Limits...
The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.

Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM - J. Cell Biol. (2003)

Bottom Line: We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation.Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B.Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

ABSTRACT
The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Show MeSH
Hesperadin causes polyploidy in HeLa cells. (A) Chemical structure of Hesperadin. (B) Hesperadin (50 nM) was added to logarithmically growing HeLa cells (day 0). At the indicated time points, the DNA content was determined by flow cytometry, and phase contrast micrographs were taken. From day 4 on, the DNA content could not be measured by flow cytometry (n.a., not applicable). (C) HeLa cells were synchronized by double thymidine treatment and released either into 100 nM Hesperadin (right) or a corresponding concentration (0.01%) of the solvent DMSO (left). Cells were harvested at the indicated time points after release, and the DNA content was analyzed by flow cytometry. 330 nM nocodazole was added to one sample of each series, and these cells were harvested at 14.5 h after release from thymidine. log, logarithmically growing untreated HeLa cells. (D) Samples from the same experiment as in C were analyzed by SDS-PAGE and immunoblotting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172906&req=5

fig1: Hesperadin causes polyploidy in HeLa cells. (A) Chemical structure of Hesperadin. (B) Hesperadin (50 nM) was added to logarithmically growing HeLa cells (day 0). At the indicated time points, the DNA content was determined by flow cytometry, and phase contrast micrographs were taken. From day 4 on, the DNA content could not be measured by flow cytometry (n.a., not applicable). (C) HeLa cells were synchronized by double thymidine treatment and released either into 100 nM Hesperadin (right) or a corresponding concentration (0.01%) of the solvent DMSO (left). Cells were harvested at the indicated time points after release, and the DNA content was analyzed by flow cytometry. 330 nM nocodazole was added to one sample of each series, and these cells were harvested at 14.5 h after release from thymidine. log, logarithmically growing untreated HeLa cells. (D) Samples from the same experiment as in C were analyzed by SDS-PAGE and immunoblotting.

Mentions: In the course of a synthesis program for novel indolinones, we tested the effect of several compounds on cell proliferation. One of these, Hesperadin (Fig. 1 A; Walter et al., 2002), had dramatic effects; HeLa cells treated with 50 nM of Hesperadin stopped proliferating but did not stop growing, and over a 6-d period, the cell diameter increased more than sevenfold (from ∼20 to >150 μm; Fig. 1 B). During this time, the cells acquired enlarged lobed nuclei (see Fig. 4 i). FACS® analysis revealed that the increase in nuclear size correlated with polyploidization, reaching a 32C DNA content on day 3 (Fig. 1 B; unpublished data). At later stages, FACS® could not be used to analyze these cells, presumably because they had grown too big to enter the measuring capillary.


The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint.

Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM - J. Cell Biol. (2003)

Hesperadin causes polyploidy in HeLa cells. (A) Chemical structure of Hesperadin. (B) Hesperadin (50 nM) was added to logarithmically growing HeLa cells (day 0). At the indicated time points, the DNA content was determined by flow cytometry, and phase contrast micrographs were taken. From day 4 on, the DNA content could not be measured by flow cytometry (n.a., not applicable). (C) HeLa cells were synchronized by double thymidine treatment and released either into 100 nM Hesperadin (right) or a corresponding concentration (0.01%) of the solvent DMSO (left). Cells were harvested at the indicated time points after release, and the DNA content was analyzed by flow cytometry. 330 nM nocodazole was added to one sample of each series, and these cells were harvested at 14.5 h after release from thymidine. log, logarithmically growing untreated HeLa cells. (D) Samples from the same experiment as in C were analyzed by SDS-PAGE and immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172906&req=5

fig1: Hesperadin causes polyploidy in HeLa cells. (A) Chemical structure of Hesperadin. (B) Hesperadin (50 nM) was added to logarithmically growing HeLa cells (day 0). At the indicated time points, the DNA content was determined by flow cytometry, and phase contrast micrographs were taken. From day 4 on, the DNA content could not be measured by flow cytometry (n.a., not applicable). (C) HeLa cells were synchronized by double thymidine treatment and released either into 100 nM Hesperadin (right) or a corresponding concentration (0.01%) of the solvent DMSO (left). Cells were harvested at the indicated time points after release, and the DNA content was analyzed by flow cytometry. 330 nM nocodazole was added to one sample of each series, and these cells were harvested at 14.5 h after release from thymidine. log, logarithmically growing untreated HeLa cells. (D) Samples from the same experiment as in C were analyzed by SDS-PAGE and immunoblotting.
Mentions: In the course of a synthesis program for novel indolinones, we tested the effect of several compounds on cell proliferation. One of these, Hesperadin (Fig. 1 A; Walter et al., 2002), had dramatic effects; HeLa cells treated with 50 nM of Hesperadin stopped proliferating but did not stop growing, and over a 6-d period, the cell diameter increased more than sevenfold (from ∼20 to >150 μm; Fig. 1 B). During this time, the cells acquired enlarged lobed nuclei (see Fig. 4 i). FACS® analysis revealed that the increase in nuclear size correlated with polyploidization, reaching a 32C DNA content on day 3 (Fig. 1 B; unpublished data). At later stages, FACS® could not be used to analyze these cells, presumably because they had grown too big to enter the measuring capillary.

Bottom Line: We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation.Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B.Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

ABSTRACT
The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. We have identified the small molecule Hesperadin as an inhibitor of chromosome alignment and segregation. Our data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, our data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling.

Show MeSH