Using Organotypic Epithelial Tissue Culture to Study the Human Papillomavirus Life Cycle

Denis Lee1, Kathryn Norby2, Mitchell Hayes1, Ya‐Fang Chiu1, Bill Sugden1, Paul F. Lambert1

1 McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, 2 Flow Cytometry Laboratory, Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.8
DOI:  10.1002/cpmc.4
Online Posting Date:  May, 2016
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Abstract

Human papillomaviruses (HPVs) are small double‐stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent “dead ends” for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus. In order to study the full viral life cycle, from the establishment to maintenance to productive stages, our lab makes use of the organotypic epithelial tissue culture system. This system allows us to mimic the three‐dimensional structure of epithelia whose differentiation is tightly linked to the completion of the HPV viral life cycle. In this chapter we describe how various aspects of the HPV life cycle are monitored in raft cultures making use of an immortalized keratinocyte cell line. © 2016 by John Wiley & Sons, Inc.

Keywords: keratinocyte; life cycle; organotypic epithelial tissue culture; papillomavirus

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generating the Organotypic Epithelial Raft Cultures
  • Basic Protocol 2: Analysis of Unscheduled DNA Synthesis by BrdU Staining Using Immunohistochemical Detection
  • Alternate Protocol 1: Analysis of Unscheduled DNA Synthesis by BrdU Staining Using Immunofluorescent Detection
  • Basic Protocol 3: Analysis of Viral DNA Amplification By Fluorescence In Situ Hybridization (FISH)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generating the Organotypic Epithelial Raft Cultures

  Materials
  • Early passage human foreskin fibroblasts for embedding in dermal equivalent
  • Fibroblast medium (see recipe)
  • 10× F12 medium (Thermo Fisher Scientific, cat. no. 21700‐075)
  • 10 N NaOH
  • Penicillin‐streptomycin (pen‐strep; Thermo Fisher Scientific, cat. no. 15140‐122)
  • Fetal bovine serum (FBS; Thermo Fisher Scientific, cat. no. 16000‐044)
  • Rat tail collagen type 1 (Millipore, cat. no. 08‐115)
  • Keratinocytes harboring HPV genome of interest
  • Keratinocyte plating medium (see recipe)
  • Cornification medium 1 (see recipe)
  • 1,2‐dioctanoyl‐sn‐glycerol (C8:O; Cayman Chemicals, cat. no. 62225)
  • Cornification medium 2 (if needed; see recipe)
  • 5‐bromo‐2′‐deoxyuridine (BrdU; Sigma, cat. no. B5002)
  • Bacto‐agar
  • 10% buffered formalin
  • Transwell inserts (Costar, cat. no. 3450)
  • Deep‐well trays (Corning, cat. no. 355467)
  • 50‐ml conical tubes
  • Cotton pads (VWR, cat. no. 21427‐393), cut into 1‐inch squares and autoclaved
  • Tissue‐Tek cassettes (Sakura, cat. no. 4118‐01)
  • Additional reagents and equipment for trypsinization and cell counting (Phelan, ) and paraffin embedding and sectioning of tissues (see unit 14.4; Venkatesh et al., ; Zeller, )

Basic Protocol 2: Analysis of Unscheduled DNA Synthesis by BrdU Staining Using Immunohistochemical Detection

  Materials
  • Slides mounted with formalin‐fixed and paraffin‐embedded sections of raft cultures (see protocol 1)
  • Xylenes
  • 70%, 80%, 95%, and 100% ethanol
  • 1× PBS ( appendix 2A)
  • 3% H 2O 2 (see recipe)
  • 10 mM citrate buffer, pH 6.0 (see recipe)
  • TE, pH 9.0 ( appendix 2A)
  • Proteinase K
  • Pepsin
  • 2 N HCl
  • Blocking serum (same host species as secondary antibody), included in VECTASTAIN kit
  • anti‐BrdU antibody (Millipore, cat. no. NA‐61)
  • VECTASTAIN Elite ABC Kit, Universal (Vector Labs, cat. no. PK‐6200)
  • DAB detection kit (Vector Labs, cat. no. SK‐4100)
  • Vector Hematoxylin QS (Vector Labs, cat. no. H‐3404)
  • Cytoseal XYL (Richard‐Allen Scientific, cat. no. 8312‐4)
  • Coplin jar or staining dish
  • Hydrophobic marker or PAP pen
  • Humidified chamber
  • Coverslips

Alternate Protocol 1: Analysis of Unscheduled DNA Synthesis by BrdU Staining Using Immunofluorescent Detection

  Materials (also see protocol 2)
  • Anti‐mouse AlexaFluor 488‐conjugated secondary antibody (Thermo Fisher Scientific, cat. no. A11001)
  • 2000× Hoescht dye
  • Anti‐fade mounting medium

Basic Protocol 3: Analysis of Viral DNA Amplification By Fluorescence In Situ Hybridization (FISH)

  Materials
  • Xylenes
  • 70%, 80%, 95% and 100% ethanol
  • 10 mM citrate buffer, pH 6.0 (see recipe)
  • 10× NT buffer (see recipe)
  • 0.1 M β‐mercaptoethanol (see recipe)
  • 10× nucleotide stock (see recipe)
  • E. coli DNA polymerase I (NEB, cat. no. M0209S)
  • DNase I stock solution (see recipe)
  • 0.4 mM dTTP (see recipe)
  • 1 mM Biotin‐16‐dUTP or DIG‐11‐dUTP (Roche, cat. no. 11093070910 or cat. no. 11573152910)
  • HPV probe template DNA
  • Human Cot‐1 DNA (Life Technologies, cat. no. 15279‐011)
  • Salmon sperm DNA (Life Technologies, cat. no. AM9680)
  • 3 M NaOAc
  • Pre‐hybridization solution (see recipe)
  • Denaturation solution (see recipe)
  • CEP hybridization buffer (Abbott Molecular, cat. no. 06L44‐01)
  • FISH detection solution (see recipe)
  • VECTAShield with DAPI (Vector Labs, cat. no. H‐1200)
  • Coplin jars
  • Microwave‐safe staining dish
  • PAP or hydrophobic pen
  • Humidified chamber
  • Coverslips, 22‐mm × 40‐mm (Fisher Scientific, cat. no. 12‐543A)
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Figures

Videos

Literature Cited

Literature Cited
  Allen‐Hoffmann, B.L., Schlosser, S.J., Ivarie, C.A., Sattler, C.A., Meisner, L.F., and O'Connor, S.L. 2000. Normal growth and differentiation in a spontaneously immortalized near‐diploid human keratinocyte cell line, NIKS. J. Invest. Dermatol. 114:444‐455. doi: 10.1046/j.1523‐1747.2000.00869.x.
  Brake, T., Connor, J.P., Petereit, D.G., and Lambert, P.F. 2003. Comparative analysis of cervical cancer in women and in a human papillomavirus‐transgenic mouse model: Identification of minichromosome maintenance protein 7 as an informative biomarker for human cervical cancer. Cancer Res. 63:8173‐8180.]
  Burnett, L. C., Lunn, G., and Coico, R. 2009. Biosafety: Guidelines for working with pathogenic and infectious microorganisms. Curr. Protoc. Microbiol. 13:1A.1.1–1A.1.14.
  Collins, A.S., Nakahara, T., Do, A., and Lambert, P.F. 2005. Interactions with pocket proteins contribute to the role of human papillomavirus type 16 E7 in the papillomavirus life cycle. J. Virol. 79:14769‐14780. doi: 10.1128/JVI.79.23.14769‐14780.2005.
  Flores, E.R., Allen‐Hoffmann, B.L., Lee, D., Sattler, C.A., and Lambert, P.F. 1999. Establishment of the human papillomavirus type 16 (HPV‐16) life cycle in an immortalized human foreskin keratinocyte cell line. Virology 262:344‐354. doi:10.1006/viro.1999.9868.
  Flores, E.R., Allen‐Hoffmann, B.L., Lee, D., and Lambert, P.F. 2000. The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle. J. Virol. 74:6622‐6631. doi: 10.1128/JVI.74.14.6622‐6631.2000.
  Genther Williams, S.M., Disbrow, G.L., Schlegel, R., Lee, D., Threadgill, D.W., and Lambert, P.F. 2005. Requirement of epidermal growth factor receptor for hyperplasia induced by E5, a high‐risk human papillomavirus oncogene. Cancer Res. 65:6534‐6542. doi:10.1158/0008‐5472.CAN‐05‐0083.
  Lorenz, L.D., Rivera Cardona, J., and Lambert, P.F. 2013. Inactivation of p53 rescues the maintenance of high risk HPV DNA genomes deficient in expression of E6. PLoS Pathog. 9:e1003717. doi: 10.1371/journal.ppat.1003717.
  Nakahara, T., Peh, W.L., Doorbar, J., Lee, D., and Lambert, P.F. 2005. Human papillomavirus type 16 E1circumflexE4 contributes to multiple facets of the papillomavirus life cycle. J. Virol. 79:13150‐13165. doi:10.1128/JVI.79.20.13150‐13165.2005.
  Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
  Venkatesh, A., Ma, S., Langellotto, F., Gao, G. and Punzo, C. 2013. Retinal gene delivery by rAAV and DNA electroporation. Curr. Protoc. Microbiol. 28:14D.4.1–14D.4.32.
  Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. Curr. Protoc. Mol. Biol. 7:14.1.1‐14.1.8.
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