A novel nuclear protein which binds to G gamma and A gamma globin promoters and modulates hemoglobin synthesis in K562 cells.

Nuclear extract of human erythroleukemic cell line K562 contains a 70 kDa protein which is gradually reduced when cells are induced to express globin genes by 25 microM hemin. When globin synthesis was inhibited by cycloheximide (100 micrograms/ml) or Actinomycin D (1 microgram/ml), the disappearance of this protein was prevented. The 70 kDa nuclear protein exhibited strong binding to G gamma and A gamma globin promoters but not to beta-globin promoter. This suggests that this 70 kDa nuclear protein may be involved in the regulation of fetal globin gene expression.     

Isolation of mutants of<Emphasis Type="Italic"> Vibrio anguillarum</Emphasis> defective in haeme utilisation and cloning of<Emphasis Type="Italic"> huvA</Emphasis>, a gene coding for an outer membrane protein involved in the use of haeme as iron source

The isolation of Vibrio anguillarum mutants lacking the ability to use haemin and haemoglobin as the only iron sources, as well as the identification of a gene involved in haeme utilisation are described. One of the isolated mutants defective in haeme utilisation lacked an iron-regulated outer membrane protein of 79-kDa. Although growth on haeme as iron source was completely abolished, the haemin and haemoglobin binding activities remained intact in the mutant, suggesting that the absent protein is not the only one involved in haeme binding. The wild-type phenotype in this mutant was restored by transformation with a cosmid clone (pML1) containing a 21-kb DNA fragment isolated from a gene library derived from the parental strain of V. anguillarum. Sequence analysis of pML1 subclones led to the finding of an ORF, huvA, that codes for a 79-kDa protein (HuvA) and whose sequence shows high identity with haeme receptors from Vibrio choleare (HutA) and Vibrio vulnificus (HupA). The sequence of huvA from the V. anguillarum haeme-utilisation mutant revealed a single mutation, leading to the synthesis of a truncated HuvA protein of 70 kDa. The parental strain and the cosmid-complemented mutant showed a higher degree of virulence for fish than the mutant strain in experimental infections in which fish were previously overloaded with haemin. This finding suggests that haeme uptake plays an important role in V. anguillarum multiplication in fish tissues when free haeme is available.     

Mechanism of differentiation of human erythroleukaemic cell line K562 by hemin

The human erythroleukaemic cell line K₅₆₂, in response to various chemical agents, undergoes differentiation and exhibits exclusive production of fetal and embryonic haemoglobins. In this study we have compared the efficiency of natural growth factors interleukin-3 and erythropoietin and three chemical inducers such as dimethyl sulfoxide (DMSO, 1.9%), phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) and hemin (25 μm) on growth and differentiation of these cells. Erythropoietin significantly stimulated the growth of K₅₆₂ cells (P<0.0001), while interleukin-3 did not (P= 0.2783). However, neither of these growth factors individually or together induced differentiation of K₅₆₂ cells. Hemin appears to be more efficient than DMSO or PMA in differentiation of K₅₆₂ cells as measured by benzidine positive cells (70% or more). The differentiation of K₅₆₂ cells by hemin occurs independently of protein kinase-C activation and the arrest of DNA synthesis. In contrast, hemin significantly stimulated RNA and protein synthesis (P<0.0001) as measured by [³H]-uridine and [³H]-leucine incorporation respectively. Analysis of hemin-treated K₅₆₂ nuclear extract on sodium dodecylsulphate gel electrophoresis showed that one protein band of molecular weight 70 kDa decreased after 48 h of incubation in the presence of 25 μm hemin. The disappearance of this protein can be prevented by cycloheximide (100 μg/ml) and actinomycin D (0.1 μg/ml) and thus indicating that the removal of 70 kDa protein seems to be dependent on RNA and protein synthesis. The regulatory role of 70 kDa protein in hemin-induced differentiation of K₅₆₂ cells is discussed.     

IL——3和羟基脲协同诱导K562细胞凋亡

The effect of IL-3 and hydroxyurea on human erythroleukemia cell line (K562 cells) was demonstrated by using the electro-microscopy and flow cytometry. Our data showed that neither IL-3 nor hydroxyurea could induce the apoptosis of K562 cells alone. However, the IL-3 and hydroxyurea could induce the apoptosis of K562 cells cooperatively. Analysis with flow cytometry showed that the percentage of apoptotic cells was about 31.90% after K562 cells were induced by IL-3 and hydroxyurea cooperatively for 5 days, and the sub-G1 peak (apoptotic peak) was detected in the induced K562 cells. Meanwhile, the percentage of S-phase in the IL-3 and hydroxyurea induced K562 cells was increased, and the proliferation of the induced K562 cells was inhibited significantly. Furthermore, the IL-3 and hydroxyurea induced K562 cells showed chromatin condensation with regular crescents at the nuclear edges and apoptotic bodies. It suggested that IL-3 could enhance the sensitivity of K562 cells to hydroxyurea and the apoptosis of K562 cells could be induced by IL-3 and hydroxyurea cooperatively.     

IL-3和羟基脲协同诱导K562细胞凋亡

本文应用流式细胞分选仪和电子显微镜研究了IL-3和羟基脲对人红白血病细胞株(K562细胞)凋亡的影响.结果显示IL-3和羟基脲分别诱导K562细胞,不能引起细胞凋亡;而IL-3和羟基脲协同诱导K562细胞,可以引起细胞凋亡.用流式细胞仪检测到IL-3和羟基脲协同诱导K562细胞后,DNA含量低于二倍体的细胞数达31.90%,并产生明显的凋亡小峰.同时,IL-3和羟基脲协同诱导K562细胞,可抑制细胞周期中的S期,阻止细胞从S期进入G2/M期,使细胞周期延长,对K562细胞的生长和增殖具有抑制作用.在电镜下可观察到IL-3和羟基脲协同诱导的K562细胞,出现典型的凋亡细胞形态,细胞核内染色质浓缩、凝聚,紧靠在核膜边沿,形成新月形或环状的染色质结构,产生凋亡小体.提示IL-3和羟基脲具有协同效应,IL-3可提高K562细胞对羟基脲的敏感性,并可协同羟基脲诱导K562细胞凋亡.     

A protein factor binding to an octamer motif in the gamma-globin promoter disappears upon induction of differentiation and hemoglobin synthesis in K562 cells.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human gamma-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erythroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the gamma-globin octamer containing fragment. The possible role of these nuclear proteins in gamma-globin gene regulation and/or cell differentiation is discussed.     

Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters

Corynebacterium diphtheriae and Corynebacterium ulcerans use haemin and haemoglobin as essential sources of iron during growth in iron-depleted medium. C. diphtheriae and C. ulcerans mutants defective in haemin iron utilization were isolated and characterized. Four clones from a C. diphtheriae genomic library complemented several of the Corynebacteria haemin utilization mutants. The complementing plasmids shared an approximately 3 kb region, and the nucleotide sequence of one of the plasmids revealed five open reading frames that appeared to be organized in a single operon. The first three genes, which we have termed hmuT, hmuU and hmuV, shared striking homology with genes that are known to be required for haemin transport in Gram-negative bacteria and are proposed to be part of an ABC (ATP-binding cassette) transport system. The hmuT gene encodes a 37 kDa lipoprotein that is associated with the cytoplasmic membrane when expressed in Escherichi coli and C. diphtheriae. HmuT binds in vitro to haemin- and haemoglobin-agarose, suggesting that it is capable of binding both haemin and haemoglobin and may function as the haemin receptor in C. diphtheriae. This study reports the first genetic characterization of a transport system that is involved in the utilization of haemin and haemoglobin as iron sources by a Gram-positive bacterium.     

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.     

The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence

The Neisseris meningitidis haemoglobin receptor gene, hmbR, was cloned by complementation in a porphyrin-requiring Escherichia coli mutant. hmbR encodes an 89.5 kDa outer membrane protein which shares amino acid homology with the TonB-dependent receptors of Gram-negative bacteria. HmbR had the highest similarity to Neisseria transferrin and lactoferrin receptors. The utilization of haemoglobin as an iron source required internalization of the haemin moiety by the cell. The mechanism of haemin internalization via the haemoglobin receptor was TonB-dependent in E. coli. A N. meningitidis hmbR mutant was unable to use haemoglobin but could still use haemin as a sole iron source. The existence of a second N. meningitidis receptor gene, specific for haemin, was shown by the isolation of cosmids which did not hybridize with the hmbR probe, but which were able to complement an E. coli hemA aroB mutant on haemin-supplemented plates. The N. meningitidis hmbR mutant was attenuated in an infant rat model for meningococcal infection, indicating that haemoglobin utilization is important for N. meningitidis virulence.     

羟基脲（Hydroxyurea）对细胞周期与珠蛋白基因表达的影响（简报）

Although the hydroxyurea (HU) has been extensively studied, little is known of its molecular mechanism in controlling the expression of human globin gene and in modulating the progression of cell-cycle in K 562 cell. In the present study, the effect of hydroxyurea on proliferative kinetics of K 562 cells was examined by monitoring the number of cells during a period of 8 day's cell culture. Our results showed that there was a dose related decrease in cell growth when K562 cells were incubated with HU. Moreover, cell-cycle analysis demonstrated that HU had profound effect on cell-cycle distribution. In the case of the induced K 562 cells, there was an increased accumulation of cells in S phase and a decreased fraction of cells in G 1 and G 2 + M phase. Furthermore, HU could induce the expression of human beta-globin gene in the induced K 562 cells. Our results indicate that HU has a potential to inhibit the proliferation of K 562 cells and to stimulate the terminal differentiation of this cell.     

Identification and characterization of a T cell growth inhibitory factor produced by K562 erythromyeloid cells

Cells of the human erythroleukemia cell line K562 constitutively secrete a factor that inhibits human T lymphocyte proliferation induced via CD3/Ti. The factor, termed K-TIF (K562-derived T cell inhibitory factor) is produced in either the presence or absence of fetal calf serum in cultures of K562 cells and can be precipitated by 70% NH4SO4. Gel filtration chromatography on Superose 12 resin by FPLC showed that the inhibitory factor has a molecular weight of approximately 30-35 kDa. A protein of this size, metabolically labeled with [35S]methionine, specifically bound human peripheral blood mononuclear cells. Chromatofocusing with Mono P by FPLC (pH gradient 7.2-5) indicates that the inhibitory factor has an isoelectric point of 6.0-6.4.     

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.     

In vivo DNA-protein interactions at hypersensitive site 3.5 of the human beta-globin locus control region.

Using ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the status of DNA-protein interactions at hypersensitive site 3.5 (HS3.5) of the locus control region in K562 and HEL cells, we found that there was protein occupancy in vivo at HS3.5 in both cell lines and the status of DNA-protein interaction was different between K562 and HEL. These data provide direct evidence that specific nuclear factor-DNA complexes form in vivo at functionally important sequence motifs of the HS3.5 in erythroid cells. This indicates that HS3.5 may play an important role in the regulation of the beta-globin gene cluster. K562 is a human erythroleukemia cell line in which the embryonic epsilon-globin gene is predominantly expressed, while the HEL cell line expresses predominantly the fetal beta-globin genes. Thus, HS3.5 might also be involved in the regulation of developmental stage-specific expression of beta-globin genes. Our results are also consistent with the model that each hypersensitive site acts as a functional unit and HS3.5 may facilitate the formation of the HS3 functional unit.