Murine Erythroleukemia Cells Research Articles

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.

Direct association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression.

PU.1, a member of the Ets family of transcription factors, is implicated in hematopoietic cell differentiation through its interactions with other transcriptional factors and cofactors. To identify a novel protein(s) binding to PU.1, we carried out affinity purification using a column of Glutathione-Sepharose beads bound to GST-PU.1 fusion protein and isolated several individual proteins using murine erythroleukemia (MEL) cell extracts. Sequence analysis of these proteins revealed that one was MeCP2 a methyl CpG binding protein. GST-pull-down assay and immunoprecipitation assay showed that PU.1 bound directly to MeCP2 via its Ets domain and MeCP2 bound to PU.1 via either its amino terminal domain or trans-repression domain. MeCP2 repressed transcriptional activity of PU.1 on a reporter construct with trimerized PU.1 binding sites. This downregulation was recovered in the presence of histone deacetylase inhibitor, trichostatin A (TSA). MeCP2 was integrated in PU.1-mSin3A-HDAC complex but not in PU.1-CBP complex. Chromatin immunoprecipitation (ChIP) assays showed that PU.1 and MeCP2 were collocated at the PU.1 binding site on the reporter construct and the PU.1 binding site of the intervening sequence 2 (IVS2) region in the intron of the beta-globin gene, which has been proposed to regulate expression of the gene, in undifferentiated MEL cells. The complex disappeared from the region during the course of erythroid differentiation of MEL cells. Our results suggest that MeCP2 acts as a corepressor of PU.1 probably due to facilitating complex formation with mSin3A and HDACs.

Identification of a TAL1 target gene reveals a positive role for the LIM domain-binding protein Ldb1 in erythroid gene expression and differentiation.

The TAL1 (or SCL) gene, originally identified from its involvement by a recurrent chromosomal translocation, encodes a basic helix-loop-helix transcription factor essential for erythropoiesis. Although presumed to regulate transcription, its target genes are largely unknown. We show here that a nuclear complex containing TAL1, its DNA-binding partner E47, zinc finger transcription factor GATA-1, LIM domain protein LMO2, and LIM domain-binding protein Ldb1 transactivates the protein 4.2 (P4.2) gene through two E box GATA elements in its proximal promoter. Binding of this complex to DNA was dependent on the integrity of both E box and GATA sites and was demonstrated to occur on the P4.2 promoter in cells. Maximal transcription in transiently transfected cells required both E box GATA elements and expression of all five components of the complex. This complex was shown, in addition, to be capable of linking in solution double-stranded oligonucleotides corresponding to the two P4.2 E box GATA elements. This DNA-linking activity required Ldb1 and increased with dimethyl sulfoxide-induced differentiation of murine erythroleukemia (MEL) cells. In contrast, enforced expression in MEL cells of dimerization-defective mutant Ldb1, as well as wild-type Ldb1, significantly decreased E box GATA DNA-binding activities, P4.2 promoter activity, and accumulation of P4.2 and beta-globin mRNAs. These studies define a physiologic target for a TAL1- and GATA-1-containing ternary complex and reveal a positive role for Ldb1 in erythroid gene expression and differentiation.

From discovery to the coming generation of histone deacetylase inhibitors.

Trichostatin A (TSA) is a Streptomyces metabolite that causes differentiation of murine erythroleukemia cells as well as specific inhibition of the cell cycle of some lower eukaryotes and mammalian cells. The targeted molecule of TSA has been shown by genetic and biochemical analyses to be histone deacetylases (HDACs). Histone acetylation is a key modification to control transcription, and HDACs are profoundly involved in pathogenesis of cancer through removing acetyl groups from histones and other transcriptional regulators. Trapoxin (TPX) and FK228 (also known as FR901228 and depsipeptide because FK228 = FR901228 = depsipeptide), structurally unrelated microbial metabolites, were also shown to inhibit HDACs. These HDAC inhibitors cause cell cycle arrest, differentiation and/or apoptosis of many tumors, suggesting their usefulness for chemotherapy and differentiation therapy. In addition, HDAC inhibitors play important roles in identifying the specific function of the enzymes. Indeed, we identified tubulin as one of the substrates of HDAC6 by means of differential sensitivity to HDAC inhibitors. Since recent studies have revealed that HDACs are structurally and functionally diverse, it should be important to develop inhibitors specific to individual enzymes as more promising agents for cancer therapy. We have synthesized novel TSA/TPX hybrids, which will serve as a basis for developing enzyme-specific HDAC inhibitors.

Opposite effects of inhibitors of mitogen-activated protein kinase pathways on the egr-1 and β-globin expression in erythropoietin-responsive murine erythroleukemia cells

The effect of erythropoietin (Epo) on the expression of mitogen-activated protein kinase (MAPK) target genes egr-1 and c- fos was investigated in Epo-responsive murine erythroblastic cell line ELM-I-1. Epo induced a transient rise in egr-1 mRNA without a similar effect on c- fos expression. The induction of egr-1 correlated with a rapid ERK1/2 phosphorylation and was prevented with MEK1/2 inhibitors PD 98059 and UO126. The p38 inhibitor SB 203580 enhanced ERK1/2 phosphorylation and egr-1 mRNA levels. Longer incubations of ELM-I-1 cells with Epo revealed a second later phase of increase in egr-1 expression which was also prevented by MEK1/2 inhibitors, whereas SB 203580 had a stimulatory effect. In contrast, the β-globin mRNA production was enhanced in the presence of PD 98059 and UO126 and reduced by SB 203580. The results suggest a regulatory role of egr-1 expression in Epo signal transduction and provide pharmacological evidence for the negative modulation of differentiation-specific gene expression by the ERK1/2 pathway in murine erythroleukemia cells.

Wild type p53 gene causes reorganization of cytoskeleton and, therefore, the impaired deformability and difficult migration of murine erythroleukemia cells.

We studied the role of p53 gene in the biophysics and biology in murine erythroleukemia cell line (MEL), with the goal of understanding the influence of this tumor suppressor gene on the deformability and metastasis of tumor cells. Experiments were performed on MEL and p53-transfected MEL (MEL-M with mutant p53 gene and MEL-W with wild-type p53 gene). The cell growth curves indicated that the over-expression of wild-type p53 gene significantly suppressed the growth of MEL, with G(0)-G(1) arrest and apoptosis shown by flow cytometric assays. Confocal laser scanning microscopy revealed that the MEL-W had a more compact organization of the F-Actin cytoskeleton than MEL and MEL-M. Fluorescence polarization measurement indicated a higher membrane fluidity of MEL-W than the other two groups. Fourier transform infrared spectroscopy (FT-IR) showed changes in the composition and/or structure of membrane lipids in MEL-W, with decreases in secondary structures of proteins such as alpha-helix, turns and bends and random coil, in comparison to MEL and MEL-M. The osmotic fragility curves indicated that MEL-W was more fragile and micropipette experiments showed that they had increased elasticity and reduced deformability in comparison to MEL and MEL-M. The adhesion assay with the use of the flow chamber revealed a lower adhesion rate of MEL-W to endothelial cells at high shear stress. The present study on the molecular biology with biophysics of MEL cells contributes to our knowledge on the tumor suppressor gene p53.

CDK6 blocks differentiation: coupling cell proliferation to the block to differentiation in leukemic cells.

Cell proliferation and differentiation are highly coordinated during normal development. Many tumor cells exhibit both uncontrolled proliferation and a block to terminal differentiation. To understand the mechanisms coordinating these two processes, we have investigated the relation between cyclin-dependent kinase (CDK) activities and the block to differentiation in murine erythroleukemia (MEL) cells. We found that CDK6 (but not CDK4) is rapidly downregulated as MEL cells are induced to re-enter erythroid differentiation and that maintenance of CDK6 (but not CDK4) activity by transfection blocks differentiation. Moreover, we found that PU.1, an Ets transcription factor that is oncogenic in erythroid cells and also can block their differentiation, controls the synthesis of CDK6 mRNA. These results suggest a mechanism for coupling proliferation and the block to differentiation in these leukemic cells through the action of an oncogenic transcription factor (PU.1) on a key cell cycle regulator (CDK6). Our findings suggest that studying the relative roles of CDK6 and CDK4 in other types of malignant cells will be important in designing approaches for cell cycle inhibition and differentiation therapy in cancer.

P53-induced inhibition of protein synthesis is independent of apoptosis.

Activation of a temperature-sensitive form of p53 in murine erythroleukaemia cells results in a rapid impairment of protein synthesis that precedes inhibition of cell proliferation and loss of cell viability by several hours. The inhibition of translation is associated with specific cleavages of polypeptide chain initiation factors eIF4GI and eIF4B, a phenomenon previously observed in cells induced to undergo apoptosis in response to other stimuli. Although caspase activity is enhanced in the cells in which p53 is activated, both the effects on translation and the cleavages of the initiation factors are completely resistant to inhibition of caspase activity. Moreover, exposure of the cells to a combination of the caspase inhibitor z-VAD.FMK and the survival factor erythropoietin prevents p53-induced cell death but does not reverse the inhibition of protein synthesis. We conclude that the p53-regulated cleavages of eIF4GI and eIF4B, as well as the overall inhibition of protein synthesis, are caspase-independent events that can be dissociated from the induction of apoptosis per se.

Prevention of PU.1-induced growth inhibition and apoptosis but not differentiation block in murine erythroleukemia cells by overexpression of CBP

PU.1 is a member of Ets family of transcription factors, some of which play critical roles in cell growth and development of hematopoietic cells in cooperation with other transcription factors and cofactors. We previously reported that overexpression of PU.1 in murine erythroleukemia (MEL) cells results in growth inhibition, differentiation block and apoptotic cell death in conjunction with DMSO or HMBA treatment. We also showed that PU.1 interacts with the transcriptional co-activator CREB binding protein (CBP). In this study, we have investigated whether CBP is involved in PU.1-induced growth inhibition, differentiation block and apoptosis in MEL cells. Overexpression of CBP rescued MEL cells from PU.1-induced growth inhibition and apoptosis, while it did not release the cells from PU.1-induced block of erythroid differentiation. These results suggest that sequestration of CBP is involved in PU.1-induced growth inhibition and apoptosis but not differentiation block in MEL cells. These results also suggest that the molecular mechanisms on PU.1-induced growth inhibition and apoptosis are different from the mechanism on PU.1-induced differentiation block in MEL cells.

Oxidative stress is involved in hydroxyurea-induced erythroid differentiation.

Hydroxyurea (HU), an inhibitor of DNA synthesis, can also induce haemoglobinization in certain erythroid cell lines. In this study, we report that intracellular peroxides levels were increased in HU-treated murine erythroleukaemia (MEL) cells and that l-acetyl-N-cysteine (LNAC), a potent reducing reagent, had a significant inhibitory effect on the HU-mediated induction of beta-globin, delta-aminolaevulinate synthase mRNA expression and haemoglobinization of MEL cells. In contrast, the addition of LNAC to dimethyl sulphoxide (DMSO)-treated MEL cells had a much smaller effect on the number of haemoglobinized cells. These findings suggest that oxidative stress is involved in HU-mediated induction of erythroid differentiation and that HU induces MEL cell differentiation by a mechanism different to that involved in DMSO-mediated differentiation. Our findings also suggest that the induction of MEL cell differentiation by HU does not involve RAS-MAP (mitogen-activated protein) kinase signalling.

Bcl-XLis required for heme synthesis during the chemical induction of erythroid differentiation of murine erythroleukemia cells independently of its antiapoptotic function

Bcl-X(L) is essential for the survival and normal maturation of erythroid cells, especially at the late stage of erythroid differentiation. It remains unclear whether Bcl-X(L) serves only as a survival factor for erythroid cells or if it can induce a signal for differentiation. We have previously shown that dimethyl sulfoxide (DMSO) induction of erythroid differentiation in murine erythroleukemia (MEL) cells correlates with delay of apoptosis and specific induction of Bcl-X(L). In this study, we investigate the contribution of Bcl-2 and Bcl-X(L) to survival and erythroid differentiation by generating stable MEL transfectants expressing these antiapoptotic regulators. Overexpression of Bcl-2 completely prevented apoptosis of MEL cells before and after DMSO induction, whereas overexpression of Bcl-X(L) only delayed it. Overexpression of Bcl-2 or Bcl-X(L) neither induced spontaneous erythroid differentiation nor accelerated DMSO-induced differentiation. Inhibition of Bcl-X(L) by antisense transcripts accelerated apoptosis in DMSO-treated MEL cells and blocked the synthesis of hemoglobin without altering the growth arrest associated with terminal erythroid differentiation. An antisense oligonucleotide to Bcl-X(L) did not induce apoptosis in MEL cells overexpressing Bcl-2 but greatly decreased their hemoglobin synthesis when treated with DMSO, suggesting that Bcl-X(L) is necessary for erythroid differentiation independently of its antiapoptotic function. Importantly, Bcl-X(L) antisense transcripts prevented heme synthesis but not globin mRNA induction in DMSO-treated MEL cells. Furthermore, inhibition of hemoglobin synthesis by Bcl-X(L) antisense was reversed by addition of exogenous hemin. Finally, Bcl-X(L) localized to mitochondria during MEL erythroid differentiation, suggesting that it may mediate a critical mitochondrial transport function related to heme biosynthesis.

Synthesis and cytotoxicity of platinum(II) complexes of 3-aminocyclopentanespiro-5-hydantoin and 3-aminocycloheptanespiro-5-hydantoin.

Four new platinum(II) complexes of 3-aminocyclopentanespiro-5-hydantoin (acpsh) and 3-aminocycloheptanespiro-5-hydantoin (achpsh) were synthesized and characterized by elemental analysis, IR and 1NMR spectra. The spectral analyses indicated a cis-square planar structure of the complexes with ligands coordinated via the NH2 group. The complexes were evaluated for in vitro cytotoxicity in murine erythroleukemia (MEL) cells, clone F4N, using cell-growth and macromolecular synthesis assay. The compounds, with exception of [Pt(NH3)(achpsh)Cl2] (IV), exhibited much lower cytotoxicity than that of cisplatin (DDP). Compound IV was nearly as cytotoxic as DDP. The new complexes exerted low antibacterial activity as assessed by seven bacterial strains.

Tumor necrosis factor-alpha expressed constitutively in erythroid cells or induced by erythropoietin has negative and stimulatory roles in normal erythropoiesis and erythroleukemia

Binding of erythropoietin (EPO) to its receptor (EPOR) on erythroid cells induces the activation of numerous signal transduction pathways, including the mitogen-activated protein kinase Jun-N-terminal kinase (JNK). In an effort to understand the regulation of EPO-induced proliferation and JNK activation, we have examined the role of potential autocrine factors in the proliferation of the murine erythroleukemia cell line HCD57. We report here that treatment of these cells with EPO induced the expression and secretion of tumor necrosis factor alpha (TNF-alpha). EPO-dependent proliferation was reduced by the addition of neutralizing antibodies to TNF-alpha, and exogenously added TNF-alpha induced proliferation of HCD57 cells. EPO also could induce TNF-alpha expression in BAF3 and DA3 myeloid cells ectopically expressing EPOR. Addition of TNF-alpha activated JNK in HCD57 cells, and the activity of JNK was partially inhibited by addition of a TNF-alpha neutralizing antibody. Primary human and murine erythroid progenitors expressed TNF-alpha in either an EPO-dependent or constitutive manner. However, TNF-alpha had an inhibitory effect on both immature primary human and murine cells, suggestive that the proliferative effects of TNF-alpha may be limited to erythroleukemic cells. This study suggests a novel role for autocrine TNF-alpha expression in the proliferation of erythroleukemia cells that is distinct from the effect of TNF-alpha in normal erythropoiesis.

Lowering the Barriers to Random Walks on the Cell Surface

We used fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques to compare diffusion of class I major histocompatibility complex molecules (MHC) on normal and α-spectrin-deficient murine erythroleukemia (MEL) cells. Because the cytoskeleton mesh acts as a barrier to lateral mobility of membrane proteins, we expected that diffusion of membrane proteins in α-spectrin-deficient MEL cells would differ greatly from that in normal MEL cells. In the event, diffusion coefficients derived from either FRAP or SPT analysis were similar for α-spectrin-deficient and normal MEL cells, differing by a factor of ∼2, on three different timescales: tens of seconds, 1–10 s, and 100 ms. SPT analysis showed that the diffusion of most class I MHC molecules was confined on both cell types. On the normal MEL cells, the mean diagonal length of the confined area was 330 nm with a mean residency time of 40s. On the α-spectrin-deficient MEL cells, the mean diagonal length was 650 nm with a mean residency time of 45s. Thus there are fewer barriers to lateral diffusion on cytoskeleton mutant MEL cells than on normal MEL cells, but this difference does not strongly affect lateral diffusion on the scales measured here.

Engagement of PI-3-kinase mediated Protein kinase C ζ activation in protecting Friend cells from ionizing radiation-induced apoptosis

Murine erythroleukemia cells (Friend) respond to ionizing radiation with the activation and nuclear translocation of p85alpha subunit of phosphatidylinositol-3-kinase (PI-3-kinase) which mediates the downstream activation and nuclear translocation of atypical Protein kinase C zeta (PKC zeta). This event occurs mainly upon high dose of ionizing radiation (15 Gy) and is concomitant to an increase in BrdU incorporation, which probably accounts for a predominant repair DNA synthesis. Following treatment with wortmannin, a relatively specific inhibitor of PI-3-kinase, both an increased number of apoptotic cells and the inhibition of protein kinase C zeta translocation were detected. Altogether the evidence suggests a potential role of the PI-3-kinase/PKC zeta pathway in protecting Friend cells from ionizing radiation-induced apoptosis offering PKC zeta for consideration as possible target of pharmacological treatments.

Hypoxic up-regulation of erythroid 5-aminolevulinate synthase

AbstractThe erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis. The hypoxia-inducible factor–1 (HIF-1) transcriptionally up-regulates erythropoietin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O2) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an increase in cellular heme content. A fragment of the ALAS2 promoter ranging from −716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct in transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1–binding site (−323/−318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is independent of this putative HIF-1 DNA-binding site.

The developmental program of murine erythroleukemia cells.

Hematopoietic stem cells (HSCs) or early progenitors respond to external stimuli in bone marrow and differentiate into cell-restricted lineages of blood cells of limited life span. In leukemias, however, early hematopoietic progenitors self-renew themselves, fail to respond to differentiation signals, and do not undergo programmed cell death (apoptosis). The basic mechanisms of differentiation and apoptosis of leukemia cells have been the long-term objective of our work. By exploiting widely studied murine and human leukemic cell systems as models of hematopoietic cell differentiation, we explored the mechanisms by which pharmaceutical agents initiate differentiation in leukemic systems. In this article, we present the developmental program of MEL cells with emphasis given on the role of commitment to terminal maturation. Commitment is initiated via inducer-receptor-mediated processes and leads to discrete patterns of expression of several genes that contribute to growth arrest at the G1 phase, expression of differentiated phenotype, and differentiation-dependent apoptosis (DDA). Overall, MEL erythroid cell differentiation represents a developmental program with a highly coordinated set of processes that is "triggered" by an inducer and functions via a network of genes and proteins interacting with each other harmonically to give birth to lineage-restricted phenotype.

Specific Destruction of Hybridoma Cells by Antigen-toxin Conjugates Demonstrate an Efficient Strategy for Targeted Drug Therapy in Leukemias of the B Cell Lineage

Many types of leukemia including multiple myeloma remain essentially incurable despite recent developments in immuno- and chemotherapy. The effectiveness of these therapies might be greatly enhanced by targeting cell surface proteins unique to the malignant clone, which for leukemias of the B cell lineage means clonotypic surface immunoglobulin (sIg). As this immunoglobulin (Ig) is necessarily epitope specific, we are developing ligand-toxin conjugates (LTCs) as a strategy for delivering toxins and other drugs to clonotypic tumor cells. Here we report in vitro studies that illustrate the effectiveness of this approach. LTC comprising the DNP hapten conjugated to ricin A toxin (DNP-RTA) were shown to specifically and effectively kill anti-DNP secreting murine hybridoma (U7.6) cells but not other hybridoma cells (1B12), a murine erythroleukemia cell line (Friend's Leukemia or) normal mouse spleen cells. In addition to direct toxicity, LTC treatment negatively affected the growth characteristics of the few surviving cells as reflected in decreased growth index and an increase in growth inhibition over 72 h post treatment. Interestingly, U7.6 cells that survived one or two LD 90 dose(s) of LTC showed no alteration in their dose response to a subsequent attack of LTC indicating that this treatment strategy may not induce drug resistance. These data suggest that LTC therapy may be a new and effective strategy for specific destruction of tumor cells such as myeloma plasma cells and could be extended to other tumors where clonotypic receptors can be identified.

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.

Cloning and characterization of the SIL promoter

The SIL gene undergoes a site-specific rearrangement with the SCL gene in 25% of patients with T cell acute lymphoblastic leukemia (ALL). The functional result of this rearrangement is that the SIL regulatory elements aberrantly drive expression of the SCL gene . We have cloned and sequenced the human SIL promoter, cloned a murine homolog, found the sequence to be highly conserved, and defined a minimal promoter region. Both the cloned murine and human sequences were found to be highly active in either human or murine cells. SCL mRNA, driven by a cloned SIL promoter, could be downregulated by DMSO in stably transfected F4-6 murine erythroleukemia cells. The SIL promoter was found to be partially unmethylated in proliferating tissues, in which it is highly expressed, and more highly methylated in post-mitotic tissues, in which SIL is not expressed. The isolation of the SIL promoter provides an important tool for the study of both the SIL gene expression as well as the role of the SIL promoter in leukemogenesis.