Induction Of Hemoglobin Synthesis Research Articles

GPS2 promotes erythroid differentiation in K562 erythroleukemia cells primarily via NCOR1.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.

Transcriptional Induction of Transferrin Receptors By Heme in Erythroid Cells

The transferrin receptor (TfR) is a membrane glycoprotein whose only clearly defined function is to mediate cellular uptake of iron (Fe) from a plasma glycoprotein, transferrin. Iron uptake from diferric transferrin (Tf) involves the binding of transferrin to the TfR followed by internalization of Tf within an endocytic vesicle by receptor-mediated endocytosis. Iron is then released from transferrin within endosomes by a combination of Fe3+ reduction by Steap3 (likely when transferrin is still bound to TfR) and a decrease in pH (~pH 5.5). Following this, Fe2+ is transported across the endosomal membrane by DMT1. Transferrin receptors are highly expressed on immature erythroid cells, placental tissue, and rapidly dividing cells, both normal and malignant. In proliferating nonerythroid cells the expression of TfR is negatively regulated post-transcriptionally by intracellular iron through iron responsive elements (IREs) in the 3' untranslated region (UTR) of transferrin receptor mRNA. IREs are recognized by specific cytoplasmic proteins (iron regulatory proteins; IRPs) that, in the absence of iron in the labile pool, bind to the IREs of transferrin receptor mRNA, preventing its degradation. On the other hand, the expansion of the labile iron pool leads to a rapid degradation of transferrin receptor mRNA that is not protected, since IRPs are not bound to it. However, some cells and tissues with specific requirements for iron probably evolved mechanisms that can override the IRE/IRP-dependent control of transferrin receptor expression. We previously documented that the TfR gene promoter contains an erythroid active element that stimulates the receptor gene transcription upon induction of hemoglobin synthesis (1).In this study we have demonstrated that incubation of erythroid cells with 5-aminolevulinic acid (ALA) increased TfR expression as well as iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is primarily a result of increased mRNA synthesis, since the effect of ALA can be abolished by actinomycin D. Moreover, we found that the TfR promoter was activated in vitro by the addition of ALA or hemin to murine erythroleukemia (MEL) cells induced to differentiate using DMSO. Furtehermore, site-directed mutation of erythroid active element (1) in the TfR promoter abolished the effects of ALA or hemin. These results indicate that heme may directly or indirectly interact with the TfR promoter, consequently enhancing the gene expression. Hence, our results show that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis. In conclusion, erythroid cells, which are the most avid consumers of iron in the organism, use a transcriptional mechanism to maintain very high transferrin receptor levels.1 Chun-Nam Lok Ponka P. (2000) Identification of an Erythroid Active Element in the Transferrin Receptor Gene. J. Biol. Chem. 275: 24185-24190. DisclosuresNo relevant conflicts of interest to declare.

Characterization of a Transcript Found within the HBS1L-MYB Intergenic Region

Fetal hemoglobin (HbF, α2γ2) is the main hemoglobin in the fetus, but its levels decline after birth to <1% in adults. However, there are individuals who continue to have high levels of HbF well into adulthood. High HbF in sickle cell anemia is often associated with milder disease hence the effort to understand more about its regulation for possible therapeutic gain. Some of the variation in HbF among patients with sickle cell anemia can be accounted for by differences in three major HbF quantitative trait loci (QTL) that were found in genome-wide association studies to be associated with high HbF. However, depending on the population studied, these QTLs only account for up to 10-50% of HbF variance. Therefore, determining other factors for high HbF levels is critical in understanding the regulation of HbF expression and finding other possible therapeutic targets to induce HbF production in patients with hemoglobinopathies. Previously, we found that HbF variability is associated with a 3-bp deletion within the HBS1L-MYB intergenic region (Farrell et al, Blood 2011) and this work has been recently confirmed (Stadhouders et al, JCI, 2014). This deletion was found to be in strong linkage disequilibrium (LD) with the QTL HMIP, the HBS1L-MYB intergenic region on chr6q23. Functional analyses of the region around the 3-bp deletion using ChIP assay in K562 cells, which are heterozygous for the 3-bp deletion, found that four transcription factors that are involved in erythroid cell differentiation—GATA-2, TAL1, E47 and RUNX1—all bind in the region of the 3-bp deletion. ENCODE datasets show that RNA polymerase II and a 50 bp RNA transcript are found in this same area. Based on these observations, we set out to determine the extent of the 50-bp transcript found by ENCODE. Using cDNA from K562 cells and the 50-bp transcript as a guide, we were able to amplify by PCR a fragment greater than 1 kb, which includes the 3-bp deletion and the binding sites for the erythroid-specific transcription factors mentioned above. Sequencing of this PCR product confirmed that the transcript is transcribed from the HBS1L-MYB intergenic region. Next, 5′- and 3′-rapid amplification of cDNA ends (RACE) reactions were done to determine the full length of this transcript. Sequencing results of the RACE products show that the 3′-end of the transcript is more than 100 bp downstream from what was originally found by PCR. Results for the 5′-end are pending. By knowing the sequence of this transcript, we were able to make multiple transcript-specific primers and use them in qPCR to determine if expression of this transcript changes with induction of hemoglobin synthesis in K562 cells. Preliminary data show after the induction of HBG2 expression with butyrate, various areas of this novel transcript increased as well. Because globin synthesis in K562 cells is fixed at the fetal stage of development we also looked for expression of this transcript in iPSC-derived erythroid progenitor cells from a disease-free individual and an individual with sickle cell anemia on day 25 of differentiation, and in CD34+ cord blood-derived erythroid cells on days 7 and 14 of differentiation. qPCR showed that in these primary erythroid cells the transcript is expressed at similar levels as in K562 cells. This novel transcript within the HBS1L-MYB intergenic region seems to increase with hemoglobin induction in K562 cells, and is also found in other primary erythroid cells derived from iPSCs and CD34+ cells. This transcript is a candidate for being a long noncoding RNA (lncRNA). LncRNAs are transcripts of 200 nucleotides or greater that can be transcribed from any region of the genome. They have been shown to be involved in gene regulation and various biological processes including hematopoiesis. HMIP has a primary effect on hematopoiesis. As this fragment is transcribed from a region known to be associated with HbF variability, it will be of interest to determine its role in HbF expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ALA Induced Heme Synthesis: Fine Tuning Mechanisms of PBG Deaminase and ALA Dehydratase

During erythroid differentiation and maturation, three critical components α-globin, β-globin, and heme are critical for the formation of stable α2β2 hemoglobin (Hb) complexes. Heme synthesis enhances globin formation, not only because it is the precursor of Hb but also by modulating the machinery of gene expression and globin synthesis and coordinating erythroid cell maturation. Exogenous 5-aminolevulinic acid (ALA), the first precursor in the heme biosynthesis pathway, circumvents the rate limiting enzyme ALA synthase, and accelerates the heme synthesis pathway. ALA induced erythroid differentiation of human myelogenous leukemia K562 and murine erythroleukemia MEL cells, leads to hemoglobinization and cellular typical maturation. Only two key enzymes where known as regulator of the accumulation of PpIx: Porphobilinogen deaminase and Ferrochelatase. Recently we demonstrated that downregulation or over expression of ALA dehydratase (ALAD) and porphobilinogen deaminase (PBGD), the second and the third enzymes of the pathway, by specific shRNAs caused a marked alteration in PpIX synthesis in K562 erythroleukemic cells. PBGD down regulation induced an elevation in ALAD activity, while over expression of PBGD reduced ALAD activity, indicating a novel regulation feedback of PBGD on ALAD activity. This feedback mechanism enabled partial PpIX synthesis under PBGD silencing, whereas ALAD silencing reduced PpIX production to a minimum, only ALAD loss resulted in reduced PpIX and Hb synthesis. Hemoglobin synthesis and assembly is largely dependent on coordinated gene expression control, it is well established that the histone deacetylase inhibitor butyric acid activates transcription of heme synthesis an globin mRNA expression, leading to changes in cell morphology and induction of erythroid differentiation of erythroleukemic cell lines. We have demonstrated that the activity of the multitargeting ALA prodrug, AlaAcBu in the induction of protoporphyrin IX (PpIX) and anti-cancer activity following light irradiation. The prodrug AlaAcBu undergos enzymatic intracellular hydrolysis releasing ALA, butyric acid and acetaldehyde and these active components induce accelerated heme and hemoglobin synthesis. Erythroid differentiation was characterized by cellular maturation characterized by cytoplasm hemoglobinization, expression of the marker glycophorin A and polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis is presented in this report.

Depletion of glutamine enhances sodium butyrate-induced erythroid differentiation of K562 cells

Human erytholeukemia K562 cells are induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, sodium butyrate and 1-β-d-arabinofuranosylcytosine. We have investigated the induction of erythroid differentiation of K562 cells by glutamine depletion. When K562 cells were cultured in glutamine-minus medium, the induction of hemoglobin synthesis, accompanied by those of heme-biosynthetic enzymes and erythroid transcriptional factors, was observed. This induction was dependent on the temporally marked decrease of intracellular level of glutathione, followed by the marked activation of p38MAPK and SAPK/JNK, but not ERK. Under glutamine-deficient conditions, the treatment of K562 cells with sodium butyrate resulted in the marked enhancement of the induction of heme biosynthesis. Glutamine depletion also accelerated the expressions of erythroid-related factors including α-globin and heme-biosynthetic enzymes, GATA-1 and NF-E2, in sodium butyrate-induced K562 cells. The transcriptional activity of β-globin gene promoter-reporter was markedly enhanced by these treatments, indicating that glutamine deficiency in combination with sodium butyrate treatment gives high efficiency of chemical-induced differentiation in the hematopoiesis process.

A multifunctional 5-aminolevulinic acid derivative induces erythroid differentiation of K562 human erythroleukemic cells

Anemia is a major clinical symptom of a wide variety of pathological conditions a common related to reduced erythropoiesis. Whereas erythropoietin treatment showed an improvement in the patients’ condition, it revealed increased risks of thromboembolic and cardiovascular events. Herein we describe stimulation of erythropoiesis by the multifunctional 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu), a 5-aminolevulinic-acid (ALA) derivative, which undergoes metabolic hydrolysis yielding two erythroid differentiation inducers, ALA and butyric acid (BA), each acting through a different mechanism. ALA, the first precursor in the heme biosynthesis, accelerates heme synthesis and BA, a histone deacetylase inhibitor (HDACI) that activates the transcription of globin mRNA.Our results show that the AlaAcBu mutual prodrug is a potent chemical differentiation inducer of K562 human erythroleukemia cells manifested by augmentation of heme and globin synthesis and assembly of hemoglobin. Exposure of K-562 cells to AlaAcBu resulted in an increase in heme synthesis and globin expression. Stimulation of the heme pathway was evident by the over-expression of porphobilinogen deaminase (PBGD) and ferrochelatase. AlaAcBu promoted cellular erythroid differentiation depicted by the expression of the marker glycophorin A and cellular maturation characterized by cytoplasm hemoglobinization, polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis presented in this report.

Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

Pulmonary Stromal Cells Expressing CC-Chemokine Receptor 5 Promote Metastasis Via Erythroid Differentiation Regulator 1.

Abstract 3601Poster Board III-538 BACKGROUNDThe protein erythroid differentiation regulator 1 (ERDR-1) is a stromal-derived factor that was previously found to induce hemoglobin synthesis in erythroleukmia cells and proliferation in hematopoietic progenitors (Dormer et al. Cytokine. 2004). However, previous work had not suggested a role for ERDR-1 in either local or distant tumor growth. We have previously shown that stromal cells, and in particular fibrocytes, which are CD45+ monocyte-derived stromal cell precursors, promote metastasis and this effect is partly dependent on expression of CC-chemokine receptor 5 (CCR5). Here, we sought to determine a mechanism for this finding. METHODSWe used Affymetrix Mouse Arrrays to identify genes that were differentially expressed in lung homogenates from wild type (WT) and CCR5 −/− mice 6, 24 and 48 hours after the intravenous (i.v.) injection of B16-F10 tumor cells. After identification of ERDR-1 as a candidate gene, we evaluated WT and CCR5 −/− primary stromal cells for expression of ERDR-1. We also meausred ERDR-1 expression in mouse embryonic fibroblasts (MEFs), and knocked-down the gene in these cells using 2 different shRNA sequences specific for ERDR-1. A third line of MEFs expressing a scrambled (“mock”) shRNA sequence was generated as a control for non-specific effects. To model the effect of stromal ERDR-1 on metastasis, we transferred ERDR-1 knockdown or mock knockdown MEFs into CCR5 −/− or WT mice 48 hours prior to injection of B16-F10 cells. Lung nodules were counted 14 days after tumor injection. We also evaluated the effect of ERDR-1 knockdown on in vitro proliferation and survival. RESULTSERDR-1 was overexpressed in the lungs of wild type (WT) compared to CCR5 −/− mice by 3.7, 2.7, and 3.5 fold at 6, 24, and 48 hours after tumor injection, respectively. Semi quantitative end-point PCR confirmed this observation in all mice tested (n = 30). Real Time RT-PCR revealed that the in vitro expression of ERDR-1 was higher in primary stromal cells from WT compared to CCR5 −/− mice by 5.2 fold (3.3 to 11.2, p < 0.001, results pooled from 3 separate primary cultures). This difference was most pronounced in the CD45+ (fibrocyte) population. There was a substantial increase in the expression of ERDR-1 mRNA and protein in MEFs compared to primary WT stromal cells (15.86 +/− 6.9 fold greater expression by Real Time RT-PCR, p < 0.001) and this correlated with a greater number of metastases in the lungs of mice receiving MEFs compared to WT primary stromal cells prior to B16-F10 injection. In CCR5 −/− mice receiving ERDR-1 knockdown MEF lines prior to tumor injection we found a 37% and 50% decrease in the number of metastases compared to mice receiving the mock knockdown MEFs ( p = 0.06 and p < 0.05, respectively). When MEF lines transduced with 43.1% lower efficiency were used, mice receiving the knockdown lines had a 24% and 26% reduction in the number of metastases compared to mice receiving the mock knockdown MEFs (p < 0.05 for both groups). Conversely, when ERDR-1 knockdown lines were transferred to WT mice prior to tumor injection, there was no significant reduction in metastases compared to the group receiving mock knockdown MEFs. This was similar to our previous finding that numbers of metastases were similar for WT mice receiving either CCR5 −/− or WT stromal cells prior to tumor cells. These results indicated that endogenous ERDR-1 expression in WT but not CCR5 −/− mice was sufficient to compensate for lower ERDR-1 expression in transferred stromal cells. Interestingly, we also found that knockdown of ERDR-1 in MEFs led to enhanced apoptosis in vitro (1.7 and 2.8 –fold increase in Annexin-V+ cells) compared to mock knockdown MEFs, suggesting that ERDR-1 acts as a stromal cell survival factor. CONCLUSIONSThe relative inability of primary stromal cells from CCR5 −/− mice to promote metastatic tumor growth in the lung was found to correlate with decreased expression of ERDR-1. Knocking down ERDR-1 substantially decreased the number of lung metastasis induced by WT MEFs. Thus, the production of ERDR-1 by stromal cells appears to be important for the CCR5-dependent promotion of metastatic tumor growth. Disclosures:No relevant conflicts of interest to declare.

Relocation of Cell Iron. A Mode of Chelation with Application to Diseases of Regional Iron Accumulation.

Iron raises to toxic levels in mitochondria of excitable cells in some forms of neuro-degeneration with brain accumulation (NBIA), in sideroblastic anemia and in Friedreich's ataxia (FA), often leaving the cytosol iron-depleted. In anemia of chronic disease (ACD) iron is withheld by macrophages, while iron levels in extracellular fluids (e.g. plasma) are drastically reduced. Although excessive iron deposition occurring in organs of iron overloaded (IO) patients can be reduced with iron chelators, it is uncertain whether this is applicable to conditions where iron accumulates within selected tissues/cells in the absence of systemic IO.Objective. We assessed whether deferiprone (DFP), a membrane-permeant bidentate chelator in clinical use for treating systemic IO, might serve as an iron relocating agent in settings of regional iron accumulation by a. capturing labile iron accumulated in cell compartments and b. conveying the chelated iron either to other cell locations for metabolic integration or to transferrin for systemic reutilization.Methods. DFP capacity to shuttle iron intracellularly and transcellularly was assessed in macrophages (J774) and heart (H9c2) cell models using organelle-targeted fluorescent iron-sensors in conjunction with fluorescence microscopy imaging. We employed pairs of sensors targeted to different cell compartments and iron-evoked quenching and chelator-evoked dequenching as means to trace DFP mediated iron transfer. Mitochondrial iron accumulation was generated with succinylacetone or by silencing genes affecting mitochondrial iron metabolism.Results. DFP facilitated iron transfer: a. from iron-laden cell organelles to other cell compartments or to medium (and vice versa) and b. from iron loaded macrophages to pre-erythroid MEL cells (for chemically induced hemoglobin synthesis) either directly or via transfer to extracellular transferrin.Discussion The results of this study indicate that relocation of cell accumulated iron can be used as a modality of chelation for treating conditions of regional iron accumulation by applying chelators able to permeate into cell compartments but also to transfer the chelated iron to cell acceptors or to the extracellular iron carrier transferrin.

Erythroid differentiation regulator (EDR), a novel, highly conserved factor: I. Induction of haemoglobin synthesis in erythroleukaemic cells

In serum-free WEHI-3 supernatants an activity was detected inducing haemoglobin synthesis in human and murine erythroleukaemia cell lines. The absolute numbers of benzidine-positive cells induced with either DMSO or WEHI-3-conditioned medium were comparable. Terminal differentiation was not observed. An expression library from WEHI-3 RNA aided by PCR cloning revealed an open reading frame corresponding to a 209 amino acid protein. This was 100% identical to a sequence from human stimulated peripheral blood mononuclear cells. In contrast to human RNA, mouse RNA exhibited multiple bands of pre-mRNA in Northern blots. The gene was provisionally termed erythroid differentiation regulator ( edr). In mammalian cells EDR is mostly expressed as a 56 kDa dimer showing higher activity than the recombinant monomer. The activity profile is bell-shaped. Expression was observed in many normal mouse tissues, yet in haematopoiesis it was largely confined to CD34 + cells. It was enhanced by a series of stimuli such as phorbol ester, and transformed cells generally showed a higher level of EDR expression than normal ones. The protein is localized at the inner side of the cytoplasmic membrane and is released in part via vesicles. In view of the broad range of EDR-expressing tissues the function obviously exceeds haemoglobin synthesis induction. Involvement in cell survival and growth control has been observed and will be dealt with in detail elsewhere.

Different roles of p38 MAPK and ERK in STI571‐induced multi‐lineage differentiation of K562 cells

STI571 is a specific tyrosine kinase inhibitor of Abl kinase. It was previously reported that STI571 induced hemoglobin synthesis in the chronic myelogenous leukemia (CML) cell line K562. However, its mechanisms remain unknown. In this study, we demonstrated that STI571 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and dephosphorylation of extracellular signal-regulated kinase (ERK) in K562 cells. In contrast, the phosphorylation of c-Jun N-terminal kinases (JNK) in K562 cells was not altered by STI571. We also found that STI571 induced all the myeloid (CD11b, CD13), megakaryocytic (CD41a, CD42), and erythroid (glycophorin-A) markers on K562 cells. A p38 MAPK-specific inhibitor, SB203580, inhibited the STI571-induced multi-lineage differentiation of K562 cells, indicating that p38 MAPK is crucial for this differentiation. In contrast, SB203580 did not overcome the inhibitory effect for proliferation of K562 cells, indicating that p38 MAPK activation by STI571 does not affect cell numbers. Among the hematopoietic transcription factors, the expression level of c-myb mRNA was clearly downregulated after incubation with STI571 in K562 cells. STI571-induced downregulation of c-myb mRNA was prevented by the pretreatment of K562 cells by SB203580. Our data provides insights into how p38 MAPK and ERK pathways are involved in STI571-induced differentiation of K562 cells.

Identification of an Erythroid Active Element in the Transferrin Receptor Gene

Hemoglobin synthesis consumes most of the iron that is taken up by cells from plasma transferrin, and this process requires very high expression of transferrin receptors (TfR) at the membranes of erythroid cells. Studies in our and other laboratories indicate that a dramatic increase in TfR levels during erythroid differentiation occurs at the transcriptional level. In this study, we investigated the transcriptional regulation of the TfR in terms of its promoter activity and DNA-protein binding in murine erythroleukemia cells. Reporter gene assays revealed that the TfR promoter activity was stimulated 6-8-fold in murine erythroleukemia cells induced to differentiate into hemoglobin-synthesizing cells by either Me(2)SO or N,N'-hexamethylene-bis-acetamide. A minimal region (-118 to +14) was required for the differentiation-induced promoter activity. Mutation of either an Ets-binding site or an activator protein-1/cyclic AMP-response element-like motif within this region, but not disruption of the adjacent GC-rich/specificity protein-1 sequence, inhibited the inducible promoter activity. Electrophoresis mobility shift assays suggest that the cyclic AMP-response element-binding proteins/activating transcription factor-like factors and Ets-like factors bind constitutively to this bipartite element. Upon induction of differentiation, a shift in the pattern of the cyclic AMP-response element-binding protein/activating transcription factor-like binding factors was observed. Our data indicate that the TfR gene promoter contains an erythroid active element that stimulates the receptor gene transcription upon induction of hemoglobin synthesis.

Effects of thymidine and phorbol 12-myristate 13-acetate on erythroid differentiation of K562 cells and their sensitivity to nonspecific lysis by rat splenocytes

Induction of hemoglobin synthesis in K562 cells by thymidine (2 mM)in vitro did not significantly enhance major histocompatibility complex antigen-unrestricted lysis of splenocyte-exposed K562 cells. Inhibition of thymidine-induced hemoglobin synthesis by simultaneous incubation of cells with thymidine and phorbol 12-myristate 13-acetate (100 nM) decreased cytolytic activity of splenocytes against K562 cells. Preincubation of tumor cells with phorbol ester alone did not affect major histocompatibility complex antigen-unrestricted lysis induced by rat splenocytes but decreased the basal level of hemoglobin synthesis.

Human acute myeloblastic leukemia-ascites model using the human GM-CSF- and IL-3-releasing transgenic SCID mice.

To generate an appropriate model for human acute myeloblastic leukemia (AML), we have successfully established a human hematopoietic growth factor-dependent AML cell line (TF-1 and UT-7/GM)-ascites model using human granulocyte-macrophage colony-stimulating factor (hGM-CSF)- and human interleukin 3 (hIL-3)-releasing transgenic (Tg)-SCID mice. When 1 x 10(7) cells of TF-1, a human erythroleukemia cell line, were transplanted into the peritoneum of irradiated Tg-SCID mice (TF-1 ip/Tg-SCID mice), TF-1 cells grew in both the single cell suspension form (asTF-1) and solid form in ascites and invaded various tissues: lungs, liver, pancreas, and genitals, 3-6 weeks following transplantation. Subsequently, 0.5-1 x 10(7) cells of UT-7/GM, a subline of the UT-7 human megakaryoblastic leukemia cell line, grown in the back of hGM-CSF Tg-SCID mice after subcutaneous inoculation, were transplanted into the peritoneum of other irradiated hGM-CSF Tg-SCID mice. After 4 weeks, UT-7/GM cells (asUT-7/GM) also grew in the same manner as TF-1 cells in hGM-CSF Tg-SCID mice. Analysis of the cells from the peritoneum and tissues by PCR amplifying ALU and human GM-CSF receptor beta sequences and by immunohistochemical staining using anti-human CD45 revealed that they possessed the original characteristics of the parental cells. To confirm the usefulness of this human AML-ascites model, experimental treatment of AML cells grown in these mice was carried out with a differentiation inducer, delta-aminolevulinic acid (deltaALA), which induces hemoglobin synthesis for TF-1 in vitro and is thus regarded as an anti-leukemia drug candidate. Unexpectedly, growth promotion of TF-1 cells was observed in the treated TF-1 ip/hIL-3 Tg-SCID mice without differentiation to erythroid cells after treatment with delta-ALA (5 mM) for 7 days. These results indicate that Tg-SCID mice can support the growth of human hematopoietic growth factor-dependent AML cell lines which are usually rejected by SCID mice, without modification of the parental cell characteristics. In addition, this Tg-SCID leukemia-ascites model may become a useful preclinical tool for estimation of drug efficacy in vivo, since the drug candidate which was promising in vitro did not act in the same manner in vivo.

Signalling mechanisms and the role of calcineurin in erythropoiesis

Erythropoietin (Epo) is the principal regulator of the production of circulating erythrocytes by controlling the proliferation, the differentiation and the survival of the erythroid progenitor cells. Early down-regulation of c-myb expression in erythroleukemia cells is a common feature of the action of Epo and chemical inducers of differentiation such as DMSO. Previously we have shown that in our Epo-responsive murine erythroleukemia cell line ELM-I-1, [Ca 2+] i increasing agents can mimic the effect of Epo on c-myb expression and activate nuclear signal transduction processes involved in the induction of hemoglobin synthesis. These results also indicated that the Ca 2+-induced down-regulation of c-myb expression and hemoglobin synthesis are mediated by the Ca 2+/calmodulin dependent serine/threonine-specific protein phosphatase PP2B, calcineurin, but the Epo induced processes are not mediated by PP2B. In spite of this, we demonstrated in this paper that in ELM-I-1 cells the Epo-induced down-regulation of c-myb expression and hemoglobin production can be effectively enhanced by the simultaneously added [Ca 2+] i -increasing agent, cyclopiazonic acid (CPA). This observation further supports the existence of at least two independent signalling pathways in the mechanism of Epo and [Ca 2+] i increasing agents and the strong correlation between c-myb expression and hemoglobin production in differentiating cells. Although the c-AMP-response element binding protein (CREB) could be the common target of both calcium-dependent and -independent dephosphorylation, our results do not support the involvement of CREB in the regulation of c-myb gene expression. In addition to the calcineurin mediated down-regulation of c-myb expression, we have found a negative regulatory effect in the Ca 2+-mediated transcriptional activation of certain genes. In response to [Ca 2+] i -increasing agents in ELM-I-1 cells, both, egr-1 and c-fos mRNA expression increased significantly after the inhibition of calcineurin by cyclosporine A. Cyclosporin A exerted stimulatory effects on the egr-1 and c-fos expression also at lower (150–400 nM) intracellular Ca 2+ levels. This potential co-regulation of c-myb, egr-1 and c-fos expression by calcineurin suggests that the negative modulation of egr-1 and c-fos expression may also be important for the induction of erythroid differentiation by [Ca 2+] i -increasing agents. This negative modulation may also contribute to the Epo-induced differentiation in the case of a moderate increase of [Ca 2+] i .

Induction of differentiation in erythroleukemic K562 cells by γ-irradiation

Various agents have been shown to induce differentiation in neoplastic cells. The present study aimed at investigating comparable phenomena induced by high doses of γ-irradiation in the presence of physiological factors. The erythroleukemic K562 cells were γ-irradiated or treated with cytosine-arabinoside (Ara-C), and examined for cell size, protein content, acetylcholinesterase (AChE)-activity and hemoglobin synthesis in relation to mitotic activity. At doses above 10 Gy, differentiation was induced, as recognized by elevated AChE-activity, accompanied by an increase in cell size and protein content and cessation of cell proliferation. Moreover, irradiation, as well as Ara-C, induced hemoglobin synthesis when cultures were supplemented with hemin prior to treatment. It is suggested that the basic mechanisms of differentiation induction are similar for ionizing radiation and certain chemical agents and are related to continued growth of essential cytoplasmic constituents during inhibition of mitotic activity.

Augmentation of γ-Globin Gene Promoter Activity by Carboxylic Acids and Components of the Human β-Globin Locus Control Region

AbstractButyric acid increases fetal hemoglobin synthesis in adult animals and in erythroid cells in culture and induces the γ-globin gene promoter in transient expression experiments in K562 cells (McDonagh KT, Nienhuis AW, Blood 78:255a, 1992 [abstr, suppl 1]). We compared the effect of butyrate and other short-chain carboxylic acids in transient expression studies with K562 cells using an expression plasmid bearing a luciferase reporter gene driven by the normal human Aγ-globin gene promoter. Butyrate (4 carbons) increased the activity of the human Aγ-globin gene promoter up to 123 times. Marked augmentation of the normal γ-promoter activity was also noted with 5-carbon valeric acid (up to 394 times) and 3-carbon propionic acid (up to 129 times). The branched isobutyric acid as well as phenylacetate showed less ability to increase promoter activity. Addition of the tandemly repeated AP-1/NF-E2 (AP) enhancer sequences from hypersensitive site 2 (HS2) of the locus control region (LCR) increased γ-promoter activity up to 24 times. Addition of a nearby 16-bp conserved motif (CM) in HS2 (Safaya S, Rieder RF, Blood 78:146a, 1992 [abstr, suppl 1]) to the AP-containing plasmid construct further increased γ-promoter activity. In the presence of butyrate, the plasmid bearing both the AP and CM sequences showed gene expression up to 477 times greater than that of the basal γ-promoter-driven luciferase plasmid in the absence of inducer. A plasmid bearing the herpes simplex thymidine kinase promoter was also tested and gene expression was markedly increased by the same organic acids. MEL cells responded to butyrate, valerate, and propionate with induction of hemoglobin synthesis. Responses to isobutyrate and 6-carbon caproate required higher concentrations of the compounds. Thus, other short-chain organic acids as well as butyrate increase γ-promoter activity in the transient expression system, and this activity can be further augmented by incorporating LCR elements into the expression vector. Nonglobin promoters also respond to the same carboxylic acids.

Effects of thyroid hormone on carbonic anhydrase I levels in human erythroid (YN-1) cells.

We recently have reported that patients with hyperthyroidism have red blood cell carbonic anhydrase I (CAI) levels that reflect the patient's integrated thyroid hormone level over the preceding few months. In the present study, the effect of T3 on CAI concentrations in the human erythroleukemic cell line, YN-1, was studied. The presence of high affinity (1.9 x 10(9) L/mol), low capacity (20.4 fmol/10(6) cells) T3 binding sites was demonstrated in YN-1 cell nuclei. T3 at a physiological free T3 concentration (0.54 nmol/L) significantly (32 +/- 7%, P < 0.01) decreased CAI concentrations in YN-1 cells before induction of differentiation. The effect of T3 on CAI levels was more obvious (42 +/- 7%, P < 0.01) in the presence of transforming growth factor-beta 1 (TGF-beta 1), which induces hemoglobin synthesis and differentiation in YN-1 cells. These results suggest that the YN-1 cell line may be used to study T3 action on human red blood cell CAI.

Induction of somatostatin immunoreactivity in cultured ciliary ganglion neurons by activin in choroid cell-conditioned medium

We have previously shown that the expression of somatostatin-like immunoreactivity in cultured ciliary ganglion neurons is stimulated by a macromolecule found in choroid cell-conditioned medium (ChCM). Here, we present the following evidence that this somatostatin-stimulating activity (SSA) is activin: human recombinant activin induces somatostatin-like immunoreactivity in CG neurons; ChCM induces hemoglobin synthesis in K562 cells, a biological activity characteristic of activin; activin A-specific antibodies recognize a protein in ChCM; cultured choroid cells contain activin RNA; and SSA is inhibited by follistatin, a specific activin-binding protein. Thus, activin is likely to be a neurodifferentiation factor for CG neurons in vivo.

Induction of hemoglobin synthesis by downregulation of MYB protein with an antisense oligodeoxynucleotide

Reduced expression of the proto-oncogene c-myb appears necessary for erythroid differentiation induced by chemical agents and by the natural regulator, erythropoietin (Epo). Treatment of Epo-responsive Rauscher erythroleukemia cells with an anti-sense oligodeoxynucleotide to c-myb in the absence of other inducers downregulated myb protein markedly and caused hemoglobinization of the cells within 48 hours. Epo treatment, which downregulates c-myb in these cells, induced hemoglobinization to the same degree. Epo also induced the appearance of anion transport protein on the plasma membrane, consistent with terminal differentiation. In contrast, antisense c-myb did not induce this erythroid marker. The results are consistent with a role for myb protein in the regulation of hemoglobin synthesis.