Figure 1.
Induction of cell cycle arrest and cyclin B1/Cdc2 activation in MCF-7 cells by nocodazole (Noco).
A. Changes in cell viability (MTT assay) after treatment of cells with 250 nM nocodazole for different lengths of time. Each data point is the mean ± S.D. from four replicate measurements from one representative experiment. B. Time-dependent induction of G2/M cell cycle arrest following treatment with nocodazole. Cells were seeded at 5×104 cells/mL and then treated with 250 nM nocodazole for 3, 6, 14, 24, 48 and 72 h. Cells were harvested and analyzed using flow cytometry. C. Cells were treatment with 250 nM nocodazole for 12 or 24 h, stained with Hoechst-33342, and examined in a phase contrast (PC) microscope (upper panel) or a fluorescence microscopy (lower panel) (at ×200 magnification). As shown, many cells are arrested in mitosis (prometaphase) after 250 nM treatment. D. Cells were treated with 250 nM nocodazole for 3, 6, 14, 24, 48 and 72 h. The morphology of cells arrested in prometaphase (based on 200 or more nuclei in each sample) was scored by fluorescence microscopy. Each bar is the mean ± S.D. value from three separate experiments. * P<0.05, ** P<0.01 versus vehicle-treated control. E (upper part). Time-dependent changes in cyclin B1 and Cdc2 protein levels following nocodazole treatment. Cells were treated with nocodazole (250 nM) for the length of time as indicated, and whole cell lysates were prepared. An equal amount of protein lysates was electrophoretically separated on the 10% SDS-polyacrylamide gel, and transferred to nitrocellulose membrane. Western blots were detected using specific antibodies against cyclin B1, Cdc2 (CDK1), MAD2, and Cdc20 on an enhanced chemiluminescence (ECL) apparatus. Membrane was stripped for determining the levels of GAPDH as a loading control. E (lower part). The relative protein levels for cyclin B1, Cdc2, MAD2, and Cdc20 were calculated according to their densitometry readings, which were normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate determinations. * P<0.05, ** P<0.01 versus vehicle-treated control. F. Cells were treatment with 250 nM nocodazole for 12 h and analyzed using immunofluorescence staining for cyclin B1 and Cdc2. Representative photographs were taken under a fluorescence microscope (original magnification, ×200).
Figure 2.
Effect of cyclin B1 knockdown on the development of nocodazole (Noco)-induced prometaphase arrest in MCF-7 cells.
A. Cells were transfected with cyclin B1 siRNA (si-cyclin B1) and the negative control siRNAs (si-Con), 24 h later, cells were exposed to 250 nM nocodazole for additional 12 h. Then the whole cell lysates were analyzed for the levels of cyclin B1 and Cdc2by Western immunoblotting. B. Relative protein levels of cyclin B1 and Cdc2 are calculated according to densitometry readings, which are then normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate determinations. * P<0.05, ** P<0.01 versus vehicle-treated control; # P<0.05, ## P<0.01 versus nocodazole treatment. C. Cells were transfected with si-cyclin B1 or siRNA negative control and then further treated with nocodazole (250 nM) for 12 h. Cells with cyclin B1 knockdown were analyzed using immunofluorescent staining for cyclin B1. Representative photographs were taken using a fluorescence microscope (original magnification, ×200) or a phase contrast (PC) microscope (×200). D. Quantitative data on prometaphase-arrested cells. Each bar is a mean ± S.D. value from three separate experiments. * P<0.05, ** P<0.01 versus the vehicle-treated control; # P<0.05 versus nocodazole treatment. E. The DNA content of cells was analyzed using flow cytometry as described in the Material and Methods section.
Figure 3.
Effect of Cdc2 knockdown on the development of nocodazole (Noco)-induced prometaphase arrest in MCF-7 cells.
A. Cells were transfected with siRNA Cdc2 (si-Cdc2) and the negative control siRNAs (si-Con), and 24 h later, cells were exposed to 250 nM nocodazole for additional 12 h. Then the whole cell lysates were analyzed for Cdc2 and cyclin B1 levels using Western immunoblotting. B. The relative protein levels of Cdc2 and cyclin B1 are calculated according to their densitometry readings, which are normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate measurements. * P<0.05 versus vehicle-treated control; # P<0.05 versus nocodazole treatment. C. Cells were transfected with si-cyclin B1 or siRNA negative control and then further treated with nocodazole (250 nM) for 12 h. Cells with Cdc2 knockdown were analyzed using immunofluorescent staining. Representative photographs were taken using a fluorescence microscopy (original magnification, ×200) or a phase contrast microscope (×200). D. Quantitative data on prometaphase-arrested cells. Each bar is a mean ± S.D. value from three separate experiments. ** P<0.01 versus the vehicle-treated control; ## P<0.01 versus nocodazole treatment.
Figure 4.
Effect of MAD2 knockdown on the development of nocodazole (Noco)-induced prometaphase arrest in MCF-7 cells.
A. Cells were transfected with siRNA MAD2 (si-MAD2) and the negative control siRNAs (si-Con), and 24 h later, cells were exposed to 250 nM nocodazole for additional 12 h. Then the whole cell lysates were analyzed for Cdc2, cyclin B1, MAD2, and Cdc20 levels using Western immunoblotting. B. The relative protein levels of Cdc2, cyclin B1, MAD2, and Cdc20 are calculated according to their densitometry readings, which are normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate measurements. * P<0.05 versus vehicle-treated control; # P<0.05 versus nocodazole treatment. C. Cells were transfected with si-MAD2 or siRNA negative control and then further treated with nocodazole (250 nM) for 12 h. Cells with Cdc2 knockdown were analyzed using immunofluorescent staining. Representative photographs were taken using a fluorescence microscopy (original magnification, ×200). D. Quantitative data on prometaphase-arrested cells. Each bar is a mean ± S.D. value from three separate experiments. ** P<0.01 versus the vehicle-treated control; # P<0.05 versus nocodazole treatment. E. The DNA content of cells was analyzed using flow cytometry as described in the Material and Methods section.
Figure 5.
Effect of rescorvitine (Rosc) on nocodazole (Noco)-induced prometaphase arrest in MCF-7 cells.
A (upper panel). Cells were pre-treated for 2 h with roscovitine (10, 20, and 30 µM) and then stimulated for additional 12 h with 250 nM nocodazole. Total cell lysates were analyzed by Western immunoblotting for cyclin B1. A (lower panel). The relative protein levels cyclin B1 are calculated according to their densitometry readings, which are normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from three replicate measurements. ** P<0.01 versus vehicle-treated control; # P<0.05, ## P<0.01 versus nocodazole treatment. B. Cells were pre-treated for 2 h with roscovitine (10, 20, and 30 µM) and then stimulated for additional 12 h with 250 nM nocodazole. The DNA content of cells was analyzed using flow cytometry as described in the Material and Methods section. C. Nuclei were stained with Hoechst-33342, and examined using a fluorescence microscope for prometaphase cells (original magnification, ×200). D. Quantitative data on prometaphase-arrested cells. Each bar is a mean ± S.D. value from three separate experiments. ** P<0.01 versus the vehicle-treated control; ## P<0.01 versus nocodazole treatment.
Figure 6.
Effect of cycloheximide (CHX) on nocodazole-induced prometaphase arrest in MCF-7 cells.
A (left panel). Cells were pre-treated for 2 h with cycloheximide (5 µg/mL) and then stimulated for additional 12 h with 250 nM nocodazole. Total cell lysates were analyzed by Western immunoblotting for cyclin B1 and Cdc2. A (right panel). The relative protein levels of cyclin B1 and Cdc2 are calculated according to their densitometry readings, which are normalized according to the corresponding readings for the GAPDH protein bands. Each value is mean ± S.D. from triplicate measurements. * P<0.05 versus vehicle-treated control; # P<0.05 versus nocodazole treatment. B (upper panel). Cells were pre-treated for 2 h with cycloheximiden (5 µg/mL) and then stimulated for additional 12 h with 250 nM nocodazole. The DNA content of cells was analyzed using flow cytometry as described in the Material and Methods section. B (lower panel). Nuclei were stained with Hoechst-33342, and examined using a fluorescence microscope for prometaphase cells (original magnification, ×200). C. Quantitative data on prometaphase-arrested cells. Each bar is a mean ± S.D. value from three separate experiments. ** P<0.01 versus the vehicle-treated control; ## P<0.01 versus nocodazole treatment.
Figure 7.
Schematic explanation of the contribution of an early cyclin B1/Cdc2 up-regulation to the development of prometaphase arrest in cells treated with nocodazole.
Treatment of cancer cells with nocodazole causes microtubule disruption and thereby prevents microtubules from attaching to kinetochores in prometaphase cells. The unattached kinetochores will be bound by MAD2, which prevents the progression from prometaphase to metaphase and anaphase. It is speculated that the kinetochore-bound MAD2 protein plays an important role in mediating the up-regulation of cyclin B1 and Cdc2 proteins in prometaphase-arrested cells. The rapid increase of these two cell cycle proteins in prometaphase cells and particularly their accumulation in the nuclei are expected to be largely responsible for the development of characteristic nuclear phenotypes. Following a prolonged prometaphase arrest, the nocodazole-treated cells are expected to undergo cell death via intrinsic apoptosis pathways.