Induced Friend Cells Research Articles

Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization

Friedreich ataxia (FA) is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (beta-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2 messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and beta-globin mRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis.

Ferrochelatase, glutathione peroxidase and transferrin receptor mRNA synthesis and levels in mouse erythroleukemia cells.

Mouse erythroleukemia Friend cells induced to undergo erythroid differentiation by treatment with hexamethylenebisacetamide (HMBA) were shown to increase cytoplasmic ferrochelatase mRNA, transferrin receptor (TfR) mRNA and glutathione peroxidase (GSHPx) mRNA levels. Inhibition of heme synthesis at the level of 5-aminolevulinic acid synthase by isonicotinic acid hydrazide (INH) and D,L-penicillamine (PA) or at the level of 5-aminolevulinic acid dehydratase by succinylacetone (SA) decreased the expression of ferrochelatase mRNA and TfR mRNA. In contrast with these mRNAs, the synthesis and the levels of glutathione peroxidase mRNA increased by the addition of these inhibitors of heme synthesis. The amount of iron in the intracellular low molecular mass iron pool detected in the post-mitochondrial supernatant of Friend cells treated with heme synthesis inhibitors was increased. On the other hand, iron levels in this pool declined with the preincubation of Friend cells with iron chelator pyridoxal isonicotinoylhydrazone (PIH). Further treatment with PIH or desferrioxamine (Desferal) increased the synthesis of TfR mRNA in induced Friend cells. The synthesis of ferrochelatase mRNA declined by the same treatment. The opposite was observed when the iron level in the low molecular mass intracellular nonheme iron pool was elevated by treatment with either diferric transferrin (Fe-Tf) or ferric pyridoxal isonicotinoylhydrazone (Fe-PIH). Exogenously supplied hemin stimulated the synthesis of ferrochelatase mRNA in uninduced Friend cells, while the synthesis of this mRNA in Friend cells taken on the fifth day after induction was inhibited by the addition of hemin.

Transcriptional regulation of ferritin H and L subunits in adult erythroid and liver cells from the mouse: Unambiguous identification of mouse ferritin subunits and in vitro formation of the ferritin shells

Abstract Ferritin H and L subunits present cell-specific features of structure, function, and transcriptional regulation. Mouse Friend erythroleukemia cells offer an interesting model to analyze the erythroid-specific expression of ferritin genes for comparison with the liver, an iron-storing tissue. cDNA clones for mouse ferritin H and L subunits have been isolated and sequenced. The two subunits have very similar calculated masses, 20.9 and 20.6 kDa for H and L, respectively. Electrophoretic analysis of the subunits encoded by the cDNA 1) allows unambiguous identification of mouse ferritin subunits; 2) clearly shows that mouse H and L chains can make heteropolymers in vitro; and 3) demonstrates that, at least in vitro, free subunits can coexist with subunits polymerized into complete shells. The mouse ferritin gene family displays a variable degree of complexity, ranging from three homologous sequences for the H genes to 10-14 homologous loci for the L genes. Transcription of ferritin genes exhibits tissue-specific difference. Nuclear transcriptional run-off experiments show that the L gene is more actively transcribed in the liver than in Friend erythroleukemia cells at different stages of maturation. The accumulation of the H subunit mRNA which results from dimethyl sulfoxide induction of Friend cells is the consequence of an increase in the transcription rate of the H gene. However, the H gene mRNA is transcribed at a similar rate in the liver and in induced Friend cells although 5-fold more mRNA accumulates in these cells. Therefore, there is a tissue-specific regulation of mouse ferritin expression at both the transcription and mRNA stability levels.

Immunofluorescent localization of histone H1 0 in the nuclei of proliferating and differentiating friend cells

By using affinity-purified antibodies to H1 0 and to H1AB the localization of these histones was studied by indirect immunofluorescence in the nuclei of proliferating (EAT and uninduced Friend cells) and of differentiating (induced Friend cells) cell populations. While with H1AB antibodies a bright fluorescence all over the chromatin was obtained, the localization of H1 0 varied depending on the state of the cell population. In the proliferating EAT cells it was localized strictly in the nucleoli. The Friend cell population revealed a heterogeneous picture with two types of H1 0 localization—nucleolar predominating in uninduced cell populations and peripheral predominating in induced cells. A comparison with literature data suggests that H1 0 seems to be associated with chromatin regions containing active genes.

Control of heme synthesis during Friend cell differentiation: role of iron and transferrin.

In many types of cells the synthesis of delta-aminolevulinic acid (ALA) limits the rate of heme formation. However, results from our laboratory with reticulocytes suggest that the rate of iron uptake from transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis during erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe-SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels of Fe-Tf (20 microM). Furthermore, in induced Friend cells 100 microM Fe-SIH stimulated 2-14C-glycine incorporation into heme up to 3.6-fold as compared to the incorporation observed with saturating concentrations of Fe-Tf. In contrast, Fe-SIH, even when added in high concentrations, did not stimulate heme synthesis in uninduced Friend cells but was able to do so as early as 24 to 48 h following induction. In addition, contrary to previous results with rabbit reticulocytes, Fe-SIH also stimulated globin synthesis in induced Friend cells above the level seen with saturating concentrations of transferrin. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells.

Nuclear matrix and transcriptional activity of the mouse α-globin gene

The association of the mouse α-globin gene with the nuclear matrix was studied when the gene was permanently repressed in Ehrlich ascites cells, potentially active in uninduced Friend cells or actively transcribed in induced Friend cells. Matrix-associated DNA was obtained by two methods, differing in the order of treatment of the nuclei with high salt and DNase I. By using a cloned α-globin probe, no enrichment in α-globin sequences was found in the matrix-associated DNA after DNase I digestion of high-salt treated nuclei from Ehrlich ascites and uninduced Friend cells. In induced Friend cells, a high enrichment (up to 20 times) of α-globin sequences was detected in the DNA left with the nuclear matrix structures. The size of the DNA fragments obtained by this procedure indicated a random attack and did not correspond to a progressive top-to-bottom cleavage model. No enrichment in α-globin sequences was found in induced Friend cells if nuclear matrices were obtained by DNase I digestion of the nuclei before the treatment with high salt. Our data suggest that the enrichment in actively transcribed genes of matrices from nuclei treated with high salt does not reflect a localization of these genes close to the attachment sites of the chromatin loops but rather their artefactual association with some high salt-insoluble proteins of the transcriptional complexes.

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 38,000 mol wt. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with cloned mouse beta-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the beta-globin gene. Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32P-labeled cloned mouse beta-globin DNA reveals the presence in hnRNP of two size classes of beta-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S beta-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S beta-globin mRNA. These results establish, for the first time, that the nuclear transcripts of a specific, well-defined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing.

Dimethyl sulphoxide induction of transferrin receptors on friend erythroleukemia cells

Friend erythroleukemia cells induced to differentiate with DMSO showed a 6-fold increase in the level of transferrin receptors with a commensurate increase in iron uptake Uninduced cells nevertheless had low levels of transferrin binding sites which showed saturability, specificity and a quantitative correlation with iron uptake, properties associated with specific transferrin receptors. Induced Friend erythroleukemia cells had more transferrin receptors than mouse reticulocytes; 440 000/cell compared with 86 000/cell. However, the rate of iron uptake per receptor was two-fold greater in mouse reticulocytes. Receptor levels in induced Friend cells decreased after prolonged culture in DMSO. The internalisation of transferrin which had previously been shown to occur with reticulocytes was also demonstrated with Friend cells using gold-labelled transferrin.

Frequency distribution of mRNA and pre-mRNA in growing and differentiated Friend cells

The frequency distribution of poly(A)+-mRNA in growing and in differentiated Friend cells has been measured by mRNA-cDNA hybridization and their differences established by heterologous hybridization of mRNA of one type and cDNA of the other. It was shown that induction of Friend cells involves an increase in abundance of a small number of mRNAs, while no specific pattern of messenger disappearance could be detected. The frequency distribution of pre-mRNA was determined by hybridizing nuclear RNA with the cDNA probes complementary to mRNA. In uninduced Friend cells, it was shown that most precursor messenger sequences are present at a single frequency of about 3 molecules per nucleus, independently of their final frequency in polysomal mRNA. In induced Friend cells, the frequency distribution of pre-mRNA is more heterogeneous and correlated to some extent with the corresponding mRNA frequency distribution.

Purification of globin messenger RNA from dimethylsulfoxide-induced friend cells and detection of a putative globin messenger RNA precursor

Procedures are described that permit the detection and isolation of a specific messenger RNA as well as its precursor from total cell extracts. DNA complementary to the mRNA was elongated by the addition of dCMP residues and annealed with labeled cell RNA. The elongated DNA with RNA hybridized to it was isolated by chromatography on a poly(I)-Sephadex column. The method was used to isolate 32P-labeled globin mRNA from labeled Friend cells, a mouse erythroleukaemic cell line, induced with dimethylsulfoxide to synthesize hemoglobin. 32P-labeled globin mRNA isolated by this procedure was estimated to be 80% pure by hybridization analysis and sedimented as a single peak at 10 S. Partial sequences were determined for 16 oligonucleotides derived from the purified 32P-labeled globin mRNA by RNAase T 1 digestion. The partial sequences for nine oligonucleotides corresponded to those predicted from the amino acid sequences of α and β globin; the other oligonucleotides were presumably derived from non-translated regions. In order to detect a possible precursor to globin mRNA, RNA from induced Friend cells pulse-labeled with [ 32P]phosphate for 20 minutes was centrifuged through a sucrose gradient and the resulting fractions were analyzed for globinspecific sequences. Two peaks of globin-specific RNA were detected, a larger one at 10 S, the position of mature globin mRNA, and a smaller one at 15 S.