Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC

Science

Volumn 275, Issue 5307, 1997, Pages 1787-1790

  Morin, Patrice J ^a   Sparks, Andrew B ^a   Korinek, Vladimir ^b   Barker, Nick ^b   Clevers, Hans ^b   Vogelstein, Bert ^a   Kinzler, Kenneth W ^a  

^a JOHNS HOPKINS UNIVERSITY   (United States)

^b UNIVERSITY MEDICAL CENTER UTRECHT   (Netherlands)

Author keywords

[No Author keywords available]

Indexed keywords

BETA CATENIN; TRANSCRIPTION FACTOR;

ARTICLE; COLON CANCER; COLON POLYPOSIS; HUMAN; HUMAN CELL; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; TRANSACTIVATION; TUMOR SUPPRESSOR GENE;

ADENOMATOUS POLYPOSIS COLI PROTEIN; BETA CATENIN; COLONIC NEOPLASMS; CYTOSKELETAL PROTEINS; GENE EXPRESSION REGULATION, NEOPLASTIC; GENES, APC; GENES, REPORTER; GERM-LINE MUTATION; HUMANS; MUTATION; PHOSPHORYLATION; SIGNAL TRANSDUCTION; TCF TRANSCRIPTION FACTORS; TRANS-ACTIVATORS; TRANSCRIPTION FACTORS; TRANSCRIPTION, GENETIC; TRANSFECTION; TUMOR CELLS, CULTURED;

EID: 0030949463     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5307.1787     Document Type: Article

References (43)

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39. Lipofectamine was used to cotransfect SW480 cells with an internal control (0.5 μg of pCMV-βgal), a reporter construct (0.5 μg of pTOPFLASH or pFOPFLASH), and the indicated amount of the various APC expression vectors. The pTOPFLASH reporter contained an optimized Tcf-binding site 5′ of a luciferase reporter gene, whereas pFOPFLASH contained a mutated site that does not bind Tcf (12). The amount of DNA in each transfection was kept constant by the addition of an appropriate amount of empty expression vector (pCEP4). Luciferase and β-galactosidase activities were determined 16 hours after transfection. Luciferase activity was corrected for transfection efficiency (by using the control β-galactosidase activity) and non-specific transcription (by using the pFOPFLASH control).
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41. Overlapping segments constituting the entire CTNNB1 were amplified by reverse transcriptase (RT)-PCR from SW480, DLD1, HCT116, and SW48 cells and sequenced directly with ThermoSequenase (Amersham). In the case of HCT116, a PCR product containing the deleted region was also cloned into pCl-neo (Promega, Madison, WI), and multiple clones corresponding to each allele were individually sequenced. Sequences of the PCR and sequencing primers used are available on request.
42. β-Catenin expression constructs were prepared as follows. WT CTNNB1 was amplified by RT-PCR from SW480 cells and cloned into the mammalian expression vector pCl-neo (Promega) to produce pCl-neo β-cat. The pCl-neo-β-cat Δ45 and S33Y mutants were generated by replacing codons 1 to 89 in pCl-neo-β-cat with a PCR product encoding the equivalent region from HCT116 or SW48 cDNA, respectively. The structures of all constructs were verified by sequence analysis. Details concerning the constructs and the primer sequences are available on request. Lipofectamine was used to cotransfect 293 cells with an internal control (0.1 μg of CMV-βgal), a reporter (0.5 μg of pTOPFLASH or pFOPFLASH), a Tcf-4 expression vector (0.5 μg of pCDNA-TCF4), and β-catenin (0.5 μg) or dominant-negative hTcf-4 (1.0 μg) (12) expression vectors. CRT was determined as in (25).
43. We thank D. Levy for construction of APC vectors. Supported by the Clayton Fund and by NIH grant CA57345.