Monoclonal antibodies against recombinant-MAGE-1 protein identify a cross-reacting 72-kDa antigen which is co-expressed with MAGE-1 protein in melanoma cells.

Abstract

The MAGE-1 gene codes for tumor-associated peptides recognized by cytolytic T lymphocytes in association with MHC-class-1 molecules such as HLA-A1 and HLA-Cw16. In the course of a study aiming at the immunohistochemical detection of the MAGE-1 gene product in tumor samples, 2 mouse monoclonal antibodies (MAbs) directed against a full-length recombinant MAGE-1 fusion protein were found to react strongly not only with the 46-kDa MAGE-1 protein, but also with a 72-kDa product in immunoblots of lysates obtained from several MAGE-1-mRNA-positive melanoma cell lines. Pre-incubation of the antibodies with the recombinant MAGE-1 fusion protein abolished their reactivity both with MAGE-1 protein and with the 72-kDa product, thus confirming the occurrence of antigenic determinant(s) shared by the 2 proteins. The 72-kDa protein is not an alternative product of MAGE-1, since it was still detected in lysates of a MAGE-1 loss variant derived from a MAGE-1-positive melanoma cell line. Moreover, the 72-kDa protein does not appear to be a product of the other members of the MAGE gene family known to be expressed in tumors (such as MAGE-2, -3, -4 and -12). Interestingly, expression of the 72-kDa protein was found to be correlated with that of MAGE-1 protein. Thus, in 30 tumor cell lines analyzed by immunoblotting and RT-PCR, the 72-kDa protein was never detected in MAGE-1-mRNA-negative cell lines, while it was co-expressed with MAGE-1 protein in 12 out of 15 cell lines expressing MAGE-1. Furthermore, the 72-kDa protein was detected in lysates of human testis, the only normal tissue known to express MAGE-1. Finally, treatment of MAGE-1-mRNA-negative cell lines with 5-Aza-2'-deoxycytidine, a hypomethylating agent known to induce MAGE-1 expression, resulted in the expression of the 72-kDa protein. Taken collectively, these findings suggest that expression of the gene encoding the 72-kDa protein identified in this study through antigenic determinant(s) shared with MAGE-1 protein is regulated in a way similar to that of MAGE-1.