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

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 (RPE) cells. Since cilia are derived from centrosomes, and aneugens may 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 extranumerarycilia.

Introduction

Primary cilia are microtubule-based organelles. Although scientists have known about the existence of primary cilia since the late 1800s, recent studies have discovered new functions for the organelles. Primary cilia, as well as cilia in general, originate from centrosomes, from which they extend upon maturation. It is now believed that primary cilia function as cellular antennae that receive signals from the environment and transmit them to the cell body to control specific cellular functions.

Aneugenic chemical agents induce DNA damage that translates into imbalanced distribution of chromosomes (aneuploidy) following mitosis (Parry and Sors, 1993). One of the mechanisms that precede aneuploidy is the induction of centrosomal amplification or production of multiple centrosomes that pull the chromosomes in multiple directions resulting in a multipolar cell. Since cilia originate from centrioles, it is expected that cells bearing multiple centrosomes generate multiple cilia.

Previous reports from our laboratory have shown that the antiretroviral drug zidovudine (AZT) induces centrosomal amplification in vivo and in vitro (Olivero et al., 2006; Borojerdi et al., 2009; Olivero et al., 2013). In this report, we used AZT to induce centrosomal amplification and consequent multiple cilia. The protocol could be used to test potential aneugens that induce centrosomal amplification (Figure 1).

Figure 1.

Schematic representation of centrosomes during cell cycle, centrosome amplification in presence of AZT (aneugen): (a) cell showing centrosome with two centrioles, mother centriole (red), daughter centriole (orange), pericentrin (light purple background). (b) cell showing newly formed daughter centrioles. (c) cell exposed to AZT showing centrosomal amplification (more than 2 centrosomes). (d) A normal serum starved (cell cycle arrested) cell showing primary cilium. (e) An AZT exposed cell starved for 48 hours showing two primary cilia.

USE OF A DERIVED CENTROSOME PRIMARY CILIA MODEL TO EVALUATE THE ANEUGEN ZIDOVUDINE (AZT)

Basic Protocol I

Basic protocol I provides the details on detection of abnormal (multiple) ciliogenesis by agents that induce multiple centrosomes and unbalanced chromosomal distribution. Cilia emerging from pre-existing centrosomes will be used as a marker of aneugenesis.

Materials

Cell line: RPE Cells, Human telomerase-immortalized retinal pigment epithelial [hTERT-RPE-1 (RPE1)] cells are one of the most frequently used models to study ciliogenesis. Cells are a gift form Dr. Christopher Westlake (also commercially available from American Type Culture Collection, ATCC Number CRL-4000, Manassas, VA)

Culture medium: Dulbecco's Modified Eagle Medium with Nutrient mixture F12 (1:1) 1X media (DMEM, Gibco by Life Technologies, Cat# 11039-021, Grand Island, NY)

Other reagents

Penicillin, 10,000 units/mL (Gibco by Life Technologies, Cat#15140-122, Grand Island, NY)

Streptomycin, 10,000 µg/mL (Gibco by Life Technologies, Cat#15140-163, Grand Island, NY)

Dulbecco's Phosphate-Buffered Saline without calcium and magnesium(DPBS, Gibco by Life Technologies, Cat# 14190-144, Grand Island, NY)

Trypsin-EDTA, 0.05%, (Gibco by Life Technologies, Cat# 25300-054)

3’-Azido-3’ deoxythymidine (AZT, Sigma, Cat# A2169, St. Louis, MO)

Paraformaldehyde 16% solution, EM grade, (Electron Microscopy Sciences, Cat# 15710-S, Hatfield, PA)

Triton®X-100 (Sigma, Cat# T8787, St. Louis, PA)

Tween 20 (Bio-Rad, Cat#170-6531)

Sucrose (MP Biomedical, Cat# 821713, Illkrich, France)

Albumin Bovine Fraction V (BSA, MP BioMedicals, LLC. Cat# 160069) (2%)

Horse Serum (ATCC, Cat# 30-2040) (20%)

Canted neck polystyrene flasks with PE vented cap, (T75, Cat# 83.1813.002, SARSTEDT, Newton, NC)

Four chamber Polystyrene Vessel Tissue Culture treated glass slides (Falcon, Cat# 354114)

Mouse Anti-Acetylated Tubulin (Sigma, Cat# T7451)

Rabbit Anti-Pericentrin (Covance, Cat# PRB-432C-200)

Goat anti-mouse Alexa Fluor®488 (Life Technologies, Cat#A11001)

Goat anti-Rabbit Alexa Fluor®488 (Life Technologies, Cat#A11012)

VectaSheild® mounting medium with 4',6-Diamidino-2-phenylindole, (DAPI, Vector Laboratories, Cat# H-1200)

Incubator (Heraeus, HERA Cell 240)

Nexcelom bioscience, Cellometer Vision trio 5

Nikon Eclipse E-400

Zeiss LSM 510 scanning laser microscope

Detaching cells from culture flasks, preparation of cell suspensions, and seeding cells in 4-chambered Labtek slides

Cell culture

• 1.Culture Human hTERT immortalized Retinal Pigment Epithelial (RPE) cells at 37°C in a 5% CO₂ atmosphere in DMEM/F12 media supplemented with 10% bovine serum. Culture cells to sub confluence (~80%) in T75 culture flasks.
• 2.Add 2 mL of 0.5% trypsin-EDTA 1X and return the flask to the incubator for 2–3 minutes. Monitor the detachment of the cells from the flask with inverted microscope.
• 3.Neutralize the trypsin by adding 2 mL of media. Collect the cells into a 15 mL Falcon tube. Centrifuge at 1,500 rpm for 5–7 minutes. Aspirate the supernatant leaving small amount of the medium. Rake the falcon tube to dislodge the pellet. Add 5 mL of complete medium and gently pipette the cell mixture up and down to ensure a single cell suspension.
• 4.Load a volume of 20 µL of cell suspension on to a cellometer cell counting chamber. Enumerate the cell using Nexcelom Bioscience Cellometer Vision Trio 5 or use any other procedure to count the cells such as coulter counter or a hemacytometer.
• 5.
  Dilute cells with complete media using the example shown below:

  • a)
    For example, if you have a cell count of 3.98 × 10⁵ cells /mL, and you need 10 mL of cells at 7,000 cell/mL, add 175 µL of cells to 9.825 mL media. If you want 3,500 cells/mL and you need 10 mL of cells, add 87.5 µL of cells to 9.912 mL of complete media.
• 6.Add 1 mL of the above diluted cell mixture (3500 cells/mL) in the control chamber (chamber 1), and 1 mL of the cell suspension (7000 cells/mL) in the treatment chambers (chambers 2, 3, and 4 of the slide).
• 7.Place the slides in an incubator (maintained at 37°C with 5% CO₂ and 95% humidity) for the cells to attach and replicate.

Addition of the Drug to RPE Cells

• 8.At 24 hours (approximately after1 cell cycle) of seeding, remove the media. Refeed the cells in chambers 1 and 2 with DMEM/F12 containing 10% FBS. Refeed the cells in the treatment chambers, 3 and 4 with 1 mL media containing AZT at a final concentration of 20 µM.
• 9.Return the slides to incubator for 72 hours.

Senescence (starvation)

• 10.Seventy two hours post treatment, aspirate off the medium from both control and experimental wells and wash with 1X PBS. Refeed the control chamber (chamber 1) with DMEM/F12 containing 10% FBS to serve as control for cell cycle (non senescencing cells). Refeed chambers 2, 3, and 4 with serum free DMEM/F12 media (no FBS, starvation to induce senescence (Vorobjev and Chentsov Yu, 1982). Return the slides to incubator for additional 48 hours.

Fixation and permeabilization

• 11.Forty eight hours post starvation, wash cells three times for 5 minutes (3 × 5) each, with PBS containing 0.1% Tween-20. Add 1mL of fixative containing 4% paraformaldehyde containing 2% sucrose to each well and incubate for 10 minutes at room temperature (Brock et al., 1999).
• 12.
  Following fixation, aspirate off the fixative and add 1 mL of permeabilizing solution containing 0.1% Triton X-100 in PBS and incubate for 1 minute at room temperature (Goldenthal et al., 1985).

  At this point, slides can be processed immediately for staining or can be stored at 2–8°C. For future staining, wash the slide chambers 3 × 5 times with PBS containing 0.1% Tween-20. Add 1 mL of PBS to each chamber and store at 2–8°C.

• 13.For immediate processing, wash cells 3 × 5 minutes with PBS containing 0.1% Tween-20 and proceed for immunostaining.

Immunofluorescence Staining of Centrosomes and Primary Cilia

• 14.Add 1 mL of blocking buffer containing 2% BSA, 20% horse serum and 0.1% Tween-20 in PBS to each chamber, and incubate for 1 hour at room temperature.
• 15.Wash the cells 3 × 5 minutes each with wash buffer.

Incubation with Primary Antibodies

• 16.To stain cilia, dilute mouse anti-acetylated tubulin (primary antibody I) 1:10,000 in dilution buffer containing 2% BSA, 0.2% horse serum with 0.1% Tween-20. Add 1 mL of the diluted antibody to each chamber. Allow the slides sit at room temperature on a rocking platform for 30–60 minutes and then transfer the slides to 2–8°C and incubate overnight (Graser et al., 2007).
• 17.Next day, wash the cells 3 × 5 minutes each with PBS containing 0.1% Tween-20.
• 18.To stain centrosomes, dilute rabbit anti-pericentrin antibody (primary antibody II) 1:500 dilution with dilution buffer containing 2% BSA, 0.2% horse serum with 0.1% Tween-20 in PBS. Add 1mL of the diluted antibody to each chamber. Incubate the slide for 2 hours at room temperature (Kawamura et al., 2006).
• 19.Following the incubations with primary antibodies, wash cells 3 × 5 minutes each with wash buffer.

Incubation with Secondary Antibodies

Staing of Cilia

• 20.Dilute anti mouse Alexa 488 (secondary antibody I) in goat 1:666 in dilution buffer, containing 2% BSA, 0.2% horse serum with 0.1% tween-20 in PBS. Add 1 mL of the diluted antibody to each chamber and incubate at room temperature for 1 hour.
• 21.Wash cells 3 × 5 minutes each with wash buffer: PBS with 0.1% Tween-20.

  Staining of Centrosomes

• 22.
  Dilute anti rabbit Alexa 594 in goat 1:1000 in dilution buffer containing 2% BSA, 0.2% horse serum with 0.1% tween-20 in PBS.

  Carry out primary or secondary antibody incubations one at a time for optimal staining.

• 23.Wash cells 3 × 5 minutes each with wash buffer containing PBS with 0.1% Tween-20. Separate the slide from the chambers using removal key prongs and key channels and immerse slides in deionized water for 2 minutes. Air dry the slides protected from light.
• 24.Apply a drop of VectaSheild® mounting medium containing 4',6-Diamidino-2-phenylindole (DAPI) and spread to cover the entire area using a pipette tip gently, without applying pressure on the surface of the slide. After five minutes, mount slides using 24 × 50 mm coverslips. Protect slides from light until visualized under the microscope.

Immunofluorescence Imaging via Confocal Microscopy (Figure 2)

Figure 2.

(A) Normal RPE cell showing primary cilium and centrosome. Cells are stained with anti-acetylated tubulin to stain cilia (green, arrow), anti-pericentrin to stain centrosomes (red, arrow head), and DAPI to stain nuclei (blue). (B) RPE cells treated with 20µM AZT for 72 consecutive hours showing more than one cilium per cell. Cells are stained with anti-acetylated tubulin to stain cilia (green, arrow), anti-pericentrin to stain centrosomes (red, arrow head), and DAPI to stain nuclei (blue).

• 25.Visualize the cells using a conventional epi-illumination fluorescence microscope; Nikon Eclipse E-400 (Nikon, Inc. Melville, NY) fitted with a Plan Apo 100X objective with a 1.40 numerical aperture. Photograph the cells using a Zeiss Axiovert 100M microscope equipped with a Zeiss Plan Apochromat 100X/1.4 oil Dichromatic objective (Coling and Kachar, 2001).
• 26.Cells containing more than two centrosomes are considered positive for centrosome amplification and cells containing more than one cilium are considered positive for abnormal ciliogenesis.

Reagents and Solutions

Culture Media

• 27.Culture cells in DMEM/F12 with no phenol red supplemented with 10% fetal bovine serum and 80 Units/mL of Penicillin and 80 µg/mL of Streptomycin. Use complete media was used within one month of preparation.

Preparation of AZT Solution

• 28.
  Prepare a stock solution, dissolve AZT (Sigma, A2169) in PBS as follows:

  • a)
    Add 10 mL of sterile PBS to an AZT vial 100 mg. This gives an expected concentration of 10 mg/mL or 0.037 M (37 mM). Allow the drug to dissolve overnight at 37°C. Make a 1:400 dilution of the stock solution in a separate vial. Read the absorbance of this solution at 230–400 nm, scan. Expect a peak at 266.5 nm. Read three times and take an average.
  • b)
    Molar coefficient absorption of AZT is 11.650
    Molecular weight is AZT = 267.2
    Therefore, a solution of 267.2 µg/mL makes a 1M solution.
    Calculate the concentration of your solution using the equation
    267.2 µg/mL/11.650 = x µg/mL in AZT vial/absorbance reading
  • c)
    Multiply your concentration by dilution factor, 400.
    Divide the final concentration by the AZT molecular weight to obtain the final molarity of the solution in the vial. Store the stock AZT solution at 2–8°C and use within 6 months from the date of preparation.
  • d)
    Dilute the stock solution in complete media, using the formula V1N₁ = V2N₂. For example, dilute 30 µL of the above stock solution with 9970 µL of complete media to obtain a 20 µM solution.

Fixative solution

• 29.Prepare a solution of 4% paraformaldehyde with 2% sucrose in PBS fresh on the day of use, and use immediately after preparation.

Permeabilizing solution

• 30.Prepare a solution of 0.1% Triton X-100 in PBS fresh and use immediately

Washing Solution/Buffer

• 31.Prepare Phosphate buffered saline with 0.1% Tween-20. Store at room temperature until six months from the date of preparation.

Blocking Buffer

• 32.Prepare 2% BSA with 20% horse serum in PBS containing 0.1% Tween-20 on the day of use.

Dilution Buffer

• 33.Prepare two percent BSA with 2% horse serum in PBS containing 0.1% Tween-20 on the day of use.

Commentary

Ciliopathies and implications

The primary cilium, is a solitary organelle that emanates from the cell surface of most mammalian cell types during growth arrest. Increasing evidence suggests that primary cilia are key coordinators of signaling pathways during development and in tissue homeostasis, and when defective, are a major cause of human diseases and developmental disorders commonly referred to as ciliopathies. Some of the Ciliopathies are Autosomal dominant polycystic kidney disease, Retinal dystrophy, Hydrocephalus, Bardet-Biedl syndrome, Meckel syndrome, Joubert syndrome.

During mitosis, two centrosomes form spindle poles and direct the formation of bipolar mitotic spindles, which is an essential event for accurate chromosome segregation into daughter cells. Drugs that are able to induce changes in the number of chromosomes by disturbing the balance distribution of genetic material in mitosis are called aneugens. There are other mechanisms of induction of aneuploidy, for example, colchicine inhibits the formation of mitiotic spindle, whereas Taxol inhibits mitotic spindle to disintegrate. This protocol identifies a subset of aneugens.

Cilia arise from the mother centriole of a quiescent cell. Then it is expected that in a cell with extranumerary centrosomes, cilia will be also present in excess. Detection of multiple cilia could be used as a surrogate marker to identify drugs suspected to have aneugenic capacity.

Troubleshooting

Background color intensity is common for any immunostaining. Double checking any reagents for shelf-life is the key. Maintaining the incubation timings, especially that of blocking translates in reproducible results.

Extreme care has to be paid to cell confluence, and to prevent contact inhibition so that cells are continually cycling. This is the reason the control well is seeded with half the number of cells.

Anticipated Results

It has been shown in the literature that AZT induces centrosomal amplification (Borojerdi et al., 2009). Since primary cilia are present in all mammalian cells, and arise from centrosomes, the hypothesis is that multiple cilia will be visualized as a result of multiple centrosomes. We have been able to show that centrosomal amplification induces multiple cilia.

Time Considerations (Figure 3)

Figure 3.

Primary cilium development time line in Retinal Pigment Epithelial Cells exposed to 20 µM AZT. Cells are seeded and allowed to go through approximately one doubling time (24 hours). Cells exposed to 20 µM AZT for 72 consecutive hours. (96 hours). Cells were withdrawn from treatment, washed with PBS and cultured without serum (starvation) for 48 hours to induce primary ciliogenesis. (144 hours). Cells were immunostained to visualize primary cilia and centrosomes.

The optimal time requirement for this experiment from start to end is 8 days. A 24 hours of culture after seeding, 72 hours of drug exposure, 48 hours of starvation and 48 hours to complete immunostaining. Authors found it optimal to incubate overnight with acetylated tubulin (primary antibody 1 to stain cilia), and other incubations with one antibody at a time. The protocol calls for a 48 hour starvation period. Typically, cells produce cilia under these conditions, but will not apoptose. Rather they become very active in transmitting messages from the environment to the interior of the cell. This is achieved by re-populating the surface of the cilia with numerous receptors.