ICANCKR RESEARCH 58. 5176-5181, November 15, I

Hidewaki Nakagawa, Yoji Murata, Kumiko Koyama, Asao Fujiyama, Yasuo Miyoshi, Morito Monden, Tetsu Akiyama, and Yusuke Nakamura2

Division of Clinictil Genetics. Department of Genetics. Biomédical Research Center. Osaka Universitv Medical School. Suita City. Osaka 565 ¡H.N.. K. K., Y. M., Y. N.J: Department of Research. Institute for Microhidl Diseases. Osaka University, Osaka 565 ¡Y.M., T. A. I; Department of Genetics. National Institute of Genetics. Mishima City. Shizuttka ¡A.F.I; Department of Surgery 11, Osaka University Medicai School ¡H.N.. M.MJ; Laboratory of Molecular Medicine, Hitman Genome Center, Institute of Medical Science. The University of Tokyo, Minato-ku, Tokyo 108 [Y. NJ, Japan

ABSTRACT pressing transfected APC (9). and a series of heptad repeats in its NH2-terminal domain can form homo- or heterodimers through the We isolated a novel gene, !/'(/. that showed significant homology to formation of a-helical rods (10). or tumor suppressor the adenomatous polyposis coli (APC} . This novel genes often belong to families whose members show sequence simi gene, located on I9pl3.3, encodes a of 2303 amino larities with each other (11); for example, the mvc family of onco- acids that is expressed specifically in the brain. The predicted protein of APCL contains five copies of a 20-amino-acid motif (FXVEXTPXCFSRX- genes consists of c-, N-myc, and L-myc (12), and p2l/Ras genes SSLSSLS). Like APC, this domain of APCL was able to bind to ß-catenin constitute a small group within the large family of G- (13). and deplete the intracellular ß-catenin pool. A reporter-gene assay re Among the products of known tumor suppressor genes, Rb shows vealed that APCL could also regulate interaction of ß-cateninwith T cell- significant similarities to pl07 (14) and pl30 (15), the pl6 tumor specific , although less actively than APC. These re suppressor to pl5 (16), and to p73 (17). sults suggest that the APCL protein may be involved in the \ViilAVmgless With this point in mind, we searched for novel genes related to signal pathway, and the identification of a novel relative of APC may APC. Here we report isolation and mapping of a gene, termed APCL, provide new insights into the function of APC. that shows significant structural and functional similarities to APC.

INTRODUCTION MATERIALS AND METHODS The APC? tumor suppressor was isolated by positional cloning from chromosome 5c¡2i(1.2) when mutations were found in patients Screening of a Human Brain cDNA Library. We searched the EST carrying familial adenomatous polyposis. an autosomal dominant database for the DNA sequence similar to APC and found two ESTs (H50182 hereditary cancer syndrome characterized by numerous adenomatous and H50183) similar to the DNA sequence of APC. A lambda ZAPII human brain cDNA library (Stratagene) was screened by using as a probe a polyps in the colorectum (3). Because APC is mutated somatically in |'2P]dCTP-labeled reverse transcription-PCR product generated from brain two-thirds of sporadic colorectal carcinomas as well as in many cDNA according to the sequence of EST H50183. After hybridi/ation at 50°C adenomas, such events are considered to be crucial early steps in overnight, the membranes were washed in 2x SSC at room temperature and colorectal tumorigenesis (4). then twice for 15 min in 2 X SSC containing 0.1% SDS at 55°C.The cDNA The amino acid sequence of APC protein has little similarity to any clones obtained from the library screening were sequenced using a Perkin- other proteins that would yield clues to its biophysiological functions. Elmer-Cetus 377 automated DNA sequencer and the Dye-Terminator Cycle However, molecules that interact with separate domains of APC have Sequencing FS Ready Reaction (Perkin-Elmer) and assembled into a been identified. In particular, its interaction with ß-cateninis thought full-length cDNA. which we designated APCL. to be very important because APC protein depletes intracellular Northern Analysis. A multiple-tissue Northern blot (Clontech) was hy- ß-catenin when a domain of seven 20-amino-acid repeats in the bridi/.ed at 42°C with a 2-kb Noll fragment (Fig. 1C) of the assembled, full-length cDNA representing APCL. labeled with [12P]dCTP. The blot was middle portion of APC binds to this target protein (5). If normal APC is not present, or if the ß-catenin itself is mutated, ß-catenin is washed in 2x SSC at room temperature, then twice for 20 min in 0.1 x SSC containing 0.1% SDS at 55°C.The membrane was exposed to X-ray film for stabilized and accumulates within the cell (5). Furthermore, an excess 4 days at -80°C. of cytoplasmic ß-cateninenhances interaction with members of the Isolation of Genomic Clones. A human cosmid library equivalent to Tcf family; the ß-catenin-Tcfcomplex appears to stimulate the Wng/ approximately five genomes was screened with a 1-kb fragment of the new Wingless signal pathway (6, 7). cDNA. labeled with (3-P]dCTP. Two clones that hybridized to this probe With respect to other activities, the COOH terminus of the APC (94G06 and 37F12) were isolated, purified, and compared with the full-length protein interacts with hDLG (human homologue of the Drosophila cDNA to identify exon-intron junctions. These cosmids were used as probes discs large tumor suppressor), a process that may regulate for FISH. progression and neuronal function (8). Moreover, the basic region of Chromosomal Localization by FISH Analysis. FISH was performed as normal APC associates with microtubules in cells that are overex- described previously (18). Human were prepared by the thymidine synchronization/bromodeoxyuridine release technique for the delineation of G-banding patterns. Before hybridi/ation. metaphase cells were Reeeivcd 6/19/98; accepted 9/18/98. stained with Hoechst 33258 and irradiated with UV light. Cosmids 94G06 and 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 37F12 were labeled with biotin-16-dUTP (Boehringer) by nick translation and 18 U.S.C. Section 1734 solely to indicate this fact. hybridized to denatured metaphase chromosomes. Hybridization signals were 1This work was supported in part by a Grant-in-Aid from the Ministry of Education. detected with FITC-avidin (Boehringer). Precise localization of the signals was Science. Sports and Culture of Japan, and by "Research for the Future" Program Grant determined by visualization of the replication G-bands. 96LOOI02 of The Japan Society for the Promotion of Science. 3 To whom requests for reprints should be addressed, at Laboratory Molecular Med Binding of APCL with ß-Catenin in Vitro. Predicted amino acids 627- icine. Center. Institute of Medical Science. The University of Tokyo. 1666 of APCL were synthesized by in vitro transcription-translation in the 4-6-1 Shirokanedai, Minato. Tokyo 108-8639, Japan. Phone: 81-3-5449-5372: Fax: 81- presence of [35S]methionine using the TNT rabbit reticulocyte lysate system 3-5449-5433: E-mail: [email protected]. 1The abbreviations used are: APC. adenomatous polyposis coli; APCL. APC-like; Tcf. (Promega). GST-/3-catenin was generated by subcloning into pGEX5X-l T cell-specific transcription factor; EST, expressed sequence tag; FISH, fluorescence in (Pharmacia), a fragment of ß-catenincDNA encoding the armadillo repeats. situ hybridisation; GST. glulalhionc .V-lransferase; 0-gal. ß-galactosidase. expressing them in Escherichia coli, and isolating them by absorption to 5176

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Fig. 2. Schematic representation of the genomic structure of the APCL gene. The boxen represent exons, numbered 1-14. The coding sequence is represented hy 123 45 67 8 shading. 1kb glutathione-Sepharose. The immobili/ed GST fusion protein was mixed with from EST clones H50182 and H50183. We obtained four cDNA in v/'/w-translated proteins in buffer A (10 mM Tris-HCI (pH 7.5), 150 mM clones and assembled them. The assembled cDNA, termed APCL, NaCl. 1 mM EGTA, 1 HIMNaVO4, and 50 mM NaF] containing 0.1% Triton consisted of 6912 nucleotides including an 6909-bp open reading X-100 tor l h at 4°Cand then washed extensively with buffer A. Proteins frame encoding a protein of 2303 amino acids (predicted molecular adhering to the beads were analyzed by 8% SDS-PAGE. Immunofluorescent Detection of /¡-(alenili in SW480 Cells Expressing weight, M, 240,000). The NH2-terminal half of the predicted protein the APCL Construct. SW480 cells (3 x IO5cells/well) were cotransfected revealed a high degree of similarity to APC, but the COOH-terminal with 1 jig of an effector plasmid [pCDNA3. pCDNA3-FLAG-APCL (627- half showed no similarity to any known protein (Fig. \A). The heptad 1666). or pMKITNeo-APC (1-1864)] and 1 /ng of a pCMV-ß-gal plasmid by repeat domain of APC was well conserved in this novel protein (45% lipotection, using Lipofectamine (Life Technologies, Inc.). Cells were fixed identical), indicating that APCL is also likely to form homo- or 48 h after transfection in 4% formaldehyde and dehydrated with 100% cold methanol. After blocking in PBS containing 10% fetal bovine serum, ß-catenin heterodimers. The armadillo domain was also well conserved between and ß-galwere detected with anti-ß-catenin monoclonal antibody (Transduc- APC and APCL (76% identical). The middle portion of APCL con tion Laboratories, Lexington, KY) or anti-ß-galpolyclonal antibody (5'—»3', sisted of five copies of this 20-amino-acid motif (FXVEXTPXCFS- Inc.), respectively. For secondary antibodies, fluorescein-conjugated goat-anti- RXSSLSSLS; Fig. \B), and the seven copies of this motif present in rabbit antibody (Cappel) or rhodamine-conjugated goat-anti-mouse antibody APC form a domain known to interact with ß-catenin(5). However, (Cappel) was used at dilutions of 1:50. a 15-amino-acid motif of APC [EX(D/E)XPXNYSX(K/R)Y(D/E)E] Reporter-Gene Assays of APCL Expression in SW480 Colon Cancer that is also thought to associate with ß-catenin(19), and the S/TXV Cells. SW480 cells (5 X IO5 cells) were transfected with 2 /Ag of effector motif in the COOH-terminal end where APC interacts with hDLG (8), plasmid |pCDNA3, pCDNA3-APCL (627-1666), pMKITNeo or pMKITNeo- APC (1-1864)], 0.3 /ig of reporter plasmid (pFOPFLASH or pTOPFLASH). were not present in the novel protein (Fig. 1C). and 0.3 ng of internal control pRL-TK (Promega) by lipofection. using Genomic Structure, Chromosomal Localization, and Brain- Lipofectamine (Life Technologies. Inc.). After 24 h, cell extracts were assayed specific Expression of APCL. We isolated two genomic cosmid for lucifera.se activity according to the manufacturer's instructions. pTOP- clones (94G06 and 37F12) for APCL to determine its genomic struc FLASH is a reporter containing an optimi/ed Tcf-binding site; we used ture and chromosomal localization. This gene spanned a genomic pFOPFLASH, a control reponer containing a mutated site that does not bind region of —¿40kb,smaller than APC. However, like APC it contained Tcf, to measure nonspecific transcription (6). one large exon at the 3' end (Fig. 2); this last exon encoded 1685 amino acids. Using each of the two cosmid clones as a probe, we RESULTS performed FISH analysis and mapped APCL to chromosome 19p]3.3 Isolation and Characterization of a Novel Homologue of APC. (Fig. 3). To examine expression of this APC homologue in various We isolated a gene showing significant similarity to APC, a tumor normal human tissues, we performed Northern blot analysis. A tran suppressor gene responsible for familial adenomatous polyposis, by script larger than 10 kb was detected specifically and abundantly in searching the EST database for similar DNA sequences and subse the brain (Fig. 4). In contrast, APC is expressed ubiquitously, al quently screening u human brain cDNA library with probes derived though its expression level in the brain is especially high.

Fig. 3. Chromosomal location of APCL. The G- band pattern of the metaphase chromosomes indi cates that cosmid 94G06 containing APCL hybrid izes only to 19pI3.3 (arrowheads}.

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DISCUSSION

We have reported here the identification of a novel gene that encodes a protein with a striking similarity to the APC tumor suppressor and have shown that this gene, APCL, indeed is a functional homologue of APC. The APCL gene product is predicted to form homo- or heterodimers by virtue of its NH2-terminal coiled-coil domain. The armadillo domain, which showed 76% identity between APC and APCL, is thought to interact with other proteins (20), although it is not clear which proteins actually do 9.5 kb I interact with this domain of the two members of the APC family. 7.5 kb APC can bind to ß-catenin through its three-copy, 15-amino-acid motif [EX(D/E)XPXNYSX(K/R)YX(D/E)E; Ref. 19) and its sev 4.4 kb en-copy 20-amino-acid motif (FXVEXTPXCFSRXSSLSSLS; Ref. 5). We were unable to find in APCL any regions corresponding to the former but detected five copies of the 20-amino-acid motif. In 2.4 kb vitro and in vivo experiments demonstrated that the APCL protein could in fact bind to ß-catenin through this motif, and that APCL was able to decrease the intracellular concentration of ß-catenin. 1.35kb Our reporter-gene assay yielded evidence that APCL is also capa ble of down-regulating the signaling pathway stimulated by the ß-catenin-Tcf complex. These results suggest that APCL, like APC, may be a key factor in regulating the ß-catenin and Wnt/ Wingless signal pathway(s). Fig. 4. Expression of APCL in adult normal human tissues. Northern hlols contained ~2 /ig of poly(A)+/lane. Left, a molecular weight marker. A transcript larger than 10 kb Although a high level of expression of APC in brain has been appears specifically and strongly in brain tissue. recognized for several years, the role of APC in the central nervous system remains unclear. Bhat et al. (21) showed by in situ hybridiza- tion that APC expression was induced soon after neurogenesis and APCL-mediated Reduction of 0-Catenin Level in SW480 Cells. maintained at high levels until the late stage of maturation of neuronal A 20-amino-acid motif present in APC is known to interact with connections and suggested that APC might contribute to suppression ß-catenin.Because five copies of a similar 20-amino-acid motif were of neuronal proliferation. An immunohistochemical study showed that present in the predicted sequence of APCL protein, we investigated a APC is localized at presynaptic sites along dendrites in cultured possible interaction of APCL with ß-catenin.We expressed a peptide hippocampal neurons, and that it plays a significant role in neuronal corresponding to the five-copy motif of APCL in vitro and tested its cell function (8). These results implicated APC in neuronal differen ability to interact with a GST-ß-catenin fusion protein. As shown in tiation. Fig. 5, the APCL 20-amino-acid motif coprecipitated with ß-catenin. The functional similarity of APCL to APC and the specific expres It has been shown previously that the 20-amino-acid repeat region sion of APCL in the central nervous system suggest that APCL may also play a significant role in neuronal proliferation, differentiation, or of APC down-regulates cytoplasmic ß-catenin level in the colon function by regulating the cytoplasmic level of ß-catenin.Although carcinoma cell line SW480. To examine whether APCL has a similar no somatic APC mutations have been found in sporadic brain tumors function, we constructed a plasmid to express a partial APCL frag (22, 23), somatic mutations of ß-cateninhave been reported in medul- ment containing the 20-amino-acid repeat region (amino acids 627- loblastomas (24), an indication that activation of ß-cateninmay be 1666) and transiently transfected into SW480. Cells transfected with involved in the development of some brain tumors. APCL, APC (amino acids 1-1864), or control vector were identified From an evolutionary point of view, it is notable that, like APCL, by cotransfection with ß-gal.At 48 h after transfection, cells were stained with anti-ß-catenin antibody. Similar to APC, overexpression of APCL resulted in a drastic reduction of the level of ß-catenin. whereas the control vector did not show any effect on the level of ß-catenin (Fig. 6), suggesting that APCL also may function as a —¿. .ÃŒ. ± negative regulator of the Wnt/Wingless signaling pathway. Interference with Interaction of 0-Catenin and Tcf. hTcf-4 is i I if thought to transactivate transcription only when associated with <£,<° <5 ? ß-catenin;for example, a stable ß-catenin-hTcf-4 complex has been d d d ? * i *§ detected in the nuclei of APC(-/-) colon cancer cell line SW480. A 200kDa— transcriptional study using a Tcf-reporter gene had demonstrated that —¿ «1 introduction of normal APC into APC-defective cells depleted the 97.4kDa— pool of intracellular ß-cateninand reduced the transcriptional activity 69kDa— of Tcf (6). To examine whether APCL can suppress Tcf in a similar manner, we performed the Tcf reporter-gene assay ourselves. Cotrans fection of pCDNA3-APCL (627-1666) and the reporter plasmid, 46kDa— pTOPFLASH, into SW480 cells did reduce transcriptional activity, Fig. 5. Association of APCL with ß-cateninin vitro. In l'i'rrw-iranslaled. l~\S-labeled APCL (amino acids 627-1666; Lane I) was incubated with GST-Sepharose (Lane 2) or although the efficiency of APCL in this process seemed to be lower GST-ß-catenin-Sepharose (Lune 3). and the absorbed proteins were separated by 8*2 than that reported for APC (Fig. 7). SDS-PAGE. 5179

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h-gal

Incateni n

Vector APCL(627-1666) APC(1-1864) Fig. 6. Immunofluorcsccnl detection of ß-caleninin SW4XO cells transiently transfected with control vector. APCL construct (627-1666), or ARC construct (1-1864). Upper panels. expression of ß-galalone (green staining). Cells expressing 0-gal plus APCL construct or APC construct do not show red slain, an indication that intraccllular ß-catcninin these cells has been down-regulated.

the Dmxopliilu homologue of APC (D-APC) contains only five copies 125 of the 20-amino-acid repeat motif and lacks the hDLG-hinding region (25). Because the genomic structure of D-APC is also similar to that of APCL, we suspect an evolutionary relationship between them. Although the biological functions of the APC and APCL proteins are 100 -l T T not fully characterized, identification of a novel relative of APC may provide new insights into the function of APC and the Wnt/Wingless signaling pathway.

t> 75 J ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance of Keiko Okui.

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Hidewaki Nakagawa, Yoji Murata, Kumiko Koyama, et al.

Cancer Res 1998;58:5176-5181.

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