Inhibition of de novo pyrimidine nucleotide and DNA synthesis and growth of cultured Novikoff rat hepatoma cells and other cell lines by pyrazofurin (NSC 143095).
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Pyrazofurin (PYF), a C-riboside, inhibited the replication of cultured Novikoff rat hepatoma cells, HeLa cells, and mouse L-cells at concentrations as low as 0.1 to 10 muM, but Novikoff cells were more sensitive than the cells of the other two cell lines. Inhibition of cell replication was completely prevented by the presence of 0.1 to 1 mM uridine in the medium, and partly by the presence of other pyrimidine, but not purine nucleosides. A 2- to 4-hr treatment of the cells with 10 muM PYF resulted in a 2-fold increase in the rate of incorporation of uridine into the acid-soluble pool and nucleic acids, while the rate of incorporation of adenosine into RNA was reduced about 85%. The incorporation of adenosine and deoxyuridine into DNA were reduced about 85 and 50%, respectively. The results are consistent with the view that PYF inhibits the de novo synthesis of pyrimidine nucleosides. The inhibition of cell replication seems to be due mainly to an inhibition of DNA rather than RNA synthesis, resulting from a rapid depletion of the pyrimidine deoxynucleotide pool, since addition of thymidine and deoxycytidine reversed the inhibition of DNA synthesis and cell replication by PYF. PYF must enter the cells to exert its toxicity since the toxicity of PYF was reduced 70 to 80% by the presence of 8 muM Persantin, a potent inhibitor of the facilitated and simple diffusion of various substrates, in the medium. If PYF is incorporated via normal nucleoside salvage pathways, its affinity for the nucleoside transport system(s) and kinases, must be low since, even at a concentration of 1 mM, it had only a slight effect on the initial rates of incorporation of various nucleosides into the nucleotide pool. (+info)
In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine-pyrimidine junctions.
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Uridine triphosphate deficiency, growth inhibition, and death in ascites hepatoma cells induced by a combination of pyrimidine biosynthesis inhibition with uridylate trapping.
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A selective deficiency of uridine triphosphate (UTP) was induced in AS-30D rat ascites hepatoma cells by the synergistic action of D-galactosamine and 6-azauridine. The resistance of these hepatoma cells to low concentrations of D-galactosamine (less than 2 mM) was due to their active de novo pyrimidine synthesis which compensated the trapping of uridylate in the form of uridine diphosphate-amino sugars derived from D-galactosamine. The additional blockage of de novo pyrimidine synthesis led to noncompensated uridylate trapping with a UTP content of less than 0.05 mmole/kg of cell wet weight as compared to the control level of 0.66 mmole/kg. The induction of UTP deficiency by incubating the cells with low concentrations of D-galactosamine and 6-azauridine (0.5 mM each) was not accompanied by significant changes in the content of adenine and guanine nucleotides, uridine diphosphate glucose, and uridine diphosphate galactose. The depletion of UTP pools could be reversed within 10 min by the addition of uridine; orotate or uracil were completely ineffective in these hepatoma cells. A UTP content in the range of 0.1 to 0.4 mmole/kg, induced by either 6-azauridine or D-galactosamine, was associated with a reversible depression of cell growth in suspension culture. A UTP content below 0.05 mmole/kg led to irreversible growth inhibition and to necrocytosis in culture, as well as to a loss of transplantability in vivo. Uridine reversal studies indicated that the percentage of cells able to resume growth in culture decreased with an increasing time period of UTP deficiency. The deficiency period required for irreparable or lethal damage in these hepatoma cells ranged from 3 to 20 hr. The principle of noncompensated uridylate trapping can be extended to other inhibitors of nucleotide synthesis combined with various nucleotide-trapping sugar analogs. Noncompensated nucleotide trapping may be useful for an induction of selective nucleotide deficiencies in tumor cells. (+info)