Dependence of Paclitaxel Sensitivity on a Functional Spindle Assembly Checkpoint

Cell Lines and Cell Culture.

All human cell lines used in this study—HEK 293 cells, for development of the recombinant plasmid; MCF-7 breast cancer and MCF-10A normal mammary cells, which are known to have a functional spindle assembly checkpoint; and T47D breast cancer and Ovca432 ovarian cancer cells, which have a defective checkpoint because of low Mad2 expression—were obtained from the American Type Culture Collection (Rockville, MD). HEK 293, MCF-7, and T47D cells were grown in DMEM:Ham’s F-12 medium. Ovca432 cells were maintained in RPMI 1640. Both DMEM:Ham’s F-12 medium and RPMI 1640 were supplemented with 2 mm l-glutamine, 10% fetal bovine serum (100 IU/ml), and penicillin-streptomycin (100 mg/ml). MCF-10A cells were maintained in DMEM:Ham’s F-12 medium supplemented with 5% horse serum, 0.02 μg/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 0.1 μg/ml cholera toxin, 100 IU/ml penicillin, and 100 mg/ml streptomycin.

Small-Interfering RNA (siRNA) Transfection.

Twenty-one nucleotide siRNA duplexes were synthesized by Dharmacon Research, Inc. (Lafayette, CO), to target the Mad2 sequence 5′-AAACCTTTACTCGAGTGCAGA-3′ and the BubR1 sequence 5′-AACAATACTCTTCAGCAGCAG-3′. Transfections of MCF-7 cells were performed in accordance with the protocol provided by Dharmacon Research using oligofectamine transfection reagent (Invitrogen, Carlsbad, CA). For the control experiments, cells were transfected with a siRNA-scrambled duplex (Dharmacon Research). The final concentration for the siRNAs was 200 nm.

Production of Replication-Defective Recombinant Adenovirus.

The adenovirus was produced in accordance with the protocol described by Dr. Vogelstein’s group (23) and Stratagene (La Jolla, CA). Briefly, the gene of cDNA Mad2 was first cloned into a shuttle vector, pAdTrack-cytomegalovirus. The resultant plasmid was linearized by digestion with restriction endonuclease Pme I and subsequently cotransformed into Escherichia coli BJ5183 cells using an adenoviral backbone plasmid, pAdEasy-1 (Stratagene). Recombinants were selected for kanamycin resistance, and recombination was confirmed by restriction endonuclease analyses. Finally, HEK 293 cells were transfected with the linearized recombinant plasmid. For our study, an infection efficiency of 80–90%, with no cytopathic effect, was obtained in each cell.

Western Blot Analysis.

At 24, 48, and 72 h after transfection, cells were harvested and subjected to protein immunoblot analysis. Cells were washed once in ice-cold PBS and lysed with lysis buffer [1% NP40, 150 mm NaCl, and 50 mm Tris-HCl (pH 7.5)] containing protease inhibitors (1 mm phenylmethane sulfonyl fluoride and 10 μg/ml aprotinin) and phosphatase inhibitors (20 mm β-glycerophosphate, 5 mm NaF, and 100 μm Na₃VO₄). After 30 min on ice, cells were subjected to centrifugation at 13,000 rpm for 15 min at 4°C. For Western blotting, equal amounts of proteins were dissolved using SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with polyclonal anti-Mad2 antibody (1:500; Covance, Princeton, NJ), monoclonal anti-BubR1 antibody (1:500; Chemicon, Temecula, CA), and monoclonal anti-α-tubulin (1:5000; Sigma-Aldrich Chemical Co., St. Louis, MO) for 1 h at room temperature (or overnight at 4°C), followed by incubation with horseradish peroxidase-conjugated antibodies. The results were visualized with the enhanced chemiluminescence detection system.

Drug Sensitivity Assays.

Cells were detached by trypsinization, seeded at 2.0 × 10³ cells/well in a 96-well microtiter plate, and treated with various concentrations of paclitaxel (1, 5, 10, 50, 100, and 1000 nm). Seventy-two h later, the effects on cell growth were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (5 mg/ml in PBS; Sigma-Aldrich) were added to each well, and the cells were incubated for 4 h at 37°C. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-formazan formed by metabolically viable cells was dissolved in 100 μl of cell lysis buffer, and fluorescence was monitored using a microplate at a wavelength of 570 nm. The percentage of cell growth was calculated by defining the absorption of cells not treated with paclitaxel (control) as 100%.

Calculation of Mitotic Indices.

Cells with mitotic condensed chromatin were visualized by staining with 10 μm Hoechst 33342 dye (Aventis Pharmaceuticals Inc., Bridgewater, NJ) in conjunction with 10 μg/ml propidium iodide; the propidium iodide was incorporated into dead cells only. Therefore, dead cells were stained with both propidium iodide and Hoechst 33342 dye, whereas mitotic cells showed the condensed chromatins with Hoechst 33342 dye only. The cells were harvested at 12, 24, and 36 h after transfection and the mitotic indices calculated. Results are presented after at least three independent experiments performed in triplicate.

Cell Death Analysis.

Cell death was evaluated using the trypan blue dye exclusion assay. Briefly, cells were harvested using trypsin and stained with 0.4% trypan blue dye (Sigma-Aldrich). Trypan blue-positive and -negative cells were counted using a hemacytometer (Hausser Scientific, Horsham, PA) under a phase-contrast microscope (Fisher Scientific, Pittsburgh, PA). The results of each assay were expressed in terms of the percentage of dead cells relative to the total number of cells. Individual experiments were performed in triplicate. The results were reported as the mean values ±SDs. Presented results reflect at least three independent experiments performed in triplicate.

Cdk1 Kinase Assay.

The Cdk1 protein kinase assay was performed using the SignaTECT cdc2 protein assay system (Promega, Madison, WI). Briefly, the harvested cells were lysed with the extraction buffer [50 mm Tris (pH 7.4), 150 mm NaCl, 0.1% Triton X-100, and 1 mm EDTA] containing protease inhibitors (100 μg/ml aprotinin and 0.5 mm phenylmethane sulfonyl fluoride) and phosphatase inhibitors (50 mm NaF). These lysates were conjugated with a substrate consisting of cdc2-specific biotinylated peptide derived from histone H1 and [γ-³²P]ATP, and incubated at 30°C for 10 min. These radiolabeled, phosphorylated substrates were recovered with streptavidin matrix biotin capture membrane (SAM; Promega). After several washings, each captured membrane was placed into a separate vial and analyzed using a liquid scintillation counter (Beckman Coulter, Palo Alto, CA). Presented results reflect at least three independent experiments performed in triplicate.

Inactivation of Spindle Assembly Checkpoint and Correlation with Suppression of Cdk1 Activity.

Transfection of MCF-7 cells, which have a functional checkpoint, with the 21-nucleotide siRNA duplex homologous to a portion of the Mad2 and BubR1 sequences resulted in dramatic reduction of Mad2 and BubR1 protein levels; these levels remained low at 24, 48, and 72 h after transfection in each cell line. Cotransfection of MCF-7 cells with siRNA/Mad2 and siRNA/BubR1 also reduced both expressions, yielding results similar to those obtained with single transfection (Fig. 1A) ⇓ . The scrambled siRNA duplex (siRNA/control) did not affect the expression level of Mad2 or BubR1, verifying the specificity of the siRNA approach.

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Fig. 1.

Loss of spindle assembly checkpoint in cells with suppression of Mad2, BubR1, or both. A, reduction of BubR1 and Mad2 levels after transfection with small-interfering siRNA (siRNA). MCF-7 cells were transfected with siRNA/Mad2 or siRNA/BubR1, resulting in a final concentration of 200 nm for the siRNAs. At the indicated times after transfection, cells were harvested and subjected to protein immunoblot analysis with each antibody and α-tubulin antibody to control for gel loading. B, no difference in cell cycle distribution by suppression of BubR1, MAD2, or both. Cell cycle distribution of cells with suppression of Mad2, BubR1, or both, as measured by DNA content by fluorescence-activated cell sorting as described in “Materials and Methods.” MCF-7 cells were transfected and harvested 72 h after transfection. Numbers indicate percentages of cells in each phase. Nonstatistical significance was determined in each cell line by χ² test (P = 0.0514). C, reduction of mitotic indices of each cell by suppression of BubR1, MAD2, or both after treatment with paclitaxel. Twenty-four h after transfection, cells were treated with paclitaxel (100 nm) and harvested at the indicated times. Bars, SDs. D, suppression of cyclin-dependent kinase-1 (Cdk1) activity by suppression of BubR1, MAD2, or both after treatment with paclitaxel in each cell line. Twenty-four h after transfection, cells were treated with paclitaxel (100 nm) and harvested at the indicated times. Activity of Cdk1 in the lysates was determined as described in “Materials and Methods.” Bars, SDs.

Using flow cytometry, we attempted to determine whether transient suppression of mitotic checkpoint genes by siRNA affects the cell cycle distribution. Seventy-two h after transfection, there were no statistically significant differences in cell cycle distributions in neither the Mad2- nor the BubR1-suppressed cells (P = 0.514; Fig. 1B ⇓ ). To test the effects of suppression of Mad2, BubR1, or both on the spindle assembly checkpoint activated by paclitaxel, we determined the mitotic indices and measured the Cdk1 activity, both of which reflect the status of this checkpoint. Twenty-four h after paclitaxel treatment, at least 80% of the control cells were arrested at mitosis and showed a dramatic increase in Cdk1 activity, thus verifying activation of the spindle assembly checkpoints (Fig. 1, C and D) ⇓ . In contrast, the accumulation of mitotic indices and activation of Cdk1 were suppressed in Mad2- and BubR1-suppressed cells at 12 and 24 h (P < 0.05; Fig. 1, C and D ⇓ ). However, at 36 h, there is a reduction of Cdk1 activity in control cells attributable to induction of cell death by paclitaxel. Interestingly, concurrent suppression of Mad2 and BubR1 showed loss of accumulation of mitotic indices and activation of Cdk1, results similar to those obtained with suppression of either Mad2 or BubR1 alone (Fig. 1, C and D) ⇓ . These results indicate that Mad2 or BubR1 alone was sufficient to abolish the function of the spindle assembly checkpoint. This abolishment was reflected by suppression of Cdk1 activity.

Loss of Spindle Assembly Checkpoint and Paclitaxel Resistance.

To determine the effect of loss of the spindle assembly checkpoint attributable to suppression of Mad2, BubR1, or both on paclitaxel sensitivity, we compared the cell viability of paclitaxel using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. As shown in Fig. 2A ⇓ , MCF-7 cells in which Mad2, BubR1, or both were suppressed were more resistant to paclitaxel than were control cells. Next, to determine whether these resistances were attributable to the reduction of cell death induced by paclitaxel, we assessed the population of cell death using trypan blue exclusion. Forty-eight h after treatment with paclitaxel, levels of cell death were high in control cells but reduced in the cells in which Mad2, BubR1, or both were suppressed (P < 0.05; Fig. 2B ⇓ ). These data demonstrate that loss of the spindle assembly checkpoint increases paclitaxel resistance.

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Fig. 2.

Induction of paclitaxel resistance via loss of spindle assembly checkpoint. A, increased paclitaxel resistance by suppression of BubR1, MAD2, or both. MCF-7 cells transfected with small-interfering RNA (siRNA)/Mad2, BubR1, or both were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to determine the effects of paclitaxel on cell growth. Twelve h after transfection, the cells were detached by trypsinization. Twelve h after seeding, cells were treated with paclitaxel at various concentrations. Bars, SDs. B, decreased paclitaxel-induced cell death by suppression of BubR1, MAD2, or both. MCF-7 cells transfected with siRNA/Mad2, BubR1, or both were examined for cell death induced by paclitaxel. Twenty-four h after transfection, cells were treated with paclitaxel (100 nm) for 48 h. Cell viability was assessed using trypan blue exclusion assay. Bars, SDs.

Effect of Mad2 Overexpression on Cdk1 Activity and Paclitaxel Sensitivity in Mad2-Dependent, Checkpoint-Defective Cells.

Although Mad2 mutations have not been detected in cancer cell lines with checkpoint defects (24) , the expression level of Mad2 protein appears to correlate with the competence of the spindle assembly checkpoint (25 , 26) . Therefore, we sought to determine whether overexpression of Mad2 restores spindle assembly checkpoint activation in cells in which low Mad2 expression renders the spindle assembly checkpoint nonfunctional. To express Mad2 effectively, we generated the recombinant adenovirus that expresses Mad2 (Ad-EGFP/Mad2). This adenovirus contains two independent cytomegalovirus-driven transcription units (one for GFP and one for Mad2), allowing direct observation of the efficiency of infection. Ad-EGFP/Mad2 induced high expression of exogenous Mad2 (Fig. 3A) ⇓ and did not affect the distribution of cells (MCF-7, T47D, Ovca432) among the various phases of the cell cycle statistically (χ test, P > 0.05 in all three cell lines; Fig. 3B ⇓ ).

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Fig. 3.

Restoration of function of spindle assembly checkpoint and enhancement of paclitaxel sensitivity by overexpression of Mad2 in Mad2-dependent checkpoint-defective cells. A, exogenous Mad2 expression in MCF-7, MCF-10A, T47D, and Ovca432 cells by infection with Ad-EGFP/Mad2 (AdMad2). Twenty-four h after infection at multiplicity of infection (MOI) values of 25 and 50, 20 μg of each lysate sample were applied and subjected to protein immunoblot analysis with anti-Mad2 antibody. B, no difference in cell cycle distribution by overexpression of Mad2. Cell cycle distribution of cells with overexpression of Mad2 as measured by DNA content by fluorescence-activated cell sorting. MCF-7, T47D, and Ovca432 cells were harvested 48 h after infection and subsequently stained with propidium iodide to detect DNA content. Numbers show the percentages of cells in each phase. Nonstatistical significance was determined in each cell line by χ² test (MCF-7, P = 0.1011; T47D, P = 0.0789; Ovca432, P = 0.9581). Top of C, schedule for combined small interfering RNA (siRNA)/Mad2 transfection and adenovirus infection. Twenty-four h after transfection of siRNA/Mad2, adenoviral vectors (MOI of 50) were delivered over a 24-h period, and cells were exposed to paclitaxel (100 nm). Bottom of C, expression of Mad2 in Mad2-suppressed MCF-7 cells infected with Ad-EGFP/Mad2 or Ad-EGFP/Luc (AdLuc). Cells were harvested at 0, 24, and 48 h after paclitaxel treatment and subjected to protein immunoblot analysis with each antibody and α-tubulin antibody to control for gel loading. D, increased cyclin-dependent kinase-1 (Cdk1) activity after treatment with paclitaxel in Mad2-suppressed MCF-7 cells infected with Ad-EGFP/Mad2 compared with Ad-EGFP/Luc-infected cells. Twenty-four h after infection with either Ad-EGFP/Mad2 or Ad-EGFP/Luc at a MOI of 50, cells were treated with paclitaxel (100 nm) and harvested at the indicated times. Cdk1 activity in the lysate was determined as described in “Materials and Methods.” E, recovered paclitaxel-induced cell death in Mad2-suppressed MCF-7 cells infected with Ad-EGFP/Mad2. Twenty-four h after infection, cells were harvested. Forty-eight h after treatment with paclitaxel, cell viability was assessed using trypan blue exclusion assay. Bars, SDs. F, increased Cdk1 activity after treatment with paclitaxel in Ad-EGFP/Mad2-infected T47D and Ovca432 cells. Twenty-four h after infection with either Ad-EGFP/Mad2 or Ad-EGFP/Luc at a MOI of 50, cells were treated with paclitaxel (100 nm) and harvested at the indicated times. Cdk1 activity in the lysate was determined as described in “Materials and Methods.” G, increased paclitaxel-induced cell death in T47D and Ovca432 cells infected with Ad-EGFP/Mad2. Cells were harvested 24 h after infection. Forty-eight h after treatment with paclitaxel, cell viability was assessed using trypan blue exclusion assay. Bars, SDs.

First, we used Mad2-knockdown MCF-7 cells, which were shown to have a nonfunctional checkpoint (Fig. 1, C and D) ⇓ . The expression of Mad2 was restored in Mad2-knockdown cells by infection of Ad-EGFP/Mad2 (Fig. 3C) ⇓ . Then, to determine whether the function of the checkpoint can be restored by re-expression of Mad2, we assessed the activation of Cdk1 in Ad-EGFP/Mad2-infected Mad2-knockdown cells after paclitaxel treatment. The Cdk1 activity did not increase in the cells that had been infected with recombinant adenovirus, which expressed luciferase (Ad-EGFP/Luc) even after exposure to paclitaxel; these findings are consistent with the data presented in Fig. 1 ⇓ . In contrast, Cdk1 activity was restored in the Ad-EGFP/Mad2-infected cells, indicating that overexpression of Mad2 can restore the function of the spindle assembly checkpoint (P < 0.05; Fig. 3D ⇓ ). We then sought to determine whether this restoration enhanced the level of cell death induced by paclitaxel and found that paclitaxel-induced cell death (10, 100 nm) was, indeed, significantly higher in the Ad-EGFP/Mad2-infected cells than in the Ad-EGFP/Luc-infected cells (P < 0.05; Fig. 3E ⇓ ).

We also used T47D and Ovca432 cells, which are known to show the defective checkpoint because of low expression of Mad2 (12) . Infection of these cell lines with Ad-EGFP/Mad2 induced high expression levels of exogenous Mad2 and did not affect the cell cycle distribution (Fig. 3, A and B) ⇓ . The Cdk1 activity and paclitaxel sensitivity were higher in the Ad-EGFP/Mad2-infected cells than in the Ad-Luc-infected cells (P < 0.05; Fig. 3, F and G ⇓ ). These data demonstrate that exogenous Mad2 expression can restore the function of the checkpoint and enhance cell death in Mad2-dependent checkpoint-defective cells.

Effect of Mad2 Overexpression on Checkpoint Function and Paclitaxel Sensitivity in BubR1-Suppressed Cells.

We then sought to determine whether overexpression of Mad2 could overcome spindle assembly checkpoint defects attributable to molecules other than Mad2. We used BubR1-knockdown MCF-7 cells, which were shown to be defective at the checkpoint (Fig. 1, C and D) ⇓ . Ad-EGFP/Mad2 induced high expression of exogenous Mad2 and did not affect the expression of BubR1 in BubR1-knockdown cells (Fig. 4A) ⇓ . However, Cdk1 was not up-regulated in Ad-EGFP/Mad2-infected cells, and paclitaxel-induced cell death was not enhanced (Fig. 4, B and C) ⇓ . These data indicate that overexpression of Mad2 did not overcome the function of the checkpoint and paclitaxel sensitivity in cells with a Mad2-independent defective checkpoint.

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Fig. 4.

Inability of Mad2 overexpression to enhance checkpoint function and paclitaxel sensitivity in cells with Mad2-independent defective or functional checkpoint. Top of A, schedule for combined small-interfering RNA (siRNA)/BubR1 transfection and adenovirus infection. Twenty-four h after transfection of siRNA/BubR1, adenoviral vectors (multiplicity of infection of 50) were delivered over a 24-h period, and cells were exposed to paclitaxel (100 nm). Bottom of A, expression of Mad2 and BubR1 in BubR1-suppressed MCF-7 cells infected with Ad-EGFP/Mad2 or Ad-EGFP/Luc. Cells were harvested at the indicated times after paclitaxel treatment and subjected to protein immunoblot analysis with each antibody and α-tubulin antibody to control for gel loading. B, cyclin-dependent kinase-1 (Cdk1) activity after treatment with paclitaxel in BubR1-suppressed MCF-7 cells infected with Ad-EGFP/Mad2 or Ad-EGFP/Luc. Cells were harvested at the indicated times after paclitaxel treatment. Activity of Cdk1 in the lysate was determined as described in “Materials and Methods.” C, paclitaxel-induced cell death in BubR1-suppressed MCF-7 cells infected with Ad-EGFP/Mad2 or Ad-EGFP/Luc. Cells were harvested 48 h after treatment with paclitaxel. Cell viability was assessed by trypan blue exclusion. Bars, SDs. D, Cdk1 activity after treatment of paclitaxel in infected MCF-7 and MCF-10A cells. Twenty-four h after infection with Ad-EGFP/Mad2 or Ad-EGFP/Luc at a multiplicity of infection of 50, cells were treated with paclitaxel (100 nm) and harvested at the indicated times. Cdk1 activity in the lysate was determined as described in “Materials and Methods.” E, paclitaxel-induced cell death in MCF-7 and MCF-10A cells infected with Ad-EGFP/Mad2 or Ad-EGFP/Luc. Cells were harvested 24 h after infection. Forty-eight h after treatment with paclitaxel, cell viability was assessed using trypan blue exclusion assay. Bars, SDs.

Effect of Overexpression of Mad2 on Checkpoint Function and Paclitaxel Sensitivity in Cells with a Functional Checkpoint.

Finally, we sought to determine whether overexpression of Mad2 enhances the function of the spindle assembly checkpoint, a finding that could translate into enhanced paclitaxel-induced cell death in checkpoint-intact cells. In MCF-10A cells and MCF-7 cells, both of which are known to have a functional checkpoint, Ad-EGFP/Mad2 effectively induced high expression levels of Mad2 (Fig. 3A) ⇓ . However, Cdk1 activity was not increased in Ad-EGFP/Mad2-infected cells of either cell line, and there was therefore no enhancement of paclitaxel-induced cell death (Fig. 4, D and E) ⇓ . Thus, overexpression of Mad2 failed to enhance the checkpoint functionality and paclitaxel sensitivity in cells with a basically functional spindle assembly checkpoint.