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 μM, 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10529-010-0277-x) contains supplementary material, which is available to authorized users.

Toxicological evaluation of the topoisomerase inhibitor, etoposide, in the model animal Caenorhabditis elegans and 3T3‐L1 normal murine cells

“…Next, we investigated the cell cycle distribution of 3T3‐L1 cells treated with etoposide using flow cytometry DNA content analysis. Etoposide is known to induce G2/M phase arrest in various cancer cells, which is a typical property of topoisomerase II inhibitors . Similar to the results observed in the cancer cells, etoposide potently induced G2/M phase arrest in 3T3‐L1 cells (Figure A).…”

“…Ten gonads were evaluated for each treatment. The nuclear size of the mitotic germ cells was determined using the circle measurement tool of the ProgRes Capture Pro 2.5 software (Jenoptik) as described previously . Two hundred mitotic cells within 20 cells from distal tip cells were randomly selected and evaluated for each treatment.…”

“…The morphology of the 3T3‐L1 cells treated with etoposide was observed using a phase contrast microscope (CK40, Olympus, Tokyo, Japan). We also evaluated the distribution of FSC‐H values, which correspond to the cell size, using flow cytometry analysis according to the previously described method …”

“…After staining with propidium iodide, we observed the nuclear morphology of the 3T3‐L1 cells using a TE2000U fluorescence microscope. The nucleus size (diameter of nucleus) was determined using the circle measurement tool of the microscope software as previously described . We also analyzed the flow cytometric data and the distribution of the FL2‐W values to confirm the changes of the nuclear size determined by microscopy as previously described …”

Daurinol, a catalytic inhibitor of topoisomerase IIα, suppresses SNU-840 ovarian cancer cell proliferation through cell cycle arrest in S phase

“…To evaluate the effect of daurinol and etoposide on cell and nuclear size, we performed fluorescence pulse signal analysis of PI-stained SNU-840 cells as previously described (15,16). The treatment and flow cytometric DNA content analysis were performed as described above in the section of cell cycle analysis.…”