Differentiation Of Friend Cells Research Articles

An early decrease in phosphatidylinositol turnover occurs on induction of Friend cell differentiation and precedes the decrease in c-myc expression.

Phosphatidylinositol turnover has recently been implicated in the regulation of proliferation and transformation. Its role in differentiation has now been investigated using Friend erythroleukemia cells, which can be induced to differentiate along the erythroid pathway by dimethylsulfoxide and certain other agents. We have found that levels of the phosphatidylinositol metabolites inositol-trisphosphate and diacylglycerol significantly decrease within 2 hr of induction of Friend cell differentiation. These decreases precede decreased expression of the c-myc proto-oncogene and its protein product. Phorbol 12-myristate, 13-acetate, which can mimic diacylglycerol, blocked differentiation without reversing the decrease in phosphatidylinositol metabolite levels. Two synthetic diacylglycerols, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol and sn-1,2-dioctanoylglycerol, also blocked differentiation and commitment. Diacylglycerol regulation of kinase C activity may play a key role in control of c-myc expression and Friend cell differentiation.

Effect of the antiglucocorticoid agent RU 38486 on the dexamethasone inhibition of Friend cell differentiation

Glucocorticoid hormones are known to inhibit the erythroid differentiation of Friend cells. The mechanism of action of these hormones has been questioned, and results suggesting an action not involving the nuclear binding of the receptors have been published. We have used the antiglucocorticoid RU 38486 to block the inhibitory effect of dexamethasone on the induced differentiation of Friend cells. Our results strongly suggest a glucocorticoid action involving the binding of classical receptors to the cell nucleus.

Peripheral-type benzodiazepines influence ornithine decarboxylase levels and neurite outgrowth in PC12 cells.

A number of the benzodiazepines (BZDs) inhibit nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner in PC12 cell cultures. The rank order of potency of a series of BZDs for inhibition of neurite outgrowth does not correlate with the order of their affinity constants for the so-called peripheral BZD sites present on PC12 cells. Whereas the inhibition of neurite extension is stereospecific, the binding to the peripheral site is not. The inhibition of neurite outgrowth is not attributable to a blockade of NGF binding to either its fast or slow receptors. Additionally, several other characteristics of NGF stimulation of PC12 cells, such as autoadhesion and the induction of ornithine decarboxylase (OrnDCase), remain unaltered in the presence of BZDs. Many BZDs increase OrnDCase levels in PC12 cells in the absence of NGF. The OrnDCase response to BZDs is blocked by actinomycin D. Furthermore, the structural requirements of BZDs for induction of OrnDCase activity is not identical to that for the inhibition of NGF-induced neurite extension. Thus, unlike attenuation of neurite extension, BZD induction of OrnDCase is not stereospecific. Also, some BZDs block neurite growth but do not induce OrnDCase. We propose that there are at least three sites of action of BZDs on PC12 cells. The first is the well-characterized high-affinity peripheral site. The second distinct locus of action results in a blockade of NGF-induced neurite outgrowth. The structural requirements for the latter effect are essentially indistinguishable from those for BZD induction of hemoglobin synthesis in Friend erythroleukemia cells. The third site of action results in an induction of OrnDCase. Structural requirements for this activity are not identical to those for either the differentiation of Friend cells or the inhibition of neurite outgrowth.

Phenotypic Heterogeneity Among Stromal Cell Lines From Mouse Bone Marrow Disclosed in Their Extracellular Matrix Composition and Interactions With Normal and Leukemic Cells

Abstract Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA-2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

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.

Induction and modification of erythropoiesis in a suspension culture system derived from long-term bone marrow culture of Syrian hamster

Cells in a subculture system derived from long-term hamster bone marrow culture were induced to differentiate into hemoglobin-producing cells in response to added erythropoietin. Treatment of the subculture with dimethyl sulfoxide or other ‘Friend inducers’ but not 5-azacytidine effected no remarkable induction, though they enhanced the effect of externally added erythropoietin. 3-Aminobenzamide, a weak inducer of Friend cell differentiation [Morioka, K., K. Tanaka, M. Ishizawa and T. Ono: Dev. Growth Differ. 24, 507–512 (1982)], had a strong inhibitory effect on erythropoiesis in the subculture system. Phorbol esters and glucocorticoids (both are known to be strong inhibitors of Friend cell differentiation) acted as strong inhibitors of the differentiation in our system as well. The present system was shown to provide a useful in vitro model to compare the differentiation of normal erythroid progenitors with the previously established systems of neoplastic cells.

The role of Ca2+ in dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells.

Cultured Friend cells can be induced by dimethyl sulfoxide (Me2SO) and several other agents to mature along the erythroid pathway. Evidence has been presented that an increase in Ca2+ influx is an early and necessary prelude to the commitment to maturation by these cells (Levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5948-5952). The simplest hypothesis supporting all the available data is that Me2SO and other inducers elevate the cytosolic Ca2+ concentration. We have now measured cytosolic Ca2+ using the fluorescent indicator quin-2, and find, contrary to expectation, a small decrease upon treatment of cells with Me2SO. Cytosolic Ca2+ was increased by raising the Ca2+ in the medium, but was not dramatically altered by addition of ouabain or monensin or by incubation in Na+-free medium. Measurement of total cell Ca2+ by a triple-labeling technique using 3H2O and 125I-albumin to determine cell water and extracellular space, respectively, revealed no significant change upon treatment with Me2SO for up to 40 h. A decrease in the initial rate of 45Ca2+ influx was observed in Me2SO-treated cells, when measured at 4 degrees C. These data do not support the hypothesis that an increase in cell Ca2+ is necessary for the induction of Friend cell differentiation or that Na+/Ca2+ exchange is a significant regulator of cytosolic Ca2+ in Friend cells.

Increase in histone acetylation and transitions in histone variants during friend cell differentiation

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [ 3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.

The effect of induction of hemoglobin synthesis in cultured friend cells on the number of initiation sites for replication and transcription

It was found that differentiation of murine erythroleukemia cells can be induced by 5-fluorodeoxyuridine (FudR), amethopterin and α-aminoisobutyrate. Each of these compounds is believed to delay the onset of DNA synthesis. Since relief of the FudR block to DNA synthesis by addition of thymidine can increase the number of initiation sites for replication (Taylor, 1977), the effect of various inducers and inhibitors of differentiation of Friend cells upon the relative number of initiation sites for replication and transcription was investigated. Very efficient inducers of hemoglobin synthesis, hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (DMSO), increase the number of initiation sites for transcription and HMBA also increases the number of functional initiation sites for replication. Two other compounds that induce differentiation of Friend cells, low levels of actinomycin D and FudR, did not increase the number of initiation sites for transcription. Compounds that prevent induction of hemoglobin synthesis by HMBA and DMSO include 5-bromodeoxyuridine (BrdU) and novobiocin. Both of these compounds were found to decrease the number of functional initiation sites for transcription and it is known that both compounds reduce the number of initiation sites for replication. The relation between initiation of replication and transcription, and its effect upon differentiation of erythroleukemia cells is discussed.

Effects of differentiation inducers on diphenylhexatriene fluorescence polarization in intracytoplasmic and plasma membranes from friend erythroleukemia cells

Treatment of Friend leukemia cells with dimethylsulfoxide or hexamethy-lenbisacetamide, which induced erythroid differentiation, resulted in enhancement of fluorescence polarization of diphenylhexatriene in not only plasma membranes, but also in intracellular membranes. In a cell variant resistant to induction, the polarization values of intracellular membranes were not affected by the inducing agents, whereas plasma membranes had the same enhancement of polarization values as in sensitive cells. Therefore, Friend cell differentiation can be associated with the effect of the inducers on intracellular membranes, but not with the effect on plasma membranes.

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 Freind 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 of inducer-treated cells to continue differentiation after a discontinuous exposure to inducer). Treatment of Friend cells with dexamethasone did not prevent the occurence of DMSO-induced alterations in cell volume. However, dexamethasone treatment prevented a series of biochemical events 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 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 earyl steps of DMSO-induced Friend cell differentiation.

Indirect induction of erythroid differentiation in mouse Friend cells: evidence for two intracellular reactions involved in the differentiation.

The mechanism of in vitro erythroid differentiation in mouse Friend cells was studied by employing cell fusion between two genetically marked Friend cells and other nonerythroid cells, including BHK (baby hamster kidney) and FM3A (mouse mammary gland) cells. We were able to induce erythroid differentiation indirectly by fusing Friend cells that had been exposed briefly to dimethyl sulfoxide prior to fusion with nonerythroid cells that had been treated with ultraviolet light (or other DNA-damaging agents). The results suggest that two distinct reactions are involved in erythroid differentiation in Friend cells in vitro. One reaction, originating from the damaged DNA (or inhibition of DNA replication as a consequence), exhibits an inducible nature, is nonspecific to Friend cells, and is trans-acting. The other reaction is specific to Friend cells and most likely is cis-acting. We also present evidence from the cell fusion experiments that a typical tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, inhibits erythroid differentiation by affecting the latter reaction. The biological significance of these findings is discussed.

The inhibitory effect of some lectins on the differentiation of Friend erythroleukemia cells

Growth and differentiation of Friend cells can be inhibited by treatment with concanavalin A and wheat-germ agglutinin. This inhibition is specific for cells that are in the early stages of the differentiation process (24–48 h after the addition of dimethyl sulfoxide) and is reversible upon treatment with the sugars competitive for these lectins. These results suggest a regulatory role for some plasma membrane-bound glycoproteins early in the differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide.

Regulation of cAMP-dependent protein kinase subunit levels in Friend erythroleukemic cells. Effects of differentiation and treatment with 8-Br-cAMP and methylisobutyl xanthine.

Friend erythroleukemic cells contain both type I and type II cAMP-dependent protein kinases. The amounts of type I and type II regulatory subunits (R I and R II) were established by a combination of the equilibrium binding of cyclic [3H]AMP and specific immunoprecipitation. In control cells the total specific cAMP-binding activity was 9.8 pmol/mg of cytosolic protein and the ratio of R II to R I was 1.2. After the induction of differentiation by growth in 2% dimethyl sulfoxide, the total cAMP-binding capacity doubled, but larger and opposing changes in R II and R I levels were observed. The concentration of R II tripled (to 16.6 pmol/mg) while R I content (1.5 pmol/mg) declined to one-third of the control cell level, resulting in an R II:R I value of 11.1. Treatment of Friend cells with 0.5 mM 8-bromoadenosine 3':5'-monophosphate (8-Br-cAMP) and 0.2 mM methylisobutyl xanthine for 2 days did not stimulate cell differentiation. however, the cAMP-binding capacity nearly doubled and the cells accumulated a high level of R II while R I declined. This resulted in a distribution of R subunits closely resembling that of differentiated cells. Cyclic AMP-stimulated histone kinase activity increased in proportion to cAMP-binding activity during differentiation; no such increase was seen following 8-Br-cAMP treatment. Fractionation of cytosolic extracts by DEAE-cellulose chromatography revealed that both R I and R II were fully associated with the catalytic subunit (C) of protein kinase before and after differentiation. In extracts of 8-Br-cAMP-treated cells all of R I and 40 to 60% of R II were not associated with C. Free R II isolated from these cells was indistinguishable from RII present in holoenzyme. Thus, erythroid differentiation of Friend cells results in a coordinate accumulation of R II and C subunits as well as a decline in R I content. In contrast, chronic exposure to 8-Br-cAMP elicits similar changes in R subunit levels but disrupts the coordinate regulation of R and C content.

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.

The effect of dexamethasone on the initiation of beta-globin gene transcription in differentiating Friend cells.

Plasmid subclones of the 5' and 3' regions of the beta major-globin gene have been used as probes to assay the level and character of the transcripts of this gene in differentiating Friend erythroleukemia cells treated with dexamethasone. Using an S1 nuclease protection protocol, it is shown that dexamethasone reduces the amount of precursor-specific sequences in differentiating Friend cells within 30 min without significantly affecting the nuclear turnover of these molecules. The sites of initiation and termination of transcription remained the same during the shutdown of transcription by the hormone. In contrast to the situation in Friend cells, dexamethasone failed to inhibit globin gene transcription in three normal erythroid tissues. The manner in which the Friend virus complex might render the globin genes sensitive to glucocorticoids is discussed.

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.

Membrane transport during erythroid differentiation.

Transport, unidirectional flux, of a monosaccharide, a nucleoside and three amino acids, all of which enter cells by independent, discrete carriers, was compared at three stages of erythroid maturation, the normal (anucleate) mouse erythrocyte, and in differentiated and undifferentiated Friend erythroleukemia cells. We found specific transport alterations during this developmental program. Transport of 3-O-methylglucose increased with each successive developmental stage. Aminoisobutyrate transport was maintained during Friend cell differentiation, but fell slightly in erythrocytes. Leucine, lysine and uridine transport began to fall two days after dimethylsulfoxide exposure, and diminished further in red cells. These studies of transport are not directly comparable to uptake studies reported by others. Median cell volume and thus surface area decreased more during differentiation than amino acid transport declined, so flux, transport past a unit area of membrane, actually increased. Monosaccharide flux also increased. Only uridine transport fell in parallel to surface area. Perhaps sites for nutrient transport required for energy production are preferentially maintained.

Lipid composition of Friend leukemia cells following induction of erythroid differentiation by dimethyl sulfoxide.

The effects of dimethyl sulfoxide (DMSO)-induced differentiation of Friend leukemia cells in vitro on the lipid composition of these cells have been examined. DMSO had no early effect on the incorporation of either [14C]glycerol or [3H]methyl choline chloride into the total lipids or individual phospholipids of Friend cells up to 240 min after addition of the inducer. Examination of DMSO-differentiated Friend cell phospholipids revealed a percentage composition which was similar to control cells, with phosphatidylcholine and phosphatidylethanolamine in both uninduced and differentiated cells accounting for over 75% of the total phospholipid. Sphingomyelin levels were significantly lower in Friend cells than in normal adult mouse erythrocytes, and differentiation of murine erythroleukemia cells resulted in a further lowering of this phospholipid. In contrast, a significant increase in the level of phosphatidylethanolamine occurred as a result of maturation. Fatty acid analysis of major lipid classes of differentiated Friend cells showed significant reduction in saturation, but no alteration in chain length in comparison to undifferentiated cells. A pronounced decrease in the cellular content of both free and esterified cholesterol, which resulted in a 45% decrease in the ratio of cholesterol/phospholipids, occurred in cells differentiated by the polar solvent. The findings indicate that erythrodifferentiation induced by DMSO results in a variety of changes in the lipid composition of the membranes of Friend leukemia cells.