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INTRODUCTION |
Serine/threonine phosphatase 5 (PP5) is an okadaic acid/calyculin
A-sensitive phosphatase that is expressed ubiquitously in mammals and
has been reported to associate with the atrial natriuretic peptide
receptor (1), the heat shock protein 90 (Hsp-90)1-glucocorticoid
receptor (GR) heterocomplex (2, 3), the CDC16/CDC27 subunits of the
anaphase-promoting complex (4), cryptochrome 2 (5), apoptosis
signal-regulating kinase1 (6), Hsp90-dependent
heme-regulated eIF2
kinase (7), and the
G
12/G
13 subunits of heterotrimeric
G proteins (8). In estrogen-responsive breast carcinoma cells, the
expression of PP5 is induced by treatment with estrogen, and the
constitutive expression of PP5 converts MCF-7 cells into an
estrogen-independent phenotype (9). Still, determining the
physiological/pathological roles of PP5 has proven difficult for many
reasons. First, in a crude cell homogenate PP5 exists predominately in
an inactive state (for review see Ref. 10). Second, the physiological
activators of PP5 are unknown. Third, when activated by the addition of
polyunsaturated lipids or protease mediated cleavage of the N-terminal
autoinhibitory domain (11, 12), the activity of PP5 cannot be
distinguished from that of PP2A and PP1, which are both ubiquitously
expressed at high levels in all eukaryotic cells. Finally, no selective small molecule inhibitors of PP5 have been reported, and genetic deletions studies in Drosophila suggest that the lack of PP5
expression results in an embryonic lethal phenotype (13). Therefore, to date there is no reliable method to measure changes in the enzymatic activity of PP5 in vivo or in crude cell homogenates.
Despite the difficulties associated with studying PP5, several studies
suggest PP5 plays a functional role in both GR- and p53-dependent growth control. PP5 associates with the
GR·Hsp-90 complex (2, 3), and mutational analysis has
implicated the N-terminal tetratricopeptide repeat domains of
PP5 in this interaction (3, 14). In GR-responsive A549 cells, treatment
with ISIS 15534, a potent and specific suppresser of PP5 expression (9, 15, 16), induces the nuclear accumulation of GRs (17). ISIS 15534 treatment also markedly increases the association of GR with its
cognate DNA binding sequence and induces GR-transcriptional activity in
the absence of glucocorticoids (18). When combined, ISIS 15534 and
dexamethasone, a potent GR agonist, have an additive effect, with
dexamethasone-mediated induction of GR reporter plasmid activity
elevated to a level that is ~10 times greater than the maximal
response obtainable in the presence of PP5 (18).
Treatment with ISIS 15534 also suppresses the growth of some, but not
all, human tumor cells (9, 15, 18). In p53 wild-type lung carcinoma
cells, treatment with ISIS 15534 produces G1 growth arrest,
which occurs with a concomitant increase in the expression of the
cyclin-dependent protein kinases inhibitor protein,
p21Waf1/Cip1 (15, 18). In contrast, in p53
/
human fibroblast or p53-defective T-24 bladder carcinoma cells, ISIS
15534 treatment does not suppress growth or induce the expression of
p21Waf1/Cip1 (18). Studies with TR9-7 cells (p53
/
human fibroblasts that contain tetracycline-regulated
transactivator and operator plasmids to control the expression of
wild-type p53) revealed that ISIS 15534-mediated induction of
p21WAF1/Cip1 requires a small amount of p53 protein, which
becomes hyperphosphorylated but does not necessarily accumulate when
PP5 expression is suppressed (15). In A549 cells both GR activation and
p53 activation have been shown to induce the expression of
p21WAF1/Cip1 and induce G1 growth arrest.
Therefore, the data obtained with ISIS 15534 is consistent with PP5
acting as a suppresser of both a GR-dependent and a
p53-dependent pathway leading to the induction of
p21Waf1/Cip1 expression. Alternatively, the data are also
consistent with PP5 acting to suppress a single pathway in which p53 is
a functional participant in GR-induced expression of
p21Waf1/Cip1.
In the present study we further explored the relationship between PP5,
GR, and p53 by developing chimeric antisense oligonucleotides that
potently and specifically suppress the expression of p53. The data
presented indicate that dexamethasone induces a response leading to
the hyperphosphorylation of p53 at serine 15 and that Ser-15-phosphorylated p53 is a functional participant in a
glucocorticoid-initiated response regulating the expression of
p21WAF1/Cip1. The data are consistent with the basal
expression of p53, in cells that have not encountered environmental
stress, playing a functional role in glucocorticoid-mediated growth
control via a pathway that was likely masked in previous studies by the
ubiquitous expression of PP5.
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EXPERIMENTAL PROCEDURES |
Materials--
Dexamethasone, all chemicals, and goat anti-mouse
Ig were purchased from Sigma. Purified mouse anti-human p53 monoclonal
antibody was purchased from BD Pharmingen. Antibodies recognizing
specific sites of phosphorylation on p53 were purchased from Cell
Signaling Technology (Beverly, MA). Phosphorothioate
deoxyoligonucleotides and
2'-O-(2-methoxy)ethylphosphorothioate oligonucleotides were synthesized and purified as described previously (19, 20). The sequence
of antisense oligonucleotides targeting p53 (ISIS 110332), PP5 (ISIS
15534), and mismatched controls are as follows: ISIS110332,
5'-CCCAGCCCGAACGCAAAGTG; ISIS 15534, 5'-GGGCCCTATTGCTTGAGTGG; ISIS 116947, 5'-CACCGCACGGCCACAAGGTA; ISIS 122205, 5'-GCCACCACGCAGCCAGAGTA; ISIS 15521, 5'-GTGCGATCGTTGCGGTTAGC.
Cell Culture and Transfection--
Tissue culture medium,
Lipofectin® and TRIzol® were purchased from Invitrogen
(Gaithersburg, MD). Human A549 lung carcinoma cells were obtained from
the American Type Tissue Collection and cultured in DMEM supplemented
with 10% fetal calf serum, streptomycin (0.1 µg/ml), and penicillin
(100 units/ml). Cell cultures were passed when 90-95% confluent
unless indicated otherwise. Antisense treatment was performed using a
1-ml solution of DMEM containing 15 µg/ml DOTMA/DOPE (Lipofectin®)
and the indicated amount of oligonucleotides for 4 h at 37 °C,
as described previously (15, 19, 20, 21). Cell numbers were determined
with a cell counter (Coulter Counter ZM) following detachment from the
dish by brief treatment with trypsin.
FACS Analysis of Cell Growth--
For cellular staining with
propidium iodine (PI), cell cultures were gently detached from the dish
with trypsin, collected by centrifugation, and resuspended in 0.5 ml of
phosphate-buffered saline containing 50 µg/ml PI and 0.5 ml of
Vindelovs reagent (10.0 mM Tris, 10 mM NaCl, 75 µM propidium iodine, 0.1% igepal, and 700 units/liter
RNase adjusted to pH 8.0). The PI-stained cells were kept in the dark
at 4 °C for 30-60 min and then analyzed with a FACS Vantage SE flow
cytometer (BD Biosciences, Immunocytometry Systems, BDIS, San
Jose, CA) using pulse processing (FL2 width and FL2 area) to gate out
cell doublets and ModFitLT cell cycle analysis software
(Verity Software House, Topsham, ME). PI fluorescence was excited at
488 nm with an argon laser (Spectra Physics, Sunnyvale, CA), and
fluorescence emission was collected with a 582/42 BP filter in front to
the FL2 detector. Multivariate data acquisition and analysis were
performed with BDIS CellQuest software.
Northern Blot Analysis--
RNA was isolated using TRIzol®
according to the methods of the manufacturer. Total RNA (15 µg) was
then fractionated on a 1% agarose gel containing formaldehyde and
transferred to a Duralon-UV (Stratagene) membrane. Following UV
cross-linking and pre-hybridization, the membrane was hybridized
overnight in the presence of 50% formamide at 42 °C with the
indicated 32P-labeled cDNA probe. cDNA probes were
generated by random labeling with a DECAprime DNA labeling kit (Ambion)
according to the protocol of the manufacturer. The membranes were
subjected to two high stringency washes with 0.1 × SSC at
55 °C and two low stringency washes with 2 × SSC at room
temperature (15). Hybridization was visualized by autoradiography. For
quantification of hybridization signals, the radiograms were scanned
and the scanned signals were integrated using Scion Image software,
beta 3b release (Scion Corp.). The membranes were then stripped and
probed a second time with a glyceraldehyde-3-phosphate dehydrogenase
cDNA probe. Signals were normalized to glyceraldehyde-3-phosphate
dehydrogenase to account for any inconsistencies in loading.
Western Blotting Analysis of Human p53--
Western analysis was
performed essentially as described previously (21). Proteins were
quantified using Bradford (Bio-Rad) protein assay with bovine serum
albumin as a standard. Protein samples (5-50 µg of total
protein/lane) were resolved using 10% SDS-PAGE and transferred to
Immobilon membranes (Millipore Corp.). Immunoblotting was performed
with primary anti-human p53 purified mouse monoclonal antibodies and
goat anti-mouse secondary antibodies (BD Pharmingen) to identify total
p53 protein. The identification of site-specific phosphorylation was
achieved with antibodies recognizing the phosphorylation of p53 at
Ser-15, Ser-37, Ser-6, and Ser-392. Goat anti-rabbit horseradish
peroxidase (Promega) was used as a secondary antibody when
phosphorylation-specific antibodies were employed. Antibody association
was detected employing ECL Western blotting detection reagents
(Amersham Biosciences, Buckinghamshire, England) essentially following
the protocols of the manufacturer.
Antisense Oligonucleotide Synthesis and Assay for Oligonucleotide
Inhibition of p53
Expression--
2'-O-(2-Methoxy)ethylphosphorothioate
oligonucleotides were synthesized and purified as described
previously (15, 19, 20). Antisense oligonucleotide-mediated inhibition
of p53 expression and Northern blot analysis were performed essentially
as described previously (15-21). Briefly, cells that reached
~60-70% confluency in 60-mm dishes were washed once with DMEM and
then treated with a solution (1 ml) of DMEM containing oligonucleotides
at the indicated concentrations and 15 µg/ml DOTMA/DOPE
(Lipofectin®). After incubating for 4 h at 37 °C, the medium
was removed and replace with fresh DMEM supplemented with 10% fetal
calf serum. At the times indicated, the cells were scraped in
TRIzol®, and RNA or proteins were prepared for Northern or Western
blot analysis, respectively, as described above.
Luciferase Assays--
A549 cells were plated at a density of
300,000 cells/60-mm tissue culture dish and incubated overnight. After
24 h the cells were transfected with p21-luciferase reporter
plasmids and treated with ISIS 110332 (to suppress p53 expression) or
ISIS 122205 (a mismatch control) as described above. After 4 h the
cells were washed with media and plated in fresh media containing 100 nM dexamethasone. After 24 h the cells were harvested
and luciferase activity was measured with a Monolight 2010 luminometer
as described previously (9). The p21-luciferase reporter plasmid was
generously provided by Dr. P. Howe (Cleveland Clinic Foundation).
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RESULTS |
Antisense-mediated Inhibition of p53 mRNA and Protein
Expression--
To study the relationship of GR, p53, PP5, and
p21Waf1/Cip1 in human cells, we needed a system that would
allow us to independently regulate the expression of either p53 or PP5
in cells that are p21Waf1/Cip1 wild-type and sensitive to
dexamethasone-induced growth suppression. Although dexamethasone has
been reported to suppress growth in many types of cells, after testing
numerous cell lines we failed to find a single p53-null cell line that
was still sensitive to growth suppression by dexamethasone. Indeed, a
search of the published literature failed to reveal even a single
report documenting dexamethasone-induced growth suppression in
p53-defective or p53-null cells. Nonetheless, previous studies have
shown that treatment with either dexamethasone (18) or ISIS 15534 can
induce G1 growth arrest with a concomitant increase in the
expression of p21Waf1/Cip1 in A549 cells (15, 17). This
suggests we could use A549 cells as a model if we could develop a
method of suppressing p53 expression. Therefore, we developed potent
and highly specific "chimeric" 2'-O-(2-methoxy)ethylphosphorothioate antisense
oligonucleotides that suppressed the expression of human p53.
To identify a suppressor of p53 expression, oligonucleotides, 20 bases
in length and predicted to hybridize to different regions of human p53
mRNA, were synthesized and tested for their ability to suppress the
expression of human p53 employing Northern and Western blot analyses as
described previously (15-19). The oligonucleotides tested were
designed to target specific regions in the protein coding, the
5'-untranslated or the 3'-untranslated regions of human p53 mRNAs,
and were chimeric 2'-O-(2-methoxy)ethylphosphorothioate oligonucleotides, containing 10 central phosphorothioate oligodeoxy residues ("oligodeoxy gap") flanked by five
2'-O-(2-methoxy) residues on the 3'- and 5'-ends. These
modifications have been shown previously to enhance the potency of
antisense oligonucleotides targeting mRNAs encoding other proteins
(12, 22, 23). Because phosphorothioate oligonucleotides commonly act
through an RNase H-dependent mRNA cleavage mechanisms
in cells (24), the ability of a particular oligonucleotide to inhibit
the expression of p53 was originally identified based on their ability
to inhibit p53 mRNA expression (15, 16, 20). The initial screen
revealed that many oligonucleotides had little or no effect on the
inhibition of p53 mRNA levels, whereas others had a moderate
effect, and a few had pronounced effects. The activity of the active
antisense oligonucleotides was verified with dose-response studies,
utilizing Northern analysis to detect changes in p53 mRNA levels
and Western analysis to detect changes in the level of p53 protein. Of
the antisense oligonucleotides identified as having potent inhibitory
activity against p53 in the initial screen, two were chosen for further
characterization. Both of these antisense oligonucleotides produced a
dose-dependent inhibition of p53 expression, and
representative Northern and Western blots for ISIS 110332 are shown in
Fig. 1.

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Fig. 1.
Inhibition of p53 mRNA and protein
expression by treatment with antisense oligonucleotides.
A, inhibition of p53 mRNA levels by antisense
oligonucleotides targeting human p53 (ISIS 110332). A549 cells were
treated with increasing concentrations (0-500 nM) of ISIS
110332 or mismatched control analogues (ISIS 116947 and ISIS 122205)
that contain eight changes (mismatches) within the 110332 sequences.
Total mRNA was prepared 24 h later and analyzed for p53 and
glyceraldehyde-3-phosphate dehydrogenase mRNA levels by Northern
blot analysis. B and C, Western blot analysis of
p53 protein levels in A549 cells. Cells were treated with ISIS 110332 or a mismatched control oligonucleotide (ISIS 116947) at the
concentrations indicated (B) or 300 nM
(C). After 24 h (B) or the times indicated
(C), protein extracts were prepared and resolved by SDS-PAGE
using 10% polyacrylamide gels. Western analysis was conducted as
described under "Experimental Procedures." Each lane
contains results for 10 µg of protein.
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To test the specificity of the response, several mismatched control
oligonucleotides were also synthesized and tested. The mismatched
control analogues contain the same base composition and same chemistry
as the antisense oligonucleotides targeting p53. However, the sequences
are scrambled and noncomplementary to p53. None of the mismatched
controls inhibited p53 protein expression. Time course analysis
revealed that p53 protein levels could be effectively suppressed 4 h after the addition of antisense oligonucleotides (Fig.
1C). As observed previously with chimeric 2'-O-(2-methoxy)ethylphosphorothioate oligonucleotides that
target other proteins (15-21), protein levels remained repressed for
~3 days. The cationic lipids (DOTMA/DOPE; Lipofectin®) were used to
facilitate the uptake of the oligonucleotides (25) and had no apparent
effect on p53 mRNA or protein levels.
Suppression of p53 Expression Is Associated with an Increase in the
Rate of Cell Growth--
Having developed a method of specifically
inhibiting the expression of human p53, we next tested the effect of a
single treatment with ISIS 110332 at 300 nM on A549 cells.
Similar to results obtained in studies employing p53-deficient human
cells or cells derived from mice homozygously null for p53 (26, 27),
A549 cell growth was not dependent on p53. In contrast, the suppression
of p53 was associated with an increase (~26%) in the rate of cell
proliferation (Table I).
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Table I
Effects of ISIS 110332 on A549 cell growth
A549 cells in log phase growth were treated with a single dose (300 nM) at time zero with ISIS 110332 or the mismatched control
analogue, ISIS 116947 (as described under "Experimental
Procedures"). Cell number was determined 24 and 48 h following
treatment. Each point represents the mean of triplicate
cultures with error bars representing mean ± S.E. of
four different experiments.
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Dexamethasone Treatment Arrests A549 Cell Growth at the
G0/G1-phase of the Cell
Cycle--
The ability of glucocorticoids to suppress cell growth can
be diminished or lost as cells are passed in culture. Therefore, to
ensure our cell cultures were responding as reported previously, we
treated A459 cells with dexamethasone and measured both
p21Waf1/Cip1 mRNA levels and proliferation rates. As
reported previously, 50-100 nM dexamethasone was
sufficient to suppress A459 cell growth (18). FACS analysis 24 h
after treatment with a single dose of 100 nM dexamethasone
revealed that the majority of the cells became arrested prior to the
onset of DNA synthesis (Fig. 2), which is
similar to the effect produced by treatment with ISIS 15534, which
targets PP5 (15, 18). Northern analysis confirmed that dexamethasone
treatment produced a dose-dependent increase in the
expression of p21Waf1/Cip1 (Fig. 2, inset).

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Fig. 2.
FACS analysis of A549 cells 24 h after
treatment with dexamethasone (100 nM) or solvent
(control). G0/G1-, S-, and
G2/M-phase cells are indicated, with the percentage
of cells in each phase indicated below in
parenthesis. Inset: Northern analysis of A549
cells 24 h after treatment with the indicated concentration of
dexamethasone using a probe specific for
p21WAF1/Cip1.
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The Inhibition of p53 Expression Prevents Dexamethasone-induced
p21WAF1/Cip1
Expression--
Dexamethasone-induced p21Waf1/Cip1
expression was clearly evident after ~24 h, and maximal induction
occurred ~48 h after continuous exposure to ~100 nM
dexamethasone (Figs. 2 and 3) (18). A
single treatment with 300 nM ISIS 110332 inhibited
dexamethasone-induced p21WAF1/Cip1 expression at all
concentrations tested, even in cell cultures continuously exposed to
100 nM dexamethasone for 48 h (Fig. 3). The
inhibition of p53 expression also suppressed the basal expression of
p21WAF1/Cip1 (Fig. 3C). In contrast, treatment
with mismatched control antisense oligonucleotides did not suppress
dexamethasone-induced p21WAF1/Cip1 expression or the basal
expression of p21WAF1/Cip1. Transient transfection studies
using a p21-luciferase reporter plasmid revealed that ISIS 110332 suppressed dexamethasone-induced p21-luciferase activity, suggesting
dexamethasone-induced p21 expression is a transcriptionally regulated
event (Fig. 3D). Similar results were observed with
additional antisense oligonucleotides targeting different regions of
p53 and their respective scrambled controls. Together, these studies
suggest that the suppression of p53 expression by treatment with
antisense oligonucleotides targeting p53 suppresses the basal
expression of p21WAF1/Cip1 and prevents
dexamethasone-induced p21WAF1/Cip1 expression.

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Fig. 3.
Inhibition of p53 expression suppresses
dexamethasone-induced expression of p21WAF1/Cip1.
A, ISIS 110332 induced suppression of dexamethasone-induced
p21WAF1/Cip1 expression. A549 cells were treated with 50 or
100 nM dexamethasone, as indicated, and either 300 nM ISIS 110332 (lanes 1 and 3) or 500 nM of a mismatched control, ISIS 116947 (lanes 2 and 4). After 24 h, p21WAF1/Cip1 and
glyceraldehyde-3-phosphate dehydrogenase mRNA levels were assessed
by Northern blot analysis as described above. B, Specificity
of ISIS 110332-induced suppression of dexamethasone-induced
p21WAF1/Cip1 expression. A549 cells were treated with 100 nM dexamethasone (+) or solvent as a control ( ) and then
either 300 nM ISIS 110332 to suppress the expression of p53
or the indicate amount of two different mismatched controls, ISIS
116947 and ISIS 122205. After 48 h, total RNA was prepared and
analyzed for p21WAF1/Cip1 and glyceraldehyde-3-phosphate
dehydrogenase mRNA levels by Northern blot analysis as described
above. C, Suppression of both basal and
dexamethasone-induced p21WAF1/Cip1 expression by ISIS
110332. A549 cells in log phase growth were treated with 300 nM ISIS 110332 and/or dexamethasone as indicated (treated
+, not treated ). Total RNA was prepared after 24 or 48 h and
analyzed for p21WAF1/Cip1 and glyceraldehyde-3-phosphate
dehydrogenase mRNA levels as described under "Experimental
Procedures." For all experiments, Western blot analysis of p53
protein levels, as shown in Fig. 1, was used to confirm the suppression
of p53 protein expression following treatment with ISIS 110332 (data
not shown). D, Suppression of p53 inhibits
dexamethasone-induced p21-luciferase activity. A549 cells were plated,
transfected with p21-Luciferase reporter plasmids, and treated with
ISIS 110332 to suppress p53 expression (AS) ISIS 122205 a
mismatch control (MM) or solvent alone (C) as
described under "Experimental Procedures." After 4 h the cells
were treated with dexamethasone (100 nM), and 24 h
later luciferase activity was measured. The data were normalized to
protein and are expressed as the mean ± S.D. from three
experiments conducted in triplicate.
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Dexamethasone Induces the Phosphorylation of p53 at
Ser-15--
When A549 cells are cultured in media containing
32Pi label and then treated with ISIS 15534 (to
suppress the expression of PP5) and/or with dexamethasone (to activate
GRs), immunoprecipitation studies revealed that the suppression of PP5
expression is associated with the hyperphosphorylation of p53 (15).
However, the site at which p53 becomes phosphorylated was not
determined. Furthermore, because radiation-induced DNA damage can
activate p53, it was also possible that the observed
hyperphosphorylation of p53 resulted from the suppression of PP5 in
combination with the activation of a stress response triggered by the
32Pi used to label the cells. Therefore, in the
present study we employed antibodies that recognized specific sites of
p53 phosphorylation to assess changes in the phosphorylation status of
p53. As seen in Fig. 4, treatment with
100 nM dexamethasone produced an increase in the
phosphorylation of p53 at Ser-15 but had no apparent effect in the
phosphorylation of Ser-37, Ser-6, Ser-392, or on p53 protein levels.
Treatment with ISIS 15534, low dose UV, and adriamycin (used as a
positive control) also resulted in an increase in the phosphorylation
at Ser-15, with adriamycin producing an marked increase in both
phosphorylation and p53 protein levels. As reported previously,
adriamycin-treatment also results in the hyperphosphorylation of p53 at
several additional sites (data not shown).

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Fig. 4.
Inhibition of p53 expression restores growth
to dexamethasone-arrested cell cultures. A,
dexamethasone treatment induces the hyperphosphorylation of p53 at
Ser-15. A549 cells were treated with 100 nM dexamethasone
or solvent (controls). 24-48 h later the relative phosphorylation of
the indicated serine residues on p53 was assessed via Western analysis
using phosphorylation-specific antibodies as described under
"Experimental Procedures." B, the suppression of PP5
expression enhances the phosphorylation of p53 at Ser-15. A549 cells
were treated with 100 nM dexamethasone, ISIS 15534 (to
suppress the expression of PP5), UV light, or 100 ng/ml adriamycin (as
positive controls). 24-48 h later the relative phosphorylation of the
indicated serine residues on p53 was assessed via Western analysis
using phospho-Ser-15-specific antibodies. The blots were also probed
with an antibody that recognizes p53 to assess changes in the amount of
total p53 protein. C, continuous exposure to dexamethasone
suppresses A549 cell growth. A549 cells in log phase growth were
treated with solvent (controls), 50 nM dexamethasone, or
100 nM dexamethasone ~16 h after the cells were plated
(indicated by an arrow). Cell growth was then assessed daily
for 5 days. The media in all cultures was changed after 72 h, at
which time fresh dexamethasone was added to each dish at the
concentrations indicated. D, The suppression of p53
expression restores A549 cell growth rates to control levels. A549
cells in log phase growth were treated with 100 nM
dexamethasone as described above ~16 h after the cells were plated
(indicated by a filled arrow). After 72 h, the cells
were treated with 300 nM ISIS 110332 or a mismatched
control (indicated by an open arrow) and placed in fresh
media containing 100 nM dexamethasone. Growth rates were
assessed at the time intervals indicated. To confirm that treatment
with ISIS 110332 suppressed p53 expression, protein from identically
treated dishes in each experiment were subjected to Western analysis.
Insets show representative Western blots of samples from the
96- and 120-h time points. Each point represents the
mean ± S.D. (n = 6).
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The Suppression of p53 Expression Restores Growth to A549 Cells
Arrested by Treatment with Dexamethasone--
Treatment of A549 cells
with dexamethasone produced a dose-dependent suppression of
growth that became evident after ~48-72 h (Fig. 4C).
Because the suppression of p53 expression inhibits dexamethasone-induced p21WAF1/Cip1 expression, we tested
the ability of ISIS 110332 to influence dexamethasone-induced growth
suppression. To assess this, A549 cells in early log phase growth were
treated with 100 nM dexamethasone. After 72 h, the
growth-suppressed cell cultures were treated with 300 nM
ISIS 110332 to inhibit the expression of p53 or with 300 nM
ISIS 116947, a mismatched control for ISIS 110332. Cellular proliferation was then assessed by counting the number of cells in each
dish over a period of 5 days. As seen in Fig. 4D, treatment with ISIS 110332, but not the mismatched control, ablates the suppression of growth produced by treatment with 100 nM
dexamethasone. Together, these findings indicate that p53 can play a
functional role in a GR-mediated response leading to the expression of
p21Waf1/Cip1 and growth suppression via a mechanism that is
suppressed by PP5 and is associated with the phosphorylation of p53
at Ser-15.
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DISCUSSION |
Because the majority of tumors from late stage cancer patients
contain mutations affecting the p53-protein, considerable effort has
been devoted to determining how aberrations in the normal functions of
p53 are associated with the pathology of human cancers. From these
studies, it has become clear that p53 acts as a stress-induced transcription factor that plays a key role in activating and
integrating a multitude of adaptive cellular responses to a wide range
of environmental stresses (for review see Refs. 28-31). Nonetheless, the reason most cells continuously express low levels of p53 in the
absence of stress is not clear.
Previous studies have shown that PP5 associates with the GR·Hsp-90
complex (2, 3) and that ISIS 15534-mediated suppression of PP5 enhances
both dexamethasone-induced transcription and the expression of
p21Waf1/Cip1 (18). In addition, ISIS 15534-induced
p21Waf1/Cip1 expression does not require an increase in p53
protein levels, yet it is dependent on the presence of p53 (15, 16).
The data presented here indicate that dexamethasone can induce the
phosphorylation of p53 at Ser-15, as does ISIS 15534. When p53
expression is inhibited with ISIS 110332 in the absence of cellular
stress, dexamethasone treatment no longer induces the expression
p21WAF1/Cip1. Furthermore, GR-induced growth suppression is
ablated by the suppression of p53, and the suppression of basal p53
expression results in an increase in the rate of cell growth. Together,
these observations suggest that basal p53 does more then provide a pool of readily available protein that allows for a rapid response to
stress. Rather, p53 can act as an active participant in a GR-mediated signaling cascade that suppresses growth and is countered by the actions of PP5.
There is ample evidence that the induction of p21Waf1/Cip1
aids the onset of G1-growth arrest and that either p53 or
GR activation can elicit a response leading to the induction of
p21Waf1/Cip1 (26, 27, 32-35). Still, a functional role for
p53 in the GR response leading to p21Waf1/Cip1 expression
had not been established. In contrast, transient transfection studies
conducted with rat hepatocytes indicate dexamethasone is capable of
inducing transcriptional activity in p21Waf1/Cip1 reporter
plasmids in constructs from which the core p53 response element had
been deleted (33), possibly via an atypical GR response involving the
CCAAT/enhancer binding protein
(34). p53 has also been reported to
physically interact with GRs, and dexamethasone-mediated transcription
activity can be repressed by the co-transfection of p53 expression
plasmids (36). Similarly, p53 and GR have been shown to form a complex
that mutually inhibits the activity of both proteins, and it has been
suggested that this p53·GR complex sequesters the active forms of
both proteins in the cytoplasm (37, 38).
At first glance the above mentioned studies contradict the data
reported here. However, the previous studies addressed "cross-talk" between GR- and p53-induced responses under conditions that produced an
increase in p53 protein levels (or an increase in p53 protein was used
as an indicator of p53 activation). In contrast, the data presented
here indicates that dexamethasone-induced p21WAF1/Cip1
expression is associated with p53 hyperphosphorylation at Ser-15 and
occurs without an increase in p53 protein levels. Therefore, the
requirement of Ser-15 phosphorylated p53 in a GR-induced pathway leading to growth suppression does not exclude another role for elevated p53 protein levels in the suppression of GR-induced gene transcription. Indeed, depending on the type and the severity of
cellular stress, several studies indicate that p53 can elicit either a
protective response (that is transient and suppresses growth) or a
sustained response leading to irreversible growth arrest or apoptosis
(28-31).
Determining how p53 is able to respond "appropriately" to a given
stimuli and how p53 then drives a cellular response that is
"tailored" to a specific insult is an area of great interest, and
several studies indicate that phosphorylation is a key regulator of p53
(28, 39). For example, following extensive DNA damage, such as that
produced by ionizing radiation, p53 promotes a death response by
inducing apoptosis or irreversible growth arrest (for review see Refs.
28, 40, and 41). In such a response several protein kinases are
activated and multiple sites on p53 become phosphorylated
(42-44). Phosphorylation of Thr-18 and Ser-20 negatively regulates the interaction with Hdm2, a key regulator of p53 that binds
its N-terminal domain and suppresses transcription. Hdm2 also acts as
an E3 ubiquitin ligase, which catalyzes the ubiquitination of p53 and
targets it for proteasome-mediated degradation. Therefore, the
disruption of the interaction of Hdm2 with p53 by phosphorylation promotes both the accumulation of p53 and an increase in
p53-dependent transcriptional activity.
The role of p53 phosphorylation in cells that have not encountered
stress has been studied less extensively. Basal p53 expression can be
detected in most, if not all cells, yet p53 knock-out mice are
developmentally normal (45). The trans-expression of wild-type p53 in
unstressed p53
/
human fibroblasts is sufficient to
induce both p21WAF1/Cip1 expression and growth arrest (26),
suggesting that the transcriptional activity of p53 is not totally
dependent on the activation of stress-induced kinases. Phosphorylation
of Ser-15 has been shown to stimulate p53-dependent
transcriptional activation, to increase the association of p53 with
transcriptional co-activator proteins, and to aid the nuclear retention
of p53 by blocking nuclear export (44, 47, 48). Consistent with the
findings reported here, Bean and Stark (46) have shown that
phosphorylation of p53 at Ser-15 is crucial to p53-mediated basal
expression of p21 mRNA. Thus, although clearly additional studies
will be needed to clarify the role of PP5 in GR- and p53-mediated
processes, the data obtained to date are consistent with PP5 acting to
suppress a GR-induced signaling cascade that influences p53
phosphorylation at Ser-15 and the basal expression of
p21WAF1/Cip1.