Expression In K562 Cells Research Articles

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.

ZNF16 (HZF1) promotes erythropoiesis and megakaryocytopoiesis via regulation of the c-KIT gene

We previously characterized the zinc finger protein gene HZF1 [also known as ZNF16 (zinc finger protein 16)] and demonstrated its important roles in erythroid and megakaryocytic differentiation of K562 cells. In the present study, we investigated its effect on erythroid and megakaryocytic differentiation of HSPCs (haemopoietic stem/progenitor cells). We observed up-regulation of ZNF16 during erythroid and megakaryocytic differentiation of the CD34+ HSPCs, and demonstrated that ZNF16 promotes erythroid and megakaryocytic differentiation by gain-of-function and loss-of-function experiments. Using a luciferase reporter and ChIP assays ZNF16 was demonstrated to bind to the c-KIT gene promoter and inhibit its expression in K562 cells. Enforced expression and knockdown of ZNF16 down-regulated and up-regulated the expression of the c-KIT gene in K562 cells and HSPCs respectively. Significantly decreased levels of the c-Kit protein were observed following erythroid and megakaryocytic differentiation of K562 and CD34+ cells. The knockdown of c-KIT partially rescued the differentiation inhibition caused by ZNF16 knockdown. The knockdown of c-KIT also blocked the activity of the c-Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK/c-Jun signal pathway and reduced further the level of HEY1 (hes-related family bHLH transcription factor with YRPW motif 1), a repressor of GATA1 (GATA-binding protein 1) transcription, which finally up-regulated the expression of GATA1, a central regulator of erythroid and megakaryocytic differentiation. In conclusion the results of the present study demonstrate that ZNF16 plays an important role in erythropoiesis and megakaryocytopoiesis via its regulation of the c-Kit/c-Raf/MEK/ERK/c-Jun/HEY1/GATA1 cascade.

Regulation of Gγ-Globin Gene by ATF2 and Its Associated Proteins through the cAMP-Response Element

The upstream Gγ-globin cAMP-response element (G-CRE) plays an important role in regulating Gγ-globin expression through binding of ATF2 and its DNA-binding partners defined in this study. ATF2 knockdown resulted in a significant reduction of γ-globin expression accompanied by decreased ATF2 binding to the G-CRE. By contrast, stable ATF2 expression in K562 cells increased γ-globin transcription which was reduced by ATF2 knockdown. Moreover, a similar effect of ATF2 on γ-globin expression was observed in primary erythroid progenitors. To understand the role of ATF2 in γ-globin expression, chromatographically purified G-CRE/ATF2-interacting proteins were subjected to mass spectrometry analysis; major binding partners included CREB1, cJun, Brg1, and histone deacetylases among others. Immunoprecipitation assays demonstrated interaction of these proteins with ATF2 and in vivo GCRE binding in CD34+ cells undergoing erythroid differentiation which was correlated with γ-globin expression during development. These results suggest synergism between developmental stage-specific recruitments of the ATF2 protein complex and expression of γ-globin during erythropoiesis. Microarray studies in K562 cells support ATF2 plays diverse roles in hematopoiesis and chromatin remodeling.

Early Growth Response-1 (EGR-1) and Nuclear Factor of Activated T Cells (NFAT) Cooperate to Mediate CD40L Expression in Megakaryocytes and Platelets

Increasing evidence implicates circulating platelets as mediators of chronic inflammatory and autoimmune diseases via the expression and release of CD40L, an important modulator of inflammation and adaptive immune responses traditionally associated with activated T cells. Emerging evidence suggests that platelet CD40L is dynamically regulated in several chronic inflammatory and autoimmune diseases and may mediate progression and secondary pathology associated with those disease states. The present study identifies NFATc2 as a key transcriptional modulator of CD40L expression in megakaryocytes and inflammatory activity of platelets. Furthermore, the current data show that EGR-1, a member of the early growth response family of zinc finger transcription factors, modulates NFATc2-dependent regulation of CD40L expression in megakaryocytes. Our novel demonstration that in vivo biochemical or genetic inhibition of NFATc2 activity in megakaryocyte diminishes platelet CD40L implicates the NFATc2/EGR-1 axis as a key regulatory pathway of inflammatory and immunomodulatory activity in platelets and represents a target for the development of therapeutics for the potential treatment of chronic inflammatory and autoimmune diseases.

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-l-methionine or its demethylated product S-adenosyl-l-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-l-homocysteine partly explained catechol-enhanced erythroid differentiation.

Enhanced adhesion/migration and induction of Pyk2 expression in K562 cells following imatinib exposure

Concern about extramedullary relapse (EMR) despite favorable response in the bone marrow has been raised with the use of imatinib for treatment of chronic myeloid leukemia (CML). In the present study we show an increase in adhesion, migration and invasion capabilities of the CML cell line K562 following imatinib administration. Imatinib induced upregulation of Pyk2 mRNA and protein levels. Pyk2 inhibition resulted in a reduction of K562 cells' adhesion and migration subsequent to imatinib treatment. This effect was similar to that shown by us previously with all trans retinoic acid (ATRA) in the acute promyelocytic leukemia (APL) cell line NB4. Our data pinpoint Pyk2 as a shared key mediator of targeted-therapy induced adhesion and migration and may implicate that targeting Pyk2 may serve as an effective therapeutic strategy to reduce EMR in APL and CML.

Synonymous nucleotide polymorphisms influence Dombrock blood group protein expression in K562 cells

To gain further insight into ART4 (DO) gene alleles (DO*A, DO*JO1, DO*A-WL, DO*DOYA, DO*B, DO*B-WL, DO*B-SH-Q149K, DO*B-(WL)-I175N, DO*HY1, DO*HY2, DO*DOMR) and evaluate the impact of synonymous nucleotide polymorphisms on protein expression and mRNA accumulation of DO*A-HA, DO*A-SH and DO*B-SH alleles, human erythroleukaemic K562 cells were transducted with variant DO-lentiviral particles and analysed by flow cytometry and quantitative reverse transcription polymerase chain reaction. Monoclonal antibody (MoAb) detection of DO*A-HA and DO*JO1 transductants was lower than DO*A transductants, while detection of DO*A-SH, DO*A-WL and DO*DOYA transductants was higher. Variant DO*B alleles, i.e. DO*B-SH, DO*B-WL, DO*HY1, DO*HY2 and DO*DOMR, showed reduced MoAb binding. The unexpected modifications of protein expression of the DO*A-HA, DO*A-SH and DO*B-SH alleles that differ from the DO*A or DO*B alleles by a single synonymous polymorphism were abolished by reversion, thus implying involvement of these polymorphisms. Depending on the Leu208 codon used, detection level ranged from 1 to 4·14. In the variant alleles resulting from single synonymous polymorphism, mRNA accumulation correlated roughly with MoAbs detection levels, suggesting post-transcriptional regulation. Other than a few reports involving aberrant splicing, the experiments described herein provide the first evidence that synonymous nucleotide polymorphisms can influence Dombrock blood group expression. Such polymorphisms should be taken into account for molecular screening and potential impact on transfusion.

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.

Identification of the C/EBPα C-terminal tail residues involved in the protein interaction with GABP and their potency in myeloid differentiation of K562 cells

The CCAAT/enhancer-binding protein α (C/EBPα) is the member of a family of related basic leucine zipper (bZIP) transcription factors and is critical for granulopoiesis. We previously demonstrated that C/EBPα interacts with the ETS domain of widely expressed GABPα, which leads to cooperative transcriptional activation of the myeloid-specific promoter for human FCAR encoding the Fc receptor for IgA (FcαR, CD89) in part by facilitating recruitment of C/EBPα to the promoter. The C/EBPα molecule contains transactivation domains (TADs) at its N-terminus and a DNA-binding and dimerization bZIP structure at its C-terminus. We demonstrate here that GABPα interacts with the last 18 residues of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerizing region. Deletion of this C-terminus resulted in loss of GABPα interaction but not affecting its DNA binding ability, indicating that it is not required for homodimer formation. Moreover, the C-terminus confers the ability to functionally synergize with GABP on a heterologous TAD when fused to the C-terminus of the VP16 TAD. We identified a three-amino acid stretch (amino acids 341–343) that is important for both functional and protein interactions with GABP. Ectopic expression in K562 cells of C/EBPα mutant incapable of interacting with GABPα does not induce expression of granulocytic differentiation markers including CD15, CD11b, GCSF-R and C/EBPε, and does not inhibit proliferation, whereas wild type does. These results demonstrate the functional importance of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerization region, which may mediate cooperative activation by C/EBPα and GABP of myeloid-specific genes involved in C/EBPα-dependent granulopoiesis.

PPARγ-dependent pathway in the growth-inhibitory effects of K562 cells by carotenoids in combination with rosiglitazone

BackgroundCarotenoids have been found to play roles in the prevention and therapy of some cancers which PPARγ was also discovered to be involved in. The present studies were directed to determine the inhibitory effects of carotenoids in combination with rosiglitazone, a synthetic PPARγ agonist, on K562 cell proliferation and elucidate the contribution of PPARγ-dependent pathway to cell proliferation suppression. MethodsThe effects of carotenoid and rosiglitazone combination on K562 cell proliferation were evaluated by trypan blue dye exclusion assay and MTT assay. When PPARγ has been inhibited by GW9662 and siRNA, cycle-related regulator expression in K562 cells treated with carotenoid and rosiglitazone combination was analyzed by Western blotting. ResultsRosiglitazone inhibited K562 cell proliferation and augmented the inhibitory effects of carotenoids on the cell proliferation greatly. Specific PPARγ inhibition attenuated the cell growth suppression induced by carotenoid and rosiglitazone combination. GW9662 pre-treatment attenuated the enhanced up-regulation of PPARγ expression caused by the combination treatment. Moreover, GW9662 and PPARγ siRNA also significantly attenuated the up-regulation of p21 and down-regulation of cyclin D1 caused by carotenoids and rosiglitazone. ConclusionsPPARγ signaling pathway, via stimulating p21 and inhibiting cyclin D1, may play an important role in the anti-proliferative effects of carotenoid and rosiglitazone combination on K562 cells. General significanceCarotenoids in combination with rosiglitazone are hopeful to provide attractive dietary or supplementation-based and pharmaceutical strategies to treat cancer diseases.

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.

Inhibitory effect of all-trans retinoic acid combined with SBA-Na on K562 and Kasumi-1 cell lines in vitro

This study was aimed to investigate the effect of all-trans retinoic acid (ATRA) combined with SBA-Na on the biologic activities of human leukemia K562 and Kasumi-1 cell lines and their mechanism. The ATRA solution of 10(-6) mol/L (W1), 10(-4) mol/L (W2) and the SBA-Na solution of 100 µg/ ml (Z1) and 200 µg/ml (Z2) were prepared respectively. The K562 and Kasumi-1 cells were treated with W1, W2, Z1, Z2, W1 + Z1 and W2 + Z2 respectively, at same time, the blank control was set up. The cell morphology and growth in different treated groups were observed under light microscope. The CCK-8 method was used to detect the proliferation ability of cells, the cell growth curves were drawn, the inhibitory rate of cells was calculated. The flow cytometry with PI single staining and PI/Annexin V double stainings was used to detect the change of cell cycle and apoptosis of 2 cell lines treated with different drugs. The RQ-PCR was used to detect the change of Cyclin A mRNA expression in K562 cells. The results showed both ATRA and SBA-Na displayed inhibitory effect on cell proliferation, and the combination of these two drugs had stronger effect. As compared with the control group, the cell cycle distribution were changed obviously, and the apoptosis increased more significantly in treated groups, especially in group of ATRA combined with SBA-Na. The Cyclin A mRNA expression was up-regulated in Z1 group, while Cyclin A mRNA expression was down-regulated in other groups. It is concluded that both ATRA and SBA-Na can inhibit the proliferation of K562 and Kasumi-1 cell lines and promote their apoptosis. This effect may be stronger when both drugs combined. For K562 cells, the inhibitory effect may be accomplished through down-regulation of Cyclin A mRNA.

The role of intracellular pathways in the proliferation of human K562 cells mediated by muscarinic receptors

Muscarinic acetylcholine receptors (mAChRs) are members of the superfamily of G protein coupled receptors (GPCRs). Muscarinic receptors are relatively abundant in the central nervous system and in the peripheral parasympathetic nervous system. Several studies have suggested that muscarinic receptors also mediate some cellular events in hematopoietic cells. K562 erythroleukemia cells contain muscarinic receptors M2, M3 and M4, and activation of muscarinic receptors changes cell proliferation. We examined the effects of several compounds on cell proliferation in K562 erythroleukemia cells. These included a muscarinic receptor agonist carbachol (CCh), a protein kinase inhibitor staurosporine; the phospholipase C inhibitor U73122, the MEK 1-2 inhibitor UO126, the PI3-kinase inhibitor wortmannin, the Ca(2+) chelators BAPTA/AM and 2-aminoethoxy-diphenylborate (2APB). In addition, we also investigated muscarinic receptor mediated protein kinase C (PKC) expression in K562 cells. CCh caused a decrease in DNA synthesis in K562 cells supplemented with 1% fetal bovine serum after starvation. Pre-treatment of K562 cells with U73122 and BAPTA/AM antagonized the inhibitory effect of CCh, suggesting that phospholipase C and intracellular calcium are involved in CCh-mediated inhibition of proliferation in K562 cells. Our data also suggest that the regulatory roles of protein kinase C and the MAPK/ERK pathways in K562 cell proliferation are independent of cholinergic activation.

Inactivation of HDAC3 and STAT3 is Critically Involved in 1-Stearoyl-sn-Glycero-3-Phosphocholine-Induced Apoptosis in Chronic Myelogenous Leukemia K562 Cells

We here investigated the anticancer mechanism of 1-stearoyl-sn-glycero-3-phosphocholine (LPC), one of the lysophosphatidylcholines, in chronic myelogenous leukemia (CML) K562 cells. LPC significantly showed cytotoxicity at 80 μM and induced apoptosis by sub-G1 accumulation, increase in Annexin V positive and caspase activation. LPC enhanced histone H3 acetylation but decreased histone deacetylase (HDAC) activity and HDAC3 expression. LPC also inhibited phosphorylation of STAT3, its DNA binding activity and nuclear co-localization of HDAC3 and STAT3. In addition, LPC effectively attenuated the expression of survival genes such as Cyclin D1, Cyclin E, Bcl-xL, Bcl-2 and survivin but did not affect COX-2 expression in K562 cells. Furthermore, LPC suppressed phosphorylation of Src and Janus activated kinase 2 while promoted the expression of tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1). Consistently, silencing SHP-1 and pervanadate, an inhibitor of protein tyrosine phosphatase, reversed inactivation of HDAC and STAT3, cleavages of caspase 3 and poly (ADP-ribose) polymerase in LPC-induced apoptosis. Of note, chromatin immunoprecipitation assay revealed that LPC suppressed the binding of HDAC3 and STAT3 to Bcl-xL, Bcl-2 and survivin promoter. Overall, our findings indicate that inactivation of STAT3 and HDAC mediates LPC-induced apoptosis in CML K562 cells.

Abstract 1826: A critical role of LincRNA-Up6 in Bcr-Abl-induced tumorigenesis.

Abstract Long noncoding RNAs (lncRNAs) have been implicated in numerous human malignancies, including leukemia. Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) is a stem cell disease induced by Bcr-Abl hybrid gene. In an attempt to provide better understanding of the mechanisms by which Bcr-Abl mediates cellular transformation, lncRNA microarray was used to determine the differentially expressed non protein-coding genes in K562 human leukemic cells in response to disruption of Bcr-Abl expression. All the lncRNA genes whose expressions were altered by at least 2-fold were clustered and displayed. We further investigated the functional involvement of selected lncRNA genes in regulating cell proliferation, cell survival and tumor formation. Interestingly, expression of a long intergenic noncoding RNA which we named as lincRNA-Up6 was found to be greatly up-regulated by silencing the Bcr-Abl expression in K562 cells. We showed that forced over-expression of lincRNA-Up6 sensitized imatinib-induced apoptosis in Bcr-Abl transformed cells. In addition, we observed that over-expression of lincRNA-Up6 significantly inhibited K562 xenograft growth in nude mice. Furthermore, we found that lincRNA-Up6 was involved in negative regulation of STAT5-dependent expression of anti-apoptotic Bcl-XL protein. Importantly, our experiments showed that expression of lincRNA-Up6 had profound effects on signaling pathways associated with development of leukemia stem cells of CML, resulting in inhibition of Bcr-Abl-mediated tumor formation. Together, these results suggest that lincRNA-Up6 suppresses Bcr-Abl-induced tumorigenesis, and the tumor suppressor function of lincRNA-Up6 may be of significance for the development of novel therapeutic strategies for treating CML. Citation Format: Jilong Chen, Guijie Guo, Qingzheng Kang. A critical role of LincRNA-Up6 in Bcr-Abl-induced tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1826. doi:10.1158/1538-7445.AM2013-1826 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The new iodoacetamidobenzofuran derivative TR120 decreases STAT5 expression and induces antitumor effects in imatinib-sensitive and imatinib-resistant BCR–ABL-expressing leukemia cells

The identification of novel compounds modulating the expression/activity of molecular targets downstream to BCR-ABL could be a new approach in the treatment of chronic myeloid leukemias (CMLs) resistant to imatinib or other BCR-ABL-targeted molecules. Recently, we synthesized a new class of substituted 2-(3,4,5-trimethoxybenzoyl)-2-N,N-dimethylamino-benzo[b]furans, and among these 3-iodoacetylamino-6-methoxybenzofuran-2-yl(3,5-trimethoxyphenyl)methanone (TR120) showed marked cytotoxic activity in BCR-ABL-expressing cells. Interestingly, TR120 was more potent than imatinib in cell growth inhibition and apoptosis induction in both BCR-ABL-expressing K562 and KCL22 cells. Moreover, it showed antitumor activity in imatinib-resistant K562-R and KCL22-R cells at concentrations similar to those active in the respective sensitive cells. Further, TR120 induced a marked decrease in signal transducer and activator of transcription 5 (STAT5) expression in K562 cells. Consistent with this effect, it determined a block of cells in the G0-G1 phase of the cell cycle, a decrease in the level of cyclin D1, and a reduction in Bcl-xL expression; however, it did not cause modifications in the Bcl-2 level. Of interest, TR120 had synergistic effects when used in combination with imatinib in both sensitive and resistant cells. Considering that STAT5 is a BCR-ABL molecular target that plays a key role in the pathogenesis of CML as well as in BCR-ABL-mediated resistance to apoptosis, TR120 could potentially be a useful novel agent in the treatment of imatinib-resistant CML.

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.

Enhanced Adhesion, Migration and Pyk2 Expression in K562 Cells Following Imatinib Treatment

AbstractAbstract 4426 Background/Aims:Despite improved prognosis of CML patients with the use of imatinib (IM), its administration is associated with extramedullary disease (EMD) occurrence. We postulate that, like in the metastatic processes, changes in migration and adherence potential may enable leukemic cells to inhabit extramedullary sites. Focal adhesion complexes linking between extracellular matrix and the cell cytoskeleton are likely to play an important role in these processes.Pyk2 is a tyrosine kinase highly expressed in hematopoietic cells, localized to focal adhesion complexes, and known to participate in adhesion and migration processes. We have previously shown that Pyk2 participates in NB4 (an acute promyelocytic leukemia [APL] cell line) cells' adhesion and migration following exposure to the APL targeted therapy ATRA.We postulate that similar to the effect of ATRA on NB4 cells, IM being also a targeted therapy, might also be associated with enhanced adhesion and migration abilities of the CML cell line K562.Our objectives were to identify the effect of IM administration on pyk2 expression and on K562 cell adhesion and migration ability and to establish the role of these changes in treatment-associated EMD. Results:We found a 2.6-fold increase in pyk2 mRNA expression in K562 cells following exposure to IM. We also found that 30% of IM-treated K562 cells adhered to fibronectin (FN) compared to untreated cells having no adhesion ability. In addition, a 3-fold induction in migration was seen in K562 cells following treatment. Furthermore, K562 cells treated with IM demonstrated a 2-fold increase in invasion potential as compared to untreated cells. In order to assess whether Pyk2 is essential for IM-dependent adhesion and migration of K562 cells, these cells were infected with pyk2 specific shRNAs. While 30% of the non-infected NB4 cells adhered to FN following IM treatment, only 12% of the pyk2-shRNA–infected K562 cells exhibited adhesion potential (Pvalue<0.002). In addition, we witnessed over a 3-fold reduction in the ability of pyk2-shRNA–infected K562 cells to migrate following exposure to IM when compared to parental K562 cells. These data support the role of Pyk2 in IM-mediated adhesion and migration.Finally, we found that IM treatment induced an in-vivo increase in pyk2 mRNA expression level in leukocytes derived from 3 out of 5 CML patients studied. Conclusions:IM induces K562 cell adhesion, migration and invasion accompanied by increased pyk2 expression. Pyk2 is one of the key proteins regulating IM-induced cell migration and adhesion. Collectively our data suggest a critical role of Pyk2 in adhesion and migration initiated by the targeted therapy IM and a possible role in EMD development. These data support a common mechanism for the development of EMD in hematological malignancies treated by targeted therapies via pyk2 expression. Disclosures:No relevant conflicts of interest to declare.

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.

Role of DNA Methylation of the G0/G1 Switch Gene 2 (G0S2) in the Proliferation of Myeloid Leukemia Cells

Abstract 3520The G0/G1 switch gene 2 (G0S2) was originally identified in human blood mononuclear cells as an early response gene. We recently reported that G0S2 maintains quiescence in hematopoietic stem cells (Yamada et al., Plos One, 2012). In addition to hematopoietic stem cells, G0S2 also inhibits proliferation of lymphocytes and hematopoietic progenitor cells. Methylation of the G0S2 gene has been reported in lung, and head and neck cancer, suggesting that G0S2 has potential tumor suppressor function in solid tumors. However, the role of G0S2 in hematological malignancies has not been studied yet. To evaluate the impact of DNA demetylation of the G0S2 gene in human leukemia, we cultured a panel of myeloid (HEL, K562, HL-60, Kasumi) and lymphoid human leukemia cell lines (Jurkat, DND41, and H9) with 10 μM of 5-Azacytidine (5-Aza). Treatment with 5-Aza led to a significant increase of G0S2 expression in K562 cells (24.1-fold) and HL-60 cells (4.9-fold) and reduced proliferation. We show that retroviral overexpression of G0S2 in K562 cells caused inhibition of proliferation. Conversely, gene silencing of G0S2 in 5-Aza-treated K562 cells increased proliferation. Differentiation of K562 with hemin and HL-60 with all-trans retinoic acid was associated with an increase of G0S2 expression of 6.7-fold and 9.9-fold, respectively. Taken together, gain- and loss-of function studies revealed that G0S2 regulates cell proliferation and differentiation in human myeloid leukemia cells. Since G0S2 does not have a direct known function on the cell cycle, we hypothesized that G0S2 interacts with proteins involved in the control of cell proliferation. Mass spectrometric analysis of proteins pulled down with G0S2 in hematopoietic cells revealed that nucleolin, a molecule known to regulate ribosome biogenesis, physically interacts with G0S2 resulting in a perinuclear retention of nucleolin. This interaction was confirmed in K562 cells overexpressing G0S2 by reciprocal co-immunoprecipitation. This data suggest that silencing of G0S2 in leukemic cells prevents sequestration of nucleolin in the cytosol inhibiting its pro-proliferation functions. Collectively, our studies uncovered an important inhibitory role of G0S2 in the proliferation of leukemic cells, suggesting a possible tumor suppressor function in myeloid malignancies. Disclosures:No relevant conflicts of interest to declare.