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Phosphatase of regenerating liver-3 (Prl-3) was shown not long ago to be involved in metastasis of human colorectal cancers and to promote invasiveness and motility ([Saha et al., 2001] and [Zeng et al., 2003]); therefore, it must have been with some curiosity that the Stanford group led by Laura Attardi spotted Prl-3 as a p53 target gene upregulated in primary cells subjected to DNA damage (Basak et al., 2008). Undaunted, and indeed intrigued by the seeming paradox of a metastasis gene induced by perhaps the world's favorite tumor suppressor, Basak et al. have gone on to show that Prl-3 can indeed mediate a growth-arrest signal in response to p53 activation. Thus, despite many years of study since p53's role as a transcriptional activator became clear, new and interesting target genes continue to be identified, and there is no reason to think our understanding of the p53 network is nearing completion. Indeed, as early work on p21 null mice indicated, even deletion of this best-known and highly cytostatic p53 target gene results in only a partial loss of sensitivity to p53 induction, predicting the ongoing identification of p53 targets such as Prl-3 (Brugarolas et al., 1995 J. Brugarolas, C. Chandrasekaran, J.I. Gordon, D. Beach, T. Jacks and G.J. Hannon, Nature 377 (1995), pp. 552–557. Full Text via CrossRef[Brugarolas et al., 1995] and [Deng et al., 1995]).

How might a gene like Prl-3 live such a dual life? Here the work of Basak et al. (2008) is also informative and provides a key indication that it is perilous to view any protein function in isolation rather than as a component of a complex network of integrated sensors in a fine-tuned machine. Prl-3 can act as a stimulator of PI3K-Akt signaling (Wang et al. [2007] and confirmed by Basak et al. [2008]), but this action appears to require “basal” levels of Prl-3, as increased levels of Prl-3 led to inhibition of Akt activation and concomitant accumulation of the Cdk2 inhibitors p21 and p27 in the current study. Thus, as has been suggested with oncogene-induced senescence, too much stimulation can lead to shutdown of the very pathways normally stimulated by the offending component ([Chen et al., 2005] and [Courtois-Cox et al., 2006]). Most intriguingly, however, reduction of Prl-3 was also found to be cytostatic, via a p38-dependent stimulation of p19Arf, a “classic” p53 induction scheme that underscores the cell's dependence on careful regulation of genes like Prl-3. Therefore, the transition into S phase can be limited by either induction or loss of a single stimulator of the PI3K-Akt pathway, illustrating the precision of homeostasis in this potential checkpoint pathway. Perhaps similarly, later-acting checkpoints may also be subject to such precise control, as evidenced, for example, by the observation that either loss or activation of the spindle checkpoint protein Bub1 can be linked to growth arrest and senescence (Williams et al., 2007). Directly implied by results such as these, and they are growing in number, is the concept that suppressive and proliferative pathways have multiple “sensor” points that act to titrate signaling inputs, such that minor tweaks may have major consequences.

Finally, Basak et al. (2008) have gained insight into the requirements for Prl-3-mediated, and thus p53-mediated, inhibition of proliferation in damaged cells. Through the astute use of MEFs variously lacking Akt1 and Akt2, Cdk2, Cdk4, or all three retinoblastoma protein (pRb) family members, the authors demonstrated that Prl-3 could arrest cells without any pRb activity but could not arrest in the absence of Akt or Cdk2. Thus, another seeming paradox is that the post-restriction point inhibition of cell cycle by Prl-3 (as evidenced by insensitivity to pRb loss) depends on the very target kinases shut down by highly induced Prl-3. One is compelled to conclude that, in the absence of these centrally acting, proproliferative kinases, the knockout cells have adapted by activating or expressing Prl-3-insensitive targets that now drive proliferation in a less regulatable manner. Indeed, such a result is consistent with the authors' observation that several tumor cell lines are insensitive to Prl-3 overexpression, and indeed this may in turn explain the favored retention of Prl-3 expression in metastatic cells. Here, too much of a good thing (Prl-3) becomes bad for the patient, as the checks and balances on pathway hyperactivation may be compromised in favor of unrelenting proliferation. A consequence of these concepts combined may be that tumor therapeutic regimens will need to be carefully controlled as they begin to increasingly target these pathways in disease. In any event, these studies from Basak et al. highlight the need to study proliferation control genes in detail on a case-by-case basis, as genomic sciences have yet to provide an alternative approach.

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The p53 tumor suppressor plays a critical role in protecting organisms from developing cancer (Vousden and Lu, 2002). The ability of p53 to inhibit tumorigenesis is attributed to p53's activity as a cellular stress sensor, as diverse stress signals, including DNA damage, hypoxia, and oncogene expression, lead to p53 activation (Ryan et al., 2001). Activation of p53 can provoke different cellular responses, including G1 cell-cycle arrest, senescence, or apoptosis, any of which can impede tumor development by preventing the expansion of neoplastic cells.

p53 is a transcription factor that activates and represses a multitude of target genes proposed to participate in the cell-cycle arrest or apoptotic responses (Vousden and Lu, 2002). Genetic data obtained from mice lacking specific p53 target genes, such as p21 or Bax, indicate their importance as mediators of p53-dependent cell-cycle arrest and apoptosis, respectively ([Brugarolas et al., 1995], [Deng et al., 1995] and [Knudson et al., 1995]). Further, the requirement for transactivation by p53 for triggering at least some of its cellular responses is emphasized by the inability of a transactivation-deficient p53 mutant to activate apoptosis in response to DNA damage ([Chao et al., 2000] and [Johnson et al., 2005]). Thus, the identification of p53 target genes is key for elucidating how p53 engages specific cellular programs, including cell-cycle arrest, senescence, and apoptosis.

Although a number of transcriptional targets of p53 have been identified, our knowledge of p53 effector functions remains incomplete (Vousden and Lu, 2002). For example, although p21 clearly participates in the G1 checkpoint response, p21−/− mouse embryonic fibroblasts (MEFs) are only partially deficient in the G1 arrest elicited by p53, suggesting the existence of additional p53-dependent targets involved in cell-cycle regulation (Brugarolas et al., 1995 J. Brugarolas, C. Chandrasekaran, J.I. Gordon, D. Beach, T. Jacks and G.J. Hannon, Radiation-induced cell cycle arrest compromised by p21 deficiency, Nature 377 (1995), pp. 552–557. Full Text via CrossRef[Brugarolas et al., 1995] and [Deng et al., 1995]). Similarly, other than Pml and Pai-1, the genes required for p53-induced senescence remain unknown ([de Stanchina et al., 2004] and [Kortlever et al., 2006]). Furthermore, given that cells deficient for p53 apoptotic target genes typically display only partially compromised apoptosis, it is probable that there remain to be identified other mediators critical for p53-dependent apoptosis (Ihrie and Attardi, 2004). Hence, there are likely to be additional undiscovered p53-inducible genes involved in mediating these different p53 responses.

To identify additional downstream effectors of p53, we performed a microarray analysis on primary mouse fibroblasts exposed to DNA damage. To enhance the likelihood of discovering new p53 target genes, we chose to examine the endogenous p53 response to DNA damage in primary cells, in contrast to most previous screens used to identify p53-inducible genes, which have relied on p53 overexpression in human cancer cell lines with a variety of genetic lesions. Using this approach, we describe here the identification of Prl-3 (phosphatase of regenerating liver-3) as a direct p53 target gene (Zeng et al., 1998). Previously, Prl-3 was shown to be overexpressed during the transition to metastasis in human colorectal cancer development, and ectopic expression of Prl-3 in cells promoted cell invasiveness and motility as well as metastasis in mouse models ([Saha et al., 2001] and [Zeng et al., 2003]). Thus, Prl-3 was of particular interest given the apparent paradox of being activated by p53 yet being involved in metastasis. Here, through analysis of Prl-3 activity in primary cells, we reveal an additional facet of Prl-3 function. Our studies demonstrate a pivotal role for Prl-3 in cell-cycle regulation, thereby providing fundamental insight into Prl-3's role in tumor development.

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The tremendous recent progress in identifying the parts that make up cells, and the parallel advances in identifying interactions between the parts, has led to an increasing appreciation of the complexity of biological networks. The challenge facing us now is to understand how network structure determines the functional behavior of these systems: in other words, how the concentration, location, specific interactions, and dynamical behavior of individual proteins control the processing of information within the cell. To build this level of understanding, we need quantitative information on the dynamics of key proteins under many different conditions; we also need to be able to make a variety of perturbations to the normal network structure and determine how these changes affect the dynamic behavior. The network surrounding the tumor suppressor protein p53 is a natural choice for studies of this kind: p53 is a master regulator of many essential cellular decisions, its interactions have been well studied, and it has been shown to undergo complex, tightly controlled dynamical behavior in response to DNA damage.

The p53 protein is kept at low levels in cells under normal conditions, primarily through a negative feedback loop with Mdm2; p53 positively regulates Mdm2 by activating Mdm2 transcription, and Mdm2 negatively regulates p53 by promoting its ubiquitination and degradation ([Barak et al., 1993], [Haupt et al., 1997], [Kubbutat et al., 1997] and [Wu et al., 1993]). Under various stress conditions, p53 is activated through upstream mediators. Most of these mediators induce posttranslational modification of p53 that disrupts the p53-Mdm2 interaction. For example, DNA double-strand breaks (DSBs) induce rapid autophosphorylation and activation of ataxia-telangiectasia mutated protein (ATM) (Bakkenist and Kastan, 2003), which then phosphorylates both p53 ([Banin et al., 1998] and [Canman et al., 1998]) and Mdm2 ([Khosravi et al., 1999] and [Maya et al., 2001]) as well as other substrates. These events lead to disassociation of the p53-Mdm2 complex, stabilizing p53 and increasing p53 protein levels. ATM also phosphorylates the checkpoint kinase Chk2 ([Ahn et al., 2000] and [Matsuoka et al., 2000]), which directly phosphorylates p53, further contributing to p53 stabilization (Chehab et al., 1999). After DNA damage caused by γ-irradiation, p53 and Mdm2 show repeated pulses that were originally characterized as damped oscillations (Lev Bar-Or et al., 2000). We later showed in single-cell experiments that individual cells show varying numbers of p53 pulses of fixed amplitude and duration (Lahav et al., 2004). The appearance of damped oscillations results from the averaging of the pulses across a population of cells. A recent in vivo study showed pulses of p53 activity in a transgenic mouse, suggesting that these dynamics are not limited to cultured cancer cells (Hamstra et al., 2006).

The p53/Mdm2 negative feedback loop is composed of interactions on two different timescales: a slow positive transcriptional arm and a fast negative protein-protein interaction arm (Figure 1A). Mathematical models predict that such feedback loops can exhibit oscillatory behavior ([Goodwin, 1965] and [Tyson et al., 2003]); thus, the simplest explanation of the repeated pulses we observed was that they were intrinsic oscillations of the p53/Mdm2 loop ([Lev Bar-Or et al., 2000], [Mihalas et al., 2000], [Monk, 2003] and [Tiana et al., 2002]) . Other models include additional features, such as an additional positive feedback on p53 (Ciliberto et al., 2005 A. Ciliberto, B. Novak and J.J. Tyson, Steady states and oscillations in the p53/Mdm2 network, Cell Cycle 4 (2005), pp. 488–493. View Record in Scopus | Cited By in Scopus (22)[Ciliberto et al., 2005] and [Zhang et al., 2007]), or assume high constant levels of active upstream DNA damage sensor proteins (such as ATM) to account for the sustained oscillations ([Ma et al., 2005] and [Wagner et al., 2005]). An interesting and distinct proposal, from us, Uri Alon, and colleagues, suggests that p53 oscillations might depend on pulses of the upstream damage signaling elements (Geva-Zatorsky et al., 2006) .