Study of the efficacy of a pronucleotide of 2-chloro-2'-deoxyadenosine in deoxycytidine kinase-deficient lymphoma cells.

2-Chloro-2 '-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 '-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.

2-Chloro-2'-deoxyadenosine inhibits DNA repair synthesis and potentiates UVC cytotoxicity in chronic lymphocytic leukemia B lymphocytes.

2-Chloro-2'-deoxyadenosine (CdA) is a deoxyadenosine analogue which targets enzymes involved in DNA synthesis, and hence might interfere with the resynthesis step of DNA repair. We tested this hypothesis in resting B cell chronic lymphocytic leukemia (B-CLL) lymphocytes, after firstly characterizing unscheduled DNA synthesis occurring in these cells. We observed that the spontaneous incorporation of [methyl-3H]thymidine (dThd) into DNA of B-CLL cells was not completely inhibitable by hydroxyurea (HU) which blocks DNA replication. In addition, in the presence of HU, dThd incorporation could be upregulated by UVC radiation or DNA alkylation, without re-entry of the cells into S phase. CdA was found to inhibit both spontaneous and upregulated DNA synthesis in B-CLL cells. Phosphorylation of CdA was essential to exert this effect. We finally observed a strong synergistic cytotoxicity between UV light and CdA, which was correlated with activation of caspase-3 and high molecular weight DNA fragmentation, two markers of apoptosis. Taken together, these observations indicate that in B-CLL cells CdA inhibits unscheduled DNA synthesis which represents the polymerizing step of a repair process responsive to DNA aggression. Inhibition of this process by CdA, together with a combined activation of the apoptotic proteolytic cascade by CdA and UV, may explain their synergistic cytotoxicity.

Potentiation of antitumor effects of cyclophosphamide derivatives in B-chronic lymphocytic leukemia cells by 2-chloro-2'-deoxyadenosine.

Because 2-chloro-2'-deoxyadenosine (CdA) is active in B-chronic lymphocytic leukemia (B-CLL), and may interfere with DNA repair, we investigated the potentiating effect of CdA on the cytotoxicity induced in vitro in B-CLL lymphocytes by cyclophosphamide (CP) derivatives, which induce DNA damage by DNA cross-linking. Exposure to CdA at clinically achievable concentrations for 2 h, followed by mafosfamide (MAF) or 4-hydroxycyclophosphamide (4HC) for 22 h, resulted in synergistic cytotoxicity in the majority of B-CLL samples tested. Synergy between CdA and MAF was observed in cell samples of sensitive/untreated patients, as well as in cells of resistant/pretreated patients, particularly at the highest concentrations of MAF. In the cells treated with CdA and MAF, we observed loss in ATP and hallmarks of apoptosis, as evidenced by cellular morphology and high molecular weight DNA fragmentation. The synergy could be explained neither by an influence of MAF on the phosphorylation of CdA, nor by an increase in the incorporation of CdA into DNA in the presence of MAF. The in vitro synergy between CdA and CP derivatives provides a rationale for the use of this association in B-CLL patients.

Resistance to 2-chloro-2'-deoxyadenosine of the human B-cell leukemia cell line EHEB.

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analogue toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-cell chronic lymphocytic leukemia. These cells were found to be less sensitive to CdA than B-cell chronic lymphocytic leukemia lymphocytes (approximately 25-fold) and other human lymphoblastic cell lines (10-1000-fold). Phosphorylation of CdA by deoxycytidine kinase and intracellular accumulation of 2-chloro-2'-deoxyadenosine triphosphate (CdATP) were similar in EHEB cells and in other CdA-sensitive cell lines. In contrast, the inhibitory effect of CdA on ribonucleotide reductase activity, which was investigated in situ by the conversion of cytidine into deoxyribonucleotides and its incorporation into DNA, was much less pronounced in EHEB cells than in other human lymphoblastic cells. Accordingly, concentrations of deoxynucleoside triphosphates did not decrease and even tended to rise. Unexpectedly, incorporation of thymidine and deoxycytidine into DNA was increased severalfold after a 24-h incubation with CdA. CdA also increased the activities of deoxycytidine kinase and thymidine kinase approximately 4-fold. Analysis of the cell cycle by flow cytometry showed that after 24 h, CdA provoked an increase in the proportion of cells in S phase, synthesizing DNA. We conclude that the EHEB cell line is resistant to the cytotoxic action of CdA not only because of a lack of inhibition of ribonucleotide reduction but also because CdA, in contrast with its known effects, provokes in this cell line an increase in the proportion of cells replicating their DNA. Unraveling of the mechanism of this effect may shed light on clinical resistance to CdA.

Metabolism and cytotoxic effects of 2-chloroadenine, the major catabolite of 2-chloro-2'-deoxyadenosine.

EHEB cells, a continuous cell line derived from a patient with B cell chronic lymphocytic leukemia (B-CLL), synthesized, when incubated with tritiated 2-chloro-2'-deoxyadenosine (CdA), labeled mono-, di-, and triphosphate ribonucleosides at a much higher rate than CdA deoxyribonucleotides. Further analysis revealed that these ribonucleotides were formed from labeled 2-chloroadenine (CAde), which contaminated commercial tritiated CdA at a proportion of 2-3%. Since CAde is the major catabolite of CdA measured in plasma after oral or intravenous administration of CdA to patients, its metabolism and in particular its potential cytotoxicity were investigated both in EHEB cells and in B-CLL lymphocytes. Phosphorylation of CAde was inhibited by adenine, indicating that its initial metabolism most probably proceeds via adenine phosphoribosyltransferase (EC 2.4.2.7). In both cell types, chloro-ATP was the major metabolite formed from CAde and its concentration increased proportionally at least up to 50 microM CAde. At high concentration, CAde metabolism was accompanied by a decrease in intracellular ATP. Cytotoxicity of CAde, evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showed an IC(50) of 16 microM in EHEB cells and 5 microM in B-CLL lymphocytes. At cytotoxic concentrations, apopain/caspase-3 activation and high molecular weight DNA fragmentation were observed, indicating that CAde cytotoxicity results from induction of apoptosis. However, since CAde cytotoxicity requires higher concentrations than CdA, it probably does not play a role in the therapeutic effect of CdA in the treatment of hematologic malignancies.