These results suggest that increased p27kip1 levels mediate the increased survival of -cateninexpressing cells. == -Catenin expression does not suppress the growth of DLD-1/ cells == p27kip1 AR7 inhibits cyclin E-CDK 2 activity implicated in the unfavorable regulation of G1 progression. by -catenin expression in low density cell cultures. The increased levels of p27kip1 correlated with ITSN2 both increased resistance to cell death and morphological changes in transfectants made up of deletion mutants. AR7 Transfection-mediated upregulation of p27kip1 decreases sphingosine-induced cell death in -catenindeficient cells. We postulate that -catenin mediates transduction of signals from the cadherincatenin complex to regulate the apoptotic cascade via p27kip1. Keywords:-catenin; cadherin; apoptosis; p27kip1; compaction == Introduction == The cadherin family of transmembrane glycoproteins plays an essential role in the initiation and stabilization of cellcell contacts (Takeichi, 1991;Kemler, 1993;Gumbiner, 1996;Marrs and Nelson, 1996). A conserved cytoplasmic domain name, common to these proteins, interacts with intracellular proteins termed catenins (Ozawa et al., 1989,1990;Ozawa and Kemler, 1992;Stappert and Kemler, 1994). The extracellular domain name is responsible for specific homophilic binding (Nose et al., 1990). Classical cadherins, including E-cadherin, bind to either -catenin or -catenin (plakoglobin), which links this complex to -catenin. -Catenin, a 102-kD protein, contains multiple conversation sites: actin-binding sites (Rimm et al., 1995), binding sites for other actin-binding proteins such as -actinin (Nieset et al., 1997), vinculin (Watabe-Uchida et al., 1998), and ZO-1 (Itoh et al., 1997;Imamura et al., 1999), and homodimerization sites (Koslov et al., 1997). Without -catenin, cells do not associate tightly with each other despite the expression of cadherins (Watabe et al., 1994;Ozawa, 1998;Maeno et al., 1999). Therefore, the interactions of -catenin, linking the cadherincatenin complexes to the actin cytoskeleton, may be essential to mediate the full activity of cadherins. Cadherin molecules around the cell surface transduce extracellular signals (Takeichi, 1991;Larue et al., 1996), possibly altering cell polarity (McNeill et al., 1990;Watabe et al., 1994), growth rate (Watabe et al., 1994;Bullions et al., 1997), and cellsubstratum adhesion (Miyaki et al., 1995). However, the mechanism by which cadherincatenin complexes regulate cell fate remains to be investigated. To examine the functions of -catenin, we established DLD-1/ cell clones transfected with -catenin (Ozawa, 1998). DLD-1/ is derived from the DLD-1 human colon cancer cell line, lacking endogenous expression of -catenin. Cadherin-mediated cell adhesion is usually disrupted in this variant, despite the presence of other cadherin cell adhesion com-plex components: E-cadherin, -catenin, and -catenin. We searched AR7 for the functions of AR7 -catenin in signal transduction and found a significant reduction in death of -cateninexpressing clones after treatment with sphingosine, an inducer (Sakakura et al., 1996;Sweeney et al., 1996;Shirahama et al., 1997) and endogenous mediator (Ohta et al., 1994,1995) of apoptosis. The basic molecular framework for regulating and executing apoptosis comprises a functionally ordered product of expanded gene families, including the caspases and Bcl-2 family of proteins. The cadherin/catenin-derived signal may affect the functions and activities of the molecules in the apoptotic pathways. Therefore, examination of the effects of cadherin signaling on death mediators and/or regulators may uncover the molecular mechanism underlying the signal. To determine the region of the molecule responsible for reductions in cell death induction, we examined AR7 the cellular phenotypes, resistance to cell death, and cell morphology, resulting from deletions in -catenin. We compared the effects of antiE-cadherin antibody treatment with -catenin deficiency. We then analyzed the status of death mediator molecules in the apoptosis cascade to find possible mediators of the signals from cadherincatenin complexes. Our results indicate that increases in p27kip1, an inhibitor of cyclin-dependent kinases (cdks), correlates with resistance to cell death in the -catenin transfectants, suggesting that expression of -catenin effects the cell death through the anti-apoptotic function of p27kip1. Transfection-mediated upregulation of p27kip1 levels in -catenindeficient cells decreases the levels of cell death induced by sphingosine, supporting our hypothesis. == Results == == Reduction of sphingosine-induced cell death by -catenin expression == D cells, a stable transfectant of a DLD-1/ variant, express full-length human -catenin; nD cells are a control vector transfectant (Ozawa, 1998). To induce apoptosis, cells were plated at 5 104cells per 35-mm dish for 24 h and then treated with 525 M sphingosine. Cell viability was then assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)*assay. D cells exhibited significant resistance to sphingosine-induced cell death compared with nD cells (Fig. 1A). When cells were treated with 25 M sphingosine, a relatively high concentration, almost all of the cells died within 7 h; there.