K562 Erythroleukemia Cells Research Articles

Effects of cholinergic compounds and TNF-alpha on human erythroleukemia K562 cell proliferation and caspase expression

Objective: The purpose of this study was to investigate ifstimulating auto-paracrine muscarinic receptor signalling pathwaycould change human erythroleukemia K562 cell proliferation andcaspase 3, 8 and 9 expression levels. To better understand the role ofmuscarinic receptors in cell signalling mechanism, we investigatedthe effects of several compounds on human erythroleukemiaK562 cell proliferation and caspase 3, 8 and 9 expression. Thesecompounds were M3 muscarinic receptor agonist, pilocarpine, proinflammatorycytokine, tumor necrosis factor (TNF)-alpha, andthe wortmannin which is a phosphoinositide 3-kinase inhibitor.Materials and Methods: Cell proliferation and cell viabilitywere evaluated by the trypan blue exclusion test and 5-Bromo-2-deoxy-uridine (BrdU) Labelling and Detection Kits. Caspase 3, 8and 9 expression levels were determined by immunoblot analysis.Results: Both pilocarpine and TNF-alpha caused a small increasein human erythroleukemia K562 cell proliferation. However, whenall the compounds were treated together, proliferation of humanerythroleukemia K562 cells increased significantly when compared tountreated control cells. TNF-alpha and wortmannin treatment increasedcaspase 3 and caspase 8 expression patterns significantly in humanerythroleukemia K562 cells. TNF-alpha and wortmannin treatmentincreased caspase 9 expression level (P>0.05) but it was not significant.Conclusion: These findings partly demonstrated that M3muscarinic receptor mediated an increase in K562 cell proliferation.Pilocarpine prevented TNF-alpha and wortmannin inducedcaspase 3 and 8 expression and indirectly showed apoptosis inhuman erythroleukemia K562 cells.

Efficient CRISPR/Cas9-Mediated Gene Editing of Pklr in Human Hematopoietic Progenitors and Stem Cells for the Gene Therapy of Pyruvate Kinase Deficiency

Pyruvate kinase deficiency (PKD) is the most common erythroid inherited enzymatic defect causing chronic nonspherocytic hemolytic anemia. PKD is an autosomal recessive disorder caused by mutations in the PKLR gene, which led in a total or partial reduction of the activity of the erythroid pyruvate kinase (RPK) protein. To date, more than 200 different mutations in the PKLR gene have been related with PKD. The disease is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. Treatments for PKD are mainly palliative, including regular red blood cell transfusion, splenectomy and iron chelation therapy. Allogeneic hematopoietic stem cell transplant (HSCT) represents the only curative treatment for severely affected patients, so far. Autologous HSCT of genetically corrected cells would offer a durable and curative clinical option. Over the last years, gene editing has emerged as a promising gene therapy approach for blood cell disorders, where genetic alterations can be accurately corrected. The high level of correction got in hematopoietic progenitors and stem cells without remarkable off-target effects suggests that the clinical use of gene editing therapy to correct genetic hematopoietic diseases is highly likely in a short term. Here, we present two gene editing approaches to correct PKD in human hematopoietic cells based of specific point mutation correction and in cDNA knock-in of a codon optimized version the RPK cDNA. We have designed both strategies to maintain the endogenous regulation of RPK once the gene editing has been carried out. First, we designed specific sgRNA targeting NM_000298.5(PKLR):c.359C>T mutation reported as pathogenic in PKD patients, and a single-stranded donor oligonucleotide (ssODN) to correct this mutation. When both sgRNA/Cas9 ribonucleoprotein (RNP) and ssODN were nucleofected together in a heterozygous PKD patient-lymphoblastic cell line (PKD LCL), around 5% of total alleles were correctly edited.In a second strategy, we developed a knock-in gene editing strategy at the genomic starting site of the PKLR gene by combining RNP electroporation and the adeno-associated viral vector (AAV) delivery of the recombination matrix. Specific gRNAs generating up to 60% indels at the RPK starting site were generated. Two different AAV constructs flanked by specific homologous arms were generated to delivery either a TurboGFP expression cassette or a promotor-less therapeutic coRPK. Up to 60% donor integration and stable expression of turbo EGFP and of coRPK driven by PKLR endogenous promoter was obtained in K562 erythroleukemia cells. Similar gene editing efficacies were obtained in human CB-CD34+. Specific integration and stable expression of the transgenes were detected in up to 30% colony forming units (CFUs). Moreover, gene edited cells engrafted efficiently in NSG mice. These results demonstrate the feasibility of editing the PKLR locus in hematopoietic progenitors and hematopoietic stem cells at efficiencies that could be clinically applicable to treat Pyruvate Kinase deficiency. DisclosuresBueren:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Porteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Identification of the Genetic Variant Responsible for Variable Platelet CD36 Expression By Massively Parallel Reporter Assay

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low density lipoprotein (oxLDL) results in platelet activation. Deletion of CD36 in mice fed a high fat diet results in attenuation of the pro-thrombotic state and platelet hyper-activity. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk. Platelet CD36 expression is highly variable with 3-8% of non-white populations displaying a complete lack of expression. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative.We have previously reported platelet expression Quantitative Trait Loci (eQTLs) that associate single nucleotide polymorphisms (SNPs) with platelet RNA levels, indicating genetic influence on gene expression. CD36 is one of the 612 platelet-expressed RNAs whose abundance has significant genotypic associations, suggesting a mechanism by which CD36 level is regulated. 86 eQTL SNPs located within a +/- 100kb window of the CD36 gene are associated with platelet CD36 mRNA levels at a significance of P<1x10-6, spanning a range of 125kb. Rather than prioritizing these 86 variants for functional testing based on genomic annotation, we opted to use an approach termed a Massively Parallel Reporter Array (MPRA), in which all 86 SNPs were tested simultaneously. A library of plasmids was generated in which both alleles of the variants including 150bp of surrounding genomic context were cloned 5' to a minimal promoter and unique barcode. For statistical robustness, 40 barcodes were constructed per allele, resulting in a library containing a total of ~5000 plasmids that were generated and transfected into K562 erythroleukemia cells. RNA-Seq was then conducted on the transfected cells and the abundance of individual barcodes was quantified, normalized to the DNA plasmid library, and then compared across variant alleles.The MPRA analysis led to identification of 5 SNPs located within a 35kb region 5‘ to the CD36 transcription start site that had significantly different barcode production between alleles. These 5 SNPs are in linkage disequilibrium with one another, with variants reported to be associated with CD36 level, and with variants reported to be associated with platelet count and volume by GWAS. To verify the functionality of these variants, we conducted luciferase assays in which the genomic sequence surrounding the variants were inserted 5‘ to the reporter gene. The genomic fragment containing the alternate alleles of two of these SNPs, rs2366739 and rs1194196, located within 29bp of each other, demonstrated 25-40% lower transcriptional activity than the reference alleles. Next, to determine if these two variants altered the interaction of DNA-binding proteins with the locus, we conducted electromobility-shift assays (EMSA) using probes derived from both alleles of the genomic sequence. As with the MPRA and luciferase assays, the alternative alleles of rs2366739 and rs1194196 resulted in lower levels of DNA:protein complexes, indicating an altered affinity for regulatory complexes. Finally, to further confirm the role of this genomic locus in regulating CD36 expression levels, we deleted a 596bp region containing these two SNPs from the K562 erythroleukemia cell genome using CRISPR/Cas. Deletion of this region resulted in an approximately 10-fold increase in CD36 RNA in these cells, confirming a role for this loci in the regulation of CD36 expression.These data indicate that we have identified a regulatory locus for CD36 expression, which has important implications for platelet function in cardiovascular disease. These variants are located within a retrotransposon long-terminal repeat and alter a putative GATA1 binding site, suggesting that these variants may alter binding of that transcription factor to the locus. Platelet CD36 expression level has been linked to the platelet response to oxLDL and may modulate the thrombotic response in hyperlipidemic conditions. Therefore, the mechanism described here, in which a genetic variant alters platelet CD36 levels, and thus the platelet response to hyperlipidemic conditions, may serve as an important biomarker for increased thrombotic potential. DisclosuresNo relevant conflicts of interest to declare.

The Effect of the Soluble Guanylyl Cyclase Stimulator Olinciguat on ƴ-Globin Gene Induction in K562 Cells

Induction of fetal hemoglobin (HbF: α2ƴ2) is a recognized mode of action of hydroxyurea, the sickle cell disease (SCD) standard of care in SCD, and has been shown to prevent red blood cell (RBC) sickling. Discovery of novel HbF inducers is underway and several therapeutics with the potential to increase HbF expression are currently at different stages of preclinical and clinical development. Soluble guanylyl cyclase (sGC) is a heterodimeric heme-containing enzyme whose catalytic activity is regulated by nitric oxide (NO). Binding of NO to heme activates the catalytic domain of sGC, enabling synthesis of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. sGC stimulators are small molecules that synergize with NO to boost signaling via the NO-sGC-cGMP pathway. This signaling pathway is involved in the regulation of many physiologic processes including inflammation, fibrosis, and blood flow. Perhaps less well-known, cGMP-mediated signaling has also been implicated in the regulation of the gene encoding the ƴ-globin subunit of fetal hemoglobin (Modulation of NO signaling by sGC stimulation, therefore, has the therapeutic potential to target the complex pathology of SCD at multiple levels. In this study, we focused on one potential mode of action of sGC stimulation-increasing HbF expression. We characterized the effects of the sGC stimulator olinciguat on ƴ-globin gene expression. Olinciguat is currently being investigated for the treatment of patients with SCD in a Phase II STRONG-SCD study (NCT03285178).The effect of olinciguat treatment on ƴ-globin mRNA levels was studied in the K562 erythroleukemic cell line. For short-term (8 hours) treatment with olinciguat, K562 cells were maintained in a serum-free media. For long-term (4 and 7 days) treatment, cell culture media contained 1% fetal bovine serum. Hydroxyurea was used as a positive control. Levels of ƴ-globin mRNA were expressed relative to mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase.K562 cells were treated for 8 hours with increasing concentrations of olinciguat (0.01, 0.1, 1, and 10 µM). Treatment of K562 cells with 0.1, 1, and 10 µM of olinciguat increased ƴ-globin mRNA levels by 1.43±0.08-, 1.37±0.06-, and 1.47±0.06-fold (mean±SEM), respectively. For comparison, 8 hours of treatment with hydroxyurea (800 µM) increased ƴ-globin mRNA levels by 1.25±0.03-fold. When K562 cells were cultured in the presence of olinciguat for 4 days, significant (P<0.05) induction of ƴ-globin mRNA levels was observed at 1 and 10 µM (1.13±0.03- and 1.55±0.09-fold, respectively). Induction of ƴ-globin mRNA following 4 days of incubation with hydroxyurea (800 µM) was 2.38±0.2-fold. The effects of hydroxyurea and olinciguat on ƴ-globin mRNA levels were compared following 7 days of incubation with the compounds. After 7 days of treatment of K562 cells with 0.1, 1, 3, and 10 µM of olinciguat, ƴ-globin mRNA levels were increased by 1.83±0.19-, 1.66±0.09-, 2.4±0.06-, and 2.9±0.33-fold, respectively. Treatment with 50- and 800-µM hydroxyurea increased levels of ƴ-globin mRNA by 2.33±0.15- and 3.8±0.56-fold, respectively.In conclusion, the sGC stimulator olinciguat increased the expression of mRNA for the ƴ-globin subunit of fetal hemoglobin in the erythroleukemic K562 cell line. This finding indicates that amplifying NO signaling by stimulating sGC may increase HbF expression, thereby preventing pathologic RBC sickling; this extends the potential therapeutic utility of olinciguat in SCD. Finally, the ability of olinciguat to induce HbF in SCD patients will be assessed in the ongoing Phase II STRONG-SCD study (NCT03285178). DisclosuresMiyashiro:Ironwood Pharmaceuticals: Employment. Pant:Ironwood Pharmaceuticals: Employment. Tchernychev:Ironwood Pharmaceuticals: Employment, Equity Ownership. Milne:Ironwood Pharmaceutics, Inc: Employment. Currie:Ironwood Pharmaceuticals: Employment. Graul:Ironwood Pharmaceuticals, Inc: Employment. Masferrer:Ironwood Pharmaceuticals, Inc: Employment.

KIR Expression on inVitro-Derived Natural Killer Cells Does Not Regulate Killing of Allogeneic Targets

Natural killer (NK) cells play an essential role in early innate killing of virally-infected and tumor targets. NK cell-mediated activity is regulated by a repertoire of activating and inhibitory receptors that recognize ligands on diseased, stressed, or tumor targets. Killer cell immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors that can be inhibitory or activating based on their intracellular signaling motifs. Expression of certain KIR haplotypes plays a key role in survival and relapse prevention for patients with acute myelogenous leukemia (AML) who receive allogeneic hematopoietic cell transplantation. Therefore, KIR haplotypes are an important consideration in selecting allogeneic donors for patients with AML. However, it is unclear if KIRs play a role in adoptive transfer of NK cells that are becoming more routinely utilized to treat refractory AML and other malignancies. To better address this question we used umbilical cord blood to isolate both CD34+ hematopoietic stem cells and CD45+CD56+ NK cells (UCB-56-NK) from the same umbilical cord blood unit. The CD34+ hematopoietic stem cells were then differentiated in vitro into CD56+ NK cells (UCB-34-NK). Despite originating from the same donor and sharing the same genetic background, as well as comparable expression of Fas ligand, TRAIL, NKp46, NKp44, NKG2A, and NKG2D, the UCB-34-NK cells have characteristically low KIR expression, whereas the UCB-56-NK cells have high KIR expression. This phenotype was further confirmed by mass cytometric (CyTOF) analysis of UCB-56-NK cells and UCB-34-NKcells with a panel of 36 phenotypic and functional NK cell markers. This unique system allows us to study the role of KIR expression independent of any other variations in donor or cell characteristics. The cytotoxicity and NK cell activation of UCB-34-NK cells and UCB-56-NK cells are compared to control NK cells isolated from peripheral blood (PB-NK cells) with standard in vitro cytotoxicity assays against neuroblastoma lines with varying HLA genotypes and a control K562 leukemic targets. Our data demonstrates that there is no statistical difference in NK cytotoxicity and activation of UCB-34-NK cells and UCB-56-NK cells across a spectrum of target cell HLA types, despite the differences in KIR expression. For example, at effector to target (E:T) ratios of 1:5 and 1:20 against neuroblastoma line IMR32, UCB-34-NK cells (KIR-low) demonstrated 68.5% and 84.1% maximal Caspase 3/7 activation, compared to 81.3% and 89.6% by UCB-56-NK cells (KIR-high). Additionally, we have used human induced pluripotent stem cells to derive NK cells (iPSC-NK cells) that vary in KIR expression levels. These CD45+CD56+ iPSC-NKs are differentiated from the same well-established iPSC line in the laboratory and therefore again share the same genetic background, and they have similar NK cell surface receptor expression of Fas ligand, TRAIL, NKp46, NKp44, and NKG2D, but differ in levels of KIR expression. Again in vitro cytotoxicity against hematopoietic tumor targets such as K562 and MOLM13 do not demonstrate a significant difference in killing, despite these KIR differences. For example, in targeting erythroleukemia K562 cells, iPSC-NK cells with high levels of KIR expression at E:T ratios of 1:2.5, 1:5, and 1: 10 have Caspase 3/7 activation of 21.1%, 28.2%, and 41.0%, compared to 20.1%, 22.0%, and 31.2% by iPSC-NK cells with low KIR expression. Together, these studies demonstrate that in vitro-derived NK cells do not require KIR expression to become licensed for anti-tumor activity and these cells are able to kill tumor targets whether or not they express KIRs. These studies better enable use of these allogeneic NK cell populations for off-the-shelf NK cell-based therapies without the need to optimize for KIR profiles for patients of differing HLA haplotypes. DisclosuresMalmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Kaufman:Fate Therapeutics: Consultancy, Research Funding.

Stem cell factor and NSC87877 synergism enhances c-Kit mediated proliferation of human erythroid cells

The biological mechanisms underlying the effects of stem cell factor (SCF) and an inhibitor, NSC87877 (N) of the c-Kit negative regulator (SHP-1 and SHP-2) on cell proliferation are different. Therefore, we compared the cell's response to these two either alone or in combination in K562 cells. Binding of SCF (S) to c-Kit induces dimerization that activates its kinase activity. The activated c-Kit undergoes autophosphorylation at tyrosine residues that serve as a docking site for signal transduction molecules containing SH2 domains. Predominantly, the phosphotyrosine 568 (pY568) in Juxtamembrane (JM) region of c-Kit interacts with adaptor protein APS, Src family kinase, and SHP-2, while phosphotyrosine 570 (pY570) interacts with the SHP-1 and the adaptor protein Shc. The dephosphorylation of phosphotyrosine residues by SHP-1/SHP-2 leads to inhibition of c-Kit proliferative signaling. A chemical molecule, N is reported to inhibit the enzymatic activity of SHP-1/SHP-2, but its effect on c-Kit-mediated proliferation has not been studied yet. Thus, this work aims at examining the effect of the combination of S and N on cells growth as compared to individual treatment. The present study is performed with erythroleukemic K562 cells, chosen for its mRNA expression concerning the c-Kit, and SHP-1/SHP-2. Interestingly, proliferation assay showed that combination significantly increased proliferation when G1 sorted K562 cells were used. These changes were significantly higher when K562 cells were initially treated with N followed by S treatment. Collectively, these results give mechanistic insight into the proliferation enhancement of bone marrow transplantation through the synergistic effect of S and N by inhibiting SHP-1/SHP-2. The study gives solid evidence that S and N combination can be used to enhance cell proliferation/growth.

Design, synthesis, and biological evaluation of novel nitric oxide releasing dehydroandrographolide derivatives

AbstractA series of new hybrids of dehydroandrographolide (TAD), a biologically active natural product, bearing nitric oxide (NO)-releasing moieties were synthesized and designated as NO-donor dehydroandrographolide. The biological activities of target compounds were studied in human erythroleukemia K562 cells and breast cancer MCF-7 cells. Biological evaluation indicated that the most active compound I-5 produced high levels of NO and inhibited the proliferation of K562 (IC50 1.55 μmol·L−1) and MCF-7 (IC50 2.91 μmol·L−1) cells, which were more potent than the lead compound TAD and attenuated by an NO scavenger. In conclusion, I-5 is a novel hybrid with potent antitumor activity and may become a promising candidate for future intensive study.

Benzofuran hydrazones as potential scaffold in the development of multifunctional drugs: Synthesis and evaluation of antioxidant, photoprotective and antiproliferative activity

New benzofuranhydrazones 3–12 were easily prepared and assayed for their radical-scavenging ability. Hydrazones 3–12 showed different extent antioxidant activity in DPPH, FRAP and ORAC assays. Good antioxidant activity is related to the number and position of hydroxyl groups on the arylidene moiety. High antioxidant activity is showed by the 2-hydroxy-4-(diethylamino)benzylidene derivative 11. Furthermore, hydrazones 3–12 showed photoprotective capacities with satisfactory in vitro SPF as compared to the commercial PBSA sunscreen filter. The antiproliferative effects of the hydrazones 3–12 was tested on erythroleukemia K562 and Colo-38 melanoma human cells. All the compounds showed growth inhibition in the micromolar to sub micromolar concentration range. If taken together these results points to benzofuran hydrazones as potential multifunctional molecules especially in the treatment of neoplastic diseases being the good antioxidant properties of 5, 7 and 11 correlated to their high antiproliferative activity.

Distinct Hypericum perforatum L. total extracts exert different antitumour activity on erythroleukemic K562 cells.

Total flower extracts of Hypericum perforatum L. obtained with 3 different solvent systems were tested on tumour cell line cultures by comparing two groups of plants harvested in different times and places. The extracts, characterized according to the spectroscopic profile and the hypericin content, were tested on the growth and apoptotic death of K562 cells, a human erythroleukemic cell line. Growth and apoptosis were analysed by viable cell count, flow cytometry, and fluorescence microscopy at 6, 24, and 48hr of culture following 1 hr exposure to the extracts under investigation. Here, we show that Hypericum extracts are able to reduce the growth of K562 cells and induce different degrees and kinetics of apoptosis according to the group of plants of origin. Also, we highlighted interesting differences in terms of efficacy among the extracts, with some samples losing their effectiveness along the culture time and others able to maintain or even increase their efficacy. Furthermore, the data herein obtained confirm the role of non hypericin compounds that are present in different proportions in the two plant groups and in the extracts analysed.

P-304 - Glutathione in redox regulation of the development of cancer cell resistance

The ratio of reduced glutathione to its oxidized form (GSH/GSSG) is essential for cell viability including its role in redox-dependent regulation of gene expression. The aim of our study was to estimate GSH/GSSG ratio and expression of redox-dependent genes (glutaredoxin, thioredoxin, peroxiredoxin) under development of cancer cell resistance to cisplatin (CDDP) possessed pro-oxidant action. Under development of resistance of human erythroleukemia K562 and ovarian carcinoma SKOV-3 cells to CDDP co-ordinative enhanced expression of genes encoding glutathione synthetase (GS), subunits of γ-glutamylcysteine synthetase (γ-GCSH, γ-GCSL) was found. In addition, the growth of GSH/GSSG, enhanced expression of glutaredoxin (GLRX1, GLRX2), thioredoxin (TRX2), peroxiredoxin (PRDX6) genes as well as elevated level of transcription factor Nrf2 were observed in both types of resistant cells. We suggest that mechanism of development of cancer cell resistance to CDDP can include activation of GSH synthesis de novo, redox-dependent increase in expression of GLRX1, GLRX2, TRX2, PRDX6 genes which are co-ordinative regulated by redox-dependent transcription factor Nrf2 and elevated level of GSH/GSSG. The publication was prepared with the support of the “RUDN University Program 5–100”.

NF90/ILF3 is a transcription factor that promotes proliferation over differentiation by hierarchical regulation in K562 erythroleukemia cells.

NF90 and splice variant NF110 are DNA- and RNA-binding proteins encoded by the Interleukin enhancer-binding factor 3 (ILF3) gene that have been established to regulate RNA splicing, stabilization and export. The roles of NF90 and NF110 in regulating transcription as chromatin-interacting proteins have not been comprehensively characterized. Here, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) identified 9,081 genomic sites specifically occupied by NF90/NF110 in K562 cells. One third of NF90/NF110 peaks occurred at promoters of annotated genes. NF90/NF110 occupancy colocalized with chromatin marks associated with active promoters and strong enhancers. Comparison with 150 ENCODE ChIP-seq experiments revealed that NF90/NF110 clustered with transcription factors exhibiting preference for promoters over enhancers (POLR2A, MYC, YY1). Differential gene expression analysis following shRNA knockdown of NF90/NF110 in K562 cells revealed that NF90/NF110 activates transcription factors that drive growth and proliferation (EGR1, MYC), while attenuating differentiation along the erythroid lineage (KLF1). NF90/NF110 associates with chromatin to hierarchically regulate transcription factors that promote proliferation and suppress differentiation.

High-level embryonic globin production with efficient erythroid differentiation from a K562 erythroleukemia cell line

A reliable cell line capable of robust in vitro erythroid differentiation would be useful to investigate red blood cell (RBC) biology and genetic strategies for RBC diseases. K562 cells are widely utilized for erythroid differentiation; however, current differentiation methods are insufficient to analyze globin proteins. In this study, we sought to improve erythroid differentiation from K562 cells to enable protein-level globin analysis. K562 cells were exposed to a variety of reagents, including hemin, rapamycin, imatinib, and/or decitabine (known erythroid inducers), and cultured in a basic culture medium or erythropoietin-based differentiation medium. All single reagents induced observable erythroid differentiation with higher glycophorin A (GPA) expression but were insufficient to produce detectable globin proteins. We then evaluated various combinations of these reagents and developed a method incorporating imatinib preexposure and an erythropoietin-based differentiation culture containing both rapamycin and decitabine capable of efficient erythroid differentiation, high-level GPA expression (>90%), and high-level globin production at protein levels detectable by hemoglobin electrophoresis and high performance liquid chromatography. In addition, β-globin gene transfer resulted in detectable adult hemoglobin. In summary, we developed an in vitro K562 erythroid differentiation model with high-level globin production. This model provides a practical evaluation tool for hemoglobin production in human erythroid cells.

Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9

Cancer cells are commonly more resistant to cell death activated by the membranolytic protein complex C5b-9. Several surface-expressed and intracellular proteins that protect cells from complement-dependent cytotoxicity (CDC) have been identified. In this study, we investigated the function of heat shock protein 90 (Hsp90), an essential and ubiquitously expressed chaperone, overexpressed in cancer cells, in C5b-9-induced cell death. As shown, inhibition of Hsp90 with geldanamycin or radicicol is enhancing sensitivity of K562 erythroleukemia cells to CDC. Similarly, Hsp90 inhibition confers in Ramos B cell lymphoma cells elevated sensitivity to treatment with rituximab and complement. C5b-9 deposition is elevated on geldanamycin-treated cells. Purified Hsp90 binds directly to C9 and inhibits zinc-induced C9 polymerization, indicating that Hsp90 may act directly on the C5b-9 complex. Mortalin, also known as stress protein 70 or GRP75, is a mitochondrial chaperone that confers resistance to CDC. The postulated cooperation between Hsp90 and mortalin in protection from CDC was tested. Geldanamycin failed to sensitize toward CDC cells with knocked down mortalin. Direct binding of Hsp90 to mortalin was shown by co-immunoprecipitation in cell extracts after triggering with complement as well as by using purified recombinant proteins. These results provide an insight into the protective mechanisms utilized by cancer cells to evade CDC. They suggest that Hsp90 protects cells from CDC by inhibiting, together with mortalin, C5b-9 assembly and/or stability at the plasma membrane.

Design and synthesis of new potent N,N-bis(arylalkyl)piperazine derivatives as multidrug resistance (MDR) reversing agents

A series of 1,4-substituted arylalkyl piperazine derivatives were synthesized and studied with the aim to obtain potent P-gp-dependent multidrug-resistant (MDR) reversers. The new compounds were designed on the basis of the structures of our previous arylamine ester derivatives endowed with high P-gp-dependent multidrug resistance reversing activity. All new compounds were active in the pirarubicin uptake assay on the doxorubicin–resistant erythroleukemia K562 cells (K562/DOX). In particular, compounds bearing methoxy aromatic moieties showed fairly high reversal activities. The most potent compounds, 8, 9, 10 and 13, were further studied by evaluating their doxorubicin cytotoxicity enhancement (reversal fold, RF) and the inhibition of P-gp-mediated rhodamine-123 (Rhd 123) efflux on the K562/DOX cell line. The results of all pharmacological assays indicated that the combination of a basic piperazine scaffold with arylalkyl residues allowed us to obtain very interesting P-gp modulating compounds. Two long-lasting P-gp pump modulators (9 and 10) were identified; they were able to inhibit remarkably the P-gp substrate rhodamine-123 efflux on the resistant K562/DOX cell line after 60 min. Overall compound 9 appeared the most promising compound being a potent and long-lasting P-gp–dependent MDR modulator.

Psychoneuroimmunology and Natural Killer Cells: The Chromium-Release Whole-Blood Assay.

Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter describes a chromium (51Cr)-release bioassay designed to measure to the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 erythroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4-h in vitro assay.

Genomic landscape of human erythroleukemia K562 cell line, as determined by next-generation sequencing and cytogenetics

We have performed detailed analysis of the genomic landscape of commercially available K562 cells, employing targeted enrichment of nearly 1300 cancer-related genes followed by next-generation sequencing (NGS) and also classical cytogenetics. Deep sequencing revealed 88 variants of potentially biological significance. Among them we have detected alterations in genes already known to be mutated in K562, such as TP53 but also in several other genes, which are implicated in tumorigenesis and drug resistance, such as MLH1, ASXL1 and BRCA1 as the most prominent examples. Fluorescence in situ hybridization (FISH) of interphases of K562 cells revealed multiplication of the BCR and ABL1 gene copies, as well as the amplification of the BCR-ABL1 fusion gene. Our results may help to better understand genomic instability of the blastic phase of CML represented by the K562 cell line and can help researchers who want to employ this cell line in various experimental settings.

Functional interrelationship between TFII-I and E2F transcription factors at specific cell cycle gene loci.

Transcription factor TFII-I is a multifunctional protein implicated in the regulation of cell cycle and stress-response genes. Previous studies have shown that a subset of TFII-I associated genomic sites contained DNA-binding motifs for E2F family transcription factors. We analyzed the co-association of TFII-I and E2Fs in more detail using bioinformatics, chromatin immunoprecipitation, and co-immunoprecipitation experiments. The data show that TFII-I interacts with E2F transcription factors. Furthermore, TFII-I, E2F4, and E2F6 interact with DNA-regulatory elements of several genes implicated in the regulation of the cell cycle, including DNMT1, HDAC1, CDKN1C, and CDC27. Inhibition of TFII-I expression led to a decrease in gene expression and in the association of E2F4 and E2F6 with these gene loci in human erythroleukemia K562 cells. Finally, TFII-I deficiency reduced the proliferation of K562 cells and increased the sensitivity toward doxorubicin toxicity. The results uncover novel interactions between TFII-I and E2Fs and suggest that TFII-I mediates E2F function at specific cell cycle genes.

Clostridium perfringens \u03b1-toxin impairs erythropoiesis by inhibition of erythroid differentiation

Clostridium perfringens α-toxin induces hemolysis of erythrocytes from various species, but it has not been elucidated whether the toxin affects erythropoiesis. In this study, we treated bone marrow cells (BMCs) from mice with purified α-toxin and found that TER119+ erythroblasts were greatly decreased by the treatment. A variant α-toxin defective in enzymatic activities, phospholipase C and sphingomyelinase, had no effect on the population of erythroblasts, demonstrating that the decrease in erythroblasts was dependent of its enzymatic activities. α-Toxin reduced the CD71+TER119+ and CD71–TER119+ cell populations but not the CD71+TER119− cell population. In addition, α-toxin decreased the number of colony-forming unit erythroid colonies but not burst-forming unit erythroid colonies, indicating that α-toxin preferentially reduced mature erythroid cells compared with immature cells. α-Toxin slightly increased annexinV+ cells in TER119+ cells. Additionally, simultaneous treatment of BMCs with α-toxin and erythropoietin greatly attenuated the reduction of TER119+ erythroblasts by α-toxin. Furthermore, hemin-induced differentiation of human K562 erythroleukemia cells was impaired by α-toxin, whereas the treatment exhibited no apparent cytotoxicity. These results suggested that α-toxin mainly inhibited erythroid differentiation. Together, our results provide new insights into the biological activities of α-toxin, which might be important to understand the pathogenesis of C. perfringens infection.

P 092 - Coordinated alteration of expression of redox-dependent genes in development of adaptive antioxidant response under formation of drug resistance of cancer cells

Aims Here we studied the expression of isoforms of redox-dependent antioxidant enzymes and a rise of adaptive antioxidant response under development of cancer cells resistance to anticancer agent cisplatin (CDDP) possessed pro-oxidant action. Results Under development of resistance of human erythroleukemia K562 and ovarian carcinoma SKOV-3 cells to CDDP co-ordinative enhanced expression of genes encoding isoforms of superoxide dismutase (SOD1, SOD2), glutathione peroxidase (GPx1, GPx4), glutathione transferase (GSTP1-1, GSTМ1-1), glutaredoxin (GLRX1, GLRX2), thioredoxin (TRX1,TRX2) was found in both types of resistant cells in compare with wild cells. In addition, growth of GSH/GSSG ratio as index of cellular redox state as well as elevated level of transcription factor Nrf2 were observed in resistant K562/DOX and SKVLB cells. Decrease of apoptotic cell death was observed under exposure to hydrogen peroxide in the CDDP-resistant cells possessed elevated antioxidant status, as evidenced by low generation of ROS. It can be concluded that the mechanism of the formation of cancer cells resistance to CDDP involves the development of adaptive antioxidant response as key in stress adaptation.

Lectin CGL from the sea mussel Crenomytilus grayanus induces Burkitt’s lymphoma cells death via interaction with surface glycan

Marine organisms are rich sources of lectins. Lectins are able to bind specifically and reversibly to different types of carbohydrates or glycoproteins. The present study reports the evaluation of glycan binding profile and anti-tumor potential of lectin CGL from the sea mussel Crenomytilus grayanus. Glycan array assay revealed that CGL was able to bind both α and β anomer of galactose, but interaction with the αGal-terminated glycans was stronger. Analysis of most common glycan motifs for CGL showed high affinity to Galα1-4Galβ1-4GlcNAc motif similar to globotriose structure (Gb3: Galα1-4Galβ1-4Glc), the epitope of globotriaosylceramide. CGL recognized Gb3 on the surface of Burkitt‘s lymphoma Raji cells (high Gb3 expression), leading to dose-dependent cytotoxic effect, G2/M phase cell cycle arrest and apoptosis. Lectin had no effect on erythroleukemia K562 cells (no Gb3 expression). The activity of CGL was specifically blocked by α-galactoside. Our findings suggest the use of CGL in cancer diagnosis and treatment.