Differences in Metabolism and Cytotoxicity between 9-ß-o Arabinofuranosyladenine and 9-/?-D-Arabinofuranosyl-2- fluoroadenine in Human Leukemic Lymphoblasts1

We compared the effects of 9-/?-o-arabinofuranosyladenine (ara-A) and its 2-fluoro derivative (F-ara-A) on DMA synthesis and viability in human leukemic lymphoblasts (CCRF-CEM). The relative DNA content and cloning capacity of the cells were determined by flow cytometry and growth in methylcellulose, respectively. Incubation with either 1 /IM ara-A in the presence of 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase, or 2.5 JIMF-ara-A alone for 18 hr stopped the progression of cells through S phase. At higher concentrations, from 2.5 to 20 JIM, both arabinonucleosides preferentially produced toxic effects in cells arrested in S phase. The DNA-inhibitory effects and cytotoxicity of F-ara-A, but not ara-A, were prevented by adding deoxycytidine to the growth medium. A CCRF-CEM variant that was unable to phosphorylate deoxycytidine proved to be highly resistant to the inhibitory effects of F-ara-A but was normally sensitive to ara-A. These results indicate that the cytotoxicity of ara-A in both sensitive and resistant cells and of F-ara-A in sensitive cells is probably mediated by the intracellular formation of nucleotides and inhibition of DNA synthesis. By contrast, the reduction in the cloning efficiency of 1-/S-Darabinofuranosylcytosine-resistant cells at high concentrations of F-ara-A may be due to some direct effect of the nucleoside. Finally, the different susceptibility of the mutant CCRF-CEM cells to the toxic effects of ara-A and F-ara-A suggests that alternative pathways phosphorylate these arabinonucleosides, one being associated with deoxycytidine kinase and the other with either deoxyadenosine kinase or, less likely, adenosine kinase. dAdo, ara-ATP can either replace these nucleotides or interfere with their metabolism in a variety of cellular reactions. For example, ara-ATP is a potent inhibitor of several enzymes, including ribonucleotide diphosphate reducÃ-ase (12, 25) and DNA polymerase (14, 26), and can be incorporated into DNA (8). Still unclear is whether the cytotoxicity of ara-A stems from inhibition of, or interference with, one step in a single metabolic reaction or whether it represents the cumulative effect of inter ference with many reactions. The antiviral and antitumor activities of ara-A are limited, inasmuch as the drug is rapidly degraded by adenosine deaminase (EC 3.5.4.4). Although agents such as erythro-9-(2hydroxy-3-nonyl)adenine and 2'-deoxycoformycin inhibit the deaminase, leading to increased biological activity by ara-A (21, 27), they are highly toxic in normal tissues (10), which may restrict their value in chemotherapy. To avoid the use of deaminase inhibitors, Montgomery and Hewson (24) synthe sized a 2-fluoro derivative of ara-A (F-ara-A). This compound is not deaminated but is phosphorylated and has activity against L1210 murine leukemia cells (5, 6). In this paper, we have compared the effects of ara-A (in the presence of deoxycoformycin) and F-ara-A on DNA synthesis and viability of human leukemic lymphoblasts (CCRF-CEM, hereafter CEM), using flow cytometry and cell cloning. Both nucleosides exerted their inhibitory and toxic effects primarily on cells that were actively synthesizing DNA. Addition of dCyd to the growth medium prevented cytotoxicity due to F-ara-A. A CEM variant deficient in deoxycytidine kinase was resistant to F-ara-A but not to ara-A, suggesting that in CEM cells, the 2 drugs are metabolized by alternate pathways.