An intracellular factor that induces erythroid differentiation in mouse erythroleukemia (Friend) cells

We have previously shown that in vitro erythroid differentiation of mouse Friend cells is a result of a synergistic action of two distinctive intracellular reactions. We now have evidence that a factor in the cell free extract is involved in one of the reactions. This factor triggers erythroid differentiation when introduced into undifferentiated mouse Friend cells, provided the cells have been briefly exposed to dimethyl sulfoxide. The factor is induced in nonerythroid cells as well following treatment of the cells by agents that affect DNA replication. Cycloheximide inhibited the induction of the factor. The factor, which is in the cytoplasm, was partially purified and proteinaceous. When introduced into the cells the partially purified factor converts 60% to 70% of undifferentiated Friend cells to erythroid cells, at an efficiency almost equivalent to the efficiencies achieved by typical inducing agents. The factor's biochemical characteristics and possible role in erythroid differentiation are also discussed.     

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.     

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.     

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.     

Glycolipids of mouse erythroleukemia cells (Friend cells) and their alteration during differentiation

Neutral and acidic glycosphingolipids of Friend cells were characterized in 1) undifferentiated Friend cells (745A), 2) differentiated Friend cells induced with dimethyl-sulfoxide, and 3) solid tumors grown in mice after subcutaneous implantation of Friend cells. The structures of the isolated glycosphingolipids were determined by means of compositional analysis, methylation analysis and enzyme treatment. Gangliosides GD1a and N-acetylgalactosaminyl-GD1a, followed by GM1a and GM2, were the main gangliosides in undifferentiated Friend cells. GD1a and N-acetylgalactosaminyl-GD1a accounted for 45 and 25% of the total gangliosides, respectively. On differentiation, ganglioside GM2 decreased significantly, from 10% to a trace amount. In solid tumors, GD1a was the major ganglioside, whereas in contrast to the situation in the cultured cells, N-acetylgalactosaminyl-GD1a was almost completely absent, and ganglioside GM1b, but not GM1a, was detected. In addition, ganglioside GD1 alpha was detected in the solid tumors. Galactosylceramide, glucosylceramide, and lactosylceramide were the main neutral components in both types of cells, while globotetraosylceramide (globoside), IV3-N-acetyl-galactosaminyl globotetraosylceramide (Forssman glycolipid) and gangliotetraosylceramide (GA1) were major in solid tumors grown in vivo.     

Inhibition of erythroid differentiation of mouse erythroleukemia cells by a macrophage product(s)

Conditioned media from established murine macrophage cell lines (RAW264.7, P388D1, and WEHI-3) incubated with endotoxin in a serum-free medium contain an erythroid inhibitory activity (EIA) that inhibited dimethylsulfoxide-induced erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells. Endotoxin itself has no EIA activity. Partial purification of EIA demonstrated that it is distinct from other macrophage products such as IL-1, TGF beta, ECGF, FGF, G-CSF, hepatocyte stimulating factor, interferon, PDGF, and cachectin/TNF. These findings indicate that EIA is a macrophage product distinct from other monokines.     

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells during their erythroid differentiation

The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post-exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis by demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin.     

Induction of erythroid differentiation in Friend murine erythroleukemic cells by inorganic selenium compounds.

Inorganic selenium compounds are shown to be inducers of hemoglobin synthesis in malignant murine erythroleukemia (MEL) cells. SeO₂ can induce hemoglobin synthesis at $\text{1}\text{20}$ the concentration of butyric acid and $\text{1}\text{5000}$ the concentration of dimethylsulfoxide (DMSO), two potent inducers of erythroid differentiation in MEL cells. SeO₂ and H₂SeO₃ showed an equivalent capacity to stimulate hemoglobin synthesis in three different MEL cell lines. The incorporation of ³H-glycine into hemoglobin was demonstrated in lysates of SeO₂-induced MEL cells.     

An intracellular factor which affects erythroid differentiation in mouse Friend cells

When permeabilized Friend cells, which had been briefly treated by DMSO, were exposed to cell-free extracts from UV irradiated cells, a small but significant number of the cells became reactive to benzidine, a characteristic of erythroid differentiation. The activity in the extracts was apparently induced following UV irradiation, reaching a maximum 25 to 30 h after irradiation. Although a similar activity was detected in the extract from mitomycin C treated cells, little activity was detected in the extract from cells treated with DMSO, a potent erythroid inducing agent. The induction of the active factor by UV irradiation was not specific of Friend cells since similar inducing activities were detected in the extract from non-erythroid murine cells irradiated by UV. The active factor in the extract seems to be a protein, judged from its heat sensitivity and high molecular weight. Significance of this finding in relation to cellular differentiation and DNA damage is discussed.     

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.     

Increased carbonic anhydrase activity in Friend erythroleukemia cells during DMSO-stimulated erythroid differentiation and its inhibition by BrdU.

Carbonic anhydrase activity is increased in Friend erythroleukemia (FL) cells during the enhancement of erythroid differentiation in the presence of dimethylsulfoxide (DMSO) or butyric acid. Untreated FL cells show an increase in enzyme activity associated with logarithmic growth. The increase in the specific activity of carbonic anhydrase in the differentiating treated cells, however, appears to be due to at least two additional general mechanisms: (1) an induction of carbonic anhydrase paralleling the stimulation of hemoglobin synthesis and (2) the stability and/or retention of active carbonic anhydrase as compared to most of the other cell proteins. The stimulation of carbonic anhydrase activity in the treated cells is inhibited by 5-bromo-2'-deoxyuridine (BrdU). This is the first demonstration of BrdU inhibition of a DMSO induced product not directly related to hemoglobin.     

Superoxide dismutase induces differentiation of Friend erythroleukemia cells

Friend erythroleukemia cells (FELC) served as a model system for cell differentiation because these cells can be triggered to differentiate by a variety of chemical agents. Treatment with the classical inducer of differentiation, hexamethylene bisacetamide (HMBA), stimulated superoxide dismutase (SOD) activity, which increased in parallel with HMBA-induced differentiation. Furthermore, FELC were shown to differentiate in response to the addition of liposomes containing SOD. Oxidative treatment with liposomes containing D-amino acid oxidase or xanthine oxidase, cumene peroxide, or potassium superoxide also induced differentiation, whereas antioxidants such as alpha-tocopherol, butylated hydroxytoluene, or beta-carotene did not induce differentiation. Also, HMBA induction of differentiation was suppressed by treatment with antioxidants.     

Specific erythroid differentiation of mouse erythroleukemia cells by activins and its enhancement by retinoic acids.

Activin A has been shown to induce hemoglobin production in various hematopoietic cells. Such activities of three structurally distinct activins (activin A, activin AB, and activin B) were compared using F5-5 mouse erythroleukemia cells. Activin A and AB had similarly potent inducing activities whereas that of activin B was much lower. The erythroid inducing activity of activins was suppressed by follistatin, an activin-binding protein but not by inhibin A and inhibin B. Retinoic acids (both all-trans and 13-cis) had weak erythroid differentiation activity. In addition, clear synergistic erythroid induction occurred when retinoic acid and activin A were mixed together. These results indicate that retinoic acid may modulate activin-induced erythropoiesis in vivo.     

Idiotype mimicry by a differentiation antigen on Friend erythroleukemia cells

We previously found that murine leukemia cells of T cell, B cell, and erythroid ontogeny express a cell membrane antigen that cross-reacts with an idiotype of an anti-retroviral antibody. In the present study, the expression of this antigen (termed AVID, for anti-viral idiotype) by murine erythroleukemia (MEL) cells was examined during chemically induced differentiation. AVID expression by MEL cells was found to be lost when they were treated with either dimethyl sulfoxide or hexamethylene bisacetamide, two chemicals that induce MEL cells to terminally differentiate. The kinetics of disappearance of AVID during inducer treatment reflected the kinetics with which the inducers caused MEL cell commitment to terminal differentiation. Loss of AVID expression by inducer-treated cells was inhibited by dexamethasone, which inhibits commitment and MEL cell differentiation. The subset of inducer-treated cells that expressed the least amount of AVID contained the greatest number of cells committed to differentiate. These results indicate that AVID identifies a novel differentiation antigen of MEL cells.     

Acceptors of poly(ADP-ribosylation) in differentiation inducer-treated and untreated Friend erythroleukemia cells

Acceptors of poly(ADP-ribosylation) were identified and compared between inducer-treated and untreated Friend erythroleukemia cells. When permeabilized Friend cells were pulse labeled with 0.6 μM [³²P]NAD for 1 min and labeled proteins analyzed by SDS-polyacrylamide gel electrophoresis, nucleosome core histones were found to be the primary acceptors, with an additional minor radioactive peak at a position corresponding to M_r = 170 000. Friend cells induced to differentiate by DMSO treatment showed a similar distribution of radioactivity, but with a 60% reduction in the overall level of poly(ADP-ribosylation) under identical labeling conditions. When isolated nuclei were pulse labeled with 0.6 μM [³²P]NAD, radioactive peaks were not restricted mainly at the positions of core histones but widely dispersed in the area from 10 to 50 kDa with another peak at 170 kDa. Increase of NAD concentration resulted in the overall shift of peaks to higher molecular weight positions. When pulse-labeled nuclei or permeable cells were chased with 1 mM NAD, radioactive peaks migrated to positions of very high molecular weight (>M_r = 180 000). Remarkable suppression of poly(ADP-ribose) synthesis was observed when DMSO, hexamethylene bisacetamide, butyric acid, or hemin were used as the inducers.     

Amiloride in differentiation and commitment of Friend erythroleukemia cells

Dimethylsulfoxide (DMSO) converts almost all of the undifferentiated murine erythroleukemia cells (MEL or Friend cells, clone 745A) in a culture to differentiated cells that contain high levels of hemoglobin and that stop growing after a limited number of cell divisions. Contrary to other reports--that amiloride strongly inhibits DMSO-induced differentiation in MEL cells--in this laboratory, inhibition by amiloride, tested with DMSO over a range of concentrations in two kinds of media and at various cell densities, was found to be only weak or absent. Similarly, amiloride did not inhibit induction by N,N'-hexamethylene bis-acetamide (HMBA). As expected from previous findings with other cell systems, amiloride inhibited protein synthesis and cell multiplication.     

Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases.

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.