scholarly journals Human erythroleukemia cells adhere to fibronectin: evidence for a Mr 190,000-receptor protein

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 578-583 ◽  
Author(s):  
I Virtanen ◽  
J Ylanne ◽  
T Vartio
Keyword(s):  
Cytochalasin B ◽  
K562 Cells ◽  
Active Role ◽  
Receptor Protein ◽  
Membrane Glycoproteins ◽  
Spreading Process ◽  
Erythroleukemia Cells ◽  
Microfilament System ◽  
Serum Free Conditions ◽  
Overlay Assay

Abstract Human erythroleukemia cells (K562) adhered rapidly on fibronectin (Fn)- coated growth substratum under serum-free conditions. The adhesion could be quantitatively inhibited by the synthetic peptide Arg-Gly-Asp- Ser (RGDS) and upon hemin-induced differentiation or trypsinization of the cells. Many of the cells also displayed rapid spreading that led to a redistribution of F-actin into spreading edges and in many cells also to a formation of typical actin fibers attaching to the ventral aspect of the cells. The spreading of the cells was inhibited by cytochalasin B but not by microtubule-disrupting drugs, suggesting an active role for the microfilament system in the spreading process. Direct overlay assay of electrophoretically separated polypeptides with 125I-Fn showed that in K562 cells there is a major Mr 190,000 Fn-binding protein that is lost upon differentiation. A similar overlay assay with purified plasma and cellular Fns followed by immunostaining with anti-Fn antibodies revealed a reaction with a similar polypeptide. The binding of Fns on the nitrocellulose sheets could be inhibited and the bound Fn eluted by using the RGDS peptide. From octylglucoside extracts of radioactively surface-labeled cells, distinct Mr 190,000/185,000 membrane glycoproteins bound to Fn-heptapeptide-Sepharose, further suggesting that the Mr 190,000 polypeptide would be the Fn-receptor of the K562 cells.

scholarly journals Human erythroleukemia cells adhere to fibronectin: evidence for a Mr 190,000-receptor protein

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 578-583
Author(s):  
I Virtanen ◽  
J Ylanne ◽  
T Vartio
Keyword(s):  
Cytochalasin B ◽  
K562 Cells ◽  
Active Role ◽  
Receptor Protein ◽  
Membrane Glycoproteins ◽  
Spreading Process ◽  
Erythroleukemia Cells ◽  
Microfilament System ◽  
Serum Free Conditions ◽  
Overlay Assay

Human erythroleukemia cells (K562) adhered rapidly on fibronectin (Fn)- coated growth substratum under serum-free conditions. The adhesion could be quantitatively inhibited by the synthetic peptide Arg-Gly-Asp- Ser (RGDS) and upon hemin-induced differentiation or trypsinization of the cells. Many of the cells also displayed rapid spreading that led to a redistribution of F-actin into spreading edges and in many cells also to a formation of typical actin fibers attaching to the ventral aspect of the cells. The spreading of the cells was inhibited by cytochalasin B but not by microtubule-disrupting drugs, suggesting an active role for the microfilament system in the spreading process. Direct overlay assay of electrophoretically separated polypeptides with 125I-Fn showed that in K562 cells there is a major Mr 190,000 Fn-binding protein that is lost upon differentiation. A similar overlay assay with purified plasma and cellular Fns followed by immunostaining with anti-Fn antibodies revealed a reaction with a similar polypeptide. The binding of Fns on the nitrocellulose sheets could be inhibited and the bound Fn eluted by using the RGDS peptide. From octylglucoside extracts of radioactively surface-labeled cells, distinct Mr 190,000/185,000 membrane glycoproteins bound to Fn-heptapeptide-Sepharose, further suggesting that the Mr 190,000 polypeptide would be the Fn-receptor of the K562 cells.

scholarly journals The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1031-1037 ◽  
Author(s):  
SM Silver ◽  
MM McDonough ◽  
G Vilaire ◽  
JS Bennett
Keyword(s):  
Leukemia Cell ◽  
K562 Cells ◽  
Platelet Membrane ◽  
Human Leukemia ◽  
Membrane Glycoproteins ◽  
In Vitro Synthesis ◽  
Calcium Dependent ◽  
Hel Cells ◽  
Von Willebrand

Abstract The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium- dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.

scholarly journals Rapid capping in alpha-spectrin-deficient MEL cells from mice afflicted with hereditary hemolytic anemia.

1994 ◽  
Vol 125 (5) ◽  
pp. 1057-1065 ◽  
Author(s):  
S C Dahl ◽  
R W Geib ◽  
M T Fox ◽  
M Edidin ◽  
D Branton
Keyword(s):  
Membrane Structure ◽  
Membrane Skeleton ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
Erythroleukemia Cells ◽  
Alpha Spectrin ◽  
Erythroleukemia Cell ◽  
The Stability ◽  
In Cells

A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin-deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes.

Functional Cell Surface Expression of Band 3, the Human Red Blood Cell Anion Exchange Protein (AE1), in K562 Erythroleukemia Cells: Band 3 Enhances the Cell Surface Reactivity of Rh Antigens

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4428-4438 ◽  
Author(s):  
Roland Beckmann ◽  
Jonathan S. Smythe ◽  
David J. Anstee ◽  
Michael J.A. Tanner
Keyword(s):  
Cell Surface ◽  
Chloride Transport ◽  
K562 Cells ◽  
Surface Expression ◽  
Band 3 ◽  
Surface Reactivity ◽  
Cell Surface Expression ◽  
Anion Exchanger 1 ◽  
Erythroleukemia Cells ◽  
Antigen Activity

Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

scholarly journals K562 human erythroleukemia cells demonstrate commitment

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 862-868
Author(s):  
PT Rowley ◽  
BM Ohlsson-Wilhelm ◽  
BA Farley
Keyword(s):  
Generation Time ◽  
K562 Cells ◽  
Maximum Size ◽  
Cell Types ◽  
Progressive Increase ◽  
Cell Number ◽  
Hemoglobin Content ◽  
Erythroleukemia Cells ◽  
Average Maximum ◽  
Friend Cells

Commitment, ie, the decision to express a differentiated phenotype and to terminate proliferation irreversibly in the absence of inducer, was investigated in K562 human erythroleukemia cells. Cells were cultured for 0, 1, 2, 3, or 4 days with inducer and then plated in medium containing methylcellulose without inducer. Daily after plating, hemoglobin content was scored by benzidine staining, and growth was assessed by estimating the cell number per colony. With all inducers used, three types of colonies were found, those containing only benzidine-positive cells, those containing only benzidine-negative cells, and those containing both cell types (mixed colonies). Thymidine produced a progressive increase in the percentage of positive and mixed colonies and a progressive fall in the percentage of negative colonies. Whereas negative colonies grew at an exponential rate with a generation time of about 20 hours, positive colonies reached an average maximum size of 16 cells, representing a total of four divisions. Butyrate had a similar effect, except that the rise was greater for mixed colonies than for positive colonies, and the plateau in positive colony size was less evident. In contrast, CO2 depletion or hemin treatment induced an increase in the fraction of cells staining benzidine positive that was lost rapidly upon removal of the inducing condition. Thus, of the four conditions, thymidine and butyrate caused commitment, whereas hemin and CO2 depletion did not. Thus K562 cells, like Friend cells, demonstrate commitment, but, unlike Friend cells, demonstrate a significant rate of commitment in the absence of inducer and hence form a significant percentage of mixed colonies with or without inducer.

Intracellular transport and secretion of fibroin in the posterior silk gland of the silkworm Bombyx mori

1981 ◽  
Vol 50 (1) ◽  
pp. 19-44 ◽  
Author(s):  
S. Sasaki ◽  
E. Nakajima ◽  
Y. Fujii-Kuriyama ◽  
Y. Tashiro
Keyword(s):  
Intracellular Transport ◽  
Cytochalasin B ◽  
Silk Gland ◽  
Luminal Surface ◽  
Golgi Bodies ◽  
Apical Cytoplasm ◽  
Radial Microtubule System ◽  
Microfilament System ◽  
Silkworm Bombyx Mori ◽  
Posterior Silk Gland

Intracellular transport and secretion of fibroin in the posterior silk gland cells of the silkworm, Bombyx mori, were investigated in relation to the radial microtubule and circular microtubule-microfilament systems of the cells. The silk glands were pulse-labelled for 3 min with [3H]glycine in vitro and then chased in media containing excess cold glycine and in some cases antimitotic reagents (colchicine or vinblastine) or cytochalasin (B or D), and the flow of the label in the glands was investigated by radioautography. It was revealed that the label initially located over the rough endoplasmic reticulum subsequently moves to the Golgi bodies to be condensed there. The secretory granules of fibroin or fibroin globules thus formed are transported via the radial microtubule system to the apical cytoplasm to be secreted there under some regulation by the circular microtubule-microfilament system. In the presence of colchicine or vinblastine, the secretion of fibroin was suppressed an marked accumulation of fibroin globules in the Golgi regions was observed, while in the presence of cytochalasin B or D the secretion was accelerated and extensive invagination of the luminal surface, which was probably due to the serial exocytosis of fibroin globules, was observed. These results suggest that the radial microtubule system and the circular microtubule-microfilament system are responsible for intracellular transport of fibroin globules from Golgi bodies to the apical cytoplasm and secretion by exocytosis at the luminal surface, respectively.

scholarly journals Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer [published erratum appears in Blood 1995 Feb 1;85(3):862]

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1900-1906 ◽  
Author(s):  
JL Miller ◽  
CE Walsh ◽  
PA Ney ◽  
RJ Samulski ◽  
AW Nienhuis
Keyword(s):  
Globin Gene ◽  
K562 Cells ◽  
Single Copy ◽  
Regulatory Elements ◽  
Globin Mrna ◽  
Adeno Associated Virus ◽  
Binding Motifs ◽  
Gamma Globin ◽  
Erythroleukemia Cells ◽  
New Strategy

Abstract The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut- NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single- copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.

delta-Aminolevulinate dehydratase in human erythroleukemia cells: an immunologically distinct enzyme

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 939-944 ◽  
Author(s):  
CS Chang ◽  
S Sassa
Keyword(s):  
Butyric Acid ◽  
Deae Cellulose ◽  
K562 Cells ◽  
Normal Adult ◽  
Erythroleukemia Cells ◽  
Normal Enzyme ◽  
Normal Human ◽  
Dehydratase Activity ◽  
Ala Dehydratase ◽  
Selenium Oxide

Abstract Physicochemical and immunologic properties of delta-aminolevulinate (ALA) dehydratase in human K562 erythroleukemia cells were examined. ALA dehydratase activity was found to increase in K562 cells after treatment with butyric acid or selenium oxide. Enzyme activity in untreated K562 cells was comparable to that in normal adult erythrocytes but was increased three- to six-fold in K562 cells treated with 1.2 mmol/L butyric acid or 0.03 mmol/L selenium oxide. The Michaelis-Menten constant (Km), the inhibitor constant (Ki), and elution profile by diethylaminoethyl (DEAE) cellulose chromatography were similar for ALA dehydratase from K562 cells and normal human adult and human fetal erythrocytes. However, ALA dehydratase from K562 cells did not react with a monospecific rabbit antibody against ALA dehydratase purified from normal adult erythrocytes, although the antibody reacted with the enzyme from normal adult and fetal red cells. These findings indicate that ALA dehydratase in K562 cells is immunologically distinct from the normal enzyme.

scholarly journals Natural killer lines and clones with apparent antigen specificity.

1990 ◽  
Vol 172 (2) ◽  
pp. 457-462 ◽  
Author(s):  
N Suzuki ◽  
E Bianchi ◽  
H Bass ◽  
T Suzuki ◽  
J Bender ◽  
...  
Keyword(s):  
Northern Blot Analysis ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Antigen Specificity ◽  
Gamma Delta ◽  
Gamma Chain ◽  
Erythroleukemia Cells ◽  
Class Ii Mhc ◽  
Specific Manner ◽  
Class Ii Mhc Antigens

Fresh CD3-, CD16+ lymphocytes that adhered to selected allogeneic lymphoblastoid cell lines (LCL) were cultured with LCL in the presence of IL-2-containing medium. The resulting lines as well as clones derived from these lines expressed CD16 and/or CD56, but lacked detectable CD3 or TCR-alpha/beta or TCR-gamma/delta complexes on the cell surface. Northern blot analysis failed to detect CD3 epsilon or TCR-beta transcripts, but revealed the presence of a TCR-gamma chain transcript in one of these lines. In addition to displaying potent cytolytic activity against K562 erythroleukemia cells (a classical NK target), the vast majority of these lines and clones lysed their specific stimulator LCL to a significantly greater extent than irrelevant LCL. This selective killing was inhibited by the addition of cold stimulator LCL or K562 cells, or anti-LFA 1 mAbs, but not by irrelevant LCL or mAbs to CD3, class I or class II MHC antigens. These results indicate that some CD3- lymphocytes, phenotypically indistinguishable from NK cells, can recognize and lyse allogeneic targets in a specific manner.

scholarly journals Different Heparan Sulfate Proteoglycans Serve asCellular Receptors for HumanPapillomaviruses

2003 ◽  
Vol 77 (24) ◽  
pp. 13125-13135 ◽  
Author(s):  
Saeed Shafti-Keramat ◽  
Alessandra Handisurya ◽  
Ernst Kriehuber ◽  
Guerrino Meneguzzi ◽  
Katharina Slupetzky ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Binding Capacity ◽  
Ectopic Expression ◽  
K562 Cells ◽  
Human Keratinocytes ◽  
Hpv Infection ◽  
Homozygous Deletion ◽  
Heparan Sulfate Proteoglycans ◽  
Receptor Protein

ABSTRACT Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of α6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than α6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.

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