Association between mitotic spindle checkpoint impairment and susceptibility to the induction of apoptosis by anti-microtubule agents in human lung cancers

Abstract

Anti-microtubule agents such as vinorelbine and paclitaxel, which are extensively used in the treatment of lung cancers, activate mitotic spindle checkpoint. Although defects of the mitotic spindle checkpoint are thought to play a role in the genesis of chromosome instability, we previously reported its frequent impairment in human lung cancer cell lines. In this study, we examined a panel of 13 human cancer cell lines comprising 11 lung and 2 other cancers and found a significant difference in the resistance to apoptosis induced by anti-microtubule agents between mitotic spindle checkpoint-impaired and -proficient cancer cell lines. This finding was in marked contrast to a lack of such correlation with a DNA damaging agent, cis-platin. Interestingly, anti-microtubule agent-induced apoptosis in mitotic spindle checkpoint-proficient cell lines, NCI-H460 and A549, was shown to be markedly reduced by staurosporine treatment in association with the shortened mitotic arrest, whereas various inhibitors of caspases seemed to have very modest effects. Taken together, these findings suggest the potential involvement of mitotic spindle checkpoint in the induction of apoptosis by anti-microtubule agents in human lung cancers, warranting further studies on the underlying mechanisms.

Figure 1.

Response of mitotic spindle checkpoint-proficient (NCI-H460) and -impaired (PC-1 and A-427) human lung cancer cell lines to nocodazole. A: Flow cytometric analysis of the cell cycle. Majority of cells are arrested at 4n DNA content in all cell lines when treated with 200 nmol/L of nocodazole for 18 hours. B: Time-dependent changes in mitotic indices. Mitotic index of mitotic spindle checkpoint-proficient NCI-H460 rises rapidly and peaks at 18 hours after the initiation of 200 nmol/L of nocodazole treatment and then declines by adaptation. In contrast, those of the mitotic spindle checkpoint-impaired cell lines show only slight increases. C: Changes in mitotic indices with respect to nocodazole concentrations. Mitotic indices of the mitotic checkpoint-impaired lines are plateaued by 100 nmol/L of nocodazole.

Figure 2.

Affirmation of mitotic arrest in response to nocodazole. A: Inhibition of entry into mitoses in synchronized cells. Cells that had been arrested at the S phase or G₁/S boundary by 16 hours of treatment with 5 μmol/L of aphidicolin were released by replacing the culture media. The initial increases in the mitotic indices are similar regardless of the presence or absence of 200 nmol/L of nocodazole, indicating the absence of entry into mitosis in certain cell lines. B: Inhibition of entry into mitoses in BrdU-labeled asynchronous cells. Simultaneous treatment with 10 μmol/L of BrdU and 200 nmol/L of nocodazole results in the similar initial increases in mitotic cells labeled with BrdU, indicating that cell lines are not differentially arrested before entering mitosis. Note that scales of the y axes are different in each cell line. C: Flow cytometric analyses of expression of cyclin B. Upper figures represent the dot plot, and lower figures represent the percentages of cells residing in each quadrant. The majority of cells having 4n DNA content express cyclin B in a mitotic spindle checkpoint-proficient cell line, but not in the impaired cell lines.

Figure 3.

Induction of apoptosis by the treatment with nocodazole or cis-platin. A: Time-dependent accumulation of apoptotic cells by the treatment with 200 nmol/L of nocodazole. Apoptotic cells become detectable at 18 hours and progressively accumulate with time in mitotic spindle checkpoint-proficient cell line, but not in the impaired cell lines. B: Significantly lower susceptibility to nocodazole-induced apoptosis in mitotic spindle checkpoint-impaired cell lines in a panel of 13 human cancer cell lines [7.1 ± 1.4 versus 38.0 ± 6.9 (mean ± SE), P = 0.01 by Student’s t-test; B]. The susceptibilities of cell lines are shown as the percentage of apoptotic cells 48 hours after the addition of 200 nmol/L of nocodazole. C: Lack of a significant relationship between the mitotic spindle checkpoint integrity and susceptibility to cis-platin-induced apoptosis in the same panel of cell lines [44.9 ± 19.7 versus 69.7 ± 7.2 (mean ± SE), NS]. Susceptibility to cis-platin is shown as the percentage of apoptotic cells at 48 hours after addition of 10 μg/ml of cis-platin.

Figure 4.

Effects of various metabolic inhibitors on the nocodazole-induced apoptosis in mitotic spindle checkpoint-proficient NCI-H460 cells. PD98059, LY294002, wortmannin, SB203580, and caspase inhibitors were added simultaneously with 200 nmol/L of nocodazole treatment, while thymidine and staurosporine were added 12 hours before and after the initiation of nocodazole treatment, respectively. Note that staurosporine treatment markedly reduces apoptotic cells 48 hours after the initiation of nocodazole treatment.

Figure 5.

Effects of staurosporine on the reduction of mitotic indices (A and C) and inhibition of the apoptosis induced by nocodazole (B and D) in mitotic checkpoint-proficient cell lines. Staurosporine at concentrations sufficient to block progression toward the mitotic phase (30 nmol/L) was subsequently added to NCI-H460 (A and B) and A549 (C and D) 12 hours after the initiation of 200 nmol/L nocodazole treatment. Note that staurosporine markedly reduced apoptosis in both cell lines, while staurosporine induces rapid reduction of the mitotic index. E: Effect of timing of the addition of staurosporine on the inhibition of the nocodazole-induced apoptosis in NCI-H460. Note that staurosporine effectively inhibits apoptosis when added early on, but that later addition of staurosporine was not effective for the suppression of apoptosis in cells that had been already exposed to nocodazole for longer than 24 hours.

Figure 6.

No reduction of nocodazole-induced apoptosis by simultaneous treatment with olomoucine in NCI-H460. A: Dose-dependent inhibition of the mitotic entry by olomoucine. Mitotic indices were counted 18 hours after the initiation of simultaneous treatment with nocodazole and olomoucine. B: No reduction in nocodazole-induced apoptosis by olomoucine. Apoptotic cells were examined after 48 hours of the treatment with nocodazole and olomoucine.

Figure 7.

Induction of phosphorylation of bcl-2 by nocodazole treatment. Apparent phosphorylation of bcl-2 is present in mitotic spindle checkpoint-proficient cell lines including nocodazole-resistant QG56.

Figure 8.

Induction of activating cleavages of caspases by nocodazole treatment. Note that complete inhibition of caspase 3 cleavage is seen despite a very modest reduction in apoptosis (see Figure 4 ▶ ) in nocodazole-sensitive, mitotic checkpoint-proficient NCI-H460.