Pharmacological and biochemical strategies to increase the accumulation of arabinofuranosylguanine triphosphatein primary human leukemia cells.

Purine nucleoside phosphorylase deficiency leads to a dGTP-mediated T-lymphopenia, suggesting that an analogue of deoxyguanosine would be selectively effective in T-cell disease. 9-beta-D-Arabinofuranosylguanine (ara-G) is relatively resistant to hydrolysis by purine nucleoside phosphorylase and selectively toxic to T cells, but its low solubility has prevented its use in the clinic. 2-Amino-6-methoxy-arabinofuranosylpurine (GW506U) serves as the water-soluble prodrug for ara-G. A Phase I trial in patients with refractory hematological malignancies demonstrated that the clinical responses to this agent were directly related to the peak levels of ara-G 5'-triphosphate (ara-GTP) in target cells. The aim of the present study was to develop and test strategies to increase intracellular accumulation of ara-GTP in primary human leukemia cells of myeloid and B-lymphoid origin. Three strategies were tested. First, incubations with 100 microM ara-G for 4 h produced a linear median accumulation rate of 19 microM/h (range, 2-45 microM/h; n = 15) in lymphoid leukemia cells and 16 microM/h (range, 0.5-41 microM/h; n = 11) in myeloid leukemia cells. Saturation of ara-GTP accumulation was achieved only after 6-8 h exposure in both lymphoid and myeloid leukemia cells, suggesting a rationale for prolonged infusion. Second, a dose-dependent increase in ara-GTP accumulation was observed with incubations of 10-300 microM ara-G for 3 h. Hence, dosing regimens that achieve high plasma levels of ara-G during therapy may increase cellular levels of ara-GTP. Finally, a biochemical modulation approach using in vitro incubation of leukemia cells with 10 microM 9-beta-D-arabinofuranosyl-2-fluoroadenine for 3 h, followed by either 50 or 100 microM ara-G for 4 h, resulted in a statistically significant median 1.3-fold (range, 1.1-9.0-fold; P = 0.034) and 1. 8-fold (range, 0.9-10.6 fold; P = 0.018) increase in ara-GTP compared to cells incubated with ara-G alone. Extension of these studies to ex vivo incubations confirmed our in vitro findings. These strategies will be used in the design of clinical protocols to increase ara-GTP accumulation in leukemia cells during therapy.

Excision of 2',2'-difluorodeoxycytidine (gemcitabine) monophosphate residues from DNA.

The activity of gemcitabine (dFdC), an effective agent against solid tumors, depends on the incorporation of its triphosphate into DNA. In vitro investigations demonstrated that, depending on the sequence of template DNA, polymerases may pause after incorporation of gemcitabine nucleotide at either the 3'-terminal or 3'-penultimate position. Proofreading enzymes such as 3'-->5' exonucleases, which are associated with DNA polymerases, can excise mismatched deoxynucleotides from DNA. To model this reaction, we evaluated excision of the gemcitabine nucleotide from oligodeoxynucleotide (19-mer) containing 3'-penultimate dFdC monophosphate (dFdCMP) or dCMP by the 3'-->5' exonuclease of the Klenow fragment. The rate of excision of the 3'-terminal deoxynucleotide was similar, with both primers resulting in formation of primers with terminal dCMP or dFdCMP. The primer containing dCMP was further excised, and by 40 min, more than 75% of total radioactivity was in excision products smaller than 18-mer. In contrast, most of the primers (90%) with terminal dFdCMP were unexcised. When primers terminated with either dFdCMP or dCMP were used as substrates, normal primer was hydrolyzed almost completely by 20 min; however, only 40% of primers containing dFdCMP had excision of dFdCMP molecule. Kinetic studies demonstrated that the enzyme had similar affinity for primers containing penultimate or terminal dFdCMP, but the apparent Vmax for excision was 4-5-fold greater for removal of a 3'-terminal deoxynucleotide than for cleavage of a dFdCMP molecule. Reaction conditions that permitted polymerization of one deoxynucleotide to primers containing either 3'-penultimate dCMP or dFdCMP were used to evaluate excision during DNA synthesis. The excised primers could not be extended because the reaction lacked the requisite deoxynucleotide triphosphate. After 5 min, more than one-half of the dCMP primers were extended, whereas only 15% had been excised. In comparison, 30% of the analogue-containing primers lost the terminal deoxynucleotide, with a proportional lower incidence of extension (30%). Lesser excision of dFdCMP-containing substrate was observed in reactions containing deoxynucleotide triphosphates required to make full-length products. Consistent with this result, in the absence of 3'-->5' exonuclease activity, both primers were extended similarly by the polymerization unit of the Klenow fragment. Taken together, these data demonstrate that dFdCMP residues are difficult to excise from DNA, and DNA polymerase can extend primers with 3'-dFdCMP. This results in the internal incorporation of dFdCMP into DNA, as observed in whole cells.