The APC Tumor Suppressor Promotes Transcription-Independent Apoptosis In vitro

Abstract

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)

APC as a mobile scaffold: Regulation and function at the nucleus, centrosomes, and mitochondria

“…In contrast, APC cancer mutants more often are connected with survival of colon tumor cell lines (31,45,46). In particular, the Arm domain of APC was linked to apoptosis, although whether its accumulation triggers or prevents apoptosis is not clear (47).…”

Caspase cleavage of the APC tumor suppressor and release of an amino-terminal domain is required for the transcription-independent function of APC in apoptosis

“…It has been used successfully by a number of laboratories to define the roles of several apoptotic mediators (Newmeyer et al, 1994;Fasschon et al, 1997). In our previous experiments, full-length APC (APC FL) protein accelerated the apoptotic time course, whereas mutant or truncated APC protein was unable to do so (Steigerwald et al, 2005). In this study, we further investigate the transcription-independent apoptosis-associated function of APC.…”

“…Several experimental lines of evidence suggest a role for APC in the regulation of cell death, including our previous work that used an in vitro assay to separate transcription-independent from transcription-dependent functions of APC in apoptosis (Steigerwald et al, 2005). This assay uses a cell-free Xenopus laevis egg extract to monitor the time course of effector caspase cleavage of a colorimetric substrate; exogenous proteins can be added or subtracted to change this time course.…”

“…(c) Representative results from an in vitro apoptosis assay (typical of at least three experiments). Xenopus laevis egg extract for in vitro apoptosis studies was prepared and reconstituted as described previously (Steigerwald et al, 2005). Apoptosis is measured by cleavage of a colorimetric substrate, Z-DEVD-pNA, by activated caspase 3 and is displayed as an increase in absorbance at 405 nm versus time in hours.…”

“…Expression of GFP-tagged proteins in adherent cells was evaluated with an anti-GFP antibody. egg extract (Steigerwald et al, 2005). To validate the in vitro result, the colon cancer cell lines SW480 and HCT116 were transiently transfected with mammalian expression vectors encoding APC aa1-777, APC aa761-1339 or APC1309 tagged with GFP.…”