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.     

The effect of heme on intracellular iron pools during differentiation of friend erythroleukemia cells

The inhibition of cellular iron uptake by hemin described previously in reticulocytes was studied in murine erythroleukemia (Friend) cells that can be induced to differentiate in culture by dimethyl sulfoxide (DMSO). Hemin had no effect on iron uptake into noninduced cells. After the induction by DMSO, hemin inhibited iron uptake into Friend cells and this effect of hemin became more pronounced with the further progress of differentiation. The reduction of cellular iron accumulation was caused mainly by inhibition of iron incorporation into heme, iron uptake into the non-heme pool was influenced by hemin treatment. Inhibition of heme synthesis by isonicotinic acid hydrazide (INH) caused an accumulation of iron in mitochondria in DMSO-induced cells but not in uninduced cells. On the basis of these results, a specific system transporting iron to mitochondria induced by DMSO treatment is suggested as a target for the inhibitory action of hemin. In Friend cells of the Fw line which are deficient in ferrochelatase, heme has no effect on iron uptake. The addition of INH to the Fw cells does not enhance the iron accumulatoni in mitochondria.     

ADP-ribosyltransferase activity during the friend virus-induced murine erythroleukemia cell differentiation

A variety of chemical agents that are known to induce erythrodifferentiation in the Friend virus-induced murine erythroleukemia (MEL) cell have been suggested to mediate DNA cleavage in cultured cells prior to differentiation. The activation of the nuclear enzyme, ADP-ribosyltransferase, depends upon the presence of single strand breaks in DNA. If dimethyl sulfoxide (Me2SO) causes DNA breakage, it would be expected that the activity of ADP-ribosyltransferase would increase. A study of ADP-ribosyltransferase activity during cell growth indicates that both Me2SO-treated and untreated MEL cells exhibit a similar increase in the enzyme activity but the increase in Me2SO-treated cells is delayed by a few hours. When examined at comparable stages of growth, both treated and untreated cells show almost identical levels of enzyme activity. The present data thus do not support the contention that Me2SO induces DNA breakage in the MEL cells.     

Changes in the glycoprotein composition of plasma membrane during the differentiation of friend erythroleukemia cells

Friend erythroleukemia cells display transient and permanent changes in the composition of their plasma membrane-bound glycoproteins during dimethyl sulfoxide-induced differentiation. The transient changes, as revealed by metabolic labeling with [¹⁴C]glucosamine, are most conspicuous around the time during which most cells become committed to terminal differentiation. Permanent changes are revealed by reductive tritiation after oxidation with NaIO₄ or galactose oxidase. In differentiated cells one glycoprotein fraction (M_r 150 000) could not be labeled by any of these methods, although it does contain neuraminic acid. We found no evidence in support of the hypothesis that the anomalous behavior of this fraction is caused by an increased degree of O-acetylated neuraminic acid in the plasma membrane of differentiated cells.     

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 PGF₁α, were completely inactive, while PGE₁ inhibited slightly and PGF₂α 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 10⁻⁶M hydrocortisone. Finally, treatment of DMSO-differentiated cells with PGA₁ (but no DMSO) prevented the return to the undifferentiated state.     

Commitment to erythroid differentiation by friend erythroleukemia cells: a stochastic analysis

A method for the clonal analysis of murine erythroleukemia cells has been developed which allows the precise characterization of the number of progeny produced by each cell and the degree of differentiation of each progeny cell. The potential of almost every cell in the culture can be monitored because a plating efficiency close to 100% has been achieved. The effects of treatment with an inducer of differentiation (DMSO) on the proliferative capacity of the treated cells have been studied with this technique. Cells from a mass culture treated with inducer give rise to colonies of differentiated progeny when subsequently cloned in the absence of inducer. Colonies exhibiting this phenotype represent the progeny of cells committed to the differentiation pathway by treatment with inducer. We observe that the commitment decision limits the subsequent proliferative capacity of the cell to four additional cell divisions. A quantitative analysis suggests that the commitment decision for each cell is made in a stochastic manner. Irreversible commitment to the expression of differentiated functions occurs with discrete probability per cell generation for many cell generations. The value for this probability is a function of the concentration of inducer (DMSO). A correlative biochemical study suggests that an irreversible commitment decision by a significant proportion of the population precedes or accompanies increases in cytoplasmic globin mRNA levels, one of the earliest detectable biochemical markers for erythroid differentiation in this system.A specific kinetic model based on these considerations has been developed to predict clonal phenotypes as a function of time and probability of commitment. Quantitative predictions based on this model are in excellent agreement with experimental observations. The effectiveness of a stochastic model in predicting the behavior of this system is discussed in relation to the stochastic behavior of normal hematopoiesis and the biochemical mechanisms which control these differentiation programs.     

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells

Binding experiments with the specific muscarinic ligand [³H]N-methylscopolamine (³H-NMS) have shown the presence of constitutive muscarinic acetylcho-line receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1–M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K_D) for ³H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B⁺) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B⁺ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells. J. Cell. Physiol. 178:333–340, 1999. © 1999 Wiley-Liss, Inc.     

Methylation of a middle repetitive DNA sequence class during differentiation in Friend erythroleukemia cells

We have examined the methylation patterns within middle repetitive sequences in Friend erythroleukemia cells. Mouse-interspersed-family-1 (MIF-1) and a group characterized by a 1350-bp Eco-Bam fragment cloned into pBR322 as plasmid pFS-13, are both less modified in Friend cell DNA than in normal tissue DNA. The pattern of methylation present in pFS-13 homologous sequences was found to be stable during cell division, i.e., somatically inherited, and stable during differentiation induced by HMBA.     

New gene expression in dimethylsulfoxide-treated friend erythroleukemia cells

Our previous studies had shown that a small amount of single-stranded DNA (ssDNA) separated from the bulk nuclear DNA of different animal cells by an improved method of hydroxylapatite chromatography (HAC) contains two distinct molecular fractions. The major fraction consists of non self-reassociating sequences that are reassociable to the unique component of bulk DNA and in great part hybridizable to homologous RNA. The minor fraction consists of self-reassociable sequences also reassociable to moderately repetitious bulk DNA. In the present work ssDNA from Friend leukemia cells induced to differentiate (ind FLC) by DMSO was compared with ssDNA from untreated control Friend cells (cont FLC). It was shown that the relative amount of ssDNA is greater in ind FLC than in cont FLC (1.5 – 1.6% and 1.2 – 1.3% of the total cell DNA respectively after a second step of HAC purification). The ind FLC-ssDNA contained a greater proportion of self-reassociable sequences (33–35%) as compared with cont FLC-ssDNA (18–20%). Also the relative amounts of ssDNA hybridizable to cytoplasmic RNA from homologous cells was slightly but constantly higher in ind FLC-ssDNA (33–34%) than in cont FLC-ssDNA (29–30%). Cross hybridizations were carried out between highly radioactive ssDNA and cellular RNAs in great excess, whether total cytoplasmic RNAs or polyadenylated mRNAs. At saturation levels, the hybridized ssDNA fraction was separated from the non-hybridized fraction, and both fractions were rehybridized to RNA from ind FLC or cont FLC. The results indicated that about 10% of ind FLC-ssDNA appeared to be specific for DMSO-treated cells. This may correspond to the expression of 1000–2000 different cytoplasmic mRNAs mostly belonging to the low abundance class.     

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.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

DNA strand scission and ADP-ribosyltransferase activity during murine erythroleukemia cell differentiation

The accumulation of DNA strand breaks and activation of ADP-ribosyltransferase (ADPRT) have recently been associated with cellular differentiation. Murine erythroleukemia (MEL) cells undergo erythropoietic differentiation when exposed to dimethyl sulfoxide (Me2SO) and several studies have suggested that DNA strand scission induced by this agent is a prerequisite for expression of the differentiated phenotype. Me2SO induction of MEL cells has also been associated with increases in ADPRT activity in one study, but not in another. We have monitored the effects of Me2SO on DNA strand breaks in preformed and replicating MEL cell DNA. The results clearly demonstrate that DNA fragmentation is not detectable during Me2SO induction of MEL differentiation, even in the presence of 3-aminobenzamide, an inhibitor of ADPRT. Further, these results are consistent with an absence of detectable changes in both endogenous and total potential ADPRT activity during Me2SO-induced MEL differentiation. These findings would argue against Me2SO induction of DNA strand scission and ADPRT in MEL cells undergoing differentiation.     

Tyrphostin-induced differentiation of mouse erythroleukemia cells

Inhibitors of protein-tyrosine kinases (TPKs) from the tyrphostins family induce terminal erythroid differentiation of mouse erythroleukemia (MEL) cells. The most potent tyrphostin was found to be AG-568 which was therefore investigated in more detail. Just prior to differentiation the inhibition of tyrosine phosphorylation of a pp⁹⁷ protein band was noted. We also found that AG-568 treatment induces the appearance of a putative differentiation factor which could induce tyrphostin-independent differentiation in untreated cells. Our study suggests that the inhibition of tyrosine phosphorylation by AG-568 leads to the production of differentiating factor(s) which induce the MEL cells to differentiate.     

Changes in proteinase activities during the differentiation of murine erythroleukemia cells

Changes in intracellular proteinase activities were examined during DMSO-induced differentiation of murine erythroleukemia cells. Suc-APA-MCA hydrolytic activity was significantly decreased, and apparent ATP-dependent multicatalytic proteinase activity was also decreased with MEL cell differentiation. Cathepsin B and L activity was mainly present in the microsomal fraction of control cells, but a part of this activity had shifted to the lysosomal fraction of differentiated cells. With the translocation of cathepsin B from the microsomal to the lysosomal fraction, the pro-enzyme form of cathepsin B was converted into the mature enzyme. These results suggest that the lysosomal pathway contributes to the degradation of specific proteins with cell differentiation.