pyrazofurin and Carcinoma--Hepatocellular

5-Azacytidine transport into cells was measured in the absence of metabolism in adenosine triphosphate-depleted and uridine kinase-deficient Novikoff cells. Azacytidine is transported with about the same efficiency as uridine and cytidine by the facilitated nucleoside transport system of these cells. The phosphorylation of azacytidine in untreated, wild-type cells, however, is much more inhibited by uridine and cytidine than is its transport into the cell. This inhibition seems to be responsible for the specific protection of cells by these nucleosides from azacytidine toxicity. Azacytidine is incorporated by Novikoff and P388 cells into both RNA and DNA, and this incorporation seems to be responsible for its cytotoxicity; an inhibition of de novo pyrimidine nucleotide synthesis is not a major contributory factor. Incorporation of azacytidine into nucleic acids is relatively slow, but it is enhanced 3 to 4 times when cells are preincubated with pyrazofurin. Pyrazofurin inhibits de novo pyrimidine synthesis and thus causes a depletion of cellular pyrimidine nucleotides. Azacytidine is largely cytostatic for Novikoff and P388 cells, but a sequential treatment with pyrazofurin and azacytidine markedly increases the cytotoxicity over that observed with drug alone or when administered together with drug, even at higher concentrations. Increased cytotoxicity correlates with the increased incorporation of azacytidine into nucleic acids.

Topics: Amides; Animals; Antibiotics, Antineoplastic; Azacitidine; Carcinoma, Hepatocellular; Cell Division; Cell Survival; Cytidine; DNA, Neoplasm; Drug Synergism; Leukemia, Experimental; Liver Neoplasms; Neoplasm Proteins; Neoplasms, Experimental; Phosphates; Pyrazoles; Ribonucleosides; Ribose; RNA, Neoplasm; Uridine

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.

Topics: Adenosine; Amides; Anti-Bacterial Agents; Carcinoma, Hepatocellular; Cell Division; Cells, Cultured; Deoxycytidine Monophosphate; Dipyridamole; DNA; DNA, Neoplasm; HeLa Cells; L Cells; Liver Neoplasms; Neoplasms, Experimental; Pyrazoles; Pyrimidine Nucleotides; Ribonucleosides; Ribose; RNA; Thymidine; Uridine