[CANCER RESEARCH 58. 5662-5666. December 15. 1998]

Advances in Brief

Differential Mucin MUC7 Gene Expression in Invasive Bladder Carcinoma in

Contrast to Uniform MUC1 and MUC2 Gene Expression in Both NormalUrothelium and Bladder Carcinoma1

Margitta Ret/,2 Jan Lehmann, Christian Roder, BjörnPlötz,JürgenHarder, Jan Eggers, Jan Pauluschke,

Holger Kalthoff, Michael Stöckle

Section of Experimental Urology, Department of Urology ¡M.R., J. L., J. E.. J. P.. M. S.j. Section of Molecular Oncology, Department of General Surgery antl Thoracic Surgery1C. R., B. P., H. K.Ì,anil Clinical Research Unii. Department t>fDermatology [J. H.¡,University Hospital Kiel. 24105 Kiel, Germany

Abstract

Mucins i Ml l 'si are high molecular weight membrane glycoproteins.

The gene expression of MUCs (MUC1-MUC8) may change characteristi

cally during malignant transformation of epithelial tissues. Total RNAwas isolated from the four bladder cancer cell lines RT4, 647V, HT1376,and 486P (pathological gradings between Gl and G4) and 17 samples oftransitional cell carcinomas, as well as 16 samples of normal humaniimllic limn of the bladder from surgically removed specimens. The RNAsamples were studied with MUC1-, MUC2- and Afi/C7-specific nestedreverse transcription-PCRs. Gene expression of MUCI and MUC2 was

found positive in all normal, as well as in malignant, tissue samples and inthe tumor cell lines. In contrast, gene expression of Ml < 7 was onlydetected in bladder cancer cell lines and samples of invasive transitionalcell carcinomas, but neither in superficial, noninvasive bladder tumorsnor normal bladder urothelium. Only one of the samples of normalurothelium obtained from 16 different tumor-bearing bladders was pos

itive for MVC7 gene expression. These results suggest a differential MUC7gene expression with the onset of malignant transformation of the bladderurothelium.

Introduction

MUCs' are high molecular weight glycoproteins that are synthe

sized by glandular epithelial cells of the gastrointestinal, respiratory,and urogenital tracts. Thus far, nine human MUC genes have beenidentified and designated MUCI, MUC2, MUC3, MUC4, MUC5AC,MUC5B, MUC6, MUC7, and MUC8 in the international nomenclature(1). A number of functions have been proposed including protection,lubrication, and formation of a selective barrier of the epithelium (2).In the bladder, MUCs provide a physical barrier between urothelialcells and urine. The MUC layer plays a role in protecting the urothelial cells against changes in urinary pH and osmolarity. Furthermore,an intact MUC layer is a prerequisite for inhibiting adhesion ofbacteria and urinary crystals (3). The aberrant expression of MUCs inmalignantly transformed epithelial tissues is also well known. Severalstudies described altered synthesis, secretion, and structure of MUCsin different cancer cell types. The up-regulation of MUC expression

and an abnormal glycosylation pattern may be associated with tumor

Received 9/8/98: accepled 10/28/98.The costs of publication of this article were defrayed in part by the payment of page

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

1Supported by the "GüntherVoges Gesellschaft" (Kiel. Germany) and in parts by the"Paul-Bliimel Stiftung" (Hannover. Germany I and the "Schleswig-Holsteinische Krebs

gesellschaft e.V. (Kiel, Germany).2 To whom requests for reprints should be addressed, at Section of Experimental

Urology, Department of Urology. University Hospital Kiel, Arnold-Heller-Stras.se 7,

24105 Kiel. Germany.' The abbreviations used are: MUC. mucin: RT-PCR, reverse transcription-PCR;

GAPDH, glyceraldehyde-3-phosphate dehydrogenase: TCC, transitional cell carcinoma:TNM, tumor-node-metastasis; VNTR. variable number of tandem repeats: EMBL. European Molecular Biology Laboratory: BCG, Bacillus Calmette-Guerin.

invasion and metastatic potential (4). Little is known about the expression of MUC genes in normal urothelium and TCC of the bladder.Immunohistochemical studies described MUCI expression in normalurothelium predominantly at the apical membranes of the umbrellacell layer, whereas in TCC MUCI stained to membranous and/orcytoplasmic cells of all urothelial cell layers, depending on tumorgrade and stage (5). MUC2 was not detected immunohistochemicallyin normal urothelium, but was found in 40% of cases of TCC (6). Insummary, only MUCI and MUC2 have been detected in normalurothelium or in bladder carcinoma by immunohistochemical techniques, but no comprehensive studies have analyzed their mRNAexpression yet. To our knowledge thus far, no reports have focused onthe investigation of any other MUC expression in human urotheliumand TCC of the bladder. We have screened a set of gastrointestinaland other tumor cell lines for the expression of MUCs. Colorectal andbladder cancer cell lines exhibited positive results for MUC7 in thisscreening. The structure and chromosomal localization of the humansalivary MUC gene MUC7 was first described by Bobek et al. (7) in1993 (8). Since then, MUC7 gene expression has also been detected inthe pancreas, in submucosal glands of the bronchus, and in cholan-giocarcinomas (9-11). On the basis of these findings, the aim of this

study was to investigate the gene expression of three MUCs (MUCI,MUC2, and MUC7) by a nested RT-PCR in cell lines of TCC, benign,

and malignant tissues of urothelial origin.

Materials and Methods

Cell Lines. Four cell lines of TCCs of the bladder (RT4 with the pathological grade Gl. 647V with grade G2, HT 1376 with grade G3. and 486P withgrade G4) were investigated (12). The colorectal carcinoma cell line WIDR(13) served as a positive control for the expression of MUCI, MUC2, andMUC7. RT4, 467V, HT 1376, 486P, and WIDR were obtained from theAmerican Type Culture Collection (Manassas, VA). Cell lines were cultured asa monolayer in RPMI 1640 (Biochrom, Berlin. Germany) supplemented with10% heat-inactivated PCS, 2 mmol/liters glutamine. and 1 mmol/liter pyruvate.HT-1376 cells were grown in Eagle's MEM (Biochrom) with the same

additives. Cultures were maintained at a temperature of 37°Cin a humidified

5% CO2 atmosphere. All cell lines were routinely tested for mycoplasmacontamination using a PCR mycoplasma detection kit (TaKaRa: BiomédicalEurope S. A., Genevilliers, France), and contaminated cultures were discarded.

Tissues. Four samples of superficial, noninvasive tumors and one samplewith severe urothelial dysplasia were obtained by transurethral resection of thebladder. Eleven samples of invasive bladder tumors and two samples ofcarcinoma in situ, as well as 16 samples of normal urothelial tissue, wereobtained from cystectomy specimens. All samples were immediately immersedin liquid nitrogen within 10 min after removal from the patients. A representative sample was taken from each tumor for histopathological assessment, andan adjacent piece was frozen in liquid nitrogen and stored at -80°C for

subsequent RNA extraction. Tumors were graded according to criteria asrecommended by the WHO and staged according to the TNM classification.The pathology of the tumors was confirmed by the consensus of two pathol-

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MVC7 GENE EXPRESSION IN INVASIVE BLADDER CANCER

ogists. The histopathological tumor stage of the superficial, noninvasive bladder tumors ranged between pTaGl and pTaG2. All invasive bladder tumorswere classified as TCCs. The histopathological tumor stage of the invasivebladder carcinomas ranged between pTl G3 and pT4 G3 (see Table 2).

Isolation of Total RNA. Total RNA was isolated from tumor cell lines andtissue samples (100-500 mg) based on the guanidium-thiocyanate-phenol-chloroform single-step isolation method (14). The RNA from tumor cell lineswas extracted using RNA-Clean (AGS-Chemie. Heidelberg. Germany), andfrozen tissue specimens were extracted for total RNA by RNA/ol (WAK-

CHEMIE MEDICAL GMBH. Bad Homburg. Germany). To optimize the yieldof RNA. 5 Lig of glycogen (Boehringer Mannheim. Mannheim, Germany)were added to the aqueous phase before precipitation with isopropanol. TheRNA preparations were dissolved in 40 LÃœof RNase-free water. RNA recovery

and purity were controlled by absorption measurement at 260 nm and 280 nm(Gene Quant II: Pharmacia Biotech. Freiburg, Germany), respectively. Samples of 2 Lig of the extracted total RNA were separated in 1% agarose gel(Small DNA agarose: Biozym, Hessisch Oldendorf, Germany). The integrityof RNA was determined, and samples with evidence of rRNA degradationwere discarded.

RT-PCR. The total RNA (2 /xg) of each sample was dissolved in a volumeof 10 id, denatured for 10 min at 70°Cand then quickly chilled on ice. The

cDNA was synthesized in a total volume of 20 /nl containing 4 Lilof 5 x first-

strand buffer, 2 mM DTT. 200 units of Superscript II (all purchased from LifeTechnologies. Inc., Eggenstein, Germany), 20 units of RNase inhibitor. 5 LIMrandom hexamers. and 1 mM deoxynucleotide triphosphate mix (all purchasedfrom Perkin-Elmer Applied Biosystems GmbH. Weiterstadt, Germany). Thereaction mix was incubated for 10 min at 24°Cand 60 min at 42°C.Reversetranscriptase was then inactivated for 3 min at 94°C.The MUC1-. MUC2-, and

MUC7-specific PCR was performed as nested PCR. Primers were synthesizedby MWG-Biotech (Ebersberg. Germany).

MUC1, MUC2, and MUC7 Nested RT-PCR. MUC1-Õ sense, 5'-TCAATTCCCAGCCAC CACTCT; MUC1-B antisense. 5'-AGTTCTTTCG-GCGGCACTGAC: MUC1-C sense, 5'-GCCAGCCATAGCACCAAGA CT:MUC1-D antisense, 5'-TGACAGACAGCCAAGGCAA TG. External MUCl

RT-PCR resulted in a 595-bp fragment: the internal PCR yielded a 539-bp

product.MUC2-A sense, 5'-CCTACGTGCTGGTGGAGGAGA; MUC2-B anti-

sense, 5'-CAGCCTGA CGGAGAAGGACAG: MUC2-C sense, 5'-GAGAT-

CAGCCCCTCCGTGGAC: MUC2-D antisense. .V-CACCGCCTGCCTGT-TCACCTG. External MUC2 RT-PCR resulted in a 317-bp fragment: theinternal PCR yielded a 180-bp product. MUC7-A sense, 5'-TTGGCTAAAAGCAAGCAACTGG: MUC7-B antisense, 5'-TTGGTGGAAGCTGATGG-GAAAG: MUC7-C sense, 5'-CACACTGCACCAGAGACATCAGAA:MUC7-D antisense, 5'-AGCCACTAAG GTAGGGTTGACCAC. External

MUC7 RT-PCR resulted in a 439-bp fragment; the internal PCR yielded a348-bp product.

For each specific MUCl, MUC2. or MUC7 nested RT-PCR, 20 til of the

cDNA synthesis mix were used in a final volume of 50 LI!.The reaction mixcontained 3 Ltl of 10 x tricine buffer III (15). 200 LIM deoxynucleotidetriphosphate mixture, 1.5 LIMAft/C-A sense. 1.5 /MMMUC-B antisense primer,and 1 unit of Taq-DNA-polymerase (Life Technologies, Inc.). The cyclingprotocols for the MUCl, MUC2, and MUC7 nested RT-PCRs are listed in

Table 1. From the first PCR reaction mix. 1 LI!was formulated with 1.5 til ofthe MUC-C sense and MUC-D antisense primers in a final volume of 50 jul.

To monitor the cDNA synthesis, a PCR for GAPDH was performed additionally. A 50-/xl PCR reaction mix contained 1 Lil of the external PCRreaction mix, l LIM of the primers GAPDH sense 5'-CCAGCCGAGCCA-CATCGCTC and GAPDH antisense 5'-ATGAGCCCCAGCCTTCTCCAT

(sequences kindly provided by C. Schliiter. Department of Dermatology,University Hospital Kiel). 3 Lil of 10 X tricine buffer III. 200 LIMdeoxynucle

otide triphosphate mix, and 1 unit of Taq DNA Polymerase. The cyclingprotocol for GAPDH PCR corresponds to the MUC7-PCR protocol describedin Table 1. The PCR product migrated as a 359-bp fragment in agarose gel

electrophoresis.As a positive control for MUCl, MUC2, and MUC7 PCR we used 2 /xg of

total RNA from the colon carcinoma cell line WIDR. The absence of contamination was routinely checked by RT-PCR assay of negative samples, in which

RNA samples were replaced with sterile water (water control) and by omittingSuperscript II. All RT-PCR products were separated by electrophoresis in a

Table 1 Cycling protocol for MUCl, MUC2. and MUC7 nested RT-PCR

StepMUCl123456MUC212345MUC71234567Denaturationlime/

temperature2min/95°Clmin/95°Clmin/95°Clmin/95°C1

min/95°C2min/95°Clmin/95°Clmin/95°Clmin/95°C2min/94°C1

min/94°C1min/94°Clmin/94°Clmin/94°Clmin/94°CAnnealing

time/temperaturelmin/6l°Clmin/60°Clmin/59°Clmin/58°Clmm/57°Clmin/61°Clmin/60°Clmin/59°Clmin/58°Clmin/61°CImin/60°Clmin/59°Clmin/58°CImin/57°Clmin/56°CExtension

time/temperature2min/72°C2min/72°C2min/72°C2min/72°C2min/72°CIOmin/72°C2min/72°C2min/72°C2min/72°C2min/72°CIOmin/72°C2min/72°C2min/72°C2min/72°C2min/72°C2min/72°C2min/72°C10min/72°CNumber

ofcycles111130111130111111291

2% agarose gel (Small DNA agarose: Biozym. Hessisch Oldendorf. Germany)in Tris-acetate-EDTA buffer and visualized by ethidium-bromide staining. The

molecular weights were determined using a DNA molecular weight marker(l(K)-bp DNA ladder; Life Technologies, Inc.).

Sequencing of the RT-PCR Products. Representative fragments of thespecific MUCl. MUC2, and MUC7 nested RT-PCR were extracted fromagarose gels by QIAEX II Gel Extraction-Kit (QIAGEN GmbH. Hilden.

Germany). The isolated cDNA product was sequenced by an automatic sequencer ALFexpress (Pharmacia. Freiburg. Germany).

Results

The gene expression of MUCl, MUC2, and MUCl was investigated in four cell lines of TCCs and in 17 samples of bladder tumors,which were obtained from cystectomy specimens (n = 13) or fromtransurethral resections (n = 4). Results were compared with isolatesfrom 16 normal urothelial tissues from tumor-bearing bladders as

matched tissue samples.RT-PCR. A GAPDH-specific RT-PCR producing a 359-bp frag

ment demonstrated the integrity of isolated RNA and success of thecDNA synthesis procedure for all MUC nested RT-PCRs (lower bandof Fig. \,A-O.

RT-PCR of MUCl. Products of 539 bp amplified by a MUClnested RT-PCR were detected in all isolates from the four bladder

carcinoma cell lines RT4, 647V, HT 1376, and 486P with pathologicalgradings between Gl and G4 (Fig. 1/4, Lanes i8-21). The colorectalcell line WIDR serving as a Mi/C7-positive control also showed anested RT-PCR product of 539 bp (Fig. \A, Lane 1). MUCl gene

expression could be detected in all tissue samples of bladder carcinomas with tumor stages ranging from pTaGl (two of four selectedsamples in Fig. IA, Lanes 9 and 10) to pT4G3 (Fig. IA, Lane 17), aswell as in two samples with carcinoma in situ of the bladder (Fig. \A,Lanes 11 and 12). All isolates of normal urothelium were also foundpositive for MUCl gene expression (6 of 16 selected samples in Fig.IA, Lanes 2-7). The corresponding negative controls (amplification

without reverse transcription) showed no MUCl gene expressionthroughout all investigated samples (data not shown). The analyzedsequence of one of the MUCl-specific nested RT-PCR products

(derived from cell line HT 1376) of 539 bp showed 100% identity withthe published MUCl mRNA sequence (GenBank accession No.J05581).

RT-PCR OÕMUC2.The expected A/(/C2-specific nested RT-PCR

product of 180 bp was amplified from extractions of the four differentbladder cancer cell lines and of the control cell line WIDR (Fig. Iß,

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MUC7 GENE EXPRESSION IN INVASIVE BLADDER CANCER

2345678 9 10 11 12 13 14 IS 16 17 18 19 20 21 M

M 12345678 ') 10 11 12 13 14 15 16 1718 l (CO 21 M

MUC1 (539 bp)

—GAPDII(3S9bp)

—MUC2(796 bp)

—MUC2(317bp)—MUCZ(lSObp)

—GAPDH(359bp)

M 12345678 9 Kl 1112 1314 15 16 1718 192021 M

-MUC7(348bp)

-GAPDH (359bp)

Fig. I. MUCÌ-,MUC2-. and Mt/C7-specific nested RT-PCR of normal urolhelium ofIhe bladder, bladder lumor. and bladder carcinoma cell lines. A, MUC I-specific nestedRT-PCR (internal PCR product, 539 bp). B. A/I7C2-specific nested RT-PCR (MUC2

intron product. 796 bp; external PCR product, 317 bp; internal PCR product, 180 bp). CMt/C7-.specific nested RT-PCR (internal PCR product, 348 bp). Samples were separatedby 2% agarosc gel electrophoresis and stained with ethidium bromide. Representative datafrom a panel of 20 specimens are presented. The colon carcinoma cell line W1DR servedas positive control for MUC1, MUC2, and MUC7 gene expression (Lane I), normalurothelial tissues of the urinary bladder (Lanes 2-7), severe urothelial dysplasia (Lane 8),superficial, noninvasive bladder tumors (Liines 9 and 10}. carcinoma in situ (Lanes II and12). invasive bladder carcinoma with T1G3 (Lime 13], T2G3 (Lane 14). T3G2 (Lane 15),T3G3 (Lane 16). T4G3 (Lane 17). and bladder carcinoma cell lines with RT4 (Lane 18).647V (Lane IV). HTI376 (Lane 20). and 468P (Lane 21). M. molecular weight marker.The corresponding control reaction was performed as GAPDH RT-PCR with a 359-bpproduct and is shown in the lower band. Data of corresponding negative control (amplification without reverse transcription) are not shown.

Lane I). MUC2 gene expression was detected in all tissue samples ofbladder carcinomas including two carcinomas in situ (selected samples in Fig. Iß,Lanes 9-20) as well as in all samples of normalurothelium (selected samples in Fig. Iß,Lanes 2-7) and in urothelialdysplasia (Fig. Iß,Lane 8). Sequencing of one of the 180-bp fragments of the Mi/C2-specific nested RT-PCR (derived from cell line

HT1376) proved 100% similarity with known sequences from theGenBank nucleotide database (GenBank accession Nos. L21998,M94132, and M86523). The majority of samples also displayed the317-bp long external product on completion of the Aft/C2-specificnested RT-PCR (Fig. Iß,Lanes 6-10 and 72-27). Correspondence ofthe 317-bp fragments, as seen in Fig. Iß,with the expected MUC2

external product was also confirmed by automatic sequencing of onesample (derived from cell line HT 1376). Furthermore, an additional

amplified fragment of 796 bp was found in 13 extracted tissuesamples (Fig. Iß,Lanes 2-5, 7, 9-11, 13-15, 17, and 19). This

fragment was identified as a genomic DNA contamination because italso appeared as a single fragment in the otherwise negative controlsthat were run without reverse transcription (data not shown). Performing a PCR with DNA from a commercially available genomic library(Promega, Madison, WI) with the MUC2-C sense and MUC2-D

antisense primers confirmed once more the genomic origin of thedetected fragment by yielding the same 796-bp fragment (data not

shown). This fragment was identified by automatic sequencing ashaving included a novel intron of 616 bp (EMBL accession No.AJ007575), located between positions 1098 and 1099 of GenBankaccession No. M94132, between positions 13710 and 13711 of Gen-

Bank accession No. L21998, and between positions 209 and 210 ofGenBank accession No. M86523, respectively. Therefore, it wasconcluded that intron spanning primers were established for theA/£7C2-specificnested RT-PCR applied in this study. The correspond

ing negative control (amplification without reverse transcription)showed no MUC2 gene expression throughout all investigated samples (data not shown).

RT-PCR of MUC7. RT-PCR products of 348 bp amplified by aMUC7 nested RT-PCR were detected in all isolates from the four

different bladder cancer cell lines and in the control cell line WIDR.Furthermore, all tissue samples of invasive TCCs, including twosamples of carcinoma in situ of the urinary bladder, showed a MUC7gene expression. In contrast to these samples and to the findings withMUCÃŒand MUC2, no MUC7 amplification was detected in thesuperficial, noninvasive bladder tumors (Fig. 1C, Lanes 9 and 70) orin the sample with severe urothelial dysplasia (Fig. 1C, Lane 8).Except for one sample of normal urothelium that was obtained froma tumor-bearing bladder (Fig. 1C, Lane 2), all 15 samples of normal

urothelium were repeatedly found negative for MUC7 gene expression (5 of 15 selected samples in Fig. 1C, Lanes 3-7). The fragmentlength and analyzed sequence of one Mt/C7-specific nested RT-PCR

product of 348 bp was in accordance with the known MUC7 cDNAnucleotide sequence (GenBank accession No. L13283). The corresponding negative control (amplification without reverse transcription) showed no MUC7 gene expression in all cases (data not shown).Table 2 gives an overview of the MUC7 gene expression in all tissuesamples of the bladder in regard to the tumor stage.

Discussion

During recent years, a number of biochemical studies on the structure and the organ specificity of several MUC proteins have beenpublished. Due to extensive research, it is now possible to identifynine different MUCs of the MUC family (1). MUC1 is the bestcharacterized MUC and is expressed on the apical surface of mostpolarized epithelial cells (2). MUC2 production and secretion was firstdemonstrated in endoderm-derived epithelial cells, mainly in the

Table 2 MUC7 gene expression in tissue samples tif the bladder with different TNMstages assessed by MUC7-specifìcnested RT-PCR

Histology of thebladderNormal

urotheliumUrothelialdysplasiaNoninvasivecarcinomaPreinvasivecarcinomaInvasive

carcinomaUICC"

classification(TNMstage)Stage

OaStageOisStage

IStageIIStageIIIStage

IVNo.

of samples withMUC7 gene expression/

total no. ofsamples1/16(VI0/42/21/12/2Wh2/2

' UICC, Union Internationale contre le Cancer.

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MUC7 GENE EXPRESSION IN INVASIVE BLADDER CANCER

gastrointestinal and respiratory tracts (16). A/t/C7has been cloned andsequenced from a salivary cDNA library (7). We investigated thethree MUCs (MUCI, MUC2, and MUC7) that are among the fewMUCs that have been fully sequenced at the cDNA level (1). MUCsequences like MUC5B and MUC6 consist mainly of a VNTRs, whichmakes them inaccessible for specific PCR detection (17). BecauseMUCI, MUC2, and MUC7 have distinct mRNA sequences apart fromVNTR regions, investigation of these MUCs by PCR is feasible. Theexact kinds and number of MUC glycoproteins expressed by thehuman urothelium of the bladder are presently unknown. Thus far,only MUCI. MUC2, and MAUB (MUC antigen of the urinary bladder; Ref. 18) have been detected in the human bladder urothelium,mainly by immunohistochemical techniques. MUCI expression innormal and neoplastically transformed urothelium of the bladder wasdescribed previously (5), which is in accordance with our findings ona larger scale of 34 surgically removed samples of normal urotheliumand TCCs as well as in four bladder cancer cell lines. MUC2 geneexpression was uniformly found in all samples of TCCs as well as innormal urothelium of the bladder. Besides the A/[/C2-specific nestedRT-PCR product of 180 bp, an additional fragment of 796 bp was

found in 13 tissue samples. It was identified as derived from genomiccontamination, containing a novel intron that was sequenced andsubmitted to the EMBL nucleotide database (EMBL accession No.AJ007575).

In contrast to our results, a differential MUC expression for MUC2has been described previously by immunohistochemistry in normalhuman urothelium of the bladder and its neoplastic transformation. Ina series of 89 human TCCs of the bladder, MUC2 expression wasdetectable by the monoclonal antibody 4F1 in approximately 40% ofthe cases, whereas normal urothelium showed no MUC2 expression(6). The antibody 4F1 is directed against a VNTR region of the coreprotein epitope of the MUC2 glycoprotein, which is located in intra-

cellular compartments before finished glycosylation. The authors concluded that due to the characteristics of the 4F1 antibody, a number ofTCCs were probably missed by immunohistochemical staining methods because the antibody might not bind to the glycosylated matureform of MUC2. This shortcoming in studying MUC expression byimmunohistochemistry is avoided by applying a RT-PCR technique

that detects MUC gene expression independent of cellular localizationor posttranslational processing.

The investigation of MUC7 gene expression in normal and malignantly transformed urothelium has not been described thus far. Thiswork demonstrates, for the first time, the differential gene expressionof MUC7 detected by a nested RT-PCR in invasive bladder carcino

mas and in TCC cell lines. Particularly, MUC7 gene expression wasfound positive in tissue samples of carcinoma in situ of the bladder,which is a preinvasive morphological alteration of epithelial cellscommonly preceding invasive tumor growth. These findings indicatea high specificity of MUC7 gene expression also with respect topreinvasive TCC of the bladder. In contrast to these results, no MUC7gene expression was detected in superficial, noninvasive bladdertumors. Except for one tissue sample, all 15 samples of normalurothelium of the bladder were found negative for MUC7 gene expression. The one positive sample of normal urothelium was obtainedfrom a tumor-bearing bladder. Although histological examinationclassified this sample as normal urothelial tissue, the up-regulation of

MUC7 gene expression might point to a premalignant genetic transformation of the bladder urothelium that was not detectable by his-

topathological criteria. Our results confirm that neoplastic transformation of the urothelium is accompanied by switching on the geneexpression of MUC7. The analysis of MUC7 gene expression by anested RT-PCR technique may be of value in the early diagnosis of

neoplastic transformation of the bladder urothelium. therefore, adding

a sensitive molecular diagnostic parameter to the disease of TCC ofthe urinary bladder. These data encourage additional studies investigating the detection of Mf/C7-positive cells in voided urine samplesby RT-PCR technique and immunocytochemistry.

Cancer-related dysregulation of MUC gene expression has been

studied extensively. The functions of MUCs in tumor cells mayinclude protection from hostile environments, protect the tumor cellsfrom the immune system by acting as a blocking agent, and sterichindrance for cell surface antigens that are involved in immunerecognition. Therefore, tumors that express higher amounts of MUCsmay have a better chance of survival and a higher tendency tometastasize once they have reached the blood and lymphatic system.Consistent with these theories, it was previously shown that theup-regulation of MUCI expression in breast cancer is associated with

tumor invasion and progressive disease ( 19). Taken together, it maywell be possible that MUC quantity and alterations in their composition may be influential on the biological behavior of a tumor cell and,consequently, on the clinical outcome.

An interesting clinical aspect of MUC expression involves theinteraction of BCG as a immunotherapeutic agent for superficialbladder carcinoma and MUC-expressing urothelial cells. Badalament

et al. (20) found in animal experiments that attachment of the BCGcells to the urothelium. which is a prerequisite for effective therapy,depends on the state of glycosylation of the urothelial MUC layer. Theauthors hypothesize that removal of the urothelial MUC layer may beassociated with increased BCG adherence to the bladder surface and,therefore, enhancement of the antitumoral effect. It is concluded thataltered MUC expression in neoplastic changes of the bladder urotheliummight influence the effectiveness of this established form of therapy.

In conclusion, this study revealed a differential MUC gene expression for MUC7 in preinvasive and invasive bladder carcinomas incontrast to superficial, noninvasive bladder tumors and normal urothelium. These results might indicate a high sensitivity of MUC7 geneexpression in premalignant transformation of the bladder urothelium.The clinical implication of the differential MUC7 gene expression inTCC and its role as a potential tumor marker warrant additionalinvestigations and studies on a larger scale of patients.

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1998;58:5662-5666. Cancer Res   Margitta Retz, Jan Lehmann, Christian Röder, et al.   Expression in Both Normal Urothelium and Bladder Carcinoma

GeneMUC2 and MUC1Carcinoma in Contrast to Uniform Gene Expression in Invasive BladderMUC7Differential Mucin

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