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Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.

Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS - J. Cell Biol. (2003)

Bottom Line: Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension.Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment.Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK.

ABSTRACT
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

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p53 restrains endoreduplication in the presence of ZM447439. (A) DNA content histograms of U20S cells with (p53+) or without (p53−) a functional p53 response treated with ZM447439 for the times indicated in hours. (B) Growth-arrested or proliferating MCF-7 cells were exposed to ZM447439 for 72 h, and were then assayed for the ability to form colonies in the absence of ZM447439. (C) Bar graph quantitating the cloning efficiency of MCF-7 cells treated with a range of ZM447439 concentrations. Each value represents the mean and SD from three independent experiments.
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fig2: p53 restrains endoreduplication in the presence of ZM447439. (A) DNA content histograms of U20S cells with (p53+) or without (p53−) a functional p53 response treated with ZM447439 for the times indicated in hours. (B) Growth-arrested or proliferating MCF-7 cells were exposed to ZM447439 for 72 h, and were then assayed for the ability to form colonies in the absence of ZM447439. (C) Bar graph quantitating the cloning efficiency of MCF-7 cells treated with a range of ZM447439 concentrations. Each value represents the mean and SD from three independent experiments.

Mentions: Although a significant fraction of A549 and HME cells endoreduplicated in the presence of ZM447439, after 48 h virtually all the cells arrested with either 4N or 8N DNA contents (Fig. 1 C). In contrast, HeLa cells, which lack a functional p53 response, continued DNA synthesis and rapidly lost viability. This raises the possibility that the 4N/8N arrest exhibited by the A549 and HME cells was not directly due to ZM447439, but was rather due to activation of the p53-dependent post-mitotic checkpoint that occurs after an aberrant mitosis and/or cytokinesis (Andreassen et al., 2001). Consistently, in the presence of ZM447439, U2OS cells expressing a dominant-negative p53 mutant endoreduplicated efficiently such that by 48 h, 78% had 8N DNA contents (Fig. 2 A). In contrast, only 38% of the p53-proficient parental U2OS cells had 8N DNA contents, demonstrating that p53 does restrain cell cycle progression after ZM447439-induced division failure. To test if cell cycle progression in the presence of ZM447439 may account for why the HeLa cells lost viability, we analyzed the colony-forming potential of cells after a 72-h exposure to ZM447439. MCF7 cells were selected for this experiment, as they can be arrested in G0 by treatment with anti-estrogens. Cells that were growth-arrested during exposure to ZM447439 gave rise to more colonies than those that were proliferating (Fig. 2 B). Consistently, at 1.25 and 2.5 μM ZM447439, the cloning efficiency of the proliferating cells was reduced to below 40%, whereas the cloning efficiency of the growth-arrested cells was largely unaffected (Fig. 2 C). Although continued cell cycle progression in the presence of ZM447439 leads to loss of viability, the p53-deficient U2OS cells did not appear to lose viability as rapidly as the HeLa cells. The reason for this is unclear, but it suggests that p53-independent mechanisms may also affect cell fate after ZM447439-induced tetraploidization.


Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.

Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS - J. Cell Biol. (2003)

p53 restrains endoreduplication in the presence of ZM447439. (A) DNA content histograms of U20S cells with (p53+) or without (p53−) a functional p53 response treated with ZM447439 for the times indicated in hours. (B) Growth-arrested or proliferating MCF-7 cells were exposed to ZM447439 for 72 h, and were then assayed for the ability to form colonies in the absence of ZM447439. (C) Bar graph quantitating the cloning efficiency of MCF-7 cells treated with a range of ZM447439 concentrations. Each value represents the mean and SD from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172902&req=5

fig2: p53 restrains endoreduplication in the presence of ZM447439. (A) DNA content histograms of U20S cells with (p53+) or without (p53−) a functional p53 response treated with ZM447439 for the times indicated in hours. (B) Growth-arrested or proliferating MCF-7 cells were exposed to ZM447439 for 72 h, and were then assayed for the ability to form colonies in the absence of ZM447439. (C) Bar graph quantitating the cloning efficiency of MCF-7 cells treated with a range of ZM447439 concentrations. Each value represents the mean and SD from three independent experiments.
Mentions: Although a significant fraction of A549 and HME cells endoreduplicated in the presence of ZM447439, after 48 h virtually all the cells arrested with either 4N or 8N DNA contents (Fig. 1 C). In contrast, HeLa cells, which lack a functional p53 response, continued DNA synthesis and rapidly lost viability. This raises the possibility that the 4N/8N arrest exhibited by the A549 and HME cells was not directly due to ZM447439, but was rather due to activation of the p53-dependent post-mitotic checkpoint that occurs after an aberrant mitosis and/or cytokinesis (Andreassen et al., 2001). Consistently, in the presence of ZM447439, U2OS cells expressing a dominant-negative p53 mutant endoreduplicated efficiently such that by 48 h, 78% had 8N DNA contents (Fig. 2 A). In contrast, only 38% of the p53-proficient parental U2OS cells had 8N DNA contents, demonstrating that p53 does restrain cell cycle progression after ZM447439-induced division failure. To test if cell cycle progression in the presence of ZM447439 may account for why the HeLa cells lost viability, we analyzed the colony-forming potential of cells after a 72-h exposure to ZM447439. MCF7 cells were selected for this experiment, as they can be arrested in G0 by treatment with anti-estrogens. Cells that were growth-arrested during exposure to ZM447439 gave rise to more colonies than those that were proliferating (Fig. 2 B). Consistently, at 1.25 and 2.5 μM ZM447439, the cloning efficiency of the proliferating cells was reduced to below 40%, whereas the cloning efficiency of the growth-arrested cells was largely unaffected (Fig. 2 C). Although continued cell cycle progression in the presence of ZM447439 leads to loss of viability, the p53-deficient U2OS cells did not appear to lose viability as rapidly as the HeLa cells. The reason for this is unclear, but it suggests that p53-independent mechanisms may also affect cell fate after ZM447439-induced tetraploidization.

Bottom Line: Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension.Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment.Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK.

ABSTRACT
The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

Show MeSH
Related in: MedlinePlus