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Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells.

Stingele S, Stoehr G, Peplowska K, Cox J, Mann M, Storchova Z - Mol. Syst. Biol. (2012)

Bottom Line: We found that whereas transcription levels reflect the chromosome copy number changes, the abundance of some proteins, such as subunits of protein complexes and protein kinases, is reduced toward diploid levels.For example, the DNA and RNA metabolism pathways were downregulated, whereas several pathways such as energy metabolism, membrane metabolism and lysosomal pathways were upregulated.In particular, we found that the p62-dependent selective autophagy is activated in the human trisomic and tetrasomic cells.

View Article: PubMed Central - PubMed

Affiliation: Group of Maintenance of Genome Stability, Max Planck Institute of Biochemistry, Martinsried, Germany.

ABSTRACT
Extra chromosome copies markedly alter the physiology of eukaryotic cells, but the underlying reasons are not well understood. We created human trisomic and tetrasomic cell lines and determined the quantitative changes in their transcriptome and proteome in comparison with their diploid counterparts. We found that whereas transcription levels reflect the chromosome copy number changes, the abundance of some proteins, such as subunits of protein complexes and protein kinases, is reduced toward diploid levels. Furthermore, using the quantitative data we investigated the changes of cellular pathways in response to aneuploidy. This analysis revealed specific and uniform alterations in pathway regulation in cells with extra chromosomes. For example, the DNA and RNA metabolism pathways were downregulated, whereas several pathways such as energy metabolism, membrane metabolism and lysosomal pathways were upregulated. In particular, we found that the p62-dependent selective autophagy is activated in the human trisomic and tetrasomic cells. Our data present the first broad proteomic analysis of human cells with abnormal karyotypes and suggest a uniform cellular response to the presence of an extra chromosome.

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Analysis of autophagy in trisomic and tetrasomic cell lines. (A) Tri- and tetrasomic cells accumulate p62-positive foci. This effect was further increased by treatment with Bafilomycin A1. Right panel: Quantification of the fluorescence intensity changes; significance was evaluated by non-parametric T-test (***P<0.001), ‘+' designates addition of Bafilomycin A1. (B) p62-positive foci co-localize with ubiquitin-positive foci and their fluorescence intensity is increased in aneuploid cell lines. Plots represent the signal intensity along the indicated gray line. Red line—p62, green line—ubiquitin. Source data is available for this figure in the Supplementary Information.
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f6: Analysis of autophagy in trisomic and tetrasomic cell lines. (A) Tri- and tetrasomic cells accumulate p62-positive foci. This effect was further increased by treatment with Bafilomycin A1. Right panel: Quantification of the fluorescence intensity changes; significance was evaluated by non-parametric T-test (***P<0.001), ‘+' designates addition of Bafilomycin A1. (B) p62-positive foci co-localize with ubiquitin-positive foci and their fluorescence intensity is increased in aneuploid cell lines. Plots represent the signal intensity along the indicated gray line. Red line—p62, green line—ubiquitin. Source data is available for this figure in the Supplementary Information.

Mentions: Markedly, the expression of p62/sequestosome (SQSTM1) was enhanced in all analyzed trisomic and tetrasomic cells (Figure 5C). p62 is a cytoplasmic stress response receptor that is activated by various cellular stresses such as oxidative stress (for review, see Lamark and Johansen, 2009). Misfolded or damaged ubiquitylated proteins are sequestered by p62 into aggregates, and targeted to autophagy via a direct interaction with LC3 (Pankiv et al, 2007). Immunoblotting (Figure 5C) as well as immunofluorescence staining showed a significant increase in p62 levels, which could be further enhanced by treatment with Bafilomycin A1 (Figure 6A). Moreover, indirect immunofluorescence revealed an increased co-localization of p62- and ubiquitin-positive foci in both the HCT116- and the RPE-1-derived cell lines, which is also enhanced by Bafilomycin (Figure 6B; Supplementary Figure S7A and B). Thus, our results suggest that cells with supernumerary chromosomes accumulate ubiquitylated proteins in the cytoplasm, likely as a consequence of the protein imbalance. The increase of p62-dependent autophagy could provide a new understanding of the pathways that allow aneuploid cells to maintain protein homeostasis despite chronic elevated expression of multiple genes.


Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells.

Stingele S, Stoehr G, Peplowska K, Cox J, Mann M, Storchova Z - Mol. Syst. Biol. (2012)

Analysis of autophagy in trisomic and tetrasomic cell lines. (A) Tri- and tetrasomic cells accumulate p62-positive foci. This effect was further increased by treatment with Bafilomycin A1. Right panel: Quantification of the fluorescence intensity changes; significance was evaluated by non-parametric T-test (***P<0.001), ‘+' designates addition of Bafilomycin A1. (B) p62-positive foci co-localize with ubiquitin-positive foci and their fluorescence intensity is increased in aneuploid cell lines. Plots represent the signal intensity along the indicated gray line. Red line—p62, green line—ubiquitin. Source data is available for this figure in the Supplementary Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472693&req=5

f6: Analysis of autophagy in trisomic and tetrasomic cell lines. (A) Tri- and tetrasomic cells accumulate p62-positive foci. This effect was further increased by treatment with Bafilomycin A1. Right panel: Quantification of the fluorescence intensity changes; significance was evaluated by non-parametric T-test (***P<0.001), ‘+' designates addition of Bafilomycin A1. (B) p62-positive foci co-localize with ubiquitin-positive foci and their fluorescence intensity is increased in aneuploid cell lines. Plots represent the signal intensity along the indicated gray line. Red line—p62, green line—ubiquitin. Source data is available for this figure in the Supplementary Information.
Mentions: Markedly, the expression of p62/sequestosome (SQSTM1) was enhanced in all analyzed trisomic and tetrasomic cells (Figure 5C). p62 is a cytoplasmic stress response receptor that is activated by various cellular stresses such as oxidative stress (for review, see Lamark and Johansen, 2009). Misfolded or damaged ubiquitylated proteins are sequestered by p62 into aggregates, and targeted to autophagy via a direct interaction with LC3 (Pankiv et al, 2007). Immunoblotting (Figure 5C) as well as immunofluorescence staining showed a significant increase in p62 levels, which could be further enhanced by treatment with Bafilomycin A1 (Figure 6A). Moreover, indirect immunofluorescence revealed an increased co-localization of p62- and ubiquitin-positive foci in both the HCT116- and the RPE-1-derived cell lines, which is also enhanced by Bafilomycin (Figure 6B; Supplementary Figure S7A and B). Thus, our results suggest that cells with supernumerary chromosomes accumulate ubiquitylated proteins in the cytoplasm, likely as a consequence of the protein imbalance. The increase of p62-dependent autophagy could provide a new understanding of the pathways that allow aneuploid cells to maintain protein homeostasis despite chronic elevated expression of multiple genes.

Bottom Line: We found that whereas transcription levels reflect the chromosome copy number changes, the abundance of some proteins, such as subunits of protein complexes and protein kinases, is reduced toward diploid levels.For example, the DNA and RNA metabolism pathways were downregulated, whereas several pathways such as energy metabolism, membrane metabolism and lysosomal pathways were upregulated.In particular, we found that the p62-dependent selective autophagy is activated in the human trisomic and tetrasomic cells.

View Article: PubMed Central - PubMed

Affiliation: Group of Maintenance of Genome Stability, Max Planck Institute of Biochemistry, Martinsried, Germany.

ABSTRACT
Extra chromosome copies markedly alter the physiology of eukaryotic cells, but the underlying reasons are not well understood. We created human trisomic and tetrasomic cell lines and determined the quantitative changes in their transcriptome and proteome in comparison with their diploid counterparts. We found that whereas transcription levels reflect the chromosome copy number changes, the abundance of some proteins, such as subunits of protein complexes and protein kinases, is reduced toward diploid levels. Furthermore, using the quantitative data we investigated the changes of cellular pathways in response to aneuploidy. This analysis revealed specific and uniform alterations in pathway regulation in cells with extra chromosomes. For example, the DNA and RNA metabolism pathways were downregulated, whereas several pathways such as energy metabolism, membrane metabolism and lysosomal pathways were upregulated. In particular, we found that the p62-dependent selective autophagy is activated in the human trisomic and tetrasomic cells. Our data present the first broad proteomic analysis of human cells with abnormal karyotypes and suggest a uniform cellular response to the presence of an extra chromosome.

Show MeSH