Differential Regulation of MDR1 Transcription by the p53 Family Members
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m414646200
ISSN1083-351X
AutoresRobert A. Johnson, Erica M. Shepard, Kathleen W. Scotto,
Tópico(s)RNA modifications and cancer
ResumoAlthough the p53 family members share a similar structure and function, it has become clear that they differ with respect to their role in development and tumor progression. Because of the high degree of homology in their DNA binding domains (DBDs), it is not surprising that both p63 and p73 activate the majority of p53 target genes. However, recent studies have revealed some differences in a subset of the target genes affected, and the mechanism underlying this diversity has only recently come under investigation. Our laboratory has demonstrated previously that p53 represses transcription of the P-glycoprotein-encoding MDR1 gene via direct DNA binding through a novel p53 DNA-binding site (the HT site). By transient transfection analyses, we now show that p63 and p73 activate rather than repress MDR1 transcription, and they do so through an upstream promoter element (the alternative p63/p73 element (APE)) independent of the HT site. This activation is dependent on an intact DNA binding domain, because mutations within the p63DBD or p73DBD are sufficient to prevent APE-mediated activation. However, neither p63 nor p73 directly interact with the APE, suggesting an indirect mechanism of activation through this site. Most interestingly, when the p53DBD is replaced by the p63DBD, p53 is converted from a repressor working through the HT site to an activator working through the APE. Taken together, these data indicate that, despite considerable homology, the DBD of the p53 family members have unique properties and can differentially regulate gene targeting and transcriptional output by both DNA binding-dependent and -independent mechanisms. Although the p53 family members share a similar structure and function, it has become clear that they differ with respect to their role in development and tumor progression. Because of the high degree of homology in their DNA binding domains (DBDs), it is not surprising that both p63 and p73 activate the majority of p53 target genes. However, recent studies have revealed some differences in a subset of the target genes affected, and the mechanism underlying this diversity has only recently come under investigation. Our laboratory has demonstrated previously that p53 represses transcription of the P-glycoprotein-encoding MDR1 gene via direct DNA binding through a novel p53 DNA-binding site (the HT site). By transient transfection analyses, we now show that p63 and p73 activate rather than repress MDR1 transcription, and they do so through an upstream promoter element (the alternative p63/p73 element (APE)) independent of the HT site. This activation is dependent on an intact DNA binding domain, because mutations within the p63DBD or p73DBD are sufficient to prevent APE-mediated activation. However, neither p63 nor p73 directly interact with the APE, suggesting an indirect mechanism of activation through this site. Most interestingly, when the p53DBD is replaced by the p63DBD, p53 is converted from a repressor working through the HT site to an activator working through the APE. Taken together, these data indicate that, despite considerable homology, the DBD of the p53 family members have unique properties and can differentially regulate gene targeting and transcriptional output by both DNA binding-dependent and -independent mechanisms. By virtue of its function as a transcriptional regulator, p53 plays an integral role in cell cycle arrest and apoptosis. It is mutated or inactivated in the vast majority of human cancers (1.Irwin M.S. Kaelin W.G. Cell Growth & Differ. 2001; 12: 337-349PubMed Google Scholar, 2.Haupt S. Berger M. Goldberg Z. Haupt Y. J. Cell Sci. 2003; 116: 4077-4085Crossref PubMed Scopus (936) Google Scholar), and engineered loss of p53 in animal models results in a tumor-prone phenotype (3.Donehower L.A. Harvey M. Slagle B.L. McArthur M.J. Montgomery Jr., C.A. Butel J.S. Bradley A. Nature. 1992; 356: 215-221Crossref PubMed Scopus (3988) Google Scholar, 4.Jacks T. Remington L. Williams B.O. Schmitt E.M. Halachmi S. Bronson R.T. Weinberg R.A. Curr. Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar), confirming the tumor suppressor function of this protein. Considerable excitement was generated by the more recent discovery of two p53 family members, p63 and p73, because their similarity to p53 with respect to protein structure (5.Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar, 6.Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1821) Google Scholar) (Fig. 1) and certain cellular functions suggested an analogous role for these proteins as transcriptional regulators and tumor suppressors. The central DNA binding domain (DBD) 1The abbreviations used are: DBD, DNA binding domain; APE, alternative p63/73 element; RT, reverse transcription; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; TAD, transcriptional activation domain; Pgp, P-glycoprotein; G3PDH, glyceraldehyde-3-phosphate dehydrogenase. 1The abbreviations used are: DBD, DNA binding domain; APE, alternative p63/73 element; RT, reverse transcription; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; TAD, transcriptional activation domain; Pgp, P-glycoprotein; G3PDH, glyceraldehyde-3-phosphate dehydrogenase. shares the highest degree of homology among family members (∼60%) and facilitates their interaction with the p53 DNA consensus element in target genes. Given this high degree of homology, it is not surprising that p63 and p73 bind to the p53 consensus sequence and activate a large subset of p53 target genes, albeit with somewhat different efficiencies (7.Kartasheva N.N. Contente A. Lenz-Stoppler C. Roth J. Dobbelstein M. Oncogene. 2002; 21: 4715-4727Crossref PubMed Scopus (125) Google Scholar, 8.Levrero M. De Laurenzi V. Costanzo A. Gong J. Wang J.Y. Melino G. J. Cell Sci. 2000; 113: 1661-1670Crossref PubMed Google Scholar, 9.Melino G. Lu X. Gasco M. Crook T. Knight R.A. Trends Biochem. Sci. 2003; 28: 663-670Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Moreover, both p63 and p73 have been shown to cause cell cycle arrest and apoptosis when overexpressed (9.Melino G. Lu X. Gasco M. Crook T. Knight R.A. Trends Biochem. Sci. 2003; 28: 663-670Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 10.Stiewe T. Putzer B.M. Apoptosis. 2001; 6: 447-452Crossref PubMed Scopus (49) Google Scholar, 11.Yang A. McKeon F. Nat. Rev. Mol. Cell. Biol. 2000; 1: 199-207Crossref PubMed Scopus (420) Google Scholar). Despite these structural and functional similarities, several observations underscore fundamental differences among these family members with respect to their roles in oncogenesis and tissue development. First, although p63 and p73 were initially described as tumor suppressor proteins because of their localization to regions of the chromosome either deleted or altered in several cancer types (12.Herranz M. Urioste M. Santos J. Martinez-Delgado J.B. Rivas C. Benitez J. Fernandez-Piqueras J. Leukemia (Baltimore). 2000; 14: 1325-1327Crossref PubMed Scopus (20) Google Scholar, 13.Irwin M.S. Kaelin Jr., W.G. Apoptosis. 2001; 6: 17-29Crossref PubMed Scopus (71) Google Scholar, 14.Kaelin Jr., W.G. Oncogene. 1999; 18: 7701-7705Crossref PubMed Scopus (158) Google Scholar), few mutations of these homologues have been identified in human tumors, and animals null for either protein do not demonstrate an increase in tumor formation (15.Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (870) Google Scholar, 16.Yang A. Schweitzer R. Sun D. Kaghad M. Walker N. Bronson R.T. Tabin C. Sharpe A. Caput D. Crum C. McKeon F. Nature. 1999; 398: 714-718Crossref PubMed Scopus (1877) Google Scholar, 17.Mills A.A. Zheng B. Wang X.J. Vogel H. Roop D.R. Bradley A. Nature. 1999; 398: 708-713Crossref PubMed Scopus (1674) Google Scholar). Second, unlike p53, both homologues exist as an array of isoforms differing at both their amino and carboxyl termini due to alternative splicing/differential promoter utilization (Fig. 1) (6.Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1821) Google Scholar, 11.Yang A. McKeon F. Nat. Rev. Mol. Cell. Biol. 2000; 1: 199-207Crossref PubMed Scopus (420) Google Scholar, 18.Ishimoto O. Kawahara C. Enjo K. Obinata M. Nukiwa T. Ikawa S. Cancer Res. 2002; 62: 636-641PubMed Google Scholar, 19.De Laurenzi V. Costanzo A. Barcaroli D. Terrinoni A. Falco M. Annicchiarico-Petruzzelli M. Levrero M. Melino G. J. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (360) Google Scholar, 20.Ueda Y. Hijikata M. Takagi S. Chiba T. Shimotohno K. Oncogene. 1999; 18: 4993-4998Crossref PubMed Scopus (131) Google Scholar). Alternative splicing introduces novel domains, such as the sterile α-motif, into certain isoforms. Differential promoter utilization results in the loss of the amino-terminal transcriptional activation domain (TAD) (the ΔN forms) and the creation of dominant negative isoforms that can inhibit transcriptional activation by other family members on at least a subset of target genes (21.Grob T.J. Novak U. Maisse C. Barcaroli D. Luthi A.U. Pirnia F. Hugli B. Graber H.U. De Laurenzi V. Fey M.F. Melino G. Tobler A. Cell Death Differ. 2001; 8: 1213-1223Crossref PubMed Scopus (306) Google Scholar, 22.Fillippovich I. Sorokina N. Gatei M. Haupt Y. Hobson K. Moallem E. Spring K. Mould M. McGuckin M.A. Lavin M.F. Khanna K.K. Oncogene. 2001; 20: 514-522Crossref PubMed Scopus (117) Google Scholar, 23.Zaika A.I. Slade N. Erster S.H. Sansome C. Joseph T.W. Pearl M. Chalas E. Moll U.M. J. Exp. Med. 2002; 196: 765-780Crossref PubMed Scopus (297) Google Scholar). Most interestingly, high levels of certain p63 and p73 isoforms have been observed in some tumors, suggesting that these proteins may act as oncogenes rather than classic tumor suppressor proteins (24.Westfall M.D. Mays D.J. Sniezek J.C. Pietenpol J.A. Mol. Cell. Biol. 2003; 23: 2264-2276Crossref PubMed Scopus (287) Google Scholar, 25.De Laurenzi V. Melino G. Ann. N. Y. Acad. Sci. 2000; 926: 90-100Crossref PubMed Scopus (104) Google Scholar, 26.Pozniak C.D. Radinovic S. Yang A. McKeon F. Kaplan D.R. Miller F.D. Science. 2000; 289: 304-306Crossref PubMed Scopus (403) Google Scholar, 27.Barlev N.A. Liu L. Chehab N.H. Mansfield K. Harris K.G. Halazonetis T.D. Berger S.L. Mol. Cell. 2001; 8: 1243-1254Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 28.King K.E. Ponnamperuma R.M. Yamashita T. Tokino T. Lee L.A. Young M.F. Weinberg W.C. Oncogene. 2003; 22: 3635-3644Crossref PubMed Scopus (132) Google Scholar). Another notable difference among the homologues lies in their apparent roles in development, with p63 playing a critical role in epithelial stem cell renewal and epithelial homeostasis, and p73 contributing to neurogenesis (1.Irwin M.S. Kaelin W.G. Cell Growth & Differ. 2001; 12: 337-349PubMed Google Scholar). Among the first evidence for these roles was the observation that p63–/– and p73–/– mice each exhibit a distinct pattern of developmental defects that had not been observed in p53–/–animals. The p63–/–animals were born with severe craniofacial, limb, and epithelial cell defects (16.Yang A. Schweitzer R. Sun D. Kaghad M. Walker N. Bronson R.T. Tabin C. Sharpe A. Caput D. Crum C. McKeon F. Nature. 1999; 398: 714-718Crossref PubMed Scopus (1877) Google Scholar, 17.Mills A.A. Zheng B. Wang X.J. Vogel H. Roop D.R. Bradley A. Nature. 1999; 398: 708-713Crossref PubMed Scopus (1674) Google Scholar), whereas p73–/–animals have congenital hydrocephalus, hippocampal dysgenesis, and defects in pheromone detection (15.Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (870) Google Scholar). These phenotypic differences demonstrate a subset of unique roles for each family member in both development (p63 and p73) and oncogenesis (p53). Wild-type p53 is best known as a transcriptional activator, an activity that is dependent on its interaction with a DNA consensus element consisting of two half-sites, each composed of two copies of the sequence PuPuPuC(A/T) (where Pu indicates purine) separated by 0–13 nucleotides. The role of p53 in transcriptional repression, although less well studied, has also been shown to be critical to its function in the apoptotic pathway (29.Zhu J. Zhang S. Jiang J. Chen X. J. Biol. Chem. 2000; 275: 39927-39934Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30.Venot C. Maratrat M. Dureuil C. Conseiller E. Bracco L. Debussche L. EMBO J. 1998; 17: 4668-4679Crossref PubMed Scopus (251) Google Scholar). Yet there have been very few queries of a similar role for p63 and p73. Nevertheless, due to clearly demonstrated functional differences among the homologues with respect to development and tumorigenicity, it seems important to identify and investigate any fundamental differences at the level of transcriptional regulation, including the effect of these homologues on p53-repressed genes. Our laboratory has a long standing interest in the transcriptional regulation of the P-glycoprotein (Pgp)-encoding MDR1 gene, which was first identified by virtue of its overexpression in multidrug-resistant tumor cells (31.Riordan J.R. Ling V. J. Biol. Chem. 1979; 254: 12701-12705Abstract Full Text PDF PubMed Google Scholar). More recently, it has been shown that Pgp plays a general anti-apoptotic role by conferring resistance to a variety of caspase-mediated apoptotic inducers in addition to chemotherapeutics (32.Johnstone R.W. Cretney E. Smyth M.J. Blood. 1999; 93: 1075-1085Crossref PubMed Google Scholar, 33.Smyth M.J. Krasovskis E. Sutton V.R. Johnstone R.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7024-7029Crossref PubMed Scopus (326) Google Scholar, 34.Robinson L.J. Roberts W.K. Ling T.T. Lamming D. Sternberg S.S. Roepe P.D. Biochemistry. 1997; 36: 11169-11178Crossref PubMed Scopus (138) Google Scholar, 35.Ruth A.C. Roninson I.B. Cancer Res. 2000; 60: 2576-2578PubMed Google Scholar). Moreover, expression of Pgp in critical organs such as the blood-brain barrier and colon epithelium impacts on drug pharmacokinetics and biodistribution (36.Schinkel A.H. Semin. Cancer Biol. 1997; 8: 161-170Crossref PubMed Scopus (438) Google Scholar, 37.Liu Y. Hu M. Clin. Chem. Lab. Med. 2000; 38: 877-881PubMed Google Scholar). Given the multiple potential roles of MDR1/Pgp in tumor progression and drug response, an understanding of its regulation in normal and tumor cells is essential. MDR1 was one of the first promoters to be shown to be repressed by p53 (38.Chin K.V. Ueda K. Pastan I. Gottesman M.M. Science. 1992; 255: 459-462Crossref PubMed Scopus (706) Google Scholar). More recently, we have dissected the mechanistic basis for this repression, and we identified a region within the MDR1 promoter consisting of an atypical p53 consensus sequence (the HT site) that mediates p53 repression by direct DNA binding (39.Johnson R.A. Ince T.A. Scotto K.W. J. Biol. Chem. 2001; 276: 27716-27720Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). This site differs from the p53 consensus (activator) motif in that the quarter sites, which are arranged in a head-to-head (HH) configuration in the consensus motif, are arranged in a head-to-tail (HT) orientation within the MDR1 promoter. When p53 binds to this HT site, MDR1 transcription is repressed; when the HT site is converted to a consensus HH site within the context of the MDR1 promoter, transcription is activated. Thus, the orientation of the quarter sites within this promoter dictates the effect of the promoter-bound p53 on MDR1 transcription. In light of the high degree of homology within the DBDs of the p53 family members, and the numerous studies that have shown that the p53 homologues are capable of activating genes that contain the p53 consensus (HH) site, we sought to investigate the role of p63 and p73 on the p53-repressed MDR1 promoter. Most surprisingly, we found that both p63 and p73 activate, rather than repress, the MDR1 promoter. Moreover, this activation occurs independent of the HT site and is indirectly mediated by a novel upstream element. Finally, the difference in transcriptional effects of the p53 family members on MDR1 transcription is due, at least in part, to differences among their DNA binding domains. Cell Culture and Transfections—The human neuroblastoma cell line SK-N-AS (ATCC CRL-2137), the hepatocellular carcinoma HepG2 cell line (ATCC HB-8065), and the lung adenocarcinoma cell line H1299 (ATCC CRL-5803) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units of penicillin/streptomycin per ml. Human osteosarcoma Saos-2 cells (ATCC HTB-85) and the colorectal adenocarcinoma cell line SW620 (ATCC CCL-227) were maintained in RPMI 1640 with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. All cells were grown at 37 °C in a 5% CO2 humidified atmosphere. Cells were transfected with FuGENE 6 (Roche Applied Science) at a ratio of 1:3 (DNA/FuGENE 6). For all transfections, DNA was added at a concentration of 2 μg/well (salmon sperm DNA was used as a carrier when needed). Luciferase activity was measured as described previously (39.Johnson R.A. Ince T.A. Scotto K.W. J. Biol. Chem. 2001; 276: 27716-27720Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), using the Luminoskan Ascent plate reader (ThermoLabSystems, Finland). Luciferase activity was normalized to protein concentration, determined using the Coomassie Plus Protein Assay Reagent (Pierce). The luciferase activity of reporter constructs in the absence of exogenous p53, p63, or p73 was arbitrarily set at 1. Experiments were performed a minimum of three times in triplicate. Plasmids and Site-directed Mutagenesis—All mutant constructs were created using the QuickChange site-directed mutagenesis kit (Stratagene, CA) according to the manufacturer's suggested protocol. The reporter constructs included pMDR1-HT (containing sequences from –1202 to +118 of the MDR1 promoter), pMDR1/–221 (–221 to +118), pMDR1/–136 (–136 to +118) (40.Jin S. Scotto K.W. Mol. Cell. Biol. 1998; 18: 4377-4384Crossref PubMed Google Scholar), and pMDR1-HTmut (pMDR1-HT promoter mutated within the p53 HT site) (39.Johnson R.A. Ince T.A. Scotto K.W. J. Biol. Chem. 2001; 276: 27716-27720Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The pMDR1/–190 construct was engineered by inserting a SmaI site at position –190 of pMDR1-HT using the following oligonucleotide: 5′-GGAATGTTCTGGCTTCCGTccCgggTCTCTAGAAAGGGCAAG-3′ (the SmaI site is shown in boldface and mutated bases are in lowercase). The resulting construct was digested with SmaI and recircularized to generate pMDR1/–190. The p63 and p73 isoform expression vectors have been previously described (23.Zaika A.I. Slade N. Erster S.H. Sansome C. Joseph T.W. Pearl M. Chalas E. Moll U.M. J. Exp. Med. 2002; 196: 765-780Crossref PubMed Scopus (297) Google Scholar, 41.Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Nat. Med. 1998; 4: 839-843Crossref PubMed Scopus (471) Google Scholar, 42.Ueda Y. Hijikata M. Takagi S. Chiba T. Shimotohno K. Biochem. J. 2001; 356: 859-866Crossref PubMed Scopus (29) Google Scholar, 43.Dietz S. Rother K. Bamberger C. Schmale H. Mossner J. Engeland K. FEBS Lett. 2002; 525: 93-99Crossref PubMed Scopus (59) Google Scholar). pRC-p63γ (R304H) and pCDNA-p73α (R293H) were created using the following oligonucleotides: R304H, 5′-CGACGCTGCTTTGAGGCCCACATCTGTGCTTGCCCAGGAAG-3′; R293H, 5′-CGCCGGTCCTTTGAGGGCCACATCTG CGCCTGTCCTGGCCGC-3′. The p53/63DBD and p53/73DBD chimeric constructs were engineered as follows. NheI sites were placed at the 5′ and 3′ boundaries of the p63, p53, and p73DBD via site-directed mutagenesis. Mutant plasmids were digested with NheI, and the DBD fragments and vectors lacking the respective DBDs were isolated using the QIAquick gel extraction kit (Qiagen) following gel electrophoresis. Relevant vectors were ligated to a heterologous DBD, and the NheI sites were converted to wild-type sequences. The plasmids p53/63DBD and p53/73DBD include amino acids 141–320 of p63 or 131–310 of p73, respectively, in place of amino acids 112–290 of the p53DBD. Electromobility Shift Assays—p63γ and p73α proteins were prepared in vitro using the TnT Quick-coupled Transcription/Translation System as described by the manufacturer (Promega, WI). Gel shift assays were performed as described previously with a few modifications (39.Johnson R.A. Ince T.A. Scotto K.W. J. Biol. Chem. 2001; 276: 27716-27720Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). SK-N-AS whole cell extract was prepared as follows. SK-N-AS cells were transfected with 12 μg of expression plasmid as described previously. Plates were washed twice with ice-cold PBS. Cells were lysed with ice-cold buffer (20 mm Hepes, pH 7.5, 20% glycerol, 10 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.1% Triton X-100,1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture tablet (Roche Applied Science)) and centrifuged at 10,000 × g for 20 min, and the supernatant was collected. Protein concentration was determined using the Coomassie Plus Protein Assay Reagent (Pierce). When indicated, 200 ng of one of the following antibodies was added: mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA), p73 Ad-2 (Oncogene Research Products, MA), p63 (4A4) (Santa Cruz Biotechnology), or HA (HA-(Y11) (Santa Cruz Biotechnology)). Oligonucleotides used as gel shift probes or for competition were as follows: con53, 5′-TTGGCTGGACATGCCCGGCGCCGGGGCGTGGGCATGTCCCAGCGC-3′; APE, 5′-GATGCGCGTTTCTCTACTTGCCCTTTCTAGAGAGGTGCA-3′; and HT, 5′-CGGCTGTGCTCAGCCCACGCCCCGGCGCTGTTCTGCCCAGCCAA-3′. RT-PCR—SK-N-AS cells were plated in 100-mm dishes and transfected with 12 μg of expression construct; total RNA was extracted using Trizol as recommended by the vendor (Invitrogen). RT-PCRs were performed using SuperScript One-step RT-PCR as described by the manufacturer (Invitrogen). RNA at a concentration of 0.1 and 1.0 μg was used for G3PDH and MDR1 PCRs, respectively. The primers used for the PCRs are as follows: MDR1 forward, 5′-GGGATAAAGAAAGCTATTACAGCC-3′, and MDR1 reverse, 5′-ATCTGGTTTGTGCCCACTCT-3′; G3PDH (BD Biosciences), 5′-ATCTGGTTTGTGCCCACTCT-3′. Western Blot Analysis—5 × 105 SK-N-AS cells were plated in 100-mm dishes and transfected with 12 μg of expression construct. Plates were washed with ice-cold PBS, and cells were lysed with ice-cold RIPA buffer (1× PBS, 0.5% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, 1 mm EDTA, pH 7.5), 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture tablet (Roche Applied Science). Cells were centrifuged at 10,000 × g for 20 min, and supernatant was collected. Protein concentration was determined using the BCA protein assay system (Pierce). Western blotting was performed using 30 μg of protein electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The following antibodies were used: p73(H-79) (Santa Cruz Biotechnology), p63(4A4) (Santa Cruz Biotechnology), p53(Ab-6), p73(Ab-2), and actin (Santa Cruz Biotechnology). Primary antibodies were diluted 1:1000, and anti-mouse, anti-rabbit (Amersham Biosciences), or anti-goat (Santa Cruz Biotechnology) secondary antibodies were diluted 1:5000. Blots were developed using ECL Western blotting detection reagents (Amersham Biosciences). p63 and p73 Activate Rather than Repress MDR1 Transcription—We have shown previously that wild-type p53 represses transcription of MDR1 through direct binding to a novel head-to-tail (HT) element within the proximal MDR1 promoter (39.Johnson R.A. Ince T.A. Scotto K.W. J. Biol. Chem. 2001; 276: 27716-27720Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To assess a role for the p53 family members p63 and p73 on MDR1 transcription, we evaluated the effect of different isoforms of each protein on MDR1 promoter activity in a transient transfection assay. The human neuroblastoma cell line SK-N-AS (which does not express either p63 or p73) was co-transfected with the wild-type MDR1 luciferase expression construct pMDR1-HT and either empty vector or one of the predominant p63 or p73 isoforms (Fig. 2A). Western blot analysis verified equivalent concentrations of both p63 isoforms (Fig. 2B, upper panel) and p73 isoforms (Fig. 2C, upper panel) in the transfectants. Most surprisingly, all three p73 isoforms (α, β, and γ) activated rather than repressed the MDR1 promoter; the relative strength of activation by the various p73 isoforms was consistent with what has been observed in the analyses of the effects of these isoforms on other promoters (7.Kartasheva N.N. Contente A. Lenz-Stoppler C. Roth J. Dobbelstein M. Oncogene. 2002; 21: 4715-4727Crossref PubMed Scopus (125) Google Scholar, 8.Levrero M. De Laurenzi V. Costanzo A. Gong J. Wang J.Y. Melino G. J. Cell Sci. 2000; 113: 1661-1670Crossref PubMed Google Scholar, 44.Melino G. De Laurenzi V. Vousden K.H. Nat. Rev. Cancer. 2002; 2: 605-615Crossref PubMed Scopus (498) Google Scholar). p63γ was also shown to activate MDR1 transcription, whereas p63α was a very weak activator; again, this difference in activation potential for p63 isoforms is consistent with what was observed for regulation of the promoter of p21 and other p53 target genes (45.Serber Z. Lai H.C. Yang A. Ou H.D. Sigal M.S. Kelly A.E. Darimont B.D. Duijf P.H. Van Bokhoven H. McKeon F. Dotsch V. Mol. Cell. Biol. 2002; 22: 8601-8611Crossref PubMed Scopus (167) Google Scholar). The failure of p63α to efficiently activate the MDR1 promoter suggested that MDR1 activation is dependent upon a transcriptionally competent form of the protein. To address this more fully, the ΔN forms of p63 and p73 that lack the amino-terminal TAD and are therefore transcriptionally compromised (Fig. 1) were co-transfected into SK-N-AS cells along with pMDR1-HT and protein expression quantitated by Western blot (Fig. 2, B and C, lower panels). As shown in Fig. 2D, ΔNp63γ was impaired in its ability to activate the MDR1 construct, resulting in approximately half the level of activation as was observed with p63γ; a similar reduction in activity was observed in a co-transfection analysis of the p21 promoter (data not shown and see Ref. 28.King K.E. Ponnamperuma R.M. Yamashita T. Tokino T. Lee L.A. Young M.F. Weinberg W.C. Oncogene. 2003; 22: 3635-3644Crossref PubMed Scopus (132) Google Scholar). That all activity is not lost upon deletion of the TAD is not completely unexpected, because it has been suggested that ΔNp63 may contain a second TAD and can in fact activate the transcription of a subset of genes (6.Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1821) Google Scholar, 46.Wu G. Nomoto S. Hoque M.O. Dracheva T. Osada M. Lee C.C. Dong S.M. Guo Z. Benoit N. Cohen Y. Rechthand P. Califano J. Moon C.S. Ratovitski E. Jen J. Sidransky D. Trink B. Cancer Res. 2003; 63: 2351-2357PubMed Google Scholar, 47.Ghioni P. Bolognese F. Duijf P.H. Van Bokhoven H. Mantovani R. Guerrini L. Mol. Cell. Biol. 2002; 22: 8659-8668Crossref PubMed Scopus (189) Google Scholar). The effect of deletion of the TAD was even more striking with respect to p73α, because ΔNp73α was unable to activate either the MDR1 promoter (Fig. 2D) or the p21 promoter (data not shown and see Ref. 21.Grob T.J. Novak U. Maisse C. Barcaroli D. Luthi A.U. Pirnia F. Hugli B. Graber H.U. De Laurenzi V. Fey M.F. Melino G. Tobler A. Cell Death Differ. 2001; 8: 1213-1223Crossref PubMed Scopus (306) Google Scholar). Taken together, these data indicate that activation of the MDR1 promoter by the p53 family members is dependent on the transcriptional activation competence of the protein. Most interestingly, when a similar experiment was performed in p53-deficient H1299 cells (Fig. 2E), neither p63 nor p73 isoforms were able to activate MDR1, indicating that activation may be cell line/tissue-specific. Although one difference between the SK-N-AS and H1299 cells is the status of p53 (wild-type and null, respectively), this does not appear to be the primary basis for the difference in activation by p63 and p73 because the SW620 cell line expressing mutant p53 (R273H) was able to support p63 and p73 activation (Fig. 2F), whereas the wild-type p53-expressing HepG2 cell line could not (Fig. 2G). To evaluate activation of the endogenous MDR1 gene by the p53 family members, either p63γ or p73α expression vectors were transiently transfected into SK-N-AS cells. RT-PCR analysis of MDR1 mRNA from transfected cells lines showed a substantial increase in MDR1 RNA in cells that expressed either p63γ (Fig. 2H, lane 2) or p73α (lane 5) as compared with the empty vector control (lane 1). However, no increase in MDR1 levels was observed in ΔNp63γ (lane 3) or ΔNp73α (lane 4) transfectants. G3PDH expression was unaffected by these proteins (Fig. 2H, lower panel). Thus, both the endogenous MDR1 gene as well as a transfected MDR1 promoter construct can be activated by the p53 homologues, and this activation is dependent on the amino-terminal activation domain. p63 and p73 Activate MDR1 Transcription through a Novel Upstream Element (the APE)—To addr
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