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PubMed

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

Virchows Archiv. 2013 Sep ; 463 (3) : 415-25. [epub] 20130726

Virchows Arch
ISSN 1432-2307 | 0945-6317
Source

Language English Country Germany Media print-electronic

Document type Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't

Persistent link https://www.medvik.cz/link/pmid23887585

PubMed 23887585
DOI 10.1007/s00428-013-1459-4
Knihovny.cz E-resources

MeSH
Adenocarcinoma diagnosis immunology MeSH
DNA-Binding Proteins immunology MeSH
Immunohistochemistry methods standards MeSH
Nuclear Proteins immunology MeSH
Rats MeSH
Cells, Cultured MeSH
Humans MeSH
Membrane Proteins chemistry immunology MeSH
Antibodies, Monoclonal * immunology MeSH
Mice MeSH
Cell Line, Tumor MeSH
Tumor Suppressor Proteins immunology MeSH
Tumor Suppressor Protein p53 immunology MeSH
Uterine Cervical Neoplasms diagnosis immunology MeSH
Lung Neoplasms diagnosis immunology MeSH
Breast Neoplasms diagnosis immunology MeSH
Carcinoma, Non-Small-Cell Lung diagnosis immunology MeSH
Protein Isoforms MeSH
Tumor Protein p73 MeSH
Antibody Specificity immunology MeSH
Carcinoma, Squamous Cell diagnosis immunology MeSH
Cross Reactions MeSH
Animals MeSH
Check Tag
Rats MeSH
Humans MeSH
Mice MeSH
Female MeSH
Animals MeSH
Publication type
Journal Article MeSH
Evaluation Study MeSH
Research Support, Non-U.S. Gov't MeSH
Names of Substances
CKAP4 protein, human MeSH Browser
DNA-Binding Proteins MeSH
Nuclear Proteins MeSH
Membrane Proteins MeSH
Antibodies, Monoclonal * MeSH
Tumor Suppressor Proteins MeSH
Tumor Suppressor Protein p53 MeSH
Protein Isoforms MeSH
Tumor Protein p73 MeSH
TP73 protein, human MeSH Browser
Trp73 protein, mouse MeSH Browser

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.

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Zluty kopec 7 656 53 Brno Czech Republic

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