Paclitaxel-induced aberrant mitosis and mitotic slippage efficiently lead to proliferative death irrespective of canonical apoptosis and p53

Abstract

Spindle poisons elicit various cellular responses following metaphase arrest, but how they relate to long-term clonogenicity has remained unclear. We prepared several HeLa lines in which the canonical apoptosis pathway was attenuated, and compared their acute responses to paclitaxel, as well as long-term fate, with the parental line. Three-nanomolar paclitaxel induced brief metaphase arrest (<5 h) often followed by aberrant mitosis, and about 90% of the cells of each line had lost their clonogenicity after 48 h of the treatment. A combination of the same concentration of paclitaxel with the kinesin-5 inhibitor, S-trityl-L-cysteine (STLC), at 1 µM led to much longer arrest (∼20 h) and predominance of subsequent line-specific responses: mitochondrial outer membrane permeabilization (MOMP) in the apoptosis-prone line, or mitotic slippage without obvious MOMP in the apoptosis-reluctant lines. In spite of this, combination with STLC did not lead to a marked difference in clonogenicity between the apoptosis-prone and -reluctant lines, and intriguingly resulted in slightly better clonogenicity than that of cells treated with 3 nM paclitaxel alone. This indicates that changes in the short-term response within 3 possible scenarios - acute MOMP, mitotic slippage or aberrant mitosis - has only a weak impact on clonogenicity. Our results suggest that once cells have committed to slippage or aberrant mitosis they eventually undergo proliferative death irrespective of canonical apoptosis or p53 function. Consistent with this, cells with irregular DNA contents originating from mitotic slippage or aberrant mitosis were mostly eliminated from the population within several rounds of division after the drug treatment.

Keywords: Aberrant mitosis; apoptosis; metaphase arrest; mitotic catastrophe; mitotic slippage; paclitaxel; spindle assembly checkpoint; spindle poison.

Figure 1.

Typical responses of the prepared HeLa cell lines to paclitaxel. (A) Time-lapse observation of MOMP-prone (GI23) and MOMP-reluctant (#44) HeLa cells treated with 10 nM paclitaxel. Contrast of bright field images (BF) is computer-enhanced. The time after drug addition is indicated below the panels. Exit from mitosis was judged by the loss of GMMN-Venus fluorescence. Cells a and b (encircled with dotted lines) of GI23 terminated metaphase arrest with MOMP, followed by loss of mCherry fluorescence (CF) and blebbing (white arrows). Cells e and f of line #44 slipped out of metaphase arrest and underwent blebbing (white arrows) while retaining mCherry fluorescence until the end of observation. (B) Switch-like loss of mCherry fluorescence in each of 4 GI23 cells treated with 10 nM paclitaxel. Cells a and b are identical to those shown in (A). (C). The number of cells retaining mCherry fluorescence is plotted against time after addition of 10 nM paclitaxel. All cells in a single microscopic field were counted for each respective cell line. The slight increase in the number of #44 cells is due to mitosis of the minor fraction.

Figure 2.

Effect of paclitaxel treatment on mitosis and survival of apoptosis-prone and -reluctant cell lines. (A) Duration of mitotic arrest of the 4 cell lines and their behavior on mitotic exit after treatment with 3 and 10 nM paclitaxel. Horizontal lines represent mitotic arrest of individual cells sorted by the type of behavior on mitotic exit and the time of arrest onset. Black, (apparently) symmetric division; red, MOMP during arrest; orange, exit with no division (mitotic slippage); green, asymmetric division; blue, ternary division; purple, miscellaneous. (B) Cell survival immediately after paclitaxel treatment for 48 h measured using the WST assay (see Materials and Methods). (C) Colony-forming ability after paclitaxel treatment for 48 h. The results are shown as mean ± SEM (n = 3).

Figure 3.

Duration of mitotic arrest of the 4 cell lines and their behavior on mitotic exit after treatment with 1 µM STLC with or without 3 nM paclitaxel. Horizontal lines represent duration of arrest and type of behavior on mitotic exit, as in Fig. 2A.

Figure 4.

Effect of STLC on cell survival after paclitaxel treatment. (A) Bulk cell growth after drug treatment. Two thousand or 2 × 10⁴ cells were plated into each well of 24-well plates, treated with appropriate drugs for 48 h, and incubated for a further 5–7 days in fresh medium. Upper two rows represent cells treated with paclitaxel alone and lower 2 rows represent cells treated with paclitaxel and 1 µM STLC. (B) Cell survival immediately after paclitaxel and STLC treatment for 48 h measured using the WST assay (see Materials and Methods). (C) Colony-forming ability after treatment with paclitaxel and STLC for 48 h. The results are shown as mean ± SEM (n = 3).

Figure 5.

Mutual suppressive effect of STLC and paclitaxel on cell killing. (A) Innately apoptosis-reluctant HMVII cells were treated with paclitaxel with (lower 2 rows) or without (upper 2 rows) 1 µM STLC, incubated for 7 days in fresh medium, and then stained. Note that the combination of 1 µM STLC and 0.5 nM paclitaxel resulted in better growth than with 1 µM STLC alone. (B) Growth of HeLa-based cell lines treated with an increasing dose of STLC with (lower 2 rows) or without (upper 2 rows) 0.75 nM paclitaxel. Addition of paclitaxel clearly suppressed cell killing by STLC.

Figure 6.

DNA contents of apoptosis-prone and -reluctant cells during and after drug treatment. Surviving cells were cultured in fresh medium for 5–7 days after the treatment, then fixed and analyzed. Anomaly of the DNA content, probably due to aberrant mitosis or mitotic slippage, is indicated by dotted boxes.