INTRODUCTION

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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.

	EXPERIMENTAL PROCEDURES

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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).

	RESULTS

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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.

View larger version (100K): [in this window] [in a new window]   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).

View larger version (65K): [in this window] [in a new window]   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).

View larger version (33K): [in this window] [in a new window]   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.

View larger version (39K): [in this window] [in a new window]   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.

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.

View larger version (27K): [in this window] [in a new window]   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.

Temperature Shift Does Not Induce Peroxide Formation-- ROIs such as superoxide anion (O₂), 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.

View larger version (26K): [in this window] [in a new window]   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.

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.

View larger version (23K): [in this window] [in a new window]   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 (SSR), 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.

View larger version (35K): [in this window] [in a new window]   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.

	DISCUSSION

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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 (O₂), 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.

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).

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.