monensin and Leukemia--Myeloid

The effects of brefeldin A, monensin, and the weak base NH4Cl on the biosynthesis and processing of cathepsin G and neutrophil elastase of myeloid cells were investigated. Monoblast-like U-937 cells were biosynthetically labeled with [35S]methionine, followed by subcellular fractionation, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Brefeldin A inhibited proteolytic processing, intracellular transport, and secretion. The effects were reversible inasmuch as removal of brefeldin A resulted in a normal pattern of processing and transfer to high-density organelles, corresponding to lysosomes, and restitution of constitutive secretion of precursor forms. Both cathepsin G and neutrophil elastase acquired resistance to endoglycosidase-H, suggesting that conversion to complex oligosaccharide side chains also occurs in the presence of brefeldin A. Monensin and NH4Cl inhibited final proteolytic processing, indicating either that acidification is necessary for directing cathepsin G and neutrophil elastase to lysosomal-like organelles or that the protease(s) responsible for processing requires an acid pH. We conclude that pH-dependent proteolytic processing of cathepsin G and neutrophil elastase occurs in post-Golgi structures and that transfer to lysosomes or an immediately prelysosomal compartment is mandatory for complete processing.

Topics: Ammonium Chloride; Antifungal Agents; Biological Transport; Brefeldin A; Cathepsin G; Cathepsins; Cyclopentanes; Humans; Hydrogen-Ion Concentration; Leukemia, Myeloid; Leukocyte Elastase; Monensin; Pancreatic Elastase; Peroxidase; Serine Endopeptidases; Tumor Cells, Cultured

The Na+/H+ ionophore monensin (M) has been used widely to study intracellular pH gradients and acidic subcellular compartments. In the present study, cultured myeloid leukemia HL60 cells, directly sampled bone marrow cells, and peripheral blood neutrophils were exposed to 1-5 microM monensin for 0.5-20 hours. The effects were evaluated using ultrastructural, cytochemical, and biochemical methods. In HL60 cells and marrow promyelocytes treated with monensin, progressive vacuolation of the trans then the cis Golgi was observed. These vacuoles lacked diaminobenzidine (DAB) reactive peroxidase, high iron diamine (HID) reactive sulfated glycoconjugates, and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) reactive vicinal glycol containing complex carbohydrates, but some cis Golgi elements retained osmium zinc iodide reactive reducing groups. The number of normal intensely stained HID reactive granules decreased and an incomplete granule that was DAB-positive/HID-negative, PA-TCH-SP-negative with flocculent matrix density increased in frequency as a function of time and concentration of monensin. Treatment of HL60 cells with monensin markedly reduced 35SO4 incorporation but myeloperoxidase labeling and activity per cell remained constant, although it shifted to lower density granule fractions consistent with the persistent DAB staining of endoplasmic reticulum and synthesis of a DAB-positive, HID-negative granule in intact HL60 cells. The Golgi complex of monensin-treated myelocytes and segmented neutrophils was also vacuolated. A subpopulation of preformed primary granules in promyelocytes, myelocytes, and segmented neutrophils appeared to increase in size and peripheral or central electron lucency. These selective effects of monensin indicate that granule components may be packaged into DAB-positive organelles that are deficient in trans Golgi-derived elements (HID- and PA-TCH-SP-negative) and that some preformed primary granules contain a monensin sensitive Na+/H+ gradient.

Topics: Bone Marrow; Cytoplasmic Granules; Golgi Apparatus; Granulocytes; Histocytochemistry; Humans; Immunoenzyme Techniques; Leukemia, Myeloid; Microscopy, Electron; Monensin; Neutrophils; Tumor Cells, Cultured