Dynamic Proteomics of Individual Cancer Cells in Response to a Drug

Cohen, Ariel; Geva‐Zatorsky, Naama; Eden, Eran; Frenkel‐Morgenstern, Milana; Issaeva, Irina; Sigal, Alex; Milo, Ron; Cohen-Saidon, Cellina; Liron, Yuvalal; Kam, Zvi; Cohen, Lydia; Danon, Tamar; Perzov, Natalie; Alon, Uri

doi:10.1126/science.1160165

Cited by 542 publications

(540 citation statements)

References 36 publications

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“…The presence of rRNA and hub proteins, such as NCL and NPM1 is likely the critical factor for the nucleolar accumulation of many proteins not uniquely related to ribosome biogenesis (27,40). Among these, many DNA repair factors have been shown to localize within the nucleolus (Table 1), which acts as a stress response organelle and responds, in a unique damage-specific manner, to different cellular stresses (5,17,30,106). During a stress response a broad reorganization of the nucleolar proteome occurs; interestingly, the vast majority of nucleolar proteins migrate toward the nucleoplasm and not vice versa, indicating that the nucleolus may act as a reservoir able to release critical factors upon DNA damage (108,139).…”

Section: Dynamics Of Dna Repair Proteins During Genotoxic Damage: Nucmentioning

confidence: 99%

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

Antoniali

Lirussi

Poletto

et al. 2014

Antioxidants & Redox Signaling

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Significance: An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. Recent Advances: After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. Critical Issues: A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. Future Directions: A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world. Antioxid. Redox Signal. 20, 621-639.

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Section: Dynamics Of Dna Repair Proteins During Genotoxic Damage: Nucmentioning

confidence: 99%

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

Antoniali

Lirussi

Poletto

et al. 2014

Antioxidants & Redox Signaling

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“…In fact, cell cycle dependence appears a widespread phenomenon among nuclear proteins (57). Other factors that contribute to the heterogeneity might be the result of differences in complexity of damage clusters (58) or stochasticity in gene expression (59,60).…”

Section: Discussionmentioning

confidence: 99%

High content image cytometry in the context of subnuclear organization

et al. 2009

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The organization of proteins in space and time is essential to their function. To accurately quantify subcellular protein characteristics in a population of cells with regard for the stochasticity of events in a natural context, there is a fast-growing need for image-based cytometry. Simultaneously, the massive amount of data that is generated by image-cytometric analyses, calls for tools that enable pattern recognition and automated classification. In this article, we present a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear spots using automated fluorescence mosaic microscopy, optimized image processing tools, and supervised classification. We demonstrate the efficiency of our analysis by determination of differential DNA damage repair patterns in response to genotoxic stress and radiation, and we show the potential of data mining in pinpointing specific phenotypes after transient transfection. The presented approach allowed for systematic analysis of subnuclear features in large image data sets and accurate classification of phenotypes at the level of the single cell. Consequently, this type of nuclear fingerprinting shows potential for high-throughput applications, such as functional protein assays or drug compound screening. ' 2009 International Society for Advancement of Cytometry

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“…Characterizing such cellular differences is key to understanding normal physiology, combating cancer recurrence 1,2 , and enhancing targeted differentiation for regenerative therapies 3 ; it demands quantifying the proteomes of single cells.…”

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confidence: 99%

Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

Budnik

Levy

Slavov

2017

Preprint

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Cellular heterogeneity is important to biological processes, including cancer 1,2 and development 3 . However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem Quantification of TMT-labeled peptides relies on reporter ions (RI) whose levels reflect both peptide abundances and noise contributions, such as coisolation interference and background noise 8,9 .To evaluate the contribution of background noise to single-cell RI quantification, we estimated the signal-to-noise ratio (SNR), Extended Data Fig. 1. The estimates indicated that RI intensities are proportional to the amount of labeled single-cell proteomes, and very low for channels left empty.These data suggest that the signal measured in single cells exceeds the background noise by 10-fold or more. As an added SNR control for every TMT set, SCoPE-MS leaves the 130N channel empty, thus simultaneously avoiding isotopic cross-contamination from the carrier cells in channel 131 and having a channel that reflects the background noise.To evaluate the ability of SCoPE-MS to distinguish different cell types, we prepared two labelswapped and interlaced TMT sets with alternating single Jurkat and U-937 cells, two blood cancer cell lines with average cell diameter of only 11 µm (Fig. 1b). The levels of all 583 proteins quantified in single cells were projected onto their principle components (PC). The three-dimensional projections of single-cell proteomes clustered by cell type (Fig. 1c), suggesting that SCoPE-MS can identify cell types based on their proteomes. Next, we identified proteins whose levels vary less within a cell type than between cell types. Among the 117 proteins showing such trends at FDR < 2%, we plotted the distributions for seven in Fig. 1d. Some of these proteins are expected to be cell type specific, such as the higher abundance of Complement C3 in the U-937 cells, which are myeloid lineage precursors for macrophages. The consistency of protein fold-changes between Jurkat and U-937 cells is also reflected in the positive correlations among fold-changes estimated from different cells and TMT channels, Extended Data Fig. 2.Next, we quantified single-cell proteome heterogeneity and dynamics during ES cell differentiation. To initiate differentiation, we withdrew leukemia inhibitor factor (LIF) from ES cell cultures and transitioned to suspension culture; LIF withdrawal results in complex and highly heterogeneous differentiation of epiblast lineages in embryoid bodies (EB). We used SCoPE-MS to quantify over a thousand proteins at FDR = 1 % (Extended Data Fig. 3a) and their pair-wise cor- relations (averaging across single cells) in days 3, 5, and 8 after LIF withdrawal (Fig. ...

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Dynamic Proteomics of Individual Cancer Cells in Response to a Drug

Cited by 542 publications

References 36 publications

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

High content image cytometry in the context of subnuclear organization

Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

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