Abstract The tumor-suppressor protein p53 has recentlybeen shown to belong to a family that includes twostructurally related proteins, p63 and p73. In contrast top53, p63 and p73 play an essential role in epithelial de-velopment, stem cell identity and cellular differentiation.Salivary gland tumors carry a wide spectrum of histo-pathological forms, which may share a common single-cell origin from the epithelial progenitor basal duct cellsand have a different tendency of malignant progression.This study was performed to examine the expression ofp53, p63, and p73 in benign salivary gland tumors. Ex-pression and mutation of p53, p73, and p63 were exam-ined by direct DNA sequencing, reverse transcriptionPCR using isoform-specific primers, and by immunohis-tochemistry in normal parotid tissue (n=10), and varioustumors of the salivary gland (42 pleomorphic adenomas,12 myoepitheliomas, 8 basal cell adenomas, 5 oncocyto-mas, 5 canalicular adenomas, and 20 adenolymphomas).In normal parotid tissue the expression of p63 and p73was restricted to few basal and myoepithelial cells. Duc-tal luminal and acinus cells were completely negative forthe expression of all three family members. In contrast,in salivary gland tumors, strong nuclear staining for p63and p73 was observed. Myoepithelial and basaloid cellsand the basal epithelial layer of adenolyphomas andoncocytomas were positive for p63 and also, to a lesserextent, to p73. Mutations of p53 were detected in 4 of 42(10%) pleomorphic adenomas, in 3 of 12 (25%) myoepi-theliomas, and in 1 of 8 (13%) basal cell adenomas butnot in other tumors. We failed to detect specific muta-tions of p63 and p73. Using isoform-specific PCR, we

found that all isoforms of p63 were expressed in normalparotid tissue whereas the pleomorphic adenomas, myo-epitehliomas, and basal cell adenomas dominantly ex-pressed the transactivation-incompetent truncated iso-forms. Our data indicate that p63 and p73 are upregulat-ed in salivary gland tumors and may serve as a marker ofepithelial and myoepithelial progenitor cells in salivaryglands. The prevalence of p53 mutations and the obser-vation of the expression of ∆Np63 isoforms only in pleo-morphic adenomas, myoepitheliomas, and also basal celladenomas may reflect their possible malignant potential.

Keywords Salivary gland tumor · Pleomorphic adenoma · p53 · p63 · p73 · Isoforms

Introduction

Tumors of the salivary gland represent 2–3% of head andneck neoplasms and usually occur in the major salivaryglands [16]. The fact that these tumors arise in organs lo-cated in an ontogenetic transitional zone, a region whereendoderm and ectoderm meet, might be one of the rea-sons for the often-problematic histopathological classifi-cation. It is known from epidemiological studies thatsome salivary gland tumors have a tendency towards ma-lignant transformation (e.g., pleomorphic adenoma,myoepithelioma), whereas others never transform intomalignant tumors (e.g., adenolymphoma) [16, 17].

Due to the fact that salivary gland tumors are relative-ly rare diseases little is known about the underlying mo-lecular mechanisms leading to tumorigenesis [3]. Muta-tions of p53, the by far best examined tumor suppressorgene, have been reported in a subset of benign and ma-lignant salivary gland tumors [6, 9, 13]. Recently twop53-related genes, p63 and p73, have been discovered.Both genes encode multiple proteins arising from alter-native promoter use and splicing, with transactivation,DNA binding, and tetramerization domains [4]. Whilesome isoforms of p63 (TAp63) and p73 (TAp73) are ca-

A. Weber · A. Gerstner · F. BootzDepartment of Oto-Rhino-Laryngology, Head and Neck Surgery,University of Leipzig, Liebigstrasse 18a, 04103 Leipzig, Germany

L. Langhanki · A. Schütz · C. Wittekind · A. Tannapfel (✉)Institute of Pathology, University of Leipzig, Liebigstrasse 26,04103 Leipzig, Germanye-mail: tana@medizin.uni-leipzig.deTel.: +49-341-9715000, Fax: +49-341-9715069

Virchows Arch (2002) 441:428–436DOI 10.1007/s00428-002-0705-y

O R I G I N A L A RT I C L E

Anette Weber · Larissa LanghankiAlexander Schütz · Andreas GerstnerFriedrich Bootz · Christian WittekindAndrea Tannapfel

Expression profiles of p53, p63, and p73 in benign salivary gland tumors

Received: 11 April 2002 / Accepted: 10 July 2002 / Published online: 11 September 2002© Springer-Verlag 2002

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pable of transactivating p53 target genes and inducingapoptosis, other isoforms (∆Np63 and Np73) act in adominant-negative fashion to counteract the transactiva-tion-competent isoforms of not only p63 and p73 but p53as well [3, 8]. It has been reported that Np63-encodingtranscripts are down-regulated during the irreversiblegrowth arrest and differentiation of human keratinocytes[15].

No data on p63 and p73 expression in salivary glandtumors are available as yet. We therefore analyzed p53,p63, and p73 in a broad spectrum of salivary gland tu-mors.

Materials and methods

Patients

This retrospective study was carried out between 1991 and 2001and included 42 patients with pleomorphic adenoma of the parotidsalivary gland (ICD-O-C 8940/0), 12 with myoepithelioma (ICD-O-C 8982/0) of the parotid gland, 8 with basal cell adenoma (ICD-O-C 8147/0), 5 with oncocytoma (ICD-O- 8290/0), 5 with canalic-ular adenoma, and 20 with adenolymphoma (Warthin tumor, ICD-O-C 8561/0) undergoing partial parotidectomy. Each tumor wasreevaluated with regard to typing [16]. In all cases hematoxylinand eosin stained slides prepared from eight different tumor areaswere examined. Immunohistochemistry was performed on paraf-fin-embedded sections of tissue microarrays. For microdissection,reverse transcriptase (RT) polymerase chain reaction (PCR) anddirect sequencing of p63 and p73 we used fresh-frozen material.Paraffin-embedded tissue was used for direct sequencing of p53.

DNA/RNA samples

For each salivary gland tumor sample the histopathological lesionsof interest were first identified on routinely stained rapid frozensections. The 12-µm sections cut from frozen-tissue blocks weremounted on glass slides with a thickness of 0.17 mm (very thinglass slides are needed to prevent laser energy from being dis-persed before reaching the section of tissue). An ultraviolet lasermicroscope system was then used to isolate particular cell popula-tions (UV-laser microbeam; PALM, Bernried, Germany). In brief,a pulsed UV laser of high beam quality (nitrogen laser, wave-length 337 nm, maximum frequency 20 pulses/s, pulse duration3 ns) was combined with an inverse microscope and focused

through an objective of high numerical aperture into the tissueplane. Beam spot diameter measured approximately 0.3–0.5 µm.Because of the extremely high energy density within the focalpoint (laser energy at object plane approximately 5 µJ) all biologi-cal material is completely destroyed. Using the UV laser beam at ahigh repetition rate (approximately 20 pulses/s) a circle was cutaround the target cells. This resulted in complete separation of thetarget population from neighboring tissues (Fig. 1). The approxi-mate number of cells was estimated to be at least 1000 per samplefor PCR analysis. After microdissection the tissue samples werethen put into Eppendorf tubes and incubated with proteinase K at37°C overnight. Proteinase K activity was inactivated by heatingat 95°C for 10 min. For DNA extraction standard methods wereused; after incubation with proteinase K at 37°C overnight the tis-sue was extracted twice in phenol and twice in chloroform, fol-lowed by ethanol precipitation.

Sequencing analysis

DNA was extracted according to standard procedures [19]. Of thep53 gene mutations in diverse types of cancers 98% have beenfound in exons 5–8 [2]; we therefore focused our study exons 4–9.Each exon, 4–9, of the p53 gene was amplified by 35 cycles ofPCR using 5′-end labeled primers and Taq polymerase (Perkin El-mer/Cetus, Norwalk, Conn., USA). The primers that we used toamplify p53 exons 4–9 have been described in detail previously[18]. The primer sequences for p73 were adopted from those de-scribed by Kaghad et al. [5] and Nomoto et al. [12]. Mutationanalysis was performed sequencing both strands of the entire p73RT-PCR fragments from parotid gland tumor and correspondingnonneoplastic tissue. For p63 six sets of primers according to [7]covering the whole coding region of Tap63γ were used. DNA se-quencing of the PCR products were performed using the DNA-Sequenase-Kit (Amersham, Germany) and an automatic sequenc-ing analyzer (ABI 373; Applied Biosystems, Perkin-Elmer).

RNA isolation and RT-PCR

Total RNA was extracted from microdissected tissue samples byTRIZOL reagent (Life Technologies, Grand Island, N.Y., USA)according to manufacturer’s specifications. Total RNA (1 µg) wasthen amplified using p63 isoform-specific primers with the Super-script One-Step RT-PCR Kit with Platinum Taq (Life Technolo-gies) according to the manufacturer’s protocol (50-µl reaction vol-ume). All reverse transcription reactions were carried out for30 min at 50°C and then 3 min at 94°C, followed by isoform-spe-cific PCR conditions for each primer set:

● TAp63 (nucleotides 3–269 of TAp63), 40 cycles at 94°C for30 s, 57°C for 40 s, and 72°C for 30 s, using SKO32 (sense; 5′-GTCCCAGAGCACACAGACAA-3′) and SKO33 (antisense;5′-GAGGAGCCGTTCTGAATCTG-3′) primers

● Np63 (nucleotides 10–207 of Np63), 40 cycles at 94°C for 30 s,57°C for 40 s, and 72°C for 30 s, using SKO26 (sense; 5′-CTGGAAAACAATGCCCAGAC-3′) and SKO27 (antisense;5′-GGGTGATGGAGAGAGAGCAT-3′) primers

Fig. 1 Microdissection of the tumor compartments. Microdissec-tion of the epithelial tumor component of a pleomorphic adenomaof the parotid gland. Outlined areas Microdissected by the lasersystem (Palm microbeam system) as described in the text

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● p63-tail (nucleotides 1380–1568 of Np63), 2 cycles at 94°C for30 s, 57°C for 40 s, and 72°C for 30 s, followed by 38 cycles at94°C for 30 s, 55°C for 40 s, and 72°C for 30 s, using SKO28(sense; 5′-GAGGTTGGGCTGTTCATCAT-3′) and SKO29 (an-tisense; 5′-AGGAGATGAGAAGGGGAGGA-3′) primers

● p63β-tail (nucleotides 1345–1550 of TAp63β), 2 cycles at 94°Cfor 30 s, 57°C for 40 s, and 72°C for 30 s, followed by 38 cyclesat 94°C for 30 s, 55°C for 40 s, and 72°C for 30 s, using SKO30(sense; 5′-AACGCCCTCACTCCTACAAC-3′) and SKO31 (an-tisense; 5′-CAGACTTGCCAGATCCTGA-3′) primers

● p63-tail (nucleotides 1057–1270 of TAp63), 2 cycles at 94°Cfor 30 s, 57°C for 40 s, and 72°C for 30 s; 2 cycles at 94°C for30 s, 55°C for 40 s, and 72°C for 30 s, and 36 cycles of 94°Cfor 30 s, 53°C for 40 s, and 72°C for 30 s, using SKO22 (sense;5′-ACGAAGATCCCCAGATGATG-3′) and SKO23 (antisense;5′-GCTCCACAAGCTCATTCCTG-3′) primers.

The GAPDH gene was chosen as an endogenous expression RT-PCR standard and was amplified using SKO36 (sense; 5′-GA-AGGTGAAGGTCGGAGT-3′) and SKO37 (antisense; 5′-GA-AGATGGTGATGGGATTTC-3′) primers. Isoform-specific RT-PCR (including GAPDH and a one primer-only control) was per-formed in triplicate. Of the RT-PCR products 25 µl was resolvedin 1.8% agarose gels.

Tissue microarray construction und immunohistochemistry

Tumor and normal tissues were embedded in paraffin, and 5-µmsections stained with hematoxylin and eosin were obtained toidentify viable, representative areas of the specimen. From the de-fined areas core biopsy specimens were taken with a precision in-strument (Beecher Instruments, Silver Spring, Md., USA) as de-scribed previously [20]. Tissue cores with a diameter of 0.6 mmwere punched from each specimen and arrayed in triplicate on arecipient paraffin block [20]; 5-µm sections of these tissue arrayblocks were cut and placed on charged polylysine-coated slides

(Fig. 2). These sections were used for immunohistochemical anal-ysis. Tissues and cell lines known to express p63 were used aspositive controls.

For immunohistochemical analysis of p73, p53, and p63 thetissue microarrays were covered with normal goat serum for20 min and then incubated with the primary antisera against p73,p63, or the p53-antibody, respectively (p53: clone DO-7, dilution1:100, Dianova, Hamburg, Germany, p63: clone 4A4, dilution: 1:60, Santa Cruz, Calif., Biotechnology, USA, p73: dilution: 1: 10,Santa Cruz, Biotechnology) [19].

After blocking and incubation with the primary antibody thesections were washed with phosphate-buffered saline, incubatedwith biotinylated goat anti-mouse (for p53, p63) and anti-rabbit(p73) immunoglobulin G (BioGenex, Germany) for 30 min, andcovered with peroxidase-conjugated streptavidin (Dako, Den-mark). The peroxidase reaction was allowed to proceed for 8 min,with 0.05% 3,3 diaminobenzidine tetrahydrochloride solution assubstrate. Slides were counterstained with hematoxylin and finallymounted. The slides were examined and scored independently bytwo of us (A.T., L.L) who were blinded to clinical and pathologi-cal information.

Results

Normal parotid tissue

Using an antibody that detects all p63 and p73 isoformsin normal salivary gland (parotid), both proteins werefound in very few basal cells and in the myoepithelialcells. Both acinus cell and luminal duct cells were nega-tive for p63 and for p73. No p53-positive cells werefound. A strong nuclear staining of both basal and myo-epithelial cells surrounding intercalated and striatedducts and excretory ducts was observed (Fig. 3A–D). Agradual diminution of p63 and p73 staining in the moreterminally differentiated cell layers occurred. Cells local-ized to the most superficial layers of intercalated ductshad undetectable p63/p73 levels. RT-PCR for p63 iso-forms displayed an equal distribution of all six isoforms(Fig. 5, Table 1).

Pleomorphic adenomas

In pleomorphic adenomas epithelial and modified myo-epithelial elements intermingle with tissue of mucoid,chondroid, squamous, or myxoid appearance. A strongp63 nuclear staining of nearly all spindle-shaped or sol-id-sheets forming myoepithelial cells was observed(Fig. 4A). To a lesser extent the myoepithelial cells werealso positive for p73. Epithelial duct forming cells wereonly occasionally positive for p63 and for p73. We failedto detect specific immunostaining in those areas withmucoid, chondroid, or myxoid differentiation. Squamouscell metaplasia exhibited specific p63 but not p73 stain-ing. Mutations of the p53 gene were detected in 4 of 42cases (10%). In all cases the mutations were point muta-tions, clustered within evolutionary conserved regions,especially in exons 4, 5, 6, and 8 (Tables 1, 2).

Fig. 2 Tissue microarray of pleomorphic adenoma; stained withhematoxylin-eosin (above) and with p63 (immunohistochemistry)(below)

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Myoepithelioma

Mutations of p53 were detected in 3 of 12 myoepitehlio-mas examined (25%). In all cases point mutations oc-curred in exons 4, 6, and 7 (Table 2). Those tumors ex-hibited a strong nuclear p53 immunostaining (Fig. 4B).Neither p63 nor p73 mutations were detected in myoepi-theliomas. p63-protein positive cells were observed in all

Fig. 3A–D Immunostaining of p73 and p63 in normal parotidgland. A p73 immunostaining of the normal parotid gland. Posi-tive cell nuclei (brown) at the basal layer of cells within a striatedduct; original magnification ×20. B p73 immunostaining of an ex-tralobular duct. Few positive basal ductal cells; original magnifi-cation ×20. C p63 immunostaining of the normal parotid gland.Positive cell nuclei (brown) at the basal ductal cells of an interca-lated duct and positive myoepithelial cells; original magnification×25. D p63 immunostaining within basal cell nuclei of a striatedduct; original magnification ×30

Table 1 Immunohistochemicalexpression of p53, p63, andp73 in benign salivary gland tu-mors

Tumor no of cases p53 (mutation) p63 p73

Pleomorphic adenomas n=42 4/42 + +Myoepithelial cells – + +Duct cells + – –Basal cells – + +Metaplastic: squamous – + +Metaplastic: mucoid, chondroid – – –

Myoepitheliomas n=12 3/12Myoepithelial cells + + +

Basal cell adenomas n=8 1/8Basaloid cells + + +

Oncocytomas n=5 – – –Canalicular adenomas n=5 – – –Adenolymphomas n=20 – +a –a

a Lymphoid follicles

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Fig. 4A–F Legend see page 434

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12 cases examined, especially within tumor areas exhib-iting a solid growth pattern (Fig. 4B). Spindle cells with-in tumors with a more reticular pattern were p63 nega-tive. p73 immunostaining was observed in 10 of 12 myo-epitheliomas, predominantly within the more epitheloidcells. p73 immunoreactivity was observed to a lesser de-gree than p63 immunoreactivity.

Basal cell adenoma

The isomorphic basaloid cells were positive for p63 and,to a lesser extent, for p73. The strongest immunoexpres-sion of both p63 and p73 occurred in solid tumor areasconsisting of uniform-appearing, small palisaded cells(Fig. 4C). In the trabecular subtype both glandular andtubular differentiated tumor areas were occasionally p63and p73 positive. We failed to detect specific p53 muta-tions or p53 immunostaining within the basal cell adeno-mas.

Oncocytoma and canalicular adenoma

We failed to detect specific mutations of p53 and its ho-mologues within these rare tumors of the salivary gland.In canalicular adenomas p63 and p73 expression oc-curred within the columnar beading pattern forming epi- thelial cells (Fig. 4D). In oncocytoma very few p63- and

p73-positive cells were observed. Few eosinophilic cellsexhibited a faint nuclear expression of p63 but not ofp73 (Fig. 4E). Due to the intense eosinophilic cytoplasm,a strong nonspecific staining was observed.

Adenolymphoma

We detected specific p63 and p73 staining within veryfew basal cells of the typically eosinophilic double-lay-ered epithelium of adenolymphoma. p63 immunoreactiv-ity was also observed occasionally within the lymphoidtissue (Fig. 4F). Neither p53, p63, nor p73 mutationswere observed.

p53 Mutation analysis Immunohistochemistry

Diagnosis Exon Codon Mutation Amino acid p53 p63substitution

Pleomorphic adenoma 8 273 CGT→AGT Arg-Ser ++ ++Pleomorphic adenoma 4 82 CCG→CTG Pro-Leu ++ ++Pleomorphic adenoma 5 163 TAC→TGC Tyr-Cys ++ ++Pleomorphic adenoma 6 209 del(–2) Frameshift – ++Myoepithelioma 4 82 CCG→CTG Pro-Leu ++ ++Myoepithelioma 6 179 CAT→CTT His-Leu ++ ++Myoepithelioma 7 245 GGC→AGC Gly-Ser ++ ++

Table 2 Mutation pattern ofp53 in benign salivary gland tu-mors

Fig. 4A–F Immunostaining of p73 and p63 in salivary gland tu-mors. A p63 and p73 immunostaining of the epithelial componentof a pleomorphic adenoma. Positivity of myoepithelial and, to alesser extent, of some epithelial (ductal) cells. Inset Positive spin-dle-formed myoepithelial cells. B p63 and p73 immunostaining ofmyoepithelioma. Strong p63 and p73 immunoreactivity of spindle-shaped epitheloidlike cells. C p63 and p73 immunostaining of abasal cell adenoma (trabecular variant). p63 and p73 immuno-staining visible in the basaloid cells. p73 staining occurred at theouter layer of glandular differentiated trabecular cells. D p63 andp73 immunostaining of a canalicular adenoma: positive cell nucleiof the basal layer of the columnar-appearing epithelial cells. E p63and p73 immunostaining of an oncocytoma. Very few positivecells observed with a high degree of staining due to intense eosi-nophilic cytoplasm. F p63 and p73 immunostaining of an adeno-lymphoma. Very few basal cells of the typically eosinophilic dou-ble-layered epithelium were positive for p63 and for p73. Positivelymphocytes served as built-in positive control

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Fig. 5 Expression of p63 in normal parotid tissue and salivarygland tumors: RT-PCR analysis for p63 using isoform-specificprimers. Lane 1 Normal parotid gland; lanes 2, 3 pleomorphic ad-enoma; lanes 4, 5 myoepithelioma; lanes 6, 7 basal cell adenoma;lanes 8, 9 adenolymphoma. GAPDH was used as an endogenousstandard. A representative result from four experiments is shown

p63 isoforms

To determine which p63 isoforms were present in oursamples we performed RT-PCR using isoform-specificprimers on total RNA from normal parotid tissue andpleomorphic adenomas, myoepitheliomas, adenolympho-ma, basal cell adenoma, and oncocytoma after microdis-section. All six splice variants of p63 were found in nor-mal parotid tissue and also in adenolymphomas. We de-tected the truncated Np63 isoforms as the dominant spe-cies expressed in all pleomorphic adenomas, myoepithe-liomas, and basal cell adenomas but not in adeno-lymphomas or nomal parotid tissue (Fig. 5).

Discussion

The identification of two homologues, p63 and p73, re-vealed that p53 is a member of a family of related tran-scription factors. Since they share amino acid sequenceidentity of 63% in the DNA-binding domain, p53, p63,and p73 could have redundant functions in the regulationof protein expression. In contrast to p53, somatic muta-tions of the p63 gene are very rare, while germline muta-tions in TP63 have been reported for patients with limbmammary syndrome, split-hand/split-foot malformation,ankyloblepharo-ectodermal dysplasia-clefting syndrome,and EEC syndrome [10, 23]. We also failed to detect spe-cific mutations of p63 or p73 in all salivary gland tumorsexamined. It appears that alterations in p63 expressionare epigenetic in nature. Our data confirm earlier resultsshowing that p63 and p73 expression follows a restrictedpattern in normal tissues [1, 3]. We observed intense p63and p73 nuclear localization only within the myoepithe-lial cells and the basal cells of the normal salivary gland,with a gradual diminuition of nuclear intensity in themore terminally differentiated duct cells within striatedand excretory ducts, while the most apical cells had unde-tectable p63 levels. Acinus as well as luminal cells werenegative for both p63 and p73. The identification of bothproteins in myoepithelial and basal cells is significant be-cause some of these cells may act as progenitors of thesuprabasal cells, which undergo differentiation and celldeath. Recent data from p63 knockout mice elucidate p63as a key regulator of proliferation and differentiation pro-grams acting to maintain the regenerative or “immortal”quality of epithelial stem cells [10, 23]. Data from otherstudies regarding p63 overexpression in tumor cells sup-port an important role for p63 in the development of hu-man primary tumors, including squamous and transitionalcell carcinomas [11, 14, 21]. Our observation of an in-creased expression of p63 and p73 pleomorphic adenoma,myoepithelioma, and basal cell adenoma, all benign sali-vary gland tumors, supports the concept of a neoplasticand proliferative potential of aberrant p63/p73 expressioneven in nonmalignant tumors.

It has been reported that p63/p73 overexpression canmimic p53 activities by binding DNA, activating tran-scription, and inducing apoptosis [4]. Unlike p53, both

p63 and p73 can undergo alternate splicing. For p63three C terminal variants (α, β, and γ), two N terminalvariants (TA and ∆N), and an interstitial splice variant inwhich 12 bases (encoding four amino acids) may be de-leted from exon 9, leading to six different isoforms thathave been reported [4]. The three aminoterminal truncat-ed isoforms of p63 are unable to transactivate and to in-duce apoptosis. Furthermore, the same truncated iso-forms appear to act in a dominant-negative manner to in-hibit the transactivation potential of wild-type p53 andp63 [22]. Our observation that the ∆N form of p63 de-void of the transcriptional activation domain is the pre-dominant form of p63 in a subset of salivary gland tu-mors with a malignant potential, i.e., pleomorphic ade-noma, myoepithelioma, and basal cell adenoma but notin normal parotid tissue, may suggest that this representsan endogenous dominant negative regulator of p53 activ-ity, an endogenous “anti-p53 protein.”

In this context, one may speculate that when certainp63 isoforms are expressed, such as dominant-negative∆Np63, they could bind to and inhibit transactivation byp53 and TAp63. Alternatively, these Np63 isoforms, bybinding to specific promoter elements, could block thetranscription of otherwise critical genes, such as thoseinvolved in the apoptotic response and terminal differen-tiation. The observation that ∆Np63 is present in pleo-morphic adenoma, myoepithelioma, and basal cell ade-noma – benign tumors with a possible malignant poten-tial – but not in adenolymphoma and normal parotid tis-sue might underline this hypothesis.

This is the first report concerning the expression ofp53 and relatives in benign salivary gland tumors. Wefound an greater expression of p63 and p73 in pleomor-phic adenoma, myoepithelioma, and basal cell adenomathan in normal parotid gland. Furthermore, the dominantisoform of p63 expressed within these tumors was the∆N-isotype. We speculate that these isoforms suppresswild-type p53 functions and lead to an abrogation of p53downstream targets, including apoptosis, transientgrowth arrest, and sustained growth arrest or senescence.Further studies are necessary to elucidate the exact mo-lecular mechanisms of p63/p73 overexpression not onlyin malignant but also in benign tumors.

Acknowledgements This research was supported by the Bun-desministerium für Bildung und Forschung, Interdisciplinary Cen-ter for Clinical Research at the University of Leipzig(01KS9504/1, project D01).

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