Authors: <div class="autor_fcen" id="8534">Thomas, M.G.</div>; <div class="autor_fcen" id="5196">Luchelli, L.</div>; Pascual, M.; Gottifredi, V.; <div class="autor_fcen" id="982">Boccaccio, G.L.</div>
Publication Date: 2012.
The p53 tumor suppressor protein is an important regulator of cell proliferation and apoptosis. p53 can be found in the nucleus and in the cytosol, and the subcellular location is key to control p53 function. In this work, we found that a widely used monoclonal antibody against p53, termed Pab 1801 (Pan antibody 1801) yields a remarkable punctate signal in the cytoplasm of several cell lines of human origin. Surprisingly, these puncta were also observed in two independent p53-null cell lines. Moreover, the foci stained with the Pab 1801 were present in rat cells, although Pab 1801 recognizes an epitope that is not conserved in rodent p53. In contrast, the Pab 1801 nuclear staining corresponded to genuine p53, as it was upregulated by p53-stimulating drugs and absent in p53-null cells. We identified the Pab 1801 cytoplasmic puncta as P Bodies (PBs), which are involved in mRNA regulation. We found that, in several cell lines, including U2OS, WI38, SK-N-SH and HCT116, the Pab 1801 puncta strictly colocalize with PBs identified with specific antibodies against the PB components Hedls, Dcp1a, Xrn1 or Rck/p54. PBs are highly dynamic and accordingly, the Pab 1801 puncta vanished when PBs dissolved upon treatment with cycloheximide, a drug that causes polysome stabilization and PB disruption. In addition, the knockdown of specific PB components that affect PB integrity simultaneously caused PB dissolution and the disappearance of the Pab 1801 puncta. Our results reveal a strong cross-reactivity of the Pab 1801 with unknown PB component(s). This was observed upon distinct immunostaining protocols, thus meaning a major limitation on the use of this antibody for p53 imaging in the cytoplasm of most cell types of human or rodent origin. © 2012 Thomas et al.
Author affiliation: Thomas, M.G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Author affiliation: Luchelli, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Author affiliation: Boccaccio, G.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
Keywords: cell marker; cycloheximide; decapping enzyme 1a; decapping enzyme 1b; decapping enzyme 2; epitope; exoribonuclease; exoribonuclease 1; messenger RNA; monoclonal antibody; pantropic antibody 1801; protein 4ET; protein Hedls; protein p53; protein p54; small interfering RNA; unclassified drug; epitope; protein p53; TP53 protein, human; animal cell; animal tissue; article; cell component; cell disruption; cell line; cell stimulation; cell strain HCT116; cellular distribution; concentration (parameters); controlled study; cross reaction; dissolution; Drosophila; embryo; fetus; gene control; gene silencing; genetic transfection; human; human cell; image analysis; immunohistochemistry; intracellular signaling; nonhuman; polysome; processing body; protein analysis; protein localization; rat; upregulation; animal; antibody specificity; chemistry; cytoplasm; immunology; metabolism; Sprague Dawley rat; tumor cell line; Rattus; Rodentia; Animals; Antibodies, Monoclonal, Murine-Derived; Antibody Specificity; Cell Line, Tumor; Cytoplasm; Epitopes; Humans; Immunohistochemistry; Rats; Rats, Sprague-Dawley; Tumor Suppressor Protein p53.
Repository: Biblioteca Digital (UBA-FCEN). Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales