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Virchows Archiv

, Volume 463, Issue 3, pp 415–425 | Cite as

Characterization of specific p63 and p63-N-terminal isoform antibodies and their application for immunohistochemistry

  • Marta Nekulova
  • Jitka Holcakova
  • Rudolf Nenutil
  • Rembert Stratmann
  • Pavla Bouchalova
  • Petr Müller
  • Lucie Mouková
  • Philip J. CoatesEmail author
  • Borivoj VojtesekEmail author
Original Article

Abstract

The TP63 gene gives rise to protein isoforms with different properties and functions due to the presence (TAp63) or absence (ΔNp63) of an N-terminal p53-like transactivation domain. Immunohistochemistry for p63 has clinical value for certain tumour types, but investigations have been hampered by a lack of well characterized antibodies and the inability to discriminate between these N-terminal isoforms with opposite functional properties. We have extensively characterized a series of monoclonal antibodies to recombinant human TAp63 and two commercial p63 monoclonals by Western blot, immunostaining and phage display epitope mapping. Twenty-eight of 29 (96.6 %) novel monoclonals that recognized all p63 isoforms showed substantial cross-reactivity with p73, as did the commercial antibody, 4A4. One novel clone, PANp63-6.1, showed slight cross-reaction with p73 by Western blotting but not immunohistochemistry and the SFI-6 monoclonal did not cross-react with p73 or p53. Phage display revealed that the PANp63-6.1 epitope has one amino acid difference between p63 and p73, the 4A4 epitope is identical in both, whereas the SFI-6 epitope is unique to p63, accounting for these findings. We also produced and characterized a TAp63-specific clone that does not recognize p53 or p73, and we prepared polyclonal sera specific for ΔNp63 isoforms. Immunohistochemistry demonstrated that TAp63 is expressed in a variety of epithelial and other cell types during development, often in a converse pattern to ΔNp63, but has a very limited expression in normal adult tissues and is independent of ΔNp63. TAp63 was expressed in 17.6 % of squamous cancers of cervix that expressed p63, unlike normal cervix where TAp63 was not expressed. TAp63 did not associate with proliferative index, but cervical carcinomas with TAp63 expression showed improved survival. These data highlight the need for rigorous antibody characterization and indicate that p63-isoform identification may improve the clinical value of p63 expression analyses.

Keywords

p63 TAp63 Development Cervical cancer 

Abbreviations

FFPE

Formalin-fixed paraffin-embedded

SCC

Squamous cell carcinoma

TA

Transactivation domain

Notes

Acknowledgments

We are grateful to the patients for allowing their tissues to be used for research. This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101). BV, RN and PM were supported with IGA MZ CR number NT13794-4/2012 and GACR P301/11/1678 and PJC was supported by the Association for International Cancer Research.

Competing interests

Borivoj Vojtesek and Petr Müller are consultants, and Rudolf Nenutil is a co-owner of Moravian Biotechnology; R. Stratmann is employed at DCS Innovative Diagnostik-Systeme, the companies that developed the novel monoclonal antibodies and polyclonal sera used in this study. The companies did not provide financial support for the studies reported herein and had no influence on the design or execution of the reported studies.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Marta Nekulova
    • 1
  • Jitka Holcakova
    • 1
  • Rudolf Nenutil
    • 1
  • Rembert Stratmann
    • 2
  • Pavla Bouchalova
    • 1
  • Petr Müller
    • 1
  • Lucie Mouková
    • 1
  • Philip J. Coates
    • 3
    Email author
  • Borivoj Vojtesek
    • 1
    Email author
  1. 1.Regional Centre for Applied Molecular OncologyMasaryk Memorial Cancer InstituteBrnoCzech Republic
  2. 2.DCS Innovative Diagnostik-SystemeHamburgGermany
  3. 3.Tayside Tissue BankUniversity of DundeeDundeeUK

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