A change in the stability of globin mRNA during the induction of murine erythroleukemia cells

The stability of globin mRNA in murine erythroleukemia cells (Friend cells) before and during DMSO-induced differentiation was investigated. Cells were exposed to 3H-uridine for 2 hr and then transferred to medium without the radioactive precursor. The loss of radioactivity in total RNA, poly(A)-containing RNA and globin mRNA was followed. The globin mRNA was isolated using a highly specific globin cDNA column. In uninduced cells and cells early in differentiation, the globin mRNA decays with a half-life of less than 50 hr. After 4 days of induction, the globin mRNA decays with a half-life of 17 hr, demonstrating a change in stability during the induction process. Although the stability of globin mRNA changes during induction, this is not true for total poly(A)-containing RNA. At all times of induction, the poly(A)-containing RNA decays as two populations, one with a half-life of 6 hr and the other with a half-life of 36 hr. The half-life of the rRNA also remains unchanged during differentiation.     

Terminal differentiation of murine erythroleukemia cells: physical stabilization of end- stage cells

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.     

Biosynthesis and stability of globin mRNA in cultured erythroleukemic friend cells

Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with ³H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA covalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2–4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16–17 hr. The rest of the poly(A)-containing RNA was composed of two kinetic populations: 85–90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but if subsequently declined gradually.Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.     

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.     

Differential effect of hemin-controlled eIF-2 alpha kinases from mouse erythroleukemia cells on protein synthesis

Cultured mouse erythroleukemia (MEL) cells can be induced to erythroid differentiation by a variety of chemical agents. This differentiation process is marked by the onset of globin mRNA and hemoglobin synthesis. In rabbit reticulocytes, globin synthesis is regulated by a hemin-controlled translational inhibitor (HCI) which acts via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2). From both uninduced and induced MEL cells, hemin-controlled eIF-2 alpha kinases have been partially purified. They resemble HCI with respect to their chromatographic behaviour and their sensitivity towards physiological concentrations of hemin (5-10 microM). Further purification on phosphocellulose, however, reveals that the eIF-2 alpha kinase from uninduced MEL cells is chromatographically distinct from HCI, whilst the eIF-2 alpha kinase activity from induced MEL cells represents a mixture of the former and the HCI-type eIF-2 alpha kinase. The latter inhibits protein synthesis in a fractionated system from rabbit reticulocytes which is free of, but sensitive to, HCI, whereas the eIF-2 alpha kinase from uninduced MEL cells does not show any inhibitory activity. This observation is supported by the finding that induced MEL cells respond in vivo to iron depletion with a shut-off of protein synthesis (as do rabbit reticulocytes), whilst uninduced MEL cells do not.     

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.     

Synthesis and stability of developmentally regulated dictyostelium mRNAs are affected by cell-cell contact and cAMP

Postaggregation Dictyostelium discoideum cells contain 3000 mRNA species that are absent from preaggregation cells; these aggregation-dependent sequences comprise 30% of the mass of mRNA in these cells. We show that the synthesis and stability of these regulated mRNA sequences are affected by both cell-cell contact and cAMP. Three independent assays are used to quantitate these mRNAs: in vitro translation followed by two-dimensional gel analysis of the protein products; hybridization of gel-separated RNAs to cloned DNAs; and hybridization of mRNA to a cDNA probe specific for the population of regulated sequences. In postag-gregation cells, the half-life of both the developmentally regulated mRNAs and the constitutive mRNAs present throughout growth and differentiation is the same—about 4 hr. Following disaggregation, all of the late mRNA sequences are degraded and decay with a half-life of 25 to 45 min. The constitutive species are unaffected; 2.5 hr after disaggregation, the ratio of late to constitutive mRNAs is about 6% that of normal plated cells. Addition of cAMP to cells that have been disaggregated for 2.5 hr (or longer) restores the level of most late mRNAs within 3 hr. We conclude that cAMP stimulates the synthesis of these mRNAs and may also act to stabilize them in the cytoplasm. This effect of cAMP is dependent on the cells having been in contact with other cells; cAMP has no effect on the levels of mRNA in suspension-starved, aggregation-competent cells that have never formed cell-cell aggregates.     

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.     

Nuclear proteins preferentially bound to the beta-globin gene: cellular specificity and variation during terminal differentiation of mouse erythroblasts

Restriction fragments of the mouse beta major globin gene and of the long terminal repeat (LTR) DNA fragment of the mouse mammary tumor provirus as a control, were used to analyze the specificity of DNA-protein interactions in nuclear extracts of mouse erythroleukemia (MEL) cells and of other differentiated mouse cultured cell lines. After gel electrophoresis and transfer to nitrocellulose, DNA-binding proteins with a preferential affinity for the cloned beta-globin genomic sequence were characterized and related to the level of globin gene expression during induction of differentiating mouse erythroblasts. Two proteins (110 K and 75 K) appear in differentiated MEL cells while another one (100 K), for which we have localized the binding site on the beta-globin gene, is present only in immature MEL cells.     

Stabilization of mRNA following serum-induction of quiescent 3T3 cells

The stability of mRNA has been measured in 3T3 cells in the resting and the growing states, and also during the transition from the resting to the growing state. Pulse labeled poly(A)⁺ mRNA chased with uridine and cytidine supplemented growth medium decayed with a half-life of 6.5 hr in the resting state, 26 hr during the transition from the resting to the growing condition, and 18 hr during serum-stimulated growth. The half-life of poly(A)⁺ mRNA determined by steady state labeling yielded similar results in resting and serum-stimulated 3T3 cells. Thus during the transition from resting to serum-stimulated growth in 3T3 cells poly(A)⁺ mRNA becomes more stable.