Use of Ciliogenesis to Detect Aneugens: The Role of Primary Cilia

Kathyayini V. Divi1, Yvona Ward2, Miriam C. Poirier1, Ofelia A. Olivero1

1 LCBG, CCR, NIH, Bethesda, Maryland, 2 NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.13
DOI:  10.1002/0471140856.tx0313s66
Online Posting Date:  November, 2015
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Abstract

Primary cilia arise from the centrosomes of quiescent or post‐mitotic cells, and serve as sensory organelles that communicate mechanical and chemical stimuli from the environment to the interior of the cell. Cilium formation may, therefore, become a useful end point signaling exposure to genotoxins or aneugens. Here we have used the aneugen, zidovudine (AZT), an antiretroviral drug that induces DNA replication arrest and centrosomal amplification (>2 centrosomes per quiescent cell), to evaluate cilia formation in retinal epithelial (pigmented) cells. Since cilia are derived from centrosomes, and aneugens can induce centrosomal amplification, the production of multiple cilia arising from multiple centrosomes may reveal the aneugenic nature of the agents. Cells were exposed to AZT to induce centrosomal amplification, cultured without serum to allow the centrioles to develop cilia, and immunostained to visualize cilia and centrosomes. Nuclear DNA was stained with DAPI. Preliminary observations suggest that cells with multiple centrosomes are able to generate extra cilia. © 2015 by John Wiley & Sons, Inc.

Keywords: primary cilia; AZT; centrosomal amplification; aneugens

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Use of a Derived Centrosome Primary Cilia Model to Evaluate the Aneugen Zidovudine (AZT)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Use of a Derived Centrosome Primary Cilia Model to Evaluate the Aneugen Zidovudine (AZT)

  Materials
  • Retinal pigmented epithelial (RPE) cells immortalized with hTERT (hTERT RPE‐1; American Type Culture Collection, cat. no. CRL‐4000), frequently used to study ciliogenesis
  • Complete culture medium (see recipe)
  • 0.05% trypsin‐EDTA, (Life Technologies, cat. no. 25300‐054)
  • AZT stock solution (see recipe)
  • Phosphate‐buffered saline (PBS) without calcium and magnesium ( appendix 2A, or Life Technologies, cat. no. 14190‐144)
  • Serum‐free culture medium (see recipe)
  • Wash buffer (see recipe)
  • Fixative solution (see recipe)
  • Permeabilizing solution (see recipe)
  • Blocking buffer (see recipe)
  • Mouse anti‐acetylated tubulin (Sigma, cat. no. T7451)
  • Rabbit anti‐pericentrin (BioLegend, cat. no. 923701)
  • Goat anti‐mouse Alexa Fluor 488 (ThermoFisher Scientific, cat. no. A‐11001)
  • Goat anti‐rabbit Alexa Fluor 594 (ThermoFisher Scientific, cat. no. A‐11012)
  • VectaSheild mounting medium (Vector Laboratories, cat. no. H‐1200), antifade mounting medium with 4′,6‐diamidino‐2‐phenylindole (DAPI)
  • T75 culture flasks (Sarstedt, cat. no. 83.3911.002), polystyrene flask with canted neck and vented polyethylene cap
  • Incubator (e.g., HERAcell 240, Thermo Scientific Heraeus)
  • 15‐ml conical tubes
  • Cellometer Vision trio 5 with counting chamber (Nexcelom Bioscience)
  • Four‐chamber culture slides (Corning, cat. no. 354114)
  • 24 × 50‐mm coverslips
  • Nikon Eclipse E‐400
  • Zeiss LSM 510 laser scanning confocal microscope
  • Additional reagents and equipment for mammalian cell culture techniques ( appendix 3A; Phelan, ) and for fluorescence staining and imaging (Smith, )
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Figures

Videos

Literature Cited

Literature Cited
  Borojerdi, J.P., Ming, J., Cooch, C., Ward, Y., Semino‐Mora, C., Yu, M., Braun, H.M., Taylor, B.J., Poirier, M.C., and Olivero, O.A. 2009. Centrosomal amplification and aneuploidy induced by the antiretroviral drug AZT in hamster and human cells. Mutat. Res. 665:67‐74. doi: 10.1016/j.mrfmmm.2009.03.004.
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  Olivero, O.A., Borojerdi, J.P., Semino‐Mora, C., Ward, Y., and Poirier, M.C. 2006. Genomic instability induced by AZT in cultured normal human mammary epithelial cells (NHMECs) generates aneuploidy. Retrovirology 3(Suppl 1):48. doi: 10.1186/1742‐4690‐3‐S1‐P48.
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  Parry, J.M. and Sors, A. 1993. The detection and assessment of the aneugenic potential of environmental chemicals: The European Community Aneuploidy Project. Mutat. Res. 287:3‐15. doi: 10.1016/0027‐5107(93)90140‐B.
  Phelan, M.C. 2007. Techniques for mammalian cell tissue culture. Curr. Protoc. Toxicol. 33:A.3B.1–A.3B.18. doi: 10.1002/0471140856.txa03bs33.
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