The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). We show that disruption of beta-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21(CIP1/WAF1) promoter. Following disruption of beta-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21(CIP1/WAF1) transcription, which in turn mediates G1 arrest and differentiation. Thus, the beta-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells.

RNAi Double-stranded RNAs (dsRNAs) were made using gld-2 cDNAs (pJK830, exons 2-8 or pJK831, exons 16-18) as templates. Young adults were either injected with 2 mg ml 21 gld-2 dsRNA or soaked in 10 ml of 2 mg ml 21 gld-2 dsRNA for 12 h at 20 8C or mock-treated by injection with M9 buffer. Embryos were collected at defined intervals after treatment and processed together. Poly(A) polymerase assayProteins were in vitro translated using the TNT coupled transcription-translation system (Promega), and assayed using buffer conditions essentially as described 26 . For scintillation counting, poly(A) (Roche) was used as substrate. For gel assays, we used RNA oligo, C 35 A 10 (Dharmacon), a 45-nucleotide and supplemental 1 mM MgCl 2 . Products were analysed on 12% sequencing gels.

The Forkhead transcription factors AFX, FKHR and FKHR-L1 are orthologues of DAF-16, a Forkhead factor that regulates longevity in Caenorhabditis elegans. Here we show that overexpression of these Forkhead transcription factors causes growth suppression in a variety of cell lines, including a Ras-transformed cell line and a cell line lacking the tumour suppressor PTEN. Expression of AFX blocks cell-cycle progression at phase G1, independent of functional retinoblastoma protein (pRb) but dependent on the cell-cycle inhibitor p27kip1. Indeed, AFX transcriptionally activates p27kip1, resulting in increased protein levels. We conclude that AFX-like proteins are involved in cell-cycle regulation and that inactivation of these proteins is an important step in oncogenic transformation.