Cyclic Adenosine Monophosphate Inhibits Nitric Oxide-Induced Apoptosis in Human Leukemic HL-60 Cells

Abstract

Previously we reported that phorbol ester, a protein kinase C (PKC) activator, exhibits a unique pattern of potentiation of nitric oxide (NO)-related apoptosis in HL-60 human promyelocytic leukemia cells. Here we show that elevation of intracellular cAMP could protect HL-60 cells from NO- or NO plus PMA-induced DNA damage. Exposure of cells to sodium nitroprusside (SNP; 0.5 to 4 mM), a NO-generating agent, induced apoptotic cell death as monitored by morphological means, gel electrophoresis, andin situTdT-apoptosis assay. However, concomitant incubation of the cells with DB-cAMP markedly inhibited SNP-induced apoptotic cell death in a dose-dependent manner. Similar results were obtained with other commonly used cAMP analogs such as CPT-cAMP and 8-Cl-cAMP and the intracellular cAMP-elevating agent such as forskolin. In contrast, pretreatment of HL-60 cells with H89 or KT5720, which are known to inhibit cAMP-dependent protein kinase (PKA), abolished the protective effect of cAMP analogs and forskolin on SNP-induced apoptosis. Synergism between SNP and phorbol ester to induce apoptosis was also inhibited by prior treatment of HL-60 cells with DB-cAMP or forskolin. The effect of DB-cAMP in maintaining cell viability was not associated with the onset of G0/G1cell cycle arrest. In addition, neither dimethyl sulfoxide nor retinoic acid (which produce granulocyte differentiation) could produce cAMP effect. Under the same conditions, DB-cAMP also inhibited NO- or NO plus phorbol ester-induced apoptosis in another transformed cell line, U-937 cells. Taken together, these findings suggest that exposure of HL-60 cells to cAMP analogs renders them more resistant to NO-induced DNA damage and further suggest the existence of specific down-modulatory mechanisms related to NO-induced apoptotic DNA fragmentation.