concanavalin-a and pyrazofurin

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.

Topics: Amides; Animals; Antibiotics, Antineoplastic; Cells, Cultured; Concanavalin A; DNA; Guinea Pigs; Intracellular Fluid; Lymphocyte Activation; Lymphocytes; Male; Nucleotides; Orotidine-5'-Phosphate Decarboxylase; Pyrazoles; Pyrimidines; Ribonucleosides; Ribose; Uridine

The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea pig lymphocytes incorporate [3H]dThd, [3H]dCyd, [3H]dUrd, [3H]Cyd and [3H]Urd into DNA-thymines and DNA-cytosines in a highly conserved distribution pattern. DNA-thymines were labeled only by dThd and dUrd, while DNA-cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA-thymines and DNA-cytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.

Topics: Amides; Animals; Cell Division; Concanavalin A; Cytidine Deaminase; DCMP Deaminase; DNA Replication; Guinea Pigs; Lymphocyte Activation; Male; Models, Biological; Pyrazoles; Pyrimidines; Ribonucleosides; Ribose; T-Lymphocytes

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10(-5) M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [3H]thymidine incorporation. Proliferation is completely restored by 5 X 10(-5) M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 X 10(-5) M each, fail to re-establish proliferation but produce an isotropic dilution of [3H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (less than 0.001 nmol X min-1 X 10(-6) cells).

Topics: Amides; Animals; Concanavalin A; Cytidine Deaminase; Deoxycytidine; DNA; Lymphocyte Activation; Pyrazoles; Pyrimidines; Rats; Ribonucleosides; Ribose; T-Lymphocytes; Uridine