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Cytoplasmic ?-catenin in esophageal cancers

1999; Wiley; Volume: 84; Issue: 2 Linguagem: Inglês

10.1002/(sici)1097-0215(19990420)84

1097-0215

Yutaka Kimura, Hitoshi Shiozaki, Yuichiro� Doki, Makoto Yamamoto, Takehiro Utsunomiya, Kenshu Kawanishi, Nariaki Fukuchi, Masatoshi Inoue, Toshimasa Tsujinaka, Morito Monden,

Kruppel-like factors research

International Journal of CancerVolume 84, Issue 2 p. 174-178 Human CancerFree Access Cytoplasmic β-catenin in esophageal cancers Yutaka Kimura, Corresponding Author Yutaka Kimura Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanDepartment of Surgery II, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka, 565–0871, Japan. Fax: +81–6–6879–3259.Search for more papers by this authorHitoshi Shiozaki, Hitoshi Shiozaki Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorYuichiro Doki, Yuichiro Doki Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorMakoto Yamamoto, Makoto Yamamoto Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorTakehiro Utsunomiya, Takehiro Utsunomiya Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorKenshu Kawanishi, Kenshu Kawanishi Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorNariaki Fukuchi, Nariaki Fukuchi Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorMasatoshi Inoue, Masatoshi Inoue Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorToshimasa Tsujinaka, Toshimasa Tsujinaka Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorMorito Monden, Morito Monden Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this author Yutaka Kimura, Corresponding Author Yutaka Kimura Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanDepartment of Surgery II, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka, 565–0871, Japan. Fax: +81–6–6879–3259.Search for more papers by this authorHitoshi Shiozaki, Hitoshi Shiozaki Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorYuichiro Doki, Yuichiro Doki Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorMakoto Yamamoto, Makoto Yamamoto Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorTakehiro Utsunomiya, Takehiro Utsunomiya Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorKenshu Kawanishi, Kenshu Kawanishi Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorNariaki Fukuchi, Nariaki Fukuchi Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorMasatoshi Inoue, Masatoshi Inoue Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorToshimasa Tsujinaka, Toshimasa Tsujinaka Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this authorMorito Monden, Morito Monden Department of Surgery II, Osaka University Medical School, Suita, Osaka, JapanSearch for more papers by this author First published: 10 November 1999 https://doi.org/10.1002/(SICI)1097-0215(19990420)84:2 3.0.CO;2-ECitations: 34AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract β-Catenin has 2 distinct roles in E-cadherin-mediated cell adhesion and carcinogenesis through APC gene mutation. One occurs at cell-adhesion sites, where cadherins become linked to the actin-based cytoskeleton. The others occur in the cytoplasm and nuclei and are thought to regulate cell transformation. We studied these different β-catenins and evaluated their significance in carcinogenesis. Fresh surgical specimens were obtained from 22 patients with squamous-cell carcinoma of the esophagus. β-Catenin in the free soluble fraction and the insoluble fraction was immunoblotted separately. At the same time, its localization was observed by immuno-histochemical techniques. In the normal esophageal epithelium, 91% of β-catenin was detected in the insoluble fraction and β-catenin staining occurred at the cell membrane, in co-existence with E-cadherin. In cancerous tissues, the amount of soluble β-catenin was significantly (about 4-fold) higher than in normal tissues. Also, in cancerous tissues with higher amounts of soluble β-catenin, immuno-histochemical techniques revealed the presence of β-catenin in the cytoplasm and nuclei, as well as in the cell membrane. However, in samples with lower amounts of β-catenin, expression was found only at the cell boundaries. The amount of soluble β-catenin was not associated with the clinico-pathological grading of the tumors. Our results show that the accumulation of free soluble β-catenin in the cytoplasm and nuclei frequently occurs during carcinogenesis of the squamous epithelium of the esophagus. Int. J. Cancer (Pred. Oncol.) 84:174–178, 1999. © 1999 Wiley-Liss, Inc. β-Catenin, a central component of the cadherin adhesion system, binds to both the cytoplasmic domain of cadherin and the amino-terminal domain of α-catenin and mediates cell adhesion. Disorders of β-catenin have been reported in association with impaired intercellular adhesion of cancer cells. For example, mutated β-catenin does not link E-cadherin and α-catenin in gastric cancer cell lines (Oyama et al., 1994). Transformation by v-src or treatment with growth factors induces tyrosine phosphorylation of β-catenin and loss of cadherin-mediated cell adhesion (Behrens et al., 1993). β-Catenin has attracted attention for having functions other than cell adhesion. Armadillo, a Drosophila homologue of β-catenin, was revealed to be a component of the Wingless/Wnt signaling pathway, involved in embryonic morphogenesis and development (Miller and Moon, 1996). In untransformed cells, a small amount of β-catenin in the cytoplasm was observed to form a complex with the adenomatous polyposis coli (APC) tumor-suppressor gene product (Rubinfeld et al., 1993). A serine/threonine glycogen synthase kinase-3β (GSK-3β) phosphorylates the APC–β-catenin complex; then, the phosphorylated β-catenin is degraded by proteasomes (Aberle et al., 1997). The Wingless/Wnt signal inactivates this GSK-3β and results in post-translational stabilization of β-catenin in the cytoplasm (Papkoff et al., 1996). Axin, which suppresses axis formation in the embryo, forms a complex with β-catenin and GSK-3β and modulates degradation of β-catenin (Behrens et al., 1998). Disorders in this process, including β-catenin or APC mutation and Wnt stimulation, are associated with carcinogenesis and accumulation of cytoplasmic β-catenin. In particular, mutation of the APC gene is largely involved in the carcinogenesis of various organs (Powell et al., 1992), and it is therefore striking that the final result of APC mutation is to accumulate β-catenin in the cytoplasm (Munemitsu et al., 1995; Korinek et al., 1997; Rubinfeld et al., 1997; Inomata et al., 1996; Alman et al., 1997). The role of cytoplasmic β-catenin has been revealed to be the formation of complexes with DNA-binding proteins of the T-cell factor– lymphoid enhancer factor (Tcf-Lef) family and their translocation to the nucleus, where this complex may regulate cell proliferation or inhibition of apoptosis. (Korinek et al., 1997) Thus, free cytoplasmic β-catenin behaves as an oncoprotein (Peifer, 1997). In vivo, cytoplasmic and nuclear accumulation of β-catenin has been observed in colorectal polyps and cancers, gastric cancers and desmoid tumors. Such up-regulation of β-catenin is associated with the presence of mutant APC (Inomata et al., 1996; Alman et al., 1997). However, mutations of the β-catenin gene are not common in colorectal cancers (Kitaeva et al., 1997), and there are no mutations in β-catenin in gastric and breast cancers (Candidus et al., 1996). We have previously described the reduction of membranous β-catenin in esophageal cancers in an immuno-histochemical study (Takayama et al., 1996); we observed, but did not evaluate, cytoplasmic β-catenin expression. In the present study, we demonstrate the accumulation of free soluble β-catenin using a fractionation technique. Since APC gene mutation is rare in esophageal cancers (Powell et al., 1994), our observation may provide an important clue for elucidating the involvement of β-catenin in carcinogenesis. MATERIAL AND METHODS Tissue samples and cells Cancer tissue and adjacent non-cancerous mucosa were obtained with informed consent from 22 patients with squamous-cell carcinoma of the esophagus. All patients underwent surgery at our department. None had received pre-operative anti-cancer therapy. Part of the tissue was snap-frozen as soon as possible after surgical excision and stored in a deep freezer (or liquid nitrogen) and the remaining sample embedded in paraffin after being fixed in buffered formalin. The dog kidney cell line MDCK and the human colon cancer cell lines SW480 and HCT116 were obtained from the ATCC (Rockville, MD). MDCK cells were maintained in DMEM and the others in RPMI 1640 medium supplemented with 5% FBS. A431 was used to obtain the standard values for E-cadherin and β-catenin. Antibodies The following antibodies were used in this study: mouse monoclonal antibody (MAb) against β-catenin (Transduction Labs, Lexington, KY), mouse MAb against human E-cadherin (Takara Shuzo, Shiga, Japan) and rabbit polyclonal antibody against pan-cadherin (Sigma, St. Louis, MO). Immunoblotting Cells (2 × 106) were cultured in 10 cm dishes for 3 days and washed 3 times with 0.05 M PBS. Alternatively, 50 mg of the tumor and normal mucosa were minced and washed 3 times with PBS. Cells and tissue samples were collected by gentle centrifugation. Samples were soaked in 500 μl of hypotonic buffer (1 mM NaHCO3) containing 2 mM PMSF and 1 μg/ml of aprotinin for 30 min and centrifuged at 15,000 g for 30 min. The supernatant was mixed with half the amount of 3 × loading buffer [30% glycerol, 6% SDS, 125 mM Tris-HCl (pH 6.8): soluble lysate]. The pellet was mixed with 750 μl of 1 × loading buffer [10% glycerol, 2% SDS, 62.5 mM Tris-HCl (pH 6.8)] and clarified by centrifugation at 15,000 g for 15 min (insoluble lysate). Both soluble and insoluble lysates were boiled for 5 min in the presence of 2-mercaptoethanol and their protein concentrations measured with a protein assay kit (Bio-Rad, Hercules, CA). Protein (50 μg) was collected from the soluble and insoluble fractions and divided according to relative protein concentrations. Samples were subjected to electrophoresis separately on 7.5% SDS-polyacrylamide gels and transferred to a Protran nitrocellulose transfer membrane (Schleicher and Schuell, Dassel, Germany). After 5% skim-milk blocking, membranes were first incubated with the primary antibodies, then with secondary antibodies coupled with HRP (Amersham, Arlington Heights, IL) for anti-β-catenin antibody and HECD-1 or with alkaline-phosphatase (Promega, Madison, WI) for anti-pan-cadherin antibody and finally visualized with enhanced chemiluminescence (ECL) reagent (Amersham) or the ProtBlot NBT/BCIP color-development system (Promega). Bands on ECL-exposed X-OMAT AR film (Eastman Kodak, Rochester, NY) were analyzed by densitometric scanning using Image Quant (Molecular Dynamics, Sunnyvale, CA). Each membrane contained 25 μg of total cell extract from A431 cells, which was used to standardize the amount of E-cadherin and β-catenin to 1,000 and 800 arbitrary units (a.u.) on a densitometer. Immuno-histochemistry The avidin–biotin peroxidase complex (ABC) technique was used for immuno-histochemical staining. Sections were cut at 4 μm thickness, kept in xylene, rehydrated and washed with water. They were treated with 0.3% hydrogen peroxidase in methanol for 30 min to inhibit endogenous peroxidase and microwaved in a citrate buffer solution (0.1 M sodium citrate, pH 6.0) at 500 W for 40 min. After incubation of 3% normal horse serum to block non-specific binding, sections were incubated with the primary antibodies against β-catenin and E-cadherin at 4°C overnight with biotinylated anti-mouse IgG (Vecstain ABC kit; Vector, Burlingame, CA) for 30 min at room temperature and with ABC Elite reagent (Vector) for 30 min at room temperature. Between incubations, sections were washed with PBS. Color was developed with diaminobenzidine tetrahydrochloride supplemented with 0.04% hydrogen peroxidase and counterstained with Mayer's hematoxylin (Chroma, Stuttgart, Germany). Clinico-pathological features and statistical analysis Clinico-pathological features were classified according to the TNM system. Statistical analysis was performed using the Spearman rank correlation test or Welch's t-test. A p value below 0.05 was regarded as statistically significant. RESULTS First, the association of the localization and fractionation of β-catenin was examined using cultured cell lines, which had been well characterized in previous reports (Munemitsu et al., 1995; Ilyas et al., 1997). According to Western blot results, MDCK cells contained most of the β-catenin in the insoluble fraction prepared with hypotonic buffer, and the proportion of the soluble fraction was only 3% (Fig. 1). In SW480 and HCT116 cells, which have mutated APC or β-catenin, the β-catenin was strongly expressed in the soluble fraction and the proportion was 41% (742 a.u.) and 39% (561 a.u.), respectively (Figs. 1, 2a). E-cadherin, or pan-cadherin, was detected only in the insoluble fraction in all cells (Fig. 1). The technical errors for this quantitative assay were less than 5% for all cell lines. Most of the cytosolic protein would be in the soluble fraction since in SW480 cells 83% of the total activity of lactate dehydrogenase (LDH), a representative cytosolic enzyme, was detected in the soluble fraction, and this proportion was similar to those found in the cell extraction with 1% Triton X-100 (87%). Figure 1Open in figure viewerPowerPoint Detection of β-catenin and E-cadherin in soluble and insoluble fractions: cultured cell lines (MDCK and SW480) and esophageal tissues (case 1–5). I, insoluble fraction; S, soluble fraction for preparation of hypotonic buffer; T, esophageal cancer tissue; N, normal esophageal tissue. The 92 kDa band (arrow) indicates full-sized β-catenin and the 120 kDa band (arrowhead) represents full-sized E-cadherin. Anti-pan-cadherin antibody was used in MDCK cells. The additional bands below the β-catenin band represent degradation products. Figure 2Open in figure viewerPowerPoint Comparison of β-catenin amounts of esophageal cancers and normal epithelium in soluble (a) and insoluble (b) β-catenin. MDCK (█), SW480 (X), HCT116 (▴), normal epithelium (•) and cancer tissues (⋄, □, ○ or ▵). (a) Mean values (open rectangles) of soluble β-catenin in normal and tumor tissues are 101 ± 86 [mean ± SD (bars), n = 22] and 408 ± 346 (n = 22), respectively. *Statistical significance (p < 0.001, Welch's t-test). (b) Mean values (open rectangles) of insoluble β-catenin in normal and tumor tissues are 1,052 ± 284 (n = 22) and 869 ± 529 (n = 22), respectively. In normal esophageal epithelium, much less β-catenin was in the soluble fraction than in the insoluble fraction (Fig. 1). The amount of soluble β-catenin was maximal at 244 a.u., and the average was 101 ± 86 a.u. in normal esophageal epithelium (Figs. 1, 2a). E-cadherin was detected only in the insoluble fraction at a similar amount in each case. Immuno-histochemistry showed that β-catenin was expressed at the cell–cell boundaries in the same manner as E-cadherin (Fig. 3). Figure 3Open in figure viewerPowerPoint E-cadherin and β-catenin expression in normal and cancer tissues in the esophagus. In normal esophageal epithelium, E-cadherin (a) and β-catenin (b) were expressed at the cell boundaries. In cancer tissues, β-catenin (d) was expressed at the cell boundaries but reduced as observed for E-cadherin (c) (case 2 in Fig. 1). β-Catenin (f) was detected in the cytoplasm and nuclei, while E-cadherin expression (e) was reduced (case 3 in Fig. 1). Staining, ABC method. Scale bar: 200 μm. In some cancerous tissue samples, only trace amounts of soluble β-catenin were detected as in normal epithelium or MDCK cells (Fig. 1, cases 1 and 2). In others, the band of soluble β-catenin was as obvious as in SW480 cells (Fig. 1, cases 3–5). Thus, the cancer samples can be roughly divided into 2 groups. In 11 cases (50%), the amount of soluble β-catenin was similar to that in the normal epithelium (average 94 ± 81 a.u., open diamonds in Fig. 2a) and ranged within the average ± 2 SD of the normal epithelium. The remaining 11 cases (50%) had more soluble β-catenin (open squares in Fig. 2a). Their amounts ranged from 329 a.u. to 914 a.u. (average 721 ± 171 a.u.), which were close to that of SW480 or HCT116. Taken together, the amount of soluble β-catenin was about 4-fold higher in cancerous tissues (average 408 ± 346 a.u.) than in normal tissues (average 101 ± 86 a.u., p <0.001, Fig. 2a). As for β-catenin in the insoluble fraction, although there was no statistically significant difference among normal (average 1,052 ± 284 a.u.) and cancer (average 869 ± 529 a.u.) tissues, one-third of the tumors showing a reduction (open circles in Fig. 2b) and the others an amount similar to that of normal tissues (open triangles in Fig. 2b). Among these 22 cases, we could not detect the truncated β-catenin. E-cadherin was always detected only in insoluble fractions, such as that of the normal epithelium, although their amounts were frequently lower than those in normal epithelium, as previously reported (Fig. 1). In cases where the amount of soluble β-catenin was low, it tended to be expressed at the cell boundaries as observed for normal epithelium (Fig. 3d). However, when the amount of soluble β-catenin was high, β-catenin staining was observed in the cytoplasm as well as at the cell membrane. In 3 cases, β-catenin staining was observed in the nuclei in addition to the cytoplasm (Fig. 3f). There was an interesting trend of cytoplasmic staining of β-catenin being stronger at the invading edge than in the center of the tumor. E-cadherin was always detected at the cell–cell boundary, despite translocation of β-catenin. The 22 tumors were classified into 2 groups according to the amount of soluble β-catenin, and the relationship with clinico-pathological factors was evaluated. However, there was no significant correlation between soluble β-catenin and clinico-pathological features, including histological grade, tumor invasion and metastasis (Table I). Table I. CLINICAL FINDINGS AND SOLUBLE β-CATENIN IN ESOPHAGEAL CANCERS 1 The amount of soluble β-catenin in esophageal cancers was measured as arbitrary units using a densitometer. 2 G1, well differentiated; G2, moderately differentiated; G3, poorly differentiated. 3 T1, tumor has invaded the lamina propria or submucosa; T2, muscularis propria; T3, adventitia; T4, adjacent structures. 4 N0, no regional lymph node metastasis; N1, metastasis. 5 M0, no distant metastasis; M1, metastasis. 6 Stage of the TNM classification. 7 N.S., not significant. DISCUSSION The involvement of β-catenin disorders in cancers has been studied in association with cadherin-mediated cell adhesion, tumor invasion and metastasis (Takayama et al., 1998). However, since the physical interaction of β-catenin with APC was found, other aspects of β-catenin function needed to be carefully investigated. Finally, APC was shown to serve as a tumor-suppressor gene by down-regulating cytoplasmic β-catenin through protein degradation (Papkoff et al., 1996; Munemitsu et al., 1995). However, the true function of cytoplasmic β-catenin has been not fully elucidated. In association with Tcf, it is translocated to the nucleus, where it is thought to promote transcription of genes involved in differentiation and proliferation (Peifer, 1997). β-Catenin in a cadherin complex and in the free form has been separated by gel filtration by Munemitsu et al. (1995). Furthermore, the former localized at the cell membrane and the latter in the cytoplasm. We have noted, using immuno-histochemistry, the existence of cytoplasmic β-catenin in cancers of various organs (Inomata et al., 1996; Alman et al., 1997). In the present study, we quantitatively measured the amount of free soluble β-catenin which accumulates in the cytoplasm using simple fractionation with hypotonic buffer. This method may be adequate for separating β-catenin in cytosol since cadherin was detected only in the insoluble fraction and most of the LDH activity was detected in the soluble fraction. After such fractionation, soluble β-catenin amounted to only 3% in MDCK cells but approximately 40% in SW480 and HCT116. MDCK cells contain no mutation in the APC gene, have full-length β-catenin and express β-catenin at the cell–cell boundaries as described previously (Munemitsu et al., 1995). SW480 cells, derived from colon cancer, have a mutation in the APC gene but not in the β-catenin gene. Thus, β-catenin is over-expressed in the cytoplasm (Munemitsu et al., 1995; Ilyas et al., 1997). HCT116 cells, however, contain the wild-type APC gene and mutated β-catenin gene (Ilyas et al., 1997). Based on these findings, we applied this method to studying esophageal cancers and found that soluble β-catenin frequently increased to the level of cancer cells with APC or β-catenin mutation. As APC mutation has frequently been found in various malignant tumors, disorders of β-catenin have also come under investigation. Mutations of the APC gene have been found in 60% to 80% of colorectal polyps and early during colorectal tumorigenesis (Powell et al., 1992). β-Catenin accumulates in the cytoplasm and nuclei, in association with APC gene mutation (Inomata et al., 1996). The same relationship was reported for desmoid tumors (Alman et al., 1997). However, as for esophageal cancers, APC gene mutations are rare and not important in tumorigenesis (Powell et al., 1994). β-Catenin mutations have been found in melanoma cells and colorectal cancer cells and may be important factors in tumorigenesis, in the same pathway as APC gene mutations (Rubinfeld et al., 1997; Ilyas et al., 1997). However, we did not find any abnormal β-catenin bands in our Western blots. Actually, the mutation of β-catenin is not frequent in esophageal cancer, as judged by PCR-SSCP results (data not shown). Another mechanism for cytoplasmic β-catenin accumulation might exist in esophageal cancers. An important clue is the observation, by immuno-histochemical methods, that cytoplasmic β-catenin is more evident at the invading front of a cancer nest. This is difficult to explain by genomic disorders such as mutation of APC or β-catenin. Some members of the Wnt family have been reported to be involved in carcinogenesis of the mammary gland (Tsukamoto et al., 1988). Proteasome degradation is another factor that cannot be ignored in the regulation of cytoplasmic β-catenin (Aberle et al., 1997). The tyrosine phosphorylation of β-catenin, which is induced by growth factor stimulation and is involved in cancer invasion, needs evaluation. Cytoplasmic β-catenin expression at the invading edge is also observed in colon cancers and gastric cancers (data not shown). The third mechanism involved in cytoplasmic β-catenin accumulation thus requires investigation. We have reported the reduction of membranous β-catenin in various cancers based on immuno-histochemical findings (Takayama et al., 1996). In the present study, we quantitatively showed the decrease of insoluble β-catenin in about one-third of esophageal cancers we examined. There was no significant correlation between the decrease of insoluble β-catenin and the increase of soluble β-catenin. Thus, we cannot propose that the accumulation of free soluble β-catenin results from translocation or overflow of membranous β-catenin. In the present study, we found a tendency for the amount of membranous β-catenin to be correlated with that of E-cadherin. This correlation was significant in colorectal cancers according to immuno-histochemical findings (Takayama et al., 1996, 1998). The reduction of membranous β-catenin is important for cell adhesion, invasion and metastasis, but its mechanism remains unknown. The β-catenin disorders presented here were not associated with clinico-pathological features of the tumors, including differentiation, depth of invasion and metastasis. Although it is not possible to conclude from our small number of cases, free soluble β-catenin in the cytoplasm may be involved in early stages of carcinogenesis, like APC , but not in the progression of tumors. Thus, β-catenin may well play a central role in tumorigenesis in association with APC. Our results suggest the novel possibility that β-catenin is more likely involved in carcinogenesis than has been expected, particularly since tissue fractionation facilitates the study of free β-catenin in the cytoplasm. Acknowledgements The critical reading of the manuscript by Dr. Y. Miyoshi and the technical assistance of Ms. K. Tamura are gratefully acknowledged. REFERENCES Aberle, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R., β-Catenin is a target for the ubiquitin-proteasome pathway. EMBO J, 16, 3797– 3804 (1997). 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