Gene Expression In K562 Cells Research Articles

30P Targeting of BCR-ABL gene expression in K562 cells using cell-penetrating peptide nanocomplexes carrying siRNAs

Gene silencing by small interfering RNA (siRNA) is a promising therapeutic approach for a wide range of disorders, including cancer. siRNAs against BCR-ABL can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitors (TKIs) therapies, especially given frequently-noted clinical TKI resistance. The main challenge for such approaches remains the development of effective RNAi systems for intracellular delivery. Cell-penetrating peptides (CPPs) are oligopeptides which have the ability to deliver various cargo molecules.

Characterization of a HBB -Locus Fetal Chromatin Domain Region during Adult Erythropoiesis

The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common genetic disorders in the world producing significant morbidity and mortality. Drug-mediated fetal hemoglobin (HbF) induction ameliorates the clinical severity of SCD and improves long-term survival. To define mechanisms of γ-globin regulation, previously we demonstrated the role of the Gγ-globin cAMP response element, in drug-mediated HbF induction (Sangerman et al., Blood 2006). Subsequently, data generated by the ENCODE project led us to investigate a region 4 kb upstream of Gγ-globin, which harbors a DNase hypersensitive site and enrichment of the histone 3 lysine 4, mono-methylated (H3K4Me1) enhancer mark, that we named the Fetal Chromatin Domain (FCD). Recently, a synthetic zinc finger DNA-binding domain targeting this region was shown to reduce γ-globin gene expression in K562 cells (Shen et al., Blood 128:320, 2016). Initially, we performed multispecies sequence alignments which revealed the FCD is located in a genomic region shared by Old World monkeys (humans and Baboons) with a delayed fetal to adult globin switch that occurs after birth. By contrast, the FCD region is absent in New World monkeys where the switch occurs during the fetal developmental stage. These findings support involvement of the FCD region in γ-globin regulation during hemoglobin switching.Subsequent studies were performed to define the functional role of the FCD during erythropoiesis. ENCODE long RNA-sequencing assay detected PolyA RNA species throughout the FCD region in K562 cells. Based on these findings, we designed two small-interference RNA molecules targeting the core FCD (siFCD). Both siFCD molecules were transfected into KU812 cells and individual γ-globin gene expression measured using RT-qPCR with gene-specific primers. The level of Gγ-globin mRNA increased by 64%, while Aγ-globin transcription was reduced 38%. These results suggest the FCD facilitates differential regulation of the γ-globin genes. To further characterize FCD function, a series of γ-globin promoter luciferase reporters were constructed with the sense or antisense 500-bp core FCD region, cloned downstream of the luciferase gene. In KU812 stable cell lines, the sense FCD construct enhanced Aγ-globin promoter activity 10-fold, whereas the antisense construct repressed promoter activity. By contrast, the FCD in both orientations silenced Gγ-globin promoter activity. These results support unidirectional gene-specific enhancer properties of the FCD. Subsequent pulldown assay with a 34-bp biotinylated FCD probe confirmed NFE2, BHLHE40 and cMyc binding, which was abolished by mutation of the E-box sequence (CACGTG) in the FCD core.To build on the KU812 cell data, we performed μChIP assay using primary erythroid progenitors generated from adult CD34+ stem cells (Zhu et al., Haematologica 2017). In our system, the γ-globin to β-globin gene switch occurred on day 4 in culture. Initially, we measured levels of acetylated histone H3 (AcH3), H3K4Me1, and TFIID binding in the β-globin locus control region hypersensitivity site 2 (HS2) and FCD. As expected, high AcH3 and H3K4Me1 marks occurred in HS2 consistent with its enhancer function. Similar patterns of histone marks and binding of TFIID, NFE2 and BHLHE40 occurred in HS2 and FCD region. It is known that HS2 participates in DNA looping to accomplish developmentally regulated globin gene expression during hemoglobin switching therefore chromosome conformation capture (3C) assay was performed in primary erythroid progenitors to determine if the FCD facilitates changes in chromatin structure. We observed strong long-range interactions between the FCD and HS2 with 4.4-fold maximal chromatin enrichment on day 5; likewise the FCD showed interactions with Aγ-globin (4.7-fold) and Gγ-globin (3.5-fold) by day 3 before switching occurred. Of note, low level interactions occurred between the FCD and β-globin gene at all time points tested.Collectively, these data support the FCD as a unidirectional enhancer element involved in γ-globin regulation during erythropoiesis. To further define mechanisms of FCD function, the effects of CrisprCas9-mediated deletions in the FCD on globin gene expression will be explored. DisclosuresNo relevant conflicts of interest to declare.

P.727 Early change in working-memory related dorsal prefrontal activity and improved executive functions following Action-Based Cognitive Remediation in remitted bipolar disorder

The BCL2L12, one of the latest discovered members of the BCL2 family, has both pro- and anti-apoptotic roles that are cell-type-dependent. Its role in tumorigenesis is highly implicated. Sixty-three splice variants of this gene have been identified so far, with significant differences in expression patterns between various cancer cell lines. Presently, little is known regarding the regulation of expression of the BCL2L12 gene. For the vast majority of BCL2L12 gene splice variants, the 5′- and 3′-untranslated regions as well as their transcriptional regulation have not been determined yet. The aim of this study was to get insight into the regulation of the BCL2L12 gene transcription in human chronic myelogenous leukemia (K562) cell line. Our results point to the activity of novel transcription start site of the BCL2L12 gene and indicate that Sp1 and GATA-1 transcription factors could be involved in the regulation of BCL2L12 gene expression in K562 cells. The previously reported active promoter of BCL2L12 gene differs from the one we described in our study. If this novel BCL2L12 promoter is confirmed to be active in other malignancies, transcripts generated from this region could be considered as new cancer-specific biomarkers. The results of our study contribute to the better understanding of the transcriptional regulation of the BCL2L12 gene.

Complex transcriptional regulation of the BCL2L12 gene: Novel, active promoter in K562 cells.

The BCL2L12, one of the latest discovered members of the BCL2 family, has both pro- and anti-apoptotic roles that are cell-type-dependent. Its role in tumorigenesis is highly implicated. Sixty-three splice variants of this gene have been identified so far, with significant differences in expression patterns between various cancer cell lines. Presently, little is known regarding the regulation of expression of the BCL2L12 gene. For the vast majority of BCL2L12 gene splice variants, the 5'- and 3'-untranslated regions as well as their transcriptional regulation have not been determined yet. The aim of this study was to get insight into the regulation of the BCL2L12 gene transcription in human chronic myelogenous leukemia (K562) cell line. Our results point to the activity of novel transcription start site of the BCL2L12 gene and indicate that Sp1 and GATA-1 transcription factors could be involved in the regulation of BCL2L12 gene expression in K562 cells. The previously reported active promoter of BCL2L12 gene differs from the one we described in our study. If this novel BCL2L12 promoter is confirmed to be active in other malignancies, transcripts generated from this region could be considered as new cancer-specific biomarkers. The results of our study contribute to the better understanding of the transcriptional regulation of the BCL2L12 gene.

Inhibitory Effect of NPM Gene Knockdown on Proliferation of Chronic Myeloid Leukemia Cell Line K562 and Its Mechanism

To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology. shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins. The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G0/G1 phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex. Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.

Co-activation of WT1 and AP-1 proteins on WT1 gene promoter to induce WT1 gene expression in K562 cells

Earlier studies have revealed one function of the inhibitory mechanism of curcumin. Activating PKCα induces WT1 gene expression via signalling through downstream JNK and c-JUN. In the present study, the effect of c-JUN/AP-1 binding and transcriptional regulation of the WT1 gene promoter was investigated in K562 leukaemic cells. The non-cytotoxic dose (IC20 values) of curcumin (WT1 and AP-1 inhibitors) was employed to examine its effect on WT1 gene-mediated WT1 and AP-1 protein expression. Non-cytotoxic doses of both tanshinone IIA (AP-1 DNA-binding inhibitor) and SP600125 (JNK inhibitor) were used to test the role of c-JUN/AP-1 in WT1 gene expression. Curcumin, tanshinone IIA, and SP600125 inhibited WT1 protein expression in a dose-dependent manner (5–15 μM) at 24 h as shown by immunoblotting. A ChIP assay showed that curcumin and tanshinone IIA inhibited AP-1 and WT1 binding to the proximal WT1 promoter (−301 bp), and a luciferase reporter assay showed that the WT1 luciferase gene reporter activity was decreased after curcumin, tanshinone IIA, and SP600126 treatments. Furthermore, depletion of c-JUN abrogated WT1 gene expression. In summary, AP-1 contributes to the WT1 autoregulation of WT1 gene expression in leukaemic K562 cells.

ShRNA targeting of ferritin heavy chain activates H19/miR-675 axis in K562 cells

PurposeThe heavy subunit of the iron storage protein ferritin (FHC) is essential for the intracellular iron metabolism and, at the same time, it represents a central hub of iron-independent pathways, such as cell proliferation, angiogenesis, p53 regulation, chemokine signalling, stem cell expansion, miRNAs expression. In this work we have explored the ability of FHC to modulate gene expression in K562 cells, through the up-regulation of the lncRNA H19 and its cognate miR-675. Materials and methodsTargeted silencing of FHC was performed by lentiviral-driven shRNA strategy. FHC reconstitution was obtained by full length FHC cDNA transfection with Lipofectamine 2000. ROS amounts were determined with the redox-sensitive probe H2DCFDA. H19, miR-675, miR-107, Twist1, ID3, EPHB6, GNS, ANK1 and SMAD6 mRNA amounts were quantified by Taqman assay and qPCR analysis. ResultsFHC silencing in K562 cells modulates gene expression through the up-regulation of the lncRNA H19 and its cognate miR-675. Experimental findings demonstrate that the molecular mechanism underlying this phenomenon is represented by an FHC knock-down-triggered increase in reactive oxygen species (ROS) production. ConclusionsIn this paper we uncover a so far not described function of the ferritin heavy subunit in the control of lncRNA pathways.

Functional Characterization of a Dnase I Hypersensitive Site Located 4Kbp Upstream of the Gγ-Globin Gene Using a Synthetic Zinc Finger DNA-Binding Domain

The human β-globin genes are expressed in a developmental stage-specific manner and regulated by many cis- and trans-regulatory components including a locus control region (LCR) and proximal promoter and enhancer elements. The two γ-globin genes, Gγ and Aγ, are expressed in the fetal period. Persistent expression of γ-globin in the adult ameliorates the symptoms associated with mutations of the adult β-globin gene, such as sickle cell disease or β-thalassemia. Thus, understanding the mechanisms by which the fetal globin genes are activated and silenced during development may lead to new avenues for the treatment of hemoglobinopathies. Using data from the human ENCODE project we identified a DNase I hypersensitive site located 4 Kbp upstream of the Gγ-globin gene (Gγ -4Kb DHS) in K562 cell line. Gγ -4Kb DHS is characterized by the presence of histone modifications typical for enhancer elements (H3K4 monomethylation and H3K27 acetylation) and binding of ubiquitous (USF, E2F, YY1, c-Myc, Egr1, and MafK) or tissue-restricted (NF-E2) transcription factors in K562 cells which express high levels of the γ-globin genes (Figure 1). The presence of USF and NF-E2 is interesting as both proteins have been implicated in the recruitment of transcription complexes to the β-globin gene locus (Crusselle-Davis et al., 2006, Mol. Cell. Biol.; Liang et al., 2009 Mol. Cell. Biol.; Zhou et al., 2010, J. Biol. Chem.; Stee & Hossain et al., 2015, Mol. Cell. Biol.).We generated and expressed in K562 cells a synthetic Zinc Finger DNA-Binding Domain (ZF-DBD) designed to specifically target the Gγ -4Kb DHS and interfere its activities (ZF@Gγ-4KbDHS). The target site for the ZF-DBD overlaps with a CCCAC Egr1 motif and is close to an E-box sequence, which is predicted to bind USF and c-Myc (Figure 1). Figure 2A and C shows the Western blot results for cells transfected with empty vector or cells expressing the ZF@Gγ-4KbDHS in cell pools or a single cell clone selected from the pool of transfected cells (Figure 2A and C, respectively). We analyzed the binding of ZF@Gγ-4KbDHS at the globin locus in K562 cells using Chromatin Immunoprecipitation (ChIP). The data demonstrate that the ZF@Gγ-4KbDHS efficiently interacted with the Gγ -4Kb DHS and less efficiently with the γ-globin promoters (Figure 2B). Expression of the ZF@Gγ-4KbDHS led to a significant reduction in expression of the γ-globin genes but had no effect on expression of the GATA-1 gene (Figure 2D).The data suggest that the Gγ -4Kb DHS contributes to high-level γ-globin gene expression in K562 cells. Additionally, a SNP (rs11036496, Figure 1) within the Gγ -4Kb DHS has been reported to be associated with a disorder of γ-globin gene expression in African Americans and Chromatin Conformation Capture (3C) experiments showed that the Gγ-globin upstream region participates in interactions with the LCR and γ-globin genes (Shriner et al., 2015, BMC Medical Genetics; Kiefer et al., 2011, Blood). Therefore, further characterization of the Gγ -4Kb DHS will enhance understanding molecular mechanism(s) regulating hemoglobin switching. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Erfe-Encoding FAM132B in Congenital Dyserythropoietic Anemia Type II

Recessive mutations in SEC23B gene cause congenital dyserythropoietic anemia type II (CDAII), a rare hereditary disorder hallmarked by ineffective erythropoiesis, iron overload, and reduced expression of hepatic hormone hepcidin (Iolascon, 2013). The most recently described hepcidin regulator is the erythroblast-derived hormone erythroferrone (ERFE), a member of TNF-α superfamily that specifically inhibits hepcidin production in experimental models (Kautz, 2014). However, the function of ERFE in humans remains to be investigated.To determine whether dysregulation of ERFE expression is associated with ineffective erythropoiesis and iron-loading in CDAII, we studied the ERFE-encoding FAM132B gene expression in 48 SEC23B-related CDAII patients and 29 age and gender matched healthy controls (HCs). Twelve new cases and four novel SEC23B mutations were described. Samples were obtained after informed consent, according to the Declaration of Helsinki. Genomic DNA, mutational screening, RNA isolation, cDNA preparation, and qRT-PCR were performed as previously described (Russo, 2013).All patients were young adults (17.0±2.5 years at diagnosis), with increased serum ferritin (395.4±67.6 ng/mL) and transferrin saturation (71.9±5.4 %). We observed a statistically significant overexpression of FAM132B gene in peripheral blood mononuclear cells from CDAII patients (9.09±0.08) compared to HCs (8.32±0.12, p<0.0001). A similar trend was obtained when evaluating FAM132B expression in reticulocytes from a subset of patients and HCs. Of note, a statistically significant correlation between peripheral blood and reticulocyte FAM132B expression from the same patients was observed (Spearman ρ= 0.78, p=0.02). Although the role of ERFE in peripheral blood is still unknown, our observations suggested that the evaluation of FAM132B mRNA in peripheral blood is a reliable and easy-to-measure marker of ERFE levels.When we divided CDAII patients into two sub-groups accordingly to FAM132B gene expression, we observed a statistically significant reduction in hemoglobin (Hb) level in the high-FAM132B subset (8.6±0.4 g/dL) respect to low-FAM132B one (10.1±0.5 g/dL, p=0.02). Of note, the expression level of FAM132B did not correlate with the transfusion regimen. The higher amount of ERFE reflects the increased iron demand for Hb production as well as the expanding abnormal erythropoiesis, as attested by the increased RDW and sTfR (although not significant) in high-FAM132B patients. This in turn leads to reduced hepcidin in high-FAM132B group (4.2±1.8 nM) compared to low-FAM132B one (5.9±1.8 nM, p=0.05), resulting in augmented iron delivery to the erythron. Although the iron balance data do not differ significantly between the two groups, a tendency to decreased hepcidin/ferritin ratio and increased transferrin saturation was observed in high-FAM132B patients. Thus, FAM132B overexpression seems to contribute to the inappropriate suppression of hepcidin with subsequent hemosiderosis observed in CDAII.Consistent with our previous studies, we observed a reduced SEC23B expression in our patients compared to HC. Indeed, FAM132B and SEC23B gene expression exhibited an inverse correlation (Spearman ρ=-0.36, p=0.01). We confirmed the ex vivo data about inverse correlation between FAM132B and SEC23B expression observed in our patients by establishing K562 SEC23B-silenced cells. To knockdown SEC23B gene expression in K562 cells two different pGIPZ Lentiviral shRNAmir for SEC23B (shSEC23B-70/-74) were used. We observed a higher expression of FAM132B at 5 days of erythroid differentiation in K562 SEC23B-silenced cell compared to not-silenced ones. Conversely, SEC23B expression was lower in both shSEC23B compared to sh-CTR at 2 and 5 days of differentiation. Although the mechanisms of hemin-induced differentiation are quite different from EPO-induced ones, we can hypothesize that FAM132B over-expression is related to the maturative arrest and the subsequent increased number of erythroid precursors.This study provides the first analysis on ERFE regulation in humans. Our data suggest that ERFE over-expression in CDAII patients is the result of both physiological and pathological mechanisms leading to hepcidin suppression in condition of dyserythropoiesis. Nevertheless, it seems that ERFE cannot be the main erythroid regulator of hepcidin suppression, at least in CDAII patients. DisclosuresNo relevant conflicts of interest to declare.

Functional Analysis of an Aγ-Globin Gene Promoter Variant (HBG1: g.-225_-222delAGCA) Underlines Its Role in Increasing Fetal Hemoglobin Levels Under Erythropoietic Stress

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by persistent γ-globin gene expression and synthesis of high levels of fetal hemoglobin (Hb F; α2γ2) during adult life. It is usually caused by promoter variants or large deletions affecting the human fetal globin (HBG1 and HBG2) genes. Some of these HPFH-causing variants, such as HBG2: g.-158 C > T, exert their effect only under conditions of erythropoietic stress, typical for β-thalassemia (β-thal) patients. Namely, the presence of HBG2: g.-158 C > T favors a higher Hb F response, while it has little effect in healthy individuals. We analyzed a previously reported deletion residing in the promoter region of the HBG1 gene (HBG1: g.-225_-222delAGCA), both in normal conditions and under conditions of erythropoietic stress. Our results indicate that this deletion is responsible for decreased HBG1 gene expression. Specifically, this deletion was shown to result in drastically reduced reporter gene expression in K562 cells, compared to the wild-type sequence but only under conditions of erythropoietic stress, mimicked by introduction of erythropoietin (EPO) into the cell culture. Also, electrophoretic mobility shift analysis showed that the HBG1: g.-225_-222delAGCA deletion creates additional transcriptional factors’ binding sites, which, we propose, bind a transcriptional repressor, thus decreasing the HBG1 gene promoter activity. These results are consistent with in silico analysis, which indicated that this deletion creates a binding site for GATA1, known to be a repressor of the γ-globin gene expression. These data confirm the regulatory role of the HBG1: g.-225_-222 region that exerts its effect under conditions of erythropoietic stress characteristic for β-thal patients.

H-ferritin-regulated microRNAs modulate gene expression in K562 cells.

In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC) comparing it with K562 transduced with scrambled RNA (K562shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.

An Unexpected Role for Ribonuclease Inhibitor (RNH1) in Erythropoiesis

Ribonuclease Inhibitor (RNH1) is a ubiquitously expressed leucine-rich repeat protein. The human RNH1 gene evolved via gene duplication and is conserved among mammalian species. RNH1 binds to and inhibits pancreatic type ribonucleases. Further, RNH1 contains numerous cysteine residues whose sulfhydryl groups might play key structural roles and protect from oxidative damage (Dickson et al Prog. Nucleic Acid Res. Mol. Biol 2005). Despite of all these observations, the precise biological role of RNH1in vivo remains unexplored. Here, we describe an essential role for Rnh1 in the regulation of erythropoiesis by controlling erythroid differentiation.To understand the biological function of Rnh1, Rnh1-deficient (Rnh1-/-) mice were generated. Rnh1-/- embryos die between embryonic days E8.5 to E10 due to severe decrease in erythroid cells. Similar percentages of c-Kit+CD41+ cells (Hematopoietic stem/progenitor cells) were present in Rnh1-/- yolk sacs compared to control genotypes, however differentiation of mature erythroid cells was impaired. Rnh1 is expressed in erythroid cells and its expression coincides with the site of primitive erythropoiesis in the yolk sac. Gene expression studies revealed that levels of hematopoietic transcription factors (TF) in Rnh1-deficient yolk sacs were normal, but their target genes were down-regulated. These results indicate that a post-transcriptional mechanism that affects TF gene function. Supporting this, protein levels of the erythroid transcription factor GATA1 and PPARγ, previously shown to control the proliferation and differentiation of erythroid progenitors, were selectively impaired. Whereas myeloid transcription factors C/EBPa and C/EBPb were not affected in Rnh1-/- embryos, suggesting that Rnh1 deficiency specifically affects the translation of erythroid transcription factors.At the molecular level, using the human erythroid K562 cell line, we show that RNH1 is recruited to the ribosome complex and binds to the ribosomal proteins. RNH1-deficiency decreased polysome formation and conversely its overexpression increased polysome formation. Increased expression of RNH1 also increased globin gene expression in K562 cells. These results suggest that RNH1 associates with ribosomes and regulates the translation of erythroid-specific genes, which are necessary for erythroid differentiation. Furthermore, Rnh1 haploinsufficiency leads to decreased erythropoiesis in the spleen of adult mice.Ribosomal haploinsufficiency in several ribosomal genes is known to impair ribosome function and cause macrocytic anemia in Diamond–Blackfan anemia (DBA), a congenital bone marrow failure syndrome, and the 5q- syndrome, a subtype of myelodysplastic syndrome (Narla et al Int. J. Hematol 2011). Recently it has been shown that ribosomal haploinsufficiency can specifically cause a decrease in GATA1 mRNA translation (Ludwig et al Nature Med 2014). Similar to these ribosomal genes, we demonstrate that Rnh1 associates with ribosomes and its deficiency impairs the translation of Gata1 and other erythroid-specific transcription factors, which leads to arrest in erythroid maturation. Collectively our results unravel the important biological function of Rnh1 in the regulation of erythropoiesis, and point to novel therapeutic targets for disorders of erythropoiesis involving ribosomal defects. [Display omitted] Summary Figure:RNH1 is recruited to ribosomal complex and is involved in translation of erythroid specific transcription factors (TF) e.g.GATA1. These TFs are necessary for differentiation of progenitor cells in to erythroid cells. RNH1 deficiency impairs the translation of GATA1 and other erythroid-specific transcription factors, which leads to arrest in erythroid maturation. DisclosuresNo relevant conflicts of interest to declare.

Arsenic Trioxide Inhibits DNA Methyltransferase and Restores TMS1 Gene Expression in K562 Cells

Background: Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor genes in human cancers. The demethylating, dose-dependent effect of arsenic trioxide (As₂O₃) on several tumor-related genes has already been postulated. However, whether such a demethylating effect also applies to the TMS1 gene in chronic myeloid leukemia cell line K562 cells has not been studied so far. The aim of the present study was to detect the methylation status of the TMS1 gene in K562 cells and the demethylation effect of As₂O₃ on TMS1 as well as TMS1 apoptosis-associated protein Bcl-2/Bax and DNA methyltransferase (DNMT) expression. Methods: TMS1 mRNA expression in K562 cells and normal bone marrow was determined by reverse transcription (RT) polymerase chain reaction (PCR), and the DNA methylation status of the TMS1 promoter in K562 cells treated with different concentrations of As₂O₃ for 48 h was determined by methylation-specific PCR. RT-PCR and Western blot were used to detect TMS1 and DNMT expression. We also assessed TMS1-associated apoptosis protein Bcl-2/Bax expression by Western blot and apoptosis rates by flow cytometry using annexin V/propidium iodide double staining. Results: In K562 cells, TMS1 was completely methylated and both TMS1 mRNA and protein showed a low expression, but 2 μmol/l As₂O₃ could significantly restore the expression of the TMS1 gene both at mRNA and protein level (p < 0.01) by fully reversing DNA methylation. As₂O₃ decreased mRNA and protein expression of DNMT1 (p < 0.05) in a dose-dependent manner. Flow cytometry showed that in the experimental group (2 μmol/l As₂O₃), cell apoptosis was significantly increased compared with the control group (no As₂O₃; p < 0.05). In the experimental group, Western blot showed that the expression of the anti-apoptotic protein Bcl-2 was significantly decreased; however, the proapoptotic protein Bax was markedly increased and the Bcl-2/Bax ratio was markedly reduced (p < 0.01). Conclusions: As₂O₃ could restore the expression of TMS1 by inhibiting DNMT to reverse the hypermethylation and induced apoptosis of K562 cells by downregulation of Bcl-2/Bax expression.

Effect of arsenic trioxide and 5-aza-2'-deoxycytidine on SHP-1, JAK3, TYK2 gene expression in K562 cells

This study was purposed to explore the effects of a methylation inhibitor arsenic trioxide (As2O3, ATO) and 5-Aza-2'-deoxycytidine (5-aza-CdR) on the expression of JAK-STAT signal transduction pathway in family members JAK3, TYK2 and hematopoietic cell phosphatase SHP-1 in chronic myeloid leukemia cell line K562 and their roles in pathogenesis of leukemia. The K562 cells were divided into 3 groups:single drug-treated group, combined 2 drugs-treated group, group without drug treatment as control. The concentration of 5-aza-CdR were 0.5, 1, 2 µmol/L; the concentration of ATO was 1, 2.5, 5 µmol/L; the concentration of combined drugs was ATO 1 µmol/L + 5-aza-CdR 0.5 µmol/L, ATO 2.5 µmol/L + 5-aza-CdR 1 µmol/L, and ATO 5 µmol/L + 5-aza-CdR 2 µmol/L. The K562 cells were treated with above-mentioned concentration of drugs for 24, 48 and 72 hours, then the total RNA of cells was extracted, the JAK3, TYK2 and SHP-1 expressions were detected by real-time quantitative-PCR. The results showed that after the K562 cells were treated with ATO and 5-aza-CdR alone and their combination, the expression of SHP-1 mRNA increased, the expressions of JAK3 mRNA and TYK2 mRNA decreased along with increasing of concentration and prolonging of time, displaying the concentration and time-dependency. The SHP-1 negatively related with JAK3 and TYK2. The effect of SHP-1 on JAK3 was significantly higher than that on TYK2. It is concluded that when the K562 cells are treated with ATO and 5-aza-CdR alone and their combination, the expression of SHP-1 is up-regulated and the expressions of JAK3, TYK2 are down-regulated in concentration-and time-dependent manners, moreover the ATO and 5-aza-CdR show synergies demethylation effect. The SHP-1 gene exert effect possibly through inhibiting the JAK/STAT pathway, the JAK3 is affected more than TYK2, the JAK3 may exert more important role in TAK/STAT pathway.

A Feedback Loop Consisting of MicroRNA 23a/27a and the β-Like Globin Suppressors KLF3 and SP1 Regulates Globin Gene Expression

The developmental stage-specific expression of the human β-like globin genes has been studied for decades, and many transcriptional factors as well as other important cis elements have been identified. However, little is known about the microRNAs that potentially regulate β-like globin gene expression directly or indirectly during erythropoiesis. In this study, we show that microRNA 23a (miR-23a) and miR-27a promote β-like globin gene expression in K562 cells and primary erythroid cells through targeting of the transcription factors KLF3 and SP1. Intriguingly, miR-23a and miR-27a further enhance the transcription of β-like globin genes through repression of KLF3 and SP1 binding to the β-like globin gene locus during erythroid differentiation. Moreover, KLF3 can bind to the promoter of the miR-23a∼27a∼24-2 cluster and suppress this microRNA cluster expression. Hence, a positive feedback loop comprised of KLF3 and miR-23a promotes the expression of β-like globin genes and the miR-23a∼27a∼24-2 cluster during erythropoiesis.

Effect of proteasome inhibitor bortezomib on proliferation, apoptosis and SHIP gene expression in K562 cells

This study was aimed to investigate the effects of proteasome inhibitor bortezomib on proliferation, apoptosis and the SHIP expression of K562 cells. K562 cells were treated with bortezomib of different concentrations. Cell proliferation was analyzed by MTT assay, cell apoptosis was detected by flow cytometry and SHIP mRNA expression was assayed by RT-PCR.The results showed that after being treated with 10, 20, 50 and 100 nmol/L bortezomib for 24 h, the inhibitory rates of K562 cells were (5.76 ± 1.47)%, (10.55 ± 1.59)%, (17.14 ± 2.05)% and (27.69 ± 3.57)% respectively, and were higher than that in control (1.30 ± 0.10); when K562 cells were treated with 20 nmol/L bortezomib for 24, 48 and 72 h, the inhibitory rates of cell proliferation were (10.55 ± 1.59)%, (16.33 ± 2.53)% and (19.78 ± 1.56)% respectively, there was statistic difference of cell proliferation rate between 24 h group and 48 h group (P < 0.05). After being treated with 10,20,50,100 nmol/L bortezomib for 24 h, the apoptotic rates of K562 cells were (12.7 ± 0.6)%, (26.9 ± 0.9)%, (32.6 ± 1.2)% and (72.5 ± 1.5)% respectively,and all higher than that in control (1.0 ± 0.5)% (P < 0.05). According to results of RT-PCR detection, the expression level of SHIP mRNA was obviously up-regulated after treatment with bortezomib, and showed statistical difference in comparison with control. It is concluded that bortezomib inhibits proliferation of K562 cells in time and concentration-dependent manner and induces apoptosis through up-regulation of SHIP gene.

Functional analysis of a novel KLF1 gene promoter variation associated with hereditary persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) is a rare hereditary condition resulting in elevated levels of fetal hemoglobin (HbF) in adults. Typical HPFH is associated with promoter mutations or large deletions affecting the human fetal globin (HBG1 and HBG2) genes, while genetic defects in other genes involved in human erythropoiesis, e.g. KLF1, also result in atypical HPFH. Here, we report the first KLF1 gene promoter mutation (KLF1:g.-148G > A) that is associated with increased HbF level. This mutation was shown to result in drastically reduced CAT reporter gene expression in K562 cells, compared to the wild-type sequence (p = 0.009) and also in reduced KLF1 gene expression in vivo. Furthermore, consistent with in silico analysis, electrophoretic mobility shift analysis showed that the KLF1:g.-148G > A mutation resides in a Sp1 binding site and further that this mutation leads to the ablation of Sp1 binding in vitro. These data suggest that the KLF1:g-148G > A mutation could play a role in increasing HbF levels in adults and further underlines the role of KLF1 as one of the key transcription factors involved in human fetal globin gene switching.

PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells.

AbstractAbstract 2393Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures:No relevant conflicts of interest to declare.

Analyzing Gene Expression Profile in K562 Cells Exposed to Sodium Valproate Using Microarray Combined with the Connectivity Map Database

To explore the mechanism underlying antileukaemia effect of sodium valproate, the growth and survival of the K562 cell line were investigated. Global profiles of gene expression in K562 cells exposed to sodium valproate were assessed and validated. The differentially expressed genes identified were further used to query the connectivity map database to retrieve a ranked list of compounds that act on the same intracellular targets as sodium valproate. A significant increase in cell apoptosis and a change in gene expression profile were observed in valproate-exposed K562 cells. The significant enrichment analysis of gene ontology terms for the differentially expressed genes showed that these genes were involved in many important biological processes. Eight differentially expressed genes involved in apoptosis were verified by quantitative real-time PCR. The connectivity map analysis showed gene expression profile in K562 cells exposed to sodium valproate was most similar to that of HDACi and PI3K inhibitors, suggesting that sodium valproate might exert antileukaemic action by inhibiting HDAC as well as inhibiting PI3K pathway. In conclusion, our data might provide clues to elucidate the molecular and therapeutic potential of VPA in leukaemia treatment, and the connectivity map is a useful tool for exploring the molecular mechanism of drug action.

Histone Deacetylase 9 Activates γ-Globin Gene Expression in Primary Erythroid Cells

Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies probably involve protein modifications by histone deacetylases (HDACs) that mediate γ-globin gene regulation. However, the role of individual HDACs in globin gene expression is not very well understood; thus, the focus of our study was to identify HDACs involved in γ-globin activation. K562 erythroleukemia cells treated with the HbF inducers hemin, trichostatin A, and sodium butyrate had significantly reduced mRNA levels of HDAC9 and its splice variant histone deacetylase-related protein. Subsequently, HDAC9 gene knockdown produced dose-dependent γ-globin gene silencing over an 80-320 nm range. Enforced expression with the pTarget-HDAC9 vector produced a dose-dependent 2.5-fold increase in γ-globin mRNA (p < 0.05). Furthermore, ChIP assays showed HDAC9 binding in vivo in the upstream Gγ-globin gene promoter region. To determine the physiological relevance of these findings, human primary erythroid progenitors were treated with HDAC9 siRNA; we observed 40 and 60% γ-globin gene silencing in day 11 (early) and day 28 (late) progenitors. Moreover, enforced HDAC9 expression increased γ-globin mRNA levels by 2.5-fold with a simultaneous 7-fold increase in HbF. Collectively, these data support a positive role for HDAC9 in γ-globin gene regulation.