Erythroid K562 Cells Research Articles

Non-Selective Cation Channel-Mediated Ca2+-Entry and Activation of Ca2+/Calmodulin-Dependent Kinase II Contribute to G2/M Cell Cycle Arrest and Survival of Irradiated Leukemia Cells

Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca²⁺ signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by fura-2 Ca²⁺ imaging. Nuclear activity of Ca²⁺/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G₂/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G₂/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca²⁺ entry as evident from fura-2 Ca²⁺ imaging. Similarly, IR stimulated a Ca²⁺-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca²⁺ entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G₂ phase was delayed upon buffering [Ca²⁺]_i with the Ca²⁺ chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca²⁺ entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.

Mechanisms of Erythropoietic Failure in Shwachman Diamond Syndrome Caused by Loss of Ribosome-Related Protein SBDS.

Abstract 3203Poster Board III-140Anemia occurs in 60% of patients with Shwachman Diamond Syndrome (SDS). Although bi-allelic mutations in SBDS cause SDS, it is unclear whether SBDS is critical for erythropoiesis and what the pathogenesis of anemia is in SDS. We hypothesize that SBDS protects early erythroid progenitors from apoptosis by promoting ribosome biosynthesis and translation. During early erythroid differentiation of human K562 cells and primary CD133+ cells, a prominent upregulation of SBDS by RT-qPCR was found. SBDS deficiency by vector-based shRNA led to impaired cell expansion of differentiating K562 cells due to accelerated apoptosis and a mild reduction in proliferation. Furthermore, the cells showed general reduction of 40S, 60S, 80S ribosomal subunits, loss of polysomes and impaired global translation during differentiation. Both cell expansion and translation defects were rescued upon re-introduction of SBDS in K562 cells. Interestingly, leucine partly corrected the cell expansion and translational defects of non-differentiating SBDS-deficient K562 cells, while differentiating SBDS-deficient K562 cells showed improved cell expansion in the presence of additional translation stimulators such as IGF-1. SBDS-knockdown CD133+ cells showed increased BFU-E colony formation under conditions with leucine and a combination of leucine and IGF-1 treatment. Although the erythroid cell expansion defect in K562 cells is independent of p53 as these cells do not express the gene, an upregulation of TAp73, was found in resting SBDS deficient K562 cells. However expression of TAp73 was lost during differentiation. DNp63 was also not upregulated in SBDS-deficient K562 erythroid cells. These results demonstrate that the role of SBDS in non-differentiated cells versus differentiated cells represents two dynamic scenarios and that SBDS plays a critical role in erythroid expansion by promoting survival of early erythroid progenitors and in maintaining ribosome biogenesis during erythroid maturation through a pathway independent of p53 family members. DisclosuresNo relevant conflicts of interest to declare.

Hydroxyurea Generates Nitric Oxide in Human Erythroid Cells: Mechanisms for γ-Globin Gene Activation

Hydroxyurea (HU) induces fetal hemoglobin synthesis through activation of cyclic guanine monophosphate (cGMP) signaling. Studies in sickle cell patients demonstrated increased circulating nitric oxide (NO) levels after oral HU treatment. However, the direct measurement of NO in erythroid cells and its role in fetal hemoglobin induction have not been defined. Therefore, we quantified the level of nitrate and nitrite (NOx) generated by HU in human erythroid progenitors in the presence of three nitric oxide synthase inhibitors (NOS), including N(G)-monomethyl-L-arginine (L-NMMA). In addition, cGMP levels were measured in the presence or absence of the pathway inhibitor 1H-(1,2,4)ox-adiazolo(4,3-a)quinoxalin-1-one, which blocks soluble guanylyl cyclase formation. HU treatment increased NOx levels and gamma-globin transcription in K562 and primary erythroid cells, which was augmented when HU was combined with L-NMMA. Pretreatment with the cGMP pathway inhibitor reversed gamma-gene activation by HU. These data demonstrate the direct stimulation of cellular NO and cGMP signaling in erythroid progenitors by HU as a possible mechanism for gamma-globin gene activation.

Expression of miR-210 during erythroid differentiation and induction of γ-globin gene expression

MicroRNAs (miRs) are a family of small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, inducing translational repression or mRNA degradation. In this paper we have first analyzed by microarray the miR-profile in erythroid precursor cells from one normal and two thalassemic patients expressing different levels of fetal hemoglobin (one of them displaying HPFH phenotype). The microarray data were confirmed by RT-PCR analysis, and allowed us to identify miR-210 as an highly expressed miR in the erythroid precursor cells from the HPFH patient. When RT-PCR was performed on mithramycin-induced K562 cells and erythroid precursor cells, miR-210 was found to be induced in time-dependent and dose-dependent fashion, together with increased expression of the fetal gamma-globin genes. Altogether, the data suggest that miR-210 might be involved in increased expression of gamma-globin genes in differentiating erythroid cells.

DYRK3 Dual-specificity Kinase Attenuates Erythropoiesis during Anemia

During anemia erythropoiesis is bolstered by several factors including KIT ligand, oncostatin-M, glucocorticoids, and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia, however, reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia, both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments, DYRK3-/- progenitors also supported enhanced erythroblast formation, and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo, DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice, in contrast, produced fewer reticulocytes during hemolytic anemia, and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally, as studied in erythroid K562 cells, DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia.

Erythropoietin Receptor Expression: A Role for the Tal1/SCL and GATA-1 Complex

Tal1/SCL is required for hematopoietic stem cell development and is essential for normal erythropoiesis. Although Tal1-DNA binding is not required for hematopoietic development, Tal1 participates in a complex with other binding partners GATA-1, LMO2, Ldb1 and possibly Sp1. During erythropoiesis, Tal1 can activate expression via direct association with E-box-GATA DNA sequence motifs for lineage specific genes such as Protein 4.2. In mice, loss of Tal1 in adult erythropoiesis also affects TER119 expression and BFU-E growth resulting in anemia. We found that knock down of Tal1 expression in primary erythropoietin (EPO) stimulated hematopoietic progenitor cells in culture inhibited EPO receptor (EPOR) expression and erythroid differentiation, consistent with the anemia observed in mice with targeted deletion of adult Tal1 expression. In contrast, overexpression of Tal1 in erythroid cells increased both EPOR expression and erythroid differentiation. In fact, overexpression of EPOR was sufficient to increase differentiation in erythroid progenitor cell cultures. Increased EPOR expression by Tal1 was mediated by 3 conserved E-boxes in the 5′ UTR. In reporter gene assays in K562 erythroid cells, EPOR promoter activity was lost with the mutation of these E-boxes, which are located 75 bp downstream of the required GATA-1 binding motif in the human EPOR proximal promoter. Tal1/E2A dimer binding to the E-box region was demonstrated by gel mobility shift assay. Tal1 transactivated EPOR mRNA and chromatin immunoprecipitation (ChIP) analysis confirmed that Tal1 bound directly to the E-box region in intact erythroid cells. We previously showed that GATA-1 also transactivates EPOR gene expression and is required for high level of EPOR transcription activity. In ChIP assays, an antibody to Tal1 also pulled down chromatin containing the GATA-1 binding site as well as the E-box region and conversely, antibodies to GATA-1 pulled down chromatin corresponding to the GATA-1 binding site and the E-box region. These data show that the complex containing Tal1 and GATA-1 bound to the E-box region and to the GATA-1 site in the EPOR proximal promoter and that the complex has greater occupancy at the GATA-1 site in the proximal promoter. High-level Tal1 expression increased chromatin containing Tal1 or GATA-1 associated with the GATA-1 binding site as well as the down stream E-box region, with a greater proportionate increase in the E-box region. Hence, in addition to the GATA-1 binding site, the downstream E-box region is necessary for high-level EPOR expression. These data suggest that transactivation of the EPOR promoter via these sites is mediated by a Tal1 and GATA-1 containing complex and that during erythropoiesis EPO induction of both Tal1 and GATA-1 regulates the high expression of its own receptor. We hypothesize that the high binding of GATA-1 and Tal1 at the GATA site opens the upstream chromatin structure. The over-expression of Tal1 promotes the open status and facilitates the spreading of open chromatin along the chromatin to the downstream E-box region. Subsequent E-box binding by Tal1 and its interaction with GATA-1 maintains the open chromatin and activates transcription.

Local and Distant Elements Regulate Tissue-Specific Expression of ANK-1 Gene Transcripts

Fewer than 20,000 protein-coding genes in the human genome generate more than 100,000 proteins. This diversity results from the selective use of alternative promoters and alternative mRNA splicing. Ankyrins are multifunctional linker/adapter proteins with isoforms expressed in cell-, tissue-, and developmental stage-specific patterns. The ANK-1 gene, which encodes a series of proteins that connect the red blood cell (RBC) membrane to the RBC skeleton, is an excellent system to study how specific promoters are selected for expression and others suppressed. The human ANK-1 locus has two tissue-specific promoters/first exons (erythroid, 1E; brain/muscle, 1B) and one ubiquitous promoter/first exon (1A). We have previously shown that the ANK-1E promoter sequences are contained in the 300 base pairs (bp) immediately upstream of exon 1E (including a critical GATA-1 binding site) are necessary for erythroid-specific expression in transgenic mice. We have recently reported a novel 9 base consensus sequence ([G/T][G/C][G/C]GGTGAG) located between +7 and +15 that serves as a binding site for the transcription initiation complex. This consensus is present in the other ANK-1 promoters, 30% of all mammalian promoters, and is highly enriched in those that lack known consensus elements (i.e, TATA box; Laflamme et al. submitted). We hypothesized that variation within this consensus sequence controls the level of mRNA transcription. We evaluated altered consensus sequences in the ANK-1E promoter linked to luciferase or gamma-globin reporter genes in transient transfection assays in erythroid K562 cells or transgenic mice, respectively. In both assays, the GCGGGTGAG sequence generated 7-fold higher levels of expression than the wild type sequence (TGCGGTGAG; p<0.01), while other variations gave similar or lower levels of expression. We concluded that while erythroid specificity of the minimal ANK-1E promoter is conferred by GATA-1 binding, the level of expression is controlled by the ([G/T][G/C][G/C]GGTGAG) box. In transient transfection assays in vitro, where the constraints of chromatin are released, the sequences adjacent to ANK-1E and ANK-1A promoters directed equivalent levels of expression in both erythroid and non-erythroid cells. We hypothesized that the activity of the ANK-1E promoter in vivo is controlled by both the core promoter sequence and the local chromatin architecture. Transcriptionally active regions of chromatin show increased sensitivity to DNase I digestion, which we have analyzed across a 200 kb region encompassing all three ANK-1 promoters. A region between the ANK-1E and ANK-1A promoters was sensitive to DNase I digestion only in erythroid cells, while the upstream (1B) and downstream (1A) regions were DNase I resistant. The 1E to 1A region is flanked by DNase hypersensitive sites (HS): one immediately 5′ to 1E (5′HS), and two adjacent HS (3′HS1, 3′HS2) located ~6 kb downstream. Histone acetylation is also associated with active chromatin. Chromatin Immunoprecipitation (ChIP) of the ANK-1E region showed erythroid-specific histone acetylation of the 6kb region between 5′HS and 3′HS1&2, with hyperacetylation at all three HS in all cell types. Barrier elements are found at the boundary between open and condensed chromatin. 5′HS provides a barrier against transgene silencing in cell lines and transgenic mice (p<0.01). 3′HS2 contains barrier activity in transfected cells (p<0.01), while the combination of 3′HS1 and 3′HS2 prevents silencing in transgenic mice (p<0.02). ChIP, EMSA (Mobility Shift Assay) and in vitro DNase I footprinting demonstrated that 3′HS1 binds the erythroid transcription factor NF-E2. In transient assays in erythroid cells, 3′HS1 increased reporter gene activity 5-fold when adjacent to the ANK-1E promoter. We hypothesized that NF-E2 could be translocated to the ANK-1E promoter by the formation of an internal chromatin loop. Chromatin Conformation Capture (3C) demonstrated the formation of a loop structure in which 5′HS and 3′HS1&2 are brought into physical proximity in erythroid, but not non-erythroid cells. In agreement with the 3C results, ChIP demonstrated that both ends of the ANK-1E chromatin loop bind GATA-1, NF-E2 and RNA Pol II. Our current model predicts that the 5′ HS barrier allows the ANK-1E promoter to function in transgenic mice, but in the native locus, ANK-1E promoter activity requires the formation of a chromatin loop mediated by GATA-1 and NF-E2.

Variable Inhibitory Effects on the Formation of Dinitrosyl Iron Complexes by Deferoxamine and Salicylaldehyde Isonicotinoyl Hydrazone in K562 Cells

A prerequisite of dinitrosyl iron complexes (DNIC) formation is the presence of nitric oxide (NO), iron (Fe) and thiol/imidazole groups. The aim of this study was to investigate the influence of Fe chelators on the formation of DNIC in erythroid K562 cells. The cells were treated with lipophilic salicylaldehyde isonicotinoyl hydrazone (SIH) (0.1 mM) and hydrophilic deferoxamine mesylate (DFO) (1 mM), a membrane permeable and non permeable Fe chelator, respectively. Dinitrosyl Fe complexes were generated by addition of 0.07 mM diethylamine NO. The DNIC formation was recorded using electron paramagnetic resonance (EPR). Both chelators inhibited DNIC formation up to 50% after 6 hours of treatment. Taken together, our data suggest that an intracellular low molecular weight labile Fe pool (LIP) and protein-bound Fe participate in DNIC formation in K562 cells to a similar extent.

The lipocalin α1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species

α1-Microglobulin is a 26 kDa plasma and tissue glycoprotein that belongs to the lipocalin protein superfamily. Recent reports show that it is a reductase and radical scavenger and that it binds heme and has heme-degrading properties. This study has investigated the protective effects of α1-microglobulin against oxidation by heme and reactive oxygen species in the human erythroid cell line, K562. The results show that α1-microglobulin prevents intracellular oxidation and up-regulation of heme oxygenase-1 induced by heme, hydrogen peroxide and Fenton reaction-generated hydroxyl radicals in the culture medium. It also reduces the cytosol of non-oxidized cells. Endogeneous expression of α1-microglobulin was up-regulated by these oxidants and silencing of the α1-microglobulin expression increased the cytosol oxidation. α1-microglobulin also inhibited cell death caused by heme and cleared cells from bound heme. Binding of heme to α1-microglobulin increased the radical reductase activity of the protein as compared to the apo-protein. Finally, α1-microglobulin was localized mainly at the cell surface both when administered exogeneously and in non-treated cells. The results suggest that α1-microglobulin is involved in the defence against oxidative cellular injury caused by haemoglobin and heme and that the protein may employ both heme-scavenging and one-electron reduction of radicals to achieve this.

Chromatin Structure and Transcription of the Human alpha Globin Locus in Erythroid and Non-Erythroid Environments.

The ζ, α-1 and α-2 are the major alpha like globin genes. The ζ gene is expressed in the embryonic stage, while α-1 and α-2 genes are expressed throughout the adult life. Although the alpha like globin genes are flanked by genes that are transcribed in many cell types, their expression is restricted to the erythroid cells. Since the alpha globin genes are situated amidst the actively transcribing genes, they are supposed to be in the open chromatin configuration, even when they are transcriptionally silent in non-erythroid cells. Hence, understanding the structure of the chromatin of the alpha globin locus in erythroid and non-erythroid cells is needed to delineate the cell type and developmental stage specific regulation of expression of these genes. In the present study, we have undertaken a comparative analysis of the chromatin structure of the alpha globin locus, recruitment of transcription factors, and the transcriptional activity of the locus in enrythroid and non-erythroid cells. We have taken advantage of the availability of genomic tiling microarrays that include 50 base oligonucleotides spaced at 38 base pair intervals throughout extended regions embedding and flanking the alpha globin cluster and performed ChIP-chip analysis. The data obtained from these studies suggest that in erythroid K562 cells, Histone 3 of the alpha globin locus is acetylated at Lys 9 and dimethylated at Lys4 throughout the locus. The trimethyl Lys 4 marker was present on the promoters of transcribed genes, but not on the active HS40 enhancer. However, Pol II and its phosphory-lated forms were present on both the actively transcribing genes and the HS40 enhancer. Among the transcription factors, NF-E2 was predominantly associated with the HS40 sequences while GATA-1 was present on the alpha like promoters as well as the HS40 enhancer. The insulator binding CTCF was detected at several flanking regions of the HS40 enhancer in K562 and HeLa cells. We speculate that differential interaction among CTCF sites may play a role in regulating the effects of the HS-40 enhancer. In erythroid K562 cells, a strong HS40 enhancer formed by the virtue of the recruitment of the enhancer factors can overcome blocking by the downstream flanking CTCF site and, in analogy to suggestions in studies of Drosophia insulating elements, this might be mediated by specific interactions between upstream and downstream insulators. In the non-erythroid cells, the alpha globin locus was hypoacetylated. Along with the absence of trimethylation of the Lys 4 marker for active transcription, the methylations at Lys 9, and Lys 27 that are associated with the inactive genes were also absent. We also observed a lack of Lys 36 marker associated with the body of the transcribing genes in HeLa cells. In contrast to these observations, we have detected a robust presence of Pol II and Brg1 on the entire locus. Surprisingly, we have detected significant amount of transcriptional activity associated with parts of the theta and zeta genes and intergenic regions in HeLa, NB4 and 06990 lymphoblastoid cells. Initial studies indicate the generation of spliced polyadenylated RNA of the alpha globin locus in HeLa cells. The transcription of the locus was not uniform, but it was localized to certain regions, suggesting that the alpha globin transcription is not just a uniform leaky transcription, but that there may be hitherto unappreciated transcriptional regulatory elements within the locus.

Identification of a TFIID Binding Sequence That Increases Expression from the Ankyrin-1 Promoter: Application to Globin Gene Therapy

Effective Gene Therapy of the hemoglobin β-chain disorders β-thalassemia or Sickle Cell Disease (SCD), requires that viral vectors deliver β-like globin gene to hematopoietic stem cells (HSC) and express β-globin at levels >20% of that of endogenous α-globin. However, the β-globin gene is poorly expressed without sequences from the Locus Control Region (LCR). The LCR contains sequences that contribute to inefficient production and low titers of the recombinant virus, and pose a significant risk of insertional activation of leukemia genes. We have taken an alternative approach using enhancer independent promoters from genes expressed in erythrocytes to express sufficient β-like globin. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the four different tissue specific promoters used to express the ANK-1 gene. We have shown that a double copy of Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. We have also demonstrated that in mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein were expressed at a uniform level of 7.5% of α-globin per vector copy. To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG dinucleotide in the transcribed region (+4 or +32 relative to the proximal or distal mRNA start sites) of the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID. We hypothesized that introducing sequences with higher TFIID binding affinity into the region of the ANK-1E promoter would result in increased level of ANK-1E/γ-globin transcription needed for an effective therapy for β-thalassemia and SCD. An ANK-1E promoter library was generated using degenerate sequence in the TFIID binding region while preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained: wild type (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones ((G/T)(G/C)(G/C)GGTGAG) was found at two other locations in the ANK-1E promoter (−5 and −54 relative to the distal mRNA start site) as well as in 25% of 5′UTR across the human genome (p=2.2e−16). 64% of GC rich promoters contain the consensus sequence, which is enriched at +50 and −70 relative to the mRNA start site. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase than the wild type promoter (p=0.001; 0.005 respectively). Electrophoretic mobility shift assays demonstrated that the two more active promoters bind more TFIID than the wild type promoter. We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

A GC Box Element Is Critical for Transcriptional Regulation of the K-Cl Cotransporter Isoform KCC3a in Hematopoetic Cells.

The KCl Cotransporter (KCC) is a key component of the volume regulation system of human reticulocytes, and its excessive activity in sickle cells contributes to cellular dehydration and therefore to sickling pathology. Three of the four KCC genes, are expressed in erythroid cells (Crable et al. Exp. Hematol. 2005; 33:624). Although the relative contribution of the three KCC isoforms to KCC fluxes and volume regulation in red cells remains unknown, KCC3 appears to be the dominant transcript in late erythroid cells. Heterogenity of the 5′ ends of the KCC3 mRNA transcripts has been described by Mount and colleagues (Mercado et al. Am J Physiol 289:F1246, 2005), including two untranslated exons (ex1D and 1C) 1.2 kb upstream from ex1A of KCC3a, which contains the originally described translation initiation site. We used RT-PCR, 5′ RACE (rapid amplification of cDNA ends), and primer extension analyses to study the 5′ ends of KCC3 cDNA transcripts in erythroid cells. The large exon 1 of KCC3 was identified in fetal liver and bone marrow RNA as a 1718bp region (containing exons 1A, 1C, and 1D described by Mercado et al) that undergoes complex patterns of alternate splicing to generate 10 different transcripts. Six major splicing isoforms are expressed in hematopoietic cell RNA. One isoform incorporates the first 167bp of the exon (exon 1D) as 5′ untranslated sequence which splices to the last 208bp of exon 1, including additional 5′ untranslated sequence and an alternative in-frame initiator methionine. The translated protein exhibits a 59 amino acid N-terminal truncation of KCC3a lacking several potential phosphorylation sites (called KCC3a-Short by Mercado et al). This transcript was the most abundant isoform in hematopoietic cell RNA. A second major isoform contains 629bp of the exon immediately 3′ of exon 1D as 5′ untranslated sequence (exon 1C), then splices out the next 735bp to join the last 243bp of the exon, including additional 5′ untranslated sequence and the alternate initiator methionine of KCC3a-Short. A third novel transcript includes the entire exon, utilizing the first initiator methionine of full-length KCC3a. Two other novel transcripts were found, both of which code for KCC3a-Short. These transcripts were also identified in EST databases. We examined the genomic region around exon 1 for promoter activity using luciferase promoter constructs expressed in erythroid K562 cells. Promoter activity was minimal with constructs which spanned from exon 1D to the beginning of exon 1A, but increased substantially in constructs that included this region plus 900 bp 5′ sequence. Further deletion analysis shows that a minimal promoter containing less than 125 base pairs yields full promoter activity. A mutation of a GC box within this region reduces activity over 15 fold, implicating SP-1 as a trans-regulatory factor. Thus, KCC3 exhibits a complex pattern of alternative splicing in erythroid cells, producing several novel transcripts, some of which encode an N-terminal truncation of KCC3a. Identifying the factors modulating transcriptional and translational control of KCC3 expression and the functional behavior of this truncated protein in erythroid cells is important to understanding volume regulation in reticulocytes and its abnormalities in sickle cells.

Mechanisms of human γ-globin transcriptional induction by apicidin involves p38 signaling to chromatin

Histone deacetylase (HDAC) inhibitors are one of promising drugs to induce fetal hemoglobin (HbF) for treatment of sickle cell disease (SCD) and β-thalassemia. The HDAC inhibitor apicidin was recently reported as a powerful inducer of HbF via a mechanism involving p38 signaling. In this study, we further investigated the signaling effects on the transcriptional activation of γ-globin gene. First, we compared histone 3 (H3) acetylation patterns of ∼70 kb β-globin loci in K562 erythroid versus HeLa cells upon apicidin treatment by chromatin immunoprecipitation assays. The results showed that the level of H3 acetylation was globally increased from the LCR to the promoter of γ-globin gene in K562 cells, but not in non-erythroid, HeLa cells. Inhibition of p38 signaling blocks the effects of apicidin-induced γ-globin expression and H3 acetylation. In parallel, we assessed the recruitment of transcriptional complex to β-globin locus following apicidin treatment. The binding of GATA-1, Sp1 and RNA polymerase II (pol II) were observed to increase over several regulatory regions of β-globin locus. Inhibitor study revealed that p38 pathway was not involved in their recruitments by apicidin. Collectively, our results provide a molecular basis to elucidate the underlying mechanisms involving p38 signaling pathway in the inducement of γ-globin transcriptional expression by apicidin.

Crucial role of lysosomal iron in the formation of dinitrosyl iron complexes in vivo

Dinitrosyl non-heme-iron complexes (DNIC) are found in many nitric oxide producing tissues. A prerequisite of DNIC formation is the presence of nitric oxide, iron and thiol/imidazole groups. The aim of this study was to investigate the role of the cellular labile iron pool in the formation of DNIC in erythroid K562 cells. The cells were treated with a nitric oxide donor in the presence of a permeable (salicylaldehyde isonicotinoyl hydrazone) or a nonpermeable (desferrioxamine mesylate) iron chelator and DNIC formation was recorded using electron paramagnetic resonance. Both chelators inhibited DNIC formation up to 50% after 6 h of treatment. To further investigate the role of lysosomal iron in DNIC formation, we prevented lysosomal proteolysis by pretreatment of whole cells with NH4Cl. Pretreatment with NH4Cl inhibited the formation of DNIC in a time-dependent manner that points to the importance of the degradation of iron metalloproteins in DNIC formation in vivo. Fractionation of the cell content after treatment with the nitric oxide donor revealed that DNIC is formed predominantly in the endosomal/lysosomal fraction. Taken together, these data indicate that lysosomal iron plays a crucial role in DNIC formation in vivo. Degradation of iron-containing metalloproteins seems to be important for this process.

Identification of Apoptosis-Related Genes Regulated by the PLZF Protein.

The PLZF gene was identified in acute promyelocytic leukemia (APL, AML-M3). PLZF expression is altered in chronic lymphocytic leukemia (CLL). PLZF is a transcriptional repressor involved in the control of cell proliferation, differentiation and survival. Overexpression of PLZF induces cell cycle arrest and growth suppression, whereas alteration of its normal function due to the presence of the reciprocal fusion proteins, PLZF-RARA and RARA-PLZF, resulting from the t(11;17) traslocation, is associated to the development of APL. We have established stable inducible PLZF expressor clones from different hematopoietic cell lines, using the tet-off system. PLZF induced inhibition of cell growth in T lymphocytic Jurkat cells, but not in erythroid K562 cells, or B lymphocytic DG75 cells. Growth suppression in Jurkat cells was dependent on its levels of expression, since it was observed only in the highest PLZF expressor clones. Moreover, it was inversely proportional to the initial density of the cultures, suggesting a dependence on exogenous mitogens. Deletion of the BTB/POZ domain abrogated growth suppression. Cell cycle and apoptosis analysis suggest that cell death may be the primary and predominant mechanism responsible for growth suppression in Jurkat cells. Since PLZF may mediate its biological effects by transcriptional control, we conducted a search of potential transcriptional targets by microarray analysis (CodeLink, human whole genome, 55K). Biological duplicates of a high PLZF expressor Jurkat clone, cultured at low cell density (25.000 cells per ml) in the presence and absence of doxycycline, were analysed at 24, 48, 72, and 96 hours. As a result, we have identified around 70 genes, involved in diverse cellular functions, whose expression was significantly modulated by PLZF. Some of them, which have been implicated in cell death, were verified using real time RT-PCR: TERT, which has been described as anti-apoptotic, was repressed; conversely, TP53INP1, ID1 and ID3, which have been described as pro-apoptotic, were induced. The identification of these genes is consistent with the pro-apoptotic phenotype observed for PLZF, suggesting that they could be major mediators of PLZF function. Other genes confirmed by real time RT-PCR included ZNF496 and IGLL1, significantly down-regulated, and MYCN, TBL1X, and TRB2, significantly up-regulated, respectively. None of these targets were significantly modulated when cultures were established at high cell density (250.000 cells per ml). At this cell density, PLZF expression did not induce growth suppression or apoptosis, suggesting that PLZF may integrate external signals before initiating specific genetic programmes that lead to growth arrest and cell death. These findings could contribute to elucidate the mechanism by which PLZF induces apoptosis, and to assess the relevance that structural alterations or abnormal expression of PLZF, respectively, may have in APL and CLL.

Identification of a Novel Core Promoter Element That Enhances Transcription: Application to Ankyrin Promoters for Globin Gene Therapy.

Gene Therapy for the hemoglobin β-chain disorders, Sickle Cell Disease (SCD) or beta thalassemia (β-thal), requires both efficient globin gene transfer into hematopoietic stem cells (HSC) as well as expression of globin mRNA and protein at levels that are >20% of the level of α-globin. Expression of the β-like globin genes in either transgenic mice or from RNA virus vectors integrated into the genome of erythroid cells requires sequences from the Locus Control Region (LCR). The LCR sequences contain cryptic splicing and polyadenylation sequences that contribute to inefficient production and low titers of recombinant virus particles. In addition, the powerful LCR enhancer elements pose a significant risk of insertional activation of leukemia genes. We hypothesized that enhancer-independent promoters from other genes expressed in erythrocytes could be used to express sufficient amounts of β-like globin to treat SCD or β-thal. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the 4 different tissue-specific promoters used to express the ANK-1 gene. We have previously demonstrated that a “double copy” Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. In mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein was expressed at uniform level of 7.5 % of α-globin per vector copy (Sabatino et al. PNAS 97:13294–9, 2000). To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG (−73/−72) dinucleotide in the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID (Gallagher et al. Hum Mol Gen 14:2501–9, 2005). We hypothesized that altering the sequence in the TFIID binding region of the ANK-1E promoter would increase TFIID binding, resulting in the increased level of ANK-1E/γ-globin transcription needed for an effective therapy for SCD and β-thal. We generated a library of ANK-1E promoters with degenerate sequence in the TFIID binding region, preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained from the sequencing results: the wild type sequence (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones: (T/G)(G/C)(G/C)GG(T/A)GAG was found in similar locations relative to the transcriptional start site in the three other ANK-1 promoter regions, as well as in 22% of >4000 human loci with GC-rich promoters that lack TATA boxes and other consensus promoter sequences. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase mRNA and protein than the wild type promoter (p=0.001; 0.005 respectively). We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

Alternative Splicing within Exon 1 of the KCl Cotransporter-3 (KCC3) Gene Results in Novel Transcripts in Erythroid Cells.

The KCl Cotransporter (KCC) is a key component of the volume regulation system of human reticulocytes, and its excessive activity in sickle cells contributes to cellular dehydration and therefore to sickling pathology. Three of the four KCC genes, including KCC3, are expressed in erythroid cells (Exp.Hematol. 2005; 33:624), but their relative contribution to KCC fluxes and volume regulation in red cells remains unknown. Heterogenity of the 5′ ends of the KCC3 mRNA transcripts has been described by Mount and colleagues (Mercado et al. Am J Physiol 289:F1246, 2005), including two untranslated exons (ex1D and 1C) 1.2 kb upstream from ex1A of KCC3a, which contains the originally described translation initiation site. We used RT-PCR, 5′ RACE (rapid amplification of cDNA ends), and primer extension analyses to study the 5′ ends of KCC3 cDNA transcripts in erythroid cells. The large exon 1 of KCC3 was identified in fetal liver and bone marrow RNA as a 1646 bp region (containing exons 1A, 1C, and 1D described by Mercado et al) that undergoes complex patterns of alternate splicing to generate 5 different transcripts. Three major splicing isoforms are expressed in hematopoietic cell RNA. One isoform incorporates the first 103bp of the exon (exon 1D) as 5′ untranslated sequence which splices to the last 208bp of the exon, including additional 5′ untranslated sequence and an alternative in-frame initiator methionine. The translated protein exhibits a 59 amino acid N-terminal truncation of KCC3a lacking several potential phosphorylation sites (KCC3a-Short of Mercado et al). This transcript was the most abundant isoform in hematopoietic cell RNA. A second major isoform contains the first 668bp of the exon (including both exons 1D and 1C) as 5′ untranslated sequence, then splices out the next 735bp to join the last 243bp of the exon, including additional 5′ untranslated sequence and the alternate initiator methionine of KCC3a-Short. A third novel transcript includes the entire exon, utilizing the first initiator methionine of ‘full-length' KCC3a. Two other novel transcripts were found, both of which code for KCC3a-Short. These transcripts were also identified in EST databases. We examined the genomic region around exon 1 for promoter activity using luciferase promoter constructs expressed in erythroid K562 cells. Promoter activity was minimal with constructs which spanned from exon 1D to the beginning of exon 1A, but increased substantially in constructs that included this region plus 900 bp 5′ sequence. Reporter activity increased 3 fold upon removal of the 590 bp 3′ segment of such constructs, suggesting the presence of negative regulatory elements within the exon immediately upstream of exon 1A. Thus, KCC3 exhibits a complex pattern of alternative splicing in erythroid cells, producing several novel transcripts, some of which encode an N-terminal truncation of KCC3a. Identifying the factors modulating transcriptional control of KCC3 expression and the functional behavior of this truncated protein in erythroid cells is important to understanding volume regulation in reticulocytes and its abnormalities in sickle cells.

Development of Improved Lentiviral ‘Suicide' Gene Therapy for the Management of GvHD and GvL/GvT Responses in Allogeneic BMT.

Donor Lymphocyte Infusion (DLI) is employed in the treatment of various malignancies, as donor-derived allogeneic T lymphocytes elicit strong anti-tumor immune responses (GvL/GvT). Unfortunately, these are often accompanied by GvHD, mediated by donor T cells that are allo-activated against host tissues. GvHD manifests with immunosupression, multi-organ dysfunction, severe morbidity and high mortality. It is suggested that CD4+Th1 and CD8+Tc1 cell subsets mediate both strong GvL/GvT and strong GvHD, while being cross-regulated by CD4+Th2 and CD8+Tc2 cells that mediate only moderate effects. Patients transplanted with T cell-depleted BM experience little GvHD, but have high rates of cancer relapse. It is possible to maintain beneficial GvL/GvT responses while controlling GvHD by transferring a drug-activating ‘suicide' gene into donor T cells prior to transplant. Cells expressing the ‘suicide' gene-product convert a non-toxic prodrug into a cytotoxic antimetabolite, and are eliminated by apoptosis. We have constructed 3rd generation lentiviral vectors (LVs) for the expression of human converting enzymes. We are using novel enzymes, endogenously present in human cells, to avoid unwanted immune responses against vector-transduced cells, recently proven a major drawback in clinical trials. Our approaches are based on the human thymidylate kinase (Tmpk), which acts on Zidovudine (AZT), and the human deoxycytidine kinase (dCK), which potently activates a number of prodrugs, including Cytarabine (AraC), Gemcitabine and Cladribine. We have developed active-site engineered mutants of Tmpk and dCK that are up to two orders of magnitude more catalytically active than wild-type (wt) enzymes. Our LVs efficiently transduce both immortalized and primary cells (up to 99%). The LV expression cassette also encodes a non-signaling form of human CD19 molecule that serves as a marker for ex vivo enrichment and in vivo tracking of transduced cells. We demonstrate efficient, selective and prompt killing of transduced cells in a dose-dependent manner by a number of prodrugs in cell lines in vitro and in a murine solid tumor model in vivo. For example, R16GLL Tmpk mutant-transduced Jurkat cells (human T cell leukemia line) are completely killed by apoptosis within 4 days of culture in media containing 100uM AZT (IC50 of 2μM), while wt cells are unaffected at the same conditions. In a NOD/SCID murine tumor model, K562 erythroid leukemia cells transduced with either empty vector, wt Tmpk, and R16GLL or F105Y Tmpk mutants were implanted subcutaneously. Mice bearing tumor cells expressing either R16GLL or F105Y Tmpk and receiving a low daily dose of 2.5mg/kg of AZT have developed significantly reduced tumors following 2 weeks of treatment (tumor size of 24–200mm2), compared to mice bearing wt Tmpk and empty vector transduced cells, or untreated mice (tumor size of over 1000mm2). We are currently evaluating the efficiency with which transduced cells can be cleared from circulation in a murine leukemia model and are evaluating the complete GvHD-treatment strategy in a murine model of GvHD/GvT based on the CD45 congenic strains. This much improved therapeutic strategy may offer a safe and complete solution for GvHD in patients undergoing BMT.

A Facilitated Tracking and Transcription Mechanism of the HS2 Enhancer in the β-Globin Locus Control Region.

The 100 kb human β-globin gene locus contains the embryonic ε-, the fetal Gγ- and Aγ-, and the adult δ- and β-globin genes arranged in the transcriptional order of 5′ ε-Gγ-Aγ-δ-β 3′. The β-globin Locus Control Region (LCR), defined by DNase I hypersensitive sites HS1, 2, 3, 4 and 5 located far upstream of the globin genes, is absolutely required for transcriptional activation of the globin genes in erythroid cells. The HS2 site, located respectively 11 and 55 kb upstream of the ε- and β-globin genes, possesses prominent enhancer activity and is able to activate transcription of the globin genes over the long distance. How the HS2 enhancer acts over the long distance is not fully understood. Three mechanisms have been proposed to explain long-range enhancer function: The looping model, the protein tracking/linking model and the facilitated tracking model. We have shown earlier that a transcription mechanism of the RNA polymerase II (pol II)-complex assembled by the HS2 enhancer mediates long-range HS2 enhancer function. In the present study, we used the chicken HS4 insulator to further study the mechanism of long-range HS2 enhancer function. We created the LCR(+I) and LCR(−I) lines in human erythroid K562 cells using Cre-loxP mediated in situ recombination and in zebrafish. The integrated LCR(+I) and LCR(−I) plasmids spanned the natural 11 kb human ε-globin gene locus either with or without the chicken HS4 insulator inserted between the LCR HS2 enhancer and the ε-globin promoter. Analysis of enhancer function in the integrated plasmids and the endogenous ε-globin gene locus provided evidence for a novel facilitated tracking and transcription mechanism of long-range enhancer function: The HS2 enhancer complex containing both the enhancer DNA and the associated pol II tracked and transcribed through the 10 kb intervening DNA to loop with and activate the ε-globin promoter. The interposed insulator interrupted this facilitated tracking and transcription mechanism of the enhancer complex through the intervening DNA, thereby blocking long-range enhancer function.

Histone acetyltransferase p300 regulates the transcription of human erythroid-specific 5-aminolevulinate synthase gene

Erythroid-specific 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis of erythroid cells. Here, we show that treatment of erythroid K562 cells with HDAC inhibitors sodium butyrate or Trichostatin A gave rise to a significant increase in ALAS2 gene transcripts, with a concurrent increase in acetylation level of histone H4 at the ALAS2 gene promoter. Histone acetyltransferase p300 bound with ALAS2 promoter and overexpression of p300 increased both the promoter reporter expression and endogenous mRNA level of ALAS2. Additionally, two functional Sp1 sites located in ALAS2 promoter were identified. Both of the GATA-1 sites and all the Sp1 sites at the ALAS2 promoter contributed to the transcription synergistic action with p300. These data implicated a close relationship between the acetylation modification of histone at the ALAS2 promoter and the regulation of this gene. Meanwhile, this work identified that ALAS2 is a novel target gene for p300/CBP action as histone acetyltransferases.