Cell growth and division in relation to differentiation of mouse erythroleukemia cells

Addition of certain inhibitors of RNA or protein synthesis to cells cultured in 20% serum, but not 5% serum, induces the differentiation of mouse erythroleukemia cells. Differentiation also was induced by culture at a low serum concentration (0.1%) to starve cells to quiescence, then inducing division by exposing cells to either of two high-pH regimes.     

Possible role of H1 histone in the differentiation of mouse erythroleukemia cells

Serum-starved mouse erythroleukemia cells, stationary phase cells or cells cultured in dibutyryl cAMP (1 mM) can be induced to differentiate by addition of 20% fetal calf serum plus cycloheximide. Culturing unstarved log phase cells in 20% fetal calf serum plus low levels of cycloheximide and histone H1 also causes a significant level of differentiation. These same concentrations of cycloheximide and H1 histone employed separately with 20% fetal calf serum do not induce differentiation. The role these procedures may have in causing an accumulation of histone H1 and cell differentiation is discussed.     

Proteases stimulate mouse erythroleukemia cell differentiation and multiplication

Seventeen different commercially available proteases stimulate both mouse erythroleukemia (MEL) cell differentiation and multiplication. These are the first enzymes shown to stimulate these processes in Friend leukemia virus-infected MEL cells. The induction of differentiation by proteases can be synergistically enhanced by the addition of low concentrations of dimethyl sylfoxide or other low molecular weight inducers. Since proteases are the first inducers of differentiation with a known biochemical function, their study should facilitate the understanding of the molecular mechanism of this process.     

Cytoplasmic factors involved in erythroid differentiation in mouse erythroleukemia (MEL) cells

In order to identify and characterize intracellular factors involved in in vitro differentiation of mouse erythroleukemia (MEL) cells, the differentiation process was analyzed by cell and cytoplast fusion. The results suggested that the process is not a single cascade of molecular chain reactions, but a synergistic result of two different inducible intracellular reactions. One reaction is induced following damage to DNA (inhibition of DNA replication) and is not specific to MEL cells. The other reaction, which is specific to MEL cells, is fully induced by typical erythroid inducing agents such as dimethylsulfoxide or hexamethylenebisacetamide even at concentrations suboptimal for the erythroid induction. Based upon these data, we searched for the putative trans-acting differentiation-inducing factors and detected two proteinaceous factors (DIF-I and DIF-II) in the cytosol fraction which apparently correspond to these reactions. When, partially purified, either one of these factors was introduced into undifferentiated MEL cells, it triggered erythroid differentiation, provided that the recipient cells had been potentiated by the induction of the other reaction. In this article, we summarize the basic characteristics of these cytoplasmic factors involved in erythroid differentiation in MEL cells.     

Modulation of cyclic AMP levels and differentiation by adenosine analogs in mouse erythroleukemia cells

Friend virus-transformed mouse erythroleukemia (MEL) cells can be induced to undergo erythroid differentiation by a variety of compounds, including dimethyl sulfoxide (DMSO) and the adenosine analog xylosyladenine. The present studies have monitored the effects of the stable adenosine receptor ligand N6-phenylisopropyladenosine (PIA) on induction of MEL cell differentiation. PIA has been previously shown to stimulate adenylate cyclase activity in rat hepatic and mouse Leydig 1-10 cells as well as inhibit adenylate cyclase in adipocytes. In the present study, PIA was ineffective as an inducer of the differentiated MEL cell phenotype. However, the results demonstrate that PIA inhibits the induction of MEL cell differentiation by DMSO and xylosyladenine. The extent of this inhibition as determined by benzidine staining, induction of globin RNA, and loss of self-renewal capacity was dependent on PIA concentration. The results also demonstrate that PIA induces a rapid and sustained increase in cyclic AMP (cAMP) levels. Furthermore, there was a highly significant correlation between cAMP levels and inhibition of xylosyladenine-induced differentiation (r = 0.962, P less than 0.0005). This relationship is further supported by the demonstration that prostaglandins E1 and E2 increase MEL cell cAMP levels and inhibit induction of the differentiated MEL cell phenotype. Moreover, PIA inhibited induction of MEL cell differentiation by butyric acid, diazepam, hypoxanthine, and the aminonucleoside analog of puromycin. These results suggest that cAMP may act as a negative regulatory signal in the induction of MEL cell differentiation.     

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.     

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.     

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.     

Lack of evidence for activation of a serum factor in protease-induced differentiation of mouse erythroleukemia cells

summary The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induced up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B⁺). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B⁺ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B⁺ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B⁺ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B⁺ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells. This study was supported by U.S. Public Health Service Grants CA 24403 and CA 37874; American Cancer Society Grant CH-303; the Spingold Foundation; the Chemotherapy Foundation, Inc; the Gar Reichman Foundation; an Irving Alpert Cancer Research Award; and institutional general research funds. Part of this work was presented at the 1984 meeting of the American Society of Biological Chemists and American Association of Immunologists, St. Louis, MO (21).     

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.     

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.     

Messenger RNA changes during differentiation of murine erythroleukemia cells

During differentiation of murine erythroleukemia cells, the levels of certain mRNA were observed to change. To characterize the various patterns of changes that occur during differentiation, cDNA libraries made from RNA isolated from uninduced and differentiating cells were screened with labeled cDNA or RNA labeled in vivo for different periods of time. cDNA clones that corresponded to individual mRNAs whose level remained constant, increased, or decreased during differentiation were identified. These clones were used to analyze Northern blots containing RNA from uninduced and differentiated cells. A number of characteristic changes in individual mRNAs in differentiating murine erythroleukemia cells could be identified, such as no change, increase in concentration, increase in concentration and slight change in size, decrease in concentration, decrease in concentration and change in size, appearance of new band(s) of entirely different size, and change in relative concentrations of two related mRNAs. Measurements of rates of mRNA synthesis and degradation suggest that both parameters change during differentiation and that these changes are instrumental in establishing cellular concentration of specific mRNAs. It seems that the changes in mRNA stability observed in differentiating murine erythroleukemia cells may be associated with changes in the primary structure of the transcribed portion of mRNA. The observation that specific mRNA synthesized before and after induction may have very different stabilities at the same point in differentiation supports this hypothesis.     

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.     

Characterization of erythropoietin receptor on erythropoietin-unresponsive mouse erythroleukemia cells

A membrane receptor for erythropoietin was identified in various erythropoietin-unresponsive mouse erythroleukemia cells. Scatchard analyses of the binding of human 125I-labeled erythropoietin to T3C1-2-0, K-1, GM86 and 707 cells showed the presence of a single class of binding sites with apparent Kd values of 0.27-0.78 nM, which are slightly higher than those of erythropoietin-responsive cells. The number of binding sites varied from 110 to 930 per cell. Crosslinking of 125I-erythropoietin to its binding sites with disuccinimidyl suberate revealed the existence of a single binding protein with molecular mass of 63 kDa. No binding sites with higher molecular mass, as observed in erythropoietin-responsive cells, were detected, nor was any specific binding observed to the non-erythroid hematopoietic cell or to the human erythroleukemia cells examined.     

Induction of differentiation of friend erythroleukemia cells with L histidinol

Addition of an analog of histidine, histidinol, together with lowering the level of histidine in the medium, can induce hemoglobin synthesis in murine erythroleukemia cells.     

Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells.

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.