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When the genome plays dice: circumvention of the spindle assembly checkpoint and near-random chromosome segregation in multipolar cancer cell mitoses.

Gisselsson D, Håkanson U, Stoller P, Marti D, Jin Y, Rosengren AH, Stewénius Y, Kahl F, Panagopoulos I - PLoS ONE (2008)

Bottom Line: The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle.Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells.The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Genetics, Lund University Hospital, Lund, Sweden. david.gisselsson@med.lu.se

ABSTRACT

Background: Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes. Cancer cells, however, occasionally divide into three or more directions. Such multipolar mitoses have been proposed to generate genetic diversity and thereby contribute to clonal evolution. However, this notion has been little validated experimentally.

Principal findings: Chromosome segregation and DNA content in daughter cells from multipolar mitoses were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells. The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent isomies in the daughter cells. Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition. The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle. Centromeric AURKB and MAD2 foci were observed frequently to remain on the centromeres of multipolar ana-telophase chromosomes, indicating that multipolar mitoses were able to circumvent the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase. Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells.

Conclusion: The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells. Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations.

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Related in: MedlinePlus

Sister chromatid separation and spindle assembly checkpoint proteins.FISH detection (A) of the chromosome 17 centromere (green) combined with immunofluorescence for beta tubulin (red) shows separation of the sister centromeres of one (arrow) but not of the other homologue (arrowhead). Immunofluorescence detection of separase (orange) and beta tubulin (green) shows localization of separase to spindle poles at metaphase (B) and early anaphase (C) and weak intensity staining in the midbody at telophase (D). Co-localisation of separase (red) and centrosomes (gamma tubulin in green) is observed at early bipolar anaphase (E, upper left) while several additional separase foci are present in an adjacent tetrapolar early anaphase cell (E, lower right). Co-labelling of separase (orange) and beta-tubulin (green) confirms this finding (F, tetrapolar at upper left and bipolar at lower right), and shows depletion of separase in a bipolar telophase cell (G, right) while several foci remain in a tetrapolar telophase cell (G, left). Immunofluorescence for CCNB1 (green) and AURKA (red) yields cytoplasmic staining in bipolar (H) and multipolar (J) metaphase cells while no staining is observed in bipolar (I) and multipolar (K) anaphase cells; AURKA stains the pericentrosomal regions at both metaphase and anaphase. Immunofluorescence for beta tubulin (green) and AURKB (red) shows localization of AURKB exclusively to centromeres at bipolar (L) and multipolar (M) metaphase, and exclusively to the spindle midzone at bipolar anaphase (N); in contrast, multipolar ana-telophase cells (O, P) exhibit AURKB in the midzone as well as on several chromosomes, indicating failure to separate the sister chromatids of some chromosomes (O, arrow). Immunofluorescence for beta tubulin (green) and MAD2L1 (red) shows MAD2L1 localization to centromeres at bipolar prometaphase (Q); at bipolar ana-telophase MAD2L1 foci is only present in chromosomes not incorporated in the mitotic process (R, arrow), whereas, at multipolar ana-telophase, multiple MAD2L1 foci are present on chromosomes (S).
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pone-0001871-g003: Sister chromatid separation and spindle assembly checkpoint proteins.FISH detection (A) of the chromosome 17 centromere (green) combined with immunofluorescence for beta tubulin (red) shows separation of the sister centromeres of one (arrow) but not of the other homologue (arrowhead). Immunofluorescence detection of separase (orange) and beta tubulin (green) shows localization of separase to spindle poles at metaphase (B) and early anaphase (C) and weak intensity staining in the midbody at telophase (D). Co-localisation of separase (red) and centrosomes (gamma tubulin in green) is observed at early bipolar anaphase (E, upper left) while several additional separase foci are present in an adjacent tetrapolar early anaphase cell (E, lower right). Co-labelling of separase (orange) and beta-tubulin (green) confirms this finding (F, tetrapolar at upper left and bipolar at lower right), and shows depletion of separase in a bipolar telophase cell (G, right) while several foci remain in a tetrapolar telophase cell (G, left). Immunofluorescence for CCNB1 (green) and AURKA (red) yields cytoplasmic staining in bipolar (H) and multipolar (J) metaphase cells while no staining is observed in bipolar (I) and multipolar (K) anaphase cells; AURKA stains the pericentrosomal regions at both metaphase and anaphase. Immunofluorescence for beta tubulin (green) and AURKB (red) shows localization of AURKB exclusively to centromeres at bipolar (L) and multipolar (M) metaphase, and exclusively to the spindle midzone at bipolar anaphase (N); in contrast, multipolar ana-telophase cells (O, P) exhibit AURKB in the midzone as well as on several chromosomes, indicating failure to separate the sister chromatids of some chromosomes (O, arrow). Immunofluorescence for beta tubulin (green) and MAD2L1 (red) shows MAD2L1 localization to centromeres at bipolar prometaphase (Q); at bipolar ana-telophase MAD2L1 foci is only present in chromosomes not incorporated in the mitotic process (R, arrow), whereas, at multipolar ana-telophase, multiple MAD2L1 foci are present on chromosomes (S).

Mentions: The irregular and rapid transition from metaphase to telophase-interphase in MM, and the absence of clear anaphase configurations in some cells indicated that sister-chromatid separation might not occur in a regular fashion. To monitor sister chromatid separation in further detail in MM, we used the centromeric probe system described above and selected late metaphase/early anaphase cells for analysis in which at least one pair of sister centromeres in the metaphase/early anaphase plate had separated, as evidenced by split FISH-signals in a double-dot formation. In both WiT49 and HEK293, the vast majority (97% and 98%, respectively; Table 2) of the cells with a bipolar configuration in which one centromere had separated showed separation also of the other centromere(s), indicating a well coordinated metaphase-anaphase transition in these cells. In multipolar cells in WiT49 and HEK293, selected by the same criteria, less than half of the analyzed cell divisions were similarly coordinated. In fact, 59% and 55%, respectively, of these cells exhibited separation of only some of the probed sister centromeres while the others remained unseparated (Figure 3A). Sister chromatid in MM was thus often unsynchronized, compared to normal cell divisions.


When the genome plays dice: circumvention of the spindle assembly checkpoint and near-random chromosome segregation in multipolar cancer cell mitoses.

Gisselsson D, Håkanson U, Stoller P, Marti D, Jin Y, Rosengren AH, Stewénius Y, Kahl F, Panagopoulos I - PLoS ONE (2008)

Sister chromatid separation and spindle assembly checkpoint proteins.FISH detection (A) of the chromosome 17 centromere (green) combined with immunofluorescence for beta tubulin (red) shows separation of the sister centromeres of one (arrow) but not of the other homologue (arrowhead). Immunofluorescence detection of separase (orange) and beta tubulin (green) shows localization of separase to spindle poles at metaphase (B) and early anaphase (C) and weak intensity staining in the midbody at telophase (D). Co-localisation of separase (red) and centrosomes (gamma tubulin in green) is observed at early bipolar anaphase (E, upper left) while several additional separase foci are present in an adjacent tetrapolar early anaphase cell (E, lower right). Co-labelling of separase (orange) and beta-tubulin (green) confirms this finding (F, tetrapolar at upper left and bipolar at lower right), and shows depletion of separase in a bipolar telophase cell (G, right) while several foci remain in a tetrapolar telophase cell (G, left). Immunofluorescence for CCNB1 (green) and AURKA (red) yields cytoplasmic staining in bipolar (H) and multipolar (J) metaphase cells while no staining is observed in bipolar (I) and multipolar (K) anaphase cells; AURKA stains the pericentrosomal regions at both metaphase and anaphase. Immunofluorescence for beta tubulin (green) and AURKB (red) shows localization of AURKB exclusively to centromeres at bipolar (L) and multipolar (M) metaphase, and exclusively to the spindle midzone at bipolar anaphase (N); in contrast, multipolar ana-telophase cells (O, P) exhibit AURKB in the midzone as well as on several chromosomes, indicating failure to separate the sister chromatids of some chromosomes (O, arrow). Immunofluorescence for beta tubulin (green) and MAD2L1 (red) shows MAD2L1 localization to centromeres at bipolar prometaphase (Q); at bipolar ana-telophase MAD2L1 foci is only present in chromosomes not incorporated in the mitotic process (R, arrow), whereas, at multipolar ana-telophase, multiple MAD2L1 foci are present on chromosomes (S).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2289843&req=5

pone-0001871-g003: Sister chromatid separation and spindle assembly checkpoint proteins.FISH detection (A) of the chromosome 17 centromere (green) combined with immunofluorescence for beta tubulin (red) shows separation of the sister centromeres of one (arrow) but not of the other homologue (arrowhead). Immunofluorescence detection of separase (orange) and beta tubulin (green) shows localization of separase to spindle poles at metaphase (B) and early anaphase (C) and weak intensity staining in the midbody at telophase (D). Co-localisation of separase (red) and centrosomes (gamma tubulin in green) is observed at early bipolar anaphase (E, upper left) while several additional separase foci are present in an adjacent tetrapolar early anaphase cell (E, lower right). Co-labelling of separase (orange) and beta-tubulin (green) confirms this finding (F, tetrapolar at upper left and bipolar at lower right), and shows depletion of separase in a bipolar telophase cell (G, right) while several foci remain in a tetrapolar telophase cell (G, left). Immunofluorescence for CCNB1 (green) and AURKA (red) yields cytoplasmic staining in bipolar (H) and multipolar (J) metaphase cells while no staining is observed in bipolar (I) and multipolar (K) anaphase cells; AURKA stains the pericentrosomal regions at both metaphase and anaphase. Immunofluorescence for beta tubulin (green) and AURKB (red) shows localization of AURKB exclusively to centromeres at bipolar (L) and multipolar (M) metaphase, and exclusively to the spindle midzone at bipolar anaphase (N); in contrast, multipolar ana-telophase cells (O, P) exhibit AURKB in the midzone as well as on several chromosomes, indicating failure to separate the sister chromatids of some chromosomes (O, arrow). Immunofluorescence for beta tubulin (green) and MAD2L1 (red) shows MAD2L1 localization to centromeres at bipolar prometaphase (Q); at bipolar ana-telophase MAD2L1 foci is only present in chromosomes not incorporated in the mitotic process (R, arrow), whereas, at multipolar ana-telophase, multiple MAD2L1 foci are present on chromosomes (S).
Mentions: The irregular and rapid transition from metaphase to telophase-interphase in MM, and the absence of clear anaphase configurations in some cells indicated that sister-chromatid separation might not occur in a regular fashion. To monitor sister chromatid separation in further detail in MM, we used the centromeric probe system described above and selected late metaphase/early anaphase cells for analysis in which at least one pair of sister centromeres in the metaphase/early anaphase plate had separated, as evidenced by split FISH-signals in a double-dot formation. In both WiT49 and HEK293, the vast majority (97% and 98%, respectively; Table 2) of the cells with a bipolar configuration in which one centromere had separated showed separation also of the other centromere(s), indicating a well coordinated metaphase-anaphase transition in these cells. In multipolar cells in WiT49 and HEK293, selected by the same criteria, less than half of the analyzed cell divisions were similarly coordinated. In fact, 59% and 55%, respectively, of these cells exhibited separation of only some of the probed sister centromeres while the others remained unseparated (Figure 3A). Sister chromatid in MM was thus often unsynchronized, compared to normal cell divisions.

Bottom Line: The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle.Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells.The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Genetics, Lund University Hospital, Lund, Sweden. david.gisselsson@med.lu.se

ABSTRACT

Background: Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes. Cancer cells, however, occasionally divide into three or more directions. Such multipolar mitoses have been proposed to generate genetic diversity and thereby contribute to clonal evolution. However, this notion has been little validated experimentally.

Principal findings: Chromosome segregation and DNA content in daughter cells from multipolar mitoses were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells. The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent isomies in the daughter cells. Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition. The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle. Centromeric AURKB and MAD2 foci were observed frequently to remain on the centromeres of multipolar ana-telophase chromosomes, indicating that multipolar mitoses were able to circumvent the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase. Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells.

Conclusion: The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells. Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations.

Show MeSH
Related in: MedlinePlus