POTENTIATION OF THE ACTIVITY OF NITRIC-OXIDE BY THE PROTEIN-KINASE-C ACTIVATOR PHORBOL ESTER IN HUMAN MYELOID LEUKEMIC HL-60 CELLS - ASSOCIATION WITH ENHANCED FRAGMENTATION OF MATURE GENOMIC DNA

Nitric oxide (NO) has been known to induce programmed cell death or ap optosis in murine macrophages, mouse splenocytes, and thymocytes. We d emonstrate 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 co ndensation of nuclear chromatin, nuclear fragmentation, and the apopto tic peak of propidium iodide-stained nuclei by how cytometry. Phorbol ester alone had a small effect on inducing DNA damage, whereas SNP in combination with phorbol ester at all concentrations tested markedly i ncreased the extent of fragmentation. Maximal potentiation of fragment ation (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 agen ts 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-gam ma but not by 1.5% (v/v) DMSO or 1 mu M retinoic acid. The stage-2 tum or promotor mezerein also mimicked the effect of phorbol ester to indu ce NO-induced apoptosis. In contrast, PKC inhibitors such as staurospo rine and 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine partially bloc ked high concentration of SNP (2-3 mM)-induced apoptosis, suggesting t hat activation of PHC closely relates to the potentiation of the activ ity of NO on HL-60 cell apoptosis. Under the same conditions, SNP in c ombination with phorbol ester caused apoptosis in another transformed cell line, U-937 cells, but was ineffective at inducing apoptosis in n ormal peripheral blood mononuclear cells. Taken together, these findin gs suggest that exposure of HL-60 cells to phorbol ester renders them more susceptible to NO-induced DNA damage and that this phenomenon con tributes to the cytotoxic effects of the NO-PKC combination in myeloid leukemia cells. (C) 1997 Academic Press.