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Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

Ly T, Endo A, Lamond AI - Elife (2015)

Bottom Line: Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014).For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication.The protein data are available in the Encyclopedia of Proteome Dynamics (

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, United Kingdom.

ABSTRACT
Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

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The proteomic response to cell cycle arrests.(A) The final proteomic dataset after quality control filteringconsisted of 3,068 proteins identified with two or more peptides per proteinand quantitated in all three replicates within a treatment group. 484 proteinsvary in abundance between asynchronous arrested cells using cutoffs based oneffect size (≥twofold change between any two conditions) and statisticalrobustness (p < 0.05, ANOVA). The scaled and clustered abundances of these484 arrest regulated proteins are illustrated as a heatmap. Each protein isrepresented by a horizontal line, and the colour (red: high, blue: low)represents the scaled abundance in three treatments. (B) Weidentify four clusters based on peak expression, which are differentiallyenriched in gene ontology (GO) biological functions. (C) Theproportions of arrest regulated proteins in each cluster. (D)Comparison of the protein abundance changes measured in the cell cycle arrestdata set vs the elutriation data set (Ly etal., 2014) for selected proteins (MT–ND5, RRM2, TK1, andCCNB1). Error bars indicate the standard error of the mean log-transformedabundances.DOI:http://dx.doi.org/10.7554/eLife.04534.003
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fig2: The proteomic response to cell cycle arrests.(A) The final proteomic dataset after quality control filteringconsisted of 3,068 proteins identified with two or more peptides per proteinand quantitated in all three replicates within a treatment group. 484 proteinsvary in abundance between asynchronous arrested cells using cutoffs based oneffect size (≥twofold change between any two conditions) and statisticalrobustness (p < 0.05, ANOVA). The scaled and clustered abundances of these484 arrest regulated proteins are illustrated as a heatmap. Each protein isrepresented by a horizontal line, and the colour (red: high, blue: low)represents the scaled abundance in three treatments. (B) Weidentify four clusters based on peak expression, which are differentiallyenriched in gene ontology (GO) biological functions. (C) Theproportions of arrest regulated proteins in each cluster. (D)Comparison of the protein abundance changes measured in the cell cycle arrestdata set vs the elutriation data set (Ly etal., 2014) for selected proteins (MT–ND5, RRM2, TK1, andCCNB1). Error bars indicate the standard error of the mean log-transformedabundances.DOI:http://dx.doi.org/10.7554/eLife.04534.003

Mentions: In summary, the DNA content and immunoblot analyses indicate that the arrested NB4 cellpopulations show high enrichment for the targeted cell cycle phases. Moreover, theenrichment profiles obtained by arrest are similar to those obtained by centrifugalelutriation (cf. [Ly et al., 2014] Figure 2). Therefore, any differences observed inprotein abundance between cell cycle phases in the respective arrested and elutriatedsamples will not be due primarily to differences in enrichment efficiency.10.7554/eLife.04534.003Figure 2.The proteomic response to cell cycle arrests.


Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

Ly T, Endo A, Lamond AI - Elife (2015)

The proteomic response to cell cycle arrests.(A) The final proteomic dataset after quality control filteringconsisted of 3,068 proteins identified with two or more peptides per proteinand quantitated in all three replicates within a treatment group. 484 proteinsvary in abundance between asynchronous arrested cells using cutoffs based oneffect size (≥twofold change between any two conditions) and statisticalrobustness (p < 0.05, ANOVA). The scaled and clustered abundances of these484 arrest regulated proteins are illustrated as a heatmap. Each protein isrepresented by a horizontal line, and the colour (red: high, blue: low)represents the scaled abundance in three treatments. (B) Weidentify four clusters based on peak expression, which are differentiallyenriched in gene ontology (GO) biological functions. (C) Theproportions of arrest regulated proteins in each cluster. (D)Comparison of the protein abundance changes measured in the cell cycle arrestdata set vs the elutriation data set (Ly etal., 2014) for selected proteins (MT–ND5, RRM2, TK1, andCCNB1). Error bars indicate the standard error of the mean log-transformedabundances.DOI:http://dx.doi.org/10.7554/eLife.04534.003
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Related In: Results  -  Collection

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fig2: The proteomic response to cell cycle arrests.(A) The final proteomic dataset after quality control filteringconsisted of 3,068 proteins identified with two or more peptides per proteinand quantitated in all three replicates within a treatment group. 484 proteinsvary in abundance between asynchronous arrested cells using cutoffs based oneffect size (≥twofold change between any two conditions) and statisticalrobustness (p < 0.05, ANOVA). The scaled and clustered abundances of these484 arrest regulated proteins are illustrated as a heatmap. Each protein isrepresented by a horizontal line, and the colour (red: high, blue: low)represents the scaled abundance in three treatments. (B) Weidentify four clusters based on peak expression, which are differentiallyenriched in gene ontology (GO) biological functions. (C) Theproportions of arrest regulated proteins in each cluster. (D)Comparison of the protein abundance changes measured in the cell cycle arrestdata set vs the elutriation data set (Ly etal., 2014) for selected proteins (MT–ND5, RRM2, TK1, andCCNB1). Error bars indicate the standard error of the mean log-transformedabundances.DOI:http://dx.doi.org/10.7554/eLife.04534.003
Mentions: In summary, the DNA content and immunoblot analyses indicate that the arrested NB4 cellpopulations show high enrichment for the targeted cell cycle phases. Moreover, theenrichment profiles obtained by arrest are similar to those obtained by centrifugalelutriation (cf. [Ly et al., 2014] Figure 2). Therefore, any differences observed inprotein abundance between cell cycle phases in the respective arrested and elutriatedsamples will not be due primarily to differences in enrichment efficiency.10.7554/eLife.04534.003Figure 2.The proteomic response to cell cycle arrests.

Bottom Line: Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014).For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication.The protein data are available in the Encyclopedia of Proteome Dynamics (

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, United Kingdom.

ABSTRACT
Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

Show MeSH
Related in: MedlinePlus