monensin and Leukemia--Promyelocytic--Acute

Platelet-activating factor (PAF) acetylhydrolase catalyzes the conversion of PAF to lyso-PAF and acetate. In this study we show that induced cellular differentiation of HL-60 cells grown in chemically defined media by dimethylsulfoxide (DMSO) to granulocytic cells increases the acetylhydrolase activity with a concomitant increased secretion of the enzyme into the media. This increase in acetylhydrolase activity is blocked by the presence of actinomycin D (1 microM) or cycloheximide (1-2 microM) in the culture media. Acetylhydrolase is located both in the cytosolic and particulate fractions; the relative distribution of acetylhydrolase activity in the particulate fraction and cytosol increases and decreases respectively, as the differentiation progresses. The addition of an intracellular protein transport inhibitor, monensin, causes further accumulation of acetylhydrolase activity in the particulate fraction and a decrease in the media, with no effect on the acetylhydrolase activity in the cytosol. Acetylhydrolase in differentiated HL-60 cells acquires properties similar to those of the plasma acetylhydrolase in that it becomes less sensitive to 5,5'-dithiobis-2-nitrobenzoic acid and p-bromophenacylbromide inhibition than the acetylhydrolase in undifferentiated cells. The acetylhydrolase secreted into the media by the differentiated cells was almost totally insensitive to these inhibitors, whereas the acetylhydrolase from the particulate fraction gave an intermediate response; the cytosolic acetylhydrolase was sensitive to both inhibitors. However, the acetylhydrolase secreted by differentiated HL-60 cells has a different electrophoretic mobility, temperature sensitivity, and association with lipoproteins when compared to that of human plasma acetylhydrolase. Collectively, these results indicate cellular differentiation induces intracellular acetylhydrolase activity through a mechanism involving both transcriptional and translational events. Furthermore, the acetylhydrolase synthesized during the DMSO-induced differentiation of HL-60 cells is then secreted into the media via the intracellular membrane transport system for proteins. Based on results obtained with HL-60 cells as a cell model, it is likely that more than one isoform of acetylhydrolase exists in the extracellular milieu.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Amino Acid Sequence; Cell Differentiation; Cycloheximide; Dactinomycin; Dimethyl Sulfoxide; Electrophoresis; Enzyme Induction; Enzyme Stability; Golgi Apparatus; Granulocytes; Humans; Leukemia, Promyelocytic, Acute; Lipoproteins; Molecular Sequence Data; Monensin; Neutrophils; Phospholipases A; Protein Biosynthesis; Sensitivity and Specificity; Subcellular Fractions; Temperature; Transcription, Genetic; Tumor Cells, Cultured

The two tumor necrosis factor (TNF) receptors (TNF-R55 and TNF-R75) can release soluble TNF-binding proteins (TNF-R55-BP and TNF-R75-BP) by proteolytic cleavage. The proteolytic processing of the TNF receptors was investigated in monoblastic THP-1 and promyelocytic HL-60-10 leukemic cell lines. The release of soluble forms of both receptors was rapidly stimulated by staurosporine-sensitive protein kinase C activation by phorbol myristate acetate (PMA) and more slowly stimulated by TNF. No receptor release was seen below a temperature of 16 degrees C. NH4Cl (10 mmol/liter) and monensin (1 mumol/liter), known to increase intracellular pH, inhibited to some extent PMA- and TNF-induced release of both TNF-R55-BP and TNF-R75-BP. The inhibitory effect of monensin might be explained by a diminished translocation of newly synthesized receptor to the plasma membrane. The weak inhibitory effect of NH4Cl on PMA-induced release of soluble receptor forms could be due to effects on a pH-sensitive compartment. PMA-induced down-regulation of receptors was not dependent on acidity as it occurred also in the presence of monensin and NH4Cl when the release of TNF-BPs is partially blocked. Dibutyryl cAMP inhibited the PMA-induced release of TNF-R55-BP but not of TNF-R75-BP in both cell lines investigated. In addition, dibutyryl cAMP alone stimulated the release of both receptors but only in THP-1 cells. Our data show that the generation of soluble forms of both TNF receptors can be regulated by both PKC and PKA.

Topics: Alkaloids; Ammonium Chloride; Bucladesine; Carrier Proteins; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Endopeptidases; Enzyme-Linked Immunosorbent Assay; HeLa Cells; Humans; Leukemia, Monocytic, Acute; Leukemia, Promyelocytic, Acute; Monensin; Neoplasm Proteins; Protein Kinase C; Protein Processing, Post-Translational; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Solubility; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tumor Necrosis Factor Decoy Receptors

Human neutrophil promyelocytes synthesize, process, and package several microbicidal proteins, including the highly abundant defensins, into their azurophil granules. As deduced from their cDNA sequences, defensins are initially synthesized as 94 amino acid (aa) precursors that must undergo extensive processing. We performed metabolic labeling studies of defensin synthesis in the human promyelocytic cell line HL-60 and in chronic myeloid leukemia cells, and showed that preprodefensins are processed to mature 29 to 30 aa defensins over 4 to 24 hours via two major intermediates: a 75 aa prodefensin generated by the cleavage of the signal sequence, and a 56 aa prodefensin that results from a subsequent preaspartate proteolytic cleavage. Almost all of the 75 aa form was found in the cytoplasmic/microsomal fraction, whereas the 56 aa prodefensin and mature defensins predominated in the granule-enriched fraction. The 75 aa prodefensin was also selectively released into the culture supernatant. Treatment of HL-60 cells with monensin, chloroquine, or ammonium chloride, substances that neutralize acidic subcellular compartments, partially blocked conversion of the 75 aa prodefensin into 56 aa prodefensin, but did not increase the extracellular release of the 75 aa form. Further studies will be required to determine the role of this processing pathway in subcellular targeting to azurophil granules and avoidance of autocytotoxicity.

Topics: Amino Acid Sequence; Blood Proteins; Defensins; Humans; Hydrogen-Ion Concentration; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Molecular Sequence Data; Molecular Weight; Monensin; Neutrophils; Peroxidase; Protein Precursors; Protein Processing, Post-Translational; Tumor Cells, Cultured

HL60 cells isolated for resistance to Adriamycin are multidrug resistant and defective in the cellular accumulation of drug. These cells do not contain detectable levels of P-glycoprotein. At the present time the mechanism by which HL60/Adr cells reduce drug levels is not known. To gain insight into the molecular basis of this system we have analyzed transport pathways and the distribution of daunomycin in drug-resistant HL60 cells. Using a cell fractionation technique we find that the major portion of daunomycin accumulates in the nucleus of both sensitive and resistant cells. Further studies reveal, however, that under efflux conditions drug is retained in the nuclei of sensitive cells but rapidly removed from the nuclei of the resistant isolate. Essentially identical results are obtained when daunomycin distribution and transport are analyzed by fluorescence microscopy. A number of agents which alter transport processes have been tested for their effect on drug accumulation in resistant cells. Thus we find that brefeldin A, which disassembles Golgi, and various lysosomotropic agents such as chloroquine and methylamine do not affect drug levels. In contrast the protonophores nigericin and monensin induce an increase in drug accumulation and inhibit efflux. The results of this study thus suggest that resistance in HL60/Adr cells is related to a mechanism whereby drug is transported to the nucleus and thereafter rapidly redistributed to the extracellular space. The molecular basis of this transport pathway is not known.

Topics: Biological Transport; Cell Line; Cell Membrane; Cell Nucleus; Cytosol; Daunorubicin; Doxorubicin; Drug Resistance; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Microscopy, Fluorescence; Monensin; Nigericin

Binding and uptake of transcobalamin II-bound cobalamin by HL-60 promyelocytic leukemia cells proceed through receptor-mediated endocytosis. The affinity constant of the receptor for transcobalamin II-cobalamin was found to be 6.1 liter/nmol and the maximal rate of uptake 12 pmol/10(9) cells/h. This uptake is mediated by about 3000 receptor sites per cell. Evidence is presented that the receptor recirculates from the cell surface to the lysosomes and vice versa. Upon differentiation induction of the cells by either DMSO in granulocytic direction or by 1,25-dihydroxy-vitamin D3 in monocytic direction a rapid decline in cellular uptake and cell surface binding of the protein-bound vitamin ensues. In particular the internalization of the complex decreases faster than all other observed signs of the ongoing differentiation process, such as reduction in the OKT9-reactive transferrin receptor, increase in lineage-specific surface markers, and decrease in [3H]thymidine incorporation and actual cell proliferation. The transcobalamin II receptor on the cell surface appears to be a proliferation-associated membrane component in human leukemic cells.

Topics: Calcitriol; Cell Line; Cell Transformation, Neoplastic; Cycloheximide; Dimethyl Sulfoxide; Endocytosis; Humans; Leukemia, Promyelocytic, Acute; Monensin; Receptors, Cell Surface; Transcobalamins; Tumor Cells, Cultured; Vitamin B 12