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.     

Prostaglandin A1 induces differentiation in Friend erythroleukemia cells.

The effect of different prostaglandins and prostaglandin-metabolites on the growth and differentiation of Friend erythroleukemia cells (FLC) was evaluated. The prostaglandin-metabolites, thromboxane B2 and 6-keto PGF1 alpha, were completely inactive, while PGE1 inhibited slightly and PGF2 alpha stimulated the replication of FLC. PGA1 was found to be the most active compound. It profoundly inhibited the replication of both DMSO-treated and undifferentiated FLC. Most importantly, PGA1 alone induced differentiation in FLC, stimulating hemoglobin production over a five-day period. PGA1-stimulated differentiation was completely suppressed by the addition of 10(-6)M hydrocortisone. Finally, treatment of DMSO-differentiated cells with PGA1 (but no DMSO) prevented the return to the undifferentiated state.     

Folate utilization in Friend erythroleukemia cells

In order to study the generation, factors controlling endogenous folate pools, and their functional importance, Friend erythroleukemia cells were grown in media containing 100; 1,000; and 10,000 ng/ml of tritiated pteroylglutamic acid (3H)PteGlu1 and then studied in unlabeled media with varying amounts of PteGlu1. The intracellular folate pool was directly proportional to the PteGlu1 in which the cells were incubated. At equilibrium, greater than 95% of the labeled intracellular folate pool chromatographed as polyglutamyl folate, regardless of the exogenous folate concentration. The functional importance of the intracellular folate pool was studied by varying the endogenous pool and the exogenous (media) supply. The ability of the cells to replicate in the absence of exogenous folate was directly proportional to the intracellular polyglutamyl folate pool. The maximal rate of replication, however, required exogenous PteGlu1 in addition. The cell doubling time was the most important determinant of intracellular folate turnover; changes in the intracellular pool size and the extracellular folate concentration had no effect on the turnover time. In a rapidly proliferating tissue, the onset of functional folate deficiency will be determined by dilution of intracellular polyglutamates among progeny until a critical level is reached.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Lipophilic proteins of Friend erythroleukemia cells

Lipophilic proteins can be extracted from Friend mouse erythroleukemia cells (MELC) with acidic chloroform-methanol. The acidic extract contains at least 4 polypeptides of apparent M. W. 5, 9.5, 14 and 17 kdaltons as determined by SDS-polyacrylamide gel electrophoresis (PAGE). Delipidation of the extract with ether causes the formation of polymers of an apparent molecular weight ranging from 25 to 85 kdaltons, and strong binding of aminoacids, sugars and phospholipids, in particular phosphatidylinositol and phosphatidylethanolamine, to the polypeptides. Though the majority of the lipophilic proteins are of cellular origin, part of the polypeptides of M.W. 14 and 17 kdaltons may be viral components.     

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.     

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.     

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.     

Role of fibronectin in the adhesion of Friend erythroleukemia cells

Friend Erythroleukemia Cells (FLC) (745 A and FW clones), normally growing in suspension, tend to adhere to fibroblast monolayers, but not to epithelial cells. Co-cultivation of FLC with Human Embryo Fibroblasts (HEF) resulted in the selection of adhesive Friend Cells. After 16 subcultures, we were able to isolate clones of adhesive FLC that grow in monolayer on plastic tissue culture plates. Both the binding of FLC to fibroblasts and of the adhesive clones to the plastic surfaces is completely suppressed by fibronectin antiserum, thus suggesting that fibronectin is responsible for FLC adhesion. Adhesive FLC clones maintain the ability to differentiate upon induction by DMSO.     

Arabinosylcytosine downregulates thymidine kinase and induces cross-resistance to zidovudine in T-lymphoid cells

The aim of this study was to determine molecular mechanism(s) responsible for the reduced thymidine kinase activity (TK) observed earlier in an arabinosylcytosine (araC) resistant lymphoid cell line (H9-araC cells), which was obtained following continuous cultivation of H9 cells in the presence of 0.5 microM araC. Compared to H9 cells, in H9-araC cells TK1 and TK2 gene expressions were reduced to 17.7% and 2.5%, respectively, and the cellular AZT accumulation was diminished to 35.8%. These cells were also found cross-resistant to azidothymidine (>42-fold). There was no significant difference in the expression of MDR1, MRP4 or TK protein. The lack of correlation between the expressions of TK protein and TK1 and TK2 suggests that post-translational factors may also play a role in the reduced TK activity in H9-araC cells. These findings suggest that araC affects TK expression at the genetic level.     

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.     

Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line

Erythroid cells from mice infected with the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP), unlike normal erythroid cells, can proliferate and differentiate in apparent absence of the erythroid hormone erythropoietin (Epo). The unique envelope glycoprotein encoded by SFFV has been shown to be responsible for this biological effect. The recent isolation of an Epo-dependent erythroleukemia cell line, HCD-57, derived from a mouse infected at birth with Friend murine leukemia virus, afforded us the opportunity to study the direct effect of SFFVP on a homogeneous population of factor-dependent cells. The introduction of SFFVP in complex with various helper viruses into these Epo-dependent cells efficiently and reproducibly gave rise to lines which expressed high levels of SFFV and were factor independent. SFFV appears to be unique in its ability to abrogate the factor dependence of Epo-dependent HCD-57 cells, since infection of these cells with retroviruses carrying a variety of different oncogenes had no effect. The induction of Epo independence by SFFV does not appear to involve a classical autocrine mechanism, since there is no evidence that the factor-independent cells synthesize or secrete Epo or depend on it for their growth. However, the SFFV-infected, factor-independent cells had significantly fewer receptors available for binding Epo than their factor-dependent counterparts had, raising the possibility that the induction of factor independence by the virus may be due to the interaction of an SFFV-encoded protein with the Epo receptor.     

Inhibition of DNA methylation in Friend erythroleukemia cells by actinomycin D

Actinomycin D caused the production of hypomethylated DNA in cultured Friend erythroleukemia cells at cell culture concentrations of 1-4 ng per ml. Inhibition of DNA methyltransferase in cell-free assays was kinetically complex, with mixed-type inhibition. Cornish-Bowden graphical analysis was used to derive a Ki of about 35 nmol Act D per mg DNA. Although nuclei from drug-treated cells were found to contain hypomethylated DNA and DNA methyltransferase could be extracted from the nuclei, the methyl-accepting ability of DNA in whole nuclei themselves was not elevated. We conclude that the low level of Act D bound to DNA in the nuclei is sufficient to prevent the remethylation of hypomethylated sites.     

Erythroid differentiation factor stimulates hydrolysis of polyphosphoinositide in Friend erythroleukemia cells

We examined an early action of erythroid differentiation factor (EDF), a polypeptide which induces differentiation of Friend murine erythroleukemia (MEL) cells. (Eto et al., Biochem. Biophys. Res. Commun. 142: 1095-1103, 1987). In MEL cells, EDF caused a rapid and transient increase in cytoplasmic concentration of free calcium, [Ca2+]c. EDF increased [Ca2+]c even in the absence of extracellular calcium. When [3H]inositol-labeled MEL cells were incubated with EDF, EDF rapidly increased radioactivity in inositol trisphosphate, bisphosphate and monophosphate. EDF also increased [3H] diacylglycerol in [3H]arachidonate-labeled MEL cells. These results indicate that EDF increases [Ca2+]c by stimulating hydrolysis of polyphosphoinositide.     

Subcellular localization of the env-related glycoproteins in Friend erythroleukemia cells.

A scheme was developed for the subcellular fractionation of murine erythroleukemia cells transformed by Friend leukemia virus. The subcellular localization of the env-related glycoproteins was determined by immune precipitation with antiserum against gp70, the envelope glycoprotein of the helper virus, followed by gel electrophoresis. In cells labeled for 2 h with [35S]methionine, the glycoprotein encoded by the defective spleen focus-forming virus, gp55SFFV, was found primarily in the nuclear fraction and in fractions containing dense cytoplasmic membranes such as endoplasmic reticulum. A similar distribution was noted for gp85env, the precursor to gp70. The concentration of viral glycoproteins in the nuclear fraction could not be accounted for by contamination with endoplasmic reticulum. In pulse-chase experiments, neither glycoprotein underwent major redistribution. However, labeled gp85env disappeared from intracellular membranes with a half-time of 30 min to 1 h, whereas labeled gp55SFFV was stable during a 2-h chase. In plasma membrane preparations with very low levels of contamination with endoplasmic reticulum, gp70 was the major viral env-related glycoprotein detected; a minor amount of gp55SFFV and no gp85env could be detected. The unexpected result of these experiments is the amount of viral glycoproteins found in the nuclear fraction. Presence of viral proteins in the nucleus could be relevant to the mechanism of viral leukemogenesis.     

Modification of lipophilic proteins in Friend erythroleukemia cells during their differenciation

In mouse erythroleukemia cells (MELC) a lipophilic protein of apparent M. W. 9.5 kdaltons increases during differentiation. This increase is due either to an increase of biosynthesis or to a structural alteration impairing the capacity of the protein to form polymers of apparent high M.W. or favoring its extractability. The increase is related to differentiation and precedes hemoglobin synthesis by at least 1 day. It is not related cells (TFA-11) in which dimethylsulfoxide (DMSO) causes an increase in virus production. As it occurs in cells treated with 4 different inducers, and as the increase is less marked when antagonists of the inducers are also present, it is unlikely that the increase of the 9.5 Kdalton protein is due to an effect of the inducers unrelated to differentiation.     

Nucleosome-associated proteins and phosphoproteins of differentiating Friend erythroleukemia cells.

Mononucleosomes derived from brief digestion of uninduced Friend cell nuclei with micrococcal nuclease contain a set of non-histone chromosomal proteins which are partly or altogether missing in the oligomeric nucleosomes. On the other hand, the latter contain a protein of Mr 190,000 not seen in the mononucleosomes. Longer digestion removes most of these non-histone proteins, excepting the Mr 190,000 protein. Brief digestion of nuclei from Friend cells induced by DMSO or by n-butyrate removes most of the non-histone proteins from the nucleosomes, as did the prolonged digestion of uninduced nuclei. The Mr 190,000 protein remains, while a protein of Mr 27,000 is increased. The rate of phosphorylation of histone H1 associated with mononucleosomes was 3 to 4-fold greater in cells induced with DMSO. The major phosphoprotein and most of the other phosphorylated non-histones were modified at the same rate in control and induced cells. However, a Mr 95,000 protein was less phosphorylated in the induced cells.     

Electron-spin resonance studies of lipid-modified microsomes from Friend erythroleukemia cells

The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5-nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.