Chromosome shattering: a mitotic catastrophe due to chromosome condensation failure

Original Paper

Abstract

Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a “shattered” chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.

Keywords

Live-cell microscopy UV irradiation and DNA repair Chromosome shattering Chromatin condensation Mitotic catastrophe Fixation procedure 

Abbreviations

FISH

Fluorescence in situ hybridization

GCS

Generalized chromosome shattering

H2BmRFP

Histone H2B conjugated to red fluorescent protein

H3pS10

Histone H3 with phosphorylated serine at position 10

MAA

Methanol/acetic acid

PALCC

Parachute-like chromatin configuration

PCC

Premature chromosome condensation

PCS

Partial chromosome shattering

PFA

Paraformaldehyde

SON

Supplemental online

Supplementary material

249_2009_496_MOESM1_ESM.tif (3 mb)
Fig. 12_SONa, b Three-color painting of Chinese hamster chromosomes 1 (green), 2 (blue), and 5 (red) in V79-H2BmRFP cells. a Apoptotic cells in PFA-fixed cultures after UV/caffeine treatment show a diffuse hybridization signal with strong intermingling of fluorescence signals reflecting a true fragmentation of chromosomes with a concomitant loss of the territorial structure. b Shadow-like chromatin patterns in MAA-fixed cultures lack any detectable hybridization signals. Scale bar = 5 μm Supplementary material 1 (tif 3043 kb)
249_2009_496_MOESM2_ESM.mpg (56.2 mb)
movie_1 Complete image sequence of the live-cell observation of V79-H2BmRFP control cells showing undisturbed cell proliferation up to 48 h. Images are taken each 15 min. The blurring after time point 1,860 min is due to a slight shifting of the z-stage after changing the medium. Picture sharpness in the late time points decreases due to the increasing cell density that hampers the autofocus function. The appearance of some apoptotic cells beyond time point 41 h is most probably due to the increased cell density. Scale bar = 10 µm Supplementary material 1 (MPG 57526 kb)
249_2009_496_MOESM3_ESM.mpg (13.3 mb)
movie_2 Complete image sequence of the live-cell observation of V79-H2BmRFP after UV irradiation/caffeine posttreatment. Scale bar = 10 µm Supplementary material 2 (MPG 13601 kb)

References

  1. Beaudouin J, Gerlich D, Daigle N, Eils R, Ellenberg J (2002) Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108:83–96. doi:10.1016/S0092-8674(01)00627-4 PubMedCrossRefGoogle Scholar
  2. Bezrookove V, Smits R, Moeslein G, Fodde R, Tanke HJ, Raap AK, Darroudi F (2003) Premature chromosome condensation revisited: a novel chemical approach permits efficient cytogenetic analysis of cancers. Genes Chromosomes Cancer 38:177–186. doi:10.1002/gcc.10268 PubMedCrossRefGoogle Scholar
  3. Bignold LP (2002) Hypothesis for the influence of fixatives on the chromatin patterns of interphase nuclei, based on shrinkage and retraction of nuclear and perinuclear structures. Br J Biomed Sci 59:105–113PubMedGoogle Scholar
  4. Blank M, Shiloh Y (2007) Programs for cell death: apoptosis is only one way to go. Cell Cycle 6:686–695PubMedGoogle Scholar
  5. Blank M, Lerenthal Y, Mittelman L, Shiloh Y (2006) Condensin I recruitment and uneven chromatin condensation precede mitotic cell death in response to DNA damage. J Cell Biol 174:195–206. doi:10.1083/jcb.200604022 PubMedCrossRefGoogle Scholar
  6. Blasina A, Price BD, Turenne GA, McGowan CH (1999) Caffeine inhibits the checkpoint kinase ATM. Curr Biol 9:1135–1138. doi:10.1016/S0960-9822(99)80486-2 PubMedCrossRefGoogle Scholar
  7. Brown EJ, Baltimore D (2000) ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev 14:397–402PubMedGoogle Scholar
  8. Chen ES, Sutani T, Yanagida M (2004) Cti1/C1D interacts with condensin SMC hinge and supports the DNA repair function of condensin. Proc Natl Acad Sci USA 101:8078–8083. doi:10.1073/pnas.0307976101 PubMedCrossRefGoogle Scholar
  9. Chowdhury I, Tharakan B, Bhat GK (2006) Current concepts in apoptosis: the physiological suicide program revisited. Cell Mol Biol Lett 11:506–525. doi:10.2478/s11658-006-0041-3 PubMedCrossRefGoogle Scholar
  10. Chu EH (1965) Effects of ultraviolet radiation on mammalian cells. I. Induction of chromosome aberrations. Mutat Res 2:75–94. doi:10.1016/0027-5107(65)90010-2 PubMedGoogle Scholar
  11. Cortez D (2003) Caffeine inhibits checkpoint responses without inhibiting the ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases. J Biol Chem 278:37139–37145. doi:10.1074/jbc.M307088200 PubMedCrossRefGoogle Scholar
  12. Cremer C, Cremer T (1986) Induction of chromosome shattering by ultraviolet light and caffeine: the influence of different distributions of photolesions. Mutat Res 163:33–40. doi:10.1016/0027-5107(86)90055-2 PubMedGoogle Scholar
  13. Cremer T, Cremer C (2006) Rise, fall and resurrection of chromosome territories: a historical perspective. Part II. Fall and resurrection of chromosome territories during the 1950s to 1980s. Part III. Chromosome territories and the functional nuclear architecture: experiments and models from the 1990s to the present. Eur J Histochem 50:223–272PubMedGoogle Scholar
  14. Cremer C, Gray JW (1982) DNA content of cells with generalized chromosome shattering induced by ultraviolet light plus caffeine. Mutat Res 94:133–142. doi:10.1016/0027-5107(82)90175-0 PubMedGoogle Scholar
  15. Cremer C, Cremer T, Simickova M (1980a) Induction of chromosome shattering and micronuclei by ultraviolet light and caffeine. I. Temporal relationship and antagonistic effects of the four deoxyribonucleosides. Environ Mutagen 2:339–351. doi:10.1002/em.2860020304 PubMedCrossRefGoogle Scholar
  16. Cremer T, Cremer C, Zimmer J, Zorn C (1980b) UV-micro-irradiation of Chinese hanster cells and posttreatment with caffeine: indication for clastogenic effects remote from the irradiation site. In: Altmann H, Riklis E, Slor H (eds) DNA-repair and late effects. NRCN Publication, Israel, pp 53–62Google Scholar
  17. Cremer C, Cremer T, Jabbur G (1981a) Laser-UV-microirradiation of Chinese hamster cells: the influence of the distribution of photolesions on unscheduled DNA synthesis. Photochem Photobiol 33:925–928. doi:10.1111/j.1751-1097.1981.tb05514.x PubMedCrossRefGoogle Scholar
  18. Cremer C, Cremer T, Zorn C, Zimmer J (1981b) Induction of chromosome shattering by ultraviolet irradiation and caffeine: comparison of whole-cell and partial-cell irradiation. Mutat Res 84:331–348. doi:10.1016/0027-5107(81)90202-5 PubMedGoogle Scholar
  19. Cremer T, Cremer C, Baumann H, Luedtke EK, Sperling K, Teuber V, Zorn C (1982a) Rabl’s model of the interphase chromosome arrangement tested in Chinese hamster cells by premature chromosome condensation and laser-UV-microbeam experiments. Hum Genet 60:46–56. doi:10.1007/BF00281263 PubMedCrossRefGoogle Scholar
  20. Cremer T, Cremer C, Schneider T, Baumann H, Hens L, Kirsch-Volders M (1982b) Analysis of chromosome positions in the interphase nucleus of Chinese hamster cells by laser-UV-microirradiation experiments. Hum Genet 62:201–209. doi:10.1007/BF00333519 PubMedCrossRefGoogle Scholar
  21. Cremer M, Grasser F, Lanctot C, Muller S, Neusser M, Zinner R, Solovei I, Cremer T (2008) Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes. Methods Mol Biol 463:205–239. doi:10.1007/978-1-59745-406-3_15 PubMedCrossRefGoogle Scholar
  22. Ghosh S, Paweletz N, Schroeter D (1992) Failure of kinetochore development and mitotic spindle formation in okadaic acid-induced premature mitosis in HeLa cells. Exp Cell Res 201:535–540. doi:10.1016/0014-4827(92)90307-T PubMedCrossRefGoogle Scholar
  23. Gotoh E (2007) Visualizing the dynamics of chromosome structure formation coupled with DNA replication. Chromosoma 116:453–462. doi:10.1007/s00412-007-0109-5 PubMedCrossRefGoogle Scholar
  24. Gotoh E (2009) Drug-induced premature chromosome condensation (PCC) protocols: cytogenetic approaches in mitotic chromosome and interphase chromatin. Methods Mol Biol 523:83–92PubMedCrossRefGoogle Scholar
  25. Greubel C, Hable V, Drexler GA, Hauptner A, Dietzel S, Strickfaden H, Baur I, Krucken R, Cremer T, Dollinger G, Friedl AA (2008) Competition effect in DNA damage response. Radiat Environ Biophys 47:423–429. doi:10.1007/s00411-008-0182-z PubMedCrossRefGoogle Scholar
  26. Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD (1997) Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106:348–360. doi:10.1007/s004120050256 PubMedCrossRefGoogle Scholar
  27. Hepperger C, Otten S, von Hase J, Dietzel S (2007) Preservation of large-scale chromatin structure in FISH experiments. Chromosoma 116:117–133. doi:10.1007/s00412-006-0084-2 PubMedCrossRefGoogle Scholar
  28. Hittelman WN, Pollard M (1984) Visualization of chromatin events associated with repair of ultraviolet light-induced damage by premature chromosome condensation. Carcinogenesis 5:1277–1285. doi:10.1093/carcin/5.10.1277 PubMedCrossRefGoogle Scholar
  29. Hoeijmakers JH (2001) Genome maintenance mechanisms for preventing cancer. Nature 411:366–374. doi:10.1038/35077232 PubMedCrossRefGoogle Scholar
  30. Huang X, Kurose A, Tanaka T, Traganos F, Dai W, Darzynkiewicz Z (2006) Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis. Cytometry A 69:222–229. doi:10.1002/cyto.a.20257 PubMedGoogle Scholar
  31. Hurley PJ, Bunz F (2007) ATM and ATR: components of an integrated circuit. Cell Cycle 6:414–417PubMedGoogle Scholar
  32. Ianzini F, Mackey MA (1997) Spontaneous premature chromosome condensation and mitotic catastrophe following irradiation of HeLa S3 cells. Int J Radiat Biol 72:409–421. doi:10.1080/095530097143185 PubMedCrossRefGoogle Scholar
  33. Ismail IH, Hendzel MJ (2008) The gamma-H2A.X: is it just a surrogate marker of double-strand breaks or much more? Environ Mol Mutagen 49:73–82. doi:10.1002/em.20358 PubMedCrossRefGoogle Scholar
  34. Johansson F, Lagerqvist A, Filippi S, Palitti F, Erixon K, Helleday T, Jenssen D (2006) Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines. DNA Repair (Amst) 5:1449–1458. doi:10.1016/j.dnarep.2006.07.005 CrossRefGoogle Scholar
  35. Karagiannis TC, El-Osta A (2007) Chromatin modifications and DNA double-strand breaks: the current state of play. Leukemia 21:195–200. doi:10.1038/sj.leu.2404478 PubMedCrossRefGoogle Scholar
  36. Kiernan J (2000) Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microsc Today 8:8–12Google Scholar
  37. Lau CC, Pardee AB (1982) Mechanism by which caffeine potentiates lethality of nitrogen mustard. Proc Natl Acad Sci USA 79:2942–2946. doi:10.1073/pnas.79.9.2942 PubMedCrossRefGoogle Scholar
  38. Legagneux V, Cubizolles F, Watrin E (2004) Multiple roles of condensins: a complex story. Biol Cell 96:201–213. doi:10.1016/j.biolcel.2004.01.003 PubMedCrossRefGoogle Scholar
  39. Lehmann AR (2005) The role of SMC proteins in the responses to DNA damage. DNA Repair (Amst) 4:309–314. doi:10.1016/j.dnarep.2004.07.009 CrossRefGoogle Scholar
  40. Lin JJ, Dutta A (2007) ATR pathway is the primary pathway for activating G2/M checkpoint induction after re-replication. J Biol Chem 282:30357–30362. doi:10.1074/jbc.M705178200 PubMedCrossRefGoogle Scholar
  41. Lovelace R (1954) Chromosome shattering by ultraviolet radiation (2650A). Pro Natl Acad Sci USA 40:1129–1135Google Scholar
  42. McManus KJ, Hendzel MJ (2005) ATM-dependent DNA damage-independent mitotic phosphorylation of H2AX in normally growing mammalian cells. Mol Biol Cell 16:5013–5025. doi:10.1091/mbc.E05-01-0065 PubMedCrossRefGoogle Scholar
  43. Meaburn KJ, Misteli T (2007) Cell biology: chromosome territories. Nature 445:379–781. doi:10.1038/445379a PubMedCrossRefGoogle Scholar
  44. Musk SR, Downes CS, Johnson RT (1988) Caffeine induces uncoordinated expression of cell cycle functions after ultraviolet irradiation. Accelerated cycle transit, sister chromatid exchanges and premature chromosome condensation in a transformed Indian muntjac cell line. J Cell Sci 90(Pt 4):591–599PubMedGoogle Scholar
  45. Newport J, Spann T (1987) Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes. Cell 48:219–230. doi:10.1016/0092-8674(87)90425-9 PubMedCrossRefGoogle Scholar
  46. Nghiem P, Park PK, Kim Y, Vaziri C, Schreiber SL (2001) ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation. Proc Natl Acad Sci USA 98:9092–9097. doi:10.1073/pnas.161281798 PubMedCrossRefGoogle Scholar
  47. Nitta M, Kobayashi O, Honda S, Hirota T, Kuninaka S, Marumoto T, Ushio Y, Saya H (2004) Spindle checkpoint function is required for mitotic catastrophe induced by DNA-damaging agents. Oncogene 23:6548–6558. doi:10.1038/sj.onc.1207873 PubMedCrossRefGoogle Scholar
  48. Okada H, Mak TW (2004) Pathways of apoptotic and non-apoptotic death in tumour cells. Nat Rev Cancer 4:592–603. doi:10.1038/nrc1412 PubMedCrossRefGoogle Scholar
  49. Pollard TD, Earnshaw W (2004) Cell biology. Elsevier, PhiladelphiaGoogle Scholar
  50. Roninson IB, Broude EV, Chang BD (2001) If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Drug Resist Updat 4:303–313. doi:10.1054/drup.2001.0213 PubMedCrossRefGoogle Scholar
  51. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73:39–85. doi:10.1146/annurev.biochem.73.011303.073723 PubMedCrossRefGoogle Scholar
  52. Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM, Abraham RT (1999) Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 59:4375–4382PubMedGoogle Scholar
  53. Schlegel R, Pardee AB (1986) Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science 232:1264–1266. doi:10.1126/science.2422760 PubMedCrossRefGoogle Scholar
  54. Solimando L, Luijsterburg MS, Vecchio L, Vermeulen W, van Driel R, Fakan S (2009) Spatial organization of nucleotide excision repair proteins after UV-induced DNA damage in the human cell nucleus. J Cell Sci 122:83–91. doi:10.1242/jcs.031062 PubMedCrossRefGoogle Scholar
  55. Solovei I, Cavallo A, Schermelleh L, Jaunin F, Scasselati C, Cmarko D, Cremer C, Fakan S, Cremer T (2002) Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH). Exp Cell Res 276:10–23. doi:10.1006/excr.2002.5513 PubMedCrossRefGoogle Scholar
  56. Sperling K, Rao PN (1974) The phenomenon of premature chromosome condensation: its relevance to basic and applied research. Humangenetik 23:235–258. doi:10.1007/BF00272508 PubMedCrossRefGoogle Scholar
  57. Stevens JB, Liu G, Bremer SW, Ye KJ, Xu W, Xu J, Sun Y, Wu GS, Savasan S, Krawetz SA, Ye CJ, Heng HH (2007) Mitotic cell death by chromosome fragmentation. Cancer Res 67:7686–7694. doi:10.1158/0008-5472.CAN-07-0472 PubMedCrossRefGoogle Scholar
  58. Telenius H, Pelmear AH, Tunnacliffe A, Carter NP, Behmel A, Ferguson-Smith MA, Nordenskjold M, Pfragner R, Ponder BA (1992) Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 4:257–263. doi:10.1002/gcc.2870040311 PubMedCrossRefGoogle Scholar
  59. Van Den Berg HW, Roberts JJ (1976) Inhibition by caffeine of post-replication repair in Chinese hamster cells treated with cis platinum (II) diamminedichloride: the extent of platinum binding to template DNA in relation to the size of low molecular weight nascent DNA. Chem Biol Interact 12:375–390. doi:10.1016/0009-2797(76)90052-1 CrossRefGoogle Scholar
  60. Zorn C, Cremer T, Cremer C, Zimmer J (1976) Laser UV microirradiation of interphase nuclei and post-treatment with caffeine. A new approach to establish the arrangement of interphase chromosomes. Hum Genet 35:83–89. doi:10.1007/BF00295622 PubMedCrossRefGoogle Scholar
  61. Zorn C, Cremer C, Cremer T, Zimmer J (1979) Unscheduled DNA synthesis after partial UV irradiation of the cell nucleus. Distribution in interphase and metaphase. Exp Cell Res 124:111–119. doi:10.1016/0014-4827(79)90261-1 PubMedCrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  1. 1.Department Biology II (Anthropology and Human Genetics)LMU BiozentrumMartinsriedGermany
  2. 2.Munich Center for Integrated Protein Science Munich (CIPSM)MunichGermany
  3. 3.Institute of Human GeneticsUniversity of MunichMunichGermany

Personalised recommendations