Arnauld C. Verschuur

Cyclopentenyl cytosine inhibits cytidine triphosphate synthetase in paediatric acute non-lymphocytic leukaemia: a promising target for chemotherapy.

Cytidine triphosphate (CTP) synthetase is a key enzyme in the anabolic pathways of cytosine and uracil ribonucleotide metabolism. The enzyme catalyses the conversion of uridine triphosphate (UTP) into CTP, and has a high activity in various malignancies, which has led to the development of inhibitors of CTP synthetase for therapeutic purposes. We studied both CTP synthetase activity and ribonucleotide concentrations in leukaemic cells of 12 children suffering from acute non-lymphocytic leukaemia (ANLL), and performed incubation experiments with cyclopentenyl cytosine (CPEC), a nucleoside analogue that is capable of inhibiting CTP synthetase. The CTP synthetase activity in ANLL cells (5.1+/-2.3 nmol CTP/mg/h) was significantly higher compared with granulocytes of healthy controls (0.6+/-0.4 nmol CTP/mg/h, P=0.0002), but was not different from the CTP synthetase activity in non-malignant CD34+ bone marrow cells (5. 6+/-2.4 nmol CTP/mg/h). Major shifts were observed in the various ribonucleotide concentrations in ANLL cells compared with granulocytes: the absolute amount of ribonucleotides was increased with a substantial rise of the CTP (2.4 versus 0.4 pmol/microg protein, P=0.0007) and UTP (8.7 versus 1.6 pmol/microg protein, P=0. 0007) concentrations in ANLL cells compared with granulocytes. Treatment of ANLL cells in vitro with CPEC induced a major depletion (77% with 2.5 microM of CPEC) in the concentration of CTP, whilst the concentrations of the other ribonucleotides remained unchanged. Therefore, the high activity of CTP synthetase in acute non-lymphocytic leukaemic cells can be inhibited by CPEC, which provides a key to a new approach for the treatment of ANLL.

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

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.

Stromal and epithelial predominant Wilms tumours have an excellent outcome: the SIOP 93 01 experience.

Wilms tumour (WT) has various subtypes that are correlated with prognosis and require distinct therapy. Stromal predominant (SpWT) and epithelial WT (EpWT) have previously been associated with a good outcome. The current analysis describes the outcome and (tumour) characteristics of all patients with SpWT, EpWT, including highly differentiated epithelial type (HDET), treated according to the International Society of Pediatric Oncology (SIOP) 93‐01 study.

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 concentrations of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in leukemic cells by inhibiting the enzyme CTP synthetase, resulting in a decreased synthesis of RNA and DNA. Low concentrations of dCTP facilitate 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 flow cytometric detection of fluorescence-labeled Annexin V in a human T-lymphoblastic MOLT-3 cell-line after 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 observed necrosis proved to be secondary to apoptosis as the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) completely blocked the CPEC-induced apoptosis and necrosis. Coincubation of various concentrations of CPEC and araC for 16h showed a significant additive effect on the occurrence of apoptosis and (secondary) necrosis. In contrast, a preincubation with 37.5 nM of CPEC for 24 h, which by itself caused only minor apoptosis (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 apoptosis (27%, P<0.001). Growth-inhibition experiments with CPEC and araC under various conditions showed an additive effect on the araC-induced growth-inhibition after 48 h. The results indicate that the cytotoxicity of araC can be increased in T-lymphoblasts by CPEC.

Cyclopentenyl cytosine increases the phosphorylation and incorporation into DNA of 1‐β‐D‐arabinofuranosyl cytosine in a human T‐lymphoblastic cell line

The cytotoxic effect of 1‐β‐D‐arabinofuranosyl cytosine (araC) depends on the intracellular phosphorylation into its active compound araCTP, on the degree of degradation of araCTP and on the incorporation of araCTP into DNA. Deoxycytidine triphosphate (dCTP) inhibits the phosphorylation of araC (by feedback inhibition of the enzyme deoxycytidine kinase) and the incorporation of araCTP into DNA (by competition for DNA polymerase). In a T‐lymphoblastic cell line, we studied whether the cytotoxicity of araC (2 nM–50 μM) could be enhanced by decreasing the concentration of dCTP, using the nucleoside‐analogue cyclopentenyl cytosine (CPEC), an inhibitor of the enzyme CTP synthetase. Preincubation of the cells with CPEC (100–1,600 nM) for 2 hr increased the concentrations of araCMP 1.6–9.5‐fold, which was significant for each concentration of CPEC used. The concentration of araCDP remained low, whereas the concentration of araCTP changed depending on the concentration of araC used. With 2–15 μM of araC and a preincubation with 400 nM of CPEC, the araCTP concentration increased by 4–15% (not significant), and the total amount of araC nucleotides increased significantly by 21–45%. When using a concentration of araC of 2 nM after a preincubation with CPEC of 100 nM, the concentration of araCMP increased by 60% (p = 0.015), whereas that of araCTP decreased by 10% (p = 0.008). This was compensated by an increase of 41% (p = 0.005) of araCTP incorporation into DNA, which represented 43% of all araC metabolites. Moreover, by performing pulse/chase experiments with 400 nM of CPEC and 2μM of araC, the retention of cytosolic araCTP and the incorporated amount of araCTP into DNA were increased by CPEC. The modulation by CPEC of araC metabolism was accompanied by a synergistic increase of araC‐induced apoptosis and by an additive effect on the araC‐induced growth inhibition.

Cyclopentenyl Cytosine Induces Apoptosis and Secondary Necrosis in a T-Lymphoblastic Leukemic Cell-Line

Cyclopentenyl cytosine (CPEC) induced a depletion of CTP and dCTP in cell-lines of hematological malignancies by inhibiting the enzyme CTP synthetase , which was accompanied by a decreased synthesis of DNA and RNA . CPEC proved to have a cytostatic effect in Molt-4 and L1210 leukemic cells 1,3 and increased the life-span in mice suffering from lymphocytic leukemia 1. We have shown that a high activity of CTP synthetase is present in malignant blasts of children suffering from acute (non)-lymphocytic leukemia 4,5 and have demonstrated that CPEC depleted in vitro the concentrations of CTP and dCTP in these samples 4,5 . So far, the mechanism of cytotoxicity of CPEC has been less elucidated. Therefore, in this study we analyzed whether CPEC could induce apoptosis and/or (secondary) necrosis in a Molt-3 lymphocytic leukemic cell-line.

Increased cytotoxicity of 2', 2'-difluoro-2'-deoxycytidine in human leukemic cell-lines after a preincubation with cyclopentenyl cytosine

The in vitro modulating effect of Cyclopentenyl cytosine (CPEC) on the metabolism of gemcitabine was studied in lymphocytic and myeloid leukemic cell‐lines. In MOLT‐3 cells, that were pretreated with CPEC, the incorporation of 2′,2′‐difluoro‐2′‐deoxycytidine triphosphate (dFdCTP) into DNA was significantly increased by 57–99% in comparison with cells that were only treated with gemcitabine. The increased incorporation of dFdCTP into DNA in CPEC pretreated cells was paralleled by an increase in apoptotic and necrotic cells of 17–34%. In HL‐60 cells that were preincubated with CPEC, increased concentrations of the mono‐/di‐ and triphosphate form of gemcitabine were observed, as well as an increased incorporation of dFdCTP into DNA (+ 773%). This increased incorporation was paralleled by a significant increase in apoptosis and necrosis. We conclude that CPEC enhances the incorporation of dFdCTP into DNA and thus increases the cytotoxicity of gemcitabine in lymphocytic and myeloid leukemic cell‐lines.

Cytidine Triphosphate Synthase Activity and mRNA Expression in Normal Human Blood Cells

Abstract Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP × mg−1 × h−1), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes.

Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography

A non-radiochemical assay procedure for CTP synthetase was developed in which CTP is detected at 280 nm after separation with anion-exchange HPLC. A complete separation of all nucleoside triphosphates was achieved within 11 min and the minimum amount of CTP which could be accurately determined proved to be 5 pmol. Therefore, our assay procedure is ten-fold more sensitive compared to the frequently used radiochemical assays. The assay was linear with time and protein concentration, although at low protein concentration a lag phase was observed. An amount of 2 x 10(6) cells was already sufficient to determine the specific activity of CTP synthetase in HL-60 cells, lymphocytes and in lymphoblasts obtained from pediatric patients suffering from acute lymphoblastic leukemia.

Burkitt lymphoma in a child with Joubert syndrome

Joubert syndrome is a rare disorder, characterized by hypoplasia, or aplasia of the cerebellar vermis, hypotonia, ataxia, and psychomotor retardation. The molecular basis underlying the disease is still unknown. There are various syndromes, which are associated with malignancies. Previously known associations between Joubert syndrome and tumors, are benign soft tissue tumors of the tongue and laryngeal lymphangioma. This report describes a 17‐year‐old boy known with Joubert syndrome, who was diagnosed with Burkitt lymphoma. The boy received chemotherapy, which successfully induced complete remission.