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-c atenin/T-cell factor (Tcf)-lymphoid enhancer factor (I,EF) complex formatio n have been frequently found in colon cancer, In this context, overproducti on of nitric oxide (NO) attributable to inflammatory stimuli in diseases su ch as ulcerative colitis and Crohn's disease may contribute to colonic carc inogenesis. Therefore, we examined the modulation by NO of cytoplasmic beta -catenin levels and the formation of the nuclear beta-catenin/LEF-1 DNA bin ding complex in conditionally immortalized mouse colonic epithelial cells t hat differed in adenomatous polyposis coli (Apc) genotype, namely young adu lt 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 c ultured under non-permissive and nonproliferative conditions. Using electro phoretic mobility shift assays, se found that NO-releasing agents (E)-methy l-2-[(E)-hydroxy- imino]-5-nitro-6-methoxy-3-hexeneamide and S-nitroso-N-ac etylpenicillamine 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 n uclear beta-catenin/LEF-1 DNA binding complex in response to NO. Super shif t by anti-beta-catenin antibody confirmed the presence of beta-catenin in t he complex. Western blot analysis of the soluble cytoplasmic fractions demo nstrated that these NO donors caused differential accumulation of cytoplasm ic beta-catenin in YAMC and LR ICE. In conclusion, this study indicates tha t the defective beta-catenin degradation machinery attributable to Apc(Min/ +) mutation in LR;ICE cells not only affects basal levels but also contribu tes to NO-induced dysregulation of cytoplasmic beta-catenin and nuclear bet a-catenin/LEF-1 DNA binding complex formation.