RESUMO
BACKGROUND & AIMS: The APC tumor suppressor is well known for its ability to regulate Wnt signaling through mediation of beta-catenin levels in the cell. Transient over expression of the tumor suppressor gene APC in colon cancer cells prevents entry into S phase of the cell cycle, a phenotype only partially restored by cotransfection of a transcriptionally active form of beta-catenin. In an attempt to define its transcription-independent tumor suppressor functions, we tested whether APC directly affects DNA replication. METHODS: A transcriptionally quiescent in vitro DNA replication system, the polymerase chain reaction, DNA binding assays, and transient transfections in colon cancer cell lines were used to determine the effects of APC on DNA replication and the mechanism by which it works. RESULTS: We report that exogenous full-length APC inhibits replication of template DNA through a function that maps to amino acids 2140-2421, a region of the protein commonly lost by somatic or germline mutation. This segment of APC directly interacts with DNA, while mutation of the DNA-binding S(T)PXX motifs within it abolishes DNA binding and reduces inhibition of DNA replication. Phosphorylation of this segment by cyclin-dependent kinases also reduces inhibition of DNA replication. Furthermore, transient transfection of an APC segment encoding amino acids 2140-2421 into a colon cancer cell line with mutant APC prevents cell cycle progression into or through S phase. CONCLUSIONS: Our results suggest that APC can negatively regulate cell cycle progression through inhibition of DNA replication by direct interaction with DNA.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/genética , Replicação do DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteína da Polipose Adenomatosa do Colo/química , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/fisiopatologia , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G1/fisiologia , Humanos , Oócitos/fisiologia , Fosforilação , Estrutura Terciária de Proteína , Fase S/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Xenopus laevisRESUMO
The APC tumor suppressor is found in nonproliferating epithelial cells of the colonic crypts and is mutated in most colorectal tumors. To understand the function of APC in normal epithelium and how its loss leads to tumor formation, we tested whether APC is a mediator of apoptosis using an in vitro assay that monitors caspase-3-mediated cleavage of lamin B protein or a colorimetric substrate in a cell-free Xenopus egg extract. Recombinant APC protein accelerates apoptosis-associated caspase activity independently of ongoing transcription and protein synthesis. Conversely, the addition of mutant APC and immunodepletion of Xenopus APC decelerates apoptosis-associated caspase activity. Acceleration of apoptosis by APC is abolished by the caspase-8 inhibitor Z-IETD-FMK, demonstrating that caspase-8 is an essential component of APC-mediated apoptosis. These results suggest that the induction of apoptosis may be one role of APC in tumor suppression and that this mechanism is independent of beta-catenin-mediated effects on transcription.
