Cyclopentenyl cytosine induces apoptosis and increases cytarabine-induced apoptosis in a T-lymphoblastic leukemic cell-line

Cyclopentenyl cytosine (CPEC) is a nucleoside-analogue that decreases the c oncentrations of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in leukemic cells by inhibiting the enzyme CTP synthetase, resultin g in a decreased synthesis of RNA and DNA. Low concentrations of dCTP facil itate the phosphorylation Of 1-beta -D arabinofuranosyl cytosine (araC) and the incorporation of arabinofuranosyl cytosine triphosphate (araCTP) into DNA. Apoptosis and necrosis were analyzed by flowcytometric detection of fl uorescence-labeled Annexin V in a human T-lymphoblastic MOLT-3 cell-line af ter incubations with CPEC and/or araC. CPEC induced apoptosis and necrosis in a concentration- (50-300 nM) and time-dependent (8-16 h) way. The observ ed necrosis proved to be secondary to apoptosis as the caspase inhibitor be nzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) completely blocke d the CPEC-induced apoptosis and necrosis. Coincubation of various concentr ations of CPEC and araC for 16h showed a significant additive effect on the occurrence of apoptosis and (secondary) necrosis. In contrast, a preincuba tion with 37.5 nM of CPEC for 24 h, which by itself caused only minor apopt osis (4%), followed by a coincubation for 16 h with 62.5 nM of araC (7% of apoptotic. cells), showed a synergistic effect on the induction of apoptosi s (27%, P < 0.001). Growth-inhibition experiments with CPEC and araC under various conditions showed an additive effect on the araC-induced growth-inh ibition after 48 h. The results indicate that the cytotoxicity of araC can be increased in T-lymphoblasts by CPEC. (C) 2001 Elsevier Science Ltd. All rights reserved.