Identification of an apoptotic cleavage product of BARD1 as an autoantigen: a potential factor in the antitumoral response mediated by apoptotic bodies.
We have shown previously that rats can be cured from induced peritoneal colon carcinomatosis by injections of apoptotic bodies derived from tumor cells and interleukin 2. This curative treatment generated a tumor-specific cytotoxic T-cell response associated with a humoral response. Autoantibodies from sera of cured rats strongly recognized a Mr 67,000 protein from apoptotic bodies and weakly reacted with a protein of Mr approximately 97,000 in PROb parental cells. We now show that these autoantibodies are directed against BARD1, originally identified as a protein interacting with the product of the breast cancer gene 1, BRCA1. We demonstrate that the Mr 67,000 antigen is a cleaved form of BARD1 present in apoptotic bodies derived from rat and human colon and mammary carcinoma cell lines. Moreover, we show that the cleavage site of BARD1 is located NH2 terminally but downstream of the RING domain essential for BARD1 and BRCA1 protein interaction. In vitro studies using [35S]methionine-labeled human BARD1 and apoptotic cellular extracts derived from SW48 carcinoma cells indicate that BARD1 proteolysis occurs at an early stage of apoptosis and in a cell cycle-dependent manner. This hydrolysis is inhibited by EGTA, and the calpain inhibitor I, N-acetyl-leu-leu-norleucinal, but not by several caspases inhibitors, suggesting that BARD1 is hydrolyzed by the calcium-dependent cysteine proteases, calpains. Thus, the highly immunogenic form of cleaved BARD1 could contribute to the antitumoral response mediated by apoptotic bodies.
Gautier
F
F
Institut de Biologie, INSERM U419, 44035 Nantes, France.
Irminger-Finger
I
I
Grégoire
M
M
Meflah
K
K
Harb
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017209:Apoptosis; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism*; D019313:BRCA1 Protein / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D002458:Cell Fractionation; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015723:Gene Library; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D015674:Mammary Neoplasms, Animal / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D019313:BRCA1 Protein; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007976:Leupeptins; D025521:Tumor Suppressor Proteins; C063261:acetylleucyl-leucyl-norleucinal; D004533:Egtazic Acid; C102449:BARD1 protein, human; C491163:Bard1 protein, mouse; D044767:Ubiquitin-Protein Ligases; D002154:Calpain