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Oncogene (1997) 14, 1093 ± 1098 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Duplication of an approximately 1.5 Mb DNA segment at chromosome 5q22 indicates the locus of a new tumour gene in nonpapillary renal cell carcinomas Chistiane Kenck, Peter Bugert, Monica Wilhelm and Gyula Kovacs Clinical Research Group Molecular Oncology, Department of Urology, Ruprecht-Karls-University of Heidelberg, Im Neuenheimer Feld 365, D-69120 Heidelberg, Germany Previous karyotyping showed an unbalanced translocation between chromosome 3p11.2-p13 and chromosome 5q22 leading to duplication of chromosome 5q22-qter region in nonpapillary renal cell carcinomas. In order to determine the breakpoint at chromosome 5q22 at the molecular level, we have investigated 50 sporadic nonpapillary renal cell carcinomas from consecutive nephrectomies and 24 renal cell carcinomas obtained from two patients with von Hippel ± Lindau disease. We used seven DNA markers mapped to and around the APC and MCC genes to detect allelic imbalance. We observed a duplication of chromosome 5q sequences in 11 of 23 informative sporadic tumours and in 18 of 24 hereditary tumours. We determined a breakpoint cluster between the APC and MCC genes at chromosome 5q22. In addition we have found a partial duplication of the smallest overlapping region of about 1.5 Mb sequences including the MCC gene in seven tumours without visible alteration of chromosome 5q in the karyotype. We suggest that this DNA segment harbours a gene or gene cluster, the altered dosage of which is important for the growth of nonpapillary renal cell carcinomas. Keywords: kidney cancer; chromosome 5q22; duplication Introduction Mutation of one allele and deletion of the wild type allele is a well known mechanism that contributes to the inactivation of tumour suppressor genes. The loss of wild type alleles is indicated by gross chromosomal deletions and by loss of constitutional heterozygosity (LOH) detected by Southern hybridisation and polymorphic microsatellites in tumour cells. Constitutional and/or somatic chromosomal deletions and subsequent LOH analysis pinpointed loci of tumour suppressor genes, and resulted in the cloning of RB, WT1, APC, MCC, DCC and DPC4 genes. Assuming that both deletion and duplication of chromosomes, chromosomal segments and genes can be produced by the same genetic mechanism, such as nondisjunction and mitotic recombination, duplications are expected to occur at the same frequency as deletions. Trisomies or partial trisomies of speci®c chromosomes are frequent cytogenetic ®ndings in human cancer but duplications of chromosomal segments detected by Southern blots, Correspondence: G Kovacs Received 18 July 1996; revised 28 October 1996; accepted 28 October 1996 microsatellite assays or FISH were described in only a few cases (Amler et al., 1995; Corvi et al., 1995; Fujiwara et al., 1993; Kovacs and Kung, 1991; Tiainen et al., 1992). A possible explanation for this controversy is that the absence of hybridisation signal (deletion) is easier to detect than increase of signal intensity for one of the alleles (duplication). An increased signal intensity might easily be misread as a LOH in tumour DNA contaminated with normal DNA. Comprehensive chromosome analysis has identi®ed highly speci®c monosomies and deletions, unbalanced translocations as well as trisomies or partial trisomies in distinct types of renal cell tumours (Kovacs, 1993). In nonpapillary renal cell carcinomas (RCC), which make up about 80% of parenchymal kidney tumours, deletions of chromosome 3p13-pter, 6q23-qter, 8p11pter, 9 and 14q22-qter regions imply the presence of tumour suppressor genes. Loss of chromosome 3p sequences occur in nearly 100% of sporadic nonpapillary RCCs as well as in renal tumours associated with von Hippel ± Lindau (VHL) disease (Zbar et al., 1987; Kovacs and Frisch, 1989; Kovacs et al., 1991). The VHL gene, a putative tumour suppressor gene, has already been cloned from the chromosome 3p25.3 region. Its inactivation by mutation occurs in about 50% of sporadic nonpapillary RCCs (Gnarra et al., 1994; Kenck 1996). Cytogenetic analysis of sporadic and hereditary nonpapillary RCCs has also revealed trisomy or partial trisomy of chromosome 5 in about 50% of cases with the smallest overlapping region of duplication for chromosome 5q22-qter region. An unbalanced translocation involving the chromosomal bands 5q22 and 3p11.2-p13 resulted in loss of chromosome 3p and trisomy of chromosome 5q22qter sequences in several cases (Kovacs and Frisch, 1989; Kovacs et al., 1991; Presti et al., 1991). In this paper we describe the identi®cation of a breakpoint cluster between the APC and MCC genes at chromosome 5q22 region in a cytogenetically wellcharacterized series of nonpapillary RCCs. We also demonstrate that an approximate 1.5 Mb DNA sequence distal to the critical breakpoint region is duplicated in tumours without visible cytogenetic alteration of chromosome 5q region. Results The chromosome 5q region is frequently duplicated in nonpapillary RCCs In order to estimate the frequency by which the chromosome 5q region is duplicated, we have studied Chromosome 5q22 duplication in RCC C Kenck et al 1094 Table 1 Allelic imbalance at the chromosome 5q22 region in nonpapillary RCCs Cytogenetic Tumour Allelic assignments with 5q probes alteration of CB86.3 FB54D L5.71-3 YN5.48-4 MC5.61 JO205HC Chromosome 5 D5S122 APC MCC D5S81 D5S372 D5S22 (a) sporadic nonpapillary RCC 295 +5q22-qter - 12 122 - 122 122 320 +5q22-qter 12 - - 122 - 122 344 +5q22-qter 12 - 12 12 - - 366 +5q22-qter 12 12 122 122 112 - 400 +5q22-qter - - - 122 122 - 407 +5q22-qter 12 12 - 122 122 122 284 ± 12 - 112 122 12 - 314 ± 12 12 112 12 - 12 319 ± - 12 - 112 - 12 324 ± - - 122 12 12 - 359 ± 12 12 122 122 12 - 379 ± 12 12 112 122 - 12 391 ± - - - 112 - - (b) VHL-associated nonpapillary RCC 606 +5q22-qter - 12 112 - ND 112 607 +5q22-qter - 12 122 - ND 112 608 +5q22-qter - 12 122 - ND 122 623 +5q22-qter - 12 112 - ND 122 625 +5q22-qter - 12 112 - ND 122 628 +5q22-qter - 12 122 - ND 112 Alleles at each locus were designated 1 or 2 and refer to the larger and smaller restriction fragments, respectively. The designation 12 indicates that the constitutional heterozygosity was retained in the karotype with an allelic dosage of 1:1, whereas 112 or 122 indicates that either allele 1 or allele 2 is present in two copies in tumour cells, as estimated from visual and densitometric analysis of autoradiographs. The entry `-' indicates that both normal and tumour tissues were homozygous for this locus. ND, not determined matched normal and tumour DNAs from 50 sporadic and 24 VHL-associated nonpapillary RCCs by Southern hybridisation. A DNA probe for locus D5S22 at the distal end of chromosome 5q was used in this assay to detect allelic changes at critical chromosome 5q22qter segment. Twenty three out of 50 patients with sporadic nonpapillary RCCs were heterozygous for the locus D5S22 and 11 tumours from these patients showed a duplication of one allele at chromosome 5q. Twenty-four tumours obtained from two VHL patients were included in this study. Both individuals showed constitutional heterozygosity at locus D5S22. Eighteen of the 24 small nonpapillary RCCs showed a duplication for one allele at the distal region of chromosome 5q. We did not detected deletion of chromosome 5q sequences among 74 nonpapillary RCCs. Recombination breakpoint cluster between the APC and MCC genes Previous karyotyping localised the most common breakpoint to the 5q22 band in nonpapillary RCCs (Kovacs and Frisch, 1989; Kovacs et al., 1991; Presti et al., 1991). In order to re®ne the breakpoints at the molecular level, we have analysed the matched normal and tumour DNAs by markers recognizing polymorphisms at the chromosome 5q22 band including those known for the APC and MCC genes (Table 1). All tumours with full trisomy of chromosome 5 or partial trisomy of chromosome 5q11-qter or 5q15-qter segments in the karyotype showed a duplication of DNA sequences for all informative loci at the chromosome 5q22 band (data not shown). Among tumour samples used for this approach, six cases (295, 320, 344, 366, 400, 407) with an unbalanced translocation and partial duplication of the chromosome 5q22-qter segment in karyotype are of particular interest (Table 1a). The duplication of one allele of the MCC gene and/or one allele at loci D5S81, D5S372 and D5S22 has been seen in ®ve out of six tumours. Both alleles of the APC gene as well as of the D5S122 locus were retained in all informative cases without any sign of allelic duplication. One of the VHL patients with multiple bilateral tumours showed constitutional heterozygosity at APC and MCC loci. Six out of nine tumours showed duplication of one allele of the MCC gene (Table 1b, and Figure 1a), whereas all tumours retained constitutional heterozygosity without duplication of DNA sequences at the APC gene. Tumours 606, 607 and 608 were obtained from the left kidney, tumours 623, 625 and 628 from the right kidney of one patient. Dierent alleles of the MCC gene were duplicated in tumours even from the same kidney. All six tumours showed an unbalanced translocation 3p13;5q22 in karyotype by previous cytogenetic analysis (Kovacs et al., 1991). Duplication of the smallest overlapping region of approximate 1.5 Mb DNA sequences involving the MCC gene While searching for allelic imbalance at chromosome 5q22, we have detected a duplication of DNA sequences in seven sporadic nonpapillary RCCs (284, 314, 319, 324, 359, 379 and 391), which by karyotyping did not show any structural or numerical alterations of chromosome 5 (Table 1a). An increased intensity of the autoradiographic signal resulting in an allelic dosage of 2:1 was detected for one allele of MCC gene in each informative case and for one allele of locus D5S81 in ®ve out of seven cases. The constitutional hetero- Chromosome 5q22 duplication in RCC C Kenck et al 1095 324 N T 608 607 606 628 627 625 N 623 a —1 —2 379 324 T N #3 Figure 2 Result of PCR-ampli®cation of microsatellites from the chromosome 3p region, which demonstrates the low amount of contaminating normal DNA in the tumour DNA samples. The same DNA samples were used as in the Southern hybridization experiments. The results from cases 324 and 379 are shown as representative examples b N 379 N T T —1 —2 YN5.48-4 —1 — 2 5.71-3 Figure 1 Representative autoradiographs showing the duplication of 5q sequences in RCCs. (a) MspI digested DNA from normal kidney (N) and multiple tumours (623, 625, 627 and 628 from the left kidney, 606, 607 and 608 from the right kidney) of a VHL patient was hybridized with L5.71-3 (D5S141). All but one tumour (627) showed an allelic inbalance. Note that dierent alleles were duplicated even in tumours obtained from the same kidney. (b) MspI digested DNA from normal kidney (N) and from sporadic nonpapillary RCCs (T) with two apparently normal chromosome 5 (cases 324 and 379) were hybridized simultaneously with DNA probes YN5.48-4 and L5.71-3. The constitutional heterozygosity was retained in both tumours. In tumour 324, L5.71-3 revealed a duplication of allel 2, whereas the allelic dosage at locus D5S81 (YN5.48-4) remained 1:1 in tumour cells. Tumour 379 showed the duplication of one allele with both probes. The loading of the amount of normal and tumour DNA was controlled by ethidium bromide staining of the gels before blotting. The degree of contamination of tumour DNA with normal nucleic acid was controlled by ampli®cation of microsatellites (see Figure 2) zygosity at all other informative loci proximal (APC and locus D5S122) and distal (loci D5S372 and D5S22) to the duplicated segment was retained with allelic dosage of 1:1 as seen in normal tissue. The proximal breakpoint was localized between the APC and the MCC gene in tumours 314, 359 and 379. The distal breakpoint of the duplication was determined between loci D5S81 and D5S372 in tumours 284 and 359. Duplication of the MCC gene with retained constitutional heterozygosity and an allelic dosage of 1:1 at locus D5S81 was seen in tumours 314 and 324, indicating that a smaller DNA fragment is duplicated. Two examples are shown in Figure 1b. To estimate contamination of tumour DNA with nucleic acid from normal cells, the same DNA samples as used in the Southern hybridization experiments were analyzed by PCR-ampli®cation of polymorphic microsatellites from chromosome 3p region, which region is speci®cally deleted in this type of tumour. The amount Figure 3 Map of the chromosome 5q22 region with the approximate locations of the probes used in this study. The duplicated segment is indicated by a solid bar. The arrows symbolize the locations and directions of transcription of the APC and MCC genes of contaminating normal DNA was low in each tumour DNA and representative results from two cases are shown in Figure 2. To estimate the size of the smallest duplicated region, we used two additional DNA polymorphisms in order to determine the distal end of duplication in these tumours. Both DNA probes, which recognize loci D5S1170 and D5S135 between D5S421 and D5S81, revealed a duplication of one allele in both tumours. Therefore, the telomeric end of the smallest overlapping duplication lies between the loci D5S81 and D5S135 (Figure 3). The distance between the APC gene and locus D5S81 is estimated to be about 2 Mb. Thus, our study determined the smallest overlapping region of duplication to be an approximate 1.5 Mb DNA segment including the MCC gene at chromosome 5q22 in nonpapillary RCCs. Chromosome 5q22 duplication in RCC C Kenck et al 1096 FISH con®rms the site of breakpoint and the duplication of a chromosome 5q22 segment A duplication of chromosome 5q22 sequences containing the MCC gene was detected by two-colour FISH in interphase cells. Tumour 359 showed a karyotype 43,X,7Y,73,78,712,714,+der(8)t(8;14)(p11.2; q11.2),+der(12)t(3; 12)(q13.2; q24.1), i.e. two apparently normal chromosomes 5 and two copies of the long arm of chromosome 8. Two-colour FISH revealed one, two, three and four copies of the C-MYC gene in one, 15, one and three cells, respectively, and two, three, four, ®ve and six copies of the MCC gene in two, 12, one, four and one interphase nuclei, respectively, suggesting a duplication of a chromosome 5q22 segment (Figure 4a). Tumour 391 had the a karyotype 46,XY,73,+7,79,+12,714,+16 without visible structural alteration at chromosome 51. In twocolour FISH the BAC clone 83c11 harbouring the MCC gene revealed two, three, ®ve and six signals, in two, 13, three and two cells, respectively, whereas the BAC clone 6e10 containing the APC gene showed two, three, four, ®ve and six signals in three, 12, one, two and two cells, respectively, including one weak signal (Figure 4b). This demonstrates that DNA segments including the MCC gene are duplicated, and also suggest that the breakpoint might be at the telomeric end of the BAC clone 6e10, downstream of the APC gene. The MCC gene is not rearranged in nonpapillary RCCs We have localized a breakpoint cluster a chromosome 5q22 between the APC and MCC genes which bracket an approximate 150 kb DNA segment. The two genes are positioned head to head along chromosome 5q. As we used the DNA probe L5.71-3, which encompasses the ®rst two exons of the MCC gene (nt 133 ± 1918) to detect an allelic duplication, we cannot exclude a rearrangement within the 3' region. Therefore, we have hybridized the 3' end of the MCC cDNA (probe MCC 40cI, nt 1634 ± 3969) to normal and tumour DNA digested with EcoRI. We did not ®nd any dierences in autoradiographic signals between paired normal and tumour samples. Therefore, a rearrangement within the MCC gene in nonpapillary RCCs showing the t(3;5) could be excluded with a high probability. Discussion b Figure 4 (a) Two-colour FISH on an interphase nucleus of tumour 359 shows two copies of the C-MYC (green spots) and three copies of the MCC gene (red spots). (b) Two-colour FISH on an interphase nucleus from tumour 391 is shown. Probe 83c11 (green spots) recognizes three copies of the MCC gene whereas probe 6e10 (red spots) detects only two copies of the APC gene and a small overlapping signal with probe 83c11 at the third copy of the MCC locus Previous cytogenetic studies showed that an unbalanced translocation between chromosomes 3q and 5q leads to loss of chromosome 3p13-pter and duplication of chromosome 5q22-qter sequences (Kovacs and Frisch, 1989; Presti et al., 1991; Kovacs et al., 1991). This cytogenetic ®nding was explained as a result of nonhomologous mitotic recombination between chromosome 3p11.2-13 and 5q22 bands (Kovacs and Kung, 1991). We present evidence in this study that the site of mitotic recombination at the molecular level is restricted to an approximate 150 kb DNA fragment between the APC and MCC genes at chromosome 5q22. We have shown in this study that the same recombination site is involved in the duplication of the smallest overlapping region of approximately 1.5 Mb DNA fragment. Taking into account the results of earlier cytogenetic analysis as well as of the present study on the same tumour samples, the duplication of chromosome 5q sequences occurs in about 70% of nonpapillary RCCs. A duplication of 5q sequences was also detected in 17% of renal cell carcinomas by comparative genomic hybridization (Moch et al., 1996). Strangely, not duplication but deletion of chromosome 5q22 region has been suggested by other investigators. LOH at chromosome 5q22 has been described in 33% of renal cell tumours analysed by Southern hybridization (Morita et al., 1991). Applying microsatellites for allelotyping of nonpapillary RCCs, Foster et al. (1994), Crossey et al. (1994) and Trash-Bingham et al. (1995) found LOH at chromosome 5q each in one out of 35, nine and 19 nonpapillary RCCs, respectively. Chromosome 5q22 duplication in RCC C Kenck et al Chromosome 5q21-22 region in involved in genetic changes of other types of cancer as well. Several studies have demonstrated loss of one allele at chromosome 5q21-22 bands and mutation of the APC gene in the germ line of patients with familial adenomatous polyposis coli and also in sporadic colon cancer (Kinzler et al., 1991; Groden et al., 1991; Nishisho et al., 1991). Apart from colorectal cancers, frequent LOH in the APC/MCC gene region has been found in tumours of the liver (Fujimori et al., 1991), oesophagus (Boynton et al., 1992) and lung (Ashton-Rickardt et al., 1991; D'Amico et al., 1992; Fong et al., 1995). Hosoe et al. (1994) determined the most common region of deletions at loci D5S141 and D5S81, which is approximately the same region found to be duplicated in nonpapillary RCCs in this study. No mutations of the APC and MCC genes have been detected neither in the aforementioned tumours nor in RCCs (Horii et al., 1992), which suggest that another tumour gene is located in this region. The eect of deletion in one type of cancer and duplication in another type is probably dependent on gene dosage for one or more critical genes within the 1.5 Mb region. It is also possible that the same gene or genes are aected by duplication and deletion at chromosome 5q22 in nonpapillary RCCs as well as in the tumours of lung, liver and colon. Cloning this gene will contribute to a better genetic characterisation of nonpapillary RCCs and some other types of tumours as well. Materials and methods Tumour samples and DNA extraction Fifty sporadic nonpapillary RCCs were obtained after nephrectomy from the Department of Urology, at the Medical School of Hannover. Nine nonpapillary RCCs obtained from one patients suering from von Hippel ± Lindau disease were obtained from the Department of Urology, at the Memorial Hospital, Worchester, MA, USA and 15 tumours from another VHL patient from the Department of Urology, at the University of Innsbruck, Austria. A homogeneous part of the tumour tissue was excised immediately after nephrectomy. One third of the specimen was used for cell culture and subsequent chromosome analysis, one third was immediately frozen in liquid nitrogen and stored at 7808C and the rest was ®xed in buered formaldehyd for reference histology. In the case of small tumours (2 ± 5 mm in diameter) from VHL patients, DNA was isolated from short term cultures of tumour cells. DNA was extracted from frozen tumour tissue or tumour cell pellet after proteinase K digestion, puri®ed with phenol and chloroform, precipitated with ethanol and dissolved in TE. DNA was isolated by the same method from normal kidney tissues. two independent observers. DNA probes used in this study were homologous to loci D5S122 (CB86.3), APC (FB54D), MCC (L5.71-3), D5S1170 (f5.12), D5S135 (EF5.44), D5S81 (YN5.48-4) and D5S22 (JO205HC). The cDNA clone FB54D includes nucleotides 6640 to 8954 of the APC gene. Microsatellite-PCR DNA samples used for Southern hybridization experiments were also analysed by microsatellite-PCR with polymorphic markers (D3S1300, D3S1766) from the chromosome 3p region. PCR reactions were performed in a 20 ml volume with 10 mmol/L Tris/HCl pH 8.3, 50 mmol/L Kcl, 1.5 mmol/L MgCl2, 0.1% bovine serum albumin, 200 mmol/L each of dATP, dCTP, dGTP and dTTP, 0.5% Tween 20, 2 pmol forward primer (5'-labelled with 1 mCi 32P), 2 pmol reverse primer, 0.5 U Taq polymerase (Promega), and 100 ng genomic DNA as template. PCR was carried out in a PTC100 thermal cycler (MJ Research Inc.) with a progam as follows: initial DNA denaturation for 2 min at 948C, 28 cycles of 948C for 40 s and 558C for 30 s and ®nal extension for 10 min at 728C. After ampli®cation 10 ml sequencing stop solution was added and the PCR-products were separated on 5% sequencing gels. The dried gels were exposed to X-ray ®lms for 6 h without using screens. FISH-analysis of tumour cells DNA-pools of a human BAC-library (Research Genetics) were screened by PCR using primers for ampli®cation of MCC (3'-end) and APC (exon 10). The BAC clones 83c11 and 6e10 were positive for MCC alone and for APC alone, respectively. For the use in FISH-experiments the BACs 83c11 and 6e10 were labelled with biotin-11-dUTP and digoxigenin-11-dUTP, respectively, using a nick translation kit (Boehringer Mannheim). In another experiment the BAC clone 83c11 was labelled with digoxigenin-11-dUTP and cosmid clone cos-myc72, encompassing the c-myc oncogene an chromosome 8 with biotin-11-dUTP. For double colour FISH 60 ng probe DNA were precipitated with 10 mg COT-1 DNA and 30 mg salmon testes DNA and resuspended in 11 ml hybridization mixture containing 50% formamid, 10% dextran sulphate, 26SSC, and 5 mM sodium phosphate. Hybridization of the probes was based on the standard Imagenetics (Naperville, Illinois) protocol with an extended incubation time over 3 days at 378C. The slides were then incubated ®rst with avidin-FITC (10mg / ml) for 30 min, then with biotinylated goat anti-avidin (5 mg/ml) and anti-digoxygenin-rhodamin (10 mg/ml) for 75 min and ®nally with avidin-FITC (10 mg/ml) for 30 min, each step at 378C. The nuclei were stained with 50 ng/ml DAPI for 8 min and mounted with 30 ml antifade solution. The results were visualized under a Leitz DMRBE ¯uorescens microscope equipped with a Photometrics NU200 CCD camera using IPLab Imagining Processing and Multiprobe software. Twenty metaphases were counted for hybridizations signals. Southern-blotting, hybridisation and DNA probes Eight mg of normal and tumour DNA were digested with appropriate restriction enzymes according to the manufacturer's instructions (Boehringer Mannheim) and fractionated by electrophoresis on 0.8 ± 1.0% agarose gels. After denaturation and neutralisation DNA was transferred to a charged nylon membrane (Hybond N+, Amersham). The DNA probes were labeled with [a 32P]dCTP by random primer extension. After prehybridization ®lters were hybridized with the labelled probes in the same solution. Autoradiographs were assessed for allelic imbalance by Acknowledgements This work was supported by the German Research Council and in part by the EMBO (short term fellowship to GK). The authors thank Sir Walter Bodmer for his support. The cDNA probes FB54D, MCC 40cI were kindly provided by Bert Vogelstein (The Johns Hopkins University), the L5.71-3, EF5.44, YN5.48-4 and MC5.61 probes by Yusuke Nakamura (Tokyo University) and the probe f5.12 by Lynn Jorde (University of Utah Health Sciences Center). 1097 Chromosome 5q22 duplication in RCC C Kenck et al 1098 References Ashton-Rickardt PG, Wyllie AH, Bird CC, Dunlop MG, Steel CM, Morris RG, Piris J, Romanowski P, Wood R, White R and Nakamura Y. (1991). 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