Gene and Protein Isoform Expression Analysis of the BCL-6 Associated Transcriptional Corepressor MTA3 in B- lineage Cells.
Deborah V Spencer, Cora L Foulks, Cissy M Geigerman, Pascale S Akl, Anne Y Lai, Paul A Wade, David L Jaye, Charles E Hill. Emory University, Atlanta, GA; NIEHS, Research Triangle Park, NC
Background: Metastasis-associated protein 3 (MTA3) is a transcriptional co-repressor expressed by germinal center (GC) B cells. MTA3 interacts with the proto-oncogene BCL6 and Mi-2/NURD complex to maintain B cell differentiation and preclude plasma cell differentiation. A role in facilitating the oncogenic potential of BCL6 is postulated. Long and short MTA3 isoforms exist, however, differences in expression and function have not been described. Thus, we designed a novel assay to assess expression of the mRNA's encoding the isoforms across mature B cell differentiation and in B cell neoplasms, and correlated with protein expression.
Design: We designed a RT-PCR assay to quantify MTA3 short and long mRNA isoforms using ABL1 as a control. Reverse primers were designed to selectively bind the unique 3' regions of the short and long mRNA's. Relative isoform quantification employed the delta-Ct method. RT-PCR and immunoblot analyses were performed on >90% pure, FACS-sorted B lineage cells (naïve, GC, memory, plasma cells) of tonsil and B cell leukemia and lymphoma cells.
Results: GC B cells express significantly more MTA3-short (MTA3-S) than benign naïve, memory, or plasma cells (mean 0.84, p<0.02). Plasma cells show higher mean MTA3-S/ABL1 values (0.29) than memory B cells (0.07), but not to statistical significance (p=0.06). Expression of MTA3-long (MTA3-L) mRNA does not differ significantly across cells types but trended higher in GC B cells. Among malignant cells, Burkitt lines express the highest levels of MTA3-S (mean MTA3-S/ABL1= 0.09) and have the highest short/long ratios (mean= 37) that are statistically different from plasma cell myeloma values (p <0.004). Immunoblot data confirm protein isoform expression in proportions consistent with RT-PCR data.
Conclusions: In B cell ontogeny, MTA3 isoform levels vary, with MTA3-S highest at the GC B cell stage. Differentiation into memory B or plasma cells yields decreased MTA3 mRNA with MTA3-S levels decreasing in memory B cells to near-naïve B cell levels. However, MTA3-S levels remain relatively higher in plasma cells, consistent with BCL6-independent functions. Burkitt cell lines, similar to their benign GC counterparts, express high levels of MTA3-S, with very high short/long ratios. Such differential isoform usage suggests functionally important differences in B cell differentiation and B cell oncogenesis for this BCL6-binding cofactor.
Monday, February 28, 2011 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 179, Monday Morning