Regulation of protein synthesis and accumulation during murine erythroleukemia cell differentiation

We have examined the repertoire of cytoplasmic proteins present at different times during murine erythroleukemia (MEL) cell differentiation. Our laboratory has developed an improved differentiation system in which the use of rapidly inducing MEL subclones and culture conditions which stabilize terminally differentiated cells results in highly synchronous differentiation and the accumulation of large numbers of cells in the end stages of differentiation. Using two-dimensional gel electrophoresis, the proteins of MEL cell cytoplasm have been fractionated at different times of induction in the improved system. The protein composition of MEL cell cytoplasm changes dramatically during the differentiation program, in contrast to previously reported results. We observe patterns of changes that are consistent with alterations in the relative degradative rates as well as the relative synthetic rates of the different proteins. We find that the rate of incorporation of labeled amino acid into protein is reduced in induced cultures of MEL cells. We demonstrate that the contribution of uninduced cells to the protein patterns observed late in differentiation is minor in our system, and argue that the results previously obtained for differentiating MEL cells were influenced by the heterogeneity of the induced populations.     

Kinetics of histone H10 accumulation and commitment to differentiation in murine erythroleukemia cells

The accumulation of histone H10 (also denoted IP 25) in murine erythroleukemia cells, induced to differentiate with hexamethylene bis-acetamide, was shown to precede by 15-20 h the appearance in the culture of cells irreversibly committed to differentiate. In addition the rates of accumulation of H10 and of committed cells vary in a similar manner with the HMBA concentration. Flow microfluorimetric analysis demonstrated that the accumulation of H10 did not occur simultaneously in all the cells. This accumulation of histone H10 was initiated first in cell in the G2 phase of the cell cycle and subsequently in the cells situated in all the phases of the cell cycle.     

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.     

Probability that the commitment of murine erythroleukemia cell differentiation is determined by the c-myc level

During the commitment of mouse erythroleukemia cell differentiation, c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the commitment of these cells, we have introduced the rat c-myc gene driven by inducible, heterologous (human metallothionein IIA) gene promoter into murine erythroleukemia cells and we have examined the ability of the transformed cells to undergo commitment to terminal differentiation. The induction of the exogenous c-myc gene expression inhibited the commitment of these cells. Time-dependent inhibition of the commitment was observed with the addition of zinc at an appropriate time after the induction with dimethyl sulfoxide. The result clearly indicated that late decline, not early decline, is required for the commitment. By examining the transformants expressing the exogenous c-myc mRNA at different levels, and the induction of the exogenous c-myc mRNA by varying the concentration of zinc, we demonstrated that the commitment may be determined by a stoichiometric amount of c-myc in the defined period. The data also suggest that the probability value for the commitment process occurring in a stochastic manner is well-correlated with the amount of c-myc mRNA.     

Kinetics of histone H1 ° accumulation and commitment to differentiation in murine erythroleukemia cells

The accumulation of histone H1 ° (also denoted IP 25) in murine erythroleukemia cells, induced to differentiate with hexamethylene bis-acetamide, was shown to precede by 15–20 h the appearance in the culture of cells irreversibly committed to differentiate. In addition the rates of accumulation of H1 ° and of committed cells vary in a similar manner with the HMBA concentration. Flow microfluorimetric analysis demonstrated that the accumulation of H1 ° did not occur simultaneously in all the cells. This accumulation of histone H1 ° was initiated first in cell in the G2 phase of the cell cycle and subsequently in the cells situated in all the phases of the cell cycle.     

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.     

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.     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

Changes in surface architecture during murine erythroleukemia cell differentiation as detected by lectin binding and agglutination

Cell surface alterations occurred during murine erythroleukemia cell (clone 745) differentiation that were detected by both agglutination and lectin binding. Agglutination of erythroleukemia cells was produced by wheat germ agglutinin; whereas, concanavalin A, Ricin, soybean agglutinin and fucose-binding protein were either ineffective or much less efficacious. Treatment of leukemia cells with the inducer of erythroid differentiation dimethylsulfoxide (DMSO) caused a progressive accumulation of hemoglobin-containing cells in culture and a decrease in the rate of agglutination by wheat germ agglutinin, which began at 24 h after exposure to the polar solvent, reached a nadir at 48 h, and remained essentially constant thereafter. The binding of radioactive wheat germ agglutinin by untreated control erythroleukemia cells increased with time in culture, reaching a maximum value at 48 h, and decreased progressively thereafter. Although an increase in ³H-labeled wheat germ agglutinin binding also occurred in DMSO-treated cells, the level bound was significantly lower than that observed in control cells at 24–96 h. The treatment of erythroleukemia cells with various concentrations of DMSO resulted in a decrease in the number of wheat germ agglutinin receptor sites. Other inducers of differentiation (i.e., dimethylformamide, bis(acetyl)diaminopentane) also inhibited the rate of wheat germ agglutinin-induced agglutination of erythroleukemia cells while, in contrast, the inducer tetramethylurea did not. These studies indicate that membrane changes occur during differentiation and suggest that there may be more than one mechanism involved in the initiation of maturation which ultimately leads to the common pathway of erythroid development.     

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.     

Induction of murine erythroleukemia cell differentiation by proteinases is inhibited by aromatic poly-amidines

The effects of the tetra benzamidine serine-proteinase inhibitor 1,3-di-(p-amidinophenoxy) -2,2- bis- (p-amidinophenoxymethyl)propane (TAPP-H) and related compounds, including halo-derivatives, were determined on the erythroid differentiation of murine erythroleukemic cells induced by trypsin and kallikrein. These aromatic poly-amidines and their halo derivatives were found to be strong inhibitors of both trypsin and kallikrein mediated induction of commitment of MEL cells to erythroid differentiation, hemoglobin synthesis and accumulation, globin mRNA production. No inhibitory effects were detected by treating proteinase-induced MEL cells with benzamidine. Only slight inhibitory activity was found after treatment of trypsin-induced MEL cells with other antiproteinase compounds widely used in the control of proteinase-dependent functions, including leupeptin, antipain and Bowman-Birk proteinase inhibitor. MEL cells induced to erythroid differentiation by proteinases could be proposed as an experimental system to test the biological activity of proteinase inhibitors.     

Effect of pyridoxine on the inhibition by dexamethasone of murine erythroleukemia cell differentiation

Glucocorticoids are known to inhibit the induced differentiation of murine erythroleukemia cells. I show here that the binding of glucocorticoid receptors to the intact nuclei of these cells is reduced by exogenous extracellular pyridoxine (1–4 mM). This reduction of glucocorticoid-receptor binding did not abrogate the inhibition by dexamethasone of the induced differentiation.     

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.