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.     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

Microarray profiling of genes differentially expressed during erythroid differentiation of murine erythroleukemia cells

Murine erythroleukemia (MEL) cells are widely used to study erythroid differentiation thanks to their ability to terminally differentiate in vitro in response to chemical induction. At the molecular level, not much is known of their terminal differentiation apart from activation of adult-type globin gene expression. We examined changes in gene expression during the terminal differentiation of these cells using microarray-based technology. We identified 180 genes whose expression changed significantly during differentiation. The microarray data were analyzed by hierarchical and k-means clustering and confirmed by semi-quantitative RT-PCR. We identified several genes including H1f0, Bnip3, Mgl2, ST7L, and Cbll1 that could be useful markers for erythropoiesis. These genetic markers should be a valuable resource both as potential regulators in functional studies of erythroid differentiation, and as straightforward cell type markers.     

Emodin can induce K562 cells to erythroid differentiation and improve the expression of globin genes

In China, the traditional Chinese medicine “YiSui ShenXu Granule” has been used for treating β-thalassemia over 20 years and known to be effective in clinic. Several purified components from “YiSui ShenXu Granule” are tested in K562 cells to reveal its effect on globin expression and erythroid differentiation, and one of the purified components, emodin, was demonstrated to increase the expression of α-, ε-, γ-globin, CD235a, and CD71 in K562 cells. Moreover, the increase of their expression is emodin concentration-dependent. The mRNA and microRNA (miRNA) expression profiles are further analyzed and 417 mRNAs and 35 miRNAs with differential expression between untreated and emodin-treated K562 cells were identified. Among them, two mRNAs that encode known positive regulators of erythropoiesis, ALAS2, and c-KIT respectively, increased during emodin-induced K562 erythroid differentiation, meanwhile, two negative regulators, miR-221 and miR-222, decreased during this process. These results indicate that emodin can improve the expression of globin genes in K562 cells and also induce K562 cells to erythroid differentiation possibly through up-regulating ALAS2 and c-KIT and down-regulating miR-221 and miR-222.     

Biphasic ordered induction of heme synthesis in differentiating murine erythroleukemia cells: role of erythroid 5-aminolevulinate synthase.

During dimethyl sulfoxide (DMSO)-stimulated differentiation of murine erythroleukemia (MEL) cells, one of the early events is the induction of the heme biosynthetic pathway. While recent reports have clearly demonstrated that GATA-1 is involved in the induction of erythroid cell-specific forms of 5-aminolevulinate synthase (ALAS-2) and porphobilinogen (PBG) deaminase and that cellular iron status plays a regulatory role for ALAS-2, little is known about regulation of the remainder of the pathway. In the current study, we have made use of a stable MEL cell mutant (MEAN-1) in which ALAS-2 enzyme activity is not induced by DMSO, hexamethylene bisacetamide (HMBA), or butyric acid. In this cell line, addition of 2% DMSO to growing cultures results in the normal induction of PBG deaminase and coproporphyrinogen oxidase but not in the induction of the terminal two enzymes, protoporphyrinogen oxidase and ferrochelatase. These DMSO-treated cells did not produce mRNA for beta-globin and do not terminally differentiate. In addition, the cellular level of ALAS activity declines rapidly after addition of DMSO, indicating that ALAS-1 must turn over rapidly at this time. Addition of 75 microM hemin alone to the cultures did not induce cells to terminally differentiate or induce any of the pathway enzymes. However, the simultaneous addition of 2% DMSO and 75 microM hemin caused the cells to carry out a normal program of terminal erythroid differentiation, including the induction of ferrochelatase and beta-globin. These data suggest that induction of the entire heme biosynthetic pathway is biphasic in nature and that induction of the terminal enzymes may be mediated by the end product of the pathway, heme. We have introduced mouse ALAS-2 cDNA into the ALAS-2 mutant cell line (MEAN-1) under the control of the mouse metallothionein promoter (MEAN-RA). When Cd and Zn are added to cultures of MEAN-RA in the absence of DMSO, ALAS-2 is induced but erythroid differentiation does not occur and cells continue to grow normally. In the presence of metallothionein inducers and DMSO, the MEAN-RA cells induce in a fashion similar to that found with the wild-type 270 MEL cells. Induction of the activities of ALAS, PBG deaminase, coproporphyrinogen oxidase, and ferrochelatase occurs. In cultures of MEAN-RA where ALAS-2 had been induced with Cd plus Zn 24 h prior to DMSO addition, onset of heme synthesis occurs more rapidly than when DMSO and Cd plus Zn are added simultaneously. This study reveals that induction of ALAS-2 alone is not sufficient to induce terminal differentiation of the MEAN-RA cells, and it does not appear that ALAS-2 alone is the rate-limiting enzyme of the heme biosynthetic pathway during MEL cell differentiation.     

Kinetics of histone H10 accumulation and commitment to differentiation in murine erythroleukemia cells

The accumulation of histone H10 (also denoted IP 25) in murine erythroleukemia cells, induced to differentiate with hexamethylene bis-acetamide, was shown to precede by 15-20 h the appearance in the culture of cells irreversibly committed to differentiate. In addition the rates of accumulation of H10 and of committed cells vary in a similar manner with the HMBA concentration. Flow microfluorimetric analysis demonstrated that the accumulation of H10 did not occur simultaneously in all the cells. This accumulation of histone H10 was initiated first in cell in the G2 phase of the cell cycle and subsequently in the cells situated in all the phases of the cell cycle.     

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.     

Characterization of cellular receptors for erythroid differentiation factor on murine erythroleukemia cells

Erythroid differentiation factor (EDF), which is structurally related to transforming growth factor-beta family and induces differentiation of murine erythroleukemia cell clone F5-5, has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-EDF to F5-5 cells is time- and temperature-dependent, specific, saturable, and reversible. Transforming growth factor-beta 1 has no significant effects on growth of F5-5 cells and binding of 125I-EDF to F5-5 cells. Scatchard analysis of the binding data indicated that F5-5 cells have a single class of binding sites (3,200/cell) with an apparent Kd of 3.1 X 10(-10) M. Affinity cross-linking experiments demonstrated three radiolabeled components of 140,000, 76,000, and 67,000 daltons under both reducing and nonreducing conditions. Labeling of these three components has been inhibited by incubation of the cells with excess unlabeled EDF. These results imply molecular weights of 115,000, 51,000, and 42,000 for the EDF receptors on this cell line.     

Isolation and characterization of murine erythroleukemia cell variants nonresponsive to hemin for the expression of globin genes

Thirty-four cloned lines of mouse erythroleukemia (MEL) cells that showed impairment in hemoglobin sy nthesis induced by hemin were isolated from line 745. Among these lines, 75% showed a reduced or nil response to induction by dimethyl sulfoxide (DMSO) and/or hypoxanthine. Four of these clones (three lines nonresponsive to hemin and one not responsive to either hemin or DMSO) were further characterized for the amount of globin mRNA accumulated in the cytoplasm and for the rate of globin synthesis upon exposure to hemin or DMSO. None of the four were induced by hemin to accumulate globin mRNA and all had a reduced rate of globin synthesis by comparison with control line 745. Two of the four lines had a high uninduced level of cytoplasmic globin mRNA not matched by a corresponding rate of globin synthesis, suggesting that they may accumulate defective globin mRNAs.     

DNA synthesis is not required for the commitment of murine erythroleukemia cells

We have assessed the relationship between DNA synthesis and the differentiation of MEL cells induced by DMSO. Under conditions where the rate of incorporation of ³H-deoxyadenosine into DNA was inhibited by 99%, the rate at which MEL cells become committed to terminal erythroid differentiation was identical to that of a culture treated with inducer alone. We conclude that commitment of MEL cells does not require concomitant DNA synthesis.     

Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation.

Cell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G1 phase of the cell cycle, followed by a re-entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G1 accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G1 block and re-enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells were unable to differentiate in the absence of seric factors, as measured by the number of benzidine-positive cells during induction with hexamethylene-bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the G1 block. The analysis of two molecular markers of cell differentiation confirmed these results.     

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 μM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.     

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 μM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.     

Amiloride in differentiation and commitment of Friend erythroleukemia cells

Dimethylsulfoxide (DMSO) converts almost all of the undifferentiated murine erythroleukemia cells (MEL or Friend cells, clone 745A) in a culture to differentiated cells that contain high levels of hemoglobin and that stop growing after a limited number of cell divisions. Contrary to other reports--that amiloride strongly inhibits DMSO-induced differentiation in MEL cells--in this laboratory, inhibition by amiloride, tested with DMSO over a range of concentrations in two kinds of media and at various cell densities, was found to be only weak or absent. Similarly, amiloride did not inhibit induction by N,N'-hexamethylene bis-acetamide (HMBA). As expected from previous findings with other cell systems, amiloride inhibited protein synthesis and cell multiplication.     

Kinetics of histone H1 ° accumulation and commitment to differentiation in murine erythroleukemia cells

The accumulation of histone H1 ° (also denoted IP 25) in murine erythroleukemia cells, induced to differentiate with hexamethylene bis-acetamide, was shown to precede by 15–20 h the appearance in the culture of cells irreversibly committed to differentiate. In addition the rates of accumulation of H1 ° and of committed cells vary in a similar manner with the HMBA concentration. Flow microfluorimetric analysis demonstrated that the accumulation of H1 ° did not occur simultaneously in all the cells. This accumulation of histone H1 ° was initiated first in cell in the G2 phase of the cell cycle and subsequently in the cells situated in all the phases of the cell cycle.