Interference of aluminium on iron metabolism in erythroleukaemia K562 cells

It has been suggested that aluminium (Al) has a deleterious effect on erythropoiesis. However, there is still uncertainty as to its action mechanism. The present work was designed to determine how Al could affect the iron (Fe) metabolism in the human erythroleukaemia cell line K562. These cells, that express surface transferrin receptors (TfRs), were induced to erythroid differentiation by either haemin or hydroxyurea in 72 h cultures in media containing apotransferrin (apoTf). In the presence of aluminium citrate, the number of benzidine-positive cells decreased 18% when the cultures were induced by haemin, and 30% when hydroxyurea was the inducer. Cell viability was always unaffected. From competition assays, surface binding of 125I-Tf-Fe2 was found to be inversely related (p < 0.05) to Tf-Al2 concentration (from 2.5 to 10 nM). The dissociation constants (Kd) of the binding reaction between TfRs and the ligands Tf-Fe2 and Tf-Al2 were calculated. Kd values of the same order of magnitude demonstrated that TfR has a similar affinity for Tf-Fe2 (Kd = 1.75 x 10(-9) M) and Tf-Al2 (Kd = 1.37 x 10(-9) M). The number of surface TfRs, measured by kinetic 125I-Tf-Fe2 binding assays, was higher in induced cells cultured in the presence of Al. Nevertheless, in spite of the inhibition of cell haemoglobinization observed, 59Fe incorporation values were not different from those measured in control cultures for 72 h. As a consequence, it can be suggested that cellular Fe utilisation, and not Fe uptake, might be the main metabolic pathway impaired by Al.     

Aluminum exposure affects transferrin-dependent and -independent iron uptake by K562 cells

Aluminum (Al) and iron (Fe) share several physicochemical characteristics and they both bind to transferrin (Tf), entering the cell via Tf receptors (TfR). Previously, we found similar values of affinity constant for the binding of TfR to Tf carrying either Al or Fe. The competitive interaction between both metals prevented normal Fe incorporation into K562 cells and triggered the upregulation of Fe transport. In the present work we demonstrated that Al modified Fe uptake without affecting the expression of Tf receptors. Both TfR and TfR2 mRNA levels, evaluated by RT-PCR, and TfR antigenic sites, analyzed by flow cytometry, were found unchanged after Al exposure. In turn, Al did induce upregulation of non-Tf bound Fe (NTBI) uptake. This modulation was not due to intracellular Fe decrease since NTBI transport proved not to be regulated by Fe depletion. Unlike its behavior in the presence of Tf, Al was unable to compete with NTBI uptake, suggesting that both metals do not share the same alternative transport pathway. We propose that Al interference with TfR-mediated Fe incorporation might trigger the upregulation of NTBI uptake, an adaptation aimed at incorporating the essential metal required for cellular metabolism without allowing the simultaneous access of a potentially toxic metal.     

Transferrin and iron uptake by human lymphoblastoid and K-562 cells

Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.     

Metabolic depletion inhibits the uptake of nontransferrin-bound iron by K562 cells

The effect of metabolic inhibitors on nontransferrin bound iron transport by K562 cells was investigated. Incubation with 1 microM rotenone, 10 microM antimycin, or 0.5 mM 2,4-dinitrophenol effectively reduced ATP levels by approximately 50%. Both the rate and extent of Fe+3 uptake were impaired in ATP-depleted cells, which display a reduced Vmax for uptake. K562 cell ferrireductase activity was also lowered by metabolic inhibitors, suggesting that the apparent energy requirements for transport reside in the reduction of Fe+3 to Fe+2. However, ATP depletion was found to inhibit the rate and extent of Fe+2 uptake as well. Thus, the transbilayer passage of Fe+2 and/or Fe+3 appears to be an energy-requiring process. These features possibly reflect properties of the transport mechanism associated with a recently identified K562 cell transport protein, called SFT for "Stimulator of Fe Transport," since exogenous expression of its activity is also affected by ATP depletion.     

Complexation of ytterbium to human transferrin and its uptake by K562 cells.

There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin-receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV-Vis spectroscopy and stopped-flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N-lobe is kinetically favored while the C-lobe is thermodynamically favored. The first Yb3+ bound to the C-lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+-transferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mechanism is a possible pathway for Yb3+ accumulation in cells.     

Receptor-mediated heme uptake from hemopexin by human erythroleukemia K562 cells

Using human erythroleukemia K562 cells, existence of receptors for hemopexin has been investigated. Hemopexin was bound to the cells in saturable, time- and temperature-dependent manner. The cells exhibited approximately 8,400 binding sites/cell for hemopexin and apohemopexin. The dissociation constants (Kd) for hemopexin and apohemopexin were 4.79 nM and 10.8 nM, respectively. Specific binding of labeled hemopexin was inhibited with increasing concentrations of unlabeled hemopexin and apohemopexin, but unaffected by transferrin and serum albumin. Heme bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that heme in hemopexin was taken up by K562 cells via the receptors for hemopexin.     

The effect of lead on iron uptake from transferrin in human erythroleukemia (K562) cells

The effect of lead on cellular iron metabolism has been investigated using human erythroleukemia (K562) cells. When the cells were cultured with 100 m Pb²⁺ for 48 h, the rate of cellular iron uptake from transferrin decreased to 46% of that in untreated cells. Scatchard analysis of the binding data revealed that this reduction was the result of a decrease in the number of transferrin receptors rather than an alteration in ligand-receptor affinity. The results of immunoprecipitation of transferrin receptors on the cell surface also confirmed the decreased expression of transferrin receptors by lead-treated cells. The down-regulation of transferrin receptors by treatment with lead did not result from a decrease in the total amount of the receptor, as determined by immunoblotting. Moreover, the biosynthesis of the receptor was unaffected by lead treatment. Thus, the down-regulation of surface transferrin receptors in lead-treated cells might be due to a redistribution of receptors rather than an actual loss of receptors from the cell. Using kinetic analysis, it was shown that redistribution of the receptor did not result from the alteration in the rates of transferrin receptor recycling. A comparison of the amounts of transferrin receptor on the cell surface and in the cycling pool revealed that the sequestration of the receptor from normal flow through the cycle might cause down-regulation of the surface receptor.     

Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells

K562 erythroleukemia cells import non-transferrin-bound iron (NTBI) by an incompletely understood process that requires initial iron reduction. The mechanism of NTBI ferrireduction remains unknown but probably involves transplasma membrane electron transport. We here provide evidence for a novel mechanism of NTBI reduction and uptake by K562 cells that utilizes transplasma membrane ascorbate cycling. Incubation of cells with dehydroascorbic acid, but not ascorbate, resulted in (i) accumulation of intracellular ascorbate that was blocked by the glucose transporter inhibitor, cytochalasin B, and (ii) subsequent release of micromolar concentrations of ascorbate into the external medium via a route that was sensitive to the anion channel inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Ascorbate-deficient control cells demonstrated low levels of ferric citrate reduction. However, incubation of the cells with dehydroascorbic acid resulted in a dose-dependent stimulation of both iron reduction and uptake from radiolabeled [(55)Fe]ferric citrate. This stimulation was abrogated by ascorbate oxidase treatment, suggesting dependence on direct chemical reduction by ascorbate. These results support a novel model of NTBI reduction and uptake by K562 cells in which uptake is preceded by reduction of iron by extracellular ascorbate, the latter of which is subsequently regenerated by transplasma membrane ascorbate cycling.     

Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells.

We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythroleukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N -acetylcysteine (NAC). Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases. However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythroleukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAC, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells. In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data. The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphoryl-ation and DNA binding of GATA-1 are already optimalin these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor. Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding. Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.     

Additive stimulatory effect of extracellular calcium and potassium on non-transferrin ferric iron uptake by HeLa and K562 cells

Elongation factor Ts (EF-Ts) from Thermus thermophilus forms a stable, functionally active homodimer in solution. Its monomer is composed of two domains: amino-terminal domain containing 50 amino acid residues and a larger, 146 residues long, C-domain which participates in dimerization of EF-Ts. Effect of removal of the N-domain on the conformational stability of EF-Ts has been studied. For comparison, the stabilities of both the full-length EF-Ts and its C-domain were studied by differential scanning calorimetry, electronic absorption and fluorescence spectroscopies over a pH range from 4 to approximately 13. Thermal denaturation of EF-Ts and of C-domain, followed by circular dichroism at 222 nm, at pH 7.0, and the pH dependence of the fluorescence of the single tryptophan 30 residue indicate a conformational instability of the N-domain. While N-domain does not affect the stability of full-length EF-Ts at acidic pH, its removal leads to stabilization of the rest of the protein at basic pH. This is reflected by higher values of transition temperatures and calorimetric enthalpies of C-domain as compared to the full-length EF-Ts. High mobility of the N-domain in alkaline pH conditions decreased the thermal stability of covalently linked C-domain of EF-Ts. An increase in intramolecular interactions at acidic pH together with a decrease of conformational entropies of the thermally denatured proteins most likely diminishes this destabilization effect.     

Effect of iron chelators on the transferrin receptor in K562 cells

Delivery of iron to K562 cells by diferric transferrin involves a cycle of binding to surface receptors, internalization into an acidic compartment, transfer of iron to ferritin, and release of apotransferrin from the cell. To evaluate potential feedback effects of iron on this system, we exposed cells to iron chelators and monitored the activity of the transferrin receptor. In the present study, we found that chelation of extracellular iron by the hydrophilic chelators desferrioxamine B, diethylenetriaminepentaacetic acid, or apolactoferrin enhanced the release from the cells of previously internalized 125I-transferrin. Presaturation of these compounds with iron blocked this effect. These chelators did not affect the uptake of iron from transferrin. In contrast, the hydrophobic chelator 2,2-bipyridine, which partitions into cell membranes, completely blocked iron uptake by chelating the iron during its transfer across the membrane. The 2,2-bipyridine did not, however, enhance the release of 125I-transferrin from the cells, indicating that extracellular iron chelation is the key to this effect. Desferrioxamine, unlike the other hydrophilic chelators, can enter the cell and chelate an intracellular pool of iron. This produced a parallel increase in surface and intracellular transferrin receptors, reaching 2-fold at 24 h and 3-fold at 48 h. This increase in receptor number required ongoing protein synthesis and could be blocked by cycloheximide. Diethylenetriaminepentaacetic acid or desferrioxamine presaturated with iron did not induce new transferrin receptors. The new receptors were functionally active and produced an increase in 59Fe uptake from 59Fe-transferrin. We conclude that the transferrin receptor in the K562 cell is regulated in part by chelatable iron: chelation of extracellular iron enhances the release of apotransferrin from the cell, while chelation of an intracellular iron pool results in the biosynthesis of new receptors.     

Regulation of intracellular iron distribution in K562 human erythroleukemia cells

Following a pulse with 59Fe-transferrin, K562 erythroleukemia cells incorporate a significant amount of 59Fe into ferritin. Conditions or manipulations which alter the supply of iron to cells result in changes in the rate of ferritin biosynthesis with consequent variations in the size of the ferritin pool. Overnight exposure to iron donors such as diferric transferrin or hemin increases the ferritin level 2-4- or 6-8-fold above that of the control, respectively. Treatment with the anti-human transferrin receptor antibody, OKT9 (which reduces the iron uptake by decreasing the number of transferrin receptors) lowers the ferritin level by approximately 70-80% with respect to the control. The fraction of total cell-associated 59Fe (given as a pulse via transferrin) that becomes ferritin bound is proportional to the actual ferritin level and is independent of the instantaneous amount of iron taken up. This has allowed us to establish a curve that correlates different levels of intracellular ferritin with corresponding percentages of incoming iron delivered to ferritin. Iron released from transferrin appears to distribute to ferritin according to a partition function; the entering load going into ferritin is set for a given ferritin level over a wide range of actual amounts of iron delivered.     

Intracellular Ca(2+) regulates the cellular iron uptake in K562 cells

Fluorescence quenching was used to study the kinetics of the transferrin receptor (TfR)-mediated iron uptake in the calcein-loaded K562 cells. It was found that elevation of intracellular free Ca(2+) ([Ca(2+)](i)) by thapsigargin (TG) speeds up the initial rate of iron uptake and increases the overall capacity of the cells in taking up iron. Depletion of intracellular Ca(2+) or complete chelation of extracellular Ca(2+) results in complete inhibition of the iron uptake in cells. To gain insight into molecular mechanism, IANBD-labeled transferrin (Tf) and microscopic fluorescence imaging were used to observe the endocytosis and recycling of the Tf-TfR complex in single live cells. The study showed that the preincubation of cells with TG or phorbol myristate acetate (PMA), the direct activator of protein kinase C (PKC), accelerated the endocytosis and recycling of the complex in a dose-dependent manner. W-7, the calmodulin antagonist, and GF109203X, a selected cell-permeant inhibitor of PKC, can reverse the acceleration. Analysis of actin polymerization in controlled, [Ca(2+)](i)-elevated and W-7-treated cells revealed that the actin polymerization is enhanced as [Ca(2+)](i) is raised, but reduced by W-7. The results suggest that the regulation of actin polymerization by intracellular Ca(2+) may play a central role in Ca(2+)-dependent iron uptake.     

Disturbance of cellular iron uptake and utilisation by aluminium.

Aluminium (Al) affects erythropoiesis but the real mechanism of action is still unknown. Transferrin receptors (TfR) in K562 cells are able to bind Tf, when carrying either iron (Fe) or Al, with similar affinity. Then, the aim of this work was to determine whether Al could interfere with the cellular Fe uptake and utilisation. K562 cells were induced to erythroid differentiation by either haemin (H) or sodium butyrate (B) and cultured with and without Al. The effect of Al on cellular Fe uptake, Fe incorporation to haem and cell differentiation was studied. H- and B-stimulated cells grown in the presence of 10 microM Al showed a reduction in the number of haemoglobinised cells (by 18% and 56%, respectively) and high amounts of Al content. Al(2)Tf inhibited both the (59)Fe cellular uptake and its utilisation for haem synthesis. The removal of Al during the (59)Fe pulse, after a previous incubation with the metal, allowed the cells to acquire Fe quantities in the normal range or even exceeding the amounts incorporated by the respective control cells. However, the Fe incorporated to haem could not reach control values in B-stimulated cells despite enough Fe acquisition was observed after removing Al. Present results suggest that Al might exert either reversible or irreversible effects on the haemoglobin synthesis depending on cellular conditions.     

The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells

The ZIP superfamily of transporters plays important roles in metal ion uptake in diverse organisms. There are 12 ZIP-encoding genes in humans, and we hypothesize that many of these proteins are zinc transporters. In this study, we addressed the role of one human ZIP gene, hZIP1, in zinc transport. First, we examined (65)Zn uptake activity in K562 erythroleukemia cells overexpressing hZIP1. These cells accumulated more zinc than control cells because of increased zinc influx. Moreover, consistent with its role in zinc uptake, hZIP1 protein was localized to the plasma membrane. Our results also demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent (65)Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous (65)Zn uptake activity. The observation that hZIP1 is the major zinc transporter in K562 cells, coupled with its expression in many normal cell types, indicates that hZIP1 plays an important role in zinc uptake in human tissues.     

Kinetic parameters for the uptake of anthracycline by drug-resistant and drug-sensitive K562 cells.

Fluorescence-emission spectra from anthracycline-treated cells suspended in buffer have been used to measure the uptake of three anthracycline derivatives: adriamycin, 4'-O-tetrahydropyranyladriamycin and aclacinomycin in drug-sensitive and drug-resistant K562 cells. The initial rate of uptake and the kinetics of active efflux under the effect of an integral membrane glycoprotein, P-glycoprotein, have been measured as a function of temperature. The activation energies for the passage of the drugs through the plasma membrane have been calculated. In the case of 4'-O-tetrahydropyranyladriamycin, the activation energies for the passive diffusion of the drug equal 45 kJ.mol-1 and 37 kJ.mol-1 for sensitive and resistant cells, respectively. The activation energy for the active efflux of 4'-O-tetrahydropyranyladriamycin equal 25 kJ.mol-1.     

Glycophorins of human erythroleukemic K562 cells

Glycophorins related to alpha glycophorin, of the human erythrocyte membrane, were isolated from human erythroleukemic K562 cells. The glycophorins were purified using sodium dodecyl sulfate (SDS)/trichloroacetic acid fractionation and Folch and hot phenol extractions. 0.1-0.2 micrograms was obtained/10(8) cells, or approximately a 15% yield. SDS-gel electrophoresis revealed a pattern similar to erythrocyte alpha glycophorin except for the slower mobility of the glycophorin monomer. Two populations of K562 glycophorins, present in nearly equivalent amounts, were distinguished by their binding to Lens culinaris lectin agarose. The two populations exhibited similar gel electrophoretic patterns except for the presence of delta-like glycophorin exclusively in the population that did not bind to L. culinaris lectin. Immunoblotting revealed a lack of reaction of the major alpha and delta-like glycophorin bands in all K562 glycophorins with M or N erythrocyte glycophorin-specific monoclonal antibodies. Only minor species of intermediate electrophoretic mobility in glycophorins not binding to L. culinaris showed a reaction with these antibodies. Both populations of glycophorins incorporated radiolabeled glucosamine, mannose, and fucose and contained O-glycosidically linked tri- and tetrasaccharides, present in a ratio of approximately 1:1 indicating a significant degree of hyposialylation when compared to erythrocyte alpha glycophorin. No precursor/product relationship was demonstrated between the major forms of two populations. K562 cell surface labeling with lactoperoxidase revealed that only the glycophorins that exhibited binding to L. culinaris were accessible to iodination and could be the only species expressed at the cell surface.     

Ferritin iron kinetics and protein turnover in K562 cells

The binding, incorporation, and release of iron by ferritin were investigated in K562 cells using both pulse-chase and long term decay studies with 59Fe-transferrin as the labeled iron source. After a 20-min pulse of labeled transferrin, 60% of the 59Fe was bound by ferritin with the proportion increasing to 70% by 4 h. This initial binding was reduced to 35% when the cells were exposed to the chelator desferrioxamine (5 mM) for an additional 30 min. By 4 h the association of 59Fe with ferritin was unaffected by the presence of the chelator, and levels of 59Fe-ferritin were identical to those in control cells (70%). Between 4-10h there was a parallel decline in 59Fe-ferritin in both control and desferrioxamine-treated cells. When incoming iron was bound by ferritin it was, therefore, initially chelatable but with time progressed to a further, nonchelatable compartment. In turnover studies where ferritin was preloaded with 59Fe by overnight incubation, 50% of the label was released from the protein by 18 h, contrasting with a t 1/2 for cellular iron release of approximately 70 h. The half-time of 59Fe release from ferritin was accelerated to 11 h by the presence of desferrioxamine. The half-time for ferritin protein turnover determined by [35S]methionine labeling was approximately 12 h in the presence or absence of the chelator. Thus, when the reassociation of iron with ferritin was prevented by the exogenous chelator there was a concordant decay of both protein and iron moieties. The direct involvement of lysosomes in this turnover was demonstrated by the use of the inhibitors leupeptin and methylamine which stabilized both 59Fe (t 1/2 = 24 h) and 35S (t 1/2 = 25.6 h) labels. We conclude that in this cell type the predominant mechanism by which iron is released from ferritin is through the constitutive degradation of the protein by lysosomes.