A hematopoietic-specific transmembrane protein, Art-1, is possibly regulated by AML1

The functions of AML1 in hematopoietic differentiation are repressed by AML1-mutants including the AML1/ETO chimeric protein, which is seen in t(8;21) acute myeloid leukemia. Erythroid progenitors of the patients with t(8;21) AML expressed AML1/ETO. To investigate the effect of AML1/ETO in erythroid cells, we made a tetracycline-regulated AML1/ETO overexpression system in mouse erythroleukemic (MEL) cells. Enforced AML1/ETO repressed the terminal erythroid differentiation. Furthermore, we performed representational difference analysis using this MEL cell system to clone the downstream targets of AML1 in erythroid cell differentiation. We cloned a novel transmembrane protein, Art-1 (AML1-regulated transmembrane protein 1), which is a member of tetramembrane spanning superfamily. Art-1 expression was restricted in hematopoietic cells. It was upregulated by AML1 and downregulated by AML1/ETO in both erythroid and myeloid cells, and increased during erythroid cell differentiation. Art-1 may play an important role in the differentiation of erythroid cells, possibly as a direct downstream target of AML1.     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.     

Maturation of murine erythroleukemia cells committed to differentiation requires protein kinase C.

Treatment of murine erythroleukemia cells (MELC) attached to fibronectin-coated dishes with dimethyl sulfoxide causes the cells to become committed to the erythroid differentiation pathway. These cells mature extensively and acquire the characteristics of erythroid cells. The cells lose their cell-surface fibronectin receptors and accumulate red cell-specific membrane proteins, such as band 3, in amounts comparable to those in erythrocytes. Previous studies of MELC have shown that the presence of protein kinase C (PKC) is required for commitment to differentiation, but that the level of PKC activity declines progressively during maturation. In this study, we have established a role for PKC in the maturation of MELC committed to differentiation. Our results show that down-regulation of PKC by addition of phorbol 12-myristate 13-acetate (PMA) to committed MELC blocks subsequent maturation of the cells. Treatment of MELC with the PKC inhibitors H7 and sphingosine had similar effects. Down-regulation of PKC was assayed by measuring cytosolic PKC activity as well as by Western blotting using PKC antibodies. MELC maturation was monitored by loss of the cell-surface fibronectin receptor, release of cells from fibronectin plates, and accumulation of the band 3 anion transport protein. Immunoprecipitation of surface-labeled proteins by an anti-fibronectin receptor (integrin) antibody showed that PMA-treated cultures had more fibronectin receptor protein than untreated cultures 6 days post-induction. As a result, cultures of committed MELC treated with PMA remained attached to fibronectin-coated plates, whereas non-PMA-treated cells were released into the culture medium. Furthermore, PKC-depleted cells accumulated much smaller amounts of band 3 protein and band 3 mRNA than did non-PKC-depleted controls. Our results show that although PKC activity declines progressively during post-commitment maturation of MELC, its continued presence is critical for the process of cellular maturation.     

Molecular properties of a galaptin and its activity in normal and leukemic erythroid tissue

Recently, the generic term “galaptins” was proposed for the group of low molecular weight, acidic, β-galactoside-specific protein lectins that have been isolated from a wide variety of animal tissues and are thought to have a role in cell-cell recognition and adhesion. A molecule of this type, called erythroid developmental agglutinin (EDA), has been isolated from rabbit bone marow where it seems to mediate the intererythroblast adhesion seen in erythroblastic islands during erythropoiesis in vivo. Here, we show that after purification, EDA shows 95%-100% Coomassie blue staining as a single component on electrophoresis in native, urea, and SDS polyacrylamide gels and electrofocuses as a single band at pH 5.6. EDA has a subunit molecular weight of 13,000 in SDS gels and, unlike the majority of other galaptins, which arc dimeric, native EDA is monomeric in solution. Another monomeric galaptin, chicken lactose lectin II, has been described recently, and it therefore seems that there may be two classes of galaptin distinguishable by their aggregation state in solution. We have previously reported that EDA agglutinates rabbit erythroblasts in vitro and that this reaction is inhibited by β-galactoside-containing sugars and by anti-EDA Fab fragments suggesting that EDA bridges directly between cell surface glycoproteins. The insensitivity of this reaction to cooling, or to the disruption of cellular metabolism or the cytoskeleton demonstrated here further supports this hypothesis. EDA-mediated erythroblast agglutination was also shown to be independent of divalent cations. Since galaptins are thought to be important in cohesion between normal cells, the possibility that EDA is not active in leukemic erythroid tissue was examined. The murine erythroleukemia cell line (MELC) provided an excellent system for this study since MELC are thought to be derived from an erythroid committed cell transformed at an early stage of development and can be induced by a number of chemical agents to differentiate terminally along the erythroid developmental pathway in culture. EDA of rabbit origin was found to agglutinate mouse erythroblasts in vitro and was used to investigate the response of MELC to EDA. It was found that the transformed cells were not readily agglutinated by EDA but on induction, and the concomitant loss of many of their transformed characteristics, MELC gained aggregation competence for EDA. The possible causes of these differences are discussed.     

Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells

Background

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells.

Methodology/Principal Findings

We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells.

Conclusion/Significance

To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells.     

Lipocalin 2-mediated growth suppression is evident in human erythroid and monocyte/macrophage lineage cells

Lipocalin 2 (LCN2), a secreted protein of the lipocalin family, induces apoptosis in some types of cells and inhibits bacterial growth by sequestration of the iron-laden bacterial siderophore. We have recently reported that LCN2 inhibits the production of red blood cells in the mouse. Here we analyzed the role of LCN2 in human hematopoiesis. Expression of LCN2 was observed not only in mature cells such as those of the granulocyte/macrophage and erythroid lineages but also in hematopoietic stem/progenitor cells. We also examined expression of two candidate receptors for LCN2, brain type organic cation transporter (BOCT) and megalin, in various cell types. BOCT showed relatively high levels of expression in erythroid and hematopoietic stem/progenitor cells but lower levels in granulocyte/macrophage and T lymphoid cells. Megalin was expressed at high levels in T lymphoid and erythroid cells but at lower levels in granulocyte/macrophage lineage cells. LCN2 suppressed the growth of erythroid and monocyte/macrophage lineages in vitro, but did not have this effect on cells of other lineages. In addition, immature hematopoietic stem/progenitor cells were not sensitive to LCN2. These results demonstrate a lineage-specific role for LCN2 in human hematopoiesis that is reminiscent of its effects upon mouse hematopoiesis and strongly suggest an important in vivo function of LCN2 in the regulation of human hematopoiesis.     

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells

Binding experiments with the specific muscarinic ligand [³H]N-methylscopolamine (³H-NMS) have shown the presence of constitutive muscarinic acetylcho-line receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1–M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K_D) for ³H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B⁺) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B⁺ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells. J. Cell. Physiol. 178:333–340, 1999. © 1999 Wiley-Liss, Inc.     

Cell volume changes in relation to the cell cycle of differentiating erythroleukemic cells

Murine erythroleukemic cells (MELC) were synchronized by sequential exposure to thymidine and hydroxyurea. Upon removal from hydroxyurea, cells cultured with or without agents that induce erythroid differentiation, such as hexamethylene bisacetamide (HMBA) or dimethylsulfoxide (Me₂SO), proceed through S, G2 and mitosis with the same kinetics: S phase averages 5 h and G2 plus mitosis, 2 h. Cells cultured with HMBA and Me₂SO remain in the subsequent G1 for 5–7 h, compared with an average of only 3 h for cells cultured without inducer. Modal cell volume doubles as the cells proceed from G1 to G2. During the inducer-mediated prolonged G1, MELC retain a small cell volume. In cultures of non-synchronous MELC, inducers also increase the G1 fraction, as well as the proportion of small cells. An Me₂SO-resistant MELC variant (DR10), cultured with Me₂SO, shows little prolongation of G1 and little difference in the modal cell volume compared with cells without inducer. However, HMBA, which induces differentiation of DR10 cells, prolongs G1 and increases the proportion of small cells. These studies indicate that early changes in cell volume associated with induction of MELC to differentiate, in large part reflect alterations in the cell cycle. Evidence is presented which suggests that only one round of DNA synthesis in the presence of inducer may be necessary to initiate differentiation.     

Patterns of expression of erythroblast non-globin mRNAs

Three classes of erythroblast non-globin mRNAs have been identified using cDNA recombinants isolated from a mouse foetal liver cDNA library. One erythroid cell-specific 12S mRNA coding for a 16-19,000 dalton protein has been identified using two independent cDNA recombinants (pFC5 and pFA6). The gene encoding this mRNA is unique in the genome and its organisation seems to be the same in both erythroid and non erythroid cell genomic DNA as judged by digestion with restriction enzymes and Southern blotting. Another erythroblast mRNA of size 16S represented by recombinant pFD12 is expressed in brain cells as well as a variety of haemopoietic cell types, but not in adult liver or fibroblasts. Two other erythroblast mRNAs of size 8S and 12S represented by recombinants pD6 and pA4 are expressed in many differentiated cell types with the exception of non-dividing cells such as reticulocytes, peripheral white blood cells, adult liver and brain cells. These mRNAs are, therefore, presumed to be involved ubiquitously in cell proliferation or general cell metabolism.