Induction of murine erythroleukemia cell differentiation by proteinases is inhibited by aromatic poly-amidines

The effects of the tetra benzamidine serine-proteinase inhibitor 1,3-di-(p-amidinophenoxy) -2,2- bis- (p-amidinophenoxymethyl)propane (TAPP-H) and related compounds, including halo-derivatives, were determined on the erythroid differentiation of murine erythroleukemic cells induced by trypsin and kallikrein. These aromatic poly-amidines and their halo derivatives were found to be strong inhibitors of both trypsin and kallikrein mediated induction of commitment of MEL cells to erythroid differentiation, hemoglobin synthesis and accumulation, globin mRNA production. No inhibitory effects were detected by treating proteinase-induced MEL cells with benzamidine. Only slight inhibitory activity was found after treatment of trypsin-induced MEL cells with other antiproteinase compounds widely used in the control of proteinase-dependent functions, including leupeptin, antipain and Bowman-Birk proteinase inhibitor. MEL cells induced to erythroid differentiation by proteinases could be proposed as an experimental system to test the biological activity of proteinase inhibitors.     

Differential effect of hemin-controlled eIF-2 alpha kinases from mouse erythroleukemia cells on protein synthesis

Cultured mouse erythroleukemia (MEL) cells can be induced to erythroid differentiation by a variety of chemical agents. This differentiation process is marked by the onset of globin mRNA and hemoglobin synthesis. In rabbit reticulocytes, globin synthesis is regulated by a hemin-controlled translational inhibitor (HCI) which acts via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2). From both uninduced and induced MEL cells, hemin-controlled eIF-2 alpha kinases have been partially purified. They resemble HCI with respect to their chromatographic behaviour and their sensitivity towards physiological concentrations of hemin (5-10 microM). Further purification on phosphocellulose, however, reveals that the eIF-2 alpha kinase from uninduced MEL cells is chromatographically distinct from HCI, whilst the eIF-2 alpha kinase activity from induced MEL cells represents a mixture of the former and the HCI-type eIF-2 alpha kinase. The latter inhibits protein synthesis in a fractionated system from rabbit reticulocytes which is free of, but sensitive to, HCI, whereas the eIF-2 alpha kinase from uninduced MEL cells does not show any inhibitory activity. This observation is supported by the finding that induced MEL cells respond in vivo to iron depletion with a shut-off of protein synthesis (as do rabbit reticulocytes), whilst uninduced MEL cells do not.     

Two different pathways link G-protein-coupled receptors with tyrosine kinases for the modulation of growth and survival in human hematopoietic progenitor cells

The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells. CXCL12 promoted growth in multipotent cells by activating the RhoA-Rho kinase pathway. Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins. Rho activation required a G(i)-mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively. By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCbeta activity. The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCbeta. Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by G-protein-coupled receptor agonists.     

Presence of haemin-controlled eIF-2 alpha kinases in both undifferentiated and differentiating mouse erythroleukaemia cells.

In rabbit reticulocytes, globin synthesis is regulated by a haemin-controlled translational inhibitor (HCI) which acts by phosphorylating the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). With purified eIF-2 as substrate, haemin-controlled eIF-2 alpha kinases could be partially purified from cultured mouse erythroleukaemia cells (MEL cells), which can be induced in vivo to erythroid differentiation. The eIF-2 alpha kinases from both uninduced and induced MEL cells are clearly distinct from the double-stranded-RNA-activated eIF-2 alpha kinase described for many mammalian cell types. A rough quantitative estimation indicates that, on a per-cell basis, induced MEL cells contain the same amount of haemin-controlled eIF-2 alpha kinase activity as rabbit reticulocytes, whereas uninduced MEL cells contain about one-tenth as much. As to their chromatographic behavior on CM-Sephadex and DEAE-cellulose and their sensitivity towards physiological concentrations of haemin (5-10 microM), the eIF-2 alpha kinases from MEL cells are indistinguishable from HCI. They differ from HCI with respect to their response towards activating stimuli such as prolonged incubation at 37 degrees C or brief exposure to the thiol reagent N-ethylmaleimide.     

Cytoplasmic factors involved in erythroid differentiation in mouse erythroleukemia (MEL) cells

In order to identify and characterize intracellular factors involved in in vitro differentiation of mouse erythroleukemia (MEL) cells, the differentiation process was analyzed by cell and cytoplast fusion. The results suggested that the process is not a single cascade of molecular chain reactions, but a synergistic result of two different inducible intracellular reactions. One reaction is induced following damage to DNA (inhibition of DNA replication) and is not specific to MEL cells. The other reaction, which is specific to MEL cells, is fully induced by typical erythroid inducing agents such as dimethylsulfoxide or hexamethylenebisacetamide even at concentrations suboptimal for the erythroid induction. Based upon these data, we searched for the putative trans-acting differentiation-inducing factors and detected two proteinaceous factors (DIF-I and DIF-II) in the cytosol fraction which apparently correspond to these reactions. When, partially purified, either one of these factors was introduced into undifferentiated MEL cells, it triggered erythroid differentiation, provided that the recipient cells had been potentiated by the induction of the other reaction. In this article, we summarize the basic characteristics of these cytoplasmic factors involved in erythroid differentiation in MEL cells.     

Inhibitors for protein-tyrosine kinases, ST638 and genistein, induce differentiation of mouse erythroleukemia cells in a synergistic Manner

In order to investigate the biochemical nature of intracellular cascades leading to cellular differentiation in vitro, we examined the effect of inhibitors of protein phosphorylation on terminal erythroid differentiation of mouse erythroleukemia (MEL) cells. We have found that specific inhibitors of protein phosphorylation at tyrosine residues, ST638 and genistein, effectively induce differentiation in a synergistic manner with an agent which blocks DNA replication such as mitomycin C (MMC). Based upon these findings, the possible involvement of protein phosphorylation (and dephosphorylation) at tyrosine residues in differentiation is discussed.     

Mannosylerythritol lipid induces granulocytic differentiation and inhibits the tyrosine phosphorylation of human myelogenous leukemia cell line K562

Mannosylerythritol lipid (MEL), which induced granulocytic differentiation of human promyelocytic leukemia cell line HL60, also induced differentiation of human myelogenous leukemia cell line K562. MEL inhibited insulin-dependent cell proliferation and induced leukocyte esterase activity of K562 cells. MEL markedly increased the differentiation-associated characteristics in granulocytes, such as nitroblue tetrazolium (NBT) reducing ability, expression of Fc receptors, and phagocytic activity of K562 cells. The tyrosine phosphorylation in K562 cells inhibited by MEL. These results suggest that MEL directly down-regulates the tyrosine kinase activities in K562 cells to inhibit the cell proliferation and to induce the differentiation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tyrphostin-induced differentiation of mouse erythroleukemia cells

Inhibitors of protein-tyrosine kinases (TPKs) from the tyrphostins family induce terminal erythroid differentiation of mouse erythroleukemia (MEL) cells. The most potent tyrphostin was found to be AG-568 which was therefore investigated in more detail. Just prior to differentiation the inhibition of tyrosine phosphorylation of a pp⁹⁷ protein band was noted. We also found that AG-568 treatment induces the appearance of a putative differentiation factor which could induce tyrphostin-independent differentiation in untreated cells. Our study suggests that the inhibition of tyrosine phosphorylation by AG-568 leads to the production of differentiating factor(s) which induce the MEL cells to differentiate.     

Involvement of the p38 mitogen-activated protein kinase pathway in tissue inhibitor of metalloproteinases-1-induced erythroid differentiation

We examined the role of the mitogen-activated protein (MAP) kinase pathway in tissue inhibitor of metalloproteinases-1 (TIMP-1)-mediated cellular effects in a human erythroleukemic cell line UT-7. We show that TIMP-1 induced both UT-7 cell erythroid differentiation and proliferation and tyrosine phosphorylation of many intracellular proteins. Using a panel of phosphospecific antibodies, we also demonstrate that phosphorylation of the p38 and c-Jun N-terminal kinases is increased by TIMP-1 whereas phosphorylation of extracellular signal-regulated kinase 1/2 is not induced. Moreover, inhibition of the p38 activity by SB203580 significantly reduces erythroid differentiation induced by TIMP-1, suggesting that the p38 MAP kinase pathway is involved in TIMP-1-induced erythroid differentiation.     

ERK signaling pathway is differentially involved in erythroid differentiation of K562 cells depending on time and the inducing agent

K562 cells can be induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, butyrate, cisplatin and ara-C. Differential signaling through MAP kinases has been suggested to be involved in this differentiation process. We have investigated the involvement of ERK activation/inhibition in hemin-, butyrate-, cisplatin- and ara-C-induced erythroid differentiation using the K562 cell line. ERK activity decreased for 2-4h after administration of either inducing agent. ERK was then activated by hemin and cisplatin, while ERK phosphorylation remained decreased during incubation with butyrate and ara-C. There was no activation of JNK or p38. The MEK-1 inhibitors UO126 or PD98059 induced erythroid differentiation in K562 cells and acted additively with butyrate. Inhibition of MEK-1 reduced the hemoglobin accumulation by hemin and cisplatin; erythroid differentiation by ara-C was unchanged. The results suggest that inhibition of signaling through ERK in K562 cells may be needed to enter the erythroid differentiation process, while after initiation both activation and inhibition of signaling through ERK enhance erythroid differentiation, which, however, is dependent on the inducing compound.     

Study of the PTEN gene expression and FAK phosphorylation in human hepatocarcinoma tissues and cell lines

K562 cells contain a Bcr-Abl chimeric gene and differentiate into various lineages in response to different inducers. We studied the role of the mitogen-activated protein kinase (MAPK) kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK) pathway during the erythroid differentiation of K562 cells induced by tyrosine kinase inhibitors (herbimycin A or STI571), using genetically modified cells (constitutively MEK1-activated K562: K562/MEK1, and inducible ERK-inactivated K562: K562/CL100). Basal expression of glycophorin A was markedly reduced in K562/MEK1 cells compared with that in parental cells, while it was augmented in K562/CL100 cells. Herbimycin A and STI571 differentiated K562 cells accompanying with the transient down-regulated ERK. Moreover, the erythroid differentiation was markedly suppressed in K562/MEK1 cells, and early down-regulation of ERK activity was not observed in these cells. In contrast, the induction of ERK-specific phosphatase in K562/CL100 cells potentiated erythroid differentiation. Once the phosphatase was induced, the initial ERK activity became repressed and its early down-regulation by the inhibition of Bcr-Abl was marked and prolonged. These results demonstrate that the erythroid differentiation of K562 cells induced by herbimycin A or STI571 requires the down-regulation of MEK1/ ERK pathway.     

Induction of in vitro differentiation of mouse embryonal carcinoma (F9) and erythroleukemia (MEL) cells by herbimycin A, an inhibitor of protein phosphorylation

Herbimycin A is one of the benzenoid ansamycin antibiotics isolated from a culture of Streptomyces species (Omura, S., A. Nakagawa, and N. Sadakane. 1979. Tetrahedron Lett. 1979: 4323-4326). Recent studies have shown that the antibiotic not only inhibits the phosphorylation of p60src in Rous sarcoma virus- (RSV) infected cells, but also reverses the cellular phenotypes acquired by transfection with tyrosine kinase oncogenes (Uehara, Y., M. Hori, T. Takeuchi, and H. Umezawa. 1985. Jpn. J. Cancer Res. 76:672-675; Uehara, Y., M. Hori, T. Takeuchi, and H. Umezawa. 1986. Mol. Cell. Biol. 6: 2198-2206; Uehara, Y., Y. Murakami, S. Mizuno, and S. Kawai. 1988. Virology. 164: 294-298). These studies and other evidence indicate that the antibiotic inhibits a reaction(s) closely associated with the function of cellular tyrosine kinases. We have found that herbimycin A is an effective inducing agent capable of triggering differentiation in two typical mouse in vitro differentiation systems, which have been considered to be quite different in their mechanism of induction: endoderm differentiation of embryonal carcinoma (F9) cells and terminal erythroid differentiation of erythroleukemia (MEL) cells. The results suggest that there is a common step in the intracellular differentiation cascade which is, directly or indirectly, associated with phosphorylation at specific (tyrosine) residues of cellular proteins. The significance of this finding with respect to the molecular mechanism of in vitro differentiation is discussed.     

Activin A/erythroid differentiation factor induces thromboxane A2 synthetic activity in murine erythroleukemia cells

Activin A, a protein homologous to transforming growth factor beta, was shown to induce hemoglobin synthesis in murine erythroleukemia (MEL) cells and was also termed erythroid differentiation factor (EDF) (Eto, Y., Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y., and Shibai, H. (1987) Biochem. Biophys. Res. Commun. 142, 1095-1103). We found that activin A/EDF also induced thromboxane (TX) A2 synthetic activity in these cells. Synthesis of TXA2 from arachidonic acid is catalyzed by cyclooxygenase and TX synthase. Activin A/EDF induced the latter TX synthase activity, whereas the cyclooxygenase activity was constitutively expressed. The induction of this enzyme activity was inhibited by cycloheximide, suggesting that activin A/EDF induced de novo protein synthesis of TX synthase. Furthermore, we studied the relationship between the induction of TXA2 synthetic activity and erythroid differentiation in MEL cells, since the former is not an erythroid phenotype. We found 1) that the two responses to activin A/EDF were distinctly affected by the initial cell density; 2) that the dose-response curves for activin A/EDF were similar (ED50 = approximately 100 pM), whereas the time course of induction of TXA2 synthetic activity was much faster; and 3) that other erythroid differentiation inducers of MEL cells, namely dimethyl sulfoxide and hexamethylene bisacetamide, had little or no effect on TXA2 synthesis. These results indicate that activin A/EDF induces TXA2 synthetic activity independently of erythroid differentiation.     

Alteration of phosphotyrosine-containing proteins at the early stage of erythroid differentiation of mouse erythroleukemia (MEL) cells.

Following treatment of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide and other typical erythroid inducing agents, the profile of cellular phosphotyrosine-containing proteins was drastically altered. We found that the level of almost all of the phosphotyrosine-containing proteins was either decreased or disappeared at a very early stage of differentiation. Addition of sodium orthovanadate (Na3VO4), a specific inhibitor of phosphotyrosine phosphatases, prevented the alteration as well as erythroid differentiation. Mutant MEL cells, which are resistant to differentiation by dimethyl sulfoxide, were apparently insensitive to the alteration. These results indicate that dephosphorylation of phosphotyrosine residues in cellular proteins is coupled with a reaction(s) which is responsible for triggering differentiation of MEL cells.     

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Tyrosine kinase expression profiles of chicken erythro-progenitor cells and oncogene-transformed erythroblasts

Tyrosine kinases are implicated in the growth and differentiation of erythroid cells. Aberrant expression and structural alterations of certain tyrosine kinases, such as erbB and sea, are known to trigger erythroleukemia development. To facilitate our understanding of the signal transduction pathways involved in erythroid differentiation and leukemic transformation, we have applied a recently developed tyrosine kinase profile technique to identify the tyrosine kinases and some novel serine/threonine kinases expressed in normal chicken erythroid progenitor cells that respond to TGF (TGF-EB), and erythroblasts transformed by viruses encoding v-erbB (v-erbB-EB) and v-sea (v-sea-EB). Our results reveal that the non-receptor tyrosine kinases, Abl, Fyn, Lyn, Btk and Csk, are expressed in all three cell types. The expression level of Btk, a tyrosine kinase implicated in Bruton's syndrome, is exceptionally high in the erythroblastoid cell line 6C2, transformed by the v-erbB carrying avian erythroblastosis virus, AEV-ES4. We have also uncovered a new STE-20-related serine/threonine kinase, KFC, which is abundantly expressed in both the TGF-stimulated erythroid progenitor cells and v-sea-transformed erythroblasts. Based on sequence homology of the kinase domain, KFC appears to be the first member of a new subfamily of STE-20-like kinases.     

Murine erythroleukemia cells possess an active ubiquitin- and ATP-dependent proteolytic pathway

The ubiquitin (Ub)-dependent proteolytic pathway may function in selective elimination of cellular proteins during erythroid differentiation. Murine erythroleukemia (MEL) cells, which can be induced to differentiate to reticulocytes in culture, may provide a convenient system for studying the role of Ub-dependent proteolysis in erythroid differentiation. The following observations indicate that MEL cells possess an active Ub-dependent proteolytic pathway. (i) Addition of purified Ub to MEL cell fraction II (Ub-depleted lysate) stimulated ATP-dependent degradation of radioiodinated proteins. (ii) Covalent conjugation of carboxyl termini of Ub molecules to substrate protein amino groups is a necessary step in Ub-dependent degradation. Des-glygly-Ub (Ub lacking its carboxyl-terminal glygly moiety) did not stimulate protein degradation in MEL cell fraction II. (iii) The Ub-dependent component of protein degradation in MEL cell fraction II was specifically inhibited by amino acid derivatives that are inhibitors of Ub-protein ligase. (iv) MEL cell fraction II contained apparent homologs of all of the rabbit reticulocyte Ub carrier proteins (E2's) except E2(20K) and E2(230K). Ub-dependent proteolysis was seen only in MEL cell lysates prepared in the presence of leupeptin; an enzyme of the proteolytic pathway was inactivated if leupeptin was omitted.