c-Jun-like immunoreactivity in apoptosis is the result of a crossreaction with neoantigenic sites exposed by caspase-3-mediated proteolysis.
Previous reports in various cells and species have shown that apoptotic cells are specifically and strongly labeled by certain c-Jun/N-terminal antibodies, such as c-Jun/sc45. This kind of immunoreactivity is confined to the cytoplasm. It is not due to c-Jun but appears to be related to c-Jun-like neoepitopes generated during apoptosis. This study was planned to gain further information about c-Jun-like immunostaining during apoptosis and to evaluate these antibodies as possible tools for characterizing cell death. Most of the experiments were performed in chick embryo spinal cord. When the apoptotic c-Jun-like immunoreactivity and caspase-3 immunostaining patterns were compared, we found that both antibodies immunostained the same dying cells in a similar pattern. In contrast to TUNEL staining, which reveals a positive reaction in both apoptotic and necrotic dying cells, active caspase-3 and c-Jun/sc45 antibodies are more selective because they stained only apoptotic cells. When cytosolic extracts from normal tissues were digested in vitro with caspase-3, c-Jun/sc45 immunoreactivity was strongly induced in several proteins, as demonstrated by Western blotting. Similar results were found when normal tissue sections were treated with caspase-3. Our results show that c-Jun/sc45 antibodies react with neoepitopes generated from cell proteins cleaved by activated caspases during apoptosis. We conclude that c-Jun/sc45 antibodies may be useful for detecting apoptosis. They can even be used in archival paraffin-embedded tissue samples.
Ribera
Joan
J
Unitat de Neurobiologia Cellular, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Lleida, Catalunya, Spain.
Ayala
Victoria
V
Esquerda
Josep E
JE
D000818:Animals; D000906:Antibodies / Q000276:immunology; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000276:immunology / Q000378:metabolism*; D002384:Catalysis; D002642:Chick Embryo; D003429:Cross Reactions; D003593:Cytoplasm / Q000378:metabolism; D000939:Epitopes; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D009336:Necrosis; D014066:Palatine Tonsil / Q000378:metabolism; D016612:Paraffin Embedding; D010446:Peptide Fragments / Q000276:immunology; D016755:Proto-Oncogene Proteins c-jun / Q000276:immunology / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism / Q000648:ultrastructure
D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D016755:Proto-Oncogene Proteins c-jun; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases