Notch(ic)-ER chimeras display hormone-dependent transformation, nuclear accumulation, phosphorylation and CBF1 activation.
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 Nic 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.
Ronchini
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Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, Ohio 45267-0524, USA.
Capobianco
A J
AJ
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004965:Estrogen Antagonists / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006728:Hormones / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011959:Receptors, Estradiol / Q000235:genetics / Q000378:metabolism*; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D004965:Estrogen Antagonists; D005656:Fungal Proteins; D006728:Hormones; D008565:Membrane Proteins; D011959:Receptors, Estradiol; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013629:Tamoxifen; C016601:afimoxifene