Mutations of the P53 Tumor Suppressor Gene Occur Infrequently in Wilms' Tumor' David Malkin,2 Elizabeth Sexsmith, Herman Yeger, Bryan It G

[CANCER RESEARCH54, 2077-2079, April 15, 1994] Advances in Brief

Mutations of the p53 Tumor Suppressor Gene Occur Infrequently in Wilms' Tumor' David Malkin,2 Elizabeth Sexsmith, Herman Yeger, Bryan it G. Williams, and Max J. Coppes

Division of Hematology/Oncology, Department of Pediatrics fD. M., E. S.], and Department of Pathology [H. }â€˜j,The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; Department of Cancer Biology, Research Institute, The Cleveland Clinic Foundation@ Cleveland Ohio 44195 [B. R. G. WJ; and the Pediatric Oncology Program, Alberta Children's Hospita4 Calgary, Alberta, Canada T2T 5C7 (M. J. C.]

Abstract ample, nephrogenic rests, foci of persistent embryonal nonmalignant remnants, are demonstrated within the kidneys of approximately 30â€” Mutations of the p53 tumor suppressor gene occur frequently in a 40% of children with WI' (14). These lesions are apparent precursors variety ofadult-onset tumors, including colon, breast, lung, and brain, yet of WT (15). Wi' is also associated with specific congenital abnormal are infrequently identified In pediatric malignancies. Wilma' tumor, a common solid tumor ofchildhood, can be associated with mutations of the ities, including genitourinary anomalies, sporadic aniridia, mental w'ri gene.Alterationsofthep53genehavebeenshownto modulatethe retardation, and hemihypertrophy (16). Genetic predisposition to WT ability of WT1 to transactivate its targets. Although positive p5.3 immu is recognized in two distinct syndromes with urogenital malforma nostaining has been demonstrated in Wilms' tumors, the correlation to tions (WAGR syndrome and Denys-Drash syndrome), as well as in p53 genemutations is not clear. We examinedWilms' tumor samplesfor BWS, a congenital overgrowth syndrome characterized by growth p53 mutatIons utilizing polymerasechain reaction-single-strandconfor abnormalities and a predisposition to several embryonal neoplasms, mation polymorphism analysis and single-strand DNA sequencing. Muta including Wi' (16). These congenital defects have been linked with dons in the coding region of the p5.3 gene were demonstrated in 2 of 21 specific genetic loci implicated in Wilms' tumorigenesis. They in (9.5%) Wilms' tumors. Each mutation yielded a substitution ofamino acid dude the WAGR locus at chromosome 1lpl3 and the BWS locus at residues. One mutation was located in exon 6 and the other in exon 7. Both mutations were found in tumors from patients with advanced stage dis chromosome lipiS. The WAGR locus encompasses several contigu ease. Focal anaplasia was demonstrated in one of these tumors. Our data ous genes, including the Wilms' tumor suppressor gene W7'l, while suggest that although p53 mutatIons occur infrequently In Wilma' tumor, the BWS locus includes the gene implicated in BWS as well as a they may be associated with advanced disease. putative second Wilms tumor suppressor gene, W72 (reviewed in Refs. 17â€”19). Introduction Although alterations of p53 have not been implicated in Wilms' tumorigenesis, recent observations by Maheswaran et a!. (20) have The p53 tumor suppressor gene is located on the short arm of demonstrated that p53 can physically interact with WTJ in transfected human chromosome 17 and encodes a 53-kDa nuclear phosphoprotein cells and that this interaction modulates the ability of each protein to that appears to act as a negative regulator of cell proliferation (1, 2). transactivate their respective targets. The association of these two The protein probably achieves its function by blocking the progres factors is also implicated by the rare occurrence of Wilms' tumors in sion of cells through the cell cycle late in G1 (3). In addition, p53 some Li-Fraumeni families who carry constitutional alterations of the seems to act as a transcription activator that suppresses abnormal cell p53 gene, predisposing affected members to cancer (21). We set out proliferation by acting as a G1 cell cycle checkpoint control for DNA to determine the frequency of p53 alterations in an unselected series damage (4). Alterations of the p53 gene and its encoded protein are of Wilms' tumors of varying histological subtypes and stage. the most frequently encountered genetic events in human cancer, Genomic DNA extracted from Wilms' tumor samples were screened having been reported in almost every type of sporadic neoplasm (5, 6). using PCR and SSCP as described previously (22). Those samples that Few studies, however, have reported the frequency ofp53 alterations showed altered electrophoretic mobility compared to the wild-type in tumors of pediatric origin. Missense mutations of p53 in neuro controls by SSCP were sequenced to further characterize the specific blastoma (7, 8), neuroblastoma cell lines (9), rhabdomyosarcoma (10), mutations. These results are discussed in the context of the histopatho rhabdomyosarcoma cell lines (10), Ewing's sarcoma (9), and pediatric logical features of the tumor, as well as clinical characteristics of the acute lymphoblastic leukemia (11) have been demonstrated in a small patient. number of studies. WT,3 or nephroblastoma, is an embryonal malignancy that arises Materials and Methods from remnants of immature kidney. It affects approximately 1 in 10,000 children, usually before the age of 6 years (median age at Thirty-two consecutive diagnostic Wilms tumor specimens were obtained diagnosis, 3.5 years) (12). Approximately 5â€”10%of children are (21 at pretreatment biopsy, 11 at resection postchemotherapy), immediately affected bilaterally, either at presentation (synchronous bilateral Wi') snap-frozen in liquid nitrogen, and then stored at â€”70Â°C.Beforeprocessing for or with unilateral disease initially, followed by the development of a DNA analysis, the tumor tissue was ground to a fme powder under liquid contralateral tumor (asynchronous or metachronous bilateral WT) nitrogen. Age at diagnosis, sex, tumor histopathology (favorable histology versus anaplastic), clinical stage according to the National Wilms' Tumor (13). Wi' is often associated with certain peculiar features. For cx Study (23), and clinical outcome were documented and recorded for each patient sample. Genomic DNA that had been isolated and stored previously Received 12/28/93; accepted 3/4/94. using standard procedures was resuspended in 10 mr@iTris-1mMEDTA, pH The costs of publication of this article were defrayed in part by the payment of page 8.0, to a concentration of 50 ng/pJ. Inadequate quality or quantity of DNA charges. This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section1734solelyto indicatethisfact. precluded 6 samples from being analyzed, while five additional samples were 1 This work was supported by the Medical Research Council of Canada. excludedbecauseof incompleteclinical data. 2 To whom requests for reprints should be addressed, at Division of Oncology, The PCR:SSCP and Sequencing Analysis. Twenty-one W'F samples were Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. available for analysis (14 were diagnostic samples, 7 were postchemotherapy). 3 The abbreviations used are: WT, Wilma' tumor; BWS, Beckwith-Wiedemann syn drome;PCR, polymerasechain reaction;SSCP, single-strandconformationalpolymor Nine sets of primers were generated to amplify DNA fragments spanning phism; WAGR, Wilms'-aniridia-genitourinary anomalies. exons 2 and 4 through 11 of the p53 gene. These primers have been published 2077

previously (22, 24). PCR was performed using 100â€”500ng of template DNA mutationsTwoTable2 Characteristicsofsomaticp.5.3 in 50 mMTris-HC1 (jH 8.6) with 1.5 mM MgCI2,0.2 mMconcentrations of aneutralbase-pairalterationswere identifiedin Wit-34. One at codon 213 represents each deoxynucleotide triphosphate, 250 ng of each primer, 1 pAof [32P]dC'FP polymorphism.Sample (3000 Ci/mmol) diluted 1:10, and 2.5 units of Taq polymerase (AmpliTaq; switchWit-34Exon Codon Sequencechange Aminoacid Cetus) in a 50-pA total reaction volume. The reaction conditions for the Gly-'ArgWit-346 199 OOA-'AGA Perkin-Elmer 480 thermocycler were: 94Â°C(45 s); 55Â°C(45 s); and 72Â°C(45 Argâ€”*ArgWit-286 213 CGAâ€”@GGA s) for 35 cycles. The reaction was initiated with one 10-mm incubation at 85Â°C 7 257 CFG-@CAG Leuâ€”*Gln and terminated with a 7-min incubation at 72Â°Cfollowedby 3 mm at 94Â°C.An equal volume of the PCR product was added to a loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene samples. Repeated sequence analysis of 3 of these failed to identify cyanol). The samples were denaturedat 85Â°Cfor5 mm and loaded immedi base-pair alterations. However, two Wilms' tumor samples (Wit-28 ately onto an acrylamide-tris buffered EDTA nondenaturing gel. The gels and Wit-34) demonstrated abnormal band-shift patterns on SSCP contained 4.5â€”9.0%acrylamideand 2â€”10%glycerol,depending on the exon analysis that were subsequently confirmed to represent abnormalities fragment being analyzed. Electrophoresis was performed at 25 W for 6 to 7 h of the nucleotide sequence. In the exon 6 fragment of sample Wit-34, or 10 W for 15â€”17hat room temperature. The gel was dried and exposed to a transition from A to 0 in the third position of codon 213 was X-OMAT film (Kodak) with an intensifying screen at â€”70Â°Cfor4 to 72 h. Each fragment was run under two or three electrophoretic conditions, again identified. This base substitution does not change the amino acid depending on the specific fragment being analyzed. Each gel carried both encoded (arginine) and thus represents either a polymorphism (26) or positive and negative controls, to be sure that known mutations could consis silent mutation. However, this sample also contains a true mutation in tently be appreciated. DNA samples determined to be abnormal by detectable exon 6 as represented by a base-pair substitution from G to A in the band shifts on SSCP were reamplified with the SSCP primers encompassing first position of codon 199 yielding an amino acid change from the abnormal region. Fragments were subcloned directly into a T-tailed pBSK glycine to arginine. In the exon 7 fragment of sample Wit-28, a vector and sequenced in both directions by the Sanger dideoxide method with transition from T to A in the second position of codon 257 was a Sequenase 2.0 kit (United States Biochemicals/Amersham). identified. This base substitution yields an encoded amino acid change Results from leucine to glutamine. Wit-28 was derived from a stage V primary tumor with favorable histology, while Wit-34 was derived from a The clinical and histopathological data of the 21 patients are sum stage HI primary that demonstrated focal areas with hyperdiploid marized in Table 1. The mean age at diagnosis was 37 months (range, mitotic figures, nuclear enlargement, or nuclear hyperchromasia con 4â€”95months). The male:female ratio was 10:11. Histological subtype sistent with focal anaplasia. These data are outlined in Table 2. was favorable in 18, focal anaplasia in 2, and diffuse anaplasia in 1. Histopathology of the posttreatment samples was not different from Discussion their respective primary biopsies; in particular, all tumors with favor Based on studies of many different tumor types, it has been sug able histology at biopsy did not show anaplastic features at nephrec gested that alterations of the p53 gene and its encoded protein may tomy and vice versa. The tumor stage was I in 5, II in 4, III in 4, IV represent the most frequent genetic event in human cancer (6, 27). in 4, and V in 4. We examined thep53 coding sequence in exons 2 and These studies have utilized either direct examination of mutations in 4 through 11. These 9 exons include all phylogenetically conserved the genomic sequence ofp53 or immunostaining ofp53 protein using sequences in the coding regions of the p53 gene and comprise all a variety of antibodies. Point mutations of the p53 gene frequently exons with published mutations. The most highly conserved region, give rise to an altered protein with a significantly increased half-life, encompassing exons 5 through 8, contains >80% of all reported allowingp53 to be detected in affected cells using immunohistochem mutations (6, 25). Abnormal band-shift patterns were noted in 6 of 21 ical methods. In the absence of a complexing viral protein, detection of significant amounts ofp53 in a cell has been generally taken to be Table 1 Clinical, histopathologica4 and WI'l characteristics of Wilms' tumor samples indicative of a p53 gene mutation (27), particularly when monoclonal Samples Wit-i and Wit-34 demonstrate focal anaplasia, while Wit-36 shows diffuse antibodies that specifically recognize mutant epitopes are used (28). anaplasia. Histopathology and stage are based on National Wilma' Tumor Study classi fications. I'Tfl expression data have been reported previously (30) as normal (N), low (L) With the exception of recent studies reporting a low frequency of p53 or absent (A). W1'l expression status was not available on 5 samples (â€”),andpercentage gene alterations in neuroblastomas (8, 9), few data are available on the blastema was not available on 9 samples (â€”). frequency and characteristics of p53 abnormalities in other pediatric at tumors of mesenchymal origin. diagnosis Recent evidence suggests that the WT1 tumor suppressor gene, the blastemaWit-2F54â€”70Wit-7F34AnaplasiaVN-Wit-8M26FHIVL80Wit-12F22FEVN45Wit-13F24FHIIIA60Wit-18M30HIIIâ€”30Wit-24M43FEHIL70Wit-25F27FEIVN70Wit-26F25FH/NRIIL80Wit-28M66FHVL35Wit-32F6FHIN70Wit-34F57AnaplasiaIIIâ€”50Wit-36M95AnaplasiaIVâ€”65Wit-40F22FHIVN-Wit-42M34FHIN-Wit-47F91FEIIINâ€”Wit-52F4FEV-â€”Wit-60M25FEVN-Wit-67M26FH/NRIIN-Wit-72F79FHIINâ€”Wit-76F4FHINâ€”aSampleSexAge (months)HistopathologyStageWJ'l expression% inactivation of which leads to the development of certain Wilms' tumors (17), may modulate the ability of p53 to transactivate target genes and vice versa (20). While V(fl enhances transcriptional acti vation by p53, wild-type p53 appears to convert 1471 from a tran scriptional activator to a transcriptional repressor (20). To date, only Lemoine et a!. (29) have studied p53 in Wilms' tumor. These investigators observed immunoreactivity in all of 34 Wilms' tumors that were analyzed with at least one of three specific antibodies (monoclonal antibody PAb24O, polyclonal antibodies CM1 and JG8) (29), suggesting that p53 mutations are very common in Wilms' tumor. Since p53 overexpression is not exclusively associated with the presence of point mutations in the coding region of the p53 gene (27, 28), we used PCR-SSCP analysis and standard single-strand sequencing to detect p53 mutations in 21 randomly selected Wilms' tumor specimens. Three base pair substitutions, two of which (9.5%) yielded missense mutations that resulted in codon alterations, were @ favorablehistology;NR, nephrogenic rests. identified. Both samples Wit-28 and Wit-34 demonstrated loss of the 2078

wild-type p53 allele, as indicated by the presence of only mutant Weston, A., Modali, R., Harris, C. C., and Vogelstein, B. Mutations in the p53 gene clones for sequencing. It is not likely that the low frequency of p53 occur in diverse human tumor types. Nature (Lond.), 342: 705â€”708,1989. 6. Hollstein, M., Sidransky, D., Vogestein, B., and Harris, C. C.p53 mutations in human mutations in this study reflects false-negative results (22). All samples cancers. Science (Washington DC), 253: 49-53, 1991. were analyzed under either two or three different electrophoretic 7. Imamura, J., Bartram, C. R., Berthold, F., Harms, D., Nakamura, H., and Koeffler, H. P. Mutationof the p53 gene in neuroblastomaand its relationshipwith N-myc conditions on the presumption that certain mutations might be less amplification. Cancer Res., 53: 4053-4058, 1993. discernible under one particular set of electrophoretic or glycerol! 8. Vogan, K., Bernstein, M., Leclerc, J-M., Brisson, L, Brossard, J., Brodeur, 0. M., Pelletier, J., and Gros, P. Absence ofp53 gene mutations in primary neuroblastomas. acrylamide gel content concentrations. Our PCR primers encom Cancer Res., 53: 5269â€”5273,1993. passed intron-exon junctions, but it is possible, of course, that muta 9. Komura,H., Hayashi,Y., Kawamura,M., Hayashi,K., Kaneko,Y., Kamoshita,S., tions might occur within introns or perhaps in the promoter region of Hanada,R., Yamamoto,K., Hongo, T., Yamada,M., andTsuchida,Y. Mutationsof the p53 gene are involved in Ewing's sarcomas but not in neuroblastomas. Cancer the p53 gene, areas that were not covered by our screening technique. Res., 53: 5284â€”5288,1993. In addition, altered function of the p53 protein might occur as a result 10. Felix, C. A., Kappel, C. C., Mitsudomi, T., Nau, M. M., Tsokos, M., Crouch, 0. D., of posttranscriptional alterations or complexing with other intracellu Nisen, P. D., Winick, N. J., and Helman, L. J. Frequency and diversity of p53 mutations in childhood rhabdomyosarcoma. Cancer Res., 52: 2243â€”2247,1992. lar factors leading to inactivation of the p53 product. Neither Wit-28 11. Felix, C. A., Nau, M. M., Takahashi, T., Mitsudomi, T., Chiba, I., Poplack, D. 0., nor Wit-34 carry mutations in the Wfl gene zinc fmger region (30), Reaman, 0. H., Cole, D. E., Letterio, J. J., Whang-Peng, J., Knutsen, T., and Minna, J. D. Hereditary and acquired p53 gene mutations in childhood acute lymphoblastic although we have noted previously (31) that Wit-28 expresses a low leukemia. J. ain. Invest., 89: 640â€”647, 1992. amount of WT1 mRNA. It will be of interest to determine whether the 12.Breslow,N.,Beckwith,J.B.,Ciol,M.,andSharples,K.AgedistributionofWilma' mutations inpS3 in these tumors affect its ability to interact with Wfl. tumor: report from the National Wilma' Tumor Study. Cancer Res., 48: 1653â€”1657, 1988. Although the number of Wilms' tumor specimens analyzed was 13. Coppes, M. J., De Kraker, J., Van Dijken, P. J., Perry, H. J., Delemarre, 3. F., limited, our findings suggest that Wilms' tumor samples carrying Tournade, M. F., Lemerle, J., and Voute, P. A. Bilateral Wilma' tumor: long-term homozygous p53 mutations are not distinct in mean age at diagnosis survival and some epidemiological features. J. Clin. Oncol., 7: 310â€”315,1989. 14. Beckwith, J. B., Kiviat, N. B., and Bonadio, J. F. Nephrogenic rests, nephroblasto or in histological features from those carrying wild-type p53 (Table matosis, and the pathogenesis of Wilms' tumor. Pediatr. Pathol., 10: 1â€”36,1990. 1). Although one of the samples (Wit-34) shows features of focal 15. Park, S., Bernard, A., Bove, K. E., Sans, D. A., Hazen-Martin, D. J., Garvin, H. A., and Haber, D. A. Inactivation of Wfl in nephrogenic rests, genetic precursors to anaplasia, two other anaplastic tumors (Wit-7 and Wit-36) carried Wilms' tumor. Nat. Genet., 5: 363â€”367,1993. wild-type p53. Therefore, there is no conclusive evidence from this 16. Clericuzio, C. L Clinical phenotypes and Wilma' tumor. Med. Pediatr. Oncol., 21: study that p53 mutations in sporadic Wilms' tumor are more likely to 182â€”187,1993. 17. Coppes, M. J., Campbell, C. E., and Williams, B. R. 0. The role of WfI in Wilma' be associated with the more aggressive anaplastic subtype. It is of tumorigenesis. FASEB J., 7: 886â€”895, 1993. interest that both tumors with p53 mutations were from patients with 18. Rauscher, F. J., III. The WT1 Wilma tumor gene product: a developmentally regu advanced disease. lated transcription factor in the kidney that functions as a tumor suppressor. FASEB J., 7: 896â€”903,1993. Our data seem to be in contrast to that of Lemoine et aL who, on the 19. Coppes, M. J., Haber, D., and Grundy, P. Genetic events in the development of Wilma basis of positive immunostaining, concluded that the frequency of p53 tumor. New Engl. J. Med., in press, 1994. 20. Maheswaran,S.,Park,S.,Bernard,A, Morris,J.F., Rauscher,F.J., III,Hill,D.E., point mutations was very high in Wilms' tumors. The apparent dis and Haber, D. A. Physical and functional interaction between WTI and p53 proteins. crepancy between the frequency of p53 mutations identified at the Proc. Nail. Aced. Sci. USA, 90: 5100-5104, 1993. gene level and positive immunostaining has been reported in several 21. Malkin, D., Li, F. P., Strong, L C., Fraumeni, J. F., Jr., Nelson, C. E., Kim, D. H., Kassel, J., Gryka, M. A., Biachoff, F. Z., Tainsky, M. A., and Friend, S. H. Germ line other tumor systems, including testis cancer (0 of 22 versus 38 of 40, p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neo respectively) (22, 27), metastatic melanoma (1 of 9 versus 7 of 10) plasma. Science (Washington DC), 250: 1233â€”1238,1990. (32, 33), and neuroblastoma (2 of 86 versus 4 of 4) (7, 8). This could 22. Peng, H-Q., Hogg, D., Malldn, D., Bailey, D., Gallie, B. L, Bulbul, M., Jewett, M., Buchanan, J., and Goaa, P. E. Mutations of the p53 gene do not occur in testis cancer. indicate that the presence of p53 protein in some tumors may reflect Cancer Rca., 53: 3574â€”3578, 1993. stabilization of wild-type protein. 23. D'Angio, 0. J., Breslow, N., Beckwith, 1. B., Evans, A., Baum, H., DeLorimier, A., Fembach, D., Hrabovsky, E., Jones, B., Kelalis, P., Othersen, B., Teffi, M., and The lack ofp53 mutations in Wilms' tumor is in striking contrast to Thomas, P. R. M. Treatment of Wilma' tumor. Results of the third National Wilma' almost all other reported cancers (6) but is not entirely unexpected. Tumor Study. Cancer (Phila.), 64: 349â€”360,1989. Several other childhood cancers, including medulloblastoma (34), 24. Mashiyama, S., Murakami, Y., Yoshimoto, T., Sekiya, T., and Hagiwara, K. Detec tion of p53 gene mutations in human brain tumors by single-strand conformation neuroblastoma (7, 8), and retinoblastoma,4 and tumors of the urogen polymorphism analysis of polymeraae chain reaction products. Oncogene, 6: 1313â€” ital system, particularly testis (22), are known to demonstrate a lack of 1318, 1991. mutations in the p53 coding sequence. Our observations suggest that 25.CarondeFromentel,C.,andSouasi,T.TP53suppressorgene:amodelforinveati gating human mutagenesia. Genes Chromosomes Cancer, 4: 1â€”15,1992. p53 gene mutations may be relatively unimportant in the genesis of 26. Carbone, D., Chiba, I., and Mitsudomi, T. Polymorphism at codon 213 within the p53 Wilms' tumors. In light of the recognized association of WT1 gene gene. Oncogene, 6: 1691â€”1692,1991. 27. Bartek, J., Bartkova, J., Vojtesek, B., Staakova, Z., Lukas, J., Rejthar, A., Kovarik, I., alteratiofis with the genesis of sporadic Wilms' tumor (17), the genetic Midgley, C. A., Gannon, J. V., and Lane, D. P. Aberrant expression of the p53 implications of the WTJ/p53 interaction remain to be defined, and the oncoprotein is a common feature of a wide spectrum of human malignancies. role ofp53, although critical in most types of carcinogenesis, presum Oncogene, 6: 1699â€”1703,1991. 28. Gannon, J. V., Greavea, R., Iggo, R., and Lane, D. P. Activating mutations in p53 ably plays a less significant role in the development of this tumor. produce a common conformational effect. A monoclonal antibody specific for the mutant form. EMBO J., 9: 1595â€”1602,1990. References 29. Lemoine, N. R., Hughes, C. M., and Cowell, J. K. Aberrant expression of the tumor suppressor gene p53 is very frequent in Wilma' tumors. J. Pathol., 168: 237â€”242, 1. Levine,A. J., Momand,J., andFinlay,C. A. Thep53 tumorsuppressorgene.Nature 1992. (Lond.),351:453â€”456,1991. 30. Coppea, M. J., Liefera, 0. J., Paul, P., Yeger, H., and Williams, B. R. 0. Homozygous 2. Vogelstein, B., and Kinzler, K. W. p.5.3function and dysfunction. Cell, 70: 523â€”526, somatic Wfl point mutations in sporadic unilateral Wilma tumor. Proc. NatI. Acad. 1992. Sci. USA, 90: 1416â€”1419, 1993. 3. Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V., and Kastan, M. B. Wild-type p53 is a 31. Yeger, H., Cullinane, C., Flenniken, A., Chilton-MacNeill, S., Campbell, C., Huang, cell cycle checkpointdeterminantfollowingirradiation.Proc.Natl. Acad. Sd. USA, A., Bonetta, L, Coppea, M. J., Thorner,P., and Williams, B. R. 0. Coordinate 89: 7491â€”7495,1992. expression of Wilma' tumor genes correlates with Wilma' tumor phenotypes. Cell 4. Kastan, M. B., Onyekwere, 0., Sidransky, D., Vogelstein, B., and Craig, R. W. Growth & Differ., 3: 855-864, 1992. Participation ofp53 protein in the cellular response to DNA damage. Cancer Res., 51: 32. Laasam, N. J., From, L., and Kahn, H. 1. Overexpreaaion ofp53 is a late event in the 6304â€”6311, 1991. development of malignant melanoma. Cancer Res., 53: 2235â€”2238,1993. 5. Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hostetter, R., Q@, K., 33. Stretch, J. R., Gatter, K. C., Ralfkiaer, E., Lane, D. P., and Harris, A. L. Expression Bigner, S. H., Davidson, N., Baylin, S., Devilee, P., Glover, T., Collins, F. C., of mutant p53 in melanoma. Cancer Rca., 51: 5976â€”5979, 1991. 34. Saylora, R. L, Sidranaky, D., Friedman, H. S., Bigner, S. H., Bigner, D. D., Vogelatein, B., and Brodeur, 0. M. Infrequent p53 gene mutations in medulloblas 4 B. Gallie, personalcommunication. tomas. Cancer Rca., 51: 4721â€”4723, 1991.

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