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Identification of 6-Azauridine Triphosphate in L1210 Cells and Its Possible Relevance to Cytotoxicity¹

Departments of Pharmaceutical Chemistry [L. L. W.] and Medicinal Chemistry [L. B. T.]. College of Pharmacy, and Department of Chemistry [L. B. T.], The University of Michigan, Ann Arbor, Michigan 48109-1065.

L1210 cells treated with 1 mM 6-azauridine (AzUrd) (concentration causing 50% inhibition of cell growth, 3 µM) continued to divide at a reduced rate for 72 h before stopping. However, a 24-h treatment was lethal to 99% of the cells, as determined by colony formation. To investigate the mechanism for this delayed cytotoxicity, the metabolism of AzUrd was studied. Cells incubated with AzUrd contained a new 254 nm-absorbing component, not found in control cells. It appeared to be 6-azauridine-5'-triphosphate, since it was the only peak in the triphosphate region of the chromatogram which contained ³H after incubation of cells with [³H]AzUrd. Incorporation of [³H]AzUrd into the acid-insoluble fraction (nucleic acids) was also detected. A role for this incorporation in the mechanism of AzUrd cytotoxicity was strongly suggested by the observation that cordycepin (0.01 mM) partially protected cells from the lethality of AzUrd, presumably by preventing its incorporation into RNA. The previously known inhibition of pyrimidine de novo synthesis by AzUrd was confirmed by a decrease in the intracellular contents of UTP and CTP in AzUrd-treated cells. Therefore, we propose that the inhibition of pyrimidine de novo synthesis and the incorporation into nucleic acid(s) may act in concert to produce the cytotoxic effects of AzUrd.

¹ This investigation was supported in part by Grant CH-312 from the American Cancer Society.

² To whom requests for reprints should be addressed.

Received 5/ 6/88. Revised 10/14/88. Accepted 10/19/88.