Abstract
The bcl-2 gene is overexpressed in the absence of gene rearrangements in most cases of B-cell chronic lymphocytic leukaemia (B-CLL) and the proto-oncogene product Bcl-2 has been shown to be a regulator of apoptosis. The activity of this protein is opposed by Bax, a homologous protein that accelerates the rate of cell death. B-lymphocyte Bcl-2 and Bax protein levels were found to be significantly altered in B-CLL and increased Bcl-2/Bax ratios were observed in both the treated and untreated patients compared with those of normal controls. These alterations were particularly pronounced in those treated patients found to be clinically unresponsive to chemotherapy. In order to determine whether Bcl-2/Bax ratios affected cell survival via an anti-apoptotic mechanism, cell death was induced in B-CLL cells in vitro using chlorambucil, and apoptosis was monitored by Annexin V and propidium iodide staining. Confirmation that the labelled cells were apoptotic was achieved by morphological assessment of cytospin preparations of cell-sorted populations. Drug-induced apoptosis in B-CLL cells was inversely related to Bcl-2/Bax ratios.
Highlights
Bcl-2 and Bax protein expression was determined by triple-colour immunofluorescence of peripheral blood lymphocyte samples from 22 B-cell chronic lymphocytic leukaemia (B-CLL) patients using flow cytometry
The results were expressed as MESF of the antibody staining and were compared with levels of Bcl-2 and Bax protein determined previously in peripheral blood B-lymphocytes from healthy donors (Pepper et al, 1996)
The ten treated patients were assessed for Bcl-2 and Bax protein expression in this present study
Summary
Cell isolation and incubation conditionsPeripheral blood was obtained from 22 B-CLL patients after informed consent was obtained. Clinical responsiveness was assessed in accordance with the National Cancer Institute Working Group Guidelines (Cheson et al, 1988), and clinical staging was based on the Binet system (Binet et al, 1981). All previously treated patients had received chlorambucil therapy and four were defined as refractory based on failure to meet standard response criteria (Cheson et al, 1988). Peripheral blood lymphocytes were isolated by density centrifugation on Ficoll-Hypaque (Sigma, UK) and were washed three times in phosphate-buffered saline (PBS). Aliquots of the lymphocytes to be cultured were resuspended in Eagle medium (Gibco, UK) at a concentration of 1x106 cells ml-1 and incubated at 37°C in a 5% carbon dioxide atmosphere for 48 h
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