Differentiation Of Mouse Erythroleukemia Cells Research Articles

Inhibition of expression of a mouse alpha-globin gene by plasmids that include antisense oligonucleotides.

Plasmid-borne DNAs, corresponding to 68-base oligodeoxynucleotides, synthesized in the antisense or sense configuration and based on the nucleotide sequences of various regions of the mouse alpha-globin mRNA, were introduced with the gene for xanthine-guanine phosphoribosyl transferase from E. coli (Ecogpt) into mouse erythroleukemia (MEL) cells by protoplast fusion. Specific inhibition of the synthesis of alpha-globin was observed only in the cells transformed with the plasmids with antisense 68-mers that corresponded to the cap site as well as the site of initiation of translation of alpha-globin mRNA (Oligo-A); Other plasmids with antisense 68-mers that corresponded to the regions of the exon/intron junctions, the individual exons, or the 3' untranslated region were ineffective. This antisense RNA efficiently reduced the production of alpha-globin to 9-18% of the endogenous level after induction with hexylmethylene-bis-acetoamide (HMBA). Moreover, most of the antisense transformants did not show any decrease in the expression of the c-myc gene at the early phases of differentiation of MEL cells. Thus, we propose a hypothesis that the early decline in levels of c-myc mRNA may be independent of and uncoupled from the program of globin synthesis during the differentiation of MEL cells.

SCL and related hemopoietic helix-loop-helix transcription factors.

The helix-loop-helix (HLH) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals. Five members of this family (MYC, SCL, TAL-2, LYL-1 and E2A) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations. Although activated in T cell leukemias, expression of SCL and LYL-1 is low or undetectable in normal T cell populations. SCL is expressed in erythroid, megakaryocyte and mast cell populations (the same cell lineages as GATA-1, a zinc-finger transcription factor). In addition, both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells, thus implying a role for SCL in erythroid differentiation events. However, in contrast to GATA-1, SCL is expressed in the developing brain. Studies of the function of SCL suggest it is also important in proliferation and self-renewal events in erythroid cells.

53. Induction of H1 histone gene expression during mouse erythroleukemia cell differentiation

This chapter focuses on structure and expression of interleukin-6 (IL-6), which is a cytokine with pleiotropic activities that plays a central role in host defense. IL-6 can exert growth-inducing, growth-inhibitory, and differentiation-inducing activities, depending on the target cells. These activities include (1) terminal differentiation (secretion of immunoglobulins) in B cells and (2) growth promotion on various B cells. IL-6 has been implicated in the pathology of many diseases including multiple myeloma, mesangial proliferative glomerulonephritis, rheumatoid arthritis, and acquired immunodeficiency syndrome (AIDS). Selective inhibition of the synthesis or of the action of IL-6 may have therapeutic benefit against the IL-6-associated diseases. On the other hand, IL-6 has potent antitumor activity against certain types of tumors. Application of IL-6 is promising in cancer treatment as well as in treatment of radiation- or chemotherapy-induced myelosuppression. The cell biology of the intracellular events that link transduction to gene regulation is an important area, and work on these topics helps to understand such phenomena as multifunction of IL-6 and bidirectional effects of cell growth depending on the cell type.

26. Precommitment gene expression changes during mouse erythroleukemia cell differentiation and their relationship to c- myc

26. Precommitment gene expression changes during mouse erythroleukemia cell differentiation and their relationship to c- myc

Derepression of mouse beta-major-globin gene transcription during erythroid differentiation.

Functional analysis of the mouse beta-major-globin gene promoter has revealed a negative regulatory element (-100 to -250 bp) which represses promoter activity in mouse erythroleukemia (MEL) cells. Promoter activity is induced 14-fold during terminal differentiation of MEL cells. Three major in vitro binding sites for NF1 (-250 bp), GATA-1 (-212 bp), and a sequence at -165 bp (BB1) have been defined in this region. Site-directed mutagenesis of any one of the three sites resulted in a five- to sixfold up-regulation of promoter activity in uninduced MEL cells, but only three- to fourfold stimulation was observed from the mutant promoters during MEL cell terminal differentiation. This finding suggests that all three sites are required for repressor activity in uninduced MEL cells and that derepression occurs during MEL cell differentiation. BB1 DNA-binding activity decreases during MEL cell differentiation, suggesting a central role for this factor in modulating the effects of the repressor element. The BB1-binding factor also competes with the CCAAT-binding protein for binding the CCAAT motif. The fact that a reduced but significant stimulation of promoter activity during differentiation is observed in the absence of the repressor element raises the possibility that the BB1 factor also down-regulates transcription in undifferentiated MEL cells by displacing binding of CCAAT-binding protein to the proximal CCAAT motif.

Derepression of Mouse β-Major-Globin Gene Transcription during Erythroid Differentiation

Functional analysis of the mouse beta-major-globin gene promoter has revealed a negative regulatory element (-100 to -250 bp) which represses promoter activity in mouse erythroleukemia (MEL) cells. Promoter activity is induced 14-fold during terminal differentiation of MEL cells. Three major in vitro binding sites for NF1 (-250 bp), GATA-1 (-212 bp), and a sequence at -165 bp (BB1) have been defined in this region. Site-directed mutagenesis of any one of the three sites resulted in a five- to sixfold up-regulation of promoter activity in uninduced MEL cells, but only three- to fourfold stimulation was observed from the mutant promoters during MEL cell terminal differentiation. This finding suggests that all three sites are required for repressor activity in uninduced MEL cells and that derepression occurs during MEL cell differentiation. BB1 DNA-binding activity decreases during MEL cell differentiation, suggesting a central role for this factor in modulating the effects of the repressor element. The BB1-binding factor also competes with the CCAAT-binding protein for binding the CCAAT motif. The fact that a reduced but significant stimulation of promoter activity during differentiation is observed in the absence of the repressor element raises the possibility that the BB1 factor also down-regulates transcription in undifferentiated MEL cells by displacing binding of CCAAT-binding protein to the proximal CCAAT motif.

Regulation of β-adrenergic responses during in vitro differentiation of mouse erythroleukemia cells

Dimethyl sulfoxide (DMSO)-induced erythroid differentiation of Friend mouse erythroleukemia (MEL) cells is associated with a marked transient modulation of catecholamine sensitivity. Within 24 h after induction and well before the onset of hemoglobin synthesis, we observed a 3-fold increase in β-receptor density and a more than 10-fold increase in receptor-coupled cAMP formation. During the following 4 days, in parallel with the development of normoblast-like cells, receptor numbers returned to preinduction levels while catecholamine-dependent cAMP formation remained significantly elevated. Simultaneously, the apparent potency of the β-adrenoceptor agonist isoprenaline increased 10-fold. Improved receptor—cyclase coupling is probably due to a major shift in the expression of G i and G s regulatory proteins. Bacterial toxin-mediated ADP-ribosylation of membrane proteins suggests that the dominating species in native cells is G i (G sα:G iα = 1:7). By contrast, G s predominates in differentiated cells (G sα:G iα = 1.8:1). Receptor-independent forskolin-stimulated cAMP formation showed a pronounced, albeit transient, decrease during differentiation. We suggest that these changes in cellular cAMP responses may be important for transient positive or negative cooperative interactions between hormones and growth factors in the course of erythroid cell development.

Differentiation of Mouse Erythroleukemia Cells Enhanced by Alternatively Spliced c- myb mRNA

C-myb, the normal cellular homolog of the retroviral transforming gene v-myb, encodes a nuclear, transcriptional regulatory protein (p75c-myb). C-myb is involved in regulating normal human hematopoiesis, and inhibits dimethyl sulfoxide-induced differentiation of Friend murine erythroleukemia (F-MEL) cells. An alternately spliced c-myb mRNA encodes a truncated version of p75c-myb (mbm2) that includes the DNA binding region and nuclear localization signal present in the c-myb protein, but does not contain the transcriptional regulatory regions. Constitutive expression of mbm2, in contrast to c-myb, here resulted in enhanced differentiation of F-MEL cells. These data suggest that the c-myb protooncogene encodes alternately spliced mRNA species with opposing effects on differentiation.

Possible role of H1 histone in the differentiation of mouse erythroleukemia cells

Serum-starved mouse erythroleukemia cells, stationary phase cells or cells cultured in dibutyryl cAMP (1 mM) can be induced to differentiate by addition of 20% fetal calf serum plus cycloheximide. Culturing unstarved log phase cells in 20% fetal calf serum plus low levels of cycloheximide and histone H1 also causes a significant level of differentiation. These same concentrations of cycloheximide and H1 histone employed separately with 20% fetal calf serum do not induce differentiation. The role these procedures may have in causing an accumulation of histone H1 and cell differentiation is discussed.

Differentiation of Mouse Erythroleukemia Cells Is Blocked by Late Up-Regulation of a c-myb Transgene

During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-induced differentiation of these transfectants was inhibited in proportion to the level of exogenous c-myb mRNA expression. By adding or removing ZnCl2 at different times during the induction process, it was possible to show that up-regulation of exogenous c-myb limited to the first 2 days of induction had little or no effect on differentiation. In contrast, continuous expression of exogenous c-myb beginning at any time during the period of induction blocked further differentiation. These results suggest that during HMBA induction of MEL cells, the early down-regulation of c-myb mRNA is not necessary for terminal differentiation, whereas the down-regulation of c-myb at a later time is necessary.

Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene.

During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-induced differentiation of these transfectants was inhibited in proportion to the level of exogenous c-myb mRNA expression. By adding or removing ZnCl2 at different times during the induction process, it was possible to show that up-regulation of exogenous c-myb limited to the first 2 days of induction had little or no effect on differentiation. In contrast, continuous expression of exogenous c-myb beginning at any time during the period of induction blocked further differentiation. These results suggest that during HMBA induction of MEL cells, the early down-regulation of c-myb mRNA is not necessary for terminal differentiation, whereas the down-regulation of c-myb at a later time is necessary.

Inhibitors for protein-tyrosine kinases, ST638 and genistein, induce differentiation of mouse erythroleukemia cells in a synergistic Manner

In order to investigate the biochemical nature of intracellular cascades leading to cellular differentiation in vitro, we examined the effect of inhibitors of protein phosphorylation on terminal erythroid differentiation of mouse erythroleukemia (MEL) cells. We have found that specific inhibitors of protein phosphorylation at tyrosine residues, ST638 and genistein, effectively induce differentiation in a synergistic manner with an agent which blocks DNA replication such as mitomycin C (MMC). Based upon these findings, the possible involvement of protein phosphorylation (and dephosphorylation) at tyrosine residues in differentiation is discussed.

Molecular analysis of the human beta-globin locus activation region.

Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human epsilon-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human beta-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase beta-globin mRNA levels 8- to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed.

Continued withdrawal from the cell cycle and regulation of cellular genes in mouse erythroleukemia cells blocked in differentiation by the c-myc oncogene.

Constitutive expression of the c-myc oncogene blocks dimethyl sulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (MEL) cells. During the first 12 h of treatment with DMSO, MEL cells undergo a temporary decrease in the level of c-myc mRNA, followed by a temporary withdrawal from the cell cycle. We found the same shutoff of DNA synthesis during the first 12 to 30 h after DMSO induction in normal MEL cells (which differentiate) and in c-myc-transfected MEL cells (which do not differentiate). We also examined whether deregulated c-myc expression grossly interfered with the regulation of gene expression during MEL cell differentiation. We used run-on transcription assays to monitor the rate of transcription of four oncogenes (c-myc, c-myb, c-fos, and c-K-ras); all except c-K-ras showed a rapid but temporary decrease in transcription after induction in both c-myc-transfected and control cells. Finally, we found the same regulation of cytoplasmic mRNA expression in both types of cells for four oncogenes and three housekeeping genes associated with growth. We conclude that in the MEL cell system, the effects of deregulated c-myc expression do not occur through a disruption of cell cycle control early in induction, nor do they occur through gross deregulation of gene expression.

Continued withdrawal from the cell cycle and regulation of cellular genes in mouse erythroleukemia cells blocked in differentiation by the c-myc oncogene.

Constitutive expression of the c-myc oncogene blocks dimethyl sulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (MEL) cells. During the first 12 h of treatment with DMSO, MEL cells undergo a temporary decrease in the level of c-myc mRNA, followed by a temporary withdrawal from the cell cycle. We found the same shutoff of DNA synthesis during the first 12 to 30 h after DMSO induction in normal MEL cells (which differentiate) and in c-myc-transfected MEL cells (which do not differentiate). We also examined whether deregulated c-myc expression grossly interfered with the regulation of gene expression during MEL cell differentiation. We used run-on transcription assays to monitor the rate of transcription of four oncogenes (c-myc, c-myb, c-fos, and c-K-ras); all except c-K-ras showed a rapid but temporary decrease in transcription after induction in both c-myc-transfected and control cells. Finally, we found the same regulation of cytoplasmic mRNA expression in both types of cells for four oncogenes and three housekeeping genes associated with growth. We conclude that in the MEL cell system, the effects of deregulated c-myc expression do not occur through a disruption of cell cycle control early in induction, nor do they occur through gross deregulation of gene expression.

Tiazofurin Induction of Mouse Erythroleukemia Cell Hemoglobin Production in the Absence of Commitment or Changes in Protooncogene Expression

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC 286193), is a synthetic nucleoside inhibitor of inosine monophosphate dehydrogenase and blocks guanine nucleotide biosynthesis. In the present study, we examined the effects of tiazofurin on mouse erythroleukemia (MEL) cell differentiation and protooncogene expression. Tiazofurin induced hemoglobin production in MEL cells in a concentration-dependent manner, as measured by an increase in benzidine staining. Northern blot analysis of MEL cells treated with 7 mumol/L tiazofurin demonstrated accumulation of both alpha- and beta-globin RNA transcripts. This induction of differentiation was blocked by the presence of exogenous guanosine (100 mumol/L). In contrast to the down-regulation of c-myc and c-myb RNA in MEL cells induced by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), there was no detectable change in levels of these transcripts after tiazofurin treatment. Furthermore, MEL cells induced by tiazofurin did not commit to terminal differentiation. These results suggest a role for guanine nucleotides, at least in part, in the regulation of MEL cell differentiation.

Tiazofurin induction of mouse erythroleukemia cell hemoglobin production in the absence of commitment or changes in protooncogene expression

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC 286193), is a synthetic nucleoside inhibitor of inosine monophosphate dehydrogenase and blocks guanine nucleotide biosynthesis. In the present study, we examined the effects of tiazofurin on mouse erythroleukemia (MEL) cell differentiation and protooncogene expression. Tiazofurin induced hemoglobin production in MEL cells in a concentration-dependent manner, as measured by an increase in benzidine staining. Northern blot analysis of MEL cells treated with 7 mumol/L tiazofurin demonstrated accumulation of both alpha- and beta-globin RNA transcripts. This induction of differentiation was blocked by the presence of exogenous guanosine (100 mumol/L). In contrast to the down- regulation of c-myc and c-myb RNA in MEL cells induced by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), there was no detectable change in levels of these transcripts after tiazofurin treatment. Furthermore, MEL cells induced by tiazofurin did not commit to terminal differentiation. These results suggest a role for guanine nucleotides, at least in part, in the regulation of MEL cell differentiation.

Relationship between transient DNA hypomethylation and erythroid differentiation of murine erythroleukemia cells.

The state of DNA methylation in mouse erythroleukemia (MEL) cells has been analyzed in relation to commitment to differentiation in response to treatment with hexamethylenebisacetamide (HMBA). Previous experiments have shown that induction by HMBA involves transient genome-wide hypomethylation of DNA that is achieved by replacement of 5-methylcytosine with cytosine residues. The experiments described in the present communication revealed that hypomethylation is a very early event in the process of differentiation. Exposure of the cells to 3-deazaadenosine, an adenosine analog, in combination with homocysteine, resulted in the intracellular accumulation of 3-deazaadenosylhomocysteine, which caused an inhibition of HMBA-induced hypomethylation that was correlated with a comparable inhibition of differentiation. While these experiments suggest that hypomethylation is a necessary step in the process of differentiation, other experiments reported here indicate that hypomethylation of DNA may be necessary but not sufficient to trigger the whole program of differentiation in MEL cells. We found, for example that exposure of the cells to cycloheximide during the first 24 hr of induction by HMBA resulted in complete inhibition of differentiation without significant effect on the HMBA-induced hypomethylation. This result also indicates that the enzymatic machinery required for the hypomethylation of DNA is present in uninduced cells.

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.