Figure 1.
(A) Fucci-based small molecule screening for overriding SAC. HeLa. S-Fucci2 cells were arrested by single thymidine block (STA) and released in medium containing 1 μM nocodazole for 16 hr. Most cells showed green fluorescence, and nocodazole-arrested cells (left) were treated with the MPS1 inhibitor, reversine for 5 hr. Cells show red fluorescence due to overriding SAC. (B) Identification of ROCK inhibitors as small molecules bypassing SAC. 30 μM Y-27632, 1 μM H-1152, and 30 μM fasudil overrode SAC as cells show red fluorescence. The population of the cells in G1 (red) was counted after 5 hr treatment. Data is mean and standard error from three independent experiments. (C) ROCK inhibitors bypassed SAC by a microtubule-dependent mechanism, not by inhibiting the SAC directly. (i) Immunofluorescent images of microtubules at low or high concentration of nocodazole. At 0.3 μM nocodazole, microtubules still remained, whereas they were completely disrupted at 3.3 μM nocodazole. Bar represents 10 μm. (ii) Reversine triggered cyclin B1 degradation in both conditions, whereas ROCK inhibitors failed to trigger cyclin B1 degradation at 3.3 μM nocodazole. Relative amount of Cyclin B1 was measured by densitometry.
Figure 2.
ROCK inhibitors induce chromosome mis-segregation and microtubule-dependent centrosome fragmentation.
(A) Chromosome mis-segregation induced by ROCK inhibitors. HeLa cells stably expressing H2B-GFP were treated with indicated concentrations of ROCK inhibitors for 48 hr. Red and white arrowheads indicate chromosome alignment defect and chromosome-bridge, respectively. (B) Centrosome fragmentation induced by ROCK inhibitors. HeLa cells were arrested by a double thymidine block and released and arrested at metaphase by 10 μM MG132. Mitotic spindles and centrosomes were observed by immunofluorescent microscopy. (i) Representative images of centrosome fragmentation by ROCK inhibitors. Bar represents 10 μm. (ii) Quantification of centrosome fragmentation by ROCK inhibitors. At least 50 cells were counted from 3 independent experiments for each sample. Error bar represents standard error. (C) ROCK depletion induced centrosome fragmentation similar to that by ROCK inhibitors. (i) Depletion of ROCK1 and ROCK2 by siRNAs. (ii) Quantification of centrosome fragmentation by ROCK depletion. At least 50 cells were counted from 3 independent experiments for each sample. Error bar represents standard error. (D) Depolymerization of microtubules abolished the centrosome fragmentation by ROCK inhibitors. HeLa cells were arrested with 3.3 μM nocodazole and treated with 30 μM Y-27632 for 3 hr. Bar represents 10 μm. (E) Centriole fragmentation by ROCK inhibitors. Representative images of centrin localization during metaphase. Cells were arrested at metaphase as in Figure 2B and centrin (centriole)-pericentrin (PCM) (i) or centrin (centriole)-γ-tubulin (PCM) (ii) were visualized by immunofluorescence. Bar represents 10 μm.
Figure 3.
Multimodal effects of ROCK and LIMK inhibitors on mitotic spindles and centrosomes.
(A) Centrosome fragmentation induced by LIMK inhibitor BMS-5. Cells were treated with 3 μM BMS-5 and arrested at metaphase. (i) Representative images of immunofluorescent microscopy. Bar represents 10 mm. (ii) Quantification of centrosome fragmentation by BMS-5. At least 50 cells were counted for each sample from 3 independent experiments. Error bar represents standard error. (iii) Centriole fragmentation by LIMK inhibitor. Centrin-γ-tubulin and centrin-pericentrin were visualized by immunofluorescent microscopy. Bar represents 10 μm. (B) Hyper-stabilization of microtubules by ROCK and LIMK1 inhibitors. Cells were arrested by double thymidine block and released. Then cells were treated with 30 μM Y-27632, 1 μM H-1152, 30 μM fasudil, 3 μM BMS-5, or 50 nM MLN8237 for 2 hr and were placed at 4°C for 30 min. Microtubules and centrosomes were visualized by tubulin and γ-tubulin immunofluorescence. (i) Representative images of microtubules after cold treatment. (ii) Quantification of cold-stable microtubules in the cells. At least 50 cells were counted for each sample from 3 independent experiments. Error bar represents standard error. (C) Inhibition of ROCK or LIMK impaired activation of Aurora-A. Cells were arrested by double thymidine block and released in 3.3 μM nocodazole, then treated with the indicated concentrations of inhibitors for 3 hr. Cell lysate was subjected to SDS-PAGE with Phos-tag acrylamide. Aurora-A was detected by western blotting. The ratio (phospho-Aurora-A/total Aurora-A) is shown. ND: not determined. Percentage of mitotic cells (round cells) is shown. Data represents means of triplicates of at least two independent experiments.
Figure 4.
ROCK and LIMK inhibitors suppress T cell leukemia growth in vitro and induced chromosome instability.
(A) Sensitivity of T cell leukemia cell lines to ROCK and LIMK inhibitors. H-1152 (i), Fasudil (ii), BMS-5 (iii). Cell viability was determined by MTT assay. Data are means and standard deviation from triplicates. (B) Sensitivity of PBMC to ROCK and LIMK inhibitors. H-1152 (i), Fasudil (ii), BMS-5 (iii). Cell viability of PBMC from three different individuals (Donor 1-3) was determined by MTT assay. Data are means and standard deviation from triplicates. (C) Centrosome fragmentation in T cell leukemia cell lines by ROCK and LIMK inhibitors. Cells were treated with the indicated concentrations of inhibitors and α-tubulin and γ-tubulin were immunostained. (i) Representative images of mitotic cells treated with inhibitors. Bar represents 10 μm. (ii) Centrosome fragmentations in 3 cell lines. At least 30 cells were counted from 3 independent experiments. Error bar represents standard error.
Figure 5.
Induction of apoptosis to T cell leukemia cells by ROCK and LIMK inhibitors. (A) ROCK and LIMK inhibitors induced apoptosis to Jurkat and ATN-1.
Cells were treated with inhibitors for 60 hr. Apoptosis was detected by the staining of PI and Annexin V. Data are representative of at least 2 independent experiments. (B) Induction of a p73 target gene in ROCK or LIMK inhibitor-treated cells. Expression of PUMA was examined by real time PCR. Data represents mean and standard error from three independent experiments. (C) Proposed mechanism of action of ROCK and LIMK inhibitors on T cell leukemia cells.