The interaction between mitotic checkpoint proteins, CENP-E and BubR1, is diminished in epothilone B-resistant A549...

Centromere associated protein-E (CENP-E), a mitotic checkpoint protein, is required for efficient, stable microtubule capture at kinetochores during mitosis. Absence of CENP-E results in misaligned chromosomes leading to metaphase arrest. Microtubule-interacting agents such as Taxol and epothilone B (EpoB), at concentrations that induce mitotic arrest, transiently increase expression of CENP-E in a variety of cancer cell lines. The CENP-E level in an EpoB-resistant A549 cell line, EpoB40, is ~ 2-fold higher than in A549 cells. CENP-E overexpression, after transfection with CENP-E cDNA into drug sensitive cells, does not alter Taxol or EpoB sensitivity. However, suppression of CENP-E expression by CENP-E siRNA results in a moderate increase in drug sensitivity, suggesting that a minimal quantity of CENP-E is required for maintaining its function. It is known that CENP-E binds to BubR1 and enhances its recruitment to each unattached kinetochore. Suppression of CENP-E results in a decrease in BubR1 levels in EpoB40 cells. During metaphase, both targeting of CENP-E and BubR1 to the kinetochores and the interaction between CENP-E and BubR1 are significantly reduced in EpoB40 cells, compared to A549 cells. In addition, the distance between the two centrosomes during metaphase is shorter in EpoB40 than in A549 cells, suggesting that defects in the spindle-assembly checkpoint have occurred in EpoB40 cells during the development of drug resistance. These results indicate that defects in the mitotic checkpoint may have a role in, or be the result of, the development of EpoB resistance.