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 col on carcinomatosis by injections of apoptotic bodies derived from tumor cell s and interleukin 2, This curative treatment generated a tumor specific cyt otoxic T-cell response associated with a humoral response. Autoantibodies f rom sera of cured rats strongly recognized a M-r 67,000 protein from apopto tic bodies and weakly reacted with a protein of M-r similar to 97,000 in PR Ob parental cells. We now show that these autoantibodies are directed again st BARD1, originally identified as a protein interacting with the product o f the breast cancer gene 1, BRCA1, We demonstrate that the M-r 67,000 antig en 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 t he cleavage site of BARD1 is located NH2 terminally but downstream of the R ING domain essential for BARD1 and BRCA1 protein interaction. In vitro stud ies using [S-35]methionine-labeled human BARD1 and apoptotic cellular extra cts derived from SW48 carcinoma cells indicate! that BARD1 proteolysis occu rs at an early stage of apoptosis and in a cell cycle-dependent manner. Thi s hydrolysis is inhibited by EGTA, and the calpain inhibitor I, N-acetyl-le u-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.