Effects of Tetrahydrouridine on the Uptake and Metabolism of 1-β-d-Arabinofuranosylcytosine in Human Normal and Leukemic Cells

Abstract The effects of a deaminase inhibitor, tetrahydrouridine (THU), on cellular levels of 1-β-d-arabinofuranosylcytosine (ara-C) and its metabolites were studied in vitro in human normal marrow, peripheral white blood cells, and leukemic cells from patients with chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), and acute myelocytic leukemia (AML). Intracellular ara-C and 1-β-d-arabinofuranosyluracil (ara-U) levels were negligible, regardless of THU treatment. Prior to 2 hr of ara-C (1 µg/ml) exposure, preincubation of AML cells for 30 min with or without THU (0.2 mm) did not affect the ara-U formation in media or intracellular ara-C nucleotides. If the exposure time to ara-C was extended to 24 hr without prior THU, the ara-C almost completely converted to ara-U; the amount of 1-β-d-arabinofuranosylcytosine 5′-monophosphate, 1-β-d-arabinofuranosylcytosine 5′-diphosphate, and 1-β-d-arabinofuranosylcytosine 5′-triphosphate (ara-CTP) formed intracellularly was minimal. When THU was preincubated with AML cells, the ara-U formed in the media decreased 2.5-fold, and the ara-C nucleotide pool was increased. More importantly, the ara-CTP level increased to 0.46 µg in the 5-ml culture with 5 × 10 7 cells, 7.5-fold higher than in the cells not pretreated with THU. Similar increases were not observed with normal marrow or peripheral white blood cells. Washing AML cells after preincubating them for 30 min with THU partially reversed the effects of THU. Preincubation of CML cells with THU for 24 hr increased intracellular 1-β-d-arabinofuranosylcytosine 5′-monophosphate, 1-β-d-arabinofuranosylcytosine 5′-diphosphate, and ara-CTP 3-, 8-, and 12-fold, respectively. In CLL cells, however, the addition of THU in the incubation media for up to 48 hr did not affect the formation of ara-C nucleotides. The ratio of ara-C deaminase to kinase activities in CLL cells is less than 0.5, whereas the ratios for CML and AML cells, due to the very high deaminase activity, are 146 and 30, respectively. Similarly, the ratios of deaminase activity to the amount of ara-CTP formed are only 0.7 for CLL cells and 284 and 65 for CML and AML cells. The high deaminase activity in CML and AML cells and the low activity in CLL cells could explain the differential THU effect on these cells. These results suggest that THU, in combination with ara-C, could be useful in the treatment of patients with CML or AML.