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High-intensity Raf Signals Convert Mitotic Cell Cycling into Cellular Growth¹

Institut für medizinische Strahlenkunde und Zellforschung (MSZ), University of Würzburg, 97078 Würzburg, Germany

The selection of NIH 3T3 cells expressing a hydroxytamoxifen-inducible c-Raf-1-estrogen receptor fusion protein (c-Raf-1-BxB-ER^TM) in the absence or presence of the inducer results in dramatic differences in the expression levels of the fusion protein. Hydroxytamoxifen-mediated constitutive activation of the Raf signal favors the selection of cells expressing low levels of c-Raf-1-BxB-ER^TM. Cells selected in the absence of hydroxytamoxifen express up to 20 times higher levels of the inducible Raf kinase. Activation of the oncogenic Raf kinase in cells expressing low levels leads to a weak activation of the Raf/Mek/Erk cascade and the induction of S phase in confluent cells. The activation of cells expressing high levels of the kinase leads to a strong persistent signal and inhibits DNA synthesis and mitosis in proliferating cells. The inhibition of DNA synthesis and cell division is presumably due to the elevated expression of the cyclin-dependent kinase inhibitor p21^cip1, similar to cells exposed to ionizing radiation. Despite the inhibition of DNA synthesis and mitosis, the constitutive activity of the Raf signaling pathway is still able to initiate cell growth. Activation of the high-intensity Raf signal in arrested serum-starved cells induces cell growth up to a size corresponding to that of M-phase cells in the absence of DNA synthesis. High-intensity Raf signals in proliferating cells consistently lead to an accumulation of cells with the size of M-phase cells and the DNA content of G₁ cells or G₂-M-phase cells. Therefore, the activation of Raf kinase is sufficient to drive cell growth, even in the presence of high levels of the cyclin-dependent kinase inhibitor p21^cip1.

¹ Supported by the Wilhelm Sander-Stiftung and the SFB 456.

² To whom requests for reprints should be addressed, at Institut für medizinische Strahlenkunde und Zellforschung, University of Würzburg, Versbacher Strasse 5, 97078 Würzburg, Germany. Phone: 49-931-201-5140; Fax: 49-931-201-3835.

Received 2/25/98. Accepted 3/ 2/98.

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