Bromodeoxyuridine Enhancement of 1-β-d-Arabinofuranosylcytosine Metabolic Activation and Toxicity in HL-60 Leukemic Cells¹

We tested whether bromodeoxyuridine (BrdUrd), an analogue of thymidine (dThd), enhances 1-β-d-arabinofuranosylcytosine (ara-C) metabolic activation, as does dThd. HL-60 cells were exposed to 10, 100, or 1000 nm ara-C for 3 h. Simultaneous exposure of log phase HL-60 cells to BrdUrd (1–1000 µm) and ara-C for 3 h resulted in enhancement of ara-C incorporation into DNA, with a doubling of incorporation in response to 10 nm ara-C occurring at concentrations of BrdUrd >100 µm. Preexposure of cells to BrdUrd for 16 h followed by addition of ara-C for 3 h resulted in even greater ara-C incorporation into DNA. This increase was most marked at the lower concentrations of ara-C (10 and 100 nm), where approximately 3-fold enhancement of ara-C incorporation was observed in response to BrdUrd concentrations >100 µm. Intracellular pools of 1-β-d-arabinofuranosyl-CTP increased significantly (up to 3-fold) following 16-h exposure to BrdUrd (30, 100, or 300 µm) at all concentrations of ara-C tested. The ara-C phosphorylating activity of cell-free extracts obtained following 16-h exposure of cells to BrdUrd increased 1.5- to 2.3-fold over control. Intracellular dCTP pools fell to approximately 50% of control after exposure to 750 µm BrdUrd or dThd. Exposure to BrdUrd for 16 h caused a concentration-dependent increase in cells with S-phase DNA content, as assessed by flow cytometry, with a doubling of cells in S phase (to 60%) observed in response to 500 µm BrdUrd. HL-60 cells exposed to identical conditions of BrdUrd for 3 h showed no significant alteration in cell cycle phase distribution. Thus, although BrdUrd does increase cells in S phase, the increased ara-C incorporation caused by BrdUrd cannot be explained solely on a cytokinetic basis since enhancement of incorporation was observed after a 3-h exposure of cells to BrdUrd and ara-C. The combination of ara-C (100 nm) and BrdUrd (100–1000 µm) exhibited cytotoxic synergism, as measured by the fluorescein diacetate/propidium iodide method. These data demonstrate a clear potential for BrdUrd modulation of ara-C metabolism in human leukemia. Additionally, the interaction of BrdUrd and ara-C should be considered in the interpretation of studies of the effects of ara-C on DNA synthesis as measured by flow cytometric quantification of incorporated BrdUrd.