Differentiation Of Human Erythroleukemia Cells Research Articles

Involvement of autophagy in diosgenin‑induced megakaryocyte differentiation in human erythroleukemia cells.

Natural agents have been used to restart the process of differentiation that is inhibited during leukemic transformation of hematopoietic stem or progenitor cells. Autophagy is a housekeeping pathway that maintains cell homeostasis against stress by recycling macromolecules and organelles and plays an important role in cell differentiation. In the present study, an experimental model was established to investigate the involvement of autophagy in the megakaryocyte differentiation of human erythroleukemia (HEL) cells induced by diosgenin [also known as (25R)-Spirosten-5-en-3b-ol]. It was demonstrated that Atg7 expression was upregulated from day 1 of diosgenin-induced differentiation and was accompanied by a significant elevation in the conversion of light chain 3 A/B (LC3-A/B)-I to LC3-A/B-II. Autophagy was modulated before or after the induction of megakaryocyte differentiation using 3-methyladenine (3-MA, autophagy inhibitor) and metformin (Met, autophagy initiation activator). 3-MA induced a significant accumulation of the LC3 A/B-II form at day 8 of differentiation. It was revealed that 3-MA had a significant repressive effect on the nuclear (polyploidization) and membrane glycoprotein V [(GpV) expression] maturation. On the other hand, autophagy activation increased GpV genomic expression, but did not change the nuclear maturation profile after HEL cells treatment with Met. It was concluded that autophagy inhibition had a more prominent effect on the diosgenin-differentiated cells than autophagy activation.

Two ellagic acids isolated from roots of Sanguisorba officinalis L. promote hematopoietic progenitor cell proliferation and megakaryocyte differentiation.

Using a bioassay-directed chromatographic separation, two ellagic acids were obtained from the ethyl acetate extract of the roots of Sanguisorba officinalis L. On the basis of chemical and spectroscopic methods, the two ellagic acids were identified as 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside and 3,3',4-tri-O-methylellagic acid. Stimulation of cell proliferation was assayed in hematopoietic progenitor cells using the Cell Counting kit-8 method. The megakaryocyte differentiation was determined in human erythroleukemia (HEL) cells using Giemsa staining and flow cytometry analysis. The ellagic acids significantly stimulated the proliferation of Baf3/Mpl cells. Morphology analysis and megakaryocyte specific-marker CD41 staining confirmed that the ellagic acids induced megakaryocyte differentiation in HEL cells. This is the first time that 3,3',4-tri-O-methylellagic acid or 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside are reported to induce megakaryopoiesis, suggesting a class of small molecules which differ from others non-peptidyl, and appears to have potential for clinical development as a therapeutic agent for patients with blood platelet disorders.

Sonic Hedgehog activation is implicated in diosgenin-induced megakaryocytic differentiation of human erythroleukemia cells.

Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells.

Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation

AbstractHow components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization. We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation.

α1-adrenergic drugs modulate differentiation and cell death of human erythroleukemia cells through non adrenergic mechanism

Preliminary data showed that α1-adrenergic antagonists induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells. To test the hypothesis whether survival and differentiation of erythroleukemia cells are under control of α1-adrenergic signalling, we examined α1-adrenoceptor expression of erythroleukemia cells and compared the in vitro effects of α-adrenergic antagonists with those of agonists. We discovered that α1-adrenergic agonists suppress both erythroid differentiation and growth of erythroleukemia cells concomitant with lipofuscin accumulation, autophagy and necrotic cell death. α1-adrenergic agonists also inhibit the in vitro growth of physiologic hematopoietic progenitors obtained from umbilical cord blood with high selectivity for the erythroid lineage. Interestingly, the observed effects could not be related to α1-adrenoceptors, even though agonists and antagonists displayed opposing effects regarding cellular growth and differentiation of erythroleukemia cells. Our data suggest that the effects of α1-adrenergic drugs are related to a non-adrenoceptor binding site, controlling the fate of erythroid progenitor cells towards differentiation and cell death. Since the observed effects are not mediated through adrenoceptors, the physiologic relevance of our data remains unclear, so far. Nevertheless, the identification of the still unknown binding site(s) might disclose new insights into regulation of erythroid differentiation and cell death.

Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy.

The α1-adrenergic receptor antagonists, benoxathian and prazosin, induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells

The erythroleukemia cell lines K562 and human erythroleukemia (HEL) are established models to study erythroid and megakaryocytic differentiation in vitro. In this study, we show that the alpha1-adrenergic antagonists, benoxathian and prazosin, inhibit the proliferation and induce apoptosis in K562 and HEL cells. Furthermore, both tested substances induced the expression of the megakaryocytic marker CD41a, whereas the expression of the erythroid marker glycophorin-a was decreased or unchanged. Even though the expression of differentiation markers was similar after benoxathian and prazosin treatment in both cell lines, endomitosis of erythroleukemia cells was observed only after prazosin treatment. So far, benoxathian and prazosin are the first described extracellular ligands, which cause megakaryocytic differentiation in K562 and HEL cells. In summary, these results indicate a possible role of alpha1-adrenergic receptor signaling in the regulation of erythroid and megakaryocytic differentiation, even though the receptor dependence of the observed effects needs further investigation.

Complete Hematologic and Cytologic Remission in M6A Erythroleukemia Using Decitabine Hypomethylation Monotherapy

Hypomethylating drugs (e.g. Azacitadine, Decitabine) are becoming increasingly attractive choices for the treatment of acute myelogenous leukemia (AML), particularly in populations deemed inappropriate for standard anthracycline-based induction chemotherapy (i.e., 7+3), such as the elderly. Acute erythroleukemia (French-American- British M6) is a rare, heterogeneous disorder involving increased red cell precursors and myeloblasts, that accounts for 3–5% of de novo AML and 20–30% of secondary AML. Three subsets have been described: M6A (myeloblast-rich erythroleukemia); M6B (proerythroblast-rich erythroleukemia); and M6C (myeloblast- and proerythroblast-rich mixed variant). Little data exists on the efficacy of hypomethylation therapy in this uncommon subtype of AML. We report the rapid induction of a complete hematologic and cytologic remission in a patient with the M6A variant erythroleukemia using decitabine monotherapy. A 75 year-old white male with a history of coronary artery disease, was admitted for elective cardiac catheterization because of recurrent exertional chest pain. A pre-catheterization complete blood count (CBC) revealed pancytopenia with a white blood cell (WBC) count of 1400/mm3, hemoglobin of 6.2 g/dL (mean cell corpuscular volume of 113 fL), and a platelet count of 51,000/mm3. The differential showed neutropenia, but was otherwise normal with no circulating blasts. Blood transfusion completely relieved his chest pain, and his catheterization was uneventful. A bone marrow aspirate and biopsy demonstrated a hypocellular marrow (15–20%) with erythroid hyperplasia (70% of total cellularity) and increased blasts (10% of total cellularity, 30% of non-erythroid cells), most compatible with acute M6A erythroid/myeloid leukemia. Cytogenetic studies revealed a normal male karyotype, but a single cell with a 5q- deletion. Subsequent fluorescent in situ hybridization (FISH) analysis using an LSI DNA probe (Vysis Inc.) for detection of the EGR1 gene on chromosome 5q, failed to confirm the 5q deletion, consistent with normal cytogenetics. Because of the patient's advanced age and co-morbidities, induction with hypomethylation monotherapy was begun with intravenous decitabine (Dacogen®) 20 mg/m2 daily for five consecutive days, repeated every four weeks. Growth factor support (Neupogen® 375 mcg daily) and antibiotic prophylaxis (ciprofloxaxin, acyclovir, fluconazole) were only required after the first cycle of decitabine because neutropenia did not occur with subsequent cycles. WBC and platelet counts normalized shortly after cycle one, while the hemoglobin normalized by cycle five. An interim bone marrow aspirate/biopsy was repeated after cycle three, showing normocellularity (30%), and no evidence of residual leukemia. Other than a localized herpetic reactivation following cycle one, the patient has tolerated hypomethylation therapy extremely well. He has now completed eight cycles of therapy, and remains in complete remission. Epigenetic alterations (such as DNA methylation) may play a role in AML leukemogenesis by inducing the inhibition of tumor suppressor genes. Recent studies have also shown that high estrogen receptor-α and p15INK4B methylation levels in AML patients in clinical remission, were associated with a high risk for leukemia relapse and poor relapse-free survival. However, little data exist for epigenetic dysregulation in the etiology of human erythroleukemia. Cell culture studies using human erythroleukemia cell lines indicate that DNA methylation may have functional consequences for gene activity, including globin gene expression and cellular differentiation. The current case indicates a potential role for DNA methylation in the pathogenesis of human erythroleukemia, and argues for further investigation into this potential mechanism of leukemogenesis. Future studies will include the effect of hypomethylating compounds on the growth and differentiation of human erythroleukemia cells, and the evaluation of DNA methylation levels in bone marrow biopsy samples from M6 patients.

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 μM, diosgenin induced megakaryocytic differentiation, in contrast to 40 μM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

Levels of Smad7 regulate Smad and mitogen activated kinases (MAPKs) signaling and controls erythroid and megakaryocytic differentiation of erythroleukemia cells

Smad and MAPK signaling cascades are involved in erythroid and megakaryocytic differentiation. The inhibitory Smad for TGF-β/activin signaling, Smad7, may directly or indirectly affect these signaling pathways. By modulating Smad7 expression, we attempted to delineate the relevance of Smad7 during erythro-megakaryocytic (E/M) differentiation of human erythroleukemia cells. Smad7 transcripts were detected at low levels in different erythroleukemia cell lines (TF-1, HEL and K562). Reduction of expression of endogenous Smad7 by RNA interference enhanced erythroid differentiation of K562 cells in response to physiological doses of activin-A/TGF-β1. Stable over-expression of Smad7 in K562 cells (K562/7) prevented activation of Smad2/3 and MAPK (ERK1/2, p38 and JNK1/2) proteins by activin-A/TGF-β1and subsequent induction of erythroid differentiation. High levels of Smad7 also interfered with hydroxyurea- and butyrate-, but not hemin-induced erythroid differentiation. Interestingly, K562/7 cells were found to harbor a significant proportion (about 35%) of large ploy nucleated cells compared to fewer than 12% in control cells. K562/7 cells treated with phorbol 12-myristate 13-acetate (PMA), showed a great shift in ploidy towards high ploidy classes (≥8N) accompanied with an increase in the expression of the maturation marker CD42b. We showed here that: (a) low levels of endogenous Smad7 in erythroleukemia cells are physiologically relevant, and (b) high levels of Smad7 interferes with TGF-β/activin-induced Smad/MAPK signaling and erythro-differentiation and promotes megakaryocytic differentiation, possibly by blocking autocrine TGF-β.

Transcriptional and post-transcriptional regulation of glutathione S-transferase P1 expression during butyric acid-induced differentiation of K562 cells

Over-expression of glutathione S-transferase P1 is related to chemotherapeutic drug resistance as well as to differentiation of human erythroleukemia cells. In opposition to previously described differentiating inducers which enhance the GST-resistance phenotype, time- and concentration-dependent activation of both erythroid and megakaryocytic differentiation pathways by butyric acid progressively diminished GSTP1 mRNA expression. GSTP1 mRNA expression decreased by 25% ( p < 0.01) and 64% ( p < 0.01) in 1 mM and 2 mM butyric acid-differentiated K562 cells, respectively. These results were associated to both a reduction of GATA-1 binding activity to the GSTP1 promoter and to a posttranscriptional destabilization of GSTP1 mRNA in a concentration dependent manner. Indeed, GSTP1 mRNA half-life decreased from 43.8 to 36.2 h and 12.6 h in 1 mM- and 2 mM-treated cells, respectively.

Modulation of MDM2/p53 and cyclin-activating kinase during the megakaryocyte differentiation of human erythroleukemia cells.

This study was undertaken to address the involvement of CDK activating kinase (CAK), p53, and MDM2 proteins in the mitotic arrest associated with the acquisition of a polyploid DNA content during megakaryocyte differentiation of human erythroleukemia (HEL) cells. To evaluate this mechanism we investigated HEL cells as a model system in which there is a marked increase in DNA content during megakaryocyte differentiation induced by phorbol-diesters. Specific cell-cycle phases were separated by centrifugal elutriation and SDS PAGE and Western analysis were performed to determine the relative abundance of these proteins. Kinase assays were carried out following immunoprecipitation of cellular lysates with the antibodies to the proteins. Polyploid HEL cells show an increase in the abundance of the CAK complex proteins, CDK7 and cyclin H, and a sixfold increase in CAK-specific activity. Increased CAK activity in polyploid HEL cells follows both the downregulation of p53 protein and its decreased association with CAK complex. Consistent with the reduction of p53, polyploid HEL cells undergo a dramatic increase in MDM2 protein abundance that in turn facilitates increased interaction of this protein with p53. These observations demonstrate that deregulated expression of MDM2 and p53 during megakaryocyte differentiation allow a relaxation of the control over genomic stability, allowing further replicative rounds of DNA synthesis.

Protein Kinase Cα Is an Effector of Hexamethylene Bisacetamide-Induced Differentiation of Friend Erythroleukemia Cells

The program of biochemical and molecular events necessary for commitment to erythroid cell differentiation is particularly well characterized in murine Friend erythroleukemia cell lines. Commitment to hemoglobin synthesis in response to a variety of chemical inducers, including hexamethylene bisacetamide and dimethyl sulfoxide is completed by 24 h and proceeds to terminal differentiation by 96 h. Phorbol 12-myristate 13-acetate, a classical tumor promoter phorbol ester that binds to protein kinase C, blocks differentiation in a reversible manner, suggesting an important role for protein kinase C signaling pathways. The classical protein kinase C isoforms α, βI, and βII, play distinct roles in the transduction of proliferative and differentiative signals in human, as well as in murine, erythroleukemia cells. Protein kinase Cα has been implicated in differentiation of human erythroleukemia cells although its translocation to the nucleus has not been observed. Taking advantage of the ability of phorbol 12-myristate 13-acetate to block differentiation in Friend erythroleukemia cells, we determined the localization of the predominant protein kinase C isoforms α and βI during differentiation and in response to their blockade. The ability of phorbol myristate acetate to preferentially diminish protein kinase Cα-protein localization to the nucleus by 24 h and thereby block differentiation induced by hexamethylene bisacetamide was paralleled by the ability of protein kinase Cα antisense transfection to block differentiation. In addition, β-globin transcription, assessed by polymerase chain reaction, was significantly decreased in protein kinase Cα antisense-transfected cells compared to that seen in vector transfected ones. Taken together, these data suggest an important temporal role for nuclear protein kinase Cα localization in Friend erythroleukemia cell differentiation.

New aspects in the vertebrate heat shock factor system: Hsf3 and Hsf4.

In response to higher temperature, all organisms induce a set of proteins known as heat shock proteins (Hsps) to protect themselves from cell death. The expression of Hsps are also activated by changes in cellular metabolic processes, such as cell cycle progression, differentiation, development, neuroendocrinological stress, infection, trauma, exercise and the expression of oncoproteins, and by stresses that cause protein denaturation, such as heat shock, oxidative stress and exposure to heavy metals and anti-cancer drugs (Morimoto et al. 1994). These expressions are controlled mainly at the level of transcription by heat shock transcription factor (Hsf) that binds to the heat shock element (HSE) located upstream of all heat shock genes (Wu 1995). Considerable research in eukaryotes such as yeast and fly in the late 1980s had led to this simple and clarifying view (Sorger 1990; Lis and Wu 1993). The molecular cloning of three tomato Hsfs (Scharf et al. 1990) and two mammalian Hsfs, Hsfl and Hsf2, focused the next generation of research on the role of each Hsf (Rabindran et al. 1991; Schuetz et al. 1991; Sarge et al. 1991). Biochemical analyses using specific antibodies clearly demonstrated that mammalian Hsfl is the factor activated by heat shock (Baler et al. 1993; Sarge et al. 1993). Hsfl has thus been considered to be the functional homologue of yeast and fly Hsf. Hsf2 is activated during the differentiation of human erythroleukemia cells induced by hemin (Sistonen et al. 1992) and its activation is also related to development (Sarge et al. 1994; Ralla et al. 1997). The identification of an avian Hsf3 (Nakai and Morimoto 1993) and a

Evidence for distinct regulation processes in the aclacinomycin- and doxorubicin-mediated differentiation of human erythroleukemic cells

Human erythroleukemic K 562 cells were induced to differentiate along the erythroid lineage by anthracycline antitumor drugs, such as aclacinomycin (ACLA) and doxorubicin (DOX). Subsequent stimulation of heme and globin synthesis led to a differential quantitative expression of hemoglobins. Gower 1 ( ϵ 2, ζ 2) was the major type for ACLA and X ( ϵ 2, γ 2) for DOX. Although ACLA and DOX increased both the expression of γ-globin and porphobilinogen deaminase mRNAs, striking differences were observed in the expression of erythropoietin receptor mRNAs and in erythroid transcription factors GATA-1 and NF-E2, known to play a key role in erythroid gene regulation. Indeed, ACLA induces an increase either in the binding capacity of GATA-1 and NF-E2 or in the accumulation of erythropoietin receptor, GATA-1 and NF-E2 transcripts. In contrast, their expression with DOX was not significantly modified compared to uninduced cells, except for a slight decrease in NF-E2 expression on day 3. In conclusion, these data show that: 1. increased expression of erythroid transcription factors and erythroid genes are associated only with ACLA treatment, and 2. although cytotoxicity of both ACLA and DOX is certainly dependent on DNA intercalation, regulation of differentiation processes by these two drugs involves distinct mechanisms.

Erythroid-specific processing of human beta spectrin I pre-mRNA

Erythroid cells express a unique form of beta spectrin I as a result of tissue-specific alternative pre-mRNA processing. Nonerythroid cells that express the beta spectrin I gene include four additional exons at the 3′ end of the mature transcript, leading to elongation of the carboxyl terminus of the protein. The nonerythroid beta spectrin I isoform is not present in the red blood cell membrane skeleton; the erythroid isoform is not detected in other cell types. Therefore, developing erythroid cells acquire this tissue-specific pre-mRNA processing activity during differentiation. In the present study, we investigated the developmental timing of erythroid-specific pre-mRNA processing in human erythroid precursors. Partially purified human peripheral blood burst forming uniterythroid (BFU-E) cells were grown in culture for 5 to 12 days. beta Spectrin I mRNA transcripts were analyzed at different time points by S1 nuclease mapping. The processing of beta spectrin I transcripts was found to be exclusively erythroid from day 5 onward, indicating that erythroid-specific processing is not linked temporally to assembly of the mature erythroid membrane skeleton. Human erythroleukemia (HEL) cells had both erythroid and nonerythroid transcripts, indicating that both processing patterns can coexist. Induction of erythroid differentiation in HEL cells using hemin resulted in a partial switch toward the erythroid processing pattern of beta spectrin I transcripts. Using a genomic S1 probe that spans the erythroid polyadenylation signal, we found that a substantial portion of the transcripts detected by the erythroid cDNA S1 probe (in both cultured BFU-E and HEL cells) is incompletely processed pre-mRNA precursors. Poly(A) RNA selection before S1 analysis showed that the unprocessed transcripts are not polyadenylated. We conclude that (1) erythroid-specific pre-mRNA processing activity is present early in erythroid differentiation; (2) beta spectrin I transcripts that are unprocessed at the 3′ end accumulate, awaiting either erythroid or nonerythroid processing pathways, from which observation we infer that the regulated alternative pathways are both inefficient; and (3) HEL cells offer a human cell culture model in which to study the balance between the two pre-mRNA processing pathways. We speculate that erythroid cells evolved this tissue-specific pre-mRNA processing machinery for other erythroid genes in addition to beta spectrin I.

Erythroid-Specific Processing of Human β Spectrin I Pre-mRNA

Erythroid cells express a unique form of beta spectrin I as a result of tissue-specific alternative pre-mRNA processing. Nonerythroid cells that express the beta spectrin I gene include four additional exons at the 3' end of the mature transcript, leading to elongation of the carboxyl terminus of the protein. The nonerythroid beta spectrin I isoform is not present in the red blood cell membrane skeleton; the erythroid isoform is not detected in other cell types. Therefore, developing erythroid cells acquire this tissue-specific pre-mRNA processing activity during differentiation. In the present study, we investigated the developmental timing of erythroid-specific pre-mRNA processing in human erythroid precursors. Partially purified human peripheral blood burst forming uniterythroid (BFU-E) cells were grown in culture for 5 to 12 days. beta Spectrin I mRNA transcripts were analyzed at different time points by S1 nuclease mapping. The processing of beta spectrin I transcripts was found to be exclusively erythroid from day 5 onward, indicating that erythroid-specific processing is not linked temporally to assembly of the mature erythroid membrane skeleton. Human erythroleukemia (HEL) cells had both erythroid and nonerythroid transcripts, indicating that both processing patterns can coexist. Induction of erythroid differentiation in HEL cells using hemin resulted in a partial switch toward the erythroid processing pattern of beta spectrin I transcripts. Using a genomic S1 probe that spans the erythroid polyadenylation signal, we found that a substantial portion of the transcripts detected by the erythroid cDNA S1 probe (in both cultured BFU-E and HEL cells) is incompletely processed pre-mRNA precursors. Poly(A) RNA selection before S1 analysis showed that the unprocessed transcripts are not polyadenylated. We conclude that (1) erythroid-specific pre-mRNA processing activity is present early in erythroid differentiation; (2) beta spectrin I transcripts that are unprocessed at the 3' end accumulate, awaiting either erythroid or nonerythroid processing pathways, from which observation we infer that the regulated alternative pathways are both inefficient; and (3) HEL cells offer a human cell culture model in which to study the balance between the two pre-mRNA processing pathways. We speculate that erythroid cells evolved this tissue-specific pre-mRNA processing machinery for other erythroid genes in addition to beta spectrin I.

Downregulation of GATA-1 expression during phorbol myristate acetate- induced megakaryocytic differentiation of human erythroleukemia cells

Phorbol myristate acetate (PMA) induces the expression of megakaryocyte and/or platelet proteins during terminal differentiation of human erythroleukemia (HEL) cells. However, it is not established whether megakaryocytic differentiation is accompanied by the downregulation of the major erythroid transcription factor GATA-1 and the concomitant loss of the erythrocytic phenotype. Studies of the molecular mechanism of PMA-induced differentiation in HEL cells showed that when HEL cells are treated with PMA, they dramatically decrease the expression of the erythroid-specific gene glycophorin A at the mRNA level but apparently not at the steady-state protein level. In addition, a gel mobility shift assay was used to demonstrate that GATA-1, a major erythroid transcription factor normally present at high levels in HEL cells is downregulated after treatment with PMA. In contrast, the DNA-binding activities of transcription factors AP-1 and SP-1 are upregulated by PMA treatment of HEL cells. Furthermore, Northern blot analysis shows that PMA also downregulates the steady-state level of GATA-1 mRNA in HEL cells. The coordinated negative regulation of glycophorin A mRNA and GATA-1 expression after PMA treatment suggests that downregulation of GATA-1 expression may be partially responsible for the loss of the erythroid phenotype during megakaryocytic differentiation. The reported data also suggest that GATA-1 activity may not be essential for obtaining megakaryocytic phenotype during terminal differentiation in HEL cells.

Downregulation of GATA-1 Expression During Phorbol Myristate Acetate-Induced Megakaryocytic Differentiation of Human Erythroleukemia Cells

Phorbol myristate acetate (PMA) induces the expression of megakaryocyte and/or platelet proteins during terminal differentiation of human erythroleukemia (HEL) cells. However, it is not established whether megakaryocytic differentiation is accompanied by the downregulation of the major erythroid transcription factor GATA-1 and the concomitant loss of the erythrocytic phenotype. Studies of the molecular mechanism of PMA-induced differentiation in HEL cells showed that when HEL cells are treated with PMA, they dramatically decrease the expression of the erythroid-specific gene glycophorin A at the mRNA level but apparently not at the steady-state protein level. In addition, a gel mobility shift assay was used to demonstrate that GATA-1, a major erythroid transcription factor normally present at high levels in HEL cells is downregulated after treatment with PMA. In contrast, the DNA-binding activities of transcription factors AP-1 and SP-1 are upregulated by PMA treatment of HEL cells. Furthermore, Northern blot analysis shows that PMA also downregulates the steady-state level of GATA-1 mRNA in HEL cells. The coordinated negative regulation of glycophorin A mRNA and GATA-1 expression after PMA treatment suggests that downregulation of GATA-1 expression may be partially responsible for the loss of the erythroid phenotype during megakaryocytic differentiation. The reported data also suggest that GATA-1 activity may not be essential for obtaining megakaryocytic phenotype during terminal differentiation in HEL cells.

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells.

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.