Differential Induction of Apoptosis by Fludarabine Monophosphate in Leukemic B and Normal T Cells in Chronic Lymphocytic Leukemia

Abstract

Fludarabine (F-ara-A), an adenine nucleoside analog with efficacy in B-cell chronic lymphocytic leukemia (B-CLL), has also been shown to have 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 transferase, 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 lymphocyte subsets from CLL patients and normal controls treated with F-ara-A in vitro. Apoptosis was also correlated to bcl-2 protein levels. We observed 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 Rai stage–dependent, 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 apoptosis of normal T cells in B-CLL, although T lymphocytes in late-stage disease were more sensitive to F-ara-A–induced apoptosis. Incubation with cyclosporin A did not affect B-CLL and T-lymphocyte survival compared with control cultures. Results suggested a direct apoptotic effect of F-ara-A on B-CLL lymphocytes that decreases with increasing clinical stage. No correlation was found between bcl-2 and spontaneous or F-ara-A–induced apoptosis. Apoptosis occurred at all cell-cycle stages and was not restricted to cells in S phase. The mechanisms of this stage-dependent apoptosis in CLL remain to be elucidated.

Early induction of apoptosis in B‐chronic lymphocytic leukaemia cells by hydroxychloroquine: activation of caspase‐3 and no protection by survival factors

“…In our study, comparable results were obtained on leukaemic B lymphocytes. The apoptotic effect of HCQ was observed early at 1 h after treatment compared with others drugs, such as fludarabine, in which significant apoptosis was not noted for a few days (Consoli et al, 1998). The HCQ-induced apoptosis of B-CLL cells involves the activation of caspase-3, as previously described for glucocorticoid-, fludarabine-, and mitoxantrone-induced apoptosis (Bellosillo et al, 1997;Chandra et al, 1997;Stoetzer et al, 1999).…”

Reduced susceptibility to apoptosis correlates with kinetic quiescence in disease progression of chronic lymphocytic leukaemia

“…These data are in agreement with those of Vermes et al (1997), who similarly reported no real increase in apoptosis among CLL cells. Consoli et al (1998) confirmed this concept by evaluation of fludarabine‐induced apoptosis. Using the in situ end labelling technique, these authors found a higher percentage of cells with DNA strand breaks associated with apoptosis in normal CD19 + B cells compared with CLL cells or other lymphocyte subpopulations.…”

“…Based on the clinical evidence that CLL is characterized by the accumulation of long‐lived lymphocytes, several studies have investigated whether apoptosis of CLL cells could play a role in the disease (Collins et al , 1989; Hanada et al , 1993; Aguilar‐Santelises et al , 1994; Robertson et al , 1996; Binet et al , 1996; Pepper et al , 1997; Consoli et al , 1998). The involvement of survival regulators, such as cytokines and soluble molecules has also been extensively evaluated in CLL (for a review see Orsini et al , 2000).…”

Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by human deoxyribonucleoside kinases and ribonucleoside kinases

“…Other enzyme targets of F-AraA include DNA primase, DNA polymerase a, DNA ligase, and DNA topoisomerase II (Tseng et al, 1982). Thus, the mechanism of action of F-AraA in proliferating cells is mainly cell cycle specific, and incorporation of F-AraA into DNA during S phase is required for the induction of apoptosis (Consoli et al, 1998;Galmarini et al, 2001Galmarini et al, , 2002. In nondividing cells, the inhibition of cellular DNA repair is the most likely explanation for cytotoxicity of F-AraA and CdA.…”