Transient endoreplication down-regulates the kinesin-14 HSET and contributes to genomic instability

Abstract

Polyploid cancer cells exhibit chromosomal instability (CIN), which is associated with tumorigenesis and therapy resistance. The mechanisms that induce polyploidy and how these mechanisms contribute to CIN are not fully understood. Here we evaluate CIN in human cells that become polyploid through an experimentally induced endoreplication cycle. When these induced endoreplicating cells (iECs) returned to mitosis, it resulted in aneuploidy in daughter cells. This aneuploidy resulted from multipolar divisions, chromosome missegregation, and failure in cytokinesis. The iECs went through several rounds of division, ultimately spawning proliferative cells of reduced ploidy. iECs have reduced levels of the kinesin-14 HSET, which likely accounts for the multipolar divisions, and overexpression of HSET reduced spindle multipolarity. However, HSET overexpression had only mild effects on CIN, suggesting that additional defects must contribute to genomic instability in dividing iECs. Overall our results suggest that transient endoreplication cycles generate a diverse population of proliferative aneuploid cells that have the potential to contribute to tumor heterogeneity.

FIGURE 1:

Antimitotic drugs induce polyploidy. (A) Representative FACS profiles showing the DNA content of propidium iodide–stained cells with the indicated treatments. (B) Quantification of FACS data from three independent experiments showing the percentage of cells with different ploidy under the indicated treatments. For each experiment, 10,000 cells were analyzed for DNA content by propidium iodide staining, and the mean ± SD is graphed. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Micrographs of HeLa cells stained with Hoechst to visualize DNA after the indicated treatments. Scale bar, 20 μm. (D) Dot plots showing the quantification of the nuclear size from three independent experiments in which at least 300 cells were scored per experiment. Bar and whiskers indicate the mean and SD. ***p < 0.001.

FIGURE 2:

Antimitotic drugs induce polyploidy through endocycle/endomitosis. (A) Time line showing experimental procedure; t = 0 indicates the time of drug addition and corresponds to the timing in B–D. (B, C) Representative time-lapse images of HeLa cells treated with 6 μM RO3306 (B) or 6 μM SU6656 (C) for 24 or 40 h, respectively. Time is marked in the upper left, and the red arrow marks the same cell at different time points. Scale bar, 10 μm. (D) Cell fate profiles of HeLa cells exposed to indicated treatment; t = 0 corresponds to time of drug addition.

FIGURE 3:

SU6656 induces polyploidy through endomitosis. (A) Control DMSO or SU6656 treated cells were fixed at ∼14 h after drug addition and processed for immunofluorescence to visualize MTs (green) and DNA (blue). Scale bar, 10 μm. (B) Quantification of the percentage of mitotic cells at different mitotic stages from the experiment in A. Data are mean ± SD from three independent experiments in which 900 cells were scored per condition. **p < 0.01, ***p < 0.001. (C) Representative time-lapse images of HeLa cells expressing GFP-labeled histone H2B treated with DMSO or SU6656 treatment. Time is marked in the upper left. Scale bar, 10 μm.

FIGURE 4:

RTM in polyploid cells results in multipolar spindles and asymmetric cell division. (A) Time line showing experimental procedure. (B) Representative images of HeLa cells after SU6656 washout (t = 0). Time after washout is marked in the upper left corner. The orange arrow represents one cell that divides into two cells (yellow and red arrows). The purple arrow represents the two daughters from the previous division of the cell indicated by the red arrow. Scale bar, 20 μm. (C) Cell morphology profiles of HeLa cells; t = 0 indicates the time of drug washout. (D) Cell fate analysis of HeLa iECs from high-throughput imaging from 7 to 16 h after drug washout. HeLa cells were synchronized, treated with SU6656 for 48 h, and then imaged at 5-min intervals starting from 9 to 16 h after drug washout. A total of 2168 cells were analyzed, 694 of which entered mitosis. The number in parentheses represents the total number of mitotic cells with the indicated cell fate.

FIGURE 5:

RTM in highly polyploid cells is error-prone and results in aneuploidy. (A–D) HeLa cells expressing GFP-CENP-A and GFP-γ-tubulin were fixed at 14 h after drug washout, labeled to visualize kinetochore/spindle poles (green), DNA (blue), and microtubules (red), and imaged by superresolution microscopy. The red arrows indicate abnormally located chromosomes during anaphase (B) or cytokinesis (C). (E, F) IMARIS 3D software was used to detect and quantify the number of GFP-CENP-A–labeled kinetochores from images in C and D. Clusters of individual kinetochores in different daughter cells are indicated by different colors, and the number of kinetochores detected is indicated by the number with the corresponding color. Scale bar, 20 μm. (G, H) HeLa cells expressing GFP-CENP-A and GFP-γ-tubulin were synchronized, treated with SU6656 for 48 h, and then imaged at 5-min intervals starting from 5 h after drug washout. Selected frames are shown, and the time is marked in the upper left corner. Scale bar, 20 μm. One example iEC divided into three daughter cells (G) and another failed in division (H). (I) Quantification of the percentage of cells with failed or successful cytokinesis among all anaphase/telophase cells with GFP-Cenp-A dots in the midzone; 62 cells. (J) Dot plot showing the quantification of the number of kinetochores in each individual cell taken from the time-lapse images. The dots represent the number of kinetochores, and the bar and whiskers indicate the mean ± SD; 18 cells. ***p < 0.001.

FIGURE 6:

RTM in polyploid cells correlates with high missegregation rate. (A, B) Representative micrographs of HeLa cells labeled with FISH probes for chromosome 2 and 7. Cells were synchronized, treated with DMSO (A) or SU6656 (B) for 48 h, and then cultured in drug-free medium for 20 h. Cells were fixed and hybridized with probes specific for centrometric satellite DNA of chromosome 2 or 7 and stained with Hoechst to visualize DNA. Scale bar, 10 μm. (C) Histogram showing the relative frequency of cells with the indicated number of chromosomes (>480 cells). (D) Percentage of anaphase/telophase cells that have an unequal distribution of the indicated chromosomes, represented as mean ± SD from three independent experiments (200 cells total). ***p < 0.001.

FIGURE 7:

Polyploid cells can resume cell proliferation and reduce their genome content over time. (A, B) HeLa cells were treated with SU6656 for 48 h and then cultured in drug-free medium for various amounts of time before the cells were counted or processed for FACS. (A) Quantification of cell density at selected days after drug washout. Each point represents mean ± SD from four independent experiments. (B) Quantification of FACS data from four independent experiments showing the percentage of cells with different ploidy after drug washout on the indicated days. For each experiment, 5000 cells were measured for DNA content, and the mean ± SD is graphed. (C, D) HeLa GFP–CENP-A cells were synchronized, treated with SU6656 for 48 h, and imaged at 1-d intervals (C) or fixed and stained every other day (D); t = 0 is time after drug washout. Selected frames are shown, and the time is marked in the upper left corner. Scale bar, 25 μm. (D) The dot plot shows the quantification of kinetochore number in individual cells on selected days after drug washout. The dots represent the number of kinetochores. The bars and whiskers indicate the mean ± SD; >120 total cells counted for controls and >240 for drug-treated cells. (E) Dot plot showing the quantification of the number of kinetochores in individual cells within a single colony of cells. The dots represent the number of kinetochores, and the bar and whiskers indicate the mean ± SD. **p < 0.01, ***p < 0.001.

FIGURE 8:

HSET overexpression partially rescues mitotic defects during RTM in polyploid cells. (A) Western blot of HeLa cells treated with DMSO (control) or SU6656 with washout for RTM and probed with anti-HSET (top) or anti-tubulin (bottom) antibodies. (B) Quantification of the number of spindle poles in either HeLa control or GFP-HSET–overexpressing iECs at 12 h after drug washout. Bars indicate the mean ± SD from three independent experiments in which at least 300 cells were scored in each condition. (C) Representative micrographs showing the inverse relationship between GFP-HSET intensity and number of spindle poles in different cells. Microtubule (MT) labeling (top) and level of GFP-HSET (bottom). Scale bar, 20 μm. (D) Average fluorescence intensity of GFP-HSET relative to number of poles from 116 cells from three independent experiments. (E, F) HeLa cells or GFP-HSET–overexpressing HeLa cells were synchronized, treated with SU6656 for 48 h, and cultured in drug-free medium for 20 h. Cells were then fixed and stained with probes specific for centrometric satellite DNA of chromosome 2 or 7 and stained with Hoechst to visualize DNA. (E) Histogram showing the relative frequency of the number of chromosomes (>450). (F) Percentage of anaphase/telophase cells that have an unequal distribution of the indicated chromosomes represented as mean ± SD from three independent experiments (200 total cells counted). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 9:

Model. After treatments that perturb cell cycle progression, a fraction of the cells escape apoptosis and instead enter the alternative cell cycle state of endoreplication (endocycle/endomitosis). Switching to endoreplication may provide a mechanism for therapy resistance. On discontinuation of the perturbation, many cells die, but a fraction of these polyploid cells resume mitotic divisions, which are highly error-prone, contributing to tumor heterogeneity and leading to cancer relapse.