Activation of β-Catenin-Tcf Signaling in Colon Cancer by Mutations in β-Catenin or APC

American Association for the Advancement of Science (AAAS) - Tập 275 Số 5307 - Trang 1787-1790 - 1997
Patrice J. Morin1, Andrew B. Sparks2, Vladimír Kořínek3, Nick Barker3, Hans Clevers3, Bert Vogelstein1, Kenneth W. Kinzler2
1P. J. Morin and B. Vogelstein, Howard Hughes Medical Institute and Johns Hopkins Oncology Center, 424 North Bond Street, Baltimore, MD 21231, USA.
2A. B. Sparks and K. W. Kinzler, Johns Hopkins Oncology Center, 424 North Bond Street, Baltimore, MD 21231, USA.
3V. Korinek, N. Barker, H. Clevers, Department of Immunology, University Hospital, 35008 GA, Utrecht, The Netherlands.

Tóm tắt

Inactivation of the adenomatous polyposis coli ( APC ) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by β-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of β-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of β-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or β-catenin.

Từ khóa


Tài liệu tham khảo

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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 nonspecific transcription (by using the pFOPFLASH control).

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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 pCI-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.

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 pCI-neo (Promega) to produce pCI-neo-β-cat. The pCI-neo-β-cat Δ45 and S33Y mutants were generated by replacing codons 1 to 89 in pCI-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).

We thank D. Levy for construction of APC vectors. Supported by the Clayton Fund and by NIH grant CA57345.