Abstract
The involvement of whole-chromosome aneuploidy in tumorigenesis is the subject of debate, in large part because of the lack of insight into underlying mechanisms. Here we identify a mechanism by which errors in mitotic chromosome segregation generate DNA breaks via the formation of structures called micronuclei. Whole-chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and often extensive fragmentation of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for ‘chromothripsis’ in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Gordon, D. J., Resio, B. & Pellman, D. Causes and consequences of aneuploidy in cancer. Nature Rev. Genet. http://dx.doi.org/10.1038/nrg3123 (in the press)
Weaver, B. A. & Cleveland, D. W. Aneuploidy: instigator and inhibitor of tumorigenesis. Cancer Res. 67, 10103–10105 (2007)
Schvartzman, J. M., Sotillo, R. & Benezra, R. Mitotic chromosomal instability and cancer: mouse modelling of the human disease. Nature Rev. Cancer 10, 102–115 (2010)
Ricke, R. M., van Ree, J. H. & van Deursen, J. M. Whole chromosome instability and cancer: a complex relationship. Trends Genet. 24, 457–466 (2008)
Weaver, B. A., Silk, A. D., Montagna, C., Verdier-Pinard, P. & Cleveland, D. W. Aneuploidy acts both oncogenically and as a tumor suppressor. Cancer Cell 11, 25–36 (2007)
Sotillo, R. et al. Mad2 overexpression promotes aneuploidy and tumorigenesis in mice. Cancer Cell 11, 9–23 (2007)
Baker, D. J., Jin, F., Jeganathan, K. B. & van Deursen, J. M. Whole chromosome instability caused by Bub1 insufficiency drives tumorigenesis through tumor suppressor gene loss of heterozygosity. Cancer Cell 16, 475–486 (2009)
Fujiwara, T. et al. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 437, 1043–1047 (2005)
Cimini, D. Merotelic kinetochore orientation, aneuploidy, and cancer. Biochim. Biophys. Acta 1786, 32–40 (2008)
Ganem, N. J., Godinho, S. A. & Pellman, D. A mechanism linking extra centrosomes to chromosomal instability. Nature 460, 278–282 (2009)
Terradas, M., Martin, M., Tusell, L. & Genesca, A. Genetic activities in micronuclei: is the DNA entrapped in micronuclei lost for the cell? Mutat. Res. 705, 60–67 (2010)
Gisselsson, D. et al. Abnormal nuclear shape in solid tumors reflects mitotic instability. Am. J. Pathol. 158, 199–206 (2001)
Thompson, S. L. & Compton, D. A. Chromosome missegregation in human cells arises through specific types of kinetochore-microtubule attachment errors. Proc. Natl Acad. Sci. USA 108, 17974–17978 (2011)
Hoffelder, D. R. et al. Resolution of anaphase bridges in cancer cells. Chromosoma 112, 389–397 (2004)
Géraud, G. et al. Three-dimensional organization of micronuclei induced by colchicine in PtK1 cells. Exp. Cell Res. 181, 27–39 (1989)
Cimini, D. et al. Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells. J. Cell Biol. 153, 517–528 (2001)
Dalton, W. B., Nandan, M. O., Moore, R. T. & Yang, V. W. Human cancer cells commonly acquire DNA damage during mitotic arrest. Cancer Res. 67, 11487–11492 (2007)
Quignon, F. et al. Sustained mitotic block elicits DNA breaks: one-step alteration of ploidy and chromosome integrity in mammalian cells. Oncogene 26, 165–172 (2007)
Orth, J. D., Loewer, A., Lahav, G. & Mitchison, T. J. Prolonged mitotic arrest induces partial activation of apoptosis, DNA damage and p53 activation. Mol. Biol. . Cellhttp://dx.doi.org/10.1091/mbc.E11-09-0781 (2011)
Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. & Bonner, W. M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998)
Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501 (1992)
Singh, N. P., McCoy, M. T., Tice, R. R. & Schneider, E. L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191 (1988)
Thompson, S. L. & Compton, D. A. Proliferation of aneuploid human cells is limited by a p53-dependent mechanism. J. Cell Biol. 188, 369–381 (2010)
Li, M. et al. The ATM-p53 pathway suppresses aneuploidy-induced tumorigenesis. Proc. Natl Acad. Sci. USA 107, 14188–14193 (2010)
Branzei, D. & Foiani, M. Maintaining genome stability at the replication fork. Nature Rev. Mol. Cell Biol. 11, 208–219 (2010)
Kline-Smith, S. L., Khodjakov, A., Hergert, P. & Walczak, C. E. Depletion of centromeric MCAK leads to chromosome congression and segregation defects due to improper kinetochore attachments. Mol. Biol. Cell 15, 1146–1159 (2004)
DeLuca, J. G., Moree, B., Hickey, J. M., Kilmartin, J. V. & Salmon, E. D. hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells. J. Cell Biol. 159, 549–555 (2002)
Nakano, M. et al. Inactivation of a human kinetochore by specific targeting of chromatin modifiers. Dev. Cell 14, 507–522 (2008)
Binz, S. K., Sheehan, A. M. & Wold, M. S. Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair 3, 1015–1024 (2004)
O’Keefe, R. T., Henderson, S. C. & Spector, D. L. Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences. J. Cell Biol. 116, 1095–1110 (1992)
DePamphilis, M. L. & Bell, S. D. Genome Duplication (Garland Science, 2011)
Dimitrova, D. S., Prokhorova, T. A., Blow, J. J., Todorov, I. T. & Gilbert, D. M. Mammalian nuclei become licensed for DNA replication during late telophase. J. Cell Sci. 115, 51–59 (2002)
Mendez, J. & Stillman, B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612 (2000)
Terradas, M., Martin, M., Tusell, L. & Genesca, A. DNA lesions sequestered in micronuclei induce a local defective-damage response. DNA Repair 8, 1225–1234 (2009)
Obe, G., Beek, B. & Vaidya, V. G. The human leukocyte test system. III. Premature chromosome condensation from chemically and x-ray induced micronuclei. Mutat. Res. 27, 89–101 (1975)
Johnson, R. T. & Rao, P. N. Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature 226, 717–722 (1970)
Smith, L., Plug, A. & Thayer, M. Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations. Proc. Natl Acad. Sci. USA 98, 13300–13305 (2001)
Nichols, W. W., Levan, A., Aula, P. & Norrby, E. Chromosome damage associated with the measles virus in vitro . Hereditas 54, 101–118 (1965)
Kato, H. & Sandberg, A. A. Chromosome pulverization in human binucleate cells following colcemid treatment. J. Cell Biol. 34, 35–45 (1967)
Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. & Miyawaki, A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl Acad. Sci. USA 99, 12651–12656 (2002)
Janssen, A., van der Burg, M., Szuhai, K., Kops, G. J. & Medema, R. H. Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations. Science 333, 1895–1898 (2011)
Stephens, P. J. et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 144, 27–40 (2011)
Liu, P. et al. Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements. Cell 146, 889–903 (2011)
Hastings, P. J., Lupski, J. R., Rosenberg, S. M. & Ira, G. Mechanisms of change in gene copy number. Nature Rev. Genet. 10, 551–564 (2009)
Sen, S., Hittelman, W. N., Teeter, L. D. & Kuo, M. T. Model for the formation of double minutes from prematurely condensed chromosomes of replicating micronuclei in drug-treated Chinese hamster ovary cells undergoing DNA amplification. Cancer Res. 49, 6731–6737 (1989)
Bekker-Jensen, S. et al. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J. Cell Biol. 173, 195–206 (2006)
Franz, C. et al. MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly. EMBO Rep. 8, 165–172 (2007)
Peterson-Roth, E., Reynolds, M., Quievryn, G. & Zhitkovich, A. Mismatch repair proteins are activators of toxic responses to chromium-DNA damage. Mol. Cell. Biol. 25, 3596–3607 (2005)
Prasanth, S. G., Mendez, J., Prasanth, K. V. & Stillman, B. Dynamics of pre-replication complex proteins during the cell division cycle. Phil. Trans. R. Soc. Lond. B 359, 7–16 (2004)
Dultz, E. et al. Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J. Cell Biol. 180, 857–865 (2008)
Acknowledgements
We thank A. D’Andrea, M. E. McLaughlin, T. A. Rapoport, J. Walters and Pellman laboratory members for discussions and/or comments on the manuscript; L. Cameron for advice and help on microscopy; H. Li for irradiation of cells; M. Nitta and M. Hennessy for preliminary experiments; and V. Larionov, B. Stillman, J. Ellenberg, I. Mattaj, A. Miyawaki and T. Kuroda for reagents. D.P. was supported by the Howard Hughes Medical Institute and the NIH (GM083299); K.C. was a fellow of A*STAR Singapore; N.J.G. was a fellow of the Leukemia and Lymphoma Society; Y.P. and D.C. were funded by the NIH (1R01CA142698-01).
Author information
Authors and Affiliations
Contributions
D.P. conceived the project; K.C., N.J.G., R.D., A.B.L., D.C. and D.P. designed the experiments; D.P., K.C. and N.J.G. wrote the manuscript with edits from all authors; K.C. contributed Figs. 1–3, 4a, b and Supplementary Figs 2–8 with help from R.D. N.J.G. contributed Fig. 5j, Supplementary Fig. 12, Supplementary Table 1 and Supplementary Movies. R.D., E.V.I. and A.P. contributed Fig. 5a–i and Supplementary Figs 1 and 11; A.B.L. contributed Fig. 4c, d and Supplementary Figs 9 and 10; Y.P. and D.C. contributed Supplementary Fig. 3d; L.N. contributed Fig. 2a.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
The file contains Supplementary Figures 1-12 with legends, Supplementary References, Supplementary Table 1 and legends for Supplementary Movies 1-4. (PDF 11345 kb)
Supplementary Movie 1
The movie shows live-cell imaging of a MN in a U2OS cell expressing H2B-mRFP -see Supplementary Information file for full legend. (MOV 6862 kb)
Supplementary Movie 2
The movie shows live-cell imaging of a MN in a U2OS cell expressing H2B-mRFP -see Supplementary Information file for full legend. (MOV 9785 kb)
Supplementary Movie 3
The movie shows live-cell imaging of a MN in a U2OS cell expressing H2B-Kaede (corresponds to the cell shown in Figure 4g, top row) - see Supplementary Information file for full legend. (MOV 16698 kb)
Supplementary Movie 4
The movie shows live-cell imaging of a MN in a U2OS cell expressing H2B-Kaede (cell is located in the bottom-left portion of the video and corresponds to the cell shown in Figure 4g, bottom row) - see Supplementary Information file for full legend. (MOV 7152 kb)
Rights and permissions
About this article
Cite this article
Crasta, K., Ganem, N., Dagher, R. et al. DNA breaks and chromosome pulverization from errors in mitosis. Nature 482, 53–58 (2012). https://doi.org/10.1038/nature10802
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature10802
This article is cited by
-
Inheritance of H3K9 methylation regulates genome architecture in Drosophila early embryos
The EMBO Journal (2024)
-
PRAME induces genomic instability in uveal melanoma
Oncogene (2024)
-
Mitotic tethering enables inheritance of shattered micronuclear chromosomes
Nature (2023)
-
Heritable transcriptional defects from aberrations of nuclear architecture
Nature (2023)
-
Antecedent chromatin organization determines cGAS recruitment to ruptured micronuclei
Nature Communications (2023)
Karel H.M. van Wely
The authors correctly identify a desynchronization of DNA synthesis in the micronucleus compared to the main nucleus. Indeed the observations made by the authors are easily explained by an initial mitotic defect (concomittant with lagging chromosomes and micronucleus formation), causing a delay in DNA synthesis in the micronucleus:
1) Please note that mitotic DNA damage has been linked to micronuclei two years ago by Alonso Guerrero et al: "Centromere-localized breaks indicate the generation of DNA damage by the mitotic spindle", 2010, PNAS 107, 4159-4164. Just as in the original paper, the current paper shows DNA damage adjacent to the centromere (Figure 1, ACA, S phase), and therefore again hints at centromere fission as the mechanism that yields the inital break. An intial break prior to S phase, but specific for the micronucleus, provides an easy explanation why the DNA therein shows a delay in DNA synthesis.
2) The authors observe that the micronucleus frequently is excluded from the next mitosis; if the micronucleus has not completed DNA synthesis, this is a logical consequence.
3) The autors observe that Thymide block allieves the DNA damage. Since a Thymidine block delays S phase progression, this treatment effectively helps to resynchronize DNA synthesis in the main nucleus and micronucleus. It would be nice to determine if this treatment reduces the number of micronuclei excluded from the following mitosis.
Since the TUNEL signal is supressed both in the primary nucleus and micronucleus, Thymidine treatment might not only inhibit S phase but also suppress DNA damage processing (Figure 3C). The DNA thus cannot be attributed automatically to S phase alone. Since sindle poisons are now regularly used to induce DNA damage which is analyzed afterwards (This paper and Janssen et al, 2011, Science), centromere fission must be considered seriously as the initial trigger of DNA damage under thse experimental conditions.
4) The authors show a reduced import of repair proteins, inparticular the proteins involved in homologous recombination. Since homologous recombination is more active towards the end of S phase and G2, a reduction of import in a structure that is not yet engaged in S phase such as the micronucleus is an obvious consequence. If the micronucleus is desynchronized with respect to DNA synthesis, other related processes will also suffer desynchronization! It would have been nice to include a proteins involved in non-homolgous end-joining, which show a more constant activity throughout the cell cycle.
5) An important question that remains to be addressed is the relevance of these results for real life. Although micronuclei might suffer a delay in rapidly dividing cells in vitro, even cancers cells in our bodies likely replicate more slowly, which allows for completion of DNA synthesis before the next mitosis. Even though micronuclei are a found frequently in tumor samples, chromothrispis is seen only in a minute proportion of tumor samples. Thus, the frequent event of centromere fission in mitosis (Martinez and van Wely, Carcinogenesis, 32:6, 796-803) does not seem to cause widespread chromothripis in real life even though it leads to whole arm translocations.