Characterization of the p53 Response to Oncogene- Induced Senescence Lidia Ruiz1., Magali Traskine2., Irene Ferrer1., Estrella Castro1, Juan F. M. Leal1, Marcelline Kaufman 2 , Amancio Carnero1* 1 Experimental Therapeutics Programme, Centro Nacional De Investigaciones Oncolo´ gicas (CNIO), Madrid, Spain, 2 Unit of Theoretical and Computational Biology, Faculte´ des Sciences, Universite´ Libre de Bruxelles (U.L.B.), Bruxelles, Belgium Abstract Background: P53 activation can trigger various outcomes, among them reversible growth arrest or cellular senescence. It is a live debate whether these outcomes are influenced by quantitative or qualitative mechanisms. Furthermore, the relative contribution of p53 to Ras-induced senescence is also matter of controversy. Methodology/Principal Findings: This study compared situations in which different signals drove senescence with increasing levels of p53 activation. The study revealed that the levels of p53 activation do not determine the outcome of the response. This is further confirmed by the clustering of transcriptional patterns into two broad groups: p53-activated or p53- inactivated, i.e., growth and cellular arrest/senescence. Furthermore, while p53-dependent transcription decreases after 24 hrs in the presence of active p53, senescence continues. Maintaining cells in the arrested state for long periods does not switch reversible arrest to cellular senescence. Together, these data suggest that a Ras-dependent, p53-independent, second signal is necessary to induce senescence. This study tested whether PPP1CA (the catalytic subunit of PP1a), recently identified as contributing to Ras-induced senescence, might be this second signal. PPP1CA is induced by Ras; its inactivation inhibits Ras-induced senescence, presumably by inhibiting pRb dephosphorylation. Finally, PPP1CA seems to strongly co- localize with pRb only during senescence. Conclusions: The levels of p53 activation do not determine the outcome of the response. Rather, p53 activity seems to act as a necessary but not sufficient condition for senescence to arise. Maintaining cells in the arrested state for long periods does not switch reversible arrest to cellular senescence. PPP1CA is induced by Ras; its inactivation inhibits Ras-induced senescence, presumably by inhibiting pRb dephosphorylation. Finally, PPP1CA seems to strongly co-localize with pRb only during senescence, suggesting that PP1a activation during senescence may be the second signal contributing to the irreversibility of the senescent phenotype. Citation: Ruiz L, Traskine M, Ferrer I, Castro E, Leal JFM, et al. (2008) Characterization of the p53 Response to Oncogene-Induced Senescence. PLoS ONE 3(9): e3230. doi:10.1371/journal.pone.0003230 Editor: Mikhail V. Blagosklonny, Ordway Research Institute, United States of America Received June 30, 2008; Accepted August 24, 2008; Published September 18, 2008 Copyright: 2008 Ruiz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by grants from the Ministerio de Educacion y Ciencia (SAF2005-00944), Fundacio´ n Mutua Madrilen˜ a and the EU (from VI framework, Project COMBIO, Project NETSENSOR). IF is supported by a fellowship from the Ministerio de Educacion y Ciencia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: acarnero@cnio.es . These authors contributed equally to this work. Introduction Among the different methods cells have to monitor external or internal stresses, the surveillance mechanism associated with the p53 gene is central. Numerous molecular studies over the years have presented p53 as an essential controller of cellular and genome integrity [1]. p53 is a master transcription factor, functionally inactive under normal conditions due to its rapid degradation by the ubiquitin ligase MDM2. A chain of events triggered in response to cellular stress upsets this precise balance, leading to the uncoupling of MDM2-driven degradation and to the ultimate accumulation and activation of p53 [2]. p53 works mostly as a transcriptional activator, with few molecules in each cell [3]. However, p53 might also act as a repressor in some instances [4]. The p53 transcriptional program includes the activation of a number of cell cycle inhibitors and proapoptotic proteins, which results in apoptosis, reversible proliferative arrest or cellular senescence [5,6,7]. In principle, the various outcomes of p53 activation might be influenced by quantitative or qualitative mechanisms [8]. Some studies suggest that the level of p53 output determines whether cells will enter cell cycle arrest or apoptosis. Consistent with this view, only a subset of the genes induced by high p53 levels are induced by lower p53 levels [9]. Introduction of high p53 levels into tumor cell lines induces apoptosis, while the introduction of lower levels induces only cell cycle arrest [10]. However, other studies suggest that the outcome of p53 activation is determined by factors controlled by the tissue type or by the cell genotype. Oncogenic Ras can activate p53 to promote cellular senescence, limiting the transforming potential of excessive signalling [11–16]. This study and others have demonstrated that conditional activation of p53 in mouse embryonic fibroblast cells (MEFs) PLoS ONE | www.plosone.org 1 September 2008 | Volume 3 | Issue 9 | e3230