Proteomic Analysis of Aneuploidy

Aneuploidy is the most common cause of early miscarriage and a cause of serious disabilities.¹ To better understand the cellular response of aneuploidy in human cells, work from Stingele et al. presents the first proteomic analysis of human cells with abnormal karyotypes.²

To study cellular changes of aneuploid cells, trisomic and tetrasomic cells were generated from chromosomally stable cell lines, HCT116 or the GFP-expressing HCT116 H2B-GFP. Micronuclei were employed to transfer additional copies of chromosome 3 into HCT116 (or chromosome 5 into HCT116 and HCT116 H2b-GFP), creating the trisomic and tetrasomic cells. To further strengthen the analysis and make up for any possible aberrances with this specific cell line, Stingele et al. also generated cell lines trisomic for chromosomes 5 and 12, as well as a cell line trisomic for chromosome 21 from the epithelial cell line RPE-1.

HCT116 cells tetrasomic for chromosome 5 (HCT116 5/4) was the most extensive cell line analyzed. To compare protein levels with the diploid parental cells, stable isotope labeling with amino acids in culture (SILAC) was used, followed by high-resolution mass spectrometry on an LTQ-Orbitrap XL or LTQ-Velos Instrument (Thermo Scientific). By this method, approximately 6,000 proteins were quantified. Six replicates of HCT116 5/4 were studied, along with up to three replicates of the other cell lines, which demonstrated a high accuracy for quantification and reproducibility for corresponding experiments. DNA levels were quantified using CGH.

Analysis of the aneuploid cells revealed cell growth was remarkedly slowed for the trisomic cell lines and even more so for the tetrasomic cell lines, in particular, the G1 and S phase. This has been previously confirmed in individuals with Down syndrome,³ as well as in studies with yeast models.^4,5

Comparison of DNA, RNA, and mRNA levels revealed 27% of proteins coded on HCT116 5/4 were at or near levels expected for the diploid parental lines.

This also proved to be true for trisomic chromosome 3. Approximately 25% of proteins coded were less abundant than the amounts expected based on the levels of mRNA present. Among those decreased proteins were kinases and subunits of protein complexes. A previous study by Torres et al. found that a decrease in protein levels can compensate for an increase in gene copy number, and as a result, the stoichiometry of protein subunits is maintained.⁶ To explore this possibility, the Stingele group examined 14 macromolecular complexes with one subunit coded on the tetrasomic chromosome. Eight of the 14 complexes (57%) were able to maintain stoichiometry by decreasing the protein levels.

The global cellular response to aneuploidy included downregulation of DNA and RNA metabolism pathways and upregulation of energy metabolism, membrane metabolism and lysosomal pathways. P62-dependant autophagy was activated, likely as an attempt to regulate the imbalance of proteins.

Further understanding of aneuploidy, and the global effects involved within the cell, could lead to possible treatments for trisomy syndromes and lead to important applications in cancer research.

References

1. Hassold, T., Hall, H., and Hunt, P. (2007) ‘The origin of human aneuploidy: where we have been, where we are going.‘, Human Molecular Genetics, 16 (R2), (R203-R208)

2. Stingele, S., et al. (2012) ‘Global analysis of genome, transcriptome and proteome reveals the response to Aneuploidy in human cells‘, Molecular Systems Biology, 8 (608), published online September 11, 2012. doi: 10.1038/msb.2012.40

3. Segal, D.J. and McCoy, E.E. (1974) ‘Studies on Down’s syndrome in tissue culture. I. Growth rates and protein contents of fibroblast cultures‘, Journal of Cellular Physiology, 83 (1), (pp. 85-90)

4. Pavelka, N., et al. (2010) ‘Aneuploidy confers quantitative proteome changes and phenotypic variation in budding yeast‘, Nature, 468 (7321), (pp. 321-325)

5. Torres, E.M., et al. (2007) ‘Effects of aneuploidy on cellular physiology and cell division in haploid yeast‘, Science, 317 (5840), (pp. 916-924)

6. Torres, E.M., et al. (2010) ‘Identification of aneuploidy-tolerating mutations‘, Cell, 143 (1), (pp. 71-83)