Fluctuations in p53 Signaling Allow Escape from Cell-Cycle Arrest.

Biological signals need to be robust and filter small fluctuations yet maintain sensitivity to signals across a wide range of magnitudes. Here, we studied how fluctuations in DNA damage signaling relate to maintenance of long-term cell-cycle arrest. Using live-cell imaging, we quantified division profiles of individual human cells in the course of 1 week after irradiation. We found a subset of cells that initially establish cell-cycle arrest and then sporadically escape and divide. Using fluorescent reporters and mathematical modeling, we determined that fluctuations in the oscillatory pattern of the tumor suppressor p53 trigger a sharp switch between p21 and CDK2, leading to escape from arrest. Transient perturbation of p53 stability mimicked the noise in individual cells and was sufficient to trigger escape from arrest. Our results show that the self-reinforcing circuitry that mediates cell-cycle transitions can translate small fluctuations in p53 signaling into large phenotypic changes.

The effect of dust storm particles on single human lung cancer cells.

Exposure to dust particles during dust storms can lead to respiratory problems, diseases, and even death. The effect of dust particles at the cellular level is poorly understood. In this study, we investigated the impact that dust storm particles (Montmorillonite) have on human lung epithelial cells (A549) at the single cell level. Using live-cell imaging, we continuously followed individual cells after exposure to a wide range of concentrations of dust particles. We monitored the growth trajectory of each cell including number and timing of divisions, interaction with the dust particles, as well as time and mechanism of cell death. We found that individual cells show different cellular fates (survival or death) even in response to the same dust concentration. Cells that died interacted with dust particles for longer times, and engulfed more dust particles, compared with surviving cells. While higher dust concentrations reduced viability in a dose-dependent manner, the effect on cell death was non-monotonic, with intermediate dust concentration leading to a larger fraction of dying cells compared to lower and higher concentrations. This non-monotonic relationship was explained by our findings that high dust concentrations inhibit cell proliferation. Using cellular morphological features, supported by immunoblots and proinflammatory cytokines, we determined that apoptosis is the dominant death mechanism at low dust concentrations, while higher dust concentrations activate necrosis. Similar single cell approaches can serve as a baseline for evaluating other aerosol types that will improve our understanding of the health-related consequences of exposure to dust storms.

Recurrent initiation: a mechanism for triggering p53 pulses in response to DNA damage.

DNA damage initiates a series of p53 pulses. Although much is known about the interactions surrounding p53, little is known about which interactions contribute to p53's dynamical behavior. The simplest explanation is that these pulses are oscillations intrinsic to the p53/Mdm2 negative feedback loop. Here we present evidence that this simple mechanism is insufficient to explain p53 pulses; we show that p53 pulses are externally driven by pulses in the upstream signaling kinases, ATM and Chk2, and that the negative feedback between p53 and ATM, via Wip1, is essential for maintaining the uniform shape of p53 pulses. We propose that p53 pulses result from repeated initiation by ATM, which is reactivated by persistent DNA damage. Our study emphasizes the importance of collecting quantitative dynamic information at high temporal resolution for understanding the regulation of signaling pathways and opens new ways to manipulate p53 pulses to ask questions about their function in response to DNA damage.

The p53 response in single cells is linearly correlated to the number of DNA breaks without a distinct threshold.

BACKGROUND: The tumor suppressor protein p53 is activated by cellular stress. DNA double strand breaks (DSBs) induce the activation of the kinase ATM, which stabilizes p53 and activates its transcriptional activity. Single cell analysis revealed that DSBs induced by gamma irradiation trigger p53 accumulation in a series of pulses that vary in number from cell to cell. Higher levels of irradiation increase the number of p53 pulses suggesting that they arise from periodic examination of the damage by ATM. If damage persists, additional pulses of p53 are triggered. The threshold of damage required for activating a p53 pulse is unclear. Previous studies that averaged the response across cell populations suggested that one or two DNA breaks are sufficient for activating ATM and p53. However, it is possible that by averaging over a population of cells important features of the dependency between DNA breaks and p53 dynamics are missed. RESULTS: Using fluorescent reporters we developed a system for following in individual cells the number of DSBs, the kinetics of repair and the p53 response. We found a large variation in the initial number of DSBs and the rate of repair between individual cells. Cells with higher number of DSBs had higher probability of showing a p53 pulse. However, there was no distinct threshold number of breaks for inducing a p53 pulse. We present evidence that the decision to activate p53 given a specific number of breaks is not entirely stochastic, but instead is influenced by both cell-intrinsic factors and previous exposure to DNA damage. We also show that the natural variations in the initial amount of p53, rate of DSB repair and cell cycle phase do not affect the probability of activating p53 in response to DNA damage. CONCLUSIONS: The use of fluorescent reporters to quantify DNA damage and p53 levels in live cells provided a quantitative analysis of the complex interrelationships between both processes. Our study shows that p53 activation differs even between cells that have a similar number of DNA breaks. Understanding the origin and consequences of such variability in normal and cancerous cells is crucial for developing efficient and selective therapeutic interventions.

A synthetic-natural hybrid oscillator in human cells.

Recent studies have shown that many cell-signaling networks contain interactions and feedback loops that give rise to complex dynamics. Synthetic biology has allowed researchers to construct and analyze well-defined signaling circuits exhibiting behavior that can be predicted and quantitatively understood. Combining these approaches--wiring natural network components together with engineered interactions--has the potential to precisely modulate the dynamics of endogenous signaling processes and control the cell decisions they influence. Here, we focus on the p53 signaling pathway as a template for constructing a tunable oscillator comprised of both natural and synthetic components in mammalian cells. We find that a reduced p53 circuit implementing a single feedback loop preserves some features of the full network's dynamics, exhibiting pulses of p53 with tightly controlled timing. However, in contrast to the full natural p53 network, these pulses are damped in individual cells, with amplitude that depends on the input strength. Guided by a computational model of the reduced circuit, we constructed and analyzed circuit variants supplemented with synthetic positive and negative feedback loops and subjected to chemical perturbation. Our work demonstrates that three important features of oscillator dynamics--amplitude, period, and the rate of damping--can be controlled by manipulating stimulus level, interaction strength, and feedback topology. The approaches taken here may be useful for the rational design of synthetic networks with defined dynamics, and for identifying perturbations that control dynamics in natural biological circuits for research or therapeutic purposes.

Stimulus-dependent dynamics of p53 in single cells.

Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input-output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to γ-radiation. We further found that while γ-triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input-sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus-dependent dynamics of p53, including excitability and input-duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network.

A Switch in p53 Dynamics Marks Cells That Escape from DSB-Induced Cell Cycle Arrest.

Cellular responses to stimuli can evolve over time, resulting in distinct early and late phases in response to a single signal. DNA damage induces a complex response that is largely orchestrated by the transcription factor p53, whose dynamics influence whether a damaged cell will arrest and repair the damage or will initiate cell death. How p53 responses and cellular outcomes evolve in the presence of continuous DNA damage remains unknown. Here, we have found that a subset of cells switches from oscillating to sustained p53 dynamics several days after undergoing damage. The switch results from cell cycle progression in the presence of damaged DNA, which activates the caspase-2-PIDDosome, a complex that stabilizes p53 by inactivating its negative regulator MDM2. This work defines a molecular pathway that is activated if the canonical checkpoints fail to halt mitosis in the presence of damaged DNA.

p53 elevation in human cells halt SV40 infection by inhibiting T-ag expression.

SV40 large T-antigen (T-ag) has been known for decades to inactivate the tumor suppressor p53 by sequestration and additional mechanisms. Our present study revealed that the struggle between p53 and T-ag begins very early in the infection cycle. We found that p53 is activated early after SV40 infection and defends the host against the infection. Using live cell imaging and single cell analyses we found that p53 dynamics are variable among individual cells, with only a subset of cells activating p53 immediately after SV40 infection. This cell-to-cell variabilty had clear consequences on the outcome of the infection. None of the cells with elevated p53 at the beginning of the infection proceeded to express T-ag, suggesting a p53-dependent decision between abortive and productive infection. In addition, we show that artificial elevation of p53 levels prior to the infection reduces infection efficiency, supporting a role for p53 in defending against SV40. We further found that the p53-mediated host defense mechanism against SV40 is not facilitated by apoptosis nor via interferon-stimulated genes. Instead p53 binds to the viral DNA at the T-ag promoter region, prevents its transcriptional activation by Sp1, and halts the progress of the infection. These findings shed new light on the long studied struggle between SV40 T-ag and p53, as developed during virus-host coevolution. Our studies indicate that the fate of SV40 infection is determined as soon as the viral DNA enters the nucleus, before the onset of viral gene expression.

High mitochondrial priming sensitizes hESCs to DNA-damage-induced apoptosis.

Human embryonic stem cells (hESCs) are highly sensitive to DNA damage and have low survival ability relative to differentiated cells. We investigated the source of this difference by comparing damage response pathways in hESCs and differentiated cells. We found that hESCs undergo more rapid p53-dependent apoptosis after DNA damage than differentiated cells do. However, p53 localization and function are similar between hESCs and differentiated cells, suggesting that p53 alone cannot explain the difference in sensitivity. Instead, we show that mitochondrial readiness for apoptosis, known as mitochondrial priming, differs between hESCs and differentiated cells. Specifically, the balance between proapoptotic and antiapoptotic proteins is shifted closer to the apoptotic threshold in hESCs than in differentiated cells. Altering this balance in differentiated cells increases their sensitivity and results in cell death, suggesting that manipulation of mitochondrial priming could potentially alter the sensitivity of other stem cells, including cancer stem cells.

p53 dynamics control cell fate.

Cells transmit information through molecular signals that often show complex dynamical patterns. The dynamic behavior of the tumor suppressor p53 varies depending on the stimulus; in response to double-strand DNA breaks, it shows a series of repeated pulses. Using a computational model, we identified a sequence of precisely timed drug additions that alter p53 pulses to instead produce a sustained p53 response. This leads to the expression of a different set of downstream genes and also alters cell fate: Cells that experience p53 pulses recover from DNA damage, whereas cells exposed to sustained p53 signaling frequently undergo senescence. Our results show that protein dynamics can be an important part of a signal, directly influencing cellular fate decisions.

Phosphorylation by casein kinase I promotes the turnover of the Mdm2 oncoprotein via the SCF(beta-TRCP) ubiquitin ligase.

Mdm2 is the major negative regulator of the p53 pathway. Here, we report that Mdm2 is rapidly degraded after DNA damage and that phosphorylation of Mdm2 by casein kinase I (CKI) at multiple sites triggers its interaction with, and subsequent ubiquitination and destruction, by SCF(beta-TRCP). Inactivation of either beta-TRCP or CKI results in accumulation of Mdm2 and decreased p53 activity, and resistance to apoptosis induced by DNA damaging agents. Moreover, SCF(beta-TRCP)-dependent Mdm2 turnover also contributes to the control of repeated p53 pulses in response to persistent DNA damage. Our results provide insight into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2 results in attenuated p53 activity, thereby facilitating tumor progression.