From the Department of Cell and Tumor Biology, The Beckman Research
Institute at City of Hope, Duarte, California 91010-3000
Pyrrolidine dithiocarbamate (PDTC) is a thiol
compound widely used to study the activation of redox-sensitive
transcription factors. Although normally used as an antioxidant, PDTC
has been shown to exert pro-oxidant activity on proteins both in
vitro and in vivo. Because p53 redox status has been
shown to alter its DNA binding capability, we decided to test the
effect of PDTC on p53 activation. In this communication, we report that
PDTC inhibits the activation of temperature-sensitive murine
p53Val-135 (TSp53) in the transformed rat embryo fibroblast
line, A1-5, as well as wild-type human p53 in the normal diploid
fibroblast line, WS1neo. In A1-5 cells, PDTC abrogated UV- and
temperature shift-induced TSp53 nuclear translocation and p53-mediated
transactivation of MDM2. PDTC also blocked UV-induced
accumulation of wild-type p53 in WS1neo cells. Continual presence of
PDTC was required for its effect as both UV-induced nuclear
translocation and accumulation resumed after PDTC removal. We next
investigated whether PDTC treatment altered the p53 redox state. We
found that PDTC increased p53 cysteine residue oxidation in
vivo. This represents the first direct evidence showing that the
p53 redox state can be altered in vivo and that increased
oxidation correlates with its inability to perform its downstream
functions.
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INTRODUCTION |
The p53 tumor suppressor protein is believed to play an important
role in maintaining genomic integrity and preventing tumorigenesis. A
high frequency of gene-inactivating mutations observed in a wide
variety of human cancers demonstrates the importance of functional inactivation of p53 in cell malignancy (1). Part of the mechanism of
its function is based on its transcription regulation of some crucial
genes, such as WAF1/CIP1/SDI1, a
cyclin kinase inhibitor that leads to cell growth suppression (2, 3)
and MDM2, a p53 feedback inhibition gene (4, 5). Nuclear
localization of p53 appears to be essential to mediate downstream
events (6). Nuclear accumulation of p53 is mediated by three specific
nuclear localization signals inherent in the primary structure of the protein, which encompass residues 310-319, 369-375, and 379-384 (6).
Mutations in the first nuclear localization signal (residues 310-319)
hinder its nuclear translocation and result in inactivation of its
transformation suppressor function (7, 8). However, some mechanisms of
p53 inactivation appear to prevent the ability of p53 to reside in the
nucleus without mutating the p53 gene. For example, in some
inflammatory breast cancers, undifferentiated neuroblastomas and
retinoblastoma cells expressing wild-type p53, the protein appears to
be partially inactivated by cytoplasmic sequestration (9-11).
Furthermore, the high tumorigenesis rate in the livers of transgenic
mice expressing the hepatitis B viral HBx protein is probably linked to
the sequestration and functional inactivation of p53 in the cytoplasm
by the HBx protein (12).
Other types of p53 inactivation have been reported in cancers. A
well-characterized example is the degradation of p53 by human papillomavirus (HPV)1 E6
protein in cervical cancer. The E6 oncoprotein, expressed by oncogenic
subtypes of HPV, binds p53 and directs its destruction through a
ubiquitin-mediated pathway (13). Furthermore, a defective p53 response
to ionizing radiation is observed in cells lacking the ATM
gene, the gene mutated in ataxia-telangiectasia patients (14). After
-radiation, p53 in such cells is not correctly induced, thus,
impairing its G1 arrest function (14, 15). Finally, the
MDM2 cellular oncoprotein appears to be required to regulate p53 levels
and its transactivation activity. Abnormal overexpression of
MDM2 can lead to p53 inhibition in a variety of cancers (16,
17).
Various forms of stress, such as ionizing radiation, UV radiation,
medium depletion, hypoxia, heat shock, ribonucleotide depletion, and
calcium phosphate treatment, lead to the induction of p53 protein level
and the accumulation of transcriptionally active p53 inside the nucleus
(18-24). Several other transactivators, such as NF-
B, AP-1, and
Egr-1, can also be activated by UV radiation and other types of
stressors (25-27). How stressors stimulate cellular responses is not
completely known. One hypothesis is that reactive oxygen intermediates
(ROIs), commonly produced by many of these stressors, act as second
messengers for the activation of these transactivators. Paradoxically,
the DNA binding activity of some of these transactivators, including
p53, is dependent on maintaining a low redox potential of these
proteins (28-30). In order to properly control the activation of these
important transactivators, it is possible that the redox state of these
proteins is highly regulated inside the cell.
In this study, we investigated p53 activation in intact cells by
analyzing the effects of pyrrolidine dithiocarbamate (PDTC), a widely
used compound in redox regulation studies of NF-
B and AP-1 (25,
31-32). PDTC contains two thiol moieties that can chelate metal ions
and may exert either antioxidant or pro-oxidant effects (33). Here we
demonstrate that PDTC inhibits p53 nuclear translocation and p53
induction. The inhibitory action of PDTC is not mediated by scavenging
peroxides, but rather through alteration of the p53 redox
state.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
The A1-5 rat embryo fibroblast cell line
was maintained and grown in 90% Dulbecco's modified Eagle's medium
supplemented with 4500 mg/liter glucose and 2 mM
L-glutamine (Irvine Scientific, Irvine, CA), 10%
heat-inactivated fetal bovine serum (Gemini Bioproducts), and
penicillin (100 units/ml)-streptomycin (100 mg/ml) solution (Irvine
Scientific) with 5% CO2 at 37 °C. WS1neo and WS1E6 cell lines were kind gifts from Drs. Geoffrey Wahl and Steven Linke at the
Salk Institute, La Jolla, CA. Cells were maintained at 37 °C in
modified Eagle's medium (Irvine Scientific) with 1× nonessential amino acids (Irvine Scientific), 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 200 µg/ml G418
(Life Technologies, Inc.). PDTC and N-ethylmaleimide (NEM)
were purchased from Sigma. Dihydrorhodamine 123 (DHR),
3-(maleimidopropioryl)biocytin (MPB) and NutrAvidin were purchased from
Molecular Probes (Eugene, OR). Dithiothreitol (DTT) was purchased from
Fisher Biotech (Fair Lawn, NJ). Slide chambers were from Nunc, Inc.
(Naperville, IL). All antibodies used in this study (except PAb421)
were purchased from Oncogene Research Products (Cambridge, MA).
UV Treatment--
A1-5 cells were seeded in 10-cm plates (5 × 105 cells) or 2-well slide chambers (3 × 104 cells) and grown at 37 °C overnight followed by
incubation at 39 °C for another 24 h. Medium was removed, and
cells were exposed to UV radiation from a germicidal lamp (254 nm) at
1.7-1.9 J/m2/sec monitored by a radiometer (UVP Inc.,
Upland, CA). After UV treatment, prewarmed fresh medium was applied to
the cultures, and they were returned to the incubator. For human
diploid fibroblast lines, a 1:3 dilution of WS1neo and 1:5 dilution of
WS1E6 from a confluent 10-cm plate were used to seed the plates 1 day
prior to the treatment.
Indirect Immunofluorescence (IF) Staining--
IF staining was
carried out as described previously (34). The intensity of fluorescence
in the cytoplasm and nucleus was quantified from film negatives using
IPLab Gel software (Signal Analytics Corp., Vienna, VA). For each
densitometric value, 20 cells were counted, and the S.D. was calculated
using Microsoft Excel software (version 2.0). Percentage of nuclear
intensity was calculated by dividing the nuclear fluorescence level by
the nuclear plus the cytoplasmic fluorescence level.
Temperature Shift Experiment--
A1-5 cells were grown at
37 °C overnight followed by incubation at 39 °C (or 32 °C as
control) for another 24 h. Prewarmed (32 °C) medium with or
without PDTC was then supplied to the cells before switching them to
32 °C for further incubation at time periods indicated in the figure
legends.
Cell Harvesting and Western Blotting--
At each indicated time
point, cells were washed once with 5 ml of PBS (137 mM
NaCl, 2.7 mM KCl, 8 mM
Na2HPO4·7H2O, 1.4 mM KH2PO4, pH 7.2) and harvested in 2 ml of cold
PBS with a cell scraper. Cell pellets were obtained by centrifugation
at 1600 rpm in a tabletop centrifuge (Beckman model T J-6) for 5 min
and stored at
80 °C. Pellets from 10-cm plates were ruptured by
sonication in 1 ml (if A1-5 cells were used) or 100 µl (if WS1neo or
WS1E6 were used) of lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 8.0, 150 mM sodium chloride, 0.5%
Nonidet P-40) freshly supplied with proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µM E64, 1 µM leupeptin, and 1 µM aprotinin). Soluble
protein concentration was determined by Bio-Rad protein assay
(Bio-Rad), and 40 µg (or other amount as indicated) of total protein
was resolved on a 10 or 12% SDS-PAGE gel. Protein transfer to
Immobilon-P membrane (Millipore Co., Bedford, MA) and Western blotting
were performed as described previously (35). Purified PAb421 hybridoma supernatant (34) or DO-1 was used as the primary antibody to detect p53
from A1-5 cells or human fibroblasts respectively. IPLab Gel software
was used to quantify band intensities.
FACS Analysis of Peroxide Levels--
Levels of peroxides were
determined by FACS analysis as described elsewhere (26). Briefly, A1-5
cells, growing at 39 °C, were preincubated with 4 µM
DHR for 30 min followed by 50 J/m2 UV radiation or changing
of the incubation temperature to 32 °C. After UV radiation or
temperature shift, DHR incubation was continued for 30 min more in the
presence or absence of PDTC. The intensity of fluorescence of rhodamine
123 (wavelength 500-540 nm), which was converted intracellularly from
DHR, was assessed from 50,000 cells by flow cytometry with an
excitation source of 488 nm.
Selective Labeling of Oxidized Cysteine Residues--
This
procedure is a modified form of the method described by Bayer et
al. (36). Cells (1.4 × 106) were seeded into
15-cm plates. For each condition, two 15-cm plates were used. Frozen
cell pellets were lysed by sonication in 1 ml of SEE (0.1 M
sodium phosphate, pH 7.0, 5 mM EDTA, and 5 mM
EGTA) with 20 mM NEM and 1 mM
phenylmethylsulfonyl fluoride. After centrifugation to remove insoluble
material and subsequent protein concentration measurements, soluble
lysates were diluted to 0.6 mg/ml with SEE plus NEM and
phenylmethylsulfonyl fluoride. After incubation on wet ice for 30 min,
diluted lysates were individually dialyzed against SEE overnight with
one change of buffer after the first 4 h. To reduce oxidized
sulfhydryl groups, DTT was added to 1.5 ml of sample to a concentration
of 20 mM. After 30 min of incubation on wet ice, the
samples were individually dialyzed as described above. MPB (10 µg/ml)
was added to DTT-treated or DTT mock-treated samples at 4 °C for 30 min followed by dialysis. Samples were again measured for protein
concentration. Immunoprecipitation of 50 µg of each sample with a
p53-specific antibody mixture (1.8 µg of PAb421 and 0.6 µg of
PAb240) or 2.4 µg of anti-E1A antibody (negative control) was
performed as described previously (34). The immunoprecipitated proteins
were analyzed by 8% SDS-PAGE. Duplicate immunoprecipitations of each
sample and SDS-PAGE were performed to determine the immunoprecipitation
efficiency. After electroblotting to Immobilon-P membranes, one
membrane was probed with NutrAvidin (avidin conjugated with horseradish
peroxidase) to detect proteins modified by MPB, and the other membrane
was probed with PAb421 to determine the amount of p53 in each sample. To ensure that MPB modified proteins with disulfide linkages and not
proteins with free sulfhydryl groups, two purified proteins were used
as controls. One was bovine pancreas chymotrypsinogen (Worthington,
Freehold, NJ), which has five disulfide linkages (37). The second was
rabbit muscle aldolase (Worthington), which has eight free sulfhydryl
groups/subunit but no disulfide linkages (38).
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RESULTS |
PDTC Inhibits p53 Nuclear Translocation and
Transactivation--
To study the mechanism of p53 activation, we used
a transformed rat embryo fibroblast cell line, A1-5 (39), which
expresses a high level of a temperature-sensitive mutant p53, TSp53.
TSp53 is a protein that expresses a valine residue at codon 135 instead of alanine. This TSp53 is located in the cytoplasm at the nonpermissive temperature, 39 °C, but in the nucleus when cells are incubated at
32 °C (Fig. 1, far left
panels) (39, 40). To demonstrate that the normal upstream p53
signaling pathway in this cell line is intact, we tested whether TSp53
was capable of translocating into the nucleus in response to UV
radiation at the nonpermissive temperature. The ability of TSp53 to
accumulate in the nucleus in response to UV radiation was tested by
treating cells with 50 J/m2 of UVC light. As shown in Fig.
1 (top row), at 2 h post-irradiation, p53 started to
appear in the nucleus, and by 6 h post-irradiation, 60% of the
total cellular p53 was detected in the nucleus as compared with 29%
prior to radiation (Figs. 1 and
2B). A time course study was
also conducted to determine p53 nuclear accumulation after temperature
shift from 39 to 32 °C (Fig. 1, bottom right three panels). After 2 h, p53 nuclear accumulation was apparent,
and by 6 h, p53 nuclear accumulation was complete, with very
little cytoplasmic p53 remaining. Although no change in p53 steady
state level was observed after UV radiation or temperature shift (data not shown), the ability of TSp53 to respond to UV at the nonpermissive temperature suggests that the upstream pathway for p53 activation in
response to DNA damage is intact in this cell line.

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Fig. 1.
UV radiation and temperature shift from 39 to
32 °C trigger p53 nuclear translocation in
A1-5 cells. A1-5 cells were grown at 39 °C and treated with 50 J/m2 UV light followed by further incubation at 39 °C
(top row). Cell incubation temperature was shifted from 39 to 32 °C for 2, 4, and 6 h (bottom row, three right
panels). Cells were incubated at 32 °C for 24 h
(bottom left panel). At indicated time points, cells were
fixed with acetone and stained with PAb421 antibody against p53
followed by FITC-conjugated secondary antibody (magnification, × 100).
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Fig. 2.
p53 nuclear translocation was suppressed in
the presence of PDTC. A and B, at 39 °C,
A1-5 cells were treated with 50 J/m2 UV radiation followed
by further incubation at 39 °C in the presence or absence of 25 µM PDTC. A, IF analysis at 2 (a) and 4 (b) h after UV radiation; IF analysis at 2 (c)
and 4 (d) h after UV radiation in the presence of 25 µM of PDTC. B, percentage of nuclear p53 in
the presence or absence of PDTC after UV radiation. (hpi, hours of
incubation post-irradiation). Stippled bar, in the presence
of 25 µM PDTC after UV radiation; clear bar,
mock PDTC-treatment after UV radiation. C and D,
A1-5 cells were first incubated at 39 °C overnight. Cells were then
moved to the 32 °C incubator after replacing the medium with fresh
medium or medium containing 25 µM PDTC. C, the
subcellular localization of p53 was detected by IF. IF analysis at 2 (a) and 4 (b) h after temperature shift to
32 °C; IF analysis at 2 (c) and 4 (d) h after
temperature shift to 32 °C in the presence of 25 µM of
PDTC. D, the percentage of nuclear p53 in the presence or
absence of 25 µM PDTC during temperature shift from 39 to
32 °C. Cells were incubated in the presence or in the absence of
PDTC for the indicated periods after temperature shift. After
incubation with PDTC, cells were either immediately fixed or washed and
allowed to incubate further at 32 °C (hrs pts, hours of
incubation post-temperature shift).
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The effect of the thiol-containing agent PDTC on p53 activation was
then investigated. When PDTC was applied to A1-5 cells immediately
after UV treatment, TSp53 nuclear translocation was completely
abrogated (Fig. 2A). In the presence of PDTC, the proportion of cellular p53 residing inside the nucleus (25 and 30% at 2 and 4 h postirradiation, respectively) after UV treatment was similar to that of non-UV treated cells (29%) (Fig. 2B).
Interestingly, PDTC also prevented TSp53 nuclear translocation induced
by temperature shift. No increase of nuclear p53 was observed if PDTC
was present during temperature shift from 39 to 32 °C, whereas a
4-fold increase was observed in the absence of PDTC (Fig. 2,
C and D). The inhibition of TSp53 nuclear
translocation by PDTC during temperature shift was
dose-dependent. The presence of 5 µM PDTC had
no effect on TSp53 nuclear translocation, whereas 10 µM
showed moderate inhibition. Only upon application of 20 µM or more PDTC was complete inhibition of translocation
observed (data not shown). In sum, we observe the prevention of both
UV- and temperature shift-mediated TSp53 nuclear translocation by
PDTC.
In order to confirm that p53 activity was inhibited, the expression of
a p53 downstream effector, MDM2, was examined. To do this,
we shifted the incubation temperature of A1-5 cells from 39 to 32 °C
to induce MDM2 expression (4, 5). In Fig.
3 we show that p53 normally activates
MDM2 protein expression within 2 h after shifting the incubation
temperature (Fig. 3, lanes 4 and 5). However, in
the presence of PDTC, temperature shift failed to induce
MDM2 expression (Fig. 3, lanes 8 and
9). This suggests that by excluding p53 from the nucleus,
PDTC is able to prevent p53-mediated transactivation of
MDM2. It should be noted that we found no induction of MDM2
after UV treatment. The lack of MDM2 induction by the high dose of UV
may be due to transcriptional repression of MDM2 (41, 42) or
because p53 fails to adopt a transcriptionally competent state inside
the nucleus at the nonpermissive temperature.

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Fig. 3.
Continual presence of PDTC is required for
its inhibitory effect on p53-mediated expression of
MDM2. A1-5 whole cell soluble lysate (40 µg) was
separated by 12% SDS-PAGE and transferred to Immobilon-P membrane. The
membrane was cut horizontally at the 68-kDa marker. The lower portion
was probed with PAb421 for p53 detection ( p53), and the
upper portion was probed with 2A10 antibody against MDM2 ( mdm-2). Lane 1, cells grown at 32 °C overnight;
lane 2, cells grown at 39 °C overnight; lane
3, cells grown at 39 °C overnight, mock-UV treated, and further
incubated for another 8 h; lanes 4-6, the incubation
temperature of the cells was shifted from 39 to 32 °C for 2, 4, and
8 h, as indicated; lane 7, blank; lanes 8 and 9, cells were first incubated at 39 °C and then
incubated at 32 °C in the presence of 25 µM PDTC for 2 and 4 h, respectively; lane 10, cells were first grown
at 39 °C and then incubated at 32 °C in the presence of PDTC for
2 h. The medium containing PDTC was subsequently removed, and
cells were washed once and supplied with fresh medium while they
remained at 32 °C for another 6 h. Lane 11, cells
were first grown at 39 °C and treated with 50 J/m2 UV,
after which cells were further incubated at 39 °C for 8 h.
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To further characterize the inhibitory effect, we tested whether the
continual presence of PDTC was required to prevent temperature shift-induced p53 nuclear translocation. PDTC was added to cells immediately after temperature shift to prevent p53 translocation but
removed after 2 h. The cells were then maintained for another 6 h at the permissive temperature with fresh medium. When both the
p53 localization and the expression of MDM2 were examined, we found that the removal of PDTC led to an increase in nuclear p53
level (Fig. 2D, last column) and MDM2 expression
(Fig. 3, lane 10). This demonstrates that p53 activity
recovers once PDTC is removed. We then tested whether PDTC was
continually required to prevent p53 nuclear translocation after UV
treatment. Cells were pretreated with PDTC for 30 min prior to UV
radiation. After UV treatment, fresh medium was added without PDTC. As
shown in Fig. 4 (columns 4 and
5) p53 accumulated into the nucleus within 4 h. All of
the data demonstrate that PDTC prevents p53 translocation and its
transactivation function and that PDTC must be continually present for
its effect.

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Fig. 4.
Inhibition of UV-induced p53 nuclear
translocation requires continual presence of PDTC. At 39 °C,
A1-5 cells were treated with 25 or 50 µM PDTC for 30 min.
Cells were then UV treated, subsequently washed with fresh medium, and
incubated for another 6 h at 39 °C with fresh medium without
PDTC. After IF analysis, the percentage of nuclear p53 was calculated.
Column 1, untreated cells; column 2, cells were
treated only with medium containing PDTC but not UV; column
3, cells were UV-treated but not treated with PDTC; columns
4 and 5, cells were treated with 25 or 50 µM PDTC followed by UV treatment.
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PDTC Inhibits UV-induced p53 Level Increase and HPV
E6-dependent p53 Degradation in Normal Human
Fibroblasts--
Because all previous studies were conducted with
TSp53, it was important to determine whether PDTC could also inhibit
wild-type p53 activity in normal human diploid cells. The human
fibroblast cell line WS1neo, expressing a retrovirally inserted
neomycin resistance gene, was used for this study (24). Previous work demonstrated that ionizing radiation and nucleotide depletion induce
p53 expression and cell cycle arrest in these cells. As shown in Fig.
5, within 4 h after UV treatment,
p53 levels increased 140% (2.4-fold). When PDTC was added to WS1neo
cells, the UV-induced increase in p53 level was almost completely
inhibited after 4 h (Fig. 5, lane 4 versus lane 5). In
the presence of PDTC, p53 increased only 30% (1.3-fold) after UV
treatment. Similar to A1-5 cells, inhibition by PDTC was reversible.
Subsequent removal of PDTC after 4 h, followed by continual
incubation in fresh medium, restored the p53 UV response. Thus, in
UV-treated cells, the p53 level at 20 h after PDTC removal was
identical to the p53 level in cells not treated with PDTC (Fig. 5,
lanes 8 and 9). This demonstrates that, as in
A1-5 cells, PDTC was able to temporarily inhibit the ability of p53 to
respond to UV radiation. Curiously, after 24 h, we observed an
intermediate p53 increase of approximately 90-150% in cells that were
not exposed to UV light, both in the presence and the absence of a 4-h
PDTC treatment (Fig. 5, lanes 6 and 7). The cause
of this intermediate p53 induction may be due to the mock treatment
(medium removal and replenishment). However, the fact that the level of
p53 induction was almost identical in these two samples indicates that
PDTC alone had little effect on p53 induction. This experiment also
shows that the UV-induced signal is maintained for at least 4 h
during PDTC treatment, although p53 is not able to respond within this
period due to the presence of PDTC. Whether this means that the damage
elicited by UV is maintained throughout this period or a UV-mediated
signal is stable throughout this period is unclear. Our data indicate
that p53 is able to respond to UV up to 4 h post-irradiation.

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Fig. 5.
PDTC inhibited UV-induced accumulation and
HPV E6-dependent degradation of wild-type p53 in normal
human fibroblasts. WS1neo or WS1E6 soluble whole cell lysate (40 µg) was separated by 10% SDS-PAGE and transferred to Immobilon-P
membrane. The membrane was probed with antibodies to detect p53 as
described under "Experimental Procedures." PDTC (10 µM) was supplied in the medium for 4 h (+). +, UV
treatment (20 J/m2) prior to PDTC addition; , cells mock
treated with UV; hpi, hours of incubation post-irradiation. The
relative level of p53 was quantified by comparing the p53 band
intensity with that from cells harvested before any treatment
(lane 1). In lanes 1-9, WS1neo cells were used,
and in lanes 10-13, WS1E6 cells were used.
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Surprisingly, we also observed an inhibition of HPV E6-mediated
degradation of p53 by PDTC. Human fibroblasts expressing E6 (WS1E6)
have no detectable p53 because E6 mediates rapid p53 degradation (43,
44). As expected, no p53 was present before or after UV radiation in
cells expressing E6 (Fig. 5, lanes 10 and 12). Nevertheless, p53 was observed in WS1E6 during PDTC treatment independent of UV radiation (Fig. 5, lanes 11 and
13), and p53 degradation resumed within 10 h after the
removal of PDTC (data not shown). The ability of PDTC to prevent p53
nuclear translocation, p53 induction, p53-mediated transactivation, and
E6-mediated p53 rapid degradation suggests that PDTC may be able to
directly interfere with p53 protein itself.
Temperature Shift Does Not Induce Peroxide Formation--
ROIs
such as superoxide anion (O
2), peroxides (ROOR), or hydroxyl
radicals (OH·) are believed to act as secondary messengers in the
signal transduction pathway of several transactivators. Because
hydrogen peroxide alone was shown to induce nuclear accumulation of p53
(Refs. 23 and 45 and data not shown), it was possible that PDTC
inhibits p53 activation through scavenging peroxides generated during
UV treatment or temperature shift. If this is the case, a change of
intracellular peroxide level should be found in all situations in which
PDTC inhibits p53 activation. The intracellular peroxide level after UV
treatment or temperature shift was then determined by the ability of
the membrane permeable nonfluorescent substrate, DHR, to react with
peroxide within the cell and oxidize to rhodamine 123, a membrane
impermeable fluorescent product (46, 47). A1-5 cells were preincubated
at 39 °C for 30 min with DHR, UV treated or temperature shifted, and
then harvested after another 30 min. As expected, the intracellular
level of peroxides increased 50-100% after UV radiation (Fig.
6). However, no alteration of peroxide
level was observed during the temperature shift (Fig. 6). These
observations show that PDTC inhibits p53 nuclear translocation independent of the peroxide level. We also found that PDTC, at p53
inhibitory concentration, only slightly inhibited UV-induced peroxide
formation. The effect of PDTC on p53 appears to involve mechanisms
distinct from the signal generated by peroxides.

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Fig. 6.
Temperature shift does not lead to a
detectable increase in peroxide level. For determination of the
peroxide level generated after UV radiation, at 39 °C, A1-5 cells
were preincubated with 4 µM DHR for 30 min followed by
irradiation with 50 J/m2 UV radiation and continual
incubation at 39 °C in the presence or absence of 25 µM PDTC for 30 min. For determination of peroxide
generation during temperature shift, A1-5 cells were preincubated with
4 µM DHR for 30 min at 39 °C. Cells were then placed
in a 32 °C incubator for 30 min and harvested. For measurement of
basal level of peroxide, cells were incubated at 39 °C with DHR for
a total of 60 min. FACS analysis was used to measure the mean
fluorescence intensity, which corresponds to the peroxide level inside
the cell. Hatched bar, experiment 1; clear bar,
experiment 2. Note that for experiment 2, a measurement of the basal
peroxide level was not conducted.
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PDTC Treatment Correlates with the Oxidation of Cysteine Residues
on p53--
PDTC has been shown to exert both pro- and antioxidant
effects in cell-free and biological systems (33). Because PDTC appears to inhibit p53 activity in a peroxide-independent manner, we tested whether PDTC could alter the p53 oxidation state in vivo.
Specifically, we checked whether p53 cysteine residues could undergo an
S-thiolation reaction, defined as any disulfide bond formation (48).
Such bonds could be formed intermolecularly or intramolecularly. Fig. 7 shows the experimental design for the
detection of cysteine oxidation on p53. Endogenous protein free
sulfhydryl groups were blocked by lysing the cells in the presence of
NEM, a reagent that forms nonreducible thioether bonds with free
sulfhydryl groups. After NEM treatment, proteins were treated with DTT
to free disulfide-linked cysteine residues, and newly formed sulfhydryl
groups were covalently modified with MPB, a biotin-conjugated maleimide
that, like NEM, forms a nonreducible thioether bond. MPB-modified p53
was then immunoprecipitated with an antibody mixture (PAb421 and
PAb240) followed by SDS-PAGE analysis. After electroblotting onto
polyvinylidene difluoride membrane, MPB-modified p53 was then detected
with peroxidase-conjugated avidin followed by ECL. Immunoprecipitation
efficiency was assessed by duplicate immunoprecipitations followed by
standard p53 Western analysis.

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Fig. 7.
Experimental design to selectively detect p53
cysteine disulfide bonds. P1 and P2
represent cellular proteins, other than p53, which contain free
sulfhydryl groups ( SH) and disulfide bonds
( S S R), respectively. p53 protein is shown with two
cysteine residues, one in a disulfide linkage and the other with a free
sulfhydryl group. The circled A represents protein
A-Sepharose. The upside-down Y-shaped line represents the
p53-specific antibodies used for immunoprecipitation. See under
"Experimental Procedures" for more details.
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A1-5 cells at 39 °C were treated with UV light. PDTC was immediately
added, and the cells were incubated for 1.5 h prior to harvesting.
As shown in Fig. 8A, cysteine
residues on p53 were oxidized in the presence of PDTC (compare
lanes 1 and 3 with lane 5). The upper
blot (Fig. 8A, a) is a chemiluminescence image showing the
level of p53 that undergoes thiol modification. Lanes 1, 3, and 5 show that p53 and several p53-associated proteins are
modified by MPB. Lanes 2, 4, and 6 show that MPB
protein modification in lanes 1, 3, and 5 requires DTT treatment to expose sulfhydryl groups. UV treatment did
not significantly alter the level of p53 S-thiolation (compare
lane 1 and lane 3). However, PDTC treatment consistently showed a 2-3-fold increase in p53 cysteine residue S-thiolation. We did not detect an increase in S-thiolation on p53-associated proteins (denoted with asterisks).
Immunoprecipitation efficiency did not vary by more than 20% in any
lane (Fig. 8A, b). We also tested whether PDTC increased p53
cysteine S-thiolation in A1-5 cells incubated at 32 °C. In this
experiment cells, growing at 39 °C were shifted to 32 °C for
1.5 h in the presence of PDTC. We found that p53 cysteine
S-thiolation was increased only with PDTC treatment and not with
temperature shift alone (Fig. 8B). The data show that PDTC
treatment promotes p53 S-thiolation. Therefore, it is possible that
direct modification of p53 cysteine residues may lead to abrogation of
p53 nuclear translocation, p53 level-increase, HPV-E6 mediated
degradation of p53 and MDM2 transactivation.

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Fig. 8.
PDTC oxidizes p53 cysteine residues.
A1-5 cells were either UV-treated at 39 °C or incubated at 32 °C
in the presence or absence of 25 µM PDTC. Cells were then
harvested at 1.5 h post-treatment and lysed in the presence of 20 mM NEM. Oxidized cysteine residues on the proteins were
selectively labeled with MPB followed by immunoprecipitation with
p53-specific antibodies. A, soluble lysate from A1-5 cells
mock-treated or UV-treated in the presence or absence of PDTC.
a, probed with avidin-peroxidase to detect MPB labeled p53.
b, Western blot to assess the p53 level after each
treatment. B, soluble lysate from A1-5 cells incubated at
39 °C or temperature shifted to 32 °C in the presence or absence
of PDTC. a, probed with avidin-peroxidase to detect MPB
labeled p53. b, Western blot to assess the p53 level after
each treatment. Lanes 1 and 2 are soluble lysates
from mock-treated cells; lanes 3 and 4 are
soluble lysates from UV-treated or temperature shifted cells;
lanes 5 and 6 are soluble lysates from UV-treated
or temperature shifted cells in the presence of PDTC; lane 7 is soluble lysate without MPB treatment; lanes 8 and
10 are sham immunoprecipitations without cell lysate;
lane 9 is an immunoprecipitation of the soluble lysate used
in lane 3 with the non-p53 specific antibody anti-E1A.
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Interestingly, we also observed endogenous cysteine S-thiolation in the
absence of any treatment (Fig. 8, A and B, lanes
1 and 3). The nature and the biological function of
this cysteine modification are not known at this point.
 |
DISCUSSION |
In this study, PDTC was used to investigate redox regulation of
p53. UV light induces p53 levels, p53 nuclear accumulation, and
p53-mediated transactivation of several downstream effector genes,
resulting in a delay in cell cycling (22, 49-50). PDTC has radical
scavenging as well as pro-oxidant properties (25, 51). The evidence
presented here shows that PDTC inhibits p53 induction, p53 nuclear
translocation and p53-mediated transactivation of MDM2.
Three principal findings of this study are: 1) PDTC inhibits p53
in vivo, 2) PDTC increases p53 cysteine residue oxidation, and 3) endogenous p53 contains oxidized cysteine residues.
PDTC Inhibits p53 Activation--
The p53 protein exists in a
latent state in cycling cells (52). Upon exposure to environmental
stress, the p53 level increases and p53 accumulates in the nucleus (22,
49). Because superoxide anions (O
2), peroxides (ROOR), and
perhaps hydroxyl radicals (OH·) are produced by UV treatment (53),
the possibility that these ROIs are required for p53 nuclear
accumulation was explored. We observed that the ROI scavenger PDTC
inhibits p53 nuclear trafficking and prevents an increase in the steady
state level of p53 after UV radiation. PDTC, however, does not inhibit
p53 function by preventing peroxide generation. Evidence for this
conclusion is based on the fact that PDTC blocked p53 nuclear
translocation induced by temperature shift, a mechanism that does not
produce detectable peroxides. Furthermore, because UV production of
ROIs is very transient (53) (t1/2 = 30 min) a 4-h
PDTC treatment should be sufficient to permanently prevent p53
activation. Instead, we found that upon PDTC withdrawal 4 h after
UV treatment, the p53 response resumed. The data are inconsistent with
the radical scavenging property of PDTC. Similarly, another study found
that hydrogen peroxide could activate
WAF1/CIP1/SDI1, another downstream effector gene of p53 (54).
WAF1/CIP1/SDI1 activation was delayed in the presence of PDTC but resumed after PDTC withdrawal. The data
suggest that PDTC acts through a mechanism other than ROI scavenging to
inhibit p53 translocation and up-regulation.
It was recently shown that the antibiotic geldanamycin can also prevent
mutant p53 nuclear translocation (55). Geldanamycin probably inhibits
p53 translocation by binding to hsp90 and interrupting the p53-hsp90
function. Geldanamycin also promotes rapid p53 destabilization (56,
57). PDTC does not act in the same manner as geldanamycin because it
does not elicit p53 destabilization. Other studies have shown that p53
phosphorylation correlates with p53 induction after DNA damage (58,
59). One consequence of phosphorylation is inhibition of MDM2-p53
complex formation. MDM2-p53 complex formation leads to rapid p53
degradation (60, 61). PDTC, however, appears to act on p53 in a manner
that is independent of MDM2 because it prevents p53 nuclear
translocation in A1-5 cells at the nonpermissive temperature. At this
temperature, little or no MDM2 is present.
PDTC Increases p53 Cysteine Residue S-thiolation--
When the p53
redox state was examined, increased cysteine residue S-thiolation was
observed during PDTC treatment. Because this modification was reversed
by a reducing agent, it is likely that p53 cysteine residues undergo
S-thiolation or thioesterification. This could explain why
PDTC also prevented E6-mediated degradation of p53. If PDTC modifies a
p53 cysteine residue critical for E6 binding, then p53 degradation
would be blocked. In this regard, it previously was shown that a
cysteine to tyrosine substitution at cysteine 135 of human p53
abolished binding between E6 and p53 and alleviated p53 degradation
(62). Recently, p53 activation of WAF1/CIP1/SDI1 and p53-DNA
complex formation was shown to be blocked by PDTC via a mechanism that
depends on Cu2+ (54). It was proposed that PDTC mediates a
pro-oxidant effect by chelating Cu2+ and transporting it
across the plasma membrane (51). Increased intracellular
Cu2+ levels have been detected in cells treated with PDTC
(51, 54).
How Cu2+ plus PDTC promotes p53 cysteine residue oxidation
is not clear. Hainaut et al. (63) have shown that p53
directly binds Cu2+ in vitro (63). Thus, one
mechanism could be that Cu2+ directs one-electron oxidation
of the cysteine residue sulfhydryl bond, resulting in thiyl radical
formation (64-66). This reactive radical could eventually lead to
glutathione S-thiolation or intramolecular disulfide bond formation. If
p53 cysteine residues critical for activation are modified by such a
reaction, one might expect that reduction would increase p53
activities. In fact, DTT was observed to increase p53 DNA binding
in vitro (29, 30). Ref-1, a protein that can regulate the
redox state of a number of different proteins, was found to stimulate
p53 DNA binding activity in a redox-dependent manner (67).
In a recent study, a transgenic Schizosaccharomyces pombe
strain that expresses transcriptionally active human p53 was created
(68). Optimal transcriptional activation depended on the thioredoxin
reductase gene, TRR1, the gene product of which has protein
disulfide reductase activity. It appears that oxidation of p53 cysteine
residues leads to inhibition of several of its activities.
Endogenous Cysteine Oxidation of p53--
Interestingly, prior to
PDTC treatment, we observe cysteine residue oxidation on p53 and on
four or five uncharacterized p53 associated proteins. It is not clear
whether such modifications are necessary for p53 activity. Cysteine to
serine substitutions can model cases in which cysteines are in the
reduced state. Cysteine to serine substitutions at conserved residues
within the p53 central domain have delineated three sets of cysteine
residues (69). One set, at codons 173, 235, and 238 of mouse p53, is
critical for optimal gene transactivation and cell transformation
suppression activity. These residues directly interact with the zinc
ion and therefore are probably necessary for structural integrity (70). A second set, at positions 121, 132, 138, and 272, is required for
optimal gene transactivation and cell transformation but not DNA
binding. A third set of cysteine residue substitutions, at positions
179 and 274, does not disturb any measured p53 activities. It is this
second set of residues in which cysteine residue modification may
actually be necessary for some p53 activity. Characterization of the
molecule that modifies p53 cysteine residues and mapping of its site
will give us more insight into the mechanism of p53 redox control.
We thank Dr. Paul Salvaterra for the use of
his fluorescence microscope and Drs. Susan Kane and Gargi Dasgupta for
critical reading of the manuscript. We thank Drs. Geoffrey Wahl and
Steven Linke (Salk Institute, La Jolla, CA) for the WS1neo and WS1E6 cell lines. We also graciously acknowledge the assistance of Robert Barber and Richard Wetts of the Department of Neurosciences at the
Beckman Research Institute at City of Hope.