CHARACTERIZING THE NUCLEAR LOCALIZATION OF ADENOMATOUS POLYPOSIS COLI PROTEIN

Mutation of tumor suppressor protein Adenomatous Polyposis Coli (APC) has been shown to be an early, if not rate limiting step in the progression of both hereditary and sporadic colorectal cancers. The observation that APC can shuttle in and out of the nucleus, and the identification of two highly conserved nuclear localization sequences (NLSs) strongly support a role for APC in the nuclear compartment. Little is currently known about the function(s) of nuclear APC or how nuclear functions contribute to tumor suppression. To further evaluate the role of APC in the nucleus we generated the APC mNLS mouse. The APC mNLS mice carry amino acid changes in both APC NLSs designed to inhibit interaction with importin-α without disrupting APC's other cellular functions. Generation of the mNLS mice is ongoing, with fully congenic animals expected in early 2009. In the interim, I have conducted a preliminary analysis of the phenotype of the non-congenic APC mNLS animals. Evaluation of the subcellular localization of APC in the intestinal epithelium of the APC mNLS animals revealed that APC retains its ability to shuttle in and out of the nucleus, even in the homozygous mutant mice. While a mild defect in nuclear import could be detected in the differentiated cells at the colonic luminal surface, the more undifferentiated cells in the lower regions of the intestinal crypts displayed a normal localization of APC. The APC mNLS-/- mice were viable and did not display intestinal polyposis. However, subtle differences were detected between mice that carry the APC mNLS mutations and their wild type (WT) littermates. These differences included higher levels of β-catenin in the intestinal epithelium of the APC mNLS mutant mice and an increased incidence of lymphoid nodules compared to their littermates with WT APC. To further evaluate the need for APC in the nuclear compartment, I exposed cultured cells expressing WT APC or APC with mutant NLSs to ultraviolet (UV) light. UV-irradiated cells displayed a nuclear accumulation of both the mutant and WT forms of APC with implications for an importin-α independent mechanism of nuclear import. Further characterization of the nuclear accumulation of APC revealed that nuclear translocation was transient and cell cycle dependent. Finally, the nuclear localization of APC could be partially suppressed using inhibitors of G2/M checkpoint kinases