulation of the other isoform.
The results on the effect of TBH on apoptosis of wild-type PC-12 cells
and PC-12 clones that stably overexpress Mn-SOD or CuZn-SOD are
summarized in Fig. 3. TBH induced ~32%
apoptosis in cells transfected with the pRetro-Off vector, a value that was similar to wild type (35%), indicating that the introduction of
the vector per se did not influence the effect of TBH.
Notably, Mn-SOD-overexpressing cells (hereafter termed Mn-SOD-S cells) were significantly protected from TBH-induced apoptosis, whereas apoptosis was exacerbated in CuZn-SOD-overexpressing cells (hereafter termed CuZn-SOD-S cells). These results suggest that the differential effects of TBH on apoptosis of PC-12 cells overexpressing Mn-SOD or
CuZn-SOD were associated with the increases in the respective enzymes
in these cells.
TBH Induces Differential Kinetics of Caspase-3 Activation in
Mn-SOD- and CuZn-SOD-overexpressing Cells--
The base-line
expression of CPP32 in pRetro-Off vector control and Mn-SOD-S cells
(Fig. 4, A and B,
respectively) was low, in agreement with wild type PC-12 cells (22,
23). In contrast, base-line procaspase-3 expression was elevated in
CuZn-SOD-S cells (Fig. 4C). TBH treatment caused an increase
in CPP32 levels in Mn-SOD-S cells at 30 min followed by a decrease at
6 h (Fig. 4B), similar to pRetro-Off control (Fig.
4A) and wild type PC-12 cells (22). In CuZn-SOD-S cells, TBH
challenge resulted in a marked decrease in procaspase-3 levels at 30 min, consistent with a rapid cleavage of CPP32 to active caspase-3. The
time course of caspase-3 activation directly correlated with the
cleavage of its target substrate, PARP, from a native 116-kDa protein
to an 85-kDa product. Significant cleavage of PARP occurred at 6 h
in pRetro-Off and Mn-SOD-S cells (Fig. 4, A and
B, respectively) and 30 min in CuZn-SOD-S cells (Fig.
4C).
TBH-induced ROS Production Is Attenuated in Mn-SOD-S but Not in
CuZn-SOD-S Cells--
Fig. 5 shows that
TBH caused 4-fold increases in ROS production in wild type PC-12 and
pRetro-Off vector controls, indicating that introduction of a plasmid
per se did not promote ROS formation. Mn-SOD overexpression
attenuated DHR oxidation, consistent with a role for mitochondrial
derived ROS in PC-12 apoptosis caused by TBH. Interestingly, CuZn-SOD
overexpression was without effect on TBH-induced cellular ROS
production, consistent with a minor contribution of cytosolic ROS to
cell apoptosis.
Two additional strategies were used to confirm that mitochondrial
derived oxidants were responsible for PC-12 cell apoptosis induced
by TBH. First, we pretreated pRetro-Off control cells with known
inhibitors of mitochondrial sites of ROS production, namely, rotenone,
an inhibitor of Complex I (NADH dehydrogenase), and antimycin A, an
inhibitor of Complex III (cytochrome bc1 complex). The
results (Fig. 6) show that rotenone as
well as antimycin A treatment significantly attenuated TBH-induced
apoptosis, indicating that blockade of electron flux at Complex I and
Complex III can effectively prevent the apoptotic outcome, in agreement
with previous observations (22). Second, the mitochondrial source of
oxidant production was directly assessed using Mito Tracker Red, a
mitochondria-specific ROS sensitive fluorescence probe, and verified
with Mito Tracker Green co-incubation. In untreated cells, mitochondria
predominantly exhibited green fluorescence, consistent with low ROS
generation (Fig. 7, A and
E). TBH challenge in pRetro-Off control cells caused rapid
and time-dependent increases in red fluorescence relative to green (15-45 min; Fig. 7, B-D), consistent with
enhanced ROS production. In contrast, red fluorescence was not
detectable at 15 min and minimally increased at 30 min in TBH-treated
Mn-SOD-S cells (Fig. 7, F and G), indicating
attenuated and kinetically delayed ROS formation. Consistent with this
suggestion, the extent of Mito Tracker Red fluorescence at 45 min in
Mn-SOD-S cells (Fig. 7H) was similar to control cells at 15 min after oxidant exposure (Fig. 7B). Parallel cell
incubations of pRetro-Off controls and Mn-SOD-S cells with
2',7'-dichlorofluorescein acetate, a cytoplasmic specific
ROS probe, gave no detectable fluorescence at all time points (data not
shown), indicating a lack of ROS generation in the cytoplasm. Taken
together, these results demonstrate that mitochondrial ROS production
is a critical early step in TBH-induced apoptotic signaling in PC-12
cells.
Glutathione Levels Are Differentially Affected by TBH in Mn-SOD-S
and CuZn-SOD-S Cells--
To determine whether the TBH-induced cell
apoptosis was associated with altered cellular redox, we determined the
cellular concentrations of GSH and GSSG. The overexpression of Mn-SOD
or CuZn-SOD alone in stably transfected PC-12 cells significantly elevated cellular GSH (3-fold above base-line values) (Fig.
8A), consistent with an
increase in GSH synthesis as an adaptive response to elevated SOD
levels. TBH challenge in pRetro-Off controls transiently decreased
cellular GSH in 5 min but recovered to base-line levels by 30 min (Fig.
8A). Similar kinetics of GSH responses were observed in
CuZn-SOD-S cells exposed to TBH (Fig. 8A). In contrast, TBH caused a small decrease in cell GSH in Mn-SOD-S cells at 30 min (Fig.
8A); this delay in GSH loss is consistent with
reduced ROS formation in these cells (see Fig. 7). The time course of
GSSG increases in pRetro-Off controls (at 5 min) and Mn-SOD-S cells (at
30 min) (Fig. 8B) corresponded to the decreases in GSH in these cells (see Fig. 8A). By 1 h, GSSG levels returned
to base-line values. In contrast, TBH caused a marked and sustained
elevation in cellular GSSG levels from 5 min to 2 h in CuZn-SOD-S
cells (Fig. 8B), indicating an induction of an exaggerated
oxidized state in cells that stably overexpress the cytoplasmic form of SOD. The ratio of GSH to GSSG is high in Mn-SOD-stable transfectants, indicative of a highly reduced intracellular milieu that was minimally altered with TBH challenge (Fig. 8C). A substantially lower
GSH/GSSG ratio was found in CuZn-SOD-stable transfectants (Fig.
8C), which largely reflected the exaggerated GSSG levels
despite high GSH contents, consistent with a high level of base line
oxidative stress in these cells. Treatment with TBH further exacerbated the cellular GSH/GSSG imbalance (Fig. 8C). Cells were
subjected to digitonin fractionation (24) to determine the mitochondria or cytoplasmic sites of GSH oxidation. Initial results showed that
Mn-SOD-S cells exhibited higher mitochondrial GSH content than
pRetro-Off vector controls and were minimally altered by TBH treatment.
Unfortunately, the values were near the limit of detection (at the
optimal cell density of 106/ml for this method), which
precluded accurate quantification and meaningful conclusions regarding
specific intramitochondrial GSH/GSSG changes. Current efforts are aimed
at optimizing the digitonin method for handling larger quantities of
cells without compromising the effective separation of mitochondrial
and cytoplasmic compartments.
Transient Expression of Mn-SOD and CuZn-SOD Does Not Induce GSH
Increases--
To test whether the elevation in GSH content per
se secondary to stable Mn-SOD expression mediated the protection
against TBH-induced apoptosis in Mn-SOD-S cells, we performed transient transfections with Mn-SOD to elevate enzyme activity without a concomitant adaptive increase in cellular GSH content. Cells
transiently transfected with Mn-SOD (hereafter termed Mn-SOD-T)
exhibited 1.8-fold increases in mitochondrial enzyme activity (2.4 units/mg protein versus 1.2 units/mg protein in pRetro-Off),
whereas cells transiently transfected with CuZn-SOD (hereafter termed
CuZn-SOD-T) exhibited 1.5-fold increases in cytoplasmic enzyme activity
(3.1 units/mg protein versus 2 units/mg protein in
pRetro-Off). The cellular GSH levels in Mn-SOD-T were not different
from vector controls or wild type cells (22) but were significantly
lower than those in stable Mn-SOD transfectants (Fig.
9A). Furthermore, the ratio of
GSH to GSSG in Mn-SOD-T resembled that in pRetro-Off vector controls
and wild type cells (Fig. 9B). CuZn-SOD-T exhibited base-line levels of GSH and GSH/GSSG ratio similar to controls (Fig. 9,
A and B) and wild type cells (22). Whereas the
cellular GSH content in stable CuZn-SOD transfectants was elevated, the GSH/GSSG ratio was decreased (Fig. 9B), a consequence of the
exaggerated GSSG level in the CuZn-SOD-overexpressing cells.
TBH-induced Apoptosis Is Attenuated in Mn-SOD-T and Exacerbated in
CuZn-SOD-T Cells--
Fig. 10
summarizes the results on the effect of TBH on apoptosis of Mn-SOD-T
and CuZn-SOD-T cells. The transient expression of Mn-SOD resulted in
significant protection against TBH-induced apoptosis, but apoptotic
death was potentiated in cells transiently transfected with CuZn-SOD,
similar to the responses in cells stably transfected with the
respective SOD isoforms (see Fig. 3).
In a recent study, we have provided evidence that the induction of
apoptosis in undifferentiated PC-12 cells by tert-TBH
follows a sequence of events that were consistent with
mitochondrial signaling in apoptosis (namely TBH-induced ROS
generation, loss of redox imbalance, mitochondrial cytochrome
c release, and activation of caspase-3) (22). Our current
results support a role for mitochondrial ROS in mediating PC-12
apoptosis. This contention is supported by several lines of
evidence. The finding that transfection with Mn-SOD afforded protection
against TBH-induced apoptosis that directly correlated with a decrease
in ROS generation is consistent with the quenching of mitochondrially
generated ROS by Mn-SOD. In comparison, the overexpression of CuZn-SOD
was without effect. Using mitochondria specific ROS probes, we have
verified that mitochondria are the important sites of ROS formation.
Moreover, blockade of electron flow at NADH dehydrogenase (Complex I)
and cytochrome bc1 complex (Complex III)
effectively prevented TBH-induced cell apoptosis, consistent with
mitochondrial ROS production being a critical step in apoptotic
signaling, in agreement with previous studies (22). We found similar
attenuation of lipid hydroperoxide-induced apoptosis in CaCo-2
intestinal cells by pretreatment of cells with rotenone or antimycin A
but not with thenoyltrifluoroacetate, an inhibitor of succinate
dehydrogenase (Complex II).2
These findings support current paradigms that Complexes I and III, but
not Complex II, are major intramitochondrial sources of superoxide
formation (25, 26). Taken together, the data support our contention
that enhanced mitochondrial production of ROS induced by TBH challenge
is an important trigger of the mitochondrial redox signaling events
that subsequently lead to PC-12 cell apoptosis. At present, the trigger
for mitochondrial ROS generation is unknown, but evidence in the
literature suggests a linkage to mitochondrial cytochrome c
loss (27). Regardless of the mechanism, the current results demonstrate
that the time window for ROS signaling was within 30-60 min after TBH
treatment, since the addition of NAC at 1 h following TBH exposure
failed to rescue the cells from the apoptotic outcome at 24 h.
In mitochondrial apoptotic signaling, the release of cytochrome
c functions as the initiator and mediator of the
apoptotic cascade via the activation of caspase-3 (28, 29).
Previous studies have shown that the channel-mediated mitochondrial
exit of cytochrome c via function of the BcL-2 families of
pro- and antiapoptotic proteins (29) subsequently leads to the
activation of caspase-3. Notably, the kinetics of caspase-3 activation
and PARP cleavage at 30 min after oxidant challenge in CuZn-SOD-S cells
is consistent with the rapid initiation of apoptotic signaling and the
exacerbation of apoptosis in these cells. The activation kinetics of
caspase-3 and PARP cleavage in Mn-SOD-S cells were substantially slower
at 6 h downstream of TBH-induced cellular GSH/GSSG shifts and in
conjunction with delayed mitochondrial ROS production. These results
support the paradigm that activation of mitochondrial signaling
determines the point of no return for the activation of cellular
apoptosis for a variety of death signals like oxidants and DNA damage
(30).
It is notable that stable transfection with Mn-SOD or CuZn-SOD resulted
in significant increases in intracellular GSH concentrations (3-4-fold), consistent with an adaptive synthesis of GSH secondary to
the stable overexpression of the SOD isoforms, since transient overexpression of the enzymes did not elicit GSH increases. Increases in GSH with SOD overexpression have been demonstrated (5, 31). Interestingly, despite a similar magnitude of GSH increases, only Mn-SOD-overexpressing and not CuZn-SOD-overexpressing cells were found
to be protected against TBH-induced apoptotic death. This indicates
that PC-12 cell survival against TBH stress is related to the
expression of the mitochondrial SOD isoform rather than to the direct
antioxidant effect of elevated GSH content associated with SOD
overexpression. However, while independent of changes in cell GSH
per se, our current data show that cell survival is linked
to the cellular GSH/GSSG ratio status. For instance, the susceptibility
of CuZn-SOD-overexpressing cells to TBH-induced apoptosis corresponded
to the finding that CuZn-SOD-stable transfectants exhibited high GSSG
contents and low cellular GSH/GSSG ratios despite elevated GSH levels,
consistent with an exaggerated oxidized state that is exacerbated by
TBH challenge. This conclusion is in agreement with previous studies in
that the apoptotic end point in wild type PC-12 cells mediated by TBH
is dictated by a shift in the redox status toward oxidation
(i.e. increase in GSSG relative to GSH) rather than simply
by an alteration in cell GSH content per se (22). One
explanation for the susceptibility of CuZn-SOD-S cells to
oxidative stress may be that overexpression of this SOD isoform results
in enhanced ROS production as suggested by earlier studies (3, 32, 33).
However, our results (Fig. 5) did not support this contention.
A consistent observation in our study is the low protein expression of
procaspase-3 in unchallenged pRetro-Off controls and Mn-SOD-S cells,
which was elevated within 30 min of TBH treatment (Fig. 4), an
observation similar to wild type PC-12 cells exposed to TBH (22). The
reason for the unusual kinetics is unclear and may be related to
increased gene induction or, more likely, increased mRNA
translation, given the rapidity of protein expression. It is noteworthy
that the time course of CPP32 elevation appeared to be directly
correlated with the extent of oxidative stress within cells and how
readily these cells ultimately succumbed to apoptosis. For instance,
the overexpression of CuZn-SOD exacerbated cell apoptosis and caused a
more oxidized intracellular environment than Mn-SOD overexpression or
pRetro-Off vector controls, and this phenotype was associated with an
elevated basal CPP32 expression even prior to TBH stress (at 0 min)
(Fig. 4). In a related study, we similarly found an accelerated CPP32
elevation (within 5 min) in association with exposure of PC-12 cells to
lipid hydroperoxides, a more potent oxidant than
TBH,3 consistent with the
notion that the potency of the apoptotic stimuli and the severity of
the induced oxidative stress within target cells dictates the kinetics
of procaspase-3 up-regulation. Alternately, our results may also be
explained by the possibility that CPP32 is more easily extractable from
oxidatively stressed cells. At present, we have no evidence to support
this latter suggestion.
In summary, we have shown that overexpression of Mn-SOD afforded
protection of PC-12 cells against TBH-induced apoptosis that is
related to enhanced mitochondrial ROS elimination, thus underscoring the contribution of mitochondrial derived ROS to initiation of apoptotic signaling. The lack of cytoprotection by CuZn-SOD
overexpression despite adaptive increases in cell GSH indicates that
elevation in GSH content per se was not a key determinant of
cell survival or cell death. However, the susceptibility of CuZn-SOD-S
cells does correspond to their elevated GSSG content and low cellular GSH/GSSG ratio, consistent with a role for redox in cell apoptosis. Given the different effects that the acute and chronic gene
manipulations have on the cellular GSH antioxidant pool, future
distinctions are warranted between studies using transient or stable
overexpression of SOD.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/+) exhibited a decrease of 30-50%
reduced GSH in several tissues, such as the lung, brain, and muscle
(12). Notably, as these animals age, Mn-SOD transgenic mice exhibited
increased muscle apoptosis when compared with age-matched controls
(12). In other studies, an overall increase in tissue GSH content was
found to be associated with transgenic mice overexpressing CuZn-SOD
(13). Thus, the relationship between overexpression of the two
different isoforms of SOD and the intracellular GSH status and how
these changes contribute to oxidant-induced initiation of cell
apoptosis has not been thoroughly investigated.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
X174 HaeIII and
EcoRI-HindIII). Fetal calf serum and horse serum were obtained from Atlanta Biologicals (Norcross, GA). Monoclonal antibodies against Bax, BcL-2, and CPP32 were acquired from
Transduction Laboratories (Lexington, KY), and the monoclonal antibody
against
-actin was bought from Oncogene (Cambridge, MA). Dulbecco's
modified Eagle's medium was obtained from Invitrogen.
Nitrocellulose membranes were acquired from Bio-Rad. The enhanced
chemiluminescence system for Western immunoblot (ECL) and the hyperfilm
were purchased from Amersham Biosciences. Fluorescent mounting medium
was obtained from DAKO Corp. (Carpinteria, CA). 12-mm circle number 1 glass cover slips used for DAPI staining were procured from Fisher. The
cDNAs for human Mn-SOD and CuZn-SOD were generously provided by Dr.
Sonia Flores (Webb-Waring Institute, Denver, CO). The pRetro-Off expression vectors were obtained from Clontech
(Palo Alto, CA). Restriction enzymes were from New England Biolabs
(Beverly, MA).
20 °C for at
least 1 h, and stained with 1 µg/ml DAPI for 30 min in the dark.
The coverslips were washed three times with PBS and mounted using DAKO
fluorescent mounting fluid onto microscope slides. Cells were viewed
and counted using a fluorescent Olympus Bx50 microscope with the ×20
objective. At least six fields of total and apoptotic cells were
counted on each slide. A total of 200 cells were counted.
20 °C until Western analyses. Twenty µg of total
cellular protein was resolved on 12% acrylamide gels (100 V, 90 min)
and blotted onto 0.2-µm nitrocellulose membranes. The membranes were probed with anti-CPP32 (1:500). The secondary antibody used
corresponded to the primary antibody (goat and mouse) and was
conjugated to horseradish peroxidase (1:1000). Detection of
chemiluminescence was performed with an ECL Western blotting detection
reagent according to the manufacturer's recommendation. The membrane
was stripped in 6.25 mM Tris, pH 6.7, 2% SDS, and 100 mM mercaptoethanol at 50 °C for 15 min and probed again
for
-actin to verify equal protein loading in each lane.
20 °C for Western analyses of PARP and its
degradation product. Twenty µg of the protein was resolved on an 8%
acrylamide gel and blotted onto nitrocellulose membranes. The membranes
were probed with 1:1000 dilution of PARP (rabbit anti-mouse polyclonal
antibody) and secondary IgG antibody 1:1000 and then treated with ECL
and exposed to film.
20 °C. For each
experiment, stock DHR was diluted fresh with dimethyl formamide and
added to cells at a final concentration of 5 µM. Cells
were then exposed to TBH for 30 min, washed twice with PBS, harvested into 2 ml of PBS, and sonicated (Braun-Sonic sonicator, Braun Biotech
International, Allentown, PA). Rhodamine 123 accumulation was
quantified using a luminescence spectrophotometer (AMINCO Bowman Series
2, Thermo Spectronic, Rochester, NY), at excitation and emission
wavelengths of 500 and 536 nm, respectively. Results are expressed as
relative fluorescence units/mg of protein.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TBH-induced ROS production and apoptosis in
wild type PC-12 cells and the abrogation by NAC. Wild type cells
were treated with 100 µM TBH, and ROS generation was
determined at 30 min by DHR oxidation (A) or cell apoptosis
was determined at 24 h by DAPI staining (B). NAC,
whenever present, was added to incubations at 30 min prior to TBH
treatment (+NAC) or at 1 h after TBH challenge
(+NAC1hr). Results are expressed as mean ± S.E. for
five separate experiments. *, p < 0.05 versus no TBH treatment (control); #, p < 0.05 versus TBH treatment; o, p < 0.05 versus TBH + NAC.
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Fig. 2.
SOD activities in stable Mn-SOD and CuZn-SOD
transfectants in cellular extracts (A) and in
mitochondrial or cytoplasmic fractions (B).
Mitochondrial and cytoplasmic compartments were separated by standard
differential centrifugation procedures. Total SOD activity (CuZn-SOD
and Mn-SOD) was determined by cytochrome c oxidation. Mn-SOD
activity was determined in the presence of cyanide, and CuZn-SOD
activity was calculated as the difference between the total SOD
activity and that of Mn-SOD. Results are expressed as units/mg protein.
Results are expressed as mean ± S.E. for four separate
experiments. A, *, p < 0.05 versus respective enzyme activities in wild type PC-12
(Wt) and pRetro-Off vector control cells; B, *,
p < 0.05 versus respective enzyme
activities in pRetro-Off controls.
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Fig. 3.
TBH-induced apoptosis in PC12 cells:
Attenuation or exacerbation by stable overexpression of Mn-SOD and
CuZn-SOD, respectively. Wild type (Wt), pRetro-Off
vector, and Mn-SOD- or CuZn-SOD-overexpressing PC12 cells were treated
for 24 h with 100 µM TBH, and cell apoptosis was
determined by DAPI staining as described under "Experimental
Procedures." Results are expressed as mean ± S.E. for six
separate experiments. Mn-SOD-S and CuZn-SOD-S,
cells with stable overexpression of Mn-SOD and CuZn-SOD, respectively.
*, p < 0.05 versus wild type control; #,
p < 0.05 versus TBH-treated wild type cells
and cells transfected with pRetro-Off vector alone; o,
p < 0.05 versus Mn-SOD-S.
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Fig. 4.
TBH-induced caspase-3 activation and PARP
cleavage. pRetro-Off vector and Mn-SOD-S and CuZn-SOD-S PC-12
cells were treated with 100 µM TBH, and at various times,
samples were collected and total protein cell lysates were prepared and
processed for Western immunoblot of CPP32 expression. In parallel
experiments, extracts were collected as described by Stefanis et
al. (18) (see "Experimental Procedures") and analyzed for PARP
(116 kDa) and its cleavage product (85 kDa). The immunoblots of CPP32
and PARP are one representative of three separate experiments for
pRetro-Off vector control (A), Mn-SOD-S (B), and
CuZn-SOD-S (C). Each immunoblot for CPP32 and PARP was
reprobed for -actin, and the results verified equal loading in each
lane. Mn-SOD-S and CuZn-SOD-S, cells with stable
overexpression of Mn-SOD and CuZn-SOD, respectively.
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Fig. 5.
Effect of TBH on ROS generation in wild type
PC-12 cells and PC-12 clones overexpressing CuZn-SOD or Mn-SOD.
PC12 wild type and clones were treated with 100 µM TBH
for 30 min in the presence of 5 µM DHR. The oxidation of
DHR (a measure of ROS production) was determined as the increase in the
fluorescence of rhodamine 123, the oxidation product, at
excitation/emission wavelengths of 500 and 536 nm, respectively.
Results are expressed as relative fluorescence units
(RFU)/mg of protein and presented as mean ± S.E. for
six separate preparations. Mn-SOD-S and
CuZn-SOD-S, cells with stable overexpression of Mn-SOD and
CuZn-SOD, respectively. *, p < 0.05 versus
corresponding zero time control; #, p < 0.05 versus TBH-treated wild type, pRetro-Off vector control, and
CuZn-SOD-S cells.
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Fig. 6.
Effect of mitochondrial inhibitors on
TBH-induced apoptosis in pRetro-Off vector controls. Cells were
grown on cover slips and treated with 100 µM TBH in the
absence or presence of 50 µM rotenone (Rot) or
1 µM antimycin A (Anti A) for 24 h. Cells
were stained with DAPI, and apoptotic cells were counted. Results are
mean ± S.E. for four separate experiments. *, p < 0.05 versus control; #, p < 0.05 versus TBH treatment.
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Fig. 7.
Time course of TBH-induced mitochondrial ROS
production in pRetro-Off vector controls and in cells with stable
Mn-SOD overexpression. PC-12 clones were grown on cover slips and
treated with 100 µM TBH for varying times. Cells were
incubated with Mito Tracker Red and co-stained with Mito Tracker Green
(see "Experimental Procedures"). The fluorescence images represent
overlaid images of the two fluorescence probes. Images
A-D and E-H, represent kinetics of ROS
production at 0, 15, 30, or 45 min for pRetro-Off and Mn-SOD-S,
respectively.
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Fig. 8.
Kinetics of changes in intracellular GSH and
GSSG induced by TBH. PC12 clones (pRetro-off vector control,
CuZn-SOD-S and Mn-SOD-S) were treated with 100 µM TBH,
and at 0-6 h, samples were collected and derivatized for analyses of
GSH and GSSG by HPLC as described under "Experimental Procedures."
A, GSH; B, GSSG; C, GSH-to-GSSG ratio.
Cellular concentrations of GSH and GSSG are expressed as nmol/mg of
protein and presented as mean ± S.E. for six separate
experiments. Mn-SOD and CuZn-SOD, cells with
stable overexpression of Mn-SOD or CuZn-SOD, respectively. A
and B, *, p < 0.05 versus
pRetro-Off vector control; #, p < 0.05 versus zero time. C, *, p < 0.05 versus pRetro-Off vector control; #, p < 0.05 versus Mn-SOD-S.
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Fig. 9.
Base-line GSH concentrations and GSH-to-GSSG
ratio in cells stably or transiently overexpressing SOD. Control
(mock transfection with no DNA) and CuZn-SOD- and Mn-SOD-overexpressing
PC-12 cells were treated with 5% trichloroacetic acid, and the acid
supernatants were derivatized for analyses of GSH and GSSG by HPLC as
described under "Experimental Procedures." A, GSH;
B, GSH-to-GSSG ratio. Cellular concentrations of GSH are
expressed as nmol/mg protein and presented as mean ± S.E. for
four separate experiments. S and T, stable and
transient expression, respectively. A, *, p < 0.05 versus control; #, p < 0.05 versus respective stable transfectants. B, *,
p < 0.05 versus control; #,
p < 0.05 versus Mn-SOD-S; o,
p < 0.05 versus CuZn-SOD-S.
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Fig. 10.
TBH-induced apoptosis in PC-12 cells
transiently overexpressing Mn-SOD or CuZn-SOD. PC-12 cells were
transiently transfected with no DNA (wild type), pRetro-Off vector,
Mn-SOD DNA, or CuZn-SOD DNA using electroporation as described under
"Experimental Procedures." Cells were allowed to recover from the
transfection for 2 days and then seeded onto coverslips. Cells were
then exposed to 100 µM TBH for 24 h and processed
for DAPI staining as described under "Experimental
Procedures." Results are expressed as mean ± S.E. for 12 separate experiments. Mn-SOD-T and CuZn-SOD-T,
cells transiently overexpressing Mn-SOD or CuZn-SOD, respectively; *,
p < 0.05 versus control without TBH;
#, p < 0.05 versus TBH-treated wild type,
pRetro-Off vector control, and CuZn-SOD-T.
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DISCUSSION
REFERENCES
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grant DK 44510.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 Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Tel.: 318-675-6032; Fax: 318-675-4217; E-mail: taw@lsuhsc.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M208670200
2 T. G. Wang and T. Y. Aw, unpublished data.
3 E. K. Pias and T. Y. Aw, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: SOD, superoxide dismutase; TBH, tert-butyl-hydroperoxide; DHR, dihydrorhodamine 123; NAC, N-acetylcysteine; DAPI, 4,'6-diamidino-2-phenylindole; PARP, poly(ADP-ribose) polymerase; HPLC, high performance liquid chromatography.
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