Commitment to differentiation in Friend cells and initiation of globin mRNA synthesis occurs during the G1 phase of the cell cycle

We have measured the kinetics of specific globin mRNA and Friend virus (FV) RNA synthesis by hybridization to immobilized cDNA after induction of differentiation of two erythroleukemia cell lines (F4N, B8) by butyrate and Me₂SO. The induction with butyrate in these cell lines occurs very rapidly (16–24 h). Cell cycle analysis was made of the populations throughout induction by flow cytofluorometry. The kinetics of commitment of cell populations to terminal differentiation by butyrate was determined by removal of inducer at various times and scoring of benzidine staining cells (hemoglobin producing). In addition, the cell cycle dependence of commitment was determined by flow sorting out of G1 and S+G2 cells various times after addition of inducer and scoring benzidine-stained colonies after growth in methylcellulose. Cells exposed to inducer were also sorted by cell cycle phase using an elutriator rotor. The amount of globin mRNA synthesis in the different cell populations was then determined.

1. 1. It was found that an 8–12 h period in butyrate was required before (a) globin specific mRNA was synthesized; and (b) commitment to differentiation occurred. The time course of globin mRNA synthesis was positively correlated with G1 arrest, as has been also found by others.

2. 2. The increase of FV RNA synthesis was not found during G1 arrest. It occurred early and before commitment.

3. 3. Commitment of cells to irreversible differentiation upon butyrate induction occurs only during the G1 phase of the cell cycle.

4. 4. Globin mRNA synthesis occurs first only in G1 cells.

5. 5. Globin mRNA is synthesized later in all phases of the cell cycle.

These data suggest that (a) commitment to differentiation and globin mRNA accumulation are coupled; and (b) that both events occur only in G 1 cells after a pre-commitment phase of about 12 h.     

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells infected with vesicular stomatitis virus or herpes simplex virus.

We have compared the effects of infection with herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) on the protein synthetic apparatus of Friend erythroleukemia cells. Previous studies demonstrated that infection with HSV rapidly shuts off the synthesis of globin and other cellular polypeptides (Y. Nischioka and S. Silverstein, 1977, Proc. Natl. Acad. Sci. U.S.A. 74: 2370-2374). In contrast to these findings, globin synthesis persists in Friend erythroleukemia cells infected with VSV. Physical measurements of the size of bulk-infected cell mRNA, using hybridization with polyuridylic acid, demonstrated that there was no detectable change in the size of mRNA's after infection with VSV. A comparison of the kinetics of hybridization of cytoplasmic RNA extracted from cells infected with either HSV or VSV with globin complementary DNA revealed that by 4 h postinfection with HSV only about 15% of the globin mRNA sequences remained, whereas there was no discernible change in the sequence abundance of globin mRNA in VSV-infected cells.     

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.     

Hexamethylenebisacetamide-mediated induction of murine erythroleukemia cells: relationship between expression of beta major and beta minor globin genes, globin mRNA synthesis and commitment to erythroid differentiation

A switch in beta globin gene expression is operated in murine Friend erythroleukemia cells due to the inducing agent used. The competence of Friend cells to express beta major globin genes is operated within 8 hours exposure to hexamethylenebisacetamide. This early feature of induced differentiation is expressed in the absence of beta globin mRNA synthesis and is not suppressed by the corticosteroid hormone dexamethasone, which by contrast inhibits later stages of induced-mediated commitment to erythroid differentiation such as globin mRNA accumulation and heme synthesis.     

Hypomethylation of DNA during differentiation of friend erythroleukemia cells

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.     

Induction of globin mRNA accumulation by hemin in cultured erythroleukemic cells

The role of heme in erythroid development is investigated in erythroleukemic (Friend) cells. Exogenous hemin induces the accumulation of globin mRNA and globin protein in T3-Cl2 erythroleukemia cells to levels comparable to those induced by polar solvents, such as dimethylsulfoxide (DMSO). The hemin concentration required for maximal induction (10^?4 M) is the same as that which stimulates globin message translation in reticulocytes or cell-free reticulocyte lysates. Hemin and DMSO together cause T3-Cl2 cells to accumulate 8–9 fold more globin mRNA than either inducer individually. The kinetics of globin mRNA induction in hemin as compared to DMSO are very different: globin message accumulation begins 4 hr after hemin addition, but not until 30–40 hr after DMSO addition. Biliverdin induces 20–40 fold less hemoglobin than hemin; delta-aminolevulinic acid and porphobilinogen do not induce.     

The major heat-shock protein hsp 68 is not induced by stress in mouse erythroleukemia cell lines

Most mammalian cells respond to brief incubation at elevated temperatures by enhanced or new synthesis of a set of heat-shock proteins (hsp). In mouse cells, as determined by SDS--one-dimensional gel electrophoresis, the most prominent hsps have molecular masses of approximately 89,000, 70,000, and 68,000 Da. When the heat-shock response of the mouse erythroleukemia cell line D1B, or two other DBA/2 cell lines (707C1 and 745C2), was examined by [35S]methionine labelling, following heat shocks of 10 min at 42 or 44 degrees C, or 1 h at 45 degrees C, no protein band corresponding to hsp 68 was observed. However, the synthesis of both hsp 89 and hsp 70 was enhanced. Northern blot analysis of cytoplasmic RNA extracted from control and stressed cells indicated that hsp 68 mRNA was absent, even after stresses of up to 1 h at 45 degrees C. Differentiation induced by dimethyl sulphoxide (DMSO) (monitored by the induction of globin synthesis) had no effect on hsp 68 expression in D1B cells; also, hsp 68 could not be induced at various stages of differentiation (0-72 h). Southern blot analysis showed that all three hsp-68 genes were present and not rearranged, and apparently did not carry any deletion in their 5' ends. To determine whether methylation could be involved in maintaining the genes in their silent state, we treated cells with 10 microM 5-azacytidine for 48 h. No hsp 68 expression was observed following such treatment in either undifferentiated or DMSO-induced differentiated D1B cells. Furthermore, Southern blot analysis of MspI/HpaII-digested genomic D1B DNA did not display any differences in methylation patterns around the promoter region of the probed gene compared with control cells, indicating that methylation is not involved in hsp-68 repression. When chimeric plasmids carrying the bacterial chloramphenicol acetyl transferase gene under regulation of the mouse hsp-68 or Drosophila hsp-70 promoters were transfected into D1B cells, minimal (2-fold) or no induction was observed, in contrast with the 60-fold induction seen in a control myeloma cell line. These results suggest a trans-acting mechanism of hsp-68 repression in erythroleukemia cells.     

细胞DNA低甲基化是否为化学致癌启动过程的必经途径

There seems to be a current view that the inhibition of DNA methylation may be a mechanism of initiation of carcinogenesis or one of the steps required in the carcinogenic process. However, because of the deficiencies in research works on cellular level, there is a long way to make such a general relationship between carcinogen and the inhibition of cellular DNA methylation. In this paper the effects of some chemical carcinogens on methylation of newly replicated DNA in human FL cells was analyzed by comparing the weight average length (Lw) of the DNA digests after complete digestion by restriction endonuclease Hpa II. It was observed that two non-genotoxic carcinogens (5-azacytidine and L-ethionine) and two genotoxic carcinogens (MNNG and aflatoxin B1) used in this study all caused obvious cytotoxicity on FL cells at the test concentration. Five days after the termination of 5-azacytidine treatment (2 x 10(-6) M, 24 hours), Lw (kb) of the Hpa II digests of cellular DNA was smaller than that of control (8.0 +/- 0.1 vs 10.9 +/- 1.0, P less than 0.01), the Lw change rate was -27%. When DNA was analyzed from FL cells after 9 days continuous treatment of L-ethionine (2 x 10 M), the digestibility of Hpa II was also increased as compared with that of the control, the Lw values (kb) showed a decrease of about 8% (9.8 +/- 0.3 vs 10.6 +/- 0.3, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

2-Aminobenzamide, an inhibitor of ADP-ribosylation, antagonizes induced DNA hypomethylation during differentiation of murine Friend erythroleukemia cells by N''-methylnicotinamide

The ADP-ribose synthesis inhibitor 2-aminobenzamide was found to reduce the induction of haemoglobin synthesis in murine Friend erythroleukemia cells, cultured continuously for 96 h with 5 mM N'-methylnicotinamide, with over 50% inhibition at equimolar doses. 2-Aminobenzamide was optimally effective as an inhibitor of differentiation if present only during the time period 24-48 h after initial N'-methylnicotinamide exposure. A transient, genome-wide DNA hypomethylation accompanies induction by N'-methylnicotinamide. Although 2-aminobenzamide does not seem to affect normal DNA methylation in cultured cells, the observed DNA hypomethylation in cells cultured for 24 h with 5 mM N'-methylnicotinamide was antagonized over 50% by equimolar 2-aminobenzamide. We suggest that ADP-ribosylation has a positive modulatory role in erythroid differentiation, and possibly in the process(es) leading to DNA hypomethylation following inducer exposure.     

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.