EFFECTS OF 2-CHLORO-9-(2-DEOXY-2-FLUORO-BETA-D-ARABINOFURANOSYL)ADENINE ON K562 CELLULAR-METABOLISM AND THE INHIBITION OF HUMAN RIBONUCLEOTIDE REDUCTASE AND DNA-POLYMERASES BY ITS 5'-TRIPHOSPHATE

2-Chloro-9-(2-deoxy-2-fluoro-beta-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 [H-3]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10-mu-M 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-alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase-alpha. Inhibition of DNA polymerase-alpha was competitive with respect to dATP (K(i) of 1-mu-M). The three analogue triphosphates were incorporated into the DNA by DNA polymerase-alpha 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-beta, DNA polymerase-gamma, 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-alpha 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.