LUMINOL AND DIAZOLUMINOMELANIN AS INDICATORS OF HL-60 CELL-DIFFERENTIATION

This paper describes use of a novel substituted melanin which is usefu l in detection of differentiating leukemia cells an their membranes. C omparisons of luminol-(5-amino-2,3-dihydro-1,4-phthalazinedione) and d iazoluminomelanin (DALM)-mediated chemiluminescence (CL) were made wit h various types of differentiated and undifferentiated HL-60 whole cel ls, cell lysates, and membrane fractions. Luminol had a greater CL res ponse than DALM with HL-60 promyelocytic stem cells and differentiated macrophage-like or neutrophil-like whole cell and cell lysate prepara tions. However, DALM showed markedly greater CL than luminol for membr ane fractions derived from each cell type. The greatest luminol-depend ent CL was observed for cell types high in myeloperoxidase (MPO). The greatest DALM-mediated CL was seen with cell types that are high in MP O or strong producers of superoxide (O2-) anions. In some cases, signi ficant differences in CL could also be distinguished on the basis of i nducing agent used [i.e. dimethylsulfoxide, all-trans retinoic acid or 12-o-tetradecanoylphorbol-13-acetatel. Both luminol- and DALM-depende nt CL were strongly inhibited by preincubation of cellular preparation s with 3-amino-L-tyrosine (a component of DALM). Taken together, these data suggest that the reaction mechanism of luminol favors interactio n with cytoplasmic MPO whereas that of DALM favors membrane interactio ns. Thus, both reagents may be of use in assays to detect differentiat ing leukocytes or their cellular components.