growth arrest. However, a second Ras-dependent signal seems to be required to stabilize the arrest as irreversible senescence. PPP1CA contributes to growth arrest stabilization in senescence The application of a retroviral-based genetic screen yielded an antisense RNA fragment against PPP1CA, the catalytic subunit of PP1a. Loss of PPP1CA function bypasses Ras/p53-induced growth arrest and senescence [18]. It was found that oncogenic Ras promotes an increase in the intracellular level of ceramides, which may increase PPP1CA activity, contributing to senescence. PP1a has been identified as the protein phosphatase responsible for the dephosphorylation of pRb [29]; this has been related to the growth arrest response [30–32]. When cells are actively growing, the hyperphosphorylated form of the Rb protein (ppRb) predominates. On the contrary, when cells are delayed in their growth, the hypophosphorylated form of the Rb protein (pRb) is the most abundant. Thus, enforced pRb dephosphorylation might contribute to the arrest to senescence transition [27,33]. PPP1CA protein levels increase upon Ras activation (Figure 5A) [34,18], but not mRNA (Figure 5B). PP1 phosphatase activity also increases upon oncogenic ras expression (Figure 5C), paralleling protein levels of PPP1CA. Expression of a specific shRNA against PPP1CA impairs pRb dephosphorylation, thus bypassing p53- Table 2. Condition-specific genes. P5 Replicative p53ts(32u) Growth Ras Oncogenic Stress arrest IGF-R MAP4 ZAP70 Wig1 PIG8 IL6 P73 Lats2 Bax Jun Pthlh Waf1 Igfbp3 MST1 RB1 Hic1 LRDD/PIDD p63 CycB1 Krt2-8 Krt1-15 Pmaip1 DKK1 PUMA Mgmt Pold1 Lats2 GML Bak Bax PTGF Igfbp6 Bcl6 PPM1D Tyr MDR1 Thbs1 Kai1 Btg2 MAP4 Tst RB1 IGFR p14-ARF This table represents the genes that are the most representative (relative to their median level–based on all conditions) in each particular arrested condition. A threshold equal to 2.30 was chosen for the ratio. Genes are arranged from the lowest to highest ratio. doi:10.1371/journal.pone.0003230.t002 p53 Response to Senescence induced arrest. When p53ts-Ras cells were shifted at 32uC, pRb became hypophosphorylated, in accordance with the growth- arrest induced by thermo-sensitive p53 at this permissive temperature. In contrast, p53ts-Ras cells stably transduced with shRNA against PPP1CA showed an increase in the hyperpho- sphorylated form of Rb protein when kept for 24 h at 32uC (Figure 5D). These data show that downregulation of PPP1CA maintains pRb in the hyperphosphorylated state, even in the presence of active p53, therefore allowing cell growth (Figures 5D and 5E). While p53ts-Ras cells at 32uC show mostly the senescent phenotype, only 22% of cells carrying the PPP1CA shRNA showed senescence features, confirming the relevance of PP1a activity to the senescence phenotype (Figure 5F). This was further confirmed by immunofluorescence studies (Figure 6). In prolifer- ating cells, PPP1CA and pRb levels are low, increasing slightly upon growth arrest. However, these proteins showed diffuse distribution (Figure 6). Under conditions inducing senescence (p53ts-Ras at 32uC), cells increase pRb and PPP1CA levels, which showed nuclear co-localization, strengthening evidence for their functional relationship to senescence. Oncogenic stress enhances p53-dependent transcription We also observed that oncogenic Ras enhances p53-dependent transcription (Figures 2A and 2C). To study this effect in detail, we selected three different p53-responsive promoters, Bax, p21waf1 and the synthetic p53 response element (x13). We engineered a construct fusing the different promoters 59 of the luciferase reporter gene and compared the effect of p53 alone to the effect of the combination of p53 and Ras-val12 (Figure 7A). Oncogenic Ras enhances p53-dependent transcription in all cases, but does not alter transcription when transfected alone (Figure 7A). These effects are dependent on p53 and Ras doses (Figure S2). To further study this effect, we selected the Bax promoter and investigated dependence of the phenomenon on Ras. To that end, we tested the N17 mutant of Hras-val12. This mutant lacks the ability to bind to Ras effectors and therefore acts as a dominant negative mutant. The N17 mutant does not alter the p53 response (Figure 7A), indicating that the Ras effect is dependent upon activation of Ras effectors. To directly discriminate between the two main effector pathways involved in this effect, the same experiment was performed with active PI3K or Raf pathway mutants. We co-transfected p53 and an active mutant of AKT (AKT-CA) (PI3K pathway), or an active mutant of Raf (BXB-Raf- CAAX). We were able to reproduce the ras-enhancing effect (Figure 7B), indicating that a strong activation of either pathway may provoke the enhancement of p53 transcription. Ras, acting through the Raf pathway, may activate p53 through p19ARF, either dependent upon or independently of MDM2, while PI3K may inhibit p53 through MDM2 phosphorylation [7,35,36]. To determine whether MDM2 or p19 was involved in the effect, the experiment was performed in p19-null or MDM2- cells (Figure 7C). We observed that the p53-enhancing effect observed in the Ras oncogenic signal was dependent upon p19ARF but not on MDM2. A similar observation was made with the activated Raf oncogene. However, activated AKT showed p53-enhancing effects independent of p19ARF and D) Feature selection. Since the number of genes is much greater than the number of conditions, we used penalized regression methods. See text and M & M for more details. E) Validation of the feature selection by quantitative PCR. See text and M&M. We determined Lats2, DKK1, pRb and PTEN mRNA levels after 24 hts treatment of HCT116 cells with the indicated treatment, by quantitative PCR. Cyclophilin (ref. 4326316E), an endogenous control, was used to normalize variations in cDNA quantities from different samples. Each reaction was performed in triplicate with cDNA from normal and tumor tissue from each patient studied. C shows untreated samples. E: Etoposide, D: Doxorubicin, T:Taxol, U: UCN-01, F:Flavopiridol. Data shows average of three determinations. doi:10.1371/journal.pone.0003230.g002 PLoS ONE | www.plosone.org 6 September 2008 | Volume 3 | Issue 9 | e3230