Antiproliferative effects of 9-beta-d-arabinofuranosyladenine in a mammalian cell line devoid of adenosine deaminase activity

Abstract

The effect of ara-A on cellular growth, DNA synthesis, and RNA synthesis, and RNA synthesis was measured in an established cell line (B-mix K-44/6) devoid of adenosine deaminase activity. Cells adapted to growth in a medium supplemented with horse serum provided an environment totally lacking adenosine deaminase activity whereas cultivation of cells in a medium supplemented with calf serum provided a system capable of deaminating ara-A to ara-H (half-life = 14 hours). Under deaminase-free conditions early log phase cells underwent 1.5 population doublings during 28 hours compared with 0.25 doublings in the presence of 37 micronM ara-A. When cells were grown in medium supplemented with calf serum the additionof 37 to 225 micronM ara-A resulted in a cessation of mitosis for periods of 5 to 30 hours respectively. Following this quiescent period growth resumed at the original rate. With 600 micronM ara-A mitosis was reversibly inhibited up to 35 hours after drug addition. The effects of ara-A on RNA and DNA synthesis were monitored by continuously or pulse labeling B-mix K-44/6 cells with [3H]-uridine or [3H]thymidine. Ara-A did not influence RNA synthesis as judged by labeled uridine incorporation. Under deaminase-free conditions, 5.4 micronM ara-A inhibited labeled thymidine incorporation by 50%. In the presence of the enzyme, approximately twice the ara-A concentration was required for the same inhibition; furthermore the initial inhibition was followed by a partial recovery in the rate of thymidine incorporation. Examination of thymidine incorporation. Examination of thymidine nucleotide pools during ara-A treatment revealed to changes in the labeling of dTMP, dTDP, and dTTP. Thus inhibition of [3H]thymidine incorporation by ara-A accurately reflected inhibition of DNA synthesis. We conclude that, in spite of an initial inhibition of DNA synthesis and mitosis by ara-A, B-mix K-44/6 cells recover from the inhibitory effects if the drug is removed either by a change in the culture medium or by metabolism to ara-H.