Human colon-carcinoma cells were exposed to D-glucosamine at 2.5, 5 and 10 mM, concentrations that were growth-inhibitory but not cytocidal in the presence of a physiological glucose concentration. Labelling of these HT-29 cells with D-[14C]-glucosamine, followed by nucleotide analyses, demonstrated that UDP-N-acetyl-hexosamines represented the major intracellular nucleotide pool and the predominant metabolite of the amino sugar. D-[14C]Glucosamine was not a precursor of UDP-glucosamine. After 4h exposure to D-glucosamine (2.5 mM), the pool of UDP-N-acetylhexosamines was increased more than 6-fold, whereas UTP and CTP were markedly decreased. UDP-glucuronate content increased by more than 2-fold, whereas purine nucleotide content was little altered. Uridine (0.1 mM) largely reversed the decrease in UTP, CTP, UDP-glucose and UDP-galactose, while intensifying the expansion of the UDP-N-acetylhexosamine pool. Uridine did not reverse the D-glucosamine-induced retardation of growth in culture. A 50% decrease in growth also persisted when uridine and cytidine, cytidine alone, or UDP, were added together with D-glucosamine. The growth-inhibitory effect of the amino sugar could therefore be best correlated with the quantitative change in the pattern of sugar nucleotides, and, in particular, with the many-fold increase in UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine.
The interference of nucleotide metabolism induced by ~-arabino-hexos-2-u~ose (glucosone), 2-deoxy-2-fluoro-D-glucose (fluoroglucose) and, for comparison, 2-amino-2-deoxy-~-glucose (glucosamine) was studied in suspension cultures of TA3 mammary tumor cells. High-performance liquid chromatography, [I4C]uridine labeling and enzymatic analyses revealed that glucosone is metabolized to the U D P derivative leading to a diversion of [I4C]uridylate from UTP into UDP-glucosone. The accumulation of UDP-glucosone, that was measured in TA3 as well as AS-30D hepatoma cells, was associated with a depletion of UTP pools to less than 15'%; of control and a reduction of CTP contents to one third. UTP and CTP deficiency could be rapidly rcversed by addition of uridine to the cell suspension.Fluoroglucose acted both as a uridylate-trapping and as a guanylate-trapping hexose analog lowering the cellular contcnts of UTP and GTP to 5 % and 27 of control, respectively. The lowering of GTP was accompanied by an increase in total acid-soluble guanine nucleotides and a diversion of 84% of the [14C]guanylate into GDP-fluoroglucose. As compared to fluoroglucose and glucosone, glucosamine was less effective in TA3 cells in decreasing the content of UTP. The uridylate-trapping effect of glucosamine was in part compensated by the most active expansion of the acid-soluble uridylate pool; this reflects an enhanced rate of dc m v o pyrimidine synthesis that could be suppressed by the inhibitor 6-azauridine.As a side effect probably resulting from phosphate trapping, glucosone, fluoroglucose, and glucosamine depressed the levels of ATP and of total adenine 5'-nucleotides to a similar though moderate extent.The depletion of UTP, induced by these C-2-modified D-glucose analogs, and additionally of GTP in the case of fluoroglucose, represent a metabolic lesion that may explain their growth inhibitory and cytotoxic effects.Several C-2-modified D-glucose analogs are phosphorylated by hexokinase [l-31; some of them are metabolized further to U D P and/or GDP derivatives [4-61. Tntracellular formation and accumulation of the respective sugar 6-phosphate can result in a fall of inorganic phosphate triggering adenine nucleotide catabolism as one of its consequences [7 -91. Diversion of uridylate for the synthesis of UDP-sugar analogs leads to a lowering of cellular UTP levels and to a consecutive enhancement of the rate of de novo pyrimidine synthesis [lo]. The UTP depletion is followed by an inhibition of RNA synthesis [I I], while some of the C-2-modified UDPDedicated to Professor H. Holzer on the occasion of his 60th birthday. Abhreviutions. Glucosone, ~-arahino-hexos-2-ulose ; FGlc, fluoroglucose, 2-deoxy-2-fluoro-~-g~ucose; GlcN, glucosamine, 2-amino-2-deoxy-D-glucose; 6-AzaUrd, 6-azauridine; CAMP, CGMP, CUMP, sum of total acid-soluble adenine, guanine, and uracil 5'-nucleotides, respectively ; GDPMan, GDP-mannose; UDPGlc, UDP-glucose; UDPHexNAc, UDP-N-acetylhexosamines.Enzymes. Alkalinc phosphatase (EC 3.1.3.1): glucose-6-phosphate deliydrogenase (...
d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [(14)C]Uridine labelling of hepatoma cells indicated a division of [(14)C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmolxh(-1)x(kg AS-30D or liver wet wt.)(-1). As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of (14)CO(2) into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5'-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37 degrees C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue.
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