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INTRODUCTION |
Human p53 is a 393-amino acid nuclear phosphoprotein and
transcription factor. Structurally, the p53 molecule consists of three
major domains: an oligomerization domain in its carboxyl terminus which
mediates homotetramer formation; a DNA binding domain in the center
portion of the molecule which specifically binds to its consensus
binding site; and a transactivation domain in the amino terminus which
mediates transactivation of many downstream target genes (1-3). As a
typical tumor suppressor, p53 has been shown to inhibit tumor cell
growth and suppress transformation through induction of G1
arrest or apoptosis (4-6). As a "genome guard," p53 prevents gene
amplification, thus maintaining genome stability (7-9). In addition,
p53 is also involved in differentiation, senescence, and antiogenesis
(10-12).
Most of the functions of p53 involve its activity as a transcription
factor. p53 binds to its consensus binding sequence (two copies of the
10-bp1 motif
5'-PuPuPuC(A/T)(T/A)GPyPyPy-3', separated by 0-13 bp) (13) and
transactivates expression of target genes. Several biologically significant genes were found to contain this consensus sequence and to
be subjected to p53 regulation. Among those commonly studied are
Waf-1/p21 (14), Mdm2 (15), Gadd45
(16), Bax (17), and the genes encoding proliferating cell
nuclear antigen (18, 19), cyclin G (20), epidermal growth factor
receptor (21), thrombospondin (22), and matrix metalloproteinases-2
(23), among others. The p53 mutations found in many human cancers were clustered in the specific DNA binding domain of the p53 molecule (24).
This leads to an inactivation of p53 function through abolishing
p53-specific DNA binding and transactivation.
Cellular glutathione peroxidase, GPX (EC 1.11.1.9), is one of the
primary antioxidant enzymes that scavenges hydrogen peroxide and
organic hydroperoxides with glutathione as the hydrogen donor (25). The
enzyme was first described in 1957 and is found mainly in cytoplasm
(26). GPX is a selenium-dependent enzyme that exists as a
homotetramer with each 22-kDa subunit containing a selenium atom
incorporated within a catalytically active selenocysteine residue (27,
28). There are three other members of the
selenium-dependent GPX family, although cytosolic GPX
(GPX1) is the predominant form (29). The gene encoding GPX was mapped
on chromosome 3q11-13 (30). Because GPX decomposes hydrogen peroxide
and organic hydroperoxides produced during normal metabolism and after
oxidative insults, GPX prevents peroxide-induced DNA damage, lipid
peroxidation, and protein degradation (31).
Using DNA chip technology, we have found recently that GPX is inducible
by etoposide, a topoisomerase II inhibitor, an apoptosis inducer, and a
p53 activator. (32). We report here the characterization of
GPX as a novel p53 target gene, determined by assays for DNA binding, transcriptional activation, and endogenous gene induction. This finding links p53 to an antioxidant enzyme for the first time and
may shed light on a better understanding of p53 signaling pathways and
redox regulation.
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MATERIALS AND METHODS |
Cell Culture and Drug Treatment
Two human osteogenic sarcoma cell lines, U2-OS and Saos-2, were
grown in 10% McCory or 10% Dulbecco's modified Eagle's medium, respectively. U2-OS cells harbor a wild type p53 (23), whereas Saos-2
cells have the p53 gene deletion (33). The doubling time is
about 24 h for U2-OS and 40 h for Saos-2 under these culture conditions. For drug treatment, U2-OS and Saos-2 cells were exposed to
etoposide (25 µM, Sigma) for various periods of time up
to 48 h.
Gel Shift Assay
The assay was performed as described previously (34, 35).
Briefly, a 20-bp synthetic oligonucleotide, GPX01
(5'-GGGCCAGACCAGACATGCCT-3'), which consists of the putative p53
binding site found in the GPX promoter and its complementary
strand, were annealed and labeled with 32P using T4
polynucleotide kinase and [
-32P]ATP. A DNA-binding
reaction mixture of 20 µl contained 20 mM Tris-HC1, pH
7.5, 4% Ficoll-400, 2 mM EDTA, 0.5 mM
dithiothreitol, 0.2 µg of poly(dI·dC), 32P-labeled
oligonucleotide (20,000 cpm), and 3 µg of partially purified
recombinant p53 or nuclear extract from U2-OS cells after etoposide
treatment (34, 35). The recombinant p53 protein used was produced in
insect cells infected with a baculovirus vector carrying human p53
cDNA and partially purified through DNA affinity chromatography
(34). In certain cases, 0.2 µg of anti-p53 antibody pAb421 and a
100-fold excess of unlabeled specific or nonspecific oligonucleotide
were also included. The mixture was incubated at room temperature for
45 min and loaded onto a 3.5% polyacrylamide gel. The gel was run in
0.5 × TBE buffer at 60 V, dried, and exposed to Kodak film.
Luciferase Reporter Constructions
The luciferase reporter constructs driven by the GPX
promoter were made as follows.
GPX W/p53BS--
A 262-bp DNA fragment of the GPX
promoter containing a p53 binding site was generated by PCR
amplification of human placenta DNA (Oncor) with primers GPX01 and
GPX04 (5'-GGCGCAATTGTCCAAGAAGC-3').
GPX W/O p53BS--
A 242-bp DNA fragment of the GPX
promoter without a p53 binding site was generated with primers GPX03
(5'-GCTGCTCCTTCCGGCTTAGG-3') and GPX04. The PCR fragments were gel
purified, subcloned into a TA cloning vector (Invitrogen), and seven
independent clones were sequenced by an automatic DNA sequenator to
verify the orientation and freedom of mutations. The recombinant clones
were digested with HindIII and XhoI and ligated
into a predigested pGL-Basic luciferase reporter-3 (Promega).
DNA Transfection and Luciferase Assay
Dispersed cells were seeded into 24-well plates at a cell
concentration of 105/well (for Saos-2) or 2 × 105/well (for U2-OS) 16-24 h before transfection. The
calcium phosphate method was used for transient transfection of Saos-2
cells as described previously (35), whereas the LipofectAMINE method (Life Technologies, Inc.) was used for U2-OS transfection according to
the manufacturer's instructions. The luciferase reporters described above, along with the control plasmid, were cotransfected with a
-galactosidase construct in the presence or absence of constructs expressing wild type or mutant p53 proteins. The ratio of the DNA
amounts for p53-expressing vector versus luciferase reporter was 1:1 or 1:2. 38 h post-transfection, cells were lysed and
assayed for luciferase/
-galactosidase activities as detailed
previously (35). The results are presented as the fold activation of
the empty reporter after normalization with
-galactosidase activity.
Northern Analysis
U2-OS and Saos-2 cells were treated with 25 µM
etoposide, a known p53 activator, for various time periods up to
48 h. Total RNA was isolated using RNAzol solution (Tel-Test), and
15 µg of total RNA was subjected to Northern analysis as described
previously (36). The probe used was an 800-bp human GPX cDNA
fragment (37). The human housekeeping gene encoding
glyceraldehyde-3-phosphate dehydrogenase (GADPH; Ambion) was
used as loading controls for densitometric quantitation.
Western Blot Analysis
Subconfluent human Saos-2 cells were cotransfected with GPX
W/p53BS construct and various constructs expressing wild type or mutant
p53 proteins along with the vector control by the calcium phosphate
method as described (35). Cells were harvested 38 h
post-transfection and lysed on ice for 30 min in a lysis buffer (phosphate-buffered saline containing 2% Nonidet P-40, 0.3% SDS, 1 mM sodium orthovanadate, 5 mM sodium fluoride,
5 mM sodium pyrophosphate, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 0.2 unit/ml
aprotinin. The lysate was centrifuged at 14,000 rpm for 30 min. Equal
amounts of proteins (60 µg) in supernatant were loaded onto a 12%
SDS-polyacrylamide gel and run at 100 V for 2 h. The proteins were
transferred onto a nitrocellular membrane and probed with p53
antibodies pAb421 and pAb240 (1:500 dilution). The p53 proteins were
detected by horseradish peroxidase-conjugated secondary antibody
coupled with Enhanced Chemiluminescence (ECL) Western blotting
detection reagents (Amersham Pharmacia Biotech).
GPX Enzyme Activity Assay
Subconfluent U2-OS and Saos-2 cells were treated with 25 µM etoposide for 24 h. Cells were then cultured in
drug-free medium for another 24 h. All media contained 30 nM selenium to stimulate GPX activity. The cells were
harvested, sonicated three times for 10 s each using a Vibra Cell
Sonicator (Sonics and Materials, Inc.) at full power and 40% duty
cycle, and then assayed for GPX activity using the method of Paglia and
Valentine (38). Briefly, GPX were measured in potassium phosphate
buffer, pH 7, containing glutathione, glutathione reductase, and NADPH
with hydrogen peroxide as substrate. 1 unit of enzyme activity was
defined as the amount of cell lysate that oxidized 1 µmol of
NADPH/min.
Protein Assay and Statistical Analysis
Protein concentrations were measured with a Bio-Rad protein
assay kit. Statistical differences between etoposide-treated and untreated cells were analyzed by Student's t test. Groups
were considered statistically different if p < 0.05.
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RESULTS |
Identification of a p53 Binding Site in the Promoter of the GPX
Gene--
In an attempt to identify genes responsible for
etoposide-induced apoptosis, we utilized DNA chip technology and
identified 62 genes responsive to etoposide. One of the
etoposide-inducible genes was GPX (32). Because we have
shown previously that etoposide activates p53 (23), we wondered whether
etoposide-induced GPX expression is mediated by p53. After searching
the promoter sequence of the GPX gene deposited in GenBank
(Accession No. M83094) for a p53 consensus sequence
(5'-PuPuPuC(A/T)(A/T)GPyPyPyPuPuPuC(A/T)(A/T)GPyPyPy-3'), we identified
a putative p53 binding site,
5'-GGGCCAGACCAGACATGCCT-3', located at
257 bp
upstream from the translation initiation site. Like the p53 binding
site found in most of the known p53 target genes (2), this site
contains two mismatches (underlined) but no space between the two 10-bp motifs.
Specific Binding of p53 to the Putative p53 Binding Site Identified
in the GPX Promoter--
We next examined whether p53 binds to this
putative p53 binding site. We used both purified p53 protein (34) and
nuclear extract from p53-positive U2-OS cells treated with 25 µM etoposide for 6 or 24 h. As shown in Fig.
1, a 20-bp oligonucleotide, consisting of
the p53 binding site found in the GPX promoter, binds to
both purified p53 (lane 2) and endogenous p53 in nuclear
extract treated with etoposide for 6 and 24 h, respectively
(lanes 8 and 10) only in the presence of p53
antibody, pAb421. It is well known that pAb421 enhances and stabilizes
p53 DNA binding (39). In nuclear extract from untreated cells, p53
binding was hardly detectable (lane 6) but was significantly
enhanced by etoposide (lanes 8 and 10),
consistent with our previous observation (23). The binding is
sequence-specific because it can be largely competed away by 100-fold
excess of cold oligonucleotide (lanes 3 and 11) but not by a sequence nonspecific competitor (lanes 4 and
12). The results indicate that p53 did bind to the putative
p53 binding site found in the GPX promoter.

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Fig. 1.
p53 binds to a putative p53 binding site in
the promoter of the GPX gene. The synthetic
oligonucleotides of the p53 binding consensus sequence (GPX01:
5'-GGGCCAGACCAGACATGCCT-3') and its complementary strand were annealed
and labeled with 32P using T4 polynucleotide kinase and
[ -32P]ATP. The gel shift assay was performed as
detailed under "Materials and Methods." Lanes 1-4,
partially purified p53 (3 µg) with or without pAb421 antibody;
lanes 5-12, nuclear extracts (8.5 µg) prepared from cells
treated with etoposide (25 µM) for 0 h (lanes
5 and 6), 6 h (lanes 7 and
8), and 24 h (lanes 9-12). The nonspecific
oligonucleotide is mT3SF (5'-GGGGTTGCTTGAAGAGCGTC-3') (74). The
p53/Ab-supershifted bands are indicated by the arrows. The
band shown on the bottom of the gel is
free-labeled probe. Also, in nuclear extracts, several nonspecific
bands were detected which do not respond to etoposide treatment.
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PCR Cloning of the GPX Promoter--
To examine whether p53 would
transactivate the GPX promoter, we cloned a 262-bp promoter
fragment containing the p53 binding site and a 242-bp fragment with the
p53 binding site deleted, by PCR amplification of genomic DNA from
human placenta (Oncor). The DNA sequence from seven independent PCR
clones revealed three disagreements with the published sequence (40;
Accession No. M83094). As shown in Fig.
2, in the PCR-cloned GPX
promoter, there is a C insertion at nucleotides 2401 and 2421, respectively, and a 3-nucleotide (CCG) insertion at 2470. The
disagreement could be derived from the sequencing errors in the GC-rich
region in the published data. Computer analysis of this 262-bp fragment
revealed that in addition to a p53 binding site, there are sites for
SP-1, AP-2, PEA-3, c-Myc, among others (data not shown).

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Fig. 2.
Comparison of the PCR-cloned GPX
promoter with a published sequence. The GPX
promoter was cloned by PCR amplification of human placenta DNA using
GPX01 and GPX04, as detailed under "Materials and Methods." The
cloned GPX promoter was sequenced and compared with a
published sequence (Accession No. M83094) using a GCG program.
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p53-dependent Transactivation of the GPX
Promoter--
To examine potential p53-dependent
transactivation of the GPX promoter, we transfected
luciferase reporters driven by the GPX promoter sequence
with (GPX W/p53BS) or without (GPX-W/O p53BS) the p53 binding site into
p53-negative Saos-2 cells along with p53 expression plasmid or an empty
vector as the control. As shown in Fig.
3, in the absence of p53 (vector control)
the PCR-cloned promoter fragment contains a strong promoter activity.
An 80-fold higher luciferase activity was observed compared with the
empty vector, pGL-Basic-3. The luciferase activity was similar in both p53 binding site-containing (GPX W/p53BS) or -deleted (GPX W/O p53BS)
constructs. Cotransfection of wild type p53 with GPX W/p53BS construct
induced a 4-fold activation of the luciferase activity compared to that
with GPX W/O p53BS, indicating a p53 binding site-dependent
activation of the promoter. Cotransfection of p53 also induced a slight
repression of the promoter activity that lacks a p53 binding site (GPX
W/O p53BS), a phenomenon also being observed in p53 regulation of the
matrix metalloproteinases-2 gene promoter (23) but not in that of the
S100A2 promoter (41). The results indicated that a 262-bp fragment
immediately upstream of the translation initiation site of the
GPX gene confers a strong promoter activity and that p53
can transactivate the GPX promoter, and transactivation is
p53 binding site-dependent.

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Fig. 3.
p53-dependent activation of the
GPX promoter. Two luciferase reporter constructs
driven by the GPX promoter with (GPX W/p53BS) or without
(GPX W/O p53BS) p53 binding site were transiently cotransfected,
respectively, with a p53-expressing plasmid or an empty vector as the
control. The recipient cells are p53-negative human Saos-2 cells. The
luciferase activity was measured. The results were expressed as fold
activation ± S.E. derived from three independent transfections
and assays, each run in duplicate, after normalization with
-galactosidase activity for transfection efficiency. The vector
controls in the absence of p53 were set arbitrarily as 1 to calculate
the fold activation.
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p53 Binding Site-independent Transactivation of the GPX Promoter by
p53 Mutants--
We next examined whether the GPX promoter
was also regulated by p53 mutants. Plasmid DNAs encoding p53 mutant
proteins were individually cotransfected with luciferase reporters into
Saos-2 cells. They are the p53-143A, p53-175H, p53-248W, p53-273H,
p53-281G, five of the most commonly found p53 mutants in human cancers
(23, 24), and p53-280T, a dominant negative p53 mutant found in
nasopharyngeal carcinomas (42-44). As shown in Fig.
4, top panel, compared with the vector control, some p53 mutants, such as 248W, 273H, and 281G
induced a 6-9-fold activation of the GPX promoter. Mutant 143A conferred a 3-fold activation, comparable to wild type p53. Other
mutants (175H and 280T) had little effect. Moreover, activation by
these p53 mutants was p53 binding site-independent because it can be
detected at a comparable level in both p53 site-containing or -deleted
luciferase constructs. It is noteworthy that there is no significant
difference in
-galactosidase activity when cotransfected with either
wild type p53 or various p53 mutants, indicating a similar transfection
efficiency (not shown). The results demonstrated that some p53 mutants
can also transactivate the GPX promoter and that
transactivation was mediated by a downstream sequence from the p53
binding site.

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Fig. 4.
Top panel, some of tumor-derived p53
mutants also transactivate the GPX promoter, but in a p53
binding site-independent manner. GPX W/p53BS and GPX W/O p53BS were
transiently cotransfected, respectively, with plasmids expressing
various p53 mutants into human Saos-2 cells. The luciferase activity
was measured. The results were expressed as fold activation ± S.E. derived from three independent transfections and assays, each run
in duplicate, after normalization with -galactosidase activity for
transfection efficiency. The vector controls in the absence of p53 were
set arbitrarily as 1 to calculate the fold activation. WT,
wild type. Bottom panel, protein expression of transfected
wild type p53 and p53 mutants. Saos-2 cells were transfected with
plasmid constructs expressing wild type p53 as well as p53 mutants by
the calcium phosphate method. Cells were lysed 38 h
post-transfection and subjected to Western analysis as detailed under
"Materials and Methods." Equal amounts of cellular protein (60 µg) were loaded. The location of the p53 protein is indicated by an
arrow.
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To determine the protein levels of wild type p53 and p53 mutants after
transient transfection into Saos-2 cells, Western blot analysis was
performed. As shown in Fig. 4, bottom panel, all constructs,
except the vector control, expressed p53 protein. The protein level
was, however, higher in mutants p53-143A, 273H, and 281G than that of
wild type, mutants p53-175H, 248W, and 280T. The actual level of the
fold activation may therefore vary among the mutants.
p53-dependent Transactivation of the GPX Promoter Was
Enhanced by Etoposide, a p53 Activator--
Etoposide has been shown
previously to activate p53 in U2-OS cells (23). We therefore examined
whether etoposide would induce p53-dependent
transactivation of the GPX promoter. As shown in Fig.
5, transactivation of the luciferase
reporter driven by the p53 site-containing promoter (GPX W/p53BS) was
induced by etoposide, and a 9-fold induction was achieved at 24 or
48 h. Slight induction (3-fold) of luciferase activity was also
seen in the p53-siteless promoter (GPX W/O p53BS), which appeared to be
p53-independent. To confirm that etoposide-induced transactivation is
largely p53-dependent, we transfected p53-280T, a known
dominant negative p53 mutant (42-44), into U2-OS cells followed by
etoposide treatment and luciferase assay. As a control, the empty
vector was used. As shown in Fig. 5, etoposide-induced,
p53-dependent activation is abolished by p53-280T (GPX
W/p53BS+p53-280T) to a level comparable with that of GPX W/O p53BS, but
it is only slightly inhibited by the vector control
(GPX-W/p53BS+vector). The results demonstrated that transactivation of
the GPX promoter by etoposide is largely
p53-dependent.

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Fig. 5.
Induction of p53-dependent
transactivation of the GPX promoter by etoposide.
Subconfluent U2-OS cells were cotransfected with
-galactosidase-expressing construct and GPX W/p53BS or W/O p53BS
luciferase reporters individually or in combination with a construct
expressing a dominant negative p53 mutant (p53-280T) or the vector
control by the LipofectAMINE method. Cells were treated with etoposide
(25 µM) 24 h post-transfection for 0, 2, 6, 12, 24, and 48 h followed by luciferase assay. Three independent
transfections and luciferase assays, each run in duplicate, were
performed, and the results are expressed as fold activation ± S.E. after normalization with -galactosidase activity for
transfection efficiency. To calculate the fold activation, the
luciferase activity from the GPX W/O p53BS construct after 0 h
etoposide treatment was arbitrarily set as 1.
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To determine whether etoposide causes p53 accumulation or p53
activation (either mechanism would lead to increased DNA binding and
transactivation), we performed Western blot analysis with U2-OS cells
after exposure to etoposide for 0, 6, or 24 h. p53 protein can
only be detected in the sample after a 24-h etoposide treatment,
indicating that the drug modifies the synthesis of endogenous p53
protein that leads to p53 accumulation (data not shown). We have also
examined whether etoposide affects the activity of wild type or p53
mutants in transactivating GPX promoter when transfected
into the Saos-2 cells that lack endogenous p53. p53-281G, a mutant that
induces a p53 binding site-independent transactivation of the
GPX promoter, was selected to be a representative of p53 mutants, and empty vector was used as the control. After transfection, Saos-2 cells were treated with 25 µM etoposide for 0, 2, 6, 12, and 24 h followed by luciferase assay. No induction of
transactivation activity by etoposide can be observed in either wild
type p53 or p53-281G mutant (data not shown). The result indicates that etoposide has no effect on preexisting p53, regardless of the status of
p53. Thus, etoposide, as a topoisomerase II inhibitor and DNA-damaging
reagent, induces p53 accumulation rather than p53 activation.
Induction of GPX mRNA Expression by Etoposide in p53-positive,
but Not p53-negative Cells--
To examine whether endogenous GPX is
subjected to p53 regulation, we treated p53-positive U2-OS cells and
p53-negative Saos-2 cells with etoposide. As shown in Fig.
6, both cells express a detectable basal
level of GPX mRNA with a higher level in U2-OS cells
(first and seventh lanes from left).
In U2-OS cells, expression of GPX started to increase 6 h after
etoposide treatment, gradually being induced thereafter, and reached a
peak at 12-24 h (first five lanes). A bigger induction
(2.7-fold, sixth lane) was seen at 48 h which, however,
might be error-prone because of the decline in mRNA synthesis
resulting from cell death. No significant induction of GPX expression
was observed in Saos-2 cells up to 24 h (seventh through ninth lanes). This is consistent with the results
shown in Fig. 5 and clearly demonstrates that expression of the
endogenous GPX is regulated by p53.

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Fig. 6.
Induction of endogenous GPX mRNA
expression by etoposide in p53-positive cells. Subconfluent
p53-positive U2-OS or p53-negative Saos-2 cells were subjected to
etoposide (25 µM) treatment for various times up to
48 h followed by total RNA isolation and Northern analysis (with
15 µg of total RNA) using GPX cDNA as a probe. The housekeeping
gene encoding glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as a loading control. The level of
induction of GPX expression was densitometrically quantitated, and fold
induction after normalization was expressed by arbitrarily designating
control cells to be 1 as indicated.
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Induction of GPX Enzymatic Activity by Etoposide in p53-positive,
but Not p53-negative Cells--
Lastly, we examined GPX enzymatic
activity after etoposide treatment in U2-OS and Saos-2 cells. As shown
in Table I, etoposide caused a 78%
increase in GPX activity in U2-OS cells at 24 h after etoposide
removal. The groups were statistically different at the
p < 0.05 level. In contrast, no increase was measured
in the Saos-2 cells. This suggests that GPX protein be increased in
p53-positive but not p53-negative cells after exposure to
etoposide.
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DISCUSSION |
The finding reported here indicates that GPX, an antioxidant
enzyme, is subjected to p53 regulation and is a novel p53 target gene.
This is evident for several reasons. First, p53 binds to the p53
binding site in the promoter of the GPX gene. Second, p53
transactivates the GPX promoter in a p53 binding
site-dependent manner. Third, p53 binding and
transactivation of the GPX promoter were enhanced by
etoposide, a p53 activator. Fourth, etoposide/p53-induced transactivation of the GPX promoter can be blocked by a
dominant negative p53 mutant. Fifth, expression of endogenous GPX was
induced significantly by etoposide only in p53-positive U2-OS cells but not in p53-negative Saos-2 cells. In addition, some of the p53 mutants
commonly found in human cancers also transactivate the GPX
promoter, but in a p53 binding site-independent manner.
p53, after being activated by DNA-damaging reagents, has been shown
either to induce G1 growth arrest or apoptosis (5, 6). The
p53 target genes that mediate or associate with p53-induced apoptosis
include Bax (45), Fas/APO1 (46),
KILLER/DR5 (47) as well as those involving generation of ROS
(48). We have shown previously that etoposide could activate p53 and
subsequently induce apoptosis in U2-OS cells (23, 32). Now we have
identified and characterized that GPX, an antioxidant enzyme, is also
induced by p53. It appears paradoxical that p53, on one hand, induces genes responsible for ROS generation, which mediates apoptosis (48),
and on the other hand, induces expression of a protective antioxidant
enzyme, GPX, which has been shown to protect cells from oxidative
damage and apoptosis (49-52). The fact that induction of GPX
immediately followed p53 activation (Figs. 1, 5, and 6) suggests that
p53 may induce GPX expression at an early stage of apoptosis. It is
known that p53-induced ROS generation is a rather later event (48).
Thus, p53 may regulate cellular redox status in a
time-dependent manner: it increases antioxidant synthesis at an early stage followed by an increase in ROS generation.
The regulation of GPX expression is rather complex and is controlled at
several levels. First, GPX is expressed differentially in a wide range
of tissues (53). Second, GPX is subjected to developmental and hormonal
regulation (54-56). Third, GPX activity can be induced by oxygen
tension, diet, and some xenobiotics (25, 54, 57) but is inactivated by
superoxide (58) and nitric oxide (59). A 3.0-kilobase promoter fragment
of the GPX gene has been cloned and characterized. Three
possible cis-acting regulatory regions as well as
oxygen-responsive elements were defined (40, 60). We showed here that a
262-bp fragment located immediately upstream from the translational
initiation site of GPX has strong promoter activity and that p53
positively regulates this promoter fragment. Our findings, along with a
recent identification of GPX as a p53-responsive gene in a
DNA microarray screening experiment by others (61), indicated that in
addition to those listed above which regulated GPX, p53 also positively
regulates GPX. Transactivation of GPX by p53 links the p53 signaling
pathway to the antioxidant pathway. Because p53 is activated by
DNA-damaging agents, this finding would implicate a role of antioxidant
enzymes in the cellular response not only to oxidative stress, but also
to DNA damage.
p53 mutations have been found to be the most common genetic alteration
in human cancers, and the majority of p53 mutations were clustered in
the DNA binding domain (24). p53 mutants either gain oncogenic
function, lose tumor suppressor function, or function dominant
negatively to suppress wild type function (43). Most p53 mutants lose
activity to bind to a p53 consensus binding site as well as lose
transcriptional activity (62, 63). Intriguingly, we showed here that
several p53 mutants commonly found in human cancers also transactivate
the GPX promoter, and transactivation is, however,
independent of the p53 binding site. Some p53 mutants confer a 9-fold
activation of the GPX promoter of 242 bp immediately upstream from the initiation codon (Fig. 4). Up-regulation of the same
gene promoter by both wild type p53 and p53 mutants has also been
reported in the genes encoding the epidermal growth factor receptor
(21), proliferating cell nuclear antigen (18, 19, 64), and multidrug
resistance-1 (65, 66). Because it is unknown for p53 mutants to
function as sequence-specific transcriptional activators, it will be of
great interest in defining whether such a cis-element exists
within this short (242 bp) GPX promoter region.
Alternatively, p53 mutants may only secondarily affect GPX
promoter activity.
The biological importance of GPX regulation by p53 mutants is not clear
at the present time. However, it has been observed that elevated GSH or
GPX is associated with acquired drug resistance (67-70), and some
human cancers with an increased GPX expression are more resistant to
chemotherapy (71, 72). Because cancer cells with p53 mutations are
often more resistant to chemotherapeutic drugs (73), GPX could mediate
drug resistance conferred by some p53 mutants. Moreover, the activity
of GPX is altered in many human cancers, being either elevated or
decreased (31). It will be of interest to correlate GPX activity with
p53 mutations in these cancers. Upon establishment of such a
correlation, the p53 mutation as well as GPX level may serve as an
index for potential drug resistance.