ICANCHR RKSKARCH 56. 1214-121«. March 15,

Advances in Brief

Genomic Organization of the Human p57Klp2 Gene and Its Analysis in the G401Wilms' Tumor Assay1

Laura Hink Reid,2 Shyra J. Crider-Miller, Ande West, Mong-Hong Lee, Joan Massagué,and Bernard E. Weissman

Department of Pathology and Laboratory Mediane, University »fNorth Carolina, Chapel Hill, North Carolina 275W ¡L.H. K., S. J. C-M., A. W., H. K. W./. unii Cell Biologyand Genetic* Program, Howard Hughe* Metlicul Invitine. Memorial Sloan-Ketlering Cancer Center, New York, New York 10021 ¡M-H.L, J. M.j

Abstract

The p57KII>2gene encodes an inhibitor of cyclin-depcndcnt kinasc ac

tivity, which negatively regulates cell cycle progression. The human p57gene is located in I Ipl5.5, a region of DNA frequently altered in neoplasia.We have isolated a human genomic clone and mapped the p57 gene to a2.2-kb region between DIIS6-Õ8 and Ü1IS679. Sequence analysis revealedthat the coding DNA of the human p57 gene is divided by a 0.5-kb intron.

A second intron was detected in the .V untranslated region, indicating thatthe human p57 gene contains at least three exons. Our previous work withsomatic cell hybrids mapped a tumor suppressor gene for the (.4(11Wilms' tumor cell line to a ~500-kb region of HplS.5 that includes p57.

Northern blot analysis detected a O.S-kh p57 transcript in several of the

(.4(11 hybrid lines. However, p57 expression did not correlate with tumorsuppression. These results suggest that p57 is not responsible for thetumor suppression observed in our somatic cell hybrid assay.

Introduction

Cellular proliferation involves an orderly progression through thecell cycle that is controlled by protein complexes of cyclins andCDKs' (reviewed in Refs. 1-3). The CDK subunits phosphorylate cell

cycle-regulatory proteins, such as the retinoblastorna protein, to re

lease cells from cell cycle arrest. The CDKs are counterbalanced by afamily of negative cell cycle regulators. These CDKIs bind with thecyclin-CDK complexes to inactivate their catalytic domains. Muta

tions in the CDKI proteins result in uncontrolled proliferation typicalof neoplasia. Therefore, CDKI proteins are excellent candidates fortumor suppressor genes.

The CDKI proteins are divided into two families based on theirsequence homology. One family includes the pl61NK4A, pl5INK4B,

and pl9i proteins whjch conta¡na related series ofankyrin repeats. The second family of CDKI proteins includesp2]cipi/wAn p27K»'1. and a recently identified third member.p57K"" (4. 5). These proteins have homologous CDK binding do

mains that are required for the inhibitory activity. The p57 proteininhibits several G, and S-phase CDKs. and overexpression of p57

causes a complete cell cycle arrest in G, (4, 5).The human p57 gene is located in 1Ipl5.5 (5). Mutations in this

region of DNA are observed in several forms of cancer, includingWilms' tumor, and in patients with BWS (6-8). In addition, func

tional studies by our lab (9-1 1) and others (12) have localized a tumor

Received 12/11/95; accepted 1/31/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore he hereby marked tul\rnisi'tm'ni in accordance with

18 U.S.C. Section 1734 solely to indicale this fact.' This research was supported by NIH Grant CA63176 (to B. E. W.) and by the

Howard Hughes Medical Institute. L. H. R. was an American Cancer Society PostdoctoralFellow. S. J. C-M. is an NIH Predocloral Fellow supported by Environmental PathologyTraining Grant ES07017.

: To whom requests for reprints should be addressed, at Department of Pathology and

Laboratory Medicine, 345 Lineberger Cancer Center. CB#7295, University of NorthCarolina. Chapel Hill. NC 27599.

' The abbreviations used are: CDK. cyclin-dependent kinase: CDKI. cyclin-dependentkinasc inhibitor; BWS. Beckwith-Wiedemann syndrome.

suppressor gene to a —¿�500-kbregion of 1Ipl5.5 between DIISftOI

and DI 1S724. In this report, we present the genomic structure of thehuman p57 gene based on restriction mapping and sequence analysisof a cosmid clone. Although the gene mapped within our candidatedomain. Northern blot analysis indicated that p57 expression did notcorrelate with the tumor suppression observed in our functional assay.

Materials and Methods

Cell Lines. Jl-4a. JI-49. Jl-43a. and J1-9 are part of a panel of deletion

hybrids derived by mutugenesis of Jl, a hamster cell line that stably retainshuman chromosome 11 (13. 14). MCH 701.8 is a mouse cell line that containsa human t(X;ll) (llpter > llql3::Xq21 > Xqter) chromosome. XMCH708.20 and XMCH 708.25 are mouse cell lines that contain radiation-reducedt(X:ll) chromosomes derived from MCH 701.8 (15). G40I.6TG.6 is a hypox-anthine-guanine phosphoribosyl-lransl'erase-deticient derivative of the G401

Wilms- tumor cell line (16). MCH 48ft. 1. MCH 486.2G. MCH 486.2J, and

MCH 48ft.2L are G401 hybrid lines that contain the t(X;l I ) chromosome fromXMCH 708.25 and do not form tumors when injected into nude mice (II).MCH 369.18. MCH 485.1, MCH 485.2A, and MCH 485.3 are G40I hybridlines that contain the t(X:ll) chromosomes from XMCH 708.24 or XMCH708.26 (10, II). These cell lines do form tumors alter mouse inoculation.

PCR. Genomic DNA from the somatic cell hybrid panel was amplifiedusing the following primers from the human ¡>57gene: 5'-CCG TGT CCCTCT CCA AGC-3' and 5'-CGG GGC TCT TTG GGC TCT-3'. They gener

ated a 180-bp amplicon after 30 cycles of denaluralion at 94°Cfor 1 min.annealing at 58"C for I min, and extension at 72"C tor 1 min. Kach reaction

contained 400 ng template. 1 unit Tat) polyinerase, 10 HIMTris (pH 9). 50 IHMKCI, 1.0 HIMMgCI,. and 0.2 Mof each deoxyribonucleosidc triphosphatc. PCRproducts were visualised in ethidium bromide-stained 2r/< agarose gels.

Isolation and Mapping of Cosmids. A human />57 partial cDNA clone

was identified in a human cDNA library by hybridisation with the mouse p57cDNA clone. A 0.7-kb fragment from the partial cDNA clone was used to

identify cosmid 14F5 (cl4F5) in the Los Alamos human chromosome 11cosmid library. Cosmids cCIl 1-395 (c395) and cCIl 1-469 (c469) had been

identified previously in a different chromosome 11 cosmid library (17). Thelocation of the p57 gene within cl4F5 was determined by restriction mappingand hybridizations with fragments from a full-length cDNA clone. hp57.Nonradioactive hybridisations were performed at 65"C. according to the manufacturer's protocol (Genius system; Boehringer Mannheim).

DNA Sequencing. The 1.2-kb Noi\ and 1.0-kb Nol\-Hind\U fragments of

genomic DNA were isolated from cl4F5 and suhcloned into pBLUESCRlPT(Sfratacene). Plasmid DNA was isolated (Qiagen) and sequenced at theUNC-CH Automated DNA Sequencing Facility on a Model 373A DNA

Sequencer (Applied Biosystems). Either external universal primers or internalprimers designed from genomic sequence were used.

Northern Analysis. Polyadenylatcd RNA (2 /xg) was isolated using anoligo-dT kit (Collaborative Research), separated on 1% agarose-formaldehyde/

MOPS gels, and transferred to nylon membranes (Boehringer Mannheim) in10x SSC ( 1X SSC = 3.0 M sodium chloride. 0.3 M sodium). mRNA samples

from adult skeletal muscle and kidney tissue (Clonlech) were included aspositive controls. RNA blots were hybridised to i:P-lahcled DNA probes at

42°Cin a 50<7cformumide solution. The p57 probe was the 1.5-kb EcoRl insertfrom the human cDNA clone, hp57. The -y-actin probe was the 1.8-kb Xlwl

insert from the mouse cDNA clone. pHFl.

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CHARACTERIZATION OF THE HUMAN pS7 GENE

Results

Localization of the Human pS7 Gene and Characterization ofCosmid Clones. Previous analysis mapped the human p57 gene toI Ipl5.5 using fluorescent in situ hybridization (5). To localize thegene within this chromosomal band, we looked for the presence ofp57 in a panel of deletion hybrids that contain fragments of humanchromosome II in a rodent background (Fig. 1/4). DNA from thedeletion hybrid panel was amplified by PCR using primers from thehuman p57 gene. As shown in Fig. \B, amplified bands were generated in all samples except Jl-9. Identical results were observed when

Southern blots containing DNA from the hybrid panel were hybridized to human p57 cDNA probes (data not shown). These resultsindicate that the p57gene is located in a region of DNA absent in Jl-9

but present in XMCH 708.25 and the remaining hybrid lines. TheJl-9 cell line contains the DIIS724 locus but not the more centra

mene DIISI probe (14. 17). The XMCH 708.25 line contains theDI IS64X locus but not the DI 1S601 probe (11). Therefore, the humanp57 gene must be located between the DìÌSou I and DI IS724 loci.This region of 1Ipl5.5 includes cosmids c395 and c469 that containthe DÌIS648and D11S679 loci, respectively (17). Neither of thesecosmids hybridized to p57 cDNA probes (data not shown).

To isolate genomic clones containing the p57 gene, we screened ahuman chromosome 11 cosmid library with a p57 partial cDNAprobe. This analysis identified one cosmid cl4F5 that contained thep57 gene. Noti restriction analysis and hybridization to genomicfragments mapped cl4F5 between c395 and c469 (Fig. 2/4). Thisanalysis also indicated that cl4F5 and c395 had substantial overlap in

aVIHRASH19

IGF2D11S724D11S1

D11S679D11S648D11S601

D11S517RRM1D11S12HBB>

<sCO<:

S il ;?««T

^r

S•1

>l2* ->|P57KIP*

B

» T T T TO i- i- *- f-

Fig. I. Local /ution of the human p57 gene in l Ipl5.5 using somatic cell hybrids. A,vertical burs ind cate regains of I Ipl5.5 DNA retained in the deletion hybrid panel. Mostlines contain ad itional human chromosome 11 DNA (not shown). Probes are listed inorder with telóneric loci on top and centromeric loci on honom. H, ethidium bromide-stained 1ck agar

cell hybrid linecontains human DNA

ise gel showing the human p57 PCR products amplified in the somaticThe GM346A9 sample contains mouse DNA. The IMR90 sample

10kb

NC395.N1C14F5

_NIIINN

NNIIII<-4RQII

BN N,

10kbP57KIP2

Fig. 2. Restriction maps of genomic clones containing the human ;>57 gene. A.long-range map of cosmids showing the location of Null sites (A7)and I Ipl5.5 loci. Left,

centromeric DNA. Right, lelomeric DNA. ß.detailed map of cosmid cl4F5 showing thelocation of AV»I(/V). Ea>Rl (R), and HMill (Hi sites. The horiymtal armw underscoresthe 1.2-kb Null and 1.0-kh jV»/l-W»idlIIfragments that contain p57 coding sequence.

genomic sequences. Blots containing EcoRi. Hindiii. and Noll digestsof cl4F5 were hybridized to a p57 full-length cDNA probe. Thisanalysis detected an 11.4-kb EcoRi band and a 10.0-kb Wmdlll bandin cl4F5. Similarly-sized EcoRl and Hiniiili fragments were observed

in Southern blots of genomic DNA (data not shown), suggesting thatthe cl4F5 cosmid contained an intact p57 gene. Double digests withthese enzymes indicated that the gene was located within a 4.8-kbEcnRI-Wmdlll fragment. This fragment contained two Noll sites thatdivided it into three smaller sections: a 2.6-kb EcoRi-Noti fragment;a 1.2-kb Noti fragment; and a 1.0-kb Notl-Hindlil fragment (Fig. 2ß).p57 cDNA probes hybridized to the 1.2-kb Noti and 1.0-kb Notl-

Hindiii fragments (data not shown) but not to flanking DNA, suggesting that the p57 coding DNA is contained within this 2.2-kb

region.Genomic Organization of the Human p57 Gene. The i.2-kb Noti

and 1.0-kb Noti-Hindiil fragments from cl4F5 were subcloned and

sequenced (Fig. 3). To determine the genomic organization of thehuman p57 gene, the sequences derived from the two genomic fragments were compared to published cDNA sequence (5). This analysisrevealed that the coding region of the human p57 gene is divided atcodon 275 into two exons that are separated by a 0.5-kb intron. Asecond, 83-bp intron was detected in the 3' untranslated region. Some

single base pair differences in the untranslated regions of the p57 genewere observed between the genomic and cDNA sequences. No differences were detected in the coding DNA.

Human p57 Expression in G401 Hybrid Cell Lines. We haveused somatic cell hybrids in a functional assay to identify regions ofchromosome 11 that suppress tumorigenicity in the G401 Wilms'

tumor cell line (9-11 ). The G401 cells form tumors when injected s.c.into nude mice. They have a pseudo-diploid karyotype with two

cytogenetically normal copies of chromosome 11 but presumably lackexpression of a tumor suppressor gene. In our assay, we introducedt(X;l 1) chromosomes into G401 cells and analyzed the tumorigenicpotential of the hybrid cell lines in nude mice. In some cases, theintroduced t(X;ll) chromosome complemented the G401 defect sothat the hybrid lines did not form tumors after inoculation. In othercases, the introduced chromosome had no influence on the tumorigenicity of the G40I cells. This functional assay has enabled us tolocalize a tumor suppressor gene to a region of 1Ipl5.5 telomeric tothe DIIS60I locus (11). Since p57 maps to this same region, weinvestigated whether p57 expression contributed to the tumor suppression observed in our assay.

Previous experiments demonstrated that four G401 hybrid lines.

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CHARACTERIZATION OF THE HUMAN ¡>57GENE

1 gcggccgcca atcgccgtgg tgttgttgaa actgaaaata ctacattatg 1301 ctcgggggcc tcgctgggtt cccgcctcct cccgtggcat taaagggccc

51 ctaatcgcgg ccgggcccgc gcgcacgggg gtggggcccg cgcgtaCaaa 1351 gcagcgctca gggcgcggct gcgcccttac ccccctcccc gccccgttgt

101 gggggcgcag gcgggctggg cgttccacag gccaagtgcg ctgtgctcga 1401 tgtgccctcc agcggcttcg cgcgggcggg gtgggaggct gaatcccggc

151 ggggtgccgg ccaggcctGA GCGAGCGAGC TAGCCAGCAG GCATCGAGGG 1451 cgcgaccccc cgggagcgca gttttcgccc ccggccgcgg gagcccctcc

201 GGCGCGGCTG CCGTCCGGAC GAGACAGGCG AACCCGACGC AGAAGAGTCC 1501 ccgggcgcgg ccgggcgccg tgagcacggc gtggaggggg ttaagcgcgg

251 ACCACCGGAC AGCCAGGTAG CCGCCGCGTC CCTCGCACAC GCAGAGTCGG 1551 cggcggcccc ggggggcttg gccgcgggac aaggggaaat gcttacacag

301 GCGGCGCGGG GTCTCCCTTG CGCCCGGCCT CCGCCCTCTC CTCCTCTCCT 1601 cacattgcgc ggcgacgtaa acaaagctga cccgccgcgg acctcggcgc

351 TTCCCCTTCT TCTCGCTGTC CTCTCCTCTC TCGCTGCCCG CGTTTGCGCA 1651 gggcggggac ggcgccccca ccccggccgg cccgcgcccc gcgccctctc

401 GCCCCGGGCC ATGTCCGACG CGTCCCTCCG CAGCACATCC ACGATGGAGC 1701 ccggccccct ctcggttctc cgggccggcc ccgccctgac cggccgcgcg

451 GTCTTGTCGC CCGTGGGACC TTCCCAGTAC TAGTGCGCAC CAGCGCCTGC 1751 cgctgtcgcc cgcagATTTC TTCGCCAAGC GCAAGAGATC AGCGCCTGAG

501 CGCAGCCTCT TCGGGCCGGT GGACCACGAG GAGCTGAGCC GCGAGCTGCA 1801 AAGTCGTCGG GCGATGTCCC CGCGCCGTGT CCCTCTCCAA GCGCCGCCCC

551 GGCCCGCCTG GCCGAGCTGA ACGCCGAGGA CCAGAACCGC TGGGATTACG 1851 TGGCGTGGGC TCGGTGGAGC AGACCCCGCG CAAGAGGCTG CGG^CAGCCA

601 ACTTCCAGCA GGACATGCCG CTGCGGGGCC CTGGACGCCT GCAGTGGACC 1901 Agtgagtaca gcgcacctgg gggggcgcgg agggccgacc cgccgggtcc

651 GAAGTGGACA GCGACTCGGT GCCCGCGTTC TACCGCGAGA CGGTGCAGGT 1951 ccgccggctt tgctgaccgc ccctctcctc gcagTTTAGA GCCCAAAGAG

701 GGGGCGCTGC CGCCTGCTGC TGGCGCCGCG GCCCGTCGCG GTCGCGGTGG 2001 CCCCGAGGGA ACCTGCCGGG GCAGCGGACG TTGGAAGGGC GCTGGGCCTC

751 CTGTCAGCCC GCCCCTCGAG CCGGCCGCTG AGTCCCTCGA CGGCCTCGAG 2051 GGCTGGGACC GTTCATGTAG CAGCAACCGG CGGCGGCTGC CGCAGAGCAG

801 GAGGCGCCGG AGCAGCTGCC TAGTGTCCCG GTCCCGGCCC CGGCGTCCAC 2101 CGTTCGGTTT TGTTTTTAAA TTTTGAAAAC TGTGCAATGT ATTAATAACG

851 CCCGCCCCCA GTCCCGGTCC TGGCTCCAGC CCCGGCCCCG GCTCCGGCTC 2151 TCTTTTTATA TCTAAATGTA TTCTGCACGA GAAGGTACAC TGGTCCCAAG

901 CGGTCGCGGC TCCGGTCGCG GCTCCGGTCG CGGTCGCGGT CCTGGCCCCG 2201 GTGTAAAGCT T

951 GCCCCGGCCC CGGCCCCGGC TCCGGCTCCG GCCCCGGCTC CAGTCGCGGC

1001 CCCGGCCCCA GCCCCGGCCC CGGCCCCGGC CCCGGCCCCC GCCCCGGCCC

1051 CGGCCCCGGA CGCGGCGCCT CAAGAGAGCG CCGAGCAGGG CGCGAACCAG

1101 GGGCAGCGCG GCCAGGAGCC TCTCGCTGAC CAGCTGCACT CGGGGATTTC

1151 GGGACGTCCC GCGGCCGGCA CCGCGGCCGC CAGCGCCAAC GGCGCGGCGA

1201 TCAAGAAGCT GTCCGGGCCT CTGATCTCCG gtgagccccg cacggccccg

1251 ccccggcccg gcccggcccc gctttgtccg gccggccggt ccccccagcc

Fig. 3. Nucleolide sequence of Ihe human />57 gene. Exon sequences present in cDNA clones are shown in uppercase tellers. Intron and flanking DNA sequences are printed inlowercase tellers. The translation initiation and termination sites are underlined. Nucleotides at positions 151-168. 263. and 2200 differ from the cDNA sequence published previously

(5). The Genhank accession number is U4SS69.

MCH 486.1, MCH 486.2G, MCH 486.2J, and MCH 486.2L, weresuppressed for tumorigenicity (11). Four other G401 hybrid lines.MCH 369.18, MCH 485.1, MCH 485.2A, and MCH 485.3, weretumorigenic (10, 11). Each of these G401 hybrid lines contains anintroduced t(X;l I) chromosome with an intact p57 gene. No mutations were detected by Southern blot analysis in the p57 alíelesin theG401 cell line (data not shown). To determine the level of p57expression in G40I and the microcell hybrid lines. Northern blotscontaining polyadenylated RNA isolated from the G401, MCH 369,MCH 485. and MCH 486 cell lines were hybridized with ap57 cDNAprobe. As shown in Fig. 4. the intact 1.5-kb p57 transcript was notdetected in G401 or in the hybrid lines. However, a 0.8-kb transcriptwas detected in all of the tumorigenic lines and some of the nontu-

morigenic G401 hybrid lines. This reduced transcript was also observed in adult skeletal muscle and adult kidney mRNA samples.These results indicate that p57 expression does not correlate withtumor suppression in the G40I hybrid lines.

Discussion

example, the breakpoint sites of balanced chromosome rearrangements in five BWS patients and one rhabdoid tumor map to a~320-kb region that includes p57 (8). These alterations probably do

G401 Hybrid Linestumorigenic nontumorigenic

!=»

<(N T- (N

«o

p57KIP2

7-Act in

ill-1.5-1.0-0.8

mm mWe have mapped the p57 gene to a 2.2-kb region in llplS.5 Fig. 4. p57 expression in the G40l hybrid lines. Northern blot of polyadenylated RNA

7->iic-/c<u j r\itc¿-rn mi i i u A . . ,) isolated froin the G40l.6TG.c6 parental cell line, nonlumorigenic CUOIhybrid lines, andbetween DI1S648 and DI 75679. Molecular analyses have detected |imK)rigenicG4(), hybrid lines 'hybridized ,„a p57 cDNA ££. mRNA samp,es from

frequent loss of heterozygosity in I Ipl5.5 in a variety of human normal adult skeletal muscle and kidney were included as controls. The O.S-kb />57tumors (6), Suggesting that a Common tumor suppressor gene may transcript was observed in Ihe MCH 486.1 and MCH 4X6.2L samples after longer

, . , exposure. Hybridization to a v-actin probe indicates relative RNA loading. Ihe smallerreside in this area. Moreover, many of the chromosome alterations ac{jn (ranscr-pt ¡sohserved frequcn,|V ¡nskcielai muscle tissue. RNA sizes are given in

observed in BWS patients map to this same region of Ilpl5.5. For kilobases.

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CHARACTERIZATION OF THE HUMAN pS7 GENE

not disrupt the small p57 gene but may alter the activity of nearbyregulatory elements.

Several studies have suggested that the 1Ipl5.5 tumor suppressorgene may be imprinted. Loss of heterozygosity analyses have detecteda preferential loss of the maternal alíelein BWS. sporadic Wilms'

tumor, and rhabdomyosarcoma patients (18-21). In addition, bal

anced translocations in BWS patients are generally transmittedthrough the maternal germline (22). The human llplS.5 band contains two imprinted genes, HI9 and insulin-like growth factor II(23-27). These genes have reciprocal expression and methylationpatterns. That is, H19 is expressed from the maternally-derived chro

mosome, which has a hypomethylated H19 promoter, whereas insulin-like growth factor II is expressed from the paternally-derived

chromosome, which has a hypermethylated H19 promoter (28, 29).Alterations in these expression and methylation patterns have beenobserved in BWS patients, Wilms' tumors, and rhabdomyosarcomas(26, 27, 30-32). These loss-of-imprinting mutations result in a "paternal" appearance on both copies of chromosome 11, suggesting that

the Ilpl5.5 tumor suppressor gene is imprinted so that only thematernal alíeleis normally expressed. The mouse p57 gene displaysthis type of monoallelic expression (33). If the human p57 gene is alsoimprinted, then loss of imprinting may be one method used to inactivate the p57 gene.

Many CDKI proteins map to chromosomal regions that are alteredfrequently in cancer cells ( 1-3). These locations, along with their role

in negative growth control, suggest that CDKI proteins may be inactivated during tumor development. Deletions, mutations, and methylation changes that inactivate pl5 and pl6 are detected frequently ina variety of tumors (reviewed in Ref. 34). In contrast, mutations in thefamily of CDKI proteins including p21, p27, and p57 are extremelyrare. Few mutations have been reported in p21 (35, 36),4 and no

structural alterations have been detected after extensive analysis ofp27 (37-39). Similar analyses have not yet revealed mutations in thep57 gene after examining 51 Wilms' tumors and 75 soft tissue

sarcomas (40). These results suggest that alternative methods ofinactivation are required if the loss of these CDKI proteins contributesto tumor development.

We have used a somatic cell hybrid assay to identify a genetic elementin 11p 15.5. which suppresses tumor formation in the G401 Wilms' tumor

line (9-11). These experiments, in combination with similar functional

analyses by Koi et al. (12), localized the suppressive element to a~500-kb region between DI1S601 and DI1S724 (11). We have now

mapped the p57 gene within this region of 1Ipl5.5. If p57 expressionmodulated the tumorigenic potential of the G401 hybrid lines, we wouldexpect to observe p57 mRNA in all of the nontumorigenic lines but notin the tumorigenic lines. Northern analysis, however, detected a 0.8-kb

p57 transcript in both tumorigenic and nontumorigenic samples. Therefore, the p57 gene does not appear to be responsible for the tumorsuppression observed in our functional assay.

Interestingly, the 0.8-kb transcript observed in the G401 hybrid

panel is too small to encode the intact p57 gene. This reduced messagehas been detected previously in normal human tissues (5) and is mostlikely generated by alternative splicing. Differential use of three spliceacceptor sites and an exon containing 5' untranslated sequences was

detected in the mouse p57 gene (4). Our sequence analysis identifiedan 83-bp intron downstream of the translation termination signal,indicating an exon containing 3' untranslated sequences. Further

investigation may reveal additional untranslated exons in the humanp57 gene. Our sequence results also demonstrated that the codingregion in the human p57 gene is divided by a 0.5-kb intron. The splice

site disrupts an aspartic acid residue (codon 275) near a putativenuclear localization site. Introns are present in homologous positionsin the p2I and p27 genes (38).s The similar exon-intron boundaries

between these three CDKI proteins suggests that they may be evolu-

tionarily related.The combination of its inhibitory role in the cell cycle, location in

human chromosomal band 1Ipl5.5, and imprinted expression in micemakes p57 an excellent candidate for a tumor suppressor gene. However, p57 expression did not correlate with tumor suppression in ourfunctional assay, and no p57 mutations have been detected in a varietyof human tumors. Therefore, the contribution of p57 inactivation totumor progression remains unclear. In this report, we present thegenomic organization of the p57 gene. This information will be animportant tool for future studies examining the expression and mutation of this intriguing protein.

Acknowledgments

We thank Michael Higgins for helpful discussions; Stephen Elledge for thehuman p57 cDNA clone; Mary Lou McMaster for the y-actin probe; theJapanese Cancer Research Resources Bank for cosmids cCl 11-395 andcCll 1-469; David McElligott and Glen Evans for the chromosome 11 cosmid

library; Laura Livingstone and Jenifer Langdon for sequence analysis: CarolJones for the Jl series cell lines; Steve Dowdy and Eric Stanhridge for theMCH 701.8, XMCH 708.20, and XMCH 708.25 cell lines; and Karen Smithfor excellent technical assistance.

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1996;56:1214-1218. Cancer Res   Laura Hink Reid, Shyra J. Crider-Miller, Ande West, et al.   Analysis in the G401 Wilms' Tumor Assay

Gene and ItsKIP2p57Genomic Organization of the Human

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