p53 Response to Senescence Table 1. Summary of cell lines and conditions used in this study. Cell line p532/2 p3 p5 P3+Ras (Ras) p532/2;ts (p53ts) p532/2;ts-ras (p53ts-ras) doi:10.1371/journal.pone.0003230.t001 Genotype p532/2 MEFs p532/2 MEFs with p53val135 p532/2 MEFs with p53val135 na¨ıve MEFs, 6 population doublings na¨ıve MEFs, 10 population doublings na¨ıve MEFs+Ras-val12, 6 population doublings phenotype Growth at 32u and 39u Growth at 39u reversible arrest at 32u Growth at 39u; senescence at 32u Growth at 37u senescence at 37u senescence at 37u Therefore, arrest vs. senescence is not determined by the relative levels of p53 activity alone. Specificity of the senescence response To study the expression pattern of p53-responsive genes during arrest or senescence in order to compare both processes and to ascertain what gene or genes may play a crucial role in the proliferating or arrested cell phenotypes, we performed a hierarchical clustering of the different cellular conditions on the basis of pattern similarity (see Materials and Methods). In Figures 2B and 2C we observed that the conditions are separated into two groups corresponding to the arrested (right side) and proliferating (left side) phenotypes. The cell lines are grouped together on the cluster dendogram by the activation or inactivation of p53 and not by the presence or absence of the Ras oncogene. This is clear in wild-type MEFs growing at passage 3 (P3), which have low levels of p53 activation compared to arrested wild-type MEFs in passage 5 (P5), which have p53 highly activated. However, it is interesting that the most extreme condition, p53 activation in the presence of oncogenic Ras, triggers an enhanced transcriptional response (Figures 2A and 2C, lane p53ts-Ras [32u]). See below. Although all the physiological conditions that lead to growth- arrest onset are clustered together and all the transcripts considered are p53-dependent, it is clear that there are some genes whose enhanced activation (relative to their median expression level over all cellular conditions) is specific to each particular condition (Figure 2C and Table 2). These genes might serve as specific marker genes. However, no concurrent senescence signature could be observed, indicating that the senescence program is not determined by the specificity of the p53 response. Next, applying a penalized least-squares regression technique with an L1-type penalty to the expression data (see Materials and Methods) it was possible to identify four p53 target genes among the 122 genes studied as the most relevant markers for predicting the proliferating or arrested phenotype of each cellular condition. These four relevant genes are: MAP4, PTEN, Lats2 and Rb1 (Figure 2D). Furthermore, combining L1- and L2-norm penalties allowed small subgroups of additional genes that are highly correlated with the main predictors to be extracted. This study identified five more genes closely related to MAP4 behavior: p63, caspase1, DKK1, Bcl2 and Gtse1; as well as LRDD, related to Rb1. This robust set of p53 target genes molecularly defines a minimal footprint to identify a p53-dependent arrest. In order to confirm the p53-dependent arrest footprint defined by these markers, we measured the p53-dependent transactivation of 4 among the selected genes by qRT-PCR in HCT 116 p53+/+ cells treated with different DNA-damaging agents. p53 protein is present at low levels in resting cells but after exposure to those agents as well as to other stressing stimuli, it is stabilized and activated by a series of post-translational modifications. These modifications leave p53 free from mdm2, an E3 ubiquitin ligase that ubiquitinates it and facilitates its degradation by the proteasome [5]. p53 stabilization and activation is followed by cell-cycle arrest. To ascertain whether the transcription of this set of genes also depends on other chemotherapeutic drugs that act through p53-independent mechanisms, we also treated the cells with compounds that do not directly cause DNA breaks. Only the treatment with the topoisomerase inhibitors Etoposide and Doxorubicin induced an activation of the transcription of PTEN, Lats2, Rb1 and MAP4 (Figure 2E). However, we did not detect increase of these genes by Taxol, flavopiridol or UCN-01. Downregulation of p53 response without senescence P53 transcription seems to define only arrest, and not senescence, suggesting the existence of a p53-independent signal necessary to convert the reversible arrest into senescence. To explore this, we analyzed whether sustained p53 activation might induce senescence without a second signal. In the same p53-induced transcriptional setting, we analyzed the activation of p53 during long periods and its correlation with the appearance of senescence. After an initial activation, general p53-induced transcription seems to decay at 24 hrs; this downregulation is maintained for long periods even in the presence of Ras activation (Figure 3A). However, senescence features are only maintained in the p53ts-Ras cells incubated at 32uC (Figure 3B). We found that in cells carrying activated p53 only, senescence is not induced despite a long period of growth arrest (up to 6 days). These data support the finding of Ferbeyre et al. [17], that growth arrest and senescence are two independent phenotypes; the permanence of growth arrest does not induce senescence unless another signal is involved. Finally, to confirm that the cells in long-term arrest have not suffered molecular changes that might indicate a switch to senescence, we analyzed 53BP1 and cH2AX phosphorylation at the senescence-associated DNA foci. As before, p53ts and p53ts- Ras cells were cultured at 39uC, then were moved to 32uC and maintained for up to 6 days at restrictive temperature. Cells were taken at different time points and analyzed for the presence of DNA-damage foci labeled by 53BP1 and cH2AX phosphorylation as markers for cellular senescence (Figure 4). One or two 53BP1 and cH2AX foci appear with cell proliferation, and the same number of foci was maintained in p53ts arrested cells even after 6 days of growth arrest. However, p53ts-Ras cells showed a strong increase in the number of foci per nuclei after 48 hrs of arrest (Figure 4); this was maintained despite p53-transcriptional downregulation. These data, which are consistent with previous observations [17,26], indicate that initial p53 activation is required to induce PLoS ONE | www.plosone.org 4 September 2008 | Volume 3 | Issue 9 | e3230