Induced MEL Cells Research Articles

BILIVERDIN REDUCTASE B/ FLAVIN REDUCTASE (BLVRB/FLR) EXHIBITS HEME-REGULATED, NADPH-DEPENDENT REDUCTASE ACTIVITY AND CONFERS CYTOPROTECTION IN DEVELOPING ERYTHROID CELLS

Biliverdin reductase B (BLVRB), also known as flavin reductase (FLR), is especially expressed in cells having high levels of heme synthesis (e.g. developing erythroid cells, fetal and adult liver). BLVRB exhibits NADPH-dependent reductase activity toward non-alpha isomers of biliverdin, flavins and redox cycling agents, and also has pharmacological importance in the treatment of methemoglobinemia. Nonetheless, the physiological function of BLVRB is obscure. In this work, the functional role of BLVRB in developing erythroid cells in relation to its heme-associated properties is elucidated. Recombinant mouse BLVRB binds ferric heme with a submicromolar affinity in association with inhibition of the NADPH-dependent reductase activity while displaying heme peroxidase activity. The interaction of BLVRB with heme in developing erythroid cells (MEL) was demonstrated by proteomic analysis of heme peroxidase-associated proteins. Upon induction of erythroid differentiation of MEL cells, the BLVRB protein expression is markedly up-regulated. Knockdown of BLVRB in the induced MEL cells aggravates intracellular oxidation and cytotoxicity mediated by thiol depleting agent and heme synthesis inhibitor, suggesting that BLVRB may maintain the cell viability under oxidative stress or heme deficient conditions. Our study suggests that BLVRB/FLR, which is usually regarded as a biliverdin reducing enzyme in the heme degradation pathway, is an erythroid active, heme-regulated NADPH-dependent reductase. It confers cytoprotection in developing erythroid cells under oxidative stress conditions in connection with cellular heme levels. This work was supported by General Research Fund (17160916) and Health and Medical Research Fund (04151846).

The Role of HDAC1 Acetylation in Hematopoiesis and GATA-1 Function.

Abstract 1459Poster Board I-482Histone deacetylases (HDACs) play important roles in transcriptional regulation in eukaryotic cells. Several lines of evidence also link HDACs to cancer. HDAC inhibitors have shown preclinical promise when combined with other therapeutic agents in the treatments of leukemia and solid tumors. HDAC1 often presents in repressor complexes, such as Sin3, NuRD and CoREST complexes. We previously found that HDAC1 can be acetylated in vivo by acetyltransferase p300 and that acetylated HDAC1 completely loses its deacetylase activity (Qiu et al, 2006). In this study, we investigate the role of HDAC1 during erythroid differentiation. HDAC1 deacetylase activity gradually reduced upon the induction of differentiation. However, the HDAC1 protein level remains unchanged, suggesting that HDAC1 deacetylase activity, but not its protein level, is regulated. To further test the role of HDAC1 in erythroid differentiation, stable cell lines that overexpress HDAC1 and mutants mimicking acetylated or unacetylated HDAC1 were established. We also generated stable HDAC1 and HDAC2 knock down cell lines. The results showed that HDAC1 deacetylase activity is required to promote erythroid proliferation and to inhibit differentiation. Next, we studied whether HDAC1 modulates erythroid differentiation through regulating the activity of key erythroid transcription factor GATA-1. GATA-1 interacts with many other key hematopoietic transcription factors, as well as chromatin remodeling/modifying coactivators and corepressors. It is suggested that GATA-1 mediates gene activation through its association with coactivator complexes. However, recent studies indicated that GATA-1 associates with HDAC1/2 containing corepressor complexes (NuRD) throughout differentiation of erythroid cells. We investigated the deacetylase activity of the GATA-1 associated NuRD complex during erythroid differentiation. We found that the deacetylase activity of the complex decreased and further diminished at day 5 of DMSO induced MEL cells. Accordingly, the acetylated form of HDAC1 within the GATA-1 complex increased during erythroid differentiation. We further demonstrated the role of HDAC1 in GATA-1 mediated gene transcription in transient transfection assays. These studies indicate that HDAC1 plays an important role in regulating GATA-1 activity and the deacetylase activity of the GATA-1 associated NuRD complex is also regulated. This complex may play different roles in undifferentiated and differentiated erythroid cells. Thus, our results suggest a novel but rather general regulatory mechanism of histone deacetylase containing protein complexes. DisclosuresNo relevant conflicts of interest to declare.

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the alpha- and beta-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both alpha- and beta-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

Coactivation of human α1- and α2-globin genes in single induced MEL cells containing one human α-globin locus

We developed a reverse-transcription polymerase chain reaction assay, performed on single isolated cells, to demonstrate the coexpression of human α1- and α2-globin mRNA in induced mouse erythroleukemic cells containing a single human α-globin locus. These results indicate that both α1 and α2 genes are activated from the same α-globin gene locus implying that HS-40-dependent transcriptional activation is mediated, either by a simultaneous interaction of HS-40 with both α1 and α2-globin gene promoters, or by a dynamic process characterized by alternative, but short-lived, interactions with each α-globin gene promoter. © 1997 Elsevier Science B.V. All rights reserved.

Two periods of sensitivity to mutagens in induced Mel cells with different outcomes

Mouse erythroleukemia (Mel) cells are particularly sensitive to mutagenic agents between 18 and 24 h from the start of induction (Foresti, M.L. Gaudio, G. Geraci and P. Manduca (1986) Inhibition of dimethyl sulfoxide induced erythropoietic differentiation of murine erythroleukemia cells in culture, Cancer Res., 46, 6260–6263). We show here the occurrence of another period of sensitivity during the initial 5 h after the addition of the inducer dimethyl sulfoxide (DMSO) to the culture medium. The sensitivity to the mutagenic action of a sublethal 3-s pulse of UV light (13.5 J/m 2 ) was monitored on the progeny of the irradiated cells at day 5 after the start of induction. The effects were analysed on functions strictly linked to the final expression of the differentiated phenotype: hemoglobin concentration, percent cells producing hemoglobin (%B+), activity of δ-amino levulinic acid dehydrase (ALA-DH) and presence of globins. Each function appeared differently and selectively affected in the progeny of the cells depending on the exact time of irradiation during the period of sensitivity. Specifically, cells irradiated at hour 3 after induction show both hemoglobin concentration and ALA-DH activity values increased by a factor 3 over controls. Cells irradiated at hour 5 show an almost complete halt in cell induction and the other tested functions show minimal values. Cells are nearly insensitive to irradiation at later times, until hour 20, after which a second period of sensitivity with peak value at hour 22 occurs at which time hemoglobin concentration in the progeny of irradiated cells is increased by a factor 3 over controls, ALA-DH activity is increased by a factor 15 while percent B+ value is at its minimum. The differential effects of UV irradiation on Mel cell functions in the first and in the second period of sensitivity to mutagens confirm the hypothesis that the consequences of a mutational event are strictly dependent on the functional state of the cell. The 1–5 h period of sensitivity in which Mel cells fix the effects of the mutagen in their genome corresponds to increased thymidine incorporation not correlated with cell duplication.

Properties of the mouse α‐globin HS‐26: Relationship to HS‐40, the major enhancer of human α‐globin gene expression

HS-26, the mouse homologue of HS-40, is the major regulatory element of the mouse α-globin gene locus. Like HS-40, HS-26 is located within an intron of a house-keeping gene; comparison of the nucleotide sequences of HS-26 and HS-40 reveals conservation of the sequences and positions of several DNA binding motifs in the 5′ regions of both elements (3 GATA, 2 NFE-2, and 1 CACCC sites) and the absence in HS-26 of three CACCC sites and one GATA site that are present in the 3′ region of HS-40, suggesting that the two elements might not be identical. We report here that when HS-26 is linked to a 1.5 kb Psti human α-globin gene fragment, it has a weak enhancer activity in induced MEL cells and in transgenic embryos, and it does not have any detectable activity in adult transgenic mice. This suggests that HS-26 does not have Locus Control Region (LCR) activity but can act as an enhancer during the embryonic life when integrated at a permissive locus. To further test the importance of HS-26 at its natural locus, we have generated embryonic stem cells and chimeric animals in which 350 bp containing HS-26 have been replaced by a neomycin resistance gene by homologous recombination. The sizes of the chimeras' red cells were then estimated by measuring forward scattering on a FacsScan apparatus in hypotonic conditions. This revealed that a fraction of the chimeric animals' red cells were smaller than normal mouse red cells and were very similar to cells from mice heterozygous for α-thalassemia. Density gradient analysis also suggested the presence of thalassemic cells. These results indicated that despite its lack of LCR activity, HS-26 is important for the regulation of the mouse α-globin gene locus. Am. J. Hematol. 54:30–39, 1997 © 1997 Wiley-Liss, Inc.

Antisense RNA and p53 regulation in induced murine cell differentiation.

p53 expression is strongly modulated during the process of induced differentiation, at the same time as both cell cycle and genetic expression become modulated, giving rise to a commitment to terminal differentiation. We took advantage of two murine cell lines inducible for differentiation, an erythroleukemia and a melanoma cell line, to outline common features of the regulation of p53 expression during the differentiation process. We found that p53 mRNA decreased early after induced differentiation and that regulation was controlled at a posttranscriptional level. Our data showed that this regulation affects p53 pre-mRNA maturation. Because, in both systems used, actinomycin D treatment abolished the inducer-mediated decrease of p53 mRNA, we looked for induced RNAs potentially involved in this process. Using different parts of the p53 gene and flanking regions as probes, we identified three RNA species whose expression is modulated during induced differentiation. A first species is made of high molecular weight RNAs that accumulate in the nuclear compartment and seem to represent antisense transcripts of the p53 gene. A second species, 1.3-kb long, was found to accumulate in the nucleus of induced MEL cells and was homologous to a restricted part of the first intron of the p53 gene due to the presence of a B1 repetitive element in an antisense orientation with respect to the p53 pre-messenger RNA. Finally, a family of B2-containing RNAs was observed in both cytoplasmic and nuclear compartments. The variation in the amounts of sense and antisense RNAs, respectively, suggested an interesting speculative model for the maturation of B2-containing pre-messenger RNAs.

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.

Affinity Isolation of Transcriptionally Active Murine Erythroleukemia Cell DNA Using a Cleavable Biotinylated Nucleotide Analog

We have developed an affinity technique to obtain active gene domains from murine erythroleukemia cell nuclei, based on the differential sensitivity of potentially active and inactive chromatin to DNase I. Nuclei isolated from potentially active noninduced cells and transcriptionally active induced MEL cells were treated with DNase I at concentrations which did not digest the beta-globin gene, followed by repair using a typical nick translation reaction during which a cleavable biotinylated nucleotide analog, 5-[N-biotinamido)hexanoamido-ethyl-1,3-dithiopropionyl -3-aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), was inserted into DNA sequences. Following purification and digestion with EcoRI restriction endonuclease, biotinylated sequences were affinity isolated by sequential binding to streptavidin and biotincellulose. The streptavidin/biotin-cellulose complex bound up to 80% of the nick-translated DNA, which comprised a small percentage of the total nuclear DNA. Cleavage of the disulfide bond in the linker arm of the biotinylated nucleotide resulted in elution of virtually all of the affinity isolated sequences. Hybridization analysis of this fraction of DNA revealed up to a 16-fold enrichment for the active beta-globin gene, as compared with DNA which did not bind to the biotincellulose. Conversely, the inactive alpha-fetoprotein gene was barely detectable in affinity isolated DNA from noninduced cells and was 2-fold depleted in samples from induced cells.

Structure of a mouse erythroid 5-aminolevulinate synthase gene and mapping of erythroid-specific DNAse I hypersensitive sites.

The enzyme 5-aminolevulinate synthase (ALA-S) catalyzes the first step in heme biosynthesis. In this study, the mouse erythroid gene has been cloned and analyzed in order to investigate the regulation of ALA-S expression during erythroid differentiation. The gene spans approximately kbp and consists of 11 exons and 10 introns. The first exon is 37 bp, non-coding, and followed by a 6kb intron. The mRNA capsite was mapped by primer extension and defines a promoter that contains no apparent TATA element. S1 nuclease analysis detects the presence at low levels of a 45 bp-deleted form of the ALA-S mRNA created by the use of an alternative splice site at the intron 2/exon 3 junction. Five DNAse I hypersensitive sites were detected in chromatin from uninduced and induced MEL cells. One site is at the promoter; the others are in the body of the gene. No significant differences were observed in the patterns or intensity of the hypersensitive sites in the uninduced and induced MEL cells, however, no sites in ALA-S were observed in NIH 3T3 cells or in deproteinized DNA. Thus, these sites are specific for erythroid chromatin but appear to be established at an earlier stage of differentiation than represented by the uninduced MEL cell.

A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced murine erythroleukemia cells.

We report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and polyadenylation signals were included to produce a mature transcript coding for t-PA, whose activity can be detected in single cells by a fibrin-agarose plaque assay. Stable murine L-cell transfectants of the gamma.t-PA and beta.t-PA hybrid genes were fused to various cell lines to show that t-PA expression is increased specifically by erythroid MEL, HEL, and K562 cell fusion. The analogous H-2Kb.t-PA construct was not inducible under the same conditions. Interestingly, uninduced MEL cells increased beta.t-PA expression to the same extent as induced MEL cells. Chemiosmotic permeabilization of the beta-globin tester cell line and exposure to nuclear extracts were used to assay for trans-acting factors capable of stimulating beta.t-PA expression. Such factors were shown to be present in the nuclei of uninduced MEL cells.

A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced murine erythroleukemia cells.

We report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and polyadenylation signals were included to produce a mature transcript coding for t-PA, whose activity can be detected in single cells by a fibrin-agarose plaque assay. Stable murine L-cell transfectants of the gamma.t-PA and beta.t-PA hybrid genes were fused to various cell lines to show that t-PA expression is increased specifically by erythroid MEL, HEL, and K562 cell fusion. The analogous H-2Kb.t-PA construct was not inducible under the same conditions. Interestingly, uninduced MEL cells increased beta.t-PA expression to the same extent as induced MEL cells. Chemiosmotic permeabilization of the beta-globin tester cell line and exposure to nuclear extracts were used to assay for trans-acting factors capable of stimulating beta.t-PA expression. Such factors were shown to be present in the nuclei of uninduced MEL cells.

A (re)initiation‐dependent cell‐free protein‐synthesis system from mouse erythroleukemia cells

Cultured mouse erythroleukemia cells (MEL cells) can be induced in vivo to erythroid differentiation which is marked by the onset of globin mRNA and haemoglobin synthesis. When these cells are briefly exposed to hypertonic growth medium prior to lysis, the resulting post-mitochondrial supernatants show a high in vitro protein-synthesis activity. Amino acid incorporation is linear up to 60 min; more than 80% of this is due to (re)initiation, as shown by the inhibition with edeine. Extracts from induced cells reach only a third of overall incorporation as compared to extracts from uninduced cells. This reduction of the protein-synthesizing capacity is also observed in vivo. Polyacrylamide gel electrophoresis shows that extracts from uninduced cells faithfully translate their endogenous mRNA, whereas in extracts from induced cells, non-globin protein synthesis is reduced and globin is preferentially synthesized. Haemin (40 microM) as well as purified eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes enhance amino acid incorporation in both kinds of extracts, which suggests that both uninduced and induced MEL cells contain a haemin-controlled eIF-2 alpha kinase. This system should be useful for studying the mechanisms controlling protein synthesis in a nucleated differentiating cell.

Activated murine α-globin gene is not preferentially associated with the nuclear matrix

1. 1. The association of the murine α-globin gene with the nuclear matrix was studied in three different states of the gene: inactive (EAT cells), potentially active (MEL cells) and active (induced MEL cells). 2. 2. When “native” nuclei were digested with DNase I it was found that the nuclear matrix was not enriched in α-globin DNA sequences in all three different types of cells. 3. 3. A nuclease-hypersensitive site in the 5'-flanking region of the α-globin gene was detected in the induced MEL-cells.

Presence of nucleosomal repeat in the transcribed α globin gene of induced murine erythroleukemia cells

The sensitivity of the mouse α-globin gene to micrococcal nuclease and its nucleosomal repeat were studied in three different functional states of the gene: inactive in EAT cells, potentially active in uninduced MEL cells and active in induced MEL cells. The results show that: 1. The nuclease sensitivity of the gene differs in the three different functional states. 2. Both the coding and the 5′-flanking regions of the induced actively transcribed gene show a typical nucleosomal repeat pattern. 3. Hypersensitive sites for micrococcal nuclease and for an endogenous nuclease appear upstream of the gene after induction of differentiation.

Appearance of blebs on the surface of differentiating friend erythroleukemia cells

The scanning electron microscope has been employed to examine the surfaces of Friend murine erythroleukemia cells (MEL cells) induced to differentiate in culture with dimethylsulfoxide (DMSO). Whereas immature, undifferentiated cells appear to be relatively smooth with a uniform density of small microvilli, fully differentiated cells are covered with large blebs or lumps having diameters in the range of 0.5–2.0 μm. Greater than 85% of induced MEL cells have ‘bleby’ surfaces after four days of growth in the presence of DMSO when significant hemoglobin synthesis has also taken place. In contrast, only 10% of uninduced MEL cells exhibit surface blebs and hemoglobin synthesis is less than 10% of that observed in induced cells. A control, non-erythroleukemia cell line did not express cell surface blebs in the presence or absence of DMSO. Analysis of three variant MEL cell clones that fail to synthesize hemoglobin in response to DMSO showed that two clones express surface blebs while one does not. This indicates that appearance of cell surface blebs is not functionally or causally linked to hemoglobin synthesis in induced MEL cells.