Somatic cell hybrids between Friend erythroleukemia cells and mouse hepatoma cells.

Somatic cell hybrids between hepatoma and Friend erythroleukemia parental cells were studied for the expression of liver-specific and erythroid properties. Several independent clones were isolated using HAT selection and were shown to be true hybrids by isozyme and chromosome analysis. All displayed a complete extinction of hemoglobin and globin mRNA production, but a retention of albumin and transferrin secretion. The data suggest that erythroid differntiation is being actively inhibited by the hepatoma genome. Possible mechanisms that might explain these results are discussed in the light of current hypotheses regarding the mechanism of cell differentiation.     

Hydrocortisone inhibits DMSO - induced differentiation of Friend erythroleukemia cells.

Hydrocortisone (10^?6 – 10^?7M) completely inhibited the production of hemoglobin by DMSO- and DMF-treated Friend erythroleukemia cells (FLC) in vitro without affecting either cell replication or general protein synthesis. Only 11, 17-dihydroxycorticosteroids were effective in inhibiting this expression of differentiation. Addition of hydrocortisone as late as 48 hours after the addition of DMSO (at a time at which cells were committed to differentiation) still resulted in significant inhibition of hemoglobin synthesis. Although the mechanism of this action is unknown, since it was not reversed by the addition of arachidonic acid nor a number of prostaglandins, it appears to be unrelated to the ability of corticosteroids to inhibit endogenous prostaglandin synthesis.     

Nuclear inositol lipids in Friend erythroleukemia cells. Changes related to differentiation induced by hexamethylenebisacetamide

Subcellular distribution of inositol lipids has been studied in Friend Erythroleukemia Cells following induction to erythroid differentiation with hexamethylenebisacetamide, after labelling with [3H]myo-inositol. In situ autoradiography indicated that inositol-derived molecules were present also in the nuclear compartment of uninduced and induced cells. Fractionation studies showed that the nuclear polyphosphoinositides were deeply changed after short induction times, while the whole cell inositol lipids resulted only slightly modified by the inducer. The nuclear recovery of phosphatidylinositol 4,5-bisphosphate was largely increased after 2 hrs of induction, suggesting that inositol lipid metabolism is involved in the early differentiation events occurring at the nuclear level.     

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.     

Hemin control of heme biosynthesis in mouse Friend virus-transformed erythroleukemia cells in culture.

Hemin treatment of mouse Friend virus-transformed cells in cultured caused a dose-dependent increase in hemoglobin synthesis. By the addition of radioactively labeled hemin and by the analysis of the radioactive heme in hemoglobin, only 60 to 70% of heme in the newly synthesized hemoglobin was accounted for by the exogenously added hemin. In keeping with this finding, hemin treatment increased the activity of two enzymes in the heme biosynthetic activity, i.e. delta-aminolevulinate (ALA) dehydratase and uroporphyrinogen-I (URO) synthase in these cells. Incorporation of [2(-14C)]glycine, [14C]ALA, and 59Fe into heme was also significantly increased in the cells treated with hemin, suggesting that essentially all enzyme activities in the heme biosynethetic pathway were increased after hemin treatment. These results indicate that heme in the newly synthesized hemoglobin in hemin-treated Friend cells derives both from hemin added to the culture and from heme synthesized intracellularly. In addition, these results suggest that the stimulation of heme biosynthesis by hemin in Friend virus-transformed cells is in contrast to the hemin repression of heme biosynthesis in liver cells.     

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.     

Specific binding of 125I-rErythropoietin to Friend polycythemia virus-transformed erythroleukemia cells purified by centrifugal elutriation

We have used countercurrent centrifugal elutriation (CCE) to determine the distribution of cells with respect to cell volume and buoyant density for an erythroleukemia cell line (JG6) transformed by the polycythemia strain of Friend virus (FV-P), and to determine the effect of inducing the cells to differentiate with dimethylsulfoxide (DMSO) on this distribution. CCE made it possible to obtain suspensions of modal JG6 populations virtually free of dead cells and uniform with respect to volume and buoyant density. These modal populations were assayed for specific binding of erythropoietin (Epo). Between 500 and 550 Epo receptors per cell were detected. These belonged to a single class having a dissociation constant of 0.36 nM. DMSO induction of differentiation of the JG6 cells had no effect on the number of Epo receptors expressed.     

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.     

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.     

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.     

Stimulation of protein synthesis by hemin in extracts of Friend erythroleukemia cells.

Extracts prepared from Friend erythroleukemia cells were highly active in translating endogenous mRNA and a consistent 2-fold stimulation by hemin was observed. When extracts were treated with micrococcal nuclease and incorporation was dependent on exogenous globin mRNA, there was more significant stimulation by 37.5 micron hemin and greater than 10-fold stimulation by 75 or 150 micron hemin. The effects of hemin were not strikingly different in extracts of dimethyl-sulfoxide-induced or uninduced cells. The results could reflect an effect on initiation of protein synthesis analogous to that in rabbit reticulocytes.     

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.     

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.     

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.     

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.     

Interferon transiently modulates intranuclear signalling system in erythroleukemia Friend cells.

The effect of human recombinant DNA interferon alpha type A on nuclear inositol lipids, diacylglycerol (DAG) and DNA metabolism has been investigated in Friend erythroleukemia cells. A transient enhancement of phosphatidylinositol (4,5) - bisphosphate (PIP2) phosphorylation together with an increase of diacylglycerol mass were observed in nuclei isolated from cells treated with interferon alpha for 90 min. At the same time, a marked reduction of DNA polymerase alpha activity was observed, suggesting a possible involvement of nuclear inositol fraction in the response of the cell nucleus to interferon treatment.     

Toxoplasma gondii: penetration into differentiating Friend erythroleukemia cells.

The ability of Toxoplasma gondii tachyzoites to penetrate erythroid-differentiating Friend erythroleukemia cells (FL cells) was examined in vitro. The parasites were incubated for 4 hr with FL cells which had been induced to synthesize spectrin, a major erythrocyte membrane protein, or hemoglobin by culturing the cells in the presence of dimethylsulfoxide (DMSO) or sodium butyrate. The penetration rate, in terms of both the average number of T. gondii per cell and the percentage of cells containing parasities, observed in the DMSO-treated FL cells was depressed as compared with that found in untreated cells. However, this depression was not related to the presence of spectrin or hemoglobin. This conclusion was based on the fact that the penetration rate in the butyrate-treated cells was the same as that in untreated cells. These results suggest that it is not the presence of spectrin and hemoglobin that inhibits penetration by T. gondii. The failure of T. gondii to enter mammalian erythrocytes is discussed in relation to surface changes in FL cells undergoing erythroid differentiation.     

Effects of metalloporphyrins on hemoglobin formation in mouse Friend virus-transformed erythroleukemia cells. Stimulation of heme biosynthesis by cobalt protoporphyrin

Mouse Friend virus-transformed erythroleukemia cells in culture undergo erythroid differentiation when treated with a variety of compounds including iron protoporphyrin IX, i.e. hemin. Exogenous hemin is not only incorporated into hemoglobin in these cells but also stimulates heme biosynthesis (Granick, J. L., and Sassa, S. (1978) J. Biol. Chem. 253, 5402-5406). In this study, we examined whether metalloporphyrins other than hemin can also induce differentiation, and if so, whether they can also be incorporated into hemoglobin. Among eight metalloporphyrins examined in culture of these cells, i.e. Co, Mn, Cu, Mg, Ni, Zn, Sn, and Cd protoporphyrin IX, only Co protoporphyrin (10(-4) M) was found to significantly increase the biosynthesis of heme and hemoglobin. In contrast to hemin-mediated induction of erythroid differentiation, Co protoporphyrin was not incorporated into hemoglobin in Friend cells. These data indicate that Co protoporphyrin induces the formation of heme and hemoglobin in Friend cells and that these increases are due to the enhancement of heme biosynthetic activity.