Abstract
Notch genes encode a family of transmembrane proteins that are involved in many cellular processes such as differentiation, proliferation, and apoptosis. Although it is well established that all four Notch genes can act as oncogenes, the mechanism by which Notch proteins transform cells remains unknown. Previously, we have shown that transformation of RKE cells can be conditionally induced by hormone activation of Notchic-estrogen receptor (ER) chimeras. Using this inducible system, we show that Notchicactivates transcription of the cyclin D1 gene with rapid kinetics. Transcriptional activation of cyclin D1 is independent from serum-derived growth factors and de novo synthesis of secondary transcriptional activators. Moreover, hormone activation of Notchic-ER proteins induces CDK2 activity in the absence of serum. Upregulation of cyclin D1 and activation of CDK2 by Notchic result in the promotion of S-phase entry. These data demonstrate the first evidence that Notchic proteins can directly regulate factors involved in cell cycle control and affect cellular proliferation. Furthermore, nontransforming Notchic proteins do not induce cyclin D1 expression, indicating that the mechanism of transformation involves cell cycle deregulation through constitutive expression of cyclin D1. Finally, we have identified a CSL [stands for CBF1, Su(H), and Lag-1] binding site within the human and rat cyclin D1 promoters, suggesting that Notchic proteins activate cyclin D1 transcription through a CSL-dependent pathway.
ACKNOWLEDGMENTS
We thank members of the Capobianco laboratory for support and technical assistance. We thank David Robbins and Yolanda Sanchez for insightful comments on our work. We are grateful to C. J. Der (University of North Carolina), K. Keyomarsy (M. D. Anderson Cancer Center), P. D. Ling (Baylor College of Medicine), E. Bresnick (University of Winsconsin), and J. A. Diehl (University of Nebraska) for kindly providing reagents used in this study.
This work was funded in part by grants from the American Cancer Society (RPG LBC-99465 to A. J. Capobianco) and the National Cancer Institute (ROI CA 83736 to A. J. Capobianco).