Differentiation Of Friend Leukemia Cells Research Articles

Role of Ets-1 in transcriptional regulation of transferrin receptor and erythroid differentiation.

High expression of transferrin receptor (TfR) on the membrane of erythroid cells accounts for the high level of iron required to sustain heme synthesis. Several studies indicate that during erythroid differentiation TfR expression is highly dependent on transcriptional regulation. In this study we characterized the minimal region able to confer transcriptional regulation during erythroid differentiation in Friend leukemia cells (FLC). This region of 120 bp, upstream the transcription start site, contains an overlapping consensus recognition sequence for AP1/CREB/ATF transcription factors and for proteins of the Ets family and a GC rich region. Here, we report that both the Ets and the Ap1/CRE like sites are essential for promoter activity during erythroid differentiation. We showed that Ets-1 binds to the EBS-TfR and its binding activity decreases in FLC induced to differentiate and during normal erythroid differentiation. Consistent with this, FLC constitutively expressing Ets-1 show a decrease in TfR gene expression, globin mRNA and hemoglobin synthesis. We conclude that Ets-1 binding activity is modulated during erythroid maturation and that a deregulated expression of this transcription factor interferes with terminal erythroid differentiation.

Induction of erythroid differentiation by the anthracycline antitumor antibiotic pyrromycin.

The oligosaccharide-anthracyclines, aclacinomycin A, marcellomycin and musettamycin, are potent inducers of erythroid differentiation in hemopoietic cells lines of rodent and human origin. The present studies revealed that pyrromycin, a closely related monosaccharide-anthracycline, induced erythroid differentiation in Friend leukemia cells and in the human leukemia cell line K 562. Pyrromycin, marcellomycin and musettamycin, which possess an identical aglycone structure containing a Cl-hydroxyl group, exhibited relatively low optimal inductive concentrations. In contrast, the optimal inductive concentration of aclacinomycin A, which lacks the Cl-hydroxyl group, was markedly higher, i.e., the differentiation inducing capacity was lower. It should be noted, however, that the yield of differentiated cells following treatment with the monosaccharide-anthracycline pyrromycin was distinctly lower than that after treatment with the oligo-saccharide-anthracyclines, aclacinomycin A, marcellomycin or musettamycin. Thus, our data indicate that the efficacy of anthracyclines to induce erythroid differentiation is related to a) the presence of a Cl-hydroxyl group in the aglycone and b) the presence of an oligosaccharide side chain.

In vitro and in vivo studies of poly-L-lysine as inducer of Friend leukemic cells differentiation.

Poly-L-lysine, a synthetic cationic polypeptide known for its ability to bind to cell membranes, was found to induce differentiation of Friend leukemia cells "in vitro". Studies were extended to the same "in vitro" model, in order to examine the therapeutic potential of this new differentiating agent. The i.p. administration of the polymer (Mw 2700) at the maximal tolerated dose resulted in major alterations of disease-related parameters. In particular, a multiple treatment schedule on the advanced disease resulted in a successful reduction of target organ weight and peripheral white blood cell count and appreciable differentiation of spleen and bone marrow cells. Apparently, the effects of poly-L-lysine were superior to those produced by N-methyl-acetamide, a potent inducer of differentiation "in vitro".

Differentiation of Friend leukemia cells induced by .BETA.-, .GAMMA.-, .DELTA.- and aza-.BETA.-lactam, and thiaziadine compounds.

β-, γ-, δ- and aza-β-lactam, and thiaziadine compounds induced the differentiation of Friend leukemia cells and hemoglobin was produced. Modification of the lactam rings of γ- and (δ-lactam compounds with aromatic side chains and bromine atoms enhanced their differentiation-inducing-activities to Friend leukemia cells. On the other hand, 4 thiaziadine compounds (AKT-1, AKT-2, AKT-3 and AKT-4), which commonly contain a -N(CH3)-N(CH3)-moiety, also promoted the differentiation of Friend leukemia cells. They had specific differentiation-inducing-abilities without toxic effects to Friend leukemia cells. 5-Aza-β-lactam compounds showed differentiation-inducing-activity toward all of three types of Friend leukemia cells, two of which were dimethylsulfoxide (DMSO)-sensitive and the other of which was DMSO-resistant. But we did not find any correlation between the differentiation-inducing-activity and the chemical group at the 1-imino position. The presence of an amino bond, which commonly exist in differentiation...

Subcellular distribution of 2′,5′-oligoadenylate synthetase in differentiating Friend leukemia cells

The occurrence of distinct (2′–5′)(A) n-synthetase activities has recently been documented in cytoplasmic and nuclear extracts of several interferon (IFN)-treated cell lines. Since a role has been proposed for (2′–5′)(A) n synthetase in the control of cell growth and differentiation, we examined the subcellular distribution of (2′–5′)(A) n-synthetase activity both in IFN-treated undifferentiated Friend leukemia cells (FLCs) and during dimethyl-sulfoxide (DMSO)-induced erythroid differentiation of FLCs. Both the nuclear and cytoplasmic (2′–5′)(A) n activities were modulated to the same extent by IFNs and DMSO. No evidence for a causal relationship between enzyme activation and FLC differentiation was found.

A New Differentiation Inducer of Friend Leukemia Cells, Trichostatic Acid

FL-657B, which induced differentiation of Friend leukemia cells, was isolated from the culture fluid of Streptomyces sioyaensis and identified with trichostatic acid, a hydrolysis product of trichostatin A and C. FL-657B induced hemoglobin biosynthesis of both dimethyl sulfoxide-sensitive and -resistant Friend leukemia cells. FL-657B caused approximately 90% of the cells to be benzidine positive and reduced the growth to approximately 30~70% of the control at 2.42 μ/ml. Hemoglobin newly biosynthesized by the induction of FL-657B showed a UV absorption pattern similar to that from the normal mouse.

A new differentiation inducer of Friend leukemia cells, trichostatic acid.

FL-657B, which induced differentiation of Friend leukemia cells, was isolated from the culture fluid of Streptomyces sioyaensis and identified with trichostatic acid, a hydrolysis product of trichostatin A and C. FL-657B induced hemoglobin biosynthesis of both dimethyl sulfoxide-sensitive and -resistant Friend leukemia cells. FL-657B caused approximately 90% of the cells to be benzidine positive and reduced the growth to approximately 30~70% of the control at 2.42 μ/ml. Hemoglobin newly biosynthesized by the induction of FL-657B showed a UV absorption pattern similar to that from the normal mouse.

Changes in tRNA levels during the induction of hemoglobin synthesis in Friend leukemia cells

There is a three- to four-fold decrease in the content/cell of tRNAs for ten different amino acids four days after the induction of erythroid differentiation in Friend leukemia cells, consistent with the decrease in cell volume that occurs. Surprisingly, there is an approximately two-fold increase in the cellular content of each of these tRNAs between day 4 and day 6 after induction, indicating the net synthesis of tRNA late in induction. The tRNA changes affect all species and do not result in tRNA specialization for hemoglobin synthesis, as occurs in normal erythroid development. The tRNA content of imidazole-treated cells, which do not synthesize hemoglobin although they undergo other changes of erythroid differentiation, decreases initially as described above, but shows no increase from day 4 to day 6.

Role of interferon and 2‘,5‘-oligoadenylate synthetase in erythroid differentiation of Friend leukemia cells. Studies with interferon-sensitive and -resistant variants.

It has been suggested that the interferon (IFN)-induced 2',5'-oligoadenylate (2-5A) synthetase, which polymerizes ATP into a series of 2',5'-linked oligomers with the general formula pppA(2'p5'A)n, plays a general role in cell growth and terminal differentiation. For instance, an increase in 2-5A synthetase activity has been described during dimethyl sulfoxide (Me2SO)-induced erythroid differentiation of Friend leukemia cells (FLC). 2-5A synthetase has been measured in two Friend leukemia cell sublines by various techniques including a radioimmunoassay of its products which would detect 10(-16) mol of 2-5A cores. Although cells of both sublines fully differentiate (as measured by benzidine staining), only in one subline was there an increase in 2-5A synthetase activity upon treatment with Me2SO. Hexamethylenebisacetamide, another potent agent of differentiation in this system, did not increase 2-5A synthetase activity in either of these two sublines. An IFN-resistant FLC variant differentiated normally upon treatment with Me2SO or hexamethylenebisacetamide while it was noninducible for 2-5A synthetase activity by exogenous IFN or by the inducers themselves. A similar situation has been observed with regard to the level of phosphorylation of the IFN-induced Mr = 67,000 protein band. In addition, treatment of IFN-sensitive and resistant FLC sublines with mouse alpha beta IFN antiserum did not affect differentiation. Even though we have duplicated previous findings on the increase of 2-5A synthetase activity in Me2SO-induced FLC, the lack of any such increase with other inducers or other sublines indicates that there is no causal relationship between the enzyme activation and FLC differentiation.

Immunosuppressive properties of polar organic compounds that induce cellular differentiation in Friend erythroleukemia cells.

We studied the effect of several polar organic compounds, known to induce erythroid differentiation in Friend leukemia (FL) cells, on in vitro human lymphocyte responses and on skin graft survival in mice. The short chain fatty acids, butyric acid (BA), propionic acid (PA), valeric acid, and the polar organic solvents, dimethyl sulfoxide, dimethylformamide, and dimethylacetamide, all mediated significant inhibition of alloantigen-induced proliferation and generation of cytotoxic T lymphocytes (CTLs) in human primary and secondary mixed lymphocyte culture (MLC) reactions. Hexamethylenebisacetemide, another potent inducer of differentiation in FL cells, also mediated significant suppression. Inhibition of MLC with polar organic compounds was accomplished at concentrations known to induce differentiation in FL cells and that are not cytotoxic to peripheral blood mononuclear cells. In distinct contrast, agents that are structurally related to BA, but that do not induce differentiation in FL cells, such as caproic acid, beta-OHBA, gamma-amino BA, and isobutyric acid, did not exhibit immunosuppressive properties. Pokeweed mitogen-driven polyclonal B cell activation was also suppressed by agents that induce erythroid differentiation in FL cells. In addition to potent in vitro immunosuppressive properties, supplementation of drinking water with BA or PA resulted in prolongation of full-thickness skin grafts in DBA/2 (H-2d) to C57BL/6 (H-2b) donor-recipient combinations. Our findings indicate that polar organic compounds that induce differentiation in FL cells are potent immunosuppressive agents.

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.

Changes in cyclic nucleotide levels during erythroid differentiation in Friend leukemia cells

Changes in cyclic nucleotide levels were observed in Friend leukemia cells exposed to the inducers of erythroid differentiation dimethylsulfoxide (DMSO), sodium butyrate and hypoxanthine. The most extensive changes in cAMP levels occurred during the initial 24 h growth period. In uninduced cultures, cAMP concentration increased 35-fold compared with a 7-fold increase in the presence of inducer. During the same period. cGMP levels rose about 3-fold in uninduced cells compared with a 20-fold increase in induced cells.

Cholera toxin and its B subunit inhibit interferon effects on virus production and erythroid differentiation of Friend leukemia cells

Treatment of Friend leukemia cells with cholera toxin or with its purified B subunit inhibits the interferon (IFN)-induced reduction of Friend virus release and of virus yields after exogenous infection with vesicular stomatitis virus. Likewise, the “enhancement” of erythroid differentiation of DMSO-treated Friend cells induced by low doses of crude or semipurified IFN is completely abolished. These results, confirmed with pure IFN preparations, are in keeping with the hypothesis that an early interaction with the cell membrane is a necessary step for the establishment of these different IFN effects. In addition, they indicate that the increased cyclic AMP levels, induced by the whole toxin, do not play a significant role in these phenomena; rather they may be due to the lectin-like activity of the B subunit. Evaluation of reciprocal dose-response curves of IFN and cholera toxin shows that treatment with toxin concentrations as low as 10 13 M induces a major inhibition of IFN effects. A millionfold higher toxin concentration does not substantially increase the toxin actions. The toxin-induced redistribution of membrane gangliosides is discussed as a possible mechanism of the observed inhibition of IFN activities.

Effect of calcium on differentiation of Friend leukemia cells

Induction of hemoglobin synthesis of Friend leukemia cells is inhibited by changing the ratio between internal and external Ca 2+ concentrations. The concentration ratio can be successfully manipulated by the addition of the growth medium of (1) Ca 2+ channel blocker D600 (90 n M-4 × 10 2 n M), (2) Ca 2+ ionophore A23187 (1 × 10 2–2 × 10 2 n M), and (3) EGTA at molar concentrations comparable to the Ca 2+ concentration of the medium formulation (3 × 10 2 μ M). The observations suggest that a specific ratio between intra- and extracellular Ca 2+ is required for erythroid differentiation to proceed.

A new class of inhibitors of lymphocyte mitogenesis: agents that induce erythroid differentiation in Friend leukemia cells.

Addition of the polar organic compounds, dimethylsulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, and butyric acid, to human lymphocyte cultures stimulated with the tumor-promoting agent, phorbol myristate acetate, results in greater than 90% inhibition of lymphocyte proliferation. Inhibition is achieved at concentrations of the organic compounds reported to be optimal for induction of erythroid differentiation in Friend leukemia cells. Butyric acid is the most potent compound tested. Compounds that are structurally related to butyric acid, but that do not induce erythroid differentiation, do not inhibit lymphocyte mitogenesis. Lymphocyte responses to other mitogens are also suppressed by the polar organic compounds, although higher concentrations are required. These agents are much less inhibitory when added 24 hr after initiation of the cultures, indicating that they may affect an early phase of lymphocyte activation. Compounds that induce erythroid differentiation in Friend leukemia cells constitute a new class of inhibitors of lymphocyte mitogenesis.

Alterations in tRNA isoaccepting species during erythroid differentiation of the Friend leukemia cell

The chromatographic profiles of isoaccepting tRNAs were analyzed at five time points during the 96 hr, dimethylsulfoxide induced, erythroid-like differentiation of Friend leukemia cells. Sixty-four isoaccepting species of tRNA for 16 amino acids were resolved by RPC-5 chromatography. The relative amounts of tRNAphe, tRNAile, and tRNAval species were maintained by the cells during differentiation; whereas the relative amounts of some of the isoacceptor tRNAs for the other 13 amino acids changed significantly. Fluctuations in amounts of isoacceptors occurred between 36 and 72 hr after addition of dimethysulfoxide, corresponding to globin mRNA appearance and hemoglobin synthesis, respectively. In most cases, thepredominant tRNA isoacceptors of uninduced cells were retained throughout differentiation. Notable exceptions were tRNA species for threonine, proline, and methionine. Some of the isoacceptors occurring in relatively smaller amounts were not expressed at all times. These changes possibly reflect the cell's functional adaptation of tRNA in differentiation for hemoglobin synthesis.

Isozyme patterns of pyruvate kinase and differentiation of Friend leukemia cells.

The isozyme patterns of glycolytic enzymes of Friend leukemia cells (FLC) were compared with those of erythrocytes and erythroblasts. Erythrocyte-specific R types of pyruvate kinase (PK) were clearly observed in phenylhydrazine-induced mouse erythroblast, and much less amount of them was also observed in Friend leukemia cells. When FLC were induced to differentiate by hexamethylene-bisacetamide (HMBA), the R types were slightly reduced. When the induction of differentiation was inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA), the R types and M2-R hybrids rather increased. These results are reverse of those obtained when hemoglobin production is used as a marker of differentiation. Isozyme patterns of lactic dehydrogenase and aldolase did not change during differentiation of FLC induced by HMBA, and were the same as those of mouse erythroblasts and erythrocytes.

Early changes in ornithine decarboxylase activity during friend leukemia cell differentiation

Agents such as dimethylsulfoxide, N, N′-dimethylformamide and bisacetyldiaminopentane that induce erythroid differentiation of Friend leukemia cells, cause a rapid increase in ornithine decarboxylase (EC 4.1.1.17) activity in intact cells during the ‘latent’ period preceding the accumulation of hemoglobin-containing cells. Blockage of erythroid differentiation with 5-bromo-2′-deoxyuridine did not prevent these alterations in enzyme activity. Addition of each chemical inducer in the extracts of these cells stimulate the basal levels of ornithine decarboxylase activity. These data indicate that the chemical inducers of differentiation modify the normal pattern of ornithine decarboxylase activity in this system.

Water permeability changes studied by 17O nuclear magnetic resonance during differentiation of Friend leukemia cells.

Water permeability of Friend leukemia cells was studied by 17O nuclear magnetic resonance during differentiation induced by dimethyl sulfoxide (Me2SO). While in noninduced cells water permeability was essentially constant during the growth period, in the Me2SO-induced cells there were two distinct periods at which the water permeability was increased by at least an order of magnitude. These periods correspond to approximately one doubling time and 6 days of growth. This change in water permeability is not due to direct interaction of Me2SO with the membrane but must be ascribed to structural changes in the membrane during the course of differentiation.

Erythroid differentiation of Friend leukemia cells as studied by acridine orange staining and flow cytometry.

Free accessAbstractFirst published January 1979Erythroid differentiation of Friend leukemia cells as studied by acridine orange staining and flow cytometry.F Traganos, Z Darzynkiewicz, […], T K Sharpless, and M R Melamed, +1 -1Volume 27, Issue 1https://doi.org/10.1177/27.1.86567