Transferrin and iron uptake by human lymphoblastoid and K-562 cells

Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.     

Human leukemic K562 cells: suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor

The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression.     

Effect of extracellular hemin on hemoglobin and ferritin content of erythroleukemia cells

Mouse (MEL) and human (K-562) erythroleukemia cell lines can be induced to undergo erythroid differentiation, including hemoglobin (Hb) synthesis, by extra cellular hemin. In order to study the effect of extracellular hemin on intracellular ferritin and Hb content, we have used Mossabauer spectroscopy to measure the amount of 57Fe incorporated into ferritin or Hb and a fluorescent enzyme-linked immunosorbent assay (ELISA) to measure the ferritin protein content. When K-562 cells were cultured in the presence of a 57Fe source either as transferrin or citrate, in the absence of a differentiation inducer, all the intracellular 57Fe was detected in ferritin. When the cells were cultured in the presence of 57Fe-hemin, 57Fe was found in both ferritin and Hb. 57Fe in ferritin increased rapidly, and after 2 days it reached a plateau at 5 X 10(-14) g/cell. 57Fe in Hb increased linearly with time and reached the same value after 12 days. Addition of other iron sources such as iron-saturated transferrin, iron citrate, or iron ammonium citrate caused a much lower increase in ferritin protein content as compared to hemin. When K-562 cells were induced by 57Fe-hemin in the presence of 56Fe-transferrin, 57Fe was found to be incorporated in equal amounts into both ferritin and Hb. However, when the cells were induced by 56Fe-hemin in the presence of 57Fe-transferrin, 57Fe was incorporated only into ferritin, but not into Hb, which contained 56Fe iron. These results indicate that in K-562 cells, when hemin is present in the culture medium it is preferentially incorporated into Hb, regardless of the availability of other extra- or intracellular iron sources such as transferrin or ferritin. In MEL cells induced to differentiate by dimethylsulfoxide (DMSO) a different pattern of iron incorporation was observed; 57Fe from both transferrin and hemin was found to incorporate in ferritin as well as in Hb.     

Dynamic immunological and cytochemical markers in the induced differentiation of the K-562 cell line

The possibility of TPA-induced differentiation of K-562 cell line was studied. Monoclonal antibodies against differentiating antigens of HAE erythroid lineage and against myelomonocytic ICO lineage raised in the USSR Cancer Research Centre were used. Changes in immunological and cytochemical indexes suggest that K-562 cell differentiation goes in the erythroid direction. The cells lost early differentiation and acquired late differentiation markers.     

The effect of topoisomerase inhibitors on the expression of differentiation markers and cell cycle progression in human K-562 leukemia cells.

Treatment of human K-562-J leukemia cells for 1 h with the topoisomerase II-reactive drugs VP-16, VM-26, or mAMSA resulted in a dose-dependent inhibition of proliferation and in an increase in the percentage of cells staining positive for hemoglobin, a marker of erythroid differentiation. Staining for hemoglobin of up to about 60% of the cells was observed at 20 microM VP-16, 1 microM VM-26, and 8 microM mAMSA. Such treatment also caused a G2/M arrest in the cell cycle. Incubation of the cells with radiolabeled VP-16 indicated that the induced erythroid differentiation was not due to continuous cell exposure to a residual amount of the drug. VP-16-induced erythroid differentiation was also not affected by DNA, RNA, or protein synthesis inhibitors. Differentiation induction and the G2/M arrest evoked by VP-16, VM-26, and mAMSA were, however, reduced in the presence of novobiocin. Our results indicate that topo-reactive drugs that cause G2/M arrest in the K-562-J cell cycle can induce in these cells erythroid differentiation after a short and irreversible interaction with their target molecule(s).     

Effect of 5-azacytidine on the differentiation of human leukemia K-562 cells

The treatment of K-562 cells with 10(-5) M to 10(-7) M 5-azacytidine induced a marked increase in benzidine-positive cells. Similarly, the exposure of K-562 cells to 2 X 10(-3) M butyric acid or 5 X 10(-7) M 1-beta-arabinofuranosylcytosine or 1 X 10(-3) M hydroxyurea induced an erythroid differentiation of K-562 cells. The activity of DNA-methyltransferase and the level of methylcytosine in newly synthesized DNA were significantly decreased when the cells were treated with 5-azacytidine or butyric acid, while 1-beta-arabinofuranosylcytosine or hydroxyurea had no inhibitory effect on DNA-methylation of K-562 cells. These results suggest that the inhibition of DNA-methylation is not necessarily a specific phenomenon for erythroid differentiation of K-562 cells.     

Expression of transferrin receptors during erythroid maturation

A monoclonal antibody, F111/2Dl, produced after immunisation of C3H/He mice with the human erythroleukemia cell line, K562, is described. It detects cell surface determinants of similar distribution to those characterised by the OKT-9 monoclonal antibody, which has been shown to identify the transferrin receptor. The F111/2Dl antibody, as well as OKT-9, has been used to investigate the distribution of transferrin receptors during erythroid maturation in normal bone marrow and peripheral blood, and on the K562 cell line during erythroid differentiation, induced in vitro.     

Human leukemia K-562 cells: induction of erythroid differentiation by 5-azacytidine

In this article we show that the cytidine analog 5-azacytidine is able to induce differentiation of the human leukemia K-562 cell line. Erythroid induction is associated with (a) an increase of the overall globin synthesis and globin mRNA accumulation, (b) a relative increase of fetal with respect to embryonic globins, and (c) a decrease of the proliferative capacity of hemoglobin-containing cells. In addition, we have analysed the DNA methylation pattern at the cleavage sites of MspI and HpaII restriction enzymes, which are known to cleave differently CCGG DNA sequences when 5-methylcytosine is present. These experiments indicate that in K-562 cells treated with 5-azacytidine, DNA becomes hypomethylated, suggesting that genetic programmes leading to an erythroid phenotype may be activated by a reduction of DNA methylation.     

Loss and reappearance of transferrin receptors in human leukemic cell lines

Serum transferrin (the iron binding protein) exerts its iron carrier function at the cell surface after binding to the appropriate receptor (TrR). In this work it is demonstrated that differentiating agents induce loss of TrR from the surface of three leukemic cell lines (Molt-3, HL-60 and K-562). Loss of TrR correlates with change in morphology and induction of phenotypic markers of the differentiated cells. Removal of the differentiating agent from the culture is followed by reexpression of TrR on the cell surface. The data presented in this paper suggest that TrR may play a regulatory role in cell differentiation and malignant transformation.     

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.     

Exposure to sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristine-13-acetate alters sensitivity of K562 cells to nonspecific lysis by human or rat leukocytes

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to nonspecific lysis (nonrestricted with respect to antigens of the major histocompatibilty complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.     

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     

Changes in the K562 cell sensitivity to nonspecific lysis by human and rat leukocytes under the influence of sodium butyrate, dimethyl sulfoxide and phorbol-12-myristate-13-acetate

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to non-specific lysis (non-restricted with respect to antigens of the major histocompatibility complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.     

Induction of differentiation in mouse erythroleukaemia cells by the action of papain at the cell surface

The addition of one of several proteases to cultures of mouse erythroleukaemia (MEL) or human K-562 leukaemia cells can induce a substantial portion of the cells to undergo erythroid differentiation. This effect is due, at least in part, to the proteolytic action of these enzymes. The critical substrate(s) for this proteolytic action is not a component of the medium or a long-lived substance(s) released from the cells. In order to determine if the substrate(s) is located on the cell surface or intracellularly, a comparison of the ability of non-immobilized papain and immobilized papain (i.e. covalently linked to Sepharose beads which were larger than the cells) to induce MEL cell differentiation was undertaken. Both papain preparations induced the same level of differentiation. The proteolytic activity of the bead-linked papain remained associated with the beads. Therefore, proteases induce erythroid differentiation in these cells by acting proteolytically on a substrate(s) that is exterior to the cell.     

氯化高铁血红素诱导K562细胞红系分化对其细胞周期的影响

细胞周期的测量是细胞增殖动力学的研究基础。通过添加30μmol·L-1氯化高铁血红素(Hemin)诱导人慢性髓系白血病K562细胞红系分化,利用5-溴脱氧尿嘧啶核苷(BrdU)与7-AAD双染的方法检测Hemin诱导的K562红系分化细胞对细胞周期各期比例的影响,未诱导的K562细胞周期各期比例作为对照,检测发现Hemin诱导的K562红系分化细胞对其细胞周期相对值无明显影响。应用BrdU间隔染色结合流式细胞术的方法,通过分析BrdU间隔染色后BrdU阳性细胞群的动态变化规律,从而推算出K562红系分化细胞的倍增时间及细胞周期各期时长。根据测量结果发现,未诱导的K562细胞总倍增时间约为20 h,与通过生长曲线公式法计算倍增时间的结果相符,Hemin诱导的K562细胞的细胞周期倍增时长约为23 h。Hemin诱导的K562红系分化细胞较未诱导的K562细胞倍增时间与各期时长无明显差异。因此,Hemin诱导K562细胞红系分化对其细胞周期绝对值及相对值均无明显影响。     

Induction of differentiation in mouse erythroleukaemia cells by the action of papain at the cell surface

Abstract The addition of one of several proteases to cultures of mouse erythroleukaemia (MEL) or human K-562 leukaemia cells can induce a substantial portion of the cells to undergo erythroid differentiation. This effect is due, at least in part, to the proteolytic action of these enzymes. The critical substrate(s) for this proteolytic action is not a component of the medium or a long-lived substance(s) released from the cells. In order to determine if the substrate(s) is located on the cell surface or intracellularly, a comparison of the ability of non-immobilized papain and immobilized papain (i.e. covalently linked to Sepharose beads which were larger than the cells) to induce MEL cell differentiation was undertaken. Both papain preparations induced the same level of differentiation. The proteolytic activity of the bead-linked papain remained associated with the beads. Therefore, proteases induce erythroid differentiation in these cells by acting proteolytically on a substrate(s) that is exterior to the cell.     

Segregation of the NK-sensitive phenotype in human x mouse somatic cell hybrids reveals separate genetic control of recognition and postrecognition determinants

In an attempt to investigate the nature of tumor cell-derived membrane surface determinants involved in natural killer cell (NK) recognition or postrecognition events, we have constructed human X mouse interspecies somatic cell hybrids. Highly NK-sensitive (NKs) human tumor cells were fused with NK resistant (NKr) mouse fibroblasts (LMTK-) in polyethylene glycol and selected in hypoxanthine/aminopterin/thymidine medium and ouabain. Hybrids generated from NKs erythroleukemia cells (K-562) or NKs retinoblastoma cells (Y-79) with LMTK- displayed an intermediate NK-sensitive phenotype. One Y-79 X LMTK- hybrid (YL-22) retained a high level of susceptibility to NK binding and cytolysis, as determined by 51Cr release and in cold-target inhibition assays. On the other hand, human NKr RAJI cells generated NK-resistant hybrids when fused with LMTK- fibroblasts. Four hybrids (KL-12, YL-2, YL-22, and YL-43) displaying consistent NK sensitivity were subsequently cloned by limiting dilution. Various hybrid clones derived from the KL-12 hybrid (K-562 X LMTK-) demonstrated a range of NK-sensitive phenotypes. However, the uncloned KL-12 and most cloned lines derived from this hybrid competed against 51Cr-labeled K-562 targets as well as unlabeled K-562 parental cells, regardless of their NK-sensitive phenotype. These findings raise the possibility that chromosomal segregation may be affecting a postbinding step in this hybrid system. The NK-sensitive hybrids exhibited a limited number of human chromosomes as assessed by quinacrine banding. Furthermore, human transferrin receptor (TfR) expression, as monitored by flow cytometry using the B3/25 monoclonal antibody, demonstrated no clear correlation with NK sensitivity or competitive ability in either KL or YL hybrid clones, thus arguing against the involvement of the TfR in human NK recognition. These results suggest that the NK-sensitive phenotype in human tumor cells may be regulated by genes encoded by a limited number of human chromosomes.