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Isolation and Characterization of a Novel Human Gene, DRCTNNB1A, the Expression of Which Is Down-Regulated by ß-Catenin

Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan [T. K., Y. F., Y. D., T. N., H. I., M. F., S. S., N. M., Y. Nak.]; Division of Clinical Genetics, Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, Osaka 565-0871, Japan [Y. Nag., Y. M.]; and Department of Surgery II, Kumamoto University Medical School, Kumamoto 860-8556, Japan [T. K., M. O.]

	ABSTRACT

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ß-Catenin plays significant roles in cell-to-cell adhesion and the Wnt/Wg signal transduction pathway. Accumulation of this protein in the cytoplasm and nucleus as a result of mutations of the adenomatous polyposis coli tumor suppressor gene or of the ß-catenin gene itself is often seen in a wide variety of tumors including carcinomas of the colon, liver, uterus, and brain. Interaction of accumulated ß-catenin with Tcf/Lef transcription factors is known to deregulate expression of some downstream genes, but the precise mechanisms whereby ß-catenin contributes to carcinogenesis remain to be disclosed. Here we report isolation of a novel murine gene, Drctnnb1a (down-regulated by Ctnnb1, a), the expression of which was experimentally down-regulated in response to the activated form of ß-catenin. To investigate a possible role of DRCTNNB1A in cancers, we also isolated the human homologue, DRCTNNB1A, the deduced product of which was 91% identical to the murine protein. The transcript was expressed in all human tissues examined, and we assigned the genomic location of DRCTNNB1A to chromosomal band 7p15.3 by in situ hybridization. Expression of DRCTNNB1A in SW480 colon cancer cells was significantly increased in response to reduction of intracellular ß-catenin by adenovirus-mediated transfer of the ß-catenin-binding domain of the adenomatous polyposis coli gene into the cells. Furthermore, we documented reduced expression of DRCTNNB1A in 12 of 15 primary colorectal cancers examined, compared with corresponding adjacent noncancerous mucosae. Our results implied that DRCTNNB1A is one of the genes involved in the ß-catenin-Tcf/Lef signaling pathway, and that reduced expression of DRCTNNB1A may have some role in colorectal carcinogenesis.

	Introduction

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ß-Catenin is a multifunctional protein that plays crucial roles in cell-to-cell adhesion and signal transduction (1, 2, 3) . At adherens junctions, ß-catenin associates with the cytoplasmic domains of cadherin and -catenin, anchoring the adhesion complex to the cytoskeleton (2) . In Drosophila and Xenopus, ß-catenin functions in the Wnt/Wg signal transduction pathway during development and morphogenesis (4 , 5) .

Abnormal accumulation of ß-catenin in the cytoplasm or nucleus occurs often in several types of cancers, including many colon carcinomas. Without a Wnt/Wg signal, cytoplasmic ß-catenin is phosphorylated through binding with APC,² glycogen synthase kinase 3ß, and Axin/Conductin, and in that form is rapidly degraded via the ubiquitin-proteosome pathway. However, genetic alterations of the APC or ß-catenin genes that abrogate the degradation pathway result in abnormal accumulation of ß-catenin and deregulation of Wnt/Wg signal transduction. Accumulated ß-catenin interacts with Tcf/Lef, thereby transactivating target genes of the transcription complex, but the precise mechanisms and the specific target molecules involved remain to be elucidated. Thus far, c-myc (6) , cyclin D1 (7) , matrilysin (8) , WISP (9) , c-jun, fra-1, uPAR, ZO-1 (10) , and NBL4 (11) have been identified as targets of the ß-catenin/Tcf complex.

We previously reported establishing a murine cell line, LMT, in which expression of exogenously introduced ß-catenin was easily regulated by doxycyclin. Moreover, LMT cells acquired some malignant properties, including the ability to grow in low-serum medium and the loss of contact inhibition of growth (12) . To isolate genes regulated by ß-catenin, we applied a fluorescent differential display method using LMT cells and parental L cells. Among the genes that were differently expressed between these two cell lines, we identified a novel murine gene, the expression of which was down-regulated in response to accumulation of ß-catenin. To investigate its possible association with ß-catenin in colon carcinogenesis, we isolated and characterized the human homologue, DRCTNNB1A. Here we report isolation and characterization of both novel genes, Drctnnb1a and DRCTNNB1A, and present evidence for involvement of the human homologue in colon carcinogenesis.

	Materials and Methods

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Cell Lines and Primary Tumor Samples.
We previously established a cell line of mouse fibroblasts, LMT, by introducing into murine L cells a mutant ß-catenin transgene, which lacks a region corresponding to exon 3 (12) . COS-7 cells and a human colon-cancer cell line, SW480, were obtained from the American Type Culture Collection. LMT, L, and COS-7 cells were cultured in DMEM (Sigma Chemical Co., St. Louis, MO) and SW480 cells in Leibovitz’s L-15 (Life Technologies, Inc., Grand Island, NY), each supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma Chemical Co., St. Louis, MO). Cultures were maintained at 37°C in an atmosphere of humid air containing 5% CO₂.

Cancerous tissues and matched noncancerous mucosal samples were excised during surgery from 15 patients with colon cancer, after informed consent had been obtained.

RNA Extraction and Fluorescent Differential Display.
Total RNA was extracted from L cells, LMT cells, colon cancer tissues, and matched noncancerous mucosal tissues of the dissected specimens, using TRIzol reagent (Life Technologies, Inc.), according to the manufacturer’s protocol. The fluorescent differential display procedure was performed essentially as described previously (13) . PCR products were resuspended in formamide sequencing dye and run for 3 h at 1800 V on a sequencing gel containing 4% acrylamide (19:1) with 7 M urea. Gel images were analyzed with an FMBIO II Multi-View fluoroimage analyzer (HITACHI). Bands that showed differential expression between L cells and LMT cells were excised from the gels, and DNA was extracted in each case by boiling the gel fragment in Tris-EDTA buffer. Each DNA sample was reamplified in 30 cycles with the same primer set used for the first PCR products. The reamplified products were cloned into pBluescript II SK (-) vector (Stratagene) and sequenced using T3, T7 primers and an ABI PRISM Dye Terminator Cycle Sequencing FS Ready kit (PE Applied Biosystems), according to the protocol provided by the supplier.

Isolation of Mouse and Human DRCTNNB1A.
Among several differently expressed bands detected by the fluorescent differential display method, we identified one fragment (F13T), the expression of which was decreased in response to the accumulation of ß-catenin. By searching databases for homologies, we identified six murine ESTs (AA271147, AA260040, AA189431, AA170341, AA189501, and AA21738) that contained the sequence of F13T. To obtain a full-length cDNA sequence of this gene, termed Drctnnb1a, we prepared a murine cDNA library using 5 µg of poly(A) RNA extracted from spleen and a ZAPII cDNA synthesis kit (Stratagene), and screened 10⁶ clones using a PCR product amplified with a primer set (5'-GGATTGACTCAACCCTTGCA-3' and 5'-AGCACATAATGATATCCAAGAT-3') as a probe. Among seven positive clones, five contained sequences corresponding to the murine ESTs. To investigate a potential involvement of this gene in human cancers, we searched the databases for homologous human sequences. Although 17 human ESTs (AA186951, AA188347, AA236600, AA282218, AA282281, AA283728, AA290947, AA456245, AA748393, AA776721, AA782980, AA905096, AA923713, AI080337, AW082765, T12324, and T34135) showed high degrees of similarity with Drctnnb1a, the assembled sequence was shorter in size and lacked the 5' part compared with Drctnnb1a. Thus, we amplified a part of the sequence using a primer set (5'-CTCTATAAAGTTATCCAGGAGC-3' and 5'-AAGTTCAGTTCTTTCTGAAACG-3') and screened 10⁶ clones from human fetal brain cDNA library (Stratagene) using the PCR product as a probe.

Northern Blot Analysis.
Human multiple-tissue blots (Clontech) were hybridized with the cDNA fragment of DRCTNNB1A, labeled by the random oligonucleotide-priming method. Prehybridization, hybridization, and washing were performed according to the supplier’s recommendations. The blots were autoradiographed with intensifying screens at -80°C for 48 h.

FISH.
A human genomic library cloned in BAC was screened in accordance with the manufacturer’s instructions (Genome Systems, Inc.). To determine the chromosomal location of the human gene, we performed FISH using B13T, a clone isolated from the BAC library.

Immunocytochemical Analysis.
To achieve a HA-tagged version of DRCTNNB1A, we constructed pcDNA3.1(+)/DRCTNNB1A/HA, which contained HA-epitope sequences (YPYDVPDYA) at the COOH-terminal of DRCTNNB1A protein and transfected the construct into COS-7 cells. Transiently transfected COS-7 cells replated on chamber slides were fixed with PBS containing 4% paraformaldehyde and then rendered permeable by incubating the slides for 3 min at 4°C with PBS containing 0.1% Triton X-100. Cells were covered with 2% BSA in PBS for 12 h at 4°C to block nonspecific antibody binding sites. After the cells were incubated with a mouse anti-HA antibody (MBL), antibodies were stained with a goat antimouse secondary antibody conjugated to FITC (Jackson Immuno Research). In addition, nuclei were stained with 4',6'-diamidine-2'-phenylindole dihydrochloride (Boehringer Mannheim), and the stained preparations were observed under a fluorescence microscope (Nikon; Eclipse E800). To confirm the expression of DRCTNNB1A/HA-tagged protein in transfected cells, we also performed immunoblotting.

Depletion of ß-Catenin by Adenovirus-mediated Gene Transfer.
The portion of the APC gene encoding the 20-amino acid-repeat domain that binds ß-catenin had been reported to down-regulate ß-catenin (14) . We constructed an adenovirus vector containing this domain (Ad-APC) by inserting a 2.5-kb HindIII fragment of APC cDNA into the HindIII site of the pAd-BglII vector, which contains the cytomegalovirus promoter/enhancer and a bovine growth hormone polyadenylation signal flanked by Ad5 E1 sequences. The recombinant adenoviruses were constructed as described previously (15) , propagated in the human embryonic kidney 293 cell line, and purified by two rounds of CsCl density centrifugation. Viral titers were measured by a limiting-dilution bioassay using the kidney cells. Cell monolayers were infected with the viral solutions and incubated at 37°C for 1 h, with brief agitation every 15 min. Culture medium was added, and the infected cells were maintained at 37°C for 48 h.

Gel Retardation Assay.
A gel retardation assay was performed as described elsewhere (16) . Briefly, nuclear extracts were prepared from intact nuclei that were washed four times to avoid contamination with cytoplasmic ß-catenin. As the optimal Tcf4 probe, we used a double-stranded 15-nucleotide oligomer, CCCTTTGATCTTACC. A typical binding reaction contained 10 µg of nuclear protein, 0.1 ng of radiolabeled probe, and 100 ng of deoxyinosine-deoxycytidine in 25 µl of binding buffer (60 mM KCl, 1 mM EDTA, 1 mM DTT, and 10% glycerol). Samples were incubated for 20 min at room temperature, followed by addition of anti-ß-catenin antibody and incubation for 20 min more. Electrophoresis was performed at 4°C on 4% nondenaturing polyacrylamide (29:1) gels with 0.25x Tris-boric/EDTA buffer. The gels were dried and autoradiographed overnight.

Semiquantitative RT-PCR Analysis.
RT-PCR experiments were carried out using cDNA reversely transcribed from 0.2 µg of total RNA from each resected specimen. The PCR exponential phase was determined on 20–32 cycles to allow comparison among cDNAs developed from identical reactions. Quantification and equalization of the amount of cDNA was achieved using primers for amplifying GAPDH as an internal control. All reactions involved initial denaturation at 94°C for 2 min, followed by 20–32 cycles at 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min, on a Gene Amp PCR system 9600 (PE Applied Biosystems). Primer sequences were as follows: 1315F (5'-TTCTCTTCAGTCACTGTGTC-3') and 1315R (5'-TGCATTAGCAACCAGCAATG-3') for DRCTNNB1A; m1315F (5'-CTCTCTTCAGTCACTGTGTC-3') and m1315R (5'-TGCATTAGCAACCAGCAGAG-3') for Drctnnb1a; ßCATF1 (5'-GCGTGGACAATGGCTACTCA-3') and ßCATR2 (5'-GAGTTGTAATGGCATAAAACAAC-3') for human ß-catenin; GAPDH3F (5'-ACAACAGCCTCAAGATCATCAG-3') and GAPDH3R (5'-GGTCCACCACTGACACGTTG-3') for human GAPDH; and mACTBF (5'-ATTGAACATGGCATTGTTACCAAC-3') and mACTBR (5'-GTCTAGAGCAACATAGCACAGC-3') for mouse ß-actin. The products were electrophoresed in 3% agarose gels and visualized by fragment Southern blot analysis, followed by the transfer to nylon membranes (Amersham, Cleveland, OH). The membranes were hybridized with ³²P-labeled internal oligonucleotide probes. Each internal oligonucleotide sequence used for Southern blot analysis as a probe was 5'-GGTATAAGTAGCAGGATACCAGT-3' for human DRCTNNB1A, 5'-GGTGTGAGCAGCAGGATACCGAT-3' for mouse Drctnnb1a, 5'-ATTGCACGTGTGGCAAGTTC-3' for human ß-catenin, 5'-CTCATGACCACAGTCCATGC-3' for human GAPDH, and 5'-CCTCTACAATGAGCTGCGTGTG-3' for mouse ß-actin.

	Results

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Isolation of DRCTNNB1A.
To isolate genes involved in the mammalian ß-catenin/Wnt signaling pathway, we looked for different expression patterns between mouse fibroblasts (L cells) and a derivative line (LMT) in which expression of activated ß-catenin could be induced by withdrawal of doxycyclin. By means of the fluorescent differential display method, we isolated a DNA segment, F13T, corresponding to part of a mouse gene termed Drctnnb1a, the expression of which was suppressed in relation to the accumulation of ß-catenin in the cells (Fig. 1) . A search for homology between F13T and known genes in the public databases revealed that parts of six murine ESTs were identical to the sequence of F13T. Subsequently, we screened a mouse-spleen cDNA library with F13T and isolated five cDNA clones. Assembling the DNA sequences of these clones and the ESTs yielded a cDNA consisting of 6572 nucleotides, with an open reading frame of 1563 nucleotides encoding 521 amino acids (DNA sequences are available from GenBank; the accession number is AB030242).

View larger version (55K): [in this window] [in a new window]   Fig. 1. A, Northern blot analysis of Drctnnb1a with RNAs extracted from L cells and LMT cells with or without doxycyclin. L cells and LMT cells were grown with or without doxycycline at various concentrations for 48 h. Expression of ß-actin served as an internal control. B, Western blot analysis of ß-catenin in the protein extracts from cells corresponding to the same lanes in A. C, the time course repression of Drctnnb1a and induction of ß-catenin by deprivation of doxycycline. LMT cells were incubated with 20 ng/ml of doxycycline for 48 h and deprived of doxycycline for the indicated time.

Expression and Chromosomal Assignment of DRCTNNB1A.
Northern blot analysis using the DRCTNNB1A cDNA clone as a probe revealed that a 7.1-kb transcript was expressed in all human tissues examined (Fig. 2A) . FISH using a BAC clone as a probe showed clear twin-spot signals specifically on chromosomal band 7p15.3. No signals could be detected on any other chromosomes in 100 metaphase cells examined (Fig. 2, B and C) .

View larger version (37K): [in this window] [in a new window]   Fig. 2. A, Northern blot analysis of DRCTNNB1A. Left, molecular size (kb) of the transcript. B, chromosomal mapping of the DRCTNNB1A gene by FISH. Metaphase chromosomes were stained with propidium iodide to show twin-spot signals (arrows). C, G-band pattern of the same metaphase chromosomes, delineated through an UV-2A filter (Nikon, Tokyo, Japan), indicates that the DRCTNNB1A gene hybridized specifically to chromosomal band 7p15.3. D, Western blot analysis of extracts from COS7 cells transfected with the plasmid containing DRCTNNB1A/HA. E, cytoplasmic localization of DRCTNNB1A. Cells were stained with anti-HA antibodies and 4',6-diamidino-2-phenylindole.

View larger version (35K): [in this window] [in a new window]   Fig. 3. A, gel retardation assay in colon cancer cell line SW480. A Tcf4-specific, double-stranded oligonucleotide was used as the probe. Arrow, supershift of the ß-catenin/Tcf4 complex after incubation with anti ß-catenin monoclonal antibody. B and C, immunocytochemical staining of ß-catenin in SW480 cells after 72 h of infection with either adenovirus containing the 20-amino acid repeat region of the wild-type APC gene (Ad-APC) at 100 multiplicity of infection or LacZ (Ad-LacZ). D, expression of DRCTNNB1A in SW480 cells. Cells were untreated or transfected for 72 h with either Ad-LacZ or Ad-APC. Lower panel, Western blot analysis of ß-catenin in corresponding cells.

Expression of DRCTNNB1A in Colorectal Cancers.
We examined expression of DRCTNNB1A in colorectal cancer tissues and their adjacent normal mucosae by means of semiquantitative RT-PCR and found reduced expression of DRCTNNB1A in cancer tissues compared with matched noncancerous tissues in 12 of the 15 cases examined (Fig. 4) . In the remaining three cases, expression of DRCTNNB1A in tumors was similar to that of paired normal tissues.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Expression of DRCTNNB1A in colon cancer tissues and adjacent noncancerous mucosae. Expression of DRCTNNB1A was reduced in tumors (T) 1, 2, 6, 9, 10, 13, and 15, compared with their adjacent noncancerous tissues (N).

	Discussion

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Because the predicted amino acid sequence of DRCTNNB1A bears no significant homology to known proteins, it is difficult to speculate about its function. But because Drctnnb1a is down-regulated in LMT cells that have acquired the ability to grow in a low-serum environment as well as in multi-layers, reduced expression of DRCTNNB1A might render cells able to grow under conditions of low blood supply and in the absence of contact inhibition. Both of these features can confer a growth advantage on affected cells. Moreover, an analysis of motifs using a computer program (PROSITE) predicted a putative site for phosphorylation by tyrosine kinase between codons 258 and 265. Tyrosine phosphorylation is involved in a variety of signal-transduction pathways associated with growth control, differentiation, or adhesion. Therefore, we suggest that DRCTNNB1A is also likely to play a role in some signal-transduction pathway associated with proliferation or cell adhesion.

Because impaired degradation of ß-catenin by mutations of APC or ß-catenin itself is often observed in colorectal cancers, our data showing that DRCTNNB1A is down-regulated by ß-catenin prompted us to examine expression of this gene in colon cancer tissues. We documented decreased expression of DRCTNNB1A in cancer tissues compared with paired noncancerous mucosal cells in more than half of the tumors examined. These observations led us to examine whether DRCTNNB1A normally exerts tumor-suppressive activity in colorectal tissues. Although we screened the entire coding region of DRCTNNB1A by RT-PCR, followed by direct sequencing, in 12 colon cancer cell lines and 20 primary tumors, we detected no mutations (data not shown). Moreover, the chromosomal locus (7p15.3) of the DRCTNNB1A gene never has been reported to show frequent loss of heterozygosity in colorectal cancers. Nevertheless, we know that dysfunction of several tumor suppressor genes including transforming growth factor-ß receptors (23) , E-cadherin (24) , KAI1 (25) , and Cdx-2 (26) follows their reduced expression. Further investigations will be necessary to clarify the possibility that DRCTNNB1A, in fact, may have a tumor-suppressive role.

Malignant transformation and progression of colorectal tumors involve multiple genetic events that include inactivation of tumor suppressor genes and activation of proto-oncogenes. Accumulated ß-catenin in colon epithelium can confer a transformed phenotype by up-regulating target molecules such as cyclin D1, which mediates constitutive cell growth; matrilysin, which is involved in tumor formation; and fra-1, which transactivates down-stream genes such as urokinase plasminogen activator via coupling with c-jun. However, all of these molecules exert their functions through the transactive role of ß-catenin. On the other hand, the ras proto-oncogene was found recently to down-regulate two types of homeobox protein, i.e., Cdx-1 and Cdx-2 (27) , and Cdx-2 was revealed as a tumor suppressor because heterozygous Cdx-2 knockout mice developed multiple colonic polyps (28) . Therefore, it is worth noting that oncogenic molecules are capable of both activating target genes and repressing tumor-suppressive activities. The findings reported here shed some light on the diverse mechanisms involving ß-catenin and underscore the importance of investigating a potential role of DRCTNNB1A in human tumorigenesis.

	FOOTNOTES

 
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 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: 81-3-5449-5433; E-mail: yusuke{at}ims.u-tokyo.ac.jp

² The abbreviations used are: APC, adenomatous polyposis coli; FISH, fluorescence in situ hybridization; Tcf/Lef, T-cell factor/lymphoid enhancer-binding factor; EST, expressed sequence tag; BAC, bacterial artificial chromosome; HA, hemagglutinin A; RT-PCR, reverse-transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Received 12/28/99. Accepted 5/18/00.

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