Constitutive transcriptional activation by a β-catenin-Tcf complex in APC(-/-) colon carcinoma

Science

Volumn 275, Issue 5307, 1997, Pages 1784-1787

  Korinek, Vladimir ^a   Barker, Nick ^a   Morin, Patrice J ^a   Van Wichen, Dick ^a   De Weger, Roel ^a   Kinzler, Kenneth W ^a   Vogelstein, Bert ^a   Clevers, Hans ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

BETA CATENIN; TRANSCRIPTION FACTOR;

ARTICLE; CELL STRAIN HT29; CELL TRANSFORMATION; COLON CARCINOMA; COLON POLYPOSIS; HUMAN; HUMAN CELL; PRIORITY JOURNAL; TRANSACTIVATION; TUMOR SUPPRESSOR GENE;

ADENOMATOUS POLYPOSIS COLI PROTEIN; AMINO ACID SEQUENCE; ANIMALS; BETA CATENIN; CELL LINE; CELL TRANSFORMATION, NEOPLASTIC; CLONING, MOLECULAR; COLON; COLONIC NEOPLASMS; CYTOSKELETAL PROTEINS; GENE EXPRESSION REGULATION, NEOPLASTIC; GENES, APC; GENES, REPORTER; HUMANS; INTESTINAL MUCOSA; MICE; MOLECULAR SEQUENCE DATA; SIGNAL TRANSDUCTION; TCF TRANSCRIPTION FACTORS; TRANS-ACTIVATION (GENETICS); TRANS-ACTIVATORS; TRANSCRIPTION FACTORS; TRANSFECTION; TUMOR CELLS, CULTURED;

EID: 0030975671     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5307.1784     Document Type: Article

References (28)

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18. A genomic fragment encoding the HMG-box region of hTcf-4 (7) was used to probe a human 12-week fetal cDNA library in Lambda GT-11. Positive clones were subcloned into pBluescriptSK and sequenced.
19. Northern blot hybridizations (7) were performed with full-length hTcf-1, hLef-1, and hTcf-4 cDNA. Colon epithelial cells were freshly prepared from a mucosal preparation dissected from a healthy surgical colon sample. The sample was minced and incubated with 1 mM dithiothreitol (DTT) in Hanks' medium to remove mucus. Single-cell suspensions were prepared by incubation at room temperature in 0.75 mM EDTA in Hanks' medium. Epithelial cells were separated from lymphocytes by Percoll gradient centrifugation.
20. In situ hybridization of 6 μm frozen sections of healthy colon biopsy samples was performed as described [E. van Hoffen et al., Am. J. Pathol. 149, 1991 (1996)]. hTcf-4 cDNA encoding amino acids 200 to 310 was amplified and labeled with Dig-11-dUTP (Boehringer Mannheim) by PCR. After hybridization and washing, the sections were sequentially incubated with mouse antibody to Dig (Boehringer) and a horseradish peroxidase-conjugated rabbit antibody to mouse immunoglobulin (Dako, Glostrup, Denmark). The signal was visualized with diaminobenzidine, which produces a reddish-brown precipitate. Blue counterstaining was performed with hematoxylin.
21. 2. Complementary DNAs encoding Myc-tagged versions of β-catenin and hTcf-4 were inserted into the mammalian expression vector pCDNA (Invitrogen). Sequences of pTOPCAT, pFOPCAT, pTOPFLASH, and pFOPFLASH are available upon request. pCATCONTROL, encoding the CAT enzyme under the control of the SV40 promoter, was purchased from Promega.
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26. Gel retardation assays were performed as described (7). Extracts were prepared from intact nuclei that were washed four times to avoid contamination with cytoplasmic β-catenin. As the optimal Tcf-Lef probe, we used a double-stranded 15-nucleotide oligomer CCCTTTGATCTTACC; the control probe was CCCTTTGGCCTTACC. All oligonucleotides were from Isogen (Maarssen, Netherlands). The β-catenin antibody was purchased from Transduction Laboratories (Lexington, KY). A typical binding reaction contained 3 μg of nuclear protein, 0.1 ng of radiolabeled probe, and 100 ng of deoxyinosine-deoxycytidine (dldC) 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, antibody was added, and the samples were incubated for a further 20 min.
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28. We thank M. Reifer for reading the manuscript, H. C. Asheim for the cDNA library, and A.-R. v.d.Vuurst de Vries and J. C. Koningsberger for preparation of colon samples. Supported by grants to H.C. from the Dutch Organization of Scientific Research and by NIH grant CA57345.