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.     

Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (Friend) cells

An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed.     

Effects of murine tumor necrosis factor on Friend erythroleukemic cells

Tumor necrosis inducing factor (TNF), a 140,000 molecular weight glycoprotein present in the serum of Corynebacterium parvum endotoxin-treated mice, was cytotoxic toward Friend virus-transformed erythroleukemic cells (FELC). These cells grow in culture as undifferentiated pro-erythroblasts but can be induced to differentiate in a limited fashion along the erythroid pathway to orthochromatic normoblasts by various agents such as dimethylsulfoxide (DMSO). Partially and highly purified preparations of TNF were cytotoxic toward logarithmically growing FELC whereas a comparable serum protein fraction from C. parvum treated mice or endotoxin from E. coli had no effect upon FELC viability. DMSO-induced cells were more sensitive to the action of TNF requiring only about half the concentration needed to produce 50% kill in noninduced cells. Inhibition of hemoglobin formation was TNF dose-related and could be decreased by 94%. TNF was also cytotoxic toward DMSO-induced cells in stationary phase and mitomycin C treated noninduced FELC. Neuraminidase modification of the surface of FELC increased the cytotoxicity of TNF by 50%. These results demonstrate that TNF destroys FELC whether they are nondividing, dividing or partially differentiated and suggest that TNF may accomplish this by affecting cell metabolism after internalization.     

Analysis of erythroid differentiation in Friend cells using noninducible variants

A series of Friend cell variants has been isolated by selecting for resistance to different inducers of Friend cell differentiation. This procedure selects for cells which have lost the capacity to differentiate terminally in the presence of inducer. Fluctuation analysis shows that these variants arise during culture and are not induced by the selective conditions. Moreover, mutagenesis of parental cells increases the frequency of occurrence of DMSO-resistant variants. Our evidence suggests that these resistant variants arise by two mechanisms. Some arise spontaneously at a relatively high rate (5 × 10^?5?5 × 10^?6 per cell per generation), but their phenotypes are not necessarily stable on removal of the selective conditions. Stable variants arise spontaneously at a lower frequency which is consistent with a true mutational origin.Screening of these stable resistant variants shows that they have different phenotypes. Some fail to respond to any inducer; others respond to all inducers tested except the one used for selection, whereas others respond to some but not all inducers. Most of the DMSO-resistant variants are noninducible by DMSO for all aspects of Friend cell differentiation tested (that is, globin mRNA, hemoglobin, spectrin and the ability to undergo terminal differentiation). Two variants, however, are inducible for an early marker of differentiation, the erythrocyte membrane protein spectrin, but not for other markers such as hemoglobin, globin RNA or terminal differentiation. This implies that the regulation of the globin pathway can be uncoupled from that of spectrin.     

Induction of interferon and erythropoietic differentiation in cells transformed by Friend virus.

Two lines of Friend virus (FV)-transformed mouse spleen cells have been analyzed in respect to their interferon production capacity: neither F4 cells, which liberate infectious FV when kept under tissue culture conditions, nor the thymidine kinase-deficient B8 cells, which do not produce significant amounts of FV, release detectable amounts of autogenous interferon into cell supernatants. However, interferon is produced in these cells in amounts comparable to that in L-929 cells when interferon induction is initiated with UV-inactivated Newcastle disease virus. Conversely poly(I)-poly(C), a potent interferon inducer in L-929 cells, proved ineffective in this capacity in F4 or B8 cells. When erythropoietic differentiation is induced in these cells by dimethyl sulfoxide, no autogenous interferon production occurs, but with NDV-induction a four- to fivefode increase of interferon production is observed. A similar elevation of interferon production is achieved during 5-bromodeoxyuridine stimulation of differentiation in the thymidine kinase-deficient B8 cells. The refractiveness against poly(I)-poly(C) displayed in unstimulated cells is not overcome at any stage of differentiation, indicating major differences of Newcastle disease virus and poly(I)-poly(C) induction mechanisms.     

Early transport changes during erythroid differentiation of Friend leukemic cells

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx. The transport of two amino acid analogues, alpha-aminoisobutyric acid and 2-aminobicyclo [2,2,1]-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast, the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes--those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.     

Butyric acid,a potent inducer of erythroid differentiation in cultured erythroleukemic cells

Butyric acid is an unusually potent inducer of erythroid differentiation in cultured erythroleukemic cells. It is effective at one hundredth the concentration required of dimethylsulfoxide, a most effective inducing agent. Studies using a variety of analogues and metabolites suggest that the structural features of butyric acid are rather stringently required for induction. This effect is considered in view of the fact that butyric acid is a naturally occurring fatty acid, is effective in relatively low concentrations, and is widely used to form derivatives of cAMP.     

Nuclear lipids in Friend cells. Shifted profile of diacylglycerol during erythroid differentiation induced by DMSO

Friend cells were labelled for 90 min. with [3H]-Glycerol and the radioactivity in DAG# and TG was measured. The relative percentage of radiolabelled DAG differs in isolated nuclei as compared to intact cells. Moreover the level of newly synthesized DAG decreases in nuclei isolated from cells treated with DMSO for 24 hrs. as well as in nuclei from cells treated for 96 hrs. and terminally differentiated in the erythroid pathway. Since these changes are not seen in intact cells, the results are consistent with the hypothesis that nuclear lipids and namely the products of polyphosphoinositide hydrolysis, such as DAG, are involved in the regulation of the processes leading to cell differentiation.     

Surface changes in differentiating Friend erythroleukemic cells in culture.

The sensitivity to agglutination by several plant lectins has been studied during the induced erythroid differentiation of Friend erythroleukemic cells in culture. In addition, the number of lectin receptors on the cell has been measured. It is shown that early during the differentiation, there is an increase in agglutinability while the receptor density remains constant. In the later phase of the differentiation process, the cells lose their sensitivity to agglutination while the receptor number and density increases. These changes were not observed on nonerythroid mastocytoma culture cells. Two nondifferentiating variants of the FL cells were shown to have altered sensitivities to agglutination by ConA.     

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.     

Changes in acid proteolytic activity during differentiation of murine erythroleukemic cells

Proteolytic activity was measured in murine erythroleukemic 745 cell line grown in culture, before and after the addition of agents which promote differentiation. The 36,000 × g soluble fraction of the cells degraded [¹⁴C]globin with maximal activity at pH 3.6, while the insoluble fraction failed to degrade [¹⁴C]globin within a pH range of 2.5–9.0.The acid protease activity in the soluble fraction of the undifferentiated murine erythroleukemic cells increased during the first 2 days in culture and remained constant during the following 4 days. We suggest that this activity resides in the lysosomes since it migrates together with the lysosomal marker α-mannosidase on colloidal silica gradients, shows maximum activity at acid pH and is sensitive towards inhibition by pepstatin. Induced differentiation of the cells by dimethyl sulfoxide, butyric acid or hexamethylene bisacetamide was concomitantly associated with a marked reduction in protease activity and the accumulation of hemoglobin within the cells. In contrast, in a non-inducible variant of 745 cell line DMSO failed to affect proteolysis. It is suggested that in murine erythroleukemic cells changes in acid protease activity are associated with the cellular triggered by chemical inducers.     

Modulation of globin gene expression by a tumor promoter during induced differentiation of Friend erythroleukemic cells

Friend erythroleukemic cells were induced to differentiate by dimethylsulfoxide (Me2SO) in the absence or presence of the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate. The effects of the latter on the molecular parameters related to globin mRNA metabolism were examined. When differentiation was scored by benzidine staining, it had an inhibitory effect on Me2SO-treated cells. On the other hand, when differentiation was followed by determination of globin mRNA accumulation, it had a pleiotropic effect on Me2SO-treated cells. At the early phase of differentiation (2--3 days) the rate of globin mRNA accumulation was higher in the promoter-treated cells than in the control. This unexpectedly high level of accumulation was followed by a sharp reduction and most of the globin RNA sequences disappeared at later stages of differentiation (days 4--5). The reduction can be related to the effect of the promoter on the stability of globin RNA in the cytoplasm which was reduced from a half-life of 16 h to that of 8 h only. Other parameters, such as the rate of globin mRNA synthesis and its capability to serve as a template for cell-free protein synthesis were not affected by treatment with the promoter throughout the differentiation process.     

Effect of interferon on growth and division cycle of Friend erythroleukemic murine cells in vitro

The administration of appropriate doses of interferon to cultures of Friend leukemia cells causes a pronounced inhibition of cell growth. Several lines of evidence indicate that this effect is due to interferon itself, rather than to unknown contaminants of interferon preparations. Autoradiograph analysis of growth parameters of Friend leukemia cells during treatment with interferon demonstrates that the rate of entry into the S phase, the percent decline of unlabeled mitoses, and the mitotic indexes are significantly lower in interferon- treated cell cultures than in control untreated cultures when tritiated thymidine was added 12 h after the administration of interferon. These data indicate that fractions of interferon-treated cell population are delayed in both G1 and in G2 phases of the cell cycle. This was confirmed by exact measurements of the length of the various phases of the cycle. The interferon-induced inhibition of growth of Friend leukemia cells is reversible after removal of the compound. Autoradiograph data obtained from control cultures and from cultures previously treated with interferon that had been washed free of interferon and reseeded in interferon-free medium, demonstrate that during the first 12 h after removal of interferon, a large majority of the cells previously treated with interferon had a deranged flow into the S phase, a high number of unlabeled mitoses, and a low mitotic index. These data provide further evidence for the above-mentioned prolongations of G1 and G2 phases of the cell cycle. All growth parameters tested reverted to normal values within 12 h after washing out interferon.