[CANCER RESEARCH 56. 3030-3037. July I. 1996] Deoxynucleotide Pool Depletion and Sustained Inhibition of Ribonucleotide ReducÃ-aseand DNA Synthesis after Treatment of Human Lymphoblastoid Cells with 2-Chloro-9-(2-deoxy-2-fluoro-/3-D-arabinofuranosyl)adenine1 Kevin Chunxi Xie and William Plunkett2 Department uf Clinical Investigation, The Uni\~ersit\ of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 ABSTRACT The action of the new adenine nucleoside analogue 2-chloro-9-(2-deoxy- 2-fluoro-ß-D-arabinofuranosyl)adenine (Cl-F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro assay systems with purified DNA polymerases. pHJThymidine incorporation into DNA in human lymphoblastoid CEM cells was inhibited by Cl-F-ara-A in a concentra tion-dependent manner that was not reversed 72 h after removal of Cl-F-ara-A from the medium. Deoxynucleotide pools were depressed after incubation of Cl-F-ara-A for 3 h and only partially recovered following washing the cells into drug-free medium. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the dATP, dGTP, and dTTl' pools. This was in concordance with the results of in situ assays of ribonucleotide reducÃ-ase, which demonstrated profound inhibition of CDP reduction in cells incubated with Cl-F-ara-A; reduction of ADP, GDP, and 11)1' were affected to lesser extents. ReducÃ-ase activity was inversely correlated with the cellular CI-F-ara-ATP level, and inhibition of the enzyme was saturated when cellular Cl-F-ara-ATP reached 25 JUM. In vitro DNA primer extension assays indicated that CI-F-ara-ATP competed with dATP for incorporation into A sites of the extending DNA strand catalyzed by both human DNA polymerases a and E. The incorporation of Cl-F-ara-AMP into DNA inhibited DNA strand elongation; the most pronounced effect was observed at CI-F-ara-ATP:dATP values >1. The sustained inhibition of ribonucleotide reductase and the consequent de pletion of deoxynucleotide triphosphate pools result in a cellular concen tration ratio of dATP to CI-F-ara-ATP, which favors analogue incorpo ration into DNA, an action that has been strongly correlated with loss of viability. INTRODUCTION Nucleoside antimetabolites have been developed into some of the most effective anticancer and antiviral agents. Cl-F-ara-A3 is a new deoxyadenosine analogue that shows potent cytotoxicity in cell lines such as CEM, K562, Hep2, and murine leukemia L1210 (1-3). Cl-F-ara-A exhibited therapeutic activity in murine tumor models (P388 leukemia, colon 36, and mammary 16/c) and was curative against both early- and advanced-stage colon 36 (3, 4). The chloro group at the 2-carbon of the purine ring confers resistance to deami- nation by adenosine deaminase (5), whereas substitution of a fluorine at the C-2' position in the arubino configuration decreases the sus ceptibility of Cl-F-ara-A to phosphorolytic cleavage by bacterial purine nucleoside phosphorylase (6) to roughly one-third that of arabinosyl- or 2'-deoxynucleosides (4). Experiments using isolated Received 1/25/96; accepted 5/1/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely lo indicate this fact. ' This work was supported in part by Grant DHP-1 from the American Cancer Society and Grant CA28596 from the National Cancer Institute. Department of Health and Human Services. K. C. X. is a Rosalie B. Hite predoctoral fellow. 2 To whom requests for reprints should be addressed, at Department of Clinical Investigation. Box 71. The University of Texas M. D. Anderson Cancer Center. 1515 Holcombe Boulevard. Houston. TX 77030. Phone: (713) 792-3335: Fax: (713) 794-4316. 1 The abbreviations used are: Cl-F-ara-A, 2-chloro-9-(2-deoxy-2-fluoro-ß-D-arabino- furanosylladenine; CI-F-ara-ATP. the triphosphaie of Cl-F-ara-A; Cl-dAdo, 2-chloro-2'- deoxyadenosine; CEM. human lymphoblastoid cell line CCRF-CEM: dNTP, deoxyribo- nucleoside triphosphate; pol, DNA polymerase; HPLC. high-pressure liquid chromatography. perfused rat liver demonstrated that Cl-F-ara-A is more stable than Cl-dAdo against deglycosylation by hepatic enzymes (7). The 2'- arabino-fluoro substitution also results in an increased acid stability of Cl-F-ara-A relative to dAdo and Cl-dAdo (2). Qian el al. (8) reported that the oral bioavailability of Cl-F-ara-A in rats could reach 50%, suggesting that Cl-F-ara-A may have potential as an oral drug. Previous studies have demonstrated that to be cytotoxic to cultured leukemia cells, Cl-F-ara-A required membrane transport to enter cells (9), after which it is phosphorylated to mono-, di-, and triphosphates of Cl-F-ara-A (10). The monophosphate and triphosphate are the predominant metabolites (11). Inhibition of DNA synthesis is the most prominent activity of Cl-F-ara-A observed in cultured cells, with little effect on RNA or protein synthesis (10). Using a synthetic oligode- oxynucleotide template, the effect of Cl-F-ara-ATP on poi a was shown to be more potent than pol ß, pol y, or DNA primase (10). Although DNA polymerases do not appear to be inhibited directly by Cl-F-ara-ATP, incorporation of the analogue into DNA impedes fur ther polymerization. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA, suggesting that the incorporation of Cl-F-ara-AMP into DNA is critical for the cytotoxicity of the compound (11). In these studies, the site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the nat ural substrate dATP. At low Cl-F-ara-ATP:dATP values, incorpora tion was mainly in phosphodiester linkages at internal sites, whereas at higher Cl-F-ara-ATP:dATP values, Cl-F-ara-AMP was principally detected at terminal sites, suggesting that the cellular deoxynucleotide pools significantly affect DNA termination in whole cells. Incubation of K562 cells with Cl-F-ara-A resulted in depletion of dATP, dCTP, and dGTP pools but did not affect the dTTP pool. Cl-F-ara-ATP potently inhibited the reduction of ADP to dADP in extracts of K562 cells, suggesting that ribonucleotide reductase is a primary target of this compound. However, the regulation of this enzyme activity is complex in whole cells (12, 13), and the purifica tion of the enzyme disrupts its regulated nature. Because investiga tions of ribonucleotide reductase activity in intact cells are likely to provide a clearer picture of the effects of metabolite inhibitors, in situ assays have now been performed to evaluate the inhibitory effect of Cl-F-ara-A metabolites on this enzyme. Furthermore, we investigated the impact of the ratio of the concentrations of Cl-F-ara-ATP and dATP upon the incorporation of Cl-F-ara-AMP into DNA and its actions on DNA strand elongation catalyzed by both human poi a and poi e in an M13 DNA primer extension system. A preliminary report of these studies has been published (14). MATERIALS AND METHODS Materials. Cl-F-ara-A was supplied by Dr. John A. Secrist III. Southern Research Institute. Birmingham, AL 35205. Cl-F-ara-ATP was chemically synthesized by the procedure described by Ludwig (15). [8-3H]Cl-F-ara-A (specific activity, 4 Ci/mmol) and [l4C]cytidine (55 mCi/mmol) were prepared by Moravek Biochemicals. Inc. (Brea, CA). [m£r/iv/-3H|Thymidine (specific activity. 52 Ci/mmol). [6-3H]uridine (27.5 Ci/mmol), [2-3H]adenosine (40 Ci/mmol), [8-3H)guanosine (8 Ci/mmol), and [7-32P]ATP (3000 Ci/mmol) 3030