Deoxynucleotide pool depletion and sustained inhibition of ribonucleotide reductase and DNA synthesis after treatment of human lymphoblastoid cells with 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) adenine.
The action of the new adenine nucleoside analogue 2-chloro-9-(2-deoxy-2-fluoro-beta-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. [3H]Thymidine incorporation into DNA in human lymphoblastoid CEM cells was inhibited by Cl-F-ara-A in a concentration-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 dTTP pools. This was in concordance with the results of in situ assays of ribonucleotide reductase, which demonstrated profound inhibition of CDP reduction in cells incubated with Cl-F-ara-A; reduction of ADP, GDP, and UDP were affected to lesser extents. Reductase activity was inversely correlated with the cellular Cl-F-ara-ATP level, and inhibition of the enzyme was saturated when cellular Cl-F-ara-ATP reached 25 microM. In vitro DNA primer extension assays indicated that Cl-F-ara-ATP competed with dATP for incorporation into A sites of the extending DNA strand catalyzed by both human DNA polymerases alpha and epsilon. The incorporation of Cl-F-ara-AMP into DNA inhibited DNA strand elongation; the most pronounced effect was observed at Cl-F-ara-ATP:dATP values >1. The sustained inhibition of ribonucleotide reductase and the consequent depletion of deoxynucleotide triphosphate pools result in a cellular concentration ratio of dATP to Cl-F-ara-ATP, which favors analogue incorporation into DNA, an action that has been strongly correlated with loss of viability.
Xie
K C
KC
Department of Clinical Investigation, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Plunkett
W
W
D000227:Adenine Nucleotides; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001087:Arabinonucleosides / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D000077866:Clofarabine; D004247:DNA / Q000096:biosynthesis*; D006801:Humans; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*
D000227:Adenine Nucleotides; D000964:Antimetabolites, Antineoplastic; D001087:Arabinonucleosides; D009711:Nucleotides; D000077866:Clofarabine; D004247:DNA; D012264:Ribonucleotide Reductases