Activation of β-Catenin-Tcf Signaling in Colon Cancer by Mutations in β-Catenin or APC
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About 50% of the Western population develop colorectal adenomas by the age of 70 [D. Ransohoff and C. Lang N. Engl. J. Med. 325 37 (1991) and at least 85% of these tumors contain APC mutations [(13)
Y. Miyoshi et al . Hum. Mol. Genet. 1 229 (1992)
J. Jen et al . Cancer Res. 54 5523 (1994).
Su L.-K., et al., Cancer Res. 53, 2728 (1993).
Munemitsu S., et al., Cancer Res. 54, 3676 (1994)K. J. Smith et al., ibid., p. 3672.
Su L. K., et al., ibid. 55, 2972 (1995).
Rubinfeld B., et al., ibid.1023.
Eccles D. M., et al., Am. J. Hum. Genet. 59, 1193 (1996);
WT and mutant APC constructs (2 μg) were transfected into 293 SW480 and HCT116 cells with lipofectamine (Gibco-BRL Gaithersburg MD). Protein was harvested 24 hours later and subjected to immunoblot analysis with APC monoclonal antibody FE9 (23). In HCT116 and 293 cells exogenous WT APC comigrated with the endogenous APC. In SW480 cells APC1309Δcomigrated with the endogenous mutant APC. In all other cases the nonfunctional APC constructs (APC331Δ APC1309Δ and APC1941Δ) produced as much or more protein than the CRT-functional forms of APC (WT APC and APC2644Δ).
Genomic DNA was isolated from paraffin-embedded normal and tumor tissue from the patient from whom the HCT116 cell line was derived [S. E. Goelz S. R. Hamilton B. Vogelstein Biochem. Biophys. Res. Commun. 130 118 (1985). A 95-bp polymerase chain reaction (PCR) product encompassing the mutation was then amplified by PCR and directly sequenced with ThermoSequenase (Amersham). The 3-bp deletion was observed in tumor but not in normal tissue.
Genomic DNA was isolated from frozen-sectioned colorectal cancers (13) and a 1001-bp PCR product containing exon 3 of CTNNB1 was then amplified by PCR and directly sequenced with ThermoSequenase (Amersham). An ACC to GCC change at codon 41 (T41A) and a TCT to TTT at codon 45 (S45F) was observed in one and two tumors respectively.
Joslyn G., et al., ibid., p. 601;
Nishisho I., et al., ibid., p. 665.
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).
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.