Erythroid properties of K562 cells : Effect of hemin, butyrate and TPA induction

Two sublines of the human leukemia cell line K562 including the original cell line and three clones have been investigated for their erythroid features. All of them produce embryonic and fetal hemoglobins, glycophorin A, spectrin and true acetylcholinesterase, but to a varying extent among the cell lines. The Hb and glycophorin contents were correlated in the different K562 cell lines, whereas acetylcholinesterase was independently expressed from these two other erythroid markers. Hb accumulation is enhanced by exposure of the cells to 100 microM hemin without a significant modification of the expression of the other erythroid markers. Butyrate greatly increased the activity of acetylcholinesterase, slightly enhanced the production of hemoglobin, but did not modify the expression of glycophorin and spectrin. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced an almost complete disappearance of glycophorin, reduced the synthesis of Hb by K562 cells and also abolished the action of hemin on Hb accumulation. Therefore, all the different K562 cell lines exhibit clear erythroid features including acetylcholinesterase. Butyrate or hemin did not induce terminal differentiation of K562 cells, whereas TPA significantly diminished the erythroid phenotype.     

Induction of erythroid differentiation of human K562 cells by 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives

In this paper we report the synthesis of twelve 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives and the results obtained on their effects in inducing erythroid differentiation of human leukemic K562 cells. The data obtained demonstrate that two of the newly synthetized compounds are able to induce erythroid differentiation of K562 cells. In addition, these same compounds potentiate K562 erythroid differentiation induced by cytosine arabinoside, retinoic acid and mithramycin. Inducers of erythroid differentiation stimulating fetal gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.     

Doppel-induced Purkinje cell death is stoichiometrically abrogated by prion protein

Activin A can induce erythroid differentiation, whereas basic fibroblast growth factor (bFGF) can maintain the undifferentiated status of erythroid progenitors. How these two factors together can affect the regulation of erythroid differentiation in hematopoietic cells has not been elucidated. This study demonstrates that bFGF antagonizes activin A-mediated growth inhibition and hemoglobin (Hb) synthesis in K562 cells. Analyses of mitogen-activated protein kinases revealed that activin A-induced p38 phosphorylation and inhibited ERK1/2 phosphorylation. In contrast, bFGF worked antagonistically to induce ERK1/2 phosphorylation and inhibited p38 phosphorylation in K562 cells. Furthermore, co-treatment of cells with activin A and bFGF decreased p38 phosphorylation and increased ERK1/2 phosphorylation. SB203580 inhibition of p38 activity eliminated activin A-mediated growth inhibition and Hb synthesis, whereas U0126 inhibition of ERK1/2 activity augmented the effects of activin A on K562 cells. These results suggest that bFGF can negatively modulate p38 and positively modulate ERK1/2 to antagonize activin A-mediated growth inhibition and Hb synthesis in K562 cells.     

Disialoganglioside GD3 synthase expression recruits membrane transglutaminase 2 during erythroid differentiation of the human chronic myelogenous leukemia K562 cells

By employing proteomics analysis tool, we examined the effects of GD3 synthase expression on the differentiation properties of chronic myelogenous leukemia (CML)-derived leukemia cells K562. Forced expression of GD3 synthase induced erythroid differentiation as determined by an increase in glycophorin A expression and synthesis of hemoglobins. The proteomic analysis revealed that 15 proteins were increased by GD3 synthase. In contrast, we observed three protein gel spots decreased in contents in the cell membranes of GD3 synthase-transfected K562 cells. Among the increased proteins, membrane transglutaminase 2 (TG2) was specifically increased in the cell membrane of GD3 synthase-transfected K562 cells. Then, we generated the GD3 synthase-transfected cells in the K562 cells. Interestingly, the TG2 level was increased in GD3 synthase-transfected cells compared with vector- and plasma membrane-associated ganglioside sialidase (Neu3)-transfected cells. In addition, its ability to be photoaffinity-labeled with [alpha-(32)P]GTP was also increased in the GD3 synthase- and TG2-transfected cells. Moreover, small interfering RNA (siRNA) analysis for the GD3 synthase showed the decrease or abolishment of the membrane TG2. Finally, GD3 synthase-transfected cells accelerated the erythroid differentiation. Therefore, we propose that the recruitment of TG2 into membranes by GD3 might play an important role in the erythroid differentiation in K562 cells.     

Menin interacts directly with the homeobox-containing protein Pem

Scalarane-type sesterterpenes, PHC-1-PHC-7, which have been isolated from a marine sponge, increased hemoglobin production in human chronic myelogenous leukemia cell line K562 at the concentration of 0.1-5 microg/ml. PHC-1, the major constituent, induced the expression of glycophorin A and the enucleation for K562 cells. These sesterterpenes were found to induce erythroid differentiation in K562 cells. In addition, PHC-1 induced G1 arrest of the cell cycle of K562 cells.     

Activin A induces erythroid gene expressions and inhibits mitogenic cytokine-mediated K562 colony formation by activating p38 MAPK

Activin A, a member of the transforming growth factor (TGF)-beta superfamily, is involved in the regulation of erythroid differentiation. Previous studies have shown that activin A inhibited the colony-forming activity of mouse Friend erythroleukemia cells, however, the mechanism remains unknown. First, we show herein that activin A induced the expression and activated the promoters of alpha-globin and zeta-globin in K562 cells, confirming that activin A induces erythroid differentiation in K562 cells. The p38 mitogen activated protein kinase (MAPK) inhibitor, SB203580, inhibited and the extracellular signal regulated kinase (ERK) inhibitor, PD98059, enhanced the expression and promoter activities of alpha-globin and zeta-globin by activin A, indicating that p38 MAPK and ERK are crucial for activin A-induced erythroid genes expression. Second, SB203580 inhibited the inhibitory effect of activin A on the colony-forming activity of K562 cells using the methylcellulose colony assay, indicating that activin A inhibits K562 colony formation by activating p38 MAPK. In addition, mitogenic cytokines SCF, IL-3, and GM-CSF induced colony formation of K562 cells that could be inhibited by PD98059 or enhanced by SB203580, respectively, indicating that these mitogenic cytokines induce K562 colony formation by activating ERK and inactivating p38 MAPK. Furthermore, activin A reduced the induction effect of these mitogenic cytokines on K562 colony formation in a dose-dependent manner. The inhibition of p38 MAPK reverted the inhibitory effect of activin A on mitogenic cytokine-mediated K562 colony formation. We conclude that activin A can regulate the same pathway via p38 MAPK to coordinate cell proliferation and differentiation of K562 cells.     

Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion

The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increasedCD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.     

Stem cell factor and interleukin-3 induce stepwise generation of erythroid precursor cells from a basic fibroblast growth factor-dependent hematopoietic stem cell line, A-6

A multipotent immature myeloid cell population was produced from a basic fibroblast growth factor (bFGF)-dependent hematopoietic stem cell line, A-6, when cultured with stem cell factor (SCF) replacing bFGF. Those cells were positive for stem cell markers, c-kit and CD34, and a myeloid cell marker, F4/80. Some cell fractions were also positive for Mac-1, a macrophage marker or Gr-1, a granulocytic maker, but negative for an erythroid marker TER119. They also showed the expression of mRNA for the myeloid-specific PU.1 but did not that for the erythroid-specific GATA-1. Among various cytokines, interleukin-3 (IL-3) induced erythroid precursor cells that expressed the erythroid-specific GATA-1 and beta-major globin. The quantitative analysis showed that erythroid precursor cells were newly produced from the immature myeloid cells by cultivation with IL-3. SCF and IL-3 induced stepwise generation of erythroid precursor cells from an A-6 hematopoietic stem cell line.     

hERG Potassium Channel Blockage by Scorpion Toxin BmKKx2 Enhances Erythroid Differentiation of Human Leukemia Cells K562

Background

The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.

Principal Findings

The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC₅₀ of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca²⁺ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process.

Conclusions/Significance

Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs.     

Induction to erythroid differentiation of K562 cells by 1-beta-D-arabinofuranosylcytosine is inhibited by iron chelators: reversion by treatment with hemin

Erythroid differentiation of human leukemic K 562 cells is inhibited by the iron chelator desferrioxamine (DF). In addition, desferrioxamine induces an increase of uptake of hemin. When hemin is added to the culture medium, the DF-mediated inhibitory effects on erythroid induction are reversed. Briefly, hemin allows hemoglobin synthesis by K 562 cells induced to erythroid differentiation by 1-beta-D-arabinofuranosylcytosine (ara-C) and treated with 12.5 micrograms/ml DF. In addition, it was found that hemin treatment leads to a reversion of inhibition of K 562 cell proliferation mediated by 50-75 micrograms/ml DF. This effect of hemin was also detected in other cultured human tumor cell lines (B-lymphoid, erythroleukemic and from breast carcinomas, melanomas and kidney carcinomas).     

Study of the PTEN gene expression and FAK phosphorylation in human hepatocarcinoma tissues and cell lines

K562 cells contain a Bcr-Abl chimeric gene and differentiate into various lineages in response to different inducers. We studied the role of the mitogen-activated protein kinase (MAPK) kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK) pathway during the erythroid differentiation of K562 cells induced by tyrosine kinase inhibitors (herbimycin A or STI571), using genetically modified cells (constitutively MEK1-activated K562: K562/MEK1, and inducible ERK-inactivated K562: K562/CL100). Basal expression of glycophorin A was markedly reduced in K562/MEK1 cells compared with that in parental cells, while it was augmented in K562/CL100 cells. Herbimycin A and STI571 differentiated K562 cells accompanying with the transient down-regulated ERK. Moreover, the erythroid differentiation was markedly suppressed in K562/MEK1 cells, and early down-regulation of ERK activity was not observed in these cells. In contrast, the induction of ERK-specific phosphatase in K562/CL100 cells potentiated erythroid differentiation. Once the phosphatase was induced, the initial ERK activity became repressed and its early down-regulation by the inhibition of Bcr-Abl was marked and prolonged. These results demonstrate that the erythroid differentiation of K562 cells induced by herbimycin A or STI571 requires the down-regulation of MEK1/ ERK pathway.     

Human leukemic K562 cells: suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor

The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression.     

KLF1和KLF9对K562细胞红系分化的协同调控作用

Krüppel样因子(Krüppel-like factors,KLFs)是锌指蛋白超家族的一个亚家族,参与细胞内的多种生理、病理过程,该家族成员在红细胞分化发育过程中发挥非常重要的作用,但是家族成员间对红系分化的协同调控作用还鲜有报道。本课题组前期研究发现,KIF家族成员KLF1和KLF9在已分化的红系细胞中的表达水平显著高于造血干细胞。为进一步探讨二者在红系分化中是否存在协同作用,本研究在K562细胞中分别过表达/敲低表达KLF1和KLF9,检测二者表达的相关性,发现KLF1和KLF9的基因表达呈现正相关,且二者共表达可以显著促进K562细胞红系分化,特异地增强β-珠蛋白的表达。通过对KLF1、KLF9单独和共同过表达、敲低表达的K562细胞转录组数据的分析发现二者可能通过PI3K-Akt和FoxO通路协同调控红系分化,FOS、TF、IL8是协同调控的候选靶基因。本研究结果为后续深入研究KLF1和KLF9协同调控红系分化的分子机制奠定了基础。