Differential formation of beta-catenin/lymphoid enhancer factor-1 DNA binding complex induced by nitric oxide in mouse colonic epithelial cells differing in adenomatous polyposis coli (Apc) genotype

increased cytoplasmic beta-catenin levels and the associated nuclear beta-catenin/T-cell factor (Tcf)-lymphoid enhancer factor (I,EF) complex formation have been frequently found in colon cancer, In this context, overproduction of nitric oxide (NO) attributable to inflammatory stimuli in diseases such as ulcerative colitis and Crohn's disease may contribute to colonic carcinogenesis. Therefore, we examined the modulation by NO of cytoplasmic beta-catenin levels and the formation of the nuclear beta-catenin/LEF-1 DNA binding complex in conditionally immortalized mouse colonic epithelial cells that differed in adenomatous polyposis coli (Apc) genotype, namely young adult mouse colon (YAMC; Apc(+/+)) and immortal mouse colon epithelium (IMCE; Apc-(Min/+)). Unlike most colon canter cell lines, this pair of cell lines has either nondetectable or low basal level of beta-catenin when they are cultured under non-permissive and nonproliferative conditions. Using electrophoretic mobility shift assays, se found that NO-releasing agents (E)-methyl-2-[(E)-hydroxy- imino]-5-nitro-6-methoxy-3-hexeneamide and S-nitroso-N-acetylpenicillamine greatly enhanced the formation of beta-catenin/LEF-1 DNA binding complex in a concentration- and time-dependent fashion in YAMC and IMCE cells, Significantly, IMCE cells showed a markedly greater amount of nuclear beta-catenin/LEF-1 DNA binding complex in response to NO. Super shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Western blot analysis of the soluble cytoplasmic fractions demonstrated that these NO donors caused differential accumulation of cytoplasmic beta-catenin in YAMC and LR ICE. In conclusion, this study indicates that the defective beta-catenin degradation machinery attributable to Apc(Min/+) mutation in LR;ICE cells not only affects basal levels but also contributes to NO-induced dysregulation of cytoplasmic beta-catenin and nuclear beta-catenin/LEF-1 DNA binding complex formation. [References: 29]

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