Globin Transcripts Research Articles

Quantitative analysis of globin gene induction in single human erythroleukemic cells.

The mechanisms involved in the normal developmental regulation of globin gene expression, and the response to pharmacological agents that elevate fetal hemoglobin, may be expected to involve either changes in each cell or a selection process affecting subsets of differentiating erythroid cells. To study these mechanisms we have developed assays to measure mRNA levels in single erythroid cells. The assay involved the use of globin-specific probes, with no detectable cross-reactivity, in real-time, fluorescence-based quantitative PCR (Q-PCR). We had previously used this Q-PCR method to measure globin mRNA levels in cultures of primary erythroid cells demonstrating that drugs like hydroxyurea, 5-azacytidine and butyric acid each yielded increases in gamma/( gamma + ss) mRNA ratios, with differential effects on ss-globin levels. We have now extended this approach to measure globin mRNA levels in single K562 cells, a human erythroleukemic cell line, with and without 30 microM hemin treatment. Hemin exposure increases total hemoglobin levels by approximately 9-fold and total alpha-, epsilon- and gamma-globin mRNA levels by 1.5-2.3-fold. Single cell analyses showed initial wide distributions of each of the three individual globin mRNA levels with most cells having detectable but very low levels of each globin transcript. Hemin induction shifted the distributions to higher levels, with a tendency to residual left skewing as some cells remained with very low expression levels despite the effect of hemin in increasing expression in most of these low expressing cells. Thus transcriptional heterogeneity remains a crucial variable, even in this extensively used model of human erythroid biology, and clearly influences strongly the response to inducing agents. These methods may enable us to define better possible molecular and/or cellular models of globin gene modulation.

Levels of GATA-1/GATA-2 transcription factors modulate expression of embryonic and fetal hemoglobins

GATA transcription factors bind the consensus sequence WGATAR, present in the flanking regions of most erythroid specific genes. GATA-1 and GATA-2, coexpressed in erythroid cells, are important for expression of erythroid genes. To elucidate the role of specific GATA transcription factors on globin gene expression, we examined the human α- and β-globin gene clusters for all GATA sites. Conserved GATA sites were found in each of the hypersensitive sites in both β-and α clusters and in proximal regulatory regions of the ζ-, ε- and γ-globin but not the α, δ or β-globin genes. We then tested the effect of increasing levels of GATA-1 and GATA-2 on the expression of endogenous globin genes in human erythroid cells. Increasing GATA-1 levels in K562 cells decreased the levels of ε-globin mRNA but had no effect on the levels of expression of γ, ζ or α-globin genes. Increasing GATA-2 levels increased ε-globin and γ-globin transcripts. Increasing levels of GATA-1 also caused a decrease in the expression of endogenous GATA-2, while increased levels of GATA-2 had no effect on GATA-1 mRNA. Our results indicate a differential role of GATA-1 and -2 transcription factors on globin transcripts and suggest a correlation between the conservation of GATA sites in the regulatory regions and the ability of endogenous globin genes to respond to GATA transcription factors. They also suggest that quantitative changes in the levels of GATA-1 or GATA-2 can result in alterations of globin target gene expression and may participate in the ontogenic control of the globin genes.

Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA.

Recent reports have demonstrated that trans-splicing ribozymes can be employed to repair mutant RNAs. One key factor that influences RNA repair efficiency is the accessibility of the substrate RNA for ribozyme binding, which is complicated by the fact that RNAs may assume multiple conformations and have proteins bound to them in vivo. Here we describe a strategy to map accessible sites on sickle beta-globin (beta(s)-globin) transcripts in vitro and in vivo and to use this information to enhance RNA repair efficiency. Two sites upstream of the sickle mutation were identified as accessible in some fraction of the beta-globin RNA by mapping with a ribozyme library and the accessibility of those sites was assessed by in vitro cleavage analyses. Ribozymes targeting either site could only convert a certain fraction of the beta(s)-globin RNA to product but not drive the reaction to completion. However, cleavage and splicing reactions were driven further toward completion when the two ribozymes were both added to the reactions, suggesting that the substrate RNA is present in multiple conformations in vitro. These two ribozymes were each able to repair beta(s)-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Moreover, the relative accessibility of the targeted sites in vivo is as predicted by mapping and in vitro analyses. These results demonstrate that this novel RNA mapping strategy represents an effective means to determine the accessible regions of target RNAs and that combinations of trans-splicing ribozymes can be employed to enhance RNA repair efficiency of clinically relevant transcripts such as beta(s)-globin RNA.

Competition between the signal sequence and a 3'UTR localisation signal during redirection of beta-globin mRNA to the endoplasmic reticulum: implications for biotechnology.

Secretion of an intracellular protein from a cell factory requires as a first step the redirection of the mRNA for synthesis of the protein on the endoplasmic reticulum. The feasibility of retargeting a mRNA coding for an intracellular protein to the endoplasmic reticulum was investigated using Ltk- fibroblasts stably transfected with gene constructs in which rabbit beta-globin coding region and 5'UTR was linked to albumin signal sequence and different 3'untranslated regions. Globin transcripts with the native globin 3'untranslated region or with the 3'untranslated region of c-myc are present in free/cytoskeletal-bound polysomes. The addition of the signal sequence from rat albumin redirects both these globin transcripts to membrane-bound polysomes but the presence of the c-myc 3'UTR reduces the extent of redirection. Globin transcripts with both the signal sequence and 3'untranslated region from the albumin gene are efficiently redirected to membrane-bound polysomes. The results suggest competition between 5' and 3' localising signals. The addition of the signal sequence does not destabilise the mRNA nor affect translational efficiency. It is concluded that it is possible to retarget an mRNA to the endoplasmic reticulum while maintaining stability and translational capacity. This has important implications for the development of vectors to promote secretion of intracellular proteins from cell factories.

GATA-1 dominantly activates a program of erythroid gene expression in factor-dependent myeloid FDCW2 cells.

Erythrocyte development has previously been shown to depend upon the expression of the lineage-restricted trans-acting factor GATA-1. Despite predicted roles for this factor during early development, GATA-1-deficient cells in chimeric mice and embryonic stem cell cultures mature to a late proerythroblast stage and express at least certain genes that normally are thought to be regulated by GATA-1 (including erythroid Krüppel-like factor [EKLF] and the erythropoietin [Epo] receptor). Opportunities to test roles for GATA-1 in erythroid gene activation in these systems therefore are limited. In the present study, in an alternate approach to test the function of GATA-1, GATA-1 has been expressed together with the Epo receptor in myeloid FDCW2 cells and the resulting effects on cytokine-dependent proliferation and erythroid gene expression have been assessed. GATA-1 expression at low levels delayed FDCW2ER cell cycle progression at the G1 phase specifically during Epo-induced mitogenesis. Upon expression of GATA-1 at increased levels, proliferation in response to Epo, interleukin-3 (IL-3), and stem cell factor was attenuated and endogenous GATA-1, EKLF and betamaj-globin gene expression was activated. Friend of GATA-1 (FOG) transcript levels also were enhanced, and ets-1 and c-mpl but not Epo receptor gene expression was induced. Finally, in FDCW2 cells expressing increased levels of GATA-1 and a carboxyl-terminally truncated Epo receptor, Epo (with respect to IL-3 as a control) was shown to markedly promote globin transcript expression. Thus, novel evidence for select hierarchical roles for GATA-1 and Epo in erythroid lineage specification is provided.

The 3′ untranslated region plays a role in the targeting of metallothionein-I mRNA to the perinuclear cytoplasm and cytoskeletal-bound polysomes

The mechanism of localisation of metallothionein-I (MT-I) mRNA was studied in transfected cells by in situ hybridisation and cell fractionation. Hepatoma cells were transfected with the 5′-untranslated region and coding region of the β-globin gene alone or linked to either the β-globin 3′-untranslated region (3′-UTR) or the MT-I 3′-UTR. The wild-type β-globin mRNA and the β-globin mRNA lacking its native 3′-UTR were present in free and cytoskeletal-bound polysomes to a similar extent and showed no localisation. Chimaeric globin-metallothionein transcripts were significantly enriched in cytoskeletal-bound polysomes and were localised in the perinuclear cytoplasm. Chimaeric globin-metallothionein and wild-type globin transcripts were of similar stability. Chinese Hamster Ovary cells were transfected with constructs in which the MT-I 5′-untranslated region and coding sequences were linked to either the endogenous 3′-UTR or the glutathione peroxidase 3′-UTR. Wild-type MT-I transcripts were localised in the perinuclear cytoplasm but the chimaeric MT-I-glutathione peroxidase transcripts showed no distinct localisation. The results indicate that the 3′-UTR of MT-I mRNA contains a localisation signal which promotes both the association of the mRNA with the cytoskeleton and its perinuclear localisation.

EVIDENCE FOR A LOCALIZATION SIGNAL IN THE 3′UNTRANSLATED REGION OF MYOSIN HEAVY CHAIN MESSENGER RNA

Localization signals in the 3′untranslated region (3′UTR) of myosin heavy chain mRNA were investigated using hybrid gene constructs. In myoblasts transfected with constructs containing either both coding sequences and 3′UTR of the rabbit β-globin gene or the β-globin coding sequences alonein situhybridization showed that globin transcripts were distributed throughout the cytoplasm with no localization. In contrast, in myoblasts transfected with β-globin coding sequences linked to the myosin heavy chain 3′UTR there was strong perinuclear localization of the hybrid mRNA; this was maintained in myotubes. We conclude that myosin heavy chain 3′UTR contains a localization signal.

NF-E2p18/mafK is required in DMSO-induced differentiation of Friend erythroleukemia cells by enhancing NF-E2 activity.

When Friend murine erythroleukemia (F-MEL) cells are induced to differentiate by dimethylsulfoxide (DMSO), erythroid-specific genes are transcriptionally activated. The erythroid transcription factor NF-E2 is essential for enhancer activity of the globin locus control regions. NF-E2 functions as a heterocomplex consisting of a 45-kDa subunit (NF-E2p45) and a 18-kDa subunit (NF-E2p18). The larger subunit NF-E2p45 is tissue-restricted and is believed to play a role in globin gene expression in F-MEL cells. The expression of the smaller subunit NF-E2p18, which is a Maf family member (MafK), is cell type- and developmental stage-specific. We have investigated the possible role of NF-E2p18 in Friend erythroid differentiation by stably transfecting either sense and antisense p18 constructs into differentiation-sensitive 745A and partially defective-differentiation TFP10 cell lines. Overexpression of NF-E2p18 induced expression of globin transcripts in both cell lines and increased their sensitivity to erythroid differentiation when exposed to DMSO. Conversely, inhibition of p18 expression by antisense transcripts resulted in the inhibition of DMSO-induced differentiation in both cell lines. These results indicate that NF-E2p18 is necessary for globin expression in F-MEL cells and that it is the predominant gene of the Maf family involved in DMSO-induced erythroid differentiation. Moreover F-MEL clones overexpressing NF-E2p18 showed an increase in specific NF-E2 DNA-binding activity whereas this activity was decreased in clones expressing antisense p18. Finally, studies using transient transfection assays showed that p18 activated NF-E2 site-dependent transcription in F-MEL cells. These data suggest that NF-E2p18 can participate in DMSO-induced erythroid differentiation of F-MEL cells by enhancing NF-E2 activity.

Modifications of RNA processing modulate the expression of hemoglobin genes.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.

Constitutive Expression of GATA-1, EPOR, α-Globin, and γ-Globin Genes in Myeloid Clonogenic Cells From Juvenile Chronic Myelocytic Leukemia

Juvenile chronic myelocytic leukemia (JCML) is a rare disorder of early childhood. Characteristic of JCML are the progressive appearance of high levels of fetal hemoglobin (HbF), reflecting a true reversion to a fetal type of erythropoiesis, and the presence of colony-forming cells able to grow in vitro spontaneously in the absence of growth factors. To better understand the relationship between the erythroid abnormalities and the leukemic process, we analyzed the expression pattern of specific genes related to erythroid differentiation--GATA-1, EPOR, alpha-globin, beta-globin, and gamma-globin genes--in JCML peripheral blood (PB) cells and in vitro-derived colonies. Northern blot analysis of PB cells from five JCML patients indicated levels of GATA-1 transcripts much higher than those usually found in other types of leukemic cells, and S1 nuclease protection assay detected significantly increased expression of gamma-globin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single granulocyte-macrophage colony-forming unit (CFU-GM) colonies, obtained in vitro in the absence of added growth factors from four JCML patients, detected GATA-1, EPOR, and globin (alpha and gamma) transcripts in most of the colonies tested, in contrast with control CFU-GM from normal bone marrow, which were positive only for GATA-1. Single JCML colonies were tested for the presence of two different transcripts; whereas alpha- and gamma-globin genes appeared mostly coexpressed, beta-globin mRNA was detected only in a minority of the gamma-globin-positive colonies, indicating that the leukemic pattern of hemoglobin synthesis is mainly fetal. In addition, the leukemic cells occurring during blast crisis of one of our patients displayed the typical features of a stem cell leukemia (CD34+, CD19-, CD2-, myeloperoxidase-). In this sorted CD34+ population, we detected the presence of a marker chromosome, der(12)t(3;12), previously identified in bone marrow cells at diagnosis and an expression pattern superimposable to that of the JCML colonies, consistently displaying a high gamma-globin:beta-globin mRNA ratio. The expression of erythroid markers within populations of leukemic cells, both in vivo and in vitro, supports the hypothesis that abnormal JCML erythroid cells may originate from the same mutated progenitor that sustains the growth of the leukemic cells.

Erythropoietin receptor signals both proliferation and erythroid-specific differentiation.

Ectopic expression of the erythropoietin receptor (EPO-R) in Ba/F3, an interleukin 3-dependent progenitor cell line, confers EPO-dependent cell growth. To examine whether the introduced EPO-R could affect differentiation, we isolated Ba/F3-EPO-R subclones in interleukin 3 and assayed for the induction of beta-globin mRNA synthesis after exposure to EPO. Detection of beta-globin mRNA was observed within 3 days of EPO treatment, with peak levels accumulating after 10 days. When EPO was withdrawn, expression of beta-globin mRNA persisted in most clones, suggesting that commitment to erythroid differentiation had occurred. Although EPO-R expression also supports EPO-dependent proliferation of CTLL-2, a mature T-cell line, those cells did not produce globin transcripts, presumably because they lack requisite cellular factors involved in erythrocyte differentiation. We conclude that the EPO-R transmits signals important for both proliferation and differentiation along the erythroid lineage.

Regulation of globin gene expression in human K562 cells by recombinant activin A

Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot- blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells.

Regulation of Globin Gene Expression in Human K562 Cells by Recombinant Activin A

AbstractRecent studies indicate that a purified protein, activin A, belongs to the transforming growth factor β(TGF-β) superfamily. Similar to TGF-β, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of a, 0, γ, and e globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for γ and є globins (209 nucleotides) as well as a (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of β globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic β-like and a globin transcripts also confirmed earlier studies of Northern and slot-blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an € globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells. © 1992 by The American Society of Hematology. 0006–4971/92/7903-0009S3.00/0

Effect of Activin A on Globin Gene Expression in Purified Human Erythroid Progenitors

AbstractThe regulatory control of human erythropoiesis through a purified protein, activin A, was examined. Previous studies using mixed populations of bone marrow cells suggested that activin A has an indirect effect on cellular proliferation and DNA synthesis of erythroid progenitors through the mediation of accessory cells. In present studies, the cultures of purified erythroid progenitors were used to examine the effect of activin A on globin gene expression. Human erythroid burst-forming units (BFU-E) were partially purified from peripheral blood, and after 8 days of culture the cells generated consisted mainly of erythroid colony-forming units (CFU-E). It was found that the subsequent 7-day cultures of these purified progenitors yielded similar numbers and size distributions of erythroid colonies, regardless of the presence of activin A in the cultures. In addition, these erythroid progenitor cells were responsive, in terms of stimulation of DNA synthesis, to the addition of erythropoietin, but not to treatment by activin A. Therefore, once the erythroid progen- itors are depleted of accessory cells, activin A has little effect on both the proliferation and the DNA synthesis of these progenitors. However, when these purified erythroid progenitors were cultured in the presence of activin A, the levels of all α, β, and e globin transcripts and hemoglobins were significantly increased. In addition, disuccinimidyl suberate was found to chemically cross-link 125l-activin A to cell surface binding proteins (45 to 54 Kd) in both purified erythroid progenitors and K562 cells. The labeling of these binding proteins was specifically inhibited by the presence of unlabeled activin A, but not transforming growth factor-β. These results suggest that, in addition to its indirect effect on DNA synthesis and cellular proliferation of erythroid progenitors, activin A directly affects the levels of globin mRNAs and hemoglobins in developing human erythroid cells through its specific surface binding receptor(s). ©1992 by The American Society of Hematology. 0006–4971/92/7903-0008$3.00/0

Effect of activin A on globin gene expression in purified human erythroid progenitors

The regulatory control of human erythropoiesis through a purified protein, activin A, was examined. Previous studies using mixed populations of bone marrow cells suggested that activin A has an indirect effect on cellular proliferation and DNA synthesis of erythroid progenitors through the mediation of accessory cells. In present studies, the cultures of purified erythroid progenitors were used to examine the effect of activin A on globin gene expression. Human erythroid burst-forming units (BFU-E) were partially purified from peripheral blood, and after 8 days of culture the cells generated consisted mainly of erythroid colony-forming units (CFU-E). It was found that the subsequent 7-day cultures of these purified progenitors yielded similar numbers and size distributions of erythroid colonies, regardless of the presence of activin A in the cultures. In addition, these erythroid progenitor cells were responsive, in terms of stimulation of DNA synthesis, to the addition of erythropoietin, but not to treatment by activin A. Therefore, once the erythroid progenitors are depleted of accessory cells, activin A has little effect on both the proliferation and the DNA synthesis of these progenitors. However, when these purified erythroid progenitors were cultured in the presence of activin A, the levels of all alpha, beta, and epsilon globin transcripts and hemoglobins were significantly increased. In addition, disuccinimidyl suberate was found to chemically cross-link 125I-activin A to cell surface binding proteins (45 to 54 Kd) in both purified erythroid progenitors and K562 cells. The labeling of these binding proteins was specifically inhibited by the presence of unlabeled activin A, but not transforming growth factor-beta. These results suggest that, in addition to its indirect effect on DNA synthesis and cellular proliferation of erythroid progenitors, activin A directly affects the levels of globin mRNAs and hemoglobins in developing human erythroid cells through its specific surface binding receptor(s).

115. All- trans retinoic acid drug development at the national cancer institute (NCI)

Transcriptional properties of the mouse globin genes and their promoters were examined in COS cells using transient expression assays. Transfected intact mouse α1-globin genes generate transcripts, whereas βmaj and βmin globin transcripts are detected only if the genes are associated with a strong exogenous (SV40) enhancer sequence. Under these conditions the ratio of accumulated βmaj and βmin mRNA sequences approximates the 4:1 ratio observed in reticulocytes and murine erythroleukemia cells (MELC) induced to differentiate by hexamethylene bisacetamide (HMBA). As determined using hybrid genes consisting of globin gene 5′ regions fused to the chloramphenicol acetyltransferase (CAT) structural gene, this 4:1 ratio appears dependent upon the relative activities of the two 5′ promoter regions.

Granulocyte-Macrophage Colony-Stimulating Factor-Dependent Growth and Erythropoietin-Induced Differentiation of a Human Cell Line MB-02

AbstractPeripheral blood blasts from a patient with acute megakaryoblastic leukemia were placed into liquid cultures with recombinant growth factors. Growth, but not differentiation, was supported by interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for the first 30 days of culture. Sustained growth occurred only with GM-CSF and gave rise to the cell line MB-02, which has been in continuous culture for over 1 year. The cell line retained the surface phenotype of the leukemic megakaryoblasts except for the loss of glycoproteins lb and IIb/IIIa, which were induced after exposure to phorbol esters. The induction of erythropoiesis occurred when GM-CSF–deprived cells were cultured with erythropoietin (Epo). Well-defined morphologic stages of differentiation ranging from primitive erythroblasts to nucleiextruding normoblasts were seen. Transforming growth factor-β inhibited GM-CSF- and Epo-dependent growth, but not erythroid maturation. Indirect immunofluorescence using globin chain-specific monoclonal antibodies detected fetal, but not adult hemoglobin in the uninduced cells, β-globin was induced and γ-globin was increased after Epo exposure. Both globin species accumulated in the developing erythrocytes until terminal differentiation. Quantitative S1 analysis of β-like globin transcripts showed very low levels of ϵ- and β-globin expression and high levels of γ-globin expression in cells maintained in GM-CSF. Five days after induction with Epo, e message decreased to barely detectable levels while γ and β transcripts increased threefold and 20-fold, respectively. This novel cell line not only retains many characteristics of the leukemic megakaryoblasts from which it was derived, but also can be induced to recapitulate apparent normal erythropoiesis.

Granulocyte-macrophage colony-stimulating factor-dependent growth and erythropoietin-induced differentiation of a human cell line MB-02

Peripheral blood blasts from a patient with acute megakaryoblastic leukemia were placed into liquid cultures with recombinant growth factors. Growth, but not differentiation, was supported by interleukin- 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for the first 30 days of culture. Sustained growth occurred only with GM-CSF and gave rise to the cell line MB-02, which has been in continuous culture for over 1 year. The cell line retained the surface phenotype of the leukemic megakaryoblasts except for the loss of glycoproteins Ib and IIb/IIIa, which were induced after exposure to phorbol esters. The induction of erythropoiesis occurred when GM-CSF- deprived cells were cultured with erythropoietin (Epo). Well-defined morphologic stages of differentiation ranging from primitive erythroblasts to nuclei-extruding normoblasts were seen. Transforming growth factor-beta inhibited GM-CSF- and Epo-dependent growth, but not erythroid maturation. Indirect immunofluorescence using globin chain- specific monoclonal antibodies detected fetal, but not adult hemoglobin in the uninduced cells. beta-globin was induced and gamma-globin was increased after Epo exposure. Both globin species accumulated in the developing erythrocytes until terminal differentiation. Quantitative S1 analysis of beta-like globin transcripts showed very low levels of epsilon- and beta-globin expression and high levels of gamma-globin expression in cells maintained in GM-CSF. Five days after induction with Epo, epsilon message decreased to barely detectable levels while gamma and beta transcripts increased threefold and 20-fold, respectively. This novel cell line not only retains many characteristics of the leukemic megakaryoblasts from which it was derived, but also can be induced to recapitulate apparent normal erythropoiesis.

The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562

We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3′ enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3′ enhancer is erythroid-specific but not developmental stage- or gene-specific.

The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562

We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3' enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3' enhancer is erythroid-specific but not developmental stage- or gene-specific.