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;lB) translocation, encodes an inner mitochondria1 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 XlOO 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 Bcell 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. 0 1992 Wiley-Liss, Inc.

Role of the CD40 and CD95 (APO‐1/Fas) antigens in the apoptosis of human B‐cell malignancies

“…Mean fluorescence intensity (MFI) of MoAb staining was determined using the flow cytometer and expressed in arbitrary units. Immunofluorescence staining for intracellular Bcl-2 protein was performed as described by Aiello et al (1992). Cells were washed twice with cold PBS, centrifuged, resuspended in 2 ml of 2% paraformaldehyde, and incubated for 10 min on ice.…”

Damaged microtubules can inactivate BCL-2 by means of the mTOR kinase

“…The biochemical events triggered by rapamycin were studied in three human follicular B-cell lymphoma lines that contain a high concentration of BCL-2 protein (Aiello et al, 1992;Steube et al, 1995) and are most sensitive to rapamycin. The lowest doses of rapamycin inhibited growth and arrested the cell lines in the G 1 phase.…”

The antisense bcl-2 – IgH transcript is an optimal target for synthetic oligonucleotides

“…To determine BCL-2 protein cellular level, 2 ϫ 10 6 ODN-treated cells were fixed for 10 min in 2% paraformaldehyde plus 1% Triton X-100, washed in Tris-buffered saline, and incubated with anti-BCL-2 mAb or normal mouse serum (for negative control) after preincubation for 10 min with 2% heat-inactivated human AB serum. Indirect immunofluorescence staining for BCL-2 was analyzed by flow cytometry with an EPICS-C instrument (Coulter), as published elsewere in detail (37).…”