Flow cytometric fluorescence pulse width analysis of etoposide-induced nuclear enlargement in HCT116 cells

Abstract

Fluorescence pulse width can provide size information on the fluorescence-emitting particle, such as the nuclei of propidium iodide-stained cells. To analyze nuclear size in the present study, rather than perform the simple doublet discrimination approach usually employed in flow cytometric DNA content analyses, we assessed the pulse width of the propidium iodide fluorescence signal. The anti-cancer drug etoposide is reportedly cytostatic, can induce a strong G2/M arrest, and results in nuclear enlargement. Based on these characteristics, we used etoposide-treated HCT116 cells as our experimental model system. The fluorescence pulse widths (FL2-W) of etoposide-treated (10 microM, 48 h) cells were distributed at higher positions than those of vehicle control, so the peak FL2-W value of etoposide-treated cells appeared at 400 while those of vehicle control cells appeared at 200 and 270. These results were consistent with our microscopic observations. This etoposide-induced increase in FL2-W was more apparent in G2/M phase than other cell cycle phases, suggesting that etoposide-induced nuclear enlargement preferentially occurred in G2/M phase cells rather than in G0/G1 or S phase cells.

Fig. 1

Schematic diagram of fluorescence pulse signal (height, width, and area)

Fig. 2

Effects of etoposide on viability, cell cycle distribution, and cellular morphology of HCT116 cells. Cells were treated with etoposide (0–10 μM) for 24 or 48 h. a Cell proliferation was determined using the MTT assay. Data are expressed as the mean ± SD of triplicate experiments. ** P < 0.001 and * P < 0.01 compared to vehicle control. Graph is a representative from three independent experiments. b Cell cycle distribution determined by measuring PI-labeled DNA content using flow cytometry. C vehicle control. Graph is a representative from three independent experiments. c Phase contrast microscope images of HCT116 cells (bar = 20 μm). Representative pictures are shown from at least three independent experiments

Fig. 3

Enlargement of cell and nucleus by etoposide treatment in HCT116 cells. a Confocal microscope images of HCT116 cells treated with etoposide (10 μM) for 24 or 48 h. Cells were fixed and stained with DAPI. Differential interference contrast (DIC) images were superimposed on DAPI fluorescence images (bar = 20 μm). b Measurement of nuclear size. HCT116 cells treated with etoposide (0–10 μM) for 24 or 48 h, fixed, and stained with PI. Nuclei size was determined by fluorescence microscopy and the circle measurement algorithm of the microscope software. Each bar represents the mean ± SD (n = 100). * P < 0.001 compared to vehicle control, C. c Expressions of ATM, lamin B1, β-actin, α-tubulin, and GAPDH in HCT116 cells determined by Western blot analysis. ATM phosphorylation was also measured. Cells were treated with etoposide (ET, 10 μM) for 24 h. C vehicle control. Representative data are shown from two independent experiments

Fig. 4

Flow cytometric analysis of PI fluorescence pulse width to evaluate etoposide-induced nuclear enlargement. HCT116 cells were treated with etoposide (10 μM) for 48 h, harvested, fixed in 70% ethanol, and stained with PI. Representative data are shown from three independent experiments. a FL2-A vs. FL2-W dot plot (R1, G0/G1 phase cells; R2, S phase cells; R3, G2 + M phase cells; R4, overall cells, R1 + R2 + R3). b Histogram plots of FSC-H, SSC-H, FL2-A, and FL2-W with gating on R4. Overlay histograms are schematized using vehicle control (dark) and etoposide-treated (48 h) (gray) cell data. Mean values of FSC-H, SSC-H, FL2-A, and FL2-W were calculated using the histogram statistics tool of CellQuest Pro software

Fig. 5

Effects of cell cycle phase on etoposide-induced nuclear enlargement. FL2-W distributions were compared between vehicle control and etoposide-treated cells (10 μM, 48 h). Representative data sets are shown from three independent experiments. a Histogram plots of FL2-W, schematized from cells in each cell cycle phase (left: R1 gated, G0/G1 phase cells; middle: R2 gated, S phase cells; right: R3 gated, G2 + M phase cells; explained in Fig. 4). Overlay histograms schematized using vehicle control (dark) and etoposide-treated (gray) cell data. Mean values of FL2-W are represented in each plot. b Kolmogorov–Smirnov statistics were used to quantitatively compare the FL2-W distribution between vehicle control (dark) and etoposide-treated cells (gray) shown in (a). D/s(n), index of similarity for the two curves. If D/s(n) = 0, the curves are identical. D, greatest difference between the two curves. Channel, channel number of D. P values <0.001 for all data, which is the probability of D being as large as it is assuming that the two selected histogram are from the same population. Each value was calculated using the Kolmogorov–Smirnov statistics tool of CellQuest Pro software