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Tumor Biology |
Radiation Research Laboratory, B180 Medical Laboratories, College of Medicine, The University of Iowa, Iowa City, Iowa 52242 [S. L., T. Y. J-Q. Y., L. W. O.]; Pathology Service, William S. Middleton Veterans Memorial Hospital, Madison, Wisconsin 53705 [T. D. O.]; and Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53705 [T. D. O.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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of nuclear
factor-
B. These results suggest that hydrogen peroxide or
other hydroperoxides appear to be key reactants in the tumor
suppression by MnSOD overexpression, and growth inhibition correlates
with the intracellular redox status. This work suggests that
manipulations that inhibit peroxide removal should enhance the tumor
suppressive effect of MnSOD overexpression. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In this system,
SODs3
convert superoxide radical (O
2) into hydrogen peroxide
(H2O2), whereas GPXs and
CATs convert H2O2 into
water. Therefore, two toxic species, O and
H2O2, are converted into
the harmless product water. Unlike SOD and CAT, GPX requires several
secondary enzymes (GR and G-6-PDH) and cofactors (GSH, NADPH,
and glucose 6-phosphate) to function at high efficiency. The
relationships between these various proteins and cofactors are shown in
Fig. 1
.
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to
H2O2 and
O2 within the mitochondrial matrix. A variety of
cancer cells have reduced levels of antioxidant enzymes, especially
MnSOD, when compared with their normal counterpart (1)
.
Studies have demonstrated that transfection of MnSOD cDNA
into various types of tumor cells leads to a decrease in their
tumorigenicity (2–7)
, which suggests that
MnSOD is a tumor suppressor gene; however, the mechanism(s)
by which this antioxidant enzyme suppresses cancer development is
currently unclear. The main purpose of the present study was to examine
the mechanism of tumor suppression by MnSOD; in particular, we sought
to determine the molecular species responsible for the tumor
suppression. Cytosolic GPX (GPX1; EC 1.11.1.9), a selenoprotein, was first described by Mills as an enzyme that protects hemoglobin from oxidative degradation in RBCs (8) . There are at least five GPX isoenzymes found in mammals (9) . Among them, GPX1 is considered as the major enzyme responsible for removing H2O2. Overexpression of this enzyme was observed to protect cells against oxidative damage (10–12) . Elevation of GPX1 activity in both FL5 (13) and MDBK cells (14) suppressed apoptosis induced by H2O2. This evidence indicates that GPX1 is a major antioxidant enzyme that protects cells against lethal oxidative stress.
It has been suggested that overexpression of SOD without concomitant increase in the level of GPX1 results in the accumulation of H2O2 that can participate in the Fenton reaction, leading to the formation of noxious hydroxyl radicals. This highly ROS oxidizes DNA, protein, and lipids, directly affecting cell survival (15, 16) . Amstad et al. (15) found that the SOD and CAT double transfectant SOCAT 3 was well protected from oxidant damage because of its increased content of CAT, which counterbalances the increase in CuZnSOD. Furthermore, they reported that GPX1 also compensated for the hypersensitivity of CuZnSOD overproducers to oxidant stress (17) . This evidence implied that the balance of SOD and GPX plus CAT is more important to overall oxidant sensitivity than the level of SOD alone.
Redox (reduction-oxidation) reactions regulate signal transduction
(18–22)
. H2O2, O,
GSH/GSSG, and NADPH/NADP+ are considered to be
important players in the cellular redox system. The GSH/GSSG couple is
thought to be the major redox buffer in the cell (23, 24)
.
As shown in Fig. 1
, GPX1, in addition to affecting GSH/GSSG,
controls the cellular content of
H2O2 (or other organic
hydroperoxides) and NADPH/NADP+, so it may
regulate the cellular redox status. In HIV-infected T-cells
(25)
and similarly in T-cell lines (26)
,
selenium supplementation increased GPX activity and decreased
H2O2-induced NF-B
activation. A human breast cancer cell line (T47D) overexpressing GPX1
responded poorly to NF-B activation by TNF- or
H2O2; both B-dependent
gene transactivation and NF-B DNA binding were reduced
(27)
.
In this paper, we hypothesize that overexpression of MnSOD in human
glioma cells results in an accumulation of
H2O2 or other peroxides,
causing a decrease in glioma cell growth. Furthermore, we hypothesize
that transfection of these cells with the human GPX1 gene
will rescue the growth of these cells through detoxification of
hydrogen peroxide or hydroperoxides and/or changes in cellular redox
status. To test our hypotheses, we transfected the human
GPX1 cDNA into human glioma MnSOD-overexpressing cells. The
results indicate that overexpression of GPX1 rescues the growth
suppression by MnSOD, which suggests that
H2O2 or some other
hydroperoxide is involved in the cell growth suppression. It is
uncertain whether H2O2
increases after SOD overexpression or whether, instead, other
hydroperoxides increase. Omar and McCord (28)
have
postulated that increased SOD does not cause an increase in
H2O2, but instead an
increase in lipid hydroperoxides. GPX1 can act on both
H2O2 and lipid
hydroperoxides, so it is not clear which hydroperoxide is being acted
on by GPX1. We also found that overexpression of GPX1 alters the
cellular contents of GSSG, ratios of GSH/GSSG, and levels of ROS and
inhibits IB degradation, which suggests
that GPX1 can modulate the intracellular redox status. Immunogold
ultrastructural staining showed that GPX1 protein exists in the
mitochondria and nucleus, as well as in the cytosol. Thus, GPX1 protein
overexpression may remove hydroperoxides produced by MnSOD in the
mitochondria.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B
primary antibody was purchased from New England Biolabs, Inc. CAT
primary antibody was from Calbiochem-Novabiochem Corp. (CAB).
Blocking Reagent®, Anti-Digoxigenin-AP®, and CSPD®
were purchased from Roche Molecular Biochemicals. Primary
antibodies against MnSOD, CuZnSOD, and GPX1 were raised by our
laboratory. Human GPX1 cDNA was a gift from Dr. James H.
Doroshow.
Cell Lines.
The human glioma P U118–9 cells were cloned from wild-type U118 cells
(5)
. Neo is a vector control line that serves as the MnSOD
single transfection control and SOD2 is a MnSOD overexpression
transfectant. Both Neo and SOD2 were derived from the P cell line by
transfection and cloning. SOD2 had been shown previously to have about
a 5-fold increase in MnSOD compared to the P cell line
(5)
. These cell lines have been described in detail
previously (5)
. Zeo35 is also a vector control line that
serves as MnSOD-GPX1 double transfection control. S-GPXs are MnSOD-GPX1
double transfectants. The Zeo35 and S-GPX cell lines were derived from
SOD2 by transfection and cloning. Thus, the lineages of these lines are
as follows.
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Cell Culture.
P cells were routinely grown on 60-mm plastic tissue culture dishes in
DMEM high-glucose medium supplemented with 10% fetal bovine serum and
1% penicillin/streptomycin at 37°C in a humidified atmosphere of
95% air and 5% CO2. Medium was changed every 4
days, and the cells were subcultured with 0.25% trypsin and 1% EDTA
whenever the cultures reached confluence. Passage numbers were limited
to 30 passages, because it has been demonstrated that antioxidant
enzymes did not change in tumor cells up to 50 passages
(29)
. Both Neo and SOD2 cells were cultured in medium
containing G418 (500 mg/liter). Both Zeo35 cells and S-GPX cells were
cultured in medium containing G418 (500 mg/liter) and zeocin (100
mg/liter). To eliminate the influence of antibiotics on experimental
results, 3–5 days before an analysis, cells were cultured in medium
without any antibiotic supplement. In all assays of our study, the
cells were cultured in medium containing 60 nM selenium.
Cells were regularly examined for Mycoplasma contamination
and used only if Mycoplasma-free.
Construction of Expression Vector.
We synthesized the 89-mer polynucleotides
5'-CATCCCAAGCTTACAGTGCTTGTTCGGGGCGCTCGGCTGGCTTCTTGGACAATTGCGCCATGTGTGCTGCTCGGCTAGCTAGTAGTAGC-3'
and its complementary strand. This synthesized DNA fragment contained
two restriction enzyme sites, HindIII and NheI
(shown underlined). The nucleotides between the two sites are
the same sequence, corresponding to 48 nucleotides of the 5'-noncoding
region and 20 nucleotides of the coding region from the ATG start codon
of the published human GPX1 cDNA sequence (30)
.
There is also a NheI site at nucleotide 20 from the ATG
start codon in the GPX1 cDNA. We annealed the two 89-mer
polynucleotide strands, and then this DNA fragment was cut with
HindIII and NheI. GPX1 cDNA was cut
with NheI and BamHI. The plasmid
[pcDNA3.1/Zeo(+)] was cut with HindIII and
BamHI. Finally, the three enzyme-digested fragments were
ligated by T4 DNA ligase. We designated this new construct
pcDNA3.1/Zeo(+)/GPX1. The new construct has 41 more nucleotides in the
5'-noncoding region of GPX1 cDNA than the original
GPX1 cDNA from Dr. Doroshow, which has only 5 nucleotides in
the 5'-noncoding region; we constructed this new plasmid because we had
difficulty in using the original plasmid for GPX1
transfection in our cell lines. The construction was confirmed by DNA
sequencing by the University of Iowa DNA Facility.
Cell Transfection.
The SOD2 cells were transfected with the pcDNA3.1/Zeo (+)/GPX1 plasmid.
Zeocin was the selection marker for the transfection. The transfection
of vector control was performed by using the plasmid pcDNA 3.1/Zeo(+)
without the GPX1 cDNA insert. The LipofectAMINE method was
used in this study. The protocol for transfection with LipofectAMINE as
recommended by the manufacturer (Life Technologies) was used with some
modification. Cells were seeded at a density of 5 x 105 cells/well in 6-well plates, allowed to grow
overnight, and then rinsed three times with serum-free medium before
the transfection. The transfection solution was prepared by diluting 2
µg of plasmid DNA into 100 µl of serum-free medium to make solution
A and diluting 10 µl of LipofectAMINE into 100 µl of serum-free
medium to make solution B. The two solutions were combined, mixed
gently, and incubated at room temperature for 45 min to allow
DNA-liposome complex formation. Finally, for each transfection, 0.8 ml
of serum-free medium was added to the tube containing 200 µl of
DNA-liposome complexes, mixed gently, overlaid onto the rinsed cells,
and then incubated with the cells for 24 h at 37°C. After
transfection, the DNA-lipid complexes were removed and replaced with
the fresh medium without supplement of G418 and zeocin. Forty-eight h
later, the cells were subcultured and subsequently incubated in the
selection medium (500 mg/liter G418 and 100 mg/liter zeocin) for 15
days to allow antibiotic-resistant colony growth. Resistant colonies
were isolated by cloning rings.
Cell Homogenization and Protein Quantification.
To prevent antioxidant enzyme denaturation, all of the procedures for
cell sample preparation were performed on ice. The culture medium was
removed before cell harvesting, and the cells were washed with PBS (pH
7.0) three times. Cells then were removed from the dish using a plastic
scraper and collected into a microcentrifuge tube. The harvested cells
were pelleted by centrifugation, and then the PBS supernatant was
removed. The cell pellets were resuspended in 0.05 M
phosphate buffer (pH 7.8) and sonicated three times for 15 s each
using a Vibra cell sonicator (Sonics and Materials, Inc., Danbury, CT)
at full power and 40% duty cycle. Total protein concentrations were
measured with the Bio-Rad protein assay kit.
GPX Activity Assay.
GPX activity were measured as described by Lawrence and Burk
(31)
. Cell sonicated samples (700 µg/100 µl) were
incubated in 0.7 ml of a mixture containing 50 mM potassium
phosphate buffer (pH 7.8), 1 mM EDTA, 1 mM
NaN3 10 mM GSH, and 2.4 units/ml GR
for 10 min. After addition of 100 µl of 1.5 mM of NADPH,
NADPH oxidation was determined at 340 nm for 3 min at 30 s
intervals, which is the independent sample as a reference. NADPH
oxidation was then measured after addition of 100 µl of 1.5
mM H2O2 at 340
nm for 3 min. One unit of GPX activity is defined as the amount of
protein that oxidized 1 µM NADPH per min. It should be
emphasized that this assay is run with high GSH levels and measures the
GPX activity at that high GSH level. If the cell has low GSH levels,
the actual GPX activity will then be lower than measured by this assay.
One cannot use low GSH levels in this assay because the activity will
then be too small to measure. For this reason, we defined "effective
GPX activity" as measured GPX activity times cellular GSH
concentration.
Native Activity Gel Assay for GPX1 and GR.
Before loading the samples, the gel was run in preelectrophoresis
buffer (22.76 g/liter Tris-HCl, 0.38 g/liter disodium EDTA, pH 8.8) for
1 h at 4°C. For GPX1 native activity gel assay, 700 µg of
total protein were separated in an 8.0% native polyacrylamide gel with
5% stacking gel. The electrophoresis process was performed in two
steps: the gel with samples was run first for 3 h in the
preelectrophoresis buffer at 4°, and then run in the sample running
electrophoresis buffer (6.06 g/liter Tris-HCl, 22.50 g/liter glycine,
0.68 g/liter disodium EDTA, pH 8.3) for 4 h at 4°C. After
electrophoresis, the gel was rinsed with 1 mM GSH three
times for 7 min each and then incubated in 75 ml of
ddH2O containing 0.008% cumene hydroperoxide
plus 1 mM GSH for 10 min with gently shaking. After briefly
rinsing twice with ddH2O, the gel was stained
with a 1% ferric chloride and a 1% potassium ferricyanide solution,
which was made fresh and mixed immediately before use from equal
volumes of a 2% stock solution of each. The achromatic band
corresponding to GPX activity appears on a blue background. For native
activity gel assay for GR, 500 µg of total protein were separated in
a 7.0% native polyacrylamide gel with 5% stacking gel. The process of
electrophoresis was the same as native gel assay for GPX1. After
electrophoresis, the gel was placed in a freshly made dye solution
[3.4 mM GSSG, 0.36 mM NADPH, 0.052
mM dichlorophenol-indophenol, 1.1 mM 3
(4,5-dimethythiazolyl-2)-2,5-diphenyl tetrazolium acid, prepared in 250
mM Tris, pH 8.0]. GR activity was indicated by the
presence of a purple precipitate in the gel. The gel was rinsed
repeatedly and photographed.
Western Blotting Assay.
The method used for Western blotting assay was described previously
(32)
and modified. Sample preparation for Western blotting
was the same as that of enzyme activity assays. After proteins were
electrotransferred onto a nitrocellulose membrane (Schleicher &
Schuell, Keene, NH), the membrane was blocked in 5% dry fat-free milk
in TBST (0.02 M Tris-HCl buffer, pH 7.5, 0.137
M NaCl, and 0.1% Tween 20) for 1 h at room
temperature and probed with primary antibody overnight at 4°C. The
MnSOD primary antibody raised in our laboratory was diluted 1:1000, the
CuZnSOD primary antibody was diluted 1:250, the CAT primary antibody
was diluted 1:1000, and the IB primary
antibody was diluted 1:2000. After the immunoreaction with primary
antibody, the membrane was incubated with horseradish peroxidase
conjugated with goat antirabbit (Sigma) IgG (1:10000) for 1 h at
room temperature. After a final wash of the membrane with TBST, the
bands were revealed in films by ECL staining (Amersham Pharmacia
Biotech). Densitometry was performed by the Eagle Eye II still
video system (Stratagene, La Jolla, CA).
Native Immunoblotting Assay.
We developed an immunoblotting method to determine the GPX1 protein
levels in different clones. The protein separation method was the same
as for the GPX activity gel assay. The native proteins were transferred
onto the nitrocellulose membrane using the same method as in the
Western blotting assay. The detection method is also the same as that
for the Western blotting. The GPX1 primary antibody was diluted 1:100.
Northern Blotting.
The total RNA were extracted from 80-90% confluent cells using a
RNeasy Mini Kit according to the manufacturer’s directions (Qiagen).
Twenty µg of the total RNA were resolved in a 1.5% agarose gel
[0.75 g of agarose, 42.2 ml of ddH2O, 5 ml of
10x 3-(N-morpholino)propanesulfonic acid (200
mM morpholinopropanesulfonic acid, 50
mM sodium acetate, 10 mM
EDTA, pH 7.0), and 2.5 ml of formaldehyde] and then transferred and
fixed onto a nylon membrane (Roche Molecular Biochemicals,
Indianapolis, IN). The human GPX1 cDNA were labeled with
digoxigenin (Roche Molecular Biochemicals) by the random-priming
technique. Prehybridization was performed in the standard hybridization
buffer [5x SSC (0.75 M NaCl, 0.75
M sodium citrate, pH 7.8), 0.1%
N-lauroylsarcosine, 0.02% SDS, 1% Blocking
Reagent®] for at least 4 h at 68°C. Hybridization was
carried out in the same standard hybridization buffer as
prehybridization containing a human GPX1 cDNA probe
overnight at 68°C. After hybridization, the membrane was washed
twice, 15 min per wash, in 2x wash solution [2x SSC (0.3
M NaCl, 0.3 M sodium
citrate, pH 7.8), 0.1% SDS] at room temperature and then twice with
0.5x wash solution [0.5x SSC (75 mM NaCl, 75
mM sodium citrate, pH 7.8), 0.1% SDS], 15 min
per wash, at 68°C. After the posthybridization washes, the membrane
was equilibrated in the washing buffer [maleic acid buffer (0.1
M maleic acid, 0.15 M NaCl,
pH 7.5), 0.3% (v/v) Tween 20] for 1 min, and then the membrane was
blocked by gentle agitation in the blocking solution (dilute Blocking
Reagent® stock solution 1:10 with maleic acid buffer) for 1 h at
room temperature. After pouring off the blocking solution, the membrane
was incubated in the antibody solution Anti-Digoxigenin-AP®;
blocking solution, 1:10,000) for 30 min at room temperature. After
discarding the antibody solution, the membrane was washed gently twice,
15 min per wash, in washing buffer. Finally, the detection of the
membrane was performed with CSPD® diluted 1:100 in detection buffer
(100 mM Tris-HCl, pH 9.5, 100
mM NaCl) according to the instructions provided
by the manufacturer (Roche Molecular Biochemicals). Ethidium
bromide-stained bands from both 18S (
2 kb) and 28S (5 kb) rRNA
bands served as markers for RNA sample loading.
RT-PCR.
The total RNA was isolated as described in the Northern blotting assay.
RT of cDNA was carried out from 5 µg of total RNA in a 30-µl
reaction solution [2 µg of oligo(dT) (18-mers), 2 µl of 10
mM dNTP (mixture of dATP, dGTP, dTTP, and dCTP), 3 µl of
100 mM DTT, 1 µl of RNase inhibitor (RNasin), 0.3
µl of BSA (100x), 1.5 µl of Moloney murine leukemia virus-reverse
transcriptase (200 units/µl), 6 µl of 5x first strand buffer (50
mM Tris-HCl, pH 8.5, 3 mM
MgCl2, 75 mM KCl, 10 mM
DTT)]. The mixture was incubated to synthesize cDNA for 50 min at
42°C, to elongate cDNA for 10 min at 52°C, and then to inactivate
the enzyme for 15 min at 70°C.
PCR primers were selected according to the human complete GPX1 cDNA sequence. The sequences of oligonucleotide primers were as follows: sense, 5'-AAGGTACTACTTATCGAGAATGTG-3'; antisense, 5'-GTCAGGCTCGATGTCAATGGTCTG-3'. The PCR product was approximately 0.5 kb long. The PCR for one reaction was carried out in a 20-µl mixture [2 µl of 10x PCR buffer, 0.5 µl of 10 mM deoxynucleotide triphosphate (mixture of dATP, dGTP, dCTP, and dTTP), 0.2 µl of 5 units/µl Taq DNA polymerase, 2 µl of cDNA (dilution of RT products, 1:100), 0.1 µl of 1 µg/µl sense primer, 0.1 µl of 1 µg/µl antisense primer, 15.1 µl of ddH2O)]. PCR was conducted in a Perkin-Elmer Cetus thermal cycler for 30 cycles. After a 5-min denaturation at 94°C, amplification was carried out with a cycling profile consisting of denaturation for 40 s at 94°C, annealing for 40 s at 55°C, and extension for 1 min at 72°C, followed by a final extension for 7 min at 72°C. Fifteen µl of PCR products were resolved in a 1.2% agarose gel with ethidium bromide staining and compared with 1-kb DNA ladders.
Immunogold Staining.
Cells were scraped from tissue culture dishes, fixed in
Carson-Millonig’s fixative (4% formaldehyde in 0.16 M
monobasic phosphate buffer, pH 7.2) and processed for immunogold
immunohistochemistry as described previously (33)
. Cell
cultures stained with anti-GPX1 antibody were photographed using a
Hitachi electron microscope (Hitachi, Ltd., Tokyo, Japan). As a
control, normal rabbit serum was used in place of the primary antibody.
Plating Efficiency.
A single cell suspension was plated at 500 cells/dish in 60-mm dishes
for each clone. The cells were kept in culture medium in an incubator
for 14 days to allow colony formation and then fixed and stained by
0.1% crystal violet and 2.1% citric acid. The colonies containing 50
cells or more were scored. The plating efficiency (PE) was calculated
as follows: PE = (Colonies formed/Cells
seeded) x 100%.
Growth Curve.
The growth rate of cells was determined by counting the number of cells
as a function of time. Cells were seeded in 24-well plates at
2 x 104 cells/well containing 1
ml of culture medium. Three parallel wells for each clone were seeded
for each time point. Cells were counted every 48 h for 12 days
using a hemocytometer. Doubling time
(Td) was calculated from the growth
curve by using the formula Td = 0.693t/ln(Nt/N0), where
t is time in hours, Nt is the cell number
at time t, and N0 is the cell number at
the initial time.
Tumorigenicity in Nude Mice.
Four- to 5-week-old female nude mice were used in this assay. Cells
were harvested by trypsinization and washed in serum-free medium.
Twenty million cells of each clone were resuspended in serum-free
medium. Two million cells were injected s.c. into the back of the neck
of each nude mouse. Four nude mice were used for each group. When the
tumor appeared, it was measured by a vernier caliper every week. Tumor
volume (TV) was calculated as follows (34)
, TV
(mm3
) = (L x W2)/2, where L is
the longest dimension of the tumor in mm, and W is the
shortest dimension of the tumor in mm.
GSH and GSSG Measurement.
The method for GSH and GSSG measurement is adapted from Anderson
(35)
. The total glutathione of cells was measured by the
colorimetric reaction of DTNB with GSH to form TNB. The rate of the
formation of TNB, which is proportional to the total GSH concentration,
was followed spectrophotometrically at 412 nm. Cellular GSSG was
reduced to GSH by specific GR. To prepare a sample for total
glutathione measurement, the cell pellet was frozen and thawed once,
then homogenized in 50 mM potassium phosphate buffer
containing 1.34 mM diethylenetriaminepentaacetic
acid (to give a protein concentration of 2–10 mg/ml) by running the
mixture up and down in the tip of a P-1000 Pipetman. For the total
glutathione measurement, the sample was diluted with 5-sulfosalicylic
acid (5%, w/v, in ddH2O) to yield values that
fall on the standard curve (usually between 1:2 and 1:10). The total
glutathione was determined by mixing 700 µl of working buffer [6
mM NADPH in stock buffer (0.143 M sodium
phosphate, 6.3 mM EDTA)], 100 µl of DTNB (6
mM DTNB in stock buffer), 100 µl of
ddH2O, 50 µl of sample, 50 µl of GR (40
units/ml) at room temperature. The rate of TNB formation was determined
at 412 nm, every 20 s for 3 min using a Beckman DU-70
spectrophotometer. The total glutathione of a sample was calculated
from a standard curve of the concentration of glutathione
versus rate. For the GSSG measurement, 30–50 µl of
homogenate were taken, 2 µl of 2-vinypyridine in 100% of ethanol
(1:1) were added, and the sample was vortexed and incubated at 4°C
for 1.5 h to remove GSH. The GSSG was measured as described in the
total glutathione measurement. The GSH content was calculated by
subtracting the GSSG content from the total glutathione content.
Because GSH is a derived quantity, errors for GSH were calculated by
propagation of error theory.
Intracellular ROS Measurement.
The level of intracellular ROS was determined by a microplate reader
(Bio-Tek Instruments, Inc., Winooski, VT) using DCFH-DA
(Molecular Probes Inc., Eugene, OR; Ref. 36
). DCFH-DA is a
nonpolar and nonfluorescent compound that can permeate cells freely.
Once inside cells, it is hydrolyzed by esterase to form the polar and
nonfluorescent DCFH, and trapped inside cells. Upon interaction of DCFH
with ROS, DCFH gives rise to cellular fluorescence. Cells (2 x 105 from each clone) were seeded and
cultured for 24 h in a 24-well dish prior to the measurement. On
the day of assay, the cells were washed twice with serum-free medium
and then incubated with 0.5 ml of serum-free medium containing a 30
µM DCFH-DA solution for 90 min at 37°C. After the
incubation, the cells were washed twice with PBS buffer (pH 7.8) and
then lysed in 0.5 ml of 0.5% SDS solution. Finally, the intensity of
the 485/530 nm fluorescence corresponding to the levels of
intracellular ROS in the lysates were recorded with a microplate reader
(Bio-Tek Instruments, Inc.) using FL500 software. The relative
fluorescence was calculated by the equation,
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To test for differences in endogenous esterase activity, the already oxidized probe (Molecular Probes C369), which fluoresces without ROS reaction, was also tested. The results were similar among all of the cell lines and indicated no significant difference in esterase activity (data not shown).
Statistics.
Both Student’s t test and the ANOVA-Tukey test were used in
the comparison of different groups. The null hypothesis was rejected at
the 0.05 level of significance. Linear regression analysis was
performed with SigmaPlot 5.0 software. All assays of Western
blots, Northern blots, RT-PCR, and activity gels were done at
least twice.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) demonstrated that the immunoreactive protein levels of
GPX1 were higher in all GPX transfectants compared with SOD2 cells and
Zeo35 cells.
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) showed the expected new band ( 1.8 kb)
from the GPX1 transfectants as well as a band from the known endogenous
GPX1 mRNA (1 kb). The new band is exogenous GPX mRNA, the size of
which corresponded to that of the GPX cDNA 1-kb fragment
plus part of the sequence from the vector. We also demonstrated
GPX1 gene overexpression at the mRNA level using the RT-PCR
method (Fig. 2c
). Brighter bands were formed in the GPX1
transfectants, indicating that the mRNA levels of GPX1 were higher in
the GPX transfectants than those in the control groups.
To examine whether the increased GPX1 protein was active, we measured
the enzyme activity by spectrophotometric assay (Fig. 3a
) and native gel electrophoresis analysis (Fig. 3b
). The GPX1 activities of five clones (S-GPX70, S-GPX86,
S-GPX146, S-GPX182, and S-GPX220) were 62.6 ± 6.0,
65.7 ± 4.5, 55.7 ± 3.5, 30.7 ± 2.8, and 45.9 ± 2.2 milliunits/mg of protein,
respectively. The five clones have significant increases in GPX
activity compared with both SOD2 (18.7 ± 1.2
milliunits/mg of protein) and Zeo35 (22.0 ± 5.0
milliunits/mg of protein). The results from native activity gels showed
that the brightness of an activity gel band corresponds to the GPX1
activity of that clone (Fig. 3b
). All GPX1 transfectants had
much brighter bands than the control groups, SOD2 and Zeo35. The native
gel result correlated with that of enzymatic activity assay. All
activity results agreed with those found by Northern and Western
blotting. We also ran a native activity gel assay for GR because its
activity could be affected by GPX1 transfection; we saw little
difference in GR activity among the different cell lines (data not
shown).
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). The immunogold staining indicated that GPX1 distributes not only to
the cytoplasm (data not shown) but also to the mitochondria and the
nucleus, which agrees with our previous results in adult hamster kidney
(37)
. Thus, GPX1 was found in all three locations in the
control cells, and the levels were increased in all three locations in
the GPX1-overexpressing cells. Increases in the mitochondria and
nucleus were unexpected because targeting sequences for these locations
have not been noted.
|
. There was a significant increase in the amount of total
glutathione in Neo, S-GPX86, and S-GPX146 cells compared with the SOD2
clone. The amount of GSH increased in Neo and S-GPX146 cells compared
with the SOD2 clone. GSSG significantly increased in S-GPX86 and
S-GPX146 cells compared with the SOD2 clone, but GSSG dramatically
decreased in P and S-GPX70 cells compared with SOD2 cells.
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. There
were similar trends as the samples harvested at 24 h after
subculture in the changes of total glutathione, GSH, and GSSG. The GSSG
levels were significantly increased in all clones compared with their
corresponding clones at 24 h except in S-GPX70.
Correlation analysis was performed on many of the data from this study.
We first performed this analysis using the measured GPX activity
values. We found poor correlations because P, Neo, and S-GPX70 were
outliers. When we removed these cell lines, good correlations were
obtained. The removal of P and Neo can easily be rationalized because
they have a different genetic background; in particular, P and Neo have
lower MnSOD levels than the other cell lines, which were all derived
from the same MnSOD-overexpressing cell line (see Fig. 8
). However,
S-GPX70 has the same genetic background as other lines. In examining
the data, we noticed that this line had much lower GSH levels than the
others; thus, we hypothesized that S-GPX 70 was different from other
GPX overexpressors because it did not have enough GSH for the GPX to
maximally function. Because the rate of peroxide removal should be
directly proportional to concentrations of both GPX and GSH, we thought
that S-GPX70 data could be more congruent with other clones if we
considered measured GPX activity times intracellular GSH as the
dependent variable in all correlations. We call this the effective GPX
activity and found the that the biological variables measured
correlated very well with effective GPX. Thus, if correlation analysis
was carried out with measured GPX activity as the dependent variable,
much poorer correlations resulted than when effective GPX activity was
used. We think that this reflects the actual biological situation in
which the activity of GPX is limited by the levels of GSH. Because at
normal physiological concentrations of GSH, GPX cannot saturated by
GSH, this definition of effective GPX activity should be valid at all
physiological concentrations of GSH (24)
|
and d, we plotted GSSG
versus effective GPX activity and found a good correlation.
The GSH levels did not give a significant correlation (data not shown).
Both [GSH]/[GSSG] and [GSH]2/[GSSG] have
been used to indicate the redox state of the cell. Neither ratio gave
us statistically significant correlations (data not shown). This could
be because other factors, such as glucose utilization and
NADP+/NADPH ratios, need to be considered when
measuring a redox balance.
The Changes in ROS Levels.
The intracellular ROS levels were measured by the DCFH fluorescence
method (Fig. 6a
). This assay measures oxidation by various ROS, including
those derived from hydrogen peroxide. The intracellular ROS levels in
SOD2 were dramatically increased to about 5-fold greater than P and
3-fold greater than Neo. The levels of intracellular ROS in the GPX
transfectants, S-GPX86 and S-GPX146, were decreased about 3-fold and
2.5-fold, respectively, compared with SOD2, but there was no
significant difference between S-GPX70 and SOD2. The levels of
intracellular ROS were correlated to the levels of GPX1 and SOD
activities. When DCFH fluorescence was plotted against effective GPX
activity (Fig. 6b
), a significant negative correlation was
found (r = -0.9192; P = 0.0273).
|
B Degradation.B in the
different clones by Western blotting assay (Fig. 7
). IB degradation is known to be activated
by H2O2. There was a
decrease in the level of IB in SOD2
compared with both P and Neo and an increase in the level of
IB in all GPX-overexpressing clones
compared with SOD2. S-GPX70 cells, which had increased GPX but also
with a high level of intracellular ROS, also had high level of
IB. Densitometry was performed on this
Western blot; all of the GPX1-overexpressing clones had
increased amounts of IB immunoreactive
protein when compared to the SOD2 cell line (Fig. 7)
. However,
correlation analysis did not show a statistically significant
correlation between the intensity of the Western bands and effective
GPX activity (data not shown).
|
). As expected, because they were derived from a MnSOD-overexpressing
line, GPX1 transfectants and Zeo35 cells still kept the higher level of
MnSOD overexpression compared with P cells and Neo cells. The level of
MnSOD immunoreactive protein declined a small amount in GPX1
transfectants with passaging, but it was still higher than P and Neo
cells (data not shown). The CuZnSOD level was slightly higher in Zeo35
cells and GPX1 transfectants than that in P, Neo, and SOD2 cells except
for S-GPX86, which had very similar CuZnSOD levels to the SOD2 cells.
For CAT, there was no great difference among the GPX1 transfectant
groups and control groups except for Neo cells, which had a lower level
of CAT protein, and S-GPX146, which had a slightly higher level of CAT
protein. As mentioned earlier, the GPX overexpressors also showed
little changes in GR activity. These data show that GPX1 overexpression
does not cause great changes in other antioxidant proteins.
The Effect of GPX1 Overexpression on Tumor Cell Growth in
Vitro.
Tumor cell growth can be suppressed by MnSOD overexpression. We
hypothesized that overexpression of MnSOD in human glioma cells results
in an accumulation of H2O2
or other hydroperoxides, causing a decrease in glioma cell growth. We
also hypothesized that transfection of these cells with the
GPX1 gene will rescue the growth inhibition of these cells
through detoxification of
H2O2 and/or changes in
cellular redox status. To test these hypotheses, we measured cell
growth rate in vitro by a growth curve (Fig. 9a
), cell population doubling time (Fig. 9, b and c)
, and plating efficiency (Fig. 10
, a and b).
|
|
shows that SOD2 and Zeo35 have the lowest growth rates, P and S-GPX70
have medium growth rates, and the other three clones (Neo,
S-GPX86, and S-GPX146) have the highest growth rates. Neo also had a
higher growth rate compared with P. One possible explanation for the
difference in growth between P and Neo is the slight elevation of
intracellular ROS concentration in Neo cells as shown in Fig. 6
. In a
broad sense, P is probably different from Neo because Neo was derived
from P by selection and cloning. Somewhere in this process, a cell line
with different properties arose. Cell population doubling times were
calculated from cell growth curves (Fig. 9b
). The cell
population doubling times were 46.0 (P), 41.4 (Neo), 54.8 (SOD2), 52.4
(Zeo35), 48.6 (S-GPX70), 42.9 (S-GPX86), and 41.6 h (S-GPX146). As
shown previously (5)
, SOD2 showed significant increases in
the cell population doubling time compared with P and Neo. There were
significant decreases in the cell population doubling times in S-GPX86
and S-GPX146 cells compared with SOD2. S-GPX70 also had a lower
doubling time than SOD2, but the difference was not
statistically significant. However, when correlation analysis was run
on population doubling times versus effective GPX activity,
a highly significant negative correlation was found (Fig. 9
c; r = -0.9747;
P = 0.0048). Population doubling time also
showed highly significant correlation with DFCH fluorescence (Fig. 9
d; r = 0.7819;
P = 0.0378).
Plating efficiency is a measure of clonigenic ability of cells. For
this assay, 500 cells were seeded in a 60-mm culture dish, and colonies
were allowed to form for 2 weeks (Fig. 10a
). The plating
efficiency significantly decreased in the SOD2 line compared with P and
Neo cells. There were significant increases in plating efficiency in
S-GPX86 and S-GPX146 compared with SOD2. The plating efficiency of
S-GPX70 was higher than that of SOD2, but the difference was not
statistically significant. There was also no significant
difference in colony formation ability between SOD2 and Zeo35. In
general, the GPX1 transfection groups had higher ability to form
colonies than SOD2 and Zeo35. Moreover, when plating efficiency was
plotted against effective GPX activity (Fig. 10b
) and
correlation analysis was performed, a highly significant positive
correlation was again found (r = 0.9384;
P = 0.0182).
The Effect of GPX1 Overexpression on Tumor Cell Growth in
Vivo.
Tumorigenicity was determined by transplanting tumor cells in the back
of the neck of nude mice. This experiment was designed to test the role
of GPX1 redox regulation in the suppression of tumor cell growth by
MnSOD in vivo. The results of the tumor growth experiment
are shown in Fig. 11a
and Table 1
. The MnSOD-overexpressing cell line formed tumors that grew much slower
than the P or Neo cell line. S-GPX86 and S-GPX146 had the highest
growth rates (Fig. 11a
). All of the GPX-overexpressing cell
lines grew faster than the SOD2 line from which they were derived. When
the tumor volume at day 63 was plotted against effective GPX activity
and correlation analysis was performed, a highly significant positive
correlation was found (r = 0.9899;
P = 0.002; Fig. 11b
). These
results show convincingly that GPX overexpression inhibits the growth
suppression caused by MnSOD overexpression. Tumor incidence showed a
similar picture (Table 1)
. P and Neo had a high tumor incidence, which
was suppressed by MnSOD overexpression. GPX and MnSOD double
overexpressors again had a high tumor incidence, showing that GPX can
inhibit the tumor suppressive ability of MnSOD.
|
|
|
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GPX1 belongs to the family of selenoproteins and plays an important role in the defense mechanisms of mammals against damage by catalyzing the reduction of H2O2 and a large variety of hydroperoxides (such as DNA hydroperoxides and lipid hydroperoxides) into water and alcohols, respectively, with GSH as the hydrogen donor (40) . GPX1 is usually thought of as a cytosolic protein. We have shown previously that GPX1 is also found in the nucleus and mitochondria (37) . In the present study all these subcellular compartments stained more intensely after GPX1 overexpression; this shows the specificity of our antibody as well as multiple subcellular localizations. Utsunomiya et al. (41) and Asayama et al. (42) have shown immunohistostaining of mitochondria, nuclei, and cytosol with polyclonal antirat GPX1 antiserum, which also suggested that GPX1 has multiple subcellular localizations. Using GPX1 knockout mice, Esworthy et al. (43) provided direct evidence suggesting that the GPX1 gene encodes mitochondrial GPX in mouse liver. Taken together, it is most likely that the GPX1 has mitochondrial, nuclear, and cytosolic subcellular locations in mammalian cells. Unlike phospholipid hydroperoxide GPX, there in no presequence that mediates GPX1 translocation into mitochondria. Therefore, the mechanism whereby GPX1 is imported into mitochondria is unclear. The same is true for import into the nucleus.
Aerobic organisms generate toxic ROS during oxidative metabolism or
under pathological conditions. Organisms have evolved antioxidant
defenses to protect against ROS, predominant among which is the
enzymatic antioxidant pathway (Fig. 1)
. This pathway suggests that
overexpression of SOD without concomitant increase in the level of GPX1
results in the accumulation of
H2O2 that not only changes
the cellular redox status but also can participate in the Fenton
reaction, leading to production of noxious hydroxyl radicals. In this
study, we hypothesized that overexpression of MnSOD in human glioma
cells results in an accumulation of
H2O2 or other
hydroperoxides causing a decrease in glioma cell growth. We further
hypothesized that transfection of these cells with the human
GPX1 gene will rescue the growth of these cells through
detoxification of H2O2
and/or changes in cellular redox status.
The findings here indicate that overexpression of GPX1 rescues the
growth suppression by MnSOD (Fig. 9a
). The growth
characteristics of different clones correlated to their intracellular
ROS levels (Fig. 9
d; r = 0.7819;
P = 0.0378). SOD2 cells with the highest ROS
levels had the lowest growth rate, longest doubling time (Fig. 9b
), and lowest plating efficiency (Fig. 10a
),
whereas GPX1 transfectants (S-GPX86. S-GPX146) and Neo cells with lower
levels of ROS had the highest growth rates, shortest doubling times,
and highest plating efficiencies. Similar results were found in
vivo in nude mice studies; MnSOD overexpression inhibited tumor
cell growth and lowered tumor incidence; GPX1 overexpression inhibited
such tumor suppression. SODs convert O into
H2O2, whereas GPX1 and CAT
convert H2O2 into water. In
this way, two toxic species, O and
H2O2, are converted into
the harmless product water. Higher activities of GPX1 resulted in
decreased levels of ROS in the S-GPX86 and S-GPX146 cells. S-GPX70 also
had high GPX1 activity, but these cells could not remove
H2O2 efficiently as shown
by high DCFH fluorescence. This may result from the fact that there was
not enough GSH in the S-GPX70 cells (Fig. 5, a and c)
. The removal of
H2O2 by GPX1 requires GSH
as cofactor. These results indicate that GPX1 overexpression can
dramatically abolish the growth suppression by MnSOD overexpression
when there is enough GSH in cells and suggest that
H2O2 or other
hydroperoxides are involved in such growth suppression. The observation
that S-GPX70 line did not behave as the other GPX-overexpressing lines
let us to formulate the effective GPX activity concept: effective GPX
activity equals measured GPX activity times intracellular GSH
concentration. This concept is based on the simple principle that the
rate of an enzymatic reaction is proportional to the substrate
concentration until the enzyme becomes saturated; because GPX does not
become saturated with GSH under normal physiological conditions, in
cells the reaction rate will generally be proportional to GSH levels
(24)
. When we used effective GPX activity, S-GPX70 was no
longer an outlier in any of our measurements. These results suggest
that effective GPX activity should be used to quantify the actual
peroxide removed by this system. Our results suggest that effective GPX
activity is what governed the biological response of all
GPX-overexpressing cell lines, although we cannot rule out the
possibility that the small changes in MnSOD observed in these lines had
some effect on the observed phenotype.
In mammalian cells, overexpression of SOD in cells bearing extra copies
of the SOD gene produced a higher sensitivity to ROS. In these cases,
it has been postulated that SOD overexpression would lead to
accumulation of H2O2
(15, 44)
. In addition, overexpression of CuZnSOD can cause
different responses in different systems. A compensatory increase in
GPX1 occurred as a consequence of the introduction of the CuZnSOD
expression vector into L-cells, neuroblastoma cells and
primary mouse cells (45, 46)
. Transfection of human glioma
cells with human MnSOD can also induce a GPX1 activity
increase (5)
. Transfection of a CuZnSOD
expression vector into 3T3 murine fibroblasts resulted in two classes
of transfectants, characterized by the presence or absence of an
increase in endogenous GPX1. In the transfectants with an absence of an
increase in endogenous GPX1, accumulation of
H2O2 was increased
(47)
. Fullerton et al. (48)
found
that transgenic brain accumulated
H2O2 after perinatal
hypoxia ischemia in CuZnSOD transgenic mice. In JB6 cells and Chinese
hamster fibroblasts, overexpression of CuZnSOD resulted in increased
DNA breakage upon exposure to oxidants (15, 16)
. Amstad
et al. (15)
studied the roles of and the
interaction of CuZnSOD and CAT in transfectants with human cDNAs of
mouse epidermal JB6 clone 41 cells. The CuZnSOD overproducers SOD 15
and SOD 3 were hypersensitive to the formation of DNA single strand
breaks, growth retardation, and killing by an extracellular burst of
O plus H2O2,
whereas CAT overproducers were protected from oxidant damage relative
to the parent clone JB6 clone 41. The double transfectant SOCAT
3 was better protected from oxidant damage because of its increased
content of CAT, which counterbalances the increase in CuZnSOD. In 1994,
Amstad et al. (17)
reported that GPX1 also
compensated for the hypersensitivity of CuZnSOD overproducers to
oxidant stress. Taken together, our results and those of others clearly
indicate that after overexpression of SOD,
H2O2 appears to be a key
player in the growth of SOD transfectants; the balance of SOD and GPX
or CAT is more important for overall sensitivity than the level of SOD
alone, and growth stimulation occurs when cells are protected from
excessive oxidative stress.
In the growth rate results, we also found that Neo cells grew faster
than P cells. This might result from the fact that Neo cells had higher
intracellular ROS levels than P cells and suggests that intracellular
ROS can stimulate cell growth at certain levels. A number of normal
cells and tumors can produce
H2O2 and O
in vitro either in response to various stimulus or
constitutively (15)
. Experiments in which either CAT or
SOD was added exogenously to the growth medium of hamster, rat
fibroblasts (49)
, and HeLa cells (50)
resulted in a depressed rate of cell proliferation. Other experiments
(51, 52)
also indicated low concentrations of O
and H2O2 (10
nM-1 µM) to be effective
in stimulating the in vitro growth of hamster and rat
fibroblasts when added to the culture medium. Using xanthine/xanthine
oxidase (100 µM xanthine and 5 microunits/ml
xanthine oxidase) to generate ROS, Rao and Berk (53)
demonstrated that H2O2 can
stimulate rat vascular smooth muscle cell growth. This evidence
suggests that H2O2 and
O can stimulate growth and growth responses in a variety of
cultured mammalian cell types when produced endogenously or added
exogenously.
GPX1 converts H2O2 to
H2O and catalyzes GSH to GSSG simultaneously. Our
results show that overexpression of GPX1 modulated the content of
intracellular GSH and GSSG (Fig. 5)
. The amount of GSSG in
S-GPX70 cells was the lowest among all of the clones. This result was
not correlated to the measured GPX activity of S-GPX70. As discussed
earlier, this result may be from lower amount of GSH in S-GPX70
compared with other transfectants. Because GPX requires GSH as a
cofactor to exert its function, the lower GSH will limit the maximum
activity of GPX. When GSSG was plotted against effective GPX activity,
good correlation resulted (Fig. 5, b and d)
, and
S-GPX70 was no longer an outlier.
We also found evidence to suggest that metabolism also affected the content of glutathione. The content of GSSG significantly increased in all clones except S-GPX70, whereas the ratio (data not shown) between [GSH]2/[GSSG] was dramatically decreased in all clones at 48 h compared with those at 24 h. Because glucose will be probably depleted during this time period, these results suggest that glucose can influence the levels of intracellular glutathione. We hypothesize that the concentration of glucose is becoming lower with time after feeding. The lower concentration of glucose leads the cells to produce less NADPH through the pentose phosphate pathway. This does not enable GR to fully function without enough NADPH, which causes the accumulation of GSSG. Blackburn et al. (54) recently demonstrated that glucose deprivation significantly increased the steady state levels of GSSG.
GSH and GSSG are important components in the cellular redox system. The
GSH/GSSG couple is the major redox buffer in the cell (23, 24)
. GSH is a tripeptide of 
-glutamate, cysteine, and
glycine, which is found ubiquitously in eukaryotic cells at a
concentration between 1 and 10 mM (24)
. The
relationship among intracellular GSH, GSSG, and GSH/GSSG or
[GSH]2/[GSSG] ratio is more complicated. They
are governed by the rate of synthesis of GSH, the prevailing oxidative
state, the activities of GPX and GR, the degree of export of GSH and
GSSG from the cell, and compartmentalization of these molecules. Most
cell types do not import GSH (55)
, and synthesis occurs
intracellularly in sequential, ATP-dependent reactions catalyzed by
-glutamylcysteine synthetase and GSH synthetase. The rate of
synthesis is influenced primarily by the availability of cysteine but
also by feedback inhibition of synthesis by GSH. Conversion of GSH to
GSSG occurs during GPX1-catalyzed reduction of
H2O2 and other peroxides
and in spontaneous reactions with free radicals (56)
. GSSG
is restored to a reduced form by GR (57)
. Irreversible
loss of GSH occurs through conjugation to endogenous and exogenous
electrophilic centers in reactions catalyzed by GSH transferases
(58, 59)
.
The intracellular ROS levels as measured by DCFH-DA fluorescence were
also modulated by GPX1 overexpression (Fig. 6)
. The results of
intracellular ROS levels suggest that MnSOD overexpression leads to
accumulation of H2O2, which
causes high fluorescence intensity of DCFH-DA. GPX1 can remove
H2O2, so there was low
fluorescence intensity in both S-GPX86 and S-GPX146 clones. Although
S-GPX70 has high GPX activity, there was no significant difference
between SOD2 and S-GPX70 in ROS levels. This result implies that GPX1
in S-GPX70 cells cannot exert its full function because this clone has
lower GSH content compared with the other clones. This result also
implies that GSH is very important to make GPX function. Again, this
conclusion was strengthened by a correlation analysis; a high
correlation resulted when DCFH fluorescence was plotted against
effective GPX activity (Fig. 6b
).
Our results also indicated that overexpression of GPX1 inhibited
IB degradation (Fig. 7)
. The levels of
intracellular ROS as measured by DCFH fluorescence correlated well
with the effective GPX activity (Fig. 6b
); this, along with
the Western blot of IB (Fig. 7)
,
suggests that ROS can cause IB degradation
and that GPX1 can inhibit this process. These results are consistent
with the results reported by Kretz-Remy et al.
(27)
. They demonstrated that
IB degradation was abolished in
GPX1-overexpressing T47D cells exposed to TNF- and
H2O2. There was also an
exception to the general result shown in Fig. 6a
. S-GPX70
cells with high levels of ROS also have high levels of
IB, which implies that other factors such
as GSSG besides ROS can cause IB
degradation. Droge et al. (60)
have
found that T cells fail to activate NF-B in response to appropriate
stimuli at low levels of cytosolic GSSG. Recently, Kroll et
al. (61)
reported that inducible degradation of
IB by the proteasomes requires interaction
with the F-box protein h-ßTrCP. Theoretically, GPX1 can regulate the
cellular redox state by modulating the concentrations of the
H2O2, GSH, GSSG, NADPH, and
NADP+ molecules that are critical players in the
cellular redox system (Fig. 1)
. In HIV-infected T-cells
(25)
and in similar T-cell lines (26)
,
selenium supplementation increased GPX activity and decreased
H2O2-induced NF-B
activation. A human breast cancer cell line (T47D) overexpressing GPX1
responded poorly to NF-B activation by TNF- or
H2O2; both B-dependent
gene transactivation and NF-B DNA binding were reduced
(27)
. Taken together, our findings and those of others
demonstrate that GPX1 plays a key role in the regulation of cellular
redox state.
In conclusion, our results show the following. First, GPX1
overexpression in MnSOD-overexpressing cells can rescue cell growth
suppressed by MnSOD, which suggests that
H2O2 or other
hydroperoxides are critical reactants in the glioma tumor suppression
by MnSOD overexpression. Second, GPX1 overexpression can modulate
intracellular redox status and inhibit IB
degradation. Third, GPX1 exists not only in cytosol but also in the
mitochondria and nucleus. Our results also suggest that effective GPX
activity should be used to quantify the actual peroxide removed by this
system. Taken together, our data show that MnSOD overexpression
inhibits tumor growth because of the production of hydroperoxides and
suggest that inhibition of peroxide removal should increase the tumor
suppressive effect of MnSOD overexpression.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This research was supported by NIH Grant CA
66081 (to L. W. O.) and by the Department of Veterans Affairs
Research Service (to T. D. O.). 
2 To whom requests for reprints should be
addressed. Phone: (319) 335-8015; Fax: (319) 335-8039;
E-mail: larry-oberley{at}uiowa.edu
3 The abbreviations used are: SOD, superoxide
dismutase; CAT, catalase; CuZnSOD, copper-and zinc-containing SOD;
DCFH, 2',7'-dichlorodihydrofluorescein; DCFH-DA, DCFH diacetate;
ddH2O, double-distilled water; DTNB,
5,5'-dithiobis(2-nitrobenzoic acid); G-6-PDH, glucose-6-phosphate
dehydrogenase; GPX, glutathione peroxidase; GR, glutathione reductase;
GSH, reduced glutathione; GSSG, glutathione disulfide;
IB, inhibitory subunit of NF-B; MnSOD,
manganese-containing SOD; NF-B, nuclear factor-B; P, parental;
ROS, reactive oxygen species; RT, reverse transcription; TNB,
5-thio-2-nitrobenzoic acid; TNF-, tumor necrosis factor .
Received 10/20/99. Accepted 5/16/00.
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C. A. Davis, A. S. Hearn, B. Fletcher, J. Bickford, J. E. Garcia, V. Leveque, J. A. Melendez, D. N. Silverman, J. Zucali, A. Agarwal, et al. Potent Anti-tumor Effects of an Active Site Mutant of Human Manganese-Superoxide Dismutase: EVOLUTIONARY CONSERVATION OF PRODUCT INHIBITION J. Biol. Chem., March 26, 2004; 279(13): 12769 - 12776. [Abstract] [Full Text] [PDF]
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S. Y. Park, I. Chang, J.-Y. Kim, S. W. Kang, S.-H. Park, K. Singh, and M.-S. Lee Resistance of Mitochondrial DNA-depleted Cells against Cell Death: ROLE OF MITOCHONDRIAL SUPEROXIDE DISMUTASE J. Biol. Chem., February 27, 2004; 279(9): 7512 - 7520. [Abstract] [Full Text] [PDF]
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Y. Qian, J. Luo, S. S. Leonard, G. K. Harris, L. Millecchia, D. C. Flynn, and X. Shi Hydrogen Peroxide Formation and Actin Filament Reorganization by Cdc42 Are Essential for Ethanol-induced in Vitro Angiogenesis J. Biol. Chem., April 25, 2003; 278(18): 16189 - 16197. [Abstract] [Full Text] [PDF]
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H. J. Zhang, W. Zhao, S. Venkataraman, M. E. C. Robbins, G. R. Buettner, K. C. Kregel, and L. W. Oberley Activation of Matrix Metalloproteinase-2 by Overexpression of Manganese Superoxide Dismutase in Human Breast Cancer MCF-7 Cells Involves Reactive Oxygen Species J. Biol. Chem., May 31, 2002; 277(23): 20919 - 20926. [Abstract] [Full Text] [PDF]
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Y. Zhang, W. Zhao, H. J. Zhang, F. E. Domann, and L. W. Oberley Overexpression of Copper Zinc Superoxide Dismutase Suppresses Human Glioma Cell Growth Cancer Res., February 1, 2002; 62(4): 1205 - 1212. [Abstract] [Full Text] [PDF]
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J. C.-m. Ho, S. Zheng, S. A. A. Comhair, C. Farver, and S. C. Erzurum Differential Expression of Manganese Superoxide Dismutase and Catalase in Lung Cancer Cancer Res., December 1, 2001; 61(23): 8578 - 8585. [Abstract] [Full Text] [PDF]
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