Mechanism of Resistance to Vidarabine By a Mammalian Cell Line Devoid of Adenosine Deaminase Activity (Anti-Herpes Drug).

Abstract

Vidarabine (ara-A) has a firmly established role in the management of certain human herpesvirus infections, and a potential use as a carcinostatic agent. Although the mode of action of ara-A and the biochemical basis for its antiviral selectivity are not completely understood, it is known that for the compound to be active, it must be metabolized to the corresponding 5'-triphosphate (ara-ATP). Furthermore, deamination of ara-A to arabinosyl hypoxanthine by adenosine deaminase drastically reduces the cytotoxic and antiviral effects of the drug. The problem of development of resistance to ara-A has been investigated by passaging an established rat embryo cell line (B-mix K-44/6), which is devoid of adenosine deaminase activity, in the presence of increasing concentrations of ara-A (up to 400 (mu)M). At this drug level, cells were cloned and two of four isolated clones were found to be highly resistant to ara-A. One of the two selected clones retained its high resistance to ara-A throughout the study and was compared to wild type (WT) B-mix cells in a series of pharmacological and biochemical studies designed to determine the mode of resistance to ara-A. The results of these experiments demonstrated that the mechanism of resistance involved neither catabolism of the drug by an induction of adenosine deaminase activity nor a change in the sensitivity of DNA polymerases (alpha) and (beta) to ara-ATP. Dose-response effects of ara-A and other nucleoside analogs on {('3)H}thymidine incorporation into DNA of whole cells suggested that the mechanism of resistance involved altered nucleoside kinases. Time and concentration studies of ara-A uptake and phosphorylation by WT and resistant cells confirmed that ara-ATP accumulated in mutant cells to the extent of only 10 to 30% of the amount in WT cells. In addition, ara-ATP formation in WT cells was reduced below the residual level seen in mutant cells by the adenosine kinase inhibitor 5-iodotubercidin and by 10 (mu)M adenosine. Experiments conducted with cell extracts and partially purified kinases demonstrated directly that there was a significant reduction in the capacity of a mutant kinase to phosphorylate ara-A to its cytotoxic product, ara-ATP. These experiments established that adenosine kinase, specifically, is either absent or significantly reduced in the mutant cells, which renders them resistant to ara-A.