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.     

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.     

Nitric oxide levels during erythroid differentiation in K562 cell line

Nitric oxide (NO) is endogenous mediator of numerous physiological processes that range from regulation cardiovascular function and neurotransmission to antipathogenic and tumoricidal responses. This study was designed to investigate the possible role of NO during erythroid differentiation in K562 erythroleukemia cells. The chronic myelogenous leukemia (K562) cell line can be triggered in culture to differentiate along the erythrocytic pathway, in response to a variety of stimulatory agents. In this study, K562 cells were induced to synthesize hemoglobin by hemin. We investigated NOx (nitrate+nitrite) levels in uninduced (control) and hemin-induced K562 cell lysates during erythroid differentiation. Our results showed that NO levels decreased significantly on fourth and sixth day both in hemin-induced and control cells; the decrease was, however, more in hemin-induced group than in control group.     

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.     

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.     

cDNA cloning and function analysis of two novel erythroid differentiation related genes

Our previous studies showed that some nuclear proteins that wereexpressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen l-gtll human cDNA expression library of fetal liver in order to obtain the rele-vant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' func-tions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leucine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigat-ed the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid develop-ment, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.     

cDNA cloning and function analysis of two novel erythroid differentiation related genes

Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen λ-gtll human cDNA expression library of fetal liver in order to obtain the relevant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' functions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a prote     

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.     

Activation of the MKK4-JNK pathway during erythroid differentiation of K562 cells is inhibited by the heat shock factor 2-beta isoform

In this study we report the activation of c-Jun N-terminal kinase (JNK) in human K562 erythroleukemia cells undergoing hemin-mediated erythroid differentiation, which occurs concomitantly with activation of heat shock factor 2 (HSF2) and leads to a simultaneous in vivo phosphorylation of c-Jun. The activation of JNK occurs through activation of mitogen-activated protein kinase kinase (MKK) 4 and not by activation of MKK7 or inhibition of JNK-directed phosphatases. We have previously shown that overexpression of the HSF2-beta isoform inhibits the activation of HSF2 upon hemin-induced erythroid differentiation. Here we demonstrate that HSF2-beta overexpression blocks the hemin-induced activation of the MKK4-JNK pathway, suggesting an erythroid lineage-specific JNK activation likely to be regulated by HSF2.     

Control of hemoglobin synthesis in erythroid differentiating K562 cells: II. Studies of iron mobilization in erythroid cells by high-performance liquid chromatography–electrochemical detection

We have demonstrated that iron controls hemoglobin (Hb) synthesis in erythroid differentiating K562 cells by enhancing the activity of a key enzyme of the Hb synthesis, δ-aminolevulinate synthase (ALAS). In the present study, we studied iron mobilization and the role of iron in erythroid differentiating cells by measuring the level of iron by means of high-performance liquid chromatography using electrochemical detection (HPLC–ED). After treatment of K562 cells with sodium butyrate, the expression of transferrin receptor (TfR) increased initially, followed by an increase in the levels of both total iron and Hb as well as the ALAS activity. However, no increase could be found in the levels of non-heme iron, low-molecular-mass iron (LMMFe) and ferritin. Addition of diferric transferrin (FeTf) enhanced both δ-aminolevulinic acid (ALA) and Hb synthesis. In contrast, addition of hemin elevated the levels of all iron species as well as the Hb synthesis but reduced the TfR expression and ALA contents in both butyrate treated and untreated cells. These results suggest that Hb synthesis is controlled by TfR expression, and that the ALA synthesis is suppressed by iron released from heme and/or Hb due to lowered expression of TfR.     

利用K562红细胞分化模型和cDNA芯片技术筛选参与红细胞分化的新基因(英文)

以氯高铁血红素 (hemin)诱导K5 6 2分化作为体外红细胞分化模型 ,结合cDNA大规模测序、生物信息学分析、基因芯片杂交和NorthernBlot分析等技术 ,筛选红细胞分化相关的新基因 .首先利用大规模测序技术从人胚肾cDNA文库中随机挑选克隆测得 192个EST(expressedsequencetags)片段 ,经在线生物信息学分析 ,得到 79个代表新基因的未知EST片段 ,并在NCBI(NationalCenterofBiotechnologyInformation)dbEST库中登录 .利用 79个ESTcDNA片段制备了基因芯片 .提取分化前后的K5 6 2细胞的mRNA作为荧光标记反转录的模板 ,反转录后的探针用于DNA芯片杂交 .分析杂交后的结果 ,得到了 2个差异表达较明显的基因 ,GenBank登录号分别为AF147772 (187bp)和AF4 776 2(6 30bp) ,并分别命名为EDRG1和EDRG2 (erythroiddifferentiationrelatedgene 1and 2 ) ,相似性检索表明它们属全新基因 ,基因组草图测序数据库检索表明了两个基因的染色体定位 .随后的Northern印迹用于验证了在分化前后的K5 6 2细胞中差异表达 .提示这两个基因参与了红细胞分化过程 .RT PCR检测了EDRG1和EDRG2在人胚胎多组织中的表达 .结果提示 ,EDRG1可能与多种胚组织的正常发育相关 ,尤其在胚脑中高丰度表达 ,而EDRG2则可能参与了胚心和胚肾的组织生成 .生物     

Overexpression of transglutaminase 2 accelerates the erythroid differentiation of human chronic myelogenous leukemia K562 cell line through PI3K/Akt signaling pathway

Transglutaminase 2 (TG2) is a GTP-binding protein with transglutaminase activity. Despite advances in the characterization of TG2 functions and their impact on cellular processes, the role of TG2 in Human chronic myelogenous leukemia K562 cell line is still poorly understood. To understand the biological significance of TG2 during the differentiation of K562 cells, we established and characterized K562 cells that specifically express TG2. Non-transfected K562 cells showed the increase of membrane-bound-TG2 level after 3 days in the response to Hemin and all trans-retinoic acid (tRA), indicating that membrane recruitment of TG2 is occurred during the erythroid differentiation. However, membrane recruitment of TG2 in TG2-transfected cells revealed within earlier time period, compared with that in vector-transfected cells. The ability of membrane-bound-TG2 to be photoaffinity-labeled with [alpha-32P]GTP was also increased in TG2-transfected cells. TG2-transfected cells activated Akt phosphorylation and inactivated ERK1/2 phosphorylation, compared with vector-transfected cells. Furthermore, phosphorylation of CREB, one of the Akt substrates, was increased in TG2-transfected cells and this phenomenon was confirmed by RT-PCR analysis of several marker genes related with erythroid lineage in the absence of PI3K specific inhibitor, Wortmannin, indicating that PI3K/Akt signaling pathway also involved in the differentiation of the cell. Finally, as results of benzidine positive staining as well as hemoglobinization analysis, overexpression of TG2 revealed acceleration of the erythroid differentiation of K562 cells. Taken together, there was no increased TG2 expression level in the response of Hemin/tRA and delayed differentiation in vector transfected cells than in TG2-transfected cells, suggesting that suppression of TG2 expression may retard the erythroid differentiation of K562 cells. Therefore, our study may give a new insight for another aspect of the development of this disease.     

Systematic RNAi studies on the role of Sp/KLF factors in globin gene expression and erythroid differentiation

Sp/KLF family of factors regulates gene expression by binding to the CACCC/GC/GT boxes in the DNA through their highly conserved three zinc finger domains. To investigate the role of this family of factors in erythroid differentiation and globin gene expression, we first measured the expression levels of selected Sp/KLF factors in primary cells of fetal and adult stages of erythroid development. This quantitative analysis revealed that their expression levels vary significantly in cells of either stages of the erythroid development. Significant difference in their expression levels was observed between fetal and adult erythroid cells for some Sp/KLF factors. Functional studies using RNA interference revealed that the silencing of Sp1 and KLF8 resulted in elevated level of gamma globin expression in K562 cells. In addition, K562 cells become visibly red after Sp1 knockdown. Benzidine staining revealed significant hemoglobinization of these cells, indicating erythroid differentiation. Moreover, the expression of PU.1, ETS1 and Notch1 is significantly down-regulated in the cells that underwent erythroid differentiation following Sp1 knockdown. Overexpression of PU.1 or ETS1 efficiently blocked the erythroid differentiation caused by Sp1 knockdown in K562 cells. The expression of c-Kit, however, was significantly up-regulated. These data indicate that Sp1 may play an important role in erythroid differentiation.     

Embryonic and fetal hemoglobin synthesis in K562 cell line

K562 cell line was grown in liquid suspension and in plasma clot cultures. Morphological studies revealed the presence of a minority of cells, which were identified as erythroblasts. However, the majority of the cells remained unidentified. Biochemical studies confirmed the synthesis of hemoglobin by K562 cells. The pattern of hemoglobin (Hb) production was of the embryonic type, with the presence of small amount of fetal Hb. The addition of several inducers, like Epo and butyrate, was unable to modify the pattern of Hb production of K562. In contrast, the addition of hemin increased the synthesis of Hb and stimulated the synthesis of fetal Hb and probably adult Hb.     

Annexin1 regulates the erythroid differentiation through ERK signaling pathway

K562 cells can be used as a model of erythroid differentiation on being induced by hemin. We found that the level of annexin1 gene expression was notably increased during this indicated process. To test the hypothesis that annexin1 can regulate erythropoiesis, K562 cell clones in which annexin1 was stably increased and was knocked down by RNAi were established, respectively. With analysis by hemoglobin quantification, benzidine staining, and marker gene expression profile determination, we confirmed that hemin-induced erythroid differentiation of K562 cells was modestly stimulated by overexpression of annexin1 while it was significantly blocked by knock down of annexin1. Further studies revealed that the mechanisms of annexin1 regulation of the erythroid differentiation was partially related to the increased ERK phosphorylation and expression of p21(cip/waf), since specific inhibitor of MEK blocked the function of annexin1 in erythroid differentiation. We concluded that annexin1 exerted its erythropoiesis regulating effect by ERK pathway.     

Rescue of K562 cells from MDM2-modulated p53-dependent apoptosis by growth factor-induced differentiation

The wild-type human MDM2 protooncogene was tested for its ability to modulate apoptotic activity of the de novo expressed p53 tumor suppressor gene in K562 cells. We also studied the role of some cytokines in this phenomenon. K562, a human myeloid leukemia cell line, does not express p53 at the mRNA or protein level. In this study, we stably transfected K562 with eukaryotic vectors containing either normal p53 cDNA (pC53-SN3) or mutated p53 (143Val-->Ala) cDNA (pC53-SCX3). Transfectants expressing WT p53 or those expressing mutant p53 are called K562 SN and K562 SM respectively. Many leukemic cell lines undergo apoptosis when de novo WT p53 is expressed alone. In contrast, while the resulting clones (K562 SN and K562 SM) expressed p53, they did not undergo apoptosis. However, when treated with MDM2 mRNA antisense (MDM2 AS) oligodeoxynucleotides (ODNs), K562 SN demonstrated apoptotic features at both molecular and morphological levels. No change was observed when the other clones (K562 and K562 SM) were treated with MDM2 AS. Apoptosis induced in this manner was associated with a relatively small increase in intracellular calcium [Ca2+]i. Cells cultured in medium previously supplemented with recombinant human (rh) interleukin (IL)-3 and rh-erythropoietin (Epo) did not undergo apoptosis. Moreover, K562 SN cells were induced to differentiate. This differentiation was evaluated by measuring hemoglobin (Hb) level in cellular extracted proteins and by analyzing erythroid colony number and morphology. High Hb synthesis was obtained when K562 SN cells were cultured with cytokines (IL-3 + Epo) combined with MDM2 AS. Our results are consistent with the hypothesis that the function of the proto-oncogene MDM2 is to provide a 'feedback' mechanism for the p53-dependent pathway of apoptosis that could be shunted toward differentiation.     

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.