DIFFERENTIAL INDUCTION OF APOPTOSIS BY FLUDARABINE MONOPHOSPHATE IN LEUKEMIC B-CELL AND NORMAL T-CELL IN CHRONIC LYMPHOCYTIC-LEUKEMIA

Fludarabine (F-ara-A), an adenine nucleoside analog with efficacy in B -cell chronic lymphocytic leukemia (B-CLL), has also been shown to hav e a long-lasting suppressive effect on T lymphocytes. In heterogeneous clinical samples, apoptosis cannot be detected by standard methods in small cellular subsets. We developed, therefore, a combined assay of in situ end-labeling of nicked DNA by terminal deoxynucleotide transfe rase, with measurements of cellular DNA content and surface antigens ( CD3, CD4, CD8, and CD19) by multiparametric flow cytometry. This assay was used to determine F-ara-A-induced apoptosis in different lymphocy te subsets from CLL patients and normal controls treated with F-ara-A in vitro. Apoptosis was also correlated to bcl-2 protein levels. We ob served a direct effect of F-ara-A on both B-CLL and T lymphocytes. The response to F-ara-A in B-CLL lymphocytes in vitro was pal stage-depen dent, the early-stages being more responsive (P = .01). Higher levels of spontaneous apoptosis were observed in B-CLL lymphocytes from early stage patients (P = .02). No difference was observed in spontaneous a poptosis of normal T cells in B-CLL, although T lymphocytes in late-st age disease were more sensitive to F-ara-A-induced apoptosis. Incubati on with cyclosporin A did not affect B-CLL and T-lymphocyte survival c ompared with control cultures. Results suggested a direct apoptotic ef fect of F-ara-A on B-CLL lymphocytes that decreases with increasing cl inical stage. No correlation was found between bcl-2 and spontaneous o r F-ara-A-induced apoptosis. Apoptosis occurred at all cell-cycle stag es and was not restricted to cells in S phase. The mechanisms of this stage-dependent apoptosis in CLL remain to he elucidated. (C) 1998 by The American Society of Hematology.