Dynamics of th p53 signaling network and its role in cell fate decisions
Final Report Abstract
The tumor suppressor p53 is one of the most import safeguards of our cells against cancer formation. It is inactivated in the vast majority of human tumors, either directly through mutations or indirectly by changes in its critical regulators. p53 reacts to cellular stress ranging from DNA damage to oncogenic transformation and initiates an appropriate response, for example cell cycle or apoptosis. p53 is regulated by a complex network of interactions, which have been largely identified in the thirty years since its discovery. We are now challenged to understand how the p53 network acts dynamically in individual living cells, which interactions control its dynamic response and how information about the stress is encoded and decoded. DNA damage is extremely dangerous for cells, as a single unrepaired lesion can lead to the genetic changes that drive tumor formation. Therefore, the p53 pathway must be highly sensitive to ensure that cells react appropriately to damage. However, proliferating cells often encounter transient damage during normal growth through intrinsic processes like DNA replication. In these cases, induction of a full p53 stress response including 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? To address this question, we used quantitative time-lapse microscopy of individual human cells expressing a fluorescent reporter for p53 and found that proliferating cells show spontaneous pulses of p53. These pulses were similar in amplitude and duration to pulses after externally induced DNA damage. We could show that pulses during normal proliferation are induced by intrinsic transient DNA double strand breaks during specific phases of the cell cycle. They are triggered by an excitable mechanism, which explains the similar dynamics after transient and sustained damage. However, in the absence of sustained damage, target genes of p53 are not expressed, as the network contains a filtering mechanism based on post-translational modifications that keeps p53 inactive in response to transient inputs. Our approach of quantifying basal dynamics in individual cells can now serve as a paradigm for studying how other pathways in human cells achieve sensitivity in noisy environments. p53 can be activated by various form of DNA damage. How does the pathway distinguish between these different inputs? To address this, we quantified the dynamics of p53 in individual cells in response to UV radiation and after the induction of DNA double strand breaks. After UV radiation, we observed a single pulse that increases in amplitude and duration in proportion to the dose. This graded response contrasts with the series of uniform pulses in response to DNA double strand breaks. We further found that the p53 response to UV is not excitable and depends on continuous signaling from the inputsensing 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. This 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 common therapy for human cancers are drugs that induce mitotic arrest. It is known that p53 is important for the cellular response to these drugs, but what is the molecular mechanism for its induction? Using live cell imaging in human cancer cells, we revealed that p53 induction is correlated with DNA damage during mitotic arrest. We could show that the DNA damage was induced by partial activation of the apoptotic pathway, which led to the activation of a specific endonuclease. This surprising finding may explain the DNA- damaging effects of diverse cellular stresses that do not immediately trigger apoptosis. For cancer formation, cells have to escape tumor suppression by the p53 network. This work has furthered our understanding of the induction and dynamics of the p53 network and may thus support the identification of novel strategies for therapeutic intervention.
Publications
- (2010) “Basal dynamics of p53 reveals transcriptionally attenuated pulses in cycling cells”, Cell 142(1): 89-100
Loewer, A., Batchelor, E., Gaglia, G., Lahav, G.
- (2011), “We are all individuals: causes and consequences of nongenetic heterogeneity in mammalian cells.”, Curr Opin Genet Dev. 21 (6): 753-758
Loewer, A., Lahav, G.
- (2011), „Stimulus-dependent dynamics of p53 in single cells.“, Mol. Syst. Biol. 7: 488
Batchelor, E., Loewer, A., Mock, C., Lahav, G.
