Generation of trisomies in cancer cells by multipolar mitosis and incomplete cytokinesis

Abstract

One extra chromosome copy (i.e., trisomy) is the most common type of chromosome aberration in cancer cells. The mechanisms behind the generation of trisomies in tumor cells are largely unknown, although it has been suggested that dysfunction of the spindle assembly checkpoint (SAC) leads to an accumulation of trisomies through failure to correctly segregate sister chromatids in successive cell divisions. By using Wilms tumor as a model for cancers with trisomies, we now show that trisomic cells can form even in the presence of a functional SAC through tripolar cell divisions in which sister chromatid separation proceeds in a regular fashion, but cytokinesis failure nevertheless leads to an asymmetrical segregation of chromosomes into two daughter cells. A model for the generation of trisomies by such asymmetrical cell division accurately predicted several features of clones having extra chromosomes in vivo, including the ratio between trisomies and tetrasomies and the observation that different trisomies found in the same tumor occupy identical proportions of cells and colocalize in tumor tissue. Our findings provide an experimentally validated model explaining how multiple trisomies can occur in tumor cells that still maintain accurate sister chromatid separation at metaphase-anaphase transition and thereby physiologically satisfy the SAC.

Fig. 1.

Chromosome missegregation in bipolar and multipolar mitoses. (A) FISH with centromeric probes for chromosomes 7 (red), 12 (green), and 18 (violet) shows amphitelic chromosome segregations at anaphase in an F-N fibroblast. Homologous chromosomes equidistant from the cellular equator have been classified in probable sister chromosome pairs (broken white lines). (B and C) Probes for chromosomes 13 (green), 18 (violet), and 21 (red) shows 3–1 missegregation at telophase of chromosome 21 (arrows) in an F-N fibroblast (B) and an MVA28 fibroblast (C). (D) Time-lapse series (t is time in minutes) showing a tripolar metaphase (green broken lines; t = 0) followed by tripolar ana-telophase (t = 14–36 min). Cytokinesis is initiated along two cleavage furrows in this cell division (t = 70 min), but one of the cleavage furrows regresses and only two daughter cells are formed (t = 306 min), of which the larger is binucleate as evidenced by two clusters of nucleoli. The larger cell (t = 3,192 min) enters mitosis (red broken lines), forming a single mitotic plate and divides into two daughters (t = 3,586 min). Daughter cells from both cell divisions remained without evidence of cell death or degeneration throughout the observation time (139-h total time lapse; Movies S1 and S2). (E and F) Immunofluorescence staining for β-tubulin (green) and MAD2L1 (red) in WiT49 cells shows retention of MAD2L1 foci in a complex tetrapolar anaphase cell with lagging chromosomes (F, arrows), whereas no MAD2L1 foci are present in a tripolar anaphase cell (E); note the absence of a β-tubulin-positive midbody between the two upper poles in E. (G) Immunofluorescence stain655555ing for β-tubulin (red) combined with FISH for the centromeres of chromosomes 4 (green), 7 (violet), and 9 (yellow) in a postmitotic HEK293D cell shows 3–1 segregation of chromosome 7, resulting in trisomy 7 in the binucleated daughter cell (nuclei a and b) and monosomy in the mononucleated daughter cell (c). (H) β-Tubulin staining combined with FISH for the centromeres of chromosomes 3 (yellow), 11 (violet), and 15 (green) in a telophase HEK293D cell shows concurrent formation of trisomies for chromosomes 3 and 11 in the binucleated daughter cell (nuclei a and b) and monosomies for these chromosomes in the mononucleated daughter cell (c); note the absence of midbody between a and b. (I) Time-lapse fluorescence/phase contrast microscopy in HEK293D H2B-GFP cells shows a tripolar anaphase (t = 0) resulting in one binucleated (a, b) and one mononucleated (c) daughter cell (t = 1,555 min; Movie S4).

Fig. 2.

Models for the generation of trisomies and tetrasomies. (A) A tripolar nuclear division with amphitelic sister chromatid separation and segregation, followed by incomplete cytokinesis, will generate tetrasomies in one daughter cell (blue membrane, Right) for chromosomes (blue) of which both homologues are located on the metaphase axis (blue line, Left), along which the cleavage furrow fails to ingress completely (red arrow, Right), whereas trisomies will be generated in the same daughter cell for homologues (red) located on this axis and on either of the other axes (green lines, Left); disomies will be retained when both homologues (green) are present on the axes (green) along which cytokinesis is complete. (B) A bipolar mitosis with missegregation of one chromosome (red) will generate one trisomic and one monosomic daughter cell. Another missegregation event in the trisomic cell population involving the same (red) chromosome will result in tetrasomic and disomic daughter cells (Lower Left), whereas missegregation involving another chromosome will result in two trisomies (Lower Right). (C) The frequency of tetrasomic tumors (blue plot) in 152 WTs with at least two whole chromosome gains is well in accordance with the model in A (green plot) but differs significantly (χ² test) from the distribution predicted from the model in B (red plot). The proportion of cells carrying specific chromosomal imbalances in primary WT biopsies WT-F (D), WT-G (E), and WT-H (F), estimated from B-allele frequencies at SNP-array analysis. Trisomies (+) are present in an equal proportion of cells (D and E; red demarcations), whereas segmental imbalances are typically present in clones of different sizes (E and F). Abnormalities present at similar proportions are signified by identical colors (red, green, or blue bars), whereas abnormalities present in significantly different proportions of cells are signified by different colors. Gray bars indicate populations for which the proportion confidence intervals overlapped with that of at least one other population. del, hemizygous deletion; dup, duplication; trp, triplication; upd, uniparental disomy.

Fig. 3.

Spatial distribution of trisomic cells in tumor tissue. (A) Tissue section from WT-G with trisomies 7 and 12 in a subpopulation of tumor cells, previously detected by SNP-based array comparative genomic hybridization. Most nuclei (stained by diaminophenylindol; blue) are sectioned, leading to a reduced number of probe signals. Therefore, the distribution of trisomic cells was mapped by calculating copy number ratios in foci of 30 to 100 cells by FISH with centromeric probes for chromosomes 7 (A, II) and 12 (A, III), using centromere 16 as a reference for disomy (Fig. S6E shows signal number ratios). This allowed demarcation of areas containing cells with trisomies (red borders in A, I) and disomies (blue borders) in the corresponding H&E section. Blue and red filled circles in A, II, and A, III, indicate cell populations classified as disomic and trisomic, respectively. The adjacent normal kidney (green borders) contains only disomic areas. The asterisk represents disomic stromal tissue surrounding a blood vessel. (B) Representative FISH image of normal kidney tubules (area B in A, I; rectangular area in B, I, is shown at higher magnification in B, II) with disomy for chromosomes 7 (red), 12 (green), and 16 (violet; arrow in B, II). (C and D) Trisomic cells were detected in epithelial (C in A, I) and stromal (D in A, I) tumor elements, as exemplified by cells showing three signals for each of chromosomes 7 and 12 (arrows in high-power images C, II, and D, II).