(CANCER RESEARCH54, 3101—3106,June 15, 19941

Advances in Brief

Wilms' Tumor Suppressor Gene Expression in Rat and Human Mesothelioma'

Cheryl Walker,2 Fons Rutten,3 Xiaoquin Yuan, Harvey Pass, Daphne M. Mew, and Jeff EverittDepartment of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957 fC. W., X. YJ; ChemicalIndustry Institute of Toxicology, Research Triangle Park, North Carolina 27709 [F. R., J. E.J; and National Cancer Institute, NIH, Bethesda, Maryland 20892 [H. P., D. M. M.J

Abstract

Induction of mesothelioma in the rat is an important animal model forassessing the carcinogenic potential of fibers and for understanding themolecular basis underlying the development of these tumors. Mesotheliomas and nephroblastoma (Wilma' tumor) have many developmental,biochemical, and histological similarities; however, the expression of theWilma' tumor suppressor gene, WT-1, has not been well characterized inthe rat, and its expression pattern in rat or human mesothelioma has notbeen described. We report that WT-1 transcripts (3.2 kilobases) could bedetected by Northern analysis in adult rat testis, spleen, kidney, lung,heart, and glomerular mesangial cells. Normal adult mesothelial cells alsoexpressed this gene. Rat mesothelioma cell lines expressed WT-1 traitscripts of 3.2 kilobases and an additional 2.8-kilobase transcript, previously only reported to be expressed in the testis. Normal and transformedrat mesothelial cells expressed all four of the WT-1splice variants, excepttestis, which only expressed WT-1 splice variants containing exon 5. Sevenof seven human mesothelloma cell lines examined also expressed WT-1transcripts, suggestingthat expressionof this gene may be useful in thediagnosis of these tumors.

Introduction

Mesotheliomas are tumors that arise from the epithelial cell layer(mesothelium) that lines the serosal surfaces (reviewed in Ref. 1).Serosal mesothelial cells form a simple squamous epithelium whosefunction is to reduce friction on the outer surfaces of the lungs andviscera and act as a selective barrier for fluid transport to the bodycavities. During embryogenesis, the mesothelium develops from thelayer of mesoderm cells exposed to the intraembryonic coelum afterthis cavity forms within the embryonic mesoderm (2). The mesenchymal origin of these cells distinguishes them from the vast majorityof epithelial cells, which arise from embryonic ectoderm or endoderm.

In the past few decades, mesothelioma has received much attentionbecause of its association with asbestos exposure. The term asbestosis used to describe a large family of fibrous silicates that vary in theirphysico-chemical properties and carcinogenic potencies (3). In the1950s and 1960s, asbestos was shown to be associated with thedevelopment of pleural tumors in asbestos miners and workers occupationally exposed to asbestos (Ref. 4; reviewed in Ref. 5). Whetherthe nonoccupational levels of exposure to asbestos that we encounterin our daily lives are a significanthealth risk has been a subjectofheated debate (6).

It is known that several intrinsic factors affect the carcinogenicityof asbestos fibers including their size, aspect ratio, surface properties,chemical composition, and durability (Ref. 7; reviewed in Ref. 8).

Received 3/2/94; accepted 5/4/94.The costs of publicationof this articlewere defrayedin partby the paymentof page

charges.This articlemust thereforebe herebymarkedadvertisementin accordancewith18 U.S.C. Section 1734 solely to indicatethis fact.

1 @l'his work was supported in part by the Department of Health and Human Services,

NIEHS Grant ES 06658-01 (to C. W.) and by a grant from the North American InsulationManufacturers Association (to J. E.).

2 To whom requests for reprints should be addressed, at Department of Carcinogenesis,

The Universityof Texas M. D. AndersonCancerCenter,Science Park-ResearchDivision,P.O. Box 389, Smithville,TX 78957.

3 Present address: Toxicology and Nutrition Institute, Zeist, The Netherlands.

However, the mechanism by which asbestos and other mineral fibersinduce tumors in humans and animals is largely unknown (9). Thelack of available information on the molecular alterations involved inthe genesis of mesothelioma has contributed to a dilemma in thediagnosis of these tumors since these neoplasms can have a diversephenotypic appearance. The histology of these tumors can be epithehal, sarcomatous (fibrous), or biphasic, containing a mixture of bothof these components (1). Mesotheliomas coexpress keratin and vimentin, a reflection of the mesenchymal origin of these epithelial cells(10—12).

Wilms' tumor is a pediatric nephroblastoma that arises from themetanephric blastema of the kidney (13, 14). The histological appearance of Wilms' tumors is variable, reflecting the multipotential natureof the renal stem cells from which they arise (15, 16). Like mesothehal cells, the epithelial cells of the renal tubule and corpuscle aremesenchymal in origin, having undergone a rare mesenchymal-epithelial transition. Wilms' tumors have a triphasic histology and contam mesenchymal stromal cells, poorly differentiated blastemal cells,and differentiated epithelial cells. The mixed histology of these tumom is reminiscent of mesotheliomas, which also differentiate alongboth mesenchymal and epithelial pathways.

The Wilms' tumor suppressor gene (WT-1) consists of 10 exons and

is 50 kilobases in length (17, 18). It has been cloned and sequenced inmice and rats as well as humans (19, 20). WT-1 is expressed as a

3.2-kilobase transcript, in most tissues, and an additional 2.5-kilobasetranscript is expressed in the testis (17, 21). The function of theadditional transcript in these cells is not known, and the event orevents responsible for alternative processing or initiation of thistranscript have not been identified. The WT-1 protein is a zinc-fingerprotein with DNA-binding activity to the EGR1 consensus sequence(17, 22—24).In normal cells, there are two alternative splice siteswithin the WT-1 gene (18). One alternative splice includes all 51 basepairs of exon 5, and a second alternative splice contains 9 base pairsinserted between zinc fingers 3 and 4. The functional significance ofthe various splice forms is not completely understood. Insertion of the9-base pair splice (which code for the amino acids KTS) abrogatesbinding to the EGRJ consensus site; and while the wild-type W1'-lprotein represses transcription, the KTh splice variant lacks thisactivity (25). In addition, another splice variant that lacks exon 2 wasrecently found in Wilms' tumors and acts as a transcriptional activator

(26). Thus, regulation of IVF-1 gene function may occur at the level ofsplicing of WT-1 transcripts, and aberrant splicing may play a role in

the transformation of cells expressing this gene.Expression of WT-1 is regulated in both a tissue-specific and a

developmentally specific fashion (19, 21, 27). In the developingkidney, WT-1 is expressed by the mesonephric blastema and isswitched off during differentiation of these cells into the renal epithelia of the mature nephron. WT-1 is also expressed during embryogenesis in the genital ridge, fetal gonads, and the mesothelium liningthe coelomic cavity (27). In the adult, expression of this gene has beendocumented in the Sertoli cells in the testis, the granulosa cells of theovary, and the myometrium of the uterus (21). The function of thisresidual expression in the mature genitourinary system is not known.

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In addition, there is a low level of expression in adult spleen, heart,lung, and thymus, which is expressed to different degrees in mice andhumans (17, 19, 21).

It is clear from the embryological studies described above that inaddition to being expressed in the kidney, Wf-1 is expressed by arestricted group of tissues and cell types during development. Although the role of W7'-l in extrarenal tumorigenesis has not beenestablished, the many developmental and histological similarities between mesothelioma and nephroblastoma suggests that WT-1 mayplay a role in the genesis of mesothelioma. Both nephroblastoma andmesothelioma arise from cells that undergo a rare mesenchymalepithelial cell transition during embryogenesis, and embryonic mesothelial cells express WT-1. The tumors themselves are histologicallysimilar in that they can be comprised of epithelial and/or mesenchymal elements and coexpress keratins and vimentin (28). In a recentstudy by Park et aL (29), 32 human mesotheliomas were examined formutations in the W1'-l gene, and only one tumor contained alterationsdetectableby single-strandconformationpolymorphismanalysisanddirect sequencing of genomic DNA from these tumors. However,there is presently no information on: (a) the frequency with whichwi:-i is expressed in mesotheliomas, (b) how WT-1 transcripts arespliced in these tumors, or (c) the WT-1 expression pattern in normaltissues in the rat, which is an important animal model for studyingmesothelioma (1). To address these questions, we examined normalrat tissues and rat and human mesotheliomas for the expression ofWT-1. We report here that the WT-1 gene is expressed in the adult ratin a tissue-specific manner, similar to the pattern of expression inhuman tissues, but is ubiquitously expressed by both rat and humanmesotheliomas. In addition, human and rat mesotheliomas and normalrat tissues expressed all four WT-1 splice variants with the exceptionof rat testis, which expressed only those WT-1 splice variants thatcontained exon 5.

Materials and Methods

Rat Cell Lines. Cell lines isolated from asbestos-induced rat mesotheliomas 11-14,11-45,111-2(30) T08, and T10 (31); spontaneous rat mesotheliomalines Mel, Me3, and MelT (32); and spontaneously transformed normal ratmesothelial cell lines NRM2 and NRM4 (33) have been described previously.11-14,11-45,and 111-2were culturedin 1:1 Ham's F-12: Dulbecco's modifiedEagle's medium with 10% FcS,4 0.1 ,.@g/mlhydrocortisone, 2.5 pg/ed insulin,2.5 @tg/mltransferrin, and 2.5 ng/ml selenium (HITS). T08 and Tl0 werecultured in Dulbecco's modified Eagle's medium with 10% FCS. Mel, MelT,and Me3 were cultured in Fl2 with 10% FCS and HITS. NRM2 and NRM4were cultured in Fl2 with 10% FCS. All cell lines were maintained at 37°Cina humidified atmosphere with 5% CO2. Primary cultures of normal rat mesothelial cells were obtained from the pleural cavity by collagenase treatmentand gentle scraping of the intracostal parietal pleura as described previously(33). Normal rat mesotheial cells were cultured in Fl2 plus 10% FCS.

Human Mesotheioma Cell Lines. A 69-year-oldpreviouslyhealthymalewith a known exposure to asbestos had a debulking resection of a biphasicpleural mesothelioma. Cells from the associated pleural effusion (calledGATES) were pelleted by centrifugation at 1200 rpm for 10 mm followed byerythrocyte lysis with ACK lysing buffer (B&B Research, Fiskeville, RI). Thewashed cells were then plated into F-b nutrient mixture medium (Biofluids,Rockville, MD) at 37°Cin a humidified 5% CO2atmosphere until a monolayerformed. Partial trypsinizations were performed as necessary. FCS was thenadded in 1% increments to the medium over the ensuing weeks, and cytospinanalysis revealed malignant-appearing cells that stained uniformly for lowmolecular weight cytokeratin by immunohistochemistry. The cell line isadapted for growth in RPM! medium with 10% F@Sand has a doubling timeof 53 h. Cell line ROBINETTE, developed under similar conditions to

4 Abbreviations used are: F@S, fetal calf serum; PCR, polymerase chain reaction; RT,

reverse transcription; PDGF, platelet-derived growth factor, IGF, insulin-like growthfactor; poly(A), polyadenylated.

GATES, was derived from a 56-year-old previously healthy male with noknown exposure to asbestos. This line was first grown in DFCI media,

followed by addition of F@S. Cell line MILOT, developed from a mincedtumor from a 59-year-old male with a known asbestos exposure, was originallydeveloped in Rheinwold medium.

The H-MESO-l cell line has been described previously (34) and wasmaintained as monolayer cultures in RPM! with 10% FCS.

RNA Isolation. Total RNA was isolated from log phase cultures of rat celllinesusingthe methodofChirgwin (35).Total RNA was isolatedfrom humancelllines with a @diniumthiocyanate-phenol-chioroformsingle step extractionaccording to manufacturer's instructions (Stratagene, La Jolla, CA). Poly(A)@

RNA was isolatedby oligothymidylatecellulosechromatography(36).Northern Analysis. Poly(A)@ RNA (5 @g)was denatured, separated by

electrophoresis in 1.0% formaldehyde-agarose gels, and transferred to nitrocellulose membranes by capillary blotting after baking at 80°Cfor 2 h asdescribed (36). Membranes were prehybridized and hybridized at 42°Cfor20—48 h in 50% formamide/5X standard saline citrate (lX standard salinecitrate is 0.15 M NaC1 and 0.015 M sodium citrate), 1X Denhart's solution,0.25% SDS, and 250 ,i.g/ml single-strand DNA to a complementary DNAprobe wt-33 (17) 32P-labeled to a specific activity of >1 X 108 t@p@4@gbyrandom priming. Alternatively, PCR probes generated by RT-PCR from rattestis RNA using primers 4581 and 4585 were used as probes where indicated.After hybridization, membranes were washed with four changes of 0.5 X SSC

and 0.1% SDS at 40°Cfor a total of 2 h, dried, and exposed at —70°Cto X-rayfilm with an intensifying screen.

RT-PCR. Poly(A)@ RNA (1 @.tg)or total RNA (20 @g)was reversetranscribed using Moloney murine leukemia virus reverse transcriptase

(GIBCO BRL, Gaithersburg,MD) in 1X RT buffer [50 msi KG, 20 instTris-HCI(pH 8.4), and 2.5 mMMg2C1],0.4 msi of each deoxynucleotidetriphosphate, and 10 pM/mlrandom hexanucleotide primers (New England BioLabs, Beverly, MA); and 80 units of RNase inhibitor (Promega, Madison, WI)in a volume of 20 @fl.One-halfofthe reverse transcribed product was then usedfor PCR amplification with a DNA thermocycler (Perkin-Elmer-Cetus) programmed to cycle at 94°Cfor 1 mm, 50—60°Cfor 1 min, and 72°Cfor 1 ruinfor 30 or 35 cycles; and after the last cycle, the reaction was incubated at 72°Cfor 5 min. The resulting PCR product was then electrophoresed on a 1%agarose or 3% NuSieve agarose gel, along with appropriate size markers. Theprimers used were: 4581 sense, 5'-CCACCCCACTCC1TCATCAAA-3',(nucleotides 189—219); 4585 antisense, 5'-AAGAGTI'GOGGCCACTCCAGATA-3', (nucleotides906-929); 4887 antisense,5'-mCFCFGATOCATGTFG-3' (nucleotides 1303-1321); P1, 5'-ATGAGGATCCCATGGGC

CAGCA-3'; P2, 5'-AAGGGUITHCACffGflTFAC-3'; and P3, 5'-AAGGQC1TVFCACCTGTATGAG-3'.

Results

Expression of WT-1 in Normal Rat Tissues. Mesothelioma is along latency tumor usually resulting from occupational exposure toasbestos; therefore, cells of the adult mesothelium are the presumptivetarget for asbestos-induced mesothelioma. WT-1 has been reported tobe expressed during development in several organs, but the expressionpattern in adult rat tissues has not been characterized. Since rats are animportant animal model for studying the biology of mesothelioma (1),

it is important to determine the expression pattern of this gene in theadult rat. Poly(A)@ RNA isolated from normal rat lung, liver, brain,heart, kidney, stomach, testis, and spleen was examined by Northern

analysis for expression of WT-1. As shown in Fig. 1, in addition to thekidney, WT-1 transcripts were detected in testis, spleen, heart, andlung. Expression of 137-1 in rat testis, spleen, and kidney is similar tothe pattern of expression previously reported for this gene in otherspecies. In the case of the lung and heart, contaminating mesothelialcells of the visceral pleura and pericardium surrounding these organsmay have been responsible for the low levels of WT-1 transcriptsdetected in these tissues.

To determine whether WT-1 is expressed by normal adult ratmesothelial cells, RNA isolated from a tertiary rat mesothelial cellculture was examined by RT-PCR using primers specific for exons

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oma cell lines (11-14, 11-45, and 111-2), which expressed abundantWI-i transcripts, produced tumors with an epithelioid histology (datanot shown). In cell lines in which WT-1 transcripts were abundantlyexpressed, both the 3.5-kilobase transcript, which is expressed in allcell lines that express the WT-1 gene, and a 2.5-kilobase transcript,previously only reported in the testis, were detected.

Expression of WT-1 Splice Variants in Normal and NeoplasticRat Cells. The WT-1 gene is alternatively spliced in Wilms' tumorsand normal kidney cells, and four alternatively spliced forms of thisgene have been reported. Transcripts that result from this alternativesplicing can be distinguished from each other on the basis of thepresence or absence of the KTh insert in exon 9 by RT-PCR usingprimers specific for this region, as shown in Fig. 4. When the PCRreaction products are then separated by gel electrophoresis, PCRproducts containing the exon 5 alternative splice can be discriminatedfrom those that lack this insert on the basis of the 51-base pairdifference in size. Using primer set P1 + P2 to amplify the KTS (+)forms and primer set P1 + P3 to amplify the KTS (—)forms, theexpression of the four splice variants in various adult rat tissues wasexamined. As shown in Fig. 5, a and b, neonatal and adult kidneyexpressed all four of the alternatively spliced forms of the WT-1 geneas did spleen, lung, and normal glomerular mesangial cells. No WI-iexpression was detected in brain or liver, confirming the results of theNorthern analysis shown in Fig. 1. Interestingly, in the testis, the onlyWI-i splice forms observed contained the 51-base exon S insert;neither the KTS+ nor KTS—transcripts that lacked exon S could bedetected in these cells.

As observed in normal rat tissues, in the transformed mesothelialcells,all fourspliceformswereexpressed(Fig.6), althoughthe spliceform containing the 51-base pair insert appeared to be more abundantthan alternativelysplicedformsthat lackedexon5. Normalmesothelialcellsalsoexpressedall fourof the WI-i alternativesplicevariants,as shown in Fig. 6. In addition to the four alternative splice formsresulting from the presence or absence of exon 5 and the KTh splice,transcripts lacking exon 2 have also been reported in Wilms' tumors

Fig@3. Expression of WJ'-l transcripts in transformed mesothelial cells. Northernanalysis of poly(A)@ RNA isolated from transformed rat mesothelial cell lines Lane I,11-14; Lane 2, 11-45; Lane 3, 111-2;Lane 4, T08; Lane 5, T10; Lane 6, NRM2; Lane 7,NRM4; Lane 8, Mel; Lane 9, Me3; and Lane 10, MelT hybridized to the human wt-33WT-1 probe.

P1 —., @— P2

—@ 51 bp @@9@bp

@— P3

Zinc Finger Region

Fig. 4. Location of primers relative to alternative splices. Antisense PCR primersspecific for WI-i splice variants that contain the KTS splice (P2) or lack this splice (P3)in exon 9 and the exon 1 P1 sense primer used to discriminate the four Wf-1 splice forms.

ATG

1—7of the WI-i gene. As shown in Fig. 2a, the expected 732-basepair WI-i PCR fragment was amplified from the normal rat mesothehal cells. As shown by Southern analysis (Fig. 2b), this PCR producthybridized to the wt-33 probe, confirming that this fragment wasderived from the amplification of WI-i gene transcripts.

Expression of WT-i in Transformed Rat MesothelialCells. Toinvestigate the role of WI-i expression in rat mesothelioma, a panelof rat cell lines isolated from spontaneous and asbestos-transformedrat mesotheial cells was examined for expression of this gene.Poly(A)@ RNA isolated from cell lines derived from mesothelial cellsspontaneously transformed in vitro (NRM2 and NRM4), spontaneousmesotheliomas (Mel, Me3, and MelT), and asbestos-induced mesotheliomas(11-14,11-45,111-2,T08, and TlO) was examined byNorthern analysis for expression of WI-i transcripts using the humanwt-33 probe. As shown in Fig. 3, all the cell lines expressed WI-i

transcripts to some extent, although expression levels varied amongthe lines. Using RT-PCR, WI-i gene transcriptscould be readilyamplified from all 10 cell lines (data not shown). Interestingly, of thetumorigenic cell lines, T08 and TlO which were among the lines thatexpressed the lowest level of WI-i transcripts, produce tumors of asarcomatous morphology, whereas the other tumorigenic mesotheli

Mi 2 3 4 5 78

.ø..i@....1A

B

Fig. 5. RT-PCR of W1'-l alternative splice variants in normal rat tissues. RT-PCR wasperformed on rat tissues and cell lines using primers P1+P2 (+KTS splice; odd lanes) orP1+P3(—K1'Ssplice; evenlanes); the PCR products were separated on a 4% NuSeive gelto discriminate between splice variants containing or lacking exon 5. The resulting 709-and 700-base pair products (respectively) were separatedby gel electrophoresisandvisualized by EtBr staining.A. Lanes 1 and 2, neonatal kidney; Lanes 3 and 4, glomerularmesangial cells; Lanes 5 and 6, testis; Lanes 7 and 8, spleen. B. Lanes 1 and 2, brain;Lanes 3 and 4, adult kidney; Lanes 5 and 6, lung; Lanes 7 and 8, liver.

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nKLu Li B H K S T S@!:@iRp‘@wri

.@ .iii@@ .Fig. 1. Expressionof WI-i transcriptsin rat tissues. Northernanalysis of poly(A)@

RNA isolated from normal rat tissues. nK neonatal kidney; Lu, lung; Li, liver; B, brain;H, heart; A; kidney; S, stomach; T, tmtis Sp@spleen hybridized to a W2'-l rat PCR probe.

Fig. 2. RT-PCR amplification of W1'-l transcripts from normal rat mesotheial cells. A,

RT-PCR amplification ofWT-1 transcripts usingprimers458l/4585 to amplify a 732-basepair fragment (exons 1—7).B, Southern analysis of the 732-base pair fragment hybridizedto the wt-33 human WT-1 probe.

1 2 3 4 5 6 7 8 9 10

I •S@4Wr1

— GAPDH

GlutamineProline Rich Region

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WILM5' TUMOR SUPPRESSOR GENE IN MESOTHELIOMA

(26). RT-PCR using primers 4581 and 4585 which amplify a 732-basepair product from exons 1—7of the rat mesothelioma cell lines did notamplify any splice forms lacking exon 2 (data not shown). However,in the transformedcellsbut not in thenormalcells,an additionalband,intermediate in size between PCR fragments that contained or lackedexon 5 and those that lacked it, was frequently observed (see Fig. 6,Lane 5, for an example). Whether this band represents a transformation-specific splice variant of the WI-i gene or is an artifact of thePCR reaction is not clear at this time.

Relationship to Human Mesothelioma. To determine whetherWI-i expression was specific for rat mesothelioma or was a generalattribute of mesothelioma with potential relevance to the humandisease, WI-i expression in human mesotheliomas was examined byNorthern analysis. As shown in Fig. 7, human mesotheliomas alsoexpressed the WI-i gene, although only the 3.2-kilobase transcriptwas detected in these cell lines. RT-PCR using primers specific forKTS+ and KTS—alternative splice forms revealed that all fouralternative splices were expressed in the human mesothelioma celllines, similar to what was observed in the transformed rat cells(Fig. 7). In contrast to human mesothelioma cell lines, a panel of celllines from other types of human tumors were negative for WI-iexpression by RT-PCR. A431 (epidermoid carcinoma), SCC15 (squamous cell carcinoma), DV14S (prostate carcinoma), LNCaP (prostateadeno carcinoma), and MCF (breast carcinoma) cell lines did notexpress WI-i, whereas expression of Prad-1 (positive control) waseasily detected in these cells by RT-PCR (data not shown). Of seven

human mesothelioma cell lines examined, seven of seven expressedWI-i transcripts as determined by RT-PCR (Fig. 8). Thus, human aswell as rat mesotheliomas express WI-i transcripts, suggesting that

Fig. 8. Wf-1 expression in human mesotheioma cell lines determined by RT-PCR.RT-PCR was performed on human mesothelioma cell lines using primers 4581 and 4887;the expected 1124-base pair PCR product was visualized by EtBr staining following gelelectrophoresis. Lane 1, GATES; Lane 2, ROBINETFE; Lane 3, MILOT; Lane 4,H-MESO; Lane 5, MILLERS; Lane 6, CONROY; Lane 7, GARDNER.

this gene may be ubiquitously expressed in these tumors acrossdifferent species.

Discussion

The pattern of expression of the WI-i Wilms' tumor suppressorgene in the adult rat is similar to that reported previously for adulthuman and mouse tissues. Our data indicate that, in addition to thekidney, testis, lung, heart and spleen, normal adult mesothelial cellsalso express this gene. WI-i has been previously reported to beexpressed by the mesothelial cells lining the coelmic cavity duringdevelopment (27), and our data indicate that expression of this genecontinues in the mesothelium in the adult rat. In situ hybridization of18-day mouse embryos has localized WI-i expression in the developingembryoto the pericardiallining(but not the heartmuscleitsell)and the pleural mesothelium lining the lung (but not parenchymal lungtissue; Ref. 29). In both the lungs and the hearts of adult rats,

mesothelial cells of the mesothelium and pericardium, respectively,are a significant fraction of the total cell mass, and the low level ofWI-i expression detected by Northern analysis in these tissues maybe due to the presence of this subpopulation of WI-i-expressing cells.Interestingly, in the adult rat testis, only the WI-i splice formscontaining exon 5 were detected by PCR amplification of this gene.This result contrasts with data previously reported for human testis inwhich all four WI-i splice forms were observed (18). This pattern ofexpression was unique to the testis among the other rat tissues examined, which expressed all four splice forms. The biological function ofthis specific splicing pattern of WI-i transcripts is not clear, but it isimportant to note that both the 3.5- and 2.8-kilobase WI-i transcriptspreviously reported to be expressed in human testis were also detectedby Northern analysis in rat testis, suggesting that alternative splicingof exon 5 is not directly related to the presence of the additional2.8-kilobase WT-i transcript detected in this tissue.

In both rat and human mesothelioma cell lines, WI-i was ubiquitously expressed. In the rat, asbestos-transformed and spontaneouslytransformed mesothelial cells expressed WI-i transcripts, althoughmesothelioma cell lines that give rise to tumors that have a sarcomatous histological appearance expressed very low levels of this generelative to lines that give rise to tumors that are more epithelial inappearance. A similar pattern of expression has been observed inWilms' tumors in which the level of expression of WI-i transcriptswas highest in the epithelioid portions of tumors and low to undetectable in the more sarcomatous regions (15, 37—39).Expression ofWT-i would, therefore, appear to be regulated during differentiationin both nephroblasts and mesothelial cells.

The different splice forms of the WI-i gene product differ in theirtranscriptional activity, but no differences were observed in the pattern of expression of these transcripts in transformed versus normalmesothelial cells. All four splice forms were detected in transformedrat mesothelial cells; the splice form containing exon S was the

predominant species in all of the cell lines examined. No splice formswithout exon 2 were detected. In normal rat tissues, this same pattern

1 2 3 4 5 6 7 8

@@@@@ —a@@@@@ °@

KTS + — + — + - 1Fig. 6. RT-PCR of 147-1 alternative splice variants in normal and transformed

mesothelial celis. RT-PCR was performed on normal and transformed rat mesotheial cellsusing primers P1+P2 (+KTS splice; odd lanes) or P1 + P3 (—KTSsplice; even lanes);the PCR products were separated on a 4% NuSeive gel to discriminate between splicevariants containing or lacking exon 5. The resulting 709- and 700-base pair products(respectively) were separated by gel electrophoresis and visualized by EtBr staining.Lanes I and 2, normal rat mesothelial cells; Lanes 3 and 4, 11-14; Lanes 5 and 6, MelT;Lanes 7 and 8, NRM4. Similar patterns were observed in the other cell lines (11-45, 111-2,T08, Tb, Me3, Mel, and NRM2) with all four splice forms expressed (data not shown).

1234WT1

.. .i. —3.2kb

__Gates H-mesa

Fig. 7. Expression of Y,@f@1transcripts by human mesothelioma cell lines. Northernanalysis (upper panel) of poly(A)@ RNA isolated from human mesothelioma cell lineshybridized to the human wt-33 WT-1probe. RT-PCR (lower panel) of alternative spliceforms of W1'.l transcripts in human mesotheioma cell lines GATES and H-MESO. Lanes1 and 3, P1 +P2 (KTS+); Lanes 2 and 4, P1 +P3 (KTS—).

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of expression was observed. Transcripts containing the 51-base pairalternatively spliced exon 5 predominated, except in the testis, inwhich alternatively spliced forms of the WI-i gene without exon Swere not detected. Although PCR is not quantitative, our PCR resultssuggested that, in the transformed mesothelial cells, the ratio of themRNAs with exon 5 to mRNAs without exon S was higher than innormal rat cells. Therefore, while the pattern of expression may besimilar, quantitative differences in the expression of the various spliceforms that could have important biological consequences may haveoccurred in the transformed cells.

The human mesothelioma cell lines also expressed WI-i transcripts. In contrast to the rat cell lines, only the 3.2-kilobase transcriptwas detected by Northern analysis. This suggests that expression ofthe 2.8-kilobase “testis―form of this gene may be regulated in both atissue-specific and a species-specific fashion. In addition, all fouralternatively spliced forms of the WI-i gene were expressed by thehuman mesothelioma lines, and as observed in the rat, no splice formswithout exon 2 were detected. Interestingly, these splice variantsappeared to be produced in approximately equal amounts, in contrastto rat mesotheiomas in which splice forms containing the exon Salternative splice seemed to predominate. More precise quantitationwill be necessary to determine if there is a significant difference in theratio of the various splice variants between the two species.

Other comparisons of the molecular alterations in rat and humanmesotheliomas have identified interesting differences in the tumorsthat develop in the two species. For example,whereas human mesotheiomas expressabundantPDGFtranscripts(40,41),PDGFis notexpressed by transformed rat mesothelial cells (42). Expression ofIGF-I has also been reported in human mesotheliomas (43), but thisgene is expressed at only a very low frequency in rat mesotheliomas,whereas IGF-II and transforming growth factor a are expressed byspontaneously transformed and asbestos-induced rat mesotheliomas,respectively.5 Interestingly, both IGF-II and PDGF are transcriptionally regulated by WI-i (44—46),although a direct link between WI-ialterations and aberrant expression of these growth factors in mesotheliomas remains to be established.

Expression of WI-i by human mesotheliomas may be a usefuldiagnostic marker for these tumors. Park et aL (29) reported that WI-iis a marker for cells of mesenchymal lineage. The mesenchymalepithelial switch that mesothelial cells undergo during developmentdistinguishes them from the parenchymal lung epithelial cells that arethe precursor for other types of lung tumors that are difficult todistinguish histologically from mesotheliomas. This would predictthat expression of WI-i by tumors arising in the pleural cavity shouldbe restrictedto mesotheliomas,andothertypesof lungtumorssuchasadenocarcinomas should be negative for WI-i expression. Studies arenow in progress to test this hypothesis and determine if WI-i expression can be used to discriminate between mesotheliomas (particularlythe epitheliod variants) and other types of undifferentiatedlungcarcinomas.

Acknowledgments

We thankE. Bermudezforpreparationsof primaryratmesothelialcell culturesas well as for many helpful discussions; S. Sukumar for assistance andthoughtful discussions in the early part of these studies; K. Ramos for mesangial cell preparations; J. Mangum and W. Stewart for technical assistance;C. Conti andJ. DiGiovannifor RNA from humancell lines; M. Gardinerforpreparation of this manuscript; and P. Ferriola and C. Corton for helpfulcomments during the preparation of this manuscript. The wt-33 complementary DNA clone was kindly provided by D. Housman, and rat mesothelioma

5 C. Walker and F. Rutten, submitted for publication.

lines were the generous gift of J. Craighead (11-14, 11-45, and 11-2) and D.

Coffin (T08 and Tb).

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