Pyrrolidine Dithiocarbamate Prevents p53 Activation and Promotes p53 Cysteine Residue Oxidation
1998; Elsevier BV; Volume: 273; Issue: 30 Linguagem: Inglês
10.1074/jbc.273.30.18898
ISSN1083-351X
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoPyrrolidine 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. 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. 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 (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6727) Google Scholar). 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 (2el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7916) Google Scholar, 3Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-815Abstract Full Text PDF PubMed Scopus (5230) Google Scholar) and MDM2, a p53 feedback inhibition gene (4Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1632) Google Scholar, 5Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1176) Google Scholar). Nuclear localization of p53 appears to be essential to mediate downstream events (6Shaulsky G. Goldfinger N. Ben-Ze'ev A. Rotter V. Mol. Cell. Biol. 1990; 10: 6565-6577Crossref PubMed Scopus (292) Google Scholar). 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 (6Shaulsky G. Goldfinger N. Ben-Ze'ev A. Rotter V. Mol. Cell. Biol. 1990; 10: 6565-6577Crossref PubMed Scopus (292) Google Scholar). Mutations in the first nuclear localization signal (residues 310–319) hinder its nuclear translocation and result in inactivation of its transformation suppressor function (7Shaulsky G. Goldfinger N. Tosky M.S. Levine A.J. Rotter V. Oncogene. 1991; 6: 2055-2065PubMed Google Scholar, 8Slingerland J.M. Jenkins J.R. Benchimol S. EMBO J. 1993; 12: 1029-1037Crossref PubMed Scopus (106) Google Scholar). 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 (9Moll U.M. Riou G. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7262-7266Crossref PubMed Scopus (535) Google Scholar, 10Moll U.M. LaQuaglia M. Benard J. Riou G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4407-4411Crossref PubMed Scopus (390) Google Scholar, 11Schlamp C.L. Poulsen G.L. Nork T.M. Nickells R.W. J. Natl. Cancer Inst. 1997; 89: 1530-1536Crossref PubMed Scopus (66) Google Scholar). 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 (12Ueda H. Ullrich S.J. Gangemi J.D. Kappel C.A. Ngo L. Feitelson M.A. Jay G. Nat. Genet. 1995; 9: 41-47Crossref PubMed Scopus (328) Google Scholar). Other types of p53 inactivation have been reported in cancers. A well-characterized example is the degradation of p53 by human papillomavirus (HPV) 1The abbreviations used are: HPV, human papillomavirus; DHR, dihydrorhodamine 123; DTT, dithiotheitol; FACS, fluorescence activated cell sorter; IF, indirect immunofluorescence; MPB, 3-(maleimidopropioryl)biocytin; NEM, N-ethylmaleimide; PDTC, pyrrolidine dithiocarbamate; ROI, reactive oxygen intermediate; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: HPV, human papillomavirus; DHR, dihydrorhodamine 123; DTT, dithiotheitol; FACS, fluorescence activated cell sorter; IF, indirect immunofluorescence; MPB, 3-(maleimidopropioryl)biocytin; NEM, N-ethylmaleimide; PDTC, pyrrolidine dithiocarbamate; ROI, reactive oxygen intermediate; PAGE, polyacrylamide gel electrophoresis. 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 (13Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (743) Google Scholar). Furthermore, a defective p53 response to ionizing radiation is observed in cells lacking the ATMgene, the gene mutated in ataxia-telangiectasia patients (14Xu Y. Baltimore D. Genes Dev. 1996; 10: 2401-2410Crossref PubMed Scopus (350) Google Scholar). After γ-radiation, p53 in such cells is not correctly induced, thus, impairing its G1 arrest function (14Xu Y. Baltimore D. Genes Dev. 1996; 10: 2401-2410Crossref PubMed Scopus (350) Google Scholar, 15Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2927) Google Scholar). Finally, the MDM2 cellular oncoprotein appears to be required to regulate p53 levels and its transactivation activity. Abnormal overexpression ofMDM2 can lead to p53 inhibition in a variety of cancers (16Piette J. Neel H. Maréchal V. Oncogene. 1997; 15: 1001-1010Crossref PubMed Scopus (240) Google Scholar,17Momand J. Zambetti G.P. J. Cell Biochem. 1997; 64: 343-352Crossref PubMed Scopus (168) Google Scholar). 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 (18Dover R. Jayaram Y. Patel K. Chinery R. J. Cell Sci. 1994; 107: 1181-1184PubMed Google Scholar, 19Renzing J. Lane D.P. Oncogene. 1995; 10: 1865-1868PubMed Google Scholar, 20Leonardo A.D. Linke S.P. Clarkin K. Wahl G.M. Genes Dev. 1994; 8: 2540-2551Crossref PubMed Scopus (1025) Google Scholar, 21Zhan Q. Carrier F. Fornace A.J. Mol. Cell. Biol. 1993; 13: 4242-4250Crossref PubMed Scopus (443) Google Scholar, 22Malzman W. Czyzyk L. Mol. Cell. Biol. 1984; 4: 1689-1694Crossref PubMed Scopus (815) Google Scholar, 23Sugano T. Nitta M. Ohmori H. Yamaizumi M. Jpn. J. Cancer Res. 1995; 86: 415-418Crossref PubMed Scopus (53) Google Scholar, 24Linke S.P. Clarkin K.C. Leonardo A.D. Tsou A. Wahl G.M. Genes Dev. 1996; 10: 934-947Crossref PubMed Scopus (480) Google Scholar). Several other transactivators, such as NF-κB, AP-1, and Egr-1, can also be activated by UV radiation and other types of stressors (25Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar, 26Huang R.-P. Wu J.-X. Fan Y. Adamson E.D. J. Cell Biol. 1996; 133: 211-220Crossref PubMed Scopus (252) Google Scholar, 27Lo Y.Y.C. Cruz T. J. Biol. Chem. 1995; 270: 11727-11730Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). 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 (28Toledano M.B. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4328-4332Crossref PubMed Scopus (577) Google Scholar, 29Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Nucleic Acids Res. 1993; 21: 3167-3174Crossref PubMed Scopus (185) Google Scholar, 30Hainaut P. Milner J. Cancer Res. 1993; 53: 4469-4473PubMed Google Scholar). 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-κB and AP-1 (25Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar,31Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1267) Google Scholar, 32Pinkus R. Weiner L.M. Daniel V. J. Biol. Chem. 1996; 271: 13422-13429Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). PDTC contains two thiol moieties that can chelate metal ions and may exert either antioxidant or pro-oxidant effects (33Orrenius S. Nobel C.S.I. van den Dobbelsteen D.J. Burkitt M.J. Slater F.G. Biochem. Soc. Trans. 1996; 24: 1032-1038Crossref PubMed Scopus (114) Google Scholar). 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. 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 mml-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 mml-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). 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. IF staining was carried out as described previously (34Sepehrnia B. Paz I.B. Dasgupta G. Momand J. J. Biol. Chem. 1996; 271: 15084-15090Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). 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. 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. At each indicated time point, cells were washed once with 5 ml of PBS (137 mmNaCl, 2.7 mm KCl, 8 mmNa2HPO4·7H2O, 1.4 mmKH2PO4, 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 (35Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2787) Google Scholar). Purified PAb421 hybridoma supernatant (34Sepehrnia B. Paz I.B. Dasgupta G. Momand J. J. Biol. Chem. 1996; 271: 15084-15090Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) 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. Levels of peroxides were determined by FACS analysis as described elsewhere (26Huang R.-P. Wu J.-X. Fan Y. Adamson E.D. J. Cell Biol. 1996; 133: 211-220Crossref PubMed Scopus (252) Google Scholar). Briefly, A1-5 cells, growing at 39 °C, were preincubated with 4 μmDHR 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. This procedure is a modified form of the method described by Bayer et al. (36Bayer E.A. Safars M. Wilchek M. Anal. Biochem. 1987; 161: 262-271Crossref PubMed Scopus (53) Google Scholar). 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 msodium phosphate, pH 7.0, 5 mm EDTA, and 5 mmEGTA) with 20 mm NEM and 1 mmphenylmethylsulfonyl 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 (34Sepehrnia B. Paz I.B. Dasgupta G. Momand J. J. Biol. Chem. 1996; 271: 15084-15090Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). 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 (37Matthews B.W. Sigler P.B. Henderson R. Blow D.M. Nature. 1967; 214: 652-656Crossref PubMed Scopus (419) Google Scholar). The second was rabbit muscle aldolase (Worthington), which has eight free sulfhydryl groups/subunit but no disulfide linkages (38Heyduk T. Moniewska A. Kochman M. Biochim. Biophys. Acta. 1986; 874: 337-346Crossref PubMed Scopus (9) Google Scholar). To study the mechanism of p53 activation, we used a transformed rat embryo fibroblast cell line, A1-5 (39Martinez J. Georgoff I. Martinez J. Levine A.J. Genes Dev. 1991; 5: 151-159Crossref PubMed Scopus (493) Google Scholar), 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) (39Martinez J. Georgoff I. Martinez J. Levine A.J. Genes Dev. 1991; 5: 151-159Crossref PubMed Scopus (493) Google Scholar, 40Ginsberg D. Michael-Michalovitz D. Ginsberg D. Oren M. Mol. Cell. Biol. 1991; 11: 582-585Crossref PubMed Scopus (123) Google Scholar). 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 and2 B). 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.Figure 2p53 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 Large Image Figure ViewerDownload (PPT) 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. 2 A). 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. 2 B). 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 μmshowed 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 (4Wu X. Bayle J.H. Olson D. Levine A.J. Genes Dev. 1993; 7: 1126-1132Crossref PubMed Scopus (1632) Google Scholar, 5Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1176) Google Scholar). 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 induceMDM2 expression (Fig. 3, lanes 8 and9). This suggests that by excluding p53 from the nucleus, PDTC is able to prevent p53-mediated transactivation ofMDM2. 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 (41Saucedo L.J. Carstens B.P. Seavey S.E. Albee II L.d. Perry M.E. Cell Growth Differ. 1998; 9: 119-130PubMed Google Scholar, 42Vichi P. Coin F. Renaud J.-P. Vermeulen W. Hoeijmakers J.H.J. Moras D. Egly J.-M. EMBO J. 1997; 16: 7444-7456Crossref PubMed Scopus (152) Google Scholar) or because p53 fails to adopt a transcriptionally competent state inside the nucleus at the nonpermissive temperature. 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. 2 D, 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 and5) 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. 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 (24Linke S.P. Clarkin K.C. Leonardo A.D. Tsou A. Wahl G.M. Genes Dev. 1996; 10: 934-947Crossref PubMed Scopus (480) Google Scholar). 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. 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 (43Crook T. Tidy J.A. Vousden K.H. Cell. 1991; 67: 547-556Abstract Full Text PDF PubMed Scopus (450) Google Scholar,44Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3456) Google Scholar). 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 and13), 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. 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. 23Sugano T. Nitta M. Ohmori H. Yamaizumi M. Jpn. J. Cancer Res. 1
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