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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.

Forty-three fresh tumor specimens of human neuroblastoma belonging to different clinical stages were analyzed for the expression of 2 proto-oncogenes: trk, which encodes a tyrosine-kinase receptor for nerve growth factor (NGF) and ret, another receptor-type tyrosine kinase whose ligand is unknown. The mRNA expression of the trk gene was detected in 67.4% of cases, with increased frequency in I, II and IVs Evans' stages and in patients with favorable prognosis according to the Shimada classification. Moreover, trk expression inversely correlated with Nmyc-gene amplification. ret mRNA was found in 36.8% of cases and equally distributed in the different stages. In addition, ngfR (low-affinity NGF receptor)-gene expression was present in 9 out of 25 cases. The simultaneous presence of mRNA related to both forms of the NGF receptor, while not proving the presence of a functional receptor, indicates the existence of a sub-set of neuroblastoma cells potentially responsive to NGF.

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