Intrinsic and chemically-induced daughter number variations in cancer cell lines

Abstract

Multipolar mitosis was observed in cancer cells under mechanical stress or drug treatment. However, a comprehensive understanding of its basic properties and significance to cancer cell biology is lacking. In the present study, live-cell imaging was employed to investigate the division and nucleation patterns in four different cell lines. Multi-daughter divisions were observed in the three cancer cell lines HepG2, HeLa and A549, but not in the transformed non-cancer cell line RPE1. Multi-daughter mother cells displayed multi-nucleation, enlarged cell area, and prolonged division time. Under acidic pH or treatment with the anti-cancer drug 5-fluorouracil (5-FU) or the phytochemical compound wogonin, multi-daughter mitoses were increased to different extents in all three cancer cell lines, reaching as high as 16% of all mitoses. While less than 0.4% of the bi-daughter mitosis were followed by cell fusion events under the various treatment conditions, 50% or more of the multi-daughter mitoses were followed by fusion events at neutral, acidic or alkaline pH. These findings revealed a "Daughter Number Variation" (DNV) process in the cancer cells, with multi-daughter divisions in Stage 1 and cell fusions leading to the formation of cells containing up to five nuclei in Stage 2. The Stage 2-fusions were inhibited by 5-FU in A549 and HeLa, and by wogonin in A549, HeLa and HepG2. The parallel relationship between DNV frequency and malignancy among the different cell lines suggests that the inclusion of anti-fusion agents exemplified by wogonin and 5-FU could be beneficial in combinatory cancer chemotherapies.

Keywords: 5-fluorouracil; Cancer; cell fusion; multi-daughter division; wogonin.

Figure 1.

Mitosis of HeLa cells under neutral pH condition. (a) HeLa cell nuclei were stained with Hoechst 33,342 dye and recorded by time-lapse imaging. (a) Mono-nucleate mother cell, (b) undergoing metaphase, (c) reaching anaphase, and (d) dividing into two daughters at 60 minutes. (e) Another mono-nucleate mother cell, (f) a triangular metaphase plate, (g) reaching anaphase, and (h) dividing into three daughters at 150 minutes. (b) Mother cells shown by phase contrast (left panels) or staining with Hoechst 33,342 (right panels). Colored arrows indicate mono-nucleate (yellow), bi-nucleate (white), tri-nucleate (red) and tetra-nucleate (green) cells. See Supplementary Figure S1 for snapshots of HeLa, HepG2 and A549 cells under different treatments. Also see videos 1 and 2 corresponding to a-d and e-h, respectively, in Part A

Figure 2.

Multi-daughter production in different cell lines under varied treatment conditions. (a) HepG2, HeLa, A549 and RPE1 cells showing bi-daughter, tri-daughter, tetra-daughter and penta-daughter cell divisions. (b) Semi-quantitative representation of multi-daughter production under different treatment conditions. Bi-daughter divisions were commonly observed in all the cell lines; tri-daughter divisions were observed at neutral, acidic and alkaline pHs, and under wogonin and 5-FU treatments in HepG2, HeLa and A549 cells; tetra-daughter ones in HepG2 and HeLa cells; and penta-daughter ones only in HepG2 cells at neutral and acidic pHs and under wogonin treatment. Magnification ×100, with 10 µm scale bars. See Videos 3–12 for the ten panels in Part A

Figure 3.

Daughter number variations (DNVs) in cancer cells. Schematic representation of two-staged DNV process showing Stage 1 (division) followed by Stage 2 (fusion). The mono-nucleate, bi-nucleate, tri-nucleate or tetra-nucleate mother cells (blue labels) give rise to different division products in Stage 1 (red labels), consisting of mono-nucleate daughters, or two mono-nucleates plus one bi-nucleate daughters. Some of these Stage 1 cell products undergo cellular fusions to form different nucleate pattern P3-P8 (green labels). The final nucleate patterns of DNV daughters therefore include the P1-P8 patterns. The P1 pattern consists of mono-nucleate daughters, and the P2 pattern consists of two mono-nucleate and one bi-nucleate daughters. The bi-nucleate cell in P3 stems from the fusion of two mono-nucleate Stage 1 DNV daughters. In P5, either one of the two bi-nucleate cells in the pattern is mitosis-born while the other arises from the fusion of two Stage 1 mono-nucleate DNV daughters, or both the bi-nucleate cells stem from the fusion of two Stage 1 mono-nucleate DNV daughters. The tri-nucleate cell in P4 arises by fusion of three Stage 1 mono-nucleate cells; P6 consists of one tri-nucleate cell derived from cell fusion and one mono-nucleate cell; and the tetra-nucleate cell in P7as well as the penta-nucleate cell in P8 arise mainly from the fusion of Stage 1 mono-nucleate DNV daughters. In all instances, fusion is indicated by enclosure within a dashed circle (white). See Supplementary Figure 2–18 and Videos 13–29 for illustration of the formation of P1-P8 nucleate patterns

Figure 4.

DNV patterns produced under different treatment conditions. HepG2, HeLa and A549 were treated with neutral, acidic or alkaline pH, or with either wogonin or 5-FU for 24 hours. The color-coded pie charts show the percentages of nucleate patterns P1-P8 under varied treatment conditions, respectively. See numerical percentages in Supplementary Table 2 and Supplementary Figure 23

Figure 5.

Mother cell characteristics. (a) Percentages of mother cells containing different numbers of nuclei. (b) Fusion origin versus mitosis-born origin of binucleate mother (BM) cells found under the different treatment conditions as indicated on x-axis below Part D. (c) Area of mother cells giving rise to bi-daughters (i.e. normal division, green circle) or multi-daughters (i.e. yielding three or more daughters in Stage 1, red circle) under different treatment conditions. (d) Division times of mother cells giving rise to bi-daughters (green circle) or multi-daughters (red circle). Statistical analysis was performed using binomial distribution in Part B, and t test with GraphPad prism software in Parts C and D (* p < 0.05, ** p < 0.001 and *** p < 0.0001). All data sets were generated by three independent experiments, and the mean is represented by horizontal bar