Article Figures & Data
Figures
- Figure 1.
Effect of BMS-777607 on growth and survival of breast cancer cells. A, the effect of BMS-777607 on survival and proliferation of MCF-7, ZR-75-1, and T-47D cells was determined by clonogenic assay. Briefly, cells (8,000 cells per well) in RPMI-1640 with 5% FBS were cultured in duplicate in a 24-well plate and then treated with different amounts of BMS-777607 for 10 days. Clonogenic cells were stained with Hema-3 staining solution (Fisher Scientific) and photographed using an Olympus BK71 microscope equipped with CCD camera. B, numbers of clonogenic cells in duplicate from 3 cell lines were counted. C, apoptotic cell death was measured by the Annexin V/propidium iodide labeling method (28). Cells were treated with 5 μmol/L BMS-777607 for 3 days, labeled, and then analyzed by flow cytometer as previously described (28). Results shown here are from 1 of 2 experiments with similar results.
- Figure 2.
Induction by BMS-777607 of polyploidy in breast cancer cells. A, BMS-777607 induces polyploidy in breast cancer cells in a dose-dependent manner. Cells (5,000 cells per well) were incubated at 37°C in RPMI-1640 with 5% FBS and treated with different amounts of BMS-777607 for 72 hours. Polyploid cells were photographed as described in Fig. 1A. B, time-dependent induction of polyploidy by BMS-777607. Cells were treated with 5 μmol/L BMS-777607 for various time intervals. Stained cells were photographed. C, knockdown of RON expression in breast cancer cells by specific siRNA. Cells (2 × 106 cells in a 60-mm diameter culture dish) were transiently transfected with 1 nmol/L scramble or RON-specific siRNA for 48 hours. Cell lysates (50 μg per sample) were subjected to Western blot analysis using rabbit anti-RON IgG antibody. D, effect of RON expression on BMS-777607–induced polyploidy. T-47D cells were transiently transfected with scramble or RON-specific siRNA for 48 hours as described in C and then treated with 5 μmol/L BMS-777607 for 72 hours. Polyploidy was monitored and photographed. E, fate of polyploid cells in culture. Polyploid T-47D cells were isolated, washed, and then cultured in BMS-777607–free Dulbecco's Modified Eagle Media with 10% FBS for 10 days. Cells were observed and photographed at various times. Results shown here are from 1 of 2 experiments with similar results.
- Figure 3.
Effect of BMS-777607 on breast cancer cell cycle and chromosome contents. A, analysis of DNA/chromosome contents was conducted as previously described (25). Cells (2 × 106 cells per sample) in RPMI-1640 with 5% FBS were treated with 5 μmol/L BMS-777607. After incubation for 24, 48, and 72 hours, cells were collected, fixed, and then stained with propidium iodide. DNA/chromosome contents in individual samples were analyzed by FACScan flow cytometer. B, chromosome spreading was conducted by treatment of T-47D cells with 5 μmol/L BMS-777607 for 72 hours. After treatment, cells were collected for chromosome spreading analysis according to a previously described method (25). The number of chromosomes was counted and photographed.
- Figure 4.
Disruptive effect of BMS-777607 on mitotic spindle assembly in breast cancer cells. A, effect of BMS-777607 on α-tubulin and γ-tubulin expression and localization. T-47D cells (1 × 106 cells per dish) in RPMI-1640 with 5% FBS were treated with or without 5 μmol/L BMS-777607 for 24, 48, and 72 hours. Cells were then fixed with cold acetone. Specific mouse IgG antibodies were used to detect α-tubulin or γ-tubulin, respectively, followed by FITC-coupled anti-mouse IgG. Cells also were stained for 4′,6-diamidino-2-phenylindole (DAPI) to detect DNAs. Analysis of immunofluorescence was conducted using an Olympus BK71 inverted microscope equipped with DSU/fluorescence apparatus and CCD camera. Image was taken at magnification of ×200. B and C, T-47D cells were treated 37°C with 5 μmol/L of R0-3306 (B) or BMS-777607 (C) for 24 hours, extensively washed, and then cultured in Dulbecco's Modified Eagle Media with 10% FBS for additional 30, 60, and 120 minutes. Expression of α-tubulin and γ-tubulin was monitored by immunofluorescent analysis as described above. Image was made at magnification of ×400. Results shown here are from 1 of 2 experiments with similar results.
- Figure 5.
Regulatory effect of BMS-777607 on aurora kinase B expression and histone H3 phosphorylation in breast cancer T-47D cells. A and B, AURK-B expression and histone H3 Ser10 phosphorylation in polyploid cells were analyzed by immunofluorescence using specific antibodies. T-47D cells (5,000 cells per culture chamber) were treated at 37°C with 5 μmol/L BMS-777607. After treatment for 24, 48, and 72 hours, cells were fixed with cold acetone and used to detect AURK-B expression and histone H3 Ser10 phosphorylation using specific antibodies to AURK-B and phospho-Ser10 of histone H3, respectively. Cells stained with 4′,6-diamidino-2-phenylindole (DAPI) for DNAs were used as the control. Immunofluorescence was observed using the Olympus BK71 microscope equipped with DSU/fluorescence apparatus. C, Western blot analysis of AURK-B and histone H3 was carried out by treatment of cells with 5 μmol/L BMS-777607 followed by use of specific antibodies to detect AURK-B, histone H3 phospho-Ser10, and histone H3, respectively. The membrane was also reprobed with anti-β-actin antibody as the loading control. D, preventive effect of lactacystin on BMS-777607–induced reduction of AURK-B. T-47D and ZR-75-1 cells were treated for 72 hours with 5 μmol/L BMS-777607 in the presence or absence of 5 μmol/L of lactacystin. Expression of AURK-B was measured by Western blot analysis using AURK-B- specific mouse IgG antibody. Actin was used as the loading control. Results shown here are from 1 of 2 experiments with similar results.
- Figure 6.
Cytotoxic effects of different chemotherapy agents on BMS-777607–induced breast cancer polyploid cells. T-47D and ZR-75-1 cells (3 × 106 cells in 100-mm diameter culture dishes) were treated with 5 μmol/L BMS-777607 for 72 hours. Polyploid cells were collected, washed, and cultured at 5,000 cells per well in a 96-well culture plate. Regular T-47D and ZR-75-1 cells (5,000 cells/well) without BMS-777607 treatment were used as the control. Cells were treated for 72 hours with different amounts of doxorubicin, bleomycin, cisplatin, paclitaxel, or methotrexate in triplicate followed by the MTS assay to determine cell viability (27). The IC50 values from individual drugs were calculated as previously described (27). The calculated IC50 values of bleomycin for control T-47D and ZR-75-1 cells were 5,783 and 6,134 nmol/L, respectively. Results shown here are from 1 of 3 experiments with similar results.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file - 71K
- Supplementary Figure 1 - PDF file - 63K, Figure S1: Expression of RON and MET by breast cancer MCF-7, T-47D and ZR-75-1 cells.
- Supplementary Figure 2 - PDF file - 68K, Figure S2: Effect of BMS-777607 on breast cancer cell growth and apoptotic death.
- Supplementary Figure 3 - PDF file - 59K, Figure S3: Effect of PHA665752, Compound-I, and BMS-777607 on induction of polyploidy in breast cancer cells.
- Supplementary Table 1 - PDF file - 66K, Supplementary Table 1 Inductive Effect of BMS-777607 on Breast Cancer Polyploid Cells with Different Numbers of Chromosomes.
- Supplementary Table 2 - PDF file - 57K, Supplementary Table 2 Increased resistance of BMS-777607-induced breast cancer polyploid cells in response to cytotoxic chemotherapy agents.
Volume 12, Issue 5
