Transcriptional activation of tyrosinase and TRP-1 by p53 links UV irradiation to the protective tanning response

Original Paper

Transcriptional activation of tyrosinase and TRP-1 byp53 links UV irradiation to the protective tanningresponse

Karin Nylander1,2,*, Jean-Christophe Bourdon3, Susan E. Bray1, Neil K. Gibbs4, Richard Kay1, Ian Hart5 and

Peter A. Hall11 Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK2 Department of Medical Biosciences/Pathology, UmeaÊ University, UmeaÊ, Sweden3 CRC Cell Transformation Research Group, Department of Biochemistry, University of Dundee, Dundee, DD1 4HN, UK4 Department of Photobiology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK5 Richard Dimbleby Department of Cancer Research, UMDS, St Thomas's Campus, Lambeth Palace Road, London, UK

*Correspondence to:Dr K. Nylander, Department ofMedical Biosciences/Pathology,UmeaÊ University, S-901 87UmeaÊ, Sweden.E-mail:[email protected]

Received: 19 May 1999

Revised: 2 August 1999

Accepted: 23 August 1999

Abstract

We are exposed constantly to potentially harmful compounds and radiations. Complex adaptive

protective responses have evolved to prevent such agents causing cellular damage, including

potentially oncogenic mutation. The p53 tumour suppressor appears to have a role in co-

ordinating such responses: it is activated by diverse insults and it acts as a transcriptional

regulator of downstream genes that facilitate cellular adaptation. Ultraviolet (UV) light is a

particularly potent inducer of p53 expression. In addition, UV light induces the production of

melanin as a protection against further irradiation-induced damage. This study shows that the

promoters of the genes coding for the enzymes crucial in melanin biosynthesis, namely tyrosinase

and tyrosinase-related protein-1 (TRP-1), are activated by wild-type p53. Both promoters have

p53-responsive elements and are activated in vivo in a dose-dependent manner by wild-type p53, as

well as by the p53 homologues p73a and p63a. Copyright # 2000 John Wiley & Sons, Ltd.

Keywords: TRP-1; tyrosinase; p53; DNA damage; promoter; UV light; melanogenesis

Introduction

Chronic exposure to physiologically relevant doses ofultraviolet (UV) irradiation induces a tanning responsein skin, with the synthesis of melanin by melanocytesand the export of this pigment into adjacent keratino-cytes. Accumulation of melanin is recognized as anadaptive response that protects the DNA from furtherdamage and can act as a scavenger of reactive oxygenspecies [1±3]. Clear evidence for the protective functionof melanin comes from the observation that albinoindividuals with homozygous inactivation of tyrosi-nase, one of the crucial enzymes in melanogenesis,have a much increased incidence of skin cancer [4]. Theprotective effects of melanin also extend beyond theepidermis, since heavily pigmented individuals showmuch lesser degree of decomposition of the collagen®bres than those with fair skin [5]. Biosynthesis ofmelanin is limited to speci®c compartments, melano-somes, within the melanocytes and involves severalenzymes, such as tyrosinase and the tyrosinase-relatedproteins-1 (TRP-1) and -2 (TRP-2). The rate-limitingstep in the process is the enzyme tyrosinase (EC1.14.18.1), which catalyses three different reactions,the most critical being the hydroxylation of tyrosine toDOPA in the initiation of melanogenesis [5,6]. Tyro-sinase is essential for the synthesis of both eu- (blackand/or brown) and pheo- (red and/or yellow) melanin;

differences in the activity and expression of the enzymehave been shown between the two processes [7]. Theother two enzymes, TRP-1 and TRP-2, regulate criticalsteps in eu-melanogenesis, whereas their expressionduring pheo-melanogenesis is down-regulated [7].TRP-2 converts DOPA-chrome into DHICA, whichis then oxidized by TRP-1 [8]. TRP-1 also interactswith tyrosinase and in this way signi®cantly stabilizesthe catalytic function of tyrosinase [9] (Figure 1).TRP-1 is also important in the prevention of pre-mature melanocyte death [10].

The p53 tumour suppressor gene encodes a tran-scription factor which, by sequence speci®c transacti-vation of downstream target genes, integrates signalsthat facilitate adaptive responses to cellular stress [11].The best-characterized inducer of p53 activity isgenotoxic insult, but other insults such as hypoxia[12], cytokines and growth factors [13], loss of cellanchorage [14], metabolic alterations [15], and onco-genes [16] have also been shown to induce p53 activity.The activity of the p53 protein is regulated post-transcriptionally by interaction with a number of othercellular proteins and by controlled phosphorylationand acetylation [17,18]. Critical in this is the interac-tion with the mdm2 protein, which can inhibit thetransactivating activity of p53 as well as targeting thep53 protein for ubiquitination and subsequent degra-dation [19].

Journal of PathologyJ Pathol 2000; 190: 39±46.

Copyright # 2000 John Wiley & Sons, Ltd.ccc 0022-3417/2000/010039±08$17.50

For p53 to exert an ef®cient transactivation, a

speci®c binding site is required in the gene to be

transactivated [20,21]. At present, apart from mdm2,

relatively few downstream targets of p53 are well

characterized [22,23]; these include p21waf1 [24], bax

[25], Gadd45 [26], cyclin G [27], PCNA [28], IGF-BP3

[29], B99 [30], and 14-3-3s [31], but the number is

increasing with the use of SAGE [32], differential

display, [33,34] and CHIP techniques. However, the

physiological relevance of many putative p53-

responsive genes remains unclear.As UV light-stimulated skin shows the accumula-

tion of p53 protein as well as a marked increase in

tyrosinase activity [35±38], we reasoned that the

induction of melanogenesis by UV might, in part,

be mediated by the activation of the p53 pathway. In

this paper we provide evidence that tyrosinase and

TRP-1 are examples of cell type-speci®c p53-

responsive genes.

Materials and methods

PCR ampli®cation

The promoter regions of tyrosinase and TRP-1 were

analysed for the presence of p53 binding sites [20].

From the mouse TRP-1 promoter, an area comprising

697 bp (starting at position 797 in GenBank sequence

accession M84966), including a six-decamer p53 bind-

ing site, was ampli®ed using the primers 5k TGTC-

AGTCAAAGCAGAAGAAC and 3k CTCCTT-

CAGAGCTAGTGTTG. From the human TRP-1

promoter, an area comprising 306 bp (starting at

position 2414 in GenBank sequence accession

L38952) was ampli®ed using the primers 5k CCAAAT-

TAGT-GCTTCTGGCC and 3k CCTCACAATCCT-

GAAGGATG. After sequencing, fragments were

ligated upstream of the luciferase gene in the promo-

terless plasmid pGL3 basic (Promega).

Plasmids

Apart from the above constructs, the followingplasmids were also used: 2.5 kb of the tyrosinasepromoter, in pGL3 vector [39]; Adluc, minimalpromoter of the major late promoter of the adenovirus,in pGL3 vector; IGFBP3 promoter, in pGL3 vector[20]; pGL3 basic vector without promoter insert; pRLSV40 encoding for the renilla luciferase expressedunder SV40 promoter (Promega); p53 cDNA underSV40 promoter (gift of Dr E. May), and mutant p53(ala 143) under CMV promoter (gift of Dr C.Midgley). Mdm2 luc was also used as a positivecontrol in transfections (data not shown). Wild-type(wt) p53, p63a, and p73a (gift of Drs C. Midgley, H.Schmale, and C. Jost) were cut out of pcDNA3plasmid and ligated into plasmid under SV40 promoterand used in parallel transfections comparing thetransactivating capacity of p53, p63a, and p73a onthe tyrosinase and TRP-1 promoters.

Cells

The human osteosarcoma cell line Saos-2 was grown inDulbecco's Modi®ed Eagle's Medium with 10% fetalcalf serum in a humidi®ed atmosphere with 10% CO2.The human p53 null melanoma cell line UISO-MEL-6(kindly provided by Dr T. Das Gupta), was grown inEarle's Modi®ed Eagle's Medium with 18% FCS, 5 mlof 100X glutamine (Gibco), 5 ml of 100X non-essentialamino acids (Gibco), and 6 ml of 100X antibiotics/antimycotics, in a humidi®ed atmosphere with 5%CO2.

For transfection, cells were split and seeded into six-well plates.

Reporter assays

Wild-type p53 under SV40 promoter at varyingconcentrations (0, 0.05, 0.1, 0.2, 0.5, 0.75, and1.0 mg/ml) as well as the mutant p53 (ala 143) underCMV promoter (at 0.1 mg/ml) was co-transfected intoSaos-2 cells with 0.5 mg of each of the above-describedreporter genes. As an internal control for the transfec-tion ef®ciency, 0.005 mg of the pRL-SV40 vector withan SV40 promoter upstream of renilla luciferase wasused. Saos-2 cells were transfected using Lipofectamine(GIBCO) and promoter activity was assayed using thePromega Dual-Luciferase Reporter Assay.

Transfections were performed at least four times,and for each, a total of 2 mg of DNA was resuspendedin serum-free DMEM and mixed with Lipofectamine.Cells were incubated with the DNA/Lipofectaminesolution for 5 h, when medium with 20% serum wasadded. After a total incubation of 20±24 h, cells weregiven new medium and left for another 24 h. Cells werethen harvested with Lysis buffer (Dual-LuciferaseReporter Assay System, Promega), and luciferaseactivity was measured using a Turner Designs Lumin-ometer model TD-20/20. A ratio between ®re¯y- andrenilla-luciferase activity was calculated and the results

Figure 1. Simpli®ed scheme of the melanogenic pathway.Modi®ed after Kobayashi et al. [8] and Jimbow et al. [10] Thearrow symbolizes the interaction between TRP-1 and tyrosinase,which has a stabilizing effect on the catalytic function of thelatter

40 K. Nylander et al.

Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 190: 39±46.

were shown as the mean of different ratios achievedfrom all separate transfections.

Electrophoretic mobility shift assay (EMSA) forp53 sequence-speci®c DNA binding

Wild-type p53 protein was produced in a baculovirusexpression system in Sf9 cells (kindly provided byAshley Craig; as described in ref. 40), and puri®edfractions active for DNA binding were then used [41].From the promoter region of the TRP-1 reporter geneconstruct, a 113 bp fragment comprising the p53binding site was ampli®ed by the polymerase chainreaction (PCR), using the same primers as beforefollowed by restriction digest with the enzymes AccIand NdeI, gel puri®cation (Qiaex Gel Extraction Kit,Promega), and labelling using 32P dATP and Klenowpolymerase.

For the EMSA, 7 ml of a buffer consisting of 25%(v/v) glycerol, 150 mM KCl, 60 mM HEPES (pH 7.5),0.3 mM EDTA, 0.2% Triton X-100, 30 mM MgCl2,3.0 mg/ml BSA, and 15 mM DTT was incubated with14 ng of super coiled plasmid DNA, 1±2 ng of labelledoligonucleotide, 3 ml of pure p53 protein diluted 1/5,and H2O to a ®nal volume of 12 ml for 20±30 min atroom temperature. For competition experiments,unlabelled double-stranded oligonucleotides either con-taining a perfect p53 consensus sequence (consensus[42]) at 12- and 25-fold molar access or non-speci®c(non-consensus; pBluescript plasmid) at 25- and 40-fold molar access were added to the binding reaction.For a supershift assay, the monoclonal antibody DO1was added to the reaction mixture. Samples were runon a 4% non-denaturing polyacrylamide gel at 200 Vfor 2 h at 4uC, and dried prior to exposure to X-ray®lm.

Deletion analysis of the TRP-1 promoter

The six-decamer region in the TRP-1 promoter was cutout with restriction enzymes Asp718/NdeI and afterseparation on gel, the remaining vector was repairedand self-ligated, and used in transfections as describedabove.

Deletion analysis of the tyrosinase promoter

Two different deletion constructs of the tyrosinasepromoter were made. The ®rst, using the restrictionenzymes AvrII/SpeI and self-ligation, left around 1000basepairs (bp) of the promoter in the reporter gene.The second deletion construct comprised around300 bp of the basal promoter located 43 bp upstreamof the ATG and was made in two steps. First thevector was cut with SpeI/XhoI and after gel separation,the longer fragment (4805 bp) was cut out. Anotheraliquot of the vector was cut with XbaI/XhoI,generating four fragments. The smallest of thesefragments (305 bp), after separation on gel, was ligatedwith the 4805bp fragment from the other restrictiondigest.

Results

Potential p53 binding sites exist in the promotersof the TRP-1 genes

When analysing the promoters of the human andmouse TRP-1 gene and the mouse tyrosinase gene, aregion comprising six decamers (each consisting ofPuPuPuCA/TT/AGPyPyPy) was found in the mouseTRP-1 promoter (Figure 2). A region of two clusters ofthree decamers separated by 14 bp was found in thehuman TRP-1 promoter at position x183 from theATG site (Figure 3). For the mouse TRP-1 promoter,the decamers are separated by 4, 5, 2, 8, and 5 bases,respectively, and localized at x601 bp from the ATG(Figure 2).

Using a computerized search algorithm [20], no p53binding site ful®lling the criteria for ef®cient transacti-vation was found in the tyrosinase promoter. Never-theless, to investigate fully any potential role for p53 inmelanogenesis, we included 2.5 kb of the mousetyrosinase promoter in a reporter construct upstreamof the luciferase gene in the promoterless plasmidpGL3 basic. The control promoters were also ligatedupstream of the luciferase gene in the same kind ofpromoterless plasmid.

Wt p53 transactivates both the tyrosinase andthe TRP-1 promoters in vivo

On sequencing of the TRP-1 promoter, a differenceconsisting of a deletion of one base (C) and exchange ofCAG into GTC at one position to the reportedGenBank sequence was found and accordingly reportedto GenBank (accession number AF087673). Thesechanges did not affect the p53-responsive element.

Twenty to twenty-four hours after co-transfection,luciferase ®re¯y and luciferase renilla activities wereanalysed. Renilla luciferase is used as an internalcontrol, taking into account the difference in transfec-tion ef®ciency. In comparison to pGL3 basic, it isclearly shown that wt p53, in a dose-dependentmanner, transactivates mouse and human TRP-1promoters. Transactivation is speci®c for wt p53, asno activation was caused by mutant p53 (Figure 4).For the tyrosinase promoter, we also observed a dose-dependent induction by p53 (29-fold activation for thehighest amount of p53 used). No transactivation wasfound by the mutant p53, suggesting that transactiva-tion is speci®c for wt p53, despite the absence of acanonical p53 binding site (Figure 4). All results wereconsistent and reproducible in at least four separatetransfections, made in duplicate and performed withdifferent plasmid preparations.

Binding of wt p53 to the six-decamer site in theTRP1 promoter shown by EMSA

To see whether wt p53 actually binds to the six-decamer region in the mouse TRP-1 promoter, afragment comprising this site was used in a binding

Tyrosinase and TRP-1 are p53-responsive genes 41


reaction, showing clear binding of wt p53 to this site.

The speci®city of the p53 binding was con®rmed by

adding DO1 antibody to the binding reaction, causing

the formation of a larger complex and accordingly a

supershifted complex at gel separation. Furthermore,

the addition of a cold sequence containing a perfect

p53 consensus sequence [42] completely competed the

labelled probe, whereas the addition of an unlabelled

sequence, devoid of p53 binding sites, did not compete

the labelled probe. These results con®rm that p53 binds

speci®cally to the TRP-1 promoter within the six-

decamer region (Figure 5).

Figure 2. Sequence of the p53 binding site in the mouse TRP-1 promoter. The sequence of the TRP-1 promoter with the sixdecamers is boxed. Putative transcription start sites are marked with arrows (GenBank accession X59513)

Figure 3. Sequence of the p53 binding site in the human TRP-1 promoter. The sequence of the TRP-1 promoter with the p53binding site is boxed. The transcription start site is marked with an arrow (GenBank accession L38952)



Deletion of the six decamers from the TRP-1reporter construct completely deletedtransactivation by wt p53

To assess the importance of the decamer region in themouse TRP-1 promoter for transactivation by p53, thewhole area comprising the six decamers was cut out,and in the transfection assay this construct showed aresidual promoter activity not inducible by p53 (Figure6). This con®rms that the p53 binding site that weidenti®ed is functional and required for transactivationby wt p53.

Deletion analysis of the tyrosinase promotershowed the presence of a p53-responsiveelement within 300 bp of the promoter

To pinpoint the region containing a potential p53-responsive element in the tyrosinase promoter, twodeletion constructs were made. The results fromtransfection showed that 300 bp of the promoterstarting at position x347 bp is still fully p53-responsive. When comparing transactivation of theentire promoter to transactivation of the deletionconstructs, the basal activity of the whole promoterwas weaker, indicating the presence of a repressor sitewithin the 1.5 kb furthest upstream of the ATG site(Figure 7).

p63a and p73a also transactivate the tyrosinaseand the TRP-1 promoters in vivo

To assess whether p63a and p73a had similar transacti-vating effects on the promoters studied, paralleltransfections with p53, p63a, and p73a were performed,showing equivalent transactivating capacity on thetyrosinase promoters by all three (Figure 8). For the

TRP-1 promoter, p73a showed better transactivatingcapacity at low doses of transfection, whereas at highdoses no signi®cant difference could be seen betweenthe three (Figure 9). The results were con®rmed in threeindependent experiments. Each of these three sets oftransfections was performed on the same day under thesame conditions and transfections were also normalizedas before with renilla activity. The physiologicalsigni®cance of this small difference is unclear.

Discussion

The induction of p53 protein levels and activity byrecreational doses of solar simulated light [35] and

Figure 4. Activation of the TRP-1 and tyrosinase promoters bywt p53. The results from four independent transfections in Saos-2 cells were averaged. In all cases, the mean is shown andstandard deviations are indicated with error bars. The y-axisshows the ratio between ®re¯y and renilla luciferase. Each barrepresents a different amount (0, 0.25, 0.5, 0.75, and 1.0 mg/ml)of SV40-p53 expression vector. The amount of CMV mutant p53(ala 143) was constant, 0.1 mg/ml

Figure 5. Speci®c binding of wt p53 protein to the six-decamerregion in the TRP-1 promoter. Binding of pure p53 protein tothe six-decamer region in the TRP-1 promoter is shown in lane1. The arrows show different bands of p53 bound to DNA. Theformation of more than one band could be due to differentstates of conformation or oligomerization of p53 bound to thesix-decamer p53 binding site. By adding the antibody DO1, asupershift was obtained (lane 2). Competitions were made withunlabelled double-stranded oligonucleotides, either the p53consensus sequence (cons; at 12- and 25-fold excess; lanes 3and 4) or non-speci®c (pBluescript plasmid DNA) (ns; at 25- and40-fold excess; lanes 5 and 6)



induction of melanin by DNA damage [36,37] led us tohypothesize that p53 might be a physiological mediatorof the tanning response. The experiments describedhere show that both genes coding for the crucialenzymes in melanin biosynthesis, tyrosinase and TRP-1, are potential p53-responsive genes. Accordingly, wedemonstrated that p53 can transactivate both TRP-1and tyrosinase promoters and also bind to a six-decamer site in the TRP-1 promoter. The region of thetyrosinase promoter which might contain a p53-responsive element mapped to within the 300 bplocated 43 bp upstream of the ATG site. Data fromtransfections also showed the presence of a repressorsite within the 1500 bp furthest upstream of the ATGsite. We are currently investigating whether thetyrosinase promoter is directly or indirectly transacti-vated by wt p53.

Based on the present results, we propose a model fortanning in response to UV irradiation in which UVinduces p53, which then targets and activates thepromoters of tyrosinase and TRP-1. The tyrosinaseprotein then initiates the melanogenic process, and is inthis task being stabilized, equalling stimulated, in itscatalytic activity by the TRP-1 protein (Figure 10).

Earlier Kichina et al. [43] reported that p53suppresses tyrosinase expression. In those experiments,a shorter fragment (270 bp compared with our2500 bp) of the mouse tyrosinase promoter was used.In contrast to Kichina et al., we transfected smallamounts of p53 expression vector driven by SV40promoter (weaker than CMV promoter) in order toreduce promoter quenching (TRP-1 and tyrosinase arevery weak promoters compared with CMV) and celldeath induced by p53. Moreover, we used renilla totake into account the variation in transfection ef®-ciency. All those different experimental conditions mayexplain our opposite results. p53 is further known tocause a dose-related response; these differentialresponses are of physiological relevance, since differinglevels of p53 protein have quite different effects ondifferent promoters [44] and there are clear differences

in the consequences of varying levels of p53 expression[45,46].

The cells employed in this study, Saos-2 (usedbecause they lack endogenous p53 protein), may notcontain the cell type-speci®c transcription factorstypically seen in melanocytes. Unfortunately, it wasnot possible transiently to transfect with wt p53, thep53 null melanocytic cell line which we have (UISO-MEL-6; kindly provided by Dr T. das Gupta), becauseeven at low doses of p53 the cells died.

Both promoters were also transactivated by the p53homologues p63a and p73a. Earlier studies of malig-nant melanomas and melanocytic cell lines have shownthat inactivation of p73 by mutation is uncommon inthese cell lines [47,48].

An important point that arises from this study isthat both tyrosinase and TRP-1 are examples of cell

Figure 6. Deletion of the site comprising the six decamers inthe TRP-1 promoter. After deletion of the six-decamer region inthe TRP-1 promoter, no p53 transactivation could be measured.Each bar represents a different amount of p53. The y-axis showsrelative luciferase activity, calculated as the ratio luciferase/renilla

Figure 7. Deletion analysis of the tyrosinase promoter. Bydeleting different parts of the tyrosinase promoter, the areacomprising the p53-responsive element was mapped to lie within300 bp located 43 bp upstream of the ATG site (construct XhoI)(ATG starting +1). Each bar represents a different amount ofp53 as in Figure 6. The size of the initial promoter, as well as thetwo deletion constructs, is also shown

Figure 8. Parallel transfections with p53, p63a and p73a and thetyrosinase promoter. In parallel transfections, p53, p63a, andp73a showed similar transactivating capacity on the tyrosinasepromoter. The x-axis shows varying levels of p53, p63a, andp73a, and the y-axis the ratio between luciferase and renillasignals (relative luciferase activity). The ®gure represents two ofthe three experiments performed, each done in triplicate.Results from the third experiment showed equivalent results



type-speci®c p53-regulated genes, rather than the moreglobal responses previously reported. Such a view isemphasized by the remarkable tissue restriction of p53protein accumulation and its transcriptional activitiesin vivo [49,50]. Recently, the presence of both thetyrosinase and the TRP-1 enzymes has also beenshown within the central nervous system, and dataindicate that tyrosinase might be involved in theproduction of neuromelanin. In neurodegenerativedisorders such as Parkinson's disease, neuromelanin isdecreased, suggesting that tyrosinase could possibly beinvolved in or contribute to these processes [51].

In conclusion, we have demonstrated that p53 cantransactivate the tyrosinase and TRP-1 promoters.This identi®es tyrosinase and TRP-1 as potential p53-responsive genes with an obvious physiological role inthe adaptive response to exogenous stress, such asexposure to UV light. The data presented here provideclear evidence that a wider perspective of the adaptiveroles of p53 is warranted, above and beyond growtharrest and cell death [11,52]. Finally, our observationshighlight the need to reconsider the strategiesemployed for the identi®cation of p53-responsivegenes, with an increased focus on both differentiation-speci®c and developmental stage-speci®c regulation ofgenes by p53.

Acknowledgements

We thank Dr Evelyne May, Dr Carol Midgley, Dr Hartvig

Schmale, and Dr C. Jost for plasmids; Ashley Craig for wild-type

p53 protein; Dr Tapas das Gupta for the melanoma cell line; and

Dr Philip Coates, Dr David Meek, and members of the Hall

Laboratory for invaluable discussions. We gratefully acknowl-

edge the ®nancial support of the CEC (Contract 362899), The

Lion's Cancer Research Foundation, UmeaÊ University, Tore

Nilson's Foundation for Medical Research, and the Swedish

Dental Society. KN has a UICC International Cancer Technol-

ogy Transfer Fellowship, ICRETT No. 866. SB has a Student-

ship from the Pathological Society of Great Britain and Ireland.

Work in the Hall Laboratory is supported by the EU, the

Department of Health, the AICR, and the Pathological Society

of Great Britain and Ireland.

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Figure 9. Parallel transfections with p53, p63a, and p73a andthe TRP-1 promoter. In parallel transfections, p73a, for loweramounts of protein, showed higher transactivating capacity thanboth p53 and p63a. p53 and p63a showed fairly similartransactivational capacity. The x-axis shows varying amounts ofp53, p63a, and p73a transfected. The y-axis shows fold activationof TRP-1 promoter activity. Mean fold activation and standarddeviation are indicated. The ®gure represents three independentexperiments, two made in triplicate and one in duplicate

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Transcriptional activation of tyrosinase and TRP-1 by p53 links UV irradiation to the protective tanning response