Commitment Of Murine Erythroleukemia Cells Research Articles

Genetic manipulation of RPS5 gene expression modulates the initiation of commitment of MEL cells to erythroid maturation: Implications in understanding ribosomopathies.

Impairment of ribosome biogenesis contributes to the molecular pathophysiology of ribosomopathies by deregulating cell-lineage specific proliferation, differentiation and apoptosis decisions of haematopoietic progenitor cells. Here, using pro-erythroblast-like murine erythroleukemia (MEL) cells, a model system of erythroid maturation, we aimed to investigate whether genetic manipulation of RPS5 expression affects the capacity of cells to grow and differentiate in culture. Parental MEL cells stably transfected with full length RPS5 cDNA in sense (MEL-C14 culture) or antisense (MEL-antisenseRPS5 culture) orientation, as well as MEL cells transiently transfected with siRNAs specific for RPS5 gene silencing (MEL-RPS5siRNA culture) were assessed for their ability to fully execute their erythroid maturation program in culture. The data obtained thus far indicate that: a) MEL-antisenseRPS5 exhibit a pronounced delay in the initiation of differentiation, as well as an impairment of commitment, since the continuous presence of the inducer in culture is required for the cells to fully execute their erythroid maturation program. b) RNAi-mediating silencing of RPS5 gene expression resulted in the inability of MEL cells to differentiate; however, when these cells were allowed to recapitulate normal RPS5 gene expression levels they regained their differentiation capacity by accumulating high proportion of erythroid mature cells. c) Interestingly the latter, is accompanied by morphological changes of cells and an impairment of their proliferation and apoptosis potential. Such data for the first time correlate the RPS5 gene expression levels with the differentiation capacity of MEL cells in vitro, a fact that might also have implications in understanding ribosomopathies.

Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells

Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA). To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-induced cells by differential hybridization and isolated GTPase Ran as a down-regulated gene. We observed that Ran was expressed in a biphasic mode. Following a decrease in mRNA level during the initial hours of induction, Ran re-expressed at 24–48 h, and gradually declined again. To investigate the role of Ran during MEL differentiation we constructed MEL transfectants capable to express or block Ran mRNA production constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL cell differentiation resulting in a decrease of cell survival in the committed population.

Secretion and binding of HMG1 protein to the external surface of the membrane are required for murine erythroleukemia cell differentiation

We show here that murine erythroleukemia (MEL) cells, following induction with hexamethylene bisacetamide, accumulate high mobility group (HMG)1 protein onto the external surface of the cell in a membrane-associated form detectable by immunostaining with a specific anti-HMG1 protein antibody. This association is maximal at a time corresponding to cell commitment. At longer times, immunostainable cells are progressively reduced and become almost completely undetectable along with the appearance of hemoglobin molecules. Binding to MEL cells does not affect the native molecular structure of HMG1 protein. The type of functional correlation between HMG1 protein and MEL cell differentiation is suggested by the observation that if an anti-HMG1 protein antibody is added at the same time of the inducer almost complete inhibition of cell differentiation is observed, whereas if the antibody is added within the time period in which cells undergo through irreversible commitment, inhibition progressively disappears. A correlation between MEL cell commitment and the biological effect of HMG1 protein can thus be consistently suggested.

Nuclear inositol lipid cycle and differentiation

Previous investigations from our laboratory and others have shown the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and PLC which in PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, rat liver and Swiss 3T3 cells is the isoform β1, which in the latter cells is activated upon IGF-I stimulation. The behavior of the nuclear inositol lipid cycle has been investigated in nuclei of Friend erythroleukemia cells. These nuclei possess both lipid kinases and PLC. The cycle upon treatment with differentiating agents (i.e., DMSO and tiazofurin) is characterized by an accumulation of polyphosphoinositides and a decrease of DAG due to down-regulation of a specific PLC. Indeed, even if both β1 and -γ1 isoforms are present in these nuclei, when Friend cells undergo terminal erythroid differentiation only the PLC β1 isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by direct determination of enzymatic activity and in the presence of neutralizing monoclonal antibodies as well as by Northern blot for PLC β1 message, whilst the amount of PLC γ1 and its activity are unaffected by erythroid differentiation. In conclusion, the presence of a specific nuclear PLC whose activity and expression are down-regulated during differentiation of erythroleukemia cells points out a role for nuclear phosphoinositide signalling in the processes of cell differentiation and hints at the nuclear PLC β1 as an important step of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

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.

Different signalling pathways are used in the commitment of murine erythroleukemia cells (TSA 8) to differentiate, and in the erythropoietin action on progenitor cells.

The murine erythroleukemia (MEL) cell line, TSA8, becomes responsive to erythropoietin after induction with dimethyl sulfoxide (DMSO). We examined the signalling pathways involved in the commitment of TSA8 cells to become the erythroid progenitor cells responsive to erythropoietin, comparing them with the pathway used in an erythropoietin-induced change of the progenitor cells. Amiloride, an inhibitor of the Na+/H+ antiporter, completely blocked the commitment of TSA8 cells to become responsive to erythropoietin at a concentration that did not affect cell proliferation, while it showed no effect on the differentiation or proliferation of the erythroid progenitor cells derived from TSA8 cells by erythropoietin. Ethyleneglycol-bis (beta-aminoethyl ether) N,N,N',N'-tetra acetic acid (EGTA) inhibited the commitment of TSA8 cells to CFU-E-like cells without affecting colony formation. In contrast, EGTA did not inhibit erythropoietin-induced differentiation of the progenitor cells, but did inhibit their proliferation. These results indicate that erythropoietin uses different signalling pathways from those used in the induction of the commitment of TSA8 cells.

Idiotype mimicry by a differentiation antigen on Friend erythroleukemia cells.

We previously found that murine leukemia cells of T cell, B cell, and erythroid ontogeny express a cell membrane antigen that cross-reacts with an idiotype of an anti-retroviral antibody. In the present study, the expression of this antigen (termed AVID, for anti-viral idiotype) by murine erythroleukemia (MEL) cells was examined during chemically induced differentiation. AVID expression by MEL cells was found to be lost when they were treated with either dimethyl sulfoxide or hexamethylene bisacetamide, two chemicals that induce MEL cells to terminally differentiate. The kinetics of disappearance of AVID during inducer treatment reflected the kinetics with which the inducers caused MEL cell commitment to terminal differentiation. Loss of AVID expression by inducer-treated cells was inhibited by dexamethasone, which inhibits commitment and MEL cell differentiation. The subset of inducer-treated cells that expressed the least amount of AVID contained the greatest number of cells committed to differentiate. These results indicate that AVID identifies a novel differentiation antigen of MEL cells.

Commitment to differentiation of murine erythroleukemia cells involves a modulated plasma membrane depolarization through Ca2+‐activated K+ channels

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.

Induction of commitment of murine erythroleukemia cells (TSA8) to CFU-E with DMSO

The commitment of novel mouse erythroleukemic (MEL) cells (TSA8) to colonyforming units of erythroid (CFU-E) by dimethylsulfoxide (DMSO) was investigated. After exposure to the inducer in liquid culture, the cells were transferred to a semi-solid culture to examine their ability to form erythroid colonies which were dependent on erythropoietin. Exposure to DMSO for 2 days is optimum for CFU-E type colony formation and colonies induced in this manner are equivalent to CFU-E. The induction occurred in a synchronous manner. Partly stained colonies appeared prior to CFU-E formation and are thought to be a result of asymmetric cell division. Appearance of these partly stained colonies suggested that the number of erythropoietin receptors is important in the complete responsiveness to erythropoietin. TSA8 cells constitute a suitable model system in which to analyse the mechanism of commitment in early erythropoiesis.

Inducer-mediated commitment of murine erythroleukemia cells to terminal cell division: the expression of commitment.

Murine erythroleukemia cells (MELC) are transformed cells that can be induced to differentiate by a variety of agents, such as hexamethylenebisacetamide (HMBA) and dimethyl sulfoxide. Dexamethasone suppresses HMBA-mediated MELC differentiation, but MELC retain a memory for their exposure to HMBA since, on transfer from culture with HMBA and dexamethasone to medium without additions, a portion of the cells express characteristics of terminal differentiation. This study characterizes the steroid suppressed steps in the multi-step process of inducer-mediated MELC terminal differentiation. MELC in culture with HMBA and dexamethasone show low levels of commitment to terminal cell division; upon transfer to culture with inducer alone there is a rapid increase in the proportion of committed cells. The magnitude of this rapid or "step-up" expression of commitment increased with the length of prior culture with inducer and steroid. This step-up expression is not inhibited by actinomycin D or cordycepin but is blocked by cycloheximide. HMBA is required for step-up expression of commitment. In the absence of inducer, there is a rapid decay in the capacity for step-up expression. Thus, HMBA initiates a series of changes leading to the accumulation of factors--which may be mRNAs--whose expression is blocked by dexamethasone. Hemin, which induces MELC accumulation of globin mRNA but not commitment to terminal cell division, cannot, as does HMBA or dimethyl sulfoxide, cause step-up expression of commitment.

Alterations in globin gene chromatin conformation during murine erythroleukemia cell differentiation.

Adult beta-globin gene chromatin in murine erythroleukemia (MEL) cells acquired increased sensitivity to both micrococcal nuclease and DNase I during hexamethylenebisacetamide-induced erythoid differentiation. The DNase I hypersensitivity of the globin genes accompanied their actual transcription and was strongly correlated with commitment events. On the other hand, the rate of micrococcal nuclease digestion was closely related to the rate of globin gene transcription. Two distinct DNase I hypersensitive sites were found on the 5' side of the beta-major globin gene in HMBA-induced cells. One site was located near the 5' side of the beta-major globin gene and the second site was located approximately 3 kilobases upstream of the beta-major cap site. Following the commitment of MEL cells to differentiate, DNase I sensitivity was stably inherited in the absence of inducer. In contrast to HMBA, another inducer, hemin, known to cause the accumulation of globin-specific mRNA in MEL cells by a post-transcriptional mechanism, did not elicit alterations of beta-globin gene chromatin. The addition of dexamethasone, a hormone known to inhibit MEL cell commitment, blocked the formation of general and site-specific nuclease sensitivity of beta-globin gene chromatin prior to but not after cell commitment.

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.

Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation

The level of cytoplasmic calcium ions appears to be important in the control of murine erythroleukemia (MEL) cell differentiation. Our interest in this study focuses on the relationship between the regulation of calcium concentration and differentiation. We used the fluorescent membrane probe DiOC 6 to examine the relationship between MEL cell mitochondria and changes in cytoplasmic calcium levels occurring at the initiation of commitment. Fluorescence microscopy reveals the selective association of DiOC 6 with MEL cell mitochondria, where an enhanced fluorescence is observed. Treatment of cells with dimethylsulfoxide (DMSO) or other inducers causes a decrease in mitochondria-associated fluorescence levels that occurs with the initiation of commitment. A decrease in DiOC 6 fluorescence is caused by agents that reduce mitochondrial membrane potential, but is only slightly affected by agents that alter plasma membrane potential. Amiloride and EGTA, agents that prevent commitment and inhibit calcium uptake, also prevent the decrease in DiOC 6 uptake caused by DMSO. The effect of DMSO on MEL cell mitochondria is mimicked by FCCP, a proton ionophore that dissipates mitochondrial membrane potential. FCCP also caused MEL cell mitochondria to release calcium into the cytoplasm. When MEL cells are treated with DMSO plus FCCP, commitment is initiated without the lag period observed when cells are treated with DMSO alone. These results are consistent with the hypothesis that mitochondrial transmembrane potential is important in the regulation of cytoplasmic calcium levels at the time of commitment of MEL cells to terminal differentiation.

Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process.

There are a number of agents which, when added to cultures of murine erythroleukemia cells (MELC), markedly increase the probability of commitment to express the characteristics of terminal erythroid differentiation, including loss of proliferative capacity and increased accumulation of globin mRNA and hemoglobin. Some characteristics of inducer-mediated commitment of MELC to terminal erythroid differentiation were examined by determining the effects of dexamethasone (an inhibitor of inducer-mediated MELC differentiation) and of hemin (an inducer of globin mRNA accumulation). Previously, it was shown that exposure of MELC to hexamethylene-bisacetamide (HMBA) leads to commitment, detectable within 12 hr. MELC cultured with both HMBA and dexamethasone do not express commitment. MELC transferred from culture with HMBA and dexamethasone to cloning medium without these agents express commitment to terminal erythroid differentiation, indicating that MELC retain a "memory" for some early HMBA-mediated changes leading to commitment which occur even in the presence of the inhibitory steroid. The kinetics of commitment in experiments in which exposure to HMBA is interrupted, or dexamethasone is added to the culture in HMBA, suggest that there is a rate-limiting step early in the commitment process. The memory for this step persists for more than one cell cycle. Addition of hemin to cultures with HMBA and dexamethasone initiated accumulation of globin mRNA but does not reverse the steroid-mediated inhibition of terminal cell division (that is, the cells retain their proliferative capacity). Inducer-mediated MELC commitment is associated with accumulation of the chromatin protein IP25; dexamethasone does not inhibit this accumulation. Accumulation of IP25 may be inducer-related, but it is not sufficient to cause expression of terminal erythroid differentiation.

Evidence that a Na+/Ca2+ antiport system regulates murine erythroleukemia cell differentiation.

The Na+ and Ca2+ transport properties of cultured murine erythroleukemia (MEL) cells have been investigated. We have previously shown that amiloride prevents dimethyl sulfoxide-induced MEL cell differentiation via inhibition of an essential Ca2+ influx (levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5948-5952). Here we show that external Na+ inhibits Ca2+ influx and stimulates Ca2+ efflux from uninduced MEL cells. Increasing the internal Na+ concentration by a brief incubation of cells with ouabain stimulates the rate of 45Ca2+ influx. Amiloride (40 microM) completely blocks the external Na+-stimulated 45Ca2+ efflux and external Na+-inhibitable 45Ca2+ influx. The same concentration of amiloride had no significant effect on net Na+ uptake. These results suggest that a significant fraction of Ca2+ flux across the MEL cell plasma membrane occurs via a Na+/Ca2+ antiport system and that amiloride prevents differentiation by blocking Ca2+ influx through this system. The importance of a Na+/Ca2+ antiport system for MEL cell differentiation is supported by the following observation: increasing the cellular Na+ level by a brief treatment with ouabain plus monensin accelerates MEL cell commitment as effectively as adding the Ca2+ ionophore A23187. We suggest that dimethyl sulfoxide induces MEL cell differentiation by inhibiting the Na+ pump and consequently allowing Ca2+ influx through the Na+/Ca2+ antiport.

Calcium regulates the commitment of murine erythroleukemia cells to terminal erythroid differentiation.

An alteration in the rate of calcium transport appears to be the rate-limiting event for the commitment of murine erythroleukemia (MEL) cells to initiate a program of terminal erythroid differentiation. The dimethyl sulfoxide (DMSO)-induced commitment of MEL cells to erythroid differentiation can be inhibited by treatment of cells with the calcium-chelating agent EGTA. Upon removal of EGTA, cells initiate commitment without the 12-h lag normally observed after treatment with DMSO alone. Treatment of cells with DMSO in the presence of calcium ionophore A23187 causes cells to initiate commitment from time zero with no lag. These results suggest that the lag is the time required for DMSO to alter the calcium transport properties of the cell.

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.

Developmental program of murine erythroleukemia cells. Effect of the inhibition of protein synthesis.

The relationship between protein synthesis and commitment to terminal erythroid differentiation by dimethylsulfoxide-treated murine erythroleukemia (MEL) cells has been studied. Treatment with cycloheximide blocks the commitment of MEL cells. The effects of cycloheximide are completely reversible, however. Treatment of MEL cells before commitment delays commitment for a period of time equal to the length of inhibitor treatment. Puromycin exerts a similar effect on the commitment of MEL cells. These results indicate that there is a continuous requirement for protein synthesis before the commitment event.

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.