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 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 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 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 using the GENSCAN program and the some 22q plays an important role in the acquisition of RT ± PCR method. The deduced SEZ6L was a aggressive phenotypes in lung cancers. transmembrane protein of 1024 amino acids with Two known tumor suppressor , NF2 and multiple domains involved in protein ± protein interaction SNF5/INI1, have been mapped to 22q12.2 and and . 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 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 . 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 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). In the course of cDNA cloning, we found that al., 1995; Herbst and Nicklin, 1997). Thus, we exons 12 ± 15 are alternatively spliced in human cells. considered SEZ6L as a candidate tumor suppressor That is, PCR products ampli®ed by a primer pair, gene, since the SEZ6L gene aberrations possibly play a encompassing exons 12 and 15, showed multiple bands role in human lung carcinogenesis through alterations in agarose electrophoresis (Figure 3b). We subcloned in cell ± cell interaction and/or intracellular signaling. PCR products into a plasmid vector by the TA cloning Thus, we characterized the SEZ6L gene in the present method and sequenced the inserts of 32 clones, which study. The mouse SEZ-6 gene consists of 17 exons and were randomly selected. The 32 clones represented nine encodes a transmembrane protein with an N-terminal di€erent types of alternatively spliced transcripts signal sequence, while SEZ6L protein deduced from (Figure 3b, panels 1a ± 4a). These di€ered in the the deposited SEZ6L cDNA lacked an N-terminal presence or absence of exons 13 and/or 14 and in the signal sequence, in spite of the presence of a putative choice of the splice acceptor sites for exon 15 among transmembrane region, indicating that the deposited three cryptic sites, which are 0, +3 and +6 bp of exon SEZ6L cDNA was partial. Therefore, we analysed the 15 (Figure 2a). Since another isoform (Figure 3b, panel genomic sequence corresponding to the deleted region 4b), which was not present in the 32 clones, had been in Lu24 (GenBankTM accession numbers AL080245, deposited in the database (GenBankTM accession Z98949, AL079300, AL022337, AL080273, AL023513 number AL035545.1), it was indicated that at least 10 and AL978460) by the GENSCAN gene prediction isoforms are transcribed from the SEZ6L gene, due to program. Then, in addition to the 16 exons already . The same reading frame was identi®ed, a candidate ®rst exon was detected in a CpG maintained in all the 10 isoforms identi®ed, although island where SacII and BssHII sites are clustered the absence of exons 13 and/or 14 caused the lack of a (Figure 1a). To con®rm that these 17 exons were SCR from SEZ6L protein. Based on the sizes of PCR transcribed as a transcript, we performed exon- products and the contents of isoforms among the 32 connection PCR using human brain cDNA as a subclones, types 1 and 2 were considered as being template with ®ve sets of primer pairs, which were major transcripts (Figure 3b). predicted to amplify ®ve overlapping cDNA fragments. PCR products of the expected sizes were ampli®ed by Genetic alterations and expression of the SEZ6L gene in PCR. Sequence analysis of the PCR products revealed human lung cancers that ampli®ed cDNA fragments overlapped each other and created a transcript of 3146 bp in size, which was Genomic PCR analysis revealed that exons 1 ± 10 of derived from the predicted 17 exons. The SEZ6L the SEZ6L gene were homozygously deleted, while cDNA contained an open reading frame (ORF) of exons 11 ± 17 were retained, in the Lu24 cell line 3072 bp encoding 1024 amino acids with a predicted (Figure 1a). To examine whether the SEZ6L gene is a Mw of 110 kD (Figure 2a, deposited in GenBankTM target of genetic alterations in human lung cancer cells, accession number AB041736). Thus, it was concluded other than in the Lu24 cell line, the remaining 45 lung that the SEZ6L gene consisted of 17 exons and was cancer cell lines and 46 surgical specimens of lung dispersed within a 210 kb region (Figure 1a). Exon ± cancer were examined for genetic alterations of the intron boundaries of all the exons agreed with the GT- SEZ6L gene. The entire coding region of the SEZ6L AG rule. gene encompassing 17 exons was ampli®ed by PCR The hydropathy pro®le of the deduced SEZ6L using 17 sets of intron-based primers. Genetic varia- protein obtained by the SOSUI program analysis tions in exon 1 and exons 3 ± 17 were examined by (Hirokawa et al., 1998) indicated the presence of two SSCP analysis followed by direct sequencing, while hydrophobic regions that probably function as a signal those in exon 2 were examined by direct sequencing. sequence and a transmembrane domain, respectively Thirteen di€erent types of nucleotide substitutions were (Figure 2a). In addition, there were 11 potential N- detected in lung cancer cell lines and surgical specimens linked glycosylation sites in a possible extracellular (Figure 4a, Table 1). Seven of them, G178C, G563A, domain. BLAST program analysis (Altschul et al., G664A, G1290T, A1381G, G1576A, G1889T, were 1997) revealed that the structure of SEZ6L protein was non-synonymous (associated with change) similar to that of the mouse SEZ-6 protein (Figure 2b), substitutions, while the remaining six consisted of ®ve and the level of amino acid identity between the human synonymous (silent) substitutions and an intronic SEZ6L protein and the mouse SEZ-6 protein was substitution. 46.1%. SEZ6L protein contained ®ve short consensus Among the seven non-synonymous substitutions, repeats (SCRs) and two N-terminal halves of the CUB G563A was detected as a homozygous substitution in (Complement subcomponents C1r/C1s, Uegf, Bmpl) a cell line, while G178C and G1889T, respectively, domain (Figure 2c). As in the case of mouse SEZ-6 were detected as heterozygous substitutions in a single (Herbst and Nicklin, 1997), SEZ6L has a putative cell line. These substitutions were not detected in short C-terminal cytoplasmic domain with the sequence surgical specimens and normal DNA samples of 50 of Asn-Pro-X-Tyr, which is a potential target for unrelated individuals (100 alleles), therefore, they were

Oncogene 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6254 a

bc

Figure 2 Characterization of the SEZ6L gene. (a) Full-length cDNA sequence and deduced amino acid sequence of SEZ6L. The putative signal peptide is thickly underlined, the possible transmembrane domain is underlined, and the potential cleavage site is indicated by an open arrowhead. Eleven potential N-linked glycosylation sites are boxed. Potential target for tyrosine phosphorylation site is double underlined. Closed arrowheads indicate positions of SEZ6L splice sites. Two other splice acceptor sites, which were functional for exon 15, are indicated by closed arrowheads in parentheses. (b) A schematic diagram showing the domain structure of human SEZ6L (upper) and mouse SEZ-6 (lower). The homology of each domain between SEZ6L and SEZ-6 is shown as a percentage. (c) Comparison of SCRs and N-terminal half of the CUB domains. Upper: SCRs. A consensus sequence is shown at the bottom of the ®gure and shaded in the comparison. The shaded boxes indicate amino acid residues that were present in at least eight out of 16 sequences at that position. Lower: N-terminal half of the CUB domains. The shaded boxes indicate amino acid residues that were present in at least ®ve out of 10 sequences at that position. CR 1; complement receptor 1, CR 2; complement receptor 2, BMP-1; bone morphogenetic protein 1, TSG-6; tumor necrosis factor-inducible protein, C1r; complement component C1r, C1s; complement component C1s

likely to be somatic mutations. However, since the were rare polymorphisms could not be excluded. The corresponding normal tissue DNAs for those cell lines remaining four non-synonymous substitutions, G664A, were not available, the possibility that the substitutions G1290T, A1381G and G1576A, were detected in both

Oncogene 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6255 a SEZ6L was expressed in the NCI-H526 and NCI-H23 cell lines with heterozygous SEZ6L missense muta- tions, while it was expressed neither in the EBC-1 cell line with a homozygous missense mutation nor in the Lu24 cell line with a homozygous deletion of the SEZ6L gene. SEZ6L expression was further examined in four primary lung tumors, a primary culture of bronchial epithelial cells, SAEC, a bronchial epithelial cell line, tpc16B, and a lung ®broblast cell line, WI-38 (Figure 4c). SEZ6L expression was detected in two of the four tumors. SEZ6L expression was detected in the cultured bronchial epithelial cells, but not in the b bronchial epithelial cell line and the lung ®broblast cell line.

Discussion

In spite of several lines of evidence that chromosome 22q contains tumor suppressor gene(s) for lung cancer, target regions for the 22q deletions in lung cancer have not been mapped to date. Therefore, we searched for homozygous deletions using 58 DNA markers dis- persed on chromosome 22q. A homozygous deletion causing the lack of the genomic segment of 428,131 bp of the 22q12.1 region was detected in a SCLC cell line, Lu24. Homozygous deletions are often observed at known tumor suppressor loci (Kohno and Yokota, 1999), therefore, it is possible that the deleted region in Lu24 cells contains a tumor suppressor gene. Thus, we further searched for genes mapped to the region of the homozygous deletion in Lu24 cells. We found that the homozygous deletion in Lu24 cells disrupted the SEZ6L gene. The structure of the SEZ6L gene has Figure 3 Expression of the SEZ6L gene. (a) SEZ6L expression in normal human tissues detected by RT ± PCR analysis. RT ± been partially determined by the e€orts of the human PCR ampli®cation was performed for 30 and 35 cycles using a set chromosome 22 sequencing project, and it was of primer pairs, SEZ6L-set6F and SEZ6L-set6R. GAPDH (25 indicated that SEZ6L is a homolog of the mouse cycles) was analysed to standardize the amounts of RNA. (b) SEZ-6 gene. Thus, we cloned and characterized the Alternative splicing of the SEZ6L gene. Upper: RT ± PCR analysis of the SEZ6L gene in lung cancer cell lines and normal full-length cDNA for the SEZ6L gene. It was lung and brain. PCR ampli®cation was performed using a set of con®rmed that this gene encodes a polypeptide primer pairs, SEZ6Lsp-s and SEZ6Lsp-as, whose sequences were homologous (46.1%) to the mouse SEZ-6 protein. derived from exons 12 and 15, respectively Since a partial cDNA sequence, HFBDF14, encoding a polypeptide showing 91.7% homology with mouse SEZ-6 protein, has been identi®ed (Shimizu-Nishikawa of the DNA samples from primary tumors and et al., 1995), it is likely that HFBDF14 is the human corresponding normal tissues. Therefore, they were counterpart of the SEZ-6 gene and that SEZ6L is a considered as genetic polymorphisms. The ®ve synon- homolog of the mouse SEZ-6 gene. Similar to mouse ymous substitutions were also considered as being SEZ-6, deduced human SEZ6L protein has a trans- genetic polymorphisms, although the presence of the membrane domain, ®ve SCRs, two N-terminal halves same substitutions in normal tissues was con®rmed in of the CUB domain, and a cytoplasmic region. The four of them. The intronic substitution, which had SCR repeat motif and the N-terminal half of the CUB occurred in a surgical specimen of SCLC, S181T, was a domain are present in the complement-related proteins, homozygous one base deletion at the 73*75 and both protein motifs are thought to be involved in nucleotide before the splice acceptor site of exon 4. signaling as well as structural associations. SEZ6L has The deletion was somatic, since it was not detected in a short C-terminal cytoplasmic domain with the the corresponding normal lung tissue (Figure 4a). sequence of Asn-Pro-X-Tyr. This motif is a potential Expression of the SEZ6L gene in human lung target for tyrosine phosphorylation by Src family cancer cell lines was assessed by RT ± PCR analysis. proteins. Phosphorylation of this motif is believed to SEZ6L expression was detected in 14 (30%) of 46 cause the association of the motif with signal lung cancer cell lines (Figure 4b, Table 2), and these transduction adaptor molecules, Shc (Src homologous 14 cell lines expressed multiple alternatively spliced and collagen) and/or IRS-1 (insulin receptor substrate- isoforms with di€erent ratios (Figure 3b). SEZ6L 1), and to relay the signals (Sudol, 1998). SEZ6L was expression was preferentially detected in SCLCs, and abundantly expressed in the brain, and also expressed the di€erence in the incidence of SEZ6L expression in a variety of human tissues, including lung epithelial between SCLC (9/14) and NSCLC (5/32) was cells. Therefore, SEZ6L protein is considered to be a statistically signi®cant (P=0.0018, Fisher's exact test). transmembrane protein functioning as an intracellular

Oncogene 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6256

Figure 4 Mutations and expression of the SEZ6L gene in human lung cancer. (a) Nucleotide substitutions of the SEZ6L gene. Nucleotide changes are apparent when compared to unrelated normal DNA samples (EBC1 and H23) or to a matched normal DNA sample (S181T). In EBC1 and S181T, histograms for sequencing in the reverse direction are shown. The mutated nucleotides are underlined. (b) SEZ6L expression in human lung cancer cell lines detected by RT ± PCR analysis. RT ± PCR ampli®cation was performed for 35 cycles using two sets of primer pairs, SEZ6L-set3F and SEZ6L-set3R, and SEZ6L-set6F and SEZ6L-set6R. GAPDH (25 cycles) was analysed to standardize the amount of RNA of each sample. SCC; small cell lung carcinoma, AdC; adenocarcinoma, AdSqC; adenosquamous carcinoma, SqC; squamous cell carcinoma, LCC; large cell carcinoma. (c) SEZ6L in cancerous and non-cancerous lung cells detected by RT ± PCR. RT ± PCR ampli®cation was performed for 40 cycles using a set of primer pair, SEZ6L-set6F and SEZ6L-set6R. GAPDH (25 cycles) was analysed to standardize the amount of RNA of each sample

Oncogene 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6257 Table 1 Mutations and polymorphisms of the SEZ6L gene in human lung cancers Allele frequency Nucleotide substitution Predicted effect Cell lines Primary tumors Unrelated individuals Cell lines with SEZ6L variantsd

Mutationsa G178C Glu60Gln 0.99/0.01 1.00/0.00 1.00/0.00 H526 G563A Arg188Gln 0.98/0.02 1.00/0.00 1.00/0.00 EBC1 G1889T Gly630Val 0.99/0.01 1.00/0.00 1.00/0.00 H23 Del C at 73 to 5 from exon 4 Unknown 1.00/0.00 0.98/0.02 ND Polymorphismsb T124C silent 0.96/0.04 0.96/0.04c 1.00/0.00 EBC1, H23 C561T silent 0.97/0.03 1.00/0.00 0.92/0.08 H526, N417, H69 G664A Gly222Arg 1.00/0.00 0.97/0.03c 1.00/0.00 G828A silent 0.97/0.03 1.00/0.00 0.92/0.08 H526, N417, H69 G1290T Met430lle 0.90/0.10 0.95/0.05c ND A1381G Ile461Val 1.00/0.00 0.99/0.01c ND A1419C silent 0.89/0.11 0.90/0.10c ND G1576A Glu526Lys 0.99/0.01 0.99/0.01c ND Ma26 C1713T silent 0.99/0.01 1.00/0.00 ND Lu135 aNon-synonymous substitutions, which were not detected in DNA from non-cancerous tissues, and a somatic deletion; bNon-synonymous substitutions, which were detected in DNA from non-cancerous tissues, and synonymous substitutions; cNucleotide substitutions were also detected in the correpsonding normal tissues; dThe cell lines with minor allele less than 10% are listed. The cell lines with SEZ6L expression by RT ± PCR are underlined. ND, Not done

Table 2 Incidence of SEZ6L expression in human lung cancer cell specimen was not available. These results indicated lines that the SEZ6L gene is genetically altered in a subset Type of tumor Expression (%) of lung cancer. Non-small cell lung carcinoma 5/32 (16)* Although the SEZ6L gene was expressed in human Adenocarcinoma 2/20 (10) lung tissue, SEZ6L expression was detected only in a Squamous cell carcinoma 1/5 (20) portion (14/46, 30%) of the lung cancer cell lines Large cell carcinoma 2/6 (33) examined. Loss of SEZ6L expression was also Adenosquamous carcinoma 0/1 (0) observed in the primary lung cancers. At present, the Small cell carcinoma 9/14 (64)* Total 14/46 (30) molecular mechanisms for the di€erential SEZ6L expression in lung cancer cells are unclear. Loss or *The di€erence in the incidence between non-small cell lung reduction of SEZ6L expression could be caused by carcinoma and small cell carcinoma was statistically signi®cant epigenetic changes, such as hypermethylation of the (P=0.0018, Fisher's exact test) CpG island, as in the cases of the VHL and p16 genes (Herman et al., 1994; Merlo et al., 1995; Hamada et al., 1998), or genetic alterations in the promoter region signal transducer via protein ± protein interactions in a and intron sequences. We preliminarily examined the variety of human cells. e€ect of 5-aza-2' deoxycytidine on SEZ6L expression We then examined the genetic and expression status in lung cancer cell lines without SEZ6L expression, of the SEZ6L gene in 46 lung cancer cell lines and 46 however, re-expression of the SEZ6L gene was not surgical specimens of lung cancer. In addition to the observed in these cell lines (data not shown). There- homozygous deletion in the Lu24 cell line, 13 di€erent fore, hypermethylation is unlikely to be a cause of the types of nucleotide substitutions were identi®ed, and loss of SEZ6L expression. It is also possible that the seven of them were non-synonymous. Three of the status of SEZ6L expression in lung cancer cell lines seven non-synonymous substitutions, which were re¯ects that in their precursor cells, since SEZ6L detected in three di€erent cell lines, NCI-H526, EBC1 expression was observed in one of the two cultured and NCI-H23, were considered as somatic mutations. lung epithelial cells. Di€erential expression was also Especially, a missense substitution at codon 630, observed between SCLC and NSCLC. However, since observed as heterozygous mutation in NCI-H23, precursor cells for lung cancer are still obscure at caused substitution of a relatively conserved amino present, more detailed analysis of SEZ6L expression in acid, glycine, in the second N-terminal half of the CUB cancerous and non-cancerous lung tissues will be domain, to valine. Thus, it is possible that the non- necessary to clarify the mechanism(s) for the di€er- synonymous substitutions of the SEZ6L gene have ential expression of SEZ6L in lung cancer cells. some e€ects on the physiological function of the The results of the present study indicated that SEZ6L protein. A homozygous 1 bp deletion in the SEZ6L is genetically altered in a small subset of lung polypyrimidine tract preceding exon 4 was also cancer in spite of the fact that LOH on 22q is observed detected in a case of primary SCLC. The polypyr- in a large portion of lung cancer. Therefore, gene(s) on imidine tract is critical for appropriate splicing 22q, other than SEZ6L, could be a major tumor (Roscigno et al., 1993; Norton, 1994), and mutations suppressor gene inactivated by 22q deletions. Interest- in the polypyrimidine tract often cause abnormal ingly, two other genes have been predicted to be splicing (Wilmotte et al., 1999; Magnusson et al., present in the homozygously deleted region of the Lu24 2000). Therefore, it is possible that the 1 bp deletion cell line by a computer-based analysis of the genomic also leads to abnormal splicing of the SEZ6L gene. sequence of this region (Dunham et al., 1999). However, we could not examine whether this mutation Therefore, we are currently isolating full-length cDNA caused abnormal splicing because mRNA from this clones for these two genes to investigate whether

Oncogene 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6258 alterations of these two genes are involved in human large cell carcinomas and two adenosquamous cell carcino- lung carcinogenesis. Recently, it was reported that the mas. The material to be analysed was selected by a EP300 gene at 22q13, encoding a histone acetyltrans- pathologist to ensure that the samples were macroscopically ferase that regulates transcription via chromatin entirely tumorous and chosen from an area devoid of necrotic remodeling, is mutated in a subset of lung cancer and tissue. Tumor and normal tissue were frozen and stored at 7708C until DNA extraction. High-molecular-weight DNA several other cancers (Gayther et al., 2000). Therefore, was prepared from the cell lines, tumors and adjacent non- it is likely that 22q contains several genes that can be cancerous tissues, as described previously (Kohno et al., targets for genetic alterations. Such a prediction is also 1999). High-molecular-weight DNA prepared from noncan- supported by the fact that LOH on 22q occurs cerous tissues of 50 unrelated patients with several cancers frequently in several types of human cancers and was also used in this study. Poly (A)+ RNA was prepared multiple regions have been mapped as common regions from 46 lung cancer cell lines, a primary culture of normal of LOH on 22q (Ueki et al., 1999; Castells et al., 2000). human small airway epithelial cells (SAEC) (Clonetics, Therefore, further investigations on the alterations of Walkersville, USA), a bronchial epithelial cell line (tpc16B) genes and regions on 22q are necessary for the and a lung ®broblast cell line (WI-38) by the Fast Track elucidation of the pathogenetic signi®cance of chromo- mRNA isolation kit (Invitrogen, Carlsbad, CA, USA). Total RNA was prepared from primary tumors obtained from four some 22q deletions in the development and/or NSCLC patients who were treated at the National Cancer progression of lung cancer and other cancers. Center Hospital, using TRIZOL Reagent (Life Technologies, Lastly, we cloned the breakpoint junctions of the Inc., Tokyo, Japan). Poly (A)+ RNA from human normal Lu24 deletion. Up to the present, breakpoint junctions tissues was purchased from CLONTECH Laboratories Japan of chromosomal deletions have been cloned from Ltd. (Tokyo, Japan). Randomly primed cDNAs were reverse- several human cancer cells (Canning and Dryja, 1989; transcribed from 0.5 mg mRNAs or 10 mg total RNAs by Pomykala et al., 1994; Kohno et al., 1996; Inoue et al., using SuperScriptII RNase H Reverse Transcriptase (Life 1997). DNA sequences surrounding the deletion break- Technologies, Inc.) according to the supplier's protocol. points often contain short direct repeats and the breakpoint junctions of the rearranged DNAs have Screening for homozygous deletions 1 ± 6 bp overlaps of germline sequences. Based on these results, it has been hypothesized that chromosomal Homozygous deletions were examined by PCR ampli®cation of STSs on chromosome 22q. STSs used for the screening of deletions occur through short homology-mediated homozygous deletions were 15 microsatellite markers, recombination after double strand breaks (Chen et D22S264, D22S301, D22S345, D22S310, D22S275, al., 1998; Ueda et al., 1999). However, there were no D22S280, D22S685, D22S283, IL2RB, D22S284, D22S276, direct repeats in the sequence surrounding the break- D22S270, D22S282, D22S274 and D22S526 (according to points for Lu24 deletion, and germline sequences were Genome Data Base) and 43 STSs developed, based on the not overlapped in the junction (Figure 1c). A similar genomic sequences of chromosome 22q (Dunham et al., feature was also observed in the breakpoint junction of 1999). The novel STSs were derived from exon 1 or 2 of 43 a chromosome 3p21.3 deletion in a case of breast genes (BID, TOP3b, D87003.1, BCRL4, BCR, BCRL6, MN1, cancer (Sekido et al., 1998). The present results suggest ESWR1, RFPL1, OSM, PES1, bK963H5.1, LIMK2, DRG1, that there are several molecular mechanisms generating RFPL2, RFPL3, TIMP3, MCM5, RBM9, bK212A2.1, MYH9, NCF4, IL2RB, PSCD4, SH3BP1, PLA2G6, genomic deletions. Information on the sequence at the dj449O17.1, DDX17, GTPBP, TAB1, GRAP2, ST13, RBX1, breakpoint junction of the Lu24 deletion would be EP300, TROB2, NHP2L1, TCF20, dJ437M21.2, dJ100N22.1, helpful to understand the mechanism underlying the ARHGAP8, SMC1L2, PPARA and CELSR1). Detailed occurrence of genomic deletions during carcinogenesis. information on these STSs can be obtained upon request. PCR was carried out by using 50 ng of genomic DNA as a template in a total volume of 20 ml reaction mixture containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM Materials and methods MgCl2 300 nM of each primer, 200 mM of each deoxynucleo- tide triphosphate, and 0.5 units of Taq DNA polymerase Samples (Amersham Pharmacia Biotech, Tokyo, Japan). PCR condi- Forty-six lung cancer cell lines consisting of 14 SCLCs and 32 tions were 958C for 1 min, 558Cor608C for 1 min and 728C NSCLCs were used in this study. The 14 SCLC cell lines were for 2 min for 30 cycles, followed by 728C for 10 min. PCR NCI-H69, NCI-H82, NCI-H209, NCI-H526, NCI-H774, products were fractioned on a 3% agarose gel and stained NCI-H841, N417, RERF-LCMA, Lu24, Lu134, Lu135, with ethidium bromide. Homozygous deletions were con- Lu139, SBC-5, and MS18, while the 32 NSCLCs consisted ®rmed by comparative multiplex PCR using the TP53 (exons of 20 adenocarcinomas (A427, A549, PC3, PC7, PC9, PC14, 7 ± 8) locus as a reference, as described previously (Kohno et RERF-LCOK, VMRC-LCD, ABC1, LCMS, NCI-H23, al., 1998). NCI-H322, NCI-H441, Ma1, Ma10, MA12, Ma17, Ma24, Ma26 and Ma29), ®ve squamous cell carcinomas (PC10, Cloning of the breakpoint junction of the homozygous deletion LC1-Sq, EBC1, NCI-H157 and NCI-H520), six large cell carcinomas (PC13, Lu99, Lu65, NCI-H1155, Ma2 and Sixteen known STSs (D22S926, D22S538, D22S650, Ma25), one adenosquamous cell carcinoma (NCI-H596) D22S538, D22S429, D22S1148, D22S574, D22S642, (Hamada et al., 1998). Forty-six surgical specimens (16 D22S46, D22S564, D22S351, D22S300, D22S560, D22S1, SCLCs and 30 NSCLCs) of lung tumors and adjacent D22S193, and a298yb5) and four novel STSs developed, noncancerous tissues were obtained from patients with based on the genomic sequences of chromosome 22q NSCLC and SCLC who were treated at the National Cancer (Dunham et al., 1999), were analysed for Lu24 in order to Center Hospital, Tokyo. Eleven primary tumors and ®ve narrow down the site of the centromeric and telomeric metastases were obtained from 16 patients with SCLC, while breakpoints of the Lu24 deletion. The primer pair, 15 primary lung tumors and 15 brain metastases were bk125H2sts#1-F, 5'-AGTTTCTTGTCCCTTTACATTGCT- obtained from 30 patients with NSCLC. NSCLCs consisted 3' and 11as, 5'-CCTAGAGGAAGGAAACAAGAC-3', was of 19 adenocarcinomas, ®ve squamous cell carcinomas, four used for the cloning of the 2092 bp fragment containing the

Oncogene 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6259 breakpoints of the Lu24 deletion. PCR fragments were sequences: 1s (5'-TCCGCGCCCCCTGCAG-3') and 1as puri®ed by using a QIA quick-spin PCR puri®cation kit (5'-TGCCTGAGTCGCCCTGGTCA-3') for exon 1, 2s (5'- (QIAGEN). DNA sequencing was performed bidirectionally TCCCCAAACTAACTGGTGTC-3') and 2as (5'-GTTTCA- by using Thermo Sequence dye terminator cycle sequencing GGATGGCTTCAGGA-3') for exon 2, 3s (5'-CATCCAA- pre-mix kits (Amersham Pharmacia Biotech) and the ABI GTTGTCTTTGGTGTC-3') and 3as (5'-CCTCTGTGAGG- 373S DNA Sequence System (PE Biosystems Japan, Tokyo, TACTCTCCT-3') for exon 3, 4s (5'-CCTCTCTGTCTGCT- Japan). The primer pair, bk125H2#2-F, 5'-CGGGGTTTT- TCTGC-3') and 4as (5'-GCAAACGCCTATGTTTCATCAG- ATACCCTCATG-3', and 11as, was used for the ampli®ca- 3') for exon 4, 5s (5'-GACTGAGTCTGCCTCTGCAT-3') tion of a 758 bp genomic DNA fragment containing the and 5as (5'-ATCAGGTTTGGTCCGGCTGA-3') for exon 5, Lu24 breakpoints to examine whether the same allele for the 6s (5'-ATTGCCCACCCACCCCTTTCT-3') and 6as (5'-GG- interstitial deletion was present in normal DNA samples of TTGGGTGTGGACCTTCAG-3') for exon 6, 7s (5'-CAG- 50 unrelated individuals. GAAGATCTGTCAGTGTC-3') and 7as (5'-CCACAAGA- GAAAGGGAAGCT-3') for exon 7, 8s (5'-CTGCATCATC- TGAACTGCTG-3') and 8as (5'-TGGGAAGAGCGTTGT- Isolation of full-length human SEZ6L cDNA GGATC-3') for exon 8, 9s (5'-TTAGCCGCTATGCCA- Genome DNA sequences obtained from GenbankTM CATTG-3') and 9as (5'-GGCCACCCATTTTAAACACT-3') (AL080245, Z98949, AL079300, AL022337, AL080273, for exon 9, 10s (5'-GCCCATTCTCTATTTCACATG-3') and AL023513 and AL978460) were analysed by the GENSCAN 10as (5'-CTAGAGGAAACCACCTTAAC-3') for exon 10, gene-prediction program (Burge and Karlin, 1997). Predicted 11s (5'-TTCCCCCATCTAAGACTGAC-3') and 11as (5'- exons were connected by exon-connection PCR using human CCTAGAGGAAGGAAACAAGAC-3') for exon 11, 12s (5'- brain cDNA as a template. RT ± PCR ampli®cation was GTCCTCTTGGTCTGGTTTCAT-3') and 12as (5'-AGTGA- performed at 958C for 1 min, 608C for 1 min and 728C for ATCTGCTTCTACCTAC-3') for exon 12, 13s (5'-CATCA- 2 min for 35 cycles, followed by 728C for 10 min using ®ve GAGCTCCTTCTGGCT-3') and 13as (5'-TGGACCCTGT- primer sets, SEZ6L-set1F (5'-TCCGCGCCCCCTGCAG-3') GTGGTGAGTA-3') for exon 13, 14s (5'-CTTCACCAAAG- and SEZ6L-set1R (5'-AGCACTTCCGCCGACTGTGA-3'), CATGGCATG-3') and 14as (5'-CTTTGGCAGTCTGAAG- SEZ6L-set2F (5'-ATCTCGCTGTTCCTCGCTCTG-3') and GTAG-3') for exon 14, 15s (5'-AAATGAGGACCTCACT- SEZ6L-set1R, SEZ6L-set3F (5'-TCTTCCCGAGGGAGAT- GGCT-3') and 15as (5'-TGGGTGCTTGGCAGTCATCT-3') GCTA-3') and SEZ6L-set3R (5'-AGTCGCTGGAGTCAA- for exon 15, 16s (5'-CTAGCTGGGGAGAAGTAACT-3') TGTAC-3'), SEZ6L-set4F (5'-CTGATGGACAAAGGTGA- and 16as (5'-TGGGAAGAGACGAGAGAGAC-3') for exon GAATG-3') and SEZ6L-set4R (5'-GCAGGTGAACTCCAC- 16 and 17s (5'-AGCGTTTGACAAATCCGTGC-3') and TATAGT-3') or SEZ6L-set5F (5'-ACTACATCCGACCC- 17as (5'-CACAAGTCCCCTTCTCAAGT-3') for exon 17. GACCTA-3') and SEZ6L-set5R (5'-TCCCCTTCTCAAG- Fifty ng of genomic DNA were suspended in a total volume TGTAGCT-3'). The presence of splicing variants in SEZ6L of 20 ml of PCR bu€er containing 10 mM Tris-HCl (pH 9.0), mRNA was examined by RT ± PCR analysis using a primer 50 mM KCl, 1.5 mM MgCl2, 300 nM each primer, 200 mM pair, SEZ6Lsp-s (5'-CACCATCCAATACACCTGC-3') and deoxynucleotide triphosphate, 1.5 mCi of [a-32P]dCTP (Amer- SEZ6Lsp-as (5'-TGAAGATAGCCAGGGCCATG-3'). Am- sham Pharmacia Biotech), and 0.5 unit of Taq DNA pli®ed cDNA fragments were either directly sequenced or polymerase (Amersham Pharmacia Biotech). PCR bu€er subcloned into the pGEM T-Easy vector (Promega, Madison for exon 2 did not contain [a-32P]dCTP. The PCR conditions WI) prior to sequencing. PCR fragments and plasmids were 958C for 1 min, 558C, 608Cor658C for 1 min, and containing PCR fragments were puri®ed and sequenced in 728C for 2 min for 30 cycles, followed by 728C for 10 min. both directions as described in Cloning of the breakpoint SSCP analysis was performed in a low pH bu€er system that junction of the homozygous deletion section. showed improved separation of long mutant fragments of up to 800 bp (Kukita et al., 1997). Gels were dried and exposed to Kodak XAR ®lms for 24 ± 48 h at 7808C. PCR products RT ± PCR analysis for exon 2 and products showing di€erent mobilities in the One microliter of cDNA conversion mixture was ampli®ed SSCP analysis were puri®ed and directly sequenced bidir- under the following conditions; 958C for 1 min, 608C for ectionally as described in Cloning of the breakpoint junction 1 min, 728C for 1.5 min for 30, 35 or 40 cycles (for SEZ6L) of the homozygous deletion section. and for 25 cycles (for GAPDH), followed by 728C for 10 min. Two sets of primers used in this study, SEZ6L-set3F and SEZ6L-set3R and, SEZ6L-set6F, 5'-TACATCACAAGATG- TCGCTA-3' and SEZ6L-set6R, 5'-TTAGATAGAAACCT- Acknowledgments CATACTCTCTG-3', were used for the ampli®cation of This work was supported in part by a Grant-in-Aid from 790 bp (nucleotides 99 ± 889) and 144 bp (nucleotides 2932 ± the Ministry of Health and Welfare for the 2nd-term 3075) SEZ6L cDNA fragments, and another set of primers, Comprehensive 10-year Strategy for Cancer Control and 5'-AAGGTCATCCATGACAAC-3' and 5'-CACCCTGTTG- Grants-in-Aid from the Ministry of Health and Welfare CTGTAGCCA-3', was used for the GAPDH gene (nucleo- and from the Ministry of Education, Science, Sports and tides 544 ± 1032 of GenbankTM M33197). The SEZ6L and Culture of Japan. We thank the following scientists for GAPDH primer pairs were designed to amplify cDNA providing cell lines: Dr Y Hayata of Tokyo Medical fragments encompassing two adjacent exons. The PCR College; Drs T Terasaki and S Hirohashi of the National products were electrophoresed on a 2.0% agarose gel and Cancer Research Institute, Japan; Dr M Takada of stained with ethidium-bromide. Izumisano Municipal Hospital; and Drs AF Gazder and JD Minna of the University of Texas, Southwestern Medical Center. Cell lines were also obtained from ATCC Mutation analysis of the SEZ6L gene and JCRB. We are grateful to Dr M Noguchi of the Seventeen exons of the SEZ6L gene were ampli®ed for SSCP University of Tsukuba for providing us with a human analysis (exons 1 and 3 ± 17) or direct sequencing (exon 2) bronchial epithelial cell line, tpc16B. M Nishioka is a with 17 sets of primers designed to amplify each exon recipient of the Research Resident Fellowship from the together with some nucleotides ¯anking the 5' and 3' intron Foundation for Promotion of Cancer Research.

Oncogene 22q12.1 homozygous deletion in human lung cancer M Nishioka et al 6260 References

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Oncogene