René Leen

Proteomic analysis of mouse kidney peroxisomes : identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity

Proteomic analysis of mouse kidney peroxisomes resulted in the identification of a novel nudix hydrolase designated RP2p, which is encoded by the D7RP2e gene. RP2p consists of 357 amino acids and contains two conserved domains: a nudix hydrolase domain and a CoA-binding domain. In addition, a PTS (peroxisomal targeting signal) type 1 (Ala-His-Leu) was found at the C-terminus. Analysis of the enzyme characteristics revealed that RP2p is a CoA diphosphatase with activity towards CoA, oxidized CoA and a wide range of CoA esters, including choloyl-CoA and branched-chain fatty-acyl-CoA esters. The enzymatic properties of RP2p indicate that at low substrate concentrations medium and long-chain fatty-acyl-CoA esters are the primary substrates. Enzyme activity was optimal at pH 9 or above, and required the presence of Mg2+ or Mn2+ ions. Subcellular fractionation studies revealed that all CoA diphosphatase activity in mouse kidney is restricted to peroxisomes.

Fenretinide induces mitochondrial ROS and inhibits the mitochondrial respiratory chain in neuroblastoma

Fenretinide induces apoptosis in neuroblastoma by induction of reactive oxygen species (ROS). In this study, we investigated the role of mitochondria in fenretinide-induced cytotoxicity and ROS production in six neuroblastoma cell lines. ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells). In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain. However, inhibition of the mitochondrial respiratory chain was not required for ROS production. Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II. The cytotoxicity of fenretinide was exerted through the generation of mitochondrial ROS and, at higher concentrations, also through inhibition of the mitochondrial respiratory chain.

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 (6·5u2003±u20033·9u2003nmol CTP/mg/h) was detected in ALL cells than in lymphocytes of healthy controls (1·8u2003±u20030·9u2003nmol CTP/mg/h, Pu2003<u20030·001) 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 (5·6u2003±u20032·4u2003nmol 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 (0·63u2003µm) induced a depletion of CTP of 41u2003±u200320% and a depletion of dCTP of 27u2003±u200321%. 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.

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

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.

Absence of cardiotoxicity of the experimental cytotoxic drug cyclopentenyl cytosine (CPEC) in rats

The experimental anticancer drug cyclopentenyl cytosine (CPEC) was associated with cardiotoxicity in a phase I study. The aim of the present study was twofold; first we investigated whether the observed effects could be reproduced in in-vitro and in-vivo rat models. Second, we intended to investigate the underlying mechanism of the possible cardiotoxicity of CPEC. Effects on frequency and contractility were studied on the isolated atria of 18 male Wistar rats. Atria were incubated with 0.1xa0mmolxa0L−1 (n=6) or 1xa0mmolxa0L−1 (n=6) CPEC for 1.5xa0h and compared with control atria (incubation with buffer solution, n=6). The cardiac apoptosis-inducing potential was studied in-vivo on 66 rats by 99mTc-Annexin V scintigraphy, followed by postmortem determination of radioactivity in tissues, histological confirmation with the TUNEL assay (late-phase apoptosis), and immunohistochemical staining for cleaved caspase 3 and cytochrome C (early-phase apoptosis). Serum levels of the necrotic cardiomyopathy marker troponin T were also determined. No effect on heart frequency was found in the isolated atria after CPEC treatment. A trend towards a decrease of contraction force was observed. However, the differences were not statistically significant. 99mTc-Annexin V scintigraphy showed no increase in cardiac uptake ratio upon CPEC treatment in the in-vivo rat model, which was confirmed by determination of radioactivity in heart versus blood ratios. At each section a few individual isolated late apoptotic cells (<5) could be identified by the TUNEL assay in the highest CPEC dose group (90xa0mgxa0kg−1) but not in controls or in rats treated with 60xa0mgxa0kg−1 CPEC. Staining for the early apoptosis markers cleaved caspase 3 and cytochrome C did not reveal any significant differences between treated and control rats. Cardiac troponin T levels were not increased after CPEC treatment. CPEC does not affect heart frequency or contraction force in our cardiotoxicity models. Moreover, we did not find an indication of CPEC-induced apoptosis in heart tissue.

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.

Synergistic interaction between cisplatin and gemcitabine in neuroblastoma cell lines and multicellular tumor spheroids

The efficacy and mechanism of action of cisplatin and gemcitabine were investigated in a panel of neuroblastoma cell lines and multicellular tumor spheroids. In neuroblastoma spheroids, the combination of cisplatin and gemcitabine induced a complete cytostasis at clinical relevant concentrations. A synergistic effect was observed when cells were coincubated with both drugs or preincubated with gemcitabine first. These administration sequences resulted in NASS cells in decreased ERCC1 and XPA expression, two key proteins of the NER DNA repair system, and increased platinum adduct formation in DNA. Most of these phenomena were not observed in SJNB8 cells which might explain the lack of synergy between cisplatin and gemcitabine in SJNB8 cells. Our results showed favorable interactions between cisplatin and gemcitabine in 4 out of 5 cell lines. Therefore, we feel that inclusion of gemcitabine into cisplatin-containing regiments might be a promising new strategy for the treatment of neuroblastoma.