Molecular Cloning of a Candidate Tumor Suppressor Gene, DLC1, from Chromosome 3P21.31
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[CANCER RESEARCH 59, 1966–1972, April 15, 1999] Molecular Cloning of a Candidate Tumor Suppressor Gene, DLC1, from Chromosome 3p21.31 Yataro Daigo, Tadashi Nishiwaki, Teru Kawasoe, Mayumi Tamari, Eiju Tsuchiya, and Yusuke Nakamura2 Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108, Japan [Y. D., T. N., T. K., M. T., Y. N.], and Department of Pathology, Saitama Cancer Center Research Institute, Saitama, Japan [E. T.] ABSTRACT MATERIALS AND METHODS The short arm of chromosome 3 is thought to contain multiple tumor Cell Lines and Primary Tumor Samples. Fourteen human esophageal suppressor genes, because one copy of this chromosomal arm frequently is carcinoma cell lines [TE series: gifts from Dr. Tetsuro Nishihira, Tohoku missing in carcinomas that have arisen in a variety of tissues. We have University (Miyagi); Ref. 12], six lung cancer cell lines [LC319, a gift from isolated a novel gene encoding a 1755-amino acid polypeptide, through Dr. Takashi Takahashi, Aichi Cancer Center (Aichi); A549, NCI-H23, -H226, large-scale sequencing of genomic DNA at 3p21.3. Mutational analysis of -H460, -H522, gifts from Dr. Takao Yamori, Cancer Institute (Tokyo)], and this gene by reverse transcription-PCR revealed the lack of functional two renal cancer cell lines (RXF631L and ACHN, gifts from Dr. Takao transcripts and an increase of nonfunctional RNA transcripts in a signif- Yamori) were grown in monolayers in RPMI 1640 supplemented with 5–10% icant proportion (33%) of cancer cell lines and primary cancers (4 of 14 fetal bovine serum. esophageal cancer cell lines, 2 of 2 renal cancer cell lines, 11 of 30 primary Tumors and corresponding normal tissue samples were obtained from a total non-small cell lung cancers, and 3 of 10 primary squamous cell carcino- of 48 patients with NSCLCs and 10 patients with primary esophageal squa- mas of the esophagus). However, no alterations of the gene itself were mous cell carcinomas, during surgery at the Cancer Institute Hospital (Tokyo) detected in any of the cancers examined. Introduction of the cDNA or the Osaka Medical Center for Cancer and Cardiovascular Diseases (Osaka). significantly suppressed the growth of four different cancer cell lines, two Of the 48 lung cancers, 35 were adenocarcinomas and 13 were squamous cell of which produced no normal transcript on their own. No such effect carcinomas. Total RNA was extracted from each of the 22 cell lines, from 30 occurred when antisense cDNA, cDNA corresponding to an aberrant of the frozen paired lung tissues, and from all 10 paired esophageal specimens, transcript, or the vector DNA alone were transfected. These data suggest using TRIzol Reagent (Life Technologies, Inc.), according to the manufactur- that aberrant transcription of this gene, designated DLC1 (deleted in lung er’s protocol. Extraction of DNA from the cancer cell lines and primary tissue cancer 1), may be involved in carcinogenesis of the lung, esophagus, and samples was carried out as described previously (13). kidney. DNA Sequencing and Isolation of cDNA. Five cosmid clones (306, 308, 603, 602, and 594; Fig. 1), each of which contains part of the genomic DNA of chromosome 3p21.3 in YAC936c1, were completely sequenced by means of INTRODUCTION the shot-gun method detailed previously (6). We analyzed genomic DNA The short arm of chromosome 3 is thought to include multiple sequences from the target region with an exon-prediction computer program, GRAIL2 (14), and performed an exon-connection experiment (15) by RT-PCR tumor suppressor genes, because one allele of this chromosomal arm to investigate whether the predicted candidate exons were actually transcribed. often has been lost in carcinomas of various tissues. Toward isolation The RT-PCR technique was performed as described previously (7). Then we of the putative tumor suppressor gene(s), we earlier performed de- screened human testis cDNA libraries (nearly 1 million plaques) using the tailed deletion mapping of this chromosomal arm using dozens of exon-connected product as a probe and obtained a full-length cDNA. polymorphic DNA probes and a large number of primary cancer Northern Blot Analysis. Human multiple-tissue blots (Clonetech) were materials. That effort identified candidate regions at 3pter–p25, p22- hybridized with a fragment of DLC1 cDNA, labeled by random-oligonucle- p21.3, and p21.1-p14 (1–3). We subsequently found a homozygous otide priming. Prehybridization, hybridization, and washing were performed deletion at 3p22-p21.3 in a lung cancer cell line (4) and performed according to the supplier’s recommendations. The blots were autoradiographed large-scale genomic DNA sequencing of this 685-kb region because at and analyzed with a BAS 1000 image analyzer (FUJI). least one tumor suppressor gene was likely to be present there (5). All Immunocytochemical Analysis. To achieve c-myc-tagged DLC1, we con- structed pcDNA3.1(1)/DLC1SS that contained c-myc epitope sequences four genes identified in the homozygously deleted region were sub- (LDEESILKQE) at the COOH-terminal of the DLC1 protein and transfected to sequently excluded as candidates for tumor suppressor functions (6). COS-7 cells. Transiently transfected COS-7 cells replated on chamber slides However, because the homozygous deletion might have exerted a were fixed with PBS containing 4% paraformaldehyde, then rendered perme- positional effect on expression of genes in the close vicinity, we able with PBS containing 0.1% Triton X-100 for 3 min at 4°C. Cells were extended our DNA sequencing further and have been characterizing covered with blocking solution (2% BSA in PBS) for 30 min at room genomic structures within a 515-kb segment lying distal to the deleted temperature to block nonspecific antibody-binding sites. Then the cells were region (7–11). Here, we report identification of a possible candidate incubated with a mouse anti-c-myc antibody (diluted 1:800 in blocking solu- gene, DLC1,3 that showed aberrant splicing patterns in one-third of tion). Antibodies were stained with a goat anti-mouse secondary antibody the carcinomas of esophagus, lung, and kidney we examined. The conjugated to rhodamine and viewed with an ECLIPSE E800 microscope DLC1 cDNA exerted growth-suppressive activity in vitro. (Nikon). To confirm the expression of DLC1/c-myc-tagged protein in trans- fected cells, we also performed Western blotting, in a manner described previously (16). Received 12/4/98; accepted 2/16/99. Mutational Analysis. SSCP analysis was performed to screen tumors for The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with genetic alterations in the DLC1 gene. DNA samples extracted from 35 ade- 18 U.S.C. Section 1734 solely to indicate this fact. nocarcinomas and 13 squamous cell carcinomas of the lung were used as 1 This work was supported by the Japan Science and Technology Corporation and by templates. In 31 of the 48 cases, LOH was confirmed at 3p21.3 using either “Research for the Future” Program Grant 96L00102 from The Japan Society for the DNA marker D3S685 or F56-CA1, a microsatellite located within the DLC1 Promotion of Science. 32 2 To whom requests for reprints should be addressed, at Laboratory of Molecular gene. Primers to amplify each exon were end-labeled with [ P]ATP, and the Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, PCR products were analyzed by electrophoresis in 5% acrylamide gels con- 4-6-1 Shirokanedai, Minato, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: 81- taining 5% glycerol. 3-5449-5433; E-mail: [email protected]. Comparative RT-PCR was performed as described elsewhere (17). The 3 The abbreviations used are: DLC1, deleted in lung cancer 1; NSCLC, non-small cell lung cancer; RT-PCR, reverse transcription-PCR; SSCP, single-strand conformation poly- cDNAs obtained from all 22 cancer cell lines mentioned above, 30 of the lung morphism; LOH, loss of heterozygosity. cancers, all 10 esophageal cancers, and samples of normal tissue adjacent to 1966 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. MOLECULAR CLONING OF DLC1 Fig. 1. Detailed physical map of the 1200-kb sequence corresponding to CEPH YAC936c1 on chromosome 3p21.3, and organization of the DLC1 gene relative to the 3p21.3 region homozygously deleted in a lung cancer cell line. A, locations of the overlapping cosmid clones subjected to nucleotide sequencing. B, all genes identified in the region thus far. Arrows, transcriptional direction of each gene. C and D, exon-intron organization and alternative transcripts of the DLC1 gene. Numbered boxes, locations of exons. each primary tumor served as templates for the PCR in a thermal cycler RESULTS (Perkin-Elmer). Six primer sets were designed to amplify the entire coding region of DLC1 cDNA, from nucleotides 23 to 5373. Isolation of a cDNA Clone. GRAIL2 computer analysis of DNA Tumor materials that showed significant reduction of expression of normal sequences in the region covered by cosmid clone 594 (Fig. 1) pre- DLC1 were examined for 59 CpG island methylation of the DLC1 gene by dicted seven possible exons with “excellent” scores. To examine PCR-based assay. Genomic DNA digested with one of the methylation- whether these candidate exons were transcribed in human tissues, we sensitive restriction enzymes (AccII, CfoI, HaeII, or HapII) was PCR amplified synthesized oligonucleotides corresponding to the possible exon re- according to the methods described previously (18). A methylation-insensitive gions and performed exon-connection experiments. We confirmed by restriction enzyme, MspI, was used as a control enzyme. Colony-Formation Assay. Plasmids designed to express DLC1 were con- RT-PCR that the seven exon candidate segments were parts of the structed by cloning the entire coding region of DLC1 cDNA into the same transcript and temporarily designated the 800-bp PCR product pcDNA3.1(1) vector (Invitrogen), which carries a cytomegalovirus promoter as 594E17.