produces reversible cell cycle arrest, whereas activation of p53 in the presence of oncogenic Ras leads to a permanent cell cycle arrest with features of cellular senescence [17,18]. Although oncogenic Ras may increase p53 levels, it is not clear whether this increase is sufficient to explain the induction of senescence. Two different, though not mutually exclusive, models have been proposed to explain the different biological outcomes associated with p53 activation. The quantitative model implies that p53 levels are sufficient to determine the outcome. Thus, low p53 levels induce a reversible cell cycle arrest while higher p53 levels induce senescence or apoptosis. This model is supported by studies in which p53 levels may be artificially controlled with the appropriate expression systems [9,10]. One potential mechanism that could explain such an effect is based on differential p53 affinity for p53 response elements, such that genes required for a reversible cell cycle arrest have protein products with greater affinities than those required for senescence or apoptosis. A qualitative model of p53 action implies that non-quantitative factors controlled by a stimulus, either the tissue origin or the cell genotype influence the outcome of p53 activation. Again, two non- mutually exclusive mechanisms might support the published data. First, certain collateral signals might directly modulate p53 activity by changing the conformation of p53 or its association with various coactivators, perhaps leading to the expression of different subsets of p53 target genes. Consistent with this possibility, ionizing radiation and UV light have been shown to induce expression of different subsets of p53-dependent target genes in the same cell type [9]. Interestingly, these two stimuli induce different p53 modifications [19–21], raising the possibility that the activating signal may modulate p53 activity in a qualitative manner by directing p53 to different promoters [22]. Similarly, the ability of oncogenes to promote either apoptosis or senescence is correlated with different p53 modifications. Furthermore, oncogenic Ras induces p53 phosphorylation on serine 15 and induces senescence, whereas the E1A oncoprotein does not induce serine 15 phosphorylation and promotes apoptosis. The E1A effect is dominant, since cells coexpressing E1A and Ras do not contain p53 that has been phosphorylated on serine 15, and these cells are prone to apoptosis [23,24]. Whether this effect leads to the expression of different p53 target genes has yet to be determined. Second, it is possible that the signal produced by p53 activation is the same in different contexts and that the outcome of p53 activation is determined by how this signal is interpreted by the cell. One may envision several mechanisms by which this might occur, but an obvious possibility involves the combined action of p53 and other transcription factors such that the action of p53 on outcome-specific targets is influenced by the presence or absence of these other factors. These other factors, in turn, would be the targets for the hypothetical collateral signal. One precedent for this involves the integration of p53 and interferon signaling on the p21 promoter, which contains both p53 and IRF-1 response elements that act to synergistically induce p21 expression during a DNA damage response [25]. How different signal transduction pathways integrate to produce new biological outcomes is an important biological problem that may also have an impact on the understanding of p53. How does oncogenic Ras convert p53 to a senescence inducer? Although it seems likely that a component of this response results from the ability of oncogenic Ras to produce quantitative increases in p53 activity via ARF-mediated inhibition of MDM2, there is compelling evidence for collateral signals that modify the outcome of p53 activation leading to senescence [17,26]. Following the discussion above, it is formally possible that oncogenic Ras directly modulates p53 activity or, instead, produces cellular changes that reinterpret the p53 signal. p53 Response to Senescence One potential mechanism may involve the ability of Ras to induce PPP1CA (the catalytic subunit of PP1a) expression, regulating senescence in a pRb-dependent manner [18]. pRb is involved in the SAHF, maintaining long-term inhibition of E2F- dependent transcription through changes in the packaging status of chromatin [27]. To characterize the p53 response during growth arrest and senescence, this series of experiments compares p53-dependent transcription in different situations involving proliferation, revers- ible arrest, replicative senescence or Ras-induced senescence. Results p53 levels and phenotype To assess if differences in p53-dependent transcription play a role in reversible arrest or senescence, this study took advantage of the mouse embryo fibroblast (MEF) cell system that allows easy manipulation of cellular stresses in otherwise homogeneous conditions. For instance, conditional activation of the p53 pathway in MEFs is known to trigger reversible cell cycle arrest, whereas activation of p53 in the presence of oncogenic Ras leads to permanent cell cycle arrest with features of replicative senescence (Figure 1A) [16]. To induce replicative senescence, wild-type and p53-null (p53 2/2) embryos were generated from crosses between heterozygous p53 knock-out mice. From wild-type embryos, MEFs were generated and grown until replicative senescence was reached (approximately at passage 5, corresponding to 10 population doublings). We extracted mRNA under these conditions, i.e., terminally arrested with senescence features (P5), and also from exponentially growing MEFs (early passage, P3). Other stress conditions leading to senescence were produced as follows. Wild- type MEFs growing at early passage were infected with retroviruses carrying oncogenic Ras (Val12-Hras). Cells were selected for retrovirus insertion and once they reached senescence (corresponding to approximately passage 3), mRNA was extracted (P3+ras). P53-null MEFs were infected with viruses carrying the 135V thermosensitive mutant of p53 that induces cellular arrest at permissive temperature (32u) [28]. These cells (p53ts), while maintained at restrictive temperature (39uC), were infected with viruses carrying oncogenic Ras (p53ts-ras), which induces senescence when shifted to permissive temperature [17]. For a summary of conditions and the resulting phenotypes see Table 1. The abundance of p53 did not change among different passages reaching replicative senescence, or between the restrictive or permissive status in the case of the overexpression of the thermosensitive mutant of p53 (Figure 1C). Therefore, this study first measured broad p53-dependent transcription (Figure 1B). We measured the expression of 122 p53 target genes using Dot Blot arrays in the different proliferating and arrested cellular scenarios discussed above (See Figure S1 for a list of the 122 genes analyzed). The increased transcription rates of important p53 target genes such as Bax, GADD45, p21 and PIG8 confirm the activation of p53 in both senescence systems (Figure 1C). We observed that the arrest of MEFs at senescence (P5) and after Ras-induced senescence (P3+ras) correlated with a net increase in p53-dependent transcription (Figure 2A). Similarly, cells arrested after p53 activation (ts and ts-Ras at 32uC) also showed, as expected, a significant increase in p53-dependent transcription (Figure 2A). Therefore, we had a genetically homogeneous system with different levels of p53 activity measured with respect to 122 p53 transcriptional targets. It was possible to ascribe a phenotype to each level of p53 activity (Table 1). There were three conditions of proliferating cells: (1) P3, (2) cells with mutant p53 at restrictive temperature (null p53), PLoS ONE | www.plosone.org 2 September 2008 | Volume 3 | Issue 9 | e3230