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.
Bontemps
F
F
Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology, Brussels, Belgium. bontemps@bchm.ucl.ac.be
Delacauw
A
A
Cardoen
S
S
Van Den Neste
E
E
Van Den Berghe
G
G
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D017338:Cladribine / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D009711:Nucleotides / Q000096:biosynthesis; D014316:Tritium; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D009711:Nucleotides; D014316:Tritium; C069484:2-chloroadenine; D017338:Cladribine; D000225:Adenine