Induction of erythroid differentiation in Friend murine erythroleukemic cells by inorganic selenium compounds.

Inorganic selenium compounds are shown to be inducers of hemoglobin synthesis in malignant murine erythroleukemia (MEL) cells. SeO₂ can induce hemoglobin synthesis at $\text{1}\text{20}$ the concentration of butyric acid and $\text{1}\text{5000}$ the concentration of dimethylsulfoxide (DMSO), two potent inducers of erythroid differentiation in MEL cells. SeO₂ and H₂SeO₃ showed an equivalent capacity to stimulate hemoglobin synthesis in three different MEL cell lines. The incorporation of ³H-glycine into hemoglobin was demonstrated in lysates of SeO₂-induced 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.     

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.     

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.     

Quantitative Changes in Unscheduled DNA Synthesis in Rat Muscle Cells after Differentiation

THE incorporation of tritiated thymidine (³H-thymidine) into cells not engaged in normal DNA replication has been called unscheduled DNA synthesis¹. The phenomenon has been observed after X-irradiation¹, ultraviolet irradiation² and after exposure to the monofunctional alkylating agent methyl methane sulphonate³ (MMS) and other carcinogens⁴. In all published reports the cells showing unscheduled DNA synthesis had retained their proliferative capacity (and hence at least their potential ability to synthesize DNA). We have investigated whether differentiated cells—that is, cells which presumably will never have to initiate normal DNA synthesis—are still capable of unscheduled DNA synthesis. We used multinucleated rat muscle cells in vitro. Myotubes have been found to form by fusion of separate, mononucleated cells^5,6, the nuclei of which no longer synthesize DNA. YalTe and Gershon⁷ have shown that such cells can reinitiate DNA synthesis after viral infection. They found it necessary, however, for fusion to continue during viral infection; in the absence of further fusion no new DNA synthesis was observed. The trigger for DNA synthesis after viral infection must therefore have come from cells which had been transformed before differentiation and fusion. This left open the question of whether differentiated cells could initiate DNA synthesis in the absence of trigger from transformed cells.     

Procaine inhibits the erythroid differentiation of MEL cells by blocking commitment: Possible involvement of calcium metabolism

The action of procaine on the terminal erythroid differentiation of murine erythroleukemia (MEL) cells has been investigated at the level of individual cells. At concentrations (7 × 10^?4 M) which had no inhibitory effect on cell growth, pretreatment of these cells with procaine for 12–24 hr caused a pronounced inhibition (> 90%) of commitment to terminal erythroid differentiation of dimethyl sulfoxide (DMSO)-treated cells. Simultaneous treatment of MEL cells with DMSO and procaine, however, resulted to only slight inhibition (< 20%) of commitment. Blockade of commitment by procaine pretreatment appears to be general since it was observed in cells treated with other inducers (6-thioguanine, dimethylformamide). Procaine pretreatment did not abolish the ability of MEL cells to complete the “latent period” and commit upon the removal of the block. Reversal of procaine inhibition of commitment was obtained by the addition of either CaCl₂ (1.0 mM), calcium ionophore A23817 (1 μg/ml), but not of MgCl₂ (1.0 mM). From these data we conclude that procaine inhibits the terminal erythroid differentiation of MEL cells by blocking an event or process required for commitment which occurs prior to commitment itself. Our results suggest that this process involves calcium metabolism.     

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.     

Activin A/erythroid differentiation factor induces thromboxane A2 synthetic activity in murine erythroleukemia cells

Activin A, a protein homologous to transforming growth factor beta, was shown to induce hemoglobin synthesis in murine erythroleukemia (MEL) cells and was also termed erythroid differentiation factor (EDF) (Eto, Y., Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y., and Shibai, H. (1987) Biochem. Biophys. Res. Commun. 142, 1095-1103). We found that activin A/EDF also induced thromboxane (TX) A2 synthetic activity in these cells. Synthesis of TXA2 from arachidonic acid is catalyzed by cyclooxygenase and TX synthase. Activin A/EDF induced the latter TX synthase activity, whereas the cyclooxygenase activity was constitutively expressed. The induction of this enzyme activity was inhibited by cycloheximide, suggesting that activin A/EDF induced de novo protein synthesis of TX synthase. Furthermore, we studied the relationship between the induction of TXA2 synthetic activity and erythroid differentiation in MEL cells, since the former is not an erythroid phenotype. We found 1) that the two responses to activin A/EDF were distinctly affected by the initial cell density; 2) that the dose-response curves for activin A/EDF were similar (ED50 = approximately 100 pM), whereas the time course of induction of TXA2 synthetic activity was much faster; and 3) that other erythroid differentiation inducers of MEL cells, namely dimethyl sulfoxide and hexamethylene bisacetamide, had little or no effect on TXA2 synthesis. These results indicate that activin A/EDF induces TXA2 synthetic activity independently of erythroid differentiation.     

Differentiation of two mouse cell lines is associated with hypomethylation of their genomes.

Methyl-accepting assays and a sensitive method for labeling specific CpG sites have been used to show that the DNA of F9 embryonal carcinoma cells decreases in 5-methylcytosine content by ca. 9% during retinoic acid-induced differentiation, whereas the DNA of dimethyl sulfoxide-induced Friend murine erythroleukemia (MEL) cells loses ca. 3.8% of its methyl groups. These values correspond to the demethylation of 2.2 X 10(6) and 0.9 X 10(6) 5'-CpG-3' sites per haploid genome in differentiating F9 and MEL cells, respectively. Fluorography of DNA restriction fragments methylated in vitro and displayed on agarose gels showed that demethylation occurred throughout the genome. In uninduced F9 cells, the sequence TCGA tended to be more heavily methylated than did the sequence CCGG, whereas this tendency was reversed in MEL cells. The kinetics of in vitro DNA methylation reactions catalyzed by MEL cell DNA methyltransferase showed that substantial numbers of hemimethylated sites accumulate in the DNA of terminally differentiating F9 and MEL cells, implying that a partial loss of DNA-methylating activity may accompany terminal differentiation in these two cell types.     

Melatonin as a central molecule connecting neural development and calcium signaling

Melatonin (MEL) is a neuroendocrine hormone secreted by the pineal gland in association with the suprachiasmatic nucleus and peripheral tissues. MEL has been observed to play a critical role in the reproductive process and in the fetomaternal interface. Extrapineal synthesis has been reported in mammalian models during pregnancy, especially by the placenta tissue. MEL can regulate intracellular processes (e.g., G-proteins) and the activity of second messengers (e.g., cAMP, IP_3, Ca²⁺). During neurodevelopment, these activities regulated by melatonin have an important role as an intracellular signaling for gene expression regulation. To review the role of MEL in neurodevelopment, we built interactome networks of different proteins that act in these processes using systems biology tools. The analyses of interactome networks revealed that MEL could modulate neurodevelopment through the regulation of Ca²⁺ intracellular levels and influencing BMP/SMAD signaling, thus affecting neural gene responses and neuronal differentiation.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.