Flow cytometric detection of the mitochondrial BCL-2 protein in normal and neoplastic human lymphoid cells.
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 greater than 80% of normal T-and 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Aiello
A
A
Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Delia
D
D
Borrello
M G
MG
Biassoni
D
D
Giardini
R
R
Fontanella
E
E
Pezzella
F
F
Pulford
K
K
Pierotti
M
M
Della Porta
G
G
D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D002453:Cell Cycle; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D015321:Gene Rearrangement; D006801:Humans; D008213:Lymphocyte Activation / Q000235:genetics; D008228:Lymphoma, Non-Hodgkin / Q000473:pathology*; D008928:Mitochondria / Q000737:chemistry* / Q000648:ultrastructure; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D013601:T-Lymphocytes / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D014178:Translocation, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2