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 h h-catenin-mediated effects on transcription. (Mol Cancer Res 2005; 3(2):78 -89)
Background & Aims-The APC tumor suppressor is well known for its ability to regulate Wnt signaling through mediation of β-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 co-transfection of a transcriptionally active form of β-catenin. In an attempt to define its transcription-independent tumor suppressor functions, we tested whether APC directly affects DNA replication.
The adenomatous polyposis coli (APC) tumor suppressor is inactivated by mutation in most colorectal tumors. APC is a component of the Wnt signaling pathway and is best known for its ability to downregulate b-catenin and consequent effects on transcriptional regulation. Previous work demonstrated that APC accelerates apoptosis-associated caspase activity independently of transcription, and suggested novel tumor suppressor functions of APC. In this work, we have mapped the APC apoptosis-accelerating region to amino acids (aa) 1-760 by testing a series of nonoverlapping APC segments. Interestingly, this segment corresponds to a stable group II caspase cleavage product of APC released during apoptosis that includes the aminoterminal aa1-777. Mutation of the APC aspartic acid residue at position 777 to an alanine completely abolished in vitro cleavage of APC by a recombinant group II caspase and rendered the full-length protein unable to accelerate apoptosis in vitro. A truncated APC protein associated with familial and sporadic colorectal cancer, also unable to accelerate apoptosis in vitro and in vivo, is resistant to group II caspase cleavage. These results demonstrate that cleavage of APC and the subsequent release of an aminoterminal segment are necessary for the transcriptionindependent mechanism of APC-mediated apoptosis.
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