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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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Activation of autophagy in trisomic and tetrasomic cell lines. (A) Abundance changes of the sub-category autophagy (GOBP) in all analyzed cell lines are depicted. CGH and proteome data are shown. (B) Fluorescence intensity of LC3-positive foci in HCT116 and HCT116 5/4. Right panel: quantification of the fluorescence intensity (non-parametric T-test, **P<0.01). (C) Western blot of LC3-II shows an increase in all analyzed aneuploids. Similarly, levels of p62/SQSTM1 are increased in aneuploids. Note that p62 is coded on chromosome 5, but its levels are increased in all aneuploids. Tun—diploid HCT116 treated with tunicamycin that activates unfolded protein response and hence autophagy. (D) Representative images of cells after transfection with the double-tagged mRFP-GFP-LC3. Yellow foci represent phagosomes (both GFP and mRFP signals visible), red foci represent lysosomes (only mRFP signal is insensitive to the acidic pH in lysosomes). Bar 10 μm. (E) Total number of LC3 foci within a defined area of each cell (2500 voxels). There are significantly more foci in the HCT116 3/3 cell line (non-parametric T-test, P<0.01), the levels in HCT116 5/4 are higher, but the difference from HCT116 is not statistically significant. See also Supplementary Figure S5. Source data is available for this figure in the Supplementary Information.
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f5: Activation of autophagy in trisomic and tetrasomic cell lines. (A) Abundance changes of the sub-category autophagy (GOBP) in all analyzed cell lines are depicted. CGH and proteome data are shown. (B) Fluorescence intensity of LC3-positive foci in HCT116 and HCT116 5/4. Right panel: quantification of the fluorescence intensity (non-parametric T-test, **P<0.01). (C) Western blot of LC3-II shows an increase in all analyzed aneuploids. Similarly, levels of p62/SQSTM1 are increased in aneuploids. Note that p62 is coded on chromosome 5, but its levels are increased in all aneuploids. Tun—diploid HCT116 treated with tunicamycin that activates unfolded protein response and hence autophagy. (D) Representative images of cells after transfection with the double-tagged mRFP-GFP-LC3. Yellow foci represent phagosomes (both GFP and mRFP signals visible), red foci represent lysosomes (only mRFP signal is insensitive to the acidic pH in lysosomes). Bar 10 μm. (E) Total number of LC3 foci within a defined area of each cell (2500 voxels). There are significantly more foci in the HCT116 3/3 cell line (non-parametric T-test, P<0.01), the levels in HCT116 5/4 are higher, but the difference from HCT116 is not statistically significant. See also Supplementary Figure S5. Source data is available for this figure in the Supplementary Information.

Mentions: The analysis of the altered pathways indicates that lysosome proteins are upregulated in all aneuploid cell lines (Figure 4C; Supplementary Figure S5). The lysosome is essential for autophagy, a pathway involved in removal of damaged or superfluous proteins and organelles (He and Klionsky, 2009). A detailed analysis of the proteins involved in autophagic processes suggested that autophagy might be more active in aneuploid cell lines (Figure 5A). To substantiate this finding, we performed functional analysis of autophagy in aneuploid cells in comparison with parental diploid cell lines. By immunofluorescence, we observed an increased number of LC3 foci in HCT116 5/4 in comparison with HCT116 (Figure 5B). Similarly, immunoblot analysis of protein lysates from aneuploid clones showed accumulation of the autophagy marker LC3-II (Figure 5C), which is the lipidated form of LC3 that is conjugated to phosphatidylethanolamine (PE) when integrated into the membrane destined for autophagosomes (Kabeya et al, 2000). By monitoring the doubly tagged mRFP-GFP-LC3 (Kimura et al, 2007) we found that the turnover of LC3 in aneuploid clones is similar to that of the diploid control (Figure 5D and E) and can be blocked by inhibition of autophagy with Bafilomycin A1 (Supplementary Figure S6), a drug inhibiting the acidification of lysosomes and fusion of autophagosomes to lysosomes (Klionsky et al, 2008). This confirms that the elevated numbers of autophagosomes are not due to a defect in autophagosome-to-lysosome fusion, but indeed due to autophagy activation. In conclusion, our data suggest that autophagy is activated in trisomic and tetrasomic human cells.


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)

Activation of autophagy in trisomic and tetrasomic cell lines. (A) Abundance changes of the sub-category autophagy (GOBP) in all analyzed cell lines are depicted. CGH and proteome data are shown. (B) Fluorescence intensity of LC3-positive foci in HCT116 and HCT116 5/4. Right panel: quantification of the fluorescence intensity (non-parametric T-test, **P<0.01). (C) Western blot of LC3-II shows an increase in all analyzed aneuploids. Similarly, levels of p62/SQSTM1 are increased in aneuploids. Note that p62 is coded on chromosome 5, but its levels are increased in all aneuploids. Tun—diploid HCT116 treated with tunicamycin that activates unfolded protein response and hence autophagy. (D) Representative images of cells after transfection with the double-tagged mRFP-GFP-LC3. Yellow foci represent phagosomes (both GFP and mRFP signals visible), red foci represent lysosomes (only mRFP signal is insensitive to the acidic pH in lysosomes). Bar 10 μm. (E) Total number of LC3 foci within a defined area of each cell (2500 voxels). There are significantly more foci in the HCT116 3/3 cell line (non-parametric T-test, P<0.01), the levels in HCT116 5/4 are higher, but the difference from HCT116 is not statistically significant. See also Supplementary Figure S5. 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

f5: Activation of autophagy in trisomic and tetrasomic cell lines. (A) Abundance changes of the sub-category autophagy (GOBP) in all analyzed cell lines are depicted. CGH and proteome data are shown. (B) Fluorescence intensity of LC3-positive foci in HCT116 and HCT116 5/4. Right panel: quantification of the fluorescence intensity (non-parametric T-test, **P<0.01). (C) Western blot of LC3-II shows an increase in all analyzed aneuploids. Similarly, levels of p62/SQSTM1 are increased in aneuploids. Note that p62 is coded on chromosome 5, but its levels are increased in all aneuploids. Tun—diploid HCT116 treated with tunicamycin that activates unfolded protein response and hence autophagy. (D) Representative images of cells after transfection with the double-tagged mRFP-GFP-LC3. Yellow foci represent phagosomes (both GFP and mRFP signals visible), red foci represent lysosomes (only mRFP signal is insensitive to the acidic pH in lysosomes). Bar 10 μm. (E) Total number of LC3 foci within a defined area of each cell (2500 voxels). There are significantly more foci in the HCT116 3/3 cell line (non-parametric T-test, P<0.01), the levels in HCT116 5/4 are higher, but the difference from HCT116 is not statistically significant. See also Supplementary Figure S5. Source data is available for this figure in the Supplementary Information.
Mentions: The analysis of the altered pathways indicates that lysosome proteins are upregulated in all aneuploid cell lines (Figure 4C; Supplementary Figure S5). The lysosome is essential for autophagy, a pathway involved in removal of damaged or superfluous proteins and organelles (He and Klionsky, 2009). A detailed analysis of the proteins involved in autophagic processes suggested that autophagy might be more active in aneuploid cell lines (Figure 5A). To substantiate this finding, we performed functional analysis of autophagy in aneuploid cells in comparison with parental diploid cell lines. By immunofluorescence, we observed an increased number of LC3 foci in HCT116 5/4 in comparison with HCT116 (Figure 5B). Similarly, immunoblot analysis of protein lysates from aneuploid clones showed accumulation of the autophagy marker LC3-II (Figure 5C), which is the lipidated form of LC3 that is conjugated to phosphatidylethanolamine (PE) when integrated into the membrane destined for autophagosomes (Kabeya et al, 2000). By monitoring the doubly tagged mRFP-GFP-LC3 (Kimura et al, 2007) we found that the turnover of LC3 in aneuploid clones is similar to that of the diploid control (Figure 5D and E) and can be blocked by inhibition of autophagy with Bafilomycin A1 (Supplementary Figure S6), a drug inhibiting the acidification of lysosomes and fusion of autophagosomes to lysosomes (Klionsky et al, 2008). This confirms that the elevated numbers of autophagosomes are not due to a defect in autophagosome-to-lysosome fusion, but indeed due to autophagy activation. In conclusion, our data suggest that autophagy is activated in trisomic and tetrasomic human cells.

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