Complete inhibition of transferrin recycling by monensin in K562 cells

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.     

Autophagy inhibition enhances daunorubicin-induced apoptosis in K562 cells

Anthracycline daunorubicin (DNR) is one of the major antitumor agents widely used in the treatment of myeloid leukemia. Unfortunately, the clinical efficacy of DNR was limited because of its cytotoxity at high dosage. As a novel cytoprotective mechanism for tumor cell to survive under unfavorable conditions, autophagy has been proposed to play a role in drug resistance of tumor cells. Whether DNR can activate to impair the sensitivity of cancer cells remains unknown. Here, we first report that DNR can induce a high level of autophagy, which was associated with the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Moreover, cell death induced by DNR was greatly enhanced after autophagy inhibition by the pharmacological inhibitor chloroquine (CQ) and siRNAs targeting Atg5 and Atg7, the most important components for the formation of autophagosome. In conclusion, we found that DNR can induce cytoprotective autophagy by activation of ERK in myeloid leukemia cells. Autophagy inhibition thus represents a promising approach to improve the efficacy of DNR in the treatment of patients with myeloid leukemia.     

A proteomic investigation into adriamycin chemo-resistance of human leukemia K562 cells

This study aimed to explore the mechanism of adriamycin resistance in human chronic myelogenous leukemia cells. Proteomic approach was utilized to compare and identify differentially expressed proteins between human chronic myelogenous leukemia K562 cells and their adriamycin-resistant counterparts. The differentially expressed proteins were analyzed by 2-DE (two-dimensional gel electrophoresis), and protein identification were performed on ESI-Q-TOF MS/MS instrument. Out of the 35 differentially expressed proteins between the two cell lines, 29 were identified and grouped into 10 functional classes. Most of identified proteins were related to the categories of metabolism (24%), proteolysis (13%), signal transduction (21%) and calcium ion binding (6%), suggesting that alterations of those biological processes might be involved in adriamycin resistance of K562 cells. We believe this study may provide some clues to a better understanding of the molecular mechanisms underlying adriamycin resistance.     

Effects of a moderate-intensity static magnetic field and adriamycin on K562 cells

The aim of this study was to investigate whether a moderate‐intensity static magnetic field (SMF) can enhance the killing effect of adriamycin (ADM) on K562 cells, and to explore the effects of SMF combined with ADM on K562 cells. We analyzed the metabolic activity of cells, cell cycle distribution, DNA damage, change in cell ultrastructure, and P‐glycoprotein (P‐gp) expression after K562 cells were exposed continuously to a uniform 8.8 mT SMF for 12 h, with or without ADM. Our results showed that the SMF combined with ADM (25 ng/ml) significantly inhibited the metabolic activity of K562 cells (P < 0.05), while neither ADM nor the SMF alone affected the metabolic activity of these cells. Cell ultrastructure was altered in the SMF + ADM group. For example, cell membrane was depressed, some protuberances were observable, and vacuoles in the cytoplasm became larger. Cells were arrested at the G2/M phase and DNA damage increased after cells were treated with the SMF plus ADM. ADM also induced the P‐gp expression. In contrast, in the SMF group and SMF + ADM group, the P‐gp expression was decreased compared with the ADM group. Taken together, our results showed that the 8.8 mT SMF enhanced the cytotoxity potency of ADM on K562 cells, and the decrease in P‐gp expression may be one reason underlying this effect. Bioelectromagnetics 32:191–199, 2011. © 2010 Wiley‐Liss, Inc.     

Expression of sialidase Neu2 in leukemic K562 cells induces apoptosis by impairing Bcr-Abl/Src kinases signaling

Chronic myeloid leukemia is a hematopoietic stem cell cancer, originated by the perpetually "switched on" activity of the tyrosine kinase Bcr-Abl, leading to uncontrolled proliferation and insensitivity to apoptotic stimuli. The genetic phenotype of myeloid leukemic K562 cells includes the suppression of cytosolic sialidase Neu2. Neu2 transfection in K562 cells induced a marked decrease (-30% and -80%) of the mRNA of the anti-apoptotic factors Bcl-XL and Bcl-2, respectively, and an almost total disappearance of Bcl-2 protein. In addition, gene expression and activity of Bcr-Abl underwent a 35% diminution, together with a marked decrease of Bcr-Abl-dependent Src and Lyn kinase activity. Thus, the antiapoptotic axis Bcr-Abl, Src, and Lyn, which stimulates the formation of Bcl-XL and Bcl-2, was remarkably weakened. The ultimate consequences of these modifications were an increased susceptibility to apoptosis of K562 cells and a marked reduction of their proliferation rate. The molecular link between Neu2 activity and Bcr-Abl signaling pathway may rely on the desialylation of some cytosolic glycoproteins. In fact, three cytosolic glycoproteins, in the range 45-66 kDa, showed a 50-70% decrease of their sialic acid content upon Neu2 expression, supporting their possible role as modulators of the Bcr-Abl complex.     

Albumin inhibition of transferrin low-affinity binding to K562 cells

Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin.     

Non-competitive inhibition of P-glycoprotein-associated efflux of THP-adrimycin by verapamil in living K562 leukemia cells

The decrease of the intracellular concentration of drug in resistant cell as compared to sensitive cells is, in most of cases, correlated with the presence, in the membrane of resistant cells, of a 170-kDA P-glycoprotein (P-gp) responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to follow the P-gp-associated efflux of these drugs in the absence or presence of verapamil. In the present study, 4′-o-tetrahydro-pyranyladriamycin (THP-adriamycin) was used. Two different methods were used to determined the kinetics of active efflux of THP-adriamycin: (1) at the steady-state, (2) directly, after the addition of glucose to cells first incubated with THP-adriamycin in the presence of N₃^? and in the absence of glucose. Kinetic analysis indicates: (1) a saturation of the active efflux when the cytosolic free drug concentration increased (the Michaelis constant K_m = 0.5 ± 0.3 μM) and (2) that the inhibitory effect of verapamil on P-gp associated efflux of THP-adriamycin in living cells in living cells is non-competitive.     

Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity.

The natural polyphenolic compound resveratrol (trans-3,4', 5-trihydroxystilbene) is shown to prevent apoptosis (programmed cell death) induced in human erythroleukemia K562 cells by hydrogen peroxide and other unrelated stimuli. Resveratrol reversed the elevation of leukotriene B4 (from 6.40 +/- 0.65 to 2.92 +/- 0.30 pmol.mg protein-1) and prostaglandin E2 (from 11.46 +/- 1.15 to 8.02 +/- 0.80 nmol.mg protein-1), induced by H2O2 challenge in K562 cells. The reduction of leukotriene B4 and prostaglandin E2 correlated with the inhibition of the 5-lipoxygenase activity, and the cyclooxygenase and peroxidase activity of prostaglandin H synthase, respectively. Resveratrol also blocked lipoperoxidation induced by hydrogen peroxide in K562 cell membranes. Resveratrol was found to act as a competitive inhibitor of purified 5-lipoxygenase and 15-lipoxygenase and prostaglandin H synthase, with inhibition constants of 4.5 +/- 0.5 microM (5-lipoxygenase), 40 +/- 5.0 microM (15-lipoxygenase), 35 +/- 4.0 microM (cyclooxygenase activity of prostaglandin H synthase) and 30 +/- 3.0 microM (peroxidase activity of prostaglandin H synthase). Altogether, the results reported here suggest that the anti-apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate-metabolizing enzymes.     

The bcr-c-abl tyrosine kinase activity is extinguished by TPA in K562 leukemia cells

Tyrosine kinase activity is associated with the transforming potential of several oncogenes. Human chronic myeloid leukemia (CML) cells and cell lines have been shown to contain an active bcr-c-abl p210 tyrosine kinase as a consequence of the Philadelphia chromosomal translocation. In the present work the activity of the c-abl and c-src oncogene-encoded tyrosine kinase was investigated during phorbol diester (TPA) induced differentiation of the K562 CML cells. The high tyrosine kinase activity of p210bcr-c-abl is strongly reduced during the initial 24 h of TPA treatment. In contrast, the activity of the c-src tyrosine kinase is not changed. No change occurs in the expression of the c-abl-specific RNAs during this period. Following the reduction of bcr-c-abl kinase activity, cell proliferation is arrested and megakaryoblastic antigens appear on the cells. Sodium butyrate caused a slight decrease in growth rate and of bcr-c-abl kinase activity during erythroid differentiation whereas no changes in c-src or c-abl tyrosine kinase activities were seen in DMSO-treated control cells.     

O-glycosylation of leukosialin in K562 cells. Evidence for initiation and elongation in early Golgi compartments

The O-glycosylation of leukosialin, a major sialoglycoprotein found on leukocytes, has been studied in the human erythroleukemic cell line K562. The appearance of its O-linked chains has been followed in pulse-chase experiments with [35S]methionine by immunoprecipitation with an anti-peptide antiserum as well as with a lectin from Salvia sclarea seeds (SSA) specific for GalNAc-Ser/Thr and the peanut (Arachis hypogaea) agglutinin (PNA) which recognizes Gal beta 1----3GalNAc-Ser/Thr structures. An O-glycan-free precursor was converted into the fully O-glycosylated mature form within the 10-min labeling period and no intermediates carrying only GalNAc-Ser/Thr structures could be detected. The ionophore monensin was used in order to slow down intracellular traffic and thus O-glycan synthesis. The drug partly inhibited the transport from rough endoplasmic reticulum (RER) to the Golgi and also the cell-surface expression of leukosialin. It was found to have a marked effect on the synthesis of O-linked carbohydrate structures of leukosialin since the amount of O-glycans containing only GalNAc or NeuNAc alpha 2----6GalNAc was significantly increased after monensin treatment. Under these conditions the biosynthesis of the N-glycan on leukosialin was completely arrested in an endoglycosidase-H-sensitive step of processing, whereas the O-glycans already contained galactose and sialic acid although at a reduced level. On the other hand, the small amounts of leukosialin expressed on the cell surface of monensin-treated cells carried the same glycans as those remaining blocked inside the cell. In addition, immunocytochemical studies using SSA and PNA on untreated K562 cells suggested the absence of detectable amounts of GalNAc-Ser/Thr-bearing glycoproteins in the RER as well as in the Golgi. In contrast Gal beta 1----3GalNAc structures could be detected on intracellular membranes which were tentatively identified as the cis-Golgi. Together these results lead us to the following conclusions: N-glycan transfer occurs in the RER before the initiation of O-glycans which takes place at the entrance of the protein into the Golgi; further elongation of O-glycans with galactose and sialic acid follows very rapidly, probably before the final processing of N-glycans to complex-type structures.     

Induction of apoptosis in K562 cells by jolkinolide B

Jolkinolide B, a bioactive diterpene isolated from the roots of Euphorbia fischeriana Steud, has various biological and pharmacological properties. In this study, the cytotoxicity of highly purified jolkinolide B was tested in human chronic myeloid leukemia (K562) and 2 other cell lines (human esophageal carcinoma Eca-109 and human hepatoma HepG2). The results indicate a significant decrease in the proliferation of all the 3 cell lines when treated with jolkinolide B for 24 h; the IC50 value of cytotoxicity was 12.1 microg/mL (for K562 cells), >50.0 microg/mL (for HepG2 cells), and 23.7 microg/mL (for Eca-109 cells). Further study of K562 cells involving fluorescence and transmission electron microscopy revealed characteristic apoptotic features, such as cell shrinkage, membrane blebbing, loss of microvilli, and nuclear condensation. Agarose electrophoresis of genomic DNA showed a typical fragmentation pattern for apoptotic cells. A kinetic cell-cycle analysis demonstrated that the cell cycle was arrested in the G1 phase. All these results suggest that the anti-proliferation effect of jolkinolide B on K562 cells is achieved by arresting the cell cycle in the G1 phase and subsequently inducing apoptosis.     

Apoptosis induced by 3,7-dinitrodibenzobromonium salts in K562 cells

We evaluated the inhibitory effect of 3,7-dini-trodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K562 cell by trypan blue exclusion test and MTT colorimetric assay.The degree of DNA damage in K562 cells treated with cBr,was detected by isotopic tracer method (3H-TdR).The morphological changes of these K562 cells were examined by fluorescence and electron microscopy.Biochemical characteristics of K562 cells were detected by flow cytome-try and 3H-thymidine incorporation assay.Findings indi-cated that cBr could significantly inhibit cell proliferation and result in DNA damage of K562 cells,cBr is a new type of immunostimulant and can induce cell apoptosis.     

Reduction of extracellular dehydroascorbic acid by K562 cells

K562 erythroleukaemic cells produced ascorbate when incubated with dehydroascorbic acid. The reduction depended on the number of cells and on the concentration of dehydroascorbic acid. The observed rate consists of a high affinity (apparent) K_m 7 μM , V_max 3·25 pmol min^?1 (10⁶ cells)^?1 and a low affinity component, which was non-saturable up to 1 mM of DHA (rate increase of 0·1 pmol min^?1 (10⁶ cells)^?1 (1 μM of DHA^?1). The rate was dependent on temperature and was stimulated by glucose and inhibited by phloretin, N-ethylmaleimide, parachloro-mercuribenzoate and thenoyltrifluoroacetone. Although uptake of DHA proceeded at a higher rate than its extracellular reduction, the generation of extracellular ascorbate from DHA cannot be accounted for by intracellular reduction and the release of ascorbate, since the latter was not linear with time and had an initial rate of approximately 3 pmol min^?1 (10⁶ cells^?1). At a concentration of DHA of 100 μM this is 25 per cent of the observed reduction.     

Stimulation of the Na+,K(+)-ATPase activity of K562 human erythroleukemia cells by triiodothyronine.

The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human erythroleukemic cell was studied to understand why the erythrocyte sodium pump activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell lysates was assayed by measuring the release of inorganic phosphate (Pi) from ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48 hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1 mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The Na+,K(+)-ATPase activity could be stimulated in a time- and concentration-dependent manner; maximum stimulatory effect of T3 was seen at a concentration of 10(-7) mol/L. When an inducer [cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium, the K562 cells showed signs of differentiation and synthesised haemoglobin. At the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3 stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3 during differentiation, the enzyme activity remains high.     

Involvement of Na+, K+-ATPase inhibition in K562 cell differentiation induced by bufalin

Human leukemia K562 cell differentiation induction by naturally occurring bufadienolides purified from the Chinese drug Senso and synthetic bufalin derivatives was examined by a nitro blue tetrazolium reduction assay. Bufalin showed the strongest activity among all the bufadienolides tested in this study. The degree of the induction of nitro blue diformazan positive cells by the bufadienolides correlated well with their inhibitory activities against Na⁺, K⁺ -ATPase prepared from K562 cells in vitro. N⁺, K⁺ -ATPases from a variant K562 clone (ouabain resistant, OuaR) and murine leukemia cell line M1-T22, which were insensitive to the bufadienolides in terms of growth inhibition and cell differentiation, appeared to be refractory to bufalin in vitro. A binding study of ³H-bufalin and ³H-ouabain revealed that saturated levels of both ligands associated with K562 cells were virtually similar; however, affinity of ³H-bufalin was considerably higher than ³H-ouabain. The saturated level of ³H-bufalin observed in the OuaR cells was approximately half of that observed in K562 cells without a change in its affinity. Association of ³H-bufalin with K562 cells was completely blocked by pretreatment of the cells with cold ouabain at concentrations saturating the binding sites. These results suggest that bufalin acts on the cells by binding to sites on the cell membrane which also bind ouabain. It is thus proposed that N⁺, K⁺ -ATPase inhibition is closely related to the initiation process in the induction of K562 cell differentiation induced by bufalin. © 1994 Wiley-Liss, Inc.     

Spontaneous mitochondrial membrane potential change during apoptotic induction by quercetin in K562 and K562/adr cells

Natural products from plants such as flavonoids are potential drugs to overcome multidrug resistance (MDR) in cancer treatments. However, their modes of action are still unclear. In this study, the effects of quercetin on mitochondrial membrane potential (DeltaPsim) change as well as quercetin's ability to induce apoptosis and inhibit Pgp-mediated efflux of 99mTc-MIBI in K562/adr cells were investigated. Quercetin exhibits cytotoxicity against erythroleukemic cells: IC50 are 11.0 +/- 2.0 micromol/L and 5.0 +/- 0.4 micromol/L for K562 and K562/adr, respectively. Quercetin induces cell death via apoptosis in both K562 and K562/adr cells and does not inhibit Pgp-mediated efflux of 99mTc-MIBI. Quercetin (10 micromol/L, 3 h) and etoposide (100 micromol/L, 24 h) induce similar levels of apoptosis in K562 and K562/adr cells. Quercetin induces an increase followed by a decrease in |DeltaPsim| value depending on its concentration. A decrease in the |DeltaPsim| value is associated with an increase in the percentage of early apoptotic cells. It is clearly shown that quercetin results in a spontaneous DeltaPsim change during apoptotic induction. Therefore, quercetin is potentially an apoptotic-inducing agent, which reacts at the mitochondrial level.     

CFTR in K562 human leukemic cells

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 microM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.     

Iron metabolism in K562 erythroleukemic cells

Iron delivery to K562 cells is enhanced by desferrioxamine through induction of transferrin receptors. Experiments were performed to further characterize this event with respect to iron metabolism and heme synthesis. In control cells, up to 85% of the iron taken up from iron-transferrin was incorporated into ferritin, 7% into heme, and the remainder into compartments not yet identified. In cells grown with desferrioxamine, net accumulation of intracellular desferrioxamine (14-fold) was observed and iron incorporation into ferritin and heme was inhibited by 86% and 75%, respectively. In contrast, complete inhibition of heme synthesis in cells grown with succinylacetone had no effect on transferrin binding or iron uptake. Exogenous hemin (30 microM) inhibited transferrin binding and iron uptake by 70% and heme synthesis by 90%. These effects were already evident after 2 h. Thus, although heme production could be reduced by desferrioxamine, succinylacetone, and hemin, cell iron uptake was enhanced only by the intracellular iron chelator. The effects of exogenous heme are probably unphysiologic and the greater inhibition of iron flow into heme can be explained by effects on early steps of heme synthesis. We conclude that in this cell model a chelatable intracellular iron pool rather than heme synthesis mediates regulation of iron uptake.     

Erythroid induction of K562 human leukemia cells: enhancement by purines

K562 cells are human leukemia cells inducible for hemoglobin synthesis by a variety of agents. This report demonstrates that hypoxanthine, which alone has no inductive effect, enhances induction by thymidine, resulting in a greater absolute, as well as relative, percentage of benzidine positive cells. This effect is seen over a 20-fold concentration range for both thymidine and hypoxanthine. This enhancement involves commitment, i.e., a process in which the induction of hemoglobin synthesis is coupled to a limitation in the number of subsequent cell divisions. Although thymidine alone increases the percentage of cells in S phase, hypoxanthine does not augment this. Purines other than hypoxanthine also enhance induction by thymidine. This enhancement by hypoxanthine of thymidine induction is inhibited by pyrimidine nucleosides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, itself an effective K562 inducer, is not additive to thymidine and hypoxanthine, suggesting that hypoxanthine may act by reducing the supply of guanosine nucleosides.