Incorporation of fludarabine and 1-β-D-arabinofuranosylcytosine 5'- triphosphates by DNA polymerase α: Affinity, interaction, and consequences

Fludarabine and 1-β-D-ambinofuranosylcytosine (ara-C) are effective nucleoside analogues for the treatment of leukemias when used as single agents or together. Recent trials of the fludarabine and ara-C therapy with or without growth factors suggested an improved clinical response by combining fludarabine and ara-C. The activity of these antimetobolites depends on their phosphorylation to the respective triphosphates, F-ara-ATP and ara-CTP. The principal mechanism through which these triphosphatase cause cytotoxicity is incorporation into DNA and inhibition of further DNA synthesis. A model system of DNA primer extension on a defined template sequence was used to quantitate the consequences of incorporation of one or two analogues by human DNA polymerase α (pol α). The template (31-mer) was designed so that DNA pol α incorporated six deoxynucleotides (alternately G and T) on the 17-mer primer, followed by insertion of an A and then a C. The pruner was then elongated with G and T to the full-length product. The apparent K(m)s of DNA pol α to incorporate these analogues (0.053 and 0.077 μM, respectively) were similar to the K(m) for dCTP (0.037 μM) and dATP (0.044 μM), suggesting that the enzyme recognized these analogues and incorporated them efficiently on the growing DNA primer. The velocity of extension (V(max)) of these primers ranged between 0.53 and 0.77%/min when normal nucleotides were present. Once inserted at the 3'-terminus, F-ara-AMP or ara-CMP were poor substrates for extension. However, in reactions lacking dCTP and dATP and with high concentrations of ara-CTP, ara-CMP was inserted by pol α after incorporation of the F-ara-AMP residue. This tandem incorporation of the two analogues resulted in almost complete inhibition (99.3%) of further extension of the primer. In the presence of competing deoxynucleotides, each analogue resulted in a dose-dependent inhibition of DNA synthesis. When present together, inhibition of the primer elongation was more than additive at low concentrations of analogue triphosphates. Based on these results and the intracellular pharmacokinetics of ara-CTP and F-ara- ATP in leukemia blasts, we propose a pharmacodynamic model to explain interactions between these analogues during combination chemotherapy.