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.

Metabolic studies in HEp-2/MP,MIR cells (an adenosine kinase, hypoxanthine phosphoribosyltransferase negative mutant) indicated the presence of adenosine phosphorylase activity. This activity, unknown in established mammalian cell lines, resulted in the glycosidic cleavage of both adenosine and the antiviral drug arabinosyladenine. The activity was observed readily in the presence or absence of the adenosine deaminase inhibitor conformycin. Isopycnic separation of [3H] thymidine-labeled DNA species in CsCl density gradients resulted in the appearance of two distinct peaks. The heavier peak coincided with [14C]thymidine-labeled marker DNA of human origin, whereas the lighter peak was within the range associated with mycoplasmal DNA. Testing by commercial laboratories confirmed the presence of mycoplasma in HEp-2/MP,MIR cells. The contaminant was identified as Mycoplasma hyorhinis, a porcine mycoplasma. Following gamma-irradiation (3000 rads) to block cellular mitosis, the mucoplasma-contaminated HEp-2/MP,MIR cells were cocultivated with mycoplasma-free wild-type HEp-2 cells which did not exhibit adenosine phosphorylase activity. Following serial cocultivation in a medium designed to favor the survival of the wild-type cells, adenosine phosphorylase activity was found in the previously uninfected cells. Studies of this nature emphasize the need for investigators to carefully monitor their cell lines for mycoplasma.

Studies were performed to test whether 9-/l-D-arabinofuranosyladenine (ara-A) would accumulate in erythrocytes as a result of phosphorylation to the nucleotide level. When C3H]ara-A was incubated with whole blood from rat, monkey or man for 2 hr at 37", the drug rapidly equilibrated between erythrocytes and plasma but did not concentrate in the cells. Incubation of C3H]ara-A with rat and human erythrocyte lysates for 2 hr followed by chromato~aphic analysis showed that 2-5 per cent of ara-A was converted to nucleotides. In contrast, 10-35 per cent of ['YY]adenosine was converted to adenine nucteotides under the same conditions. Incubation of C3H]ara-A with human erythrocyte lysates for 18 hr resulted in a conversion of approx. 40 per cent of the labeled drug to nucleotides. Additional chromatography revealed, however, that the nucleotide fraction contained almost no arabinosyl nucleotides. Rather, 90 per cent of the label in the nucleotide fraction was identified as IMP. These results indicate that only a minor amount, if any, of ara-A was phosphorylated by erythrocyte enzymes to yield arabinosyl nucleotides. An alternative pathway converted much of the labeled drug to ribosyl nucleotides via the deamination of ara-A to ara-hypoxanthine, cleavage to hypoxanthine and conversion of the free hypoxanthine to IMP.