Hemoglobin Synthesis In K562 Cells 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.

Effects of Atpif1 gene on hemoglobin synthesis in K562 cells

To investigate the effects of mitochondrial ATPase inhibitory factor 1 (Atpif1) on hemoglobin synthesis. Firstly, the K562 cells were divided into 2 groups, hypoxia-treated group and normoxic control group. The K562 cells in hypoxia-treated group were treated with 2% oxygen. The K562 cells in the two groups were collected after cultured for 24, 48 and 72 hours. The proliferation-inhibitory rates of cells were detected by CCK-8 assay. The apoptosis rates of K562 cells were analyzed by flow cytometry. The hemoglobin synthesis of K562 cells was induced by hemin. The gene expressions of Atpif1, Aladelta-aminolevulinate synthase 2 (Alas2) and nuclear factor kappa B (NF-κB) were detected by qRT-PCR. Then, the K562 cells were cultured in hypoxic incubator and divided into blank control group, negative control group and si-Atpif1 group. After sliencing Atpif1 gene, the hemoglobin synthesis and the levels of NF-κB and Alas2 were determined. Compared with the normoxic control group, the proliferation activity of K562 cells was inhibited, the apoptosis rate was increased, and the hemoglobin synthesis was also increased in hypoxia-treated groups. The expressions of Atpif1, Alas2 and NF-κB mRNA of K562 cells were upregulated. Compared with blank control group and negative control group, the content of hemoglobin was decreased, and the levels of NF-κB and Alas2 mRNA were also decreased in si-Atpif1 group. Atpif1 gene is involved in the regulation of hemoglobin synthesis. Exploring its roles in the development of high altitude polycythemia (HAPC) can provide new ideas and therapeutic targets for the prevention and treatment of HAPC.

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.

Phenolic metabolites of benzene inhibited the erythroid differentiation of K562 cells

Benzene is a common occupational hazard and a ubiquitous environmental pollutant. Benzene exposure at the levels even below 1 ppm still showed hematotoxicity. It is widely accepted that the metabolites of benzene play important roles in the benzene toxicity to the hematopoietic system, but little is known about the effects of benzene metabolites on erythropoiesis. In present study, erythroid progenitor-like K562 cells were used to determine the effects of phenolic metabolites of benzene, including phenol, hydroquinone and 1,2,4-benzenetriol, on the erythroid differentiation. After the treatment with these benzene metabolites at the concentrations with no obvious cytotoxicity, the hemin-induced hemoglobin synthesis in K562 cells decreased in a concentration- and time-dependent manner, and the expression of CD71 and GPA protein on the surface of K562 cells was also inhibited. The reverse transcription-PCR was used to determine the mRNA level of the erythroid related genes in the K562 cells that were treated with benzene metabolites. The hemin-induced expression of globin genes, including α-, β- and γ-globin genes, and the gene encoding the heme synthesis enzyme porphobilinogen deaminase was inhibited by benzene metabolites. When the K562 cells were pretreated with benzene metabolites, the hemin-induced expression of two transcription factor genes GATA-1 and NF-E2 was distinctly reduced, and the pre-treatment with benzene metabolites promoted the decrease of the mRNA level of transcription factor gene GATA-2 by hemin. These results indicated that benzene metabolites inhibited the hemin-induced erythroid differentiation through affecting the transcription of the erythroid related genes.

Cucurbitacin D induces fetal hemoglobin synthesis in K562 cells and human hematopoietic progenitors through activation of p38 pathway and stabilization of the γ-globin mRNA

The search for novel therapeutic candidates targeting fetal hemoglobin (HbF) activation to reduce the imbalance of globin genes is regarded as a promising approach for the clinical management of sickle cell disease and β-thalassemia. For the first time, we identified cucurbitacin D (CuD), an oxygenated tetracyclic triterpenoid, as a molecular entity inducing γ-globin gene expression and HbF synthesis in K562 cells and human hematopoietic progenitors from a β-thalassemia patient. CuD demonstrated a higher potency in HbF induction when compared with hydroxyurea, which was revealed by the evidence that CuD results in a higher fetal cell percentage and greater HbF content in K562 cells, in addition, to being less cytotoxic. Moreover, CuD also promotes higher HbF expression in primary erythroid cells. In the study to elucidate the molecular mechanisms of CuD's action, our data indicated that CuD-stimulated HbF synthesis was mediated by p38 pathway activation. At the post-transcriptional level, CuD treatment led to a significant elongation of the γ-globin mRNA half-life in K562 cells. Taken together, the results suggest that CuD may be a potential therapeutic agent for β-hemoglobinopathies, including sickle cell anemia and β-thalassemia.

Effects of Astragalus polysaccharide on the erythroid lineage and microarray analysis in K562 cells

Ethnopharmacological relevance Astragalus polysaccharide (APS), obtained from Astragalus membranaceus, displays a range of activities in many systems, including the promotion of immune responses, anti-inflammation, and the protection of vessels. It possesses potent pharmacological activity on differentiation to the erythroid lineage. Aim of the study To investigate the effects of APS on the erythroid differentiation and the mechanism of action by microarray analysis in K562 cells. Materials and methods Benzidine staining, semi-quantitative RT-PCR, Western blot and microarray methods were used to survey the effects of APS on inducing erythroid differentiation and the changes of gene expression profile in K562 cells. Results Of the 13.2% positive cells detected by benzidine staining, the induction was the highest with 200 μg/ml APS on 72 h. Gγ-mRNA expression and fetal hemoglobin synthesis were significantly up-regulated. Microarray analysis showed that 31 genes were up-regulated and 108 genes were down-regulated. These differential expression genes generally regulate protein binding, cellular metabolic process, the cell proliferation, and transcriptional activator activity. The γ-globin gene was up-regulated, the genes related with erythroid differentiation such as LMO2, Runx1 and GTF2I were up-regulated, while Bklf, Eklf, EPHB4 and Sp1 were down-regulated. Conclusions Our studies indicate that APS indicate potent activities on the erythroid differentiation by modulating genes of LMO2, Klf1, Klf3, Runx1, EphB4 and Sp1, increasing γ-globin mRNA expression and fetal hemoglobin synthesis in K562 cells.

Basic fibroblast growth factor antagonizes activin A-mediated growth inhibition and hemoglobin synthesis in K562 cells by activating ERK1/2 and deactivating p38 MAP kinase

Activin A can induce erythroid differentiation, whereas basic fibroblast growth factor (bFGF) can maintain the undifferentiated status of erythroid progenitors. How these two factors together can affect the regulation of erythroid differentiation in hematopoietic cells has not been elucidated. This study demonstrates that bFGF antagonizes activin A-mediated growth inhibition and hemoglobin (Hb) synthesis in K562 cells. Analyses of mitogen-activated protein kinases revealed that activin A-induced p38 phosphorylation and inhibited ERK1/2 phosphorylation. In contrast, bFGF worked antagonistically to induce ERK1/2 phosphorylation and inhibited p38 phosphorylation in K562 cells. Furthermore, co-treatment of cells with activin A and bFGF decreased p38 phosphorylation and increased ERK1/2 phosphorylation. SB203580 inhibition of p38 activity eliminated activin A-mediated growth inhibition and Hb synthesis, whereas U0126 inhibition of ERK1/2 activity augmented the effects of activin A on K562 cells. These results suggest that bFGF can negatively modulate p38 and positively modulate ERK1/2 to antagonize activin A-mediated growth inhibition and Hb synthesis in K562 cells.

Cyclosporin A induces erythroid differentiation of K562 cells through p38 MAPK and ERK pathways.

We studied the effects of cyclosporin A (CsA) on the erythroid differentiation of human erythroid leukemia cell line K562. After K562 was treated with CsA for 4 days, the percentage of hemoglobinized cells was increased by 3.3 times. Because it was reported p38 MAPK (p38) and ERK are involved in erythropoietin-induced erythroid differentiation, we studied their roles using specific inhibitors. p38 inhibitor (SB203580) prevented CsA-induced hemoglobin synthesis in K562 cells, although MEK/ERK inhibitor (U0126) enhanced it by 3.3 times in K562 cells. These results indicate activation of p38 and inactivation of ERK are involved in CsA-induced erythroid differentiation of K562 cells.

Initial function analysis of a novel erythroid differentiation related geneEDRF1

Erythroid differentiation depends on the establishment of specific patterns of gene expression. Hypersensitive site 2 (HS2, serving as a major enhancer of globin genes)-binding proteins may be involved in its natural open chromosomal environment formation. Previously we prepared monoclonal antibodies against HS2-binding nuclear proteins of terminal differentiated erythroid cells. By utilizing the monoclonal antibodies, we screened lambda-gt11 human fetal liver cDNA expression library and obtained one cDNA clone, which was named erythroid differentiation related gene (EDRF1, Genbank accession number AF040247), encompassing an entire open reading frame. We investigated the expression pattern of EDRF1 by RT-PCR technique. And a clue to the function of EDRF1 has been found from confirmation of high levels of EDRF1 mRNA in differentiated K562 and human fetal liver tissue. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. alpha-globin mRNA was down-regulated and EpoR (erythropoietin receptor) mRNA expression was increased in antisense transfected cells. Cells transfected with sense construct grew more slowly than control cells suggested by [(3)H] thimidine incorporation experiments. Suppression of K562 proliferation was accompanied by increased spontaneous hemoglobin synthesis demonstrated by spectrometry. K562 cells transfected with sense construct exhibited reduced clongenicity compared with control cells in methycellulose culture. These data provided the evidence that EDRF1 can influence globin expression and hemoglobin synthesis in K562 cells and modulated self-renewal in K562 cells.

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.

Basic fibroblast growth factor antagonizes transforming growth factor beta-mediated erythroid differentiation in K562 cells

Basic fibroblast growth factor (bFGF) and transforming growth factor- beta 1 (TGF-beta) have both been shown to act on hematopoietic progenitor cells. bFGF is a hematopoietic cytokine that acts on progenitor cells in concert with other cytokines to promote their proliferation. TGF-beta induces erythroid differentiation in K562 cells. To determine whether bFGF might act on progenitor cells by antagonizing the effects of cytokines that induce differentiation, we determined the effects of bFGF on the TGF-beta-mediated induction of hemoglobin synthesis in K562 cells. bFGF antagonized the TGF-beta- mediated induction of hemoglobin in a dose-dependent manner, with 0.1 ng/mL bFGF inhibiting hemoglobin induction by 40% and 10 ng/mL bFGF completely abrogating hemoglobin production. bFGF was most effective at antagonizing the TGF-beta-mediated induction of hemoglobin if it and TGF-beta were added simultaneously to K562 cells, but delayed addition of bFGF to TGF-beta-treated cultures still resulted in significant inhibition of hemoglobin synthesis. The inhibitory effects of bFGF on hemoglobin production were fully reversible, showing that bFGF did not permanently alter the phenotype of K562 cells. The hemin-mediated induction of hemoglobin synthesis in K562 cells was only partially negated by bFGF. bFGF also diminished the expression of glycophorin A on the surface of K562 cells. These results indicate that bFGF might increase progenitor/stem cell numbers by antagonizing the effects of cytokines that induce differentiation, thereby increasing the pool of proliferating progenitor/stem cells.

Basic Fibroblast Growth Factor Antagonizes Transforming Growth Factor β-Mediated Erythroid Differentiation in K562 Cells

Basic fibroblast growth factor (bFGF) and transforming growth factor-beta 1 (TGF-beta) have both been shown to act on hematopoietic progenitor cells. bFGF is a hematopoietic cytokine that acts on progenitor cells in concert with other cytokines to promote their proliferation. TGF-beta induces erythroid differentiation in K562 cells. To determine whether bFGF might act on progenitor cells by antagonizing the effects of cytokines that induce differentiation, we determined the effects of bFGF on the TGF-beta-mediated induction of hemoglobin synthesis in K562 cells. bFGF antagonized the TGF-beta-mediated induction of hemoglobin in a dose-dependent manner, with 0.1 ng/mL bFGF inhibiting hemoglobin induction by 40% and 10 ng/mL bFGF completely abrogating hemoglobin production. bFGF was most effective at antagonizing the TGF-beta-mediated induction of hemoglobin if it and TGF-beta were added simultaneously to K562 cells, but delayed addition of bFGF to TGF-beta-treated cultures still resulted in significant inhibition of hemoglobin synthesis. The inhibitory effects of bFGF on hemoglobin production were fully reversible, showing that bFGF did not permanently alter the phenotype of K562 cells. The hemin-mediated induction of hemoglobin synthesis in K562 cells was only partially negated by bFGF. bFGF also diminished the expression of glycophorin A on the surface of K562 cells. These results indicate that bFGF might increase progenitor/stem cell numbers by antagonizing the effects of cytokines that induce differentiation, thereby increasing the pool of proliferating progenitor/stem cells.

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.

Differential expression of amino acid transport systems A and ASC during erythroleukemia cell differentiation

The human erythroleukemic cell K-562 serves as an in vitro model to study changes in cell surface antigens and mechanisms regulating globin gene expression associated with in vivo erythropoiesis. In this report we have examined the regulation of amino acid transport systems, in particular, systems A and ASC, during differentiation of erythroleukemic cells. For additional comparison we examined the uptake of leucine, 3-aminoendobicyclo-(3,2,1)-octane-3-carboxylic acid (BCO), arginine, and glutamate. Hexamethylene-bis-acetamide (HMBA), dimethyl sulfoxide, and butyrate induce cell differentiation with a block in G1-G0 phase of the cell cycle. These agents caused a significant downregulation of 2-(methylamino)isobutyric acid uptake by system A. In contrast, the Na(+)-dependent threonine uptake by system ASC remained unaltered. The uptake of leucine, BCO, arginine, and glutamate by as yet unidentified systems was, however, stimulated after HMBA treatment. Hemin, a potent inducer of hemoglobin synthesis in K-562 cells, does not block cell cycle events and, interestingly, had no significant effect on both systems A and ASC. These differences in inducer actions suggest that system A activity may be related to specific stages of cell differentiation and perhaps to other cellular signals.

A protein factor binding to an octamer motif in the gamma-globin promoter disappears upon induction of differentiation and hemoglobin synthesis in K562 cells.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human gamma-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erythroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the gamma-globin octamer containing fragment. The possible role of these nuclear proteins in gamma-globin gene regulation and/or cell differentiation is discussed.

Induction of hemoglobin synthesis in k562 cells by carbon dioxide deficiency

Proliferation and differentiation are inversely related in many cell culture systems. The study of inducible systems is facilitated by optimal growth conditions in order that whatever differentiation is observed may be attributed to a specific effect of the inducer, rather than to a nonspecific effect of adverse growth conditions. To investigate the role of CO2 supply in an inducible system, the K562 human leukemia cell line inducible for hemoglobin synthesis was studied at 10%, 5% and 1.5% CO2 concentrations. The lower the CO2 concentration, the higher the percentage of benzidine-positive cells but the slower the growth rate. This increase in benzidine positivity reflected hemoglobin synthesis as indicated by incorporation of 3H-leucine into globin chains. If, in addition to reducing CO2 concentration, the complete medium was replaced by a bicarbonate-free medium, the percentage of benzidine-positive cells was further increased and growth further slowed. However, if endogenously produced CO2 was retained by sealing the culture vessel, these effects were mitigated. Since addition of ribosides blocked these effects, the mechanism for these effects appears to be inhibition of riboside biosynthesis due to the depletion of CO2 as a substrate. The implication of this work is that, for reproducibility in studies of inducible systems in which reduction of proliferation may itself increase the probability of differentiation, the CO2 tension, the bicarbonate concentration in the medium and the rate of egress of endogenously produced CO2 must be kept constant.

Control of oxygen affinity of hemoglobin in K562 cells induced by hemin

The oxygen affinity of hemoglobin in K562 cells induced by hemin and the relationship between levels of 2,3-diphosphoglycerate (2,3-DPG) and hemoglobin have been investigated. Absorption spectra of induced cells indicate that the hemoglobin is oxygenated; oxygen dissociation curves are symmetric, with a P50 of 20 +/- 0.9 mm Hg, Hill coefficient of 2.5, and a normal temperature dependence. The intracellular pH measured by phosphorus 31 nuclear magnetic resonance (NMR) was 7.3. The amount of 2,3-DPG was determined by an enzymatic method and by 31P NMR. The level of 2,3-DPG in uninduced K562 cells, containing 0.5 pg of hemoglobin per cell, was low (5 +/- 0.5 mumole/10(8) cells), but increased to 64 +/- 5 mumole/10(8) cells upon induction of hemoglobin accumulation (to a final level of 20 pg hemoglobin/cell). For several experiments, there was a closely coordinated relationship between 2,3-DPG and hemoglobin levels, at about 1:1 stoichiometry of the two molecules. The time course of induction of hemoglobin, and of 2,3-DPG levels, are very similar; both processes are reversible. These data suggest that induction of hemoglobin synthesis in K562 cells by hemin results in hemoglobin-containing cells with normal oxygenation properties and that 2,3-DPG and hemoglobin levels are coordinately controlled in these cells. Elucidation of the mechanism of this effect should be of importance in understanding the erythroid-like differentiation of these cells.

Control of Oxygen Affinity of Hemoglobin in K562 Cells Induced by Hemin

The oxygen affinity of hemoglobin in K562 cells induced by hemin and the relationship between levels of 2,3-diphosphoglycerate (2,3-DPG) and hemoglobin have been investigated. Absorption spectra of induced cells indicate that the hemoglobin is oxygenated; oxygen dissociation curves are symmetric, with a P50 of 20 +/- 0.9 mm Hg, Hill coefficient of 2.5, and a normal temperature dependence. The intracellular pH measured by phosphorus 31 nuclear magnetic resonance (NMR) was 7.3. The amount of 2,3-DPG was determined by an enzymatic method and by 31P NMR. The level of 2,3-DPG in uninduced K562 cells, containing 0.5 pg of hemoglobin per cell, was low (5 +/- 0.5 mumole/10(8) cells), but increased to 64 +/- 5 mumole/10(8) cells upon induction of hemoglobin accumulation (to a final level of 20 pg hemoglobin/cell). For several experiments, there was a closely coordinated relationship between 2,3-DPG and hemoglobin levels, at about 1:1 stoichiometry of the two molecules. The time course of induction of hemoglobin, and of 2,3-DPG levels, are very similar; both processes are reversible. These data suggest that induction of hemoglobin synthesis in K562 cells by hemin results in hemoglobin-containing cells with normal oxygenation properties and that 2,3-DPG and hemoglobin levels are coordinately controlled in these cells. Elucidation of the mechanism of this effect should be of importance in understanding the erythroid-like differentiation of these cells.

Embryonic erythroid differentiation in the human leukemic cell line K562.

K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.