Epithelial proliferation induces novel changes in APC expression

The role of wild-type adenomatous polyposis coli (APC) protein in native epithelia is poorly understood. The present study examined the relationships between wild-type APC and beta-catenin expression in an established model of hyperproliferation, transmissible murine colonic hyperplasia (TMCH). Distal colonic crypts isolated from normal or TMCH mice were: (i) fractionated into cytosolic and nuclear components for Western blotting and immunoprecipitation (IP), (ii) extracted for total RNA isolation for Northern blotting and, (iii) analysed immunohistochemically by confocal microscopy. Western blots performed sequentially through day 12 TMCH with N-terminal APC antibodies revealed increased abundance of approximately 312 kDa (p312) protein by day 6 (4.0 +/- 0.75-fold, n = 6) that peaked by day 9, before declining by day 12. A approximately 130 kDa (p130) band appeared at day 9 and increased by day 12 (1.5 +/- 0.11-fold, n = 6). A C-terminal antibody detected only p312. APC mRNA level did not change during TMCH and appearance of p130 was not due to alternative splicing. Co-IP with N-terminal anti-APC antibodies, revealed APC's association with beta-catenin both at day 6 and day 12. p130, but not p312, associated predominantly with beta-catenin at day 12 during co-IP with anti-beta-catenin. p130 also selectively accumulated in the nucleus, bound to nuclear beta-catenin at day 12. Immunocytochemistry with N-terminal antibodies revealed an increasing crypt base : surface gradient of APC within the apical pole/apical-lateral membranes at day 6. At day 12, intense apical/cytoplasmic and occasional nuclear staining along the longitudinal crypt axis was observed. Full-length APC increases during epithelial hyperproliferation and may represent a homoeostatic response. The dramatic increase in cytoplasmic and sporadic nuclear APC staining at day 12 with N-terminal antibodies may represent p130. The nuclear accumulation of p130 may be a novel mechanism regulating nuclear beta-catenin function during TMCH.