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INTRODUCTION |
Many chemotherapeutic drugs, including Vp16 and mitomycin C,
induce cells to undergo apoptosis through damage of nuclear DNA. The
activation of wild type p53, a major tumor suppressor protein, which is
implicated in cell cycle control, DNA repair, and apoptosis (1-3), has
been reported to be involved in the process of apoptosis induced by
DNA-damaging agents (3, 4). The increase in p53 levels after DNA damage
results in large part from stabilization of the p53 protein against
degradation, and accumulation of p53 occurs because of this increased
protein stability in the presence of ongoing translation (4). Acute
overexpression of wild type p53 mediated by adenovirus facilitates the
ability of DNA-damaging drugs to induce death of cancer and normal
cells (5). As a transcription factor, p53 protein enhances the
transcription rate of several genes including p21WAF1,
GADD45, and 14-3-3
, which are genes involved in cell
cycle arrest (6-8), and Bax, CD95, Noxa, PUMA, and p53AIP1, which are
related to p53-dependent apoptosis (9-13).
Studies in a variety of cell types have suggested that ROS formation
may be directly related to the ability of DNA-damaging agents to induce
apoptosis (14). Other studies indicate that ROS are downstream
mediators of p53-dependent apoptosis (15, 16). p53 acts to
regulate the intracellular redox state and induces apoptosis by a
pathway that is dependent on ROS production (17). p53 results in
apoptosis through a multi-step process, including the transcriptional
induction of redox-related genes, the generation of ROS, and the
oxidative degradation of mitochondrial components, leading to apoptosis
(15). There is also evidence that ROS could regulate apoptosis as a
signal transmitter or mediator (16, 18, 19). However, other studies
indicate that apoptosis can be induced in the absence of any detectable
oxidative stress when cells are treated with VP-16 and cisplatin (20),
and oxidative stress can inhibit apoptosis in human lymphoma cells
(21).
Catalase, an anti-oxidative enzyme, is able to degrade hydrogen
peroxide (H2O2), which is an important ROS
related to p53 activation. Catalase has been shown to protect a variety
of cells against oxidative stress-induced toxicity (22). Our laboratory has utilized adenoviral catalase expression systems to study the ability of catalase to prevent CYP2E1-dependent, and
ROS-dependent toxicity to HepG2 cells (23, 24). In the
current study, catalase adenovirus was used to infect HepG2 cells to
study the effects of catalase on the regulation of apoptosis and
the modulation of p53 expression induced by DNA-damaging agents.
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MATERIALS AND METHODS |
Reagents--
Propidium Iodide was purchased from Molecular
Probes (Eugene, OR). Polyclonal antibody raised in rabbit against human
catalase was obtained from Calbiochem, and antibodies raised in rabbit against p53, phosphorylated p53 on serine 20, p21, Bcl-2, and Bcl-xL
were obtained from Santa Cruz (Santa Cruz, CA). Horseradish peroxidase
conjugated to goat anti-rabbit IgG,
MEM,1 and fetal bovine serum
were purchased from Sigma.
Cell Culture and Recombinant Adenovirus Infection--
HepG2
cells were cultured in MEM containing 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine in a humidified atmosphere in 5% CO2 at
37 °C. Ad-LacZ, a
-galactosidase-expressing replication-deficient
adenovirus, and Ad-Cat, a human catalase-expressing
replication-deficient adenovirus, were constructed as described
previously (23). Before infection, cells were seeded onto dishes or
plates, grown to 60% confluence, and infected with Ad-LacZ or Ad-Cat
with 100 m.o.i. Forty-eight hours after infection, cells were
collected and assayed for catalase expression or treated with
DNA-damaging agents.
Cell Transfection
HepG2 cells were seeded onto 10-cm
culture dishes and grown to 80% confluence. The expression plasmid
vector pCMV-p53mt135 (Clontech), containing
mutant p53 or empty vector pCMV-neo, were transfected into HepG2 cells
using Effectene transfection reagent (Qiagen) according to the
instructions provided by the manufacturer. This mutant p53 expression
vector encodes a mutant p53 protein that combines with wild type p53
protein in a dominant-negative manner and blocks the DNA binding domain
on wild type p53, therefore inhibiting the activity of wild type p53.
Cells were selected by MEM containing 1 mg/ml G418. Two weeks after
transfection, the surviving clones were isolated and grown to large
scale. Stable cell lines with overexpression of mutant p53
(HepG2-mtp53) as well as cells transfected with empty vector
(HepG2-neo) were selected and maintained in MEM containing 1 mg/ml G418.
Western Blotting--
Cell lysates were prepared by sonicating
cells followed by centrifugation. The protein concentration of the
supernatant was measured (DC protein assay reagent, Bio-Rad), and ten
µg of denatured protein was resolved on 10% SDS-PAGE and
electroblotted onto nitrocellulose membranes (Bio-Rad). The membrane
was incubated with rabbit anti-human catalase polyclonal antibody
(1:1000) followed by incubation with horseradish peroxidase conjugated
to goat anti-rabbit IgG (Sigma) (1:5000). Detection by the
chemiluminescence reaction was carried out for 1 min using the ECL kit
(Amersham Biosciences) followed by exposure to Kodak X-Omat x-ray film
(Eastman Kodak Co.). Similar Western blots were carried out to detect
P53, phosphorylated p53 (pp53), P21, Bcl-2, and Bcl-xL.
Catalase Activity Assay--
To validate expression of
functional catalase, catalase activity of fresh sonicated cell extracts
was determined at 25 °C. The decomposition of hydrogen peroxide by
catalase was followed by ultraviolet spectroscopy at 240 nm. The
reaction was performed using a solution of 20 mM hydrogen
peroxide in 50 mM K2HPO4, pH 7.0, containing 10 µg of total cellular protein in a final volume of 1 ml.
Specific activity of catalase was calculated from the equation:
specific activity (units/mg of protein/min) =
A240 nm (1 min) × 1000/43.6 × mg
of protein.
Intracellular Measurement of Reactive Oxygen Species
(ROS)--
Fluorescence spectrophotometry was used to measure
production of intracellular ROS with 2',7'-DCF-DA as the probe
as previously described (24). Briefly, HepG2 cells were uninfected or
infected with Ad-Cat or Ad-LacZ at 100 m.o.i. for 48 h with
or without further treatment with 50 µM Vp16 for 16 h followed by incubation with 5 µM DCF-DA in MEM for 30 min at 37 °C in the dark. The cells were washed in PBS, trypsinized,
and resuspended in 3 ml of PBS, and the intensity of fluorescence was
immediately read in a fluorescence spectrophotometer (PerkinElmer Life
Sciences 650-10S, Hitachi, Ltd.) at 503 nm for excitation and at 529 nm for emission.
DNA Fragmentation Assay--
The DNA fragmentation pattern (DNA
ladder) was carried out by agarose gel electrophoresis. Cells (1 × 106) treated with various reagents were scraped and
centrifuged at 1200 rpm for 10 min. The cell pellet was resuspended in
1 ml of lysis buffer consisting of 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10 mM EDTA, 100 µg/ml proteinase
K, and 0.5% SDS and incubated for 2 h at 50 °C. DNA was
extracted with 1 ml of phenol, pH 8.0, followed by extraction with 1 ml
of phenol/chloroform (1:1) and chloroform. The aqueous phase was
precipitated with 2.5 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate, pH 5.2, at 20 °C overnight. The
precipitates were collected by centrifugation at 13,000 × g for 10 min. The pellets were air-dried and resuspended in
50 µl of Tris/EDTA (10 mM Tris, 1 mM EDTA, pH
8.0) buffer supplemented with 100 µg/ml RNase A. DNA was loaded onto
a 1.5% agarose gel containing ethidium bromide, electrophoresed in
Tris acetate/EDTA buffer for 2 h at 50 V, and photographed under
UV illumination.
DNA Analysis by Flow Cytometry--
Flow cytometry DNA analysis
was used to quantify the percentage of apoptotic cells. Cells (5 × 105) were seeded onto six-well plates and incubated with
various reagents. At different time points, cells were harvested by
trypsinization and washed with PBS, followed by centrifugation at 2000 rpm for 10 min. The cell pellet was resuspended in 80% ethanol and
stored at 4 °C for 24 h. Cells were washed twice with PBS. The
pellet was resuspended in PBS containing 100 µg/ml RNase A,
incubated at 37 °C for 30 min, stained with propidium iodide (50 µg/ml), and analyzed by flow cytometry DNA analysis as described
previously (24).
P53 Synthesis and Degradation--
HepG2 cells were plated
separately in 10% fetal bovine serum, MEM; after overnight incubation,
cells were infected with Ad-Cat and Ad-LacZ adenovirus with 100 m.o.i. Forty-eight hours latter, 50 µM Vp16 was added,
and the cells incubated for a further 16 h. The medium was
replaced with methionine-cysteine-free MEM plus 10% dialyzed fetal
bovine serum and 50 µM Vp16, and the cells were incubated
for 2 h, after which they were pulse-labeled with 150 µCi of
EasyTagTM Expre35S35S Protein
Labeling Mix (PerkinElmer Life Sciences) for 0.5, 2, and 4 h to
study the rate of p53 synthesis. One set of samples was pulse-labeled
for 2 h in the presence of 40 µM cycloheximide, an
inhibitor of protein synthesis, as a control for the analysis of p53
synthesis. After the pulse period, cells were washed in 1×
phosphate-buffered saline and lysed at the indicated time points with
150 µl of 10 mM Tris-HCl buffer, pH 7.4, 0.5% Triton
X-100, 1 mM EDTA, 150 mM NaCl, 0.5% sodium
deoxycholate, 1% SDS, and 1 mM phenylmethylsulfonyl
fluoride and immunoprecipitated with anti-p53 IgG as described below.
To study the rate of p53 degradation, HepG2 cells were treated as above
but pulse-labeled with the Expre35S35S mix for
4 h. The cells were then washed 3 times and chased with complete
MEM supplemented with 300 µg/ml cold methionine. Cells were washed in
1× phosphate-buffered saline and lysed at chase times of 0, 2, and
4 h in the same lysis buffer. In all cases, p53 was
immunoprecipitated with rabbit-anti-human p53 IgG-protein G-agarose as
follows; 40 µl of cell lysate (100 µg of protein) was first
incubated with 10 µl of preimmune rabbit serum for 15 min followed by
the addition of 30 µl of a 50% (v/v) suspension of protein
G-agarose. After centrifugation for 2 min at 13,000 rpm, the
supernatant was incubated with anti-p53 IgG and shaking overnight at
4 °C followed by the addition of 30 µl of a suspension of protein
G-agarose. Samples were centrifuged for 1 min at 13,000 rpm, and the
pellets were washed 3 times with lysis buffer, once with lysis buffer
plus 2% SDS, and 3 times with 0.1 M Tris-HCl buffer, pH
6.8. p53 was eluted by boiling for 5 min in Laemmli buffer, and samples
were centrifuged for 2 min at 13,000 rpm to remove the protein
G-agarose, resolved on 8% SDS-PAGE, and dried. The radioactive signal
was detected by exposure to Kodak X-Omat x-ray film.
Northern Blotting--
Total RNA was isolated using the Trizol
reagent (Invitrogen). Ten micrograms of RNA was electrophoresed under
denaturing conditions on formaldehyde-containing 1% agarose gels and
transferred onto GeneScreen nylon membranes (PerkinElmer Life
Sciences). RNA was UV-cross-linked at 1200 J × 100 for 30 s
and prehybridized in a hybridization oven for 2 h at 42 °C in
5× SSC (1× SSC = 0.15 M NaCl and 0.015 M
sodium citrate), 50% formamide, 50 mM potassium phosphate (pH 6.5), and 1× Denhardt's solution containing 0.1 mg/ml
denatured salmon sperm DNA. cDNA probes were radiolabeled with
[32P]dCTP (specific activity >3000 Ci/mmol; Amersham
Biosciences) using a random primer DNA labeling kit (Amersham
Biosciences). Immobilized RNA was hybridized in 0.1% SDS, 5× SSC,
50% formamide, 50 mM potassium phosphate, pH 6.5, 1×
Denhardt's solution, and 12.5× dextran sulfate containing
106 cpm/ml labeled cDNA and 0.1 mg/ml denatured salmon
sperm DNA for 16 h at 42 °C. Blots were washed 4 times for 15 min with 2× SSC and 0.5% SDS at 65 °C and exposed to Kodak x-ray film.
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RESULTS |
Overexpression of Catalase in HepG2 Cells--
To examine the
capacity of Ad-Cat to enhance the expression and activity of catalase
in cells, HepG2 cells were infected with 100 m.o.i. of Ad-Cat or
Ad-LacZ. By
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
assay it was determined that more than 98% of the HepG2 cells were
infected with the adenovirus under these reaction conditions. 48 h
later, cell lysates were prepared and subjected to Western blot
analysis. Fig. 1A shows the
expression of catalase in the total cell extract from these cells.
Results from densitometric analyses of the intensity of the various
bands indicated that the expression of catalase in total cell extracts
of cells infected with Ad-Cat was 7-10-fold higher than that in
uninfected HepG2 cells or HepG2 cells infected with Ad-LacZ. Similar
results were obtained by measuring catalase activity (Fig.
1B). In total cellular extracts of HepG2 cells infected with
Ad-Cat, catalase activity was 7-10-fold higher than that in uninfected
HepG2 cells or HepG2 cells infected with Ad-LacZ.

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Fig. 1.
Expression of catalase in HepG2 cells
infected with adenovirus. HepG2 cells were infected with Ad-Cat or
Ad-LacZ at 100 m.o.i. 48 h later, a cell extract was
prepared, and a cell extract from uninfected HepG2 cells was also
prepared as control. Ten micrograms of protein was loaded onto each
lane for 10% SDS-polyacrylamide gel electrophoresis followed by
Western blot analysis with polyclonal rabbit anti-human catalase
antibody as described under "Materials and Methods" (A).
Lane 1, uninfected HepG2 cells as control; lane
2, HepG2 cells infected with Ad-LacZ; lane 3, HepG2
cells infected with Ad-Cat. Ten micrograms of protein from cell
extracts was used to measure the catalytic activity of catalase
(B). Specific catalase units (units/mg of protein/min) were
calculated as described under "Materials and Methods." First
bar, uninfected HepG2 cells as control; second bar,
HepG2 cells infected with Ad-LacZ; third bar, HepG2 cells
infected with Ad-Cat. (mean ± S.D.; n = 3).
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Intracellular ROS--
We evaluated ROS generation by measuring
dichlorofluorescein fluorescence after loading the cells with 5 µM DCF-DA. It is recognized that
dihydrochlorofluorescin may be oxidized by several oxidants
including H2O2, superoxide radical, lipid
hydroperoxides, lipid peroxyl and alkoxyl radicals, and by cellular
peroxidases (25, 26) and is not specific for e.g.
H2O2 but serves as a general index for ROS
production. In the absence of VP16, control fluorescence was 150 arbitrary units (Fig. 2). Infection with Ad-LacZ had no effect on DCF production, but infection with Ad-Cat decreased fluorescence by about 45%. The addition of 50 µM VP16 increased DCF production by about 60%. Ad-LacZ
again had no effect, whereas Ad-Cat decreased fluorescence by about
40%. Thus, VP16 increased production of ROS by HepG2 cells, and
catalase decreased the basal as well as the VP16-stimulated ROS
generation.

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Fig. 2.
Determination of intracellular ROS;
fluorescence spectrophotometry was used to measure intracellular ROS
with 2',7'-DCF-DA as the probe. HepG2 cells were uninfected or
infected with Ad-Cat or Ad-LacZ at 100 m.o.i. for 48 h with
or without further treatment with 50 µM Vp16 for 16 h followed by incubation with 5 µM DCF-DA in MEM for 30 min at 37 °C in the dark. The cells were washed in PBS, trypsinized,
and resuspended in 3 ml of PBS, and the intensity of fluorescence was
immediately read in a fluorescence spectrophotometer at 503 nm for
excitation and at 529 nm for emission. The results were expressed as
arbitrary units of the fluorescence intensity. Data are the
mean ± S.D. of triplicate experiments.
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Overexpression of Catalase Protects HepG2 Cells from Apoptosis
Induced by DNA-damaging Reagents--
HepG2 cells were plated
separately in 10% fetal bovine serum, MEM; after overnight incubation,
cells were infected with Ad-Cat and Ad-LacZ adenovirus with 100 m.o.i. Forty-eight hours later, 500 µM Vp16 or 10 µg/ml
mitomycin C were added, and the cells were incubated for a further
24 h. DNA fragmentation and DNA analysis by flow cytometry as well
as cellular morphology changes were carried out as described under
"Material and Methods." Fig.
3A shows the results of DNA
fragmentation. No DNA ladder was seen in either Ad-LacZ (lane
2)- or Ad-Cat (lane 3)-infected HepG2 cells without any
treatment. When treated with Vp16 (lanes 4 and 5)
or mitomycin C (lanes 6 and 7), HepG2 cells
infected with Ad-LacZ showed a clear DNA ladder (lanes 4 and
6), whereas only a very weak DNA ladder was seen in HepG2
cells infected with Ad-Cat (lanes 5 and 7).

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Fig. 3.
Expression of catalase protects HepG2 cells
from apoptosis induced by Vp-16 or mitomycin C. HepG2 cells were
infected with Ad-Cat and Ad-LacZ adenovirus at 100 m.o.i.
Forty-eight hours later, 500 µM Vp16 or 10 µg/ml
mitomycin C were added and incubated for a further 24 h. DNA
fragmentation and DNA analysis by flow cytometry as well as morphology
changes. were carried out as described under "Material and
Methods." A, DNA fragmentation. Lane 1, 100-bp
DNA marker; lane 2 (Ad-LacZ) and lane 3 (Ad-Cat)
were HepG2 cells infected with adenovirus without any treatment of
Vp-16 or mitomycin C; lane 4 (Ad-LacZ) and lane 5 (Ad-Cat) were HepG2 cells treated with Vp16 after infection; lane
6 (Ad-LacZ) and lane 7 (Ad-Cat) were HepG2 cells
treated with mitomycin C after infection. B, DNA analysis by
flow cytometry. Panels a (AD-LacZ) and b (AD-Cat)
were HepG2 cells infected with adenovirus without any treatment of
Vp-16 or mitomycin C; panels c (Ad-LacZ) and d
(Ad-Cat) were HepG2 cells treated with Vp16 after infection;
panels e (Ad-LacZ) and f (Ad-Cat) were HepG2
cells treated with mitomycin C after infection. One of the three
independent experiments is shown. C, morphologic change.
Panels a (AD-LacZ) and b (AD-Cat) were HepG2
cells infected with adenovirus without any treatment of Vp-16 or
mitomycin C; panels c (Ad-LacZ) and d (Ad-Cat)
were HepG2 cells treated with Vp16 after infection; panels e
(Ad-LacZ) and f (Ad-Cat) were HepG2 cells treated with
mitomycin C after infection (100×).
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DNA analysis by flow cytometry after staining with propidium iodide was
used to measure the percentage of apoptotic cells (Fig. 3B).
After 48 h infection with either Ad-LacZ or Ad-Cat, the percentage
of apoptotic cells was less than 3% in the absence of any treatment
with DNA-damaging agents (Fig. 3B, panels a and b). The percentage of apoptotic cells increased to 25 and
21% after 24 h of treatment with 500 µM Vp16 or 10 µg/ml mitomycin C, respectively (Fig. 3B, panels
c and e). The HepG2 cells infected with Ad-Cat were
resistant to 500 µM Vp16 or to 10 µg/ml mitomycin C-induced apoptosis, displaying only 5 and 7% apoptotic cells, respectively (Fig. 3B, panels d and
f). Similar results were observed when studying morphologic
changes (Fig. 3C). Cells infected with Ad-LacZ lost normal
morphology when treated with 500 µM Vp16 or 10 µg/ml
mitomycin C for 24 h, becoming round in shape and shrinking in
size (panels c and e), whereas most of the cells
infected with Ad-Cat retained their shape and structure (panels
d and f).
Overexpression of Catalase Down-regulates p53 and p21 Protein but
Not Bcl-2 and Bcl-xL in HepG2 Cells--
We investigated the
expression of selected proteins related to apoptosis in cells infected
with Ad-LacZ and Ad-Cat for 48 h with or without further treatment
with 50 µM Vp16 or 5 µg/ml mitomycin C for 16 h.
The basal level of p53 was low in HepG2 cells, most likely due to the
short half-life of wild type p53. This basal level of p53 was even
lower in cells infected with Ad-Cat than in cells infected with Ad-LacZ
(Fig. 4, panel a). After
treatment with Vp16 or mitomycin C, the p53 protein level was
dramatically increased in cells infected with Ad-LacZ but only slightly
increased in the cells infected with Ad-Cat. Because p21 is one of the
downstream genes of p53, which is induced by activation of
p53, p21 protein level was also detected by Western blot.
Coincident with p53, the p21 protein level was dramatically increased
in cells infected with Ad-LacZ after treatment with Vp16 or mitomycin C
(Fig. 4, panel b). Infection with catalase adenovirus
lowered the basal as well as the stimulated levels of p21, analogous to
results with p53 levels. Bcl-2 and Bcl-xL are two anti-apoptotic genes.
The expression of these proteins was not different between Ad-LacZ and
Ad-Cat infection with or without treatment by Vp16 or mitomycin C (Fig.
4, panels c and d).

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Fig. 4.
Expression of catalase modulates p53 and p21
but not Bcl-2 and Bcl-xL protein level. HepG2 cells were infected
with Ad-Cat (C) or Ad-LacZ (Z) at a m.o.i of 100 for 48 h with or without further treatment by adding 50 µM Vp16 or 5 µg/ml mitomycin C (Mito) for
16 h. 25 µg of protein were loaded onto each lane for 10%
SDS-polyacrylamide gel electrophoresis followed by Western blot
analysis with polyclonal rabbit anti-human p53 (a), p21
(b), Bcl-2 (c), and Bcl-xL (d)
antibody as described under "Materials and Methods." Three
independent experiments were carried out.
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Apoptosis Induced by Vp16 Is p53-dependent--
To
evaluate whether catalase protects HepG2 cells from apoptosis induced
by DNA-damaging agents through down-regulation of p53 protein, it was
important to first demonstrate that p53 was required for the developing
apoptosis. HepG2-mtp53 and HepG2-neo cell lines were set up by stable
transfection of HepG2 cells with the expression plasmid vector
containing mutant p53 or empty vector. This mutant p53 expression
vector encodes a mutant p53 protein, which combines with wild type p53
protein in a dominant-negative manner and blocks the DNA binding domain
on wild type p53, therefore inhibiting the activity of wild type p53.
After treatment with different concentrations (50, 250, and 500 µM) of Vp16 for 24 h, HepG2 cells transfected with
the empty vector (HepG2-neo) underwent DNA fragmentation (Fig.
5). However, no or very weak DNA
fragmentation was observed in HepG2 cells transfected with the mutant
p53 expression vector (HepG2-mtp53), even at the maximum concentration
of Vp16 (500 µM) studied (Fig. 5). These results indicate
that p53 is necessary for the Vp16-induced apoptosis in the HepG2 cells
and suggest that one mechanism by which catalase protects against this
apoptosis is to lower the intracellular levels of p53.

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Fig. 5.
A decrease in normal p53 function confers
resistance to Vp16-induced apoptosis. HepG2-neo and HepG2-mtp53
cell lines were produced as described under "Materials and
Methods." Cells were treated with 0, 50, 250, or 500 µM
Vp16 for 24 h. DNA fragmentation was carried out as described in
Fig. 3A. One of two independent experiments is shown.
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Overexpression of Catalase Down-regulates p53 Protein by
Potentiating p53 Protein Degradation but Not by Decreasing p53 Protein
Synthesis or mRNA Levels--
The catalase down-regulation of the
expression of p53 protein under basal conditions or after treatment
with DNA-damaging agents could be due to an effect on p53 transcription
or translation or protein degradation. HepG2 cells were infected with
Ad-LacZ and Ad-Cat for 48 h with or without further treatment by
50 µM Vp16 for 16 h. RNA was isolated, and a
Northern blot was performed using [32P]dCTP-labeled p53
or glyceraldehyde-3-phosphate dehydrogenase cDNA probes. There was
no significant difference in p53 mRNA levels between cells infected
with Ad-LacZ and Ad-Cat either under basal conditions or after
treatment with Vp16 (Fig. 6). Vp16
treatment, which dramatically elevated p53 protein levels (Fig. 4), did
not have any effect on p53 mRNA levels (Fig. 6). This result
indicates that overexpression of catalase does not decrease
transcription of the p53 gene into mRNA; therefore, changes in
mRNA are not responsible for the inhibitory effect of catalase on
the level of p53 protein.

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Fig. 6.
Expression of catalase and/or treatment with
Vp-16 does not change the p53 mRNA levels. Wild type HepG2
cells (W) and HepG2 cells infected with Ad-Cat
(C) or Ad-LacZ (Z) for 48 h were incubated
in the absence or presence of 50 µM Vp16 for 16 h.
RNA was isolated, and Northern blot analysis was performed using
[32P]dCTP-labeled p53 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA probes. Two independent
experiments were carried out.
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Two other possibilities that might relate to the inhibitory effect of
catalase on the level of p53 protein were considered, which are that
catalase inhibits p53 protein synthesis or catalase increases the
degradation of p53 protein. Pulse-labeling and pulse-chase assays were
performed to investigate these possibilities. HepG2 cells were infected
with Ad-LacZ and Ad-Cat for 48 h and then treated with 50 µM Vp16 for 16 h followed by pulsing with
35S-labeled methionine for 0.5, 2, and 4 h. p53
protein was immunoprecipitated, and the incorporated label was
quantified by SDS-PAGE and recorded on x-ray film. The intensity of the
p53 signal increased after 2 h of pulsing as compared with
0.5 h (Fig. 7A). To
validate the assay, the effect of cycloheximide was determined; p53
synthesis was inhibited by adding 40 µM cycloheximide.
There was no decrease in the p53 signal intensity in cells infected
with Ad-Cat compared with cells infected with Ad-LacZ at any time point
(Fig. 7A). This indicates that overexpression of catalase
does not change the rate of p53 protein synthesis.

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Fig. 7.
Effects of expression of catalase on p53
protein synthesis and degradation. HepG2 cells were infected with
Ad-LacZ (Z) and Ad-Cat (C) for 48 h followed
by treatment with 50 µM Vp16 for 16 h. The cells
were then pulse-labeled with 35S-labeled methionine for
0.5, 2, and 4 h, p53 protein was immunoprecipitated, and the
amount of incorporated label was quantified by SDS-PAGE and recorded on
x-ray film as described under "Materials and Methods." One set of
experiments in which cells were incubated with 40 µM
cycloheximide was included at 2 h (A). To determine the
rate of p53 protein degradation, a chase was performed by adding cold
methionine for 0, 2, and 4 h after a 4-h pulse with
35S-labeled methionine (B). C is a
bar graph of the arbitrary densitometric units of the p53
bands in B. Two independent experiments were carried
out.
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To determine the rate of p53 protein degradation, pulse-chase was
performed by adding cold methionine for 0, 2, and 4 h after a 4-h
pulse with 35S-labeled methionine. Compared with cells
infected with Ad-LacZ, the p53 signal in cells infected with Ad-Cat was
considerably lower after the 2- and 4-h chase (Fig. 7B;
quantification in Fig. 7C). This indicates that the rate of
p53 degradation was potentiated by overexpression of catalase. In the
presence of Vp16, p53 levels were maintained over the 4-h chase period
in the Ad-LacZ-infected HepG2 cells.
The Potentiated p53 Protein Degradation Caused by Catalase
Overexpression Is Prevented by Proteasome Inhibitors--
The
proteasome complex plays an important role in the turnover of p53;
therefore, the effect of proteasome inhibitors such as 5 µM lactacystin or 10 µM MG115 as well as
the calpain inhibitor calpeptin on p53 protein levels in cells infected
with Ad-LacZ or Ad-Cat was determined (Fig.
8). The administration of lactacystin or
MG115 increased the p53 protein level 3-4-fold in Ad-LacZ-infected cells not treated with Vp16. This likely reflects the prevention of p53
degradation. The elevated level of p53 in the Vp16-treated cells was
not further increased by lactacystin or MG115, which may reflect that
both treatments result in decreasing p53 turnover (further described
below, Fig. 9). Calpeptin did not affect the basal level of p53
or the Vp16-stimulated levels in the Ad-LacZ-infected cells (Fig. 8).
In the Vp16-treated cells, catalase lowered p53 levels by 50-60% in
the control or calpeptin-treated cells but only 20-25% in the
lactacystin- or MG115-treated cells. Thus, the lowering of p53 levels
by catalase was partially prevented or diminished by administration of
proteasome inhibitors but not by calpeptin. Because of toxicity, higher
concentrations of lactacystin or MG115 could not be evaluated; however,
these results are consistent with role for the proteasome in p53
turnover and in the catalase enhancement of this turnover.

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Fig. 8.
The effect of proteasome inhibitors on the
catalase mediated down-regulation of p53 protein levels. HepG2
cells were infected with Ad-LacZ (Z) or Ad-Cat
(C) for 48 h followed by treatment with
(VP16+) or without (Vp16 ) 50 µM
Vp16 in the absence (Contl) or presence of the proteasome
inhibitors 5 µM lactacystin (Lacta) or 10 µM MG115 or the calpain inhibitor 50 µM
calpeptin for 8 h, and their effects on the catalase-mediated
lowering of p53 protein was evaluated. A cell extract was prepared, and
25 µg of protein was loaded onto each lane for 10%SDS-polyacrylamide
gel electrophoresis followed by Western blot analysis with polyclonal
rabbit anti-human p53 antibody as described under "Materials and
Methods." One of three independent Western blot results is shown. The
blots were scanned, and the band intensity numbers are shown
below the specific band.
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Catalase Overexpression Decreases the Phosphorylation of
p53--
p53 is activated through phosphorylation. Once
phosphorylated, p53 is imported into the nucleus and can now activate
downstream genes such as p21 and Bax, acting as a transcription factor.
In addition, phosphorylated p53 has a longer half-life than
unphosphorylated p53, therefore leading to p53 accumulation. This
phosphorylation can occur on several serine residues including serine
9, 15, 20, and 392 of the p53 protein. One possible mechanism by which
catalase increases turnover of p53 could be by decreasing formation of the more stable phosphorylated p53. Using anti-p53 antibody and a
specific antibody for recognition of phosphorylation of p53 on serine
20, the pp53 level was compared with the total p53 level in cells
treated with Vp16 or lactacystin. Both Vp16 and lactacystin increase
the p53 level; however, only Vp16 increased the pp53 level (Fig.
9A). This suggests that
although lactacystin and Vp16 each increase the p53 level by decreasing
the degradation of p53, they do so by different mechanisms. Lactacystin
inhibits the degradation of p53, whereas Vp16 results in formation of
the more stable pp53. To determine the effect of catalase on p53
phosphorylation, HepG2 cells infected with Ad-LacZ or Ad-Cat were
treated with Vp16 for different times (0, 8, 16, and 24 h), and
immunoblots to detect p53 and pp53 were carried out. Catalase
overexpression decreased the phosphorylated form of p53 in association
with the decrease in total p53 levels as compared with Lac Z expression
(Fig. 9B).

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Fig. 9.
Expression of catalase decreases the
phosphorylation of p53 protein induced by Vp16. A,
Western blot analysis of p53 and pp53 levels in HepG2 cells treated
with or without 50 µM Vp16 in the absence or presence of
5 µM lactacystin. B, HepG2 cells were infected
with Ad-LacZ (Z) or Ad-Cat (C) for 48 h
followed by treatment with 50 µM Vp16 for 0, 8, 16, and
24 h. A cell extract was prepared, and Western blot analysis was
carried out with anti-human p53 and anti-pp53-20 antibody as described
above. Three independent experiments were carried out.
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The Decrease in p53 and Phosphorylated p53 Levels Caused by
Catalase Overexpression Is Not Prevented by the Phosphatase Inhibitor
Okadaic Acid--
Phosphorylated p53 can be dephosphorylated by
multiple protein phosphatases. To determine whether phosphatases may be
involved in catalase-mediated down-regulation of p53 and pp53, a
broad-spectrum phosphatase inhibitor, okadaic acid, was used. HepG2
cells were infected with Ad-LacZ and Ad-Cat for 48 h followed by
treatment without or with 50 µM Vp16 in the presence of
100 nM okadaic acid. After incubating with okadaic acid for
0, 2, 4, and 8 h, Western blot analysis was carried out to detect
the level of p53 and pp53. Okadaic acid did not prevent the effect of
catalase on down-regulation of p53 and pp53 levels (Fig.
10).

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Fig. 10.
The effect of the phosphatase inhibitor
okadaic acid on catalase-mediated down-regulation of p53 and pp53
protein levels. HepG2 cells were infected with Ad-LacZ
(Z) or Ad-Cat (C) for 48 h followed by
treatment with or without 50 µM Vp16 in the presence of
100 nM okadaic acid for different periods (0, 2, 4, and
8 h). Western blot analysis on cell extracts was carried out to
detect the level of p53 and pp53 as described above. Three independent
experiments were carried out.
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DISCUSSION |
Overexpression of Catalase Protects HepG2 Cells from Apoptosis
Induced by DNA-damaging Agents--
DNA ladder and flow cytometry
experiments as well as morphology changes indicated that HepG2 cells
infected with Ad-Cat were less sensitive to Vp16 and mitomycin
C-induced apoptosis than HepG2 cells infected with Ad-LacZ. This
suggests that ROS, especially H2O2, might be
involved in the process of apoptosis induced by these DNA-damaging
drugs. Studies in a variety of cell types suggest that cancer
chemotherapeutic drugs induce tumor cell apoptosis in part by
increasing formation of ROS (14). However, other studies indicate that
apoptosis can be induced in the absence of any detectable oxidative
stress when tumor cells were treated with VP-16 and cisplatin (20). ROS
may not necessarily be the direct factor to cause apoptosis induced by
DNA-damaging reagents, but intracellular ROS may modulate apoptotic
gene expression, which subsequently regulates apoptosis. Thus,
although catalase was protective against Vp16 or mitomycin C-induced
apoptosis, it is not clear if H2O2 is directly
responsible for inducing toxicity or H2O2 is
upstream of signaling events that alter the pro- and anti-apoptotic
balance in the HepG2 cells.
Catalase Protects HepG2 Cells from Apoptosis by Lowering the p53
Protein Level as a Consequence of Increased Degradation--
P53 is a
critical apoptotic gene that is activated before and during apoptosis.
The overexpression of wild type p53 constructs in tumor cells was able
to inhibit cell growth or to initiate apoptosis. p53 gene status
modulates the chemosensitivity of non-small cell lung cancer cells. DNA
damage induced by the DNA-damaging agents Vp16 or mitomycin C activates
p53 mainly through protein modification events such as phosphorylation,
which results in p53 protein stabilization and accumulation of p53
protein due to the decrease of its degradation (4) (Fig. 9,
A and B). HepG2 cells transfected with a mutant
p53 expression vector (HepG2-mtp53), which encodes a mutant p53
protein, decreasing wild type p53 function, are more resistant to
Vp16-induced apoptosis than HepG2 cells transfected with empty vector
(HepG2-neo) (Fig. 5). This indicates that apoptosis induced by Vp16 in
HepG2 cells is p53-dependent. HepG2 cells overexpressing
catalase as mediated by adenovirus-mediated gene transfer (Ad-Cat) show
a lower level of basal or induced p53 protein than control (Ad-LacZ)
cells. Lowering of p53 levels is likely to be the mechanism whereby
catalase overexpression protects HepG2 cells from apoptosis induced by
the DNA-damaging agents, whereas the latter induce apoptosis by
elevating p53 levels. There are no differences in p53 mRNA levels
between catalase-overexpressing cells and control cells with or without
treatment by DNA-damaging agents, and the p53 mRNA level did not
change after treatment with Vp16 even though p53 protein levels were
strongly increased. Thus, the decrease in p53 protein levels by
catalase overexpression or the increase in p53 levels by treatment with
Vp16 is due to posttranscriptional effects. P21 is a downstream gene
target of p53 and is regulated by p53 (27). HepG2 cells overexpressing catalase also display a lower level of p21 protein than control cells
either in the absence or presence of treatment with DNA-damaging agents. Lower levels of p21 protein in the presence of catalase may
reflect the lower levels of p53 protein in the presence of catalase.
The results of synthesis experiments indicate that overexpression of
catalase does not change the rate of p53 protein synthesis; however,
pulse-chase experiments did reveal that p53 protein degradation was
increased in cells infected with Ad-Cat compared with cells infected
with Ad-LacZ.
By adenovirus-mediated gene transfer, Johnson et al. (17)
find that cells sensitive to p53-mediated apoptosis produced ROS concomitantly with p53 overexpression, whereas cells resistant to p53
failed to produce ROS. In sensitive cells, both ROS production and
apoptosis were inhibited by antioxidant treatment. These results suggest that p53 acts to regulate the intracellular redox state and
induces apoptosis by a pathway that is dependent on ROS production (17). In our experiments, removing H2O2 by
expression of catalase causes the p53 protein level to decrease, and
this is associated with resistance to apoptosis induced by Vp16 or
mitomycin C. This suggests that, at least in HepG2 cells,
H2O2 regulates apoptosis by modulating p53
levels, and H2O2 acts upstream of p53.
As a result of a high turnover rate, the p53 protein has a half-life of
~30-60 min and is maintained at low levels in normal proliferating
cells (28). In response to genotoxic stress or oncogenic signaling, p53
levels rapidly increase, mainly through protein stabilization. An
important regulator of the p53 level is MDM2, which possesses intrinsic
E3 ligase activity and, thus, promotes p53 ubiquitination and
subsequent degradation via proteasome-mediated proteolysis (29).
Overexpression of catalase did increase p53 protein degradation, and
the proteasome inhibitors lactacystin and MG115 but not the calpain
inhibitor calpeptin could partially block the p53 protein lowering
caused by overexpression of catalase. This is consistent with the
overall mechanism that catalase lowers p53 levels by accelerating p53 degradation.
How overexpression of catalase specifically down-regulates p53 protein
or enhances p53 turnover is not clear. Because
H2O2 is a specific substrate of catalase,
catalase down-regulation of p53 protein would appear to involve some
function or action of H2O2. One hypothesis is
the H2O2 may increase the level of phosphorylated p53 by stimulating certain protein kinases implicated in
the phosphorylation of p53 or by inhibiting the activity of protein
phosphatases that convert pp53 to the less stable nonphosphorylated state. Once p53 protein is phosphorylated, its half-life is increased, which causes p53 accumulation (30). If ROS indeed modulate the actions
of p53 kinases or p53 phosphatases, overexpression of catalase may
decrease p53 accumulation by removing ROS such as H2O2. Using a specific antibody for detecting
phosphorylated p53 on serine 20, we found that overexpression of
catalase did prevent phosphorylation of p53 induced by Vp16, and low
levels of pp53 were associated with low levels of total p53. However,
the general phosphatase inhibitor, okadaic acid, did not prevent the
effect of catalase on down-regulation of p53 or pp53. This suggests
that catalase may inhibit activity of p53 kinases, which may be
activated by H2O2. Recent studies indicate that
ROS such as H2O2-induced phosphorylation of p53
can be mediated in part by polo-like kinase-3 (30). Further studies
will be necessary on the effects of ROS and of catalase on the
activities of protein kinases implicated in phosphorylation of p53 and
on protein phosphatases implicated in the dephosphorylation of pp53.
Chromium and vanadate metals that can cause oxidative stress are
believed to modulate p53 through H2O2 (31, 32).
H2O2 may also cause certain modifications of the p53 protein structure, e.g. thiol oxidation, such that
the protein is not an effective substrate for proteolysis. Such
putative, stabilizing modifications would be prevented by rapid removal of H2O2 by catalase.
In summary, these results demonstrate that catalase protects HepG2
cells from apoptosis induced by DNA-damaging agents in association with
decreasing the level of pp53, which leads to accelerating the
degradation of p53 protein. The level of catalase may therefore play an
important role in cell-induced resistance to the effects of anti-cancer
drugs by modulating the cellular levels of p53 and p53 targets such as p21.