Genomic Organization of the Human P57klp2gene and Its Analysis In

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

Advances in Brief

Genomic Organization of the Human p57Klp2 Gene and Its Analysis in the G401 Wilms' Tumor Assay1

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

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

Abstract suppressor gene to a â€”¿500-kbregion of 1Ipl5.5 between DIISftOI The p57KII>2gene encodes an inhibitor of cyclin-depcndcnt kinasc ac and DI 1S724. In this report, we present the genomic structure of the human p57 gene based on restriction mapping and sequence analysis tivity, which negatively regulates cell cycle progression. The human p57 of a cosmid clone. Although the gene mapped within our candidate gene is located in I Ipl5.5, a region of DNA frequently altered in neoplasia. domain. Northern blot analysis indicated that p57 expression did not We have isolated a human genomic clone and mapped the p57 gene to a 2.2-kb region between DIIS6-Ã•8 and Ãœ1IS679. Sequence analysis revealed correlate with the tumor suppression observed in our functional assay. that 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 that Materials and Methods the human p57 gene contains at least three exons. Our previous work with somatic cell hybrids mapped a tumor suppressor gene for the (.4(11 Cell Lines. Jl-4a. JI-49. Jl-43a. and J1-9 are part of a panel of deletion Wilms' tumor cell line to a ~500-kb region of HplS.5 that includes p57. hybrids derived by mutugenesis of Jl, a hamster cell line that stably retains Northern blot analysis detected a O.S-kh p57 transcript in several of the human chromosome 11 (13. 14). MCH 701.8 is a mouse cell line that contains (.4(11 hybrid lines. However, p57 expression did not correlate with tumor a human t(X;ll) (llpter > llql3::Xq21 > Xqter) chromosome. XMCH suppression. These results suggest that p57 is not responsible for the 708.20 and XMCH 708.25 are mouse cell lines that contain radiation-reduced t(X:ll) chromosomes derived from MCH 701.8 (15). G40I.6TG.6 is a hypox- tumor suppression observed in our somatic cell hybrid assay. 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 Introduction MCH 48ft.2L are G401 hybrid lines that contain the t(X;l I ) chromosome from Cellular proliferation involves an orderly progression through the XMCH 708.25 and do not form tumors when injected into nude mice (II). cell cycle that is controlled by protein complexes of cyclins and MCH 369.18. MCH 485.1, MCH 485.2A, and MCH 485.3 are G40I hybrid CDKs' (reviewed in Refs. 1-3). The CDK subunits phosphorylate cell lines that contain the t(X:ll) chromosomes from XMCH 708.24 or XMCH cycle-regulatory proteins, such as the retinoblastorna protein, to re 708.26 (10, II). These cell lines do form tumors alter mouse inoculation. PCR. Genomic DNA from the somatic cell hybrid panel was amplified lease cells from cell cycle arrest. The CDKs are counterbalanced by a using the following primers from the human Â¡>57gene: 5'-CCG TGT CCC family of negative cell cycle regulators. These CDKIs bind with the TCT CCA AGC-3' and 5'-CGG GGC TCT TTG GGC TCT-3'. They gener cyclin-CDK complexes to inactivate their catalytic domains. Muta ated a 180-bp amplicon after 30 cycles of denaluralion at 94Â°Cfor 1 min. tions in the CDKI proteins result in uncontrolled proliferation typical annealing at 58"C for I min, and extension at 72"C tor 1 min. Kach reaction of neoplasia. Therefore, CDKI proteins are excellent candidates for contained 400 ng template. 1 unit Tat) polyinerase, 10 HIMTris (pH 9). 50 IHM tumor suppressor genes. KCI, 1.0 HIMMgCI,. and 0.2 Mof each deoxyribonucleosidc triphosphatc. PCR The CDKI proteins are divided into two families based on their products were visualised in ethidium bromide-stained 2r/< agarose gels. sequence homology. One family includes the pl61NK4A, pl5INK4B, Isolation and Mapping of Cosmids. A human />57 partial cDNA clone and pl9i proteins whjch contaÂ¡na related series of was identified in a human cDNA library by hybridisation with the mouse p57 cDNA clone. A 0.7-kb fragment from the partial cDNA clone was used to ankyrin repeats. The second family of CDKI proteins includes p2]cipi/wAn p27KÂ»'1. and a recently identified third member. identify cosmid 14F5 (cl4F5) in the Los Alamos human chromosome 11 p57K"" (4. 5). These proteins have homologous CDK binding do cosmid library. Cosmids cCIl 1-395 (c395) and cCIl 1-469 (c469) had been identified previously in a different chromosome 11 cosmid library (17). The mains that are required for the inhibitory activity. The p57 protein location of the p57 gene within cl4F5 was determined by restriction mapping inhibits several G, and S-phase CDKs. and overexpression of p57 and hybridizations with fragments from a full-length cDNA clone. hp57. causes a complete cell cycle arrest in G, (4, 5). Nonradioactive hybridisations were performed at 65"C. according to the man The human p57 gene is located in 1Ipl5.5 (5). Mutations in this ufacturer's protocol (Genius system; Boehringer Mannheim). region of DNA are observed in several forms of cancer, including DNA Sequencing. The 1.2-kb Noi\ and 1.0-kb Nol\-Hind\U fragments of Wilms' tumor, and in patients with BWS (6-8). In addition, func genomic DNA were isolated from cl4F5 and suhcloned into pBLUESCRlPT tional studies by our lab (9-1 1) and others (12) have localized a tumor (Sfratacene). Plasmid DNA was isolated (Qiagen) and sequenced at the UNC-CH Automated DNA Sequencing Facility on a Model 373A DNA Sequencer (Applied Biosystems). Either external universal primers or internal Received 12/11/95; accepted 1/31/96. primers designed from genomic sequence were used. 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 Northern Analysis. Polyadenylatcd RNA (2 /xg) was isolated using an 18 U.S.C. Section 1734 solely to indicale this fact. oligo-dT kit (Collaborative Research), separated on 1% agarose-formaldehyde/ ' This research was supported by NIH Grant CA63176 (to B. E. W.) and by the MOPS gels, and transferred to nylon membranes (Boehringer Mannheim) in Howard Hughes Medical Institute. L. H. R. was an American Cancer Society Postdoctoral 10x SSC ( 1X SSC = 3.0 M sodium chloride. 0.3 M sodium). mRNA samples Fellow. S. J. C-M. is an NIH Predocloral Fellow supported by Environmental Pathology Training Grant ES07017. from adult skeletal muscle and kidney tissue (Clonlech) were included as : To whom requests for reprints should be addressed, at Department of Pathology and positive controls. RNA blots were hybridised to i:P-lahcled DNA probes at Laboratory Medicine, 345 Lineberger Cancer Center. CB#7295, University of North 42Â°Cin a 50<7cformumide solution. The p57 probe was the 1.5-kb EcoRl insert Carolina. Chapel Hill. NC 27599. from the human cDNA clone, hp57. The -y-actin probe was the 1.8-kb Xlwl ' The abbreviations used are: CDK. cyclin-dependent kinase: CDKI. cyclin-dependent kinasc inhibitor; BWS. Beckwith-Wiedemann syndrome. insert from the mouse cDNA clone. pHFl. 1214

Results

Localization of the Human pS7 Gene and Characterization of NC395.N1C14F5 NNII II<-4RQII Cosmid Clones. Previous analysis mapped the human p57 gene to I Ipl5.5 using fluorescent in situ hybridization (5). To localize the gene within this chromosomal band, we looked for the presence of 10kb _NIIINN p57 in a panel of deletion hybrids that contain fragments of human chromosome II in a rodent background (Fig. 1/4). DNA from the deletion hybrid panel was amplified by PCR using primers from the human p57 gene. As shown in Fig. \B, amplified bands were gener B ated in all samples except Jl-9. Identical results were observed when N N, Southern blots containing DNA from the hybrid panel were hybrid ized to human p57 cDNA probes (data not shown). These results P57KIP2 indicate that the p57gene is located in a region of DNA absent in Jl-9 10kb but present in XMCH 708.25 and the remaining hybrid lines. The Fig. 2. Restriction maps of genomic clones containing the human ;>57 gene. A. Jl-9 cell line contains the DIIS724 locus but not the more centra long-range map of cosmids showing the location of Null sites (A7)and I Ipl5.5 loci. Left, mene DIISI probe (14. 17). The XMCH 708.25 line contains the centromeric DNA. Right, lelomeric DNA. ÃŸ.detailed map of cosmid cl4F5 showing the DI IS64X locus but not the DI 1S601 probe (11). Therefore, the human location of AVÂ»I(/V).Ea>Rl (R), and HMill (Hi sites. The horiymtal armw underscores the 1.2-kb Null and 1.0-kh jVÂ»/l-WÂ»idlIIfragmentsthat contain p57 coding sequence. p57 gene must be located between the DÃ¬ÃŒSouI and DI IS724 loci. This region of 1Ipl5.5 includes cosmids c395 and c469 that contain the DÃŒIS648and D11S679 loci, respectively (17). Neither of these genomic sequences. Blots containing EcoRi. Hindiii. and Noll digests cosmids hybridized to p57 cDNA probes (data not shown). of cl4F5 were hybridized to a p57 full-length cDNA probe. This To isolate genomic clones containing the p57 gene, we screened a analysis detected an 11.4-kb EcoRi band and a 10.0-kb Wmdlll band human chromosome 11 cosmid library with a p57 partial cDNA in cl4F5. Similarly-sized EcoRl and Hiniiili fragments were observed probe. This analysis identified one cosmid cl4F5 that contained the in Southern blots of genomic DNA (data not shown), suggesting that p57 gene. Noti restriction analysis and hybridization to genomic the cl4F5 cosmid contained an intact p57 gene. Double digests with fragments mapped cl4F5 between c395 and c469 (Fig. 2/4). This these enzymes indicated that the gene was located within a 4.8-kb analysis also indicated that cl4F5 and c395 had substantial overlap in EcnRI-Wmdlll fragment. This fragment contained two Noll sites that divided 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. 2ÃŸ). p57 cDNA probes hybridized to the 1.2-kb Noti and 1.0-kb Notl- aVIHRASH19 |P57KIP* >l gesting 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 IGF2 and 1.0-kb Noti-Hindiil fragments from cl4F5 were subcloned and D11S724D11S1 sequenced (Fig. 3). To determine the genomic organization of the human p57 gene, the sequences derived from the two genomic frag D11S679D11S648D11S601 ments were compared to published cDNA sequence (5). This analysis revealed that the coding region of the human p57 gene is divided at D11S517RRM1D11S12HBB> codon 275 into two exons that are separated by a 0.5-kb intron. A second, 83-bp intron was detected in the 3' untranslated region. Some single base pair differences in the untranslated regions of the p57 gene were observed between the genomic and cDNA sequences. No dif ferences were detected in the coding DNA. Human p57 Expression in G401 Hybrid Cell Lines. We have used somatic cell hybrids in a functional assay to identify regions of B chromosome 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 lack Â» T T T T O i- i- *- f- expression of a tumor suppressor gene. In our assay, we introduced t(X;l 1) chromosomes into G401 cells and analyzed the tumorigenic potential of the hybrid cell lines in nude mice. In some cases, the introduced t(X;ll) chromosome complemented the G401 defect so that the hybrid lines did not form tumors after inoculation. In other cases, the introduced chromosome had no influence on the tumorige nicity of the G40I cells. This functional assay has enabled us to Fig. I. Local /ution of the human p57 gene in l Ipl5.5 using somatic cell hybrids. A, localize a tumor suppressor gene to a region of 1Ipl5.5 telomeric to vertical burs ind cate regains of I Ipl5.5 DNA retained in the deletion hybrid panel. Most lines contain ad itional human chromosome 11 DNA (not shown). Probes are listed in the DIIS60I locus (11). Since p57 maps to this same region, we order with telÃ³neric loci on top and centromeric loci on honom. H, ethidium bromide- investigated whether p57 expression contributed to the tumor sup stained 1ck agar ise gel showing the human p57 PCR products amplified in the somatic cell hybrid line The GM346A9 sample contains mouse DNA. The IMR90 sample pression observed in our assay. contains human DNA Previous experiments demonstrated that four G401 hybrid lines. 1215

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 in lowercase 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, were example, the breakpoint sites of balanced chromosome rearrange suppressed for tumorigenicity (11). Four other G401 hybrid lines. ments in five BWS patients and one rhabdoid tumor map to a MCH 369.18, MCH 485.1, MCH 485.2A, and MCH 485.3, were ~320-kb region that includes p57 (8). These alterations probably do tumorigenic (10, 11). Each of these G401 hybrid lines contains an introduced t(X;l I) chromosome with an intact p57 gene. No muta tions were detected by Southern blot analysis in the p57 alÃelesin the G401 Hybrid Lines G401 cell line (data not shown). To determine the level of p57 tumorigenic nontumorigenic !=Â» expression in G40I and the microcell hybrid lines. Northern blots containing polyadenylated RNA isolated from the G401, MCH 369, < Â«o (N T- (N MCH 485. and MCH 486 cell lines were hybridized with ap57 cDNA probe. As shown in Fig. 4. the intact 1.5-kb p57 transcript was not detected in G401 or in the hybrid lines. However, a 0.8-kb transcript was detected in all of the tumorigenic lines and some of the nontu- -1.5 p57KIP2 -1.0 morigenic G401 hybrid lines. This reduced transcript was also ob -0.8 served in adult skeletal muscle and adult kidney mRNA samples. These results indicate that p57 expression does not correlate with ill tumor suppression in the G40I hybrid lines. 7-Act in mm m Discussion

We 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 between DI1S6487->iic-/c57 tumors (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 smaller reside in this area. Moreover, many of the chromosome alterations ac{jn(ranscr-pt Â¡sohservedfrequcn,|V Â¡nskcielaimuscle tissue. RNA sizes are given in observed in BWS patients map to this same region of Ilpl5.5. For kilobases. I216

not disrupt the small p57 gene but may alter the activity of nearby site disrupts an aspartic acid residue (codon 275) near a putative regulatory elements. nuclear localization site. Introns are present in homologous positions in the p2I and p27 genes (38).s The similar exon-intron boundaries Several studies have suggested that the 1Ipl5.5 tumor suppressor gene may be imprinted. Loss of heterozygosity analyses have detected between these three CDKI proteins suggests that they may be evolu- a preferential loss of the maternal alÃelein BWS. sporadic Wilms' tionarily related. tumor, and rhabdomyosarcoma patients (18-21). In addition, bal The combination of its inhibitory role in the cell cycle, location in anced translocations in BWS patients are generally transmitted human chromosomal band 1Ipl5.5, and imprinted expression in mice through the maternal germline (22). The human llplS.5 band con makes p57 an excellent candidate for a tumor suppressor gene. How tains two imprinted genes, HI9 and insulin-like growth factor II ever, p57 expression did not correlate with tumor suppression in our (23-27). These genes have reciprocal expression and methylation functional assay, and no p57 mutations have been detected in a variety patterns. That is, H19 is expressed from the maternally-derived chro of human tumors. Therefore, the contribution of p57 inactivation to mosome, which has a hypomethylated H19 promoter, whereas insu tumor progression remains unclear. In this report, we present the lin-like growth factor II is expressed from the paternally-derived genomic organization of the p57 gene. This information will be an chromosome, which has a hypermethylated H19 promoter (28, 29). important tool for future studies examining the expression and muta Alterations in these expression and methylation patterns have been tion of this intriguing protein. observed in BWS patients, Wilms' tumors, and rhabdomyosarcomas (26, 27, 30-32). These loss-of-imprinting mutations result in a "pa Acknowledgments ternal" appearance on both copies of chromosome 11, suggesting that We thank Michael Higgins for helpful discussions; Stephen Elledge for the the Ilpl5.5 tumor suppressor gene is imprinted so that only the human p57 cDNA clone; Mary Lou McMaster for the y-actin probe; the maternal alÃeleis normally expressed. The mouse p57 gene displays Japanese Cancer Research Resources Bank for cosmids cCl 11-395 and this type of monoallelic expression (33). If the human p57 gene is also cCll 1-469; David McElligott and Glen Evans for the chromosome 11 cosmid imprinted, then loss of imprinting may be one method used to inac library; Laura Livingstone and Jenifer Langdon for sequence analysis: Carol tivate the p57 gene. Jones for the Jl series cell lines; Steve Dowdy and Eric Stanhridge for the Many CDKI proteins map to chromosomal regions that are altered MCH 701.8, XMCH 708.20, and XMCH 708.25 cell lines; and Karen Smith frequently in cancer cells ( 1-3). These locations, along with their role for excellent technical assistance. in negative growth control, suggest that CDKI proteins may be inac tivated during tumor development. Deletions, mutations, and methy References lation changes that inactivate pl5 and pl6 are detected frequently in 1. Hunter. T.. and Pines. J. Cyclins and cancer II: cyclin D and CDK inhibitors come of a variety of tumors (reviewed in Ref. 34). In contrast, mutations in the age. Cell, 79: 573-582. 1994. 2. Cordon-Cardo. C. Mutation of cell cycle regulators: biological and clinical implica family of CDKI proteins including p21, p27, and p57 are extremely tions for human neoplasia. Am. J. Pathol., 147: 545-560. 1995. rare. Few mutations have been reported in p21 (35, 36),4 and no 3. MacLachlan. T. K., Sang. N.. and Giordano. A. Cyclins, cyclin-dependent kinases and structural alterations have been detected after extensive analysis of cdk inhibitors: implications in cell cycle control and cancer. Crii. Rev. Eukaryotic Gene Expr., 5: 127-156, 1995. p27 (37-39). Similar analyses have not yet revealed mutations in the 4. Lee. M-H., Reynisdottir. I., and MassaguÃ©.J.Cloning of p57KIP2. a cyclin-dependent p57 gene after examining 51 Wilms' tumors and 75 soft tissue kinase inhibitor with unique domain structure and tissue distribution. Genes & Dev., 9: 639-649. 1995. sarcomas (40). These results suggest that alternative methods of 5. Matsuoka. S.. Edwards. M. C.. Bai. C., Parker. S.. Zhang, P., Baldini, A., Harper, inactivation are required if the loss of these CDKI proteins contributes J. W., and Elledge. S. J. p57KIP2, a structurally distinct member of the p2lcipl Cdk to tumor development. inhibitor family, is a candidate tumor suppressor gene. Genes & Dev., 9: 650-662, We have used a somatic cell hybrid assay to identify a genetic element 1995. in 11p 15.5. which suppresses tumor formation in the G401 Wilms' tumor 6. Seizinger, B. R.. Klinger. H. P.. Junien. C., Nakamura. Y.. Le Beau. M., Cavenee. W., Emanuel. B.. Ponder, B., Naylor. S.. Mitelmun. F.. Louis. D.. Menon, A., Newsham, line (9-11). These experiments, in combination with similar functional I.. Decker. J.. Kaelbling. M.. Henry. !.. and Deimling. A. v. Report of the committee on chromosome and gene loss in human neoplasia. Cytogenet. Cell Genet., 58: analyses by Koi et al. (12), localized the suppressive element to a 1080-1096. 1991. ~500-kb region between DI1S601 and DI1S724 (11). We have now 7. Sail. S. N. J.. Nowak. N. J.. Singh-Kahlon, P.. Weksberg. R., Squire. J., Shows, T. B., and Higgins. M. J. Localization of Beckwith-Wiedemann and rhabdoid tumor chro mapped the p57 gene within this region of 1Ipl5.5. If p57 expression mosome rearrangements to a defined interval in chromosome band 1Ipl5.5. Genes modulated the tumorigenic potential of the G401 hybrid lines, we would Chromosomes & Cancer, //: 97-105. 1994. expect to observe p57 mRNA in all of the nontumorigenic lines but not 8. Hoovers. J. M. N.. Kalikin. L. M.. Johnson, L. A.. Alders. M., Redeker, B., Law, D. J., Bliek. J.. Sleenman. M.. Benedict, M.. Wiegant. J.. Lcngauer. C.. Taillon-Miller, P., in the tumorigenic lines. Northern analysis, however, detected a 0.8-kb Schlessinger. D.. Edwards. M. C.. Elledge. S. J.. Ivens. A., Westerveld, A.. Little, P., p57 transcript in both tumorigenic and nontumorigenic samples. There Mannens. M.. and Feinberg. A. P. Multiple genetic loci within Ilpl5 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal fore, the p57 gene does not appear to be responsible for the tumor transferable fragments. Proc. Nati. Acad. Sci. USA, 92: 12456-12460, 1995. suppression observed in our functional assay. 9. Weissman, B. E., Saxon, P. J.. Pasquale, S. R., Jones, G. R., Geiser, A. G.. and Interestingly, the 0.8-kb transcript observed in the G401 hybrid Stanbridge. E. J. Introduction of a normal human chromosome 11 into a Wilms' tumor cell line controls its tumorigenic expression. Science (Washington DC). 236: panel is too small to encode the intact p57 gene. This reduced message 175-180, 1987. has been detected previously in normal human tissues (5) and is most 10. Dowdy. S. F.. Fasching. C. L., Araujo. D., Lai, K-M., Livanos, E., Weissman, B. E., and Stanbridge. E. J. Suppression of tumorigenicity in Wilms' tumor by the pl5.5-pl4 likely generated by alternative splicing. 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Our sequence results also demonstrated that the coding 13. Gerhard, D. S., Jones. C.. Morse, H. G.. Handelin. B., Weeks. V., and Housman. D. region in the human p57 gene is divided by a 0.5-kb intron. The splice Analysis of human chromosome 11 by somatic cell genetics: reexamination of derivatives of human-hamster cell line Jl. Somat. Cell Mol. Genet.. 13: 293-304. 1987. "L. LaCombe, I. Orlow, W. Gerald, W. R. Fair, and C. Cordon-Cardo. personal communication. 1W. S. EI-Deiry and J. Pietenpohl, personal communication. 1217

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Laura Hink Reid, Shyra J. Crider-Miller, Ande West, et al.

Cancer Res 1996;56:1214-1218.

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