Cellular Response to Oncogenic Ras Involves Induction of the Cdk4 and Cdk6 Inhibitor p15^INK4b

Abstract

The cell cycle inhibitor p15^INK4b is frequently inactivated by homozygous deletion together with p16^INK4a and p19^ARF in some types of tumors. Although the tumor suppressor capability of p15^INK4b is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16^INK4aalterations. Here we show that, in vitro, p15^INK4b is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15^INK4bis induced in cultured cells by oncogenic Ras to an extent similar to that of p16^INK4a, and their expression is associated with premature G₁ arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15^INK4band p16^INK4a promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15^INK4b, by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15^INK4b knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15^INK4b-deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15^INK4b is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell.

ACKNOWLEDGMENTS

We thank J. Altschmied, M. Barbacid, J. L. Bos, D. A. Brenner, P. Crespo, C. J. Der, L. A. Feig, M. Kasuga, D. Levy, J. Massagué, M. Pagano, U. R. Rapp, K. Reif, P. Rodríguez-Viciana, M. Serrano, R. A. Weinberg, and Y. Xiong for kindly providing some of the plasmids and reagents used in this work. We are specially indebted to E. Latres and M. Barbacid for providing us with the p15^INK4b-deficient MEFs.

M.M., M.I.H., and I.P.C. received fellowships from the Ministerio de Educación (Madrid, Spain). This work was supported by grants CA 36327 and CA 50434 from NIH to A.P.