p53 Response to Senescence Figure 3. P53 activity is downregulated maintaining senescence. Cells were plated in 10-cm-diameter plates. Cells were grown at 39uC (i.e., never incubated at 32uC) or arrested for the indicated times at 32uC. Cells were harvested and RNA collected for A, or stained for SA b-GAL for B. A) Comparison of the levels of 122 transcriptional targets of p53 at different times after p53 activation. Data normalized against b-actin was compared to the proliferative conditions at 39uC to evaluate statistical significance. Statistical analysis was performed by paired t-test. (*) = p,0.05; (**) = p,0.005; (***) = p,0.001. B) More than 400 cells were visually analyzed for SA b-GAL staining as described in Figure 1A. Data represent the percentage of cells showing SA b-GAL staining. doi:10.1371/journal.pone.0003230.g003 MDM2 (Figure 7C). These data match those previously reported [11,37–39]; Ras and Raf oncogenes require the p19ARF protein to activate p53. Discussion The data presented in this study elucidate the regulation of p53- responsive genes during proliferation and senescence. We have clearly demonstrated that Ras effects on p53-dependent transcrip- tional activation result in quantitative rather than qualitative changes. Therefore, the senescence response depends on factors other than p53 activation. p53 activation seems to be necessary but not sufficient to induce senescence, as other signals may be needed for the full onset of senescence. We have shown that Ras-induced activation of PPP1CA, the catalytic subunit of PP1a, is necessary to induce Ras-dependent senescence [18]. It is therefore possible to split the senescence response into two physiological processes. The first of these involves induction of growth arrest and is dependent on p53 activation or other physiological signals activating a prolifer- ative brake similar to that of p53, such as p73 or p63. The second PLoS ONE | www.plosone.org 7 September 2008 | Volume 3 | Issue 9 | e3230