High-does nitric oxide induces apoptosis in HL-60 human myeloid leukemia cells

Abstract

The treatment of human promyeloid leukemia cell line HL-60 cells with nitric oxide (NO) is associated with loss of proliferative apacity and induction of monocytic differentiation. The present results demonstrate that treatment of asynchronous human HL-60 leukemia cells with sodium nitroprusside (SNP), a NO generating agent, is also associated with oligonucleosomal DNA cleavage, including morphological changes, condensation of nuclear chromatin, nuclear fragmentation, and the apoptotic peak of propidium iodide-stained nuclei in flow cytometric analysis. SNP-induced apoptosis was associated with a steep inhibition of the proliferation of the cells in a time- and concentration- dependent manner. Endonucleolytic cleavage was prevented by addition of ZnSO4 or -acetylcysteine, a free radical-scavenging thiol compound. In addition, potassium ferricyanide, which is structurally similar to SNP except for the absence of a nitroso group, did not induce growth inhibition and apoptosis in HL-60 cells. However, the cells which did not undergo apoptosis displayed characteristics of monocytic differentiation, including spreading, vacuolization, expression of monocyte marker CD14, and increased capacity to produce hydrogen peroxide. Since it was of interest to examine whether NO also induce apoptosis in the cells which was undergone differentiation, DNA fragmentation was studied in the cells pretreated with phorbol ester, a monocytic differentiation agent, for 48 h and then monitored for 18 h in the presence of SNP. Flow cytometric analysis revealed no characteristic apoptotic pattern in phorbol 12-myristate 13 acetate-pretreated cells, implying that a more efficient defense mechanism may exist in the differentiating cells. Collectively, these data suggest that NO elaborated in the bone marrow microenvironment might have biphasic regulatory roles in normal and malignant hematopoietic programmed cell death in addition to its already known role as a cell differentiating molecule in HL-60 cells.