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Lentiviral vectors have been used for gene therapy in the clinical phase in recent years. These vectors provide a tool for gene insertion, deletion, or modification in organisms. The K562 human cell line has been used extensively in hematopoietic research. Despite its broad application, it is hard-to-transfection and transduction. So, this study presents a simple method to increase the transduction efficiency of K562 cells with a low multiplicity of infection (MOI) of the virus particle. For this purpose, 24-well plate was coated by 300 μ l fetal bovine serum (FBS) before seeding. Then 2×10 4 K562 cells were seeded in each FBS coated plate. After 24h, K562 cells were attached and doubled. Different amount of lentivirus-based GFP vector according to MOI (5, 10, 15, and 20) along with 8 μ g polybrene was added to the attached K562 cells and after 6h cells and viral particle complex were spinfected. Then cells were returned to the plate and incubated in 37°C overnight. After 48h transduction efficiency was established by measuring the GFP-expressing cells by flow cytometry. Flow cytometry analysis showed that, after plate treatment by FBS, 64.5% transduction rate in K562 cells was achieved at MOI=20. Therefore, this method can be an effective and simple way to increase the lentiviral transduction rate for suspended cells such as K562.

Background: Flavonoids are natural compounds with antioxidant, anticarcinogenic, and anti-inflammatory effects., Aims: To determine the cytotoxic effects of flavonoids and drug resistance related to P-gp on K562 human chronic myeloid leukemia cells. We also aimed to evaluate the therapeutic potential of imatinib and flavonoid combinations., Study Design: Cell culture study., Methods: In this study, K562 cells were treated with apigenin, luteolin, 5-desmethyl sinensetin and the anticancer drug imatinib mesylate. The effect of flavonoids on K562 cell proliferation was detected using the 3-(4,5-dimethylthiazolyl)2,5‑diphenyl‑tetrazolium bromide assay. Concentrations of apigenin, luteolin, and 5-desmethyl sinensetin ranging from 25 to 200 μM and of imatinib from 5 to 50 μM administered for 72 h were studied. Apoptosis/necrosis and P-gp activity were measured using flow cytometry. The combined effects of different concentrations of flavonoids with imatinib were evaluated according to combination index values calculated using CompuSyn software., Results: In our study, the IC 50 values for apigenin, luteolin, and 5-desmethyl sinensetin were found to be 140 μM, 100 μM, and >200 μM, respectively. Luteolin (100 μM) had the highest cytotoxic activity of these flavonoids. These results were statistically significant (p<0.05). Among the flavonoids studied, the combination of luteolin and imatinib was the most effective and is therefore recommended for its cytotoxic activity in the K562 cell line. After 72 h of incubation at their respective IC 50 concentrations, all flavonoids were associated with an apoptosis rate of approximately 50%. P-glycoprotein activity was increased in all groups. Combination treatment may provide better outcomes in terms of cytotoxicity and thus reduce the dosages of imatinib used., Conclusion: The combination of some flavonoids and imatinib mesylate may increase the cytotoxic effect; However, the antagonistic effect should be considered in combined use on k562 cells.

The human K562 chronic myeloid leukemia cell line has long served as an experimental paradigm for functional genomic studies. To systematically and functionally annotate the human genome, the ENCODE consortium generated hundreds of functional genomic data sets, such as chromatin immunoprecipitation coupled to sequencing (ChIP-seq). While ChIP-seq analyses have provided tremendous insights into gene regulation, spatiotemporal insights were limited by a resolution of several hundred base pairs. ChIP-exonuclease (ChIP-exo) is a refined version of ChIP-seq that overcomes this limitation by providing higher precision mapping of protein-DNA interactions. To study the interplay of transcription initiation and chromatin, we profiled the genome-wide locations for RNA polymerase II (Pol II), the histone variant H2A.Z, and the histone modification H3K4me3 using ChIP-seq and ChIP-exo. In this Data Descriptor, we present detailed information on parallel experimental design, data generation, quality control analysis, and data validation. We discuss how these data lay the foundation for future analysis to understand the relationship between the occupancy of Pol II and nucleosome positions at near base pair resolution.

Objective: To study the function of ten-eleven translocation 2 (Tet2) in γ globin gene expression in patients with β- thalassemia. Methods: Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line. The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit. 5hmC level of γ globin gene was quantified by sulfite sequencing. The mRNA level of Tet2, γ globin, and related transcription factors Nfe4 and Klf1 were quantified by real-time PCR. Results: Tet2 knockdown resulted in a decreased global 5hmC level from 0.14% to 0.03% as of the control group in K562 cells. The expression of γ globin was enhanced after 5-azacytidine treatment in vitro. However, γ globin mRNA level in Tet2 knockdown cells was only 55% as that in control group. The CG sites on γ globin gene were unmethylated. As Tet2 was down-regulated, the expression levels of Nfe4 and Klf1 decreased by about 80% and increased to 3.5 folds, respectively. Conclusions: Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation. Moreover, Tet2 more likely regulates γ globin expression via affecting transcription factors rather than the gene itself. Thus, Tet2 could be a potential therapeutic target for β thalassemias.

Objective: To investigate the K562 cells biological function and related molecular changes in PTEN-PI3K/AKT signaling pathway of leukemia K562 cells by inhibiting the miRNA-21 expression to explore its pathogenesis of leukemia. Methods: The chemical synthetic miRNA-21 inhibitor was transfered into K562 cells by electrotransfection. RT-PCR was used to detect the miRNA-21 expression changes. Cell proliferation and apoptosis were determined by using MTT and flow cytometry. Western-blot were used to detect the protein expression changes of PTEN, PI3K and p-AKT respectively. Results: The relative expression of miRNA-21 in experimental group was (8.070 ± 5.138)% at 24 hours, which was lower than control groups ( P <0.05). The apoptotic rate of (13.370±0.250)% at 24 hours in experimental group was obviously higher than control groups. The cellular proliferation were significantly different at 24 hours. The proliferation inhibition rate was (8.1±0.9)% at 24 hours, which was up to (43.1±2.1)% at 60 hours, but the control groups showed no difference. K562 cell proliferation significantly decreased, while cell apoptosis markedly increased by inhibiting miRNA-21 expression ( P <0.01). Western-blot analysis revealed up-regulation of PTEN and down-regulation of PI3K and p-AKT protein expressions after successfully suppressed miRNA-21 expression ( P <0.01). Conclusion: Inhibiting miRNA-21 expression in K562 cell could suppress the PI3K/AKT pathway by up-regulation of PTEN expression and promote cell antiproliferative and pro-apoptosis effects.

We aimed to investigate the role of CCAAT enhancer-binding protein α (C/EBPα) in the pathogenesis of chronic myeloid leukemia (CML) and the mechanism underlying its effect. Bone marrow specimens from 50 patients with CML and peripheral blood specimens from 20 healthy individuals were collected. K562 cells were treated with imatinib. Subsequently, a stable cell line, K562-C/EBPα, was constructed. Cell proliferation was assayed with cell counting kit-8, and mRNA levels of C/EBPα, forkhead transcription factor FKHRL1 (Foxo3a) and Bim were detected by semiquantitative PCR. The correlation of C/EBPα and BCR-ABL was assessed by Spearman's correlation analysis. The results showed that C/EBPα mRNA levels were significantly reduced in CML patients compared with healthy subjects (p < 0.001) and were negatively correlated with BCR-ABL1 (r = -0.5046, p < 0.01). Additionally, imatinib enhanced the expression of C/EBPα in K562 cells compared with untreated cells (p < 0.05). Overexpression of C/EBPα significantly decreased cell proliferation and upregulated the expressions of the apoptosis-related genes Foxo3a (p < 0.01) and Bim (p < 0.05) in K562 cells. In conclusion, C/EBPα expression was decreased in patients with CML. Imatinib enhances the expression of C/EBPα in K562 cells, and the overexpression of C/EBPα inhibits cell proliferation and increases apoptosis via the Foxo3a-Bim pathway., (© 2016 S. Karger AG, Basel.)

p21(CIP) is a potent cell cycle inhibitor often up-regulated in differentiation. Protooncogene MYC induces cell growth and proliferation, inhibits differentiation and represses p21(CIP). However, both molecules are involved in processes of polyploidisation, cell size increase, differentiation and senescence. It is unclear why MYC has a dual role in differentiation. We have previously shown that overexpression of p21(CIP) in K562 myeloid cells induces megakaryocytic differentiation with polyploidy. We have now investigated the requirements for p21(CIP) to block mitosis and induce differentiation in the presence of overactivated MYC. Silencing and over-expression studies showed that p21(CIP) is required to induce differentiation. However, the expression of p21(CIP) needs to be transient to irreversibly inhibit mitosis but not DNA replication, what leads to polyploidy. Transient overexpression of p21(CIP) caused early down-regulation of mitotic Cyclins and up-regulation of G1/S Cyclins D and E, changes typical of endoreplication. Interestingly, over-activation of MYC did not release the proliferative block imposed by p21(CIP) and instead, accelerated cell size increase, megakaryocytic differentiation and polyploidisation. Our data suggests that in some systems p21(CIP) takes part in a mitosis control driving MYC-induced cellular growth into differentiation., (Copyright © 2011 Wiley Periodicals, Inc.)

The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A(*)1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A(*)1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3'terminal of the HLA-A(*)1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A(*)1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A(*)1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A(*)1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A(*)1101-T2A-EGFP plasmid was successfully constructed; after G418 selection for 2 months, two sublines of K562 cells (HLA-A(*)1101(+)K562, pEGFP-N3(+)K562) expressing GFP were constructed. The expression of HLA-A*A1101 gene could be determined in HLA-A(*)1101(+)K562 cell line by RT-PCR, while the pEGFP-N3(+)K562 cells could not express HLA-A*A1101 gene. HLA-A(*)1101 protein and GFP double positive HLA-A(*)1101(+)K562 cells were up to 88.5%, which was obviously higher than pEGFP-N3(+)K562 cells (0.698%) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A(*)1101(+)K562 cells has been established and a subline of K562 cell expressing HLA-A(*)1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.

Our previous study demonstrates that Bcr-Abl fusion oncogene frequently found in chronic myeloid leukemia (CML) cells can up-regulate Skp2 expression via transcriptional activation. However, Bcr-Abl also modulates Skp2 protein stability in these cells. Treatment of Bcr-Abl kinase inhibitor imatinib led to G1 growth arrest accompanied with reduced Skp2 expression. Interestingly, reduction of Skp2 protein occurred prior to down-regulation of Skp2 mRNA suggesting a post-translational control. The half-life of Skp2 protein was significantly attenuated in imatinib-treated cells. These effects are not cell line specific because similar results were also found in CML cells obtained from patients. Knockdown of Bcr-Abl similarly caused Skp2 protein instability. The decrease of Skp2 was induced by increased protein degradation through the ubiquitin/proteasome pathway. Imatinib treatment or Bcr-Abl knockdown reduced Emi1, an endogenous inhibitor of the E3 ligase APC/Cdh1 which mediated Skp2 degradation. We found that Emi1 stability was regulated by phosphorylation and mutation of tyrosine 142 reduced the stability. Our data suggested Bcr-Abl-induced Emi1 phosphorylation might be mediated by Src kinase. Firstly, Src inhibitor SU6656 inhibited Emi1 tyrosine phosphorylation in K562 cells. Secondly, transfection of v-Src rescued the reduction of Emi1 by imatinib. Thirdly, mutation of tyrosine 142 to phenylalanine (Y142F) abolished the phosphorylation of Emi1 by recombinant Src kinase. In addition, ectopic expression of wild type but not Y142F mutant Emi1 counteracted imatinib-caused growth arrest. Collectively, our results suggest that Bcr-Abl increases Emi1 phosphorylation and stability to prevent Skp2 protein degradation via APC/Cdh1-induced ubiquitination and to enhance proliferation of CML cells., (© 2010 Wiley-Liss, Inc.)

Background: A promising approach to optimize the disposition of daunorubicin-loaded magnetic nanoparticles (DNR-MNPs) was developed to minimize serious side effects of systematic chemotherapy for cancer., Methods: The physical properties of DNR-MNPs were investigated and their effect on leukemia cells in vitro was evaluated by a standard WST-1 cell proliferation assay. Furthermore, cell apoptosis and intracellular accumulation of DNR were determined by FACSCalibur flow cytometry., Results: Our results showed that the majority of MNPs were spherical and their sizes were from 10 to 20 nm. The average hydrodynamic diameter of DNR-MNPs in water was 94 nm. The in vitro release data showed that the DNR-MNPs have excellent sustained release property. Proliferation of K562 cells was inhibited in a dose-dependent manner by DNR in solution (DNR-Sol) or by DNR-MNPs. The IC(50) for DNR-MNPs was slightly higher than that for DNR-Sol. DNR-MNPs also induced less apoptosis in K562 cells than did DNR-Sol. Detection of fluorescence intensity of intracellular DNR demonstrated that DNR-MNPs could be taken up by K562 cells and persistently released DNR in cells., Conclusion: Our study suggests that optimized DNR-MNPs formulation possesses sustained drug-release and favorable antitumor properties, which may be used as a conventional dosage form for antitumor therapy.

Objective: To construct a eukaryotic expression vector containing aldehyde dehydrogenase-2 (ALDH2) gene and investigate the effects and its possible mechanisms of ALDH2 gene on cell proliferation and anti-oxidative damage in the K562 cells., Methods: An eukaryotic expression vector containing the ALDH2 gene cloned from human hepatocytes was constructed and transfected into K562 cells by liposome. RT-PCR and Western blot were used to evaluate the expression of ALDH2. MTT assay was used to check the cell proliferation and trypan blue exclusion to check K562 cells damage induced by hydrogen peroxide (H2O2). RT-PCR and fluorescence spectrophotometry were used to determine the expression of heme oxygenase-1 (HO-1) and the generation of intracellular reactive oxygen species (ROS) respectively., Results: RT-PCR and Western blot analysis showed distinct higher ALDH2 protein expression in gene transfected group. The latter group had a higher cell proliferation (P < 0.05) and survival rate against H2O2 induced-oxidative damage, being increased by 7.8 times (IC(50) was 12.3 µmol/L and 1.4 µmol/L for K562-pcDNA3.1-ALDH2 and control cells, respectively, P < 0.01). The HO-1 mRNA expression and the generation of intracellular ROS were downregulated at a specific concentration of H2O2 in the ALDH2 gene transfected group., Conclusion: ALDH2 gene transfection can protect K562 cells against oxidative damage, and the downregulation of HO-1 expression and intracellular ROS may be involved in this process.

In order to establish K562 line with stable expressions of hermap and hermap-siRNA, amplified hermap and hermap-siRNA were cloned into pEGFP-c1 and pRNAT to acquire hermap-pEGFP-c1 and hermap-siRNA-pRNAT, respectively. These two plasmids were electrotransferred into K562 cells, then were followed by culturing with G418. The result showed that the transfer rate of hermap-pEGFP-c1-K562 and hermap-siRNA-pRNAT-K562 plasmids were 10.0% and 9.3%, respectively. After selective culture by G418, these two cell lines were still able to express GFP. It is concluded that the eukaryotic expression plasmids containing hermap and hermap-siRNA have been successfully constructed, and the cell lines of hermap-K562 and hermap-siRNA-K562 are established, definitely contributing to further functional investigation on HERMAP and its interaction with other proteins.

Objective: To investigate the effect of tyrosine kinase inhibitor ZD6474 (Vandetanib) on the proliferative inhibition of K562 cells and its derived imatinib-resistant K562/G cells and its mechanism., Methods: Imatinib-resistant K562/G cells were obtained by culturing cells in gradually increasing concentrations of imatinib. The changed factors related to drug-resistance were tested by Western blot. ZD6474 and imatinib affected K562/G and parental K562 cells proliferation were analyzed by WST assay. Flow cytometry was used to analyze cell cycle. Direct inhibition of Src activity by ZD6474 was measured by a colorimetric ELISA assay with recombinant human Src kinase., Results: 10 µmol/L imatinib failed to inhibit K562/G cells proliferation or induce cell cycle arrest. Compared with that in parental K562 cells, there were marked high levels of p-Src and Src protein in K562/G cells. The expression of Bcl-2 and p-STAT3 also increased in K562/G cells. After 48 hours incubation, the IC(50) values of ZD6474 in K562 and K562/G cells were 1.61 µmol/L and 3.18 µmol/L, respectively. ZD6474 treatment caused accumulation of cells in the G(0)/G(1) fraction and cell apoptosis in K562 and K562/G cells. ZD6474 decreased the expression of p-Src and Src at post-transcriptional level. Moreover, ZD6474 increased the ratio of Bax/Bcl-2 and decreased the expression of p-STAT3 at the same concentration for inducing apoptosis., Conclusions: ZD6474 is effective in inhibiting the proliferation of imatinib-resistant K562/G cells and parental K562 cells, and induces their apoptasis by significant inhibition of Src kinase activity. Our study provides a reliable experimental basis for chronic myeloid leukemia treatment with ZD6474.

Objective: To investigate the role of Ara-C in regulating anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells., Methods: The diabody of anti-CD3/anti-Pgp was purified by E-tag affinity chromatography. K562 and K562/A02 cells were treated with Ara-C. The expressions of B7-1 and B7-2 on K562 and K562/AO2 cells were detected by FACS. The cytotoxicity of T-lymphocytes combined with anti-CD3/anU-Pgp plus Ara-C was analyzed by CytoTox 96 nonradioactive method., Results: The expressions of B7-1 and B7-2 on K562 and K562/A02 cells treated by Ara-C was significantly higher than those untreated. The effect/target ratio was from 0.39:1 to 25:1, and the killing rate of activated T cells to anti-drug-resistant leukemia cells was from (16.44 +/- 1.20)% to (60.49 +/- 2.90)%. The killing rates were increased gradually, with both the effect/target ratio and the antibody concentration increasing (P < 0.05)., Conclusion: Ara-C may be an important adjuvant for improving anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.

Objective: To study the effect of adenovirus vector-mediated siRNA targeting vascular endothelial growth factor(VEGF) on apoptosis and the expression of survivin in K562 cells., Methods: K562 cells were infected with recombinant adenovirus Ad5-VEGFsi for 72 hours as experimental group (K562/Ad5-VEGFsi), and empty vector group (K562/Ad5) and blank control group (K562) as controls. VEGF mRNA and survivin mRNA expression were determined by RT-PCR. The protein levels of VEGF and survivin were measured by ELISA and Western blot, respectively. The apoptosis of K562 cells was detected by flow cytometry., Results: The levels of VEGF and survivin mRNA expression in experimental group cells were significantly decreased (P < 0.01). The protein concentration of VEGF in experimental group supernatant was (1121 +/- 15) pg/ml, being lower than that in empty vector group \[(1290 +/- 28) pg/ml\] and black control group \[(1303 +/- 28) pg/ml\] (P < 0.01), and the level of survivin protein in experimental group (0.26 +/- 0.11) was significantly reduced compared with that in blank control group (0.74 +/- 0.10) (P < 0.01). The apoptosis rate of K562/Ad5-VEGFsi cells (16.45 +/- 0.14)% was higher than those of K562/Ad5 cells (3.54 +/- 0.17)% and K562 cells (2.56 +/- 0.20)% (P < 0.01)., Conclusions: VEGF can up-regulate the expression of survivin. After inhibition of VEGF by RNAi, the expression of survivin is decreased subsequently and the rates of cell apoptosis are increased.

We have shown previously that iron deprivation significantly stimulates the uptake of non-transferrin ferric iron from ferric citrate by erythroleukemia K562 cells and that this stimulation depends on protein synthesis. However, we have not detected increased expression of any known iron transport protein (Kovar J. et al. (2006) Blood Cells Mol Dis 37:95-99). Therefore, in order to identify membrane proteins of K562 cells with increased expression under iron deprivation, we employed the isolation of membrane proteins by two-phase partitioning system, protein separation by high-resolution 2D electrophoresis, computer differential analysis, and tandem mass spectrometry. Employing these techniques we identified two proteins with statistically significant upregulation, i.e., aldolase A (ALDA) and voltage-dependent anion channel 2 (VDAC2). The upregulation of aldolase A and VDAC2 in K562 cells under iron deprivation was also confirmed by western blot analysis. This is the first time when the control of aldolase A and VDAC2 levels by iron status of the cell is demonstrated.

Objective: Results of previous studies have suggested that tipifarnib (Zarnestra), a farnesyltransferase inhibitor, is useful for treating various hematological disorders, including chronic myeloid leukemia. However, acquisition of resistance may be a problem for patients being treated with tipifarnib., Methods: We generated a tipifarnib-resistant BCR/ABL-positive cell line, K562/RR, and examined its characteristics., Results: While levels of cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and cleaved poly (ADP-ribose) polymerase were significantly increased in K562 cells, the levels were not changed in K562/RR cells with tipifarnib treatment, indicating that induction of apoptosis signaling mediated by tipifarnib is much less in K562/RR cells than in K562 cells. In addition, tipifarnib-mediated induction of cell-cycle blockage was abrogated in K562/RR cells. No mutation of farnesyltransferase alpha and beta genes was found and the level of unprocessed HDJ-2, which is a substrate of farnesyltransferase, was increased by tipifarnib treatment in K562/RR cells, suggesting that tipifarnib inhibits protein farnesylation in K562/RR cells in the same manner as in K562 cells and that mechanisms independent of farnesyltransferase activity are involved in the acquisition of resistance to tipifarnib in these cells. By DNA microarray analyses using a cDNA microarray comprising 25,000 genes, we identified 5 genes with higher expression levels in K562/RR cells than in K562 cells. These genes include beta-globin, calcium channel Caveolin 2, and FEN1, which is involved in DNA replication and repair, and CUGBP2, which may affect expression of cyclooxygenase 2., Conclusion: The results of this study provide useful information for clarification of the mechanisms of resistance to tipifarnib.

Polymorphonuclear neutrophils (PMN) have a remarkable capacity for generation of large amounts of reactive oxygen species in response to a variety of infectious or inflammatory stimuli, a process known as the respiratory burst that involves activation of a multicomponent NADPH oxidase. Given their short life span, PMN are not amenable to most molecular biology methods for studying activation of this oxidant-generating system. We have explored a variety of methods for introduction of components of the phagocytic oxidase (phox system) into the promyelocytic erythroleukemia cell line, K-562. Here, we describe a series of cloned K-562 cell lines that were retrovirally transduced for stable production of one or more essential components of the phagocytic oxidase (phox) complex. We outline methods for the use of these transfectable cells for investigating structure, function, and signaling requirements for assembly and activation of the phox system. These versatile lines can be used to examine effects of genetic polymorphisms or mutations in phox components associated with chronic granulomatous disease, to serve as a system for testing gene therapy vectors designed to correct the defective oxidase, to study cross-functioning with recently described phox component homologs, or to explore signaling components involved in regulation of the respiratory burst.

Background: Natural Killer (NK) cells are key actors of innate immunity that supervise the organism's cells, and fight against viral infections and cancer development through their cytotoxic activity. This cytotoxic activity is modulated by cytokines and hormones and could be influenced by physiological or pathological conditions. New techniques for measuring NK cytotoxic activity by flow-cytometry have recently been developed, and they correlated strongly with the standard chromium ((51)Cr) release assay. Our aim was to implement a previously published enhanced green fluorescent protein (EGFP)-K562 flow cytometric method and use it to evaluate NK cytotoxic activity under different nutritional conditions., Methods: NK effector cells were isolated from peripheral blood mononuclear cells, and a K562 cell line stably transfected by EGFP was used as target cells. Different analytical parameters, including cell ratios and incubation times, were studied to improve the EGFP-K562 flow cytometric NK test conditions., Results: The optimized test was then used to determine the effect of fasting and refeeding on NK cell numbers and activity in a physiological situation. NK cytotoxic activity in fasted conditions (30.4 +/- 4.4%) increased by a factor 1.7 +/- 0.2 (P = 0.0025) in nourished conditions (45.0 +/- 4.6%) in healthy elderly people., Conclusion: Therefore, this method provides a reliable, reproducible and rapid test for analyzing NK cytotoxicity under various conditions., ((c) 2006 International Society for Analytical Cytology.)

New derivatives of daunorubicin (DRB), doxorubicin (DOX), and epidoxorubicin (EDOX) with an amidine group bonded to C-3' of daunosamine moiety with either morpholine or hexamethyleneimine ring attached to the amidine group are studied in this paper. We have shown that all of these newly synthesized anthracycline derivatives inhibit human leukemia K562 cell line proliferation but only some of them induce erythroid differentiation when used at subtoxic concentrations. Morpholine derivative of DOX has the greatest potential to inhibit proliferation and to induce differentiation in vitro. The correlation between these two cellular processes was also significant for other tested compounds. In cell cycle analysis, we have demonstrated that those anthracycline derivatives that exert the greatest cytostatic potential caused G(2)/M arrest, which in turn, might contribute to the development of a differentiating phenotype. The concentrations of the compounds used in the study are pharmacologically relevant. These new potent inducers of differentiation might be exploited as anticancer drugs for treatment of leukemia by differentiation therapy.

Several agents are known to induce differentiation in the human erythroleukemic cell line K562. In this work we have studied the ability of hyperthermia to induce differentiation in the K562 cell line. K562 cells were treated with hyperthermia in the range of 41-45 degrees C. Cell proliferation and the plating efficiency of heat treated cells along with their hemoglobin synthesis was measured and compared with controls. Hyperthermia severely inhibited the growth of K562 cells in the suspension culture in a time- and temperature-dependent manner. Sixty minutes of heating and 44 and 40 min of heating at 45 degrees C totally inhibited the growth of the cells. The number of clonogenic cells also decreased as a result of heat treatment. Extended periods of heating for more than 2 h at 41 degrees C resulted in thermal adaptation. Hyperthermia-induced hemoglobin synthesis by these cells, only at 42 and 43 degrees C. Maximum induction was observed after heat treatment for 80 min at 43 degrees C and 180 min at 42 degrees C. At lower temperature, although the fraction of surviving cells was high, but no signs of hemoglobin synthesis could be observed. At temperatures higher than 43 degrees C, the fraction of surviving cells decreased rapidly and also no signs of hemoglobin synthesis could be detected. At the two selective temperatures, hemoglobin synthesis started 4 days after heat treatment. The results showed that hyperthermia caused cytotoxicity and growth arrest and induced differentiation as judged by hemoglobin synthesis and reduced clonogenicity in this cell line. This is the first time that a physical agent has been shown to induce differentiation in erythroleukemic cell lines.

The delta-globin gene produces the delta chain of Hb A2 which represents less than 3% of the hemoglobin (Hb) in normal individuals. The delta-globin gene is also expressed in the human erythroleukemia cell line K562. The expression of the delta-globin gene in this cell line is unexpected since K562 shows an embryonic-fetal globin gene expression pattern with no expression of the adult beta-globin gene. delta-Globin gene activation has been proposed as a potential therapeutic tool for the cure of delta-thalassemia (thal). In order to shed some light on the delta-globin gene activation in K562 the present study has: (1) determined the complete nucleotide sequence of the delta- and beta-globin genes; (2) assessed, by reverse transcription-polymerase chain reaction (RT-PCR), the relative delta- and beta-globin mRNA level; and (3) analyzed the exact level of the endogenous expression delta-globin gene by S1 mapping. No sequence variations were identified in the (delta- and beta-globin genes when compared to the normal sequences. delta-Globin mRNA represent more than 95% of the total delta + beta-mRNA content. The level of expression of the delta-globin gene is 12.3% (+/- 1.2) compared to the endogenous alpha-globin gene. These results indicate that the high expression of the delta-globin gene in K562 is most likely due to the transacting environment. Therefore, the presence and/or absence of specific transacting factors are able to specifically activate the human delta-globin gene. The level of expression of the delta-globin gene in this cell line suggests that it could be of relevance to identify the transacting factor(s) responsible for this selective activation in order to better understand the molecular mechanisms undergoing gene activation.

Cell line K562 is the defacto forensic control material for forensic restriction fragment length polymorphism (RFLP) DNA profiling in the U.S. Fifty-one proficiency tests conducted from 1991 through 1997 enable a detailed description of RFLP measurement performance during this period. Sufficient data are available to define reference distributions for all commonly utilized and many less commonly reported genetic loci, for both HaeIII- and HinfI-based RFLP systems. The average measured size of HaeIII locus D1S7 and D5S110 bands has varied slightly over time; while relatively small, these temporal changes add to the overall interlaboratory measurement uncertainty. The characteristic standard deviation for HinfI RFLP system measurements has a nearly identical dependence on expected band size as does the standard deviation for HaeIII measurements. The ellipsoidal distance, K, is suggested for use as an RFLP data quality metric; the critical threshold value that on average excludes 1% of plausibly valid proficiency data for a given polymorphic locus is K1% = 14.2.

Fast, nondestructive three-dimensional (3D) imaging of live suspension cells remains challenging without substrate treatment or fixation, precluding scalable single-cell morphometry with minimal alterations. While optical sectioning techniques achieve 3D live cell imaging, lateral versus depth resolution differences further complicate analysis. We present a scalable microfluidic method capable of 3D fluorescent isotropic imaging of live, nonadherent cells suspended inside picoliter droplets with high-speed single-cell volumetric readout (800 to 1,200 slices in 5 to 8 s) and near-diffraction limit resolution (~216 nm). The platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intradroplet microvortices, which rotate single cells on their axis to enable optical projection tomography (OPT)-based imaging. This allows gentle (~1 mPa shear stress) observation of cells encapsulated inside nontoxic isotonic buffer droplets, facilitating scalable OPT acquisition by simultaneous spinning of hundreds of cells. We demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells-small cells prone to movement in their suspended phenotype. Our fully suspended, orientation-independent cell morphometry, driven by isotropic imaging and spherical harmonic analysis, enabled the study of primary T cells across various immunological activation states. This approach unveiled six distinct nuclear content distributions, contrasting with conventional 2D images that typically portray spheroid and bean-like nuclear shapes associated with lymphocytes. Our arrayed-droplet OPT technology is capable of isotropic, single live-cell 3D imaging, with the potential to perform large-scale morphometry of immune cell effector function states while providing compatibility with microfluidic droplet operations.

IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner.

Genome-wide association studies (GWAS) have become well-powered to detect loci associated with telomere length. However, no prior work has validated genes nominated by GWAS to examine their role in telomere length regulation. We conducted a multi-ancestry meta-analysis of 211,369 individuals and identified five novel association signals. Enrichment analyses of chromatin state and cell-type heritability suggested that blood/immune cells are the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6 or POP5 and demonstrated that both lengthened telomeres. CRISPR/Cas9 deletion of the predicted causal regions in K562 blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5. Our results demonstrate the utility of telomere length GWAS in the identification of telomere length regulation mechanisms and validate KBTBD6 and POP5 as genes affecting telomere length regulation.

Piezo1 is a Ca2+‐permeable mechanically activated ion channel that is involved in various physiological processes and cellular responses to mechanical stimuli. The study of biophysical characteristics of Piezo1 is important for understanding the mechanisms of its function and regulation. Stretch activation, a routine approach that is applied to stimulate Piezo1 activity in the plasma membrane, has a number of significant limitations that complicate precise single‐channel analysis. Here, we aimed to determine pore properties of native Piezo1, specifically to examine permeation for physiologically relevant signaling divalent ions (calcium and magnesium) in human myeloid leukemia K562 cells using Piezo1‐specific chemical agonist, Yoda1. Using a combination of low‐noise single‐current patch‐clamp recordings of Piezo1 activity in response to Yoda1, we have determined single‐channel characteristics of native Piezo1 under various ionic conditions. Whole‐cell assay allowed us to directly measure Piezo1 single currents carried by Ca2+ or Mg2+ ions in the absence of other permeable cations in the extracellular solutions; unitary conductance values estimated at various concentrations of Mg2+ revealed strong saturation effect. Patch clamp data complemented with fluorescent imaging clearly evidenced Ca2+ and Mg2+ entry via native Piezo1 channel in human leukemia K562 cells. Mg2+ influx via Piezo1 was detected under quasi‐physiological conditions, thus showing that Piezo1 channels could potentially provide the physiological relevant pathway for Mg2+ ion transport and contribute to the regulation of Mg2+‐dependent intracellular signaling. [ABSTRACT FROM AUTHOR]

Objective: We aimed to establish capsaicin as a potent natural chemotherapeutic agent in chronic myeloid leukemia by unveiling anti-cancer mechanisms, concentrating at pro-apoptotic and anti-proliferative effects on K562 cells. Methodology: After REC approval, this research proceeded with culturing K562 cells and treatment with serial concentrations of capsaicin for calculation of subsequent 50% Inhibitory Concentration (IC50) values via MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, expression analysis of gene, comparative analysis of relative gene fold (intrinsic biomarkers caspase-3, caspase-9) as apoptosis mediator biomarkers followed by Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR). Anti-proliferative activity was confirmed via Nitric Oxide (NO) releasing Assay. Results: For K562 cells, the IC50 of capsaicin (12.1 µM) showed high gene expression of intrinsic pro-apoptotic biomarkers caspase-3 and caspase-9 with the relative gene fold of 12.1 and 13.9 respectively. Persistent peaks of NO levels confirmed the anti-proliferative potential of the compounds. Strongest growth inhibitory activity was seen at the peak time of 100-120 min. Conclusion: Capsaicin proved to be a strong pro-apoptotic and anti-proliferative phytochemical agent leading to therapeutic effects against K562 cells. Further studies would help in identifying key molecular intricacies by which capsaicin exhibits diversified anti-cancer potential. [ABSTRACT FROM AUTHOR]

LL-37 can inhibit the growth of K562 cancer cells when it is conjugated with iron oxide nanoparticles. In this study, Fe3O4 nanoparticles were synthesized using the co-precipitation method and then modified with the LL-37 peptide through an NH2 bridge. The accuracy of the synthesis process was confirmed through various analytical tests, including FTIR, XRD, FESEM, and EDX. To assess the treatment's effectiveness, a viability test was carried out on K562 leukemia cells and normal peripheral blood mononuclear cells. In addition, flow cytometry and Hoechst staining were used to investigate the mechanism of action of the drug. The expression levels of the Bcl-2, Bax, and TP53 genes in the treated cells and the control group were measured using qRT-PCR. The results indicated that the size of the nanoparticles ranged between 34 and 40 nm. The NH2@LL-37@Fe3O4 nanoparticles more effectively inhibited the growth of cancer cells in a concentration-dependent manner, as compared to Fe3O4 alone. Further analysis revealed that apoptosis occurred through increased expression of TP53 and Bax genes compared to the Bcl-2 gene. Therefore, induction of apoptosis and inhibition of growth in K562 cells was attributed to the impact of iron oxide magnetic nanoparticles conjugated with the LL-37 peptide through the TP53/Bax/Bcl-2 pathway. [ABSTRACT FROM AUTHOR]

Abstract β-Ionone is the end-ring counterpart of β-carotenoids, which are widely found in fruits and vegetables. Recent studies have illustrated the antimetastatic, anti-proliferative, and apoptosis-inducing activities of β-ionone both in vitro and in vivo. We aimed to explore the anti-cancer potency of β-Ionone-derived ester, (E)-4-(2,6,6-trimethylcyclohex-1-enyl) but-3-en-2-ylpyrazine-2-carboxylate (4-TM.P). The cytotoxic effects of the compound on K562 cells were evaluated by MTT assay. The mechanisms of apoptosis induction were investigated by acridine orange/ethidium bromide (AO/EtBr) double staining, cell cycle analysis, and Annexin V/PI staining. Furthermore, the 4-TM.P-DNA interactions have been thoroughly elucidated by various methods, such as ultraviolet–visible spectroscopy, fluorescence assays, viscosity measurements, molecular docking, and dynamic simulation. The MTT cytotoxicity assay revealed that the growth of K562 cells was inhibited by treatment with β-ionone-derived ester, with an IC50 of 25 ± 5.0 µM at 72 h. Morphological studies revealed the occurrence of apoptosis in treated cells, and G0/G1 cell cycle arrest was observed after treatment of the cells with the IC50 value of the compound. Analyses of multi-spectroscopy and viscosity assays revealed that 4-TM.P binds to DNA in the minor groove mode, which was supported by molecular docking studies. The dynamic stability of the complex was also confirmed using molecular dynamic simulation analyses.

Abstract LL-37 can inhibit the growth of K562 cancer cells when it is conjugated with iron oxide nanoparticles. In this study, Fe3O4 nanoparticles were synthesized using the co-precipitation method and then modified with the LL-37 peptide through an NH2 bridge. The accuracy of the synthesis process was confirmed through various analytical tests, including FTIR, XRD, FESEM, and EDX. To assess the treatment's effectiveness, a viability test was carried out on K562 leukemia cells and normal peripheral blood mononuclear cells. In addition, flow cytometry and Hoechst staining were used to investigate the mechanism of action of the drug. The expression levels of the Bcl-2, Bax, and TP53 genes in the treated cells and the control group were measured using qRT-PCR. The results indicated that the size of the nanoparticles ranged between 34 and 40 nm. The NH2@LL-37@Fe3O4 nanoparticles more effectively inhibited the growth of cancer cells in a concentration-dependent manner, as compared to Fe3O4 alone. Further analysis revealed that apoptosis occurred through increased expression of TP53 and Bax genes compared to the Bcl-2 gene. Therefore, induction of apoptosis and inhibition of growth in K562 cells was attributed to the impact of iron oxide magnetic nanoparticles conjugated with the LL-37 peptide through the TP53/Bax/Bcl-2 pathway.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells. [ABSTRACT FROM AUTHOR]

Objective: To explore the role and mechanism of IL-39 in K562 cells. Methods: The expressions of IL-39R and STAT1/STAT3 in K562 cells were detected via qRT-PCR； the protein levels of phospho-STAT1/STAT3 and STAT1/STAT3 in K562 cells were detected by Western blot； the mRNA regulated by IL-39 in K562 cells was predicted by RNA sequencing. Results: rIL-39 significantly promoted the expression of IL-39R in K562 cells； IL-39 affected K562 cells through STAT1 pathway； IL-39 significantly regulated the expression of mRNA in K562 cells， thus potentially affecting a series of biological processes. Conclusion: IL-39 can directly act on K562 cells， alter gene transcriptional expression and inhibit tumor growth through the STAT1 pathway. IL-39 may similarly modulate gene expression profile in human leukemia cells. [ABSTRACT FROM AUTHOR]

Introduction: The Vel– phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the Vel protein was detected using Western blot on single-cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high-efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin. [ABSTRACT FROM AUTHOR]

Oncological diseases represent a significant global health challenge, with high mortality rates. Early detection is crucial for effective treatment, and aptamers, which demonstrate superior specificity and stability compared to antibodies, offer a promising avenue for diagnostic advancement. This study presents the design, development and evaluation of a quartz crystal microbalance (QCM) sensor functionalized with the T2-KK1B10 aptamer for the sensitive and specific detection of Chronic Myeloid Leukemia (CML) K562 cells. The research focuses on optimizing the biorecognition layer by adjusting the aptamer conditions, demonstrating the sensor's ability to detect these CML cells with high specificity and sensitivity. The aptamer-modified QCM sensor operates on the principle of mass change detection upon binding of target cells. By employing the Langmuir isotherm model, the performance of the sensor was optimized for the capture of CML cells from biological samples with LOD of 263 K562 cells. The sensor was also successfully regenerated multiple times without sensitivity loss. Validation of the sensor's performance was conducted under controlled laboratory settings, followed by extensive testing utilizing human lyophilized plasma and clinical samples from patients. The sensor exhibited high sensitivity and specificity in the detection of CML cells within clinical specimens, thereby illustrating its potential for practical clinical deployment. This research presents a novel approach to the early diagnosis of CML, facilitating timely intervention and enhanced patient outcomes. The developed aptasensor demonstrates potential for broader application in cancer diagnostics and personalized medicine.

Hydroquinone (HQ), one of the phenolic metabolites of benzene, is widely recognized as an important participant in benzene-induced hematotoxicity. However, there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn't been fully understood yet. In this study, we treated K562 cells with 40 μmol/L HQ for 72 h, examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring (PRM), and performed bioinformatics analysis to identify interaction networks. One hundred and eighty-seven upregulated differentially expressed proteins (DEPs) and 279 downregulated DEPs were identified in HQ-exposed K562 cells, which were involved in neutrophil-mediated immunity, blood microparticle, and other GO terms, as well as the lysosome, metabolic, cell cycle, and cellular senescence-related pathways. Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways, we constructed the network of protein interactions and determined 6 DEPs (STAT1, STAT3, CASP3, KIT, STAT5B, and VEGFA) as main hub proteins with the most interactions, among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity. Our work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification. [ABSTRACT FROM AUTHOR]

Dinitrosyl iron complexes (DNICs) stabilize nitric oxide in cells and tissues and constitute an important form of its storage and transportation. DNICs may comprise low-molecular-weight ligands, e.g., thiols, imidazole groups in chemical compounds with low molecular weight (LMWDNICs), or high-molecular-weight ligands, e.g., peptides or proteins (HMWDNICs). The aim of this study was to investigate the role of low- and high-molecular-weight ligands in DNIC formation. Lysosomal and proteasomal proteolysis was inhibited by specific inhibitors. Experiments were conducted on human erythroid K562 cells and on K562 cells overexpressing a heavy chain of ferritin. Cell cultures were treated with •NO donor. DNIC formation was monitored by electron paramagnetic resonance. Pretreatment of cells with proteolysis inhibitors diminished the intensity and changed the shape of the DNIC-specific EPR signal in a treatment time-dependent manner. The level of DNIC formation was significantly influenced by the presence of protein degradation products. Interestingly, formation of HMWDNICs depended on the availability of LMWDNICs. The extent of glutathione involvement in the in vivo formation of DNICs is minor yet noticeable, aligning with our prior research findings. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) of HLA‐E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA‐E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti‐tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA‐E mRNA transcription and membrane HLA‐E (mHLA‐E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA‐E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA‐E (sHLA‐E) was not influenced by both rs1264457 and rs76971248. The higher HLA‐E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA‐E molecules played a significant inhibitory effect on the killing activity of NK‐92MI cells (p < 0.05). In conclusion, the higher HLA‐E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK‐92MI cells' killing. [ABSTRACT FROM AUTHOR]

Background: Imatinib resistance remains a major obstacle in the treatment of chronic myelogenous leukemia (CML). Crocin (CRC) and astaxanthin (ATX) are phytochemicals with anti-cancer properties. Aims: This study aimed to explore the effects of combination treatment of Imatinib with CRC and ATX on Imatinib-resistant K562 (IR-K562) cells. Methods and results: After the establishment of IR-K562 cells, growth inhibitory activity was determined by the MTT assay. To test the regeneration potential, a colony formation assay was performed. Cell cycle analyses were examined by flow cytometry. Cell injury was evaluated by lactate dehydrogenase (LDH) leakage. Real-time PCR was applied to assess the expression of IL6, TNF-α, STAT3, BAD, CASP3, TP53, and Bcl-2 genes. Caspase-3 activity was determined by a colorimetric assay. Antioxidant activity was measured using a diphenylpicrylhydrazyl (DPPH) assay. After 48 h of treatment, ATX (IC50 = 30µM) and CRC (IC50 = 190µM) significantly inhibited cell proliferation and colony formation ability, induced G1 cell cycle arrest and cell injury, upregulated the expression of apoptosis-associated genes, and downregulated the expression of anti-apoptotic and inflammatory genes. The combination of IM with ATX and/or CRC synergistically reduced cell viability (combination index [CI] < 1). Conclusion: Our data suggest that IM shows better therapeutic efficacy at lower doses when combined with ATX and/or CRC. [ABSTRACT FROM AUTHOR]

BACKGROUND: Natural killer (NK) cell-based immunotherapy is a promising treatment approach for multiple myeloma (MM), but obtaining a sufficient number of activated NK cells remains challenging. Here, we report an improved method to generate ex vivo expanded NK (eNK) cells from MM patients based on genetic engineering of K562 cells to express OX40 ligand and membrane-bound (mb) IL-18 and IL-21. METHODS: K562-OX40L-mbIL-18/-21 cells were generated by transducing K562-OX40L cells with a lentiviral vector encoding mbIL-18 and mbIL-21, and these were used as feeder cells to expand NK cells from peripheral blood mononuclear cells of healthy donors (HDs) and MM patients in the presence of IL-2/IL-15. Purity, expansion rate, receptor expression, and functions of eNK cells were determined over four weeks of culture. RESULTS: NK cell expansion was enhanced by short exposure of soluble IL-18 and IL-21 with K562-OX40L cells. Co-culture of NK cells with K562-OX40L-mbIL-18/-21 cells resulted in remarkable expansion of NK cells from HDs (9,860-fold) and MM patients (4,929-fold) over the 28-day culture period. Moreover, eNK cells showed increased expression of major activation markers and enhanced cytotoxicity towards target K562, U266, and RPMI8226 cells. CONCLUSIONS: Our data suggest that genetically engineered K562 cells expressing OX40L, mbIL-18, and mbIL-21 improve the expansion of NK cells, increase activation signals, and enhance their cytolytic activity towards MM cells.

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5 bp increase to >30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2's dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five 'aromatic fingers'. NSs binding preserves eIF2B activity by maintaining eIF2B's conformation in its active A-State.

RNA molecules are known to fold into specific structures which often play a central role in their functions and regulation. In silico folding of RNA transcripts, especially when assisted with structure profiling (SP) data, is capable of accurately elucidating relevant structural conformations. However, such methods scale poorly to the swaths of SP data generated by transcriptome-wide experiments, which are becoming more commonplace and advancing our understanding of RNA structure and its regulation at global and local levels. This has created a need for tools capable of rapidly deriving structural assessments from SP data in a scalable manner. One such tool we previously introduced that aims to process such data is patteRNA, a statistical learning algorithm capable of rapidly mining big SP datasets for structural elements. Here, we present a reformulation of patteRNA's pattern recognition scheme that sees significantly improved precision without major compromises to computational overhead. Specifically, we developed a data-driven logistic classifier which interprets patteRNA's statistical characterizations of SP data in addition to local sequence properties as measured with a nearest neighbour thermodynamic model. Application of the classifier to human structurome data reveals a marked association between detected stem-loops and RNA binding protein (RBP) footprints. The results of our application demonstrate that upwards of 30% of RBP footprints occur within loops of stable stem-loop elements. Overall, our work arrives at a rapid and accurate method for automatically detecting families of RNA structure motifs and demonstrates the functional relevance of identifying them transcriptome-wide.

Chronic myeloid leukemia (CML) is an uncommon type of white blood cells cancer that originates from bone marrow stem cells. Progesterone (P4) and berberine (BBR) are bioactive compounds that inhibit the growth of tumor cells. The present study aimed to assess the simultaneous effect of P4 and BBR on the level of reactive oxygen species (ROS) in K562 cells. The K562 cells were cultured in a complete cell culture medium and simultaneously exposed to IC50 different concentrations of P4 and BBR at 24 h (P4:102.4 μM; BBR:125 μM), 48 h (P4:78.4 μM; BBR:114 μM), and 72 h (P4:70 μM; BBR:45 μM). Then, the cell viability and cellular ROS level were determined using MTT assay and 2′, 7′-dichlorofluorescin diacetate (DCF) by flow cytometry, respectively. Our results showed that the combination of P4 and BBR inhibited the cells more effectively than P4 and BBR alone at 72 h, as well as P4 and their combination reduced ROS level in the cells compared to BBR and untreated cells at 24 h and 72 h. Taken together, P4 through a ROS-dependent pathway and BBR through a ROS-independent pathway may be inhibited cell growth.

To electrochemically measure human myeloid leukemia cells (K562 cells), we constructed a probe consisting of peptide/single‐strand (ss) DNA. Ac‐H6Y4C with an acetylated N‐terminal of peptide was used to enhance the probe to allow electrode responses that could detect target cells. A ss‐DNA was selected as the target cell recognition moiety. The probe exhibits properties that combine the functionalities of both DNA and peptides. The measurement principle is based on changes in the peak currents of the peptide moieties that are caused by interactions between the ss‐DNA and target cells. The peak currents were proportional to the concentration of K 562 cells that ranged from 10 to 2,000 cells/mL with a LOD of 3 cells/mL. [ABSTRACT FROM AUTHOR]

Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the Philadelphia chromosome. Therefore, K562 cells have been widely used for investigating the BCR/ABL1 oncogene and the tyrosine kinase inhibitor, imatinib-mesylate. Further, K562 cells overexpress transferrin receptors (TfR) and have been used as a model for targeting cytotoxic therapies, via receptor-mediated endocytosis. Here, we have characterized K562 cells focusing on the karyotype of cells in prolonged culture, regulation of expression of TfR in wildtype (WT) and doxorubicin-resistant cells, and responses to histone deacetylase inhibition (HDACi). Karyotype analysis indicates novel chromosomes and gene expression analysis suggests a shift of cultured K562 cells away from patient-derived leukemic cells. We confirm the high expression of TfR on K562 cells using immunofluorescence and cell-surface receptor binding radioassays. Importantly, high TfR expression is observed in patient-derived cells, and we highlight the persistent expression of TfR following doxorubicin acquired resistance. Epigenetic analysis indicates that permissive histone acetylation and methylation at the promoter region regulates the transcription of TfR in K562 cells. Finally, we show relatively high expression of HDAC enzymes in K562 cells and demonstrate the chemotoxic effects of HDACi, using the FDA-approved hydroxamic acid, vorinostat. Together with a description of morphology, infrared spectral analysis, and examination of metabolic properties, we provide a comprehensive characterization of K562 cells. Overall, K562 cell culture systems remain widely used for the investigation of novel therapeutics for CML, which is particularly important in cases of imatinib-mesylate resistance. [ABSTRACT FROM AUTHOR]