Aberrant BUB1 Overexpression Promotes Mitotic Segregation Errors and Chromosomal Instability in Multiple Myeloma

Abstract

Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.

Keywords: BUB1; chromosomal instability; chromosome segregation error; clonogenicity; multiple myeloma.

Figure 1

BUB1 is highly expressed in advanced multiple myeloma cells. (a) BUB1 transcriptional level in human myeloma-derived cell lines (HMCLs). Normal plasma cells (PCs) were obtained from the bone marrow of 3 healthy donors (PC1-3) and used as the control. Data in the graph are means ± SD of four independent experiments. (b) BUB1 transcriptional level in normal PCs, CD138-positive myeloma cells from 39 multiple myeloma (MM) and 19 monoclonal gammopathy of undetermined significance (MGUS) cases, and 10 HMCLs. * p < 0.05, ** p < 0.01, N.S.: not significant, by one-way ANOVA with a Tukey multiple comparison test. The mRNA expression levels of BUB1 were normalized to those of β-actin in each sample, and their relative values were calculated considering the mean BUB1 transcription levels in normal PCs to be 1.0. (c) Immunoblot of BUB1 in HMCLs and normal PCs. The expression level of BUB1 relative to that of β-actin in each HMCL was measured by the densitometric analysis.

Figure 2

Generation of BUB1-reduced HMCLs. (a,b) KMS-18, AMO-1, and RPMI-8226 cells were transduced with mock, shBUB1#1, or shBUB1#2. BUB1 mRNA expression (Y-axis: % of control) was determined by qRT-PCR (a) and Western blotting (b) after puromycin selection. The expression level of BUB1 relative to that of β-actin in each HMCL was measured by densitometric analysis; the BUB1 to the β-actin ratio in controls was defined as 1.0. The protein level relative to the control is shown below each band.

Figure 3

BUB1 expression did not affect the short-term proliferation of HMCLs. (a) Association between cell proliferation rates and BUB1 mRNA levels in HMCLs. Fold changes in cell numbers over three days of culture are plotted on the horizontal axis, and BUB1 mRNA levels relative to those in normal plasma cells (PCs) are on the vertical axis. Ten HMCLs were examined. (b) Cells were seeded at 1 × 10⁵ cells/mL and grown in the complete liquid culture medium. Cell number was measured by Trypan blue dye exclusion assay over time. Dotted lines show growth of control (yellow circle) and mock-treated (red square) cells, and solid lines show BUB1-reduced cells using shBUB1#1 (green diamonds) and shBUB1#2 (blue triangles). Assays were performed in triplicate, and cell counts are expressed as means ± SD. N.S.: not significant. (c) BUB1 knockdown had no effect on cell cycle distribution in HMCLs. Data shown are the representative results with KMS-18 cells. The percentages of cell populations in the G1, S, and G2/M phases of the cell cycle are shown. Data are means ± SD from three independent experiments.

Figure 3

BUB1 expression did not affect the short-term proliferation of HMCLs. (a) Association between cell proliferation rates and BUB1 mRNA levels in HMCLs. Fold changes in cell numbers over three days of culture are plotted on the horizontal axis, and BUB1 mRNA levels relative to those in normal plasma cells (PCs) are on the vertical axis. Ten HMCLs were examined. (b) Cells were seeded at 1 × 10⁵ cells/mL and grown in the complete liquid culture medium. Cell number was measured by Trypan blue dye exclusion assay over time. Dotted lines show growth of control (yellow circle) and mock-treated (red square) cells, and solid lines show BUB1-reduced cells using shBUB1#1 (green diamonds) and shBUB1#2 (blue triangles). Assays were performed in triplicate, and cell counts are expressed as means ± SD. N.S.: not significant. (c) BUB1 knockdown had no effect on cell cycle distribution in HMCLs. Data shown are the representative results with KMS-18 cells. The percentages of cell populations in the G1, S, and G2/M phases of the cell cycle are shown. Data are means ± SD from three independent experiments.

Figure 4

Involvement of BUB1 in chromosome segregation errors in myeloma cells. (a) Two types of anaphase chromosome segregation errors were defined: attachment defects, such as a lagging chromosome with a centromere (left), and structural defects (right), such as a chromosome break (right upper) and a chromosomal bridge (right lower). Cells were stained for centromeres with anti- centromere protein (CENP)-C antibody (Ab) with a secondary Ab conjugated with Alexa Fluor 546 (red), and for DNA using DAPI (blue). (× 300) (b) Percentage of anaphase cells with chromosome segregation errors in parental, mock-treated, and BUB1-reduced (transfected with shBUB1#1 or shBUB1#2) KMS-18 and AMO-1 cells. A total of 100 anaphase cells were counted in each experiment. Data are means ± SD from three independent experiments. * p < 0.05, ** p < 0.01 vs. control cells in a two-tailed t-test. Gray, blue, and orange bars show the ratios of cells with attachment errors, structural errors, and concomitant attachment and structural errors, respectively.

Figure 5

Involvement of BUB1 in the emergence of aneuploidy in myeloma cells. (a) Percentage deviations from the modal chromosome number for chromosome (chr) 3 and chr 7 in 10 clones of KMS-18 and AMO-1 cells. A total of 100 interphase cells were evaluated for the numbers of the chr 3 and chr 7 in each clone. Gray and white bars represent parental and BUB1-knockdown cells, respectively. Means ± SD of ten independent clones are shown. * p < 0.05, ** p < 0.01 by two-tailed t-test. (b) Distribution of whole chromosome numbers of 50 metaphase cells of control (gray), mock-transduced (blue), and BUB1-reduced (red) KMS-18 (upper) and AMO-1 (lower) cells. Numbers are the average chromosome number, SD, and p values vs. control cells in an F test.

Figure 6

BUB1 knockdown impaired colony-forming capacity in KMS-18 and RPMI-8226 cells. (a,b) Control, mock-treated, or BUB1-reduced KMS-18 (left) and RPMI-8226 (right) cells were subjected to a colony-forming assay. Triplicate assays were performed in two independent experiments. Data are shown as means ± SD. * p < 0.05, ** p < 0.01 vs. control cells in a two-tailed t-test. N.S.: not significant.