Nitric oxide (NO) has been known to induce programmed cell death or apoptosis in murine macrophages, mouse splenocytes, and thymocytes. We demonstrate here that phorbol ester, a protein kinase C (PKC) activator, synergistically augments the antileukemic actions of the NO in HL-60 human promyelocytic leukemia cells. Exposure of cells to sodium nitro-prusside (SNP; 0.5 to 2 mM), a NO-generating agent, induced time- and concentration-related increases in morphological changes, including condensation of nuclear chromatin, nuclear fragmentation, and the apoptotic peak of propidium iodide-stained nuclei by flow cytometry. Phorbol ester alone had a small effect on inducing DNA damage, whereas SNP in combination with phorbol ester at all concentrations tested markedly increased the extent of fragmentation. Maximal potentiation of fragmentation (e.g., four- to fivefold greater than that obtained with 0.5 mM SNP alone) was observed with simultaneous treatment of phorbol ester. Similar results were obtained with another commonly used NO donor agents such as SNAP (0.5 mM) and GSNO (0.5 mM). DNA fragmentation of HL-60 cells was also augmented by 100 U/ml human recombinant interferon-gamma but not by 1.5% (v/v) DMSO or 1 microM retinoic acid. The stage-2 tumor promotor mezerein also mimicked the effect of phorbol ester to induce NO-induced apoptosis. In contrast, PKC inhibitors such as staurosporine and 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine partially blocked high concentration of SNP (2-3 mM)-induced apoptosis, suggesting that activation of PKC closely relates to the potentiation of the activity of NO on HL-60 cell apoptosis. Under the same conditions, SNP in combination with phorbol ester caused apoptosis in another transformed cell line, U-937 cells, but was ineffective at inducing apoptosis in normal peripheral blood mononuclear cells. Taken together, these findings suggest that exposure of HL-60 cells to phorbol ester renders them more susceptible to NO-induced DNA damage and that this phenomenon contributes to the cytotoxic effects of the NO-PKC combination in myeloid leukemia cells.