Activation of AXIN2 Expression by β-Catenin-T Cell Factor
2002; Elsevier BV; Volume: 277; Issue: 24 Linguagem: Inglês
10.1074/jbc.m200139200
ISSN1083-351X
AutoresJanet Y. Leung, Frank T. Kolligs, Rong Wu, Yali Zhai, Rork Kuick, Samir Hanash, Kathleen R. Cho, Eric R. Fearon,
Tópico(s)Kruppel-like factors research
ResumoThe Wnt pathway regulates cell fate, proliferation, and apoptosis, and defects in the pathway play a key role in many cancers. Although Wnts act to stabilize β-catenin levels in the cytosol and nucleus, a multiprotein complex containing adenomatous polyposis coli, glycogen synthase kinase 3β, and Axin1 or its homolog Axin2/Axil/conductin promotes β-catenin phosphorylation and subsequent proteasomal degradation. We found that the rat Axil gene was strongly induced upon neoplastic transformation of RK3E cells by mutant β-catenin or γ-catenin or after ligand-induced activation of a β-catenin-estrogen receptor fusion protein. Expression of Wnt1 in murine breast epithelial cells activated the conductin gene, and human cancers with defective β-catenin regulation had elevated AXIN2 gene and protein expression. Expression ofAXIN2/Axil was strongly repressed in cancer cells by restoration of wild type adenomatous polyposis coli function or expression of a dominant negative form of T cell factor (TCF)-4. TCF binding sites in the AXIN2 promoter played a key role in the ability of β-catenin to activate AXIN2 transcription. In contrast to AXIN2/Axil, expression of human or rat Axin1 homologs was nominally affected by β-catenin-TCF. Because Axin2 can inhibit β-catenin abundance and function, the data implicate AXIN2 in a negative feedback pathway regulating Wnt signaling. Additionally, although Axin1 and Axin2 have been thought to have comparable functions, the observation that Wnt pathway activation elevates AXIN2 but not AXIN1 expression suggests that there may be potentially significant functional differences between the two proteins. The Wnt pathway regulates cell fate, proliferation, and apoptosis, and defects in the pathway play a key role in many cancers. Although Wnts act to stabilize β-catenin levels in the cytosol and nucleus, a multiprotein complex containing adenomatous polyposis coli, glycogen synthase kinase 3β, and Axin1 or its homolog Axin2/Axil/conductin promotes β-catenin phosphorylation and subsequent proteasomal degradation. We found that the rat Axil gene was strongly induced upon neoplastic transformation of RK3E cells by mutant β-catenin or γ-catenin or after ligand-induced activation of a β-catenin-estrogen receptor fusion protein. Expression of Wnt1 in murine breast epithelial cells activated the conductin gene, and human cancers with defective β-catenin regulation had elevated AXIN2 gene and protein expression. Expression ofAXIN2/Axil was strongly repressed in cancer cells by restoration of wild type adenomatous polyposis coli function or expression of a dominant negative form of T cell factor (TCF)-4. TCF binding sites in the AXIN2 promoter played a key role in the ability of β-catenin to activate AXIN2 transcription. In contrast to AXIN2/Axil, expression of human or rat Axin1 homologs was nominally affected by β-catenin-TCF. Because Axin2 can inhibit β-catenin abundance and function, the data implicate AXIN2 in a negative feedback pathway regulating Wnt signaling. Additionally, although Axin1 and Axin2 have been thought to have comparable functions, the observation that Wnt pathway activation elevates AXIN2 but not AXIN1 expression suggests that there may be potentially significant functional differences between the two proteins. glycogen synthase kinase 3β adenomatous polyposis coli estrogen receptor β-galactosidase glyceraldehyde-3-phosphate dehydrogenase human embryonic kidney luciferase ovarian endometriod adenocarcinoma 4-hydroxytamoxifen reverse transcription PCR T cell factor The Wnt signaling pathway plays an important role in cellular proliferation, differentiation, and morphogenesis, and control of β-catenin stability is central to Wnt signaling (1Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2248) Google Scholar, 2Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1760) Google Scholar, 3Peifer M. Polakis P. 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Christensen E. Schmidt S.S. Roche P.C. Smith D.I. Thibodeau S.N. Nat. Genet. 2000; 26: 146-147Crossref PubMed Scopus (447) Google Scholar). A prime consequence of the mutational defects in β-catenin regulation is constitutive activation of downstream β-catenin-TCF-regulated target genes, particularly genes with major effects on cell growth regulation and tumorigenesis, such as c-myc, CCND1, and MMP-7 (4Bienz M. Clevers H. Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1321) Google Scholar,5Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). In an effort to understand better the effects of Wnt-β-catenin-TCF pathway activation in cancer cells, we undertook studies to identify novel β-catenin-TCF-regulated target genes. We used oligonucleotide microarrays to identify transcripts with elevated expression after neoplastic transformation of the rat E1A-immortalized RK3E cell line by mutant β-catenin or γ-catenin or after ligand-induced activation of a β-catenin-estrogen receptor (ER) fusion protein. We found that expression of the rat Axil gene was strongly induced in the RK3E cell line in all three of these settings. Further studies established that the mouse and human homologs of Axil, known as conductin and AXIN2, respectively, were consistently induced by Wnt pathway activation. TCF proteins played a key role in AXIN2 induction. Unlike AXIN2, AXIN1 was not found to be a β-catenin-TCF-regulated gene. Prior studies have shown that the Axin1 and Axin2 proteins have roughly 45% amino acid identity and essentially identical functions in regulating β-catenin levels (7Zeng L. Fagotto F. Zhang T. Hsu W. Vasieck T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar, 8Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1114) Google Scholar, 9Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1127) Google Scholar, 10Yamamoto H. Kishida S. Uochi T. Ikeda S. Koyama S. Asashima M. Kikuchi A. Mol. Cell. Biol. 1998; 18: 2867-2875Crossref PubMed Scopus (175) Google Scholar, 28Kikuchi A. Cytokine Growth Factor Rev. 1999; 10: 255-265Crossref PubMed Scopus (69) Google Scholar). In addition to showing thatAXIN2 functions in a feedback repressor pathway regulating Wnt signaling, our findings on the differential effects of Wnt pathway activation on AXIN2 versus AXIN1expression suggest that potentially significant functional differences may exist between their protein products. Expression vectors for wild type and mutant (codon 33 substitution of tyrosine for serine, S33Y) forms of β-catenin and dominant negative Tcf-4 (Tcf-4ΔN31) have been described previously (29Kolligs F.T., Hu, G. Dang C.V. Fearon E.R. Mol. Cell. Biol. 1999; 19: 5696-5706Crossref PubMed Google Scholar). The pBabe-S33Y-ER-puro expression vector encoding a chimeric β-catenin-ER protein, in which full-length S33Y β-catenin sequences are fused in-frame to a mutated ER ligand binding domain, was generated by cloning the S33Y β-catenin cDNA into the BamHI and EcoRI sites of the retroviral plasmid pBabe-puro (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (713) Google Scholar). The reporter constructs pTOPFLASH, which contains three copies of an optimal TCF binding motif (CCTTTGATC), and pFOPFLASH, which contains three copies of a mutant motif (CCTTTGGCC), have been described previously (31Korinek V. Barker M. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2977) Google Scholar). Plasmid pCH110 (Amersham Biosciences) contains a functional lacZ gene cloned downstream from a cytomegalovirus early region promoter-enhancer element. The Axin2pcDNA3.1mycHis(−)B expression vector was a kind gift from Wanguo Liu (Mayo Clinic, Rochester, MN) (27Liu W. Dong X. Mai M. Seelan R.S. Taniguchi K. Krisnadath K.K. Halling K.C. Cunningham J.M. Boardman L.A. Qian C. Christensen E. Schmidt S.S. Roche P.C. Smith D.I. Thibodeau S.N. Nat. Genet. 2000; 26: 146-147Crossref PubMed Scopus (447) Google Scholar). DNA fragments containing human AXIN2 promoter sequences cloned upstream from a luciferase reporter gene were obtained by PCR amplification of genomic DNA, using primers generated from AXIN2 sequences in GenBank (accession no. AC00485). AXIN2 genomic DNA fragments were subcloned upstream from the luciferase reporter gene in the pGL3Basic reporter vector (Promega, Madison, WI), using the KpnI and NheI sites. The reporter gene vector AX2(1078WT)/Luc contains AXIN2 sequences from −1078 to +5 relative to the presumed transcription start site, and the vector AX2(181WT)/Luc contains AXIN2 sequences from −181 to +5. The forward primer for generating the AX(1078WT)/Luc vector was 5′-CCCGTTCAGCCCCTACCCTTCTTAG-3′, and the forward primer for the AX(181WT)/Luc vector was 5′-CAGCGCCTGATACTTAGATGAGC-3′; the reverse primer for generating both vectors was 5′-CAAGTCAGCAGGGGCTCATCTG-3′. Mutations in a presumptive TCF DNA binding site at bp −108 to −102 were obtained in vitro via a standard PCR-based mutagenesis strategy, generating the reporter gene vectors AX2(1078Mut)/Luc and AX2/(181Mut)/Luc. All plasmid sequences were confirmed by automated sequencing of double-stranded DNA templates. All cell lines were obtained from American Type Culture Collection (Rockville, MD), with the exception of the following: the amphotropic Phoenix packaging cell line, which was obtained from G. Nolan (Stanford University School of Medicine); the RAC311, RAC311/Wnt-1, RAC311/Wnt-1 9, C57/Vect, and C57/Wnt-1 lines (32Howe L. Subbaramaiah K. Chung W.J. Dannenberg A.J. Brown A.M.C. Cancer Res. 1999; 59: 1572-1577PubMed Google Scholar), all of which were obtained from L. Howe (Weill Medical College of Cornell University); Gli-transformed RK3E cells (33Luoro I.D. McKie-Bell P. Gosnell H. Brindley B.C. Bucy R.P. Ruppert J.M. Cell Growth Differ. 1999; 10: 503-516PubMed Google Scholar), which were obtained from J. M. Ruppert (University of Alabama at Birmingham); and the HT29/β-Gal and HT29/APC lines (34Morin P.J. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 93: 7950-7954Crossref Scopus (448) Google Scholar), which were obtained from B. Vogelstein (Johns Hopkins University School of Medicine). All cells were grown in 5% CO2 with medium containing 10% fetal bovine serum and penicillin/streptomycin, unless otherwise stated. HEK293, Phoenix, parental RK3E cells, RK3E cells neoplastically transformed by K-ras, Gli, β-catenin, and γ-catenin (29Kolligs F.T., Hu, G. Dang C.V. Fearon E.R. Mol. Cell. Biol. 1999; 19: 5696-5706Crossref PubMed Google Scholar, 35Kolligs F.T. Kolligs B. Hajra K.M., Hu, G. Tani M. Cho K.R. Fearon E.R. Genes Dev. 2000; 14: 1319-1331PubMed Google Scholar), RAC311 lines, C57 lines, and all human colon cancer lines, except for HT29, LS174T, RKO, and SW48 cells, were grown in Dulbecco's modified Eagle's medium (Invitrogen). LS174T cells were grown in minimum Eagle's medium α (Invitrogen), and SW48 cells were grown in L15 medium (Invitrogen) in the absence of CO2. RKO, HT29, HT29/β-Gal, and HT29/APC cells were cultured in McCoy's medium (Invitrogen). Hygromycin B (Sigma) was included at a concentration of 0.6 mg/ml for the HT29/β-Gal and HT29/APC cells. Insulin (10 μg/ml; Sigma) was added to the media for the C57 lines. A clonal RK3E cell line expressing the β-catenin S33Y-ER fusion protein was obtained after retroviral transduction of RK3E cells with supernatants from amphotrophic Phoenix cells transfected with pBabe-S33Y-ER-puro. Drug selection on the pBabe-S33Y-ER-puro-transduced RK3E cells was carried out in puromycin (Sigma) at a concentration of 1.0 μg/ml. A single resistant colony was isolated by ring cloning and expanded into a stable cell line, termed RK3E/S33Y-ER. The RK3E/S33Y-ER line was maintained subsequently in 0.5 μg/ml puromycin. To activate the S33Y-ER fusion protein, the RK3E/S33Y-ER cells were treated with medium supplemented with 0.5 μm 4-hydroxytamoxifen (4-OH-T) (Sigma), made from a stock concentration of 100 μm 4-OH-T in 100% ethanol. To inhibit new protein synthesis in RK3E/S33Y-ER cells, the medium was supplemented with cycloheximide (Sigma) at a concentration of 1 μg/ml. To assess the effects of dominant negative TCF-4 on AXIN1 and AXIN2 gene expression, a retroviral TCF-4ΔN31 expression construct (29Kolligs F.T., Hu, G. Dang C.V. Fearon E.R. Mol. Cell. Biol. 1999; 19: 5696-5706Crossref PubMed Google Scholar) was used to transduce two RK3E lines that had been transformed neoplastically by mutant β-catenin (RK3E/ΔN47-B and RK3E/ΔN132-A) (29Kolligs F.T., Hu, G. Dang C.V. Fearon E.R. Mol. Cell. Biol. 1999; 19: 5696-5706Crossref PubMed Google Scholar) as well as the SW480 and DLD1 colon cancer lines. Empty vector (pPGS-Neo) control transductions of the two RK3E and two colon lines were carried out in parallel. The TCF4ΔN31- and empty vector-transduced cells were subsequently selected for 7–10 days in 1.0–1.5 mg/ml G418 (Sigma). To assess the effects of wild typeAPC gene function on AXIN1 and AXIN2gene expression, HT29/β-Gal and HT29/APC cells were treated with 150 μm ZnCl2 for induction of the controllacZ and wild type APC genes, respectively. Trizol (Invitrogen) extraction and purification with the RNeasy Cleanup Kit (Qiagen, Chatsworth, CA) was used to prepare total RNA from five samples: parental RK3E cells; RK3E/S33Y-ER cells either mock (ethanol)-treated or 4-OH-T-treated for 24 h; a pool of equal masses of RNA from seven clonal RK3E lines transformed neoplastically by mutant β-catenin (29Kolligs F.T., Hu, G. Dang C.V. Fearon E.R. Mol. Cell. Biol. 1999; 19: 5696-5706Crossref PubMed Google Scholar); and a pool of equal masses of RNA from five clonal RK3E lines transformed neoplastically by γ-catenin (35Kolligs F.T. Kolligs B. Hajra K.M., Hu, G. Tani M. Cho K.R. Fearon E.R. Genes Dev. 2000; 14: 1319-1331PubMed Google Scholar). Gene expression analyses on the five samples were carried out with commercial high density oligonucleotide arrays (Affymetrix, Santa Clara, CA), using protocols and methods developed by the supplier. Arrays were scanned using the GeneArray scanner (Affymetrix), and image analysis was performed with GeneChip 4.0 software (Affymetrix), which stores the results for each feature in .CEL files. Each RG_U34A chip consists of 534 × 534 probes (24 × 24 μm each) that are 25-base long single-stranded DNA sequences. There are typically 16 pairs of features (probe pairs) for each of the transcripts (probe sets) and a total of 8,799 probe sets. Half of the features are complementary to a specific sequence (perfect match = PM features), the other half have an identical match except a central base has been altered (mismatch = MM features). We have developed software to read .CEL files and perform some processing of the data, available at dot.ped.med.umich.edu:2000/ourimage/pub/shared/Affymethods.html. The chip for the parental RK3E sample was selected as a standard. Probe pairs for which PM-MM < −100 on the standard were excluded from the analysis. One-sided signed rank tests of the PM-MM values for each probe set on each chip were obtained to help judge whether transcripts were detectable. The average intensity for each probe set was computed as the mean of the PM-MM differences, after trimming away the 25% highest and lowest differences. A set of 3,692 reference probe sets was selected for use in normalization, these being the probe sets that gave p < 0.05 for all five chips for the test of detectability. A normalization factor for each chip was obtained using the reference probe sets by computing the antilogarithm of the mean log ratios of the average intensities for the selected chip divided by the standard. The average intensities were divided by this factor to obtain the normalized intensities for the probe sets. When computing fold change indices, we replaced intensities less than 10 by 10 before forming ratios to avoid negative or spuriously large fold change numbers. Total RNA was extracted from cells with Trizol, and Northern blot analysis was performed. Approximately 15–20 μg of total RNA was separated on a 1.2% formaldehyde-agarose gel and transferred to Zeta-Probe GT membranes (Bio-Rad) by capillary action. cDNA probes to detect rat Axil, mouseconductin, rat Axin1, and human AXIN1expression were generated by RT-PCR, using primers derived from sequences in GenBank. The probe to detect AXIN2 was generated by PCR using the Axin2pcDNA3myc3.1 plasmid (provided by W. Liu; Mayo Clinic). The sequences of all PCR products probes were confirmed by automated sequencing. All probes were random labeled with [α32P]dCTP using Rediprime (Invitrogen) and hybridized to the membrane with RapidHyb Buffer (Invitrogen) according to the manufacturer's protocol. All Northern blots were stripped and hybridized to a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe to control for RNA loading and transfer efficiency. Whole cell lysates were prepared in radioimmunoprecipitation assay buffer (Tris-buffered saline (TBS), 0.5% deoxycholic acid, 0.1% SDS, and 1% Nonidet P-40 with complete protease inhibitors (Roche Molecular Biochemicals)). Protein concentration was determined by the bicinchoninic acid assay (Pierce Biochemicals), and 50 μg of total protein from each sample was separated on 10% SDS-polyacrylamide gels. Proteins were transferred to Immobilon P membranes (Millipore, Bedford, MA) by semidry electroblotting. Immunoblot analyses were carried out with the anti-conductin (S-19) or anti-conductin (M-20) goat polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:500 dilution in 1× TBS with 5% dry milk and 0.5% Tween followed by incubation with a horseradish peroxidase-conjugated donkey anti-goat antibody (Pierce Biochemicals) at a 1:10,000 dilution. To verify equal loading of the samples, membranes were incubated with a rabbit polyclonal antibody against β-actin (Sigma) followed by a horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (Pierce Biochemicals). Antibody complexes were detected with the ECL Western blot kit (Amersham Biosciences) and exposure to X-Omat-AR film (Eastman Kodak). Total RNA was isolated with Trizol from 42 snap frozen, primary ovarian endometrioid type adenocarcinomas (OEAs) that had been analyzed in detail previously for β-catenin nuclear localization and mutational defects in the β-catenin, APC, AXIN1, and AXIN2 genes (36Wu R. Zhai Y. Fearon E.R. Cho K.R. Cancer Res. 2001; 61: 8247-8255PubMed Google Scholar). The RNA was used for real time RT-PCR studies of AXIN2 and HPRT gene expression. In brief, first strand cDNA was synthesized from DNase I-treated mRNA samples using random hexamer primers (Amersham Biosciences) and Superscript II (Invitrogen). For PCR with a Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA), 5 ng of cDNA from each tumor sample was used in each reaction. For AXIN2, PCR was performed in 96-well plates in a 25-μl reaction volume containing 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 0.2 μmAXIN2 forward primer (5′-CAAGGGCCAGGTCACCAA-3′), 0.2 μmAXIN2reverse primer (5′-CCCCCAACCCATCTTCGT-3′), and 0.2 μmdye-labeled AXIN2 probe (5′-CCCATGTCTGTCTCTTCCAACACCAGG-3′) (Synthetic Genetics, San Diego). For HPRT, the 25-μl reaction contained 1× TaqMan Universal PCR Master Mix, 0.2 μm forward HPRT primer (5′-TTCCTCGAGATGTGATGAAGGA-3′), 0.2 μm reverseHPRT primer (5′-CCAGCAGGTCAGCAAAGAATT-3′), and 0.2 μm dye-labeled HPRT probe (5′-CCATCACATTGTAGCCCTCTGTGTGCTC-3′) (Applied Biosystems). TheAXIN2 probe had a carboxyfluorescein label at its 5′-end, and the HPRT probe had a VICTM label at its 5′-end. Both probes had carboxytetramethyl rhodamine labels at their 3′-ends. The AXIN2 and HPRT PCRs were performed in duplicate for each tumor sample, and AXIN2 and HPRT reactions were performed in adjacent wells. The following PCR conditions were used: 2 min at 50 °C and 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Using the software accompanying the Prism 7700 detector, theHPRT signals were used for normalization. Student'st test was used to determine the significance of differences in AXIN2 expression between the 12 OEAs with strong nuclear staining for β-catenin and mutations in the β-catenin, APC, AXIN1, or AXIN2 genes and the 30 OEAs lacking strong nuclear β-catenin staining and pathway mutations. Immunohistochemcial analysis of AXIN2 expression in OEAs was performed as described previously (36Wu R. Zhai Y. Fearon E.R. Cho K.R. Cancer Res. 2001; 61: 8247-8255PubMed Google Scholar). In brief, 5-μm sections of formalin-fixed, paraffin-embedded tissues were mounted on Probe-On slides (Fisher Scientific), deparaffinized in xylene, and then rehydrated into distilled water through graded alcohols. Antigen retrieval was enhanced by microwaving the slides in citrate buffer (pH 6.0; Biogenex, San Ramon, CA) for 15 min. Endogenous peroxidase activity was quenched with 6% hydrogen peroxide in methanol, and the slides were blocked with 1.5% normal horse serum for 1 h. Sections were then incubated with the anti-conductin (M-20) goat polyclonal antibody (Santa Cruz Biotechnology) at a 1:500 dilution overnight at 4 °C followed by a biotinylated horse anti-goat secondary antibody at a 1:200 dilution for 30 min at room temperature. Antigen-antibody complexes were detected with the avidin-biotin peroxidase method using 3,3′-diaminobenzidine as a chromogenic substrate (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Immunostained sections were lightly counterstained with hematoxylin and then examined by light microscopy. For all luciferase reporter assays, cells were plated in 35-mm six-well plates 12–24 h before transfection. Transfections were performed with FuGENE 6 (Roche Molecular Biochemicals) for 24–36 h according to the manufacturer's protocol. Lysates were collected in 1× Reporter Lysis Buffer (Promega). TCF transcriptional activity was measured as the ratio of luciferase activity from the pTOPFLASH vector to the pFOPFLASH vector. All luciferase activities were normalized for transfection efficiency by cotransfection with pCH110 and measurement of β-galactosidase activity. To assess the effects of AXIN2 on wild type and mutant β-catenin-induced TCF activity, 293 cells were cotransfected with 0.25 μg of pTOP- or pFOPFLASH, 0.5 μg of a pcDNA3 vector encoding wild type or S33Y mutant β-catenin (29Kolligs F.T., Hu, G. Dang C.V. Fearon E.R. Mol. Cell. Biol. 1999; 19: 5696-5706Crossref PubMed Google Scholar), 1 μg of Axin2pcDNA3.1mycHis(−)B, and 0.25 μg of pCH110. To confirm stable expression of TCF-4ΔN31in β-catenin-transformed RK3E cells as well as SW480 and DLD1 cells, cells were cotransfected with 1 μg of pTOPFLASH or pFOPFLASH and 1 μg of PCH110. For reporter gene assays with AXIN2 promoter constructs, DLD1 cells were cotransfected with 1 μg of AX2(1078WT)/Luc or AX2(1078Mut)/
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