G 1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: Phenotypes reversed by a tumorigenic mutation

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Abstract
The tumor suppressor Smad4/Dpc4 is a transcription activator that binds specific DNA sequences and whose nuclear localization is induced after exposure to type β transforming growth factor-like cytokines. We explored an inducible system in which Smad4 protein is activated by translocation to the nucleus when cell lines that stably express wild-type or mutant Smad4 proteins fused to a murine estrogen receptor domain are treated with 4-hydroxytamoxifen. This induced Smad4-mediated transcriptional activation and a decrease in growth rate, attributable to a cell cycle arrest at the G 1 phase and an induction of apoptosis. A tumor-derived mutation (Arg-100 → Thr) affecting a residue critical for DNA-binding demonstrated an “oncogenic” phenotype, having decreases in both the G 1 fraction and apoptosis and, consequently, an augmentation of population growth. This model should be useful in the exploration and control of components that lie further downstream in the Smad4 tumor-suppressor pathway.