Dynamics of the DNA damage response: insights from live-cell imaging

Karanam, Ketki; Loewer, Alexander; Lahav, Galit

doi:10.1093/bfgp/els059

Cited by 16 publications

(13 citation statements)

References 67 publications

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“…Further investigation of the components of the DDR pathway is beyond the scope of this study, however it is acknowledged that additional cell cycle and DNA damage biomarkers could be included in subsequent studies to expand our understanding of the mode of action of DDR targeting oncology therapies 43 44 . Studies have been published using live cell imaging and demonstrated that detailed information can be obtained for complex dynamics of intracellular signal transduction pathways 42 , however implementing large mechanistic models, such as those published by Tyson and Novák, would add considerable numbers of model reactions that would require significant experimental resource to parameterize in a relevant cell background 45 46 . Our model was therefore constructed at a resolution corresponding with the experimental data which could be generated within the time-lines relevant to therapeutic drug discovery, and was appropriate to adding mechanistic detail without digressing from the intended functionality of the tumor model.…”

Section: Discussionmentioning

confidence: 99%

“…Also, detailed studies of single cell dynamics such as the work published by, e.g. the White and Lahav groups, have made substantial contribution to understanding the dynamical behaviors of the interactions within biochemical networks, however it is difficult to translate these complex dynamics into a response in a multi-cellular in vivo tumor model, in order to link mode of action and drug efficacy in a cell population for clinical trial design 41 42 .…”

mentioning

confidence: 99%

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Bridging the gap between in vitro and in vivo: Dose and schedule predictions for the ATR inhibitor AZD6738

Checkley

MacCallum

Yates

et al. 2015

Sci Rep

153

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Understanding the therapeutic effect of drug dose and scheduling is critical to inform the design and implementation of clinical trials. The increasing complexity of both mono, and particularly combination therapies presents a substantial challenge in the clinical stages of drug development for oncology. Using a systems pharmacology approach, we have extended an existing PK-PD model of tumor growth with a mechanistic model of the cell cycle, enabling simulation of mono and combination treatment with the ATR inhibitor AZD6738 and ionizing radiation. Using AZD6738, we have developed multi-parametric cell based assays measuring DNA damage and cell cycle transition, providing quantitative data suitable for model calibration. Our in vitro calibrated cell cycle model is predictive of tumor growth observed in in vivo mouse xenograft studies. The model is being used for phase I clinical trial designs for AZD6738, with the aim of improving patient care through quantitative dose and scheduling prediction.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Bridging the gap between in vitro and in vivo: Dose and schedule predictions for the ATR inhibitor AZD6738

Checkley

MacCallum

Yates

et al. 2015

Sci Rep

153

View full text Add to dashboard Cite

show abstract

“…Experimental induction of DNA damage foci in living cells became an ideal method to analyze in time and space the recruitment and binding properties of repair factors at DNA lesions ( 138 ). Live cell imaging of DNA damage foci has been used to reveal the mobility of repair proteins, their assembly timing into repair sites, and the movement of damaged chromatin ( 139 – 141 ).…”

Section: Laser Microirradiationmentioning

confidence: 99%

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

et al. 2018

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The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

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“…Due to the prominent role of positive feedback on ATM, we decided to explicitly consider the generation and repair of DSBs in our model. Recently, we measured DSB dynamics in single cells over time and observed considerable variability in their number and half-life, highlighting the stochastic nature of DSB repair [15,39]. We represent DSB kinetics by a data based modeling approach involving stochastic birth-death processes [40].…”

Section: Atm Kinase Is Involved In a Positive Feedback Driving The P5mentioning

confidence: 99%

Excitability in the p53 network mediates robust signaling with tunable activation thresholds in single cells

Moenke¹,

Christiano

Finzel

et al. 2016

Preprint

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Cellular signaling systems precisely transmit information in the presence of molecular noise while retaining flexibility to accommodate the needs of individual cells. To understand design principles underlying such versatile signaling, we analyzed the response of the tumor suppressor p53 to varying levels of DNA damage in hundreds of individual cells and observed a switch between distinct signaling modes characterized by isolated pulses and sustained oscillations of p53 accumulation. Guided by dynamic systems theory we show that this requires an excitable network structure comprising positive feedback and provide experimental evidence for its molecular identity. The resulting data-driven model reproduced all features of measured signaling responses and is sufficient to explain their heterogeneity in individual cells. We present evidence that heterogeneity in the levels of the feedback regulator Wip1 sets cell-specific thresholds for p53 activation, providing means to modulate its response through interacting signaling pathways. Our results demonstrate how excitable signaling networks can provide high specificity, sensitivity and robustness while retaining unique possibilities to adjust their function to the physiology of individual cells.To ensure reliable information processing, cellular signaling systems need to faithfully sense inputs in noisy environments while maintaining the flexibility to adjust their function to different physiologies. A commonly observed strategy to enable robust signal detection is the pulsed activation of signaling pathways in a digital-like response 1 . To understand how pulsatile dynamics can mediate robust yet versatile signal processing, it is necessary to identify the design principles that enable molecular networks to switch between different dynamic states and the mechanisms that allow modulation of their activity.A well-known example of a pulsatile signaling pathway in mammalian cells is the tumor suppressor p53. As a central hub of the cellular stress response, p53 maintains genomic integrity in proliferating cells and during tissue homeostasis 2 . In healthy cells, p53 levels are low due to poly-ubiquitination by the E3-ligase Mdm2 and subsequent proteasomal degradation 3,4 . Upon stress, p53 is activated by kinases that serve as primary damage sensors. One particularly dangerous insult is DNA damage in the form of double strand breaks (DSB), which may cause genomic rearrangements such as translocations, deletions and chromosome fusions. The primary sensor for DSBs is the PI3K-like kinase ataxia telangiectasia mutated (ATM) 5 , which gets phosphorylated and activated within minutes after damage induction 6 . Active ATM then stabilizes p53 by at least two distinct mechanisms: it phosphorylates Mdm2, which induces its auto-ubiquitination and subsequent degradation 7 , and p53, which interferes with Mdm2 binding 8,9 . As a consequence, p53 accumulates in the nucleus, where it acts as a transcription factor activating the expression of hundreds of target genes 10 .A key feature ...

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Dynamics of the DNA damage response: insights from live-cell imaging

Cited by 16 publications

References 67 publications

Bridging the gap between in vitro and in vivo: Dose and schedule predictions for the ATR inhibitor AZD6738

Bridging the gap between in vitro and in vivo: Dose and schedule predictions for the ATR inhibitor AZD6738

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

Excitability in the p53 network mediates robust signaling with tunable activation thresholds in single cells

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