Type I MAGE Proteins: Novel Markers of Proliferation in Multiple Myeloma Progenitor Cells.

CT7 (MAGE-C1) and MAGE-A3, two members of the type I MAGE family of Cancer-Testis antigens, are commonly expressed at both the mRNA and protein levels in primary tumor specimens from multiple myeloma patients. In previous analyses, tumors with higher percentages of type I MAGE-expressing cells had a positive correlation with abnormally elevated plasma cell proliferation. These data support the hypothesis that type I MAGE proteins are molecular markers of proliferating myeloma progenitor cells (the so-called “myeloma stem cell”) and may play a role in the pathobiology of this disease. To test this hypothesis, we examined the expression of type I MAGE in proliferating myeloma cells by flow cytometry. Human multiple myeloma cell lines U266, RPMI-8226, and KMS-11 were co-cultured for 12 or 24 hours with the nucleoside analog bromodeoxyuridine (BrdU), then fixed, permeabilized, and stained with CT7-33, a monoclonal antibody (mAb) to CT7, or M3H67 (to MAGE-A3), followed by a phycoerythrin (PE)-conjugated secondary mAb. The cells were then treated with DNAse and stained with a fluoroisothiocyanate (FITC)-conjugated mAb against BrdU. Proliferating cells that incorporated BrdU into their DNA exhibited high FITC fluorescence. For mAb M3H67, dual color analysis of this population showed that greater than 99% demonstrated a significant shift in PE fluorescence in all three of these cell lines as measured by Mean Fluorescence Index (MFI= geometric mean fluorescence [specific primary antibody]/mean fluorescence [no primary antibody], table 1). For CT7-33 mAb, greater than 85% demonstrated a shift in two of three lines (U266 and KMS-11), but not in RPMI-8226. For all three of these cell lines, dual color analysis of the BrdU-low population demonstrated less than 65% staining with either type I MAGE mAb. Interestingly, RT-PCR with CT7-specific primers of total RNA from RPMI-8226 revealed a product of lower molecular weight than expected, suggesting that a gene deletion occurred in this cell line possibly resulting in a stop codon, decreased translation, or decreased protein stability. This PCR product is being sequenced to determine the nature of the deletion. These results demonstrate that type I MAGE proteins are expressed in proliferating myeloma cells and are molecular markers of this population. These data suggest that novel therapeutics such as vaccines that target type I MAGE may preferentially eliminate the cycling myeloma cells, resulting in long-term cures.