Influence of calcium and calcium-modulating agents on differentiation of murine erythroleukaemia cells

Since the involvement of calcium ions in the regulation of cell division and differentiation has been proposed, in this study we have examined the effect of extracellular calcium and of calcium-modulating agents on the DMSO-induced differentiation of murine erythroleukaemia cells. Neither proliferation nor differentiation of these cells was affected by calcium deprivation in the culture medium. Moreover, calcium-chelating agents or agents blocking intracellular calcium uptake induced a marked inhibition of cell differentiation. Intracellular calcium antagonists induced inhibition when cells were grown in a calcium-deprived medium. In contrast, murine erythroleukaemia cell differentiation was unaffected by agents that increased intracellular concentration of calcium. Our results indicate that a mobilization of calcium is indispensable for eliciting full cellular response, but the increase in intracellular level of this cation is not sufficient for complete signal transduction. It is likely that a marked alteration of the intracellular calcium system and availability could be responsible for the independence of our cell system from calcium modulation.     

Characterization of ferritin in murine erythroleukaemia cells

Murine erythroleukaemic cells were studied to determine whether different isoferritins have different functions. The cells were labelled with radioactive iron and the pattern of isoferritins was analysed by chromatofocussing. No change was found after iron-loading the cells but after inducing erythroid differentiation with dimethyl sulphoxide (DMSO), iron was incorporated into both more basic and more acidic isoferritins. This was compared to ferritin subunit synthesis; DMSO induced the synthesis of a third, minor subunit whereas iron-loading had no effect. The fate of murine erythroleukaemic cell ferritin iron was followed after incubations in iron-deficient medium containing DMSO; some, but not all, of the ferritin iron was mobilized and used for haem synthesis, and the remaining iron was found amongst the more basic isoferritins. Finally, sequential radioactive iron labels were used to demonstrate that the movement of iron from ferritin to haem was compatible with the 'last-in-first-out' principle, but this could not be related to different isoferritins. These results show firstly that DMSO changes the pattern of isoferritins and ferritin subunits in murine erythroleukaemic cells. Secondly, iron associated with more basic isoferritins seems to be less easily mobilized for haem synthesis. These results support the concept that different isoferritins have different functions.     

Analysis of the growth kinetics of murine erythroleukaemia cells following commitment to terminal differentiation

Abstract Differentiation of murine erythroleukaemia cells by various inducers involves a step of irreversible commitment, after which the presence of the inducer is not required for completion of the process. Some cells become partially committed and give rise to differentiated as well as undifferentiated progeny. Commitment occurs asynchronously; under suboptimal inducing conditions, such as low concentration of inducer or short duration of exposure, both committed and uncommitted cells co-exist. In the present study the growth of these subpopulations was compared. Murine erythroleukaemia cells were exposed to the inducer hexamethylene-bisacetamide for 24 hr, then the inducer was removed by washing and the rate of proliferation of committed and uncommitted cells was measured. Commitment was scored by cloning the cells in inducer-free semi-solid medium and determining the cellular composition of the colonies with respect to haemoglobin content. The results indicated that following removal of the inducer the rate of proliferation was retarded similarly for both committed and uncommitted cells. Partially committed cells disappeared rapidly due to assymetrical cell division into fully committed and uncommitted cells. Both committed and uncommitted cells resumed logarithmic growth at 53 hr, but while uncommitted cells continued this pace until saturation was achieved, committed cells stopped multiplying earlier as a result of terminal differentiation.     

Analysis of the growth kinetics of murine erythroleukaemia cells following commitment to terminal differentiation

Differentiation of murine erythroleukaemia cells by various inducers involves a step of irreversible commitment, after which the presence of the inducer is not required for completion of the process. Some cells become partially committed and give rise to differentiated as well as undifferentiated progeny. Commitment occurs asynchronously; under suboptimal inducing conditions, such as low concentration of inducer or short duration of exposure, both committed and uncommitted cells co-exist. In the present study the growth of these subpopulations was compared. Murine erythroleukaemia cells were exposed to the inducer hexamethylene-bisacetamide for 24 hr, then the inducer was removed by washing and the rate of proliferation of committed and uncommitted cells was measured. Commitment was scored by cloning the cells in inducer-free semi-solid medium and determining the cellular composition of the colonies with respect to haemoglobin content. The results indicated that following removal of the inducer the rate of proliferation was retarded similarly for both committed and uncommitted cells. Partially committed cells disappeared rapidly due to assymetrical cell division into fully committed and uncommitted cells. Both committed and uncommitted cells resumed logarithmic growth at 53 hr, but while uncommitted cells continued this pace until saturation was achieved, committed cells stopped multiplying earlier as a result of terminal differentiation.     

Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation.

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.     

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.     

Distribution of interdivisional times in proliferating and differentiating Friend murine erythroleukaemia cells

The interdivisional times of Friend murine erythroleukaemia cells which are growing continuously, or during terminal erythroid differentiation after exposure to dimethyl sulphoxide (DMSO), were determined by time lapse video photography. The median interdivisional times were found to increase from 11.75 hr before exposure to DMSO, to 24.0 hr at 72 hr after exposure. This increase in median interdivisional time was accompanied by an increase in heterogeneity of interdivisional times (%CV = 8.5----40.8), by an increase in the similarity of sister interdivisional times (ryy = 0.622----0.925), and by a decrease in the fraction of cells observed to divide (F = 1.0----0.807). Cells exposed to DMSO for 72 hr can be induced to divide at least once with nearly normal interdivisional times, if they are resuspended at a tenfold higher cell concentration. Computer simulations of cell cycle regulation, based on the opposing reactions model of Murphy, generate interdivisional time distributions which resemble the experimental data better than the single transition probability model of Smith and Martin.     

Hepatic differentiation of murine embryonic stem cells.

Murine embryonic stem (ES) cells can replicate indefinitely in culture and can give rise to all tissues, including the germline, when reimplanted into a murine blastocyst. ES cells can also be differentiated in vitro into a wide range of cell types. We have utilized a liver-specific marker to demonstrate that murine ES cells can differentiate into hepatocytes in vitro. We have used ES cells carrying a gene trap vector insertion (I.114) into an ankyrin repeat-containing gene (Gtar) that we have previously shown provides an exclusive beta-galactosidase marker for the early differentiation of hepatocytes in vivo. beta-Galactosidase-positive cells were differentiated from I.114 ES cells in vitro. The identity of these cells was confirmed by the expression of the proteins alpha-fetoprotein, albumin, and transferrin and by the fact that they have an ultrastructural appearance consistent with that of embryonic hepatocytes. We propose that this model system of hepatic differentiation in vitro could be used to define factors that are involved in specification of the hepatocyte lineage. In addition, human ES cells have recently been derived and it has been proposed that they may provide a source of differentiated cell types for cell replacement therapies in the treatment of a variety of diseases.     

26S multicatalytic proteinase complexes decrease during the differentiation of murine erythroleukemia cells.

Changes in multicatalytic proteinase activity during differentiation were investigated using Me2SO-induced differentiation of murine erythroleukemia cells as a model. The apparent ATP-dependent multicatalytic proteinase activity decreased in the Me2SO-treated cells with ATP-dependent incorporation of [3H]diisopropyl fluorophosphate decreasing notably after Me2SO-treatment. This decrease in activity does not seem to arise from a cessation of cell-proliferation, because no significant changes in proteinase activity were observed under different culture conditions. Hydroxyapatite column chromatography was employed to analyze the form of multicatalytic proteinase. It was clearly demonstrated that the 26S form of the proteinase decrease in the differentiated cells relative to normal cells. Multicatalytic proteinase-associated proteins that bind to the proteinase in an ATP-dependent manner were purified on an anti-multicatalytic proteinase IgG conjugated column. Only a small amount of protein was recovered from the differentiated cells. These results suggest that the decrease in multicatalytic proteinase-associated proteins that occurs upon cell-differentiation abolishes the ATP-dependent activity of the proteinase.     

Regulation of glycolipid synthesis during differentiation of clonal murine muscle cells

The two clonal murine muscle cell lines G7 and G8, originally derived from the M114 line [20], represent unique models for comparative studies of myogenesis. Glycolipid synthesis was examined during differentiation using [³H]-galactose and [³H]-glucosamine as precursors. Upon G7 contact glucosylceramide labeling increased and nLcOse₅Cer labeling stopped. During membrane fusion, glucosylceramide labeling stopped and lactosylceramide became the major synthetic product. G8 cells presented a different pattern, with increased labeling of GbOse₃Cer during myogenesis. The major ganglioside synthesized by both myoblasts was GM3, and more complex structures were observed following completion of myotube formation. Total glycopeptide labeling increased when G8 myoblasts fused and remained elevated in myotubes, whereas no differences during fusion of G7 cells were noted. Upon comparison of the two clonal lines, the only consistent observation was a significant increase in the synthesis of total gangliosides and neutral glycolipid during cell contact and membrane fusion (p < 0.02). The results suggest that changes in the synthesis of specific glycolipid structures during myogenesis are unique to each muscle cell line examined. However, transient increases in synthesis of total myoblast gangliosides and neutral glycolipids may be a more general phenomenon, possibly by curbing proliferation or by altering myoblast membrane fluidity characteristics during differentiation.Abbreviations MG6 VI³NeuAc-V⁴Gal-IV³GlcNAc-nLcOse₄Cer - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - Gal galactose - GlcNH glucosamine - PBS phosphate buffered saline - CK creatine kinase     

Changes in acid proteolytic activity during differentiation of murine erythroleukemic cells

Proteolytic activity was measured in murine erythroleukemic 745 cell line grown in culture, before and after the addition of agents which promote differentiation. The 36,000 × g soluble fraction of the cells degraded [¹⁴C]globin with maximal activity at pH 3.6, while the insoluble fraction failed to degrade [¹⁴C]globin within a pH range of 2.5–9.0.The acid protease activity in the soluble fraction of the undifferentiated murine erythroleukemic cells increased during the first 2 days in culture and remained constant during the following 4 days. We suggest that this activity resides in the lysosomes since it migrates together with the lysosomal marker α-mannosidase on colloidal silica gradients, shows maximum activity at acid pH and is sensitive towards inhibition by pepstatin. Induced differentiation of the cells by dimethyl sulfoxide, butyric acid or hexamethylene bisacetamide was concomitantly associated with a marked reduction in protease activity and the accumulation of hemoglobin within the cells. In contrast, in a non-inducible variant of 745 cell line DMSO failed to affect proteolysis. It is suggested that in murine erythroleukemic cells changes in acid protease activity are associated with the cellular triggered by chemical inducers.     

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.     

The synthesis of murine ferrochelatase in vitro and in vivo.

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.     

Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation.

Cell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G1 phase of the cell cycle, followed by a re-entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G1 accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G1 block and re-enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells were unable to differentiate in the absence of seric factors, as measured by the number of benzidine-positive cells during induction with hexamethylene-bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the G1 block. The analysis of two molecular markers of cell differentiation confirmed these results.     

Enhanced translation and increased turnover of c-myc proteins occur during differentiation of murine erythroleukemia cells.

To determine whether regulation of c-myc proteins occurs during the differentiation of murine erythroleukemia cells, we examined c-myc protein synthesis and accumulation throughout dimethyl sulfoxide (DMSO)- or hypoxanthine-induced differentiation. c-myc protein expression exhibited an overall biphasic reduction, with an initial concomitant decrease in c-myc RNA, protein synthesis, and protein accumulation early during the commitment phase. However, as the mRNA and protein levels recovered, c-myc protein synthesis levels dissociated from the levels of c-myc mRNA and protein accumulation. This dissociation appears to result from unusual translational and posttranslational regulation during differentiation. Translational enhancement was suggested by the observation that relatively high levels of c-myc proteins were synthesized from relatively moderate levels of c-myc RNA. This translational enhancement was not observed with c-myb. Under certain culture conditions, we also observed a change in the relative synthesis ratio of the two independently initiated c-myc proteins. Posttranslational regulation was evidenced by a dramatic postcommitment decrease in the accumulated c-myc protein levels despite relatively high levels of c-myc protein synthesis. This decrease corresponded with a twofold increase in the turnover of c-myc proteins. The consequence of this regulation was that the most substantial decrease in c-myc protein accumulation occurred during the postcommitment phase of differentiation. This result supports the hypothesis that the reduction in c-myc at relatively late times is most important for completion of murine erythroleukemia cell terminal differentiation.     

Sulfhydryl groups and the differentiation of murine erythroleukemia cells.

The importance of cysteine and sulfhydryl groups has been demonstrated in relation to the differentiation and respiration of Friend erythroleukemia cells (FLC). The respiratory rate of undifferentiated FLC was higher basally (5.06 ± 0.16 vs. 3.10 ± 0.09 nmoles 02/min/10⁶ cells) and was further 70% stimulated by addition of cysteine, whereas DMSO-induced differentiated cells were insensitive. A sulfhydryl blocking agent (PCMS) was capable of maintaining the differentiated state of FLC cultured in the absence of DMSO and this effect appeared to be reversible upon removal of the PCMS.