Skip to main content
SpringerLink
Log in

Micronucleus formation during chromatin condensation and under apoptotic conditions

  • The many ways of apoptotic cell death
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

In early S phase the newly replicated DNA is folded back to increasingly compact structures. The process of chromatin condensation inside the nucleus starts with the formation of a micronucleus observed in five established cell lines (K562, CHO, Indian muntjac, murine preB and SCC). Supercoiling of chromatin generates a polarized end-plate region extruded from the nucleus. The extruded chromatin is turned around itself forming the head portion (micronucleus) visible by fluorescence microscopy until the middle of S phase when chromatin structures are succeeded by distinguishable early forms of chromosomes. The generation of micronuclei upon apoptotic treatment was achieved by the methotrexate (MTX) treatment of cells. A close correlation was found between the frequency of micronucleus and MTX concentration, with low frequency at low (0.1 µM) and increasingly higher frequency between 1 and 100 µM concentrations. Characteristic deformation and shrinkage of nuclei indicated apoptosis. High MTX concentration (100 µM) caused the enlargement and necrotic disruption of nuclei. Inhibition of DNA synthesis during replicative DNA synthesis by biotinylated nucleotide prevented the formation of metaphase chromosomes and elevated the frequency of early intermediates of chromosome condensation including micronucleus formation. Based on these observations the micronucleus is regarded as: (a) a regularly occuring element of early chromatin condensation and (b) a typical sign of nuclear membrane damage under toxic conditions. Explanation is given why the micronucleus is hidden in nuclei under normal chromatin condensation and why chromatin motifs including micronuclei become visible upon cellular damage.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Sears DA, Udden MM (2011) Howell–Jolly bodies: a brief historical review. Am J Med Sci 343:407–409

    Article  Google Scholar 

  2. Fenech M, Morley AA (1985) Measurement of micronuclei in lymphocytes. Mutat Res 147:29–36

    Article  CAS  PubMed  Google Scholar 

  3. Countryman PI, Heddle JA (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutat Res 41:321–332

    Article  CAS  PubMed  Google Scholar 

  4. Wenkert D, Allis CD (1984) Timing of the appearance of macronuclear-specific histone variant hv1 and gene expression in developing new macronuclei of Tetrahymena thermophila. J Cell Biol 98:2107–2117

    Article  CAS  PubMed  Google Scholar 

  5. Allis CD, Glover CVC, Bowen JK, Gorovsky MA (1980) Histone variants specific to the transcriptionally active, amitotically dividing macronucleus of the unicellular eucaryote, Tetrahymena thermophila. Cell 20:609–617

    Article  CAS  PubMed  Google Scholar 

  6. Allis CD, Ziegler YS, Gorovsky MA, Olmsted JB (1982) A conserved histone variant enriched in nucleoli of mammalian cells. Cell 31:131–136

    Article  CAS  PubMed  Google Scholar 

  7. Collins K, Gorovsky MA (2005) Tetrahymena thermophyla. Curr Biol 15:R317–R318

    Article  CAS  PubMed  Google Scholar 

  8. Norppa H, Falck GC (2003) What do human micronuclei contain? Mutagenesis 18:221–233

    Article  CAS  PubMed  Google Scholar 

  9. Abend M, Rhein A, Gilbertz KP, Blakely WF, Van Beuningen D (1995) Correlation of micronucleus and apoptosis assays with reproductive cell death. Int J Radiat Biol 67:315–326

    Article  CAS  PubMed  Google Scholar 

  10. Schupp N, Stopper H, Heidland A (2016) DNA damage in chronic kidney disease: evaluation of clinical biomarkers. Oxidative Med Cell Longev 3592042

  11. Matsuoka Y, Matsuoka Y, Shibata S, Yoneda Y (2002) Bovine liver chromatin fraction contains actin polymerization activity inducing micronuclei formation when injected into prometaphase cultured cells. Cell Struct Funct 27:39–45

    Article  CAS  PubMed  Google Scholar 

  12. Li M, Deugarte CM, Surrey M, Danzer H, Decherney A, Hill DL (2005) Fluorescence in situ hybridization reanalysis of day-6 human blastocysts diagnosed with aneuploidy on day 3. Fertil Steril 84:1395–1400

    Article  PubMed  Google Scholar 

  13. Briton-Jones C, Yeung Q, Steinberg J (2006) Different perceptions of similar observations. Fertil Steril 86:e3–e3 C

    Article  Google Scholar 

  14. Evsikov S, Verlinsky Y (1998) Mosaicism in the inner cell mass of human blastocysts. Hum Reprod 13:3151–3155

    Article  CAS  PubMed  Google Scholar 

  15. Nagy G, Gacsi M, Rehak M, Basnakian AG, Klaisz M, Banfalvi G (2004) Gamma irradiation-induced apoptosis in murine pre-B cells prevents the condensation of fibrillar chromatin in early S phase. Apoptosis 9:765–776

    Article  CAS  PubMed  Google Scholar 

  16. Gacsi M, Nagy G, Pinter G, Basnakian AG, Banfalvi G (2005) Condensation of interphase chromatin in nuclei of Chinese hamster ovary (CHO-K1) cells. DNA Cell Biol 24:43–53

    Article  CAS  PubMed  Google Scholar 

  17. Banfalvi G, Gacsi M, Nagy G, Kiss BZ, Basnakian AG (2005) Cadmium induced apoptotic changes in chromatin structure and subphases of nuclear growth during the cell cycle in CHO cells. Apoptosis 10:631–642

    Article  CAS  PubMed  Google Scholar 

  18. Banfalvi G (2006) Linear connection of condensing chromosomes in nuclei of synchronized CHO cells. DNA Cell Biol 25:541–545

    Article  CAS  PubMed  Google Scholar 

  19. Hwang-Bo J, Park JH, Chung IS (2015) Tumstatin induces apoptosis mediated by Fas signaling pathway in oral squamous cell carcinoma SCC-VII cells. Oncol Lett 10:1016–1022

    PubMed  PubMed Central  Google Scholar 

  20. O’Malley BW Jr, Cope KA, Johnson CS, Schwartz MR (1997) A new immunocompetent murine model for oral cancer. Arch Otolaryngol Head Neck Surg 123:20–24

    Article  PubMed  Google Scholar 

  21. Kanazawa H, Rapacchietta D, Kallman RF (1988) Schedule-dependent therapeutic gain from the combination of fractionated irradiation and cis-diamminedichloroplatinum (II) in C3H/Km mouse model systems. Cancer Res 48:3158–3164

    PubMed  Google Scholar 

  22. Sperling K, Ludtke EK (1981) Arrangement of prematurely condensed chromosomes in cultured cells and lymphocytes of the Indian muntjac. Chromosoma 83:541–553

    Article  CAS  PubMed  Google Scholar 

  23. Röhme D (1974) Prematurely condensed chromosomes of the Indian muntjac: a model system for the analysis of chromosome condensation and banding. Hereditas 76:251–258

    Article  PubMed  Google Scholar 

  24. Pillidge L, Musk SRR, Johnson RT, Waldren CA (1986) Excessive chromosome fragility and abundance of sister-chromatid exchanges induced by UV in an Indian muntjac cell line defective in postreplication (daughter strand) repair. Mutat Res 166:265–273

    CAS  PubMed  Google Scholar 

  25. Lozzio CB, Lozzio BB (1975) Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45:321–334

    CAS  PubMed  Google Scholar 

  26. Banfalvi G (2008) Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation. Nat Protoc 3:663–673

    Article  CAS  PubMed  Google Scholar 

  27. Banfalvi G (2011) Synchronization of mammalian cells and nuclei by centrifugal elutriation. Cell Cycle Synchron Methods Mol Biol 761, 25–45

    Article  CAS  Google Scholar 

  28. Offer H, Zurer I, Banfalvi G, Rehak M, Falcovitz A, Milyavsky M, Goldfinger N, Rotter V (2001) p53 modulates base excision activity in a cell cycle-specific manner after genotoxic stress. Cancer Res 61:88–96

    CAS  PubMed  Google Scholar 

  29. Basnakian A, Banfalvi G, Sarkar N (1989) Contribution of DNA polymerase δ to DNA replication in permeable CHO cells synchronized in S phase. Nucleic Acids Res 17:4757–4767

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Banfalvi G, Sooki-Toth A, Sarkar N, Csuzi S, Antoni F (1984) Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes. Eur J Biochem 139:553–559

    Article  CAS  PubMed  Google Scholar 

  31. Halldorson H, Gray DA, Shall S (1978) Poly (ADP-ribose) polymerase activity in nucleotide permeable cells. FEBS Lett 85:349–359

    Article  Google Scholar 

  32. Kucera R, Paulus H (1982) Studies on ribonucleoside-diphosphate reductase in permeable animal cells: II. Catalytic and regulatory properties of the enzyme in mouse L cells. Arch Biochem Biophys 214:114–123

    Article  CAS  PubMed  Google Scholar 

  33. Lesh RE, Somlyo AP, Owens GK, Somlyo AV (1995) Reversible permeabilization: a novel technique for the intracellular introduction of antisense oligodeoxynucleotides into intact smooth muscle. Circ Res 77:220–230

    Article  CAS  PubMed  Google Scholar 

  34. Kobayashi S, Kitazawa T, Somlyo AV, Somlyo AP (1989) Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling. J Biol Chem 264:17997–18004

    CAS  PubMed  Google Scholar 

  35. Banfalvi G (1993) Fluorescent analysis of replication and intermediates of chromatin folding in nuclei of mammalian cells. In: Bach PH, Reynolds CH, Clark JM, Mottley J, Poole PL (eds) Biotechnology applications of microinjection, microscopic imaging, and fluorescence. Plenum Press, New York, pp 111–119

    Chapter  Google Scholar 

  36. Banfalvi G, Wiegant J, Sarkar N, van Duijn P (1989) Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells. Histochemistry 93:81–86

    Article  CAS  PubMed  Google Scholar 

  37. Banfalvi G, Nagy G, Gacsi M, Roszer T, Basnakian AG (2006) Common pathway of chromosome condensation in mammalian cells. DNA Cell Biol 25:295–301

    Article  CAS  PubMed  Google Scholar 

  38. Banfalvi G, Ujvarosi K, Trencsenyi G, Somogyi C, Nagy G, Basnakian AG (2007) Cell culture density dependent toxicity and chromatin changes upon cadmium treatment in murine pre-B-cells. Apoptosis 12:1219–1228

    Article  CAS  PubMed  Google Scholar 

  39. Iwano M, Fukui K, Tkaichi S, Isogai A (1997) Globular and fibrous structure in barley chromosomes revealed by highresolution scanning electron microscopy. Chromosome Res 5:341–349

    Article  CAS  PubMed  Google Scholar 

  40. Liang H, Salinas RA, Leal BZ, Kosakowska-Cholody T, Michejda CJ, Waters SJ, Herman TS, Woynarowski JM, Woynarowska BA (2004) Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells. Mol Cancer Ther 3:1385–1396

    CAS  PubMed  Google Scholar 

  41. Banfalvi G, Sarvari A, Nagy G (2012) Chromatin changes induced by Pb and Cd in human cells. Toxicol In Vitro 26:1064–1071

    Article  CAS  PubMed  Google Scholar 

  42. Yamamoto K, Kikuchi Y (1981) Studies on micronuclei time response and on the effects of multiple treatments of mutagens on induction of micronuclei. Mutat Res 90:163–173

    Article  CAS  PubMed  Google Scholar 

  43. Hayashi M, Sofuni T, Ishidate M (1984) A pilot experiment for the micronucleus test, the multi-sampling at multi-dose levels method. Mutat Res 141:165–169

    Article  CAS  PubMed  Google Scholar 

  44. Kasahara Y, Nakai Y, Miura D, Yagi K, Hirabayashi K, Makita T (1992) Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate. Mutat Res 280:117–128

    Article  CAS  PubMed  Google Scholar 

  45. Yamamoto KI, Kikuchi Y (1980) A comparison of diameters of micronuclei induced by clastogens and by spindle poisons. Mutat Res 71:127–131

    Article  CAS  PubMed  Google Scholar 

  46. Tinwell H, Ashby J (1991) Micronucleus morphology as a means to distinguish aneugens and clastogens in the mouse bone marrow micronucleus assay. Mutagenesis 6:193–198

    Article  CAS  PubMed  Google Scholar 

  47. Vanderkerken K, Vanparys P, Verschaeve L, Kirsch-Volders M (1989) The mouse bone marrow micronucleus assay can be used to distinguish aneugens from clastogens. Mutagenesis 4:6–11

    Article  CAS  PubMed  Google Scholar 

  48. Sekeroğlu ZA, Sekeroğlu V (2012) Effects of Viscum album L. extract and quercetin on methotrexate-induced cyto-genotoxicity in mouse bone-marrow cells. Mutat Res 746:56–59

    Article  PubMed  Google Scholar 

  49. Trencsenyi G, Bako F, Nagy G, Kertai P, Banfalvi G (2015) Methotrexate induced apoptotic and necrotic chromatin changes in rat myeloid leukemia cells. Inflamm Res 64:193–203

    Article  CAS  PubMed  Google Scholar 

  50. Heslop-Harrison JS, Huelskamp M, Wendroth M, Atkinson MD, Leicht AR, Benett MD (1988) Chromatin and centromeric structures in interphase nuclei. New chromosome conference III in PE Brandham ed, Allan & Unwin, London, pp 209–217

  51. Banfalvi G (2014) Apoptotic agents inducing genotoxicity-specific chromatin changes. Apoptosis 19:1301–1316

    Article  CAS  PubMed  Google Scholar 

  52. Tanaka T, Watanabe T (2003) Spatiotemporal sites of DNA replication in macro- and micronuclei of the ciliate Paramecium caudatum. Chromosome Res 11:153–164

    Article  CAS  PubMed  Google Scholar 

  53. Dopp E, Hartmann LM, Florea AM, Von Recklinghausen U, Pieper R, Shokouhi B, Rettenmeier AW, Hirner AV, Obe G (2004) Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells. Toxicol Appl Pharmacol 201:156–165

    Article  CAS  PubMed  Google Scholar 

  54. Kallio M, Lähdetie J (1993) Analysis of micronuclei induced in mouse early spermatids by mitomycin C, vinblastine. Mutagenesis 8:561–567

    Article  CAS  PubMed  Google Scholar 

  55. Surrallés J, Catalán J, Creus A, Norppa H, Xamena N, Marcos R (1995) Micronuclei induced by alachlor, mitomycin-C and vinblastine in human lymphocytes: presence of centromeres and kinetochores and influence of staining technique. Mutagenesis 10:417–423

    Article  PubMed  Google Scholar 

  56. Çelik A, Mazmanci B, Çamlica Y, Ali Aşkin, Ükü Çömelekoğlu (2005) Induction of micronuclei by lambda-cyhalothrin in Wistar rat bone marrow and gut epithelial cells. Mutagenesis 20:125–129

    Article  PubMed  Google Scholar 

  57. Bertsche U (1985) Micronucleus induction in mammalian cell cultures treated with ionizing radiations. Radiat Environ Biophys 24:27–44

    Article  CAS  PubMed  Google Scholar 

  58. Huber R, Schraube H, Nahrstedt U, Braselmann H, Bauchinger M (1994) Dose response relationships of micronuclei in human lymphocytes induced by fission neutrons and by low LET radiations. Mutat Res 306:135–141

    Article  CAS  PubMed  Google Scholar 

  59. Hovhannisyan G, Aroutiounian R, Babayan N, Harutyunyan T, Liehr T (2016) Comparative analysis of individual chromosome involvement in micronuclei induced by mitomycin C and bleomycin in human leukocytes. Mol Cytogenet 9:49

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the OTKA Grant T042762 to GB and GINOP-2.3.2-15-2016-00005 Grant to IJ. The work/publication is supported by the GINOP-2.3.2-15-2016-00005 project. The project is co-financed by the European Union under the European Regional Development Fund.

Author information

Authors and Affiliations

  1. Department of Biotechnology and Microbiology, University of Debrecen, 1 Egyetem Square, Debrecen, 4010, Hungary

    Gabor Kiraly, Athene S. Simonyi, Melinda Turani, Gabor Nagy & Gaspar Banfalvi

  2. Department of Dermatology, University of Debrecen, Debrecen, 4032, Hungary

    Istvan Juhasz

Authors
  1. Gabor Kiraly
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Athene S. Simonyi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Melinda Turani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Istvan Juhasz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gabor Nagy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gaspar Banfalvi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gabor Nagy.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kiraly, G., Simonyi, A.S., Turani, M. et al. Micronucleus formation during chromatin condensation and under apoptotic conditions. Apoptosis 22, 207–219 (2017). https://doi.org/10.1007/s10495-016-1316-4

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-016-1316-4

Keywords

Search

Navigation