Induction of delta-aminolevulinic acid dehydratase in mouse Friend virus-transformed erythroleukemia cells during erythroid differentiation

The activity of delta-aminolevulinic acid (ALA) dehydratase, an enzyme involved in heme biosynthesis, has been shown to increase in Friend virus-transformed murine erythroleukemia (MEL) cells during erythroid differentiation. In this study, the nature of the increase in ALA dehydratase activity in MEL cells was examined using a monospecific antibody directed to the enzyme. A sevenfold increase in ALA dehydratase activity was observed after cells had been treated with 1.5% Me2SO for 5 days. Ouchterlony double immunodiffusion analysis showed that lysates from untreated and Me2SO-treated MEL cells formed a single precipitin line with rabbit IgG directed to the normal mouse liver ALA dehydratase. A single arc of identity was also observed with the lysates from normal mouse erythrocytes, spleen, liver, and lysates from both uninduced and induced MEL cells. Rocket immunoelectrophoresis demonstrated that lysates from both uninduced and induced cells formed rockets with the IgG and that the peak height of the rocket was proportional to the ALA dehydratase activity applied. The slope of linear plots of rocket peak heights v ALA dehydratase activity was identical for lysates from uninduced and Me2SO-induced cells. Succinylacetone, a potent inhibitor of ALA dehydratase, was shown to markedly inhibit the activity of the enzyme, but did not interfere with the synthesis of ALA dehydratase induced by Me2SO treatment. Me2SO- induced increases in ALA dehydratase activity and the enzyme protein were both blocked by the simultaneous treatment of cells with 5-bromo- 2'-deoxyuridine (BrdU). BrdU-mediated repression of ALA dehydratase was partially overcome by treating the cells with thymidine. These data demonstrate that increased ALA dehydratase activity in MEL cells undergoing erythroid differentiation after Me2SO treatment is due to de novo synthesis of the same enzyme protein present in uninduced MEL cells as well as in normal erythrocytes. This represents the first direct demonstration of an increase in a heme biosynthetic pathway enzyme protein in erythroid cells undergoing differentiation.     

Dissociation of iron transport and heme biosynthesis from commitment to terminal maturation of murine erythroleukemia cells.

Treatment of murine erythroleukemia (MEL) cells with imidazole in the presence of dimethyl sulfoxide (Me2SO) has been shown to dissociate hemoglobin accumulation from commitment to terminal maturation. To explore the mechanism(s) of this effect, we studied iron transport and heme and hemoglobin synthesis in Me2SO-induced MEL cells that were then exposed to imidazole. Imidazole treatment (i) causes moderate inhibition of 125I-labeled transferrin binding to both control and Me2SO-treated MEL cells; (ii) markedly suppresses Me2SO-induced activation of iron uptake into MEL cells; (iii) markedly decreases the incorporation of iron into ferritin; and (iv) abolishes heme biosynthesis from [2-14C]glycine and hemoglobin accumulation in Me2SO-treated cells. Imidazole treatment does not inhibit other aspects of cellular maturation; cells treated with Me2SO in the presence or absence of imidazole exhibit similar changes in proliferative activity and protein synthesis and, as shown previously, in cell morphology. Inhibition of hemoglobin accumulation in MEL cells is reversible on withdrawal of imidazole but is not altered by exogenous hemin. These data indicate that commitment to terminal maturation is regulated independently from the systems for iron transport and heme biosynthesis during early phases of erythroid cell differentiation.     

Synthesis and turnover of globin mRNA in murine erythroleukemia cells induced with hemin

When murine erythroleukemia (MEL) cells are induced with hemin, they carry out several early functions of the erythroid program. However, they do not become committed to terminal differentiation nor do they become benzidine positive. This is in contrast to MEL cells induced with dimethyl sulfoxide (Me(2)SO) which undergo a more complete program of erythroid differentiation. In order to determine the relationship between commitment and various events in the erythroid program, we compared the induction of MEL cells with hemin and with Me(2)SO. The amount of globin mRNA accumulated in inducing MEL cells and the rate of its synthesis and turnover were quantitated. Although MEL cells induced with hemin accumulated significantly less globin mRNA than did cells induced with Me(2)SO, the rate of synthesis of globin mRNA was the same in fully induced cells, irrespective of inducer. Therefore, there is no evidence that induction with hemin produces an early program that is different or altered from that which is part of Me(2)SO induction. MEL cells induced with Me(2)SO specifically destabilize their globin mRNA after 4 days of induction. This raises the question of whether this destabilization of globin mRNA is an independently programmed late event, as suggested by the time of its occurrence, or, alternatively, whether it might be the inevitable consequence of an early event(s). For instance, destabilization might be linked to the synthesis or translation of globin mRNA. Because MEL cells induced with hemin do not destabilize their globin mRNA, we have concluded that this turnover of globin mRNA is a late event, occurring only in a committed cell population.     

Proteomic analysis of erythroid differentiation induced by hexamethylene bisacetamide in murine erythroleukemia cells

OBJECTIVE: Murine erythroleukemia (MEL) cells are transformed erythroid precursors that are arrested in an immature and proliferating state. These leukemic cells can be grown in cell cultures and induced to terminal erythroid differentiation by a treatment with a specific chemical inducer such as N,N'-hexamethylene bisacetamide. MEL cells then re-enter their original erythroid program and differentiate along the erythroid pathway into non-dividing hemoglobin-rich cells resembling orthochromatophilic normoblasts. To deepen our understanding of the molecular mechanisms underlying and erythroid differentiation and leukemia we monitored changes in protein expression in differentiating MEL cells. METHODS: In our effort to find new candidate proteins involved in the differentiation of MEL cells, we embraced a proteomic approach. Employing two-dimensional (2D) electrophoresis combined with mass spectrometry, we compared protein expression in non-induced MEL cells with MEL cells exposed to N,N'-hexamethylene bisacetamide for 48 h. RESULTS: From 700 proteins spots observed, 31 proteins were differentially expressed. We successfully identified 27 of the differentially expressed molecules by mass spectrometry (MALDI-MS). CONCLUSION: In addition to proteins involved in heme biosynthesis, protein metabolism, stress defense and cytoskeletal organization, we identified 3 proteins engaged in regulation of cellular trafficking and 7 proteins important for regulation of gene expression and cell cycle progression including 3 components of chromatin remodeling complexes. Many of the identified molecules are associated with erythroid differentiation or leukemia for the first time. To our knowledge, this is the first study applying a modern proteomic approach to the direct analysis of erythroid differentiation of leukemic cells.     

Regulation of hemoglobin synthesis and proliferation of differentiating erythroid cells by heme-regulated eIF-2alpha kinase

Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2alpha kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2alpha. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.     

Lengthening of the G1 phase is not strictly correlated with differentiation in Friend erythroleukemia cells.

Friend murine erythroleukemia cells (Friend cells) undergo erythroid differentiation in vitro with an increased probability when cells are cultured in the presence of dimethyl sulfoxide (Me2SO) or other agents. Exponentially growing Friend cells, after dilution into medium containing Me2SO, underwent a transient lengthening of the G1 phase of the cell cycle before they became committed to erythroid differentiation. For nine inducing agents, a positive correlation was found between the percentage of cells that had differentiated and synthesized heme, and the percentage of progenitor cells in which a lengthened G1 phase had previously been observed. This correlation was not found, however, with two other potent inducing agents, hypoxanthine and actinomycin D. Moreover, cells that underwent a lengthened G1 phase did not always terminally differentiate. One such example was a Me2SO-resistant, variant Friend cell line (520a) grown in the presence of Me2SO. These results imply that the prolonged G1 phase, although observed with many inducers, is not a prerequisite for erythroid differentiation with all inducers.     

Human ABC7 transporter: gene structure and mutation causing X-linked sideroblastic anemia with ataxia with disruption of cytosolic iron-sulfur protein maturation

The human protein ABC7 belongs to the adenosine triphosphate-binding cassette transporter superfamily, and its yeast orthologue, Atm1p, plays a central role in the maturation of cytosolic iron-sulfur (Fe/S) cluster-containing proteins. Previously, a missense mutation in the human ABC7 gene was shown to be the defect in members of a family affected with X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). Here, the promoter region and the intron/exon structure of the human ABC7 gene were characterized, and the function of wild-type and mutant ABC7 in cytosolic Fe/S protein maturation was analyzed. The gene contains 16 exons, all with intron/exon boundaries following the AG/GT rule. A single missense mutation was found in exon 10 of the ABC7 gene in 2 affected brothers with XLSA/A. The mutation was a G-to-A transition at nucleotide 1305 of the full-length cDNA, resulting in a charge inversion caused by the substitution of lysine for glutamate at residue 433 C-terminal to the putative sixth transmembrane domain of ABC7. Expression of normal ABC7 almost fully complemented the defect in the maturation of cytosolic Fe/S proteins in a yeast strain in which the ATM1 gene had been deleted (Deltaatm1 cells). Thus, ABC7 is a functional orthologue of Atm1p. In contrast, the expression of mutated ABC7 (E433K) or Atm1p (D398K) proteins in Deltaatm1 cells led to a low efficiency of cytosolic Fe/S protein maturation. These data demonstrate that both the molecular defect in XLSA/A and the impaired maturation of a cytosolic Fe/S protein result from an ABC7 mutation in the reported family.     

A novel system to identify Myb target promoters in friend murine erythroleukemia cells

Friend murine erythroleukemia (MEL) cells provide an early erythroid precursor model that can be induced to terminally differentiate in cell culture and has been used to study erythroid differentiation as well as multistage tumorigenesis. During the chemically induced differentiation of MEL cells, expression of the c-myb protooncogene is downregulated in a biphasic fashion and forced expression of c-myb is able to block the differentiation process, suggesting that c-myb activity may be limiting for differentiation in MEL cells. We have recently produced stable transfectants in the C19 MEL cell line that carry a dominant interfering myb allele (MEnT) under the control of an inducible mouse metallothionein I (MTH) promoter. Upon inducing expression of MEnT, transfected cells enter a differentiation program and begin to produce alpha-globin mRNA, assemble hemoglobin, and stop proliferating. Differential display was used to compare mRNA expression between parental C19 MEL cells induced to differentiate with hexamethylene bisacetamide (HMBA) and stable transfectants induced to differentiate via expression of MEnT to identify potential Myb target promoters. We identified six candidate cDNAs in this fashion and present evidence that two of these represent genes that are dependent on c-Myb activity for maximal expression in MEL cells.     

Erythroid cell differentiation: murine erythroleukemia cell variant with unique pattern of induction by polar compounds.

The murine-virus-infected erythroleukemia cell system provides an opportunity to examine regulatory mechanisms controlling cytodifferentiation. A cloned cell line (DR10c3) resistant to the erythropoiesis-inducing effect of dimethylsulfoxide (Me2SO) was isolated from the Me2SO-sensitive line DS19. DR10c3 is characterized as follows: (1) the uptake of [3H]Me2SO is similar to that in DS19; (2) cell growth with and without Me2SO is similar to that of DS19; (3) resistance is relatively stable; (4) the karyotype of DR10c3 reveals an average loss of five chromosomes per cell, but is otherwise similar to that of DS19; (5) total protein and globin synthesis by cells cultured 4 days with or without Me2SO is similar to these syntheses in DS19 cultured without Me2SO; (6) virtually no globin mRNA is detectable after 3 days in Me2SO, as assayed both by RNA-complementary DNA hybridization and by the heterologous cell-free protein-synthesizing system; (7) other polar compounds, N-methylpyrrolidinone, 1-methyl-2-piperidone, N, N-dimethylacetamide, and N-methylacetamide, induce erythroid differentiation in DR10c3, and the accumulation of alpha- and beta-globin chains is indistinguishable from that in DS19; and (8) the concentration optima for induction of differentiation by all these compounds are identical for DR10c3 and DS19.     

Melatonin inhibits glucocorticoid receptor nuclear translocation in mouse thymocytes

The antiapoptotic effect of melatonin (MEL) has been described in several systems. In particular, MEL inhibits glucocorticoid-mediated apoptosis. Our group previously demonstrated that in the thymus, MEL inhibits the release of Cytochrome C from mitochondria and the dexamethasone-dependent increase of bax mRNA levels. In this study we analyzed the ability of MEL to regulate the activation of the glucocorticoid receptor (GR) in mouse thymocytes. We found that even though the methoxyindole does not affect the ligand binding capacity of the receptor, it impairs the steroid-dependent nuclear translocation of the GR and also prevents transformation by blocking the dissociation of the 90-kDa heat shock protein. Coincubation of the methoxyindole with dexamethasone did not affect the expression of a reporter gene in GR-transfected Cos-7 cells or HC11 and L929 mouse cell lines that express Mel-1a and retinoid-related orphan receptor-alpha (RORalpha) receptors. Therefore, the antagonistic effect of MEL seems to be specific for thymocytes, in a Mel 1a- and RORalpha-independent manner. In summary, the present results suggest a novel mechanism for the antagonistic action of MEL on GR-mediated effects, which involves the inhibition of 90-kDa heat shock protein dissociation and the cytoplasmic retention of the GR.     

Aberrance and modification of alpha-1- and alpha-2-globin gene expression in human and mouse erythroleukemia cells

In contrast to normal human erythroid tissues where the alpha 2:alpha 1-globin mRNA ratio is about 72:28, in human erythroleukemia K562 cells this ratio was found to be quite low, i.e. about 8:92. The ratio was moderately increased by hemin induction and approached almost normal levels after chromosomal transfer from K562 to the mouse erythroleukemia (MEL) cells. We suggest that operationally positive regulatory factors may exist in erythroleukemia cells, modifying the relative alpha 1- and alpha 2-globin gene expression by events following induction and by the adult MEL environment. These factors may act by altering the relative rate of alpha 1- and alpha 2-globin mRNA synthesis.     

Oxidative stress is involved in hydroxyurea-induced erythroid differentiation

Hydroxyurea (HU), an inhibitor of DNA synthesis, can also induce haemoglobinization in certain erythroid cell lines. In this study, we report that intracellular peroxides levels were increased in HU-treated murine erythroleukaemia (MEL) cells and that l-acetyl-N-cysteine (LNAC), a potent reducing reagent, had a significant inhibitory effect on the HU-mediated induction of beta-globin, delta-aminolaevulinate synthase mRNA expression and haemoglobinization of MEL cells. In contrast, the addition of LNAC to dimethyl sulphoxide (DMSO)-treated MEL cells had a much smaller effect on the number of haemoglobinized cells. These findings suggest that oxidative stress is involved in HU-mediated induction of erythroid differentiation and that HU induces MEL cell differentiation by a mechanism different to that involved in DMSO-mediated differentiation. Our findings also suggest that the induction of MEL cell differentiation by HU does not involve RAS-MAP (mitogen-activated protein) kinase signalling.