Antiproliferative Effects of 9-P-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.

Tritiated Thymidine Incorporation Does Not Measure DNA Synthesis in Ribavirin-Treated Human Cells

“…In addition, duplicate flasks without drug were held at 20C during the 12-hour incubation period to provide data on the amount of DNA present prior to the incubation period. After incubation, cells from all cultures were harvested and portions of suspended cells were assayed for cell number and incorporated [3HJthymidine by techniques described previously (5,17). The remainder of the cells were pelleted by centrifugation, lysed by resuspension in 0.1I percent sodium citrate containing propidium diiodide (50 RigIml), and examined in a Coulter model TPS-I flow cytometer according to the procedure of Krishan (18).…”

Chemotherapy of Viral Infections

“…Adenosine deaminase was found and isolated as the enzyme which converts ara-A to ara-Hx (CORY and SUHADOLNIK 1965;FREDERIKSEN 1966;BLOCH et al 1967). DRACH et al (1977) also demonstrated that ara-A significantly inhibited cellular growth and DNA synthesis in adenosine deaminase-negative cell lines (B-mix K-44/6). They also showed that when adenosine deaminase-containing calf serum was added to the media, the cytotoxic effect of ara-A was almost reversed.…”

Iontophoretic Application of Adenine Arabinoside Monophosphate to Herpes Simplex Virus Type 1-Infected Hairless Mouse Skin

“…However, Whitely et al (30) have reported the successful control of biopsy-proven HSV encephalitis by systemic administration of ara-A. Topical application of antiviral agents has shown moderately successful results in the eye but less than satisfactory results on skin and mucocutaneous junctions (14,15 Recently, ara-AMP was synthesized to eliminate some of the disadvantages of ara-A, i.e., extremely low water solubility (24) and metabolic instability (1,5,24). Even though ara-AMP has been used successfully against HSV in vitro (26,28) and in animal models (15,17,27,28) the transport of ara-AMP across the cell membrane is highly limited because of its charged phosphate group.…”