Differentiation Of Mouse Erythroleukemia Cells Research Articles

Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton.

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3's function during ED, we generated a Tmod3knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,β1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.

Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis.

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.

Cancer Differentiating Agent Hexamethylene Bisacetamide Inhibits BET Bromodomain Proteins.

Agents that trigger cell differentiation are highly efficacious in treating certain cancers, but such approaches are not generally effective in most malignancies. Compounds such as DMSO and hexamethylene bisacetamide (HMBA) have been used to induce differentiation in experimental systems, but their mechanisms of action and potential range of uses on that basis have not been developed. Here, we show that HMBA, a compound first tested in the oncology clinic over 25 years ago, acts as a selective bromodomain inhibitor. Biochemical and structural studies revealed an affinity of HMBA for the second bromodomain of BET proteins. Accordingly, both HMBA and the prototype BET inhibitor JQ1 induced differentiation of mouse erythroleukemia cells. As expected of a BET inhibitor, HMBA displaced BET proteins from chromatin, caused massive transcriptional changes, and triggered cell-cycle arrest and apoptosis in Myc-induced B-cell lymphoma cells. Furthermore, HMBA exerted anticancer effects in vivo in mouse models of Myc-driven B-cell lymphoma. This study illuminates the function of an early anticancer agent and suggests an intersection with ongoing clinical trials of BET inhibitor, with several implications for predicting patient selection and response rates to this therapy and starting points for generating BD2-selective BET inhibitors. Cancer Res; 76(8); 2376-83. ©2016 AACR.

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation.

Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult βmajor-globin gene promoter but did not affect expression of the βminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmajor-globin gene promoter during erythroid cell differentiation.

K-Cl Cotransporter Gene Expression during Human and Murine Erythroid Differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.

Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate.

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol®). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.

A Novel Splicing Factor RBM-9 Regulates Protein 4.1R Exon 16 Splicing.

Protein 4.1R (4.1R), a vital component of the red cell membrane cytoskeleton, stabilizes the spectrin-actin lattice and attaches it to embedded membrane proteins. A regulated splicing event, the inclusion of exon 16 that encodes for peptides critical for spectrin/actin binding, occurs during late erythroid differentiation. We showed earlier that an intricate combination of enhancer and silencer elements direct exon 16 splicing. Regulated expression of splicing factors, SF2/ASF and hnRNP A/B, has also been implicated in mediating exon 16 splicing. In this study, we attempted to characterize the mechanism involved in exon 16 splicing through UGCAUG, an intronic splicing enhancer present in three copies in the intron downstream of exon 16. We first used a wild-type minigene construct consisting of exons 13, 16, 17 and their respective flanking introns that mimics endogenous exon 16 splicing during the induced differentiation of mouse erythroleukemia cells (MELC). Mutational analysis showed a dose-dependent effect of UGCAUG on exon 16 splicing: the presence of all three copies had the most effect. These results were recapitulated with an internal chimeric exon in a test neutral reporter system “DUP4-1”, suggesting that the enhancing effect could be attributed directly to UGCAUG. Furthermore, we identified a novel splicing factor, RBM-9, from MELC that enhanced the internal exon splicing in an UGCAUG-dependent manner in both the exon 16 minigene and DUP4-1 reporter systems. Our characterization of RBM-9 revealed that diverse isoforms of RBM-9 are generated by the utilization of alternative translation initiation sites and tissue-specific alternative splicing; different isoforms from various tissues exhibited differential exon 16 splicing enhancing activities. MELC-RBM-9 enhanced exon 16 splicing the most among all RBM-9 isoforms tested. Inhibition of RBM-9 expression by RBM-9-shRNA reversed its enhancing activity on exon 16 inclusion in MELC. RBM-9-shRNA also reduced exon 16 splicing in a dose-dependent manner in HeLa cells. Furthermore, purified RBM-9 specifically binds to the UGCAUG sequence in a gel-mobility shift assay. Finally, expression of RBM-9 is upregulated and correlates with exon 16 inclusion during MELC differentiation. These results suggest that a novel splicing factor, RBM-9, enhances erythroid differentiation stage-specific exon 16 splicing by interacting with the splicing enhancer UGCAUG.

Histone H4-Lysine 20 Monomethylation Is Increased in Promoter and Coding Regions of Active Genes and Correlates with Hyperacetylation

Methylation and acetylation of position-specific lysine residues in the N-terminal tail of histones H3 and H4 play an important role in regulating chromatin structure and function. In the case of H3-Lys(4), H3-Lys(9), H3-Lys(27), and H4-Lys(20), the degree of methylation was variable from the mono- to the di- or trimethylated state, each of which was presumed to be involved in the organization of chromatin and the activation or repression of genes. Here we investigated the interplay between histone H4-Lys(20) mono- and trim-ethylation and H4 acetylation at induced (beta-major/beta-minor glo-bin), repressed (c-myc), and silent (embryonic beta-globin) genes during in vitro differentiation of mouse erythroleukemia cells. By using chromatin immunoprecipitation, we found that the beta-major and beta-minor promoter and the beta-globin coding regions as well as the promoter and the transcribed exon 2 regions of the highly expressed c-myc gene were hyperacetylated and monomethylated at H4-Lys(20). Although activation of the beta-globin gene resulted in an increase in hyperacetylated, monomethylated H4, down-regulation of the c-myc gene did not cause a decrease in hyperacetylated, monomethylated H4-Lys(20), thus showing a stable pattern of histone modifications. Immunofluorescence microscopy studies revealed that monomethylated H4-Lys(20) mainly overlaps with RNA pol II-stained euchromatic regions, thus indicating an association with transcriptionally engaged chromatin. Our chromatin immunoprecipitation results demonstrated that in contrast to trimethylated H4-Lys(20), which was found to inversely correlate with H4 hyper-acetylation, H4-Lys(20) monomethylation is compatible with histone H4 hyperacetylation and correlates with the transcriptionally active or competent chromatin state.

MRG15 Regulates Embryonic Development and Cell Proliferation

MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15-/- embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15-/- mouse embryonic fibroblasts. The hearts of the Mrg15-/- embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15-/- embryos appeared pale, and microarray analysis revealed that alpha-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the alpha-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.

Histone H4 Hyperacetylation Precludes Histone H4 Lysine 20 Trimethylation

Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non- and monoacetylated H4 lysine 20 (H4-Lys(20)) trimethylation. An increase in H4-Lys(20) trimethylation, however, to a clearly lesser extent, was also found when cells accumulated in the stationary phase. Since we show that trimethylated H4-Lys(20) is localized to heterochromatin, the increase in H4-Lys(20) trimethylation observed indicates an accumulation of chromatin-dense and transcriptionally silent regions during differentiation and during the accumulation of control cells in the stationary phase, respectively. When using the deacetylase inhibitors butyrate or trichostatin A, we found that H4 hyperacetylation prevents H4-Lys(20) trimethylation, but not mono- or dimethylation, and that the nonacetylated unmethylated H4-Lys(20) is therefore the most suitable substrate for H4-Lys(20) trimethylase. Summarizing, histone H4-Lys(20) hypotrimethylation correlates with H4 hyperacetylation and H4-Lys(20) hypertrimethylation correlates with H4 hypoacetylation. The results provide a model for how transcriptionally active euchromatin might be converted to the compacted, transcriptionally silent heterochromatin.

PU.1 and pRb Bind GATA-1 on DNA and Recruit a Histone H3K9 Methyl Transferase-Containing Complex to Repress the Erythroid Transcription Program.

Current work indicates that transcriptional repression is at least as important as transcriptional activation in normal development. Inappropriate or untimely transcriptional repression in immature hematopoietic cells is often the basis for a block to differentiation in hematologic malignancies. Activation of PU.1, a myeloid and B-cell specific transcription factor, in erythroid cells plays a key role in Friend virus-induced mouse erythroleukemia (MEL). Previous results from our laboratory showed that PU.1 blocks the erythroid differentiation-promoting activity of GATA-1 by binding directly to GATA-1 on DNA and inhibiting its transcriptional function. PU.1-mediated repression of GATA-1 on transiently transfected GATA-1 target genes is dependent on the corepressor pRb that also binds to PU.1 (Rekhtman et al., Genes & Dev 1999 and Mol Cell Biol 2003). To further investigate the mechanism of PU.1-mediated repression of GATA-1 in chromatin, we examined the occupancy of several GATA-1 target genes by PU.1 and pRb, as well as the state of core histone modifications at these loci in MEL cells by quantitative chromatin immunoprecipitation. These studies included both endogenous GATA-1 target genes and an exogenous GATA-1 target gene (alpha globin) integrated at a specific locus in MEL cells by Recombinase-Mediated Cassette Exchange. We found that GATA-1 sites at both the exogenous, integrated gene as well as at endogenous genes (including the regulatory regions of the alpha globin, beta globin, alas-e, eklf, p45 nf-e2) are occupied by a GATA1 - PU.1 - pRb complex in undifferentiated MEL cells. The presence of all three components of the complex is dependent on intact GATA-1 binding sites in the exogenous, integrated gene. The histone methyltransferase Suv39H1 and the histone H3MeK9 binding protein, HP1alpha, are also present at the repressed loci. During induced differentiation of MEL cells, PU.1, pRb, Suv39H1 and HP1alpha occupancy at these sites declines but GATA-1 continues to be present at its binding sites. The disruption of the repression complex at these loci during differentiation as well as during siRNA-mediated PU.1 knock down is associated with conversion of methylated H3K9 to acetylated H3K9 and significant transcriptional derepression of these GATA-1 target genes. These findings support a model for repression of GATA-1 by PU.1 at endogenous loci through recruitment of the corepressor pRb and associated histone methyltransferase (Suv39H1) and H3MeK9 binding (HP1alpha) activities.

Spi-1/PU.1 but not Fli-1 inhibits erythroid-specific alternative splicing of 4.1R pre-mRNA in murine erythroleukemia cells.

The inclusion of exon 16 in mature protein 4.1R mRNA arises from a stage-specific splicing event that occurs during late erythroid development. We have shown that mouse erythroleukemia (MEL) cells reproduce this erythroid-specific splicing event upon induction of differentiation. We here found that this splicing event is regulated specifically in erythroleukemic cells that have the potential to differentiate and produce hemoglobin, regardless of the nature of the differentiation inducer. Knowing that dysregulated expression of spi-1/pu.1 and fli-1 oncogenes is involved in MEL cell differentiation arrest, we looked at their effect on exon 16 erythroid splicing. We found that exon 16 inclusion requires Spi-1/PU.1 shutdown in MEL cells, and that enforced expression of Spi-1/PU.1 inhibits exon selection, regardless of the presence or absence of a chemical inducer. By contrast, endogenous overexpression or enforced expression of Fli-1 has no effect on exon selection. We further showed that Spi-1/PU.1 acts similarly on the endogenous and on a transfected exon 16, suggesting a promoter-independent effect of Spi-1/PU.1 on splicing regulation. This study provides the first evidence that Spi-1/PU.1 displays the unique property, not shared with Fli-1, to inhibit erythroid-specific pre-mRNA splicing in erythroleukemia cell context.

Leukemia-related transcription factor TEL accelerates differentiation of Friend erythroleukemia cells.

TEL belongs to a member of the ETS family transcription factors that represses transcription of target genes such as FLI-1. Although TEL is essential for establishing hematopoiesis in neonatal bone marrow, its role in erythroid lineage is not understood. To investigate a role for TEL in erythroid differentiation, we introduced TEL into mouse erythroleukemia (MEL) cells. Overexpressing wild-type-TEL in MEL cells enhanced differentiation induced by hexamethylene bisacetamide or dimethylsulfoxide, as judged by the increased levels of erythroid-specific delta-aminolevulinate synthase and beta-globin mRNAs. TEL bound to a corepressor mSin3A through the helix-loop-helix domain. A TEL mutant lacking this domain still bound to the ETS binding site, but lost its transrepressional effect. This mutant completely blocked erythroid differentiation in MEL cells. Moreover, it showed dominant-negative effects over TEL-mediated transcriptional repression and acceleration of erythroid differentiation. Endogenous TEL mRNA was found to increase during the first 3 days in differentiating MEL cells and drastically decrease thereafter. All these data suggest that TEL might play some role in erythroid cell differentiation.

Chromatin remodeling gene SMARCA5 is dysregulated in primitive hematopoietic cells of acute leukemia

We identified a subset of genes involved in chromatin remodeling whose mRNA expression changes in differentiating mouse erythroleukemia (MEL) cells. We furthermore tested their mRNA expression patterns in normal and malignant CD34+ bone marrow cells. SMARCA5, imitation switch gene homologue, was rapidly silenced during in vitro erythroid differentiation of MEL cells whereas it was up-regulated in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Moreover, SMARCA5 mRNA levels decreased in AML CD34+ progenitors after the patients achieved complete hematologic remission. We detected high levels of SMARCA5 mRNA in murine bone marrow and spleen and monitored its expression in these hematopoietic tissues during accelerated hematopoiesis following hemolytic anemia induced by phenylhydrazine. SMARCA5 expression levels decreased after the onset of accelerated erythropoiesis. Our data suggest that both in vitro and in vivo induction of differentiation is followed by down-regulation of SMARCA5 expression. In CD34+ AML progenitors over-expression of SMARCA5 may thus dysregulate the genetic program required for normal differentiation.

Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells.

Malignant transformation usually inhibits terminal cell differentiation but the precise mechanisms involved are not understood. PU.1 is a hematopoietic-specific Ets family transcription factor that is required for development of some lymphoid and myeloid lineages. PU.1 can also act as an oncoprotein as activation of its expression in erythroid precursors by proviral insertion or transgenesis causes erythroleukemias in mice. Restoration of terminal differentiation in the mouse erythroleukemia (MEL) cells requires a decline in the level of PU.1, indicating that PU.1 can block erythroid differentiation. Here we investigate the mechanism by which PU.1 interferes with erythroid differentiation. We find that PU.1 interacts directly with GATA-1, a zinc finger transcription factor required for erythroid differentiation. Interaction between PU.1 and GATA-1 requires intact DNA-binding domains in both proteins. PU.1 represses GATA-1-mediated transcriptional activation. Both the DNA binding and transactivation domains of PU.1 are required for repression and both domains are also needed to block terminal differentiation in MEL cells. We also show that ectopic expression of PU.1 in Xenopus embryos is sufficient to block erythropoiesis during normal development. Furthermore, introduction of exogenous GATA-1 in both MEL cells and Xenopus embryos and explants relieves the block to erythroid differentiation imposed by PU.1. Our results indicate that the stoichiometry of directly interacting but opposing transcription factors may be a crucial determinant governing processes of normal differentiation and malignant transformation.

Notch-1 inhibits apoptosis in murine erythroleukemia cells and is necessary for differentiation induced by hybrid polar compounds

Strikingly increased expression of notch-1 has been demonstrated in several human malignancies and pre-neoplastic lesions. However, the functional consequences of notch-1 overexpression in transformed cells remain unclear. We investigated whether endogenously expressed notch-1 controls cell fate determination in mouse erythroleukemia (MEL) cells during pharmacologically induced differentiation. We found that notch-1 expression is modulated during MEL cell differentiation. Premature downregulation of notch-1 during differentiation, by antisense S-oligonucleotides or by enforced expression of antisense notch-1 mRNA, causes MEL cells to abort the differentiation program and undergo apoptosis. Downregulation of notch-1 expression in the absence of differentiation inducer increases the likelihood of spontaneous apoptosis. We conclude that in MEL cells, endogenous notch-1 expression controls the apoptotic threshold during differentiation and growth. In these cells, notch-1 allows differentiation by preventing apoptosis of pre-committed cells. This novel function of notch-1 may play a role in regulating apoptosis susceptibility in notch-1 expressing tumor cells.

Dephosphorylation of Vav is associated with the induction of mouse erythroleukemia cell differentiation: effects of orthovanadate and levamisole.

Mouse erythroleukemia (MEL) cell erythroid differentiation induced by dimethyl sulfoxide or hexamethylene bisacetamide (HMBA) is accompanied by the production of hemoglobin, terminal cell division and decreases in lactate production and fructose 2,6-bisphosphate levels. A number of studies have suggested that decreases in the cellular level of protein phosphotyrosine content may play a role in MEL cell differentiation. In particular, it was shown that the expression of several protein tyrosine phosphatase genes accompany this process and that the transfection of one of these genes into MEL cells followed by its subsequent expression induced eythroid differentiation. However, none of the physiological substrates for these protein tyrosine phosphatases have been identified. It is shown here that MEL cell differentiation is accompanied by decreases in tyrosine phosphorylation of Vav and possibly of the erythropoietin receptor (EpoR). Immunoprecipitation of the EpoR and analysis of co-precipitated proteins, indicates that the EpoR associates with Vav, STAT5 and an unidentified 60 Kd protein, . HMBA-induced erythroid differentiation abrogates these associations. The phosphatase inhibitors, Na3VO4 and levamisole, inhibit HMBA-induced differentiation as well as the association of the EpoR with Vav, STAT5 and the 60 Kd protein. This is of interest since Na3VO4, at the concentrations used here, has been shown to be a potent inhibitor of the activity of protein tyrosine phosphatases. These results suggest that levamisole, at least indirectly, acts by a molecular mechanism similar to that of Na3VO4 and that the loss of the association of the EpoR with Vav, STAT5, and and/or the reduction in the level of tyrosine phosphorylation of these proteins may play a role in MEL cell differentiation.

Induction of erythroid differentiation is associated with inhibition of glycolysis and a decrease in fructose 2,6-bisphosphate levels

The fraction of glucose metabolized to lactate is dramatically reduced during erythroid differentiation of mouse erythroleukemia (MEL) cells induced by dimethyl sulfoxide (DMSO), hexamethylene bisacetamide (HMBA), or sodium butyrate treatment. In order to determine the mechanism of the reduction in lactate production, several enzymatic steps in glucose catabolism were investigated. No changes in glycolytic enzyme levels were found during differentiation that could account for the alteration in lactate production and alterations in pyruvate kinase activity are known not to occur during MEL cell differentiation. Further, utilizing D-mannoheptulose, a specific inhibitor of hepatic-/tumor-specific glucokinase, no dependence on the activity of this enzyme for growth or differentiation was observed. Therefore, the possibility was entertained that the decrease in lactate production reflected a decrease in fructose 2,6-bisphosphate (F-2,6-P-2) which is a major regulator of the lactate production due to its ability to allosterically stimulate phosphofructokinase-1 (PFK-1) activity. PFK-1 cannot function in the absence of F-2,6-P-2 when only a suboptimal concentration of one of its substrates, fructose-6-phosphate (F-6-P), is present. When assayed under limiting F-6-P concentrations, it was found that following DMSO- or HMBA-induced differentiation, PFK-1 activity was decreased 7-20-fold. This finding suggested that F-2,6-P-2 levels might be controlling lactate production in this system. In keeping with this idea, marked decreases in F-2,6-P-2 levels were found to occur during DMSO- or HMBA-induced differentiation. These data suggest that decreasing F-2,6-P-2 levels account for the decrease in lactate accumulation that occurs during MEL cell differentiation.

Regulation of the ferrochelatase gene expression during differentiation of mouse erythroleukemia cells.

Ferrochelatase [EC 4.99.1.1], the last step enzyme of heme biosynthesis, is transcribed from a single promoter. The enzyme is ubiquitously expressed in all cells, while the transcription is induced during erythroid differentiation. In a transient transfection assay, we identified two cis-acting elements involved in regulating the human ferrochelatase gene in mouse erythroleukemia (MEL) cells; one is the distal region (-108 to -95) for the basic expression in erythroid and non-erythroid cells, and the other is the proximal region (-80 to -72) corresponding to the induced expression during differentiation of dimethylsulfoxide-treated MEL cells. DNase I footprinting and gel mobility shift analyses revealed the presence of nuclear protein binding to the distal and the proximal regions, in both induced and uninduced MEL cells. These mean that two promoter regions play an important role in regulating the induced expression of ferrochelatase.

Expression of the Protein Tyrosine Phosphatase β2 Gene in Mouse Erythroleukemia Cells Induces Terminal Erythroid Differentiation

We have cloned cDNA for protein tyrosine phosphatase beta2, which had been implicated in erythroid differentiation of mouse erythroleukemia cells. Expression of cDNA constructs, in which beta2 cDNA is placed under the control of mouse metallothionein-I promoter, by ZnCl2 converted a significant portion (20 to 38%) of the cells to erythroid-like cells, which is 25-50% of the erythroid differentiation efficiency observed by conventional erythroid-inducing agents. Furthermore, introduction and expression of altered protein tyrosine phosphatase beta2 cDNA constructs designed to produce the enzyme lacking the phosphatase activity inhibited erythroid differentiation by 100-20%, depending upon the concentration of erythroid-inducing agents employed. These results strongly suggest that protein tyrosine phosphatase beta2 is involved in triggering erythroid differentiation in mouse erythroleukemia cells.