In vitro inhibition of cytidine triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia

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Abstract
Cytidine triphosphate (CTP) synthetase is a key enzyme for the synthesis of cytosine (deoxy)ribonucleotides, catalysing the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in several malignancies. In this preclinical study, the enzyme activity and mRNA expression of the enzyme and (deoxy)ribonucleotide concentrations were analysed in leukaemic cells of 57 children suffering from acute lymphocytic leukaemia (ALL). In addition, in vitro experiments were performed with the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC). A significantly higher activity of CTP synthetase (6·5 ± 3·9 nmol CTP/mg/h) was detected in ALL cells than in lymphocytes of healthy controls (1·8 ± 0·9 nmol CTP/mg/h, P < 0·001) that was independent of white blood cell (WBC) count, blast percentage, age, gender or type of ALL. The enzyme activity was not correlated with the CTP synthetase mRNA expression. The activity of CTP synthetase in ALL cells compared with non‐malignant CD34+ bone marrow controls (5·6 ± 2·4 nmol CTP/mg/h) was not statistically different. In vitro treatment of ALL cells with CPEC induced a dose‐dependent decrease of the CTP concentration. The lowest concentration of CPEC (0·63 µm) induced a depletion of CTP of 41 ± 20% and a depletion of dCTP of 27 ± 21%. The degree of CTP depletion of ALL cells after treatment with CPEC was positively correlated with the activity of CTP synthetase. The inhibition of CTP synthetase in situ was confirmed by flux studies using radiolabelled uridine. From these results, it can be expected that CPEC has a cytostatic effect on lymphoblasts of children with ALL.