ORIGINAL ARTICLE

Altered expression of UPIa, UPIb, UPII, and UPIIIaduring urothelial carcinogenesis inducedby N-butyl-N-(4-hydroxybutyl)nitrosamine in rats

Daša Zupančič & Zdenka Ovčak & Gaj Vidmar &

Rok Romih

Received: 24 October 2010 /Revised: 21 December 2010 /Accepted: 10 January 2011 /Published online: 8 February 2011# Springer-Verlag 2011

Abstract In normal urothelium, superficial umbrella cellsexpress four major integral membrane proteins, uroplakinsUPIa, UPIb, UPII, and UPIIIa, which compose urothelialplaques. In the apical plasma membrane, urothelial plaquesform microridges. During neoplastic changes, microridges arereplaced by microvilli, while uroplakin expression is retained.We correlated individual uroplakin expression with apicalplasma membrane structure, cytokeratin 20 expression, andurothelial cell proliferation (Ki-67). Male Wistar rats weretreated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN) in drinking water, which caused flat hyperplasia withmild dysplasia, low-grade papillary urothelial carcinoma,invasive low- and high-grade papillary urothelial carcinomaand invasive squamous cell carcinoma with extensivekeratinization, grade 2. During urothelial carcinogenesis, UPIIexpression was the most decreased in all urothelial lesions,while UPIa, UPIb, and UPIIIa expression was differentlyaltered in different types of lesions. Superficial cells werecovered with microvilli and ropy ridges, while microridges

were disappearing. The expression of cytokeratin 20 wasdecreased and limited to superficial urothelial cells. Prolifer-ation indices were increased, except for invasive squamouscell carcinoma with extensive keratinization. Our resultsindicate that during urothelial carcinogenesis the expressionof UPII is diminished, suggesting that UPIb/UPIIIa hetero-dimer can still be formed, while heterodimer UPIa/UPIIformation is disrupted. Correlation between decreased level ofUPII expression and changed apical plasma membranestructure suggests that diminished expression of UPII hindersthe urothelial plaque formation.

Keywords Urothelium . Uroplakins . Carcinogenesis .

Plasma membrane . CK 20 . Ki-67

Introduction

The epithelium of the urinary bladder, known as urothe-lium, acts as a constant permeability barrier and protects theblood from toxic urinary substances [1]. The apical surfaceof normal urothelium is covered by large, highly differen-tiated, superficial umbrella cells [2]. Umbrella cells elabo-rate membrane specialization called the urothelial plaquethat contribute to functional blood-urine barrier [3, 4].Urothelial plaques are rigid-looking, concave-shaped bio-membrane structures that occupy almost complete urothe-lial apical surface, interrupted by narrow “hinge” regions[5]. On cross-sections, the plaques (0.3–1 μm in diameter)exhibit outer membrane leaflets twice as thick as the innerones, hence the name asymmetric unit membrane (AUM)[3]. This unique apical plasma membrane of umbrella cellsform microridges observed by scanning electron microsco-py [6, 7]. Each microridge corresponds to hinge regionencircling one urothelial plaque [8].

Electronic supplementary material The online version of this article(doi:10.1007/s00428-011-1045-6) contains supplementary material,which is available to authorized users.

D. Zupančič (*) :R. RomihInstitute of Cell Biology, Faculty of Medicine,Lipičeva 2,1000 Ljubljana, Sloveniae-mail: dasa.zupancic@mf.uni-lj.si

Z. OvčakInstitute of Pathology, Faculty of Medicine,Korytkova 2,1000 Ljubljana, Slovenia

G. VidmarUniversity Rehabilitation Institute,Linhartova 51,1000 Ljubljana, Slovenia

Virchows Arch (2011) 458:603–613DOI 10.1007/s00428-011-1045-6

Urothelial plaques consist of two-dimensional, hexag-onally packed 16-nm protein particles [5, 9] that containfour major proteins uroplakins UPIa (27 kDa), UPIb(28 kDa), UPII (15 kDa) and UPIIIa (47 kDa) [10, 11].Among uroplakins, UPIa and UPIb belong to a tetraspaninfamily of integral membrane proteins [12], while UPII andUPIIIa both have a single transmembrane domain [13, 14].Within the plaques, these four major uroplakins form twoheterodimers consisting of UPIa/UPII and UPIb/UPIIIa, asdemonstrated by chemical cross-linking [15] and proteinisolation [16]. In addition, transfection experimentsshowed that when individual uroplakins are expressed in239 T cells, only UPIb can exit from the endoplasmicreticulum (ER) and move to the plasma membrane,whereas UPII and UPIIIa reach the plasma membraneonly when they form heterodimeric complexes with UPIaand UPIb, respectively [17].

The assembly of 16-nm particles and urothelial plaquesare interesting and still unresolved issues. Ablation of genesencoding UPII or UPIIIa inhibited the formation of UPIa/UPII and UPIb/UPIIIa pairs, respectively [18, 19]. UPIIIaknockout yielded small plaques and UPII knockout abol-ished plaque formation, indicating that both uroplakinheterodimers are required for normal plaque formation [19].

The fact that uroplakins are the major differentiationproducts of urothelium implies that they would besignificantly downregulated during urothelial dedifferentia-tion, transformation, and carcinogenesis. This was provento be the case in cultured normal urothelial cells and humanbladder cancer cell lines [20, 21]. Surprisingly, however,this idea is not supported by data from urothelial carcino-mas. For example, Ogawa et al. showed that in N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urothelial car-cinogenesis of rats, the expression of total uroplakins insuperficial cells is decreased but the expression in someintermediate cells is increased [22]. Their results, combinedwith data of human, mouse, canine, and cow carcinogenesis[22–25], suggest that uroplakin downregulation does notstrictly parallel urothelial tumorigenesis and progression.All of the existing studies were limited to the use ofantibodies that mainly recognize UPIIIa, i.e., anti-AUM oranti-UPIIIa antibody [22–25].

Besides altered uroplakin expression, bladder carcino-genesis is accompanied by other characteristics, such as: (1)the appearance of microvilli on the urothelial apical surface[26, 27], (2) the disappearance of AUM [28] and micro-ridges [29, 30], (3) altered expression pattern of cytokeratin20 (CK 20) [31, 32] and (4) increased urothelial cellproliferation, as determined by Ki-67 antigen [33, 34].

In the present study, the expression patterns of individualuroplakin were investigated during urothelial carcinogene-sis induced by BBN in rats. Since uroplakins on urothelialapical surface form microridges, we correlated their

expression with apical plasma membrane structure. Foradditional bladder carcinogenesis characterization, CK 20and Ki-67 expression was evaluated to determine differen-tiation state and proliferation activity of urothelial cells,respectively.

Materials and methods

Animals and treatment

Eighteen adult male Wistar rats were used in this study inaccordance with European guidelines and Slovenian legis-lation. Rats were divided into six groups by simple randomsampling. They were housed in plastic cages at 23±2°Cand 50–60% relative humidity. Basal diet was available adlibitum.

The BBN was obtained from Tokyo Chemical IndustryCo., Ltd. (Tokyo, Japan) and diluted to 0.05% with tapwater. This mixture was then provided as drinking water(ad libitum) for 5 weeks (group BBN 5 weeks), 10 weeks(group BBN 10 weeks), 15 weeks (group BBN 15 weeks)or 20 weeks (groups BBN 20 weeks and BBN 20+15 week). For the BBN 20+15-week group, animals dranktap water for 15 weeks after withdrawal of BBN adminis-tration. Control group (C) of animals had tap wateravailable ad libitum (for 5, 10, and 15 weeks, one animalunder each condition). At specified time-points, the ratswere euthanized by CO2 inhalation. Urinary bladder of eachanimal was cut sagittaly into three parts, designated forparaffin embedding, scanning electron microscopy, andwestern blot analysis.

Paraffin embedding, histological examination,and classification of urothelial lesions

The urinary bladder samples were fixed in 4% parafor-maldehyde overnight and embedded in paraffin. Allparaffin blocks were cut into serial sections and everytenth section was stained with hematoxylin and eosin andexamined by a uropathologist. Histological changes wereclassified according to WHO classification of tumors ofthe urinary tract [35].

Immunohistochemical detection of Ki-67, CK 20, UPIa,UPIb, UPII, and UPIIIa

Paraffin sections were deparaffinized and hydrated, theendogenous peroxidase activity was blocked with 3% H2O2

in methanol. For detection of Ki-67, UPIa, UPIb, UPII, andUPIIIa sections were microwave heated and for detection ofCK 20, they were treated with 0.1% trypsin (Sigma,Taufkirchen, Germany). Non-specific labeling was blocked

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by 5% fetal calf serum and 1% bovine serum albumin.Sections were incubated overnight at 4°C with thefollowing primary antibodies: monoclonal anti-Ki-67 (cloneMIB-5, Dako, Glostrup, Denmark; diluted 1:50), monoclo-nal anti-CK 20 (Dako, Glostrup, Denmark; diluted 1:20),polyclonal anti-UPIa (diluted 1:1,000), polyclonal anti-UPIb (diluted 1:2,000), polyclonal anti-UPII (diluted1:2,000), or monoclonal anti-UPIIIa (diluted 1:20; all foururoplakin antibodies were a kind gift from Prof. T.T. Sun,Department of Dermatology, New York University MedicalSchool, New York, NY 10016). Uroplakin labeling wasdone on sequential serial sections. For negative controls,the incubation with primary antibody was omitted or thespecific primary antibody was replaced by a non-relevantantibody. For secondary antibodies, biotinylated rabbit anti-mouse (Dako, Glostrup, Denmark; diluted 1:100) or swineanti-rabbit immunoglobulins (Dako, Glostrup, Denmark;diluted 1:400) were applied for 1 h at room temperature,followed by ABC/HRP complex (Vector Laboratories,Burlingame, CA) incubation. After the standard DAB(Sigma, Taufkirchen, Germany) development procedure,sections were counterstained with hematoxylin, examinedwith a Nikon Eclipse TE300 (Nikon Corporation, Tokyo,Japan) and classified by a uropathologist.

To determine optimal dilutions for all anti-uroplakinantibodies, immunohistochemical reactions described abovewere performed with dilution series for each antibody.Dilutions 1:20, 1:200, 1:1,000, 1:2,000, and 1:20,000 weretested on control normal urothelium and on urothelialcarcinomas.

Scanning electron microscopy

The urinary bladder samples were cut transversally intothree parts (each with apical area approximately 4 mm2)and fixed in 4.5% paraformaldehyde and 2% glutaralde-hyde for 3 h. The samples were postfixed in osmiumtetroxide and critical-point dried. After sputter-coating withgold, they were examined at 15 kV with a Jeol JSM 840 Ascanning electron microscope (Jeol Ltd., Tokyo, Japan).

Quantitative analysis

Quantitative analysis was performed on all samples from allanimals in each group. Changes of the apical plasmamembrane structure were analyzed by scanning electronmicroscopy. On three bladder samples (each with apicalarea approximately 4 mm2) of each animal, the presence ofmicrovilli, ropy ridges, and microridges was evaluated byfour-point scale (1=none, 2=weak, 3=moderate, 4=strongpresence).

To estimate proliferative activity, cells with a positiveimmunohistochemical reaction against Ki-67 were used for

scoring of proliferation indices. All urothelial cells pertissue section were counted on two paraffin sections peranimal and the indices for each tissue section were definedas a percentage ratio of labeled urothelial cells to the totalnumber of counted cells.

To evaluate differentiation state and uroplakin expressionin urothelial cells, results of CK 20, UPIa, UPIb, UPII, andUPIIIa staining were evaluated on two paraffin sections peranimal. The intensity of staining in superficial, in intermediateand in basal urothelial cells was rated on a four-point scale(1=none, 2=weak, 3=moderate, 4=strong staining) [36].

Statistical analysis

Data from scanning electron microscopy were analyzedusing one-way analysis of variance with Scheffe post-hoccomparisons as a compromise between simplicity (since allthree bladder samples from each animal were used asindependent data because the samples were too small formultilevel modeling) and power (since parametric analysiswas chosen despite discrete limited-range data). It shouldbe noted that other multiple comparisons methods, as wellas nonparametric analyses, lead to equivalent conclusions.Controls were pooled into a single group because all valueswere equal between animals.

For Ki-67, proliferation index was compared between theBBN and the control group using exact Mann–Whitney testat each of the three time-points for which the controls wereavailable. Association between lesion severity and prolifer-ation index was tested using exact Kruskal–Wallis test.

For CK 20, the mean of the two sections was calculatedfor superficial and intermediate cells and analyzed the sameway as data from scanning electron microscopy.

Analyses of uroplakins immunohistochemistry data werefirst performed for each uroplakin separately for superficialand intermediate cells in the same way as for the data fromscanning electron microscopy, including using the twosections from each animal as independent and pooling thecontrols into a single group. Then, the four uroplakins werecompared within each stage of tumor evolution usingrepeated-measures analysis of variance with simple contrasts.

Association of uroplakin expression with proliferationindex and CK 20 expression in BBN-treated animals wasassessed using Spearman rank-correlation (Rho) takinganimal as the unit of analysis, i.e., after calculating themedian across sections and sections for each animal.

All statistical analyses were performed using SPSS forWindows 15.0 (SPSS Inc., Chicago, IL, 2007).

Western blot

Urothelial cells were scrubbed from urinary bladders andhomogenized in ice-cold buffer (0.8 M Tris-HCl, 7.5%

Virchows Arch (2011) 458:603–613 605

SDS, 1 mM phenylmethylsulfonyl fluoride). The lysate wascentrifuged and the protein concentration in the supernatantwas determined by using a BCA™ protein assay kit(Pierce, Rockford, IL). Sample proteins (50 μg/lane) weresize fractionated on 12% or 15% SDS-polyacrylamide gelsand then transferred to Hybond ECL nitrocellulose mem-branes (Amersham Biosciences, Buckinghamshire, UK) byelectroblotting. After blocking overnight at 4°C in 5% skimmilk in PBS-Tween, membranes were incubated for 2 h atroom temperature with the polyclonal anti-UPIa (diluted1:2,000), polyclonal anti-UPIb (diluted 1:3,000), polyclonalanti-UPII (diluted 1:3,000), or monoclonal anti-UPIIIa(diluted 1:1,000). After washing in PBS-Tween, mem-branes were incubated for 1 h with adequate secondaryantibodies horseradish peroxidase-conjugated goat anti-mouse (Dako, Glostrup, Denmark; diluted 1:1,000), or goatanti-rabbit (Sigma, Taufkirchen, Germany; diluted 1:1,000).Membranes were finally probed with enhanced chemo-luminescence reagent (ECL; Amersham Biosciences, Buck-inghamshire, UK) and exposed to X-ray films. To confirmequal protein loading, the blots were striped with RestoreWestern Blot Stripping Buffer (Pierce, Rockford, IL) andreprobed with anti-actin antibody (Sigma, Taufkirchen,Germany; diluted 1:2000).

Results

The urothelial apical surface changes after BBNadministration

In the control urothelium, scanning electron microscopyshowed large polygonal superficial cells, covered withmicroridges (Fig. 1a). Administration of BBN for 5, 10, and15 weeks resulted in strong presence of ropy ridges andmoderate presence of microvilli, while the presence ofmicroridges was weak (Fig. 1b and Online Resource 1). At20 weeks of BBN administration, the presence of microvilliand ropy ridges was strong, while the presence of micro-ridges was still weak (Online Resource 1). At 20 weeks ofBBN followed by 15 weeks of tap water administrationresulted in strong presence of microvilli (Fig. 1c) andmoderate presence of ropy ridges, while microridges werenot observed (Online Resource 1). In all samples, cells withmicrovilli and ropy ridges were smaller than those coveredwith microridges (Fig. 1).

BBN induces various urothelial lesions

In control group of rats, urothelium showed normalhistology, consisting of three cell layers (Fig. 2a, 3a–d).Administration of BBN to rats for 5 and 10 weeks resultedin flat hyperplasia accompanied by mild dysplasia in 2/3

animals. In 1/3 animals, low-grade papillary urothelialcarcinoma was noted. At 15 weeks of BBN administration,flat hyperplasia with mild dysplasia were observed in 2/3animals, against a background of low-grade papillaryurothelial carcinoma in 3/3 animals. At 20 weeks of BBNadministration, low-grade papillary urothelial carcinomadeveloped in all three animals sacrificed at this time point.At 20 weeks of BBN followed by 15 weeks of tap wateradministration, low-grade papillary urothelial carcinomawas noted in 1/3 animals. Lamina propria invasion wasobserved in 1/3 animals with low-grade papillary urothelialcarcinoma and in 2/3 animals with high-grade papillaryurothelial carcinoma. Furthermore, at this stage, squamouscell carcinoma with extensive keratinization and laminapropria invasion, grade 2 were noted in 2/3 animals. Theresults are summarized in Table 1 and representativecarcinomas are shown in Fig. 2b–c and 3e–p.

BBN increases proliferation indices in urothelium

In control urothelium, Ki-67 positive cells were extremelyrare and exclusively limited to the basal cell layer, withproliferation indices below 1% (Online Resource 2A, 3, 4).After BBN administration, substantial elevation of prolifer-ation indices (Online Resource 2B, 2C, 3, 4) was observed,whereby proliferation indices were significantly higher afterBBN administration than in the control group (p<0.001 at5, 10, and 15 weeks). Proliferation index was statisticallysignificantly associated with lesion severity (p<0.001) ina curvilinear way (Online Resource 4): mean proliferationindex (across animals, pooled over sections within eachanimal) of flat hyperplasia with mild dysplasia was 12.9%,in low-grade papillary urothelial carcinoma it was 20.5%,the highest proliferation index was observed in invasivelow- and high-grade papillary urothelial carcinoma (mean29.7% and 28.7%, respectively), while the mean prolifer-ation index of invasive squamouse cell carcinoma withextensive keratinization, grade 2, was 5.0%. In allurothelial lesions, a Ki-67 reaction positive signal wasobserved not only in the nuclei of basal cells, but also inthe nuclei of intermediate and superficial cells (OnlineResource 2B, 2C).

CK 20 expression in urothelial cells decreases after BBNadministration

CK 20 positive staining was observed in all superficialurothelial cells of control urothelium, while intermediateand basal cells were completely negative (Fig. 2a). AfterBBN administration, CK 20 labeling was statisticallysignificantly decreased in superficial cells (p<0.001) andkept decreasing until the end of the experiment (OnlineResource 5). In intermediate cells, statistically significant

606 Virchows Arch (2011) 458:603–613

increase in CK 20 labeling was also observed (p=0.001),but the values rose only at 10 weeks of BBN administrationand return to baseline at the end of the experiment (OnlineResource 5).

Intensity of staining was statistically significantly asso-ciated with lesion severity for superficial (p<0.001) as wellas intermediate cells (p=0.020).Flat hyperplasia with milddysplasia and low-grade papillary urothelial carcinomaexhibited CK 20-positive staining only in some individualsuperficial urothelial cells and rare intermediate cells(Fig. 2b and Online Resource 5). All three carcinomaswith lamina propria invasion showed mainly negativelabeling in all cell layers (Fig. 2c and Online Resource 5).

Uroplakins expression is altered after BBN administration

Highest specific staining for each anti-uroplakin antibodywas obtained by dilution series. They were 1:1,000 for anti-UPIa, 1:2,000 for anti-UPIb, 1:2,000 for anti-UPII (OnlineResource 6) and 1:20 for anti-UPIIIa. These optimaldilutions were further used to estimate expression ofindividual uroplakin.

The typical pattern for all four individual uroplakinsin control rat urothelium was limited to staining ofsuperficial cells (Fig. 3a–d, Table 2, and Online Resource7). In the flat hyperplasia with mild dysplasia almost allsuperficial cells showed strong UPIa, UPIb, and UPIIIastaining, while UPII staining was weak to moderate(Fig. 3e–h and Table 2). Considerable staining of UPIa,UPIb, and UPIIIa was also observed in intermediate cells,whereas UPII staining of intermediate cells was weak(Fig. 3e–h and Table 2). Similar pattern of uroplakinsstaining appeared in low-grade papillary urothelial carci-noma. In superficial cells, UPIa staining was strong, UPIband UPIIIa staining was moderate and UPII staining wasweak or absent (Fig. 3i–l and Table 2). In intermediatecells, UPIa and UPIb staining was moderate, while UPIIand UPIIIa staining was weak or negative (Fig. 3i–l andTable 2). All invasive carcinomas showed decreasedexpression of all uroplakins. In superficial cells ofinvasive low-grade papillary urothelial carcinoma, UPIaand UPIb staining was weak or absent, whereas staining ofUPII and UPIIIa was completely negative (Table 2). Inintermediate cells, UPII and UPIIIa staining was com-

Fig. 2 CK 20 immunohistochemistry. a All superficial urothelial cellsof control urothelium are CK 20 positive. b Some superficial cells areCK 20 positive in the low-grade papillary urothelial carcinoma (at20 weeks of BBN administration). c Negative CK 20 staining of the

invasive squamous cell carcinoma with extensive keratinization (K),grade 2, (at 20 weeks of BBN followed by 15 weeks of tap wateradministration)

Fig. 1 Scanning electron microscopy. a Large polygonal superficialcell covered with microridges in control urothelium. b Smallsuperficial cells with ropy ridges (large star) and microvili (small

star) at 10 weeks of BBN administration. c Small superficial cells withmicrovili at 20 weeks of BBN followed by 15 weeks of tap wateradministration

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pletely negative, while UPIa and UPIb staining was weak insome intermediate cells (Table 2). In BBN-induced invasivehigh-grade papillary urothelial carcinoma, some superficialcells were stained with anti-UPIa and anti-UPIb antibody,rare superficial cells were stained with anti-UPIIIa antibody,while UPII staining was negative (Fig. 3m–p and Table 2).Rare intermediate cells of invasive high-grade papillaryurothelial carcinoma were UPIa and UPIb positive, whileUPII and UPIIIa staining was negative (Fig. 3m–p andTable 2). In invasive squamous cell carcinoma withextensive keratinization, grade 2, all uroplakins stainingwas negative in all cells (Table 2).

Statistically significantly differences in immunohisto-chemistry of all uroplakins between control normalsamples and the samples with different duration ofBBN administration were observed in superficial andintermediate cells (Online Resource 7). Simultaneouslywith longer BBN administration, the intensity of stainingof all uroplakins was decreased, with UPII staining beingthe most diminished (Online Resource 7). Results wereconfirmed by Western blot analysis of urothelial proteinsamples obtained from control animals and at 5, 10, 15,20 weeks of BBN administration and at 20 weeks ofBBN followed by 15 weeks of tap water administration(Fig. 4). At the same time, staining intensity of alluroplakins was significantly associated with lesion sever-ity for both superficial and intermediate cells (Table 2). Inboth superficial and intermediate cells, staining of UPIIwas less intense than that of UPIa, UPIb, and UPIIIawithin each lesion type, except within squamous cellcarcinoma with extensive keratinization (where there wasno staining of any uroplakin) and within invasive high-grade papillary urothelial carcinoma in intermediate cells(Table 2).

Correlations between uroplakins, CK 20, and proliferationindices

Among BBN-treated animals, expression of different uro-plakins was highly positively correlated within superficialcells, within intermediate cells and between superficial andintermediate cells. Expression of all uroplakins in superficialand intermediate cells was highly positively correlated withexpression of CK 20 in superficial cells, while there could beno association with CK 20 expression in intermediate cellssince the median value of the later was 1 (i.e., no expression)in all animals. Expression of all uroplakins in superficial andintermediate cells tended to be negatively correlated with Ki-67 proliferation index, though not statistically significantly.There was also a weak negative association between Ki-67proliferation index and CK 20 expression in superficial cells.The correlations are summarized in Online Resource 8.

Discussion

In our study, we demonstrate alterations in individualuroplakin expression during carcinogenesis in rat bladderurothelium. BBN-induced carcinogenesis model was usedas it mimics the morphological, biological, and molecularfeatures of human bladder cancer [37–40]. Flat hyperplasiawith mild dysplasia and low-grade papillary urothelialcarcinomas develop at 5, 10, and 15 weeks of BBNadministration. At 20 weeks, low-grade papillary urothelialcarcinomas occur in all animals. Invasive tumors appear at20 weeks of BBN followed by 15 weeks tap wateradministration and they are either low- or high-gradepapillary urothelial carcinomas or squamous cell carcinomawith extensive keratinization. Taking together, prevailing

Table 1 Urothelial lesions during carcinogenesis induced by BBN administration

Weeks of BBN Urothelial lesion

flat hyperplasia with mild dysplasia (2/3) 5

papillary urothelial carcinoma, low grade (pTa) (1/3)

flat hyperplasia with mild dysplasia (2/3) 10

papillary urothelial carcinoma, low grade (pTa) (1/3)

flat hyperplasia with mild dysplasia (2/3) 15

papillary urothelial carcinoma, low grade (pTa) (3/3)

20 papillary urothelial carcinoma, low grade (pTa) (3/3)

papillary urothelial carcinoma, low grade (pTa) (1/3)

papillary urothelial carcinoma, low grade, with lamina propria invasion (pT1) (1/3)

papillary urothelial carcinoma, high grade, with lamina propria invasion (pT1) (2/3) 20

+ 15

squamous cell carcinoma with extensive keratinization, grade 2, with lamina propria invasion (pT1) (2/3)

Darker shading indicates more severe lesions

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types of cancers are papillary carcinomas and invasivenessappears at the end of the experiment, which is consistentwith previously published data.

Since identification of uroplakins as the major differen-tiation products of normal urothelium [11], several studieshave been made to determine whether these proteins areexpressed in urothelial carcinomas [22–25]. Most of thepublished studies have evaluated the expression of totaluroplakins [22, 23, 25]. Our findings suggest that duringprogression from hyperplasia to invasive carcinomas, UPIIis the most diminished of all uroplakins in superficialurothelial cells and the least present in intermediateurothelial cells. UPII is the only uroplakin synthesized as

a precursor (pro-UPII), which presumably undergo furin-mediated removal of the prosequence before oligomeriza-tion of heterotetramers (UPIa/UPII and UPIb/UPIII) andformation of 16-nm protein particles could occur [41]. Thelack of mature UPII in BBN-induced urothelial lesions canhamper the assembly of urothelial plaques. This isconsistent with the fact that after BBN administration,microridges are almost completely replaced by microvilliand ropy ridges. Similarly, in UPII knockout mouseurothelial plaque formation is completely abolished andapical surface is smooth [19]. Moreover, cultured urothelialcells, which fail to form properly glycosylated pro-UPII, areunable to assemble uroplakins into particles and plaques

Fig. 3 Uroplakins immunohistochemistry on sequential serial sec-tions of normal control urothelium (a–d), of flat hyperplasia with milddysplasia (5 weeks of BBN administration) (e–h), of low-gradepapillary urothelial carcinoma (20 weeks of BBN administration) (i–l)and of invasive high-grade papillary urothelial carcinoma (20 weeksof BBN followed by 15 weeks of tap water administration) (m–p). Innormal control urothelium, UPIa (a), UPIb (b), UPII (c), and UPIIIa(d) staining of all superficial cells is strong. In flat hyperplasia withmild dysplasia, UPIa (e), UPIb (f), and UPIIIa (h) staining of almostall superficial cells and some intermediate cells is strong, while UPII

(g) staining is weak. In low-grade papillary urothelial carcinoma, UPIa(i) staining is strong to moderate in superficial cells and moderate insome intermediate cells. UPIb (j) and UPIIIa (l) staining is moderatein some superficial cells and weak to moderate in some intermediatecells; UPII (k) staining is weak or negative in superficial andintermediate cells. In invasive high-grade papillary urothelial carcino-ma, some superficial and intermediate cells are UPIa (m) and UPIb (n)positive; rare superficial and none intermediate cells are UPIIIa (p)positive; UPII (o) staining is completely negative

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Tab

le2

Statisticsforurop

lakins

immun

ohistochem

istrydata

bylesion

severity

Mean

(range)

UP

Normal

control

urotheliu

m(C)

Flat

hyperplasia

with

mild

dysplasia(f)

Papillary

urothelial

carcinom

a,low-grade

q(p)

Papillaryurothelial

carcinom

a,low

grade,

with

laminapropria

invasion

(l)

Papillaryurothelial

carcinom

a,high

grade,

with

laminapropria

invasion

(h)

Squam

ouscell

carcinom

awith

extensivekeratin

ization,

grade2,

with

lamina

propriainvasion

(e)

Pfor

difference

between

grou

ps

Hom

ogeneous

subsets

Superficial

cells

UPIa

3.9(3–4)

3.7(2–4)

3.2(1–4)

1.5(1–2)

1.3(1–3)

1.0(1–1)

<0.001

e,h,

l<p,

f<C

UPIb

3.7(3–4)

3.0(2–4)

2.7(1–4)

1.6(1–3)

1.3(1–3)

1.0(1–1)

<0.001

e,h,

l<p,

f<C

UPII

3.9(3–4)

2.2(1–3)

1.7(1–3)

1.0(1–1)

1.0(1–1)

1.0(1–1)

<0.001

e,h,

l<p,

f<C

UPIII

3.9(3–4)

3.0(1–4)

2.4(1–4)

1.0(1–1)

1.1(1–2)

1.0(1–1)

<0.001

e,l,h<p<f<C

Pfordifference

betweenUPs

0.703

<0.00

1<0.001

0.003

0.06

4NA

Hom

ogenous

subsets

allUPsequal

II<III,Ib

<Ia

II<III<Ib

<Ia

II,III<Ia,Ib

II,III<Ia,Ib

allUPsequal

Interm

ediate

cells

UPIa

1.3(1–2)

2.8(1–4)

2.6(1–4)

1.0(1–1)

1.1(1–2)

1.0(1–1)

<0.001

e,l,h,

C<p,

f

UPIb

1.2(1–2)

2.2(1–3)

2.1(1–3)

1.3(1– 2)

1.1(1–2)

1.0(1–1)

<0.001

e,h,

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f

UPII

1.2(1–2)

1.6(1–2)

1.2(1–2)

1.0(1–1)

1.0(1–1)

1.0(1–1)

<0.001

e,h,

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1.2(1–2)

1.9(1–3)

1.6(1–3)

1.0(1–1)

1.0(1–1)

1.0(1–1)

<0.001

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f

Pfordifference

betweenUPs

0.144

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0.020

0.40

0NA

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allUPsequal

II<III<Ib

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allUPsequal

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Post-ho

ccomparisons

aresummarized

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means

across

grou

ps

NAno

tapplicable

610 Virchows Arch (2011) 458:603–613

[41] and do not exhibit microridges [42–44]. Furthermore,expression of CK 20 is also decreased. As both, micro-ridges and CK20 are markers of terminally differentiatedumbrella cells [7, 45], negative CK 20 staining and theappearance of microvilli and ropy ridges indicate lowdifferentiation state of superficial urothelial cells in urothe-lial lesions. Ki-67 immunohistochemistry show that prolif-eration indices are increasing, except for invasive squamouscell carcinoma with extensive keratinization, which issimilar as in human bladder carcinogenesis [46–48]. Thesedata show that BBN-induced carcinogenesis is accompa-nied by increased proliferation and altered differentiation ofurothelial cells. Furthermore, decreased expression of UPIIduring urothelial carcinogenesis results in diminishedurothelial plaque formation and changed apical plasmamembrane structure.

In BBN-induced flat hyperplasia with mild dysplasiaexpression of UPIIIa is similar to that of UPIa and UPIb.This indicates: (1) persistent strong expression of UPIa,UPIb, and UPIIIa in superficial cells; and (2) the appear-ance of many intermediate cells that express UPIa and UPIband some of those that express UPIIIa. Changes inexpression of UPIIIa, UPIa, and UPIb are in accordancewith previously published data obtained by anti-AUMantibody, which reacts strongly with UPIIIa and moderatelywith UPIa and UPIb [22]. Expression of uroplakins in

intermediate cells is in contrast to the fact that uroplakinsare synthesized primarily in the umbrella cells of thenormal urothelium and may reflect aborted attempts atterminal differentiation during carcinogenesis [22].

In low-grade papillary urothelial carcinomas, the mostlyexpressed uroplakin in superficial and intermediate cells isUPIa, closely followed by UPIb and UPIIIa. In contrast toUPII-deficient urothelium, where UPIa is entrapped in ER[19], in BBN-induced urothelial carcinogenesis UPIadespite the lack of UPII can reach the urothelial surface(Fig. 3m, o).

In invasive low- and high-grade papillary urothelialcarcinomas, the expression of UPIa and UPIb in superficialand intermediate cells is very low, while UPII and UPIIIaare not expressed. It seems that, despite the lack of theirpartners, UPIa and UPIb can reach the urothelial surface(Fig. 3m, n). This is not unexpected for UPIb, as it is theonly uroplakin that, when expressed alone in 293 T cells,can exit from the ER to reach the cell surface [17]. Similarto noninvasive low-grade papillary urothelial carcinomas,UPIa immunohistochemistry again indicates that UPIacould reach the urothelial surface without its partner UPII.These data indicate that the assembly of uroplakins andtheir transport to apical plasma membrane is altered incancer urothelial cells.

Invasive squamous cell carcinoma with extensive keratini-zation exhibits no uroplakin expression. This is not surprisingas squamous cell carcinomas closely resemble epidermalkeratinocytes both in morphology and in protein expressionprofiles [49, 50]. Obviously, the neoplastic transformation ofthe urothelium leading to squamous cell carcinoma is sosevere that uroplakin expression is completely lost.

An important finding of this study is that duringurothelial carcinogenesis UPII expression is the mostdiminished in all urothelial lesions, while the expressionof UPIa, UPIb, and UPIIIa is differently altered in differenttypes of lesions. We assume that decreased level of UPIIexpression hinders the urothelial plaque formation andtherefore changes apical plasma membrane structure. WhileBBN model of urothelial carcinogenesis has morphological,biological, and molecular parallels to human bladdercancer, our results about individual uroplakin expressionand apical plasma membrane structure could be valuablefor further human bladder cancer research.

Acknowledgment We are grateful to Prof. T.T. Sun for carefulreading of the manuscript and valuable suggestions. We thank NadaPavlica, Sabina Železnik, Sanja Čabraja, and Linda Štrus for theirtechnical assistance. This study was supported by a grand fromMinistry of Higher Education, Science and Technology, Republic ofSlovenia (P3-0108).

Conflict of interests The authors declare that they have no conflictof interest.

Fig. 4 Western blot analysis of UPIa, UPIb, UPII, UPIIIa, and actinin control normal urothelium and at 5, 10, 15, 20 weeks of BBNadministration and at 20 weeks of BBN followed by 15 weeks of tapwater administration. Bands correspond to expected molecularweights (27, 28, 15, 47, and 43 kDa for UPIa, UPIb, UPII, UPIIIa,and actin, respectively), that confirm specificity of used antibodies.All four uroplakins are detected in control normal urothelium. AfterBBN administration, the intensity of all bands decrease, with UPIIbands being the most diminished. Western blot experiments wereperformed twice with similar results. Molecular weight standards areshown on the right

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