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.     

ERK signaling pathway is differentially involved in erythroid differentiation of K562 cells depending on time and the inducing agent

K562 cells can be induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, butyrate, cisplatin and ara-C. Differential signaling through MAP kinases has been suggested to be involved in this differentiation process. We have investigated the involvement of ERK activation/inhibition in hemin-, butyrate-, cisplatin- and ara-C-induced erythroid differentiation using the K562 cell line. ERK activity decreased for 2-4h after administration of either inducing agent. ERK was then activated by hemin and cisplatin, while ERK phosphorylation remained decreased during incubation with butyrate and ara-C. There was no activation of JNK or p38. The MEK-1 inhibitors UO126 or PD98059 induced erythroid differentiation in K562 cells and acted additively with butyrate. Inhibition of MEK-1 reduced the hemoglobin accumulation by hemin and cisplatin; erythroid differentiation by ara-C was unchanged. The results suggest that inhibition of signaling through ERK in K562 cells may be needed to enter the erythroid differentiation process, while after initiation both activation and inhibition of signaling through ERK enhance erythroid differentiation, which, however, is dependent on the inducing compound.     

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.     

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.     

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.     

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).     

Characterization of "fetal-type" acetylcholinesterase in hemin-treated K562 cell culture

The alteration of acetylcholinesterase (ACHE) activity, a marker enzyme of erythroid differentiation, was studied during the hemin-induced erythroid differentiation of K562 human leukemia cells in suspension culture. The kinetics of postinduction differentiation was followed by determining the hemoglobin (Hb) content and the ACHE activity of cells. Embryonic hemoglobins as well as small quantities of fetal Hb (HbF) were synthetized by stimulated cells. The peaks of ACHE activity preceded the highest level of Hb content and, following induction, reached their pinnacles at 72 and 120 hours, respectively. These data indicate that ACHE activity is an earlier and more sensitive marker for hemin-induced erythroid differentiation of K562 cells than is elevated Hb content. Electrophoretic mobility of ACHE from hemin-treated cells proved to be the fetal type, but after incubation with neuraminidase, the rate of migration decreased to the level of the adult type enzyme.     

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.     

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     

Induction of calcium-activated potassium channel activity by hemin in human erythroleukemia cells

The agent hemin has been demonstrated to be able to initiate a coordinated differentiation program in several cell types. In the present study, we examined the ability of hemin on inducing cell differentiation and Ca(2+)-activated K(+) channel activity in erythroleukemic K562 cells. Treating undifferentiated K562 cells with hemin (0.1 mM) for five days caused these cells to display differentiation-like characteristics including chromatin aggregation, nuclear degradation, pseudopod extension of the membrane and increased hemoglobin production. However, overall cell viability was not significantly changed by the presence of hemin. After hemin treatment for different periods, the Ca(2+)-activated K(+) channel was activated by the addition of ionomycin (1 microM), and was inhibited by either clotrimazole, charybdotoxin, or EGTA. Before hemin treatment there was no significant Ca(2+)-activated K(+) channel activity present in undifferentiated K562 cells. After hemin treatment for 5 days, a significant Ca(2+)-activated K(+) channel activity was detected. This increasing Ca(2+)-activated K(+) channel activity may be contributed from a subtype of Ca(2+)-activated K(+) channel, KCNN4. These results suggest that the ability of hemin to induce increasing Ca(2+)-activated K(+) channel activity may contribute to the mechanism of hemin-induced K562 cell differentiation.     

Involvement of p38 kinase in hydroxyurea-induced differentiation of K562 cells.

Hydroxyurea is a differentiation-inducing agent of human erythroleukemia K562 cells. However, the cellular mechanisms by which hydroxyurea exerts its effects on tumor cells, leading to the inhibition of cell growth and the induction of differentiation markers, are largely unknown. This study examined the role of different mitogen-activated protein kinase signal transduction pathways in hydroxyurea-induced erythroid differentiation of K562 cells. Using a panel of anti-extracellular signal-related kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 phosphospecific antibodies, we demonstrated that phosphorylation of ERK and JNK is decreased after the treatment of cells with hydroxyurea, whereas phosphorylation of p38 is increased. Moreover, inhibition of ERK activity by PD98059 induced erythroid differentiation, and it acted synergistically with hydroxyurea on hemoglobin synthesis, whereas inhibition of p38 activity by SB203580 inhibited induction of hemoglobin production by hydroxyurea. These findings suggest that the activation of p38 kinase may play important roles in the signal transduction mechanisms of hydroxyurea leading to erythroid differentiation.     

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.     

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.     

Humanin delays apoptosis in K562 cells by downregulation of P38 MAP kinase

Humanin (HN) is a newly identified neuroprotective peptide. In this study, we investigated its antiapoptotic effect and the potential mechanisms in K562 cells. Upon serum deprivation, expression of HN in K562 cells decreased and its intracellular distribution changed from cytoplasm to cell membrane. In HN stably transfected K562 cells, apoptosis was delayed compared with control vector transfected cells as measured by flow cytometry. Furthermore, analysis of different mitogen-activated protein (MAP) kinases activity revealed that extracellular signal-regulated kinase (ERK) pathway was inhibited while p38 signaling was activated following serum deprivation in K562 cells. And in HN transfected K562 cells, ERK downregulation was not affected, but p38 activation was suppressed, which may responsible for the delayed apoptosis in these cells. Activation of the ERK signaling pathway by phorbol myristate 13-acetate (PMA) and sorbitol protected K562 cells from serum deprivation induced apoptosis. Additionally, overexpression of HN reduced megakaryocytic differentiation of K562 cells. The present data outline the role of ERK and p38 MAP kinases in serum deprivation induced apoptosis in K562 cells and figure out p38 signaling pathway as molecular target for HN delaying apoptosis in K562 cells.     

An essential role for mitogen-activated protein kinases, ERKs, in preventing heat-induced cell death.

Stimulation of mitogen-activated protein kinases (MAPKs) or extracellular signal regulated protein kinases (ERKs) after exposure of mammalian cells to ultraviolet (UV) and X-irradiation occurs through activation of receptor tyrosine kinases via Ras/Raf/Mek/ERKs cascade. This activation of MAPKs is proposed to play a role in the replacement of damaged proteins during these stresses. Heat shock also activates MAPKs; however, the signaling cascade and the biochemical and physiological links between activation by heat and downstream effects are unknown. In this report we demonstrate that, unlike irradiation, heat induces MAPKs through ceramide metabolism to sphingosine with stimulation of Raf-1 protein kinase. The activation of MAPKs by heat does not occur in all cell types, because the step(s) downstream of ceramide to activation of Raf-1 protein kinase is missing in myeloid leukemic cells such as HL-60, U937, and K562, while it is present in NIH3T3 fibroblasts. Heat-induced MAPK activation may enhance the ability of cells to survive a severe heat shock. Blocking 60-70% of the activity of MAPK (ERK1) by stable overexpression of the dominant negative allele ERK1-KR renders NIH3T3 and K562 cells up to 100-fold more sensitive to cytotoxic effects of heat. Conversely, NIH3T3 and K562 cells stably overexpressing the wild-type ERK1 develop resistance to killing by heat. These results suggest that increased thermal sensitivity of leukemic cells to thermal stress or other cancer therapy regimens could be attributable to lack of pertinent activation of the MAPK pathway by such stresses.     

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     

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.