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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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Chromosome dynamics.A chromosome 12 specific alpha-satellite probe (green) combined with immunofluorescence for beta tubulin (red) and DNA-counterstaining by DAPI (blue) shows tetrasomic/bipolar cell division at metaphase/early anaphase (A) and late anaphase (B) and a disomic/tripolar cell division at metaphase (C) and telophase (D); the segregation pattern is 4-4 in (B) and 3-1-0 in (D). Time-lapse microscopy of H2B/GFP transfected HEK293 cells shows succession of a tripolar metaphase configuration (E; poles denoted a-c) through two successive chromosome segregation events to four daughter nuclei and from a tetrapolar metaphase configuration (F) through a tripolar ana-telophase to three daughter nuclei shown by three-dimensional reconstruction of a confocal image stack at T = 40 min (T, time from first image).
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pone-0001871-g001: Chromosome dynamics.A chromosome 12 specific alpha-satellite probe (green) combined with immunofluorescence for beta tubulin (red) and DNA-counterstaining by DAPI (blue) shows tetrasomic/bipolar cell division at metaphase/early anaphase (A) and late anaphase (B) and a disomic/tripolar cell division at metaphase (C) and telophase (D); the segregation pattern is 4-4 in (B) and 3-1-0 in (D). Time-lapse microscopy of H2B/GFP transfected HEK293 cells shows succession of a tripolar metaphase configuration (E; poles denoted a-c) through two successive chromosome segregation events to four daughter nuclei and from a tetrapolar metaphase configuration (F) through a tripolar ana-telophase to three daughter nuclei shown by three-dimensional reconstruction of a confocal image stack at T = 40 min (T, time from first image).

Mentions: To test whether segregation patterns of individual chromosomes at human multipolar cell divisions conformed to the principles of euploid segregation, we used two well studied cancer cell lines in which mitotic multipolarity has been associated with chromosomal instability (CIN), i.e. WiT49 from an anaplastic Wilms tumor with 7% MM [10], [26] and SW480 from a colorectal carcinoma with 4% MM [27], [28]. In order to compare cancer cell lines to non-neoplastic immortalized cells, we also included the adenovirus-transformed human embryonal cell line HEK293 with a complex karyotype and 1% MM [29]. In these cell lines, cross-labeling of DNA and spindle poles by beta-tubulin antibodies showed that the vast majority of multipolar cell divisions were either tripolar or tetrapolar, while <10% of MM had a higher polarity number (Figure 1A–D). In each cell line, the centromeres of two chromosomes which had showed little intercellular structural heterogeneity [10], [28] were labeled by fluorescence in situ hybridization (FISH): chromosomes 12 and 17 in WiT49, X and 18 in SW480, and 3 and 4 in HEK293, respectively. This selection was done to minimize confounding from anaphase bridging and other mitotic abnormalities primarily leading to abnormalities in chromosome structure. We then screened for the distribution of these chromosomes at anaphase in bipolar mitosis and MM.


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)

Chromosome dynamics.A chromosome 12 specific alpha-satellite probe (green) combined with immunofluorescence for beta tubulin (red) and DNA-counterstaining by DAPI (blue) shows tetrasomic/bipolar cell division at metaphase/early anaphase (A) and late anaphase (B) and a disomic/tripolar cell division at metaphase (C) and telophase (D); the segregation pattern is 4-4 in (B) and 3-1-0 in (D). Time-lapse microscopy of H2B/GFP transfected HEK293 cells shows succession of a tripolar metaphase configuration (E; poles denoted a-c) through two successive chromosome segregation events to four daughter nuclei and from a tetrapolar metaphase configuration (F) through a tripolar ana-telophase to three daughter nuclei shown by three-dimensional reconstruction of a confocal image stack at T = 40 min (T, time from first image).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001871-g001: Chromosome dynamics.A chromosome 12 specific alpha-satellite probe (green) combined with immunofluorescence for beta tubulin (red) and DNA-counterstaining by DAPI (blue) shows tetrasomic/bipolar cell division at metaphase/early anaphase (A) and late anaphase (B) and a disomic/tripolar cell division at metaphase (C) and telophase (D); the segregation pattern is 4-4 in (B) and 3-1-0 in (D). Time-lapse microscopy of H2B/GFP transfected HEK293 cells shows succession of a tripolar metaphase configuration (E; poles denoted a-c) through two successive chromosome segregation events to four daughter nuclei and from a tetrapolar metaphase configuration (F) through a tripolar ana-telophase to three daughter nuclei shown by three-dimensional reconstruction of a confocal image stack at T = 40 min (T, time from first image).
Mentions: To test whether segregation patterns of individual chromosomes at human multipolar cell divisions conformed to the principles of euploid segregation, we used two well studied cancer cell lines in which mitotic multipolarity has been associated with chromosomal instability (CIN), i.e. WiT49 from an anaplastic Wilms tumor with 7% MM [10], [26] and SW480 from a colorectal carcinoma with 4% MM [27], [28]. In order to compare cancer cell lines to non-neoplastic immortalized cells, we also included the adenovirus-transformed human embryonal cell line HEK293 with a complex karyotype and 1% MM [29]. In these cell lines, cross-labeling of DNA and spindle poles by beta-tubulin antibodies showed that the vast majority of multipolar cell divisions were either tripolar or tetrapolar, while <10% of MM had a higher polarity number (Figure 1A–D). In each cell line, the centromeres of two chromosomes which had showed little intercellular structural heterogeneity [10], [28] were labeled by fluorescence in situ hybridization (FISH): chromosomes 12 and 17 in WiT49, X and 18 in SW480, and 3 and 4 in HEK293, respectively. This selection was done to minimize confounding from anaphase bridging and other mitotic abnormalities primarily leading to abnormalities in chromosome structure. We then screened for the distribution of these chromosomes at anaphase in bipolar mitosis and MM.

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