Flow cytometric detection of the mitochondrial BCL‐2 protein in normal and neoplastic human lymphoid cells

Abstract

The bcl‐2 proto‐oncogene, rearranged and deregulated in B‐cell lymphomas bearing the t(14;18) translocation, encodes an inner mitochondrial membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single‐ and multicolor flow cytometric analysis of bcl‐2 protein in relation to other markers and cell cycle, based on a fixation‐permeation step of cells with paraformaldehyde and Triton X100 and the use of a bcl‐2 specific monoclonal antibody (MoAb). As an application of this method, we have examined the expression of bcl‐2 in normal and neoplastic lymphoid cells. We have found that > 80% of normal Tand B‐cells are bcl‐2 positive; following in vitro mitogen activation, the bcl‐2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl‐2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two‐color analysis. On lymphoblastoid cell lines, the bcl‐2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl‐2 gene. Among fresh B‐cell non‐Hodgkin's lymphomas (B‐NHL), most sporadic Burkitt's cases were bcl‐2 negative. Of four centroblastic‐centrocytic cases with rearrangements of the bcl‐2 gene, only two presented elevated amounts of bcl‐2 protein, indicating that the levels of bcl‐2 are not diagnostic of the translocation. The flow cytometric analysis of bcl‐2 protein allows study and quantification, as the single cell level and in selected cell subsets, of the expression of the bcl‐2 gene and provides an important tool for assessing its role in hematopoietic cell development, proliferation, and neoplastic conversion.