¹Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2 Nitona, Chiba 260-8717, Japan

²Laboratory of Gene Structure 1, Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan

³Department of Cancer Chemotherapy, Saitama Cancer Center Hospital, Saitama 362, Japan

⁴Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

⁵Sanofi-Recherche, 31676 Labe'ge Cedex, France

^aAuthor for correspondence

p73, a novel p53 family member, is a recently identified candidate neuroblastoma (NBL) suppressor gene mapped at chromosome 1p36.33 and was found to inhibit growth and induce apoptosis in cell lines. To test the hypothesis that p73 is a NBL suppressor gene, we analysed the p73 gene in primary human NBLs. Loss of heterozygosity (LOH) for p73 was observed in 19% (28/151) of informative cases which included 92 mass-screening (MS) tumors. The high frequency of p73 LOH was significantly associated with sporadic NBLs (9% vs 34%, P<0.001), N-myc amplification (10% vs 71%, P<0.001), and advanced stage (14% vs 28%, P<0.05). Both p73 and p73 transcripts were detectable in only 46 of 134 (34%) NBLs at low levels by RT - PCR methods, while they were easily detectable in most breast cancers and colorectal cancers under the same conditions. They found no correlation between p73 LOH and its expression levels (P>0.1). We found two mutations out of 140 NBLs, one somatic and one germline, which result in amino acid substitutions in the C-terminal region of p73 which may affect transactivation functions, though, in the same tumor samples, no mutation of the p53 gene was observed as reported previously. These results suggest that allelic loss of the p73 gene may be a later event in NBL tumorigenesis. However, p73 is infrequently mutated in primary NBLs and may hardly function as a tumor suppressor in a classic Knudson's manner.

p73; neuroblastoma; loss of heterozygosity; 1p36; mutation; p53

Introduction

p73, a new member of the p53 family, has been mapped to chromosome 1p36.33, a locus where loss of heterozygosity (LOH) is frequently observed in neuroblastoma (NBL) (Kaghad et al., 1997; Schwab et al., 1996). Using cell lines of NBL and other cancers, Kaghad et al. (1997) showed that the expression level of p73 in NBL is low compared with that of other cancers. Although mutations in the p73 coding region were absent in eight NBL cell lines, they found an LOH in six of the eight lines and evidence for monoallelic expression of p73. In addition, stable transfection of p73 inhibited the growth of the SK-N-AS NBL cell line, and its transient expression promoted apoptosis in Saos-2 osteosarcoma cells (Jost et al., 1997). Those results implied that p73 may be a candidate NBL suppressor gene localized at chromosome 1p36.33. However, studies using cell lines are sometimes not definitive, and it is critical to examine primary tumors to confirm whether or not the candidate gene is a real tumor suppressor gene. In the present report, we have studied 272 primary neuroblastomas for loss of heterozygosity (LOH) and 140 tumors for the analysis of p73 expression and mutation. Our results suggest that the p73 mutation in the primary NBLs is infrequent, although its allelic loss is associated with advanced stage NBL.

Results and Discussion

Extensive analyses of the distal region of 1p in NBL suggest the presence of multiple tumor suppressor loci (Schwab et al., 1996). However, recent reports of 1p LOH in NBL have defined the smallest region of overlap (SRO) between D1S244 and D1S214 at 1p36.2-p36.3 (so-called NB1 locus, Figure 1) (White et al., 1995; Martinsson et al., 1995; Amler et al., 1995). p73 is mapped distal to D1S214 (reference 1 and unpublished data). In order to determine whether p73 represents a unique SRO at chromosome 1p36.3-pter, we performed an LOH analysis on NBL tumors using four markers, D1Z2, p73, D1S80 and D1S214. As a microsatellite marker for p73, we used a CT repeat in intron 9 which proved polymorphic in 50% of normal individuals. The p73 LOH was examined in 272 paired DNA samples (tumor tissue and peripheral blood leukocytes) and the allelic loss was found in 28 of 151 (19%) informative cases (Table 1). Of 59 sporadic tumors, it was observed in 20 (34%), while the tumors detected by the mass-screening program in Japan, which may represent congenital tumors, susceptible to spontaneous regression, showed the LOH in eight out of 92 (9%) (²-test: P<0.001). The p73 LOH was also observed in 13 of 128 (10%) tumors without amplification of N-myc oncogene, while it was found in 15 of 21 (71%) tumors with the amplification (P<0.001). Furthermore, p73 LOH was significantly high in advanced stage tumors (P<0.05). These suggest that the allelic loss of p73 might occur at low frequency at an early stage of the NBL genesis and increase as the tumor progresses.

Allelic loss of at least one of four loci was detected in 41 out of 214 (19%) tumors (Figure 1). Twenty-nine tumors (22 with N-myc amplification and seven without the amplification) appeared to have a large terminal deletion including the proximal region of D1S214. Seven tumors (CC-4, CC-5, CC-14, CC-16, CC-20, CC-21 and CC-22) had small terminal deletion and another tumor (CC-2) had larger terminal deletion distal to D1S80. Intriguingly, a stage 1 tumor (CC-1) and a stage 4 s tumor (CC-9) had a small interstitial deletion at the p73 site with retention of D1Z2 and D1S80 loci. One tumor (CC-6) had no LOH for D1Z2, but had allelic loss of p73. One tumor (CC-7) had a possible interstitial deletion proximal to D1S80 which was the distal end marker of the constitutional deletion case reported by White et al. (1995). Although the SRO at distal 1p in NBL is currently assumed to be between D1S244 and D1S214 (NB1 locus) which may span 5 - 10 Mb (White et al., 1995; Martinsson et al., 1995; Amler et al., 1995), other regions of chromosome 1p32-pter (such as an NB2 locus (Schwab et al., 1996) in Figure 1) could also harbor candidate loci, because most of the aggressive NBLs with N-myc amplification usually have a very large allelic loss in this region (Ohtsu et al., 1997; Fong et al., 1992; Takeda et al., 1994; Schleiermacher et al., 1994; Caron et al., 1995). The chromosomal region for the putative NB2 gene(s) is still ambiguous. However, our present results suggest that the region between D1Z2 and p73, including p73 gene itself, could be a locus (named as a NB3 locus) for one of the NBL suppressor genes (Figure 1). Since most of the advanced NBLs, especially those with N-myc amplification, have large allelic losses at the distal 1p region, including the NB3 locus, the loss of the tumor suppressor in the locus may give advantage to the tumor progression, in accordance with the result of the p73 LOH.

It is also possible that p73 is localized between D1S80 and D1S214. In that case, p73 seems to be excluded from the consensus region of LOH on 1p36. However, the result reported by Amler et al. which defined the distal end of SRO as at D1S214, was obtained from a translocated and duplicated cell line (NGP) which has not been shown to be deleted for this region. This suggests that the generally accepted SRO is from D1S244 to D1S80, a region that likely includes p73. In addition, even in such case, the region between D1Z2 and D1S80 could still be a possible SRO based on the results in Figure 1.

We next examined levels of p73 expression in primary NBLs using reverse transcriptase (RT) - PCR procedures. The p73 mRNA message was detected in 46 of 134 (34%) primary tumors, while it was easily detectable in all 35 primary colon and breast cancers tested (Figure 2a). The results were similar to those found in cell lines as reported by Kaghad et al. (1997). There was no significant difference in p73 expression between favorable disease stages (1+2+4 s, signals detectable in 29 out of 84) and unfavorable disease stages (3+4, signals detectable in 17 out of 50) (²-test: P>0.1). The results obtained by RT - PCR with primers flanking exon 13, which is spliced out in p73 isoform, has revealed that both p73 and p73 transcripts were expressed with decreased levels of expression of the latter (Figure 2b). These suggest that expression of p73 is extremely low in primary NBLs. In addition, there was no correlation between p73 mRNA expression and its LOH (²-test: P>0.1).

To search for mutations of p73 in 140 primary NBLs, PCR-single strand conformation polymorphism (PCR - SSCP) (Mashiyama et al., 1991) and DNA sequencing was performed on tumor RNA and genomic DNA templates prepared from the paired tumorous tissues and peripheral blood leukocytes. We found two tumors (1.4%) with amino acid substitutions at the C-terminal region. The case 78 (3-years-old, stage 4, diploidy, and adrenal origin) had a mutation of Pro405Arg (CCGCGG) which was determined to be somatic (Figure 3). The case 119 (1-month-old, stage 3, diploidy+pentaploidy, and mediastinal origin) had a mutation of Pro425Leu (CCCCTC), which was germline (Figure 3). It is intriguing that both mutations are localized at the C-terminal region containing a proline repeat and a glutamine repeat, a possible transactivation regulatory domain. Our preliminary result indicate that this region is important for transcriptional activation, and both mutations have revealed a loss of function mutation (Takada et al., manuscript under submission). We found four polymorphisms with silent mutation in the p73 gene, as described elsewhere (Mihara et al., 1998). No mutation of p73 has been observed in cancers of prostate (Takahashi et al., 1998), lung (Nomoto et al., 1998; Mai et al., 1998), esophagus (Nimura et al., in press), colorectum (Sunahara et al., 1998), and liver (Mihara et al. in press).

To determine whether p73 is expressed monoallelically or biallelically in NBL, we subcloned and sequenced several cDNA fragments amplified from tumor RNA at polymorphic sites. In some tumors with p73 heterozygosity, the gene appeared to be expressed from both alleles (Figure 3b). However, the tumorous tissues contain variable amounts of stromal cell components with mRNA transcribed from constitutional DNA. The imprinting as well as the loss of imprinting at the chromosome 1p distal region in NBL is still unclear and should be studied further (Caron et al., 1995; Cheng et al., 1993).

Although our data suggest that p73 is expressed biallelically in neuroblastomas at low levels, the comparison with the corresponding normal tissues (infant's adrenal medulla or sympathetic ganglia) is impossible to do because of the difficulty to obtain them. If p73 expression is down-regulated in neuroblastomas independent of disease stage or allele-specific expression, the inhibitory mechanism of the p73 promoter/enhancer system may be related to the tumorigenesis at its early stage. On the other hand, if p73 expression is up-regulated compared with that in normal tissues with the monoallelic expression, activation of the p73 silenced allele could be one of the possible mechanisms, as Mai et al. (1998) suggested. Nevertheless, based on our data showing low frequency of p73 expression independent of the LOH and disease stage, it may be rather reasonable to hypothesize that there may be another tumor suppressor gene(s) at the distal region of 1p36.3 (NB3 locus in Figure 1).

As p73 can interact with p53 in yeast two-hybrid system (Kaghad et al., 1997), we examined p53 expression in those tumors. Intriguingly, p53 in NBL cell lines has been shown to be localized to the cytoplasm (Moll et al., 1995; Ostermeyer et al., 1996). Expression of p53 in 51 primary NBLs was examined by Northern blot analysis with expression of -actin for the normalization. The p53 transcripts were detectable in 36 (71%) tumors. There was no significant difference in p53 expression between stages 1+2+4s (22/27) and stages 3+4 (14/24) (²-test: P>0.05) (data not shown). Furthermore, as in previous reports (Vogan et al., 1993; Komuro et al., 1993), no p53 mutations (exons 4 to 11) were observed by PCR - SSCP in 140 primary NBLs.

In conclusion, we have found that frequent p73 LOH is associated with sporadic NBLs, N-myc amplification, and advanced stage tumors, suggesting that the allelic loss of p73 is a later event in tumorigenesis of NBL. The p73 gene may be localized at the consensus region for NBL suppressor at 1p36.3-pter. However, mutations in p73 were rare, although we found two loss of function mutations at the C-terminal region. Thus, p73 could still be a candidate NBL suppressor gene, but the infrequent mutation hardly support the idea that p73 is a tumor suppressor in a classic Knudson's manner.

Materials and methods

Tissue and blood specimens

Fresh, frozen tumorous tissues as well as corresponding peripheral blood samples were sent to the Division of Biochemistry, Chiba Cancer Center Research Institute, from various hospitals in Japan. Staging was according to the International Neuroblastoma Staging System (Brodeur et al., 1993). Tissues were store at -80°C until use. DNA and RNA were extracted as previously described (Nakagawara et al., 1993).

Southern and Northern blot analyses

N-myc amplification was measured by Southern analysis, as reported previously (Nakagawara et al., 1992). For p73 expression, 20 g of total RNA was subjected to electrophoresis in 1% formamide gels as described previously (Nakagawara et al., 1994). The gels were soaked for 30 - 60 min in 20´SSC and transferred to Hybond N⁺ blotting membrane (Amersham). The membranes were hybridized with an -³²P-dCTP-labeled human p73 probe (1299 bp PCR product). Washing was started at 65°C for 15 min in 2´SSC and 0.1% SDS, and then the salt concentration was decreased stepwise to 0.5´SSC. For p53 expression, Northern analysis was performed as described previously (Nakagawara et al., 1993).

RT - PCR assays

Five g of total RNA was converted to cDNA by using random primers (TAKARA) with Superscript II reverse transcriptase (GIBCO - BRL). One twentieth of the cDNA was used for PCR amplification. Oligonucleotide primers for detecting human p73 transcripts were p73-c6F (5'-TACCTTCAGGTGCGAGGC-3') and p73-c6R (5'-CCCAGGTTGGGTGTAGCT-3'). To detect the transcripts of p73 and p73, a primer pair flanking exon 13 was designed: p73/-F; 5'-CAGTCCATGGTCTCGGG-3' and p73/-R; 5'-GATGGTCATGCGGTACTGC-3'. The 20 l PCR reaction mixture contained 1 M each primer, 200 M dNTP, 1 ´Taq buffer, 1.5 mM MgCl₂ and 1.4 units Taq and Pwo DNA polymerase (Boehringer Mannheim). As a control, GAPDH primers (forward primer; 5'-ACCTGACCTGCCGTCTAGAA-3', reverse primer; 5'-TCCACCACCCTGTTGCTGTA-3') were also added to the mixture. The reaction was conducted for 35 cycles of 30 s at 96°C, 30 s at 55°C and 1 min at 72°C, using a Perkin-Elmer Cetus thermocycler. After gel electrophoresis, amplified DNA was visualized by UV illuminator.

P1 genomic clone and its sequencing

A human genomic P1 library (DMPC-HFF # 1 series B: Du Pont; Shepherd et al., 1994) was screened by a PCR-based strategy using 3'-end specific primers generated from p73 cDNA sequences. P1 DNAs were prepared by using an automated plasmid isolation apparatus (PI-100, Kurabo, Japan) and used for sequencing. To obtain intronic DNA sequences, vectorette libraries were constructed from EcoRI- and PvuII-digested P1 DNA fragments and subjected to PCR amplification using a cDNA primer and a vectorette specific primer as described (Ohira et al., 1996). The PCR products were sequenced using an ABI 377 automated DNA sequencer. Genomic DNA primers to amplify exon sequences were designed from intronic sequences (Nimura et al., in press).

PCR - SSCP and DNA sequencing

PCR - SSCP was performed as previously described (Mashiyama et al., 1991). In brief, PCR products amplified with primers for p73 genomic DNA and -[³²P]dCTP were diluted 1 : 10 with loading buffer, denatured at 98°C for 5 min and separated on 5% polyacrylamide gel with 5% glycerol at 200 V for 12 - 14 h at room temperature. After electrophoresis, gels were dried and exposed to X-ray film overnight to visualize migrated bands. To confirm the presence or absence of mutation, PCR products were subcloned into the pGEM-T easy vector (Promega) and subsequently sequenced on an ABI 377 DNA sequencer.

LOH analysis

Tumor and peripheral blood leukocyte DNA from 214 patients with neuroblastoma were analysed for LOH. N-myc amplification was observed in 34 tumors. For p73 LOH, 58 additional samples were analysed. The p73 microsatellite marker containing CT repeat was found in intron 9 using the sequence information of the p73 P1 genomic DNA described above. The primer sequences were as follows: forward primer; 5'-CCTCTTCCTCCCCTACCAAC-3', and reverse primer; 5'-TAGGCGACAGAGCAAGACG-3'. The PCR reaction mixture contained 1´Taq buffer, 1.5 mM MgCl₂, 100 M dNTP, 0.5 Ci [-³²P]dCTP, 2 M each primer, 0.25 units Taq polymerase (TAKARA), 90 ng TaqSTART (Clontech) and 2 ng template DNA. PCR conditions were as follows: initial denaturation at 96°C for 3 min, followed by 35 cycles of 94°C for 20 s, 62°C for 30 s, 72°C for 30 s and a final extension of 72°C for 9 min. Allelic loss analyses using the PCR reactions with D1S80 (VNTR marker) and D1S214 (microsatellite marker) were performed with primers published in the literature (White et al., 1995).

Interphase FISH analysis

FISH experiments were carried out on the fixed samples used for chromosome analysis (Christiansen et al., 1992). To determine the copy number of chromosome 1 and presence or absence of 1p deletions, we used repetitive DNA probes D1Z1 (pUC1.77) and D1Z2 (p1-79) specific for the pericentromeric region (1q12) and the subtelomeric region (1p36.33), respectively (Christiansen et al., 1992). D1Z1 was labeled with digoxigenin-11-dUTP, and D1Z2 with biotin-16-dUTP (Boehringer Mannheim) by nick translation. Interphase cells were stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI). Two-color FISH was performed as previously described (Kobayashi et al., 1996). One hundred interphase cells were counted in each tumor.

Acknowledgements

The authors thank Eiji Hanaoka for experimental support, and Makoto Kasama, Aiko Morohashi and Naoko Sugimitsu for technical assistance. The authors also thank the following institutes for providing surgical samples and clinical data: First Department of Surgery, Hokkaido University; Department of Pediatrics, National Sapporo Hospital; Department of Pediatric Surgery, Tohoku University; Department of Pediatric Surgery, Iwate Prefectural Central Hospital; Department of Surgery, Gunma Children's Medical Center; Department of Pediatric Surgery, Jichi Medical University; Department of Hematology and Oncology, Saitama Children's Medical Center; Department of Pediatric Surgery, Tsukuba University; Department of Pediatrics, Juntendo University; Department of Surgery, Kiyose Metropolitan Children's Hospital; Departments of Surgery and Pathology, Chiba Children's Hospital; Department of Pediatric Surgery, Matsudo Municipal Hospital; Department of Pediatric Surgery, Chiba University; Department of Pediatric Surgery, Kimitsu Central Hospital; Department of Pediatric Surgery, Niigata University; Department of Pediatric Surgery, Niigata Municipal Hospital; Department of Pediatrics, Aichi Medical University; Department of Pediatrics, Kyoto Prefectural Medical University; Department of Pediatric Surgery, Osaka City General Medical Center; Department of Pediatrics, Osaka Medical University; Tumor Board, Hyogo Children's Hospital; Department of Pediatric Surgery, Kumamoto University; Departments of Pediatrics and Pediatric Surgery, Kagoshima University. This work was supported in part by a grant-in-aid from the Ministry of Health and Welfare for a New Comprehensive 10-Year Strategy for Cancer Control, Japan, a grant from the Naito Foundation, and a grant from the Japanese Foundation for Multidisciplinary Treatment of Cancer. S.I. is an awardee of Research Resident Fellowship from the Foundation for the Promotion of Cancer Research.

Amler LC, Corvi R, Praml C, Savelyeva L, Le Paslier D and Schwab M. (1995). Oncogene 10, 1095-1101. MEDLINE

Brodeur GM, Pritchard J, Berthold F, Carlsen NL, Castel V, Castelberry RP, De Bernardi B, Evans AE, Favrot M, Hedborg F, Kaneko M, Kemshead J, Lampert F, Lee REJ, Look T, Pearson ADJ, Philip T, Roald B, Sawada T, Seeger RC, Tsuchida Y and Voute PA. (1993). J. Clin. Oncol. 11, 1466-1477. MEDLINE

Caron H, Peter M, van Sluis P, Speleman F, de Kraker J, Laureys G, Michon J, Brugieres L, Voute PA, Westerveld A, Slater R, Delattre O and Versteeg R. (1995). Hum. Mol. Genet. 4, 535-539. MEDLINE

Cheng JM, Hiemstra JL, Schneider SS, Naumova A, Cheung NK, Cohn SL, Diller L, Sapienza C and Brodeur GM. (1993). Nature Genet. 4, 191-194. MEDLINE

Christiansen H, Schestag J, Christiansen NM, Grzeschik K and Lampert F. (1992). Genes Chromosom. Cancer 5, 141-149. MEDLINE

Fong CT, White PS, Peterson K, Sapienza C, Cavenee WK, Kern SE, Vogelstein B, Cantor AB, Look AT and Brodeur GM. (1992). Cancer Res. 52, 1780-1785. MEDLINE

Jost CA, Marin MC and Kaelin Jr WG. (1997). Nature 389, 191-194. Article MEDLINE

Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Lelias JM, Dumont X, Ferrara P, McKeon F and Caput D. (1997). Cell 90, 809-819. MEDLINE

Kobayashi H, Satake N, Maseki N, Sakashita A and Kaneko Y. (1996). Br. J. Haematol. 94, 105-111. MEDLINE

Komuro H, Hayashi Y, Kawamura M, Hayashi K, Kaneko Y, Kamoshita S, Hanada R, Yamamoto K, Hongo T, Yamada M and Tsuchida Y. (1993). Cancer Res. 53, 5284-5288. MEDLINE

Mai M, Yokomizo A, Qian C, Yang P, Tindall DJ, Smith DI and Liu W. (1998). Cancer Res. 58, 2347-2349. MEDLINE

Martinsson T, Sjoberg R-M, Hedborg F and Kogner P. (1995). Cancer Res. 55, 5681-5686. MEDLINE

Mashiyama S, Murakami Y, Yoshimoto T, Sekiya T and Hayashi K. (1991). Oncogene 6, 1313-1318. MEDLINE

Mihara M, Nimura Y, Ichimiya S, Sakiyama S, Kajikawa S, Adachi W, Amano J and Nakagawara A. (1998). Br. J. Cancer in press.

Moll UM, LaQuaglia M, Benard J and Riou G. (1995). Proc. Natl. Acad. Sci. USA 92, 4407-4411. MEDLINE

Nakagawara A, Arima-Nakagawara M, Scavarda NJ, Azar CG, Cantor A and Brodeur GM. (1993). N. Engl. J. Med. 328, 847-854. . MEDLINE

Nakagawara A, Arima M, Azar CG, Scavarda NJ and Brodeur GM. (1992). Cancer Res. 52, 1364-1368. MEDLINE

Nakagawara A, Azar CG, Scavarda NJ and Brodeur GM. (1994). Mol. Cell. Biol. 14, 759-767. MEDLINE

Nimura Y, Mihara M, Ichimiya S, Sakiyama S, Seki N, Ohira M, Nomura N, Fujimori M, Adachi W, Amano J, He M, Ping Y-M and Nakagawara A. (1998). Int. J. Cancer in press.

Nomoto S, Haruki N, Kondo M, Konishi H, Takahashi T, Takahashi T and Takahashi T. (1998). Cancer Res. 58, 1380-1383. MEDLINE

Ohira M, Ichikawa H, Suzuki E, Iwaki M, Suzuki K, Saito-Ohara F, Ikeuchi T, Chumakov I, Tanahashi H, Tashiro K, Sakaki Y and Ohki M. (1996). Genomics 33, 65-74. MEDLINE

Ohtsu K, Hiyama E, Ichikawa T, Matsuura Y and Yokoyama T. (1997). Clin. Cancer Res. 3, 1221-1228. MEDLINE

Ostermeyer AG, Runko E, Winkfield B, Ahn B and Moll UM. (1996). Proc. Natl. Acad. Sci. USA 93, 15190-15194. Article MEDLINE

Schleiermacher G, Peter M, Michon J, Hugot JP, Vielh P, Zucker JM, Magdelenat H, Thomas G and Delattre O. (1994). Genes Chromosomes Cancer 10, 275-281. MEDLINE

Schwab M, Praml C and Amler LC. (1996). Genes Chrom. Cancer 16, 211-229. MEDLINE

Shepherd NS, Pfrogner BD, Coulby JN, Ackerman SL, Vaidyanathan G, Sauer RH, Balkenhol TC and Sternberg N. (1994). Proc. Natl. Acad. Sci. USA 91, 2629-2633. MEDLINE

Sunahara M, Ichimiya S, Nimura Y, Takada N, Sakiyama S, Sato Y, Todo S, Adachi W, Amano J and Nakagawara A. (1998). Int. J. Oncol. 13, 319-323. MEDLINE

Takahashi H, Ichimiya S, Nimura Y, Watanabe M, Furusato M, Wakui S, Yatani R, Aizawa S and Nakagawara A. (1998). Cancer Res. 58, 2076-2077. MEDLINE

Takeda O, Homma C, Maseki N, Sakurai M, Kanda N, Schwab M, Nakamura Y and Kaneko Y. (1994). Genes Chromosomes Cancer 10, 30-39. MEDLINE

Vogan K, Bernstein M, Leclerc JM, Brisson L, Brossard J, Brodeur GM, Pelletier J and Gros P. (1993). Cancer Res. 53, 5269-5273. MEDLINE

White PS, Maris JM, Beltinger C, Sulman E, Marshall HN, Fujimori M, Kaufman BA, Biegel JA, Allen C, Hilliard C, Valentine MB, Look AT, Enomoto H, Sakiyama S and Brodeur GM. (1995). Proc. Natl. Acad. Sci. USA 92, 5520-5524. MEDLINE

Figure 1 Loss of heterozygosity at the region of chromosome 1p36.3-pter found in 41 out of 214 primary NBLs. (a) The deletion map based on 41 tumors with LOH at 1p36.3-pter is demonstrated. open circle: absence of LOH, closed circle: presence of LOH, NI: not informative. The markers used are D1Z2, p73, D1S80 and D1S214. The region between D1Z2 and p73 including the p73 gene itself may be one of SROs for NBL suppressor gene (NB3 locus), which is distal to the previously identified SRO between D1S244 and D1S214 (NB1 locus; Refs. 12, 15, 16). (b) The results of LOH at D1Z2, p73 and D1S80 in tumors of CC-9 and CC-20. D1Z2 and D1Z1 are FISH markers at 1p36.3 and at 1q centromere, respectively. For p73 LOH, we used a microsatellite marker with CT repeat (see text). The PCR product sizes of D1S80 are 530 bp and 490 bp. In the CC-9 tumor with pentaploidy, D1Z2 and D1S80 are retained but the allelic loss of p73 is observed. In the CC-20 tumor with diploidy, D1Z2 is lost but p73 is retained

Figure 2 Expression of p73 and its isoforms, p73 and p73, in primary NBLs. (a) Expression of p73 mRNA was examined by RT - PCR procedure as described in Materials and methods. The representative data from the analysis of 158 NBL samples are shown. Three cases of each colorectal cancer and breast cancer were also assessed for p73 expression. The presence or absence of N-myc amplification is depicted as+or -, respectively. PCR primers were designed to amplify 272 bp cDNA fragment at exon 9 - 11. The PCR to amplify 247 bp DNA fragment of GAPDH was also performed by using the same cDNA samples. (b) Expression of p73 and p73 in primary NBLs was determined by RT - PCT method. The NBL tissues preferentially expressed p73 with significant levels of p73 transcript

Figure 3 A somatic and a germline mutation at the C-terminal region of p73 found in primary NBLs. (a) Results of PCR - SSCP analysis for p73 with primer pairs flanking exon 11. L and T mean DNA from peripheral blood leukocytes and from tumorous tissue, respectively. The common and aberrant bands are indicated with black and white arrows, respectively. The data show the presence of a somatic mutation in case 78 and a germline mutation in case 119 at exon 11. (b) The results of sequences of genomic DNA from both leukocytes (L) and tumorous tissue (T) and of cDNA from the tumor (T) in both case 78 and case 119. The number of wild type and mutant p73 sequences are shown on the right side. The results also show a somatic mutation P405R(CCGCGG) of p73 in the case 78 tumor and a germline mutation P425L(CCCCTC) in the case 119 tumor. (c) Heterozygosity for p73 in tumors of case 78 and case 119

Table 1 Table 1