Molecular Cloning of a Candidate Tumor Suppressor Gene, DLC1, from Chromosome 3P21.31

[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 constructed pcDNA3.1(ϩ)/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 transfected 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

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 5Ј 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(ϩ) vector (Invitrogen), which carries a cytomegalovirus promoter as 594E17. Northern blot analysis using 594E17 as a probe revealed and a gene conferring resistance to neomycin (G418). We constructed two that 6.0- and 8.0-kb transcripts were expressed in all human tissues plasmid clones, one that was designed to express a sense transcript examined, including lung and kidney; prostate and testis seemed to [pcDNA3.1(ϩ)/DLC1SS], and the other to express an anti-sense transcript express this gene more abundantly than other tissues (data not shown). ϩ [pcDNA3.1( )/DLC1AS] that inserted the same cDNA in the opposite direc- The 6.0-kb transcript of the DLC1 gene was expressed more abun- tion. In addition, we constructed a plasmid clone designed to express the sense dantly (2–3-fold) than the 8.0-kb transcript. strand of an aberrant transcript, the predicted protein of which was truncated at exon 13 [pcDNA3.1(ϩ)/DLC1T]. Because the 594E17 PCR product was smaller than either of the The cancer cell lines used for colony-formation assays included human transcripts indicated by Northern analysis, we screened a human testis esophageal cancer line TE14, renal cancer lines RXF631L and ACHN, and cDNA library and identified cDNA sequences consisting of 5615 lung cancer lines LC319 and NCI-H23. Each of these five cell lines was plated nucleotides, including an open reading frame of 5265 nucleotides in 25-cm2 flasks (2 ϫ 105 cells/flask) and transfected at 24 h later with the encoding a 1755-amino acid peptide (Fig. 2). A comparison of plasmid [pcDNA3.1(ϩ)/DLC1SS] designed to express the sense strand of the genomic sequences with cDNA sequences revealed that the 6.0-kb ϩ DLC1 cDNA. As controls, we also transfected the pcDNA3.1( ) vector alone, transcript spanned ϳ59 kilobases of genomic DNA and consisted of the pcDNA3.1(ϩ)/DLC1AS, or the pcDNA3.1(ϩ)/DLC1T to each of the five 37 exons (nucleotide sequences of cDNA and exon-intron boundaries cell lines. Five ␮g of plasmid DNA and 25 ␮g of TransIT-LT1 (PanVera Corp.) were used for each transfection. Cells were diluted 1:8 after6hof are not shown here but are available from GenBank; accession num- transfection and cultured for 14 days in the presence of 400–800 ␮g/ml of bers are AB020522 and AB010443, respectively). geneticin (G418). The transfection experiments were independently repeated Analysis using the FASTA program (19) indicated that the Mr three times. 166,000 protein sequence showed no significant homology to any 1967

versely transcribed the mRNAs from the 22 cancer cell lines (14 esophageal, 6 lung, and 2 renal) as well as from tumors and corresponding normal tissues of the lung, esophagus, and kidney. We amplified the entire coding region of DLC1 cDNA in each case using six primer sets and sequenced the PCR products. In addition to the cDNA corresponding to the 6.0-kb transcript, we detected six alternatively spliced transcripts in normal lung, esophagus, and kidney tissues (Fig. 1D). Among them, three (DLC1-N2, -L1, and -L2) included an additional exon corresponding to part of intron 12 or 13 or all of intron 13; these transcripts had involved aberrant splice acceptor/donor sites. The largest transcript, DLC1-L1 contained an additional exon between exons 13 and 14, and its open reading frame was disrupted within this additional exon. The 8.0-kb transcript detected on Northern blots seemed to correspond to the DLC1-L1 by the following criteria: (a) about 2–3-fold difference in expression level between normal transcript (DLC1-N1) and DLC1-L1 observed by RT-PCR was compatible with that between 6.0-kb and 8.0-kb transcript; and (b) no cDNA clone corresponding to the larger transcript except for DLC1-L1 has been obtained by screening human cDNA libraries. The remaining three alternative transcripts (DLC1-S1, -S2, and -S3) lacked exons 11, 13, or 36, respectively. The proteins predicted by all of the aberrant transcripts except DLC1-N2 would be truncated close to the alternatively spliced sites. We subsequently examined these various transcripts in cancer cell lines and found that the patterns varied; some lines apparently ex- Fig. 2. Amino acid sequence of the DLC1 gene product. The cDNA and genomic pressed no normal transcript, and some showed a significant reduction sequence data will appear in the DDBJ, EMBL, and GenBank databases with accession or total absence of any DLC1 expression. Esophageal cancer-derived ء numbers AB020522 and AB010443, respectively. , stop codon. TE2, TE7, and TE13 expressed no DLC1 at all; TE15 lacked a normal product. TE1 and TE14 showed significant reduction of expression of normal DLC1 transcript in comparison to the other types of transcript. known proteins in the public database. However, the PROSITE data- Renal cancer-derived RXF631L did not express the DLC1 gene in any base (20) identified three possible cyclic AMP-dependent protein form, and ACHN showed only aberrant products (Fig. 4a). kinase phosphorylation sites, 24 protein kinase C phosphorylation We also examined DLC1 transcripts in 30 of the primary NSCLCs sites, 27 casein kinase II phosphorylation sites, 12 N-glycosylation in our panel and their adjacent normal lung tissues and found abnor- sites, and 15 N-myristoylation sites. Because this gene was isolated mal expression patterns in 11 of the tumor samples. Of those, eight from the commonly deleted region at 3p21.3 defined by the LOH produced no detectable DLC1 transcripts. In the remaining three study of lung cancers and its gene product was able to suppress cases, no normal transcript was detected, but aberrant transcripts were growth of some cancer cells (see below), we designated the novel (Fig. 4b). Furthermore, 3 of the 10 primary esophageal carcinomas gene DLC1. examined lacked normal products (data not shown). However, anal- Localization of the Gene Product in Mammalian Cells by Im- ysis of genomic sequences with SSCP and direct DNA sequencing of munofluorescence. To investigate the cellular localization of DLC1 lost exons in the cell lines or primary tumors that exhibited abnormal protein in mammalian cells, we transfected COS-7 cells with patterns of DLC1 transcripts revealed no alterations in the gene itself. pcDNA3.1(ϩ)/DLC1SS, a plasmid that contained c-myc epitope se- Analysis of 5؅ CpG Island Methylation of DLC1 Gene. To quences (LDEESILKQE) at the COOH-terminal of DLC1 protein. We investigate the possibility of hypermethylation-based inactivation of confirmed expression of DLC1 in transfected cells by immunoblot- the DLC1 gene, we examined 20 cancer materials that showed sig- ting. After transient expression of the DLC1SS/c-myc protein in nificant reduction of expression of normal DLC1 transcript for 5Ј CpG COS-7 cells, the proteins were extracted and separated by SDS- island methylation of the DLC1 gene by PCR-based assay. However, Ј PAGE. The Mr 166,000 DLC1SS/c-myc protein was detected by we detected no 5 CpG island methylation in the tumor samples Western blot analysis using anti-c-myc antibodies (Fig. 3a). Using the examined (data not shown). same antibodies, we detected the DLC1SS/c-myc protein mainly in Colony-Formation Assay. We performed colony-formation as- cytoplasm, when the COS-7 cells were transfected with the DLC1 says to investigate whether the DLC1 gene can act as a growth expression plasmid (Fig. 3b). suppressor in transfected cells. Among the five cell lines tested, the SSCP Analysis. Because the DLC1 gene is located within a region number of geneticin-resistant colonies was significantly reduced in that is deleted in many lung cancers, we considered that mutated dishes containing TE14 (18.3% in an average of three independent forms might contribute to the etiology of lung carcinoma. To inves- experiments), RXF631L (19.6%), ACHN (40.6%), or LC319 (36.2%) tigate this possibility, we performed SSCP analyses involving all cells that had been transfected with the sense-strand of cDNA corre- coding exons in 48 NSCLCs, including 31 that had shown LOH at the sponding to the normal transcript, in comparison to cells transfected DLC1 locus on 3p21.3. Apart from a few probable polymorphisms, with the mock vector or with plasmids designed to express anti-strand we detected no genetic alterations that should cause dysfunction of the cDNA or a sense-strand cDNA encoding a protein truncated at exon gene product. 13 (Fig. 5). However, no difference in the colony numbers was Comparative RT-PCR and cDNA Sequence Analysis of Tumor- observed when we transfected these plasmids to NCI-H23. NCI-H23 derived mRNAs. To look for abnormalities in DLC1 transcripts from expressed the functional transcript of the DLC1 gene relatively abun- the cancer materials that showed frequent LOH at 3p21.3, we re- dantly (Table 1). 1968

Fig. 3. a, Western blot analysis of extracts from COS7 cells transfected with the plasmid containing DLC1SS/c-myc. b, cytoplasmic localization of DLC1. Cells were stained with anti-c-myc antibodies and 4Ј,6-diamidino-2-phenylindole.

DISCUSSION of DLC1 has no significant homology to known proteins or domains, we were unable to speculate on the function of this protein. However, The DLC1 cDNA encodes a novel Mr 166,000 protein. Northern the PROSITE database identified a total of 54 putative phosphoryla- blot analysis detected 6.0- and 8.0-kb transcripts in several normal tion sites including 27 casein kinase (CSNK) II phosphorylation sites, human tissues, including lung and kidney, although prostate and testis 12 putative N-glycosylation sites, and 15 putative N-myristoylation seemed to express this gene more abundantly than other tissues. The sites. CSNK II is a ubiquitous, highly conserved enzyme consisting of minor 8.0-kb transcript detected seemed to correspond to the largest subunits ␣, ␣-prime, and ␤. A ubiquitous, messenger-independent transcript, DLC1-L1, which contained an additional exon between serine/threonine kinase, CSNK II is localized in both the cytoplasm exons 13 and 14, and its open reading frame was disrupted within this and the nucleus and functions as a protease (21–23). The kinase additional exon (Fig. 1). Because the predicted amino acid sequence domain of human CSNK I delta, a serine/threonine-specific protein 1969

Fig. 4. a, RT-PCR analysis of the DLC1 transcript in various carcinoma cell lines. Primers allowed amplification from exons 12–14; glycer- aldehyde-3-phosphate dehydrogenase (G3PDH) amplification served as a control for cDNA qual- ity. Arrowheads, cancer materials that produced no detectable normal transcripts. b, RT-PCR analysis of the DLC1 transcript in lung cancers and adjacent normal tissues. Primers allowed amplification from exons 12 to 19. T and N, samples of tumor tissue and adjacent normal tissue, respectively; PC, positive control.

kinase, may function in DNA metabolism through excision and reduction of the cDNA corresponding the normal transcript of DLC1 combinational repair (24). Using immunostaining, we determined that into several cancer cell lines caused significant suppression of growth DLC1 is localized only in cytoplasm. Therefore, the DLC1 protein suggest that aberrant patterns of DLC1 transcription may play impor- may act as a downstream gene in the serine/threonine kinase pathway. tant roles in carcinogenesis of those tissues. Lung cancers exhibit multiple genetic lesions including mutations The proportion of nonfunctional RNA transcripts (36%) in lung activating the dominant oncogenes in the myc and ras families, as well cancer materials was less than the frequency of LOH at 3p21.3. We as those inactivating the tumor suppressor genes. With respect to selected 48 tumor samples from many NSCLCs, which had been tumor suppressor genes in NSCLCs, LOH studies suggested possible analyzed for LOH and considered to be less contaminated with normal involvement of multiple tumor suppressor genes on chromosomal cells than the others; however, in some of these cases, the normal arms 1p, 1q, 3p, 5q21, 9p, 9q, 13q, 16q, and 17p that include the loci transcripts derived from admixed normal cells could still have been containing the p53, RB, or p16 genes (13). At present, several differ- detected in the RT-PCR amplification. Hence, the frequency of loss of ent regions of chromosome 3p are considered to contain tumor sup- the normal DLC1 transcripts in noncultured tumor samples is likely to pressor genes: 3p12, 3p14, 3p21.3, and 3p25. One candidate, the be underestimated. FHIT gene, was isolated from 3p14.2 (25) and another, the von Tumor suppressor genes can be inactivated by genetic or epigenetic Hippel-Lindau (VHL) disease gene, was isolated from 3p25 (26). To changes. Genetic changes may consist of: (a) mutations in regulatory our knowledge, several genes that have reduced expression but no regions that cause elimination or suppression of mRNA expression; mutations in small cell lung cancers or other tumors were identified on (b) deletions of part of or an entire gene; and (c) missense, nonsense, chromosome 3p21. Except for BAP1 (27), most of them (28–33), such frameshift, or splice-site mutations resulting in absence of a functional as PTPG, UBEIL, semaphorin family [III/F, IV, A(V)], UNP, and protein. The causes and mechanisms involved in epigenetic changes, ACY1, were not analyzed by detailed LOH studies or re-introducing such as abnormal methylation, deregulation of imprinting, or aberrant gene products into malignant cells. We previously observed LOH on splicing, are still not well understood. However, dysfunction of a gene 3p21.3 in 22 of 27 (81%) squamous cell carcinomas and 34 of 86 occasioned by epigenetic changes is often observed in cancer cells. (40%) adenocarcinomas of the lung (13). We have now identified and The abnormal transcription patterns of DLC1 in the tumors examined characterized a novel transcriptional unit, DLC1, lying within the here can be divided into two categories: (a) absence of normal commonly deleted region at 3p21.3 defined by that LOH study of lung transcript but presence of transcripts lacking one exon (exon 11 or 13) cancers (3, 4). The frequent appearance of aberrant transcripts of or containing an additional exon corresponding to part or all of intron DLC1 in lung, esophageal, and renal cancers and the fact that intro- 13, both mechanisms that disrupt the intact open reading frame; and 1970

Fig. 5. Colony-formation assay. The photo- graphs are of flasks containing cells from esophageal cancer cell line TE14 and renal cancer cell line RXF631L with pcDNA3.1(ϩ) vector, pcDNA3.1(ϩ)/DLC1T (truncated) cDNA, or pcDNA3.1(ϩ)/DLC1SS (full-length), after geneticin (G418) selection for 14 days.

(b) complete absence of DLC1 transcripts of any kind. In this study, which of several genes within a minimal region of LOH or homozy- we detected alteration of DLC1 expression in 20 of our primary gous deletion may function as a tumor suppressor gene, because most cancer materials; among them, 15 were classified as category (b) and of them did not fulfill the criteria of classical tumor suppressor. Haber five belonged to category (a), although no genetic alterations were and Harlow (35) drew attention to an increasing vagueness in the use detected in 5Ј noncoding region, exons, or flanking introns, and no of the term “tumor suppressor gene.” They suggested that the sim- hypermethylation of the 5Ј CpG island was detected in any of these plest, most inclusive, and cleanest genetic definition would be “genes cases. The mechanism that caused the loss of transcript(s) or aberrant that sustain loss-of-function mutations in the development of cancer.” splicing detected in our experiments remains unclear. However, we At the simplest level, mutations associated with cancer can be split suggest several possible explanations for category (b) alterations: (i) informatively into gain-of-function and loss-of-function mutations. mutations may have occurred within intronic sequences or in the 3Ј Additional studies might be required to confirm whether DLC1 meets noncoding region of DLC1;or(ii) genetic or epigenetic mechanisms this simpler definition. not yet identified might have inactivated this gene. p27Kip is a candidate tumor suppressor protein, because it blocks Most of the tumor suppressor genes described to date are inacti- cell proliferation and abnormally low levels of the p27 protein are vated by global loss of a region containing one allele (detected by frequently found in human carcinomas. However, only rare instances LOH) and subtle mutation in the other. Genes that are commonly of homozygous inactivating mutations of the p27 gene have been inactivated by such “two-hit” events have been termed class I tumor found in human tumors. Recently, Fero et al. (36) showed that both suppressors (34). Other genes, such as the protease inhibitor maspin, p27 nullizygous and p27 heterozygous mice were predisposed to have been proposed as tumor suppressors of class II on the basis of tumors in multiple tissues when challenged with ␥-irradiation or a low expression in tumors combined with a decrease in tumorigenicity chemical carcinogen. Molecular analyses of tumors in the p27 het- when the normal cDNA is transfected into cancer cell lines. On the erozygous mice show that the remaining wild-type allele is neither basis of these criteria, DLC1 belongs to class II. mutated nor silenced. Hence, p27 is haplo-insufficient for tumor Some candidate tumor suppressors were identified from many suppression. The assumption that null mutations in tumor suppressor chromosomal regions; however, it is especially difficult to determine genes are recessive excludes those genes that exhibit haplo-insuffi-

Table 1 Summary of results of colony-formation assaysa Number of colonies (%; Vector ϭ 100.0)

pcDNA3.1(ϩ) pcDNA3.1(ϩ)/DLC1AS pcDNA3.1(ϩ)/DLC1T pcDNA3.1(ϩ)/DLC1SS Cell lines (vector) (antisense cDNA) (truncated cDNA) (full-length cDNA) TE14 100.0 96.8 92.9 18.3 RXF631L 100.0 90.1 91.2 19.6 ACHN 100.0 104.2 102.3 40.6 LC319 100.0 105.2 98.1 36.2 NCI-H23 100.0 93.3 95.3 90.5 a Geneticin-resistant colonies were counted after 14-day selection. Each transfection was independently repeated three times. The fraction of colonies (in percent) in each flask compared with the vector-transfected cells is indicated. The vector transfectants contained an average of 50–200 colonies for each experiment. 1971

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Yataro Daigo, Tadashi Nishiwaki, Teru Kawasoe, et al.

Cancer Res 1999;59:1966-1972.

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