Terminal differentiation of human erythroleukemia cell line K562 induced by aphidicolin

To analyze the relationship between differentiation and DNA replication, the effect of aphidicolin, a specific inhibitor for DNA polymerase alpha, was measured with respect to erythroid differentiation and activities of DNA polymerases alpha, beta, and gamma. Five micromolar aphidicolin completely blocked the growth of K562 cells and caused 80% of cells to become hemoglobin positive after 5 days exposure. The cessation of K562 cell growth induced by aphidicolin was irreversible, whereas the inhibition of HeLa cell growth was completely reversible. The enzyme activity of DNA polymerase alpha of K562 cells showed a 50-110% increase with aphidicolin treatment as compared to control K562 cells; activities of DNA polymerases beta and gamma were not affected. These features sharply contrasted with the erythroid induction of the same cells by hemin, where cell growth was not suppressed and DNA polymerase alpha was not increased but rather decreased. The enzyme activity of DNA polymerase alpha remained high even after removal of aphidicolin from the culture medium. These results suggest that treatment with aphidicolin might induce an accumulation of protein factors for replication and/or differentiation, causing rapid cell differentiation of cells without cell division.     

The binding of insulin-like growth factor II to the erythroleukemia cell line K562

The erythroleukemia cell line K562 was previously shown to have specific binding sites for insulin but not for insulin-like growth factor I (IGF-I). In this study the presence of specific receptors for insulin-like growth factor II (IGFqI) is established. Scatchard analysis of the competition curve for IGF-II disclosed a non-cooperative binding kinetic with a calculated affinity constant of 2.4×108 M^–1 and a receptor number of 4.8×l0⁴ sites/cell. IGF-I displayed 10% crossreactivity over the IGF-II receptor but insulin did not crossreact at all. Instead insulin, present in high concentrations, enhanced the binding of IGF-II. The presence of IGF II but not IGF-I receptors makes t h e K562 cell line suitable for studying properties of the type-2 receptor.     

Phenotypes of HeLa S3 variant cell lines resistant to growth inhibition by sodium butyrate

Summary HeLa cell variants capable of multiplying in the presence of sodium butyrate were used to study the relationship of cell cycle position to human chorionic gonadotropin (hCG) production and regulation of the genes encoding hCG α- and β-subunits. The butyrate-resistant variants exhibit several different stable phenotypes. In wild-type HeLa cells, butyrate arrests cell division and modulates synthesis of α- and β-subunits of glycoprotein hormones by coordinately regulating steady-state levels of their respective mRNAs. Because the variant cell lines replicate, in addition to producing hCG subunits in the presence of butyrate, cell cycle arrest does not seem to be a requirement for expression of glycoprotein hormone genes. Studies of histone modification suggest that neither hyperacetylation of histones H3 and H4 nor dephosphorylation of histones H1 and H2A mediates inhibition of cell replication. In the variants, α-subunit and hCGβ levels are independently regulated, as a consequence of independent regulation of α- and β-hCG mRNA levels. Long-term effects of butyrate include derepression of some genes (hCGβ in the variant AO) and repression of others (hCGα in variant AO). Moreover, hormone production correlates with the steady-state levels of mRNA for each of the subunits, suggesting that regulation occurs before translation. These findings indicate that the butyrate-resistant variant cell lines are valuable for studies of the molecular mechanisms involved in regulation of expression of ectopic hormones.     

Characterization of insulin binding to the erythroleukemia cell line K 562

The K 562 is a transformed human erythroid stemcell and is used as a target cell for NK-T-cells. In this study the presence of insulin receptors in K 562 is established.The best binding and negative cooperativity was found in the two Hepes containing buffers whereas no cooperativity was obtained in the Krebs-Ringer buffer. The calculated affinity constants and receptor number per cell varied according to the buffer. Preincubation with insulin caused a down-regulation of the insulin binding capacity. 10 ng/ml caused a lowering of the affinity, with an unchanged number of receptors. 100 ng/ml caused a decrease in receptor number with unchanged affinity. These results were found in both Hepes and Krebs-Ringer phosphate buffer. IGF-I shows cross-reactivity with the insulin receptor, with a potency of 12 and 100 times less than insulin in Krebs-Ringer phosphate buffer and G-buffer respectively. However, no specific IGF-I receptors were found.The presence of receptors on K 562 cells suggests a biological role for insulin. The different results in the different buffers, indicate that a buffer containing Hepes and/or Tris, is required to expose negative cooperativity and make the receptors more accessible to insulin.     

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.     

Immunochemical detection of cell cycle synchronization in a human erythroleukemia cell line, K562

Cells of the human erythroleukemic line K562 can be induced by manipulation of culture conditions to arrest within the G1 phase of the cell cycle, and subsequently to enter S phase synchronously after release from G1. Cell cultures subjected to serum deprivation and hydroxyurea (HU) treatment demonstrated less than 5% of the cells to be in S phase. Four hours after release from HU, 63% of the cells were in S phase, as detected by immunofluorescent staining. This protocol offers a method for synchronization of K562 cells at the G1/S border and a technique for detection of S-phase cells without the use of radioisotopes or flow cytometry instrumentation.     

Increase in histone acetylation and transitions in histone variants during Friend cell differentiation

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.     

Biogenesis of glycophorin A in K562 human erythroleukemia cells

A monoclonal antibody (mAb-233) directed against an epitope in the nonglycosylated carboxyl-terminal region of human erythrocyte glycophorin A (GPA) was used in combination with metabolic labeling, the modification of N- and O-linked oligosaccharide processing by tunicamycin and monensin, and digestions with neuraminidase and O-glycanase to elucidate the pathway of GPA biogenesis in K562 human erythroleukemia cells. Cell-surface GPA is derived from two obligatory precursors in a stepwise manner. The initial GPA precursor has a Mr of 27,000 and appears to contain one N-linked high mannose oligosaccharide chain. In tunicamycin-treated cells, the initial precursor is similar in size (Mr = 24,000) to deglycosylated GPA from human erythrocytes. The 27-kDa initial precursor is rapidly converted to a transient 31-kDa intermediate by the addition of N-acetylgalactosamine residues to serine/threonine hydroxyl groups. Subsequent maturation involves the conversion of the high mannose chain to a complex-type oligosaccharide and the concomitant addition of galactose and sialic acid to internal N-acetylgalactosamine residues to extend the O-linked chains. These results define a single, stepwise processing pathway for the generation of all cell-surface GPA molecules and document for the first time the occurrence of both a unique initial precursor that contains a high mannose N-linked oligosaccharide chain but no O-linked sugars and a transient intermediate that appears to contain the same N-linked group and N-acetylgalactosamine at multiple serine/threonine residues. The properties of the intracellular GPA precursors and the relatively simple nature of the processing pathway reported herein contrast markedly with the characteristics of three intermediates and the complexity of two independent pathways in previously postulated schemes for GPA biogenesis (Gahmberg, C. G., Jokinen, M., Karhi, K. K., Kampe, O., Peterson, P. A., and Andersson, L. C. (1983) Methods Enzymol. 96, 281-298; Jokinen, M., Andersson, L. C., and Gahmberg, C. G. (1985) J. Biol. Chem. 260, 11314-11321).     

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.     

Preapoptotic chromatin changes induced by ultraviolet B irradiation in human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.     

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.     

Cell mechanisms for apoptosis induction in K562 human erythroleukemia cell line treated with quinoline-N-oxide derivatives

The effect of two quinoline-N-oxide derivatives (2-(4′-nitrostyryl)-quinoline-l-oxide (2-NSQO) and 4-(4′-nitrostyryl)-quinoline-1-oxide (4-NSQO)) on modulation of microsomal NADPH oxidoreductase activity, nicotinamide coenzyme concentrations, and induction of apoptosis in K562 human erythroleukemia cells has been. 4-NSQO at the concentration of (10 μM) and 2-NSQO (10 μM) inhibited the activity of microsomal NADPH cytochrome c reductases in tumor cells by 15 and 50% respectively. Treatment of cells with these compounds for two days resulted in the activation of caspase-9 and caspase-3, the increase in ethidium bromide and 4′,6-diamidino-2-phenylindole (DAPI) fluorescence upon DNA binding, and induction of apoptosis. The latter was preceded by the decrease in intracellular nicotinamide coenzyme concentrations. The results obtained allow considering 4-NSQO (and its structural analogs) as perspective compounds for further experimental studies as antitumor agent with low toxic effect on tissues of an organism.     

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.     

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.     

Transferrin binding to K562 cell line : Effect of heme and sodium butyrate induction

Experiments demonstrating the existence of receptors for iron-saturated transferrin on K562 cells are described. Binding of ¹²⁵I-labelled transferrin is rapid, saturable and reversible, and can be specifically inhibited by unlabelled transferrin, but not by other proteins. The number of receptors determined by Scatchard analysis significantly decreased when K562 cells moved from the exponential to the quiescent phase of growth. Induction by hemin or sodium butyrate resulted in a marked reduction of transferrin binding. This phenomenon was due entirely to reduction in the number of receptors and was without effect on the affinity of interaction. The effect of butyrate and hemin on the number of transferrin receptors in other hematopoietic cell lines was investigated. Butyrate on the various cell lines was variable in its effect, whereas hemin constantly elicited a significant reduction in the number of transferrin receptors.     

Butyrate-induced GPR41 activation inhibits histone acetylation and cell growth

Butyrate has been recently identified as a natural ligand of the G-protein-coupled receptor 41(GPR41).In addition,it is an inhibitor of histone deacetylase(HDAC).Butyrate treatment results in the hyperacetylation of histones,with resultant multiple biological effects including inhibition of proliferation,induction of cell cycle arrest,and apoptosis,in a variety of cultured mammalian cells.However,it is not clear whether GPR41 is actively involved in the above-mentioned processes.In this study,we generated a stable cell line expressing the hGPR41 receptor in order to investigate the involvement of GPR41 on butyrate-induced biochemical and physiologic processes.We found that GPR41 activation may be a compensatory mechanism to counter the increase in histone H3 acetylation levels induced by butyrate treatment.Moreover,GPR41 had an inhibitory effect on the anti-proliferative,pro-apoptotic effects of butyrate.GPR41 expression induced cell cycle arrest at the G1-stage,while its activation by butyrate can cause more cells to pass the G1 checkpoint.These results indicated that GPR41 was associated with histone acetylation and might be involved in the acetylation-related regulation of cell processes including proliferation,apoptosis,and the cell cycle.