Mode of action of 9-β- d-arabinosyladenine and 1-β- d-arabinosylcytosine on DNA synthesis in human lymphoblasts

Abstract

The effects of 9-β- d-arabinosyladenine (AraAde), 1-β- d-arabinosylcytosine (AraCyt) and 2′-deoxyadenosine on DNA replication in cultured human lymphoblasts (CCRF-CEM line) were studied by pulse-labeling cells with [ 3H]-thymidine and analyzing the nascent DNA by velocity sedimentation in alkaline sucrose gradients. At doses that reduced the overall rate of DNA synthesis to 50–70% of control values, both AraAde and AraCyt profoundly inhibited the formation of new replicons, with secondary effects on chain elongation contributing to the total inhibition of DNA synthesis. In contrast, the suppression of DNA synthesis by 2′-deoxyadenosine stemmed mainly from an inhibition of chain elongation. These studies also disclosed that about 100 times more AraAde than AraCyt was required to produce a similar inhibition of DNA replication in CCRF-CEM cells. Determination of intracellular concentrations of the nucleoside triphosphates (AraCTP and AraATP) indicated that 90% inhibition of DNA synthesis was achieved at 1.6 and 25 pmol/l · 10 6 cells, respectively. Studies with cell lysates revealed that the replicative machinery in CCRF-CEM cells is more sensitive to AraCTP than to AraATP. This finding contrasts with earlier research, in which the inhibition of purified DNA polymerase by either AraATP of AraCTP yielded essentially the same K i value. The difference in sensitivity of the cell lysate to these arabinonucleotides may reflect either a target enzyme other than DNA polymerase or, more plausibly, some subtle interaction of the polymerase with other components of the replicative process.