High-performance liquid chromatographic analysis of biogenic amines in cells and in culture media using on-line dialysis and trace enrichment.

A highly sensitive method is presented for the automatic quantitative detection of DOPA metabolites in low concentrations in cells derived from the neural crest using reversed-phase HPLC in combination with fluorescence and electrochemical detection. The HPLC system was combined with on-line dialysis and on-line trace enrichment for the detection of small quantities of DOPA metabolites in culture media. Parameters like detector settings, pH, dialysis time and flow-rates are evaluated and optimized. Static-continuous dialysis can be performed at a low flow-rate concomitant with a high dialysis efficiency (up to >65%) depending on the type of DOPA metabolite. Counterflow dialysis can be used to analyse, with low efficiencies (17-29%), samples consisting of large volumes. Samples containing up to at least 7% (w/v) protein can be analysed in the low flow-rate static-continuous mode. In this last mode of dialysis, limits of detection for dopamine, norepinephrine, epinephrine and n-methyldopamine in DMEM/HAMF12 medium samples are 100 fmol or even lower. Serotonin is detectable at 10 fmol at a signal/noise ratio of 3. Biogenic amines were detectable at a concentration of 10 fmol/microl in a volume of 100 microl medium with an intra- and inter-assay imprecision <6.4%. This method is applied to study the differentiation level of tumour cells in culture and slices of a tumour derived from the neural crest. With this system, we also detected the excretion of DOPA metabolites from PC-12 cells after treatment with prenylamine.

In-situ cytidine-deaminase activity and chromosome 1P deletion in human neuroblastoma cells.

Cytidine deaminase can cause the deamination of cytotoxic analogues of cytidine or rescue cells from the cytotoxicity of uracil analogues. Therefore, cytidine deaminase influences the cytotoxicity exerted by these compounds. We investigated the activity of this enzyme in situ in neuroblastoma cell-line cells. The in-situ cytidine deaminase activity in human neuroblastoma cell-line SK-N-SH cells appeared to be two-fold higher than in the human neuroblastoma N-myc amplified cell-line SK-N-BE(2)-C cells. The observed activity correlated with the presence of both alleles in SK-N-SH cells versus one allele of cytidine deaminase in SK-N-BE(2)-C cells. This observation may be exploited for the treatment of neuroblastoma patients having a tumour with a chromosome Ip deletion including the domain containing the gene encoding cytidine deaminase.

Quantitative analysis of the pyrimidine metabolism in pheochromocytoma PC-12 cells.

A detailed quantitative study of pyrimidine metabolism in exponentially growing rat pheochromocytoma PC-12 cells has been performed. The sizes of ribonucleotide pools have been analysed and the pathways and the rates of metabolism of uridine, cytidine and aspartic acid have been determined, based on the incorporation of radioactive label. The fluxes of radioactive label through uridine-cytidine kinase, cytidine deaminase. CTP synthetase, nucleoside monophosphate kinase and nucleoside diphosphate kinase were obtained, as well as the flux through the pyrimidine de novo pathway. Also, the fluxes of radioactive label towards UDP-sugars, CDP-compounds, DNA and RNA were quantified in situ under steady-state conditions in intact PC-12 cells. From these fluxes of radioactivity, distribution ratios at the branch points of the metabolism were obtained. The pyrimidines synthesised via the de novo pathway were preferentially used for the synthesis of UDP-N-acetylhexosamines and UDP-hexoses, whereas the salvage of precursors from the medium contributed, to a large extent, to the synthesis of RNA. Therefore, we postulate that at least two different UTP pools exist in these cancer cells derived from the neural crest. Furthermore, after metabolism of radiolabeled cytidine and uridine into UTP, radiolabel was distributed in a similar manner from UTP towards UDP-N-acetylhexosamines, UDP-hexoses and RNA-UMP. Uridine, as well as cytidine, was channelled towards nucleic acids via small compartmented ribonucleotide pools.

The effect of cyclopentenyl cytosine on human SK-N-BE(2)-C neuroblastoma cells.

Human neuroblastoma SK-N-BE(2)-C cell-line cells were cultured in the presence of various concentrations of cyclopentenyl cytosine (CPEC). In the absence of cytidine, the IC50 value of CPEC for SK-N-BE(2)-C cells was 100 nM after 72 hr drug exposure. The IC20 value was 1 microM after 24 hr of exposure to CPEC in the presence of 10 microM cytidine, whereas in the absence of cytidine, CPEC at 1 microM resulted in an IC40 value after 24 hr. Therefore, cytidine partially prevented the cytostatic effect of CPEC. Cells cultured with 1 microM CPEC for 72 hr were enriched by approximately 410% with mono- and oligonucleosomes in comparison with cells cultured without CPEC. This enrichment was partially prevented with 10 microM deoxycytidine and completely prevented with 10 microM cytidine.

Cyclopentenyl cytosine and neuroblastoma SK-N-BE(2)-C cell line cells.

We studied the effect of cyclopentenyl cytosine (CPEC) on human neuroblastoma SK-N-BE(2)-C cell line cells. CPEC had an IC50 value of 100 nM for non-synchronised SK-N-BE(2)-C cells. These cells were arrested in G0/G1-phase or early S-phase of the cell cycle upon treatment with CPEC. After treatment of synchronised S-phase cells with 1 microM CPEC, the number of cells present after 3 days was less than 10% of that observed for the untreated cells. S-phase synchronised cells treated with CPEC and deoxycytidine showed an increased viability in comparison with cells treated with CPEC alone. Approximately 15% of the cells treated with CPEC and deoxycytidine traversed through one cell cycle. The amount of CTP declined to undetectable levels within 3 h after addition of 1 microM CPEC. The presence of cytidine prevented, to a large extent, the cytostatic effect of CPEC.