Oral Oncology (2008) 44, 156–161

ava i lab le at www.sc iencedi rec t . com

journal homepage: ht tp : / / in t l .e lsevierheal th .com/ journals /oron/

Expression of novel p53 isoforms in oral lichen planus

Majid Ebrahimi a,b,*, Linda Boldrup b, Philip J. Coates c, Ylva-Britt Wahlin a,Jean-Christophe Bourdon d, Karin Nylander b

a Department of Odontology, Umea University, SE – 901 85 Umea, Swedenb Department of Medical Biosciences/Pathology, Building 6M, 2nd floor, Umea University, SE – 901 85 Umea, Swedenc Division of Pathology and Neurosciences, University of Dundee, Ninewells Hospital and Medical School,Dundee DD1 9SY, UKd Department of Surgery and Molecular Oncology, University of Dundee, Ninewells Hospital and Medical School,Dundee DD1 9SY, UK

Received 5 December 2006; received in revised form 17 January 2007; accepted 17 January 2007Available online 5 April 2007

Summary Oral lichen planus (OLP) is a chronic inflammatory disease of unknown origin, show-ing little spontaneous regression. WHO classifies OLP as a premalignant condition, however, theunderlying mechanisms initiating development of cancer in OLP lesions are not understood. Thep53 tumour suppressor plays an important role in many tumours, and an increased expression ofp53 protein has been seen in OLP lesions. Recently it was shown that the human TP53 geneencodes at least nine different isoforms. Another member of the p53 family, p63, comprisessix different isoforms and plays a crucial role in the formation of oral mucosa, salivary glands,teeth and skin. It has also been suggested that p63 is involved in development of squamous cellcarcinoma of the head and neck (SCCHN). In contrast to p53, a decreased expression of p63 pro-tein has been seen in OLP lesions. In this study, we mapped the expression of five novel p53isoforms at RNA and protein levels in OLP and matched normal controls. In the same sampleswe also measured levels of p63 isoforms using quantitative RT–PCR. Results showed p53 tobe expressed in all OLP lesions and normal tissues. The p53b and D133p53 isoforms wereexpressed in the majority of samples whereas the remaining three novel isoforms analysed wereexpressed in only a few samples. Levels of p63 isoforms were lower in OLP lesions comparedwith normal tissue, however, changes were not statistically significant.

�c 2007 Elsevier Ltd. All rights reserved.

KEYWORDSp53;p63;Isoforms;OLP

1d

e8

368-8375/$ - see front matter �c 2007 Elsevier Ltd. All rights reservedoi:10.1016/j.oraloncology.2007.01.014

* Corresponding author. Address: Department of Medical Biosci-nces/Pathology, Building 6M, 2nd floor, Umea University, SE – 9015 Umea, Sweden. Tel.: +46 90 785 2787; fax: +46 90 785 2829.E-mail address: majid.ebrahimi@medbio.umu.se (M. Ebrahimi).

Introduction

Oral lichen planus (OLP) is a chronic inflammatory disease ofskin and mucosa affecting approximately 1–2% of the adultpopulation. The disease occurs more frequently in women

.

Expression of novel p53 isoforms in oral lichen planus 157

than in men (1.4:1) and is more common in middle aged andelderly than in young people.1 In contrast to lichen of theskin, OLP seems to have a chronic cause with low tendencyto spontaneous regression and high resistance to topicaltreatment.2 Despite numerous studies the origin remainsunknown. However, an autoimmune cause has been sug-gested3 and we have recently demonstrated occurrence ofauto antibodies against p63 and p73 in sera from patientsdiagnosed with OLP.4

OLP lesions have a higher cell turnover than normal tis-sue5 and WHO classifies OLP as a premalignant conditionmaking ‘‘oral mucosa more sensitive to exogenous carcin-ogens and thus to develop oral carcinoma’’.6 Malignanttransformation to squamous cell carcinoma has been seenin 2–3% of OLP patients. Females with OLP run a 50% high-er risk of developing squamous cell carcinoma of the headand neck (SCCHN) compared to a control group.6 Theunderlying mechanisms initiating development into SCCHNhave not been clarified, however, an association betweenchronic inflammation and a variety of cancers isknown.7–9

Approximately two decades ago the p53 tumour suppres-sor was discovered. Activation of p53 after DNA damage oroncogenic signalling is an important protective mechanismenabling DNA repair, induction of cell cycle arrest or apop-tosis. Mutation within the human TP53 gene is found inapproximately half of all human tumours studied and a highprevalence of TP53 mutations has been seen in patientsdiagnosed with OLP, particularly females.10 We and otherinvestigators have previously shown an increased expressionof p53 protein in OLP lesions compared with normal tis-sue.10–18

Recently it was shown that human p53 encodes at leastnine different isoforms through alternative splicing, alter-native initiation sites for translation and the use of aninternal promoter: p53, p53b, p53c, D133p53, D133p53b,D133p53c, D40p53, D40p53b and D40p53c.19 In the firststudy of these novel p53 isoforms in SCCHN it was shownthat p53 variant mRNAs were expressed in both normal oralepithelium and SCCHN. The most common of these isoformsbeing p53b, which was detected in the majority of samples.Of the other isoforms studied all but Dp53 could be de-tected in at least some samples from tumour and normalepithelium.20

The p53 relative p63 is encoded by a gene located onchromosome 3q27–29. In humans this gene encodes six dif-ferent isoforms: TAp63a, TAp63b, TAp63c, DNp63a, DNp63band DNp63c.21,22 p63 is essential for development of ecto-dermal structures, and p63 �/� mice lack oral mucosa,skin, teeth, hair follicles, salivary and mammary glands.23,24

The exact function of each individual isoform is, however,not clear. It is known that each of the p63 proteins have dif-ferent characteristics and functions, where some resembleits relative the tumour suppressor protein p53, while othershave the opposite function.25 We have previously shown adecreased expression of the different p63 proteins in OLPlesions compared with normal oral mucosa.18 ConcerningRNA levels of the different isoforms, no data is as of todayavailable.

The aim of the present study was to clarify if the ob-served increased expression of p53 protein seen in OLP le-sions represents a mixture of the different novel p53

isoforms or the original p53 isoform only. Furthermorewe wanted to see in the same samples, if there was anycorrelation between RNA levels of the different p63 iso-forms, and the decrease in protein expression that wehave previously seen in these lesions. In order to judgewhether OLP in this aspect resembles normal or tumourtissue, normal matched control tissue was analysed in par-allel, and results compared with data from analysis ofSCCHN.

Materials and methods

Patients and specimens

Two 4 mm punch biopsies were, after informed consent, ta-ken from the buccal mucosa on 20 consecutive patients withOLP referred to the specialist clinic at the department ofOdontology, Umea University. All patients were clinicallyand histologically diagnosed with oral lichen planus. Four-teen were females with a mean age of 63.5 (range 42–78)and six were males with a mean age of 59.5 (range 48–75). Biopsies were also collected from twenty sex and agematched healthy controls. All samples were immediatelyfrozen and stored at �80 �C. Permission for the study hadebeen granted by the Ethical Committee at Umea University(dnr 05-010 M).

RNA extraction

One of the frozen biopsies was cut into pieces in a Petridish placed on dry ice and transferred into a 1.5 ml tubecontaining 250 ll TRIzol reagent (Invitrogen). Sampleswere homogenized using a pellet mixer (MARCK Eurolab),a further 750 ll of TRIzol added and samples incubatedat room temperature for 5 min. Mixtures were then centri-fuged for 15 min at 13,000 rpm at 4 �C. The upper liquidphase was moved to a new tube, diluted with an equalamount of isopropyl alcohol, incubated for 10 min andagain centrifuged for 10 min at 4 �C. The upper layer wascarefully removed and pellets washed in 70% ethanol be-fore drying and dissolved in DePc-H2O by pipetting 10–20times, heated to 60 � C for 10 min and pipetted another10–20 times. Extracted RNA was then kept at �80 �C untiluse.

cDNA synthesis and RT–PCR for analysis of p53isoforms

cDNA was synthesized using a Cloned AMW First-Stand cDNASynthesis kit (Invitrogen) according to the manufacturersinstructions. One microgram of RNA was added into a totalreaction volume of 20 ll. The cDNA was amplified in tworounds of PCR (nested PCR) in order to detect the differentisoforms of p53. The following p53-specific primer sets wereused: for the first PCR-round of p53, p53b, and p53c: e2.1/RT1. For D133p53, D133p53b, and D133p53c: i4f1/ RT2were used. For the nested PCR the primers for p53 were:e2/RT2; for p53b: e2/p53b; for p53c: e2/p53g; forD133p53: i4f2/RDNp53; for D133p53b: i4f2/p53b, and forD133p53c: i4f2/p53g.

Primer sequences : e2:1 ð50-GTCACTGCCATGGAGGAGCCGCA-30Þe2 ð50-ATGGAGGAGCCGCAGTCAGAT-30Þi4f1 ð50-CTGAGGTGTAGACGCCAACTCTCTCTAG-30Þi4f2 ð50-GCTAGTGGGTTGCAGGAGGTGCTTACAC-30ÞRT1 ð50 � GACGCACACCTATTGCAAGCAAGGGTTC-30ÞRT2 ð50-AATGTCAGTCTGAGTCAGGCCCTTCTGTC-30Þp53b ð50-TTTGAAAGCTGGTCTGGTCCTGA-30Þp53g ð50-TCGTAAGTCAAGTAGCATCTGAAGG-30ÞRDNp53 ð50-CTCACGCCCACGGATCTGA-30Þ

158 M. Ebrahimi et al.

The PCR programme comprised 35 cycles with 94 �C for30 s, 60 �C for 45 s and 72 �C for 90 s. PCR products wereloaded on a 1% agarose gel.19 All samples were done induplicate and most of them in triplicate, and considered po-sitive when positive in all replicates, or positive/variablewhen isoforms were detected in at least one PCR run.

Quantitative PCR for analysis of p63 isoforms

Quantitative RT–PCR for analysis of p63 isoforms was per-formed using a human p63/b-actin multi-parametric kit(Search LC, Heidelberg, Germany) and analysed on a Light-cyclerTM from Roche. In brief, cDNA was diluted 12 timesand thenmixedwith the different primer sets to amplify eachof the p63 splice variant mRNAs individually and, simulta-neously amplify b-actin to serve as an internal control forcDNA integrity and relative cDNA quantity in the different tis-sue samples. Primers were selected to bridge intronicsequences in order to discriminate between amplification ofcDNA and contaminating genomic DNA. Reactions were ana-lysed on the LightcyclerTM with one denaturation cycle, 50amplification cycles, one melting curve analysis cycle, and,finally, one cooling cycle. Reactions were run in duplicate,and a mean value of the two samples was calculated.26

Statistical analysis

For analysis of p63 mRNA levels in OLP lesions and corre-sponding clinically normal oral mucosa an independent sam-ples T-test was performed using Computer software SPSSversion 12.0.

Protein extraction

From one frozen biopsy protein was extracted using a Micro-dismembrator (B. Braun. Biotech International) for pulveriz-ing the sample. One hundred microliter of lysis buffer con-taining 0.5% NP-40, 0.5% Na-doc, 0.1% SDS, 150 mM NaCl,50 mM Tris pH 7.5, 1 mM EDTA, 1 mM NaF and proteaseinhibitor cocktail (Sigma-Aldrich Chemie, Steinheim, Ger-many) was added and the pulverized samples homogenized.Protein concentrations were determined using BCA ProteinAssay Kit (Pierce, Rockford, IL, USA).

Immunoblot analysis and quantification

Bio-Rad PROTEAN II xi ΠXL Vertical Electrophoresis systemor vertical mini-gels were used according to the user man-

ual. Thirty microgram of protein was mixed with 2· loadingbuffer, boiled for 10 min and electrophoresed on 10% SDS-polyacrylamide gels. Proteins were transferred to nitrocel-lulose membranes (Hybond ECL, Amersham Biosciences, Lit-tle Chalton, NA, USA) and stained with ponceau red forevaluation of transfer efficiency and loading. Membraneswere blocked in 5% milk with 0.4% Tween in 1X PBS, andantibodies diluted in the same blocking solution. Primaryantibodies used in this study were DO-1 for detection ofp53, p53b, p53c.27, and DO-12 for detection of p53, p53b,p53c, D133p53, D133p53b, D133p53c, D40p53, D40p53b,and D40p53c isoforms.28

Results

Expression of p53 isoforms using RT–PCR

By the use of RT–PCR, six different p53 isoforms could beamplified from OLP tissue as well as normal tissue: p53,p53b, p53c, D133p53, D133p53b, and D133p53c. All sampleswere analysed as matched pairs, meaning that one OLP le-sion and corresponding matched control were analysed inparallel, though coded, as to hide which sample was lesionand which was control. p53 mRNA could be detected in rep-licate analyses of all but 1 of the 20 OLP samples as well asall but 1 of the 20 controls. These two samples were positivein one analysis, but negative in the other. The majority ofOLP and control samples also expressed the p53b andD133p53 isoforms in at least one of the replicate analyses.Expression of p53c, D133p53b and D133p53c was, however,limited to only a few samples. In general the novel p53 iso-forms studied were detected in fewer OLP samples com-pared to matched controls (Fig. 1).

Expression of p63 isoforms

Using quantitative RT–PCR analysis of p63 showed that p63ahad highest and DNp63 second highest level of all p63mRNAs. TAp63 had lowest levels in both OLP and corre-sponding normal tissue. For all isoforms individual variationscould be seen (results not shown). Results also showed ageneral decrease in levels of p63 isoforms in OLP comparedto normal tissue. The most pronounced decrease seen inexpression of p63c, p63a and DNp63 (P = 0.116; 0.187 and0.205, respectively). The level of TAp63 was unchangedwhile a slight reduction in p63b was found in OLP lesionscompared to normal tissue. None of these changes were,however, statistically significant (Fig. 2).

Figure 1 Results from nested RT–PCR analysis of six p53 isoforms. Bars show percentage of positive samples. In total 40 sampleswere analysed, 20 OLP lesions and 20 sex and aged matched normal tissues. P53 mRNA was detected in all OLP lesions and normaltissue, either in all replicates analysed (positive) or in one or two of the replicates (positive/variable). Of the five novel p53 isoformsanalysed, p53b and D133p53 were detected in the majority of samples, while the remaining three novel isoforms only were detectedin a few samples.

Figure 2 Quantitative RT/PCR of the different p63 isoforms in OLP lesions and controls. Black bars represent OLP lesions andwhite bars normal controls. Results are expressed as mean for all samples analysed in each group and are based on the relativeamount of p63 RNA for each sample analysed adjusted for the level of internal control amplification (b-actin) obtained for eachsample. The most pronounced decrease was seen in levels of DNp63, p63a and p63c. Levels of TAp63 were basically unchanged whencomparing OLP lesions and controls, while a slight reduction in p63b was seen in OLP. Note that different scales are used for the twogroups TAp63, p63b, p63c and DNp63, p63a, respectively.

Expression of novel p53 isoforms in oral lichen planus 159

Immunoblot analysis

In order to detect different p53 isoforms also at proteinlevels, two antibodies were used. DO-1 recognising an epi-tope located at amino acids 20–25, and therefore not rec-ognising the N-terminal truncated D133p53 or D40p53isoforms and DO-12 recognising the p53, p53b, p53c,D133p53, D133p53b, D133p53c, D40p53, D40p53b, andD40p53c isoforms.20 All samples were run in duplicate andresults showed detectable levels of p53 protein in onlyone of the OLP samples. This protein had an identicalmolecular weight to p53 and bands that might representp53-isoforms were not detected (data not shown).

Discussion

Increased expression of the p53 tumour suppressor proteinhas been seen in studies of oral lichen planus, simulta-neously with a decreased expression of its sibling p63.10–18

With the discovery of several novel p53 isoforms we there-fore wanted to see if this increased expression of p53 pro-tein in fact represented different isoforms which could beimportant in OLP lesions. By using RT/PCR we could detectthree of the isoforms in the majority of OLP and controlsamples, whereas the remaining three isoforms were de-tected in only a few samples. In a previous study of p53 iso-forms in SCCHN we have shown that the method used,20

160 M. Ebrahimi et al.

nested RT/PCR, is very sensitive and identifies extremelylow levels of mRNA. This was clearly shown when comparingRT/PCR results with just one single round of PCR. Using thesingle round PCR only the p53, p53b and D133p53 isoformswere detected.20 These three isoforms were also the onesthat could be amplified in the majority of our samples.The inconsistencies seen between replicates of the samesamples, using different batches of cDNA, is, as in the studyof SCCHN, compatible with low levels of the novel p53 iso-form mRNAs. Also, based on the comparatively higher incon-sistency in amplification of the p53b and D133p53 isoformsseen in this study, we suggest that levels of these isoformsare in fact much lower in OLP lesions compared to tumour-and clinically normal tumour adjacent tissue in patientswith SCCHN. This could in turn indicate a more tumour re-lated role for these isoforms. The fact that we in the pres-ent study used three times as much RNA for cDNA synthesis,1 lg of total RNA compared to 300 ng from the tumours, fur-ther supports the theory that levels of the novel p53 iso-forms really are lower in OLP lesions compared to SCCHN.

By using immunoblotting and antibodies detecting p53/p53b/p53c and all isoforms analysed respectively, only thep53 protein could be detected in one of the OLP samples,whereas around half of the tumours and a little fewer ofthe normal tumour adjacent tissue had detectable levelsof p53 protein.20 However, none of the novel p53 proteinswere expressed at detectable levels in the present or previ-ous study. It should be kept in mind that wild type p53, andmaybe also these novel isoforms, is predominantly regu-lated by post-translational mechanisms, and therefore a di-rect correlation to RNA levels cannot be expected.29

However, with the reagents available today, none of the no-vel isoforms could be detected. Other possible explanationscould be short half life of the isoforms, in concordance withwild type p53, or tissue specific expression. In the lattercase translation of RNA is dependent on tissue specificcofactors and with these absent, RNA is not translated intoprotein.

For the p53 sibling p63 we could see a down regulation ofall isoforms at the RNA level in OLP lesions, though not sig-nificant. This is in clear contrast to SCCHN tumours wherelevels of all isoforms were upregulated compared to clini-cally normal tumour adjacent tissue. Levels of p63b andDNp63 further showed a statistically significant upregula-tion.26 In OLP lesions levels of the TAp63 isoforms were un-changed compared to the controls (0.0010 versus 0.0011). Adiscrepancy between RNA and protein levels for the TA iso-form specifically has been shown previously, for example inpsoriasis lesions.30

As chronic inflammation is known to cause DNA damage,the decreased levels of p63 seen in the chronic inflamma-tory condition OLP are comparable to changes seen in epi-thelia after exposure to UV light. In this DNA damagedepithelia decreased DNp63 expression is needed for induc-tion of apoptosis.31 This may, as previously suggested, indi-cate a protective mechanism enabling removal of DNAdamaged cells in OLP.18

In conclusion, we have been able to detect RNA of 3 p53isoforms in the majority of OLP lesions and normal controlsanalysed. Two of these belong to the group of recently de-tected novel p53 isoforms. The function of these novel iso-forms is, so far, not known but the same isoforms have also

been amplified in samples of SCCHN, at seemingly higherlevels. This could indicate a more tumour related role forthese isoforms. None of them could, however, be detectedat protein level with the reagents available today. There-fore, in order to clarify the role of these novel p53 isoformsin OLP lesions, further studies with preferably more isoformspecific reagents are needed.

Conflict of interest statement

None declared.

Acknowledgements

We thank Bodil Backlund and Inger Cullman for excellentand skilful technical assistance.This study was supported by grants from The Swedish Can-

cer Society Grant number 4569-B05-05XAC; Lion’s CancerResearch Foundation, Umea University; The County Councilof Vasterbotten and the Swedish Dental Society.

References

1. Axell T, Rundquist L. Oral lichen planus – a demographic study.Community Dent Oral Epidemiol 1987;15(1):52–6.

2. Dissemond J. Oral lichen planus: an overview. J Dermatol Treat2004;15(3):136–40.

3. Sugerman PB, Savage NW. Oral lichen planus: causes, diagnosisand management. Aust Dent J 2002;47(4):290–7.

4. Ebrahimi M, Wahlin YB, Coates PJ, Wiik A, Roos G, Nylander K.Detection of antibodies against p63 and p73 isoforms in serafrom patients diagnosed with oral lichen planus. J Oral PatholMed 2007;36(2):93–8.

5. Epstein JB, Wan LS, Gorsky M, Zhang L. Oral lichen planus:progress in understanding its malignant potential and theimplications for clinical management. Oral Surg Oral Med OralPathol Oral Radiol Endod 2003;96(1):32–7.

6. Pindborg JJRP, Smith CJ, van der Waal I. Histological typing ofcancer and precancer of the oral mucosaWorld Health Orga-nization international histological classification of tumours.2nd ed. Berlin: Springer; 1997.

7. Clevers H. At the crossroads of inflammation and cancer. Cell2004;118(6):671–4.

8. Coussens LM, Werb Z. Inflammation and cancer. Nature2002;420(6917):860–7.

9. Philip M, Rowley DA, Schreiber H. Inflammation as a tumorpromoter in cancer induction. Semin Cancer Biol2004;14(6):433–9.

10. Ogmundsdottir HM, Hilmarsdottir H, Astvaldsdottir A, Johanns-son JH, Holbrook WP. Oral lichen planus has a high rate of TP53mutations. A study of oral mucosa in icelanD. Eur J Oral Sci2002;110(3):192–8.

11. Acay RR, Felizzola CR, Soares de Araujo N, Machado de SousaSO. Evaluation of proliferative potential in oral lichen planusand oral lichenoid lesions using immunohistochemical expres-sion of p53 and Ki67. Oral Oncol.

12. Chaiyarit P, Ma N, Hiraku Y, Pinlaor S, Yongvanit P, JintakanonD, et al. Nitrative and oxidative DNA damage in oral lichenplanus in relation to human oral carcinogenesis. Cancer Sci2005;96(9):553–9.

13. Hirota M, Ito T, Okudela K, Kawabe R, Yazawa T, Hayashi H,et al. Cell proliferation activity and the expression of cell cycle

Expression of novel p53 isoforms in oral lichen planus 161

regulatory proteins in oral lichen planus. J Oral Pathol Med2002;31(4):204–12.

14. Lee JJ, Kuo MY, Cheng SJ, Chiang CP, Jeng JH, Chang HH, et al.Higher expressions of p53 and proliferating cell nuclear antigen(PCNA) in atrophic oral lichen planus and patients with arecaquid chewing. Oral Surg Oral Med Oral Pathol Oral RadiolEndod 2005;99(4):471–8.

15. Schifter M, Jones AM, Walker DM. Epithelial p53 gene expres-sion and mutational analysis, combined with growth fractionassessment, in oral lichen planus. J Oral Pathol Med1998;27(7):318–24.

16. Girod SC, Cesarz D, Fischer U, Krueger GR. Detection of p53 andMDM2 protein expression in head and neck carcinogenesis.Anticancer Res 1995;15(4):1453–7.

17. Girod SC, Kramer C, Knufermann R, Krueger GR. p53 expressionin the carcinogenesis in the oral mucosa. J Cell Biochem1994;56(4):444–8.

18. Ebrahimi M, Wahlin YB, Coates PJ, Sjostrom B, Nylander K.Decreased expression of p63 in oral lichen planus and graft-vs.-host disease associated with oral inflammation. J Oral PatholMed 2006;35(1):46–50.

19. Bourdon JC, Fernandes K, Murray-Zmijewski F, Liu G, Diot A,Xirodimas DP, et al. p53 isoforms can regulate p53 transcrip-tional activity. Genes Dev 2005;19(18):2122–37.

20. Boldrup L, Bourdon J, Coates PJ, Sjostrom B, Nylander K.Expression of p53 isoforms in squamous cell carcinoma of thehead and neck. Eur J Cancer 2007;43(3):617–23.

21. Osada M, Ohba M, Kawahara C, Ishioka C, Kanamaru R, Katoh I,et al. Cloning and functional analysis of human p51, whichstructurally and functionally resembles p53. Nat Med1998;4(7):839–43.

22. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V,et al. p63, a p53 homolog at 3q27–29, encodes multiple

products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998;2(3):305–16.

23. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A. p63 isa p53 homologue required for limb and epidermal morphogen-esis. Nature 1999;398(6729):708–13.

24. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT,et al. p63 is essential for regenerative proliferation in limb,craniofacial and epithelial development. Nature1999;398(6729):714–8.

25. Ghioni P, Bolognese F, Duijf PH, Van Bokhoven H, Mantovani R,Guerrini L. Complex transcriptional effects of p63 isoforms:identification of novel activation and repression domains. MolCell Biol 2002;22(24):8659–68.

26. Thurfjell N, Coates PJ, Uusitalo T, Mahani D, Dabelsteen E,Dahlqvist A, et al. Complex p63 mRNA isoform expressionpatterns in squamous cell carcinoma of the head and neck. Int JOncol 2004;25(1):27–35.

27. Stephen CW, Helminen P, Lane DP. Characterisation ofepitopes on human p53 using phage-displayed peptide libraries:insights into antibody-peptide interactions. J Mol Biol1995;248(1):58–78.

28. Vojtesek B, Dolezalova H, Lauerova L, Svitakova M, Havlis P,Kovarik J, et al. Conformational changes in p53 analysed usingnew antibodies to the core DNA binding domain of the protein.Oncogene 1995;10(2):389–93.

29. Prives C, Hall PA. The p53 pathway. J Pathol 1999;187(1):112–26.

30. Gu X, Lundqvist EN, Coates PJ, Thurfjell N, Wettersand E,Nylander K. Dysregulation of TAp63 mRNA and protein levels inpsoriasis. J Invest Dermatol 2006;126(1):137–41.

31. Hall PA, McKee PH, Menage HD, Dover R, Lane DP. High levels ofp53 protein in UV-irradiated normal human skin. Oncogene1993;8(1):203–7.