Identification of a 428-Kb Homozygously Deleted Region
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Oncogene (2000) 19, 6251 ± 6260 ã 2000 Nature Publishing Group All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Identi®cation of a 428-kb homozygously deleted region disrupting the SEZ6L gene at 22q12.1 in a lung cancer cell line Michiho Nishioka1,3, Takashi Kohno1, Mina Takahashi1, Toshiro Niki2, Tesshi Yamada2, Saburo Sone3 and Jun Yokota*,1 1Biology Division National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; 2Pathology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; 3Third Department of Internal Medicine, Tokushima University, School of Medicine, 3-18, Tokushima-shi Kuramoto-cho, Tokushima 770-8503, Japan Frequent allelic losses on chromosome 22q in small cell et al., 1997) and comparative genomic hybridization lung carcinomas (SCLCs) and advanced non-small cell (Levin et al., 1994), indicating the presence of tumor lung carcinomas indicate the presence of tumor suppressor gene(s) on this chromosome. We previously suppressor gene(s) on this chromosome arm. We detected reported that the incidence of LOH on 22q in a homozygous deletion at 22q12.1 in a SCLC cell line, advanced-stage NSCLC (460%) was signi®cantly Lu24. Cloning of the breakpoints of the Lu24 deletion higher than that in early-stage NSCLC (530%) revealed that the deletion was interstitial and 428, 131 bp (Shiseki et al., 1994, 1996). In addition, SCLC, the in size. The deleted region contained the SEZ6L (Seizure most aggressive histological subtype of lung cancer, 6-like) gene, whose structure had been partially due to its early and wide dissemination, showed determined by the chromosome 22 sequencing project. frequent (460%) LOH irrespective of clinical stages We determined the full length cDNA sequence for the (Kawanishi et al., 1997). Therefore, it is likely that SEZ6L gene based on the genomic sequence for the inactivation of tumor suppressor gene(s) on chromo- SEZ6L locus using the GENSCAN program and the some 22q plays an important role in the acquisition of RT ± PCR method. The deduced SEZ6L protein was a aggressive phenotypes in lung cancers. transmembrane protein of 1024 amino acids with Two known tumor suppressor genes, NF2 and multiple domains involved in protein ± protein interaction SNF5/INI1, have been mapped to 22q12.2 and and signal transduction. SEZ6L expression was detected 22q11.2, respectively. The NF2 gene, which encodes a in a variety of human tissues, including lung, while its protein similar to the ERM (ezrin, radixin, and expression was detected in 14 (30%) of 46 lung cancer moesin) family of proteins linking cytoskeletal compo- cell lines examined. Missense mutations were detected in nents with proteins in the cell membrane, was identi®ed three (7%) of the 46 cell lines, and a 1 bp deletion in the as the gene responsible for neuro®bromatosis type 2 polypyrimidine tract preceding exon 4 was detected in (NF2), an autosomal dominant disorder characterized one (2%) of 46 primary lung cancers. Therefore, it is by tumors of neural-crest origin (Rouleau et al., 1993; possible that genetic and/or epigenetic SEZ6L altera- Trofatter et al., 1993). Biallelic inactivation of the NF2 tions are involved in the development and/or progression gene has been detected in NF2-related tumors, in a subset of lung cancer, although functional analysis including schwannoma and meningioma (Ueki et al., of the SEZ6L gene as well as molecular analysis of other 1999). The hSNF5/INI1 gene, which encodes a member genes in the homozygously deleted region is necessary to of the chromatin-remodeling SWI/SNF multiprotein understand the pathogenetic signi®cance of 22q deletions complexes, was identi®ed as a gene frequently in human lung carcinogenesis. Oncogene (2000) 19, inactivated in malignant rhabdoid tumors by homo- 6251 ± 6260. zygous deletions and mutations (Versteege et al., 1998). However, we and others showed that genetic and/or Keywords: lung cancer; tumor suppressor gene; chro- epigenetic alterations of these two genes are infrequent mosomal deletion; chromosome 22q; SEZ6L in SCLC and NSCLC (Sekido et al., 1995; Manda et al., 2000). Therefore, the target gene(s) for 22q deletions in human lung cancer are still unknown. In Introduction addition, to our knowledge, common regions of chromosome 22q deletions have not been mapped in Frequent allelic losses on chromosome 22q in small cell human lung cancer. lung carcinoma (SCLC) and non-small cell lung In the present study, we searched for homozygous carcinoma (NSCLC) have been detected by several deletions in 46 lung cancer cell lines to de®ne tumor approaches, such as loss of heterozygosity (LOH) suppressor loci on chromosome 22q. Fifteen micro- analysis (Shiseki et al., 1994, 1996; Kawanishi et al., satellite markers and 43 STS (sequence tagged site) 1997; Virmani et al., 1998; Stanton et al., 2000), DNA markers, which were selected from the chromosome ®ngerprinting (Anami et al., 2000; Yamada et al., 22q sequence (Dunham et al., 1999), were used for the 2000), cytogenetic analysis (Testa et al., 1994; Mertens screening of homozygous deletions. A homozygous deletion at chromosome 22q12.1 was detected in a SCLC cell line, Lu24. The Lu24 deletion was *Correspondence: J Yokota interstitial and 428,131 bp in size. The deleted region Received 11 August 2000; revised 18 October 2000; accepted 19 did not include any well-known genes, but included the October 2000 SEZ6L gene and two putative genes, whose structure 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6252 had been partially determined or predicted by the The homozygous deletion at the D22S310 locus in the chromosome 22 sequencing project (Dunham et al., Lu24 cell line was con®rmed by multiplex PCR (Figure 1999). We determined the structure of the full-length 1a). SEZ6L cDNA based on the data obtained by the Proximal and distal boundaries of the homozygous GENSCAN gene prediction program analysis of the deletion in the Lu24 cell line were accurately mapped genomic sequence for the SEZ6L locus in conjunction by the analysis of an additional 20 STS markers. The with the RT ± PCR analysis. We then examined the proximal breakpoint was mapped between the genomic and expression status of the SEZ6L gene in bk125H2sts#1 and D22S429 loci, while the distal human lung cancer cells to investigate the role of one was mapped between exons 10 and 11 of the SEZ6L alterations in human lung carcinogenesis. SEZ6L gene (Figure 1a). Proximal and distal primers were designed to amplify the breakpoint junction of the deletion, and PCR was performed against genomic DNA from Lu24 cells and three normal lung tissues. A Results DNA fragment of approximately 2.0 kbp in size was ampli®ed from Lu24 DNA, but not from normal lung Detection and characterization of a homozygous deletion DNAs (Figure 1b). Comparison of the sequence of the in the Lu24 cell line PCR fragment with that of genomic DNA revealed Homozygous deletions on 22q were searched for in 46 that the Lu24 deletion was a simple interstitial one of lung cancer cell lines, consisting of 14 SCLCs and 32 428,131 bp from the chromosome 22q12.1 region NSCLCs, using 58 STS markers dispersed in the (Figure 1c). Since the same deletion was not observed 22q11.2-q13.3 region with intervals of 75 kb to in additional normal DNA samples of 50 unrelated 3.7 Mb. The STS markers consisted of 15 known individuals (100 alleles), the deletion was considered to microsatellite markers and 43 newly developed ones, be a somatic alteration rather than a genetic poly- which were derived from exons of 43 genes on 22q. The morphism. D22S310 locus mapped to 22q12.1 was not ampli®ed in the SCLC cell line Lu24, while the remaining 57 loci Cloning and characterization of the SEZ6L gene were ampli®ed in this cell line, indicating that Lu24 cells have a homozygous deletion of the region The sequence data of chromosome 22 (Dunham et al., containing the D22S310 locus. On the other hand, all 1999) indicated that the region deleted in the Lu24 cell the 58 loci were ampli®ed in the remaining 45 cell lines. line contains the SEZ6L gene consisting of 16 exons Figure 1 Homozygous deletion at 22q12.1 in the Lu24 cell line. (a) Physical map of the homozygously deleted region. Restriction sites and STS markers in this region are marked on the map. The homozygously deleted region in the Lu24 cell line is indicated by thick black bar. Lane 1; normal lung, lane 2; Lu24, lane 3; Lu135, S; SacII, B; BssHII. (b) PCR ampli®cation of the rearranged DNA containing the breakpoint junction of the interstitial deletion. The PCR products were fractionated on a 1% agarose gel, and the gel was stained with ethidium bromide. The size marker of DNA is indicated on the left. (c) Alignment of the germline sequences and the breakpoint junction sequence from Lu24 Oncogene 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6253 (GenBankTM accession number AB023144). In addition, tyrosine phosphorylation by the Src family of tyrosine two other genes have been predicted to be present in kinases (Figure 2a). this region by a computer-based analysis of the Northern blot hybridization analysis indicated that genomic sequence (Dunham et al., 1999). The SEZ6L SEZ6L transcripts of 7.5 kb in size were expressed in gene was identi®ed as a gene homologous to the mouse adult and fetal brains but not in other tissues including SEZ-6 gene (Shimizu-Nishikawa et al., 1995). The lung (data not shown). However, RT ± PCR analysis mouse SEZ-6 gene encodes a polypeptide with multiple indicated that the SEZ6L gene is expressed in diverse domains possibly involved in cell adhesion and human tissues, including adult and fetal lungs (Figure protein ± protein interaction (Shimizu-Nishikawa et 3a).