Human Erythroleukemia Cells Research Articles

PPARα Activation Bypasses the Transcription Elongation Defect in Mutant tif1γ Erythroid Differentiation

Aberrant erythroid differentiation can give rise to anemias and leukemias. Regulated transcription elongation at erythroid loci is vital for specific differentiation steps during blood development. Transcriptional intermediary factor 1 gamma (TIF1γ), whose gene is mutated in the blood deficient zebrafish moonshine mutant, recruits positive elongation factors to erythroid genes, thus relieving paused Pol II. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we have performed a chemical suppressor screen in the bloodless moonshine mutant. Progeny of heterozygous moonshine mutants were treated at 50% epiboly with 4,000 individual chemical compounds with mostly known functional targets. A rescue in blood formation was assessed for by in situ hybridization for β-globin e3 expression in primitive erythrocytes at 22 hours post fertilization (hpf). Using this strategy we have identified peroxisome proliferator-activated receptor alpha (PPARα) agonists, most importantly, Clofibrate to rescue βe3 globin expression at 22 hpf in 70 - 90% of moonshine embryos in a dose-dependent manner. To address whether the rescue by Clofibrate is due to its activation of the PPARα receptor, we either knocked down PPARα using morpholinos or treated zebrafish embryos with the PPARα antagonist GW6471. In both cases we observed a significant reduction in Clofibrate-mediated rescue. To identify the PPARα-interacting proteome in an erythroid progenitor context, human K562 erythroleukemia cells expressing doxycyclin-inducible Flag-PPARα were generated. In these cells, PPARα target genes such as ANGPTL4 and PDK4 are activated starting four hours after doxycyclin addition and this activation is significantly reduced in the presence of the PPARα antagonist GW6471. Large-scale Flag-immunoprecipitation followed by mass spectrometric analysis identified the heterodimerization partner of PPARα, RXR, co-activators (NCOA1, NCOA6) and co-repressors (NCOR2), furthermore 24 subunits of the mediator complex, six subunits of the cohesin (loading) complex, seven RNA polymerase (Pol) II subunits as well as the Cyclin T1 subunit of P-TEFb and both subunits (SUPT5H, SUPT4H1) of DSIF, two proteins with a positive role in transcription elongation. In co-immunoprecipitation experiments using K562 cells, doxycyclin-induced PPARα activation leads to an enhanced interaction of Pol II with both, the Cyclin T1 and CDK9 subunits of P-TEFb. Morpholino-mediated knockdown of the mediator complex subunit med1 prevents Clofibrate-mediated rescue of βe3 globin expression in moonshine embryos. Together these data suggest that PPARα functionally interacts with the mediator complex in hematopoietic progenitors, leading to increased recruitment of the transcription elongation factors to lineage-specific genes. Our studies provide a basic understanding of the processes that regulate transcription elongation in the differentiation of hematopoietic cells, and could lead to novel therapeutic strategies for the treatment of blood diseases and leukemia. DisclosuresZon:FATE Therapeutics, Inc.: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Recombinant Human IL-12 Exerts Differential Effects on Circulating CD56bright and CD56dim NK Cells in Healthy Human Subjects

Interleukin-12 (IL-12) has potent immunoregulatory and hematopoietic properties, and exerts significant biological effects on natural killer (NK) cells, inducing IFNγ production and enhancing cytotoxicity. Two distinct NK cell populations correlate with their immunoregulatory functions. Mature CD56dimCD16bright NK cells represent 90% of the NK cells resident in the blood and can exert cytotoxic effects on transformed cells. Cytokine producing immature CD56brightCD16+/- NK cells exist in the blood (10% of total circulating NK cells) but are most prominently located in secondary lymphoid tissues.In the continued clinical development of recombinant human IL-12 (HemaMax™, rHuIL-12), to be used in combination with radiotherapy or chemotherapy for the treatment of cancer patients, we have performed a clinical safety study in healthy human subjects. A single subcutaneous (sc) dose of rHuIL-12 (12μg) was administered to 17 healthy human subjects. Placebo was administered to 5 healthy subjects. Peripheral blood samples were collected before rHuIL-12 administration, and up to Day 14 post administration. Immunophenotyping of blood cell populations was conducted by FACS.rHuIL-12 caused a transient decrease in peripheral blood CD56dimCD16bright NK cells, with a nadir (60% reduction from baseline) reached on Day 2 following rHuIL-12 administration. CD56dimCD16bright NK cell levels returned almost to baseline levels on Day 5. Placebo was without effect. Conversely rHuIL-12 caused an elevation in peripheral blood CD56brightCD16+/- NK cells, particularly between Days 2 and 3 after rHuIL-12 administration, which was sustained until a peak was reached on Day 5 (265% above baseline). Levels returned to baseline by Day 11, while placebo was without effect. rHuIL-12 did not impact the less functional CD56-CD16bright NK cell subset. CD56dimCD16bright NK cells expressing the IL-12 receptor β2 subunit (IL-12Rβ2+) showed a substantial, and transient, decrease in levels on Day 2. The plasma concentration of IFNγ was elevated to a peak over 35 fold above baseline level at 10hr. after rHuIL-12 administration.Human NK cells were negatively selected from highly enriched leukapheresis-derived blood and stimulated in vitro with 10 pM rHuIL-12. After 16hr. incubation these predominantly CD56dimCD16brightNK cells showed enhanced release of IFNγ and the increased killing of K562 cells, a human erythroleukemic cell line, when compared with vehicle controls. qPCR analysis of the human NK cell lysates showed rHuIL-12-induced elevation of CD56 (302%) and IL-12Rβ2 (587%) mRNA, when compared with vehicle controls. rHuIL-12 did not influence CD16 mRNA expression, but did increase the level of CD62L (L selectin, 206%) mRNA.The rapid 60% fall in circulating mature CD56dimCD16bright NK cells after rHuIL-12 administration to healthy human subjects suggests their immediate exit from peripheral blood into the tissue compartments. This could be mediated by the observed increase in NK cell CD62L mRNA expression seen in vitro. The sustained increase in immature CD56brightCD16+/- NK cell levels between Day 3 and 6 suggests their IL-12-induced development from CD34+ hematopoietic progenitor cells. In summary rHuIL-12 administration to healthy human subjects demonstrates differential effects on the two key NK cell populations in peripheral blood, increasing CD56brightCD16+/- NK cell numbers, potentially stimulating IFNγ release from and enhancing the cytotoxicity of the CD56dimCD16bright NK cells, and preparing this population for migration into tissues. rHuIL-12 thus shows excellent potential as an immunotherapeutic and hematopoietic agent for the treatment of cancer patients, by impacting the maturation, activation, immunoregulation, and cytolytic properties of NK cells. DisclosuresThomas:Neumedicines: Employment, Equity Ownership. Lawrence:Neumedicines: Employment, Equity Ownership. Mar:Neumedicines: Employment, Equity Ownership. Kha:Neumedicines: Employment, Equity Ownership. Basile:Neumedicines: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Targeting KLF1 for the Treatment of Sickle Cell Disease Using Antisense Oligonucleotides

Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein in the fetus during the last months of embryonic development and the first few months of life after birth. HbF has a slightly greater oxygen binding affinity than adult hemoglobin (HbA, α2β2) and inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD.Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of KLF1, we can promote production of HbF through the upregulation of γ-globin expression.Since rodents don’t express γ-globin, we have employed both human and engineered mouse cell lines to demonstrate upregulation of γ-globin mRNA expression in vitro. We used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse KLF1, and treated with antisense oligonucleotides (ASOs) targeting mouse KLF1. After 7 days of free uptake with the ASOs, we observed a 6-fold increase of human γ-globin mRNA expression after achieving 65% mRNA reduction of mouse KLF1 compared to the untreated control. We were also able to demonstrate significant upregulation of human γ-globin protein expression in these cells by western blot. We have shown similar results in a human erythroleukemia cell line, K562, using ASOs targeting human KLF1. K562 cells were electroporated with the KLF1 ASOs and 4 days later, we observed a 5-fold increase of human γ-globin mRNA expression after achieving 40% mRNA reduction of human KLF1 compared to the untreated control. These data indicate that targeting mouse or human KLF1 with ASO treatment can cause an increase in human γ-globin expression in vitrothat is necessary for the upregulation of fetal hemoglobin.We have also shown that we are able to target the bone marrow in both mice and rats through subcutaneous administration of our KLF1 ASOs. In wild type mice, at a dose of 100 mpk/wk for 4 weeks, we observed KLF1 target reduction of 88% and a β-globin reduction of 58% compared to the saline control in whole bone marrow. In Sprague-Dawley rats, at a dose of 50 mpk/wk for 4 weeks, we observed KLF1 target reduction of 83% and a β-globin reduction of 77% compared to the saline control in whole bone marrow. Therefore, we are able to achieve significant β-globin mRNA reduction in the bone marrow in both mice and rats after subcutaneous administration of KLF ASOs. These data indicate that reducing KLF1 with antisense oligonucleotides is a viable option for the treatment of sickle cell anemia. DisclosuresPeralta:Isis Pharmaceuticals, Inc.: Employment. Low:Isis Pharmaceuticals, Inc.: Employment. Booten:Isis Pharmaceuticals, Inc.: Employment. Zhou:Univeristy of Alabama at Birmingham: Employment. Kim:Isis Pharmaceuticals, Inc.: Employment. Freier:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Murray:Isis Pharmaceuticals, Inc.: Employment. Townes:University of Alabama at Birmingham: Employment. Hung:Isis Pharmaceuticals, Inc.: Employment.

Codanin-1 Binds to Key Erythroid Genes and Its Knockdown Coupled with Ectopic Mutant Expression Recapitulates the Congenital Dyserythropoietic Anemia Type I (CDA I) Phenotype

Congenital dyserythropoietic anemia type I (CDA I) is an autosomal recessive disorder marked by specific morphological abnormalities of erythroblasts in the bone marrow, resulting in ineffective erythropoiesis and anemia. CDA I principally affects the erythroid lineage, while other non-erythroid hematopoietic lineages are unaffected. Light microscopy of bone marrow shows erythroid hyperplasia and binucleated erythroblasts connected by internuclear bridges. Electron microscopy of bone marrow demonstrates erythroblasts with spongy heterochromatin and invagination of the nuclear membrane. The gene responsible for causing CDA I, CDAN1, encodes for a ubiquitous protein, codanin-1, with unknown function. The goal of our study is to understand how codanin-1 regulates erythropoiesis and how codanin-1 defects result in CDA I.To determine codanin-1 expression pattern during erythroid differentiation, we analyzed codanin-1 RNA and protein levels in erythroid primary cells and cell lines. RNA-seq conducted in erythroid-induced human primary CD34+ cells revealed codanin-1 RNA expression is maintained in erythroid lineage. Similarly, immunoblotting shows codanin-1 protein is expressed at high levels in human erythroleukemia cell line stimulated down the erythroid lineage. Codanin-1 has been identified as a chromatin-binding protein. Thus, we conducted ChIP-seq analysis to determine the location of DNA binding sites for codanin-1 in K562 cells. The ChIP-seq results identified 2534 binding sites for codanin-1. Interestingly, many of these sites belong to key erythroid genes. Furthermore, 28% of these DNA binding sites overlap with GATA-1 bound sites.Because putative loss of function mutations in CDAN1 result in CDA I, we studied the effects of codanin-1 gene silencing on erythroid differentiation. shRNA-mediated knockdown reduced codanin-1 mRNA by 50% and protein expression levels by 90%. To assess the effects of codanin-1 perturbation on erythroid differentiation, hemoglobin synthesis was quantified using Drabkin’s reagent in which the absorbance measured at 540 nm is proportional to hemoglobin content in the sample. Erythroid-induced knockdown cells displayed decreased hemoglobin content when compared to non-targeting control shRNA (0.20 versus 0.48, measured in 3 separate experiments; p<0.01). In order to assess binuclearity of the knockdown cells, we stained cytospin slides with Wright Giemsa to assess nuclear morphology. Codanin-1 knockdown cells showed increased binuclearity when compared to control shRNA cells (6% versus 1%, measured in 3 separate experiments; p<0.01). Furthermore, we quantified changes in gene expression induced by codanin-1 gene silencing. Quantitative PCR showed that knockdown of codanin-1 decreased expression of key erythroid genes.To establish a robust CDA I cell system, the first well-characterized CDAN1 mutation (R1042W) was studied because codanin-1 deletion mutants are embryonic lethal in mice and do not occur in humans. Endogenous codanin-1 knockdown cells were transduced with mutant or wild type codanin-1 and induced down the erythroid lineage. Because CDA I is characterized by abnormal heterochromatin appearance, we conducted electron microscopy studies to examine the ultrastructural features. Studies show that knockdown of endogenous codanin-1 coupled with ectopic expression of mutant codanin-1 results in recapitulation of the CDA I phenotype in the K562 cell line model.Because CDA I is almost exclusively restricted to the erythroid lineage, understanding its pathophysiology has the potential to identify critical pathways in erythropoiesis. Here we show that the full phenotype of CDA I is recapitulated upon not only the loss of function of the codanin-1 protein but also on the expression of the mutant form of codanin-1. Our analysis demonstrates that codanin-1 binds to important erythroid loci, suggesting that codanin-1 affects the expression of erythroid genes. Further work will focus on studying codanin-1 function in human primary erythroid cells and in animal models. DisclosuresNo relevant conflicts of interest to declare.

Opposing regulation of megakaryopoiesis by LPA receptors 2 and 3 in K562 human erythroleukemia cells

Erythrocytes and megakaryocytes (MK) are derived from a common progenitor that undergoes lineage specification. Lysophosphatidic acid (LPA), a lipid growth factor was previously shown to be a regulator for erythropoietic process through activating LPA receptor 3 (LPA3). However, whether LPA affects megakaryopoiesis remains unclear. In this study, we used K562 leukemia cell line as a model to investigate the roles of LPA in MK differentiation. We demonstrated that K562 cells express both LPA2 and LPA3, and the expression levels of LPA2 are higher than LPA3. Treatment with phorbol 12-myristate 13-acetate, a commonly used inducer of megakaryopoiesis, reciprocally regulates the expressions of LPA2 and LPA3. By pharmacological blockers and knockdown experiments, we showed that activation of LPA2 suppresses whereas, LPA3 promotes megakaryocytic differentiation in K562. The LPA2-mediated inhibition is dependent on β-catenin translocation, whereas reactive oxygen species (ROS) generation is a downstream signal for activation of LPA3. Furthermore, the hematopoietic transcriptional factors GATA-1 and FLI-1, appear to be involved in these regulatory mechanisms. Taken together, our results suggested that LPA2 and LPA3 may function as a molecular switch and play opposing roles during megakaryopoiesis of K562 cells.

OCT4 mutations in human erythroleukemic cells: implications for multiple drug resistance (MDR) phenotype.

The OCT4 transcription factor is a crucial stem cells marker and it has been related to the cancer stem cells concept. Moreover, it has also been associated to the multiple drug resistance (MDR) phenotype. Our first results pointed out a straight relation between OCT4 and ABC transporters in K562-derivative MDR (Lucena) cells. Sequencing of ABC promoters did not reveal any mutation that could explain the differential expression of OCT4 in Lucena cells. Furthermore, sequencing of the homeobox domain region from the OCT4 gene isolated from both cell lines evinced, for the first time, that this transcription factor is a target of mutations and might be related to the MDR phenotype. The encountered mutations implied in several amino acids substitutions in both cell lines. K562 had seven amino acids substituted (three of them exclusive), while Lucena had 13 substitutions (nine of them exclusive). In addition, an in silico search for phosphorylation motifs within the amino acid stretch compared showed that human normal OCT4 has seven potential phosphorylation motifs. However, K562 has lost one phosphorylation motif and Lucena two of them. These findings bring OCT4 as an important target for cancer treatment, especially those resistant to chemotherapy.

A novel DAG-dependent mechanism links PKCɑ and Cyclin B1 regulating cell cycle progression.

Through the years, different studies showed the involvement of Protein Kinase C (PKC) in cell cycle control, in particular during G1/S transition. Little is known about their role at G2/M checkpoint. In this study, using K562 human erythroleukemia cell line, we found a novel and specific mechanism through which the conventional isoform PKCα positively affects Cyclin B1 modulating G2/M progression of cell cycle. Since the kinase activity of this PKC isoform was not necessary in this process, we demonstrated that PKCα, physically interacting with Cyclin B1, avoided its degradation and stimulated its nuclear import at mitosis. Moreover, the process resulted to be strictly connected with the increase in nuclear diacylglycerol levels (DAG) at G2/M checkpoint, due to the activity of nuclear Phospholipase C β1 (PLCβ1), the only PLC isoform mainly localized in the nucleus of K562 cells. Taken together, our findings indicated a novel DAG dependent mechanism able to regulate the G2/M progression of the cell cycle.

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its d,l-polylactide microparticle formulation

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this “depowered” GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP . This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators.

Somatostatin analogues for receptor targeted photodynamic therapy.

Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K3-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K3-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst2 compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst2-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst2 + AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst2 targeting photosensitizer conjugate.

Superior activity of a new histone deacetylase inhibitor (ZYJ-34c) in inhibiting growth of human leukemia cells by inducing p21 WAF1 expression and cell cycle arrest

Histone deacetylase inhibitors are a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis and cell cycle arrest in various cancer cells. Recently, we identified ZYJ-34c, a modified histone deacetylase inhibitor that showed significantly higher antitumor activity in vivo than the FDA-approved drug suberoylanilide hydroxamic acid. We aimed to investigate its exact mechanism of action. Activity of ZY-34c against human erythroleukemic (K562) cells, human myeloid leukemia (HL-60) cells, and primary leukemic cells were investigated in vitro using proliferation assays, cell cycle assays, apoptosis assays, RNA interference, promoter acetylation assays, and assays of transcription of related molecules. ZYJ-34c strongly inhibited the proliferation of leukemia cells compared with suberoylanilide hydroxamic acid. Primary leukemic cells isolated from patients with acute myeloid leukemia were more sensitive to ZYJ-34c. The 50% growth-inhibitory concentrations of ZYJ-34c and suberoylanilide hydroxamic acid were 2.95±0.75 and 4.45±0.29 μmol/l for K562 cells treated for 48 h, respectively. We found that ZYJ-34c caused more significant G1 cell cycle arrest than suberoylanilide hydroxamic acid, in a time-dependent manner. ZYJ-34c-induced hyperacetylation of histone H3 and H4 around the promoter region of p21. P21 RNA interference markedly impaired ZYJ-34c-induced or suberoylanilide hydroxamic acid-induced G1 cell cycle arrest. In addition, levels of bcr-abl mRNA and protein in K562 cells decreased after treatment with either suberoylanilide hydroxamic acid or ZYJ-34c; moreover, ZYJ-34c had a higher inhibition activity. ZYJ-34c could represent a novel pharmacological agent with potential benefit for patients with leukemia.

Cbl negatively regulates erythropoietin-induced growth and survival signaling through the proteasomal degradation of Src kinase.

We examined the biological functions of the gene Cbl in erythropoietin (EPO) signaling using Cbl-deficient F-36P human erythroleukemia cells by the introduction of the Cbl siRNA expression vector. Knockdown of Cbl promoted EPO-dependent proliferation and survival of F-36P cells, especially at a low concentration of EPO (0.01U/mL), similar to serum concentrations of EPO in healthy volunteers (0.005-0.04U/mL). We found that Src was degraded mainly by the proteasomal pathway because the proteasome inhibitor MG-132 but not the lysosome inhibitor NH4Cl suppressed the EPO-induced degradation of Src in F-36P cells and that knockdown of Cbl inhibited EPO-induced ubiquitination and degradation of Src in F-36P cells. The experiments using the Src inhibitor PP1 and co-expression experiments further confirmed that Cbl and the kinase activity of Src are required for the EPO-induced ubiquitination of Src. In addition, the co-expression experiments and in vitro kinase assay demonstrated that the EPO-induced tyrosine phosphorylation and ubiquitination of Cbl were dependent on the kinase activity of Src but not Jak2. Thus, Cbl negatively regulates EPO signaling mainly through the proteasome-dependent degradation of Src, and the E3 ligase activity of Cbl and its tyrosine phosphorylation are regulated by Src but not Jak2.

Mpl traffics to the cell surface through conventional and unconventional routes.

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER-tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling.

In silico identification of natural product inhibitors of JAK2

Emodic acid (1) and 6-chloroemodic acid (2) have been identified from a natural product database as useful scaffolds for the future development of novel JAK2 inhibitors using structure-based high-throughput virtual screening. Low-energy binding conformations of 1 and 2 in the JAK2 PTK domain were generated by virtual ligand docking and were found to overlap considerably with the binding pose of CMP6, a known JAK2 inhibitor. Compounds 1 and 2 displayed low micromolar efficacies against JAK2 enzyme activity and JAK2 autophosphorylation in human erythroleukemia cells, and inhibited STAT3 DNA-binding activity in a human hepatocarcinoma cell line.

PRKAA1/AMPKα1 is required for autophagy-dependent mitochondrial clearance during erythrocyte maturation

AMP-activated protein kinase α1 knockout (prkaa1−/−) mice manifest splenomegaly and anemia. The underlying molecular mechanisms, however, remain to be established. In this study, we tested the hypothesis that defective autophagy-dependent mitochondrial clearance in prkaa1−/− mice exacerbates oxidative stress, thereby enhancing erythrocyte destruction. The levels of ULK1 phosphorylation, autophagical flux, mitochondrial contents, and reactive oxygen species (ROS) were examined in human erythroleukemia cell line, K562 cells, as well as prkaa1−/− mouse embryonic fibroblasts and erythrocytes. Deletion of Prkaa1 resulted in the inhibition of ULK1 phosphorylation at Ser555, prevented the formation of ULK1 and BECN1- PtdIns3K complexes, and reduced autophagy capacity. The suppression of autophagy was associated with enhanced damaged mitochondrial accumulation and ROS production. Compared with wild-type (WT) mice, prkaa1−/− mice exhibited a shortened erythrocyte life span, hemolytic destruction of erythrocytes, splenomegaly, and anemia, all of which were alleviated by the administration of either rapamycin to activate autophagy or Mito-tempol, a mitochondria-targeted antioxidant, to scavenge mitochondrial ROS. Furthermore, transplantation of WT bone marrow into prkaa1−/− mice restored mitochondrial removal, reduced intracellular ROS levels, and normalized hematologic parameters and spleen size. Conversely, transplantation of prkaa1 −/− bone marrow into WT mice recapitulated the prkaa1−/− mouse phenotypes. We conclude that PRKAA1-dependent autophagy-mediated clearance of damaged mitochondria is required for erythrocyte maturation and homeostasis.

Distinct effects of Disabled-2 on transferrin uptake in different cell types and culture conditions.

Iron uptake by the transferrin (Tf)-transferrin receptor (TfR) complex is critical for erythroid differentiation. The mechanisms of TfR trafficking have been examined, but the adaptor proteins involved in this process are not fully elucidated. We have investigated the role of the adaptor protein, Disabled-2 (Dab2), in erythroid differentiation and Tf uptake in the cells of hematopoietic lineage. Dab2 was upregulated in a time-dependent manner during erythroid differentiation of mouse embryonic stem cells and human K562 erythroleukemic cells. Attenuating Dab2 expression in K562 cells diminished TfR internalization and increased surface levels of TfR concomitantly with a decrease in Tf uptake and erythroid differentiation. Dab2 regulated Tf uptake of the suspended, but not adherent, cultures of K562 cells. In contrast, Dab2 is not involved in TfR trafficking in the HeLa cells with epithelial origin. These differential effects are Dab2-specific because attenuating the expression of adaptor protein 2 μ subunit inhibited the uptake of Tf regardless of culture condition. We offer novel insight of Dab2 function in iron uptake and TfR internalization for the suspended culture of hematopoietic lineage cells.

Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress.

The ubiquitously expressed transcription factor TFII-I exerts both positive and negative effects on transcription. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I in the human erythroleukemia cell line K562. This analysis revealed that TFII-I binds upstream of the transcription start site of expressed genes, both upstream and downstream of the transcription start site of repressed genes, and downstream of RNA polymerase II peaks at the ATF3 and other stress responsive genes. At the ATF3 gene, TFII-I binds immediately downstream of a Pol II peak located 5 kb upstream of exon 1. Induction of ATF3 expression increases transcription throughout the ATF3 gene locus which requires TFII-I and correlates with increased association of Pol II and Elongin A. Pull-down assays demonstrated that TFII-I interacts with Elongin A. Partial depletion of TFII-I expression caused a reduction in the association of Elongin A with and transcription of the DNMT1 and EFR3A genes without a decrease in Pol II recruitment. The data reveal different interaction patterns of TFII-I at active, repressed, or inducible genes, identify novel TFII-I interacting proteins, implicate TFII-I in the regulation of transcription elongation and provide insight into the role of TFII-I during the response to cellular stress.

Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.

PLC-beta 1 regulates the expression of miR-210 during mithramycin-mediated erythroid differentiation in K562 cells.

PLC-beta 1 (PLCβ1) inhibits in human K562 cells erythroid differentiation induced by mithramycin (MTH) by targeting miR-210 expression. Inhibition of miR-210 affects the erythroid differentiation pathway and it occurs to a greater extent in MTH-treated cells. Overexpression of PLCβ1 suppresses the differentiation of K562 elicited by MTH as demonstrated by the absence of γ-globin expression. Inhibition of PLCβ1 expression is capable to promote the differentiation process leading to a recovery of γ-globin gene even in the absence of MTH. Our experimental evidences suggest that PLCβ1 signaling regulates erythropoiesis through miR-210. Indeed overexpression of PLCβ1 leads to a decrease of miR-210 expression after MTH treatment. Moreover miR-210 is up-regulated when PLCβ1 expression is down-regulated. When we silenced PKCα by RNAi technique, we found a decrease in miR-210 and γ-globin expression levels, which led to a severe slowdown of cell differentiation in K562 cells and these effects were the same encountered in cells overexpressing PLCβ1. Therefore we suggest a novel role for PLCβ1 in regulating miR-210 and our data hint at the fact that, in human K562 erythroleukemia cells, the modulation of PLCβ1 expression is able to exert an impairment of normal erythropoiesis as assessed by γ-globin expression.

Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene.

The mammalian Shc family, composed of p46, p52, and p66 isoforms, serves as an adaptor protein in cell growth and stress response. p66Shc was shown to be a negative lifespan regulator by acting as a prooxidant protein in mitochondria; however, the regulatory mechanisms of p66Shc expression and function are incompletely understood. This study provides evidence for new features of p66Shc serving as an antioxidant and critical protein in cell differentiation. Unique among the Shc family, transcription of p66Shc is activated through the antioxidant response element (ARE)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in K562 human erythroleukemia and other cell types after treatment with hemin, an iron-containing porphyrin. Phosphorylated p66Shc at Ser-36, previously reported to be prone to mitochondrial localization, is increased by hemin treatment, but p66Shc remains exclusively in the cytoplasm. p66Shc knockdown inhibits hemin-induced erythroid differentiation, in which reactive oxygen species production and apoptosis are significantly enhanced in conjunction with suppression of other ARE-dependent antioxidant genes. Conversely, p66Shc overexpression is sufficient for inducing erythroid differentiation. Collectively these results demonstrate the isoform-specific regulation of the Shc gene by the Nrf2-ARE pathway and a new antioxidant role of p66Shc in the cytoplasm. Thus p66Shc is a bifunctional protein involved in cellular oxidative stress response and differentiation.

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.