Detection of Mitotic Figures and Components of the Mitotic Machinery

Jane Bayani1, Jeremy A. Squire1

1 Princess Margaret Hospital and The Ontario Cancer Institute, Toronto, Ontario
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.8
DOI:  10.1002/0471143030.cb2208s25
Online Posting Date:  January, 2005
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Abstract

Mitotic figures have provided important information on the mechanisms behind observed chromosomal aberrations. Variations in cell culturing and fixation methods enhance the visualization of mitotic figures. Furthermore, immunohistochemistry using a host of antibodies against protein structures involved in the cell cycle, as well as against those components involved in anchoring chromosomal structures, can reveal changes in chromosomal segregation.

Keywords: mitotic figures; mitotic machinery; immunohistochemistry; chromosomal segregation

     
 
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Table of Contents

  • Basic Protocol:
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol:

 Materials
  • Cells growing on coated glass slides, coverslips, or chambered tissue culture slides (unit 22.2), 75% confluent
  • Phosphate-buffered saline (PBS; appendix 2A), room temperature and cold
  • 4% (w/v) paraformaldehyde in PBS (see recipe), cold
  • 70% and 100% ethanol, cold
  • Permeabilizing solution (see recipe)
  • Blocking solution (see recipe)
  • Primary antibody: mouse anti -tubulin antibody (Sigma) at working concentration recommended by manufacturer
  • 0.1% (v/v) NP-40 in PBS (prepare fresh)
  • Secondary antibody: rhodamine-conjugated anti-mouse IgG antibody (Sigma) at working concentration recommended by manufacturer
  • DAPI in antifade (see recipe in unit 22.4 or purchase from Vector Laboratories)
  • Nail polish
  • Petri dishes
  • Coplin jars
  • 37°C dry incubator or oven
  • Hybridization box: e.g., black videocassette box containing slightly dampened moist paper towel or gauze
  • Fluorescence microscope and appropriate filters (unit 4.2 and appendix 1E)
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Figures

  •  FigureFigure 22.8.1 Examples of aberrant mitotic figures. Shown are examples of mitotic cells stained with -tubulin to detect centrosomes (red) and the spindle apparatus. Counterstaining with DAPI (blue) was used to visualize DNA. (A) and (B) show cells undergoing mitosis, where duplicated chromosomes are being pulled from the metaphase plate to their respective poles. (C) In this image, the cells were stained with antibodies against centromere/kinetichore proteins and illustrate the formation of an anaphase bridge. Abnormal mitotic figures are shown in (D), (E) and (F), where centrosome amplification has occurred, resulting in multipolar mitotic figures (D and E) and functional centrosomes. In (F), centrosome amplification has occurred, but the extra centrosome is apparently nonfunctional, and the cell maintains normal division. Amplification of centrosomes are also seen in interphase cells. (G) Mouse metastatic osteosarcoma cells derived from the lung show amplified centrosomes. (H) Amplified centrosomes are present in a clear cell ovarian cancer cell. (I) A multi-nucleated ovarian cancer cell shows amplified centrosomes.
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Literature Cited

Literature Cited
    Al-Romaih, K., Bayani, J., Vorobyova, J., Karaskova, J., Park, P.C., Zielenska, M., and Squire, J.A. 2003. Chromosomal instability in osteosarcoma and its association with centrosome abnormalities. Cancer Genet. Cytogenet. 144:91-99.
    Andreassen, P.R., Lacroix, F.B., Lohez, O.D., and Margolis, R.L. 2001. Neither p21WAF1 nor 14-3-3 prevents G2 progression to mitotic catastrophe in human colon carcinoma cells after DNA damage, but p21WAF1 induces stable G1 arrest in resulting tetraploid cells. Cancer Res. 61:7660-7668.
    Cahill, D.P., Lengauer, C., Yu, J., Riggins, G.J., Willson, J.K., Markowitz, S.D., Kinzler, K.W., and Vogelstein, B. 1998. Mutations of mitotic checkpoint genes in human cancers. Nature 392:300-303.
    Difilippantonio, M.J., Zhu, J., Chen, H.T., Meffre, E., Nussenzweig, M.C., Max, E.E., Ried, T., and Nussenzweig, A. 2000. DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature 404:510-514.
    Doxsey, S.J. 2001. Centrosomes as command centres for cellular control. Nat. Cell Biol. 3:E105-E108.
    Ghadimi, B.M., Sackett, D.L., Difilippantonio, M.J., Schrock, E., Neumann, T., Jauho, A., Auer, G., and Ried, T. 2000. Centrosome amplification and instability occurs exclusively in aneuploid, but not in diploid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations. Genes Chromosomes Cancer 27:183-190.
    Nigg, E.A. 2002. Centrosome aberrations: Cause or consequence of cancer progression? Nat. Rev. Cancer. 2:815-825.
    Pihan, G.A., Wallace, J., Zhou, Y., and Doxsey, S.J. 2003. Centrosome abnormalities and chromosome instability occur together in pre-invasive carcinomas. Cancer Res. 63:1398-1404.
    Skoufias, D.A., Andreassen, P.R., Lacroix, F.B., Wilson, L., and Margolis, R.L. 2001. Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints. Proc. Natl. Acad. Sci. U.S.A. 98:4492-4497.
 Internet Resources
    http://www.abcam.com/

Abcam Web site.

    http://bethyl.com/

Bethyl Laboratories Web site.

    http://www.ihcworld.com/

IHC World Web site.

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