Extinction of globin gene expression in human fibroblast x mouse erythroleukemia cell hybrids.

We have chosen human fibroblast x mouse erythroleukemia hybrid cells as a model system to examine regulation of unique genes. The globin genes were studied as a marker of erythroid differentiation. Three separate hybrid cell lines were incubated in 2% dimethylsulfoxide, an agent which induces erythroid differentiation of the parental erythroleukemia cells. Neither human nor mouse globin mRNA sequences could be detected by a sensitive molecular hybridization assay which utilized globin complementary D N A. However, td n a from one of the cell lines was shown to contain both the mouse and humand globin genes. Thus, loss of the genes by chromosomal segregation did not account for their failure to be expressed. Cocultivation of the mouse erythroleukemia cells with excess human fibroblasts did not prevent erythroid differentiation of the erythroleukemia cells in the presence of dimethylsulfoxide. Similarly globin gene expression was preserved in tetraploid cells generated by fusion of two erythroleukemia lines. Thus, extinction of globin geneated by fusion of two erythroleukemia lines. Thus, extinction of blobin gene expression in the human fibroblast x erythroleukemia hybrids occurred at the level of mRNA production and appeared to be due to the presence of the fibroblast genome within the hybrial cell.     

Correlation between hypomethylation of DNA and expression of globin genes in Friend erythroleukemia cells.

This report identifies L-ethionine as an inducer of differentiation in murine erythroleukemia cells. When Friend erythroleukemia cells are grown in the presence of 4mM L-ethionine, globin mRNA accumulates and in 4-5 days, 25-30% of the cells in the culture contain hemoglobin. Incubation of the cells with bromodeoxyuridine prevents both ethionine-induced accumulation of globin mRNA and erythroide differentiation. At the concentration where L-ethionine acts as an inducer of FL cell differentiation it inhibits methylation of DNA and tRNA in vivo but does not prevent macromolecular synthesis or cell division. To establish whether a link existed between inhibition of a specific methyltransferase and activation of globin synthesis in FL cells, we examined the degree of hypomethylation of DNA and tRNA from FL cells induced to differentiate with dimethylsulfoxide and butyrate. In contrast to the tRNA from ethionine-treated cells, tRNA from cells induced by butyrate or Me2SO cannot be methylated in vitro using homologous enzymes. DNA isolated from cells exposed to any of the three inducers, however, was significantly hypomethylated when compared with DNA from uninduced cells. These data suggest that methylation of DNA may play a role in the regulation of gene expression.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

Presence of nucleosomal repeat in the transcribed alpha globin gene of induced murine erythroleukemia cells

The sensitivity of the mouse alpha-globin gene to micrococcal nuclease and its nucleosomal repeat were studied in three different functional states of the gene: inactive in EAT cells, potentially active in uninduced MEL cells and active in induced MEL cells. The results show that: 1. The nuclease sensitivity of the gene differs in the three different functional states. 2. Both the coding and the 5'-flanking regions of the induced actively transcribed gene show a typical nucleosomal repeat pattern. 3. Hypersensitive sites for micrococcal nuclease and for an endogenous nuclease appear upstream of the gene after induction of differentiation.     

Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.     

Stability of globin mRNA in terminally differentiating murine erythroleukemia cells

The stability of globin mRNA in terminally differentiating MEL cells has been reevaluated. Previously, it had been reported that globin mRNA has a half-life of approximately 17 hr in terminally differentiating MEL cells. We show that the previous measurements of this parameter were confounded by physical instability of differentiating MEL cells. By using culture conditions that physically stabilize end-stage cells we show that the stability of globin mRNA in terminally differentiating MEL cells is equal to the value observed for ribosomal RNA, a half-life greater than 60 hr.