Activation of Wild Type p53 Function by Its Mortalin-binding, Cytoplasmically Localizing Carboxyl Terminus Peptides

The Hsp70 family member mortalin (mot-2/mthsp70/GRP75) binds to a carboxyl terminus region of the tumor suppressor protein p53. By in vivo co-immunoprecipitation of mot-2 with p53 and its deletion mutants, we earlier mapped the mot-2-binding site of p53 to its carboxyl terminus 312-352 amino acid residues. In the present study we attempted to disrupt mot-2-p53 interactions by overexpression of short p53 carboxyl-terminal peptides. We report that p53 carboxyl-terminal peptides (amino acid residues 312-390, 312-352, 323-390, and 323-352) localize in the cytoplasm, whereas 312-322, 337-390, 337-352, and 352-390 locate mostly in the nucleus. Most interestingly, the cytoplasmically localizing p53 peptides harboring the residues 323-337 activated the endogenous p53 function by displacing it from p53-mortalin complexes and relocating it to the nucleus. Such activation of p53 function was sufficient to cause growth arrest of human osteosarcoma and breast carcinoma cells. The Hsp70 family member mortalin (mot-2/mthsp70/GRP75) binds to a carboxyl terminus region of the tumor suppressor protein p53. By in vivo co-immunoprecipitation of mot-2 with p53 and its deletion mutants, we earlier mapped the mot-2-binding site of p53 to its carboxyl terminus 312-352 amino acid residues. In the present study we attempted to disrupt mot-2-p53 interactions by overexpression of short p53 carboxyl-terminal peptides. We report that p53 carboxyl-terminal peptides (amino acid residues 312-390, 312-352, 323-390, and 323-352) localize in the cytoplasm, whereas 312-322, 337-390, 337-352, and 352-390 locate mostly in the nucleus. Most interestingly, the cytoplasmically localizing p53 peptides harboring the residues 323-337 activated the endogenous p53 function by displacing it from p53-mortalin complexes and relocating it to the nucleus. Such activation of p53 function was sufficient to cause growth arrest of human osteosarcoma and breast carcinoma cells. p53, the “guardian of the genome,” is a major player in cell cycle arrest and apoptosis in response to the diverse endogenous and exogenous stress signals (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6740) Google Scholar). Since its first identification three decades ago, it has been studied widely and has been recognized as a most frequently mutated gene in a variety of cancers (2Levine A.J. Wu M.C. Chang A. Silver A. Attiyeh E.F. Lin J. Epstein C.B. Ann. N. Y. Acad. Sci. 1995; 768: 111-128Crossref PubMed Scopus (98) Google Scholar, 3Lane D. Carcinogenesis. 2004; 25: 1077-1081Crossref PubMed Scopus (34) Google Scholar). Loss of p53 function is one of the early events in immortalization of human cells (4Kaul S.C. Wadhwa R. Sugihara T. Obuchi K. Komatsu Y. Mitsui Y. Biochim. Biophys. Acta. 1994; 1201: 389-396Crossref PubMed Scopus (15) Google Scholar, 5Duncan E.L. Wadhwa R. Kaul S.C. Biogerontology. 2000; 1: 103-121Crossref PubMed Scopus (34) Google Scholar). In addition, it confers resistance to radiation and chemotherapy of tumors (6Lee T.K. Man K. Poon R.T. Lo C.M. Ng I.O. Fan S.T. Oncol. Rep. 2004; 12: 25-31PubMed Google Scholar, 7Koivusalo R. Krausz E. Ruotsalainen P. Helenius H. Hietanen S. Cancer Res. 2002; 62: 7364-7371PubMed Google Scholar, 8Brown J.M. Wouters B.G. Cancer Res. 1999; 59: 1391-1399PubMed Google Scholar). Therefore, studies on the regulation of p53 function and its reactivation are extremely important for cancer therapeutics. p53 is synthesized in the cytoplasm and must translocate to the nucleus to exert its sequence-specific transcription factor function (9Vousden K.H. Woude G.F. Nat. Cell Biol. 2000; 2: E178-E180Crossref PubMed Scopus (70) Google Scholar). Another function of p53, independent to its nuclear localization, is to regulate mitochondrial membrane potential by interactions with the mitochondrial proteins Bcl2 and mortalin/mthsp70 (10Mihara M. Erster S. Zaika A. Petrenko O. Chittenden T. Pancoska P. Moll U.M. Mol. Cell. 2003; 11: 577-590Abstract Full Text Full Text PDF PubMed Scopus (1464) Google Scholar, 11Murphy M.E. Leu J.I. George D.L. Cell Cycle. 2004; 3: 836-839Crossref PubMed Scopus (61) Google Scholar, 12Perfettini J.L. Kroemer R.T. Kroemer G. Nat. Cell Biol. 2004; 6: 386-388Crossref PubMed Scopus (70) Google Scholar, 13Javelaud D. Besancon F. J. Biol. Chem. 2002; 277: 37949-37954Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 14Merrick B.A. He C. Witcher L.L. Patterson R.M. Reid J.J. Pence-Pawlowski P.M. Selkirk J.K. Biochim. Biophys. Acta. 1996; 13: 57-68Crossref Scopus (58) Google Scholar, 15Sugrue M.M. Tatton W.G. Biol. Signals Recept. 2001; 10: 176-188Crossref PubMed Scopus (52) Google Scholar, 16Dumont P. Leu J.I. Della Pietra III, A.C. George D.L. Murphy M. Nat. Genet. 2003; 33: 357-365Crossref PubMed Scopus (1113) Google Scholar, 17Leu J.I. Dumont P. Hafey M. Murphy M.E. George D.L. Nat. Cell Biol. 2004; 6: 443-450Crossref PubMed Scopus (650) Google Scholar). Nuclear import and export of p53 is regulated by three nuclear localization signals (NLSs) 2The abbreviations used are:NLSnuclear localization signalaaamino acidsGFPgreen fluorescent proteinYFPyellow fluorescent protein , two nuclear export signals, and its binding to nuclear and cytoplasmic binding partners (9Vousden K.H. Woude G.F. Nat. Cell Biol. 2000; 2: E178-E180Crossref PubMed Scopus (70) Google Scholar, 18Stommel J.M. Marchenko N.D. Jimenez G.S. Moll U.M. Hope T.J. Wahl G.M. EMBO J. 1999; 18: 1660-1672Crossref PubMed Scopus (604) Google Scholar, 19Zhang Y. Xiong Y. Cell Growth 12: 175-186PubMed Google Scholar). The tetrameric state of p53 in the nucleus masks its nuclear export signal (18Stommel J.M. Marchenko N.D. Jimenez G.S. Moll U.M. Hope T.J. Wahl G.M. EMBO J. 1999; 18: 1660-1672Crossref PubMed Scopus (604) Google Scholar) and, thus, favors its nuclear retention. Transcriptional activation of p53 in the nucleus leads to the expression of MDM2 (one of its downstream regulators and antagonists) that results in its degradation by one or both of the following mechanisms: (i) physical export of p53 to the cytoplasm and its subsequent degradation by proteasome pathway; (ii) ubiquitination of p53 in the nucleus, unmasking of the nuclear export signal that favors its export from the nucleus, and subsequent proteasome-mediated degradation (20Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3077-3080Crossref PubMed Scopus (297) Google Scholar, 21Haupt S. Louria-Hayon I. Haupt Y. J. Cell. Biochem. 2003; 88: 76-82Crossref PubMed Scopus (38) Google Scholar, 22O'Brate A. Giannakakou P. Drug Resist. Updat. 2003; 6: 313-322Crossref PubMed Scopus (181) Google Scholar). Other proteins that regulate p53 function include p14ARF, CARF, PML, Parc, mthsp70/mortalin, Bcl2, and cytoskeleton proteins (actin, vimentin, and microtubules) (20Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3077-3080Crossref PubMed Scopus (297) Google Scholar, 22O'Brate A. Giannakakou P. Drug Resist. Updat. 2003; 6: 313-322Crossref PubMed Scopus (181) Google Scholar, 23Stott F.J. Bates S. James M.C. McConnell B.B. Starborg M. Brookes S. Palmero I. Ryan K. Hara E. Vousden K.H. Peters G. EMBO J. 1998; 17: 5001-5014Crossref PubMed Scopus (1012) Google Scholar, 24Wadhwa R. Sugihara T. Taira K. Kaul S.C. Histol. Histopathol. 2004; 19: 311-316PubMed Google Scholar, 25Hasan K. Yaguchi T. Sugihara T. Kumar P.K. Taira K. Reddel R. Kaul S.C. Wadhwa R. J. Biol. Chem. 2002; 277: 37765-37770Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 26Nikolaev A.Y. Li M. Puskas N. Qin J. Gu W. Cell. 2003; 112: 29-40Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 27Kaul S.C. Taira K. Pereira-Smith O.M. Wadhwa R. Exp. Gerontol. 2002; 37: 1157-1164Crossref PubMed Scopus (85) Google Scholar). In some tumors, wild type p53 is rendered nonfunctional by its cytoplasmic sequestration, which is mediated by its enhanced nuclear export, binding to cytoplasmic proteins, and degradation (19Zhang Y. Xiong Y. Cell Growth 12: 175-186PubMed Google Scholar, 26Nikolaev A.Y. Li M. Puskas N. Qin J. Gu W. Cell. 2003; 112: 29-40Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 28Zhao L.Y. Liao D. J. Virol. 2003; 77: 13171-13181Crossref PubMed Scopus (42) Google Scholar, 29Moll U.M. LaQuaglia M. Benard J. Riou G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4407-4411Crossref PubMed Scopus (390) Google Scholar, 30Tweddle D.A. Pearson A.D. Haber M. Norris M.D. Xue C. Flemming C. Lunec J. Cancer Lett. 2003; 197: 93-98Crossref PubMed Scopus (140) Google Scholar, 31Wadhwa R. Yaguchi T. Hasan M.K. Mitsui Y. Reddel R.R. Kaul S.C. Exp. Cell Res. 2002; 274: 246-253Crossref PubMed Scopus (155) Google Scholar). Cytoplasmic confinement of p53 contributes to the rebelliousness of these tumors to radiotherapy and chemotherapy (29Moll U.M. LaQuaglia M. Benard J. Riou G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4407-4411Crossref PubMed Scopus (390) Google Scholar, 32Bosari S. Viale G. Roncalli M. Graziani D. Borsani G. Lee A.K. Coggi G. Am. J. Pathol. 1995; 147: 790-798PubMed Google Scholar, 33Sembritzki O. Hagel C. Lamszus K. Deppert W. Bohn W. Neurooncology. 2002; 4: 171-178Crossref PubMed Google Scholar). nuclear localization signal amino acids green fluorescent protein yellow fluorescent protein We had previously reported that mortalin causes cytoplasmic sequestration of p53 by binding to its carboxyl terminus amino acid residues 312-352 (31Wadhwa R. Yaguchi T. Hasan M.K. Mitsui Y. Reddel R.R. Kaul S.C. Exp. Cell Res. 2002; 274: 246-253Crossref PubMed Scopus (155) Google Scholar, 34Wadhwa R. Takano S. Robert M. Yoshida A. Reddel R.R. Nomura H. Mitsui Y. Kaul S.C. J. Biol. Chem. 1998; 273: 29586-29591Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 35Kaul S.C. Reddel R.R. Mitsui Y. Wadhwa R. Neoplasia. 2001; 3: 110-114Crossref PubMed Scopus (61) Google Scholar). In the present study, we report an activation of p53 function by overexpression of mortalin binding, cytoplasmically localizing the carboxyl terminus region peptides of p53. Plasmid Constructions—Full-length and deletion mutants of mouse p53 were obtained by PCR using p53-specific primers and cloned into the EYFPC1, EGFPC1 (Clontech), and pTOPO/V5 (Invitrogen) mammalian expression vectors. The integrity of the plasmids encoding various deletion mutant proteins was confirmed by sequencing. Proteins expressed in cells were visualized on a Carl Zeiss microscope and were also examined by Western blotting with GFP, V5, and mouse p53-specific antibodies. Cell Culture and Transfections—Human osteosarcoma (U2OS) or breast carcinoma (MCF7) cells were cultured in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal bovine serum. Transfections were performed using Lipofectamine™ (Invitrogen). Typically, 3 μg of plasmid was used per 6-cm dish. After 24-48 h of transfection, protein expression was visualized by Western blotting or immunostaining as described below. Cell Fractionation—Transfected cells were harvested and fractionated into nuclear and cytoplasmic fractions using a nuclear/cytosol fractionation kit from BioVision (Mountain View, CA). Protein (20 μg) from each fraction was resolved on a 12% SDS-polyacrylamide gel and Western-blotted with anti-YFP antibody for detection of YFP-tagged p53 proteins. Immunoprecipitation and Immunodepletion—Cell lysates (600 μg) were incubated with anti-p53 polyclonal antibody for immunoprecipitation of an endogenous p53 protein as described (31Wadhwa R. Yaguchi T. Hasan M.K. Mitsui Y. Reddel R.R. Kaul S.C. Exp. Cell Res. 2002; 274: 246-253Crossref PubMed Scopus (155) Google Scholar). p53 immunocomplexes were examined for the presence of mortalin by Western blotting with an anti-mortalin monoclonal antibody. Equal amounts of the lysate taken for immunoprecipitation were ensured by probing with an anti-actin antibody. For mortalin immunodepletion, lysates were immunoprecipitated with polyclonal anti-mortalin antibody for two rounds of precipitation. Supernatants were analyzed for endogenous p53 and YFP-p53 fragments by Western blotting with specific monoclonal anti-GFP and anti-p53 antibodies. Endogenous p53 was quantitated by image analysis software. Western Blotting—Cells transfected with various deletion mutants of mouse p53 were lysed with lysis buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, and protease inhibitors (Roche Applied Science Protease Inhibitor Cocktail). The protein concentration in cell lysate was determined by a standard dye binding assay (BioRad). 10 μg of total protein was resolved on a 12.5% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan), and probed with anti-GFP, anti-p53, anti-histone H1, or anti-mortalin antibodies. Immunocomplexes were observed with horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody (ECL kit, Amersham Biosciences). Immunostaining—Cells were fixed with ice cold methanol/acetone (1:1) for 5 min and stained for endogenous p53 with a human p53-specific antibody (p53-DO1; Santa Cruz Biotechnology) or mortalin with an anti-mortalin antibody (36Wadhwa R. Kaul S.C. Ikawa Y. Sugimoto Y. J. Biol. Chem. 1993; 268: 6615-6621Abstract Full Text PDF PubMed Google Scholar). Transfected YFP- or GFP-tagged mouse p53 proteins were visualized by their autofluorescence at the same time. The cells were examined on a Carl Zeiss microscope (Axiovert 200 m) in either conventional fluorescence or the ApoTome mode for high optical resolution attached to Photomerics Sensys and AxioCam MRm monochrome charge-coupled device cameras. The extent to which the two proteins overlapped was assessed by combining the two images using either Metamorph or AxioVison software. p53-dependent Reporter Assays—U2OS cells were stably transfected with the p53-responsive luciferase reporter plasmid PG-13luc (kindly provided by Dr. Bert Vogelstein (Howard Hughes Medical Institute)). Cells stably expressing a p53-dependent reporter were then transfected with expression plasmids encoding p53 deletion mutants. As a control, a pRL-TK vector (Promega) was co-transfected in each assay to correct for variations in transfection Cells were and luciferase was by using the reporter assay are the of three were by of a plasmid encoding was per μg of protein and as the of with cells taken as Cell Growth growth of cells was by Transfected cells were by of plasmid and subsequent in medium for cells were in a 6-cm and to for the with a of medium were fixed in stained with and of and YFP-tagged of plasmids encoding or YFP-tagged and various deletion mutants of p53 were by PCR by into the or vector Cells transfected with expression plasmids were fixed and visualized the microscope for the localization of the exogenous The of cells with and nuclear p53 were on the the were into three as (i) encoding nuclear p53, YFP-p53 protein and its carboxyl mutant (ii) cytoplasmic p53, YFP-p53 312-352, 323-390, and 323-337 and nuclear p53 with some localization in the cytoplasm, 312-322, 337-390, 337-352, and 352-390 the following (i) The carboxyl terminus of p53 is for its cytoplasmic (ii) acid residues 323-337 are for its cytoplasmic In the of 323-337 the carboxyl mutants locate to the nucleus We also confirmed the presence of YFP-tagged p53 mutants in the cytoplasm or the nucleus by cell fractionation and Western The nuclear and cytoplasmic fractions were probed with for proteins for the cytoplasmic fraction and for nuclear to the cell YFP-p53 was two mutants and 312-352 were cytoplasmic and one mutant was nuclear with a in the cytoplasmic fraction with the p53 amino acid residues 323-337 to for its cytoplasmic with we had reported earlier (31Wadhwa R. Yaguchi T. Hasan M.K. Mitsui Y. Reddel R.R. Kaul S.C. Exp. Cell Res. 2002; 274: 246-253Crossref PubMed Scopus (155) Google Scholar) that p53 amino acid residues 312-352, to mortalin in immunoprecipitation performed on cell lysates of the amino acid residues as 312-322, 337-390, and also to mortalin in immunoprecipitation (31Wadhwa R. Yaguchi T. Hasan M.K. Mitsui Y. Reddel R.R. Kaul S.C. Exp. Cell Res. 2002; 274: 246-253Crossref PubMed Scopus (155) Google in vivo of their nuclear Nuclear and of earlier (31Wadhwa R. Yaguchi T. Hasan M.K. Mitsui Y. Reddel R.R. Kaul S.C. Exp. Cell Res. 2002; 274: 246-253Crossref PubMed Scopus (155) Google Scholar, 34Wadhwa R. Takano S. Robert M. Yoshida A. Reddel R.R. Nomura H. Mitsui Y. Kaul S.C. J. Biol. Chem. 1998; 273: 29586-29591Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 35Kaul S.C. Reddel R.R. Mitsui Y. Wadhwa R. Neoplasia. 2001; 3: 110-114Crossref PubMed Scopus (61) Google Scholar) that mortalin with p53 and it in the We p53 peptides with the endogenous p53 and its cytoplasmic sequestration from the in nuclear of p53 Such nuclear also to its and enhanced activation As we that the cells transfected with p53 fragments 312-352, and of endogenous p53. by or to p53 337-390, or was and of p53 to mortalin was examined by Cell lysates with anti-mortalin antibody were examined for the presence of YFP-p53 and endogenous p53. As in lysates for mortalin a in in or with the of endogenous p53 was by in two rounds of mortalin immunoprecipitation of immunoprecipitation is in each in cells transfected with or the of endogenous p53 in cells expressing amino acid residues The the that the p53 with the endogenous p53, in its We then the p53 by p53-dependent reporter in cells stably transfected with luciferase reporter with and the obtained as described cells expressing p53 fragments harboring amino acid residues 323-337 activation of p53 The p53 was We also examined the of overexpression of mortalin in these The cells expressing each of the two peptides that region and a of mortalin in p53 immunocomplexes as with an vector or the 352-390 of cells and 3 with and in and Most in cells the expression of p53 fragments and cause a in mortalin in p53 The that the p53 peptides disrupt interactions by the endogenous p53 from complexes as in and cause and activation of p53 function We to the nuclear of endogenous p53 by the transfected protein was the p53, it was to the exogenous and endogenous proteins by the of a specific anti-p53 antibody that to the human Transfected p53 deletion mutants to the p53 antibody We transfected the cells with YFP-tagged p53 deletion mutants and examined the localization of endogenous p53 by anti-p53 antibody were also taken subsequent to the transfection of 312-352 and 352-390 fragments We that of the cells transfected with p53 deletion mutants and in the nuclear for endogenous p53 In cells transfected with vector or the deletion mutants that locate in the nucleus 337-390, and 352-390 nuclear for p53 in of the cells and also p53 in a fraction of cells. Most in p53 to the cell cycle the of cells endogenous p53 was 10% transfected with localizing p53 peptides as with in the of cells transfected with peptides localizing in the confirmed that the p53 cause the of endogenous p53 to the nucleus, whereas the was We also performed Western analysis on nuclear fractions of p53 The confirmed that the p53 peptides as 312-352 and in an in nuclear p53 to the in was 312-352 and in its these that the p53 peptides as and that with mortalin and and with endogenous p53, binding, and in of the endogenous p53 protein to the nucleus as was in results were obtained in both and cells. The that the and activation of p53 function by p53 peptides nuclear of endogenous p53. analysis of mortalin to the family of it of the three and present in three of Therefore, it is that the cytoplasmic of p53 by mortalin its degradation by the proteasome degradation of interactions in the cytoplasm by as of endogenous p53 to the nucleus and it the degradation in the cytoplasm as by an in the of endogenous p53 and its and results were obtained cells were transfected with MDM2 expression by the a from Dr. of with the p53 peptides as and 323-337 that the nuclear of p53 by these p53 peptides and was in it from degradation in the nuclear of endogenous p53 by the 312-352 was by overexpression of it is that the 312-352 is a or 323-337 in the endogenous p53 to the nucleus and the in a of endogenous p53 in cells transfected with with 312-352 or these results the following two (i) The p53 residues 312-322, which localize in the nucleus present with cytoplasmically localizing residues as disrupt their as a to (ii) The p53 activation of endogenous p53 in study by the MDM2 at in of p53 the activation of endogenous wild type p53 the growth of cells. and cells transfected with cytoplasmically localizing p53 peptides growth as with the cells. peptides 312-352 and 323-337 were were also performed in cells. Cells were transfected with p53 deletion mutants and by of a resistance The cells were to whereas p53 peptides and and p53 peptides 337-352, and had a on the of cells. these that the cytoplasmically localizing deletion mutants of p53 endogenous p53 from in its nuclear and activation of growth arrest of p53 is a of tumors P. T. M. M. E. R. Res. 1997; 25: PubMed Scopus Google Scholar, M. T. G. M.C. H. Cancer Res. 1997; PubMed Scopus Google and the include the following three (i) of p53 that its binding or activation (ii) expression of proteins, that in degradation of the wild type or of mutant and nuclear of wild type p53 A. Giannakakou P. Drug Resist. Updat. 2003; 6: 313-322Crossref PubMed Scopus (181) Google Scholar). the first two been in the the The of p53 include an amino terminus a sequence-specific a carboxyl terminus and a J. Natl. Cancer 1996; 88: PubMed Scopus Google Scholar). The localization of p53 has been to determined by nuclear localization signals, amino acid residues as and nuclear export signals (18Stommel J.M. Marchenko N.D. Jimenez G.S. Moll U.M. Hope T.J. Wahl G.M. EMBO J. 1999; 18: 1660-1672Crossref PubMed Scopus (604) Google Scholar, G. K. S. A. M. U. Peters R. 1997; PubMed Scopus Google and its interactions with proteins MDM2 and some of the Hsp70 family (18Stommel J.M. Marchenko N.D. Jimenez G.S. Moll U.M. Hope T.J. Wahl G.M. EMBO J. 1999; 18: 1660-1672Crossref PubMed Scopus (604) Google Scholar, W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3077-3080Crossref PubMed Scopus (297) Google Scholar, 31Wadhwa R. Yaguchi T. Hasan M.K. Mitsui Y. Reddel R.R. Kaul S.C. Exp. Cell Res. 2002; 274: 246-253Crossref PubMed Scopus (155) Google Scholar, R. Sugihara T. Yoshida Nomura H. Reddel R.R. R. H. Kaul S.C. Cancer Res. 2000; Google Scholar). are three nuclear signals and III, at the carboxyl terminus of p53. As the deletion mutants that in the nucleus the amino acid residues 323-337 by or with terminus and in the the deletion mutants amino acid residues and in the nucleus. In vivo activation of p53 by of MDM2 that the interactions of p53 and MDM2 was D. F. N. U. C. C. N. 2004; PubMed Scopus Google Scholar). with an for p53 enhanced resistance to tumors and early of S. J. S. N. H. G. C. S. T. G. A. 2002; PubMed Scopus Google for the of wild type p53 function by carboxyl p53. We reported that the overexpression of cytoplasmically localizing carboxyl-terminal p53 peptides harboring amino acid residues 323-337 in nuclear and activation of wild type p53. for of tumors with wild type p53.