We have examined the interaction between 1-β-d-arabinofuranosylcytosine (ara-C) and the macrocyclic lactone protein kinase C activator bryostatin 1 in the human promyelocytic leukemia cell line HL-60. Preexposure of cells to 10 nm bryostatin 1 for 24 h, followed by an additional 24-h incubation with 10 µm ara-C, resulted in greater than additive inhibitory effects toward clonogenic HL-60 cells. In a series of alkaline elution assays, cells preincubated with bryostatin 1 and prelabeled with [3H]thymidine exhibited a significant increase in DNA fragmentation following exposure to ara-C in comparison to cells exposed to ara-C alone. This increase in DNA damage was apparent at both neutral and alkaline pH and was not protein associated. In contrast, studies using cells pulse-labeled with [3H]thymidine immediately before analysis suggested that bryostatin 1 pretreatment did not increase the ability of ara-C to interfere with DNA replicative intermediates. Additional studies demonstrated that the increase in DNA fragmentation induced by bryostatin 1 and ara-C preceded both loss of cell membrane integrity (as determined by trypan blue exclusion) as well as depletion of intracellular ATP and NAD pools. Furthermore, the enhanced inhibitory effects of bryostatin 1 and ara-C toward clonogenic HL-60 cells did not appear to result from the induction of cellular differentiation. Finally, agarose gel electrophoresis of DNA obtained from cells exposed to both bryostatin 1 and ara-C revealed a pattern of integer multiples of 180- to 200-base pair fragments commonly associated with endonucleolytic cleavage; the extent of this fragmentation was considerably greater than that observed in cells exposed to ara-C alone. Taken together, these findings suggest that exposure of HL-60 cells to bryostatin 1 renders them more susceptible to ara-C-related DNA damage and that this phenomenon contributes to the cytotoxic effects of this drug combination. They also raise the possibility that bryostatin 1, perhaps through modulation of intracellular signaling events in leukemic cells, has the capacity to potentiate ara-C-related apoptosis or programmed cell death.

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This work was supported by Grant CH-523 from the American Cancer Society (S. G.) and Cancer Center Support Core Grant CA-16059 from the National Cancer Institute, by the Medical College of Virginia Grants-in-Aid Program and A. D. Williams Foundation, the Fannie E. Rippel Foundation, the Robert B. Dalton Endowment Fund, the Thomas F. Jeffries and Kate Miller Memorial Trust, and the Bone Marrow Transplantation Core Research Laboratory of the Medical College of Virginia; G. R. P. is a recipient of Outstanding Investigator Award CA 44344-03A1; and W. D. J. is recipient of a Cancer Biology Fellowship supported by USPHS Training Grant CA-09564-05. Portions of this material were presented in preliminary form at the 82nd Meeting of the American Association for Cancer Research, Inc.

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