Ultrastructural, immunochemical, and cytochemical study of myeloperoxidase in myeloid leukemia HL-60 cells following treatment with succinylacetone, an inhibitor of heme biosynthesis.
Myeloperoxidase (MPO) is a heme-containing glycoprotein found in the primary granules (or azurophilic granules) of human polymorphonuclear leukocytes. In the present study, cultured myeloid leukemia HL-60 cells were exposed for 0-72 h to 250 microM 4,6-dioxoheptanoic acid (succinylacetone, SA), a specific inhibitor of heme biosynthesis, and the effects were evaluated using ultrastructural, immunochemical, and cytochemical methods. En bloc peroxidase staining of glutaraldehyde-fixed cells was accomplished with a 30-min exposure to 3,3'-diaminobenzidine (DAB) tetrahydrochloride. Ultrastructural examination revealed that peroxidase reactivity in the endoplasmic reticulum (ER) was relatively unchanged for 8 h and decreased between 12 and 24 h; however, ER lacked DAB-reactive peroxidase at 48-72 h. Peroxidase reactivity in the ER reappeared within 4 h after removal of SA. Seventy-two hours after exposure to SA the number of condensed cytoplasmic granules stained with DAB was significantly decreased, and many of the granules had a "target" appearance with a central DAB-reactive dense core. Staining of mitochondria was observed with overnight exposure to DAB and persisted in HL-60 cells treated 72 h with SA. Mitochondrial and nuclear morphology appeared unaltered. Immunostaining of MPO in thin sections of paraformaldehyde/glutaraldehyde-fixed unosmicated HL-60 cells, embedded in Lowicryl K4M, was accomplished with sequential exposure to an affinity-purified monospecific rabbit antibody to HL-60-MPO and protein A conjugated to 5- or 10-nm colloidal gold. Compared to untreated control HL-60 cells, cells exposed to SA for 48 h exhibited comparable to increased immunoreactive MPO in the ER, despite the absence of heme-dependent peroxidase reactivity. The data indicate that SA inhibits formation of enzymatically active MPO and that in the presence of SA, the ER contains a form(s) of MPO that lacks enzymatic reactivity.
Castañeda
V L
VL
Department of Pediatrics, University of Texas Health Science Center, San Antonio 78284-7810.
Parmley
R T
RT
Pinnix
I B
IB
Raju
S G
SG
Guzman
G S
GS
Kinkade
J M
JM
D006418:Heme / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D006537:Heptanoates / Q000494:pharmacology*; D006651:Histocytochemistry; D006801:Humans; D007150:Immunohistochemistry; D007951:Leukemia, Myeloid / Q000201:enzymology* / Q000473:pathology*; D008854:Microscopy, Electron; D009504:Neutrophils / Q000187:drug effects; D009195:Peroxidase / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000648:ultrastructure
D006537:Heptanoates; D006418:Heme; C020804:succinylacetone; D009195:Peroxidase