Notch(ic)-ER chimeras display hormone-dependent transformation, nuclear accumulation, phosphorylation and CBF1 activation

Abstract

Notch genes encode a family of evolutionarily conserved transmembrane receptors that are involved in many distinct cellular processes such as differentiation, proliferation and apoptosis. Notch function has been shown to be required both during development and in adult life. Moreover, several studies on spontaneous human tumors and in experimental models demonstrate that three of the four mammalian Notch genes can act as oncogenes. The mechanism by which Notch proteins induce neoplastic transformation is not known. In order to determine the early signaling events mediated by Notch during cellular transformation we constructed several inducible alleles of Notch(ic) by fusing portions of N(ic) to the hormone-binding domain of the estrogen receptor. Here we show that Notch(ic)-ER chimeras are conditionally activated by 4-Hydroxytamoxifen (OHT) in a dose-dependent manner. Clonal RKE cell lines expressing Notch(ic)-ER chimeras display hormone-dependent transformation in vitro. Transformation mediated by Notch(ic)-ER is reversible and chronic stimulation is necessary for the maintenance of the transformed phenotype. In response to hormone activation Notch(ic)-ER chimeras become hyperphosphorylated and accumulate in the nucleus of the cell; indicating that both phosphorylation and nuclear localization are required for Notch transforming activity.