Normal Human Fibroblasts Are Resistant to RAS-Induced Senescence

Abstract

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated β-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS.

We thank Thomas Fazzio, Carla Grandori, Beatrice Knudsen, and Joseph Carter for critical reading of the manuscript; Jim Roberts for advice and support; Kristin Robinson for establishing HFF cultures; Greg Wipf for cloning hTert; and members of the Galloway laboratory for their continued support and suggestions. In addition, we thank Magnus von Knebel Doeberitz for the E6H4 antibody, Scott Lowe for his gift of the p16 shRNA vector prior to publication, and Manuel Serrano for RAS expression vectors.

This work was supported by grant number CA64795 from the NCI to D.A.G. J.A.B. was supported in part by grant PHS NRSA T32 GM07270 from NIGMS.