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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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DNA distribution and timing.Reconstructed three-dimensional multiphoton cross sectioning images of a bipolar (A) and a multipolar (B) telophase cell in WiT49 show asymmetrical DNA-distribution in the latter configuration. Quantification of the relative amount of chromatin (C) in bipolar (black), tripolar (red) and tetrapolar (green) ana-telophase cells confirms a wide variation in the DNA content of multipolar daughter cells (X axis shows rank order according to DNA content). Measurement of the proportion of BrdU-positive bipolar (blue) and multipolar (red) mitotic cells at different time points after labelling (D) indicate delayed exit from mitosis for multipolar cell divisions in WiT49 and HEK293 (error bars indicate standard deviation). Time lapse imaging of H2B/GFP HEK293 cells show a prolonged metaphase-anaphase interval, and a reduced anaphase-interphase interval in multipolar compared to bipolar cell divisions (E); single and double asterisks indicate significance at the <0.05 and <0.01 levels respectively (Mann-Whitney U-test). One-per-minute time lapse imaging (F) of 12 multipolar mitoses, each corresponding to a flow of identically colored arrows, shows frequent polarity transformations (I = interphase, PM = prometaphase, M2 = bipolar metaphase, M3 = tripolar metaphase, M4 = tetrapolar metaphase, A = anaphase, T = telophase). Quantification of total intensity in arbitrary fluorescence units relative to centrosomal gamma tubulin fluorescence (G) shows a strong reduction of separase levels in telophase compared to metaphase in bipolar but not in multipolar cell divisions (error bars show standard deviation). Diplochromosomes (H, arrows) in G-banded partial metaphase spreads from WiT49.
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pone-0001871-g002: DNA distribution and timing.Reconstructed three-dimensional multiphoton cross sectioning images of a bipolar (A) and a multipolar (B) telophase cell in WiT49 show asymmetrical DNA-distribution in the latter configuration. Quantification of the relative amount of chromatin (C) in bipolar (black), tripolar (red) and tetrapolar (green) ana-telophase cells confirms a wide variation in the DNA content of multipolar daughter cells (X axis shows rank order according to DNA content). Measurement of the proportion of BrdU-positive bipolar (blue) and multipolar (red) mitotic cells at different time points after labelling (D) indicate delayed exit from mitosis for multipolar cell divisions in WiT49 and HEK293 (error bars indicate standard deviation). Time lapse imaging of H2B/GFP HEK293 cells show a prolonged metaphase-anaphase interval, and a reduced anaphase-interphase interval in multipolar compared to bipolar cell divisions (E); single and double asterisks indicate significance at the <0.05 and <0.01 levels respectively (Mann-Whitney U-test). One-per-minute time lapse imaging (F) of 12 multipolar mitoses, each corresponding to a flow of identically colored arrows, shows frequent polarity transformations (I = interphase, PM = prometaphase, M2 = bipolar metaphase, M3 = tripolar metaphase, M4 = tetrapolar metaphase, A = anaphase, T = telophase). Quantification of total intensity in arbitrary fluorescence units relative to centrosomal gamma tubulin fluorescence (G) shows a strong reduction of separase levels in telophase compared to metaphase in bipolar but not in multipolar cell divisions (error bars show standard deviation). Diplochromosomes (H, arrows) in G-banded partial metaphase spreads from WiT49.

Mentions: To quantify the total distribution of DNA in multipolar ana-telophases, WiT49 was selected for further analysis because this cell line contained the highest frequency of MM. Previous studies of DNA-content in tumor cell mitoses have been performed by Feulgen staining in a two-dimensional setting [30]. However, MM often have a larger chromatin volume than normal mitoses with a diameter of up to 100 µm and a complex three-dimensional structure [31]. To be able to quantify the relative DNA-content of daughter ana-telophases under these conditions, we applied fluorescence quantification of DAPI-labeled DNA by multiphoton cross sectioning microscopy. This technique provides high spatial resolution imaging in a three dimensional setting due to the nonlinear signal generation at the laser focus [32]. The localized excitation greatly reduces out-of-focus fluorescence and photo-bleaching. To objectify the analysis further, a range of different thresholds for DAPI-intensity was chosen, extending from near the noise floor. For each threshold value, the ratio of the amount of fluorescence in each region to that in the first region was calculated. A mean relative DNA content for each ana-telophase pole was then calculated by averaging the results for all thresholds (Figure 2A–C).


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)

DNA distribution and timing.Reconstructed three-dimensional multiphoton cross sectioning images of a bipolar (A) and a multipolar (B) telophase cell in WiT49 show asymmetrical DNA-distribution in the latter configuration. Quantification of the relative amount of chromatin (C) in bipolar (black), tripolar (red) and tetrapolar (green) ana-telophase cells confirms a wide variation in the DNA content of multipolar daughter cells (X axis shows rank order according to DNA content). Measurement of the proportion of BrdU-positive bipolar (blue) and multipolar (red) mitotic cells at different time points after labelling (D) indicate delayed exit from mitosis for multipolar cell divisions in WiT49 and HEK293 (error bars indicate standard deviation). Time lapse imaging of H2B/GFP HEK293 cells show a prolonged metaphase-anaphase interval, and a reduced anaphase-interphase interval in multipolar compared to bipolar cell divisions (E); single and double asterisks indicate significance at the <0.05 and <0.01 levels respectively (Mann-Whitney U-test). One-per-minute time lapse imaging (F) of 12 multipolar mitoses, each corresponding to a flow of identically colored arrows, shows frequent polarity transformations (I = interphase, PM = prometaphase, M2 = bipolar metaphase, M3 = tripolar metaphase, M4 = tetrapolar metaphase, A = anaphase, T = telophase). Quantification of total intensity in arbitrary fluorescence units relative to centrosomal gamma tubulin fluorescence (G) shows a strong reduction of separase levels in telophase compared to metaphase in bipolar but not in multipolar cell divisions (error bars show standard deviation). Diplochromosomes (H, arrows) in G-banded partial metaphase spreads from WiT49.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2289843&req=5

pone-0001871-g002: DNA distribution and timing.Reconstructed three-dimensional multiphoton cross sectioning images of a bipolar (A) and a multipolar (B) telophase cell in WiT49 show asymmetrical DNA-distribution in the latter configuration. Quantification of the relative amount of chromatin (C) in bipolar (black), tripolar (red) and tetrapolar (green) ana-telophase cells confirms a wide variation in the DNA content of multipolar daughter cells (X axis shows rank order according to DNA content). Measurement of the proportion of BrdU-positive bipolar (blue) and multipolar (red) mitotic cells at different time points after labelling (D) indicate delayed exit from mitosis for multipolar cell divisions in WiT49 and HEK293 (error bars indicate standard deviation). Time lapse imaging of H2B/GFP HEK293 cells show a prolonged metaphase-anaphase interval, and a reduced anaphase-interphase interval in multipolar compared to bipolar cell divisions (E); single and double asterisks indicate significance at the <0.05 and <0.01 levels respectively (Mann-Whitney U-test). One-per-minute time lapse imaging (F) of 12 multipolar mitoses, each corresponding to a flow of identically colored arrows, shows frequent polarity transformations (I = interphase, PM = prometaphase, M2 = bipolar metaphase, M3 = tripolar metaphase, M4 = tetrapolar metaphase, A = anaphase, T = telophase). Quantification of total intensity in arbitrary fluorescence units relative to centrosomal gamma tubulin fluorescence (G) shows a strong reduction of separase levels in telophase compared to metaphase in bipolar but not in multipolar cell divisions (error bars show standard deviation). Diplochromosomes (H, arrows) in G-banded partial metaphase spreads from WiT49.
Mentions: To quantify the total distribution of DNA in multipolar ana-telophases, WiT49 was selected for further analysis because this cell line contained the highest frequency of MM. Previous studies of DNA-content in tumor cell mitoses have been performed by Feulgen staining in a two-dimensional setting [30]. However, MM often have a larger chromatin volume than normal mitoses with a diameter of up to 100 µm and a complex three-dimensional structure [31]. To be able to quantify the relative DNA-content of daughter ana-telophases under these conditions, we applied fluorescence quantification of DAPI-labeled DNA by multiphoton cross sectioning microscopy. This technique provides high spatial resolution imaging in a three dimensional setting due to the nonlinear signal generation at the laser focus [32]. The localized excitation greatly reduces out-of-focus fluorescence and photo-bleaching. To objectify the analysis further, a range of different thresholds for DAPI-intensity was chosen, extending from near the noise floor. For each threshold value, the ratio of the amount of fluorescence in each region to that in the first region was calculated. A mean relative DNA content for each ana-telophase pole was then calculated by averaging the results for all thresholds (Figure 2A–C).

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