scholarly journals Expression and modulation of specific, high affinity binding sites for erythropoietin on the human erythroleukemic cell line K562

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 104-109 ◽  
Author(s):  
JK Fraser ◽  
FK Lin ◽  
MV Berridge
Keyword(s):  
Cell Line ◽  
K562 Cell ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Receptor Expression ◽  
Target Cells ◽  
Epo Receptor ◽  
High Affinity ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

Abstract Erythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 125I-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor- potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.

scholarly journals Expression and modulation of specific, high affinity binding sites for erythropoietin on the human erythroleukemic cell line K562

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 104-109 ◽  
Author(s):  
JK Fraser ◽  
FK Lin ◽  
MV Berridge
Keyword(s):  
Cell Line ◽  
K562 Cell ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Receptor Expression ◽  
Target Cells ◽  
Epo Receptor ◽  
High Affinity ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

Erythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 125I-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor- potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.

scholarly journals Effects of butyrate on the erythropoietin receptor of cell line IW201

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 811-814 ◽  
Author(s):  
O Hermine ◽  
YJ Dong ◽  
E Goldwasser
Keyword(s):  
Cell Line ◽  
Sodium Butyrate ◽  
Actinomycin D ◽  
Erythropoietin Receptor ◽  
Affinity Binding ◽  
High Affinity ◽  
Receptor Mrna ◽  
Receptor Number ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

Abstract The murine erythroleukemic cell line, IW201, normally expresses only low-affinity erythropoietin receptors. Exposure of these cells for 48 hours to sodium butyrate results in a change in receptor kd from about 600 pmol/L to 100 to 200 pmol/L. This change in affinity is accompanied by downregulation of both receptor number and receptor mRNA. Cells exposed to sodium butyrate for 2 hours show a similar change in kd but no change in receptor number. The butyrate effect on kd at 2 hours is abrogated by either cycloheximide or actinomycin D. These data indicate that an accessory protein induced by sodium butyrate is responsible for high-affinity binding of erythropoietin.

The Transcription Factor SCL/TAL-1 Plays a Positive Role in the Erythropoietic Differentiation Via MEK/ERK Pathway in EPO-Induced K562 Cell Line

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4799-4799
Author(s):  
Yuping Gong ◽  
Ruiqing Zhou ◽  
Ting Niu
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
K562 Cell ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Erk Pathway ◽  
Rt Pcr ◽  
K562 Cell Line ◽  
Positive Role

Abstract Abstract 4799 Objective In the present study, EPO-induced K562 cell line was used to be the cell model of erythroid differentiation, the role and mechanism of transcript factor SCL/TAL-1 in the erythropoiesis was investigated. Methods Three plasmids, which included pTRIPdU3-RNAiTALh -EF1a-GFP (SCL/TAL1 shRNA to reduce the expression of SCL/TAL-1), pTRIP-EF1a-TAL1 (SCL/TAL-1 cDNA to enhance the expression of SCL/TAL-1) and control plasmid pTRIP-dU3-RNAiluc-EF1-GFP expressing EGFP gene, were transfected into K562 cell line via lentiviral vector system, and K562 SCL/TAL-1low, K562 SCL/TAL-1high and K562 LUC (control) were established and the effect of reducing or enhancing the expression of SCL/TAL-1 on the erythropoiesis of these three cell lines was investigated. After incubated with EPO-RPMI1640 medium in which EPO induced K562 cell line into erythropoiesis for 5 day, the mRNA levels of SCL/TAL-1 and erythroid related RhD, GPA, CD47 were detected by RT-PCR assay and erythroid antigen CD71, CD235a were examined by flow cytometry in the three cell lines. Effect of SCL/TAL-1 on key phosphorylated proteins, including p-PTEN, p-Akt, p-mTOR, p-P70 and p-4EBP-1 from PI3K/Akt/mTOR pathway and p-c-Raf, p-MEK and p-ERK1/2 from Raf/MEK/ERK pathway, in the downstream of EGFR signaling pathway were checked by Western Blot assay. Effect of MEK-ERK 1/2 inhibitor U0126 on the expression of SCL/TAL1 also examined. Results 1. After 48h of transfect, more than 95% of K562 cells were GFP positive under the fluorescence microscope, indicating that infection rate of the plasmids in the K562 cells was more than 95%. 2. The results of RT-PCR showed SCL/TAL-1 mRNA expression in the K562 SCL/TAL-1low was significantly lower than that in the K562 LUC control (P <0.05). The mRNA levels of CD47 and RhD was also significantly lower and however, GPA just decreased slightly in comparison with the control. The mRNA levels of above erythroid antigens increased a little in K562-SCL/TAL-1high. 3. The FCM results showed the expression of CD71, CD235a obviously reduced in the K562 SCL/TAL-1low and positive rates were 10.4% and 76.5%, while the positive rates in the LUC control were 94.3% and 83.6%. The expression of CD71 and CD235a in K562-SCL/TAL-1high was similar to the control. 4. The level of p-MEK and p-ERK1/2 increased with transfect of SCL/TAL-1 cDNA and decreased after SCL/TAL-1 RNA interference. However, there were no obvious changes to be observed in PI3K-Akt-mTOR pathway, another important signal pathway. 5. There was no obvious alteration in SCL/TAL-1 level after treatment of MEK-ERK1/2 inhibitor, although MEK-ERK1/2 level reduced. Conclusion Our findings suggest that transcription factor SCL/TAL-1 plays a positive role in erythroid differentiation in EPO-induced K562 cell line. SCL/TAL-1 is located in the upstream of MEK-ERK1/2 and may regulate erythroid differentiation by affecting the phosphorylation levels of MEK-ERK1/2 pathway. Grant support: National Natural Science Foundation of China (No.30770912), Foundation of the Science & Technology Department of Sichuan Province (No.2008SZ0017). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activation of erythroid-specific promoters during anthracycline-induced differentiation of K562 cells

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2885-2890 ◽  
Author(s):  
A Aries ◽  
C Trentesaux ◽  
S Ottolenghi ◽  
JC Jardillier ◽  
P Jeannesson ◽  
...  
Keyword(s):  
Cell Line ◽  
Binding Sites ◽  
Gene Promoter ◽  
K562 Cells ◽  
Mrna Levels ◽  
Gene Promoters ◽  
Regulatory Regions ◽  
Gamma Globin ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

Anthracycline antitumor drugs such as aclacinomycin (ACM) and doxorubicin (DOX) used in subtoxic concentrations induce erythroid differentiation of the erythroleukemic cell line K562. To elucidate the possible role of erythroid genes of the erythropoietin receptor (EpoR) and the transcription factor GATA-1 in this effect, the regulatory regions of the above genes and human epsilon- and gamma-globin and porphobilinogen deaminase (PBGD) genes were fused to the firefly luciferase gene. The resulting reporter constructs were tested in a transfection assay of the erythroleukemic cell line K562 stimulated to differentiate by treatment with the anthracycline drugs ACM and DOX or hemin (HEM). The results showed activation of the tested promoters after cell treatment with ACM, but not with DOX or HEM. In contrast to the mouse EpoR gene promoter, the activity of the human EpoR gene promoter (-659/-60) in the reporter construct was not modified by addition of the first intron sequence. In ACM-treated K562 cells, EpoR gene promoter activity completely correlated with EpoR and GATA-1 mRNA levels and the degree of erythroid maturation. In addition, ACM strongly activated the erythroid gene promoters that contain GATA binding sites. Nevertheless, less activation was also observed for the GATA-1 gene promoter (-312/-31) lacking any known GATA binding sites. Insertion of the GATA-1 gene enhancer with two canonic GATA binding sites, stimulated the ACM activation effect for EpoR and GATA-1 promoter-containing constructs. Mutation of the enhancer GATA binding sites abolished this effect. All the regulatory regions tested (except gamma-globin promoter) were completely inactive in nonerythroid COS7 cells. These data indicate that (1) two structurally different anthracycline analogues, DOX and ACM, differ in their differentiation mechanisms, and (2) ACM switches on the erythroid program of K562 cells, at least in part because of interaction with a factor(s) that recognizes the GATA binding sites in the promoter region of erythroid genes leading to their activation.

scholarly journals Effects of butyrate on the erythropoietin receptor of cell line IW201

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 811-814 ◽  
Author(s):  
O Hermine ◽  
YJ Dong ◽  
E Goldwasser
Keyword(s):  
Cell Line ◽  
Sodium Butyrate ◽  
Actinomycin D ◽  
Erythropoietin Receptor ◽  
Affinity Binding ◽  
High Affinity ◽  
Receptor Mrna ◽  
Receptor Number ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

The murine erythroleukemic cell line, IW201, normally expresses only low-affinity erythropoietin receptors. Exposure of these cells for 48 hours to sodium butyrate results in a change in receptor kd from about 600 pmol/L to 100 to 200 pmol/L. This change in affinity is accompanied by downregulation of both receptor number and receptor mRNA. Cells exposed to sodium butyrate for 2 hours show a similar change in kd but no change in receptor number. The butyrate effect on kd at 2 hours is abrogated by either cycloheximide or actinomycin D. These data indicate that an accessory protein induced by sodium butyrate is responsible for high-affinity binding of erythropoietin.

scholarly journals Absorption Characteristics of Combination Medication of Realgar and Indigo Naturalis: In Vitro Transport across MDCK-MDR1 Cells and In Vivo Pharmacokinetics in Mice after Oral Administration

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Miao Zhang ◽  
Lin Guo ◽  
Long-Fei Lin ◽  
Chang-Hai Qu ◽  
Xing-Bin Yin ◽  
...  
Keyword(s):  
Cell Line ◽  
K562 Cell ◽  
Computational Study ◽  
Arsenic Concentration ◽  
K562 Cells ◽  
Cell Permeability ◽  
K562 Cell Line ◽  
Apparent Permeability ◽  
Indigo Naturalis

Realgar and indigo naturalis are clinically combined to treat varieties of leukemia. Exploring the drug-drug interactions might be beneficial to find active substances and develop new targeted drugs. This study aimed at exploring the change of arsenic concentration in mice and across MDCK-MDR1 cells and the cytotoxicity on K562 cells when realgar and indigo naturalis were combined. In the presence or absence of indigo naturalis, pharmacokinetics and cell-based permeability assays were used to evaluate the change of arsenic concentration, and K562 cell line was applied to evaluate the change of cytotoxicity. The drug concentration-time profiles exhibited that the combination medication group generated higher AUC, thalf, and longer MRT for arsenic, compared with the single administration of realgar. The apparent permeability coefficients (Papp) of bidirectional transport in MDCK-MDR1 cell permeability experiments showed that arsenic permeability obviously went up when indigo naturalis was incubated together. The combination medication significantly decreased the cell viability of K562 cells when both the concentration of realgar and the concentration of indigo naturalis were nontoxic. The pharmacokinetic research, the MDCK-MDR1 based permeability study, and the K562 cytotoxicity study were united together to verify the combination medication of realgar and indigo naturalis enhanced the absorption and the permeability across cells for arsenic and effectively inhibited the proliferation of K562 cell line. The molecular binding of As4S4 and indirubin was analyzed by computational study. It is predicted that the formation of the complex [As4S4…Indirubin] involves noncovalent interaction that changes the concentration of arsenic.

scholarly journals The influence of fullerenol on the cell number, cell area and colony forming unit ability in irradiated human erythroleukemic cell line

2007 ◽  
Vol 61 (3) ◽  
pp. 167-170 ◽  
Author(s):  
Ivana Icevic ◽  
Visnja Bogdanovic ◽  
Dragan Zikic ◽  
Slavica Solajic ◽  
Gordana Bogdanovic ◽  
...  
Keyword(s):  
Colony Formation ◽  
K562 Cells ◽  
Cell Number ◽  
Cell Area ◽  
X Rays ◽  
Colony Forming Unit ◽  
Erythroleukemic Cell ◽  
Analysis Of The Images ◽  
Erythroleukemic Cell Line ◽  
Cell Colony

DET (dye exclusion test) cell count and cell area by computer analysis of the images were determined in cell lines of human eritroleukemia (K562), which were irradiated with X-rays in one dose of 24 Gy and pretreated with 10 nmol/mL fullerenol (Cgo(OH)24). Cell samples obtained using a citocentrifuge and May-Gr?nvald Giemsi (MGG) during, were analyzed. The cell colony formation ability was monitored using quantative CFU (colony forming unit) test. Irradiation decreases the number of K562 cells, but fullerenol significantly increases cell number on 24th and 48th hour of the experiment. Cell area is larger, and the number of formed cell colonies after irradiation is significantly smaller compared to pretreated groups during the whole experiment. Pretreatment with fullerenol maintains a smaller cell area, and the number of colony formed units was larger compared to the irradiated cells.

Differential MAGE-1 Gene Expression in Two Variants of an Erythroleukemic Cell Line (K562)

Immunobiology ◽  
1995 ◽  
Vol 194 (4-5) ◽  
pp. 449-456 ◽  
Author(s):  
Alfonso Serrano ◽  
Angel Garcia ◽  
Eduardo Abril ◽  
Federico Garrido ◽  
Francisco Ruiz-Cabello
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

scholarly journals Stem cell factor influences the proliferation and erythroid differentiation of the MB-02 human erythroleukemia cell line by binding to a high-affinity c-kit receptor

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 436-444 ◽  
Author(s):  
VC Broudy ◽  
DA Morgan ◽  
N Lin ◽  
KM Zsebo ◽  
FW Jacobsen ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Stem Cell ◽  
Growth Factors ◽  
Cell Line ◽  
Stem Cell Factor ◽  
Erythroid Differentiation ◽  
Gm Csf ◽  
High Affinity ◽  
Kit Receptor ◽  
Erythroleukemia Cell

Abstract Stem cell factor (SCF) acts in synergy with other growth factors such as erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3), to stimulate the growth of primitive hematopoietic cells. Because of the prominent role of CSF in the maintenance of normal erythropoiesis in vivo, we examined the effects of SCF on the Epo-inducible human erythroleukemia cell line MB- 02, and characterized the c-kit receptor in these cells. MB-02 cells cultured in serum-containing media do not survive in the absence of exogenous growth factors, but the addition of SCF, Epo, or IL-3 as a single factor enhanced MB-02 survival. Furthermore, in the presence of Epo, SCF (5 to 25 ng/mL) enhanced MB-02 proliferation in a dose- dependent manner, and increased the relative and absolute number of benzidine-positive cells generated. SCF also enhanced cell proliferation in the presence of either IL-3 or low concentrations of GM-CSF. A neutralizing anti-c-kit receptor monoclonal antibody (SR-1) blocked binding of 125I-SCF to MB-02 cells by 98%, and the effect of SCF on MB-02 growth, c-kit receptor-binding parameters were quantitated by equilibrium-binding experiments with 125I-SCF. MB-02 cells display a single class of high-affinity (50 pmol/L) c-kit receptors, with approximately 8,000 receptors per cell. The molecular weight of the c- kit receptor was determined by affinity cross-linking 125I-SCF to MB-02 cells. 125I-SCF-c-kit receptor complexes of approximately 155,000 and approximately 310,000 daltons were found, likely representing the monomeric and dimeric forms of the c-kit receptor. The binding affinity and molecular weight of the c-kit receptor on MB-02 cells are similar to those of normal human marrow cells. These results suggest that SCF synergizes with Epo to influence not only the proliferation but the erythroid differentiation of MB-02 cells. Thus, the MB-02 cell line may be a useful model in which to investigate the molecular mechanisms of SCF action.

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