J. Bierau

The pharmacokinetic effect of discontinuation of mesalazine on mercaptopurine metabolite levels in inflammatory bowel disease patients

Background :u2002In vitro studies suggest interactions between mesalazine (mesalamine) and thiopurines by thiopurine S‐methyltransferase (TPMT) inhibition, influencing the balance of hepatotoxic 6‐methylmercaptopurine ribonucleotide and immunosuppressive tioguanine (thioguanine) metabolites.

The cytostatic- and differentiation-inducing effects of cyclopentenyl cytosine on neuroblastoma cell lines

This paper describes the effects of cyclopentenyl cytosine (CPEC) on the proliferation and cell-cycle distribution of the SK-N-BE(2)c and SK-N-SH neuroblastoma cell lines, as well as their ability to recover from treatment with CPEC. The IC50 value of SK-N-BE(2)c for CPEC, determined after 48 hr was 80 nM. SK-N-BE(2)c cells showed a time- and concentration-dependent accumulation in the S-phase of the cell cycle after 2 and 3 days of incubation with 50-250 nM CPEC, followed by a G0/G1-phase arrest after 4 days. After incubation with 50 nM CPEC for 2 days, SK-N-BE(2)c cells fully recovered and resumed logarithmic proliferation. In contrast, a complete and persistent growth arrest occurred when SK-N-BE(2)c cells were incubated for 2 days with 100 or 250 nM CPEC. The IC50 value of SK-N-SH, determined after 48 hr, for CPEC was > or =1 microM. SK-N-SH cells incubated with 250 nM or 1 microM CPEC showed a time-dependent accumulation in the S-phase of the cell cycle, followed by an accumulation in the G0/G1-phase, which reached a maximum of 84.1% after 7 days of incubation with 1 microM CPEC. SK-N-SH cells did not resume proliferation after removal of the drug. In addition, CPEC strongly induced differentiation in SK-N-SH cells. After 48 hr incubation with 250 nM CPEC, 90% of the cell population was differentiated. Both neuronal type and Schwannian type cells were observed. We conclude that at very low concentrations, CPEC has profound cytostatic- and differentiation-inducing effects on the neuroblastoma cells studied.

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.

Retinoic acid reduces the cytotoxicity of cyclopentenyl cytosine in neuroblastoma cells

In this paper, it is demonstrated that all‐trans, 9‐cis and 13‐cis retinoic acid (RA) decreased the sensitivity of SK‐N‐BE(2)c neuroblastoma cells towards the chemotherapeutic agent cyclopentenyl cytosine (CPEC), a potent inhibitor of cytosine‐5′‐triphosphate synthetase. Retinoic acid attenuated CPEC‐induced apoptosis as reflected by a decreased caspase‐3 induction. Retinoic acid decreased the accumulation of CPEC, whereas the salvage of cytidine was strongly increased. Metabolic labeling studies using [3H]uridine showed a strongly decreased biosynthesis of CTP via CTP synthetase. Retinoic acid likely confers resistance of neuroblastoma cells to CPEC in part by slowing down proliferation, and in part by shifting the synthesis of CTP towards the salvage of cytidine, thereby bypassing CTP synthetase.

Dihydropyrimidine Dehydrogenase Deficiency Caused by a Novel Genomic Deletion c.505_513del of DPYD

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine degradation pathway. In a patient presenting with convulsions, psychomotor retardation and Reye like syndrome, strongly elevated levels of uracil and thymine were detected in urine. No DPD activity could be detected in peripheral blood mononuclear cells. Analysis of the gene encoding DPD (DPYD) showed that the patient was homozygous for a novel c.505_513del (p.169_171del) mutation in exon 6 of DPYD.

Recovery of SK-N-BE(2)c cells from treatment with cyclopentenyl cytosine.

Neuroblastoma is one of the most common extra cranial solid tumours of childhood. The tumours are derived from the neural crest and have a high degree of heterogeneity1. Success of current therapies is modest. New pharmaceuticals have to be developed and their mechanism of action evaluated. As neuroblastoma cells are rapidly proliferating cells, they rely to a great extend on CTP synthetase for the synthesis of CTP. CTP synthetase catalyses the conversion of UTP into CTP and an increased CTP synthetase activity may cause an imbalance in the pyrimidine ribonucleotide pool2. The gene coding for CTP synthetase is located on chromosome 1p343, which is often deleted in neuroblastoma causing LOH. Despite 1p-deletion neuroblastoma cells have a high CTP synthetase activity, making CTP synthetase an attractive target for chemotherapy. Cyclopentenyl cytosine (CPEC) is a cytidine analogue that is readily metabolised to its active form: CPEC-triphosphate, which potently inhibits CTP synthetase causing rapid depletion of the intracellular cytidine nucleotides4 . In this study, we