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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell. Author manuscript; available in PMC Jul 9, 2011.
Published in final edited form as:
PMCID: PMC3003696

Basal dynamics of p53 reveals transcriptionally attenuated pulses in cycling cells


The tumor suppressor p53 is activated by stress and leads to cellular outcomes such as apoptosis and cell cycle arrest. Its activation must be highly sensitive to ensure that cells react appropriately to damage. However, proliferating cells often encounter transient damage during normal growth, where cell cycle arrest or apoptosis may be unfavorable. How does the p53 pathway achieve the right balance between high sensitivity and tolerance to intrinsic damage? Using quantitative time-lapse microscopy of individual human cells we found that proliferating cells show spontaneous pulses of p53, which are triggered by an excitable mechanism during cell cycle phases associated with intrinsic DNA damage. However, in the absence of sustained damage, post-translational modifications keep p53 inactive, preventing it from inducing p21 expression and cell cycle arrest. Our approach of quantifying basal dynamics in individual cells can now be used to study how other pathways in human cells achieve sensitivity in noisy environments.


A multicellular organism must keep a delicate balance: on one hand, cells must preserve the integrity of their genome and other essential structures to prevent aberrant cell behavior, which could lead to failure of tissue function or the formation of cancer. To this end, complex sensing mechanisms detect cellular damage with high sensitivity and activate the appropriate response such as cell cycle arrest or apoptosis (Bakkenist and Kastan, 2004). On the other hand, cells are constantly subjected to intrinsic stress caused by normal physiological processes such as growth and division. Halting the cell cycle, or killing the cell, in response to each of these transient events might be problematic. How do signaling pathways, and specifically stress response pathways, achieve the right balance between high sensitivity and tolerance to transient spontaneous damage during normal growth?

A central protein in the mammalian stress response is the tumor suppressor p53 (Vogelstein et al., 2000; Vousden and Lane, 2007). p53 is activated by upstream kinases that respond to different forms of cellular stress (Fig. 1A). For example, DNA double strand breaks (DSBs) lead to activation of the kinases ataxia telengiectesia mutated (ATM) and checkpoint kinase 2 (Chk2). This activation mechanism is highly sensitive; one or two breaks in the human genome were found to be sufficient for partial induction of ATM , and full ATM activation is achieved by less than 20 DSBs (Bakkenist and Kastan, 2003; Huang et al., 1996). Active ATM and Chk2 phosphorylate p53 (Fig. 1A), leading to its stabilization and accumulation in the nucleus. p53 then acts as a transcription factor for numerous target genes involved in stress response pathways ranging from DNA repair to apoptosis (Riley et al., 2008). In addition, p53 regulates the expression of proteins that modulate its own activation and stability, forming multiple positive and negative feedback loops (Harris & Levine, 2005). The most prominent feedback loop is between p53 and the E3 ubiquitin ligase mouse/human double minute 2 (Mdm2/Hdm2) (Kruse and Gu, 2009); p53 positively activates Mdm2 transcription and Mdm2 negatively regulates p53 stability by targeting it for proteasomal degradation (Wu et al., 1993). p53, ATM and Chk2 form additional negative feedback loops which are mediated by p53 dependent expression of the phosphatase Wip1 (Batchelor et al., 2008; Fiscella et al., 1997; Lu et al., 2007; Shreeram et al., 2006) (Fig. 1A).

Figure 1
p53 levels vary in populations of non-stressed cells. A) Diagram showing key species of the p53 signaling network. B–C) p53 levels in non-stressed conditions (B) or after DNA damage induced by neocarzinostatin (NCS, 400ng/ml) (C) as measured by ...

Post-translational modifications of p53 are important modulators of its function and stability (Bode and Dong, 2004) . Currently, modifications have been detected on at least 30 different sites on the p53 protein. The function of several of these modifications is well understood. For example, Mdm2-mediated ubiquitination of p53’s C-terminal lysine residues targets p53 to degradation. For others, it is less clear what role they play in regulating p53. It has been proposed that the specific combination of modifications on p53 may provide selectivity toward a particular cellular response. However, it is still unclear which of the enormous number of possible p53 modification patterns occur in living cells in different conditions, and what their specific function is (Toledo and Wahl, 2006).

In addition, even though the p53 network and the DNA damage response are among the most investigated signaling pathways in mammalian cells, most studies focus on the induction of p53 after stress. Very little is known about the basal dynamics of p53 in proliferating cells. In most studies, the basal level of p53 was simply determined by averaging over cell populations prior to a specific treatment. Based on this information, it has been suggested that p53 is kept at low levels in basal conditions (Michael and Oren, 2003). However, it is possible that by averaging over a population of cells important features of p53 dynamics are missed. For example, population studies suggested that p53 levels show damped oscillations after the induction of DSBs (Lev Bar-Or et al., 2000). Recent studies using live single cell imaging have shown that individual human cells show varying numbers of undamped p53 pulses of fixed amplitude and duration, which are independent of the amount of damage (Batchelor et al., 2008; Geva-Zatorsky et al., 2006; Lahav et al., 2004). The appearance of damped oscillations in the population studies resulted from averaging cells with different numbers of p53 pulses. Similarly, it is conceivable that averaging p53 levels across a population of proliferating cells masks cell-cell variations such as asynchronous events that can reveal the basal behavior of this pathway and the way it responds to spontaneous damage during normal growth.

Basal p53 dynamics during normal cell growth are of specific interest when one considers that cells are constantly confronted with various forms of stress. For example, DNA damage is a common event: spontaneous hydrolysis leads to DNA depurination; reactive oxygen species induce base damage and DNA breaks; and collapsing replication forks leave the DNA backbone broken (Branzei and Foiani, 2008; Sancar et al., 2004). It has been estimated that the frequencies of DNA lesions range from 10 DSBs to as much as 104 bases damaged by oxidative stress per cell per day. In addition, cells are confronted with other types of insults, such as ribosomal or metabolic stress, which are also known to activate p53 (Vousden and Lane, 2007; Vousden and Prives, 2009).

In the present study, we focused on characterizing the basal dynamics and function of p53 using quantitative time-series measurements in individual cells. We found that proliferating human cells show transient induction of p53 in non-stressed conditions giving the appearance of spontaneous pulses. These pulses correlate with cell cycle events associated with intrinsic DNA damage and depend on a functional ATM/DNA-PK pathway, suggesting that they represent the cellular response to spontaneous double strand breaks. We show that p53 is activated by an excitable mechanism that leads to similar pulses in non-stressed conditions as in response to severe damage caused by irradiation or radiomimetic drugs. However, p53 pulses in non-stressed conditions do not lead to induction of p21 or cell cycle arrest, as sustained damage signaling is required to change p53’s post-translational modifications to an active state. Our study demonstrates how the interplay between the DNA damage sensing pathway and the p53 pathway enables cells to distinguish between transient low damage during normal growth - which does not justify arrest or cell death - and severe sustained damage that requires induction of the full stress response.


Basal dynamics of p53 in non-stressed conditions

To identify the basal level of p53 in individual cells, we first measured p53 by immunofluorescence in HCT116 colon cancer cells. Surprisingly, p53 levels were not uniform across the population: most cells showed low levels of p53 as expected; however, several cells showed high p53 levels that were comparable to those seen after the induction of DSBs by the radiomimetic drug neocarzinostatin (NCS (Shiloh et al., 1983), Fig. 1B-C). We determined the distribution of basal p53 levels in populations of additional cancer (MCF7) and non-cancerous cells (MCF10A and RPE-hTERT) using automated image analysis (Figs. 1D-E and S1A-H). In all cell lines analyzed, the basal distribution of p53 showed a long right tail that overlapped with the distribution of p53 in cells exposed to NCS.

To verify that high basal p53 levels are not restricted to cells in culture, we measured p53 levels in thin sections of normal human tissues using immunofluorescence. Similar to cultured cells, we found a high variability in p53 levels in regenerating tissues like epidermis or small intestine (Figs. 1F and S1I-J). In post-mitotic tissues like breast epithelium or cerebral cortex, a relatively small fraction of cells showed high basal p53 levels (Fig. S1K-L).

At least two scenarios can explain the observed variation in p53 levels: individual cells have different constant steady-state levels of p53 or p53 levels change dynamically in each cell. To distinguish between these possibilities we followed p53 levels in individual cells over time using a fluorescently-tagged p53 (Batchelor et al., 2008). In previous work we showed that fluorescently-tagged p53 faithfully mimics the dynamics of endogenous p53 in response to DNA damage, and is functional in activating its target genes and in inducing apoptosis (Batchelor et al., 2008; Lahav et al., 2004). We quantified the mean nuclear fluorescent intensity in individual damaged cells and in cells growing in non-stressed conditions and analyzed the resulting trajectories to extract statistical parameters describing p53 dynamics (Fig. S2A-B and Supplemental Material). As previously reported, DNA damage induced a series of uniform p53 pulses (Fig. 2A,C) (Batchelor et al., 2008; Geva-Zatorsky et al., 2006; Lahav et al., 2004). Surprisingly, individual cells with no extrinsic damage showed p53 pulses as well (Fig 2B,D and Movie S1). Almost all cells show at least one p53 pulse in 24 hours, and about a third of the cells show a series of three to five pulses (Fig. 2G). Interestingly, the amplitude and width of p53 pulses in non-stressed conditions were similar to those after DNA damage induced by NCS (Fig. 2E-F) or γ-irradiation (data not shown). However, as opposed to the synchronized pulses post-damage, the pulses in non-stressed conditions were asynchronous (Fig. 2H), explaining why they could not be detected in assays that average p53 levels over a population of cells.

Figure 2
p53 dynamics in living cells. A–B) Time-lapse microscopy images of cells expressing p53-Venus following treatment with NCS (200ng/ml, (A)) or in non-stressed conditions (B). C–D) Individual cells were tracked and the average fluorescence ...

Our results show that in response to DNA damage p53 does not shift from a low steady state into a pulsing mode, but instead it shifts from asynchronous, spontaneous pulses into a series of regular, high frequency, and synchronized pulses (Fig. S2C-E). With increasing amount of damage, more cells show p53 pulses with a detectable frequency and the pulsatile dynamics persist for longer (Fig. S2F-G).

Spontaneous p53 pulses depend on proliferation and the ATM/DNA-PK pathway

We next asked whether p53’s spontaneous pulses in non-stressed conditions result from random activation of this pathway or are linked to specific internal processes within the cell such as cell cycle progression. To answer this question, we synchronized cells in-silico by aligning individual trajectories to the time of consecutive cell divisions and normalizing them to the length of the cell cycle (Fig. S3A-B) (Sigal et al., 2006). Using flow-cytometry, we were able to map the corresponding cell cycle phases to the synchronized trajectories (Fig. S3C-E) (Toettcher, 2008). We found that most proliferating cells (>90%) induce a p53 pulse during one cell cycle (Fig. 3A), and that the pulses are correlated with specific phases of the cell cycle: the distribution of the first p53 pulse peaked during G1, at 20% of the cell cycle post mitosis (Fig. 3B); the occurrence of the second pulse was more widely distributed around the boundary between S- and G2 phases (Fig. 3C).

Figure 3
p53 induction in non-stressed conditions correlates with the cell cycle and depends on the activity of upstream kinases. A–C) Cells were imaged for 48h in non-stressed conditions and synchronized in-silico (see Suppl. Material). (A) The cumulative ...

To further determine whether there is a causal relationship between cell cycle progression and p53 pulses, we arrested cells in G2 phase using the Cdk inhibitor RO3306 (Vassilev et al., 2006) and measured p53 dynamics by time-lapse microscopy. Prior to drug addition, we detected spontaneous p53 pulses as before. However, after the Cdk inhibitor was added, cells showed only residual pulses while progressing into S or G2-phase, and stopped pulsing once the arrest was established (Fig. 3D and S3F-G). This led to a strong reduction in the mean number of p53 pulses compared to freely cycling cells (Fig. 3E), suggesting that spontaneous p53 pulses are causally related to cell cycle progression.

How might normal progression through the cell cycle activate the p53 pathway? Proliferating cells are constantly confronted with various forms of stress. The observation that cell cycle events associated with intrinsic DNA damage (e.g. mitosis and replication) (Dart et al., 2004; Ichijima et al., 2005; Su, 2006; Tanaka et al., 2006) increase the probability of a p53 pulse suggests that p53 induction may be mediated by damage-activated regulators. To test this hypothesis we inhibited different kinases upstream of p53 (Fig. 1A) (Bode and Dong, 2004) and followed p53 dynamics in individual cells in non-stressed conditions (Fig. 3F). Inhibition of the stress kinase JNK or the kinases ATR and Chk1, which participate in the response to single-stranded DNA, had little effect (Fig. 3F). In contrast, inhibition of the kinases that dominate the response to double strand breaks, such as ATM, DNA-PK or Chk2, reduced the number of p53 pulses significantly (Fig. 3F). Similarly, inhibition of these kinases in a non-cancerous breast epithelial cell line (MCF10A) led to a specific reduction of p53 levels as measured by immunofluorescence (Fig. 3G-H). Specifically, the right-sided tail, representing cells with high p53 levels, was lost. This suggests that the activity of DNA damage-activated kinases is required for inducing p53 pulses in non-stressed conditions, both in cancerous and in untransformed cells. Most likely, these kinases are activated by transient damage that occurs during normal progression through the cell cycle.

p53 pulses result from an excitable mechanism

Next, we asked how a transient low input (such as intrinsic damage during growth) could lead to p53 pulses similar to the ones seen in cells after severe damage by NCS or IR, especially since ATM activity might be lower, or of shorter duration, under these conditions. One possibility is that the p53 network behaves as an excitable system where a transient input is sufficient for full activation, similar to action potentials in neurons (Hodgkin and Huxley, 1952). To test this hypothesis, we induced DNA damage with NCS and inhibited the upstream kinases that activate p53 at different times post-damage, thereby creating transient input signals. If the network behaves as an excitable system, transient and sustained inputs are expected to trigger a similar p53 pulse. The resulting trajectories show that indeed p53 levels continue to increase after inhibition of upstream kinases, leading to a full p53 pulse (Fig. 4A). Addition of the inhibitor at early time points reduced the number of cells showing a p53 pulse (Fig. 4B). However, once a p53 response was initiated, the amplitude and width of the pulse remained unchanged (Fig. 4C-D). We obtained similar results for p53 pulses in non – stressed conditions; all cells that initiated a p53 pulse before addition of the inhibitor completed a full pulse (Fig. 4E). These results suggest that p53 pulses, in both non-stressed conditions and in response to extrinsic damage, are excitable and independent of the damage duration. Such a mechanism fulfills the requirement for a highly sensitive response since it ensures that the pathway is fully activated even in response to low level transient damage.

Figure 4
p53 pulses are triggered by an excitable mechanism A–D) Cells expressing p53-Venus were damaged with 400ng/ml NCS and imaged for 6h. Wortmannin (100μM) was added at the indicated time points and subsequently refreshed every hour. p53 dynamics ...

Spontaneous p53 pulses are filtered by a network of posttranslational modifications

In damaged cells, p53 leads to cell cycle arrest or apoptosis (Vogelstein et al., 2000). We did not observe cell death or arrest in response to spontaneous p53 pulses in non-stressed conditions (see for example Fig. S3A and F), suggesting that the corresponding target genes are not expressed. To take a closer look at the function of spontaneous p53 pulses, we measured the transcription of p21, a well-characterized p53 target gene involved in cell cycle arrest (el-Deiry et al., 1993). By expressing a destabilized version of the red-fluorescent protein mCherry under the control of the p21 promoter (Fig. 5A), we were able to measure p21 induction and p53 dynamics simultaneously in individual cells (Fig. 5B-C). Following DNA damage, the p21 reporter was induced in about a third of the cells (Figs. 5D,F and S4A). In contrast, the p21 reporter stayed at basal levels in non-stressed conditions, even in cells showing multiple p53 pulses at a comparable level to those in damaged cells (Figs. 5E,G and S4B). In agreement, measurements of both p53 and p21 proteins in normal human epidermis using immunofluorescence, revealed that cells with high p53 levels did not have detectable levels of p21 (Fig. 5H).

Figure 5
p53 pulses in non-stressed conditions do not activate p21 expression. A) Schematic drawing of the p53 and p21 reporter constructs. B–C) Time-lapse images of cells expressing the p53 and p21 reporters after damage (400ng/ml NCS, (B)) or in non-stressed ...

The observation that spontaneous p53 pulses do not activate p21 expression suggests that p53 is inactive as a transcriptional activator in non stressed conditions, and that transient low damage, like the one cells encounter during normal growth, is insufficient to convert it to its active form. To test this hypothesis we generated transient input signals as explained above (Fig. 4) and followed p53 levels and p21 expression simultaneously in live cells. We found that transient input signals (for 30 min post-damage) excite a full p53 pulse, but do not allow p21 activation (Fig. 6B and S5A). If we allowed the upstream signal to persist for longer (adding the inhibitor 60min post damage), more cells showed p21 activation (Fig. 6C). These results suggest that in the absence of sustained kinase activation even full induction of total p53 protein is insufficient for inducing target genes such as p21. Accordingly, we found that induction of p53 using a zinc-inducible promoter, which leads to high levels of p53 (about 1.5 fold higher than post damage) did not result in p21 activation (Fig. S5B-D), further supporting that p53 levels are uncoupled from its transcriptional activity.

Figure 6
Posttranslational modifications control p53 activity in non-stressed cells. A) Schematic drawing of the p53 protein domains including selected posttranslational modifications in the N- and C-terminal domains. The modified residues and the responsible ...

Uncoupling of p53’s accumulation and transcriptional activity could be achieved by the numerous posttranslational modification on p53 (Bode and Dong, 2004) (Kruse and Gu, 2009). Specifically, modifications in the C-terminal domain have been suggested to control p53’s activity as a transcription factor; acetylation at residues K373 and K382 by the transcriptional co-activator CBP/p300 (Fig. 6A) (Barlev et al., 2001; Li et al., 2007; Tang et al., 2008) were shown to increase p53’s transcriptional activity, while methylation of these lysines can play an inhibitory role. Methylation of K382 by the methyl-transferase SET8, for instance, has been reported to keep p53 in a transcriptionally inactive state (Shi et al., 2007).

To determine whether posttranslational modifications play a role in uncoupling p53 accumulation from its transcriptional activity, we analyzed the state of C-terminal lysines in response to sustained and transient activation, using antibodies against acetylated K373 and K382 as readout for p53’s acetylation in general. Sustained damage by NCS led to acetylation of both residues (Fig. 6D). In contrast, almost no acetylation was detected in response to a transient input (when the inhibitor was added 30min post-damage), although total p53 levels continued to increase (Fig. 6D).

To directly analyze acetylation of p53 in non-stressed conditions, we synchronized cells using the Cdk1 inhibitor RO3306 (Vassilev, 2006) and determined the modification state of p53 during its accumulation in G1 phase. No acetylation was detected in p53’s spontaneous accumulation while cells reenter the cell cycle, in contrast to its accumulation in response to sustained damage (Fig. S5E). This lack of acetylation correlated with no induction of p21 mRNA in non-stressed conditions (Fig. S5E). These results show that although accumulation of p53 is excitable and independent of the input duration, the shift in its acetylation state requires continuous input from the upstream kinases post damage (Fig. S5F).

We next sought to change the acetylation state of p53 in non-stressed conditions and test the effect on its ability to activate p21. We used the deacetylase inhibitor JW1521, which, in population measurements, induced the accumulation of acetylated p53 (Fig. 6E) (Roy et al., 2005). We found both in western blot (Fig. 6E) (Roy and Tenniswood, 2007) and in individual living cells that this change in modification state led to activation of p21 expression in the absence of extrinsic damage in a p53 dependent manner (Figs. 6F and S5G). This indicates that changing the acetylation pattern of p53 during its spontaneous pulses increases its transcriptional activity and allows induction of p21.

As deacetylase inhibitors might affect various pathways in the cell, we next aimed to alter p53 modifications in a more specific way. We chose to inactivate the methyl-transferase SET8, which has been shown to mediate the inhibitory methylation of p53 at K382 (Shi et al., 2007). We established stable SET8 shRNA-expressing cell lines and followed p53 dynamics and p21 induction (Fig. 6G-I and S5H-K). We found that p21 induction was about two-fold more frequent in the SET8 knockdown cells compared with induction in control cells (Figs. 6I and S5J). We suspect that SET8-shRNA did not lead to p21 induction in all cells due to an incomplete knockdown of SET8 and the presence of other compensatory inhibitory modifications, for example, methylation at K370 by the methyl-transferase Smyd2 (Huang et al., 2006). Interestingly, the same number of cells induced p21 after extrinsic damage in the SET8 shRNA and control cell lines (Fig. S5L), indicating that the inhibitory methylation is overridden by the strong damage stimulus.

Taken together, our results show that the interplay between inhibitory and activating modification at p53’s C-terminal domain provides a filtering mechanism that distinguishes p53 pulses after sustained damage, caused by irradiation or drugs, from spontaneous p53 pulses caused by transient and low-level damage during normal proliferation. In both cases p53 is excited and shows a full pulse. However up-regulation of p53’s transcriptional activity is not excitable; sustained damage is required to change p53’s modification profile and evoke the corresponding cellular responses, for example, cell cycle arrest or apoptosis.


One major objective of signaling pathways is to cope with, and filter, noisy environments, which often produce transient low-level inputs. This goal is especially challenging when dealing with dangerous inputs such as DNA damage. Cells must ensure they react appropriately in response to damage, but keep growing in response to transient spontaneous insults. How is this delicate balance achieved? The results presented in this study suggest that the p53 pathway meets these requirements by combining a highly sensitive activation mechanism with an intricate filtering mechanism. Transient intrinsic damage during normal growth is detected by damage sensors, including the kinases ATM and DNA-PK. This triggers a pulse of p53 accumulation (Fig. 7A). However, p53’s ability to arrest the cell cycle and activate its target genes such as p21 depends on specific post-translational modifications, which occur only in response to severe extrinsic damage (Fig. 7A).

Figure 7
p53’s dynamics and regulation in response to sustained and transient damage. A) A highly sensitive mechanism activates p53 in response to low transient DNA damage during normal growth and in response to severe damage caused by irradiation or drugs. ...

The level of spontaneous damage during growth is much lower than the damage in irradiated cells. However, our results show that p53 pulses in non-stressed conditions are as strong as the pulses in irradiated cells. How is this possible? Here, we provide evidence that this dynamical behavior results from an excitable system. Perturbation of the network over a certain threshold leads to full p53 accumulation. As a result, transient low damage leads to a pulse with amplitude and duration similar to those in response to sustained severe damage (Fig. 7A). The level of DNA damage therefore does not affect the shape of p53 pulses but only their number in each cell. In addition, such an excitable behavior contributes to, and fits with, the requirements for a sensitive damage sensing mechanism in response to DSBs, which are among the most dangerous types of DNA damage.

While excitability ensures that p53 reacts to low levels of damage, it creates the need to filter pulses that result from low intrinsic damage (“false alarms”), which can be fixed promptly and do not justify cell cycle arrest. One way to achieve this goal is to suppress p53’s activity during the response to transient low damage and to allow its activation only after severe extrinsic damage (Fig. 7A). Our results suggest that such a switching mechanism may be implemented through C-terminal modifications of p53. Two lines of evidence support the idea of interplay between inhibitory and activating modifications: First, increasing the level of activating acetylation led to p53-mediated expression of the target gene p21 even in the absence of damage. Second, reducing the activity of the inhibitory methyl-transferase SET8 had a similar effect. Interestingly, it has been shown that upon DNA damage, the activity of the inhibitory enzymes SET8 and Smyd2 is reduced, while activating factors like SET7/9 or CPB/p300 show increased activity (Huang et al., 2006; Ivanov et al., 2007; Li et al., 2007; Shi et al., 2007). DNA damage signaling therefore seems to play a dual role in regulating p53 levels and activity. Transient DNA damage triggers p53 accumulation. However, sustained damage signaling is necessary to change the p53 modification state from inhibitory to active. In this scenario, damage-activated kinases such as ATM would (i) directly modify p53 to increase its stability and (ii) induce p53’s acetylation/demethylation indirectly via regulation of intermediate enzymes, forming a coherent feed forward network motif (Fig. 7B). Similarly, Alon and colleagues have previously shown that in bacterial transcriptional networks coherent feed forward loops can act as persistence detectors that filter brief spurious signals in noisy environments (Alon, 2007; Mangan et al., 2003). In the p53 network, accumulation of p53 soon becomes independent of kinase input through an excitable mechanism (Fig. 7B). In contrast, the change of the modification pattern happens on a slower time scale and requires sustained upstream signaling (Fig. 7B). A plausible explanation for the delay in acetylation is the slow accumulation and nuclear transport of co-factors like Strap that are necessary for p300/CBP-mediated modification of p53 (Adams et al., 2008; Demonacos et al., 2004).

In the present study, we focus on the interplay of methylation and acetylation in the C-terminal domain. However, it is conceivable that additional modifications play a role in regulating p53 activity. The need for intricate processing beyond p53 stabilization may indeed be one explanation for the overwhelming complexity of p53 modifications. This also highlights the importance of studying the behavior of signaling pathways not only after external stimulation, but also in response to basal internal processes during normal growth. If the p53 network is viewed only in light of induced DNA damage, its architecture might seem overly complicated. However if one considers its frequent activation in proliferating cells, the need for further complexity becomes obvious.

Although our results indicate that p53 pulses in non-stressed conditions do not lead to expression of canonical target genes involved in cell cycle arrest, it is possible that the suggested filtering mechanisms are gene specific, allowing the expression of other target genes by spontaneous p53 pulses (Riley et al., 2008). For example, recent studies identified genes, including Sestrin 1 and Glutathione Peroxidase 1, with expression levels that are influenced by p53 knockdown in normally proliferating cells (Godar et al., 2008; Sablina et al., 2005). In addition, the downregulation of p53 at the end of a pulse indicates that the Mdm2 negative feedback is active. It has been previously reported that Mdm2 levels change dynamically even in non-stressed conditions (Geva-Zatorsky et al., 2006). Accordingly, we see an increase in Mdm2 protein levels after p53 accumulation in G1 phase and in respond to induction of p53 using an inducible promoter in non-stressed conditions (data not shown), in agreement with studies indicating that the architecture of the Mdm2 promoter allows its activation independent of C-terminal p53 acetylation (Kaku et al., 2001; Tang et al., 2008).

Our study provides an example of how analysis of the basal dynamics of a signaling pathway can provide new insights into the system’s behavior. The p53 network might be just one example of a signaling network showing complex dynamics in the absence of extrinsic stimuli. Our approach of looking at basal dynamics in individual cells can now be extended to other signaling pathways and especially to other pulsatile or oscillatory transcription factors, such as NFκB (Nelson et al., 2004). It will be important to determine the basal dynamics of these signaling pathways; whether dynamic changes also occur in the absence of extrinsic input, and what this can teach us about information processing in response to extrinsic inputs.

Experimental Procedures

Cell culture

MCF7 cells were grown in RPMI + 10% fetal bovine serum (FBS) supplemented with selective antibiotics (400μg/ml G418, 5μg/ml blasticidin, 0.5μg/ml Puromycin) when needed. HCT116 cells were grown in McCoy’s + 10% FBS and RPE-hTERT in DMEM/F12 + 10% FBS. MCF10A cells were cultured in DMEM/F12 + 5% horse serum with 20ng/ml EGF, 0.5μg/ml hydroxycortisone, 100ng/ml cholera toxin and 10μg/ml insulin. All media contained penicillin, streptomycin and fungizone. The MCF7 p53 reporter cell line has been previously described (Batchelor et al., 2008). The dual p53/p21-promoter reporter cell line was created using a 2.4 kb fragment of the p21 promoter (el-Deiry et al., 1993), the SET8-shRNA cell line by cloning specific oligonucleotides into pRetroSuper.puro (Brummelkamp et al., 2002a). See Supplemental Material for details. VSV-G pseudotyped retroviral particles expressing SET8-shRNA or p53-shRNA (Brummelkamp et al., 2002b) were produced in 293T cells

Antibodies and reagents

We used antibodies against p53 (DO1 and FL-393, Santa Cruz), acK373-p53 and acK382-p53 (Abcam), p21 (Calbiochem), Actin (Sigma) and Lamin A/C (T. Mitchison). Neocarzinostatin was obtained from the National Cancer Institute or Sigma, the ATM inhibitor KU55933 (used at 5μM), the Chk1 inhibitor SB218078, Chk2 inhibitor II and JNK inhibitor II (all used at 10μM) from Calbiochem, the DNA-PK inhibitor NU7026 (used at 10μM) from Sigma, the Cdk1 inhibitor RO3306 (used at 9μM) and wortmannin (used at 100μM) from Alexis Biochemicals and JW1521 from Errant Gene Therapeutics (used at 7.5–10μM). DharmaFect 1 and SMARTpool siRNA from Dharmacon were used for ATM and ATR knockdowns.

Western blot analysis

We harvested cells, obtained protein extract by lysis in the presence of protease and deacteylase inhibitors, and quantified total protein amount using the BCA assay (Pierce). Equal protein amounts were separated by electrophoreses on 4–12% Bis-Tris gradient gels (Invitrogen) and transferred to PVDF membranes by electroblotting. We blocked membranes with 5% non-fat dried milk, incubated them overnight with primary antibody, washed, incubated with secondary antibody coupled to peroxidase and detected protein levels using chemoluminiscence (ECL plus, Amersham) after additional washing steps.

Live cell Microscopy

For live cell time-lapse microscopy, cells were plated in RPMI without phenol red and riboflavin supplemented with 10%FBS in poly-D-lysine coated glass-bottom plates (MatTek Coporation). Cells were imaged on a Nikon Eclipse TE-2000 inverted microscope with a 40x plan apo objective (NA 0.95) using a Hammamatsu Orca ER camera. See Supplemental Material for filter sets used. The microscope was enclosed with an environmental chamber controlling temperature, atmosphere (5% CO2) and humidity. Images were acquired every 15 or 20min for 6–48h. Image acquisition was controlled by MetaMorph Software (Molecular Devices); image analysis was done with ImageJ (NIH) and Matlab (MathWorks).


Cells were grown on coverslips coated with poly-L-lysine and fixed with 4% paraformaldehyde. Cells were permeabilized in PBS/0.2% Triton, blocked with 10% goat serum, incubated with primary antibody, washed, and incubated with secondary antibody coupled to either Alexa488 or Alexa647. After washing, cells were stained with Hoechst and embedded in Prolong Antifade (Invitrogen). Images were acquired with a 20x plan apo objective (NA 0.75) using appropriate filter sets. Automated segmentation was performed in Matlab (MathWorks) using algorithms from CellProfiler (Carpenter et al., 2006). 2–6×104 cells were measured per condition.

Thin-sections from paraffin-fixed normal human tissues (Imgenex) were heated to 62°C for 1h, washed 5×4min in xylene, 2×3min in 100% ethanol, 2×3min in 95% ethanol, 2×3min in 75% ethanol and 1×5min in tap water. For antigene recovery, slides were microwaved 3×5min in 10mM citrate buffer (pH 6.0) and subsequently immersed in cold PBS. Staining was continued as described above.


  • p53 levels show pulses during the cell cycle in response to damage-activated kinases.
  • An excitable mechanism leads to similar p53 pulses during proliferation and after severe damage.
  • p53 pulses in proliferating cells do not induce a down-stream p53 response .
  • Posttranslational modifications keep p53 inactive during proliferation.

Supplementary Material




We thank G. Hornung and M. Oren for the p21 reporter construct; T. Mitchison for the Lamin A/C antibody and for comments; K. Janes and J. Brugge for MCF10A; B. Vogelstein for HCT p53−/−; S. Elledge for RPE-hTERT, and R. Agami for MCF7+p53shRNA cell lines; O. Gozani and E. Appella for antibodies, Martin Tenniswood for help with obtaining JW1521; J. Alzate and M. DiBona for technical assistance; J. Waters (NIC@HMS) for advice on live cell imaging; J. Paulsson, U. Alon, Y. Pilpel, M. Springer, B. Ward, M. Jain and S. Loewer for comments and discussions.

This research was supported by the National Institute of Health (NIH) grant GM083303. A.L. was supported by fellowships from the German Research Foundation and the Charles A. King Trust. E.B. was supported by the American Cancer Society, P. and E. Taft Postdoctoral Fellowship.


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