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.

Synergism between fludarabine and rituximab revealed in a follicular lymphoma cell line resistant to the cytotoxic activity of either drug alone

“…As one of the major mechanisms of action of fludarabine is thought to be induction of apoptosis (Huang & Plunkett, 1995; Adkins et al , 1997; Bellosillo et al , 1997; Consoli et al , 1998; Kitada et al , 1998; Pettitt et al , 1999), we next verified whether fludarabine on its own could trigger the apoptotic cascade in Karpas 422 cells. Apoptosis was measured at different times by monoparametric Annexin V or biparametric TUNEL/DNA assay, using FACS analysis.…”

“…The molecular basis for the resistance or sensitivity to fludarabine‐induced apoptosis was investigated by analysing the expression of several molecules previously implicated in this process in Western blots, in particular fas, bcl‐2 and p53 (Fisher, 1994; Adkins et al , 1997; Bellosillo et al , 1997; Consoli et al , 1998; Kitada et al , 1998; Pettitt et al , 1999). No significant changes in levels of expression of these molecules was observed up to 48 h after fludarabine treatment in any of the cell lines and no correlation could be observed between expression levels and sensitivity to fludarabine‐induced apoptosis (data not shown).…”

“…Present evidence suggests that it acts through inhibition of DNA synthesis after incorporation of the active metabolite into DNA during replication and through inhibition of ribonucleotide reductase, DNA primase and DNA ligase (Plunkett et al , 1993; Adkins et al , 1997). Furthermore, evidence suggests that induction of apoptosis is an important mechanism of action of fludarabine (Huang & Plunkett, 1995; Adkins et al , 1997; Bellosillo et al , 1997; Consoli et al , 1998; Kitada et al , 1998; Pettitt et al , 1999). Our data show that different cell lines can show quite varied response to the same chemotherapeutic agent, as all four showed inhibition of cell growth, two (BJAB and WSU‐NHL) were induced into apoptosis and only one showed a strong response at RNA levels (Karpas 422).…”

Review of Prodigiosin, Pigmentation in Serratia marcescens

“…PNU156804 blocks IL-2dependent NF-kB and AP-1 activation [91] and it is a selective inhibitor of Janus tyrosine kinase 3(Jak3) [111] . Current B-CLL therapies do not demonstrate specificity, thus fludarabine is cytotoxic for T lymphocytes [109,110] . Interestingly, in a subset of samples, prodigiosin is less toxic than fludarabine for normal T cells, while having similar effect in B-CLL cells.…”

Population Pharmacokinetics of Fludarabine in Children and Adults during Conditioning Prior to Allogeneic Hematopoietic Cell Transplantation

“…Subsequently, intracellular phosphorylation takes place to the active metabolite fludarabine triphosphate (F-ara-ATP), which is built into the DNA and RNA, thereby inhibiting DNA/RNA synthesis. This leads to apoptosis in both chronic lymphocytic leukemia cells [ 5 ] and (with different susceptibility) in cell types targeted in the HCT setting [ 6 , 7 ].…”