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Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest.

Dikovskaya D, Cole JJ, Mason SM, Nixon C, Karim SA, McGarry L, Clark W, Hewitt RN, Sammons MA, Zhu J, Athineos D, Leach JD, Marchesi F, van Tuyn J, Tait SW, Brock C, Morton JP, Wu H, Berger SL, Blyth K, Adams PD - Cell Rep (2015)

Bottom Line: ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects.Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16.In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Sciences, CR-UK Beatson Laboratories, University of Glasgow, Glasgow G61 1BD, UK. Electronic address: ddikovskaya@gmail.com.

No MeSH data available.


Related in: MedlinePlus

Activated H-RasV12 Dysregulates a Subset of Mitotic Genes in Pre-senescent Cells(A) Experimental layout. 4-day-induced (+4OHT) or control (−4OHT) ERRAS cells were arrested in mitosis with DME (12–16 hr). Mitotic (detached) cells were selectively collected by a washout, and >90% mitotic index (MI) was confirmed by microscopic scoring. Total RNA was isolated and validated on the Bioanalyzer. A cDNA library was constructed from poly(A) RNA and subjected to RNA-seq. The experiment was independently repeated three times. See Table S1 for alignment statistics.(B) Total number of significantly upregulated genes (up) and downregulated genes (down) genes (5% FDR) in Ras-induced mitotic cells compared to control mitotic cells.(C) PCA of Ras-induced (R1, R2, and R3) and control (C1, C2, and C3) mitoses based on expression of each known coding gene by FPKM. PC1 and PC2, principal components 1 and 2, respectively.(D) Heatmap of significantly differentially expressed genes (5% FDR) within the mitotic-related gene set (collated from several GSAE/MSigDB entries, Broad Institute) between Ras-induced mitotic cells and control mitotic cells. Genes are represented in columns, and samples are represented in rows. In the FPKM-based column Z-score, intensity represents higher (red) to lower (blue) expression.(E) DAVID GO analysis of differentially expressed genes between Ras-induced mitotic cells and control mitotic cells. The top ten most enriched ontologies are shown (FDR <5%).(F) Heatmap of gene expression for genes within mitotic-spindle-related GO terms, derived and represented as in (D).(G) Heatmap of gene expression for genes within the chromatin gene set derived and represented as in (D).See also Tables S1 and S2.
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fig3: Activated H-RasV12 Dysregulates a Subset of Mitotic Genes in Pre-senescent Cells(A) Experimental layout. 4-day-induced (+4OHT) or control (−4OHT) ERRAS cells were arrested in mitosis with DME (12–16 hr). Mitotic (detached) cells were selectively collected by a washout, and >90% mitotic index (MI) was confirmed by microscopic scoring. Total RNA was isolated and validated on the Bioanalyzer. A cDNA library was constructed from poly(A) RNA and subjected to RNA-seq. The experiment was independently repeated three times. See Table S1 for alignment statistics.(B) Total number of significantly upregulated genes (up) and downregulated genes (down) genes (5% FDR) in Ras-induced mitotic cells compared to control mitotic cells.(C) PCA of Ras-induced (R1, R2, and R3) and control (C1, C2, and C3) mitoses based on expression of each known coding gene by FPKM. PC1 and PC2, principal components 1 and 2, respectively.(D) Heatmap of significantly differentially expressed genes (5% FDR) within the mitotic-related gene set (collated from several GSAE/MSigDB entries, Broad Institute) between Ras-induced mitotic cells and control mitotic cells. Genes are represented in columns, and samples are represented in rows. In the FPKM-based column Z-score, intensity represents higher (red) to lower (blue) expression.(E) DAVID GO analysis of differentially expressed genes between Ras-induced mitotic cells and control mitotic cells. The top ten most enriched ontologies are shown (FDR <5%).(F) Heatmap of gene expression for genes within mitotic-spindle-related GO terms, derived and represented as in (D).(G) Heatmap of gene expression for genes within the chromatin gene set derived and represented as in (D).See also Tables S1 and S2.

Mentions: To identify potential cause(s) of mitotic defects during the establishment of OIS, we performed RNA sequencing (RNA-seq) gene expression profiling of cells captured in mitosis 4 days after Ras activation (Figure 3A). Our isolation procedure yielded >90% mitotic cells from both induced and control ERRAS cells. RNA-seq revealed that Ras induction significantly altered the abundance of approximately 2,000 gene transcripts in mitotic cells (Figure 3B). Principal-component analysis (PCA) showed that replicates were highly consistent (Figure 3C). Consistent with Ras-induced defects in mitosis, we found that, out of 371 genes included in mitosis-related Gene Ontology (GO) terms, 74 were significantly (5% false discovery rate [FDR]) altered in mitoses with activated Ras (Figure 3D; Table S2). This constituted a statistically significant (empirical p value < 0.0001) 2.17-fold enrichment of alterations in this gene set over randomly expected changes. Furthermore, out of 328 transcripts that were highly up- or downregulated in normal mitosis compared to the unsynchronized control ERRAS cell population [that are potentially relevant to mitotic processes (Cho et al., 2001)], 64 (approximately 20%) were significantly (5% FDR) altered by H-RasV12 in mitotic cells (2.11-fold increase over random, empirical p value < 0.0001). More importantly, mitotic spindle-related gene ontologies (namely, “mitotic spindle organization,” “spindle localization,” and “establishment of spindle localization”) were the top three most altered GO terms (Figures 3E and 3F; Table S2), consistent with the diverse spindle defects in induced ERRAS cells (Figures 2A and 2B). Changes in the spindle-related gene set were, significantly (empirical p < 0.0001), 3.18-fold enriched over randomly expected. In line with the observed chromatin defect in these cells (Figures 2C–2E), the expression of chromatin regulators was also significantly (5% FDR) changed (Figure 3G; Table S2) (1.26-fold enrichment, empirical p = 0.0318). Underscoring the specificity of these changes, the spindle checkpoint GO term was not significantly altered in Ras-induced mitotic cells (empirical p = 0.75), consistent with efficient mitotic arrest in these cells (see Figures 4B and 4C). Thus, H-RasV12 activation dysregulates expression of a specific subset of mitotic genes linked to the observed mitotic abnormalities in pre-senescent cells.


Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest.

Dikovskaya D, Cole JJ, Mason SM, Nixon C, Karim SA, McGarry L, Clark W, Hewitt RN, Sammons MA, Zhu J, Athineos D, Leach JD, Marchesi F, van Tuyn J, Tait SW, Brock C, Morton JP, Wu H, Berger SL, Blyth K, Adams PD - Cell Rep (2015)

Activated H-RasV12 Dysregulates a Subset of Mitotic Genes in Pre-senescent Cells(A) Experimental layout. 4-day-induced (+4OHT) or control (−4OHT) ERRAS cells were arrested in mitosis with DME (12–16 hr). Mitotic (detached) cells were selectively collected by a washout, and >90% mitotic index (MI) was confirmed by microscopic scoring. Total RNA was isolated and validated on the Bioanalyzer. A cDNA library was constructed from poly(A) RNA and subjected to RNA-seq. The experiment was independently repeated three times. See Table S1 for alignment statistics.(B) Total number of significantly upregulated genes (up) and downregulated genes (down) genes (5% FDR) in Ras-induced mitotic cells compared to control mitotic cells.(C) PCA of Ras-induced (R1, R2, and R3) and control (C1, C2, and C3) mitoses based on expression of each known coding gene by FPKM. PC1 and PC2, principal components 1 and 2, respectively.(D) Heatmap of significantly differentially expressed genes (5% FDR) within the mitotic-related gene set (collated from several GSAE/MSigDB entries, Broad Institute) between Ras-induced mitotic cells and control mitotic cells. Genes are represented in columns, and samples are represented in rows. In the FPKM-based column Z-score, intensity represents higher (red) to lower (blue) expression.(E) DAVID GO analysis of differentially expressed genes between Ras-induced mitotic cells and control mitotic cells. The top ten most enriched ontologies are shown (FDR <5%).(F) Heatmap of gene expression for genes within mitotic-spindle-related GO terms, derived and represented as in (D).(G) Heatmap of gene expression for genes within the chromatin gene set derived and represented as in (D).See also Tables S1 and S2.
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fig3: Activated H-RasV12 Dysregulates a Subset of Mitotic Genes in Pre-senescent Cells(A) Experimental layout. 4-day-induced (+4OHT) or control (−4OHT) ERRAS cells were arrested in mitosis with DME (12–16 hr). Mitotic (detached) cells were selectively collected by a washout, and >90% mitotic index (MI) was confirmed by microscopic scoring. Total RNA was isolated and validated on the Bioanalyzer. A cDNA library was constructed from poly(A) RNA and subjected to RNA-seq. The experiment was independently repeated three times. See Table S1 for alignment statistics.(B) Total number of significantly upregulated genes (up) and downregulated genes (down) genes (5% FDR) in Ras-induced mitotic cells compared to control mitotic cells.(C) PCA of Ras-induced (R1, R2, and R3) and control (C1, C2, and C3) mitoses based on expression of each known coding gene by FPKM. PC1 and PC2, principal components 1 and 2, respectively.(D) Heatmap of significantly differentially expressed genes (5% FDR) within the mitotic-related gene set (collated from several GSAE/MSigDB entries, Broad Institute) between Ras-induced mitotic cells and control mitotic cells. Genes are represented in columns, and samples are represented in rows. In the FPKM-based column Z-score, intensity represents higher (red) to lower (blue) expression.(E) DAVID GO analysis of differentially expressed genes between Ras-induced mitotic cells and control mitotic cells. The top ten most enriched ontologies are shown (FDR <5%).(F) Heatmap of gene expression for genes within mitotic-spindle-related GO terms, derived and represented as in (D).(G) Heatmap of gene expression for genes within the chromatin gene set derived and represented as in (D).See also Tables S1 and S2.
Mentions: To identify potential cause(s) of mitotic defects during the establishment of OIS, we performed RNA sequencing (RNA-seq) gene expression profiling of cells captured in mitosis 4 days after Ras activation (Figure 3A). Our isolation procedure yielded >90% mitotic cells from both induced and control ERRAS cells. RNA-seq revealed that Ras induction significantly altered the abundance of approximately 2,000 gene transcripts in mitotic cells (Figure 3B). Principal-component analysis (PCA) showed that replicates were highly consistent (Figure 3C). Consistent with Ras-induced defects in mitosis, we found that, out of 371 genes included in mitosis-related Gene Ontology (GO) terms, 74 were significantly (5% false discovery rate [FDR]) altered in mitoses with activated Ras (Figure 3D; Table S2). This constituted a statistically significant (empirical p value < 0.0001) 2.17-fold enrichment of alterations in this gene set over randomly expected changes. Furthermore, out of 328 transcripts that were highly up- or downregulated in normal mitosis compared to the unsynchronized control ERRAS cell population [that are potentially relevant to mitotic processes (Cho et al., 2001)], 64 (approximately 20%) were significantly (5% FDR) altered by H-RasV12 in mitotic cells (2.11-fold increase over random, empirical p value < 0.0001). More importantly, mitotic spindle-related gene ontologies (namely, “mitotic spindle organization,” “spindle localization,” and “establishment of spindle localization”) were the top three most altered GO terms (Figures 3E and 3F; Table S2), consistent with the diverse spindle defects in induced ERRAS cells (Figures 2A and 2B). Changes in the spindle-related gene set were, significantly (empirical p < 0.0001), 3.18-fold enriched over randomly expected. In line with the observed chromatin defect in these cells (Figures 2C–2E), the expression of chromatin regulators was also significantly (5% FDR) changed (Figure 3G; Table S2) (1.26-fold enrichment, empirical p = 0.0318). Underscoring the specificity of these changes, the spindle checkpoint GO term was not significantly altered in Ras-induced mitotic cells (empirical p = 0.75), consistent with efficient mitotic arrest in these cells (see Figures 4B and 4C). Thus, H-RasV12 activation dysregulates expression of a specific subset of mitotic genes linked to the observed mitotic abnormalities in pre-senescent cells.

Bottom Line: ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects.Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16.In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Sciences, CR-UK Beatson Laboratories, University of Glasgow, Glasgow G61 1BD, UK. Electronic address: ddikovskaya@gmail.com.

No MeSH data available.


Related in: MedlinePlus