monensin and Leukemia--Myeloid--Acute

The master transcriptional regulator MYB is a key oncogenic driver in several human neoplasms, particularly in acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). MYB is therefore an attractive target for drug development in MYB-activated malignancies. Here, we employed a MYB-reporter cell line and identified the polyether ionophores monensin, salinomycin, and nigericin as novel inhibitors of MYB activity. As a proof of principle, we show that monensin affects the expression of a significant number of MYB-regulated genes in AML cells and causes down-regulation of MYB expression, loss of cell viability, and induction of differentiation and apoptosis. Furthermore, monensin significantly inhibits proliferation of primary murine AML cells but not of normal hematopoietic progenitors, reflecting a high MYB-dependence of leukemic cells and underscoring the efficacy of monensin in MYB-activated malignancies. Importantly, monensin also suppressed the viability and non-adherent growth of adenoid cystic carcinoma (ACC) cells expressing MYB-NFIB fusion oncoproteins. Our data show that a single compound with significant MYB-inhibitory activity is effective against malignant cells from two distinct MYB-driven human neoplasms. Hence, monensin and related compounds are promising molecular scaffolds for development of novel MYB inhibitors.

Topics: Animals; Carcinoma, Adenoid Cystic; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mice; Monensin; Nigericin; Proteolysis; Proto-Oncogene Proteins c-myb; Pyrans; THP-1 Cells

Monensin, an Na(+) ionophore, regulates many cellular functions including apoptosis. However, there has been no report about the antitumoral effect of monensin on acute myelogenous leukemia (AML). Here, we investigated the antiproliferative effect of monensin on AML cells in vitro and in vivo. Monensin efficiently inhibited the proliferation of all of 10 AML cell lines, with IC(50) of about 0.5 microM. DNA flow cytometric analysis indicated that monensin induced a G(1) and/or a G(2)-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of monensin, we examined the effect of monensin on cell cycle-related proteins in HL-60 cells. The levels of CDK6, cyclin D1 and cyclin A were decreased. In addition, monensin not only increased the p27 level but also enhanced its binding with CDK2. Furthermore, the activities of CDK2- and CDK6-associated kinases reduced by monensin were associated with hypophosphorylation of Rb protein. Monensin also induced apoptosis in AML cells including HL-60 cells. The apoptotic process of HL-60 cells was associated with changes in Bax, caspase-3, caspase-8 and mitochondria transmembrane potential (Deltapsi(m)). In particular, monensin (i.p. at a dose of 8 mg/kg thrice weekly) significantly reduced the tumor size of BALB/c mice that were inoculated s.c. with its derived cell line, WEHI-3BD cells (69% growth inhibition relative to control group; p < 0.05). Tumors from monensin-treated mice exhibited increased apoptosis, and these tumor were immunohistochemically more stained with Bax, Fas and p53 antibodies than control tumors. In conclusion, this is the first report that monensin potently inhibits the proliferation of AML cells.

Topics: Animals; Apoptosis; Blotting, Western; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Female; Humans; Immunoenzyme Techniques; Ionophores; Leukemia, Myeloid, Acute; Membrane Potentials; Mice; Mice, Inbred BALB C; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Monensin; Poly(ADP-ribose) Polymerases; Tumor Cells, Cultured; Tumor Suppressor Proteins

We have used laser-assisted confocal microscopy to evaluate the intracellular distribution of daunorubicin (DNR) in acute myeloid leukemia (AML) cell lines and fresh AML cells according to their differentiation phenotype. In KG1a, KG1, TF-1 and HEL cells, which express the early differentiation marker CD34, DNR was distributed in perinuclear vesicles which could be associated with the Golgi apparatus, as suggested by the distribution of fluorescent probes specific for intracellular organelles. In contrast, U937 and HL-60 cells, which display a more mature phenotype, exhibited nuclear and diffuse cytoplasmic DNR fluorescence. DNR sequestration was not correlated with P-glycoprotein (P-gp) or multidrug resistance protein expression. Furthermore, PSC833, a potent P-gp blocker, had little effect on drug sequestration in CD34+ AML cells. We also tested the effect of metabolic inhibitors, cytoskeleton inhibitors and carboxy-ionophores on DNR distribution in both CD34- and CD34+ AML cells. However, only non-specific metabolic inhibitors restored nucleic/cytoplasmic distribution in CD34+ cells. In these cells, the intracellular distribution of doxorubicin and idarubicin was very similar to that of DNR, while the distribution of methoxymorpholinyl-doxorubicin was nuclear and diffusely cytoplasmic. In fresh AML cells, DNR was also concentrated in the perinuclear region in CD34+ but not in CD34- cells. However, DNR sequestration was not observed in normal CD34+ cells. Finally, our results show that DNR is sequestered in organelles in CD34+ AML cells via an active mechanism which appears to be different from P-gp-mediated transport. Abnormal DNR distribution may account for the natural resistance of immature AML cells to anthracyclines.

Topics: Antibiotics, Antineoplastic; Antigens, CD34; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Blood Cells; Cell Nucleus; Colchicine; Cytochalasin B; Cytoplasm; Daunorubicin; Deoxyglucose; Fluorescent Antibody Technique, Indirect; Humans; Leukemia, Myeloid, Acute; Microscopy, Confocal; Monensin; Nigericin; Sodium Azide; Temperature; Tumor Cells, Cultured

The uptake of 67Ga by HL60 cells requires binding of 67Ga-transferrin (Tf) to cell surface Tf receptors. To further examine this process, we have studied early events in the cellular uptake of 67GaTf. Cell surface-bound 67GaTf and 59FeTf displayed similar kinetics during the first 10 min of uptake. Thereafter, approximately 10% of intracellular 67Ga was released by cells while 59Fe internalization continued to increase with time. In pulse-chase studies of 125I-Tf-67Ga uptake, internalized 125I-Tf, but not 67Ga, was chased out of cells by nonradioactive Tf-Ga. Exposure of cells to monensin, a carboxylic ionophore, during initial uptake decreased the internalization of both 125I-Tf and 67Ga. Exposure to monensin at a later time, after cells had incorporated 125I-Tf-67Ga or 59FeTf, caused an increase in the release of 67Ga and 59Fe with a decrease in the release of 125I-Tf. Ammonium chloride inhibited the internalization of both 67Ga and 59Fe. 67GaTf uptake by HL60 cells involves initial internalization into an acidic receptosome. This is followed by dissociation of 67Ga and Tf and subsequent trafficking of each to separate intracellular compartments. Disruption of this process by monensin results in the release of 67Ga from cells.

Topics: Gallium Radioisotopes; Humans; Hydrogen-Ion Concentration; Iron; Leukemia, Myeloid, Acute; Monensin; Receptors, Transferrin; Time Factors; Transferrin; Tumor Cells, Cultured

The biosynthesis of myeloperoxidase in human promyelocytic leukemia HL-60 cells was studied by pulse-chase and immunoprecipitation methods and separation of subcellular organelles using Percoll density gradient fractionation. These studies revealed that in control and monensin (1 microM) treated cells, more than 85% of the total immunoprecipitable radiolabeled myeloperoxidase was present predominantly in precursor form (Mr 91,000) and resided in lower density compartments after an initial 3-h labeling period. Using biochemical and ultrastructural techniques, the lower density regions of the gradient were found to contain elements of the endoplasmic reticulum and the Golgi complex. Following a 16-h chase period, about 70% of the radiolabeled myeloperoxidase in untreated cells was found predominantly in denser regions of the gradient and was present mainly in the form of the mature large subunit (Mr 63,000). These dense regions were shown to contain azurophilic granules by means of the distribution of beta-glucuronidase and myeloperoxidase activities and by electron microscopy. Processing of myeloperoxidase and its deposition into dense granules were blocked by monensin treatment. Following a 16-h chase period in the presence of monensin, approximately 80% of the radiolabeled myeloperoxidase continued to reside in lower density compartments and was predominantly in precursor (Mr 91,000) and intermediate (Mr 81,000 and 74,000) forms. Only about 10% of the radiolabeled myeloperoxidase was associated with dense azurophilic granules. Monensin treatment produced large, Golgi-derived vacuoles which were isolated using Percoll density centrifugation and identified by electron microscopy. These vacuoles were found to be essentially devoid of peroxidase activity and pulse-labeled, newly synthesized radiolabeled myeloperoxidase species. The effects of monensin on transport and processing were reversible after a 3-h exposure and 16-h chase period in the absence of monensin. Taken together, these data indicate that maturation of myeloperoxidase is closely linked to its deposition into dense azurophilic granules via a monensin-sensitive process(es). The lower density compartments within which immature myeloperoxidase species accumulate in the presence of monensin appear to be functionally related to or associated with Golgi or endoplasmic reticulum structures distinct from the large monensin-induced vacuoles.

Topics: Cell Line; Humans; Kinetics; Leukemia, Myeloid, Acute; Monensin; Organoids; Peroxidase; Protein Processing, Post-Translational; Subcellular Fractions

The observation that myeloperoxidase precursor and larger intermediate (Mr 91,000 and 81,000, respectively) were extracted in the presence of detergent from isolated granule fractions of human promyelocytic leukemia HL-60 cells under mildly acidic conditions was investigated. In contrast, under conditions of neutral pH, only the Mr 74,000 intermediate and mature species were extracted. Extraction of the Mr 91,000 and 81,000 forms was also enhanced in the presence of EDTA. Kinetic studies of the processing of the different myeloperoxidase species confirmed the intermediate nature of the Mr 81,000 and 74,000 forms. Support for a role of an acidic intracellular compartment was obtained through evidence that the acid-extractable precursor and intermediates accumulated in HL-60 cells which had been treated with 1 microM monensin. Under these conditions, the production of mature heavy (Mr 63,000) and light (Mr 13,500) subunits of myeloperoxidase was consistently inhibited by greater than 40% over a 16-h period. The effects of monensin on processing of myeloperoxidase were completely reversed if monensin was removed during this 16-h period. These data support the idea that an acidic compartment may be involved in the transport of myeloperoxidase precursors to azurophil granules and/or their processing to a smaller intermediate form (Mr 74,000) of the enzyme.

Topics: Cell Line; Cytoplasmic Granules; Humans; Kinetics; Leukemia, Myeloid, Acute; Lysosomes; Molecular Weight; Monensin; Peroxidase; Protein Processing, Post-Translational

Myeloperoxidase is a major component of specialized lysosomes known as azurophil granules in polymorphonuclear leukocytes or neutrophils. The processing of myeloperoxidase in human HL-60 promyelocytic leukemia cells was studied by pulse-labeling cells in culture with [35S]methionine followed by immunoprecipitation and identification of myeloperoxidase polypeptides from cell fractions after various chase intervals. These studies revealed the presence of a previously unidentified intermediate with Mr 74,000 which kinetically followed the appearance of a larger Mr 81,000 intermediate. Using an in vitro lysosomal preparation the newly identified Mr 74,000 intermediate was directly converted within protected granules to mature forms of myeloperoxidase (Mr 63,000 and 60,000). This conversion occurred optimally at pH 7.5 and was not inhibited by lysosomotropic agents (chloroquine, NH4Cl) or protonophores (monensin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Furthermore, the uptake of radiolabeled amines indicated a neutral intragranular environment (pH 7.35-7.67) which remained unchanged in the presence and absence of 1 mM ATP or 2.5 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone. We conclude that, in contrast to other lysosomal pathways, the final proteolytic cleavage of myeloperoxidase does not require an acidic environment.

Topics: Ammonium Chloride; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Chloroquine; Cytoplasmic Granules; Hexosaminidases; Humans; Hydrogen-Ion Concentration; Ionophores; Leukemia, Myeloid, Acute; Molecular Weight; Monensin; Peroxidase; Protein Processing, Post-Translational

Monensin blocks human transferrin recycling in a dose-dependent and reversible manner in K562 cells, reaching 100% inhibition at a noncytocidal dose of 10(-5) M, whereas transferrin recycling is virtually unaffected by noncytocidal doses of chloroquine. The intracellular pathway of human transferrin in K562 cells, both in the presence and absence of 10(-5) M monensin, was localized by indirect immunofluorescence. Monensin blocks transferrin recycling by causing internalized ligand to accumulate in the perinuclear region of the cell. The effect of 10(-5) M monensin on human transferrin kinetics was quantitatively measured by radioimmunoassay and showed a positive correlation with immunofluorescent studies. Immunoelectron microscopic localization of human transferrin as it cycles through K562 cells reveals the appearance of perinuclear transferrin-positive multivesicular bodies within 3 min of internalization, with subsequent exocytic delivery of the ligand to the cell surface via transferrin-staining vesicles arising from these perinuclear structures within 5 min of internalization. Inhibition of ligand recycling with 10(-5) M monensin causes dilated transferrin-positive multivesicular bodies to accumulate within the cell with no evidence of recycling vesicles. A coordinated interaction between multivesicular bodies and the Golgi apparatus appears to be involved in the recycling of transferrin in K562 cells. Cell-surface-binding sites for transferrin were reduced by 50% with 10(-5) M monensin treatment; however, this effect was not attenuated by 80% protein synthesis inhibition with cycloheximide, supporting the idea that the transferrin receptor is also recycled through the Golgi.

Topics: Cell Line; Chloroquine; Fluorescent Antibody Technique; Furans; Humans; Kinetics; Leukemia, Myeloid, Acute; Microscopy, Electron; Monensin; Receptors, Cell Surface; Receptors, Transferrin; Transferrin

NH4Cl and monensin, two agents which neutralize intracellular acidic compartments, block the segregation of iron from transferrin after endocytosis, while neither of these reagents affects internalization of diferric transferrin into the cell. In conclusion the molecular separation of iron from transferrin inside the cell requires a non-lysosomal acidic compartment.

Topics: Ammonium Chloride; Cell Line; Endocytosis; Humans; Hydrogen-Ion Concentration; Iron; Kinetics; Leukemia, Myeloid, Acute; Microbodies; Monensin; Organoids; Protein Binding; Transferrin