Chromosome mis-segregation and cytokinesis failure in trisomic human cells

Abstract

Cancer cells display aneuploid karyotypes and typically mis-segregate chromosomes at high rates, a phenotype referred to as chromosomal instability (CIN). To test the effects of aneuploidy on chromosome segregation and other mitotic phenotypes we used the colorectal cancer cell line DLD1 (2n = 46) and two variants with trisomy 7 or 13 (DLD1+7 and DLD1+13), as well as euploid and trisomy 13 amniocytes (AF and AF+13). We found that trisomic cells displayed higher rates of chromosome mis-segregation compared to their euploid counterparts. Furthermore, cells with trisomy 13 displayed a distinctive cytokinesis failure phenotype. We showed that up-regulation of SPG20 expression, brought about by trisomy 13 in DLD1+13 and AF+13 cells, is sufficient for the cytokinesis failure phenotype. Overall, our study shows that aneuploidy can induce chromosome mis-segregation. Moreover, we identified a trisomy 13-specific mitotic phenotype that is driven by up-regulation of a gene encoded on the aneuploid chromosome.DOI: http://dx.doi.org/10.7554/eLife.05068.001

Quantitative proteomic and phosphoproteomic comparison of human colon cancer DLD-1 cells differing in ploidy and chromosome stability

“…Compared to the diploid parental line, the frequencies of chromosome missegregation and micronuclei formation were significantly elevated in most PTA clones ( Figure 2A ) but not in the tetraploid line ( Figure 2A ). In agreement with previous work ( Nicholson et al , 2015 ), the trisomic clones showed similar aberrations, albeit to a lesser extent (Supplemental Figure S2B). Furthermore, we observed an increase of structural aberrations in PTA lines and, consistent with earlier work ( Kuznetsova et al , 2015 ; Passerini et al , 2016 ), also in trisomic clones ( Figure 2B ).…”

p53 induces senescence in the unstable progeny of aneuploid cells

“…To independently confirm the observed chromosome instability, RPE +18+18 aneuploid cells were treated with dihydrocytochalasin B (DCB) to disrupt cytokinesis followed by FISH labelling using specific probes to identify chromosomes 13, 18 and 21 in the binucleated cells (Fig 2E and F). This method reveals the reciprocal distribution of labeled chromosomes between daughter nuclei immediately after chromosome segregation and can be applied to the analysis of several hundred cells in tandem [10]. As shown in Fig 2E and F, RPE +18+18 cells displayed a significant increase in chromosome mis-segregation rates, consistent with the results of our live-cell imaging analysis.…”

“…Aneuploid karyotypes display different degrees of genome instability between model systems [8][9][10][11]. We therefore proceeded to test whether karyotype stability of the aneuploid lines obtained here was due to a high overall level of chromosomal integrity or could be attributed to growth restriction of the aberrant daughter cells.…”

“…Despite impairing the proliferative capacity of individual cells, aneuploidy correlates with cancer evolution and therapy resistance [2], possibly due to the fitness advantages this confers under specific environmental and genetic conditions [3][4][5][6][7]. Moreover, since aneuploidy has been shown to cause genetic and chromosomal instability (CIN) in both yeast and mammalian cells [8][9][10][11], this state may promote the generation of heterogeneous populations that support cancer development and tumor acquisition of drug resistant traits [7,12,13]. Given this risk of potential pathological changes, multicellular organisms have evolved a range of different strategies to curb the proliferation of aneuploid cells.…”

The impact of monosomies, trisomies and segmental aneuploidies on chromosomal stability

“…This observation might seem surprising at first as there are many studies that showed that aneuploidy leads to CIN. However, the specific effects of monosomies versus trisomies on CIN have not been extensively explored before as most model systems studying the effects of aneuploidy solely involve chromosome gains [24,25,28,30]. Also, in contrast to our findings, it was recently documented that certain monosomies can in fact trigger a CIN phenotype [60].…”

“…While aneuploidy is a consequence of CIN, it remains controversial if aneuploidy per se can be an underlying cause of CIN. Some studies have indeed suggested that deviating karyotypes can instigate instability [24][25][26][27][28][29]. For instance, CIN has been suggested to be the result of specific gains or losses of chromosomes that contain key regulators of proper segregation in mitosis [25,30].…”

“…Some studies have indeed suggested that deviating karyotypes can instigate instability [24][25][26][27][28][29]. For instance, CIN has been suggested to be the result of specific gains or losses of chromosomes that contain key regulators of proper segregation in mitosis [25,30]. Furthermore, it has been shown that the presence of extra chromosomes disrupts proteostasis and saturation of the protein folding machinery can result in misfolded proteins, aggregate formation and activation of protein degradation pathways [31][32][33][34].…”

The Dynamic Instability of the Aneuploid Genome

“…In line with the data obtained in yeast, work in Chinese hamster embryo and human cells indicates that aneuploidy is associated with genome instability also in higher eukaryotes ( Li et al, 1997 ; Nawata et al, 2011 ; Nicholson et al, 2015 ). Indeed, HE35 cells with an extra copy of chromosome 8 display an increase in structural chromosomal aberrations ( Nawata et al, 2011 ).…”

“…Indeed, HE35 cells with an extra copy of chromosome 8 display an increase in structural chromosomal aberrations ( Nawata et al, 2011 ). Also, a systematic comparison between trisomic and diploid human cells (both untransformed amniotic fibroblasts and colorectal cancer cells DLD1) has uncovered that aneuploidy is associated with increased frequency of anaphase lagging chromosomes and cytokinesis failure ( Nicholson et al, 2015 ).…”

Quantifying chromosomal instability from intratumoral karyotype diversity using agent-based modeling and Bayesian inference

“…Importantly, these putative rates of chromosome mis-segregation fall within the range of approximated per chromosome rates experimentally observed in cancer cell lines and human tumors (Bakhoum et al, 2014(Bakhoum et al, , 2011(Bakhoum et al, , 2009Dewhurst et al, 2014;Nicholson et al, 2015;Orr et al, 2016;Thompson and Compton, 2008;Worrall et al, 2018;Zasadil et al, 2014). Higher inferred mis-segregation rates significantly correlated with lower inferred selection experienced in these samples (Figure 6F).…”

Living in CIN: Mitotic Infidelity and Its Consequences for Tumor Promotion and Suppression

“…Molecularly, cytokinesis failure in DLD1 cells containing an extra copy of chromosome 13 was attributed to overexpression of the gene encoding Spartin, a gene previously implicated in cytokinesis, which is located on chromosome 13. Consistent with this, overexpression of Spartin caused cytokinesis failure in control DLD1 cells while partial depletion of Spartin rescued cytokinesis failure in DLD1 cells and amniotic fibroblasts containing an extra copy of chromosome 13 (Nicholson et al, 2015). Thus, though there are common effects of aneuploidy, individual aneuploid karyotypes also have specific defects depending on the genes on the imbalanced chromosome(s).…”

“…Meiosis in yeast strains with an uneven ploidy (3n or 5n) results in the production of both stably aneuploid and CIN yeast strains, with CIN yeast strains predominating (Pavelka et al, 2010; Sheltzer et al, 2011; Zhu et al, 2012). Similarly, in human cells the addition of one copy of chromosome 7 (in the chromosomally stable colorectal cancer cell line DLD1) or one copy of chromosome 13 (in DLD1 cells or in amniotic fibroblasts) leads to increased rates of chromosome missegregation, predominantly of the triploid chromosome (Nicholson et al, 2015). Gain of chromosome 13 but not 7 causes an increase in tetraploidy due to cytokinesis failure.…”

“…Interphase fibroblasts from human tissue with constitutional gain of one copy of chromosome 8 (Warkany syndrome 2), 13 (Patau syndrome), 18 (Edwards syndrome), or 21 (Down syndrome) had no increase in copy number alterations of chromosomes 2 or 17 (Valind et al, 2013), suggesting that aneuploidy did not result in CIN in this context. During mitosis, the primary chromosome missegregated was found to be the trisomic chromosome itself (Nicholson et al, 2015), which was not examined in the interphase study (Valind et al, 2013), suggesting that trisomic cells without additional mitotic defects, such as cells trisomic for chromosome 7, revert to a diploid state. Consistent with this, cells trisomic for chromosome 3 were found to have late or incomplete replication of the pericentromeric region of the third copy of chromosome 3, which after division can produce one trisomic and one diploid daughter cell.…”

Clonal origin and development of high hyperdiploidy in childhood acute lymphoblastic leukaemia

“…Thus, scWGS strongly supports that HeH ALL is chromosomally stable, in line with cytogenetic and SNP array data. Notably, this also shows that aneuploidy in cancer does not lead to CIN per se; something that has also been debated 33,34 .…”

Growth disadvantage associated with centrosome amplification drives population-level centriole number homeostasis

“…Although we have not characterized the mechanism of the cell cycle arrest and/or delay that many CA-cells undergo, we note that previous findings by other groups showed that CA-cells divide aberrantly to generate aneuploidy at higher frequency than cells without extra centrioles ( Ganem et al. , 2009 ; Nicholson et al. , 2015 ) which can result in cell cycle arrest or cell death ( Nicholson et al.…”

“…, 2009 ; Nicholson et al. , 2015 ) which can result in cell cycle arrest or cell death ( Nicholson et al. , 2015 ),…”

The impact of mitotic errors on cell proliferation and tumorigenesis

“…Consistently, analysis in yeast has shown that aneuploid cells are less genomically stable and show increased rates of chromosome missegregation, mitotic recombination, and defective DNA damage repair (Sheltzer et al 2011;Zhu et al 2012). In human cells, specific aneuploidies have also been shown to increase mitotic error frequency (Nicholson et al 2015;Passerini et al 2016). In addition, while human cell lines with defined trisomies show reduced cell growth in vitro and in xenograft tumor assays, spontaneous karyotype evolution occurs during prolonged growth and improves cellular fitness (Sheltzer et al 2017).…”

“…In addition, aneuploidy can also generate karyotype-specific phenotypic changes that lead to mitotic defects. For example, trisomy of chromosome 13 results in a cytokinesis defect because of increased expression of a gene encoded on the aneuploid chromosome (Nicholson et al 2015). However, CIN is clearly not a necessary outcome of aneuploidy, as cells from individuals with specific trisomies exhibit rates of chromosome missegregation similar to those of euploid cells in vitro, and singlecell sequencing of neurons from an individual with Down syndrome failed to reveal additional aneuploidies (Valind et al 2013;van den Bos et al 2016).…”

Selective advantage of trisomic human cells cultured in non-standard conditions

“…This observation suggests that the advantage conferred by these two chromosomes may depend on a common phenotypic effect observed for both trisomies. Such an effect may be the increased rate of chromosome mis-segregation recently reported for DLD1 + 7, DLD1 + 13, and AF + 13 cells compared to diploid DLD1 and AF cells 27 . In DLD1 + 7, DLD1 + 13, and AF + 13 cells, high rates of chromosome mis-segregation are associated with high rates of karyotypic heterogeneity, which become apparent in the population even when cells are examined at relatively low passages 27 ; this is consistent with findings from other studies showing a link between aneuploidy and chromosome instability (CIN) in both yeast and human cells 39 41 43 44 45 46 .…”

“…Such an effect may be the increased rate of chromosome mis-segregation recently reported for DLD1 + 7, DLD1 + 13, and AF + 13 cells compared to diploid DLD1 and AF cells 27 . In DLD1 + 7, DLD1 + 13, and AF + 13 cells, high rates of chromosome mis-segregation are associated with high rates of karyotypic heterogeneity, which become apparent in the population even when cells are examined at relatively low passages 27 ; this is consistent with findings from other studies showing a link between aneuploidy and chromosome instability (CIN) in both yeast and human cells 39 41 43 44 45 46 . Cell populations with high rates of karyotypic heterogeneity are expected to display high degrees of phenotypic heterogeneity 33 47 and this karyotypic/phenotypic heterogeneity will allow aneuploid cells to be more “adaptable” 33 47 .…”

“…To explore whether aneuploidy confers a selective advantage to cancer cells, we made use of two trisomic cell lines derived from the diploid (2N = 46), chromosomally stable CRC cell line DLD1 24 . The DLD1-derived trisomic cell lines used in this study carried an extra copy of either chromosome 7 (DLD1 + 7) or chromosome 13 (DLD1 + 13) 26 27 . This experimental set-up is advantageous for several reasons.…”

“…Similarly, the acquisition of trisomy 7 in colon epithelial cells cultured in serum-free conditions was associated with overexpression of the epidermal growth factor receptor, encoded on chromosome 7 39 . Finally, a specific cytokinesis failure phenotype was recently shown to be associated with trisomy 13 and to be caused by overexpression of Spartin, a protein encoded by a gene (SPG20) on chromosome 13 27 . The selective advantage we observed in our study seems unlikely to depend on overexpression of specific genes on the aneuploid chromosomes.…”

“…In several of our assays, DLD1 + 13 cells performed better than DLD1 + 7 cells, despite both displaying an advantage over DLD1 cells. DLD1 + 13 cells were recently shown to fail cytokinesis and acquire tetraploid/near-tetraploid karyotypes at significantly higher rates compared to the other two CRC cell lines 27 . Interestingly, tetraploidy was recently shown to increase the tolerance for mitotic errors and CIN 52 53 .…”

“…The DLD1 cell line was obtained from American Type Culture Collection (ATCC, VA, USA); the DLD1 + 7 and DLD1 + 13 cell lines were generated previously by microcell-mediated chromosome transfer 26 , and DLD1 + 13 cells were sub-cloned as previously described 27 . All the DLD1 cell lines were maintained in RPMI 1640 medium (ATCC, VA, USA) supplemented with 10% FBS (Gibco, NY, USA) and antibiotic/antimycotic mixture (Gibco, NY, USA).…”

“…Finally, we extended our investigation to primary human cells by performing cell proliferation experiments in diploid amniocytes (AF) and amniocytes with trisomy 13 (AF + 13). The trisomic cell lines used here (DLD1 + 7, DLD1 + 13, and AF + 13) were recently shown to display higher rates of whole-chromosome mis-segregation and to rapidly accumulate chromosome number heterogeneity compared to their diploid counterparts 27 .…”