A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes

Erythroid differentiation of murine erythroleukemia (MEL) cells is far more extensive when the cells are attached to fibronectin-coated dishes than in suspension culture. Cells induced in suspension culture for 4 d become arrested at a late erythroblast stage and do not undergo enucleation. Incubation of cells in suspension beyond 4 d results in lysis. In contrast, cells induced by DMSO on fibronectin-coated dishes for 7 d differentiate into enucleating cells, reticulocytes, and erythrocytes. As determined by quantitative immunoblotting, cells induced in suspension culture accumulate approximately 33% of the amount of the major erythroid membrane protein Band 3 present in erythrocyte, whereas cells induced on fibronectin-coated dishes accumulate 80-100% of the amount present in erythrocytes. Both suspension-induced cells and cells induced on fibronectin-coated dishes accumulate approximately 90% of the amount of spectrin and ankyrin present in erythrocytes. As revealed by immunofluorescence microscopy during enucleation of MEL cells, both Band 3 and ankyrin are sequestered in the cytoplasmic fragment of the emerging reticulocyte. Enucleated and later-stage cells detach from the fibronectin matrix, due to the loss of the surface fibronectin receptor; this mimics the normal release of reticulocytes from the matrix of the bone marrow into the blood. Thus a fibronectin matrix provides a permissive microenvironment within which erythroid precursor cells reside, proliferate, migrate, and express their normal differentiation program.     

Inhibition of dimethyl sulfoxide-induced Friend erythroleukemia cell differentiation in vitro by lithium chloride

This report describes the ability of ultra-pure lithium chloride (LiCl) to influence the growth kinetics and differentiation of Friend erythroleukemia cells in vitro. LiCl (0.2-50 mEq/l) was effective in reducing the ability of Friend cells to grow in liquid suspension culture (p less than or equal to 0.001). In addition, the capacity of these erythroleukemic cells to respond to the inducing agent dimethyl sulfoxide (DMSO) was also significantly reduced (p less than or equal to 0.001). These results demonstrate that LiCl can influence not only the proliferation of erythroleukemia cells but also their subsequent differentiation after exposure to such chemical inducers.     

Inhibitors of protein glycosylation inhibit the differentiation of Friend erythroleukemia cells

Tunicamycin, 2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose inhibit dimethyl sulfoxide-induced differentiation of Friend cells. This inhibition, characterized by inhibition of hemoglobin synthesis, is accompanied by a specific inhibition of protein glycosylation. The results of cloning experiments indicate that this inhibition specifically affects cells in the period preceding their commitment. These results suggest that glycoprotein synthesis is a requirement for Friend erythroleukemia cells in order to initiate the expression of the terminal differentiation program.     

Dexamethasone-sensitive and -insensitive responses during in vitro differentiation of Friend erythroleukemia cells

We have investigated the mechanism(s) by which dexamethasone inhibit DMSO-induced Friend erythroleukemia cell differentiation in vitro. In particular, we examined the effects of dexamethasone on (a) the early events of differentiation such as cell volume alterations and 'memory response' and (b) the onset of biochemical events associated with terminal erythroid cell differentiation. By analysing kinetics of commitment of Friend cells to terminal erythroid differentiation on a clonal basis, we have observed that dexamethasone inhibited the completion of the latent period (time elapsed prior to commitment) and impaired "memory" (ability to inducer-treated cells to continue differentiation after a discontinuous exposure to inducer). Treatment of Friend cells with dexamethasone did not prevent the occurrence of DMSO-induced alterations in cell volume. However, dexamethasone treatment prevented a series of biochemical events associated with terminal Friend cell differentiation. These include the decrease in the rate of both cytoplasmic and nuclear RNA synthesis as well as the induction of cytidine deaminase activity and hemoglobin synthesis. These data indicate that the dexamethasone-sensitive process(es) operate during the early stages of Friend cell differentiation and that they are responsible for the inhibition of terminal erythroid maturation. These dexamethasone-sensitive processes, however, appear to be different from those regulating cell volume alterations during the early steps of DMSO-induced Friend cell differentiation.     

Synthesis and intracellular distribution of cathepsins E and D in differentiating murine Friend erythroleukemia cells.

The synthesis, accumulation, and cellular distribution of cathepsins E and D during the dimethyl sulfoxide (DMSO)-induced differentiation of Friend erythroleukemia cells were investigated. The cellular levels of cathepsins E and D rapidly increased within 1 day of DMSO induction and then sharply decreased over the next 7 days. Since the cells during 1 day of differentiation were morphologically the same as uninduced cells, the results suggest the importance of these enzymes in more cellular proteolysis for the following committed differentiation. While cathepsin D was present mostly in the sedimentable fraction of cells throughout the differentiation period, the distribution of cathepsin E varied to the stage of differentiation. The ratio of the soluble/sedimentable cathepsin E content was 1.1, 1.4, 0.9, and 0.7 in cells after 0, 1, 4, and 7 days of DMSO treatment, respectively. The maturation of reticulocytes to erythrocytes was accompanied by complete loss of the soluble cathepsin E and of all of the cellular cathepsin D. Immunoblotting analyses revealed that both uninduced and induced cells contained two forms of cathepsin E; a high molecular weight form (82 kDa) which was mainly associated with the sedimentable fraction and a low molecular weight form (74 kDa) which was found largely in the soluble fraction. The distribution of these two forms was not significantly changed throughout the differentiation period, but the 74-kDa protein was completely eliminated with maturation of reticulocytes to erythrocytes. Cathepsin D also appeared in two forms in both uninduced and induced cells; a minor (46 kDa) and a major (42 kDa) form which appear to have a precursor-product relationship.     

Phorbol ester tumor promoters block the transition from the early to the heme-dependent late program of friend cell differentiation

In this study, the mechanism of inhibition of differentiation of Friend erythroleukemia cells by the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has been examined. These studies indicate that some early events associated with Friend cell differentiation, including an early change in ⁸⁶Rb⁺ transport and a decrease in cell volume, still occur in the presence of TPA. However, several late events in the program of Friend cell differentiation, including the induction of heme synthesis and the loss of proliferative capacity, are inhibited by TPA. These effects of TPA can be reversed by hemin, which alone does not induce Friend cells to differentiate. The addition of hemin to cultures grown in the presence of inducer plus TPA for several days results in the rapid restoration of hemoglobin synthesis, and also causes a parallel decrease in colony-forming ability. These results suggest that tumor promoters may inhibit only heme-dependent events, rather than the entire program of Friend cell differentiation.     

Correlation between protein 4.1a/4.1b ratio and erythrocyte life span

Erythrocyte membranes from various healthy mammals contained a doublet of protein 4.1a and 4.1b, which appeared to differ by 2-3 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The ratio of protein 4.1a/4.1b showed much variety among animal species, and the 4.1a/4.1b ratio correlated to the mean erythrocyte life span, that is, the mean cell age in circulating blood. We also found that the 4.1b is the predominant form in the immature erythroid cells such as reticulocytes and K562 cells. In addition, the 4.1b but not 4.1a protein was metabolically labeled with [35S]methionine in the erythropoietic cells from anemic mouse. Immunological detection showed that there is a doublet of minor variants of protein 4.1 with apparent molecular masses slightly more than those of 4.1a and 4.1b. The ratio of these minor isoforms designated as 4.1a + and 4.1b + revealed the alteration during erythrocyte senescence as observed in 4.1a/4.1b ratio. These results show that protein 4.1 may be synthesized as 4.1b and 4.1b + and intercalated into membrane skeletons at an early stage of erythroidal differentiation, and that the posttranslational modification into 4.1a and 4.1a + appears to occur by a common mechanism in many mammalian species. Feline erythrocytes, however, appeared to lack such a postsynthetic processing of protein 4.1, and exhibited one major component of 4.1b with the other minor variant of 4.1b +.     

Actions of lipoxins A4 and B4 on signal transduction events in Friend erythroleukemia cells.

Earlier studies in our laboratory suggested a role for 15-lipoxygenase products of arachidonic acid, such as 15-hydroperoxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid, in supporting proliferative events in Friend erythroleukemia cells. Because lipoxins are also products of the same lipoxygenase enzyme, we tested their actions on signal transduction events related to DNA synthesis. Lipoxins A4 and B4 (10 nM) significantly enhanced [3H]thymidine incorporation into Friend cells in the absence of fetal bovine serum without affecting cell differentiation or cell number. Lipoxin B4 increased the duration of time that cells spent in the S phase of the cell cycle, and also significantly enhanced protein kinase C activity in nuclei, whereas c-fos expression was unaffected by either of the lipoxins tested. The novel, intracellular actions of lipoxins A and B on Friend erythroleukemia cells documented in this study represent a unique spectrum of effects of lipoxins on signal transduction events as compared with other eicosanoids.     

Perturbation of growth and differentiation of Friend murine erythroleukemia cells by 5-bromodeoxyuridine incorporation in early S phase.

Cultured Friend murine erythroleukemia cells (Friend cells) are induced to undergo erythroid differentiation when grown in the presence of dimethylsulfoxide (DMSO) and other compounds. The effects of unifilar substitution of bromouracil (BU) for thymidine in the DNA (BU-DNA) of Friend cells were examined. Cells were grown in the presence of 5-bromodeoxy-uridine (BrdU) for one generation, then centrifuged and resuspended in medium containing DMSO without BrdU. These cells exhibited a delay in the appearance of heme-producing, benzidine-reative (B+) cells and a decreased rate of cell proliferation in comparison to the control not containing BU-DNA. A transient inhibition of entry into S phase was observed when control cells or cells containing BU-DNA were grown in the presence of DMSO) for 10 to 20 hours. This transient inhibition was increased in the BrdU culture. Thus BU-substitution in Friend cells alters other cellular functions in addition to erythroid differentiation. The rate of increase in the percent of cells committed to differentiate (those forming B+ colonies in plasma clots) was similar in the BrdU and control cultures until 40 to 50 hours. After this time, a delay in the appearance of committed cells was observed in the BrdU culture. The effect of BrdU on the appearance of B+ cells was more pronounced and occurred earlier than its effect on the rate of commitment. Therefore, the delay in the appearance of B+ cells in the BrdU culture was due primarily to perturbation of post-commitment events such as the accumulation of hemoglobin. We also examined the effect on growth and differentiation after BrdU was incorporated during different intervals of S phase in cells synchronized by centrifugal elutriation or by double thymidine block and hydroxyurea treatment. The delay in the appearance of B+ cells and inhibition of cell proliferation were only observed when BrdU was incorporated in the first half of S phase. BrdU (10 muM) had no effect on growth or differentiation when present during late S or G1 and G2. These results, using two very different methods to achieve cell synchrony, indicate that the effects of BrdU on growth and differentiation described above are due to its incorporation into DNA sequences replicating during early S.     

15-Lipoxygenase products affect protein phosphorylation in Friend erythroleukemia cells

Endogenous arachidonic acid metabolism and protein phosphorylation have been examined in Friend erythroleukemia cells in response to the induction of differentiation by dimethyl sulfoxide and hexamethylene bisacetamide. 15-Hydroxyeicosatetraenoic acid levels were elevated in cells differentiated with hexamethylene bisacetamide or dimethyl sulfoxide compared with undifferentiated cells. Protein phosphorylation decreased markedly in differentiated cells compared with undifferentiated cells and the addition of 15-hydroperoxyeicosatetraenoic acid specifically decreased the phosphorylation of a 28-kilodalton protein. These findings indicate that products of 15-lipoxygenase may act as intracellular messengers in Friend erythroleukemia cells by affecting protein phosphorylation.     

Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation

Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half-life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.     

Factors involved in supporting the growth and steroidogenic functions of bovine adrenal cortical cells maintained on extracellular matrix and exposed to a serum-free medium

Bovine adrenal cortex cells maintained on extracellular matrix (ECM)-coated dishes will proliferate actively when serum is replaced by HDL (25 micrograms protein/ml), insulin (10 ng/ml), and FGF (100 ng/ml). The cells have an absolute requirement for HDL in order to survive and grow. The omission of insulin, FGF, or both results in a slower growth rate and lower final cell density of the cultures. A requirement for transferrin (1 microgram/ml) becomes apparent only when cells have been grown for at least four generations in the absence of serum. Early passage (P1-P3) bovine adrenal cortex cells cultured in serum-free medium responded to ACTH (10(-8)M) with increased 11-deoxycortisol production; this effect was not observed in later passage cells (P7-P15). The cells' ability to utilize LDL-derived cholesterol and to respond to db cAMP (1mM) by increased steroid release was preserved in cells cultured for over 60 generations in the serum-free medium. HDL, although also able to increase steroid production in early-passage cultures exposed to ACTH or to ACTH and dibutyryl cyclic AMP (db cAMP), was 10 fold less potent than LDL. It did not support steroidogenesis in cultures not exposed to these trophic agents. The life span of bovine adrenal cortex cells grown in the serum-free medium on fibronectin (FN)- versus ECM-coated dishes was compared. Cells seeded in serum-containing medium and grown in serum-free medium had a life span of 34 versus 60 generations when maintained on fibronectin- or ECM-coated dishes, respectively. Cells seeded in the complete absence of serum in the serum-free medium on ECM- or fibronectin-coated dishes could be passaged for 26 or 13 generations, respectively. While FGF was an absolute requirement for cells cultured on fibronectin-coated dishes, it was not required when cells were maintained on ECM. These observations demonstrate the influence of the ECM not only in promoting cell growth and differentiation but also on the life span of cultured cells.     

Cell surface molecules of friend erythroleukemias: Decrease in T200 glycoprotein expression after induction

Monoclonal antibodies against the Thy-1 and T200 glycoproteins were used to study the expression of cell surface molecules on mouse hematopoietic cell lines. Friend erythroleukemias express T200 glycoprotein but do not express significant amounts of Thy-1 glycoprotein on their cell surface. The rate of T200 glycoprotein synthesis in maximally-induced Friend erythroleukemia 745.6 cells is less than 10% that in noninduced cells, although total protein synthesis shows only a twofold decline and induced cells express 2-6-fold less T200 glycoprotein on their surface compared to noninduced cells. T200 glycoprotein expression is reduced in a variant cell line obtained by selection for growth in dimethylsulfoxide, showing that the reduction in T200 glycoprotein synthesis characteristic of induced cells is an event that can be dissociated from commitment and hemoglobin synthesis. Analysis of T200 glycoprotein negative cell lines, isolated by cytotoxic immunoselection against T200 glycoprotein, indicates that the presence of T200 glycoprotein on the cell surface is not necessary for induction of hemoglobin synthesis and terminal differentiation of Friend erythroleukemias.     

Multimeric structure of the membrane erythropoietin receptor of murine erythroleukemia cells (Friend cells). Cross-linking of erythropoietin with the spleen focus-forming virus envelope protein.

In erythroleukemia cells infected with the polycythemia strain of the Friend virus complex, erythropoietin could be cross-linked mainly to a protein of 63 kDa when using disuccinimidyl suberate. In contrast, erythropoietin in other erythroleukemia cells cross-linked to two proteins of 85 and 100 kDa. When native erythropoietin receptor complexes were immunoprecipitated, the 63-kDa erythropoietin-cross-linked protein could be precipitated both by antibodies directed against the intracellular part of the cloned chain of the erythropoietin receptor and by antibodies directed against the envelope proteins of the Friend virus. However, after denaturation of the complexes, the 63-kDa protein was only precipitated by antibodies directed against the envelope proteins of the Friend virus. Enzymatic deglycosylation confirmed that erythropoietin was cross-linked with the envelope protein of the defective virus and bidimensional diagonal gel electrophoresis analyses showed that some of the erythropoietin cross-linked envelope proteins were dimerized by disulfide bonds. Thus, the main erythropoietin-receptor complex in the plasma membrane of these cells consisted of a molecule of the cloned chain of the erythropoietin receptor noncovalently associated with one or two disulfide-bonded molecule(s) of the envelope protein of the defective virus. Moreover, our results also showed that the viral envelope protein associated with the cloned chain of the erythropoietin receptor at a site distinct from the erythropoietin binding site.     

Terminal differentiation in cultured Friend erythroleukemia cells.

Two populations of differentiated, hemoglobin-containing cells have been identified in cultures of Friend murine erythroleukemia cells (Friend cells): terminally differentiated benzidine-positive (B+) cells that are no longer capable of proliferation and are arrested in the G1 phase of the cell cycle, and their precursors, traversing B+ cells which undergo two or three cell divisions before reaching their terminally differentiated state. Thus Friend cells in suspension culture retain a limited capacity to synthesize DNA and divide after commitment to erythroid differentiation. We identified terminally differentiated cells using autoradiography after benzidine staining. We also developed a quantitative flow microfluorometric assay to distinguish cells that are terminally differentiated from those cells committed to differentiation but still capable of proliferation.We developed a purification procedure to isolate terminally differentiated Friend cells. Their DNA content was the same as that of the undifferentiated cells in G1 by both the diphenylamine reaction and a fluorescence assay. No loss of DNA was detected during the differentiation of Friend cells. As many as 72% of the total cells in a culture induced with DMSO (88% B+) were differentiated cells arrested in G1. As a control, a DMSO-resistant line derived from 745A neither differentiated nor arrested in G1 after growth in the presence of DMSO. The results of these studies were obtained using several compounds that induce differentiation and three independently isolated clones of 745A. We also observed arrest of differentiated cells in G1 with the two other well characterized, independently derived erythroleukemia cell lines, F4-1 and T3-C1-2.     

Hemoglobin synthesis in cell hybrids formed between teratocarcinoma and friend erythroleukemia cells

Viable hybrid cells have been isolated following fusion of Friend erythroleukemia cells and undifferentiated teratocarcinoma cells. The hybrids formed between near-diploid parental cells resembled Friend cells in their ability to grow in suspension and to synthesize hemoglobin in the presence of the chemical inducers dimethyl sulfoxide (DMSO) and ouabain. Erythropoietin (EPO) was effective in inducing hemoglobin synthesis in some of the hybrid cell lines. The hemoglobins synthesized by the hybrids were of the adult forms, but were quantitatively different from those hemoglobins synthesized by the parental Friend cells, suggesting that the fusion event modulated the expression of the hemoglobin chain genes.     

Correlation between hypomethylation of DNA and expression of globin genes in Friend erythroleukemia cells.

This report identifies L-ethionine as an inducer of differentiation in murine erythroleukemia cells. When Friend erythroleukemia cells are grown in the presence of 4mM L-ethionine, globin mRNA accumulates and in 4-5 days, 25-30% of the cells in the culture contain hemoglobin. Incubation of the cells with bromodeoxyuridine prevents both ethionine-induced accumulation of globin mRNA and erythroide differentiation. At the concentration where L-ethionine acts as an inducer of FL cell differentiation it inhibits methylation of DNA and tRNA in vivo but does not prevent macromolecular synthesis or cell division. To establish whether a link existed between inhibition of a specific methyltransferase and activation of globin synthesis in FL cells, we examined the degree of hypomethylation of DNA and tRNA from FL cells induced to differentiate with dimethylsulfoxide and butyrate. In contrast to the tRNA from ethionine-treated cells, tRNA from cells induced by butyrate or Me2SO cannot be methylated in vitro using homologous enzymes. DNA isolated from cells exposed to any of the three inducers, however, was significantly hypomethylated when compared with DNA from uninduced cells. These data suggest that methylation of DNA may play a role in the regulation of gene expression.