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.     

Dissociation of hemoglobin accumulation and commitment during murine erythroleukemia cell differentiation by treatment with imidazole

The effect of imidazole on DMSO-induced murine erythroleukemia (MEL) cell differentiation has been examined. While imidazole does inhibit heme, globin mRNA, and hemoglobin accumulation in DMSO-induced MEL cells, it does not affect the commitment of MEL cells to the specific limitation of proliferative capacity associated with the in vitro differentiation program. Furthermore, imidazole treatment does not affect DMSO-induced changes in cell volume, in the relative proportion of nuclear protein IP25, and in the specific activity of the enzyme cytidine deaminase. A clonal analysis in the presence of imidazole indicated that the drug prevents heme accumulation even in MEL cells already committed to terminal differentiation. These observations suggest that imidazole effectively dissociates two aspects of the erythroid differentiation program of MEL cells: globin gene expression and commitment to loss of proliferative capacity.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Maturation of murine erythroleukemia cells committed to differentiation requires protein kinase C.

Treatment of murine erythroleukemia cells (MELC) attached to fibronectin-coated dishes with dimethyl sulfoxide causes the cells to become committed to the erythroid differentiation pathway. These cells mature extensively and acquire the characteristics of erythroid cells. The cells lose their cell-surface fibronectin receptors and accumulate red cell-specific membrane proteins, such as band 3, in amounts comparable to those in erythrocytes. Previous studies of MELC have shown that the presence of protein kinase C (PKC) is required for commitment to differentiation, but that the level of PKC activity declines progressively during maturation. In this study, we have established a role for PKC in the maturation of MELC committed to differentiation. Our results show that down-regulation of PKC by addition of phorbol 12-myristate 13-acetate (PMA) to committed MELC blocks subsequent maturation of the cells. Treatment of MELC with the PKC inhibitors H7 and sphingosine had similar effects. Down-regulation of PKC was assayed by measuring cytosolic PKC activity as well as by Western blotting using PKC antibodies. MELC maturation was monitored by loss of the cell-surface fibronectin receptor, release of cells from fibronectin plates, and accumulation of the band 3 anion transport protein. Immunoprecipitation of surface-labeled proteins by an anti-fibronectin receptor (integrin) antibody showed that PMA-treated cultures had more fibronectin receptor protein than untreated cultures 6 days post-induction. As a result, cultures of committed MELC treated with PMA remained attached to fibronectin-coated plates, whereas non-PMA-treated cells were released into the culture medium. Furthermore, PKC-depleted cells accumulated much smaller amounts of band 3 protein and band 3 mRNA than did non-PKC-depleted controls. Our results show that although PKC activity declines progressively during post-commitment maturation of MELC, its continued presence is critical for the process of cellular maturation.     

Alterations in cell tetrahydrobiopterin levels may regulate queuine hypomodification of tRNA during differentiation of murine erythroleukemia cells

The base at the first anticodon ("wobble") position of certain eukaryotic tRNA species is either guanine or the hypermodified base queuine. These tRNA species are synthesized with guanine in the wobble position (tRNAG); this guanine can then be replaced with queuine by the action of the enzyme tRNA-guanine ribosyltransferase. In the present report, we show that tRNAG levels increased in response to the induction of erythroid differentiation of murine erythroleukemia (MEL) cells. We also found that tRNA-guanine ribosyltransferase was significantly inhibited by tetrahydrobiopterin. MEL cells showed a transient threefold increase in tetrahydrobiopterin levels 6 to 12 h after exposure of the cells to inducers such as DMSO or tetramethylurea. The increase in tetrahydrobiopterin preceded the increase in tRNAG which in turn preceded the appearance of phenotypic changes characteristic of differentiation. By contrast, a mutant MEL cell line unable to differentiate in response to inducers showed no change in the level of tetrahydrobiopterin or of tRNAG upon exposure to DMSO. N-acetylserotonin, a well-characterized inhibitor of tetrahydrobiopterin synthesis, prevented the DMSO-mediated increase in tetrahydrobiopterin in normal MEL cells. N-acetylserotonin also inhibited the increase in tRNAG levels and the appearance of phenotypic differentiation in these cells.     

Microarray profiling of genes differentially expressed during erythroid differentiation of murine erythroleukemia cells

Murine erythroleukemia (MEL) cells are widely used to study erythroid differentiation thanks to their ability to terminally differentiate in vitro in response to chemical induction. At the molecular level, not much is known of their terminal differentiation apart from activation of adult-type globin gene expression. We examined changes in gene expression during the terminal differentiation of these cells using microarray-based technology. We identified 180 genes whose expression changed significantly during differentiation. The microarray data were analyzed by hierarchical and k-means clustering and confirmed by semi-quantitative RT-PCR. We identified several genes including H1f0, Bnip3, Mgl2, ST7L, and Cbll1 that could be useful markers for erythropoiesis. These genetic markers should be a valuable resource both as potential regulators in functional studies of erythroid differentiation, and as straightforward cell type markers.