(CANCER RESEARCH 57. 4493-4497, October 15, ¡9971

Advances in Brief

GOK: A Gene at llplS Involved in Rhabdomyosarcoma and RhabdoidTumor Development1

Silvia Sabbioni, Giuseppe Barbanti-Brodano, Carlo M. Croce, and Massimo Negrini2

Department of Experimental and Diagnostic Medicine. Section of Microbiology, and Inlerdepartment Center fur Biotechnology. University of Ferrara. Ferrara, 44100 Italy ¡S.S..G. B-B.. M. N.I. cimi Kiniinel Cancer institute and Kiininel Cancer Center. Tilomas Jefferson Uni\'ers¡t\. Philadelphia, Pennsylvania 19107 1C. M. C.i

Abstract

The expression of GOK, a gene recently identified at llplS.5, wasstudied in breast cancer, rhabdomyosarcoma, and rhabdoid tumor celllines. In these neoplasms, deletions at llplS and suppression of tumori-

genicity induced by a normal human chromosome II were previouslydemonstrated. Whereas breast cancer cell lines express readily detectablelevels of GOK mRNA, expression is absent in rhabdomyosarcoma andrhabdoid tumor cell lines. This is in contrast with the high expression ofGOK in skeletal muscle, the normal tissue of origin of rhabdomyosarco-mas, suggesting that down-regulation of GOK expression could be involved in tumor development. In agreement with this hypothesis, trans-

fection of GOK cDNA into G401 derived from a rhabdoid tumor and RDcells derived from a rhabdomyosarcoma that do not express detectablelevels of GOK mRNA, induced cell death. Because GOK expression is notcompatible with growth of these tumor cells, these results support thehypothesis that loss of GOK expression plays a role in tumor establishmentor progression and suggest that GOK may act as a recessive tumorsuppressor gene in rhabdomyosarcomas and rhabdoid tumors. On thecontrary, transfection of GOK cDNA into the breast cancer cell lineUlti 10(1 produced no detectable effects, indicating that the growth-

suppressive effect of GOK in RD and G401 cells was specific. Becauserhabdomyosarcomas have been observed in cases of Beckwith-Wiede-

mann syndrome, a genetic disorder linked to 1IplS, a role of GOK in thisdisease cannot be excluded.

Introduction

LOH3 and functional studies have associated the chromosomal

region Ilpl5 with neoplastic diseases. In addition, linkage studieshave mapped the locus for the BWS, an overgrowth genetic disorder,to chromosomal region 11pi5 (1). Because BWS is associated withincreased predisposition to the development of various pediatrie tumors, including adrenal carcinoma, nephroblastoma (Wilms' tumor),

hepatoblastoma, and rhubdomyosarcoma (2), and because of the identification of several chromosomal rearrangements at 1Ipl5 (3), it hasbeen suggested that genes involved in BWS might also be implicatedin tumorigenesis. Compiled results from these studies (3-23) also

indicated the possibility that more than one locus at 1Ipl5 could beinvolved in tumorigenesis and/or BWS. Indeed, a number of genes,including CDKNIC, KVLQT1, HÌ9,and TSG101, were associatedwith neoplastic diseases or BWS (24-27). For clarity, Fig. 1 schematically summarizes the compiled results of the above-mentioned

Received 7/24/97; accepted 8/29/97.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 Telethon Grant E.366. Associazione Italiana per la Ricerca sul Cancro.Consiglio Nazionale delle Ricerche. Progetto Finalizzato "Applicazioni Cllniche dellaRicerca Oncologica" and by National Cancer Institute Grant CA56366.

2 To whom requests for reprints should be addressed, at Dipartimento di Medicina

Sperimentale e Diagnostica. Sezione di Microbiologia. Università di Ferrara, via LuigiBorsari. 46. Ferrara. 44100 Italy. Phone: 39-532-291395: Fax: 39-532-247618; E-mail:ngm@ifeuniv.unife.it.

•¿�'The abbreviations used are: LOH. loss of heterozygosity; BWS. Beckwith-

Wiedemann syndrome: poly(A| RNA. polyadcnylated RNA.

studies. However, these genes do not completely clarify all of theaspects that connect this chromosomal region to neoplastic diseases orBWS; therefore, it seems possible that additional genes mapping atllplS might be involved. In summary, biological and genetic evidence indicates that region 1Ipl5 harbors several tumor susceptibilitygenes, which may coincide with BWS genes, and some of them areyet to be discovered.

In this report, we describe the analysis of GOK, a gene recentlyidentified at Ilpl5.5, near the 5' end of the gene encoding the

ribonucleotide reducíasesubunit l (RRMl; Ref. 28), in close proximity to the loci DI1S12-D1IS860. which are located within the LOH

region 2 (Fig. 1). Based on its primary sequence, the gene encodes atransmembrane protein whose function is not yet known. Our resultssuggest that the absence of GOK gene expression in rhabdomyosarcomas and rhabdoid tumors may affect functions that play a role intumor development.

Materials and Methods

Cell Lines. All of the cell lines analyzed were obtained from the AmericanType Culture Collection. Rhabdomyosarcoma cell lines analyzed were A2()4,A673, Hs729, RD. SJCRH30. TEI25.T. and TE611. Breast cancer cell linesanalyzed were BT-474, HBL100, MCF-7. MDA-MB-231. MDA-MB-361,MDA-MB-436, and T47-D. The rhabdoid tumor cell line G401 was alsoanalyzed. Cells were grown according to the supplier's specifications.

Probes and GOK Expression Vectors. Two GOK cDNA clones, pDR2-GOKIA and pDR2-GOK10A, were isolated from a human testis cDNA library

(Clontech. Palo Alto. CA). Nucleotide sequence of clone IA indicated that the3.8-kb insert was identical to the published sequence from nucleotides 10-

3800 (28). which includes the entire coding sequence, whereas clone 10Acontained an insert from nucleotides 790-1534 cloned in antisense orientation.Shorter probes were generated by PCR amplification with specific oligonu-

cleotide primers.DNA Transfection. The eukaryotic expression vector pDR2 (Clontech)

was used to drive the expression of the inserted GOK cDNA in tumor cells,under the control of the Rous sarcoma virus long terminal repeat, a strongubiquitously active promoter. The selectable marker hygro1 induces resistance

to hygromycin B (Sigma. St. Louis. MO). Transfections were carried out usingLipofectAMINE (Life Technologies, Inc., Gaithersburg. MD). G401-trans-

fected cells were selected with 25 /xg/ml hygromycin B. Both growing anddegenerating cell clones were picked up using glass cylinders. DNAs werepurified at the very early stages of clone development ( UK)-300 cells) usingthe InstaGene Matrix (Bio-Rad. Hercules, CA). Genomic DNA from well-

growing clones was also analyzed after several passages by Southern blotanalysis, using standard procedures (29).

PCR DNA Analysis. PCR analysis was performed on 10 /*! (1/20) of theInstaGene Matrix-purified genomic DNAs using the following primers:GOK551F, 5'-CTGAGACCTAGAGTCATGGA-3f; and GOK2820R, 5'-GCAGGCCATGGTGGTCAGTT-3'. Long PCR (30) was carried out using the

Expand Long Template PCR System (Boehringer Mannheim). Three stepswere used for 35 cycles: (a) a denaturation step at 94°Cfor 15 s: (b) anannealing step at 58°Cfor 30 s; and (r) an extension step at 68°Cfor 4 min.

The expected PCR product is about 2.3 kb and includes the entire codingregion. PCR products were separated on a 1% agarose gel in 0.5 X Tris acetaterunning buffer.

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GOK INVOLVEMENT IN TUMOR DEVELOPMENT

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Fig. 1. Schematic representation of the regions involved in human malignancies and in the genetic disorder BWS at chromosome I lp!5. Regions defined by LOH and functionalstudies (5. 7) are shown ut the bottom of the diagram. LOH regions 1-3 were compiled from a number of studies (9-23). Regions involved in chromosomal rearrangements of BWS(BWSCRI-BWSCR3; Ref. 3) are shown at the lap. The breakpoint called Tm87-l6 was detected in a rhahdoid tumor cell line with the same name (3). The diagram is not drawn lo

scale. Double slashes on the map, large gaps between the loci.

Northern Blot Analysis. RNA was purified using the RNA-STAT30 (Tel-

Test Inc., Friendswood. TX I purification method. Northern blots were prepared from either total RNA (20 fig of each sample) or poly(A) RNA (2 ¿¿g)transferred to Hybond N+ membranes (Amersham). Hybridization was performed using standard procedures (29). Membranes containing poly(A) RNAfrom normal human tissues were purchased from Clontech.

Results

Northern blot analysis of poly(A) RNA from a variety of humanadult tissues with a GOK cDNA probe showed that GOK mRNA wasexpressed at various levels in all of the tissues, with the highestexpression in skeletal muscle and lung. The gene was transcribed asa 4-kb mRNA, except in the brain, in which a 4.5-kb mRNA was

detected (Fig. 2). In spite of the evidence that GOK is highly expressed in skeletal muscle. GOK expression was not detectable byNorthern blot analysis in any of seven rhabdomyosarcoma cell lines,

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Fig. 2. Analysis of GOK expression in human adult tissues. Northern blot (Clontech)containing poly(A) RNA from various human adult tissues was hybridi/ed with a GOKcDNA probe containing the segment from nucleolides 561-1120. A transcript of about 4kb expressed at various levels is visible in all of the tissues, except in brain, in which a4.5-kb transcript is detected.

which originate from precursors of striated muscle cells, and in therhabdoid tumor cell line G401 (Fig. 3, a and b). It is interesting to notethat functions associated with chromosome llplS were found tosuppress tumorigenicity and growth of G401 and RD cell lines,respectively (5-7), in which GOK expression is undetectable. GOK

mRNA expression was readily detectable in breast cancer cell linesand normal breast tissue (Fig. 3r), suggesting a specificity for the lossof GOK expression in tumors derived from striated muscle cells.

To understand whether the absence of GOK expression in rhabdomyosarcoma and rhabdoid tumor cell lines could affect charactersassociated with the neoplastic phenotype, such as in vivo tumorigenicity or in vitro growth control, the eukaryotic expression vectorspDR2-GOKl A, which contains a full-length GOK cDNA, and pDR2-GOK10A, which contains a truncated version of COA"cDNA (nucle-

otides 790-1534), were cloned from a human testis cDNA library.

These two vectors and the pDR2 vector alone were transfected intoG401 and RD cells. Many hygromycin-resistant clones appeared after2 weeks from transfection. However, in most of the pDR2-GOKlA

cell clones, the cells became round and detached from the platesurface, and the remaining attached cells became enlarged and died in3-4 days (Fig. 4, a-d). Only 10-20% of the clones continued to grow

as immortal cell lines. On the contrary, none of the clones transfectedwith the vector pDR2 alone or pDR2-GOK10A showed the growth-

suppressed phenotype. Because of these differences, several degenerating clones were picked up at an early stage (100-300 cells) andanalyzed for the presence of intact full-length GOK cDNA. All 10 ofthe degenerating G401 clones analyzed contained the full-length

cDNA, strongly suggesting that cell degeneration was specifically dueto the expression of GOK (Fig. 5a). This conclusion was supported bythe observation that none of the immortal clones contained an intactfull-length exogenous GOK cDNA (Fig. 5, b and c), confirming adirect relationship between expression of the transfected full-length

GOK cDNA and cellular degeneration. Completely overlapping results were obtained in cell line RD. These results prove that loss of the

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COK INVOLVEMENT IN TUMOR DEVELOPMENT

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4.0 kb GOK

Fig. 3. a. analysis of GOK expression in rhabdomyosarcoma and rhabdoid tumor celllines. A Northern blot containing poly(A) RNA from various human rhabdomyosarcomacell lines and the rhabdoid tumor cell line G40I was hybridized with the GOK cDNAprobe containing the segment from nucleotides 561-1120. No expression was detected inany of the analy/.ed cell lines after I week of exposure, b. ß-actinmRNA was detected in

all of the samples after 2 h of exposure, r, analysis of GOK expression in breast cancercell lines. A Northern blot containing 20 fj.g of total RNA from seven human breast cancercell lines was hybridized with the GOK cDNA segment from nucleotides 561-1120. Allof the cell lines express detectable levels of GOK mRNA after an overnight exposure.Normal breast tissue and peripheral blood lymphocytes (PBL) were also included in thisNorthern hybridization.

transfected full-length GOK cDNA confers a selective growth advan

tage to tumor cells and suggest that reduction or extinction of GOKmRNA expression in rhabdomyosarcomas and rhabdoid tumors mayrepresent a critical event in tumorigenesis. To exclude the possibilitythat overexpression or uncontrolled expression of GOK causes celldegeneration in any cell type, we transfected the GOK-expressingvector into the HBL100 breast cancer cells. The number of hygromy-cin-resistant clones obtained with pDR2-GOKlA or the vector alonewere comparable, and six well-growing randomly picked HBL100GOK-transfected clones showed the presence of the intact transfected

GOK cDNA (Fig. 5d). This result indicates that the growth suppressoractivity of GOK is specific to RD or G401 cells.

The mechanism that leads to GOK mRNA extinction is not understood. However, apparently, it cannot be ascribed to gene rearrangement, because no abnormal bands were detected by Southern blotanalysis (data not shown), or to genomic imprinting, because doubleallelic expression was detected in various human cell lines analyzed inthis study (data not shown). It seems that an epigenetic mechanismoperating at the transcriptional level is involved in the down-regula

tion of GOK expression. These results also indicate that this mechanism, down-regulation of GOK expression, may be involved in rhab

domyosarcomas and rhabdoid tumors but not in breast cancer.

Discussion

Expression of GOK driven by the eukaryotic vector pDR2 in G401and RD cells induced growth arrest and degeneration in these rhabdomyosarcoma and rhabdoid tumor cell lines, suggesting that down-

modulation of GOK expression could be a mechanism leading to cellimmortalization. In agreement with this hypothesis, the undetectableexpression of GOK mRNA in all rhabdomyosarcoma cell lines testedsuggests that the loss or down-regulation of GOK expression is a

general phenomenon occurring in rhabdomyosarcoma and that GOKmay act as a tumor or growth suppressor gene involved in theestablishment or progression of this tumor type. The possibility thatGOK functions as a tumor suppressor gene is supported by theobservation that other tumor suppressor genes, such as p53 and KB-/.

Fig. 4. Morphological appearance of degenerating pDR2-GOKlAG40I cell clones, a. parental cell line G40I. b. G401 cell clone beginning to degenerale; cells become loose, round, and detach from thesubstrate, r and </. the few cells thai remain attached to the plasticsubstrate enlarge and die in 2-4 days. Pictures were taken at X200

enlargement.

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COÕÕ[NVOLVEMKNT IN TUMOR DEVELOPMENT

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induce growth arrest when transfected into cells lacking expression ofthe endogenous gene (31-33).

GOK Growth arrest and degeneration induced by GOK transfection recallexo a similar effect described in the RD rhabdomyosarcoma cell line after

transfer of subchromosomal transferable fragments from chromosomellplS (7). On the contrary, whereas tumorigenicity was suppressed,no in vitro effect was observed when a normal chromosomal regionllplS was introduced into the G401 cells (4, 5). These results areapparently in contrast with ours. However, GOK introduced with anormal chromosome 11 may be under the same transcriptional inhibition of the endogenous GOK gene, therefore producing no in vitrophenotype. Forcing the expression of GOK by a different promotercould instead reveal in vitro phenotypes otherwise impossible toobserve. Identification and analysis of the natural GOK promoterregion may reveal the mechanisms leading to its negative regulation inrhabdomyosarcoma and rhabdoid tumor cells. Although the growth-inhibiting and tumor-suppressing regions at 1Ipl5 are partially over

lapped between loci D11S601 and DIIS724 (Refs. 5 and 7; Fig. 1)and it was suggested that this common area could contain a generesponsible for the effects observed in both cell lines (5), our resultssuggest that functions inducing growth arrest and suppression oftumorigenicity are distinct. Indeed, GOK (a growth-suppressing gene)

and H19 (a tumor suppressor gene; Ref. 26) map to the unique portionof each region (Fig. 1).

The mechanism by which GOK causes cell death is unknown andwas not investigated in the present work. However, the results suggestspeculative ideas. The structure of the GOK product, deduced from itsprimary amino acid sequence, indicates that it is a typical membraneprotein with NH2 terminus outside and the COOH terminus in thecytosol. These features suggest that GOK might be a receptor connected to a pathway that leads to cell growth arrest. Because uncontrolled proliferation is an intrinsic property of tumor cells, the simultaneous presence of inhibitory signals mediated by GOK may result incell death, which was observed in degenerating cell clones containingthe full-length transfected GOK cDNA. Alternatively, the high ex

pression of GOK mRNA in muscle cells, which is lost in rhabdomyosarcoma cells, might reflect a role of GOK in cell differentiation.This last hypothesis is in line with the absence of effect in breastcancer cells.

The localization of GOK at 1Ipl5 and its involvement in pediatrietumors associated with BWS, such as rhabdomyosarcomas and rhabdoid tumors, suggest that a role of this gene in BWS cannot be

GOK excluded. If lack of GOK expression proves to be a critical step inendo human carcinogenesis, and eventually in BWS. anti-GOK antibodies

could be important diagnostic tools in the analysis of pediatrie tumorsand possibly that of other human neoplasms. Furthermore, the determination of the molecular pathway in which GOK is involved could

GOKexo

Fig. 5. Analysis of ihe stalus of the transl'ecled COK cDNA in G401 cell clones, a,

PCR analysis of degenerating cell clones; hybridization of PCR products (primersGOK551F and GOK2820R ) from 10 degenerating clones with GOK cDNA segment fromnucleotides 561-1 120. The expected product has a size of 2.3 kb. The positive control (P)was O.I pg of the pDR2-GOK vector used for transfection. The negative control (AOwasthe parental G401 genomic DNA. In all 10 of the clones analyzed, the specific 2.3-kb

cDNA product was detected. The PCR product is specific for the transfected cDNA.because the gene is split in several exons spanning more than 200 kb.4 h and c. Southern

blot analysis of genomic DNA from various immortal G401 hygromycin-resistant cell

4 Unpublished results.

clones digested with the restriction enzymes BaniHl and Xhal and hybridized with thevector pDR2 alone (b) or with the COK cDNA segment from nucleotides 1374-1990 (c).Lane G401 contains DNA from the parental cell line. The other lanes contain DNAs fromG40I-GOK1A cell clones 16. 17. and 22 and G401-GOK10A cell clones 1 and 2. LaneC contains one genome equivalent of pDR2-GOKl A mixed with 10 ^ig of sheared salmonsperm DNA. The probe GOK 1374-1990 was chosen because it allows a clear distinction

between the hybridizing fragments from the endogenous (enilo) and transfected (exo}COK DNA. Arrows, the expected fragments (pDR2. GOK exo, and COK endo) detectedby the probes. -. an empty lane. The results show that none of the clones contains the bandcorresponding to the fragment of the transfected GOK cDNA. whereas vector sequenceswere clearly delected, indicating that the specific deletion of the segment containing GOKcDNA provided a selective growth advantage to cultured cells, d. Southern blot analysisof genomic DNA from HBL100 hygromycin-resistant clones transfected with pDR2-GOK1A and hybridized with GOK cDNA segment from nucleolides 1374-1990. All of

the clones contain the band corresponding to the transfected GOK cDNA. indicating thatthe growth suppressor activity of GOK is specific to RD or G401 cells. The presence ofa nonspecific band obscures the GOK endogenous band of samples c.3, c4. and c5.

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(¡OK INVOLA l.MI.M IN TUMOR DKVELOPMENT

lead to the understanding of new pathogenetic mechanisms related tothe tumorigenic process.

Acknowledgments

We thank Augusto Bevilacqua and Pietro Zucchini for excellent technicalassistance.

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1997;57:4493-4497. Cancer Res   Silvia Sabbioni, Giuseppe Barbanti-Brodano, Carlo M. Croce, et al.   Rhabdoid Tumor Development

: A Gene at 11p15 Involved in Rhabdomyosarcoma andGOK

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