Basic–alimentary tractThe Intestinal Wnt/TCF Signature
Section snippets
Cell Culture
CRC cell lines LS174T and DLD1, stably expressing inducible dominant-negative (dn)TCF1 or dnTCF4, were generated as previously described.8 The Wnt pathway activity in the CRC cells was determined as described previously3 using the optimized TCF reporter pTopGlow and its negative control pFopGlow, constructed in our laboratory.9
Oligonucleotide Microarray Analysis of CRC Cell Lines
RNA was isolated after 10 and 20 hours induction of the dnTCFs. RNA quality was assessed using capillary gel electrophoresis (BioAnalyzer; Agilent Technologies).
Inhibition of the Constitutively Active Wnt Pathway in CRC Cells
N-terminally truncated TCFs do not bind β-catenin and act as potent inhibitors of endogenous β-catenin/TCF complexes.12 Tcf4 is expressed physiologically in the intestine.13 In our hands, the DNA-binding characteristics of TCF4 and TCF1 are essentially identical. For TCF target gene identification, we generated a panel of cell clones from the CRC cell lines LS174T (mutationally activated allele of the CTNNB1 gene encoding β-catenin) and in DLD1 (mutant APC), in which the Wnt cascade could be
Discussion
The current study builds on previous cell line–based work from our laboratory.8 Here, we provide a comprehensive identification of TCF4 targets in 2 different cell lines carrying 2 different dnTCF genes, and by performing differential gene-expression analysis on a genome-wide oligonucleotide array platform. Moreover, we relate these findings to expression profiles of a set of human adenomas and adenocarcinomas. The Wnt/TCF signature gene set defines the core program activated by TCF4 in
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L.G.V.D.F. and J.S.-B. contributed equally to this article.