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。     

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.     

Fluctuation of gene expression for poly(ADP-ribose) synthetase during hemin-induced erythroid differentiation of human leukemia K562 cells and its reversion process

We have studied the regulation of gene expression for poly(ADP-ribose) synthetase during erythroid differentiation and its reversion process. When human leukemia K562 cells were incubated in the presence of 80 microM hemin, benzidine-positive cells appeared at day 2 and 90% of the cells became positive at day 6. However, RNA blot analysis reveals that mRNA for gamma-globin was already abundant in untreated K562 cells and the level of the message was slightly increased by hemin-treatment. Spectroscopic analysis and polyacrylamide gel electrophoresis of the induced cell extracts indicate that hemoglobin molecules were not detected in untreated cells, and increased successively up to day 6. The hemin-induced cells were thoroughly washed, and then recultured in the absence of hemin. The benzidine-positive cells mostly disappeared 3 days after the elimination of the inducer. During the hemin-induced erythroid differentiation, the activity and mRNA for poly(ADP-ribose) synthetase decreased to 50% and 20% of the initial level at day 3 and a low level of the gene expression was maintained afterwards, whereas the activity and mRNA returned to the initial value 1 day after hemin elimination. The results indicate that the hemin-induced erythroid differentiation of K562 cells is a reversible process and depression of the synthetase may be involved in the progress of differentiation.     

Externalization and binding of galectin-1 on cell surface of K562 cells upon erythroid differentiation

Galectin 1 (GAL1) is a β-galactoside-binding lectin involvedin cell cycle progression. GAL1 overexpression is associatedwith neoplastic transformation and loss of differentiation.The gene encoding for human GAL1 resides on chromosome 22(ql2;ql3), and its expression is devel-opmentally regulated. Althoughdevoid of signal peptide GAL1 can be externalized from cellsby a mechanism independent of the normal secretory process.We report here on a study of the effects of erythroid differentiationof the human leukemia cell line K562 on GAL1 protein expression.In undifferentiated K562 cells, GAL1 was expressed into thecytosol. However, the amount of GAL1 was surprisingly weakerin K562 cells than in other leukemia cell lines such as TF-1or KGla. Treatment of K562 cells with erythropoietin (EPO) orwith aphidicolin (APH), an inhibitor for DNA polymerase , inducedan erythroid pheno-type and led to the externalization of cytosolicGAL1 which was then bound to ligands on cell surface in a galactoside-inhibitablefashion. Our results demonstrate that acquisition of an erythroidphenotype is associated with an exter-nalization of GALL Theautocrine binding of GAL1 to cell surface ligands of non adherentcells such as K562 suggest that GAL1 is implicated rather insignal transduction than in cell-cell or cell-matrix interaction.Moreover, the reciprocal translocation involving chromosomes9 and 221(9;22) present in K562 cells might explain the weakexpression of GAL1 in K562 leukemia cells. galectin-l K562 cells differentiation glycoconjugates     

Role of ERK activation in growth and erythroid differentiation of K562 cells

Inhibition of signaling through Ras in BCR-ABL-positive pluripotent K562 cells leads to apoptosis and spontaneous differentiation. However, Ras-induced activation of the mitogen-activated protein kinase ERK has been suggested to play a critical role in either growth or differentiation in different model systems. We studied the role of ERK activation in the growth-promoting and anti-apoptotic effect of Ras and its involvement in hemin-induced nonterminal erythroid differentiation using the BCR-ABL-positive K562 cell line as a model. K562 cells were stably transfected with ERK1 or the dominant inhibitory mutant of ERK1 (ERK1-KR). Overexpression of ERK1-KR inhibited cell growth with an approximately fourfold increase in doubling time and induced apoptosis in K562 cells. Incubation with the MEK1 inhibitor UO126 inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner as well. In the presence of exogenously added hemin, K562 cells differentiate into erythroblasts, as indicated by the production of large amounts of fetal hemoglobin. We examined the activation of MAP kinases during hemin-induced differentiation. The ERK1 and 2 activity increased within 2 h post hemin treatment and remained elevated for 24-48 h. During this time, fetal hemoglobin synthesis also increases from 0.8 to 10 pg/cell. There was no activation of JNK or p38 protein kinases. The hemin-induced accumulation of hemoglobin was inhibited in ERK1-KR overexpressing cells and was enhanced in the wild-type ERK1 transfectants. Our results suggest that ERK activation is involved in both growth and hemin-induced erythroid differentiation in the BCR-ABL-positive K562 cell line.     

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.     

Essential role for ALCAM gene silencing in megakaryocytic differentiation of K562 cells

Background  

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is expressed by hematopoietic stem cells. However, its role in hematopoietic differentiation has not previously been defined.     

Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells

Heterotrimeric G proteins which couple extracellular signals to intracellular effectors play a central role in cell growth and differentiation. The pluripotent erythroleukemic cell line K562 that acquires the capability to synthesize hemoglobin in response to a variety of agents can be used as a model system for erythroid differentiation. Using Western blot analysis and RT-PCR, we studied alterations in G protein expression accompanying hemin-induced differentiation of K562 cells. We demonstrated the presence of G(alpha s), G(alpha i2) and G(alpha q) and the absence of G(alpha i1), G(alpha o) and G(alpha 16) in K562 cells. We observed the short form of G(alpha s) to be expressed predominantly in these cells. Treatment of K562 cells with hemin resulted in an increase in the levels of G(alpha s) and G(alpha q). On the other hand, the level of G(alpha i2) was found to increase on the third day after induction with hemin, followed by a decrease to levels lower of those of uninduced cells. The mitogen-activated protein kinase ERK1/2 pathway is crucial in the control of cell proliferation and differentiation. Both Gi- and Gq-coupled receptors stimulate MAPK activation. We therefore examined the phosphorylation of ERK1/2 during hemin-induced differentiation of K562 cells. Using anti-ERK1/2 antibodies, we observed that ERK2 was primarily phosphorylated in K562 cells. ERK2 phosphorylation increased gradually until 48 h and returned to basal values by 96 h following hemin treatment. Our results suggest that changes in G protein expression and ERK2 activity are involved in hemin-induced differentiation of K562 cells.     

ERK1/2 inactivation and p38 MAPK-dependent caspase activation during guanosine 5'-triphosphate-mediated terminal erythroid differentiation of K562 cells

Since differentiation therapy is one of the promising strategies for treatment of leukemia, universal efforts have been focused on finding new differentiating agents. In that respect, it was recently shown that guanosine 5'-triphosphate (GTP) induced the differentiation of K562 cells, suggesting its possible efficiency in treatment of chronic myelogenous leukemia (CML). However, further investigations are required to verify this possibility. Here, the effects of GTP on activation of mitogen-activated protein kinases (MAPKs) and caspases in K562 cells were examined. Exposure of K562 cells to 100muM GTP markedly inhibited growth (4-70%) and increased percent glycophorin A positive cells after 1-6 days. GTP-induced terminal erythroid differentiation of K562 cells was accompanied with activation of three key caspases, i.e., caspase-3, -6 and -9. More detailed studies revealed that mitochondrial pathway is activated along with down-regulation of Bcl-xL and releasing of cytochrome c into cytosol. Among MAPKs, ERK1/2and p38 were modulated after GTP treatment. Western blot analyses showed that sustained phosphorylation of p38 MAPK was accompanied by a decrease in ERK1/2 activation. These modulatory effects of GTP were observed at early exposure times before the onset of differentiation (3h), and followed for 24-96h. Interestingly, inhibition of p38 MAPK pathway by SB202190 impeded GTP-mediated caspases activation and differentiation in K562 cells, suggesting that p38 MAPK may act upstream of caspases in our system. These results point to a pivotal role for p38 MAPK pathway during GTP-mediated erythroid differentiation of K562 cells and will hopefully have important impact on pharmaceutical evaluation of GTP for CML treatment in differentiation therapy approaches.     

Emodin can induce K562 cells to erythroid differentiation and improve the expression of globin genes

In China, the traditional Chinese medicine “YiSui ShenXu Granule” has been used for treating β-thalassemia over 20 years and known to be effective in clinic. Several purified components from “YiSui ShenXu Granule” are tested in K562 cells to reveal its effect on globin expression and erythroid differentiation, and one of the purified components, emodin, was demonstrated to increase the expression of α-, ε-, γ-globin, CD235a, and CD71 in K562 cells. Moreover, the increase of their expression is emodin concentration-dependent. The mRNA and microRNA (miRNA) expression profiles are further analyzed and 417 mRNAs and 35 miRNAs with differential expression between untreated and emodin-treated K562 cells were identified. Among them, two mRNAs that encode known positive regulators of erythropoiesis, ALAS2, and c-KIT respectively, increased during emodin-induced K562 erythroid differentiation, meanwhile, two negative regulators, miR-221 and miR-222, decreased during this process. These results indicate that emodin can improve the expression of globin genes in K562 cells and also induce K562 cells to erythroid differentiation possibly through up-regulating ALAS2 and c-KIT and down-regulating miR-221 and miR-222.     

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.     

K-Cl cotransporter gene expression during human and murine erythroid differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.     

Carbon monoxide induced erythroid differentiation of K562 cells mimics the central macrophage milieu in erythroblastic islands

Growing evidence supports the role of erythroblastic islands (EI) as microenvironmental niches within bone marrow (BM), where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb) is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule - CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage.Based on the above, the study hypothesized CO to have a role in erythroid maturation. Thus, the effect of CO gas as a potential erythroid differentiation inducer on the common model for erythroid progenitors, K562 cells, was explored. Cells were kept under oxygen lacking environment to mimic BM conditions. Nitrogen anaerobic atmosphere (N₂A) served as control for CO atmosphere (COA). Under both atmospheres cells proliferation ceased: in N₂A due to cell death, while in COA as a result of erythroid differentiation. Maturation was evaluated by increased glycophorin A expression and Hb concentration. Addition of 1%CO only to N₂A, was adequate for maintaining cell viability. Yet, the average Hb concentration was low as compared to COA. This was validated to be the outcome of diversified maturation stages of the progenitor''s population.In fact, the above scenario mimics the in vivo EI conditions, where at any given moment only a minute portion of the progenitors proceeds into terminal differentiation. Hence, this model might provide a basis for further molecular investigations of the EI structure/function relationship.     

Comparison of different methods for erythroid differentiation in the K562 cell line

Objective

To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis.

Results

Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001).

Conclusions

Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

     

K562 leukemia cells transfected with the human c-fes gene acquire the ability to undergo myeloid differentiation

Expression of the proto-oncogene p93c-fes and its associated tyrosine kinase activity is marked in mature granulocytes, monocytes, differentiated HL-60 leukemia cells, and leukemia cell lines KG-1, THP-1, HEL, and U-937, which can be induced to differentiate along the granulocyte/monocyte pathway. Conversely, p93-c-fes expression is absent in the K562 cell line, which is resistant to myeloid differentiation. Upon transfection and clonal selection of K562 cells using a mammalian expression vector containing the 13-kilobase pair c-fes gene, c-fes mRNA was transcribed and p93-c-fes tyrosine activity kinase was expressed. Clones expressing c-fes underwent myeloid differentiation as assessed by the appearance of phagocytic activity, Fc receptors, nitro blue tetrazolium reduction, Mac-1 immunofluorescence, and lysozyme production. These results indicate that the expression of the c-fes protooncogene and its associated tyrosine kinase activity plays a major role in the initiation of myeloid differentiation.