CP-31398 Restores DNA-binding Activity to Mutant p53 in Vitro but Does Not Affect p53 Homologs p63 and p73
2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês
10.1074/jbc.m401854200
ISSN1083-351X
AutoresMark J. Demma, Serena Wong, Eugene Maxwell, Bimalendu Dasmahapatra,
Tópico(s)DNA Repair Mechanisms
ResumoThe p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effective in vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elements in vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kd value) of wild type p53 for DNA and only increases Bmax slightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73. The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effective in vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elements in vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kd value) of wild type p53 for DNA and only increases Bmax slightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73. The p53 tumor suppressor protein belongs to a superfamily of transcription factors that includes its homologs p63 and p73. p53 is involved in a wide range of cellular activities that help ensure the stability of the genome, whereas p63 and p73 are involved in ectodermal morphogenesis, limb morphogenesis, neurogenesis, and homeostatic control and are not considered tumor suppressor genes (1Bernard J. Douc-Rasy S. Ahomadegbe J.-C. Human Mutat. 2003; 2: 182-191Crossref Scopus (192) Google Scholar). p53 is involved in DNA damage repair, cell cycle arrest, and apoptosis via transcriptional regulation of genes involved in these activities or by direct interaction with other proteins (2Lane D.P. Hupp T.R. Drug Discov. Today. 2003; 8: 347-355Crossref PubMed Scopus (77) Google Scholar, 3Vousden K. Cell. 2000; 103: 691-694Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 4Willis A.C. Chen X. Curr. Mol. Med. 2002; 2: 329-345Crossref PubMed Scopus (45) Google Scholar). Mutations that inactivate p53 are present in over 50% of all cancers and are indicative of aggressive cancers that are difficult to treat by chemotherapy or ionizing radiation (2Lane D.P. Hupp T.R. Drug Discov. Today. 2003; 8: 347-355Crossref PubMed Scopus (77) Google Scholar, 5Bullock A.N. Fersht A.R. Nat. Rev. Cancer. 2001; 1: 68-76Crossref PubMed Scopus (487) Google Scholar). The majority of inactivating mutations reside in the central core DNA binding domain (DBD) 1The abbreviations used are: DBD, DNA binding domain; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; 6FAM, 6-carboxy fluorescein.1The abbreviations used are: DBD, DNA binding domain; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; 6FAM, 6-carboxy fluorescein. of p53 (2Lane D.P. Hupp T.R. Drug Discov. Today. 2003; 8: 347-355Crossref PubMed Scopus (77) Google Scholar, 5Bullock A.N. Fersht A.R. Nat. Rev. Cancer. 2001; 1: 68-76Crossref PubMed Scopus (487) Google Scholar). These mutations can be divided into two main classes, DNA contact mutants, like R273H, where the mutation alters a residue involved in contact with DNA, and structural mutants, like R249S, which result in structural changes in the p53 core domain (6Wong K.-B. DeDecker B.S. Freund S.V. Proctor M.R. Bycroft M. Fersht A.R. Proc. Nat. Acad. Sci U. S. A. 1999; 96: 8348-8442Crossref PubMed Scopus (73) Google Scholar, 7Bullock A.R. Henckel J. DeDecker B.S. Johnson C.M. Nikolova P.V. Proctor M.R. Lane D.P. Fersht A.R. Proc. Nat. Acad. Sci. U. S. A. 1997; 94: 14338-14343Crossref PubMed Scopus (349) Google Scholar, 8Bullock A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (325) Google Scholar). These mutations affect the function of p53 by distorting the structure and reducing the thermal stability of the protein (6Wong K.-B. DeDecker B.S. Freund S.V. Proctor M.R. Bycroft M. Fersht A.R. Proc. Nat. Acad. Sci U. S. A. 1999; 96: 8348-8442Crossref PubMed Scopus (73) Google Scholar, 7Bullock A.R. Henckel J. DeDecker B.S. Johnson C.M. Nikolova P.V. Proctor M.R. Lane D.P. Fersht A.R. Proc. Nat. Acad. Sci. U. S. A. 1997; 94: 14338-14343Crossref PubMed Scopus (349) Google Scholar, 8Bullock A.N. Henckel J. Fersht A.R. Oncogene. 2000; 19: 1245-1256Crossref PubMed Scopus (325) Google Scholar). This can alter the ability of p53 to bind to various p53 response elements in a variety of genes, hampering its transcriptional regulation (9Nicholls C.D. McLure K.G. Shields M.A. Lee P.W.K. J. Biol. Chem. 2002; 277: 12937-12945Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In addition, these mutations may alter p53 structure, so that p53 can no longer induce apoptosis by binding to BclXL, thereby inhibiting its anti-apoptotic function (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 (1458) Google Scholar). One potential therapeutic approach to cancer would be restoration of growth suppression activity to mutant p53. Several approaches have been tried, ranging from micro-injection of monoclonal antibody 421, C-terminal peptide of p53 and small molecules (11Abarzua P. LoSardo J.E. Gubler M.L. Spathis R. Lu Y.-A. Felix A. Neri A. Oncogene. 1996; 13: 2477-2482PubMed Google Scholar, 12Abarzua P. LoSardo J.E. Gubler M.L. Neri A. Cancer Res. 1995; 55: 3490-3494PubMed Google Scholar, 13Halazonetis T.D. Davis L.J. Kandil A.N. EMBO J. 1993; 12: 1021-1028Crossref PubMed Scopus (182) Google Scholar, 14Wieczorek A.M. Waterman J.L.F. Waterman J.F. Halazonetis T.D. Nat. Med. 1996; 2: 1143-1146Crossref PubMed Scopus (83) Google Scholar, 15Selinova G. Ryabchenko L. Jansson E. Iotsova V. Wiman K.G. Mol. Cell Biol. 1999; 19: 3395-3402Crossref PubMed Scopus (129) Google Scholar, 16Peng Y. Li C. Sebti S. Chen J. Oncogene. 2003; 22: 4478-4487Crossref PubMed Scopus (123) Google Scholar). Recently, small molecules and peptides, such as CP-31398, PRIMA1, and CDB3 peptide, have been shown to be effective in restoring p53 function (17Foster B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (688) Google Scholar, 18Bykov V.J.N. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selinova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (881) Google Scholar, 19Friedler A. Hansson L.O. Veprintsev D.B. Freund S.M.V. Rippin T.M. Nikolova P.V. Proctor M.R. Rudiger S. Fersht A.R. Proc. Nat. Acad. Sci. U. S. A. 2002; 99: 937-942Crossref PubMed Scopus (233) Google Scholar, 20Freidler A. Verprintsev D.B. Hansson L. Fersht A.R. J. Biol. Chem. 2003; 278: 24108-24112Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 21Issaeva N. Friedler A. Bozko P. Wiman K.G. Fersht A.R. Selivanova G. Proc. Nat. Acad. Sci. U. S. A. 2003; 100: 13303-13307Crossref PubMed Scopus (130) Google Scholar, 22Luu Y. Bush J. Cheung Jr., K.-J. Li G. Exp. Cell Res. 2002; 276: 214-222Crossref PubMed Scopus (72) Google Scholar, 23Wang W. Takimoto R. Ratinejad F. El-Diery W. Mol. Cell Biol. 2003; 23: 2171-2181Crossref PubMed Scopus (127) Google Scholar, 24Wischhusen J. Naumann U. Ohgaki H. Rastinejad F. Weller M. Oncogene. 2003; 22: 8233-8245Crossref PubMed Scopus (127) Google Scholar, 25Takimoto R. Wang W. Dicker D.T. Rastinejad F. Lyssikatos J. El-Diery W.S. Cancer Biol. Ther. 2002; 1: 47-55Crossref PubMed Scopus (92) Google Scholar). Both PRIMA1 and CDB3 have been shown to restore p53 DNA-binding activity in vitro (18Bykov V.J.N. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selinova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (881) Google Scholar, 19Friedler A. Hansson L.O. Veprintsev D.B. Freund S.M.V. Rippin T.M. Nikolova P.V. Proctor M.R. Rudiger S. Fersht A.R. Proc. Nat. Acad. Sci. U. S. A. 2002; 99: 937-942Crossref PubMed Scopus (233) Google Scholar, 20Freidler A. Verprintsev D.B. Hansson L. Fersht A.R. J. Biol. Chem. 2003; 278: 24108-24112Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 21Issaeva N. Friedler A. Bozko P. Wiman K.G. Fersht A.R. Selivanova G. Proc. Nat. Acad. Sci. U. S. A. 2003; 100: 13303-13307Crossref PubMed Scopus (130) Google Scholar), whereas the effects for CP-31398 have been shown primarily in cell-based assays (17Foster B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (688) Google Scholar, 22Luu Y. Bush J. Cheung Jr., K.-J. Li G. Exp. Cell Res. 2002; 276: 214-222Crossref PubMed Scopus (72) Google Scholar, 23Wang W. Takimoto R. Ratinejad F. El-Diery W. Mol. Cell Biol. 2003; 23: 2171-2181Crossref PubMed Scopus (127) Google Scholar, 24Wischhusen J. Naumann U. Ohgaki H. Rastinejad F. Weller M. Oncogene. 2003; 22: 8233-8245Crossref PubMed Scopus (127) Google Scholar, 25Takimoto R. Wang W. Dicker D.T. Rastinejad F. Lyssikatos J. El-Diery W.S. Cancer Biol. Ther. 2002; 1: 47-55Crossref PubMed Scopus (92) Google Scholar). Both CP-31398 and PRIMA1 have been shown to reduce tumor size in animal models (17Foster B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (688) Google Scholar, 18Bykov V.J.N. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selinova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (881) Google Scholar). It is postulated that the two molecules perform similar tasks, but by different mechanisms. PRIMA1 has been suggested to work more broadly to restore p53 DNA-binding activity, but the specific mechanism is not known (18Bykov V.J.N. Issaeva N. Shilov A. Hultcrantz M. Pugacheva E. Chumakov P. Bergman J. Wiman K.G. Selinova G. Nat. Med. 2002; 8: 282-288Crossref PubMed Scopus (881) Google Scholar). CP-31398, on the other hand, has been suggested to stabilize p53 as a protectant against thermal denaturation and maintain monoclonal antibody 1620 epitope conformation in newly synthesized p53 (17Foster B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (688) Google Scholar). Recently, CP-31398 has also been shown to stabilize wild type p53 in cells by inhibiting Mdm2-mediated ubiquitination and degradation (23Wang W. Takimoto R. Ratinejad F. El-Diery W. Mol. Cell Biol. 2003; 23: 2171-2181Crossref PubMed Scopus (127) Google Scholar). Reports from other studies suggest that CP-31398 interacts with DNA and not with p53 in vitro, and it is proposed to act as a DNA-damaging agent (26Rippin T.M. Bykov V.J.N. Freund S.M.V. Selivanova G. Wiman K.G. Fersht A.R. Oncogene. 2002; 21: 2119-2129Crossref PubMed Scopus (146) Google Scholar). Although it has been shown that the effects of CP-31398 on p53 do not depend on the DNA damage pathway (23Wang W. Takimoto R. Ratinejad F. El-Diery W. Mol. Cell Biol. 2003; 23: 2171-2181Crossref PubMed Scopus (127) Google Scholar), its p53-independent cellular growth suppression effects at higher concentrations and lack of demonstrated binding to p53 protein raise confusion about its mechanism of action (24Wischhusen J. Naumann U. Ohgaki H. Rastinejad F. Weller M. Oncogene. 2003; 22: 8233-8245Crossref PubMed Scopus (127) Google Scholar, 25Takimoto R. Wang W. Dicker D.T. Rastinejad F. Lyssikatos J. El-Diery W.S. Cancer Biol. Ther. 2002; 1: 47-55Crossref PubMed Scopus (92) Google Scholar, 26Rippin T.M. Bykov V.J.N. Freund S.M.V. Selivanova G. Wiman K.G. Fersht A.R. Oncogene. 2002; 21: 2119-2129Crossref PubMed Scopus (146) Google Scholar). Here we demonstrate that CP-31398 can promote mutant p53 to bind to p53 response elements in vivo using a chromatin immunoprecipitation (ChIP) assay. In addition, using purified p53 core domain, we clearly demonstrate that CP-31398 can restore DNA-binding activity to mutant p53 in vitro. We also show that CP-31398 does not have any effects on the DNA-binding activity of the p53 homologs, p63 and p73. In contrast, PRIMA1 was ineffective in restoring DNA-binding activity to isolated mutant p53 DNA binding domain. Cell Culture—WiDr cells, which contain an R273H hotspot mutation, were grown in Dulbecco's modified Eagle's medium from Invitrogen, supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. H1299 cells were grown in RPMI medium from Invitrogen, supplemented with 10% FBS and penicillin and streptomycin. SkBr3 (mt.p53) cells were grown in McCoy's medium from Invitrogen, supplemented with 10% FBS and penicillin/streptomycin. Cell Transfection—WiDr cells were transfected with either pSuper or pSuper-p53 (gift of Dr. J. Wischhusen) (24Wischhusen J. Naumann U. Ohgaki H. Rastinejad F. Weller M. Oncogene. 2003; 22: 8233-8245Crossref PubMed Scopus (127) Google Scholar) by electroporation using a Bio-Rad GenePulser. Flow Cytometry Analysis—H1299 (p53-null) and SkBr3 cells were treated for 24 h with either 0 or 2.5 μg/ml CP-31398. After 24 h, the cells were trypsinized, fixed in 70% methanol, and then washed in phosphate-buffered saline. The fixed cells were stained with 50 μg/ml propidium iodide and analyzed on a BD Biosciences FACS Vantage instrument. Cell Lysis and Western Blotting—Cell lysis of WiDr cells was carried out using the method of Foster et al. (17Foster B.A. Coffey H.A. Morin M.J. Rastinejad F. Science. 1999; 286: 2507-2510Crossref PubMed Scopus (688) Google Scholar). 40 mg of cell lysate was run either on a 10% polyacrylamide gel (for p53 and actin) or a 4–20% polyacrylamide gradient gel (p21 and bax). The gels were transferred to nitrocellulose paper, and probed with antibodies to p53 (DO1), actin (Sigma), Bax (Santa Cruz Biotechnology), or p21 (Calbiochem). Chromatin Immunoprecipitation Assay—WiDr cells were plated at 107 cells/ml and treated for 24 h with either 1% Me2SO (untreated) or 10 μg/ml CP-31398 in 1% Me2SO final. The ChIP assay was performed as described by Frank et al. (27Frank S.R. Schroeder M. Fernandez P. Taubert S. Amati B. Genes Dev. 2001; 15: 2069-2082Crossref PubMed Scopus (420) Google Scholar). Immunoprecipitation was performed in the presence or absence of p53 antibody DO1 (Calbiochem). PCR was performed with 10 μl of DNA, 800 nm primers, and fluorescent probe (28Dignam J.D. Lebowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1474-1489Crossref Scopus (9153) Google Scholar) diluted in a final volume of 30 μl. The accumulations of fluorescence products were monitored on an ABI Prism 7700 sequence detector. The primer/probe pairs used were as follows. Bax: forward, 5′-TCCCCCCGAGAGGTCTTTT-3′; reverse, 5′-CGGCCCCAGTTGAAGTTG-3′; probe (6FAM): 5′-TCAGAAAACATGTCAGCTGCCACTCGG-3′; p21: forward, 5′-TGGAGACTCTCAGGGTCGAAA-3′; reverse, 5′-GGCGTTTGGAGTGGTAGAAATC-3′; probe (6FAM): 5′-CGGCGGCAGACCAGCATGAC-3′; S9: forward, 5′-GGCTCCGGAACAAACGTG-3′; reverse, 5′-GCGGCCTTGCGGATCT-3′; and probe (6FAM) 5′-TCTGGAGGGTCAAATTTACCCTGGCC-3′. Cloning, Expression, and Purification—The DNA binding domain of p53 (aa92–312) was generated via PCR from plasmid pCTK53 (Isabella Atencio, Canji Inc.) using gene-specific primers 5′-ATGGATCCATGTCATCTTCTGTCCCTTCC-3′ and 5′-TAAAGCTTTCAGGTGTTGTTGGGCAGTGC-3′. The resulting product was cloned into the BamHI/HindIII site of plasmid pET42a (Novagen), to generate a recombinant GST-5X His fusion protein (GST-His p53DBD wt). A mutant p53R249S was generated by the same method from pCTMR249S and called pGST-His p53DBDR249S. The p53 mutant, R273H was generated by mutagenesis of pCTK53 by polymerase chain reaction. The subsequent mutant was then used to generate the mutated DNA binding domain by the same procedure as for the wild type DNA binding domain. The resulting PCR product was also cloned into BamHI/HindIII site in pET42a to generate the recombinant GST-5X His fusion protein (GST-His p53DBDR273H). The DNA binding domains of p63 and p73 were generated by polymerase chain reaction from plasmids pCUB 412 (p63) and pCUB370 (p73) (gifts of Dr. Charles DiComo) using gene-specific primers 5′-CATGCCATGGCAAGCTCCACCTTCGATGCTCTC-3′ and 5′-CCCAAGCTTTCATGTCATCTGGATACCATGTC-3′ for p63 and 5′-CATGCCATGGCAAGCTCCACCTTCGACACCATGTC-3′ and 5′-CCCAAGCTTACTCTTCTTCACACCGGCACC-3′ for p73. The resulting PCR products were cloned similarly into pET42a and expressed as a GST-5X His fusion protein, GST-His p63DBD, and GST-His p73DBD. The various plasmids were transformed into BL21(DE3) bacteria for expression. p53 containing plasmid-transformed bacteria were initially grown at 30 °C until they reached an A600 of 0.8–1.2 and then were cooled to 18 °C. After 1 h at low temperature, they were induced with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside (Sigma) and grown overnight at 18 °C. p63 and p73, containing plasmid-transformed bacteria, were initially grown at 37 °C, and then shifted to 28 °C and induced with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside. The cultures were grown overnight at 28 °C. Bacteria were harvested by centrifugation at 10,000 × g for 10 min and then resuspended in buffer containing 20 mm Hepes (pH 7.5), 100 mm NaCl, 1 mm β-mercaptoethanol, and protease inhibitor tablets (Roche Applied Science). After resuspension, Triton X-100 (Sigma) was added to 1% in volume, and the bacterial paste was lysed by sonication (Kontes). The lysate was rotated at 4 °C for 30 min and then centrifuged at 15,000 × g for 1 h. The lysate was filtered with a Whatman Puradisc filter and applied to a 20-ml Q-Sepharose Fast Flow (AP Biotech) column, equilibrated with 20 mm Hepes, pH 7.5, 100 mm NaCl, and 10 μm ZnCl2. The flow-through and the first column wash were collected and applied to an HR16/10 glutathione-Sepharose column (AP Biotech) equilibrated in the above buffer. The protein was eluted with a single step of 40 mm glutathione in 50 mm Hepes, pH 8.3, 100 mm NaCl, and 10 μm ZnCl2. The eluted peak was then applied to a Mono S HR5/5 column (AP Biotech) equilibrated in 20 mm Hepes, pH 7.9, 100 mm NaCl, 10 μm ZnCl2, and 10 mm β-mercaptoethanol. The protein was eluted with a salt gradient from 0.1 to 1 m NaCl in the same buffer. The material was then pooled and frozen at –80 °C for later use in assays. Nuclear Extract Preparation—Nuclear extracts of WiDr cells were prepared according to a published method (28Dignam J.D. Lebowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1474-1489Crossref Scopus (9153) Google Scholar). Cells were grown to 80–90% confluence on 100-cm2 plates and treated with either 10 mg/ml CP-31398, in a final concentration of 1% Me2SO, or 1% Me2SO for 24 h. The cells were then washed with 10 ml of cold phosphate-buffered saline and scraped off the plate in 10 ml of phosphate-buffered saline, and centrifuged at 500 × g at 4 °C for 5 min. The volume of the packed pellet was estimated and was added to four volumes of Nonidet P-40 lysis buffer (50 mm Tris-HCl, 10 mm NaCl, 5 mm MgCl2, 0.5% Nonidet P-40, with protease inhibitor tablet) while the pellet was being vortexed. The resuspended pellet was left on ice for 5 min, followed by centrifugation at 500 × g for 5 min. The supernatant was discarded, and the pellet was resuspended in four volumes of Nonidet P-40 lysis buffer and re-centrifuged. The supernatant was discarded, and the packed nuclei volume was estimated. Three volumes of nuclear extraction buffer (20 mm Hepes, pH 7.9, 0.5 m NaCl, 1 mm EDTA, 20% glycerol, 1mm dithiothreitol, and protease inhibitor) were added while vortexing. The extract was left at 4 °C for 30 min and then centrifuged at 25, 000 × g for 30 min. The supernatant was collected and stored at –80 °C until use. Electrophoretic Mobility Shift Assay—The EMSA (gel shift) assay was done with both nuclear extracts and recombinant proteins. The protein was added to a mixture containing 20 mm Hepes, pH 7.9, 1 mm MgCl2, 10 μm ZnCl2, 5% glycerol, 1 μg/ml bovine serum albumin, 200 ng of deoxyinosine/deoxycytodine, 1 mm dithiothreitol, and 1 mm spermidine in the presence or absence of CP-31398 and incubated for 15 min at room temperature. A 25-mer double-stranded oligonucleotide containing the p53 consensus DNA binding sequence (5′-AGCTGGACATGCCCGGGCATGTCC-3′) and end labeled with either 32P or 33P was added into the reaction, and the mixture was incubated for an additional 15 min. The mixture was loaded onto a 6% polyacrylamide gel in TBE buffer (1× TBE is 100 mm Tris, 90 mm boric acid, and 1 mm EDTA, pH 8.4) and run at 150 V in 0.5× TBE (Invitrogen). The gel was dried and exposed on a Fuji Fluorescent Image Analyzer with the image being quantitated with the phosphorimaging software. DNA Binding Assay—To quantitate binding of p53 to either p53 consensus sequence or bax promoter sequence (5′-AGCACAAGCCTGGGCGTGGGC-3′), biotinylated double-stranded oligonucleotides were synthesized (Research Genetics and IDT, respectively). First, protein and compound mixtures were incubated in reaction buffer (20 mm Hepes, pH 7.9, 1 mm MgCl2, 10 μm ZnCl2, 1 μg/ml bovine serum albumin, 200 ng of deoxyinosine/deoxycytodine, 1 mm dithiothreitol, and 0.1% Nonidet P-40) for 10 min at room temperature in a volume of 45 μl. Then, 5 μl of DNA was added to the mixture for a final volume of 50 μl, and the mixture incubated for an additional 10 min. Then 167 fmol of primary antibody (Anti-Penta His monoclonal antibody, Qiagen) and 12.5 μg of streptavidin magnetic beads (Dynal) in antibody buffer (20 mm Hepes, pH 7.9, 1 mm MgCl2, 10 μm ZnCl2, 0.5 mm EDTA, 0.1% Nonidet P-40) were added, and the mixture was shaken for 1 h at room temperature. At 1 h, 333 fmol of secondary goat anti-mouse antibody (Jackson Laboratories) labeled with Ruthenium oxide (IGEN) was added according to the manufacturer's instructions, and the mixture was incubated with shaking for an additional hour. At the end of the incubation, 75 μl of antibody buffer was added to bring the final volume to 200 μl. The reaction mixture was then assayed on either an M8 analyzer or M384 analyzer workstation (IGEN) using IGEN read and stop buffer. Data were analyzed using Prism 3.0 software from Graph-Pad Software. CP-31398 Restores p53 Activity to Mutant p53 in Vivo—To evaluate the effects of CP-31398 in restoring wild type p53 function to mutant p53, we tested to see if the compound could restore p53-mediated apoptosis to cells harboring mutant p53. We treated both H1299 cells, which are p53-null cells, and SkBr3 tumor cells, which have the p53 mutation R175H, with or without 2.5 μg/ml CP-31398 and assayed for apoptosis via fluorescence-activated cell sorting analysis (Fig. 1A). Apoptosis is only seen in the SkBr3 cells that are treated with 2.5 μg/ml CP-31398. The H1299 cells show no apoptosis effect when treated with CP-31398 and are similar to the untreated H1299 cells. Previous reports (22Luu Y. Bush J. Cheung Jr., K.-J. Li G. Exp. Cell Res. 2002; 276: 214-222Crossref PubMed Scopus (72) Google Scholar, 23Wang W. Takimoto R. Ratinejad F. El-Diery W. Mol. Cell Biol. 2003; 23: 2171-2181Crossref PubMed Scopus (127) Google Scholar, 24Wischhusen J. Naumann U. Ohgaki H. Rastinejad F. Weller M. Oncogene. 2003; 22: 8233-8245Crossref PubMed Scopus (127) Google Scholar, 25Takimoto R. Wang W. Dicker D.T. Rastinejad F. Lyssikatos J. El-Diery W.S. Cancer Biol. Ther. 2002; 1: 47-55Crossref PubMed Scopus (92) Google Scholar) have shown that CP-31398 can induce p53-mediated apoptosis in mutant p53 tumor cells and that it has maximal effectiveness at 10 μg/ml, which is the concentration that we used in subsequent experiments. Because the effect of CP-31398 seems to be dependent upon the presence of p53, we treated WiDr cells, a colon tumor cell line that harbors an R273H hotspot mutation, with either 0 or 10 μg/ml CP-31398 for 24 h, and then made a nuclear extract, which we then used in an electrophoretic mobility shift assay (EMSA) using a consensus p53 DNA binding site (Fig. 1B) to see if CP-31398 could restore sequence-specific binding to mutant p53. Treatment with CP-31398 causes a shift of labeled probe that can be competed away with unlabeled p53 consensus oligonucleotide, in the CP-31398-treated nuclear extract, whereas no such band occurs in the untreated extract, indicating that the compound was capable of inducing mutant p53 to bind p53-specific DNA. To further demonstrate that the effect of CP-31398 was specific for p53-responsive genes, we treated WiDr cells with 0 or 10 μg/ml CP-31398 for 24 h, then made a cell lysate, and Western blotted for p53, bax, p21, and actin. Treatment with CP-31398 did not alter the level of either p53 or actin, but it significantly increased the level of both p21 and bax (Fig. 1C). In addition, we transiently transfected WiDr cells either with pSuper-p53, which encodes p53 SiRNA, or with the control empty vector, pSuper, then treated transfected cells with either 0 or 10 μg/ml CP-31398 for 24 h, made a cell lysate, and Western blotted for p53, actin, p21, and bax. The cells transfected with pSuper behaved similarly to the untransfected WiDr cells (Fig. 1C). The level of p53 and actin were unaltered, but there was a significant increase in the levels of both p21 and bax in the presence of CP-31398. With the pSuper-p53-transfected cells, actin levels were unaffected, but p53 levels were significantly decreased with or without CP-31398 (Fig. 1C). In addition, there was no induction of p21 or bax in the pSuper-p53-transfected cells, indicating that the effects of CP-31398 were dependent on the presence of p53. To prove that the induction of bax and p21 was due to promoter binding of mutant p53 reactivated by CP-31398 in vivo, we performed a chromatin immunoprecipitation (ChIP) assay in WiDr cells treated with either 0 or 10 μg/ml CP-31398 for 24 h (Fig. 1D). Treatment with CP-31398 specifically increased the amount of both p21 and bax promoter DNA that was immunoprecipitated with p53-specific antibody DO1, whereas there was no increase in the amount of ribosomal protein S9 promoter DNA, whose promoter does not have a p53 response element. This indicates that the effect of CP-31398 is specific for p53 and promotes the reactivation of full-length mutant p53 into functional “wild type” p53, which is fully capable of binding DNA in vivo. Characterization of CP-31398 Effects on p53 in Vitro—Given that CP-31398 appears to be capable of restoring DNA-binding activity to full-length mutant p53 in vivo, we wanted to see if CP-31398 would be equally effective in a system using purified protein, free of any potential cellular contaminants. Previous reports (19Friedler A. Hansson L.O. Veprintsev D.B. Freund S.M.V. Rippin T.M. Nikolova P.V. Proctor M.R. Rudiger S. Fersht A.R. Proc. Nat. Acad. Sci. U. S. A. 2002; 99: 937-942Crossref PubMed Scopus (233) Google Scholar) have indicated that CP-31398 protects the DNA binding domain of p53 from thermo-denaturation in vitro. We then cloned and expressed the DNA binding domain of wild type and mutant p53 as GST fusion proteins (Fig. 2A). To see if the recombinant material was capable of binding to DNA, we performed an EMSA using wild type p53 DNA binding domain (GST-His p53DBD wt) (Fig. 2B). The wild type protein bound to a labeled double-stranded oligonucleotide containing a p53 consensus binding sequence. Binding could be competed away with excess unlabeled p53-specific oligonucleotide but not with excess unlabeled nonspecific oligonucleotide. With wild type protein, a doublet is normally seen. The slower migrating band corresponds to the GST dimer of the protein, which is also observed with the mutant protein. The faster migrating lower band is monomeric GSTp53. Similar effects with GST fusion proteins have been previously observed (29Klein C. Georges G. Kunkele K.-P. Huber R. Engh R.A. Hansen S. J. Biol. Chem. 2001; 276: 37390-37401Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). To see the effect on mutant p53 protein, we tested the binding of mutant R273H GST-His p53 DNA binding domain protein to both a consensus p53 DNA binding site oligonucleotide and an altered p53 DNA binding site (Fig. 2C). Binding w
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