the transcriptional activities of p53 and its homologue p51/p63: similarities … · show...

[CANCER RESEARCH 59, 2781–2786, June 15, 1999]

Advances in Brief

The Transcriptional Activities of p53 and Its Homologue p51/p63: Similaritiesand Differences1

Akira Shimada, Shunsuke Kato, Kentaro Enjo, Motonobu Osada, Yoji Ikawa, Kenji Kohno, Masuo Obinata,Ryunosuke Kanamaru, Shuntaro Ikawa, and Chikashi Ishioka2

Departments of Clinical Oncology [A. S., S. K., R. K., C. I.] and Cell Biology [K. E., M. Os., M. Ob., S. I.], Institute of Development, Aging and Cancer, Tohoku University,Sendai 980-8575; Department of Retroviral Regulation, Tokyo Medical and Dental University, Medical Research Division, Tokyo 113-8519 [Y. I.]; and Nara Institute of Scienceand Technology, Nara 630-01 [K. K.], Japan

Abstract

p51/p63 is a novel p53 homologue that has been shown to act as atranscriptional activator through the p53-binding sequence of thep21/WAF1 promoter and to induce apoptosis when it is expressed transientlyin a human tumor cell line. We developed transcription assay systems forthese two related genes in bothSaccharomyces cerevisiaeand mammaliancells and used them to investigate the functional similarities and differ-ences of these genes. We found that p51/p63trans-activated the previouslyidentified p53 target genes, but the degree of the transactivation byp51/p63 differed from that by p53. These results suggest that the cellularsignal on p51/p63 cross-talks partially but not completely with that of thep53 pathway.

Introduction

Thep53tumor suppressor gene is the most frequently mutated genepresent in a variety of human cancers (1). It serves to maintain geneticstability by inducing cell cycle arrest in late G1 and/or apoptosis inresponse to genotoxic stress (for reviews see Refs. 2–4). The biolog-ical effects of p53 are controlled by p53-dependenttrans-activationthrough a p53RE3 that regulates the expression of downstream targetgenes, although apoptosis is due, at least in part, to a mechanism thatis independent oftrans-activation (5).

The p53 genes are highly conserved in lower species (such assquid) and higher mammals, and for a long time, it was thought therewas nop53 family (6). The first mammalianp53 homologue to beidentified was ratKet (7), after which it was considered likely therewould be humanp53homologues. Two humanp53homologues,p73and p51/p63 (hereafter referred to asp51), were identified by adegenerate PCR method (8, 9), and subsequently, they were alsoidentified in other laboratories (10–13). The predicted protein struc-tures of the two human homologues are similar to that of human p53protein, and;60% of their amino acids in the region correspondingto the p53 DNA-binding domain are identical to those of p53 (8, 9).In contrast, only;30% of the amino acids of the NH2- and COOH-terminal portions of the homologues are identical to those of p53protein (8, 9), although the COOH-terminal oligomerization domain isrelatively well conserved among human p53, p73, and p51 and squidp53 (8, 9, 14). The NH2- and COOH-terminal portions of p53 encodethe regulatory domains of p53 that are involved in p53 activation by

the upstream signal (15–18). In 1997, the upstream biological signalthat activates p73 was shown to be different from the signal thatactivates p53 (7, 8). On the basis of these observations, we predict thatthe upstream signals of p51 and p53 are also different and that p53 andits homologues partially or exclusively share downstream target genesand, therefore, both play roles in biological events such as G1 arrestand apoptosis. In fact, initial studies showed that both p73 and p51proteins, when overexpressed in human cells, can up-regulatep21/WAF1transcription and induce apoptosis (9, 19). Furthermore, initialmutation screening ofp73 andp51 in a variety of human tumors (9,20–23) and cell lines (9) revealed rare mutations in the open readingframes. All the evidence to date suggests that the p53 homologuesshow functional similarities and differences.

In view of the structural and functional similarities of p51 and p53,it is obviously important to study the biological pathway through p51that contains unknown upstream and downstream signals and comparethis pathway with the known p53 pathway. In this study, we used bothyeast and mammalian cell systems to examine the ability of p51 totrans-activate p53-inducible promoters other than thep21/WAF1pro-moter, i.e., BAX, MDM2, and14-3-3s promoters.

Materials and Methods

Plasmids. For the yeast-based transcription assay, the p53 expression vec-tor pLSC53A and the p51 expression vector pCIP51-2 were constructed byinserting theBamHI/HindIII PCR fragment containing the open reading framesof p53cDNA andp51AcDNA, respectively, into pCI53Y3 (also called pLSX;Ref. 14). We also constructed the HA-tagged p53 expression vector pHA53and HA-tagged p51A expression vector pHA51-2 by inserting theBamHI/XhoIfragments of p53/CMV and p51/CMV-2 (see below), respectively, into pRS-PGK (24). These expression vectors were used for both the yeast-basedtranscription and immunoblotting assays. These low-copy centromeric vectorsare maintained stably in yeast grown on medium lacking leucine and expresswild-type p53 and p51A under the control of theADH1 (pLSC53A andpCIP51-2) orPGK (pHA53 and pHA51-2) promoter. The reporter plasmidpSS1 contains a p53-binding RGC sequence upstream of theGAL1 minimalpromoter (25). The reporter plasmids pCI-WAFP(HIS), pCI-MDMPs(HIS),pCI-BAXPs(HIS), and pCI-SIGMAPs(HIS) were identical to pSS1, exceptthat the RGC sequence inserted in the uniqueEcoRI site of pSS1 has beenreplaced by the partial promoter sequences of thep21/WAF1(GenBank ac-cession no. U24170, nucleotides 2241–3258),MDM2 (GenBank accession no.U28935, nucleotides 686–791),BAX (GenBank accession no. U17193, nucle-otides 487–574), and14-3-3s (EMBL accession no. AF029081, nucleotides6017–6797) genes, respectively. All these fragments contain the p53RE(s), asshown in Fig. 1. These low-copy centromeric vectors are maintained stably inyeast grown on medium lacking tryptophan and express the yeastHIS3 gene,depending on the wild-type p53 expression.

GFP reporter plasmids were constructed as follows. AnEcoRI/SalI frag-ment containing theHIS3gene of pSS1 was ligated into theEcoRI/SalI site ofpRS424DB, a plasmid identical to pRS424 (26), except that theBamHI site hadbeen disrupted by the Klenow enzyme, producing pAS01. ABamHI PCRfragment ofGFPcDNA (codon 2, termination codon) derived from pQB2 (27)

Received 3/18/99; accepted 4/29/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Grants-in-Aid from the Ministry of Education, Science, Sportsand Culture and the Ministry of Health and Welfare.

2 To whom requests for reprints should be addressed, at Department of ClinicalOncology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8547; Fax: 81-22-717-8548;E-mail: [email protected].

3 The abbreviations used are: p53RE, p53-responsivecis-acting element; HA, hemag-glutinin; RGC, ribosomal gene cluster; GFP, green fluorescent protein.

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was inserted into theBamHI sites of theHIS3 gene of pAS01, which wasprepared using an inverse PCR technique, generating pAS01G. Then, theEcoRI fragments containing the p53RE from pCI-WAFP(HIS), pCI-MDMP-s(HIS), pCI-BAXPs(HIS), and pCI-SIGMAPs(HIS) were inserted into theEcoRI site of pAS01G, generating pAS03G, pAS05G, pAS07G, and pAS09G,respectively. These high-copy2m vectors are maintained stably in yeast grownon medium lacking tryptophan and express GFP, depending on the wild-typep53 expression. The GFP protein thus produced is a variant form, with S65Tand S147P mutations (27), and emits a stronger fluorescent signal than wild-type GFP and variant GFP with only the S65T mutation.

For the luciferase assay, the p53 expression vector p53/CMV and p51Aexpression vector p51/CMV-2 were constructed by inserting theBamHI/EagIfragments derived from the pLSC53A- and pCIP51-2-containing open readingframes ofp53 cDNA and p51A cDNA, respectively, into pcDNA1.1/Amp(Invitrogen, Carlsbad, CA) with the 39untranslated region of thep53gene. Wealso constructed the HA-tagged p53 expression vector pHA53/CMV andHA-tagged p51A expression vector pHA51/CMV-2 by replacing thep53 andp51A sequences of p53/CMV and p51/CMV-2 withHindIII fragments con-taining HA-taggedp53 and HA-taggedp51A sequences, respectively. Thesevectors were used for both the luciferase and immunoblotting assays.Fireflyluciferase reporter plasmids were constructed as follows. A double-strandlinker containing theSpeI-EcoRI-SacII-PstI sequence was inserted into theBglII sites of pGL3-Basic and pGL3-Promoter (Promega, Madison, WI),generating pGL3E-Basic and pGL3E-Promoter, respectively. TheEcoRI frag-ments derived from pCI-WAFP(HIS), pCI-MDMPs(HIS), pCI-BAXPs(HIS),and pCI-SIGMAPs(HIS) were inserted into theEcoRI site of pGL3E-pro-moter, generating p21Ps luc, pMDMPs luc, pBAXPs luc, and pSIGMAPs luc,respectively. The p21Luc-1 plasmid was constructed by inserting theHindIIIfragment containing thep21/WAF1promoter (GenBank accession no. U24170,nucleotides 2256–4594) into theHindIII site of pGL3-Basic, and pMDMPlluc, BAXP12 luc, and pSIGMAP1 were constructed by inserting theEcoRIfragment containing the promoters of theMDM2 (GenBank accession no.U28935, nucleotides 314–982),BAX (GenBank accession no. U17193, nucle-otides 288–646), and14-3-3s (EMBL accession no. AF029081, nucleotides6040–8610) genes, respectively, into pGL3E-Basic. The promoter sequencesof p21Luc-1, pMDMPl luc, pBAXP12 luc, and pSIGMAP1 luc each contain ap53RE, TATA box, and transcription initiation sites. The promoter sequencesinserted into all the reporter plasmids were obtained by subjecting normalgenomic DNA to the PCR using a set of appropriate primers, except for thep21/WAF1promoter sequences, which were derived from pWWP-CAT (a giftfrom Bert Vogelstein, Johns Hopkins University, Baltimore, MD). TheRenillaluciferase expression vector pRL-CMV (Promega) was used as an internal

control to correct values according to the transfection efficiency of the dualluciferase assay.

Yeast Strains and Media. Basic yeast manipulation was carried out asdescribed previously (28). The three haploid yeast strains used for the p53 andp51A transcription assays were ySS5 (MATa, ura3-1, ade2-1, trp1-1, his3-11,leu2-3, 112, can1-100, pep4<URA3, pSS1; Ref. 25), YSIS (MATa, ura3-1,ade2-1, trp1-1, his3-11, leu2-3, 112, can1-100, pep4<URA3; Ref. 29), andYPH499 (MATa, his3D200, ade2-101, leu2D1, ura3-52, trp1-289, lys2-801;Stratagene, La Jolla, CA). Frozen competent yeast cells were prepared asdescribed previously (30), and the solid media used for prototrophic selectionof appropriate plasmids and the His phenotype assay were the syntheticcomplete media lacking leucine and tryptophan (SC2leu 2trp) and lackinghistidine (SC2his 2leu 2trp), respectively, as described previously (25).

Yeast-based Transcription Assay.TheHIS3transcription assay describedpreviously (25) was used. Briefly, cells of the strain YSIS containing thep53-inducibleHIS3 reporter plasmid were transformed with the p53 or p51Aexpression vector on SC2leu 2trp, and the resulting transformants wereassayed for histidine prototrophy (His phenotype) on SC2his 2leu 2trpplates. For the GFP reporter assay, cells of the strains YSIS and YPH499 werecotransformed with the p53 or p51A expression vector and a GFP reporterplasmid (see above). The resulting colonies on SC2leu 2trp were assayeddirectly for GFP expression using a fluorescence microscope (MZ8; Leica)equipped with a GFP Plus filter.

Quantification of the Fluorescent Signal of GFP.The fluorescence in-tensities of GFP were determined by analyzing, using a fluoroscanmeter(Fluoroskan Ascent FL, Dainippon, Tokyo, Japan), living yeast cells on96-well microtiter plates containing SC2leu 2trp medium.

Cell Lines and Transfection. The p53-null human osteosarcoma cell lineSaos-2 and the p53-deficient lung cancer cell line EBC-1 were obtained fromthe American Type Culture Collection (Manassas, VA) and the JapaneseCancer Research Resource Bank, respectively, and grown in 12-well tissueculture plates containing RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum at 37°C in the presence of 5% CO2. Transienttransfections were performed using the Effectene (Qiagen, Hilden, Germany)transfection reagent. For the luciferase assay, Saos-2 and EBC-1 cells grown to;70% confluence in 12-well culture plates were cotransfected with the p53 orp51A expression vector (15 or 150 ng) and the p53-responsive luciferasereporter plasmid (0.4mg) as well as pRL-CMV (0.4mg) and incubated for afurther 24–48 h. For immunoprecipitation, 4.5mg of the required expressionvector were transfected into Saos-2 cells grown in tissue culture dishes(60 3 15 mm) and incubated for a further 36 h.

Fig. 1. The p51 and p53trans-activation systems for yeast and mammalian cells.A, diagram of the expression and reporter plasmids used for the yeast assay.ADHp, ADH1promoter;CYC1t, CYC1terminator;LEU2, yeast selectable marker for leucine;CEN/ARS, CEN6/ARSH4sequences for stable and low-copy replication;PGKp, PGK1promoter;PGKt, PGK1terminator;DUAS-GAL1p, GAL1 promoter lacking the upstream activating sequence;HIS3, yeast-assayable marker for histidine;TRP1, yeast-selectable marker for tryptophan;2m,sequences for stable and high-copy replication.B, diagram of the expression and reporter plasmids used for the mammalian cell assay.CMVp, human cytomegalovirus promoter andenhancer;intron/pA, SV40 splice segment and polyadenylation signal;SV40p, SV40 promoter;luc, Firefly luciferase gene;SV40polyA, SV40 polyadenylation signal.p andpp, promotersequences shown inC. C, structures of the p53 target gene promoters. The numbers correspond to the nucleotide numbers recorded in genetic databases (see “Materials and Methods”).59-UTR, 59 untranslated region;ORF, open reading frame;p, partial promoter sequence containing p53RE(s);pp, promoter sequence containing the p53RE and transcription initiationsite.

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TRANSCRIPTIONAL ACTIVITIES OF p53 AND p51/p63



Luciferase Assay.The activities ofFirefly luciferase expressed by thep53-responsive reporter plasmids were measured using the Dual-Luciferasereporter assay system (Promega) and a Fluoroskan Ascent FL (Dainippon) andcorrected according to theRenilla luciferase activities derived from pRL-CMVfor assessment of the transfection efficiency. The relative luciferase activitywas calculated using the formula: (Fireflyluciferase activity)/(Renillalucifer-ase activity).

Immunoprecipitation and Immunoblotting. Yeast and Saos-2 cell ly-sates were prepared as described previously (31, 32). To detect HA-tagged p53and p51A proteins in Saos-2 cells, the lysates were immunoprecipitated witha rat high-affinity anti-HA monoclonal antibody (Boehringer Mannheim, In-dianapolis, IN), fractionated by SDS-PAGE, and transferred electrophoreti-cally to an Immobilon SQ filter (Millipore, Bedford, MA). The HA-taggedproteins were detected using the same antibody. The yeast lysates werefractionated directly by SDS-PAGE and transferred to the same membrane,and the HA-tagged p53 or p51A were detected using a mouse anti-HAmonoclonal antibody (Boehringer Mannheim). The HA-tagged proteins werevisualized using an enhanced chemiluminescence kit (Amersham Life Science,Buckinghamshire, United Kingdom).

Results

Characterization of Expression and Reporter Plasmids.Theplasmids used in this study are summarized in Fig. 1. Thep51 genehas two major splicing variants,p51A (also calledp63g) and p51B(p63a), which are similar to the two major splicing variants ofp73,p73b, and p73a, respectively (9). We chosep51A cDNA to berepresentative ofp51because this form possesses the strongest trans-activation activity of the reported splicing variants ofp51 (12). Thebasic structures of the p51 expression vectors with and without theHA sequence are identical to those of p53 in both yeast and mamma-lian expression systems (Fig. 1,A andB). To determine whether thesevectors expressed p51 and p53 proteins, we transformed the p51 andp53 yeast expression vectors with HA sequences in YSIS yeast cellsand subjected the cell lysates to immunoblotting with an anti-HAantibody. As shown in Fig. 2A, both HA-tagged p51 and HA-taggedp53 proteins in the yeast cells were detected clearly, and their levelswere comparable, although the level of p51 was significantly lowerthan that of p53. We also examined the expression of these proteins inSaos-2 cells subjected to transient transfection with the HA-taggedp51 or p53 expression vector. The cell lysates were immunoprecipi-tated and immunoblotted with an anti-HA antibody and, as shown inFig. 2B, p51 protein was detected clearly, although its level was

significantly lower than that of p53 protein. Although the differentprotein levels may have been attributable to differences in antibodyaccessibility, the transcription/translation efficiency of the reportergenes and/or posttranslational events between p51 and p53, we did notpursue this issue in this study. We observed that the electrophoreticmobilities of p51 from both yeast and Saos-2 cells on a SDS-poly-acrylamide gel were lower than those of p53 and that Saos-2 cellsyielded two separate p51 bands (Mr ;60,000 and 66,000).

To produce reporter plasmids, we introduced the partial promotersequences containing p53REs derived from thep21/WAF1, MDM2,BAX, and14-3-3s (Fig. 1A) genes previously identified asp53 targetgenes into the upstream region of theGAL1minimal promoter lackingthe upstream activating sequences of theHIS3or GFP reporters usedfor the yeast assay. Identical fragments were also introduced into theenhancerless SV40 promoter of the luciferase reporters for mamma-lian cells (“enhancer reporters”). We also constructed a series ofreporter plasmids with promoter sequences containing transcriptioninitiation sites as well as the p53-binding sequences by inserting thefragments into promoterless/enhancerless luciferase reporter plasmids(“promoter reporters”).

In Yeast, p51 Acts as a Transcriptional Activator through ap53-binding Sequence.In a previous study, we showed that thesequence-specific transcriptional activity of p53 can be monitored bya simple yeast growth assay (25). Therefore, to examine whether p51can regulate the p53 target promotersp21/WAF1, MDM2, BAX, and14-3-3s, we used a similar yeast system. The p51 and p53 expressionvectors were cotransformed with a series ofHIS3 reporter plasmids(see above) as well as an artificial p53-responsive sequence, RGC(pSS1; Ref. 25), and the growth of the resulting transformants onplates lacking histidine was assayed to determine the His phenotype.As shown in Table 1, all the transformants harboring p51 or p53showed the His1 phenotype to varying degrees, indicating that p51also acts as a sequence-specific transcriptional activator in yeastthrough the previously reported p53REs.

Comparison of Transactivation by p51 and p53 of p53 TargetGene Promoters in Yeast.During the HIS3 reporter assay, weobserved that the growth patterns of the transformants on histidine-lacking medium differed, suggesting that the transcriptional activitiesthrough the p53-binding elements of p51 and p53 may differ. Unfor-tunately, theHIS3assay is basically an all-or-none growth assay andis not suitable for evaluating subtle differences among transcriptionalactivities of yeast transformants. Therefore, we chose a variant formof GFP with two missense mutations (S65T/S147P) as a reporter genebecause GFP expression in yeast is not toxic and the fluorescenceintensity correlates with the level of GFP expressed. Furthermore, theS65T/S147P variant shows strong fluorescent signals at 30°C and37°C (27), the temperatures at which p51 and p53 functions in yeastshould be monitored. To evaluate the transcriptional abilities of p51and p53 quantitatively, we transformed a yeast haploid strain (YSIS)

Fig. 2. Detection of p51 and p53 proteins.A, protein lysates were extracted from yeastcells harboring a null, HA-tagged p51, or HA-tagged p53 expression vector and subjectedto immunoblotting with a mouse anti-HA antibody. A yeast protein that cross-reacted withthe anti-HA antibody was detected (p). B, Saos-2 cells were transfected transiently witha null, HA-tagged p51, or HA-tagged p53 expression vector. The cell lysates wereimmunoprecipitated with a rat anti-HA antibody and then subjected to immunoblottingwith the same antibody. Rat immunoglobulin heavy (pp) and light (ppp) chains were alsodetected.

Table 1 Transcriptional activation of p51 through p53-responsive HIS3 reportersin yeasta

Reporter plasmidb

Expression vectorc

Null p51 p53

RGC 2 11 111MDM2 2 111 111BAX 2 11 1p21 2 111 11114-3-3s 2 11 11

a His phenotype:2, no growth; 1, growth; 11, moderate growth;111, goodgrowth.

b A series ofHIS3 reporter plasmids containing p53-responsive promoter sequencesderived from indicated genes.

c p51 and p53 expression vectors and a control null vector.

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harboring one of a series of GFP reporter plasmids (Fig. 1) with eitherthe p53 or p51 expression vector. The fluorescence intensities of theresulting transformants were analyzed at 37°C, the physiologicaltemperature for human proteins, and 30°C, the temperature that mayalter the conformations of human proteins expressed in yeast (Fig. 3).As expected, at 37°C (Fig. 3,A and B), p53 activated all four p53target promoters tested in this study, whereas p51 activated only threeof the four promoters, as follows. TheMDM2 reporter that wasactivated strongly by p53 was also activated strongly by p51, theBAXreporter that was activated moderately by p53 was activated stronglyby p51, thep21/WAF1reporter that was activated moderately by p53was activated weakly by p51, and the14-3-3s reporter that wasactivated moderately by p53 was not activated significantly by p51. At30°C (Fig. 3,C and D), significant reductions of the fluorescenceintensity relative to that at 37°C were observed only in cultures thatexpressed p53 with theBAXreporter (76% reduction) and the14-3-3sreporter (77% reduction), whereas no significant changes in GFPexpression levels occurred in cultures expressed p51 (Fig. 3D). These

results suggest that the conformations of the DNA-binding domains ofp51 and p53 that bind to each p53RE differ slightly. Similar resultswere observed when HA-tagged p53 and p51 and a different yeaststrain (YPH499) were used (data not shown).

Comparison of Transactivation by p51 and p53 of p53 TargetGene Promoters in Mammalian Cell Lines.To establish whetherp51 also has the ability to transactivate p53 target promoters inmammalian cells and, if so, whether p51 activates differentially p53target promoters, we cotransfected human osteosarcoma Saos-2 cellswith the p51 or p53 expression vector and a reporter plasmid as wellas an internal control plasmid and subjected them to the dual-lucif-erase assay after incubation for 24 h. The results of representativeexperiments are shown in Fig. 4,A. We used a series of promoterreporters and found that p51 significantly activated thep21/WAF1,MDM2, andBAX promoters but not the14-3-3s promoter. Similarresults were obtained when we used a series of enhancer reporters(Fig. 4A). These results indicate that p51 has the ability to activatep21/WAF1, MDM2, andBAX through the known p53-binding se-

Fig. 3. Transcriptional activation of p53 target genes by p51 in yeast. Yeast (YSIS) cells were cotransformed with a null, p51, or p53 expression vector and a GFP reporter plasmid,which contained p53RE, derived from the indicated gene.A andC, quantitative analysis of the fluorescent signal of GFP. The representative transformants were cultured in 96-wellmicrotiter plates containing SC2leu 2trp solid medium and incubated at 37°C (A) or 30°C (C) for 12 h, and the fluorescence intensity of each plate was analyzed automatically usinga fluoroscanmeter (see “Materials and Methods”).B andD, yeast expressing GFP. Representative transformants were incubated at 37°C (B) or 30°C (D) for 12 h on SC2leu 2trpsolid medium and photographed using a fluorescence microscope.

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quences within the promoters. Similar results were obtained when wesubjected human lung cancer EBC-1 cells (Fig. 4B) to the enhancerreporter assay. The levels of luciferase activity induced by p51 weresignificantly lower than those induced by p53 with all the reporterstested except theBAXreporter. However, we cannot conclude that thetranscriptional activity of p51 was lower than that of p53 because theexpression level of the p51 protein in Saos-2 cells was lower than thatof p53 (Fig. 2) and because our experiments involved overexpression,which means that the data may not reflect physiological conditions.Even so, theBAXpromoter in both cell lines was activated by p51 atlevels comparable with those of p53. We confirmed these observa-tions in experiments using different incubation times (36 and 48 h) fortransfection, higher levels (10-fold) of the expression vector DNAsand HA-tagged p51 and p53 expression vectors (data not shown).These results suggest strongly that the abilities of p51 and p53 totransactivate distinct p53-responsive promoters differ.

Discussion

In this study, we examined the ability of human wild-type p51 toactivate a variety of p53-responsive artificial promoters inSaccharo-myces cerevisiaeand p53-responsive artificial and natural promotersin human cell lines. We found that wild-type p51 activated signifi-cantly distinct p53 target promoters in both yeast and human cells.The results also indicate that transcriptional activation by p51 is directand sequence specific and suggest that not only the structure but alsothe function are well conserved between p51 and p53. Among the

promoters tested, different levels oftrans-activation in both yeast andmammalian cells were observed. In the light of the data from the yeastassay at 30°C and 37°C, we speculate that this difference is caused bya slight difference between the conformations of the DNA-bindingdomains of the two proteins, although we have not compared thedirect interaction between p51 and DNA with that between p53 andDNA using a gel-shift assay.

Recently, two independent groups reported that p73, another p53homologue, alsotrans-activated differentially distinct p53 targetgenes. Di Comoet al. (33) carried out yeast and mammalian tran-scription assays and showed that p73 up-regulated the BAX promoteras efficiently as it did p53, but up-regulated thep21/WAF1andMDM2promoters less efficiently than it did p53. Their results are similar tothe data we obtained in this study. This is not surprising because p51and p73 are the structurally closest relatives of the p53 homologues:the homologies of the DNA-binding domains of p51 and p73, p51 andp53, and p73 and p53 are 87, 60, and 63%, respectively. Furthermore,Zhu et al. (34) showed that tetracycline-regulated p73 expression in alung cancer cell line activated differentially endogenous p53 targetgenes. Among these genes, p73 activated efficiently a subset of thegenes, including14-3-3s, which was not activated significantly in ourstudy. Although the methods used were different, it is likely there aredifferences between p51- and p73-mediatedtrans-activation of dis-tinct p53 target genes. In the light of these observations, we predictthat the two p53 homologues partially but not exclusively sharedownstream p53 target genes and, therefore, differentially regulate thecell cycle and apoptosis in response to currently unknown upstreamsignals. Alternatively, not all the genes reported as p53 targets arefunctionally relevant to p53 under physiological conditions, and someof these and also some currently unidentified genes may be specifictargets of p51 and/or p73. To explore these possibilities, it is obvi-ously important to study both the upstream and downstream signals ofp51 and p73.

Finally, it would be interesting to elucidate whether p53 homo-logues are involved in human tumorigenesis. In previous studies, weand others demonstrated that germ-line and somatic mutations in p53could be screened efficiently by performing yeast-based functionalassays (25, 35, 36). The yeast assay we carried out in this study hastechnical advantages over the previous versions, namely the use ofGFP as a reporter gene, which enables more rapid and quantitativeanalyses and, therefore, simplifies the detection of functionally subtlemutations, and the multiple reporter systems for p53 target genes,which enables the mutations to be characterized. Although there aremany possible applications of this assay for both basic and clinicalstudies of p53 and its homologues, currently we are using it for thedetection and functional evaluation of tumor-derived missense muta-tions in p51.

Acknowledgments

We thank Bert Vogelstein for providing the plasmid pWWW-CAT.

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Fig. 4. Transcriptional activation of p53 target genes by p51 in mammalian cells.A andB, Saos-2 cells were cotransfected with different combinations of a p51, p53, or nullexpression vector (15 ng) and the indicatedpromoter reporter(0.4 mg; A), enhancerreporter (0.4 mg; B), and pRL-CMV (0.4mg) and harvested 24 h after transfection, andthe relative luciferase activity was determined as described in “Materials and Methods.”Columns, means of three values;bars, SE. These mean values were compared using theStudent’s unpairedt test:p, P , 0.05;pp, P , 0.005;ppp, P , 0.0005;NS, no significantdifference.C, EBC-1 cells were subjected to the same experimental procedure as de-scribed forB. Representative data from two independent experiments are shown.

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1999;59:2781-2786. Cancer Res Akira Shimada, Shunsuke Kato, Kentaro Enjo, et al. p51/p63: Similaritiesand DifferencesThe Transcriptional Activities of p53 and Its Homologue

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