2-Chloro-9-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nm inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5′-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nm Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 µm Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2′-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nm). The effect of Cl-F-ara-ATP on human DNA polymerases α, β, and γ isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-β-d-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase α. Inhibition of DNA polymerase α was competitive with respect to dATP (K1 of 1 µm). The three analogue triphosphates were incorporated into the DNA by DNA polymerase α as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase β, DNA polymerase γ, or DNA primase. These results indicate that the inhibition of DNA synthesis by Cl-F-ara-A was due to the inhibition of ribonucleotide reductase activity and inhibition of chain elongation by DNA polymerase α and that, with respect to inhibition of these enzymes, Cl-F-ara-A incorporates the best properties of F-ara-A and 2-chloro-2′-deoxyadenosine into one compound.

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Supported by National Cancer Institute Grant CA34200.

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