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Bromodeoxyuridine Enhancement of 1-ß-D-Arabinofuranosylcytosine Metabolic Activation and Toxicity in HL-60 Leukemic Cells¹

Division of Developmental Therapeutics, University of Maryland Cancer Center, and Division of Hematology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201

We tested whether bromodeoxyuridine (BrdUrd), an analogue of thymidine (dThd), enhances 1-ß-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-ß-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.

¹ Supported in part by American Cancer Society Research Grant CH-284 and by Grant 1RO-1-CA40188-01 of the National Cancer Institute, NIH. Presented in part at the annual meeting of the American Association for Cancer Research, Los Angeles, CA, May 1986.

² Special Fellow of the Leukemia Society of America. To whom requests for reprints should be addressed, at University of Maryland Cancer Center, University of Maryland School of Medicine, Room 9-015, NFBRB, 655 West Baltimore Street, Baltimore, MD 21201.

Received 6/22/87. Revised 10/15/87. Accepted 10/29/87.