Cyclopentenyl cytosine primes SK‐N‐BE(2)c neuroblastoma cells for cytarabine toxicity

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Abstract
CPEC is a potent inhibitor of CTP synthetase and causes depletion of CTP and dCTP pools. AraC is an analog of dCyd and a chemotherapeutic agent. Here, we demonstrate that, upon incubation with CPEC, both the anabolism and cytostatic effect of AraC in SK‐N‐BE(2)c neuroblastoma cells were increased. Cotreatment of CPEC (50–250 nM) and AraC (37.5–500 nM) decreased the 4‐day ED50 value for AraC 2‐ to 8‐fold in the SK‐N‐BE(2)c cell line, while pretreatment with CPEC followed by incubation with AraC alone decreased the 4‐day ED50 value for AraC 1‐ to 19‐fold. Preincubation of SK‐N‐BE(2)c cells with 100 nM CPEC followed by incubation with 500 nM [3H]AraC increased the total amount of AraC nucleotides and incorporation of [3H]AraC into DNA by 392% and 337%, respectively, compared to non‐CPEC‐treated cells. When 20 nM [3H]AraC was used, the maximum incorporation of [3H]AraC into DNA was 1,378% compared to non‐CPEC‐treated cells. Incorporation of AraC into DNA correlated well with the accumulation of cells in S phase of the cell cycle caused by CPEC. DNA synthesis was almost completely inhibited (>91%) when 100 nM CPEC and 500 nM AraC were combined. CPEC alone and the combination of CPEC and AraC increased caspase‐3 activity 3‐fold, indicating induction of apoptosis in SK‐N‐BE(2)c cells. In contrast, AraC alone did not induce caspase‐3 activity. Our results demonstrate that low concentrations of CPEC profoundly increase the cytostatic properties of AraC toward SK‐N‐BE(2)c human neuroblastoma cells.