ATP depletion causes translational immobilization of cell surface transferrin receptors in K562 cells

We have used quantitative fluorescence microscopy and fluorescence photobleaching recovery to examine the role of metabolic energy in the translational movement of transferrin receptors in the plasma membrane of K562 erythroleukemia cells. Cellular ATP depletion caused a significant decrease in the translational mobility of cell surface transferrin receptors and a significant increase in the number of receptors on the cell surface. ATP repletion restored receptor translational mobility and cell surface expression to control values. Inhibition of ATP hydrolases by orthovanadate also immobilized cell surface transferrin receptors and altered cell surface receptor expression, in a concentration-dependent manner. Vanadate-induced changes in receptor mobility and cell surface expression were reversible upon washing out the drug. Cellular ATP depletion did not affect the translational mobility of plasma membrane glycophorins or a fluorescent phospholipid analogue. We conclude that the translational movement of cell surface transferrin receptors specifically requires metabolic energy and ATP hydrolysis. © 1996 Wiley-Liss, Inc.     

Receptor-mediated endocytosis of transferrin in K562 cells

Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell line. There are about 1.6 X 10(5) binding sites per cell surface, exhibiting a KD of about 10(-9) M. Upon warming cells to 37 degrees C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 degree C. This is accounted for by internalization of the ligand as shown by the development of resistance to either acid wash or protease treatment of the ligand-cell association. After a minimum residency time of 4-5 min, undegraded transferrin is released from the cell. Internalization is rapid but is dependent upon cell surface occupancy; at occupancies of 20% or greater the rate coefficient is maximal at about 0.1-0.2 min-1. In the absence of externally added ligand only 50% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-1. The remaining transferrin can be released from the cell only by the addition of ligand, suggesting a tight coupling between cell surface binding, internalization, and release of internalized ligand. There is a loss of cell surface-binding capacity that accompanies transferrin internalization. At low (less than 50%) occupancy this loss is monotonic with the extent of internalization. Even at saturating levels of transferrin, the loss of surface receptors upon internalization never exceeds 60-70% of the initial binding capacity. This suggests that receptors enter the cell with ligand but are replaced so as to maintain a constant, albeit reduced, receptor number on the cell surface. In the absence of ligand, the cell surface receptor number returns at 37 degrees C. Neither sodium azide nor NH4Cl blocks internalization of ligand. However, they both prevent the release of transferrin from the cell thus halting the transferrin cycle. Excess ligand can overcome the block due to NH4Cl but not azide although the cycle is markedly slower. Iron is delivered to these cells by transferrin at 37 degrees C with a rate coefficient of 0.15 to 0.2 min-1. The iron is released from the transferrin and the majority is found in intracellular ferritin. There is a large internal receptor pool comprising 70 to 80% of the total cell receptors and this may be involved in maintaining the steady state iron uptake.     

Regulation of K562 cell transferrin receptors by exogenous iron

Single-cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 micrograms/ml, by the addition of iron in the form of FeCl3, iron-saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8-fold increase in external receptors, detected as binding by both transferrin and monoclonal anti-TfR antibodies, and a 1.5-fold increase in the pool of internal receptors, as detected by anti-TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cycloheximide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.     

Single-cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells

Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.     

Phorbol ester-induced regulation of transferrin receptors in human leukemia K562 cells

The treatment of human leukemia K562 cells with 12-0-tetradecanoyl phorbol-13-acetate (TPA) caused a decrease of transferrin receptors. The mechanism of the decrease of the receptors with TPA has been investigated. In cells incubated with TPA, the rate of biosynthesis of transferrin receptors was reduced to 10-20% of that in untreated cells. Pulse-chase experiments showed that turnover of the receptors in TPA-treated cells was accelerated over that in untreated cells. These results indicated that the decrease of transferrin receptors in TPA-treated cells was caused by reduced biosynthesis and accelerated degradation of the receptors.     

3'-Azido-3'-deoxythymidine reduces the rate of transferrin receptor endocytosis in K562 cells.

K562 cells, exposed for at least 24 h to 5 microM 3'-azido-3'-deoxythymidine (AZT), gave rise to an overall increase in the number of cell surface transferrin binding receptors (18-20%). This effect was ascertained either with binding experiments by using 125I-transferrin and with immunoprecipitation by using a specific monoclonal antibody against the transferrin receptor. At higher AZT concentrations (20 and 40 microM), a further increase was found, that is, up to 23% by binding experiments and up to 110% by immunoprecipitation. However, Scatchard analysis of the binding data indicated that although the number of cell surface transferrin receptors increased, the affinity of transferrin for its receptor did not change (Ka=4.0x108 M). Surprisingly, immunoprecipitation of total receptor molecules showed that the synthesis of receptor was not enhanced by the drug treatment. The effect of AZT on transferrin internalization and receptor recycling was also investigated. In this case, data indicated that the increase in the number of receptors at the cell surface was probably due to a slowing down of endocytosis rate rather than to an increased recycling rate of the receptor to cell surface. In fact, the time during which half the saturated amount of transferrin had been endocytosed (t1/2) was 2.15 min for control cells and 3.41, 3.04, and 3.74 min for 5, 20, and 40 microM AZT-treated cells, respectively. Conversely, recycling experiments did not show any significant differences between control and treated cells. A likely mechanism through which AZT could interfere with the transferrin receptor trafficking, together with the relevance of our findings, is extensively discussed.     

Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis.

Iron distribution in subcellular fractions was investigated at different times after a single cohort of 59Fe-125 I-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients. Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis 59Fe activity in the low-molecular-weight fraction remained constant and 59Fe accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, 59Fe radioactivity in this fraction decreased and a majority of cytosolic 59Fe was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.     

A rapid redistribution of the transferrin receptor to the cell surface of HL-60 cells and K562 cells upon treatment with dimethyl sulfoxide due to slowing of endocytosis

Treatment of two human leukemia cell lines with 1.25% dimethyl sulfoxide at 37 degrees C results in a rapid increase in the number of transferrin receptors on the cell surface detected by fluorescein-labeled anti-transferrin receptor antibodies. Both HL-60 cells, a human myeloid cell line, and K562 cells, a human erythroid-myeloid cell line, showed a 25-65% increase in cell surface transferrin binding in parallel experiments. Scatchard plot analysis of the data indicates that the number of receptors increases while the affinity of transferrin for the receptor remains the same. This rapid increase in the number of receptors at the cell surface appears to be due to a slowing of endocytosis rather than an increase in externalization of the receptor.     

Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells

The intracellular movement of cell surface transferrin receptor (TfR) after internalization was studied in K562 cultured human erythroleukemia cells. The sialic acid residues of the TfR glycoprotein were used to monitor transport to the Golgi complex, the site of sialyltransferases. Surface-labeled cells were treated with neuraminidase, and readdition of sialic acid residues, monitored by isoelectric focusing of immunoprecipitated TfR, was used to assess the movement of receptor to sialyltransferase-containing compartments. Asialo-TfR was resialylated by the cells with a half-time of 2-3 h. Resialylation occurred in an intracellular organelle, since it was inhibited by treatments that allow internalization of surface components but block transfer out of the endosomal compartment. Moreover, roughly half of the resialylated molecules were cleaved when cells were retreated with neuraminidase after culturing, indicating that this fraction of the molecules had returned to the cell surface. These results suggest that TfR is transported from the cell surface to the Golgi complex, the intracellular site of sialyltransferases, and then returns to the cell surface. This pathway, which has not been previously described for a cell surface receptor, may be different from the route followed by TfR in iron uptake, since reported rates of transferrin uptake and release are significantly more rapid than the resialylation of asialo-TfR.     

Brefeldin a down-regulates the transferrin receptor in K562 cells

The fungal metabolite brefeldin A (BFA) induces profound alterations in the morphology of intracellular organelles. Although BFA promotes the formation of extensive tubular endosomal domains, our understanding of the effects of the antibiotic on vesicle traffic events associated with endocytosis is limited. Thus, alterations in the transferrin (Tf) receptor's endocytic/recycling pathway upon treatment of human erythroleukemia K562 cells with BFA were studied as a pharmacological response. Treatment of K562 cells with BFA caused a down-regulation in the number of cell surface Tf receptors. This effect is highly reminiscent of the well-known action of phorbol 12-myristate 13-acetate (PMA) on Tf receptor traffic in K562 cells. However, our results demonstrate that these two agents down-regulate the Tf receptor via different mechanisms. The effects of BFA and PMA were additive when K562 cells were incubated with both together. Using the In/Sur method, the endocytic rate constant for Tf internalization was determined and PMA was found to greatly enhance k_e, from 0.28 min^–1 to 0.43 min^–1, while BFA had little effect (K_e=0.20 min^–1). In contrast, BFA-treatment alters the exocytic rate constant for return of internalized receptors to the cell surface, with the largest effect exerted on a slow-release, monensin-sensitive, compartment. The sum of the endocytic and exocytic kinetic data support a model in which BFA and PMA down-regulate the Tf receptor in K562 cells by mechanistically distinct actions, with BFA targeting exocytic monensin-sensitive intracellular compartments and PMA acting to exert a profound influence on elements of receptor internalization.Abbreviations BFA brefeldin A - ARF ADP-ribosylation factor - HRP horseradish peroxidase - Tf transferrin - PMA phorbol 12-myristate 13-acetate - DMSO dimethyl sulfoxide - PBS phosphate-buffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin - FITC-Tf fluorescein isothiocyanate-labelled transferrin     

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.     

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.     

Characterization of transferrin receptor released by K562 erythroleukemia cells

A soluble form of transferrin receptor has been detected in human serum and has been shown recently to be a truncated form of the intact membrane bound receptor. Mechanisms governing the release of transferrin receptor by cells are poorly understood and could be better defined by tissue culture. The present investigation was undertaken to characterize the transferrin receptor released by K562 erythroleukemic cells. In contrast with maturing sheep reticulocytes, which have been shown to release transferrin receptor in small vesicles termed exosomes, we demonstrated, with a monoclonal enzyme-linked immunoassay, that less than 30% of the transferrin receptor released by K562 cells in log phase growth was in a particulate form. The relative amounts of soluble and particulate receptor released to the supernatant did not change significantly during 48 hr of incubation. Soluble receptor was purified by immunoaffinity chromatography. On polyacrylamide gel electrophoresis, its mobility was the same (85 kDa) as that of the truncated monomeric form recently identified in human serum. Further evidence that serum and soluble receptors released by K562 cells are identical was provided by amino acid sequence analysis, which demonstrated that 16 of the first 19 residues of the N-terminal sequence of soluble K562 receptor are homologous with the serum receptor. The remaining three were not identifiable. K562 cells provide a useful in vitro model for studying the production of membrane-bound and soluble forms of released transferrin receptor.     

Interferon-gamma is a strong modulator of NK susceptibility and expression of beta 2-microglobulin but not of transferrin receptors of K562 cells

The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.     

Co-migration and internalization of transferrin and its receptor on K562 cells

The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC- transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fab-antitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4 degrees C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20 degrees C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20 degrees C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.     

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.     

Biosynthetic regulation of the human transferrin receptor by desferrioxamine in K562 cells

Treatment of K562 cells with the iron chelator desferrioxamine results in the gradual increase in total cell receptors for transferrin. Receptor number rises 2.5-4.5-fold over 24 h and remains at the elevated level if the chelator is continuously present. Preincubation of the chelator with ferric chloride abolishes the effect. The drug has no effect on the 7-h half-life of the receptor. The increased number of receptors can be accounted for by a specific increase in the rate of receptor biosynthesis which reaches 3-4 times that seen in untreated cells by 6 h after the addition of the chelator. Isolation of mRNA from treated cells reveals that, after 8 h in the presence of desferrioxamine, there is a 3-fold increase in the specific translation of transferrin receptor over untreated cells. Total protein synthesis is not changed under these conditions.     

Phorbol ester induces increased expression, altered glycosylation, and reduced adhesion of K562 erythroleukemia cell fibronectin receptors

The human multipotential hematopoietic cell line K562 expresses fibronectin receptor (FNR) subunits of 160 kDa (alpha chain) and 120 kDa (beta chain). Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) led to reduced binding of K562 to immobilized fibronectin (FN), although treated cells expressed 10-fold more cell surface FNR than untreated cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed this and showed altered electrophoretic mobilities of FNR subunits from TPA-treated cells. TPA treatment affected N-linked glycosylation, as tunicamycin treatment of K562 cells abolished differences in FNR mobility. Sialidase treatment of FNR immunoprecipitates minimized and sialidase treatment of intact cells eliminated these mobility differences between subunits from control and TPA-treated cells. Reduced sialylation of FNR from TPA-treated cells was further demonstrated by chromatography with bead-coupled lectins and by the greater negative charge of untreated K562 FNR subunits in two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis. A relationship between reduced FNR sialylation and reduced FN binding was suggested by adhesion assays of sialidase-treated K562 which showed that desialylation of cell surface FNR was associated with decreased cell adhesion. Thus, TPA treatment reduces the function, increases the expression, and alters the structure of K562 FNR, and these changes appear to involve FNR sialylation.     

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.     

Induced cell surface expression of functional alpha 2 beta 1 integrin during megakaryocytic differentiation of K562 leukemic cells.

The pluripotential hematopoietic cell line K562 was studied as a model of inducible integrin expression accompanying differentiation. Differentiation along the megakaryocytic pathway was induced with phorbol 12,13-dibutyrate and differentiation along the erythroid pathway with hemin. Induction of megakaryocytic differentiation was associated with changes in cell morphology and with increased cell-cell and cell-substrate adhesion and spreading. Erythroid differentiation was not associated with changes in morphology or adhesion. Cell surface expression of the IIb-IIIa and alpha 2 beta 1 integrins increased markedly with phorbol treatment but decreased with hemin treatment. Phorbol-treated K562 cells, but not control cells or hemin-treated cells, adhered to collagen substrates in a Mg(2+)-dependent manner which was specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1 integrin. Northern blot analysis revealed that megakaryocytic differentiation of K562 cells was accompanied by de novo expression of the alpha 2 integrin mRNA with no change in the level of mRNA for the beta 1 subunit. K562 cells provide a model of differentiation-dependent, regulated integrin expression in which expression is up- or down-regulated depending upon the differentiation pathway selected.